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Volume 16
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

In Silico Mutational Analysis of Two-Component System Genes Associated with Colistin Resistance in Clinical Pseudomonas aeruginosa Isolates from Peshawar

by
Bashir Ahmad
1,
Qaisar Ali
1,
Sadiq Azam
1,*,
Muhammad Asghar
2,
Noor Rehman
2,
Gul-e-Sehra Mujib
1,
Syed Sohail Shah
1,
Jamila Javed
3,
Ibrar Khan
1,
Taj Ali Khan
4 and
Taane G. Clark
5,6,7,*
1
Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan
2
Department of Pathology, Khyber Medical College, Peshawar 25000, Khyber Pakhtunkhwa, Pakistan
3
Institute of Biotechnology Genetic Engineering, The University of Agriculture, Peshawar 25130, Khyber Pakhtunkhwa, Pakistan
4
Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan
5
Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
6
Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
7
Department of Pathology, Khyber Teaching Hospital, Khyber Medical College, Peshawar 25120, Pakistan
*
Authors to whom correspondence should be addressed.
Biomolecules 2026, 16(7), 962; https://doi.org/10.3390/biom16070962 (registering DOI)
Submission received: 27 May 2026 / Revised: 22 June 2026 / Accepted: 26 June 2026 / Published: 29 June 2026
(This article belongs to the Section Bioinformatics and Systems Biology)
Versions Notes

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen causing healthcare-associated infections. Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative bacteria. Resistance arises through mutations in two-component systems (TCS) regulating the arn operon. Data on colistin resistance in P. aeruginosa from Pakistan remain limited. A total of 3189 clinical samples (urine, blood, sputum, pus, wound swabs) were cultured. P. aeruginosa was identified by Gram staining, biochemical tests (catalase, oxidase, API 20E), and oprL gene amplification. Antibiotic susceptibility was determined by disk diffusion and MIC strips. Resistance genes (PhoP, PhoQ, PmrA, PmrB, mcr-1, oprD) were detected by PCR and Sanger sequencing. Wild-type protein structures were retrieved from PDB; mutant structures were predicted using AlphaFold3. ANP (phosphoaminophosphonic acid-adenylate ester) was docked using MOE 2019.0102. Of 3189 samples, 384 (12.0%) yielded P. aeruginosa. Wound/pus (38.0%) and surgical wards (30.0%) were the predominant sources. Colistin and polymyxin B showed 99.0% susceptibility (MIC50/MIC90 = 1 µg/mL). High resistance was observed for Piperacillin–Tazobactam (96.4%), Aztreonam (70.6%), and Gentamicin (64.2%). oprD was the most prevalent gene (87.5%), followed by PmrB (54.0%), PhoQ (44.0%), PhoP (36.0%), PmrA (18.0%), and mcr-1 (8.0%). Docking revealed the strongest binding in wild-type PhoQ (1ID0; −12.0 kcal/mol, LYS392), wild-type PmrB (2JSO; −9.8 kcal/mol, ASP37), and wild-type PhoP (2PKX; −9.1 kcal/mol, LYS87/ARG111). Mutant proteins showed reduced binding affinities and dispersed interaction networks. Mutant PhoP formed 16 contacts (strongest −4.3 kcal/mol) versus wild-type PhoP with 13 contacts (−9.1 kcal/mol). Colistin remains highly effective against P. aeruginosa in this setting (99.0% susceptibility). The presence of mcr-1 (8.0%) and high oprD prevalence (87.5%) require continued surveillance. Mutations in TCS proteins reduce ANP binding affinity and alter interaction specificity, suggesting that ATP-competitive inhibitors targeting these kinases merit further investigation and experimental validation.
Keywords: Pseudomonas aeruginosa; colistin resistance; two-component system; molecular docking; ANP; mcr-1 Pseudomonas aeruginosa; colistin resistance; two-component system; molecular docking; ANP; mcr-1

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MDPI and ACS Style

Ahmad, B.; Ali, Q.; Azam, S.; Asghar, M.; Rehman, N.; Mujib, G.-e.-S.; Shah, S.S.; Javed, J.; Khan, I.; Khan, T.A.; et al. In Silico Mutational Analysis of Two-Component System Genes Associated with Colistin Resistance in Clinical Pseudomonas aeruginosa Isolates from Peshawar. Biomolecules 2026, 16, 962. https://doi.org/10.3390/biom16070962

AMA Style

Ahmad B, Ali Q, Azam S, Asghar M, Rehman N, Mujib G-e-S, Shah SS, Javed J, Khan I, Khan TA, et al. In Silico Mutational Analysis of Two-Component System Genes Associated with Colistin Resistance in Clinical Pseudomonas aeruginosa Isolates from Peshawar. Biomolecules. 2026; 16(7):962. https://doi.org/10.3390/biom16070962

Chicago/Turabian Style

Ahmad, Bashir, Qaisar Ali, Sadiq Azam, Muhammad Asghar, Noor Rehman, Gul-e-Sehra Mujib, Syed Sohail Shah, Jamila Javed, Ibrar Khan, Taj Ali Khan, and et al. 2026. "In Silico Mutational Analysis of Two-Component System Genes Associated with Colistin Resistance in Clinical Pseudomonas aeruginosa Isolates from Peshawar" Biomolecules 16, no. 7: 962. https://doi.org/10.3390/biom16070962

APA Style

Ahmad, B., Ali, Q., Azam, S., Asghar, M., Rehman, N., Mujib, G.-e.-S., Shah, S. S., Javed, J., Khan, I., Khan, T. A., & Clark, T. G. (2026). In Silico Mutational Analysis of Two-Component System Genes Associated with Colistin Resistance in Clinical Pseudomonas aeruginosa Isolates from Peshawar. Biomolecules, 16(7), 962. https://doi.org/10.3390/biom16070962

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