Reduced amounts of LPS affect both stress tolerance and virulence of Salmonella enterica serovar Dublin

Signature-tagged mutagenesis (STM) is a widely used technique for identification of virulence genes in bacterial pathogens. While this approach often generates a large number of mutants with a potential reduction in virulence a major task is subsequently to determine the mechanism by which the mutations influence virulence. Presently, we have characterised a Salmonella enterica serovar Dublin STM mutant that, in addition to having reduced virulence, was also impaired when growing under various stress conditions. The mutation mapped to the manC (rfbM) gene of the O-antigen gene cluster involved in O-antigen synthesis. The O-antigen is a component of the lipopolysaccharide (LPS) forming a unique constituent of the outer membrane of Gram-negative bacteria. While mutations in the O-antigen genes usually eliminate the entire O-antigen side chain we found that the transposon mutant produced intact O-antigen, however, the mutation reduced the amount of LPS.     

Molecular phylogenetic typing of pandemic isolates of Salmonella enteritidis

Salmonella enteritidis is now the most common Salmonella serovar in many countries. We have used cloned DNA probes to analyze genome interrelationships between strains chosen to represent the current S. enteritidis pandemic, and included designated type strains of the seven subspecies of Salmonella in order to compare the levels of discrimination of probes. DNA sequence divergence and rearrangements were analyzed in and around the rfa, fim and umuDC loci, and around insertion sites of the Salmonella-specific DNA insertion element, IS200. The S. enteritidis isolates showed a high degree of genome homogeneity. Chromosomal genetic loci exhibited characteristic DNA sequence divergence between subspecies of Salmonella, but no intraserovar divergence or difference with the subspecies I type strain was observed for S. enteritidis. The locus umuDC was not found in S. enteritidis. S. enteritidis contains a conserved and a variable site of insertion of insertion sequence IS200 and the analysis of DNA rearrangements around the second of these sites showed that three distinct evolutionary lines or races exist within pandemic isolates associated with human gasteroenteritis. IS200 profiles of a range of U.K. isolates of the epidemic phage type PT4 showed that all belonged to a single clonal line.     

Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure

AIMS: To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces. METHODS AND RESULTS: Faeces samples collected from six adult bovines from different salmonella-negative herds were split into subpools and spiked with three strains of S. Dublin at a concentration level of c. 10 CFU g(-1) faeces. Each of the 18 strain-pools was divided into two sets of triplicates of four volumes of faecal matter (1, 5, 10 and 25 g). The two sets were pre-enriched with and without novobiocin, followed by combinations of culture media (three types) and selective media (two types). The sensitivity of each combination and sources of variation in detection were determined by a generalized linear mixed model using a split-plot design. CONCLUSIONS: Biological factors, such as faecal origin and S. Dublin strain influenced the sensitivity more than technical factors. Overall, the modified semi-solid Rappaport Vassiliadis (MSRV)-culture medium had the most reliable detection capability, whereas detection with selenite cystine broth and Mueller Kauffman tetrathionate broth combinations varied more in sensitivity and rarely reached the same level of detection as MSRV in this experiment. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that for MSRV-culture medium and xylose lysine decarboxylase agar as the indicative medium, the sensitivity of the faecal culture method may be improved by focusing on the strain variations and the ecology of the faecal sample. Detailed investigation of the faecal flora (pathogens and normal flora) and the interaction with chemical factors may result in developing an improved method for detection of S. Dublin.     

2014—2015年大连市伤寒沙门菌耐药性及分子分型研究

目的 了解大连市伤寒沙门菌的药物敏感情况及同源性特点,为临床用药和疾病预防提供科学指导。方法 选择15种抗生素、运用微量肉汤稀释法对65株伤寒沙门菌进行抗生素敏感试验;采用脉冲场凝胶电泳（PFGE）法进行聚类分析。结果 65株伤寒沙门菌对阿奇霉素100.00%敏感,对红霉素100.00%耐药,对其他13种药物有不同程度的耐药率（1.54%~73.85%）。发现1株多重耐药菌株。65株菌共产生41种PFGE带型,其中8株菌表现为同一型别。结论 大连地区临床分离伤寒沙门菌耐药形势严峻;聚类分析结果表明其PFGE型别较多,而且其中存在着优势菌株。     

Cross-contamination with Salmonella on a broiler slaughterhouse line demonstrated by use of epidemiological markers

AIMS: To investigate contamination of surfaces on a poultry slaughter line from infected poultry and subsequent cross-contamination of non-infected poultry. METHODS AND RESULTS: A broiler slaughterhouse was investigated for the presence of Salmonella on 17 defined points over two 1-week periods. Flocks supplied to slaughter and neck skin samples from processed chicken were likewise investigated. Salmonella was detected in 10 out of 18 flocks at ante-mortem inspection, while seven flocks tested positive in the finished products. Equipment at all but one control point at the slaughter line tested positive at least once during the study. The chicken receiving area was the most contaminated. By comparison of typing results from serotyping, plasmid profile typing and phage typing, direct evidence for cross-contamination with Salm. serotype Typhimurium, Salm. Serotype 4.12:b:- and Salm. serotype Virchow on the slaughter line was obtained for four of the flocks. The cleaning procedure in place did not remove all Salmonella from the contaminated areas. CONCLUSIONS: Evidence for contamination of equipment on a slaughter line and subsequent cross-contamination to non-infected chicken was provided by typing methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided detailed information on cross-contamination on a slaughter line by the use of phage typing and plasmid profiling. The study stresses the importance of controlling Salmonella in the primary production, as contamination of the equipment on the slaughter line will act as a vehicle to contaminate finished products. Cleaning procedures on slaughter lines cannot be expected to control this problem with the current equipment.     

Complete nucleotide sequence of a virulence plasmid of Salmonella enterica serovar Dublin and its phylogenetic relationship to the virulence plasmids of serovars Choleraesuis, Enteritidis and Typhimurium

The complete nucleotide sequence of pOU1113 (pSDVu), one of the two types of virulence plasmids of Salmonella enterica serovar Dublin, was determined. It contained 80 156 bp with 53.8 mol% G+C content. Approximately 70 genes could be discerned. Compared with pSTV, the virulence plasmid of serovar Typhimurium, pOU1113 was shorter owing to a missing region amounting to c. 10 kb; furthermore, except for a unique 10 849-bp region, the nucleotide as well as deduced amino acid sequences of pOU1113 were nearly identical to the corresponding regions of three S. enterica virulence plasmids, namely pSCV (virulence plasmid of Choleraesuis), pSTV and pSEV (virulence plasmids of Enteritidis), confirming their close phylogenetic relationship. Comparative analysis indicated that these virulence plasmids appeared to have descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. From a biological and evolutionary point of view, if the decreasing sizes of pOU1113 and pSCV truly reflect a process in which the virulence plasmid has been shedding unnecessary genes during evolution, our data suggest that some genes in the missing region, such as the pef and tra operons, could have a minimal role in maintaining the survival of the bacteria in their environmental niche.     

2016‒2017年辽宁省沙门菌耐药菌谱与分子分型

目的 分析2016‒2017年辽宁省沙门菌分离株的耐药特性与脉冲场凝胶电泳（PFGE）分子分型特征,为沙门菌引起的食源性疾病暴发、防控及抗生素使用提供参考数据。方法 对分离的54株沙门菌进行血清分型和药物敏感试验。根据PulseNet沙门菌标准PFGE分型技术,选取全部菌株进行PFGE分子分型分析,应用BioNumerics软件对菌株条带进行分析,确定菌株间的特征及相关性。结果 54株沙门菌血清型居首位的是肠炎沙门菌,占46.30％;其次是鼠伤寒沙门菌,占24.07％;共分为10个血清型。对13种抗生素的耐药分析显示多重耐药菌株为36株,占66.7％,其中耐3～5种的13株（24.1%）,耐6～8种的13株（24.1%）,耐9～11种的10株（18.5%）。54株沙门菌经聚类分析获得36种带型,相似度区间为49.7%～100.0％。结论 辽宁省沙门菌分离株多重耐药状况比较严重,相同血清型其PFGE带型相似度相对较高,同时具有较显著的优势带型特点;而且发现同一PFGE型菌株的耐药谱相对比较接近。     

Use of IgG avidity ELISA to differentiate acute from persistent infection with Salmonella Dublin in cattle

AIMS: To investigate whether an immunoglobulin (Ig)G avidity ELISA can be used to differentiate between acute and persistent infection with Salmonella (S.) Dublin in cattle. To determine whether the IgG isotype, IgG(1) and IgG(2) responses in acute and persistent infections differ. METHODS AND RESULTS: Animals were selected from two herds with long-term infection (years) and two herds recently infected (<3 months). Forty-seven animals were categorized into groups based on the persistence of their antibody level in milk. Based on titre from two serial dilutions the avidity index (AI) was calculated for IgG (IgG-AI), IgG(1) (IgG(1)-AI) and IgG(2) (IgG(2)-AI). The mean IgG-AI for suspected carrier animals with either persistently high (group 1) or persistently high to medium high (group 2) antibody levels was significantly (P = 0.003) higher (32.1% and 38.4%) than for acutely infected animals (21.7% and 22.3%). The probability of being a suspect carrier was associated with IgG-AI, antibody level in the sample and age. However, the effect of age could be the result of a biased sample selection. Specificities and sensitivities were calculated at a range of cut-off values for IgG-AI and IgG(1)-AI. Overall, IgG(2)-AI was high compared with IgG(1)-AI, and there was no difference in IgG(2)-AI between infection groups. There was no difference in the ratio IgG(2):IgG(1) for acute and persistent infection groups. CONCLUSIONS: Assuming that a persistently high antibody response is indicative of persistent infection with S. Dublin in cattle, it can be concluded that the IgG-AI can aid in differentiating between acute and long-term infection on herd level. However, for the test to be useful as an alternative tool to repeated sampling over time for detection of persistently infected carriers during control strategies in cattle herds, the test needs to be optimized and studied further in a larger sample of well-characterized infections in cattle. The affinity of IgG(2) is higher than IgG(1) early in the S. Dublin infection. There appears to be no difference in the IgG(2)-AI between the acute and chronic infection stages. SIGNIFICANCE AND IMPACT OF THE STUDY: For decades the strategies for detection of persistently infected cattle in S. Dublin infected herds have involved repeated bacteriological culture of faecal samples or repeated antibody measurements over several months. Both methods are time consuming and costly, leaving a new method for detection of carrier animals based on a single sampling highly desirable. This study illustrates a tool, IgG-AI, which may prove useful, although more validation of the method is required before it is used in practice.     

Evaluation of an indirect serum ELISA and a bacteriological faecal culture test for diagnosis of Salmonella serotype Dublin in cattle using latent class models

AIMS: To evaluate a conventional bacteriological test based on faecal culture and an indirect serum ELISA for detection of S. Dublin infected cattle. To compare the predictive values of the two tests in relation to the prevalence. METHODS AND RESULTS: A total of 4531 paired samples from cattle in 29 dairy herds were analysed for presence of S. Dublin bacteria in faeces and immunoglobulins directed against S. Dublin lipopolysaccharide in an indirect serum ELISA. Sensitivity and specificity were estimated at two ELISA cut-off values using a validation method based on latent class models, which presumably provides less biased results than traditional validation methods. Stratification of data into three age groups gave significantly better estimates of test performance of the ELISA. Receiver operating characteristic (ROC) curves were constructed for comparison of overall performance of the ELISA between the three age groups. The sensitivity of the faecal culture test was low (6-14%). ELISA appeared to have a higher validity for animals aged 100-299 days of age than older or younger animals. Overall, the negative predictive value of the ELISA was 2-10 times higher than for the faecal culture test at realistic prevalence of infection in the test population. CONCLUSIONS: The diagnostic sensitivity of the faecal culture test for detection of S. Dublin is poor, the specificity is 1. The superior sensitivity and negative predictive value of the serum ELISA makes this test preferable to faecal culture as an initial screening test and for certification of herds not infected with S. Dublin. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative estimate of the sensitivity of a faecal culture test for S. Dublin in a general population was provided. ELISA was shown to be an appropriate alternative diagnostic test. Preferably, samples from animals aged 100-299 days of age should be used as these give the best overall performance of the ELISA. Plots of ROC curves and predictive values in relation to prevalence facilitates optimisation of the ELISA cut-off value.     

Molecular methods for typing of Helicobacter pylori and their applications

Microbial typing is a useful tool in clinical epidemiology for defining the source and route of infection, for studying the persistence and reinfection rates, clonal selection in the host and bacterial evolution. Phenotypic methods such as biotyping, serotyping and hemagglutinin typing have little discriminatory power compared to genotypic methods concerning the typing of Helicobacter pylori. Therefore great efforts have been made to establish useful molecular typing methods. In this context, the most frequently used genotypic methods are described based on our own experience and the literature: (1) restriction endonuclease analysis, (2) endonuclease analysis using pulsed-field gel electrophoresis, (3) ribotyping, (4) polymerase chain reaction (using either random primers or repetitive DNA sequence primers), and (5) polymerase chain reaction-restriction fragment length polymorphism analysis of e.g. the urease genes. Furthermore, reproducibility, discriminatory power, ease of performance and interpretation, cost and toxic procedures of each method are assessed. To date no direct comparison of all the molecular typing methods described has been performed in the same study with the same H. pylori strains. However, PCR analysis of the urease gene directly on suspensions of H. pylori or gastric biopsy material seems to be useful for routine use and applicable in specific epidemiological situations.     

沙门氏菌的分子分型方法

沙门氏菌是一类危害人和动物健康的重要致病菌,是引起食物中毒的最常见病原菌之一.本文对目前常用的3种分子分型方法脉冲场凝胶电泳(Pulsed-field gel electrophoresis,PFGE),多位点序列分析(Multi-locus sequence analysis,MLST)及多位点可变数串联重复分析(Multiple-locus variable number tandem repeats analysis,MLVA)在沙门氏菌分子分型的应用做了阐述和比较,为沙门氏菌分型研究提供一定的参考.     

32株金黄色葡萄球菌分子分型及肠毒素分析

目的 对辽宁省内2016-2018年分离出的食源性金黄色葡萄球菌采用脉冲场电泳(PFGE)和肠毒素分型进行分析,为今后公共卫生等领域提供技术保障.方法 将32株金黄色葡萄球菌用限制性内切酶SmaI酶切以进行PFGE分析,并用BioNumerics(7.6版本)软件对分离株的指纹图谱进行聚类分析;用PCR方法对菌株进行肠...     

副溶血性弧菌肠细菌基因间共有重复序列-PCR分子分型和耐药性、血清型研究

目的对副溶血性弧菌进行ERIC-PCR分子分型、耐药性和血清型相关性研究。方法肠细菌基因间共有重复序列（ERIC）为引物,对40株菌株基因组DNA进行扩增,得到DNA指纹图谱,并利用SPSS13.0统计软件对DNA扩增图谱进行分析,做出聚类图从而分型,并与菌株血清型、耐药性比较分析。结果40株菌用ERIC-PCR分为5个型,分辨力指数为（DI）为0.5;血清分型分为4个型;对8种抗生素中的萘啶酸、头孢噻亏、头孢西丁出现了不同程度的耐药。耐药菌株均出现在ERIC-PCR方法分型A型和血清分型O3型中。结论研究显示ERIC-PCR方法可以用于该菌分型分析,具有较好的分型能力。血清分型与ERIC-PCR方法分型一致。通过ERIC-PCR分型的树状图和血清分型结果推断,血清型O3群菌株很可能起源于血清型O1群菌株,血清型O3群和O1群密切相关。     

大肠埃希氏菌的分型方法及其研究进展

大肠埃希氏菌是一种条件性致病菌,致病性的大肠埃希氏菌具有高度的传染性,会严重危害健康。快速准确地测定大肠埃希氏菌的污染来源对有效缩小疫情影响范围极有帮助,从而避免对人类健康和经济贸易造成重大损失。建立简便高效的分型方法是微生物溯源的关键,常见的大肠埃希氏菌分型方法可分为表型分型和分子分型,这些分型方法各有优劣,具有不同的适用范围。本文详细介绍了大肠埃希氏菌的分型方法,并对国内外大肠埃希氏菌分型的研究进展进行综述,为致病菌溯源方法的选择提供参考依据,对防御并控制致病菌引起的流行病传播具有重要的意义。     

2016-2019年辽宁省肠炎沙门菌分离株分子分型及其耐药性

目的 分析辽宁省肠炎沙门菌分离株的分子分型特征及耐药情况,为辽宁省肠炎沙门菌的分子流行病学及防控措施提供参考依据。 方法 采用PFGE分子分型方法对辽宁省2016-2019年肠炎沙门菌分离株进行分子分型,应用BioNumerics 7.6软件对酶切片段进行聚类分析,明确菌株的特征及同源性;采用最低抑菌浓度(MIC)法测定菌株对14种药物敏感性。 结果 共获得49株肠炎沙门菌,分子分型结果证明其呈17种PFGE带型,相似度区间为77.4%～100.0%,有2种优势带型;对萘啶酸的耐药率最高,达89.80%,其次氨苄西林的耐药率为69.39%,对3种以上抗生素的耐药率为55.10%。 结论 辽宁省肠炎沙门菌PFGE分子分型具有独特的优势带型,存在带型较多的特点;肠炎沙门菌分离株多重耐药状况比较严重,对萘啶酸的耐药率最高。