MAJOR ARTICLE Clinical Infectious Diseases β-lactam–Resistant Toxigenic C. diphtheriae • CID 2020:XX (XX XXXX) • 1 Received 7 April 2020; editorial decision 23 July 2020; accepted 3 August 2020; published online August 9, 2020. Correspondence: B. M. Forde, University of Queensland Centre for Clinical Research, 71/918 RBWH Herston, Brisbane, QLD 4029 (b.forde@uq.edu.au). Clinical Infectious Diseases ® 2020;XX(XX):1–8 © The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. DOI: 10.1093/cid/ciaa1147 Fatal Respiratory Diphtheria Caused by ß-Lactam– Resistant Corynebacterium diphtheriae Brian M. Forde, 1,2,3, Andrew Henderson, 4,5 Elliott G. Playford, 5,6 David Looke, 5,6 Belinda C. Henderson, 5 Catherine Watson, 5 Jason A. Steen, 7 Hanna E. Sidjabat, 2,4 Gordon Laurie, 8 Sharmini Muttaiyah, 9 Graeme R. Nimmo, 9 Guy Lampe, 10 Helen Smith, 11 Amy V. Jennison, 11 Brad McCall, 12 Heidi Carroll, 13 Matthew A. Cooper, 7 David L. Paterson, 2,4 and Scott A. Beatson 1,2,3 1 School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia, 2 Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Australia, 3 Australian Centre for Ecogenomics, University of Queensland, Brisbane, Australia, 4 University of Queensland Centre For Clinical Research, Brisbane, Australia, 5 Infection Management Services, Princess Alexandra Hospital, Brisbane, Australia, 6 School of Medicine, University of Queensland, Brisbane, Australia, 7 Institute for Molecular Biosciences, University of Queensland, Brisbane, Australia, 8 Intensive Care Unit, Princess Alexandra Hospital, Brisbane, Australia, 9 Department of Microbiology, Pathology Queensland, Brisbane, Australia, 10 Department of Anatomical Pathology, Pathology Queensland, Brisbane, Australia, 11 Public Health Microbiology, Forensic and Scientifc Services, Queensland Health, Brisbane, Australia, 12 Metro South Public Health Unit, Metro South Health, Brisbane, Australia, and 13 Communicable Diseases Branch, Prevention Division, Department of Health, Queensland Health, Brisbane, Australia Background. Diphtheria is a potentially fatal respiratory disease caused by toxigenic Corynebacterium diphtheriae. Although resistance to erythromycin has been recognized, β-lactam resistance in toxigenic diphtheria has not been described. Here, we report a case of fatal respiratory diphtheria caused by toxigenic C. diphtheriae resistant to penicillin and all other β-lactam antibiotics, and describe a novel mechanism of inducible carbapenem resistance associated with the acquisition of a mobile resistance element. Methods. Long-read whole-genome sequencing was performed using Pacifc Biosciences Single Molecule Real-Time sequencing to determine the genome sequence of C. diphtheriae BQ11 and the mechanism of β-lactam resistance. To investigate the phenotypic inducibility of meropenem resistance, short-read sequencing was performed using an Illumina NextSeq500 sequencer on the strain both with and without exposure to meropenem. Results. BQ11 demonstrated high-level resistance to penicillin (benzylpenicillin minimum inhibitory concentration [MIC] ≥ 256 μg/ml), β-lactam/β-lactamase inhibitors and cephalosporins (amoxicillin/clavulanic acid MIC ≥ 256 μg/mL; cefriaxone MIC ≥ 8 μg/L). Genomic analysis of BQ11 identifed acquisition of a novel transposon carrying the penicillin-binding protein (PBP) Pbp2c, responsible for resistance to penicillin and cephalosporins. When strain BQ11 was exposed to meropenem, selective pressure drove amplifcation of the transposon in a tandem array and led to a corresponding change from a low-level to a high-level meropenem-resistant phenotype. Conclusions. We have identifed a novel mechanism of inducible antibiotic resistance whereby isolates that appear to be carbapenem susceptible on initial testing can develop in vivo resistance to carbapenems with repeated exposure. Tis phenomenon could have signifcant implications for the treatment of C. diphtheriae infection, and may lead to clinical failure. Keywords. carbapenem resistance; inducible resistance; toxigenic; genome sequencing. Diphtheria is a potentially life-threatening, toxin-mediated respiratory disease primarily caused by toxigenic strains of Corynebacterium diphtheriae. Routine childhood immuniza- tion, beginning in the 1930s, has led to the dramatic reduc- tion in diphtheria incidences in most developed countries [1]. Despite waning immunity with age, adults remain relatively pro- tected by herd population immunity that limits the circulation of toxigenic C. diphtheriae [2]. However, sporadic diphtheria among nonimmunized or partially immunized individuals still occurs in countries with high childhood immunization rates [3], highlighting the emerging threat posed by the potential introduction of multidrug-resistant, toxigenic C. diphtheriae from endemic regions [4, 5]. C. diphtheriae is susceptible in vitro to a wide range of anti- biotics. Typically, penicillin or erythromycin, together with diphtheria antitoxin, is recommended for the treatment of toxigenic strains. Although resistance to erythromycin has been recognized [6, 7], to our knowledge, β-lactam resistance in toxigenic C. diphtheriae has not yet been reported. Here, we describe the case of an unvaccinated adult patient with fatal diphtheria caused by toxigenic C. diphtheriae that was resistant to penicillin and all other β-lactam antibiotics, in- cluding carbapenems. We show that the resistance to β-lactams is due to the acquisition of a mobile gene cassette that en- codes a penicillin binding protein (PBP) that is not found in penicillin-susceptible C. diphtheriae. Finally, we demonstrate that high-level carbapenem resistance in this isolate can be at- tributed to the tandem amplifcation of this resistance cassette in the C. diphtheriae genome. 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