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Journal of Microbiological Methods Feb 2019Vibrio cholerae strains producing cholera toxin (CT) and toxin co-regulated pilus (TCP) and belonging to O1 and O139 serogroups are responsible for cholera. However,...
Vibrio cholerae strains producing cholera toxin (CT) and toxin co-regulated pilus (TCP) and belonging to O1 and O139 serogroups are responsible for cholera. However, non-CT producing V. cholerae from non-O1/non-O139 serogroups have been increasingly isolated from diarrheal stools and extra-intestinal infections. In this study, we have developed a multiplex PCR for the simultaneous detection of heat-stable enterotoxin (stn), type three-secretion system (vopF), and cholix toxin (chxA), along with CT (ctx) in V. cholerae strains. As other species from genus Vibrio carries homologous virulence genes, V. cholerae specific ompW was also included to differentiate V. cholerae from other vibrios. This assay was 100% specific and sensitive, and could detect homologous virulence genes like ctxA in V. mimicus and vopF in V. parahaemolyticus. This multiplex PCR assay, which can detect four major virulence genes in V. cholerae, is novel and important for epidemiologic and environmental surveillance of pathogenic V. cholerae.
Topics: Bacterial Outer Membrane Proteins; Cholera Toxin; Enterotoxins; Environmental Monitoring; Fimbriae, Bacterial; Humans; Multiplex Polymerase Chain Reaction; Type III Secretion Systems; Vibrio cholerae; Virulence Factors
PubMed: 30576750
DOI: 10.1016/j.mimet.2018.12.012 -
Applied and Environmental Microbiology Feb 2019Atypical El Tor strains of O1 harboring variant genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. occasionally produces CT,...
Atypical El Tor strains of O1 harboring variant genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. occasionally produces CT, encoded by on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in -positive () strains (i.e., strains containing ) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTX) carrying a novel , tandem copies of environmental type pre-CTX prophage (pre-CTX), and RS1 elements, which were organized as an RS1-CTX-RS1-pre-CTX-pre-CTX array. Additionally, novel variants of and , respectively, showing phylogenetic lineage to a clade of non-O1 and to a clade of non-O139, were identified. The strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in promoter region similar to those seen with some classical strains of O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the strains were clonally related. However, their CT production and toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of hybrid strains. Natural diversification of CTXФ and genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of This report, showing the occurrence of CTX comprising a novel variant of in , points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of Identification and cluster analysis of the newly discovered alleles of and suggest their horizontal transfer from an uncommon clone of The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФ and of a CTXФ in probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.
Topics: Bacterial Proteins; Cholera; Cholera Toxin; Environmental Microbiology; Evolution, Molecular; Genetic Variation; Humans; Phylogeny; Regulon; Vibrio cholerae O1; Vibrio mimicus; Virulence Factors
PubMed: 30446560
DOI: 10.1128/AEM.01977-18 -
Letters in Applied Microbiology Aug 2018The Rio Bravo (Rio Grande) adjoins various states in the Mexican region and has a great importance in water distribution in the northeast Tamaulipas (Mexico). In this...
UNLABELLED
The Rio Bravo (Rio Grande) adjoins various states in the Mexican region and has a great importance in water distribution in the northeast Tamaulipas (Mexico). In this work 161 strains were isolated, identified and characterized from the water samples taken from the flow of the Rio Bravo and the two inner canals that cover Reynosa city. The strains were identified as Vibrio cholerae (74·5%), Vibrio spp. (1·2%) and Vibrio mimicus (0·6%). Furthermore, the detected virulence genes in the V. cholerae strains, were the hlyA, ompU, tcpA, toxR genes in 78·3, 62·5, 15·8 and 90·8% respectively. Only the ompU and vmh genes were detected in the V. mimicus strain. These results indicate the presence of multi-toxigenic V. cholerae strains in the Rio Bravo/Grande and in the water bodies from Reynosa city, which could represent a risk for the exposed population.
SIGNIFICANCE AND IMPACT OF THE STUDY
Water quality is associated with public health, as it plays an important role in the transmission and epidemiology of pathogens such as Vibrio, since this species have been responsible for human diseases around the world. This study demonstrated the presence of toxigenic Vibrio species in water bodies in Reynosa surroundings, indicating that water bodies may be a source of public health risk.
Topics: Adhesins, Bacterial; Bacterial Proteins; DNA-Binding Proteins; Fimbriae Proteins; Hemolysin Proteins; Humans; Mexico; Nitriles; Rivers; Serogroup; Transcription Factors; Vibrio cholerae; Virulence; Water Microbiology
PubMed: 29791974
DOI: 10.1111/lam.13009 -
Microbial Pathogenesis Jun 2018In this study, we evaluated the virulence factor production, biofilm-forming ability and cell surface properties of ready-to-eat shrimps associated vibrios strains. A...
In this study, we evaluated the virulence factor production, biofilm-forming ability and cell surface properties of ready-to-eat shrimps associated vibrios strains. A total of 1440 ready-to-eat (RTE) shrimp samples were purchased from open markets in southern Nigeria, from November 2016 to October 2017. Biofilm formation was carried out using the microtitre plate method. Cell-to-cell adhesion of Vibrio species was assessed via surface hydrophobicity using the bacterial adherence to hydrocarbons (BATH) and salting aggregation technique, autoaggregation and coaggregation assay. The virulence potential of the identified 120 Vibrio strains includes haemolysis 107 (89.17%), lipase 106 (88.33%), protease 108 (90%), gelatinase 111 (92.5%), the presence of surface-layer (S-layer) 109 (90.8%) and DNA degrading activity 107 (89.17%). Biofilm formation at 30 °C tryptone soy broth in dynamic conditions revealed total biofilm producers for the Vibrio species as follows: V. parahaemolyticus (95.65%), V. vulnificus (92.86%), V. fluvialis (91.67%), V. alginolyticus (87.5%), V. cholerae (100%), V. mimicus (90%), V. harveyi (66.7%), and other Vibrio spp. (84%). A total of 50 biofilm producing vibrios using BATH technique include 49 (98%) hydrophilic and 1 (2%) moderately hydrophobic. Using the modified salting aggregation technique, 50 (100%) was characterized as hydrophilic. Autoaggregation index for the 12 biofilms producing Vibrio strains ranged from 11.6 to 41.3%, while the autoaggregation index for the 12 test bacteria ranged from 26.2 to 71.3%. Coaggregation between the 12 test bacteria with the 12 Vibrio strains ranged from 9.3 to 78.5%. However most vibrios in this study were hydrophilic, their hydrophilic potential is important for their capability to autoaggregate and coaggregate. Findings on the specific process by which virulent Vibrio spp. form biofilm and adhere to shrimp surface as attached plankton may assist in monitoring epidemics of the pathogen.
Topics: Animals; Bacterial Adhesion; Bacterial Typing Techniques; Biofilms; DNA, Bacterial; Hydrophobic and Hydrophilic Interactions; Nigeria; Penaeidae; Seafood; Shellfish; Species Specificity; Vibrio; Virulence; Virulence Factors
PubMed: 29654902
DOI: 10.1016/j.micpath.2018.04.015 -
Environmental Monitoring and Assessment Feb 2018Diarrhoea remains a global public health enigma raising deep concerns for the health planners since contaminated potable water often spoils the community health...
Diarrhoea remains a global public health enigma raising deep concerns for the health planners since contaminated potable water often spoils the community health structure. We hereby report a 6-year odd continuing outbreak surveillance report based on potable water indices, during which 264 water samples were screened from different districts of West Bengal, India. Samples were analysed for the presence of different enteropathogenic bacterial species by conventional molecular tools and their sensitivity to antibiotics. 78.03% samples were positive for enteropathogenic bacterial organisms and 75% samples harbored Coliform. 45.45, 12.12, and 4.16% samples were positive for E.coli, V. cholerae, and V. mimicus, respectively. Diarrhoeagenic E.coli 7 EPEC, 10 ETEC, and 2 EIEC were isolated along with 2 V. cholerae O1 Ogawa (ctxA and tcpA ElTor positive), one each from tube well and pond. Interestingly, 4 V.cholerae non-O1/non-O139 also harbored hlyA gene. The detection of toxin genes among this bacterial pool of sampled water indicates the fallout of the potable water sources, thus enabling us to establish that it is none other than the contaminated potable water system which often wreaks havoc in the south Bengal diarrhoeal menace. The consequences are further complicated by the presence of drug-resistant pathogenic bacterial pool to fluoroquinolone, beta-lactams, and cephalosporins, in the accessible potable water, with threats of outbreaks exploding into an epidemic, given suitable environment, poor sanitation, and unhygienic practices. Therefore, we strongly recommend re-modelling of 'point-of-use water disinfection' measures and adequate personal hygiene for healthier community life.
Topics: Anti-Bacterial Agents; Cholera; Diarrhea; Disease Outbreaks; Environmental Exposure; Environmental Monitoring; Escherichia coli; Humans; India; Vibrio cholerae; Water Microbiology; Water Pollution
PubMed: 29478177
DOI: 10.1007/s10661-017-6424-2 -
International Journal of Food... Jan 2018The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods....
The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 to 10CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively.
Topics: DNA Primers; Denaturing Gradient Gel Electrophoresis; Food Microbiology; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; RNA-Directed DNA Polymerase; Reverse Transcriptase Polymerase Chain Reaction; Vibrio alginolyticus; Vibrio cholerae; Vibrio mimicus; Vibrio parahaemolyticus
PubMed: 29111407
DOI: 10.1016/j.ijfoodmicro.2017.10.014 -
International Journal of Biological... Feb 2018In this study, we reported a jellyfish-derived Kazal-type serine protease inhibitor, named CcKPI1, from Cyanea capillata. CcKPI1 has a calculated molecular mass of...
In this study, we reported a jellyfish-derived Kazal-type serine protease inhibitor, named CcKPI1, from Cyanea capillata. CcKPI1 has a calculated molecular mass of 19.02kDa and contains three typical Kazal domains. Soluble recombinant CcKPI1 (rCcKPI1) was successfully expressed and purified. rCcKPI1 exhibited significant inhibitory activities against elastase, subtilisin A and proteinase K, but not against trypsin or chymotrypsin. Kinetic studies showed that all of the inhibitory effects of rCcKPI1 were competitive, indicating that it may be a microbial serine protease inhibitor and can exhibit antimicrobial activity. As predicted, rCcKPI1 directly bound to various microorganisms, including the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram-negative bacteria Escherichia coli, marine pathogenic vibrios Vibrio vulnificus, Vibrio cholerae, Vibrio natriegens, Vibrio mimicus, Vibrio alginolyticus and Vibrio parahaemolyticus, and fungi Candida albicans, Candida parapsilokis and Candida glabrata. In addition, rCcKPI1 inhibited the growth of most of the tested microorganisms that it bound to. These findings indicate that CcKPI1 possesses marked antibacterial and antifungal activities and may play an important role in the immune defence of C. capillata, providing a novel view for the understanding of the immune system of jellyfish and also facilitating future research on antimicrobial agents from marine natural products.
Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Bacteria; Base Sequence; DNA, Complementary; Gene Expression Profiling; Kinetics; Microbial Sensitivity Tests; Phylogeny; Recombinant Proteins; Scyphozoa; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Serine Proteinase Inhibitors
PubMed: 29054522
DOI: 10.1016/j.ijbiomac.2017.10.069 -
Journal of Fish Diseases Dec 2017During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential...
During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential pathogenic and zoonotic risks caused by 53 Vibrio cholerae non-O1/non-O139 and a Vibrio mimicus strain isolated from ornamental fish species mostly originating from South-East Asia countries between 2000 and 2015 in Italy. All the strains were firstly identified at species level by biochemical, phylogenetic and mass spectrometry (matrix-assisted laser desorption ionization time of flight) methods, and then studied to reveal the presence of the main virulence and colonization-associated factors, as ctxA, ace, zot, stn/sto, toxR, rtxA, hlyA and tcpA by multiplex and single endpoint PCR assays. Findings showed that 21 of 54 strains harboured at least one virulence factor with a predominance for the toxR , rtxA and hlyAET genotype. Interestingly, the V. mimicus strain harboured the colonization factor and the CTX prophage receptor, tcpA, indicating the ability to capture and integrate it in its genome increasing its pathogenicity. Although these enterotoxins can sporadically cause gastroenteritis, the results highlight their probable involvement in causing severe implications for public health, suggesting the need for an European microbiological monitoring.
Topics: Animals; Fish Diseases; Fishes; Italy; Vibrio cholerae non-O1; Vibrio mimicus; Virulence Factors; Zoonoses
PubMed: 28677232
DOI: 10.1111/jfd.12659 -
Biomedicines Jul 2016is a member of the Arecaceae family and a common plant in the Southeast Asian region. This plant has been reported as an anti-microbial agent in recent years. Thus, we...
BACKGROUND
is a member of the Arecaceae family and a common plant in the Southeast Asian region. This plant has been reported as an anti-microbial agent in recent years. Thus, we aimed to find out the MIC (minimum inhibitory concentration) against different pathogenic microorganism.
METHODS
The leaves of were extracted and fractioned using different reagents (chloroform, -hexane and carbon tetrachloride). Disc diffusion method was implemented for the assessment of in vitro anti-microbial potency (500 and 250 µg/disc).
RESULT
The entire fraction showed good effect (with the zone of inhibition 19-25 mm) against both gram positive (, , , ) and gram negative (, , , ) bacterial pathogens and fungal strains (, ). The plants also possess effective free radical scavenging potency with an IC of 130.32 µg/mL.
CONCLUSION
This finding reflects a link between the presence of anti-oxidative material and a substantial anti-microbial activity, and substantiates all previous claims against .
PubMed: 28536384
DOI: 10.3390/biomedicines4030017 -
Antonie Van Leeuwenhoek Jun 2017The Vibrionaceae are Gram-negative bacteria present in marine and estuarine environments worldwide, including several species known as important pathogens to humans and...
The Vibrionaceae are Gram-negative bacteria present in marine and estuarine environments worldwide, including several species known as important pathogens to humans and aquatic organisms. The aim of this research was to investigate the occurrence and virulence properties of Vibrio and Salinivibrio isolated from lagoons at Cuare Wildlife Refuge and Margarita Island in the southern Caribbean Sea. Water, plankton and oyster samples were collected during October 2011 and March 2012 and examined by specific PCR and culture methods. Vibrio genus DNA was detected in 95% of samples, while the intergenic spacer region (ISR) of Vibrio cholerae and the genes that code for the thermolabile direct haemolysin (tl) of Vibrio parahaemolyticus and the haemolysin/cytolysin (vvhA) of Vibrio vulnificus were absent or amplified in low proportions (23, 5, and 0%, respectively). Nine isolates from water and plankton were confirmed as Vibrio or Salinivibrio by phenotypic tests, 16S rRNA gene sequencing and phylogenetic analysis. All the isolates presented similar patterns of virulence factors, in which the genes ctxA (encoding for cholera toxin), tl and vvhA were lacking, whereas seven isolates displayed antibiotic resistance against ampicillin and cephalosporins. The 16S rRNA phylogenetic analysis showed the clustering of Vibrio isolates in three main clades: the plankton isolate from Cuare Wildlife Refuge formed a group with V. cholerae and Vibrio mimicus while the Margarita isolates clustered with sequences from the harveyi clade and Salinivibrio. This is the first time that Salinivibrio species are reported in tropical lagoons of the Caribbean Sea with antibiotic resistance.
Topics: Animals; Caribbean Region; Humans; Phylogeny; RNA, Ribosomal, 16S; Seawater; Tropical Climate; Vibrio; Virulence; Water Microbiology
PubMed: 28324231
DOI: 10.1007/s10482-017-0856-0