Emergence of a novel FRI-type carbapenemase; blaFRI-12 in Enterobacter asburiae located on an IncR plasmid

Emergence of a novel FRI-type carbapenemase; blaFRI-12 in Enterobacter asburiae located on an IncR plasmid | 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 Short Report Emergence of a novel FRI-type carbapenemase; blaFRI-12 in Enterobacter asburiae located on an IncR plasmid Laura F. Mataseje, Florence Doualla-Bell, Ken Fakharuddin, Simon Wong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4650475/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jul, 2024 Read the published version in European Journal of Clinical Microbiology & Infectious Diseases → Version 1 posted 7 You are reading this latest preprint version Abstract Carbapenem-resistance in Enterobacter spp due to acquisition of mobile carbapenemases is of concern. An Enterobacter spp grew on ChromID CARBA medium and was positive for the mCIM carbapenemase detection assay. Susceptibility testing showed resistance to aztreonam and reduced susceptibility to imipenem. Conventional PCR using FRI primers detected a bla FRI gene. Whole genome sequencing reveled a new variant; bla FRI−12 was closest in sequence to bla FRI−5 differing by 13 amino acids and was found on a unique 110Kb IncR plasmid. Given the intrinsic nature of Enterobacter spp. to be carbapenem non-susceptible, bla FRI-types may be under reported globally. Figures Figure 1 Introduction A report on the global burden of bacterial antimicrobial resistance (AMR) from 2019 was published and indicated Enterobacter species were in the top 9 bacterial species associated with global deaths attributable to and associated with bacterial AMR in infections [ 1 ]. In 2020, Enterobacter species were found to be the top species to produce carbapenemases in Canadian hospitals, just behind Escherichia coli [ 2 ]. Enterobacter cloacae complex (ECC) are common nosocomial pathogens and often associated with multi-drug resistance including the carbapenems [ 3 ]. Though carbapenem resistance in ECC may be related to overexpression of a β-lactamase or AmpCs combined with decreased outer membrane permeability, it is the production of carbapenemases harboured on mobile genetic elements that are most concerning [ 3 ]. Minor class A carbapenemases, such as FRI-type are emerging (Bonnin et al., 2021). The first French imipenemase (FRI) variant was reported in 2015 isolated from Enterobacter cloacae [ 4 ]. Since then limited reports on FRI-type carbapenemases have been described and include those from the UK (FRI-2)[ 5 ], Germany (FRI-3)[ 6 ], Japan (FRI-4, FRI-5, FRI-7-11)[ 7 – 9 ] China (FRI-11)[ 10 ] and Canada (FRI-6, FRI-8)[ 11 , 12 ]. Here we describe a new FRI-type isolated from an Enterobacter asburiae in Canada. Materials and Methods Strain identification phenotypic and molecular testing Enterobacter spp N22-01531 was suspected to harbour a carbapenemase due to growth on ChromID CARBA medium from a rectal swab. Initial susceptibility testing determined that the isolate was resistant to ertapenem (2 mg/L) and imipenem (4 mg/L) and intermediate to meropenem (2 mg/L) by broth microdilution using CLSI guidelines (ref CLSI). Phenotypic testing was conducted using modified carbapenem inactivation method (mCIM) (Wick et al. 2017) (ref CLSI). Following a positive mCIM test this isolate was sent to the National Microbiology Laboratory (NML) for further testing. Additional phenotypic testing for carbapenemase production was done using the Rosco Neo-Rapid Carb Kit and β CARBA kit. Conventional PCR containing FRI specific primers (FRI-F-U1: 5’-TAAACTCAGCTATTCCAGGC-3’ and FRI-R-U2: 5’-ACAGGTGCCTGTTTTATCGCC-3’) was conducted yielding a positive result, Boyd et al, 2020. Antimicrobials susceptibility testing was done using the commercial Sensititre™ panel from Thermo Scientific™. Whole genome sequencing Once confirmed positive by FRI primers the isolate was sent for whole genome sequencing (WGS). DNA was extracted using Qiagen DNeasy kits (Qiagen, Toronto, Canada) and sequenced on an Illumina NextSeq™ platform using Nextera XT libraries. MinION sequencing (Nanopore Technologies, Oxford, UK) was conducted using the rapid kit (SQK-RBK 004) on R9.4.1 flowcells and run on Guppy 6.3.7 using the super accurate basecalling model. De novo hybrid assemblies were done using Unicycler 0.4.7[ 13 ]. Analysis was conducted using the StarAMR pipeline ( https://github.com/phac-nml/staramr ) for resistance gene detection, plasmid detection and multilocus sequence typing. Analysis of the genome using FastANI ( https://github.com/ParBLiSS/FastANI ) was used to determine species. Data for this project was deposited on NCBI under BioProject PRJNA865257. Results and Discussion A 91-year-old patient presenting with severe neuro-cognitive disorders entered the emergency department after suffering injuries from a fall during 2021. She was hospitalized for three weeks where she received a 2 day course of tazobactam. She had no known history of travel or acute infections or stay in palliative care. She was screened for CPE following a suspected outbreak of KPC on the floor where she is hospitalized. The result of her mCIM came back positive and no carbapenemase genes were found following testing for NDM, KPC, OXA-48-like, IMP, VIM, GES, NMC and IMI. The patient was moved to a residential and long-term care center and in June 2022 and tested positive for mCIM following routine screening practices. At this time PCR targets for FRI had been developed due to a separate FRI case reported in Canada [ 11 ]. The CPE isolate (N22-01531) was confirmed to be FRI-positive as were all retrospective isolates from this patient (data not shown). Susceptibility testing showed resistance to aztreonam only. The isolate was susceptible to meropenem and ertapenem while intermediate to imipenem (Table 1 ). Similarly to another report [ 14 ] novel β-lactam/ β-lactamase inhibitor combinations were effective against our clinical FRI containing isolate. As previously observed for FRI-6 (Boyd et al. 2020), phenotypic detection of carbapenemase using mCIM and Rosco Neo-Rapid tests exhibited positive results while β CARBA was negative. Concerning FRI-8 [ 12 ], only mCIM detected FRI production. Similar discrepancies between phenotypic assays were reported concerning other FRI-variants; indeed, while β CARBA didn’t detect FRI-1 [ 15 ], mCIM was positive for multiple variants (FRI-8, FRI-4, FRI-2, FRI-5) [ 5 , 7 , 8 , 16 ] and carba NP showed variable results (negative for FRI-8, inconsistent for FRI-11 [ 9 ] and positive for FRI-4, FRI-5, FRI-2, FRI-1 [ 4 , 5 , 7 , 8 ]). Interestingly, mCIM was positive for all FRI-variants positive, however, this test may exhibit false positive results with E. cloacae due to AmpC over production [ 17 , 18 ]. Indeed, further work needs to be done in FRI-type carbapenemase to avoid over testing E. cloacae non-carbapenemase producers using mCIM. Data from WGS revealed the presence of an Enterobacter asburiae harbouring the novel FRI-variant bla FRI−12 ( reference sequence number NG_081789.1). FRI-12 had the closest amino acid identity to FRI-5 differing by 13 amino acids (95.6% identity) (Supplementary Fig. 1). The bla FRI−12 gene was found on a 104 Kb IncR plasmid (pN22-01531, Fig. 1). The closest hit to a known plasmid was pKeioCLMIC23 harbouring bla FRI−5 (accession number AP028423.1) which shared 44% coverage at 95% identity. Indeed, when all other FRI plasmids were BLAST against pN22-01531only 5–17% coverage was observed. Previous reports have shown all bla FRI−type genes are harboured on plasmids containing the replicon IncFll-type [ 10 ]. However, some of these (FLC-1, FRI-2, FRI-4, FRI-9 and FRI-11) are multi-replicon plasmids that also contain IncR [ 10 ]. Interestingly, the IncFII-type FRI-5 plasmid (pKeioCLMIC23), which shared the closest identity to our FRI-12 plasmid did not contain the IncR replicon as observed in other FRI-variants. The alignment of FRI surrounding regions in different variants highlights a diverse arrangement of coding sequences (Supplementary Fig. 2). The FRI-12 surrounding region was most similar to FRI-5 where we observed a common 7 Kb of shared sequence representing FRI-12, FriR (a LysR transcriptional regulator), SafC and SafB (components of a saf operon for pili functionality). No additional resistance genes were found on the FRI-12 IncR plasmid. Similar to our previous reports,[ 11 , 12 ] we were able to successfully transform pN22-01531 into Escherichia coli TOP10 (labeled pFRI-12TF), however, conjugation experiments with Escherichia coli J53AzR were not successful. Other than with FRI-6 [ 11 ] this phenomenon is not uncommon in FRI-type plasmids. We cloned bla FRI−12 into pCR2.1TOPO and subsequently transformed this plasmid into E. coli TOP10 (pblaFRI-12) to test its antimicrobial susceptibility (Table 1 ). The transformant showed higher MICs than the clinical strain to aztreonam, ceftriaxone, cefoxitin, ertapenem, meropenem, imipenem, ceftolozone/tazobactam and piperacillin/tazobactam. However, the cloned bla FRI−12 gene showed higher MIC to piperacillin/tazobactam only. Further work will need to be conducted on FRI-12 plasmid copy number and gene expression to establish if they affect MIC to selected antimicrobials. To our knowledge, this is the first published report of bla FRI−12 identified globally. This study highlights the benefits of phenotypic screening of carbapenemases in Enterobacter sp. isolates exhibiting non-susceptible MICs to carbapenems despite susceptibility to cephalosporins. Table 1 Antimicrobial susceptibilities (mg/L) for strains tested in this report. Sensititre CAN1MSF E. asburiae N22-01531 E.coli TOP10 (pFRI-12TF)a E.coli TOP10 (pblaFRI-12) b E.coli TOP 10 amikacin <=8 S <=8 S <=8 S 16 R > 16 R <=1 S cefepime <=1 S <=1 S <=1 S <=1 S ceftazidime <=4 S <=4 S <=4 S <=4 S ceftriaxone <=1 S 4 R <=1 S <=1 S ceftazidime/avibactam <=4 S <=4 S <=4 S <=4 S ciprofloxacin <=0.06 S <=0.06 S <=0.06 S <=0.06 S colistin <=1 I <=1 I <=1 I <=1 I doxycycline <=4 S <=4 S <=4 S <=4 S ertapenem 2 R <=0.25 S <=0.25 S ceftolozone/tazobactam <=1 S 8 R <=1 S <=1 S gentamicin <=2 S <=2 S <=2 S <=2 S imipenem/relebactam 2 S <=1 S <=1 S <=1 S levofloxacin <=0.5 S <=0.5 S <=0.5 S <=0.5 S meropenem 0.25 S 4 R <=0.06 S <=0.06 S meropenem/vaborbactam <=1 S <=1 S <=1 S <=1 S minocycline <=4 S <=4 S <=4 S <=4 S piperacillin/tazobactam 64 R > 64 R <=8 S plazomicin <=1 S <=1 S <=1 S <=1 S tigecycline <=0.5 S <=0.5 S <=0.5 S <=0.5 S tobramycin <=2 S <=2 S <=2 S <=2 S trimethoprim/ sulfamethoxazole <=2 S <=2 S <=2 S <=2 S Etest cefoxitin 6 S 8 S 8 S < 0.25 S Imipenem 2 I 4 R 0.5 S 0.125 S Bold formating indicates resistant phenotype according to CLSI (M100:2023). S-susceptible; I-intermediate; R-resistant a E.coli TOP10 electrotransformed with pFRI-12 b bla FRI−12 and upstream region amplified from N22-01531 with primers FRI-12A (forward-GATTTCTCATTGTATACCAACC) and FRI-12B (reverse-TGGCGGACATTTCATGGCGC), cloned into pCR21.TOPO (ThermoFisher Scientific). pCR2.TOPO contains an ampicillin resistance gene ( bla TEM−1 ) and kanamycin resistance gene (aphII). Figure 1. FRI-12 plasmid generated with Proksee. Innermost two tracks represent forward and reverse open reading frames of pN22-01531. Third track represents BLASTn of FRI-12 plasmid to its closest related plasmid (pFRI-5). Outer two tracks are annotations generated by CARD RGI and mobileOG databases. Supplementary Fig. 1. Amino Acid alignment of FRI-variants and phylogenetic tree showing relatedness among FRI-type sequences. Conserved Ambler class A regions; active site motifs 70 SXXK 73 , 130 SDN 132 , 166 EXXXN 170 and 234 KTG 236 and cysteine residues C 69 and C 238 are shown [ 6 ]. Supplementary Fig. 2. Schematic representation of FRI region among all FRI variants. Black arrows are hypothetical proteins. All other arrows are labelled. Declarations The authors have no relevant financial or non-financial interests to disclose. Funding: Funding was provided by the Public Health Agency of Canada. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LFM, FD-B, KF, SW, and AY. The first draft of the manuscript and all figures were composed by LM and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement We would like to acknowledge the work of the late Dr. Michael Mulvey who championed the discovery and research of AMR in Canada and for whom this work would not have been made possible. We would like to thank the DNA Core Facility at the National Microbiology Laboratory for Illumina sequencing. Data Availability The datasets generated during and/or analysed during the current study are available in the NCBI repository under BioProject PRJNA865257. References Antimicrobial Resistance Collaborators Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022 12;399:629–55. 10.1016/S0140-6736(21)02724-0 Canada PHA of. Healthcare-associated infections and antimicrobial resistance in Canadian acute care hospitals, 2016 to 2020, CCDR 48(7/8) [Internet]. 2022 3 [cited 2023 17];doi: https://www.canada.ca/en/public-health/services/reports-publications/canada-communicable-disease-report-ccdr/monthly-issue/2022-48/issue-7-8-july-august-2022/healthcare-associated-infections-antimicrobial-resistance-canadian-acute-care-hospitals-2016-2020.html Nordmann P, Dortet L, Poirel L (2012) Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol Med 18:263–272. 10.1016/j.molmed.2012.03.003 Dortet L, Poirel L, Abbas S, Oueslati S, Nordmann P (2015) Genetic and Biochemical Characterization of FRI-1, a Carbapenem-Hydrolyzing Class A β-Lactamase from Enterobacter cloacae. Antimicrob Agents Chemother 59:7420–7425. 10.1128/AAC.01636-15 Meunier D, Findlay J, Doumith M, Godoy D, Perry C, Pike R et al FRI-2 carbapenemase-producing Enterobacter cloacae complex in the UK. J Antimicrob Chemother 2017 1;72:2478–2482. 10.1093/jac/dkx173 Schauer J, Gatermann SG, Marschal M, Pfennigwerth N (2019) Genetic and biochemical characterization of FRI-3, a novel variant of the Ambler class A carbapenemase FRI-1. J Antimicrob Chemother 174:2891–2894. 10.1093/jac/dkz295 Kubota H, Uwamino Y, Matsui M, Sekizuka T, Suzuki Y, Okuno R et al (2018) FRI-4 carbapenemase-producing Enterobacter cloacae complex isolated in Tokyo, Japan. J Antimicrob Chemother 173:2969–2972. 10.1093/jac/dky291 Uwamino Y, Kubota H, Sasaki T, Kosaka A, Furuhashi M, Uno S et al (2019) Recovery of FRI-5 carbapenemase at a Japanese hospital where FRI-4 carbapenemase was discovered. J Antimicrob Chemother 174:3390–3392. 10.1093/jac/dkz336 Gomi R, Matsumura Y, Tanaka M, Ihara M, Sugie Y, Matsuda T et al Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC and SFH-1) in Enterobacterales isolated from surface waters in Japan. J Antimicrob Chemother 2022 27;77:1237–1246. 10.1093/jac/dkac029 Wu S, He Y, Feng Y, Zong Z (2023) A rare class A carbapenemase FRI-11 in Enterobacter clinical strain. Eur J Clin Microbiol Infect Dis 42:513–517. 10.1007/s10096-023-04565-1 Boyd DA, Lefebvre B, Mataseje LF, Gagnon S, Roger M, Savard P et al Enterobacter sp. N18-03635 harbouring blaFRI-6 class A carbapenemase, Canada. J Antimicrob Chemother 2020 1;75:486–488. 10.1093/jac/dkz438 Mataseje LF, Doualla-Bell F, Boyd DA, Fakharuddin K, Felipe Garcia Jeldes H, Plante V et al (2023) Genetic and Phenotypic Characterization of the First Canadian Case of Ambler Class A Carbapenemase FRI-8. Microb Drug Resist 29:47–50. 10.1089/mdr.2022.0123 Wick RR, Judd LM, Gorrie CL, Holt KE (2017) Completing bacterial genome assemblies with multiplex MinION sequencing. Microb Genomics 3:e000132. 10.1099/mgen.0.000132 Sadek M, Bosch Duran J, Poirel L, Nordmann P Impact of Minor Carbapenemases on Susceptibility to Novel β-Lactam/β-Lactamase Inhibitor Combinations and Cefiderocol in Enterobacterales. Antimicrob Agents Chemother 2023 17;67:e0007823. 10.1128/aac.00078-23 Bernabeu S, Dortet L, Naas T Evaluation of the β-CARBA ™ test, a colorimetric test for the rapid detection of carbapenemase activity in Gram-negative bacilli. J Antimicrob Chemother 2017 1;72:1646–1658. 10.1093/jac/dkx061 Adachi F, Sekizuka T, Yamato M, Fukuoka K, Yamaguchi N, Kuroda M et al (2021) Characterization of FRI carbapenemase-producing Enterobacter spp. isolated from a hospital and the environment in Osaka, Japan. J Antimicrob Chemother 1176:3061–3062. 10.1093/jac/dkab284 Tamma PD, Simner PJ Phenotypic Detection of Carbapenemase-Producing Organisms from Clinical Isolates. J Clin Microbiol 2018 25;56: 10.1128/jcm.01140-18 . doi: 10.1128/jcm.01140-18 Muntean MM, Muntean A-A, Guerin F, Cattoir V, Creton E, Cotellon G et al Optimization of the rapid carbapenem inactivation method for use with AmpC hyperproducers. J Antimicrob Chemother 2021 12;76:2294–2301. 10.1093/jac/dkab170 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1FRIAAalignmentv3.png SupplementaryFigure2FRIregionalignmentv2.png Cite Share Download PDF Status: Published Journal Publication published 24 Jul, 2024 Read the published version in European Journal of Clinical Microbiology & Infectious Diseases → Version 1 posted Editorial decision: Accepted 19 Jul, 2024 Reviews received at journal 12 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers invited by journal 02 Jul, 2024 Editor assigned by journal 28 Jun, 2024 Submission checks completed at journal 28 Jun, 2024 First submitted to journal 27 Jun, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4650475","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":329091921,"identity":"4153c380-14da-4c21-8207-c021abba5739","order_by":0,"name":"Laura F. 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Innermost two tracks represent forward and reverse open reading frames of pN22-01531. Third track represents BLASTn of FRI-12 plasmid to its closest related plasmid (pFRI-5). Outer two tracks are annotations generated by CARD RGI and mobileOG databases.\u003c/p\u003e","description":"","filename":"Figure1pN2201531FRI12.png","url":"https://assets-eu.researchsquare.com/files/rs-4650475/v1/77146adee5e5c336dac8e21b.png"},{"id":61596132,"identity":"c7b6be3c-fd16-476f-a81a-31933d8763e2","added_by":"auto","created_at":"2024-08-01 17:25:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3106295,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4650475/v1/e9a821a0-94d8-4f64-81f3-4c0449cf0408.pdf"},{"id":60847495,"identity":"b20afc1b-5b08-4127-9612-4cb5b9e97782","added_by":"auto","created_at":"2024-07-22 19:35:26","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6660967,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1FRIAAalignmentv3.png","url":"https://assets-eu.researchsquare.com/files/rs-4650475/v1/ec94f4aae881e935d509f35d.png"},{"id":60847496,"identity":"31778169-00d1-46a5-8320-313d33b55e44","added_by":"auto","created_at":"2024-07-22 19:35:26","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3322743,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2FRIregionalignmentv2.png","url":"https://assets-eu.researchsquare.com/files/rs-4650475/v1/37785c8324ad6dc30eae666c.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emergence of a novel FRI-type carbapenemase; blaFRI-12 in Enterobacter asburiae located on an IncR plasmid","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA report on the global burden of bacterial antimicrobial resistance (AMR) from 2019 was published and indicated \u003cem\u003eEnterobacter\u003c/em\u003e species were in the top 9 bacterial species associated with global deaths attributable to and associated with bacterial AMR in infections [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In 2020, \u003cem\u003eEnterobacter\u003c/em\u003e species were found to be the top species to produce carbapenemases in Canadian hospitals, just behind \u003cem\u003eEscherichia coli\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eEnterobacter cloacae complex\u003c/em\u003e (ECC) are common nosocomial pathogens and often associated with multi-drug resistance including the carbapenems [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Though carbapenem resistance in ECC may be related to overexpression of a β-lactamase or AmpCs combined with decreased outer membrane permeability, it is the production of carbapenemases harboured on mobile genetic elements that are most concerning [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Minor class A carbapenemases, such as FRI-type are emerging (Bonnin et al., 2021). The first French imipenemase (FRI) variant was reported in 2015 isolated from \u003cem\u003eEnterobacter cloacae\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Since then limited reports on FRI-type carbapenemases have been described and include those from the UK (FRI-2)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], Germany (FRI-3)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], Japan (FRI-4, FRI-5, FRI-7-11)[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] China (FRI-11)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and Canada (FRI-6, FRI-8)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Here we describe a new FRI-type isolated from an \u003cem\u003eEnterobacter asburiae\u003c/em\u003e in Canada.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrain identification phenotypic and molecular testing\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEnterobacter spp\u003c/em\u003e N22-01531 was suspected to harbour a carbapenemase due to growth on ChromID CARBA medium from a rectal swab. Initial susceptibility testing determined that the isolate was resistant to ertapenem (2 mg/L) and imipenem (4 mg/L) and intermediate to meropenem (2 mg/L) by broth microdilution using CLSI guidelines (ref CLSI). Phenotypic testing was conducted using modified carbapenem inactivation method (mCIM)\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(Wick et al. 2017)\u003c/span\u003e (ref CLSI). Following a positive mCIM test this isolate was sent to the National Microbiology Laboratory (NML) for further testing. Additional phenotypic testing for carbapenemase production was done using the Rosco Neo-Rapid Carb Kit and β CARBA kit. Conventional PCR containing FRI specific primers (FRI-F-U1: 5\u0026rsquo;-TAAACTCAGCTATTCCAGGC-3\u0026rsquo; and FRI-R-U2: 5\u0026rsquo;-ACAGGTGCCTGTTTTATCGCC-3\u0026rsquo;) was conducted yielding a positive result, Boyd et al, 2020. Antimicrobials susceptibility testing was done using the commercial Sensititre\u0026trade; panel from Thermo Scientific\u0026trade;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome sequencing\u003c/h2\u003e \u003cp\u003eOnce confirmed positive by FRI primers the isolate was sent for whole genome sequencing (WGS). DNA was extracted using Qiagen DNeasy kits (Qiagen, Toronto, Canada) and sequenced on an Illumina NextSeq\u0026trade; platform using Nextera XT libraries. MinION sequencing (Nanopore Technologies, Oxford, UK) was conducted using the rapid kit (SQK-RBK 004) on R9.4.1 flowcells and run on Guppy 6.3.7 using the super accurate basecalling model. De novo hybrid assemblies were done using Unicycler 0.4.7[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Analysis was conducted using the StarAMR pipeline (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/phac-nml/staramr\u003c/span\u003e\u003cspan address=\"https://github.com/phac-nml/staramr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for resistance gene detection, plasmid detection and multilocus sequence typing. Analysis of the genome using FastANI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ParBLiSS/FastANI\u003c/span\u003e\u003cspan address=\"https://github.com/ParBLiSS/FastANI\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to determine species. Data for this project was deposited on NCBI under BioProject PRJNA865257.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eA 91-year-old patient presenting with severe neuro-cognitive disorders entered the emergency department after suffering injuries from a fall during 2021. She was hospitalized for three weeks where she received a 2 day course of tazobactam. She had no known history of travel or acute infections or stay in palliative care. She was screened for CPE following a suspected outbreak of KPC on the floor where she is hospitalized. The result of her mCIM came back positive and no carbapenemase genes were found following testing for NDM, KPC, OXA-48-like, IMP, VIM, GES, NMC and IMI. The patient was moved to a residential and long-term care center and in June 2022 and tested positive for mCIM following routine screening practices. At this time PCR targets for FRI had been developed due to a separate FRI case reported in Canada [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The CPE isolate (N22-01531) was confirmed to be FRI-positive as were all retrospective isolates from this patient (data not shown).\u003c/p\u003e \u003cp\u003eSusceptibility testing showed resistance to aztreonam only. The isolate was susceptible to meropenem and ertapenem while intermediate to imipenem (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similarly to another report [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] novel β-lactam/ β-lactamase inhibitor combinations were effective against our clinical FRI containing isolate. As previously observed for FRI-6 (Boyd et al. 2020), phenotypic detection of carbapenemase using mCIM and Rosco Neo-Rapid tests exhibited positive results while β CARBA was negative. Concerning FRI-8 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], only mCIM detected FRI production. Similar discrepancies between phenotypic assays were reported concerning other FRI-variants; indeed, while β CARBA didn\u0026rsquo;t detect FRI-1 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], mCIM was positive for multiple variants (FRI-8, FRI-4, FRI-2, FRI-5) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and carba NP showed variable results (negative for FRI-8, inconsistent for FRI-11 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and positive for FRI-4, FRI-5, FRI-2, FRI-1 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]). Interestingly, mCIM was positive for all FRI-variants positive, however, this test may exhibit false positive results with \u003cem\u003eE. cloacae\u003c/em\u003e due to AmpC over production [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Indeed, further work needs to be done in FRI-type carbapenemase to avoid over testing \u003cem\u003eE. cloacae\u003c/em\u003e non-carbapenemase producers using mCIM.\u003c/p\u003e \u003cp\u003eData from WGS revealed the presence of an \u003cem\u003eEnterobacter asburiae\u003c/em\u003e harbouring the novel FRI-variant \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI\u0026minus;12 (\u003c/sub\u003ereference sequence number NG_081789.1). FRI-12 had the closest amino acid identity to FRI-5 differing by 13 amino acids (95.6% identity) (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI\u0026minus;12\u003c/sub\u003e gene was found on a 104 Kb IncR plasmid (pN22-01531, Fig.\u0026nbsp;1). The closest hit to a known plasmid was pKeioCLMIC23 harbouring \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI\u0026minus;5\u003c/sub\u003e (accession number AP028423.1) which shared 44% coverage at 95% identity. Indeed, when all other FRI plasmids were BLAST against pN22-01531only 5\u0026ndash;17% coverage was observed. Previous reports have shown all \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI\u0026minus;type\u003c/sub\u003e genes are harboured on plasmids containing the replicon IncFll-type [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, some of these (FLC-1, FRI-2, FRI-4, FRI-9 and FRI-11) are multi-replicon plasmids that also contain IncR [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Interestingly, the IncFII-type FRI-5 plasmid (pKeioCLMIC23), which shared the closest identity to our FRI-12 plasmid did not contain the IncR replicon as observed in other FRI-variants. The alignment of FRI surrounding regions in different variants highlights a diverse arrangement of coding sequences (Supplementary Fig.\u0026nbsp;2). The FRI-12 surrounding region was most similar to FRI-5 where we observed a common 7 Kb of shared sequence representing FRI-12, FriR (a LysR transcriptional regulator), SafC and SafB (components of a \u003cem\u003esaf\u003c/em\u003e operon for pili functionality). No additional resistance genes were found on the FRI-12 IncR plasmid.\u003c/p\u003e \u003cp\u003eSimilar to our previous reports,[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] we were able to successfully transform pN22-01531 into \u003cem\u003eEscherichia coli\u003c/em\u003e TOP10 (labeled pFRI-12TF), however, conjugation experiments with \u003cem\u003eEscherichia coli\u003c/em\u003e J53AzR were not successful. Other than with FRI-6 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] this phenomenon is not uncommon in FRI-type plasmids. We cloned \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI\u0026minus;12\u003c/sub\u003e into pCR2.1TOPO and subsequently transformed this plasmid into \u003cem\u003eE. coli\u003c/em\u003e TOP10 (pblaFRI-12) to test its antimicrobial susceptibility (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The transformant showed higher MICs than the clinical strain to aztreonam, ceftriaxone, cefoxitin, ertapenem, meropenem, imipenem, ceftolozone/tazobactam and piperacillin/tazobactam. However, the cloned \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI\u0026minus;12\u003c/sub\u003e gene showed higher MIC to piperacillin/tazobactam only. Further work will need to be conducted on FRI-12 plasmid copy number and gene expression to establish if they affect MIC to selected antimicrobials.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first published report of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI\u0026minus;12\u003c/sub\u003e identified globally. This study highlights the benefits of phenotypic screening of carbapenemases in \u003cem\u003eEnterobacter\u003c/em\u003e sp. isolates exhibiting non-susceptible MICs to carbapenems despite susceptibility to cephalosporins.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial susceptibilities (mg/L) for strains tested in this report.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSensititre CAN1MSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. asburiae\u003c/em\u003e N22-01531\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eE.coli\u003c/em\u003e TOP10 (pFRI-12TF)a\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eE.coli\u003c/em\u003e TOP10 (pblaFRI-12)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eE.coli\u003c/em\u003e TOP 10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eamikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eaztreonam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eceftazidime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eceftriaxone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eceftazidime/avibactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eciprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecolistin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003edoxycycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eertapenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eceftolozone/tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egentamicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eimipenem/relebactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elevofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emeropenem/vaborbactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eminocycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epiperacillin/tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;64\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;64\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eplazomicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etigecycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etobramycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etrimethoprim/ sulfamethoxazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;=2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEtest\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecefoxitin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBold formating indicates resistant phenotype according to CLSI (M100:2023). S-susceptible; I-intermediate; R-resistant\u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eE.coli TOP10 electrotransformed with pFRI-12\u003c/p\u003e \u003cp\u003e \u003csup\u003eb\u003c/sup\u003e \u003cem\u003ebla\u003c/em\u003e \u003csub\u003eFRI\u0026minus;12\u003c/sub\u003e and upstream region amplified from N22-01531 with primers FRI-12A (forward-GATTTCTCATTGTATACCAACC) and FRI-12B (reverse-TGGCGGACATTTCATGGCGC), cloned into pCR21.TOPO (ThermoFisher Scientific). pCR2.TOPO contains an ampicillin resistance gene (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1\u003c/sub\u003e) and kanamycin resistance gene (aphII).\u003c/p\u003e \u003cp\u003eFigure 1. FRI-12 plasmid generated with Proksee. Innermost two tracks represent forward and reverse open reading frames of pN22-01531. Third track represents BLASTn of FRI-12 plasmid to its closest related plasmid (pFRI-5). Outer two tracks are annotations generated by CARD RGI and mobileOG databases.\u003c/p\u003e \u003cp\u003eSupplementary Fig.\u0026nbsp;1. Amino Acid alignment of FRI-variants and phylogenetic tree showing relatedness among FRI-type sequences. Conserved Ambler class A regions; active site motifs \u003csup\u003e70\u003c/sup\u003eSXXK\u003csup\u003e73\u003c/sup\u003e, \u003csup\u003e130\u003c/sup\u003eSDN\u003csup\u003e132\u003c/sup\u003e, \u003csup\u003e166\u003c/sup\u003eEXXXN\u003csup\u003e170\u003c/sup\u003e and \u003csup\u003e234\u003c/sup\u003eKTG\u003csup\u003e236\u003c/sup\u003e and cysteine residues C\u003csup\u003e69\u003c/sup\u003e and C\u003csup\u003e238\u003c/sup\u003e are shown [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSupplementary Fig.\u0026nbsp;2. Schematic representation of FRI region among all FRI variants. Black arrows are hypothetical proteins. All other arrows are labelled.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eFunding was provided by the Public Health Agency of Canada.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LFM, FD-B, KF, SW, and AY. The first draft of the manuscript and all figures were composed by LM and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to acknowledge the work of the late Dr. Michael Mulvey who championed the discovery and research of AMR in Canada and for whom this work would not have been made possible. We would like to thank the DNA Core Facility at the National Microbiology Laboratory for Illumina sequencing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available in the NCBI repository under BioProject PRJNA865257.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAntimicrobial Resistance Collaborators Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022 12;399:629\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(21)02724-0\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(21)02724-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanada PHA of. 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J Antimicrob Chemother 1176:3061\u0026ndash;3062. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jac/dkab284\u003c/span\u003e\u003cspan address=\"10.1093/jac/dkab284\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamma PD, Simner PJ Phenotypic Detection of Carbapenemase-Producing Organisms from Clinical Isolates. J Clin Microbiol 2018 25;56:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jcm.01140-18\u003c/span\u003e\u003cspan address=\"10.1128/jcm.01140-18\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. doi: 10.1128/jcm.01140-18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuntean MM, Muntean A-A, Guerin F, Cattoir V, Creton E, Cotellon G et al Optimization of the rapid carbapenem inactivation method for use with AmpC hyperproducers. J Antimicrob Chemother 2021 12;76:2294\u0026ndash;2301. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jac/dkab170\u003c/span\u003e\u003cspan address=\"10.1093/jac/dkab170\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"european-journal-of-clinical-microbiology-and-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejcm","sideBox":"Learn more about [European Journal of Clinical Microbiology \u0026 Infectious Diseases](https://www.springer.com/journal/10096)","snPcode":"10096","submissionUrl":"https://submission.nature.com/new-submission/10096/3","title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4650475/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4650475/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCarbapenem-resistance in \u003cem\u003eEnterobacter\u003c/em\u003e spp due to acquisition of mobile carbapenemases is of concern. An \u003cem\u003eEnterobacter\u003c/em\u003e spp grew on ChromID CARBA medium and was positive for the mCIM carbapenemase detection assay. Susceptibility testing showed resistance to aztreonam and reduced susceptibility to imipenem. Conventional PCR using FRI primers detected a \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI\u003c/sub\u003e gene. Whole genome sequencing reveled a new variant; \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI−12\u003c/sub\u003e was closest in sequence to \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eFRI−5\u003c/sub\u003e differing by 13 amino acids and was found on a unique 110Kb IncR plasmid. Given the intrinsic nature of \u003cem\u003eEnterobacter\u003c/em\u003e spp. to be carbapenem non-susceptible, \u003cem\u003ebla\u003c/em\u003eFRI-types may be under reported globally.\u003c/p\u003e","manuscriptTitle":"Emergence of a novel FRI-type carbapenemase; blaFRI-12 in Enterobacter asburiae located on an IncR plasmid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 19:35:22","doi":"10.21203/rs.3.rs-4650475/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2024-07-19T10:00:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T08:30:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25998704347942834884228583697557359992","date":"2024-07-02T09:13:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-02T08:16:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-28T07:55:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-28T07:54:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","date":"2024-06-27T17:53:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejcm","sideBox":"Learn more about [European Journal of Clinical Microbiology \u0026 Infectious Diseases](https://www.springer.com/journal/10096)","snPcode":"10096","submissionUrl":"https://submission.nature.com/new-submission/10096/3","title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"586228bc-75f4-4526-9dd8-89215b667fb7","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:07:36+00:00","versionOfRecord":{"articleIdentity":"rs-4650475","link":"https://doi.org/10.1007/s10096-024-04907-7","journal":{"identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isVorOnly":false,"title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases"},"publishedOn":"2024-07-24 16:15:56","publishedOnDateReadable":"July 24th, 2024"},"versionCreatedAt":"2024-07-22 19:35:22","video":"","vorDoi":"10.1007/s10096-024-04907-7","vorDoiUrl":"https://doi.org/10.1007/s10096-024-04907-7","workflowStages":[]},"version":"v1","identity":"rs-4650475","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4650475","identity":"rs-4650475","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}