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.     

Species origin of milk in Italian mozzarella and Greek feta cheese

Several animal species such as cattle, goats, sheep, and water buffalo provide milk for dairy products. We describe a simple procedure for detecting the species origin of milk used for cheese production. DNA was isolated from Italian mozzarella or Greek feta by sequential organic extractions and resin purification. This DNA was analyzed by polymerase chain reaction-restriction fragment length polymorphism as described previously for meat samples. This procedure differentiated mozzarella made from water buffalo milk and from less expensive bovine milk and also feta cheeses made from bovine, ovine, and caprine milk.     

云南省食品中金黄色葡萄球菌耐药性和mecA基因的检测及分析

目的调查2010—2016年云南省不同食品中分离的金黄色葡萄球菌的抗生素耐药情况,了解多重耐药(MDR,即耐3类或3类以上抗生素)菌株和耐甲氧西林金黄色葡萄球菌(MRSA)分布以及mecA基因的携带率,为有效防控金黄色葡萄球菌食物中毒和临床感染提供基础数据及参考。方法采用VITEK 2 Compact全自动细菌鉴定仪、GPI鉴定卡及AST-GP67进行药敏试验,聚合酶链式反应(PCR)法进行mecA基因检测。结果 325株金黄色葡萄球菌分离株耐药率为92.00%(299/325),前3位的分别是青霉素G(88.00%,286/325)、四环素(43.38%,141/325)、红霉素(40.92%,133/325),未检出耐喹奴普汀/达福普汀、万古霉素、力奈唑烷和替加环素的耐药株;MDR菌株检出率为47.69%(155/325),MRSA检出率为13.23%(43/325)。不同食品对青霉素G、四环素和呋喃妥因的耐药率差异有统计学意义(P0.05);餐饮食品、肉与肉制品、速冻米面制品中均检出九重耐药菌株;MRSA在水产品中占33.33%(1/3),肉与肉制品占17.89%(17/95),焙烤食品占16.67%(3/18),乳与乳制品占14.29%(3/21),餐饮食品占12.41%(17/137),速冻米面制品占5.71%(2/35),差异均无统计学意义(P0.05),婴幼儿食品、冷冻饮品和豆制品未检出MRSA。不同年份分离株对环丙沙星、莫西沙星和复方磺胺甲噁唑的耐药率差异有统计学意义(P0.05),2012—2015年均检出九重耐药株;2015年MRSA占比为20.00%(12/60),2014年占16.47%(14/85),2013年占14.12%(12/85),2012年占6.82%(3/44),2011年占10.00%(2/20),2010和2016年未检出MRSA。43株MRSA药敏试验显示耐药率最高的主要为青霉素G(100.00%,43/43),呈多重耐药性,未检出耐万古霉素的MRSA,mecA基因的检出率为37.21%(16/43)。结论食品中金黄色葡萄球菌的分布、抗生素耐药变化和发展趋势值得关注,有利于食源性疾病防治和指导临床合理选用抗生素,延缓细菌耐药株的产生。     

Discrimination of Staphylococcus aureus strains from different species of Staphylococcus using Fourier transform infrared (FTIR) spectroscopy

Staphylococcus aureus is a widespread opportunistic pathogen that can cause food-borne illness and is sometimes associated with raw milk and raw milk cheese products. The traditional taxonomic procedures for classification of staphylococcal species are time consuming and often several tests are required. FTIR spectroscopy offers a rapid method for the discrimination and identification of S. aureus strains isolated from raw milk and raw milk cheeses. FTIR spectroscopy was used to discriminate S. aureus from other species of Staphylococcus. This was achieved by using a model composed of 39 species and subspecies of Staphylococcus. The model was validated using a set of spectra of strains isolated from raw milk and different varieties of French raw milk cheese. S. aureus was successfully discriminated from the other species of Staphylococcus and all the strains of S. aureus isolated from raw milk and different varieties of French raw milk cheese were also successfully identified as such. These results demonstrated that FTIR spectroscopy is a rapid (results obtained within 24 h starting from a pure strain or a single colony) and robust method for the identification of S. aureus isolates of dairy origin and food-borne origin in general.     

Emergence and characterization of foodborne methicillin-resistant Staphylococcus aureus in Korea

A total of 165 Staphylococcus aureus strains, isolated from different food samples between 2003 and 2006, were tested for antimicrobial susceptibility. The mecA-positive methicillin-resistant S. aureus (MRSA) strains were further characterized by testing for various virulence genes and by molecular typing with multilocus sequence typing and pulsed-field gel electrophoresis. Of the 165 S. aureus isolates, 150 strains (90.9%) were resistant to at least one antibiotic while no strain was resistant to vancomycin. Four strains were resistant to both oxacillin and cefoxitin and were mecA positive. The mecA-positive MRSA strains were isolated from raw meat and fish samples (two beef samples and two fish samples) and were resistant to β-lactam antibiotics. Based on multilocus sequence typing analysis, the isolates were assigned to sequence type 1 (ST1), ST72, and an undetermined ST (ST72 slv). All four MRSA isolates were shown to be enterotoxigenic. The ST1 MRSA isolate harbored the sea-seh gene combination and the ST72 and ST72 slv MRSA strains harbored the seg-sei and the sea-seg-sei gene combinations, respectively. However, none of the MRSA isolates had the genes for Panton-Valentine leukocidin, toxic shock syndrome toxin 1, and exfoliative toxins. The pulsed-field gel electrophoresis patterns of the ST72 isolates in our study were highly similar, even though they were isolated from food samples in different years and from different regions of Korea.     

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。     

Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France

Enterotoxins produced by Staphylococcus aureus are responsible for staphylococcal food-poisoning outbreaks (SFPO). In France, SFPO are the second cause of food-borne diseases after Salmonella. However, very little is known about the strains involved. The objective of this study was to characterize the staphylococcal strains related to these SFPO through phenotypic and genotypic analyses. A total of 178 coagulase-positive staphylococcal isolates recovered from 31 SFPO (1981-2002) were screened through biotyping. Thirty-three strains representative of the different biotypes in each SFPO were further examined for SmaI macrorestriction-type, phage-type, resistance to various antimicrobial drugs, presence of staphylococcal enterotoxin (se) genes sea to sei, and production of enterotoxins SEA to SED. All these 33 strains were identified as S. aureus species: 27 were of human biotypes and six ovine or non-host-specific biotypes. Most (74.1%) strains reacted with group III phages. Eleven strains were resistant to at least two classes of antibiotics and among them, two were resistant to methicillin. Twenty-nine strains carried one or several of the eight se genes tested; the gene sea was most common (n=23), and often linked to sed (n=12) or seh (n=5). The novel se genes seg-i were in all cases associated with se genes sea to sed except for one strain which carried only seg and sei. Pulsed-Field Gel Electrophoresis (PFGE) of SmaI macrorestriction digests of the 33 strains discriminated 32 PFGE patterns grouped into nine biotype-specific clusters. All five strains carrying sea and seh were grouped together into the same sub-cluster. Three of the four se-gene-negative strains were in one PFGE cluster: all four should be tested for se genes not included in this study and, if negative, be further investigated for the presence of unidentified SEs.     

现制饮品中金黄色葡萄球菌耐药及遗传特征研究

目的 探究我国现制饮品中金黄色葡萄球菌抗生素耐药及遗传特征。方法 采用微量肉汤稀释法对金黄色葡萄球菌进行药敏测定,同时提取菌株基因组DNA进行全基因组测序,并使用生物信息学分析流程进行基因组特征数据挖掘。结果 现制饮品中43株金黄色葡萄球菌对10种抗生素的总体耐药率为88.4%,分属9种耐药谱,其中3株为耐甲氧西林金黄色葡萄球菌（Methicillin-resistant Staphylococcus aureus,MRSA）。多重耐药菌（Multidrug resistant,MDR）占比16.3%,主要为含乳现制饮品。耐受7类及以上抗生素金黄色葡萄球菌3株,其中1株为MRSA。全基因测序进化分析表明菌株耐药表型与基因型具有良好的相关性,MRSA及多重耐药株出现明显聚集。43株菌属于16种多位点序列分型（multilocus sequence typing,MLST）,其中MRSA菌株包括1株ST45型和2株ST398型。多重耐药株ST型别为ST5、7、15、59、121和398型。多重耐药MRSA株为ST398型。金葡肠毒素基因整体携带率为34.9%,其中sea为主要基因型。MRSA菌株均不携带毒力基因,多重耐药株肠毒素基因携带率为42.9%。3株主要来自含水果类现制饮品的ST1型菌株同时携带6种肠毒素基因,但其仅对青霉素耐药或为敏感株。结论 我国现制饮品中金黄色葡萄球菌存在MRSA及多重耐药菌,且该类菌株在遗传进化中聚类明显,虽然MRSA菌株致病性相对较弱,然而MDR菌株肠毒素基因的高携带率提示耐药金黄色葡萄球菌的潜在危害不容忽视。因此,应加强现制饮品中金黄色葡萄球菌的耐药监测与防控。     

Comparison of the enterotoxigenic types,toxic shock syndrome toxin I (TSST-1) strains and antibiotic susceptibilities for enterotoxigenicStaphylococcus aureusstrains isolated from food and clinical samples

The distribution of enterotoxin types and toxic shock syndrome toxin I (TSST-1) strains for 176Staphylococcus aureusstrains obtained from food samples and 62S. aureusstrains isolated from clinical samples were compared. It was found that for both the food and clinical isolates, staphylococcal enterotoxin A (SEA) strains accounted for the major part (75% and 45% of the total enterotoxigenic strains for food and clinical isolates, respectively) followed by SEB or SEAB, SEC and SED strains. For food isolates, none of theS. aureusstrains was TSST-1 strain while for clinical isolates, three strains (1 SEC, 1 SED and 1 SEAB strain) were found to be TSST-1 strains. When susceptibilities for these enterotoxigenicS. aureusstrains to antibiotics, such as penicillin, oxacillin, vancomycin, methicillin, streptomycin, tetracycline, gentamycin and kanamycin were compared, results showed that 51.6% of the food isolates were resistant to penicillin G only but sensitive to the other antibiotics tested. Also, 8 of the 64 enterotoxigenic strains isolated from food samples were all sensitive to antibiotics while none of the enterotoxigenic strains from clinical samples showed this antibiotic susceptibility pattern. No methicillin resistantS. aureus(MRSA) strains could be found among the food isolates. On the other hand, the majority (42.4%) of the enterotoxigenicS. aureusstrains from clinical samples were penicillin and/or other antibiotics resistant MRSA strains. Since MRSA strains often posed the therapeutic problem, the MRSA strains were further confirmed by PCR assay using the primers specific formecA gene. It was found that results obtained from the disc agar diffusion method and the PCR method were the same.     

In vitro screening of potential probiotic activities of selected lactobacilli isolated from unpasteurized milk products for incorporation into soft cheese

The aim was to select potentially probiotic lactobacilli from 88 strains isolated from unpasteurized milk and cheese products, and to incorporate these bacteria in a viable state into a soft cheese, without changing its quality. The survival of these bacteria was assessed in acidic and bile conditions, after freezing at -80 degrees C. Four strains from unpasteurized Camembert--two Lactobacillus plantarum strains and two Lb. paracasei/casei strains--were identified and typed by PCR and PFGE and were found to display potentially probiotic characteristics in addition to resistance to low pH and bile. These characteristics were resistance to lysozyme, adhesion to CACO-2 cells, antimicrobial effects against common foodborne pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli, innocuity following the ingestion of high doses by mice and appropriate antibiotic susceptibility profiles. The potential of Lb. plantarum strain UCMA 3037 for incorporation into a soft cheese (Pont-l'Eveque registered designation of origin (RDO)) was investigated. This strain grew well and survived in sufficient numbers (more than 10(7) cfu/g throughout the shelf-life of the product) in the cheese. This strain did not change the quality score of the product until the best before date (75 days after manufacture). Thus, unpasteurized Camembert is a natural source of potentially probiotic lactobacilli, which could be used as an additive in the development of potentially probiotic soft cheeses. Further work is required to demonstrate the persistence and efficacy of these strains in the human host upon ingestion.     

Biogenic Amines Content of Four Types of “Pecorino” Cheese Manufactured in Tuscany

Biogenic amines content of four types of Tuscan ewes’ milk “pecorino” cheese was evaluated using HPLC-UV analysis. All cheeses were manufactured in the same dairy factory with different combinations of milk (raw or pasteurized) and type of ripening. Total biogenic amines and tyramine levels of a raw milk “pecorino” ripened five months, partly in a traditional cave, were significantly higher than those of a pasteurized milk “pecorino” with a similar ripening; and of a two months raw milk “pecorino” ripened in the dairy plant. No statistical significant difference was found when comparing total biogenic amines and tyramine contents of the same five month ripened raw milk “pecorino” with a pasteurized milk “pecorino” ripened six months, partly in a traditional “fossa.” In raw milk cave-ripened and “fossa”-ripened cheeses, total biogenic amines exceeded 1000 mg/kg. In cheeses manufactured with raw milk and/or in particular ripening environments, specific hygienic cares are needed to limit biogenic amines formation.     

Use of duplex polymerase chain reaction (duplex-PCR) technique to identify bovine and water buffalo milk used in making mozzarella cheese

Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.     

Enterotoxin production by Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is an important cause of mastitis in cows. The ability of S. aureus strains to produce one or more enterotoxins in milk and dairy products is linked to staphylococcal food poisoning. To determine whether staphylococci causing bovine mastitis could cause human foodborne intoxication, the production of staphylococcal enterotoxins A through D (SEA, SEB, SEC, and SED) by 160 S. aureus isolates was evaluated with the use of a reverse passive latex agglutination enterotoxin kit. All S. aureus strains were isolated over a 9-month period from 2,343 routine submissions of a composite quarter collection of individual mastitic cows at 18 dairy farms in the San Joaquin Valley in California. Prior to enterotoxin detection, isolates were grown by a method that enhances the in vitro synthesis of enterotoxin. Twenty-two of 160 S. aureus isolates produced enterotoxin. Seven produced SEC, 12 produced SED, and 3 produced both SEC and SED. None of the isolates produced SEA or SEB.     

鄂西北地区食源性金黄色葡萄球菌污染及耐药性和毒力基因分析

目的 了解鄂西北地区食源性金黄色葡萄球菌的检出情况、耐药性以及毒力基因分布特征。方法 从湖北省襄阳市、十堰市、随州市三地共采集303份食物样品进行金黄色葡萄球菌筛查,通过聚合酶链式反应(PCR)法进行耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)的鉴定和毒力基因检测,并用K-B纸片扩散法进行耐药性测定。结果 303份样品中共有41份检出金黄色葡萄球菌,阳性率为13.53%,其中生肉制品检出率最高(23.91%,22/92)。在产肠毒素菌株中以携带产肠毒素基因sea的菌株最多,占87.80%(36/41)。携带表皮剥脱素基因eta和中毒性休克综合征毒素基因tst的菌株分别占97.56%(40/41)和7.32%(3/41)。同时携带3种及以上肠毒素基因的菌株占17.07%(7/41)。同时含有eta和tst的菌株占2.44%(1/41)。药敏结果显示,分离菌株对青霉素、四环素、红霉素和多西环素的耐药率分别为78.05%(32/41)、43.90%(18/41)、31.71%(13/41)和21.95%(9/41),对其他8种抗菌药物的耐药率均<20%。mecA基因检测表明,19.51%(8/41)的菌株为MRSA,且7株MRSA菌株来源于生肉制品和未加调料的素卤菜。结论 鄂西北地区食源性金黄色葡萄球菌具有较高的检出率,而且产毒株较多,并且存在多重耐药现象,相关部门需加强食品安全监测以控制该菌株的流行与扩散。     

A survey of antibiotic resistance in Micrococcaceae isolated from Italian dry fermented sausages

The transfer of bacteria that are resistant to antimicrobial agents or resistance genes from animals to humans via the food chain is increasingly a problem. Therefore, it is important to determine the species and the numbers of bacteria involved in this phenomenon. For this purpose, 148 strains of microstaphylococci were isolated from three types of Italian dry fermented sausages. Eight of 148 strains belonged to the genera Kocuria and Micrococcus. The remaining 140 strains belonged to 11 different species of the genus Staphylococcus. The species most frequently isolated was Staphylococcus xylosus, followed by Staphylococcus saprophyticus and Staphylococcus aureus. Antibiotic resistance levels differed among the species and depended on the strain origin. Microstaphylococci were generally susceptible to beta-lactams, but 12 strains were resistant to methicillin, 8 were resistant to oxacillin, and 9 were resistant to penicillin G. No resistance was observed for aminoglicosides and cephalosporines. Many strains were resistant to sulfonamide, colistin suphate, tetracyclin, and bacitracin. Two strains of S. aureus, four strains of S. xylosus, and one strain of Staphylococcus sciuri were able to grow in the presence of 8 microg of vancomycin per g, but all strains were susceptible to teicoplanin. Twenty-two microstaphylococci were resistant to at least five of the tested antibiotics. The multiresistant strain S. aureus 899 was unaffected by eight antibiotics, including vancomycin and methicillin, indicating that a more prudent use of antibiotics in animal husbandry and better hygienic conditions during production should be encouraged because they can play a major role in reducing the incidence of such multiresistant microorganisms and the possible spread of the genetic elements of their resistance.     

Sources of enterococci in Idiazábal-type cheese

Enterococci are a ubiquitous group and are natural constituents of the intestinal flora of nearly all animals and humans and can reach high levels in a variety of farmhouse cheeses. The purpose of this study was to determine the origin of the different enterococcal strains present in cheeses at different stages of ripening by typing the enterococci isolated from the raw milk, the cheeses, the cheesemaking environment, and from the faecal matter of the ewes and humans associated with cheese production. The potential presence of vancomycin-resistant enterococci (VRE) at all stages of the process and in the cheeses was also considered. The study was carried out at two separate cheesemaking dairy plants, and samples of the ewes' faeces, the shepherds' and cheesemakers' stools, teat cups, vat, brine, holding tank milk, vat milk, and the cheeses after brining and after 1, 15, and 60 days of ripening were collected. Cheesemaking procedures at the two plants were similar, yet the enterococcal levels and species observed differed at all the sample collection points, though E. faecalis predominated in all the milk and cheese samples. The traceability study performed for the species E. faecalis present at all the sample collection points suggested that the cheesemaker and the cheesemaking equipment were the source of the enterococci in the cheeses, though other possible non-faecal sources remain to be determined. VanC1 and vanC2/C3 enterococcal strains were isolated from the ovine faeces, teat cup, brine, and vat samples at cheesemaking dairy plant A, while only two vanC2/C3 strains were isolated from ovine faeces samples at dairy plant B. No VRE strains were detected in any of the milk or cheese samples.     

黑龙江耐甲氧西林金黄色葡萄球菌耐药及分子分型研究

目的了解黑龙江省健康人与屠宰场猪耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)携带情况,及其耐药和分子分型情况。方法参照黑龙江省金黄色葡萄球菌专项监测方案,对分离的金黄色葡萄球菌进行药物敏感性检测筛选MRSA,对MRSA进行spa、MLST和SCCmec基因分型,同时检测pvl基因。结果 1500份健康人样本分离出257株金黄色葡萄球菌(17.13%, 257/1500), 500份猪样本分离出149株金黄色葡萄球菌(29.8%, 149/500)。所有菌株对万古霉素、替考拉宁和利奈唑烷均敏感,对红霉素和克林霉素耐药率较高。MLST分型结果为ST9型, 1株未鉴定出型别。spa分型为t899和t2922, t899为主要型别(83%, 29/35)。SCCmec分型结果为IVb型, pvl基因均为阴性。结论黑龙江省健康人MRSA携带率比例较低,屠宰场猪携带一定程度MRSA,且猪源金黄色葡萄球菌耐药情况较重,应控制养殖动物抗生素的使用。     

PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

Polymerase chain reaction (PCR) has been applied for the specific detection of cows’ DNA in water buffalo milk and mozzarella cheese by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of specific primers for cow yielded a 346 bp fragment from cows’ milk DNA, whereas no amplification signal was obtained in sheep's, goats’ and water buffalo's milk DNA. Analysis of both buffalo milk and buffalo mozzarella cheese mixtures containing different percentages of cows’ milk or bovine mozzarella cheese, enabled the specific detection of cow's DNA with a sensitivity threshold of 0.1%. The proposed PCR assay represents a rapid and straightforward method for the detection of adulterations in water buffalo milk and mozzarella cheese.     

Development of two multiplex polymerase chain reactions for the detection of enterotoxigenic strains of Staphylococcus aureus isolated from foods

Two multiplex polymerase chain reactions were developed for the detection of enterotoxigenic strains of Staphylococcus aureus: one multiplex reaction for the simultaneous detection of enterotoxigenic strains type A (entA), type B (entB), and type E (entE) and another for the simultaneous detection of enterotoxigenic strains type C (entC) and type D (entD). Both reactions were standardized with the use of the reference enterotoxigenic strains of S. aureus: FRI 722, producer of staphylococcal enterotoxin (SE) type A (SEA); FRI 1007, producer of SEB; FRI 137, producer of SEC1; FRI 472, producer of SED; and FRI 326, producer of SEE. Optimized methods were used to determine the presence of enterotoxigenic types for 51 S. aureus strains isolated from meat (sausage, ham, and chorizo) and dairy (powdered milk and cheese) products by the Baird-Parker technique. The enterotoxigenic capacities of the strains were determined by the indirect enzyme-linked immunosorbent assay (ELISA) with the use of reference staphylococcal toxins and antitoxins. Fifty of the 51 strains isolated were enterotoxigenic and produced one to four enterotoxin types, with the most frequently produced types being SEA and SED. Levels of correlation between the presence of genes that code for the production of SE (as determined by polymerase chain reaction) and the expression of these genes (as determined by the indirect ELISA) were 100% for SEA and SEE, 86% for SEC, 89% for SED, and 47% for SEB.     

Identification of cow's milk in "buffalo" cheese by duplex polymerase chain reaction

A duplex polymerase chain reaction (PCR) was developed to identify the milk of bovine and buffalo species in cheese products, particularly in mozzarella cheese, a typical Italian cheese made from buffalo's milk. Two sets of primers were designed on the basis of the alignment of the sequence codifying mitochondrial cyt b available in the GenBank database. The primers proved to be species-specific, giving rise to 279-bp (bovine) and 192-bp (buffalo) amplified fragments. Since the amplification conditions for bovine and buffalo primers were identical, a duplex PCR was successfully applied to identify the two species in a single reaction step. This technique, when used to test cheese products from the retail trade, allowed the detection of partial or even total substitution of cow's milk for buffalo's milk, in some cases in samples of cheese misleadingly labeled "pure buffalo" mozzarella.