Short communication: Prevalence of methicillin resistance in coagulase-negative staphylococci and Staphylococcus aureus isolated from bulk milk on organic and conventional dairy farms in the United States

The objective of this study was to evaluate the presence of methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus spp. in bulk tank milk samples from 288 organic and conventional dairy farms located in New York, Wisconsin, and Oregon from March 2009 to May 2011. Due to recent publications reporting the presence mecC (a mecA homolog not detected by traditional mecA-based PCR methods), a combination of genotypic and phenotypic approaches was used to enhance the recovery of methicillin-resistant organisms from bulk tank milk. In total, 13 isolates were identified as methicillin resistant: Staph. aureus (n = 1), Staphylococcus sciuri (n = 5), Staphylococcus chromogenes (n = 2), Staphylococcus saprophyticus (n = 3), Staphylococcus agnetis (n = 1), and Macrococcus caseolyticus (n = 1). The single methicillin-resistant Staph. aureus isolate was identified from an organic farm in New York, for an observed 0.3% prevalence at the farm level. The methicillin-resistant coagulase-negative staphylococci prevalence was 2% in the organic population and 5% in the conventional population. We did not identify mecC in any of the isolates from our population. Of interest was the relatively high number of methicillin-resistant Staph. sciuri recovered, as the number of isolates from our study was considerably higher than those recovered from other recent studies that also assessed milk samples. Our research suggests that the presence of a potential methicillin-resistant Staphylococcus reservoir in milk, and likely the dairy farm population in the United States, is independent of the organic or conventional production system.     

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.     

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: 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.     

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.     

Prevalence and characterization of methicillin-resistant Staphylococcus aureus carrying mecA or mecC and methicillin-susceptible Staphylococcus aureus in dairy sheep farms in central Italy

Between January and May 2012, a total of 286 bulk tank milk samples from dairy sheep farms located in central Italy were tested for the presence of Staphylococcus aureus. One hundred fifty-three samples were positive for S. aureus (53.5%), with an average count of 2.53 log cfu/mL. A total of 679 S. aureus colonies were screened for methicillin resistance by the cefoxitin disk diffusion test, and 104 selected cefoxitin-susceptible isolates were also tested for their susceptibility to other antimicrobials representative of the most relevant classes active against Staphylococcus spp. by using the Kirby-Bauer disk diffusion method. Two methicillin-resistant Staphylococcus aureus (MRSA) isolates, carrying respectively the mecA and the mecC genes, were detected in 2 samples from 2 different farms (prevalence 0.7%). The mecA-positive MRSA isolate was blaZ positive, belonged to spa type t127, sequence type (ST)1, clonal complex (CC)1, carried a staphylococcal cassette chromosome mec (SCCmec) type IVa, and was phenotypically resistant to all the β-lactams tested and to erythromycin, streptomycin, kanamycin, and tetracycline. The mecC-positive MRSA isolate was negative for the chromosomally or plasmid-associated blaZ gene but positive for the blaZ allotype associated with SCCmec XI (blaZ-SCCmecXI), belonged to spa type 843, ST(CC)130, carried a SCCmec type XI, and was resistant only to β-lactams. Both MRSA were negative for the presence of specific immune-evasion and virulence genes such as those coding for the Panton-Valentine leucocidin, the toxic shock syndrome toxin 1, and the immune evasion cluster genes. Regarding the presence of the major S. aureus enterotoxin genes, the mecC-positive MRSA tested negative, whereas the ST (CC)1 mecA-positive MRSA harbored the seh gene. Among the 104 methicillin-susceptible S. aureus isolates examined for antimicrobial susceptibility, 63 (60.58%) were susceptible to all the antimicrobials tested, and 41 (39.42%) were resistant to at least 1 antimicrobial. In particular, 23 isolates (22.12%) were resistant to tetracycline, 16 (15.38%) to sulfonomides, 14 (13.46%) to trimethoprim and sulfamethoxazole, and 9 (8.65%) to ampicillin, whereas only 1 isolate was resistant to both fluoroquinolones and aminoglycosides. The high prevalence of S. aureus found in bulk tank milk samples and the isolation of MRSA, although at a low prevalence, underlines the importance of adopting control measures against S. aureus in dairy sheep farms to minimize the risks for animal and public health. Moreover, this study represents the first report of mecC-positive MRSA isolation in Italy and would confirm that, among livestock animals, sheep might act as a mecC-MRSA reservoir. Although this lineage seems to be rare in dairy sheep (0.35% of farms tested), because mecC-positive MRSA are difficult to detect by diagnostic routine methods employed for mecA-positive livestock-associated MRSA, diagnostic laboratories should be aware of the importance of searching for the mecC gene in all the mecA-negative S. aureus isolates displaying resistance to oxacillin, cefoxitin, or both.     

Short communication: Occurrence of methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci in dairy goat herds in Ohio,United States

In light of the scarcity of information about the occurrence and epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (MRCNS) in small ruminants in general, and particularly dairy goats, we launched this limited-scope study. The findings reported here show the detection of MRSA and MRCNS in goat milk and teat skin samples from dairy goat herds in the state of Ohio. A total of 120 milk samples and 120 teat-swab samples were collected from 5 farms. After conventional isolation and phenotypic characterization of the staphylococci colonies, bacterial isolates were tested by PCR assay targeting the genes nuc to identify Staphylococcus aureus and mecA to detect MRSA and MRCNS. The clonal complexes of MRSA isolates was also determined by multiloccus sequence typing. Fifteen (6.2%) positive S. aureus samples were found in this study: 9 from milk and 6 from teat skin samples. Four (2%) MRSA isolates were detected and, using multiloccus sequence typing genotyping, these were designated to clonal complexes CC133 (n = 2; milk samples) and CC5 (n = 2; teat skin). Three (1.25%) coagulase-negative staphylococci isolates from the teat skin also harbored the mecA gene. Although, the MRSA isolated from milk samples is not a typical human-associated lineage, the CC5 clone isolated from teat skin is a common and widespread clonal complex associated with humans, suggesting that this extramammary niche could be a relevant reservoir of methicillin-resistant staphylococci. Furthermore, the fact that 75% of MRSA were recovered from 1 farm showing poor hygiene practices strengthens the hypothesis that good hygiene practices could be useful to prevent persistence and spread of MRSA at a farm level.     

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.     

Molecular diversity of Staphylococcus aureus and the role of milking equipment adherences or biofilm as a source for bulk tank milk contamination

Staphylococcus aureus is one of the most frequent pathogens causing intramammary infections in dairy herds. Consequently, virulence factors, pathobiology, and epidemiology of Staphylococcus aureus strains have been widely assessed through the years. Nevertheless, not much has been described about the epidemiology of Staph. aureus strains from bulk tank milk (BTM) and adherences on milking equipment (AMES), even when these strains may play a role in the quality of milk that is intended for human consumption. The objective of this study was to assess the strain diversity of 166 Staph. aureus isolates collected from 3 consecutive BTM samples, and from AMES in contact with milk from 23 Chilean dairy farms. Isolates were analyzed and typed using pulsed-field gel electrophoresis. Diversity of strains, both within and among farms, was assessed using Simpson's index of diversity (SID). On farms where Staph. aureus was isolated from both AMES and BTM (n = 8), pulsotypes were further analyzed to evaluate the role of AMES as a potential source of Staph. aureus strains in BTM. Among all Staph. aureus analyzed by pulsed-field gel electrophoresis, a total of 42 pulsotypes (19 main pulsotypes and 23 subtypes) were identified. Among dairy farms, strain diversity was highly heterogeneous (SID = 0.99). Within dairy farms, Staph. aureus strain diversity was variable (SID = 0 to 1), and 18 dairy operations (81.8%) had one pulsotype that was shared between at least 2 successive BTM samples. In those farms where Staph. aureus was isolated in both AMES and BTM (n = 8), 7 (87.5%) showed a clonal distribution of Staph. aureus strains between these 2 types of samples. The overlapping of certain Staph. aureus strains among dairy farms may point out common sources of Staph. aureus among otherwise epidemiologically unrelated farms. Indistinguishable Staph. aureus strains between AMES and BTM across dairy farms suggest that Staph. aureus–containing AMES may represent a source for BTM contamination, thus affecting milk quality. Our study highlights the role of viable Staph. aureus in AMES as a source for BTM contamination on dairy farms, and also describes the overlapping and presence of specific BTM and AMES pulsotypes among farms.     

The occurrence of methicillin-resistant non-aureus staphylococci in samples from cows,young stock,and the environment on German dairy farms

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

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.     

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.     

Biofilm-producing ability of Staphylococcus aureus isolates from Brazilian dairy farms

This study aimed to investigate the in silico biofilm production ability of Staphylococcus aureus strains isolated from milking parlor environments on dairy farms from São Paulo, Brazil. The Staph. aureus isolates were obtained from 849 samples collected on dairy farms, as follows: milk from individual cows with subclinical mastitis or history of the disease (n = 220); milk from bulk tank (n = 120); surfaces of milking machines and utensils (n = 389); and milk handlers (n = 120). Thirty-one Staph. aureus isolates were obtained and categorized as pulsotypes by pulsed-field gel electrophoresis and submitted to assays for biofilm formation on polystyrene, stainless steel, rubber, and silicone surfaces. Fourteen (45.2%) pulsotypes were considered producers of biofilm on the polystyrene microplate assay, whereas 13 (41.9%) and 12 (38.7%) pulsotypes were biofilm producers on stainless steel and rubber, respectively. None of the pulsotypes evaluated produced biofilms on silicone. Approximately 45% of Staph. aureus pulsotypes isolated from different sources on dairy farms showed the ability to produce biofilms in at least one assay, indicating possible persistence of this pathogen in the milking environment. The potential involvement of Staph. aureus in subclinical mastitis cases and its occurrence in milk for human consumption emphasize the need to improve hygiene practices to prevent biofilm formation on the farms studied.     

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.     

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.     

Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction

The aim of this study was to develop and evaluate a real-time quantitative PCR (qPCR)-based method to detect and quantify Staphylococcus aureus in bronopol-preserved milk samples from subclinical intramammary infections (IMI). Serial dilutions of milk artificially inoculated with Staph. aureus ATCC 29213 were used to establish a standard curve (cfu/mL) of the qPCR assay targeting the Staph. aureus thermonuclease-encoding gene nuc according to the strain plate count. The analytical sensitivity, specificity, and repeatability of the qPCR assay were determined. A total of 60 milk samples, collected from mammary quarters without abnormal appearance and with positive isolation of Staph. aureus, were submitted to both the qPCR protocol and Staph. aureus plate counting and results from both methods were compared. Staphylococcus aureus from bronopol-preserved, subclinical IMI milk samples were not accurately enumerated by qPCR compared with plate counting of the nonpreserved, raw milk sample. The detection limit of the qPCR protocol of inoculated Staph. aureus ATCC 29213 in bronopol-preserved milk samples was 1.04 × 10¹ cfu/mL. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect Staph. aureus IMI from bronopol-preserved milk samples compared with a traditional culturing method. However, the proposed qPCR protocol is not accurate for counting of Staph. aureus in bronopol-preserved milk samples from naturally infected mammary glands.     

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.     

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.     

Molecular characterisation and typing the methicillin resistance of Staphylococcus spp. isolated from raw milk and cheeses in northwest Spain: A mini survey

During the last decade, the occurrence of methicillin-resistant staphylococci in animals and foods derived from them has been increasingly reported, but the dairy sector still requires further surveillance. For this, 150 samples of raw bovine milk, ovine and caprine chesses were analysed for identification of Staphylococcus spp. isolates based on 16S rRNA gene sequencing and additionally, typing of methicillin resistance (MR) via PCR-targeting of mec-region genes. Accordingly, 84 staphylococci isolates were identified and their 16S rRNA gene sequences were deposited in GenBank. Among these, 2 isolates of each of Staphylococcus chromogenes and Staphylococcus epidermidis were methicillin-resistant. Phenotypically, a variable multidrug-resistance was noticed for the mecA-positive isolates against eleven selected antibiotics. Results demonstrated that coagulase-negative staphylococci can be a carrier for MR, as Staphylococcus aureus does, and could exist in raw milk from lactating animals; which represents a source for MR transmission to the surrounding environment.     

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.