Genomic analysis of extended-spectrum β-lactamase-producing Enterobacter kobei ST691 strain harbouring mcr -10.1 isolated in Yaoundé, Cameroon

Antibiotic resistance, WGS, Enterobacter kobei, mcr-10.1, Cameroon. 29 Running title: ESBL-Enterobacter kobei ST 691 harbouring mcr-10.1 in Cameroon 30 List of figures: 01 31 32 33 34 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 2

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36 Enterobacter species. are opportunistic pathogens commonly responsible for serious, difficult-to-37 treat hospital-acquired infections. Extended-spectrum β -lactamase (ESBL)-producing and 38 colistin-resistant Enterobacterales are increasingly implicated in human and animal infections 39 worldwide. Here we report a first detection of colistin-resistant ESBL-producing E. kobei strain 40 belonging to ST691 and harbouring mcr-10.1. 41

42 This strain was isolated from the faecal sample of a two-year old child aged, who was diagnosed 43 with gastroenteritis in Ya oundé, Cameroon. This ESBL-E. kobei ST691 genome was sequenced 44 using Illumina Miseq (Illumina, San Diego, CA, USA). Mobile genetic elements and antibiotic 45 resistance genes were predicted using AMRFinderPlus, ARIBA and the VFDB and 46 PlasmidFinder databases respectively. 47

48 This strain exhibited phenotypic resistance to numerous antibiotics belonging to penicillin, third 49 generation of cephalosporin and carbapenem families. However, it was susceptible to 50 aminoglycoside and fluoroquinolone. Genome analysis reveals a length of 4 626 300 bp, and 51 N50 of 143 731, GC content of 54.9%. Genes conferring resistance to β -lactams ( blaACT-9), 52 polymyxin (mcr-10.1) and phenicol/quinolone (oqxA,B) were detected. Mobile genetic elements 53 including plasmid replicon type [IncFIB(pECLA) and IncFII(pECLA)] and the IS1, IS110, 54 ISEc34, IS1222SC, IS66, IS630, IS3, IS26 insertion sequences were also detected. 55

56 We report the first colistin-resistant ESBL-producing E. kobei isolated from a two-year old child. 57 It is a high priority potential pathogen showing resistance to last-resort antimicrobials to which 58 there is little access in Cameroon. This underscores the necessity to strengthen genomic 59 surveillance, antimicrobial stewardship and infection prevention and control as well as to 60 heighten awareness of the threat posed by resistant bacteria. 61 62 63 64 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 3 1. Introduction 65 Enterobacter species. are opportunistic pathogens responsible for difficult-to-treat hospital-66 acquired infections, including UTIs, skin infections and bacteraemia [1]. They overproduce 67 chromosomally encoded cephalosporinase (cAmpC) associated with the permeability reduction 68 of the outer membrane which may confer reduced susceptibility to carbapenem [2]. The 69 emergence of carbapenem-and colistin-resistant E. kobei limits therapeutic options [1]. This 70 strain is increasingly important pathogens implicated in human and animal infections [1,3] and 71 carbapenem-resistant Enterobacter spp. are listed by the World Health Organisation (WHO) as 72 critical priority pathogens for which new antimicrobials need to be developed [4]. 73 We report here the first description of a colistin-resistant and extended-spectrum beta-lactamase 74 (ESBL)-producing Enterobacter kobei strain (PR13) harbouring blaACT-9 and mcr-10.1. It was 75 isolated from the faecal sample of a two year old child aged, who was diagnosed with 76 gastroenteritis in Cameroon. Multi-drug resistance of this isolate prompted whole genome 77 sequencing with the goal of understanding the genetic basis of the observed resistance 78 phenotype. 79 2. Materials and methods 80 During a four-month period from July to October 2020, clinical specimens at two health facilities 81 were screened for ESBL-producing Enterobacterales. The specimens screened included wound 82 swabs, urine, uro-vaginal swabs, and stool samples. Specimens were collected and transported to 83 the microbiology laboratory of the Research Institute of the Centre of Expertise and Biological 84 Diagnostic of Cameroon ( CEDBCAM-RI) within two hours of collection. Stool samples were 85 cultured on Mac Conkey agar supplemented with crystal violet. ESBL screening was done using 86 CHROMagar™ ESBL according to the manufacturer’s instructions. Identification was 87 performed using biochemical profile with API 20E and confirmed with VITEK 2 system and 88 MALDI-TOF. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc 89 diffusion method and minimum inhibitory concentrations were obtained with the Vitek® 2 90 System using Gram Negative Susceptibility card (AST-N255) (BioMérieux, Marcy l’Etoile, 91 France). The European Committee on Antimicrobial Susceptibility testing guidelines 2019 was 92 used for interpretation of the results and E. coli ATCC 25922 and K. pneumoniae ATCC700603 93 were used as controls. 94 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 4 Genomic DNA (gDNA) was extracted using the Wizard® Genomic DNA Purification Kit 95 (Promega, Wisconsin, USA) according to the manufacturer’s instructions. NanoDrop 96 spectrophotometry and fluorometric analysis (Qubit®) were used to verify the integrity and 97 purity of the gDNA. Double-stranded DNA libraries were prepared with the NEBNext Ultra II 98 FS DNA Library Prep Kit for Illumina (New England Biolabs, Massachusetts, USA) following 99 Global Health Research Unit (GHRU) in-house protocols. Library concentration and fragment 100 distribution were analysed with Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher 101 Scientific, Massachusetts, USA) on a Qubitflex fluorometer and Agilent High Sensitivity DNA 102 Kit (Agilent Technologies, California, USA) on a bioanalyser. Libraries were then denatured and 103 sequenced on an Illumina Miseq using paired-end 2 by 150 bp reads (Illumina, San Diego, CA, 104 USA). The sequenced reads were assembled using the GHRU assembly pipeline with default 105 parameters and genome quality cut-offs ( https://www.protocols.io/view/ghru-genomic-106 surveillance-of-antimicrobial-resista-bpn6mmhe). The genome was annotated using the National 107 Center for Biotechnology Information's Prokaryotic Genome Automated Pipeline and 108 antimicrobial resistance genes were identified using AMRFinderPlus v3.10.24. Plasmids were 109 detected using PlasmidFinder 2.1 ( http://cge.cbs.dtu.dk/services/PlasmidFinder/) and the 110 Comprehensive Antibiotic Resistance Database (CARD) respectively. 111 3. Results 112 Among 49 non-duplicate ESBL- Enterobacterales from clinical specimens (endocervical swab, 113 blood, urine, feces and throat swab), three were Enterobacter spp. including Enterobacter kobei 114 (stool), Enterobacter hormaechei (wound swab) and Enterobacter asburiae (uro-genital swab). 115 The sole E. kobei from a two-year old child with the gastroenteritis symptoms (stomach-ache, 116 vomiting and diarrhoea), showed resistance to amoxicillin/clavulanic acid, ticarcillin/clavulanic 117 acid, cefuroxime cefotaxime, ceftazidime chloramphenicol and meropenem. However, it was 118 susceptible to imipenem, ertapenem, amikacin, gentamicin, ofloxacin, ciprofloxacin, 119 levofloxacin and sparfloxacin. 120 The genome of E. kobei isolate has a genome length of 4 626 300 bp, an N50 of 143 731, GC 121 content of 54.9% and was deposited in the European Nucleotide Archive 122 (https://www.ebi.ac.uk/ena) with accession No. ERR10862935. It was assigned to the sequence 123 type (ST) 691. Genes conferring resistance to β -lactams (blaACT-9), polymyxin ( mcr-10.1) and 124 phenicol/quinolone (oqxA,B). Two plasmid replicon types, namely IncFIB(pECLA) (Accession 125 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 5 no. CP001919; 100% identity, contig 51) and IncFII(pECLA) (Accession no. CP001919; 99.6% 126 identity, contig 56) were detected in the isolate’s genome, as were IS1, IS110, ISEc34, 127 IS1222SC, IS66, IS630, IS3, IS26, and cn_4541_ISSgsp1 insertion sequences. 128 VirulenceFinder revealed the mrkABCDF fimbrial operon promoting adherence and biofilm 129 formation among Enterobacterales. Interestingly, a BLAST analysis of the mrkABCDF contig 130 revealed that it is identical to a 2.5kb region of an E. coli IncFII plasmid (accession: 131 CP088133.1) that also carries mcr-10. 132 The Figure 1 showed selected E. kobei genomes in Pathogenwatch ( https://pathogen.watch) 133 belonging to distinct sequence types and isolated from distinct countries and constructed a 134 maximum likelihood phylogeny. This isolate was closely related to an E. kobei, recovered from a 135 human (USA) in 2003 ( https://microreact.org/project/8xhqAsqugKT6RmTAHHNFEW-136 ekobeiyaounde) [5]. 137 4. Discussion 138 We described here the first report of a colistin-resistant ESBL-producing E. kobei stool 139 harbouring mcr-10.1 and it was isolated from a child of two-year old with gastro-enteritis that 140 unlikely to have been exposed to colistin. Mobile colistin resistance ( mcr) genes confer resistant 141 to a last-line antibacterial therapy and are increasingly detected in isolates from humans, animals 142 and the environment worldwide [6]. Recent reports from the Republic of Korea showed that 143 clinical E. kobei co-harbouring mcr-4.3, mcr-9 and blaACT-64 were implicated in infections among 144 elderly patients attending emergency department [6]. It has also been detected in a Franciscana 145 dolphin in Brazil, chickens and exposed workers in Asia [6-7] . E. kobei bearing bla ACT-9 and 146 mcr-9 have recently been reported from wastewater in South Africa [8]. The strain carrying the 147 gene had mobile element signatures are indicative of transmission potential and the isolation of 148 this strain points to a human faecal reservoir of a priority pathogen. 149 5. Conclusion 150 Beta-lactam antimicrobials are a mainstay for addressing bacterial pathogens in Cameroon and 151 colistin is considered as a last resort treatment option among patients infected with resistant 152 strains. The detection of Enterobacter kobei bearing resistance genes to both classes of 153 antimicrobials emphasizes the need to reinforce antimicrobial stewardship programmes, heighten 154 awareness among physicians and general population, strengthen and implement strict infection 155 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 6 prevention and control measures in healthcare settings to curb the dissemination of colistin-156 resistant ESBL-E. kobei. 157 158

159 We are grateful to Faith I. Oni and Odion O. Ikhimiukor for technical assistance, Jola-Ade J 160 Ajiboye for administrative support and the NCBI Genbank submission staff for help with 161 genome upload, decontamination and deposition procedures. We thank other members of the 162 SEQAFRICA consortium, in particular Rene Hendricksen and Pernille Nilsson, for helpful 163 discussions and program oversight. 164 Funding 165 Whole genome sequence generation and analyses was supported thr ough the SEQAFRICA 166 project, funded by the Department of Health and Social Care’s Fleming Fund using UK aid. 167 INO is a Calestous Juma Fellow supported by the Bill and Melinda Gates Foundation INV-168 036234. The views expressed in this publication are those of the authors and not necessarily 169 those of the UK Department of Health and Social Care or its Management Agent, Mott 170 MacDonald or other funders. 171 Transparency Declarations 172 The authors declare that they have no known competing financial interests or personal 173 relationships that could have appeared to influence the work reported in this paper. 174 Ethical approval 175 Ethical approval was obtained from the institutional ethics of Research in Human Health 176 (CEIRSH) (No. 2020/020804/CEIRSH/ESS/MIM). Permission to conduct the research was 177 also granted from the Head Department of Health. The study was conducted in accordance with 178 the declaration of Helsinki. In addition, the research authorizations of the various healthcare 179 structures have been granted. We had assured the confidentiality of the patient information’s and 180 only the principal investigator had this information. Moreover, this information was anonymized 181 and all isolates were stored for further research. 182 Author contributions 183 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 7 RCF co-conceptualized the study, undertook sample collection, microbiological laboratory and 184 data analyses, prepared tables and figures, interpreted results, contributed to bioinformatics 185 analysis, and drafted the manuscript. LLF undertook sample collection, microbiological 186 laboratory analyses, contributed to bioinformatics analysis and vetted the results. EEO 187 performed whole genome sequencing analysis, prepared tables and figures, interpreted results 188 and edited the manuscript. AOO set up and oversaw the sequencing workflow and validated the 189 identity and antimicrobial susceptibility of the isolate. INO co-conceptualized the study and 190 undertook critical revision of the manuscript. All authors read and approve the final manuscript. 191

192 [1]. Kim JS, Kwon MJ, Jeon SJ, Park SH, Han S, Park SH. et al. Identification of a carbapenem-193 resistant Enterobacter kobei clinical strain co-harbouring mcr-4.3 and mcr-9 in Republic of 194 Korea. J Glob Antimicrob Resist 2021; 26, 114-116. https://doi.org/10.1016/j.jgar.2021.05.008. 195 [2]. Jousset AB, Oueslati S, Bernabeu S, Takissian J, Creton E, Vogel A. et al. False-Positive 196 Carbapenem-Hydrolyzing Confirmatory Tests Due to ACT-28, a Chromosomally Encoded 197 AmpC with Weak Carbapenemase Activity from Enterobacter kobei. Antimicrob Agents 198 Chemother 2019; 63(5). https://doi.org/10.1128/AAC.02388-18. 199 [3]. Manandhar S, Nguyen Q, Pham DT, Amatya P, Rabaa M, Dongol S. et al. A fatal outbreak 200 of neonatal sepsis caused by mcr-10-carrying Enterobacter kobei in a tertiary care hospital in 201 Nepal. J Hosp Infect 2022; 125, 60-66. https://doi.org/10.1016/j.jhin.2022.03.015. 202 [4]. World Health Organization. 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Draft genome sequences of rare 218 Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and 219 MEZEK194 carrying mobile colistin resistance gene mcr-9 isolated from wastewater in South 220 Africa. J Glob Antimicrob Resist 2023;21;33:231-237. 221 https://doi.org/10.1016/j.jgar.2023.03.007. 222 223 224 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 1 1 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 2 2 3 Figure 1 : Phylogeny based on core genome multi-locus sequence typing genes of 27 E. kobei genomes. The following information is provid isolate: name/reference, ST types (STs), country and colistin resistance. STs are highlighted as indicated in the legend and is olate name’s pr study is CM-LURIA-PR13_S16. 0.09 5 A vided for each present in the .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 3 4 5 6 Figure 1: Phylogeny based on core genome multi-locus sequence typing genes of 27 E. kobei genomes. The following information is provided for each isolate: name/reference, ST types (STs), country and colistin resistance. STs are highlighted as indicated in the legend and isolate name’s present in the study is CM-LURIA-PR13_S16. B .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint 4 7 8 9 10 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 30, 2025. ; https://doi.org/10.64898/2025.12.28.696731doi: bioRxiv preprint