国内首次发现的伊迪坎沙门菌的鉴定与探讨

目的对一株从从业人员健康体检中分离的罕见沙门菌血清型进行鉴定与探讨。方法结合API 20E系统生化反应、荧光定量PCR检测以及血清学分型进行沙门菌鉴定。用K-B纸片法检测该菌对12种抗生素的敏感性。结果该菌生化反应及PCR检测符合沙门菌属,为罕见的非A-F群沙门菌,血清学分型鉴定为伊迪坎沙门菌,抗原式为1,13,23∶i,5。结论该菌是一株罕见的发酵蔗糖的伊迪坎沙门菌,首次从从业人员健康体检中发现,国内首次报道。开展沙门菌血清型检测,对该菌引起的疾病和公共卫生事件的防控有着重要指导意义。     

多位点序列分型技术在沙门菌鉴定中的应用研究

目的:探讨多位点序列分型技术(Multilocus sequence typing,MLST)在沙门菌鉴定中的应用。方法:对1株分离自腹泻病人的沙门菌疑似菌株用肠杆菌科鉴定试条API 20E进行生化鉴定并进行血清学鉴定,应用MLST分型方法对该菌株分子分型。结果:API 20E鉴定为沙门菌属,Vi因子血清不凝集,O因子血清A-F O多价不凝集。该菌的MLST型别为ST64型,提示为鸭沙门菌。结论:MLST分子分型技术有助于沙门菌的鉴别。     

API 20E、VITEK-32在临床细菌鉴定中的应用

目的对API 20E、VITEK-32在临床细菌鉴定中的应用效果进行评价。方法利用API 20E、VITEK-32对临床收集的67株肠道杆菌进行了鉴定,并用常规生化和血清学方法对鉴定结果进行确认。结果 API 20E试条和VITEK-32对67株肠道杆菌的鉴定结果与常规鉴定法符合率为100%。结论 API 20E、VITEK-32鉴定系统快速、准确,可用于临床细菌的鉴定。     

多位点序列分型和脉冲场凝胶电泳技术辅助鉴定肠炎沙门菌

伤寒沙门菌和甲、乙、丙型副伤寒沙门菌引起肠热型感染,而肠炎沙门菌、鼠伤寒沙门菌、鸭沙门菌等则多引起急性胃肠炎型感染.准确的沙门菌菌株鉴定对于传染病的预防控制有重要意义.笔者应用肠杆菌科细菌鉴定系统ATB ID32E和API 20E对一起食源性疾病暴发事件中分离到的2株沙门属菌株及1株分离自住院患者的沙门属菌株进行生化鉴定,同时进行血清凝集试验.     

多位点序列分型和脉冲场凝胶电泳技术在肠炎沙门菌暴发事件病原学鉴定中的应用

目的探讨脉冲场凝胶电泳(pulse-field gel electrophoresis,PFGE)和多位点序列分型(multilocus sequence typing,MLST)技术在沙门菌鉴定中的应用价值。方法对分离自一起食物中毒事件中的27株沙门菌,用肠杆菌科鉴定系统API 20E和ATB ID 32E进行生化鉴定并血清分型,以脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)进行分子分型。结果 27株沙门菌的生化鉴定结果为沙门菌属细菌,其中25株血清分型结果为肠炎沙门菌,抗原模式(9,12:g,m);其余两株菌H相抗原除g和m因子凝集阳性外,d因子也有凝集反应。PFGE结果显示27株菌株中26株的带型完全一致,另1株与其他26株仅有1条条带的差异,相似度为96.3%。27株菌的MLST型别均为ST11型,提示为肠炎沙门菌。结论分子分型技术,特别是MLST技术有助于沙门菌的辅助鉴定。     

实时荧光PCR方法检测水产品中沙门菌

[目的]探索沙门菌快速检验法，加快水产品检验检疫通关速度，促进外经贸经济的发展。[方法]用深圳太太基因工程有限公司提供的沙门菌实时荧光PCR试剂盒对水产品样本进行检验。[结果]用实时荧光PCR从多个水产品样品的增菌液中检出9份样品沙门菌阳性，用传统的微生物生理生化培养法从该9份样品中分离出9株沙门菌。根据血清分型、API鉴定和荧光PCR结果，确认所分离的菌株中有沙门菌B群3株、C1群4株、D群1株、其他群1株。[结论]实时荧光PCR方法在沙门菌的检验方面较传统方法具有快速、灵敏、特异性强等优势，有利于提高沙门菌的检出率，具有广阔的运用前景。     

水产源性沙门菌的多位点序列分型

目的探讨多位点序列分型方法在沙门菌分型鉴定研究中的应用,建立水产品中沙门菌监测的分子分型数据库。方法选取沙门菌的7对管家基因aro C、dna N、hem D、his D、pur E、suc A和thr A作为靶标基因,对分离鉴定的47株沙门菌样本进行PCR扩增、测序及序列分析。结果 47株沙门菌分离株有20个ST型,其中ST11为优势ST型,占分离株的27.66%。此外,本研究新鉴定出3个ST型,分别为ST1808、ST1809和ST1810。结论多位点序列分型为水产源性沙门菌的分类研究提供了信息,对研究沙门菌的进化关系、鉴定溯源及预防控制预警提供了技术支持和数据参考。     

实时荧光PCR检测技术在沙门菌血清鉴定中的应用

目的 探究实时荧光PCR检测技术在沙门菌血清分型中的应用。方法 选取2010—2015年无锡市健康从业人员肠道分离得到的144株沙门菌，分别采用传统玻片凝集法和实时荧光PCR法进行血清分型，以前者为金标准，比较两种方法的一致性。结果 144株沙门菌利用传统玻片凝集法均可分型，共鉴定出28个型别。两种方法鉴定结果一致的有134株菌，符合率为93.06%,Kappa值为0.68,具有高度一致性。结论 实时荧光PCR法对沙门菌进行血清分型与玻片凝集法鉴定结果一致性高，且检测时间较短，操作简单；对不常见血清型或存在交叉情况时，建议两种方法联合使用，可提高沙门菌血清分型检测效率。     

广东部分零售畜禽产品沙门菌生化型和血清型分析

目的研究分析畜禽产品中沙门菌的生化型和血清型,了解沙门菌在畜禽产品中的污染状况,为确保食品安全提供依据。方法依据GB 4789.4—2010《食品安全国家标准食品微生物学检验沙门氏菌检验》,对样品进行检测,利用API 20E试剂条对菌株进行鉴定分析,采用玻片凝集法测定沙门菌的血清型。结果畜禽产品209份,检出沙门菌阳性样品42份,总检出率为20.10%,污染样品多来自于生鸭肉(检出率为69.23%),生鸡肉(37.14%)、生牛肉(20.00%)、生猪肉(16.67%)。API鉴定出46株沙门菌,分为7种不同生化谱的沙门菌API生化型,主要的2种为6704752和6704552。畜禽肉中沙门菌主要血清型为德尔卑沙门菌(21.74%)、鼠伤寒沙门菌(10.87%)、肠炎沙门菌(8.70%)、茨昂威沙门菌(8.70%)、印地安纳沙门菌(8.70%)、韦太夫雷登沙门菌(8.70%)。结论广东畜禽产品沙门菌污染情况较为严重,沙门菌菌株生化型和血清型表现为多样性的特征。     

不同方法鉴定包装饮用水中铜绿假单胞菌含量比较分析

目的:比较分析不同方法鉴定包装饮用水中铜绿假单胞菌含量。方法:采用GB8538.57—2016、API20E鉴定及VITEK鉴定3种方法对25株野生型菌株进行鉴别,使用16SrRNA基因序列分析与3种鉴别结果进行比对。结果:GB8538.57—2016鉴定结果与16SrRNA基因序列对比结果一致,API 20E鉴定5株为假阳性菌株,占比20.0%;VITEK鉴定3株为假阳性菌株,占比12.0%。结论:鉴定包装饮用水中铜绿假单胞菌需将GB8538.57—2016、API 20E鉴定及VITEK鉴定互相结合,辅佐印证,提高鉴别准确性。     

Vibrio cholerae in the environment: a simple method for reliable identification of the species

A simple screening and identification protocol was assessed for the efficient distinction of colonies of Vibrio cholerae species from others obtained on thiosulphate citrate bile salts sucrose agar after isolation from different environmental specimens. It was demonstrated here that the yellow colonies (sucrose-fermenting), which are able to grow on nutrient agar without added NaCl and which present a positive oxidase reaction, can be confidently considered as presumptive V. cholerae. Confirmation of the identification was carried out using the API 20E microtest and by species-specific ompW-based polymerase chain reaction: 809 of 925 isolates obtained by this screening procedure were identified as V. cholerae by API 20E and confirmed by PCR. The results showed that the direct use of the PCR-based method for the definite identification of the screened colonies gave better results than the API 20E method: of a selection of 100 isolates presumptively identified as V. cholerae according to the proposed screening procedure, all gave a positive result with PCR but only 94 were confirmed by API 20E. This protocol provides reliable identification of V. cholerae species and is adapted to the capabilities of routine clinical, food-testing and environmental microbiology laboratories.     

Abundance of pathogenic Escherichia coli, Salmonella typhimurium and Vibrio cholerae in Nkonkobe drinking water sources

In order to study the prevalence of enteric pathogens capable of causing infection and disease in the rural communities of Nkonkobe, bacterial isolates were collected from several surface water and groundwater sources used by the community for their daily water needs. By making use of selective culture media and the 20E API kit, presumptive Escherichia coli, Salmonella spp. and Vibrio cholerae isolates were obtained and then analysed by polymerase chain reaction assays (PCR). The PCR successfully amplified from water samples a fragment of E. coli uidA gene that codes for beta-D-glucuronidase which is a highly specific characteristic of enteropathogenic E. coli, enterotoxigenic E. coli and entero-invasive E. coli. The PCR also amplified the epsM gene from water samples containing toxigenic V. cholerae. Although E. coli was mostly detected in groundwater sources, toxigenic V. cholerae was detected in both surface and groundwater sources. There was a possibility of Salmonella typhimurium in Ngqele and Dyamala borehole water samples. The presence of these pathogenic bacteria in the above drinking water sources may pose a serious health risk to consumers.     

水产品中沙门氏菌的检测和确证

目的 在进口水产品中沙门氏菌检测中应用多种方法互相验证,以提高检测准确性。方法 对进口水产品沙门氏菌检验时,按国家标准GB／T4789、2—2003进行常规检测的同时,结合了生物梅里埃公司的miniVIDAS仪器法和API20E鉴定系统。结果检出1批鼠伤寒沙门氏菌阳性水产品。三种方法的结果相同。结论 日常检验中传统方法可与仪器法以及快速生化鉴定法结合运用,互为补充可以提高检测准确性及检测效率。     

Molecular epidemiological survey of Citrobacter freundii misidentified as Cronobacter spp. (Enterobacter sakazakii) and Enterobacter hormaechei isolated from powdered infant milk formula

A total of 75 powdered infant milk formula (PIF) samples collected from pharmacies and drugstores in Western Sicily, Italy, and representative of 12 different brands were analyzed in this study to evaluate their microbiological quality. According to the U.S. Food and Drug Administration protocol, 32 samples out of 75 were contaminated by enterobacteria. Commercial biochemical API(r) 20E-system identification method indicated that six PIF samples were presumptively contaminated by Cronobacter spp., but further characterization by alpha-glucosidase based polymerase chain reaction (PCR) assay identification strongly suggested that these strains did not belong to the genus Cronobacter. Phylogenetic analysis of partial 16S rRNA (rrs) sequences combined with the results of biochemical tests allowed to identify the six strains as Citrobacter freundii. Similarly, rrs sequence analysis identified as Enterobacter hormaechei 23 strains originally ascribed to Enterobacter cloacae by the API 20E system. Characterization of C. freundii and E. hormaechei PIF isolates by the DiversiLab(r) repetitive sequence-based PCR (rep-PCR) typing method revealed a variety of amplification patterns, but the recovery of the same rep-PCR genotype in several products might indicate a special adaptation of genetic clones to this food or cross-contamination through common ingredients. Antibiotic-resistance profiles were also determined, but none of the strains tested was resistant to third-generation cephalosporins or fluoroquinolones and extended-spectrum beta-lactamase activity was not detected. Our results confirm that E. hormaechei contamination of PIF is widespread, thus making it a cause for concern. Similarly to what was demonstrated for E. hormaechei, we suggest that C. freundii also may be an under-reported cause of bacterial infection, especially in high-risk neonates, due to misidentification.     

应用PCR法检测沙门菌

目的 研究沙门菌的PCR检测方法.方法 引物选择沙门菌的hilA基因,应用PCR法检测沙门菌.结果 PCR能检测经选择性增菌液培养的沙门菌,扩增片段在497 bp.结论 应用PCR检测沙门菌,具有快速、特异、灵敏和简便的特点.     

Molecular identification of coliform bacteria isolated from drinking water reservoirs with traditional methods and the Colilert-18 system

The accuracy of a traditional method (lactose utilization with acid and gas production) for the detection of coliform bacteria and E. coli was tested in comparison with method ISO 9308-1 (based on acid formation from lactose) and the Colilert-18 system (detection of beta-galactosidase). A total of 345 isolates were identified after isolation from water samples using API 20E strips. The Colilert-18 led to the highest number of positive findings (95% of the isolates were assigned to coliforms), whereas the ISO-9308-1 method resulted only in 29% coliform findings. With the traditional method only 15% were rated positive. Most of the isolates were identified by the API 20E system as Enterobacter spp. (species of the Enterobacter cloacae complex), Serratia spp., Citrobacter spp.and Klebsiella spp.; but species identification remained vague in several cases. A more detailed identification of 126 pure cultures by using 16S rRNA gene sequence analysis and analysis of the hsp60 gene resulted in the identification of Enterobacter nimipressuralis, E. amnigenus, E. asburiae, E. hormaechei, and Serratia fonticola as predominat coliforms. These species are beta-galactosidase positive, but show acid formation from lactose often after a prolonged incubation time. They are often not of fecal origin and may interfere with the ability to accurately detect coliforms of fecal origin.     

合肥市屠宰生猪肉样沙门菌的PCR快速检测

目的:了解合肥市屠宰生猪肉样中沙门菌的污染状况。方法:应用聚合酶链式反应(polym erase chain reaction,PCR)对200份生猪肉样进行沙门菌的快速检测,并与常规法检测结果以及国内其他11个城市生猪肉样沙门菌的检出率作比较分析。结果:200份生猪肉样中沙门菌检出率为20%(40/200),污染率处于较高水平,而常规法的检出率为13%(26/200)。结论:合肥市生猪肉样中沙门菌的污染状况较为严重,存在发生食源性沙门菌病的潜在危险,PCR技术对沙门菌检测时间仅需2 d,与常规法相比,快速、简便、准确的优势特点使其成为调查食源性沙门菌的有用工具。     

A rapid and sensitive method to identify and differentiate Salmonella enterica serotype Typhimurium and Salmonella enterica serotype 4,[5],12:i:- by combining traditional serotyping and multiplex polymerase chain reaction

Salmonella enterica subspecies enterica serotype 4,[5],12:i:- is an emerging serovar considered as a monophasic variant of Salmonella enterica serotype Typhimurium. The antigenic and genetic similarity between Salmonella 4,[5],12:i:- and Salmonella Typhimurium suggests that they may behave in a similar way and represent a comparable threat to public health. As serotyping alone does not necessarily provide for identification of Salmonella 4,[5],12:i:- and its differentiation from Salmonella Typhimurium, a method that combines traditional serotyping and a multiplex polymerase chain reaction has been tested on 208 strains serotyped as Salmonella 4,[5],12:i:-, Salmonella Typhimurium, and similar serovars of serogroup B sharing the same phase-1 antigen "i." For 191 strains, the combined method fully confirmed the results provided by traditional serotyping, whereas for 17 strains of Salmonella 4,[5],12:i:- and Salmonella Typhimurium some inconsistencies emerged between the two methods. The combined method resulted in a more accurate and faster identification of these two relevant serovars.     

Detection of Salmonella spp. in water using magnetic capture hybridization combined with PCR or real-time PCR

The removal of target DNA by magnetic capture hybridization (MCH) from constituents inhibitory to amplification by polymerase chain reaction (PCR) was evaluated using Salmonella as the test pathogen. Hybrids were subjected to both conventional and quantitative real-time PCR (qPCR). When PCR inhibitors commonly found in water were added to the reaction, MCH-PCR increased the detection sensitivity on the order of 8 to 2,000-fold compared with the system using only PCR. To determine the selectivity of MCH for target DNA (Salmonella), different amounts of non-target DNA (Escherichia coli) were added to the qPCR reaction. The highest non-target DNA concentration interfered with the amplification by qPCR alone, while MCH-qPCR was unaffected. Average recovery of Salmonella DNA by MCH-qPCR was 31% using optimized buffers, washing solutions and enzymatic digestion. A recovery function was proposed in order to calculate the real cell number based on the measured value. Preliminary testing confirmed the suitability of this method for analysis of natural waters.