A carbapenem-resistant Escherichia coli isolate (sequence type 448 [ST448]) was recovered from a urine culture of a female patient with no recent record of traveling. PCR screening identified the presence of blaNDM-5, blaTEM-1, blaOXA-1, blaCMY-42, and rmtB. blaNDM-5 was carried in a conjugative IncFII-type plasmid (90 kb) together with blaTEM-1 and rmtB. The genetic environment of blaNDM-5 showed a structure similar to those of pMC-NDM and pGUE-NDM, identified in Poland and France in E. coli of African and Indian origin, respectively

Marco Reverté, Francesc

Moreno Camacho, Ma. Asunción

Pitart, Cristina

Roca Subirà, Ignasi

Román, Angely

Solé, Mar

Vila Estapé, Jordi

Antimicrobial Agents and Chemotherapy

Diposit Digital de la Universitat de Barcelona

 1 Molecular characterization of blaNDM-5 carried in an IncFII 1 plasmid in Escherichia coli from a non-traveller patient in 2 Spain 3 Cristina Pitart1Ψ, Mar Solé1Ψ, Ignasi Roca1*, Angely Román,1 Assumpta 4 Moreno2, Jordi Vila1* and Francesc Marco1 5  6 1 Department of Clinical Microbiology, School of Medicine, CRESIB-Hospital Clinic, 7 University of Barcelona, Barcelona, Spain and 2 Department of Infectious Diseases, 8 School of Medicine, Hospital Clinic, University of Barcelona, Barcelona, Spain. 9  10 Ψ These authors equally contributed to this work. 11  12 Running title: NDM-5-producing E. coli in Spain 13 *Corresponding author 14 Ignasi Roca 15 Barcelona Centre for International Health Research (CRESIB), CEK building, Hospital 16 Clínic, Barcelona, Spain. Tel: +34 93 227 54 00; Fax: +34 93 312 94 10; E-mail: 17 Ignasi.roca@cresib.cat 18 Jordi Vila 19 Servei de Microbiologia, Centre de Diagnòstic Biomèdic, Hospital Clínic, Facultat de 20 Medicina, Universitat de Barcelona, Barcelona, Spain. Tel: +34 93 227 55 22; Fax: +34 21 93 227 93 72; E-mail: jvila@ub.edu 22 Keywords: Antibiotic resistance, metallo-β-lactamase, E. coli, plasmid replicon typing 23  2 ABSTRACT 24 A carbapenem-resistant Escherichia coli (ST448) was recovered from a urine 25 culture of a female patient with no recent travelling record. PCR screening identified the 26 presence of blaNDM-5, blaTEM-1, blaOXA-1, blaCMY-42 and rmtB. blaNDM-5 was carried in a 27 conjugative IncFII-type plasmid (90 Kb) together with blaTEM-1 and rmtB. The genetic 28 surrounding of blaNDM-5 showed structural similarities to those of pMC-NDM and 29 pGUE-NDM, identified in Poland and France in E. coli of African and Indian origin, 30 respectively. 31  3 Carbapenemase-producing Enterobacteriaceae (CPE) constitute a public health 32 problem regarding both hospital and community-acquired infections (1). Despite 33 increasing reports of CPE in Europe and, predominantly in southern European 34 countries, the prevalence of CPE in Spain still remains relatively low although is 35 steadily increasing, with only a few outbreaks or sporadic isolates harboring VIM, KPC, 36 OXA-48 or NDM-1 being reported (2). After the identification of NDM-1 in a Swedish 37 traveller returning from India (3), NDM enzymes have gathered special attention due to 38 their rapid global spread and frequent association with additional resistance genes (4). 39 There are currently twelve NDM variants (http://www.lahey.org) differing by one or 40 two amino acid substitutions that may display slightly different hydrolytic capabilities 41 (4). 42 The aim of this study was to characterize an NDM-5-producing E. coli recovered 43 in Spain from a non-traveller patient. 44 A 78-years-old Spanish Caucasian female with a history of acute pyelonephritis 45 treated with ceftriaxone (1g/24h) was positive for a carbapenem-resistant E. coli strain 46 (HC105) in urine 15 days after the end of treatment. The patient was asymptomatic at 47 this stage. Susceptibility testing using Etest strips (AB-bioMérieux, Solna, Sweden) and 48 interpreted according to EUCAST guidelines (version 4.0, 2014, http://www.eucast.org) 49 indicated that HC105 was resistant to all the antibiotics tested except tigecycline, 50 fosfomycin and colistin (MIC of 0.38 µg/ml, 0.5 µg/ml and 0.125 µg/ml, respectively) 51 with ertapenem, imipenem and meropenem MICs > 32 µg/ml (Table 1). The patient was 52 successfully treated with fosfomycin (2g/8h) and no additional CPE were recovered. 53 Screening of HC105 for carbapenemase/metallo-β-lactamase (MBL)-production yielded 54 positive results using either the modified Hodge test or imipenem-EDTA Etest strips, 55 suggesting carriage of an MBL. PCR and sequence analysis for carbapenemase, ESBL, 56  4 and plasmid-mediated AmpC cephalosporinase-encoding genes (5, 6) identified the 57 presence of blaNDM-5, blaTEM-1, blaOXA-1 and blaCMY-42. Screening for 16S rRNA-methylase 58 genes (7) also identified the rmtB gene conferring high-level resistance to all 59 aminoglycosides, in agreement with the susceptibility profile (Table 1). Multilocus 60 sequence typing (http://mlst.ucc.ie/mlst/dbs/Ecoli) and PCR-based phylogroup analysis 61 (8) assigned E. coli HC105 to sequence type 448 (ST448) and phylogroup B1, 62 respectively. 63 To study the transferability of the resistance phenotype, a biparental mating 64 between HC105 and E. coli J53AziR was conducted and transconjugants were selected 65 on LB agar plates containing 1 µg/ml meropenem and 100 µg/ml sodium azide. All 66 transconjugants acquired resistance to all aminoglycosides and β-lactams tested but 67 aztreonam (Table 1), concomitant with the acquisition of blaNDM-5, blaTEM-1 and rmtB but 68 not of blaOXA-1, and blaCMY-42. S1 nuclease–pulsed-field gel electrophoresis (PFGE) (6) 69 revealed two plasmids of circa 50 and 90kb in HC105 but only a single plasmid of 90kb 70 in the transconjugant strains. Plasmid replicon typing (9, 10) showed that HC105 71 contained plasmid-types belonging to IncFII and I1 incompatibility groups while 72 transconjugant strains had only acquired the IncFII-type. Digoxigenin-labeled probes 73 against blaNDM-5, blaTEM-1 and rmtB hybridized against blotted nylon membranes from the 74 S1-PFGE gels matched with the band corresponding to the 90kb plasmid, demonstrating 75 co-carriage of all three resistance mechanisms in a single conjugative IncFII plasmid of 76 circa 90kb, designated as pHC105-NDM. 77 Inverse PCR over genomic DNA from HC105 (6) and DNA sequencing showed 78 the presence of a remnant ISAba125 sequence immediately upstream from blaNDM-5 as 79 well as the ble, trpF and tat genes downstream from blaNDM-5 (Fig.1), altogether 80 comprising a genetic arrangement highly conserved among NDM-producing isolates 81  5 from different Gram-negative species (11). Subsequent PCR mapping of the blaNDM-5 82 genetic surrounding showed that this region was bracketed by two IS26 insertion 83 sequences constituting a putative composite transposon also containing an ISCR1 84 element and a class I integron whose int1 gene was truncated by the downstream IS26 85 copy. In addition, blaTEM-1 and rmtB were located adjacent to the upstream IS26 copy 86 (Fig.1).  87 The genetic surrounding of the blaNDM-5 gene in pHC105-NDM is almost 88 identical to that of the blaNDM-1 gene present in pMC-NDM (12), which contains an 89 additional IS26 truncating ISCR1, and is very similar to that of blaNDM-1 in pGUE-NDM 90 (13), except for the upstream blaTEM-1 and rmtB genes that are missing in pGUE-NDM. 91 Both pMC-NDM and pGUE-NDM are IncFII plasmids of approximately 90kb that were 92 identified in Poland and France from E. coli isolates of African and Indian origin and 93 belonging to ST complexes ST23 and ST131, respectively. 94 NDM enzymes encoded in IncFII plasmids, with a narrow host range, are being 95 increasingly reported (12-17), an observation that deserves further attention since IncFII 96 plasmid scaffolds have been linked to the worldwide spread of CTX-M-15 (18). Of 97 note, the genetic array blaTEM-1-rmtB-IS26 upstream from blaNDM in both pHC105-NDM 98 and pMC-NDM has typically been associated with the Tn3 transposon in IncFII 99 plasmids as well (19-22), and it is not unlikely that linkage of this array with the NDM 100 module originated from an IS26-mediated recombination event, a common mechanism 101 in the mobilization and genetic rearrangement of NDM-related structures (13, 14). 102 In this study we report the molecular characterization of an E. coli (ST448) 103 clinical isolate carrying a NDM-5 metallo-β-lactamase in a ∼90kb IncFII plasmid, 104 representing the fourth NDM-producing isolate described in Spain. Previous isolates 105 carried the NDM-1 variant and were associated with recent travels to India (6, 23, 24), 106  6 while HC105 represents the first NDM-5 reported in Spain and it was recovered from a 107 non-traveller whose close relatives reported no recent travelling history either, thus 108 suggesting that it was community-acquired and autochthonous. This is also the first 109 ST448 E. coli strain harboring an NDM enzyme. Interestingly, blaNDM-5 was harbored by 110 IncFII-type plasmids in isolates from India and the UK also bearing blaTEM-1 and rmtB 111 genes, suggesting that blaNDM-5 genes might share a common genetic platform (25, 26).  112 The nosocomial spread of NDM-producing isolates has only been sporadically 113 reported and was likely related to particular clonal lineages (17, 27-29). Multiple 114 reports, however, have identified community-acquired NDM-producing isolates, 115 suggesting the existence of a hidden reservoir and transmission among colonized 116 carriers (30). The present study highlights that, while NDM enzymes are still rarely 117 reported in the clinical setting in Spain, attention should be paid to the prevalence of 118 NDM enzymes within the community to monitor future trends and prevent their further 119 spread into epidemic clonal lineages. 120 The sequence of blaNDM-5 and its genetic environment has been deposited at the 121 NCBI GenBank under accession number KM598665. 122 This study was supported by grant 2014-SGR0653 from the Departament de 123 Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya, by the 124 Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, co-financed by 125 European Regional Development Fund (ERDF) “A Way to Achieve Europe,” the 126 Spanish Network for Research in Infectious Diseases (REIPI RD12/0015) and by 127 funding from the European Community (MagicBullet contract HEALTH-F3-2011-128 278232). 129 We would like to thank Dr. Alessandra Carattoli for kindly providing the control 130 plasmids for replicon typing of incompatibility groups. 131  7 REFERENCES 132 1. Nordmann P, Dortet L, and Poirel L. 2012. Carbapenem resistance in 133 Enterobacteriaceae: here is the storm! Trends Mol. Med. 18:263-272. 134 2. Oteo J, Calbo E, Rodríguez-Baño J, Oliver A, Hornero A, Ruíz-Garbajosa 135 P, Horcajada JP, Del Pozo JL, Riera M, Sierra R, Bou G, and Salavert M. 136 2014. [The threat of the carbapenemase-producing Enterobacteriaceae in Spain: 137 Positioning report of the SEIMC study groups, GEIH and GEMARA.]. Enferm. 138 Infecc. Microbiol. Clin. Epub 2014 Apr 21 139 3. Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, and Walsh 140 TR. 2009. Characterization of a new metallo-β-lactamase gene, bla(NDM-1), 141 and a novel erythromycin esterase gene carried on a unique genetic structure in 142 Klebsiella pneumoniae sequence type 14 from India. Antimicrob. Agents. 143 Chemother. 53:5046-5054. 144 4. Dortet L, Poirel L, and Nordmann P. 2014. Worldwide Dissemination of the 145 NDM-Type Carbapenemases in Gram-Negative Bacteria. Biomed. Res. Int. 146 Epub 2014 Mar 26 147 5. Pitart C, Solé M, Roca I, Fàbrega A, Vila J, and Marco F. 2011. First 148 Outbreak of a Plasmid-Mediated Carbapenem-Hydrolyzing OXA-48 β-149 lactamase in Klebsiella pneumoniae in Spain. Antimicrob. Agents Chemother. 150 55:4398-4401. 151 6. Solé M, Pitart C, Roca I, Fàbrega A, Salvador P, Muñoz L, Oliveira I, 152 Gascón J, Marco F, and Vila J. 2011. First description of an Escherichia coli 153 strain producing NDM-1 carbapenemase in Spain. Antimicrob. Agents. 154 Chemother. 55:4402-4404. 155  8 7. Yamane K, Wachino J, Doi Y, Kurokawa H, and Arakawa Y. 2005. Global 156 spread of multiple aminoglycoside resistance genes. Emerg. Infect. Dis. 11:951-157 953. 158 8. Clermont O, Bonacorsi S, and Bingen E. 2000. Rapid and simple 159 determination of the Escherichia coli phylogenetic group. Appl. Environ. 160 Microbiol. 66:4555-4558. 161 9. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, and Threlfall EJ. 162 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. 163 Methods. 63:219-228. 164 10. Villa L, Garcia-Fernandez A, Fortini D, and Carattoli A. 2010. Replicon 165 sequence typing of IncF plasmids carrying virulence and resistance 166 determinants. J. Antimicrob. Chemother. 65:2518-2529. 167 11. Tada T, Miyoshi-Akiyama T, Dahal RK, Sah MK, Ohara H, Shimada K, 168 Kirikae T, and Pokhrel BM. 2014. NDM-1 metallo-β-lactamase and ArmA 169 16S rRNA methylase producing Providencia rettgeri clinical isolates in Nepal. 170 BMC Infect. Dis. 14:56. 171 12. Fiett J, Baraniak A, Izdebski R, Sitkiewicz I, Zabicka D, Meler A, Filczak 172 K, Hryniewicz W, and Gniadkowski M. 2014. The first NDM metallo-β-173 lactamase-producing Enterobacteriaceae isolate in Poland: evolution of IncFII-174 type plasmids carrying the bla(NDM-1) gene. Antimicrob. Agents Chemother. 175 58:1203-1207. 176 13. Bonnin RA, Poirel L, Carattoli A, and Nordmann P. 2012. Characterization 177 of an IncFII plasmid encoding NDM-1 from Escherichia coli ST131. PLoS One. 178 7:e34752. 179  9 14. Hishinuma A, Yoshida A, Suzuki H, Okuzumi K, and Ishida T. 2013. 180 Complete sequencing of an IncFII NDM-1 plasmid in Klebsiella pneumoniae 181 shows structural features shared with other multidrug resistance plasmids. J. 182 Antimicrob. Chemother. 68:2415-2417. 183 15. Mataseje LF, Boyd DA, Lefebvre B, Bryce E, Embree J, Gravel D, Katz K, 184 Kibsey P, Kuhn M, Langley J, Mitchell R, Roscoe D, Simor A, Taylor G, 185 Thomas E, Turgeon N, and Mulvey MR. 2014. Complete sequences of a 186 novel blaNDM-1-harbouring plasmid from Providencia rettgeri and an FII-type 187 plasmid from Klebsiella pneumoniae identified in Canada. J. Antimicrob. 188 Chemother. 69:637-642. 189 16. Poirel L, Dortet L, Bernabeu S, and Nordmann P. 2011. Genetic features of 190 blaNDM-1-positive Enterobacteriaceae. Antimicrob. Agents Chemother. 55:5403-191 5407. 192 17. Voulgari E, Gartzonika C, Vrioni G, Politi L, Priavali E, Levidiotou-193 Stefanou S, and Tsakris A. 2014. The Balkan region: NDM-1-producing 194 Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece. J. 195 Antimicrob. Chemother. 69:2091-2097. 196 18. Price LB, Johnson JR, Aziz M, Clabots C, Johnston B, Tchesnokova V, 197 Nordstrom L, Billig M, Chattopadhyay S, Stegger M, Andersen PS, Pearson 198 T, Riddell K, Rogers P, Scholes D, Kahl B, Keim P, and Sokurenko EV. 199 2013. The epidemic of extended-spectrum-beta-lactamase-producing 200 Escherichia coli ST131 is driven by a single highly pathogenic subclone, H30-201 Rx. MBio. 4:e00377-00313. 202  10 19. Bercot B, Poirel L, and Nordmann P. 2008. Plasmid-mediated 16S rRNA 203 methylases among extended-spectrum β-lactamase-producing 204 Enterobacteriaceae isolates. Antimicrob. Agents Chemother. 52:4526-4527. 205 20. Deng Y, Zeng Z, Chen S, He L, Liu Y, Wu C, Chen Z, Yao Q, Hou J, Yang 206 T, and Liu JH. 2011. Dissemination of IncFII plasmids carrying rmtB and qepA 207 in Escherichia coli from pigs, farm workers and the environment. Clin. 208 Microbiol. Infect. 17:1740-1745. 209 21. Li DX, Zhang SM, Hu GZ, Wang Y, Liu HB, Wu CM, Shang YH, Chen 210 YX, and Du XD. 2012. Tn3-associated rmtB together with qnrS1, aac(6')-Ib-cr 211 and blaCTX-M-15 are co-located on an F49:A-:B- plasmid in an Escherichia coli 212 ST10 strain in China. J. Antimicrob. Chemother. 67:236-238. 213 22. Wachino J, and Arakawa Y. 2012. Exogenously acquired 16S rRNA 214 methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative 215 bacteria: an update. Drug. Resist. Updat. 15:133-148. 216 23. Gil-Romero Y, Sanz-Rodríguez N, Almagro-Molto M, and Gómez-Garces 217 JL. 2013. [New description of a NDM-1 carbapenemase producing Klebsiella 218 pneumoniae carrier in Spain]. Enferm. Infecc. Microbiol. Clin. 31:418-419. 219 24. Oteo J, Domingo-García D, Fernández-Romero S, Saez D, Guiu A, Cuevas 220 O, López-Brea M, and Campos J. 2012. Abdominal abscess due to NDM-1-221 producing Klebsiella pneumoniae in Spain. J. Med. Microbiol. 61:864-867. 222 25. Rahman M, Shukla SK, Prasad KN, Ovejero CM, Pati BK, Tripathi A, 223 Singh A, Srivastava AK, and Gonzalez-Zorn B. 2014. Prevalence and 224 molecular characterisation of New Delhi metallo-β-lactamases NDM-1, NDM-5, 225 NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India. Int. 226 J. Antimicrob. Agents. 44:30-37. 227  11 26. Hornsey M, Phee L, and Wareham DW. 2011. A novel variant, NDM-5, of 228 the New Delhi metallo-β-lactamase in a multidrug-resistant Escherichia coli 229 ST648 isolate recovered from a patient in the United Kingdom. Antimicrob. 230 Agents Chemother. 55:5952-5954. 231 27. Borgia S, Lastovetska O, Richardson D, Eshaghi A, Xiong J, Chung C, Baqi 232 M, McGeer A, Ricci G, Sawicki R, Pantelidis R, Low DE, Patel SN, and 233 Melano RG. 2012. Outbreak of carbapenem-resistant Enterobacteriaceae 234 containing blaNDM-1, Ontario, Canada. Clin. Infect. Dis. 55:e109-117. 235 28. Poirel L, Savov E, Nazli A, Trifonova A, Todorova I, Gergova I, and 236 Nordmann P. 2014. Outbreak caused by NDM-1- and RmtB-producing 237 Escherichia coli in Bulgaria. Antimicrob. Agents Chemother. 58:2472-2474. 238 29. Yoo JS, Kim HM, Koo HS, Yang JW, Yoo JI, Kim HS, Park HK, and Lee 239 YS. 2013. Nosocomial transmission of NDM-1-producing Escherichia coli 240 ST101 in a Korean hospital. J. Antimicrob. Chemother. 68:2170-2172. 241 30. Johnson AP, and Woodford N. 2013. Global spread of antibiotic resistance: 242 the example of New Delhi metallo-β-lactamase (NDM)-mediated carbapenem 243 resistance. J. Med. Microbiol. 62:499-513. 244  12  245 FIGURE LEGENDS AND TABLES 246 Fig. 1. Schematic drawing showing the genetic elements surrounding the 247 blaNDM genes in pHC105-NDM (KM598665), pMC-NDM (HG003695) and pGUE-248 NDM (JQ364967). The lengths of the arrows are not proportional to the lengths of the 249 genes or open reading frames (ORFs). Red arrow indicates the remnant ISAba125 250 fragment providing the -35 promoter region for transcription of blaNDM. Abbreviations 251 and symbols are: blaTEM, TEM β-lactamase; rmtB, 16S rRNA methylase gene; IS, 252 insertion sequence; blaNDM, New Delhi metallo-β-lactamase gene; ble, bleomycin 253 resistance gene; trpF, phosphoribosylanthranilate isomerase gene; tat, twin-arginine 254 translocation pathway signal protein gene; sul1, sulfonamide resistance gene; qac, 255 quaternary ammonium compounds resistance gene; aadA2, aminoglycoside 256 adenyltransferase gene; orf1, open reading frame; dhfrA12, dihydrofolate reductase 257 gene; int1, integrase gene; tnpM, transposition modulator gene; aacC2, aminoglycoside 258 acetyltransferase gene; IRL, inverted repeat left; IRR, inverted repeat right; Tn, 259 transposon; Δ, truncated gene. 260  261  13 Table 1. In vitro susceptibilities of E. coli HC105 and E. coli J53AziR HC105 262 transconjugant expressing NDM-5 carbapenemase.  263 Antimicrobial Agents MIC (µg/ml) in: E. coli HC105 E. coli J53 AziR HC105T E. coli J53AziR Cefoxitin > 256 > 256 2 Cefotaxime > 256 > 256 0.094 Ceftazidime > 256 > 256 0.25 Cefepime > 256 128 0.25 Imipenem > 32 > 32 0.19 Meropenem > 32 > 32 0.023 Ertapenem > 32 > 32 0.008 Aztreonam 64 0.19 0.047 Gentamicin > 256 > 256 0.19 Amikacin > 256 > 256 0.75 Tobramycin > 256 > 256 0.19 Tigecycline 0.38 0.38 0.38 Colistin 0.125 0.125 0.125 Fosfomycin 0.5 0.5 0.5 Levofloxacin >32 0.047 0.047 Ciprofloxacin > 32 0.032 0.008  264  265 

Molecular characterization of blaNDM-5 carried in an IncFII plasmid in Escherichia coli from a non-traveller patient in Spain.

Molecular characterization of blaNDM-5 carried in an IncFII plasmid in Escherichia coli from a non-traveller patient in Spain.

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