Glycyrrhizin, an inhibitor of HMGB1 induces autolysosomal degradation function and inhibits Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori is a key agent for causing gastric complications linked with gastric disorders. In response to infection, host cells stimulate autophagy to maintain cellular homeostasis. However, H. pylori have evolved the ability to usurp the host's autophagic machinery. High mobility group box1 (HMGB1), an alarmin molecule is a regulator of autophagy and its expression is augmented during infection and gastric cancer. Therefore, this study aims to explore the role of glycyrrhizin (a known inhibitor of HMGB1) in autophagy during H. pylori infection. MAIN METHODS: Human gastric cancer (AGS) cells were infected with the H. pylori SS1 strain and further treatment was done with glycyrrhizin. Western blot was used to examine the expression of autophagy proteins. Autophagy and lysosomal activity were monitored by fluorescence assays. A knockdown of HMGB1 was performed to verify the effect of glycyrrhizin. H. pylori infection in in vivo mice model was established and the effect of glycyrrhizin treatment was studied. RESULTS: The autophagy-lysosomal pathway was impaired due to an increase in lysosomal membrane permeabilization during H. pylori infection in AGS cells. Subsequently, glycyrrhizin treatment restored the lysosomal membrane integrity. The recovered lysosomal function enhanced autolysosome formation and concomitantly attenuated the intracellular H. pylori growth by eliminating the pathogenic niche. Additionally, glycyrrhizin treatment inhibited inflammation and improved gastric tissue damage in mice. CONCLUSION: This study showed that inhibiting HMGB1 restored lysosomal activity to ameliorate H. pylori infection. It also demonstrated the potential of glycyrrhizin as an antibacterial agent to address the problem of antimicrobial resistance.

Sequence Polymorphisms in Vibrio cholerae HapR Affect Biofilm Formation under Aerobic and Anaerobic Conditions.

We investigated the influence of hapR sequence mutations on the biofilm formation of Vibrio cholerae. In this study, hapR sequences from 85 V. cholerae strains belonging to both pandemic and nonpandemic serogroup were investigated through phylogenetic and sequence analyses. Biofilm formation assays under aerobic and anaerobic conditions were also performed. Sequence variations include single point mutations and insertions/deletions (indels) leading to either truncated or frameshifted HapR. Population structure analysis revealed two major hapR haplogroups, hapR1 and hapR2. Phylogenetic reconstruction displayed a hypothetical ancestral hapR sequence located within the hapR1 haplogroup. Higher numbers of single nucleotide polymorphisms and genetic diversity indices were observed in hapR1, while indels occurred dominantly in hapR2. Aerobic conditions supported more robust biofilms compared to anaerobic conditions. Strains with frameshifted HapR produced the largest amount of biofilm under both oxygen conditions. Quantitative real-time PCR assay confirmed that strains with truncated and frameshifted HapR resulted in a nonfunctional regulator as exhibited by the significantly low hapA gene expression. The present study shows that HapR mutations had a strong influence on biofilm formation and that sequence polymorphisms leading to the disruption of DNA-binding sites or dimerization of the HapR will result in more-robust V. cholerae biofilms. IMPORTANCE Our study revealed an ancestral hapR sequence from a phylogenetic reconstruction that displayed the evolutionary lineage of the nonpandemic to the pandemic strains. Here, we established hapR1 and hapR2 as major hapR haplogroups. The association of the O1 and O139 serogroups with the hapR2 haplogroup demonstrated the distinction of hapR2 in causing cholera infection. Moreover, mutations in this regulator that could lead to the disruption of transcription factor-binding sites or dimerization of the HapR can significantly affect the biofilm formation of V. cholerae. These observations on the relationship of the hapR polymorphism and V. cholerae biofilm formation will provide additional considerations for future biofilm studies and insights into the epidemiology of the pathogen that could ultimately help in the surveillance and mitigation of future cholera disease outbreaks.

Distribution of virulence factors and its relatedness towards the antimicrobial response of enterotoxigenic Escherichia coli strains isolated from patients in Kolkata, India.

AIM: Enterotoxigenic Escherichia coli (ETEC) is one of the most widely recognized diarrhoeal pathogens in developing countries. The advancement of ETEC vaccine development depends on the antigenic determinants of the ETEC isolates from a particular geographical region. So, the aim here was to comprehend the distribution of virulence determinants of the clinical ETEC strains of this region. Additionally, an attempt was made to find any correlation with the antimicrobial response pattern. METHODS AND RESULTS: Multiplex PCR was employed to identify virulence determinants followed by confirmatory singleplex PCR. For observation of antibiotic response, the Kirby-Bauer method was used. Out of 379 strains, 46% of strains harboured both the enterotoxins ST and LT, whereas 15% were LT only. Among the major colonization factors (CFs), CS6 (41%) was the most prevalent followed by CFA/I (35%) and CFA/III was the lowest (3%). Among the minor CFs, CS21 (25%) was most prevalent, while CS15 showed the lowest (3%) presence. Among the non-classical virulence factors, EatA (69%) was predominant. ETEC strains harbouring CS6 showed resistance towards the commonly used drug Ciprofloxacin (70%). CONCLUSION: CS6 and elt+est toxin genes co-occurred covering 51% of the isolates. CS21 was found in most strains with est genes (43%). EatA was found to occur frequently when ST was present alone or with LT. CS6-harbouring strains showed an independent correlation to antimicrobial resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study would aid in identifying the commonly circulating ETEC isolates of Kolkata, India, and their prevalent virulence determinants. Knowledge of antibiotic resistance patterns would also help in the appropriate use of antibiotics. Furthermore, the study would aid in identifying the multivalent antigens suitable for region-specific ETEC vaccines with maximum coverage.

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.

Iron influences the expression of colonization factor CS6 of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a major pathogen of acute watery diarrhoea. The pathogenicity of ETEC is linked to adherence to the small intestine by colonization factors (CFs) and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). CS6 is one of the most common CFs in our region and worldwide. Iron availability functions as an environmental cue for enteropathogenic bacteria, signalling arrival within the human host. Therefore, iron could modify the expression of CS6 in the intestine. The objective of this study was to determine the effect of iron availability on CS6 expression in ETEC. This would help in understanding the importance of iron during ETEC pathogenesis. ETEC strain harbouring CS6 was cultured under increasing concentrations of iron salt to assess the effect on CS6 RNA expression by quantitative RT-PCR, protein expression by ELISA, promoter activity by ß-galactosidase activity, and epithelial adhesion on HT-29 cells. RNA expression of CS6 was maximum in presence of 0.2 mM iron (II) salt. The expression increased by 50-fold, which also reduced under iron-chelation conditions and an increased iron concentration of 0.4 mM or more. The surface expression of CS6 also increased by 60-fold in presence of 0.2 mM iron. The upregulation of CS6 promoter activity by 25-fold under this experimental condition was in accordance with the induction of CS6 RNA and protein. This increased CS6 expression was independent of ETEC strains. Bacterial adhesion to HT-29 epithelial cells was also enhanced by five-fold in the presence of 0.2 mM iron salt. These findings suggest that CS6 expression is dependent on iron concentration. However, with further increases in iron concentration beyond 0.2 mM CS6 expression is decreased, suggesting that there might be a strong regulatory mechanism for CS6 expression under different iron concentrations.

The Synergistic Role of Tip α, Nucleolin and Ras in Helicobacter pylori Infection Regulates the Cell Fate Towards Inflammation or Apoptosis.

Infection with Helicobacter pylori (H. pylori) leads to a fork in the road situation where it is critical and complex to judge the fate of the cell. We propose for the first time an in silico representation of a protein level network model that can unfold the mystery behind the cell fate decision between inflammation or cell proliferation or cell death. Upon infection TNF inducible protein α (Tip α) is internalised after binding with the cell surface receptor Nucleolin which is overexpressed on the cell surface thereby activating the Ras pathway. Tip α, Nucleolin and Ras decides the cell fate for apoptosis or abnormal cell proliferation along with ulcers in the gastric tract, hence we term it as the "death triad", which otherwise triggers the inflammatory pathway through downstream signalling of NF-κß. A series of proteins involved in the signalling cascade are portrayed through compartmentalization of the bacteria and the gut wall. The depicted network works synchronously toward an overarching goal of deciding between apoptosis or inflammation or proliferation. The model has been validated by simulating it with existing transcriptomic data along with clinical findings from patients infected with H. pylori across different regions in India. The results clearly indicate that for a short period of time there is increased binding of Tip α to Nucleolin and the receptor starts to saturate. This increases the tenacity of binding and the cell triggers an inflammatory cascade reaction which involves proinflammatory cytokines such as TNF α thereby progressing to inflammation by activating NF-κß downstream. On the other hand, Ras involved in interaction with nucleolin can be present both in its activated or inactivated state. Binding of Tip α as a monomer leads to desensitization of Nucleolin leading to cell survival and proliferation.

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.

A Point Mutation in carR Is Involved in the Emergence of Polymyxin B-Sensitive Vibrio cholerae O1 El Tor Biotype by Influencing Gene Transcription.

Antimicrobial peptides play an important role in host defense against Vibrio cholerae Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carRr and carRs alleles, respectively, and replaced the carRs allele of a sensitive strain with the carRr allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarRs protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca2+ but increased with a rise in pH. The downregulation of almEFG in CarRs strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

Genome-wide mRNA-miRNA profiling uncovers a role of the microRNA miR-29b-1-5p/PHLPP1 signalling pathway in Helicobacter pylori-driven matrix metalloproteinase production in gastric epithelial cells.

Aberrant expression of microRNAs (miRNAs) is associated with tumour progression, extracellular matrix remodelling, and cell proliferation. miRNAs modulate host gene expression during infection by pathogens such as Helicobacter pylori, which is associated with varying degrees of gastric pathology. In order to gain insight into the regulation of gene expression by miRNAs during H. pylori infection of gastric epithelial cells and its likely downstream consequences, we analysed the transcriptomes and miRnomes of AGS cells infected with H. pylori. In silico analysis of miRNA-mRNA interactions suggested that miR-29b-1-5p was a likely regulator of pathways associated with gastric epithelial cell pathology. We validated PH domain leucine rich phosphatase 1 (PHLPP1), a negative regulator of the Akt signalling pathway, as a target of miR-29b-1-5p. In an in vivo mouse model, we observed that infection with H. pylori was associated with upregulation of miR-29b-1-5p and downregulation of PHLPP1. Transfection with either a mimic or an inhibitor of miR-29b-1-5p confirmed that downregulation of PHLPP1 upregulates Akt-dependent NF-κB signalling leading to activation of matrix metalloproteinases 2 and 9, players in the degradation of extracellular matrix during H. pylori infection. The secreted antigen HP0175 was associated with upregulation of miR-29b-1-5p, regulation of metalloproteinase activity, and migration of AGS cells. Our study suggests that targeting the miR-29b-1-5p/PHLPP1 signalling axis could be a potential host-directed approach for regulating the outcome of H. pylori infection.

Characterization of the clonal profile of methicillin resistant Staphylococcus aureus isolated from patients with early post-operative orthopedic implant based infections.

BACKGROUND: To analyze the molecular epidemiology and to compare between the major methicillin resistant Staphylococcus aureus biotypes for association with patient characteristics who had an implant for closed fracture and developed early post-operative wound infections (POWI) in a tertiary care hospital of India. METHODS: Pulsed-field gel electrophoresis (PFGE), antimicrobial resistance, accessory gene regulator (agr) and staphylococcal cassette chromosome mec (SCCmec) types, Paton-Valentine leukocidin (PVL) gene, toxin gene profiling, biofilm formation and patient demographics were correlated with MLST clonal complexes (CC). FINDINGS: Overall eight different sequence types (STs) were detected with a predominance of ST239 (66%), ST22 (18%) and some minor types ST772, ST30 (4% each) ST1, ST642, ST6, ST107 (2% each). All ST239 isolates belong to CC239 and SCCmec III whereas ST22 isolates belong to CC22 and SCCmec IV. The isolates varied in the distribution of various toxin genes. With 63.63% biofilm formers ST239 were all multidrug resistant with frequent resistance to erythromycin, clindamycin, gentamicin, cefuroxime, amoxyclav and ciprofloxacin indicating doxycycline, amikacin, vancomycin and linezolid can be the drug of choice. CONCLUSION: This study shows that ST239 MRSA is still most prevalent strain with new emergence of ST642 and ST107 isolates in association with orthopedic implant based POWI. As compare to other ST types ST239 strain was associated with adverse treatment outcomes. This highlights the importance of improving nosocomial infection control measures in this unit.

'Modulation of the enzymatic activities of replicative helicase (DnaB) by interaction with Hp0897: a possible mechanism for helicase loading in Helicobacter pylori'.

DNA replication in Helicobacter pylori is initiated from a unique site (oriC) on its chromosome where several proteins assemble to form a functional replisome. The assembly of H. pylori replication machinery is similar to that of the model gram negative bacterium Escherichia coli except for the absence of DnaC needed to recruit the hexameric DnaB helicase at the replisome assembly site. In the absence of an obvious DnaC homologue inH. pylori, the question arises as to whether HpDnaB helicase is loaded at theHp-replication origin by itself or is assisted by other unidentified protein(s). A high-throughput yeast two-hybrid study has revealed two proteins of unknown functions (Hp0897 and Hp0340) that interact with HpDnaB. Here we demonstrate that Hp0897 interacts with HpDnaB helicase in vitro as well as in vivo Furthermore, the interaction stimulates the DNA binding activity of HpDnaB and modulates its adenosine triphosphate hydrolysis and helicase activities significantly. Prior complex formation of Hp0897 and HpDnaB enhances the binding/loading of DnaB onto DNA. Hp0897, along with HpDnaB, colocalizes with replication complex at initiation but does not move with the replisome during elongation. Together, these results suggest a possible role of Hp0897 in loading of HpDnaB at oriC.

Molecular characterization and antimicrobial resistance profile of Clostridium perfringens type A isolates from humans, animals, fish and their environment.

The study was aimed to characterize, and determine antibiogram of C. perfringens type A isolated from the feces of human and animal diarrhoeal cases, as well as healthy animals, meat of pigs and goats, gills and intestine of fish and samples from fish pond. A total of 460 samples, including human diarrhoeal cases (n = 130); diarrhoeal cases of pig (n = 52) and goat (n = 50); fecal samples from healthy pig (n = 50) and goat (n = 50); meat samples viz. pork meat (n = 52); goat meat (n = 50) and fish including their environmental sources (n = 26) were used for isolation and identification of C. perfringens type A. All the biochemically confirmed isolates were positive for species-specific 16S rRNA and cpa genes by PCR assays. Toxinotyping of C. perfringens type A isolates showed that overall prevalence of C. perfringens type A with only cpa+ gene was 43.2%; with cpa+ and cpb2+ genes was 45.4%; with cpa+ and cpe+ genes was 4.9%; however, with cpa+, cpb2+and cpe+ genes was 6.6%. Antimicrobial susceptibility testing revealed that 83.7% of isolates were resistant to three or more antibiotics.

Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children.

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

High prevalence of cagA positive Vac A s1m1 Helicobacter pylori strains isolated from patients suffering from various gastroduodenal diseases in Guwahati, Assam, India.

AIM AND BACKGROUND: Genotyping of H. pylori strains was identified on formalin-fixed paraffin wax-embedded tissue (FFPE) sections and correlated with severity grades of gastric mucosal pathologies in biopsies from upper gastrointestinal (UGI) tract from Guwahati, Assam. MATERIALS AND METHODS: In total, 168 gastric biopsies collected from patients with UGI symptoms underwent histological evaluation as per the updated Sydney system. RESULT: H. pylori-like organisms were identified with Warthin and Starry stain, and virulent genes were amplified using polymerase chain reaction (PCR) from genomic DNA extracted from FFPE sections by using QIAamp® DNA FFPE Tissue Kit. Histological examination identified H. pylori-like organisms in 100 biopsies, of which 96 were urease + ve. The prevalence of H. pylori infection was high in age groups 71-80 (88.8%) as compared to other age groups, and it was higher in females (78.9%) when compared to males. The prevalence of virulent genes in biopsies was 88.5% cagA and vacA s1m1, 31.2% iceA1, 32.2% iceA2, and 85.2% babA2. The histological parameters mononuclear cell infiltrate (P = 0.04) and atrophy (P = 0.03), showed statistically significant association with iceA2 and intestinal metaplasia with cagA (P = 0.01) vacAs1m1 (P = 0.01) and babA (P = 0.02) genotypes. Gastric erosion due to H. pylori infection and atrophy showed a significant association. A high bacterial density score was seen with the virulent genotypes. CONCLUSION: Our work reports for the first time a high prevalence (88.5%) of H. pylori cagA vacA s1m1 genotype in Guwahati, Assam. Association of gastric atrophy and intestinal metaplasia was seen with virulent genotypes. Results show the effectiveness of the FFPE kit for DNA extraction in remote areas where transportation and storage of biopsies are otherwise difficult.

Establishment of an intragastric surgical model using C57BL/6 mice to study the vaccine efficacy of OMV-based immunogens against Helicobacter pylori.

Chronic gastritis is one of the major symptoms of gastro-duodenal disorders typically induced by Helicobacter pylori (H. pylori). To date, no suitable model is available to study pathophysiology and therapeutic measures accurately. Here, we have presented a successful surgical infection model of H. pylori-induced gastritis in C57BL/6 mice that resembles features similar to human infection. The proposed model does not require any preparatory treatment other than surgical intervention. C57BL/6 mice were injected with wild-type SS1 (Sydney strain 1, reference strain) directly into the stomach. Seven days post infection, infected animals showed alterations in cytokine responses along with inflammatory cell infiltration in the lamina propria, depicting a prominent inflammatory response due to infection. To understand the immunogenicity and protective efficacy, the mice were immunized with outer membrane vesicles (OMVs) isolated from an indigenous strain with putative virulence factors of H. pylori [A61C (1), cag+/vacA s1m1]. In contrast to the non-immunized cohort, the OMV-immunized cohort showed a gradual increase in serum immunoglobulin(s) levels on the 35th day after the first immunization. This conferred protective immunity against subsequent challenge with the reference strain (SS1). Direct inoculation of H. pylori into the stomach influenced infection in a short time and, more importantly, in a dose-dependent manner, indicating the usefulness of the developed model for pathophysiology, therapeutic and prophylactic studies.

Prevalence and changing antimicrobial resistance profiles of Shigella spp. isolated from diarrheal patients in Kolkata during 2011-2019.

BACKGROUND: The primary aim of this study was to investigate the occurrence, characteristics, and antimicrobial resistance patterns of various Shigella serogroups 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 serogroups, 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 ß-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.

The gut microbiome of the healthy population in Kolkata, India, is a reservoir of antimicrobial resistance genes emphasizing the need of enforcing antimicrobial stewardship.

Antimicrobial resistance (AMR) alleviation warrants antimicrobial stewardship (AS) entailing the indispensability of epidemiological surveillance. We undertook a small-scale surveillance in Kolkata to detect the presence of antimicrobial resistance genes (ARGs) in the healthy gut microbiome. We found that it was a reservoir of ARGs against common antibiotics. A targeted Polymerase Chain Reaction (PCR) and sequencing-based ARGs detection against tetracyclines, macrolides, trimethoprim, sulfamethoxazole, aminoglycosides, amphenicol, and mobile genetic element (MGE) markers were deployed in 25 fecal samples. Relative abundance and frequency of ARGs were calculated. We detected markers against all these classes of antibiotics. 100% samples carried aminoglycoside resistance marker and int1U. A comparison with our previously published diarrheal resistome from the same spatial and temporal frame revealed that a higher diversity of ARGs were detected in the community and a higher rate of isolation of tetC, msrA, tmp, and sul-2 was found. The presence of common markers in the two cohorts proves that the gut microbiome has been contaminated with ARGs and which are being disseminated among different ecosystems. This is an issue of discerning concern for public health. The study raises an alarming picture of the AMR crisis in low-middle and emergent economies. It emphasizes the strict enforcement of AS in the community.

Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children.

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected 12% more Shigella and 13% more ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Development and evaluation of a simple PCR assay and nested PCR for rapid detection of clarithromycin-resistant Helicobacter pylori from culture and directly from the biopsy samples in India.

BACKGROUND: Eradication of Helicobacter pylori provides the most effective treatment for gastroduodenal diseases caused by H. pylori infection. Clarithromycin, a member of the macrolide family, still remains the most important antibiotic used in H. pylori eradication treatment. But the increasing prevalence of clarithromycin resistant H. pylori strains due to point mutations in the V region of the 23S rRNA, poses a great threat in treating the ailing patients. So, we aimed for PCR-mediated rapid detection of the point mutation at 2143 position of 23S rRNA gene in H. pylori that is relevant to clarithromycin resistance from culture and simultaneously from biopsy specimens to avoid the empirical treatment. RESULTS: Newly developed PCR assay using DNA of pure culture detected point mutation in 23S rRNA gene in 21 (8.04%) of 261 clinical strains tested. The agar dilution method showed that all these 21 strains were resistant to clarithromycin indicating the perfect match of the PCR based results. Additionally, the sequencing study also identified the A to G mutation at 2143 position in 23S rRNA gene of the resistant strains only. Consequently, the newly developed Nested-ASP-PCR dealing directly with 50 biopsy specimens demonstrated 100% sensitivity and specificity with the findings of agar dilution method taken as Gold standard. Bioinformatics based analysis such as accessibility analysis and dot plot clearly stated that the base pairing probability has increased due to mutation. Computational studies revealed that the point mutation confers more stability in secondary structure due to conversion of loop to stem. Furthermore, interaction studies showed binding affinity of the CLR to the mutant type is weaker than that to the wild type. CONCLUSION: This assay outlines a rapid, sensitive and simple approach to identify point mutation that confers clarithromycin resistance as well as clarithromycin sensitive strains, providing rapid initiation of effective antibiotic treatment. Additionally, it is simple to adopt for hospital based diagnostic laboratories to evaluate the degree of regional clarithromycin resistance from biopsy specimens itself. Furthermore, in silico studies provide evidence or a signal that the prevalence of clarithromycin resistance may rise in the near future as a result of this point mutation.

Pentavalent outer membrane vesicles immunized mice sera confers passive protection against five prevalent pathotypes of diarrhoeagenic Escherichia coli in neonatal mice.

Diarrhoeagenic Escherichia coli (DEC) pathotypes are one of the major causative agents of diarrhoea induced childhood morbidity and mortality in developing countries. Licensed vaccines providing broad spectrum protection against DEC mediated infections are not available. Outer membrane vesicles (OMVs) are microvesicles released by gram-negative bacteria during the growth phase and contain multiple immunogenic proteins. Based on prevalence of infections, we have formulated a pentavalent outer-membrane vesicles (POMVs) based immunogen targeting five main pathotypes of DEC responsible for diarrhoeal diseases. Following isolation, OMVs from five DEC pathotypes were mixed in equal proportions to formulate POMVs and 10 µg of the immunogen was intraperitoneally administered to adult BALB/c mice. Three doses of POMVs induced significant humoral immune response against whole cell lysates (WCLs), outer membrane proteins (OMPs) and lipopolysaccharides (LPS) isolated from DEC pathotypes along with significant induction of cellular immune response in adult mice. Passive transfer of POMVs immunized adult mice sera protected neonatal mice significantly against DEC infections. Overall, this study finds POMVs to be immunogenic in conferring broad-spectrum passive protection to neonatal mice against five main DEC pathotypes. Altogether, these findings suggest that POMVs can be used as a potent vaccine candidate to ameliorate the DEC-mediated health burden.