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。     

广州市白云口岸航空食品中金黄色葡萄球菌凝固酶基因分型及肠毒素基因研究

目的对2013—2015年从广州市白云口岸航空食品中分离的金黄色葡萄球菌进行基因分型研究,为食源性金黄色葡萄球菌分子溯源提供基础数据。方法以血浆凝固酶和肠毒素为目标基因,采用聚合酶链式反应(PCR)方法对9株金黄色葡萄球菌进行基因分型,其中6株为航空食品分离株,1株为配餐车间大门手拭分离株,2株为标准菌株。肠毒素基因检测包括5种传统肠毒素基因(sea、seb、sec、sed、see)和6种新型肠毒素基因(ser、seg、seh、sei、sej、sep)。结果 6株航空食品分离株的血浆凝固酶基因扩增分型结果为2个PCR型,酶切后得3种亚型;肠毒素基因检测结果显示有2株航空食品分离株含有肠毒素基因,检出率为33.3%(2/6),检出的基因为2种传统肠毒素基因(sec、sed)和4种新型肠毒素基因(ser、seg、sei、sej),均同时携带2种以上肠毒素基因。结论血浆凝固酶基因扩增分型结果显示,不同时间、不同采集地点存在相同的基因型,提示金黄色葡萄球菌存在交叉污染的可能性;航空食品分离株共检出6种肠毒素基因,提示金黄色葡萄球菌基因型多样性,应加强其他新型肠毒素基因检测。     

2005—2012年宁波市食源性金黄色葡萄球菌肠毒素基因和多位点序列分型研究

目的了解宁波市食源性金黄色葡萄球菌(S.aureus)肠毒素基因分布和分子分型特征。方法收集2005—2012年宁波市食品中金黄色葡萄球菌菌株,利用聚合酶链式反应(PCR)检测肠毒素A、B、C和D基因(sea,seb,sec和sed),对肠毒素基因阳性菌株利用多位点序列分型(MLST)进行分子分型。结果 2005—2012年共分离菌株190株,肠毒素基因阳性菌株为41株,阳性率为21.58%。4种肠毒素基因阳性率分别为7.37%(14/190)、5.26%(10/190)、8.95%(17/190)和5.79%(11/190),其中13株菌株具有两个及两个以上肠毒素基因。41株菌株可分为12个序列型(ST),以ST5、ST6、ST188和ST1为主,共占75.61%(31/41),在进化树上主要形成4个分支。结论 2005—2012年宁波市食品中金黄色葡萄球菌携带肠毒素基因频率较高,需加强监测。肠毒素基因阳性菌株与中国其他地区食品株在分子分型上存在较大差异。     

北京市朝阳区食物中毒相关金黄色葡萄球菌病原学分析

目的 了解2017年北京市朝阳区11株食物中毒相关金黄色葡萄球耐药性、耐药基因、毒力基因及分子分型。方法 依据GB4789.10-2016对甲、乙饭店留样食品、餐具及厨师手涂抹样品进行金黄色葡萄球菌分离鉴定;荧光定量PCR法检测耐药基因mecA、vanA与毒力基因sea、seb、sec、sed、see;纸片法与微量肉汤稀释法进行药物敏感性试验;脉冲场凝胶电泳(PFGE)进行溯源分析。结果 甲、乙饭店留样食品、餐具涂抹及厨师手涂抹样品共检出11株金黄色葡萄球菌;耐药基因mecA、vanA与毒力基因sea、seb、sec、sed、see均未检出;青霉素100%耐药,四环素、氯霉素耐药率分别为27.3%、9.09%,未发现耐甲氧西林金黄色葡萄球菌株;PFGE分型显示：甲饭店餐具涂抹菌株、手涂抹菌株、乙饭店所有菌株为3种带型。 结论 甲乙饭店分别为两起由金黄色葡萄球菌引起的食物中毒事件,皆为携带菌株的厨师污染餐具及食品引起,菌株具有较高耐药性。     

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。     

用DiversiLab系统对食品中分离的金黄色葡萄球菌进行分型溯源研究

目的:对分离自食品样本中的44株金黄色葡萄球菌进行DiversiLab分型,研究其基因特征和聚类分布,为食源性疾病溯源及预警提供依据。方法:采用DiversiLab自动分型系统扩增广泛分布于金黄色葡萄球菌基因组中的重复序列,并在Agilent2100自动生物分析仪上对扩增产物进行微流体电泳分离,然后使用DiversiLabSoftware软件实时分析和处理数据,对44株金黄色葡萄球菌进行分子分型。结果:DiversiLab分型系统将44株金黄色葡萄球菌分为26个型别,其中P1型包含菌株n76、n78和n71;P2型包含z78、z60和g75菌株;P3型包含菌株n61、z77、g31;P4型包含菌株0868和10786;P5型包含菌株6’-3600和13-3462;菌株1167、11219和z12属于P6型;菌株n73和n75属于P7型;菌株1151和11059为P8型;菌株3-1154和0979为P9型;菌株11-2063和6-1310为P10型;P11型包含菌株z67、z63、z64、z61和z36;其余15株金黄色葡萄球菌的每一个菌株被分为一个单独的型别。结论:DiversiLab分型结果准确揭示了44株来自食品的金黄色葡萄球菌的遗传特征和亲缘关系;Diversilab自动化分型技术方便快捷、结果可靠,是监测分析食源性致病菌和对食源性疾病溯源及预警的有力工具。     

化妆品中金黄色葡萄球菌焦磷酸测序检测方法的研究

本研究采用焦磷酸测序法对nuc基因的保守片断进行测序,分别对36株金黄色葡萄球菌和30株非金黄色葡萄球菌进行了特异性和复现性实验,并在化妆品中进行了方法验证,建立了化妆品中鉴定金黄色葡萄球菌的焦磷酸测序法,结果表明,本方法具有准确、快速的特点,可满足金黄色葡萄球菌的高通量鉴定的需求。     

合肥市即食肉类食品中金黄色葡萄球菌污染情况及耐药和基因型分布

目的调查合肥市即食肉类食品中金黄色葡萄球菌的污染情况,并对分离株进行耐药性分析及多位点序列分型研究。方法 2017年10月—2018年10月自合肥市区范围内的超市和开放型菜市场共采集744份即食型肉类食品样品。根据GB 4789.10—2016《食品安全国家标准食品微生物学检验金黄色葡萄球菌检验》中的方法对样品进行金黄色葡萄球菌定性和定量分析,采用K-B纸片扩散法检测分离株的耐药性,采用多位点序列分型法对分离株进行ST分型,使用MEGA 7.0绘制聚类树状图。结果样品中金黄色葡萄球菌检出率为4.2%(31/744),其中卤肉制品检出率最高为8.1%(15/185),其次为烤肉制品(6.3%,11/174),定量分析显示超过90%的样品金黄色葡萄球菌污染水平在0.3～10 MPN/g范围内。耐药性分析结果显示80.6%(25/31)的菌株对青霉素G耐药,40%以上的菌株对链霉素、红霉素和卡那霉素耐药;多重耐药菌株占比为61.3%(19/31)。分型分析共获得8个ST型,分别为ST6、ST7、ST15、ST59、ST72、ST188、ST398和ST2196型,其中以ST188和ST72型为主。结论合肥市卤肉和烤肉制品中存在金黄色葡萄球菌污染,且多重耐药菌株比例较高,对消费者的健康有潜在威胁,有必要持续监测污染率、菌株耐药和基因型流行趋势,完善合肥市食源性金黄色葡萄球菌感染的风险评估。     

速冻水饺中金黄色葡萄球菌的DiversiLab分型研究

目的:对速冻水饺中分离的20株金黄色葡萄球菌进行DiversiLab分型,研究其基因特征和聚类分布。方法:采用DiversiLab自动分型系统扩增金黄色葡萄球菌基因组中广泛分布的重复序列,然后在Agilent2100自动生物分析仪上对扩增片段进行微流体电泳分离,并使用DiversiLab Software软件实时分析和处理数据,对20株金黄色葡萄球菌进行分子分型。结果:DiversiLab分型系统将20株金黄色葡萄球菌分为13个型别,其中P1型包含菌株L17、L14、L11,P2型包含L16、L12、L19、L4菌株,P3型包含菌株L3、L18、L13,其余10株金黄色葡萄球菌的每一个菌株被分为一个单独的型别。结论:20株金黄色葡萄球菌从基因型来看有一定的聚集性,可能来自相近的污染源。同时也有一些金黄色葡萄球菌菌株之间的基因型差异较大,遗传学上关系较远,可能来自不同的污染途经。Diversilab自动化分型技术方便快捷,是监控食品及其生产企业细菌性污染的有力工具。     

温州市食品中金黄色葡萄球菌污染状况及分子流行病学特征研究

目的了解温州市食品中金黄色葡萄球菌的污染状况,分析分离的金黄色葡萄球菌的耐药性、毒力基因分布及脉冲场凝胶电泳(PFGE)分子分型特征。方法依据GB 4789.10—2010《食品安全国家标准食品微生物学检验金黄色葡萄球菌检验》进行菌株分离鉴定,采用纸片法进行药敏试验,mini-VIDAS法和聚合酶链式反应(PCR)法分别进行肠毒素及其基因的检测,PFGE法进行分子分型。结果 4类食品388份样品中有16份样品检出金黄色葡萄球菌,检出率为4.12%,其中生畜肉和生禽肉检出率较高,分别为13.89%(5/36)和11.11%(4/36)。所有菌株均有不同程度的耐药,对青霉素耐药率最高(100.00%,16/16),其次为红霉素(56.25%,9/16),多重耐药率为18.75%(3/16),未检出对甲氧西林耐药的金黄色葡萄球菌。金黄色葡萄球菌肠毒素及其基因检测阳性率均为56.25%(9/16),其中seb、seg基因检出率较高,均为37.50%(6/16)。PFGE图谱分为12种PFGE带型。结论金黄色葡萄球菌在温州市食品中存在一定的污染率,且具有分子多态性、产肠毒素率及毒素基因携带率较高的特征,提示存在潜在的食品安全隐患。     

Genotype analysis of enterotoxin H-positive Staphylococcus aureus strains isolated from food samples in the Czech Republic

Twenty-eight enterotoxin H-positive Staphylococcus aureus strains isolated from food samples collected in eleven districts of the Czech Republic between 2000 and 2005 were genotypically characterized by pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting and prophage carriage detection. These strains accounted for about 21% of the food-derived, staphylococcal enterotoxin (SE)-positive isolates. One strain, detected in feta cheese, was implicated in a case of enterotoxinosis. Sixteen of the twenty-eight isolates carried the seh gene alone. The remaining twelve strains harbored the seh gene in combination with other enterotoxin genes, most often the seg and sei genes, followed by the sea, seb, sec and sed genes. Comparison of various genomic profiles resulted in the determination of twenty genotypes designated G-1 to G-20. Two new, to date not defined, spa types (t2000 and t2002) were identified in one strain isolated from raw meat and two strains obtained from prepacked pizza. Evidence has been given that the seh-positive S. aureus isolates from foodstuffs did not originate from a single source or a common ancestor.     

Characterization of Staphylococcus aureus isolates from white-brined Urfa cheese

The aim of this study was to investigate the presence of Staphylococcus aureus and staphylococcal enterotoxin (SE) genes in Urfa cheese samples and to characterize the enterotoxigenic potential of these isolates. From a total of 127 Urfa cheese samples, 53 isolates (from 41.7% of the samples) were identified by a species-specific PCR assay as S. aureus. Of these isolates, 40 (75.5%) gave positive PCR results for the 3' end of the coa gene. The coa-positive S. aureus strains were characterized for their population levels and enterotoxigenic properties, including slime factor, β-lactamase, antibiotic susceptibilities, production of the classical SEs (SEA through SEE), in both cheese and liquid cultures by enzyme-linked immunosorbent assay (ELISA) and for the presence of specific genes, including classical SE genes (sea through see), mecA, femA, and spa, by PCR. The genetic relatedness among the coa-positive S. aureus isolates was investigated by PCR-based restriction fragment length polymorphism (RFLP) analysis and the 23S rRNA gene spacer. The 23S rRNA gene spacer and coa RFLP analysis using AluI and Hin6I revealed 14 different patterns. SEB, SEC, and SEA and SEE were detected by ELISA in three cheese samples. Fourteen S. aureus strains harbored enterotoxin genes sea through see, and three strains carried multiple toxin genes. The most commonly detected toxin gene was sec (25% of tested strains). Of the 40 analyzed S. aureus strains, 3 (7.5%) were mecA positive. Based on tandem repeats, four coa and spa types were identified. The results of this study indicate that S. aureus and SEs are present at significant levels in Urfa cheese. These toxins can cause staphylococcal food poisoning, creating a serious hazard for public health.     

食物中毒事件中致病菌肠毒素检测和分子分型研究

目的 对一起食物中毒事件样本进行检测,查明食物中毒原因,并对分离菌株进行相关性分析。方法 参照国家标准方法GB4789.10-2016,对采集样本进行细菌学检验。应用酶联免疫吸附法(ELISA)检测葡萄球菌肠毒素。通过脉冲场凝胶电泳(PFGE)进行分子分型,分析菌株之间的相关性。结果 食物中毒事件中5名患者洗胃液、食物、分离菌株,均检测出E型葡萄球菌肠毒素。3株分离菌株PFGE分子分型提示来源不同克隆株,除了产生E型葡萄球菌肠毒素外,还有B、C、D型。结论 该起食物中毒由不同PFGE型别的产毒金黄色葡萄球菌污染食物引起,菌株产葡萄球菌肠毒素的型别不完全相同。金黄色葡萄球菌引起的食物中毒应关注多污染源及菌株肠毒素基因表达情况。     

2009—2020年北京市朝阳区113株食源性金黄色葡萄球菌耐甲氧西林和肠毒素/类肠毒素基因携带与分子分型分析

目的 了解北京市朝阳区2009—2020年113株食源性金黄色葡萄球菌甲氧西林耐药、肠毒素/类肠毒素基因携带和分子分型情况.方法 采用纸片扩散法进行头孢西丁药物敏感性实验,结合mecA基因扩增鉴定耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)...     

Prevalence of methicillin-resistant Staphylococcus aureus in a fresh meat pork production chain

The objective of this study was to investigate the prevalence of methicillin-resistant Staphylococcus aureus on different stages of a fresh pork production chain to reveal potential carryover from live animals to meat. Samples were collected at different stages of the production process in a large German abattoir with an integrated processing unit for fresh pork. Samples included nasal swabs from pigs at stunning, environmental samples from the slaughter line, surface samples from carcasses, environmental and meat samples from the processing unit, and samples from final products. Samples were analyzed with an established two-step selective enrichment method, and isolates were characterized with respect to their S. aureus protein A gene (spa) and staphylococcal cassette chromosome mec (SCCmec; which harbors the mecA gene) types. Contamination rate was highest (64.7%) in nasal swabs and lower (6.0%) on carcasses, meat at processing (4.2%), and final products (2.8%). Environmental samples were positive along the slaughter line (12%) but not in the processing unit. spa types t011 and t034 and SCCmec type V predominated the isolates. Heterogeneity of spa types was highest in nasal swabs. Results show that methicillin-resistant S. aureus can be identified at all stages of the production chain. Further studies are needed to identify potential control points to reduce the carryover from farm animals to the final products.     

2017年广州市餐饮食品中金黄色葡萄球菌的调查分析

目的 了解餐饮食品中金黄色葡萄球菌的污染状况及分布, 确定高危食品的种类, 为预防金黄色葡萄球菌引起的食物中毒提供依据。方法 按照GB 4789.10-2016《食品安全国家标准 食品微生物学检验 金黄色葡萄球菌检验》对广州市5类500批次餐饮食品进行检验, 并经VITEK 2鉴定。结果 500批次餐饮食品中检出金黄色葡萄球菌9株, 检出率为1.80%, 其中熟肉制品和凉拌菜、寿司食品检出率分别为3.33%和3.00%。结论 广州市餐饮食品整体情况良好, 但存在着一定的风险安全隐患, 需加强对易受金黄色葡萄球菌污染的熟肉制品和凉拌菜、寿司等重点品种的监管。     

Genotypes and enterotoxicity of Staphylococcus aureus isolated from the hands and nasal cavities of flight-catering employees

Hand and nasal samples of flight-catering staff were collected from 1995 to 1997 to find employees carrying Staphylococcus aureus. Altogether 153 hand samples and 136 nose samples were taken. Nasal sampling showed a higher prevalence of S. aureus among food handlers (29%) than hand sampling (9%). A high proportion of the strains (46%) were enterotoxigenic, and a considerable amount of food handlers carried enterotoxigenic S. aureus, 6% and 12% according to hand and nasal sampling, respectively. Pulsed-field gel electrophoresis macrorestriction profiles revealed a total of 32 different types associated with the 35 employees carrying S. aureus. In most cases, the same type colonized both the hand and nose of a person. Despite the wide variety of types found, one strain colonized five persons and the second most common strain was associated with four food handlers. The predominant toxin produced was B, which was produced by the most common strain. The results showed that nasal sampling is a good way to detect S. aureus carriers, whereas hand sampling may fail to reveal carriers. The high proportion of enterotoxigenic strains show that a food handler harboring S. aureus must be considered a potential source of enterotoxigenic strains for airline meals.     

Comparative sequence analysis of spa gene of Staphylococcus aureus isolated from bovine mastitis: characterization of an unusual spa gene variant

The protein A encoding gene spa of four Staphylococcus aureus strains isolated from bovine clinical mastitis was amplified by PCR and sequenced. The four strains were selected after an initial screening of spa gene of 41 strains isolated from mastitic cows and were subjected to detailed investigations. According to the sequencing results the spa gene of three strains (M1, M2, M3) appeared with gene segments encoding five (E, D, A, B and C) and four (E, A, B and C) IgG binding domains for two (M1, M3) and one (M2) strain, respectively and with gene segments encoding four, two and two repeats of the octapeptide Xr-repeats for the strains M1, M2 and M3, respectively. For the remaining Staph. aureus strain (M4) gene segments encoding IgG binding domains E, D and A and a new domain BC with a size of 219 bp could be observed. The BC domain appears, with a deletion of a 123 bp segment from the border region between both domains, as fused domain of both previously characterized domains. The Xr-region of this strain had 11 octapeptide repeats.     

2015年南宁市动物源性食品中金黄色葡萄球菌污染状况及耐药调查

目的调查2015年南宁市市售的动物源性食品中金黄色葡萄球菌的污染情况以及流行菌株的耐药情况,为食源性疾病监控提供数据支撑。方法随机采集市售的不同种类的动物源性食品383份,按照国标法进行金黄色葡萄球菌分离鉴定,并对分离到的28株金黄色葡萄球菌进行K-B纸片法耐药试验和PCR方法检测耐药基因。结果从383份动物源性食品中分离到28株金黄色葡萄球菌,耐药试验结果发现,其中10株为耐甲氧西林金黄色葡萄球菌,28株分离株对不同的抗菌药物有不同程度的耐药,并检测到了4类耐药基因。结论市售动物源性食品中有金黄色葡萄球菌污染,存在引发食物中毒的风险;分离株的耐药试验结果可以为南宁市金黄色葡萄球菌引起的食物中毒提供用药参考。