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.     

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.     

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.     

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.     

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: 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.     

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.     

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.     

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.     

Occurrence of methicillin-resistant Staphylococcus aureus in dairy cattle herds,related swine farms,and humans in contact with herds

In this study we investigated the circulation of methicillin-resistant Staphylococcus aureus (MRSA) in 2 dairy cattle farms (farm A and B), previously identified as MRSA-positive in bulk tank milk samples, and epidemiologically related to swine farms. Collected specimens included quarter milk samples and nasal swabs from dairy cows, pig nasal swabs collected at both the farm and slaughterhouse level, environmental dust samples, and human nasal swabs from the farms’ owners and workers. The prevalence of MRSA was estimated at the herd level by testing quarter milk samples. The prevalence of MRSA was 4.8% (3/63; 95% confidence interval = 0–10.2%) and 60% (33/55; 95% confidence interval = 47.05–72.95) in farm A and B, respectively. In farm A, MRSA was also isolated from humans, pigs sampled at both farm and slaughterhouse level, and from environmental samples collected at the pig facilities. The dairy cattle facilities of farm A tested negative for MRSA. In farm B, MRSA was isolated from environmental dust samples in both the cattle and pig facilities, whereas nasal swabs collected from cows and from humans tested negative. Sixty-three selected MRSA isolates obtained from different sources in farm A and B were genetically characterized by multilocus sequence typing, spa-typing, ribosomal spacer-PCR, and also tested for the presence of specific virulence genes and for their phenotypical antimicrobial susceptibility by broth microdilution method. Different clonal complex (CC) and spa-types were identified, including CC398, CC97, and CC1, CC already reported in livestock animals in Italy. The MRSA isolates from quarter milk of farm A and B mostly belonged to CC97 and CC398, respectively. Both lineages were also identified in humans in farm A. The CC97 and CC398 quarter milk isolates were also identified as genotype GTBE and GTAF by ribosomal spacer-PCR respectively, belonging to distinct clusters with specific virulence and resistance patterns. The GTBE and GTAF clusters also included swine, environmental, and human isolates from both farms. A high heterogeneity in the genetic and phenotypic profiles was observed in environmental isolates, in particular from farm B. These results demonstrate the possibility of a dynamic sharing and exchange of MRSA lineages or genotypes between different species and farm compartments in mixed-species farms. The risk of transmission between swine and related dairy cattle herds should be considered. Our findings also confirm the zoonotic potential of livestock-associated MRSA and underline the importance of applying biosecurity measures and good hygiene practices to prevent MRSA spread at the farm level and throughout the food production chain.     

Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis

The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined.     

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap−), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.     

Relationships between milk culture results and treatment for clinical mastitis or culling in Norwegian dairy cattle

In quarter milk samples from 2,492 randomly sampled cows that were selected without regard to their current or previous udder health status, the relationships between the following outcome variables were studied: treatment of clinical mastitis; the joint event of either treatment or culling for mastitis; culling for all reasons; culling specifically for mastitis; and the covariates of positive milk culture for Staphylococcus aureus, Streptococcus spp., and coagulase-negative Staphylococcus spp., or other pathogens, or of negative culture for mastitis pathogens. Microbiological diagnoses were assigned at the cow level, and altogether 3,075 diagnoses were related to the outcome variables. The relation between the absence of pathogens and rich (>1,500 cfu/mL of milk) or sparse (≤1,500 cfu/mL of milk) growth of Staph. aureus were also assessed separately for each outcome variable. The hazard of treatment of clinical mastitis was greater for cows diagnosed with Staph. aureus compared with cows with no pathogens in all analyses. Cows with sparse growth of Staph. aureus upon microbiological analysis were more likely to be treated for clinical mastitis, and cows with rich growth of the bacteria experienced a higher overall risk of culling when the models adjusted for cow composite milk somatic cell count. No difference between rich and sparse growth of Staph. aureus was found when mastitis was defined as the joint event of either culling for mastitis or treatment of clinical mastitis, and when the relationship with culling specifically for mastitis was assessed. The combined outcome of treatment and culling for mastitis was related to a positive diagnosis of Strep. spp. after cow composite milk somatic cell count was omitted from the model. Presence of Streptococcus spp. was also related to culling specifically for mastitis, whereas culling for all reasons and treatment of clinical mastitis was not related to a positive culture of Strep. spp. Presence of coagulase-negative Staph. spp. or other pathogens was not associated with either of the outcome variables.     

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.     

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.     

Streptococcus dysgalactiae isolates at calving and lactation performance within the same lactation

The objective of the study was to investigate the association between early lactation Streptococcus dysgalactiae isolates and milk yield, somatic cell count (SCC), clinical mastitis, and culling in the same lactation. The 178 commercial dairy herds were randomly placed into 3 penicillin- or penicillin-dihydrostreptomycin-based dry-cow treatments and 3 different postmilking teat disinfection groups—negative control, iodine, or external teat sealant. All cows were sampled in early lactation, and Strep. dysgalactiae-positive and culture-negative cows were followed throughout the remainder of the lactation. Mixed models, including repeated measurements, with test-day observation as dependent variable, were used to compare milk yield, SCC, and available milk quality variables throughout the remaining lactation. Survival analyses, using a positive frailty model to account for any herd random effects, were used to estimate the hazard ratio for clinical mastitis and culling. Streptococcus dysgalactiae-positive cows had a significantly higher SCC throughout the lactation compared to culture-negative cows. For primiparous or multiparous cows, respectively, the differences in the geometric mean SCC between Strep. dysgalactiae-positive and culture-negative cows was 197,000 or 280,000 cells/mL at the beginning of the lactation, 24,000 or 46,000 cells/mL in mid lactation, and 39,000 or 111,000 cells/mL at the end of the lactation. Streptococcus dysgalactiae-positive primiparous or multiparous cows produced 334 or 246 kg less milk, respectively, during a 305-d lactation compared with culture-negative cows. Compared with culture-negative cows, the hazard ratios for clinical mastitis in Strep. dysgalactiae-positive cows were 2.3 (1.9 to 2.9) and 1.6 (1.3 to 2.0) for culling. For cows with both Strep. dysgalactiae and Staphylococcus aureus isolates, the hazard ratio for culling significantly increased to 2.5 (1.9 to 3.2).     

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.     

Molecular epidemiology,antimicrobial activity,and virulence gene clustering of Streptococcus agalactiae isolated from dairy cattle with mastitis in China

Streptococcus agalactiae is a contagious pathogen that causes bovine mastitis worldwide, resulting in considerable economic losses. In this study, we isolated 42 S. agalactiae strains in 379 milk samples from cows with subclinical mastitis on 15 dairy farms in 12 Chinese provinces. Analysis based on capsular typing and multilocus sequence typing, combined with patterns of virulence gene scanning and antimicrobial resistance, identified the lineages and populations of the isolates. We grouped the 42 isolates into 7 sequence types belonging to 6 clonal complexes, mainly CC103 (31/42 isolates; 73.8%). We identified an ST-23 strain named Sa 129 for the first time on Chinese dairy farms—this strain is usually associated with human isolates. Capsular types Ia and II were predominant in capsular typing. The prevalence of virulence profile 1 (bibA, cfb, cspA, cylE, fbsA, fbsB, hylB, and pavA) was 64.3%, and represented the main trend in China. With respect to antimicrobial resistance, most isolates were susceptible to β-lactams, rifamycin, glycopeptides, and oxazolidone; resistance to several antimicrobial agents, including lincomycin, clindamycin, and doxycycline, varied in 4 different regions. Our research provides a profile for the molecular epidemiology, multilocus sequence typing, antimicrobial resistance, and virulence gene clustering of S. agalactiae, and may be beneficial for the clinical monitoring, prevention, and control of mastitis in dairy cattle.     

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.     

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.