伤寒沙门菌CT18和新疆XJ19体外培养条件下的蛋白质组差异分析

目的：分析我国新疆伤寒沙门菌分离株XJ19与全基因组测序的国际标准菌株CT18的蛋白表达差异,并推算基因差异。方法：运用二维蛋白电泳,对CT18和XJ19在体外培养基中的全菌蛋白进行分离,使用PDQuest软件找到其差异蛋白,进行质谱鉴定;对CT18差异蛋白编码基因设计引物,以XJ19DNA为模板进行PCR扩增,检测CT18差异蛋白编码基因在XJ19的存在情况。结果：菌株XJ19中存在53个特异蛋白点,鉴定出47个,但这些蛋白的编码基因在CT18中均存在,其中36个蛋白点在CT18的蛋白谱中不存在,11个蛋白在CT18中处于其他修饰状态;CT18中找到13个特异蛋白点,质谱鉴定出7个,其中6个所对应基因在XJ19中均能扩增出目的片段,但点C9蛋白的编码基因在XJ19中不存在。菌株XJ19中的多个差异蛋白参与磷酸戊糖途径的代谢及信号感应调控,此外超氧化物歧化酶、外膜蛋白OmpA呈现与CT18不同的修饰状态。结论：我们认为不同伤寒沙门菌分离株的遗传差异不仅仅是基因的有或无,还包括蛋白的不同表达和修饰所造成的不同调控机制和代谢的差异。     

沙门菌CWDMs苹果酸脱氢酶的检测

目的：了解沙门菌细菌壁缺陷突变株（CWDMs）的生物氧化及遗传特点和探讨细菌壁缺陷变异的性质与机制。方法：采用PAGE电泳法和分光光度法检测伤寒沙门菌和甲型副伤寒沙门菌及其CWDMs和伤寒沙门菌粗糙型和苹果酸脱氢酶（MDH）同工酶的活性与类型。结果：伤寒沙门菌和甲型副伤寒沙门菌的细菌型和伤寒沙门菌粗糙型经PAGE电泳可见一条MDH同工酶带,CWDMs电泳后可见两条MDH同Ⅰ酶带,在CWDMs的MDH中有一条泳动速率与细菌型及粗糙型的相同,另一条则较快。分光光度法检测证实。细菌型与粗糙型的MDH活性相似,CWDMs的MDH活性则明显较低。结论：CWDMs保留了与亲代细菌型一致的MDH和形成了一种新的MDH,并且其MDH的活性已显著降低,此特性可能与CWDMs生物氧化特性的改变有关。     

6种沙门菌单克隆抗体的制备和效能评价

目的：制备稳定、特异、高亲和性的分别针对甲型副伤寒沙门菌、乙型副伤寒沙门菌、丙型副伤寒沙门菌、肠炎沙门菌、伤寒沙门菌和猪霍乱沙门菌的单克隆抗体。方法：用甲醛灭活的菌液抗原免疫BALB／c小鼠,取脾细胞与SP2／0骨髓瘤细胞融合;用灭活的菌液包被酶标板,ELISA筛选阳性克隆株,建立细胞系;选取高效分泌杂交瘤细胞,常规制备腹水并纯化,进行单抗特异性与亲和性评价。结果：筛选得到分泌6种沙门菌相应单克隆抗体的杂交瘤细胞株,获得高亲和性单抗;所有单抗与大部分病原菌（包括7种沙门菌、3株志贺菌、2株李斯特菌、4株致病性大肠杆菌、2株霍乱弧菌）无交叉反应,但由于同类型O抗原的广泛分布,抗乙型副伤寒沙门菌单抗与鼠伤寒沙门菌、抗伤寒沙门菌单抗与肠炎沙门菌有明显的交叉反应。结论：沙门菌单抗的制备,为感染性腹泻的监测、诊断奠定了基础。     

鼠伤寒沙门菌外膜蛋白与耐药性关系的分析

目的分析鼠伤寒沙门菌外膜蛋白(OMP)与耐药性的关系。方法用消除剂丫啶橙消除耐药性,盲传测其遗传稳定性,采用超声波物理裂解法制备鼠伤寒沙门菌外膜蛋白标本,用变性聚丙烯酰胺凝胶电泳(SDA-PAGE)检测外膜蛋白,用紫外分光光度计测其吸光值,计算浓度。结果抗性消除表型能稳定遗传,耐药鼠伤寒沙门菌与敏感鼠伤寒沙门菌都含有6条主要的外膜蛋白条带,两者相比,发现耐药菌的外膜蛋白在约57、53、30 kDa处减弱或缺失,总的蛋白浓度也低于后者。结论鼠伤寒沙门菌耐药性与外膜蛋白的减弱或缺失有关。     

沙门菌属耐药机制的研究进展

伤寒、副伤寒是中国主要发生的肠道传染病之一,由于抗生素的广泛和盲目使用,使伤寒、副伤寒沙门菌的耐药性变得越来越严重,耐药菌株不断出现,而且其耐药谱也不断发生变化。现对有关沙门菌属耐药状况、耐药机制及防治等研究进展作一综述。     

甲型副伤寒沙门菌培养条件的优化

优化甲型副伤寒沙门菌的培养条件,提高菌体产量。方法通过单因素及正交试验,对影响甲型副伤寒沙门菌生长的培养温度、NaCl浓度和pH等条件进行优化。结果甲型副伤寒沙门菌在NaCl浓度0.75%、温度35℃、pH6.5时菌体产量最高。结论通过对甲型副伤寒沙门菌培养条件进行优化,获得较高的菌体产量,为后期诊断试剂盒的开发奠定了基础。     

伤寒沙门菌与甲、乙、丙副伤寒沙门菌质控血清的制备

应用免疫学原理,将伤寒沙门菌O901、H901和甲、乙、丙型副伤寒沙门菌分别制成全菌体抗原,免疫实验兔获取免疫血清。依据伤寒沙门菌和副伤寒沙门菌的抗原成分的异同性,选择适当的吸收菌除去免疫血清中的交叉反应抗体和类属凝集素,而保留其特异性的抗体。通过对诊断菌液的验证试验,证实吸收充分的免疫血清具有质控血清的特性。具备可靠性能的质控血清,适用于伤寒沙门菌与副伤寒沙门菌的菌种检定及其效价检测;亦有利于肥达氏诊断菌液的质量控制。     

沙门菌CWDMs乳酸脱氢酶同功酶的观察

采用聚丙烯酰胺凝胶电泳法检测伤寒杆菌和甲型副伤寒杆菌的ＣＷＤＭｓ及其宁代细菌型和伤寒杆菌粗糙型的乳酸脱氢酶（ＬＤＨ）同功酶,以了解沙门菌ＣＷＤＭｓ生物氧化的特点和机制,探讨ＣＷＤＭｓ变异的性质。结果表明,伤寒杆菌和甲型副伤寒杆菌的细菌型及伤寒杆粗糙型在聚丙烯酰胺凝胶电泳后显示出相同的４种具有不同泳动速率的ＬＤＨ同功酶,但ＣＷＤＭｓ仅显示２种ＬＤＨ。ＣＷＤＭｓ的２种ＬＤＨ同功酶与其亲代细菌型及伤寒杆     

基于串联质谱标签法和平行反应监测技术的氟喹诺酮耐药沙门菌蛋白质组学分析

【背景】随着沙门菌对氟喹诺酮类药物的耐药性不断增强,对其耐药机理的研究显得尤为迫切和重要,蛋白质组学分析将为沙门菌的耐药机理研究提供新的靶点和方向。【目的】对鼠伤寒沙门菌诱导获得耐药性前后进行蛋白质组学分析,为深入研究沙门菌耐药机理奠定基础。【方法】用环丙沙星对鼠伤寒沙门菌ATCC13311进行耐药性诱导,利用串联质谱标签法(Tandem mass tag,TMT)对其耐药性进行差异蛋白的筛选和生物信息学分析,并选取15个差异蛋白进行平行反应监测(Parallel reaction monitoring,PRM)靶向蛋白验证。【结果】筛选出318个差异表达蛋白,其中上调159个,下调159个,涉及的KEGG通路主要包括细菌趋药性、ABC转运蛋白、双组分系统等;PRM定量到13个验证蛋白且变化趋势与TMT一致。【结论】通过TMT定量结合PRM靶向验证对鼠伤寒沙门菌耐药前后进行蛋白质组学分析,筛选出多个差异蛋白和代谢通路,包括外排泵相关蛋白、外膜蛋白、双组分相关蛋白及通路、细菌趋化性相关蛋白及通路等,为沙门菌氟喹诺酮类耐药机理的深入研究奠定了基础。     

伤寒沙门菌野生型与粗糙型菌体蛋白质的双向电泳分析与研究

了解伤寒沙门菌野生型与粗糙型菌株的菌体蛋白质组成特点,探讨伤寒沙门菌粗糙型变异的遗传学基础。分离提取伤寒沙门菌野生型与粗糙型菌株的菌体蛋白质,聚丙烯酰胺凝胶双向电泳(2D-PAGE)和考马斯亮蓝染色,计算机分析与比较两菌株的蛋白质组成特点及其相关性。伤寒沙门菌野生型与粗糙型具有相似的蛋白质电泳图谱,相似系数为78%。多数蛋白质斑点分布于pH 3.0~6.4之间,并且分子量小于30kDa。两菌株的蛋白质电泳图谱之间的主要差异共计36处,多数差异蛋白质的分子量小于20kDa。伤寒沙门菌粗糙型与野生型菌体蛋白质组成的差异显示,粗糙型变异绝不仅仅是O抗原多糖的缺失,也可发生菌体蛋白质组成的改变与缺失。伤寒沙门菌粗糙型保留了同其亲代野生型菌株一致的绝大多数菌体蛋白质组成,在2D-PAGE中形成伤寒沙门菌特征性的基本蛋白质图谱,有助于对粗糙型菌株进行蛋白质分子同源性与变异性的分析与鉴别。     

Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal

AIMS: To study the occurrence and diversity of Salmonella serovars in urban water supply systems of Nepal. METHODS AND RESULTS: Occurrence of Salmonella was detected in 42 out of 300 water samples by enrichment culture technique in selenite F broth followed by plating on Salmonella Shigella agar. A total of 54 isolates identified to genus level by standard tests were subsequently confirmed by serotyping, phage typing and PCR detection of virulence genes (inv A and spv C). The predominant serotype was Salmonella Typhimurium, followed by Salm. Typhi, Salm. Paratyphi A and Salmonella Enteritidis. Most of the Salm. Typhi isolates were E1 phage type followed by UVS4, A and UVS1. All isolates of Salm. Paratyphi A and Salm. Enteritidis were an untypable (UT) phage type. The majority of isolates were multi-drug resistant as revealed by Kirby-Bauer disc diffusion technique. Ceftriaxone resistant isolates of Salm. Enteritidis indicated the presence of one of the ESBL genes, blaSHV, whereas the genes blaTEM and blaCTX were absent. CONCLUSIONS: The microbiological quality of the urban water supply is poor and indicates possibility of fatal outbreaks of enteric fever and related infections in Nepal. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study will be useful in water borne disease control and prevention strategy formulation in Nepal and in the global context.     

Combined high-resolution genotyping and geospatial analysis reveals modes of endemic urban typhoid fever transmission

Typhoid is a systemic infection caused by Salmonella Typhi and Salmonella Paratyphi A, human-restricted bacteria that are transmitted faeco-orally. Salmonella Typhi and S. Paratyphi A are clonal, and their limited genetic diversity has precluded the identification of long-term transmission networks in areas with a high disease burden. To improve our understanding of typhoid transmission we have taken a novel approach, performing a longitudinal spatial case-control study for typhoid in Nepal, combining single-nucleotide polymorphism genotyping and case localization via global positioning. We show extensive clustering of typhoid occurring independent of population size and density. For the first time, we demonstrate an extensive range of genotypes existing within typhoid clusters, and even within individual households, including some resulting from clonal expansion. Furthermore, although the data provide evidence for direct human-to-human transmission, we demonstrate an overwhelming contribution of indirect transmission, potentially via contaminated water. Consistent with this, we detected S. Typhi and S. Paratyphi A in water supplies and found that typhoid was spatially associated with public water sources and low elevation. These findings have implications for typhoid-control strategies, and our innovative approach may be applied to other diseases caused by other monophyletic or emerging pathogens.     

Multidrug-resistant Salmonella enterica serovar paratyphi A harbors IncHI1 plasmids similar to those found in serovar typhi

Salmonella enterica serovars Typhi and Paratyphi A cause systemic infections in humans which are referred to as enteric fever. Multidrug-resistant (MDR) serovar Typhi isolates emerged in the 1980s, and in recent years MDR serovar Paratyphi A infections have become established as a significant problem across Asia. MDR in serovar Typhi is almost invariably associated with IncHI1 plasmids, but the genetic basis of MDR in serovar Paratyphi A has remained predominantly undefined. The DNA sequence of an IncHI1 plasmid, pAKU_1, encoding MDR in a serovar Paratyphi A strain has been determined. Significantly, this plasmid shares a common IncHI1-associated DNA backbone with the serovar Typhi plasmid pHCM1 and an S. enterica serovar Typhimurium plasmid pR27. Plasmids pAKU_1 and pHCM1 share 14 antibiotic resistance genes encoded within similar mobile elements, which appear to form a 24-kb composite transposon that has transferred as a single unit into different positions into their IncHI1 backbones. Thus, these plasmids have acquired similar antibiotic resistance genes independently via the horizontal transfer of mobile DNA elements. Furthermore, two IncHI1 plasmids from a Vietnamese isolate of serovar Typhi were found to contain features of the backbone sequence of pAKU_1 rather than pHCM1, with the composite transposon inserted in the same location as in the pAKU_1 sequence. Our data show that these serovar Typhi and Paratyphi A IncHI1 plasmids share highly conserved core DNA and have acquired similar mobile elements encoding antibiotic resistance genes in past decades.     

Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.     

Development of a real-time PCR assay for detection of gyrA mutations associated with reduced susceptibility to ciprofloxacin in Salmonella enterica serovar typhi and paratyphi A

A real-time PCR assay with the cycling probe method was used to detect mutations at codons 83 and 87 in the DNA gyrase A subunit encoded by gyrA in Salmonella enterica serovar Typhi and Paratyphi A clinical isolates. The susceptibility estimated from the results of the gyrA mutation assay was consistent with that identified by the culture method using an E-test. This assay allows rapid screening of S. enterica serovar Typhi and Paratyphi A with reduced susceptibility to ciprofloxacin.     

Linkage of avian and reproductive tract tropism with sequence divergence adjacent to the 5S ribosomal subunit rrfH of Salmonella enterica

The 183 bp between the end of the 23S rrlH rRNA gene and the start of the 5S rrfH rRNA gene (ISR-1) and the 197 bp between the end of the rrfH rRNA gene and the start of the transfer RNA aspU (ISR-2) of Salmonella enterica ssp. enterica serotypes Enteritidis, Typhimurium, Pullorum, Heidelberg, Gallinarum, Typhi and Choleraesuis were compared. ISR-1s of D1 serotypes (Pullorum, Gallinarum and Enteritidis), B serotypes (Typhimurium and Heidelberg) and the C2 serotype Newport and the enteric fever pathogens serotype A Paratyphi and serotype D1 Typhi formed three clades, respectively. ISR-2 further differentiated the avian-adapted serotype Gallinarum from avian-adapted Pullorum and Salmonella bongori from S. enterica. The results suggest that serotypes Heidelberg and Choleraesuis share some evolutionary trends with egg-contaminating serotypes. In addition, ISR-1 and ISR-2 sequences that confirm serotype appear to be linked to clinically relevant host associations of the Salmonellae.     

Antimicrobial Resistance,Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata,India during 2009-2013

Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was to characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 to June 2013 and were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance and virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) and 25 Salmonella enterica serovar Paratyphi A (S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) and absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non-IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (bla _TEM-1, catA, sul1, sul2, dfrA15, strA-strB) and class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR and sequencing. Most of the study isolates were likely to be virulent due to the presence of virulence markers. Major diversity was not noticed among S. Typhi and S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles and S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR and molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology.     

Construction of an attenuated Salmonella enterica serovar Paratyphi A vaccine strain harboring defined mutations in htrA and yncD

The global epidemic features of enteric fever have changed greatly in recent years. The incidence of enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased. In some areas of Asia, infections with S. Paratyphi A have exceeded those with S. Typhi, resulting in S. Paratyphi A becoming the main causative agent of enteric fever. However, two currently licensed typhoid vaccines do not confer adequate cross‐protection against S. Paratyphi A infection. Therefore, development of specific vaccines against enteric fever caused by S. Paratyphi A is urgently needed. In the present study, an attenuated strain was constructed by double deletion of the htrA and yncD genes in a wild‐type strain of S. Paratyphi A and its safety and immunogenicity assessed. In a mouse model, the 50% lethal dose of the double deletion mutant and the wild‐type strain were 3.0 × 10⁸ CFU and 1.9 × 10³ CFU, respectively, suggesting that the double deletion resulted in remarkably decreased bacterial virulence. Bacterial colonization of the double deletion mutant in the livers and spleens of infected mice was strikingly less than that of the wild‐type strain. A single nasal administration of the attenuated vaccine candidate elicited high concentrations of anti‐LPS and anti‐flagellin IgG in a mouse model and protected immunized mice against lethal challenge with the wild‐type strain. Thus, our findings suggest that the attenuated vaccine strain is a promising candidate worthy of further evaluation both as a human enteric fever vaccine and as a vaccine delivery vector for heterologous antigens.     

O:2-CRM197 Conjugates against Salmonella Paratyphi A

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM₁₉₇, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM₁₉₇ as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.