Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a global health concern. The present study regarded 160 S. aureus strains that had been isolated from 1634 foodstuff samples of animal origin in a previous survey conducted in Italy during 2003-2005. The strains were characterized by detecting the mecA gene, the production of type A to D staphylococcal enterotoxins (SEs), and studying their resistance properties against several antibiotics; their ecological origin was determined by biotyping. Of the 160 analyzed S. aureus strains six (3.75%) were mecA positive and derived from six different samples; four isolates were from bovine milk and two from dairy products (pecorino cheese and mozzarella cheese). Two strains isolated from milk belonged to the non-host-specific biovar while the others to the ovine biovar. The strain isolated from mozzarella cheese belonged to the non-host-specific biovar and the strain isolated from pecorino cheese to the ovine biovar. All the MRSA strains isolated were enterotoxigenic; two strains synthesized SEA/SED two SED and one SEC. All the strains showed resistance to at least one of the antibiotics tested but none was resistant to glycopeptides.     

Virulence factors and genetic variability of Staphylococcus aureus strains isolated from raw sheep's milk cheese

Contamination of dairy products with Staphylococcus aureus can be of animal or human origin. The host pathogen relationship is an important factor determining genetic polymorphism of the strains and their potential virulence. The aim of the present study was to carry out an extensive characterization of virulence factors and to study the genetic variability of S. aureus strains isolated from raw ewe's milk cheese. A total of 100 S. aureus strains isolated from cheese samples produced in 10 artisan cheese factories were analyzed for the presence of enterotoxins (sea-see) and enterotoxins-like genes (seh, sek, sel, sem, seo, sep), leukocidins, exfoliatins, haemolysins, toxic shock syndrome toxin 1 (TSST-1) and the accessory gene regulator alleles (agr). Strains were also typed using pulsed-field gel electrophoresis (PFGE). AMOVA analysis carried out on PFGE and PCR data showed that the major component explaining genetic distance between strains was the dairy of origin. Of the total isolates 81% had a pathogenicity profile ascribable to "animal" biovar while 16% could be related to "human" biovar. The biovar allowed to estimate the most likely origin of the contamination. Minimum inhibitory concentrations (MICs) of nine antimicrobial agents and the presence of the corresponding genes coding for antibiotic resistance was also investigated. 18 strains carrying blaZ gene showed resistance to ampicillin and penicillin and 6 strains carrying tetM gene were resistant to tetracycline. The presence of mecA gene and methicillin resistance, typical of strains of human origin, was never detected. The results obtained in the present study confirm that S. aureus contamination in artisan cheese production is mainly of animal origin.     

Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production

Staphylococcus aureus is an important agent of bacterial mastitis in milking animals and of foodborne intoxication in humans. The purpose of this study was to examine the genetic and phenotypic diversity, enterotoxigenicity, and antimicrobial resistance of S. aureus strains isolated from raw milk used for the production of artisan cheese in Vermont. Cross-tabulations revealed that the 16 ribotypes identified among the 90 milk isolates examined were typically associated with a specific animal species and that more than half of these ribotypes were unique to individual farms. In general, specific EcoRI ribotypes were commonly associated with specific phenotypical characteristics, including staphylococcal enterotoxin production or the lack thereof. Limited antimicrobial resistance was observed among the isolates, with resistance to ampicillin (12.51%) or penicillin (17.04%) most common. Two isolates of the same ribotype obtained from the same farm were resistant to oxacillin with 2% NaCl. More than half (52.22%) of isolates produced toxin, and 31 of the 32 isolates solely produced staphylococcal enterotoxin type C. Although these data demonstrate that S. aureus strains found in raw milk intended for artisan cheese manufacture are capable of enterotoxin production, staphylococcal enterotoxin C is not typically linked to foodborne illness. Because S. aureus is a common contaminant of cheese, an understanding of the ecology of this pathogen and of the antimicrobial susceptibility and toxigenicity of various strains will ultimately contribute to the development of control practices needed to enhance the safety of artisan and farmstead cheese production.     

金黄色葡萄球菌耐药性与生物被膜能力的鉴定

本文选择常见的典型食源性微生物金黄色葡萄球菌,从食品微生物安全角度出发,对127株葡萄球菌的菌株特性、耐药性与生物被膜生长能力进行研究,包括菌株生化鉴定;PCR扩增葡萄球菌特异16S rRNA与金葡菌特异femA基因,通过对耐药基因mecA和orfX检测确定菌株的耐药特性;最后,运用结晶紫染色法进行生物被膜能力检测。127株菌株包括119株金葡菌(均携带16S rRNA与femA)与8株凝固酶阴性葡萄球菌8株(仅携带16S rRNA)。119株金葡菌中,107株携带mecA基因与orfX基因,为耐甲氧西林金葡球菌(MRSA);8株凝固酶阴性葡萄球菌中,均携带mecA基因。所有菌株均能生成生物被膜,其中能形成强、中等与弱粘附生物被膜能力菌株分别有5(3.9%)、47(37.0%)和75(59.1%)株。金葡菌中耐药性与生物被膜较为普遍,由于食源性微生物形成生物被膜后,具有逃逸常规消毒和杀菌手段的能力,成为食品安全中的潜在隐患。     

Occurrence of enterotoxic Staphylococcus aureus in raw milk from yaks and cattle in Mongolia

Staphylococcal food poisoning is considered one of the leading foodborne illnesses in humans worldwide and is associated with contaminated foods of animal origin, such as milk and dairy products. In this study, we investigated the occurrence of staphylococci and the enterotoxigenic properties of Staphylococcus aureus isolated from raw milk from yaks (Bos mutus) and cattle in Mongolia. Staphylococci were isolated from 72 (74%) of the 97 raw milk samples. Of the samples containing staphylococci, 69% (50 of 72) were from yaks and 30.5% (22 of 72) were from cattle. S. aureus was detected in 10% of yak (7 of 72) and 21% of cattle (15 of 72) milk samples. Staphylococcal enterotoxin C was detected in 23% (5 of 22) of the S. aureus strains investigated, based on the reverse passive latex agglutination technique. Three of the five enterotoxigenic strains were from yaks and two were from cattle. None of the S. aureus strains tested produced staphylococcal enterotoxins A, B, or D. To our knowledge, this is the first report of the occurrence of staphylococci and enterotoxigenic S. aureus in milk from yaks and cattle in Mongolia.     

Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.     

四川省动物性食品中金黄色葡萄球菌的分离鉴定及污染分析

目的：为分析四川省动物性食品源金黄色葡萄球菌(Staphylococcus aureus,简称SA)的耐药性,收集菌株,同时分析动物性食品中SA的污染情况。方法：本实验自2006年12月至2007年9月从四川省各地采集猪源、牛源、鸡源动物性食品样品共2560份,利用选择性培养基和生化实验等常规方法分离鉴定SA。结果：用Baird-Parker平板初步筛出疑似SA 118株,结合国标中规定的涂片染色镜检,溶血现象和血浆凝固酶实验结果,判定SA 有108株,而科玛嘉金黄色葡萄球菌显色平板和TH-16S中的双歧索引鉴定结果均判定SA有 113株;依据TH-16S中的16项生化实验结果,118株疑似SA仅有64%(76株)的菌株鉴定为SA。依据国标判定结果,2560份样品中SA的分离率为4.21%,其中生牛奶中SA的分离率最高(10.54%),其次是猪肉(7.11%),鲜鸡蛋中SA的分离率为零。结论：四川地区动物性食品源SA的生化表型复杂且存在一定差异,常规的表型分析具有一定缺陷。各个地方各类样品中SA污染情况有一定差异,与其他报道相比,四川省生肉、生奶和鲜蛋中SA的分离率较低。     

Implication of milk and milk products in food-borne diseases in France and in different industrialised countries

A study was carried out to estimate the proportion of diseases due to milk and milk products among food-borne diseases recorded in France and in other countries since 1980. Particular attention was given to whether the milk involved was heat-treated or not. Four etiologic agents were considered: Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, and pathogenic Escherichia coli. An overview of food-borne disease annual reports from seven countries indicated that milk and milk products were implicated in 1-5% of the total bacterial outbreaks; however, details about the type of product and milk involved were usually not provided. When considering 60 outbreaks and four single cases described in the literature and implicating milk and milk products, confirmed or suspected food vehicles were distributed as follows: milk, 39.1%, cheese, 53.1%, other milk products, 7.8%. Overall, 32.8% of the food vehicles were made from pasteurised milk; 37.5% from raw milk; 10.9% from milk stated as "unpasteurised"; and 18.8% from unspecified milk. Salmonella spp. were responsible for 29 outbreaks, L. monocytogenes for 10 outbreaks and four well-documented single cases, pathogenic E. coli for 11 outbreaks, and S. aureus for 10 outbreaks. Analysis of unpublished data about food-borne disease outbreaks, listeriosis excluded, collected by the coordinator of the French surveillance system from 1992 to 1997, revealed 69 documented outbreaks for which milk and milk products were confirmed as the vehicle by the isolation of the etiologic agent. The food vehicles were distributed as follows: milk, 10%; cheese, 87%; others, 3%. UHT milk accounted for 1.5%, raw milk and raw milk products for 48%, and milk and milk products from unspecified milk for 50.5% of the 69 outbreaks. S. aureus was by far the most frequent pathogen associated with these outbreaks (85.5% of the outbreaks), followed by Salmonella (10.1%). This study demonstrates the limitations of the surveillance systems and the difficulties in estimating the contribution of milk and milk products to food-borne diseases. In particular, it was not possible to find out in many outbreaks what heat treatment, if any, the milk had undergone.     

Occurrence and characterization of Staphylococcus aureus isolated from raw milk for cheesemaking

Fifty-four samples of raw milk for cheesemaking were tested for the presence of Staphylococcus aureus . Multiplex polymerase chain reactions were performed to identify presumptive isolates and the presence of enterotoxin-coding genes sea-see . The strains were tested for antibiotic resistance. Eighty strains were identified as S. aureus and 31 of these carried one or more enterotoxin genes ( sea-see ). Resistance to eritromycin, penicillin and ampicillin was widespread among isolates . Staphylococcus aureus in raw milk for cheesemaking may constitute a risk with respect to staphylococcal food poisoning from raw milk products.     

The influence of Lactobacillus plantarum culture inoculation on the fate of Staphylococcus aureus and Salmonella typhimurium in Montasio cheese.

The growth and survival of Staphylococcus aureus and Salmonella typhimurium were investigated during the manufacturing and ripening of raw milk Montasio cheese. Initial inoculated populations in the cheese milk were about 10(5) cfu/ml for S. aureus and 10(6) cfu/ml for S. typhimurium. Samples of curds and cheeses were taken during manufacturing and storage and analysed for pH and microbial populations. S. aureus increased slightly in number during the early period of ripening and attained a population of about 10(6) cfu/ml during the remaining period of storage. S. typhimurium decreased during cheesemaking and storage but persisted through 90 days. The addition of Lactobacillus plantarum culture (0.2% v/v) produced a marked reduction in populations of the test strains in 10 days of storage. Enterotoxin A was not detected in Montasio cheese even with a S. aureus population of 1.1 X 10(7) cfu/ml. L. plantarum strains were also tested by the spot method and the associative growth approach for their antagonistic activity against S. aureus and S. typhimurium. The compound excreted by L. plantarum was active only toward S. aureus. Furthermore, its activity was destroyed by protease treatment. These results indicated that while the growth of S. typhimurium is reduced by the acid production, S. aureus inhibition can be ascribed to bacteriocin production.     

Phage inactivation of Staphylococcus aureus in fresh and hard-type cheeses

Bacteriophages are regarded as natural antibacterial agents in food since they are able to specifically infect and lyse food-borne pathogenic bacteria without disturbing the indigenous microbiota. Two Staphylococcus aureus obligately lytic bacteriophages (vB_SauS-phi-IPLA35 and vB_SauS-phi-SauS-IPLA88), previously isolated from the dairy environment, were evaluated for their potential as biocontrol agents against this pathogenic microorganism in both fresh and hard-type cheeses. Pasteurized milk was contaminated with S. aureus Sa9 (about 10(6)CFU/mL) and a cocktail of the two lytic phages (about 10(6)PFU/mL) was also added. For control purposes, cheeses were manufactured without addition of phages. In both types of cheeses, the presence of phages resulted in a notorious decrease of S. aureus viable counts during curdling. In test fresh cheeses, a reduction of 3.83log CFU/g of S. aureus occurred in 3h compared with control cheese, and viable counts were under the detection limits after 6h. The staphylococcal strain was undetected in both test and control cheeses at the end of the curdling process (24h) and, of note, no re-growth occurred during cold storage. In hard cheeses, the presence of phages resulted in a continuous reduction of staphylococcal counts. In curd, viable counts of S. aureus were reduced by 4.64log CFU/g compared with the control cheeses. At the end of ripening, 1.24log CFU/g of the staphylococcal strain was still detected in test cheeses whereas 6.73log CFU/g was present in control cheeses. Starter strains were not affected by the presence of phages in the cheese making processes and cheeses maintained their expected physico-chemical properties.     

Effects of Nisin and Lysozyme on Growth Inhibition and Biofilm Formation Capacity of Staphylococcus aureus Strains Isolated from Raw Milk and Cheese Samples

Effects of nisin and lysozyme on growth inhibition and biofilm formation capacity of 25 Staphylococcus aureus strains isolated from raw milk (13 strains) and cheese (12 strains) were studied. Nisin was tested at concentrations between 0.5 and 25 μg/ml; the growth of all strains was inhibited at 25 μg/ml, but the resistances of strains showed a great variation at lower nisin concentrations. In contrast, lysozyme tested at concentrations up to 5.0 mg/ml showed no inhibition on the growth of strains. Nisin used at the growth inhibitory concentration prevented the biofilm formation of strains, but strains continued biofilm formation at subinhibitory nisin concentrations. Lysozyme did not affect the biofilm formation of 19 of the strains, but it caused a considerable activation in the biofilm formation capacity of six strains. Twelve of the strains contained both biofilm-related protease genes (sspA, sspB, and aur) and active proteases; eight of these strains were nisin resistant. These results suggest a potential risk of S. aureus growth and biofilm formation when lysozyme is used in the biopreservation of dairy products. Nisin can be used to control growth and biofilm formation of foodborne S. aureus, unless resistance against this biopreservative develops.     

Examination of Staphylococcus aureus survival and growth during cheese-making process

Inoculation tests of Staphylococcus aureus were performed to evaluate the risk of toxic hazard in cheese manufacturing processes. S. aureus was inoculated into pasteurized milk or cheese curd, and the survival and growth were examined. S. aureus grew only slightly or decreased in cell number under the manufacturing condition of semi-hard type cheese or soft-type cheese. Under the conditions of the fresh cheese making process, S. aureus slightly increased in cell number, though no enterotoxin was detected. In processed cheese, S. aureus did not grow at all. Growth inhibition of S. aureus by lactic acid produced from starter culture was suggested to be the cause of growth inhibition in the natural cheese.     

Surveillance of Staphylococcus aureus in cheese produced in Hokkaido

Although the number of cheese manufacturing units in Hokkaido had increased every year and exceeds 60, many of these units are small-scale processors. We examined the cheese produced in Hokkaido for the presence of Staphylococcus aureus for 3 years after 2002. During the study period, S. aureus was isolated from 38 cheese samples: 3.6 to 9.2% of the total cheese samples examined and 13.0 to 20.0% of the total mozzarella-type cheese samples. The largest population of S. aureus was 2.0 x 10(4) CFU/g. The isolated S. aureus strains were subjected to PCR analysis to look for seven se genes. Of the 38 isolates, 20 did not possess the se gene, but the remaining 13 isolates had seg and sei genes. No enterotoxins were detected in the cheese samples analyzed with a commercial kit.     

Development of a PCR test to differentiate between Staphylococcus aureus and Staphylococcus intermedius

The presence of Staphylococcus intermedius in food remains unclear because routine laboratory analysis does not discriminate between S. intermedius and Staphylococcus aureus, a major cause of food poisoning. Both species share many phenotypic characteristics, including coagulase and thermonuclease production. In both species, some strains can produce enterotoxin and therefore can be the cause of food poisoning outbreaks. Although the ID32 Staph System (bioMérieux, SA, Marcy l'Etoile, France), based on a miniaturized phenotypic characterization, gives satisfactory results for discriminating between these two species, some rapid molecular PCR-based methods have been developed to identify S. aureus specifically, but they do not identify S. intermedius. Here, we developed a rapid, accurate, and discriminative multiplex PCR method that targets species-specific sequences in the nuc gene, which encodes thermonuclease in the two species. The test includes an internal positive control that targets a highly conserved region of 16S ribosomal RNA gene (rDNA). A total of 116 strains were used to validate our test. The test gave no signal on the following Staphylococcus species: S. epidermidis, S. chromogenes, S. hyicus, S. warneri, S. xylosus, S. lentus, and S. sciuri. It allowed a 100% successful discrimination between S. aureus (44 strains tested) and S. intermedius (57 strains) isolated from different origins.     

金黄色葡萄球菌基因组岛对多重耐药表型与生物被膜能力的影响机制

本文选择常见的典型食源性微生物金黄色葡萄球菌,从耐药性微生物感染防控角度出发,对广州地区临床分离的127株葡萄球菌的耐药表型、基因组岛分型与生物被膜生长能力进行研究。通过微量肉汤稀释法检测确定菌株对26种抗菌药物的药敏结果;PCR扩增葡萄球菌属特异性基因16S r RNA、金黄色葡萄球菌菌株特异性基因fem A、耐药基因mec A以检测确定菌株耐药特性;多重PCR检测金葡菌基因组岛SCCmec基因元件中ccr复合物,mec复合物以对其进行分型。107株耐药型金葡菌均为多重耐药,且呈耐9种或以上抗生素占76.1%(86/113)。113株葡萄球菌SCCmec分型结果为:I型0株,II型12株,III型73株,IV型10株,V型11株,5株为无法分型;本文对基因组岛和金黄色葡萄球菌耐药表型与生物被膜能力的相关性进行分析与探讨,为进一步对各种食源性微生物引起的食品污染进行安全控制,提供了研究基础。     

Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.     

Selection and identification of protective culture for controlling Staphylococcus aureus in fresh Domiati like cheese

Antibacterial activity of forty lactic acid bacteria (LAB) isolates toward Staphylococcus aureus was evaluated. The selected strains were then used as protective culture in artificial contaminated Domiati like cheese with S. aureus. The effect of using these strains on physicochemical properties and overall acceptability of fresh cheese was evaluated. Depending on its antibacterial activity, three strains of Lactobacillus rhamnosus 130RZFAAU, 131RZFAAU, and 190RZFAAU were selected for cheese making. No negative sensory properties were observed by the panelists when LAB strains were used as a single culture in the fresh cheese making. The application of these strains as protective culture in artificial contaminated cheesemaking process give a positive results. S. aureus was detected in cheese samples by culture method and propidium mono azide–quantitative polymerase chain reaction method. The results recommended that the strain L. rahmnosus 131RZFAUU that used in this study has antimicrobial activity against S. aureus and could be used as protective culture for improving the safety of Egyptian soft cheese.

Practical applications

Detection of pathogenic bacteria by classical tests can take several days. It would be useful to have a rapid detection protocol to screen for the presence of Staphylococcus aureus in milk and cheese. Application of real‐time PCR in cheese is sufficient in characterization the S. aureus communities in raw milk and follow the dynamics of the entire populations in cheese. Recently, some scientific publications have shown that the naturally cheese microflora can efficiently prevent the growth of pathogenic or spoilage microorganisms. The control of spoilage and pathogens bacteria has been traditionally done by chemical additives, but the application of promising protective cultures, especially for traditionally cheeses made from raw milk, is limited. This work present some protective culture selected for controlling S. aureus in soft cheese. This work confirm the PMA‐q PCR method for detection live cells of S. aureus in cheese rapidly.     

Antibiotic Resistance Traits and Molecular Subtyping of Staphylococcus aureus Isolated from Raw Sheep Milk Cheese

The main objective of the present research was to evaluate the antibiotic resistance profiles of Staphylococcus aureus isolated from raw sheep milk cheese. A total of 150 strains were isolated from curd cheese samples and identified as S. aureus. The survey on antibiotic resistance was carried out on 47 strains, selected among isolates showing differences in the banding pattern after Pulsed Field Gel Electrophoresis (PFGE) screening or, belonging at the same pulsotype but isolated from different cheese samples. On selected strains antimicrobial resistance against ampicillin, penicillin, cloxacillin, tetracycline, erythromycin, and vancomycin was assessed by broth microdilution method. The presence of the genes coding for antibiotic resistance and virulence factors (agr alleles, sea‐see, and tst) was also investigated by PCR. Thirty‐one isolates belonging to agrI and agrIII groups carried at least one gene coding for enterotoxins or toxic shock syndrome toxin. Approximately 60% of the selected strains were susceptible to the tested antibiotics. Twelve of 47 isolates showed multiple resistance against ampicillin and penicillin. Only 1 strain, represented by a unique PFGE profile showed simultaneous resistance to ampicillin, penicillin and cloxacillin. Single resistance against tetracycline was found in 5 isolates belonging to 2 different pulsotypes. The results of this study suggest that the recovery of S. aureus resistant strains in raw milk cheese samples is quite common but it is limited to few antibiotic classes, mainly β‐lactams and tetracyclines. None of the strains showed resistance to erythromycin and vancomycin.     

Evaluation of three different molecular markers for the detection of Staphylococcus aureus by polymerase chain reaction

The aim of this study was to target three genes of Staphylococcus aureus-fmhA (coding for a factor of unknown function), catalase and femA (coding for a factor essential for methicillin resistance) to establish and validate a PCR assay for the detection of this pathogen. Two pairs of primers were designed for fmhA and one pair each for catalase and femA genes. The PCR assays were standardized and found to give specific amplicons under similar reaction parameters. Target specificity of the primers was confirmed by DNA sequencing of the amplicons. While the initial inclusivity and exclusivity test reactions were in agreement in case of three of the primer pairs, one pair based on fmhA gene produced a non-specific product with a template DNA used in exclusivity test reactions. Forty-five strains of S. aureus were subjected to these PCR assays for their evaluation. Three among the four pairs of primers, one against each gene detected all the 45 strains precisely whereas one of the PCR assays using primers targeting the fmhA gene did not generate the specific amplicon with several of the strains. Seven unidentified strains of Gram-positive cocci subjected to these PCR assays produced negative results for each culture. Six of the strains were identified as Staphylococcus haemolyticus and one strain as Staphylococcus arlettae by 16S ribosomal gene analyses. All the three assay systems showed a detection limit of 100 cells per 20mul reaction assay. For validation of these assay systems, 80 coded samples of 11% skimmed milk spiked with different pathogens were received from NICED (National Institute of Cholera and Enteric Diseases), Kolkata and subjected to these PCR assays. All the three assays could detect S. aureus correctly in two of the samples. Amongst 150 raw milk samples, 36 (24%) were found positive for S. aureus. We conclude that fmhA, catalase and femA genes are conserved in S. aureus and, therefore, could be used as specific targets for its detection and identification by PCR. The protocols developed herein could be used for rapid and specific detection of this pathogen in food, clinical and environmental samples, especially milk.