Silent spread of mcr-9 in ESBL-producing Enterobacteriaceae clinical isolates, Jimma, Ethiopia

Tsegaye Sewunet; Mohammad Razavi; Chaitanya Tellapragada; Christian G. Giske

doi:10.1371/journal.pone.0336440

Abstract

Background

Mobile colistin resistance (MCR) genes, have been increasingly identified worldwide, but their presence and characteristics in Africa remain poorly understood. Herein, we characterized a silent mcr-9 gene carried in Enterobacteriaceae from Ethiopia.

Methods

In a study investigating genomic epidemiology of ESBL-producing Gram-negative bacilli from clinical samples in 2016, eleven isolates were found to encode mcr-9 genes. Whole genome sequencing, combining Illumina and MinION Nanopore technologies were performed for in-depth investigation of the genome and understand the genetic contexts of mcr-9 and its associated plasmids.

Result

The mcr-9 genes were detected in Enterobacter cloacae (n = 8), Escherichia coli (n = 1), Klebsiella pneumoniae (n = 1), and K. michiganensis (n = 1). They were isolated from urine (54.5%, 6/11), wound secretions (27.3%, 3/11), and fecal samples (n = 18.2%, 2/11). In addition to mcr-9, all isolates encoded bla_CTX-M-15, and aac (6’)-Ib-cr among several other resistance genes. These isolates were susceptible to colistin (MIC ≤ 0.5 mg/L). The E. cloacae strains belong to three different sequence types (ST): ST114 (n = 3), ST184 (n = 3), ST254 (n = 1), and Enterobacter mori ST2197 (n = 1)), whereas the E. coli and the K. pneumoniae strains belonged to ST410 and ST337. IncHI2 and IncHI2A plasmid replicons were present in all isolates. Although the genetic content of plasmids carrying the mcr-9 genes varied, the genetic contexts surrounding all mcr genes within ±10kb region were largely consistent, mostly flanked by composite transposons such as IS26, and IS903B.

Conclusion

A silent mcr-9 gene was detected among ESBL-producing Enterobacteriaceae isolates. The IncHI2 plasmids encoding the mcr-9 had 25% genetic content dissimilarity.

Citation: Sewunet T, Razavi M, Tellapragada C, Giske CG (2025) Silent spread of mcr-9 in ESBL-producing Enterobacteriaceae clinical isolates, Jimma, Ethiopia. PLoS One 20(11): e0336440. https://doi.org/10.1371/journal.pone.0336440

Editor: Md Bashir Uddin, The University of Texas Medical Branch at Galveston, UNITED STATES OF AMERICA

Received: February 24, 2025; Accepted: October 24, 2025; Published: November 18, 2025

Copyright: © 2025 Sewunet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data is available in the main article and supplementary information. In addition, the sequence reads are available at a public domain: NCBI (Sequence Read Archive). The data is accessible using a BioProject ID: PRJNA1322377 or using a direct link at (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1322377).

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interest exists.

Introduction

The global spread of antimicrobial resistance (AMR) is fueled by mobile genetic elements (MGEs) [1]. They are drivers of the plasticity of bacterial genomes through intercellular mobility of necessary functions such as antibiotic resistance genes (ARGs) between different species [2,3]. The important human pathogens, including Enterobacteriaceae and other Gram-negative bacilli, have recruited mobile ARGs conferring resistance to antibiotics with broad spectrum activity including colistin and carbapenems. These antimicrobials are designated as critically important, and alternative therapeutic agents are urgently needed against resistant strains [4], particularly in low- and -middle income settings where novel antimicrobials such as the new beta-lactam/beta-lactam inhibitor combinations are not yet available.

The main colistin resistance mechanism was historically known as chromosomal mutations. However, the discovery of plasmid-mediated colistin resistance (mcr-1) in 2015 highlighted the presence of a new mechanism with ample opportunity to spread among diverse bacterial species [5]. Since the first report of mcr-1, increased prevalence of mcr-genes, and about ten different variants of mcr were reported [6]. The mcr-9 was identified in Salmonella enterica serovar Typhimurium [7], E. coli [8] and other species [6]. The mcr family has been reported from several sources including human, environmental, food, and animals [9,10]. Multiple studies detected mcr-genes from clinical samples, including bloodstream infections from countries such as Italy [11], Switzerland [12], China [13], Philippines [14], and Czeck Republic [15]. Understanding the transmission mechanisms and the spread of mcr genes could be important to contain their further dissemination.

In this study, plasmids from clinical isolates of Enterobacteriaceae and carrying mcr-9 were characterized. The genetic context of mcr-9, the antibiotic susceptibility and resistome profiles, and genomic epidemiology of the bacterial hosts were studied. In Africa, data regarding mcr-9 is limited and it is difficult to predict the situation. Also, when it occurs in low-income health care settings where colistin has not been used, or patients had no travel history, it requires more attention to understand the mechanism of transmission. In this regard, our study shed light on the co-selection and silent spread of the mcr-9 gene among ESBL-producing Enterobacteriaceae which might later revert to resistance phenotype under selection pressure when colistin is used [16].

Materials and methods

Ethical approval was given by the Ministry of Science and Technology of Ethiopia- National Ethics and Review committee (NERC) Ref No. 3–10/150/2016.

Strain collection

Isolates carrying mcr-9 were collected as part of genome-based cross-sectional survey of ESBL-producing Enterobacteriaceae at Jimma Medical Center, Ethiopia. As shown in our previous work, [17,18] we enrolled a total of 1,087 patients with suspected bacterial infection. Species identification was done using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) at the Department of Clinical Microbiology, Karolinska University Hospital, Solna. Antimicrobial susceptibility testing was done by disk diffusion using EUCAST guidelines (https://www.eucast.org/ast_of_bacteria) and broth microdilution for novel antibiotics tested against these isolates. Eleven ESBL-producing isolates were found to encode the mcr-9 gene upon resistome analysis. The genetic context surrounding the mcr-genes as well as the plasmids carrying them were studied. These isolates, and their mutant variants were tested for susceptibility to increased concentration colistin.

DNA extraction and sequencing: Genomic DNA was extracted using EZ1^®DNA Tissue Kit (QIAGEN) by using the EZ1 Advanced DNA Bacteria Card on EZ1 Advanced extraction system. NEXTRA-XT kits (Illumina) were used for library preparation and sequencing was performed on Illumina (HiSeq2500) platform at Science for Life Laboratories, Stockholm.

All the sequenced isolates were annotated as follows: First, the quality of the reads was checked using FastQC. Then, the paired-end reads with low-quality bases were trimmed and filtered using Trim-Galore tool [19] The remaining paired-end and single reads were assembled into longer contigs using SPAdes (version 3.13). Then, open reading frames (ORFs) were predicted by prodigal (version 2.6.3). For annotation, Diamond (version 2.0.4) were used to compare ORFs against the comprehensive antimicrobial resistance database (CARD, version 3.1), Virulence factor database (VFDB), Non-redundant NCBI protein database (downloaded Feb. 2022). The assembled draft genomes were also used to query bacterial genome analysis at Center for Genomic Epidemiology (https://cge.food.dtu.dk/services/MLST Last access: September 2023) to identify multi-locus sequence typing. Capsular and O-lipopolysaccharide typing were identified using Kaptive (https://kaptive-web.erc.monash.edu/ last access: September 2023).

Characterization of the plasmids: After identifying mcr-9 in the characterized isolates, nine of them were selected for sequencing with Nanopore MinION technology. The resulting long reads enabled reconstructing of the genetic contexts around mcr-9 by employing a hybrid assembly using Unicycler (version 0.4.8). The resulted drafted genomes were annotated using the previously mentioned pipeline. Plasmids were identified by mapping the contigs against the NCBI plasmid collection (downloaded Feb. 2022) using BLASTn algorithm in BLAST+ program [20]. The incompatibility groups were identified using the PlasmidFinder tool. Moreover, the content of the recovered plasmids was compared as follows: First, all the predicted ORFs on plasmids were clustered using CD-HIT tool (version 4.8.1) with 90% similarity. Then, a matrix was created, in which columns were different clusters of ORFs and rows were identified plasmids and values were the number of discovered clusters on plasmids. Using Bray-Curtis measure, the dissimilarity between plasmids were calculated. Finally, the dendrogram and PCoA plot were produced in R using the following packages: vegan (version 2.5), ggdendro (version 0.1.23), ggplot2 (version 3.3.5).

Moreover, the immediate (±5 kb) genetic context around mcr-9 were analyzed using progressiveMauve (version 2.4.0). The reference genome was a region of plasmid discovered in Salmonella enterica that was harboring mcr-9 and confer resistance to colistin (NCBI-accession: CP006057.1[11014:22633]). The visualization was preformed using an in-house Mauve-viewer program (https://github.com/xrazmo/mauve-viewer).

Antimicrobial susceptibility testing: Minimum inhibitory concentrations (MICs) were determined using a customized broth microdilution plate, MDROXF (sensititer plates from Thermo Fisher Scientific, Waltham, United States). The Sensititer plates panel contains the following antibiotics: amikacin, aztreonam, cefepime, colistin, imipenem, meropenem, piperacillin-tazobactam, tobramycin, tigecycline, ceftazidime-avibactam, meropenem, imipenem, eravacycline, ceftolozane-tazobactam, imipenem-relebactam, and meropenem-vaborbactam.

Result

Isolation of strains and patient characteristics

The mcr-9 genes were detected in four different species of Enterobacteriaceae, all the isolates were ESBL-producing. The prevalence of mcr-9 gene among ESBL-producing Enterobacterales was 3.5% (11/312), however, it varied with species, E. cloacae (12.9%, 8/62), Klebsiella spp. (K. pneumoniae and K. michiganensis) (1.8%, 2/109)), and E. coli (0.7%, 1/141). All strains were isolated from patients admitted to the hospital. Two of the strains (K. pneumoniae, and E. coli), were isolated from patients admitted at pediatric ward. Similarly, two isolates of E. cloacae were from the medical ward, and the rest of E. cloacae (n = 6), and K. michiganensis (n = 1) were isolated from the surgical ward. About 64% (7/11) were isolated from patients admitted with urinary tract infections and most of these patients had underlying chronic disease. Details of the clinical information regarding types of infection, factors such as prior use of antibiotics, and types of underlying chronic diseases are presented in Table 1.

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Table 1. Patients’ socio-demographic characteristics and clinical data.

https://doi.org/10.1371/journal.pone.0336440.t001

Minimum inhibitory concentration of isolates

In all isolates MICs in the susceptible range were observed for colistin, meropenem, imipenem, meropenem/vaborbactam, and imipenem/relebactam. Also, for tigecycline, a low MIC (<= 1 mg/L) was observed, suggesting no acquired resistance. In most of the isolates, MICs in the resistant range was observed for aztreonam 100% (11/11), tobramycin 63.6%, (7/11), piperacillin/tazobactam 63.6% (7/11), and cefepime (63.6%, 7/11). Details about antimicrobial susceptibility are presented in Table 2.

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Table 2. Minimum inhibitory concentration of the mcr-9 isolates to commonly prescribed antibiotics.

https://doi.org/10.1371/journal.pone.0336440.t002

Molecular characteristics of strains

Table 3 shows that each of the isolates carried at least 9–14 different ARGs in addition to mcr-9. Notably, they harbored ESBL genes including: bla_CTX-M-15, and bla_SHV-12. Moreover, ARGs against aminoglycosides (aac(6’)-Ib-cr), trimethoprim-sulfamethoxazole (dfrA-17, dfrA-19, dfrA-7, and dfrA-14), phenicols (catB3), and sulfonamides (sul1 and sul2) were commonly observed. Genomic analysis identified the E. coli isolates as ST410, which is associated with the pandemic extraintestinal pathogenic group (ExPEC). It belonged to the O96:H serotype, and its phylogroup was categorized as phylogroup C. Among the numerous virulence genes detected in this isolate, notable ExPEC-associated virulence genes such as papC, afra/dra, and iutA were present. The K. pneumoniae isolate was identified as ST337, with the capsular locus KL109, and the O2v2 O-lipopolysaccharides type. Similarly, K. michiganensis, when analyzed using the Kaptive/Holt lab database, was found to carry the capsular locus KL107, and the O1v1 O-lipopolysaccharides type. The E. cloacae were polyclonal including ST114 (n = 3), ST184 (n = 3), ST254 (n = 1) and E. mori ST2791 (n = 1). ST114, ST184 and ST254 were commonly associated with nosocomial infection.

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Table 3. Species identity, resistome profile, and plasmid replicon types in isolates carrying the mcr-9-genes.

https://doi.org/10.1371/journal.pone.0336440.t003

Genetic structure and content of plasmids encoding mcr-9

Multiple plasmid replicon types were identified in all the isolates which included IncHI2, IncHI2A and IncX3. The IncHI2 replicon was most prevalent found in all isolates, followed by IncHI2A as the second most prevalent type details are shown in Table 3. All the plasmids encoding the mcr-9 had similar replicon profiles, and the mcr-9 genes were located on IncHI2 replicon types. The sequenced plasmids carrying the mcr-9 had different size. The backbones of the plasmids were mapped to previously sequenced plasmids from the NCBI database (S1-S8_File.pdf in S1 File). However, the eight characterized plasmids formed three clusters regarding gene content (Fig 1), highlighting their different evolutionary path and accessibility of probable co-selection of mcr-9 with other functions.

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Fig 1. Clustering recovered plasmids that contain mcr-9 using Bray-Curtis measure of their gene-content.

A) a dendrogram showing cluster of plasmids with 25% dissimilarity in gene content. B) a PCoA plot showing the overall dissimilarity plasmids in their gene-content.

https://doi.org/10.1371/journal.pone.0336440.g001

Moreover, Fig 2 shows that the plasmids contain similar genetic contexts upstream of the mcr-9, which present in all the recovered genetic contexts (the green rectangle in Fig 2). Nonetheless, the variations observed in the downstream region underscore the occurrence of separate transpositional events involving the mcr-9–wbuC complex within distinct plasmids or their subsequent downstream regions, as illustrated by the rectangles with different colors in Fi 2. It is noteworthy that all the retrieved genetic contexts lack the specific genetic elements that encompass qseC- and qseB-like genes, as indicated in pink within the reference genome in Fig 2. However, the remnant of IS26 identified beyond qseC- and qseB-like genes region matches with the same gene downstream of mcr-9–wbuC complex, suggesting that a separate transposition event might have inserted the missing region in the Salmonella enterica genome (CP006057.1) which can express mcr-9.

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Fig 2. The immediate genetic context of mcr-9 encoding plasmids compared to standard reference plasmid from Salmonella enterica (CP006057.1).

Aligned regions are shown by colored rectangles and the annotated ORFs are plotted with arrows in forward strand. A genetic context upstream of mcr-9–wbuC complex are almost intact in all studied plasmids (the light green block). However, the downstream were quite dissimilar.

https://doi.org/10.1371/journal.pone.0336440.g002

Discussion

The current study presents the silent mcr −9 gene and its genetic context in clinical isolates of Enterobacteriaceae from Ethiopia. The spread of mcr-genes through several variants mcr-1 and mcr-10 over wider geographical areas has become a global threat. We detected the mcr-9 genes in four different species of ESBL-producing clinical isolates of Enterobacteriaceae when the use of colistin was not documented in the country. However, Ethiopia has recently included colistin in the national list of essential medicines for use in clinical setting (http://efmhaca.hcmisonline.org/wp-content/uploads/2020/12/EML-sixth-edition.pdf).

Despite encoding mcr-9, isolates in the current study were susceptible for colistin. The clinical use of colistin may apply selective pressure that induces expression of the mcr-9 gene. The core genetic context of mcr-9 and wbuC, was the same in most of the plasmids in this study. But as compared to previous studies, in the genetic context of mcr-9 (the flanking regions) qseC-like and qseB-like genes which are believed to function as regulatory genes [8,21] are missing. However, it worth mentioning that a study from United States reported S. enterica and E. coli strains with qseC and qseB genes susceptible for colistin (MIC ≤ 1) [22]. It could suggest that mcr-9 gene is silent in these contexts, and most likely the reason that all the isolates were susceptible to colistin with MIC less than 0.5 mg/L is due to the absence of this upstream region regulatory gene component.

Moreover, the presence of multiple resistance genes in addition to the mcr-9 increases risk of co-selection. Acquisition of the qseC/qseB [8] or other undetermined mechanisms (even in the absence of qseC/qseB) like the arnBCADTEF gene cassette which are genospecies specific mechanisms of resistance to colistin might interact with mcr-9 and enhance the selection and dissemination of the gene [23]. It has also been demonstrated that in a hybrid plasmid IncHI2/IncHI2A encoding bla_NDM-1, mcr-9 has shown increased expression and consequently phenotypic resistance following induction with colistin [24]. Thus, activation of the gene could occur either through mobilization of the gene by MGEs and consequently its integration into a fully functional genetic contexts or through the acquisition of novel regulatory mechanisms driven by mutations or horizonatal genetic transfer. Additionally, the coexistence of ARGs against other antibiotic classes facilitates the dissemination of mcr-9 across species and lineages via co-selection mechanisms [25].

The spread of a silent mcr-9 gene extends beyond the findings of this study. For example, a study from Ethiopia [26] identified two cases of mcr-9 in isolates obtained from two geographically distant tertiary hospitals. One isolate was an unclassified Salmonella species, and the other was Klebsiella pneumoniae, both associated with bloodstream infections. Notably, similar to the findings in the current study, both isolates remained susceptible to colistin. These findings highlight the potential for wider dissemination of mcr-9 and emphasize the need for molecular surveillance.

There are some reports of mcr-9 from human clinical samples in Africa; mainly one from South Africa [27] and the other from Egypt [28]. Despite the limited studies among clinical samples in the continent, mcr genes were reported from several other ecosystems including human carriers, animals, environment, and food products [29]. Furthermore, the limited diagnostic capacity might have obscured the actual prevalence of colistin resistance and mcr genes. On the other hand, the lack of strict control of antimicrobial usage, and lack of stringent infection prevention and control strategies can catalyze the spread of colistin resistance and/or spread of the mcr-9 genes and may continue to pose a challenge in both human and animal health.

The transmission of mcr genes is commonly through polyclonal strains, and mostly one or two species were reported from different studies. In the current study, the detection of mcr-9 from four different Enterobacteriaceae species, and one of them E. cloacae belonging to different sequence types, may indicate that there is an ongoing silent spread of the gene. Furthermore, in the apparent global perspective, E. cloacae complex seem more prone to harboring and spreading the mcr-9 mediated colistin resistance and also the silent spread of the gene [30–32].

The mechanism of spread for mcr-9 genes is mediated by plasmids, all the mcr-9 genes were located on either IncHI2 or IncHI2A replicon types. The genetic content and location of the mcr-9 gene differed between these plasmids. Clustering analysis showed that the plasmids in this study have some differences regarding their genetic content. Similar findings were reported from previous studies that IncHI2/IncHI2A were carriers of the mcr-9 genes [8,28,33]. Moreover, in the presence of multiple plasmid replicon types in all the isolates, mcr-9 can be transposed to other plasmids that may encode transcription factors necessary for expressing phenotypic resistance.

The mcr-9 gene carried on the super-plasmids IncHI2 can be co-selected with other ARGs (like the extended spectrum cephalosporins, aminoglycosides, fluoroquinolones, trimethoprim, and sulfonamides), and mobilized with a range of mobile genetic elements. A global review of literature regarding mcr-9 isolates and plasmids showed that the IncHI2 plasmids were responsible for the transmission of mcr-9 in several countries. Apparently, the silent spread of mcr-9 without phenotypic resistance seems to have followed similar trend as in other parts of the world [29]. In Africa, the lack of targeted epidemiological data may have obscured the extent of silent spread of mcr-9 and colistin resistance in general.

Despite mcr-9 being first described in 2019 and availability of sporadic reports from different regions; retrospective analysis of genomic data collected earlier offers valuable insights into the emergence of mcr-9 and its epidemiology, particularly in resource-limited settings such as Africa. Many bacterial isolates sequenced prior to the identification of mcr-9 were not screened for this gene, resulting in a critical knowledge gap. By re-examining these historical datasets using current bioinformatics tools and updated resistance gene databases, we identified previously unrecognized occurrences of mcr-9 in higher number than previously reported from a human source and at a a clinical settings in Africa, thereby establishing a more accurate timeline and geographical distribution of genetic determinant in Ethiopia. This finding is important in understanding evolutionary origins, and early reservoirs of mcr-9, which can inform a more effective surveillance and control strategies. In Africa, where active genomic surveillance remains limited, leveraging existing datasets enhance epidemiological understanding as complemenatry approach to active surveillance. Integrating such historical genomic epidemiology data into global antimicrobial resistance knowledge pool is therefore not only beneficial but also addresses the underreporting and delayed recognition of emerging resistance genes like mcr-9 across the continent.

Conclusion

The detection of the mcr-9 gene in multiple species of clinical isolates—despite their susceptibility to colistin—carried on conjugative plasmids with similar replicon types but diverse genetic content and an array of MGEs, is highly concerning. Colistin has not been included as national list of drugs in Ehtiopia in 2016 when these isolates were collected. The clinical use of colistin should be carefully regulated in settings where further mobilization and co-selection of silent mcr-9 could potentially lead to the emergence of phenotypic resistance. However, susceptibility of these strains to carbapenems, and beta-lactam/beta-lactam inhibitor combinations is important clinical information for current consideration.

Supporting information

S1 File. Sequence alignment of 40 IncHI2 mcr-9 encoding plasmids and their genetic content compared to plasmid from 8 of the isolates where we detected mcr-9.

The outer most structure in each of the eight figures below shows the genetic structure of plasmids from each of the isolates from the current study, and the gaps indicated the absence a gene.

https://doi.org/10.1371/journal.pone.0336440.s001

(PDF)

Acknowledgments

We acknowledge the technical support from Aina Iversen at the clinical microbiology laboratory, Karolinska University Hospital, Solna.

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Figures

Abstract

Background

Methods

Result

Conclusion

Introduction

Materials and methods

Strain collection

Result

Isolation of strains and patient characteristics

Minimum inhibitory concentration of isolates

Molecular characteristics of strains

Genetic structure and content of plasmids encoding mcr-9

Discussion

Conclusion

Supporting information

S1 File. Sequence alignment of 40 IncHI2 mcr-9 encoding plasmids and their genetic content compared to plasmid from 8 of the isolates where we detected mcr-9.

Acknowledgments

References