Viable but non-culturable Vibrio cholerae O1 revert to a cultivable state in the human intestine

Vibrio cholerae O1 can enter a state in which they remain viable but are non-culturable. Presumably, such bacteria can be pathogenic if they retain the capacity to proliferate in the human intestine following ingestion. Two groups of volunteeers were given inocula containing viable but non-culturable V. cholerae O1 of the attenuated vaccine strain CVD 101 (viable CVD 101 organisms readily colonize the human intestine). Volunteers in one of the two groups excreted viable CVD 101, demonstrating that, in the environment of the human intestine, previously non-culturable vibrios can regain the capacity to multiply. These observations support the proposition that viable but non-culturable bacterial enteropathogens may pose a potential threat to health.     

Cholera toxin gene polymerase chain reaction for detection of non-culturable Vibrio cholerae O1

Cholera enterotoxin is a major antigenic determinant for virulence of Vibrio cholerae O1 which can enter into a viable but non-culturable (N-C) state, not detectable by conventional culture methods, yet remain capable of producing enterotoxin and potentially pathogenic. PCR was applied in the current study to detect the chilera toxin (ctx) gene of N-C cells, thus eliminating the necessity of culture. Sets of oligonucleotide primers were designed, based on the ctxAB operon of V. cholerae O1, to detect the presence of the ctx gene. DNA from both culturable and N-C cells of V. cholerae O1 was amplified by PCR using sets of primers flanking 302-, 564- and 777-bp fragments of the ctx gene. The PCR method employed was capable of detecting the ctx gene in N-C V. cholerae in aquatic microcosms and in diarrheal stool samples from three patients who had distinct clinical symptoms of cholera but were culture-negative for V. cholerae O1 and non-O1 and enterotoxigenic Escherichia coli. Forty cycles of a two-step reaction (30 s each at 94 and 60°C) were optimal and more time efficient than a three-step PCR described previously. The procedure, from the point of heating microcosms or broth culture samples to observation on gels, requires < 4 h to complete.J.A.K. Hasan, A. Huq, M.A.R. Chowdhury, and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD, USA. M. Shahabuddin is with the National Institute of Health. Bethesda, MD, USA. L. Loomis is with New Horizons Diagnostics Corporation, Columbia, MD, USA.     

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.     

Molecular phylogenetic analysis ofVibrio cholerae O1 El Tor strains isolated before, during and after the O139 outbreak based on the intergenomic heterogeneity of the 16S-23S rRNA intergenic spacer regions

We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eightrrn operons (rrna-rrnh) ofVibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O139 outbreak. ISR classes ‘a’ and ‘g’ were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O139 serogroup and post-O139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.     

Short communication: Detection of non-culturable Vibrio cholerae O139, by PCR and fluorescent antibody methods,in laboratory microcosms

Non-culturable Vibrio cholerae O139 was detected in microcosms by PCR and fluorescent-antibody (FA) techniques. When survival of V. cholerae O139 in microcosms was assessed by viable counting on culture media, the vibrio became non-culturable after 44 days and remained non-culturable for an additional 7 weeks.     

Synthesis of the conjugation ready, downstream disaccharide fragment of the O-PS of Vibrio cholerae O:139

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-β-d-galactopyranosyluronate-β-d-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-β-d-glucopyranoside. The hydroxyl group present at C-4^II in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-β-d-galactopyranosyluronate)-β-d-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

霍乱弧菌的致病性与流行性研究进展

综述了近年来霍乱弧菌的致病性和流行性的研究进展,并讨论了流行性和致病性的关系.编码霍乱毒素CT的基因ctxAB并不是霍乱弧菌基因组自身的成分,而是CTXΦ噬菌体基因组的一部分.CTXΦ能特异性地感染并整合至霍乱弧菌的基因组,成为CTX原噬菌体;携带有CTX原噬菌体的霍乱弧菌在环境因素的诱导下,也能向外界分泌CTXΦ噬菌体.RS1基因元件与CTX基因元件在染色体位置上紧邻,它们在功能上也紧密相关,RS1元件能利用CTXΦ的基因形成一个丝状噬菌体RS1Φ,并能向细胞外分泌,进行水平传播;VPI来源于VPIΦ,VPIΦ能在霍乱弧菌的菌株之间传播;VPI是霍乱弧菌获得CTX基因元件的桥梁.最近,又发现了两个与霍乱大流行相关的两个基因区域,VSP-Ⅰ和VSP-Ⅱ.     

Response and tolerance of toxigenic Vibro cholerae O1 to cold temperatures

Survival and tolerance at cold temperatures, the differentially expressed cellular proteins, and cholera toxin (CTX) production were evaluated in Vibrio cholerae O1. Rapid loss of culturability and change to distinct coccoid morphology occurred when cultures of V. cholerae O1 were exposed to 5°C directly from 35°C. Also, cultures of V. cholerae first exposed to 15°C for 2 h and then maintained at 5°C failed to exhibit an adaptive response, instead a rapid loss of viable plate count was noticed. Results from Western blot experiments revealed the absence of a major cold shock protein, CS7.4. Also, a decreased level of CTX was noticed in V. cholerae O1 cultures exposed to 5 or 15°C after first being exposed to 15°C for 2 h, followed by transfer to 5°C. Reduced expression of CTX at cold temperatures, compared to the cultures maintained at 35°C, may be a result of decreased cellular metabolic activity. When V. cholerae O1 cultures were exposed to 15°C for 2 h, elevated expressions of 8, 26 and 194 kDa, and decreased expression of 28 and 183 kDa proteins occurred. It is suggested that these differentially expressed cold-responsive proteins are involved in regulating culturability and conversion to a coccoid cell morphology in V. cholerae O1.     

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index = 0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.     

霍乱弧菌检测技术研究进展

随着社会的发展,公共卫生及水体质量已得到明显提升,但到目前为止在发展中国家由霍乱疫情导致的死亡率依旧非常高,而霍乱弧菌即为霍乱疫情爆发的病原菌,且霍乱在我国被列为甲类传染病。目前国内外针对霍乱弧菌已经建立了多种有效的检测技术,对控制霍乱暴发作用显著。本文综述了近年来霍乱弧菌检测技术研究新进展,包括微生物学、免疫学、分子生物学等较成熟的检测方法,生物传感器、快速检测技术等新兴检测方法,并总结各种技术优缺点,展望未来霍乱弧菌检测的市场需求。     

霍乱弧菌DsbA蛋白通过增强MSHA的表达促进生物被膜的形成

【目的】阐明霍乱弧菌DsbA蛋白(VcDsbA)在生物被膜形成过程中的作用。【方法】采用Overlapping PCR的方法构建VcdsbA基因敲除质粒p MH524;采用同源重组和基因克隆的方法对霍乱弧菌dsbA(vc0034)基因进行敲除和回补;通过结晶紫染色实验,比较野生株(WT)、dsbA突变株(ΔdsbA)和回补菌株(CΔdsbA)的生物被膜形成差异;用荧光素酶基因作为报告基因分析与生物被膜形成相关的甘露糖敏感血凝素纤毛合成蛋白(Mannose-sensitive hemagglutinin,pili biogenesis protein,MSHA)在霍乱弧菌WT、ΔdsbA和CΔdsbA中表达水平的区别。【结果】成功构建霍乱弧菌dsbA基因缺失突变株和回补株;与WT相比,ΔdsbA生物被膜形成能力显著下降,且msh操纵子的表达水平显著降低。【结论】VcDsbA可能通过影响其它调控因子直接或间接增强霍乱弧菌MSHA的生物合成,从而促进霍乱弧菌生物被膜的形成。本文为进一步研究DsbA在霍乱弧菌生物被膜形成中的调控机制奠定了基础。     

Immunological Biosensor for Detection of Vibrio cholerae O1in Environmental Water Samples

Environmental surveillance for the presence of Vibrio cholerae O1 is of utmost importance for the effective public health protection of cholera. In the present study, an amperometric immunosensor was developed for detection of Vibrio cholerae in environmental samples by using disposable screen-printed electrodes (SPEs). For this purpose, the experiments done include fabrication of SPEs by using carbon ink, electrochemical characterization of electrodes, optimization of dilutions of antibodies and immobilization of antibody. V. cholerae O1 bacteria were spiked in various environmental water samples in known number. The seeded samples were filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly for detection of V. cholerae using the immunosensor. The immunosensor could detect as few as 8 c.f.u./ml in hand-pump water (ground water) and seawater, and 80 c.f.u./ml in sewer water and tap water. The total time taken in this detection assay was 55 min. Thus, the proposed method is simple and can be used for environmental monitoring of V.␣cholerae.     

Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholerae

A PCR assay has been developed based on a lolB (hemM) gene, which was found to be highly conserved among the Vibrio cholerae species but non-conserved among the other enteric bacteria. The lolB PCR detected all O1, O139 and non-O1/non-O139 serogroup and biotypes of V. cholerae. The analytical specificity of this assay was 100% while the analytical sensitivity was 10 pg/microL and 10(3) CFU/mL at DNA and bacterial level respectively. The diagnostic sensitivity and specificity was 98.5% and 100% respectively.     

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Structural organization of the transfer RNA operon I ofVibrio cholerae: Differences between classical and El Tor strains

Nine major transfer RNA (tRNA) gene clusters were analysed in variousVibrio cholerae strains. Of these, only the tRNA operon I was found to differ significantly inV. cholerae classical (sixth pandemic) and El Tor (seventh pandemic) strains. Amongst the sixteen tRNA genes contained in this operon, genes for tRNA Gln3 (CAA) and tRNA Leu6 (CUA) were absent in classical strains as compared to El Tor strains. The observation strongly supported the view that the above two pandemic strains constitute two different clones.     

A naturally occurring point mutation in the 13-mer R repeat affects the <Emphasis Type="Italic">oriC</Emphasis> function of the large chromosome of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O1 classical biotype

The genome of Vibrio cholerae consists of two circular chromosomes of different sizes. Here, a comparative analysis of the replication origins of the large chromosomes (oriCI_VC) of classical and El Tor biotypes of the pathogen is reported. Extensive nucleotide sequence analyses revealed that the oriCI_VC region has six DnaA boxes instead of the five found in Escherichia coli oriC. The additional DnaA box, designated R_V, was unique in V. cholerae as well as in other members of the family Vibrionaceae. However, R_V was not found to be essential for the autonomous replication function of the 307-bp oriCI_VC minimal region. In contrast to El Tor and the recently evolved V. cholerae O139 strains, the oriCI_VC region of the classical biotype showed only a single base transition (TG) in a highly conserved AT-rich 13-mer R repeat region. From the minichromosome copy number and its transformational efficiency analyses, it appears that the single base substitution in the oriCI_VC of the classical biotype has a significant effect on its replication initiation.     

Marine ecosystems and cholera

Historically, most of the major epidemics or outbreaks of cholera around the world have originated in coastal regions. The most dramatic of recent outbreaks of cholera occurred in India and Bangladesh in 1991, followed by an outbreak of cholera after almost a century without cholera in South America in 1991. Both of these recent epidemics were reported first in the coastal regions of India and Peru, respectively. Cholera epidemics are seasonal, occurring during the spring and fall months. Outbreaks of cholera in noncholera epidemic areas have been ascribed to travel and shipping activities, but there is compelling evidence that V. cholerae always is present in the aquatic environment and proliferates under nonepidemic conditions while attached to, or associated with, eucaryotic organisms. It is hypothesized that climate directly influences the incidence and geographic distribution of the cholera bacterium.     

Differentiation among the Vibrio cholerae serotypes O1, O139, O141 and non-O1, non-O139, non-O141 using specific monoclonal antibodies with dot blotting

Seven different monoclonal antibodies (MAbs) specific to only Vibrio cholerae were produced using a combination of five representative serotypes of V. cholerae for immunization. The first three MAbs (VC-93, VC-82 and VC-223) were specific to the V. cholerae serogroup O1 with different avidity for the serotypes O1 Inaba and O1 Ogawa. The fourth and the fifth MAbs were specific to V. cholerae O139 (VC-812) or O141 (VC-191) serogroups, respectively. The sixth MAb (VC-26) bound to all three serogroups of V. cholerae. The seventh MAb (VC-63) bound to all twenty five isolates of V. cholerae used in this study. None of the seven MAbs showed cross-reactivity with other Vibrio spp. or closely-related V. cholerae species, V. mimicus or other gram-negative bacteria. The eighth MAbs (VC-201) specific to almost all Vibrio spp. was also obtained. In dot blotting, these MAbs can be used to detect a diluted pure culture of V. cholerae in solution with a sensitivity range of from 10⁵ to 10⁷ CFU ml^− 1. However, the detection capability could be improved equivalent to that of PCR technique after preincubation of samples in alkaline peptone water (APW). Thus, these MAbs constitute convenient immunological tools that can be used for simple, rapid and simultaneous direct detection and differentiation of the individual serotypes of V. cholerae in complex samples, such as food and infected animals, without the requirement for bacterial isolation or biochemical characterization.