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A N Ghosh

Publications and source records attributed to A N Ghosh.

At least 19 recordsLinked to original sources
PubMed2006

Epidemiological, microbiological & electron microscopic study of a cholera outbreak in a Kolkata slum community.

BACKGROUND & OBJECTIVE: Epidemics of cholera caused by Vibrio cholerae O1 or O139 have been reported from different parts of India. Factors such as unsafe water supply, poor environmental sanitation, indiscriminate defaecation and lack of personal hygiene are mainly responsible for continued transmission of this disease. We report here epidemiological and microbiological findings of a localized outbreak of cholera, which occurred during March and April 2004 in the eastern part of Kolkata city. METHODS: The affected slum area has a population of 4409, predominantly muslims. Patients suffering from acute watery diarrhoea attended the health outposts organized by National Institute of Cholera and Enteric Diseases, Kolkata and International Vaccine Institute, South Korea as part of a routine surveillance programme at the locality as well as the emergency medical camp organized by the Kolkata Municipal Corporation. Stool and water samples were collected and tested for diarrhoeagenic pathogens in the laboratory. Bacteriophages specific for V. cholerae were isolates and studied electron microscopically for morphology. RESULTS: A total of 89 diarrhoea cases were reported giving an attack rate of 2 per cent. V. cholerae O1 biotype ElTor, serotype Ogawa was isolated as a sole pathogen from 15 (15.8%) of 89 stool samples screened. Water samples (2 from tube wells, 3 from municipal taps and 1 from well) showed presence of coliform bacilli with high MPN (Most Probable Number) count. Bacteriophages specific to V. cholerae were isolated from 2 of 6 water samples examined. A leakage was detected in the main pipeline supplying drinking water to that area. INTERPRETATION & CONCLUSION: The outbreak was caused by V. cholerae O1 (Ogawa) biotype ElTor. The presence of phages in the water samples was an additional indicator for V. cholerae contamination in this community. Occurrences of such outbreaks support vaccination against cholera as an alternative strategy.

Bacteriophage Typing↗
PubMed2005

A new small temperate DNA phage BcP15 isolated from Burkholderia cepacia DR11.

A Burkholderia cepacia DR11 strain was isolated during the survey of microorganisms from coastal water of deltaic Sunderbans. This strain always released temperate phage BcP15 into culture supernatant. UV irradiation of the strain also induced phage induction. The phage titer was 2.3 x 10(8). New temperate phage BcP15 has unusual structure. It has a hexagonal head, 65 nm in diameter and a tail 200 nm long, attached with single thick wavy tail fiber (424-705 nm). Phage DNA is double stranded 11.9 kb long. Southern hybridization result indicated that the phage DNA was in lysogenic state into the B. cepacia DR11 genome. SDS-PAGE of phage protein showed two major bands of molecular weight 20 kDa and 40 kDa.

Bacteriophages↗
PubMed2005

Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages.

The relationship among (i) the local incidence of cholera, (ii) the prevalence in the aquatic environment of Vibrio cholerae, and (iii) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh. Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V. cholerae strain than both (P < 0.00001). The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V. cholerae strains in water samples that otherwise lacked detectable cholera phages. Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria. Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V. cholerae pandemic serogroups or clones.

Bacteriophages↗
PubMed2005

Reduction in capsular content and enhanced bacterial susceptibility to serum killing of Vibrio cholerae O139 associated with the 2002 cholera epidemic in Bangladesh.

Vibrio cholerae O139 emerged in 1992 as a major cause of epidemic cholera. However, the incidence of disease due to this new serogroup subsequently decreased for almost a decade. In April 2002, there was a dramatic resurgence of V. cholerae O139 in Bangladesh. We compared the phenotypic properties of the bacterial isolates and the immunological responses in patients with disease due to V. cholerae O139 during the 2002 epidemic with those dating to the emergence of this disease in 1993 to 1995. Strains isolated from patients in the two time periods were compared with respect to capsular polysaccharide, their resistance to the bactericidal effect of serum, and their capacity to be used as target strains in complement-mediated vibriocidal assays. Phase-contrast microscopy showed that strains isolated in 2002 had less capsular material than those isolated from 1993 to 1995 (P = <0.001), a finding confirmed by electron microscopic studies. Strains isolated in 2002 were more susceptible to the bactericidal activity of serum compared to strains from 1993 to 1995 (P = 0.013). Compared to results using a standard O139 strain, a modified vibriocidal assay utilizing a 2002 strain, CIRS 134, as the target organism detected higher vibriocidal responses in both O139-infected cholera patients as well as O139 vaccine recipients. The vibriocidal assay utilizing the less encapsulated 2002 strain, CIRS 134, is a more sensitive indicator of adaptive immune responses to recent infection with V. cholerae O139. Consequently, this assay may be useful in studies of both O139-infected patients and recipients of O139 vaccines.

Adult↗
PubMed2005

Genomic sequence and receptor for the Vibrio cholerae phage KSF-1phi: evolutionary divergence among filamentous vibriophages mediating lateral gene transfer.

KSF-1phi, a novel filamentous phage of Vibrio cholerae, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1phi infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the mshA gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1phi infection, naturally occurring MSHA-negative strains and an mshA deletion mutant were resistant. Furthermore, d-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1phi. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1phi, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXphi, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of V. cholerae strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to V. cholerae evolution.

Bacteriophages↗
PubMed2003

Shigella dysenteriae type 1-specific bacteriophage from environmental waters in Bangladesh.

Shigella dysenteriae type 1 is the causative agent of the most severe form of bacillary dysentery, which occurs as epidemics in many developing countries. We isolated a bacteriophage from surface water samples from Bangladesh that specifically lyses strains of S. dysenteriae type 1. This phage, designated SF-9, belongs to the Podoviridae family and has a 41-kb double-stranded DNA genome. Further screening of water samples for the prevalence of the phage revealed 9 of 71 (12.6%) water samples which were positive for the phage. These water samples were also positive in PCR assays for one or more S. dysenteriae type 1-specific genes, including ipaBCD and stx1, and live S. dysenteriae type 1 was isolated from three phage-positive samples. The results of this study suggest that phage SF-9 may have epidemiological applications in tracing the presence of S. dysenteriae type 1 in environmental waters.

Bacterial Proteins↗
PubMed2002

RS1 element of Vibrio cholerae can propagate horizontally as a filamentous phage exploiting the morphogenesis genes of CTXphi.

In toxigenic Vibrio cholerae, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTXPhi morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXPhi is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC. We identified a single-stranded circularized form of the RS1 element, in addition to the CTXPhi genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km(r)) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC. Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Km(r) transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmPhi). Analysis of V. cholerae strains for susceptibility to RS1-KmPhi showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX(-)) O1 and O139 strains which carried genes encoding the CTXPhi receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXPhi, the RS1Phi genome also integrated into the host chromosomes by using the attRS sequence. However, only transductants of RS1-KmPhi which also harbored the CTXPhi genome produced a detectable level of extracellular RS1-KmPhi. This suggested that the core genes of CTXPhi are also required for the morphogenesis of RS1Phi. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXPhi.

Bacterial Proteins↗
PubMed2001

Multiple copies of the upstream regulatory region of the major capsid protein gene of bacteriophage MB78 inhibit phage morphogenesis.

The 2.311 kb EcoRI F fragment of bacteriophage MB78 has been cloned in multicopy vectors pUC19 and pCR90. Salmonella typhimurium strains carrying such plasmids cannot support development of phage MB78 while other Salmonella phages like P22 and 9NA grow normally. Most of the phage MB78 induced functions are normal in such transformed hosts but proper maturation of the phage particles does not take place. Deletion of 138 bp from the 3' end of the cloned fragment reverses the inhibitory effect. Analysis of nucleotide and the deduced amino acid sequence of a 1.2 kb HindIII-SalI fragment of the phage genome which overlaps the 138 bp confirms that this part contains the upstream regulatory region of the major structural protein gene. It seems that in presence of multiple copies of the upstream regulatory region (which includes a number of promoter like sequence) of the coat protein gene, the maturase gene is down regulated and this is effective only in cis, a situation quite similar to that of Qbeta RNA phages.

Amino Acid Sequence↗
PubMed2000

Conversion of Vibrio eltor MAK757 to classical biotype: role of phage PS166.

Temperate phage PS166 infection of Vibrio eltor MAK757 resulted in complete changes in all biotype-specific determinants. About 10% of the PS166 lysogens of MAK757 lost their eltor-specific determinants, namely, the ability to produce soluble hemolysin, cell-associated hemagglutinin for chicken erythrocytes, and resistance to polymyxin B, as well as resistance to Mukherjee's group IV phage and sensitivity to eltor phage e4. These lysogens were found to have acquired the properties of classical strains, most significantly becoming sensitive to group IV phage but resistant to eltor-specific e4. The remainder of these lysogens, however, retained their parental biotype and serotype but acquired auxotrophy for glycine and histidine. The differential behavior of the two types of lysogen was due to the integration of the phage PS166 genome at different locations in the host chromosome. A 800-bp BglII fragment was found to contain the attP site. Phage PS166 has a polyhedral head (95 nm in diameter) and a contractile tail (98 nm in length). The phage chromosome is a linear double-stranded DNA of 110 kb and a G + C content of 58.7%.

Absorption↗
PubMed2000

Virulence genes in environmental strains of Vibrio cholerae.

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.

Amino Acid Sequence↗
PubMed2000

Development and evaluation of a phage typing scheme for Vibrio cholerae O139.

The scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic Vibrio cholerae O139 in India. The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country. A total of five newly isolated phages lytic to V. cholerae O139 strains were used for the development of this phage typing scheme. These phages differed from each other and also differed from the existing O1 phages in their lytic patterns, morphologies, restriction endonuclease digestion profiles, and immunological criteria. With this scheme, 500 V. cholerae O139 strains were evaluated for their phage types, and almost all strains were found to be typeable. The strains clustered into 10 different phage types, of which type 1 (38.2%) was the dominant type, followed by type 2 (22.4%) and type 3 (18%). Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during the period between 1993 and 1994, whereas phage type 2 (32. 1%) was the next major type during the period from 1996 to 1998. This scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V. cholerae O139.

Bacteriophage Typing↗
PubMed1999

Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid.

A Shigella flexneri strain, cured of the large 220-kb virulence plasmid, expresses adhering and invading ability in confluent monolayers of HeLa cells similar to its parent strain. Invasion by both the parent and the cured strains resulted in alteration of the monomeric actin (G) in the total actin pool of HeLa cells. Other indicators of invasive characteristics of virulent Shigella strains such as production of keratoconjunctivitis in guinea pig eye in vivo, Congo red binding and expression of contact hemolysin however, indicated loss of invasive properties in the plasmid cured strain. Further, pretreatment of bacterial cells with para-bromophenacyl bromide (p-BPB), a specific chemical inhibitor of phospholipase A, adversely affected adhesion to and invasion of HeLa cells in vitro, irrespective of the presence of the 220-kb plasmid indicating the possible involvement of the enzyme phospholipase A in the invasion process. Adherence of both the strains to guinea pig colonic epithelial cells (CECs) in vitro was reduced significantly on pretreatment of bacteria or CECs with p-BPB. Expression of exocellular enzymes viz. protease, elastase, phospholipase A and phospholipase C were not related to the large plasmid.

Acetophenones↗
PubMed1999

Complementary role of two fragments of domain V of 23 S ribosomal RNA in protein folding.

We have shown that the domain V of bacterial 23 S rRNA could fold denatured proteins to their active state. This segment of 23 S rRNA could further be split into two parts. One part containing mainly the central loop of domain V could bind denatured human carbonic anhydrase I stably. This association could be reversed by adding the other part of domain V. The released enzyme was directed in such a way by the central loop of domain V that it could now fold by itself to active form. This agrees with our earlier observation that proteins fold within the cell posttranslationally, a process that is completed after release of the newly synthesized polypeptide from the ribosome (Chattopadhyay, S., Pal, S., Chandra, S., Sarkar, D., and DasGupta, C. (1999) Biochim. Biophys. Acta 1429, 293-298).

Animals↗
PubMed1999

Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on int 407 and HeLa cells.

Vibrio cholerae produces a non-membrane damaging cytotoxin (NMDCY), also known as cell rounding factor, which causes rapid rounding of cultured cells like HeLa, CHO and Vero and reportedly elicits enterotoxic activity in the rabbit ileal loop assay. Pursuing the concept that NMDCY might be an accessory factor contributing to the diarrhea caused by V. cholerae, we investigated the effect of NMDCY on Int 407 (intestinal cell line) and HeLa (non-intestinal cell line) cells using light, fluorescent and electron microscopy to gain insight into the cellular response evoked by NMDCY. Binding assays showed that NMDCY has affinity for both Int 407 and HeLa cells. Changes in the internal organelles and cytoskeletal structures of the cell lines were documented indicating changes in the secretory and metabolic function of the toxin-treated cells. Toxin-treated cells visualized under the electron microscope revealed retraction of cell body, formation of blebs on cell surface, changes in mitochondria having dilated and rarefied matrix and an extensively developed Golgi apparatus, endoplasmic reticulum and lysosomes compared to those in normal cells. Immunofluorescence study showed restructuring of microfilament network represented by actin, filamin and vinculin, as also of the microtubular component, tubulin and the intermediate filament, vimentin. Immunogold study further revealed that the toxin is internalized even within the nucleus. Moreover, a rise in the intracellular calcium level of the NMDCY-treated cells leads us to hypothesize that a cascade of events results in the final impairment of the cell machinery.

Calcium↗
PubMed1999

Antibodies to the truncated (short) form of 'O' polysaccharides (TFOP) of Vibrio cholerae O139 lipopolysaccharides protect mice against experimental cholera induced by encapsulated O139 strains and such protection is mediated by inhibition of intestinal colonization of vibrios.

An antiserum raised against the lipopolysaccharides (LPS) of an encapsulated Vibrio cholerae O139 strain was shown to induce passive protection against challenge with O139, but not O1, organisms. Subsequent experiments, however, revealed that the purified LPS, obtained by the conventional phenol-water extraction method, contained capsular polysaccharide (CPS) material. Therefore, another antiserum was raised by immunization with electrophoresed gel-cut material containing only the truncated (short) form of 'O' polysaccharides (TFOP) linked to the core of O139 LPS. Anti-TFOP antibodies and their Fab (IgG) fragments induced passive protection against challenge with colonial variants of encapsulated O139 strains and such protection was mediated by inhibition of intestinal colonization. These results suggest that it is possible to engender protection against encapsulated O139 strains by using TFOP material (devoid of CPS) as the immunogen.

Animals↗
PubMed1999

Purification of a mannose/glucose-specific hemagglutinin/lectin from a Vibrio cholerae O1 strain.

A cell-associated mannose/glucose-specific hemagglutinin (MSHA) has been purified from a strain of Vibrio cholerae O1 by chromatography on a chitin column followed by affinity purification on Sephadex G100. The purified protein gave a single stained band of 40 kDa by SDS-PAGE, exhibited high affinity towards D-mannose and D-glucose but was resistant to L-fucose and N-acetyl-D-glucosamine. The purified MSHA was revealed as a globular form of protein under electron microscope. In immunodiffusion tests the purified MSHA produced a single precipitin band against homologous antisera and antisera raised against the whole cell bacteria without any reactivity towards antisera raised against the purified N-acetyl-D-glucosamine-specific lectin of the same bacterial strain. Immunogold labelling confirmed the location of hemagglutinin on the surface of the bacteria. Purified MSHA reacted strongly with sera from convalescent cholera patients in immunodiffusion tests.

Animals↗
PubMed1997

Isolation of Vibrio cholerae 0139 phages to develop a phage typing scheme.

Five V. cholerae 0139 phages isolated from different parts of India have been used for phage typing study. A strain isolated from Nagpur city (NPR-4) was used as the host for phage propagation. All but one of the 260 strains of V. cholerae 0139 were found to be typeable and could be clustered into 8 distinct phage types as revealed by lytic patterns. Phage type 1 was the predominant type (61.15%) followed by type 2 (18.46%). The strains isolated from Madras exhibited 7 out of 8 phage types. These newly isolated phages could be adopted for phage typing of V. cholerae 0139 strains as an epidemiological tool.

Bacteriophage Typing↗