Emergence of mcr-1 mediated colistin resistant Escherichia coli from a hospitalized patient in Bangladesh

Introduction: The emergence of plasmid mediated mcr in bacteria has become global public health threat. Herein, we report a mcr-1 positive E. coli in normal human flora from a patient admitted in Dhaka Medical College Hospital (DMCH). Methodology: In total, 700 non-duplicate rectal swabs were collected from DMCH during 13 th May to 12 th June 2018. E. coli from rectal swabs were isolated on chromogenic UTI media containing vancomycin 10mg/l (Liofilchem, Italy) and confirmed by MALDI-TOF. Minimum inhibitory concentrations (MIC) were determined by agar dilution and interpreted according to EUCAST breakpoints. Genomic analysis of mcr positive E. coli (MCRPEC) was performed by Illumina MiSeq sequencing and pulsed field gel electrophoresis (PFGE) using S1 nuclease DNA digests and bla mcr-1 probing. Transferability of bla mcr-1 were determined by conjugation assays. Results: We found one MCRPEC from 700 rectal swab screening which was isolated from the rectal swab culture of a 17-year boy who was admitted to the burns ICU, DMCH with 53% flame burn involving much of the trunk and face. Genome sequencing revealed that mcr-1 was present on an IncH12 plasmid of 257,243 bp and flanked by IS ApaI1 . The colistin resistance can be transferred to the recipient Klebsiella varricola with a frequency of 8.3 × 10 -5 . Transconjugants were more resistant to colistin than donor (MIC 32 µg/mL). Conclusions: This is the first human associated mcr in Bangladesh. These data indicate the need for a systematic “one health” surveillance in the country.


The Study
The emergence of the plasmid mediated colistin resistance gene, mcr in bacteria of environmental, animal, food and human origin has become global public health threat and radically limits therapeutic options for multidrug resistant (MDR) Gram-negative bacterial infections [1,2].There are few studies on the prevalence of mcr in Southeast Asia.Recently, mcr has been detected in the food-chain and environment from Pakistan, India and Bangladesh [3][4][5].Here, we report the presence of mcr-1 in an E. coli strain retrieved from human faecal flora in a Bangladeshi hospital and provide evidence of its emergence into the clinical sector.
In total, 700 non-duplicate rectal swabs were collected from both inpatients and the outpatient department (OPD) of Dhaka Medical College Hospital (DMCH) from the 13 th May to 12 th June 2018.Informed consent was taken from each participant and the project was ethically approved by Ethical Review Committee of DMCH (MEU-DMC/ECC/2017122).E. coli from rectal swabs were isolated on chromogenic UTI media containing vancomycin 10mg/Liter (Liofilchem, Roseto degli Abruzzi, Italy) and confirmed by MALDI-TOF.Minimum inhibitory concentrations (MIC) were determined by agar dilution and interpreted according to EUCAST breakpoints [6].E. coli resistant to colistin were examined by PCR for mcr variants.Genomic analysis of mcr positive E. coli (MCRPEC) was performed by Illumina MiSeq (Illumina Inc., San Diego, CA) (please see Supplementary methods) and minION (Oxford Nanopore technologies, Oxford, UK) sequencing (please see Supplementary methods) and pulsed field gel electrophoresis (PFGE) using S1 nuclease DNA digests and bla mcr-1 probing.Conjugation assays used a Klebsiella varricola strain (accession number: PJQN00000000) as the recipient.Transconjugants were selected on UTI agar (colistin 4mg/Liter) and investigated for mcr-1 by PCR (please see Supplementary methods).
The MCRPEC (RS571; accession no: CP034389-CP034392) was isolated from the rectal swab culture of a 17-year boy who was admitted to the burn's ICU, DMCH with 53% flame burn involving much of the trunk and face.He suffered a smoke inhalation injury and became severely hypoxic.The patient worked as a laborer in a garment factory in Munshiganj district and the injury was sustained due to a gas pipe leak.Rectal swabs were collected 2 days after admission so RS571 could have been acquired in the community or the hospital.Clindamycin and ceftriaxone treatment were commenced early in the hospital stay.The patient died on day 5 of the admission, with the cause of death given as septicemia.No clinical specimens were referred for culture during his hospital stay so it is not known whether RS571 was colonising flora only or played a pathogenic role, although it was resistant to all of the antibiotics received during the admission.

Conclusion
This is the first comprehensive report of mcr in humans from South-Asia, and detected on a MDR IncH12 plasmid that is widespread throughout China [8,9].However, mcr positive strains were detected from the food-chain in India and Pakistan those were related to IncH12 and IncI2 plasmids, respectively [3,4].These data indicate the need for a "one health" surveillance system to prevent the spread of mobile colistin resistance.
1 MIC values for the antibiotics are in µg/mL.