First Identification of blaNDM-1 in High-Risk Klebsiella pneumoniae ST147 Clone in Cameroon: A Genomic Insight

First Identification of blaNDM-1 in High-Risk Klebsiella pneumoniae ST147 Clone in Cameroon: A Genomic Insight | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First Identification of bla NDM -1 in High-Risk Klebsiella pneumoniae ST147 Clone in Cameroon: A Genomic Insight Roméo Tsayem Fouéméné, Madjid Morsli, Thierry Ngouana Kammalac, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8231079/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The emergence and spread of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) represent a serious clinical challenge due to limited treatment options. This pilot study aimed to track the evidence of CP-Kp genes in Yaounde, Cameroon where antimicrobial resistance (AMR) surveillance data remain scarce. Methods The resistance profile of clinical Klebsiella pneumoniae isolated in Yaoundé, Cameroon was investigated using routine Antibiotic Susceptibility Testing (AST) and Whole Genome Sequencing (WGS). Carbapenemase production was phenotypically confirmed using the selective chromogenic medium designed for the detection of carbapenemase-producing Enterobacterales (CHROMagar™ Modified SuperCARBA™), and the Klebsiella pneumoniae carbapenemase (KPC)/Metallo-β-lactamase (MBL)/Oxacillinase-48 (OXA-48) confirmatory kit. Results Three carbapenemase-producing Klebsiella pneumoniae strains, isolated from urine and lower limb wounds were found to belong to the high-risk sequence type ST147 and harbored the bla NDM −1 carbapenemase gene on an IncHI1B (pNDM-MAR) plasmid. Whole Genome Sequencing concurrently detected additional β-lactamase genes bla CTX-M − 15 , bla DHA −1 , bla OXA −1 , bla TEM −1 , bla SHV −67 and other resistance genes conferring resistance to aminoglycosides, phenicols, fosfomycin, macrolides, quinolones, sulphonamides, and tetracyclines. Plasmid analysis identified three plasmids, one (10,634 bp) which carried aadA1 and dfrA15 genes. Phylogenetic analysis revealed a high degree of sequence similarity among the three isolates, with a SNP distance of only 2, suggesting a recent clonal dissemination. Conclusions These findings represent the first report of the bla NDM −1 gene in Cameroon, identified in the high-risk clone K. pneumoniae ST147. A large-scale epidemiological investigation is warranted to determine the prevalence of this clone and to establish robust antimicrobial stewardship strategies to contain these multidrug-resistant pathogens. Molecular Epidemiology Carbapenemase-producing Klebsiella pneumoniae blaNDM−1 ST147 Whole-Genome Sequencing Carbapenem resistance Cameroon Figures Figure 1 Figure 2 Figure 3 Figure 4 1. BACKGROUND Antimicrobial resistance (AMR) is a major global public health threat, contributing to an estimated 4.95 million deaths in 2019, with a disproportionate burden in low- and middle-income countries [ 1 ]. The World Health Organization (WHO) has prioritized certain pathogens in response to the growing AMR crisis, including Gram-negative bacteria of the order Enterobacterales [ 2 ]. Klebsiella pneumoniae , a member of the Enterobacteriaceae family, is a leading cause of healthcare-associated infections such as urinary tract infections, pneumonia, bacteremia, and liver abscesses, accounting for approximately one-third of Gram-negative infections [ 3 , 4 ]. Klebsiella pneumoniae readily acquires antimicrobial resistance, particularly to β-lactam antibiotics [ 5 ]. Carbapenems, a class of β-lactams with a broad spectrum of activity and stability against many resistance mechanisms, are often reserved as last-line agents for treating multidrug-resistant (MDR) Gram-negative infections [ 6 ]. However, the emergence of carbapenemase-producing Enterobacteriaceae (CPE), which hydrolyze carbapenems, poses a severe clinical challenge due to limited therapeutic options [ 6 ]. The primary mechanism of carbapenem resistance in K. pneumoniae is the production of carbapenemases, enzymes commonly encoded on mobile genetic elements that facilitate their rapid dissemination [ 5 , 6 ]. The most clinically significant carbapenemases belong to Ambler classes A (e.g., KPC), B (e.g., NDM, VIM, IMP), and D (e.g., OXA-48-like) [ 7 ]. In Africa, NDM-type and OXA-48-type enzymes are the most prevalent, frequently identified in K. pneumoniae [ 8 ]. In Cameroon, few studies have phenotypically reported the presence of carbapenemase-producing K. pneumoniae (CP-Kp) [ 9 , 10 ]. However, data on molecular characterization and WGS are lacking, despite widespread antibiotic use and significant gaps in AMR surveillance. Herein, we report the first genomic characterization of CP-Kp clinical isolates in Yaoundé, Cameroon, revealing the emergence of the bla NDM −1 gene within the high-risk ST147 clone. 2. METHODS 2.1. Patients and Isolates The K. pneumoniae isolates were collected between January 2022 and September 2023 from consenting inpatients at the Yaounde Central Hospital (YCH) and relevant clinical data (e.g., collection date, specimen type, patient clinical status) were recorded. Bacterial identification was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, VITEK® MS, bioMérieux, France). Isolates were stored at -30°C in brain-heart infusion broth with 15% glycerol until further analysis. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar and results interpreted according to EUCAST recommendations [ 11 ]. Carbapenem-resistant isolates were phenotypically screened for carbapenemase production using CHROMagar™, mSuperCARBA™ (CHROMagar, France), and the KPC/MBL/OXA-48 combination disk test (Rosco Diagnostica, Denmark), following manufacturers' instructions. 2.2. DNA Extraction and Whole Genome Sequencing Genomic DNA was extracted using the DNeasy UltraClean Microbial Kit (Qiagen, Germany), eluted in 30 µL, and stored at -20°C. DNA concentration was quantified using a Qubit fluorometer with the dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, France). Libraries were prepared from 250 ng of DNA using the Illumina DNA Prep kit and sequenced on an Illumina MiSeq platform (Illumina, USA) in a 2×250 bp paired-end run. 2.3. Bioinformatic Analysis Raw readings quality was assessed using FastQC v0.23.2. De novo genome assembly was performed with SPAdes v3.15.5. Sequence types (STs), antimicrobial resistance genes, and virulence factors were identified using the PubMLST and Center for Genomic Epidemiology (CGE) platforms ( https://cge.cbs.dtu.dk/services/ ). Genomes were annotated with DFAST v1.2.0. A phylogenetic tree was constructed based on whole genome alignment of the three CP-Kp isolates and 25 African K. pneumoniae genomes from NCBI, using Seaview software and visualized with iTol ( https://itol.embl.de ). SNP distances were calculated using snp-dists v0.8.2, and genome representations were generated with Proksee ( https://proksee.ca/ ). 3. RESULTS 3.1. Patient Characteristics and Strain Isolation From a biobank of clinical Enterobacteriaceae, 30 MALDI-TOF-MS-confirmed K. pneumoniae isolates from various sources were investigated, including: urine (13%, 4/30), urinary catheters (7%, 2/30), diabetic foot ulcers (23%, 7/30), ear discharge (17%, 5/30), and wounds (40%, 12/30). Three CP-Kp isolates were identified and selected for further investigation, including: Isolate 033Kp was collected from the urine of a 69-year-old woman admitted at Yaounde Central Hospital (YCH) in January 2022 for a urinary tract infection (UTI). Her history included recurrent UTIs, hypertension, and prior episodes of self-medication and treatment at under-resourced facilities without susceptibility testing. The current infection was persistent and unresponsive to levofloxacin and sulfamethoxazole, leading to a presentation consistent with acute pyelonephritis. The patient was subsequently treated using pivmecillinam. Isolate 260Kp was obtained from a chronic foot wound in a 27-year-old woman inpatient (YCH) in November 2023. The wound originated from a motorcycle accident and was initially managed with unsupervised antibiotics and traditional medicinal plants. After surgical debridement and amoxicillin-clavulanate treatment failed, the wound worsened with purulent discharge and fever, prompting admission and initiation of fosfomycin treatment. Isolate 304Kp was collected from a heel wound swab from a 65-year-old woman presenting at YCH in July 2023. The patient had a long history of type 2 diabetes, hypertension, and chronic kidney failure. Despite multiple courses of broad-spectrum antibiotics, including imipenem, the wound failed to heal, and the patient deceased before culture results were available. 3.2. Antimicrobial Susceptibility Profiles The three CP-Kp isolates (033Kp, 260Kp, 304Kp) were multidrug-resistant (MDR), as they exhibited resistance to all tested antibiotics except pivmecillinam and fosfomycin. They were all resistant to a panel of eight β-lactams (ampicillin, amoxicillin-clavulanate, ticarcillin, piperacillin, cephalexin, cefixime, cefotaxime, cefuroxime) and to other antibiotic classes (Figs. 1 & 2 ). Screening for carbapenemase production indicated isolates 033Kp, 260Kp, and 304Kp as metallo-β-lactamases (MBLs) producers.. 3.3. Genomic Analysis of Antimicrobial Resistance Following WGS, bioinformatic analysis of the three clinical isolates predicted a comprehensive repertoire of resistance genes, including the key determinant carbapenemase gene bla NDM −1 (Table 1 ). Additional β-lactamase genes included the extended-spectrum β-lactamase (ESBL) genes bla CTX-M − 15 , bla SHV −67 , bla OXA −1 , and bla TEM −1B , as well as the AmpC gene bla DHA −1 . The 16S rRNA methyltransferase gene rmtB , conferring high-level aminoglycoside resistance, was also detected, alongside other aminoglycoside resistance genes ( aac(3)-IIa , aac(6')-Ib-cr , aadA1 ). Other genes conferring resistance to tetracyclines [ tet(G) ], macrolides [ mph(A) ], fosfomycin ( fosA ), trimethoprim ( dfrA15 ), phenicols ( catB3 , floR ), quinolones ( oqxA, oqxB, qnrB4 ), and sulfonamides ( sul1 ) were detected. Table 1 Patient demographics, sequence types, and resistance genes in K. pneumoniae isolates Isolate ID Demographics Sample type K. pneumoniae Phenotype ARGs Plasmid Incompatibility Group MLST Sex Age (Years) β-lactam resistance genes Others ARGs 033Kp Female 69 urine MBL (metallo-beta-lactamase) bla TEM −1B , bla CTX-M − 15 , bla DHA −1 , bla NDM −1 , bla OXA −1 , bla SHV −67 . aac(3)-IIa, aac(6')-Ib-cr, aadA, rmtB , tet(G) , mph(A) , fosA, dfrA15 , catB3, floR2, oqxA , oqxB, qnrB4, sul1 IncHI1B, IncFIB(K), IncR ST147 260Kp Female 27 Wound swab 304Kp Female 65 Wound swab ARGs: Antibiotic resistance genes; MLST: Multilocus Sequence Typing. 3.4. Virulence Factors and Genotyping Virulome analysis identified genes associated with adherence (type 1 and type 3 fimbriae : fim and mrk clusters), biofilm formation, capsule synthesis, siderophore-mediated iron acquisition (enterobactin: ent , fep clusters), and efflux pumps. The hypermucoviscosity regulator gene rmpA was absent. Multilocus sequence typing (MLST) assigned all three isolates to the sequence type ST147. 3.5. Plasmid and Mobile Genetic Element Analysis Three distinct plasmids were identified, including plasmid 1 (33,991 bp) showing 100% coverage and 99.95% identity to K. pneumoniae plasmid pSCKLB555-2 (CP043934), plasmid 2 (10,634 bp) exhibiting 99% identity to plasmid pCRKP-1 (CP118902) and carrying the aadA1 and dfrA15 resistance genes (Fig. 3 ), and plasmid 3 (7,098 bp) that showed no significant matches in public databases, suggesting a novel plasmid variant. All isolates carried three plasmid replicons simultaneously: IncHI1B, IncFIB(K), and IncR. The bla NDM −1 gene was associated with the IncHI1B (pNDM-MAR) replicon. 3.6. Clonal Relationship and Clinical Correlation The patients' clinical outcomes were likely influenced by the multidrug-resistance (MDR) profile of the infecting strains, prior empirical antibiotic use, and underlying comorbidities in a resource-limited setting. These factors may have facilitated the emergence and persistence of the high-risk ST147 clone in the actual AMR hotspot. Genomic comparison confirmed a close relationship between the three isolates, which clustered together in the phylogenetic analysis. This was further supported by a minimal SNP distance of 2, indicating a recent clonal expansion and likely common source or recent transmission event (Fig. 4 , Table 1 ). 4. DISCUSSION Infections caused by carbapenemase-producing K. pneumoniae (CP-Kp) represent a critical threat to global health, a concern underscored by its WHO priority pathogen status [ 12 ]. This study provides the first genomic evidence of the bla NDM −1 gene in Cameroon, identified within the high-risk global clone ST147. The CP-Kp prevalence in this study was 10% (3/30), which is lower than the 25% (26/104) and 19.3% (6/31) reported in previous studies in Cameroon [ 9 , 10 ]. This discrepancy may be attributed to differences in detection methods (phenotypic vs. genomic), geographic variation, or local antibiotic stewardship practices. Nonetheless, our findings confirm the circulation of CP-Kp and highlight the necessity of incorporating molecular surveillance into AMR programs in Cameroon. The convergence of the bla NDM −1 gene with the ST147 clone is a major public health concern. Indeed, ST147 is a well-documented, high-risk clone responsible for nosocomial outbreaks and is increasingly reported in sub-Saharan Africa [ 13 – 15 ]. While K. pneumoniae clones ST11, ST15, and ST307 have been previously detected in Cameroon [ 16 ], this is the first report of ST147. Its emergence could be due to regional transmission or local selection pressure from uncontrolled antibiotic use. The extensive resistome, including genes conferring resistance to nearly all major antibiotic classes, aligns with the MDR phenotype observed. The association of bla NDM −1 with an IncHI1B plasmid is particularly noteworthy, as this plasmid type is known for its stability and ability to carry multiple resistance determinants, facilitating the dissemination and persistence of MDR strains [ 12 , 17 ]. The identification of a potentially novel plasmid (plasmid 3— 7,098 bp) in Yaounde underscores the ongoing evolution of mobile genetic elements in this region. The observed minimal genetic diversity (2 SNPs) among the isolates might suggest recent clonal dissemination. While the patients had no documented epidemiological link, the findings point to a possible common reservoir or healthcare-associated transmission chain, warranting immediate investigation. 5. CONCLUSION This pilot study reports the first identification of bla NDM -1 -harbouring K. pneumoniae ST147 in Cameroon. The genomic data reveal a clonal cluster of extensively drug-resistant pathogens, highlighting a serious and emerging threat. Our findings necessitate an urgent, large-scale epidemiological study to determine the true prevalence and spread of this high-risk clone. Abbreviation ST: Sequence Type; MLST: Multi-Locus Sequence Type; AMR: Antimicrobial Resistance; WGS: Whole Genome Sequencing; MDR: MultuDrug resistance; CPE: Carbapenemase-Producing Enterobacteriaceae; MALDI-TOF-MS: Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry; YCH: Yaounde Central Hospital; EUCAST: European Committee on Antimicrobial Susceptibility Testing; UTI: Urinary-Tract Infection, SNP: Single Nucleotide Polymorphism; pCRKP-1: plasmid Carbapenem-Resistant Klebsiella pneumoniae- 1; pNDM-MAR: plasmid New Delhi Metallo-beta-lactamase resistance gene-Morroco. Declarations Ethics approval and consent to participate Ethical approval for this study was obtained from the Cameroon National Ethics Committee for Human Health Research (Comité National d’Éthique de la Recherche pour la Santé Humaine, CNERSH ), Ministry of Public Health, Cameroon, under Reference No. 2024/01/684/CE/CNERSH/SP. Consent for publication The patient's written and signed consent was obtained prior to participation and personal information was removed from the manuscript. Availability of data and materials The data generated are presented in this manuscript. Patients’ personal data are confidential and cannot be disclosed. Competing interests There is no competing interest to declare. Funding No specific funding was received for this study. Authors contribution F.F.B. and T.N.K. conceptualized the study; R.T.F., M.M., C.P., C. M. and A.P. performed the experiments; M.M. and A.P. performed the bioinformatics analyses; R.T.F. and M.M. visualized the data; F.F.B., C.D-R., P.F.M., and J-P.L. supervised the study; R.T.F. and M.M. wrote the original manuscript draft; M.M., A.P., T.N.K, J-P. L., and F.F.B. edited and reviewed the manuscript; All authors reviewed and approved the final manuscript. Acknowledgement We are grateful to the laboratory of Microbiology and Hospital Hygiene of the Nîmes University Hospital, as well as the Bacterial Virulence and Chronic Infections Unit teams, for their invaluable administrative and technical support to this study. References Medina-Pizzali ML, Venkatesh A, Riveros M, Cuicapuza D, Salmon-Mulanovich G, Mäusezahl D et al (2022) Whole-Genome Characterisation of ESBL-Producing E. coli Isolated from Drinking Water and Dog Faeces from Rural Andean Households in Peru. 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Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/EUCAST_SOPs/2025/EUCAST_SOP_9.4_Disk_diffusion_breakpoints_and_QC_final_250710.pdf Marais G, Moodley C, Claassen-Weitz S, Patel F, Prentice E, Tootla H et al (2024) Antimicrobial Resistance Carbapenem-resistant Klebsiella pneumoniae among hospitalized patients in Cape Town, South Africa: molecular epidemiology and characterization. JAC Antimicrob Resist 6(2):dlae050 Ofosu-Appiah F, Acquah EE, Mohammed J, Sakyi Addo C, Agbodzi B, Ofosu DAS et al (2024) Klebsiella pneumoniae ST147 harboring blaNDM-1, multidrug resistance and hypervirulence plasmids. Microbiol Spectr 12(3):e0301723 Dinda V, Kimang'a AN, Kariuki D, Sifuna AW, O'Brien TJ, Welch M et al (2024) Whole genome sequencing and genotyping Klebsiella pneumoniae multidrug-resistant hospital isolates from Western Kenya. 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09:59:12","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74219,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8231079/v1/fae1bf37aeaa7c81270455c9.html"},{"id":97234091,"identity":"45fabbcc-fe88-41e4-aec8-b394b6406b16","added_by":"auto","created_at":"2025-12-02 09:59:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":107668,"visible":true,"origin":"","legend":"\u003cp\u003eSusceptibility profile of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates to β-lactams\u003c/p\u003e\n\u003cp\u003eAMP = Ampicillin; AMC= Amoxicillin + clavulanic acid; TIC = Ticarcillin; TCC = Ticarcillin + clavulanic acid; PIL = Piperacillin; CXN = Cefalexin; FIX = Cefixime; FOX = Cefoxitine; TEM = Temocillin; COX = Cefotaxime, CZD = Ceftadizime; FEP = Cefepime; ETP = Ertapenem; IPM = Imipenem; MEM = Meropenem; MEC = Mecillinam; TPZ = Piperacillin + tazobactam; CXM = Cefuroxime; CAZ / AVI = Ceftazidime + avibactam; ATM = Aztreonam.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8231079/v1/8f51abcdcba25463a2023738.png"},{"id":97234090,"identity":"886213bb-bf4f-4b48-9a85-2338c4de4308","added_by":"auto","created_at":"2025-12-02 09:59:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87797,"visible":true,"origin":"","legend":"\u003cp\u003eSusceptibility profile of \u003cem\u003eK. pneumoniae \u003c/em\u003eisolates to other antibiotics\u003c/p\u003e\n\u003cp\u003eNFE = Nitrofurantoine; NTM = Netilmicine; AKN = Amikacin; GMN = Gentamicin; TMN = Tobramycin; NAL = nalidixic acid; OFX = Ofloxacin; NXN = Norfloxacin; CIP = Ciprofloxacin; FOS = Fosfomycin; SXT = Trimethoprim + sulfamthoxazole.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8231079/v1/c4bebc2b43209ccf1e717b4c.png"},{"id":97250690,"identity":"c993887e-f72e-4004-8644-4071329c6c7e","added_by":"auto","created_at":"2025-12-02 13:15:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318080,"visible":true,"origin":"","legend":"\u003cp\u003ePlasmid analysis and annotation. A) (33,991 bp) exhibited 100% sequence coverage and 99.95 identity with \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003eplasmid pSCKLB555-2 (GenBank accession number: CP043934). B) plasmid-2 (10,634 bp) exhibited 99% sequence identity with CRKP plasmid pCRKP-1 (GenBank: CP118902). C) Plasmid-3 (7,098 bp) not matched with any sequence available on the GenBank database.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8231079/v1/5c1da93731ccb3f49462fbbb.png"},{"id":97251168,"identity":"f8ca4c98-32d2-4b58-a796-7487d9713148","added_by":"auto","created_at":"2025-12-02 13:16:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":283053,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on whole genome alignment of the 033Kp, 260Kp, and 304Kp \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates and other African isolates available on the NCBI GenBank database. Despite the different origins of the three clinical isolates, they portrayed high genome similarity to the environmental isolate CPCKP10.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8231079/v1/1f183c2993c816ec49d8c98d.png"},{"id":97665235,"identity":"2833e280-685d-4270-a9cc-d2a7914dbaa7","added_by":"auto","created_at":"2025-12-08 09:17:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1473893,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8231079/v1/39d47b85-4cb9-415d-ba26-bbe02c8b33d6.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eFirst Identification of \u003c/strong\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003ebla\u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cstrong\u003eNDM\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e-1 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003ein High-Risk \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKlebsiella pneumoniae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eST147 Clone in Cameroon: A Genomic Insight\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. BACKGROUND","content":"\u003cp\u003eAntimicrobial resistance (AMR) is a major global public health threat, contributing to an estimated 4.95\u0026nbsp;million deaths in 2019, with a disproportionate burden in low- and middle-income countries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The World Health Organization (WHO) has prioritized certain pathogens in response to the growing AMR crisis, including Gram-negative bacteria of the order Enterobacterales [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, a member of the Enterobacteriaceae family, is a leading cause of healthcare-associated infections such as urinary tract infections, pneumonia, bacteremia, and liver abscesses, accounting for approximately one-third of Gram-negative infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e readily acquires antimicrobial resistance, particularly to β-lactam antibiotics [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Carbapenems, a class of β-lactams with a broad spectrum of activity and stability against many resistance mechanisms, are often reserved as last-line agents for treating multidrug-resistant (MDR) Gram-negative infections [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the emergence of carbapenemase-producing Enterobacteriaceae (CPE), which hydrolyze carbapenems, poses a severe clinical challenge due to limited therapeutic options [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The primary mechanism of carbapenem resistance in \u003cem\u003eK. pneumoniae\u003c/em\u003e is the production of carbapenemases, enzymes commonly encoded on mobile genetic elements that facilitate their rapid dissemination [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The most clinically significant carbapenemases belong to Ambler classes A (e.g., KPC), B (e.g., NDM, VIM, IMP), and D (e.g., OXA-48-like) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In Africa, NDM-type and OXA-48-type enzymes are the most prevalent, frequently identified in \u003cem\u003eK. pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In Cameroon, few studies have phenotypically reported the presence of carbapenemase-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e (CP-Kp) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, data on molecular characterization and WGS are lacking, despite widespread antibiotic use and significant gaps in AMR surveillance. Herein, we report the first genomic characterization of CP-Kp clinical isolates in Yaound\u0026eacute;, Cameroon, revealing the emergence of the \u003csub\u003ebla\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e gene within the high-risk ST147 clone.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Patients and Isolates\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were collected between January 2022 and September 2023 from consenting inpatients at the Yaounde Central Hospital (YCH) and relevant clinical data (e.g., collection date, specimen type, patient clinical status) were recorded. Bacterial identification was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, VITEK\u0026reg; MS, bioM\u0026eacute;rieux, France). Isolates were stored at -30\u0026deg;C in brain-heart infusion broth with 15% glycerol until further analysis.\u003c/p\u003e\u003cp\u003eAntibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar and results interpreted according to EUCAST recommendations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Carbapenem-resistant isolates were phenotypically screened for carbapenemase production using CHROMagar\u0026trade;, mSuperCARBA\u0026trade; (CHROMagar, France), and the KPC/MBL/OXA-48 combination disk test (Rosco Diagnostica, Denmark), following manufacturers' instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. DNA Extraction and Whole Genome Sequencing\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted using the DNeasy UltraClean Microbial Kit (Qiagen, Germany), eluted in 30 \u0026micro;L, and stored at -20\u0026deg;C. DNA concentration was quantified using a Qubit fluorometer with the dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, France). Libraries were prepared from 250 ng of DNA using the Illumina DNA Prep kit and sequenced on an Illumina MiSeq platform (Illumina, USA) in a 2\u0026times;250 bp paired-end run.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Bioinformatic Analysis\u003c/h2\u003e\u003cp\u003eRaw readings quality was assessed using FastQC v0.23.2. De novo genome assembly was performed with SPAdes v3.15.5. Sequence types (STs), antimicrobial resistance genes, and virulence factors were identified using the PubMLST and Center for Genomic Epidemiology (CGE) platforms (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.cbs.dtu.dk/services/\u003c/span\u003e\u003cspan address=\"https://cge.cbs.dtu.dk/services/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Genomes were annotated with DFAST v1.2.0. A phylogenetic tree was constructed based on whole genome alignment of the three CP-Kp isolates and 25 African \u003cem\u003eK. pneumoniae\u003c/em\u003e genomes from NCBI, using Seaview software and visualized with iTol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de\u003c/span\u003e\u003cspan address=\"https://itol.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). SNP distances were calculated using snp-dists v0.8.2, and genome representations were generated with Proksee (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca/\u003c/span\u003e\u003cspan address=\"https://proksee.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Patient Characteristics and Strain Isolation\u003c/h2\u003e\n \u003cp\u003eFrom a biobank of clinical Enterobacteriaceae, 30 MALDI-TOF-MS-confirmed \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from various sources were investigated, including: urine (13%, 4/30), urinary catheters (7%, 2/30), diabetic foot ulcers (23%, 7/30), ear discharge (17%, 5/30), and wounds (40%, 12/30). Three CP-Kp isolates were identified and selected for further investigation, including:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate 033Kp\u003c/strong\u003e was collected from the urine of a 69-year-old woman admitted at Yaounde Central Hospital (YCH) in January 2022 for a urinary tract infection (UTI). Her history included recurrent UTIs, hypertension, and prior episodes of self-medication and treatment at under-resourced facilities without susceptibility testing. The current infection was persistent and unresponsive to levofloxacin and sulfamethoxazole, leading to a presentation consistent with acute pyelonephritis. The patient was subsequently treated using pivmecillinam.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate 260Kp\u003c/strong\u003e was obtained from a chronic foot wound in a 27-year-old woman inpatient (YCH) in November 2023. The wound originated from a motorcycle accident and was initially managed with unsupervised antibiotics and traditional medicinal plants. After surgical debridement and amoxicillin-clavulanate treatment failed, the wound worsened with purulent discharge and fever, prompting admission and initiation of fosfomycin treatment.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate 304Kp\u003c/strong\u003e was collected from a heel wound swab from a 65-year-old woman presenting at YCH in July 2023. The patient had a long history of type 2 diabetes, hypertension, and chronic kidney failure. Despite multiple courses of broad-spectrum antibiotics, including imipenem, the wound failed to heal, and the patient deceased before culture results were available.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Antimicrobial Susceptibility Profiles\u003c/h2\u003e\n \u003cp\u003eThe three CP-Kp isolates (033Kp, 260Kp, 304Kp) were multidrug-resistant (MDR), as they exhibited resistance to all tested antibiotics except pivmecillinam and fosfomycin. They were all resistant to a panel of eight \u0026beta;-lactams (ampicillin, amoxicillin-clavulanate, ticarcillin, piperacillin, cephalexin, cefixime, cefotaxime, cefuroxime) and to other antibiotic classes (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Screening for carbapenemase production indicated isolates 033Kp, 260Kp, and 304Kp as metallo-\u0026beta;-lactamases (MBLs) producers..\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Genomic Analysis of Antimicrobial Resistance\u003c/h2\u003e\n \u003cp\u003eFollowing WGS, bioinformatic analysis of the three clinical isolates predicted a comprehensive repertoire of resistance genes, including the key determinant carbapenemase gene \u003csub\u003ebla\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Additional \u0026beta;-lactamase genes included the extended-spectrum \u0026beta;-lactamase (ESBL) genes \u003csub\u003ebla\u003c/sub\u003eCTX-M\u003csub\u003e\u0026minus;\u0026thinsp;15\u003c/sub\u003e, \u003csub\u003ebla\u003c/sub\u003eSHV\u003csub\u003e\u0026minus;67\u003c/sub\u003e, \u003csub\u003ebla\u003c/sub\u003eOXA\u003csub\u003e\u0026minus;1\u003c/sub\u003e, and \u003csub\u003ebla\u003c/sub\u003eTEM\u003csub\u003e\u0026minus;1B\u003c/sub\u003e, as well as the \u003cem\u003eAmpC\u003c/em\u003e gene \u003csub\u003ebla\u003c/sub\u003eDHA\u003csub\u003e\u0026minus;1\u003c/sub\u003e. The 16S rRNA methyltransferase gene \u003cem\u003ermtB\u003c/em\u003e, conferring high-level aminoglycoside resistance, was also detected, alongside other aminoglycoside resistance genes (\u003cem\u003eaac(3)-IIa\u003c/em\u003e, \u003cem\u003eaac(6\u0026apos;)-Ib-cr\u003c/em\u003e, \u003cem\u003eaadA1\u003c/em\u003e). Other genes conferring resistance to tetracyclines [\u003cem\u003etet(G)\u003c/em\u003e], macrolides [\u003cem\u003emph(A)\u003c/em\u003e], fosfomycin (\u003cem\u003efosA\u003c/em\u003e), trimethoprim (\u003cem\u003edfrA15\u003c/em\u003e), phenicols (\u003cem\u003ecatB3\u003c/em\u003e, \u003cem\u003efloR\u003c/em\u003e), quinolones (\u003cem\u003eoqxA, oqxB, qnrB4\u003c/em\u003e), and sulfonamides (\u003cem\u003esul1\u003c/em\u003e) were detected.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePatient demographics, sequence types, and resistance genes in K. pneumoniae isolates\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eIsolate ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDemographics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSample type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e Phenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eARGs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePlasmid\u003c/p\u003e\n \u003cp\u003eIncompatibility Group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMLST\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (Years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026beta;-lactam resistance genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOthers ARGs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e033Kp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eurine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eMBL (metallo-beta-lactamase)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eTEM\u003csub\u003e\u0026minus;1B\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eCTX-M\u003csub\u003e\u0026minus;\u0026thinsp;15\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eDHA\u003csub\u003e\u0026minus;1\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eOXA\u003csub\u003e\u0026minus;1\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eSHV\u003csub\u003e\u0026minus;67\u003c/sub\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eaac(3)-IIa, aac(6\u0026apos;)-Ib-cr, aadA, rmtB\u003c/em\u003e, \u003cem\u003etet(G)\u003c/em\u003e, \u003cem\u003emph(A)\u003c/em\u003e, \u003cem\u003efosA, dfrA15\u003c/em\u003e, \u003cem\u003ecatB3, floR2, oqxA\u003c/em\u003e, \u003cem\u003eoqxB, qnrB4, sul1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eIncHI1B,\u003c/p\u003e\n \u003cp\u003eIncFIB(K),\u003c/p\u003e\n \u003cp\u003eIncR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eST147\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e260Kp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWound swab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e304Kp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWound swab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eARGs: Antibiotic resistance genes; MLST: Multilocus Sequence Typing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Virulence Factors and Genotyping\u003c/h2\u003e\n \u003cp\u003eVirulome analysis identified genes associated with adherence (type 1 and type 3 \u003cem\u003efimbriae\u003c/em\u003e: \u003cem\u003efim\u003c/em\u003e and \u003cem\u003emrk\u003c/em\u003e clusters), biofilm formation, capsule synthesis, siderophore-mediated iron acquisition (enterobactin: \u003cem\u003eent\u003c/em\u003e, \u003cem\u003efep\u003c/em\u003e clusters), and efflux pumps. The hypermucoviscosity regulator gene \u003cem\u003ermpA\u003c/em\u003e was absent. Multilocus sequence typing (MLST) assigned all three isolates to the sequence type ST147.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Plasmid and Mobile Genetic Element Analysis\u003c/h2\u003e\n \u003cp\u003eThree distinct plasmids were identified, including plasmid 1 (33,991 bp) showing 100% coverage and 99.95% identity to \u003cem\u003eK. pneumoniae\u003c/em\u003e plasmid pSCKLB555-2 (CP043934), plasmid 2 (10,634 bp) exhibiting 99% identity to plasmid pCRKP-1 (CP118902) and carrying the \u003cem\u003eaadA1\u003c/em\u003e and \u003cem\u003edfrA15\u003c/em\u003e resistance genes (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), and plasmid 3 (7,098 bp) that showed no significant matches in public databases, suggesting a novel plasmid variant. All isolates carried three plasmid replicons simultaneously: IncHI1B, IncFIB(K), and IncR. The \u003csub\u003ebla\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e gene was associated with the IncHI1B (pNDM-MAR) replicon.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. Clonal Relationship and Clinical Correlation\u003c/h2\u003e\n \u003cp\u003eThe patients\u0026apos; clinical outcomes were likely influenced by the multidrug-resistance (MDR) profile of the infecting strains, prior empirical antibiotic use, and underlying comorbidities in a resource-limited setting. These factors may have facilitated the emergence and persistence of the high-risk ST147 clone in the actual AMR hotspot. Genomic comparison confirmed a close relationship between the three isolates, which clustered together in the phylogenetic analysis. This was further supported by a minimal SNP distance of 2, indicating a recent clonal expansion and likely common source or recent transmission event (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eInfections caused by carbapenemase-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e (CP-Kp) represent a critical threat to global health, a concern underscored by its WHO priority pathogen status [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This study provides the first genomic evidence of the \u003csub\u003ebla\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e gene in Cameroon, identified within the high-risk global clone ST147.\u003c/p\u003e\u003cp\u003eThe CP-Kp prevalence in this study was 10% (3/30), which is lower than the 25% (26/104) and 19.3% (6/31) reported in previous studies in Cameroon [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This discrepancy may be attributed to differences in detection methods (phenotypic vs. genomic), geographic variation, or local antibiotic stewardship practices. Nonetheless, our findings confirm the circulation of CP-Kp and highlight the necessity of incorporating molecular surveillance into AMR programs in Cameroon.\u003c/p\u003e\u003cp\u003eThe convergence of the \u003csub\u003ebla\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e gene with the ST147 clone is a major public health concern. Indeed, ST147 is a well-documented, high-risk clone responsible for nosocomial outbreaks and is increasingly reported in sub-Saharan Africa [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While \u003cem\u003eK. pneumoniae\u003c/em\u003e clones ST11, ST15, and ST307 have been previously detected in Cameroon [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], this is the first report of ST147. Its emergence could be due to regional transmission or local selection pressure from uncontrolled antibiotic use.\u003c/p\u003e\u003cp\u003eThe extensive resistome, including genes conferring resistance to nearly all major antibiotic classes, aligns with the MDR phenotype observed. The association of \u003csub\u003ebla\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e with an IncHI1B plasmid is particularly noteworthy, as this plasmid type is known for its stability and ability to carry multiple resistance determinants, facilitating the dissemination and persistence of MDR strains [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The identification of a potentially novel plasmid (plasmid 3\u0026mdash; 7,098 bp) in Yaounde underscores the ongoing evolution of mobile genetic elements in this region.\u003c/p\u003e\u003cp\u003eThe observed minimal genetic diversity (2 SNPs) among the isolates might suggest recent clonal dissemination. While the patients had no documented epidemiological link, the findings point to a possible common reservoir or healthcare-associated transmission chain, warranting immediate investigation.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThis pilot study reports the first identification of \u003cem\u003e\u003csub\u003ebla\u003c/sub\u003e\u003c/em\u003eNDM\u003csub\u003e-1\u003c/sub\u003e-harbouring \u003cem\u003eK. pneumoniae\u003c/em\u003e ST147 in Cameroon. The genomic data reveal a clonal cluster of extensively drug-resistant pathogens, highlighting a serious and emerging threat. Our findings necessitate an urgent, large-scale epidemiological study to determine the true prevalence and spread of this high-risk clone.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviation","content":"\u003cp\u003eST: Sequence Type; MLST: Multi-Locus Sequence Type; AMR: Antimicrobial Resistance; WGS: Whole Genome Sequencing; MDR: MultuDrug resistance; CPE: Carbapenemase-Producing Enterobacteriaceae; MALDI-TOF-MS:\u0026nbsp;Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry; YCH: Yaounde Central Hospital; EUCAST: European Committee on Antimicrobial Susceptibility Testing; UTI: Urinary-Tract Infection, SNP: Single Nucleotide Polymorphism; pCRKP-1: plasmid Carbapenem-Resistant \u003cem\u003eKlebsiella pneumoniae-\u003c/em\u003e 1; pNDM-MAR: plasmid New Delhi Metallo-beta-lactamase resistance gene-Morroco.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was obtained from the Cameroon National Ethics Committee for Human Health Research \u003cem\u003e(Comité National d’Éthique de la Recherche pour la Santé Humaine, CNERSH\u003c/em\u003e), Ministry of Public Health, Cameroon, under Reference No. 2024/01/684/CE/CNERSH/SP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient's written and signed consent was obtained prior to participation and personal information was removed from the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated are presented in this manuscript. Patients’ personal data are confidential and cannot be disclosed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no competing interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo specific funding was received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors contribution\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.F.B. and T.N.K. conceptualized the study; R.T.F., M.M., C.P., C. M. and A.P. performed the experiments; M.M. and A.P. performed the bioinformatics analyses; R.T.F. and M.M. visualized the data; F.F.B., C.D-R., P.F.M., and J-P.L. supervised the study; R.T.F. and M.M. wrote the original manuscript draft; M.M., A.P., T.N.K, J-P. L., and F.F.B. edited and reviewed the manuscript; All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the laboratory of Microbiology and Hospital Hygiene of the Nîmes University Hospital, as well as the Bacterial Virulence and Chronic Infections Unit teams, for their invaluable administrative and technical support to this study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMedina-Pizzali ML, Venkatesh A, Riveros M, Cuicapuza D, Salmon-Mulanovich G, M\u0026auml;usezahl D et al (2022) Whole-Genome Characterisation of ESBL-Producing E. coli Isolated from Drinking Water and Dog Faeces from Rural Andean Households in Peru. Antibiot (Basel) 11(5):692\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSati H, Carrara E, Savoldi A, Hansen P, Garlasco J, Campagnaro E et al (2025) The WHO Bacterial Priority Pathogens List 2024: a prioritisation study to guide research, development, and public health strategies against antimicrobial resistance. Lancet Infect Dis 25(9):1033\u0026ndash;1043\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRamsamy Y, Mlisana KP, Allam M, Ismail A, Singh R, Amoako DG et al (2018) Whole-Genome Sequence of a Novel Sequence Type 3136 Carbapenem-Resistant Klebsiella pneumoniae Strain Isolated from a Hospitalized Patient in Durban, South Africa. Microbiol Resour Announc 7(23):e01300\u0026ndash;e01318\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEnany S, Zakeer S, Diab AA, Bakry U, Sayed AA (2022) Whole genome sequencing of Klebsiella pneumoniae clinical isolates sequence type 627 isolated from Egyptian patients. 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Am J Trop Med Hyg 108(2):268\u0026ndash;274\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbderrahim A, Djahmi N, Loucif L, Nedjai S, Chelaghma W, Gameci-Kirane D et al (2022) Dissemination of OXA-48- and NDM-1-Producing Enterobacterales Isolates in an Algerian Hospital. Antibiot (Basel) 11(6):750\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEbongue C, Simo G, Nda Mefo\u0026rsquo;o J, Ngondi G, Mengue E, Ngaba G et al (2021) Detection of the Production of Klebsiella Pneumoniae Carbapenemase, New Delhi Metallo-Beta-Lactamase and Oxacillinase-48-Type Carbapenemases by Gram-Negative Bacilli in Resource-Limited Setting. Adv Microbiol 11:579\u0026ndash;590\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDjuikoue C, Djoulako P, Wouambo R, Tomi C, Kiyang C, Tchitchoua M et al (2023) Phenotypic Characterization of Carbapenemase-Producing Enterobacteriaceae Strains in a Referral Teaching Hospital in Yaound\u0026eacute;, Cameroon. Open J Med Microbiol 13:52\u0026ndash;67\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEuropean Committee on Antimicrobial Susceptibility Testing (EUCAST) Procedure for establishing zone diameter breakpoints and quality control criteria for new antimicrobial agents. EUCAST SOP 9.4. 2025 [cited 2025 Mar 26]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/EUCAST_SOPs/2025/EUCAST_SOP_9.4_Disk_diffusion_breakpoints_and_QC_final_250710.pdf\u003c/span\u003e\u003cspan address=\"https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/EUCAST_SOPs/2025/EUCAST_SOP_9.4_Disk_diffusion_breakpoints_and_QC_final_250710.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarais G, Moodley C, Claassen-Weitz S, Patel F, Prentice E, Tootla H et al (2024) Antimicrobial Resistance Carbapenem-resistant Klebsiella pneumoniae among hospitalized patients in Cape Town, South Africa: molecular epidemiology and characterization. JAC Antimicrob Resist 6(2):dlae050\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOfosu-Appiah F, Acquah EE, Mohammed J, Sakyi Addo C, Agbodzi B, Ofosu DAS et al (2024) Klebsiella pneumoniae ST147 harboring blaNDM-1, multidrug resistance and hypervirulence plasmids. Microbiol Spectr 12(3):e0301723\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDinda V, Kimang'a AN, Kariuki D, Sifuna AW, O'Brien TJ, Welch M et al (2024) Whole genome sequencing and genotyping Klebsiella pneumoniae multidrug-resistant hospital isolates from Western Kenya. Access Microbiol 6(1):000667v4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDikoumba AC, Onanga R, Mangouka LG, Boundenga L, Ngoungou EB, Godreuil S (2023) Molecular epidemiology of antimicrobial resistance in central africa: A systematic review. Access Microbiol 5(8):acmi000556v5\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFounou LL, Founou RC, Allam M, Ismail A, Djoko CF, Essack SY (2018) Genome Sequencing of Extended-Spectrum β-Lactamase (ESBL)-Producing Klebsiella pneumoniae Isolated from Pigs and Abattoir Workers in Cameroon. Front Microbiol 9:188\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMadni O, Amoako DG, Abia ALK, Rout J, Essack SY (2021) Genomic Investigation of Carbapenem-Resistant Klebsiella pneumoniae Colonization in an Intensive Care Unit in South Africa. Genes (Basel) 12(7):951\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Yaounde 1","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carbapenemase-producing Klebsiella pneumoniae, blaNDM−1, ST147, Whole-Genome Sequencing, Carbapenem resistance, Cameroon","lastPublishedDoi":"10.21203/rs.3.rs-8231079/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8231079/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe emergence and spread of carbapenemase-producing \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (CP-Kp) represent a serious clinical challenge due to limited treatment options. This pilot study aimed to track the evidence of CP-Kp genes in Yaounde, Cameroon where antimicrobial resistance (AMR) surveillance data remain scarce.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe resistance profile of clinical \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e isolated in Yaound\u0026eacute;, Cameroon was investigated using routine Antibiotic Susceptibility Testing (AST) and Whole Genome Sequencing (WGS). Carbapenemase production was phenotypically confirmed using the selective chromogenic medium designed for the detection of carbapenemase-producing Enterobacterales (CHROMagar\u0026trade; Modified SuperCARBA\u0026trade;), and the \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemase (KPC)/Metallo-β-lactamase (MBL)/Oxacillinase-48 (OXA-48) confirmatory kit.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThree carbapenemase-producing \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains, isolated from urine and lower limb wounds were found to belong to the high-risk sequence type ST147 and harbored the \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e carbapenemase gene on an IncHI1B (pNDM-MAR) plasmid. Whole Genome Sequencing concurrently detected additional β-lactamase genes \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eCTX-M\u003csub\u003e\u0026minus;\u0026thinsp;15\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eDHA\u003csub\u003e\u0026minus;1\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eOXA\u003csub\u003e\u0026minus;1\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eTEM\u003csub\u003e\u0026minus;1\u003c/sub\u003e, \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eSHV\u003csub\u003e\u0026minus;67\u003c/sub\u003e and other resistance genes conferring resistance to aminoglycosides, phenicols, fosfomycin, macrolides, quinolones, sulphonamides, and tetracyclines. Plasmid analysis identified three plasmids, one (10,634 bp) which carried \u003cem\u003eaadA1\u003c/em\u003e and \u003cem\u003edfrA15\u003c/em\u003e genes. Phylogenetic analysis revealed a high degree of sequence similarity among the three isolates, with a SNP distance of only 2, suggesting a recent clonal dissemination.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThese findings represent the first report of the \u003csub\u003e\u003cem\u003ebla\u003c/em\u003e\u003c/sub\u003eNDM\u003csub\u003e\u0026minus;1\u003c/sub\u003e gene in Cameroon, identified in the high-risk clone \u003cem\u003eK. pneumoniae\u003c/em\u003e ST147. A large-scale epidemiological investigation is warranted to determine the prevalence of this clone and to establish robust antimicrobial stewardship strategies to contain these multidrug-resistant pathogens.\u003c/p\u003e","manuscriptTitle":"First Identification of blaNDM-1 in High-Risk Klebsiella pneumoniae ST147 Clone in Cameroon: A Genomic Insight","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 09:59:07","doi":"10.21203/rs.3.rs-8231079/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9e41669-e025-4148-bef9-e1289cf20f97","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58779351,"name":"Molecular Epidemiology"}],"tags":[],"updatedAt":"2025-12-02T09:59:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 09:59:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8231079","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8231079","identity":"rs-8231079","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}