Received 1 November 2011; returned 25 November 2011; revised 30 November 2011; accepted 1 December 2011

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1 J Antimicrob Chemother 2012; 67: doi: /jac/dkr553 Advance Access publication 29 December 2011 Characterization of plasmids encoding extended-spectrum b-lactamases and their addiction systems circulating among Escherichia coli clinical isolates in the UK Michel Doumith 1 *, Hiran Dhanji 1, Matthew J. Ellington 2, Peter Hawkey 3,4 and Neil Woodford 1 1 Antibiotic Resistance Monitoring & Reference Laboratory, HPA Colindale, 61 Colindale Avenue, London NW9 5EQ, UK; 2 Cambridge Microbiology and Public Health Laboratory, HPA Addenbrooke s Hospital, Hills Road, Cambridge CB2 0QW, UK; 3 West Midlands Public Health Laboratory, HPA, Birmingham B9 5SS, UK; 4 School of Immunity and Infection, University of Birmingham, Edgbaston B15 2TT, UK *Corresponding author. Tel: +44-(0) ; Fax: +44-(0) ; michel.doumith@hpa.org.uk Received 1 November 2011; returned 25 November 2011; revised 30 November 2011; accepted 1 December 2011 Objectives: To characterize plasmids encoding extended-spectrum b-lactamases (ESBLs) from a recent UK collection of clinical Escherichia coli isolates. Methods: The isolates comprised 118 ESBL producers referred from 54 laboratories. Plasmids were transferred by electroporation, and their incompatibility groups, associated addiction systems and resistance genes with the flanking genetic environments were identified by PCR or sequencing. Results: Seventy isolates had plasmids encoding CTX-M-15 (n¼53), CTX-M-14 (n¼9), CTX-M-27 (n¼1), CTX-M-3 (n¼2) and SHV-12 (n¼5) ESBLs that were transformable; non-transformable ESBLs were mainly CTX-M enzymes (42/48). Most transformable bla CTX-M-15 genes (43/53) were harboured on single replicon or multireplicon IncF plasmids, with IncFIA4-FIB1-FII31 (n¼11) and IncFIA1-FII2 (n¼15) being most frequent; the latter included eight pek499 plasmids, typical of UK epidemic strain A. Plasmids harbouring bla CTX-M-14 belonged variously to IncF, IncI1 and IncHI2 types, and 16 encoding CTX-M or SHV enzymes were non-typeable. Only IncF plasmid types carried the addiction systems sought and those with bla CTX-M-15 frequently harboured bla OXA-1 and aac(6 )-Ib-cr, and often transferred trimethoprim and tetracycline resistance; those with bla CTX-M-14 encoded trimethoprim, sulphonamide, streptomycin and tetracycline resistance. Most ESBL genes were associated with the well-known mobile elements ISEcp1 and IS26, but nearly half (23/55) of the ISEcp1 sequences upstream of bla CTX-M-15 were interrupted by an IS26 at various positions. Conclusions: Most ESBLs (70/118) were encoded by transformable plasmids, although a sizable minority could not be transformed. The majority of transformable plasmids (51/70; 72.9%) were diverse multiresistant IncF types possessing multiple addiction systems. The spread of bla CTX-M-15 can be attributed not just to clonal expansion, but also to the horizontal dissemination of related plasmids. Keywords: replicon typing, toxin antitoxin systems, multiresistance, ST131 Introduction Plasmid-encoded extended-spectrum b-lactamases (ESBLs), along with carbapenemases, are among the most important acquired resistance determinants spreading worldwide in members of the Enterobacteriaceae. ESBLs confer resistance to oxyimino-cephalosporins, but producers are often multidrug resistant, leaving only limited therapeutic options. 1 Most early ESBLs evolved from TEM and SHV b-lactamases and were largely restricted to healthcare facilities, occurring mainly in Klebsiella spp. Over the last decade, CTX-M-type ESBLs have proliferated and are now the most common ESBLs in most areas of the world. 1 3 Escherichia coli producing CTX-M ESBLs have become a major cause of infections in both the community and hospitals. In the UK, ESBL-producing E. coli isolates, mostly with the CTX-M-15 enzyme, have become prevalent since The epidemiology of these isolates is partly clonal, including numerous PFGE-defined variants of the international O25:H4-ST131 uropathogenic lineage. 4 6 One variant of this clone, designated strain A, often harbours bla CTX-M-15 on the multidrug resistance non-conjugative IncFII-IncFIA plasmid pek499, which confers resistance to eight different antibiotic classes. However, other diverse plasmid types have been associated with CTX-M-type ESBLs in humans, animals and the environment in the UK The present study sought to characterize # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oup.com 878

2 Molecular characterization of ESBL plasmids in E. coli JAC ESBL-encoding plasmids from a recent collection of UK E. coli clinical isolates from humans. Materials and methods Bacterial isolates and transformants The clinical isolates comprised 118 ESBL-producing E. coli isolates referred to the Antibiotic Resistance Monitoring & Reference Laboratory (ARMRL) from 54 different UK laboratories; they were selected from isolates referred to ARMRL in for confirmation of ESBL production. Plasmids were extracted using an alkaline lysis method and transferred by electroporation into the E. coli a-select strain (Bioline, London, UK), as described in the manufacturer s protocol, using a GenePulser electroporator (Bio-Rad, Hemel Hempstead, UK). 11 Transformants were cultured on Luria Bertani agar supplemented with 2 mg/l cefotaxime or ceftazidime to select plasmid-encoded CTX-M and SHV enzymes, respectively. Antibiotic susceptibility testing MICs were determined by agar dilution and interpreted according to BSAC guidelines ( The antibiotics tested in this study are listed in Table 3. Isolate typing methods E. coli isolates were assigned to phylogenetic groups A, B1, B2 and D, according to the rapid multiplex PCR method described by Clermont et al. 12 All group B2 isolates were screened by PCR for ST131-associated single nucleotide polymorphisms in the pabb gene. 13 Selected isolates were typed by PFGE of XbaI-digested genomic DNA. Patterns were analysed using Bionumerics software (Applied Maths, Sint Martens-Latem, Belgium) to generate a dendrogram based on the unweighted pair group method with an arithmetic average from the Dice coefficient. 14 Identification of plasmid addiction systems Plasmid-mediated CcdA-CcdB (coupled to cell division), PemK-PemI (for plasmid emergency maintenance), RelB-RelE (relaxed control of stable RNA), ParD-ParE (DNA replication), VagC-VagD (virulence-associated protein), Hok-Sok (host-killing), SnrB-SnrC (RNA stability) and PndA-PndC (promotion of nucleic acid) were sought by PCR. Primers and amplification conditions were as described in Mnif et al. 22 Results Clinical isolates and transformants The 118 ESBL-positive E. coli clinical isolates variably produced CTX-M group 1 (n¼85) or group 9 (n¼22), SHV (n¼9) and TEM (n¼2) enzymes. They divided among all four major phylogenetic groups: B2 (n¼73, 61.9%), D (n¼33, 28.0%), A (n¼10, 8.5%) and B1 (n¼2, 1.7%). Most group B2 isolates (60/73) belonged to the internationally disseminated uropathogenic clone, O25:H4-ST131. Only 70/118 (59.3%) ESBL determinants were transferable by electroporation into E. coli a-select; these encoded CTX-M-15 (n¼53), CTX-M-3 (n¼2), CTX-M-14 (n¼9), CTX-M-27 (n¼1) and SHV-12 (n¼5) variants (Table 1). Forty-eight ESBL determinants, including CTX-M groups 1 and 9 (n¼42), SHV (n¼4) and TEM (n¼2) types, were nontransformable despite repeated transformation attempts and therefore not studied further; they were distributed among E. coli of all four phylogenetic groups (Table 1). The high proportion of non-transformable plasmids might be related to their large size and inability to propagate in the host strain used for electroporation experiments or might indicate a chromosomal location for the ESBL genes. Characterization of resistance genes and their surrounding environments The bla TEM-1 and bla OXA-1 genes, and the aminoglycoside and fluoroquinolone resistance genes aac(6 )-Ib-cr, qnra, qnrb, qnrc, qnrd and qnrs were detected by PCR Detection of bla SHV and bla CTX-M-15 at group levels was carried using previously described methods. 18,19 The genetic environments of the bla CTX-M genes were identified by PCR and sequencing using combinations of the forward primers ISEcp1, IS26a or IS26b with the reverse primer ORF477 for CTX-M group 1 and reverse primers IS903 or CTX-M-9-rev for those of group 9. Combinations of IS26a- or IS26b-forward and SHV-reverse primers were used to characterize the environment of bla SHV genes. All these primer sequences and amplification conditions were as described by Dhanji et al., 8,9 and all the PCR products were treated with exonuclease I (Biolabs, Hitchin, UK) and alkaline phosphatase (Roche, Burgess Hill, UK) prior to sequencing using an ABI Genetic Analyser capillary platform 3130XL (Applied Biosystems, CA, USA). Plasmid replicon typing Plasmids were first rep typed as described by Carattoli et al., 20 and those found to harbour IncF replicons were further characterized using primers and amplification conditions according to the recent IncF replicon sequencing scheme. 21 IncF sequence types were assigned using the plasmid multilocus sequence typing (MLST) database ( org/plasmid/). Plasmid replicon (rep) types Fifty-one of the 70 transformable ESBL plasmids (72.8%) belonged to IncF replicon types, mostly (45/51) built as combinations of IncFIA, IncFII and/or IncFIB replicons (Table 2). These included the majority of plasmids encoding CTX-M-15 ESBL (43/53); 5 carried single FIA1 (n¼3) or FII2 (n¼2) rep types and 38 harboured multiple IncF replicons, occurring in 13 different combinations, but with types FIA1-FII2 (n¼15) and FIA4-FIB1-FII31 (n¼11) being most frequent (Table 2). Even though the latter included plasmids harbouring FIA4-FIB1-FII31 (n¼7), FIA4-FIB1 (n¼3) or FIA4-FII31 (n¼1) rep types, they were associated as a group of 11 closely related plasmids (Table 2). The FIA1-FII2 and FII2 rep types corresponded to those of the bla CTX-M-15 -encoding plasmids pek499 and pek516 from UK epidemic strains A and D, respectively. 23 Plasmids with bla CTX-M-14 variably belonged to rep types FIA2-FII2 (n¼4), FIA1-FIB20 (n¼1), FIB6-FII31 (n¼1), IncI1 (n¼2) and HI2 (n¼1), and the single plasmid with the bla CTX-M-27 (CTX-M group 9) enzyme was an FIA2-FIB20-FII1 type. Only one plasmid with bla SHV-12 was assigned a rep type, FIB10 (Table 2). Sixteen of 70 transformable plasmids (22.9%) were nontypeable for the 18 incompatibility groups sought by the PCR-based replicon typing; 10 of these encoded the CTX-M-15 enzyme, 4 encoded SHV-12 and 2 encoded CTX-M-3 (Table 2). 879

3 Doumith et al. Table 1. Phylogenetic groups, ESBL types and plasmid transformability for the 118 ESBL-producing E. coli isolates Transformable ESBLs Non-transformable ESBLs Phylogroups CTX-M-gp1 CTX-M-gp9 SHV TEM CTX-M-gp1 CTX-M-gp9 SHV TEM Total B2 39 (37) 6 (3) 4 (2) 18 (17) (1) 73 (60) D A B Total Numbers within brackets indicate B2 isolates of the uropathogenic clone ST131. gp1, group 1; gp9, group 9. Addiction systems of IncF plasmids Among the seven addiction systems sought, only the ccdab, pemki, vagcd, snrbc and hok-sok systems were detected; none of the plasmids harboured parde or pndac. Addiction systems were identified exclusively on IncF plasmids and mainly in combinations of two (n¼6), three (n¼13), four (n¼29) or five (n¼1) different modules; one IncFII2 plasmid harbouring only the pemki system and one IncFIB10 lacked any, as did the IncI1 (n¼2), IncHI2 (n¼1) and non-typeable (n¼16) plasmids (Table 2). There were no clear relationships between the numbers or the combination of the addiction modules and the ESBL types. Nevertheless, all plasmids of the FI4-FIB1-FII31 rep group (n¼11) carried an identical combination of four addiction modules, namely ccdab, pemki, vagcd and snrbc. The combination of these systems was less conserved among plasmids belonging to the FIA1-FII2 rep group (n¼15); nine had a combination of ccdab, pemki, vagcd and hok-sok systems, as found on the pek499 plasmid, five harboured only three of these systems and another had four addiction modules, but with the vagcd system replaced by snrbc. 23 Only one of the two plasmids harbouring the IncFII replicon alone had the pemki, hok-sok combination found on pek516; the other had only the pemki system (Table 2). 23 ESBL environments The bla CTX-M-15 gene was flanked on 30 of 53 plasmids by the international genetic environment, with ORF477 downstream and an intact copy of insertion sequence ISEcp1 48 bp upstream of the bla gene. The ISEcp1 element in the other 23 plasmids was interrupted by IS26: (i) inserted in a transcriptional orientation opposite to the bla gene, 24 bp from the 3 end of ISEcp1, as in UK epidemic strain A (n¼10); (ii) 545 bp from the 3 end of the mobile element (n¼7); or (iii) inserted in the same orientation as the bla gene, and 203 bp (n¼1), 276 bp (n¼1) or.700 bp from the 3 end of ISEcp1 (n¼4) (Table 2). All 10 plasmids harbouring the strain A bla CTX-M-15 genetic environment originated from isolates that belonged to the B2-ST131 clone and clustered at.85% similarity with strain A by PFGE, suggesting that these were pek499-like plasmids. However, only eight had the FIA1-FII2 rep type of pek499, as originally defined; the two others had just the IncFIA1 replicon. Plasmids belonging to the major FIA4-FIB1-FII31 multireplicon group (n¼11) exhibited three different bla CTX-M-15 environments: six plasmids from B2-ST131 isolates had the ISEcp1 element interrupted by IS26 inserted at 545 bp from the end of ISEcp1; three from phylogroup A isolates had IS26 inserted.700 bp from the end of the element; and two had an intact ISEcp1 upstream of the bla gene (Table 2). The bla CTX-M-14 gene was flanked, on three of nine plasmids, by ISEcp1 upstream and IS903 downstream; another had IS26 upstream of the bla gene, as did the single plasmid harbouring bla CTX-M-27, and five lacked these insertion sequences. All five bla SHV-12 -carrying plasmids had IS26 inserted upstream of the bla gene (Table 2). Antibiotic resistance genes and phenotypes In addition to the ESBL-mediated resistance to penicillins and cephalosporins, the majority of these plasmids conferred multidrug resistance phenotypes, as described in Table 3. Although high resistance to ciprofloxacin was not transferred, many plasmids conferring an increase in the MICs from 0.03 to 0.06 (n¼36), 0.25 (n¼1) or 0.5 mg/l (n¼1) were associated with the presence of the aac(6 )-Ib-cr and qnr genes, respectively (Table 2). The bla TEM-1 gene was identified on 20 ESBL plasmids, but with no association to particular types of plasmids; those hosting it belonged to various IncF types (n¼15) or were nontypeable (n¼5). Among the 43/53 IncF plasmids encoding bla CTX-M-15, 34 harboured the aac(6 )-Ib-cr and bla OXA-1 genes mediating resistance to amikacin, tobramycin and piperacillin/ tazobactam and increasing ciprofloxacin MICs from 0.03 to 0.06 mg/l; these included all 11 members of the FIA4-FIB1-FII31 rep type group and 13/15 of those belonging to the FIA1-FII2 rep group. The antibiotic resistance phenotypes of the bla CTX-M-15 transformants also showed a frequent transfer of coresistance to trimethoprim and tetracycline, with 28/53 plasmids transferring resistance to both antibiotics and a further 14 transferring resistance to one or the other (Table 3). All members of the FI4-FIB1-FII31 rep type group (n¼11) conferred resistance to trimethoprim and five of these also transferred resistance to tetracycline (Table 2). Differences in antibiotic resistance phenotypes were observed amongst related plasmids harbouring the same replicon types. For example, pek499 conferred resistance to eight different antibiotics, but resistance to tetracycline, trimethoprim and sulphonamides was not conferred by all FIA1-FII2 rep type, pek499-like plasmids; only one conferred resistance to 880

4 881 Table 2. Molecular characteristics and antibiotic resistance phenotypes of 70 ESBL-encoding plasmids Plasmid type Addiction system n bla genetic environment bla TEM-1 aac(6 )-Ib-cr bla OXA-1 Resistance profile Phylogenetic group CTX-M-15 FIA1 ccdab, pemki, vagcd, hok-sok 1 A TZP, TET, SUL, TMP B2-ST131 C42 ccdab, pemki, vagcd 1 A TZP, AMK, TOB, TET, TMP, CIP B2-ST131 C23 ccdab, vagcd, hok-sok 1 B TZP, AMK, TOB, GEN, TET, TMP, CIP B2-ST131 C33 FIA1-FII2 ccdab, pemki, vagcd 2 A TZP AMK, TOB, TET, SUL, TMP, CIP B2-ST131 C43 ccdab, pemki, vagcd, hok-sok 6 A TZP, AMK, TOB, TET, STR, SUL, TMP, CIP B2-ST131 C33 A TZP, AMK, TOB, TMP, CIP B2-ST131 C50 A TZP, AMK, TOB, TET, SUL, TMP, CIP B2-ST131 C27 A TZP, AMK, TOB, TET, TMP, CIP B2-ST131 C34 A TZP, AMK, TOB, TET, SUL, TMP, CIP B2-ST131 C16 A TZP, AMK, TOB, TET, SUL, CIP B2-ST131 C3 ccdab, pemki, vagcd, hok-sok 3 B TZP, AMK, TOB, TET, SUL, TMP, CIP B2-ST131 C20 B TZP, AMK, TOB, TET, STR, SUL, TMP, CIP B2-ST131 C41 C TZP, AMK, TOB, TET, TMP, CIP B2-ST131 C20 ccdab, vagcd, hok-sok 1 B GEN, STR, CHL B2-ST131 C20 ccdab, pemki, snrbc, hok-sok 1 B TZP, AMK, TOB, GEN, TET, STR, SUL, TMP, CIP D C32 ccdab, pemki, hok-sok 1 B TZP, AMK, TOB, TET, TMP, CIP B2-ST131 C40 pemki, vagcd, hok-sok 1 B none B2-ST131 C33 FIA1-FIB20 ccdab, pemki, snrbc 1 B TZP, AMK, TOB, GEN, TET, SUL, TMP, CIP B2-ST131 C11 FIA1-FIB9 ccdab, pemki 1 B TZP, AMK, TOB, TET, CHL, TMP, CIP B2-ST131 C18 ccdab, pemki, snrbc, hok-sok 1 B TZP, AMK, TOB, GEN, STR, SUL, TMP, CIP A C34 ccdab, pemki, snrbc 1 D TZP, AMK, TOB, TET, TMP, CIP D C25 FIA1-FIB1-FII1 ccdab, pemki, vagcd, snrbc, hok-sok 1 B GEN, TET B2-ST131 C23 ccdab, pemki, snrbc, hok-sok 1 B GEN, TET, TMP D C17 FIA1-FIB1-FII2 ccdab, pemki, vagcd, hok-sok 1 E TZP, AMK, TOB, TET, TMP, CIP B2-ST131 C5 FIA1-FIB9-FII1 ccdab, pemki, snrbc, hok-sok 1 B GEN, TET, TMP D C17 FIA1-FIB9-FII2 ccdab, pemki, hok-sok 1 F TZP, AMK, TOB, GEN, TET, CIP B2-ST131 C20 FIA1-FIB16-FII1 ccdab, pemki, snrbc, hok-sok 1 B TET, STR, SUL, TMP D C24 FIA1-FIB20-FII22 ccdab, pemki, snrbc 1 B TZP, AMK, TOB, GEN, TMP, CIP B2-ST131 C36 FIA4-FIB1 ccdab, pemki, vagcd, snrbc 2 D TZP, AMK, TOB, TMP, CIP B2-ST131 C7 1 C TZP, AMK, TOB, TET, TMP, CIP A C28 FIA4-FII31 ccdab, pemki, vagcd, snrbc 1 B TZP, AMK, TOB, TMP, CIP B2-ST131 C1 FIA4-FIB1-FII31 ccdab, pemki, vagcd, snrbc 4 D TZP, AMK, TOB, GEN, TET, TMP, CIP B2-ST131 C24 TZP, AMK, TOB, GEN, TMP, CIP B2-ST131 C19 TZP, AMK, TOB, SUL, TMP, CIP B2-ST131 C48 TZP, AMK, TOB, TMP, CIP B2-ST131 C51 2 C TZP, AMK, TOB, GEN, TET, TMP, CIP A C12-C34 1 B TZP, AMK, TOB, GEN, TET, CHL, TMP, CIP A C23 FIA4-FIB20-FII1 ccdab, pemki, snrbc, hok-sok 1 B GEN, TET, TMP D C36 FII2 pemki, hok-sok 1 B TZP, AMK, TOB, GEN, TET, CIP B2-ST131 C46 pemki 1 B TZP, AMK, TOB, CIP B2-ST131 C10 Centre Continued Molecular characterization of ESBL plasmids in E. coli JAC

5 882 Table 2. Continued Plasmid type Addiction system n bla genetic environment bla TEM-1 aac(6 )-Ib-cr bla OXA-1 Resistance profile Phylogenetic group ND none 1 a B TZP, AMK, TOB, GEN, TET, STR, SUL, TMP, CIP B2 C29 7 B none B2-ST131/D C32-C33-C37-C43 1 B STR, SUL B2 C37 1 B TET, SUL D C23 Centre Doumith et al. CTX-M-3 ND none 1 B TZP B2-ST131 C15 1 B TZP, AMK D C2 CTX-M-14 FIA1-FIB20 ccdab, pemki, snrbc, hok-sok 1 ND GEN, TET, STR, SUL, CHL, TMP D C45 FIA2-FII2 ccdab, pemki 2 ND TET, STR, SUL, TMP B2-ST131 C24-C35 1 ND TZP B2-ST131 C35 1 G TET, STR, SUL, TMP D C32 FIB6-FII31 pemki, snrbc, hok-sok 1 G TET, STR, SUL, TMP D C38 I1 none 1 G none A C21 1 ND none B2 C6 HI2 none 1 H none B2 C43 CTX-M-27 FIA2-FIB20-FII1 ccdab, pemki, snrbc 1 H TET, STR, SUL, TMP B2-ST131 C23 SHV-12 FIB10 none 1 H TZP, AMK, TOB B2 C4 ND none 1 H AMK, TOB, GEN, SUL, CHL, TMP B2-ST131 C44 1 H TZP, TOB, GEN A C31 1 H TZP, AMK, TOB, GEN, STR B2-ST131 C22 1 b H TZP, STR, CIP B2 C13 AMK, amikacin; TOB, tobramycin; GEN, gentamicin; CIP, ciprofloxacin; CHL, chloramphenicol; SUL, sulfamethoxazole; STR, streptomycin; TET, tetracycline; TMP, trimethoprim; TZP, piperacillin+4 mg/l tazobactam; ND, not determined. bla genetic environment: A, ISEcp1 interrupted by IS26 inserted in opposite orientation to bla 24 bp from end; B, ORF477 downstream and intact ISEcp1 upstream of bla; C, ISEcp1 interrupted by IS26 inserted in same orientation to bla.700 bp from end; D, ISEcp1 interrupted by IS26 inserted in opposite orientation to bla 545 bp from end; E, ISEcp1 interrupted by IS26 inserted in same orientation to bla 276 bp from end; F, ISEcp1 interrupted by IS26 inserted in same orientation to bla 203 bp from end; G, IS903 downstream and intact ISEcp1 upstream of bla; and H, IS26 upstream of bla. a Plasmid harboured a qnrb1. b Plasmid harboured a qnrs1.

6 Molecular characterization of ESBL plasmids in E. coli JAC Table 3. Number of transformants for which MICs of comparator antibiotics were raised on acquisition of ESBL plasmids Antibiotic Recipient E. coli a-select MIC (mg/l) Transformants MIC range (mg/l) any (n¼70) Number of transformants acquiring reduced susceptibility or resistance CTX-M-15 (n¼53) CTX-M-3 (n¼2) CTX-M-14 (n¼9) CTX-M-27 (n¼1) SHV-12 (n¼5) Cefotaxime to Ceftazidime Ciprofloxacin Piperacillin/tazobactam Amikacin Tobramycin Gentamicin Trimethoprim Tetracycline Chloramphenicol Streptomycin Sulfamethoxazole 2 8 to streptomycin and none transferred resistance to chloramphenicol. 23 Moreover, the bla TEM-1 gene harboured by pek499 was only detected on three of these plasmids. Seven of the 12 non-typeable plasmids harbouring bla CTX-M conferred only ESBL-mediated resistance to b-lactams; among those containing multiresistance phenotypes (5/12), one harboured qnrb1 and conferred resistance to nearly all the antibiotics tested (9/10; Table 2). Plasmids encoding bla CTX-M-14 consistently lacked bla OXA-1 and aac(6 )-Ib-cr, and only five of the six harbouring IncF rep types conferred multiresistance phenotypes; these encoded resistance to streptomycin, sulphonamides, tetracycline and trimethoprim, and one also conferred resistance to chloramphenicol and gentamicin. The sixth remaining IncF plasmid had only reduced susceptibility to piperacillin/tazobactam, although it lacked bla OXA-1. Resistance to aminoglycosides was also rarely detected; only one plasmid conferred reduced susceptibility to gentamicin. In contrast with 7/10 non-typeable plasmids with bla CTX-M-15, the 4 harbouring bla SHV-12 conferred multiresistance phenotypes. Of these, two conferred resistance to amikacin, tobramycin and gentamicin; one of these also transferred resistance to sulphonamides, trimethoprim and chloramphenicol, and one also to piperacillin/tazobactam and streptomycin. One plasmid harboured bla OXA-1 and qnrs1 genes, and conferred resistance to ciprofloxacin, piperacillin/tazobactam and streptomycin (Table 2). Discussion Even though most (60/118) of the ESBL-producing E. coli used in this study, in particular those producing CTX-M-15 enzyme, belonged to the B2-ST131 clone, the plasmids carrying their b-lactamase genes were variably transformable. Most of the bla genes (51/70) that could be transformed were located on IncF plasmids, but these included 20 different single and multireplicon IncF combinations. In addition, bla CTX-M-14 was carried on IncI1 (n¼2) and IncHI2 (n¼1) plasmids, whereas 16 plasmids harbouring bla CTX-M-15, bla CTX-M-3 and bla SHV-12 were nontypeable for the 18 replicon types described in Carattoli et al. 20 The possibility that these non-typeable plasmids harboured other defined replicons, such as ColE, IncR or IncU types, cannot be excluded since these were not sought in our repliconbased PCR testing. 24 As expected, most IncF plasmid types conferred antibiotic multiresistance profiles. In particular, CTX-M-15-encoding plasmids mostly conferred resistance to piperacillin/tazobactam, amikacin, tobramycin and ciprofloxacin due to the frequent presence of the bla OXA-1 and aac(6 )-Ib-cr genes; many also conferred resistance to trimethoprim and tetracycline. In contrast, plasmids with bla CTX-M-14 lacked the bla OXA-1 and aac(6 )-Ib-cr genes and transformants often remained susceptible to aminoglycosides, whilst acquiring resistance to tetracycline, trimethoprim, streptomycin and sulphonamides. This is consistent with bla CTX-M-14 plasmids reported from Canada, France, Portugal and Spain, but is in marked contrast to those reported from the Far East where this bla variant is dominant and its producers multiresistant The IncI, IncHI and the majority of the nontypeable plasmids lacked resistance determinants to non-b-lactams. IncF plasmid types are thought to be well-adapted to proliferate in E. coli, but their successful retention in E. coli populations may also be attributed to the presence of addiction systems; nearly all (95%) were found to harbour at least two different modules, promoting maintenance and success even in the absence of antibiotic selection. Although some of these systems, such as pndac, parde and snrbc, have previously been identified on non-incf plasmids, none was identified on the three IncI1 or HI2 plasmids or those that were non-typeable. Genes encoding CTX-M-15, the most widespread ESBL amongst clinical E. coli clinical isolates in the UK and worldwide, were harboured on 15 different IncF plasmid types. More than half belonged to FIA4-FIB1-FII31 and IncFIA1-FII2 multireplicon types, suggesting that the spread of CTX-M-15 in the UK is in part attributable to the horizontal dissemination of related plasmids. The 11 of the FIA4-FIB1-FII31 rep group, received from

7 Doumith et al. different laboratories, shared the same combination of four addiction systems, harboured bla OXA-1 and aac(6 )-Ib-cr, and conferred resistance to trimethoprim. However, 4 of the 11 related plasmids lacked the IncFIB1 or IncFII31 replicons and thus carried only two rep types; they variably conferred resistance to gentamicin and tetracycline, and exhibited three different bla CTX-M-15 genetic environments. Amongst the plasmids harbouring the IncFIA1-FII2 rep-type group and the characteristic pek499 bla CTX-M-15 environment with its IS26 insertion, three lacked hok-sok, which is one of the four different addiction systems identified on pek499; moreover, their antibiotic resistance profiles were slightly different from that associated with pek499. All the pek499-like plasmids originated from isolates sharing.85% similarity in PFGE banding patterns with strain A; they were received from seven different centres, suggesting that the clonal spread of strain A is still in part responsible for CTX-M-15 dissemination across the UK, 7 years after its initial description. Similar alteration of the pek499 antibiotic profile throughout the acquisition of gentamicin resistance has been also described in strain A isolates from the West Midlands. 31 Such variations at the origin of IncF plasmid diversification, in particular those affecting the addiction systems and/ or origins of replication that are part of the plasmid backbone, are more likely the result of interplasmid recombination events, which seem to be accentuated in plasmids harbouring multiple IncF replicon types; it has been proposed that one replicon is strongly conserved as a result of the selection pressure imposed by plasmid duplication, whereas the others are free to diverge through diverse recombination events, mutations and the uptake or loss of genes, including resistance determinants in response to different antibiotic selection pressure. 32 The majority of plasmids carrying CTX-M-15 that were typeable (42/43) harboured only 3 of the 52 alleles described for the IncFII replicon: 19 harboured FII2 alone (like PEK516) or in combination with FIA1 (like in pek499), including 2 also with FB1 or FB9; 8 plasmids had FII31 in combination with FIA4, of which 7 had also FIB1; and 5 plasmids had the FII1 in combination with FIA1 and variable FIBs. The remaining 10 plasmids lacked the IncFII replicon, but harboured FIA1 (n¼3) or FIA4 (n¼7) along with various FIB types. The low variability of the IncFII types may suggest that there are three major scaffolds of CTX-M-15 plasmids circulating in the UK, consisting of FII2-FIA1, FII31-FIA4-FIB1 or FII1-FIA1 replicons, and diversifying by an FII replicon loss and/or an FIB replicon exchange. However, the validity of these suggestions remains to be confirmed. Even though most of the regions surrounding bla CTX-M-15 corresponded to the international genetic environment with an intact copy of ISEcp1 upstream and ORF477 downstream of the bla gene, different rearrangements of the ISEcp1 element by IS26 insertions occurring at different positions within the ISEcp1 sequences were identified. Most of these bla CTX-M-15 environments were recently identified also in E. coli isolates from the faeces of travellers returning to the UK, predominantly from the Indian subcontinent. Nevertheless, the plasmids were not characterized in detail, making the link between these two populations difficult to establish. The frequency and site of insertion of IS26 in our study were comparable to those described in a study of 294 CTX-M-15 ESBL-producing clinically significant E. coli from the West Midlands isolated in In that study, 14.6% (43/294) of the isolates had the typical strain A IS26 insertion and 6.5% (19/294), compared with 10% in our study, had IS26 inserted in a transcriptional orientation opposite to the bla gene, 545 bp from the end of ISEcpI; those with IS26 inserted in the same orientation as the bla gene were not explored in the study and thus could not be compared. The IS26 insertion at position 545 bp from the end of ISEcpI is the most common IS26 insertion in CTX-M-15-producing E. coli isolated from India, where only the CTX-M-15 variant was found across three separate geographical regions. 33 The West Midlands has a significant population from India/Pakistan, which has recently been shown to have much higher community faecal carriage rates of CTX-M-15-producing E. coli than Caucasians (22.4% versus 8.1%) (N. Wickramasinghe, L. Xu, A. Eustase, S. Shabir, T. Saluja and P. Hawkey, unpublished results). The evolution of CTX-M-15-carrying plasmids in the UK is likely to be complex, as there must be a significant importation of plasmids from India/Pakistan where CTX-M-15 is carried by up to 60% of E. coli. 33 The rearrangements caused by IS26 insertion have been revealed to be particularly valuable for plasmid identification, especially when associated with the new IncF-typing scheme. Due to the mode of transposition of IS26, the remnant of the ISEcp1 sequences located between IS26 and bla CTX-M-15 are likely to be conserved and may well be used, as in the case of pek499, to identify specific plasmids and/or to track their evolution. In summary, the majority of the bla ESBL genes were mobilized on various multiresistance IncF-type plasmids, harbouring multiple addiction systems that presumptively contribute to their maintenance. The IncF replicon-typing scheme allowed the identification of related plasmids contributing to bla CTX-M-15 dissemination. These results suggest that the spread of CTX-M ESBLs in the UK is in part due to the horizontal transfer of clonally related plasmids as well as the clonal spread of strain A, which remains widely scattered across the UK. Acknowledgements This work was commissioned by the National Institute for Health Research. Funding This project was funded by the Health Protection Agency s Strategic Research and Development Fund, project number Transparency declarations P. H. and N. W. have received research and conference support from numerous pharmaceutical companies. Other authors: none to declare. Disclaimer The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health. 884

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