Emergence of a novel Enterobacter kobei clone carrying chromosomal encoded CTX M 12 with diversified pathogenicity in northeast China Accepted Manuscript Emergence of a novel Enterobacter kobei clone[.]
Accepted Manuscript Emergence of a novel Enterobacter kobei clone carrying chromosomal-encoded CTXM-12 with diversified pathogenicity in northeast China Kai Zhou, Wei Yu, Richard Bonnet, Vincent Cattoir, Ping Shen, Baohong Wang, John W Rossen, Yonghong Xiao PII: S2052-2975(17)30006-9 DOI: 10.1016/j.nmni.2017.01.006 Reference: NMNI 294 To appear in: New Microbes and New Infections Received Date: 21 September 2016 Revised Date: January 2017 Accepted Date: 11 January 2017 Please cite this article as: Zhou K, Yu W, Bonnet R, Cattoir V, Shen P, Wang B, Rossen JW, Xiao Y, Emergence of a novel Enterobacter kobei clone carrying chromosomal-encoded CTX-M-12 with diversified pathogenicity in northeast China, New Microbes and New Infections (2017), doi: 10.1016/ j.nmni.2017.01.006 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Article category: original article Emergence of a novel Enterobacter kobei clone carrying chromosomal-encoded RI PT CTX-M-12 with diversified pathogenicity in northeast China Kai Zhou1,4, Wei Yu1, Richard Bonnet2, Vincent Cattoir3, Ping Shen1, Baohong State Key Laboratory for Diagnosis and Treatment of Infectious Diseases; Collaborative M AN U SC Wang1, John W Rossen4, Yonghong Xiao1* Innovation Center for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital of Medicine School, Zhejiang University, Hangzhou, China; Clermont Université, Université d'Auvergne; Inserm U1071; INRA USC2018, TE D Clermont-Ferrand, France Centre Hospitalier Universitaire, Clermont-Ferrand, France; CHU de Caen, Service de Microbiologie, F-14033 Caen, France; Université de Caen Basse-Normandie, EA4655 (équipe "Antibiorésistance"), F-14032 Caen, France; CNR de la EP Résistance aux Antibiotiques, Laboratoire Associé "Entérocoques et résistances particulières AC C des bactéries Gram positif", F-14033 Caen, France; Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, Netherlands *Corresponding author Address: The first affiliated hospital of Zhejiang University, Qingchun Road 79, 330001 Hangzhou, Zhejiang, China ACCEPTED MANUSCRIPT Telephone: +86-571-87236427 Fax: +86-571-87236427 RI PT E-mail addresses: xiao-yonghong@163.com AC C EP TE D M AN U SC Running title: Characterization of an ESBL-producing E kobei clone ACCEPTED MANUSCRIPT Abstract A rare CTX-M gene blaCTX-M-12 used to be carried on plasmids was detected in the chromosome of three clinical E kobei strains They shared a new sequence type assigned as ST591, and were differed by 57 single-nucleotide polymorphisms An intra-clonal diversification on the capacity of biofilm formation was detected Keywards: E cloacae, E kobei, CTX-M-12, chromosomal integration, biofilm SC M AN U RI PT TEXT Enterobacter cloacae complex (ECC), comprising at least five species (E cloacae, E kobei, 11 E asburiae, E hormaechei, and E ludwigii) with different subspecies, has emerged as one of 12 the important nosocomial pathogens in the last decade, responsible for 65-75% of all 13 infections due to Enterobacter spp (1) Recently, some potentially high-risk international 14 clones causing nosocomial infections were revealed in an European-wide survey (2) In this 15 study, we characterized a CTX-M-12-producing ECC clone responsible for severe infections 16 circulating in northeast China EP AC C 17 TE D 10 18 Three ECC strains were isolated from broncho-alveolar lavage (ECC3018), blood (ECC3026) 19 and abscesses (ECC3047) of patients admitted to a secondary hospital in a northeast 20 province (Liaoning) in China, respectively The isolates were further identified as E kobei by 21 hsp60 typing (3) The three strains exhibited similar antimicrobial profiles determined by the 22 agar dilution method (Table 1) They showed an ESBL-positive and AmpC-overexpression ACCEPTED MANUSCRIPT phenotype detected as previously reported (2) Multilocus sequence typing with use of loci 24 (dnaA, fusA, gyrB, leuS, pyrG, rplB, and rpoB) detected a new profile (3-3-110-32-19-16-17) 25 for all isolates, and was assigned as ST591 by PubMLST database 26 (https://pubmlst.org/ecloacae/) RI PT 23 27 Isolates were sequenced by Illumina Hiseq2500 (Illumina, San Diego, CA, USA) using 29 2x125-bp pair-end libraries Genomes were assembled by CLC genomic workbench v8.0, and 30 annotated by to RAST sever (http://rast.nmpdr.org/) To analyze the resistome, genomic 31 sequences were uploaded to Resfinder (https://cge.cbs.dtu.dk) The three isolates shared an 32 identical resistome comprising 19 genes, including: aac(3)-IId, aac(6')-II, aadA2, aadA16, 33 aph(3')-Ic and armA for aminoglycoside resistance; blaCTX-M-12, blaCARB-2, blaTEM-1, and an 34 unnamed blaACT gene for beta-lactam resistance; and other genes for various drug resistance 35 (fosA, msr(E), mph(A), mph(E), ere(B), sul1, sul2, cmlA1, dfrA1) The genotypes can fully 36 explain the results of susceptibility tests M AN U TE D EP 37 SC 28 The blaCTX-M-12 is a rare CTX-M gene, and its genetic environment was identical in the three 39 strains (Figure 1A) An ISEcp1 located 48-bp upstream of blaCTX-M-12, and the structure was 40 identical to that of E coli isolates (DQ658220) identified in Korean The transposition unit 41 ISEcp1-blaCTX-M-12-orf477 was highly similar to the well-reported typical blaCTX-M-15 42 transposition unit with length of 2971bp The unit was integrated into the chromosome, 43 locating upstream of a pseudo gene and downstream of a gene encoding an asparaginyl-tRNA 44 synthetase Two 5-bp direct repeats TATTA were identified adjacent to the flanking left AC C 38 ACCEPTED MANUSCRIPT 45 inverted repeat (IRL) and putative right inverted repeat (IRR) (Figure 1A), suggesting that 46 ISEcp1 mediated the chromosomal integration of blaCTX-M-12 47 The blaCTX-M-12 gene was first detected from a K pneumoniae outbreak clone in Kenya in 49 2001 (4), and later identified in E coli and K pneumoniae isolates from Colombia and Korea 50 (5,6) To our knowledge, this is the first report of blaCTX-M-12 identified in E kobei As the 51 geographic area where our strains isolated is bounded on the south by Korea, it is possible 52 that the emergency of blaCTX-M-12 may be due to the cross-border spread Further surveillance 53 for blaCTX-M-12 should be carried out to test this hypothesis Additionally, among 54 Enterobacteriaceae, chromosomal-encoded CTX-Ms are frequently found in E coli, K 55 pneumoniae and Proteus mirabilis, but very rare in ECC (7) Additionally, this is the first 56 evidence of chromosomal integration of blaCTX-M-12, which has been exclusively detected in 57 plasmids for now This suggests the role of chromosomal locations in the spread of 58 blaCTX-M-12 SC M AN U TE D EP 59 RI PT 48 The three isolates showed high MIC values of amikacin (>256 mg/L) A 16S rRNA methylase 61 gene armA detected in a Tn1548-like segment in the isolates responsible for the amikacin 62 resistance (Figure 1B) This Tn1548-like segment consisted of a 3’-end conserved region and 63 a 5’-end class integron The conserved region was structured as 64 ISCR1-ISEc28-armA-ISEc29-msr(E)-mph(E), and the variable region of class integron 65 carried aac(6')-II and aadA16 Notably, the region from an IS26-disrupted intl1 gene to an 66 ISAba24 was identical to that identified in Acinetobacter baumannii strain A071 (KT317079) AC C 60 ACCEPTED MANUSCRIPT (Figure 1B) Both ends of the Tn1548-like segment were disrupted by IS26 indicating that 68 IS26 mediated the mobilization of this composite transposon cross species The concomitance 69 of ESBLs and 16S rRNA methylases raises clinical concern and may become a major 70 therapeutic threat in the future RI PT 67 71 The genetic diversity of the three strains was determined by single-nucleotide polymorphism 73 (SNP) analysis as described previously (8), and they were differed by 57 SNPs (Table S1) 74 This excludes the possibility of a recent transmission among the three patients suggesting a 75 clonal dissemination in the region To investigate whether the genetic differences were 76 associated with alterations of biological function, biofilm formation was tested by microtiter 77 plate assay as described previously (9) Intriguingly, ECC3018 could form ca 4~10-fold more 78 biofilm (24h: 0.12±0.02; 48h: 0.18±0.04) than the other two (24h: 0.012±0.003, 0.011±0.002; 79 48h: 0.048±0.03, 0.037±0.02) (P < 0.05) at 37°C The discrepancy could be explained by 80 multiple non-synonymous SNPs identified in the genes involving in biofilm formation (Table 81 S1), including barA encoding a sensory histidine kinase (10), kefA encoding a potassium 82 efflux system (11), and malT encoding a transcriptional activator of maltose regulon (12) 83 Additionally, more non-synonymous SNPs than synonymous ones (36 vs 12) were identified 84 in the clone frequently associated with genes involving in metabolism, membrane, and 85 pathogenicity This implicates that the clone underwent positive selections resulting in 86 pathogenicity diversification 87 AC C EP TE D M AN U SC 72 ACCEPTED MANUSCRIPT 88 In summary, this study raises the concern that wide repertoire of resistance mechanism and 89 enhanced pathogenicity detected in the novel E kobei clone increases its epidemic potential, 90 and highlights the necessity of surveillance on the potential high-risk clone in the future RI PT 91 Nucleotide Sequence Genbank Accession Numbers 93 The Whole Genome Shotgun BioProject for E kobei isolates has been deposited at 94 DDBJ/EMBL/GenBank under the accession of LYUR00000000, LYUS00000000, 95 LYUT00000000, respectively M AN U SC 92 96 Funding 98 This study was supported by the Fundamental Research Funds for the Central Universities 99 (2016FZA7008), the National Basic Research Program of China (973 program, TE D 97 2015CB554201), the National Natural Science Foundation of China (81361138021), and the 101 Key Project of Science and Technology & Social Development of Zhejiang Province 102 (2014C03039) AC C 103 EP 100 104 Transparency declarations 105 None to declare 106 107 References 108 109 Eugene Sanders WE, Sanders CC Enterobacter spp.: Pathogens poised to flourish at the turn of the century Clin Microbiol Rev 1997;10:220–41 ACCEPTED MANUSCRIPT 110 Izdebski R, Baraniak A, Herda M, Fiett J, Bonten MJM, Carmeli Y, et al MLST 111 reveals potentially high-risk international clones of Enterobacter cloacae J 112 Antimicrob Chemother 2015;70:48–56 114 115 Hoffmann H, Roggenkamp A Population genetics of the nomenspecies Enterobacter cloacae Appl Environ Microbiol 2003;69:5306–18 RI PT 113 Kariuki S, Corkill JE, Revathi G, Musoke R, Hart CA Molecular characterization of a novel plasmid-encoded cefotaximase (CTX-M-12) found in clinical Klebsiella 117 pneumoniae isolates from Kenya Antimicrob Agents Chemother 2001;45:2141–3 M AN U 118 SC 116 Villegas MV, Correa A, Perez F, Zuluaga T, Radice M, Gutkind G, et al CTX-M-12 119 beta-lactamase in a Klebsiella pneumoniae clinical isolate in Colombia Antimicrob 120 Agents Chemother 2004;48:629–31 Bae IK, Lee YN, Hwang HY, Jeong SH, Lee SJ, Kwak HS, et al Emergence of TE D 121 122 CTX-M-12 extended-spectrum β-lactamase-producing Escherichia coli in Korea J 123 Antimicrob Chemother 2006;58:1257–9 125 126 127 128 129 Zhao W-H, Hu Z-Q Epidemiology and genetics of CTX-M extended-spectrum EP β-lactamases in Gram-negative bacteria Crit Rev Microbiol 2013;39:79–101 AC C 124 Zhou K, Ferdous M, de Boer RF, Kooistra-Smid AMD, Grundmann H, Friedrich AW, et al The mosaic genome structure and phylogeny of Shiga toxin-producing Escherichia coli O104:H4 is driven by short-term adaptation Clin Microbiol Infect 2015;21:468.e7-468.e18 ACCEPTED MANUSCRIPT 130 Nyenje ME, Green E, Ndip RN Evaluation of the effect of different growth media and 131 temperature on the suitability of biofilm formation by Enterobacter cloacae strains 132 isolated from food samples in South Africa Molecules 2013;18:9582–93 10 Sahu SN, Acharya S, Tuminaro H, Patel I, Dudley K, LeClerc JE, et al The bacterial RI PT 133 adaptive response gene, barA, encodes a novel conserved histidine kinase regulatory 135 switch for adaptation and modulation of metabolism in Escherichia coli Mol Cell 136 Biochem 2003;253:167–77 11 Zhang W, McLamore ES, Wu R, Stensberg M, Porterfield DM, Banks MK M AN U 137 SC 134 138 Glutathione-gated potassium efflux as a mechanism of active biofilm detachment 139 Water Environ Res 2014;86:462–9 140 12 Lone AG, Deslandes V, Nash JHE, Jacques M, MacInnes JI malT knockout mutation invokes a stringent type gene-expression profile in Actinobacillus pleuropneumoniae 142 in bronchoalveolar fluid BMC Microbiol 2009;9:195 EP 144 AC C 143 TE D 141 ACCEPTED MANUSCRIPT Table MICs of some antibiotic agents for E kobei isolates ECC3018 ECC3026 ECC3047 Ampicillin >512 >512 >512 Ampicillin-sulbactam 64/32 32/16 32/16 Ciprofloxacin 006 006 003 Levofloxacin 03 03 256 >256 >256 Gentamycin 128 128 128 Cefepime 8 Cefotaxime 4 Cefoperazone-sulbactam 64/32 64/32 32/16 Ceftriaxone 32 16 32 Ceftazidime 1 Cefoxitin 128 128 128 Cefazolin >128 >128 >128 Cefuroxime >128 >128 >128 Meropenem