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Background Worldwide, noroviruses are the leading cause of acute gastroenteritis (AGE) in people of all age groups. In India, norovirus rates between 1.4 to 44.4% have been reported. Only a very few complete norovirus genome sequences from India have been reported. Objective To perform full genome sequencing of noroviruses circulating in India during 2017-2021, identify circulating genotypes, assess evolution including detection of recombination events. Methodology Forty-five archived norovirus-positive samples collected between October 2017 to July 2021 from patients with AGE from two hospitals in Kolkata, India were processed for full genome sequencing. Phylogenetic analysis, recombination breakpoint analysis and comprehensive mutation analysis were also performed. Results Full genome analysis of norovirus sequences revealed that strains belonging to genogroup (G)I were genotyped as GI.3[P13]. Among the different norovirus capsid-polymerase combinations, GII.3[P16], GII.4 Sydney[P16], GII.4 Sydney[P31], GII.13[P16], GII.16[P16] and GII.17 were identified. Phylogenetic analysis confirmed phylogenetic relatedness with previously reported norovirus strains and all viruses were analyzed by Simplot. GII[P16] viruses with multiple residue mutations within the non-structural region were detected among circulating GII.4 and GII.3 strains. Comprehensive mutation analysis and selection pressure analysis of GII[P16] viruses showed positive as well as negative selection sites. A GII.17 strain (NICED-BCH-11889) had an untypeable polymerase type, closely related to GII[P38]. Conclusion This study highlights the circulation of diverse norovirus strains in eastern India. These findings are important for understanding norovirus epidemiology in India and may have implications for future vaccine development., Author(s): Mahadeb Lo[sup.1], Yen Hai Doan[sup.2], Suvrotoa Mitra[sup.1], Ritubrita Saha[sup.1], Shin-ichi Miyoshi[sup.3,4], Kei Kitahara[sup.3], Shanta Dutta[sup.5], Tomoichiro Oka[sup.6] and Mamta Chawla-Sarkar[sup.1] Introduction Noroviruses are a major cause of acute gastroenteritis [...]

Objective To evaluate the clinical accuracy of rapid diagnostic tests for the detection of Ebola virus. Methods We searched MEDLINE[R], Embase[R] 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. Objectif Evaluer la precision clinique des tests de diagnostic rapide pour depister le virus Ebola. Methodes Nous avons explore les bases de donnees MEDLINE[R], Embase[R] et Web of Science a la recherche d'articles, publies entre 1976 et octobre 2021, qui evoquaient des etudes cliniques mesurant les performances des tests de diagnostic rapide du virus Ebola compares a une reaction en chaine par polymerase apres transcription inverse (RT-PCR). Nous avons determine la qualite de ces etudes a l'aide des criteres QUADAS-2. Enfin, pour estimer la sensibilite et la specificite regroupees de ces tests de diagnostic rapide, nous avons eu recours a une meta-analyse bivariee a effets aleatoires. Resultats Nos recherches nous ont permis d'identifier 113 etudes uniques, dont neuf correspondaient aux criteres d'inclusion. Ces etudes avaient ete menees en Guinee, au Liberia, en Republique democratique du Congo et en Sierra Leone, et portaient sur douze tests de diagnostic rapide. Nous avons inclus huit etudes dans notre meta-analyse. La sensibilite et la specificite combinees des tests rapides s'elevaient respectivement a 86% (intervalle de confiance de 95%, IC: 80-91) et 95% (IC de 95%: 91-97). Neanmoins, la sensibilite combinee baissait a 83% (IC de 95%: 77-88) apres retrait des valeurs aberrantes. Lorsque l'analyse se limitait aux etudes utilisant la RT-PCR d'altona Diagnostics en guise de reference, la sensibilite combinee augmentait jusqu'a 90% (IC de 95%: 82-94). Elle atteignait meme 99% (IC de 95%: 67-100) quand l'analyse se limitait aux etudes basees sur des echantillons de sang total ou capillaire. Conclusion Les tests de diagnostic rapide pris en compte ne detectaient pas tous les cas de maladie a virus Ebola. Neanmoins, en depit d'une sensibilite et d'une specificite moderees, ils demeurent de precieux outils, en particulier pour le triage et le depistage du virus Ebola dans les zones reculees. Objetivo Evaluar la precision clinica de las pruebas diagnosticas rapidas para la deteccion del virus del Ebola. Metodos Se realizaron busquedas en MEDLINE[R], Embase[R] y Web of Science de articulos publicados entre 1976 y octubre de 2021 que informaran sobre estudios clinicos en los que se evaluara el rendimiento de las pruebas diagnosticas rapidas para detectar el virus del Ebola en comparacion con la reaccion en cadena de la polimerasa con retrotranscripcion (RT-PCR). Se evaluo la calidad de los estudios mediante los criterios QUADAS-2. Para estimar la sensibilidad y la especificidad combinadas de estas pruebas diagnosticas rapidas, se utilizo un metanalisis bivariante de efectos aleatorios. Resultados La busqueda identifico 113 estudios singulares, de los que nueve cumplian los criterios de inclusion. Los estudios se realizaron en la Republica Democratica del Congo, Guinea, Liberia y Sierra Leona y evaluaron doce pruebas diagnosticas rapidas. Se incluyeron ocho estudios en el metanalisis. La sensibilidad y la especificidad agrupadas de las pruebas rapidas fueron del 86 % (intervalo de confianza del 95%, IC: 80-91) y del 95% (IC del 95%: 91-97), respectivamente. Sin embargo, la sensibilidad agrupada disminuyo al 83 % (IC del 95%: 77-88) tras eliminar los valores atipicos. La sensibilidad agrupada aumento al 90% (IC del 95%: 82-94) cuando el analisis se restringio a los estudios que utilizaban la RT-PCR de altona Diagnostics como criterio de referencia. La sensibilidad agrupada aumento al 99 % (IC del 95%: 67-100) cuando el analisis se restringio a los estudios que utilizaban muestras de sangre total o capilar. Conclusion Las pruebas diagnosticas rapidas incluidas no detectaron todos los casos de enfermedad por el virus del Ebola. Aunque la sensibilidad y la especificidad de estas pruebas son moderadas, siguen siendo herramientas valiosas, sobre todo utiles para el triaje y la deteccion del virus del Ebola en zonas remotas., Introduction Ebola virus disease was first discovered in 1976 in the Democratic Republic of the Congo and South Sudan. (1) This highly pathogenic disease is often fatal in humans; in [...]

Bacteria in the genus Vibrio are ubiquitous in estuarine and coastal waters. Some species (including Vibrio cholerae and Vibrio vulnificus) are known human pathogens causing ailments like cholera, diarrhea, or septicemia. Notably, V. vulnificus can also cause a severe systemic infection (known as vibriosis) in eels raised in aquaculture facilities. Water samples were periodically collected from the estuary of the Asahi River, located in the southern part of Okayama City, Japan. These samples were directly plated onto CHROMagar Vibrio plates, and colonies displaying turquoise-blue coloration were selected. Thereafter, polymerase chain reaction was used to identify V. cholerae and V. vulnificus. A total of 30 V. cholerae strains and 194 V. vulnificus strains were isolated during the warm season when the water temperature (WT) was higher than 20 °C. Concurrently, an increase in coliforms was observed during this period. Notably, V. vulnificus has two genotypes, designated as genotype 1 and genotype 2. Genotype 1 is pathogenic to humans, while genotype 2 is pathogenic to both humans and eels. The loop-mediated isothermal amplification method was developed to rapidly determine genotypes at a low cost. Of the 194 strains isolated, 80 (41.2%) were identified as genotype 1 strains. Among the 41 strains isolated when the WTs were higher than 28 °C, 25 strains (61.0%) belonged to genotype 1. In contrast, of the 32 strains isolated when the WTs were lower than 24 °C, 27 strains (84.4%) belonged to genotype 2. These results suggest that the distribution of the two genotypes was influenced by WT. [ABSTRACT FROM AUTHOR]

Background: The primary aim of this study was to investigate the occurrence, characteristics, and antimicrobial resistance patterns of various Shigella serotypes isolated from patients with acute diarrhea of the Infectious Diseases Hospital in Kolkata from 2011–2019. Principal findings: During the study period, Shigella isolates were tested for their serotypes, antibiotic resistance pattern and virulence gene profiles. A total of 5.8% of Shigella spp. were isolated, among which S. flexneri (76.1%) was the highest, followed by S. sonnei (18.7%), S. boydii (3.4%), and S. dysenteriae (1.8%). Antimicrobial resistance against nalidixic acid was higher in almost all the Shigella isolates, while the resistance to β-lactamases, fluoroquinolones, tetracycline, and chloramphenicol diverged. The occurrence of multidrug resistance was found to be linked with various genes encoding drug-resistance, multiple mutations in the topoisomerase genes, and mobile genetic elements. All the isolates were positive for the invasion plasmid antigen H gene (ipaH). Dendrogram analysis of the plasmid and pulsed-field electrophoresis (PFGE) profiles revealed 70–80% clonal similarity among each Shigella serotype. Conclusion: This comprehensive long-term surveillance report highlights the clonal diversity of clinical Shigella strains circulating in Kolkata, India, and shows alarming resistance trends towards recommended antibiotics. The elucidation of this study's outcome is helpful not only in identifying emerging antimicrobial resistance patterns of Shigella spp. but also in developing treatment guidelines appropriate for this region. Author summary: Shigella spp. is one of the WHO-designated "antibiotic-resistant priority pathogens" that is the leading cause of bacterial diarrhea among children worldwide. The distribution of Shigella species varies depending on the geographic location, particularly in regions where environmental risk factors like substandard water quality, limited healthcare accessibility, and insufficient sanitation are widespread. The presence of serotype diversity and drug resistance combinations often leads to treatment failures. Therefore, it is crucial to comprehend the prevailing serogroups and antimicrobial resistance patterns of Shigella species in different geographical areas to implement effective treatment strategies. We have identified 535 Shigella positive cases from 9276 diarrheal cases. Multidrug-resistant (MDR) Shigella globally requires a profound understanding of its genetic diversity, as there is significant regional variation in MDR strains. In our study, we observed a broad spectrum of resistance among the Shigella strains, with over 53.6% of the isolates demonstrating multidrug resistance. Representative isolates were evaluated for antimicrobial resistance encoding and virulence-associated genes as they enact significantly in disease development and interaction with the host immune system. This report marks the initial presentation of the outcomes from long-term surveillance and molecular characterization of antimicrobial resistances in clinical Shigella strains in Kolkata since 2011. The outcome of this investigations will guide in identifying emerging antimicrobial resistance trend, formulating suitable treatment guidelines tailored to the community's needs, and establishing baseline data for comparison with potential outbreak strains in the future. [ABSTRACT FROM AUTHOR]

Human Salmonella infections pose significant public health challenges globally, primarily due to low diagnostic yield of systemic infections, emerging and expanding antibiotic resistance of both the typhoidal and non-typhoidal Salmonella strains and the development of asymptomatic carrier state that functions as a reservoir of infection in the community. The limited long-term efficacy of the currently licensed typhoid vaccines, especially in smaller children and non-availability of vaccines against other Salmonella serovars necessitate active research towards developing a multivalent vaccine with wider coverage of protection against pathogenic Salmonella serovars. We had earlier reported immunogenicity and protective efficacy of a subunit vaccine containing a recombinant outer membrane protein (T2544) of Salmonella Typhi in a mouse model. This was achieved through the robust induction of serum IgG, mucosal secretory IgA and Salmonella-specific cytotoxic T cells as well as memory B and T cell response. Here, we report the development of a glycoconjugate vaccine, containing high molecular weight complexes of Salmonella Typhimurium Ospecific polysaccharide (OSP) and recombinant T2544 that conferred simultaneous protection against S. Typhi, S. Paratyphi, S. Typhimurium and cross-protection against S. enteritidis in mice. Our findings corroborate with the published studies that suggested the potential of Salmonella OSP as a vaccine antigen. The role of serum antibodies in vaccine-mediated protection is suggested by rapid seroconversion with high titers of serum IgG and IgA, persistently elevated titers after primary immunization along with a strong antibody recall response with higher avidity serum IgG against both OSP and T2544 and significantly raised SBA titers of both primary and secondary antibodies against different Salmonella serovars. Elevated intestinal secretory IgA and bacterial motility inhibition by the secretory antibodies supported their role as well in vaccine-induced protection. Finally, robust induction of T effector memory response indicates long term efficacy of the candidate vaccine. The above findings coupled with protection of vaccinated animals against multiple clinical isolates confirm the suitability of OSP-rT2544 as a broad-spectrum candidate subunit vaccine against human infection due to typhoidal and nontyphoidal Salmonella serovars. [ABSTRACT FROM AUTHOR]

We have previously reported 1,2,6,7-tetraoxaspiro [7.11]nonadecane (N-89) as a promising antimalarial compound. In this study, we evaluated the effect of transdermal therapy (tdt) of N-89 in combination (tdct) with other antimalarials as an application for children. We prepared ointment formulas containing N-89 plus another antimalarial drug, specifically, mefloquine, pyrimethamine, or chloroquine. In a 4-day suppressive test, the ED50 values for N-89 alone or combined with either mefloquine, pyrimethamine, or chloroquine were 18, 3, 0.1, and 3 mg/kg, respectively. Interaction assays revealed that N-89 combination therapy showed a synergistic effect with mefloquine and pyrimethamine, but chloroquine provoked an antagonistic effect. Antimalarial activity and cure effect were compared for single-drug application and combination therapy. Low doses of tdct N-89 (35 mg/kg) combined with mefloquine (4 mg/kg) or pyrimethamine (1 mg/kg) gave an antimalarial effect but not a cure effect. In contrast, with high doses of N-89 (60 mg/kg) combined with mefloquine (8 mg/kg) or pyrimethamine (1 mg/kg), parasites disappeared on day 4 of treatment, and mice were completely cured without any parasite recurrence. Our results indicated that transdermal N-89 with mefloquine and pyrimethamine provides a promising antimalarial form for application to children. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Shigella remains one of the most common causes of diarrhoea in South Asia. Current estimates of the prevalence of Shigella are critical for guiding control measures. We estimated the prevalence of Shigella species and serogroups in South Asia.Methods: We performed a systematic review using PubMed, EMBASE, Google Scholar, and Web of Science for peer-reviewed studies published between 2000 and June 19, 2022. We also manually searched the reference lists of the reviewed studies to identify additional studies. We included studies that detected the presence of Shigella in stool by culture or polymerase chain reaction (PCR). Studies associated with outbreaks were excluded. Two investigators independently reviewed the studies, extracted the data, and performed quality assessment. A random-effects meta-analysis was performed to determine the pooled prevalence of Shigella.Results: Our search yielded 5707 studies, of which 91 studies from five South Asian countries were included in the systematic review, 79 in the meta-analysis of Shigella prevalence and 63 in the meta-analysis of Shigella serogroups prevalence. The pooled prevalence of Shigella was 7% (95% CI: 6-7%), with heterogeneity (I2 = 98.7; p < 0.01). The prevalence of Shigella was higher in children aged < 5 years (10%; 95% CI: 8-11%), in rural areas (12%; 95% CI: 10-14%), and in studies using PCR (15%; 95% CI: 11-19%). Shigella flexneri (58%) was the most abundant serogroup, followed by Shigella sonnei (19%), Shigella boydii (10%), and Shigella dysenteriae (9%). S. flexneri 2a was the most frequently isolated serotype (36%), followed by serotype 3a (12%), serotype 6 (12%), and serotype 1b (6%). The prevalence of non-typeable Shigella was 10.0%.Conclusions: Although the prevalence of Shigella in South Asia remains generally high, it varies by age group and geographical area, with data lacking in some countries. Effective Shigella vaccines would be advantageous for both endemic communities and travellers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Linear alkylbenzene sulfonate (LAS) and polyoxyethylene lauryl ether (POLE) are the major surfactants in washing detergents. In the present study, we isolated surfactant‐resistant bacteria from soil samples collected from a sports ground and a farm field. The samples were treated with 2.0% LAS or POLE at 25°C for 30 min and cultivated on agar plates at 25°C for several days, after which manifold bacterial colonies were isolated. Thereafter, we tested the ability of each bacterial isolate to resist the antibacterial activity of the surfactant. Ten LAS‐resistant strains were isolated, and all were found to be Gram‐negative bacteria such as Enterobacter and Pseudomonas. On the other hand, 18 POLE‐resistant strains were isolated, of which 14 were Gram‐positive bacteria including Bacillus and Microbacterium. Notably, one POLE‐resistant strain was identified as Bacillus cereus, a potential causative agent for foodborne illness. The genera of LAS‐ and POLE‐resistant bacteria did not overlap. Therefore, the combination of LAS and POLE could be more effective to eliminate soil bacteria from clothes and/or daily necessities. [ABSTRACT FROM AUTHOR]

Purpose: The multidrug resistance Enterobacteriaceae cause many serious infections resulting in prolonged hospitalization, increased treatment charges and mortality rate. In this study, we characterized blaNDM-5-positive multidrug resistance commensal Escherichia coli (CE) isolated from diarrheal patients in Kolkata, India. Methods: Three CE strains were isolated from diarrheal stools, which were negative for different pathogroups of diarrheagenic E. coli (DEC). The presence of carbapenemases encoding genes and other antimicrobial resistance genes (ARGs) was detected using PCR. The genetic arrangement adjoining blaNDM-5 was investigated by plasmid genome sequencing. The genetic relatedness of the strains was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) methods. Results: In addition to colistin, the blaNDM-5-positive CE strains showed resistance to most of the antibiotics. Higher MICs were detected for ciprofloxacin (> 32 mg/L) and imipenem (8 mg/L). Molecular typing revealed that three CE strains belonged to two different STs (ST 101 and ST 648) but they were 95% similar in the PFGE analysis. Screening for ARGs revealed that CE strains harbored Int-1, blaTEM, blaCTX-M3, blaOXA-1, blaOXA-7, blaOXA-9, tetA, strA, aadA1, aadB, sul2, floR, mph(A), and aac(6´)-Ib-cr. In conjugation experiment, transfer frequencies ranged from 2.5× 10− 3 to 8.4x10− 5. The blaNDM-5 gene was located on a 94-kb pNDM-TC-CE-89 type plasmid, which is highly similar to the IncFII plasmid harboring an IS26-IS30-blaNDM-5-bleMBL-trpF-dsbd-IS 91-dhps structure. Conclusion: To the best of our knowledge, this is the first report on carbapenem resistance involving the blaNDM-5 gene in CE from diarrheal patients. The circulation of blaNDM-5 gene in CE is worrisome, since it has the potential to transfer blaNDM-5 gene to other enteric pathogens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, L-alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE, constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon, pvsC, encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE, encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA. Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

A complex virulence-regulatory cascade controls expression of the cholera toxin genes (ctxAB) in Vibrio cholerae , which eventually leads to the production and secretion of choleragen (CT), responsible for rice watery diarrhoea in infected individuals. The cholera toxin promoter (P ctxAB) contains a series of heptad repeats (5′-TTTTGAT-3′), which has previously been shown to play a crucial role in transcriptional regulation of ctxAB by recruiting the transcriptional activators ToxT, ToxR and the nucleoid-associated protein H-NS along the ctx promoter. The number of these repeats differs not only between the two biotypes of V. cholerae O1 strains, but also among the strains belonging to the same biotype. In this study, we examined if regulation of P ctxAB is influenced in any way by the number of these repeats. Based on our observations, we posit that ctx activation indeed depends on the number of TTTTGAT heptad repeats within P ctxAB , and occupation of the distal repeats by H-NS could prevent transcriptional activation of the ctx genes in V. cholerae O1 pandemic isolates. Our results suggest that ToxT-dependent transcriptional activation may not require entire displacement of H-NS and supports a recently described revised model of ToxT and H-NS mediated P ctxAB transcriptional regulation. [ABSTRACT FROM AUTHOR]

The Fur target genes from Vibrio parahaemolyticus reveal a siderophore utilization gene containing operon that encodes a TonB-dependent receptor and the ferric vibrioferrin receptor. Insertion mutation and genetic complementation analyses indicate that the ferric vibrioferrin receptor mediates iron transport in V. parahaemolyticus.

Cholera toxin (CT)-producing Vibrio cholerae O1 and O139 cause acute diarrheal disease and are proven etiological agents of cholera epidemics and pandemics. On the other hand, V. cholerae non-O1/non-O139 are designated as non-agglutinable (NAG) vibrios and are not associated with epidemic cholera. The majority of NAG vibrios do not possess the gene for CT (ctx). In this study, we isolated three NAG strains (strains No. 1, 2, and 3) with ctx from pond water in Kolkata, India, and examined their pathogenic properties. The enterotoxicity of the three NAG strains in vivo was examined using the rabbit ileal intestinal loop test. Strain No. 1 induced the accumulation of fluid in the loop, and the volume of fluid was reduced by simultaneous administration of anti-CT antiserum into the loop. The volume of fluid in the loop caused by strains No. 2 and 3 was small and undetectable, respectively. Then, we cultured these three strains in liquid medium in vitro at two temperatures, 25°C and 37°C, and examined the amount of CT accumulated in the culture supernatant. CT was accumulated in the culture supernatant of strain No.1 when the strain was cultured at 25°C, but that was low when cultured at 37°C. The CT amount accumulated in the culture supernatants of the No. 2 and No. 3 strains was extremely low at both temperature under culture conditions examined. In order to clarify the virulence properties of these strains, genome sequences of the three strains were analyzed. The analysis showed that there was no noticeable difference among three isolates both in the genes for virulence factors and regulatory genes of ctx. However, vibrio seventh pandemic island-II (VSP-II) was retained in strain No. 1, but not in strains No. 2 or 3. Furthermore, it was revealed that the genotype of the B subunit of CT in strain No. 1 was type 1 and those of strains No. 2 and 3 were type 8. Histopathological examination showed the disappearance of villi in intestinal tissue exposed to strain No. 1. In addition, fluid accumulated in the loop due to the action of strain No. 1 had hemolytic activity. This indicated that strain No. 1 may possesses virulence factors to induce severe syndrome when the strain infects humans, and that some strains of NAG vibrio inhabiting pond water in Kolkata have already acquired virulence, which can cause illness in humans. There is a possibility that these virulent NAG vibrios, which have acquired genes encoding factors involved in virulence of V. cholerae O1, may emerge in various parts of the world and cause epidemics in the future. [ABSTRACT FROM AUTHOR]