Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in Bacillus subtilis.

The lipopeptide antibiotic daptomycin exhibits bactericidal activity against Gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanisms of bacterial resistance to daptomycin has gained great importance. In this study, we aimed to identify the genetic factors contributing to daptomycin resistance in Bacillus subtilis, a model Gram-positive bacterium. Our findings demonstrated that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis. Specifically, overexpression of ugtP resulted in increased levels of diglucosyldiacylglycerol (Glc2DAG) and decreased levels of acidic phospholipids cardiolipin and PG, as well as the basic phospholipid lysylphosphatidylglycerol. However, ugtP overexpression did not alter the cell surface charge and the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide. Furthermore, by serial passaging in the presence of daptomycin, we obtained daptomycin-resistant mutants carrying ugtP mutations. These mutants showed increased levels of Glc2DAG and a >4-fold increase in the minimum inhibitory concentration of daptomycin. These results suggest that increased Glc2DAG levels, driven by ugtP overexpression, modify the phospholipid composition and confer daptomycin resistance in B. subtilis without altering the cell surface charge of the bacteria.IMPORTANCEDaptomycin is one of the last-resort drugs for the treatment of methicillin-resistant Staphylococcus aureus infections, and the emergence of daptomycin-resistant bacteria has become a major concern. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis, a model Gram-positive bacteria. The overexpression of UgtP increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection and management.

Effect of physicochemical and microbiological factors on the development of viable but non-culturable and resuscitation states of Vibrio cholerae.

BACKGROUND: Vibrio cholerae can endure harsh environmental conditions by transitioning into viable but non-culturable (VBNC) form and resuscitate upon return of appropriate conditions. METHOD: In this study, we assessed the impact of physicochemical and microbiological factors, on the development of low temperature-induced VBNC state and subsequent recovery by temperature upshift. RESULTS: In estuarine water, Vibrio cholerae exhibits a slower decline in culturability over a period of 77 days as compared to 10 days in fresh water. When variable cell numbers from different growth phases were used for VBNC induction, it was observed that the higher inoculum size (106-107 cfu ml-1) from the late log phase culture appears to be crucial for entering the VBNC state. Conversely, starved cells could enter the VBNC state with an initial inoculum of 104-105 cfu ml-1, followed by resuscitation as well. The addition of glucose, GlcNAc and mannitol differentially affects progression into VBNC, while the addition of tryptone, yeast extract and casamino acid facilitated early entry into the VBNC state and shortened the length of the recovery period. CONCLUSION: Altogether these findings demonstrated that the ionic strength of water, inoculum size and the availability of nutrients played distinct roles during VBNC induction and resuscitation.

Foodborne Outbreak by Salmonella enterica Serovar Weltevreden in West Bengal, India.

Foodborne gastroenteritis outbreaks owing to Salmonella enterica serovar Weltevreden (Salmonella Weltevreden) represent a significant global public health problem. In the past two decades, Salmonella Weltevreden has emerged as a dominant foodborne pathogen, especially in South-East Asian countries. This report describes a community foodborne outbreak of gastroenteritis caused by Salmonella Weltevreden in August 2022 following consumption of panipuri from a street vendor in the Polba block in Hooghly district, West Bengal, India. This food item was consumed by 185 people, of whom 129 had acute watery diarrhea with other clinical symptoms and 65 of them were admitted to different District hospitals for treatment. Stool specimens collected from hospitalized cases were positive for S. enterica, and further serotyped as Salmonella Weltevreden. All the Salmonella Weltevreden strains possessed the Salmonella pathogenicity islands associated genes (invA/E, orgA, ttrc, ssaQ, mgtC, misL, spi4D), the enterotoxin (stn), and hyperinvasive locus gene (hilA). Except erythromycin, all the strains were susceptible for commonly used antimicrobials in the treatment of diarrhea. The XbaI-based pulsed-field gel electrophoresis analysis indicated that all the isolates responsible for the recent outbreak were similar, but diverged from other Salmonella Weltevreden that were previously reported in West Bengal. This report indicates that foodborne infection is a major public health concern in India and demands to strengthen capacity-building measures at the local health care levels for linking causative agents of outbreaks.

Flavobacterium okayamense sp. nov. isolated from surface seawater.

Strain KK2020170T, a Gram-stain negative, yellow colony-forming bacterium, was isolated from surface seawater sampled in Kojima Bay, Okayama, Japan. Phylogenetic analysis based on the 16S rRNA gene revealed that strain KK2020170T belongs to the genus Flavobacterium, with Flavobacterium haoranii LQY-7T (98.1% similarity) being its closest relative, followed by Flavobacterium sediminis MEBiC07310T (96.9%) and Flavobacterium urocaniciphilum YIT 12746T (96.0%). Whole-genome shotgun sequencing showed that strain KK2020170T, when paralleled with F. haoranii LQY-7 T, had 81.3% average nucleotide identity, and 24.6% in silico DNA-DNA hybridization values, respectively. The DNA G + C content of strain KK2020170T was 31.1 mol%. The most abundant fatty acids (> 10%) of strain KK2020170T were iso-C15: 0, iso-C17: 0 3-OH and iso-C15: 1 G. The dominant respiratory quinone of the strain was menaquinone MK-6. Based on the phylogenetic and phenotypic analysis results, we propose that strain KK2020170T represents a novel species, for which the name Flavobacterium okayamense sp. nov. has been proposed. The type strain is KK2020170T (= ATCC TSD-280 T = NBRC 115344 T).

Asiatic acid inhibits intracellular Shigella flexneri growth by inducing antimicrobial peptide gene expression.

AIMS: A rapid rise in resistance to conventional antibiotics for Shigella spp. has created a problem in treating shigellosis. Hence, there is an urgent need for new and non-conventional anti-bacterial agents. The aim of this study is to show how Asiatic acid, a plant-derived compound, inhibits the intracellular growth of Shigella flexneri. METHODS AND RESULTS: Shigella flexneri sensitive and resistant strains were used for checking antimicrobial activity of Asiatic acid by gentamicin protection assay. Asiatic acid inhibited the intracellular growth of all strains. Gene expression analysis showed antimicrobial peptide (AMP) up-regulation by Asiatic acid in intestinal cells. Further western blot analysis showed that ERK, p38, and JNK are activated by Asiatic acid. ELISA was performed to check IL-8, IL-6, and cathelicidin secretion. The antibacterial effect of Asiatic acid was further verified in an in vivo mouse model. CONCLUSIONS: The reason behind the antibacterial activities of Asiatic acid is probably over-expression of antimicrobial peptide genes. Besides, direct antimicrobial activities, antimicrobial peptides also carry immunomodulatory activities. Here, Asiatic acid increased IL-6 and IL-8 secretion to induce inflammation. Overall, Asiatic acid up-regulates antimicrobial peptide gene expression and inhibits intracellular S. flexneri growth. Moreover, Asiatic acid reduced bacterial growth and recovered intestinal tissue damages in in vivo mice model.

Environmental water in Kolkata is suitable for the survival of Vibrio cholerae O1.

Many patients with cholera emerge in Kolkata, India throughout the year. Such emergency indicates that cholera toxin-producing Vibrio cholerae O1 (toxigenic V. cholerae O1) are widespread in Kolkata. This suggests that the suitable conditions for replication of toxigenic V. cholerae O1 is provided in Kolkata. In previous studies, we found that the replication rate of toxigenic V. cholerae O1 is low in the low ionic aqueous solution. Then we measured the ion concentration in the environmental water of Kolkata. As a control, we measured them in Japanese environmental water. The ion concentration in the environmental water of Kolkata was significantly high. Then, we examined the survival of toxigenic V. cholerae O1 in groundwater from Kolkata and found that V. cholerae O1 survive for long time in the solution but not in the solution diluted with Milli Q water. In addition, we found that V. cholerae O1 proliferated in environmental water of Kolkata to which a small amount of nutrient was added, but did not grow in the environmental water diluted with water to which the same amount of nutrient was added. These results indicate that the environmental water from Kolkata is suitable for survival of V. cholerae O1.

Rapid diagnostic tests versus RT-PCR for Ebola virus infections: a systematic review and meta-analysis.

Objective: To evaluate the clinical accuracy of rapid diagnostic tests for the detection of Ebola virus. Methods: We searched MEDLINE®, Embase® and Web of Science for articles published between 1976 and October 2021 reporting on clinical studies assessing the performance of Ebola virus rapid diagnostic tests compared with reverse transcription polymerase chain reaction (RT-PCR). We assessed study quality using the QUADAS-2 criteria. To estimate the pooled sensitivity and specificity of these rapid diagnostic tests, we used a bivariate random-effects meta-analysis. Findings: Our search identified 113 unique studies, of which nine met the inclusion criteria. The studies were conducted in the Democratic Republic of the Congo, Guinea, Liberia and Sierra Leone and they evaluated 12 rapid diagnostic tests. We included eight studies in the meta-analysis. The pooled sensitivity and specificity of the rapid tests were 86% (95% confidence interval, CI: 80-91) and 95% (95% CI: 91-97), respectively. However, pooled sensitivity decreased to 83% (95% CI: 77-88) after removing outliers. Pooled sensitivity increased to 90% (95% CI: 82-94) when analysis was restricted to studies using the RT-PCR from altona Diagnostics as gold standard. Pooled sensitivity increased to 99% (95% CI: 67-100) when the analysis was restricted to studies using whole or capillary blood specimens. Conclusion: The included rapid diagnostic tests did not detect all the Ebola virus disease cases. While the sensitivity and specificity of these tests are moderate, they are still valuable tools, especially useful for triage and detecting Ebola virus in remote areas.

Epidemiology of major entero-pathogenic viruses and genetic characterization of Group A rotaviruses among children (≤5 years) with acute gastroenteritis in eastern India, 2018-2020.

AIMS: This study was carried out from January 2018 to March 2020 in Kolkata, eastern India to determine the prevalence rates and epidemiological patterns associated with the major viral agents of gastroenteritis among children ≤5 years of age. Molecular characterization of GARV, the predominant agent of viral gastroenteritis, was done to understand their genotype diversity. METHODS AND RESULTS: 1284 of 3157 stool samples (~40%) from children (≤5 years) with acute gastroenteritis tested positive for one or more enteric viruses with positivity rates 25.11%, 8.74%, 6.62% and 6.11% for GARV, HAdV-F, AstV and NoV respectively. Co-infection was observed in 5.31% of cases. Associated clinical/meteorological variables like age, sex, symptoms, temperature and precipitation were assessed to find any correlation between these and enteric virus infection rates. >70% of viral gastroenteritis cases were observed in 6-24 months' age group. GARV and AstV infection occurred mostly during cooler months while HAdV-F infection mostly occurred during warmer periods. No definite seasonality was observed for NoV infections. Clinical severity associated with GARV infection was higher compared to other enteric viruses. Genotyping of rotavirus positive samples revealed G3P[8] was the predominantly circulating GARV genotype throughout the study period. CONCLUSIONS: GARV remained the predominant viral agent of acute gastroenteritis among children though its prevalence rates in this region declined significantly compared to the previous years (2010-2016). The prevalence of other enteric viruses was below 10%. SIGNIFICANCE AND IMPACT OF STUDY: This study provides valuable insights regarding the current burden of viral gastroenteritis in Eastern India. The 2-year study in children will provide the baseline data for future surveillance studies in evaluating the impact of the introduced GARV vaccine on the overall prevalence of viral gastroenteritis.

Altered molecular attributes and antimicrobial resistance patterns of Vibrio cholerae O1 El Tor strains isolated from the cholera endemic regions of India.

AIMS: The present study aimed to document the comparative analysis of differential hypervirulent features of Vibrio cholerae O1 strains isolated during 2018 from cholera endemic regions in Gujarat and Maharashtra (Western India) and West Bengal (Eastern India). METHODS AND RESULTS: A total of 87 V. cholerae O1 clinical strains from Western India and 48 from Eastern India were analysed for a number of biotypic and genotypic features followed by antimicrobial resistance (AMR) profile. A novel polymerase chain reaction was designed to detect a large fragment deletion in the Vibrio seventh pandemic island II (VSP-II) genomic region, which is a significant genetic feature of the V. cholerae strains that have caused Yemen cholera outbreak. All the strains from Western India belong to the Ogawa serotype, polymyxin B-sensitive, hemolytic, had a deletion in VSP-II (VSP-IIC) region and carried Haitian genetic alleles of ctxB, tcpA and rtxA. Conversely, 14.6% (7/48) of the strains from Eastern India belonged to the Inaba serotype, polymyxin B-resistant, nonhemolytic, harboured VSP-II other than VSP-IIC type, classical ctxB, Haitian tcpA and El Tor rtxA alleles. Resistance to tetracycline and chloramphenicol has been observed in strains from both regions. CONCLUSIONS: This study showed hypervirulent, polymyxin B-sensitive epidemic causing strains in India along with the strains with polymyxin B-resistant and nonhemolytic traits that may spread and cause serious disease outcomes in future. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcomes of this study can help to improve the understanding of the hyperpathogenic property of recently circulating pandemic Vibrio cholerae strains in India. Special attention is also needed for the monitoring of AMR surveillance because V. cholerae strains are losing susceptibility to many antibiotics used as a second line of defence in the treatment of cholera.

Characterization of diarrhoeagenic Escherichia coli with special reference to antimicrobial resistance isolated from hospitalized diarrhoeal patients in Kolkata (2012-2019), India.

AIMS: This study analyses the prevalence and antimicrobial resistance (AMR) of major diarrhoeagenic Escherichia coli (DEC) pathotypes detected in hospitalized diarrhoeal patients in Kolkata, India, during 2012-2019. METHODS AND RESULTS: A total of 8891 stool samples were collected from the Infectious Diseases Hospital, Kolkata and screened for the presence of enteric pathogens. Multiplex PCR identified the presence of DEC in 7.8% of the samples, in which ETEC was most common (47.7%) followed by EAEC (38.4%) and EPEC (13.9%). About 54% cases were due to sole DEC infections. Majority of the mixed DEC infections were caused by the Vibrio spp. (19.1%) followed by Rotavirus (14.1%) and Campylobacter spp. (8.4%). ETEC and EAEC were associated significantly with diarrhoea in children <5 years of age, whereas EPEC and also ETEC were prevalent in patients aged between 5 and 14 years. AMR profile showed high prevalence of multidrug resistance (MDR) among DEC (56.9%) in which 9% were resistant to antibiotics of six different antimicrobial classes. Screening of the AMR conferring genes of DEC showed the presence of blaCTX-M3 (30.2%) in highest number followed by blaTEM (27.5%), tetB (18%), sul2 (12.6%), strA (11.8%), aadA1 (9.8%), blaOXA-1 (9%), dfrA1 (1.6%) and blaSHV (1.2%). CONCLUSIONS: These findings highlighted the high prevalence of MDR in major DEC pathotypes that could be considered as the leading aetiological bacterial agent responsible for diarrhoea and suggests a significant public health threat. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can help to improve the understanding of the epidemiology of DEC infections in patients with diarrhoea. Monitoring of AMR surveillance needs special attention because the DEC isolates were highly resistant to commonly used antimicrobials in the treatment of diarrhoea.

Chitin degradation and its effect on natural transformation: a systematic genetic study in Vibrio parahaemolyticus.

The degradation of polymeric chitin by chitinase liberates soluble N-acetyl glucosamine oligosaccharides (GlcNAcn≥2), a source of nutrition that can also induce a state of natural genetic competence in Vibrio parahaemolyticus. This analysis revealed that among seven predicted chitinases, the synergistic action of VPA0055 (ChiA2), VP0619 (ChiB), and VPA0832 (Cdx) were essential for the robust growth and high transformation frequency on chitin. The endochitinase, ChiA2, and periplasmic chitinase, Cdx were indispensable for chitin degradation. ChiB was not essential for growth on chitin but did have an effect on the rate of chitin degradation. Interestingly, the loss of Cdx drastically reduced growth on insoluble chitin, but growth on soluble GlcNAc5/6 remained same. The utilization of GlcNAc5/6 was only inhibited when there was mutation of Cdx with the other periplasmic chitinases VP0755 and VP2486. This suggests that Cdx might also be involved in extracellular degradation of chitin, in addition to its role as a periplasmic chitinase. Moreover, the periplasmic chitin oligosaccharide-binding protein (CBP) was found to be essential for the efficient utilization of chitin. The CBP was specifically needed for the processing of GlcNAc4-6 during growth on chitin. Overall, this study provides detailed analysis of the machinery behind chitin degradation in V. parahaemolyticus.

Second extracellular protease mediating maturation of Vibrio mimicus hemolysin.

Vibrio mimicus is a bacterium that causes gastroenteritis in humans. This pathogen produces an enterotoxic hemolysin called V. mimicus hemolysin (VMH), which is secreted extracellularly as an inactive 80-kDa protoxin and converted to a 66-kDa mature toxin through cleavage between Arg151 and Ser152. The 56-kDa serine protease termed V. mimicus trypsin-like protease (VmtA) is known to mediate this maturating process. However, some strains including strain ES-20 does not possess the vmtA gene. In the present study, the vmtA-negative strains were found to have a replaced gene that encodes a 43-kDa (403 aa) precursor of a serine protease designated by VmtX (V. mimicus trypsin-like protease X). To examine whether VmtX is also involved in the maturation of VMH, VmtX was isolated from the culture supernatant of V. mimicus strain NRE-20, a metalloprotease-negative mutant constructed from strain ES-20. Concretely, the culture supernatant was fractionated with 70% saturated ammonium sulfate and subjected to affinity column chromatography using a HiTrap Benzamidine FF column. The analysis of the N-terminal amino acid sequences of the proteins in the obtained VmtX preparation indicated that the 39-kDa protein was active VmtX consisting of 371 aa (Ile33-Ser403). The VmtX preparation was found to activate pro-VMH through generation of the 66-kDa protein. Additionally, treatment of the VmtX preparation with serine protease inhibitors, such as leupeptin and phenylmethylsulfonyl fluoride, significantly suppressed the activities to hydrolyze the specific peptide substrate and to synthesize the 66-kDa toxin. These findings indicate that VmtX is the second protease that mediats the maturation of VMH.

Genetic characterization and evolutionary analysis of norovirus genotypes circulating among children in eastern India during 2018-2019.

Noroviruses are significant etiological agents of acute gastroenteritis (AGE) across all age groups, especially in children under 5 years of age. Although the prevalence of norovirus infection is known to have increased in various countries, in India there are few reports pertaining to the norovirus disease burden. We investigated the epidemiology and molecular characteristics of noroviruses in children seeking health care at two hospitals in Kolkata, Eastern India. Faecal specimens were collected between January 2018 and December 2019 from 2812 children under 5 years of age with acute gastroenteritis. Noroviruses were detected in 6.04% (170/2812) of the samples, and 12.9% (22/170) of these were cases of coinfection with rotavirus. Among children (≤5 years), a higher infection rate (8.2%, n = 94/1152) was observed in the 6 to 12 month age group. GII.4 Sydney 2012 was the dominant norovirus capsid genotype (n = 75/90, 83.3%), followed by GII.3 (n = 10/90, 11.1%). Other capsid types GII.13 (n = 4/90, 4.4%) and GII.17 (n = 1/90; 1.1%) were also detected at low frequency. Phylogenetic analysis showed that the GII.P16 polymerase of strains in this region clustered with those of the phylogenetically distinct monophyletic clade of GII.P16 strains, whose members have been circulating worldwide since 2014. Inter-genotypic norovirus recombinants such as GII.P16-GII.3 (n = 10) and GII.P16-GII.13 (n = 4) were also observed among the circulating strains. In comparison to previous studies from eastern India, the present study shows a higher detection rate of norovirus infection in the paediatric population suffering from acute gastroenteritis. Continuous surveillance is required for predicting the emergence of novel genotypes and recombinant strains and for future vaccine development.

Molecular characterization and antibiotic resistance of Vibrio parahaemolyticus from Indian oyster and their probable implication in food chain.

Vibrio parahaemolyticus is one of the leading causes of diarrhoea and gastroenteritis in human on consumption of raw or insufficiently cooked seafood. This study was aimed at isolating and characterizing the pathogenic and pandemic V. parahaemolyticus from oysters (n = 90) in coastal parts of West Bengal, India; their antibiotic resistance and potential for involvement in the food chain. During bacteriological culture, typical V. parahaemolyticus colony was recovered in 88.9% samples followed by presumptive identification in 71 (78.9%) samples by characteristic biochemical (K/A) test. All the presumptive isolates (n = 71) were confirmed by species specific Vp-toxR PCR assay. Of these, 10 (14.08%) were tdh+ and none for the trh. Further, 5 (50%) of these tdh+ isolates were found to carry the pandemic potential gene in PGS-PCR assay; however, none in GS-PCR. Majority (80%) of these pathogenic (tdh+) isolates belonged to pandemic serovars (OUT: KUT; OUT: K24; O1: KUT; O1:K25; O10: KUT) and only 20% to non-pandemic serovars (OUT: K15; O9:K17). All the isolates (100%) exhibited resistance to cefpodoxime followed by ampicillin and cefotaxime (90%), ceftizoxime (60%), tetracycline (50%), ceftriaxone (40%), ciprofloxacin and nalidixic acid (10% each). Overall, the study findings suggested that 11.1% (10/90) of commonly marketed oysters in this area were harbouring pathogenic V. parahaemolyticus. Moreover, 5.5% (5/90) of the oyster population were harbouring pandemic strains of this pathogen. Besides, the pathogenic isolates from oysters were exhibiting a considerable genetic relatedness (53 to 70%) to human clinical isolates in PFGE analysis that relates to a substantial public health risk. Further, their multidrug resistance added gravity to the antimicrobial resistance (AMR), a globally growing public health threat and this is a critical area of concern especially during the treatment of foodborne gastroenteritis.

Spirulina platensis and its ingredient biopterin glucoside improved insulin sensitivity in non-alcoholic steatohepatitis model.

Non-alcoholic steatohepatitis is the chronic liver disease leading to cirrhosis and cancer and its prevalence is increasing. Some agents are under clinical trials for non-alcoholic steatohepatitis treatment. We previously reported Spirulina (Arthrospira) platensis effectively prevented non-alcoholic steatohepatitis progression in our model rats. The contribution of phycocyanin, an ingredient of Spirulina (Arthrospira) platensis, was limited. We, therefore, have looked for more active components of Spirulina (Arthrospira) platensis. In this study, we pursued the effect of biopterin glucoside, another bioactive ingredient of Spirulina (Arthrospira) platensis. We found Spirulina (Arthrospira) platensis and biopterin glucoside oral administrations effectively alleviated oxidative stress, inflammation and insulin signal failure, and prevented fibroblast growth factor 21 gene overexpression in non-alcoholic steatohepatitis rat livers. We concluded biopterin glucoside is a major component of Spirulina (Arthrospira) platensis action.

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation.

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.

Genomic characterization of antibiotic resistance-encoding genes in clinical isolates of Vibrio cholerae non-O1/non-O139 strains from Kolkata, India: generation of novel types of genomic islands containing plural antibiotic resistance genes.

Non-O1/non-O139 nontoxigenic Vibrio cholerae associated with cholera-like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non-O1/non-O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole-genome sequences of these strains were determined. From the analysis of these sequences, it becomes clear that all quinolone resistance isolates have mutations in quinolone resistance-determining regions. Further research on the genome sequence showed that four strains possess Class 1 integrons in their genomes, and that three of the four integrons are found to be located in their genomic islands. These genomic islands are novel types. This indicates that various integrons containing drug resistance genes are spreading among V. cholerae non-O1/non-O139 strains through the action of newly generated genomic islands.

Low Viability of Cholera Toxin-Producing Vibrio cholerae O1 in the Artificial Low Ionic Strength Aquatic Solution.

It has been well known that Vibrio cholerae inhabit in environmental water. As many patients infected with cholera toxin-producing V. cholerae O1 (toxigenic V. cholerae O1) emerge in Kolkata, India, it has been thought that toxigenic V. cholerae O1 is easily detected in environmental water in Kolkata. However, we could not isolate toxigenic V. cholerae O1 from environmental water in Kolkata, though NAG Vibrio (generic name of V. cholerae non-O1/non-O139) is constantly detected. To clear the reason for the non-isolation of toxigenic V. cholerae O1, we examined the viability of V. cholera O1 and NAG Vibrios in the artificial low ionic strength aquatic solution. We found that the viability of toxigenic V. cholerae O1 in the solution is low, but that of NAG Vibrios is high. Subsequently, we examined the viability of NAG Vibrios possessing cholera toxin gene (ctx) in the same condition and found that the viability of these NAG Vibrios is low. These results indicate that the existence of ctx in V. cholerae affects the viability of V. cholerae in the aquatic solution used in this experiment. We thought that there was closely relation between the low viability of toxigenic V. cholerae O1 in the artificial low ionic strength aquatic solution and the low frequency of isolation of the strain from environmental water.

Comparative proteomic analysis to characterize temperature-induced viable but non-culturable and resuscitation states in Vibrio cholerae.

Vibrio cholerae can survive environmental adversities by entering into a viable but non-culturable (VBNC) state and is able to resuscitate under favourable conditions. In this study, an environmental strain of V. cholerae (AN59) showed a decrease in culturability from 4×107 to ≤ 3 c.f.u. ml -1 in artificial seawater media at 4 °C within 35 days. During the course of VBNC progression, viability was confirmed by real-time RT-PCR which showed reduced but stable expression of molecular chaperones groEL and dnaK. Resuscitation was induced in VBNC microcosm by a temperature increase from 4 to 37 °C for 24 h. The results obtained from resuscitation and growth experiments suggest that 103-104 c.f.u. ml -1 of VBNC cells should recover upon temperature increase and grow to attain 107 c.f.u. ml -1. We used comparative proteomics to differentiate recovery from the VBNC state and selected 19 proteins whose expression was significantly variable between these two states. These proteins were mainly related to carbohydrate metabolism, phosphate utilization, stress response, transport and translation. The main difference in the proteome profile was higher protein expression in the recovery state compared to VBNC state. However, during recovery Pi-starvation led to expression of PhoX, PstB and Xds, which might help in utilization of extracellular DNA to promote growth after resuscitation. In addition, the expression of EctC suggests that osmotic adaptation is necessary to grow at high salinity. Detection of AhpC in the VBNC and recovery state indicates the significance of the oxidative stress response. A temperature-induced VBNC and recovery state is a combination of adaptive and survival responses under nutrient limitation.

Functional analysis of N-terminal propeptide in the precursor of Vibrio vulnificus metalloprotease by using cell-free translational system.

Vibrio vulnificus is a human pathogen causing fatal septicemia with edematous and hemorrhagic skin damage. Among multiple virulence factors, an extracellular metalloprotease termed as V. vulnificus protease (VVP) is known to play a crucial role in eliciting the skin damage. The mature VVP (413 aa) is composed of two domains, the N-terminal core domain with proteolytic activity and the C-terminal domain mediates efficient attachment to protein substrates. However, VVP is produced as an inactive precursor (609 aa) with a signal peptide (24 aa) and propeptide (172 aa). In order to clarify the function of propeptide, a series of DNA fragments encoding the VVP precursor and its various domains were designed and the proteins were expressed in vitro by using cell-free translational system. The results indicated that the propeptide might function as an intramolecular chaperon to promote the proper folding of both N-terminal and C-terminal domains. The obtained results also suggest that the propeptide, itself was unstable and thus digested easily by the enzymes present in cell lysate used for cell-free system. Additionally, the C-terminal domain in VVP found to inhibit the folding of the N-terminal domain in absence of propeptide.