Multidrug Resistant Vibrio Cholerae Research Articles

Whole Genome Sequencing and Pan-Genomic Analysis of Multidrug-Resistant Vibrio cholerae VC01 Isolated from a Clinical Sample.

Cholera, a disease caused by the Vibrio cholerae bacteria, threatens public health worldwide. The organism mentioned above has a significant historical record of being identified as a prominent aquatic environmental pollutant capable of adapting its phenotypic and genotypic traits to react to host patients effectively. This study aims to elucidate the heterogeneity of the sporadic clinical strain of V. cholerae VC01 among patients residing in Silvasa. The study involved conducting whole-genome sequencing of the isolate obtained from patients exhibiting symptoms, including those not commonly observed in clinical practice. The strain was initially identified through a combination of biochemical analysis, microscopy, and 16s rRNA-based identification, followed by type strain-based identification. The investigation demonstrated the existence of various genetic alterations and resistance profiles against multiple drugs, particularly chloramphenicol (catB9), florfenicol (floR), oxytetracycline (tet(34)), sulfonamide (sul2), and Trimethoprim (dfrA1). The pan-genomic analysis indicated that 1099 distinct clusters were detected within the genome sequences of recent isolates worldwide. The present study helps to establish a correlation between the mutation and the coexistence of antimicrobial resistance toward current treatment.

Clinical and socio-environmental determinants of multidrug-resistant vibrio cholerae 01 in older children and adults in Bangladesh

ObjectivesFew studies have evaluated determinants of multidrug-resistant (MDR) Vibrio cholerae O1 in older children and adults. This study aimed to characterize the prevalence of MDR V. cholerae O1 and associated risk factors among patients over five years of age in Bangladesh. MethodsStool culture and antimicrobial susceptibility testing were performed as a part of a larger study at Dhaka Hospital in Bangladesh from March 2019–March 2020. Univariate statistics and multiple logistic regression were used to assess the association between a range of variables and MDR V. cholerae O1. ResultsMDR was found in 175 of 623 (28.1%) V. cholerae O1 isolates. High levels of resistance were found to erythromycin (99.2%), trimethoprim-sulfamethoxazole (99.7%), and ampicillin (88.9%), while susceptibility was high to tetracyclines (99.7%), azithromycin (99.2%), ciprofloxacin (99.8%), and cephalosporins (98.6%). MDR was associated with prior antibiotic use, longer transport time to hospital, higher income, non-flush toilet use, greater stool frequency, lower blood pressure, lower mid-upper arm circumference, and lower percent dehydration. ConclusionsMDR V. cholerae O1 was common among patients over five in an urban hospital in Bangladesh. Significant factors associated with MDR may be actionable in identifying patients with a high likelihood of MDR.

Antibacterial activity of polyphenolic fraction of Kombucha against Vibrio cholerae: targeting cell membrane.

The emergence of multidrug-resistant Vibrio cholerae strains has hindered an efficient anti-Vibrio therapy. This study has demonstrated the membrane damage-mediated antibacterial mechanism of Kombucha, a popular fermented beverage of sugared tea, which is mostly attributed to its polyphenolic content. This study also implies the exploitation of Kombucha as a potential new source of bioactive polyphenols against V. cholerae.

Comparison of untagged and his-tagged dihydrodipicolinate synthase from the enteric pathogen Vibrio cholerae

Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7), which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V. cholerae (Vc-DHDPS) and provide comparative structural and kinetic analyses. Structural studies employing circular dichroism spectroscopy and analytical ultracentrifugation demonstrate that the recombinant enzymes are folded and exist as dimers in solution. Kinetic analyses of untagged and His-tagged Vc-DHDPS show that the enzymes are functional with specific activities of 75.6 U/mg and 112 U/mg, KM (pyruvate) of 0.14 mM and 0.15 mM, KM (L-aspartate-4-semialdehyde) of 0.08 mM and 0.09 mM, and kcat of 34 and 46 s−1, respectively. These results demonstrate there are no significant changes in the structure and function of Vc-DHDPS upon the addition of an N-terminal His tag and, hence, the tagged recombinant product is suitable for future studies, including screening for new inhibitors as potential anti-cholera agents. Additionally, a polyclonal antibody raised against untagged Vc-DHDPS is validated for specifically detecting recombinant and native forms of the enzyme.

Antibacterial-guided isolation of constituents from <i>Senna alata </i>leaves with a particular reference against Multi-Drug-Resistant <i>Vibrio cholerae </i>and <i>Shigella flexneri</i>

Senna alata is widely used in Cameroon for the treatment of several infections which include gonorrhoea, gastro-intestinal and skin diseases. Therefore, its leaves were investigated for antibacterial principles. Extraction of plant material was done with methanol, follow by partition with hexane. Separation and purification of compounds was done using a combination of chromatographic techniques. Isolated compounds were identified by means of spectroscopic methods and comparison with literature data. The antibacterial activity of extracts, fractions and compounds was assessed by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Multi-Drug-Resistant (MDR) Vibrio cholerae and Shigella flexneri . Three secondary metabolites namely kaempferol, luteolin and aloeemodin were isolated from methanol residue active extract. The antibacterial results showed that the MeOH residue extract, fractions A and C, as well as compounds 1-3 exhibited variable MIC values, depending on the bacterial strains. Aloe-emodin (MIC = 4 to 128 μg/mL) exhibited the highest antibacterial activity against MDR Vibrio cholerae and Shigella flexneri . From these results obtained, S. alata leaf could be considered as a natural antibacterial source due to the presence of flavonoid and anthraquinone compounds. Keywords : Senna alata, Fabaceae, kaempferol, luteolin, aloe-emodin, MDR strains.

Cholera outbreak caused by drug resistant Vibrio cholerae serogroup O1 biotype ElTor serotype Ogawa in Nepal; a cross-sectional study.

BackgroundCholera is a major cause of mortality and morbidity in underdeveloped countries including Nepal. Recently drug resistance in Vibrio cholerae has become a serious problem mainly in developing countries. The main objectives of our study were to investigate the occurrence of Vibrio cholerae in stool samples from patients with watery diarrhea and to determine the antimicrobial susceptibility patterns of V. cholerae isolates.MethodsA total of 116 stool samples from patients suffering from watery diarrhea during July to December 2012 were obtained from outbreak areas from all over Nepal. Alkaline peptone water and thiosulphate citrate bile salt sucrose agar (TCBS) were used to isolate the Vibrio cholerae. The isolates were identified with the help of colony morphology, Gram’s staining, conventional biochemical testing, serotyping and biotyping. Antimicrobial susceptibility testing was performed by determining the minimum inhibitory concentration (MIC) by agar dilution method.ResultsVibrio cholerae was isolated from 26.72 % of total samples. All isolated Vibrio cholerae were confirmed to be Vibrio cholerae serogoup O1 biotype El Tor and serotype Ogawa. All isolates were resistant to ampicillin and cotrimoxazole. Twenty nine isolates were resistant toward two different classes of antibiotics, one strain was resistant to three different classes of antibiotics and one strain was resistant to four different classes of antibiotics. According to the definition of the multidrug resistant bacteria; 6.45 % of the strains of Vibrio cholerae were found to be multidrug resistant.ConclusionsCholera due to multidrug resistant Vibrio cholerae is also possible in Nepal. According to the antimicrobial susceptibility pattern of Vibrio cholerae in our study we recommend to use any antibiotics among tetracycline, doxycycline, levofloxacin, azithromycin, chloramphenicol and ciprofloxacin for preliminary treatment of cholera in Nepal.

Multidrug resistant Vibrio cholerae O1 from clinical and environmental samples in Kathmandu city.

BackgroundCholera, an infectious disease caused by Vibrio cholerae, is a major public health problem and is a particularly burden in developing countries including Nepal. Although the recent worldwide outbreaks of cholera have been due to V. cholerae El Tor, the classical biotypes are still predominant in Nepal. Serogroup O1 of the V. cholerae classical biotype was the primary cause of a cholera outbreak in Kathmandu in 2012. Thus, this study was designed to know serotypes and biotypes of V. cholerae strains causing recent outbreak with reference to drug resistant patterns. Moreover, we also report the toxigenic strains of V. cholerae from both environmental and clinical specimens by detecting the ctx gene.MethodsTwenty four V. cholerae (n = 22 from stool samples and n = 2 from water samples) isolated in this study were subjected to Serotyping and biotyping following the standard protocols as described previously. All of the isolates were tested for antimicrobial susceptibility patterns using the modified Kirby-Bauer disk diffusion method as recommended by CLSI guidelines. The screening of the ctx genes (ctxA2-B gene) were performed by PCR method using a pair of primers; C2F (5′-AGGTGTAAAATTCCTTGACGA-3′) and C2R (5′-TCCTCAGGGTATCCTTCATC-3′) to identify the toxigenic strains of V. cholerae.ResultsAmong twenty four V. cholerae isolates, 91.7% were clinical and 8.3% were from water samples. Higher rate of V. cholerae infection was found among adults of aged group 20–30 years. All isolates were serogroups O1 of the V. cholerae classical biotype and sub serotype, Ogawa. All isolates were resistant to ampicillin, nalidixic acid and cotrimoxazole. 90.9% were resistant to erythromycin however, tetracycline was found to be the most effective drug for the isolates. All isolates were multidrug resistant (MDR) and possessed a ctx gene of approximately 400 base pairs indicating the toxigenic strains.ConclusionHundred percent strains of V. cholerae were MDR possessing a ctx gene. It suggests that toxigenic strains be identified and proper antibiotic susceptibility testing be conducted. This will allow effective empirical therapy to be used to treat and control cholera.

Meddling Vibrio cholerae Murmurs: A Neoteric Advancement in Cholera Research.

Cholera, a known diarrheal disease is associated with various risk factors like hypovolemic shock, rice watery stools, and death in developing countries. The overuse of antibiotics to treat cholera imposed a selective pressure for the emergence and spread of multi-drug resistant Vibrio cholerae strains. The failure of conventional antimicrobial therapy urged the researchers to find an alternative therapy that could meddle the cholera murmurs (Quorum Sensing). It seems to effectively overcome the conventional cholera therapies in parallel to decrease the morbidity and mortality rate in the developing countries. The paramount objective of this review essentially focuses on the different Quorum Sensing (QS) regulatory switches governing virulence and pathogenicity of Vibrio cholerae. This review also provides an insight into the plausible QS targets that could be exploited to bring about a breakthrough to the prevailing cholera therapy.

Anethole inhibits growth of recently emerged multidrug resistant toxigenic Vibrio cholerae O1 El Tor variant strains in vitro.

To search natural compounds having inhibitory effect on bacterial growth is important, particularly in view of growing multidrug resistant (MDR) strains of bacterial pathogens. Like other bacterial pathogens, MDR Vibrio cholerae, the causative agent of diarrheal disease cholera, is becoming a great concern. As an approach of searching new antimicrobial agents, here, we show that anethole, a well-studied natural component of sweet fennel and star anise seeds, could potentially inhibit the growth of MDR O1 El Tor biotype, the ongoing 7th cholera pandemic variant strains of toxigenic V. cholerae. The minimum inhibitory concentration (MIC) of anethole against diverse O1 El Tor biotype strains is evaluated as 200 µg/ml. Moreover, the effect of anethole is bactericidal and exerts rapid-killing action on V. cholerae cells. This study is the first report which demonstrates that anethole, purified from natural compound, is a potent inhibitor of growth of toxigenic V. cholerae. Our data suggest that anethole could be a potential antimicrobial drug candidate, particularly against MDR V. cholerae mediated infections.

Worldwide occurrence of integrative conjugative element encoding multidrug resistance determinants in epidemic Vibrio cholerae O1.

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.

Cholera outbreaks (2012) in three districts of Nepal reveal clonal transmission of multi-drug resistant Vibrio cholerae O1.

BackgroundAlthough endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal.MethodsV. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE).ResultsAll V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498–511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.ConclusionsIn 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistant V. cholerae El Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.

Edible ice in Jakarta, Indonesia, is contaminated with multidrug-resistant Vibrio cholerae with virulence potential

Consumption of street food is considered a major health risk in the absence of public-health inspection programmes in Indonesia. It is hypothesized that ice used in street food could be one of the major sources of Vibrio cholerae contamination. This study documented V. cholerae contamination in edible ice from different areas of Jakarta, the capital city of Indonesia, and attempted to characterize the virulence potential of the strains. A selective medium was used to isolate 98 V. cholerae strains and their identity was confirmed using biochemical assays. Serological tests classified the majority (78%) in the non-O1 serogroup. Multiplex PCR was used to detect the presence of V. cholerae virulence genes, namely ctxA, ompU, tcpA, ace, zot and toxR. The toxR, ctxA, ompU and zot genes were detected in 75, 26, 15 and 1% of isolates, respectively. The ace and tcpA genes were not detected in any of the isolates. The ctxA gene encoding the cholera toxin subunit A, which has been associated only with clinical strains of O1, here was present in both serogroups. The antibiotic-resistance profile showed that 65, 60, 52, 39, 37, 19 and 3% of the isolates were resistant to ampicillin, streptomycin, kanamycin, sulfamethoxazole-trimethoprim, erythromycin, tetracycline and ciprofloxacin, respectively. A large proportion of V. cholerae isolates came from west and south Jakarta, and these strains exhibited multidrug resistance to ampicillin, streptomycin, tetracycline, erythromycin, kanamycin and sulfamethoxazole-trimethoprim. Many of these isolates from west and south Jakarta also harboured toxR, encoding a regulator, and ctxA. The presence of multidrug-resistant V. cholerae with virulence genes in edible ice, which could cause a severe outbreak, reflects the poor water quality in Jakarta, and indicates an urgent need for better surveillance and management.

Multi-drug resistant Vibrio cholerae O1 variant El Tor isolated in northern Vietnam between 2007 and 2010

Since 2007, there has been a re-emergence of cholera outbreaks in northern Vietnam. To understand the molecular epidemiological relatedness and determine the antibiotic susceptibility profiles of responsible V. cholerae O1 outbreak strains, a representative collection of 100 V. cholerae O1 strains was characterized. V. cholerae O1 strains isolated from diarrhoeal patients in northern Vietnam between 2007 and 2010 were investigated for antibiotic susceptibility and characterized by using phenotypic and genotypic tests, including PFGE analysis. Ten clinical V. cholerae O1 isolates from Bangladesh and Zimbabwe were included for comparison. The results revealed that all isolates were resistant to co-trimoxazole and nalidixic acid, 29 % were resistant to tetracycline and 1 % were resistant to azithromycin. All strains were susceptible to ampicillin–sulbactam, doxycycline, chloramphenicol and ciprofloxacin and 95 % were susceptible to azithromycin. MIC values did show reduced susceptibility to fluoroquinolones and 63 % of the strains were intermediately resistant to tetracycline. The isolates expressed phenotypic traits of both serogroup O1 Ogawa and El Tor and harboured an rstR El Tor and ctxB classical biotype. Among the outbreak isolates, only a single PFGE pattern was observed throughout the study period. This study shows that multi-drug resistant V. cholerae altered El Tor producing classical CT strains are now predominant in northern Vietnam.

Aqueous synthesis of ZnTe/dendrimer nanocomposites and their antimicrobial activity: implications in therapeutics

The present strategy proposes a simple and single step aqueous route for synthesizing stable, fluorescent ZnTe/dendrimer nanocomposites with varying dendrimer terminal groups. In these hybrid materials, the fluorescence of the semiconductor combines with the biomimetic properties of the dendrimer making them suitable for various biomedical applications. The ZnTe nanocomposites thus obtained demonstrate bactericidal activity against enteropathogenic bacteria without having toxic effects on the human erythrocytes. The average size of the ZnTe nanoparticles within the dendrimer matrix was in the range of 2.9-6.0 nm, and they have a good degree of crystallinity with a hexagonal crystal phase. The antibacterial activities of the ZnTe/dendrimer nanocomposites (ZnTe DNCs) as well other semiconductor nanocomposites were evaluated against enteropathogenic bacteria including multi-drug resistant Vibrio cholerae serogroup O1 and enterotoxigenic Escherichia coli (ETEC). ZnTe DNCs had significant antibacterial activity against strains of V. cholerae and ETEC with minimum inhibitory concentrations ranging from 64 to 512 μg ml(-1) and minimum bactericidal concentrations ranging from 128 to 1000 μg ml(-1). Thus, the observed results suggest that these water-soluble active nanocomposites have potential for the treatment of enteric diseases like diarrhoea and cholera.

Multidrug resistant Vibrio cholerae O1 El Tor carrying classical ctxB allele involved in a cholera outbreak in South Western India

Cholera is a fatal diarrheal disease characterized with enormous fluid loss through stools. A total of 41 Vibrio cholerae isolates collected from a recent cholera outbreak in Solapur, South Western India were found to belong to serogroup O1, biotype El Tor and serotype Ogawa. Molecular analysis revealed the prevalence of different toxigenic and pathogenic genes in the isolates. All the isolates harboured rstRElTor allele indicating the presence of CTXΦElTor. However, cholera toxin (ctxB) gene sequencing and a ctxB allele specific PCR of the isolates confirmed the presence of ctxB of classical biotype. The antibiogram profile revealed the resistance for several antibiotics including nalidixic acid, polymyxin B, streptomycin, sulfamethoxazole, trimethoprim, rifampicin and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). All the isolates were PCR positive for class 1 integron and SXT elements also. Fingerprinting analysis revealed the clonal relationship among the outbreak isolates. The results suggested the involvement of multidrug resistant V. cholerae El Tor biotype isolates having ctxB gene of classical biotype in the cholera outbreak.

Evaluation of the Antimicrobial Activity of Some Medicinal Plants against Enteric Bacteria with Particular Reference to Multi-Drug Resistant Vibrio cholerae

Purpose: To evaluate the anti-enterobacterial potential of nine ethnobotanically selected plants traditionally used in different parts of India for the treatment of gastrointestinal disorders such as cholera, diarrhea or dysentery. Methods: The methanol extracts of these plants were screened for antibacterial activity against 11 strains of enteropathogenic bacteria, including multi-drug resistant Vibrio cholerae (serotypes O1, O139, and non-O1, non-O139), using broth microdilution method. Ampicillin was used as a positive reference standard to determine the sensitivity of the strains. Phytochemical screening was carried out for phenolics and flavonoids. Results: All of these plants had bactericidal activity against at least one of the test microorganisms with minimum inhibitory concentration (MIC) ranging from 0.125 to 32 mg/ml and minimum bactericidal concentration (MBC) ranging from 0.25 to 32 mg/ml. Among these, Terminalia chebula Retz. (Combretaceae ) and Syzygium cumini (L.) Skeels (Myrtaceae) showed the most promising broad spectrum antibacterial properties, inhibiting all of the strains tested, especially Vibrio cholerae, Aeromonas hydrophila and Bacillus subtilis, with MBC ranging from 0.25 to 4 mg/ml. Phenolics and flavonoids were found to be present in the extracts. Conclusion: The findings provide support for the use of this plant in traditional medicine for treatment of diarrheoa, especially cholera.

Antibiotic resistance conferred by a conjugative plasmid and a class I integron in Vibrio cholerae O1 El Tor strains isolated in Albania and Italy.

Multidrug-resistant Vibrio cholerae O1 El Tor strains isolated during the 1994 outbreak of cholera in Albania and Italy were characterized for the molecular basis of antibiotic resistance. All strains were found to be resistant to tetracycline, streptomycin, spectinomycin, trimethoprim, sulfathiazole, and the vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylteridine). Resistance genes were self-transferable by a conjugative plasmid of about 60 MDa, with the exception of spectinomycin resistance, which was conferred by the aadA1 gene cassette located in the bacterial chromosome within a class 1 integron. The resistance to trimethoprim and O/129 was conferred by the dfrA1 gene, which was present on the plasmid. Although the dfrA1 gene is known to be borne on an integron cassette, class 1, 2, or 3 intI genes were not detected as part of the plasmid DNA from the strains studied.