Characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from deep ground water in a cholera endemic area in Central India

A total of 8 out of 11 deep ground water samples collected from different villages in Central India were found contaminated with Vibrio cholerae non O1, non O139. In a multiplex PCR, isolates were found positive for ompW gene but negative for ctxAB and rfbO1 genes. However, isolates from two places were positive for tcp and zot genes, indicating their intestinal colonization and toxigenic potential. Antibiotic susceptibility studies revealed that all isolates were multidrug resistant. Although, none of the isolates was found PCR positive for the mobile genetic elements, class 1 integrons and SXT constins. The results of this study corroborated that deep ground water can also be an important reservoir of V. cholerae in plane endemic areas, suggesting a continuous monitoring of water samples for timely prevention of the disease.     

Virulence regulator AphB enhances <Emphasis Type="Italic">toxR</Emphasis> transcription in <Emphasis Type="Italic">Vibrio cholerae</Emphasis>

Background  

Vibrio cholerae is the causative agent of cholera. Extensive studies reveal that complicated regulatory cascades regulate expression of virulence genes, the products of which are required for V. cholerae to colonize and cause disease. In this study, we investigated the expression of the key virulence regulator ToxR under different conditions.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> isolates from cholera outbreaks in north India

Vibrio cholerae isolates recovered from cholera outbreaks in Bhind district of Madhya Pradesh and Delhi, Northern India were characterized. The O1 serogroup isolates from Bhind outbreak were of Inaba serotype whereas both Ogawa and Inaba serotypes were recovered from Delhi. PCR analysis revealed that only O1 serogroup V. cholerae isolates carried the virulence-associated genes like ctxA, tcpA, ace, and zot. Molecular typing by repetitive sequence based ERIC, VCR1, and VC1 PCR’s revealed similar DNA profile for both Inaba and Ogawa serotypes. A discrete VC1-PCR band identified among the El Tor strains had greater similarity (>97%) to the V. cholerae genome sequence and therefore has the potential to be used as a marker for the identification of the V. cholerae strains. Non-O1 strains recovered from Bhind region differed among themselves as well as from that of the O1 isolates. All the O1 serogroup isolates possessed SXT element and were uniformly resistant to the antibiotics nalidixic acid, polymyxin-B, furazolidone, cloxacilin, trimethoprim-sulfamethaxazole, and vibriostatic agent 0129. Inaba strains from both Delhi and Bhind differed from Ogawa strains by their resistance to streptomycin despite sharing similar DNA patterns in all the three rep-PCRs. Though Delhi and Bhind are separate geographical regions in Northern India, Inaba strains from both these places appear to be closely related owing to their similarity in antibiogram and genetic profile.     

Survival of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O1 on fomites

It is well established that the contamination sources of cholera causing bacteria, Vibrio cholerae, are water and food, but little is known about the transmission role of the fomites (surfaces that can carry pathogens) commonly used in households. In the absence of appropriate nutrients or growth conditions on fomites, bacteria have been known to assume a viable but non-culturable (VBNC) state after a given period of time. To investigate whether and when V. cholerae O1 assumes such a state, this study investigated the survival and viable quantification on a range of fomites such as paper, wood, glass, plastic, cloth and several types of metals under laboratory conditions. The fomites were inoculated with an outbreak strain of V. cholerae and its culturability was examined by drop plate count method at 30 min intervals for up to 6 h. For molecular detection, the viable/dead stain ethidium monoazide (EMA) which inhibits amplification of DNA from dead cells was used in combination with real-time polymerase chain reaction (EMA-qPCR) for direct quantitative analyses of viable V. cholerae at 2, 4, 6, 24 h and 7 day time intervals. Results showed that V. cholerae on glass and aluminum surfaces lost culturability within one hour after inoculation but remained culturable on cloth and wood for up to four hours. VBNC V. cholerae on dry fomite surfaces was detected and quantified by EMA-qPCR even 7 days after inoculation. In conclusion, the prolonged survival of V. cholerae on various household fomites may play vital role in cholera transmission and needs to be further investigated.     

Antimicrobial susceptibility and molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.     

Arctic Actinomycetes as Potential Inhibitors of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Biofilm

The aim of this study was to identify novel biofilm inhibitors from actinomycetes isolated from the Arctic against Vibrio cholerae, the causative agent of cholera. The biofilm inhibitory activity of actinomycetes was assessed using biofilm assay and was confirmed using air–liquid interphase coverslip assay. The potential isolates were identified using 16S rRNA gene sequencing. Of all, three isolates showed significant biofilm inhibition against V. cholerae. The results showed that 20% of the actinomycetes culture supernatant could inhibit up to 80% of the biofilm formation. When different extracted fractions were assessed, significant biofilm inhibition activity was only seen in the diethyl ether fraction of A745. At 200 μg ml⁻¹ of diethyl ether fraction, 60% inhibition of V. cholerae biofilm was observed. The two potential isolates were found to be Streptomyces sp. and one isolate belonged to Nocardiopsis sp. This is the first report showing a Streptomyces sp. and Nocardiopsis sp. isolated from the Arctic having a biofilm inhibitory activity against V. cholerae. The spread of drug resistant V. cholerae strains is a major clinical problem and the ineffectiveness in antibiotic treatment necessitates finding new modes of prevention and containment of the disease, cholera. The formation of biofilms during the proliferation of V. cholerae is linked to its pathogenesis. Hence, the bioactive compound from the culture supernatant of the isolates identified in this study may be a promising source for the development of a potential quorum sensing inhibitors against V. cholerae.     

In silico prediction of drug targets in <Emphasis Type="Italic">Vibrio cholerae</Emphasis>

Identification of potential drug targets is the first step in the process of modern drug discovery, subjected to their validation and drug development. Whole genome sequences of a number of organisms allow prediction of potential drug targets using sequence comparison approaches. Here, we present a subtractive approach exploiting the knowledge of global gene expression along with sequence comparisons to predict the potential drug targets more efficiently. Based on the knowledge of 155 known virulence and their coexpressed genes mined from microarray database in the public domain, 357 coexpressed probable virulence genes for Vibrio cholerae were predicted. Based on screening of Database of Essential Genes using blastn, a total of 102 genes out of these 357 were enlisted as vitally essential genes, and hence good putative drug targets. As the effective drug target is a protein which is only present in the pathogen, similarity search of these 102 essential genes against human genome sequence led to subtraction of 66 genes, thus leaving behind a subset of 36 genes whose products have been called as potential drug targets. The gene ontology analysis using Blast2GO of these 36 genes revealed their roles in important metabolic pathways of V. cholerae or on the surface of the pathogen. Thus, we propose that the products of these genes be evaluated as target sites of drugs against V. cholerae in future investigations.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Semi-nested polymerase chain reaction for detection of toxigenic <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10³ CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10⁴ CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

The sodium cycle in <Emphasis Type="Italic">Vibrio cholerae</Emphasis>: Riddles in the dark

Twenty years ago, V. P. Skulachev put forward the revolutionary concept of the chemiosmotic sodium cycle which is an integral part of the paradigm of modern bioenergetics. This fundamental concept stimulated studies in many areas and yielded plenty of sometimes quite unexpected (and thus most valuable) discoveries. In particular, variations of the sodium cycle have been found in a surprisingly large number of pathogenic microorganisms, raising the question about the possible link of sodium energetics and virulence. This brief review discusses some paradoxes related to the Na⁺ cycle in an important human pathogen, Vibrio cholerae.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 185–190.Original Russian Text Copyright © 2005 by Dibrov.This revised version was published online in April 2005 with corrections to the post codes.     

<Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and<Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>: Short generation-time marine bacteria

The growth rates of 30 different strains ofVibrio parahaemolyticus andVibrio alginolyticus at 37°C was determined. Each species consists of two major groups, one having a short generation time (12–14 min) and one with a longer generation time (20–25 min). The diversity in generation times of different strains belonging to the same species is discussed. The effect of temperature, salt, and nutrient concentrations on the growth rate of oneV. alginolyticus strain (NCMB 1803) was studied. The most striking is the effect of the temperature; at 39°C the generation time is 10–11 min, while at 21°C it is 60 min. The heat of activation for growth calculating from such data is 22,580 kcal/mole/hr⁻¹. The ecological significance of these results is discussed.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Characterization and Virulence of Hemolysin III from <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the word. A gene (hlyIII) encoding a hemolysin was cloned and sequenced from V. vulnificus. Nucleotide sequence analysis predicted an open reading frame of 642 bp encoding a 214 amino acid polypeptide that showed 48% sequence identity to the hemolysin III of Bacillus cereus. When HlyIII of V. vulnificus was expressed in Escherichia coli, crude extracts exhibited hemolytic activity similar to that of hemolysin III from Bacillus cereus. A hlyIII isogenic mutant was constructed via insertional inactivation and showed an attenuated virulence compared with the wild-type strain when this mutant was administered intraperitoneally in mice.     

Natural transformation of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> as a tool - Optimizing the procedure

Background  

Vibrio cholerae gains natural competence upon growth on chitin. This allows the organism to take up free DNA from the environment and to incorporate it into its genome by homologous recombination.