What happens when bacteria become resistant to both carbapenems and colistin, two critical last-line options? This study traces the mobile DNA that may let mcr-1 move between bacterial hosts in Thai hospitals. The global spread of carbapenem-resistant Enterobacterales has become a leading public health concern because carbapenemase gene carriage is rapidly increasing.

Most carbapenem resistance is conferred by carbapenem-degrading enzymes such as blaKPC, blaNDM, and OXA-48-like carbapenemase. In Thailand, information about the genetic context of mcr-1 plasmids and their organization in the genome is still limited. Genomic analysis had not yet determined insights into the plasmidome of carbapenem-resistant Enterobacterales harboring mcr-1.

The study therefore aimed to determine complete genomic sequences, provide insight into the plasmidome, and compare plasmids harboring mcr-1 among isolates from human patients in Thailand. The study used seventeen carbapenem-resistant Enterobacterales isolates carrying mcr-1: twelve from a previous study and five sent by hospitals in Thailand during 2016 to 2019.

The isolates included Escherichia coli and Klebsiella pneumoniae, and multiplex polymerase chain reaction confirmed mcr genes and the carbapenemase genes blaIMP, blaKPC, blaNDM, and blaOXA-48-like. A conjugation assay was performed for all seventeen mcr-1-carrying isolates, including twelve Escherichia coli and five Klebsiella pneumoniae isolates.

The mcr-harboring strains acted as donors, while streptomycin-resistant Escherichia coli UB1637 acted as the recipient, mixed at a ratio of one to twenty-five. The mixtures were plated on MacConkey agar containing streptomycin and colistin, and transconjugants harboring mcr genes were confirmed using polymerase chain reaction.

Bacterial genomic DNA was extracted using ZymoBIOMICS DNA Kits, and twelve isolates were sequenced with Oxford Nanopore Technologies while five were sequenced with Oxford Nanopore Technologies and Illumina. Oxford Nanopore library preparation used a rapid barcoding protocol without DNA size selection to preserve plasmid DNA.

Raw data were base-called and demultiplexed with Guppy, adapters were trimmed with Porechop, and read quality control used Nanoplot. Antimicrobial-resistance genes were identified with ResFinder and the Comprehensive Antibiotic Resistance Database. PlasmidFinder was used to determine the plasmid carrying mcr-1.

ClermonTyping and Kaptive provided phylogrouping for Escherichia coli and the KL type of Klebsiella pneumoniae, while MLST two point zero determined multilocus sequence types. PCR confirmed mcr-1 in all seventeen isolates, and one isolate, number 54715, also carried mcr-3.

Among five additional isolates, blaNDM-1 appeared in strains 2509, 2117, and V417, while carbapenemase genes were not detected in P24-5 and P36.8. All seventeen isolates were resistant to colistin, ampicillin, and ampicillin with sulbactam, and fifteen, or 88.24 percent, were resistant to carbapenems.

Among the five isolates tested in this study, three were resistant to a broad set of antibiotics, whereas P24-5 and P36.8 were susceptible to carbapenems. Table two reports antimicrobial susceptibility results for seventeen mcr-one-harboring carbapenem-resistant E.

coli and K. pneumoniae isolates, including five isolates from this study. The table records minimum inhibitory concentrations alongside susceptible, intermediate, or resistant interpretations: for example, ampicillin is greater than two hundred fifty-six R across the earlier isolates, while isolates P24.5 and P36.8 show less than or equal to one S for cefepime, cefotaxime, and several carbapenems.

This matters because the authors identify mcr-one in all seventeen isolates, with some also carrying carbapenemase genes, documenting clinically important resistance combinations. All seventeen mcr-1-carrying carbapenem-resistant Enterobacterales isolates could be conjugatively transferred into Escherichia coli UB1637.

The isolate carrying both mcr-1 and mcr-3, number 54715, transferred both genes. Colistin resistance appeared in fourteen recipient Escherichia coli, or 82.35 percent, with minimum inhibitory concentrations from four to sixteen micrograms per milliliter; the remaining recipients were susceptible.

These results suggested that mcr-1 in all seventeen isolates was carried on conjugative plasmids. Table 3 compares seventeen donor isolates with their E. coli transconjugants after conjugative transfer. Every donor and transconjugant retained mcr-1, while isolate 54715 carried and transferred both mcr-1 and mcr-3.

The MIC values in the transconjugants ranged from one to sixteen, with fourteen recipients showing colistin resistance; this supports the authors’ conclusion that the mcr-1 genes were carried on conjugative plasmids. Three plasmid replicon types were identified among the seventeen mcr-1-harboring isolates: IncX4, IncI2, and IncHI2 with IncN.

IncX4 was most frequent, appearing in eleven of seventeen isolates, or 64.7 percent; IncI2 appeared in four, or 23.53 percent; and IncHI2 with IncN appeared in two, or 11.76 percent. The eleven IncX4 plasmids ranged from 33,309 to 45,011 base pairs, the four IncI2 plasmids ranged from 60,960 to 67,526 base pairs, and the two IncHI2 with IncN plasmids were 270,820 and 273,765 base pairs.

IncX4 plasmids showed high similarity, although some had different mcr-1 gene positions. Figure five aligns plasmids carrying mcr-one genes, showing their open reading frames as arrows and marking mcr-one in red. Gray blocks connect homologous DNA segments identified by BLASTn, making shared and differing plasmid structures visible across the samples.

The authors report three replicon types among seventeen isolates: IncX4 in eleven, IncI2 in four, and IncHI2 or IncN in two, highlighting multiple plasmid backbones involved in mcr-one carriage. Figure 6 demonstrated diversity in the upstream and downstream coding sequences flanking mcr-1 in each plasmid type.

IncHI2 with IncN plasmids had TnpA and TraN upstream of mcr-1 and tellurium-resistance genes downstream, while IncI2 plasmids shared an integrase gene downstream but had different upstream genes. Among eleven IncX4 plasmids, nine had similar flanking sequences, including RepB family plasmid initiator replication protein upstream or downstream, or peptidyl-arginine deaminase downstream.

Two IncX4 plasmids, V417 and 56511, had different genes upstream and downstream of mcr-1. Figure six maps the coding sequences flanking mcr-1 across seventeen plasmids from E. coli and K.

pneumoniae, with arrow direction indicating gene orientation. It shows conserved patterns among the eleven IncX4 plasmids, while IncI2 plasmids share a downstream integrase but differ in upstream genes; IncHI2/IncN plasmids include upstream TnpA and TraN and downstream tellurium-resistance genes.

This diversity helps distinguish plasmid backgrounds carrying mcr-1. Plasmid phylogeny demonstrated diversity among the mcr-1-harboring plasmids, while most IncX4 plasmids grouped into three clusters. The first cluster included strain 59990 and was related to the pTB602 plasmid of Salmonella from China.

The second cluster contained strains 58967, 50220, 2509, 62122, and 2117, related to plasmids from Klebsiella pneumoniae NH54 and Escherichia coli PN42, both from Thailand. The third cluster contained 53360 and 53037, related to the pEC931 plasmid of Escherichia coli EC931 from China; strains 56511, 60000, and V417 were not clustered.

Figure seven is a Mashtree phylogenetic analysis of mcr-1-carrying plasmids, combining the study’s whole-genome sequences with reference plasmids. The branching pattern shows substantial diversity, while the authors identify most IncX4 plasmids as grouped into three clusters.

One includes strain 59990 with the pTB602 plasmid from Salmonella in China; another includes strains 58967, 50220, 2509, 62122, and 2117 alongside related reference plasmids. The conjugation experiment showed that all mcr-1-harboring plasmids were successfully transferred from donors to the Escherichia coli recipient, indicating that they were conjugative plasmids according to their Inc types.

IncX4 and IncI2 were genetically similar with the least variability, whereas IncHI2 with IncN was divergent and contained multiple antimicrobial-resistance genes. The study also states that mcr-1 was stably located on IncX4 and IncI2 without cut-and-paste transposition, which could explain why mcr-1 was commonly distributed in these plasmids.

The prevalence and dissemination of mcr-1-harboring Enterobacterales have been described globally across animals, humans, and the environment. Control of dissemination among humans, animals, and the environment requires a One Health approach.

Judicious antibiotic use is advisable to minimize the development and dissemination of colistin resistance in human isolates. Across seventeen isolates, mcr-1 sat on several conjugative plasmid types, with IncX4 most common but structurally varied. That mobility supports surveillance focused not only on bacterial strains, but also on the plasmids carrying resistance.