{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T15:12:57Z","timestamp":1783696377486,"version":"3.55.0"},"reference-count":40,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T00:00:00Z","timestamp":1782691200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"UKRI MRC","award":["IAA2129"],"award-info":[{"award-number":["IAA2129"]}]},{"name":"UKRI MRC","award":["MR\/R026297\/1"],"award-info":[{"award-number":["MR\/R026297\/1"]}]},{"name":"UKRI MRC","award":["MR\/X005895\/1"],"award-info":[{"award-number":["MR\/X005895\/1"]}]},{"DOI":"10.13039\/501100000266","name":"EPSRC","doi-asserted-by":"crossref","award":["EP\/Y018842\/1"],"award-info":[{"award-number":["EP\/Y018842\/1"]}],"id":[{"id":"10.13039\/501100000266","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>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 \u00b5g\/mL). High resistance was observed for Piperacillin\u2013Tazobactam (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; \u221212.0 kcal\/mol, LYS392), wild-type PmrB (2JSO; \u22129.8 kcal\/mol, ASP37), and wild-type PhoP (2PKX; \u22129.1 kcal\/mol, LYS87\/ARG111). Mutant proteins showed reduced binding affinities and dispersed interaction networks. Mutant PhoP formed 16 contacts (strongest \u22124.3 kcal\/mol) versus wild-type PhoP with 13 contacts (\u22129.1 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.<\/jats:p>","DOI":"10.3390\/biom16070962","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T03:06:34Z","timestamp":1782875194000},"page":"962","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["In Silico Mutational Analysis of Two-Component System Genes Associated with Colistin Resistance in Clinical Pseudomonas aeruginosa Isolates from Peshawar"],"prefix":"10.3390","volume":"16","author":[{"given":"Bashir","family":"Ahmad","sequence":"first","affiliation":[{"name":"Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1556-4987","authenticated-orcid":false,"given":"Qaisar","family":"Ali","sequence":"additional","affiliation":[{"name":"Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sadiq","family":"Azam","sequence":"additional","affiliation":[{"name":"Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad","family":"Asghar","sequence":"additional","affiliation":[{"name":"Department of Pathology, Khyber Medical College, Peshawar 25000, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noor","family":"Rehman","sequence":"additional","affiliation":[{"name":"Department of Pathology, Khyber Medical College, Peshawar 25000, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gul-e-Sehra","family":"Mujib","sequence":"additional","affiliation":[{"name":"Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Syed Sohail","family":"Shah","sequence":"additional","affiliation":[{"name":"Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jamila","family":"Javed","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology Genetic Engineering, The University of Agriculture, Peshawar 25130, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4562-9526","authenticated-orcid":false,"given":"Ibrar","family":"Khan","sequence":"additional","affiliation":[{"name":"Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taj Ali","family":"Khan","sequence":"additional","affiliation":[{"name":"Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8985-9265","authenticated-orcid":false,"given":"Taane G.","family":"Clark","sequence":"additional","affiliation":[{"name":"Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK"},{"name":"Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2117","DOI":"10.1007\/s40265-021-01635-6","article-title":"The Epidemiology and Pathogenesis and Treatment of Pseudomonas Aeruginosa Infections: An Update","volume":"81","author":"Reynolds","year":"2021","journal-title":"Drugs"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Tuon, F.F., Dantas, L.R., Suss, P.H., and Tasca Ribeiro, V.S. (2022). Pathogenesis of the Pseudomonas Aeruginosa Biofilm: A Review. Pathogens, 11.","DOI":"10.3390\/pathogens11030300"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"28","DOI":"10.15698\/mic2021.02.741","article-title":"Biofilms by Bacterial Human Pathogens: Clinical Relevance\u2014Development, Composition and Regulation\u2014Therapeutical Strategies","volume":"8","author":"Schulze","year":"2021","journal-title":"Microb. Cell"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"100030","DOI":"10.1016\/j.microb.2023.100030","article-title":"Phage Therapy as a Revolutionary Treatment for Multidrug-Resistant Pseudomonas Aeruginosa Infections: A Narrative Review","volume":"2","author":"Nawaz","year":"2024","journal-title":"Microbe"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Holger, D., Kebriaei, R., Morrisette, T., Lev, K., Alexander, J., and Rybak, M. (2021). Clinical Pharmacology of Bacteriophage Therapy: A Focus on Multidrug-Resistant Pseudomonas Aeruginosa Infections. Antibiotics, 10.","DOI":"10.3390\/antibiotics10050556"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1038\/s41392-022-01056-1","article-title":"Pseudomonas Aeruginosa: Pathogenesis, Virulence Factors, Antibiotic Resistance, Interaction with Host, Technology Advances and Emerging Therapeutics","volume":"7","author":"Qin","year":"2022","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"55","DOI":"10.5195\/jmla.2024.1780","article-title":"Alexander Fleming: A Second Look","volume":"112","author":"Gerberi","year":"2024","journal-title":"J. Med. Libr. Assoc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e03089-20","DOI":"10.1128\/AEM.03089-20","article-title":"Two-Component Signaling Systems Regulate Diverse Virulence-Associated Traits in Pseudomonas Aeruginosa","volume":"87","author":"Wang","year":"2021","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3372","DOI":"10.1128\/AAC.00242-10","article-title":"Adaptive Resistance to the \u201cLast Hope\u201d Antibiotics Polymyxin B and Colistin in Pseudomonas Aeruginosa Is Mediated by the Novel Two-Component Regulatory System ParR-ParS","volume":"54","author":"Fernandez","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e00556-19","DOI":"10.1128\/AAC.00556-19","article-title":"Resistance and Heteroresistance to Colistin in Pseudomonas Aeruginosa Isolates from Wenzhou, China","volume":"63","author":"Lin","year":"2019","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Bastidas-Caldes, C., de Waard, J.H., Salgado, M.S., Villac\u00eds, M.J., Coral-Almeida, M., Yamamoto, Y., and Calvopi\u00f1a, M. (2022). Worldwide Prevalence of Mcr-Mediated Colistin-Resistance Escherichia Coli in Isolates of Clinical Samples, Healthy Humans, and Livestock\u2014A Systematic Review and Meta-Analysis. Pathogens, 11.","DOI":"10.3390\/pathogens11060659"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Sanya, D.R.A., On\u00e9sime, D., Vizzarro, G., and Jacquier, N. (2023). Recent Advances in Therapeutic Targets Identification and Development of Treatment Strategies towards Pseudomonas Aeruginosa Infections. BMC Microbiol., 23.","DOI":"10.1186\/s12866-023-02832-x"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2046","DOI":"10.51470\/PLANTARCHIVES.2021.v21.S1.335","article-title":"Genetic study of psoriasis disease: A reveiew","volume":"21","author":"Muslim","year":"2021","journal-title":"Plant Arch."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e10089","DOI":"10.15252\/msb.202010089","article-title":"Collective Colony Growth Is Optimized by Branching Pattern Formation in Pseudomonas Aeruginosa","volume":"17","author":"Luo","year":"2021","journal-title":"Mol. Syst. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"100640","DOI":"10.1016\/j.drup.2019.07.002","article-title":"Antibiotic Resistance in Pseudomonas Aeruginosa\u2014Mechanisms, Epidemiology and Evolution","volume":"44","author":"Botelho","year":"2019","journal-title":"Drug Resist. Updates"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1868","DOI":"10.1093\/cid\/ciad012","article-title":"Deconstructing the 2023 Clinical and Laboratory Standards Institute Revised Piperacillin-Tazobactam Breakpoints against Pseudomonas Aeruginosa","volume":"76","author":"Tamma","year":"2023","journal-title":"Clin. Infect. Dis."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Cavka, M., Kvesic Ivankovic, M., Maravic, A., Dzelalija, M., Marinovic, J., Goic-Barisic, I., Tonkic, M., and Novak, A. (2026). Phenotypic and Whole-Genome Sequencing-Based Profiling of Antimicrobial Resistance and Virulence in Pseudomonas Aeruginosa Isolated from Patients with Ventilator-Associated Pneumonia and Ventilator-Associated Tracheobronchitis in a Croatian Intensive Care Unit. Genes, 17.","DOI":"10.3390\/genes17020130"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e03191-25","DOI":"10.1128\/spectrum.03191-25","article-title":"A Rapid Ionic Liquid-Based DNA Extraction Method for Molecular Diagnostics of Urinary Tract Infections","volume":"14","author":"Kreuter","year":"2026","journal-title":"Microbiol. Spectr."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Khan, Z., Ali, Q., Azam, S., Khan, I., Javed, J., Rehman, N., Ahmed, M.M., Uddin, J., Khan, A., and Al-Harrasi, A. (2025). Current Pattern of Antibiotic Resistance and Molecular Characterization of Virulence Genes in Klebsiella Pneumoniae Obtained from Urinary Tract Infection (UTIs) Patients, Peshawar. PLoS ONE, 20.","DOI":"10.1371\/journal.pone.0319273"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Khan, M.R., Azam, S., Ahmad, S., Ali, Q., Liaqat, Z., Rehman, N., Khan, I., Alharbi, M., and Alshammari, A. (2023). Molecular Characterization and Epidemiology of Antibiotic Resistance Genes of \u03b2-Lactamase Producing Bacterial Pathogens Causing Septicemia from Tertiary Care Hospitals. Antibiotics, 12.","DOI":"10.3390\/antibiotics12030617"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1403","DOI":"10.1080\/17460441.2020.1803274","article-title":"Novel Therapeutic Strategies for Treating Pseudomonas Aeruginosa Infection","volume":"15","author":"Shao","year":"2020","journal-title":"Expert Opin. Drug Discov."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"fuab026","DOI":"10.1093\/femsre\/fuab026","article-title":"Pseudomonas Aeruginosa: A Clinical and Genomics Update","volume":"45","author":"Pelegrin","year":"2021","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_23","first-page":"1953","article-title":"Comparative Analysis of Pseudomonas Aeruginosa Isolated from Different Clinical Sources Using Whole Genome Sequencing and Bioinformatics","volume":"13","author":"Khadem","year":"2026","journal-title":"J. Appl. Biotechnol. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"101736","DOI":"10.1016\/j.nmni.2026.101736","article-title":"The Emergence of Pan-Drug-Resistant Pseudomonas Aeruginosa in Iran: A Genomic and Phenotypic Analysis of Clinical Isolates, Multifactorial Resistance Mechanisms, and Its Societal Impact","volume":"71","author":"Fayyazi","year":"2026","journal-title":"New Microbes New Infect."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"10273","DOI":"10.1007\/s00210-025-03913-6","article-title":"Molecular Characterization of Metallo-Beta-Lactamase Producers among Carbapenem-Resistant Pseudomonas Aeruginosa from Clinical Samples in a Tertiary Care Hospital, Lucknow, India","volume":"398","author":"Ahmad","year":"2025","journal-title":"Naunyn-Schmiedeberg\u2019s Arch. Pharmacol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1111\/j.1469-0691.2011.03570.x","article-title":"Multidrug-Resistant, Extensively Drug-Resistant and Pandrug-Resistant Bacteria: An International Expert Proposal for Interim Standard Definitions for Acquired Resistance","volume":"18","author":"Magiorakos","year":"2012","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1007\/s11033-025-11368-4","article-title":"Polymyxin Resistance in Gram-Negative Bacteria: A Comprehensive Review with a Focus on the Role of 4-Amino-4-Doexy-l-Arabinose","volume":"53","author":"Sasal","year":"2025","journal-title":"Mol. Biol. Rep."},{"key":"ref_28","first-page":"e46","article-title":"Antibiotic Resistance Patterns of Pseudomonas Aeruginosa Isolated from Clinical Samples in Khyber Pakhtunkhwa, Pakistan","volume":"3","author":"Ali","year":"2025","journal-title":"J. Health Wellness Community Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1981","DOI":"10.4014\/jmb.2407.07005","article-title":"Evaluation and Molecular Characterization of Colistin-Resistant Isolates of Pseudomonas Aeruginosa and Klebsiella Pneumoniae from the Infected Wounds of Hospitalized Patients","volume":"34","author":"Ijaz","year":"2024","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_30","first-page":"1","article-title":"Prevalence of Pseudomonas Aeruginosa and Acinetobacter Baumanni Isolated from Clinical Samples in a Tertiary Care Hospital Islamabad","volume":"1","author":"Iqbal","year":"2023","journal-title":"J. Microb. Insights Orig. Artic."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"10531","DOI":"10.1007\/s11033-022-07930-z","article-title":"Molecular Characterization and Antibiogram of the Carbapenemase Gene Variants in Clinical Strains of Pseudomonas Aeruginosa","volume":"49","author":"Ejaz","year":"2022","journal-title":"Mol. Biol. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1097\/MRM.0000000000000348","article-title":"Detection of Some Virulence Genes among Clinical Pseudomonas Aeruginosa Isolates in Baghdad","volume":"35","author":"Khazaal","year":"2023","journal-title":"Rev. Res. Med. Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1111\/jcpt.13446","article-title":"Successfully Controlling the Incidence of Multidrug-Resistant Pseudomonas Aeruginosa through Antibiotic Stewardship and Infection Control Programmes at a Chinese University Hospital","volume":"46","author":"Li","year":"2021","journal-title":"J. Clin. Pharm. Ther."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1556\/030.2025.02715","article-title":"Antimicrobial Resistance in Major Bacterial and Fungal Pathogens in Hospital-Acquired Infections from a Tertiary Care Hospital in Lodhran, Pakistan","volume":"72","author":"Nehvi","year":"2025","journal-title":"Acta Microbiol. Immunol. Hung."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Gondal, A.J., Choudhry, N., Niaz, A., and Yasmin, N. (2024). Molecular Analysis of Carbapenem and Aminoglycoside Resistance Genes in Carbapenem-Resistant Pseudomonas Aeruginosa Clinical Strains: A Challenge for Tertiary Care Hospitals. Antibiotics, 13.","DOI":"10.3390\/antibiotics13020191"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Thabit, A.K., Alghamdi, A.M., Miaji, M.Y., Alharbi, F.S., Jawah, A.F., Alturki, F., Hosin, N., Bazuqamah, M., Almutairi, M.S., and Alhamed, H. (2024). Antibiotic Susceptibility of Pseudomonas Aeruginosa in Saudi Arabia: A National Antimicrobial Resistance Surveillance Study. Front. Public Health, 12.","DOI":"10.3389\/fpubh.2024.1436648"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"5101","DOI":"10.1038\/s41467-026-71782-5","article-title":"Carbapenem-Resistance OprD Mutations Reshape Pseudomonas Aeruginosa Host-Pathogen Interactions during Infection","volume":"17","author":"Laborda","year":"2026","journal-title":"Nat. Commun."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4247","DOI":"10.1038\/sj.emboj.7600886","article-title":"Structure of the Entire Cytoplasmic Portion of a Sensor Histidine-Kinase Protein","volume":"24","author":"Marina","year":"2005","journal-title":"EMBO J."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1021\/cb5007135","article-title":"Bacterial Histidine Kinases as Novel Antibacterial Drug Targets","volume":"10","author":"Bem","year":"2014","journal-title":"ACS Chem. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"26085","DOI":"10.1038\/srep26085","article-title":"Putative Histidine Kinase Inhibitors with Antibacterial Effect against Multi-Drug Resistant Clinical Isolates Identified by in Vitro and in Silico Screens","volume":"6","author":"Velikova","year":"2016","journal-title":"Sci. Rep."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/7\/962\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T14:19:50Z","timestamp":1783693190000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/7\/962"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,29]]},"references-count":40,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["biom16070962"],"URL":"https:\/\/doi.org\/10.3390\/biom16070962","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,29]]}}}