Discovery of a mcr-1-bearing plasmid in commensal colistin-resistant Escherichia coli from healthy broilers in Faisalabad, Pakistan

Jiali Lv, Mashkoor Mohsin, Sheng Lei, Swaminath Srinivas, Raja Talish Wiqar, Jingxia Lin, and Youjun Feng School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an, Shaanxi, China; Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan; Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China; Department of Biochemistry, University of Illinois, Urbana, USA; College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China

Letter to the editor Polymyxins, like colistin (polymyxin E), are a group of cationic antimicrobial cyclic polypeptide, which have been extensively-used as prophylactic feed additives in animal production since the 1960s [1]. A strong association has especially been drawn between antimicrobial use and resistance in poultry and pig farms [2][3][4][5]. The advent and rise of multi-drug resistant bacteria has now prompted the re-introduction of colistin as a last-resort treatment option in human medicine [6,7]. However, the recent emergence and diversity of plasmid-borne mobile colistin resistance determinants (mcr-1 to mcr-5) in Enterobacteriaceae has severely challenged its use in a clinical setting [8][9][10][11][12]. In the past two years, mcr-1 has been detected in over 40 countries across 5 of 7 continents worldwide [13][14][15]. Except rare cases of chromosomally-integrated mcr-1 [16,17], its prevalent transmission relies on the transfer by diversified plasmids of different replication incompatibilities [18,19].
Mechanistically, the mcr-1 gene that encodes phosphoethanolamine transferase mediates the modification of the lipopolysaccharide layer (LPS) of the outer membrane of Gram-negative bacteria through the addition of phosphoethanolamine (PEA) to the 1 (or 4 0 )-phosphate position of lipid A moieties [20,21]. This reduces the affinity of polymyxin antibiotics to their primary target, the LPS layer. Since its discovery during routine surveillance in China [8], increasingly-accumulated evidence suggested the presence of mcr-1-bearing bacteria in food producing animals and humans across the world. To the best of our knowledge, the leading two types of mcr-1-harboring plasmids referred to IncI2 [8,18,22,23] and IncX4 [24][25][26], have greatly facilitated global dissemination of mcr-1 colistin resistance [14,27]. In Pakistan, the mcr-1 gene has been identified in Escherichia coli (E. coli) isolates from human [28], wildlife [29] and a broiler suffering from colibacillosis [30]. However, little is known about the prevalence of mcr-1 and its genetic environment in commensal E. coli isolates from poultry in Pakistan.
From December 2016 to January 2017, cloacal swabs from a total of 100 healthy broiler chicken were obtained from four commercial farms (n = 25 each) in the Faisalabad region of Pakistan. To screen the colistin resistant E. coli, all the samples were seeded directly onto MacConkey agar supplemented with 4 mg/ml of colistin and were incubated at 37 C for 24 hours. Of 100 birds, colistin resistant E. coli were found in only 8 (8%) samples. A single colony of E. coli was selected per sample and identified using API 20E biochemical strips (bioM erieux, Marcy l'Etoile, France). The presence of mcr-1 gene was confirmed among all 8 E. coli isolates by conventional PCR as we recently conducted [19,31]. Subsequently, the minimum inhibitory concentration (MIC) of colistin among these strains was tested by micro-dilution according to the guidelines of Clinical and Laboratory Standards Institute [32,33]. The mcr-1positive E. coli gave MIC of colistin between 2-8 mg/ml (Table 1). Plasmids were extracted from mcr-1-positive E. coli using alkaline lysis method. To elucidate the genetic context of mcr-1 on these plasmids, the conventional multiplex PCR with 7 primer sets was performed (Table S1) as we recently described [3,19]. The plasmids isolated from the different strains had unexpectedly similar PCR profiles with the exception of pPK112 which lacks the tnpA loci (Fig. 1B). Genetic context of mcr-1 shows that all the plasmids lack the insertion element ISApl1 (Fig. 1A), which has been responsible for insertion of mcr-1 in previous studies [34]. Also, these plasmids (Fig. 1A) are identical to the mcr-1-carrying plasmid pE15017 isolated in China [19,22]. These bacteria were subjected to multi-locus sequence typing analyses with the Warwick method (http://mlst.warwick.ac. uk/mlst/). Diversified sequence types were detected, namely, ST10, ST2847, ST155, ST361 and ST6395. Evidently, all strains belonged to different STs with the exception of two strains of ST361 (Table 1). Despite that none of these STs have been reported from Pakistan in the past, ST10 and ST155 have been reported in mcr-1harboring E. coli isolated from chicken in China [17,35]. Subsequently, a representative mcr-1-carrying plasmid pPK105 (Table 1) was subjected to whole genome sequencing using the method of Illumina HiSeq X-ten. The plasmid sequences were annotated by RAST, and the genome maps were drawn with the Circos program. As a result, the genome size of pPK105 was determined to be 60.499 kb (Acc. no.: MG808035, Fig. 2A), encoding hundreds of open reading frames with a GC content of 42.3%. Unlike the prevalent IncX4 type plasmid reported in Pakistan, Plasmid Finder at the web server (https:// cge.cbs.dtu.dk/services/PlasmidFinder/) indicated that pPK105 is a member of IncI2-type plasmid family (Fig. 2B). Notably, mcr-1 is the only resistance gene detected in pPK105 (Fig. 2B). It is quite different from   the scenarios observed in the other mcr-1-containing Pakistan isolate coharboring ESBL (extended-spectrum b-lactamase gene) and heavy-metal resistance. Although that ISApl1 sequence is missing, pPK105 retains the two conjugative genes vir and pil (Fig. 2). Intriguingly, we noticed that the mcr-1 gene in pPK105 is next to a sdrl gene, a serine-aspartate repeat surface protein known to bind collagen (Fig. 2B). However, its relevance remains unclear. Nevertheless, it has been shown earlier that ISApl1 transposon element is highly unstable in IncI2 plasmid [36]. For further investigation, the chemical modification of lipid A by the mcr-1 protein product MCR-1, the bacterial LPS was extracted as we conducted earlier [20,37] and then subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) [38]. In comparison to E. coli MG1655 (m/z 1796.29, Fig. 3A), a shift in the predominant lipid A species (m/z 1920.136, Fig. 3A) was observed in the mcr-1expressing PK105 strain (D mass is close to 123) corresponding to the addition of a PEA moiety (Fig. 3B). This new peak corresponds to a single modification that may occur at either the 1 or the 4 0 position (Fig. 3B). This highlighted that surface remodeling by the mcr-1-encoding phosphoethanolamine transferase contributes to the resultant colistin resistance [14].
The discovery of the mcr-1 gene, prompted a shift in focus from chromosomal mutations causing colistinresistance to a transmissible plasmid-borne colistin resistance determinant. In addition to clinical isolates in humans, antimicrobial surveillance programs in Europe have identified mcr-1 in commensal bacterial populations from broilers, pigs and turkeys [39]. This study shows a similar threatening scenario in the Faisalabad region of Pakistan where high rates of mcr-1 positive E. coli were identified in healthy broilers. Retrospectively, the discovery of diverse clonal backgrounds of E. coli harboring the plasmid-borne mcr-1 is similar to scenarios observed earlier in Chinese poultry.
Similarly, data on global population structure of mcr-1-positive E. coli showed large diversity in STs but limited plasmid types, particularly with regional spread of IncHI2 plasmids in Europe and IncI2 in Asia [14]. This indicated the possible spread of a single mcr-1 colistin resistance gene across large geographical distances. It seems likely that farm animals act as a reservoir for the genetic diversity of mcr-1 [40].

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.    [20].