Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

AIM: To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.     

金黄色葡萄球菌的快速检测方法

金黄色葡萄球菌是引起食物中毒常见的病原菌之一,其传统检测方法(选择性培养、生化鉴定)步骤多,操作复杂,耗时长、灵敏性不高。近年来以抗原抗体反应为基础的免疫学方法和以DNA为基础的分子生物学技术以其快速性、准确性已经成为微生物检测方法的发展方向。     

免疫捕获PCR法快速检测金黄色葡萄球菌

旨在建立一种快速检测金黄色葡萄球菌(Staphylococcus aureus,SA)免疫捕获PCR技术,并探讨其灵敏度和特异性。SA特异性抗体包被PCR管以富集待测样品中目标菌,之后在同一PCR管里直接进行免疫捕获PCR,并和直接PCR比较。免疫捕获PCR法可特异性检测2株SA菌株,而无法检测到其它8种常见食源性致病菌,说明该方法对SA具有良好的特异性;该方法对纯菌液而言,检测灵敏度可达到2.35×102CFU/mL,是直接PCR的100倍;对5种食品模拟带菌检测发现,无需增菌培养,其灵敏度可达到2.35×103-2.35×104CFU/mL,是直接PCR的10-100倍。免疫捕获PCR法集免疫学与分子生物学检测技术于一体,具有高特异性、高灵敏度、检测快速、易于操作、成本低廉等诸多优点,是一种适合基层实验室使用的检测技术。     

Proteomic analysis of exoproteins expressed by enterotoxigenic Staphylococcus aureus strains

Pathogenic bacteria excrete a variety of virulence factors into extracellular medium and to the cell surface which have essential roles in the colonization and insurrection of the host cells, and thus reflect the degree of bacterial pathogenicity. For the exploration of virulence factors expressed in the secreted proteome fraction, different Staphylococcus aureus strains were analyzed using gel-based bottom-up proteomic approach. A total of 119 distinct proteins were identified for the enterotoxin gene cluster (egc) negative and seb gene positive S. aureus American Type Culture Collection (ATCC) 14458 strain by the use of one- and 2-DE based proteomics. Detailed analysis of enterotoxin region of the 2-D map confirmed, beside the highly expressed staphylococcal enterotoxin B (SEB), the presence of enterotoxin-like proteins SElK and SElQ previously predicted by genotyping (Sergeev et al.., J. Clin. Microbiol. 2004, 42, 2134-2143). Exoprotein patterns at the late-exponential (7 h) and stationary (24 h) phases of cellular growth show a high-level similarity in this region. Comparative analysis of enterotoxin region of five S. aureus strains including two clinical isolates (RIMD 31092 and A900322), a food derived strain (AB-8802) with highly prevalent egc positive operon and a nonenterotoxigenic reference strain (ROS) revealed the presence of different known enterotoxins and other virulence factors along with a number of core exoproteins. In addition, production of SElL (RIMD 31092) and SElP (A900322) was demonstrated for the first time at the protein level. Under the experimental conditions applied none of the enterotoxins encoded by the genes of egc operon was identified.     

Clonal distribution of enterotoxigenic Staphylococcus aureus on handles of handheld shopping baskets in supermarkets

Aims: Shopping carts and handheld shopping baskets in supermarkets are subject to accidental bacterial contamination through contacts with a variety of food. We investigated the prevalence of Staphylococcus aureus on the handles of handheld shopping baskets in four supermarkets distantly located in Osaka district, Japan. Methods and Results: Fifty two strains of Staph. aureus were isolated from 760 basket handles. Among these, six strains were positive for staphylococcal enterotoxin B (SEB) production, representing 12% of total. This SEB producer ratio is considerably higher than among Staph. aureus isolated from nasal swabs of the supermarket workers (2%) and from independently collected clinical specimens (4%). These SEB‐producing Staph. aureus strains from the basket handles are clonal and belong to ST12. Coagulase typing showed that they are in group VII, which is the most common cause of food poisoning in Japan. Biofilm assays indicated that SEB gene (seb)‐positive strains including this clone produced a significantly higher amount of biofilm than seb‐negative strains. Conclusions: The frequent isolation of seb‐positive Staph. aureus on shopping basket handles raises the possibility that they could be a hidden reservoir for Staph. aureus with a potential to cause food poisoning and draws attention to the importance of shopping basket sanitation.     

253株金黄色葡萄球菌耐药现状分析

目的 了解医院金黄色葡萄球菌临床分布情况及其对常用抗菌药物的耐药率,为临床合理使用抗菌药物提供依据.方法 回顾分析医院2010年5月至2011年4月检出的金黄色葡萄球菌,采用VITEK-AMS全自动微生物分析仪进行菌种鉴定和药敏分析.结果 共检出金黄色葡萄球菌253株,菌株的主要来源为痰130株(51.4％)、血液39株(15.4％)、创面24株(9.5％);菌株主要科室分布前3位是神内科35株(13.8％)、ICU30( 11.8％)、脑外科26株(10.3％);其中耐甲氧西林金黄色葡萄球菌( MRSA)为165株(65.2％),MRSA对多种抗菌药物耐药率＞70.0％,MSSA为88株(34.8％),对除青霉素、红霉素外的大多数抗菌药物敏感,未发现耐万古霉素菌株.结论 MRSA检出率高,耐药现状严重,应加强对金黄色葡萄球菌耐药性的监测,并根据药敏试验结果合理使用抗菌药物.     

金黄色葡萄球菌的分离及其抗药性的研究

从土壤中分离出金黄色葡萄球菌后,以其为出发菌株,采用梯度培养皿法,利用青霉素、四环素、红霉素、氯霉素和链霉素5种抗生素,对自然界中和经过NaNO2诱变的菌株进行了耐药性菌株的分离及抗性水平的确定。在自然界中分离的金黄色葡萄球菌只对青霉素(80μg/mL)和四环素(60μg/mL)有抗性,而对红霉素、氯霉素和链霉素则没有抗性。经过NaNO2诱变后,金黄色葡萄球菌对四环素(40μg/mL)的抗性降低,但对青霉素(120μg/mL)和其他3种抗生素的抗性均有所增加。     

纳米金标记-逐步银染法快速检测金黄色葡萄球菌

将纳米金探针应用于目的核酸的检测,具有与PCR相当的灵敏度和特异性.本研究建立了一种可以在微孔板上快速检测金黄色葡萄球菌的纳米金标记-逐步银染法.该方法利用已包被链霉亲和素的微孔板,将PCR扩增的金黄色葡萄球菌nuc基因与生物素探针、纳米金探针形成的三明治杂交结构锚定其上,然后在低温下逐步银染显色,通过酶标仪检测放大的银染信号.这种纳米金标记-逐步银染法可以在显著降低非特异性背景信号的同时放大银染信号,检测金黄色葡萄球菌nuc基因的灵敏度为1 pmol/L,比常温一步银染法的灵敏度提高约102倍. 51例临床标本的检测结果与PCR法一致,与培养生化鉴定法的检测结果之间无显著性差异(P >0.05). 综上所述,本研究成功构建了金黄色葡萄球菌的纳米金标记-逐步银染法,在病原微生物的快速检测领域表现出广阔的发展潜力.     

三种金黄色葡萄球菌显色培养基检测效果的比较

本文研究2种国外主流的金葡菌显色培养基和广东环凯微生物科技有限公司生产的PEN-TCF检测金葡菌的效果。通过研究3种产品对多种金葡菌菌株的回收率、最低检出限以及检测人工污染食品的回收率、特异性和抗干扰能力比较其检测效果。结果表明3种培养基的最低检出限相近, 显色培养基A和PEN-TCF的回收率较高, 但抗干扰能力较低; 显色培养基B回收率稍低, 而抗干扰能力较强; PEN-TCF和培养基B的特异性更优于培养基A。     

金黄色葡萄球菌荚膜多糖研究进展

金黄色葡萄球菌作为引起人和动物发病的一种主要的致病菌,已严重影响到人们的身体健康和畜牧业的发展。致病性金黄色葡萄球菌多数含有荚膜成分,并且这种荚膜成分与金黄色葡萄球菌的毒力和抗噬菌作用有关。主要从金黄色葡萄球菌荚膜多糖的5型和8型血清学分类、分子结构、影响因素、表达调控等方面简要介绍了目前国内外关于金黄色葡萄球菌荚膜多糖的研究进展。     

Iron depletion and virulence in Staphylococcus aureus

Abstract Staphylococcus aureus is able to grow in the presence of extremely low iron concentrations (0.04 μM). In iron-limiting conditions, this species develops alternative metabolic strategies such as highly efficient iron-uptake mechanisms which are only partially shared with S. epidermidis . Here we summarize the mechanisms induced by iron starvation in S. aureus in order to elucidate the virulence characteristics of this bacterium.     

Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain

AIMS: Control of the enterotoxigenic Staphylococcus aureus CECT 976 strain by enterocin AS-48 in laboratory cultures, and behaviour of the AS-48 activity in the presence of food preservatives. METHODS AND RESULTS: Enterocin AS-48 shows inhibitory activity on the majority of the Staphylococcus species tested. This enterocin has a bactericidal and bacteriolytic mode of action on S. aureus CECT 976, a strain selected for this study by its enterotoxigenic character (SEA production). The inhibitory effect of AS-48 was pH and temperature dependent, and enterocin activity was higher at pH 5. The minimum bactericidal concentration (MBC) of AS-48, decreased from 15 microg ml(-1) at 37 degrees C to 10 microg ml(-1) at 15 degrees C. Sublethally injured cells showed an increased sensitivity with a MBC of 5 microg ml(-1). In this way, the highest effectiveness of Ent AS-48 against S. aureus CECT 976 was obtained at 4 degrees C in combination with high concentrations of NaCl (6 and 7%). Interestingly, enterotoxin SEA production by strain CECT 976 was markedly inhibited by subinhibitory concentrations of Ent AS-48. These low concentrations also provoked a delay of bacterial growth. CONCLUSION: The results presented indicated that Ent AS-48 has a potential for application as a protective agent against S. aureus in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have established the conditions for an efficient inhibition of growth and enterotoxin production by S. aureus CECT 976 in culture media by a combination of environmental factors and Ent AS-48.     

2008-2010年舟山某院金黄色葡萄球菌的分布及耐药性变迁

目的 分析舟山医院三年来金黄色葡萄球菌分布及耐药性变迁,并对耐甲氧西林金黄色葡萄球菌(MRSA)与甲氧西林敏感金黄色葡萄球菌(MSSA)的耐药性差异做对比.方法 用ATB Expression半自动微生物分析仪进行菌株鉴定及药敏试验,用K-B法测红霉素、克林霉素、头孢西丁、苯唑西林直径,比较耐甲氧西林金黄色葡萄球菌(MRSA)与甲氧西林敏感金黄色葡萄球菌(MSSA)的耐药性差异.结果 金黄色葡萄球菌对苯唑西林、庆大霉素、红霉素、四环素和克林霉素的耐药率有上升的趋势;MRSA对苯唑西林、庆大霉素、复方新诺明、克林霉素、红霉素、青霉素、喹奴普汀-达福普汀、利福平和四环素的耐药率都明显高于MSSA的耐药率,二者间差异有统计学意义(P＜0.01),D-试验阳性71株,占72.45％.结论 金黄色葡萄球菌的耐药性逐渐升高,特别是对MRSA应引起临床的重视,检测克林霉素诱导型耐药具有重要的临床应用价值.     

金黄色葡萄球菌分子检测技术的常用靶基因

金黄色葡萄球菌是一种常见的致病菌,可以产生多种毒素并具有广泛的耐药性。从特异性基因位点、抗药性基因和毒素基因3个方面列举了检测金黄色葡萄球菌的靶基因以及利用这些靶基因的PCR检测方法。详细说明了这些基因的发现、功能和实际应用情况。将金黄色葡萄球菌分子检测的思路和方法进行汇总、比较,并对未来的相关研究趋势和研究领域进行了展望。     

FTA滤膜用于PCR检测肉中的金黄色葡萄球菌

利用FTA滤膜,采用PCR技术可直接检测肉及肉制品中的金黄色葡萄球菌,无需增菌,灵敏度高。在采用浮选与溶剂萃取相结合方法的基础上,使用FTA膜可高效地从鲜肉中提取金黄色葡萄球菌的DNA,消除PCR反应的抑制因子。以金黄色葡萄球菌耐热核酸酶基因（nuc）为靶基因,经过PCR扩增得到279bp的产物。经过DNA测序证实该产物为目的扩增产物。使用FTA滤膜处理样品,再通过PCR方法检测金黄色葡萄球菌,猪肉、牛肉、羊肉、鸡肉匀浆液的检出限均为10cfu/mL,可在6h内完成对肉中金黄色葡萄球菌的检测,比目前普遍采用的先增菌再进行PCR检测的方法缩短了12～24h 。实际检测了72份样品,同时与GB 4789.10-94方法及两种快速检测致病性金黄色葡萄球菌检测方法做比较,PCR方法的检出率为72.2％,检出时间为6h,GB 4789.10-94方法检出率为70.8%,检出时间为5d,Prfilm RSA方法的检出率为61.1％, 检出时间为18h,BairdParker R.P.F方法的检出率为69.4％,检出时间为18h,结果表明FTA滤膜用于PCR检测肉中金黄色葡萄球菌检出率高,耗时短。使用FTA滤膜法制备模板DNA,为食品中的致病菌快速检测构建了一个技术平台。     

Detection of Protein A Produced by Staphylococcus aureus with a Fiber-optic-based Biosensor

Staphylococcus aureus is a pathogen important in causing human infections and intoxication. A sensitive fiber-optic that produces evanescent waves was developed for the detection of protein A, a product secreted only by S. aureus. In the immunosensor, a 40-mV argon-ion laser that generated laser light at 488 nm was used together with plastic optical fiber and antibodies to protein A were physically adsorbed onto the fiber. The principle of the detection involved a sandwich immunoassay with fluorescein isothiocyanate conjugated with anti-(protein A) immunoglobulin G to produce signals of the antigen-antibody reaction. The detection limit was 1 ng of protein A per milliliter. The fiber-optic immunosensor could be used for rapid and specific detection of S. aureus in clinical specimens and foods.     

Lead resistance and sensitivity in Staphylococcus aureus

Abstract Five lead-resistant strains of Staphylococcus aureus were isolated. Plasmid-free lead-sensitive variants were obtained from the three plasmid-bearing strains. Lead-resistant strains tolerated an approximately 600 × higher Pb(NO₃)₂ concentration than lead-sensitive strains. Both types of strains initially bound lead, but only the resistant strains accumulated the metal as an intracellular lead-phosphate.     

黄芩素抑制金黄色葡萄球菌生物被膜的形成

细菌生物被膜的形成是导致细菌耐药和引起持续性感染的主要原因之一。本文通过检测黄芩素对金黄色葡萄球菌26112菌株（Staphylococcus aureus 26112,SA26112）多糖细胞间黏附素（polysaccharide intercellular adhesion, PIA）的合成和胞外DNA（extracellular DNA,eDNA）释放量的影响,及其对icaA和cidA基因表达量的影响,探讨黄芩素对金黄色葡萄菌生物被膜形成的抑制作用及其机制。结果显示,黄芩素能抑制SA26112生物被膜的形成,其抑杀SA26112的最低抑菌浓度和最低杀菌浓度均为0.04 mg/mL。0.16 mg/mL黄芩素和256 μg/mL环丙沙星单独作用时,均不能杀死其成熟生物被膜内的SA26112细菌,而当二者联用时则可杀死成熟生物被膜内的细菌。黄芩素能显著抑制SA26112菌株PIA的合成、eDNA的释放量及icaA和cidA基因的相对表达量。其中,0.04 mg/mL黄芩素作用SA26112菌株24 h,与对照组相比,eDNA的释放量减少97%,icaA和cidA基因的相对表达量分别减少62%和41%。上述结果表明,黄芩素能抑制SA26112菌株生物被膜的形成,其作用机制可通过降低icaA和cidA的基因表达量,进而影响PIA的合成和eDNA的释放,来抑制金黄色葡萄球菌生物被膜的形成。