fliC基因在快速分型鉴定伤寒沙门菌和甲型副伤寒沙门菌的应用

目的:探讨沙门菌1相鞭毛蛋白抗原fliC基因在快速分型鉴定伤寒沙门菌和甲型副伤寒沙门菌的应用。方法:根据伤寒沙门菌和甲型副伤寒沙门菌1相鞭毛蛋白fliC-d和fliC-a基因以及菌体抗原rfbS基因的核酸序列,设计针对fliC-d、fliC-a及rfbS基因的3对特异性引物,采用多重PCR法进行检测。结果:实验结果显示,伤寒沙门菌和甲型副伤寒沙门菌分别在750 bp和329 bp处扩增出2条特异性目的条带,在258 bp处扩增出1条相同条带,非伤寒沙门菌株和甲型副伤寒沙门菌株均为阴性。结论:fliC基因检测可用于伤寒沙门菌和甲型副伤寒沙门菌的分型鉴定,而rfbS基因则无助于分型鉴定。     

基于TaqMan探针四重荧光PCR检测甲型、乙型、丙型副伤寒和伤寒沙门菌

目的建立基于Taq Man探针四重荧光PCR检测甲型、乙型、丙型副伤寒和伤寒沙门菌的方法。方法根据甲型副伤寒沙门菌(Salmonella paratyphi A,SPA)、乙型副伤寒沙门菌(Salmonella paratyphi B,SPB)、丙型副伤寒沙门菌(Salmonella paratyphi C,SPC)和伤寒沙门菌(Salmonella typhi,ST)的特异序列分别设计引物SPAP、SPBP、SPCP和STP,在探针的5'端分别标记TET、ROX、FAM、HEX,建立基于Taq Man探针四重荧光PCR检测方法。结果 SPAP、SPBP、SPCP和STP分别扩增出5株SPA、4株SPB、7株SPC和11株ST,而其他血清型沙门菌和17株非沙门菌扩增结果阴性。SPAP、SPBP、SPCP和STP扩增效率分别为84.5%、101.8%、92.4%和90.9%,线性相关系数(R~2)分别为0.996、0.975、0.996和0.984。结论建立的方法特异性高、敏感性强,可用于SPA、SPB、SPC和ST的特异性检测。     

PCR快速检测甲型副伤寒沙门菌鞭毛抗原H-a研究

目的研究快速、特异、灵敏的检测甲型副伤寒沙门菌鞭毛特异相H-a的PCR法.方法选择甲型副伤寒沙门菌鞭毛抗原H-a基因的特异引物进行PCR扩增,检测甲型副伤寒沙门菌标准菌株和我省不同地区上送的57株甲型副伤寒沙门菌以及非甲型副伤寒沙门菌的其它菌株;将甲型副伤寒沙门菌标准菌株按10-1～10-9稀释后扩增比较PCR的检测灵敏度.结果所有甲型副伤寒沙门菌均在329bp处出现甲型副伤寒沙门菌鞭毛抗原基因产物,非甲型副伤寒沙门菌株PCR结果均为阴性;PCR检测下限为103cfu/ml.结论 PCR比传统细菌检测方法更特异、快速、灵敏、简便,为临床诊断、实验室筛查以及流行病学快速查源、溯源提供了新的手段.     

890株伤寒、甲型副伤寒沙门菌耐药性分析

目的 了解本省伤寒、甲型剐伤寒沙门菌的耐药性，指导合理用药。方法 对各市(地)疾控中心提供的890株伤寒、甲型副伤寒沙门菌株进行生化鉴定和血清学试验；药敏实验方法参照K-B琼脂扩散法。结果 我省近几年来流行优势菌株甲型副伤寒沙门菌的耐药性明显高于伤寒沙门菌，不同地区及不同年份其耐药性均有一定差异，爆发型菌株的耐药性明显高于非爆发型菌株。结论 应根据不同菌型、地区及不同流行强度的伤寒、副伤寒做出相府防治措施。     

中国四省甲型副伤寒分离株脉冲场凝胶电泳分析及分型数据库建立

目的对中国4个甲型副伤寒流行省份的分离株进行标准化脉冲场凝胶电泳（PFGE）分型分析。方法对来自4个省的甲型副伤寒沙门菌利用XbaⅠ酶切染色体DNA进行PFGE分析，利用BioNumerics建立数据库和进行相似性聚类。结果分离自1998-2002年的89株甲型副伤寒分离株共有11种PFGE带型，这些菌株中具有优势带型。有3种带型相似性在96．3％，这3种带型的菌株占到分析菌株的865％，并在不同省份和年度中出现。结论建立了中国甲型副伤寒沙门菌的用于网络监测分析的PFGE技术和数据库，近年中国部分省份的甲型副伤寒沙门菌分离株PFGE型别集中，存在同型菌株的广泛流行。     

江苏省2012-2015年伤寒/副伤寒沙门菌耐药及分子分型研究

目的 回顾性分析2012-2015年江苏省伤寒/副伤寒沙门菌的耐药表型和分子分型特征。方法 采集江苏省伤寒/副伤寒监测病例的血液和粪便标本,分离培养伤寒/副伤寒沙门菌后进行生化鉴定和血清分型;采用Kirby-Bauer纸片法检测菌株对抗菌药物的敏感性;应用PFGE技术对伤寒和甲型副伤寒沙门菌进行分子分型。结果 134株伤寒沙门菌和甲型副伤寒沙门菌对萘啶酸的耐药率最高,分别为61.2%和86.7%,其余抗生素的耐药率均低于15.0%。伤寒和甲型副伤寒沙门菌中只耐1种抗生素的菌株最多,多重耐药菌在伤寒沙门菌中占2.6%,而在甲型副伤寒沙门菌中占13.3%。2015年伤寒沙门菌的全敏感菌株构成比较2012年增加了44.3%,同年副伤寒沙门菌中出现了耐5种和6种抗生素的多重耐药株。甲型副伤寒沙门菌可分为8种PFGE型别,多重耐药株与其他菌株条带相似性低,不同型别与耐药谱有对应关系;伤寒沙门菌可分为68种PFGE型别,不同型别间变异度较大,与耐药谱无对应关系。结论 2012-2015年江苏省伤寒/副伤寒沙门菌对抗生素的耐药性总体随时间降低,耐药种类数有所增加。伤寒沙门菌株PFGE带型呈现多样性,无耐药谱对应性;而甲型副伤寒菌株PFGE带型较少,有耐药谱对应性。需要加强重点地区重点型别菌株的监测。     

多种方法同步检测海产品中甲型副伤寒沙门菌的应用

目的：为查找食源性甲型副伤寒的病因,提高检出率.方法：采用国标法、mini VIDAS仪器法和PCR 3种方法同步检测11种生吃、半生吃海产品中甲型副伤寒沙门菌.检出菌株用VITEK32全自动微生物分析系统鉴定以及血清分型.结果：mini VIDAS仪器法和PCR 2种方法从1份牡蛎样品中检测出甲型副伤寒沙门菌,检出率为0.73%.结论：多种方法同步检测有利于提高生吃、半生吃海产品中甲型副伤寒沙门菌和沙门菌的检出率.用mini VIDAS或/和PCR过筛检测,具有简便快捷、灵敏度高的特点,阳性时再用国标法分离,可及时获得菌株,以提高检测工作效率,值得应用,尤其适用于杂菌量较多、目标菌易被掩盖以及不适宜甲型副伤寒沙门菌和其它沙门菌生长的样品.     

玉溪红塔区212株甲型副伤寒沙门菌脉冲场凝胶电泳(PFGE)结果分析

[目的]了解玉溪红塔区甲型副伤寒沙门菌脉冲场凝胶电泳(PFGE)基因型和流行优势型,为探讨传播途径、追踪传染源和确定流行范围等提供依据。[方法]利用XbaⅠ酶切染色体DNA进行PFGE分析。[结果]212株甲型副伤寒沙门菌共分为13种PFGE型,其中0002型123株,占58.02%,0003型46株,占21.7%,0001型32株,占15.1%,0007型2株,占0.94%,其余9个型各1株,分别占0.47%。其中优势型为0002型、0003型和0001型,这3种带型的菌株占到分离菌株的94.8%,并在不同月份和绝大部分乡镇中出现。菌株的带型变化在l～3条范围内,表明红塔区甲型副伤寒沙门菌变异不大,有可能是同一克隆系的甲型副伤寒沙门菌。[结论]红塔区甲型副伤寒沙门菌分离株PFGE型别集中,存在同型菌株的广泛流行。另外10种菌型少见,可能与红塔区甲型副伤寒高度散发有关。     

宁波地区甲型副伤寒沙门菌基因分型

目的 了解宁波地区甲型副伤寒沙门菌的脉冲场凝胶电泳（PFGE）基因型和流行优势型，为探讨传播途径和追踪传染源等提供依据。方法 用全自动细菌鉴定仪（Vitek32）确定细菌的生物学特征；用诊断血清确定细菌的血清型；用PFGE方法对细菌的染色体进行基因分型。结果 102株甲型副伤寒沙门苗可分成9个PFGE型，其中X2型73株，占71．57％。X1型21株，占20，58％，X7型2株，占1．96％，其他6个型各1株，分别占0．98％。其中优势型为X2型，菌株的带型变化在1～3条范围内，表明宁波地区的甲型副伤寒沙门菌变异不大，有可能是同一克隆系的甲型副伤寒沙门菌。结论 PFGE分型对探讨伤寒、副伤寒疫情内在联系具有重要意义，该法特异性高、重复性好、结果容易判读，有实际应用价值。     

2008年-2010年上海市部分伤寒副伤寒沙门菌耐药分析及分子分型

目的:了解上海市2008年-2010年伤寒、副伤寒沙门菌的耐药及分子分型特点,初步掌握伤寒、副伤寒沙门菌的分子流行特点,为今后的防制工作提供科学依据。方法:采用WHO推荐的改良K-B纸片法,对18株伤寒、副伤寒沙门菌进行13种抗生素敏感性试验;运用脉冲场凝胶电泳分型(PFGE)方法对18株伤寒、副伤寒沙门菌进行分子分型及聚类分析。结果:18株伤寒副伤寒沙门菌对利福平100%耐药,对奈啶酸的耐药率为44.4%,其中伤寒沙门菌对奈啶酸的耐药率为37.5%,5株甲型副伤寒沙门菌对奈啶酸100%耐药,乙型副伤寒对奈啶酸无耐药株。所有菌株对其他11种抗生素均敏感;18株伤寒、副伤寒沙门菌共产生16种PGFE带型,有3株甲型副伤寒沙门菌表现为同一PFGE型别。结论:2008年-2010年伤寒、副伤寒沙门菌对抗生素的敏感性较高;甲型副伤寒沙门菌之间有较高的同源性,伤寒沙门菌的PFGE带型比较分散。     

Multiplex PCR for differential diagnosis of emerging typhoidal pathogens directly from blood samples

Classically Salmonella enterica serovar Typhi (S. Typhi) is associated with typhoid, a major health problem in developing countries. However, in recent years S. Paratyphi A and Vi-negative variants of S. Typhi have emerged rapidly. We have developed a nested multiplex PCR targeting five different genes for differential diagnosis of typhoidal pathogens which has been optimized to be directly applicable on clinical blood samples. Of 42 multiplex PCR-positive blood samples, 26, nine, and two were Vi-positive S. Typhi, Vi-negative S. Typhi and S. Paratyphi A, respectively, and five patients were found to have mixed infection. Seventeen patients grew Salmonella from blood culture and the remaining 25 were positive in the Salmonella-specific PCR. Tests with several common pathogens confirmed the specificity of the assay. We conclude that the proposed multiplex PCR is rapid, sensitive and specific for the diagnosis of typhoidal pathogens directly from blood samples.     

Distribution trends of Salmonella serovars in India (2001-2005)

A total of 3079 samples were received and identified at the National Salmonella and Escherichia Centre (NSEC), Central Research Institute, Kasauli, India during 2001-2005. Out of these, 2098 samples were from humans, 250 from animals, and 726 from meat, vegetables, seafood and the environment. The Salmonella strains isolated were distributed among 35 different Salmonella serovars. The most common serovars from humans were Salmonella Typhi (73%) and Salmonella Paratyphi A (24%) among typhoidal serovars, and Salmonella Worthington (28.2%) and Salmonella Typhimurium (22.5%) among non-typhoidal serovars. The other frequently isolated serovars from different sources were Salmonella Gallinarum, Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Dublin. Analysis of the prevalence of the 10 most common serovars in 18 states indicated that different serovars are distributed in different parts of India. The results from this study may be helpful in formulating preventive strategies to control the spread of infection in the country.     

Comparative genomic analysis between typhoidal and non-typhoidal Salmonella serovars reveals typhoid-specific protein families

BackgroundThe genus Salmonella contains more than 2600 serovars. While most cause a self-limiting gastroenteritis, four serovars, S. Typhi, S. Paratyphi A, B and C, elicit typhoid, a potentially fatal systemic infection. Because of the prevalence in certain regions, such as South Asia, and the disease severity of typhoidal Salmonella infections, comprehensive studies are needed to elucidate the pathogenesis of diseases caused by these typhoidal serovars.ResultsWe performed comparative genomic analyses on eight human typhoidal strains and 27 non-human typhoidal Salmonella strains to elucidate their evolutionary relationships and identify the genes specific to the four typhoidal serovars. Our results indicate that Salmonella may have an open pan-genome. A core-genome based phylogeny demonstrated that divergence between S. Paratyphi A and S. Typhi took place not long ago and S. Paratyphi B shared a recent common ancestor with S. Paratyphi C. Of great interest, the divergence between S. Paratyphi B and S. Paratyphi C was shown to be more recent than that between S. Paratyphi A and S. Typhi. Alignment and comparisons of the genomes identified unique complements of protein families to each of the typhoidal serovars. Most of these protein families are phage related and some are candidate virulence factors. Importantly, we found 88 protein families specific to two to three of the four typhoidal serovars. All but two of the 88 genes are present in S. Typhi, with a few in the three paratyphoidal serovars but none in the non-human typhoidal serovars. Most of these genes are predicted to encode hypothetical proteins and some are known to code for virulence factors such as Vi polysaccharide related proteins.ConclusionsBy comprehensive genomic comparisons, we identified protein families specific to the human typhoidal serovars, which will greatly facilitate investigations on typhoid pathogenesis.     

Status of paratyphoid fever vaccine research and development

Salmonella enterica serovars Typhi and Paratyphi (S. Paratyphi) A and B cause enteric fever in humans. Of the paratyphoid group, S. Paratyphi A is the most common serovar. In 2000, there were an estimated 5.4 million cases of S. Paratyphi A worldwide. More recently paratyphoid fever has accounted for an increasing fraction of all cases of enteric fever. Although vaccines for typhoid fever have been developed and in use for decades, vaccines for paratyphoid fever have not yet been licensed. Several S. Paratyphi A vaccines, however, are in development and based on either whole cell live-attenuated strains or repeating units of the lipopolysaccharide O-antigen (O:2) conjugated to different protein carriers. An O-specific polysaccharide (O:2) of S. Paratyphi A conjugated to tetanus toxoid (O:2-TT), for example, has been determined to be safe and immunogenic after one dose in Phase I and Phase II trials. Two other conjugated vaccine candidates linked to diphtheria toxin and a live-attenuated oral vaccine candidate are currently in preclinical development. As promising vaccine candidates are advanced along the development pipeline, an adequate supply of vaccines will need to be ensured to meet growing demand, particularly in the most affected countries.     

Emergence of Salmonella enterica serotype Paratyphi A as a major cause of enteric fever in Kathmandu, Nepal

We performed pulsed-field gel electrophoresis (XbaI) on 114 bloodstream isolates of Salmonella enterica serotype Paratyphi A and S. enterica serotype Typhi collected from febrile patients in Kathmandu, Nepal. Of the 56 S. Paratyphi A isolates, 51 (91%) were indistinguishable, which suggests the emergence of a single clone. In contrast, only 21 (36%) of the 58 S. Typhi isolates exhibited a common genotype, which is consistent with endemic disease from multiple sources.     

Salmonella serovars (others than Typhi and Paratyphi) from extra-intestinal sources. Israel, 1984-9

The purpose of this study was to examine whether certain salmonella serovars, other than Typhi and Paratyphi, may have an increased ability to enter the human body fluids. The data are based on over 20,000 salmonella isolated from humans during 1984-9, among them 436 from extra-intestinal (EI) sources. The mean percentage of EI salmonella isolated in Israel (excluding Typhi and Paratyphi), was 2.1%. In three serovars: S.9, 12:1, v:-, Virchow and Saintpaul, the percentage of blood isolations and of EI isolations in general, was high. Among the frequent serovars, ser. Infantis and Hadar had a much lower percentage of EI isolations.     

Antibiogram pattern and seasonality of Salmonella serotypes in a North Indian tertiary care hospital

The antibiogram pattern and seasonal distribution of Salmonella serotypes were analysed retrospectively over a 6-year period from January 1999 to December 2004. Blood cultures received in the Bacteriology Laboratory were processed by standard procedures and the Salmonella spp. isolates were identified with specific antisera and standard biochemical tests. Antimicrobial susceptibility testing was carried out by a standard disc diffusion method and the minimum inhibitory concentration (MIC) of ciprofloxacin for 332 representative Salmonella isolates was determined by E test. Salmonella Typhi (75.7%) was the predominant serotype among 830 Salmonella spp. isolated during the study period followed by S. Paratyphi A (23.8%). The maximum number of enteric fever cases occurred during April-June (dry season) followed by July-September (monsoon season). There was a decrease in multidrug-resistant (MDR) S. Typhi, but MDR S. Paratyphi A isolates increased. There was also a dramatic increase in nalidixic acid-resistant isolates. All isolates were susceptible to third-generation cephalosporins and ciprofloxacin except one S. Typhi strain which demonstrated high-level ciprofloxacin resistance with a MIC of 16 mug/ml. A knowledge of the seasonal distribution and antibiotic resistance pattern of Salmonella in a particular geographical region is helpful in the delineation of appropriate control measures required for prevention of enteric fever.     

Salmonella paratyphi A rates, Asia

Little is known about the causes of enteric fever in Asia. Most cases are believed to be caused by Salmonella enterica serovar Typhi and the remainder by S. Paratyphi A. We compared their incidences by using standardized methods from population-based studies in China, Indonesia, India, and Pakistan.     

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.