{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,9]],"date-time":"2026-06-09T15:29:54Z","timestamp":1781018994314,"version":"3.54.1"},"reference-count":44,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T00:00:00Z","timestamp":1766966400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009660","name":"UK Research and Innovation grants","doi-asserted-by":"publisher","award":["BBSRC BB\/X018156\/1"],"award-info":[{"award-number":["BBSRC BB\/X018156\/1"]}],"id":[{"id":"10.13039\/100009660","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009660","name":"UK Research and Innovation grants","doi-asserted-by":"publisher","award":["MRC MR\/X005895\/1"],"award-info":[{"award-number":["MRC MR\/X005895\/1"]}],"id":[{"id":"10.13039\/100009660","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009660","name":"UK Research and Innovation grants","doi-asserted-by":"publisher","award":["EPSRC EP\/Y018842\/1"],"award-info":[{"award-number":["EPSRC EP\/Y018842\/1"]}],"id":[{"id":"10.13039\/100009660","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Background: Cholera, caused by Vibrio cholerae, remains endemic in many developing countries, including Pakistan. The extensive use of antibiotics has led to the emergence of antimicrobial resistance in V. cholerae, limiting available treatment options. In this study, we performed molecular characterisation of antibiotic-resistant V. cholerae serotype Ogawa isolates from a recent cholera outbreak in Khyber Pakhtunkhwa, Pakistan. Methodology: Suspected cholera stool samples were collected from hospitalised patients at various district hospitals of Khyber Pakhtunkhwa Province (KPK), Pakistan. The samples were transported to the Public Health Reference Microbiology Laboratory at Khyber Medical University, Peshawar. V. cholerae were identified based on colonial morphology, Gram staining, and biochemical tests using EPI 10E. For serotype identification, monovalent antisera were used. Antibiotic susceptibility testing (AST) was performed using CLSI M45 and EUCAST guidelines. DNA was extracted from pure colonies of multidrug-resistant (MDR) V. cholerae and subjected to whole-genome sequencing (WGS) for genomic characterisation using an Illumina MiSeq platform. Results: Of the 350 active diarrheal cases investigated, 70 were confirmed as V. cholerae. The outbreak was initially reported in Dir and was subsequently followed by a high incidence of cholera in the Peshawar district of KPK. All strains belong to the Ogawa serotype, which shows high antibiotic resistance, particularly to ampicillin (n = 62, 88.57%), Sulfamethoxazole\/Trimethoprim (n = 60, 85.71%), Erythromycin (n = 59, 84.29%), and Tetracycline (n = 53, 75.71%). The lowest resistance was against Meropenem (n = 1, 1.4%), followed by amikacin (n = 7, 10.0%) and levofloxacin (n = 13, 18.57%). Furthermore, 34 (48.57%) of the isolates were MDR, while 13 (18.57%) were extensively drug-resistant. Six samples were selected for whole-genome sequencing. The selection of six V. cholerae samples for WGS was based on their drug resistance pattern and origin of isolation. At the genomic level, all sequenced V. cholerae strains harboured multiple antimicrobial resistance determinants. Quinolone resistance was associated with mutations and genes in gyrA, gyrB, parC, and parE; resistance to sulfamethoxazole\u2013trimethoprim with folA, folP, and dfr; tetracycline resistance with tetA and tet35; chloramphenicol resistance with catB and S10p; and aminoglycoside resistance with hns, S12p, and gigB. In addition, \u03b2-lactam resistance was linked to the presence of efflux and \u03b2-lactamase genes, including blaSHV and mox-3. Mutations were identified in gyrA at positions S83I, S177A, and S202A, and in parC at positions S85L and I231V. Collectively, the presence of these resistance determinants likely enables V. cholerae to survive exposure to high concentrations of multiple antibiotics. Conclusions: Our V. cholerae isolates showed close genetic relatedness to previously sequenced strains from Pakistan (2010 and 2022), as well as to recently reported international strains from the USA, Australia, and China. These findings highlight both the long-term persistence of these lineages within Pakistan and their international dissemination, likely facilitated by globalisation.<\/jats:p>","DOI":"10.3390\/pathogens15010039","type":"journal-article","created":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T15:30:32Z","timestamp":1767195032000},"page":"39","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Whole Genome Sequencing of Drug-Resistant Vibrio cholerae Serotype Ogawa from an Outbreak in Khyber Pakhtunkhwa"],"prefix":"10.3390","volume":"15","author":[{"given":"Aftab","family":"Ali","sequence":"first","affiliation":[{"name":"Department of Microbiology, Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar 25100, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Momin","family":"Khan","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar 25100, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taj Ali","family":"Khan","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar 25100, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sajjad","family":"Ahmad","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar 25100, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noor","family":"Rahman","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar 25100, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-6469-1377","authenticated-orcid":false,"given":"Aiman","family":"Waheed","sequence":"additional","affiliation":[{"name":"Center of Biotechnology and Microbiology, University of Peshawar, Peshawar 25120, 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 & Tropical, London WC1E 7HT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e00211","DOI":"10.1128\/cmr.00211-21","article-title":"Diagnosis, management, and future control of cholera","volume":"35","author":"Chowdhury","year":"2022","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"209","DOI":"10.2471\/BLT.11.093427","article-title":"The global burden of cholera","volume":"90","author":"Ali","year":"2012","journal-title":"Bull. World Health Organ."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Ali, M., Nelson, A.R., Lopez, A.L., and Sack, D.A. (2015). Updated global burden of cholera in endemic countries. PLoS Negl. Trop. Dis., 9.","DOI":"10.1371\/journal.pntd.0003832"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1186\/s13063-022-06601-5","article-title":"A double-blind clinical trial to compare the efficacy and safety of a multiple amino acid-based ORS with the standard WHO-ORS in the management of non-cholera acute watery diarrhea in infants and young children:\u201cVS002A\u201d trial protocol","volume":"23","author":"Das","year":"2022","journal-title":"Trials"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2466","DOI":"10.1016\/S0140-6736(12)60436-X","article-title":"Cholera","volume":"379","author":"Harris","year":"2012","journal-title":"Lancet"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e480","DOI":"10.1016\/S2666-5247(22)00131-8","article-title":"Malawi takes on cholera outbreak amid cyclone devastation","volume":"3","author":"Bagcchi","year":"2022","journal-title":"Lancet Microbe"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1038\/nrmicro2204","article-title":"Cholera transmission: The host, pathogen and bacteriophage dynamic","volume":"7","author":"Nelson","year":"2009","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1423","DOI":"10.1099\/jmm.0.001423","article-title":"JMM Profile: Vibrio cholerae: An opportunist of human crises","volume":"70","author":"Grant","year":"2021","journal-title":"J. Med. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Ramamurthy, T., Mutreja, A., Weill, F.-X., Das, B., Ghosh, A., and Nair, G.B. (2019). Revisiting the global epidemiology of cholera in conjunction with the genomics of Vibrio cholerae. Front. Public Health, 7.","DOI":"10.3389\/fpubh.2019.00203"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1111\/j.1574-6976.2002.tb00605.x","article-title":"Vibrio cholerae and cholera: Out of the water and into the host","volume":"26","author":"Reidl","year":"2002","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"703","DOI":"10.1016\/0140-6736(93)90480-5","article-title":"Emergence of novel strain of Vibrio cholerae with epidemic potential in southern and eastern India","volume":"341","author":"Ramamurthy","year":"1993","journal-title":"Lancet"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1038\/nature10392","article-title":"Evidence for several waves of global transmission in the seventh cholera pandemic","volume":"477","author":"Mutreja","year":"2011","journal-title":"Nature"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1086\/341206","article-title":"Epidemic and endemic cholera trends over a 33-year period in Bangladesh","volume":"186","author":"Longini","year":"2002","journal-title":"J. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1422","DOI":"10.1016\/0140-6736(93)92780-W","article-title":"Vibrio cholerae 0139 in Karachi, Pakistan","volume":"342","author":"Khan","year":"1993","journal-title":"Lancet"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"13","DOI":"10.3201\/eid2001.130428","article-title":"Genomic epidemiology of Vibrio cholerae O1 associated with floods, Pakistan, 2010","volume":"20","author":"Shah","year":"2014","journal-title":"Emerg. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e0088722","DOI":"10.1128\/mra.00887-22","article-title":"Genomes of Vibrio cholerae O1 Serotype Ogawa Associated with Current Cholera Activity in Pakistan","volume":"12","author":"Sim","year":"2023","journal-title":"Microbiol. Resour. Announc."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6291","DOI":"10.1038\/s41467-024-50484-w","article-title":"Genomic insights into the 2022\u20132023 Vibrio cholerae outbreak in Malawi","volume":"15","author":"Chaguza","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"10-1128","DOI":"10.1128\/mBio.03339-19","article-title":"Genome Dynamics of Vibrio cholerae Isolates Linked to Seasonal Outbreaks of Cholera in Dhaka, Bangladesh","volume":"11","author":"Baddam","year":"2020","journal-title":"mBio"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1787","DOI":"10.1038\/s41564-023-01472-1","article-title":"Genomic epidemiology reveals multidrug-resistant plasmid spread between Vibrio cholerae lineages in Yemen","volume":"8","author":"Lassalle","year":"2023","journal-title":"Nat. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Bilal, H., Khan, M.N., Rehman, T., Hameed, M.F., and Yang, X. (2021). Antibiotic resistance in Pakistan: A systematic review of past decade. BMC Infect. Dis., 21.","DOI":"10.1186\/s12879-021-05906-1"},{"key":"ref_21","first-page":"3","article-title":"Socio-Economic Implications of Terrorism on Khyber Pakhtunkhwa: A Case Study of ANP Era (2008\u20132013)","volume":"19","author":"Din","year":"2022","journal-title":"Webology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"181","DOI":"10.31033\/ijrasb.7.6.26","article-title":"Laboratory protocols for isolation and identification of toxigenic strains of Vibrio cholerae: A review","volume":"7","author":"Shrestha","year":"2020","journal-title":"Int. J. Res. Appl. Sci. Biotechnol. (IJRASB)"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Cheesbrough, M. (2005). District Laboratory Practice in Tropical Countries, Part 2, Cambridge University Press.","DOI":"10.1017\/CBO9780511581304"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e14896","DOI":"10.7717\/peerj.14896","article-title":"Emergence of colistin resistance and characterization of antimicrobial resistance and virulence factors of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from hybrid red tilapia cage culture","volume":"11","author":"Thaotumpitak","year":"2023","journal-title":"PeerJ"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"115","DOI":"10.3354\/dao03766","article-title":"Epidemiological cut-off values for non-O1\/non-O139 Vibrio cholerae disc diffusion data generated by standardised methods","volume":"156","author":"Smith","year":"2023","journal-title":"Dis. Aquat. Org."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1093\/jac\/dkad391","article-title":"Vibrio species: Development of EUCAST susceptibility testing methods and MIC and zone diameter distributions on which to determine clinical breakpoints","volume":"79","author":"Karatuna","year":"2024","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_27","unstructured":"Hindler, J., Richter, S., Bernard, K., Jones, S., Castanheira, M., Citron, D., Couturier, M., Fritsche, T., Humphries, R., and Jorgensen, J. (2016). CLSI M45-Methods for an-Timicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria, Clinical and Laboratory Standards Institute."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4065603","DOI":"10.1155\/2016\/4065603","article-title":"Multidrug resistant and extensively drug resistant bacteria: A study","volume":"2016","author":"Basak","year":"2016","journal-title":"J. Pathog."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Nayak, D.S.K., Das, J., and Swarnkar, T. (2022). Quality control pipeline for next generation sequencing data analysis. Intelligent and Cloud Computing: Proceedings of ICICC 2021, Springer.","DOI":"10.1007\/978-981-16-9873-6_20"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing","volume":"19","author":"Bankevich","year":"2012","journal-title":"J. Comput. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1038\/s41592-023-01940-w","article-title":"CheckM2: A rapid, scalable and accurate tool for assessing microbial genome quality using machine learning","volume":"20","author":"Chklovski","year":"2023","journal-title":"Nat. Methods"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2068","DOI":"10.1093\/bioinformatics\/btu153","article-title":"Prokka: Rapid prokaryotic genome annotation","volume":"30","author":"Seemann","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"D535","DOI":"10.1093\/nar\/gkw1017","article-title":"Improvements to PATRIC, the all-bacterial bioinformatics database and analysis resource center","volume":"45","author":"Wattam","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"W293","DOI":"10.1093\/nar\/gkab301","article-title":"Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation","volume":"49","author":"Letunic","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1056\/NEJMc2004773","article-title":"Highly resistant cholera outbreak strain in Zimbabwe","volume":"383","author":"Mashe","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Bitew, A., Gelaw, A., Wondimeneh, Y., Ayenew, Z., Getie, M., Tafere, W., Gebre-Eyesus, T., Yimer, M., Beyene, G.T., and Bitew, M. (2024). Prevalence and antimicrobial susceptibility pattern of Vibrio cholerae isolates from cholera outbreak sites in Ethiopia. BMC Public Health, 24.","DOI":"10.1186\/s12889-024-19621-4"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e1","DOI":"10.4102\/phcfm.v12i1.2264","article-title":"Geographical distribution and antibiotics susceptibility patterns of toxigenic Vibrio cholerae isolates from Kisumu County, Kenya","volume":"12","author":"Awuor","year":"2020","journal-title":"Afr. J. Prim. Health Care Fam. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"A93","DOI":"10.1016\/j.vaccine.2019.12.003","article-title":"Mapping cholera outbreaks and antibiotic resistant Vibrio cholerae in India: An assessment of existing data and a scoping review of the literature","volume":"38","author":"Chatterjee","year":"2020","journal-title":"Vaccine"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1016\/j.ijid.2021.02.102","article-title":"Clinical and socio-environmental determinants of multidrug-resistant Vibrio cholerae 01 in older children and adults in Bangladesh","volume":"105","author":"Garbern","year":"2021","journal-title":"Int. J. Infect. Dis."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"De, R. (2021). Mobile genetic elements of Vibrio cholerae and the evolution of its antimicrobial resistance. Front. Trop. Dis., 2.","DOI":"10.3389\/fitd.2021.691604"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Fuesslin, V., Krautwurst, S., Srivastava, A., Winter, D., Liedigk, B., Thye, T., Herrera-Le\u00f3n, S., Wohl, S., May, J., and Fobil, J.N. (2022). Prediction of Antibiotic Susceptibility Profiles of Vibrio cholerae Isolates From Whole Genome Illumina and Nanopore Sequencing Data: CholerAegon. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.909692"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Zhou, Y.-Y., Ma, L.-Y., Yu, L., Lu, X., Liang, W.-L., Kan, B., and Su, J.-R. (2023). Quinolone resistance genes and their contribution to resistance in Vibrio cholerae serogroup O139. Antibiotics, 12.","DOI":"10.3390\/antibiotics12020416"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1503","DOI":"10.1038\/s41598-024-81596-4","article-title":"Genomic insights into the dynamic antibiotic resistance landscape of Vibrio cholerae during the Cholera outbreak 2022 in Odisha, India","volume":"15","author":"Samal","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2428","DOI":"10.1093\/jac\/dkaf232","article-title":"Highly drug-resistant Vibrio cholerae harbouring bla PER-7 isolated from travellers returning to England","volume":"80","author":"Nair","year":"2025","journal-title":"J. Antimicrob. Chemother."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/1\/39\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T15:51:54Z","timestamp":1767196314000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/1\/39"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,29]]},"references-count":44,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["pathogens15010039"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15010039","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,29]]}}}