Genomic patterns and characterizations of chromosomally-encoded mcr-1 in Escherichia coli populations

The emergence and transmission of the mobile colistin resistance gene (mcr-1) threatened the extensive use of polymyxin antimicrobials. Accumulated evidence showed that the banning of colistin additive in livestock feed efficiently reduce mcr-1 prevalence, not only in animals but also in humans and environments. However, our previous study has revealed that a small proportion of Escherichia coli could continually carry chromosomally-encoded mcr-1. The chromosomally-encoded events, indicated the existence of stabilized heritage of mcr-1 and revealed a potential threat in the antimicrobial stewardship interventions, are yet to be investigated. In this study, we systematically investigated the genetic basis of chromosomally-encoded mcr-1 in prevalence and potential mechanisms of lineage, plasmid, insertion sequence, and phage. Our results demonstrated that the emergence of chromosomally-encoded mcr-1 could originate from multiple mechanisms, but mainly derived through the recombination of ISApl1/Tn6330. We reported a specific transmission mechanism, which is a phage-like region without lysogenic components, could associate with the emergence and stabilization of chromosomally-encoded mcr-1. These results highlighted the potential origin and risks of chromosomally-encoded mcr-1, which could be a heritable repository and thrive again when confronted with new selective pressures. To the best of our knowledge, this is the first study to systematically reveal the genomic basis of chromosomally-encoded mcr-1, and report a specific transmission pattern involved in phage-like region. Overall, we demonstrate the origin mechanisms and risks of chromosomally-encoded mcr-1. It highlights the need of public attention on chromosome-encoded mcr-1 to prevent from its reemergence.


Short report
The emergence and rapid dissemination of plasmidmediated mobile colistin resistance gene (mcr-1) have become a severe threat to public health [1]. The predominant carriers of mcr-1 were IncX4, IncI2, and IncHI2 plasmids, which are transferable and adaptive plasmid types with broad host range and contributed to the spread of mcr-1 among various sources and bacterial species [2][3][4]. Besides, recombination of transposons, especially Tn6330 (ISApl1-mcr-1-pap2-ISApl1), the primary vehicle for transmission of mcr-1, and phage-like sequences enable mcr-1 to transfer across plasmids and isolates. Such contributed factors facilitated high mcr-1 prevalence in several sources around the world, pushing local governments in Europe, Brazil and China to prohibit the use of colistin as growth promoter additive for livestock [5][6][7][8].

Open Access
Gut Pathogens *Correspondence: tiangb@mail.sysu.edu.cn 1 Department of Microbiology, Zhongshan School of Medicine, Sun Yatsen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China Full list of author information is available at the end of the article Accumulated evidence showed that banning of colistin in animal feed efficiently restricted mcr-1 prevalence, not only in animals but also in humans and the whole ecosystem in China [2][3][4]. However, our previous study showed that a low proportion of Escherichia coli carrying chromosomally-encoded mcr-1 continually existed in the ecosystem [4], which was sporadically reported by other studies as well [9][10][11]. On account of the plasmid that could be lost under certain circumstances due to instability, the chromosomally-encoded events could stabilize the heritage of mcr-1, threatening the intervention of colistin stewardship. In current study, we systematically investigate the epidemiological and genomic characterizations of E. coli population with chromosomally-encoded mcr-1.
Based on our previous large-scale epidemiological study from 2016 to 2018 in Guangzhou, China [4], we identified 24 (3.5%) out of 688 mcr-1-positive E. coli isolates with the chromosomally-encoded mcr-1 ( Table 1). The prevalence of chromosomally-encoded mcr-1-positive E. coli was from 0 to 9.8% for each source and from 2.2 to 4.8% for each epoch, indicating that the chromosomally-encoded mcr-1 was at a low prevalence state in different dimensions (Table 1). Additionally, the comparison of prevalence for chromosomally-encoded mcr-1 between different niches or epochs showed no significant difference (Fisher's exact test, p > 0.05 for each comparison), suggesting that the emergence of chromosomally-encoded mcr-1 was sporadic without temporal or source-specific signals.
We subsequently analyzed the genetic context of mcr-1 for each isolate to investigate the genetic model of chromosomally-encoded mcr-1, except seven isolates were excluded due to short mcr-1-harboring contigs. We found that most of the mcr-1 genes (93.6%, 44/47) were flanked by ISApl1, comprising 24 isolates harboring upstream ISApl1 and 20 isolates carrying composite Tn6330, which complied with the hypothesis of transposition-mediated chromosome insertion.
By mapping the insertion site onto the chromosome of E. coli MG1655, we noted that the distribution of chromosomally-encoded mcr-1 insertion sites was sporadic (Fig. 2a). Thirty-seven clusters of mcr-1-harboring segments were generated based on sequence clustering analysis (Fig. 2a), which included three   Fig. 1 The phylogenetic tree and annotation of epidemiological and genomic features. The red colour range on the phylogenetic tree represents sequence cluster 1 (SC1), and the blue colour range represents SC2. The heatmap is showing the presence/absence of characters for antimicrobial resistance genes (ARGs) and plasmid Inc types clusters involving more than one isolates (Fig. 2b) and 34 clusters only containing a single isolate (Additional file 2: Figure S1). The most common genetic pattern of chromosomally-encoded mcr-1 (19.1%, 9/47) involves in an insertion segment in size of ~ 25.7 kb, containing an incomplete phage-like region (score = 40 for phage Vibrio 12B8 [NC_021073] by PHASTER) and a truncated Tn6330 (ISApl1-mcr-1-pap2), which was inserted into the E. coli genome between lysN and hicB (toxin-antitoxin system) loci (Fig. 2b). The incomplete phage-like region only contains head, tail, and fiber protein, and lacks some necessary functional components (Fig. 2b), which seems unfunctional under current conditions. We used BLASTn to search this phage-like sequence in NCBI non-redundant nucleotide database, and the results showed that only five sequences, which are located on E. coli chromosome, were identified with ≥ 60% coverage and ≥ 90% identity, indicating the correlation between chromosomally-encoded mcr-1 and such phage-like region. Collectively, we heuristically concluded that such a phage-like region could mediate the emergence of chromosomally-encoded mcr-1, and then the phage may lose the lysogenic components, stabilization the genetic inheritance of chromosomally-encoded mcr-1. Additionally, the mcr-1 of two isolates showed the insertion of mcr-1 located on an integrative element region and a plasmid segment respectively, suggesting that chromosomally-encoded mcr-1 could be derived from the integration of the integrative region and plasmid segment (Fig. 2c).
In conclusion, our study comprehensively investigated the genetic basis of chromosomally-encoded mcr-1 in prevalence and potential mechanisms of lineage, plasmid, insertion sequence, and phage. Our results showed that chromosomally-encoded mcr-1 was mainly derived from ISApl1 insertion in genomic locations sporadically. Notably, we reported a new transmission mechanism, a phage-like region without functional components, could associate with the emergence and stabilization of chromosomally-encoded mcr-1. The chromosomally-encoded mcr-1 in current situations seems not a severe threat for public health, however, it could be a heritable repository and thrive again if the new selective pressure emerges, because the chromosome-mediated antimicrobial resistance genes (ARGs) might be conferred with genetic sustainability. In-depth investigations are needed to illustrate the genomic and epidemiological dynamics of chromosomally-encoded mcr-1, which may be changed after the approval of colistin in human clinical therapeutics in China [16].

Statistical analysis
The significance of prevalence variation of chromosomally-encoded mcr-1 between niches and epochs were tested by Fisher's exact test using Statistical Package for the Social Sciences (SPSS), version 20.0.
Additional file 1: Table S1. Additional file 2: Figure S1. The genetic structure of chromosomallyencoded mcr-1 patterns which included only one isolate. The number for each pattern was identical to Fig. 2a. Additional file 3: Figure S2. Flow diagram of the study selection process.