Methicillin-Resistant Staphylococci in Animals: Veterinary and Public Health Implications

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Final Program and Abstracts

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals: Veterinary and Public Health Implications

ASM Conferences Information...... 2

Conference Organization...... 3

Acknowledgments...... 3

General Information...... 4

Travel Grants...... 6

Scientific Program...... 7

Abstracts for Speakers...... 13

Abstracts for Posters...... 38

Index...... 112

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 1 ASM Conferences Committee

William E. Goldman, Chair Lora Hooper University of North Carolina, Chapel Hill University of Texas Southwestern Medical Center Sean Whelan, Vice Chair Harvard Medical School Gary Procop Cleveland Clinic Victor DiRita University of Michigan Curtis Suttle University of British Columbia Joanna Goldberg Emory University

ASM Conferences Mission

To identify emerging or underrepresented topics of broad scientific significance.

To facilitate interactive exchange in meetings of 100 to 500 people.

To encourage student and postdoctoral participation.

To recruit individuals in disciplines not already involved in ASM to ASM membership.

To foster interdisciplinary and international exchange and collaboration with other scientific organizations.

2 ASM Conferences Program Committee

Luca Guardabassi, Co-chair Engeline van Duijkeren University of Copenhagen, Denmark National Institute for Public Health, the Netherlands Robert Skov, Co-chair and ESCMID liaison Birgit Strommenger Statens Serum Institut, Denmark Robert Koch Institute, Germany

Scott Weese, ASM liaison Andreas Voss University of Guelph, Canada Canisius-Wilhelmina Hospital & University Medical Centre, the David Bemis Netherlands University of Tennessee, US Lothar Heinz Wieler Freie University Berlin, Germany

Acknowledgments he American Society for Microbiology and the European Society of Clinical TMicrobiology and Infectious Diseases gratefully acknowledge the following sponsors of the 3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals: Veterinary and Public Health Implications. On behalf both organizations, our leadership and members, we thank them for their financial support:

Silver Sponsors

ICF Thermo Fisher Scientific Zoetis

Bronze Sponsors

Bayer Dyrefondet Pfizer Denmark (in kind support of travel for Joseph Blondeau)

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 3 General Information

REGISTRATION AND NAME BADGES The posters are grouped by topic. ASM Staff will be available at the registration desk in the Ceremonial A Session Posters: Hall during session hours. Participants • Epidemiology of MRSA (1-21) may collect name badges and program • Diagnostics and Typing (22-52) materials at the registration desk. A name badge is required for entry into all B Session Posters: sessions, meals, and social events. • Epidemiology of MRSP (53-61) • Genomics, Virulence, Host-Specificity GENERAL SESSIONS and Evolution (62-79) All general sessions will be held in the • What is New (80-88) • Treatment and Control (89-102) Ceremonial Hall. CERTIFICATE OF ATTENDANCE POSTER SESSIONS Certificates of Attendance can be found Poster boards are located in the main in the registration packet received at the Foyer of the Ceremonial Hall. registration desk. Posters 1 – 52 will be displayed in Poster Note: Certificates of Attendance do not Session A on Tuesday. list session information.

Posters 53 – 102 will be displayed in CAMERAS AND RECORDINGS Poster Session B on Wednesday. POLICY Digital recorders, cameras (including Please check your assigned number in the camera phones) and video cameras abstract index. The same number is used (including video phones) are prohibited for the presentation and board number. in the poster hall and session room. Anyone found photographing, videotaping “A” session posters may be mounted or recording in the prohibited areas on the assigned board starting Monday will be asked to surrender their badge afternoon and must be mounted by no immediately and leave the conference. later than the morning coffee break on No refund will be provided. This rule is Tuesday. Posters in session “A” should be strictly enforced. removed at the end of the day on Tuesday. CHILD POLICY “B” session posters must be mounted by Children are not permitted in session no later than the morning coffee break on rooms, poster sessions, conference meals Wednesday. Posters in session “B” should or social events. be removed at the end of the conference on Thursday.

4 ASM Conferences General Information

CONFERENCE MEALS AND GUEST REGISTRATION SOCIAL EVENTS Registered participants may also register Registration includes the Welcome an accompanying guest (age 16 and older) Reception on Monday, November 4, to attend the Welcome Reception for an lunches on Tuesday, November 5 and additional fee of $100. Guests are not Wednesday, November 6, and coffee permitted in the lunches, coffee breaks, breaks throughout the meeting. All general sessions or poster sessions. events take place in the Ceremonial Hall. Guests must present their guest badge for entrance to the Welcome Reception. Non-registered guests are not permitted to attend any part of the conference, including social events.

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 5 Travel Grants

STUDENT TRAVEL GRANTS To encourage the participation of graduate students and new postdoctoral fellows at this conference, ASM and ESCMID have awarded travel grants of $500 to each of the following individuals:

Mohamed Abdelbary Ewan Harrison Matthew Saab Raghavendra Amachawadi Joost Hordijk Jisun Sun Britta Ballhausen Aneta Mroczkowska Joany Van Balen Michelle Chen Maya Nadimpalli Gianpiero Ventrella Meghan Davis Alim Nazarali Min Tao Wan Alejandro Dorado-Garcia Matthew Riley Thomas Groenthal Joana Rolo

6 ASM Conferences Scientific Program

Monday, November 4, 2013

5:00 – 6:00 pm Opening Session

Welcome Remarks Luca Guardabassi, University of Copenhagen Ulla Wewer, Dean of Faculty of Health and Medical Sciences, University of Copenhagen

Opening Keynote Lecture Staphylococci at the Human-Animal Interface Ross Fitzgerald, The Roslin Institute, University of Edinburgh, Edinburgh, UNITED KINGDOM

6:00 – 8:00 pm Welcome Reception at the Ceremonial Hall of the University of Copenhagen

Tuesday, November 5, 2013

9:00 – 11:00 am SESSION 1: Epidemiology of MRSA Chairman: Engeline van Dujikeren

9:00 – 9:30 am LA-MRSA: What Have We Learned and What Are We Still Missing? Robert Skov, Statens Serum Institut, Copenhagen, DENMARK

9:30 – 10:00 am Role of Plasmids in Antimicrobial (Multi)Resistance of LA- MRSA Stefan Schwarz, Institute of Farm Animal Genetics (FLI), Neustadt-Mariensee, GERMANY

10:00 – 10:15 am Strong Association Between MRSA Air Exposure and MRSA Carriage in Veal Calf and Pig Farmers Marian Bos, IRAS - Utrecht University, Utrecht, NETHERLANDS

10:15 – 10:30 am MRSA Contamination in the Vicinity of Poultry and Pig Farms in Germany Anika Friese, Freie Universität Berlin, Inst. for Animal Hygiene and Environmental Health, Berlin, GERMANY

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 7 Scientific Program

10:30 – 10:45 am Residential Proximity to Large Swine CAFOs is Associated with Increased Risk of MRSA Carriage at Time of Hospital Admission in Rural Iowa Veterans Margaret Carrel, University of Iowa, Iowa City, IA

10:45 – 11:00 am Environmental Contamination and Pet Colonization in the Households of People Diagnosed with a Community-acquired MRSA Infection Meghan Davis, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD

11:00 – 11:30 am Coffee Break and Poster Viewing

11:30 am – 1:00 pm SESSION 2: Epidemiology of MRSP Chairman: Scott Weese

11:30 am – 12:00 pm Evolution and Spread of Multidrug-resistant Staphylococcus pseudintermedius Strains Lothar Wieler, Institute of Microbiology and Epizootics, Berlin, GERMANY

12:00 – 12:30 pm Emergence of New MRSP Clones Stephen Kania, University of Tennessee, Knoxville, TN

12:30 – 12:45 pm Subtle Strain Variation of Staphylococcus pseudintermedius Complicates Correct Diagnostics in Infected Dogs Joost Hordijk, Utrecht University, Utrecht, NETHERLANDS

12:45 – 1:00 pm The Role of Methicillin Resistant Staphylococcus pseudintermedius Colonization as a Risk Factor for Development of Surgical Site Infections in Dogs Undergoing Tibial Plateau Leveling Osteotomy Alim Nazarali, University of Guelph, Guelph, ON, CANADA

1:00 – 2:00 pm Lunch

2:00 – 3:00 pm Poster Session A Posters 1-52 will be presented.

8 ASM Conferences Scientific Program

3:00 – 4:30 pm SESSION 3: Diagnostics and Typing Chairman: Dave Bemis

3:00 – 3:30 pm Untangling the Transmission Dynamics of MRSA Using Whole Genome Sequencing Matthew Holden, The Wellcome Trust Sanger Institute, Cambridge, UNITED KINGDOM

3:30 – 4:00 pm New MLST Scheme for S. pseudintermedius Vincent Perreten, Institute of Veterinary Bacteriology, University of Berne, Berne, SWITZERLAND

4:00 – 4:15 pm Prevalence of mecA-positive Methicillin-resistant Staphylococcus aureus in Pigs Exhibits Dose-response to Zinc Supplementation Raghavendra Amachawadi, Kansas State University, Manhattan, KS

4:15 – 4:30 pm Molecular Characterization of Methicillin-resistant and Methicillin-susceptible S. aureus (MRSA, MSSA) Clonal Complex 97 Isolates from Pigs and Cattle in Italy Antonio Battisti, IZSLT, Rome, ITALY

4:30 – 5:00 pm Coffee Break

5:00 – 6:00 pm Plenary Session on LA-MRSA Part 1: Definition of LA-MRSA Lothar Wieler and Lance Price Part 2: Control of MRSA Jaap Wagenaar and Andreas Voss

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 9 Scientific Program

Wednesday, November 6, 2013

9:00 – 11:00 am SESSION 4: Genomics, Virulence, Host Specificity and Evolution Chairman: Lothar Wieler

9:00 – 9:30 am Host Adaptation of LA-MRSA Jodi Lindsay, St. George’s, University of London, London, UNITED KINGDOM

9:30 – 9:45 am The Genome of MRSP ST71 Arshnee Moodley, University of Copenhagen, Frederiksberg, DENMARK

9:45 – 10:00 am New LA-MRSA Clones Emerging in Pigs: An Insight into ST433 Yvonne Agersø, National Food Institute, Technical University of Denmark, Lyngby, DENMARK

10:00 – 10:15 am Phylogenetics and Molecular Epidemiology of mecC-MRSA Isolates in Europe Ewan Harrison, University of Cambridge, Cambridge, UNITED KINGDOM

10:15 – 10:30 am Staphylococcus aureus ST398 Gene Expression Profiling During ex vivo Colonization of Porcine Nasal Epithelium Birgitta Duim, Utrecht University, Utrecht, NETHERLANDS

10:30 – 11:00 am Coffee Break and Poster Viewing

11:00 am – 1:30 pm SESSION 5: What’s New? Chairman: Birgit Strommenger

11:00 – 11:30 am The Population Structure of CC398 and the Emergence of Horse-associated Sub-clone Mohamed M. H. Abdelbary, Robert Koch Institute, Wernigerode, GERMANY

11:30 am – 12:00 pm Staphylococcus stepanovicii: The potential Origin of the Methicillin-resistance Encoding Gene mecC Birgit Walther, Institute of Microbiology and Epizootics, FU Berlin, Berlin, GERMANY

10 ASM Conferences Scientific Program

12:00 – 12:15 pm Methicillin-resistant Staphylococcus aureus in Urban Rats Scott Weese, University of Guelph, Guelph, ON, CANADA

12:15 – 12:30 pm The mecA Homolog mecC Confers Resistance Against beta- lactams in Staphylococcus aureus Irrespective of the Genetic Strain Background Britta Ballhausen, Institute of Medical Microbiology - University Hospital Muenster, Muenster, GERMANY

12:30 – 1:30 pm Lunch

1:30 – 2:30 pm Poster Session B

2:30 – 4:00 pm SESSION 6: Stakeholder’s Points of View Chairman: Luca Guardabassi

2:30 – 3:00 pm Multi-drug Resistant Staphylococci and Increasing Antimicrobial Resistance: What Are We Currently Learning Using Novel in vitro Measurements Joseph Blondeau, Royal University Hospital, Saskatoon, SK, CANADA

3:00 – 3:30 pm Epidemiology and Management of MRSA in Dairy Herds (Moredun Research Institute) Ruth Zadoks, University of Glasgow, Glasgow, UNITED KINGDOM

3:30 – 3:45 pm The View on LA-MRSA by the Danish Pig Industry (Pig Research Center) Poul Bækbo, Pig Research Centre, Danish Agriculture & Food Council, Kjellerup, DENMARK

3:45 – 4:00 pm Risk Management Initiatives on MRSA CC398 in the Danish Pig Production in a One Health Perspective (Danish Veterinary and Food Administration) Charlotte Thrane, Danish Veterinary and Food Administration, Glostrup, DENMARK

4:00 – 4:30 pm Coffee Break

4:30 – 5:30 pm Plenary Session on MRSP Use of Critically Important Antimicrobials (CIAs) in Companion Animals: What Should be Done? Scott Weese, Engeline van Duijkeren, Ulrika Grönlund- Andersson and Luca Guardabassi

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 11

Scientific Program

Thursday, November 7, 2013

9:00 – 11:00 am SESSION 7: Treatment and Control Chairman: Andreas Voss

9:00 – 9:30 am Towards Harmonised Monitoring of MRSA in Animals and Food in the EU Pierre Alexandre Beloeil, European Food Safety Authority (EFSA), Parma, ITALY

9:30 – 10:00 am Understanding Staphylococcus aureus Colonization in Pigs: Can Selective Breeding Be Used to Produce MRSA-Free Pigs? Luca Guardabassi, University of Copenhagen, Copenhagen, Denmark

10:00 – 10:15 am Intervention Measures Reducing Livestock-associated MRSA on Pig Farms in the Netherlands: a Longitudinal Study Alejandro Dorado-Garcia, Institute for Risk Assessment Sciences, Utrecht, NETHERLANDS

10:15 – 10:30 am Methicillin-resistant Staphylococcus aureus (MRSA) Screening at a Tertiary Veterinary Hospital: is Testing Cost- beneficial from an Integrated Human-animal Perspective? Jorge Pinto-Ferreira, SAFOSO, Bern, SWITZERLAND

10:30 am – 11:00 am Coffee Break

11:00 – 11:45 am Closing Keynote Lecture Phage Lysins May be Used to Treat and Prevent Infections in Both Human and Veterinary Applications Vincent Fischetti, Rockefeller Univ., New York, NY

11:45 am – 12:00 pm Concluding Remarks Robert Skov

12 ASM Conferences Speaker Abstracts n OS:1 n S1:1 STAPHYLOCOCCI AT THE HUMAN-ANIMAL LA-MRSA: WHAT HAVE WE LEARNED AND INTERFACE WHAT ARE WE STILL MISSING? R. Fitzgerald; R. Skov; The Roslin Institute, University of Edinburgh, Statens Serum Institut, Copenhagen, DEN- Edinburgh, UNITED KINGDOM. MARK. The genus Staphylococcus is a diverse group Since LA-MRSA CC398 was first recognized of bacterial species associated with a wide in The Netherlands in 2004 in pig farmers and array of host species. For example, Staphylo- their families, it has been reported in most coccus aureus is both a global human pathogen European countries, the Americas, Australia and a major cause of infectious disease in and Asia. Although pigs are the dominating livestock animal species. In this presenta- reservoir, CC398 has been reported in a wide tion, I will summarise recent studies which range of other animal species. LA-MRSA is have highlighted the origin and evolution of not confined to CC398 but also includes CC9 livestock strains of S. aureus, revealing the (especially in Asia) and ST5 (especially in the capacity of some clones to switch host species, US and Canada). In addition, a new variant of undergo adaptation, and then transmit widely methicillin resistance determinant (mecC) has in new host populations. Such host switching been described in specific LA-MRA lineages events may have important consequences for that are primarily associated with cows and veterinary and public health, and food security. sheep but again display a surprisingly broad Staphylococcus pseudintermedius is another host range. Altogether the LA-MRSA phenom- major pathogen from the genus, responsible enon seems to be much more complex than for the common skin infection pyoderma and anticipated at the beginning. notable for its increasing multi-drug resistance. Through eight years of intensive research we Recent population and genomic studies have have learned a lot about the evolution and opened the door towards a better understand- epidemiology of LA-MRSA but several pieces ing of how S. pseudintermedius causes disease. of the puzzle are still missing. Today we as- In particular, we have identified the family of sume that CC398 originated as a human MSSA encoded cell wall-associated (CWA) proteins which has subsequently adapted to pigs. In the which are involved in key host-pathogen new host it has acquired methicillin resistance interactions. I will summarise how we are and changed phage content by losing phage Φ3 investigating the function of the CWA proteins and acquiring phages Φ2 and Φ6. LA-MRSA and their potential application as targets for displays diverse resistance patterns and SC- controlling this important canine disease. Cmec types, suggesting that strain evolution is driven by a variety of selective pressures in the agricultural sector. Despite the considerable knowledge gained over the last years, various key aspects regarding the dynamics of transmission within and between herds are still not fully elucidated. How many of the pigs and humans that we score as positive are truly colonized and how many are just contaminated due to the high density of LA-MRSA in farm

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 13 Speaker Abstracts

environment? Trade has certainly played and is amide-streptogramin B (MLSB) resistance gene playing a very important role for the dissemi- erm(T), the pleuromutilin-lincosamide-strep- nation of LA-MRSA but is this the only factor? togramin A resistance genes vga(C), vga(E) To what extent do veterinarians and workers and lsa(E), as well as the spectinomycin moving from farm to farm act as vehicles? resistance gene spw. Usually, multi-resistance LA-MRSA CC398 is increasingly found in plasmids, which carried several resistance people without direct contact to pigs and farm genes, were detected in LA-MRSA. Most of workers, typically in areas with intense farm- these plasmids harbored the dfrK gene linked ing. As LA-MRSA can be detected in exhaust to the tetracycline resistance gene tet(L) in air from pig barns and even in soil samples at addition to one or more other resistance genes, least 300 m from the barn, to what extent does including apmA, aadD (kanamycin, neomy- carriage in these people reflect person-to-per- cin, tobramycin resistance), vga(A), or MLSB son transmission and to what extent is this due resistance genes erm(B), erm(C), or erm(T). to environmental contamination? What is the The co-location of several resistance genes on risk that further human adaptation will result in the same plasmid supports co-selection and increased human-to-human transmission? And persistence of this plasmid under the selective will the risk of foodborne transmission remain pressure imposed by any of the respective an- negligible? These and other topics of present timicrobial agents. Structural analysis of these interest will be addressed by the talk. multiresistance plasmids showed that they carried a variety of insertion sequences, such n S1:2 as IS257, IS431 and/or ISSau10, all of which are widely disseminated in S. aureus and have THE ROLE OF PLASMIDS IN been postulated to play an important role in the ANTIMICROBIAL (MULTI)RESISTANCE OF generation of mosaic plasmids by integrating LIVESTOCK-ASSOCIATED METHICILLIN- small plasmids into larger plasmids or mediat- RESISTANT STAPHYLOCOCCUS AUREUS ing interplasmid recombination processes. K. Kadlec1, S. Wendlandt1, A. T. Feßler1, S. Most recently, a large multiresistance gene Schwarz2; cluster of ca. 17 kb, which is flanked by inser- 1Institute of Farm Animal Genetics (FLI), tion sequences and most likely originated from Neustadt-Mariensee, GERMANY, 2Molecular enterococcal plasmids, was detected in MRSA Microbiology and Antibiotic Resistance, Insti- CC398 and MSSA ST9 isolates from humans, tute of Farm Animal Genetics (FLI), Neustadt- but also in MRSA ST9 isolates from pigs. A Mariensee, GERMANY. 41-kb plasmid, which carried this multiresistance gene cluster, was sequenced completely During the last decade research has focused and harbored - besides the novel resistance on livestock-associated methicillin-resistant genes lsa(E) and spw - another five resistance Staphylococcus aureus (LA-MRSA) with genes, including tet(L), erm(B), aadE (strep- particular reference to isolates of the clonal tomycin resistance), aacA-aphD (gentamicin, complex 398 and their dissemination between kanamycin, tobramycin resistance), and lnu(B) various human and animal hosts. Studies on (lincosamide resistance). the resistome of these isolates identified a In addition to small erm(C)-carrying plasmids number of novel resistance genes. Many of the of < 4 kb, plasmids of < 6 kb from LA-MRSA novel resistance genes were located on mobile and other porcine staphylococci have been genetic elements among which plasmids play detected, which carried only the genes vga(A), the most important role. vga(C), dfrK or apmA and - in part - differed The novel resistance genes included the trim- distinctly in their plasmid replication and mo- ethoprim resistance gene dfrK, the apramycin bilization genes from the corresponding genes resistance gene apmA, the macrolide-lincos- usually found on staphylococcal plasmids.

14 ASM Conferences Speaker Abstracts

These observations suggest that plasmids from of work week, age, sex, and smoking habits. other bacteria can be acquired by and stably Prevalence for farmers in population A was maintained in LA-MRSA and other staphylo- 30/78 (53%), for population B 48/67 (72%), cocci from food animal sources. and for farmers from population C prevalence was 32/104 (31%). Mean log(CFUeq) MRSA n S1:3 on EDCs per farm for population A was 1.84 (95%CI: 1.62-2.07), for population B it was STRONG ASSOCIATION BETWEEN MRSA 2.06 (95%CI: 1.92-2.20), and for popula- AIR EXPOSURE AND MRSA CARRIAGE IN tion C it was 0.97 (95%CI: 0.75-1.19). We VEAL CALF AND PIG FARMERS applied a multivariate, pooled analysis and a M. Bos1, W. Dohmen1, A. Dorado-Garcia1, meta-analysis on the combined datasets. The B. Van Cleef2, H. Graveland1, B. Duim3, K. results showed a consistent strong association Verstappen3, J. Kluytmans4, J. Wagenaar3, D. between the mean log MRSA exposure on Heederik1; a farm and nasal MRSA carriage in farm- 1IRAS - Utrecht University, Utrecht, NETH- ers, even after adjusting for working hours, ERLANDS, 2St. Elisabeth Hospital, Tilburg, smoking, sex, and age (pooled-OR: 1.98 (95% NETHERLANDS, 3Dept. Infectious Diseases CI: 1.34-2.93), meta-OR: 1.81 (95% CI:1.10- and Immunology - Utrecht University, Utrecht, 2.96)). A model with only MRSA exposure NETHERLANDS, 4Amphia Hospital, Breda, provided a better fit than one with only work- NETHERLANDS. ing hours. Model fit was not improved by including an interaction term of exposure level Around 30% of the Dutch veal calf farmers are and working hours. The findings suggest that a MRSA carrier with a lower percentage being role for MRSA transmission through air exists, persistent carrier. Prevalence in pig farmers is and give new insights into the importance of even higher. Previous studies showed a strong air exposure. This may have a large impact on association with carriage in animals and the predictive models for MRSA transmission and number of hours worked in the stables (as possible intervention measures. proxy for direct animal contact). Furthermore, MRSA was found in air samples obtained outside farms. However, airborne exposure and n S1:4 transmission are poorly studied so far. There- MRSA CONTAMINATION IN THE VICINITY OF fore, we determined MRSA air levels in stables POULTRY AND PIG FARMS IN GERMANY of pig and veal calf farms, and studied the ex- A. Friese1, J. Schulz2, B. Tenhagen3, A. Fetsch3, posure-response relationship with nasal MRSA J. Hartung2, U. Roesler1; carriage in farmers. We analysed samples from 1Freie Universität Berlin, Inst. for Animal three independent populations; population Hygiene and Environmental Health, Berlin, A consisted of 38 assumed frontrunner pig GERMANY, 2Institute for Animal Hygiene, farms, population B of 50 randomly selected Animal Welfare and Farm Animal Behaviour, pig farms, and population C of 49 randomly University of Veterinary Medicine Hannover, selected veal calf farms. Farmers were defined Foundation, Hannover, GERMANY, 3Federal as participants working on the farm at least Institute for Risk Assessment, Department 20 hours per week. Per farm 1-6 electrostatic Biological Safety, Berlin, GERMANY. dust collectors (EDCs) were analysed by qPCR to determine an equivalent to the number of Although the occurrence of methicillin-resis- colony forming units (CFUeq). Furthermore, tant Staphylococcus aureus (MRSA) is known we used culturing to analyse nasal swabs col- in different healthy livestock species, there are lected from the participants. The three human only little data on the emission amounts and populations were comparable regarding length dispersion distances of these resistant bacteria

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 15 Speaker Abstracts from farm buildings via the aerial route. There- emissions from animal husbandries. However, fore, this study determined the presence and further studies need to verify this presumption. concentration of MRSA in the ambient air and on surrounding ground surfaces of different n S1:5 animal farms in order to give an estimate of RESIDENTIAL PROXIMITY TO LARGE SWINE the environmental load. Six pig farms were in- CAFOS IS ASSOCIATED WITH INCREASED vestigated four times within one year and five RISK OF MRSA CARRIAGE AT TIME OF turkey as well as two broiler farms were ana- HOSPITAL ADMISSION IN RURAL IOWA lyzed four and three times, respectively within VETERANS. one fattening period. Different samples were collected inside (samples of animals and their M. Carrel1, M. L. Schweizer2, M. Vaughan Sar- direct surrounding including air) and outside razin2, T. C. Smith3, E. N. Perencevich2; (air, ground surfaces). All samples were ana- 1Department of Geography, University of Iowa, lyzed for the presence of MRSA. Selected iso- Iowa City, IA, 2Center for Comprehensive lates were spa typed and grouped as Livestock Access & Delivery Research and Evaluation associated (LA)-MRSA according to their (CADRE), Iowa City VA Health Care System, association to the clonal complex (CC)398. Iowa City, IA, 3Department of Epidemiology, MRSA was found regularly on ground surfaces University of Iowa College of Public Health, downwind of the pig (73% of 67 samples) and Iowa City, IA. poultry barns (44.4% of 81 samples), up to the Background: Methicillin-resistant S. aureus planned investigated distance of 500 m. MRSA (MRSA) colonization has been documented in was detected in exhaust air samples from three livestock, livestock workers and individuals pig farms and two turkey farms, however, living in areas of high livestock density in the with very low concentrations between 7 and US and Europe. Recently, the US Veterans Ad- 93 cfu/m3. Inside the barn MRSA occurred ministration (VA) implemented procedures that in samples of animals in high prevalences but screened all patients at time of admission for also in barn air with higher concentrations than MRSA colonization via nasal swabs. We aimed outside as well as regularly in dust and in some to determine whether residential proximity fecal samples. Isolates originating from inside to swine Confined Animal Feeding Opera- and outside the farms were of the same spa tions (CAFOs) was associated with increased types. The relevance of the emission of MRSA MRSA colonization in patients admitted to from livestock holdings to the environment the Iowa City VA Health Care System (IC- has to be discussed critically. Neighbouring VAHCS). Methods: Nasal swabs were taken residents, livestock and wild animals might from patients on admission to the IC-VAHCS be exposed and even contaminated via the air from 12/1/2009 -12/31/2011 and MRSA status and also via contaminated ground surfaces. was assessed. Patient addresses were geocoded However, MRSA concentration in the exhaust and assigned to either rural or urban residen- air was relatively low. This makes a direct tial status based on Census designations. The airborne colonization of animals and people number of swine within 1 mile of the patient housed or living in close vicinity of livestock household was calculated using data provided farms rather unlikely. The role of deposited by the Iowa Department of Natural Resources. MRSA and thus a potential contamination of Relative risk was estimated for rural residents crops used for food and feed are not yet well based upon residential proximity to any swine understood. This needs to be studied in more or to large numbers of swine. Generalized detail, in particular in respect to the survival estimating equations were used to determine times of deposited MRSA. We conclude that the impact of residential proximity to any or there seems to be no acute health risk for large numbers of swine on MRSA colonization neighbouring farms or residents due to MRSA

16 ASM Conferences Speaker Abstracts at time of admission, controlling for age and and related staphylococci enhances opportuni- number of prior admissions to the IC-VAHCS. ties for indirect transmission. Goals: (1) To Results: Overall MRSA colonization on hos- evaluate risk factors for pet acquisition of pital admission among 1036 rural IC-VAHCS MRSA given exposure to an infected person. patients was 7%. Residential proximity within (2) To evaluate the roles of household contami- 1 mile of large swine populations was associ- nation and pet positivity as sources of contin- ated with nearly double the risk of MRSA ued MRSA exposure for people undergoing colonization at time of admission (RR=1.8786, decolonization treatment. (3) To characterize 95% CI=1.0928, 3.2289, p=0.0239). Af- staphylococci of veterinary significance in ter adjusting for age and number of prior these homes. Methods: We sampled environ- hospital admissions, residential proximity to mental surfaces and all pets of any species large swine populations was associated with present in 95 homes of patients diagnosed with nearly triple the odds of MRSA colonization a community-acquired MRSA infection. We (OR=2.76, 95% CI=1.2728, 5.9875, p=.0101). repeated sampling in 65 homes, three months The relationship between MRSA and no after randomized administration of a decoloni- residential proximity to swine was not statisti- zation therapy to 65% of human households. cally significant.Conclusions: Residential We cultured samples for methicillin-resistant proximity to large swine CAFO populations and methicillin-susceptible S. aureus (MRSA/ in rural Iowans was associated with increased MSSA), S. pseudintermedius (MRSP/MSSP) risk of MRSA colonization at time of hospital and S. schleiferi (MRSS/MSSS) using a admission, while proximity to smaller swine broth enrichment, media-based protocol. We populations was not associated with MRSA confirmed staphylococcal species and mecA/ colonization. Healthcare facilities serving rural mecC presence via multiplex PCR. Results: At populations need to respond to increased risk baseline, MRSA prevalence was 63% of homes MRSA introduction into healthcare settings. (one or more sites contaminated) and 8% of 184 pets (including a fish tank). At follow-up, n S1:6 MRSA prevalence was 50% of homes and 5% of 130 pets, with 3% of pets persistently posi- ENVIRONMENTAL CONTAMINATION AND tive. At baseline, MSSP prevalence was 4% of PET COLONIZATION IN THE HOUSEHOLDS homes and 18% of pets. At follow-up, MSSP OF PEOPLE DIAGNOSED WITH A prevalence was 5% of homes and 15% of COMMUNITY-ACQUIRED MRSA INFECTION pets, with 9% of pets persistently positive. At M. F. Davis1, A. B. Brazil1, S. Iverson1, J. baseline, one dog each was positive for MRSP Ferguson1, A. Vasse2, P. Baron1, P. Tolomeo3, I. and MSSS. No reptiles or birds were positive Nachamkin3, S. C. Rankin4, E. L. Lautenbach3, for MRSA, but an aquatic turtle was positive D. O. Morris4; for MSSA at baseline. Pets with a history of 1Johns Hopkins Bloomberg School of Public antimicrobial use were 6.4 times more likely to Health, Baltimore, MD, 2Tufts University be positive for MRSA (p<0.05). Dogs were 20 School of Veterinary Medicine, North Grafton, times more likely than cats to be positive for MA, 3University of Pennsylvania School of MSSP (p<0.05). Home MRSA contamination, Medicine, Philadelphia, PA, 4University of MRSA prevalence in people, and household Pennsylvania School of Veterinary Medicine, randomization to a treatment group were Philadelphia, PA. positively correlated with pet carriage of MRSA. Conclusions: The patient’s home was Background: Households may serve as a commonly contaminated with MRSA and may point of transmission for methicillin-resistant serve as a reservoir for continued exposure to Staphylococcus aureus (MRSA) among people both people and pets. While household decolo- and pets. Environmental survival of MRSA nization reduced MRSA carriage in the treated

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 17 Speaker Abstracts people, it did not reduce MRSA carriage in American continent. Conventional typing pets (pets were not treated). S. pseudinter- methods like pulsed-field gel electrophoresis medius was frequently cultured from pets, (PFGE) are able to compare strains in a re- particularly dogs. Further work to characterize stricted geographical area over a limited time veterinary staphylococci from people enrolled period only, while Multi-locus sequence typ- in this study is planned. ing (MLST) enables unequivocal comparison of Sequence types (STs) of globally isolated n S2:1 strains. An initial MLST scheme stated the European-wide spread of a clone belonging to EVOLUTION AND SPREAD OF MULTIDRUG- ST71. RESISTANT STAPHYLOCOCCUS To get a deeper insight into the population PSEUDINTERMEDIUS STRAINS structure, we comparatively analyzed 100 B. Walther, T. Semmler, L. H. Wieler; MRSP and 100 MSSP from a convenience Centre for Infection Medicine, Institute of Mi- sample of strains obtained from various crobiology and Epizootics, Berlin, GERMANY. geographic origins in Germany. Representa- tive strains were analyzed by whole genome Over the last decade, an increasing preva- sequence (WGS) analysis to unravel the lence of colonization, infections and infec- population structure and genetic makeup of tious diseases caused by multidrug-resistant these clones. DNA sequences encoding the bacteria particularly in companion animals methicillin resistance determinant (mecA) like cats, dogs and horses, has been observed. seem to be highly conserved among MRSP Multidrug-resistant methicillin-resistant S. strains. Despite clearly distinct PFGE patterns, pseudintermedius (MRSP) cause infections most of the MRSP strains isolated in Germany and infectious diseases often associated with belong to ST71 by applying both, the old and clinical settings and involve mostly wound, the updated MLST scheme while MSSP gener- skin or ear infections. S. pseudintermedius is ally display more heterogeneous ST profiles. a typical cause of canine skin infections and In contrast to high relatedness of PFGE pattern until recently regarded as being host-adapted. displayed by epidemic MRSA strains, results However, the epidemic spread of MRSP to- of WGS analysis contributes to the understand- gether with the changing socio-cultural interac- ing of the limited PFGE-clonality of epidemic tion between companion animals and humans STs like ST71. In conclusion, MRSP is yet has even resulted in human cases of MRSP another example highlighting the influence of infections. multidrug-resistance speeding up microbial Methicillin-resistant variants of S. pseudin- spread. termedius (MRSP) were first reported in the late 1990s sporadically, followed by a sudden rise in incidence only a few years later. Mean- n S2:2 while diseases caused by MRSP are a huge EMERGENCE OF NEW METHICILLIN- therapeutic challenge due to their frequently RESISTANT STAPHYLOCOCCUS exhibited multi-drug resistant phenotype. PSEUDINTERMEDIUS CLONES Although knowledge on the global infection S. Kania; epidemiology of MRSP is still scarce, spread Comparative Medicine, University of Tennes- of a limited number of MRSP clones is envi- see, Knoxville, TN. sioned. Currently it is believed that the genetic background of MRSP is associated with its Studies of clonal populations of Staphylo- geographic origin, i.e. certain dominating coccus pseudintermedius provide important MRSP-lineages spread in Europe or the North information about the epidemiology of the

18 ASM Conferences Speaker Abstracts organism as well as insight into the mecha- n S2:3 nisms by which antibiotic resistance genes and SUBTLE STRAIN VARIATION OF genes associated with virulence are spread. STAPHYLOCOCCUS PSEUDINTERMEDIUS An expanding database of genomic informa- COMPLICATES CORRECT DIAGNOSTICS IN tion has facilitated molecular characterization INFECTED DOGS of bacterial populations. These molecular studies provide information about the spatial J. Hordijk, D. Goilo, J. A. Wagenaar, K. M. and temporal distribution and host specificity Verstappen, A. Timmerman, E. Broens, B. of the bacterium. Correlation of multilocus Duim; sequence types with antibiotic resistance Utrecht University, Utrecht, NETHERLANDS. phenotypes and genotypes, SCCmec types, spa Background: S. pseudintermedius is an types, optical mapping and other methods of opportunistic pathogen that colonizes the population characterization provide a broad skin and mucosal membranes of dogs and picture of the distribution of various strains of is the major cause of canine pyoderma. The S.pseudintermedius and their role in the pathol- worldwide spread of multi resistant methicil- ogy of this organism. Two clonal populations lin resistant S. pseudintermedius (MRSP) has of methicillin resistant S.pseudintermedius complicated treatment considerably. Genetic represented by ST68 in North American and diversity among strains from the same dog has ST71 in Europe were originally described been described but many questions concern- as predominant strains within their regions. ing the temporal genetic diversity in time and Recent studies, however, have identified consequences for diagnosis and treatment additional strains, some of which are ge- remain unanswered. The index case for this netically closely related, others that appear study was a dog suffering from chronic otitis to represent additional clonal complexes and externa. Differences in antimicrobial suscep- others that may have more recently acquired tibility patterns of strains isolated from the methicillin resistance. In the United States index dog during one year were noted and several new clonal complexes of methicillin questioned was if the ongoing infection was resistant S.pseudintermedius (MRSP) have caused by multiple strains or one strain that been identified suggesting independentmecA was quickly evolving. Additionally, it was acquisition events, clonal expansion and the questioned how often strain diversity would be evolution of new sequence types. In addition, observed in a single sample from other dogs. studies on other continents have revealed both Methods: At the Veterinary Microbiologi- the apparent spread of ST71 and ST68 and the cal Diagnostic Center (VMDC), during one presence of independent clonal populations year swabs were repeatedly obtained from the of resistant organisms with diverse cassette index dog. In addition, 5 epidemiologically types and antibiotic resistance profiles. Recent unrelated dogs suffering from an S. pseudinter- data suggest that MRSP clonal populations medius infection were sampled prospectively. continuously arise and spread, however, they Of each swab ten colonies were subcultured remain primarily concentrated within distinct and confirmed with pta PCR-RFLP, mecA, geographical regions. and mecALGA251 PCR as MRSP. All strains were analyzed with PFGE and the MICs were determined. A selected amount of isolates was further characterized by SCCmec typing and spa typing. Results: From the 10 swabs of the index case 5 contained multiple strains that showed 9 different antibiotic resistance patterns. PFGE analyses showed that all strains

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 19 Speaker Abstracts were highly related. Remarkable was the Unites States was undertaken. Within 24 hours finding of strains that were genetically related of admission, samples of the nares, pharynx, based on PFGE and spa-typing, but were not rectum and skin at the surgical site were ob- all positive for mecA. In the prospective study tained for MRSP screening using enrichment two dogs carried a single MSSP strain, one dog culture and mannitol salt with oxacillin agar. carried two unrelated MSSP strains, and two Isolates were typed by dry typing. Active sur- dogs carried an unrelated MSSP and MRSP veillance of all patients was performed 30 days strain that were both multi resistant with dif- post-operatively and SSIs were documented ferent β-lactam resistances. Conclusion: The according to standard definitions. In cases of finding of genetically related strains express- SSI, samples from the wound were obtained. ing a different β-lactam phenotype in one dog All patients at 2 institutions were swabbed at is suggestive for excision of SCCmec and time of radiographic re-evaluation to deter- needs further confirmation. The observation mine MRSP colonization status. The overall of multiple strains in a sample with different SSI rate was 21/338 (6.2%), with 9/21 (43%) antibiotic resistances is a risk for misidentifica- caused by MRSP. Ten of 338 (3%) dogs were tion and treatment failures. colonized with MRSP preoperatively. Two of ten (20%) MRSP colonized dogs developed n S2:4 MRSP SSI, compared to 7/323 (2.2%) dogs that were not colonized (P=0.026). 7/138 (5%) THE ROLE OF METHICILLIN RESISTANT of dogs were colonized with MRSP at time of STAPHYLOCOCCUS PSEUDINTERMEDIUS post-operative recheck, between 4 and 8 weeks COLONIZATION AS A RISK FACTOR FOR after surgery. 4/7 (57%) of these dogs were DEVELOPMENT OF SURGICAL SITE colonized at time of initial admission to the INFECTIONS IN DOGS UNDERGOING TIBIAL hospital. Isolates consisted of dt9a (n=6, 46%), PLATEAU LEVELING OSTEOTOMY. dt10h (n=3 - 23%), dt10f (n=1, 7.7%), dt11a A. Nazarali, A. M. Singh, S. Weese; (n=1, 7.7%) and dt11af (n=2, 15.4%). All dogs University of Guelph, Guelph, ON, CANADA. with pre-operative MRSP colonization carried the same type at either time of post-operative Tibial plateau leveling osteotomy (TPLO) is recheck or diagnosis with MRSP SSI. The one of the most commonly performed surgical pre-operative MRSP colonization rate was techniques to stabilize a cranial cruciate insuf- consistent with studies of similar populations, ficient stifle in dogs. Numerous studies have as was the SSI rate and commonness of MRSP reported high surgical site infection (SSI) rates SSI. Pre-operative colonization was associated for TPLO, and methicillin-resistant S. pseud- with development of MRSP SSI, something intermedius has emerged as a leading cause that indicates a need to consider pre-operative of these infections. In humans, pre-operative screening and/or peri-operative prophy- colonization with methicillin resistant Staphy- laxis measures. However, while there was a lococcus aureus (MRSA) is a risk factor for significant association, most MRSP infections subsequent MRSA SSI, but the relationship be- developed in dogs that were not identified as tween MRSP carriage and TPLO infection risk colonized pre-operatively, so further study are unknown. The objective of this study was of the epidemiology and pathophysiology of to evaluate the impact of pre-operative MRSP MRSP TPLO SSI is needed. colonization on TPLO SSIs. A prospective, multi-institutional study of dogs undergoing TPLO at 6 surgical facilities in Canada and the

20 ASM Conferences Speaker Abstracts n S3:1 infection spreading beyond the hospital into UNTANGLING THE TRANSMISSION the community. Combining the genomic data DYNAMICS OF MRSA USING WHOLE with epidemiological data we were able to GENOME SEQUENCING hypothesise that a member of staff was linked to the transmissions on the ward. Subsequent M. T. G. Holden; screening identified a single member of staff Pathogen Genomics, The Wellcome Trust who was MRSA positive and colonised with Sanger Institute, Cambridge, UNITED KING- a strain belonging to the SCBU outbreak. The DOM. staff member was decolonised, and since then there has been no reoccurrence of the outbreak. Next-generation sequencing (NGS) has These results highlight the potential of whole- changed the face of bacterial genomics, allow- genome sequencing to identify outbreaks in a ing larger numbers of samples to be sequenced healthcare setting and provide clinically rel- more rapidly with decreasing costs. This has evant data that can influence patient care and opened up new opportunities for the applica- guide initial therapy choices. The challenge tion of whole genome sequencing beyond for the future is transitioning NGS into the basic research, towards more applied areas clinical setting, and integrating the results into such as clinical microbiology diagnostics. The everyday practice. high-resolution genotyping that NGS provides can be used to distinguish closely related isolates, and also place them within a popula- n S3:2 tion structure based on data from previously NEW MLST SCHEME FOR S. sequenced isolates. Phylogenetic analysis PSEUDINTERMEDIUS of single nucleotide polymorphisms (SNPs) V. Perreten; identified in the genomes of bacterial isolates Institute of Veterinary Bacteriology, University allows us to reconstruct their evolutionary of Berne, Berne, SWITZERLAND. history, and consequently make inferences about their origins and genetic relationships. Staphylococcus pseudintermedius belongs Applying these approaches to methicillin- primarily to the normal flora of dogs and is resistant Staphylococcus aureus (MRSA), now established as one of the most common we have used NGS to investigate two MRSA causes of canine dermatitis, as well as hospital- outbreaks in a large teaching hospital. In the acquired infections, in companion animals. S. first study focusing on a Neonatal Intensive pseudintermedius has also been reported to Care Unit (NICU), we were able to demon- be a cause of infection in horses and mastitis strate that whole genome sequencing was able in dairy cows. Additionally, S. pseudinterme- to distinguish outbreak isolates from unlinked dius occasionally causes severe infections in MRSA isolates in the hospital. In addition, humans. To determine the phylogenetic diver- we used genotype information generated from sity between S. pseudintermedius strains, in NGS to characterise the antibiotic resistance 2007 Bannoehr et al. developed a Multilocus gene content of the isolates, and demonstrated Sequence Typing (MLST) method based on congruence with the antibiotic resistance pro- 4 housekeeping genes (cpn60, pta, tuf, agrD) file generated by standard phenotypic testing. and the partial 16S rRNA gene (Bannoehr et In a second study we investigated a prolonged al. 2007. J. Bacteriol. 189:8685-8692). This MRSA outbreak on a Special Care Baby Unit MLST scheme permitted the identification (SCBU). Genome sequencing identified that of predominant clones, such as e.g. ST71 in there had been multiple transmissions on the Europe and ST68 in North America. In 2013, ward, but also that there had been a series of a new MLST scheme using three of the previ- secondary transmissions that lead to the MRSA ously described alleles (cpn60, pta, tuf) as well

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 21 Speaker Abstracts as 4 new alleles (ack, fdh, purA, sar,) has been n S3:3 published for a better discrimination between PREVALENCE OF MECA-POSITIVE isolates of a related clonal lineage (Solyman METHICILLIN-RESISTANT et al. 2013. J Clin. Microbiol. 51:306-310). It STAPHYLOCOCCUS AUREUS IN PIGS is however still recommended to sequence the EXHIBITS DOSE-RESPONSE TO ZINC agrD locus which is useful additional marker SUPPLEMENTATION for the further discrimination of clones belonging to the same ST. R. G. Amachawadi, H. M. Scott, S. Niti- A website developed and sited at the Univer- kanchana, J. Vinasco, T. G. Nagaraja, M. sity of Oxford (Jolley & Maiden. 2010. BMC D. Tokach, S. S. Dritz, J. L. Nelssen, R. D. Bioinformatics 11:595) has been adapted to the Goodband; MLST scheme of S. pseudintermedius and is Kansas State University, Manhattan, KS. now available at http://pubmlst.org/spseudin- Zinc (Zn) is often supplemented at elevated termedius/. concentrations in swine diets to promote The site contains useful information including growth and to prevent enteric infections. It is methodology, submission form, publication list hypothesized that the occurrence of methicil- of the corresponding ST, as well as two data- lin-resistant Staphylococcus aureus (MRSA) bases. The first database consists of the locus/ in pigs is associated with the use of zinc in the sequence definition database which contains diet. In Europe, swine have been identified as allele sequences, allele profiles and MLST an emerging reservoir of livestock-associated profiles. This database may be used, for ex- MRSA (LA-MRSA). Previous studies from ample, for sequence, allele and ST queries. The Denmark have suggested a genetic linkage second database contains the isolates database and a phenotypic association between Zn which includes epidemiological information on resistance, encoded by czrC, and methicillin- the different S. pseudintermedius, such as e.g. resistance, encoded by mecA, in S. aureus. methicillin-susceptible S. pseudintermedius Such an association has not been reported in (MSSP) and methicillin-resistant S. pseudin- the U.S. swine population. We conducted a termedius (MRSP), country and source of the study to determine the effects of various con- isolates, health status and type of disease. In centrations of Zn, supplemented as zinc oxide September 2013, the databases contained 169 (ZnO), on the prevalence of MRSA in nursery sequences, 280 STs, and 305 isolates. Among (n=40) and finisher pigs (n=40). The basal diet them, 69 were MRSP (22.62%) and 236 were consisted of zinc as zinc sulfate at 110 and MSSP (77.38%), which came from 21 different 55 mg/kg of feed in nursery and finisher pigs, countries. All users are welcome to submit, respectively. The nursery pigs included control using the online submission form, their new (n= 20; no supplemental ZnO) and zinc groups allele sequences and the STs, if new for their (n= 20; 1,800 mg/kg of supplemental ZnO). countries, to the database for S. pseudinter- The finisher pigs included four groups, each medius, as well as the publication in which with ten animals that received 0 (no supple- they have been released. The S. pseudinter- mental ZnO), 75, 150, or 225 mg/kg of ZnO. medius MLST database will provide valuable Nasal swabs were collected from both nursery epidemiological information about this rapidly and finisher pigs. The swab samples were spreading zoonotic bacteria. inoculated on to MRSA CHROMagar and presumptive MRSA colonies (mauve or magenta color) were tested for mecA and czrC genes by PCR. Zinc susceptibility [minimum inhibitory concentration (MIC)] was determined by the agar gel dilution method. Statistical analyses

22 ASM Conferences Speaker Abstracts for binary endpoints were carried out using resistant S. aureus (MRSA) and methicillin- STATA MP (v.12.1) via the multivariable susceptible S. aureus (MSSA) Clonal Complex exact logistic regression procedure, consider- (CC)97, one of the most prevalent lineages ing categorical dose of zinc and controlling in Italian primary productions (Battisti et al., for the fixed effect of production age. Zinc 2010), detected in holdings of pigs and dairy MIC susceptibility data also were analyzed cattle in Italy in the last five years. Recently using non-parametric survival analysis. The (Spoor et al., 2013) a livestock origin for prevalence of mecA-positive MRSA was 0% human pandemic CC97 MRSA has been (0/20) in the nursery control group and 20% demonstrated. METHODS: A total 42 CC97 S. (4/20) in piglets fed an elevated concentration aureus were studied: 35 MRSA of swine and of ZnO (P = 0.05). In finisher pigs, the preva- bovine origin and 7 MSSA, with one human lence of mecA-positive MRSA was 0% (0/10), MSSA from Spain for comparison purposes. 10% (1/10), 20% (2/10), and 50% (5/10) in Genotyping was performed using multilocus groups that received 0, 75, 150, and 225 ppm sequence typing (MLST), spa-typing, SCCmec of supplemental Zn, respectively (P = 0.05). multiplex-PCR characterization. Virulence, Of the 12 mecA-positive S. aureus isolates, pathogenicity and resistance genes were in- 7 had the czrC gene (58.3%) compared to vestigated by PCR and microarray. RESULTS: none that were positive for czrC among the 68 The CC97 isolates belonged to ST97 (n=37; mecA-negative isolates. The presence of both spa-types t4795, t1730, t1236, t2112, t267), mecA (P = 0.002) and czrC (P = 0.006) genes ST71 (n=3; spa-type 524), ST352 (n=2; spa- was strongly associated with higher levels types t359). All MRSA carried SCCmec cas- of Zn supplementation. The median MICs of sette type V (5C2), and were all PVL negative. Zn for MRSA and MSSA were 8 and 4 mM, Conversely, all isolates (MRSA and MSSA) respectively (P = 0.0001). The possible genetic were positive for genes of other leukotoxin link between czrC and mecA genes suggests families: LukF-LukS-HlgA, LukD-LukE and the importance of elevated Zn supplementation LukX-LukY. One MSSA (ST97) from dairy in co-selection and propagation of antibiotic cattle carried the LukF-PV(P83) gene typical resistance. Further studies are underway to bet- of S. aureus from ruminants. One MSSA and ter understand the molecular epidemiology of one MRSA from cattle, one MRSA from pigs MRSA via genetic typing (spa typing) and the and the human isolate were positive for sak effects of feeding zinc versus other antimicro- and scn, within the immune evasion cluster bials on the prevalence of LA-MRSA in pigs. (IEC). All isolates were positive for several superantiges/enterotoxin-like genes, with few n S3:4 isolates from cattle only carrying seg (n=1), sed (n=1 MRSA), sed, sej, ser (n=1 MSSA), or MOLECULAR CHARACTERIZATION ORF CM14 (n=3 MSSA). None was positive OF METHICILLIN-RESISTANT AND for tst1. As regards antimicrobial resistance, METHICILLIN-SUSCEPTIBLE S. AUREUS all MRSA carried the tet(M) gene, and 27/35 (MRSA, MSSA) CLONAL COMPLEX 97 (77%) also the tet(K) gene. Aminoglycoside ISOLATES FROM PIGS AND CATTLE, ITALY. (GEN-KAN) resistance gene aacA-aphD was F. Feltrin1, P. Alba1, A. Ianzano1, R. Amoruso1, present in 6/42 isolates often in co-presence A. Caprioli1, M. A. Argudín2, B. Guerra3, A. with aadD. Macrolide-lincosamide resistance Battisti1, A. Franco1 ; was mediated by erm(B) (n=13), erm(C) (n=2) 1IZSLT, Rome, ITALY, 2 CODA-CERVA, Brux- or both genes (n=1). The vast majority (32/35, elles, BELGIUM. 3BfR, Berlin, GERMANY 91%) MRSA showed pleuromutilin resistance (tiamulin MIC=8 mg/L), attributed to the AIMS: The aim of this study was to provide vga(A) gene, also contributing to resistance to molecular characterization of methicillin- streptogramins A and lincosamides. All isolates

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 23 Speaker Abstracts carried sdrM, a chromosomally-encoded multi- during competition and survival on different drug efflux pump, and two porcine isolates the hosts, nor how mobile the elements are, nor the plasmid borne qacC. All MRSA isolates had potential prospects for future evolution. the same pattern of capsule and biofilm-associ- We co-inoculated a pig associated isolate ated genes, with 4 MRSA (2 bovine, 2 swine) (S0385) and a human adapted isolate (H278) and 1 MSSA isolates (bovine) which carried onto gnotobiotic piglets in isolation chambers also the bap gene. All isolates carried several to establish if the pig adapted strain was fitter genes encoding MSCRAMMs, bbp, clfA, clfB, and a more successful coloniser. Our data ebh, ebpS, eno, fib, fnbA, fnbB, map, sasG, surprisingly revealed both isolates could grow sdrC, sdrD, vwb. The two spa-types t267 had equally well, but this involved extensive gene the same genetic “core profile” (includingsak transfer and exchange of MGE between the and scn), except for acquired resistance genes/ isolates. Whole genome sequencing suggests elements (e. g. SCCmec, blaZ, aacA-aphD, MGE alone are responsible for host-adaptation fusC). CONCLUSIONS: Very few host- or and survival, and the prospects for the evolu- ST-associated differences were found among tion of strains with broader host range is high. MSSA and MRSA CC97 isolates studied. A Extensive MGE movement also has implica- minority of isolates harbour genes associated tions for our understanding of genome stability with human adaptation. MRSA CC97 from and bacterial growth and physiology. pigs and cattle possesses several virulence, pathogenicity and resistance genes towards n S4:2 other major classes of antimicrobials, and may THE GENOME OF MRSP ST71 represent a serious therapeutic challenge in case of invasive infections in humans. A. Moodley; Faculty of Life Sciences, University of Copen- n S4:1 hagen, Frederiksberg C, DENMARK. HOST ADAPTATION OF LA-MRSA Methicillin-resistant Staphylococcus pseudintermedius (MRSP) E140 was the first fully J. A. Lindsay; sequenced strain belonging to the widespread St. George’s, University of London, London, MRSP clone ST71, which has been associated UNITED KINGDOM. with infections in different animal species and CC398 isolates from pigs and humans in humans. E140 was isolated from a canine bite contact with pigs have conserved genomes but wound infection in Copenhagen, Denmark in vary in their carriage of mobile genetic ele- 2008. The draft genome was determined to be ments (MGE). Variation is partly due to host 2,77Mb, with a GC content of 38%, and pre- and partly to geography. More rarely, certain dicted to have 2678 coding sequences. Methi- CC398 isolates appear to transmit between cillin resistance was attributed to the presence humans without animal contact. These isolates of SCCmec type II-III, which is typically asso- have substantially different MGE profiles. ciated with this genetic lineage. Excluding the MGEs are important as they often encode presence of the SCCmec cassette, we observed resistance genes as well as genes involved in that E140 had a larger genome compared to the virulence and host-adaptation. Snapshots of two published methicillin susceptible S. pseud- bacterial DNA content provide important clues intermedius genomes of ED99 and HKU10- as to which genes and MGEs may be selected 03. Using PHAST, an online prophage finder for in populations of bacteria in different programme, E140 contains prophage related environments. But it does not tell us which DNA of up to 300Kb, including three intact genes are selected under controlled conditions prophages of ~222Kb, collectively represent-

24 ASM Conferences Speaker Abstracts ing 8% of the total genome. Currently, efforts ST2082 (both CC30) and lacked scn. These are underway to annotate the phage related findings suggest that in addition to an ancestral DNA for identification of novel virulence fac- population of scn-positive S. aureus CC30 in tors, as well as whole genome sequencing of humans there is a newly emerged scn-negative additional MRSP ST71 genomes isolated from subpopulation in pigs, which can spread to pig humans and different animal species to iden- farmers and be a source of S. aureus bacterae- tify common genetic traits that may explain mia in humans. the rapid emergence and global spread of this Both pulsotype A and B isolates lacked lukF- clonal lineage. lukS encoding PVL, and most (20/22) isolates were tetracycline susceptible; only two porcine n S4:3 MSSA ST433 isolates were tetracycline resistant and carried tet(K). Tetracycline is NEW LA-MRSA CLONES EMERGING IN the most commonly used antimicrobial agent PIGS: AN INSIGHT INTO ST433 in the Danish pig production. The low rate Y. Agersø; of tetracycline resistance (9%) in the pig- National Food Institute, Technical University associated S. aureus ST433/ST2082 isolates is of Denmark, Lyngby, DENMARK. in sharp contrast to the high rate of tetracycline resistance in livestock-associated S. aureus Livestock associated methicillin resistant CC398 (~100%) and may partly explain the Staphylococcus aureus (MRSA) in pigs mainly high occurrence of CC398 in pigs. belong to CC398, which is also the most com- Sequence analysis of SCCmec from ST433 mon clonal complex among methicillin suscep- and CC398 isolates, respectively showed tible S. aureus (MSSA) in pigs in Denmark. I similar SCCmec cassettes. Isolates of CC30 2009 MRSA ST433 (CC30) (spa type t1333) including ST30 and ST433 were analyzed for was detected among 4% of MRSA in pigs at mutations in the sau1 restriction-modification slaughter. ST433 is the second most common system and the result revealed an amino acid MSSA type among pigs in Denmark. change in position 45 of hsdM1 in MRSA From 2010 to 2011 pigs and farm work- ST433 isolates and an amino acid change in ers were sampled for MRSA in 39 farms in position 187 of ST433 isolates when com- Denmark. MRSA ST433 (spa type 1333) pared to residual CC30 isolates. These amino was detected in a pig and two farm workers acid changes could have made MRSA ST433 from the same farm. The MRSA isolates were isolates more capable of taken up foreign compared to three human MSSA t1333 isolates DNA and support that SCCmec cassette was identified retrospectively in the national col- exchanged horizontally. lection of S. aureus bacteraemia isolates from In conclusion, pigs constitute a zoonotic 2007-2011. Pulsed-field gel electrophoresis reservoir of S. aureus (MRSA and MSSA) (PFGE) analysis of 14 MSSA and five MRSA ST433/ST2082 and suggests a link between from pigs, the two MRSA from farm work- pigs and MSSA ST433 bacteraemia in humans. ers and the three MSSA from bacteraemia Moreover, mutations in the sauI restriction- revealed two different pulsotypes. Pulsotype modification system may have caused up take A included two MSSA bacteraemia isolates of the SCCmec cassette in MRSA ST433. that belonged to ST30 and carried the scn gene (encoding a human-specific complement inhibitor). Pulsotype B included all isolates from pigs and farm workers, as well as one MSSA bacteraemia isolate; these isolates belonged to ST433 and in one case its single-locus variant

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 25 Speaker Abstracts n S4:4 was also carried out. Finally, comparison of PHYLOGENETICS AND MOLECULAR the content of mobile genetic elements within EPIDEMIOLOGY OF MECC-MRSA ISOLATES isolates was made to further understand the IN EUROPE evolutionary history of isolates and to identify potential determinates of host specificity. E. M. Harrison1, G. K. Paterson1, F. E. Mor- 1 2 3 3 gan , M. T. Holden , M. Stegger , J. Larsen , n S4:5 A. R. Larsen3, A. Petersen3, R. Skov3, R. N. Zadoks4, S. J. Peacock1, J. Parkhill2, M. A. STAPHYLOCOCCUS AUREUS ST398 GENE Holmes1; EXPRESSION PROFILING DURING EX 1University of Cambridge, Cambridge, VIVO COLONIZATION OF PORCINE NASAL UNITED KINGDOM, 2Wellcome Trust Sanger EPITHELIUM Institute, Hinxton, UNITED KINGDOM, B. Duim1, P. Tulinski1, F. Wittink2, M. J. 3Statens Serum Institut, Copenhagen, DEN- Jonker3, T. M. Breit3, J. P. van Putten1, J. A. MARK, 4Moredun Research Institute, Penicuik, Wagenaar1, A. Fluit4; UNITED KINGDOM. 1Department of Infectious Diseases and Im- munology, Faculty of Veterinary Medicine, A novel mecA homologue (mecALGA251: Utrecht University, Utrecht, NETHERLANDS, now designated mecC) present on an SC- 2Dept of Technology, Leiden University of Cmec type XI element has been identified in Applied Sciences, Leiden, NETHERLANDS, methicillin-resistant Staphylococcus aureus 3MicroArray Department, University of (MRSA) isolates from humans and animals Amsterdam, Amsterdam, NETHERLANDS, throughout Europe. MRSA with mecC present 4Department of Medical Microbiology, a potential public health problem as they can University Medical Center Utrecht, Utrecht, be missed by current laboratory diagnostics NETHERLANDS. and have been associated with animals. More recently, transmission of mecC-MRSA be- Background: Staphylococcus aureus is a com- tween livestock and humans has been demon- mon human and animal opportunistic patho- strated in Denmark by epidemiological follow gen. In humans nasal carriage of S. aureus is a up and whole genome sequencing. mecC has risk factor for various infections. Methicillin- also been identified in a number of coagulase- resistant S. aureus ST398 is highly prevalent negative staphylococci species. To further in pigs in Europe and North America. The understand the epidemiology and evolutionary mechanism of successful pig colonization by history of mecC-MRSA, isolates from a wide MRSA ST398 is poorly understood. Here we range of sources and geographical locations in present the analysis of the transcriptome of Europe were whole genome sequenced. mecC- MRSA ST398 strain S0462 during mainte- MRSA isolates and closely related sequence nance of colonization on ex vivo nasal epi- types were subjected to SNP analysis of the thelium. Methods: Previously, we developed core genome, and high resolution phylogenetic a nasal colonization model of porcine nasal trees constructed in order to understand the mucosa explants to identify molecular traits evolutionary history, transmission and spread involved in nasal MRSA colonization of pigs. of mecC-MRSA. Phylogenetic trees from From explants colonized with MRSA strain the core genome and SCCmec elements were S0462 bacteria were isolated for RNA isolation compared to gain insight into the evolutionary in four repetitions. RNA was purified using history of the mecC carrying SCCmec type the NucleoSpin RNA II total RNA isolation XI element. Analysis of SCCmec type XI in kit. The microarray was specifically developed comparison to orfX regions of mecC-positive for multiple S. aureus strains and contained coagulase-negative staphylococci (CoNS) 121,901 probes (Nimblegen, Roche). Process-

26 ASM Conferences Speaker Abstracts ing of the data was performed using R (version an Centre for Disease Prevention and Control, 2.14.1) and the Bioconductor MAANOVA Stockholm, SWEDEN, 11Scottish MRSA package. Results: Major regulated genes were Reference Laboratory (SMRSARL), Glasgow, encoding metabolic processes and regulation UNITED KINGDOM, 12Labor Dr. Böse GmbH, of these genes represents metabolic adapta- Harsum, GERMANY. tion to nasal mucosa explants. Maintenance Methicillin-resistant Staphylococcus aureus of colonization was not accompanied by (MRSA) is a significant nosocomial and com- significant changes in transcripts of main munity acquired pathogen worldwide. The virulence associated genes or known human clonal complex 398 (CC398) was described colonization factors. We studied the regulation as livestock associated MRSA. Nevertheless, of two genes which have potential influence on various studies have reported colonisation and S. aureus colonization; cysteine extracellular infection of humans and various companion proteinase (scpA) and von Willebrand factor- animals with CC398. We investigated the binding protein (vwbp, located on SaPIbov5). population structure of 195 CC398 isolates Colonization with isogenic-deletion strains through mutation discovery. Isolates were col- (Δvwbp and ΔscpA) did not alter the nasal S. lected from various hosts (human, pigs, poul- aureus colonization compared to wild type. try, horses, and cattle) and countries (Germany, Conclusion: Our results suggest that mainte- the Netherlands, USA, UK, Canada, Belgium, nance of nasal colonization with MRSA ST398 Italy, Denmark, Austria and Thailand). Spa- is a complex event that is accompanied with and SCCmec-types were determined for all changes in bacterial gene expression regulation isolates. The denaturing high-performance and metabolic adaptation. liquid chromatography was used for the single nucleotide polymorphism (SNP) discovery at n S5:1 97 loci (≈ 40 kb of the CC398 genome). The THE POPULATION STRUCTURE OF CC398 polymorphic PCR products were sequenced AND THE EMERGENCE OF HORSE- and a minimum spanning tree (MST) was ASSOCIATED SUB-CLONE constructed by using Bionumerics 6.5. The Bayesian tip-association significance test M. M. Abdelbary1, A. Wittenberg2, C. Cuny1, F. revealed that spa types (t034, t011, t571, t108, Layer1, K. Kurt1, L. H. Wieler2, B. Walther2, R. t1457, and t899) and SCCmec types (IV, and Skov3, J. Larsen3, H. Hasman4, R. Fitzgerald5, V) were significantly associated with phylog- T. C. Smith6, J. A. Wagenaar7, A. Pantosti8, eny. We detected the φAvβ prophage-related M. Hallin9, M. J. Struelens10, G. Edwards11, R. sequences only in MSSA turkey meat isolates. Böse12, U. Nübel1, W. Witte1; In addition, immune evasion genes carried on 1Robert Koch Institute, Wernigerode, GER- the β- converting φSa3 prophage (sak, chp and MANY, 2Freie Universität Berlin, Berlin, scn) were found in 17 CC398 isolates, which GERMANY, 3Statens Serum Institut, Copen- had been from human and animal origin. hagen, DENMARK, 4Technical University of Interestingly, we revealed the spread of spe- Denmark, Lyngby, DENMARK, 5The Roslin cific MRSA-CC398 sub-clone within equine Institute, Edinburgh, UNITED KINGDOM, settings, which causes infection in horses and 6College of Public Health, the University of humans nasal colonisation. The spread of this Iowa, Iowa, IA, 7Utrecht University, Utrecht, CC398 sub-clone may be due to insufficient NETHERLANDS, 8Istituto Superiore di Sanità, hygiene practices in veterinary settings or Rome, ITALY, 9Centre de Diagnostic Molécu- unknown host specificity factors. laire, iris-Lab, Brussels, BELGIUM, 10Europe-

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 27 Speaker Abstracts n S5:2 ters, New Zealand). Results: The mecC gene STAPHYLOCOCCUS STEPANOVICII: THE of IMT27065 (EMBL: KC110686.1) shares POTENTIAL ORIGIN OF THE METHICILLIN- 99.2% nucleotide and 98.5% amino acid RESISTANCE ENCODING GENE MECC sequence identity with the mecCLGA251 of the recently described SCCmecXI in Staphy- B. Walther1, A. Lübke-Becker1, S. Vincze1, R. lococcus aureus. Furthermore, the surround- G. Ulrich2, L. H. Wieler1, S. Guenther1, E. M. ing region revealed the presence of genomic Harrison3, M. A. Holmes3, T. Semmler1; elements similar to those of the class E mec of 1Institute of Microbiology and Epizootics, FU SCCmecXI (blaZ, mecC, mecR1, mecI) and Berlin, Berlin, GERMANY, 2Friedrich-Loeffler- the presence of the arsenic resistance operon. Institut, Institute for Novel and Emerging Discussion: Here we present S. stepanovicii Infectious Diseases, Greifswald-Insel Riems, as potential origin of the mecC gene and the GERMANY, 3Dept. of Veterinary Medi- class E mec in SCCmecXI. Further compara- cine, University of Cambridge, Cambridge, tive genomic analysis including more regions UNITED KINGDOM. surrounding mecC genes harbored by CNS are needed to reconstruct the phylogeny of Introduction: Methicillin resistance in SCCmecXI in S. aureus. Our data highlights coagulase-positive (CPN) staphylococci is a the role of the environmental resistome as an major threat to both, human and veterinary important source of antibiotic resistance in op- medicine. In recent years, the origin of the portunistic bacteria such as S. aureus. methicillin-resistance encoding gene mecA has been identified among coagulase-negative staphylococci (CNS) like Staphylococcus n S5:3 fleurettii, Staphylococcus vitulinus and further METHICILLIN-RESISTANT CNS. In 2011, a novel mecA homologue STAPHYLOCOCCUS AUREUS IN URBAN RATS (mecC; EMBL FR821779) harbored by SC- C. G. Himsworth1, R. Millar1, P. Tang1, D. M. CmecXI was described for MRSA from human Patrick1, S. Weese2; and bovine origin, and later also from compan- 1University of British Columbia, Vancouver, ion animals. We suspected that mecC, similar BC, CANADA, 2Univ. of Guelph, Guelph, ON, to mecA, originated potentially from CNS. CANADA. Material and methods: The Staphylococcus stepanovicii strain IMT27065 (ODD4) was The Norway and black rat (Rattus norvegicus isolated in August 2011 from a fecal sample and Rattus rattus) are among the most common of a bank vole (Myodes glareolus) as part of a urban pest species and sources of various zoo- screening study focusing pathogens from wild notic pathogens. Little is known about MRSA rodents in Germany (Network “Rodent-Borne in rats, although it has been identified in rats Pathogens”). Rectal swabs were enriched for on farms in Europe. This study evaluated staphylococcal growth in order to prevent MRSA colonization in rats in an impoverished overgrowing gram-negative bacteria. The inner city region. Rats were trapped within the staphylococcal species of isolate IMT27065 core of the Downtown Eastside (DTES) neigh- was verified by 16S rRNA sequence analysis borhood of Vancouver, Canada, between Sept (LGC Genomics, Berlin). A positive PCR- 2011 and May 2012 as part of the Vancouver result for mecC was the reason for sequencing Rat Project. A swab of the oropharynx and the whole genome of the strain on a HiSeq (Il- nares was collected under general anesthesia. lumina, USA). The reads were assembled us- Selective, enrichment culture was performed. ing CLC Genomics Workbench 6.5 (CLC bio, Isolates were characterized by spa typing, PVL Denmark) and genomic comparative analyses PCR and whole genome sequencing (WGS). were performed using Geneious 6.1.5 (Biomat- MRSA was isolated from 22/637 (3.5%) rats, with a block prevalence ranging from 0-50%. 28 ASM Conferences Speaker Abstracts

Spa type t008/ST8 was most common (n=7, n S5:4 32%), followed by t034/ST3982 (n=5, 23%), THE MECA HOMOLOG MECC CONFERS t267/ST97 (n=5, 23%) and t002/ST105 (n=2, RESISTANCE AGAINST BETA-LACTAMS IN 9.1%). 3 other ST8 isolates (2 spa types) were STAPHYLOCOCCUS AUREUS IRRESPECTIVE also identified. WGS identified 4 clusters OF THE GENETIC STRAIN BACKGROUND of isolates, corresponding to MLST and spa types. Within 3 clusters, samples were diverse, B. Ballhausen, A. Kriegeskorte, D. Kuhn, N. with 44-107 variant positions. Rat t008 isolates Schleimer, G. Peters, K. Becker; were indistinguishable by WGS from human Institute of Medical Microbiology - University t008 isolates from the DTES, although there Hospital Muenster, Muenster, GERMANY. were distinct differences between t008 from Background: In contrast to hitherto known rats and humans from other regions. Rats SCCmec elements carrying mecA gene, caught in the spring and winter had higher SCCmec type XI harbors a mecA homolog, odds of being MRSA positive compared to designated mecC, along with the regulatory those in the fall, as did rats with greater body genes mecRI/mecI most probably conferring weight and internal fat stores. Two distinct resistance to beta-lactam antibiotics. Since clusters of high-than-expected MRSA preva- mecC shares only 70% identity on DNA level lence were identified, each corresponding to to the mecA gene of other SCCmec elements, distinct WGS clusters. The main clone found the impact of mecC on beta-lactam resistance here, the PVL-positive t008 (which corre- in Staphylococcus aureus was elucidated sponds to USA300) is an important cause of by generation of a defined mecC knock-out MRSA infection in Canada, and is frequently mutant in this study. To compare mecC and identified in humans from the DTES. The pre- mecA directly in its ability to confer resistance dominance of this common human epidemic to beta-lactams, we analyzed the MICs of clone, along with the inability of WGS to dif- oxacillin and cefoxitin in S. aureus strains with ferentiate rat isolates from those from humans different genetic backgrounds by expressing in the same area supports interspecies trans- mecA or mecC in trans regulated by its respec- mission, although the route(s) and direction(s) tive wild-type promoter or by a standardized are unknown. ST398/t034 was unexpected in promoter. Material and Methods: The mecC this urban population given its association with gene was knocked out in the MRSA isolate livestock. Most of the other isolates were rec- w44646 (spa type t843) by inserting an eryth- ognized human types, including the common romycin resistance cassette via homologues t002 (USA100/Canadian epidemic MRSA2). recombination. For homologous expression, Typing data and spatial clustering suggest mecC and mecA were cloned into the vectors that there are independent incidents of MRSA pCN47.2 and pCN68.2. The resulting plasmids introduction into rats in different blocks over were introduced into methicillin-susceptible S. time. The seasonal association might reflect aureus strains RN4220, ME131 and NE1868 differences in human-rat interactions or the as well as into the w44646∆mecC mutant. An- type of indirect contacts between humans and timicrobial susceptibility testing with oxacillin rats. The association of MRSA with higher and cefoxitin was performed by microdilution body fat could suggest that MRSA exposure is according to CLSI guidelines. The expres- influenced by rat social hierarchy, with fatter sion of mecC, mecRI and mecI of SCCmec (dominant) rats at greater risk, as has been XI in presence and absence of oxacillin was reported for Leptospira shedding. The public comparatively analyzed by real time-PCR. health consequences of MRSA shedding in Results: The mecC knock-out strain showed rats are unclear but this evidence of interspe- considerably reduced oxacillin and cefoxitin cies transmission indicates that further study is MICs compared to its wild type stain (oxacillin required.

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 29 Speaker Abstracts and cefoxitin MICs decreased from 8 to 0.25 temperature/duration and atmosphere. In vitro mg/L and from 16 to 4 mg/L, respectively). pharmacokinetic and pharmacodynamic (PK/ Complementing w44646∆mecC in trans using PD) modelling is often based on the minimum both mecC and mecA restored the resistance to inhibitory concentration (MIC) values. Lit- oxacillin and cefoxitin. Homologues expres- erature on bacterial densities during infection sion of mecC in S. aureus RN4220, ME131, show low to high (102 colony forming units per NE1868 and w44646∆mecC led to an increase milliliter –CFU/ml) to 109 CFU/ml) bacterial in cefoxitin and oxacillin MICs to 32, 256, densities in meningitis and high density (107 32-64, 64mg/L and to 32, 128, 32-64, 64mg/L, ->1010 CFUs) burdens in pneumonia. As such, respectively. As described for the mec operon, a single bacterial density used for susceptibil- oxacillin was likewise found to induce the ex- ity testing is misleading and clearly does not pression of mecC as well as mecRI and mecI. represent bug-drug interactions when low/ Sub-inhibitory concentrations of oxacillin (2 high density burdens are present. The mutant mg/L) led to a 100-, 9- and 10-fold increase prevention concentration (MPC) defines the of transcription of mecC, mecRI and mecI, antimicrobial drug concentration necessary respectively. Conclusion: This study proofs to block growth of the least susceptible cells for the first time genetically that mecC medi- present in high density (>109 CFUs) bacterial ates ß-lactam resistance in MRSA carrying the populations. MPC measurements are conduct- SCCmec type XI. We demonstrated that mecC ed on susceptible strains. MPC measurements alone mediates resistance to oxacillin and with methicillin susceptible Staphylococcus cefoxitin irrespective of the underlying genetic aureus (MSSA) and fluoroquinolones showed background. Comparing the oxacillin/cefoxitin differences in the ability of each compound MIC values of strains expressing mecC and to restrict mutant growth despite the fact the mecA in trans, we found only slight differ- compounds are clinically equivalent. MPC ences between mecA and mecC in their ability testing of quinolone susceptible, methicillin to confer ß-lactam resistance. Furthermore, resistant S. aureus (MRSA), also showed clear this study provides evidence that the mecRI/ differences in the ability to restrict mutant mecI regulatory system of SCCmec type XI growth between the quinolones tested. MPC is functional and that it can be activated by testing of Staphylococcus pseudintermedius ß-lactam antibiotics. against quinolones, aminoglycosides, beta lactams and macrolides also showed clear n S6:1 differences between compounds in the amount of drug necessary to block growth of the least MULTI-DRUG RESISTANT STAPHYLOCOCCI susceptible cell in high density inocula. Low AND INCREASING ANTIMICROBIAL MIC values were often followed by MPC val- RESISTANCE: WHAT ARE WE CURRENTLY ues above therapeutic drug concentrations for LEARNING USING NOVEL IN VITRO some compouns. With fluoroquinolones andS. MEASUREMENTS? pseudintermedius, pradofloxacin had the low- J. M. Blondeau; est MPC values of all agents tested. For MRSA Clinical Microbiology, Royal University Hos- and vancomycin (MIC 1ug/ml) MPC values pital, Saskatoon, SK, CANADA. of 16ug/ml were seen which upon retesting by MIC yielded a parental MIC value. The expla- Antimicrobial resistance has compromised nation of this observation remains unknown; every antimicrobial agent currently licensed pulsed field gel electrophoresis analysis for therapeutic use. Bacterial resistance or showed identical profiles between the parental susceptibility is based on an in vitro measure- strain and that with the high MPC value. In ment utilizing 105 organisms per milliliter vitro kill measurements based on a range of standardized for inoculum, media, incubation bacterial densities (106-109 CFU/ml) showed

30 ASM Conferences Speaker Abstracts striking differences between MIC, MPC, maxi- cillin susceptibility whereas this is considered mum serum and tissue drug concentrations and uninformative or economically not justified killing of bacteria. How this relates to resis- elsewhere. Screening for methicillin resistance, tance selection and clinical recovery is cur- whether caused by mecA or mecC, does not rently debated. Novel in vitro measurements normally take place as part of routine mastitis are adding considerable discussion around our diagnostics. In addition to mastitis with visible understanding of the emergence and spread of signs, S. aureus may cause subclinical mastitis antimicrobial resistance and how this impacts or asymptomatic intramammary infection in clinically. How such information should be dairy cattle. This condition affects milk quan- incorporated in PK/PD modelling is an intrigu- tity and milk quality, providing an economic ing but necessary question. rather than a welfare incentive for its control. Depending on herd management, S. aureus n S6:2 may spread between cows in a milking herd in a contagious manner, or even between milk- EPIDEMIOLOGY AND MANAGEMENT OF ing animals and non-milking animals. Thus, MRSA IN DAIRY HERDS control of infection in one animal may lead R. N. Zadoks; to prevention of infection in other animals. Food Science, University of Glasgow, Whether or not the use of antimicrobials is Glasgow, UNITED KINGDOM. justified under those circumstances depends on the balance of economic and societal fac- From the farm perspective, there is no such tors, e.g. the expected economic gain or the thing as the management of MRSA in dairy potential risk of selecting for antimicrobial herds because farmers or herd managers would resistance. Information on prevalence and epi- rarely be aware that they have MRSA in their demiology of MRSA in dairy herds stems from herds. Mastitis, as it presents to farmers, is the research rather than diagnostic investigations. presence of clots, flakes or other abnormali- Molecular epidemiology studies suggest that ties in a cow’s milk, possibly accompanied by MRSA in dairy herds may be a human- or pig- the classical signs of “-itis”, i.e. rubor, tumor, derived problem or a primary cattle problem, dolor and calor (redness, swelling, pain, in- with differences between countries and strains. creased temperature). In developed countries, In this presentation, an overview will be given such milk is not considered fit for human of mastitis management on dairy farms, with consumption. As a result, the affected milk is specific attention to the management ofS. discarded or fed to calves or other animals. aureus mastitis, potential beneficial or harmful Meanwhile, to protect animal welfare and to consequences of the use of antimicrobials return the cow to production of saleable milk and the feasibility or need for MRSA specific as quickly as possible, animals may or may not management strategies from the perspective of be treated with antimicrobials, anti-inflamma- farms and society. tory drugs or supportive therapy. At this stage, it will generally not be known that the disease is caused by Staphylococcus aureus, let alone n S6:3 MRSA, so clinical case management will not THE VIEW ON LA-MRSA BY THE DANISH be targeted to MRSA. Availability and opin- PIG INDUSTRY (PIG RESEARCH CENTRE) ions on the usefulness of diagnostics, including P. Baekbo; identification of bacterial species and, pos- Pig Research Centre, Danish Agriculture & sibly, antimicrobial resistance profiles, differ Food Council, Kjellerup, DENMARK. between laboratories and countries. In some countries, detection of S. aureus from milk is Denmark has a significant pig production of automatically followed by screening for peni- 30 million pigs per year. Denmark is one of

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 31 Speaker Abstracts the major players on the world pork marked, 4. Recommend a high level of personal exporting nearly 2 mill tons of pork per year. hygiene in all pig herds, whenever people are The Danish pig producers are organised in a leaving the farm pig units NGO, The Pig Research Centre (PRC) that is 5. Seeks a high degree of dialog and coopera- an integrated part of The Danish Agriculture & tion with the human and veterinary health Food Council (DAFC). DAFC brings together authorities the largest industry grouping in Denmark, rep- 6. Supports activities to reduce the overall con- resenting the Danish food and farming indus- sumption of antibiotics in the pig production try. More than 50% of the pig herds are run as (already the amount of antibiotics used per kg SPF-herds being free from some of the major produces pork is one of the lowest in the world pig diseases and all herds have a Salmonella of modern pig production) status according to the Danish Salmonella It has been discussed if the spread of infec- control program initiated in 1995. tion between herds could be controlled by an LA-MRSA CC398 was isolated from pigs in infection declaration of all pig herds (positive/ Denmark in 2006 for the first time. Surveys negative herds). Based on the present knowl- performed by the authorities in 2010 and 2011 edge PRC do not see declaration as a relevant shows that 16% of the Danish pig herds are in- option, because: fected (www.danmap.org). In humans CC398 • Too many herds already infected constituted 12.5% of the MRSA cases in 2011 • Reliable free-testing is a challenge and pre- (164 persons), of which the majority are seen sumably very costly among persons with direct contact to pigs. • False confidence by the stockmen From 2012 contact with live pigs is regarded • No proven way of elimination of MRSA on as a general risk factor in the MRSA guideline herd level for the human health sector. • Potential conflicts with other infections of MRSA CC398 is expected to be introduced interest (The SPF-pig diseases & Salmonella) to the Danish pig production from other countries, presumable from human contacts as n S6:4 the import of live pigs is very limited. MRSA RISK MANAGEMENT INITIATIVES OF is transmitted between herds by transfer of live LA-MRSA CC398 IN THE DANISH PIG pigs, whereas the significance of transmission PRODUCTION IN A “ONE HEALTH” by other routes is unknown. PERSPECTIVE The PRC regards LA-MRSA as a problem for the work environment (people working with C. Thrane; live pigs) and with no food safety aspects. Danish Veterinary and Food Administration, PRC advices all people working with live pigs Glostrup, DENMARK. to give this information whenever they are in Risk management initiatives of LA-MRSA contact with human health system, especially CC398 in the Danish pig production in a in any case of surgery. “One Health” perspective The strategy of the PRC on MRSA CC398 can LA-MRSA is present in the Danish pig pro- be put into the following 6 statements: duction. During the last years there has been 1. Takes the problem seriously, but we have to an increase in the impact of LA-MRSA on the live with it human health. Consequently, we are currently 2. Seeks full transparency and high level of reconsidering how to manage the human risk information for people working in the pig of LA-MRSA. industry The Danish Ministers of Food, Fisheries, and 3. The herd owner of infected herds must Agriculture respectively of Human Health inform their workers, visitors and byers of live decided that risk management of LA-MRSA pigs of the MRSA status

32 ASM Conferences Speaker Abstracts was best dealt with if handled in a “one health The primary route of zoonotic transmission perspective”. Thus, at the national level we of MRSA has been considered to be direct or established a Danish action group with experts indirect contact of livestock professionals with from different disciplines across veterinary, colonised animals, while the role of food as a food safety, and working environment from source of human colonisation or infection has Universities, authorities and private stake- been deemed of minor importance. Therefore, holders. This action group has been working monitoring the occurrence and diversity of together to ensure that managing the LA- MRSA in primary production, including at MRSA risk is based on “one health” principles, slaughter, seems pivotal, while monitoring and that resources to combat LA-MRSA are in food may also help with the assessment of leveling the known risk. consumers’ exposure via this route. MRSA has The first task of the action group was to point been shown to evolve continuously, and chang- out short term initiatives, that were supposed es in characteristics, such as virulence and to help to decrease the human exposure to LA- transmissibility, may occur in the future and MRSA. The prioritized short term initiatives to favour exchanges between animal and human fight the human impact of LA-MRSA will be populations, in which the organism may adapt, presented. diffuse, multiply and evolve further. Regular We still have some gaps about the physiology monitoring of MRSA is beneficial in directly and epidemiology of the LA-MRSA, which is providing information about the emergence of a basis for effectively combatting and control- strains of potential public health significance, ling the microbe. In Denmark, it was decided but can also provide important epidemiological that we cannot wait for more knowledge – we data on the spread of particular strains between need to improve measures starting in the short animal and human populations. Monitoring in term. animals should be performed in parallel with consistent monitoring in humans. n S7:1 Monitoring recommendations have proposed a harmonised definition of MRSA and prioritised TOWARDS HARMONISED MONITORING OF several different food-producing animal popu- MRSA IN ANIMALS AND FOOD IN THE EU lations previously described as MRSA reser- P. Beloeil; voirs and, to a lesser extent, food produced by European Food Safety Authority (EFSA), these animals. Regular monitoring of broiler Parma, ITALY. flocks, fattening pigs and dairy cattle, as well as in veal calves under 1 year of age, and fat- MRSA colonisation, in production animals, tening turkey flocks, is recommended every detected in recent years, has, in several cases, third year on a rotating basis. It is proposed resulted in infection in humans, and infections that breeding poultry flocks and breeding pigs, with a livestock-associated strain of MRSA as well as meat and raw milk products, are may be considered a zoonosis. An assessment monitored on a voluntary basis. Representative of the public health significance of MRSA sampling should be made within the frame- in animals and in food was issued by EFSA work of the national Salmonella control pro- in 2009. In addition, EFSA has also recently grammes for the poultry populations targeted, issued proposals, as a result of a mandate from at the slaughterhouse for calves and either on the European Commission, to improve the the farm or at the slaughterhouse for fattening harmonisation of monitoring of prevalence, pigs. Harmonised analytical methodologies for genetic diversity and antimicrobial susceptibil- identification, typing and further characterisa- ity in MRSA from food-producing animals tion of MRSA are also proposed. The use of and food derived thereof, by the EU Member the microdilution method applied to a harmon- States. ised set of antimicrobials through harmonised

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 33 Speaker Abstracts concentration ranges, and interpreted using onto SaSelect (Biorad, USA) and blood agar. EUCAST epidemiological cut-off values, for Pigs were defined as permanent carriers if antimicrobial susceptibility testing of MRSA, positive in all three sampling times, transient is recommended. carriers if positive in one or two sampling times, and non-carriers if negative in all sam- n S7:2 pling times. Data were analysed statistically using a logistic regression model for either UNDERSTANDING STAPHYLOCOCCUS multinomial or binary data (proc glimmix, AUREUS COLONIZATION IN PIGS: CAN SAS Inst.), with sampling round, swab cleanli- SELECTIVE BREEDING BE USED TO ness and MRSA-positivity for herd as fixed PRODUCE MRSA-FREE PIGS? effects when appropriate, and farm, room and L. Guardabassi; pig as random effects, when appropriate. University of Copenhagen, Copenhagen, Results: All farms were S. aureus-positive and DENMARK. in-herd prevalence of permanent carriers was on average 23% (range 0 to 75%). Among the Introduction: Staphylococcus aureus colo- 480 pigs tested, 23% were permanent carriers, nization is a complex biological phenomenon 54% were transient carriers and 23% were resulting from interaction of bacterial, host and non-carriers. There was a strong effect by farm environmental factors. S. aureus colonization (p<0.0001) and pen (p≤0.02) on in-herd preva- rates in the human population are known but lence and load of S. aureus in nasal swabs. virtually nothing is known about frequency Within farms, some pigs had a significantly and stability of colonization in pigs. This higher load than others (p=0.004), and the is because most studies to date focussed on bacterial load was significantly higher in per- MRSA only, had cross-sectional design and manent carriers than in transient carriers, when assessed MRSA carriage by use of enrichment the sample was positive (p=0.002). There was procedures, which enhance detection but do a tendency of negative animals to remain nega- not provide any quantitative data. Objec- tive during the study period. tive: This lecture is based on the results of a large longitudinal study where S. aureus nasal Conclusions: This study shows that, even if colonization rates were assessed quantitatively farm- and pen-related effects are taken into in 480 adult pigs over a period of three weeks. consideration, certain pigs are more predis- The objective of the study was to understand posed than others to S. aureus colonization. frequency and stability of colonization in adult The genomes of 50 permanent carriers and 50 pigs. non-carriers identified in this study are presently analysed to investigate whether coloniza- Material and methods: A longitudinal quan- tion is enhanced by specific host genetic traits. titative study was performed in 20 randomly If this hypothesis will be confirmed, selective selected Danish production farms. Within breeding could be used to produce pigs with each farm, 24 individual pigs in the finishing reduced susceptibility to S. aureus. Potential sections were ear-tagged and nasal swabs were use of this approach for control of MRSA in collected from each individual three times at pig farming will be discussed taking into con- weekly frequency. S. aureus was quantified sideration sustainability and risks associated by the most probable number (MPN) method changes in the current breeding programmes. following enrichment of swab serial dilutions in physiological saline and subsequent plating

34 ASM Conferences Speaker Abstracts n S7:3 the study, is given by the β estimates from the INTERVENTION MEASURES REDUCING mixed models. Univariate results, adjusted for LIVESTOCK-ASSOCIATED MRSA ON time trend, showed that farms with finish- PIG FARMS IN THE NETHERLANDS: A ing pigs and farms without external supply LONGITUDINAL STUDY. of animals had significantly (p<0.05) lower prevalence during the study (β=-17.9 and -24.4 A. Dorado-García1, M. E. Bos1, W. Dohmen1, respectively). MRSA prevalence was signifi- K. M. Verstappen2, J. A. Wagenaar2, D. J. cantly higher in farms cleaning with soaking Heederik1; agents (β=15.6), farms vaccinating piglets 1Institute for Risk Assessment Sciences, and/or fatteners (β=17.0) and farms injecting Utrecht, NETHERLANDS, 2Department of antibiotics in piglets during the first week of Infectious Diseases and Immunology, Faculty life (β=15.8). Housing sows in stable groups of Veterinary Medicine, Utrecht University, led to significantly lower prevalence (β=- Utrecht, NETHERLANDS. 13.5). Other downward trends in prevalence (borderline significant or p<0.2) were shown Livestock-associated (LA) MRSA is widely in farms where showering was mandatory (β=- spread in pig farms in the Netherlands with 7.4), delivery platform was cleaned right after approximately 70% of them being positive. animal movements (β=-9.1), no teeth clipping The aim of this study is to determine the in piglets was done (β=-10.4), and external quantitative effect of implementing interven- biosecurity score was high (β=-9.8). Addition- tion measures on pig farms in order to reduce ally, data on prescription of antimicrobials dur- MRSA transmission to humans. A longitudi- ing the study are being processed . Preliminary nal study in 40 randomly selected pig farms results show a positive and significant linear in the Netherlands was done for a period relationship between the Defined Daily Doses of 18 months. On each farm 10 pools of 6 (DDD) animal/year and pool prevalence in nasal swabs were obtained from pigs every 6 at least two sampling moments. However, months. A tailor made intervention protocol longitudinal evaluation still needs to be done. was developed by the farmer and the main vet- A final multivariate mixed model by which we erinarian targeting 3 key points: reduction of attempt to explain change in prevalence over the use of antimicrobials, improving hygiene time, as a result of the described interventions, and changing the animal contact structure. An is under development. The longitudinal nature extensive questionnaire was used to assess the of our data will enable us to give indications of course of all specific interventions. There was the most effective strategies to decrease MRSA a non-significant decrease in the overall MRSA prevalence on pig farms. pool prevalence from the beginning (42.6%) to the end of the study (36.3%). For the purpose of variable reduction, potential determinants, registered by the questionnaire, were evaluated cross-sectionally in the 4 sampling moments by a simple linear regression for pool prevalence. Those with p<0.20 in at least 2 sampling moments were further evaluated longitudinally. The selected determinants were included with sampling moment as a factor in a bivariate random intercept model to adjust for trend over time. The difference in mean prevalence between the 2 categories of a variable during

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 35 Speaker Abstracts n S7:4 n CS:1 METHICILLIN-RESISTANT PHAGE LYSINS MAY BE USED TO TREAT STAPHYLOCOCCUS AUREUS (MRSA) AND PREVENT INFECTIONS IN BOTH SCREENING AT A TERTIARY VETERINARY HUMAN AND VETERINARY APPLICATIONS HOSPITAL: IS TESTING COST-BENEFICIAL V. A. Fischetti; FROM AN INTEGRATED HUMAN-ANIMAL Lab of Bacterial Path., Rockefeller Univ., New PERSPECTIVE? York, NY. J. P. Ferreira1, T. Birkland2, K. Anderson2, M. Bacteriophage lytic enzymes (lysins) are Correa2; highly evolved molecules used by the phage 1SAFOSO, Bern, SWITZERLAND, 2North to quickly destroy the bacterial cell wall to Carolina State University, Raleigh, NC. release phage progeny. We have exploited Methicillin-resistant Staphylococcus aureus the rapid and lethal action of these enzymes (MRSA) is an antimicrobial resistant organism to destroy pathogenic bacteria on mucous of international significance to human and vet- membranes, in blood and infected tissues. In erinary medicine. In human medicine, policies general lysins are specific for the species or proposed and tested for MRSA control include bacterial group for which they were produced, variations of universal patient screening and thus avoiding destruction of the surround- surveillance programs. In veterinary medicine ing normal commensal organisms found on information on MRSA control is scarce. Objec- mucosal surfaces. Lysins have been developed tive: To develop a cost-benefit (CB) analysis that are specific forS. equi, S. suis, S. uberis, from an integrated human-animal that could be S. pyogenes, S. pneumoniae, S. aureus, B. used for policy development and including all anthracis, E. faecalis, E. faecium and group animals admitted for hospitalization to NCSU B streptococci. Our results show that in vitro CVM Veterinary Teaching Hospital. Materi- 107 bacteria can be reduced to sterility minutes als and methods: A model was developed to after enzyme contact. In animal model experi- estimate the costs of MRSA of all animals ments, we were able to colonize mice with admitted to the hospital taking into consider- either streptococcal or pneumococcal species ation the projected economic benefits of the (orally or nasally) and remove them to unde- prevention of human infections. Results: The tectable with lysins delivered to these sites us- baseline model used the most plausible inputs ing a single lysin dose. In a septicemia model and different scenarios were considered in the with S. pneumoniae, bacteria are reduced by sensitivity analysis. The cost of the screening >3-logs from the blood of infected animals policy was estimated at $320,104 and exceed- with a single intravenous dose of enzyme ed the savings of about $183,409. Variations in resulting in survival of nearly all animals. A the input assumptions, most notably the addi- model of murine pneumococcal pneumonia tional cost for treating a human case, rendered was developed demonstrating widespread in- a variety of possible outcomes. Conclusions: filtration of the lung and physiologic evidence Our study suggests that it is not beneficial to of systemic infection within 24h. When mice implement MRSA screening programs in vet- were treated intraperitoneally with either the erinary hospitals from an integrated economic anti-pneumococcal lysin Cpl-1 or amoxicil- human-animal perspective. lin every 12 hours for 3 days, bacteremia was

36 ASM Conferences Speaker Abstracts eliminated from both groups and lung function prominent effect on MRSA biofilms compared and histological findings returned to normal, to the limited or no effect when antibiotics whereas all animals treated with buffer died. were used. When we tested the S. uberis lysin Similar results were obtained in endocardi- for its activity in the presence of raw milk, we tis and meningitis models of pneumococcal found that it was not inhibited, suggesting its infection. Resistance to lysins has not been use in controlling mastitis in combination with found nor do antibodies completely neutralize the staphylococcal lysin PlySs2. Thus, phage their activity, allowing for repeated use in the lytic enzymes are a safe new reagent that may same individuals. Furthermore, a combina- be used to control both antibiotic resistant and tion of antibiotic and lysin has been shown to sensitive pathogenic bacteria in both human work synergistically resulting in significant and veterinary applications and offer a capabil- lethal activity in cases of antibiotic resistant ity previously unavailable. bacteria. We also found that lysins have a

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 37 Poster Abstracts n 1A belonged to five different MLST types, ST2, PRESENCE, DISTRIBUTION AND ST5, ST10, ST48, and ST66, with ST5 being TRANSMISSION OF METHICILLIN- predominant. All isolates were multidrug- RESISTANT STAPHYLOCOCCI IN A SMALL resistant with resistance to at least three classes ANIMAL CLINIC of antimicrobial agents. In addition to isolates that varied in their molecular characteristics, a S. Weiß, K. Kadlec, A. Feßler, S. Schwarz; single multidrug-resistant S. epidermidis clone Institute of Farm Animal Genetics, Friedrich- was detected in nine samples from cats, clinic Loeffler-Institute (FLI), Neustadt-Mariensee, environment and an employee. Members of GERMANY. this clone shared SCCmec type IV, dru type dt10a and MLST type ST5. Moreover, these Objective: The aim of this study was to nine MRSE isolates showed indistinguishable identify and to characterize methicillin- plasmid profiles and SmaI macrorestriction resistant staphylococci (MRS) isolated from patterns. Conclusion: Multiresistant MRS samples collected in a small animal clinic isolates belonging to the same species and and to investigate their distribution, focusing showing the same characteristics were isolated on a possible transmission and on resistance from patients without clinical infections, the properties. Materials and Methods: In total, clinic environment as well as from healthy 72 swabs were taken from animals (n=10), the employees, suggesting a possible transmission. environment (n=58) and employees (n=4) and Moreover, MRS were mainly found in the screened for the presence of MRS. The staphy- stationary area of the clinic. lococcal species was confirmed biochemically and by 16S rDNA sequencing. Methicillin resistance was confirmed by MIC determina- n 2A tion for oxacillin according to the recommen- MULTIDRUG- AND METHICILLIN-RESISTANT dations given by the Clinical and Laboratory STAPHYLOCOCCUS AUREUS ARE PRESENT Standards Institute and by PCR detection of IN SURFACE WATERS NEAR INDUSTRIAL mecA. Further characterization followed stan- HOG OPERATIONS IN NORTH CAROLINA dard procedures. Results: The 34 identified S. M. Hatcher1, K. Myers1, C. D. Heaney2, D. MRS isolates were Staphylococcus (S.) aureus Hall3, J. Larsen4, S. Wing1, J. Stewart1; (n=5), S. epidermidis (n=21), S. haemolyticus 1University of North Carolina at Chapel Hill, (n=6) or S. pettenkoferi (n=2). They originated Chapel Hill, NC, 2Johns Hopkins Bloomberg from cats (n=6), the environment (n=24) or School of Public Health, Baltimore, MD, 3Ru- humans (n=4). Neither the feline patients nor ral Empowerment Association for Community the employees showed clinical signs of staphy- Help, Warsaw, NC, 4Microbiology and Infec- lococcal infections. The isolates had SCCmec tion Control, Statens Serum Institut, Copenha- types IV (n=22) or V (n=8) or were non-type- gen, DENMARK. able (n=4) by the multiplex PCR assays used and dru typing showed five noveldru types Industrial animal production using sub- (dt5i, dt9bb, dt9bc, dt10cb, dt12w) in addition therapeutic levels of antibiotics for growth to five knowndru types (dt10a, dt10g, dt10r, promotion and disease prevention has raised dt11a, dt11c). Four of the five MRSA isolates concerns about antibiotic resistance in clini- belonged to the clonal complex 398, the re- cally relevant bacteria such as Staphylococcus maining MRSA had multi locus sequence type aureus. Although nasal carriage of antibiotic- (MLST) ST217. The S. epidermidis isolates resistant S. aureus in industrial animal workers

38 ASM Conferences Poster Abstracts has been documented, transmission of S. of waterborne S. aureus and MRSA collected aureus to the water environment in areas from surface waters near industrial hog opera- where waste products are sprayed has not tions. been characterized. We investigated whether S. aureus is present in surface waters near n 3A industrial animal operations that manage liquid EFFECT OF PIG POPULATION DENSITY waste with lagoons and spray fields. Surface ON METHICILLIN RESISTANT water samples (n=183) were collected over the STAPHYLOCOCCUS AUREUS PREVALENCE course of approximately one year from nine IN BULK TANK MILK OF ITALIAN DAIRY sites in southeastern NC and analyzed for the FARMS presence of S. aureus. A total of 698 presumptive Staphylococcus isolates exhibiting phe- C. Locatelli1, P. Cremonesi2, L. Bertocchi3, M. notypic antibiotic resistance were recovered Zanoni3, G. Varisco3, A. Barberio4, I. Drigo5, and archived. Culture-based, biochemical, and B. Castiglioni2, V. Bronzo1, P. Moroni1; molecular tests were used to confirm the pres- 1Università degli Studi di Milano, Milan, ence of S. aureus and methicillin-resistant S. ITALY, 2Istituto di Biologia e Biotecnologia aureus (MRSA). Confirmed S. aureus isolates Agraria, Lodi, ITALY, 3Istituto Zooprofilattico were subject to additional characterization, Sperimentale della Lombardia e dell’Emilia- including antibiotic susceptibility testing using Romagna, Brescia, ITALY, 4Istituto Zoopro- Kirby-Bauer disk diffusion, spa typing, and a filattico Sperimentale delle Venezie, Vicenza, duplex PCR to detect the tet(M) and scn genes. ITALY, 5Istituto Zooprofilattico Sperimentale Of the 698 isolates, 16 were S. aureus and 5 delle Venezie, Treviso, ITALY. were MRSA. Staphylococcus aureus isolates MRSA contamination of pig holdings in Eu- were recovered from 16 distinct samples, 9 rope with ST398 and other lineages is a fact. sampling events, and 7 sites, with an overall Dairy cows can also be colonized and suffer percent detection of 0.09 (16/183 samples). mastitis by MRSA. Professional categories By site, S. aureus and MRSA detection ranged linked to farming resulted colonized at higher from 0-30% and 0-15%, respectively. Eleven rates than general population due to exposure isolates were resistant to at least one antibiotic along all the production chains. The proxim- and four were multidrug-resistant (defined as ity to livestock itself is at risk. A Dutch recent complete resistance to ≥3 antibiotic classes). study showed that people without direct The most common spa types represented in contact with pigs or cows and living in a rural this study were t008 (5/16) and t021 (7/16), area were more likely to be LA-MRSA posi- which have been associated with clonal tive than people in urban settings. The mutual complexes 8 and 30, respectively. Other spa risk between different species in livestock has types represented include t216, t338, and been not completely investigated yet. We used t267. No spa types detected in this study have bulk tank milk (BTM) as a screening sample previously been associated with livestock, but to detect MRSA in dairy herds located within phenotypic tetracycline resistance and lack of a high-density pig farming area. Applying the scn gene--both of which have been con- Geographic Information Systems (GIS) to sidered markers of livestock association--were the area, the association between density of observed in four and six isolates, respectively. finishing pigs and MRSA status of dairy herds This study demonstrated that multidrug- and was evaluated. Bulk tank milk (BTM) samples methicillin-resistant S. aureus were present in were collected in March 2011 from 225 dairy surface waters adjacent to industrial hog opera- farms resulted positive for taphylococcus tion waste spray fields in southeastern NC. To aureus in the bulk tank at a previous control. our knowledge, this is the first documentation Each milk sample underwent a double enrich-

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 39 Poster Abstracts ment, firstly in Mueller Hinton broth with 6.5 n 4A % NaCl and then in Tryptic Soy broth with 3.5 FINDINGS OF MRSA OF THE SAME SPA- mg/l cefoxitin and 75 mg/l aztreonam. Each TYPE IN FARMER AND DAIRY COWS IN enrichment step required an incubation for SWEDEN 18-20 h at 37°C. Finally, 100 µl were plated onto MRSA Chromogenic Agar (Pronadisa, H. Ericsson Unnerstad1, B. Bengtsson1, T. Spain). After incubation of 48 h at 37°C, Hallgren2, H. Hedbäck3, H. Landin4, B. Lars- putative colonies were confirmed MRSA by son5, P. Nordmark6, Y. Persson7, K. Strand3, C. a duplex PCR targeting nuc and mecA genes. Thörn5, K. Mieziewska5; Dairy herds with at least a MRSA isolate 1National Veterinary Institute, Uppsala, were classified MRSA-positive. Isolates were SWEDEN, 2District Veterinarian Department, typed by Multi Locus Sequence Typing and Valdemarsvik, SWEDEN, 3Department of spa-typing. Each milk sample underwent also Communicable Disease Control, Östergöt- DNA extraction. Dairy herds in whose milk land, SWEDEN, 4Växa Sverige, Stockholm, the detection of mecA by PCR occurred were SWEDEN, 5Swedish Board of Agriculture, classifiedmec A-positive. All the other dairy Jönköping, SWEDEN, 6County Administra- herds were classified as negative. Geographic tive Board, Östergötland, SWEDEN, 7Växa Information System ArcVIEW (ArcGis, 9.3.1, Sverige, Uppsala, SWEDEN. Esri, Redlands, CA) computed number of Background: Methicillin resistant Staphylo- finishing pig holdings and pig heads within 3 coccus aureus (MRSA) has only been detected km range around each dairy farm. Statistical sporadically in dairy cows in Sweden. Here analyses by SPSS 19.0 (IBM, SPSS Inc., Chi- we describe the findings of MRSA of the cago IL, USA) and test U Mann-Whitney were same spa-type from both farmer and several applied to compare pigs consistency based of the cows in a dairy herd. The farmer was on the MRSA status of dairy herds. Statisti- confirmed positive withpvl -positive MRSA cal significance was accepted when P<0.05. A of spa-type t002, which is one of the most total of 53 herds (23.6%) were mecA-positive common spa-types in humans in Sweden. and 9 herds (4%) were effectively MRSA- Materials and methods: In order to inves- positive. Two STs were isolated (ST398 and tigate if also cattle in the herd were infected ST97) and fivespa -types (t899, t001, t108, with or carriers of MRSA, all 68 animals in the t4795 and t9305). The number of finishing pig herd were sampled. Composite milk samples holdings was significantly higher around both were taken from all 25 lactating cows and mecA-positive (P=0.004) and MRSA-positive one sample from bulk tank milk. Nasal swabs herds (P=0.000) than those surrounding nega- and samples from groin skin were taken from tive herds. Also the number of finishing pigs non-lactating cattle, including one dry cow was significantly higher aroundmec A-positive and 42 young cattle and calves. In addition, and MRSA-positive (P=0.019 and P=0.001, re- wounds were sampled, if present. All samples spectively). The present work give evidence to were selectively enriched over night in 37°C in the significant effect of pig population density Trypton Soy Broth with mannitol and phenol on dairy herds status regarding MRSA. STs red, 4% NaCl, 3.5 mg/L cefoxitin and 50 mg/L and spa-types confirmed this link too, as they aztreonam. Composite milk samples and 10 are most common in Italian finishing pigs. mL of the bulk tank milk were centrifuged and the supernatant discarded before enrichment. Nasal swabs and swabs from groins were pooled five and five in enrichment broth. After incubation, enrichment broth was spread on MRSA Brilliance agar (Oxoid) and bovine

40 ASM Conferences Poster Abstracts blood agar and incubated over night in 37°C. conventional swine farm. The nasal micro- Suspected colonies were confirmed with PCR biome was assessed using next generation for the nuc-, mecA-, mecC- and pvl-genes. sequencing following V3-V5 16s rRNA gene Confirmed isolates werespa -typed. Results: PCR. Species richness was high, a mean of MRSA was detected in samples from 12 cows 120 species-level OTUs (range 25-277, median and in bulk tank milk. From 10 of the cows 98) and no difference between MRSA carriers only the milk sample was positive, from one and non-carriers (P=0.94). In conventionally cow both the milk sample and the sample from fed pigs, the Proteobacteria Phylum was most groin and from one dry cow only the nasal abundant, followed by Firmicutes (largely swab. All isolates were positive for the nuc-, based on high abundances of Staphylococcus, mecA- and pvl-genes and were of spa-type Bacillus and Paenibacillus spp). Bacteroidetes, t002. MRSA was not detected in samples from Spirochaetes, Actinobacteria, Cyanobacte- young cattle and calves. One month later, all ria, Thermotogae, Tenericutes, Synergistes, previously MRSA-positive cows, except three Fibrobacteres, Fusobacteria, Elusimicrobia, that had been culled, were sampled again by Lentisphaerae and Deferribacteres were means of milk samples and nasal swabs. At present at lower abundances. There was no the second sampling, MRSA was detected in relationship between microbial population milk samples from seven cows. Conclusion: structure and MRSA carriage (P>0.05), nor Since MRSA-isolates both from the farmer was there any apparent clustering by principal and from dairy cows belonged to spa-type t002 component analysis. There were a few statisti- which is one of the most common spa-type in cally significant differences at lower taxo- humans but has never before been detected in nomical orders, including Sphingobacteriales farm animals in Sweden, it was suspected that (Bacteroidetes Phylum, P=0.005), Burkhold- transmission occurred from humans to dairy eriales order (P=0.037) and Comanadaceae cows. All lactating MRSA-positive cows that family (P=0.036)(Proteobacteria Phylum) and remained in the herd after one month were still Microbacterium Genus (P=0.021, Actinobacte- positive at the second sampling, indicating ria Phylum), the biological relevance of which that MRSA of a common human spa-type was is unclear. There was abundant staphylococcal established in lactating cows in the herd. diversity, with a total of 12 different species identified. ; S. aureus, S. epidermidis, S. n 5A equorum, S. fleuretti, S. hominis, S. pseudintermedius, S. kloosii, .S lentus, S. lugdunensis, THE EFFECT OF METHICILLIN-RESISTANT S. pasteuri, S. saprophyticus and S. schleiferi, STAPHYLOCOCCUS AUREUS CARRIAGE although care should be taken with this result AND DIET ON THE NASAL MICROBIOTA IN because of the limited species resolution with SLAUGHTER-AGE PIGS 16s rRNA gene-based studies. There was a J. Weese, M. Slifierz, M. Jalali, R. Friendship; significant impact of diet. Liquid-fed pigs had Univ. of Guelph, Guelph, ON, CANADA. significantly fewer Bacteroidetes (P=0.042) and Proteobacteria (P=0.027), and more The nasal microbiota may play a role in deter- Firmicutes (P=0.004). There were numerous mining the fate of bacteria to which it becomes differences between groups at lower taxonomic exposed, including MRSA. The objective of levels, including Paenibacillaceae (P=0.007), this study was to describe the nasal micro- Staphylococcaceae (P=0.043), Planococcaceae biome of slaughter-age pigs and to evaluate (P=0.025) and Enterococcaceae (P=0.038). the influence of the microbiome on MRSA While there was limited influence of microbial colonization. Nasal swabs were collected from population on MRSA colonization status, this 16 age-matched pigs (8 MRSA positive, 8 study indicates that management factors can negative) approaching slaughter age on one influence the nasal microbiome.

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 41 Poster Abstracts n 6A medius group, and S. hyicus. Strain types were METHICILLIN-RESISTANT determined by sequence analysis of the X re- STAPHYLOCOCCUS AUREUS ISOLATED gion of protein A (spa typing). Culture results FROM RETAIL MEAT IN THE CANADIAN from 195 samples (56 of each chicken, beef MARITIME PROVINCES. and pork, and 27 turkey) have been completed to date. The overall prevalence of MRSA M. Saab1, J. Eisnor1, B. Avery2, J. T. McClure1; in raw retail meat was 2.1% (4/195), while 1Department of Health Management, Atlantic the prevalence in chicken was 5.4% (3/56), Veterinary College, University of Prince in beef 1.8% (1/56) and in pork and turkey Edward Island, Charlottetown, PE, CANADA, 0%. All isolates displayed typical phenotypic 2Laboratory for Foodborne Zoonoses, Public characteristics of MRSA, and were PBP2’ Health Agency of Canada, Guelph, ON, positive. Molecular typing is being completed CANADA. and will be presented. These results are in contrast with a Canadian study where MRSA Food animals and their products have been was primarily isolated from pork, with samples suggested to be an important source of of chicken being the least likely to be MRSA antimicrobial resistant organisms, including positive. Determination of the strain type will methicillin-resistant Staphylococcus aureus provide insight to whether the MRSA isolated (MRSA). While MRSA has been detected in is human or livestock associated, and will lend livestock and meat products in various regions evidence to the source of the contamination. of Canada and the world, there are no esti- Factors associated with MRSA contamination mates of MRSA in livestock or retail meat in of retail meat will be explored. Atlantic Canada. Overall, this region has lower estimates of methicillin-resistant staphylococci in animals. The objective of this study was to n 7A determine the prevalence and molecular types SLAUGHTERHOUSE PERSISTENCE OF of MRSA in beef, pork and poultry in raw LIVESTOCK-ASSOCIATED METHICILLIN- retail samples collected in New Brunswick, RESISTANT STAPHYLOCOCCUS AUREUS Nova Scotia and Prince Edward Island. It was (MRSA CC398) hypothesized that the prevalence of MRSA in M. Sunde, B. Lium, J. S. Slettemeås, M. Nor- retail meats in the Canadian Maritimes would ström, A. Urdahl; be lower than reported in other Canadian Norwegian Veterinary Institute, Oslo, NOR- regions. Samples and epidemiologic data were WAY. collected as part of the Canadian Integrated Program for Antimicrobial Resistance Sur- During the last decade livestock-associated veillance, which focusses on antimicrobial methicillin-resistant Staphylococcus aureus resistance among enteric bacteria. Isolation (MRSA) clonal complex (CC) 398 has become was completed by enrichment in broth contain- pandemic. Pigs are usually asymptomatic ing mannitol and sodium chloride, followed carriers, but MRSA CC398 can spread from by culturing to chromogenic agar. Isolates animals to humans and therefore has a zoo- were tested for coagulase production, man- notic potential. Three studies on MRSA in nitol fermentation and resistance to oxacillin swine in 2008, 2008 and 2012, documented and cefoxitin. Detection of penicillin-binding a very low prevalence of MRSA CC398 in protein 2a (PBP2’) by latex agglutination Norwegian swine holdings with 0, 0 and was used to confirm methicillin-resistance. A 0.6% positive herds, respectively. However, multiplex PCR assay was used to differentiate in 2011, 1033 pigs from 207 different farms between coagulase-positive staphylococci of were sampled at ten different slaughterhouses. veterinary significance: S. aureus, the S. inter- From one slaughterhouse, MRSA CC398 was

42 ASM Conferences Poster Abstracts detected in six pooled samples (3%). An at- of Methicillin-resistant Staphylococcus aureus tempt to identify positive swine holdings was (MRSA) for patients and personnel at equine performed unsuccessfully. Follow-up sampling veterinary hospitals remains undefined, as of the environment in the slaughterhouse barn the environment is only monitored during showed that MRSA CC398 was present in the outbreak situations or for short periods of environment, and thereby may have contami- time. Therefore, the objectives of this study nated the pigs during housing at the slaugh- were to determine the monthly presence and terhouse giving an overestimation of herd distribution of MRSA in the environment of an prevalence. The objective of the present study equine hospital during one year, to characterize was to perform a follow-up sampling at the MRSA strains circulating in this setting, and to slaughterhouse now two years after to investi- establish patterns of contamination over time gate whether the slaughterhouse environment using molecular epidemiological analyses. still is contaminated with MRSA CC398. In We hypothesized that if MRSA is present in addition, the other ten largest slaughterhouses both humans working at an equine hospital, in Norway (handling more than 95% of all pigs as well as in horses admitted to such practice, slaughtered in Norway) were to be screened then this bacterium will be found frequently for MRSA the same way. MRSA was detected contaminating different contact surfaces across in one slaughterhouse, the same that was posi- the hospital throughout the year. For this tive in 2011. Samples from the remaining ten purpose, a yearlong active MRSA surveil- slaughterhouses were negative. The positive lance was performed, including a molecular slaughterhouse yielded nine positive samples epidemiological analysis of environmental and out of a total of ten samples. Genotyping iden- equine-origin MRSA isolates. Antimicrobial tified MRSA CC398spa type t034. Norway susceptibility testing, SCCmec typing, PFGE probably still has a low MRSA prevalence typing, and dendrographic analysis were used in pigs as ten of the 11 slaughterhouses were to characterize and analyze these isolates. negative for MRSA, and also based on the Overall, 8.6% of the surfaces were contami- previous performed surveys. Slaughterhouse nated, and 5.8% of the horses sampled were sampling could be a convenient and less positive for MRSA. The most common con- expensive way for early detection of MRSA taminated surfaces were: computer (16.7%), CC398 status in a country or new area, but the feed and water buckets (16.7%), and surgery results cannot be used to give estimations on tables and mats (15.6%). Characterization of herd prevalence. all MRSA isolates showed that 90.1% of the isolates were carrying SCCmec type IV, and n 8A 47.9% were classified as USA500, reflecting a low diversity among the strains circulating INTRODUCTION, CIRCULATION AND the hospital. Molecular analysis showed that MAINTENANCE OF METHICILLIN-RESISTANT new pulsotypes were constantly introduced STAPHYLOCOCCUS AUREUS IN CONTACT into the hospital throughout the year. However, SURFACES AT A LARGE EQUINE HOSPITAL: maintenance of MRSA in the environment YEARLONG MOLECULAR EPIDEMIOLOGY was also observed when unique clones were OF ENVIRONMENTAL CONTAMINATION detected for 2 consecutive months in the same J. Van Balen, J. Braman, M. Piraino, R. Nava- surfaces. It was also detected that MRSA Hoet, C. Kohn, A. E. Hoet; pulsotypes were circulating throughout several The Ohio State University, Columbus, OH. areas and different contact surfaces of the hospital. Based on these results, it is evident that The role that environmental contamination MRSA is constantly introduced and frequently might play as a reservoir and a possible source found in the equine hospital environment. In

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 43 Poster Abstracts addition, there were contact surfaces that could PCR and a CC398-specific PCR. The isolates act as “hot spots”, where either MRSA was (n=147) were further characterized using SC- capable of surviving over time or continuously Cmec typing, multiple-locus variable-number reintroduced throughout the year. These con- tandem-repeat analysis (MLVA) and antimi- taminated surfaces should be actively targeted crobial susceptibility testing. On a selection for strict cleaning and disinfection as well of isolates, Pulsed Field Gel Electrophoresis as regular monitoring. Key Words: MRSA, (PFGE) and spa typing was performed. After Surveillance, Environment, Equine, Molecular direct plating of the dilution series, a gen- Epidemiology, Veterinary Hospital eral MRSA prevalence of 8% on pork was observed. The cfu/g or cfu/cm² ranged from n 10A 6.100 to 8.104. After enrichment, MRSA was isolated from 98 out of 137 samples (72%), PRESENCE OF LIVESTOCK-ASSOCIATED mainly originating from rib, ear and forelimb. MRSA ON BELGIAN PORK There was a large genetic diversity amongst M. L. Verhegghe1, F. Crombé2, K. Luyckx3, the isolates within and between butcheries, F. Haesebrouck4, P. Butaye2, L. Herman1, M. which indicates that the obtained isolates were Heyndrickx3, G. Rasschaert1; of various origins. Twenty CC398 isolates did 1Institute for Agricultural and Fisheries Re- not show acquired antimicrobial resistance, ex- search (ILVO), Melle, BELGIUM, 2Veterinary cept to β-lactam antibiotics, which is unusual and Agrochemical Research Centre (VAR) for LA-MRSA isolates. In conclusion, MRSA - Ghent University, Faculty of Veterinary was found in a lower percentage of the pork Medicine, Brussels - Ghent, BELGIUM, 3Insti- samples after direct plating in comparison to tute for Agricultural and Fisheries Research the high percentage of positive samples after (ILVO) - Ghent University, Faculty of Veteri- enrichment, which indicates that enrichment nary Medicine, Melle - Ghent, BELGIUM, is recommended for examination of MRSA on 4Ghent University, Faculty of Veterinary pork. The genetic diversity of the isolates in- Medicine, Ghent, BELGIUM. dicated that a butchery can be considered as a reservoir that acts as a potential contamination Since the first description of livestock-asso- source for the general human population. ciated MRSA (LA-MRSA), high prevalence rates were observed in pigs. However, the importance of this reservoir for public health n 11A remains obscure. To investigate one possible EMERGENCE AND EPIDEMIOLOGY OF route of transmission, the prevalence of LA- LIVESTOCK-ASSOCIATED METHICILLIN- MRSA on Belgian pork was determined. Meat RESISTANT STAPHYLOCOCCUS AUREUS samples (chops, bacon, minced pork, ribs, CC398 IN HUMANS, DENMARK, 2004-2011 forelimbs and ears; n=137) originating from J. Larsen1, A. Petersen1, M. Sørum1, L. van four butcheries (A to D) were collected weekly Alphen1, P. Valentiner-Branth1, L. K. Knudsen1, for six weeks. Twenty-five grams of chops, L. S. Larsen2, A. R. Larsen1, R. L. Skov1; bacon and minced meat were 10 times diluted 1Statens Serum Institut, Copenhagen S, DEN- with salt-enriched broth. From the ribs, fore- MARK, 2Technical University of Denmark, limbs and ears the cm²/sample was determined Søborg, DENMARK. and a 1/1 dilution was performed. After ho- mogenization, the dilutions were spread plated Over the past decade, a livestock-associated on ChromIDTM MRSA plates both before and methicillin-resistant Staphylococcus au- after overnight enrichment. Suspect colonies reus strain belonging to clonal complex 398 were confirmed using a MRSA-specific triplex (LA-MRSA CC398) has been increasingly

44 ASM Conferences Poster Abstracts identified in food-producing animals, retail in poultry, there is paucity of information on meat, and humans. In particular, LA-MRSA the occurrence of MRSA in both live poultry CC398 has been identified as the predominant and poultry products especially in sub-Saharan strain in pigs and also as an important human Africa. The aim of this study was to determine pathogen in Europe. The main objective of the prevalence of MRSA in both live chicken this study was to investigate the epidemiology and poultry products from five poultry farms in of LA-MRSA CC398 in humans, Denmark. the Buea District in Cameroon. Materials and During 2004-2011, we noted a significant methods: A total of 100 live chickens were increase in the incidence of LA-MRSA CC398 sampled; including 50 broilers, 25 layers and carriers, including patients with LA-MRSA 25 free-ranged birds. Samples included cloacal CC398 infections, as well as in the percent- and nasal swabs from live chicken, meat from age of LA-MRSA CC398 carriers/patients slaughtered chicken and swabs from eggs. among all MRSA carriers/patients. In addition, Samples were inoculated unto mannitol salt our findings strongly suggested that spread agar (MSA) and isolates were identified using of LA-MRSA CC398 into the community biochemical tests. Antimicrobial susceptibil- is increasing due to repeated spillover from ity was tested by disk diffusion. Results: an expanding livestock reservoir, rather than MRSA was detected in chicken from all farms to emergence and geographic expansion including the free-ranged birds; however, the of distinct community-adapted LA-MRSA prevalence was higher in broilers (24/50, 48%) CC398 isolates. Consistent with this hypoth- than in free-ranged birds (5/25, 20%). MRSA esis, geographic information system-based was not detected in any of the layers’ samples. analysis showed that LA-MRSA CC398 Out of the 100 cloacal and nasal samples, carriers/patients with no livestock exposure MRSA was detected in 51 nasal swabs and 47 predominantly lived in close proximity to LA- cloacal samples. The isolation rates of MRSA MRSA CC398 carriers/patients with livestock from chicken meat and egg swabs were 12/25, exposure in rural areas where pig densities 48% and 8/25, 32%; respectively. Results of were high. These findings underscore the need susceptibility test revealed that all isolates for new strategies to control the spread of showed multiple resistance against antibiotics LA-MRSA CC398 in the livestock setting and such as penicillin, erythromycin, oxacillin, from there to the community. amoxicillin, trimethoprim-sulfamethoxazole, clindamycin and tetracycline. On the contrary, n 12A most isolates were susceptible to chloramphenicol, ciprofloxacin and rifampin.Discussion: DETECTION OF METHICILLIN-RESISTANT Results from this study confirms the occur- STAPHYLOCOCCUS AUREUS IN LIVE rence of MRSA in live chicken and other poul- CHICKEN AND POULTRY PRODUCTS IN THE try products as reported by previous studies. BUEA DISTRICT, CAMEROON. Thus, live chicken, eggs, poultry meat as well M. E. Bissong; as other poultry products might be a potential University of Buea, Buea, CAMEROON. source of infection in humans. The highest isolation rate of MRSA was observed in broil- Introduction: Methicillin-resistant Staphylo- ers while MRSA was not found in layers. In coccus aureus (MRSA) is an important patho- the present study, free-ranged birds had lower gen in the Public Health sector as diseases prevalence of MRSA than battery-caged birds. caused by this agent are more difficult to treat. This difference might be due low exposure of There is increasing evidence on the occurrence free-ranged birds to antibiotic-containing feed. of MRSA in several livestock animals and Further research is required on a larger flock the possible transmission of MRSA strains to to investigate the colonization of free-ranged humans. Although MRSA has been detected and battery-caged poultry and their role in the

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 45 Poster Abstracts transmission of multi-resistant pathogens to S. aureus were defined as any isolate demon- humans. strating ≥1 of the following: absence of the scn gene, tetracycline resistance, or CC398. n 13A Results. 327 nasal swabs from 22 workers were analyzed. During the 14-day period, PERSISTENCE OF LIVESTOCK-ASSOCIATED 10/22 participants were persistently carrying METHICILLIN AND MULTIDRUG-RESISTANT LA S. aureus, among which one persistently STAPHYLOCOCCUS AUREUS AMONG carried LA MRSA and six LA MDRSA. An INDUSTRIAL HOG OPERATION WORKERS IN additional six workers carried LA S. aureus in- NORTH CAROLINA termittently, 5/6 of whom carried LA MDRSA. M. Nadimpalli1, J. Rinsky1, D. Hall2, E. No worker carried LA MRSA intermittently. Pierce1, N. Pisanic3, J. Larsen4, K. Nachman3, CC398 was detected among eight workers, of D. Love3, S. Wing1, J. Stewart1, C. Heaney3; whom seven were persistently carrying. LA 1University of North Carolina at Chapel Hill CC9 was detected among nine workers, of Gillings School of Global Public Health, Cha- whom two were persistent carriers. Non-LA pel Hill, NC, 2Rural Empowerment Association CC30, CC8, CC15, CC20, and CC779 were for Community Help, Warsaw, NC, 3Johns also observed among participants. Despite all Hopkins Bloomberg School of Public Health, but two workers experiencing ≥24 hours away Baltimore, MD, 4Microbiology and Infection from work, we observed null associations Control, Statens Serum Institut, Copenhagen, between work-related and non-work-related DENMARK. exposures and carriage of LA S. aureus and LA MDRSA. No symptoms indicative of infection Background. Whether livestock-associated were reported during the study period. Con- (LA) Staphylococcus aureus is a persistent clusion. Although limited by a small sample nasal colonizer of individuals frequently ex- size, we observed evidence of persistent nasal posed to livestock remains unclear. Persistence carriage of LA S. aureus, LA MRSA, and LA studies have focused mainly on clonal complex MDRSA among workers at IHOs in the United (CC) 398 and methicillin-resistant S. aureus States even after ≥24 hours away from work. (MRSA) - not methicillin-susceptible or Future studies should examine dynamics of LA multidrug-resistant (MDRSA) S. aureus - and S. aureus persistence in IHO workers’ noses limited LA S. aureus persistence information is over longer periods of time with greater time available from the United States. We con- away from work. ducted a 14-day study of nasal carriage of LA S. aureus, LA MRSA, and LA MDRSA among industrial hog operation (IHO) workers in n 14A North Carolina. Methods. Healthy volunteer THE HUMAN AND COMPANION ANIMAL IHO workers anticipating ≥24 hours away MICROBIOME OF HOUSEHOLDS from work self-collected nasal swabs and com- WITH METHICILLIN-RESISTANT pleted an exposure and work activity journal STAPHYLOCOCCAL INFECTIONS in the evening on day 1, and in the morning A. Misic1, M. Davis2, A. Tyldsley1, B. Hodkin- and evening on days 2-7 and 14. Nasal swabs son1, J. Bugayev1, I. Nachamkin1, E. Lauten- were incubated overnight in enrichment broth bach1, D. Morris1, E. Grice1; at 37°C, then plated on CHROMagar™ Staph 1University of Pennsylvania, Philadelphia, PA, aureus and incubated for 24 hours at 37°C. 2Johns Hopkins University, Baltimore, MD. Resulting S. aureus isolates were confirmed by biochemical testing and assessed for presence Methicillin-resistant Staphylococcus aureus of the 16S, nuc, mecA, and scn genes, suscepti- (MRSA), a skin pathogen in pets and people, bility to 12 antibiotic classes, and spa-type. LA produces high rates of re-infection even after

46 ASM Conferences Poster Abstracts antibiotic treatment. In companion animals, 20% in the human lesion samples), indicating species such as S. aureus, S. pseudintermedius, that the total microbial community may play a and S. schleferi, are major pathogens that key role in Staphylococcal disease modulation. produce a wide array virulence factors and Conclusion: The results of this study describe result in many disease syndromes. Humans the human and animal microbiomes longitu- and pets live in the same environments, share dinally and indicate that there is transmission skin-to-fur contact, and share microbiota; both of microbes and methicillin-resistance genes pathogens and commensals. We hypothesize between family members and their pets. that companion animals share microbiota with humans, and that the microbiome has a role in n 15A modulating MRSA colonization and infection THE EVALUATION OF MOLECULAR in households with and without pets. Methods: EPIDEMIOLOGICAL RELATEDNESS WITHIN Under approved IRB and IACUC protocols, ST5 STAPHYLOCOCCUS AUREUS ISOLATED we enrolled 25 households where there was FROM SWINE VETERINARIANS IN THE USA a primary human MRSA infection for a total of 63 pets (cats, dogs, etc) and 30 humans. J. Sun; We collected animal swab samples (mouth, University of Minnesota, Saint Paul, MN. nose, and lesion if applicable) for microbiome The livestock associated Staphylococcus analysis while the humans self-sampled (a aureus (LA-MRSA) ST 398 has been isolated combined inguinal crease/axilla swab, a nasal frequently from livestock, especially pigs, swab, and a lesion swab for the index patient). worldwide since first detected from pigs We obtained samples at two time-points: and pig farmers in the Netherland in 20051. enrollment and 3 months later, for a total of Subsequent studies are revealed greater ge- 329 samples. To investigate the microbiota netic diversity in LA-MRSA using molecular present, we used next-generation sequenc- epidemiologic characteristics. Several studies ing technologies (Illumina MiSeq) to amplify reported that ST9 isolates are predominate the V4 region of the 16S rRNA gene from among MRSA isolates from pigs in Asian the extracted genomic DNA. The resulting countries, while both ST398 and ST5 lineages reads were quality filtered, binned, assigned have been found in pigs and livestock workers taxonomy, and analyzed with bioinformatics in North America3,4. Unlike ST398, which is software (QIIME and mothur). We identified rarely involved in significant human infections, bacterial taxa present and analyzed them with the occurrence of ST5 sequence type in pig alpha and beta-diversity metrics with respect industry is of some public health concern as to time, species, sample location, household, this ST5 lineage has long been associated with carriage of coagulase-positive Staphylococ- human MRSA infections related the USA100 cus (CPS), and the presence of the mecA gene group (defined by sma1 PFGE)2,5. In a pilot as determined by PCR. Results: The six most study of pig farms in Minnesota, the predomi- abundant bacterial taxa of the total oral and nant spa types found were t034 (ST398), and nasal companion animal microbiomes are: t002 (ST5), comprising 37% and 29% of iso- Streptococcus, Pasteurellaceae, Moraxellaceae, lates respectively (all MSSA). The aim of this Clostridiales, Fusobacterium, and Staphylo- study was to investigate the prevalence of ST5 coccus. The greatest determinant of microbial S. aureus nasal swabs in US swine veterinar- composition is the species and location of ians, and to evaluate the diversity of spa type sampling (Weighted Unifrac metric, Anosim t002 isolates using PFGE with SmaI. Similar test, R=0.58, p=0.001). Intriguingly, the hu- to our findings in pigs, the most prevalent spa man MRSA lesion samples had a high level of types found in US swine veterinarians were diversity and staphylococcal species were pres- t034-ST398 and t002-ST5. ST9 spa types ent at a low level (an average composition of

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 47 Poster Abstracts

(t337, t3446, t2498) were also common. Sixty total, 192 isolates were identified as S. aureus, (19%) of 308 MSSA isolates and 6 (19%) including 128 from pigs (58 MRSA and 70 of 32 MRSA isolates detected were spa type MSSA) and 64 from humans (total carriage t002 (ST5). Other ST5 MSSA spa types found 19%; 14 MRSA and 50 MSSA). All character- included t045 (14), t062 (6), t570 (3) and ized isolates belonged to 10 genetic lineages: t2049 (1). Sma1 PFGE analysis of 23 isolates CC398 (n=140), CC9 (n=26); CC30 (n=14, indicated that t002 isolates from the swine vet- CC22 (n=3), and CC1, CC8, CC15 (n=2 each), erinarians were not clonal but heterogeneous and CC12, CC5, ST182 (n=1 each). Among between isolates. Fifteen distinct pulsotypes identified genetic lineages, five (CC398, CC9, were seen, and eight isolates were classified as CC30, CC22 and CC1) were represented by USA100. Given previous reports of t002-ST5 isolates originated both from pigs and humans, MRSA in North American pigs, our findings four (CC5, CC8, CC15, and ST182) by isolates of diverse ST5 spa types as well as genetic di- from humans, while CC12 by one isolate versity within spa type t002, provide improved from pig. lukS/lukF-PV genes were not de- understanding of ST5 lineage, which may have tected. MRSA isolates belonged to two clonal long association with pigs overtime after intro- complexes only (CC398 and CC30), with duction from human. Further investigation of CC30-MRSA isolates observed exclusively in ST5 lineage is warranted to better understand pigs. CC398, the most represented CC com- their potential implications for human health. prised 94 isolates from pigs (50 MRSA and 44 MSSA) and 46 from humans (14 MRSA and n 16A 32 MSSA). Among CC398-MRSA, six spa types were assigned: t011 (n=31), t034 (n=25), MOLECULAR CHARACTERIZATION OF t108 (n=4), t4389 (n=2), and t2582, t5452 S. AUREUS ASSOCIATED WITH PIG (n=1 each); and three SCCmec types were ENVIRONMENT IN POLAND, 2010-2013 determined: Vc[zinc] (n=55), V (n=8) and IVa A. Mroczkowska1, M. Orczykowska-Kotyna1, (n=1). The most prevalent dru type was dt11a N. Marszałek1, I. Komorowska1, A. Grzesiak2, (n=26 out of 30), followed by dt11ap, dt9s, A. Nowak2, J. Żmudzki2, J. Empel1; dt6j and dt5e represented by single isolates. 1National Medicines Institute, Warsaw, PO- Among CC398-MSSA, 13 spa types were LAND, 2National Veterinary Research Institute, assigned, with the most prevalent t034 (n=36), Puławy, POLAND. followed by t4389 (n=13) and t1928 (n=8). All CC398 isolates represented sequence type Objectives: The objective of the study was ST398. The second numerous CC was CC9, to investigate the nasal carriage of S. aureus gathering only MSSA isolates, both of pig among Polish pig farmers, veterinarians (n=21) and human (n=5) origin. Among this working on selected farms and at the same CC 8 spa types were assigned, with the most time/place among pigs, and to compare on common t1430 (n=9), t337 (n=7) and t1670 molecular level recovered S. aureus. Mate- (n=4). The third CC, in terms of the number rial and methods: The study was performed of isolates, was CC30, that encompassed 10 on 339 and 2070 nasal swabs from humans isolates from pigs (8 MRSA and 2 MSSA) and pigs, respectively, taken in Polish 125 and 4 MSSA from humans. MRSA isolates farms, between 2010 and 2013. For all isolates were characterized by spa type t318, SCCmec re-identification by standard microbiological IVa and dru type dt10a. CC30-MSSA were methods, the detection of mecA and lukS/lukF- assigned to five spa types: t318 (n=2) and t018, PV genes was performed. They were char- t021, t1333, t9034 (n=1 each). Conclusion: It acterized by spa-typing, RM test typing, and seems that isolates of CC398 lineage are most SCCmec typing. MLST and dru-typing were prevalent and well settled in pig farm environ- performed for selected isolates. Results: In

48 ASM Conferences Poster Abstracts ment in Poland. Polish S. aureus belonging to to be positive for types I, II and IV. MLVA CC398 are genotypically similar to the strains analysis produced limited results and most observed worldwide. isolates gave patterns with 5 bands but could not differentiate the strains as most appeared n 17A to have identical patterns. Best results were observed using novel restriction enzymes and GENOTYPE AND PHENOTYPE PFGE. Restriction patterns for isolates exam- ANALYSIS OF METHICILLIN RESISTANT ined using EagI had typically 12 bands ranging STAPHYLOCOCCUS AUREUS ST 398 FROM in size from 30 kb to < 300 kb; Cfr9I resulted HUMANS, ANIMALS AND RETAIL MEAT. in patterns with 16-20 fragments ranging in C. M. Logue1, C. Thompson1, S. E. Lord1, V. size from 20 kb to 1100 kb, and BstZ1 analysis Velasco2, J. S. Sherwood3, T. Meng4, I. Rohl- yielded approximately 20 fragments of sizes wing1; from 20 kb to 700 kb. Virulence gene analysis 1Iowa State University, Ames, IA, 2University produced limited patterns but the presence of of Concepcion, Concepcion, CHILE, 3North adhesion genes appeared to correlate well with Dakota State University, Fargo, ND, 4Jiangsu biofilm assays.Conclusion: Multiple molecu- Agri-animal Husbandry Vocational College, lar tools provide value in assessing host source Taizhou, CHINA. of MRSA and offer alternative strategies for strain differentiation. Ongoing work is validat- Background: Methicillin resistant Staphy- ing the use of these tools for blinded isolates lococcus aureus is a significant pathogen of to determine traits for rapidly distinguishing healthcare, community and livestock associ- sources of MRSA. ated infections. Among strains of interest are those identified as multi-locus sequence type 398 because of their emergence in swine and n 18A human disease. MRSA ST 398 is unique in the RELATIONSHIP OF MRSA IN VARIOUS inability to sub-type these strains by pulse field SAMPLES FROM INSIDE AND OUTSIDE OF gel electrophoresis (PFGE) using SmaI, due to DIFFERENT POULTRY FARMS a methylase which modifies the clevage site of K. Krüger1, U. Roesler1, J. Schulz2, J. Har- the enzyme rendering the strain indigestible. tung2, A. Friese1; Methods: A collection of MRSA ST398 strains 1Freie Universität Berlin, Institut for Animal (n = 68) from humans, swine, sheep, and retail Hygiene and Environmental Health, Berlin, meats were assessed using molecular tools GERMANY, 2Institute for Animal Hygiene, to subtype them. All strains examined were Animal Welfare and Farm Animal Behaviour, identified as MLST ST398, and failed to re- University of Veterinary Medicine Hannover, strict when SmaI was used for PFGE Analysis. Foundation, Hannover, GERMANY. Molecular profiling was carried out usingspa typing, SCCmec typing, multi locus vari- Objective: The occurrence of methicillin ance analysis (MLVA), virulence genotyping, resistant Staphylococcus aureus in different biofilm assay and PFGE using EagI, Cfr9I and animal species has been investigated during BstZI. Results: Variable results were observed the last years. MRSA has a mobile genetic ele- but a combination of tools appeared to allow ment called staphylococcal cassette chromo- better differentiation of the strains examined. some mec which carries the mecA gen being spa types identified included types t011, t034, responsible for the resistance against ß-lactam t693, t1430 and t4389, some of which were antibiotics. In this study different samples found in both human and animal host strains, from animals, the barns´ interior and from the and raw meat. SCCmec typing for strains vicinity outside were taken. The aim of the identified as possessingmecA , were found presented study is the detection of epide-

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 49 Poster Abstracts miological relations between the strains of n 19A different origin. Material and Methods: Five LONGITUDINAL QUANTITATIVE STUDY OF turkey and two broiler farms were analyzed NASAL MRSA CARRIAGE IN PIGS three and four times, respectively, within one fattening period. Samples were collected from C. Espinosa-Gongora1, A. Elvstrøm2, J. Dahl3, individual animals (swabs of choana and skin), L. Guardabassi1; their direct surroundings (boot swabs, feces, 1Faculty of Health and Medical Sciences - dust, feed, air) as well as from the farms´ en- University of Copenhagen, Frederiksberg vironment by taking air samples and samples C, DENMARK, 2Odder vet. clinic, Odder, of the ground surfaces around the barns up DENMARK, 3Danish Agriculture and Food to distances of 500m. Selected isolates from Council, Copenhagen, DENMARK. in- and outside were spa typed and grouped as Introduction: The current knowledge of nasal Livestock associated (LA)-MRSA according methicillin-resistant S. aureus (MRSA) car- to their association to the clonal complex (CC) riage in pigs is based on the use of enrichment 398. Additionally, the isolates were compared methods that do not provide any quantita- by using pulsed field gel electrophoresis tive information about MRSA distribution (PFGE). Results: The investigated isolates within individual pigs and herds. Objective: predominantly belonged to the clonal complex To determine in-herd prevalence and nasal 398 and are therefore assigned to livestock load of and MRSA in adult pigs. Methods: A associated (LA) MRSA. We detected up to five longitudinal quantitative study was performed different spa types occurring in and around in 16 randomly selected Danish production one investigated barn, however, the spa types farms. Within each farm, 24 individual pigs of isolates from inside were the same as these in the finishing sections were ear tagged and from outside. This is underlined by the prelimi- sampled three times at weekly frequency nary results of the PFGE where we could find a using cotton swabs. Swabs were suspended close relationship between samples taken from in 1ml saline and serial dilutions (10-1 to animals, the barn interior and the vicinity of 10-4) were enriched in Mueller-Hinton broth one barn. However, until now this relationship 6.5% NaCl. Following incubation, aliquots was not found when comparing the isolates of the enrichment were plated onto MRSA2 from different barns with each other although Brilliance agar (Oxoid, UK) and MRSA load they belonged to the same company. Conclu- (CFU) in swabs was estimated by the most sion: Since we found the same PFGE patterns probable number (MPN) method. Data were in isolates from in- and outside of one farm the analysed statistically using a logistic regres- results show that an emission of MRSA from sion model for either multinomial or binary broiler farms definitively occurs. A preliminary data (proc glimmix, SAS Inst.), with sampling comparison of the results between spa typing round, swab cleanliness and MRSA-positivity and PFGE show a high relationship. However, for herd as fixed effects when appropriate, the PFGE method is necessary for deeper and farm, room and pig as random effects, epidemiological analysis. Until now it seems when appropriate. Results: Eleven of the 16 that the relationship of the isolates originating farms were MRSA-positive. Among positive from different sample matrices occurs within farms, 58 pigs (22%) were permanent MRSA one barn and is different between different carriers, 139 (54%) were transient carriers and farms even when they were located in the same 62 (24%) were non-carriers. MRSA load in region and belonged to the same company as positive nasal swabs was on average 2.53 log it was the case in both broiler fattening farms. 10 cfu per swab (s.d. 0.45) in permanent carriers Maybe one MRSA clone is selected and then which was significantly higher than 2.36 log distributed within the barn. More results will 10 cfu per swab (s.d. 0.52) observed in intermit- be presented on the conference.

50 ASM Conferences Poster Abstracts tent carriers (OR 1.5, p=0.005). Conclusions: (n=7) were also sampled, respectively in 12 and This study provides the first data on MRSA 7 farms. Swabs and milk were inoculated into a load in the nose of pigs, which are comparable pre-enrichment medium containing Brain-Heart to previously observed MRSA load in humans. broth with 5% NaCl broth. After overnight Prevalence of positive farms was markedly incubation at 37°C, hundred microliters were higher compared to previous data based on streaked on SAID agar (bioMérieux) and screening of slaughter pigs in Denmark. Our BrillianceTM MRSA agar (Oxoid). Suspected data showed a significant association between colonies were subcultured on blood agar and MRSA load and stability of carriage where identification was confirmed by MALDI-TOF permanent carriers harbour significant higher (Vitek MS, bioMérieux). All S. aureus isolates amounts of MRSA. were tested by PCR for the presence of nuc (a species-specific marker), mecA and mecC n 20A genes. MecC positive strains were further characterized using DNA microarray StaphyType PERSISTENCE AND DIFFUSION OF Kit (Alere Technologies). Results: Whereas MECC-POSITIVE CC130 MRSA ISOLATES S. aureus were isolated in 10 out of 17 farms, IN CATTLES IN MEURTHE-ET-MOSELLE including 39 isolates (NS (n=9), RS (n=4), M DEPARTMENT (FRANCE) (n=18), co-colonisation (NS + M; n=4)) from J. Tasse1, B. Jacques1, M. Haenni2, A. Sapin1, 35 cows, only seven isolates were MRSA. All L. Saffroy3, B. Coppe4, F. Pirart4, M. Bes1, of them harboured mecC gene. These mecC- A. Tristan1, F. Vandenesch1, J. Madec2, F. positive MRSA isolates were detected only in Laurent1; two specific farms and included respectively 1French National Reference Centre for Staphy- two out of the 10 tested cows (NS (n= 2), M lococci, International Centre for Infectiology (n=1)) and four out of the 10 tested cows (M Research - Inserm U1111, Hospices Civils (n=3), RS (n=1)). The two farms corresponded de Lyon, Lyon, FRANCE, 2Anses Lyon, Lyon, to those in which mecC-positive isolates had FRANCE, 3Clinique veterinaire du Gremillon, been detected in 2008 and 2011. All MRSA Essey lès Nancy, FRANCE, 4Clinique veteri- strains belonged to the clonal complex CC130. naire, Vézelise, FRANCE. None of the farmers and domestic animals (including those from farms with mecC-positive Background: Recently, mecC gene has been isolates in animals) were positive for MRSA. described in methicillin-resistant Staphylo- Conclusion: Our data suggest that mecC- coccus aureus (MRSA) isolated from cattle. positive strains are able to persist in farms for This mecA variant has been mainly found in a long period (> 5 years), but also to colonize Northern Europe. In France, animal cases were noses, digestive tract and/or to be involved detected only in two cows in 2008 and 2011 in in sub-clinical mastitis in cows as well as to two farms distant by less than 15 kilometers in disseminate and to induce outbreak within the Meurthe-et-Moselle department, France. cattles. Diffusion from cattle to neighbouring In 2013, the two same dairy farms were cattle and from animals to farmers appears to screened again for mecC-positive MRSA as be limited and to be restricted to specific dairy well as farms in the surrounding area to study farms. A larger study in this geographical area epidemiological dynamics of these strains. with inclusion of a higher number of farms, Methods: Seventeen dairy farms were tested sampling of all cows in mecC-positive screened from January to March 2013. We collected farms and clinical human MRSA isolates are nasal swabs (NS), rectal swabs (RS) and milk underway to further explore the epidemiology (M) from ten randomly-choosen cows in each and dynamic of mecC-positive clones. farm. Farmers (n=21) and domestic animals

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 51 Poster Abstracts n 21A Oxacillin, imipenem, ampicillin, gentamicin, SUSCEPTIBILITY OF METHICILLIN penicillin, Erytromicine, and Methicillin. The RESISTANCE STAPHYLOCOCCUS AUREUS, anterior nares swabs were taken from 173 ON VOLCANOES NATIONAL PARK Volcanoes National Park personal protecting PERSONAL PROTECTING ENDANGERED endangered mountain gorilla 4/173 (2.3%) had MOUNTAIN GORILLA IN RWANDA - 2012 Staphylococcus aureus, ᵦ lactamase positive, antibiotic sensitivity reveals: Cefotaxime was j. KABEMBA LUKUSA; 75% sensitive, Oxacillin was 25% sensitive, Mountain Gorilla Veterinary Project, Musanze, Imipenem: was 75% intermediate and 25% , RWANDA. Ampicillin: was 25% sensitive, Gentamicin: was 100% sensitive, Penicillin: was 75% Humans are natural reservoir of Staphylo- sensitive , Erytromicine: was 75% sensitive coccus aureus germs 30 to 40% of healthy and Methicillin was 100% resistant. The strain adults are carriers. Staphylococcus aureus can of Staphylococcus aureus was isolated from be found temporarily or permanently in the (2.3%) Volcanoes National Park, personal skin and mucous membranes (nasal, throat, protecting endangered mountain gorillas, has perineum, groin, navel, and armpit). Following active resistance with ᵦ lactamase production, the excessive use of antibiotics and hygienic Methicillin resistant at 100%, this aspect calls measures insufficient, some percentage of us to deepen research in molecular biology, Staphylococcus aureus become resistant to polymerase chain reaction (PCR) for specific antibiotics, the objective of this study is to identification and epidemiological surveillance evaluate the susceptibility and prevalence of of this organism present in the mountain goril- antibiotics on Staphylococcus aureus isolated las and humans. from Volcanoes National Park, personal protecting endangered mountain gorilla. In mountain gorilla population, Staphylococ- n 22A cus aureus, can be identified as a secondary RELIABILITY OF METHODS FOR DETECTING infection during respiratory virus infection. METHICILLIN-RESISTANT STAPHYLOCOCCI 173 anterior nares samples were collected in (MRS) IN ANIMALS DURING ROUTINE microbiology laboratory with a sterile swab MICROBIOLOGICAL DIAGNOSTICS (BBL Culture swab, Becton Dickinson and D. Misic, K. Aksentijevic, N. Zdravkovic, J. Company, Sparks, Maryland, USA) was Asanin; inserted approximately 1 cm inside the nostrils Faculty of Veterinary Medicine, Dept.for of each employee before the process bacte- Microbiology, Belgrade, SERBIA. riological culture, the sample was enriched in 2 ml of sodium chloride (NaCl) for 24 to Objectives: Routine microbiological diagnos- 48 hours, then inoculated on fresh blood agar tic service assumes that results are available medium at 37 C for 24 hours in an aerobic within 48 to 72 h, significantly interfering with environment. The susceptibility of pathogenic detection of MRS strains, especially when bacteria was performed⁰ by the method of laboratory processes large number of samples. diffusion on the solid medium Muller Hilton, Broth microdilution recommended by EU- as proposed in the recommendations of the CAST and CLSI, as well as PCR, are time con- committee susceptibility of the French Society suming, expensive and technically demanding of Microbiology (CASFM) and interpreted procedures to be applied in routine diagnostic, by comparing the diameters obtained critical especially when most laboratories do not diameters proposed by CASFM.les antibiot- have access to PCR. In Serbia MRS strains ics (code, load the disc). For the susceptibility are usually reported only upon resistance of test the antibiotics discs use are: cefotaxime, staphylococci to cefoxitin detected with disc

52 ASM Conferences Poster Abstracts diffusion. The subject of this research was to confirm the results of this study. In addi- to test if animal MRS strains can be detected tion, E test was deemed applicable to routine with certainty during routine diagnostic work diagnostics, since there were no differences in by using disc diffusion cefoxitin susceptibil- susceptibility results from E test and disc diffu- ity, E test and Slidex MRSA Detection kit. sion. Slidex MRSA detection kit is proper for Materials and methods: MRS strains were PBP2a detection in CoPS, not for CoNS. isolated from ear, eyes, skin, and nose swabs originated from different animal species and n 23A identified with ID32 STAPH (bioMerieux). TRENDS IN ANTIMICROBIAL RESISTANCE Although amoxicillin with clavulanic acid (A/ IN CLINICAL STAPHYLOCOCCI ISOLATED CL) is not recommended due to unreliability FROM COMPANION ANIMALS OVER A of results, since 2012 our routine laboratory 8-YEAR PERIOD work does use A/CL discs besides cefoxitin (Becton Dickinson) for the purpose of better C. Monchique, N. Couto, A. Belas, L. T. Gama, interpretation of staphylococcal susceptibility C. Pomba; to β-lactams. MIC values were determined Faculdade de Medicina Veterinária, Universi- with E test (bioMerieux). Detection of PBP2a dade de Lisboa, Lisboa, PORTUGAL. was done with Slidex MRSA agglutination kit Staphylococci are a group of bacteria with (bioMerieux). Confirmation of presence of a clinical, agricultural, and economic importance mecA gene was done by PCR using MRSA because of their ability to become resistant to ATCC 43300 as control. Results: From 2007- antimicrobials. This alarming feature should 2013, 35 MRS strains were detected in 21 be considered and antimicrobial susceptibil- dogs, 8 cats, 5 pigs and one donkey sample ity testing (AST) is important to monitor the submitted to routine diagnostics. Until 2012, spread of resistant organisms/genes and the disc diffusion revealed 31 strains with cefoxi- rise of multidrug-resistant staphylococci. tin resistance, E test showed MIC cefoxitin The aim of this study was to investigate the values >4 µg/mL, 22 strains were positive for evolution of Staphylococcus spp. resistance to PBP2a in Slidex MRSA test, and 9 CoNS were antibiotics between 1999-2006. The 384 clini- false negative. Presence of mecA gene in all cal samples were received by the Laboratory strains was detected with PCR. However, in of Clinical Analysis of the FMV-UL and were 2012, the finding of 4 CoPS that were sensitive obtained from various animal species admitted to cefoxitin (>22 mm zones, EUCAST 2103) to several hospitals in the Lisbon’s district. and resistant to A/CL (zones <20 mm, CLSI AST was performed by disk diffusion using 26 2011), was unclear since this is impossible antibiotics (AB). Staphylococcal species were resistance phenotype. E test of these strains identified by PCR amplification of the respec- showed sensitivity to cefoxitin with MIC tive nuc gene and by 16S rRNA sequencing. values of <1.5 µg/mL, except for 1 strain of The mecA and mecC genes were screened MIC = 3µg/mL, and resistance to A/CL with by PCR. Statistical analyses were performed MIC values of >8/4 µg/mL. These strains were using SAS 9.3 and differences were considered clearly positive on Slidex MRSA detection relevant if P≤0.05. In total, 251 isolates were test and had mecA gene confirmed by PCR. resistant to at least 1 AB (65.36%), with higher Conclusions: Use of cefoxitin alone in disc frequencies of resistance to penicillin and diffusion can fail to detect MRS. Based on ampicillin (53.13%) followed by tetracycline results presented above, it is suggested that A/ (31.77%). Overall, 75 isolates (19.53%) were CL is used alongside cefoxitin, because strains multidrug resistant and only 34.64 % of the that show cefoxitin sensitivity, but also display isolates were susceptible to all the AB tested. A/CL resistance may be MRS. Further studies Resistance increased over time, with the using larger number of samples are warranted

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 53 Poster Abstracts highest level observed in 2006 (79.55%). The resistance among an Australia-wide collection highest resistance to 1 AB was found in S. of coagulase positive staphylococci causing aureus and S. pseudintermedius (nearly 73% clinical infections in animals. The survey will of the isolates) followed by coagulase-negative focus on coagulase-positive staphylococci staphylococci (CNS) and S. schleiferi (about (n=1500) identified as causes of infection by 49%). Eleven CNS were resistant to oxacilin, all veterinary diagnostic laboratories (n=22) but only 8 were mecA-positive. One S. aureus across Australia from January-September had the mecA gene and was cefoxitin-resistant. 2013. All isolates will be subjected to CLSI When compared to dogs, cats had lower odds disc diffusion susceptibility testing for 16-18 to have a resistant isolate (P=0.001, OR=0.307, antimicrobials of importance to veterinary and [0.152-0.619]) and a CNS resistant to at least public health. Here we report the antimicrobial 1 AB (P=0.004, OR=0.170, [0.051-0.560]). resistance profiles on the first 193 isolates S. pseudintermedius originating from otitis (S. pseudintermedius n=120 and S. aureus had higher odds of being resistant to at least n=73). S. pseudintermedius isolates were all 1 AB than those from pyoderma (P=0.013), obtained from dogs and cats whereas S. aureus OR=0.452, [0.241-0.845]). Our results con- isolates were from dogs (n=16) and cats (n=7), firmed that antimicrobial resistance is very horses (n=9) and bovine mastitis (n=41). frequent in staphylococci, and the presence The antimicrobial resistance profiling of S. of methicillin-resistant staphylococci in sick pseudintermedius revealed that 10% of the animals highlights the importance of hori- isolates were resistant to oxacillin. In addition, zontal transfer of different SCCmec elements resistance to penicillin (80%), tetracycline and might favor the transmission of resistance (20.8%), azithromycin (12.5%), gentamycin genes from staphylococci present in animals (11.6%), clindamycin (11.6%) trimethoprim- to those from humans. Changes in antibiotic sulfamethoxazole (9.2%) and ciprofloxacin resistance require continuous monitoring for (7.5%) was also observed. The majority of adjustment of antimicrobial strategy. the oxacillin-resistant isolates exhibited a multidrug-resistant phenotype (resistance to n 24A clindamycin, trimethoprim-sulfamethoxazole, azithromycin, gentamicin and ciprofloxacin). A EPIDEMIOLOGY OF METHICILLIN slightly higher proportion of S. aureus isolates RESISTANT COAGULASE POSITIVE from dogs, cats and horses were resistant to STAPHYLOCOCCUS IN AUSTRALIA oxacillin (12.5%). The S. aureus isolates were S. Abraham1, H. S. Wong1, A. Kidsley1, M. resistant to penicillin (78.1%), tetracycline Neale2, D. J. Trott1; (28.1%), gentamicin (9.4%), azithromycin 1University of Adelaide, Roseworthy, AUSTRA- (3.1%) and ciprofloxacin (3.1%). The S. aureus LIA, 2Zoetis Australia, Sydney, AUSTRALIA. from cases of bovine mastitis were sensitive to all antimicrobials tested with the exception Coagulase-positive staphylococci are recog- of penicillin (19.5%). This study demonstrates nised as major zoonotic pathogens causing that methicillin-resistant S. pseudintermedius serious infection in both humans and animals. isolates with a multidrug resistant pheno- Recently, there has been an increase in the type have emerged in Australia but currently prevalence of methicillin-resistant Staphy- represent a small proportion of total isolates. lococcus spp. in both humans and animals Whilst a higher proportion of S. aureus isolates around the globe. Australia currently has no showed methicillin resistance these isolates national network of surveillance for monitor- were sensitive to a greater number of antimi- ing antimicrobial resistance in animals. We re- crobial classes. Whilst the study is only in its cently commenced a study sponsored by Zoetis preliminary stages, it does provide the first to determine the prevalence of antimicrobial

54 ASM Conferences Poster Abstracts national data on antimicrobial resistance in co- total infections and 11% of MRS (64/580). agulase positive staphylococcus for evaluating Undifferentiated coagulase (+) infections the public health impact of veterinary use of comprised the rest; the relative proportion of antimicrobials in both livestock and compan- staph subtypes remained consistent over the ion animals in Australia. years. Year tested was associated with MRS (p<.0001); the prevalence of MRS increased n 25A from 6% (45/722) in 2002, 11% (98/857) in 2005, 19% (181/977) in 2008 and 32% STAPHYLOCOCCUS SPP. ANTIBIOTIC (256/794) in 2011. The prevalence of MDR RESISTANCE ON THE RISE; A SOUTH EAST Staph infections also increased linearly and USA PERSPECTIVE was associated with year (p<.0001). In 2002, M. L. Keeling, S. Sanchez; 4% (2/45) of all MRS were resistant to all University of Georgia, Athens, GA. three drug classes used to indicate MDR compared to 36% (91/256) in 2011. Con- The global rise of Staphylococcal infec- versely, in 2002, 36% (16/45) of all MRS tion (Staph) poses a veterinary challenge as cases were sensitive to the three drug classes; increases in Meticillin resistant staphylococ- this dropped to 19% (48/256) by 2011. Care cus (MRS), indicating general beta-lactam center was associated with MRS (p<.0001). resistance, often corresponds with resistance Primary care contributed 79% (2091/2639) of from other drug classes making treatment pro- the cases and had an overall MRS prevalence longed and/or difficult. We characterized the of 18% (367/2091) while tertiary care centers Staph infections submitted to the University of contributed 21% (548/2639) of cases but had a Georgia’s (UGA) Athens Diagnostic Labora- MRS prevalence of 31% (168/548); 767 cases tory (ADL) from clinically ill patients over did not specify location and were removed the years 02, 05, 08 and 11 from UGA’s small from this analysis. Companion animals (dog, and large animal hospitals and those submitted cat, horse, ferret, guinea pig, rabbit) com- from private clinics. We hypothesized that the prised 97% (3301/3406) of all cases which is prevalence of MRS increased annually as has concerning for practitioners in this popula- the prevalence of multi-drug resistance (MDR) tion. Treatment and prevention options must within them. We aimed to identify differences be carefully evaluated as resistance spreads. in drug resistance between primary and tertiary This is paramount within tertiary care settings care centers and hypothesized a higher preva- which hosts a larger prevalence of MRS and lence of MRS in tertiary care settings who often contain patients in close proximity to one often perform surgical interventions and take another for longer time periods. referrals from failed primary care treatment posing higher infection risks. Over the study, 3406 of the phenotypically identified Staph n 26A infections had antimicrobial sensitivity testing. DETECTION OF MRSA DURING THE Oxacillin was used as a proxy for Meticillin BACTERIOLOGICAL ROUTINE EXAMINATION resistance and Tetracycline, Trimethoprin- M. Kotsch, M. Frank, A. Heusinger, E. Müller; sulfa and Fluoroquinolones as indicators for Laboklin GmBH & Co. KG, Bad Kissingen, MDR. S.psuedintermedius comprised 66% GERMANY. (2235/3406) of the total infections & 50% (293/580) of MRS infections. Undifferenti- Because of their antimicrobial resistance ated coagulase (-) infections comprised 21% situation and the risk of transmission to other (727/3406) of all infections & 33% (191/580) species and contamination of the environ- of MRS. S.aureus made up 9% (305/3406) of ment, infections with Methicillin resistant

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 55 Poster Abstracts

Staphylococcus aureus-species constitute a big n 27A challenge for human and veterinary medi- GENETIC RELATEDNESS, ANTIMICROBIAL cine. Therefore, the early detection of MRSA AND BIOCIDE SUSCEPTIBILITY PATTERNS infections is important for the early initiation AMONG HUMAN, ANIMAL AND of the optimal therapy and for interrupting ENVIRONMENTAL METHICILLIN-RESISTANT transmission pathways. Thereby, the quick and STAPHYLOCOCCUS AUREUS IN PORTUGAL certain diagnosis in the laboratory is of special significance. This study shows retrospectively, N. Couto1, A. Belas1, M. Centeno1, K. Kadlec2, how many MRSA could be identified during S. Schwarz2, C. Pomba1; the microbiological routine examination of 1Faculdade de Medicina Veterinária, Uni- veterinary sample materials in 2012. S. aureus versidade de Lisboa, Lisboa, PORTUGAL, are identified in clinical samples by means of 2Institute of Farm Animal Genetics, Friedrich- their culture morphology, hyaluronidase activ- Loeffler-Institut (FLI), Neustadt-Mariensee, ity and, if applicable, by using MALDI-ToF GERMANY. mass spectrometry or latex agglutination (best The extension of the host range of methicillin- bion dx GmbH, Cologne, Germany). S. aureus- resistant Staphylococcus aureus (MRSA) to isolates that show complete resistance against different animal species has raised the concern beta-lactam-antibiotics and cephalosporins for the role of animals in the epidemiology of in the antibiogram are selected and analyzed MRSA infection and colonization in humans. with an MRSA-detection test (Biomerieux, The aim of this study was to characterize the Marcy L’Etoile, France). If this test shows a genetic relatedness, antimicrobial and biocide positive result, all isolates are examined via susceptibility patterns of human, animal and PCR (McDonald, Antonishyn et al., 2005) environmental MRSA isolates (n=60). Infec- for the presence of the mecA gene. In 2012, tion (i) and colonization (c) isolates were S. aureus-species were diagnosed in a total of obtained from pigs (n=17 i+c), environmental 1.333 samples of companion animals, horses dust samples from breeding pig sheds (n=14), and farm animals. 5,93 % of these isolates humans in contact with animals (n=11 c), showed resistances to beta-lactam-antibiotics calves (n=6 c), dogs (n=5 i+c), cats (n=5 i+c) and cephalosporins and a positive aggluti- and horses (n=2 c). MICs towards 26 anti- nation result. 84,81 % of these resistant S. microbial agents, 3 biocides and 1 dye were aureus-isolates had the mecA gene. 53,16 % determined by broth microdilution according of the MRSA-suspicious S. aureus-isolates to CLSI recommendations. PCR amplification came from dogs, 27,85 % from cats, 15,19 % was used to detect antimicrobial resistance and from horses and 3,80 % from birds/rabbits. efflux-pump genes. The promoter region of The highest percentage of mecA positive S. the norA gene was sequenced. All strains were aureus-species among all suspicious isolates subjected to PFGE and spa typing. Overall, was found in horses. Wounds were the main eleven spa types were identified. Four of these localization in all animal species. This study spa types were associated with CC398 (n=40), reveals that a multi-resistant antibiogram can while five were related to CC22 (n=17) and give certain indication of the presence of the only three associated with CC5 (n=3). Three mecA gene. Even during the bacteriological different clusters, corresponding to CC5, CC22 routine examination, such S. aureus-species and CC398, were identified by PFGE (> 80% are positively detected and therefore give the similarity). All CC398 strains were resistant practitioner an early possibility of initiating an to tetracycline due to the presence of tet(M), optimal therapy and adequate hygiene management.

56 ASM Conferences Poster Abstracts tet(K), tet(L) or a combination of these genes. performing a survey in different pig farms All CC22 and bovine CC398 strains were randomly selected over Belgium, with the fluoroquinolone-resistant. One porcine and aim of monitoring the current prevalence and the six bovine CC398 MRSA, were resistant antimicrobial susceptibility of MRSA among to chloramphenicol and florfenicol due to asymptomatic pigs. Detection and identifica- the fexA gene. Genes dfrK and vga(A) were tion of MRSA was confirmed by selective present in almost all porcine and environmen- chromogenic agar (ChromID MRSA) and 16S tal MRSA CC398 strains. Twelve strains had rRNA-mecA-nuc triplex PCR. MRSA isolates high MICs to ethidium bromide (32 mg/L were characterized by susceptibility testing by in three strains and 16 mg/L in nine strains) a microbroth-dilution method using epidemio- and benzalkonium chloride (1 mg/L in four logical cut-off values (Eucast), spa-typing and strains, 2 mg/L in seven strains and 4 mg/L in sau1-hsdS1 CC398 PCR. Currently, a total of one strain) when compared to S. aureus ATCC 111 farms, out of the 141 tested, were posi- 29213 and all carried the qacG gene. Two tive (78.7%) for MRSA. One MRSA isolate of these strains had an insertion of sequence was recovered from each farm. At present, CAAT in the -10 motif of the norA promoter a total of 87 isolates have been character- gene. All strains had a low MIC to triclosan (≤ ized by susceptibility testing. They showed a 0.125 mg/L). This study showed that MRSA high prevalence of resistance to tetracycline are important reservoirs of antimicrobial and (98.9%) trimethoprim (97.7%), clindamycin biocide resistance genes. Animals and humans (75.9%), ciprofloxacin (71.3%), erythromycin that are in close contact with them share the (64.4%), and gentamicin (50.6%). Lower resis- same MRSA clone. Moreover these strains can tance levels were detected against to kanamy- stay in the environment, which can perpetuate cin (46.0%), tiamulin (40.2%), quinupristin/ the colonization/infection cycle. This raises the dalfopristin (32.2%), streptomycin (34.5%), concern for the successful treatment of human sulfamethoxazole (28.7%), rifampicin, fusidic infections and the effective control of MRSA acid (both 20.7%), chloramphenicol (18.4%), dissemination. mupirocin (10.3%), and linezolid (1.2%). Most isolates (98.9%) were multi-resistant (resis- n 28A tance to 3 or more antibiotics), but susceptible to vancomycin. A total of 67 MRSA isolates ANTIMICROBIAL RESISTANCE were typed by the CC398 PCR, and all them OF METHICILLIN-RESISTANT belonged to this lineage. Moreover, 43 CC398 STAPHYLOCOCCUS AUREUS FROM PIGS IN isolates have been already analysed by spa BELGIUM typing. Six different spa types were identified, M. A. Argudin, A. Lucchina, S. Nemeghaire, being t011 the most common (86%) in the se- P. Butaye; ries. Few isolates have other spa types: t034 (2 Veterinary and Agrochemical Research centre, isolates), t1451, t1456, t1985 and t4872 (one Brussels, BELGIUM. isolate each). These preliminary results suggest that MRSA prevalence remains high among During the last decade, an increasing number Belgian pigs, and worrisomely most isolates of studies reported the presence of methicillin- are multi-resistant. This survey is on-going and resistant Staphylococcus aureus (MRSA) in more isolates are been analysed. Acks: M.A. animals. Most studies have focused on the Argudín is supported by Fundación Alfonso asymptomatic carriage of MRSA among pigs, Martín Escudero. in which clonal complex (CC) 398 is the dominant lineage. During this year, we are

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 57 Poster Abstracts n 29A n 30A IN VITRO ANTIMICROBIAL SUSCEPTIBILITY CHARACTERIZATION AND EPIDEMIOLOGY OF S. AUREUS STRAINS FROM DAIRY OF METHICILLIN RESISTANT HERDS IN KWAZULU-NATAL STAPHYLOCOCCUS AUREUS IN BOVINES T. Schmidt, T. Schmidt; S. Nemeghaire1, M. Á. Argudín1, F. Haeseb- Allerton Provincial Veterinary Laboratory, rouck2, P. Butaye1; Pietermaritzburg, SOUTH AFRICA. 1Veterinary and Agrochemical Research centre, Brussels, BELGIUM, 2Ghent University, Staphylococcus aureus is one of the most Ghent, BELGIUM. important causes of bovine mastitis and is responsible for significant economic losses to the Methicillin-resistant Staphylococcus aureus dairy industry worldwide. One of the principal (MRSA) is considered as a growing problem approaches used in treating intramammary in human and veterinary medicine. The aim of infections is the administration of antimicro- this study was to determine the molecular epi- bials. Due to the propensity of S. aureus to demiology of MRSA in healthy bovines and to develop resistance, antimicrobial susceptibil- compare different rearing practices. Therefore, ity monitoring is necessary to ensure that in 2012, nasal swabs were collected on 432 treatment regimens are effective. As part of randomly chosen Belgian farms and tested for this investigation, a collection of 90 S. aureus the presence of MRSA. Among these farms, strains, isolated from mastitis cases submitted 141 were dairy farms, 187 reared beef cattle to Allerton Provincial Veterinary Laboratory and 104 veal calves. MRSA was isolated from during 2008 and 2009, was evaluated for their a pool of 20 nasal samples using an enrichment susceptibility to a panel of 10 antimicrobials. method. Identification ofS. aureus and methi- Only 8 of the 90 S. aureus isolates tested (8,9 cillin resistance was confirmed by a triplex %) were found to be susceptible to all of the PCR. MICs were determined using micro- antimicrobials evaluated. A very high level of broth dilution test and MRSA isolates were resistance to the beta-lactam antibiotics was characterized by means of SCCmec typing, noted: 47,8 % resistance to penicillin and 65,6 spa-type, CC398 PCR and MLST. Eighty-two % resistance to ampicillin. Minimal resistance farms (19.0%, 95% CI [15.3% -22.7%]) were to oxacillin, cephalothin and trimethoprim-sul- positive for MRSA. Among these, 14 (9.9%, famethoxazole (1,1 %) was found. Seventeen 95% CI [5.0% -14.9%]) were dairy farms, 19 (18,9 %) of the isolates tested were found to (10.2%, 95% CI [5.8% -14.5%]) were farms resistant to 3 or more antimicrobials. The need holding meat cows and 49 were (47.1%, 95% for vigilant monitoring of bacterial resistance CI [37.5% -56.7%]) farms rearing veal calves. trends in the dairy industry is warranted as the One isolate did not regrow after conservation potential public health implications hereof are and was lost. More than 90% of the isolates significant. were resistant to tetracycline and trimethoprim. A high prevalence of resistance was also observed to clindamycin, erythromycin, kanamycin and gentamicin. More than half of the isolates were also resistant to streptomycin. Lower resistance levels were detected to fusidic acid, sulfamethoxazole, quinupristin/dalfopristin, tiamulin, ciprofloxacin, rifampicin, chloramphenicol and mupirocin. No resistance was observed to linezolid and vancomycin. Among the 81 MRSA recovered, 78 were

58 ASM Conferences Poster Abstracts

ST398. One isolate was ST8 and two ST239. The aim of this cross-sectional study was Ten different spa-types were identified. Sixty- to find out MRSA presence in milk samples four were spa-type t011, and others were t037, derived from cows with clinical mastitis and t121, t388, t1451, t1456, t1985, t3423, t6228 reveal their potential Multiple Drug Resistance or non-typable (NT). MRSA spa-type t121 was patterns. For that purpose, 480 milk samples associated to MLST type ST8, t388 and t037 to with clinical mastitis out of 1600 cattle were ST239. Forty-six isolates carried SCCmec type examined in the province of Northwestern Tur- IV (2B) and eleven SCCmec type IV (2B&5). key. API-Staphy® (bioMėrieux) detection kit Sixteen isolates carried SCCmec type V (5C2) was used for identification and the results were and two SCCmec type III (3A). Six isolates evaluated by API-web® system. The isolates were non typable (NT) using Kondo’s proto- were also confirmed by Staphaureux Plus® col. Both ST239 isolates carried the NT SC- (remel) Latex Agglutination Test. Phenotypic Cmec cassette while the ST8 isolate harboured methods i.e. disk diffusion test with cephoxi- SCCmec type IV (2B). In summary, MRSA tin® (OXOID), PBP2 ‘ Latex Agglutination has been found at low prevalence in the nares Test® (OXOID), Nitrocephin Disk® (remel) of dairy and meat cows, but at elevated levels and reproductive characteristics in ORSAB in veal calves. Most isolates were resistant to medium (Oxacillin Resistant Screening Agar tetracycline and trimethoprim. The preva- Base-OXOID ®) were carried out to determine lence of acquired resistance to erythromycin, the methicillin resistance properties of the clindamycin, kanamycin and gentamicin is isolates. Kirby-Bauer disk diffusion test was extremely high compared to what has been performed to the isolates using 20 antimicro- found in MRSA from other origins in Belgium. bials belonging to 7 different groups. A PCR Although SCCmec type III was previously method was applied to the isolates in terms of described in CC398 using Zhang’s protocol, carriage of the mecA gene. Additionally, MIC these isolates were eventually found to carry Evaluators (Oxoid-Remel®) Penicillin G, Van- a novel type of SCCmec type V. Therefore, comycine, Tetracycline, Oxacillin, Ciprofloxa- further analyses have to be performed in order cin and Gentamycin were used for the accurate to confirm the SCCmec type for these isolates. detection of minimal inhibitory concentration Ackowledgment: Dr. M. A. Argudín has (MIC) of 10 selected isolates which were received a research grant from the Fundación carrying mecA. The zone diameters and MIC Alfonso Martín Escudero. breakpoints were both evaluated by the direc- tives according to EUCAST Clinical Break- n 31A point Table Version 3,0, 2013. In this study, S. aureus ATCC 25923 and the methicillin-resis- MULTIPLE DRUG RESISTANT tant strains (MRSA) ATCC 33 591, ATCC 43 STAPHYLOCOCCUS AUREUS (MDR) 300 and ATCC 700 699 were used as reference DETECTION IN METHICILLIN RESISTANT strains. As a result, 151 (31.45 %) S. aureus STAPHYLOCOCCUS AUREUS (MRSA) AND were isolated from 480 milk samples. Several STAPHYLOCOCCUS AUREUS DERIVED numbers of isolates (10 (6.62%) by PBP2 ‘; FROM BOVINE MASTITIS CASES IN TURKEY 40 (26.49%) by ORSAB and 36 (23.84%) E. Buyukcangaz1, S. Kahya1, A. Sen1, T. K. by Nitrocephin Disc) showed positivity at Carli1, K. S. Intas1, B. Mat2, H. Gocmen2, E. ß-lactamase activity. Twenty four of 151 Biyikli2, A. Eyigor1, S. Temelli1; (15.89%) S. aureus isolates were detected as 1Uludag University, Bursa, TURKEY, 2Uludag mecA gene carrier. Sixty two (41.05%) isolates University, Health Sciences Institute, Bursa, were resistant to cefoxitin by disc diffusion TURKEY. testing. Broad range of antibiogram and their resistance patterns of the isolates are; Penicil- lin group of antibiotics, cephalosporins, fluoro-

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 59 Poster Abstracts quinolones, glycopeptides, macrolide group of tion of MRSA from oral fluids with and with- antibiotics, tetracyclines and the other groups out enrichment broth, 2) compare detection of at the rates of 32.4, 38,5, 18.6, 2, 34, 44, and MRSA from pork production facilities using 10.6 %, respectively. Seventy six of isolates oral fluids, environmental sponges and nasal (50.33%) showed resistance more than one and swabs, and 3) assess prevalence of MRSA 23 (15.23%) were more than three groups of from oral fluid samples submitted for routine antibiotics. MIC results were ranged between diagnostic testing. Methods: MRSA negative 0.12-256 and five isolates showed MDR pat- swine oral fluids were spiked with three strains terns as follows; one was 6, two were 5 and of MRSA (ATCC 43300, spa t034, spa t002) two were 4 different groups of antimicrobials. at various levels. Colony counts from oral Additionally, four isolates were accepted as fluids and phosphate buffered solution (PBS) borderline Glycopeptide Intermediate Staphy- spiked with same levels were compared after lococcus aureus (GISA) and two were GISA. streaking onto chromogenic media (BioRad As a conclusion, a high rate of antibacterial MRSASelect). Additionally aliquots (50 µl) resistance was observed in S. aureus isolates from spiked oral fluids were added to 5ml of from bovine milk samples under large-scale enrichment broth (10g tryptone/L, 75g NaCl/L, investigation. This is remarkable both in terms 10g mannitol/L and 2.5g yeast extract/L), of animal and public health. incubated, streaked onto chromogenic media and presence of MRSA determined. Samples n 32A (nasal swabs, environmental sponges, and oral fluids) were collected from pork production USING ORAL FLUIDS TO DETECT facilities with known and unknown MRSA METHICILLIN-RESISTANT status. These samples were tested for MRSA as STAPHYLOCOCCUS AUREUS IN PIGS AND described using enrichment broth. Aliquots of PORK PRODUCTION FARMS oral fluids were taken from diagnostic samples T. Frana1, A. Beaman1, D. Fligg1, J. Kinyon1, submitted for routine testing and tested for S. Wardyn2, B. Hanson2, L. Layman1, L. Kar- MRSA as described using enrichment broth. In riker1, A. Ramirez1, T. Smith2; all samples S. aureus isolates were confirmed 1Iowa State University, Ames, IA, 2University as MRSA with mecA PCR and selected isolates of Iowa, Iowa City, IA. forwarded for spa typing. Results: There was no difference in level of recovery of MRSA Introduction: Methicillin-resistant Staphy- from PBS versus oral fluids. After enrichment lococcus aureus (MRSA) is a global health MRSA could be detected in spiked oral fluids concern and reservoirs of specific types have at <1 CFU/ml. MRSA could be detected from been found in swine and pork production fa- all samples of oral fluids, nasal swabs, and cilities. This has led to concern about possible environmental swabs collected at a known transmission of livestock-associated MRSA MRSA positive pork production farm. Paired (LA-MRSA) to swine workers and their oral fluids and environmental sponges were contacts. MRSA can be detected from a variety collected from 15 pork production farms of of sample matrices including nasal swabs and unknown MRSA status. Four farms were posi- environmental sponges. Oral fluids are an tive for MRSA (3 from oral fluids, one from efficient, cost-effective method of collecting environmental sponge). Thirty one (31) of 513 diagnostic samples for several swine diseases. diagnostic oral fluids (6.0%)were positive for Use of oral fluids to detect MRSA would allow MRSA. Spa types found in this study included: an effective means of determining the status of t002, t034, t548, and t111.Conclusion: The specific swine facilities as well as aiding the use of oral fluids appears to be a sensitive, ef- investigations into the prevalence of MRSA in ficient and cost-effective method for detecting swine. Objectives: 1) evaluate level of detec- MRSA in swine.

60 ASM Conferences Poster Abstracts n 33A (n=406), 6%, 25% and 73% were positive for SCCMEC METAL RESISTANCE GENES arsA, copB and czrC genes, respectively. In AMONG ANIMAL STAPHYLOCOCCUS contrast, only 5.6% and 4.3% of non-CC398 AUREUS ISOLATES FROM GERMANY isolates (n=72) were positive for copB and czrC genes, respectively. The czrC gene was M. A. Argudin1, B. Lauzat2, B. Kraushaar2, Y. associated with the presence of SCCmec V in Kelner-Burgos2, A. Fetsch2, B. A. Tenhagen2, both CC398 and non-CC398 MRSA isolates. B. Guerra2; 95% of SCCmec V and czrC positive isolates 1Veterinary and Agrochemical Research centre, carried tetK, indicating that they may have SC- Brussels, BELGIUM, 2Federal Institute for Cmec V (5C2&5)c. Interestingly, one CC398 Risk Assessment (BfR), Berlin, GERMANY. isolate with a possible new SCCmec V variant carried also czrC. czrC was found in CC398 Methicillin resistant Staphylococcus (S.) isolates from all origins, whereas among non- aureus (MRSA) has emerged as a zoonotic CC398, all czrC positive isolates were from pathogen in the animal production world- pig origin, only. The gene copB was present in wide. Most of the livestock associated (LA) isolates with different SCCmec types and was MRSA belong to the clonal complex (CC) observed in pigs, cattle, and poultry. The arsA 398, although other CCs (non-CC398) have gene was found in one MSSA and in isolates also been described. The reason for the LA- with SCCmec V, mainly from poultry. This MRSA emergence is unknown, but it has study shows that genes conferring metal-R are been suggested that tetracycline resistance (R) frequently present in LA-CC398, suggesting could have driven selection of MRSA CC398. that the use of metal containing compounds Besides antimicrobial agents used for therapy, in animal feed may contribute to the selection other substances with antimicrobial activity are of MRSA. Acks: M. A. Argudín is supported used in animal feed, including metal contain- by Fundación Alfonso Martín Escudero. Part ing compounds used for prevention of gastro- of the work was carried out in the EMIDA- intestinal diseases. Recently, metal R-genes ERA NET Project “LA-MRSA” (Grant-No. have been found in various novel SSCmec 0315868A). cassettes. The aim of this study was to assess the occurrence of metal R-genes among LA-S. aureus. A total of 478 isolates from animals n 34A (pigs, bovine, poultry and companion animals), PREVALENCE OF METHICILLIN-RESISTANT dust from farms, milk and meat products and STAPHYLOCOCCUS AUREUS (MRSA) IN humans between 2004 and 2012, were selected ITALIAN DAIRY HERDS. from the collection of the National Reference R. Piccinini1, M. Malvisi1, P. Cremonesi2, E. Laboratory for coagulase positive staphylo- Capra2, G. Bignoli2, B. Castiglioni2, L. Valen- cocci including S. aureus. They were assigned tino1, F. Pozzi3, F. Vezzoli3, M. Luini3; to CCs using spa or MLST typing, and 1Dept. Veterinary Sciences and Public Health, analysed for their SCCmec type and the pres- University of Milan, Milan, ITALY, 2Istituto di ence of R-determinants via PCR, including: Biologia e Biotecnologia Agraria, CNR, Lodi, tetK (tetracycline-R, SCCmec III, V (5C2&5) ITALY, 3Istituto Zooprofilattico Sperimentale c and plasmid associated), arsA (arsenate-R, della Lombardia e dell’Emilia, Lodi, ITALY. SCCmec IX), cadD (cadmium-R, SCCmec IX and X), copB (copper-R, SCCmec IX and X), Methicillin-resistant Staphylococcus aureus and czrC (cadmium and zinc-R, SCCmec V (MRSA) has been reported in human medicine (5C2&5)c). 75% of the isolates carried at least as a cause of nosocomial and community- one metal-R gene. Among the CC398 isolates associated infections. In veterinary medicine,

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 61 Poster Abstracts the microorganism has been identified in a n 35A wide range of animals and diseases, includ- HIGH PREVALENCE OF TETRACYCLINE ing dairy cows mastitis. Therefore, MRSA RESISTANCE IN S. EPIDERMIDIS FROM is considered an emerging threat with a high OVINE AND BOVINE SUB-CLINICAL zoonotic potential. AIM: In the present study, MASTITIS: EVIDENCE FOR ADAPTATION TO we investigated the diffusion of MRSA in Ital- ANIMALS? ian dairy herds and the prevalence of infected animals at herd level. MATERIALS AND J. Rolo, A. Havrysh, H. de Lencastre, M. METHODS: Quarter milk samples were Miragaia; aseptically taken from all lactating cows of 83 Instituto de Tecnologia Química e Biológica, S. aureus infected herds and bacteriological Oeiras, PORTUGAL. analysis was performed by culture on blood- S. epidermidis is a human commensal bac- agar plates. The hemolytic, Gram-positiv and terium that can cause opportunistic infec- coagulase-positive colonies of growth were tions mainly associated to medical devices identified as S. aureus. At least four isolates in hospitals and community. In humans, the morphologically representative for each herd rates of resistance to antimicrobials are lower were tested by oxacillin disk-diffusion method in the community setting when compared to to detect methicillin-resistance. DNA of hospitals. In animals, colonization with S. oxacillin resistant isolates was extracted and epidermidis appears to be sporadic and depen- the presence of mecA gene was investigated dent on the contact with humans; however S. by PCR. RESULTS: All oxacillin-resistant epidermidis is frequently found as a cause of isolates were confirmed as MRSA by mecA mastitis in bovine and ovine, suggesting that positivity. Prevalence of S. aureus infected they are adapted to these hosts. In the veteri- cows and frequency of MRSA isolation were nary setting huge doses of antibiotics are used registered at herd level. Overall prevalence of as growth promoters and in the treatment of mammary infections by S. aureus ranged 0,7 - infections, but the frequency of antimicrobial 62%: in 28 herds (33.7%) the prevalence was resistance as well as the epidemiology of S. higher than 25%, while in 27 herds (32.5%) epidermidis from animals is not well under- it was lower than 4.7%. Methicillin-resistant stood. To evaluate the role of S. epidermidis as strains were detected in 11 farms (13,25%), all a reservoir of antibiotic resistance genes in the belonging to the low-prevalence group. In 5 veterinary setting and study its epidemiology, out of such 11 herds, all S. aureus isolates were we analyzed a collection of 87 S. epidermidis MRSA, while in the remaining 6 herds both isolates collected from the milk of sheep and MRSA and methicillin-susceptible S. aureus cows with sub-clinical mastitis located in two (MSSA) were present. CONCLUSIONS: Our different farms in Portugal, over a period of data confirmed the potential risk of subclinical eight years (1996-2003). Antimicrobial sus- mastitis caused by MRSA and suggest a low ceptibility testing to a panel of six antibiotics diffusiveness of these strains in infected herds. (oxacillin, vancomycin, ciprofloxacin, tetracy- This characteristic could reduce the zoonotic cline, erythromycin and chloramphenicol) and potential of bovine MRSA, but the real preva- molecular characterization by pulsed-field gel lence in dairy farms could be underestimated, electrophoresis (PFGE) were performed for since bacteriological analysis of milk is usually representative isolates. Moreover, isolates were performed only when a clear problem of mam- screened for methicillin-resistant determinant mary infections affects the herd. (mecA/SCCmec) and biofilm-associated genes (atlE and aap) by PCR. The great majority of the isolates (70%) belonged to only 5 out of 11

62 ASM Conferences Poster Abstracts clonal types identified by PFGE. Almost half + RPF. For each sample, at least 5 colonies of the isolates analyzed (48%) were resistant to suspected to be S. aureus were tested for tetracycline and 18% were resistant to oxacil- susceptibility to oxacillin by disk diffusion lin. However, the mecA was present in only 11 test. The same milk samples were examined out of 87 isolates (12%). SCCmec type IV was by enrichment in MH broth + 7.5% NaCl and frequent (n=6), but SCCmec III (n=1) and non- TSB + 5mg/l of oxacillin and plating on Bril- typable cassettes (n=4) were also found. The liance MRSA agar. The presence of the mecA atlE and aap were found in a high proportion gene was confirmed by PCR. Genotyping was of isolates (76% and 44%, respectively), many performed by Multilocus Sequence Typing of which were also resistant to methicillin (MLST). RESULTS: A total of 291 bovine and tetracycline. S. epidermidis from bovine (43.2%) and 85 caprine (43.1%) samples tested sub-clinical mastitis appear to have increased positive for S.aureus. Among these, MRSA resistance to tetracycline (48%) when com- were demonstrated in 29 bovine and 4 goat pared to human isolates (<10%) from the samples (10.0 and 4.7% of the isolates respec- same geographic region, suggesting long-term tively). Five out of 33 MRSA were isolated by colonization and adaptation of S. epidermidis both by direct plating and after enrichment, 12 to animal host. only by enrichment and 16 only by direct plating,. MLST analysis of 20 bovine MRSA iso- n 36A lates found twelve belonged to Sequence Type ST398, four to ST1, three to ST97 and one to PREVALENCE OF STAPHYLOCOCCUS ST5. MLST of the 4 caprine MRSA isolates AUREUS AND MRSA IN BULK TANK MILK OF identified three ST398 and one ST1.DISCUS - BOVINE AND GOAT HERDS OF NORTHERN SION: The high prevalence of positive S. ITALY aureus bulk tank milk samples underline the M. V. Luini1, C. Cortimiglia1, F. Vezzoli1, D. importance of the infection in both bovine and Avisani2, A. Franco3, A. Battisti3; caprine farms in the investigated area. MRSA 1IZSLER, Lodi, ITALY, 2IZSLER, Brescia, were detected with significant prevalence in ITALY, 3IZSLT, Roma, ITALY. bovine samples and were also found in caprine bulk milk. The lower MRSA positivity rates AIMS: S.aureus is the most important caus- observed when testing isolates by selective ative agent of subclinical mastitis in cattle and enrichment may be due to higher susceptibility goats. Methicillin-resistant strains (MRSA) of clones to the salt concentration employed. may have zoonotic importance, especially in The genotyping of bovine and goat MRSA case of occupational exposure and were repeat- strains showed that the more frequent lineage edly isolated from bovine mastitis in Italy and was ST398, while ST97 and ST1, both associ- other countries, but very rarely in goat herds. ated with cattle and pigs in Italy, were also The aim of our study was to evaluate the confirmed to be common among herds from prevalence of MRSA in bulk milk of bovine Northern Italy. The application of more strin- and goat dairy herds from Lombardy region gent control measures against MRSA mastitis (Northern Italy) whose production of cow’s in cattle and goat herds, seems appropriate in milk represents 38% of national production order to minimize the risk of transmission of and to identify the main circulating genotypes. MRSA to humans by occupational exposure METHODS: 673 and 197 bovine and goat or through the consumption of raw milk or bulk tank milk samples from different herds products made of un-pasteurized milk. were examined respectively. Samples were plated directly on Blood agar and the S.aureus count was determined on Baird Parker Agar

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 63 Poster Abstracts n 37A software based on the method described by PROPOSAL FOR QUALITY CONTROL Turnidge and Bordash. Results: All results of RANGES OF STAPHYLOCOCCUS AUREUS the testing of S. aureus ATCC® 25923 with ATCC® 25923 FOR AGAR DISK DIFFUSION the erythromycin 15 µg disk were within the CLSI-approved QC range of 22-30 mm. For USING 30 µG TYLOSIN DISKS the tylosin 30 µg disks, zone diameters of 18 M. Buß1, A. T. Feßler1, J. Turnidge2, T. Peters3, to 26 mm, 18 to 27 mm, and 19 to 26 mm were S. Schwarz1; observed for the MH agar lots from Oxoid, 1Institute of Farm Animal Genetics (FLI), Roth and Mast, respectively. The data revealed Neustadt-Mariensee, GERMANY, 2SA Pathol- a mean of 22.16 mm and a median of 22 mm ogy at Women’s and Children’s Hospital, (20-23 mm) for all participating laboratories. North Adelaide, South Australia, AUSTRALIA, Based on the results obtained with the Range- 3Milchtierherden-Betreuungs- und Forsc- Finder software, we recommend to use 18 to hungsgesellschaft mbH (MBFG), Wunstorf, 26 mm for the 30 µg tylosin disk as QC range GERMANY. for S. aureus ATCC® 25923. This proposed QC range includes 475/480 (99.0%) of the Objective: Tylosin is a 16-membered mac- observed zone diameter values for the 30 µg rolide that is licenced for the treatment of tylosin disk. Conclusions: The proposed QC bovine mastitis, pneumonia and arthritis in range has been recently approved by the CLSI. calves, bronchitis in dogs, pneumonia, ileitis This QC range will help the routine diagnostic and erysipelas in pigs, and various diseases in laboratories to validate their results when using poultry. When testing bacteria for their sus- 30 µg tylosin disks. Moreover, this QC ranges ceptibility/resistance, approved quality control can contribute to a harmonization and stan- (QC) ranges are important parameters for the dardization of tylosin susceptibility testing. internal validation of antimicrobial susceptibility test systems. For agar disk diffusion, there have been available only QC ranges for the n 38A 60 µg tylosin disks. These disks, however, are PROPOSAL FOR CEFOPERAZONE QUALITY rarely if at all commercially available while CONTROL RANGES FOR STAPHYLOCOCCUS the 30 µg tylosin disks are commonly used AUREUS ATCC® 25923 FOR AGAR DISK in routine diagnostics. The aim of the present DIFFUSION USING 30 µG DISKS study was to determine QC ranges for 30 µg A. T. Feßler1, J. Turnidge2, S. Schwarz1; tylosin disks for the reference strain Staphylo- 1Institute of Farm Animal Genetics (FLI), coccus aureus ATCC® 25923. Materials and Neustadt-Mariensee, GERMANY, 2SA Pathol- methods: An interlaboratory trial including ogy at Women’s and Children’s Hospital, North eight laboratories was performed according Adelaide, South Australia, AUSTRALIA. to the recommendations given in the CLSI document M37-A3. Each laboratory tested Objectives: The third generation cephalospo- the S. aureus reference strain ten times by rin cefoperazone is commonly used for the agar disk diffusion using two different lots therapy of bovine mastitis. Quality control of tylosin 30 µg disks (Biolab, Mast) and (QC) ranges are important parameters for the three different Mueller-Hinton (MH) agar lots internal validation of antimicrobial susceptibil- (Oxoid, Roth, Mast). A 15 µg erythromycin ity testing in routine diagnostics. So far, there disk (Oxoid) was tested on the MH agar from are only QC ranges for the 75 µg cefopera- Roth as quality control. The tests of all eight zone disks available. The aim of the present laboratories resulted in 480 data points for the study was to determine QC ranges for 30 µg tylosin 30 µg disks. The analysis of the results cefoperazone disks for the QC reference strain was performed by using the RangeFinder Staphylococcus aureus ATCC® 25923

64 ASM Conferences Poster Abstracts based on the recommendations given in the n 39A document M37-A3 of the Clinical and Labora- CHLORAMPHENICOL AND FLORFENICOL tory Standards Institute (CLSI). Material and MIC DISTRIBUTIONS, GENETIC RESISTANCE Methods: According to the recommendations MARKERS AND TIME-KILL KINETICS of CLSI document M37-A3 an interlabora- IN CANINE CLINICAL ISOLATES OF tory trial was performed. Each of the eight STAPHYLOCOCCUS PSEUDINTERMEDIUS participating laboratories tested the S. aureus ATCC® 25923 reference strain ten S. S. Mo1, M. G. Maaland1, S. Schwarz2, L. times using two different cefoperazone 30 µg Guardabassi1; disk lots (Oxoid, Mast) and three different 1Department of Veterinary Disease Biology, Mueller-Hinton (MH) agar lots (Oxoid, Roth, Faculty of Health and Medical Sciences, Mast). A 75 µg cefoperazone disk (Oxoid) was University of Copenhagen, Frederiksberg, tested on one MH agar lot (Oxoid) as quality DENMARK, 2Institute of Farm Animal Genet- control. The test results obtained from all eight ics, Friedrich-Loeffler-Institut (FLI), Neustadt- laboratories resulted in 480 data points. The Mariensee, GERMANY. analysis of the results was performed by using Introduction: The emergence of methicillin- the RangeFinder software based on the method resistant Staphylococcus pseudintermedius described by Turnidge and Bordash. Results: (MRSP) has led to an urgent need for alterna- The results of the testing of S. aureus ATCC® 25923 for the cefoperazone 75 µg animal practice. Chloramphenicol and florfeni- disk were within the CLSI-approved QC-range col may be alternative drugs for treatment of of 24-33 mm. The testing of S. aureus ATCC® 25923 with the cefoperazone 30 µg rently not specifically approved for dogs and disk revealed zone diameters of 23 to 38 mm, cats, this veterinary drug may be used under 25 to 34 mm and 25 to 34 mm for the MH agar Animal Medicinal Drug Use Clarification Act lots from Oxoid, Roth and Mast. The zone (AMDUCA) or similar regulations. Objec- diameters for the disk lots from Oxoid and tive: The aim of the study was to elucidate Mast ranged from 23 to 38 mm and from 23 to the antibacterial activity of chloramphenicol 35 mm, respectively. The 30 µg cefoperazone and florfenicol onS. pseudintermedius and to results revealed a mean value of 28.71 mm evaluate the potential use of these phenicols in for all laboratories. The median value for all small animal medicine. Methods: MICs were laboratories was 28 mm (26 to 31 mm) and determined by broth microdilution accord- the overall mode value was 28 mm. Six of the ing to CLSI guidelines in 170 canine clinical laboratories had mode values of 27 to 30 mm, isolates of S. pseudintermedius from Denmark whereas the remaining two laboratories had 31 (n=93), US (n=30), Italy (n=28) and Germany and 32 mm, respectively. Conclusions: Based (n=19), including 30 MRSP. Isolates with on the data, we recommend the use of 23 to 34 MIC-values >8 µg/ml were screened by PCR mm as QC range for S. aureus ATCC® for the presence of the chloramphenicol resis- 25923 and the 30 µg cefoperazone disk. This tance genes cat , cat and cat . Time- new QC range has recently been approved pC194 pC221 pC223 kill kinetics at concentrations corresponding to by the CLSI and will help routine diagnostic 0.5-16 x MIC was determined for one isolate laboratories to validate their cefoperazone with MICs of 8 µg/ml (chloramphenicol) and susceptibility testing results when using 30 4 µg/ml (florfenicol).Results: The florfenicol µg disks. Moreover, they will contribute to a MIC-distribution was unimodal with MICs harmonization of the cefoperazone susceptibil- in the range 1-8 µg/ml. The chloramphenicol ity testing. MIC distribution was bimodal with MICs of 4-16 µg/ml for susceptible isolates (n=141), and 32-64 µg/ml for resistant isolates (n=29). 3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 65 Poster Abstracts

Five MRSP isolates were resistant to chlor- n 41A amphenicol. All resistant isolates harbored the TWO CASES OF CO-INFECTION WITH gene cat . At concentrations corresponding pC221 METHICILLIN RESISTANT AND METHICILLIN to 1-2 x MIC, a 0-1 log unit reduction in cfu/ SUSCEPTIBLE STAPHYLOCOCCAL ISOLATES ml was seen after 24 hours for both drugs. IN ANIMALS. At 4-16 x MIC concentrations, a 2-3 log unit reduction in cfu/ml was observed. Discussion/ M. Sunde1, Ø. Kolbjørnsen1, H. Fanuelsen2, J. conclusion: The antibacterial action of both S. Slettemeås1, K. W. Larssen3, L. Marstein3; drugs on S. pseudintermedius was bacteriostat- 1Norwegian Veterinary Institute, Oslo, ic and time-dependent, although moderately NORWAY, 2Drammen sykehus, Drammen, enhanced killing was observed at increasing NORWAY, 3The Reference Laboratory for drug concentrations. The absence of florfenicol MRSA in Norway, St Olavs Hospital, Trond- resistance and the rare occurrence of chloram- heim, NORWAY. phenicol resistance indicate that these antibiot- Co-infection with different isolates of the same ics could potentially be useful against MRSP. species, including Staphylococcus aureus with Further studies, including in vivo PK-PD target or without the mecA gene, has been described determination and Monte Carlo simulation ap- from infections in humans. Current knowledge plying MIC values and target animal pharma- about such infections in veterinary medicine cokinetics, are needed to optimize the use of is limited. In this study we have character- these drugs in small animal practice. ized isolates involved in co-infection with two different staphylococcal isolates; methicillin n 40A resistant and methicillin susceptible, in a pig MUPIROCIN RESISTANCE IN (Staphylococcus aureus) and a dog (Staphylo- STAPHYLOCOCCUS PSEUDINTERMEDIUS coccus pseudintermdius). From a pig admitted ISOLATED FROM DOGS. to autopsy two different S. aureus were isolated after culturing from the spleen. The S. M. Godbeer, S. D. Lawhon; autopsy findings and histological examination Texas A&M University, College Station, TX. showed that the pig suffered from meningitis, In the United States, veterinary use of mu- probably as consequence of tale biting. The pirocin is primarily limited to the treatment S. aureus isolates were investigated for their of canine pyoderma caused by methicillin- antimicrobial resistance profiles,mec A status resistant Staphylococcus pseudintermedius (PCR), spatype and multi locus sequence type (MRSP). In this study, only 1 of 580 (0.002%, (MLST). One of the S. aureus was an MRSA, 1/580) S. pseudintermedius isolates tested with spatype t034, belonging to ST398. The was resistant to mupirocin. Further evalua- other was mecA negative (MSSA), belonged tion of this isolate indicated that it carried the to spatype t1430 and ST9. The two S. aureus high-level mupirocin resistance gene, ileS2 on isolates displayed different resistance profiles, a plasmid. The isolate was collected from a with the MSSA being fully susceptible and the patient without clinical staphylococcal infec- MRSA resistant to a broad range of antimicro- tion that was presented for orthopedic evalua- bial agents. From a dog with otitis externa two tion. The isolate was not methicillin-resistant. different S. pseudintermedius were isolated. All isolates were collected prior to February The staphylococcal isolates occurred in a 15, 2012 in a large referral practice in Texas. mixed flora also containingPseudomonas A total of 155 isolates (27%, 155/580) were aeruginosa and Malassezia pachydermatis. methicillin-resistant. These results indicate that The staphylococcal isolates were subjected to mupirocin resistance in the patient population species determination and were further tested of the referral practice is low. for antimicrobial resistance, mecA content

66 ASM Conferences Poster Abstracts and subjected to MLST. These investigations wounds. The Atlantic Veterinary College confirmed presence of twoS. pseudinterme- (AVC) Diagnostic Bacteriology Laboratory is dius, one with mecA (MRSP) and the other a principle reference laboratory for Atlantic without (MSSP). The strains had different Canada, and has seen a significant increase in resistance profiles and belonged to different the recovery of MRSP from samples submitted STs; The MRSP to ST129 and the MSSP to for routine culture and sensitivity. Currently, ST120. In both cases there was no clear differ- there are no reports investigating MRSP in ence in colony morphology on blood agar. The dogs in this region. Sequence analysis of the presence of different isolates was discovered mec-associated direct repeat units (dru typing) when performing confirmation tests S.( aureus/ is a preferred method for strain typing MRSP. MRSA) and susceptibility testing by the use A significant difference in the distribution of of disc diffusion (S. pseudintermedius). Our predominant dru clusters has been reported findings show that co-infection with MSSA/ between Europe, 5 U.S. states, and Ontario, MRSA and MSSP/MRSP can occur in animals. Canada, with suggestion that dru types may It is important that the diagnostic laboratories be unevenly distributed in a single country. are aware of this as methicillin resistant and The objective of this study was to strain type susceptible isolates may mask each other and the MRSP cultured at the AVC Diagnostic challenge correct diagnostic. A co-infection Bacteriology Laboratory using dru typing. may also enable exchange of genetic elements Staphylococcal isolates recovered from dogs like resistance and virulence genes. This is between January 2010 and December 2012 particularly relevant for the S. aureus strains were tested. Staphylococci were identified from swine as MRSA ST9/t1430 is considered using biochemical testing and methicillin- a possible new emerging livestock associated resistance was confirmed by the presence MRSA. of penicillin-binding protein 2a (PBP2’). A multiplex PCR assay was used to iden- n 42A tify coagulase-positive staphylococci to the species-level. Isolates were typed by analyz- DIRECT REPEAT UNIT (DRU) TYPING ing sequence data from the direct repeat units. OF METHICILLIN-RESISTANT To date, 115 MRSP isolates have been typed. STAPHYLOCOCCUS PSEUDINTERMEDIUS 17 different dru types have been identified FROM DOGS AT A DIAGNOSTIC with the majority belonging to type dt11a LABORATORY IN ATLANTIC CANADA. (n=33, 28.7%), dt10h (n=27, 23.5%), dt9a M. Saab1, J. S. Weese2, C. A. Muckle3, J. T. (n=27, 23.5%), and dt11af (n=12,10.4%). The McClure1; remaining 16 (13.9%) isolates were distributed 1Department of Health Management, Atlantic between 13 different dru types, nine of which Veterinary College, University of Prince have not been previously identified: dt5k, dt6t, Edward Island, Charlottetown, PE, CANADA, dt8ag, dt9ba, dt9bd, dt10bz, dt10cc, dt10cj, 2Department of Pathobiology, Ontario Veteri- and dt11ca. Each of the previously unidentified nary College, University of Guelph, Guelph, dru types was represented by one isolate. The ON, CANADA, 3Department of Pathology and predominant dru types identified in this study Microbiology and Diagnostic Services, Atlan- are similar to those found in Ontario, Canada; tic Veterinary College, University of Prince however, a cluster analysis is needed to make Edward Island, Charlottetown, PE, CANADA. further conclusions. Further work will include epidemiologic analysis of dru types and patient Methicillin-resistant Staphylococcus pseud- information. Results from this study will intermedius (MRSP) has emerged as major provide information on the MRSP situation in pathogen in dogs, being primarily isolated Atlantic Canada, furthering the understanding from skin, ears, surgical site infections, and of the dissemination of this pathogen.

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 67 Poster Abstracts n 43A cattle isolates were of spa type t172 (ST375, CHARACTERIZATION OF METHICILLIN- SCCmec IV) and t3256 (ST130), the latter RESISTANT STAPHYLOCOCCUS AUREUS being the only mecC positive MRSA find- (MRSA) ISOLATED FROM ANIMALS IN ing in animals in Finland so far. In addition, FINLAND three MRSA isolates have been isolated from dogs (t008, t067 and t073, SCCmec IV and S. Nykäsenoja1, L. Lindholm2, S. Salmenlinna3, nontypeable) and two from cats (t324, ST72, M. Rantala4; SCCmec nontypeable). All the porcine and one 1Finnish Food Safety Authority Evira, Helsinki, equine MRSA had the czrC gene. Spa types FINLAND, 2National Institute for Health and t3256, t3933, t5103 and t6867 were unique for Welfare THL, Turku, FINLAND, 3National animal isolates. Conclusions: Several different Institute for Health and Welfare, Helsinki, spa types were present among animal MRSA FINLAND, 4Faculty of Veterinary Medicine, isolates and most of them were also found in University of Helsinki, Helsinki, FINLAND. human isolates. SCCmec types V and IV were most commonly encountered. The czrC gene Background: In Finland, there is no sys- was mainly associated with porcine MRSA. tematic surveillance for MRSA in animals, but it is recommended to send suspected isolates for verification. Intermittent surveys n 44A in pigs have revealed MRSA positive farms CHARACTERIZATION OF A CATALASE- and slaughterhouses. MRSA have also been NEGATIVE METHICILLIN-RESISTANT found from clinical and screening specimens STAPHYLOCOCCUS AUREUS ISOLATE FROM in hospitalized horses. Among cattle, MRSA A DOG have been detected from two dairy farms, both G. Ventrella1, M. Greco1, V. Martella1, A. originating from a mastitis case. Among small Parisi2, D. Buonavoglia1, M. Corrente1; companion animals, MRSA findings have been 1University of Bari, Bari, ITALY, 2Istituto sporadic including only a few isolations from Zooprofilattico della Puglia e Basilicata, cats and dogs. The aim of the present study Putignano (BA), ITALY. was to characterize the Finnish veterinary MRSA isolates and to compare the spa types Introduction: Catalase-negative staphylococci with those of human isolates. Materials and are characterised by mutation and/or deletions methods: Animal MRSA strains isolated of the katA gene that alter the functionality of between 2005 and 2013 (N=44) were spa the enzyme, and are thought to be less virulent. typed. The SCCmec type and the presence of However, catalase-negative S. aureus strains, czrC gene, conferring resistance to cadmium either resistant or susceptible to methicillin, and zinc, were determined. MLST analysis have been associated with systemic infec- was performed to selected strains. Results: tions in human patients. A dog tested positive Sixteen different spa types were found, twelve for a catalase-negative MRSA strain and a of which were common with human isolates. methicillin resistant S. pseudintermedius strain In Finnish pig farms, livestock-associated (MRSP). To our knowledge, this is the first (LA) MRSA CC398 (spa types t034, t108, report in animals on a MRSA strain with a t3933, t5103) is the most common type but catalase-negative phenotype. Methods and re- MRSA t127 (CC1) is also present. The porcine sults: Samples from a dog with a severe form MRSA isolates were mainly of SCCmec type of pododermatitis were subjected to bacterio- V. Five different spa types were found among logical analysis. Pure cultures of staphylococci equine MRSA: t026, t064, t1399 (ST125), t011 were isolated, with two distinct populations (ST398), t6867 (ST398). MRSA isolates from on Mannitol Salt Agar. The mannitol-positive horses were of SCCmec type IV or V. The two isolate (164/13a) was shown to be catalase

68 ASM Conferences Poster Abstracts negative, and identified as S. aureus by n 45A species-specific PCR. The mannitol-negative ISOLATION A NOVEL METHICLLIN isolate (164/13b) was catalase positive and it STAPHYLOCOCCUS AUREUS T11469 was identified as S. pseudintermedius with a STRAIN BELONGING TO MULTILOCUS species-specific PCR. Both strains were shown SEQUENCE TYPE STS IN CHICKEN to be methicillin resistant by PCR detection of mecA. Strain 164/13a was subsequently typed N. A. Amos1, O. Josiah2, O. Busayo2; as t002 by spa-typing and as ST5 by Multilo- 1Ebonyi State University, Abakaliki, NIGERIA, cus Sequence Typing. This isolate harboured 2Ahmadu Bello University, Zaria, NIGERIA. Staphylococcal cassette chromosome (SC- Background:In the last decade, a distinctive Cmec) type I, and tested negative using a PCR type of methicillin resistant Staphylococcus targeting Panton Valentin Leukocidin (PVL) aureus (MRSA) emerged in livestock and genes. The full-length katA gene of strain companion animals. Its isolation in chicken 164/13a was amplified as described previously has been reported in some countriesand its and the sequence was determined, revealing propensity for zoonotic transmission represents a 99.6% nt identity in the catalase gene to the potential risk for poultry farm workers and the reference strain S. aureus ATCC 12600 (ac- general population. Methodology:Nasal and cession AJ000472). A deletion at position 487 cloacae swabs of 1800 birds selected at ran- caused a frame shift and introduced a prema- dom but equally from 9 poultry farms in Eb- ture termination codon (TAA) downstream at onyi State, Nigeria were screened and analyzed position 536-538, which altered the structural using standard microbiological techniques to and functional integrity of the enzyme. Discus- determine the prevalence of MRSA amongst sion: In strain 164/13a the altered functionality chicken population in the State. Antibiotic of the catalase was related to a deletion but it resistance profiles of the isolates were deter- is not clear whether and to which extent that mined using the Kirby-Bauer disc diffusion strain played a primary role as pathogen. In (DAD) method. Molecular epidemiology of fact a MRSP was also isolated from the cutane- the isolates were determined using Polymerase ous lesions of the dog. The clonal type ST5 Chain Reaction (PCR) based techniques. All t002 has been reported in dogs in USA and in the MRSA were screened for methicillin resis- France, and also in human patients from the tance gene (mecA), Panton Valentin leukocidin same regions. SCCmecI and PVL negative determinants (lukF-lukS), a tetracycline resis- pattern are characteristic of Hospital-acquired tance gene (tetM) and spa-sequence typing of MRSA in man. Thus, a human source of that the polymorphic region of proteinA. Staphylo- strain may be hypothesized. Conclusion: The coccal Cassette Chromosome mec (SCCmec) catalase test is a rapid assay routinely em- and Multilocus sequence typing (MLST) ployed for the identification of staphylococci of the novel isolates were also carried out. in clinical microbiology laboratories. While Result: Out of 247 (13.7%) Staphylococcus providing firm evidence, for the first time, that aureusfrom 1800 chicken screened, 15 wer- catalase-negative MRSA can be harboured in eresistant to cefoxitin, representing an MRSA animals, the findings of this study may also prevalence of 0.83%. All the MRSA isolates suggest that the presence of staphylococci with were susceptible to vancomycin and carried this uncommon phenotype may be underesti- the mecA gene by multiplex PCR. Spa typing mated. Moreover, further studies are needed analysis show that 3 spa types; t002, t2304 and to assess the virulence of catalase-negative t11469 were circulating amongst poultry birds phenotypes. in Ebonyi State and all belonged to SCCmec type V. A new strain of MRSA t11469 that has not been described anywhere, to the best of our

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 69 Poster Abstracts knowledge, was isolated from poultry birds survey, four hundred and sixty-five bulk tank in Ebonyi State. Conclusion: The presence samples in England and Wales and six hundred of MRSA in this study represents a potential in Scotland were tested for mecC MRSA. Ten health risks to the poultry farm workers, their farms (2.15%) in England/Wales were positive families and the general public. The isolation for mecC MRSA but none in Scotland were of a new strain of MRSA t11469 shows the positive. Seven of these isolates belonged versatility and the changing epidemiology of to ST425 with the other three being CC130. this agent. mecC MRSA therefore appears rare among human MRSA isolates in GB at present with n 46A CC130 the predominant lineage. In bovine milk ST425 appears the predominate lineage. EPIDEMIOLOGY OF MECC MRSA IN GREAT In the UK we have also identified veteri- BRITAIN nary isolates of Staphylococcus xylosus and G. K. Paterson1, E. M. Harrison1, F. Morgan1, Staphylococcus sciuri which are mecC positive P. Sharon1, J. Parkhill2, R. Zadoks3, M. A. and have found mecC MRSA from domestic Holmes1; dog, chaffinch and common seal. The presence 1University of Cambridge, Cambridge, of mecC MRSA in other host species and the UNITED KINGDOM, 2Wellcome Trust Sanger presence of mecC in staphylococcal species Institute, Cambridge, UNITED KINGDOM, other than S. aureus needs to be considered in 3University of Glasgow, Glasgow, UNITED our understanding of mecC MRSA epidemiol- KINGDOM. ogy. Our group recently described a novel mecA homologue in MRSA from humans and n 47A dairy livestock in the United Kingdom and MSSA SPA TYPES IN US PIGS AND SWINE Denmark. This homologue, originally named VETERINARIANS CORRESPOND WITH mecALGA251 and since designated mecC, has MRSA SPA TYPES REPORTED GLOBALLY IN 69% identity to mecA at the DNA level. This SWINE divergence results in mecC MRSA producing P. R. Davies, J. Sun, L. Linhares, S. Sreevat- negative results in molecular tests to identify san, M. Yang; or confirm MRSA despite being resistant to University of Minnesota, St. Paul, MN. methicillin (oxacillin/cefoxitin). This has important implications for the correct diagnosis Since the detection of ST398 MRSA in pigs of individual patients and for the surveillance in Europe, research worldwide has identified of MRSA.To better understand the epidemi- diverse MRSA types in swine, both within the ology of mecC MRSA in Great Britain we ST398 lineage and across MLST types. Studies conducted prevalence studies among human in Asian pigs generally report a predominance MRSA isolates and bovine bulk tank milk of ST9 MRSA, and ST398 and ST5 vari- samples. In the case of human prevalence, six ants appear most common in pigs in North hospital laboratories supplied three hundred America, where ST9 MRSA have also been and fifty sequential MRSA isolates in 2011-12 found. Other MLST types detected in pigs in which were tested by PCR to determine their various countries include ST49, ST97, ST39, mec gene status. From a total of two-thousand and ST1. Despite active research on MRSA in one hundred isolates, nine were positive for livestock, there are few systematic studies of mecC (0.43%) with the remainder being mecA generic S. aureus ecology in swine herds. We positive. Eight of these mecC MRSA isolates are conducting 3 studies of S. aureus (MSSA belonged to clonal complex 130 with the ninth and MRSA) in pigs and swine veterinarians in belonging to ST425. In the bovine prevalence the USA: Study A - an intensive longitudinal

70 ASM Conferences Poster Abstracts study of 2 multiple site production systems in pigs in many countries. The apparently recent Minnesota; Study B - a cross-sectional study emergence of MRSA in swine likely reflects of 36 herds in 15 states; Study C - an 18 month sporadic acquisition of the mecA gene by longitudinal study of 67 swine veterinarians preexisting flora of pigs. in the same states. We report the distribution of spa types and MLST sequence types among n 48A 513 isolates from Study A (completed); 135 THE ASSOCIATION BETWEEN METHICILLIN- isolates from 8 farms in study B, and 254 RESISTANT STAPHYLOCOCCUS AUREUS isolates from 6 months of sampling in Study (MRSA) CARRIAGE AND THE GENE C (both ongoing). In study A, no MRSA were EXPRESSION OF GROWTH FACTORS AND detected in 384 samples from pigs (192) or the IMMUNOMODULATORY AGENTS IN PIGS environment (192) of the two systems. Among 513 S. aureus isolates, 15 spa types were M. Slifierz, A. Farzan, R. Friendship,S. detected and the most common were ST398/ Weese; t034 (36%); ST9/t337 (28%); ST9/t7331 Univ. of Guelph, Guelph, ON, CANADA. (13%);ST9/t3446 (7%); ST9/t2462 (6%) and Although very prevalent among swine herds, ST5/t002 (5%). Only 1 isolate (ST72) did not rarely caused disease in pigs. However, it is belong to ST398 (39% of isolates), ST9 (55%), unclear whether MRSA carriage elicits any or ST5 (6%). S. aureus was isolated from 65% immune response. The objective of this study of 395 veterinary samples (57% MSSA; 8% was to determine whether MRSA coloniza- MRSA) over 6 months, with 12 veterinarians tion in pigs is associated with differential (18%) consistently positive for a specific spa gene expression of C-reactive protein (CRP), type. Predominant spa types were ST398/t034 serum amyloid A (SAA), haptoglobin (Hp), (38%); ST5/t002 (18%); ST9/t337 (13%); interferon- α (IFN-α), interferon- γ (IFN-γ), other ST398 (13%); other ST5 (7%); other tumor necrosis factor- α (TNF-α), interleukin ST9 (4%). Overall, 93% of spa types from the (IL)-1β, IL-6, IL-10, and IL-18 in addition to veterinary samples belonged to ST398, ST9, several growth factors, including insulin-like or ST5. All MRSA isolates belonged to ST398 growth factor-1 (IGF-1), IGF binding protein-3 (t034, t2330, t011) or ST5 (t002, t2049). In (IGFBP-3), and growth hormone receptor 8 farms sampled in study C, ST398, ST9 and (GHR). Liver tissue, nasal swabs, and serum ST5 were the predominant lineages on all samples were collected from healthy 9-week- farms and collectively accounted for more than old pigs (n=168) from 8 commercial farrow-to- 85% of isolates (all MSSA). The absence of finish operations in southern Ontario, Canada. MRSA in all 10 swine herds tested, together Data on growth performance and herd-level with the low prevalence (8%) among swine management was also collected. Nasal swabs veterinarians supports previous data suggesting were cultured for MRSA and serum samples a relatively low prevalence of MRSA in the US were tested for porcine reproductive and swine industry. The consistent predominance respiratory syndrome virus (PRRSV) using of the ST398, ST5, and ST9 lineages across real time PCR. Reverse transcription quantita- all 3 studies is striking, and diversity of spa tive PCR (RT-qPCR) was used to quantify the types was evident within all 3 major MLST gene expression in liver of 74 samples from 4 lineages. We infer that the normal S. aureus farms. The pig-level and herd-level prevalence flora of US commercial swine, and occupation- of MRSA was 41.5% (68/164) and 75% (6/8), ally exposed veterinarians, is dominated by respectively. For PRRSV, the pig-level and diverse swine adapted MSSA largely clustered herd-level prevalence was 28.0% (46/164) and within the ST398, ST9 and ST5 lineages. The 50% (4/8), respectively. Univariable analysis same 3 lineages predominate in MRSA in pigs revealed a significant association between the globally, implying they may be widespread in

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 71 Poster Abstracts herd-level prevalence of PRRSV and MRSA number of cases started to increase during the (R2=0.635; P=0.018). MRSA colonization did upcoming years, worried dog owners contacted not affect expression of the studied cytokines, the National Veterinary Institute (SVA), Up- acute-phase proteins, and growth factors (all psala, Sweden, about MRSP. A frequently P>0.05). In contrast, PRRSV was found to be asked question was how dangerous MRSP inversely associated with IGF-1 expression were for themselves and their family members. in liver tissue (OR=0.034, P<0.001). There S. aureus and S. pseudintermedius are common was no association between MRSA carriage coagulase positive staphylococci in human and farm antibiotic use (P=0.98), with MRSA and veterinary medicine. Both are pathogens found on 3/4 antibiotic-free farms, with preva- and can cause similar types of infections. S. lences ranging from 33-71%. The association pseudintermedius belong to the normal bacte- between PRRSV and MRSA has not been rial skin flora of dogs and is common cause of previously reported. This may be the result wound and skin infections.. However, infec- of poor farm biosecurity or an indicator of tions in humans are rare but with the uncer- movement of pigs from other herds; however, tainty that these two species can be mixed up factors such as a reduction in bactericidal in routine laboratory diagnostics. There is also activity of porcine macrophages from PRRSV a probability for MRSP to be mixed up with in exposure cannot be excluded. The lack of asso- methicillin resistant S. aureus (MRSA) since ciation between MRSA and immunomodulato- the mecA gene mediating the methicillin resis- ry gene expression was expected since MRSA tance is the identical. In this study, we wanted is thought to be a relatively host-adapted to investigate if S. pseudintermedius could commensal in pigs. However, further research cause wound infection after dog bite and how could whether there are differences with dif- commonly S. pseudintermedius were misdiag- ferent MRSA strains as well as the relationship nosed as S. aureus in these cases We choose between MRSA carriage and local immunity. dog bites because of the higher likelihood of These data provide more indication that MRSA having an S. pseudintermedius involvement. carriage is not associated with a demonstrable The hypothesis was that S. pseudintermedius systemic immune response in pigs, as opposed can be misdiagnosed as S. aureus by routine to subclinical carriage of PRRSV. The associa- laboratory diagnostics in human medicine. S. tion of MRSA and PRRSV requires further aureus isolated from dogs bite wound infec- study because of the commonness of both tions in human medicine laboratories were sent pathogens, the potential influence of PRRSV to SVA for species confirmation with PCR.S. on farm MRSA status and the potential impact aureus and S. pseudintermedius were also ana- of PRRSV control on MRSA. lyzed with spa-typing. In total, 102 suspected S. aureus were sent to SVA for further analyses n 49A from 22 different laboratories in seven different counties. Of these, 13 (12.7%) were STAPHYLOCOCCUS PSEUDINTERMEDIUS misdiagnosed as S. aureus and were instead INFECTIONS AFTER DOG BITES IN HUMANS S. pseudintermedius. The S. aureus- isolate MISDIAGNOSED AS STAPHYLOCOCCUS belonged to 58 different spa-types where one AUREUS isolate represented one spa-type besides nine U. G. Andersson, S. Börjesson; isolates that all belonged to t015. They were National Veterinary Institute, Uppsala, SWE- isolated in different counties. Spa-typning was DEN. not an useful tool for analyzing relationships between the S. pseudintermedius isolates. This After the first isolates of methicillin resistant study showed that S. pseudintermedius can Staphylococcus (S.) pseudintermedius (MRSP) cause wound infections after dog bites and that were confirmed in Sweden in 2006 and the

72 ASM Conferences Poster Abstracts they can be misdiagnosed as S. aureus. There were screened for the presence of 10 pyrogenic are therefore needs for better species character- toxin genes. Nine of the 27 (90%) (27/30) ization and methods for bacterial relationship MRSA harbored 1 to 5 toxin genes. One organ- to improve epidemiological tracing and treat- ism (ST537, t437) possessed five genes sed ment together with communication between + seg + sei + sea + sej; the most predominant human and veterinary medicine. toxin genes are seg + sei (20%) (egc cluster). Toxic shock syndrome toxin genes (tsst-1) n 50A were found in two (2/30) (6.67%) MRSA and one MRS isolate (1/30) (3.33%). No toxin MOLECULAR CHARACTERIZATION genes found in three cattle isolates. This study OF METHICILLIN RESISTANT highlighted that food animals could serve as a STAPHYLOCOCCUS AUREUS ISOLATED vehicle for the transfer and disseminations of FROM CHICKENS AND CATTLE IN MALAYSIA antibiotic resistant bacteria with enterotoxi- Z. Zakaria, A. Magaji, S. Abdul Aziz, J. Abu, Y. genic potential to the public thereby making Goh, S. Radu; clinical treatment difficult and expensive. Universiti Putra Malaysia, Serdang, MALAY- SIA. n 51A Methicillin resistant Staphylococcus aureus COPS OTHER THAN S. AUREUS IN (MRSA) was recently reported from a diverse CLINICAL MICROBIOLOGY LAB: COULD group of food producing animals. The aim NEW TECHNOLOGIES PROVIDE USEFUL of this study was to investigate the genetic INFORMATIONS? background of thirty one MRSA isolates using E. Carretto1, D. Barbarini2, F. Brovarone1, R. different types of typing methods pulse field Varini1, V. Savini3; gel electrophoresis (PFGE), for multilocus 1IRCCS Arcispedale Santa Maria Nuova, sequence typing (MLST) and spa typing were Reggio Emilia, ITALY, 2Fondazione IRCCS carried out on twelve isolates. Pyrogenic toxin Policlinico San Matteo, Pavia, ITALY, 3Santo genes screening was carried out on 27 MRSA Spirito Hospital, Pescara, ITALY. and three methicillin resistant staphylococci. MLST characterized 12 MRSA isolates into Background: Coagulase positive staphylo- 11 sequence types, namely ST9, ST15, ST14, cocci (CoPS) different from Staphylococcus ST537, ST190, ST194, ST795, and ST1279 aureus (SA) have gained importance not only from chickens while ST59, ST35 and ST573 in veterinary medicine, but also in medical from cattle. These 12 isolates were grouped microbiology. Among the microorganisms into five spa types’ t437, t442, t360, t189 and belonging to the Staphylococcus intermedius t5696. The analysis of PFGE macrorestriction group (SIG), S. intermedius (SI) and S. pseud- patterns percentage of similarity identified intermedius (SP) are sometimes described as from the dendogram at 80% similarity coeffi- cause of diseases in humans. With standard cient was used to define pulsotypes. The PFGE clinical microbiology techniques, it is very analysis identified 22 pulsotypes with nine sub difficult to correctly identify these isolates types and the most common cluster is C which at species level; only molecular techniques appeared to be present in four farms. Cluster B allow to do that. Moreover, different reports was similar albeit having different spa types. emphasized that methicillin-resistant (MR) SP Diversity ensued among the isolates from isolates, if evaluated according to the CLSI/ chickens due to occurrence of more than two EUCAST criteria for SA, are susceptible to pulsotypes,’ no genetic diversity was observed cefoxitin but resistant to oxacillin (cefoxitin among the cattle isolates. Thirty staphylococ- is widely used in clinical microbiology as cal isolates (including 27 MRSA and 3 MRS) a marker of methicillin-resistance). Aim of

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 73 Poster Abstracts this study was to evaluate if two molecular Background: Staphylococcus schleiferi is a techniques that are used directly on positive Gram positive, coagulase variable bacterium blood cultures, the Staphylococcus QuickFISH that has been isolated from the skin, ears, and test (QF - AdvanDx, USA - an in situ fluo- nasal mucosa of healthy dogs and from the rescence), and Gene Xpert MRSA/SA Blood pre-axillary skin of healthy humans. In humans Culture (Cepheid, USA), a real time PCR that it has been shown to be the causative agent in is able to identify SA and to detect MR strains, a variety of infections, including prosthetic can provide information useful to discrimi- infections, wound infections, bacteremia, and nate among CoPS. Study Design: The ATCC endocarditis. In dogs it has been isolated from strains of S. delphini and of SI and 8 strains of lesions of pyoderma, otitis externa and media. SP (7 MRSP, one methicillin susceptible), pre- Reports of infection caused by S. schleiferi viously identified through molecular methods, are increasing in both human and veterinary were analyzed using QF and Xpert. Results: medicine, but the true incidence may still be All the strains analyzed through QF gave a red unknown due to the potential for misidentifica- fluorescence that is characteristic, for this test, tion of S. schleiferi as S. aureus due to their for coagulase negative staphylococci (CoNS). very similar biochemical profiles. Multiple Xpert technology identified the isolates as dif- studies have determined the prevalence of ferent from SA. It was able to detect the mecA methicillin resistance amongst S. schleiferi gene for all the 7 MRSP but their SCCmec isolates to be between 40-60%. While many of regions were not amplified.Discussion: When these isolates are susceptible to other classes of automated instruments identify CoPS as SI, it drugs multi-drug resistance strains have begun is very difficult for the clinical microbiologists to emerge. Aims: To determine the relatedness to confirm this classification. Our experience, of S. schleiferi isolates obtained from dogs even if based on a limited number of strains, from across the United States. Methods: Four demonstrates that QF and Xpert technologies hundred canine isolates that were determined could provide ambiguous results if used di- to be S. schleiferi on the basis of biochemical rectly on clinical samples (it is possible to sup- testing were used in this study. Isolates were pose the presence of CoNS in the specimens). obtained from clinical samples that were col- However, if they are used on cultured strains lected by Dermatologists in the United States that have been known to be coagulase posi- and submitted for routine diagnostic purposes. tive, they are both able to exclude SA. Xpert Isolates were frozen in Microbank Tubes prior seems also to be able to detect the mecA gene to analysis. All isolates were tested with the in MRSP, solving the problem of the cefoxitin multiplex PCR assay described by Sasaki et susceptibility if SA breakpoints are used. Both al (2010) to confirm the phenotypic identi- techniques, if available in clinical labs, could fication. Phenotypic methicillin resistance provide useful information for a reliable SIG was confirmed using the Oxoid PBP2a latex identification. agglutination test. Pulsed-field gel electrophoresis was performed with SmaI according n 52A to the CDC Pulse Net protocol for molecular typing of S. aureus. BioNumerics software was EPIDEMIOLOGY OF STAPHYLOCOCCUS used to determine pulsed-field profiles based SCHLEIFERI ISOLATES FROM DOGS IN THE on Dice coefficients of similarity. A similar- UNITED STATES (2003-2013) ity coefficient of 80% was selected to define S. C. Rankin, S. D. Cole, K. O’Shea, D. O. pulsed-field profile clonal clusters.Results: Morris, C. Cain; Three clonal clusters were identified that Univ.ofPennsylvaniaSch.ofVet.Med., Philadel- comprised the majority of isolates. Isolates phia, PA. in clonal clusters 1 and 2 were predominantly methicillin resistant but clonal cluster

74 ASM Conferences Poster Abstracts

3 comprised isolates that were predominantly NaCl overnight at 37°C, followed by plating methicillin sensitive. The rate of methicillin re- out on MRSA Brilliance agar and incubation sistance was 64% overall. Conclusions: As the at 37°C. Presumptive MRSP isolates were incidence of infections caused by S. schleiferi subjected to PCR for species identification and increases in the veterinary and medical fields PCR for mecA detection. All MRSP isolated an understanding of the population structure is from healthy dogs were subjected to multi- important. The data presented here is the most locus-sequence typing (MLST) based on a expansive performed on canine isolates of S. seven-locus technique. MRSP was recovered schleiferi to date. Methicillin resistant isolates from five dogs (2.6 %), sampled at three differ- were recovered from dogs across the United ent small animal clinics located in geographi- States and these data suggest the emergence cally distinct areas. MLST showed that the and spread of at least two successful multidrug isolates belonged to three different STs: ST89 resistant S. schleiferi clones. (one isolate), ST71 (two isolates) and the novel ST252 (two isolates). MLST of clinical MRSP n 53B isolates (n=35) isolated in our diagnostic laboratory has shown that MRSP ST89 is most METHICILLIN RESISTANT frequent followed by ST71; the same STs as STAPHYLOCOCCUS PSEUDINTERMEDIUS detected among three of five MRSP carrier FROM HEALTHY DOGS ATTENDING SMALL isolates. Our findings showed that MRSP could ANIMAL CLINICS IN NORWAY be isolated from healthy dogs, but the carrier E. E. Kjellman1, J. S. Slettemeås2, H. Small1, rate was low. The MRSP population, including M. Sunde2; carrier and clinical isolates, contained MRSP 1Follo smådyrklinikk, Ski, NORWAY, 2Norwe- belonging to several different STs. The most gian Veterinary Institute, Oslo, NORWAY. prevalent STs of clinical MRSP were also represented among the MRSP isolates recovered Methicillin resistant Staphylcoccus pseudinter- from healthy carriers. Risk factors for MRSP mdius (MRSP) is of growing concern in small colonization and possible ways of eradication animal veterinary medicine. In Norway the of MRSP in carriage in dogs should be further first MRSP was detected in 2008 and since that investigated. time the number of MRSP infections in dogs has increased steadily. The current knowledge about MRSP carrier prevalence among healthy n 54B dogs is limited. In this study we wanted to A CASE REPORT OF A CANINE LONG-TERM investigate the occurrence of MRSP among PATIENT SUFFERING FROM RECURRENT healthy dogs without infections and with no re- INFECTIONS DUE TO INDISTINGUISHABLE cent history of antimicrobial therapy. Detected METHICILLIN RESISTANT AND MRSP isolates were genotyped and compared -SUSCEPTIBLE S. PSEUDINTERMEDIUS with clinical MRSP isolates. A total of 189 GENOTYPES dogs, attending ten small animal clinics in Oslo S. Vincze1, B. Walther1, L. H. Wieler1, B. and the surrounding counties during the period Kohn2, L. Brunnberg2, A. Lübke-Becker1; February to April 2013, were included in the 1Institute of Microbiology and Epizootics, Cen- study. The clinics were invited to participate in tre for Infection Medicine FU-Berlin, Berlin, advance and instructions on how to sample and GERMANY, 2Small Animals Clinic, FU-Berlin, include dogs for the study was given. The dogs Berlin, GERMANY. were sampled at two body sites by swabbing: the mouth and perineal region. Detection of Introduction: The skin and mucosa of dogs is MRSP in the laboratory included one enrich- the natural habitat of S. pseudintermedius and ment step in Mueller Hinton broth with 6.5% individuals may carry one or more different strains over a long time. As an opportunistic 3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 75 Poster Abstracts pathogen, S. pseudintermedius is an important patient seems to be a reasonable explanation. cause of purulent infections in dogs, with skin, Further investigation including whole genome ear and soft tissue as main infection sites. sequencing is needed to reveal critical genomic Infections with methicillin resistant S. pseudin- alterations among the strains reported on here. termedius (MRSP), which have been increasingly reported during the last years, are of n 55B particular concern. This case report describes METHICILLIN-RESISTANT recurrent infections of a canine patient with STAPHYLOCOCCUS PSEUDINTERMEDIUS two distinct S. pseudintermedius-lineages over ISOLATION FROM COLOSTRUM AND MILK a five year period.Material and Methods: OF BITCHES IN A BREEDING KENNEL In total, 21 S. pseudintermedius-isolates from wound infection swabs of the canine patient A. Rota1, M. Corrò2; were sampled between August 2008 and April 1Dipartimento Scienze Veterinarie- University 2012. Species identity was confirmed by of Torino, Torino, ITALY, 2Istituto Zoopro- polymerase chain reaction-restriction length filattico Sperimentale delle Venezie, Padova, polymorphism (PCR-RFLP) and methicillin ITALY. resistance was confirmed by PCR-detection Dogs are natural hosts of Staphylococcus of the mecA-gene. The clonal relationship of pseudintermedius and methicillin-resistant all strains was investigated by pulsed field gel strains (MRSP) have been isolated with electrophoresis (PFGE) using BioNumerics increasing frequency; antimicrobials use is a 6.5 (Applied Maths, Sint-Martens-Latem, Bel- known predisposing factor for the selection of gium). Results: Investigation of wound swabs resistant strains. This study is part of a larger from different time points during five years investigation on subclinical mastitis in the revealed twelve MRSP- and six MSSP-positive bitch and is focused on the pattern of isolation cultures. Both, MRSP and MSSP were detected of MRS strains from colostrum and milk of simultaneously twice. PFGE-analysis con- postpartum bitches in a breeding kennel, also firmed the occurrence of two distinct patterns. in relation to antimicrobials use. The study While all MRSP showed identical profiles (pat- included 12 Staffordshire bull terrier/American tern A), MSSP clustered in pattern B, including Staffordshire terrier bitches, aged 2 to 5 years, five undistinguishable strains (subtype B1) belonging to a breeding kennel in Northern and a single isolate belonging to subtype B2. Italy. The incidence of Cesarean deliveries MSSP-infections occurred initially and recur- is rather high in the kennel, especially on ring infections with this strain were detected at Staffordshire terrier bitches, and antimicro- least once a year. Between each time period of bials (either amoxicillin/clavulanic acid or MSSP-detection, several infections accounted cephazolin) are administered in the following for MRSP. Discussion: This case report 5-7 days. In our study, six bitches underwent describes a canine patient with recurring S. Cesarean section and were treated with anti- pseudintermedius-infections during a five year microbials; six bitches had a natural delivery, period, mediated by strains belonging to two but the same agents were administered to two distinct PFGE-types. The alternation between of them, because of endometritis risk and of MRSP and MSSP infections associated with generalized dermatitis, respectively. On day 1, two distinct genetic lineages (PFGE-A and 7 and 15 postpartum, drops of secretion were -B) over a long period points towards several collected from the last abdominal mammary independent re-infection processes with either glands. Coagulase-positive staphylococci were MRSP or MSSP in this patient. Both lineages identified using conventional biochemical seem to be potent pathogens for wound infec- analysis and MALDI-TOF MS; antimicrobials tions in dogs. Recurrent auto-infection or/and susceptibility and presence of mecA gene were a re-infection source in the proximity of this

76 ASM Conferences Poster Abstracts also tested. Methicillin-resistant staphylococci comial infections in human and veterinary hos- were isolated from 6 out of 12 bitches, and pitals. Since 2010 an infection control program all the strains resulted mecA positive. Two at the Atlantic Veterinary College Veterinary MRS species were identified:Staphylococ - Teaching Hospital (AVC-VTH) has focused its cus pseudintermedius and Staphylococcus efforts on reducing transmission and persis- sciuri. Methicillin resistant S.sciuri was tence of MR Staphylococcus pseudintermedius isolated from a single bitch, in the second and (MRSP) in patients and the hospital environ- third samples, together with MRSP. MRSP ment. The objectives of this study were (1) to colonization was highest at D7 postpartum and determine if MRSP isolates recovered from involved 5/6 bitches under antibiotic treat- hospital environments are clonal to isolates ment, while MRSP was isolated from a single recovered from patients associated with that bitch at D1. At D15 only 1 bitch out of 4 still area, and (2) if epidemiologically related hosted MRSP. Among non-antibiotic-treated patients with MRSP have clonal strains. It was bitches, MRSP strains were isolated at every hypothesized that hospital and patient strains sampling time from a single animal, that had and isolates from epidemiologically associ- been brought in the kennel only for parturi- ated patients would be clonal. Environmental tion. Notwithstanding the limited number of specimens were collected using an electrostatic animals, some interesting observations can be cloth. Culture of cloths was accomplished by made. The presence of MRSP strains appears using an enrichment broth, followed by routine associated with antimicrobials use and it plating methodology. Patient and environmen- looks sporadic, not persisting overtime in the tal isolates were identified by colony morphol- majority of cases. The rather high frequency ogy, coagulase production, and resistance to of Cesarean deliveries in this kennel, and oxacillin. Methicillin-resistance was confirmed the following antimicrobials administration, by penicillin-binding protein 2a (PBP2’) latex induced the selection of methicillin-resistant agglutination. Species-level identification was strains and then the diffusion of MRSP appears achieved by using a multiplex PCR assay for rather high. The presence of MRSP did not af- coagulase-positive staphylococci. MR isolates fect neonatal health and survival, and neonatal were typed by sequence analysis of the mec- mortality rate was low, especially in the natu- associated direct repeat units (dru typing.) rally delivered puppies (2.2% versus 11.1%). Epidemiologically related isolates of the same Genetical characterization of the MRSP strains dru type were compared using pulsed-field gel is under way, in order to assess whether they electrophoresis (PFGE). Preliminary analysis belong to the major clone lineage dominating of isolates from 2010 to date indicated that in Europe, the ST71-J-t02-II-III. 35 patients were positive for MRSP at the AVC-VTH and MRSP was isolated from 9 en- n 56B vironmental samples. Molecular work has been completed for all isolates from 2010 to 2012. METHICILLIN-RESISTANT All isolates were confirmed to be S. pseudin- STAPHYLOCOCCUS PSEUDINTERMEDIUS termedius by PCR. Typing results have shown IN A VETERINARY TEACHING HOSPITAL IN that strains isolated from VTH areas were the ATLANTIC CANADA. same dru type as temporally associated patient M. Saab, J. Lofstedt, J. T. McClure; strains. PFGE provided further evidence to Department of Health Management, Atlantic suggest that patients were contaminating Veterinary College, University of Prince Ed- the hospital environment. There were two ward Island, Charlottetown, PE, CANADA. instances where hospital-acquired infections were suspected. In one instance, the dru types Methicillin-resistant (MR) staphylococci have of the isolates were the same. PFGE analysis been implicated as an important cause of noso- of these isolates revealed clonal strains, further

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 77 Poster Abstracts suggesting hospital-acquired transmission. (MRSP and MSSP), methicillin resistant coag- Results from this study will provide support ulase negative or other staphylococci (MRS), for the development and application of policies Escherichia coli and Enterococcus spp. MRSP in veterinary infection control programs. screening specimens were taken from the perianal, nasal and oral mucosa of 106 dogs. n 57B The CB and TSB were inoculated in parallel. After incubation all TSB and positive CB tubes THE SUITABILITY OF A COMMERCIAL (=yellow/orange color) were plated onto selec- CONTRAST BROTH MEDIA FOR tive media (MRSA Select, Bio Rad, USA). DETECTION OF METHICILLIN RESISTANT Identification of suspected MRSP-isolates was STAPHYLOCOCCUS PSEUDINTERMEDIUS done by phenotyping, oxacillin resistance and IN DOGS mecA detection. Results: All tested MRSP T. Grönthal1, H. Piiparinen1, S. Nykäsenoja2, (n=36) and MRSA-strains (n=6) produced a M. Rantala1; positive color reaction in CB. E. coli (n=1), 1University of Helsinki, Faculty of Veterinary MRS (n=5) and MSSP (n=9) isolates were Medicine, Department of Equine and Small negative (=red color). However, all tested Animal Medicine, Central laboratory, Helsinki, enterococci (n=6) and MSSA-isolates (n=6) FINLAND, 2Finnish Food Safety Authority caused a false positive color reaction. 22 of the Evira, Food and Feed Microbiology Research 106 (21%) patient specimens were found to Unit, Helsinki, FINLAND. be MRSP-positive by the TSB-method, while 16 (15%) were positive with the CB-method. Background: A number of different enrich- In 3 cases the CB-method revealed MRSP ment broths exist to increase the sensitivity of when the TSB-method did not. The sensitivity screening of methicillin resistant Staphylococ- and specificity of the CB was 59% and 96%, cus aureus (MRSA) in humans, but very little respectively. The positive predictive value was data is available about their usefulness for 81%, while the negative predictive value was screening of methicillin resistant Staphylococ- 90%. Conclusions: We cannot recommend the cus pseudintermedius (MRSP) in dogs. Some studied CB media for MRSP screening in dogs broths, as the one studied here, contain selec- due to its low sensitivity. tive agents that suppress growth of other bacteria, and an indicator that changes the color of the media when the target bacterium ferments n 58B sugars. This allows for a more rapid discrimi- COMPARISON OF OXACILLIN, CEFOXITIN nation between negative and suspected positive AND CEFPODOXIME DISK DIFFUSION samples. Aims and objectives: The purpose of SCREENING TESTS FOR DETECTION OF this study was to investigate the suitability of MEC A GENE-MEDIATED METHICILLIN a commercial contrast enrichment broth (CB) RESISTANCE IN STAPHYLOCOCCUS (Contrast MRSA Broth, Oxoid, UK) to detect PSEUDINTERMEDIUS ISOLATES FROM MRSP in screening specimens from dogs by DOGS using a trypticase soy broth (TSB) with 6.5% J. B. Everett, A. Odoi, S. A. Kania, D. A. NaCl (MRSA broth, Tammer-Tutkan Maljat Bemis; Oy, Finland) as the reference. If the CB proved Department of Biomedical and Diagnostic efficient it would reduce the reply time of Sciences, University of Tennessee College of negative samples, and decrease the amount of Veterinary Medicine, Knoxville, TN. samples requiring further testing. Methods: The functionality of the CB was tested by a The question posed in this study was: How set of bacterial isolates including S. aureus do oxacillin, cefoxitin and cefpodoxime disk (MRSA and MSSA), S. pseudintermedius diffusion susceptibility test results compare

78 ASM Conferences Poster Abstracts when used singly or in combination to detect new information regarding the correlation of methicillin resistance in Staphylococcus cefpodoxime disk diffusion test results with pseudintermedius? A retrospective analysis mec A gene detection. was performed on cumulative data from 1841 isolates of Staphylococcus pseudintermedius n 59B from dogs. Each isolate was from specimens MIC VALUES OF SELECTED ANTIBIOTICS submitted to the Clinical Bacteriology and FOR MRSP STRAINS ISOLATED FROM Mycology Laboratory at the University of DOGS IN POLAND Tennessee College of Veterinary Medicine (Tennessee, USA) from 2006 to 2012. Multiple D. Chrobak1, M. Kizerwetter-Świda1, M. Rze- isolates from a single patient were possible; wuska1, A. Moodley2, M. Binek1; however, each isolate was unique with respect 1Warsaw University of Life Sciences, Warsaw, to year isolated, body site from which it was POLAND, 2University of Copenhagen, Copen- isolated and antimicrobial susceptibility hagen, DENMARK. profile. Species identification was made by Introduction: Infections caused by methicil- signature biochemical profile and/or MboI lin resistant Staphylococcus pseudintermedius restriction endonuclease fragment profile of (MRSP) in small animal practice has been a polymerase chain reaction (PCR)-amplified observed with increasing frequency. Most locus of the phosphate-acetyltransferase (pta) MRSP strins are multidrug resistant, including gene. Disk diffusion susceptibility tests were resistance to fluoroquinolones, macrolides, performed with oxacillin (1 µg disk), cefoxitin tetracyclines, which are commonly used to (30 µg disk) and cefpodoxime (10 µg disk) ac- treat S. pseudintermedius infections. Veterinar- cording to Clinical and Laboratory Standards ians are limited to a few effective antibiotics Institute (CLSI) standard reference methods. that require careful selection based on the Diameters of the zones of growth inhibition results of antimicrobial susceptibility testing were recorded. PCR assays for detection of the making prudent use of “last resort” antibiotics mec A gene, performed by either conventional for human treatment. Objective: The objective or real time methods, were recorded as positive of this study was to determine the MIC values or negative. Receiver-operator- characteristic for randomly selected 16 MRSP isolated from analysis, was performed using commercial clinical infections in Poland against 12 antimi- software. Epidemiological cutoff values crobials that are widely used in small animals. (ECVs), that optimized both sensitivity and Methods: Sixteen clinical MRSP of canine specificity, were 16, 29 and 21 for oxacillin, origin isolated at the Diagnostic Laboratory cefoxitin and cefpodoxime, respectively. Error of the Division of Microbiology, Faculty of rates obtained when using these ECVs were Veterinary Medicine at the Warsaw University unacceptable. When adjusted to ensure very of Life Sciences were examined. Methicillin major error rates of < 2%, ECVs of 19 for resistance was confirmed by the detection of oxacillin, 33 for cefoxitin and 26 for cefpo- mecA. Additionally, all strains were SCCmec doxime were recommended. However, total typed. E-test® strips (bioMerieux) were used error rates exceeded 10% and will require for determining MIC values of enrofloxacin, further attention in the future. Senstitvities ciprofloxacin, chloramphenicol, mupirocin and specificities of multiple disk diffusion linezolid, clindamycin, kanamycin, amika- tests analyzed in series or in parallel were not cin, doxycycline, vancomycin, rifampicin significantly greater than those observed for and gentamicin according to the procedures the single disk assays. The results of this study, defined by the CLSI and Eucast.Results: The in general, support previously recommended predominant SCCmec type was II-III (n=15). ECVs for oxacillin and cefoxitin and provide

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 79 Poster Abstracts

One strain carried a novel SCCmec; mec A, ccr objective of this study was to study diversity C. This mecA-positive isolate was susceptible of clinical MRSP isolates obtained from dogs, to all tested antibiotics with the exception of including five dogs sampled on multiple oc- kanamycin. All strains were susceptible to mu- casions, in Denmark over a five-year period. pirocin, linezolid, amikacin and vancomycin. Methods: A total of 38 MRSP isolates ob- Resistance to rifampicin and chloramphenicol tained from clinical specimens of 29 dogs be- was observed in one (MIC = >32 µg/ml) and tween 2008 and 2013 were tested for resistance two (MICs = 32 µg/ml and 64 µg/ml) strains, to 22 antibiotics using broth microdilution, and respectively. Most of the MRSP were resistant were subjected to multilocus-sequence typing to 6 out of 12 tested antibiotics: kanamycin (MLST) and SCCmec typing. Results: Sixteen

MIC90 >256 µg/ml (n=16, 100%); enrofloxacin sequence types (STs) were identified. The most

MIC90 >32 µg/ml (n=15, 93%); ciprofloxacin common STs were ST71 (n=11 in 11 dogs),

MIC90 >32 µg/ml (n=15, 93%); clindamycin ST258 (n=7 in 2 dogs), ST45 (n=2 in 2 dogs),

MIC90 >256 µg/ml (n=15, 93%); gentamicin ST269 (n=2 in 2 dogs), and ST273 (n=2 in 1

MIC90 >192 µg/ml (n=15, 93%), and doxycy- dog). ST118, ST265, ST267, ST268, ST270, cline MIC90 12 µg/ml (n=13, 81%). Discussion ST271, ST272, and five of six new STs were and conclusion: Our results demonstrate the all detected once. The isolates harbored SCC- high-level of resistance to commonly used mec types IV (n=17), II-III (n=11), V (n=3) or antibiotics among MRSP and complicating non-typeable (n=7). Isolates belonging to ST71 current empiric treatment strategies of S. and the related ST270 and ST272 displayed the pseudintermedius infections. Treatment should most resistant phenotypes (resistant to 20 of be based on antibiotic resistance profiles. 22 antibiotics). Four out of the five dogs, from In our study, susceptibility to: mupirocin, which MRSP were repeatedly isolated, har- linezolid, vancomycin and rifampicin was bored two distinct strains. Conclusions: The observed but these antibiotics are not licensed European MRSP clone ST71-SCCmec II-III is for use in veterinary medicine. Chlorampheni- the most common lineage in Denmark. How- col and amikacin look promising but they are ever, the overall genotypic MRSP diversity is not widely available in veterinary hospitals in much higher than reported in other countries. Poland, and they have serious side effects. Our results indicate multiple introductions of distinct MRSP strains into Denmark. SCCmec n 60B type IV was associated with 9 STs, suggesting enhanced mobility of this genetic element HIGH GENETIC DIVERSITY across distinct S. pseudintermedius lineages. AMONG METHICILLIN-RESISTANT The isolation from four dogs of distinct STs STAPHYLOCOCCUS PSEUDINTERMEDIUS on different sampling events suggests repeated ISOLATED FROM CANINE INFECTIONS IN exposure and that some patients may DENMARK be predisposed to MRSP acquisition. Alterna- P. Damborg, A. Moodley, L. Guardabassi; tively, since the isolates obtained from these University of Copenhagen, Frederiksberg, dogs were single locus variants of the same ST, DENMARK. this finding may indicate short-term genetic evolution of MRSP strains within individual Background: Methicillin-resistant Staphy- patients. lococcus pseudintermedius (MRSP) have emerged globally in companion animals in recent years. In Denmark, MRSP constitute approximately 2% of S. pseudintermedius obtained from clinical specimens of dogs. The

80 ASM Conferences Poster Abstracts n 61B types were identified amongst the MRSP, with CHARACTERIZATION OF CLINICAL 27/41 isolates having spa type t02 associated CANINE METHICILLIN-RESISTANT with SCCmec II-III. Conversely, MSSP were AND METHICILLIN-SUSCEPTIBLE diverse with 19 different spa types. PFGE on STAPHYLOCOCCUS PSEUDINTERMEDIUS IN MRSP isolates showed the presence of two FRANCE main clusters: cluster A (n=34) associated with ST71, and cluster B (n=7) associated with non- M. Haenni1, N. Alves de Moraes1, P. Châtre1, ST71. Conclusion: This study confirms the C. Médaille2, A. Moodley3, J. Madec1; presence of the common multi-resistant Euro- 1ANSES, Lyon, FRANCE, 2Vebio, Arcueil, pean MRSP ST71 clone causing infections in FRANCE, 3Department of Veterinary Disease French dogs. Also of interest is the multi-resis- Biology, Faculty of Health and Medical Sci- tant phenotypes observed amongst genetically ences, University of Copenhagen, Copenha- diverse MSSP clinical isolates. Together, these gen, DENMARK. data support the continuous surveillance of both MSSP and MRSP, as well as the develop- Introduction: S. pseudintermedius is an ment of new therapeutic alternatives. opportunistic pathogen in dogs, and was originally susceptible to most antibiotics available for treatment. Since 2006, methicillin-resistant n 62B S. pseudintermedius (MRSP) has emerged as VIRULENCE FACTORS, AGR GROUPS an important pathogen resistant to nearly all (ALLELES), BIOFILM-FORMING classes of antibiotics. Here, we conducted a ABILITY IN METHICILLIN-RESISTANCE one-year survey in order to gain insight into AND METHICILLIN-SUSCEPTIBLE the antimicrobial susceptibility patterns and STAPHYLOCOCCUS PSEUDINTERMEDIUS genetic diversity of MRSP and methicillin-sus- N. Couto, A. Belas, R. Seixas, M. Oliveira, C. ceptible S. pseudintermedius (MSSP) clinical Pomba; isolates in France. Materials and Methods: A Faculdade de Medicina Veterinária, Universi- total of 268 non-replicate coagulase-positive dade de Lisboa, Lisboa, PORTUGAL. staphylococci were collected from diseased dogs at a veterinary referee clinic from January Staphylococcus pseudintermedius is the most to December 2010. Species were identified common opportunistic pathogen in dogs. Sev- by kat PCR-RFLP. Antimicrobial susceptibil- eral virulence factors have been described in ity testing was performed by disc diffusion S. pseudintermedius, however little is known and methicillin resistance was confirmed about its epidemiology. The aim of this study by a mecA-specific PCR. Genetic diversity was to investigate the diversity of virulence was assessed by spa typing on all isolates, factors, agr groups and the biofilm-forming plus SCCmec typing, PFGE and MLST on ability among 20 methicillin-resistant and 20 MRSP isolates. Results: S. pseudintermedius methicillin-susceptible Staphylococcus pseud- was identified in 91% of isolates, and 41 S. intermedius (MRSP and MSSP, respectively). pseudintermedius were further confirmed to be Isolates were typed by MLST. The agr group MRSP. MSSP mainly presented resistances to and the presence of 18 virulence genes were penicillin (71%), tetracycline (52%), kana- analysed by PCR, and the biofilm-forming mycin (44%) and macrolides (38%), whereas ability was evaluated by Congo red agar MRSP presented numerous co-resistances (CRA) plates and by the capacity to adhere to to macrolides, lincomsamides, aminoglyco- polystyrene microtitre plates using brain heart sides, fluoroquinolones and tetracyclines. No infusion broth (BHIB), BHIB+1% glucose and resistance was observed to pristinamycin, BHIB+4% NaCl as the growth mediums. The glycopeptides and florfenicol. Only a few spa- isolates were relatively unevenly distributed

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 81 Poster Abstracts among the four agr groups, but agr group III Staphylococcus pseudintermedius (Bannoehr was significantly associated with MRSP strains et al. 2011). We hypothesized that carriage (p=0.014). All MRSP clonal complex (CC) of the genes that encode these four proteins 71 (n=17) isolates belonged to agr group III. would vary between isolates from patients All the MSSP strains belonged to different with clinical staphylococcal infections and sequence types. Five virulence genes were those without clinical infections. Between detected among all strains, but two genes September 22, 2010 and February 15, 2012 only appeared in MSSP isolates. spsO gene we conducted a study to determine prevalence was found significantly more associated with of methicillin-resistant Staphylococcus spp. MSSP than with MRSP (p=0.04). Using CRA in dogs that presented for orthopedic evalua- 14 strains (35%) were identified as biofilm- tion to our hospital. A total of 188 isolates of producers. All 40 strains produced biofilm in S. pseudintermedius were recovered from the BHIB+4% NaCl. Two and nine strains did nares (n = 82) and perineum (n = 106) of 133 not produce biofilm on BHIB and BHIB+1% dogs. Of these isolates, 14 (7%; 14/188) were glucose, respectively. Biofilm production in methicillin-resistant of which 8 were recovered the BHIB and BHIB+1% glucose media was from the perineum and 6 from the nares from significantly higher in MSSP than in MRSP 10 dogs. Three of these 10 dogs carried meth- isolates (p=0.03 and p=0.02, respectively). icillin-resistant S. pseudintermedius (MRSP) There was a high association between results at both the nares and perineum. Of the 188 of the biofilm-producing ability on polystyrene isolates, 62% (117/188) did not carry spsF, using BHIB+4% NaCl and the presence of ica spsO, spsP, or spsQ. Carriage of these genes operon. The high frequency of virulence genes was 20% (37/188) for spsF; 12% (22/188) among the S. pseudintermedius strains ex- for spsO; 10% (19/188) for spsP, and 14% plains the high adaptation of these bacteria to (26/188) for spsQ. At the same time as this its host. Furthermore the association between prevalence study, we collected a convenience CC71, agr allele III and certain virulence fac- sample of each isolate of Staphylococcus spp. tors found in this study may explain how this from the infection site in canine patients with clone expanded so rapidly over the past few clinical infections. A total of 193 isolates of years. The dissemination of multidrug resistant S. pseudintermedius were collected from 164 clones and highly virulent strains may be a dogs. Of the isolates from clinical infections serious problem in public health. Understand- 55 (28%; 55/193) were MRSP. Of the 193 iso- ing the biological basis of S. pseudintermedius lates, 65% (125/193) did not carry spsF, spsO, virulence might allow veterinarians to more spsP, or spsQ. Carriage of these genes was successfully treat and prevent MRSP infec- 18% (34/193) for spsF; 7% (14/193) for spsO; tions. 24% (46/193) for spsP, and 24% (47/188) for spsQ. There were only 2 (4%; 2/55) MRSP iso- n 63B lates that carried spsO as compared to 12 (9%; 12/55) methicillin-susceptible isolates. There CARRIAGE OF CELL WALL-ASSOCIATED were 24% (46/193) carrying spsP and 24% PROTEINS IN STAPHYLOCOCCUS (47/193) carrying spsQ. These data suggest PSEUDINTERMEDIUS ISOLATES FROM that there were differences in carriage of spsO, DOGS. spsP, and spsQ between S. pseudintermedius R. M. Gold, C. Wolff, S. Kerwin, N. D. Cohen, isolates from patients with clinical infections S. D. Lawhon; and those without. The primary limitations of Texas A&M University, College Station, TX. this study were that the isolates from patients with clinical infections were a convenience Recent work demonstrated that 4 cell wall- sample and the demographics of these animals associated (CWA) proteins were variable in may differ from those of patients presented for

82 ASM Conferences Poster Abstracts orthopedic evaluation. Further study is needed sistant and methicillin-susceptible S. pseudin- to resolve these differences. Following these termedius (MSSP) strains were compared with studies, we retrospectively analyzed a his- special focus on the epidemic lineage ST71. torical collection of 199 S. pseudintermedius Methods: Corneocytes were collected from isolates collected between 2007 and September five dogs and six humans by applying 25-mm 22, 2010 and found that 37% (74/199) did not diameter adhesive discs on the donor skin. The carry spsF, spsO, spsP, or spsQ while 44% adherence of two MSSP isolated from dogs (88/199) carried spsF, 11% (22/199) carried and one from human, two MRSP non-ST71 spsO, 27% (53/199) carried spsP, and 24% from dogs, one dog MRSP ST71, and one carried (48/199) spsQ. This suggests that there human MRSP ST71 were evaluated by an in have been temporal changes in carriage of vitro assay. Experiments were carried out at spsF in S. pseudintermedius isolates from our two different growth phases; mid-exponential patient population. and late stationary. Adherent bacteria were counted manually at × 1000 magnification. n 64B Mean bacterial counts for all combinations were compared using a negative binomial COMPARATIVE ADHERENCE TO regression model. Results: Mean bacterial CORNEOCYTES AND HOST SPECIFICITY counts were affected by corneocyte donor type BETWEEN METHICILLIN-SUSCEPTIBLE and strain genotype whereas strain host origin AND METHICILLIN-RESISTANT was not statistically significant, in both growth STAPHYLOCOCCUS PSEUDINTERMEDIUS phases. The adherence of S. pseudintermedius ISOLATED FROM DOGS AND HUMANS was significantly greater to canine corneocytes F. Latronico1, A. Moodley1, S. S. Nielsen2, L. than to human corneocytes (p<0.0001). MRSP Guardabassi1; ST71 showed greater adherence than MSSP 1Department of Veterinary Disease Biology, and MRSP non-ST71 (p<0.0001). Overall Faculty of Health and Medical Sciences, Uni- MRSP ST71 of human origin was the best versity of Copenhagen, 1870 Frederiksberg, strain adhering to both canine and human cor- DENMARK, 2Department of Large Animal neocytes. Discussion/Conclusion: Our study Sciences, Faculty of Health and Medical supports the general notion that S. pseudinter- Sciences, University of Copenhagen, 1870 medius is a bacterial species adapted to dogs Frederiksberg, DENMARK. and indicates that under in vitro conditions MRSP ST71 adheres better compared to MSSP Introduction: Staphylococcus pseudintermedi- and MRSP non-ST71. The enhanced adherence us is the most common opportunistic bacterial properties of ST71 may facilitate coloniza- pathogen associated with otitis and pyoderma tion and transmission between dogs, thereby in dogs. Multidrug methicillin-resistant S. contributing to the epidemiological success of pseudintermedius (MRSP) are increasingly re- this MRSP lineage. The unexpected ability of ported with two dominant clonal lineages rec- MRSP ST71 to adhere to human corneocytes ognized worldwide: ST71 and ST68. Bacterial suggests possible adaptation to the human host adherence to the host epithelium is an essential as exemplified by the greater adherence of the step to colonization and subsequent infection. human ST71 isolate to human corneocytes. Corneocytes constitute the outermost layer of skin epithelium and represent a passive barrier between external environment and internal organs. Objective: To investigate the epidemiological success of specific MRSP lineages, in vitro adherence properties of methicillin-re-

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 83 Poster Abstracts n 65B reports involving MRSA, as is the association METHICILLIN AND ZINC RESISTANCE IN with SCCmecV, since crzC is known to reside STAPHYLOCOCCUS HYICUS FROM PIGS within SCCmecV. The finding that czrC is WITH EXUDATIVE EPIDERMITIS common amongst MRSH, inversely associated with antimicrobial exposure and distributed M. Slifierz, J. Park, R. Friendship,S. Weese; across multiple Ontario farms raises concern Univ. of Guelph, Guelph, ON, CANADA. because of the commonness of supplementation of swine starter ration with high levels of Staphylococcus hyicus is the leading cause zinc oxide (≥2500 ppm) to prevent post-wean- of exudative epidermitis (EE), an imporatnt ing diarrhea, particularly on antibiotic-free disease in young pigs. Anecdotally decreased farms. Exposure to zinc oxide in the starter response of EE to beta-lactams has raised con- ration may be selecting for MRSH in newly cern about the emergence of methicillin-resis- weaned pigs and the unintended consequences tance (MR) in S. hyicus (MRSH). Additionally, of zinc supplementation on MR staphylococci there has been recent concern about zinc and deserves further study. Whether MRSH could cadmium resistance, conferred by czrC, in MR act as a potential source of SCCmec for other staphylococci. The objective of this study was staphylococci is unknown. to evaluate the prevalence of methicillin-resistant S. hyicus (MRSH) in pigs with EE and the presence of czrC. Skin swabs were collected n 66B from 186 pigs with EE from 30 conventional VALIDATION OF APPROPRIATE REFERENCE and antibiotic-free farms. Staphylococcus GENES FOR QUANTITATIVE POLYMERASE hyicus was isolated using enrichment culture CHAIN REACTION (QPCR) STUDIES IN and characterized by PBP2a LAT, mecA PCR, STAPHYLOCOCCUS PSEUDINTERMEDIUS SCCmec typing and PCR detection of czrC. S. AND PRELIMINARY ASSESSMENT OF GENE hyicus was isolated from 124 (68%) pigs. 28 EXPRESSION IN BIOFILM-EMBEDDED (23%) were MRSH, for an overall MRSH pig BACTERIA. prevalence of 15%. MRSH was found on 15 E. Crawford, A. Singh, D. Metcalf, S. Weese; (50%) farms. 18 MRSH contained SCCmecV. University of Guelph, Guelph, ON, CANADA. The rest were untypable as they only contained recognized ccr or mec complex, not both. 4 Quantitative PCR (qPCR) is rapidly becom- possessed classA mec, 2 had classC mec, and 3 ing the standard method for analyzing gene had ccr5. SCC 14 (50%) MRSH from 8 farms expression in a wide variety of biological carried czrC. czrC was found in 14 isolates. All samples. It is extremely sensitive and specific, 14 czrC positive isolates possessed SCCmecV but can suffer from significant bias and error versus only 4/14 (29%) of czrC negative if stably expressed reference genes are not isolates (P<0.0001). There was an inverse as- identified. Here, three reference genes in sociation between antimicrobial exposure and Staphylococcus pseudintermedius were identi- czrC (P<0.027), with 64% of MRSH-positive fied and validated from a set of 8 potential pigs being raised without exposure to antimi- genes (proC, gyrB, rplD, rho, rpoA, ftsZ, recA, crobials. MRSA possessing SCCmecV were sodA). Two strains of Staphylococcus pseud- isolated from 5/13 (38%) farms that harboured intermedius were used, and primer specificity SCCmecV MRSH. Both MRSA (t571) and and efficiency were confirmed and measured. MRSH containing SCCmecV were isolated Ranking of the genes with respect to expres- from one pig. MRSH was common, which is sion stability revealed a combination of gyrB, consistent with anecdotal evidence of poor re- rho and recA as suitable reference genes for sponse of EE to beta-lactams. The high preva- qPCR. This combination was used to quantify lence of zinc resistance in MRSH is similar to expression of a single biofilm associated target,

84 ASM Conferences Poster Abstracts icaA, in logarithmic, stationary and biofilm quorum sensing system (agrA, agrB) were growth phases; there was significant upregula- assessed in logarithmic, stationary and biofilm tion of expression of this gene in the biofilm phases for two strains of MRSP (ST68, ST71). growth phase in both strains. We also report Testing was performed on a broad spectrum a modified nucleic acid harvesting technique of clinically relevant surfaces; stainless steel, affording significantly increased yields, par- titanium, polystyrene, polymethylmethacrylate, ticularly from biofilms. latex, silicone, polydioxanone and glass. One- way ANOVA followed by Tukey’s pairwise n 67B comparison (where appropriate) was used to identify any differences due to surface for each ASSESSMENT OF EXPRESSION OF SEVERAL gene / strain combination. GyrB, rho and recA POTENTIAL VIRULENCE GENES IN THE were used a reference genes. Genes in the ica CANINE PATHOGEN STAPHYLOCOCCUS family tended to be upregulated in biofilm PSEUDINTERMEDIUS IN PLANKTONIC phases, especially on glass. MSCRAMMs also AND BIOFILM PHASES ON A VARIETY OF tended generally to be upregulated in biofilm SURFACES USING QPCR. phases, however there were notable strain E. Crawford, A. Singh, D. Metcalf, S. Weese; variations such as where atl was significantly University of Guelph, Guelph, ON, CANADA. upregulated in the planktonic phase in one sequence type. MecA expression varied both Reasons for the rapid dissemination of methi- with strain and surface. There was some varia- cillin-resistant Staphylococcus pseudinterme- tion identified, but no trend in expression of dius (MRSP), an important canine pathogen, quorum sensing genes. Upregulation of biofilm and the leading cause of surgical site infec- and MCRAMMs in biofilms is not unexpected, tions, are unknown. Biofilm formation may be as these genes are necessary for the initiation an important factor, but evaluation of regula- and proliferation of biofilms. The other genes tion of virulence factors and biofilm-associated studied may have more complex determinants genes in MRSP is lacking. The objective of of expression. this study is to evaluate expression of a series of biofilm-associated and antimicrobial resistance genes during the planktonic and biofilm n 68B growth phases using qualitative polymerase COMPLETE SEQUENCE OF A chain reaction (qPCR). qPCR is emerging as STAPHYLOCOCCAL RESISTANCE PLASMID the standard for examining gene expression, OBTAINED FROM A SMALL ANIMAL CLINIC but requires the identification of constitutively S. Weiß, K. Kadlec, A. Feßler, S. Schwarz; expressed reference genes to obtain valid Institute of Farm Animal Genetics, Friedrich- results. Previously, reference genes have been Loeffler-Institute (FLI), Neustadt-Mariensee, identified for MRSP, providing the basis for GERMANY. qPCR expression studies in this species. Fol- lowing development and validation of suitable Objective: During analysis of methicillin- primers for qPCR, expression of a number of resistant staphylococci from samples taken microbial surface components recognizing in a small animal clinic, nine S. epidermidis adhesive matrix molecules (MSCRAMMs) (MRSE) isolates showed the same molecular including elastin, fibrinogen/fibronectin and- fi characteristics and resistance to antimicrobial bronectin binding proteins (ebpS, spsE, fnbB), agents belonging to ten different classes. The a bifunctional autolysin (atl), penicillin binding aim of this study was to investigate these protein 2a (mecA), biofilm associated genes MRSE isolates for the presence and orga- in the intracellular adhesion operon (icaA, nization of plasmid-borne resistance genes. icaC, icaD), and the accessory gene regulator Materials and Methods: The nine MRSE

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 85 Poster Abstracts isolates had SCCmec type IV, dru type dt10a, n 69B belonged to multi locus sequence type ST5 and DISTRIBUTION OF BIOFILM were resistant to aminoglycosides/aminocy- FORMATION GENES IN LIVESTOCK- clitols, β-lactams, fluoroquinolones, lincos- ASSOCIATED METHICILLIN-RESISTANT amides, macrolides, phenicols, pleuromutilins, STAPHYLOCOCCUS AUREUS FROM sulfonamides, tetracyclines and trimethoprim. SWINE AND SWINE SLAUGHTERHOUSE Plasmids were extracted by alkaline lysis and WASTEWATER transferred by protoplast transformation into Staphylococcus aureus RN4220. Transfor- M. Wan, C. Chou; mants were subjected to suceptibility testing School of Veterinary Medicine, National Tai- using broth micro- or macrodilution accord- wan University, Taipei, TAIWAN. ing to the recommendations given by the Background: Livestock-associated methicillin- Clinical and Laboratory Standards Institute resistant Staphylococcus aureus (LA-MRSA) (CLSI). Resistance genes were detected by are found in different animals and meat PCR and plasmids were subjected to restric- products. LA-MRSA ST9 has emerged as a tion analysis. One representative plasmid was potential zoonotic pathogen for humans and cloned, completely sequenced and analyzed. animals. Biofilms mediate MRSA coloniza- Results: Transformants carrying the large tion and are associated with host infection. plasmid showed increased MICs to clindamy- The occurrence of biofilm formation genes cin, tiamulin, trimethoprim, tobramycin, and the biofilm formation ability in LA-MRSA tetracycline and minocycline and harboured ST9 from different ecological niches have not the corresponding resistance genes vga(A), been reported. The aims of the study were to dfrK, aadD, tet(L) and tet(M). They showed investigate the dynamics of biofilm formation indistinguishable restriction patterns with genes (intercellular adhesion [ica], and staphy- BglII, EcoRI and HindIII. The sequence of lococcal accessory gene regulator [SarA]) and the 28,743 bp plasmid pSWS47 confirmed the biofilm expression in LA-MRSA ST9 from presence of the five antimicrobial resistance asymptomatic nasal swine and swine slaugh- genes. Sequence analysis identifiedtet (M) as terhouse wastewater. Methods: A total of 259 part of a ΔTn916-like element. Furthermore, MRSA isolates were recovered between March a functionally inactive ΔblaZ gene as part of a 2010 and August 2011, including 147 isolates ΔTn552 was detected. Moreover, several com- from asymptomatic LA-MRSA-carrying swine plete or incomplete plasmid maintenance genes (the colonization group) and 112 isolates (rep, pre, mob, rlx), as well as six complete or from swine slaughterhouse wastewater (the incomplete insertion sequences (IS257, IS431, environmental group). The distributions of ica IS1310) were identified. Sequence analysis (icaR and the icaADBC operon) and SarA loci showed in part high identity (≥98%) to other for the colonization and environmental group described resistance plasmids, which suggests were investigated independently by PCR. recombination processes. Conclusions: The Biofilm formation was quantified by microtiter presence of several resistance genes on the plate assay. Data were analyzed by chi-square multi-resistance plasmid pSWS47 from a test for homogeneity of two-group proportions. feline MRSE isolate ensures the persistence Comparisons of biofilm formation between of the plasmid under the selective pressure of groups were performed using the Mann-Whit- different antimicrobial agents used. Moreover, ney U test. Results: The ica and sarA locus to the best of our knowledge, this is the first were detected in 64.9% (168/259) and 82.6% report of a staphylococcal plasmid carrying (214/259) of the MRSA isolates, respectively. the tetracycline/minocycline resistance gene The detection rate of the ica and sarA loci in tet(M). the environmental group (48.2% and 65.2%)

86 ASM Conferences Poster Abstracts were significantly lower than in the coloniza- 1000 human MRSA isolates, collected in tion group (77.6% and 95.9%) (P < 0.05, χ2). Dresden between 2000 and 2013, were tested Of the 259 MRSA isolates, 61.8% (n=160) and their strain/clonal complex affiliations MRSA isolates exhibited the biofilm formation were determined, but no mecC-isolates were ability. Biofilm production in the colonization identified. However, three human isolates of group was higher than in the environmental CC130-MRSA-XI were referred by external group (P < 0.05, MWU test). Conclusions: units for characterisation, one originating from This study characterized the distribution of ica, a county hospital nearby and two from North- and sarA in LA-MRSA isolates from different Eastern Germany (Frankfurt/Oder). Screen- ecological niches and in biofilm formation. ing of approximately 600 veterinary isolates The high prevalence of biofilm formation gens of S. aureus from Thuringia, mostly from and biofilm production in LA-MRSA ST9 cattle and a few from goats and sheep, did not from the colonization group may improve reveal any mecC-positive isolates. In Bavaria, the persistent MRSA in the swine population milk samples from approximately 2000 cows and present a threat to the health of livestock were screened for the presence of S. aureus animals as well as farm workers. and MRSA. This resulted in the detection of CC130-MRSA-XI in milk samples from 16 n 70B cows and an udder swab of one additional cow in one herd of 56. Regarding wildlife, mecC HOST RANGE AND DISTRIBUTION OF MECC was identified in two ill hedgehogs (Erinaceus POSITIVE CC130 MRSA europaeus) from Sweden as well as in one fox S. Monecke1, D. Bandt2, D. C. Coleman3, (Vulpes vulpes) and one fallow deer (Dama D. Gavier-Widen4, H. Hotzel5, A. Lazaris3, dama) from Germany. S. aureus isolates from R. Mattsson4, M. Peters6, L. Rangstrup- other wildlife and belonging to other CCs, Christensen4, K. Schlotter7, A. C. Shore3, R. namely CC1 from fallow deer and mouflon Ehricht1; sheep (Ovis aries), CC133 from a mute swan 1Alere technologies, Jena, GERMANY, 2In- (Cygnus olor), ST425 from roe deer (Capreo- stitut für Medizinische Diagnostik Oderland, lus capreolus), red deer (Cervus elaphus), wild Frankfurt (Oder), GERMANY, 3Trinity College, boar (Sus scrofa) and badger (Meles meles), Dublin, IRELAND, 4SVA, Uppsala, SWEDEN, CC692 from a white-tailed eagle (Haliaeetus 5FLI, Jena, GERMANY, 6SVUA, Arnsberg, albicilla), ST2279 from lynx (Lynx lynx) and GERMANY, 7Bavarian Animal Health Service, reindeer (Rangifer tarandus), ST2425 from Poing, GERMANY. hare (Lepus europaeus), ST2691 from moose (Alces alces) as well as coagulase-negative Recently, a novel methicillin resistance gene staphylococci from roe deer, wild boar, least was discovered in Staphylococcus aureus and weasel (Mustela nivalis), fox and racoon designated mecC. There is growing evidence (Procyon lotor) all were found to be mecC- for a zoonotic background of mecC-positive negative. All detected mecC-positive isolates, MRSA although it also has been found in both from humans and animals, were virtually humans. In order to obtain an insight into the identical to the initially described human prevalence and distribution of mecC, clinical CC130-MRSA-XI strain from Ireland lacking isolates from a university hospital in Dresden, enterotoxin genes, PVL genes and lukM/lukF- Saxony, veterinary screening isolates from P83, but harbouring edinB and an exfoliative Thuringia and Bavaria and wildlife isolates toxin gene homolog etD2. CC130-MRSA-XI from Germany and Sweden were screened for is rare, but it can be found sporadically in the presence of mecC /SCCmec XI elements humans, livestock and wildlife, and it has the using DNA microarrays (Alere Technologies). potential to cross host species barriers and

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 87 Poster Abstracts to cause outbreaks. Thus, MRSA screening by both electrophoretic separations. The most procedures and diagnostic tests need to be abundant five to eight proteins from each 1-DE evaluated and redesigned to ensure detection gels were identified by MALDI-TOF/TOF of isolates harbouring mecC. mass spectrometry. Those identifications revealed that enrichment on the proteins of each n 71B cellular fraction was achieved. Nevertheless, some proteins were identified in all fractions, OPTIMIZING PROTEIN FRACTIONATION which could indicate that they had multiple AND CHARACTERIZING THE WHOLE subcellular locations or that they can transit PROTEOME OF STAPHYLOCOCCUS between the cytosol and the surface compart- PSEUDINTERMEDIUS ments, depending on the physiological and/ N. Couto1, J. Martins2, M. Ventosa2, C. or environmental conditions. The established Pomba1, A. V. Coelho2; fractionation protocol was shown to be ef- 1Faculdade de Medicina Veterinária, Lisboa, ficient and adequate for the characterization PORTUGAL, 2Instituto de Tecnologia Química of S. pseudintermedius proteome. The protein e Biológica, Universidade Nova de Lisboa, identification by mass spectrometry is being Oeiras, PORTUGAL. performed and will help us understand the cell physiology and, consequently, the pathogen- Pyoderma caused by Staphylococcus pseud- esis of these bacteria. intermedius is one of the most common infections seen in small-animals veterinary practice worldwide. The recent emergence n 72B of methicillin-resistant S. pseudintermedius GENETIC CHARACTERIZATION OF (MRSP) strains has renewed the interest in the METHICILLIN-RESISTANT STAPHYLOCOCCI pathogenesis and virulence of this species. The IN WALLABIES proteomic approaches can be useful to identify M. M. Chen1, I. Smith2, W. Boardman3, A. E. new potential antigens, in order to develop new Goodman1, M. H. Brown1; therapeutic strategies, namely vaccines. The 1School of Biological Sciences, Flinders Uni- aim of this study was to optimize the protein versity, Bedford Park, AUSTRALIA, 2ZoosSA, fractionation and characterize the proteome of Adelaide, AUSTRALIA, 3School of Animal and S. pseudintermedius through 1-DE-LC-MAL- Veterinary Sciences, University of Adelaide, DI-TOF/TOF and 2-DE MALDI-TOF/TOF Roseworthy, AUSTRALIA. approaches. The studied strain of S. pseudintermedius (5819/10) was isolated from a dog Methicillin-resistant staphylococci (MRS), with pyoderma. Bacterial cells were treated defined as staphylococci harboring themecA with lysostaphin, from which the supernatant, gene, have been isolated from a number of enriched in cell wall proteins (fraction I), was farm and companion animals in addition to obtained. After an osmotic shock, proteins free-living birds, rodent species, deer, boars were ultracentrifuged and two fractions were and ibexes. Few studies have examined the attained: the pellet was enriched in membrane prevalence of MRS in native endangered proteins (fraction II) and the supernatant was fauna. The objective of this study was to enriched in cytoplasmatic proteins (fraction determine and characterize the prevalence of III). The proteins were precipitated either with beta-lactam resistance in commensal staphy- trichloroacetic acid (fraction I) or trifluoro- lococcal species isolated from healthy captive ethanol/chloroform (fractions II and III). Total and wild wallabies from two distinct environ- protein in each fraction was quantified and ments in South Australia, namely indigenous separated by 1-DE and 2-DE gels. All fractions land and Monarto Zoo. Here, 89 staphylococ- differed in their protein content as visualized

88 ASM Conferences Poster Abstracts cal isolates recovered from 98 wallabies were n 73B studied. These isolates were characterized by ANTIMICROBIAL RESISTANCE AND ARRAY 16S rRNA sequencing, Kirby-Bauer disc diffu- ANALYSIS OF MECA POSITIVE COAGULASE sion and growth on oxacillin-selective media. NEGATIVE STAPHYLOCOCCI FROM VEAL Minimum inhibitory concentrations for oxacil- CALVES IN BELGIUM lin were determined by broth microdilution for MRS. These 89 isolates from 15 staphylococ- M. A. Argudin1, W. Vanderhaeghen2, P. cal species were screened for the presence of Butaye1; blaZ and mecA. Sequencing of the 23 blaZ 1Veterinary and Agrochemical Research centre, genes revealed three signature types dominated Brussels, BELGIUM, 2Department of Repro- by signature type 3 and, according to Bush duction, Obstetrics and Herd Health, Ghent classification, the amino acid sequence has in- University, Ghent, BELGIUM. ferred plasmid origins for these elements. The Non S. aureus staphylococci are usually more sequencing of mecA from 11 isolates show a resistant to antibiotics than S. aureus, and are 97-99% identity to that of the prototype N315 considered a reservoir of resistance genes. The strain. Multiplex PCR was undertaken to clas- aim of this work was to study the prevalence sify the mecA determinants into various SCC- of typical S. aureus resistance genes among mec types, revealing one SCCmec type III and 58 mecA positive coagulase negative staph- two SCCmec type V isolates. However, eight ylococci-CNS recovered from nasal swabs strains of Staphylococcus sciuri harboring a of veal calves in Belgium. tRNA intergenic class A mec region were unable to be typed spacer PCR was used for species identification. by conventional methods. These eight isolates Isolates were characterized by susceptibility contained 19 single-nucleotide polymorphisms testing by a microbroth-dilution method using and were subjected to ccrB typing which found clinical breakpoints (Clinical Laboratory Stan- a potential novel allele of ccrAB allotype 1 dards Institute and Eucast) and SCCmec PCR in a single isolate. Interestingly, ccrB typing typing. Presence of S. aureus antimicrobial also revealed a novel combination of a ccrAB resistance genes was analysed via microarray allotype 6 with the single SCCmec type III (Alere). CNS were identified as:S. lentus-22, element. Studies into determining the presence S. sciuri-20, S. epidermidis-11, S. haemolyti- of mecC in staphylococci which exhibited a cus-4, and S. equorum-1. All isolates were resistance phenotype for oxacillin and cefoxi- multi-resistant, and they showed a high preva- tin, yet were negative for mecA, blaZ and ccrB, lence of resistance to erythromycin, clindamy- are currently being undertaken. Thus, genetic cin-both 86%, tetracycline-84%, tiamulin-76%, characterization of the mec elements revealed fusidic acid-69%, and streptomycin-67%. two SCCmec types being identified in three A lower prevalence of resistances was seen MRS. Additionally, a combination of the novel against trimethoprim-59%, kanamycin-52%, ccrB allele with a class A mec region could ciprofloxacin-48%, sulfamethoxazole-47%, constitute a novel variant of the SCCmec type I synercid-45%, chloramphenicol-38%, gen- element being isolated from healthy wallabies tamicin-24%, and linezolid-5%. Phenotypic in South Australia. Importantly, irrespective of resistance to penicillin-93% and cefoxitin-76% captivity status, a similar carriage rate of MRS was lower than expected based on the positive was found in these populations. results for mecA. All CNS were susceptible to rifampicin, mupirocin and vancomycin. Most isolates-69% carried the SCCmec III and few carried other mec cassettes (7%-SCCmec IV, 9%-SCCmec V, 15%-non-typeable) as shown by the combined SCCmec PCR and array as-

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 89 Poster Abstracts say. Regarding to the antimicrobial resistance health problem for farmers, veterinarians and genes selection detected by the array, in other livestock professionals. most isolates one or more genes conferring The aim of this study was to characterise resistance to macrolides, lincosamides and MRSA strains isolated from farm animals streptogramins B-83%, aminoglycosides-78% or their environs in Germany with respect to and tetracyclines-76% were detected, with their virulence and resistance genes. In total, the individual percentages of resistance genes 125 German MRSA strains isolated between being: ermA-19%, ermB-22%, ermC-55%, 2008 and 2012 at primary production from pig mpbBM-22%, mphC-19%, msrA-7%, vgaA- (n = 56), cattle (n = 36) and turkey (n = 34) 5%, lnuA-10%, aacA-aphD-45%, aphA3-40%, were analysed using a commercially available aadD-40%, sat-40%, tetK-64% and tetM-43%. microarray (StaphyType, Alere Technologies, 40% of the isolates carried chloramphenicol Jena, Germany). The array covers about 300 resistance genes (cat-17%, fexA-24%). Less target sequences corresponding to approxi- isolates carried the bla operon-28%, trim- mately 185 distinct genes and their allelic vari- ethoprim dfrS1-19%, fusidic acid far1-5%, ants including, among others, virulence and fosfomycin fosB-3%, mercury merA-merB-5%, resistance genes, SCCmec-, agr-, and capsule quaternary ammonium compound qacA-14% typing markers. Further characterisation of and qacC-5% resistance genes. Three isolates SCCmec-, spa- and in some cases MLST-type carried the multi-resistance cfr gene. In was done by means of PCR and sequencing, conclusion, although the CNS carried mainly respectively. Most of the isolates tested were different SSCmec types than those found in associated to CC398 (90%), while the remain- the typical livestock associated CC398 MRSA, ing belonged to CC9, CC5, CC30, CC97, CC8 they share a large number of resistance genes and CC12. Isolates mainly harboured SCCmec with S. aureus. Hence, they might be an type V (80%) and exhibited spa type t011 important reservoir of antimicrobial resistance (39%) or t034 (31%). Altogether 54 different genes. There is a large discrepancy between resistance gene patterns were detected. The the presence of mecA and the expression of most frequent resistance genes beside mecA β-lactam resistance indicating that phenotypic and blaZ (100% each) were tet(M) (94%), data are an underestimation of the presence of tet(K) (73%), ermC (30%) ermA (28%) and mecA in CNS from veal calves. Acks: M.A. aadD (28%). In contrast, the virulence gene Argudín is supported by Fundación Alfonso content was low. For 87% of the strains the Martín Escudero. virulence pattern lukF-lukS-hlgA was found. All of the strains were negative for PVL, exfo- n 74B liative toxins and the ACME locus. Few strains carried the gene tst1 (2 strains), genes for the MICROARRAY-BASED GENOTYPING phage associated immune modulators sak (5 OF METHICILLIN-RESISTANT strains), scn (3 strains), chp (1 strain) or for en- STAPHYLOCOCCUS AUREUS (MRSA) FROM terotoxin genes like sea and seb (1 strain each), PRIMARY PRODUCTION IN GERMANY sec (3 strains), sek, sel and seq (3 strains each) A. Fetsch, Y. Kelner-Burgos, B. Lauzat, B. or the egc-cluster (9 strains). As expected, al- Guerra, B. A. Tenhagen, A. Kaesbohrer, B. most all of the MRSA tested harboured a high Kraushaar; prevalence of resistance encoding genes. In Federal Institute for Risk Assessment, Berlin, contrast, virulence genes were predominantly GERMANY. found in MRSA not associated with CC398. Whether these Non-CC398 strains were intro- Methicillin-resistant Staphylococcus aureus duced by humans into livestock or represent (MRSA) especially of clonal complex 398 particular adapted animal strains is not known (CC398), are considered an occupational yet. The results of the study underline, that

90 ASM Conferences Poster Abstracts molecular typing of MRSA is essential to strategy. Results: Plasmid-borne cfr genes closely characterize the risk associated with were found on either previously described MRSA in livestock and that a focus should be plasmid types pSS-02 and pSS-02-like (n= 7), on those strains that are not associated with the pSS-03-like (n=1), pJP1-like (n=3) or novel CC 398. Acknowledgement: This work was plasmid types pSS-04 (n=1) and pJP2 (n=1). carried out in the EMIDA-ERA NET Project These plasmids ranged in size between 35 and “LA-MRSA” (Grant-No. 0315868A) 50 kb. Analysis of the cfr-flanking regions on plasmids revealed that IS sequences (IS21-558, n 75B IS256, IS257, IS1216E) and other resistance genes (aacA-aphD, aadD, ble, fosD, erm(B), GENETIC ENVIRONMENT OF THE MULTI- erm(C) and/or fexA) coexisted on the respec- RESISTANCE GENE CFR IN METHICILLIN- tive plasmids. Chromosomal copies of cfr were RESISTANT COAGULASE-NEGATIVE identified in eightS. lentus isolates. The cfr STAPHYLOCOCCI FROM CHICKENS, DUCKS, gene was also bracketed either by IS elements AND PIGS IN CHINA (IS256-cfr-IS256 or ISEnfa5-cfr-ISEnfa5) or T. He1, C. Wu1, Y. Wang1, S. Schwarz2, Q. by a combination of a composite transposon Zhao1, J. Shen1; and an IS element (Tn4001-cfr-IS256) in the 1Beijing Key Laboratory of Detection Technol- chromosome of the eight S. lentus isolates. ogy for Animal-Derived Food Safety, College The chromosomal cfr-containing region, which of Veterinary Medicine, China Agricultural may have a plasmid origin, can be looped out University, Beijing, CHINA, 2Institute of Farm via IS element-mediated recombination. Con- Animal Genetics (FLI), Neustadt-Mariensee, clusions: This is the first time that the genetic GERMANY. environment of cfr has been identified on plasmids or in chromosome of CoNS isolates Objective: During a previous study, the from chickens and ducks. IS256 and ISEnfa5 multidrug-resistance gene cfr was detected elements may play an important role in the in coagulase-negative staphylococci (CoNS) dissemination of cfr, not only on plasmids, but from 3/401 pigs, 15/305 chickens, and 3/78 also in the chromosome. The co-location of ducks obtained from 31 different farms and cfr with several other antimicrobial resis- one slaughterhouse in 2 provinces of China. tance genes bears the risk of co-selection and Twenty of the 21 cfr-positive isolates were persistence of the cfr gene under the selective methicillin-resistant. The gene cfr was detected pressure imposed by these other antimicrobial on plasmids in the majority of the isolates. agents. The aim of this study was to investigate the genetic environment of the cfr gene in the 20 methicillin-resistant CoNS (MRCoNS) and n 76B the single methicillin-susceptible CoNS from COMPLETE GENOME SEQUENCE OF THE chickens, ducks and pigs in China. Materials METHICILLIN-RESISTANT PATHOGEN and methods: The cfr-carrying plasmids in 13 STAPHYLOCOCCUS PSEUDINTERMEDIUS CoNS were transferred into Staphylococcus NA45 aureus RN4220 by electrotransformation. The M. C. Riley1, F. A. Hartmann2, D. A. Bemis1, S. transformants were screened for their plasmid A. Kania1; content and resistance phenotypes. The sizes of 1University of Tennessee, Knoxville, TN, 2Uni- the cfr-carrying plasmids were determined by versity of Wisconsin-Madison, Madison, WI. BglII restriction analysis. The partial nucleotide sequences of the cfr-carrying plasmids or Introduction: Methicillin-resistant Staphy- chromosomal regions were determined by a lococcus pseudintermedius (MRSP) is an modified random primer sequencing walking important zoonotic pathogen commonly found

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 91 Poster Abstracts in canines, but has been shown to infect a found only in other genera such as Streptococ- variety of species including humans. Similar cus and Bacillus. Conclusion: Whole genome to methicillin-resistant Staphylococcus aureus sequencing has revealed that mobile ge- (MRSA), multiple drug resistance phenotypes netic elements are responsible for most of the exist within the species and cause significant genomic differences between NA45 and the clinical infection. MRSP isolate NA45 is a other genomes, and contain genes involved in clinical canine isolate with high drug resistance drug resistance and pathogenicity. As several and virulence characteristics. By sequencing of them are not conserved within the Staphy- the genome of this isolate and comparing it to lococcus lineage, movement of these elements other S. pseudintermedius genomes, we hope appears to cross species and genus level to elucidate the genetic components respon- boundaries. As more genomes are sequenced, sible for these pathogenic qualities. Here we these elements can be better tracked and used present the complete, circular genome, in com- for functional and epidemiological studies to parison with methicillin-susceptible (MSSP) determine their contribution to the evolution of isolates ED99 and HKU10-03, and methicillin- drug resistance and virulence in this important resistant (MRSP) isolate E140. Methods: S. pathogen. pseudintermedius NA45 was isolated in the United States from canine urine captured by n 77B cystocentesis and sub-cultured into pure stock EFFECT OF OPSONIZATION ON cultures. Whole Genome Sequencing (WGS) BACTERICIDAL ACTIVITY OF CANINE was performed using Illumina©, Ion Torrent™ AND HUMAN POLYMORPHONUCLEAR and 454© paired-end sequencing, and de novo LEUKOCYTES AGAINST S. assembly of contigs was performed using Ge- PSEUDINTERMEDIUS neious©. De novo contigs were aligned against a whole genome map acquired on the Argus® F. Latronico1, A. R. Porter2, A. K. Krogh3, system and a combination of BLAST align- A. Moodley1, M. Kjelgaard-Hansen3, F. R. ments and extension read mapping resulted in DeLeo4, L. Guardabassi1; a complete, circular genome without the use of 1Department of Veterinary Disease Biology, a reference genome sequence. Annotation was University of Copenhagen, Frederiksberg, performed using the RAST annotation server, DENMARK, 2Laboratory of Human Bacterial and comparative genomic analysis was per- Pathogenesis, Rocky Mountain Laboratoires, formed using progressiveMauve and GCview. National Institute of Allergy and Infection Dis- Results: S. pseudintermedius NA45 has a eases, National Institute of Health, Hamilton, genome content of 2,841,212 base pairs encod- MT, 3Small Animal Clinical Sciences, Faculty ing 2812 CDS, 76 RNAs and an average GC of Health and Medical Sciences, Univeristy content of 37.3%. It contains a novel SCCmec of Copenhagen, Frederiksberg, DENMARK, cassette type and has 4 prophage insertions, 3 4Laboratory of Human Bacterial Pathogen- of them unique sites incorporating ~200Kb. esis, Rocky Mountain Laboratories, National Phage and other mobile elements are elevated Institute of Allergy and Infection Diseases, in NA45 and other MRSPs relative to MSSPs, National Institute of Health, Hamilton, MT. and a ~120Kb prophage has inverted GC con- Introduction: S. pseudintermedius is a com- tent relative to lagging strand DNA, indicating mon pathogen in dogs. Human infections are recent incorporation. Unique genomic regions rare but have recently gained attention due contain genes for sporulation, erythrocyte to several reports of methicillin-resistant S. binding, heavy metal resistance, aminoglyco- pseudintermedius (MRSP) in human patients. side resistance and a superantigen pathogenic- Very little is known about the ability of this ity island, among others. Many of these are staphylococcal species to evade killing by not taxonomically conserved and have been

92 ASM Conferences Poster Abstracts polymorphonuclear leukocytes (PMNs) in property is associated with specific bacterial diverse hosts. PMNs are part of the innate surface-associated proteins or whether survival immune system and phagocytosis by PMNs at 3 h is related to lysis of PMNs, thereby is facilitated by opsonization that allows reducing their phagocytic effect. Regardless of leukocytes to readily recognize bacteria. opsonization, S. pseudintermedius had reduced Objective: To study the effects of opsonization ability to survive in the presence of human on bactericidal activity of canine and human PMNs, confirming that this staphylococcal PMNs against S. pseudintermedius. Methods: species is not adapted to the human host. PMNs were isolated from heparinized venous blood from two healthy dogs and ten healthy n 78B humans using Hystopaque-1077 (canine blood) FIRST DETECTION OF METHICILLIN- or Hystopaque-Ficoll (human blood) gradi- RESISTANT STAPHYLOCOCCUS AUREUS ent centrifugation. Purity and cell viability ST398 STRAIN WITH ARGININE CATABOLIC were determined by flow cytometry. ThreeS. MOBILE ELEMENT (ACME) pseudintermedius (two MRSP, one methicillin- susceptible S. pseudintermedius) were tested A. J. Sabat1, V. Akkerboom1, M. van Rijen2, B. by an in vitro phagocytosis assay using a ratio Sinha1, J. Kluytmans2, A. W. Friedrich1; of 10 bacteria per PMN. For each strain, both 1Department of Medical Microbiology and unopsonized and opsonized cultures were Infection Prevention, University of Groningen, used. Opsonization was obtained by incubat- University Medical Center Groningen, Gron- ing the strains in 50% autologous serum at ingen, NETHERLANDS, 2Amphia Academy 37°C for 30 min prior to addition of PMNs. Infectious Disease Foundation, Amphia Hospi- PMNs were allowed to phagocytise bacteria tal, Breda, NETHERLANDS. for 1 or 3 hours. Survival of bacteria was Recently, a specific clone of methicillin evaluated by comparing mean colony forming resistant Staphylococcus aureus (MRSA) has units (CFUs) of bacteria exposed to PMNs been identified in farmers and food producing and bacteria alone using paired t test. Results: animals. This so called livestock associated S. pseudintermedius (serum opsonized and MRSA (LA-MRSA) belongs to multilocus unopsonized) had significantly greater capacity sequence type 398 (ST398) or closely related to survive following phagocytic interaction STs associated with clonal complex 398 (1h) with canine PMNs compare to that with (CC398). Acquisitions of mobile genetic ele- human PMNs (p<0.05). However, following ments which harbor genes that can enhance the extended time points (3h) after phagocytosis transmission of LA-MRSA CC398 in humans with either canine and human PMNs, survival have the potential to threaten public health. was significantly greater for opsonizedS. As part of the CAM study aiming to identify pseudintermedius compared to unopsonized determinants of community-associated MRSA bacteria (p<0.05). No significant differ- (CA-MRSA) in the Netherlands, we identified ences were found between bacterial strains. an MRSA isolate of ST398 that was positive Discussion/Conclusion: This is the first study for the arginine catabolic mobile element evaluating the interaction between human and (ACME). The presence of ACME has been re- canine PMNs and S. pseudintermedius and ported to enhance colonization of the skin and the effects of serum opsonization. Although the mucosal surfaces by neutralizing the acidic our results are based on limited number of test pH of human sweat. The objective of this study subjects, they reveal an unexpected ability was to determine the genetic organization of opsonized S. pseudintermedius to evade of ACME- staphylococcal cassette chromo- phagocytic killing by human or canine PMNs. some mec composite island (SCCmec-CI) in Future research should investigate whether this

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 93 Poster Abstracts the ACME-positive isolate by whole-genome from the nares of a dog with sinusitis in May sequencing (WGS). A total of 125 MRSA 2013 in Bern (Switzerland). Minimum Inhibi- isolates recovered from patients between 2009 tory Concentration (MIC) was performed to and 2011 in The Netherlands were screened in 22 different antimicrobials. Southern-Blot the current study. The isolates were analyzed hybridization was conducted to determine the by DNA microarrays and the LA-MRSA chromosomal or plasmid location of the mecB ACME-positive isolate of ST398 was investi- gene. High-throughput sequencing of KM450 gated by WGS. Sequence analysis revealed a was performed with Roche 454 GS Titanium new organization of the ACME-SCCmec-CI chemistry and protocols (GS Junior in the ST398 isolate. ACME-SCCmec-CI Szytem, Roche Diagnostics) and sequence was composed of the J1 region of SCCmec I reads were assembled de novo using newbler adjacent to orfX, followed by a truncated form 2.6. Results: Whole Genome Sequencing of ACME type II and SCCmec type IVa. More- (WGS) of M. caseolyticus strain KM450 as over, in the J1 region of SCCmec I the gene well as Southern-Blot hybridization experi- encoding plasmin sensitive protein (Pls) was ments permitted the identification of a novel found, which has been implicated in adher- chromosomally located SCCmec cassette. ence to desquamated nasal epithelial cells and The novel SCCmec element was 39 kb in size as a virulence factor in murine septic arthritis. and only presented two discontinuous regions This is the first report of the ACME element in of homology (SCCmec coverage of 46%) to MRSA with the genotype ST398. This finding the single chromosomal SCCmec element may have implications for the transmissibility described to date in the genus Macrococcus of ST398 isolates, and thus public health. (M. caseolyticus strain JCSC7096): (i) the mec complex (98.8% identity) and (ii) the ccr n 79B region (91.8%), which also covered several orfs and a radC gene. The novel SCCmec NOVEL SCCMEC CASSETTE IN A MECB- element, named SCCmec , was integrated CARRYING MACROCOCCUS CASEOLYTICUS KM450 at the 3’ end of orfX and was delimited at both ISOLATE FROM A SINUSITIS INFECTION OF ends by imperfect direct repeats (GA[AG] A DOG. TCGTATCATAAGTGA). SCCmecKM450 con- E. Gómez-Sanz, A. Thomann, S. Schwendener, tained a mecRm-mecIm-mecB-blaZm complex V. Perreten; in the 3’ end of the cassette Neither transpo- Institute of Veterinary Bacteriology, Vetsuisse sons nor transposase genes were detected in

Faculty, University of Bern, Bern, SWITZER- the whole cassette. SCCmecKM450 contained LAND. the recombinase genes ccrA and ccrB 7.5-kb distant from the orfX. Conclusions: A novel Background: The methicillin-resistant mecB mecB-carrying SCCmec cassette is detected gene has been only detected in M. caseolyti- in a clinical alpha-hemolytic M. caseolyticus cus in three occasions, always located within strain. This observation underlines both the a mec transposon, which contains the mec role of commensal bacteria as opportunistic complex (mecRm-mecIm-mecB-blaZm), with animal pathogens and as reservoirs for novel either a plasmid or chromosomal location. SCCmec elements. We characterised the SCCmec cassette of a mecB-positive M. caseolyticus strain isolated from a clinical sample of a dog with sinusitis. Material and Methods: An alpha-hemolytic methicillin-resistant mecB-positive M. caseolyticus strain, named KM450, was isolated

94 ASM Conferences Poster Abstracts n 80B reaction to catalase and coagulase tests. They NEW SEQUENCE TYPE OF METHICILLIN underwent confirmation by PCR, antimicrobial RESISTANT STAPHYLOCOCCUS AUREUS susceptibility test and typing. S. aureus was (MRSA) FROM LIVER INFECTION IN AN isolated from nasal mucosa and liver. No other ALPINE CHAMOIS relevant bacterial growth was detected. At the post-mortem examination, animal showed a C. Locatelli1, P. Cremonesi2, L. Scaccabarozzi1, good kidney fat deposit and regular contents V. Gualdi3, R. Viganò4, G. Sironi1, M. Besozzi1, of stomach compartments. No macroscopic le- B. Castiglioni2, P. Lanfranchi1, C. Luzzago1; sions were observed. PCR revealed mecA gene 1Università degli Studi di Milano, Milan, in the liver isolate. Antimicrobial susceptibility ITALY, 2Istituto di Biologia e Biotecnologia test confirmed resistance to all beta-lactams Agraria, Lodi, ITALY, 3Parco Tecnologico tested (amoxicillin-clavulanic acid, penicillin, Padano, Lodi, ITALY, 4AlpVet, Crodo, ITALY. ampicillin, ceftiofur, cefoxitin) besides resistance to fluoroquinolones (ciprofloxacin, enro- Methicillin resistant Staphylococcus aureus floxacin, marbofloxacin) and susceptibility to (MRSA) is a well-known cause of skin and tetracycline. This resistance profile is unusual soft tissue infections in humans, both in for MRSA commonly isolated from livestock hospital and community settings. Compan- and poultry, due to coexistence of sensibility to ion animals (dogs, cats, horses) can experi- tetracycline and resistance to fluoroquinolones. ence such infections as the consequence of No antimicrobial resistance was detected in the penetrating wound and surgery. Nasal and nasal isolate. Multi Locus Sequence Typing re- skin carriage are proved predisposing factors. vealed two different new ST, namely ST 2716 Production animals are frequently healthy for MRSA from the liver and ST2715 for nasal carriers of the Livestock-associated sequence mucosa isolate. This accidental isolation of type 398 (ST398) at nasal and other body sites MRSA in a free-living wild animal opens new level. Cattle mastitis can be caused by MRSA. perspectives in MRSA spread. Surveillance Wild animals are thought to be less exposed on lineages and their prevalence should be to MRSA due to presumed uncontaminated implemented among wild animal population to environment and lack of antibiotics selective clarify host specificity and to assess zoonotic pressure. Yet, MRSA was recently detected in potential of S. aureus. free-ranging species in aquatic and terrestrial environment in North America. In wild rumi- nant, a low prevalence of ST398 MRSA car- n 81B riage was recently reported in healthy Iberian NASAL CARRIAGE OF METHICILLIN Ibex and red deer. S. aureus isolates included RESISTANT STAPHYLOCOCCUS AUREUS were obtained from a kid chamois (Rupi- IN CAPTIVE CHIMPANZEES (PAN capra r. rupicapra), that was euthanized by TROGLODYTES) gamekeepers, due to walking impairment and P. W. Hanley1, K. F. Barnhart2, J. S. Weese3; painful status, in autumn 2011 in north-western 1UT MD Anderson Cancer Center, Bastrop, Italian Alps. A post-mortem examination TX, 2Abbvie, Inc, North Chicago, IL, 3Univer- was performed. Samples for bacteriological sity of Guelph, Guelph, ON, CANADA. analysis were swabs collected from nasal cavi- ties and organs (brain, lung, liver, spleen and Methicillin resistant Staphylococcus au- kidney). After an overnight incubation in Brain reus (MRSA) in non-human primates within Hearth Infusion at 37°C, 100 µl of the pre- research facilities is reportedly widespread; enrichment were plated onto 5% sheep blood however, this is not reflected in the literature. agar. Bacteria were identified according to col- At the Keeling Center for Comparative Medi- ony morphology, hemolysis, Gram-stain and cine, we identified a trend of culturing MRSA

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 95 Poster Abstracts from clinical cases within our chimpanzee epidemic clone. Recent literature noted similar colony. Over a period of two years, sixty per- strains of S. aureus between human sanctuary cent of our S. aureus cultures were methicil- workers and chimpanzees in Africa; however, lin resistant. Based on this information, we none of the strains were methicillin resistant. investigated the overall prevalence of MRSA Further research is ongoing to determine the in chimpanzees, hypothesizing that the rate for prevalence of chimpanzee caretakers that carry captive chimpanzees may be higher due to lack MRSA and if the strains are similar to the of chimpanzee personal hygiene, close contact chimpanzees. while in captivity and antibiotic practices. Chimpanzees were trained to present their n 82B nostrils for sample collection via nasal swabs FIRST IDENTIFICATION OF that were subsequently tested for the presence CC398 METHICILLIN-RESISTANT of MRSA. Isolates were characterized by spa STAPHYLOCOCCUS AUREUS IN IRISH typing and PCR detection of Panton Valentine LIVESTOCK: VALUABLE LESSONS leukocidin genes. Of the 158 chimpanzees housed at our facility, we were able to sample F. C. Leonard1, Y. Abbott1, A. Burns1, D. Cole- 125 chimpanzees within a one-month period. man2, B. Leggett1, G. Brennan3, S. Malhotra4, MRSA was isolated from 86 (69%) chimpan- B. Markey1, J. Sabirova4, A. Shore2; zees. Three chimpanzees were sampled twice 1University College Dublin, Dublin, IRE- for a total of 89 positive samples. Fifty-seven LAND, 2Dublin Dental University Hospital, (66%) isolates were PVL positive t008, con- Trinity College Dublin, Dublin, IRELAND, sistent with the sequence type (ST) 8 USA300 3National MRSA Reference Laboratory, St clone. All but two of the remaining isolates James’s Hospital, Dublin, IRELAND, 4Univer- corresponded to six spa types that were related sity of Antwerp, Antwerp, BELGIUM. to t008 (t818 (19, 22%) t024 (4, 4.7%), t197 Methicillin-resistant Staphylococcus aureus (2, 2.3%), t2030 (2, 2.3%) and 1 (1.2)% each (MRSA) belonging to multilocus sequence t9141, t682 and t6172. Single isolates of t116 type (MLST) clonal complex (CC) 398 and t1754, which are related to each other have not been reported previously in Ireland but distinct from ST8, were also found. In the although methicillin-susceptible S. aureus three chimpanzees that were sampled twice, CC398 have been identified in Irish pigs. two of the chimpanzees had the same strain in The aim of the study was to characterize the both cultures (t008, t818) and one chimpanzee first CC398 MRSA isolates recovered from had two different but related strains on each horses and pigs in Ireland and to identify their culture(t24, t818). MRSA positivity did not probable source of introduction. Isolates were correlate with age or sex. The housing location investigated using antimicrobial susceptibil- of chimpanzees within our facility may have ity testing, pulsed-field gel electrophoresis had an effect. Testing identified groups that (PFGE) using the restriction enzyme ApaI, spa were completely positive, other groups that typing and DNA microarray profiling using were completely negative and some groups the StaphType kit (Alere, Germany) to assign that showed a mixed pattern. Further evalu- isolates to MLST CCs/sequence types, SC- ation is being completed to determine which Cmec types and to detect a range of virulence groups are more volatile or affiliative and and resistance genes. Selected isolates were if this factor correlates with the number of also analysed using whole genome mapping. positive MRSA cultures. Overall, this project In June 2012 MRSA was isolated from an um- represents the first prevalence study for MRSA bilical abscess in a foal admitted to the UCD carriage within captive chimpanzees, indicat- veterinary hospital. A second isolate obtained ing both a striking prevalence of MRSA and from a nasal swab of one of the attending clini- the predominance of an important human

96 ASM Conferences Poster Abstracts cians was indistinguishable based on PFGE n 83B and microarray analysis and differed only by PERSISTENCE OF METHICILLIN-RESISTANT 1.1% on whole genome mapping, the differ- STAPHYLOCOCCUS AUREUS (MRSA) ST 398 ence being the acquisition of a novel genomic IN AN INDUSTRIAL RABBIT HOLDING AND island by the human isolate. Both isolates were FARM RELATED PEOPLE assigned to CC398-MRSA-IV, agr type I, spa type t011, and harboured the immune evasion F. Agnoletti, E. Mazzolini, C. Bacchin, E. cluster (IEC) genes sak, chp and scn, and a Tonon, G. Berto, L. Bano, I. Drigo; range of resistance genes. CC398-MRSA-IV Istituto Zooprofilattico Sperimentale delle are common in Belgium, the country of origin Venezie, Legnaro, ITALY. of the attending clinician. CC398 MRSA Livestock associated MRSA belonging to was not isolated from any other horses in the the clonal complex CC398 was described in hospital or from nasal swabs collected from animals and farm or slaughterhouse workers of horses in the stable of origin. No evidence swine, bovine and poultry industrial primary of onward spread of this strain was found. production. So far, despite S. aureus being Coincidentally, in September 2012 CC398- commonly found in rabbits and importantly af- MRSA-V was isolated from a joint abscess in fecting production, the clone was not reported a pig submitted for post mortem examination in rabbits raised for meat. In 2012-2013 forty to the UCD veterinary hospital. The farm had Italian industrial rabbit farms were selected recently depopulated and restocked with Irish by convenience according to staphylococcosis pigs and a small number of gilts imported severity to investigate on the genetic markers from Germany. Additional CC398-MRSA-V of S.aureus virulence. Within farm 60 female isolates were subsequently obtained from nasal breeders were chosen by systematic sampling swabs of pigs from the farm and spread to pigs and clinically examined. Between 20 and 35 on a second farm was also documented; this rabbits were also sampled by skin swabbing farm had bought pigs from the original farm. and tested for S. aureus, ten isolates were The MRSA isolates were typical of livestock selected to be phenotypically and genetically strains and displayed characteristics consistent characterized within the primary research with a German origin including SCCmec V, scope, this included mecA, mecC, tetM and agr type I, spa types t011 and t034, carriage of tetK genes detection. After detecting MRSA all a variety of resistance genes and lack of IEC S. aureus isolates of one holding were geneti- and enterotoxin genes. Whole genome map- cally characterized and nasal swabs of farm- ping showed a very close relationship between workers and relatives were tested for MRSA. these isolates and other published CC398 The sampling of rabbits was repeated after five strains. These findings clearly illustrate two months and extended to farm environment by distinct routes of introduction of LA MRSA surface swabs and air samples. MRSA isola- into a country previously free of infection: tion, detection and confirmation was carried human and animal carriers. Animal infections out using standard cultural, phenotypic and caused by CC398 MRSA are not notifiable in molecular methods. PCR was used to detect Ireland and animals being imported into this mecA and mecC. Although it was not described country are not required to be tested for this in rabbits so far, the latter was added to avoid organism. Inadequate biosecurity has resulted MRSA misclassification. MRSA were typed in the introduction of CC398 MRSA to Ireland. with MLST and spa typing. With the exception Countries still free of CC398 MRSA should of air samples, where S. aureus and MRSA take note. were counted, only one S. aureus isolate per rabbit/person/environment sample was characterized. Among 40 industrial holdings

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 97 Poster Abstracts tested only the rabbits of one holding were Background: Spectinomycin resistance in found MRSA colonized/infected. MRSA was staphylococci is mostly mediated by a spec- detected in 11 (48%) of 23 S. aureus isolates tinomycin 9-O-adenyltransferase encoded by detected in 25 rabbit skin swabs during the first the gene spc. Analysis of MRSA ST398 and sampling and in 17 (28%) of 59 S. aureus iso- MSSA ST9 isolates of human origin from lates detected in 60 rabbits sampled after five Spain, but also MRSA ST9 isolates from months in same holding. Five persons (four swine in China identified multi-resistance gene farm workers and one farmer’s relative) were clusters, which were most likely of entero- carrying MRSA in their nose. MRSA collected coccal origin. A closer look at these clusters during the first rabbit sampling were ST398, revealed the presence of a reading frame for spa-type t034 and t5210 and the MRSA a putative spectinomycin resistance gene. isolates from the humans belonged to same Database searches showed identical or closely spa-types and were ST398 as well. Two out related proteins in E. faecium, E. faecalis and of ten surface samples, and two out of three L. johnsonii. The corresponding proteins were air samples were contaminated with MRSA. referred to as either streptomycin 3’’-adenyl- Air samples were carrying 5 and 15 cfu/m3 transferase, spectinomycin 9-O-adenyltrans- MRSA counts. All MRSA isolated had tetM ferase, a putative toxin-antitoxin system, or as and tetK genes and none S. aureus from animal hypothetical proteins. Material and Meth- or environment carried the mecC gene. This ods: A first PCR assay was developed which paper reports MRSA ST398 t034 and t5210 amplified the entire gene. This amplicon was circulating in rabbits raised for meat produc- cloned into the E. coli-S. aureus shuttle vector tion and involving also people in contact with pLI50 and transferred into the recipient strain rabbits. After five months from first sampling S. aureus RN4220. Susceptibility testing was MRSA was still detected in animals and farm conducted by broth macrodilution for spec- environment. The case described was the only tinomycin and streptomycin. To see whether one out of a considerable farm sample and spw is present in other staphylococci, a second may represent the initial spreading of the clone PCR assay was developed that amplified an among the rabbit meat primary production sec- internal spw segment. This PCR assay was tor. Clonal spreading of CC398 from breeders applied to four spectinomycin-resistant, but to slaughter rabbits should be prevented. spc-negative MRSA CC398 isolates from fresh turkey meat or turkey meat products. Results: n 84B In comparison with S. aureus RN4220 and S. aureus RN4220 carrying the shuttle vector IDENTIFICATION OF THE NOVEL pLI50, S. aureus RN4220 transformants carry- SPECTINOMYCIN RESISTANCE GENE SPW ing the putative spectinomycin resistance gene IN MRSA AND MSSA OF HUMAN AND exhibited an at least 16-fold increase in the ANIMAL ORIGIN MIC of spectinomycin. In contrast, the MIC S. Wendlandt1, B. Li2, C. Lozano3, Z. Ma2, C. values for streptomycin were 4 mg/L for all Torres3, S. Schwarz1; tested isolates. Of the four isolates from fresh 1Institute of Farm Animal Genetics, Friedrich- turkey meat or turkey meat products, three Loeffler-Institut (FLI), Neustadt-Mariensee, were also negative for spw while the remaining GERMANY, 2Shanghai Vetrinary Research isolate from a seasoned turkey breast schnitzel Institute, Chinese Academy of Agricultural was positive in the spw-specific PCR.Conclu - Scienes, Shanghai, CHINA, 3Biochemistry and sions: The gene in the multiresistance gene Molecular Biology, University of La Rioja, clusters of MRSA CC398 as well as MSSA/ Logrõno, SPAIN. MRSA ST9 of human and porcine origin, respectively, represents a novel spectinomycin resistance gene, designated spw. Moreover, the

98 ASM Conferences Poster Abstracts spw gene is likely of enterococcal origin and collected from all caregivers and veterinarians thus represents another example of the gene (n=22). All swabs were inoculated into a pre- flux between enterococci and staphylococci. enrichment medium containing Brain-Heart More expanded analyses of spectinomycin- broth with 5% NaCl broth. After overnight resistant staphylococci are needed to determine incubation at 37°C, hundred microliters how widespread is the spw gene in staphylo- were streaked on SAID agar (bioMérieux). cocci of different clonal lineages and different Suspected S. aureus colonies were subcultured geographical and host origins. on blood agar and identification was confirmed by MALDI-TOF (Vitek MS, bioMérieux). n 85B All S. aureus isolates were tested by PCR for the presence of nuc gene (a species-specific STAPHYLOCOCCUS AUREUS CARRIAGE IN marker), mecA and mecC genes. Genetic CAPTIVE NON-HUMAN PRIMATES AND IN characterization was performed using DNA CAREGIVERS AT THE “LE PARC DE LA TÊTE microarray (StaphyType, Alere). Fourteen out D’OR” ZOO IN LYON, FRANCE. of the 22 lemurs were positive for S. aureus J. Bietrix1, J. Tasse1, A. Sapin1, M. Bes1, (N, n=8; T, n=8; R ,n=6). All were methicillin- A. Tristan1, F. Vandenesch1, G. Douay2, F. susceptible (MSSA), belonged to the clonal Laurent1; complex CC49 and harboured the same micro- 1French National Reference Centre for Staphy- array profile. Fourteen out of the 22 caregivers lococci, International Centre for Infectiology and veterinarians were detected as S. aureus Research - Inserm U1111, Hospices Civils de carriers. Surprisingly, 12 were methicillin- Lyon, Lyon, FRANCE, 2Jardin Zoologique - La resistant S. aureus (MRSA), among which 11 parc de la Tête d’Or, Lyon, FRANCE. belonged to the CC5 pediatric clone, and one belonged to CC398. The two remaining human During the last decade, the emergence of MSSA were identified as CC398 MSSA and livestock-associated methicillin-resistant CC30 MSSA, two clones highly prevalent in staphylococcus aureus (LA-MRSA) in human MSSA French carriers in the community. Our population has raised questions about their study showed that S. aureus is able to colonize host specificity and animal/human transmis- captive lemurs and is associated to nasal, sion. If Staphylococcus aureus has been oral and/or rectal carriage. A single CC49 reported in horses, pigs, cattle, poultry and clone, classically considered as well adapted companion animals, only few data are avail- to animal, has disseminated in lemurs, but no able about carriage in captive wildlife at zoos. evidence of transmission to caregivers was The goal of our study was i) to determine observed. Conversely, the worrisome diffusion staphylococcus aureus carriage in a non- of a CC5 MRSA clone within the zoo worker human primate population of the “Le parc de population is surprising and could be poten- la Tête d’Or” zoo, Lyon, France; ii) to compare tially due to another animal reservoirs, even if Staphylococcus aureus carriage isolates from this genetic background is classically rather a wild animals and from their caregivers and vet- human-related clone. Further epidemiological erinarians; iii) to investigate potential transfer investigations are underway to determine the between both groups. Nasal (N), throat (T) and sources and the reasons for this high preva- rectal (R) swabs were collected on 22 lemurs lence of MRSA in caregivers and veterinarians belonging to different species (Lemur catta, at the “Le parc de la Tête d’Or” zoo. n=6; Varecia variegata variegata, n=6; Varecia variegata subcincta, n=6; Varecia rubra n=2, Eulemur rubriventer, n=2). Nasal samples were

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 99 Poster Abstracts n 86B ity testing of S. aureus RN4220 and S. aureus COMPLETE SEQUENCE OF THE MULTI- RN4220 carrying the recombinant shuttle RESISTANCE PLASMID PV7037 FROM A vectors did not show differences in the MIC PORCINE MRSA INCLUDING TWO NOVEL values suggesting that the ABC transporter and RESISTANCE GENES rRNA methylase genes have no function in antimicrobial resistance. Conclusions: The co- S. Wendlandt1, B. Li2, Z. Ma2, S. Schwarz1; location of seven functionally active antimicro- 1Institute of Farm Animal Genetics, Friedrich- bial resistance genes together with a cadmium Loeffler-Institut (FLI), Neustadt-Mariensee, resistance operon bears the risk of co-transfer GERMANY, 2Shanghai Vetrinary Research and co-selection of resistance genes, but Institute, Chinese Academy of Agricultural also persistence of resistance genes even if Scienes, Shanghai, CHINA. no direct selective pressure by the use of the respective antimicrobial agents is applied. The Background: The two novel resistance genes sequence of plasmid pV7037 confirms the lsa(E) and spw have been identified as parts ability of S. aureus to acquire genetic material of a multi-resistance gene cluster in MRSA/ from other bacteria and to generate novel mo- MSSA. In the porcine MRSA ST9 isolate saic plasmids with numerous resistance genes SA7037, this multi-resistance gene cluster was from different sources. identified on the ca. 41-kb plasmid pV7037 of which only 17.5 kb have been sequenced previously. The aim of this study was determine n 87B the complete sequence of plasmid pV7037 to NOVEL PSEUDO SCCMEC ELEMENT gain insight into its structure and organization. (ΨSCCMEC57395) IN METHICILLIN-RESISTANT Material and Methods: In addition to the STAPHYLOCOCCUS PSEUDINTERMEDIUS previously sequenced 17.5-kb XbaI fragment, CC45 the three remaining XbaI fragments of ca. 4.5, S. Schwendener,1 P. Chanchaithong,2 N. Pra- 6.5 and 12.5 kb cloned in the shuttle vector pasarakul,2 A. Rossano,1 S. E. Blum,3 D. Elad,3 pLI50 were sequenced by primer walking. The and V. Perreten1; entire plasmid sequence was assembled and Institute of Veterinary Bacteriology, Vetsuisse investigated for reading frames. In addition, Faculty, University of Bern, Bern, SWITZER- two reading frames, one coding for an ABC LAND1; Department of Veterinary Microbio- transporter and the other for a rRNA methyl- logy, Faculty of Veterinary Science, Chula- ase, were also cloned into pLI50 and expressed longkorn University, Bangkok, THAILAND2; in S. aureus RN4220 to investigate their poten- and Division of Bacteriology and Mycology, tial role in antimicrobial resistance. Antimicro- The Kimron Veterinary Institute, Bet Dagan, bial susceptibility testing of S. aureus RN4220 ISRAEL.3 carrying each of the two recombinant shuttle vectors was performed by broth microdilution. Genetic characterization of methicillin-re- Results: Plasmid pV7037 proved to be 40,971 sistant Staphylococcus pseudintermedius bp in size. Besides the previously determined (MRSP) from Thailand and Israel revealed resistance gene cluster, it carried a functionally the presence of a predominant atypical clonal active tet(L) gene, a complete cadDX operon lineage which was not typeable by SmaI-PFGE and also a variant of the β-lactamase transpo- and SCCmec typing. A novel pseudo staphylo- son Tn552. Two single bp deletions, which coccal cassette chromosome (ΨSCCmec57395) resulted in frame shifts, functionally deleted element was identified in MRSP strain 57395 the genes for the BlaZ β-lactamase and the (ST45) by whole genome sequencing. The signal transducer protein BlaR1 in this Tn552 12,282-bp ΨSCCmec57395 element contained variant of pV7037. Comparative susceptibil- a class C1-like mec gene complex but no ccr

100 ASM Conferences Poster Abstracts genes. In addition to the methicillin resistan- to mecC carriage in 2011, we investigated the ce gene mecA, ΨSCCmec57395 also carried presence of this novel methicillin resistance determinants of resistance to heavy metals, determinant in the isolates from mara, a large such as arsenic, cadmium, and copper. Bsu36I rodent species native to South America. Ob- restriction analysis of the ΨSCCmec57395 ele- jective: To identify mecC in S. aureus ST130 ment amplified by long range PCR revealed isolates from mara and characterize their anti- the presence of a of genetic elements similar microbial susceptibility patterns, virulence and to ΨSCCmec57395 in 29 additional isolates of resistance gene content. Material and meth- MRSP ST45, and in 2 MRSP ST179, 1 MRSP ods: All seven isolates were screened by mecC ST57, and 1 MRSP ST85 isolate(s), which all PCR. Whole genome sequencing was per- belonged to clonal complex CC45. The strains formed on two randomly selected mecC-posi- originated from healthy and diseased dogs and tive isolates to analyze SCCmec sequence and cats, as well as from the environment of one presence of virulence and resistance genes. An- clinic. Cfr9I-PFGE and dru typing allowed to timicrobial susceptibility was tested by broth further distinguish between CC45 isolates from microdilution using commercial MIC plates the two different countries. Microarray analy- (Sensititre, TREK diagnostics). Results: All sis also identified genes that confer resistance isolates harboured mecC. The mecC sequences to β-lactams (mecA; blaZ), aminoglycosides in two sequenced strains were 100% identi- [aac(6‘)-Ie–aph(2‘)-Ia; aph(3‘)-III; ant(6)-Ia], cal to that described in S. aureus LGA251. macrolides and lincosamides [erm(B)], tetracy- Both strains carried SCCmec XI with 23 SNPs clines [tet(M)], trimethoprim [dfr(G)], strepto- compared to the prototype of SCCmec XI in thricin [sat4], and chloramphenicol [catpC221]. LGA251. The only putative resistance gene Fluoroquinolone resistance was attributed to detected in addition to mecC and blaZ (as part specific amino acid substitutions, namely Ser- of the mec complex class E) was norA. The 84Leu in GyrA and Ser80Ile and Asp84Asn in following virulence factors were detected: hla,

GrlA. The ΨSCCmec57395 element represents a hlb, hlgACB, lukED, eta, etb, edin-B, set2, new class of SCCmec and has been identified set3, set4, set5, set7, set10 and a previously in MRSP of CC45, which is a predominant described variant of etd. A 3.3 kb deletion was clonal lineage in Israel and Thailand. observed in the collagen-adhesin gene (cna) as previously described in ST130. The two strains n 88B were resistant to cefoxitin but susceptible to oxacillin according to current breakpoints for METHICILLIN-RESISTANT MRSA detection, and displayed susceptibility STAPHYLOCOCCUS AUREUS CARRYING to all non-beta-lactam agents tested. Discus- MECC IN CAPTIVE MARA sion and conclusions: This study provides C. Espinosa-Gongora1, E. M. Harrison2, A. further evidence of the broad host range of Moodley1, L. Guardabassi1, M. A. Holmes2; MRSA ST130. The strains from mara had a 1Department of Veterinary Disease Biology, set of virulence factors and genomic features Faculty of Health and Medical Sciences - previously observed in ST130 originating from University of Copenhagen, Frederiksberg C, other host species, suggesting that the genome DENMARK, 2Department of Veterinary Medi- of this lineage is conserved. Methicillin resis- cine - University of Cambridge, Cambridge, tance was not accompanied by resistance to an- UNITED KINGDOM. timicrobial classes other than beta-lactams and susceptibility data matched with the resistance Introduction: Staphylococcus aureus se- genotype since norA is a housekeeping gene quence type ST130 was isolated from seven that confers fluoroquinolone resistance only captive mara (Dolichotis patagonum) at a Dan- when overexpressed. ish Zoo in 2010. After ST130 was associated

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 101 Poster Abstracts n 89B n 90B METHICILLIN-RESISTANT A NOVEL PEPTIDE-PEPTOID HYBRID STAPHYLOCOCCUS AUREUS (MRSA) ACTIVE AGAINST METHICILLIN RESISTANT SCREENING AT A TERTIARY VETERINARY STAPHYLOCOCCUS PSEUDINTERMEDIUS HOSPITAL: IS TESTING COST-BENEFICIAL I. Greco, P. P. Damborg, L. Guardabassi, P. R. FROM AN INTEGRATED HUMAN-ANIMAL Hansen; PERSPECTIVE? University of Copenhagen, Copenhagen, J. P. Ferreira1, T. Birkland2, K. Anderson2, M. DENMARK. Correa2; Introduction: Limited treatment options are 1SAFOSO, Bern, SWITZERLAND, 2North available against methicillin-resistant Staphy- Carolina State University, Raleigh, NC. lococcus pseudintermedius (MRSP) infec- Methicillin-resistant Staphylococcus aureus tions in small animals. In recent years, much (MRSA) is an antimicrobial resistant organism research has been directed towards antimicro- of international significance to human and vet- bial peptides as potential antimicrobial agents. erinary medicine. In human medicine, policies More recently, the focus of this research has proposed and tested for MRSA control include been extended to peptoids, synthetic ana- variations of universal patient screening and logs of peptides whose structure improves surveillance programs. In veterinary medicine pharmacological properties of natural peptides. information on MRSA control is scarce. Objec- Objective: To identify novel antimicrobial tive: To develop a cost-benefit (CB) analysis peptide-peptoid hybrids for topical use against from an integrated human-animal that could be pyoderma and otitis in dogs. Methods: Ten used for policy development and including all peptoids and peptide-peptoid hybrids were animals admitted for hospitalization to NCSU obtained by solid phase synthesis. Minimum CVM Veterinary Teaching Hospital. Materi- inhibitory concentrations (MICs) of the als and methods: A model was developed to purified products were determined for 50 estimate the costs of MRSA of all animals clinical S. pseudintermedius isolates by broth admitted to the hospital taking into consider- microdilution. The most potent candidate was ation the projected economic benefits of the tested against 10 methicillin resistant strains, prevention of human infections. Results: The and its hemolytic activity on human erythro- baseline model used the most plausible inputs cytes was measured by determining the half and different scenarios were considered in the maximal effective concentration (EC50) after 1 sensitivity analysis. The cost of the screening hour of exposure. Time kill kinetics of the lead policy was estimated at $320,104 and exceed- compound was studied at 1x, 2x and 4x MIC. ed the savings of about $183,409. Variations in Results: The MICs of the ten compounds the input assumptions, most notably the addi- ranged from 2 to 32 µg/ml. Compound B1, a tional cost for treating a human case, rendered peptide-peptoid hybrid, showed the highest a variety of possible outcomes. Conclusions: antimicrobial activity (MIC=2 µg/ml). The Our study suggests that it is not beneficial to hemolytic activity of B1 was low (EC50>128 implement MRSA screening programs in vet- µg/ml). The compound showed ability to kill erinary hospitals from an integrated economic MRSP within 2 hours at 2x MIC and 1 hour human-animal perspective. at 4x MIC. No difference in MIC values was observed between MRSP and MSSP. Discus- sion/Conclusions: We identified a peptide- peptoid hybrid which is active against MRSP and potentially may be used in the treatment of canine pyoderma and otitis. This molecule was

102 ASM Conferences Poster Abstracts selected as the lead candidate for further de- further tested for their ceftaroline susceptibility velopment. Optimization of drug formulation using Etest. Results: Our collection included and in vivo toxicity and efficacy studies using 23 (30%) mecC positive, phenotypically oxa- animal models will be the next steps. cillin susceptible isolates (MIC range, 0.5-2 mg/L) and all of them were resistant towards n 91B cefoxitin using disk diffusion test. With regard to ceftaroline susceptibility testing by disk dif- MECC AND CEFTAROLINE SUSCEPTIBILITY fusion, only four isolates revealed “borderline” IN CLINICAL S. AUREUS ISOLATES zone diameters of 21 mm and subsequent broth B. Strommenger1, F. Layer1, A. Kriegeskorte2, microdilution revealed a susceptible pheno- I. Klare1, C. Cuny1, K. Becker2, G. Werner1; type for all of them (MIC range, 0.5-1 mg/L). 1Robert Koch Institute, Wernigerode, GER- Among the 72 isolates isolates with zone di- MANY, 2Institute of Medical Microbiology, ameters above 22 mm, we detected three non- University Münster, GERMANY. susceptible isolates (zone diameters: 22-23 mm; MICs: 2 mg/L). MICs for the ceftaroline Objectives: Ceftaroline fosamil (ceftaroline) is susceptible isolates were 0.25 mg/L (n=3), 0.5 a novel cephalosporin with broad-spectrum ac- mg/L (n=32) and 1 mg/L (n=38), respectively. tivity against Gram-positive pathogens, includ- Subsequent Etest for selected isolates revealed ing methicillin-resistant Staphylococcus aureus MICs which were generally 2- to 3-fold lower (MRSA). It was approved in the EU in August than those generated by broth microdilution. 2012 for the treatment of complicated skin and Conclusion: Preliminary results demonstrate a soft tissue infections and community acquired low prevalence of ceftaroline non-susceptibil- pneumonia. The activity of ceftaroline against ity among mecC positive S. aureus (4% of all MRSA is attributed to its ability to inhibit the isolates investigated). biochemical activity of PBP2a more efficiently than other presently available ß-lactams. Previous studies suggested that the relatively n 92B low minimal inhibitory concentration (MIC) ANALYSIS OF SOCIETAL BENEFITS AND towards oxacillin found in mecC positive S. COSTS OF PREVENTING INTRODUCTION OF aureus is most likely a result of a higher affin- MRSA CC398 AMONG PIGS IN SWEDEN ity of the mecC encoded PBP2a for oxacillin S. Höjgård1, O. Aspevall2, B. Bengtsson3, H. compared to the cephalosporin cefoxitin. Thus, Ericsson Unnerstad3, S. Haeggman2, M. Lind- susceptibility of these isolates towards ceftaro- berg4, S. Nilsson5, D. Viske5, H. Wahlström3; line appeared as an interesting question. So far, 1Swedish University of Agricultural Sciences there is only limited data on susceptibility of and AgriFood Economics Centre, Lund, SWE- mecA and mecC positive MRSA towards cef- DEN, 2Swedish Institute for Communicable taroline, especially with respect to the German Disease Control, Stockholm, SWEDEN, 3Na- S. aureus population. Material and methods: tional Veterinary Institute, Uppsala, SWEDEN, We investigated the susceptibility of mecC 4Federation of Swedish Farmers and Swedish positive S. aureus towards ceftaroline using Animal Health Service, Uppsala, SWEDEN, a collection of 76 human isolates originating 5Swedish Board of Agriculture, Jönköping, from all over Germany. Based on spa-typing, SWEDEN. the majority of isolates was affiliated to clonal lineage ST130. Initially, all isolates were tested Background: A reservoir of MRSA CC398 for their susceptibility towards cefoxitin and in pigs implies a risk for transmission to ceftaroline by disk diffusion methodology humans in contact with live pigs. The Swedish followed by broth microdilution according to nucleus- and multiplying herds are considered EUCAST guidelines. Selected isolates were free from MRSA and monitoring in fattening

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 103 Poster Abstracts pigs at slaughter has only revealed one positive dations. These were estimated using Swedish finding. If MRSA CC398 is introduced and data on the expected number of live boars spread among pigs in Sweden and persons in (batches of semen) imported annually, duration contact with live pigs would be considered and costs of the quarantine period, costs of col- a risk group for carriage, costs would arise lecting and analyzing bacteriological samples when people in the risk group visit health care and prices of boars and semen. Results: A institutions. Trade of live animals is the most model for estimation of the annual societal important risk factor for MRSA CC398 in pigs benefits and costs has been created and values and the favourable situation in Sweden is prob- are under calculation. ably mainly due to limited import of live pigs. Since imports of live animals are expected to n 93B increase due to structural changes in breeding THE EFFECT OF ROUTINE ANTIMICROBIAL practices in Sweden, the Swedish Farmers’ THERAPY ON CANINE COMMENSAL Disease Control Programme (SDS) has issued STAPHYLOCOCCI recommendations to prevent MRSA from being introduced through import of live animals V. M. Schmidt, N. J. Williams, S. Dawson, N. or semen. The breeding companies operating McEwan, T. Nuttall; in Sweden abide to the recommendations. University of Liverpool, Neston, UNITED The present study ventures to investigate if KINGDOM. the societal benefits of abiding to the recom- Meticillin resistance is emerging at an alarm- mendations are large enough to outweigh their ing rate among staphylococci. Antimicrobial societal costs. Methods: The risk group, i. e. therapy, particularly with either ß-lactams persons in contact with live pigs, was estab- or fluoroquinoles, has been associated with lished as pig farmers, slaughterhouse workers, carriage of and/or infection with meticil- pig transporters, pig veterinarians and house- lin resistant staphylococci in man and other hold members to these groups. It was assumed animals. This study was undertaken to examine that following the import recommendations the canine mucosal staphylococcal populations would preserve Sweden free of MRSA CC398 before and following therapy with systemic in pigs. The societal benefits were considered antimicrobials. Dogs (n=126) requiring routine as the value of health care expenditures and systemic antimicrobial therapy either with production losses caused by MRSA CC398 in cephalexin (CFX; n=31), amoxicillin-clavu- the risk group that would be avoided if MRSA lanate (AC; n=29), cefovecin (CVN; n=25), was not introduced among Swedish pigs. clindamycin (CD; n=28) or a fluoroquinolone These were estimated using Swedish data on (FL; n=13) without prior antimicrobial treat- the incidence of skin infections in primary- ment for 3 months were enrolled. Mucosal and hospital care for the period 2001 - 2011, swabs (nasal and perineal) were collected from Swedish data on treatment costs and wage each dog before therapy (D0), end of therapy costs, and assuming that human prevalence of (End) and one and three months after therapy MRSA CC398 in Sweden, without the recom- (M1 and M3 respectively). Staphylococcus mendations, would have been the same as in spp. were isolated and identified phenotypi- the Netherlands or, alternatively, in Denmark. cally and tested for antimicrobial susceptibility The societal costs were considered as costs by disc diffusion. Isolates with an oxacillin of abiding to the recommendations including resistant phenotype were further investigated quarantine procedure, sampling of animals, for the carriage of the mecA gene by PCR laboratory analyses, culling of colonized assay. Outcome measures were oxacillin resis- animals, destroying of contaminated semen tance and carriage of the mecA gene (mecA), and adverse effects on profits due to the delay multidrug resistance (MDR; ≥3 antimicrobial of genetic progress caused by the recommen-

104 ASM Conferences Poster Abstracts classes), and carriage of coagulase positive n 94B (CoPS) and negative staphylococci (CoNS). ISOLATION AND CHARACTERIZATION There was an overall trend for increased car- OF PHAGES WITH LYTIC ACTIVITY riage of mecA gene positive staphylococci at AGAINST METHICILLIN-RESISTANT End following general antimicrobial therapy STAPHYLOCOCCUS AUREUS STRAINS and in the CFX, AC, CVN and FL groups BELONGING TO THE CLONAL COMPLEX 398 followed by a decline towards pre-treatment levels at M3, particularly for CoNS. However A. Fetsch, B. Kraushaar, M. D. Thanh, J. A. these findings were only statistically signifi- Hammerl, J. Reetz, S. Hertwig; cant for general antimicrobial therapy. MDR Federal Institute for Risk Assessment, Berlin, staphylococci also tended to increase at End GERMANY. following general antimicrobial therapy and Some years ago, Methicillin-resistant Staphy- in the CFX, CVN and FL groups and decline lococcus (S.) aureus (MRSA) emerged in by M3 but these findings were not statistically livestock worlwide. In Germany, 90% of these significant. Carriage of CoPS decreased at End livestock-associated MRSA (LA-MRSA) in the majority of antimicrobial groups but isolates can be attributed to the clonal complex recovered by M3, particularly in the CFX and 398 (CC398). People with occupational CD groups. These changes were significant livestock contact are at high risk of coloniza- in the CD group. On the other hand, CoNS tion hence, invasive infections with CC398 carriage increased at End in all groups other MRSA have already been reported in several than CFX and CD and decreased again by M3 countries. This calls for intervention strategies in the majority of the groups but did not reach focusing on a prevalence reduction among significance (Cochran and McNemar; P≤0.05). livestock. A reduction of MRSA CC398 in This study demonstrated an association livestock might be achieved by application between antimicrobial therapy and increased of virulent phages. However, there have yet carriage of MDR and oxacillin resistant and no reports been published on phages lysing mecA positive canine commensal staphylo- MRSA CC398 strains. In this study three cocci and their subsequent recovery to near virulent phages (PSa1, PSa2 and PSa3) with pre-treatment levels three months after the lytic activity against MRSA CC398 strains end therapy. Interestingly, whilst there was an were isolated from German pig husbandries. increase in carriage of CoNS during therapy, Morphologically the phages are members CoPS carriage decreased. Increased carriage of of the family Podoviridae and exhibited an the mecA gene and MDR was more common identical host range. They lysed 52 (60%) out among CoNS than CoPS. It is therefore likely of 86 tested MRSA CC398 strains represent- that the increase in CoNS populations during ing 18 different but most common spa types. therapy is due to the fact that CoNS are gener- While the PSa1 and PSa3 genomes have a ally more antimicrobial resistant than CoPS similar size of approximately 17.5 kb, the populations. PSa2 genome is somewhat larger (ca. 18.5 kb). Southern hybridization revealed strong DNA homologies between the phages confirmed by sequence analysis of cloned restriction fragments and PCR products. Moreover, the whole PSa3 genomic sequence (17,602 bp) showed close relationship to 44AHJD-like phages, which are not known to contain virulence-associated genes. To assess whether these phages

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 105 Poster Abstracts might be suitable candidates for applications in prevalences ranging between 0.15 and 1.15 % vivo, in vitro experiments were carried out in were achieved among pig carcasses indicat- which the number of MRSA CC398 could be ing that the pig slaughter chain generally reduced by up to four log10 units. The phages includes process steps with the capacity of were stable at a wide range of temperatures limit carcass contamination. Especially scald- and pH values. Further experiments will be ing and singeing can lead to a significant conducted to assess the potential of these lytic reduction of superficial MRSA contamination phages to reduce the total LA-MRSA burden during the first half of the slaughter process. for public health. This work was accepted for Nevertheless, scenario analyses showed that publication: Kraushaar, B. et al. (2013). Isola- the low MRSA outcome prevalence can only tion and characterization of phages with lytic be guaranteed if recontamination during the activity against methicillin-resistant Staphylo- ongoing slaughter process is obviated. In order coccus aureus strains belonging to clonal com- to ensure a low MRSA load on pig carcasses at plex 398. Arch Virol, in press: DOI: 10.1007/ the end of slaughter the abattoir should primar- s00705-013-1707-6 Acknowledgement: This ily concentrate on controlling the process pa- study has been partially supported by a grant rameters of scalding and singeing and avoiding from the Bundesministerium fuer Bildung und recontamination on subsequent process steps. Forschung, “MedVet-Staph” (01KI1014C). Acknowledgements: This work was carried out within the Project MedVet-Staph funded n 95B by the German Bundesministerium für Bildung und Forschung, Grant Nr. 01Kl1014C. MODELING THE TRANSMISSION OF LIVESTOCK ASSOCIATED METHICILLIN- RESISTANT STAPHYLOCOCCUS AUREUS n 96B ALONG THE PIG SLAUGHTER LINE THE EFFECTIVENESS OF BACTERIOPHAGES AGAINST METHICILLIN-RESISTANT B. Lassok, H. Sharp, J. Brandt, A. Fetsch, A. STAPHYLOCOCCUS AUREUS NASAL Käsbohrer, B. A. Tenhagen; COLONIZATION IN PIGS IN VITRO, EX VIVO Federal Institute for Risk Assessment, Berlin, AND IN VIVO GERMANY. K. M. Verstappen1, P. Tulinski1, B. Duim1, The study introduces a new approach for a A. C. Fluit2, J. Carney3, A. van Nes1, J. A. qualitative transmission assessment of MRSA Wagenaar1; throughout the pig slaughter process. Based 1Utrecht University, Utrecht, NETHERLANDS, on prevalence data found in literature the 2University Medical Centre Utrecht, Utrecht, MRSA contamination and elimination rates of NETHERLANDS, 3Novolytics Ltd., War- each individual slaughter step were estimated. rington, UNITED KINGDOM. The rates were used to set up a Monte Carlo simulation for modeling the propagation of Background Methicillin-resistant Staphy- MRSA along the process chain and to quantify lococcus aureus (MRSA) is widely spread the impact of a variable initial prevalence among animals. Humans in contact with on the outcome prevalence on carcasses. animals are at increased risk of becoming colo- Sensitivity analyses for the model as well nised with this opportunistic pathogen. Bacte- as three different scenarios were performed riophages are generally specific for subsets of to estimate the impact of cross contamina- strains within a species and would be a promis- tion during slaughter and to determine the ing candidate therapeutic agent for eliminating process stages where hygiene interventions colonization by MRSA. The aim of this study are most effective. Regardless of the initial was to compare the efficacy of bacteriophage extent of MRSA contamination low outcome treatment on porcine nasal colonization with

106 ASM Conferences Poster Abstracts

MRSA in vitro, ex vivo, and in vivo. Methods n 97B Phages Fred*710 and Felix (developed for use IMPACT OF GENTAMICIN AND against human MRSA strains) were provided SILVER ON METHICILLIN-RESISTANT in a proprietary gel formulation by Novolytics STAPHYLOCOCCUS PSEUDINTERMEDIUS Ltd. (Warrington, UK). The in vitro effective- BIOFILM FORMATION ON ness of these phages was assessed by incubat- POLYMETHYMETHACRYLATE ing phages with MRSA strain V0608892/1 6 A. Singh, S. Morrison, J. Rousseau, E. Craw- (ST398) at 10 CFU/mL, measuring the OD600 hourly. The strain was grown on a porcine ford, J. S. Weese; mucosa explant and phages were applied to Ontario Veterinary College, University of investigate the ex vivo efficacy of treatment. To Guelph, Guelph, ON, CANADA. study the in vivo effect, phages were adminis- Methicillin-resistant Staphylococcus pseudin- tered for 5 days to caesarean-derived piglets termedius (MRSP) has emerged as the leading (N=8), which were experimentally colonized cause of surgical site infections (SSIs) in dogs. with the aforementioned MRSA strain to Biofilm formation has been suspected as a pos- assess in vivo effectiveness. Eight piglets re- sible virulence factor and reason for its rapid ceived a placebo. MRSA was enumerated from emergence. This is of particular concern for or- explants and nasal swabs by bacterial culture. thopedic procedures involving implants, since Six days after the last phage administration 8 biofilm formation on these foreign materials piglets received a nasal ointment with mupiro- can complicate infection. Polymethylmeth- cin for 5 days, 2 doses per day. Results MRSA acrylate (PMMA) is commonly used in total did not grow after incubation with phages joint arthroplasty and a variety of antimicrobial in vitro. On the explants colonization with additives have been used in an attempt to pre- MRSA was established with approx. 108 CFU/ vent bacterial adhesion and SSI. The objective explant. However, after application of phages of this study was to evaluate the impact of no reduction of colonization was observed. In addition of gentamicin or silver to PMMA on 16 piglets, which were colonized with MRSA MRSP biofilm formation. Six MRSP isolates with approx. 105 CFU/nasal swab, the numbers from dogs were evaluated. PMMA beads were of MRSA recovered from the nose were not prepared with 1.25% gentamicin, 1% micro- reduced after application of the phages or the silver, their combination or no additive, then placebo. Phages that were re-isolated from inoculated into MRSP suspension. Beads were pig’s noses were still effective against the rinsed to remove non-adhered (planktonic) original and the re-isolated MRSA strain. Mu- bacteria and then sonicated for 5 minutes to pirocin ointment eradicated MRSA from the remove biofilm-embedded bacteria. Serial dilu- nose and the mucosal explants. Conclusions i) tions of the sonicate were plated on Columbia The MRSA strain was not able to grow in the blood agar and colony forming units (CFU) presence of the Fred*710 and Felix phages in counted. Analysis of variance (ANOVA) was vitro. ii) Phages did not reduce porcine nasal used to compare biofilm growth (log10 CFUs) colonization ex vivo and in vivo, in contrast to between the four PMMA groups. A p value mupirocin. This may be due to physiological of <0.05 was considered significant. None properties of the pig mucosa rather than lack of the PMMA additives completely inhibited of efficacy of the phages. iii) The correlation MRSP biofilm formation. Gentamicin-loaded between the results from the ex vivo and in PMMA significantly reduced log10 CFUs vivo experiments suggests the potential of the compared with no additive (p = 0.04). Silver- explant model for pre-animal screening. loaded PMMA did not significantly reduce log10 CFUs compared with PMMA alone (p =

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 107 Poster Abstracts

0.97). The combination of gentamicin + silver isolates. The minimum inhibitory concentra- loaded PMMA did not significantly reduce tions of each Staphylococcus species was less log10 CFUs compared with gentamicin alone than 20 mg/ml and it appeared that after the 24 (p = 0.85). The results of this study indicate hr contact period at which the microbroth dilu- that gentamicin-loaded PMMA was effective tion test was read, this concentration resulted in reducing in vitro MRSP biofilm formation. in killing of all isolates. Yerba mate extracts or Silver-loaded PMMA had no effect on reduc- compounds purified from such extracts may be ing MRSP biofilm formation in vitro. This may of potential use for topical treatment of canine be a result of the concentration of microsilver infections caused by multiresistant Staphylo- tested in this study. Further in vitro and in vivo coccus species. study into PMMA additives that prevent MRSP biofilm formation is warranted. n 99B IN VITRO ANTIBACTERIAL AND ANTIBIOTIC- n 98B POTENTIATION ACTIVITIES OF PIPER ANTIBIOTIC EFFECT OF YERBA MATE TEA NIGRUM AND TELFAIRIA OCCIDENTALIS EXTRACTS ON METHICILLIN-RESISTANT AGAINST MULTIDRUG-RESISTANT STAPHYLOCOCCUS ISOLATES FROM DOGS BACTERIA K. A. Miller1, K. P. Burris2, D. A. Bemis1; J. K. Noumedem1, M. Mihasan2, J. Kuiate1, D. 1University of Tennessee College of Veterinary Cojocaru2, M. Stefan2, D. Djeussi1, V. Kuete1; Medicine, Knoxville, TN, 2University of Ten- 1University of Dschang, Cameroon, Dschang, nessee College of Agricultural Sciences and CAMEROON, 2University of Alexandru Ioan Natural Resources, Knoxville, TN. Cuza, Iasi, ROMANIA. The emergence of multidrug resistance in Background and Aim: Methicillin-resistant staphylococci and other bacterial pathogens Staphylococcus aureus (MRSA) is Multi-drug has led many investigators to seek alternative resistant pathogen (MDR), over-expressing, antimicrobial compounds that might circum- different efflux pumps which causes many vent resistance through potentially unique health problems in human, animals and even mechanisms of action. In this study extracts in agriculture. Because of this problem of of yerba mate tea were tested with modified resistance to conventional antibiotics, atten- agar disk diffusion and microbroth dilution tion is now being shifted towards biologically methods for their antimicrobial activity on active components from plant species used recent clinical isolates of methicillin-resistant as herbal medicine, as these plants may offer Staphylococcus aureus (MRSA), S. pseud- a new source of antibacterial. The present intermedius (MRSP) and S. schleiferi subsp. study was designed at evaluating the antibac- coagulans (MRSS) from dogs. Preliminary terial activities of the methanol extracts of testing with the quality control strain, S. aureus black Piper nigrum and Telfairia ociidentalis, ATCC 29523, gave reproducible growth two plants locally used in Cameroon to treat inhibition over a linear dynamic range of disk microbial infections, and their synergistic ef- contents from 40 mg to 160 mg. Disk content fects with antibiotics against a panel of twenty of greater than 20 mg of yerba mate tea extract nine Gram negative bacteria including some was required to produce measurable zones of phenotypes expressing active efflux pumps. growth inhibition. Each of the canine isolates Methods: The broth microdilution method was also inhibited by these concentrations of was used to determine the minimum inhibitory extract. Zone of growth inhibition diameters concentrations (MIC) of the extracts alone were greatest among S. aureus isolates and and in the presence of Phenylalanine-Arginine smallest for S. schleiferi subsp. coagulans β-Naphtylamide (PAβN), an efflux pumps

108 ASM Conferences Poster Abstracts inhibitor (EPI), as well as the MIC of antibiot- ment of “helper drugs” that reverse methicillin ics in association with two of the most active resistance or have a synergistic effect with extracts, Piper nigrum and Telfairia occiden- β-lactams. In order to explore the latter option, talis. The preliminary screening of the extract it is important to understand MRSA response composition was conducted according to the to therapeutic concentrations of β-lactams. standard phytochemical methods. Results: Objective: To investigate the growth response Phytochemical analysis showed the presence of MRSA to therapeutic and sub-therapeutic of alkaloids, phenols, flavonoids and tannins concentrations of cephalosporins widely in both plant extracts. Other chemical classes used in clinical practice, cefuroxime and of secondary metabolites were selectively cefotaxime. Methods: Two clinical strains present. The results of the MIC determination representing epidemic MRSA lineages, ST8 indicated that the extract from P. nigrum were (USA300) and ST398, were selected. MICs able to inhibit the growth of all the twenty nine of cefuroxime and cefotaxime were deter- studied bacteria within a concentration range mined by Etest® (bioMérieux). Two in vitro of 32 to 1024 µg/ml meanwhile T. occidentalis growth experiments were performed using the prevented the growth of 93.1% of such micro- Bioscreen® in microtitre plates for 48 hr and organisms. At MIC/2 and MIC/5, synergistic in traditional flasks for seven hr. In both ex- effects were noted with both extracts on more periments, cultures in early exponential phase than 70 % of the tested bacteria for seven (Bioscreen: 106 CFU/mL, flask: 107 CFU/mL), antibiotics, namely tetracycline (TET), doxy- were exposed to either cefuroxime, cefotaxime cyclin (DOX), ciprofloxacin (CIP), norfloxacin or no antibiotic as a control. A therapeutic (NOR), kanamycin (KAN), chloramphenicol concentration of 30 µg/mL was selected for (CHL) and erythromycin (ERY). Conlusions: both drugs based on PK calculations estimat- The overall results of the present study provide ing the serum concentrations achieved 1 hr information for the possible use of the studied after standard dosage (1.5-3 g q8 hr). The edible plants extracts in the control of bacterial sub-therapeutic concentration (0.3 µg/mL) was infections involving MDR phenotypes. They arbitrarily defined as 1/100 of the therapeutic also provide a good basis for further investiga- concentration. Optical density (OD600) was tions with the plant extracts for the control of measured every 20 min (Bioscreen) or 30 MRSA. min (flask). In addition, bacterial counts were

determined for each OD600 reading in the flask n 100B experiment. All experiments were performed in triplicate. Results: The USA300 strain GROWTH DYNAMICS OF METHICILLIN displayed cefuroxime and cefotaxime MICs of RESISTANT STAPHYLOCOCCUS AUREUS 16 and 24 µg/mL, respectively. Slightly lower USA300 AND ST398 EXPOSED TO MICs were observed for the ST398 strain (6 THERAPEUTIC CONCENTRATIONS OF and 8 µg/mL, respectively). Growth was not CEFUROXIME AND CEFOTAXIME inhibited and resembled that of the control R. P. Brochmann, A. Moodley, C. Friis, L. following exposure to the sub-therapeutic con- Guardabassi; centration of both cephalosporins. Both strains Veterinary Disease Biology, Copenhagen, exhibited a marked reduction in growth 3.5 hr DENMARK. after exposure to the therapeutic concentration. However based on bacterial counts, such Introduction: Methicillin-resistant Staphy- reduction was 2.5-fold for ST398 and 0.8-fold lococcus aureus (MRSA) are resistant to for USA300 regardless of the cephalosporin traditional penicillins and cephalosporins. used. Conclusions: Under in vitro conditions, As an alternative to new antimicrobial drugs, both MRSA strains were inhibited by cefurox- resistance could be overcome by develop- ime and cefotaxime concentrations achieved

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 109 Poster Abstracts during therapy, even though their MICs were patient contact compliance was 3% (123/4377) above the clinical breakpoint. This observation and 26% (1145/4377), respectively. Soap suggests that MRSA, even if regarded as clini- and water was used for 87% (1182/1353) of cally resistant to cephalosporins, are stressed observed hand hygiene attempts with a mean in the presence of therapeutic concentrations of contact time of 4 s (median 2 s, range 1-49 s), these drugs. These data support our hypothesis while alcohol-based hand sanitizer (AHS) was that β-lactam “helper drugs” may be developed used for 7% (98/1353) of attempts with a mean to target MRSA under such stressful condi- contact time of 8 s (median 7 s, range 1-30 s). tions. Gene expression profiling studies are The presence of the posters had no significant underway to identify potential target genes that effect on compliance, although some staff are up-regulated in MRSA during therapy with reported that they felt the posters did increase cephalosporins. their personal awareness of the need to perform hand hygiene, and the posters had some n 101B effect on product contact times. Conclusions: Overall hand hygiene compliance in veterinary VIDEO OBSERVATION OF HAND HYGIENE clinics in this study was low, and contact time PRACTICES DURING ROUTINE COMPANION with hand hygiene products was frequently ANIMAL APPOINTMENTS AND THE EFFECT below the recommended 15 s. Use of AHS was OF A POSTER INTERVENTION ON HAND low despite its advantages over hand washing HYGIENE COMPLIANCE and availability in the majority of clinics. The M. E. Anderson, J. M. Sargeant, J. S. Weese; poster campaign had a limited effect on its University of Guelph, Guelph, ON, CANADA. own, but could still be used as a component of a multimodal hand hygiene campaign. Improv- Background: Hand hygiene is considered the ing the infection control culture in veterinary most important infection control measure for medicine would facilitate future campaigns preventing transmission of hospital-associated and studies in this area. pathogens, including methicillin-resistant staphylococci, but there is little information available regarding hand hygiene frequency n 102B and technique used in veterinary clinics. The PENICILLIN, THE FIRST CHOICE FOR objectives of this study were to describe hand INFECTIONS IN HORSES IN SWEDEN hygiene practices associated with routine K. E. Bergström, U. Grönlund Andersson; appointments in companion animal clinics Department of animal health and antimicro- in Ontario, and the effectiveness of a poster bial strategies, Uppsala, SWEDEN. campaign to improve hand hygiene compliance. Results: Observation of hand hygiene Introduction: The presence of antimicro- practices was performed in 51 clinics for bial-resistant bacteria (ABR) in veterinary approximately 3 weeks each using 2 small medicine might bring unpremeditated use wireless surveillance cameras: one in an exam of broad spectrum antimicrobials in infected room, and one in the most likely location for animals. Current data of causative pathogens hand hygiene to be performed outside the and their antibiogram then becomes impor- exam room following an appointment. Data tant. Therefore equine surgical site infections from 38 clinics were included in the final (SSI) and traumatic wounds with symptoms of analysis, including 449 individuals, 1139 infection on admission to hospital (TW) were appointments before and after the poster sampled for culture and antibiogram testing. intervention, and 10894 hand hygiene op- Material and methods: During approximately portunities. Overall hand hygiene compliance one year each, between May 2009 and October was 14% (1473/10894), while before and after 2011, three Swedish equine hospitals collected

110 ASM Conferences Poster Abstracts samples of SSI and TW. Culture, conventional and their antibiogram should be continuously typing of the bacterial species, antibiogram collected. In this study 63 of 83 isolates (76%) testing (VetMIC) and when relevant, extended from sampled wounds were susceptible to genotyping i.e. for MRSA were performed ac- penicillin (Figure 1). Another common disease cording to standard routine methods (National in horses in Sweden is respiratory infections, Veterinary Institute, Uppsala, Sweden). SSI predominantly caused by virus and/or beta- were defined according to Centers for Disease haemolytic streptococci, the latter constantly Control and Prevention, USA (CDC) criteria, susceptible to penicillin. Conclusion: Penicil- except for allowance to exceed the deadline of lin is still an adequate first choice in many 30 days if there was a clear link to the surgery, infected horses in Sweden if antimicrobials are i.e. funikulitis after castration. TW was defined considered necessary. Figure 1: Bacterial spe- as lesions caused by trauma having clinical cies cultured from SSI (n=45) and IW (n=38) symptoms of infection (CDC) on admission to in three Swedish equine hospitals. * Penicillin hospital. Results and discussion: If antimicro- susceptible except for one penicillinas produc- bial treatment is considered necessary to clear ing Actinobacillus; NG/USP = no growth or an infection a narrow spectrum antimicrobial unspecific growth; A =Actinobacillus spp; is most ideal. To guide veterinarians of first BHS = beta-haemolytic streptococci; CPS = choice antimicrobial pending the culture re- coagulase positive staphylococci; EC = E. coli; port, current local data of causative pathogens O = other species

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 111 Index

Abbott, Y. 82B Bignoli, G. 34A Cohen, N. D. 63B Abdelbary, M. M. S5:1 Binek, M. 59B Cojocaru, D. 99B Abdul Aziz, S. 50A Birkland, T. 89B, S7:4 Cole, S. D. 52A Abraham, S. 24A Bissong, M. E. 12A Coleman, D. 82B Abu, J. 50A Biyikli, E. 31A Coleman, D. C. 70B Agersø, Y. S4:3 Blondeau, J. M. S6:1 Coppe, B. 20A Agnoletti, F. 83B Blum, S. E. 87B Correa, M. 89B, S7:4 Akkerboom, V. 78B Boardman, W. 72B Corrente, M. 44A Aksentijevic, K. 22A Börjesson, S. 49A Corrò, M. 55B Alves de Bos, M. S1:3 Cortimiglia, C. 36A Moraes, N. 61B Bos, M. E. S7:3 Couto, N. 23A, 27A, Amachawadi, R. G. S3:3 Böse, R. S5:1 62B, 71B Amos, N. A. 45A Braman, J. 8A Crawford, E. 66B, 67B, Anderson, K. 89B, S7:4 Brandt, J. 95B 97B Anderson, M. E. 101B Brazil, A. B. S1:6 Cremonesi, P. 34A, 3A, Andersson, U. G. 49A Breit, T. M. S4:5 80B Argudin, M. A. 28A Brennan, G. 82B Crombé, F. 10A Argudín, M. Á. 30A Brochmann, R. P. 100B Cuny, C. 91B, S5:1 Argudin, M. A. 33A, 73B Broens, E. S2:3 Dahl, J. 19A Asanin, J. 22A Bronzo, V. 3A Damborg, P. 60B Aspevall, O. 92B Brovarone, F. 51A Damborg, P. P. 90B Avery, B. 6A Brown, M. H. 72B Davies, P. R. 47A Avisani, D. 36A Brunnberg, L. 54B Davis, M. 14A Bacchin, C. 83B Bugayev, J. 14A Davis, M. F. S1:6 Baekbo, P. S6:3 Buonavoglia, D. 44A Dawson, S. 93B Ballhausen, B. S5:4 Burns, A. 82B de Lencastre, H. 35A Bandt, D. 70B Burris, K. P. 98B DeLeo, F. R. 77B Bano, L. 83B Busayo, O. 45A Djeussi, D. 99B Barbarini, D. 51A Buß, M. 37A Dohmen, W. S1:3, S7:3 Barberio, A. 3A Butaye, P. 10A, 28A, Dorado-Garcia, A. S1:3 Barnhart, K. F. 81B 30A, 73B Dorado-García, A. S7:3 Baron, P. S1:6 Buyukcangaz, E. 31A Douay, G. 85B Battisti, A. 36A Cain, C. 52A Drigo, I. 3A, 83B Beaman, A. 32A Capra, E. 34A Dritz, S. S. S3:3 Becker, K. 91B, S5:4 Carli, T. K. 31A Duim, B. 96B, S1:3, Belas, A. 23A, 27A, Carney, J. 96B S2:3, S4:5 62B Carrel, M. S1:5 Edwards, G. S5:1 Beloeil, P.-A. S7:1 Carretto, E. 51A Ehricht, R. 70B Bemis, D. A. 58B, 76B, Castiglioni, B. 34A, 3A, Eisnor, J. 6A 98B 80B Elad, D. 87B Bengtsson, B. 4A, 92B Centeno, M. 27A Elvstrøm, A. 19A Bergström, K. E. 102B Chanchaithong, P. 87B Empel, J. 16A Berto, G. 83B Châtre, P. 61B Ericsson Bertocchi, L. 3A Chen, M. M. 72B Unnerstad, H. 4A, 92B Bes, M. 20A, 85B Chou, C. 69B Espinosa- Besozzi, M. 80B Chrobak, D. 59B Gongora, C. 19A, 88B Bietrix, J. 85B Coelho, A. V. 71B Everett, J. B. 58B

112 ASM Conferences Index

Eyigor, A. 31A Haeggman, S. 92B Kania, S. S2:2 Fanuelsen, H. 41A Haenni, M. 20A, 61B Kania, S. A. 58B, 76B Farzan, A. 48A Haesebrouck, F. 10A, 30A Karriker, L. 32A Ferguson, J. S1:6 Hall, D. 13A, 2A Käsbohrer, A. 95B Ferreira, J. P. 89B, S7:4 Hallgren, T. 4A Keeling, M. L. 25A Feßler, A. 1A, 68B Hallin, M. S5:1 Kelner-Burgos, Y. 33A, 74B Feßler, A. T. 37A, 38A Hammerl, J.-A. A. 94B Kerwin, S. 63B Feßler, A. T. S1:2 Hanley, P. W. 81B Kidsley, A. 24A Fetsch, A. 33A Hansen, P. R. 90B Kinyon, J. 32A Fetsch, A. 74B, 94B, Hanson, B. 32A Kizerwetter- 95B, S1:4 Harrison, E. M. 46A, 88B, Świda, M. 59B Fitzgerald, R. OS:1, S4:4, S5:2 Kjelgaard- S5:1 Hartmann, F. A. 76B Hansen, M. 77B Fligg, D. 32A Hartung, J. 18A, S1:4 Kjellman, E. E. 53B Fluit, A. S4:5 Hasman, H. S5:1 Klare, I. 91B Fluit, A. C. 96B Hatcher, S. M. 2A Kluytmans, J. 78B, S1:3 Frana, T. 32A Havrysh, A. 35A Knudsen, L. K. 11A Franco, A. 36A He, T. 75B Kohn, B. 54B Frank, M. 26A Heaney, C. 13A Kohn, C. 8A Friedrich, A. W. 78B Heaney, C. D. 2A Kolbjørnsen, Ø. 41A Friendship, R. 48A, 5A, Hedbäck, H. 4A Komorowska, I. 16A 65B Heederik, D. S1:3 Kotsch, M. 26A Friese, A. 18A, S1:4 Heederik, D. J. S7:3 Kraushaar, B. 33A, 74B, Friis, C. 100B Herman, L. 10A 94B Gama, L. T. 23A Hertwig, S. 94B Kriegeskorte, A. 91B, S5:4 Gavier-Widen, D. 70B Heusinger, A. 26A Krogh, A. K. 77B Gocmen, H. 31A Heyndrickx, M. 10A Krüger, K. 18A Godbeer, S. M. 40A Himsworth, C. G. S5:3 Kuete, V. 99B Goh, Y. 50A Hodkinson, B. 14A Kuhn, D. S5:4 Goilo, D.-M. S2:3 Hoet, A. E. 8A Kuiate, J.-R. 99B Gold, R. M. 63B Höjgård, S. 92B Kurt, K. S5:1 Gómez-Sanz, E. 79B Holden, M. T. S4:4 Landin, H. 4A Goodband, R. D. S3:3 Holden, M. T. G. S3:1 Lanfranchi, P. 80B Goodman, A. E. 72B Holmes, M. A. 46A, 88B, Larsen, A. R. 11A, S4:4 Graveland, H. S1:3 S4:4 Larsen, J. 11A, 13A, Greco, I. 90B Holmes, M. A. S5:2 2A, S4:4, Greco, M. 44A Hordijk, J. S2:3 S5:1 Grice, E. 14A Hotzel, H. 70B Larsen, L. S. 11A Grönlund Intas, K. S. 31A Larssen, K. W. 41A Andersson, U. 102B Iverson, S. S1:6 Larsson, B. 4A Grönthal, T. 57B Jacques, B. 20A Lassok, B. 95B Grzesiak, A. 16A Jalali, M. 5A Latronico, F. 64B, 77B Gualdi, V. 80B Jonker, M. J. S4:5 Laurent, F. 20A, 85B Guardabassi, L. 100B, Josiah, O. 45A Lautenbach, E. 14A 19A, 39A, KABEMBA Lautenbach, E. L. S1:6 60B, 64B, LUKUSA, j. 21A Lauzat, B. 33A, 74B 77B, 88B, Kadlec, K. 1A, 27A, Lawhon, S. D. 40A, 63B 90B, S7:2 68B, S1:2 Layer, F. 91B, S5:1 Guenther, S. S5:2 Kaesbohrer, A. 74B Layman, L. 32A Guerra, B. 33A, 74B Kahya, S. 31A Lazaris, A. 70B

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 113 Index

Leggett, B. 82B Monecke, S. 70B Perencevich, E. N. S1:5 Leonard, F. C. 82B Moodley, A. 100B, Perreten, V. 79B, 87B, Li, B. 84B, 86B 59B, 60B, S3:2 Lindberg, M. 92B 61B, 64B, Persson, Y. 4A Lindholm, L. 43A 77B, 88B, Peters, G. S5:4 Lindsay, J. A. S4:1 S4:2 Peters, M. 70B Linhares, L. 47A Morgan, F. 46A Peters, T. 37A Lium, B. 7A Morgan, F. E. S4:4 Petersen, A. 11A, S4:4 Locatelli, C. 3A, 80B Moroni, P. 3A Piccinini, R. 34A Lofstedt, J. 56B Morris, D. 14A Pierce, E. 13A Logue, C. M. 17A Morris, D. O. 52A Piiparinen, H. 57B Lord, S. E. 17A Morris, D. O. S1:6 Piraino, M. 8A Love, D. 13A Morrison, S. 97B Pirart, F. 20A Lozano, C. 84B Mroczkowska, A. 16A Pisanic, N. 13A Lübke-Becker, A. 54B, S5:2 Muckle, C. A. 42A Pomba, C. 23A, 27A, Lucchina, A. 28A Müller, E. 26A 62B, 71B Luini, M. 34A Myers, K. 2A Porter, A. R. 77B Luini, M. V. 36A Nachamkin, I. 14A, S1:6 Pozzi, F. 34A Luyckx, K. 10A Nachman, K. 13A Prapasarakul, N. 87B Luzzago, C. 80B Nadimpalli, M. 13A Radu, S. 50A Ma, Z. 84B, 86B Nagaraja, T. G. S3:3 Ramirez, A. 32A Maaland, M. G. 39A Nava-Hoet, R. 8A Rangstrup- Madec, J.-Y. 20A, 61B Nazarali, A. S2:4 Christensen, L. 70B Magaji, A. 50A Neale, M. 24A Rankin, S. C. 52A Malhotra, S. 82B Nelssen, J. L. S3:3 Rankin, S. C. S1:6 Malvisi, M. 34A Nemeghaire, S. 28A, 30A Rantala, M. 43A, 57B Markey, B. 82B Nielsen, S. S. 64B Rasschaert, G. 10A Marstein, L. 41A Nilsson, S. 92B Reetz, J. 94B Marszałek, N. 16A Nitikanchana, S. S3:3 Riley, M. C. 76B Martella, V. 44A Nordmark, P. 4A Rinsky, J. 13A Martins, J. 71B Norström, M. 7A Roesler, U. 18A, S1:4 Mat, B. 31A Noumedem, J. K. 99B Rohlwing, I. 17A Mattsson, R. 70B Nowak, A. 16A Rolo, J. 35A Mazzolini, E. 83B Nübel, U. S5:1 Rossano, A. 87B McClure, J. T. 42A, 56B, Nuttall, T. 93B Rota, A. 55B 6A Nykäsenoja, S. 43A, 57B Rousseau, J. 97B McEwan, N. 93B O’Shea, K. 52A Rzewuska, M. 59B Médaille, C. 61B Odoi, A. 58B Saab, M. 42A, 56B, Meng, T. 17A Oke, A. A. 9A 6A Metcalf, D. 66B, 67B Oke, A. J. 9A Sabat, A. J. 78B Mieziewska, K. 4A Oliveira, M. 62B Sabirova, J. 82B Mihasan, M. 99B Orczykowska- Saffroy, L. 20A Millar, R. S5:3 Kotyna, M. 16A Salmenlinna, S. 43A Miller, K. A. 98B Pantosti, A. S5:1 Sanchez, S. 25A Miragaia, M. 35A Parisi, A. 44A Sapin, A. 20A, 85B Misic, A. 14A Park, J. 65B Sargeant, J. M. 101B Misic, D. 22A Parkhill, J. 46A, S4:4 Savini, V. 51A Mo, S. S. 39A Paterson, G. K. 46A, S4:4 Scaccabarozzi, L. 80B Monchique, C. 23A Patrick, D. M. S5:3 Schleimer, N. S5:4 Peacock, S. J. S4:4 Schlotter, K. 70B

114 ASM Conferences Index

Schmidt, T. 29A, 29A Tang, P. S5:3 Vezzoli, F. 34A, 36A Schmidt, V. M. 93B Tasse, J. 20A, 85B Viganò, R. 80B Schulz, J. 18A, S1:4 Temelli, S. 31A Vinasco, J. S3:3 Schwarz, S. 1A, 27A, Tenhagen, B.-A. S1:4 Vincze, S. 54B, S5:2 37A, 38A, Tenhagen, B. A. 33A Viske, D. 92B 39A, 68B, Tenhagen, B.-A. A. 74B Wagenaar, J. S1:3 75B, 84B, Tenhagen, B. A. 95B Wagenaar, J. A. 96B, S2:3 86B, S1:2 Thanh, M. D. 94B Wagenaar, J. A. S4:5, S5:1, Schweizer, M. L. S1:5 Thomann, A. 79B S7:3 Schwendener, S. 79B, 87B Thompson, C. 17A Wahlström, H. 92B Scott, H. M. S3:3 Thörn, C. 4A Walther, B. 54B, S5:1, Seixas, R. 62B Thrane, C. S6:4 S5:2 Semmler, T. S5:2 Timmerman, A. S2:3 Wan, M. 69B Sen, A. 31A Tokach, M. D. S3:3 Wang, Y. 75B Sharon, P. 46A Tolomeo, P. S1:6 Wardyn, S. 32A Sharp, H. 95B Tonon, E. 83B Weese, J. 5A Shen, J. 75B Torres, C. 84B Weese, J. S. 101B, Sherwood, J. S. 17A Tristan, A. 20A, 85B 42A, 81B Shore, A. 82B Trott, D. J. 24A Weese, J. S. 97B Shore, A. C. 70B Tulinski, P. 96B, S4:5 Weese, S. 48A, 65B, Singh, A. 66B, 67B, Turnidge, J. 37A, 38A 66B, 67B, 97B Tyldsley, A. 14A S2:4, S5:3 Singh, A. M. S2:4 Ulrich, R. G. S5:2 Weiß, S. 1A, 68B Sinha, B. 78B Urdahl, A. 7A Wendlandt, S. 84B, 86B, Sironi, G. 80B Valentiner- S1:2 Skov, R. S1:1, Branth, P. 11A Werner, G. 91B S4:4, S5:1 Valentino, L. 34A Wieler, L. H. 54B Skov, R. L. 11A van Alphen, L. 11A Wieler, L. H. S2:1 Slettemeås, J. S. 41A, 53B, Van Balen, J. 8A Wieler, L. H. S5:1, S5:2 7A Van Cleef, B. S1:3 Williams, N. J. 93B Slifierz, M. 48A, 5A, van Nes, A. 96B Wing, S. 13A 65B van Putten, J. P. S4:5 Wing, S. 2A Small, H. 53B van Rijen, M. 78B Witte, W. S5:1 Smith, I. 72B Vandenesch, F. 20A, 85B Wittenberg, A. S5:1 Smith, T. 32A Vanderhaeghen, W. 73B Wittink, F. S4:5 Smith, T. C. S1:5, S5:1 Varini, R. 51A Wolff, C. 63B Sørum, M. 11A Varisco, G. 3A Wong, H. S. 24A Sreevatsan, S. 47A Vasse, A. S1:6 Wu, C. 75B Stefan, M. 99B Vaughan Yang, M. 47A Stegger, M. S4:4 Sarrazin, M. S1:5 Zadoks, R. 46A Stewart, J. 13A, 2A Velasco, V. 17A Zadoks, R. N. S4:4, S6:2 Strand, K. 4A Ventosa, M. 71B Zakaria, Z. 50A Strommenger, B. 91B Ventrella, G. 44A Zanoni, M. 3A Struelens, M. J. S5:1 Verhegghe, M. L. 10A Zdravkovic, N. 22A Sun, J. 15A, 47A Verstappen, K. S1:3 Zhao, Q. 75B Sunde, M. 41A, 53B, Verstappen, K. M. 96B, S2:3 Żmudzki, J. 16A 7A Verstappen, K. M. S7:3

3rd ASM-ESCMID Conference on Methicillin-resistant Staphylococci in Animals 115 American Society for Microbiology 1752 N Street, N.W. Washington, DC 20036-2904