Abbott, I.J. (Iain)

Bonten, M.M. (Marc M.)

Paganelli, F.L.

Rogers, M.R.C.

Ten Doesschate, T. (Thijs)

Top, J. (Janetta)

Willems, R.J.L. (Rob)

Erasmus University Digital Repository

Tet(X4) in A. caviae, a potential reservoir for the dissemination oftigecycline resistance into different environmental niches. Thetet(X4)-carrying element analysis suggests that the catD-tet(X4)-ISCR2 gene cassette is highly active and may further spread intodifferent bacterial species. Continuous monitoring of tet(X4) inhumans, animals and environments should be considered, to im-prove understanding and address the spread of resistance to tet-racyclines, including tigecycline.AcknowledgementsWe thank Ke Cheng, Guo-Hui Deng, Jia-Qi Song and Bo Jiang (SouthChina Agricultural University) for sample collecting.FundingThis work was jointly supported by the National Key Research andDevelopment Program of China (2016YFD0501300), the Program forInnovative Research Team in the University of Ministry of Educationof China (IRT_17R39) and the Foundation for Innovation andStrengthening School Project of Guangdong, China (2016KCXTD010).Transparency declarationsNone to declare.Supplementary dataTables S1 and S2 are available as Supplementary data at JAC Online.References1 He T, Wang R, Liu D et al. Emergence of plasmid-mediated high-level tige-cycline resistance genes in animals and humans. Nat Microbiol 2019; 4:1450–6.2 Sun J, Chen C, Cui C-Y et al. Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli. Nat Microbiol 2019;4: 1457–64.3 Batra P, Mathur P, Misra MC. Aeromonas spp.: an emerging nosocomialpathogen. J Lab Physicians 2016;8: 1–4.4 Walsh TR, Weeks J, Livermore DM et al. Dissemination of NDM-1 positivebacteria in the New Delhi environment and its implications for human health:an environmental point prevalence study. Lancet Infect Dis 2011; 11:355–62.5 Wang X, Zhai W, Li J et al. Presence of an mcr-3 variant in Aeromonas cav-iae, Proteus mirabilis, and Escherichia coli from one domestic duck. AntimicrobAgents Chemother 2018;62: e02106-17.6 Ling Z, Yin W, Li H et al. Chromosome-mediated mcr-3 variants inAeromonas veronii from chicken meat. Antimicrob Agents Chemother 2017;61: e01272-17.7 Clinical and Laboratory Standards Institute. Methods for AntimicrobialDilution and Disk Susceptibility Testing of Infrequently Isolated or FastidiousBacteria—Second Edition: M45. CLSI, Wayne, PA, USA, 2010.8 Wick RR, Judd LM, Gorrie CL et al. Unicycler: resolving bacterial genome as-semblies from short and long sequencing reads. PLoS Comput Biol 2017; 13:e1005595.9 Poirel L, Mugnier PD, Toleman MA et al. ISCR2, another vehiclefor blaVEB gene acquisition. Antimicrob Agents Chemother 2009; 53:4940–3.10 He YZ, Li XP, Miao YY et al. The ISApl12 dimer circular intermediate partici-pates in mcr-1 transposition. Front Microbiol 2019;10: 15.J Antimicrob Chemother 2019; 74: 3630–3632doi:10.1093/jac/dkz380Advance Access publication 11 September 2019In vivo acquisition of fosfomycinresistance in Escherichia coli by fosAtransmission from commensal floraThijs ten Doesschate1*, Iain J. Abbott2†,Rob J. L. Willems3, Janetta Top3, Malbert R. C. Rogers3,Marc M. Bonten3 and Fernanda L. Paganelli31Julius Center for Health Sciences and Primary Care, UniversityMedical Center Utrecht, University of Utrecht, Utrecht,The Netherlands; 2Department of Medical Microbiology andInfectious Diseases, Erasmus Medical Center, Rotterdam,The Netherlands; 3Department of Medical Microbiology,University Medical Center Utrecht, University of Utrecht, Utrecht,The Netherlands*Corresponding author. E-mail: t.tendoesschate@umcutrecht.nl†Present address: Department of Infectious Diseases, Alfred Hospitaland Central Clinical School, Monash University, Melbourne, VIC, Australia.Sir,Fosfomycin is increasingly used to treat infections caused by MDRbacteria.1 Fosfomycin acts by inhibiting UDP-N-acetylglucosamineenolpyruvyl transferase (murA), which prevents the formation of N-acetylmuramic acid, an essential component of peptidoglycan.1Although resistance to fosfomycin is still low in Escherichia coli, theacquisition of fosA may reduce future activity of fosfomycin to treatinfections caused by E. coli.2 FosA is a glutathione transferase thatinactivates fosfomycin through catalysing the addition of glutathi-one. fosA genes are often present in the chromosome of Klebsiellapneumoniae, but not in the chromosome of E. coli.2,3 Klebsiella varii-cola is closely related and often misidentified as K. pneumoniae.4While horizontal spread of fosA has been demonstrated in vitro,5we here provide evidence for in vivo fosA transmission from K. varii-cola to E. coli, resulting in development of fosfomycin resistance.VC The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creati-vecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, providedthe original work is properly cited. For commercial re-use, please contact journals.permissions@oup.comResearch letters3630Downloaded from https://academic.oup.com/jac/article-abstract/74/12/3630/5567518 by Erasmus University Rotterdam user on 27 November 2019The Medical Research Ethics Committee of the UniversityMedical Center Utrecht confirmed that the Medical ResearchInvolving Human Subjects Act does not apply to this study (refer-ence number WAG/mb/18/027282). We were not able to obtain in-formed consent because the patient died a few years ago. Allinformation including gender, age, dates and medical history thatwas not directly clinically relevant has been omitted to protect theprivacy of the patient.An aged patient had a suspicion of chronic endovascular infectionof their aortic bifurcation graft, which the patient received after anacute aortic aneurysm 22 years earlier. The patient had suffered fromrecurrent episodes of sepsis, with blood cultures yieldingPropionibacterium spp., K. variicola, Citrobacter koseri andPseudomonas aeruginosa, as determined by MALDI-TOF MS. Positronemission tomography (PET)-CT findings were compatible with pros-thetic graft infection. The patient subsequently developed septicshock with E. coli bacteraemia without a clear source of infection thatwas treated successfully with intravenous ceftriaxone. The isolatewas resistant to amoxicillin/clavulanic acid and ciprofloxacin that hadbeen used to suppress chronic infection, prompting the addition oforal fosfomycin at 3 g every 48 h. Seven months later, while still usingfosfomycin, the patient developed spondylodiscitis. Blood culturesdrawn at the time isolated E. coli with an identical resistance pattern,except being resistant to fosfomycin. Fosfomycin was discontinuedand the patient received a prolonged course of ceftriaxone.Fosfomycin susceptibility, determined by agar dilution accord-ing to CSLI guidelines,6 demonstrated a rise in the MIC from 2 mg/Lfor the initial E. coli isolate to.1024 mg/L for the second E. coli iso-late. WGS revealed five SNP differences between E. coli isolates inthe core genome, based on core genome MLST (cgMLST) analysis.7Yet, the second E. coli isolate has a 3573 bp insertion consisting ofISEcp1, a fosA gene we named fosA9 as the next available numberaccording to NCBI, syrM1 and lysN2. The insertion is flanked by 5 bpDRs (AAAAA) suggesting mobilization of this fosA9 gene cluster byISEcp1 (Figure 1).8 Genes other than fosA9 responsible for fosfomy-cin resistance were not found. At the time of the first E. coli sepsisepisode, six K. variicola had been isolated from rectum swabs andblood cultures over a period of 20 months (Table S1, available asSupplementary data at JAC Online). cgMLST analysis revealed amaximum of 16 SNP differences between K. variicola isolates.7 Thesame cluster as above containing fosA9, without the mobile ge-netic element ISEcp1, was identified in the K. variicola isolates, sug-gesting K. variicola to be the source of fosA9 acquired by E. coli(Figure 1). fosA genes were not identified in other clinical isolatesfrom this patient. Sequence information of all isolates has been de-posited in the European Nucleotide Archive (ENA) under projectnumber PRJEB32329.fosA transfer from Klebsiella spp. to E. coli, leading to fosfo-mycin resistance, has been demonstrated in vitro.3 Based onpublicly available genomes, fosA and adjacent genes are wellconserved in K. variicola (minimum 98% identity to fosA9) and K.pneumoniae (minimum 94% identity to fosA9) isolates.According to mlplasmids, PlasmidFinder and contig coverage,fosA9 was predicted to be located in the chromosome of thesecond E. coli and all K. variicola isolates.9,10 However, based onBLASTn, the contig containing fosA9 aligns to plasmid sequen-ces. The localization of fosA9 in E. coli can thus only be con-firmed by completely assembling its genome using long-readsequencing, as the mobilization of the fosA9 gene cluster by anIS element might switch its genomic background. We postulatethat fosA9 transfer from K. variicola to E. coli occurred in the gas-trointestinal tract, as K. variicola was not co-cultured in theblood at the time of E. coli bacteraemia. We hypothesize thatfosfomycin pressure played a role in this transfer; however, thisFigure 1. Schematic representation of the contig (ECO-BAB-IMI-103297_P-ACH-BAB-IMI-103242_1528359160_131_length_8653_cov_18.1163_ID_8928, 8653 bp) in the fosfomycin-resistant E. coli isolate containing a fosA9 gene cluster originating from a K. variicola isolate. TheISEcp1-syrM1-fosA9-lysN2 region is flanked by 5 bp DRs (AAAAA), suggesting mobilization from K. variicola by ISEcp1. Upstream and downstreamsequences of the insertion region align to contig ECO-BAB-IMI-103298_P-ACH-BAB-IMI-103242_1528359160_92_length_16411_cov_29.2905_ID_8090 from the first susceptible E. coli isolate. Sequence information of complete genomes of all isolates and separate sequences of the relevantcontigs (containing fosA9 in E. coli and K. variicola, and ECO-BAB-IMI-103298_P-ACH-BAB-IMI-103242_1528359160_92_length_16411_cov_29.2905_ID_8090 from the susceptible E. coli) have been deposited in the ENA under project number PRJEB32329. This figure appears in colour in the onlineversion of JAC and in black and white in the print version of JAC.Research letters JAC3631Downloaded from https://academic.oup.com/jac/article-abstract/74/12/3630/5567518 by Erasmus University Rotterdam user on 27 November 2019has to be confirmed with further experiments in vitro.Acquisition of fosA9 was associated with an 8-fold increase inthe MIC for E. coli (from 2 to 1024 mg/L) while, despite the pres-ence of fosA9 in the chromosome of the K. variicola isolates, thefosfomycin MICs were below the EUCAST susceptibility break-point of 32 mg/L (Table S1).6 This could suggest either higherdependency of E. coli growth on glutathione or a difference infosA9 expression or metabolism, i.e. higher expression by theISEcp1 promoter present upstream of the fosA9 gene cluster.8In conclusion, our case illustrates the potential of long-termuse of oral fosfomycin to promote horizontal gene transfer offosA9 from commensal gut flora to potential pathogenic microor-ganisms, such as E. coli.AcknowledgementsWe are grateful to Dr Ad C. Fluit and Dr Anita Schurch from the UniversityMedical Center Utrecht for the critical appraisal of this case report priorto submission.FundingThis study was carried out as part of our routine work.Transparency declarationsNone to declare.Supplementary dataTable S1 is available as Supplementary data at JAC Online.References1 Karageorgopoulos DE, Wang R, Yu XH et al. Fosfomycin: evaluation of thepublished evidence on the emergence of antimicrobial resistance in Gram-negative pathogens. J Antimicrob Chemother 2012;67: 255–68.2 Ito R, Mustapha MM, Tomich AD et al. Widespread fosfomycin resistance inGram-negative bacteria attributable to the chromosomal fosA gene. MBio2017;8: e00749–17.3 Guo Q, Tomich AD, McElheny CL et al. Glutathione-S-transferase FosA6 ofKlebsiella pneumoniae origin conferring fosfomycin resistance in ESBL-producing Escherichia coli. J Antimicrob Chemother 2016;71: 2460–5.4 Linson SE, Long SW, Ojeda Saavedra M et al. Whole-genome sequencingof human clinical Klebsiella pneumoniae isolates reveals misidentificationand misunderstandings of Klebsiella pneumoniae, Klebsiella variicola, andKlebsiella quasipneumoniae. mSphere 2017;2: e00290–17.5 Klontz EH, Tomich AD, Gu¨nther S et al. Structure and dynamics of FosA-mediated fosfomycin resistance in Klebsiella pneumoniae and Escherichiacoli. Antimicrob Agents Chemother 2017;61: e01572–17.6 Clinical and Laboratory Standards Institute. Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—EleventhEdition: M07. CLSI, Wayne, PA, USA, 2018.7 De Been M, Pinholt M, Top J et al. Core genome multilocus sequence typingscheme for high-resolution typing of Enterococcus faecium. J Clin Microbiol2015;53: 3788–97.8 Poirel L, Decousser JW, Nordmann P. Insertion sequence ISEcp1B is in-volved in expression and mobilization of a blaCTX-M b-lactamase gene.Antimicrob Agents Chemother 2003;47: 2938–45.9 Arredondo-Alonso S, Rogers MRC, Braat JC et al. mlplasmids: a user-friendly tool to predict plasmid- and chromosome-derived sequences for sin-gle species. Microb Genom 2018;4: e000224.10 Carattoli A, Zankari E, Garcıa FA et al. In silico detection and typing of plas-mids using PlasmidFinder and plasmid multilocus sequence typing.Antimicrob Agents Chemother 2014;58: 3895–903.J Antimicrob Chemother 2019; 74: 3632–3634doi:10.1093/jac/dkz394Advance Access publication 17 September 2019Emergence of Klebsiella pneumoniaeand Enterobacter cloacae producingOXA-48 carbapenemases from retailmeats in China, 2018Zilin Zhuang, Luchao Lv, Jiaxun Lu, Jinhang Lin andJian-Hua Liu*Guangdong Provincial Key Laboratory of VeterinaryPharmaceutics Development and Safety Evaluation, KeyLaboratory of Zoonoses of Ministry of Agriculture, College ofVeterinary Medicine, South China Agricultural University,Guangzhou, China*Corresponding author. College of Veterinary Medicine, South ChinaAgricultural University, Guangzhou 510642, P. R. China; Tel: !86-20-85283824; Fax:!86-20-85283824; E-mail: jhliu@scau.edu.cnSir,Carbapenem-resistant Enterobacteriaceae (CRE) have beenglobally reported, not only in hospitals, but also in the community,animals (including livestock, companion animals and wildlife), theenvironment and food,1,2 and they are recognized as a seriousthreat to human health. Recently, an increased prevalence ofEscherichia coli strains carrying blaNDM from food in China from2015 to 2018 has been reported, highlighting the risk of humanexposure to food polluted by strains producing NDM carbapene-mase.3,4 OXA-48-producing CRE have been frequently reportedin Europe and have been identified in many ecosystems.However, Enterobacteriaceae producing OXA-48 had so farVC The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.For permissions, please email: journals.permissions@oup.com.Research letters3632Downloaded from https://academic.oup.com/jac/article-abstract/74/12/3630/5567518 by Erasmus University Rotterdam user on 27 November 2019

In vivo acquisition of fosfomycin resistance in Escherichia coli by fosA transmission from commensal flora

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In vivo acquisition of fosfomycin resistance in Escherichia coli by fosA transmission from commensal flora

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