Apparent prevalence and selected risk factors of methicillin-resistant and non- staphylococci and mammaliicocci in bulk tank milk of dairy herds in Indiana, Ohio, and Michigan.

The purpose of this study was to determine the apparent prevalence and risk factors of methicillin-resistant and non- staphylococci and mammaliicocci (NASM) in bulk tank milk (BTM) obtained from 300 dairy farms that belong to a cooperative collecting milk from Indiana, Michigan, and Ohio. Dairy field personnel recorded information about selected farm level risk factors and collected and froze BTM samples (n = 300) that were sent to Michigan State University researchers. Milk samples were thawed at room temperature and pre-enriched by adding 1 to 4 mL of Mueller-Hinton broth supplemented with 6.5% NaCl and incubated at 37°C for 24 h. Subsequently, 10 µL was plated on mannitol salt agar and Mueller-Hinton agar supplemented with 2.5% NaCl containing 2 mg/L oxacillin and 20 mg/L aztreonam. Colonies that grew on the selective media were subcultured on blood agar and identified using MALDI-TOF mass spectrometry. Phenotypic methicillin resistance was tested using cefoxitin disk diffusion. Conventional PCR was used to detect and in phenotypically resistant isolates. Of 550 isolates that were obtained from mannitol salt agar plates and 10 isolates from Mueller-Hinton agar plates, 16 species of NASM accounted for 84% of staphylococci, while accounted for the remaining 16%. Among , 4 isolates from 4 farms (1.3%) demonstrated phenotypic resistance to methicillin resistance but none carried or genes. Among NASM, 45 isolates from 40 farms (13.3%) demonstrated phenotypic resistance to methicillin. However, only 13 NASM isolates (7 , 2 , 1 , 1 , 1 , and 1 ) from 13 farms were positive for , whereas all were negative for . Thus, the prevalence of -positive NASM in BTM was 4.3%. Based on molecular results, this study demonstrated a low prevalence of methicillin resistance NASM from BTM samples collected from farms in the Upper Midwest. Dairy farms that contained ≤200 lactating cows and had swine located on the farm had a higher prevalence of methicillin-resistant NASM than smaller farms that did not contain swine.