PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis

PulseNet is a national network of pubic health and food regulatory laboratories established in the US to detect clusters of foodborne disease and respond quickly to foodborne outbreak investigations. PulseNet laboratories currently subtype Escherichia coli O157:H7, non-typhoidal Salmonella, and Shigella isolates by a highly standardized 1-day pulsed-field gel electrophoresis (PFGE), and exchange normalized DNA "fingerprint" patterns via the Internet. We describe a standardized molecular subtyping protocol for subtyping Listeria monocytogenes that was recently added to PulseNet. The subtyping can be completed within 30 h from the time a pure culture of the bacteria is obtained.     

A pulsed-field gel electrophoresis typing scheme for Vibrio parahaemolyticus isolates from fifteen countries

Vibrio parahaemolyticus is an important foodborne pathogen in Taiwan and many other maritime Asian countries where seafood is frequently consumed. A total of 535 strains of V. parahaemolyticus were recovered mostly (97%) from clinical samples obtained in Taiwan or in 14 other countries. These strains were typed by pulsed-field gel electrophoresis following SfiI digestion and a typing scheme was generated. The 115 different patterns identified were grouped into 13 types with dissimilarity values less than 15, plus 16 miscellaneous patterns not grouped into any of the types. Types I, A, D and J contained the most patterns, with the numbers of patterns being 17, 13, 12, and 11, respectively. However, types I, B, D, A, H and C contained the most strains, with the numbers of strains being 204, 73, 71, 54, 29 and 25, respectively. Type I consisted exclusively of the pandemic O3:K6 strains and genetically closely related strains. This PFGE typing scheme for V. parahaemolyticus could be used for the characterization of pathogenic isolates.     

Antimicrobial susceptibilities and epidemiological analysis of Salmonella enteritidis isolates in Korea by phage typing and pulsed-field gel electrophoresis

A total of 81 isolates of Salmonella Enteritidis were analyzed by antibiotic susceptibility, phage typing, and pulsed-field gel electrophoresis (PFGE). Thirty-two isolates came from broiler carcasses and pig feces, and 49 isolates were from humans in Seoul and suburbs of Seoul, Korea. Antibiotic resistance was most prevalent among human isolates. Of human isolates, 89.8% were resistant to more than two antibiotics, while 64.7% of poultry isolates and 13.3% of pig isolates showed multiple resistance to more than two antibiotics. The most common phage type (PT) was PT1, followed by PT30 or 33, PT21 and PT20a. The isolates showed six PFGE patterns with XbaI or SpeI digestion, and five PFGE patterns with NotI digestion. But a single pattern, PFGE X1, S1, or N1, was predominant and the rest of the PFGE patterns differed by only one or two bands. Results indicated the spread of a genetically related clone of Salmonella Enteritidis in foods and humans in Korea and that phage typing as well as PFGE may offer an improved level of discrimination for the epidemiological investigation of Salmonella Enteritidis.     

Technical note: a rapid pulsed-field gel electrophoresis method for analysis of bifidobacteria

Pulsed-field gel electrophoresis (PFGE) is a widely used and highly discriminatory molecular typing method that has been applied to bifidobacteria. However, published PFGE protocols used with bifidobacteria require between 5 and 7 d to complete. A rapid PFGE method was developed that can be completed within 24 h.     

Use of pulsed-field gel electrophoresis to characterize the genetic diversity and clonal persistence of Salmonella senftenberg in mussel processing facilities

Salmonella senftenberg was detected in association with persistent contamination events in mussel processing facilities between 1998 and 2002 in Spain. A total of 110 isolates from 8 facilities were subjected to molecular typing by Pulsed-Field Gel Electrophoresis (PFGE). Additionally, a selection of epidemiologically unrelated isolates of this serovar originating from human, animal, feed and environmental sources was included in the study. PFGE analysis proved to be a useful tool for studying the persistence and dissemination of S. senftenberg in factory environments. Facilities that used brine in their processing lines had greater genetic diversity among their S. senftenberg populations, which supports the hypothesis that imported salt used for brine preparation could have been the origin of the contamination. The XbaI type X19 was the most prevalent among the panel, and it was found persisting exclusively in one facility during the 5-year study. In general, isolates from mussel processing plants were clearly different from those of clinical and environmental sources. However, one of the human isolates showed an indistinguishable restriction pattern to an isolate from a frozen mussel sample, this could indicate the potential for food-borne transmission of this serovar via consumption of contaminated seafood products. Isolates in the study were largely sensitive to antimicrobials. Only 9 isolates (6 from mussel processing facilities, 1 from soy flour and 2 from meat meal) showed antimicrobial resistance.     

Raw and processed fish show identical Listeria monocytogenes genotypes with pulsed-field gel electrophoresis

A total of 257 raw fish samples at two different sites were examined for the presence of Listeria monocytogenes. The prevalence of L. monocytogenes was 4%. From 11 positive samples, nine different L. monocytogenes pulsed-field gel electrophoresis genotypes were recovered. From nine pulsotypes recovered from raw fish and 32 pulsotypes shown by 101 fish product isolates, two raw fish and fish product pulsotypes were indistinguishable from each other. Although the prevalence of L. monocytogenes in raw fish is low, the range of L. monocytogenes strains entering the processing plant in large amounts of raw material is wide. This indicates that the raw material is an important initial contamination source of L. monocytogenes in fish processing plants. This postulation is supported by the identical pulsotypes recovered from both raw and processed fish. Some L. monocytogenes strains entering a plant may thus contaminate and persist in the processing environment, causing recurrent contamination of the final products via processing machines.     

Unveiling contamination sources and dissemination routes of Salmonella sp. in pigs at a Portuguese slaughterhouse through macrorestriction profiling by pulsed-field gel electrophoresis

Sixty-nine isolates of Salmonella sp. isolated from the ileum, tonsils, carcass and mandibular and ileocolic lymph nodes of individual pigs slaughtered for consumption in one abattoir were analyzed using serotyping and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (RFLP-PFGE), in order to identify clonal relationships. XbaI macrorestriction was able to distinguish 18 genotypes among the eight identified serotypes: Salmonella Typhimurium (4 genotypes), Salmonella Rissen (3), Salmonella Tennessee (2), Salmonella Enteritidis (2), Salmonella 4,[5],12:i:- (4), Salmonella Give (1), Salmonella Anatum (1), and Salmonella Derby (1). Except for one sample, the serotype and the genotype identified in the samples from the same pork were always the same, allowing to unravel possible dissemination routes of Salmonella sp. through these pork tissues and equate presumptive sources of contamination or infection. Highly significant associations (p < 0.001) were observed for the presence of Salmonella sp. in the ileum and in the ileocolic lymph nodes, as well as between the carcass contamination and the presence of Salmonella sp. in others samples of the correspondent slaughtered pig, such as the ileum, the ileocolic and mandibular lymph nodes and the tonsils. Moreover, 80% of the pigs with ileum and ileocolic lymph nodes positive samples also presented the same salmonella genotype in the correspondent tonsils and, among pigs with positive tonsils, 70% also carried the same genotype in the corresponding mandibular lymph nodes. The occurrence of cross-contamination was also detected, since a genotype identified in other pigs slaughtered in the same day was found in 31% of positive carcasses. The global analysis of the genotypes suggested three different sources of pig infection: the farm of origin, the transportation and the lairage. A particular attention should be paid to the last one, since the majority of the isolates from pig samples were related to infection in the lairage. Since the presence of Salmonella sp. in the ileum of pigs and faeces ingestion promotes tonsils infection and internal dissemination of the agent through the mandibular lymph nodes, as well as drainage to the ileocolic lymph nodes, a potential risk exists at slaughter for Salmonella sp. contamination in the carcasses during pork processing. This risk may be increased by incorrect evisceration techniques and by hygienically inappropriate meat inspection procedures, especially those concerned to the mandibular lymph nodes incisions.     

进出口食品中不同血清型沙门氏菌PFGE和MLST分型比较研究

目的通过脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)两种方法对进出口食品中不同血清型沙门氏菌的分辨力和应用价值进行了比较研究。方法采用PFGE和MLST两种方法对进出口食品中分离得到的鼠伤寒及肠炎两种血清型共30株沙门氏菌进行分型研究。结果两种血清型共30株沙门氏菌经PFGE分型可得到23种型别,DI值为0.9563;鼠伤寒和肠炎两种血清型菌分别经PFGE分型,DI值分别为1.000和0.9048。MLST对30株沙门氏菌分型得到4种ST型,DI值为0.5793;鼠伤寒和肠炎两种血清型菌分别经MLST分型,DI值分别为0.2571和0.1333。结论 在分辨力方面,PFGE更适合于同一沙门氏菌血清型的分型,MLST则适合于不同沙门氏菌血清型间的分型。MLST在替代、补充血清型分型方面有潜在的优势,PFGE在溯源研究方面更胜一筹。     

进出境食品中单核增生李斯特菌的血清分型与脉冲场凝胶电泳分型分析

目的研究进出境食品中的单核增生李斯特菌(LMO)的血清分型与脉冲场凝胶电泳(PFGE)分型的特性及其关系。方法采用标准方法对39株分离的LMO进行PFGE分型,分别用ApaI和Asc I酶切,电泳图谱进行聚类分析,以上菌株同时进行血清分型检测。结果 39株LMO分成6个血清型;经ApaI酶切后,分成29种带型,相似度在57.4%～100%;Asc I酶切后,分成34种带型,相似度在48.6%～100%。讨论 AscI酶切的PFGE分离效果优于ApaI酶切,与血清分型的一致性低于ApaI酶切;PFGE分型效果优于血清分型。     

Epidemiological analysis of Salmonella enterica from beef sampled in the slaughterhouse and retailers in Dakar (Senegal) using pulsed-field gel electrophoresis and antibiotic susceptibility testing

Seventy-eight isolates of Salmonella spp. isolated from beef sampled from the official city slaughterhouse and from retailers in Dakar, Senegal were analyzed using serotyping, antimicrobial testing and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (PFGE). These analyses were done to identify clonal relationships and potential transmission routes in beef channel. XbaI macrorestriction allowed defining 17 genotypes among the six main analyzed serotypes: Salmonella bredeney (3 genotypes), S. muenster (6), S. waycross (1), S. corvallis (3), S. kentucky (1) and S. brandenburg (3). The cross analysis of PFGE profiles and origin of the beef samples reveals a wide range of contamination sources in the beef channel in Dakar. Comparison of PFGE and antimicrobial resistance types shows that the Salmonella contamination sources are equally shared by the slaughterhouse (56% of the isolates) and by the distribution channel (44% of the isolates) by handlings and houseflies.     

Restriction fragment length polymorphisms analysis by pulsed-field gel electrophoresis for discrimination of Staphylococcus aureus isolates from foodborne outbreaks

A number of outbreaks of disease due to Staphylococcus aureus occurring in Aichi-ken, Japan, have provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. Coagulase types, enterotoxin types, phage types, and restriction fragment length polymorphisms (RFLPs) as assessed by pulsed-field gel electrophoresis (PFGE) was performed for S. aureus infections diagnosed in the area of Aichi-ken. Among the 56 isolates of S. aureus from 30 outbreaks, 15 distinctive RFLP types were found by digestion with the restriction enzyme, SmaI. A total of 32 isolates from patients, foodstuffs and cooks on six occasions had the same RFLP types, coagulase types, enterotoxin types and phage types in the same outbreaks. Moreover, the coagulase and phage types could be separated in terms of RFLP. In one outbreak, ten isolates, which were derived from six patients, two foodstuffs and two cooks, had the same coagulase type, enterotoxin type, phage type, and RFLP type. This PFGE method may therefore prove useful for subclassifying S. aureus and differentiating isolates of the same coagulase types and phage types derived from sporadic cases and those derived from foodborne outbreaks.     

Detection and differentiation of Salmonella serotypes using surface enhanced Raman scattering (SERS) technique

This research was conducted to prove that developed silver biopolymer nanoparticle substrate for surface enhanced Raman scattering (SERS) technique could detect and differentiate three different serotypes of Salmonella. Nanoparticle was prepared by adding 100 mg of silver nitrate to a 2 % polyvinyl alcohol solution, then adding 1 % trisodium citrate to reduce silver nitrate and produce silver encapsulated biopolymer nanoparticles. Then, nanoparticle was deposited on a stainless steel plate and used as SERS substrate. Fresh cultures of Salmonella typhimurium, Salmonella enteritidis and Salmonella infantis were washed and suspended in 10 mL of sterile deionized water. Approximately 5 μl of the bacterial suspensions were placed on the substrate individually and exposed to 785 nm laser excitation. SERS spectral data were recorded between 400 and 1,800 cm^?1. SERS signals were collected from 15 different spots on the substrate for each sample. PCA model was developed to classify Salmonella serotypes. PC1 identified 92 % of the variation between the Salmonella serotypes, and PC2 identified 6 % and in total 98 % between the serotypes. Soft independent modeling of class analogies of validation set gave an average correct classification of 92 %. Comparison of the SERS spectra of Salmonella serotypes indicated that both isolates have similar cell walls and cell membrane structures which were identified by spectral regions between 520 and 1,050 cm^?1. However, major differences were detected in cellular genetic material and proteins between 1,200 and 1,700 cm^?1. SERS with silver biopolymer nanoparticle substrate could be a promising tool in pathogen detection and it would potentially be used to classify them.     

Diversity of Campylobacter isolates from retail poultry carcasses and from humans as demonstrated by pulsed-field gel electrophoresis

Campylobacter spp. are a major contaminant of poultry. Eating undercooked chicken and handling raw poultry have been identified as risk factors for campylobacteriosis in humans. Previous studies have found Campylobacter spp. on 90% of poultry carcasses. In the present study, pulsed-field gel electrophoresis (PFGE) was used to assess the genetic diversity of strains on retail poultry carcasses. PFGE patterns of isolates from campylobacteriosis cases were compared to those from the poultry isolates. Over a 1-year study period (March 2000 through February 2001), whole fresh young chickens (n = 72) were obtained from three retail outlets in an urban community in the south-central United States. Campylobacter spp. were isolated from 82% of these carcasses. Strains (n = 70) were defined on the basis of their PFGE pattern. Sixty-seven percent of the carcasses from which Campylobacter spp. were isolated were contaminated with more than one PFGE-distinguishable strain. During the 1-year study period, most of the PFGE patterns (59%) were limited to isolates obtained from a single carcass. Forty-one percent of the PFGE-distinguishable strains were recovered from more than one carcass. Ninety-seven percent of the carcasses contaminated with the same strain were purchased at the same time from the same store. To examine the degree of genetic stability, four strains were followed in vitro over an estimated 1,000 doublings. The PFGE pattern of one of these isolates underwent minor changes during in vitro growth. The data indicate extensive variability in the PFGE patterns of Campylobacter spp. isolated from humans and from poultry carcasses. In spite of difficulties caused by such diversity and the fact that some carcasses are contaminated with more than one strain, the pattern variation provides a useful method for linking a particular strain to its source.     

Typing of Listeria monocytogenes isolates originating from the food processing industry with automated ribotyping and pulsed-field gel electrophoresis

A total of 486 Listeria monocytogenes isolates originating from 17 Finnish food processing plants (representing meat, poultry, fish, and dairy production) were collected and typed by automated ribotyping using EcoRI as the restriction enzyme. The isolates were divided into 16 different ribotypes (RTs). Some of these isolates (121), representing all EcoRI types and 16 food plants, were subjected to ribotyping with the PvuII enzyme, to pulsed-field gel electrophoresis (PFGE) typing with AscI and SmaI restriction enzymes, and to serotyping with O-antigen antisera. Nineteen ribotypes were generated with PvuII, 42 macrorestriction patterns were generated with AscI and 24 with SmaI, and three serotypes were generated with antisera. When the results were combined, the overall number of RTs was 23, and that of the PFGE types was 46. Thus, the overall discrimination power of PFGE was higher (discrimination index [DI] 0.966) than that of ribotyping (DI 0.906). The most common serotype (90.1% of the isolates) was 1/2, and isolates of serotype 4 (3.3%) were rare. There was no connection between food sectors and RTs or PFGE types, but PFGE indicated the single plants (78.3% of the types) better than ribotyping (56.5%). On the basis of its automation and on the availability of identification databases, automated ribotyping had some advantages over PFGE. Overall, automated ribotyping can be considered a practical and rapid tool when Listeria contamination is suspected and when screening a large number of isolates is necessary, e.g., when tracing contamination sources. However, in cases of outbreaks, the identical patterns must be confirmed by PFGE, which is a more discriminatory method.     

Variable number of tandem repeats and pulsed-field gel electrophoresis cluster analysis of enterohemorrhagic Escherichia coli serovar O157 strains

Ninety-five enterohemorrhagic Escherichia coli serovar O157 strains, including 30 strains isolated from 13 intrafamily outbreaks and 14 strains isolated from 3 mass outbreaks, were studied by pulsed-field gel electrophoresis (PFGE) and variable number of tandem repeats (VNTR) typing, and the resulting data were subjected to cluster analysis. Cluster analysis of the VNTR typing data revealed that 57 (60.0%) of 95 strains, including all epidemiologically linked strains, formed clusters with at least 95% similarity. Cluster analysis of the PFGE patterns revealed that 67 (70.5%) of 95 strains, including all but 1 of the epidemiologically linked strains, formed clusters with 90% similarity. The number of epidemiologically unlinked strains forming clusters was significantly less by VNTR cluster analysis than by PFGE cluster analysis. The congruence value between PFGE and VNTR cluster analysis was low and did not show an obvious correlation. With two-step cluster analysis, the number of clustered epidemiologically unlinked strains by PFGE cluster analysis that were divided by subsequent VNTR cluster analysis was significantly higher than the number by VNTR cluster analysis that were divided by subsequent PFGE cluster analysis. These results indicate that VNTR cluster analysis is more efficient than PFGE cluster analysis as an epidemiological tool to trace the transmission of enterohemorrhagic E. coli O157.     

Application of pulsed-field gel electrophoresis to characterise and trace the prevalence of Enterobacter sakazakii in an infant formula processing facility

Enterobacter sakazakii (E. sakazakii) contamination of powdered infant formula (PIF) and its processing environment was monitored between April 2005 and March 2006. The purpose of the monitoring programme was to locate points of contamination, investigate clonal persistence, and identify possible dissemination routes along the processing chain. A total of 80 E. sakazakii isolates were recovered from the manufacturing facility. The overall frequency of isolation of E. sakazakii in intermediate and final product was 2.5%, while specific locations in the processing environment were contaminated at frequencies up to 31%. All E. sakazakii isolates were characterised by pulsed-field gel electrophoresis (PFGE). XbaI macrorestriction digests yielded 19 unique pulse-types that could be grouped into 6 clusters of between 5 and 32 isolates. The formation of large clusters was consistent with the presence of a number of clones in the manufacturing environment. While the majority of isolates were of environmental origin (72.5%), no cluster was confined to one specific location and indistinguishable PFGE profiles were generated from isolates cultured from the manufacturing environment, sampling points along the processing chain and from intermediate and final product. These findings suggest that the manufacturing environment serves as a key route for sporadic contamination of PIF. These data will support the development of efficient intervention measures contributing to the reduction of E. sakazakii in the PIF processing chain.     

Tracing Campylobacter jejuni strains along the poultry meat production chain from farm to retail by pulsed-field gel electrophoresis, and the antimicrobial resistance of isolates

In this study Campylobacter jejuni isolates were recovered from birds, carcasses and carcass portions from two broiler chicken flocks and from equipment used for carcass and meat processing along the production chain from farms to retail stores. Isolates were subjected to pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes and their antimicrobial susceptibilities were determined. C. jejuni was recovered from product and equipment used with both flocks at each point in the production chain. The prevalence of C. jejuni in poultry products at retail stores was 58.97% (flock 1) and 69.23% (flock 2). SmaI divided 122 C. jejuni strains from flock 1 and 106 from flock 2 into 17 and 13 PFGE types, respectively. PFGE types H and F were present at all steps along the chain, from farms to retail products. Similarly, for both flocks PFGE type D was detected in crates, slaughterhouse and retail stores. Moreover, the PFGE types were highly diverse at the processing and retail steps. Most PFGE types were resistant to ciprofloxacin (95.45%) and tetracycline (81.82%); and multidrug resistant PFGE types were found in the final products. Our study showed that there were several points of cross-contamination of product along the chain, and a high diversity of PFGE types with antimicrobial resistance to ciprofloxacin and tetracycline in the retail products.     

Biofilm formation,virulence gene and multi-drug resistance in Salmonella Kentucky isolated in Tunisia

Food-borne diseases caused by Salmonella enterica are a significant public health concern around the world. Since 2002, S. enterica serovar Kentucky has shown an increase in several countries with the concurrent emergence of multidrug-resistant isolates. The spread of such strains in the environment poses a major public health problem. A total of 57 Salmonella Kentucky strains isolated from different sources during the period 2005 to 2008 in Tunisia, were characterized by their antimicrobial and mercury resistance profiles; ability to form a biofilm; virulence invA/spvC genes and quorum sensing sdiA gene. A total of 10.6% of the isolates demonstrated multidrug-resistance against 3 to 13 antibiotics with ciprofloxacin resistance occurring in 33% of human isolates. In addition, 37% of the isolates exhibited minimum inhibitory concentrations value to mercuric chloride, ranging from 8 to 32 μg ml^?1 and were considered as resistant strains. The majority of strains tested were able to form a biofilm, especially for environmental and animal derived isolates. Therefore, the biofilm seems to comprise a normal and favorable capability in the life of Salmonella Kentucky in the environment. Interestingly, all the isolates possessed the sdiA gene, 87.7% of isolates possessed the invA gene, and no isolate harbored the spvC gene.The emergence of resistance to ciprofloxacin in human Salmonella Kentucky isolates, added to the presence of invA and sdiA genes, and the production of biofilm could be the decisive factors in the dissemination of S. Kentucky strains on a large scale.     

Coagulase-negative staphylococci from non-mastitic bovine mammary gland: characterization of Staphylococcus chromogenes and Staphylococcus haemolyticus by antibiotic susceptibility testing and pulsed-field gel electrophoresis

During routine microbiological examination of milk samples from dairy cows without clinical signs of mastitis, quarter milk samples of 231 dairy cows from 12 herds were investigated for the presence of coagulase-negative staphylococci (CNS). The isolates were identified on the basis of colony morphology, Gram staining, catalase and coagulase test and the commercial kit, API Staph. CNS was detected in 29% (67/231) of the cows. A total of seven CNS species were identified with the most prevalent being Staphylococcus (Staph.) chromogenes (30%) and Staph. haemolyticus (28·8%), followed by Staph. simulans (11·2%), Staph. xylosus (11·2%), Staph. epidermidis (7·5%), Staph. hyicus (6·3%) and Staph. sciuri (5%). The predominant species, Staph. chromogenes and Staph. haemolyticus, were further characterized by antibiotic susceptibility testing using the agar disc diffusion method (Kirby-Bauer) and by pulsed-field gel electrophoresis (PFGE). Considerable resistance to ampicillin and penicillin was observed in both species. Isolates with identical or highly similar PFGE profiles were detected at the herd level despite a marked heterogeneity seen for both species. On the basis of somatic cell count, absence of clinical signs of inflammation and heterogeneity of genotypes, we assume that CNS isolated in this study could not be considered as important causative agents of the bovine mammary gland inflammation.     

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.