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Commensal bacteria play a major role in multiple skin functions by providing the first layer of defense against pathogens and maintaining the skin barrier. Staphylococcus epidermidis is one of the most common skin commensals. In this study, we showed that S. epidermidis ferments glycerol and uses it as a nutrient, while producing short-chain and organic fatty acids, with the most notable being lactic acid. Lactic acid is an alpha-hydroxy acid that inhibits the growth of pathogenic bacteria, without any negative effect on the commensal bacteria itself. Using in vivo experiments, we validated our in vitro results, showing that the skin microbiome is also capable of doing this. Finally, using 2D and 3D skin culture models, we showed that the fermentation of glycerol, mainly lactic acid, as determined by analytical methods, upregulates the expression levels of several key genes that are associated with the barrier properties of the skin. While the hydration effect of glycerol on the skin is well known, our study shows the overall benefits of glycerol on the skin microbiome, while revealing an alternate mode of action of glycerol for multiple skin benefits.

The skin microbiome varies across individuals. The causes of these variations are inadequately understood. We tested the hypothesis that inter-individual variation in facial skin microbiome can be significantly explained by variation in sebum and hydration levels in specific facial regions of humans. We measured sebum and hydration from forehead and cheek regions of healthy female volunteers (n = 30). Metagenomic DNA from skin swabs were sequenced for V3-V5 regions of 16S rRNA gene. Altogether, 34 phyla were identified; predominantly Actinobacteria (66.3%), Firmicutes (17.7%), Proteobacteria (13.1%) and Bacteroidetes (1.4%). About 1000 genera were identified; predominantly Propionibacterium (58.6%), Staphylococcus (8.6%), Streptococcus (4.0%), Corynebacterium (3.6%) and Paracoccus (3.3%). A subset (n = 24) of individuals were sampled two months later. Stepwise multiple regression analysis showed that cheek sebum level was the most significant predictor of microbiome composition and diversity followed by forehead hydration level; forehead sebum and cheek hydration levels were not. With increase in cheek sebum, the prevalence of Actinobacteria (p = 0.001)/Propionibacterium (p = 0.002) increased, whereas microbiome diversity decreased (Shannon Index, p = 0.032); this was opposite for other phyla/genera. These trends were reversed for forehead hydration levels. Therefore, the nature and diversity of facial skin microbiome is jointly determined by site-specific lipid and water levels in the stratum corneum.

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 .

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.

Five wild-type mutant strains of Vibrio cholerae serogroup O1 that lacked the CTX virulence cassette, or contained a natural deletion of a virulence gene within the CTX virulence cassette, or possessed an additional virulence gene, along with a prototype toxigenic strain representing the El Tor classical biotypes were examined by in-vivo and in-vitro methods to determine their enterotoxic potential. The ability of whole cells and culture supernates of the strains to cause fluid accumulation in the rabbit ileal loop model revealed a pattern consistent with the presence of the various virulence gene(s), with those possessing the intact CTX virulence cassette being the most secretogenic. Culture supernates of strains without the CTX virulence cassette or the strain with an incomplete cassette were also able to evoke mild to moderate fluid accumulation in the rabbit ileal loop. Of the various media used, AKI and brain heart infusion broth appeared to support the production of a hitherto unknown secretogenic factor, because culture supernates of the non-toxigenic V. cholerae O1 strains showed higher fluid accumulation ratios when grown in these media than in the others. To confirm that the fluid accumulation elicited by these strains in the ileal loop was due to enterotoxin activity, the effect of supernate of the strains was examined in rabbit small intestine mounted on Ussing chambers. Increases in short circuit current and tissue conductance, as compared with the medium control, were observed even with the strains that did not possess the CTX virulence cassette, confirming their ability to disrupt the function of intestinal tissue. From these studies, it was concluded that strains of V. cholerae O1 devoid of the CTX virulence cassette were still able to elicit a secretory response in the ileal loop and displayed enterotoxic activity in an in-vitro experimental model.

Using molecular techniques, we investigated whether the clone of Vibrio cholerae O1 biotype El Tor which appeared in Calcutta, India, in 1994 has spread to other cholera endemic areas in the country. The ribotype of 31 of the 33 strains isolated from different parts of India during 1996 and 1997 was identical to the ribotype displayed by the new clone of V. cholerae O1 which emerged in Calcutta in 1994. Likewise, 12 of the 15 strains examined by pulsed-field gel electrophoresis (PFGE) showed identical profile to that exhibited by the new clone of O1. The restriction fragment length polymorphism (RFLP) of CTX genetic element of these strains also matched with the new clone of O1 which emerged after the outbreak of V. cholerae O139 in Calcutta. However, two strains (AH042 and AH046) isolated from an outbreak in Ahmedabad (western India) showed different CTX RFLP but had the same ribotype and PFGE profile as the new clone, whereas one strain from Goa (G2) showed distinct ribotype and PFGE profile and the CTX RFLP was identical to the O1 strains which prevailed before the genesis of O139 in Calcutta. The drug resistance pattern of most of the O1 strains examined in this study, except strain G2, was similar to that of the new clone of V. cholerae O1. None of the strains in this study carried plasmids. Molecular studies clearly show that the new expanded drug resistant clone of V. cholerae O1 has spread to all cholera endemic areas in India and also provide evidence for the evolution of new clones of the O1 serogroup.

There was an inexplicable upsurge in the incidence of non-O1, non-O139 Vibrio cholerae among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996. Of the 18 strains of V. cholerae isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup. Cell-free culture supernatants of 13 representative non-O1, non-O139 V. cholerae strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin. By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the ctxA , zot , ace , and tcpA genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of Escherichia coli and the various variants of these genes. Studies on the clonality of non-O1, non-O139 V. cholerae strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes ( hlyA , hlyU , hlx , toxR , and attRS1 ) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones. Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles. It is clear from this study that some serogroups of V. cholerae can cause diarrhea by a mechanism quite different from that of toxigenic V. cholerae O1 and O139, and we have proposed the nomenclature of enteropathogenic V. cholerae to include these serogroups.

This study presents results of a surveillance on cholera conducted with hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, from January 1993 to December 1995. The O139 serogroup of Vibrio cholerae dominated in 1993 but was replaced by O1 as the dominant serogroup in 1994 and 1995. The isolation rate of V. cholerae non-O1 non-O139 did not exceed 4.9% throughout the study period, while the isolation rate of the O139 serogroup in 1994 and 1995 was below 9%. No temporal clustering of any non-O1 non-O139 serogroup was observed. With the exception of 1 strain, none of the 64 strains belonging to the non-O1 non-O139 serogroup hybridized with ctx, zot, and ace gene probes, while 97.3 and 97.7% of the O139 and O1 strains, respectively, hybridized with all the three probes. Multiplex PCR studies revealed that all the O1 strains belonged to the EIT or biotype. There was a progressive increase in the cytotoxic response on CHO and HeLa cells evoked by culture supernatants of strains of V. cholerae non-O1 non-O139 isolated during 1994 and 1995 compared with the response evoked by those isolated in 1993. Dramatic shifts in patterns of resistance to antibiotics between strains of V. cholerae belonging to different serogroups and within strains of a serogroup isolated during different time periods were observed. There was a discernible increase in the incidence of multidrug-resistant strains of V. cholerae O1 isolated in 1994 and 1995 compared with that in 1993. On the basis of the results of this study, we predict the possibility of newer variants of V. cholerae emerging in the future.

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4[ratio ]K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3[ratio ]K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3[ratio ]K6 and O4[ratio ]K68 strains and for comparison, non-O3[ratio ]K6 and non-O4[ratio ]K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3[ratio ]K6 and O4[ratio ]K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3[ratio ]K6 and O4[ratio ]K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3[ratio ]K6 and O4[ratio ]K68 strains formed a cluster with 78–91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3[ratio ]K6 and non-O4[ratio ]K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.

Culture supernatants of nontoxigenic nonepidemic clinical strains of Vibrio cholerae belonging to diverse serogroups were found to induce vacuolation of nonconfluent HeLa cells. The vacuoles became prominent 18 h after introduction of culture supernatant, and vacuolated cells survived for 48 h and then died. Only a fraction of the vacuolated cells took up neutral red dye, implying that there were differences in the vacuolar microenvironment. Further tests showed that the factor responsible for vacuolation was heat labile and proteinaceous. Vacuolating activity was completely neutralized by antibody to hemolysin of V. cholerae but not by antibody to vacuolating cytotoxin of Helicobacter pylori . Partial purification of the vacuolating factor led to elution of fractions, which showed both hemolytic and vacuolating activity. PCR amplification and cloning of the hemolysin structural gene ( hlyA ) into Escherichia coli DH5α led to isolation of clones producing cell vacuolating factor in a cell-associated form. Further, a null insertion mutation in the hlyA gene of a high-vacuolating-factor-producing strain led to complete abolition of both cell vacuolating and hemolytic activities. These analyses establish vacuolation as a potentially important but previously unrecognized property of V. cholerae El Tor hemolysin.

The non-membrane-damaging cytotoxin which causes dramatic cell rounding of cultured HeLa cells was purified to homogeneity from a clinical strain (WO5) of non-toxigenic Vibrio cholerae O1 Inaba belonging to the E1 Tor biotype. The purified protein has a denatured molecular weight of 35 kDa and a native molecular weight of approximately 37 kDa indicating the monomeric nature of the protein. The 15 N-terminal amino acid sequence of non-membrane-damaging cytotoxin showed complete homology to the hemagglutinin protease previously purified and characterized from V. cholerae O1. Purified non-membrane-damaging cytotoxin from V. cholerae O1 was immunologically and biochemically identical to that previously purified from V. cholerae O26. Non-membrane-damaging cytotoxin was found to be enterotoxic in rabbit ileal loop assay inducing accumulation of non-hemorrhagic fluid at 100 micrograms and elicited a concentration dependent increase in short circuit current and tissue conductance of rabbit ileal mucosa mounted on Ussing chambers. A significant serum immunoglobulin G response against non-membrane-damaging cytotoxin was elicited by patients infected with V. cholerae O139 but not with V. cholerae O1. These properties make non-membrane-damaging cytotoxin a potential virulence factor of V. cholerae which should be taken into consideration while making live, attenuated recombinant vaccine strains against cholera.

1992 saw the emergence in southern India of a novel serogroup currently classified as Vibrio cholerae O139 Bengal. On the basis of this microorganism's ability to spread rapidly through cholera endemic areas in India and in neighbouring countries and on the propensity of the new serogroup to replace the O1 El Tor biotype, V cholerae O139 was described as the new pandemic strain of cholera. 1 Surprisingly, however, in the subsequent years the El Tor biotype reappeared in most parts of the Indian subcontinent.