Molecular Characterisation of Biofilm-Forming, Multidrug-Resistant Bacteria from Water Distribution Systems, Choba, Rivers State, Nigeria

Technical Summary: Molecular Characterisation of Biofilm-Forming, Multidrug-Resistant Bacteria from Water Distribution Systems, Choba, Rivers State, Nigeria

Problem Statement
Access to safe drinking water is a fundamental human right and a target of the 6th Sustainable Development Goal. However, water distribution systems are recognized as environments where pathogenic bacteria can persist, often forming biofilms that protect them from disinfectants and antibiotics. While previous studies have identified pathogenic species in drinking water, there is a gap in research assessing the specific virulence determinants and drug resistance profiles of these isolates. This study addresses the need to characterize biofilm-forming, multidrug-resistant (MDR) bacteria isolated from municipal water distribution systems in Choba, Rivers State, Nigeria, to differentiate between pathogenic and non-pathogenic strains and assess public health risks.

Methodology
The study employed a combination of standard microbiological, phenotypic, and molecular techniques:

• Sample Collection: Twelve environmental tap water samples were collected aseptically from the Abuja campus of the University of Port Harcourt.
• Isolation and Identification: Twenty-eight bacterial isolates were recovered and confirmed via biochemical assays (including motility, sugar fermentation, oxidase, catalase, urease, and indole tests).
• Antimicrobial Susceptibility Testing: The Kirby-Bauer disc diffusion method was used on Mueller Hinton agar to determine resistance profiles against various antibiotics.
• Biofilm Assessment: Two phenotypic methods were utilized:
  1. Congo Red Agar (CRA): To observe black pigmentation indicative of biofilm production.
  2. Tube Method: To visualize ring formation and quantify biofilm via crystal violet staining.
• Molecular Screening: Genomic DNA was extracted using the boiling method. Polymerase Chain Reaction (PCR) was performed to detect four specific virulence-associated genes: fimH (type 1 fimbriae), papC (P fimbriae), afaCc (afimbrial adhesins), and rmpA (capsule-associated regulator).
• Statistical Analysis: Data were analyzed using Microsoft Excel 2010.

Key Results

• Bacterial Identification: The 28 isolates comprised various species, with Escherichia coli and Streptococcus sp. being the most prevalent (21.4% each). Chromobacterium violaceum accounted for 3.6% (1 isolate).
• Antibiotic Resistance:
  • Gram-negative isolates showed high resistance (>80%) to three specific antibiotics but were fully sensitive to three quinolones (ofloxacin, nalidixic acid, levofloxacin).
  • Gram-positive isolates exhibited lower resistance rates (<15% for 50% of tested antibiotics).
  • Overall, 84.6% of Gram-negative isolates were classified as MDR, compared to 33.3% of Gram-positive isolates.
• Biofilm Formation: Only one isolate, identified as Chromobacterium violaceum (designated PC19), demonstrated biofilm-forming ability. This was confirmed by black pigmentation on CRA and a visible purple ring in the tube assay.
• Molecular Findings:
  • PC19 (C. violaceum): Despite low phenotypic antibiotic resistance (resistant only to cefuroxime), this biofilm-forming isolate tested positive for the papC gene. The genes afaCc, fimH, and rmpA were not detected.
  • PC12 (Klebsiella pneumoniae): Selected for its MDR status and lack of biofilm formation, this isolate tested positive for fimH and papC but negative for afaCc and rmpA.
  • Notably, the study detected the presence of virulence genes in isolates that did not necessarily exhibit high multidrug resistance phenotypes.

Key Contributions

• Novel Isolation: This study reports the successful isolation and molecular identification of Chromobacterium violaceum from a municipal water distribution system in Nigeria, a finding not previously documented in this specific context.
• Phenotype-Genotype Correlation: The research highlights a complex interplay between antimicrobial resistance and virulence. It demonstrates that an isolate can possess virulence-associated genes (specifically papC in C. violaceum) and biofilm-forming capabilities even when it is not extensively multidrug-resistant.
• Methodological Application: The study validates the use of Congo Red Agar and tube methods alongside PCR screening for virulence genes as effective tools for assessing the pathogenic potential of waterborne bacteria.

Significance and Claims
The authors conclude that the presence of Chromobacterium violaceum with biofilm-forming potential in a potable water network implies an increased risk of microbial persistence and reduced susceptibility to disinfection processes. The study emphasizes that antimicrobial susceptibility does not necessarily equate to low pathogenic potential; even susceptible isolates may harbor virulence determinants that allow them to adhere to pipes and form biofilms.

The paper asserts that these findings underscore the necessity for continuous microbiological surveillance that integrates both phenotypic and molecular methods. This approach is deemed essential for effective water quality management and the protection of public health, particularly in distinguishing between harmless environmental bacteria and those with latent pathogenic capabilities.