Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR

Objective This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase(DGC)in Vibrio cholerae and how its transcription is regulated by Fur and Hap R.Methods The roles of VCA0560 was investigated by utilizing various phenotypic assays,including colony morphological characterization,crystal violet staining,Cyclic di-GMP(c-di-GMP)quantification,and swimming motility assay.The regulation of the VCA0560 gene by Fur and Hap R was analyzed by luminescence assay,electrophoretic mobility shift assay,and DNase I footprinting.Results VCA0560 gene mutation did not affect biofilm formation,motility,and c-di-GMP synthesis in V.cholerae,and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity.The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator Hap R.Conclusion Overexpressed VCA0560 functions as an active DGC in V.cholerae,and its transcription is repressed by Fur and Hap R.

泥鳅(Misgurnus anguillicaudatus)病原霍乱弧菌(Vibrio cholerae)的表型与分子鉴定

从江苏连云港某养殖场养殖死亡的泥鳅肝脏、血液及腹水中分离出大量优势生长的细菌,人工感染试验证明其对泥鳅具有很强的致病性。采用表观分类学及分子生物学方法,对分离菌进行了形态特征、理化特性、胞外酶活性及溶血活性等生物学性状检验;用PCR方法同时扩增其16SrRNA和gyrB基因,分析了16SrRNA和gyrB两种基因序列的同源性,并构建了系统发生树,通过基因序列分析,比较了两种基因在相似细菌的检测和鉴定能力;基于16SrRNA和gyrB基因的系统发育学分析表明分离菌(LD081008B-1)所扩增的16SrRNA和gyrB基因序列均与GenBank数据库中霍乱弧菌具有较高的相似性,且gyrB基因用于细菌种间鉴定更具优越性;16SrRNA基因序列长度为1446bp(GenBank登录号:GQ205447),gyrB基因序列长度为1207bp(GenBank登录号:GQ205452);根据分离菌的表型特征及分子特征,判定病原菌为弧菌属(VibrioPacini1854)的霍乱弧菌(Vibriocholerae)。胞外酶活性及溶血活性检测表明分离菌均具有蛋白酶、卵磷脂酶、淀粉酶、明胶酶及DNA酶活性,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血。分离菌的耐药谱测定结果显示,除对供试49种抗菌药物中的杆菌肽耐药外,对其它48种药物敏感或高度敏感。

泥鳅(Misgurnus anguillicaudatus)病原霍乱弧菌(Vibrio cholerae)PCR与LAMP检测方法的比较研究

采用分子生物学方法,以霍乱弧菌lolB为靶基因设计特异性引物,进行了霍乱弧菌的PCR和环介导等温核酸扩增(Loop-mediatedisothermal amplification,LAMP)检测技术研究,并对它们的特异性、灵敏性和实际应用进行了比较。结果表明,所建立的PCR检测霍乱弧菌的方法最低检测限为4.0×103CFU/ml;LAMP检测方法在65℃下恒温扩增60min,检测限为4.0×101CFU/ml,反应产物加入荧光染料SYBRGreenI后反应液呈现明显的绿色;以温和气单胞菌、副溶血弧菌、鳗弧菌及美人鱼弧菌为对照菌株,检测结果均为阴性;霍乱弧菌人工染菌的8种水产品进行PCR及LAMP检测,结果均为阳性,而未染菌组均为阴性;PCR及LAMP检测霍乱弧菌的方法均具有灵敏度较高、特异性强等优点,且LAMP检测霍乱弧菌的方法灵敏度是PCR方法的100倍,更适合于养殖现场检测的推广使用。

Plasticity of Regulation of Mannitol Phosphotransferase System Operon by CRP-cAMP Complex in Vibrio cholerae

Objective The complex of the cyclic AMP receptor protein （CRP） and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems （PTS） is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tot biotype. Methods The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. choleroe El Tor. Electrophoretic mobility shift assays （EMSA） and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon intl. Results In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. Conclusion The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. choleroe, indicating an elaborate and subtle CRP activation mechanism.

Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With Flow Cytometry

Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

Structural Variation of the Superintegron in the Toxigenic Vibrio cholerae O1 El Tor

Objective To understand the genetic structures and variations of the superintegron （Sl） in Vibrio cholerae isolated in the seventh cholera pandemic. Methods Polymerase chain reaction scanning and fragment sequencing were used. Sixty toxigenic V. cholerae O1 El Tor strains isolated between 1961 and 2008 were analyzed. Results Some variations were found, including insertions, replacements, and deletions. Most of the deletions were probably the result of recombination between V. cholerae repeat sequences. The majority of the variations clustered together. The Sis of the strains isolated in the 1960s and 1970s showed more diversity, whereas SI cassette variations in strains isolated in the 1990s and after were lower, with -24 kb signature sequence deletion. This indicates the predominant Sl in the host during the epidemic in the 1990s and after. The insertion cassettes suggested the mobilization from the Sls of other V. cholerae serogroups and Vibrio mimicus. Conclusion The study revealed that structural variations of Sis were obvious in the strains isolated in epidemics in different decades, whereas the divergence was based on syntenic structure of Sis in these El Tot strains. Also, the continuing cassette flows in the Sis of the host strains during the seventh cholera pandemics were displayed.

RING-OPENING COPOLYMERIZATION OF DL-LACTIDE AND POLY(ETHYLENE GLYCOL) INITIATED BY LANTHANUM ACETATE AND APPLICATION OF THE COPOLYMER AS MATRIX OF MICROSPHERES CONTAINING VIBRIO CHOLERA ANTIGENS

Poly-dl-lactide-poly(ethylene glycol) (PELA) triblock copolymers were synthesized with lanthanum acetate as the initiator. PELA microspheres with entrapped Vibrio Cholera antigen and outer membrane protein (OMP) were prepared by a double emulsion W/O/W based on solvent extraction methods. The obtained microspheres showed smooth and spherical surface and their size varied between 0.5 and 5.0 mu m, which are suitable for oral targeting delivery system. The distribution tests in rabbits and mice through scanning electronic micrography and fluorescence microscope indicated that microspheres have successfully reached the immunization-related tissues, such as the liver, spleen and intestinal peyer's patches, following oral administration. The PELA microspheres were also evaluated as an efficient antigen delivery system by enhancing a higher protective ratio against live Vibrios Cholera.

Acquisition and dissemination mechanisms of CTXΦ in Vibrio cholerae : New paradigm for dif residents

Vibrio cholerae(V. cholerae) genome is equipped with a number of integrative mobile genetic element(IMGE) like prophages, plasmids, transposons or genomic islands, which provides fitness factors that help the pathogen to survive in changing environmental conditions. Metagenomic analyses of clinical and environmental V. cholerae isolates revealed that dimer resolution sites(dif) harbor several structurally and functionally distinct IMGEs. All IMGEs present in the dif region exploit chromosomally encoded tyrosine recombinases, Xer C and Xer D, for integration. Integration takes place due to site-specific recombination between two specific DNA sequences; chromosomal sequence is called att B and IMGEs sequence is called att P. Different IMGEs present in the att P region have different attP structure but all of them are recognized by Xer C and Xer D enzymes and mediate either reversible or irreversible integration. Cholera toxin phage(CTXΦ), a lysogenic filamentous phage carrying the cholera toxin genes ctx AB, deserves special attention because it provides V. cholerae the crucial toxin and is always present in the dif region of all epidemic cholera isolates. Therefore, understanding the mechanisms of integration and dissemination of CTXΦ, genetic and ecological factors which support CTXΦ integration as well as production of virion from chromosomally integrated phage genome and interactions of CTXΦ with other genetic elements present in the genomes of V. cholerae is important for learning more about the biology of cholera pathogen.

Comparative proteomic analysis of Vibrio cholerae O139, O22, O155 and El Tor biotype epidemic strains

The purposes of this study are to compare the proteome of Vibrio cholerae O139 strains with that of O22, O155 and El Tot biotype epidemic strains, and to identifv whether O139 strains have a close relation with the latter. Proteins of two V. cholerae serogroup O139 strains, two El Tor biotype epidemic strains, one O22 strain, and one O155 strain were separated by two-dimensional gel electrophoresis （2-DE） ; the silver stained 2-DE gels were scanned with （ligital lmageScanner and analyzed with lmageMaster 2D Elite 3.10 software. Similarities of protein maps between these strains were analyzed. The two O139 strains had 86% proteins in common; the two El Tor biotype epidemie strains showed 84% proteins in common; O139 strains shared 67 % , 47 % and 45 % proteins with El Tor biotype epidemic strains, O22 and O 155 strains respectively. Although the proteome of O 139 strains have close similarity to that of El Tor biotype epidemic strains, great disparities exist. O139 strains may not originate from El Tor biotype directly, and a transition strain may exist.

Design of a multiplex PCR method for detection of toxigenicpathogenic in Vibrio cholerae

Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic and pathogenic V.cholera by chain reaction assay method. Results:According to the results of the PCR,the incidence of hlyA,tcpI,and ctxB genes in clinical isolates was obtained as 94.7%(72 sample),90.8%(69 sample),and 92.1%(70 sample), respectively.Five strains possessed all genes except ctxB,six strains possessed all genes except tcpI,four strains possessed all genes except hlyA,one strain possessed only hlyA and 60 strains contained a combination of three genes.Including hlyA,ctxB and tcpI,Conclusions:Result show that this method could be reliable to detect toxigenic-pathogenic strains of V.cholerae in Iran.

Survival and proliferation of the lysogenic bacteriophage CTXΦ in Vibrio cholerae

The lysogenic phage CTXΦ of Vibrio cholerae can transfer the cholera toxin gene both horizontally(inter-strain) and vertically(cell proliferation). Due to its diversity in form and species, the complexity of regulatory mechanisms, and the important role of the infection mechanism in the production of new virulent strains of V.cholerae, the study of the lysogenic phage CTXΦ has attracted much attention. Based on the progress of current research, the genomic features and their arrangement, the host-dependent regulatory mechanisms of CTXΦ phage survival, proliferation and propagation were reviewed to further understand the phage's role in the evolutionary and epidemiological mechanisms of V. cholerae.

Occurrence of <i>Vibrio cholerae</i>in Some Households Engaged in Livestock Farming in Some Parts of Zaria, Nigeria

The study was designed to determine the presence of Vibrio cholerae from the environment in close proximity with livestock in Zaria,Kadunastate. Three hundred and thirty six environmental samples comprising soil, water, manure and vegetables were collected from some selected households inZariacoveringZariacity, Sabo and Samaru from April to October 2006 and analyzed for the presence of Vibrio cholerae, one of the etiologic agent of gastroenteritis. Twelve Vibrios exhibiting characteristics of Vibrio cholerae were identified using biochemical techniques. Serological identification confirmed 5 (41.7%) of these isolates as Vibrio, 1 (20%) as Vibrio serogroup O1, Ogawa biotype. A 0.59% prevalence was obtained for this pathogen in the study. The 0.59% isolation rate though low is significant considering it’s source being animal, since livestock keeping is a common feature in the study location, with animals living in close proximity to man this work is imperative. Animals are a point source of contamination of enteric pathogens, therefore extensive management system and proper treatment of animal manure is recommended before its use as fertilizer.

Isolation, Identification and Antimicrobial Susceptibility Profile Analysis of <i>Vibrio cholerae</i>O1 from Stool Samples of Bangladesh

Cholera is a severe diarrheal disease which is usually caused by toxigenic strain of Vibrio cholerae O1 and O139. Cholera is still one of the major health concerns in developing countries like Bangladesh due to poor sanitation and unavailability of safe drinking water. This experiment was confronted to identify V. cholerae O1 from stool samples as well as to determine the antibiotic susceptibility pattern of the isolated strains. A total of 140 stool samples from people infected with diarrheal disease were collected from July 2016 to December 2016. Among all, 58 samples were found positive for V. cholerae which were further subjected to sero-grouping by specific anti-sera and antimicrobial sus-ceptibility test by Kirby Bauer disc diffusion method. The zones of inhibition were measured and interpreted by following the recommendations of the criteria of Clinical and Laboratory Standards Institute (CLSI). It was found that 43 (74.1%) isolates of V. cholerae were O1 serogroup of Ogawa serotype and the rest 15 (25.9%) were O1 serogroup of Inaba serotype. People aged between 41 - 50 were most susceptible to V. cholerae O1 having about 39.7% of positive cases. The isolates were highly susceptible to Ciprofloxacin and Gentamicin with 100% susceptibility whereas 100% resistant was found towards Nalidixic acid. Though most of the isolates in our study were susceptible against tested antibiotics, the continuous surveillance is required to see the changing pattern of serogroups or serotypes and antimicrobial profile in this region.

霍乱弧菌(Vibrio cholerae)的细胞形态研究——活的非可培养状态细胞

使用透射电镜和萤光抗体染色，对处于可培养和活的非可培养状态的霍乱弧菌进行了细胞形态结构比较研究。结果表明：处于非可培养状态的霍乱菌细胞，体积明显地缩小，形态由弧形变成了球形，我们共观察了３８２个非可培养状态的细胞，发现它们的细胞壁都是完整的，即不是细菌－Ｌ型。

Toxin(s), Other Than Cholera Toxin, Produced by Environmental Non O1 Non O139 Vibrio cholerae

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin （ST）, as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary （CHO） cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin （CT）. Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen （CAMP） method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

Oligomerization of Vibrio cholerae Hemolysin Induces CXCR3 Upregulation and Activation of B-1a Cell

The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation.

A new look at the mechanism of cholera endemicity caused by Vibrio cholerae biotype eltor

Objective Cholera caused by Vibrio cholerae biotype eltor (EVC) is an endemic disease, subsiding in winter and reappearing in spring and summer Investigating the state of EVC during the intermittent time is of crucial importance in controlling this disease Methods Different factors mimicking the internal and external environmental conditions of the host, including human and fish bile, bacterial phages and antibiotics were used experimentally to induce variation in EVC EVC variants were isolated from the stool of diarrhea patients and river water in old endemic areas during the winter The variants obtained were tested with gene probe hybridization, DNA restriction enzyme mapping, immunoenzyme staining and animal passaging Results Due to the loss of cell walls, 3 kinds of EVC variants were obtained during induction: the L form variant, with a complete loss of cell walls; the nonagglutinating variants, with the loss of surface O antigen; the phage resistant variants, with the loss of phage receptors Similar variants were found in field isolation This variation was proved to be phenotypic, with no change in genetic material: it was reversible and appeared in a seasonal pattern, which coincided with the endemicity of this disease Passage in animal enhanced this reversion In compensation for the loss of cell walls, cell membranes were greatly thickened, increasing the ability of the variants to survive during the unfavorable winter conditions Conclusions EVC varied in a seasonal pattern, coincident with the endemicity of this disease The compensatory thickening of the cell membranes protects the EVC variants to survive the winter

On or Off:Life-Changing Decisions Made by Vibrio cholerae Under Stress

Vibrio cholerae,the causative agent of the infectious disease,cholera,is commonly found in brackish waters and infects human hosts via the fecal-oral route.V.cholerae is a master of stress resistance as V.cholerae’s dynamic lifestyle across different physical environments constantly exposes it to diverse stressful circumstances.Specifically,V.cholerae has dedicated genetic regulatory networks to sense different environmental cues and respond to these signals.With frequent outbreaks costing a tremendous amount of lives and increased global water temperatures providing more suitable aquatic habitats for V.cholerae,cholera pandemics remain a probable catastrophic threat to humanity.Understanding how V.cholerae copes with different environmental stresses broadens our repertoire of measures against infectious diseases and expands our general knowledge of prokaryotic stress responses.In this review,we summarize the regulatory mechanisms of how V.cholerae fights against stresses in vivo and in vitro.

一株克氏原螯虾病原——霍乱弧菌的分离鉴定

从尾扇边缘溃烂的克氏原螯虾中分离到一株优势菌LK-18,并对该菌株进行种属鉴定和致病性分析。通过菌落形态观察、16S rRNA测序、血清型分析及基因特性鉴定对细菌的类别进行初步判断,并采用浸泡感染、肌肉注射和腹腔注射等方式进行人工感染实验对细菌的致病性进行评估。经16S rRNA测序分析和4个管家基因(atp A、pyr H、rec A、gyr B)串联分析,该菌株最终被鉴定并命名为VibriocholeraeLK-18。V. choleraeLK-18经PCR鉴定不属于O1和O139血清型,但含hly毒力基因和几丁质分解相关的几丁质酶(chitinase,Chi)基因。人工感染实验虽未能重现烂尾症状,但从克氏原螯虾烂尾组织中分离的V. choleraeLK-18具有致病性,能够引起克氏原螯虾的死亡。研究结果表明霍乱弧菌LK-18是一种致病菌,不仅扩大了非O1/O139群霍乱弧菌的感染宿主范围,同时预警霍乱弧菌有导致克氏原螯虾养殖业暴发疾病的可能,因此要提前做好该疾病的预防工作,从而避免霍乱弧菌感染带来的损失。