铜绿假单胞菌假阳性株的鉴定方法比较及应用

目的 探讨提高GB 8538—2016《食品安全国家标准饮用天然矿泉水检验方法》中铜绿假单胞菌的检测准确性。方法 按照GB 8538—2016，检测1份桶装饮用水样品中铜绿假单胞菌，并用16S rRNA基因测序法、API 20E试剂盒、VITEK2鉴定仪、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）对分离到的“阳性菌”ZH-1和ZH-2进行确认。同时比较42℃生长试验、硝酸盐还原产气试验、API 20E试剂盒、VITEK2鉴定仪和MALDI-TOF MS的应用。结果 经多种方法确证，菌株ZH-1为铜绿假单胞菌，菌株ZH-2为恶臭假单胞菌，是GB 8538—2016检出的假阳性菌。在42℃生长试验和硝酸盐还原产气试验中，菌株ZH-2为阴性反应，不同来源的38株铜绿假单胞菌则均为阳性。此外，API 20E试剂盒和VITEK2系统对铜绿假单胞菌鉴定准确率分别为92.1%、97.4%，但API 20E试剂盒必须进行补充试验，耗时、繁琐；MALDI-TOF MS能够将铜绿假单胞菌鉴定到属水平，但种水平一致性准确率仅为43.2%。结论 在国家标准的鉴定方法基础上，增加42℃生长试验...     

2012—2014年中国婴儿配方食品和谷基辅食类食品及临床腹泻病例来源的克罗诺杆菌种水平鉴定研究

目的建立一种基于细菌RNA聚合酶β亚基编码基因(rpo B)的克罗诺杆菌种鉴定PCR方法,对不同来源的克罗诺杆菌进行种水平鉴定。方法通过rpo B基因设计针对克罗诺杆菌7个种的引物,以9株参考菌株作为参照,对261株2012—2014年收集的我国婴儿配方食品和谷基辅食类食品及临床腹泻病例来源的克罗诺杆菌进行种水平鉴定。结果 9株参考菌株PCR结果与对应大小一致,261株克罗诺杆菌中的179株为阪崎克罗诺杆菌,56株为丙二酸盐克罗诺杆菌,13株为尤尼沃斯克罗诺杆菌,11株为都柏林克罗诺杆菌,2株为苏黎世克罗诺杆菌,分别占总菌株数的68.58%、21.46%、4.98%、4.21%和0.77%。结论建立的克罗诺杆菌PCR法种水平鉴定具有特异、便捷、灵敏等优势,可为我国食品安全克罗诺杆菌的风险监测和控制提供技术支持。     

API20E、PCR方法在沙门菌鉴定中的应用

为了评价API 20E试条、PCR方法在沙门菌鉴定中的应用，本研究利用API 20E、PcR方法对54株疑似沙门菌进行鉴定，利用常规生化和血清学方法对鉴定结果进行确认。结果发现API20E、PCR方法与常规鉴定方法的结果一致，51株为沙门菌，3株非沙门菌（1株为柠檬酸杆菌，2株为河生肠杆菌）。API20E、PCR方法具有简便、特异、敏感的特点，可适用于沙门菌鉴定。     

基质辅助激光解吸电离飞行时间质谱检测肠杆菌科细菌产碳青霉烯酶的应用价值

目的 评估基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)检测肠杆菌科细菌产碳青霉烯酶的应用价值。方法 选取2018-2021年中山市致病菌识别网分离的111株肠杆菌科细菌。所有菌株采用纸片扩散法(K-B法)、改良碳青霉烯灭活试验(mCIM)、MALDI-TOF MS进行检测，分析评估MALDI-TOF MS在检测肠杆菌科细菌产碳青霉烯酶的能力。结果 111株菌株采用mCIM和MALDI-TOF MS两种方法进行检测，均检出32株菌产碳青霉烯酶，其余79株菌不产酶。结论 mCIM和MALDI-TOF MS两种方法对肠杆菌科细菌产碳青霉烯酶检测结果一致，具有相同的灵敏度，且MALDI-TOF MS检测时间大幅减少，可实现批量检测，具有推广应用的价值。     

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。     

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。     

能与沙门菌及O157诊断血清交叉凝集的细菌分离与鉴定

目的:研究能与沙门菌、大肠埃希菌O157发生交叉凝集的细菌,为防止误诊提供参考。方法:采用观察形态、血清学凝集、以及API20E生化鉴定试剂盒进行系统生化鉴定的方法。结果:生化鉴定符合变形杆菌、阴沟肠杆菌、柠檬酸杆菌的18株肠杆菌,其中11株能与沙门菌多价血清发生交叉反应凝集,7株能与大肠埃希菌O157血清发生交叉凝集。结论:在肠杆菌科细菌的鉴定中,单凭血清学反应做出判定,容易产生错误的结论,为确保菌株鉴定的准确性,应当血清学反应和全面生化检验结合的方法。     

应用MALDI-TOF MS快速检测泛耐药鲍氏不动杆菌的研究

目的应用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)建立一种快速鉴定泛耐药鲍氏不动杆菌的方法。方法收集临床微生物实验室全自动细菌鉴定/药敏系统鉴定的鲍氏不动杆菌菌株135株,其中包括28株敏感鲍氏不动杆菌(S-AB),107株泛耐药鲍氏不动杆菌(PDR-AB),随机挑选8株敏感鲍氏不动杆菌和10株耐药鲍氏不动杆菌自行建立敏感和耐药标准菌株库,用MALDI-TOF MS鉴定余下20株敏感鲍氏不动杆菌和97株泛耐药鲍氏不动杆菌菌株,并与自建库比对。结果 20株敏感菌株中有4株直接鉴定到自建库敏感菌株,97株耐药菌株中有31株直接鉴定到自建库耐药菌株,余下菌株均鉴定到系统数据库鲍氏不动杆菌属,并且所有的鉴定结果都与PhoenixTM-100结果相符。结论应用MALDI TOF MS可以对院内敏感和泛耐药鲍氏不动杆菌进行快速鉴定药敏,这种方法快速、高效,对临床医生及时准确的用药具有重要的指导意义。     

克罗诺杆菌种间鉴定recN基因方法建立

目的建立一种基于重组和修复蛋白基因(recN)的克罗诺杆菌种间鉴定方法,将不同来源的克罗诺杆菌分离株鉴定到种。方法扩增recN基因全长序列,选取不同长度的recN基因片段,用MEGA 4.0软件对克罗诺杆菌8个参考菌株进行聚类分析,确定克罗诺杆菌种间鉴定可信度高、序列较短的片段;设计并合成该片段的引物,以8株参考菌株作为参照,构建44株克罗诺杆菌实验菌株的聚类分析图,并用生化反应鉴定对聚类结果进行佐证。结果基于recN基因上一段640 bp的片段可对克罗诺杆菌不同种的菌株进行区分;以8株参考菌株在聚类图上的位置作为参考,根据遗传距离的远近,44株分离菌中有37株阪崎克罗诺杆菌,3株丙二酸盐阳性克罗诺杆菌,3株苏黎世克罗诺杆菌,1株尤尼沃斯克罗诺杆菌,与生化反应鉴定结果一致。结论该方法与生化鉴定和基于recN全基因方法相比简便快速,PCR扩增后,一次测序反应就可以将克罗诺杆菌鉴定到种。     

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。     

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.     

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.     

MALDI-TOF mass spectrometry-based identification of group A Streptococcus isolated from areas of the 2011 scarlet fever outbreak in china

There was a dramatic increase in scarlet fever cases in China from March to July 2011. Group A Streptococcus (GAS) is the only pathogen known to cause scarlet fever. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled to Biotyper system was used for GAS identification in 2011. A local reference database (LRD) was constructed, evaluated and used to identify GAS isolates. The 75 GAS strains used to evaluate the LRD were all identified correctly. Of the 157 suspected β-hemolytic strains isolated from 298 throat swab samples, 127 (100%) and 120 (94.5%) of the isolates were identified as GAS by the MALDI-TOF MS system and the conventional bacitracin sensitivity test method, respectively. All 202 (100%) isolates were identified at the species level by searching the LRD, while 182 (90.1%) were identified by searching the original reference database (ORD). There were statistically significant differences with a high degree of credibility at species level (χ² = 6.052, P < 0.05 between the LRD and ORD). The test turnaround time was shortened 36–48 h, and the cost of each sample is one-tenth of the cost of conventional methods. Establishing a domestic database is the most effective way to improve the identification efficiency using a MALDI-TOF MS system. MALDI-TOF MS is a viable alternative to conventional methods and may aid in the diagnosis and surveillance of GAS.     

Serotypes and biochemical profiles of British hospital strains of Enterobacter cloacae in relation to site of infection and antibiotic susceptibility

Comparisons were made between the O-serotype, API 20E profile, site of isolation and antimicrobial resistance of clinical isolates of Enterobacter cloacae. Correlations were found between autoagglutinable strains and urinary-tract infection, and API 20E profile 3305573 and strains isolated from blood. The proportion of strains sensitive to amikacin, gentamicin, cefotaxime, cefuroxime and trimethoprim were 100%, 93%, 91%, 83% and 89%, respectively. No individual resistances or patterns of resistance were associated with O-serotype or biochemical profile. Strains isolated from urinary-tract infections were the most resistant, 40% being resistant to five or more antimicrobials compared to 18%, 12% and 4% for strains from blood, wounds and sputum, respectively. There were no readily identifiable phenotypes within E. cloacae that possessed unique characteristics that could contribute to infections in hospitals.     

广州地区ST17无乳链球菌的分子流行病学特征及快速鉴定

目的了解广州地区ST17无乳链球菌的分子流行病学特征,运用基质辅助激光解吸/电离飞行时间质谱(Matrix assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF MS)建立其快速筛查方法。方法收集侵袭性无乳链球菌98株,进行多位点序列分型(Multilocus sequence typing,MLST)和分子血清学分型,进行药敏试验并检测红霉素和克林霉素耐药基因(ermB、ermA/E、ermTR和linB基因),运用MALDI-TOF MS分析ST17菌株的特征质谱峰。结果所有菌株被分为10个ST,来源于7个克隆群,其中ST17占16.3%。ST17菌株均为血清学Ⅲ型。ST17菌株对红霉素与克林霉素耐药率分别为93.8%和87.5%,检出ermB和mefA/E两种耐药基因,阳性率分别为100.0%和20.0%,未检出ermTR和linB基因。ST17菌株与非ST17菌株对红霉素和克林霉素耐药性以及ermB和mefA/E携带率的差异均具有统计学意义(P<0.05)。10种ST中,仅ST17在7620 Da处有特征质谱峰。结论广州地区流行的侵袭性无乳链球菌具有较高遗传多样性。ST17/血清学Ⅲ型菌株是该地区流行的主要侵袭性菌株之一,对红霉素和克林霉素耐药率高,红霉素耐药主要由ermB介导。MALDI-TOF MS可用于ST17菌株的快速筛查。     

婴幼儿配方奶粉中阪崎肠杆菌分子检测方法探讨

目的：对婴幼儿配方奶粉中阪崎肠杆菌的分子检测方法进行探讨。方法：分别用常规PCR方法和实时荧光PCR方法对32份奶粉样品进行阪崎肠杆菌鉴定。结果：32份样品中检出2株阳性株,阳性率为6.25%,两种检测方法结果相符。结论：市售婴幼儿配方奶粉中存在阪崎肠杆菌污染,应尽快制订婴幼儿食品中阪崎肠杆菌的检测标准,分子检测方法可快速准确检测阪崎肠杆菌。     

从食物中毒患者标本中分离鉴定海藻和腐败施万菌

目的 对两起食物中毒调查中分离到的施万菌属(Shewanella spp.)菌株进行生化和分子生物学鉴定.方法 对2007年9月29日至10月3日间,马鞍山市的两起食物中毒患者的肛拭、从业人员手拭和剩余食物标本进行采集,按照国标方法(GB/T4789),对所有标本进行增菌和选择性培养基分离培养,疑似菌落用VITEK-32和AP120E系统鉴定,用辅助生化、生长、溶血和药敏实验分析菌株特性,同时扩增16S rDNA并测序,用MEGA 4.0软件建立进化树并进行分群.结果 所有标本经增菌后接种选择性培养基,共有8份标本在TCBS和BP培养基上长出单一的菌落,在三糖铁琼脂(TSI)斜面上的主要生长特征为:产硫化氢、不产气,氧化酶阳性.8株菌经VITEK-32鉴定仪鉴定为海藻施万菌(S.algae)或腐败施万菌(S.putrefaciens),经AP120E系统鉴定为腐败施万菌.在WS、SS和EMB培养基均未检测到施万菌生长.比较施万菌的16S rDNA序列表明,其中7株为海藻施万菌,1株为腐败施万菌.没有从这8份标本中检测到其他肠道病原菌,包括霍乱弧菌、沙门菌、副溶血性弧菌、变形杆菌和金黄色葡萄球菌.结论 从食物中毒患者中分离到施万菌,为该菌作为可能的食物中毒病原菌提供了线索.     

葡萄球菌属和肠球菌属耐药性监测研究

目的监测临床分离的葡萄球菌属和肠球菌属对抗菌药物的耐药率,为临床有效控制葡萄球菌属的感染提供参考。方法采用VITEK-32、GPI鉴定卡及GPS-107药敏卡进行菌种鉴定和药敏实验,并以WHONET 5软件对试验数据进行分析处理。结果1 445株葡萄球菌属和肠球菌属细菌中,金黄色葡萄球菌330株(22.8%),凝固酶阴性葡萄球菌872株(60.3%),粪肠球菌213株(14.7%),屎肠球菌30株(2.1%);金黄色葡萄球菌中,耐苯唑西林金黄色葡萄球菌(MRSA)占67.6%,凝固酶阴性葡萄球菌中,耐苯唑西林凝固酶阴性葡萄球菌(MRCNS)占82.3%,2004年MRSA和MRCNS检出率分别为75.3%和82.3%,显著高于2003年的48.4%和78.4%,未发现对万古霉素耐药的金黄色葡萄球菌和凝固酶阴性葡萄球菌;MRSA和MRCNS对各种抗菌药物耐药率明显高于MSSA和MSCNS;屎肠球菌对各种抗菌药物的耐药率均明显高于粪肠球菌。结论葡萄球菌属和肠球菌属是造成医院感染的主要病原菌;通过对耐药性监测研究使我们提高抗菌药物耐药性问题的认识,为抗菌药物使用和调控提供基础。