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

Abstract

In view of continued high clinical prevalence of infections involving extended-spectrum β-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). Cefotaximases 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 ISEcp1 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 multireplicon 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 β-lactamases (ESBLs) are among the most important resistance determinants in Enterobacteriaceae, 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 bla_CTX-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 sequences (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), ISEcp1 and ISCR1 are responsible for the mobilization of bla_CTX-M and drive their expression. CTX-M genes are frequently found downstream of ISEcp1 within modular multidrug-resistance regions (MRRs) on ESBL plasmids^5,6 or are linked to ISCR1 on complex class 1 integrons which also bear highly variable gene cassette regions encoding resistance to multiple antimicrobial classes. The surveillance of non bla_CTX-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 also found on MRRs of ESBL plasmids and have contributed to the spread of bla_CTX-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. coli 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. coli (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 bla_CTX-M and other resistance genes were associated in this collection with reference to the published literature from human and animal E. coli and (2) to determine the potential for horizontal transfer of CTX-M plasmids from clinical E. coli 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 for beta-lactamase negative, bla_TEM, bla_SHV and each of the five bla_CTX-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.coli strain J53 was the recipient for conjugations and was a gift from Prof. Martin Cormican, Department of Bacteriology, National University of Ireland, Galway. E. coli ElectroMAX™ DH5α-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 bla_TEM, bla_SHV and the five bla_CTX-M gene groups in the isolates was investigated by multiplex Polymerase chain reaction (PCR) using previously described primers and cycling conditions.^10,11 Specific bla_CTX-M alleles and their upstream genetic environments were identified by PCR mapping and were fully sequenced. Due to resource constraints, specific bla_SHV and bla_TEM alleles were identified by sequencing only in six isolates that were bla_CTX-M-negative. PCR and sequencing primers used to detect class 1 and 2 integron motifs, ISEcp1, IS26, and ISCR1 and their arrangement in relation to bla_CTX-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 et al. 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 plasmids lack the necessary conjugation machinery.⁶ Briefly, after 4 h 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 μg/mL sodium azide and 1 μg/mL cefotaxime. Presumptive transconjugants were confirmed by PCR¹³ and bla multiplex PCR¹⁰ with reference to donor and recipient strains, using 10 μL of cleared cell lysate as the template. Where conjugation was unsuccessful, transformation of ESBL plasmids into E. coli DH5α was attempted by electroporation performed at 1,700 V and at a time constant of 4.8–5.0 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 (bioMérieux).¹⁴ High-level cefotaxime resistance was defined as MIC ≥128 μg/mL. Plasmid DNA was isolated from clinical isolates and their transconjugants/transformants using the phenol-chloroform extraction method of Kado and Liu.¹⁵ The presence of bla_CTX-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 transconjugant/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 bla_{CTX-M/TEM/SHV} genes revealed that 94% of isolates were bla_CTX-M gene positive. The bla_SHV-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 bla_CTX-M, bla_SHV or bla_TEM ESBL gene, but bla_TEM-1 was identified (Supplementary Table S2). Bla_TEM genes were the second most common (46%) and 5% of clinical isolates carried a bla_SHV gene. Group 1 was the most common cefotaximase gene cluster (80/94; 85%) and comprised 66 bla_CTX-M-15, 7 bla_CTX-M-1, 4 bla_CTX-M-3, 2 bla_CTX-M-55 and 1 bla_CTX-M-32. Thirteen group 9 (13/94; 14%) were identified, comprising 8 bla_CTX-M-14, 3 bla_CTX-M-9 and 2 bla_CTX-M-27. A single isolate contained a bla_CTX-M-2 gene, but no group 8 or group 25 CTX-M genes were detected.

Specific bla_CTX-M genes and their upstream genetic environments

The arrangement of bla_CTX-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 ISEcp1 promoter was located upstream of bla_CTX-M in 53 isolates (56%). In 35 (66%) of these isolates, PCR detected the full length ISEcp1 element (1.7 kb) including the tnpA transposase gene (0.8 kb). However, PCR results suggested truncation within ISEcp1 in the other 18 isolates (no ISEcp1 amplicon was detected), which apparently occurred within tnpA for 10 isolates where no tnpA amplicon was detected either. Many of the genetic environments identified between ISEcp1 and bla_CTX-M matched the frequently described “W,” “X” and “V” common regions first described by Eckert et 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 bla_CTX-M-15 and generally associated with high-level cefotaxime resistance (MICs ≥128 μg/mL).

Table 1.

bla_CTX-M Genes and Their Genetic Environments in 94 ESBL-Escherichia coli Clinical Isolates

N	PFGE Clusters^a	bla_CTX-M allele	Upstream IS	Spacer region^b	CTX MIC^c	GenBank ID	Integrons (size in kb)
34	A, J	15	IS26 (rev)	24 bp ISEcp1 + W	2–256 (8)	GU264003	32 × dfrA17-aadA5 (1.7) 1 × dfrA1-sat1-aadA1 (2.2)
							1 × dfrA7 (0.8)
32	B–E, I, K, L, sporadic	15	ISEcp1	W	16–256 (256)	AY463958	13 × dfrA17-aadA5 (1.7)2 × dfrA1-sat1-aadA1 (2.2)
							1 × aadB-aadA1-cmlA6 (3.1)
							1 × aadA1 (1.0)
7	H, L, sporadic	1	ISEcp1	XW	12–256 (256)	AM003904	2 × dfrA1-aadA1 (1.6)
							2 × dfrA1-sat1-aadA1 (2.2)
3	E, sporadic	3	ISEcp1	W	12–256	HF549092	2 × dfrA17-aadA5 (1.7)
1	Sporadic	3	ISEcp1	VW	12	EU935740	None
1	Sporadic	55	ISEcp1	45 bp	256	KC576516	None
1	Sporadic	55	ISEcp1	W	256	GQ456159	None
1	Sporadic	32	ND	NS	256	AJ557142	None
8	I, F, sporadic	14	ISEcp1	NS	32–256	AF252622	6 × dfrA17-aadA5 (1.7)
							1 × aadA1 (1.0)
							1 × aacA4-cmlA1 (2.3)
							1 × dfrA12-orfF-aadA2 (1.9)
2	B	27	IS26	NS	48–128	AY156923	1 × dfrA17-aadA5 (1.7)
3	G	9	ISCR1	326 bp	8	AM040708	3 × drfA12-orfF-aadA8b (1.8) + [orf513-bla_CTX-M-9]
							3 × orfD-aacA4-orf105-catB8 (2.2)
1	Sporadic	2	ISCR1	498 bp	256	EF592570	1 × dfrA1-aadA1 (1.6) + [orf513-bla_CTX-M-2]

PFGE clusters as identified previously¹.

Spacer region size or letter-coded description: W = 48 bp, X = 32 bp, V = 79 bp (see text and⁹).

Cefotaxime MIC range in mg/L (mode, where distinguishable).

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

PCR and sequencing confirmed reversely oriented IS26 inserted within the terminal inverted repeat of ISEcp1 (24 bp before the 3′ end of ISEcp1) and upstream of bla_CTX-M-15, 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 bla_CTX-M-15 start codon within IS26, conferring variable levels of cefotaxime resistance as described previously.¹⁸ The remaining PFGE cluster A isolate contained bla_CTX-M-14 downstream of ISEcp1. The ISCR1 element was located upstream of bla_CTX-M-9 as part of a complex class 1 integron in all three PFGE cluster G isolates, which upon sequencing resembled the sul1-type integron In60-D.¹⁹ ISCR1 was also detected upstream of a bla_CTX-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 bla_CTX-M as described above and these are indicated in Table 1. The 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 2.2 kb (dfrA1-sat1-aadA1), five of which also contained a class 1 integron. The most common class 1 integron variable region was 1.7 kb (dfrA17-aadA5), present in 54% of isolates. Most integron variable regions identified in this study contained genes for trimethoprim (dfr) 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. coli 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 cmlA1, cmlA6 (sporadic isolates) or catB8 (cluster G isolates). Although susceptibility patterns to chloramphenicol were untested, the presence of either aacA4 or aadB correlated with phenotypic 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 phenotype through expression of bla_TEM or bla_SHV. 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- plasmids (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 dfrA17-aadA5 integron was commonly transferred by IncF plasmids.

Table 2.

Characteristics of 28 Transferable CTX-M Plasmids

Plasmid ID	Replicon sequence type	Previously found in ^a	Size (kb)	Transfer method ^b	Insertion sequence	bla_CTX-M allele	Integron transferred ^c	Donor CTX MIC	Recipient CTX MIC ^d
IncF plasmids
pBHEC91	F1:A-:B-	Novel	27.5	T	IS26	CTX-M-27	No transfer	128	12
pBHEC80	F2:A-:B-	H/A WW	78.5	C	ISEcp1	CTX-M-15	dfrA17-aadA5	>256	8
pBHEC82	F2:A1:B-	H UK	95	T	IS26	CTX-M-15	dfrA17-aadA5	32	6
pBHEC87	F2:A1:B-	H UK	115	T	IS26	CTX-M-15	dfrA17-aadA5	3	3
pBHEC32	F45:A1:B-	Novel	100	C	ISEcp1	CTX-M-15	N/A	>256	>256
pBHEC99	F45:A4:B-	Novel	151	C	ISEcp1	CTX-M-15	dfrA17-aadA5	>256	32^d
pBHEC48	FII, FIA (UTD)	N/A	137	T	IS26	CTX-M-15	dfrA17-aadA5	12	8^d
pBHEC12	FII, FIB (UTD)	N/A	122	C	ISEcp1	CTX-M-55	N/A	>256	32^d
pBHEC88	F22:A1:B20	H/A EU	157	C	ISEcp1	CTX-M-15	dfrA7	>256	64^d
pBHEC36	F31:A4:B1	H/A EU	157	C	ISEcp1	CTX-M-15	dfrA17-aadA5	192	32
pBHEC56	F31:A4:B1	H/A EU	162	T	ISEcp1	CTX-M-15	dfrA17-aadA5	>256	32
pBHEC97	F31:A4:B1	H/A EU	146.5	C	ISEcp1	CTX-M-15	dfrA17-aadA5	>256	32
IncI1 plasmids
pBHEC06	I1-ST3	H/A EU	107	T	ISEcp1	CTX-M-1	N/A	>256	>256
pBHEC17	I1-ST3	H/A EU	112.5	C	ISEcp1	CTX-M-1	N/A	12	8
pBHEC22	I1-ST3	H/A EU	112.5	C	ISEcp1	CTX-M-1	N/A	>256	64
pBHEC53	I1-ST7	H/A EU	104.5	T	ISEcp1	CTX-M-1	No transfer	>256	128
pBHEC20	I1-ST7	H/A EU	113	C	ISEcp1	CTX-M-1	dfrA1-aadA1	16	6
pBHEC90	I1-ST7	H/A EU	113	C	ISEcp1	CTX-M-1	dfrA1-aadA1	48	6
pBHEC19	I1-ST16	H UK	94	C	ISEcp1	CTX-M-3	No transfer	12	4
pBHEC52	I1-ST16	A UK	94	C	ISEcp1	CTX-M-15	N/A	>256	64
pBHEC37	I1-ST31	H/A EU	93	C	ISEcp1	CTX-M-15	No transfer	>256	64
pBHEC70	I1-ST57	H EU	84	C	ISEcp1	CTX-M-3	N/A	64	24
pBHEC16	I1-ST159	Novel	100	T	ISEcp1	CTX-M-14	N/A	48	32
Other plasmids
pBHEC39	N-ST1	H/A EU	30	C	ND	CTX-M-32	N/A	>256	>256
pBHEC71	N-ST6	H UK	31	C	ND	CTX-M-3	No transfer	>256	16
pBHEC66	N/D	N/A	62	C	ISEcp1	CTX-M-55	N/A	>256	>256
pBHEC54	N/D	N/A	116	T	ISEcp1	CTX-M-15	N/A	>256	24
pBHEC76	N/D	N/A	115	T	ISEcp1	CTX-M-15	N/A	>256	32

Plasmid 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/

Transfer Method = Transformation (T) or Conjugation (C).

Integron transferred on plasmid.

Recipient contains >1 β-lactamase plasmid.

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

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 dfrA1-aadA1 integron was transferred on two very similar IncI1-ST7 plasmids, pBHEC20 and pBHEC90. Two IncN type plasmids of ∼30 kb 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 bla_CTX-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 bla_CTX-M-15, 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 bla_CTX-M among bla_CTX-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 bla_CTX-M-15.^9,21

The endemic nature of CTX-M-15-producing E. coli is evident in Dublin, as indicated by the detection of bla_CTX-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 bla_CTX-M-15 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_{CTX-M-1, −3, −32} and bla_CTX-M-55. To the best of our knowledge, this represents the first detection of the latter two genes, or indeed the group 2 gene bla_CTX-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 bla_CTX-M-14 was associated with ISEcp1, bla_CTX-M-27 with IS26 and bla_CTX-M-9 with ISCR1 as part of the In60-D integron.¹⁹ Group 1 alleles were usually associated with ISEcp1, likely controlling bla expression and driving high-level cefotaxime resistance when associated with bla_CTX-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 bla_CTX-M was found.²³ Interestingly, dfrA17-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 Tn21-like transposons²⁶ or multiple copies of IS26 on large conjugative 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 dfrA17-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 sul 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 Gram-negative 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 traW to traX 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 bla_CTX-M genes and (7/66, 10.6%) of dfr/aad containing class 1 integrons were transferred by conjugative plasmids (Table 2).

Many of the 28 CTX-M plasmids characterised had RSTs and bla_CTX-M alleles in common with those previously identified throughout Europe in both human and animal E. coli isolates. Identical ESBL genes, plasmids and strains of E. coli 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. coli 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 epidemiological 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 bla_CTX-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 bla_CTX-M-15 and bla_CTX-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. coli may occur through contact with animals or through the food chain.

Footnotes

Acknowledgments

We are grateful to Dr. Alessandra Carratoli, Istituto Superiore 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 ( plasmid/) developed by Keith Jolley and sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioinformatics, 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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Supplementary Material

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

Abstract

Introduction

Materials and Methods

Bacterial strains and culture conditions

Characterisation of resistance genes and their associated genetic elements

Transfer of CTX-M plasmids by conjugation and transformation

Characterisation of CTX-M plasmids

Results

Genotypic characterisation of ESBL genes in E. coli clinical isolates

Specific blaCTX-M genes and their upstream genetic environments

Integron content of ESBL E. coli clinical isolates

Characterization of transferable CTX-M plasmids

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Supplementary Material

Specific bla_CTX-M genes and their upstream genetic environments