The objective of this study was to investigate the occurrence and distribution of methicillin-resistant Staphylococcus aureus (MRSA) on 20 German dairy farms. Farms were selected based on previous MRSA reports from phenotypic susceptibility testing of mastitis pathogens. Samples were collected from predefined groups of cows, young stock, farm personnel, and the environment. A high MRSA-positive test rate was detected in swab samples from milk-fed calves (22.7%; 46/203). In postweaning calves, the MRSA-positive test rate was 9.1% (17/187). From prefresh heifers, both nasal swabs and udder cleft swabs were collected if possible. Including both sample types, the MRSA-positive test rate in prefresh heifers was 13.0% (26/200). The positive test rate was 8.9% (17/191) in nasal swabs and 6.5% (11/170) in udder cleft swabs. In quarter milk samples (QMS), the MRSA-positive test rate was 2.9% (67/2347), and on cow level, 7.9% (47/597) of the dairy cows were affected. Among all cows included in this study, the geometric mean of somatic cell counts was higher in QMS that carried MRSA (345,000 cells/mL) in comparison to all QMS (114,000 cells/mL). No differences in parity or the affected mammary quarter position on the udder were observed among the 47 infected cows. Methicillin-resistant S. aureus was also detected in boot swab samples (dust), teat liners, and in suckers from automatic calf feeders. All isolates belonged to livestock-associated sequence type 398 and most common staphylococcal protein A (spa)-types were t011 and t034. Most isolates harbored the staphylococcal cassette chromosome mec (SCCmec)-type V, with the exception of some isolates with SCCmec-type IVa on 1 farm. Similar MRSA genotypes in samples from humans and dairy cows underline the possible zoonotic and reverse-zoonotic transmission of livestock-associated MRSA strains from dairy farms. Similar MRSA genotypes in pig and cattle barns were detected on only 1 of 5 farms that kept both cattle and pigs. Similar MRSA spa-types were detected in samples from different sources (dairy cows, young stock, environment, and humans), suggesting a possible contagious transmission on some of the farms. Sporadically, up to 3 different MRSA spa-types were detected in QMS from the respective farms. On MRSA-affected farms, improper milking hygiene procedures and elevated bulk-tank milk somatic cell counts (>250,000 cells/mL) were observed. The occurrence of livestock-associated MRSA ST398 in different samples from dairy farms, and especially in young calves, should be considered for future MRSA-monitoring programs and biosecurity guidelines.

Methicillin-resistant Staphylococcus aureus (MRSA) have been found on German dairy farms and may be the cause of difficult-to-treat bovine mastitis. Considering the one health approach, MRSA might be transmitted from animals to humans raising the risk for severe infections. On 17 German dairy farms with a history of MRSA detection, MRSA strains were isolated from quarter milk, bulk tank milk, and swab samples of calves, heifers, pigs, and the environment. A selection of 33 isolates was analyzed using whole-genome sequencing and antimicrobial resistance testing. All detected MRSA strains were attributed to the livestock-associated sequence type 398. Methicillin-resistance was associated with the mecA gene in the staphylococcal cassette chromosome (SCC)mec types IVa (7/33) or V (26/33). The MRSA strains across the German federal states showed large allelic differences indicating independent development and distribution. On one farm, a clonal MRSA isolate was widely spread among different animals and the milking equipment. Moreover, MRSA transmission between two dairy farms in one federal state seems to be likely. In depth studies indicated that the resistance gene prediction and phenotypic resistance are in good agreement. Twenty eight strains were determined to exhibit a non-wildtype phenotype (resistant) against up to seven antimicrobial substances with an overall resistance to β-lactams and tetracycline. Ten different phenotypic antimicrobial resistance patterns were found among the MRSA strains. The strains harbored a wide virulence gene repertoire, of which some of them are related to bovine mastitis. However, the isolates lacked typical human infection associated factors such as the immune evasion cluster genes, staphylococcal enterotoxin genes, or Panton-Valentine leukocidin genes leading to the assumption for a low risk for severe human infections and foodborne diseases.

Mammaliicocci might play a major role in antimicrobial resistance (AMR) gene transmission between organisms of the family Staphylococcaceae, such as the potentially pathogenic species Staphylococcus aureus. The interest of this study was to analyze AMR profiles of mammaliicocci from German dairy farms to evaluate the AMR transmission potential. In total, 65 mammaliicocci isolates from 17 dairy farms with a history of MRSA detection were analyzed for AMR genotypes and phenotypes using whole genome sequencing and antimicrobial susceptibility testing against 19 antibiotics. The various genotypic and phenotypic AMR profiles of mammaliicocci from German dairy farms indicated the simultaneous occurrence of several different strains on the farms. The isolates exhibited a non-wildtype phenotype to penicillin (58/64), cefoxitin (25/64), chloramphenicol (26/64), ciprofloxacin (25/64), clindamycin (49/64), erythromycin (17/64), fusidic acid (61/64), gentamicin (8/64), kanamycin (9/64), linezolid (1/64), mupirocin (4/64), rifampicin (1/64), sulfamethoxazol (1/64), streptomycin (20/64), quinupristin/dalfopristin (26/64), tetracycline (37/64), tiamulin (59/64), and trimethoprim (30/64). Corresponding AMR genes against several antimicrobial classes were detected. Linezolid resistance was associated with the cfr gene in the respective isolate. However, discrepancies between genotypic prediction and phenotypic resistance profiles, such as for fusidic acid and tiamulin, were also observed. In conclusion, mammaliicocci from dairy farms may carry a broad variety of antimicrobial resistance genes and exhibit non-wildtype phenotypes to several antimicrobial classes; therefore, they may represent an important source for horizontal gene transfer of AMR genes to pathogenic Staphylococcaceae.

Methicillin-resistant Staphylococcus aureus (MRSA) are a major threat to human and animal health, causing difficult-to-treat infections. The aim of our study was to evaluate the intra-farm transmission of livestock-associated (LA) MRSA sequence type (ST) 398 isolates on German dairy farms. A total of 115 LA-MRSA ST398 isolates originating from animals, humans and the environment of six dairy farms were analyzed by whole-genome sequencing and core genome multilocus sequence typing. Phylogenetic clusters of high allelic similarity were detected on all dairy farms, suggesting a MRSA transmission across the different niches. On one farm, closely related isolates from quarter milk samples (QMS), suckers of calf feeders and nasal cavities of calves indicate that MRSA may be transferred by feeding contaminated milk to calves. Detection of related MRSA isolates in QMS and teat cups (4/6 farms) or QMS and human samples (3/4 farms) pointed out a transmission of MRSA between cows during the milking process and a potential zoonotic risk. In conclusion, LA-MRSA ST398 isolates may spread between animals, humans and the environment on dairy farms. Milking time hygiene and other internal biosecurity measures on farms and pre-treatment of milk before feeding it to calves may reduce the risk of MRSA transmission.

This study aimed to determine the occurrence of methicillin-resistant (MR) non-aureus staphylococci (NAS) on 20 preselected German dairy farms. Farms were selected based on the detection of methicillin-resistant Staphylococcus aureus (MRSA) during previous diagnostic investigations. Bacterial culture of presumptive MR-NAS was based on a 2-step enrichment method that has been recommended for MRSA detection. Quarter milk samples (QMS), bulk tank milk, swab samples from young stock, and environmental samples were collected for bacterial culture. Methicillin-resistant NAS were detected on all study farms. The MR-NAS positive test rate was 3.3% (77/2,347) in QMS, 42.1% (8/19) in bulk tank milk, 29.1% (59/203) in nasal swabs from milk-fed calves, 18.3% (35/191) in postweaning calves, and 7.3% (14/191) in nasal swabs from prefresh heifers. In the environment, MR-NAS were detected in dust samples on 25% (5/20) of the dairy farms as well as in teat liners and suckers from automatic calf feeders. The geometric mean somatic cell count in QMS affected by MR-NAS (183,000 cells/mL) was slightly higher compared with all QMS (114,000 cells/mL). Nine MR-NAS species were identified; Staph. sciuri, Staph. lentus, Staph. fleurettii, Staph. epidermidis, and Staph. haemolyticus were the most common species. In addition, 170 NAS isolates were identified that showed reduced cefoxitin susceptibility (4 mg/L) but did not harbor the mecA or mecC genes. On some farms, similar mobile genetic elements were detected in MR-NAS and MRSA. It was suggested that resistance genes may be transferred between NAS and Staph. aureus on the respective farms.

Non-aureus staphylococci are commonly found on dairy farms. Two rarely investigated species are Staphylococcus (S.) cohnii and S. urealyticus. Since multidrug-resistant S. cohnii and S. urealyticus are known, they may serve as an antimicrobial resistance (AMR) gene reservoir for harmful staphylococcal species. In our study, nine S. cohnii and six S. urealyticus isolates from German dairy farms were analyzed by whole-genome sequencing and AMR testing. The isolates harbored various AMR genes (aadD1, str, mecA, dfrC/K, tetK/L, ermC, lnuA, fexA, fusF, fosB6, qacG/H) and exhibited non-wildtype phenotypes (resistances) against chloramphenicol, clindamycin, erythromycin, fusidic acid, rifampicin, streptomycin, tetracycline, tiamulin and trimethoprim. Although 14/15 isolates lacked the blaZ, mecA and mecC genes, they showed reduced susceptibility to a number of beta-lactam antibiotics including cefoxitin (MIC 4–8 mg/L) and penicillin (MIC 0.25–0.5 mg/L). The specificity of cefoxitin susceptibility testing for mecA or mecC gene prediction in S. cohnii and S. urealyticus seems to be low. A comparison with penicillin-binding protein (PBP) amino acid sequences of S. aureus showed identities of only 70–80% with regard to PBP1, PBP2 and PBP3. In conclusion, S. cohnii and S. urealyticus from selected German dairy farms show multiple resistances to antimicrobial substances and may carry unknown antimicrobial resistance determinants.