P 1102 Antimicrobial susceptibility monitoring of respiratory tract pathogens isolated from diseased cattle and pigs across Europe

Anno de Jong1, Shabbir Simjee1, Valérie Thomas1, Kirsty Maher2, Ian Morrissey2, Pascal Butty1, Ulrich Klein1, Hervé Marion1, Hilde Moyaert1, 22nd ECCMID Delphine Rigaut1, Brigitte Schiessl1, Michel Vallé1 London, United Kingdom Contact: 31 March – 3 April 2012 1VetPath Study Group, Brussels, Belgium; 2Quotient Bioresearch, Fordham, UK [email protected]

Introduction Results and discussion Antimicrobial resistance is of concern for the antimicrobial therapy of both humans and In total 1388 isolates were recovered from pre-treatment cattle and VetPath animals. Monitoring of antimicrobial susceptibility trends over time is important to ensure pigs suffering from an acute respiratory tract infection. For anti­ long-term antimicrobial efficacy. However, for various comparatively little is biotics having CLSI breakpoints, percentage resistance is shown 1,2 l The VetPath project is a pan-European pro- known about MIC distributions among the pathogens of food animals. Recent reviews in Table 1; MIC50 and MIC90 values for all antibiotics are presented gramme dedicated to the collection and to the and research findings in cattle and pigs3,4 highlight the importance of validated, harmo- in Table 2. monitoring of susceptibility of veteri- nized and continuous monitoring of antimicrobial susceptibility of veterinary pathogens. Cattle nary pathogens from diseased food-producing The present surveillance study is part of the VetPath programme which is co-ordinated In cattle, 343 P. multocida and 230 M. haemolytica were isolat- animals not recently exposed to antimicrobials. by the European Animal Health Study Centre (CEESA). The VetPath programme collects ed, the majority of these were susceptible to antibiotics for which l VetPath is an initiative of, and is coordinated bacteria from diseased cattle, pigs and poultry across Europe, employing a protocol with CLSI resistant breakpoints are available (Table 1). The percent- by, the European Animal Health Study Center harmonized methods of sampling and bacterial isolation. A central laboratory conducts age of intermediate category isolates was usually below 1%, and always­ <5%. (CEESA) in Brussels. It has been operational MIC determinations using a panel of licensed antimicrobials commonly used in veterinary since 2000. CEESA’s membership is composed medicine. Antimicrobial susceptibility of several major veterinary pathogens recovered The activity of all antibiotics (based on MICs) is summarized in of international pharmaceutical companies re- from cattle and pigs with respiratory tract infections across 11 EU countries predominant- Table 2; below some results for the antibiotics without CLSI break- searching and producing veterinary medicinal ly between 2002 and 2006 is presented here. points are described. For amoxicillin, 8 M. haemolytica isolate products. showed an MIC of ≥64 mg/L while the highest MIC observed for cephalexin was 8 mg/L (n=1) and 0.06 mg/L for cefquinome l Sponsors of the current VetPath project were Methods (n=2). For P. multocida, the highest MICs were ≥64 mg/L (n=4), the following companies: Bayer Animal Health 8 mg/L (n=1) and 0.12 mg/L (n=3) for amoxicillin, cephalexin and GmbH, Germany; CEVA Sante Animale, France; • Eleven European countries (Belgium, Czech Republic, Denmark, France, Germany, Ire- cefquinome, respectively. The MIC distributions for marbofloxa- Elanco Animal Health, UK; MSD Animal Health, land, Italy, The Netherlands, Poland, Spain and United Kingdom) were involved in the cin and enrofloxacin were similar for both bovine pathogens. MIC The Netherlands; Novartis Animal Health Inc., study. Samples were taken from animals with acute clinical signs, not recently exposed values for ciprofloxacin, enrofloxacin’s major metabolite, were one Switzerland; Pfizer Animal Health, Belgium; Ve- to antimicrobial treatment. These samples included lung tissue (both host species) or or two dilution steps lower than those for enrofloxacin or marbo- toquinol S. A., France; ­Virbac, France. alternatively deep nasopharyngeal swabs (cattle) and nasal swabs (pigs). The samples floxacin (data not shown). For danofloxacin, with a susceptible were processed according to standard procedures for the isolation of major patho- breakpoint of ≤0.25 mg/L, 94% of M. haemolytica and 99% of l The culture collection of VetPath was organized gens at regional laboratories. Only one isolate per farm and per outbreak was selected. P. multocida were susceptible. MIC range went up by CEESA, and subsequently from each partici- P. multocida and M. haemolytica from cattle and P. multocida, A. pleuropneumoniae to ≥256mg/L (n=8) for P. multocida. The highest MIC for tiamulin pating country transmitted to a central Europe- and S. suis from pigs were included in this study. For S. suis, recovery was from both was 32 mg/L (n=7) for P. multocida and 64 mg/L for M. haemolyt- an laboratory (Quotient Bioresearch, Fordham, brain and lung tissue. ica. MICs ranged from 1 to ≥32 mg/L for P. multocida and UK). All antimicrobial susceptibility determina- • Isolates were transferred to a central laboratory (Quotient Bioresearch, UK) for broth from 2 to >64 mg/L for M. haemolytica. For the respec- tions were performed by Quotient Bioresearch. microdilution MIC determination using 96 well TREK plates, containing a total of 16 tive ranges for both organisms was 4->16 mg/L. l The CEESA project manager is Hervé ­Marion, antibiotics/antibiotic combinations according to the Clinical and Laboratory Standard Pigs CEESA, Rue Defacqz 1, Bte 8, B-1000 Brussels, Institute guidelines.5 The antibiotics and tested ranges were: amoxicillin (0.03-64mg/L), A total of 326 P. multocida, 262 A. pleuropneumoniae and 227 Belgium. amoxicillin/clavulanic acid (AMC; 0.03/0.015-64/32mg/L), cefquinome (0.002-8mg/L), S. suis were recovered from pigs. As with the cattle isolates, the For correspondence: ceftiofur (0.002-8mg/L), cephalexin (0.03-64mg/L), enrofloxacin (0.002-8mg/L), danoflox- majority of tested isolates were susceptible, except for tetracy- Tel: +32 2 543 7572 acin (0.002-8mg/L), marbofloxacin (0.002-8mg/L), (0.12-8mg/L), cline where 20% of P. multocida, 11% of A. pleuropneumoniae e-mail: [email protected] (0.06-64mg/L), lincomycin (0.06-16mg/L), tiamulin (0.12-64mg/L), tilmicosin (0.12-64mg/L), and 77% of S. suis displayed resistance (Table 1). In general, in- tylosin (0.12-64mg/L), spectinomycin (2-256mg/L), and trimethoprim/sulfamethoxazole termediate category was usually zero and at most 4%, except (TMS; 0.008/0.15-8/152mg/L). for (P. multocida, 14%; A. pleuropneumoniae, 31%; S. suis, 7%). • The following quality control strains were used: Enterococcus faecalis ATCC 29212, ­Escherichia coli ATCC 25922, E. coli ATCC 35218, Pseudomonas aeruginosa ATCC For the antibiotics without defined CLSI breakpoint (Table 2), similar Revised abstract 27853, A. pleuropneumoniae ATCC 27090, Staphylococcus aureus ATCC 25923, MIC ranges to those obtained for cattle isolates were obtained for S. aureus ATCC 29213, and Streptococcus pneumoniae ATCC 49619. The numbers P. multocida and A. pleuropneumoniae. The highest MIC observed Objectives: VetPath is an ongoing pan-European resis- of isolates included for susceptibility testing are detailed in Tables 1 and 2. MIC and for amoxicillin (n=2) and cephalexin (n=1) was ≥64mg/L for P. mul- 50 tocida, whereas the highest value was >8 mg/L for cefquinome tance monitoring program for veterinary pathogens isolated MIC values were determined, and resistance was calculated for each drug, organism 90 and ceftiofur (n=1). The corresponding maximum MICs for A. pleu- from diseased antimicrobial-naive cattle, pigs and poultry. and country according to CLSI veterinary breakpoints where available5 (except for TMS ropneumoniae were 32 mg/L (n=6), 4 mg/L (n=1) and 0.5 mg/L Here, antimicrobial susceptibilities of isolates from cattle where the breakpoints used were for other species). (n=1), respectively. The highest MICs for spectinomycin were >256 and pig respiratory tract infections are presented. mg/L (n=9) and for tiamulin >32 mg/L (n=4) for P. multocida; for Methods: Lung samples or nasopharyngeal swabs from Table 1. Prevalence of antibiotic resistance (%) in respiratory tract pathogens of A. pleuropneumoniae maximum MICs for these antibiotics were cattle or nasal swabs from pigs were collected from animals cattle and pigs. 128 mg/L and 16 mg/L, respectively. For S. suis, the highest MICs with acute clinical signs, not recently treated with antibiot- Cattle Pigs observed for tylosin and tilmicosin were >64 mg/L and for spec- ics, in 11 EU countries, mainly in 2002-2006. Pasteurella Antimicrobial Resistance Pasteurella multo- Mannheimia Pasteurella Actinobacillus Streptococcus tinomycin >128 mg/L (n=5). ­breakpoint cida (Pm) ­haemolytica (Mh) ­multocida (Pm) pleuropneumoniae suis (Ss) multocida and Mannheimia haemolytica from cattle sam- (CLSI) (n=343) (n=230) (n=326) (App) (n=262) (n=227) Our findings for cattle and pig isolates are similar to those report- 6,7,8 ples and P. multocida, Actinobacillus pleuropneumoniae Amoxicillin/ R ≥32mg/L 0.0 0.0 0.0 0.0 0.0 ed by GERM-Vet, the German national surveillance programme. and Streptococcus suis from pigs samples were isolated clavulanic acid Similar findings were also reported for cattle from the Netherlands by standard methods (one isolate/farm/outbreak). S. suis Ceftiofur R ≥8mg/L 0.0 0.0 0.3 0.0 0.9 in the same time period with the exception of tetracycline for which were also isolated from meningitis cases. MICs of 16 anti- Enrofloxacin R ≥2mg/L; 0.6 0.4 0.0 0.4 0.4 resistance rates were higher than in this current study (14 and 40% R ≥1mg/L: 9 biotics were assessed in a central laboratory by broth mi- Pm, App (pigs)* for P. multocida and M. haemolytica, respectively). Marked coun- crodilution as per CLSI recommendations. Results were Florfenicol R ≥8mg/L 0.3 0.0 0.0 0.0 0.4 try differences were not noted in the present study, although the interpreted using CLSI resistance breakpoints (M31-A3) Spectinomycin R ≥128mg/L 3.2 0.0 - - - limited number of isolates per country make country comparisons difficult. where available. Tetracycline** R ≥8mg/L, cattle; 2.9 16.1 19.6 11.1 76.7 R ≥2mg/L, pigs Results: In all, 1388 isolates were recovered. In cattle, 343 Tiamulin R ≥32mg/L - - - 0.0 - P. multocida and 230 M. haemolytica were isolated, the Tilmicosin R ≥32mg/L - 1.7 0.6 0.0 - Conclusions majority of these were susceptible to antibiotics for which Trimethoprim/ R ≥4mg/L 2.3 6.1 2.5 3.8 5.2 The results of this pan-European survey, with standard- breakpoints are available. Resistance of P. multocida and sulfamethoxazole ized methods, show an absence or low antimicrobial -: no CLSI breakpoint available. * M31-A4 (in press). ** was teted for isolates prior to 2004. M. haemolytica to amoxicillin/clavulanic acid and ceftiofur resistance among the major respiratory tract pathogens was absent; resistance to enrofloxacin and florfenicol was isolated from non-treated, diseased cattle and pigs, with <1 %. Spectinomycin, co-trimoxazole and tetracycline re- Table 2. MIC50 and MIC90 values (mg/L) of pathogens from cattle and pigs with the exception of tetracycline. The results of VetPath are sistance varied from 2 to 16%. For amoxicillin, 4 P. multo- ­respiratory tract infections. cida and 8 M. haemolytica isolates showed MIC ≥ 64 mg/L similar to other European surveys. The VetPath network, Cattle Pigs based on quantitative and representative data, enables while the highest MIC observed for cephalexin was 8mg/L Antimicrobial Pasteurella Mannheimia Pasteurella Actinobacillus Streptococcus and 0.12 mg/L for cefquinome. MIC distributions of marbo- multocida ­haemolytica ­multocida ­pleuropneumoniae suis to reliably monitor antimicrobial susceptibility of major (n=343) (n=230) (n=326) (n=262) (n=227) floxacin and enrofloxacin were similar. With a susceptibility veterinary pathogens across Europe. MIC MIC MIC MIC MIC MIC MIC MIC MIC MIC breakpoint of ≤0.25 mg/L, 94.8% M. haemolytica and 96.5% 50 90 50 90 50 90 50 90 50 90 Amoxicillin 0.25 0.25 0.25 8 0.25 0.25 0.25 0.5 ≤0.06 ≤0.06 P. multocida were susceptible to danofloxacin. 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