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In Silico Mutational Analysis of Two-Component System Genes Associated with Colistin Resistance in Clinical

Bashir Ahmad1, Qaisar Ali1, Sadiq Azam1

  • 1Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan.

Biomolecules
|July 28, 2026
PubMed

Insights

Colistin remains highly effective against Pseudomonas aeruginosa in Pakistan, with 99% susceptibility. However, the emergence of mcr-1 and high prevalence of oprD warrant ongoing surveillance for this opportunistic pathogen.

Area of Science:

  • Clinical microbiology and infectious diseases
  • Molecular biology and genetics
  • Structural biology and drug discovery

Background:

  • Pseudomonas aeruginosa is a significant cause of healthcare-associated infections.
  • Colistin is a critical last-resort antibiotic for multidrug-resistant Gram-negative bacteria.
  • Resistance to colistin in P. aeruginosa is often linked to mutations in two-component systems (TCS) regulating the arn operon.

Purpose of the Study:

  • To investigate colistin resistance mechanisms and antibiotic susceptibility patterns of P. aeruginosa in Pakistan.
  • To identify prevalent resistance genes and analyze the structural impact of mutations on TCS proteins.
  • To evaluate the binding affinity of ANP to wild-type and mutant proteins.

Main Methods:

  • Culturing of 3189 clinical samples to isolate P. aeruginosa.
  • Antibiotic susceptibility testing using disk diffusion and MIC strips.
  • Detection of resistance genes (PhoP, PhoQ, PmrA, PmrB, mcr-1, oprD) via PCR and Sanger sequencing.
  • Structural analysis and molecular docking of ANP using AlphaFold3 and MOE.

Main Results:

  • P. aeruginosa was isolated from 12.0% of samples, predominantly from wound/pus.
  • High susceptibility (99.0%) to colistin and polymyxin B was observed, contrasting with high resistance to other antibiotics.
  • The most prevalent genes were oprD (87.5%), PmrB (54.0%), PhoQ (44.0%), PhoP (36.0%), PmrA (18.0%), and mcr-1 (8.0%).
  • Molecular docking revealed reduced ANP binding affinity in mutant TCS proteins compared to wild-type.

Conclusions:

  • Colistin remains a highly effective treatment option for P. aeruginosa infections in the studied Pakistani setting.
  • The presence of mcr-1 and high oprD prevalence necessitate continuous monitoring.
  • Mutations in TCS proteins impair ANP binding, suggesting potential for ATP-competitive inhibitors.
Keywords:
ANPPseudomonas aeruginosacolistin resistancemcr-1molecular dockingtwo-component system

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