Cholerae Isolates Research Articles

Antibacterial Spectrum and Susceptibility of Bacterial Pathogens Causing Diarrheal Illnesses: Cross Sectional Study of Patients Visiting Health Facility in Lake Victoria Region - Kenya

Diarrheal illness has been studied extensively among children under five years, however little information is known among population over five years. This study determined the spectrum and antibiotic susceptibility of bacterial pathogens causing diarrhea in above five-year-old patients in a health facility along Lake Victoria region. In a cross-sectional study, 400 patients aged five years and above with diarrhea visiting Port Victoria health facility along Lake Victoria region were recruited in this study from 2013 to 2016. Stool samples were collected and cultured following standard microbiological procedures. Bacterial agents were isolated, identified and subjected to antibiotic susceptibility test. A total of 317 organisms (283 bacteria, 31 parasites and 3 others) were identified from 303 (76%) patients. Most frequently isolated pathogens being Non O1 Vibrio cholera 98(30.9%), Salmonella species 97(30.6%), Shigella species 47(14.8.0%) and Aeromonas species 38(12.0%). Non O1 Vibrio cholerae isolates were less sensitive to tetracycline, ampicillin and nalidixic acid. Salmonella species were less sensitive to ampicillin but sensitive to nalidixic acid. Shigella species isolates were more sensitive to chloramphenicol, cipplofloxin and nalidixic acid. Similar trend was observed for Aeromonas species isolates. The susceptibilities expressed were fairly good. However, some of the resistances shown pose a threat to public health, especially in unexposed populations. With the observed trends in this study, bacterial species have responded differently to antibiotics. It may be beneficial for clinicians to consider antibiotic susceptibility testing prior to management of patients to slow down resistance development.

Genomic analysis of pathogenic isolates of Vibrio cholerae from eastern Democratic Republic of the Congo (2014-2017).

BackgroundOver the past recent years, Vibrio cholerae has been associated with outbreaks in sub-Saharan Africa, notably in Democratic Republic of the Congo (DRC). This study aimed to determine the genetic relatedness of isolates responsible for cholera outbreaks in eastern DRC between 2014 and 2017, and their potential spread to bordering countries.Methods/Principal findingsPhenotypic analysis and whole genome sequencing (WGS) were carried out on 78 clinical isolates of V. cholerae associated with cholera in eastern provinces of DRC between 2014 and 2017. SNP-based phylogenomic data show that most isolates (73/78) were V. cholerae O1 biotype El Tor with CTX-3 type prophage. They fell within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and were contained in the introduction event (T)10 in East Africa. These isolates clustered in two sub-clades corresponding to Multiple Locus Sequence Types (MLST) profiles ST69 and the newly assigned ST515, the latter displaying a higher genetic diversity. Both sub-clades showed a distinct geographic clustering, with ST69 isolates mostly restricted to Lake Tanganyika basin and phylogenetically related to V. cholerae isolates associated with cholera outbreaks in western Tanzania, whereas ST515 isolates were disseminated along the Albertine Rift and closely related to isolates in South Sudan, Uganda, Tanzania and Zambia. Other V. cholerae isolates (5/78) were non-O1/non-O139 without any CTX prophage and no phylogenetic relationship with already characterized non-O1/non-O139 isolates.Conclusions/SignificanceCurrent data confirm the association of both DRC O1 7PET (T)10 sub-clades ST69 and ST515 with recurrent outbreaks in eastern DRC and at regional level over the past 10 years. Interestingly, while ST69 is predominantly a locally endemic sequence type, ST515 became adaptable enough to expand across DRC neighboring countries.

Toxigenic Vibrio cholerae evolution and establishment of reservoirs in aquatic ecosystems

The spread of cholera in the midst of an epidemic is largely driven by direct transmission from person to person, although it is well-recognized that Vibrio cholerae is also capable of growth and long-term survival in aquatic ecosystems. While prior studies have shown that aquatic reservoirs are important in the persistence of the disease on the Indian subcontinent, an epidemiological view postulating that locally evolving environmental V. cholerae contributes to outbreaks outside Asia remains debated. The single-source introduction of toxigenic V. cholerae O1 in Haiti, one of the largest outbreaks occurring this century, with 812,586 suspected cases and 9,606 deaths reported through July 2018, provided a unique opportunity to evaluate the role of aquatic reservoirs and assess bacterial transmission dynamics across environmental boundaries. To this end, we investigated the phylogeography of both clinical and aquatic toxigenic V. cholerae O1 isolates and show robust evidence of the establishment of aquatic reservoirs as well as ongoing evolution of V. cholerae isolates from aquatic sites. Novel environmental lineages emerged from sequential population bottlenecks, carrying mutations potentially involved in adaptation to the aquatic ecosystem. Based on such empirical data, we developed a mixed-transmission dynamic model of V. cholerae, where aquatic reservoirs actively contribute to genetic diversification and epidemic emergence, which underscores the complexity of transmission pathways in epidemics and endemic settings and the need for long-term investments in cholera control at both human and environmental levels.

An outbreak of diarrhoeal disease of El Tor Vibrio cholerae O1 Ogawa in and around Yavatmal district, Maharashtra, India in 2018

Background: Epidemics of cholera have been reported from various parts of India. We investigated the epidemic of cholera that occurred in and around Yavatmal district in Maharashtra, India 2018, reported during March to July.Methods: 711 stool samples collected from diarrhea patients were bacteriologically analyzed for their identification and antibiogram of Vibrio cholera.Results: The cholera outbreak was caused by V. cholerae O1 Ogawa biotype El Tor. All the V. cholerae isolates from the stool samples were sensitive to tetracycline, doxycycline, ofloxacin, ciprofloxacin gentamycin, cotrimoxazole and resistant to ampicillin and ceftazidime.Conclusions: The present outbreak was due to V. cholerae O1 Ogawa El Tor which seems to have completely replaced O139 serogroup of the previous outbreaks during the last decade.

Genomic epidemiology of Vibrio cholerae reveals the regional and global spread of two epidemic non-toxigenic lineages.

Non-toxigenic Vibrio cholerae isolates have been found associated with diarrheal disease globally, however, the global picture of non-toxigenic infections is largely unknown. Among non-toxigenic V. cholerae, ctxAB negative, tcpA positive (CNTP) isolates have the highest risk of disease. From 2001 to 2012, 71 infectious diarrhea cases were reported in Hangzhou, China, caused by CNTP serogroup O1 isolates. We sequenced 119 V. cholerae genomes isolated from patients, carriers and the environment in Hangzhou between 2001 and 2012, and compared them with 850 publicly available global isolates. We found that CNTP isolates from Hangzhou belonged to two distinctive lineages, named L3b and L9. Both lineages caused disease over a long time period with usually mild or moderate clinical symptoms. Within Hangzhou, the spread route of the L3b lineage was apparently from rural to urban areas, with aquatic food products being the most likely medium. Both lineages had been previously reported as causing local endemic disease in Latin America, but here we show that global spread of them has occurred, with the most likely origin of L3b lineage being in Central Asia. The L3b lineage has spread to China on at least three occasions. Other spread events, including from China to Thailand and to Latin America were also observed. We fill the missing links in the global spread of the two non-toxigenic serogroup O1 V. cholerae lineages that can cause human infection. The results are important for the design of future disease control strategies: surveillance of V. cholerae should not be limited to ctxAB positive strains.

Genome Dynamics of Vibrio cholerae Isolates Linked to Seasonal Outbreaks of Cholera in Dhaka, Bangladesh.

The temporal switching of serotypes from serotype Ogawa to Inaba and back to Ogawa was identified in Vibrio cholerae O1, which was responsible for seasonal outbreaks of cholera in Dhaka during the period 2015 to 2018. In order to delineate the factors responsible for this serotype transition, we performed whole-genome sequencing (WGS) of V. cholerae O1 multidrug-resistant strains belonging to both the serotypes that were isolated during this interval where the emergence and subsequent reduction of the Inaba serotype occurred. The whole-genome-based phylogenetic analysis revealed clonal expansion of the Inaba isolates mainly responsible for the peaks of infection during 2016 to 2017 and that they might have evolved from the prevailing Ogawa strains in 2015 which coclustered with them. Furthermore, the wbeT gene in these Inaba serotype isolates was inactivated due to insertion of a transposable element at the same position signifying the clonal expansion. Also, V. cholerae isolates in the Inaba serotype dominant clade mainly contained classical ctxB allele and revealed differences in the genetic composition of Vibrioseventh pandemic island II (VSP-II) and the SXT integrative and conjugative element (SXT-ICE) compared to those of Ogawa serotype strains which remerged in 2018. The variable presence of phage-inducible chromosomal island-like element 1 (PLE1) was also noted in the isolates of the Inaba serotype dominant clade. The detailed genomic characterization of the sequenced isolates has shed light on the forces which could be responsible for the periodic changes in serotypes of V. cholerae and has also highlighted the need to analyze the mobilome in greater detail to obtain insights into the mechanisms behind serotype switching.IMPORTANCE The switching of serotype from Ogawa to Inaba and back to Ogawa has been observed temporally in Vibrio cholerae O1, which is responsible for endemic cholera in Bangladesh. The serospecificity is key for effective intervention and for preventing cholera, a deadly disease that continues to cause significant morbidity and mortality worldwide. In the present study, WGS of V. cholerae allowed us to better understand the factors associated with the serotype switching events observed during 2015 to 2018. Genomic data analysis of strains isolated during this interval highlighted variations in the genes ctxB, tcpA, and rtxA and also identified significant differences in the genetic content of the mobilome, which included key elements such as SXT ICE, VSP-II, and PLE. Our results indicate that selective forces such as antibiotic resistance and phage resistance might contribute to the clonal expansion and predominance of a particular V. cholerae serotype responsible for an outbreak.

Phenotypic Pattern of Vibrio choleraeIsolates from a Tertiary Care Hospital in Vadodara, Gujarat, India

Introduction: Cholera is an acute diarrhoeal disease caused by Vibrio cholerae (V.cholerae). Based on antigenic differences of O antigen, O1 serogroup can be divided into three serotypes. In addition, by performing various biochemical reactions, O1 Serogroup can be differentiated into two biotypes. Outbreaks of Cholera occur seasonally. It is associated with monsoon season, warm temperature, heavy rainfall and increased plankton population. Aim: The aim was to determine the trends in resistance pattern and phenotypic Pattern of Vibrio cholerae. Materials and Methods: A retrospective study was conducted during the period from June 2019-December 2019. Culture of Stool specimens were done on different agar media. Biotyping was done by conventional methods. Serotyping and phage typing was also done along with the Antibiotic susceptibility testing. Descriptive analysis was used and presented in terms of percentage. Results:V.cholerae was isolated in 72 patients and they belonged to serogroup O1 and biotype El Tor. The most common serotype was Ogawa. The predominant phage type were T2 by old scheme and T27 by new scheme of phage typing. The maximum number of V. cholerae isolates was seen in the month of November, 2019 followed by October, 2019. Conclusion: The phenotypic pattern and fluctuating seasonal trend of V. cholerae and antimicrobial resistance encourage the continued epidemiological and microbiological surveillance of the disease.

Backward compatibility of whole genome sequencing data with MLVA typing using a new MLVAtype shiny application for Vibrio cholerae.

Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is widely used by laboratory-based surveillance networks for subtyping pathogens causing foodborne and water-borne disease outbreaks. However, Whole Genome Sequencing (WGS) has recently emerged as the new more powerful reference for pathogen subtyping, making a data conversion method necessary which enables the users to compare the MLVA identified by either method. The MLVAType shiny application was designed to extract MLVA profiles of Vibrio cholerae isolates from WGS data while ensuring backward compatibility with traditional MLVA typing methods. To test and validate the MLVAType algorithm, WGS-derived MLVA profiles of nineteen Vibrio cholerae isolates from Democratic Republic of the Congo (n = 9) and Uganda (n = 10) were compared to MLVA profiles generated by an in silico PCR approach and Sanger sequencing, the latter being used as the reference method. Results obtained by Sanger sequencing and MLVAType were totally concordant. However, the latter were affected by censored estimations whose percentage was inversely proportional to the k-mer parameter used during genome assembly. With a k-mer of 127, less than 15% estimation of V. cholerae VNTR was censored. Preventing censored estimation was only achievable when using a longer k-mer size (i.e. 175), which is not proposed in the SPAdes v.3.13.0 software. As NGS read lengths and qualities tend to increase with time, one may expect the increase of k-mer size in a near future. Using MLVAType application with a longer k-mer size will then efficiently retrieve MLVA profiles from WGS data while avoiding censored estimation.

Potential pathogenicity and antibiotic resistance of aquatic Vibrio isolates from freshwater in Slovakia.

This study aimed to evaluate the potential pathogenicity and antibiotic resistance of 31 environmental Vibrio isolates obtained from surface water in southern and eastern Slovakia. Isolates were identified as Vibrio cholerae non-O1/non-O139 and Vibrio metschnikovii by biochemical tests, MALDI biotyping, and 16S RNA gene sequencing. Analysis of the susceptibility to 13 antibacterial agents showed susceptibility of all isolates to ciprofloxacin, trimethoprim/sulfamethoxazole, chloramphenicol, gentamicin, imipenem, tetracyclin, and doxycycline. We recorded high rates of resistance to β-lactams and streptomycin. Investigation of antibiotic resistance showed five different antibiotic profiles with resistance to antibacterials from three classes, but no multidrug resistance was observed. The investigation of the pathogenic potential of V. cholerae isolates showed that neither the cholera toxin coding gene ctxA nor the genes zot (zonula occludens toxin), ace (accessory cholera toxin), and tcpA (toxin-coregulated pilus) were present in any of 31 isolated samples. Gene ompU (outer membrane protein) was confirmed in 80% and central regulatory protein-coding gene toxR in 71% of V. cholerae isolates, respectively. A high prevalence of the hemolysin coding gene hlyA in all V. cholerae was observed. The data point toward the importance of systematic monitoring and comparative studies of potentially pathogenic vibrios in European countries.

Looking for the hidden: Characterization of lysogenic phages in potential pathogenic Vibrio species from the North Sea

The incidence of potentially pathogenic Vibrio species in the marine environment around Europe, is correlated with the increase of surface seawater temperature. Despite their importance, little is known about the trigger factors of potential outbreak-causing strains in this region. As prophages may compose a major reservoir of virulence traits in marine ecosystems, this study aims to identify and characterize the genomes of lysogenic Vibrio phages exemplarily from the North Sea. Therefore, 31 isolates from potentially pathogenic Vibrio species from the North Sea were screened for inducible prophages with mitomycin C. From them, one V. cholerae isolate and 40% V. parahaemolyticus isolates carried inducible prophages. Three lysogenic phages were selected for genomic characterization. The phage vB_VpaM_VP-3212 (unclassified Myoviridae) has a genome with a length of 36.81 Kbp and 55 CDS were identified. This lysogenic phage of V. parahaemolyticus contains genes related to replicative transposition mechanism, such as transposase and mobile elements similar to Mu-like viruses. The phage vB_VpaP_VP-3220 (Podoviridae, unclassified Nona33virus) has a genome length of 58,14 Kbp and contains 63 CDS. This V. parahaemolyticus phage probably uses a headful (pac) packaging replication mechanism. The phage vB_VchM_VP-3213 (unclassified Myoviridae) has a genome with a length of 41 Kbp and 63 CDS were identified, including integrase and Xer system for lysogenic recombination. This lysogenic phage of V. cholerae has similar genomic features as lambdoid phages. Although no pathogenicity genes were identified, their similarity among other phage genomes indicates that these phages can affect the development of pathogenic Vibrio strains in marine environments.

Vibrio cholerae strains with inactivated cqsS gene overproduce autoinducer-2 which enhances resuscitation of dormant environmental V. cholerae.

BackgroundToxigenic Vibrio cholerae resides in aquatic reservoirs of cholera-endemic areas mostly in a dormant form known as conditionally viable environmental cells (CVEC) in which the bacteria remain embedded in an exopolysaccharide matrix, and fail to grow in routine bacteriological culture. The CVEC can be resuscitated by supplementing culture media with either of two autoinducers CAI-1 and AI-2, which are signal molecules controlling quorum sensing, a regulatory network of bacterial gene expression dependent on cell density. This study investigated possible existence of variant strains that overproduce AIs, sufficient to resuscitate CVEC in environmental waters.MethodsEnvironmental V. cholerae isolates and Tn insertion mutants of a V. cholerae strain C6706 were screened for production of AIs using bioluminescent reporter strains. Relevant mutations in environmental strains which overproduced AI-2 were characterized by nucleotide sequencing and genetic complementation studies. Effect of AIs produced in culture supernatants of relevant strains on reactivation of CVEC in water was determined by resuscitation assays.ResultsTwo of 54 environmental V. cholerae isolates were found to overproduce AI-2. Screening of a Tn-insertion library of V. cholerae strain C6706, identified a mutant which overproduced AI-2, and carried Tn insertion in the cqsS gene. Nucleotide sequencing also revealed mutations inactivating the cqsS gene in environmental isolates which overproduced AI-2, and this property was reversed when complemented with a wild type cqsS gene. Culture of river water samples supplemented with spent medium of these mutants resuscitated dormant V. cholerae cells in water.SignificanceV. cholerae strains with inactivated cqsS gene may offer a convenient source of AI-2 in enhanced assays for monitoring bacteriological quality of water. The results also suggest a potential role of naturally occurring cqsS mutants in the environmental biology of V. cholerae. Furthermore, similar phenomenon may have relevance in the ecology of other waterborne bacterial pathogens beyond V. cholerae.

Revisiting the Global Epidemiology of Cholera in Conjuction With the Genomics of Vibrio cholerae.

Toxigenic Vibrio cholerae is responsible for 1.4 to 4.3 million cases with about 21,000–143,000 deaths per year. Dominance of O1 and O139 serogroups, classical and El tor biotypes, alterations in CTX phages and the pathogenicity Islands are some of the major features of V. cholerae isolates that are responsible for cholera epidemics. Whole-genome sequencing (WGS) based analyses of single-nucleotide polymorphisms (SNPs) and other infrequent genetic variants provide a robust phylogenetic framework. Recent studies on the global transmission of pandemic V. cholerae O1 strains have shown the existence of eight different phyletic lineages. In these, the classical and El Tor biotype strains were separated as two distinctly evolved lineages. The frequency of SNP accumulation and the temporal and geographical distribution supports the perception that the seventh cholera pandemic (7CP) has spread from the Bay of Bengal region in three independent but overlapping waves. The 2010 Haitian outbreak shared a common ancestor with South-Asian wave-3 strains. In West Africa and East/Southern Africa, cholera epidemics are caused by single expanded lineage, which has been introduced several times since 1970. The Latin American epidemics that occurred in 1991 and 2010 were the result of introductions of two 7CP sublineages. Sublineages representing wave-3 have caused huge outbreaks in Haiti and Yemen. The Ogawa-Inaba serotype switchover in several cholera epidemics are believed to be due to the involvement of certain selection mechanism(s) rather than due to random events. V. cholerae O139 serogroup is phylogenetically related to the 7CP El Tor, and almost all these isolates belonged to the multilocus sequence type-69. Additional phenotypic and genotypic information have been generated to understand the pathogenicity of classical and El Tor vibrios. Presence of integrative conjugative elements (ICE) with antibiotic resistance gene cassettes, clustered regularly interspaced short palindromic repeats-associated protein system and ctxAB promoter based ToxRS expression of cholera toxin (CT) separates classical and El Tor biotypes. With the availability of WGS information, several important applications including, molecular typing, antimicrobial resistance, new diagnostics, and vaccination strategies could be generated.

Virulence, antimicrobial and heavy metal tolerance, and genetic diversity of Vibrio cholerae recovered from commonly consumed freshwater fish

Vibrio cholerae is a leading waterborne pathogen worldwide. Continuous monitoring of V. cholerae contamination in aquatic products and identification of risk factors are crucial for assuring food safety. In this study, we determined the virulence, antimicrobial susceptibility, heavy metal tolerance, and genetic diversity of 400 V. cholerae isolates recovered from commonly consumed freshwater fish (Aristichthys nobilis, Carassius auratus, Ctenopharyngodon idellus, and Parabramis pekinensis) collected in July and August of 2017 in Shanghai, China. V. cholerae has not been previously detected in the half of these fish species. The results revealed an extremely low occurrence of pathogenic V. cholerae carrying the major virulence genes ctxAB (0.0%), tcpA (0.0%), ace (0.0%), and zot (0.0%). However, high incidence of virulence-associated genes was observed, including the RTX toxin gene cluster (rtxA-D) (83.0–97.0%), hlyA (87.8%), hapA (95.0%), and tlh (76.0%). Meanwhile, high percentages of resistance to antimicrobial agents streptomycin (65.3%), ampicillin (44.5%), and rifampicin (24.0%) were observed. Approximately 30.5% of the isolates displayed multidrug resistant (MDR) phenotypes with 42 resistance profiles, which were significantly different among the four fish species (MARI, P = 0.001). Additionally, tolerance of isolates to heavy metals Hg2+ (49.3%), Zn2+ (30.3%), and Pb2+ (12.0%) was observed. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)-based fingerprinting of the 400 V. cholerae isolates revealed 328 ERIC-genotypes, which demonstrated a large degree of genomic variation among the isolates. Overall, the results of this study support the need for food safety risk assessment of aquatic products.

Molecular Detection of Cholera Infection during the Outbreak in Thi-Qar Province/Iraq in 2015-2016

Cholera is an acute disease caused by Vibrio cholerae; it’s affected to all aged groups. Cholera infection is outbreaks in Iraq as reported for several years. The recent cholera outbreak, emerged throughout 2015-2016, was investigated by using bacteriological laboratory tests, singleplex and multiplex PCR technique for the detection of V. cholerae from stool samples. Furthermore the antibiotic susceptibility test for cholera was also investigated coupled with the toxigenic potential. A total of Twenty Vibrio cholerae isolates were isolated from diarrheal patients in Thi-Qar province. These isolates were diagnosis by conventional biochemical test, API20 E system and molecular methods by using 16SrRNA. The isolates were characterized for gene traits; antimicrobial susceptibility. The results appeared all 20 isolates were positive for 16S rRNA. Multiplex PCR analysis revealed that 65%, 35% and 15% of isolates were positive for tox R, tcp and ctx B genes respectively. The antimicrobial susceptibility testing to isolates revealed high levels of resistance to ampicillin (100%), nalidixic acid (90%), sulfamethoxazoletrimethoprim (80%), tetracycline and ciprofloxacin (55%) and chloramphenicol (45%) in addition to increase the prevalence of multidrug resistant (MDR) between Vibrio cholerae isolates.

Utilization of Small RNA Genes to Distinguish Vibrio cholerae Biotypes via Multiplex Polymerase Chain Reaction.

The diarrheal disease "cholera" is caused by Vibrio cholerae, and is primarily confined to endemic regions, mostly in Africa and Asia. It is punctuated by outbreaks and creates severe challenges to public health. The disease-causing strains are most-often members of serogroups O1 and O139. PCR-based methods allow rapid diagnosis of these pathogens, including the identification of their biotypes. However, this necessitates the selection of specific target sequences to differentiate even the closely related biotypes of V. cholerae. Oligonucleotides for selective amplification of small RNA (sRNA) genes that are specific to these V. cholerae subtypes were designed. The resulting multiplex PCR assay was validated using V. cholerae cultures (i.e., 19 V. cholerae and 22 non-V. cholerae isolates) and spiked stool samples. The validation using V. cholerae cultures and spiked stool suspensions revealed detection limits of 10-100 pg DNA per reaction and 1.5 cells/mL suspension, respectively. The multiplex PCR assay that targets sRNA genes for amplification enables the sensitive and specific detection, as well as the differentiation of V. cholerae-O1 classical, O1 El Tor, and O139 biotypes. Most importantly, the assay enables fast and cheaper diagnosis compared with classic culture-based methods.

Studies on diversity of Vibrio sp. and the prevalence of hapA, tcpI, st, rtxA&C, acfB, hlyA, ctxA, ompU and toxR genes in environmental strains of Vibrio cholerae from Port Blair bays of South Andaman, India

Vibrio species are widely distributed in the estuarine and coastal waters that possess the greatest threat to human health worldwide. In this study it is aimed to isolate and observe the abundance of Vibrio sp. and prevalence of biomarker genes and antibiotic resistance profile of V. cholerae isolated from the Port Blair bays of South Andaman. A total of 56 water samples were collected from the seven sampling stations of Port Blair bays in which maximum number of Vibrio sp. population density (1.78 × 104) was recorded in Phoenix Bay. Among the 786 isolates 57.38% of the isolates were confirmed as Vibrio sp., Vibrio cholerae and Vibrio parahaemolyticus. PCR results revealed that the prevalence of biomarker genes was recorded maximum in the isolates from Phoenix Bay and Junglighat Bay samples. Upon further analysis, it was observed that the prevalence of hlyA gene (215 bp), was found to be the most widespread biomarker determinant in 84.17% of isolates. Major virulence determinants; ctxA, ompU and toxR genes were not detected in V. cholerae isolates from Port Blair bays. Maximum antibiotic resistance pattern was observed in Phoenix Bay isolates and maximum number of V. cholerae isolates was resistance to tetracycline (60.76%). Cluster and Principal Component Analysis were employed to understand the diversity and distribution of Vibrio isolates and its biomarker genes. Upon PCA analysis seasonal influence was not much perceived in Vibrio species diversity in Port Blair bays and the lack of significant difference in the detection of species diversity in this study is due to resemblance in geographical conditions and sources of pollution.

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 of Vibrio cholerae from sub-Saharan Africa: A systematic review.

BackgroundThe World Health Assembly adopted the Global Action Plan on Antimicrobial Resistance, which includes improving the knowledge base through surveillance and research. Noteworthily, the World Health Organization has advocated a Global Antimicrobial Resistance Surveillance System to address the plan’s surveillance objective, with most African countries enrolling in or after 2017.AimThe aim of this article was to review prior data on antimicrobial resistance of Vibrio cholerae from sub-Saharan Africa with a view for future control and intervention strategies.MethodsWe used the Preferred Reporting Items for Systematic Review and Meta-Analysis (or ‘PRISMA’) guidelines to search the PubMed and African Journals Online databases, as well as additional articles provided by the Nigeria Centre for Disease Control, for articles reporting on the antibiotic susceptibility of V. cholerae between January 2000 and December 2017.ResultsWe identified 340 publications, of which only 25 (reporting from 16 countries within the sub-Saharan African region) were eligible. The majority (20; 80.0%) of the cholera toxigenic V. cholerae isolates were of the serogroup O1 of the El Tor biotype with Ogawa and Inaba serotypes predominating. Resistance was predominantly documented to trimethoprim-sulphamethoxazole (50% of the studies), ampicillin (43.3% of the studies), chloramphenicol (43.3% of the studies) and streptomycin (30% of the studies). Resistance mechanisms were reported in 40% of the studies.ConclusionOur results demonstrate a documented antimicrobial resistance of V. cholerae to multiple antibiotic classes, including cell wall active agents and antimetabolites with evidence of phenotypic/genotypic resistance to fluoroquinolones.

Comparative genomics for non-O1/O139 Vibrio cholerae isolates recovered from the Yangtze River Estuary versus V. cholerae representative isolates from serogroup O1

Vibriocholerae, which is autochthonous to estuaries worldwide, can cause human cholera that is still pandemic in developing countries. A number of V. cholerae isolates of clinical and environmental origin worldwide have been subjected to genome sequencing to address their phylogenesis and bacterial pathogenesis, however, little genome information is available for V. cholerae isolates derived from estuaries, particularly in China. In this study, we determined the complete genome sequence of V. cholerae CHN108B (non-O1/O139 serogroup) isolated from the Yangtze River Estuary, China and performed comparative genome analysis between CHN108B and other eight representative V. cholerae isolates. The 4,168,545-bp V. cholerae CHN108B genome (47.2% G+C) consists of two circular chromosomes with 3,691 predicted protein-encoding genes. It has 110 strain-specific genes, the highest number among the eight representative V. cholerae whole genomes from serogroup O1: there are seven clinical isolates linked to cholera pandemics (1937–2010) and one environmental isolate from Brazil. Various mobile genetic elements (such as insertion sequences, prophages, integrative and conjugative elements, and super-integrons) were identified in the nine V. cholerae genomes of clinical and environmental origin, indicating that the bacterium undergoes extensive genetic recombination via lateral gene transfer. Comparative genomics also revealed different virulence and antimicrobial resistance gene patterns among the V. cholerae isolates, suggesting some potential virulence factors and the rising development of resistance among pathogenic V. cholerae. Additionally, draft genome sequences of multiple V. cholerae isolates recovered from the Yangtze River Estuary were also determined, and comparative genomics revealed many genes involved in specific metabolism pathways, which are likely shaped by the unique estuary environment. These results provide additional evidence of V. cholerae genome plasticity and will facilitate better understanding of the genome evolution and pathogenesis of this severe water-borne pathogen worldwide.

1113. Real-Time Evolution of Extensively Drug-Resistant Vibrio cholerae

Background Bay of Bengal is known as the epicenter of a number of distinct waves of global transmission of cholera. Vibrio cholerae, the etiological agent of acute diarrhoeal disease cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and acclimatize them into their genome for structuring metabolic process, developing drug resistance and disease. Antimicrobial resistance (AMR) in V. cholerae is a global concern. However, little is known about the identity, source, acquisition process, and stability of the resistance traits in the genome of cholera pathogen.MethodsAntibiotic susceptibility testing of V. cholerae isolated from different parts of India during 2001–2017 was performed using Discs and E-strips. Whole-genome sequencing of resistant (R), multidrug resistant (MDR), extensively drug resistant (XDR), and pandrug (PDR) resistant V. cholerae was done by next-generation DNA sequencing. Mobile genetic elements (MGEs) linked with AMR genes were tagged by allelic exchange methods. Whole-cell proteome analysis was done by iTRAQ analysis.ResultsAlmost 99% of V. cholerae isolates (n = 438) are resistant against ≥2 antibiotics, 17.2% isolates (n = 76) are resistant against ≥10 antibiotics, and 7.5% isolates (n = 33) are resistant against ≥14 antibiotics. Highest resistance was detected against sulfamethaxozole (99.8%, n = 442). In addition, resistance to nalidixic acid (n = 429), trimethoprim (n = 421), and streptomycin (n = 409) are also very high. All the sequenced resistant isolates carrying multiple resistance genes and are linked with MGEs like integrating conjugative elements, transposons etc. Most of the resistance traits are functional and expressed even in the absence of antibiotics.ConclusionOur comprehensive analysis of 443 clinical V. cholerae isolates show that the cholera pathogen is continuously evolving to counterbalance the antimicrobial effects of antibiotics. Several MGEs linked with AMR genes and other fitness factors potentially propagate to other bacterial species through HGTs. Knowledge of the present study would be useful to understand the evolution of cholera pathogens and management of cholera by helping selection of specific drug regimen against the pathogens.Disclosures All authors: No reported disclosures.