Effect of Campylobacter jejuni extracts and culture supernatants on cell culture

This study reports the effect of culture supernatants and extracts of Campylobacter jejuni isolated in Bangladesh from patients with gastroenteritis, asymptomatic carriers, poultry, and animals on tissue culture system. Isolates from patients produced morphologic changes on HeLa, Vero and Y1 adrenal cell lines. Incorporation of polymyxin B in the culture medium enhanced the effect on cell lines significantly and sonicated extracts of the organisms had an even more significant effect. Almost all sonicated extracts of patients' isolates had effect on HeLa cells. Animal isolates, using identical conditions, had much less effect on cell lines whereas isolates from asymptomatic carriers produced almost no effect. This toxicity of the patients' isolates might indicate some relation to pathogenic strains.     

格林巴利综合征相关及单纯腹泻相关空肠弯曲菌细胞毒作用分析

目的分析格林-巴利综合征(GBS)相关空肠弯曲菌细胞毒素(CDT)作用。方法9株GBS相关空肠弯曲菌以及4株腹泻患者分离菌株的全菌蛋白分别以0.001μg～5μg/mL的浓度作用于HeLa细胞,通过观察细胞形态的改变,获得不同菌株对细胞作用的差异。结果不同菌株间产生细胞毒作用的浓度范围从0.01μg/mL到5μg/mL。结论GBS相关菌株对HeLa细胞的细胞毒素作用与腹泻患者分离株相比没有显著差异。不同空肠弯曲菌菌株对HeLa细胞的细胞毒素作用具有菌株特异性。     

Disruption of colonic barrier function and induction of mediator release by strains of Campylobacter jejuni that invade epithelial cells

AIM： To study the mechanisms by which Campylobacter jejuni （C. jejuni） causes inflammation and diarrhea. In particular, direct interactions with intestinal epithelial cells and effects on barrier function are poorly understood.METHODS： To model the initial pathogenic effects of C. jejuni on intestinal epithelium, polarized human colonic HCA-7 monolayerswere grown on permeabilized filters and infected apically with clinical isolates of C. jejuni. Integrity of the monolayer was monitored by changes in monolayer resistance, release of lactate dehydrogenase, mannitol fluxes and electron microscopy. Invasion of HCA-7 cells was assessed by a modified gentamicin protection assay, translocation by counting colony forming units in the basal chamber, stimulation of mediator release by immunoassays and secretory responses in monolayers stimulated by bradykinin in an Ussing chamber.RESULTS： All strains translocated across monolayers but only a minority invaded HCA-7 cells. Strains that invaded HCA-7 cells destroyed monolayer resistance over 6 h, accompanied by increased release of lactate dehydrogenase, a four-fold increase in permeability to [^3 H] mannitol, and ultrastructural disruption of tight junctions, with rounding and lifting of cells off the filter membrane. Synthesis of interleukin （IL）-8 and prostaglandin E2 was increased with strains that invaded the monolayer but not with those that did not.CONCLUSION： These data demonstrate two distinct effects of C. jejuni on colonic epithelial cells and provide an informative model for further investigation of initial host cell responses to C. jejuni.     

肠侵袭性大肠埃希菌O抗原多糖的致病作用

目的研究肠侵袭性大肠埃希菌(EIEC)O抗原多糖(OPS)的特异性致病作用。方法提取并纯化大肠埃希菌O29——EIEC的脂多糖(LPS)亚单位分子——OPS进行体内外试验，观察其毒性作用并与LPS作对比。结果EIEC OPS可引起Hela细胞病变；致兔回肠袢肠黏膜出血，但不引起肠腔积液；电镜观察OPS致病变的Hela细胞，明显可见细胞的超微病理损害。结论EIEC OPS有特异的致病作用，与其LPS的内毒素作用不同；大肠埃希菌O29(致病菌株)OPS毒性作用比大肠埃希菌HB101(非致病菌株)OPS毒性强烈。     

Biological and genetic characteristics of uropathogenic Escherichia coli strains.

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.     

江苏奶牛空肠弯曲菌和结肠弯曲菌流行状况及耐药性分析

目的了解江苏省奶牛空肠、结肠弯曲菌流行及耐药状况。方法采用多重PCR方法对10个奶牛场的产奶牛、育成牛和饲养环境进行空肠弯曲菌和结肠弯曲菌流行状况调查,采用琼脂扩散法测定分离株的耐药性。结果1531份样品中,119份空肠弯曲菌阳性,平均阳性率7.77%;3份结肠弯曲菌阳性,平均阳性率0.20%;空肠弯曲菌、结肠弯曲菌阳性率最高分别为30.91%、3.57%。在三类样品中,产奶牛空肠弯曲菌、结肠弯曲菌阳性率分别为5.02%、0.32%,育成牛空肠弯曲菌阳性率为8.70%、未检出结肠弯曲菌,环境样品空肠弯曲菌、结肠弯曲菌阳性率为10.28%、0.18%。35株奶牛空肠弯曲菌分离株对8大类21种抗生素高度敏感率的是:阿莫西林100%、阿齐红霉100%、链霉素97.14%、庆大霉素94.29%、红霉素91.43%、头孢噻肟82.86%、克林霉素82.86%;高度耐药率的是:头孢哌酮100%、恩诺沙星100%、环丙沙星97.14%、复方新诺明97.14%、左旋氧氟沙星94.29%、萘啶酸94.29%、头孢拉定94.29%、诺氟沙星91.43%、头孢克罗88.57%。菌株耐药谱显示,35株分离株的耐药主要集中在9耐到12耐,占88.57%,产奶牛分离株的多重耐药性较其它分离株更严重。结论我国奶牛群中空肠弯曲菌、结肠弯曲菌的流行和耐药状况呈现多样化和复杂化,研究结果为正确评价我国奶牛群弯曲菌的流行状况和制定切实可行的防控措施提供科学依据。     

Comparative phylogenomics of the food-borne pathogen Campylobacter jejuni reveals genetic markers predictive of infection source

Campylobacter jejuni is the predominant cause of bacterial gastroenteritis worldwide, but traditional typing methods are unable to discriminate strains from different sources that cause disease in humans. We report the use of genomotyping (whole-genome comparisons of microbes using DNA microarrays) combined with Bayesian-based algorithms to model the phylogeny of this major food-borne pathogen. In this study 111 C. jejuni strains were examined by genomotyping isolates from humans with a spectrum of C. jejuni-associated disease (70 strains), chickens (17 strains), bovines (13 strains), ovines (5 strains), and the environment (6 strains). From these data, the Bayesian phylogeny of the isolates revealed two distinct clades unequivocally supported by Bayesian probabilities (P = 1); a livestock clade comprising 31/35 (88.6%) of the livestock isolates and a "nonlivestock" clade comprising further clades of environmental isolates. Several genes were identified as characteristic of strains in the livestock clade. The most prominent was a cluster of six genes (cj1321 to cj1326) within the flagellin glycosylation locus, which were confirmed by PCR analysis as genetic markers in six additional chicken-associated strains. Surprisingly these studies show that the majority (39/70, 55.7%) of C. jejuni human isolates were found in the nonlivestock clade, suggesting that most C. jejuni infections may be from nonlivestock (and possibly nonagricultural) sources. This study has provided insight into a previously unidentified reservoir of C. jejuni infection that may have implications in disease-control strategies. The comparative phylogenomics approach described provides a robust methodological prototype that should be applicable to other microbes.     

Prevalence of Campylobacter jejuni and enteric bacterial pathogens among hospitalized HIV infected versus non-HIV infected patients with diarrhoea in southern India

A prevalence study on Campylobacter jejuni and other enteric bacterial pathogens was carried out in 200 HIV infected and 200 non-HIV infected subjects with diarrhoeal symptoms at an AIDS Hospital in southern India. Diarrhoeal specimens were inoculated onto standard culture media as well as onto Columbia and Campylobacter blood agar media for C. jejuni isolation. All the C. jejuni isolates were tested for antimicrobial susceptibility using Kirby-Bauer's method. A significant difference in recovery rates was observed between the 2 groups in relation to C. jejuni (p < or = 0.02; 95% CI 5.5 (1-10) and Shigella spp. (p < or = 0.02; 95% CI 6.5 (1-12). 21 isolates of Shigella spp., 16 C. jejuni, 5 Salmonella typhi, 3 Arcobacter spp., 3 Yersinia enterocolitica, and 2 Aeromonas hydrophila were recovered from the HIV infected cases. All the C. jejuni isolates were sensitive to ciprofloxacin whereas 1 strain was resistant to nalidixic acid. Interestingly, all the 29 Shigella spp. (21 from HIV and 8 from non-HIV cases) were resistant to erythromycin and most were resistant to many other antibiotics used. Our observations underline the need for epidemiological investigations to screen C. jejuni and Shigella spp. in HIV infected subjects with diarrhoea and analyse their antibiograms periodically to minimize disease burden in HIV/AIDS.     

Enteropathogenicity and enteropathogenic toxin production of Vibrio mimicus]

Enteropathogenicity and enteroreactive-toxins were examined in 66 strains of Vibrio mimicus and the following results were obtained. Frequencies of enteropathogenic strains judged by the result of suckling mouse tests were 11/13 (85%) for clinical isolates and 37/53 (70%) for fish or environmental isolates. Frequencies of preservation of cholera toxin gene and NAG-ST gene were 2 and 15%, respectively, for 48 enteropathogenic strains, and 0 and 6%, respectively, for 18 non-enteropathogenic strains. Frequencies of production of NAG-rTDH, FAF and hemolysin were 4, 63 and 100%, respectively, for 48 enteropathogenic strains, and 6, 50, and 100%, respectively, for 18 non-enteropathogenic strains. No correlation between serovar and enteropathogenicity was observed in the suckling mouse test. Six out of 12 enteropathogenic strains produced hemolysin in ligated rabbit ileal loop, while 1 out of 12 non-enteropathogenic strains did so under the same condition. A significant inhibition of fluid accumulation in the ligated rabbit ileal loop test with viable cells was noted in rabbits immunized with hemolysin of non-O1 V. cholerae. These results suggest that approximately two-thirds of environmental isolates are enteropathogenic and that hemolysin is the most important toxin in the enteropathogenic mechanism of V. mimicus strains.     

Campylobacter jejuni as a cause of acute diarrhoea in children: a study at an urban hospital in Bangladesh

The importance of C. jejuni as an aetiological agent of childhood diarrhoea was investigated at an urban children's hospital in Dhaka over a period of 1 year. C. jejuni was isolated from 25.5% of 102 diarrhoeal patients compared to 8.6% of 93 age and sex-matched healthy control children studied (P less than 0.002). The organism was isolated as a single pathogen in 17.6% of diarrhoeal patients. No C. coli was detected. The infection rate was significantly higher (P less than 0.05) amongst children up to 1 year of age (32.8%) compared to those aged over 1 year (15.9%). The clinical features of the majority of Campylobacter-positive cases resembled toxin-mediated secretory type diarrhoea. A fourfold rise of antibody titre against autologous Campylobacter strains was observed in the convalescent sera of Campylobacter-positive cases. The findings strongly suggest that C. jejuni is an important aetiological agent of childhood diarrhoea amongst Bengali children and therefore should be looked for in diarrhoeal illness.     

Susceptibility of Campylobacter isolates to the bactericidal activity of human serum

Although Campylobacter jejuni and related thermophilic organisms are more common human pathogens than are Campylobacter fetus, most bloodstream or systemic isolates are C. fetus. To understand the pathophysiology related to this observation, the authors studied susceptibility to the bactericidal activity of normal human serum of Campylobacter coli, C. jejuni, and C. fetus isolates from feces and blood. In standardized assays, 10 of 15 C. jejuni and related isolates showed 90% kill (mean, 90.6% +/- 5.9); under more stringent conditions, the relatively resistant strains were completely killed. In contrast, all C. fetus strains were highly serum resistant under both standard and stringent conditions. Killing of C. jejuni was ablated by heating serum to 56 C but restored by addition of complement. Both classical and alternative complement pathways may contribute to killing, and adsorption studies demonstrated antibody dependence. Serum resistance may permit systemic infection by C. fetus, whereas complement- and antibody-mediated serum sensitivity of C. jejuni may account for the relative infrequency of systemic invasion.     

Effects of Helicobacter pylori on endothelial cell proliferation and chemotaxis

BACKGROUND/AIM: Helicobacter pylori is associated with an increased risk of peptic ulcer disease development and recurrence. Ulcer healing is dependent upon angiogenesis, requiring endothelial cell (EC) proliferation and chemotaxis. This study determined whether extracts of H. pylori affected EC proliferation and/or chemotaxis in vitro. METHODS: The effects of water and broth extracts of three genotypically different strains of H. pylori and of single strains of Campylobacter jejuni and Escherichia coli on human dermal microvascular EC (HuDMEC) and human umbilical vein EC (HUVEC) were assessed. Tetrazolium dye (MTT) proliferation, dual staining cell viability, flow cytometry, and microchemotaxis assays were performed. RESULTS: H. pylori (all strains) and C. jejuni inhibited HuDMEC (p < 0.01) and HUVEC (p < 0.01) proliferation and decreased the proportion of HUVECs in the S phase of the cell cycle. E. coli had no effect on EC proliferation. The levels of vascular endothelial growth factor stimulated chemotaxis were significantly greater (p < 0.01) than the levels of basal migration for both control and extract-treated ECs. However, none of the bacterial extracts affected EC basal migration or chemotaxis. CONCLUSION: H. pylori extracts inhibit HuDMEC and HUVEC proliferation in vitro by a cytostatic, strain-independent mechanism. A similar antiproliferative effect of C. jejuni was observed. Our findings suggest that both H. pylori and C. jejuni have the ability to inhibit one of the key stages of angiogenesis which may have implications in peptic ulcer disease.     

福氏志贺菌临床分离株毒力基因的研究

目的研究在一种快速、特异的mPCR方法在检出福氏志贺菌的同时对其毒力进行监测。方法选取福氏志贺菌三种毒力基因ipaH,ial,set1B作为扩增目标,在同一mPCR体系中对86株福氏志贺菌临床分离株进行了检测,并选取12株进行了噬斑形成试验,观察了扩增产物不同的福氏志贺菌对Hela细胞的毒力作用。结果86株福氏志贺菌临床分离株均检测到ipaH基因,阳性率为100%;45株检测到ial基因,阳性率为52%;69株检测到set1B基因,阳性率为80%。噬斑形成试验证明,mPCR结果不同的菌株对Hela细胞的感染能力存在明显差异。结论应用上述mPCR体系在检出福氏志贺菌的同时能够对菌株的毒力进行初步判别,对于临床诊断及治疗具有重要意义。     

Invasion-related antigens of Campylobacter jejuni

A HEp-2 cell culture model was used to investigate the antigens required for epithelial cell penetration by Campylobacter jejuni. Penetration of HEp-2 epithelial cells by C. jejuni was significantly inhibited (P less than .05) with C. jejuni lysate and a monoclonal antibody (MAb 1B4) in competitive inhibition assays. Immunogold electron microscopy revealed that MAb 1B4 bound to the flagella and cell surface of low-passage (invasive) C. jejuni M 96, whereas only the flagella of high-passage (noninvasive) C. jejuni M 96 were labeled. Western blot analysis revealed that MAb 1B4 identified an epitope on antigens of 64-44 kDa in lysates prepared from invasive and noninvasive isolates. In addition, antigens of 42-38 kDa were recognized in lysates prepared from only invasive C. jejuni strains. Proteinase K and sodium meta-periodate chemical treatment of C. jejuni M 96 lysate changed the mobility of antigens recognized by MAb 1B4. The increase in mobility demonstrated a decrease in size of molecules and suggested that antigens required for HEp-2 cell invasion by Campylobacter species may be glycoprotein in nature.     

Evaluation of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the identification of Campylobacter strains isolated from diarrheal patients in Japan

We have developed a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection and identification of Campylobacter jejuni, C. coli, and C. fetus. The applicability of this assay was evaluated with 325 Campylobacter strains isolated from diarrheal patients in Japan and the results were compared with those obtained by other genetic methods, including hipO gene detection and 16S rRNA gene sequencing. Of the 325 strains analyzed, 314 and 11 were identified as C. jejuni and C. coli, respectively, by combination of hipO gene detection and 16S rRNA gene sequencing. When the multiplex PCR assay was employed, 309, 310, and 314 strains were identified as C. jejuni on the basis of cdtA, cdtB, and cdtC gene-specific primers, respectively. Similarly, 11, 11, and 10 strains were identified as C. coli on the basis of cdtA, cdtB, and cdtC gene-specific primers, respectively. Sequence analysis of the cdt gene region of 6 strains (5 C. jejuni and 1 C. coli) which did not yield specific PCR products in any of the cdt gene-based multiplex PCR assays revealed deletions or mutations of the cdt genes. Pulsed-field gel electrophoresis indicated that C. jejuni and C. coli strains were genetically diverse. Taken together, these findings suggest that the cdtC gene-based multiplex PCR seems to be a particularly simple and rapid method for differentiating between species of Campylobacter strains, such as C. jejuni and C. coli. However, combination of these multiplex PCR assays will allow more accurate identification.     

Persistent Campylobacter jejuni infections in patients infected with the human immunodeficiency virus (HIV)

We identified Campylobacter jejuni infections in four patients infected with the human immunodeficiency virus (HIV); three had persistent and severe C. jejuni infections. Multiple isolates obtained from each patient had the same biochemical and serotypic characteristics, indicating recurrent infection rather than reinfection with unrelated strains. Serum antibody responses to C. jejuni group antigens by enzyme-linked immunosorbent assay were markedly impaired in the three patients with persistent infection compared with forty-two immunocompetent C. jejuni-infected controls and with the HIV-infected patient who readily cleared the organism. One patient was bacteremic; his blood isolate was killed by normal serum but was resistant to his own serum, whereas a simultaneous stool isolate of a different serotype was sensitive. Failure of two patients to eradicate the organism and long-term administration of erythromycin therapy led to the in-vivo development of resistance to this antibiotic, which is most frequently used to treat C. jejuni infections.     

Pathogenic effects of Opolysaccharide from Shigella flexneri strain

SubjectheadingsShigellaflexneristrain;bacterialantigen;O--POlysaccharide,virulenceINTRODUCTIONShigellaflexneriisoneofthepathogenswhictcausesdiarrheaanditspathogenicmechanismissill:nuclear,eventhoughextensiveinvestigationswercarriedoutworldwide.IthasbeenknownthatthinvasiveoutermembraneproteinencodedbyitfplasmidDNAplaysaprimaryroleinbacteria;infection.Therolesofotherfactors,especiall}lipopolysaccharides(LPS),werealsobettelunderstood.ThespecificpathogenesisofOpolysaccharide(O--PS)which…     

Identification and characterization of a major subgroup of conjugative Campylobacter jejuni plasmids

OBJECTIVES: Enterocyte invasion of Campylobacter jejuni 81-176 has been reported to depend upon the virulence plasmid pVir. The objective of this study was to determine the prevalence of pVir in clinical C. jejuni isolates, to investigate DNA homologies between C. jejuni plasmids and the significance of plasmids for C. jejuni invasiveness. METHODS: DNA homologies between C. jejuni plasmids were studied by southern blot hybridization. C. jejuni invasion into human intestinal Caco-2 cells was assessed in a gentamicin exclusion assay. RESULTS: Twenty-nine percent of C. jejuni isolated from patients with bloody or watery diarrhoea harboured plasmids of various sizes. One plasmid (7%) was a pVir homologue whereas, the majority of the plasmids (53%) belonged to a subgroup distinct from pVir. The plasmids of this novel subgroup share extensive DNA sequence homology with each other, including homologues to so-called invasion-promoting genes. However, conjugative transfer of these plasmids clearly did not increase invasiveness of plasmidless recipient C. jejuni strains. CONCLUSION: This study indicates that only a small proportion of C. jejuni strains carry the virulence factor pVir and that at least one other distinctive group of plasmids in C. jejuni exists, which does not seem to be associated with invasiveness.     

猪源弯曲菌的分离鉴定及耐药性分析

目的 为调查猪肠道内空肠弯曲菌和结肠弯曲菌的流行和耐药性状况。方法 本试验自北京郊区养猪场和屠宰场采集猪肠道粪便样品,经增菌培养后利用三重PCR进行弯曲菌属、空肠弯曲菌和结肠弯曲菌的检测,进而对PCR检测阳性样品进行弯曲菌的分离鉴定,最后对分离鉴定的弯曲菌分离株进行药敏试验鉴定。结果 本试验共采集猪肠道和肛门棉拭子样品241份,PCR检测结肠弯曲菌阳性样品数为178份,阳性率为73.9%,而空肠弯曲菌阳性样品数5份,阳性率仅为2.1%。经分离鉴定后共获得结肠弯曲菌菌株63株,分离率为35.4%,未分离获得空肠弯曲菌菌株。对26种抗生素的药敏试验结果显示结肠弯曲菌分离株对氯霉素(100%)、丁胺卡那(93.65%)、强力霉素(91.48%)、庆大霉素(82.54%)和阿莫西林(82.54%)等抗生素比较敏感,而对头孢哌酮(100%)、 头孢氨苄(100%)、头孢拉啶(100%)、头孢唑啉(98.41%)、头孢克肟(92.06%)、萘啶酸(92.06%)、链霉素(90.48%)、环丙沙星(90.32%)、诺氟沙星(88.89%)和青霉素(87.30%)具有显著的耐药性;而且分离株多重耐药现象比较普遍,主要集中于14-20耐。结论 本试验结果显示猪肠道内普遍存在结肠弯曲菌,而且菌株对多种抗生素产生了显著的耐药性。     

乳胶颗粒凝集试验(LPAT)检测国内产毒性大肠杆菌

本文报告了用乳胶凝集试验(LPAT),检测国内各地送检的132株大肠杆菌,检出与核实ETEC69株(52％)。本文对在我国使用的多种检测LT的方法进行了比较研究,进一步考核LPAT的效果。发现鼠肠法误诊率达30％,因此仅可作为初筛ETEC的方法,而不宜作为肯定报告的依据。也发现Biken试验阴性而LPAT阳性的菌株,并用平板免疫溶血方法予以证实。因此再一次证明了LPAT是一个简单、可靠、灵敏、快速的方法,可用于临床实验室检测大肠杆菌的肠毒素。