Analysis of bla_CTX-M-Carrying Plasmids from Escherichia coli Isolates Collected in the BfT-GermVet Study

Abstract

In this study, 417 Escherichia coli isolates from defined disease conditions of companion and farm animals collected in the BfT-GermVet study were investigated for the presence of extended-spectrum β-lactamase (ESBL) genes. Three ESBL-producing E. coli isolates were identified among the 100 ampicillin-resistant isolates. The E. coli isolates 168 and 246, of canine and porcine origins, respectively, harbored bla_CTX-M-1, and the canine isolate 913 harbored bla_CTX-M-15, as confirmed by PCR and sequence analysis. The isolates 168 and 246 belonged to the novel multilocus sequence typing (MLST) types ST1576 and ST1153, respectively, while isolate 913 had the MLST type ST410. The ESBL genes were located on structurally related IncN plasmids in isolates 168 and 246 and on an IncF plasmid in isolate 913. The bla_CTX-M-1 upstream regions of plasmids pCTX168 and pCTX246 were similar, whereas the downstream regions showed structural differences. The genetic environment of the bla_CTX-M-15 gene on plasmid pCTX913 differed distinctly from that of both bla_CTX-M-1 genes. Detailed sequence analysis showed that the integration of insertion sequences, as well as interplasmid recombination events, accounted for the structural variability in the bla_CTX-M gene regions.

INTRODUCTION

The production of β-lactamases is the predominant resistance mechanism against β-lactam antibiotics in Enterobacteriaceae. In contrast to narrow-spectrum β-lactamases, extended-spectrum β-lactamases (ESBLs) are able to hydrolyze the amide bond in the four-membered β-lactam ring of oxyimino-cephalosporins, such as cefotaxime (4). Nowadays, ESBLs are the major cause of clinical cephalosporin resistance among Enterobacteriaceae (24, 27). During recent years, ESBL-producing Enterobacteriaceae have been identified not only in the community (11, 20) but also in animals (2, 10, 16, 22, 34). Livermore et al. (20) stated that around the year 2000, dramatic shifts in the prevalence and types of ESBLs among Enterobacteriaceae occurred in Europe and that ESBLs of the CTX-M type have become predominant since then. In human isolates, the gene bla_CTX-M-15 has spread during recent years through the successful emergence of E. coli strains belonging to the multilocus sequence type ST131 (20). A recent study also showed that human pandemic O25:H4-ST131 E. coli carrying the bla_CTX-M-15 gene emerged among companion animals (14). Although different subtypes of ESBL genes seem to be prevalent in animals and humans, the predominant subtypes detected in members of the Enterobacteriaceae from food-producing animals, bla_CTX-M-1 and bla_CTX-M-2, were detected in four hospitals in Amsterdam, The Netherlands (1). Animals, especially food-producing animals, have been assumed to represent a reservoir for ESBL-producing bacteria (5), and this observation might suggest a transmission from the animal reservoir to the community. The spread of bla_CTX-M genes was shown to be due mainly to the dissemination of plasmids (10, 12, 24, 32). Thus, the investigation of plasmids is a key component for a better understanding of the dissemination and persistence of ESBL genes (6).

To date, little is known about the prevalence of bla_CTX-M genes in Escherichia coli strains from food-producing animals or companion animals in Germany. Therefore, the aims of the present study were as follows: (i) to investigate a representative collection of E. coli isolates from defined disease conditions of companion and farm animals for the presence of ESBL producers, (ii) to identify the types of ESBL genes present, (iii) to determine the location of the ESBL genes on plasmids or in the chromosomal DNA, and (iv) to sequence the ESBL gene region to gain insight into its organization.

MATERIALS AND METHODS

Bacterial isolates and susceptibility testing.

The isolates included in this study originated from the BfT-GermVet monitoring study, which was carried out in Germany during the years 2004 to 2006 (30). In total, 1,626 bacterial isolates collected during these years from defined disease conditions of companion and farm animals have been tested for their susceptibility to 24 antimicrobial agents or combinations of antimicrobial agents by broth microdilution. This collection also included 417 E. coli isolates from diseased dogs/cats (n=228), horses (n=102), and swine (n=87) (17). Among them, 100 E. coli isolates showed ampicillin resistance. They were from respiratory (n=11), urinary/genital (n=24), or gastrointestinal tract (n=14) infections of cats/dogs, from urinary/genital tract infections, including mastitis-metritis-agalactia syndrome, of swine (n=33), or from genital tract infections of horses (n=18).

To identify potential ESBL producers, the 100 ampicillin-resistant E. coli isolates were subjected to an initial ESBL screening for growth in the presence of 1 μg/ml cefotaxime and subsequent ESBL phenotypic confirmatory tests as recommended by the Clinical and Laboratory Standards Institute (CLSI) (9).

Molecular analyses.

The ESBL genes were identified by previously described PCR assays (13) and initially confirmed by sequence analysis of the amplicons. Multilocus sequence typing (MLST) was performed for the bla_CTX-M-positive E. coli isolates (36). Plasmids were transferred by conjugation or transformation as previously described (19), with subsequent selection on Luria-Bertani agar supplemented with ampicillin (100 μg/ml) or cefotaxime (1 μg/ml). Transconjugants and transformants were tested for their antimicrobial susceptibility by broth microdilution according to CLSI recommendations (9). Plasmids were subjected to PCR-based replicon typing (8), and plasmid sizes were determined by S1 nuclease pulsed-field gel electrophoresis (PFGE) using a Chef-DR III system (Bio-Rad, Munich, Germany). The DNA-containing agarose plugs were prepared as recommended in the PulseNet protocol (29), and the slices were digested with S1 nuclease (Fermentas, St. Leon-Rot, Germany) as described previously (18). The pulse times were increased from 1 to 25 s over 17 h at a voltage of 6 V and an angle of 120°. Salmonella enterica serovar Braenderup H9812 digested with XbaI (Fermentas) and the MidRange PFG marker I (New England BioLabs, Frankfurt, Germany) were used as size markers.

The flanking regions of the bla_CTX-M genes were sequenced by primer walking using either the plasmid DNA obtained from the respective transformants and transconjugants or cloned plasmid fragments. For the latter approach, the cloning vector pBluescript(SK+) or pCR-BluntII-TOPO (both from Invitrogen, Groningen, The Netherlands) was used. Sequence comparisons were carried out using the software programs blastn and blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi), the ORF Finder (open reading frame finder) program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), and the IS finder program (http://www-is.biotoul.fr/is.html) (all last accessed 18 February 2011).

Nucleotide sequence accession numbers.

The nucleotide sequences of the bla_CTX-M-1 genes, the bla_CTX-M-15 gene, and their flanking regions have been deposited in the EMBL database under accession numbers FN806788, FN806790, and FR828676.

RESULTS AND DISCUSSION

Presence and types of ESBL genes.

According to their cefotaxime resistance and the subsequent ESBL phenotypic confirmatory tests, three ESBL-producing E. coli isolates were identified among the 100 ampicillin-resistant isolates (Table 1). Comparative analysis of the zone diameters of ceftazidime (30 μg) and ceftazidime-clavulanic acid (30/10 μg), as well as cefotaxime (30 μg) and cefotaxime-clavulanic acid (30/10 μg), showed a ≥5-mm increase in the zone diameter of either antimicrobial agent tested in combination with clavulanic acid versus its zone diameter when tested alone (9). PCR and sequence analysis confirmed that isolate 913 harbored a bla_CTX-M-15 gene while the remaining two isolates, 168 and 246, had a bla_CTX-M-1 gene.

Table 1.

Characteristics of the three ESBL-producing E. coli isolates and their bla_CTX-M-carrying plasmids

Isolate	Animal species	Disease condition	Resistance patterna	MLST	ESBL gene	ESBL-carrying plasmid	Replicon type	Approximate size (kb)	Colocated resistancesa
168	Dog	Pneumonia	BLA, CHL, ENR, GEN, NEO, SMZ, SXT, TET	ST1576	blaCTX-M-1	pCTX168	IncN	50
246	Swine	Mastitis-metritis-agalactia syndrome	BLA, SMZ, SXT, TET	ST1153	blaCTX-M-1	pCTX246	IncN	50
913	Dog	Urinary tract infection	BLA, CHL, ENR, GEN, NEO, SMZ, SXT, TET	ST410	blaCTX-M-15	pCTX913	IncF	50	GEN, TET

^aBLA, β-lactams; CHL, chloramphenicol; ENR, enrofloxacin; GEN, gentamicin; NEO, neomycin; SMZ, sulfamethoxazole; SXT, sulfamethoxazole-trimethoprim (19:1); TET, tetracycline.

Analysis of the bla_CTX-M-15 gene region of isolate 913.

E. coli 913 was obtained in 2004 from the urine sample of a dog suffering from a urinary tract infection. It showed high MIC values for cefalothin (≥64 μg/ml), cefazolin (≥64 μg/ml), cefoperazone (≥32 μg/ml), ceftiofur (≥32 μg/ml), and cefquinome (≥32 μg/ml) and for the non-β-lactams chloramphenicol (128 μg/ml), enrofloxacin (≥32 μg/ml), gentamicin (64 μg/ml), neomycin (32 μg/ml), sulfamethoxazole (≥2,048 μg/ml), trimethoprim-sulfamethoxazole (≥64/1,216 μg/ml), and tetracycline (≥128 μg/ml). The isolate exhibited the MLST type ST410, which belongs to clonal complex 23. E. coli isolates of this clonal complex and harboring bla_CTX-M-15 genes have been found to be present among human patients in the United States (31) and in Brazil (26) and very recently in clinical and food samples from Spain (21). In isolate 913, the bla_CTX-M-15 gene was located on a plasmid of approximately 50 kb, designated pCTX913, which was transferable by transformation. Replicon typing confirmed that this plasmid belonged to the incompatibility group IncF. The plasmid also conferred resistance to gentamicin and tetracycline.

Sequencing of the bla_CTX-M-15 flanking regions revealed the presence of ISEcp1 in the upstream region. Downstream of the bla_CTX-M-15 gene, the terminal 342 bp of a reading frame for a hypothetical protein of 182 amino acids (aa), previously identified on the bla_CTX-M-3-carrying plasmid pCTX-M360 from Klebsiella pneumoniae (38), were detected. This segment was followed by a truncated transposase gene, tnpA, previously found on the bla_TEM-1-carrying transposon Tn3 (Fig. 1). At the junction of both sequences, a potential recombination site was seen which might have served for a recombination event which led to the truncation of the reading frames of both the hypothetical protein and the Tn3-associated transposase.

Fig. 1.

Schematic presentation of the flanking gene regions of the bla_CTX-M-15 gene. The open reading frames are shown as arrows, with the arrowhead indicating the direction of transcription. IS elements are shown as boxes. The recombination site between the Tn3-associated tnpA gene and orf477 is shown below the map of the bla_CTX-M-15 gene region. The sequences depicted in this alignment are from Tn3 (GenBank accession no. V00613), pCTX913 (FR828676), and pCTXM360 (EU938349). Vertical bars indicate identical bases compared to the pCTX913 sequence. The area where the crossover most likely occurred is indicated by a box.

A database search revealed that identical sequences downstream of the bla_CTX-M-15 gene were present in a wide variety of bla_CTX-M-15-carrying plasmids from E. coli (3, 13, 15, 32, 37) and K. pneumoniae (33) but also Enterobacter cloacae (GenBank accession no. HM204572, 219230) and Serratia liquefaciens (GenBank accession no. HM470254). Some of these plasmids were distinctly larger than pCTX913, belonged to different replicon types (including multireplicon types), and conferred additional resistance properties (3, 32, 37). This comparison showed that identical bla_CTX-M-15 genes are present in diverse plasmid backgrounds in different members of the Enterobacteriaceae from human and animal sources in different countries. Such an observation underlines the enormous flexibility of resistance plasmids to undergo structural changes which might render them resistant to different selective pressures by acquisition of additional resistance genes and able to replicate in different hosts by acquisition of additional replicons.

Analysis of the bla_CTX-M-1 gene regions of isolates 168 and 246.

Isolate 168 was obtained in 2004 from a case of canine pneumonia. It showed high MIC values of amoxicillin combined with clavulanic acid (≥64/32 μg/ml), cefalothin (≥64 μg/ml), cefazolin (≥64 μg/ml), cefoperazone (≥32 μg/ml), ceftiofur (≥32 μg/ml), and cefquinome (≥32 μg/ml) and of chloramphenicol (128 μg/ml), enrofloxacin (≥32 μg/ml), gentamicin (64 μg/ml), neomycin (32 μg/ml), sulfamethoxazole (≥2,048 μg/ml), trimethoprim-sulfamethoxazole (≥64/1,216 μg/ml), and tetracycline (≥128 μg/ml). Isolate 246 was identified in 2004 from a cervix swab of a sow suffering from the mastitis-metritis-agalactia syndrome. It showed high MIC values of cefalothin (≥64 μg/ml), cefazolin (≥64 μg/ml), cefoperazone (≥32 μg/ml), ceftiofur (≥32 μg/ml), and cefquinome (≥32 μg/ml) and of sulfamethoxazole (≥2,048 μg/ml), trimethoprim-sulfamethoxazole (≥64/1,216 μg/ml), and tetracycline (≥128 μg/ml).

Each of the two E. coli isolates belonged to a novel MLST type, namely, ST1576 (isolate 168) and ST1153 (isolate 246). The bla_CTX-M-1-bearing plasmids, designated pCTX168 and pCTX246, were approximately 50 kb in size and proved to be closely related in their BglII, DraI, KpnI, PstI, PvuI, and SspI fragment patterns. None of them conferred other resistance properties besides β-lactam resistance. Plasmid pCTX168 was transferable by conjugation, whereas plasmid pCTX246 was transferable by transformation. PCR-based replicon typing identified both plasmids as positive for IncN.

The genetic organization of the bla_CTX-M-1 gene regions of plasmids pCTX168 and pCTX246 is shown in Fig. 2. The upstream region of the bla_CTX-M-1 genes was similar in the two plasmids. It consisted of a 214-bp fragment of the insertion sequence ISEcp1, including the right terminal repeat, which was truncated by the integration of an IS26 element. In plasmid pCTX168, the IS26 element and the region immediately downstream of it were completely sequenced, whereas it was sequenced only in part in pCTX246. Downstream of the IS26 element in pCTX168, a small reading frame for a hypothetical protein of 65 aa was found. The same reading frame was also detected on the IncN plasmid R46 (NC_003292). Immediately upstream of this open reading frame, an incomplete reading frame for a protein was detected, of which the C-terminal 186 aa corresponded exactly to the Mrr protein of the IncN plasmid pKOX105 from Klebsiella oxytoca (7).

Fig. 2.

Schematic presentation of the flanking gene regions of the bla_CTX-M-1 genes in plasmids pCTX246 and pCTX168. The open reading frames are shown as arrows, with the arrowhead indicating the direction of transcription. IS elements are shown as boxes. The recombination site between the mrx gene and orf477 is shown between the two maps, while the recombination site that played a role in the truncation of the mph(A) gene in pCTX168 is shown below the map of pCTX168. The sequences depicted in these two alignments are from pCTX168 (GenBank accession no. FN806788), pCTX246 (FN806790), pECL0701 (FJ966096), pTZ3509 (AB038042), pSE11-3 (AP009243), and 55989p (CU928159). Vertical bars indicate identical bases compared to the pCTX246 and/or pCTX168 sequences or bases that are identical between the pSE11-3 and 55989p sequences. The areas where the crossovers most likely occurred are indicated by boxes.

In the bla_CTX-M-1 downstream region, a truncated mrx gene was identified in both plasmids. This gene is part of the mph(A)-mrx-mphR(A) gene cluster, which is involved in macrolide resistance if complete (25). The truncation of the mrx gene is most likely the result of an interplasmid recombination event (Fig. 2). Sequence analysis identified two plasmids, pECL0701 from Enterobacter cloacae (GenBank accession no. FJ966096) and pTZ3509 from E. coli (25), which carried a complete mph(A)-mrx-mphR(A) gene cluster and displayed striking similarity to the sequences detected in plasmids pCTX168 and pCTXM246. A 12-bp region in plasmids pCTX168 and pCTXM246, which exhibited 75% identity to the pECL0701 sequence and 66.7% identity to the pTZ3509 sequence, was considered the recombination site. The part further upstream of the Δmrx gene differed distinctly between the two plasmids. On plasmid pCTX246, a complete mph(A) gene and another IS26 element, which is located in the same orientation as the IS26 element in the bla_CTX-M-1 upstream region, were detected. In contrast, the mph(A) gene on plasmid pCTX168 was truncated by the integration of a segment homologous to plasmids pSE11-3 (28) and 55989p (35) from E. coli (Fig. 2). Further sequencing revealed an open reading frame for a putative protein of 133 aa without known function and part of a Tn3-associated resolvase gene. Another potential recombination site was identified, which might explain the truncation of the mph(A) gene in plasmid pCTX168.

These results showed that the structural variability of the bla_CTX-M-1 gene region resulted from interplasmid recombination events and the integration of insertion sequences. The gene bla_CTX-M-1 in combination with the IS26-ΔISEcp1-structure and the truncated mph(A)-mrx-mphR(A) gene cluster, which is followed by a second IS26 element, linked to IncN plasmids, was recently found in a German human clinical E. coli ST131 isolate, proposing a novel composite transposon with both IS26 elements being flanked by 8-bp direct repeats (5′-TTACCGGT-3′) (13). An identical sequence was located upstream of the right-hand IS26 in pCTX246, too, while in pCTX168 the direct repeat was found downstream of the IS26. This observation underlines the structural relatedness of these bla_CTX-M-1 gene regions, which have not been found in animal E. coli isolates from Germany so far. IncN plasmids carrying the bla_CTX-M-1 gene have been described to be transmitted between Danish farm personnel and pigs in different E. coli lineages (23). The finding of bla_CTX-M-1 genes with similar genetic environments on IncN plasmids in different human, porcine, and canine E. coli isolates from Germany points toward a plasmid transfer among E. coli isolates from human and animal sources and a common gene pool for transferable ESBL genes.