Cooccurrence of Multiple AmpC β-Lactamases in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in Tunisia

Abstract

Over a period of 40 months, plasmid-mediated AmpC β-lactamases were detected in Tunis, Tunisia, in 78 isolates (0.59%) of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. In 67 isolates, only one ampC gene was detected, i.e., bla_CMY-2-type (n = 33), bla_ACC (n = 23), bla_DHA (n = 6) or bla_EBC (n = 5). Multiple ampC genes were detected in 11 isolates, with the following distribution: bla_MOX-2, bla_FOX-3, and bla_CMY-4/16 (n = 6), bla_FOX-3 and bla_MOX-2 (n = 3), and bla_CMY-4 and bla_MOX-2 (n = 2). A great variety of plasmids carrying these genes was found, independently of the species and the bla gene. If the genetic context of bla_CMY-2-type is variable, that of bla_MOX-2, reported in part previously, is unique and that of bla_FOX-3 is unique and new.

INTRODUCTION

AmpC β-lactamases are cephalosporinases that are poorly inhibited by clavulanic acid (1). AmpC production is one of the mechanisms of resistance to β-lactams in enterobacteria, conferring resistance to all β-lactams except fourth-generation cephalosporins and carbapenems (2). The genes encoding these enzymes are chromosome or plasmid borne (3).

Most plasmid-borne AmpC (pAmpC) β-lactamase genes are derived from chromosomal genes that have been mobilized onto plasmids. Based on sequence similarities with species-specific AmpC enzymes, pAmpC variants are classified into five evolutionary groups: the CIT variants (CMY-2 types) originating in Citrobacter freundii, the Enterobacter sp. EBC variants (ACT-1 type, MIR-1), the Morganella morganii DHA variants, the Hafnia alvei ACC variants, and the Aeromonas sp. FOX and MOX variants (1, 2). Several genetic elements are involved in the mobilization of pAmpC β-lactamase genes, e.g., the insertion sequences IS26 (CMY-13), ISEcp1 (CMY-2-type, ACC-1-type), and ISCR1, associated with complex integrons (DHA-1 type) (1, 2).

The geographic scattering of the different types of pAmpC shows that the CMY-2 type is the most frequent, particularly in Europe (e.g., in France, Spain, Italy, and Turkey [4–7]), in Canada, Argentina, and Tunisia (8–10), and in Korea and China (11, 12). These pAmpC β-lactamases were detected among Enterobacteriaceae, especially in Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enterica, and Proteus mirabilis, and also in naturally AmpC-producing species such as Enterobacter cloacae, Enterobacter aerogenes, and C. freundii (13–15).

The pAmpC genes have also been reported in animals. Indeed, in the United States, CMY-2-producing E. coli strains have been found among canine clinical isolates (16). Clinical disease associated with AmpC-producing E. coli in dogs in Australia was first reported in 2006 (17). More recently, a survey of clinical isolates of E. coli from dogs and horses in the Netherlands revealed that 2% of them were AmpC producers (8). Furthermore, a Swedish study has described CMY-2-producing E. coli isolated from broilers (18). Such enzymes have been found in Klebsiella isolates from companion animals in Italy (19). In Tunisia, CMY-2-producing E. coli isolates have been recovered from healthy food animals and feces from healthy pets (20, 21).

Cooccurrence of multiple pAmpC β-lactamases in a single strain of Enterobacteriaceae has not been reported previously but has been observed in a Tunisian collection of various species of this family of bacteria. The present study was conducted to characterize the strains and plasmids carrying multiple pAmpC genes and to explore the genetic context of these genes in an attempt to explain this new phenomenon.

MATERIALS AND METHODS

Bacterial strains, antimicrobial susceptibility, and synergy testing.

A total of 11,393 strains from three enterobacterial species. i.e., E. coli (n = 7,504), Klebsiella sp. (n = 2,905) and P. mirabilis (n = 984), were collected at the clinical microbiology laboratory of Charles Nicolle hospital, Tunis, Tunisia, from January 2006 to April 2009. Only 107 nonduplicate isolates (36 E. coli, 58 Klebsiella, and 13 P. mirabilis isolates) resistant to third-generation cephalosporins, with negative double-disc synergy tests (DDST), were investigated. Susceptibility to antibiotics was determined using standard disc diffusion on Mueller-Hinton agar (Bio-Rad, Marnes-la-Coquette, France), and MICs were determined using standard agar dilution on the same medium according to the CLSI recommendations (22). Moreover, all isolates were phenotypically screened for the production of extended-spectrum β-lactamases (ESBL) using DDST (23) with and without cloxacillin (250 mg/liter) (24).

E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923 served as controls for MIC determinations. Rifampin-resistant E. coli J-53 and azide-resistant E. coli C600 were used as recipients in conjugation, and E. coli DH10B (Invitrogen SARL, Cergy-Pontoise, France) was used in cloning and transformation experiments.

E. coli CFT073 (O6 K1 H7) and E. coli J96 (O4 K6 H6) were used as reference strains for the detection of virulence factors (25). Strains carrying FOX-3 and MOX-2 genes (26, 27) were used as positive controls for detection of FOX and MOX producers with PCR.

Molecular characterization of β-lactamases.

Total DNA preparations for PCR were obtained by thermolysis of the isolates. Initial identification was obtained with primers of a multiplex PCR protocol for the detection of ampC families as described by Pérez-Pérez et al. (3). Confirmation was obtained using simplex reactions with primers CF-1/CF-2, FOX MF/MR (20), and MOX MF/MR (MOX-MF, 5′-GCTGCTCAAGGAGCACAGGAT-3′; MOX-MR, 5′-CACATTGACATAGGTGTGGTGC-3′) for bla_CMY, bla_FOX, and bla_MOX, respectively. Amplicons of bla_AmpC were purified and sequenced. Primers OT3, OT4, and OXA-1 (forward and reverse) were used to amplify putative bla_TEM-1 and bla_OXA-1 genes, respectively.

Epidemiological studies.

Total DNA was obtained with the QIAamp DNA minikit (Qiagen, Courtaboeuf, France) to assess epidemiological relationships between isolates producing plasmid-mediated cephalosporinases using repetitive-element PCR (rep-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) as previously described (28, 29) and pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with the restriction enzyme XbaI (Biolabs).

Determination of phylogenetic groups of E. coli was performed using PCR as previously described (30). The E. coli clone O25b-ST131 of phylogenetic group B2 was detected using PCR (31). E. coli isolates were screened for the presence of 18 genes encoding putative virulence factors characteristic of extraintestinal pathogenic E. coli by multiplex PCR as previously described (32, 33, 34). These genes Included fimH, afa, sfa/foc, papG (3 alleles), cnf1, sat, hlyA, iutA, iroN, fyuA, iha, kpsMTII, ompT, malX (pathogenic island marker PAI II of the CFT073 reference strain), traT, and usp.

The clonal structure of the K. pneumoniae strains was determined by multilocus sequence typing (MLST) based on the allelic diversity of seven loci: rpoB, mdh, pgi, phoE, infB, gapA, and tonB. These housekeeping locus fragments of all K. pneumoniae isolates were also sequenced.

The primers used for amplifications were designed by Diancourt et al. (35). Sequence types (STs) were assigned at the http://www.pasteur.fr/recherche/genopole/PF8/mlst website.

The K. pneumoniae isolates were screened for the presence of nine genes encoding putative virulence factors and six capsular serotypes by PCR as previously described (36, 37, 38).

These genes included mrkD, kpn, ycfM, entB, ybtS, iroN_Kp, rmpA, magA, allS2, wcaG, and genes encoding K2, K5, K20, K54, and K57 (capsular serotypes).

Transfer of resistance and molecular characterization of plasmids.

Mating experiments were performed by mixing equal volumes (1 ml) of exponentially growing cultures of each isolate tested and E. coli J-53 or E. coli C600 in Trypticase soy broth (Bio-Rad) and incubating the mixture for 3 h at 37°C. β-Lactam-resistant E. coli J-53 or E. coli C600 transconjugants were selected on Drigalski agar (Bio-Rad) containing rifampin (250 μg/ml) or azide (100 μg/ml) and cefotaxime (2.5 μg/ml).

E. coli DH10B transformants were obtained by electroporation of plasmid DNA extracted with the High Pure plasmid isolation kit (Roche, Mannheim, Germany) in a Gene Pulser II (Bio-Rad) and selected on Drigalski agar supplemented with cefotaxime (2.5 μg/ml). Transfer of β-lactam resistance markers from all clinical isolates studied was attempted by conjugation or transformation.

Replicon typing of the plasmids of the donor strains and the corresponding transconjugants or transformants was performed by PCR under the conditions previously described (39, 40).

Plasmid DNA of the clinical isolates of P. mirabilis, E. coli, and K. pneumoniae and E. coli transconjugants was extracted by alkaline lysis, as previously described (41), to estimate plasmid number and size. RP4 (54 kb), pCFF04 (85 kb), and pIP173 (126.8 kb) were used as reference plasmids.

Genetic context analysis.

For molecular cloning, we used total DNA of strain Kp10 for the bla_MOX gene extracted using the Qiagen DNeasy blood and tissue kit and plasmid DNA from transconjugant J-53 of K. pneumoniae Kp8 for the bla_FOX gene extracted with the Qiagen large-construct kit. DNA was partially digested with Sau3A and ligated into the BamHI site of pUC18 (42). The recombinant plasmid was introduced into E. coli DH10B by transformation. Transformants were selected on Drigalski agar supplemented with cefotaxime (2.5 μg/ml). Recombinant plasmids were extracted with the High Pure plasmid isolation kit (Roche), insert sizes were estimated after digestion with EcoRI, and initial genetic environments were determined by sequencing using universal M13 primers the first time for recombinant plasmids obtained. Recombinant plasmids were extracted with the High Pure plasmid isolation kit (Roche), and insert sizes were estimated after digestion with EcoRI. Initial analysis of the genetic environments of the bla genes borne on recombinant plasmids was done by sequencing using universal M13 primers. The genetic context of bla_MOX, bla_FOX, and bla_CMY of recombinant plasmids and all nine remaining strains was further explored by PCR mapping and sequencing using primers designed for bla_MOX (A, B, D, E, and G), bla_FOX (H and I), and bla_CMY (A, B, C, D, E, F, G, H, I, J, K, and L) (see the supplemental material). Two additional primers were tested for bla_CMY: CF2/IS26-SL2 up (IS26-SL2 up, 5′-CTGGGTAAAATCCTCAACA-3′) and CF2/ISEcp1 (ISEcp1, 5′-AAAAATGATTGAAAGGTGGT-3′) (see the supplemental material).

Nucleotide sequence accession number.

The accession number of the genetic context of bla_FOX-3 is LC072710.

RESULTS AND DISCUSSION

Among the 107 enterobacterial isolates studied, multiplex PCR revealed pampC β-lactamase genes in 78 isolates, whereas in 29 isolates (E. coli), no pampC β-lactamase genes were detected. In 67 isolates, only one pampC gene was detected with bla_CMY-2-type (n = 32), bla_ACC-1-type (n = 23), bla_DHA-1-type (n = 6), and bla_EBC (n = 5); among these 67 isolates, 48 (K. pneumoniae) also produced CTX-M-15. Multiplex PCR revealed pampC β-lactamase genes in 78 of the 107 isolates studied but not in 29 isolates of E. coli. Single pampC genes were detected in 67 isolates, i.e., bla_CMY-2-type (n = 32), bla_ACC-1-type (n = 23), bla_DHA-1-type (n = 6), and bla_EBC (n = 5). Among the 67 isolates, 48 K. pneumoniae isolates also produced CTX-M-15.

In the 11 remaining strains (5 E. coli, 4 K. pneumoniae, and 2 P. mirabilis strains), at least two pampC genes were detected. At this stage, our study was focused on these 11 strains that harbored multiple pAmpC β-lactamases, as follows: MOX-type, FOX-type, and CMY-2-type enzymes in six isolates (in three E. coli, one K. pneumoniae, and two P. mirabilis isolates), MOX-type and FOX-type enzymes in three isolates (in one E. coli and two K. pneumoniae isolates), and MOX-type and CMY-2-type enzymes in two isolates (in one E. coli and one K. pneumoniae isolate). The origin and characteristics of clinical interest of these isolates are summarized in Table 1. They were resistant to all β-lactams except ertapenem, imipenem, and cefepime. MICs of ticarcillin and piperacillin ranged from 512 to >2,048 μg/ml, those of cefotaxime from 256 to >2,048 μg/ml, those of ceftazidime from 128 to 1,024 μg/ml, those of cefoxitin from 64 to 512 μg/ml, and those of cefepime 0.5 to 2 μg/ml.

TABLE 1.

Chromosomal characteristics of the 11 Enterobacteriaceae harboring multiple plasmid-borne AmpC β-lactamase genes^a

Isolate	Ward	Specimen	Yr	REP profile	ERIC profile	PFGE profile	PG	ST	Virulence profile	Virulence score	KS
Ec3	ICU	Blood	2005	A1	A1	A1	A	ND	fimH, hlyA, iha, usp, iroN	5	ND
Ec5	Med	Urine	2008	A2	A2	A2	B2	ST131	fimH, pai, fyu A, sfa/foc, pap G, hlyA, cnf1, iha, usp	9	ND
Ec6	Chir	Pus	2008	A3	A3	A3	D	ND	fimH, iutA, hlyA, iroN	4	ND
Ec7	Ped	Urine	2008	A4	A4	A4	A	ND	fimH, fyuA, sfa/foc, cnf1	4	ND
Ec11	Uro	Urine	2007	A5	A5	A5	B2	ST131	fimH, pai, fyu A, sfa/foc, iut A, hlyA, papG, afa/dra, iha, usp	10	ND
Kp2	ICU	Blood	2005	B1	B1	B1	ND	ST218	entB, ybtS, mrkD, ycfM, kpn	5	K57
Kp4	Ped	Urine	2008	B2	B2	B2	ND	ST11	entB, ybtS	2	ND
Kp8	ICU	Urine	2005	B1	B1	B1	ND	ST218	entB, ybtS, mrkD, ycfM, kpn	5	K57
Kp10	Ortho	Blood	2005	B3	B3	B1	ND	ST218	entB, ybtS, mrkD, ycfM, kpn	5	K57
Pm1	Uro	Urine	2006	C1	C1	C1	ND	ND	ND	ND	ND
Pm9	ICU	Catheter	2007	C2	C2	C2	ND	ND	ND	ND	ND

^aEc, Escherichia coli; Kp, Klebsiella pneumoniae; Pm, Proteus mirabilis; ICU, intensive care unit; MED, medicine; Surg, surgery; Ped, pediatrics; Uro, urology; Ortho, orthopedics; REP, genomic fingerprinting by repetitive-element PCR profiling; ERIC, enterobacterial repetitive intergenic consensus PCR profile; PFGE, pulsed-field gel electrophoresis profile; PG, phylogenetic groups; ST, sequence type; KS, capsular serotype; ND, not determined.

As determined by sequence analysis, bla_CMY genes were bla_CMY-4 (n = 6) and bla_CMY-16 (n = 2), bla_FOX genes were all bla_FOX-3 (n = 9), and bla_MOX genes were all bla_MOX-2 (n = 11). Seven isolates among the pAmpC producers also produced the broad-spectrum TEM-1 β-lactamase, and one of them produced OXA-1 in addition (Table 2).

TABLE 2.

Plasmid characteristics of the 11 Enterobacteriaceae harboring multiple plasmid-borne AmpC genes and exploration by PCR mapping^a

Isolate	Resistance pattern	blaAMPC	blaTEM-1, blaOXA-1	RT	PCR mapping result
					blaMOX					blaFOX		blaCMY
					A	B	D	E	G	H	I	A	B	C	F	G	H	I	J	K	L	D	E	CF2/IS26-SL2 up	CF2/ISEcp1 commune
Ec3	GM, TM, NET, TE, C, NA, SXT	FOX-3, MOX-2		A/C	+	+	+	+	+	+	+
Ec5	GM, TM, NA, OFX, CIP	CMY-4, FOX-3, MOX-2	TEM-1	FIA, FIB	+	+	+	+	+	+	+	−	−	+	+	−	−	−	−	−	+	+	+	−	−
El Ec5	TM	CMY-4		FIA
Ec6	TE	CMY-4, FOX-3, MOX-2	TEM-1	A/C, FIB	+	+	+	+	+	+	+	−	+	+	+	+	+	+	−	−	−	−	−	−	−
El Ec6	TE	CMY-4		FIB
Ec7	TE	CMY-4, MOX-2	TEM-1	Q	+	+	+	+	+			−	−	−	−	−	−	−	−	−	−	−	−	−	−
Tc Ec7		CMY-4	TEM-1	Q
Ec11	TM, NET, AN, NA, CIP, SXT, OFX	CMY-16, FOX-3, MOX-2	OXA-1	FIA	+	+	+	+	+	+	+	−	+	+	−	−	−	−	−	−	−	−	−	−	−
El Ec11	TM, SXT	CMY-16	OXA-1	FIA
Kp2	GM, TM, NET, TE, C, SXT	FOX-3, MOX-2		A/C, FIIK	+	+	+	+	+	+	+
Tc Kp2	TM, TE, SXT	FOX-3		A/C
Kp4	TE, SXT	CMY-16, FOX-3, MOX-2	TEM-1	Q	+	+	+	+	+	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−
Tc Kp4A		FOX-3		Q
El Kp4B	TE, SXT	CMY-16	TEM-1	-
Kp8	GM, TM, NET, TE, SXT	FOX-3, MOX-2		A/C, FIIK	+	+	+	+	+	+	+
Tc Kp8	TM, TE, SXT	FOX-3		A/C
Kp10	GM, TM, NET, TE, SXT	CMY-4, MOX-2	TEM-1	A/C, FIIK	+	+	+	+	+			−	+	+	+	+	−	−	−	−	−	−	−	−	−
El Kp10	TE, SXT	CMY-4		FIIK
Pm1	TE, C, NA, OFX, CIP, SXT	CMY-4, FOX-3, MOX-2	TEM-1	A/C, FIA	+	+	+	+	+	+	+	−	+	+	+	+	+	+	−	−	−	−	−	−
Tc Pm1A	C, SXT	CMY-4		A/C
Tc Pm1B	TE	FOX-3		FIA
Pm9	TM, NET, AN, TE, SXT	CMY-4, FOX-3, MOX-2	TEM-1	A/C, FIA	+	+	+	+	+	+	+	−	+	+	+	+	−	−	−	−	−	−	−	−	−
Tc Pm9A	TM, TE, SXT	CMY-4	TEM-1	A/C
Tc Pm9B		FOX-3		FIA

^aEc, Escherichia coli; Kp, Klebsiella pneumoniae; Pm, Proteus mirabilis; Tc, transconjugant; El, electroporant; RT, replicon type; GM, gentamicin; TM, tobramycin; NET, netilmicin; AN, amikacin; C, chloramphenicol; NA, nalidixic acid; OFX, ofloxacin; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole; TE, tetracycline.

CMY-4 was first identified in a clinical P. mirabilis isolate from Tunisia (43), and since then, it has been found throughout the world. CMY-16, a variant of the CMY lineage, which differs from its closest homologue, CMY-4, by a single amino acid substitution (A171S), was first identified in a P. mirabilis isolate from Italy (6).

FOX-3 and MOX-2 β-lactamases have been detected in K. pneumoniae isolates from Italy (26) and Greece (27), respectively. However, these enzymes were not previously reported in Tunisia; hence, this is the first report of FOX-3 and MOX-2 β-lactamase in this country. To our knowledge, this is also the first report of cooccurrence of various plasmid-borne bla_AmpC genes in a single strain and of such cooccurrence in several species of Enterobacteriaceae.

Based on the distinct patterns observed with molecular typing (rep-PCR, ERIC-PCR, and PFGE), there was no epidemiological link between the five E. coli isolates harboring multiple pampC genes. The phylogenetic study showed that the five E. coli isolates belonged to phylogenetic groups B2 (n = 2), A (n = 2), and D (n = 1). We found that the two B2 isolates belonged to the ST131 clone and harbored many virulence determinants leading to the highest virulence scores, i.e., 9 and 10 (Table 1). The ST131 isolates harbored an extended virulence profile and presented a virulence gene panel similar to that of other previously described members of ST131 (44), an ST often involved in the dissemination of CTX-M-15 genes (31). Moreover, a recent study showed that this clone has enhanced metabolic capacities, acts as a potent colonizer of the intestine, and displays the typical features of virulent E. coli strains (45).

ERIC and Rep-PCR typing revealed a similarity in the patterns of two K. pneumoniae isolates (Kp2 and Kp8) indicating that they are genetically related. In addition, PFGE analysis confirmed the existence of an epidemic clone represented by these strains, distinct from Kp10, while all of them belonged to ST218 (Table 1). This ST has been described recently in K. pneumoniae isolates from patients with bacteremia in China (46). Moreover, we found that the clonal K. pneumoniae strains had the capsular serotype K57 and showed important virulence profiles including five genes, i.e., entB, mrkD, ycfM, kpn, and ybtS. The latter is considered a major virulence determinant in K. pneumoniae and is required for the synthesis of yersiniabactin, which promotes biofilm formation and a reduction in the killing capacity of innate immune cells by blocking the production of reactive oxygen species (47, 48). Kp4, on the other hand, was a sporadic isolate of ST11, reported to be involved in the dissemination of various β-lactamases, including carbapenemases and pAmpC (49).

Plasmids carrying pAmpC genes were successfully transferred via conjugation and/or electroporation from 10 of the 11 isolates tested. The pAmpC gene transfer experiments showed that no more than one gene was transferred at one time, either bla_CMY or bla_FOX, indicating that they are probably located on different plasmids. However, bla_MOX could not be transferred from any isolate, suggesting its location on the chromosome.

We found that pAmpC genes were carried on plasmids of different incompatibility groups. The bla_CMY genes were carried on conjugative Inc A/C (n = 2), Inc FIA (n = 2), Inc FIB (n = 1), Inc Q (n = 1), or Inc FIIK (n = 1) plasmids, and bla_FOX genes were located on Inc FIA (n = 2), Inc A/C (n = 2), or Inc Q (n = 1) plasmids (Table 2). Our findings are consistent with previous reports noting the predominance of Inc A/C replicons among plasmids carrying bla_CMY (34). Accordingly, the Inc A/C replicon is the most commonly reported worldwide (39). The fact that pAmpC genes are carried by different plasmid types promotes their rapid spread among Enterobacteriaceae (28, 50). Furthermore, we found that pAmpC genes were generally located on plasmids of various sizes. Analysis of DNA extracted according to Kado and Liu (41) revealed that K. pneumoniae isolates Kp2, Kp4, and Kp8 harbored two large plasmids (>130 kb) and a third smaller one (ca. 50 kb), while E. coli isolate Ec7 harbored only one large plasmid (>130 kb) and one of smaller one (50 kb). The Kp10 isolate and its corresponding transformant harbored one large plasmid (>130 kb) (Fig. 1). For the remaining E. coli and P. mirabilis isolates, we obtained no clear results with the procedure of Kado and Liu (41). Our results are in agreement with reports of pAmpC genes found on plasmids of sizes varying from 7 to 180 kb (2).

FIG 1.

Plasmid analysis. (A) Lane 1, Kp2; lane 2, transconjugant (Tc) Kp2; lane 3, Kp4; lane 4, Tc Kp4 B; lane 5, pIP173 (126 kb); lane 6, RP4 (55 kb) and pCFF04 (85 kb); lane 7, Ec7; lane 8, Tc Ec7; lane 9, Kp8; lane 10, Tc Kp8. (B) Lane 1, pIP173 (126 kb); lane 2, RP4 (55 kb) and pCFF04 (85 kb); lane 3, Kp10; lane 4, transformant (Tf) Kp10.

Two plasmids of the present study carrying CMY-4 and FOX-3 genes belonged to the Q1 group. These replicons are characterized by their relatively small size (5 to 10 kb), their ability to be mobilized by several self-transmissible plasmids, and their broad host range, making them highly promiscuous (51).

Analysis of the genetic environment of bla_MOX-2 by cloning and mapping experiments showed that it was very closely related to that of the single reference strain K. pneumoniae KOL (accession number AJ276453) reported in 2002 (27). The DNA insert carrying bla_MOX-2 was sequenced and four additional open reading frames (ORFs) were identified (Fig. 2). The ORFs are on the opposite DNA strand. The first (ORF2, accession number AJ276453) is located 212 bp upstream from bla_MOX-2 and encodes a putative 375-amino-acid protein 99% identical to a transposase (ISA1 type) of the IS4 family. Nine copies of this ORF are found on the chromosome of Aeromonas media strain WS (accession number CP007567) and on the multiresistance-encoding plasmid pP2G1 from an Aeromonas sp. (accession number HE616910). Two other ORFs are located upstream from ISA1 and encode two hypothetical proteins of 150 and 129 amino acids, respectively; they are organized in tandem in many plasmids from Enterobacteriaceae, Vibrio spp., and Aeromonas spp. A 612-bp ORF lacking 5′ sequences was identified 1,010 bp upstream from ORF3. It encodes a 204-amino-acid product 100% identical to the putative transposase A of IS186 of E. coli K-12 (accession number X03123).

FIG 2.

Genetic context of bla_MOX-2 beta-lactamase genes.

Exploration of the genetic environment of bla_FOX-3 of Kp8 revealed the presence of ISApu2 upstream from bla_FOX-3 (Fig. 3). The genetic organization was different from that of the previously published genes bla_FOX-1, bla_FOX-5, bla_FOX-6, and bla_FOX-7 (accession numbers X77455, CP007732 and DQ478715, AY034848, and HG934082, respectively), suggesting different recombination events, although the insertion sequence ISApu2 also seems to play a role in the mobilization of FOX-1 and FOX-7. We noted the presence of a repeat sequence of unknown function (TGCTAA) upstream from bla_FOX-3. Using PCR mapping and sequencing, the genetic environment of bla_FOX-3 in the other strains carrying this gene was found to be the same. To our knowledge, this is the first report of the genetic environment of bla_FOX-3.

FIG 3.

Genetic context of bla_FOX-3 beta-lactamase genes.

In six isolates (Ec6, Pm1, Kp10, Pm9, Ec5, and Ec11) of eight in the present study which carried bla_CMY, at least part of ISEcp1 was present upstream from the bla_CMY-2-like gene (Fig. 4). The region between ISEcp1 and the bla_CMY-2-like gene had the same structure in only five isolates (Table 2; Fig. 4).

FIG 4.

Genetic context of bla_CMY-2-type beta-lactamase genes.

Our findings are consistent with those concerning the genetic environment of bla_CMY-2-type genes described previously in which ISEcp1 is present (42). It was suggested that ISEcp1 could be involved in mobilizing the bla_CMY-2-like gene from the C. freundii chromosome to plasmids (52, 53).

All plasmids carrying bla_CMY described here shared various structural similarities with the 13-kb type I structure described in Salmonella enterica serovar Newport (accession number DQ164214) (Fig. 4) in which bla_CMY-2 is duplicated. The second copy is adjacent to a partial copy of ISEcp1 in the opposite orientation (53). Downstream from the bla_CMY-2-like gene in this 13-kb type I structure, several ORFs were detected between the two copies of bla_CMY-2 (53). Very similar structures were found in isolates Ec6, Pm1, Pm9, and Kp10. However, no isolate harbored two copies of the bla_CMY-2-like gene. For Ec6, Pm1, Kp10, and Pm9, the sequence either downstream from traC or downstream from orf1 remains unknown (Fig. 4). In Ec5, as opposed to Ec6, Pm1, Pm9, and Kp10, there were fewer similarities with the 13-kb type I structure. Downstream from the bla_CMY-2-like gene were blc, sugE, ecnR, and orf1 only, with a truncated yafA adjacent in reverse orientation, followed by yafB. Both yaf genes were part of a segment which is 98% identical to a region of S. enterica plasmid pNF1358 (accession number DQ017661) (Fig. 4). This segment is located directly upstream from ISEcp1−bla_CMY-2 on pNF1358. In Ec5, truncation of ISEcp1 was due to the insertion of IS1294 in the reverse orientation. 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 segment comprising the repY and repZ genes involved in replication initiation is 95% identical to a region of S. enterica pNF1358. In Ec11, only ISEcp1 was present upstream from the bla_CMY-2-like gene, while the rest of the structure remains unknown, suggesting a new genetic environment.

The results of the present study are consistent with those of Barlow and Hall with respect to Ec6, Pm1, Kp10, Pm9, Ec5, and Ec11, in which ISEcp1 could be involved in the mobilization of the bla_CMY-2-like gene, irrespective of the replicon type and the plasmid backbone (54).

According to Hopkins et al., each replicon type reflects the genetic organization surrounding the bla_CMY-2-like gene, with several minor rearrangements, including small insertions or deletions (55). Our findings are not consistent with those of Hopkins et al., since we found in our collection diverse plasmids carrying bla_CMY-2-like genes in which the structure of the genetic environment of bla_CMY is different even in cases of the same replicon type. In Ec7, bla_CMY was carried on an IncQ plasmid. This is the first observation of the gene on such an extremely rare plasmid. The study of the genetic environment of bla_CMY carried on IncQ by mapping PCR was negative, suggesting a new genetic structure.