AAC Accepted Manuscript Posted Online 24 October 2016 Antimicrob. Agents Chemother. doi:10.1128/aac.01945-16 Copyright 2016, American Society for Microbiology. All Rights Reserved. 1 2 Title: Description of the First Escherichia coli Clinical Isolate Harboring Colistin- Resistance mcr-1 Gene from the Indian Subcontinent 3 4 5 Mashkoor Mohsin 1#, Shahbaz Raza 1, Nicole Roschanski 2, Sebastian Guenther 2, Aamir Ali 3,4, Peter Schierack 3 6 7 1 Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan 8 9 2 Institute of Animal Hygiene and Environmental Health, Freie Universitaet Berlin, Berlin, Germany 10 11 3 Faculty of Environment and Natural Sciences, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany 12 4 National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan 13 14 Running title: Colistin resistance from human in the Indian Subcontinent 15 16 Keywords: Polymyxins, mcr-1, ESBL, CTX-M-15, IncI2, Indian Subcontinent 17 18 19 20 # Corresponding Author: Mashkoor Mohsin, Institute of Microbiology, University of Agriculture, Faisalabad 38000, Pakistan. Tel: +92-419200161 21 E-mail: mashkoormohsin@uaf.edu.pk 1
22 23 24 25 26 The first report of plasmid-mediated colistin resistance among Enterobacteriaceae in China sounded alarms in the medical community worldwide (1). Since, E. coli isolates harboring colistin resistance encoded by the mcr-1 gene have been globally reported (2). To our knowledge, we hereby report the first detection of mcr-1 gene in a human E. coli isolate from the Indian subcontinent. 27 28 29 30 31 32 33 34 35 36 37 In February 2016, a total of 29 extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates recovered from patients related to wound infections admitted to Burn & Reconstructive Surgery Centre, Allied Hospital Faisalabad, Pakistan were tested for plasmidmediated colistin resistance. Phenotypic resistance to polymyxin B was determined by vitek-2 compact system (biomérieux, Germany) followed by determination of the colistin MIC by broth microdilution in 96-well plate test panel (Micronaut S, Merlin, Bornheim-Hersel, Germany). One colistin resistant E. coli isolate (X26) with MIC of 4 μg/ml was found from a 35-year old male patient suffering from burn-associated wounds. Presence of the mcr-1 gene was confirmed by TaqMan-based real-time PCR (3). The isolate exhibited multidrug resistance, but was susceptible to carbapenems (Table 1). The presence of the bla CTX-M-15 gene in E. coli X26 was confirmed by PCR and sequencing (4). 38 39 40 41 42 43 44 45 46 The plasmid-carriage of E. coli isolate X26 was analyzed using a conventional plasmid profile analysis (5). The conjugative transfer ability of the colistin resistance was investigated by plasmid conjugation experiments using sodium azide resistant E. coli K12- J53 as a recipient strain. Transconjugants were selected on LB agar plates supplemented with colistin (4 μg/ml) and sodium azide (100 μg/ml) and subsequently confirmed by PCR for the mcr-1 gene. PCRbased plasmid replicon typing of transconjugants showed IncI2 type. PFGE was performed to determine genetic relatedness of the E. coli X26 with the recently reported mcr-1 carrying ESBL-E. coli isolate PK-13 of ST354 recovered from wild migratory bird in Pakistan (6). Multi-locus sequence typing (MLST) was carried out using the method developed by Wirth et 2
47 al. (7). MLST results revealed E. coli X26 as a new ST. 48 49 50 51 52 53 54 55 56 57 58 59 60 Colistin has become the last line of defense for the treatment of infections caused by Gramnegative bacteria, in particular carbapenem-resistant Enterobacteriaceae (CRE). The presence of plasmid-mediated colistin resistance in Indian subcontinent where there is already burden of CRE, is worrisome as it threatened the use of last resort antibiotics. Recently, we have reported the mcr-1 gene in E. coli isolated from a wild migratory bird in Pakistan (6). The plasmid size (~ 63 kb) as well as the replicon type of E. coli X26 correspond to that described by Liu and colleagues (1) and our recent findings (6). Thus, indicating that IncI2 represent a major plasmid type involved in the spread of colistin resistance in our region. The PFGE pattern and ST type showed no genetic relatedness between E. coli X26 and our recently reported strain (6), suggesting that different clonal types are involved in the dissemination of mcr-1 gene. In conclusion, the presence of the plasmid-mediated colistin resistance in a clinical isolate, adds to the worry as this potent gene may spread to other susceptible bacteria, making pan-resistant pathogens. 61 62 63 Acknowledgements: Mashkoor Mohsin was supported by postdoctoral fellowship from the Alexander von Humboldt Foundation, Germany. 64 Funding Infromation 65 This study received no specific funding. 66 67 68 3
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114 Table 1: Antibiotic resistance profile of E. coli X26 115 Antimicrobial Agents MIC (μg/ml) 1 Amikacin 4 Aztreonam >64 Ciprofloxacin >4 Colistin 4 Cefepime >64 Ceftazidime >64 Cefotaxime >64 Fosfomycin 16 Gentamicin >16 Imipenem 0.25 Meropenem 0.25 Marbofloxacin >4 Piperacillin/Tazobactam >128 Tetracyclin 4 Tigecycline 5 6
Tobramycin >16 Trimethoprim/sulfamethoxazole >320 116 117 118 119 1 MIC s were determined using the Vitek-2 system (biomérieux) with except for colistin, which was performed using broth microdilution in 96-well plate test panel (Micronaut S, Merlin, Bornheim-Hersel, Germany). 120 7