Staphylococcus aureus related to bovine mastitis in Switzerland: Clonal diversity,virulence gene profiles,and antimicrobial resistance of isolates collected throughout 2017

Staphylococcus aureus can be associated with subclinical, acute, chronic, and toxic cases of bovine intramammary infections, leading to considerable financial losses for the dairy industry in Switzerland and worldwide. In addition, milk products are one of the most common food categories implicated in staphylococcal food poisoning in humans. Detailed information on the population structure, as well as the virulence and resistance characteristics of Staph. aureus originating from bovine mastitis milk is needed to allow for source attribution and risk assessment of Staph. aureus in a food poisoning context and to improve therapeutic approaches in cattle. Our objective was to assess the population structure, phenotypic resistance patterns, and virulence and resistance gene profiles of Staph. aureus isolates from bovine mastitis milk in Switzerland. To this end, 58 Staph. aureus strains were characterized. The DNA microarray was used to test for the presence or absence of virulence and resistance genes. In addition, minimum inhibitory concentrations of various antimicrobial agents were determined by microdilution. To assess the population structure of the isolates, we determined clonal complexes (CC) using DNA microarray hybridization profiles and performed multilocus sequence typing and spa typing. The strains were assigned to 7 clonal complexes, 10 sequence types, and 11 spa types, with CC705 (43%), CC97 (33%), and CC20 (12%) representing the most common lineages and t529 (43%) and t267 (21%) representing the most common spa types. Only 1 isolate was assigned to CC8, a clonal lineage linked to high within-herd prevalence of mastitis. A total of 14% (n = 8) of strains were classified as resistant to penicillin, and 1 strain each was classified as oxacillin and pirlimycin resistant. Although no clinical breakpoints are available for the combination of kanamycin/cefalexin, growth of all strains was inhibited by the lowest combination of kanamycin/cefalexin concentrations tested (4 µg/mL of kanamycin and 0.4 µg/mL of cefalexin). One strain assigned to CC20, ST389, and t2094 exhibited resistance to penicillin, oxacillin, and pirlimycin as well as intermediate susceptibility to erythromycin and high minimum inhibitory concentration for several antimicrobial agents, for which no breakpoints were available.     

Staphylococcus aureus adlb gene is associated with high prevalence of intramammary infection in dairy herds of northern Italy: A cross-sectional study

Staphylococcus aureus is a major mastitis pathogen in dairy cattle worldwide, responsible for substantial economic losses. Environmental factors, milking routine, and good maintenance of milking equipment have been described as important factors to prevent intramammary infections (IMI). Staphylococcus aureus IMI can be widespread within the farm or the infection can be limited to few animals. Several studies have reported that Staph. aureus genotypes differ in their ability to spread within a herd. In particular, Staph. aureus belonging to ribosomal spacer PCR genotype B (GTB)/clonal complex 8 (CC8) is associated with high within-herd prevalence of IMI, whereas other genotypes are generally associated with individual cow disease. The adlb gene seems to be strictly related to Staph. aureus GTB/CC8, and is a potential marker of contagiousness. We investigated Staph. aureus IMI prevalence in 60 herds in northern Italy. In the same farms, we assessed specific indicators linked to milking management (e.g., teat condition score and udder hygiene score) and additional milking risk factors for IMI spread. Ribosomal spacer-PCR and adlb-targeted PCR were performed on 262 Staph. aureus isolates, of which 77 underwent multilocus sequence typing. In most of the herds (90%), a predominant genotype was identified, especially Staph. aureus CC8 (30%). In 19 of 60 herds, the predominant circulating Staph. aureus was adlb-positive and the observed IMI prevalence was relevant. Moreover, the adlb gene was detected only in genotypes of CC8 and CC97. Statistical analysis showed a strong association between the prevalence of Staph. aureus IMI, the specific CCs, and carriage of adlb, with the predominant circulating CC and presence of the gene alone explaining the total variation. Interestingly, the difference in the odds ratio obtained in the models for CC8 and CC97 suggests that it is carriage of the adlb gene, rather than the circulation of these CCs per se, that leads to higher within-herd prevalence of Staph. aureus. In addition, the model showed that environmental and milking management factors had no or minimal effect on Staph. aureus IMI prevalence. In conclusion, the circulation of adlb-positive Staph. aureus strains within a herd has a strong effect on the prevalence of IMI. Thus, adlb can be proposed as a genetic marker of contagiousness for Staph. aureus IMI in cattle. However, further analyses using whole-genome sequencing are required to understand the role of genes other than adlb that may be involved in the mechanisms of contagiousness of Staph. aureus strains associated with high prevalence of IMI.     

Relationships among superantigen toxin gene profiles,genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes and SAg genes. Isolates of RAPD type III were more frequently associated with clinical and subclinical mastitis, whereas strains of type VI were mostly related to subclinical mastitis. In addition, SAg genes were related to severity of bovine mastitis. We conclude that an obvious relationship exists among RAPD genotypes, SAg toxin genes, and severity of bovine mastitis.     

Prevalence and characterization of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus isolated from retail yak butter in Tibet,China

This study aimed to investigate the prevalence, molecular characteristics and antibiotic resistance of Staphylococcus aureus isolates from yak butter in Tibet, China. A total of 218 yak butter samples were collected from retail stores in Tibet and screened for Staph. aureus. Furthermore, the virulence genes, resistance genes, antimicrobial susceptibility, and molecular typing [pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and staphylococcal protein A (spa) typing] of Staph. aureus isolates were detected. The results showed that 12.4% of yak butter samples were contaminated with Staph. aureus, including 5 samples positive for methicillin-resistant Staph. aureus (MRSA). Among all isolates, 96.3% harbored one or more virulence genes, including classical (sea and sec), novel enterotoxin-encoding genes (seh, sek, sel, and seq), and hemolysin genes (hla and hld). All isolates were resistant to at least 2 different antibiotic classes, and the isolates were most commonly resistant to sulfonamides, β-lactams, and erythromycin. For resistance genes, blaZ (74.1%) was most frequently detected, followed by dfrG (51.9%), erm(B) (22.2%), mecA (18.5%), tet(K) (14.8%), aph(2″)-Ia, aph(3′)-III, and ant(6)-Ia (11.1% for each), and erm(C) (7.4%). We detected 8 spa types, 6 sequence types (ST), and 5 clonal complex (CC) types. In addition, 1 isolate of Staph. aureus was nontypeable. We found that CC1-ST1-t559 (55.6%) was the most predominant clone, followed by CC59-ST59-t437 (11.1%), CC5-ST5-t002 (7.4%), CC1-ST1, CC1-ST1-t114, CC1-ST573-t4938, CC1-ST573-t8915, CC30-ST30-t021, and CC25-ST25-t167 (3.7% for each). For PFGE typing, a total of 5 clusters and 15 pulsotypes were generated, and some isolates from different samples showed indistinguishable pulsotypes. Our findings suggest that yak butter produced in Tibet, China, could be contaminated by Staph. aureus strains, including MRSA strains, carrying various virulence and resistance genes, representing multiple antimicrobial resistance phenotypes. The presence of potentially virulent and antibiotic-resistant Staph. aureus strains in yak butter poses a potential threat to consumers, and appropriate measures need to be taken in the production chain to reduce the occurrence of Staph. aureus in yak butter.     

Detection of methicillin-resistant coagulase-negative staphylococci and PVL/mecA genes in cefoxitin-susceptible Staphylococcus aureus (t044/ST80) from unpasteurized milk sold in stores in Djelfa,Algeria

This study was designed to determine antimicrobial resistance phenotypes and genotypes and virulence factors in Staphylococcus aureus and coagulase-negative staphylococci (CNS) in unpasteurized milk sold in Djelfa, Algeria. Eighty-two unpasteurized cow milk samples were randomly obtained from 82 retail stores in Djelfa and tested to detect staphylococci. Species were identified by biochemical tests and MALDI-TOF. Antimicrobial resistance phenotypes and genotypes were determined by disk diffusion test, PCR, and sequencing. The Staph. aureus isolates were subjected to spa typing, multilocus sequence typing, and detection of virulence genes and the scn gene by PCR and sequencing. Forty-five (54.9%) milk samples were contaminated by staphylococci and 45 isolates were recovered: 10 Staph. aureus (12.2% of total samples) and 35 CNS (42.7%). Resistance to penicillin (blaZ), tetracycline (tetL/tetK), and erythromycin (ermB/msrA/ermC) were the most common phenotypes (genotypes). Three CNS were methicillin-resistant and all were mecA-positive. The Staph. aureus isolates were ascribed to the following lineages [spa type/sequence type/associated clonal complex (number of isolates)]: t267/ST479/CC479 (n = 6), t1510/ST5651/CC45 (n = 1), t359/ST97/CC97/ (n = 1), t346/ST15/CC15 (n = 1), and t044/ST80 (n = 1). The mecA gene was detected in the cefoxitin-susceptible t044/ST80 isolate and co-harbored the lukF/lukS-PV and scn genes. The detection of mecA-PVL-positive Staph. aureus, methicillin-resistant CNS, and multidrug-resistant staphylococcal species indicates a potentially serious health issue and reveals that unpasteurized milk sold in Djelfa city could be a potential vehicle for pathogenic and antimicrobial-resistant staphylococci.     

Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil

Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis.     

Short communication: Outbreak of methicillin-resistant Staphylococcus aureus (MRSA)-associated mastitis in a closed dairy herd

Cows are probably the main source of contamination of raw milk with Staphylococcus aureus. Mammary glands with subclinical mastitis can shed large numbers of Staph. aureus in milk. Because of the risk of this pathogen to human health as well as animal health, the aim of this paper was to describe an outbreak of mastitis caused by methicillin-resistant Staph. aureus (MRSA), oxacillin-susceptible mecA-positive Staph. aureus (OS-MRSA), and methicillin-susceptible Staph. aureus (MSSA) on a dairy farm. Milk samples were obtained from all quarters, showing an elevated somatic cell count by the California Mastitis Test. The isolates were identified by phenotypic and genotypic methods. Staphylococcus spp. were isolated from 53% (61/115) of the milk samples, with 60 isolates identified as Staph. aureus (98.4%) and 1 isolate identified as Staphylococcus epidermidis (1.6%). The presence of the mecA gene was verified in 48.3% of Staph. aureus isolates. Of the Staph. aureus isolates, 23.3% were MRSA and 25.0% were OS-MRSA. The total of mastitis cases infected with MRSA was 12.2%. The detection of this large percentage of mastitis cases caused by MRSA and OS-MRSA is of great concern for the animals’ health, because β-lactams are still the most important antimicrobials used to treat mastitis. In addition, Staph. aureus isolates causing bovine mastitis represent a public health risk.     

Short communication: profile of virulence factors of Staphylococcus aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil

Staphylococcus aureus produces exoproteins that contribute to its ability to colonize the mammary gland such as hemolysins, coagulase, slime, and protein A. This study characterized phenotypically and genotypically these virulence factors in 50 Staph. aureus isolates. These isolates were obtained from milk samples from subclinical mastitis cases identified in 15 dairy cattle farms located in the state of Rio de Janeiro, Brazil. All of the confirmed Staph. aureus samples were PCR positive for the coa gene, which displayed 3 different size polymorphisms. The amplification of the spaA X region yielded a single amplicon for each isolate with the prevalent amplicon sized 315 bp. The Staph. aureus isolates were 24 and 16% positive for the hla and hlb genes, respectively, and 22 and 20% positive for the icaA and icaD genes, respectively. Amplification of the agr gene RNAIII was positive in 74% of the strains. Twenty-seven different profiles were identified among the samples, indicating a great diversity of Staph. aureus involved in the etiology of mastitis cases in the analyzed region. These findings are valuable to the comprehension of the distribution of the profiles of Staph. aureus strains isolated from subclinical mastitis cases in the state of Rio de Janeiro.     

A survey of mastitis pathogens including antimicrobial susceptibility in southeastern Australian dairy herds

The objectives for this study were to (1) describe the pathogen profile in quarters from cows with clinical mastitis and in cows with subclinical mastitis in southeastern Australia; and (2) describe antimicrobial susceptibility among isolated pathogens. As a secondary objective, we aimed to compare antimicrobial resistance prevalence in pathogens isolated from clinical and subclinical mastitis samples. A convenience sample of dairy herds (n = 65) from 4 regions in southeastern Australia (Gippsland, Northern Victoria, Tasmania, Western Victoria) were invited to submit milk samples from cows with clinical and subclinical mastitis over a 14-mo period (January 2011 to March 2012). Farmers were instructed to collect aseptic quarter milk samples from the first 10 cases of clinical mastitis for each month of the study. In addition, farmers submitted composite milk samples from cows with subclinical mastitis at 1 or 2 sampling occasions during the study period. Aerobic culture and biochemical tests were used to identify isolates. Isolates were classified as susceptible, intermediate, or resistant to a panel of antimicrobial agents based on the zone of growth inhibition around antimicrobial-impregnated disks, with antimicrobial resistance (AMR) classified as nonsusceptibility by combining intermediate and resistant groups into a single category. Generalized linear mixed models were used to compare the prevalence of AMR between clinical and subclinical mastitis isolates. For clinical mastitis samples (n = 3,044), 472 samples (15.5%) were excluded for contamination. Of the remaining samples (n = 2,572), the most common results were Streptococcus uberis (39.2%), no growth (27.5%), Staphylococcus aureus (10.6%), Escherichia coli (8.4%), and Streptococcus dysgalactiae (6.4%). For subclinical mastitis samples (n = 1,072), 425 (39.6%) were excluded due to contamination. Of the remaining samples (n = 647), the most common results were no growth (29.1%), Staph. aureus (29.1%), and Strep. uberis (21.6%). The prevalence of AMR among common isolates was low for the majority of antimicrobial agents. Exploratory analysis found that the probability of Staph. aureus demonstrating resistance to penicillin was 5.16 times higher (95% confidence interval: 1.68, 15.88) in subclinical isolates relative to clinical Staph. aureus isolates. A similar association was observed for amoxicillin with subclinical Staph. aureus isolates being 4.70 times (95% confidence interval: 1.49, 14.75) more likely to be resistant than clinical Staph. aureus isolates. We concluded that the most common bacteria causing clinical mastitis in dairy herds in Australia is likely to be Strep. uberis, whereas Staph. aureus is likely to be the most common cause of subclinical mastitis. Despite decades of antimicrobial use to control these organisms, AMR appears to be uncommon.     

Long-term persistence of specific genetic types of mastitis-causing Staphylococcus aureus on three dairies

Pulsed-field gel electrophoresis (PFGE) after SmaI digestion was used to investigate the persistence of specific genotypes of bovine mammary gland isolates of Staphylococcus aureus on 3 dairy herds. A total of 341 isolates of Staph. aureus were available from cows in 3 herds, collected over a period of 15 yr. Pulsed-field gel electrophoresis band patterns of Staph. aureus isolates were analyzed visually and with gel analysis and comparison software. Based on this analysis, isolates were classified by PFGE type. Persistence was determined as the time period from the first to the last isolation of a particular PFGE type of Staph. aureus within a herd. Specific types of mastitis-causing Staph. aureus persisted long-term on these dairies. For example, PFGE type 3 isolates persisted on farms A, B, and C for 15, 15, and 13 yr, respectively. Type 6 was found to persist for 13 yr on farm C. Despite the application of standard mastitis control practices, mastitis-causing Staph. aureus types appeared to persist long-term, as detected by PFGE, and were isolated coincident with herd problems of increased milk somatic cell counts and decreased milk production.     

Short communication: Staphylococcus aureus isolated from colostrum of dairy heifers represent a closely related group exhibiting highly homogeneous genomic and antimicrobial resistance features

In heifers, intramammary infections caused by Staphylococcus aureus affect milk production and udder health in the first and subsequent lactations, and can lead to premature culling. Not much is known about Staph. aureus isolated from heifers and it is also unclear whether or not these strains are readily transmitted between heifers and lactating herd mates. In this study, we compared phenotypic characteristics, spa types, and DNA microarray virulence and resistance gene profiles of Staph. aureus isolates obtained from colostrum samples of dairy heifers with isolates obtained from lactating cows. Our objective was to (1) characterize Staph. aureus strains associated with mastitis in heifers and (2) determine relatedness of Staph. aureus strains from heifers and lactating cows to provide data on transmission. We analyzed colostrum samples of 501 heifers and milk samples of 68 lactating cows within the same herd, isolating 48 and 9 Staph. aureus isolates, respectively. Staphylococcus aureus strains from heifers, lactating herd mates, and an unrelated collection of 78 strains from bovine mastitis milk of mature cows were compared. With 1 exception each, characterization of all strains from heifers and lactating cows in the same herd yielded highly similar phenotypic and genotypic results. The strains were Staphaurex latex agglutination test negative (Oxoid AG, Basel, Switzerland) and belonged to agr type II, CC705, and spa types tbl 2645 and t12926. They were susceptible to all antimicrobial agents tested. In contrast, the strains from mature cows in other herds were spread across different clonal complexes, spa types, and SplitsTree clusters (http://www.splitstree.org/), thus displaying a far higher degree of heterogeneity. We conclude that strains isolated from colostrum of heifers and mastitis milk of lactating cows in the same herd feature highly similar phenotypic and genomic characteristics, suggesting persistence of the organism during the first and potentially subsequent lactations or transmission between heifers and mature herd mates.     

Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese

The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S–23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an seh-carrying Staph. aureus spa type tbl 0635 (non-GTB). We conclude that control and reduction of enterotoxigenic Staph. aureus GTB in dairy herds in Switzerland will not only prevent economic losses at the farm level but also improve the safety of raw milk cheeses; distribution of methicillin-resistant Staph. aureus via raw milk cheese is of less concern.     

Prevalence,antimicrobial susceptibility,and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2”, ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China.     

Host adaptation of bovine Staphylococcus aureus seems associated with bacteriological cure after lactational antimicrobial treatment

Staphylococcus aureus causes a wide range of diseases in multiple species. Some sequence types (ST) are observed in a variety of hosts, whereas other strains are mainly associated with bovine mastitis, suggesting host adaptation. We propose that host adaptation of Staph. aureus may influence bacteriological cure of bovine subclinical mastitis after antimicrobial treatment. To test this hypothesis, multilocus sequence typing was performed on Staph. aureus isolates from 60 treated and 79 untreated control quarters that were obtained from well-defined cohorts of dairy cows from a recently conducted randomized field trial on early treatment of subclinical mastitis. Bovine-associated ST were distinguished from non-bovine-associated ST based on the literature and public databases. The association between host adaptation and bacteriological cure was investigated using population-averaged logistic regression models. Thirteen ST were identified, with approximately 80% of isolates belonging to bovine-associated ST. The odds for cure were around 2.5 times as high for non-bovine-associated ST as for bovine ST in treated quarters, whereas no difference in spontaneous cure was observed in untreated control quarters. In addition, host adaptation was related to known predictors of cure, such as penicillin susceptibility and somatic cell count. All isolates belonging to non-bovine-associated ST were resistant to penicillin, whereas the majority of isolates belonging to bovine-associated ST were penicillin susceptible. Penicillin-resistant bovine-associated strains were associated with high somatic cell counts compared with other strains. The correlation between penicillin resistance, cell counts, and host adaptation may affect the association between host adaptation and cure. For diagnostic purposes, a simple and fast alternative to multilocus sequence typing of Staph. aureus to determine host adaptation may be valuable.     

Antibiogram and coagulase diversity in staphylococcal enterotoxin-producing Staphylococcus aureus from bovine mastitis

We investigated antibiogram and coagulase gene diversity in staphylococcal enterotoxin (StE)-producing Staphylococcus aureus isolated from raw milk samples of cows infected with mastitis from 140 dairy farms in Korea between 1997 and 2004. Of the 696 Staph. aureus isolates collected in this study, 164 isolates (23.6%) produced one or more staphylococcal enterotoxins (A to D), and 19 isolates (2.7%) were methicillin-resistant. The percentage of StE-producing Staph. aureus (SES) isolates resistant to methicillin, kanamycin, neomycin, amikacin, and tetracycline was greater than that of non-SES. Ten coagulase genotype patterns were observed, including 4 main types comprising I (25.4%), II (13.9%), VII (13.2%), and VIII (17.8%). More than 4 Staph. aureus types were isolated from each of 82 dairy farms in different geographic locations, and only 1 coagulase genotype pattern was observed in 39 of the herds (47.6%). There was no significant correlation between coagulase genotypes harbored by Staph. aureus and their specific StE type. The percentage of isolates producing major StE types (A, B, AC, and ABCD) and being resistant to cephalothin and methicillin was greater among the Staph. aureus isolates with the 4 predominant coagulase genotypes (I, II, VII, and VIII) than among the isolates harboring the 6 rare coagulase types (III, IV, V, VI, IX, and X). Based on coagulase gene polymorphisms, our data indicate that a broad distribution of identical or closely related enterotoxin-producing Staph. aureus strains seem to contribute to bovine mastitis in the Republic of Korea.     

Differences between Staphylococcus aureus lineages isolated from ovine and caprine mastitis but not between isolates from clinical or subclinical mastitis

Staphylococcus aureus is an important mastitis pathogen, causing both clinical mastitis (CM) and subclinical mastitis (SCM) in small ruminants. In general, CM has a low incidence in sheep and goats but can be very severe and costly. In contrast, subclinical mastitis (SCM) is common but is associated with less cost. For both sheep and goats, S. aureus is the main cause of CM and is associated with SCM cases with a high SCC. Recently, specific lineages of S. aureus have been identified that are associated with CM rather than SCM in dairy cows. It is unknown whether specific S. aureus lineages are associated with CM in goats and sheep. The aim of this study was to compare the clonal complex (CC), staphylococcal protein A (spa) type, leukocidin lukM-lukF' presence, and potential to produce LukMF' in vitro between CM and SCM S. aureus mastitis isolates obtained from sheep and goats. Differences between isolates from different host species were also compared. Ovine (CM, n = 12; SCM, n = 29) and caprine (CM, n = 14; SCM, n = 30) isolates were obtained from 8 sheep flocks and 8 goat herds in the Netherlands. Overall, the isolates belonged to CC133 (85%), CC398 (7%), CC425 (5%), and CC45 (2%). Seventeen spa types were found, including 6 novel types; the predominant types were t2678 (34%), t544 (18%), and t3583 (18%). Although CC133 was dominant among both sheep and goat isolates, spa type CC133/t2678 was associated with ovine isolates, whereas CC133/t544 and CC133/t3583 were found mostly in goats. The presence of lukM-lukF' among the S. aureus isolates was high (87%), especially in CC133 (96%) and CC425 (100%), but the genes were absent in CC45 and CC398. In vitro-cultured lukM-lukF'-positive isolates produced LukM (71 out of 74 positive isolates tested) in the range of 0.4 to 5.0 µg/mL. Interestingly, the goat-associated lineages CC133/t544 and CC133/t3583 produced more LukM in vitro than the sheep-associated CC133/t2678. We found no difference in LukMF' production potential between CM and SCM isolates. In sheep as well as in goats, no association was found between genotype and CM or SCM, demonstrating that the same lineages of S. aureus are responsible for both CM and SCM. These results suggest that subclinically infected animals in a herd or flock likely act as the reservoir of S. aureus causing CM. This highlights the importance of early identification and control of SCM and suggests that controlling SCM within a herd is an effective intervention to prevent CM in small ruminants.     

Short communication: Prevalence and antibiotic resistance of Staphylococcus aureus isolated from bovine clinical mastitis

The aims of this study were to determine the prevalence and antibiotic resistance of Staphylococcus aureus isolated from bovine clinical mastitis in Varamin, Tehran Province, Iran. All of the isolated Staph. aureus were identified by morphology and culture and confirmed using the API Staph identification system (bioMérieux, Marcy-l’Étoile, France). Antibiotic resistance genes were detected by PCR with oligonucleotide primers specific for each gene. Staphylococcus aureus was recovered from 43 of 207 (20.1%) bovine clinical milk samples. Using disk diffusion, methicillin-resistant Staph. aureus was detected in 5 of 43 (11.6%) samples. The pathogen showed high resistance against penicillin G (86%) and tetracycline (76.7%). The blaZ (penicillin) (86%), tetM (tetracycline), and ermC (erythromycin) genes (39.5% each) were the most prevalent antibiotic resistance genes. The findings of this study are useful for designing specific control programs for bovine clinical mastitis caused by Staph. aureus in this region of Iran.     

Identification of potential sources of Staphylococcus aureus in herds with mastitis problems

Staphylococcus aureus is a common udder pathogen of dairy cows that often causes herd problems. Various mastitis control programs have been used to combat the problem but have not always been efficient in preventing new Staph. aureus infections, indicating the presence of possible sources of infection other than those traditionally considered. Therefore, the aim of the study was to identify potential sources of infection relevant for Staph. aureus mastitis within 5 dairy herds with udder health problems caused by Staph. aureus. Samples were collected from milk of lactating cows, from body sites, and from the environment of lactating cows, dry cows, late pregnant heifers, young heifers 4 to 12 mo old, and heifer calves 0 to 3 mo old. Isolates of Staph. aureus were identified and compared using pulsed-field gel electrophoresis. Four to 7 unique Staph. aureus pulsotypes were found within each herd, with one strain predominating in milk in each herd. The milk pulsotypes were also frequently isolated in body samples, especially on hock skin, and in the immediate environment of lactating cows, and were sometimes found in other animal groups, especially in dry cows and heifer calves 0 to 3 mo old. The prevalence of Staph. aureus in milk and other types of samples varied markedly between herds. Staphylococcus aureus isolates with genotypes indistinguishable from those found in milk also dominated in extra-mammary sites within the dairy herds studied, and hock skin was identified as an important reservoir of Staph. aureus. The results contribute new knowledge necessary to improve strategies for udder health control in herds.     

Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples

Mastitis is a prevalent disease in dairy cattle, and staphylococci are among the most common causative pathogens. Staphylococci can express resistance to a range of antimicrobials, of which methicillin resistance is of particular public health concern. Additionally, Staphylococcus aureus carries a variety of virulence factors, although less is understood about the virulence of non-aureus staphylococci (NAS). The aim of our study was to identify and characterize 3 collections of staphylococcal isolates from bovine milk samples regarding antimicrobial resistance, with emphasis on methicillin resistance, and their carriage of virulence genes typically displayed by Staph. aureus. A total of 272 staphylococcal isolates collected in Norway and Belgium in 2016 were included, distributed as follows: group 1, Norway, 100 isolates; group 2, Flanders, Belgium, 64 isolates; group 3, Wallonia, Belgium, 108 isolates. Species identification was performed by use of MALDI-TOF mass spectrometry. Phenotypic resistance was determined via disk diffusion, and PCR was used for detection of methicillin resistance genes, mecA and mecC, and virulence genes. Antimicrobial resistance was common in Staphylococcus epidermidis and Staphylococcus haemolyticus from all different groups, with resistance to trimethoprim-sulfonamide frequently occurring in Staph. epidermidis and Staph. haemolyticus as well as in Staph. aureus. Resistance to penicillin was most frequently observed in group 1. Ten Belgian isolates (1 from group 2, 9 from group 3) carried the methicillin resistance determinant mecA: 5 Staph. aureus from 2 different farms and 5 NAS from 3 different farms. Almost all Staph. aureus isolates were positive for at least 3 of the screened virulence genes, whereas, in total, only 8 NAS isolates harbored any of the same genes. Our study contributes to the continuous need for knowledge regarding staphylococci from food-producing animals as a basis for better understanding of occurrence of resistance and virulence traits in these bacteria.     

Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea

Staphylococcus aureus belongs to the group of major contagious mastitis pathogens, whereas the coagulase-negative staphylococci (CNS) are also capable of causing opportunistic bovine mastitis. Many of these strains are resistant to penicillin or ampicillin because of the long-term use of β-lactam antibiotics in agricultural and healthcare settings. Based on the simple and highly specific coagulase genotyping by PCR-RFLP used for discriminating among Staph. aureus strains, the relationship between phenotypic antibiogram and the polymorphism of coagulase gene was determined in this study. The staphylococci strains (835 Staph. aureus and 763 CNS) were isolated from 3,047 bovine mastitic milk samples from 153 dairy farms in 8 provinces from 1997 to 2004 in the Republic of Korea. Twenty-one (2.5%) Staph. aureus and 19 (2.4%) CNS strains were resistant to methicillin [oxacillin minimum inhibitory concentration (MIC) ≥4 μg/mL]. The mecA gene was also found in 13 methicillin-resistant Staph. aureus (MRSA) and 12 methicillin-resistant CNS (MRCNS) isolates with a significantly higher detection rate of the mecA gene in MRSA with high MIC (≥16 μg/mL) compared with those with MIC ≤ 8 μg/mL. Methicillin-resistant Staph. aureus and MRCNS were also more resistant to other antibiotics (ampicillin, cephalothin, kanamycin, and gentamicin) than methicillin-susceptible staphylococci. Among 10 different coa PCR-RFLP patterns (A to J) in 706 Staph. aureus strains, the main types were A (26.9%), B (17.0%), G (10.5%), and H (15.4%), with the frequent observation of the A and H types (6 and 10 isolates) in MRSA. This study indicates that major epidemic Staph. aureus clones may be spread between different dairy farms, and the profile of coa genotype can be applied for epidemiological investigations and control of bovine mastitis, particularly one caused by MRSA with specific prevalent coa types.