Genetic Context of Plasmid-Carried bla_CMY-2-Like Genes in Enterobacteriaceae

Abstract

Analysis of 15 European clinical Enterobacteriaceae isolates showed that differences in the genetic context of bla_CMY-2-like genes reflected the replicon type, usually IncA/C or IncI1. These bla_CMY-2 loci may originate from the same ISEcp1-mediated mobilization from the Citrobacter freundii chromosome as structures described in earlier studies.

Among plasmid-mediated AmpC enzymes, CMY-2 is the most prevalent both in France and worldwide, especially in Salmonella species (1, 17). Previous studies in the United States revealed that large plasmids carrying bla_CMY-2 have been transmitted between different genera of bacteria, and notably from enteric Salmonella strains to pathogenic Escherichia coli strains (22). American studies of plasmids encoding CMY-2 using mixed-plasmid microarrays showed that the plasmids fell into two clusters (3); likewise, replicon-typing experiments revealed two replicon types, I1 and A/C (11). Similar studies in Taiwan confirmed that plasmids from diverse isolates carrying bla_CMY-2 genes have common restriction patterns, evidence of interspecies spread (23). Further investigations of Taiwanese isolates allowed the identification of a specific transposon-like element responsible for the spread of bla_CMY-2 among members of the family Enterobacteriaceae (6, 19). In the American and Taiwanese studies, ISEcp1 has been presumed to be involved both in the mobilization of bla_CMY-2 from the Citrobacter freundii chromosome and in the expression of ampC lacking chromosomal ampR (6, 10, 12, 14, 15, 19). Poirel et al. have shown the involvement of ISEcp1B, an ISEcp1-like element, in the expression and mobilization of the β-lactamase gene bla_CTX-M-19 (18).

We characterized 15 CMY-type-producing isolates, including eight E. coli isolates, four Klebsiella pneumoniae isolates, two Proteus mirabilis isolates, and one Salmonella enterica isolate. We describe the genetic organization of bla_CMY-2-like genes in these European isolates and compare our findings with those from the United States and Taiwan.

Table 1 lists the 15 clinical isolates and their sources. Six isolates were previously reported (4, 7, 9, 13, 21). Total DNA was extracted by using a QIAamp DNA Mini kit (Qiagen, Courtaboeuf, France). Using repetitive-element PCR, we showed that three of the eight E. coli clinical isolates were clonally related (profile A: TN2106, TN38148, TN386) according to the interpretation criteria of van Belkum et al. (Table 1) (16, 20). Enterobacterial repetitive intergenic consensus PCR of the four K. pneumoniae isolates gave different amplification patterns (Table 1) (16).

TABLE 1.

Clinical isolates, origins, epidemiological features, and exploration by PCR mapping

Strain	Reference	Collection yr	Country of origin (city, hospital)a	PCR profile	CMY plasmid transferb	Replicon typing	CMY type	PCRe
								A	B	C	D	E	F	G	H	I	J	K	L
S. enterica serovar Senftenberg SENF	13	1994	Algeria	ND	Tc E. coli C1a Nalr	A/C	2	+	+	+	−	−	+	+	+	+	−	−	−
K. pneumoniae 169	7	1999	France (Paris, H2)	Wc	Tc E. coli J53	A/C	2	+	+	+	−	−	+	+	+	+	−	−	−
K. pneumoniae BM2974	4	1998	Sweden	Xc	Tc E. coli J53	A/C	4	+del	+del	+del	−	−	+	+	+	+	−	−	−
K. pneumoniae LMCMY	This study		France (Colombes, H3)	Yc	Tc E. coli J53	A/C	2	+	+	+	−	−	+	+	+	+	−	−	−
K. pneumoniae 9701	7	1997	France (Paris, H1)	Zc	Tc E. coli J53	A/C	4	+ins	+ins	+ins	−	−	+	+	+	+	−	−	−
P. mirabilis H223b	21	1996	Tunisia	ND	Tc E. coli J53	A/C	4	+	+	+	−	−	+	+	+	+	−	−	−
P. mirabilis RPCMY	This study	2002	France (Garches, H6)	ND	Tc E. coli J53	Negative	2	−	−	+	−	−	+	+	+	+	+	+	−
E. coli TN2106	This study	2003	France (Paris, H4)	Ad	Tc E. coli J53	A/C	2	+	+	+	−	−	+	+	+	−	−	−	−
E. coli TN38148	This study	2004	France (Paris, H4)	Ad	Tc E. coli J53	I1	2	−	−	+	+	+	+	−	−	−	−	−	+
E. coli TN10	This study	2002	France (Paris, H4)	Bd	Tc E. coli J53	I1	2	−	−	+	+	+	+	−	−	−	−	−	+
E. coli TN13	9	2002	France (Paris, H4)	Cd	Ep E. coli DH10B	I1	2	−	−	+	+	+	+	−	−	−	−	−	+
E. coli TN386	This study	2002	France (Paris, H4)	Ad	Tc E. coli J53	I1	2	−	−	+	+	+	+	−	−	−	−	−	+
E. coli IGR4801	This study	2005	France (Villejuif, H5)	Dd	Tc E. coli C600	Negative	2	−	−	+	+	+	+	−	−	−	−	−	+
E. coli IGR4872	This study	2005	France (Villejuif, H5)	Ed	Tc E. coli C600	Negative	2	−	−	+	+	+	+ins	−	−	−	−	−	+ins
E. coli TN44889	This study	2004	France (Paris, H4)	Fd	NT	ND	2	−	+	+	−	−	−	−	−	−	−	−	−

^aH1, Hôpital Cochin; H2, Hôpital Saint-Antoine; H3, Hôpital Louis Mourier; H4, Hôpital Tenon; H5, Institut Gustave Roussy; H6, Hôpital Raymond Poincaré.

^bAbbreviations: Tc, transconjugant; Ep, transformant; NT, no transfer; ND, not determined.

^cEnterobacterial repetitive intergenic consensus PCR profile.

^dRepetitive-element PCR profile.

^e−, negative; +, positive; +_ins, positive with an insertion; +_del, positive with a deletion.

Except for one strain, SENF, which was previously transferred in E. coli C1a Nal^r, E. coli K-12 strain J53-2 (Rif^r) or C600 (Nal^r) transconjugants were obtained by mating and selection on cefoxitin (10 μg/ml) and either rifampin (rifampicin) (250 μg/ml) or nalidixic acid (50 μg/ml). For isolates with which mating was unsuccessful, E. coli strain DH10B (Invitrogen SARL, Cergy-Pontoise, France) was transformed with plasmid DNA by electroporation (Bio-Rad, Marnes la Coquette, France). Transformants were selected with cefoxitin (10 μg/ml). All of the transformants or transconjugants were successfully typed by PCR-based replicon typing, with the exception of three isolates (5). Most of the isolates (11/15) carried an A/C or an I1 replicon plasmid (Table 1).

Except for the six isolates previously published, the bla_CMY gene was amplified by PCR experiments with primers ampC1 and ampC2, as previously described (7), and sequenced. Three isolates (BM2974, 9701, and H223b) carried bla_CMY-4, a CMY-2 variant; all of the others carried bla_CMY-2 (Table 1).

The genetic context of bla_CMY was explored both by PCR mapping and cloning experiments. A 13-kb sequence surrounding bla_CMY-2 from S. enterica serovar Newport was available in GenBank (accession number DQ164214); we used several PCRs (B, C, F, G, H, I, J, and K) to characterize the genetic context of bla_CMY in our collection of 15 isolates (see the supplemental material). Table 1 shows several PCR profiles. PCR products B and F did not have the expected length for three isolates. Four isolates (169, TN44889, TN38148, and RPCMY) were chosen as representative of most of the PCR profiles, and cloning experiments were performed to study the genetic organization of bla_CMY more extensively.

Plasmid DNA was partially digested with Sau3AI, and the fragments were ligated into the BamHI site of pACYC184. E. coli DH10B was transformed with the resulting plasmids, and transformants were selected with cefoxitin (10 μg/ml) and chloramphenicol (50 μg/ml). The largest plasmids with a bla_CMY insert were selected, and both strands were sequenced. PCR primers were designed (PCRs A, D, E, and L), and PCR experiments were performed with all of the isolates (Table 1; see the supplemental material). The sequenced regions and DNA sequence analyses are shown in Fig. 1.

FIG. 1.

Comparison of regions surrounding bla_CMY-2. IR_R, imperfect right inverted repeat.

Previous American and Taiwanese studies suggested that ISEcp1 is involved in mobilization of the bla_CMY-2-like gene from the C. freundii chromosome, with ISEcp1-IR_R consistently 117 bp upstream from bla_CMY-2 (6, 10, 12). By comparing the sequence of S. enterica serovar Newport DQ164214 with that of C. freundii chromosome U21727, a 2,823-bp region including a bla_CMY-2-like gene, blc, sugE, and ΔecnR could be delimited. Interestingly, the last 14 bp of this region, CCACACAATTCAGG, could have been recognized as an imperfect right inverted repeat-like sequence by the transposase of ISEcp1 during the mobilization process. In the 15 isolates in this study, at least a part of ISEcp1 was present upstream from the bla_CMY-2-like gene. Nevertheless, the ISEcp1-bla_CMY-2-like gene region was in the same configuration in six isolates only: 169, LMCMY, SENF, H223b, TN2106, and TN44889. For the other nine isolates, ISEcp1 was present with various deletions/insertions which do not separate the bla_CMY-2-like gene from its putative promoter in ISEcp1 (Fig. 1). These modifications are likely to have occurred after ISEcp1 had mobilized the bla_CMY-2-like gene from the C. freundii chromosome to a plasmid.

The plasmids from most of the isolates shared various structural similarities with the 13-kb type I structure described in S. enterica serovar Newport (accession number DQ164214), where the bla_CMY-2 gene is duplicated (12); the second copy follows a partial copy of ISEcp1 in the opposite orientation (Fig. 1).

Downstream from the bla_CMY-2-like gene in this 13-kb type I structure, several open reading frames (ORFs) were described between the two copies of bla_CMY-2, from blc to traC (Fig. 1). In 8 of our 15 isolates, RPCMY, 169, BM2974, LMCMY, SENF, H223b, 9701, and TN2106, the genetic organization downstream from the bla_CMY-2-like gene was very similar to this 13-kb type I structure. Only RPCMY had two copies of the bla_CMY-2-like gene. For the other isolates, the sequence remains unknown either downstream from traC or downstream from orf3 (Fig. 1).

Upstream from the bla_CMY-2-like gene, ISEcp1 was present with deletions/insertions in its sequence for three isolates. (i) In RPCMY, there is a deletion of the first 1,140 bp due to the insertion of a separate copy of ISEcp1 in the reverse orientation; this second copy has a 1,296-bp deletion due to the insertion of IS10. Interestingly, IS10 has previously been identified as being responsible for the disruption of ISEcp1 upstream from bla_CTX-M-14 in E. coli TN13, but in a different position (8). (ii) In BM2974, there is a deletion of a 104 bp-segment at the 3′ end of ISEcp1. (iii) In Kp9701, there is an insertion of IS5 at position 1509 in ISEcp1 (Fig. 1).

In 169, BM274, LMCMY, SENF, H223b, TN2106, and 9701, an additional 1,560-bp segment was characterized upstream from ISEcp1. It is 100% identical to the region comprising the traB, traV, and traA genes in S. enterica serovar Newport SL254 (accession no. CP000604).

Of the 15 isolates, 8 had a genetic organization around the bla_CMY-2-like gene highly similar to that in the 13-kb type I structure and, except for RPCMY, carried an A/C replicon plasmid.

A second group included six isolates less similar to the 13-kb type I structure (TN38148, TN10, TN13, TN386, IGR4801, and IGR4872): the bla_CMY-2-like gene was followed by blc, sugE, ecnR, and orf1 only. Indeed, orf1 had 1,078 bp deleted at the 3′ end and was followed by yafA, which had a 3′ deletion and was in the reverse orientation. The truncated yafA gene was followed by yafB, and both genes belonged to a 1,983-bp segment which is 98% identical to a region of S. enterica plasmid pNF1358 (accession number no. DQ017661) (Fig. 1). This segment in pNF1358 is located directly upstream from ISEcp1-bla_CMY-2.

In all six isolates of this second group, a deletion of the first 1,281 bp in the 5′ part of ISEcp1 was due to the insertion of IS1294 in the reverse orientation (Fig. 1). IS1294 belongs to the IS91 family and is a putative transposable element capable of mediating one-ended transposition (www-is.biotoul.fr). Downstream from IS1294, a 2,265-bp segment comprising the repY and repZ genes involved in replication initiation is 95% identical to a region of S. enterica pNF1358 (Fig. 1). In IGR4872, an additional IS1294 was inserted between sugE and ecnR, leading to the duplication of the GTTC target site.

Four isolates of this second group carried an IncI1 plasmid; two isolates from the same hospital could not be replicon typed.

In the 15th isolate, TN44889, the genetic organization of the ISEcp1-bla_CMY-2-like gene region was unique with the presence of IS4 upstream from a complete copy of ISEcp1 (Fig. 1). Downstream from the bla_CMY-2-like gene, 467 bp of blc was deleted by the insertion of a truncated ORF, yggR. That is followed by a 4,591-bp region including five ORFs, which is 95% identical to the Shigella flexneri 2a SRL pathogenicity island. This system is linked to a cluster of multiple antibiotic resistance determinants (accession number no. AF326777).

Our findings are consistent with the study of Hopkins et al. (11); there is a predominance of A/C and I1 replicons among plasmids carrying bla_CMY-2-like genes in 15 nonrepetitive Enterobacteriaceae isolates. Each replicon type reflected the genetic organization surrounding the bla_CMY-2-like gene, with several minor rearrangements, including small insertions or deletions. Using phylogenetic methods, Barlow and Hall demonstrated that all CMY-2-type β-lactamase variants had a common ancestor which was plasmid borne (2). This suggests that a region from the C. freundii chromosome (ampC, blc, sugE, ecnR) has been mobilized only once. This study is consistent with results of Barlow and Hall, as ISEcp1 seems to have been involved in the mobilization of the bla_CMY-2-like gene, irrespective of the replicon type and the plasmid backbone.

Nucleotide sequence accession numbers.

The nucleotide sequences in strains 169, 9701, RPCMY, TN38148, and TN44889 have been submitted to GenBank and have been assigned accession numbers FM246880, FM246881, FM246882, FM246883, and FM246884, respectively.