Anti-biofilm and anti-virulence potential of cell free supernatant of Akkermansia muciniphila against Salmonella

Akkermansia muciniphila is one of the commensals residing within the mammalian gut and co-evolving with the host.Numerous studies have demonstrated the benefits of A.muciniphila in ameliorating metabolic disorders,while little is known about the antimicrobial potential of A.muciniphila against pathogens.Here,we examined the antimicrobial and anti-virulence properties of cell free supernatant(CFS)of A.muciniphila against Salmonella Typhimurium.CFS retarded bacterial growth and inhibited the motility of S.Typhimurium SL1344 and S.Typhimurium 14028.CFS dose-dependently reduced cell hydrophobicity and auto-aggregation of both strains.Also,CFS from A.muciniphila significantly attenuated biofilm formation.Compared with untreated bacteria,CFS-treated bacteria significantly decreased adhesion and invasion to Caco-2 cells,and reduced intracellular survival in macrophages.CFS maintained antimicrobial properties after treatment with high temperatures and various proteases,while it lost its antimicrobial activity after pH neutralization.Gas chromatography-mass spectrometry(GC-MS)confirmed that A.muciniphila produced a certain amount of acetate and propionate,and ultra-high-performance liquid chromatography-mass spectrometry(UHPLCMS)identified other organic acids and metabolites in CFS.In summary,CFS from A.muciniphila exhibited anti-biofilm and anti-virulence properties against Salmonella and could be potentially utilized in the food industry for controlling Salmonella contamination and reducing infection.

Low interleukin-10 level indicates a good prognosis in Salmonella enterica serovar typhimurium-induced pediatric hemophagocytic lymphohistiocytosis:A case report

BACKGROUND Secondary hemophagocytic lymphohistiocytosis(sHLH)triggered by Salmonella enterica serovar Typhimurium is rare in pediatric patients.There is no consensus on how to treat S.typhimurium-triggered sHLH.CASE SUMMARY A 9-year-old boy with intermittent fever for 3 d presented to our hospital with positive results for S.typhimurium,human rhinovirus,and Mycoplasma pneumoniae infections.At the time of admission to our institution,the patient’s T helper 1/T helper 2 cytokine levels were 326 pg/mL for interleukin 6(IL-6),9.1 pg/mL for IL-10,and 246.7 pg/mL for interferon-gamma(IFN-γ),for which the ratio of IL-10 to IFN-γwas 0.04.In this study,the patient received meropenem,linezolid,and cefoperazone/sulbactam in combination with high-dose methylprednisolone therapy(10 mg/kg/d for 3 d)and antishock supportive treatment twice.After careful evaluation,this patient did not receive HLH chemotherapy and recovered well.CONCLUSION S.Typhimurium infection-triggered sHLH patient had a ratio of IL-10 to IFN-γ≤1.33,an IL-10 concentration≤10.0 pg/mL,and/or an IFN-γconcentration≤225 pg/mL at admission.Early antimicrobial and supportive treatment was sufficient,and the HLH-94/2004 protocol was not necessary under these conditions.

Antibiotic Resistance Profile of Salmonella Strains Isolated at the National Clinical Biology and Public Health Laboratory in Bangui, Central African Republic

In Africa, each year, there are estimated to be more than 91 million cases of salmonellosis and 137,000 cases of death. The problem of antibiotic resistance in Salmonella strains is a threat to public health. The objective of this study is to evaluate the antibiotic resistance profile of Salmonella strains isolated in biological products analyzed at the National Laboratory of Clinical Biology and Public Health (NLCBPH) in Bangui. This is, therefore, a cross-sectional study with a descriptive aim, running from January to December 2022. It focused on the strains of Salmonella isolated and identified in stools, urines, and blood samples. For each strain of Salmonella isolated, an antibiogram was carried out following the recommendations of the French Society of Microbiology (CASFM, 2022). A total of 93 strains of Salmonella have been recorded. The age group 0 - 9 years was 29% and that of >50 years was 11%. The median age of patients was 30 years with a minimum of 1 and a maximum of 78 years. The female gender was more represented at 52.69% than the male gender at 47.31%, i.e. a sex ratio of 0.89 (M/F). Salmonella strains were much more isolated in stools at 62% followed by urines at 29% and blood at 6%. Salmonella arizonae strains were more represented with 52%. Salmonella strains have a resistance rate to Tetracycline of 62.37% followed by Penicillins of 50%. The rate of multi-antibiotic resistance of the Salmonella strains isolated represented 48.38%. Salmonella spp. strains were multi-resistant at 58.69% followed by Salmonella arizonae strains at 47.91%. There is a significant association between the different families of antibiotics and Salmonella strains (p < 0.05). According to the results obtained, Penicillins, Phenicoles, and Cyclins had a high rate of resistance on Salmonella strains. No strain-producing Broad Spectrum Beta-lactamase has been isolated. Salmonella strains represent a zoonotic health danger, constitute a public health problem and remain a current subject. This germ is resistant to the antibiotics used. It is, therefore, essential to emphasize monitoring the resistance of these germs in the Central African Republic (CAR) to improve the health of the population.

Case Report: Bilateral Intra-Parenchymal Hematomas Caused by Ventricular Flood Complicating Neonatal Salmonella typhi Meningitis

Salmonella meningitis is an uncommon condition in neonates, and when it does occur, it is often linked to serious complications, such as subdural collections and abscesses. We present a case involving a 23-day-old neonate diagnosed with Salmonella meningitis, who developed complications including bilateral intra-parenchymal hematomas with ventricular involvement. The infant showed significant improvement following an extended course of systemic antibiotics and supportive care.

Multidrug-Resistant of Escherichia coli and Salmonella spp. Strains in Chicken Feces Intended for Consumption in Open Spaces of Ouagadougou, Burkina Faso

Resistant bacteria can be transmitted to humans through feces or contaminated meat from local chickens. Bacterial strains were isolated from the intestinal contents of 400 local chicken samples from various sales sites. These strains were then characterized using bacteriological and biochemical methods to identify resistant strains. In a study conducted in Ouagadougou, we systematically collected chicken fecal samples from 20 locations across the city, followed by isolation and identification of Salmonella spp. using specific enrichment and culture methods, as well as Escherichia coli. Bacterial strains were characterized using antibiotic resistance profiles were determined through agar diffusion tests, revealing sensitivity or resistance to a range of antibiotics based on established scientific criteria. The results showed that out of the 400 samples collected, 81.25% and 63.5% were contaminated by Escherichia coli and Salmonella spp., respectively. Among these, 86.15% of identified Escherichia coli and 50.78% of Salmonella spp. displayed resistance to at least one tested antibiotic. Among 280 Escherichia coli isolates identified resistant to at least one antibiotic, 31.07% were resistant to cefotaxime (CTX), 20.35% to ceftazidime (CAZ), 21.07% to ceftriaxone (CTR), 75% to amoxicillin clavulanic acid (AMC), 23.57% aztreoname (ATM) and 27.14% were resistant to imipenem (IMP). In the case of the 129 Salmonella spp. isolates resistant to at least one tested antibiotic, 34.88% were resistant to CTX;41.08% to CAZ;35.65% to CTR, 92% to AMC, 39.53% to ATM and finally 47.28% were resistant to IMP. Our study revealed high prevalence of resistance in bacterial strains isolated from local chickens sold outdoors in Ouagadougou. These findings raise significant public health concerns, due to the possible transmission of these resistant strains to humans through the consumption of contaminated meat, thus complicating the treatment of bacterial infections.

Salmonella hadar对喹诺酮类药物耐药性及其耐药基因分析

目的:研究新疆乌鲁木齐市部分农贸市场内检出的21株Salmonella hadar对2种喹诺酮类抗生素的药敏性及相关耐药基因,更好地了解耐药性的产生和传播途径,确保食品安全。方法:用琼脂稀释法测定Salmonella hadar的药敏性,用聚合酶链式反应和测定基因序列的方法确定耐药沙门氏菌中与喹诺酮类抗生素耐药性相关的喹诺酮类抗性决定区突变基因以及质粒携带的耐药基因。结果:21株Salmonella hadar对萘啶酮酸的耐药率达100%,对环丙沙星表现为敏感;qnrB、qnrA、qnrS基因的检出率分别为52.30%、4.76%、4.76%;21株Salmonella hadar均为gyrA和parC基因同时突变,gyrA基因的突变类型是Ser83Phe,parC基因的突变类型是Thr57Ser。结论:新疆乌鲁木齐Salmonella hadar对喹诺酮类药物的耐药状况应当予以关注,其耐药决定区突变基因及质粒携带的耐药基因在一定程度上会影响Salmonella hadar的耐药机制。

Survey of Multidrug Resistance and Class I Integron Prevalence in Chicken-derived Salmonella

[Objective] This study aimed to investigate the multidrug resistance and prevalence of class I integrons in Salmonel a. [Method] Salmonel a strains were isolated from chicken farms in Shandong Province. Kauffmann-White classification method was employed to analyze the serotypes of Salmonel a strains. Minimum in-hibition concentration （MIC） of Salmonel a strains against 19 common antimicrobial drugs was analyzed determined with microdilution method. The class I integrons and carried drug resistance gene cassettes were detected by PCR. [Result] A total of 311 Salmonel a strains were isolated and classified into two serotypes, including 133 Salmonel a Indiana strains and 178 Salmonel a Enteritidis strains. Drug sensitivity test showed that the isolated Salmonel a strains were general y resistant to sulfadiazine, sulfamethoxazole, nalidixic acid, ampicil in, tetracycline, doxycycline and trimethoprim, with a multidrug resistance rate of 91.0% （283/311）; 99% strains were sensitive to amikacin and colistin. PCR assay indicated that the detection rate of class I integrons was 65.0% （202/311）; the positive rate of class I integrons in Salmonel a strains with multidrug resistance was 92.6%; among 202 positive strains, six strains carried gene cassette dfr17-aadA5. [Conclusion] According to the above results, class I integrons exist general y in Salmonel a and are closely associated with the multidrug resistance of Salmonel a strains.

抗Salmonella minnesota R_(595)菌脂多糖杂交瘤的建立及其抗体特性的初步鉴定

将煮沸致死的R595菌体免疫BALB/C小鼠,取其脾细胞在PEG介导下与SP2/0细胞融合,经筛选获得2个阳性孔2D6、3H4。将2D6、3H4阳性孔细胞克隆,传代最终获得8株能稳定分泌抗R595内毒素单抗的杂交瘤株。经鉴定,杂交瘤细胞染色体数90余条,抗体效价（EL1SA法）104～105,2D6B1,2D6E7单抗的Ig类型为IgG1,3H42F7、3H42H3单抗Ig类型为IgG2b,轻链均为K链。类脂A抑制试验显示,单抗识别的表位为类脂A或KDO,表明抗体识别内毒素保守区域。间接EL1SA试验表明,抗体能与多种GNB交叉反应,提示抗体具广谱交叉反应性。玻片凝集试验显示,抗体仅能凝集R595菌,不能凝集其它GNB菌。

生姜中Salmonella enterica的分离与宿主氧化应答

本研究对重庆荣昌采后发病姜块进行病原菌分离鉴定,分离到一株新的生姜致病菌。通过细菌形态、生理生化、16S r DNA遗传分析鉴定为沙门氏菌(Salmonella enterica)。将沙门氏菌回接姜苗、姜块,发现姜苗叶片失绿,褶皱;姜块表面水渍并有分泌物;动态污染研究表明:沙门氏菌能在姜块中迅速繁殖,显著提高姜块超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)、过氧化氢酶(catalase,CAT)的活性。该研究表明,沙门氏菌能以生姜为寄主存活。

Isolation and Drug Resistance Analysis of Swine Salmonella

In this study, 68 fresh pork samples were collected from several major wholesale markets in Dalian City of Liaoning Province to isolate Salmonella using SS agar medium as selective medium. Salmonella isolates were identified with colony morphology observation and PCR method. The resistance of Salmonella iso- lates to 15 antibiotics was analyzed by broth microdilution susceptibility test. Accord- ing to the result, 12 Salmonella strains were isolated from 68 fresh pork samples, indicating a detection rate of 17.64%; 83.33% of Salmonella strains isolated from fresh pork samples could at least resist one antibiotic; Salmonella strains exhibited the strongest resistance to florfenicol （75.0%）, sarafloxacin hydrochloride （66.7%）, neomycin sulfate （66.7%） and doxycycline hydrochloride （66.7%）, followed by oxyte- tracycline （58.3%）, gentamicin sulphate （58.3%） and mequindox （58.3%）; Salmonella strains exhibited relatively low resistance to ciprofloxacin lactate （50.0%）, en- rofloxacin （50.0%）, ciprofloxacin hydrochloride （50.0%）, amoxicillin （16.7%） and col- istin sulphate （16.7%）. Salmonella in fresh pork samples from wholesale markets in Dalian Economic ＆ Technological Development Zone exhibited a high detection rate, and the isolated Salmonella strains showed extremely high resistance to various an- tibiotics. The strong drug-resistance of Salmonella can also be transmitted through the food chain from the food to the people, which should attract sufficient attention.

1990—2019年中国三种肠道传染病发病和死亡趋势分析及预测研究

背景肠道传染病是常见的传染性疾病之一,分析和预测其流行现状能够为肠道传染病的防治提供一定的参考。目的了解1990—2019年中国腹泻病、伤寒与副伤寒和侵袭性非伤寒沙门菌肠道感染3种肠道传染病的发病和死亡情况,并预测2020—2030年其发病率和死亡率,为肠道传染病的防控提供参考。方法基于2019全球疾病负担研究数据库(GBD),收集1990—2019年中国腹泻病、伤寒与副伤寒和侵袭性非伤寒沙门菌肠道感染3种肠道传染病的发病和死亡数据,根据变化率(%)和年估计百分比(EAPC)分析以上3种肠道感染疾病的变化趋势。利用自回归移动平均模型(ARIMA)预测2020—2030年中国以上3种肠道传染病的发病率和死亡率。结果1990—2019年腹泻病的发病率变化无统计学意义(EAPC=0.09,P>0.05),而伤寒与副伤寒和侵袭性非伤寒沙门菌肠道感染的发病率均呈下降趋势(EAPC分别为-4.0%、-0.64%,P<0.05)。1990—2019年腹泻病、伤寒与副伤寒和侵袭性非伤寒沙门菌肠道感染的死亡率均呈下降趋势(EAPC分别为-8.39%、-3.38%、-1.87%,P<0.05)。在各年龄组中,2019年≥70岁人群腹泻病的发病率在各年龄组中最高,且呈上升趋势(EAPC=0.27,P<0.05)。1990—2019年所有年龄组以上3种肠道传染病的死亡率均呈下降趋势(P<0.05)。ARIMA模型预测结果显示,2020—2030年我国腹泻病发病率呈上升趋势,伤寒与副伤寒和侵袭性非伤寒沙门菌的发病率呈下降趋势,预计以上3种疾病的发病率分别为58793.04/10万、5.26/10万、0.447/10万。此外,2020—2030年我国腹泻病、伤寒与副伤寒和侵袭性非伤寒沙门菌的死亡率均呈下降趋势,预计2030年以上3种疾病的死亡率分别为0.214/10万、0.039/10万、0.026/10万。结论2030年我国腹泻病、伤寒与副伤寒和侵袭性非伤寒沙门菌肠道感染的死亡率呈下降趋势;除腹泻病的发病率呈上升趋势外,其余两种疾病的发病率呈下降趋势,提示政府及相关卫生部门应当重视关注腹泻病,并针对不同人群采取不同防控措施。

Enrichment-ELISA for Detection of Salmonella typhi From Food and Water Samples

Objective Development of monoclonal antibody based sandwich enzyme linked immunosorbant assay （sELISA） for rapid detection of Salmonella enterica serovar typhi （S. typhi） from food and water samples and optimization of enrichment procedures for use with the developed sELISA to increase the detection sensitivity of the assay. Methods Spleen cells from BALB/c mice immunized with flagellin （H=d） antigen of S. typhi were fused with Sp2/0 myeloma cells. The hybridoma cell line specific to H=d antigen was established, characterized and ascites raised against one of these clones. The hyperimmune serum to flagellin antigen was raised in New Zealand White rabbits. An sELISA was developed using polyclonal antibody as capture and monoclonal antibody as detection antibody. To design the efficient culture strategies for use with the sELISA, different pre-enrichment and enrichment broths were evaluated. The media included buffered peptone water （BPW） and brain heart infusion broth for pre-enrichment and selenite F broth and Rappaport-Vassiliadis broth as enrichment broths. The developed sELISA with preceding enrichment step in BPW （Enrichment-ELISA） was evaluated in various food samples artificially inoculated with S. typhi bacteria. Various food （30） and water （35） samples collected from field were also tested by Enrichment-ELISA and culture method. Results Out of four specific clones to H=d antigen, one clone （# 2/56, IgG2a isotype） was used in sELISA. The sELISA had the detection limit of 10^4-10^5 cfu of S. typhi. Of the various broths used with sELISA, BPW was found to yield maximum ELISA values. Enrichment-ELISA, when tested in artificially inoculated food samples, generally, could detect 10^2 S. typhi cfu/mL within 10 h from various food rinses （meat, vegetable） and milk samples. After overnight enrichment in BPW, as less as 2 bacteria per 10 mL of milk, meat rinse, and chicken rinse could be detected. Only one of the field samples （water） gave false positive result by Enrichment-ELISA. Conclusion In comparison to culture, the Enrichment-ELISA is a rapid, sensitive, and specific method for detection of S. typhi from food or water samples. This method may be used as rapid screening procedure for environmental monitoring during outbreak situation.

Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass

Chicken is one of the most popular meat products in the world. Salmonella Typhimurium is a common foodbome pathogens associated with the processing of poultry. An optical Surface Plasmon Resonance （SPR） biosensor was sensitive to the presence of Salmonella Typhimurium in chicken carcass. The Spreeta biosensor kits were used to detect Salmonella Typhimurium on chicken carcass successfully. A taste sensor like electronic tongue or biosensors was used to basically ＂taste＂ the object and differentiated one object from the other with different taste sensor signatures. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with dif- ferent antisera or other molecular species. The selectivity of the SPR biosensor was assayed using a series of antibody con- centrations and dilution series of the organism. The SPR biosensor showed promising to detect the existence of Salmonella Typhimurium at 1 x 106 CFU/ml. Initial results show that the SPR biosensor has the potential for its application in pathogenic bacteria monitoring. However, more tests need to be done to confirm the detection limitation.

Prevalence and antibiogram study of Salmonella and Staphylococcus aureus in poultry meat

Objective:To evaluate the presence and antibiogram pattern of Salmonella and Staphylococcus aureus(S.aureus)in retail poultry meat products.Methods:Foodborne pathogens(Salmonella and S.aureus)were isolated from poultry meat and confirmed with the help of biochemical and immunological test.Antibiogram of the isolates were examined by following CLS1 methods.Results:A total number of 209 poultry meat samples were collected and studied in this study.Out of which,5.26%were found contaminated with Salmonella while 18.18%were found contaminated with S.aureus.All the Salmonella and S.aureus isolates were found resistant to at least one antibiotic.About 72.72%of the Salmonella isolates showed resistance to tetracycline,while S.aureus isolates were also found highly resistant to tetracycline equal to 44.73%.One of the Salmonella isolates showed multi-drug resistance to almost six antibiotics out of nine antibiotics used in the study.Multidrug resistant S.aureus isolates were also found in the study.Conclusions:The study confirmed the presence of Salmonella and S.aureus in retail poultry meat.It is a potential threat to consumer health.To reduce the risk of contamination,good hygiene practices are necessary from processing to storage.

Supplemental magnolol or honokiol attenuates adverse effects in broilers infected with Salmonella pullorum by modulating mucosal gene expression and the gut microbiota

Background:Salmonella pullorum is one of the most harmful pathogens to avian species.Magnolol and honokiol,natural compounds extracted from Magnolia officinalis,exerts anti-inflammatory,anti-oxidant and antibacterial activities.This study was conducted to evaluate the effects of dietary supplemental magnolol and honokiol in broilers infected with S.pullorum.A total of 360 one-day-old broilers were selected and randomly divided into four groups with six replicates:the negative control group(CTL),S.pullorum-infected group(SP),and the S.pulloruminfected group supplemented with 300 mg/kg honokiol(SPH)or magnolol(SPM).Results:The results showed that challenging with S.pullorum impaired growth performance in broilers,as indicated by the observed decreases in body weight(P<0.05)and average daily gains(P<0.05),along with increased spleen(P<0.01)and bursa of Fabricus weights(P<0.05),serum globulin contents,and the decreased intestine villus height and villus/crypt ratios(P<0.05).Notably,supplemental magnolol and honokiol attenuated these adverse changes,and the effects of magnolol were better than those of honokiol.Therefore,we performed RNA-Seq in ileum tissues and 16S rRNA gene sequencing of ileum bacteria.Our analysis revealed that magnolol increased the α-diversity(observed species,Chao1,ACE,and PD whole tree)and β-diversity of the ileum bacteria(P<0.05).In addition,magnolol supplementation increased the abundance of Lactobacillus(P<0.01)and decreased unidentified Cyanobacteria(P<0.05)both at d 14 and d 21.Further study confirmed that differentially expressed genes induced by magnolol and honokiol supplementation enriched in cytokine-cytokine receptor interactions,in the intestinal immune network for IgA production,and in the cell adhesion molecule pathways.Conclusions:Supplemental magnolol and honokiol alleviated S.pullorum-induced impairments in growth performance,and the effect of magnolol was better than that of honokiol,which could be partially due to magnolol’s ability to improve the intestinal microbial and mucosal barrier.

;~lasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

Persistent activation of Survivin and its overexpression contribute to the formation, progression and metastasis of several different tumor types. Therefore, Survivin is an ideal target for RNA interference mediated-growth inhibition. Blockade of Survivin using specific short hairpin RNAs （shRNA） can significantly reduce prostate tumor growth. RNA interference does not fully ablate target gene expression, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Survivin-specific shRNA, we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 （GRIM-19）. Then, the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium （S. typhimurium） to treat prostate cancer in vitro and in vivo. We found that the co-expressed Survivin-specific shRNA and GRIM-19 synergistically and more effectively inhibited prostate tumor proliferation and survival, when compared with treatment with either single agent alone in vitroand in vivo. This study has provided a novel cancer gene therapeutic approach for prostate cancer.

Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2

AIM： To construct a recombinant live attenuated Salmonella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS： Hpylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction （PCR） and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. co/i DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicib/of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 10^8 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 10^7 CFU of live Hpylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylon 4 wk post-challenge. RESULTS： The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistent with the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that （100%） in the control group （P 〈 0.01）. CONCLUSION： Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.

Comparative analysis of intestinal microbial community diversity between healthy and orally infected ducklings with Salmonella enteritidis by ERIC-PCR

AIM: To analyze the difference of intestinal microbial community diversity between healthy and (S. enteritidis) orally infected ducklings.METHODS: Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was applied to analyze the intestinal microbial community diversity and dynamic change including duodenum, jejunum, ileum, cecum and rectum from healthy ducklings and 7-day-old ducklings after oral infection with S. enteritidis at different time points.RESULTS: The intestinal microbial community of the control healthy ducklings was steady and the ERIC-PCR band numbers of the control healthy ducklings were the least with rectum and were the most with caecum. ERIC-PCR bands of orally inoculated ducklings did not obviously change until 24 h after inoculation (p.i.). The numbers of the ERIC-PCR bands gradually decreased from 24 h to 72 h p.i., and then, with the development of disease, the band numbers gradually increased until 6 d p.i. The prominent bacteria changed because of S. enteritidis infection and the DNAstar of staple of ERIC-PCR showed that aerobe and facultative aerobe (Escherichia coli, Shigella, Salmonella) became preponderant bacilli in the intestine of orally infected ducklings with SE.CONCLUSION: This study has provided significant data to clarify the intestinal microbial community diversity and dynamic change of healthy and S. enteritidis orally infected ducklings, and valuable insight into the pathogenesis of S. enteritidis infection in both human and animals.

Oral Immunization of Mice With Vaccine of Attenuated Salmonella typhimurium Expressing Helicobacter pylori Urease B Subunit

Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine against H. pylori infection. Methods Using genomic DNA of H. pylori Sydney strain （SS1） as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LBS000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups （including body weight, gastric inflammation, etc.） were also collected. Results The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H, pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-T and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. Conclusion The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Salmonella typhi and gallbladder cancer:report from an endemic region

BACKGROUND:Evidence exists of a link between chronic infection by Salmonella typhi(S.typhi) and the development of gallbladder cancer(GBC),but several studies from endemic regions contradict its role in the etiopathogenesis of GBC.This study used various tools to assess the prevalence of S.typhi in patients with GBC and gallstone disease(GSD) in this region with a high incidence of GBC.METHODS:S.typhi was detected in tissue and bile by PCR and culture and in serum by the Widal test and indirect hemagglutination assay(IHA).PCR with two pairs of S.typhi specific primers(flagellin gene H1d and SOP E gene) could detect 0.6 ng of S.typhi DNA.Fifty-four patients with GBC(cases) were matched with 54 patients with GSD(controls).RESULTS:Of the 54 cases,24(44.44%) were positive on the Widal test and 12(22.22%) on IHA,compared to 13(24.07%) and 5(9.26%) respectively in the controls.Eighteen(33.33%) cases showed a positive result on PCR(tissue) and 2 on PCR(bile) vs.none in the controls.Bile culture revealed no Salmonella colonies in either cases or controls.Only 3 cases were positive for Salmonella on tissue culture compared to none in the controls.The sensitivity of PCR(tissue) relative to the Widal test,IHA,culture(bile and tissue) and PCR(bile) was 100% vs.66.67%,11.11%,and 11.11%,and the specificity was 83.33% vs.100%,100%,and 100%,respectively.CONCLUSIONS:S.typhi is significantly associated with GBC compared to GSD(33% vs.0%).PCR appears to be the most specific diagnostic tool,the gold standard for S.typhi in tissue samples.