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

Abstract

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 plasmid-mediated 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].

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.

Table 1 Prevalence of chromosomally-encoded mcr-1 among 688 mcr-1-positive E. coli isolates

To systematically illustrate the genomic basis of chromosomally-encoded mcr-1-positive E. coli population, we collected other 30 E. coli genomes with chromosomally-encoded mcr-1 from published literature for subsequent analysis (Additional file 1: Table S1). Through in silico multilocus sequence typing (MLST) assignment, 32 different sequence types (STs) within 10 ST complexes were determined (Fig. 1). The most common ST among chromosomally-encoded mcr-1-positive E. coli isolates was ST10 (n = 10, 18.5%), which is consistent with the main host for plasmid-mediated mcr-1 on E. coli species [3, 4, 12]. The phylogeny demonstrated two sequence clusters (SCs), except for two isolates which were distinct from two SCs as the outgroup (Fig. 1). The sources and serotypes of these genomes were scattered on the phylogeny, suggesting that the emergence of chromosomally-encoded mcr-1 was random without source- or lineage-based specificity (Fig. 1). Since most of the chromosomally-encoded mcr-1-positive E. coli isolates have been identified in China (n = 40, 74.1%), which was attributed to the extensive screening of mcr-1 in China, the associations between locations and SCs was ambiguous (SC1 [11/16] vs SC2 [29/36], Fisher’s exact test, p = 0.49).

Fig. 1
figure1

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

The mcr-1 gene was initially found on plasmids in Enterobacteriaceae and on a transposon Tn6330, prompting that the chromosomally-encoded mcr-1 could come from recombination of plasmid segments or transposition of Tn6330 [13,14,15]. Therefore, we investigated the plasmidome of 54 genomes to illustrate the potential origin of chromosomally-encoded mcr-1. We identified 33 plasmid Inc types among all isolates, and the results showed that the most common Inc type was IncFIB(K) (45.8%, n = 22), followed by IncColRNAI (43.8%, n = 21), IncHI1 (33.3%, n = 16), IncX1 (31.3%, n = 15), IncFIB (AP001918) (27.1%, n = 13), and IncY (20.8%, n = 10). Remarkably, the common Inc types of mcr-1-harboring plasmids, such as IncX4, IncI2, IncHI2, and IncpO111 [1, 3, 4, 12], were rarely detected among these isolates (Fig. 1), indicating that the chromosomally-encoded mcr-1 may derive from ISApl1/Tn6330 through transposition, but not from the plasmid.

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 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).

Fig. 2
figure2

The insertion site and genomic patterns of chromosomally-encoded mcr-1. a The insertion patterns mapped to the Escherichia coli str. K-12 substr. MG1655 (Accession: NC_000913.2). The ring colored with orange represents the genome sequence of Escherichia coli str. K-12 substr. MG1655. The number in the outmost represents the order for each pattern, which showed in b, c and Additional file 2: Figure S1. b The genetic structure of chromosomally-encoded mcr-1 patterns which included more than one isolate. c The genetic structure of chromosomally-encoded mcr-1 which located on an integrative element region and a plasmid-like region

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].

Literature searching

We searched PubMed using the terms of “mcr-1” [MeSH]/[All Fields] AND “chromosome” [MeSH]/[All Fields] AND “Escherichia coli” [MeSH]/[All Fields] for articles published before 1th October 2020, and identified 20 publications, including 30 available E. coli genomes with chromosome-mediated mcr-1 (Additional file 3: Figure S2).

Bioinformatic analysis

Antimicrobial resistance genes screening, plasmid incompatibility typing and serotype identification were performed by Center for Genomic Epidemiology (http://www.genomicepidemiology.org/). Multilocus sequence typing (MLST) was assigned using Enterobase (http://enterobase.warwick.ac.uk/). Prophage prediction was implemented by PHASTER [17]. The phylogeny was constructed using RAxML v8.2 with GTR+G model and 1000 bootstrap [18] based on core genome single-nucleotide polymorphisms (cgSNPs) produced by Roary v3.11.2 and snp-site v2.4.1 [19]. Population structure was assessed using cgSNPs with hierBAPS [20]. The chromosome map was drawn by BRIG v0.95 and marked with insertion pattern manually by Easyfig v2.2.2 [21, 22]. The sequence clustering was performed by CD-HIT-EST [23].

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.

Availability of data and materials

The datasets generated and analysed during the current study are available in the NCBI GenBank repository. The accession number for each genome can be obtained in Additional file 4: Appendix material.

Abbreviations

mcr-1 :

Mobile colistin resistance gene

MLST:

Multilocus sequence typing

STs:

Sequence types

SCs:

Sequence clusters

cgSNPs:

Core genome single-nucleotide polymorphisms

ARG:

Antimicrobial resistance gene

Inc:

Incompatibility

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Acknowledgements

We acknowledge Ms. Lujie Liang (Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University) for assistance in writing of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Numbers 81830103, 81722030), China Postdoctoral Science Foundation (BX20200394), National Key Research and Development Program (Grant Number 2017ZX10302301), Guangdong Natural Science Foundation (Grant Number 2017A030306012), Project of high-level health teams of Zhuhai at 2018 (The Innovation Team for Antimicrobial Resistance and Clinical Infection), 111 Project (Grant Number B12003), Open project of Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education (Grant Number 2020kfkt04, 2020kfkt07), Innovative Development Program for Outstanding Graduate Students (No. 19ykyj557), and The Science and Technology Planning Project of Guangdong (2017A020215017).

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CS, GT and YD designed the study. CS and FM did the literature searching. CS, LZ, FM and GZ collected the data and genomes from NCBI database. CS and FM analyzed the genome data and visualized the results. CS write the draft manuscript. GT and MAE-GE-SA reviewed and edited the final manuscript. All author (except YD) contributed to sample collection and data collection in epidemiological study. All authors reviewed, revised and approved the final submission. All authors read and approved the final manuscript.

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Correspondence to Guo-Bao Tian.

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Supplementary information

Additional file 1: Table S1.

Additional file 2: Figure S1.

The genetic structure of chromosomally-encoded 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.

Additional file 4: Appendix.

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Shen, C., Zhong, LL., Ma, F. et al. Genomic patterns and characterizations of chromosomally-encoded mcr-1 in Escherichia coli populations. Gut Pathog 12, 55 (2020). https://doi.org/10.1186/s13099-020-00393-2

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Keywords

  • mcr-1
  • Colistin
  • Antimicrobial resistance
  • Genomic pattern
  • Chromosome
  • Insertion sequence
  • Phage