Emergence of NDM-type metallo-β-lactamase and ArmA 16S rRNA methylase co-producing Morganella morganii in Myanmar

Emergence of NDM-type metallo-β-lactamase and ArmA 16S rRNA methylase co-producing Morganella morganii in Myanmar | 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 Emergence of NDM-type metallo-β-lactamase and ArmA 16S rRNA methylase co-producing Morganella morganii in Myanmar Maiko Kirikae, Satomi Takei, Yuki Uehara, Satoshi Oshiro, Nang Sarm Hom, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7405418/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2026 Read the published version in BMC Infectious Diseases → Version 1 posted 14 You are reading this latest preprint version Abstract Background Morganella morganii is intrinsically resistant to a wide range of antibiotics and is increasingly being reported worldwide. Methods A total of five M. morganii isolates were obtained from five patients at three hospitals in Myanmar between Sept. 2023 and Oct. 2024. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. Genomic DNA was extracted and sequenced on the next generation sequencer. Drug-resistant factors were determined and the genetic environments surrounding carbapenemase and 16S rRNA methylase genes were analyzed. A phylogenetic tree was constructed using forty-six M. morganii subsp. morganii genome sequences, including the five Myanmar. Results One isolate harbored both bla NDM−1 and armA . This isolate was resistant to imipenem (MIC: 16 ug/ml) but susceptible to meropenem (MIC: 1 ug/ml). Whole genome sequencing revealed that bla NDM−1 and armA were located in close proximity on the chromosome of M. morganii strain YGH122, with the genetic region flanked by the two IS 26 elements. Twenty-four strains deposited in GenBank carry the identical IS 26 -flanked genetic structure. Conclusions This is the first report of a clinical M. morganii isolate coharboring bla NDM−1 and armA in Myanmar. These genes located on chromosome with the genetic region flanked by the two IS 26 elements. The presence of this structure suggests the possibility of horizontal co-transfer of these resistance genes from other Enterobacterales , such as Escherichia coli and Klebsiella pneumoniae . Morganella morganii carbapenemase 16S rRNA methylase molecular epidemiology Figures Figure 1 Figure 2 Background Morganella morganii , formerly classified as Bacillus morganii and later as Proteus morganii , is a facultatively anaerobic, Gram-negative rod first isolated by Morgan et al. in 1905 from the feces of children with summer diarrhea [ 1 ]. Recognized as a clinically significant opportunistic pathogen, M. morganii has been increasingly reported worldwide and is associated with a range of infections including sepsis, abscesses, urinary tract infections, purple urine bag syndrome, chorioamnionitis, cellulitis, and wound infections [ 2 , 3 ]. This species exhibits intrinsic resistance to a broad spectrum of antibiotics, including oxacillin, ampicillin, amoxicillin, amoxicillin-clavulanate, first- and second-generation cephalosporins (except cefoxitin), macrolides, lincosamides, glycopeptides, fosfomycin, fusidic acid, colistin, and tigecycline [ 4 , 5 ]. However, it remains naturally susceptible to aztreonam, aminoglycosides, antipseudomonal penicillins, third- and fourth-generation cephalosporins, carbapenems, quinolones, trimethoprim/sulfamethoxazole, and chloramphenicol [ 4 ]. The emergence and global dissemination of carbapenemase-producing Enterobacterales represents a major public health threat [ 6 ]. Multiple classes of carbapenemases have been identified in Enterobacterales , including class A Klebsiella pneumoniae carbapenemase (KPC), class B metallo-β-lactamases (MBLs) such as NDM-, VIM-, and IMP-types, and class D OXA-48-like enzymes [ 6 ]. Prior to 2014, reports of carbapenemase-producing M. morganii were rare; however, such isolates have since been detected in regions including the Amazon [ 7 ], Bulgaria [ 8 ], China [ 9 ], seven European countries [ 10 ], Kuwait [ 11 ] and Nepal [ 12 ]. Among these, NDM-type MBLs are the most frequently reported, followed by OXA-48-like enzymes and KPC [ 10 ]. High-level aminoglycoside resistance mediated by 16S rRNA methylases is also increasingly observed in Gram-negative pathogens, including Enterobacterales [ 13 , 14 ]. These enzymes confer broad-spectrum resistance to aminoglycosides, and to date, 10 classes have been described in clinical isolates, including ArmA, RmtA–H, and NpmA [ 14 ]. Among the methylase-encoding genes, armA is one of the most widely disseminated. First identified on plasmid pCTX-M3 from Citrobacter freundii in 1996 and characterized 2007 [ 15 ], armA has since been detected in various Gram-negative species, including Enterobacterales and Acinetobacter baumannii , across human, animal, and environmental reservoirs [ 14 ]. Here, we report a clinical isolate of carbapenem-resistant M. morganii from Myanmar co-producing NDM-1 and the 16S rRNA methylase ArmA. Whole-genome sequencing revealed that bla NDM−1 and armA were located in close proximity on the chromosome and were likely co-transferred from other Enterobacterales species such as Escherichia coli and K. pneumoniae . MATERIALS AND METHODS Bacterial strains and antimicrobial susceptibility testing Five M. morganii isolates were obtained from five patients at three hospitals in Myanmar between September 2023 and October 2024: two isolates from hospital A, two from hospital B, and one from hospital C. Bacteria identification was performed using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; (Shimadzu, Kyoto, Japan), and confirmed by 16S rRNA gene sequencing. All isolates were recovered from clinical urine specimens, with two obtained from inpatients and three from outpatients. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method, following guidelines of the Clinical and Laboratory Standards Institute [ 5 ]. Whole genome sequencing and identification of drug-resistant genes Genomic DNA from all isolates was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Tokyo, Japan) and sequenced on the MiSeq platform (Illumina, San Diego, CA, USA). Raw sequencing reads were assembled using CLC Genomics Workbench version 10.0.1 (CLC bio, Aarhus, Denmark). For the isolate producing a carbapenemase, genomic DNA was additionally extracted using the QIAGEN Genomic-tip 20/G and Genomic DNA Buffer Set (Qiagen) and sequenced on the MinION platform (Oxford Nanopore Technologies, Oxford, UK). Hybrid genome assembly of MiSeq and MinION reads was performed using Unicycler v0.4.7. Antimicrobial resistance genes were identified using ResFinder v4.7.2 ( http://genepi.food.dtu.dk/resfinder ). Fluoroquinolone resistance was assessed by analyzing mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes, encoding DNA gyrase and topoisomerase IV, respectively, using CLC Genomics Workbench v24.0.1 [ 16 ]. The genetic environments surrounding carbapenemase and 16S rRNA methylase genes were analyzed using the Basic Local Alignment Search Tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome ) and visualized with in silico MolecularCloning Genomics Edition (In Silico Biology, Inc, Yokohama, Japan). Phylogenetic analysis based on single nucleotide polymorphisms Forty-six M. morganii subsp. morganii genome sequences, including the five Myanmar isolates tested and the type strain, M. morganii ATCC25830 (GenBank accession no. ATCC25830), were retrieved from GenBank ( https://www.ncbi.nlm.nih.gov/genbank/ ). A phylogenetic tree was constructed using kSNP4.0. [ 17 ]. The analysis included complete genomes of M. morganii isolates from Canada, China, the Czech Republic, Germany, India, Myanmar, Taiwan, the United Kingdom, and the United States. The single nucleotide polymorphism distance between isolate YGH122 and its closest relative, AR_0057, was calculated using CSIPhylogeny v1.4 ( https://cge.food.dtu.dk/services/CSIPhylogeny/ ). GenBank accession numbers Raw reads of four isolates including NPT03, MGH29, MGH31 and YGH329, were deposited at GenBank as accession number DRA021378 (experiment; DRX671572 to 671575, run; DRR691470 to DRR691473, and BioSample SAMD00893583 to SAMD00894836). The complete genome sequence of YGH122 was deposited at GenBank as accession number AP041090 (BioSample; SAMD00894837). The BioProject of this study was deposited as PRJDB20445. RESULTS Drug susceptibilities and drug-resistant genes As shown in Table 1, all five isolates showed extremely high MICs of over 512 µg/ml for penicillin, ampicillin, and colistin. Four isolates, YGH122, YGH329, NPT03 and MGH29, were highly resistant to trimethoprim/sulfamethoxazole. YGH122 and MGH29 were highly resistant to ampicillin/sulbactam, aztreonam, ceftazidime, cefepime, and cefotaxime, whereas YGH329 and NPT03 were either intermediate or susceptible to these drugs (Table 1). Four isolates, YGH122, YGH329, MGH29 and MGH31, were resistant to imipenem, while NPT03, was intermediate. All isolates were susceptible to meropenem. YGH 122 showed MICs greater than 512 µg/ml for amikacin, arbekacin, and gentamicin, and also exhibited higher MICs for imipenem and meropenem than the other four isolates (Table 1). All five isolates were resistant to ciprofloxacin, and either resistant or intermediate to levofloxacin. MGH 29 was resistant to tigecycline with an MIC of 8 µg/ml, while the other four were susceptible (Table 1). All five isolates harbored various genes and mutations associated with resistance to several classes of antibiotics (Table 1). These included genes encoding aminoglycoside-modifying enzymes (for aminoglycoside resistance), β-lactamases (for β-lactam resistance), and enzymes involved in folic acid synthesis inhibition (for trimethoprim/sulfamethoxazole resistance), as well as genes conferring resistance to chloramphenicol and tetracycline (Table 1). All isolates carried two or three mutations in QRDRs, and MGH29 additionally harbored the plasmid-mediated quinolone resistance gene qnrD1 . YGH122 harbored bla NDM−1 encoding metallo-β-lactamase, and armA encoding a 16S rRNA methylase. Genetic environments surrounding bla NDM−1 and armA The genetic structure of M. morganii YGH122 is shown in line 3 of Fig. 1 . The bla NDM−1 and armA genes were located in close proximity on the chromosome, separated by a 7,510 bp intergenic region. The surrounding genetic environment, spanning 26,322 bp, was as follows: NADK - recN - bamE - MFS -IS 26 - bla NDM−1 - ble MBL - orf1 - ΔampC - orf2 - orf3 - orf4 - sul1 -IS 91 - Δ IS 5 - armA -IS 4 - msrD - mph - orf5 - orf6 -IS 26 - orf7 - MFS - orf8 (Fig. 1 . line 3). This region included five insertion sequence (IS) elements (IS 26 , IS 91 , Δ IS 5 , IS 4 , and IS 26 ) and resistance-related genes ( bla NDM−1 , ble MBL , ΔampC , sul1 , armA, msrD and mph ). The structure flanked by the two IS 26 elements, 17,313 bp in length, was identical to those of K. pneumoniae NDM-1saitama01, K. pneumoniae ST101:960186733, and E. coli 4974960 (Fig. 1 ). Twenty-four strains were found to harbor a genetic structure identical to that of YGH122 with > 99.0% identity (Table 2). These included three Enterobacter homaechei , seven E. coli , one Klebsiella oxytoca , and 13 K. pneumoniae . Except for YGH122 the IS 26 -flanked structures were located on plasmids (Table 2). All strains were isolated from humans, although information was unavailable for three of them. Of the 24 strains, five were isolated from urine, four from sputum, four from rectal swabs, one from bile, one from a wound, and nine from unknown sources (Table 2). These 24 were isolated in several countries/regions, including Myanmar (five strains), China (four), Canada (three), Hong Kong (two), Italy (two), Bangladesh, India, Israel, Japan, Oman, South Africa, and Taiwan (one, respectively); and one was unknown (Table 2).The genetic structure flanked by IS 26 was initially detected in a clinical isolate of NDM-1-producing E. coli in 2010 in Japan [ 18 ] (Table 2). Since then, similar genetic structures have been increasingly reported worldwide (Table 2). In the genetic environment of bla NDM−1 and armA , the two outer structures of the IS 26 -flanked genetic structure, NADK - recN - bamE - MFS (5,024 bp) and orf7 - MFS - orf8 (3,996 bp) was identical with 100% identity to two strains of M. morganii AR_0057 and OT11, of which the two structures were continuously connected (Fig. 1 ). In other words, the IS 26 -flanked segment was inserted between the two outer structures in YGH122 (Fig. 1 , line 3). AR_0057 was isolated in the USA and OT11 in China (Table S1 ). Phylogenetic analysis Phylogenetic analysis of 46 whole-genome sequences, including the type strain, five isolates from Myanmar, and 40 isolates from various countries, revealed three major clades: A, B, and C (Fig. 2 ). The five Myanmar isolates (in rectangles) belonged to Clade A, which was further divided into Subclades A1 and A2. In Subclade A1, MGH31 was from Hospital C in Mandalay; YGH329 and YGH122 were from Hospital A in Yangon; and NPT03 was from Hospital B in Naypyidaw. MGH29 in Subclade A2, was also from Hospital C. YGH122, which harbored bla NDM−1 and armA , clustered closely with M. morganii strains MM50821 (Czech Republic, 2019), MM46903 (Czech Republic, 2018), AR_0057 (USA, 2015), and OT11(China, 2019). YGH122 differed from AR_0057 by 282 snps, making AR_0057 its closest relative. DISCUSSION IS 26 likely plays an important role in the simultaneous acquisition of bla NDM−1 and armA in M. morganii YGH122. The IS 26 -flanked genetic structure, which spans 17,313 bp, contains seven drug resistance genes, including bla NDM−1 and armA (Fig. 1 ). The structure has been detected in 24 Enterobacteriaceae strains, including E. coli and K. pneumoniae , isolated across Asia, Africa, Europe, and North America (Table 2), indicating that the IS 26 -flanked region harboring bla NDM−1 and armA has spread globally among several Enterobacterales species. IS 26 , a member of the IS 6 family, is particularly significant in clinical contexts [ 19 ]. It has been identified in numerous clinical isolates of Enterobacterales as part of both chromosomal and plasmids DNA and is considered instrumental in the rearrangement and dissemination of multidrug resistance [ 19 ]. Infections by M. morganii YGH122, which produces both NDM-1 and 16S rRNA methylase, may be treated with tigecycline and/or high-dose meropenem. However, antimicrobial selection should always be guided by drug susceptibility profiles. M. morganii is intrinsically resistant to many β-lactam antibiotics; hence, third-generation cephalosporins and/or gentamicin for 10–14 days is recommended to treat M. morganii infections [ 2 ]. In contrast, the multidrug-resistant M. morganii YGH122 isolate exhibited high-level resistance to nearly all tested antibiotics except meropenem and tigecycline. CONCLUSIONS Most isolates were resistant to imipenem, but all isolates were susceptible to meropenem. Of them, an isolate was highly aminoglycoside-resistant. The isolate harbored both bla NDM−1 and armA located in close proximity on the chromosome with the genetic region flanked by the two IS 26 elements. The presence of this structure suggests the possibility of horizontal co-transfer of these resistance genes from other Enterobacterales , such as Escherichia coli and Klebsiella pneumoniae . This is the first report of a clinical M. morganii isolate coharboring bla NDM−1 and armA in Myanmar. Declarations Conflicts of interest The authors declare that there are no conflicts of interest. Funding information This research was supported by grants from Science and Technology Research Partnership for Sustainable Development (SATREPS) from Japan Agency for Medical Research and Development (AMED) and Japan International Cooperation Agency (JICA) (25jm0110026h0004), and Japan Society for the Promotion of Science (JSPS) KAKENHI (21K07031, 23K06550 and 24K10204). Ethical approval and consent to participate The study protocol was approved by: Ministry of Health and Sports, Republic of the Union of Myanmar (letter no. Ethical Committee 2016); the ethics committee of Juntendo University (number 809); and Biosafety Committee, Juntendo University (approval nos. BSL2/29-1). Written informed consent was obtained from the study participants. This study did not involve human participants, human data, or human tissue. Author contributions MK analyzed and summarized research data and drafted the manuscript. ST performed sequencing and analyzed the data. SO, NHM and PES collected samples. SH analyzed the data. TTH, SS, HHT and TK supervised the study. TT designed and supervised the study. Acknowledgements We thank Naeko Mizutani for supporting the study. References Morgan Hde R. Report XCV. Upon the bacteriology of the summer diarrhoea of infants. Br Med J. 1906 Apr 21;1(2364):908-12. doi: 10.1136/bmj.1.2364.908. PMID: 20762625; PMCID: PMC2381076. 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Supplementary Files Table1.xlsx Table2.xlsx TableS1.xlsx Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2026 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 28 Oct, 2025 Reviews received at journal 26 Oct, 2025 Reviews received at journal 25 Oct, 2025 Reviews received at journal 15 Oct, 2025 Reviews received at journal 05 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviewers agreed at journal 30 Sep, 2025 Reviewers agreed at journal 30 Sep, 2025 Reviewers agreed at journal 29 Sep, 2025 Reviewers invited by journal 29 Sep, 2025 Editor assigned by journal 01 Sep, 2025 Submission checks completed at journal 01 Sep, 2025 First submitted to journal 01 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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07:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7405418/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7405418/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-025-12482-1","type":"published","date":"2026-02-07T15:59:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93339971,"identity":"3050b387-6599-4569-b95e-1ff9bd64c56d","added_by":"auto","created_at":"2025-10-12 14:28:29","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":833712,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/bba6fd6c1ecdca60d8dd4d78.tif"},{"id":93339968,"identity":"409a21dc-73c8-46fb-8d41-8285d91b29aa","added_by":"auto","created_at":"2025-10-12 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14:36:30","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":495316,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/befa096ba966cf08a8b42f04.tif"},{"id":93339983,"identity":"8b2d7d9a-50d9-4ef6-a1b3-7ce45fe81134","added_by":"auto","created_at":"2025-10-12 14:28:30","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154267,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/d9f20a7581edfb67443b7615.png"},{"id":93339976,"identity":"1806eaca-6364-442c-ab55-9e6e67e286e1","added_by":"auto","created_at":"2025-10-12 14:28:30","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":345063,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/05eed8088b9ad664fb838227.png"},{"id":93340915,"identity":"332f2c27-3b5f-41ce-9590-fb1acad289eb","added_by":"auto","created_at":"2025-10-12 14:36:30","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67323,"visible":true,"origin":"","legend":"","description":"","filename":"c12e036f5b6f43308eddcd39b4cfdd031structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/505401d3e607c637e0e740df.xml"},{"id":93339975,"identity":"99c4b8c6-f0ab-44b4-9385-943d2030aeea","added_by":"auto","created_at":"2025-10-12 14:28:30","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80040,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/6f5b859b034f067b15090493.html"},{"id":93340912,"identity":"2ca5498f-35d5-4e1f-b610-401f3b0bb8cb","added_by":"auto","created_at":"2025-10-12 14:36:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1453898,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic environments surrounding \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e in \u003cem\u003eM. morganii\u003c/em\u003e YGH122. The genetic environments surrounding \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e were closed to \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates obtained from Japan and South Africa, and \u003cem\u003eE. coli \u003c/em\u003eisolates in Israel respectively. The outer structures of IS\u003cem\u003e26\u003c/em\u003e were resembled to \u003cem\u003eM. morganii \u003c/em\u003eisolates obtained from the United States and China. \u003cem\u003eorf1\u003c/em\u003e: phosphoribosylanthranilate isomerase, \u003cem\u003eorf2\u003c/em\u003e: LysR family transcriptional regulator AmpR, \u003cem\u003eorf3\u003c/em\u003e: hydrogenase maturation nickel metallochaperone HybF, \u003cem\u003eorf4\u003c/em\u003e: QacE family quaternary ammonium compound efflux SMR transporter, \u003cem\u003eorf5\u003c/em\u003e: DNA-binding protein, \u003cem\u003eorf6\u003c/em\u003e: RepB family plasmid replication initiator protein,\u003cem\u003e orf7\u003c/em\u003e: alternation dehydratase protein family protein, and \u003cem\u003eorf8\u003c/em\u003e: dihydrodipicolinate synthase family protein\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/432ea612cd4b8e82360ec11f.png"},{"id":93339973,"identity":"4e07a062-3cd4-41ed-86d6-f6c6262f4908","added_by":"auto","created_at":"2025-10-12 14:28:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1231042,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular phylogenetic tree of \u003cem\u003eMorganella morganii\u003c/em\u003e based on whole genome sequences. The tree included 45 strains collected from several countries, including Canada, Chaina, Czech, Germany, India, Myanmar, Taiwan, the United Kingdom, and the United States of America, and the type strain, ATCC 25830. The five strains tested in this study, obtained from Myanmar are enclosed rectangles. Asterisk (*) indicates isolates obtained from animals and environment.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/ea74c087b5574ec073ec4852.png"},{"id":102234276,"identity":"eab951f6-7a70-4afb-88f3-f622c7ff9f95","added_by":"auto","created_at":"2026-02-09 16:08:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3472508,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/d38292a3-da24-4844-ab10-d1e771f28e35.pdf"},{"id":93339965,"identity":"1e34c5aa-13e7-4917-9d66-33381f7876d8","added_by":"auto","created_at":"2025-10-12 14:28:29","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14502,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/fc89f3fb3cd40d968b52cbab.xlsx"},{"id":93339966,"identity":"1daa986a-c613-4e21-9507-f3d2e75b2a36","added_by":"auto","created_at":"2025-10-12 14:28:29","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14046,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/40c41c8b16c419b00d65d1be.xlsx"},{"id":93339969,"identity":"7c4334e4-20fa-4adc-92b4-fc65a6e8f679","added_by":"auto","created_at":"2025-10-12 14:28:29","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12687,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7405418/v1/4f7aae9118e2f89f65eca893.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEmergence of NDM-type metallo-β-lactamase and ArmA 16S rRNA methylase co-producing Morganella morganii in Myanmar\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eMorganella morganii\u003c/em\u003e, formerly classified as \u003cem\u003eBacillus morganii\u003c/em\u003e and later as \u003cem\u003eProteus morganii\u003c/em\u003e, is a facultatively anaerobic, Gram-negative rod first isolated by Morgan et al. in 1905 from the feces of children with summer diarrhea [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recognized as a clinically significant opportunistic pathogen, \u003cem\u003eM. morganii\u003c/em\u003e has been increasingly reported worldwide and is associated with a range of infections including sepsis, abscesses, urinary tract infections, purple urine bag syndrome, chorioamnionitis, cellulitis, and wound infections [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This species exhibits intrinsic resistance to a broad spectrum of antibiotics, including oxacillin, ampicillin, amoxicillin, amoxicillin-clavulanate, first- and second-generation cephalosporins (except cefoxitin), macrolides, lincosamides, glycopeptides, fosfomycin, fusidic acid, colistin, and tigecycline [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, it remains naturally susceptible to aztreonam, aminoglycosides, antipseudomonal penicillins, third- and fourth-generation cephalosporins, carbapenems, quinolones, trimethoprim/sulfamethoxazole, and chloramphenicol [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe emergence and global dissemination of carbapenemase-producing \u003cem\u003eEnterobacterales\u003c/em\u003e represents a major public health threat [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Multiple classes of carbapenemases have been identified in \u003cem\u003eEnterobacterales\u003c/em\u003e, including class A \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemase (KPC), class B metallo-β-lactamases (MBLs) such as NDM-, VIM-, and IMP-types, and class D OXA-48-like enzymes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Prior to 2014, reports of carbapenemase-producing \u003cem\u003eM. morganii\u003c/em\u003e were rare; however, such isolates have since been detected in regions including the Amazon [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], Bulgaria [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], China [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], seven European countries [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], Kuwait [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and Nepal [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Among these, NDM-type MBLs are the most frequently reported, followed by OXA-48-like enzymes and KPC [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHigh-level aminoglycoside resistance mediated by 16S rRNA methylases is also increasingly observed in Gram-negative pathogens, including \u003cem\u003eEnterobacterales\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These enzymes confer broad-spectrum resistance to aminoglycosides, and to date, 10 classes have been described in clinical isolates, including ArmA, RmtA\u0026ndash;H, and NpmA [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Among the methylase-encoding genes, \u003cem\u003earmA\u003c/em\u003e is one of the most widely disseminated. First identified on plasmid pCTX-M3 from \u003cem\u003eCitrobacter freundii\u003c/em\u003e in 1996 and characterized 2007 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], \u003cem\u003earmA\u003c/em\u003e has since been detected in various Gram-negative species, including \u003cem\u003eEnterobacterales\u003c/em\u003e and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, across human, animal, and environmental reservoirs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHere, we report a clinical isolate of carbapenem-resistant \u003cem\u003eM. morganii\u003c/em\u003e from Myanmar co-producing NDM-1 and the 16S rRNA methylase ArmA. Whole-genome sequencing revealed that \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e were located in close proximity on the chromosome and were likely co-transferred from other \u003cem\u003eEnterobacterales\u003c/em\u003e species such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eBacterial strains and antimicrobial susceptibility testing\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eFive \u003cem\u003eM. morganii\u003c/em\u003e isolates were obtained from five patients at three hospitals in Myanmar between September 2023 and October 2024: two isolates from hospital A, two from hospital B, and one from hospital C. Bacteria identification was performed using matrix-assisted laser desorption ionization\u0026ndash;time of flight mass spectrometry (MALDI-TOF MS; (Shimadzu, Kyoto, Japan), and confirmed by 16S rRNA gene sequencing. All isolates were recovered from clinical urine specimens, with two obtained from inpatients and three from outpatients. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method, following guidelines of the Clinical and Laboratory Standards Institute [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWhole genome sequencing and identification of drug-resistant genes\u003c/h3\u003e\n\u003cp\u003eGenomic DNA from all isolates was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Tokyo, Japan) and sequenced on the MiSeq platform (Illumina, San Diego, CA, USA). Raw sequencing reads were assembled using CLC Genomics Workbench version 10.0.1 (CLC bio, Aarhus, Denmark). For the isolate producing a carbapenemase, genomic DNA was additionally extracted using the QIAGEN Genomic-tip 20/G and Genomic DNA Buffer Set (Qiagen) and sequenced on the MinION platform (Oxford Nanopore Technologies, Oxford, UK). Hybrid genome assembly of MiSeq and MinION reads was performed using Unicycler v0.4.7. Antimicrobial resistance genes were identified using ResFinder v4.7.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genepi.food.dtu.dk/resfinder\u003c/span\u003e\u003cspan address=\"http://genepi.food.dtu.dk/resfinder\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Fluoroquinolone resistance was assessed by analyzing mutations in the quinolone resistance-determining regions (QRDRs) of the \u003cem\u003egyrA\u003c/em\u003e and \u003cem\u003eparC\u003c/em\u003e genes, encoding DNA gyrase and topoisomerase IV, respectively, using CLC Genomics Workbench v24.0.1 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The genetic environments surrounding carbapenemase and 16S rRNA methylase genes were analyzed using the Basic Local Alignment Search Tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn\u0026amp;PAGE_TYPE=BlastSearch\u0026amp;LINK_LOC=blasthome\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn\u0026amp;PAGE_TYPE=BlastSearch\u0026amp;LINK_LOC=blasthome\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized with in silico MolecularCloning Genomics Edition (In Silico Biology, Inc, Yokohama, Japan).\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis based on single nucleotide polymorphisms\u003c/h3\u003e\n\u003cp\u003eForty-six \u003cem\u003eM. morganii\u003c/em\u003e subsp. \u003cem\u003emorganii\u003c/em\u003e genome sequences, including the five Myanmar isolates tested and the type strain, \u003cem\u003eM. morganii\u003c/em\u003e ATCC25830 (GenBank accession no. ATCC25830), were retrieved from GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A phylogenetic tree was constructed using kSNP4.0. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The analysis included complete genomes of \u003cem\u003eM. morganii\u003c/em\u003e isolates from Canada, China, the Czech Republic, Germany, India, Myanmar, Taiwan, the United Kingdom, and the United States. The single nucleotide polymorphism distance between isolate YGH122 and its closest relative, AR_0057, was calculated using CSIPhylogeny v1.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/CSIPhylogeny/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/CSIPhylogeny/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGenBank accession numbers\u003c/h3\u003e\n\u003cp\u003eRaw reads of four isolates including NPT03, MGH29, MGH31 and YGH329, were deposited at GenBank as accession number DRA021378 (experiment; DRX671572 to 671575, run; DRR691470 to DRR691473, and BioSample SAMD00893583 to SAMD00894836). The complete genome sequence of YGH122 was deposited at GenBank as accession number AP041090 (BioSample; SAMD00894837). The BioProject of this study was deposited as PRJDB20445.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDrug susceptibilities and drug-resistant genes\u003c/h2\u003e\u003cp\u003eAs shown in Table\u0026nbsp;1, all five isolates showed extremely high MICs of over 512 \u0026micro;g/ml for penicillin, ampicillin, and colistin. Four isolates, YGH122, YGH329, NPT03 and MGH29, were highly resistant to trimethoprim/sulfamethoxazole. YGH122 and MGH29 were highly resistant to ampicillin/sulbactam, aztreonam, ceftazidime, cefepime, and cefotaxime, whereas YGH329 and NPT03 were either intermediate or susceptible to these drugs (Table\u0026nbsp;1). Four isolates, YGH122, YGH329, MGH29 and MGH31, were resistant to imipenem, while NPT03, was intermediate. All isolates were susceptible to meropenem. YGH 122 showed MICs greater than 512 \u0026micro;g/ml for amikacin, arbekacin, and gentamicin, and also exhibited higher MICs for imipenem and meropenem than the other four isolates (Table\u0026nbsp;1). All five isolates were resistant to ciprofloxacin, and either resistant or intermediate to levofloxacin. MGH 29 was resistant to tigecycline with an MIC of 8 \u0026micro;g/ml, while the other four were susceptible (Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eAll five isolates harbored various genes and mutations associated with resistance to several classes of antibiotics (Table\u0026nbsp;1). These included genes encoding aminoglycoside-modifying enzymes (for aminoglycoside resistance), β-lactamases (for β-lactam resistance), and enzymes involved in folic acid synthesis inhibition (for trimethoprim/sulfamethoxazole resistance), as well as genes conferring resistance to chloramphenicol and tetracycline (Table\u0026nbsp;1). All isolates carried two or three mutations in QRDRs, and MGH29 additionally harbored the plasmid-mediated quinolone resistance gene \u003cem\u003eqnrD1\u003c/em\u003e. YGH122 harbored \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e encoding metallo-β-lactamase, and \u003cem\u003earmA\u003c/em\u003e encoding a 16S rRNA methylase.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenetic environments surrounding\u003c/b\u003e \u003cb\u003ebla\u003c/b\u003e\u003csub\u003e\u003cb\u003eNDM\u0026minus;1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003earmA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe genetic structure of \u003cem\u003eM. morganii\u003c/em\u003e YGH122 is shown in line 3 of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e genes were located in close proximity on the chromosome, separated by a 7,510 bp intergenic region. The surrounding genetic environment, spanning 26,322 bp, was as follows: \u003cem\u003eNADK\u003c/em\u003e-\u003cem\u003erecN\u003c/em\u003e-\u003cem\u003ebamE\u003c/em\u003e-\u003cem\u003eMFS\u003c/em\u003e-IS\u003cem\u003e26\u003c/em\u003e- \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e-\u003cem\u003eble\u003c/em\u003e\u003csub\u003eMBL\u003c/sub\u003e-\u003cem\u003eorf1\u003c/em\u003e-\u003cem\u003eΔampC\u003c/em\u003e-\u003cem\u003eorf2\u003c/em\u003e-\u003cem\u003eorf3\u003c/em\u003e-\u003cem\u003eorf4\u003c/em\u003e-\u003cem\u003esul1\u003c/em\u003e-IS\u003cem\u003e91\u003c/em\u003e-\u003cem\u003eΔ\u003c/em\u003eIS\u003cem\u003e5\u003c/em\u003e-\u003cem\u003earmA\u003c/em\u003e-IS\u003cem\u003e4\u003c/em\u003e-\u003cem\u003emsrD\u003c/em\u003e-\u003cem\u003emph\u003c/em\u003e-\u003cem\u003eorf5\u003c/em\u003e-\u003cem\u003eorf6\u003c/em\u003e-IS\u003cem\u003e26\u003c/em\u003e-\u003cem\u003eorf7\u003c/em\u003e-\u003cem\u003eMFS\u003c/em\u003e-\u003cem\u003eorf8\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. line 3). This region included five insertion sequence (IS) elements (IS\u003cem\u003e26\u003c/em\u003e, IS\u003cem\u003e91\u003c/em\u003e, \u003cem\u003eΔ\u003c/em\u003eIS\u003cem\u003e5\u003c/em\u003e, IS\u003cem\u003e4\u003c/em\u003e, and IS\u003cem\u003e26\u003c/em\u003e) and resistance-related genes (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e, \u003cem\u003eble\u003c/em\u003e\u003csub\u003eMBL\u003c/sub\u003e, \u003cem\u003eΔampC\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003earmA, msrD\u003c/em\u003e and \u003cem\u003emph\u003c/em\u003e). The structure flanked by the two IS\u003cem\u003e26\u003c/em\u003e elements, 17,313 bp in length, was identical to those of \u003cem\u003eK. pneumoniae\u003c/em\u003e NDM-1saitama01, \u003cem\u003eK. pneumoniae\u003c/em\u003e ST101:960186733, and \u003cem\u003eE. coli\u003c/em\u003e 4974960 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTwenty-four strains were found to harbor a genetic structure identical to that of YGH122 with \u0026gt;\u0026thinsp;99.0% identity (Table\u0026nbsp;2). These included three \u003cem\u003eEnterobacter homaechei\u003c/em\u003e, seven \u003cem\u003eE. coli\u003c/em\u003e, one \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e, and 13 \u003cem\u003eK. pneumoniae\u003c/em\u003e. Except for YGH122 the IS\u003cem\u003e26\u003c/em\u003e-flanked structures were located on plasmids (Table\u0026nbsp;2). All strains were isolated from humans, although information was unavailable for three of them. Of the 24 strains, five were isolated from urine, four from sputum, four from rectal swabs, one from bile, one from a wound, and nine from unknown sources (Table\u0026nbsp;2). These 24 were isolated in several countries/regions, including Myanmar (five strains), China (four), Canada (three), Hong Kong (two), Italy (two), Bangladesh, India, Israel, Japan, Oman, South Africa, and Taiwan (one, respectively); and one was unknown (Table\u0026nbsp;2).The genetic structure flanked by IS\u003cem\u003e26\u003c/em\u003e was initially detected in a clinical isolate of NDM-1-producing \u003cem\u003eE. coli\u003c/em\u003e in 2010 in Japan [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] (Table\u0026nbsp;2). Since then, similar genetic structures have been increasingly reported worldwide (Table\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eIn the genetic environment of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e, the two outer structures of the IS\u003cem\u003e26\u003c/em\u003e-flanked genetic structure, \u003cem\u003eNADK\u003c/em\u003e-\u003cem\u003erecN\u003c/em\u003e-\u003cem\u003ebamE\u003c/em\u003e-\u003cem\u003eMFS\u003c/em\u003e (5,024 bp) and \u003cem\u003eorf7\u003c/em\u003e-\u003cem\u003eMFS\u003c/em\u003e-\u003cem\u003eorf8\u003c/em\u003e (3,996 bp) was identical with 100% identity to two strains of \u003cem\u003eM. morganii\u003c/em\u003e AR_0057 and OT11, of which the two structures were continuously connected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In other words, the IS\u003cem\u003e26\u003c/em\u003e-flanked segment was inserted between the two outer structures in YGH122 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, line 3). AR_0057 was isolated in the USA and OT11 in China (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003ePhylogenetic analysis of 46 whole-genome sequences, including the type strain, five isolates from Myanmar, and 40 isolates from various countries, revealed three major clades: A, B, and C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The five Myanmar isolates (in rectangles) belonged to Clade A, which was further divided into Subclades A1 and A2. In Subclade A1, MGH31 was from Hospital C in Mandalay; YGH329 and YGH122 were from Hospital A in Yangon; and NPT03 was from Hospital B in Naypyidaw. MGH29 in Subclade A2, was also from Hospital C. YGH122, which harbored \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e, clustered closely with \u003cem\u003eM. morganii\u003c/em\u003e strains MM50821 (Czech Republic, 2019), MM46903 (Czech Republic, 2018), AR_0057 (USA, 2015), and OT11(China, 2019). YGH122 differed from AR_0057 by 282 snps, making AR_0057 its closest relative.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIS\u003cem\u003e26\u003c/em\u003e likely plays an important role in the simultaneous acquisition of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e in \u003cem\u003eM. morganii\u003c/em\u003e YGH122. The IS\u003cem\u003e26\u003c/em\u003e-flanked genetic structure, which spans 17,313 bp, contains seven drug resistance genes, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The structure has been detected in 24 \u003cem\u003eEnterobacteriaceae\u003c/em\u003e strains, including \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e, isolated across Asia, Africa, Europe, and North America (Table\u0026nbsp;2), indicating that the IS\u003cem\u003e26\u003c/em\u003e-flanked region harboring \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e has spread globally among several \u003cem\u003eEnterobacterales\u003c/em\u003e species. IS\u003cem\u003e26\u003c/em\u003e, a member of the IS\u003cem\u003e6\u003c/em\u003e family, is particularly significant in clinical contexts [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It has been identified in numerous clinical isolates of \u003cem\u003eEnterobacterales\u003c/em\u003e as part of both chromosomal and plasmids DNA and is considered instrumental in the rearrangement and dissemination of multidrug resistance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Infections by \u003cem\u003eM. morganii\u003c/em\u003e YGH122, which produces both NDM-1 and 16S rRNA methylase, may be treated with tigecycline and/or high-dose meropenem. However, antimicrobial selection should always be guided by drug susceptibility profiles. \u003cem\u003eM. morganii\u003c/em\u003e is intrinsically resistant to many β-lactam antibiotics; hence, third-generation cephalosporins and/or gentamicin for 10\u0026ndash;14 days is recommended to treat \u003cem\u003eM. morganii\u003c/em\u003e infections [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In contrast, the multidrug-resistant \u003cem\u003eM. morganii\u003c/em\u003e YGH122 isolate exhibited high-level resistance to nearly all tested antibiotics except meropenem and tigecycline.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eMost isolates were resistant to imipenem, but all isolates were susceptible to meropenem. Of them, an isolate was highly aminoglycoside-resistant. The isolate harbored both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e located in close proximity on the chromosome with the genetic region flanked by the two IS\u003cem\u003e26\u003c/em\u003e elements. The presence of this structure suggests the possibility of horizontal co-transfer of these resistance genes from other \u003cem\u003eEnterobacterales\u003c/em\u003e, such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. This is the first report of a clinical \u003cem\u003eM. morganii\u003c/em\u003e isolate coharboring \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e in Myanmar.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from Science and Technology Research Partnership for Sustainable Development (SATREPS) from Japan Agency for Medical Research and Development (AMED) and Japan International Cooperation Agency (JICA) (25jm0110026h0004), and Japan Society for the Promotion of Science (JSPS) KAKENHI (21K07031, 23K06550 and 24K10204).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by: Ministry of Health and Sports, Republic of the Union of Myanmar (letter no. Ethical Committee 2016); the ethics committee of Juntendo University (number 809); and Biosafety Committee, Juntendo University (approval nos. BSL2/29-1). \u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the study participants. This study did not involve human participants, human data, or human tissue.\u003cbr\u003e \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMK analyzed and summarized research data and drafted the manuscript. ST performed sequencing and analyzed the data. SO, NHM and PES collected samples. SH analyzed the data. TTH, SS, HHT and TK supervised the study. TT designed and supervised the study.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Naeko Mizutani for supporting the study.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMorgan Hde R. Report XCV. 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Genomic Characterization of Carbapenemase-Producing \u003cem\u003eEnterobacter hormaechei\u003c/em\u003e, \u003cem\u003eSerratia marcescens\u003c/em\u003e, \u003cem\u003eCitrobacter freundii\u003c/em\u003e, \u003cem\u003eProvidencia stuartii\u003c/em\u003e, and \u003cem\u003eMorganella morganii\u003c/em\u003e clinical isolates from Bulgaria. Antibiotics (Basel). 2024 May 16;13(5):455. doi: 10.3390/antibiotics13050455. PMID: 38786183; PMCID: PMC11117914.\u003c/li\u003e\n\u003cli\u003eXiang G, Lan K, Cai Y, Liao K, Zhao M, et al. Clinical molecular and genomic epidemiology of \u003cem\u003eMorganella morganii\u003c/em\u003e in China. Front Microbiol. 2021 Sep 28;12:744291. doi: 10.3389/fmicb.2021.744291. PMID: 34650543; PMCID: PMC8507844.\u003c/li\u003e\n\u003cli\u003eBonnin RA, Creton E, Perrin A, Girlich D, Emeraud C, et al. Spread of carbapenemase-producing Morganella spp from 2013 to 2021: a comparative genomic study. Lancet Microbe. 2024 Jun;5(6):e547-e558. doi: 10.1016/S2666-5247(23)00407-X. Epub 2024 Apr 25. PMID: 38677305.\u003c/li\u003e\n\u003cli\u003eJamal WY, Albert MJ, Khodakhast F, Poirel L, Rotimi VO. Emergence of new sequence type OXA-48 carbapenemase-producing enterobacteriaceae in Kuwait. Microb Drug Resist. 2015 Jun;21(3):329-34. doi: 10.1089/mdr.2014.0123. Epub 2014 Dec 31. PMID: 25551428.\u003c/li\u003e\n\u003cli\u003eShrestha S, Tada T, Sherchan JB, Uchida H, Hishinuma T, et al. Highly multidrug-resistant \u003cem\u003eMorganella morganii\u003c/em\u003e clinical isolates from Nepal co-producing NDM-type metallo-\u0026beta;-lactamases and the 16S rRNA methylase ArmA. J Med Microbiol. 2020 Apr;69(4):572-575. doi: 10.1099/jmm.0.001160. PMID: 32100711.\u003c/li\u003e\n\u003cli\u003eWachino JI, Doi Y, Arakawa Y. aminoglycoside resistance: updates with a focus on acquired 16s ribosomal RNA methyltransferases. Infect Dis Clin North Am. 2020 Dec;34(4):887-902. doi: 10.1016/j.idc.2020.06.002. Epub 2020 Sep 30. PMID: 33011054; PMCID: PMC10927307.\u003c/li\u003e\n\u003cli\u003eDoi Y, Wachino JI, Arakawa Y. Aminoglycoside resistance: the emergence of acquired 16s ribosomal RNA methyltransferases. Infect Dis Clin North Am. 2016 Jun;30(2):523-537. doi: 10.1016/j.idc.2016.02.011. PMID: 27208771; PMCID: PMC4878400.\u003c/li\u003e\n\u003cli\u003eGołebiewski M, Kern-Zdanowicz I, Zienkiewicz M, Adamczyk M, Zylinska J, et al. Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum beta-lactamase gene blaCTX-M-3. Antimicrob Agents Chemother. 2007 Nov;51(11):3789-95. doi: 10.1128/AAC.00457-07. Epub 2007 Aug 13. PMID: 17698626; PMCID: PMC2151408.\u003c/li\u003e\n\u003cli\u003eNakano M, Deguchi T, Kawamura T, Yasuda M, Kimura M, et al. Mutations in the gyrA and parC genes in fluoroquinolone-resistant clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1997 Oct;41(10):2289-91. doi: 10.1128/AAC.41.10.2289. PMID: 9333065; PMCID: PMC164110.\u003c/li\u003e\n\u003cli\u003eHall BG, Nisbet J. Building phylogenetic trees from genome sequences with kSNP4. Mol Biol Evol. 2023 Nov 3;40(11):msad235. doi: 10.1093/molbev/msad235. PMID: 37948764; PMCID: PMC10640685. doi: 10.1093/molbev/msad235.\u003c/li\u003e\n\u003cli\u003eHishinuma A, Yoshida A, Suzuki H, Okuzumi K, Ishida T. Complete sequencing of an IncFII NDM-1 plasmid in Klebsiella pneumoniae shows structural features shared with other multidrug resistance plasmids. J Antimicrob Chemother. 2013 Oct;68(10):2415-7. doi: 10.1093/jac/dkt190. Epub 2013 May 16. PMID: 23681270.\u003c/li\u003e\n\u003cli\u003eVarani A, He S, Siguier P, Ross K, Chandler M. The IS6 family, a clinically important group of insertion sequences including IS26. Mob DNA. 2021 Mar 23;12(1):11. doi: 10.1186/s13100-021-00239-x. PMID: 33757578; PMCID: PMC7986276.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Morganella morganii, carbapenemase, 16S rRNA methylase, molecular epidemiology","lastPublishedDoi":"10.21203/rs.3.rs-7405418/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7405418/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eMorganella morganii\u003c/em\u003e is intrinsically resistant to a wide range of antibiotics and is increasingly being reported worldwide.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA total of five \u003cem\u003eM. morganii\u003c/em\u003e isolates were obtained from five patients at three hospitals in Myanmar between Sept. 2023 and Oct. 2024. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. Genomic DNA was extracted and sequenced on the next generation sequencer. Drug-resistant factors were determined and the genetic environments surrounding carbapenemase and 16S rRNA methylase genes were analyzed. A phylogenetic tree was constructed using forty-six \u003cem\u003eM. morganii\u003c/em\u003e subsp. \u003cem\u003emorganii\u003c/em\u003e genome sequences, including the five Myanmar.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOne isolate harbored both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e. This isolate was resistant to imipenem (MIC: 16 ug/ml) but susceptible to meropenem (MIC: 1 ug/ml). Whole genome sequencing revealed that \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e were located in close proximity on the chromosome of \u003cem\u003eM. morganii\u003c/em\u003e strain YGH122, with the genetic region flanked by the two IS\u003cem\u003e26\u003c/em\u003e elements. Twenty-four strains deposited in GenBank carry the identical IS\u003cem\u003e26\u003c/em\u003e-flanked genetic structure.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis is the first report of a clinical \u003cem\u003eM. morganii\u003c/em\u003e isolate coharboring \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003earmA\u003c/em\u003e in Myanmar. These genes located on chromosome with the genetic region flanked by the two IS\u003cem\u003e26\u003c/em\u003e elements. The presence of this structure suggests the possibility of horizontal co-transfer of these resistance genes from other \u003cem\u003eEnterobacterales\u003c/em\u003e, such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Emergence of NDM-type metallo-β-lactamase and ArmA 16S rRNA methylase co-producing Morganella morganii in Myanmar","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-12 14:28:25","doi":"10.21203/rs.3.rs-7405418/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-28T08:00:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-26T04:22:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-25T10:21:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T05:58:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T00:06:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305421521646257320227290570013538544492","date":"2025-10-02T10:49:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267176485872132516689211527693099123937","date":"2025-10-01T05:25:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144542993463317043533400126271603827209","date":"2025-09-30T11:31:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328730999447123634240438222393541373356","date":"2025-09-30T10:01:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208945047112337993992618254662119773565","date":"2025-09-29T23:16:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-29T17:24:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-01T11:33:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-01T05:24:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2025-09-01T05:22:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"80a1ba93-a11d-4eaf-b5e0-dd4d4173f03b","owner":[],"postedDate":"October 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:05:52+00:00","versionOfRecord":{"articleIdentity":"rs-7405418","link":"https://doi.org/10.1186/s12879-025-12482-1","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2026-02-07 15:59:37","publishedOnDateReadable":"February 7th, 2026"},"versionCreatedAt":"2025-10-12 14:28:25","video":"","vorDoi":"10.1186/s12879-025-12482-1","vorDoiUrl":"https://doi.org/10.1186/s12879-025-12482-1","workflowStages":[]},"version":"v1","identity":"rs-7405418","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7405418","identity":"rs-7405418","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}