Sabu Thomas

Inhibition of Vibrio cholerae biofilm by AiiA enzyme produced from Bacillus spp.

Vibrio cholerae is the causative agent of water-borne diarrheal disease, cholera. The formation of biofilm favors survival and persistence of V. cholerae in the aquatic environment and also inside the host. AHL lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus spp., blocks quorum sensing in Gram-negative bacteria by hydrolyzing N-acyl-homoserine lactones (AHLs). In the present investigation, AiiA-mediated inhibition of V. cholerae biofilm was studied. Two novel alleles of aiiA-encoding genes from Bacillus spp. were expressed in E. coli, and the results demonstrated that AiiA enzyme is a potent inhibitor of V. cholerae biofilm.

Resveratrol--a potential inhibitor of biofilm formation in Vibrio cholerae.

Resveratrol, a phytochemical commonly found in the skin of grapes and berries, was tested for its biofilm inhibitory activity against Vibrio cholerae. Biofilm inhibition was assessed using crystal violet assay. MTT assay was performed to check the viability of the treated bacterial cells and the biofilm architecture was analysed using confocal laser scanning microscopy. The possible target of the compound was determined by docking analysis. Results showed that subinhibitory concentrations of the compound could significantly inhibit biofilm formation in V. cholerae in a concentration-dependent manner. AphB was found to be the putative target of resveratrol using docking analysis. The results generated in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae and can be used as a novel therapeutic agent against cholera. To our knowledge, this is the first report of resveratrol showing antibiofilm activity against V. cholerae.

Phenotypic and molecular identification of Cellulosimicrobium cellulans isolated from Antarctic snow

We report for the first time the isolation of Cellulosimicrobium cellulans from Antarctic snow. This strain demonstrated physiological traits that were markedly different from that of the mesophilic C. cellulans type strain DSM 43879T. The dominant cell wall sugars in C. cellulans were glucose, galactose and mannitol whereas rhamnose was the only major sugar in the type strain. Cellular fatty acid patterns were dominated by 12-methyltetradecanoic acid (ai-C15:0), hexadecanoic acid (C16:0) and 14-methylhexadecanoic acid (ai-C17:0) but lacked iso fatty acids unlike the type strain. The ability of C. cellulans to survive in Antarctic snow could be due to these modified physiological properties that distinguish it from its mesophilic counterpart. Carbon utilization studies demonstrated that C. cellulans preferred complex carbon substrates over simple ones suggesting that it could play a potential role in carbon uptake in snow. Our study shows that this genus could be more cosmopolitan than hitherto thought of and is capable of living in extreme cold environments.

Halophilic and halotolerant actinomycetes from a marine saltern of Goa, India producing anti-bacterial metabolites.

Marine salterns are estuarine ecosystems in Goa, receiving inputs from riverine and marine waters. The Salinity fluctuates between 0 and 300 psu which makes it a conducive niche for salt tolerant and salt loving Actinomycetales. Halotolerant and halophilic Actinomycetales producing anti-bacterial metabolites were studied from crystallizer pond sediments of Ribandar saltern, Goa. Three media viz. Starch casein, R2A and Inorganic salt starch agar at four different salinities (35, 50, 75 and 100 psu) were used for isolation. R2A agar at 35 psu was the most preferred by hypersaline actinomycetes. The dominant group was halotolerant Streptomyces spp. others being rare actinomycetes viz. Nocardiopsis, Micromonospora and Kocuria spp. More than 50% of the isolates showed anti-bacterial activity against one or more of the fifteen human pathogens tested. Eight strains from 4 genera showed consistent anti-bacterial activity and studied in detail. Most halotolerant isolates grew from 0 to 75 psu, with optimum antibiotic production at 35 psu whereas halophiles grew at 20 to 100 psu with optimum antibiotic production at 35 psu. Four Streptomyces strains showed multiple inhibition against test organisms while four rare actinomycetes were specific in their inhibitory activity. This is the first report of a halophilic Kocuria sp., Nocardiopsis sp., and halotolerant Micromonospora sp. producing anti-bacterial compound(s) against Staphylococcus aureus, Staphylococcus citreus, and Vibrio cholerae, respectively. Sequential extraction with varying polarity of organic solvents showed that the extracts inhibited different test pathogens. These results suggest that halophilic and halotolerant actinomycetes from marine salterns are a potential source of anti-bacterial compounds.

Characterization of an SXT variant Vibrio cholerae O1 Ogawa isolated from a patient in Trivandrum, India

The emerging multiple drug resistance in bacterial pathogens is complicating the treatment of diseases and hence is a major public health concern. In the present study, Vibrio cholerae O1 El Tor Ogawa isolated from a patient was examined for antibiotic susceptibility pattern, presence of SXT and its transmissibility, associated drug resistance genes and variation in the int gene and the attP attachment site of SXT. The strain showed resistance to ampicillin, polymixin B, co-trimoxazole, trimethoprim, streptomycin, spectinomycin, furazolidone, tetracycline, ciprofloxacin and nalidixic acid. The sequencing of int, the SXT-specific integrase and attP attachment site indicated that it possessed a variant of SXT with trimethoprim (dfrA1), sulphamethoxazole (sul2) and streptomycin (strB) resistance genes. Its mobile nature was demonstrated by conjugation with rifampicin-resistant Escherichia coli. The emergence of such an isolate should be closely monitored because it will improve our understanding of the evolution of the multidrug resistance phenotype.

Detection of Virulence Genes in Vibrio cholerae Isolated from Aquatic Environment in Kerala, Southern India

Vibrio cholerae is the etiologic agent of cholera. It is an autochthonous inhabitant of all aquatic environments. The virulence of V. cholerae is maintained by the CTX genetic element and tcpA gene. In the present investigation, environmental strains of V. cholerae isolated from different aquatic biotopes in Kerala were identified and serotyped. The antibiotic resistance pattern and presence of virulence and regulatory genes were examined. We found the presence of toxigenic non-O1/non-O139 strains harboring the CTX genetic element, heat-stable enterotoxin, rtxA gene, El Tor hemolysin, and Vibrio pathogenicity island (VPI). The strains also produced the cholera toxin (CT) as determined by monosialoganglioside enzyme-linked immunosorbent assay. A few strains belonging to the O1 serogroup but lacking the CTX genetic element were also observed. The majority of the environmental strains belonged to non-O1/non-O139 serogroup with many possessing toxR, ompU, heat-stable enterotoxin, and rtxA gene. The toxigenic non-O1/non-O139 strains exhibited resistance to trimethoprim, ampicillin, and polymixin B and intermediate resistance to co-trimoxazole. However, all other environmental strains were found resistant to ampicillin and polymixin B. Our findings demonstrate that the virulence genes are dispersed among the environmental strains of V. cholerae and a complex aquatic environment can give rise to pathogenic V. cholerae.

Use of REP- and ERIC-PCR to reveal genetic heterogeneity of Vibrio cholerae from edible ice in Jakarta, Indonesia

BackgroundVibrio cholerae is the causative organism of waterborne disease, cholera. V. cholerae has caused many epidemics and pandemics of cholera for many years. In this study, V. cholerae recovered from edible ice were investigated for their genetic diversity using Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and Repetitive Extragenic Palindromic (REP) PCR. Isolation was done using selective medium and the presumptive isolates were confirmed through biochemical and serological assays.ResultsSeventy-five isolates of V. cholerae were recovered from ice samples collected from different locations of Jakarta. Specifically, 19 of them were identified as O1 serotype, 16 were Ogawa, 3 isolates were Inaba and the remaining isolates were non-O1. The fingerprinting profiles of V.cholerae isolated from ice samples were very diverse.ConclusionThis result showed that the ERIC sequence is more informative and discriminative than REP sequence for analysis of V. cholerae diversity.

Multiresistant Vibrio cholerae non-O1/non-O139 from waters in South India: resistance patterns and virulence-associated gene profiles

From different aquatic locations in Alleppey district, Kerala, South India a number (n = 36) of multiresistant non‐O1, non‐O139 V. cholerae strains were isolated. Water samples were filtered through 0.22 μm membrane filters, enriched in alkaline peptone water and plated onto thiosulfate‐citrate‐bile salts‐sucrose (TCBS) agar. The isolates were resistance to cefotaxime (50%), nalidixic acid (44.4%), streptomycin and tetracycline (41.6%), trimethoprim (38.8%), co‐trimoxazole (33.3%), furazolidone (27.7%), neomycin and ofloxacin (19.4%), ciprofloxacin, norfloxacin and spectinomycin (16.6%), gentamicin (8.3%) and chloramphenicol (2.7%). To our knowledge, this is the first report from Kerala, South India on the emergence of multiple drug resistance in V. cholerae isolates belonging to serogroup other than O1 and O139. Virulence‐associated gene profiling of the isolates by PCR revealed the presence of toxR (100%), rtxA (61.1%), hlyA (50%), mshA (33.3%), tcpA‐acfB (13.8%) and st (2.7%) genes. The virulence gene clusters ctxA, ompU, ace, and zot were not detected. This study demonstrates the presence of a wide array of critical virulence factors in diverse strains of Vibrio cholerae non‐O1/non‐O139. Hence, this serogroup can no longer be ignored as an environmental reservoir of virulence genes. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Wohlfahrtiimonas chitiniclastica-associated osteomyelitis: a rare case report

Wohlfahrtiimonas chitiniclastica is a rare pathogen that was first isolated from a parasitic fly, Wohlfahrtia magnifica. It is a very rare but an emerging human pathogen reported only in Europe and South America. Recently, it is reported to be an egressing zoonotic pathogen from different geographical locations. The present case represents the first report of this pathogen from a patient suffering from osteomyelitis from India and so far no reports are available regarding the W. chitiniclastica associated infections in Asian countries. Clinical awareness of such emerging human pathogens is crucial for the infectious disease containment.

Proteome profile of a pandemic Vibrio parahaemolyticus SC192 strain in the planktonic and biofilm condition

Vibrio parahaemolyticus is one of the leading causative agents of foodborne diseases in humans. In this study, the proteome profiles of the pandemic strain V. parahaemolyticus SC192 belonging to the O3:K6 serovar during the planktonic and biofilm stages were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry. This non-gel-based multidimensional protein identification technology approach identified 45.5% of the proteome in the reference genome V. parahaemolyticus RIMD 2210633. This is the largest proteome coverage obtained so far in V. parahaemolyticus and provides evidence for expression of 27% of the hypothetical proteins. Comparison of the planktonic and biofilm proteomes based on their cluster of orthologous groups, gene ontologies and KEGG pathways provides basic information on biofilm specific functions and pathways. To the authors’ knowledge, this is the first study to generate a global proteome profile of the pandemic strain of V. parahaemolyticus and the method reported here could be used to rapidly obtain a snapshot of the proteome of any microorganism at a given condition.