bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.314500; this version posted September 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Submission to the Journal of mSphere 2 Characterization of a clinical Enterobacter hormaechei strain belonging to 3 epidemic clone ST418 co-carrying blaNDM-1, blaIMP-4 and mcr-9.1 4 Wei Chena#, Zhiliang Hub,c#, Shiwei Wangd, Doudou Huange, Weixiao Wanga, Xiaoli 5 Caof*, Kai Zhoug* 6 a. Clinical Research Center, the Second Hospital of Nanjing, Nanjing University of 7 Chinese Medicine, Nanjing, 210003, China 8 b. Nanjing infectious Disease Center, the Second Hospital of Nanjing, Nanjing 9 University of Chinese Medicine, Nanjing, 210003, China. 10 c. Center for Global Health, School of Public Health, Nanjing Medical University, 11 Nanjing 211166, China. 12 d. Key Laboratory of Resources Biology and Biotechnology in Western China, 13 Ministry of Education, College of Life Science, Northwest University, Xi’an, 14 ShaanXi, 710069, China 15 e. Department of the Medical Records and Statistics Room, The Affiliated Drum 16 Tower Hospital of Nanjing University Medical School, Nanjing, 210008, China. 17 f. Department of Laboratory Medicine, Nanjing Drum Tower Hospital, the affiliated 18 Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China 19 g. Shenzhen Institute of Respiratory Diseases, the First Affiliated Hospital 20 (Shenzhen People’s Hospital), Southern University of Science and Technology; 21 Second Clinical Medical College (Shenzhen People's Hospital), Jinan University, bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.314500; this version posted September 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 22 Shenzhen, China. 23 24 #Contributed equally to this study. 25 26 * To whom correspondence should be addressed: Dr. Xiaoli Cao, 27 [email protected] and Dr. Kai Zhou, [email protected] 28 Abstract 29 An Enterobacter hormaechei isolate (ECL-90) simultaneously harboring 30 blaNDM-1, blaIMP-4 and mcr-9.1 was recovered from the secretion specimen of a 31 24-year-old male patient in a tertiary hospital in China. The whole genome 32 sequencing of this isolate was complete, and 4 circular plasmids with variable sizes 33 were detected. MLST analysis assigned the isolate to ST418, known as a 34 carbapenemase-producing epidemic clone in China. blaIMP-4 and mcr-9.1 genes were 35 co-carried on an IncHI2/2A plasmid (pECL-90-2) and blaNDM-1 was harbored by an 36 IncX3 plasmid (pECL-90-3). The genetic context of mcr-9.1 was identified as a 37 prevalent structure, “rcnR-rcnA-pcoE-pcoS-IS903-mcr-9-wbuC”, which is a relatively 38 unitary model involved in the mobilization of mcr-9. Meanwhile, blaNDM-1 gene was 39 detected within a globally widespread structure known as NDM-GE-U.S (“ISAba125– 40 blaNDM-1–blaMBL”). Our study warrants that the convergence of genes mediating 41 resistance to last-resort antibiotics in epidemic clones would largely facilitate their 42 widespread in clinical settings, thus representing a potential challenge to clinical 43 treatment and public health. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.314500; this version posted September 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 44 Importance 45 Carbapenemase-producing Enterobacteriaceae (CPE) spread at a high rate and 46 colistin is the last-resort therapeutic for the infection caused by CPE. However, the 47 emergence of plasmid-borne mcr genes highly facilitates the wide dissemination of 48 colistin resistance, thus largely threatens the clinical use of colistin. Here, we for the 49 first time characterized a clinical Enterobacter hormaechei strain co-producing 50 blaNDM-1, blaIMP-4 and mcr-9.1 belonging to an epidemic clone (ST418). The 51 accumulation of genes mediating resistance to last-resort antibiotics in epidemic 52 clones would largely facilitate their widespread in clinical settings, which may cause 53 disastrous consequence with respect to antimicrobial resistance. Understanding how 54 resistance genes were accumulated in a single strain could help us to track the 55 evolutionary trajectory of drug resistance. Our finding highlights the importance of 56 surveillance on the epidemic potential of colistin-resistant CPE, and effective 57 infection control measures to prevent the resistance dissemination. 58 59 Key words: carbapenem resistance, NDM-1, MCR-9.1, IMP-4, Enterobacter 60 hormaechei 61 62 The worldwide prevalence of carbapenem-resistant Enterobacteraceae (CRE) has 63 been well known (1), with Klebsiella pneumoniae carbapenemases (KPCs), New 64 Delhi metallo-lactamases (NDMs), imipenemases (IMPs) and OXA-48-like enzymes 65 being the most prevalent carbapenemase (2). Moreover, co-occurrence of these genes bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.314500; this version posted September 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 66 in a single strain has been frequently identified (3, 4). Currently, colistin is one of the 67 last-resort antibiotics for the CRE infections (5). However, the emergence of 68 plasmid-borne colistin resistance gene mcr frustrates colistin’s efficiency. MCR 69 encodes a phosphoethanolamine transferase which is involved in the modification of 70 lipopolysaccharide, the target of colistin. At present, the prevalence of mcr is the most 71 widespread mechanism of colistin resistance (6). As of now, ten different mcr variants 72 noted from mcr-1 to mcr-10 have been described (6-8). Of more concern, co-existence 73 of mcr and carbapenemase genes has been sporadically reported (9). For instance, 74 mcr-1, mcr-3.5, and blaNDM-5 are found in an Escherichia coli isolate (10), mcr-4.3 75 and blaNDM-1 are co-identified in a clinical E. cloacae isolate from China (11), and 76 blaVIM-4 and mcr-9 are found in an E. hormaechei isolate in USA (12). The rapid 77 emergence of such new resistance phenotypes has broken through the last defense line 78 and severely limited therapeutic options. In this report, we characterized a clinical E. 79 hormaechei isolate simultaneously harboring blaNDM-1, blaIMP-4 and mcr-9.1. The 80 structures of plasmids carrying three resistance genes were fully dissected to 81 understand their dissemination and accumulation pattern. 82 This E. hormaechei isolate (ECL-90) was recovered from the secretion specimen of 83 a 24-year-old male patient in October, 2017, who was admitted into a large tertiary 84 hospital in Nanjing because of an acute community-acquired pneumonia. The patient 85 subsequently developed a left orbital cellulitis resulting from the ascending infection 86 of the respiratory tract. After receiving endoscopic sinus opening and drainage, and an 87 anti-infective regimen including meropenem, tigecycline and fosfomycin, the patient bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.314500; this version posted September 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 88 recovered and was therefore discharged. 89 ECL-90 was first identified as E. cloacae complex by matrix-associated laser 90 desorption ionization-time of flight mass spectrometry (BioMerieux, Craponne, 91 France), and further confirmed as E. hormaechei by whole genome sequencing 92 (WGS). Antimicrobial susceptibility test using microbroth dilution method showed 93 that this strain was resistant to ertapenem (16 μg/ml), imipenem (8 μg/ml), 94 meropenem (>16 μg/ml), cefepime (>32 μg/ml), ceftazidime (>32 μg/ml), 95 cefotaxime (>32 μg/ml), cefuroxime (>64 μg/ml), cefazolin (>32 μg/ml), cefmetazole 96 (>64 μg/ml), piperacillin/tazobactam (>256 μg/ml), amikacin (8 μg/ml), gentamicin 97 (64 μg/ml), funantuoyin (128 μg/ml), trimethoprim and sulphame-thoxazole (>32 98 μg/ml), aztreonam (>128 μg/ml), piperacillin (>256 μg/ml), ciprofloxacin (8 μg/ml), 99 levofloxacin (4 μg/ml), and ceftazidime/avibactam (>32 μg/ml), while remained 100 susceptible to aztreonam/avibactam (<0.25 μg/ml), tigecycline (1 μg/ml), colistin B 101 (0.25 μg/ml) and fosfomycin (32 μg/ml). 102 To better understand the resistance mechanism and genomic characterization of this 103 strain, the genomic DNA was extracted and WGS was performed on the Hiseq 4000 104 instrument (Illumina, San Diego, CA, USA). Multi-locus sequence typing (MLST) 105 analysis using MLST 2.0 assigned ECL-90 to sequence type (ST) 418 (allelic profile 106 53-35-154-44-45-4-6) (13), which is known as one of the predominant epidemic 107 clones of carbapenemase-producing E. cloacae in China (14, 15). Antibiotic 108 resistance genes were identified by using Resfinder v2.1 109 (http://cge.cbs.dtu.dk/services/ResFinder-2.1/), including genes conferring bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.314500; this version posted September 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 110 beta-lactam resistance (blaNDM-1, blaIMP-4, blaSFO-1, blaSHV-12, blaTEM-1B, blaACT-16), 111 colistin resistance (mcr-9.1), aminoglycoside resistance [aac(3)-IId, aac(6')-IIc, 112 aac(6')-Ib3, aph(3'')-Ib, aph(3')-Ia, aph(6)-Id, aph(6)-Id], fluoroquinolone and 113 aminoglycoside resistance [aac(6')-Ib-cr], macrolide resistance (ereA, mphA), 114 phenicol resistance (catA2), sulphonamide resistance (sul1), tetracycline resistance 115 (tetD), and trimethoprim (dfrA19).