Study Notes on Vibrio cholerae O1 Research

Research Article: Investigating the Virulence Genes and Antibiotic Susceptibility Patterns of Vibrio cholerae O1 in Environmental and Clinical Isolates in Accra, Ghana

Abstract

  • Background: Cholera has been endemic in Ghana since its first detection in 1970. Long-term survival of Vibrio cholerae is possible in aquatic environments, leading to outbreaks mainly in crowded urban areas.
  • Objective: To investigate clinical and environmental isolates of V. cholerae O1 in Accra, focusing on virulence genes, antibiotic susceptibility patterns, and environmental factors promoting persistence.
  • Methods: Water samples analyzed for V. cholerae O1 via culture methods; included 40 clinical isolates from a cholera outbreak. Antibiotic susceptibility was assessed through disc diffusion. Virulence genes analyzed via PCR for genes: ctx, tcpA (tcpA El Tor, tcpA Classical), zot, ompW, rbfO1, and attRS. Physicochemical characteristics of water were measured. Statistical analysis used one-way ANOVA and Student t-test.
  • Results: Eleven V. cholerae O1 strains isolated from various water sources. All isolates exhibited resistance to at least one antibiotic, with 97% showing multidrug resistance. Approximately 82.4% of isolates had the cholera toxin gene; 81.8% tested positive for tcpA El Tor gene. Thirteen genotypes identified. Environmental parameters like temperature, salinity, total dissolved solids, and conductivity positively correlated with V. cholerae O1 isolation. The predominant circulating strain in Ghana is V. cholerae Ogawa biotype El Tor, with emerging hybrid strains.
  • Conclusions: Multidrug-resistant V. cholerae O1 with varying genotypes coexist in water sources along with non O1/O139 in Accra.

Keywords

  • Vibrio cholerae O1, Cholera, Virulence genes, Multidrug resistance, Environmental factors, Genotypes, Accra

Introduction

  • Cholera, caused by the Gram-negative bacterium Vibrio cholerae, is acute secretory diarrhea.
  • V. cholerae thrives in aquatic environments (rivers, estuaries) and can exist as free cells or attached to surfaces.
  • The ability to express virulence factors (choler toxin (CT), TCP) is crucial for epidemic potential.
  • The ctx gene, encoded by a filamentous transportable phage (ctxΦ) and the Vibrio Pathogenicity island (VPI), is important for pathogenicity.
  • Non-toxigenic strains can cause various infections (gastroenteritis, septicemia).
  • Cholera outbreaks in Ghana led to significant fatalities, with the highest number of cases in 2014. Lack of data on environmental reservoirs for V. cholerae O1 in West Africa has prompted further research.
  • Concerns over antibiotic resistance have arisen from sporadic increases in V. cholerae strain resistance to key antibiotics.

Study Site

  • Conducted across four densely populated communities in Greater Accra (Teshie, James Town, Chorkor, Nima).
  • Communities face significant potable water shortages, relying on various storage tanks.
  • Historically affected by cholera outbreaks (notably 2014).

Methods

Sample Collection
  • Water samples (244) collected between October 2015 - January 2016 from streams, wells, storage containers, and taps.
  • Samples acquired using standardized aseptic techniques to avoid contamination.
Environmental Parameter Determination
  • Recorded ambient conditions (temperature, salinity, conductivity, pH, total dissolved solids) with appropriate equipment.
Isolation and Characterization of V. cholerae
  • Enrichment of water samples in alkaline peptone water followed by colony isolation on thiosulfate citrate bile salt sucrose (TCBS) agar.
  • Pure cultures subjected to biochemical tests (Gram-staining, catalase and oxidase tests) and serotyping.
Antibiotic Susceptibility Testing
  • Performed via disc diffusion method per Clinical and Laboratory Standards Institute guidelines using eight antibiotics: tetracycline, doxycycline, trimethoprim/sulfamethoxazole, ciprofloxacin, chloramphenicol, erythromycin, azithromycin, nalidixic acid.
Detection of Virulence Genes
  • Assessed for ctx, tcp, zot, ompW, rbfO1, and attRS using PCR. Included controls for quality assurance.

Results

Physicochemical Parameters
  • Temperatures ranged from 25.6 °C (streams) to 33.2 °C (storage tanks).
  • pH values varied significantly with wells being the most acidic (pH 4.78) and tap waters least acidic (pH 8.75).
  • Statistically significant differences in parameters were recorded between various sources.
Isolated Strains
  • 51 isolates total: 40 clinical (78.4%) from past cholera outbreaks, 11 environmental (21.6%) from this study.
Antibiotic Resistance Patterns
  • Clinical isolates showed highest resistance against erythromycin (92.5%) and nalidixic acid (72.5%); resistance to ciprofloxacin (10%) and doxycycline (15%) noted in some instances.
  • Environmental strains showed comparable resistance patterns, with none resistant to both ciprofloxacin and doxycycline.
Genotyping and Virulence Gene Presence
  • ctx gene present in 90% of clinical and 63.6% of environmental isolates.
  • All isolates positive for tcpA El Tor; varying patterns found for other genes tested.
Genotype Distribution
  • Multiple genotypes identified, with predominant genotype tcpA El Tor + ompW + ctx + attRS + rbfO1 + zot + tcpA Classical constituting 55% of clinical isolates.

Discussion

  • The study reaffirms the complexity of cholera ecology, linking environmental conditions to V. cholerae prevalence.
  • Notable cholera strain patterns in Ghana; need for monitoring and improved public health responses.
  • Resistance of V. cholerae denotes challenges to treatment protocols; urgent re-evaluation necessary.
  • Recommendations for controlled antibiotic use and efforts to provide clean water supply in urban areas.

Conclusion

  • Multidrug resistant V. cholerae O1 exists in both clinical and environmental contexts.
  • Ongoing vigilance and action required to mitigate cholera threat, including potential outbreaks linked to environmental sources.
  • Effective antibiotics remain available but vigilance against increasing resistance is crucial.

Additional Files

  • Additional File 1: General physicochemical parameters of water sources.
  • Additional File 2: Effects of physicochemical parameters on V. cholerae O1 occurrence.

Abbreviations

  • ATCC: American Type Culture Collection
  • CLSI: Clinical Laboratory Standard Institutes
  • CT: Cholera Toxin
  • Ctx: Cholera toxin gene
  • MDR: Multidrug Resistance
  • NPHRL: National Public Health Reference Laboratory
  • PCR: Polymerase Chain Reaction
  • TDS: Total Dissolved Solids
  • TCP: Toxin Co-regulated Pilus
  • WHO: World Health Organization

Acknowledgements

  • Gratitude extended to collaborating institutions for support in various aspects of the research.

Funding

  • Master’s fellowship support was received from World Bank African Centres of Excellence Grant.

Authors’ Contributions

  • Contributions detailed with particular roles in study design, data collection, analysis, and manuscript drafting.

Ethics Approval and Consent

  • No formal ethics approval deemed necessary for the study according to inquiries made.