Scholarly article on topic ' The Molecular Epidemiology of Resistance in Cefotaximase-Producing Escherichia coli Clinical Isolates from Dublin, Ireland '

The Molecular Epidemiology of Resistance in Cefotaximase-Producing Escherichia coli Clinical Isolates from Dublin, Ireland Academic research paper on "Biological sciences"

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Academic research paper on topic " The Molecular Epidemiology of Resistance in Cefotaximase-Producing Escherichia coli Clinical Isolates from Dublin, Ireland "

MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2016 © Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2015.0154

EPIDEMIOLOGY

The Molecular Epidemiology of Resistance in Cefotaximase-Producing Escherichia coli Clinical Isolates from Dublin, Ireland

Liam Burke1* Hilary Humphreys1,2 and Deirdre Fitzgerald-Hughes1

In view of continued high clinical prevalence of infections involving extended-spectrum b-lactamase (ESBL)-producing Escherichia coli, this study sought to characterise the blaCTX-M genes, their associated mobile genetic elements and the integrons present in 100 ESBL-producing E. coli isolates collected in a Dublin hospital and associated community healthcare facilities. Polymerase chain reaction (PCR) mapping and sequencing was used to detect blaCTX-M alleles, their associated insertion sequences (ISs) and class 1 and 2 integrons in the collection. ESBL plasmids were characterised by PCR-based replicon typing and replicon sequence typing (RST). Cefo-taximases were harboured by 94% of isolates (66 blaCTX-M-15, 8 blaCTX-M-14, 7 blaCTX-M-1, 4 blaCTX-M-3, 3 blaCTX-M-9, 2 blaCTX-M-27, 2 blaCTX-M-55, 1 blaCTX-M-32 and 1 blaCTX-M-2). An ISEcpl promoter was linked to a group 1 blaCTX-M gene in 45% of isolates. A further 34% of isolates contained blaCTX-M-15 downstream of IS26, an arrangement typical of epidemic UK strain A. Class 1 integrons were found in 66% of isolates, most carrying trimethoprim/aminoglycoside resistance genes. CTX-M plasmids were primarily of mul-tireplicon IncF or IncI1 type, but IncN and unidentified types were also found. Novel IncF RSTs F1:A-:B-, F45:A1:B-, F45:A4:B- and a novel IncI1 sequence type, ST159, were identified. CTX-M plasmids and integrons resembled those identified recently in animal isolates from Ireland and Western Europe. The molecular epidemiology of CTX-M-producing E. coli in Dublin suggests that horizontal spread of mobile genetic elements contributes to antimicrobial resistant human infections. Further investigations into whether animals or animal products represent an important local reservoir for these elements are warranted.

Introduction

Extended-spectrum ß-LACTAMASES (ESBLs) are among the most important resistance determinants in Entero-bacteriaceae, conferring resistance to oxyimino-cephalosporins. ESBLs have increased remarkably in diversity and range recently, predominantly due to the evolution of cefotaximase (CTX-M)-type enzymes, which are now the most common ESBLs worldwide, and are often found on widespread transferable plasmids in Escherichia coli responsible for infections in both the community and hospitals.1 In addition to human-to-human transmission, the contribution of zoonotic transmission by direct contact and through the food chain, to the diversity and mobilisation of blaCTX-M genes is increasingly recognised.2-4 CTX-Ms are organized into five clusters (groups 1, 2, 8, 9, and 25) based on their amino acid se-

quences (www.lahey.org/studies/webt.stm), with group 1 (including CTX-M-1, -3 and -15) and group 9 enzymes (including CTX-M-9, -14 and -27) widespread in Europe. The insertion sequences (ISs), ISEcpl and ISCR1 are responsible for the mobilization of b/aCTX_M and drive their expression. CTX-M genes are frequently found downstream of ISEcpl within modular multidrug-resistance regions (MRRs) on ESBL plasmids5,6 or are linked to IS CR1 on complex class 1 integrons which also bear highly variable gene cassette regions encoding resistance to multiple antimicrobial classes. The surveillance of non blacTx-M-associated integrons in nosocomial ESBL is important as ESBL genes co-localised on the same plasmid may be co-selected in the gut flora of animals where these antibiotics are used and may be a source of zoonotic transfer to humans through the food chain. Multiple copies of IS26 are

department of Clinical Microbiology, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland. 2Department of Microbiology, Beaumont Hospital, Dublin 9, Ireland.

*Present address: RCSI Anatomy, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin 2, Ireland.

also found on MRRs of ESBL plasmids and have contributed to the spread of b/aCTX_M by facilitating genetic rearrangements of these regions between ESBL plasmids.6'7 The staggering propensity for recombination and transposition, afforded by this genetic arrangement' has resulted in the evolution of mosaic ESBL plasmids of narrow and broad host ranges that confer multidrug-resistance' including to the carbapenems.8 In the healthcare setting, these plasmids are likely to be vertically and horizontally disseminated, as the multi-drug resistant (MDR) phenotype they confer provides a survival advantage to bacteria in the antimicrobial laden environment. Their association with widespread successful E. co/i clones, such as the pandemic O25b-ST131 clone, further facilitates their dissemination by clonal expansion.8

We previously investigated the genetic relatedness of 100 ESBL-producing E. co/i (ESBL-EC) from North Dublin9 revealing the widespread dissemination of ST131 and other clones within Beaumont Hospital, Dublin and the local community. The aims of the present study were: (1) to characterise the mobile genetic elements, including the ISs, integrons and plasmids to which b/aCTX_M and other resistance genes were associated in this collection with reference to the published literature from human and animal E. co/i and (2) to determine the potential for horizontal transfer of CTX-M plasmids from clinical E. co/i to laboratory strains.

Materials and Methods

Bacterial strains and culture conditions

One hundred ESBL-EC clinical isolates were collected as part of a previous study between January 2009 and December 2010 in Beaumont Hospital, Dublin, Ireland and were previously subjected to routine diagnostic antimicrobial susceptibility tests and PFGE, which identified 12 clusters A-L.9 Appropriate control strains forbeta-lactamase negative, b/aTEM, b/aSHV and each of the five b/aCTX_M groups and epidemic UK strain A were sourced from the American Type Culture Collection (ATCC) and the National Collection of Type Cultures (NCTC). A sodium azide-resistant E.co/i strain J53 was the recipient for conjugations and was a gift from Prof. Martin Cormican, Department of Bacteriology, National University of Ireland, Galway. E. co/i ElectroMAX™ DH5a-E™ (Invitrogen) was the recipient for transformations. All isolates were routinely grown on Mueller-Hinton (MH) agar.

Characterisation of resistance genes and their associated genetic elements

DNA was prepared from overnight cultures using the Wizard® Genomic DNA Purification Kit (Promega). The carriage of b/aTEM, b/aSHV and the five b/aCTX_M gene groups in the isolates was investigated by multiplex Poly-merase chain reaction (PCR) using previously described primers and cycling conditions.10,1 Specific b/aCTX_M al-leles and their upstream genetic environments were identified by PCR mapping and were fully sequenced. Due to resource constraints, specific b/aSHV and b/aTEM alleles were identified by sequencing only in six isolates that were b/aCTX-M-negative. PCR and sequencing primers used to detect class 1 and 2 integron motifs, ISEcpl, IS26, and ISCR1 and their arrangement in relation to b/aCTX_M are

listed in Supplementary Table S1(Supplementary Data are available online at www.liebertpub.com/mdr). Sequencing was performed by Source BioScience and GATC Biotech and sequence analysis was carried out using CLC Main Workbench 6.6.2 (CLC Bio). Representative integron variable region amplicons of each different size were sequenced and used to perform BLAST searches on the National Centre for Biotechnology Information (NCBI) nucleotide databases.

Transfer of CTX-M plasmids by conjugation and transformation

Conjugation was attempted by broth mating following the method of Gray ef a/. with modifications12 for all ESBL-EC strains except the 33 strains that clustered with the single reference UK strain A (PFGE cluster A), whose ESBL plas-mids lack the necessary conjugation machinery.6 Briefly, after 4h aerobic growth in tryptone soya broth cultures of donor and recipient strains were mixed at 1:1 ratio and incubated at 37°C overnight. Mating mixtures were pelleted, rinsed and serially diluted in PBS. Transconjugants were selected using MH agar containing 100 mg/mL sodium azide and 1 mg/mL cefotaxime. Presumptive transconjugants were confirmed by PCR13 and b/a multiplex PCR10 with reference to donor and recipient strains, using 10 mL of cleared cell lysate as the template. Where conjugation was unsuccessful, transformation of ESBL plasmids into E. co/i DH5a was attempted by electro-poration performed at 1,700 V and at a time constant of 4.85.0 ms using an electroporator (Eporator®).

Characterisation of CTX-M plasmids

Cefotaxime (CTX) minimum inhibitory concentration ( MIC) assays were performed and interpreted using the guidelines of the Clinical and Laboratory Standards Institue (CLSI) for all clinical isolates and their transconjugants/ transformants using Etest® strips (bioMerieux).14 High-level cefotaxime resistance was defined as MIC >128 mg/mL. Plasmid DNA was isolated from clinical isolates and their transconjugants/transformants using the phenol-chloroform extraction method of Kado and Liu. 5 The presence of b/aCTX_ M genes, associated IS elements and integrons in plasmid extracts of transconjugants/transformants was investigated by PCR as described for clinical isolates.

CTX-M-containing plasmids were sized by S1 PFGE.16 Plasmid incompatibility typing was carried out on transcon-jugant/transformant plasmid preparations and clinical isolates using PCR-based replicon typing (PBRT). Transferable IncF plasmids were further characterised by replicon sequence typing (RST) and IncN and IncI plasmids were further characterised by plasmid multi-locus sequence typing (pMLST). All typing schemes were performed and interpreted using the methods and databases available at http://pubmlst.org/plasmid.

Results

Genotypic characterisation of ESBL genes in E. coli clinical isolates

Investigation of the carriage of ¿/act^m^m^^ genes revealed that 94% of isolates were b/aCTX_M gene positive. The b/aSHV-12 gene was responsible for the ESBL phenotype in a further four isolates. In the remaining two isolates the ESBL phenotype could not be explained by the presence of

a blaCTX_M, bZaSHv or blaTEM ESBL gene, but blaTEM-1 was identified (Supplementary Table S2). BlaTEM genes were the second most common (46%) and 5% of clinical isolates carried a blaSHv gene. Group 1 was the most common ce-fotaximase gene cluster (80/94; 85%) and comprised 66

^CTX-M-^ 7 ^CTX-M-! 4 W^tTX-M^ 2 blaCTX-M-55 and 1

blaCTX-M-32. Thirteen group 9 (13/94; 14%) were identified, comprising 8 blaCTX-M-14, 3 blaCTX-M-9 and 2 blaCTX-M-27. A single isolate contained a blaCTX-M-2 gene, but no group 8 or group 25 CTX-M genes were detected.

Specific blaCTX-M genes and their upstream genetic environments

The arrangement of blaCTX_M genes and associated upstream IS elements are summarised in Table 1, with reference to identical previous GenBank entries. A number of common genetic arrangements were identified within and between the PFGE cluster groups and these arrangements were grouped together for comparison with arrangements previously described in the literature. An ISEcpl promoter was located upstream of bZaCTX_M in 53 isolates (56%). In 35 (66%) of these isolates, PCR detected the full length ISEcpl element (1.7 kb) including the fnpA transposase gene (0.8 kb). However, PCR results suggested truncation within ISEcpl in the other 18 isolates (no ISEcpl amplicon was detected), which apparently occurred within fnpA for 10 isolates where no fnpA amplicon was detected either. Many of the genetic environments identified between ISEcpl and blaCTX-M matched

the frequently described ''W,'' ''X'' and ''V'' common regions first described by Eckert ef al.5 the most common of which was the 48 bp W spacer region typical of UK strains B-E,17 which was present in 32 isolates in combination with blaCTX-M-15 and generally associated with high-level cefotaxime resistance (MICs >128 mg/mL).

PCR and sequencing confirmed reversely oriented IS26 inserted within the terminal inverted repeat of ISEcpl (24 bp before the 3' end of ISEcpl) and upstream of bla^^M^, as described previously for UK strain A17 in all but one (33/34) PFGE cluster A isolates and an ST131 isolate from cluster J. These 34 isolates also contained the alternative ''promoter X'' region with -35 TTCATG and -10 GGGGATGAT sequences positioned 140 and 115 bp, respectively, upstream of the blscx-M^ start codon within IS26, conferring variable levels of cefotaxime resistance as described previously.18 The remaining PFGE cluster A isolate contained blaCTX-M-14 downstream of ISEcpl. The ISCRl element was located upstream of blaCTX-M-9 as part of a complex class 1 integron in all three PFGE cluster G isolates, which upon sequencing resembled the swll-type integron In60-D.19 ISCRl was also detected upstream of a blaCTX-M-2 gene as part of an In35-like complex class 1 integron.

Integron content of ESBL E. coli clinical isolates

Class 1 integrons were detected in 66% of isolates. Four isolates contained complex class 1 integrons bearing blaCTX-M as described above and these are indicated in Table 1. The

Table 1. biaCTX-M Genes and Their Genetic Environments in 94 ESBL-Escherichia coli Clinical Isolates

N PFGE Cl^sfe^ blaCTx_M allele Upsfream IS Spacer regionb CTX MICc GenBank ID Infegrons (size in kb)

34 A, J 15 IS26 (rev) 24 bp IS 2-256 (8) GU264003 32 x d/rAl7-aadA5 (1.7)

Ecpl + W 1 X d/rAl-safl-aadAl (2.2)

1 x d/rA7 (0.8)

32 B-E, I, K, L, 15 ISEcpl W 16-256 (256) AY463958 13 xd/rAl7-aadA5 (1.7)

sporadic 2 X d/rAl-safl-aadAl (2.2)

1 X aadB-aadAl-cmlA6 (3.1)

1 X aadAl (1.0)

7 H, L, sporadic 1 ISEcpl XW 12-256 (256) AM003904 2 x d/rAl-aadAl (1.6)

2 X d/rAl-safl-aadAl (2.2)

3 E, sporadic 3 ISEcpl W 12-256 HF549092 2x d/rAl7-aadA5 (1.7)

1 Sporadic 3 ISEcpl VW 12 EU935740 None

1 Sporadic 55 ISEcpl 45 bp 256 KC576516 None

1 Sporadic 55 ISEcpl W 256 GQ456159 None

1 Sporadic 32 ND NS 256 AJ557142 None

8 I, F, sporadic 14 ISEcpl NS 32-256 AF252622 6 x d/rAl7-aadA5 (1.7)

1 X aadAl (1.0)

1 X aacA4-cmlAl (2.3)

1 x d/rAl2-or/F-aadA2 (1.9)

2 B 27 IS26 NS 48-128 AY156923 1 x d/rAl7-aadA5 (1.7)

3 G 9 ISCRl 326 bp 8 AM040708 3 x dr/Al2-or/F-aadA8b (1.8) +

[or/5l3-blacTx-M-9]

3 X or/D-aacA4-or/l05-cafB8

1 Sporadic 2 ISCRl 498 bp 256 EF592570 1 X d/rAl-aadAl (1.6) +

[or/5l3-blacTx-M-2]

aPFGE clusters as identified previously1

bSpacer region size or letter-coded description: W = 48bp, X = 32bp, V = 79bp (see text and9). cCefotaxime MIC range in mg/L (mode, where distinguishable).

ESBL, extended-spectrum b-lactamase; IS, insertion sequence; MIC, minimum inhibitory concentration (mg/L); ND, none detected; NS, not sequenced.

remaining isolates contained class 1 integrons associated with resistance to agents such as trimethoprim, sulphonamides and aminoglycosides. Nine distinct class 1 integron variable region amplicons of different sizes were detected. Their distribution among PFGE clusters is summarised in Table 1 and details of each individual isolate are given in Supplementary Table 2.

Seven isolates produced more than one amplicon, indicating multiple class 1 integrons were present. Six isolates contained class 2 integron variable regions of 2.2kb (d/rA7-saf7-aad47), five of which also contained a class 1 integron. The most common class 1 integron variable region was 1.7 kb (d/rA77-aad45), present in 54% of isolates. Most integron variable regions identified in this study contained genes for trimethoprim (d/r) and streptomycin/spectinomycin (aad) resistance. The presence of dihydrofolate reductase-containing integrons correlated with resistance to trimethoprim and/or trimethoprim/sulfamethoxazole in all clinical E. co/i isolates. However, streptomycin or spectinomycin susceptibility was

unknown for ESBL-EC as they were not routinely tested for. Gene cassettes conferring resistance to gentamicin and tobramycin were detected infrequently, were co-localised with a chloramphenicol resistance gene: either cm/A7, cm/A6 (sporadic isolates) or catB8 (cluster G isolates). Although susceptibility patterns to chloramphenicol were untested, the presence of either aacA4 or aadB correlated with pheno-typic resistance to gentamicin in all five strains with these gene cassettes.

Characterization of transferable CTX-M plasmids

PBRT of all clinical isolates detected IncF plasmids in 90% of clinical isolates, IncI1 plasmids in 30%, IncN plasmids in 3% and two isolates carrying L/M and B/O plasmids (Supplementary Table S2). Transfer of ESBL plasmids was successful for 33 strains, 28 of which transferred a CTX-M-producing plasmid. The remaining five plasmids conferred an ESBL

Table 2. Characteristics of 28 Transferable CTX-M Plasmids

P/asm/d ID

Rep//con sequence type

Prev/ows/y fownd /na

Size (kb)

Transfer method3

Insert/on seqwence

IncF plasmids

pBHEC91 F1:A-:B- Novel 27.5 T

pBHEC80 F2:A-:B- H/A WW 78.5 C

pBHEC82 F2:A1:B- H UK 95 T

pBHEC87 F2:A1:B- H UK 115 T

pBHEC32 F45:A1:B- Novel l00 C

pBHEC99 F45:A4:B- Novel 151 C

pBHEC48 FII, FIA N/A 137 T

pBHEC12 FII, FIB N/A 122 C

pBHEC88 F22:A1:B20 H/A EU 157 C

pBHEC36 F31:A4:B1 H/A EU 157 C

pBHEC56 F31:A4:B1 H/A EU 162 T

pBHEC97 F31:A4:B1 H/A EU 146.5 C

IncIl plasmids

pBHEC06 I1-ST3 H/A EU 107 T

pBHEC17 I1-ST3 H/A EU 112.5 C

pBHEC22 I1-ST3 H/A EU 112.5 C

pBHEC53 I1-ST7 H/A EU 104.5 T

pBHEC20 I1-ST7 H/A EU 113 C

pBHEC90 I1-ST7 H/A EU 113 C

pBHEC19 I1-ST16 H UK 94 C

pBHEC52 I1-ST16 A UK 94 C

pBHEC37 I1-ST31 H/A EU 93 C

pBHEC70 I1-ST57 H EU 84 C

pBHEC16 Il-STl59 Novel 100 T

Other plasmids

pBHEC39 N-ST1 H/A EU 30 C

pBHEC71 N-ST6 H UK 31 C

pBHEC66 N/D N/A 62 C

pBHEC54 N/D N/A 116 T

pBHEC76 N/D N/A 115 T

' blaCTx_M a//e/e Integron frans/erred0 Donor CTX MIC Rec/p/ent CTXM/Cd

CTX-M-27 No transfer 128 12

CTX-M-15 d/rA77-afldA5 >256 8

CTX-M-15 d/rA77-aadA5 32 6

CTX-M-15 d/rA77-aadA5 3 3

CTX-M-15 N/A >256 >256

CTX-M-15 d/rA77-aadA5 >256 32d

CTX-M-15 d/rA77-aadA5 12 8d

CTX-M-55 N/A >256 32d

CTX-M-15 d/rA7 >256 64d

CTX-M-15 d/rA77-afldA5 192 32

CTX-M-15 d/rA77-aad45 >256 32

CTX-M-15 d/rA77-afldA5 >256 32

CTX-M-1 N/A >256 >256

CTX-M-1 N/A 12 8

CTX-M-1 N/A >256 64

CTX-M-1 No transfer >256 128

CTX-M-1 d/rA7-aadA7 16 6

CTX-M-1 d/rA7-aadA7 48 6

CTX-M-3 No transfer 12 4

CTX-M-15 N/A >256 64

CTX-M-15 No transfer >256 64

CTX-M-3 N/A 64 24

CTX-M-14 N/A 48 32

CTX-M-32 N/A >256 >256

CTX-M-3 No transfer >256 16

CTX-M-55 N/A >256 >256

CTX-M-15 N/A >256 24

CTX-M-15 N/A >256 32

ISEcpl

ISEcpl

ISEcpl

ISEcpl

ISEcpl ISEcpl ISEcpl ISEcpl

ISEcpl ISEcpl ISEcpl ISEcpl ISEcpl ISEcpl ISEcpl ISEcpl ISEcpl ISEcpl ISEcpl

ISEcpl

ISEcpl

ISEcpl

aPlasmid with same RST and CTX-M allele previously found in human (H) or animal (A) isolates worldwide (WW), in the UK (UK) or in Europe (EU), see http://pubmlst.org/plasmid/ bTransfer Method = Transformation (T) or Conjugation (C) cIntegron transferred on plasmid dRecipient contains >1 b-lactamase plasmid

N/A, not applicable; RST, replicon sequence typing; UTD, unable to determine; T, transformation; C, conjugation; sporadic, sporadically occurring strain; CTX, cefotaxime.

phenotype through expression of blaTEM or blaSHV. The CTX-M plasmids were transferred by conjugation (18) and transformation (10). The replicon types identified in these plasmids were IncF (12), IncI1 (11) and IncN (2). The remaining three CTX-M plasmids were untypeable by PBRT.

Characteristics of the transferable CTX-M plasmids are detailed in Table 2. Individual RSTs were indeterminable for two CTX-M bearing plasmids in recipient strains that harboured multiple IncF plasmids; pBHEC48 and pBHEC12. Six different RSTs were identified among the remaining ten IncF CTX-M plasmids, which included three F31:A4:B1 plasmids (146.5-162 kb), two F2:A1:B- plas-mids (95-115 kb), three other large multireplicon plasmids (>100 kb) and single replicon F1 and F2 plasmids. IncF plasmids carried group 1 CTX-M genes except for the 27.5 kb plasmid pBHEC91, which carried a group 9 CTX-M gene (CTX-M-27). The 1.7 kb d/rAl7-aad45 integron was commonly transferred by IncF plasmids.

Among 11 Incl1 CTX-M plasmids identified, six different sequence type (ST)s were identified by IncI1 pMLST, including the previously undefined ST159, 100 kb with repI1/ ardA/trbA/sogS/pilL alleles 1/2/9/1/7 (pBHEC16). The 1.5 kb dfrAl-aadAl integron was transferred on two very similar IncI1-ST7 plasmids, pBHEC20 and pBHEC90. Two IncN type plasmids of *30kb were identified which belonged to the previously identified ST1 and ST6 types. CTX-M plasmids untypeable by PBRT comprised pBHEC66 (CTX-M-55), pBHEC54 (CTX-M-15, TEM) and pBHEC76 (CTX-M-15). Cefotaxime MICs for recipient strains bearing CTX-M plasmids were often less (by between two and five doubling dilutions) than those of their corresponding clinical isolate donor strains. Data for all CTX-M plasmids were deposited in the relevant plasmid MLST database at http://pubmlst.org/plasmid.

Discussion

Similar to the situation reported in Europe and globally, our study identified blaCTX-M as the most common ESBL gene in clinical E. coli isolates collected during 2009-2010 with group 1 alleles the most common (80/94, 85%), especially blaCT^M^, which was found in two thirds (66%) of ESBL-EC. A previous nationwide 11 year study (1997-2007) reported a prevalence of 59% group 1 blaCTX-M among blaCTX-M producing E. coli. Group 9 CTX-M prevalence among E. coli was 144/348 (41.4%) compared to 13/94 (14%) in this study, however, this may reflect regional differences in prevalence or changes during the intervening period.20 The increased prevalence of group 1 genes may be partly due to E. coli ST131 dissemination in Ireland, associated with blaCT^M^.9,21

The endemic nature of CTX-M-15-producing E. coli is evident in Dublin, as indicated by the detection of blaCTX-M-15 in clonal and sporadically-occurring isolates. Isolates from the endemic PFGE cluster A were confirmed as genetically indistinguishable from UK strain A and contain blacr^M^ under the control of ''promoter X,'' as described previously.18 Despite the local dominance of CTX-M-15, four other group 1 genes were detected among 14 isolates; bla^^M^, -3, -32 and bla^^M^. To the best of our knowledge, this represents the first detection of the latter two genes, or indeed the group 2 gene blaCTX-M-2, among

human E. coli isolates in Ireland. Molecular investigation of the promoter regions by PCR mapping enabled the identification of a number of allele-specific associations. Among group 9 CTX-M genes blaCTX-M-14 was associated with ISEcpl, blaCT^M^ with IS26 and bla^^^g with ISCRl as part of the In60-D integron.19 Group 1 alleles were usually associated with ISEcpl, likely controlling bla expression and driving high-level cefotaxime resistance when associated with blaCTX-M-15 as previously described.22

Class 1 integron carriage in our Dublin ESBL-EC collection (66%) was similar to that recorded in a longitudinal study of ESBL-EC isolates from Madrid (67%)23 but lower than recorded in clinical E. coli isolates collected from 1998 to 2004 in Guangzhou, China (86%).24 As previously reported, a low prevalence of complex class 1 integrons containing blaCT^M was found.23 Interestingly, dfrAl7-aadA5 was the most common integron array in the present study (54%) and the Chinese study (36%). This integron was also detected in Madrid and is globally disseminated in E. coli.7,23-25

Class 1 integrons are frequently associated with Tn2l-like transposons26 or multiple copies of IS26 on large con-jugative plasmids in clinical Enterobacteriaceae, which facilitate the mobilisation of drug resistance elements among plasmids by homologous recombination.6,7 This may explain the presence of the dfrAl7-aadA5 integron on at least four different IncF type plasmids in our E. coli collection. The epidemic distribution of host strains, as exemplified here by UK strain A, may account for its high prevalence.

Six of the nine distinct integron variable regions identified in the present study, including all those identified in multiple isolates, were documented previously in a study carried out at the Veterinary Hospital, University College Dublin during 2007.27 The authors characterised consecutive MDR E. coli isolates from predominantly faecal samples of horses (44), cattle (17), pigs (9), dogs (3) and a sheep, reporting high carriage rates of class 1 integron gene cassette regions (76%). None of the veterinary E. coli isolates belonged to the B2 phylogenetic lineage which is mainly associated with infections in humans.27 Nonetheless, the identification of matching class 1 integron variable regions exemplifies a possible reservoir for these antimicrobial resistance determinants, which may evolve in the commensal E. coli of companion and food animals amidst the selective pressure of veterinary antimicrobial use and transfer to strains causing human disease.3,28 The widespread and lengthy use of trimethoprim and sulphonamides in veterinary medicine has been implicated in the evolution and persistence of integron-bound resistance genes of the dfr and swl families.29 Likewise, the aad genes for streptomycin resistance are positively selected for in animals, where it is used as a first-line drug for Gramnegative infections. One can speculate that co-carriage of ESBL genes on integrons or indeed plasmids containing these gene cassettes may drive their co-selection and propagation in the gut flora of animals treated with veterinary antimicrobials.

We noted a local dominance of multi-replicon IncF and IncI1 plasmids among CTX-M producing ESBL-EC in Dublin and identified four new RSTs. The range of IncF replicon sequences identified in this small cross section of ESBL-EC isolates from the same geographical location demonstrates the diversity in ESBL-bearing IncF plasmids. The diversity of STs among the ten transferred IncF CTX-M

plasmids contrasts with the phylogenetic homogeneity of the host isolates, 7/10 (70%) of which belonged to the ST131 pandemic clone. This reflects the plasticity of IncF plasmids, which contain multiple hotspots for genetic recombination.7 The success of conjugation (18) and transformation (10) of CTX-M plasmids was limited. Low success of conjugation may be explained by assuming that the replicon type FII-FIA (pEK499-like) plasmids detected here in 41 strains, including PFGE cluster A, lack the requisite fraW to fraX genes for conjugation, as has been shown previously.6 Nonetheless, horizontal transfer is important in the dissemination of resistance mechanisms, as evidenced by the fact that 19% (18/94) of b/aCTX_M genes and (7/66,10.6%) of d/r/aad containing class 1 integrons were transferred by conjugative plasmids (Table 2).

Many of the 28 CTX-M plasmids characterised had RSTs and b/aCTX-M alleles in common with those previously identified throughout Europe in both human and animal E. co/i isolates. Identical ESBL genes, plasmids and strains of E. co/i have been identified in Dutch poultry, chicken meat and humans.2'4 There are relatively few studies on the prevalence of ESBLs in Irish animals and animal food products. However, it can be speculated based on the similarity of mobile genetic elements between commensal E. co/i of animals and clinical ESBL-E from humans that they may be a reservoir for MDR plasmids. However, further investigations in this area are warranted.

Ireland is a major exporter of animal meats with 75% exported to UK and European markets and the remainder going to the rest of the world. Studies investigating epide-miological links between agricultural and human isolates of MDR Enterobacteriaceae, particularly in relation to antimicrobial resistance platforms should be investigated at least at a European level to provide an evidence base for informed policy in relation to antibiotic use in agriculture.

In conclusion, this study reveals the complex array of tools for the mobilization and expression of b/aCTX-M and other antibiotic resistance genes within ESBL-EC circulating in Dublin and highlights the importance of group 1 and 9 CTX-M genes and specifically b/aCTX_M_15 and b/aCTX_M_14. Our data supports significant roles for both horizontal transfer of ESBL and integron-bound resistance genes via conjugative IncF, I1 and N plasmids and vertical transfer via clonal spread of the pandemic ST131 clone. Zoonotic transfer of both integrons and ESBL plasmids to human-associated E. co/i may occur through contact with animals or through the food chain.

Acknowledgments

We are grateful to Dr. Alessandra Carratoli, Istituto Su-periore di Sanita, Italy and Professor Neil Woodford, Dr. Katie Hopkins and all the staff of the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, London. This publication made use of the plasmid MLST website (http://pubmlst.org plas-mid/) developed by Keith Jolley and sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioin/ormafics, 11:595). The development of this site has been funded by the Wellcome Trust. We are also grateful for the support and assistance of the staff of the Department of Microbiology, Beaumont Hospital. This work was supported by the Health Research Board in Ireland (grant number PHD/2007/11).

Disclosure Statement

No competing financial interests exist.

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Address correspondence to: Liam Bwrfce, PhD Department of Clinical Microbiology Royal College of SUrgeons in Ireland Beawmonf Hospital Dwblin 9 Ireland

E-mail: liamburke@rcsi.ie