Content of Research Report A. Project title: Antibiotic Sensitivity of Bacterial Pathogens Isolated From Bovine Mastitis Milk

B. Abstract: Antibiotic sensitivity of bacteria isolated from bovine milk samples was investigated. The 18 antibiotics that were evaluated (e.g., penicillin, novobiocin, gentamicin) are commonly used to treat various diseases in cattle, including mastitis, an inflammation of the udder of dairy cows. A common concern in using antibiotics is the increase in drug resistance with time. This project studies if antibiotic resistance is a threat to consumers of raw milk products and if these antibiotics are still effective against mastitis pathogens. The study included isolating and culturing bacteria from quarter milk samples (n=205) collected from mastitic dairy cows from farms in Chino and Ontario, CA. The isolated bacteria were tested for sensitivity to antibiotics using the Kirby Bauer disk diffusion method. The prevalence (%) of resistance to the individual antibiotics was reported.

Resistance to penicillin was 45% which may support previous data on penicillin-resistant bacteria, especially Staphylococcus and Streptococcus. Resistance rates (%) for oxytetracycline (26.8%) and tetracycline (22.9%) were low compared to previous studies but a trend was seen in our results that may support concerns of emerging resistance to tetracyclines in both gram- positive and gram-negative bacteria. Similarly, 31.9% of bacterial isolates showed resistance to erythromycin which is at least 30% less than in reported literature concerning emerging resistance to macrolides. More numbers (%) that should be noted are cefazolin (26.3%), ampicillin (29.7%), novobiocin (33.0%), polymyxin B (31.5%), and resistance ranging from 9­ 18% for the other antibiotics.

The aim for this project and the trends shown by the data are to encourage further surveillance of antimicrobial resistance. This will allow us to determine whether the antibiotics are still effective against mastitis and whether mastitic bacterial pathogens pose a threat to consumers of raw milk products.

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C. Major objective(s): The main objective of this study is to determine antibiotic sensitivity of bacteria isolated from mastitic dairy cow milk samples. Eighteen antibiotics were evaluated.

D. Background research with analysis and summary of literature review: Mastitis is an inflammation of the udder of dairy cows that can occur in one or multiple quarters. It costs the U.S. dairy industry about $1.7-2 billion/year as a result of reduced milk production, discarded milk, cattle replacements, and culling, as well as treatment, labor, and veterinary costs. If left untreated, mastitis can result in death which increases economic loss (Jones and Bailey, 2009).

These mastitis-causing pathogens present a public health concern due to possible transmission of bacteria from mastitic cattle to milk causing human infection. This is a larger concern in raw dairy products because pasteurization is meant to kill a majority of bacterial pathogens (Oliver, et al., 2009). For this reason, we decided to focus on bacterial pathogens even though mastitis is also caused by fungi and viruses. Some bacterial pathogens of interest are methicillin-resistant S. aureus, penicillin-resistant Streptococcus (e.g., S. pneumoniae), and vancomycin-resistant Enterococcus (e.g., E. faecalis) because they are gram-positive pathogens that have developed wide-spread resistance to antibiotics (Diekema and Jones, 2001).

The 18 antibiotics, cefazolin (CZ30), cefotaxime (CTX30), ampicillin (AM10), penicillin (P10), gentamicin (GM10), kanamycin (K30), spectinomycin (SPT100), ciprofloxacin (CIP5), nalidixic acid (NA30), norfloxacin (NOR10), oxytetracycline (T30), tetracycline (TE30), chloramphenicol (C30), novobiocin (NB30), trimethoprim (TMP5), polymyxin B (PB300), vancomycin (Va30), and erythromycin (E15) have a broad spectrum of use including treatment of mastitis in dairy cows. A common concern in using antibiotics is the potential increase in drug resistance and emergence of multiple drug resistance with time.

Antibiotic resistance may occur through a mutation or through exposure to exogenous DNA, such as transposons or plasmids, containing the gene(s) coding for resistance. The 18 antibiotics represent 10 families: beta-lactams, aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicols, aminocoumarins, sulfonamides, polypeptides, glycopeptides, and

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macrolides/polyketides, and understanding their mechanisms of action may help develop antibiotic combinations with synergistic effects against resistant bacteria (emedexpert.com, 2015). An example is the treatment of extended spectrum beta-lactamase producing bacteria with a combination of piperacillin and tazobactam. The goal is to develop a mixture of agents with varying modes of action and expanded activity against several species (Bradford, 2001).

The prevalence of methicillin-resistant S. aureus (MRSA) is of interest because S. aureus causes ~ 30% of mastitis cases and ≥ 90% are resistant to penicillin and other beta-lactam antibiotics due to production of beta-lactamase (Juhasz-Kaszanyitzky, et al., 2007). Methicillin, a semisynthetic penicillin, was developed to address resistance to penicillin. However, some strains of S. aureus have developed resistance to methicillin and treatment shifted to mupirocin as the last line of defense (Murinda, 2014). Another concern with MRSA is its resistance to aminoglycosides, macrolides, tetracycline, chloramphenicol, and lincosamides, showing resistance to multiple antibiotics (Nikaido, 2009). Vancomycin, a glycopeptide, is commonly used to treat MRSA due to its reliability against these multiple-drug resistant bacteria. However, prolonged treatment with vancomycin has led to rare cases of vancomycin-resistant S. aureus (VRSA) containing the transposon Tn1546; presumably acquired from vancomycin-resistant E. faecalis (Gardete and Tomasz, 2014; Diekema and Jones, 2001). Fortunately the development of oxazolidinones such as linezolid provides an alternative to vancomycin and is effective against methicillin-resistant S. aureus, penicillin-resistant Streptococcus, and vancomycin-resistant Enterococcus (Diekema and Jones, 2001).

There are several pathways to acquisition of antibiotic resistance, it is therefore important to regularly test sensitivity to these antibiotics and determine their continued efficacy in treating bacterial diseases. These antibiotics have broad clinical use in humans and animals and it is important to know whether resistant bacteria pose a threat to current mastitis treatment procedures.

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E. Methods (Experimental procedure/design): Materials & Methods About four hundred milk samples collected from dairy farms in Chino and Ontario, San Bernardino County, CA, were analyzed in a previous study. The samples were collected from mastitis studies conducted in 2009-2012 and stored frozen at -20°C. From these samples, a total of 205 samples yielded bacteria and were used in the current study to test for antibiotic sensitivity. Isolation of bacteria: Milk samples were thawed and then streaked on blood agar (5% blood; vol/vol) to isolate bacterial colonies. These colonies were purified on tryptic soy agar then confirmed for morphology and gram reaction using microscopy. Antibiotic Sensitivity: Samples were tested for antibiotic sensitivity to each of the 18 antibiotics indicated before on Mueller Hinton Agar using the Kirby Bauer disk diffusion method following the Clinical and Laboratory Standards Institute interpretive criteria (CLSI, 2002). Zones of inhibition (diameter, mm) were measured. Data analysis: The prevalence (%) of resistance to the individual antibiotics was determined.

Expansion to Project: Isolation of S. aureus: Standard methods from the National Mastitis Council’s Laboratory Handbook on Bovine Mastitis (NMC, 1999) are used for screening S. aureus. Briefly, milk samples will be thawed and then streaked on blood agar (5% blood; vol/vol) to isolate bacterial colonies. Presumptive S. aureus (n=109), determined through morphology, gram staining, and biochemical tests, will be purified on tryptic soy agar then confirmed for morphology and gram reaction using microscopy. Confirmation of MRSA will be conducted using PCR targeting unique DNA sequences associated with S. aureus and MRSA. Antibiotic resistance: After the confirmation of S. aureus, representative isolates will be tested for antibiotic resistance using the Kirby Bauer disk diffusion method following the Clinical and Laboratory Standards Institute interpretive criteria (CLSI, 2002). The most commonly used antibiotics for determination of methicillin resistance are cefoxitin, oxacillin, and methicillin (San Juan et al., 2012). Zones of inhibition (diameter, mm) will be measured. Mupirocin is one of the few antibiotics still highly active against MRSA; therefore resistance to mupirocin will be tested (Zhang et al., 2004).

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PCR tests for target genes: The primers for use in DNA amplification will include: Staph756F (5′-AAC TCT GTT ATT AGG GAA CA-3′) and 3′ primer Staph750R (5′-CCA CCT TCC GGT TTG TCA CC-3′) targeting Staphylococcus genus-specific 16S rRNA; nuc 1 (5′-GCG ATT GAT GGT GAT ACG GTT-3′) and nuc 2 (5′-AGC CAA GCC TTG ACG AAC TAA AGC-3′) for nuc, targeting S. aureus species-specific sequences; mecA1 (5′-GTA GAA ATG ACT GAA CGT CCG ATA A-3′) and mecA2 (5′-CCA ATT CCA CAT TGT TTC GGT CTA A-3′) for mecA, targeting sequences for a determinant of methicillin resistance, and mupA (5′-TAT ATT ATG CGA TGG AAG GTT GG-3′) and mupB (5′-AAT AAA ATC AGC TGG AAA GTG TTG-3′) for mupA, targeting genes for mupirocin resistance. PCR amplification of target DNA sequences will be conducted as described by Zhang et al. (2004). Data analysis: Positive and negative quality control bacterial strains are being used to establish sensitivity and specificity of all tests. Tests will be conducted in duplicate and repeated. MRSA prevalence % will be determined.

F. Results: Milk samples that tested positive for bacteria were tested for antibiotic sensitivity to 18 antibiotics. Each bacterial isolate was tested at least twice against a single antibiotic and the zones of inhibition were recorded. These measurements were compared to zones of the Quality Control bacterial strains; zones of inhibition below 14 mm were considered resistant, while those above 14 mm were considered susceptible. Zone diameters that were intermediate or partially susceptible were recorded as susceptible.

The data was organized into three tables, Tables 1-3. These tables list the individual antibiotics as well as the class they fall under. The “# of Samples” refers to the total number of samples that were tested for that single antibiotic; not all samples were tested for each antibiotic. “# of Samples R to Ab” refers to the number of samples from the total that showed resistance to the single antibiotic on both trials. The last row lists the percentage of resistance calculated using # ௢௙ ௦௔௠௣௟௘௦ ௥௘௦௜௦௧௔௡௧ ௧௢ ௔௡௧௜௕௜௢௧௜௖ ൈ 100. # ௢௙ ௌ௔௠௣௟௘௦

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Resistance to penicillin was 45% (Table 1). Resistance to cefazolin (26.3%), ampicillin (29.7%), oxytetracycline (26.8%), and tetracycline (22.9%) was noted. The bacterial isolates demonstrated resistance ranging from 9-18% for the other antibiotics in Table 1 and Table 2 (e.g. ciprofloxacin, nalidixic acid, norfloxacin).

Table 1: Resistance Data for the Beta-lactam and Aminoglycoside antibiotic classes Beta-lactams Aminoglycosides

CZ30 CTX30 AM10 P10 GM10 K30 SPT100 (cefazolin) (cefotaxime) (ampicillin) (penicillin) (gentamicin) (kanamycin) (spectinomycin) # of Bacterial Isolates 50 22 51 92 21 30 29 R to Ab # of Bacterial 190 199 191 202 202 202 189 Isolates Tested % Resistance 26.3 11.0 26.7 45.5 10.4 14.8 15.3

Notes: R- Resistant, Ab-Antibiotic Ex. CZ30: CZ- cefazolin, 30- Disc Potency (30 µg/disc)

Table 2: Resistance Data for the Fluoroquinolone, Tetracycline, and Chloramphenicol antibiotic classes Fluoroquinolones Tetracyclines Chloramphenicol

CIP5 NA30 NOR10 T30 TE30 C30 (ciprofloxacin) (nalidixic (norfloxacin) (oxytetracycline) (tetracycline) (chloramphenicol) acid) # of Bacterial Isolates 29 28 18 51 46 25 R to Ab # of Bacterial 202 185 203 190 201 136 Isolates Tested % Resistance 14.3 15.1 8.9 26.8 22.9 18.4

Notes: R- Resistant, Ab-Antibiotic Ex. NB30: CIP- ciprofloxacin, 5- Disc Potency (5 µg/disc)

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Some bacterial isolates showed high resistance (%) to novobiocin (33.0%), polymyxin B (31.5%), erythromycin (31.9%), and vancomycin (40.2%) (Table 3).

Table 3: Resistance Data for the Aminocoumarin, Sulfonamide, Polypeptide, Macrolide/Polyketide, and Glycopeptide antibiotic classes Aminocoumarin Sulfonamides Polypeptides Macrolides/Polyketides Glycopeptides NB30 TMP5 PB300 E15 Va30 (novobiocin) (trimethoprim) (polymyxin (erythromycin) (vancomycin) B) # of Bacterial Isolates 66 21 64 62 39 R to Ab # of Bacterial 200 198 203 194 97 Isolates Tested % Resistance 33.0 10.6 31.5 31.9 40.2

Notes: R- Resistant, Ab-Antibiotic Ex. NB30: NB- novobiocin, 30- Disc Potency (30 µg/disc)

G. Discussion: Colonies that were successfully isolated on blood agar and purified on tryptic soy agar were characterized by morphology, gram staining reaction, and biochemical tests, such as, catalase and KOH test. These tests suggested high prevalence of Staphylococcus and Streptococcus species in many of the milk samples. As stated before, a common concern with Staphylococcus and Streptococcus species is their acquired resistance to several beta-lactam antibiotics such as penicillin. This resistance rate of 45% (Table 1) may support previous data that penicillin- resistant bacteria are a complication in the treatment of mastitis and require new treatments. We compared our percentages to a 2012 study. The results indicated penicillin resistance of up to 91% for gram-positive bacteria, such as, Streptococcus and up to 50% for Staphylococcus, which is similar to our results (Oliver and Murinda, 2012). Similarly, the bacterial isolates showed resistance of 26.3 and 29.7% for cefazolin and ampicillin respectively (Table 1); both of these also fall under the beta-lactam family.

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In addition, the figures for oxytetracycline and tetracycline, 26.8 and 22.9% respectively (Table 2), may support concerns of emerging resistance to tetracyclines. Incidence of tetracycline resistance is seen in both gram-positive and gram-negative species. In a 1994 study, “[a]pproximately 90% of MRSA, 70% of S. agalactiae, [and] 70% of multiple-drug resistant E. faecalis” were found to be tetracycline-resistant. Our results do not fall within the range of the 1994 study, but the tetracycline-resistance genes have been seen on the same plasmids (transposons and integrons as well) as the resistance genes for other antibiotics (e.g., macrolides, chloramphenicol, aminoglycoside) (Chopra and Roberts, 2001). The implication of this is that tetracycline resistance may be an additional complication in the treatment of mastitis and should be tested for if other resistance strains are found.

The resistance rate to erythromycin, 31.9%, is less than in previous reports concerning emerging resistance to macrolides (Table 3). In the 2012 study, resistance to erythromycin (E15) ranged from 60-90% in gram-positive bacteria; this is at least 30% greater than our results (Oliver and Murinda, 2012).

Although ~ 40% of the isolates showed resistance to vancomycin (Table 3), the number of samples tested was significantly lower for vancomycin than the other microbial agents, so a comparison could not be made. However, susceptibility to vancomycin may be studied further after confirming Staphylococcus species for rare cases of vancomycin-resistant S. aureus (VRSA).

The trends shown by the data in the current study should be taken into consideration and studied further to build on current antimicrobial stewardship programs. This will allow us to determine whether the antibiotics are still effective against mastitis and whether mastitic bacterial pathogens pose a threat to consumers of raw milk products. However, it is still recommended that people drink pasteurized milk because it kills a majority of bacterial pathogens and decreases the risk of food borne illnesses.

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Further Direction: The study was expanded to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) among the isolates. As mentioned before, S. aureus is a major mastitis-causing pathogen and some strains are known to be resistant to beta-lactam antibiotics. MRSA is a pathogen of economic and public health significance, thus making it important to study the subject further. In expanding the project, presumptive S. aureus (n=109) will be genetically confirmed using PCR; confirmation of MRSA (methicillin-resistance S. aureus) will also be conducted using PCR. These PCR protocols target unique DNA sequences associated with S. aureus and MRSA (i.e., Staph and nuc, and mec).

We will test sensitivity to cefoxitin, oxacillin, methicillin, and mupirocin using the Kirby Bauer disk diffusion method and confirm mupirocin resistance using mup primers. Most published research conducted outside of the U.S. (e.g., Korea, Germany, Turkey) suggests that there is a low prevalence of MRSA and that it does not pose a significant threat to cows or to humans that consume raw milk products (reviewed by Oliver and Murinda, 2012). However, comprehensive surveys of MRSA in dairy farms and its presence in milk are lacking in the United States (Virgin, et al., 2009). Thus, we expect our results to encourage further studies so that there may be an accurate representation of the incidence of MRSA on dairy farms in California and the U.S. This will allow us to determine whether MRSA poses a potential threat on dairy farms and to consumers of raw bovine milk products.

References Becton, Dickinson and Company. “BD BBL™ Sensi-Disc™ Antibiotic Susceptibility Test Discs” product insert. Becton, Dickinson and Company (2011). PDF/Web. 15 May 2015.

Bradford, Patricia A. “Extended-Spectrum Beta-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat.” Clinical Microbiology Reviews 14 (2001): 933-951. PDF/Web. 02 Feb. 2015.

Chopra, Ian, and Marilyn Roberts. “Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance.” Microbiology and Molecular Biology Reviews 65.2 (2001): 232-260. PDF/Web. 13 Aug. 2015.

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CLSI (Clinical and Laboratory Standards Institute). “Performance Standards for Antibiotic Disk Susceptibility Tests for Bacteria Isolated From Animals.” NCCLS document M32-A2, approved standard. 2nd ed. CLSI, Wayne, PA.

Diekema, Daniel J. and Ronald N. Jones. “Oxazolidinone Antibiotics.” The Lancet 358 (2001): 1975-1982. PDF/Web. 02 Feb. 2015.

Gardete, Susana and Alexander Tomasz. “Mechanism of Vancomycin Resistance in Staphylococcus aureus.” The Journal of Clinical Investigation 124.7 (2014): 2836-2840. PDF/Web. 02 Feb. 2015.

Jones, G. M., and T. L. Bailey, Jr. “Understanding the Basics of Mastitis.” Virginia Cooperative Extension (2009): 1-5. PDF/web. 02 Feb 2015.

Juhasz-Kaszanyitzky, et al. “MRSA Transmission between Cows and Humans.” Emerging Infectious Diseases 13.4 (2007): 630-632. PDF/web. 02 Feb. 2015.

Lists of antibiotics. http://www.emedexpert.com/lists/antibiotics.shtml (Accessed 2/15/15)

Murinda, Shelton. “Methicillin-Resistant Staphylococcus aureus (MRSA) Prevalence in Mastitic Dairy Cows from San Bernardino County, CA.” Research Proposal. Word. 30 March 2014.

National Mastitis Council. Laboratory Handbook on Bovine Mastitis. Madison: NMC Inc., 1999.

Nikaido, Hiroshi. “Multidrug Resistance in Bacteria.” Annual Review of Biochemistry 78 (2009): 119-146. PDF/web. 08 Aug. 2015.

Oliver, S. P. and K. J. Boor. “Food Safety Hazards Associated with Consumption of Raw Milk.” 6.7 (2009): 793-806.

Oliver, S.P. and SE Murinda. “Antimicrobial Resistance of Mastitis Pathogens.” The Veterinary Clinics of North America: Food Animal Practice 28 (2012): 165-185.

San Juan, et al. “Occurrence of Methicillin-Resistant Staphylococcus aureus (MRSA) among the Health Workers of Rizal Provincial Hospital and Characterization for the Presence of luks-lukf PVL Gene.”

Virgin, J.E., et al. “Short Communication: Methicillin-resistant Staphylococcus aureus Detection in US Bulk Tank Milk.” Journal of Dairy Science 92 (2009): 4988-4991. PDF/web. 09 Aug. 2014.

Zhang, Kunyan. et al. “New Quadriplex PCR Assay for Detection of Methicillin and Mupirocin Resistance and Simultaneous Discrimination of Staphylococcus aureus from Coagulase­ negative Staphylococci.” Journal of Clinical Microbiology 42.11 (2004): 4947-4955. PDF/web. 01 Sept. 2014.

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