Short communication: Biofilm production characterization of mecA and mecC methicillin-resistant Staphylococcus aureus isolated from bovine milk in Great Britain

Staphylococcus aureus is an important cause of contagious intramammary infection in dairy cattle, and the ability to produce biofilm is considered to be an important virulence property in the pathogenesis of mastitis. The aim of this study was to characterize the biofilm formation capacity of methicillin-resistant Staph. aureus (MRSA), encoding mecA or mecC, isolated from bulk tank milk in Great Britain. For this purpose, 20 MRSA isolates were grown on microtiter plates to determine the biofilm production. Moreover, the spa-typing and the presence of the intercellular adhesion genes icaA and icaD were analyzed by PCR. All MRSA isolates tested belonged to 9 spa-types and were PCR-positive for the ica genes; 10 of them (50%) produced biofilm in the microtiter plate assay. This is also the first demonstration of biofilm production by mecC MRSA.     

Characterization of Staphylococcus aureus isolates in milk and the milking environment from small-scale dairy farms of São Paulo,Brazil, using pulsed-field gel electrophoresis

This research aimed to evaluate the occurrence of Staphylococcus aureus isolates in milk and in the milking environment of 10 small-scale farms (<400 L/d) located in the regions of Franca and Ribeirão Preto, state of São Paulo, Brazil. Two-hundred twenty samples of milk were collected from individual cows, along with 120 samples from bulk tank milk, 389 samples from milking equipment and utensils (teat cups, buckets, and sieves), and 120 samples from milkers’ hands. Fifty-six Staph. aureus strains were isolated from 849 analyzed samples (6.6%): 12 (5.5%) from milk samples of individual cows, 26 (21.7%) from samples of bulk tank milk, 14 (3.6%) from samples collected from equipment and utensils, and 4 (3.3%) from samples from milkers’ hands. Pulsed-field gel electrophoresis typing of the 56 Staph. aureus isolates by SmaI restriction enzyme resulted in 31 profiles (pulsotypes) arranged in 12 major clusters. Results of this study indicate a low incidence, but wide distribution of Staph. aureus strains isolated from raw milk collected from individual cows and surfaces of milkers’ hands and milking equipment in the small-scale dairy farms evaluated. However, the high percentage of bulk milk samples found with Staph. aureus is of public health concern because raw, unprocessed milk is regularly consumed by the Brazilian population.     

An improved method to culture Staphylococcus aureus from bovine milk

Staphylococcus aureus is an important udder pathogen often associated with subclinical mastitis in dairy cows. Identification of Staph. aureus-positive udder quarters and cows is an important part of control programs to reduce spread of Staph. aureus within and between dairy herds. Therefore, accurate and easy-to-perform culturing methods of Staph. aureus in milk are needed. In the present study, 8 methods for isolation of Staph. aureus in bovine milk samples were investigated. The methods involved different culturing volumes, enrichment, incubation, and freezing processes as well as sedimentation and use of the Mastistrip cassette (SVA, Uppsala, Sweden). Three different sets of milk samples were collected, and 6, 5, and 4 methods were used in each subset of samples. Our results indicate an increased probability of detecting Staph. aureus in milk samples when a simple incubation step (37°C for 18 h) without additives was included before culturing. Using this incubation method, the number of Staph. aureus-positive udder quarters and cows increased by 50 and 29%, respectively, compared with using the standard method of direct culturing of 10 μL of milk. The improved method may be especially useful for detection of low concentrations of Staph. aureus in milk; for example, when screening herds for Staph. aureus.     

Short communication: Antimicrobial susceptibility profiling and genotyping of Staphylococcus aureus isolates from bovine mastitis in Poland

Staphylococcus aureus is the predominant causative agent of bovine mastitis, a disease that remains a major economic burden for the dairy industry worldwide. In this study, the antimicrobial resistance patterns and the genetic composition of 80 S. aureus mastitis isolates collected from 14 dairy farms in Eastern Poland were determined. Of the 10 antimicrobial agents evaluated, only testing for penicillin G produced drug resistance. As 41% of the S. aureus isolates were penicillin resistant, this drug along with other β-lactamase-sensitive β-lactams, should rather not be considered for the treatment of bovine mastitis caused by S. aureus. Upon genotyping, with a triplex PCR method, a total of 11 distinct PCR types were produced. The population structure of S. aureus isolates was highly clonal, with 1 predominant genotype circulating on each farm. The observed similarities in the genotype composition of S. aureus populations from geographically distant farms underscore the significance of interfarm transmission of S. aureus in Poland. This, in turn, argues for the establishment of a nationwide surveillance program for bovine mastitis due to this pathogen.     

Multiple cases of methicillin-resistant CC130 Staphylococcus aureus harboring mecC in milk and swab samples from a Bavarian dairy herd

The discovery of a new mecA homolog, mecC, necessitates a modification of diagnostic procedures for the identification of methicillin-resistant Staphylococcus aureus (MRSA), as most assays used for the genotypic and phenotypic mecA detection cannot currently recognize mecC. Although the prevalence, distribution, and importance of mecC are not yet completely understood, an exchange of mecC-MRSA between humans and animals seems possible. All previously reported observations of mecC-positive strains have been sporadic. To the best of our knowledge, this is the first report about multiple cases of mecC-positive Staph. aureus in 1 dairy herd. Clonal complex 130 Staph. aureus harboring mecC were found in milk samples from 16 of 56 lactating cows kept in a herd in Bavaria, Germany. Almost all quarter milk samples positive for mecC-MRSA had the lowest possible California Mastitis Test score; composite somatic cell counts obtained from monthly milk recordings showed a mean of 51,600 cells/mL in mecC-MRSA affected cows. Additionally, mecC-positive clonal complex 130 Staph. aureus were detected in swab samples from the mammary skin and a teat lesion of 1 cow from this herd. This report suggests that mecC-carrying strains are able to spread among livestock, and that they have the ability to cause multiple cases in single herds. Therefore, future studies targeting MRSA in dairy cows need to consider mecC.     

Prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples and their potential as biocontrol agents

The prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples was assessed. Fourteen Staph. aureus strains were used in enrichment cultures of 75 dairy samples. All samples grew specific Staph. aureus bacteriophages. According to the host range, 8 different phages were isolated. Three of them, phages ΦH5, ΦG7, and ΦA72, were found in 89% of the samples; all the isolated phages were temperate. Phages ΦH5 and ΦA72 were used in preliminary bacterial challenge tests against Staph. aureus in milk. A phage mixture (1:1) was more effective than each single phage, most likely by preventing the survival of lysogenized cells. Phages inhibited Staph. aureus in UHT and pasteurized whole-fat milk. However, the phages were less active in semi-skimmed raw milk and little inhibition was achieved in whole, raw milk. Killing of Staph. aureus was observed at room temperature and at 37°C, but not at refrigeration temperature.     

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.     

A longitudinal study investigating the prevalence of Staphylococcus aureus genotype B in seasonally communal dairy herds

Staphylococcus aureus is a major mastitis-causing pathogen. Various genotypes have been recently identified in Switzerland but Staph. aureus genotype B (GTB) was the only genotype associated with high within-herd prevalence. The risk of introducing this Staph. aureus genotype into a herd may be increased by frequent animal movements. This may also be the case when cows from different herds of origin are commingled and share their milking equipment for a limited period of time. The aim of the present study was to determine the prevalence of Staph. aureus GTB in seasonally communal dairy herds before and after a summer period when dairy farming is characterized by mixing cows from different herds of origin in 1 communal operation. In addition, the environment was investigated to identify potential Staph. aureus GTB reservoirs relevant for transmission of the disease. A total of 829 cows from 110 herds of origin in 9 communal operations were included in the study. Composite milk samples were collected from all cows during the first or second milking after arrival at the communal operation and again shortly before the end of the season. Swab samples from the environment, involved personnel, and herding dogs present were collected before the cows arrived. At the end of the season, sampling of personnel was repeated. All samples were analyzed for the presence of Staph. aureus GTB using an established quantitative PCR. At the beginning of the season, Staph. aureus GTB-positive cows were identified in 7 out of 9 communal operations and the within-communal operation prevalence ranged from 2.2 to 38.9%. At the second sampling, all communal operations were Staph. aureus GTB positive, showing within-communal operation prevalence from 1 to 72.1%. The between-herd of origin prevalence increased from 27.3 to 56.6% and the cow-level prevalence increased from 11.2% at the beginning of the season to 29.6% at the end of the season. On 3 different communal operations, Staph. aureus GTB-positive swabs from seasonally employed personnel were identified at the end of the season. The results indicate that Staph. aureus GTB can easily spread in communal operations when cows from different herds of origin are mixed during the summer season. Effective management measures need to be designed to prevent the spread of Staph. aureus GTB in seasonally communal herds.     

Efficacy of vaccination on Staphylococcus aureus and coagulase-negative staphylococci intramammary infection dynamics in 2 dairy herds

The aim of this study was to evaluate vaccine efficacy of a commercial vaccine (Startvac, Hipra Spain) aimed at reducing intramammary infections (IMI) with Staphylococcus aureus and coagulase-negative staphylococci under field conditions. During the 21-mo duration of the study, 1,156 lactations from 809 cows were enrolled in 2 herds. During the first phase of the trial, all cows that were due to calve were vaccinated until approximately 50% of cows in the milking herd were vaccinated (at ~6 mo). At that point, when 50% vaccination coverage was reached, cows that were due to calve were randomly assigned to be vaccinated or left as negative controls. Cure rate, rate of new infection, prevalence, and duration of infections were analyzed. Vaccination resulted in a moderate reduction in incidence of new staphylococcal IMI and a more pronounced reduction in duration of IMI associated with reduction of the basic reproduction ratio of Staph. aureus by approximately 45% and of coagulase-negative staphylococci by approximately 35%. The utilization of vaccine in combination with other infection-control procedures, such as excellent milking procedures, treatment, segregation, and culling of known infected cattle, will result in an important reduction in incidence and duration of intramammary staphylococcal infections.     

Genetic relatedness and virulence factors of bovine Staphylococcus aureus isolated from teat skin and milk

The objective of this study was to assess the role of teat skin colonization in Staphylococcus aureus intramammary infections (IMI) by evaluating genetic relatedness of Staph. aureus isolates from milk and teat skin of dairy cows using pulsed-field gel electrophoresis and characterizing the isolates based on the carriage of virulence genes. Cows in 4 known Staph. aureus-positive herds were sampled and Staph. aureus was detected in 43 quarters of 20 cows, with 10 quarters positive in both milk and skin (20 isolates), 18 positive only in milk, and 15 only on teat skin. Quarters with teat skin colonized with Staph. aureus were 4.5 times more likely to be diagnosed with Staph. aureus IMI than quarters not colonized on teat skin. Three main clusters were identified by pulsed-field gel electrophoresis using a cutoff of 80% similarity. All 3 clusters included both milk and skin isolates. The majority of isolates (72%) belonged to one predominant cluster (B), with 60% of isolates in the cluster originating from milk and 40% from teat skin. Genotypic variability was observed within 10 pairs (formed by isolates originating from milk and teat skin of the same quarter), where isolates in 5 out of the 10 pairs belonged to the same cluster. Forty-two virulence factors were screened using PCR. Some virulence factors were carried more frequently by teat skin isolates than by milk isolates or isolates from quarters with high somatic cell counts. Isolates in the predominant cluster B carried virulence factors clfA and clfB significantly more often than isolates in the minor clusters, which may have assisted them in becoming predominant in the herds. The present findings suggest that teat skin colonization with Staph. aureus can be an important factor involved in Staph. aureus IMI.     

Mastitis-related subtypes of bovine Staphylococcus aureus are characterized by different clinical properties

Based on a former study from our group, one subtype of Staphylococcus aureus was associated with high within-herd prevalence of mastitis, whereas the other subtypes were associated with a low prevalence (sporadic intramammary infection). To confirm this hypothesis, a prospective study was done in 29 Swiss dairy herds. In particular, milk samples were collected from 10 herds with Staph. aureus herd problems (cases) and compared with samples from 19 herds with only sporadic cases of with Staph. aureus intramammary infection (controls). The isolates were tested for their virulence gene pattern and genotyped by PCR amplification of the 16S-23S rRNA intergenic spacer. The patterns and genotypes were then associated and compared with epidemiological and clinical data. Confirming the hypothesis, one particular subtype (genotype B) was associated with high within-herd and within-cow prevalence of intramammary infection, whereas the other subtypes were associated with low within-herd prevalence and infected single quarters. The gene patterns and genotypes were highly related, demonstrating the genetic diversity of the genotypes. The somatic cell counts were clearly increased in herds with a genotype B problem compared with herds with infections of other genotypes. Based on the different clinical properties and treatment consequences associated with these different genotypes found in Switzerland, we recommend subtyping Staph. aureus in other countries to determine if this finding is universally applicable.     

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows and in Mediterranean buffaloes. Genotype B (GTB) is contagious in dairy cows and may occur in up to 87% of cows of a dairy herd. It was the aim of this study to evaluate genotypes present, clinical outcomes, and prevalence of Staph. aureus in milk samples of primiparous Mediterranean dairy buffaloes. Two hundred composite milk samples originating from 40 primiparous buffaloes were collected from May to June 2012, at d 10, 30, 60, 90, and 150 d in milk (DIM) to perform somatic cell counts and bacteriological cultures. Daily milk yields were recorded. Before parturition until 40 to 50 DIM, all primiparous animals were housed separated from the pluriparous animals. Milking was performed in the same milking parlor, but the primiparous animals were milked first. After 50 DIM, the primiparous were mixed with the pluriparous animals, including the milking procedure. Individual quarter samples were collected from each animal, and aliquots of 1 mL were mixed and used for molecular identification and genotyping of Staph. aureus. The identification of Staph. aureus was performed verifying the presence of nuc gene by nuc gene PCR. All the nuc-positive isolates were subjected to genotype analysis by means of PCR amplification of the 16S-23S rRNA intergenic spacer region and analyzed by a miniaturized electrophoresis system. Of all 200 composite samples, 41 (20.5%) were positive for Staph. aureus, and no genotype other than GTB was identified. The prevalence of samples positive for Staph. aureus was 0% at 10 DIM and increased to a maximum of 22/40 (55%) at 90 DIM. During the period of interest, 14 buffaloes tested positive for Staph. aureus once, 6 were positive twice, and 5 were positive 3 times, whereas 15 animals were negative at every sampling. At 90 and 150 DIM, 7 (17.5%) and 3 buffaloes (7.5%), respectively, showed clinical mastitis (CM), and only 1 (2.5%) showed CM at both samplings. At 60, 90, and 150 DIM, 1 buffalo was found with subclinical mastitis at each sampling. At 30, 60, 90, and 150 DIM, 2.5 (1/40), 22.5 (9/40), 35 (14/40), and 10% (4/40) were considered affected by intramammary infection, respectively. Buffaloes with CM caused by Staph. aureus had statistically significantly higher mean somatic cell count values (6.06 ± 0.29, Log₁₀ cells/mL ± standard deviation) and statistically significantly lower mean daily milk yields (7.15 ± 1.49, liters/animal per day) than healthy animals (4.69 ± 0.23 and 13.87 ± 2.64, respectively), buffaloes with IMI (4.82 ± 0.23 and 11.16 ± 1.80, respectively), or with subclinical mastitis (5.47 ± 0.10 and 10.33 ± 0.68, respectively). Based on our knowledge, this is the first time that Staph. aureus GTB has been identified in milk samples of dairy Mediterranean buffaloes.     

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.     

Characterization for enterotoxin production,virulence factors,and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal

Staphylococcus aureus represents a public health challenge worldwide. The aim of this study was the characterization of different food isolates of S. aureus on the basis of their production of enterotoxins, hemolysins and resistance to antibiotics. A total of 148 coagulase-positive staphylococcal strains isolated from different food origins were identified to the species level. By multiplex PCR, 69% of the isolates were shown to be enterotoxigenic (SEs); the most common were sea seg, sea seg sei and seg sei. According to CLSI [CLSI, Clinical and Laboratory Standards Institute, 2007. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. CLSI document M100-S15. Clinical and Laboratory Standards Institute, Wayne, PA], 38% of the isolates were resistant to oxacillin (≥6 μg/mL; MRSA positives) but only 0.68% showed the presence of mecA gene. 70 and 73% of the S. aureus strains were resistant to β-lactams, ampicillin and penicillin, respectively. The virulence pattern was demonstrated to be origin and strain dependent. These findings emphasise the need to prevent the presence of S. aureus strains and SEs production in foods.     

Short communication: Methicillin-resistant Staphylococcus aureus detection in US bulk tank milk

Staphylococcus aureus is a major cause of mastitis in dairy cattle. This study estimated the herd prevalence of methicillin-resistant Staph. aureus (MRSA) among US dairy herds by testing bulk tank milk (BTM) samples using genotypic and phenotypic methods. A nationally representative sample of 542 operations had BTM cultured for Staph. aureus, and 218 BTM samples were positive upon initial culture. After 4 wk to 4 mo of frozen storage, 87% of 218 samples (n = 190) were still culture positive for Staph. aureus on blood agar, but none were positive for MRSA on the selective indicator medium CHROMagar MRSA. A duplex PCR was used to detect the Staph. aureus-specific nuc gene and the methicillin resistance gene, mecA, in mixed staphylococcal isolates from the 190 BTM samples that were positive for Staph. aureus after storage. Seven samples tested positive for nuc and mecA, and 2 samples tested positive for mecA only. MecA-positive Staphylococcus spp., but not MRSA, were subsequently isolated from 5 samples, whereas neither mecA-positive Staphylococcus spp. nor MRSA was isolated from the remaining 4 samples. Presence of methicillin-resistant, coagulase-negative Staphylococcus spp. may complicate the detection of MRSA by means of PCR on BTM. Bulk tank milk in the United States is not a common source of MRSA.     

Genotype-specific risk factors for Staphylococcus aureus in Swiss dairy herds with an elevated yield-corrected herd somatic cell count

Bovine mastitis is a frequent problem in Swiss dairy herds. One of the main pathogens causing significant economic loss is Staphylococcus aureus. Various Staph. aureus genotypes with different biological properties have been described. Genotype B (GTB) of Staph. aureus was identified as the most contagious and one of the most prevalent strains in Switzerland. The aim of this study was to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB in Swiss dairy herds with an elevated yield-corrected herd somatic cell count (YCHSCC). One hundred dairy herds with a mean YCHSCC between 200,000 and 300,000 cells/mL in 2010 were recruited and each farm was visited once during milking. A standardized protocol investigating demography, mastitis management, cow husbandry, milking system, and milking routine was completed during the visit. A bulk tank milk (BTM) sample was analyzed by real-time PCR for the presence of Staph. aureus GTB to classify the herds into 2 groups: Staph. aureus GTB-positive and Staph. aureus GTB-negative. Moreover, quarter milk samples were aseptically collected for bacteriological culture from cows with a somatic cell count ≥150,000 cells/mL on the last test-day before the visit. The culture results allowed us to allocate the Staph. aureus GTB-negative farms to Staph. aureus non-GTB and Staph. aureus-free groups. Multivariable multinomial logistic regression models were built to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB. The prevalence of Staph. aureus GTB herds was 16% (n = 16), whereas that of Staph. aureus non-GTB herds was 38% (n = 38). Herds that sent lactating cows to seasonal communal pastures had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 10.2, 95% CI: 1.9–56.6), compared with herds without communal pasturing. Herds that purchased heifers had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds without purchase of heifers. Furthermore, herds that did not use udder ointment as supportive therapy for acute mastitis had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 8.5, 95% CI: 1.6–58.4) or Staph. aureus non-GTB (odds ratio: 6.1, 95% CI: 1.3–27.8) than herds that used udder ointment occasionally or regularly. Herds in which the milker performed unrelated activities during milking had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds in which the milker did not perform unrelated activities at milking. Awareness of 4 potential risk factors identified in this study guides implementation of intervention strategies to improve udder health in both Staph. aureus GTB and Staph. aureus non-GTB herds.     

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.     

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.     

Incidence of Staphylococcus aureus and associated risk factors in Nham,a Thai fermented pork product

Staphylococcus aureus is one of the most prevalent bacterial pathogens causing food-borne disease worldwide. Staphylococcal food poisoning is caused by ingestion of staphylococcal enterotoxins (SEs) pre-formed in the implicated food. In this study, the incidences of S. aureus and classical SEs (SEA-SEE) contamination in ‘Nham’, a traditional Thai fermented pork product, were determined. Among 155 Nham samples tested, as high as 39.35% of the samples were positive for S. aureus (2–3500 MPN/g), but none were positive for the SEs. The risk factors for S. aureus contamination were highly correlated with the manufacturer and the pH of the product. A predictive model determined the probability of the presence of S. aureus to be ≤0.24 at the pH ≤ 4.6. During the fermentation process, the number of S. aureus slightly increased in the first day and decreased afterward. S. aureus counts continued to decrease when Nham was stored refrigerated. The negative result for enterotoxins and low counts of S. aureus in Nham surveyed in this study, and reduction of the pathogen counts during fermentation and storage suggested that there is very low risk of staphylococcal food poisoning from consuming properly fermented Nham.