Plasmidome in mcr-1 harboring carbapenem-resistant enterobacterales isolates from human in Thailand

The emergence of the mobile colistin-resistance genes mcr-1 has attracted significant attention worldwide. This study aimed to investigate the genetic features of mcr-1-carrying plasmid among carbapenem-resistant Enterobacterales (CRE) isolates and the potential genetic basis governing transmission. Seventeen mcr-harboring isolates were analyzed based on whole genome sequencing using short-read and long-read platforms. All the mcr-1-carrying isolates could be conjugatively transferred into a recipient Escherichia coli UB1637. Among these 17 isolates, mcr-1 was located on diverse plasmid Inc types, consisting of IncX4 (11/17; 64.7%), IncI2 (4/17; 23.53%), and IncHI/IncN (2/17; 11.76%). Each of these exhibited remarkable similarity in the backbone set that is responsible for plasmid replication, maintenance, and transfer, with differences being in the upstream and downstream regions containing mcr-1. The IncHI/IncN type also carried other resistance genes (blaTEM-1B or blaTEM-135). The mcr-1-harboring IncX4 plasmids were carried in E. coli ST410 (7/11; 63.6%) and ST10 (1/11; 9.1%) and Klebsiella pneumoniae ST15 (1/11; 9.1%), ST336 (1/11; 9.1%), and ST340 (1/11; 9.1%). The IncI2-type plasmid was harbored in E. coli ST3052 (1/4; 25%) and ST1287 (1/4; 25%) and in K. pneumoniae ST336 (2/4; 50%), whereas IncHI/IncN were carried in E. coli ST6721 (1/2; 50%) and new ST (1/2; 50%). The diverse promiscuous plasmids may facilitate the spread of mcr-1 among commensal E. coli or K. pneumoniae strains in patients. These results can provide information for a surveillance system and infection control for dynamic tracing.

The lack of accessible treatment has resulted in the use of colistin, an outmoded antibiotic, as a last-resort therapeutic drug for human infections by Gram-negative bacteria. The widespread use of colistin in humans and animals has led to the emergence of colistin resistance in Gram-negative bacteria, with rates of resistance continuously increasing 3,4 . A classic mechanism of colistin resistance is thought to be associated with chromosomal mediation 5 . The discovery of plasmid-mediated colistin resistance encoded by mcr genes revealed high prevalence in human and animal isolates harboring these genes and the transmission of mcr is of global concern 6 . Up to the present, 10 variants of mcr (mcr-1 through mcr-10) have been reported 7,8 . Of particular concern is the spread of mcr genes into CRE, which would create strains that are potentially pan-drug resistant. The coexistence of mcr and carbapenemase genes, such as bla NDM , bla OXA-48-like , and bla IMP , in CRE isolates has been described worldwide [9][10][11][12] .
The global prevalence of mcr genes revealed that mcr-1 (4917/5191; 94.7%) is a common gene and has a wider distribution than mcr-2 through to mcr-8 4  www.nature.com/scientificreports/ reported [10][11][12][13][14] and the prevalence of mcr-1 has been increasing in Thailand 13 . The mcr-carrying plasmids identified consist of IncX4, IncI2, IncHI2, IncF, IncP, IncY, and ColE10-like ones, most of which are conjugative plasmid 15 . Collectively, information regarding the genetic context of mcr-1 plasmid and its organization in the genome is still limited in Thailand. One study revealed the general characteristics of mcr-1 harboring CRE isolated from patients in Thailand 13 . However, genomic analysis has not yet determined insights to plasmidome in the CRE harboring mcr-1. Thus, this study aimed to determine the complete genomic sequences to provide insight into plasmidome and to compare plasmid harboring mcr-1-among CRE isolates from human patients in Thailand.
Ethical approval. Ethical review and approval were not required because no human specimens or data were used in the current study.
For the Illumina platform, the sequencing library was generated using a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, UK), following the manufacturer's recommendations. The genomic DNA was randomly fragmented to a size of 350 bp and the fragments were A-tailed and ligated with the adapter. Libraries were sequenced using the Illumina HiSeq platform with the 150 paired-end sequencing strategy. We applied Fastp v0.19.5 21 with default parameters for the quality filtering of Illumina reads. Adapters were trimmed using Skewer v0.2.2 22 . The quality checking of Illumina reads was performed using FastQC v0.11.8 (https:// www. bioin forma tics. babra ham. ac. uk/ projects/fastqc/). Hybrid assemblies with the ONT and Illumina data were performed using Unicycler v0.4.8 23 and the genome sequences of all 17 isolates were checked for quality using QUAST v5.0.2 24 . Genome sequences were submitted to the NCBI Prokaryotic Genome Annotation Pipeline (PGAP v4.12) for annotation. The default parameters were used for all software unless otherwise specified.
Bioinformatics analysis. Identification of antimicrobial resistance genes was analyzed using ResFinder 4.1 25 and the Comprehensive Antibiotic Resistance Database (CARD) 26 . Determination of the mcr-1-carrying plasmid was carried out using PlasmidFinder 27 . Phylogrouping for E. coli and the KL type of K. pneumoniae were performed using ClermonTyping 28 and Kaptive 29 , respectively. Multilocus sequence typing (MLST) analysis of mcr-1-carrying E. coli and K. pneumoniae was determined using MLST 2.0 30 .
To search for the genetically closest relatives to the mcr-carrying isolates, a modular single genome analysis was conducted following the core genome multilocus sequence typing approach by BacWGSTdb 2.0 31 . The genetically closest relatives were chosen for 5-10 strains based on small numbers of allelic differences with selection thresholds of 100-500, depending on the strains under current study. The phylogenetics of the mcr-carrying CRE isolates and the closest relatives selected from BacWGSTdb were conducted using a reference genome-based, www.nature.com/scientificreports/ single-nucleotide polymorphism (SNP) strategy with REALPHY 32 . The phylogenetic tree was visualized using the iTOL V4 software 33 . E. coli K12 substrain MG1655 (accession no. U00096) and K. pneumoniae WCHKP9G2 (accession no. NBYD01000091) were used as the reference sequences for SNP analysis. In addition, phylogenetic analysis of plasmid-harboring mcr-1 was conducted using the Mashtree program, following the program's instructions 34 .
Accession number. The assembled genomic sequences were deposited under the BioProject accession number PRJNA525849. The accession numbers for each mcr-1-harboring isolate are provided in Table 1.
The genetic relationships based on the SNPs phylogeny of these mcr-1-harboring isolates are shown in Fig. 3 and Fig. 4. E. coli strain no. 2117 was closely related with strains from China (accession no. CP035123.1). Isolate no. P36.8 was closely related with the reference strain K12 and clustered with P24-5 (new ST), as shown in Fig. 3. K. pneumoniae strain no. 2509 was closely related with the K. pneumoniae SIKP199 strain from Thailand (accession no. GCA_004833525.1) (Fig. 4).   Figure 6 demonstrated diversity in the upstream and downstream coding sequences flanking mcr-1 in each type of plasmid. The IncHI2/IncN carrying mcr-1 plasmids showed the repressor gene TnpA and the conjugative transfer gene TraN upstream and the tellurium resistance genes downstream of mcr-1. The IncI2 carrying mcr-1 plasmids revealed the same integrase gene downstream but different genes upstream. In contrast with 11 IncX4-carrying plasmids, the flanking coding sequences or genes were similar in 9 plasmids containing the gene RepB family plasmid initiator replication protein upstream (nos. 2117, 2509, 58967, 62122, 59990, 60220) and downstream (nos. 53037, 53060, 60000) or peptidyl-arginine deaminase downstream, whereas 2 IncX4 plasmids (V417 and 56511) had different genes upstream and downstream of mcr-1 (Fig. 6). Isolates with nos. 2117, 2509, 62122, and 60220 showed high genetic organization similarity in the plasmids (Figs. 5 and 6).
As shown in Fig. 7, plasmid phylogeny demonstrated diversity among the mcr-1 harboring-plasmids. Most IncX4 carrying mcr-1 plasmid were grouped in three clusters. The first cluster comprised strain 59990 which was related to the pTB602 plasmid of Salmonella sp. SSDFZ69 from China (NZ_CP034833.1). The second cluster contained strains 58967, 50220, 2509, 62122, and 2117 that related to the plasmids pKPNH54.3 (NZ_CP024919.1) of K. pneumoniae NH54, and PN42 (MG557854.1) of E. coli PN42, both from Thailand. The third cluster consisted of 53360 and 53037 which related to the plasmid pEC931 (CP049122.1) of E. coli EC931 from China. We found that the other IncX4-type plasmids carrying mcr-1 of strains 56511, 60000, and V417 were not clustered and were located on different branches (Fig. 7).

Discussion
The high prevalence of human Enterobacterales isolates harboring mcr genes is of global concern. A recent report revealed the overall average prevalence of mcr genes was 4.7% (0.1-9.3%) in 47 countries across 6 continents 4 ; as many as 10 variants of the mcr genes (mcr-1 through to mcr-10) have been documented 7 40 . According to several reports in Thailand, the major plasmid types carrying mcr-1 in Enterobacterales isolates were IncX4 and IncI2, although other plasmid replicons have been  The conjugation experiment in the current study revealed that all mcr-1-harboring plasmids were successfully transferred from the donor to the E. coli recipient; according to the plasmid Inc types, they are conjugative plasmids 39 . Among such plasmids in our study, the IncX4 and IncI2 plasmid types were genetically similar, with the least variability, whereas the IncHI2/IncN plasmid type was divergent due to the fact that this type of double-Inc type plasmid contains multiple antimicrobial-resistant genes. This was consistent with the results of the two plasmids merging to perhaps increase the range of host species, plasmid fitness, and/or the acquisition of multiple antimicrobial-resistant genes 45 . Another study demonstrated that IncHI2-type plasmids are genetically divergent due to containing an MDR region which comprises a variable combination of antimicrobial-resistance genes and insertion sequences, such as Tn6330, in the IncHI2 type that is still highly active and is often transposable 37 . Our IncHI2/IncN plasmid also showed multiple antimicrobial-resistant genes. In addition, mcr-1 was stably located on IncX4 and IncI2 without cut-paste transposition 37 , which could explain that why mcr-1 was commonly distributed in these plasmids.  www.nature.com/scientificreports/ Plasmid phylogenetic analysis in the current study showed that most of our IncX4-type plasmids carrying mcr-1 were grouped, although some were diverse. Notably, 5 isolates carrying mcr-1 on the IncX4-type plasmid were clustered with mcr-1-IncX4 plasmids from either E. coli or K. pneumoniae from Thailand, indicating that they are close relatives and this type of plasmid is circulating in Thailand. In contrast, the other mcr-1 plasmid replicon types in our study were mostly related to several plasmid carrying mcr-1 types from China, perhaps suggesting that they are widely distributed in this region and they may have originated from the same source or ancestor.
The STs of mcr-1-harboring E. coli isolates in this study were mainly disseminated through local clonal expansion with a high-risk international clone ST410 that can cause several types of infection highly resistant to antibiotics and a global distribution 46 . The mcr-1-carrying IncX4 plasmids have also been identified in E. coli ST410 recovered from human blood 47 . This may suggest a possible association between E. coli ST410 and the carriage of mcr-1-IncX4 plasmids. In contrast, the most globally common ST of E. coli carrying mcr-1 is ST10 4 . However, previous study in Thailand revealed the mcr-1 carrying E. coli isolates from humans had diverse STs 42 . In the current study, K. pneumoniae ST336 was predominant. ST336 belongs to clonal complex 17, predominant in carbapenem-resistant K. pneumoniae and is considered an international clone frequently associated with global spread [47][48][49][50] . The K. pneumoniae ST15 isolates associated with the spread of multiple drug-resistance genes include ESBLs and mcr-1 40,48 .
mcr-1 was widely distributed in many bacterial species such as E. coli, K. pneumoniae, Salmonella enterica, Shigella spp., Enterobacter cloacae, Pseudomonas spp., Aeromonas spp., Citrobacter freundii, Kluyvera ascobarta,    54 . A previous study reported that the dissemination of 26 mcr-1-carrying enterobacterial strains (23 E. coli and 3 K. pneumoniae) isolated from contact surfaces (such as handrails and vending machines) on public transportation routes suggested a possible transmission vector of these organisms from one location to another, thereby posing a broader public health risk 55 . These results demonstrated that plasmids are the major vehicle involved in the dissemination of resistance or virulence genes. Notably, mcr-1-carrying enterobacterial strains were recovered from samples collected from hospitals in the current and the previous studies 13 , indicating that these isolates could be of nosocomial origin and thus highlighting the need for strong infection control implementation to prevent transmission of mcr-gene-containing bacteria capable of causing potential outbreaks. The prevalence and dissemination of mcr-1-harboring Enterobacterales isolates from animals (food animals, pet animals, and wildlife), humans (healthy populations and patients) and the environment (farms, urban and rural communities, and natural environments) have been mentioned globally 36 . Control of their dissemination among humans, animals, and the environment based on the "One-health approach" is necessary. In addition, the judicious use of antibiotics is advisable to minimize the development and dissemination of colistin resistance in human isolates. www.nature.com/scientificreports/

Data availability
The assembled genomic sequences during the current study were deposited under the BioProject with accession number JAJBZQ000000000, JAJBZP000000000, JAJGBP000000000, JAJBZO000000000, and JAJGBO000000000.