Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Genomic and functional insights into antibiotic resistance genes floR and strA linked with the SXT element of Vibrio cholerae non-O1/non-O139.

The emergence and spread of antibiotic-resistant bacterial pathogens are a critical public health concern across the globe. Mobile genetic elements (MGEs) play an important role in the horizontal acquisition of antimicrobial resistance genes (ARGs) in bacteria. In this study, we have decoded the whole genome sequences of multidrug-resistant Vibrio cholerae clinical isolates carrying the ARG-linked SXT, an integrative and conjugative element, in their large chromosomes. As in others, the SXT element has been found integrated into the 5'-end of the prfC gene (which encodes peptide chain release factor 3 involved in translational regulation) on the large chromosome of V. cholerae non-O1/non-O139 strains. Further, we demonstrate the functionality of SXT-linked floR and strAB genes, which confer resistance to chloramphenicol and streptomycin, respectively. The floR gene-encoded protein FloR belongs to the major facilitator superfamily efflux transporter containing 12 transmembrane domains (TMDs). Deletion analysis confirmed that even a single TMD of FloR is critical for the export function of chloramphenicol. The floR gene has two putative promoters, P1 and P2. Sequential deletions reveal that P2 is responsible for the expression of the floR. Deletion analysis of the N- and/or C-terminal coding regions of strA established their importance for conferring resistance against streptomycin. Interestingly, qPCR analysis of the floR and strA genes indicated that both of the genes are constitutively expressed in V. cholerae cells. Further, whole genome-based global phylogeography confirmed the presence of the integrative and conjugative element SXT in non-O1/non-O139 strains despite being non-multidrug resistant by lacking antimicrobial resistance (AMR) gene cassettes, which needs monitoring.

Metagenomic insights into fungal community composition of the nasopharyngeal region of COVID-19 associated mucormycosis patients from India.

Coronavirus disease 2019 (COVID-19) associated mucormycosis (CAM) was reported predominantly from India during the second wave of COVID-19  and has a high mortality rate. The present study aims to understand the fungal community composition of the nasopharyngeal region of CAM-infected individuals and compare it with severe COVID-19 patients and healthy controls. The fungal community composition was decoded by analyzing the sequence homology of the internal transcribed spacer-2-(ITS-2) region of metagenomic DNA extracted from the upper respiratory samples. The alpha-diversity indices were found to be significantly altered in CAM patients (p < 0.05). Interestingly, a higher abundance of Candida africana, Candida haemuloni, Starmerella floris, and Starmerella lactiscondensi was observed exclusively in CAM patients. The interindividual changes in mycobiome composition were well supported by beta-diversity analysis (p < 0.05). The current study provides insights into the dysbiosis of the nasal mycobiome during CAM infection. In conclusion, our study shows that severe COVID-19 and CAM are associated with alteration in mycobiome as compared to healthy controls. However, the sequential alteration in the fungal flora which ultimately leads to the development of CAM needs to be addressed by future studies.

Genomic repertoires linked with pathogenic potency of arthritogenic Prevotella copri isolated from the gut of patients with rheumatoid arthritis.

OBJECTIVES: Prevotella copri is considered to be a contributing factor in rheumatoid arthritis (RA). However, in some non-Westernised countries, healthy individuals also harbour an abundance of P. copri in the intestine. This study investigated the pathogenicity of RA patient-derived P. copri (P. copri RA) compared with healthy control-derived P. copri (P. copri HC). METHODS: We obtained 13 P. copri strains from the faeces of patients with RA and healthy controls. Following whole genome sequencing, the sequences of P. copri RA and P. copri HC were compared. To analyse the arthritis-inducing ability of P. copri, we examined two arthritis models (1) a collagen-induced arthritis model harbouring P. copri under specific-pathogen-free conditions and (2) an SKG mouse arthritis model under P. copri-monocolonised conditions. Finally, to evaluate the ability of P. copri to activate innate immune cells, we performed in vitro stimulation of bone marrow-derived dendritic cells (BMDCs) by P. copri RA and P. copri HC. RESULTS: Comparative genomic analysis revealed no apparent differences in the core gene contents between P. copri RA and P. copri HC, but pangenome analysis revealed the high genome plasticity of P. copri. We identified a P. copri RA-specific genomic region as a conjugative transposon. In both arthritis models, P. copri RA-induced more severe arthritis than P. copri HC. In vitro BMDC stimulation experiments revealed the upregulation of IL-17 and Th17-related cytokines (IL-6, IL-23) by P. copri RA. CONCLUSION: Our findings reveal the genetic diversity of P. copri, and the genomic signatures associated with strong arthritis-inducing ability of P. copri RA. Our study contributes towards elucidation of the complex pathogenesis of RA.

Post-COVID-19 rhino-orbito-cerebral mucormycosis-A clinico-mycological study from North India.

The second wave of coronavirus disease 2019 (COVID-19), during the early 2021, lead to a devastating outbreak of mucormycosis in India. This study aimed to determine the aetiology, clinical features, comorbidities, and risk factors of rhino-orbito-cerebral mucormycosis (ROCM) and antifungal susceptibility pattern for the isolates. The study included all suspected cases of ROCM in post-COVID-19 patients attending the hospital from May to December 2021. A total of 70 patients were diagnosed with mucormycosis during the study period. The commonest presentations were rhino-orbital and rhino-orbito-cerebral in 35.7% of cases each. Diabetes mellitus was the commonest associated risk factor in 95.7% of all patients, while 78.5% of the patients were treated with corticosteroids in the recent past, and 25.7% presented with active COVID-19 pneumonia. The commonest isolate was Rhizopus arrhizus n = 14, followed by Aspergillus flavus n = 16, A. fumigatus n = 4, A. niger n = 3, Fusarium oxysporumn = 1, and Apophysomyces variabilisn = 1. Fungal species identification was done by phenotypic methods for all the isolates and DNA sequence analysis of 18 isolates, and antifungal susceptibility testing of 30 isolates was performed by commercially prepared HiMIC plate (HiMedia, Mumbai, India) using broth microdilution for amphotericin B, isavuconazole, itraconazole, voriconazole, and posaconazole. The MIC50 and MIC90 of amphotericin B for R. arrhizus strains were 0.25 and 4 µg/ml, respectively; and the MIC50 and MIC90 results for itraconazole, posaconazole, and isavuconazole were 8 and 8, 2 and 2, and 2 and 8 µg/ml, respectively. In vitro data showed that amphotericin B was the most effective antifungal against most species. The commercially available ready-to-use minimum inhibitory concentration plates are user-friendly for performing antifungal susceptibility, which may be useful in choosing appropriate regimens and monitoring emerging resistance.

Human Gut Microbiota and Drug Metabolism.

The efficacy of drugs widely varies in individuals, and the gut microbiota plays an important role in this variability. The commensal microbiota living in the human gut encodes several enzymes that chemically modify systemic and orally administered drugs, and such modifications can lead to activation, inactivation, toxification, altered stability, poor bioavailability, and rapid excretion. Our knowledge of the role of the human gut microbiome in therapeutic outcomes continues to evolve. Recent studies suggest the existence of complex interactions between microbial functions and therapeutic drugs across the human body. Therapeutic drugs or xenobiotics can influence the composition of the gut microbiome and the microbial encoded functions. Both these deviations can alter the chemical transformations of the drugs and hence treatment outcomes. In this review, we provide an overview of (i) the genetic ecology of microbially encoded functions linked with xenobiotic degradation; (ii) the effect of drugs on the composition and function of the gut microbiome; and (iii) the importance of the gut microbiota in drug metabolism.

Study on Effects of Probiotics on Gut Microbiome and Clinical Course in Patients with Critical Care Illnesses.

Ventilator-associated pneumonia (VAP) is a nosocomial infection contracted by ventilator patients in which bacteria colonize the upper digestive tract and contaminated secretions are released into the lower airway. This nosocomial infection increases the morbidity and mortality of the patients as well as the cost of treatment. Probiotic formulations have recently been proposed to prevent the colonization of these pathogenic bacteria. In this prospective observational study, we aimed to investigate the effects of probiotics on gut microbiota and their relation to clinical outcomes in mechanically ventilated patients. For this study, 35 patients were recruited (22 probiotic-treated and 13 without probiotic treatment) from a cohort of 169 patients. Patients in the probiotic group were given a dose of 6 capsules of a commercially available probiotic (VSL#3®:112.5 billion CFU/cap) in three divided doses for 10 days. Sampling was carried out after each dose to monitor the temporal change in the gut microbiota composition. To profile the microbiota, we used a 16S rRNA metagenomic approach, and differences among the groups were computed using multivariate statistical analyses. Differences in gut microbial diversity (Bray Curtis and Jaccard distance, p-value > 0.05) between the probiotic-treated group and the control group were not observed. Furthermore, treatment with probiotics resulted in the enrichment of Lactobacillus and Streptococcus in the gut microbiota of the probiotic-treated groups. Our results demonstrated that probiotics might lead to favorable alterations in gut microbiome characteristics. Future studies should focus on the appropriate dosages and frequency of probiotics, which can lead to improved clinical outcomes.

Molecular insights into the genome dynamics and interactions between core and acquired genomes of Vibrio cholerae.

Bacterial species are hosts to horizontally acquired mobile genetic elements (MGEs), which encode virulence, toxin, antimicrobial resistance, and other metabolic functions. The bipartite genome of Vibrio cholerae harbors sporadic and conserved MGEs that contribute in the disease development and survival of the pathogens. For a comprehensive understanding of dynamics of MGEs in the bacterial genome, we engineered the genome of V. cholerae and examined in vitro and in vivo stability of genomic islands (GIs), integrative conjugative elements (ICEs), and prophages. Recombinant vectors carrying the integration module of these GIs, ICE and CTXΦ, helped us to understand the efficiency of integrations of MGEs in the V. cholerae chromosome. We have deleted more than 250 acquired genes from 6 different loci in the V. cholerae chromosome and showed contribution of CTX prophage in the essentiality of SOS response master regulator LexA, which is otherwise not essential for viability in other bacteria, including Escherichia coli In addition, we observed that the core genome-encoded RecA helps CTXΦ to bypass V. cholerae immunity and allow it to replicate in the host bacterium in the presence of similar prophage in the chromosome. Finally, our proteomics analysis reveals the importance of MGEs in modulating the levels of cellular proteome. This study engineered the genome of V. cholerae to remove all of the GIs, ICEs, and prophages and revealed important interactions between core and acquired genomes.

Genetic and mutational analysis of virulence traits and their modulation in an environmental toxigenic Vibrio cholerae non-O1/non-O139 strain, VCE232.

Vibrio cholerae O1 and O139 isolates deploy cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause the diarrhoeal disease cholera. The ctxAB and tcpA genes encoding CT and TCP are part of two acquired genetic elements, the CTX phage and Vibrio pathogenicity island-1 (VPI-1), respectively. ToxR and ToxT proteins are the key regulators of virulence genes of V. cholerae O1 and O139. V. cholerae isolates belonging to serogroups other than O1/O139, called non-O1/non-O139, are usually devoid of virulence-related elements and are non-pathogenic. Here, we have analysed the available whole genome sequence of an environmental toxigenic V. cholerae non-O1/non-O139 strain, VCE232, carrying the CTX phage and VPI-1. Extensive bioinformatics and phylogenetic analyses indicated high similarity of the VCE232 genome sequence with the genome of V. cholerae O1 strains, including organization of the VPI-1 locus, ctxAB, tcpA and toxT genes, and promoters. We established that the VCE232 strain produces an optimal amount of CT at 30 °C under AKI conditions. To investigate the role of ToxT and ToxR in the regulation of virulence factors, we constructed ΔtoxT, ΔtoxR and ΔtoxTΔtoxR deletion mutants of VCE232. Extensive genetic analyses of these mutants indicated that the toxT and toxR genes of VCE232 are crucial for CT and TCP production. However, unlike O1 isolates, the presence of either toxT or toxR gene is sufficient for optimal CT production in VCE232. In addition, the VCE232 ΔtoxR mutant showed differential regulation of the major outer membrane proteins, OmpT and OmpU. This is the first attempt to explore the regulation of expression of major virulence genes and regulators in an environmental toxigenic V. cholerae non-O1/non-O139 strain.

Genomic plasticity associated with antimicrobial resistance in Vibrio cholerae.

The Bay of Bengal is known as the epicenter for seeding several devastating cholera outbreaks across the globe. Vibrio cholerae, the etiological agent of cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and adapt them into its genome for structuring metabolic processes, developing drug resistance, and colonizing the human intestine. Antimicrobial resistance (AMR) in V. cholerae has become a global concern. However, little is known about the identity of the resistance traits, source of AMR genes, acquisition process, and stability of the genetic elements linked with resistance genes in V. cholerae Here we present details of AMR profiles of 443 V. cholerae strains isolated from the stool samples of diarrheal patients from two regions of India. We sequenced the whole genome of multidrug-resistant (MDR) and extensively drug-resistant (XDR) V. cholerae to identify AMR genes and genomic elements that harbor the resistance traits. Our genomic findings were further confirmed by proteome analysis. We also engineered the genome of V. cholerae to monitor the importance of the autonomously replicating plasmid and core genome in the resistance profile. Our findings provided insights into the genomes of recent cholera isolates and identified several acquired traits including plasmids, transposons, integrative conjugative elements (ICEs), pathogenicity islands (PIs), prophages, and gene cassettes that confer fitness to the pathogen. The knowledge generated from this study would help in better understanding of V. cholerae evolution and management of cholera disease by providing clinical guidance on preferred treatment regimens.

Antimicrobial resistance and virulence in Helicobacter pylori: Genomic insights.

Microbes evolve rapidly by modifying their genome through mutations or acquisition of genetic elements. Antimicrobial resistance in Helicobacter pylori is increasingly prevalent in India. However, limited information is available about the genome of resistant H. pylori isolated from India. Our pan- and core-genome based analyses of 54 Indian H. pylori strains revealed plasticity of its genome. H. pylori is highly heterogenous both in terms of the genomic content and DNA sequence homology of ARGs and virulence factors. We observed that the H. pylori strains are clustered according to their geographical locations. The presence of point mutations in the ARGs and absence of acquired genetic elements linked with ARGs suggest target modifications are the primary mechanism of its antibiotic resistance. The findings of the present study would help in better understanding the emergence of drug-resistant H. pylori and controlling gastric disorders by advancing clinical guidance on selected treatment regimens.

Gut microbiome diversity in acute severe colitis is distinct from mild to moderate ulcerative colitis.

BACKGROUND AND AIM: Although the gut microbiome of patients with ulcerative colitis (UC) has been characterized, no study has characterized the gut microbiome in acute severe colitis (ASC). We compared the gut microbiome of patients with UC, ASC, and healthy controls (HCs). METHODS: Patients with mild to moderate UC (n = 24), ASC (n = 19 with 21 episodes) and HCs (n = 50) were recruited prospectively. A 16SrDNA amplicon approach was used to explore gut microbial diversity and taxonomic repertoires. UC was diagnosed using European Crohn's and Colitis Organization guidelines, and ASC was diagnosed using Truelove and Witts' criteria. RESULTS: The normalized alpha diversity was significantly lower in ASC than mild-moderately active UC (P < 0.05) or HC (P < 0.001). The gut microbiome in ASC was highly unstable, as characterized by high intracohort variation (analyzed using J-divergence measure), which was significantly greater than in UC or HC. On principal coordinate analysis, the microbiome of HC and UC were similar, with the ASC cohort being distinct from both. Comparison of ranked abundances identified four distinct clusters of genera (G1, G2, G3, and G4), with specific trends in their abundance across three groups: G1/G2A clusters had the least, whereas G3 had the highest abundance in the ASC cohort. CONCLUSIONS: Gut microbial diversity is lower in ASC than mild-moderate UC or HCs. Gut microbiome composition is increasingly unstable in ASC, with a distinct abundance of specific genera varying between HCs and ASC. Mild-moderate UC lies within the spectrum.

Vaginal Microbiome of Pregnant Indian Women: Insights into the Genome of Dominant Lactobacillus Species.

The trillions of microorganisms residing in the human body display varying degrees of compositional and functional diversities within and between individuals and contribute significantly to host physiology and susceptibility to disease. Microbial species present in the vaginal milieu of reproductive age women showed a large personal component and varies widely in different ethnic groups at the taxonomic, genomic, and functional levels. Lactobacillus iners, L. crispatus, L. gasseri, L. jensenii, and L. johnsonii are most frequently detected bacterial species in the vaginal milieu of reproductive age women. However, we currently lack (i) an understanding of the baseline vaginal microbiota of reproductive age Indian women, (ii) the extent of taxonomic and functional variations of vaginal microbiota between individuals and (iii) the genomic repertoires of the dominant vaginal microbiota associated with the Indian subjects. In our study, we analyzed the metagenome of high vaginal swab (HVS) samples collected from 40 pregnant Indian women enrolled in the GARBH-Ini cohort. Composition and abundance of bacterial species was characterized by pyrosequencing 16S rRNA gene. We identified 3067 OTUs with ≥ 10 reads from four different bacterial phyla. Several species of lactobacilli were clustered into three community state types (CSTs). L. iners, L. crispatus, L. gasseri, and L. jensenii are the most frequently detected Lactobacillus species in the vaginal environment of Indian women. Other than Lactobacillus, several species of Halomonas were also identified in the vaginal environment of most of the women sampled. To gain genomic and functional insights, we isolated several Lactobacillus species from the HVS samples and explored their whole genome sequences by shotgun sequencing. We analyzed the genome of dominant Lactobacillus species, L. iners, L. crispatus, L. gasseri, and L. paragesseri to represent the CSTs and identify functions that may influence the composition of complex vaginal microbial ecology. This study reports for the first time the vaginal microbial ecology of Indian women and genomic insights into L. iners, L. crispatus, L. gasseri, and L. paragesseri commonly found in the genital tract of reproductive age women.

The 2019 novel coronavirus disease (COVID-19) pandemic: A review of the current evidence.

A novel coronavirus (nCoV) spillover event, with its epicenter in Wuhan, People's Republic of China, has emerged as a public health emergency of international concern. This began as an outbreak in December 2019, and till February 28, 2020, there have been 83,704 confirmed cases of novel coronavirus disease 2019 (COVID-19) globally, with 2,859 deaths, resulting in an overall case fatality rate of 3.41 per cent (95% confidence interval 3.29-3.54%). By this time (February 28, 2020) 58 countries or territories and one international conveyance (Diamond Princess Cruise Ship) were affected. As a part of the global response to manage and contain the pandemic, major emphasis was placed on generating research intelligence to guide evidence-based responses to contain the virus, which was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), owing to its genetic similarities with the SARS virus. This review summarizes the emerging evidence which can help guide the public health response, particularly in India. Key areas have been identified in which research needs to be conducted to generate critical intelligence for advising prevention and control efforts. The emergence of SARS-CoV-2 has once again exposed the weaknesses of global health systems preparedness, ability to respond to an infectious threat, the rapidity of transmission of infections across international borders and the ineffectiveness of knee-jerk policy responses to emerging/re-emerging infectious disease threats. The review concludes with the key learning points from the ongoing efforts to prevent and contain COVID-19 and identifies the need to invest in health systems, community-led response mechanisms and the need for preparedness and global health security.

Serum protein signature of coronary artery disease in type 2 diabetes mellitus.

BACKGROUND: Coronary artery disease (CAD) is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). The purpose of the present study was to discriminate the Indian CAD patients with or without T2DM by using multiple pathophysiological biomarkers. METHODS: Using sensitive multiplex protein assays, we assessed 46 protein markers including cytokines/chemokines, metabolic hormones, adipokines and apolipoproteins for evaluating different pathophysiological conditions of control, T2DM, CAD and T2DM with CAD patients (T2DM_CAD). Network analysis was performed to create protein-protein interaction networks by using significantly (p < 0.05) altered protein markers in each disease using STRING 10.5 database. We used two supervised analysis methods i.e., between class analysis (BCA) and principal component analysis (PCA) to reveals distinct biomarkers profiles. Further, random forest classification (RF) was used to classify the diseases by the panel of markers. RESULTS: Our two supervised analysis methods BCA and PCA revealed a distinct biomarker profiles and high degree of variability in the marker profiles for T2DM_CAD and CAD. Thereafter, the present study identified multiple potential biomarkers to differentiate T2DM, CAD, and T2DM_CAD patients based on their relative abundance in serum. RF classified T2DM based on the abundance patterns of nine markers i.e., IL-1ß, GM-CSF, glucagon, PAI-I, rantes, IP-10, resistin, GIP and Apo-B; CAD by 14 markers i.e., resistin, PDGF-BB, PAI-1, lipocalin-2, leptin, IL-13, eotaxin, GM-CSF, Apo-E, ghrelin, adipsin, GIP, Apo-CII and IP-10; and T2DM _CAD by 12 markers i.e., insulin, resistin, PAI-1, adiponectin, lipocalin-2, GM-CSF, adipsin, leptin, Apo-AII, rantes, IL-6 and ghrelin with respect to the control subjects. Using network analysis, we have identified several cellular network proteins like PTPN1, AKT1, INSR, LEPR, IRS1, IRS2, IL1R2, IL6R, PCSK9 and MYD88, which are responsible for regulating inflammation, insulin resistance, and atherosclerosis. CONCLUSION: We have identified three distinct sets of serum markers for diabetes, CAD and diabetes associated with CAD in Indian patients using nonparametric-based machine learning approach. These multiple marker classifiers may be useful for monitoring progression from a healthy person to T2DM and T2DM to T2DM_CAD. However, these findings need to be further confirmed in the future studies with large number of samples.

Molecular Insights into Antimicrobial Resistance Traits of Commensal Human Gut Microbiota.

Antimicrobial resistance (AMR) among bacterial species that resides in complex ecosystems is a natural phenomenon. Indiscriminate use of antimicrobials in healthcare, livestock, and agriculture provides an evolutionary advantage to the resistant variants to dominate the ecosystem. Ascendency of resistant variants threatens the efficacy of most, if not all, of the antimicrobial drugs commonly used to prevent and/or cure microbial infections. Resistant phenotype is very common in enteric bacteria. The most common mechanisms of AMR are enzymatic modifications to the antimicrobials or their target molecules. In enteric bacteria, most of the resistance traits are acquired by horizontal gene transfer from closely or distantly related bacterial population. AMR traits are generally linked with mobile genetic elements (MGEs) and could rapidly disseminate to the bacterial species through horizontal gene transfer (HGT) from a pool of resistance genes. Although prevalence of AMR genes among pathogenic bacteria is widely studied in the interest of infectious disease management, the resistance profile and the genetic traits that encode resistance to the commensal microbiota residing in the gut of healthy humans are not well-studied. In the present study, we have characterized AMR phenotypes and genotypes of five dominant commensal enteric bacteria isolated from the gut of healthy Indians. Our study revealed that like pathogenic bacteria, enteric commensals are also multidrug-resistant. The genes encoding antibiotic resistance are physically linked with MGEs and could disseminate vertically to the progeny and laterally to the distantly related microbial species. Consequently, the AMR genes present in the chromosome of commensal gut bacteria could be a potential source of resistance functions for other enteric pathogens.

XerD-mediated FtsK-independent integration of TLCϕ into the Vibrio cholerae genome.

As in most bacteria, topological problems arising from the circularity of the two Vibrio cholerae chromosomes, chrI and chrII, are resolved by the addition of a crossover at a specific site of each chromosome, dif, by two tyrosine recombinases, XerC and XerD. The reaction is under the control of a cell division protein, FtsK, which activates the formation of a Holliday Junction (HJ) intermediate by XerD catalysis that is resolved into product by XerC catalysis. Many plasmids and phages exploit Xer recombination for dimer resolution and for integration, respectively. In all cases so far described, they rely on an alternative recombination pathway in which XerC catalyzes the formation of a HJ independently of FtsK. This is notably the case for CTXϕ, the cholera toxin phage. Here, we show that in contrast, integration of TLCϕ, a toxin-linked cryptic satellite phage that is almost always found integrated at the chrI dif site before CTXϕ, depends on the formation of a HJ by XerD catalysis, which is then resolved by XerC catalysis. The reaction nevertheless escapes the normal cellular control exerted by FtsK on XerD. In addition, we show that the same reaction promotes the excision of TLCϕ, along with any CTXϕ copy present between dif and its left attachment site, providing a plausible mechanism for how chrI CTXϕ copies can be eliminated, as occurred in the second wave of the current cholera pandemic.

Effect of LexA on Chromosomal Integration of CTXϕ in Vibrio cholerae.

UNLABELLED: The genesis of toxigenic Vibrio cholerae involves acquisition of CTXϕ, a single-stranded DNA (ssDNA) filamentous phage that encodes cholera toxin (CT). The phage exploits host-encoded tyrosine recombinases (XerC and XerD) for chromosomal integration and lysogenic conversion. The replicative genome of CTXϕ produces ssDNA by rolling-circle replication, which may be used either for virion production or for integration into host chromosome. Fine-tuning of different ssDNA binding protein (Ssb) levels in the host cell is crucial for cellular functioning and important for CTXϕ integration. In this study, we mutated the master regulator gene of SOS induction, lexA, of V. cholerae because of its known role in controlling levels of Ssb proteins in other bacteria. CTXϕ integration decreased in cells with a ΔlexA mutation and increased in cells with an SOS-noninducing mutation, lexA (Ind(-)). We also observed that overexpression of host-encoded Ssb (VC0397) decreased integration of CTXϕ. We propose that LexA helps CTXϕ integration, possibly by fine-tuning levels of host- and phage-encoded Ssbs. IMPORTANCE: Cholera toxin is the principal virulence factor responsible for the acute diarrheal disease cholera. CT is encoded in the genome of a lysogenic filamentous phage, CTXϕ. Vibrio cholerae has a bipartite genome and harbors single or multiple copies of CTXϕ prophage in one or both chromosomes. Two host-encoded tyrosine recombinases (XerC and XerD) recognize the folded ssDNA genome of CTXϕ and catalyze its integration at the dimer resolution site of either one or both chromosomes. Fine-tuning of ssDNA binding proteins in host cells is crucial for CTXϕ integration. We engineered the V. cholerae genome and created several reporter strains carrying ΔlexA or lexA (Ind(-)) alleles. Using the reporter strains, the importance of LexA control of Ssb expression in the integration efficiency of CTXϕ was demonstrated.

Molecular evolution and functional divergence of Vibrio cholerae.

PURPOSE OF REVIEW: The purpose of this review is to synopsize and highlight the recent subtle genetic changes in cholera causing toxigenic Vibrio cholerae with special reference to their virulence, integrating and conjugative elements and toxin-antitoxin systems. It is not intended to cover issues on the whole genome sequence and epidemiology of cholera. RECENT FINDINGS: Analyses have been made using major published works on genetic changes associated with potential virulence, integrating and conjugative elements and toxin-antitoxin systems of toxigenic V. cholerae. During the course of evolution, V. cholerae strains show evidence of genetic selection for the expression of additional virulence, better survival in the environment, colonization ability and antimicrobial resistance. Some of the critical modifications that occurred at the molecular level include CTXϕ genome, cholera toxin B-subunit, integrating and conjugative elements and toxin-antitoxin systems. Frequent changes in the genome of V. cholerae appear to be an ongoing dynamic process that is assisting the pathogen to subtly change during or after epidemics of cholera. SUMMARY: Cholera is a reemerging public health problem. Continued basic research is important to understand the changing dynamics of bacterial virulence, survival strategies and disease pathogenesis for efficient therapeutic intervention and to abort transmission of the disease.