Colistin resistant Escherichia coli carrying mcr-1 in urban sludge samples: Dhaka, Bangladesh
© The Author(s) 2017
Received: 23 November 2017
Accepted: 16 December 2017
Published: 20 December 2017
Of 48 bacteria belonging to the family Enterobacteriaceae tested from urban sludge samples, one Escherichia coli isolate was resistant to colistin and possessed the resistance marker gene mcr-1 found for the first time from Bangladesh. The colistin resistant E. coli was multidrug resistant showing resistance to 11 different antibiotics tested.
Antimicrobial resistance is a multi-sectoral problem which is now recognized as one of the most serious threats to human health globally. Resistance trends among Enterobacteriaceae are especially worrisome considering their ubiquity in the environment and animal systems, and their enhanced propensity to acquire antibiotic resistance determinants through mobile genetic elements. Indiscriminate use of antibiotics is mainly responsible for the emergence of Enterobacteriaceae resistant to multiple antibiotics including carbapenems.
Colistin, a cationic polypeptide, is considered as one of the last-resort drugs of choice for the treatment of multi-drug resistant, Gram negative bacteria such as carbapenem resistant Enterobacteriaceae (CRE). Present day increase in the incidences of multi-drug resistant bacteria has resulted in enhanced use of colistin globally, with an inevitable risk of emerging resistance. A study in Vietnam has shown increasing resistance in commensal Escherichia coli associated with the extensive use of colistin in livestock and poultry industry . A recent study has shown colistin sulfate to be the most commonly used antibiotic in poultry industry in Bangladesh . Acquired resistance to colistin is generally associated with chromosomal mutations , although a new plasmid-mediated transferable resistance determinant, the mcr-1 gene, encoding a phosphoethanolamine transferase, has been described recently in China . Since the plasmid encoding mcr-1 has been established as a marker for colistin resistance, Enterobacteriaceae carrying this gene has been reported from many parts of the world . Considering the widespread occurrence of colistin resistance and the impending danger associated with it, we screened bacteria belonging to the family Enterobacteriaceae isolated from sludge samples of Dhaka city for their resistance to colistin and for the presence of colistin resistance-related gene mcr-1. Here we report the occurrence of colistin resistant E. coli carrying mcr-1 gene in urban environment of Dhaka, Bangladesh.
After preliminary identification following standard culture methods and final biochemical confirmation with API 20 E (bioMérieux, France), 48 bacterial isolates including Escherichia coli (n = 23), Klebsiella pneumoniae (n = 15), Pseudomonas luteola (n = 6), Pseudomonas aeruginosa (n = 1), Pantoea spp. (n = 2) and Citrobacter freundii (n = 1), collected from sludge samples of Dhaka city, were tested for their response to colistin by measuring the minimum inhibitory concentration (MIC) by E-test (Biomeuriex). The results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints . Although broth microdilution assay is recommended by EUCAST for determining MIC, several studies have found an unequivocal correlation between the Etest and reference techniques [7–9].
Bacterial DNA was obtained from all isolates by the standard boiling method. Detection of mcr-1 gene was performed by polymerase chain reaction (PCR) using the primer as described elsewhere . Amplified fragment of mcr-1 was sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems) on an ABI PRISM 310 automated sequencer (Applied Biosystems). After analyzing the raw sequence with sequence analysis software (Chromas), a deduced 252 bp sequence was then searched for homology by Basic Local Alignment Search Tool (BLAST). The partial sequence of the gene was submitted to GenBank (Accession Number MF173059).
Results and discussion
Among all the tested bacteria, one of the E. coli isolates was found resistant to colistin with a determined MIC value of 4 μg/mL. This colistin resistant E. coli isolate called E3B was then tested for its response to 15 different antibiotics by Kirby–Bauer disk diffusion method , following the Clinical Laboratory Standards Institute recommendations. Our results confirmed the colistin resistant E. coli to be multidrug resistant (MDR) as the isolate E3B was resistant to 10 different antibiotics: Nalidixic acid, Ciprofloxacin, Levofloxacin, Azithromycin, Gentamicin, Erythromycin, Sulfamethoxazole/Trimethoprim, Ampicillin, Cephalothin and Tetracycline, but sensitive to Mecillinam, Fosfomycin, Ceftriaxone, Cefixime and Meropenem. This appears to be an alarming trend for a populous country like Bangladesh where infection control is becoming increasingly challenging due to the rise of multidrug resistance among members of the family Enterobacteriaceae, including E. coli [11, 12].
All of the 48 bacterial isolates, including the colistin resistant E. coli were screened by PCR for the presence of colistin resistance gene mcr-1 using specific primers. However, only the colistin resistant E. coli isolate E3B supported the amplification of a 309 bp fragment of mcr-1. The PCR amplicon of the mcr-1 gene was then sequenced. Although it was not possible for us to cover the full length sequence of the gene, BLAST homology searching demonstrated the deduced sequenced data of the 252 bp DNA fragment (Accession No. MF173059) to be identical to that of the mcr-1 reported from E. coli strain SHP45 (GenBank Accession No. KP347127) isolated from a Chinese pig farm . To our knowledge, colistin resistant E. coli carrying mcr-1 has not been isolated so far from hospital settings, and this is the first report of the occurrence of colistin resistant E. coli carrying resistant marker mcr-1 from environment of Bangladesh.
Finally, the data presented in this study show environmental dissemination of MDR E. coli carrying colistin resistance and related marker gene mcr-1 via urban sludge disposed into the water bodies. Dhaka is a densely populated city with circular river systems, and millions living in urban slums do not have access to safe drinking water. Given this, fecal–oral transmission might allow MDR enteric pathogens to transmit rapidly. Further, the high selection pressure of residual antibiotics in the urban environment, and since the colistin resistance marker mcr-1 can be transferred horizontally; there is an urgent requirement for broader surveillance in both clinical and environmental settings in Bangladesh in order to prevent further spreading of this resistance gene.
AI and MA participated in the conception and design of the study. AI, ZR and MAR performed the laboratory work. AI and MA analyzed the data and wrote the manuscript. SM, AC, NA and CMG contributed to the analysis and helped in writing the manuscript. All authors read and approved the final manuscript.
icddr,b acknowledges the following donors: the governments of Bangladesh, Canada, Sweden and United Kingdom for providing core/unrestricted support.
The authors declare that they have no competing interests.
Availability of data and materials
Data sharing not applicable to this article.
Consent for publication
Ethics approval and consent to participate
This research study was supported jointly by icddr,b, NIH Grant 1R01AI39129-01 and the National Institutes of Infectious Diseases (NIID), Japan.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Nguyen NT, Nguyen HM, Nguyen CV, Nguyen TV, Nguyen MT, Thai HQ, et al. The use of colistin and other critical antimicrobials on pig and chicken farms in southern Vietnam and their association with resistance in commensal Escherichia coli Bacteria. Appl Environ Microbiol. 2016;82(13):3727–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Islam KS, Shiraj-Um-Mahmuda S, Hazzaz-Bin-Kabir M. Antibiotic usage patterns in selected broiler farms of Bangladesh and their Public Health Implications. J Public Health Dev Ctries. 2016;2(3):276–84.Google Scholar
- Olaitan AO, Morand S, Rolain JM. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol. 2014;5:643. https://doi.org/10.3389/fmicb.2014.00643.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16:161–8.View ArticlePubMedGoogle Scholar
- Skov RL, Monnet DL. Plasmid mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Eurosurveillance. 2016;21(9):30155.View ArticlePubMedGoogle Scholar
- European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters: version 6.0. 2016. http://www.eucast.org/fleadmin/src/media/PDFs/EUCAST_fles/Breakpoint_tables/v_6.0_Breakpoint_table.pdf. Accessed 15 Mar 2016.
- Behera B, Mathur P, Das A, Kapil A, Gupta B, Bhoi S, Farooque K, Sharma V, Misra MC. Evaluation of susceptibility testing methods for polymyxin. Int J Infect Dis. 2010;14:e596–601. https://doi.org/10.1016/j.ijid.2009.09.001.View ArticlePubMedGoogle Scholar
- Lo-Ten-Foe JR, de Smet AM, Diederen BM, Kluytmans JA, van Keulen PH. Comparative evaluation of the VITEK 2, disk diffusion, Etest, broth microdilution, and agar dilution susceptibility testing methods for colistin in clinical isolates, including heteroresistant Enterobacter cloacae and Acinetobacter baumannii strains. Antimicrob Agents Chemother. 2007;51:3726–30. https://doi.org/10.1128/AAC.01406-06.View ArticlePubMedPubMed CentralGoogle Scholar
- Maalej SM, Meziou MR, Rhimi FM, Hammami A. Comparison of disc diffusion, Etest and agar dilution for susceptibility testing of colistin against Enterobacteriaceae. Lett Appl Microbiol. 2011;53:546–51. https://doi.org/10.1111/j.1472-765X.2011.03145.x.View ArticlePubMedGoogle Scholar
- Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method Bauer. Am J Clin Pathol. 1966;45:493–6.PubMedGoogle Scholar
- Monira S, Shabnam SA, Ali SI, Sadique A, Johura FT, Rahman KZ, et al. Multi-drug resistant pathogenic bacteria in the gut of young children in Bangladesh. Gut Pathog. 2017;9:19.View ArticlePubMedPubMed CentralGoogle Scholar
- Rahman Z, Islam A, Rashid MU, Johura FT, Monira S, Watanabe H, et al. Existence of a novel qepA variant in quinolone resistant Escherichia coli from aquatic habitats of Bangladesh. Gut Pathog. 2017;9:58. https://doi.org/10.1186/s13099-017-0207-8.View ArticlePubMedPubMed CentralGoogle Scholar