Mammaliicoccus spp. from German Dairy Farms Exhibit a Wide Range of Antimicrobial Resistance Genes and Non-Wildtype Phenotypes to Several Antibiotic Classes.

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.

Phylogenetic Tracking of LA-MRSA ST398 Intra-Farm Transmission among Animals, Humans and the Environment on German Dairy Farms.

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.

Multidrug-resistant Staphylococcus cohnii and Staphylococcus urealyticus isolates from German dairy farms exhibit resistance to beta-lactam antibiotics and divergent penicillin-binding proteins.

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.

Genomic Distinctions of LA-MRSA ST398 on Dairy Farms From Different German Federal States With a Low Risk of Severe Human Infections.

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 ß-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.

Risk Factors for the Occurrence of Methicillin-Resistant Staphylococcus aureus in Dairy Herds: An Update.

In dairy cows, Staphylococcus aureus is a major mastitis pathogen and methicillin-resistant S. aureus (MRSA) has been reported from dairy farms around the world. The risk of foodborne zoonotic infections with bovine MRSA strains seems to be low since MRSA prevalence is low in dairy herds and milk is commonly heat treated before consumption. However, bovine mastitis caused by MRSA is an important issue in veterinary medicine since treatment options with non-ß-lactam antibiotics are limited. For the development of effective MRSA prevention strategies, it is necessary to know which factors increase the risk for MRSA transmission into and within dairy herds. Therefore, the aim of this review is to summarize the risk factors for the occurrence of MRSA in dairy herds and to identify the respective knowledge gaps. MRSA was more frequently detected in conventional dairy farms than in organic farms and in larger farms than in smaller farms. Dairy farms housing pigs along with cattle are more frequently affected by MRSA. Moreover, humans carrying MRSA can probably infect dairy cows. Consequently, pigs and humans may introduce new MRSA strains into dairy herds. MRSA transmission within dairy herds was associated with improper milking hygiene procedures. Furthermore, methicillin-resistant coagulase-negative staphylococci (MR-CoNS) were repeatedly isolated from dairy farms. This is an important issue since MR-CoNS may transfer resistance genes to S. aureus. The role of antimicrobial exposure as a risk factor for the occurrence of MRSA within dairy herds needs to be further investigated.