Molecular antibiotic resistance mechanisms and co-transmission of the mcr-9 and metallo-β-lactamase genes in carbapenem-resistant Enterobacter cloacae complex

Carbapenem-resistant Enterobacter cloacae complex (CRECC) has increasingly emerged as a major cause of healthcare-associated infections, with colistin being one of the last-resort antibiotics of treatment. Mobile colistin resistance (mcr)-9 is a member of a growing family of mcr genes and has been reported to be an inducible gene encoding an acquired phosphoethanolamine transferase. Here, we collected 24 ECC strains from Chongqing, China from 2018 to 2021. Subsequently, antibiotic resistance genes and the transmission dynamics of the strains were determined by PCR, whole-genome sequencing, and bioinformatic analysis. The mcr-9 was identified in IncHI2/2A or IncHI2/2A + IncN plasmids from six CRECC strains and was co-located with blaNDM-1 or blaIMP-4 in 2/6 plasmids. The genetic environment of mcr-9.1 was composed of IS903B-mcr-9.1-wbuC-IS26 in the five mcr-9.1-harboring-plasmid, but IS1B was located downstream of mcr-9.2 in the pECL414-1 sequence. We also found that the pNDM-068001 plasmid carrying mcr-9.1 could be a hybrid plasmid, formed by a Tn6360-like blaNDM-1 region inserted into an mcr-9.1-positive IncHI2/2A plasmid. A conjugation assay showed that plasmids mediated the co-dissemination of mcr-9 and metallo-β-lactamase (MBL) genes. In addition, we performed induction assays with sub-inhibitory concentrations of colistin and found an increase in the relative expression levels of the mcr-9.2, qseC, and qseB genes, as well as an increase in the minimum inhibitory concentration values of colistin in the CRECC414 strain. These findings provide a basis for studying the regulatory mechanisms of mcr-9 expression and highlight the importance of effective monitoring to assess the prevalence of MBL and mcr-9 co-existing plasmids.


Introduction
The dramatic increase in the prevalence and clinical impact of infections caused by Enterobacterales producing carbapenemases, such as Klebsiella pneumoniae carbapenemase-2 (KPC-2) and New Delhi metallo-β-lactamase-1 (NDM-1), has triggered a global health crisis (Kumarasamy et al., 2010;Munoz-Price et al., 2013). Colistin belongs to the family of polymyxins, cationic polypeptides. Owing to its nephrotoxicity, the clinical use of colistin was discontinued in the 1980s; however, it was reintroduced in the 1990s because of the lack of effective antimicrobial agents for the treatment of multidrug-resistant Gram-negative pathogens (Falagas and Kasiakou, 2005). Previously reported mechanisms of colistin resistance are chromosomally mediated and involve the regulation of two regulatory systems (pmrAB, phoPQ, and its negative regulator mgrB in K. pneumoniae), resulting in the modification of lipid A by phosphoethanolamine or 4-amino-4-arabinose (Gunn, 2008;Cannatelli et al., 2013). However, a plasmid-mediated mobilized colistin resistance (mcr) gene mcr-1 was first reported in China in late 2015 (Liu et al., 2016), which largely threatens the use of colistin in clinical settings, and nine mcr homologs (mcr-2 to -10) have since been detected (Nang et al., 2019). Although the transmission of most mcr genes to carbapenem-resistant Enterobacterales (CRE) has been limited (Carroll et al., 2019;Kieffer et al., 2019;Nang et al., 2019), mcr-9, first identified in Salmonella enterica clinically isolated in the United States in 2019 (Carroll et al., 2019), has been identified in several CRE backgrounds (Chavda et al., 2019;Lalaoui et al., 2019;Liu et al., 2021). In addition, the first identified strain that carried mcr-9 was sensitive to colistin, but mcr-9 confers resistance to colistin after cloning and overexpression in the laboratory (Carroll et al., 2019). Further studies have revealed that mcr-9 expression is inducible in the presence of colistin when mcr-9 is located upstream of the qseBC two-component system (Kieffer et al., 2019). However, other studies have suggested that mcr-9 and colistin resistance may not be related (Tyson et al., 2020). These results suggest that there is uncertainty as to when mcr-9 confers elevated minimum inhibitory concentration (MIC) values for colistin, which may be related to the genetic background or differences in host and other unidentified regulatory genes (Chavda et al., 2019;Simoni et al., 2021).
In the present study, we investigated the antibiotic resistance genes (ARGs) and transmission dynamics of 24 ECC isolates collected from 2018 to 2021 using PCR, whole-genome sequencing (WGS), and bioinformatic analysis. We analyzed WGS data and compared them with publicly available data to determine the genome structure of strains carrying the mcr-9 gene. We determined the phenotypic impact of carbapenem-resistant Enterobacter cloacae complex (CRECC) strains carrying mcr-9 under the sub-inhibitory concentrations of colistin induction. Moreover, plasmid-mediated co-transmission of the mcr-9 and MBL genes was determined using a conjugation assay.

Materials and methods
Bacterial isolates, susceptibility testing, and clinical data collection Enterobacter cloacae complex (ECC) strains that were resistant to meropenem, imipenem, or ertapenem or produced carbapenemase isolated from 2018 to 2021 at Yongchuan Affiliated Hospital of Chongqing Medical University, China, were collected, in addition to one carbapenem-sensitive Enterobacter cloacae complex (CSECC) strain carrying mcr-9, identified in the screening, which was also included in this study. All 24 ECC strains were identified using a VITEK-2 Compact automatic microbiology analyzer and MALDI-TOF MS (Bruker, Billerica, MA, United States). Antimicrobial susceptibility was evaluated for all isolates following the Clinical and Laboratory Standards Institute (CLSI) guidelines (M100-S30; CLSI, 2020), and the results were interpreted according to these guidelines (Clinical and Laboratory Standards Institute (CLSI), 2020), except that colistin resistance was defined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST; version 10.0) clinical breakpoints (The European Committee on Antimicrobial Susceptibility Testing, 2020). Metadata including patients' departments, dates of specimen collection, and specimen types were recorded.
A total of 2,337 complete genomes of ECC strains were retrieved from the NCBI database (accessed on 31 December 2021) and were subjected to comparative alignments of mcr-9 positive plasmid genomes isolated from patients using BLAST and visualized using Easyfig v 2.2.5.
Core genome MLST and cg-SNP All strains contained 3,048 core genomes according to the reference genome (GenBank accession no. CP010377). The results of core genome MLST (cg-MLST) were visualized using PHYLOViZ, based on the goeBURST algorithm (Ribeiro-Goncalves et al., 2016). The results of core genome single nucleotide polymorphism (cg-SNP) were visualized using ggtree after building the evolutionary tree with FastMe (Lefort et al., 2015).

Conjugation experiment
We selected six isolates of CRECC carrying the mcr-9 and carbapenemase genes and used them as donors in a conjugation 1 https://pubmlst.org/ 2 https://cge.food.dtu.dk/services/PlasmidFinder/ 3 https://cge.food.dtu.dk/services/pMLST/ 4 https://cge.food.dtu.dk/services/ResFinder/ experiment, with rifampicin-resistant Escherichia coli EC600 as the recipient. Conjugation experiments were performed using the membrane bonding method according to previously described method (Gong et al., 2018). Briefly, donor (CRECC strain) and recipient cultures (E. coli EC600) were mixed at a 1:3 ratio in Luria-Bertani (LB) broth. The mixtures were placed on a membrane and subsequently incubated at 35°C for 24 h. Mueller-Hinton agar plates were supplemented with meropenem (1 mg/l) and rifampicin (600 mg/l) to select transconjugants. The VITEK-2 Compact system and 16S rRNA sequence were performed to confirm the transconjugants and PCR was determined to the presence of resistance genes.

Colistin induction assays
Colistin induction assays were performed as previously described with some modifications (Kieffer et al., 2019;Liu et al., 2021). Briefly, the CRECC414 strain was inoculated in 4 ml LB broth with shaking at 37°C for 2 h and then supplemented with colistin (1 and 2 mg/l) or without at a final bacterial suspension of 1.0 McFarland turbidity standard. After shaking at 37°C for 6 h, bacterial suspensions were used for mRNA extraction.

mRNA extraction and RT-qPCR
Total RNA was extracted using the PureLink TIM RNA Mini Kit (Thermo Fisher Scientific), as specified by the manufacturer. Each RNA sample was reverse-transcribed using the PrimeScript™ RT reagent kit (TaKaRa, Japan) according to the manufacturer's instructions. The relative expression level of mcr-9 was determined by quantitative real-time PCR (RT-qPCR) using SYBR Green detection reagents on the CFX96 RealTime PCR system. The primer sequences used are listed in Supplementary Table 1. Relative expression levels were calculated using the 2 -ΔΔCT method, with rpoB as a reference gene for comparison with those of samples that were not induced. Three independent replicates were performed with and without induction.

Nucleotide sequence accession number
WGS data were submitted to NCBI with accession numbers in Supplementary Table 2.

Genetic environment of mcr-9-carrying isolates
We identified 26 mcr-9-containing complete plasmid genomes isolated from patients in the NCBI database (Supplementary Figure 3). We found that most (24/26) of the mcr-9 genetic environments showed 100% homology to the Clustering analysis and resistance determinants of 24 ECC strains from our institution. The resulting structure reflected the similarity between the sequences, and resistance determinants present (in blue) in each strain are shown on the right.
Frontiers in Microbiology 06 frontiersin.org Table 4). This prompts that mcr-9 was frequently present on mobile genetic elements with other ARGs, implying that the use of these types of antibiotics has the potential to co-select for the continued presence of mcr-9.

Colistin induction assays and colistin susceptibility testing
The strains were exposed to sub-inhibitory concentrations of colistin, and the CRECC414 strain was used as the template for mRNA extraction. Notably, a 2.6-fold increase in the plasmid-located mcr-9 mRNA was detected once the culture was induced with 1 mg/l colistin; however, there was no statistically significant difference in the relative expression levels of the qseB and qseC genes located on chromosomes ( Figure 5). After induction with 2 mg/l colistin, the relative expression levels of mcr-9, qseC, and qseB mRNA increased by 8.5-, 2.6-, and 2.7-fold, respectively ( Figure 5). In parallel, the MIC value of colistin for the CRECC414 strain was 4 mg/l, and the MIC values were 64 and 128 mg/l after induction with 1 or 2 mg/l colistin, respectively.

Discussion
The IncHI2-ST1 plasmid is the main replicon type carrying mcr-9, which is a member of a growing family of mobile colistin resistance genes. The mcr-9 is predominantly present in MBL-producing strains at our institution; therefore, novel betalactam/beta-lactamase inhibitor combinations have no activity against them. The use of colistin in CRE infections remains a priority. However, the present study found an increase in the MIC value of strains mcr-9-carrying for colistin under antibiotic selection pressure, which may raise concerns regarding the use of colistin.
Notably, mcr-9-harbouring plasmids recovered from human isolates were interspersed with plasmids from animals and food (Supplementary Figure 4), and it is possible that the use of colistin in food animals largely contributes to the prevalence of mcr genes in humans and the environment (Sun et al., 2018). Interestingly, we found that mcr-9 genetic environments were major composed of IS903B-mcr-9-wbuC-IS26 in our setting (Figure 3), and this genetic environment has been reported previously (Wu et al., 2022).
Frontiers in Microbiology 08 frontiersin.org similar to that observed in mcr-9-harboring plasmid sequences, suggesting that mcr-9 may be transferred between plasmids and chromosomes in the form of a gene cassette. According to MLST typing, six CRECC isolates carrying mcr-9 were ST97 (33.3%, 2/6), ST93, ST114, ST25, and ST365 (Figure 1), which are most likely horizontal transmission rather than clonal transmission in our hospital. Interestingly, molecular epidemiology revealed that ST93 is the predominant sequence type of the CRECC strain, followed by ST171 and ST145 in China ; and ST114 is a common global CRECC strain clone (Peirano et al., 2018). The results of the current study showed that the CRECC68 and CRECC78 strains carrying mcr-9 belonged to ST93 or ST114, indicating that mcr-9 may have co-evolved with the host, which carries a high risk of transmission.
Studies have been conducted on the phenotypic effects of strains under antibiotic selection pressure. Kieffer et al. (2019) found that the expression of mcr-9 is inducible by subinhibitory concentrations of colistin and is related to the presence of qseC and qseB genes downstream of mcr-9. However, the MIC values of a few isolates in the presence of the qseB/qseC-like two-component system downstream of mcr-9 remained unchanged after colistin exposure (Kim et al., 2021;Macesic et al., 2021;Simoni et al., 2021). It was also found that the absence of qseC and qseB genes downstream of mcr-9 also does not change the MIC values of the strains after colistin exposure (Chavda et al., 2019;Bitar et al., 2020;Wu et al., 2022). The different results could be related to differences in the genetic background, host, and other undiscovered regulatory genes (Chavda et al., 2019;Simoni et al., 2021). In the present study, six CRECC strains carrying mcr-9 were isolated from six patients from different departments, all of whom had different diseases, but none of them had been treated with colistin (Supplementary Table 5). First, we found that the colistin resistance rate of six CRECC strains carrying mcr-9 was 50% (Supplementary Table 2). Nevertheless, it has been reported that few isolates carrying mcr-9 display colistin resistance at baseline (Carroll et al., 2019;Kieffer et al., 2019;Tyson et al., 2020;Macesic et al., 2021). Alignment revealed no possible mutations in chromosomal resistance genes in our strains. Second, the relative expression levels of mcr-9.2 and MIC values of the CRECC414 strain increased after colistin exposure, which is consistent with the results of K. pneumoniae (Liu et al., 2021). Although qseC and qseB are not located downstream of mcr-9.2 on the plasmid, our experiments showed an increase in the relative expression levels of qseC and qseB after colistin induction. Therefore, we hypothesized that once mcr-9 is integrated into the chromosome, it may be regulated by the qseB/ qseC-like two-component of the lysM-mdaB-qseC-qseB-parC-parF-sufl genetic background (Supplementary Figure 5), ultimately leading to a possible increase in the relative expression levels of mcr-9 and the MIC value of colistin in the CRECC strain under antibiotic selection pressure. In addition, comparative alignments of the genetic environments revealed that IS1B was located downstream of mcr-9.2 in both the CRECC414 and K. pneumoniae strains (Figure 3). Similarly, IS1 was found downstream of mcr-9 in both pN260-1 and pMCR-SCNJ07 sequences, and these strains displayed colistin resistance at baseline (Yuan et al., 2019;Umeda et al., 2021; Figure 3). We hypothesized that the induced expression of mcr-9 is related to the presence of downstream IS1. Therefore, further validation of these possible regulatory mechanisms will be the focus of future research.
The strength of the present study was the extensive use of Illumina and Nanopore read sequencing to construct highquality hybrid plasmid assemblies. Notably, two mcr-9-harboring plasmids also carried bla IMP-4 (pECL405-1) or bla NDM-1 (pNDM-068001), respectively (Supplementary Table 3). We found that bla NDM-1 and mcr-9 were located on the IncHI2/2A + IncN plasmid, and the bla NDM-1 cassette was similar to the recently reported cassette of Tn6360 (carrying bla NDM-1 and located on the IncN1 plasmid; Zhao et al., 2017), suggesting that pNDM-068001 could be a hybrid plasmid, formed by a Tn6360-like bla NDM-1 region inserted into an mcr-9-positive IncHI2/HI2A plasmid. In addition, bla IMP-4 and mcr-9 were located on the IncHI2/2A plasmid, and bla IMP-4 was characterized by the following structure: IS6-dfrA19-IntI1-bla IMP-4 -ltrA-SMR-sul1-GNAT-IS6 (IS6100), which may mediate the transfer of bla IMP-4. This indicated that horizontal gene transfer events played a significant role in plasmid evolution. Notably, the results of our study showed a co-transmission characteristic of the mcr-9 and MBL genes, and colistin for CRE infections remains an important priority. We also found that mcr-9 was inducible in the CRECC strains; therefore, this may pose some challenges for the use of colistin in the clinical environment. The current study has a minor limitation; the boiling method extracts a small amount of DNA with many impurities, and DNA breakage may occur, leading to errors. However, PCR and sequencing of the Expression of genes in clinical isolate CRECC414 with and without colistin exposure. The genes are the plasmid-located mcr-9 and the chromosome-located qseB and qseC. Fold changes in mRNA levels were determined by RT-qPCR. The data have been normalized to values for reference rpoB gene. Three independent replicates were performed and the data shown represent the means. The asterisks indicate statistical significance at different levels by Student's t-tests: *p ≤ 0.05.
Frontiers in Microbiology 09 frontiersin.org extracted nucleic acids for the primary screening of ARGs in strains still have some value.
In conclusion, the spread of mcr-9 is primarily driven by the IncHI2/2A plasmid. Our results alert physicians to the circulation of plasmid-mediated co-transmission of mcr-9 and MBL genes already in the hospital setting, which could lead to a serious public health crisis; therefore, effective monitoring is urgently needed to assess the prevalence of MBL and mcr-9 co-existing plasmids and to find an effective measure to control their spread. We found that mcr-9 showed increased expression when induced with colistin in the CRECC strain, which may be related to IS downstream of mcr-9 or qseB/qseC-like two-component systems on chromosomes. Further clarification of their regulatory mechanisms for mcr-9 gene expression will be the focus of our future research.

Data availability statement
The data presented in the study are deposited at GenBank, accession number(s) can be found in the article/Supplementary material. Further inquiries can be directed to the corresponding author.

Author contributions
XZ, CL, SJ, and XW conceived and designed the study. SJ and XW wrote the manuscript and participated in the whole experiment process. XL and KH helped with the experimental process. SJ, XW, HY, JZ, JW, and JL analyzed and interpreted the data. JW, JL, XGong, XGou, YY, SJ, and XW collected the isolates and the clinical data. All authors contributed to the article and approved the submitted version.

Funding
This work was supported by General projects of Chongqing Natural Science Foundation (cstc2020jcyj-msxm0067), Yongchuan Natural Science Foundation (2021yc-jckx20053), Talent introduction project of Yongchuan Hospital of Chongqing Medical University (YJYJ202004 and YJYJ202005), and Program for Youth Innovation in Future Medicine, Chongqing Medical University (W0113).