A Plasmid-Encoded Class 1 Integron Contains GES-Type Extended-Spectrum β-Lactamases in Enterobacteriaceae Clinical Isolates in Mexico

LETTER

Plasmid-located extended-spectrum-β-lactamase (ESBL) genes are mostly found in Enterobacteriaceae (6). A new class A ESBL was identified in Klebsiella pneumoniae. It was named GES-1, and it corresponds to the ceftazidime-hydrolyzing enzyme (8). GES-type ESBLs have emerged in a variety of countries, and there are 18 known variants (http://www.lahey.org/Studies/). In the present study, we investigated the prevalence of GES-type β-lactamases in ESBL-producing Enterobacteriaceae clinical isolates; two new alleles (GES-19 and GES-20) were identified in a plasmid-encoded class 1 integron (In724).

(This work was presented in part as an abstract at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy, abstract C1-1210, 2011.)

Between March 2005 and June 2009, 578 ESBL-producing Enterobacteriaceae clinical isolates were collected from 11 Mexican hospitals. All isolates were screened for the presence of β-lactamases from the GES family by means of PCR, using generic primers (14). Among the 578 ESBL-producing Enterobacteriaceae isolates studied, 8 (1.3%) contained the GES-type gene and were distributed as follows: 1/5 K. oxytoca isolates, 5/137 K. pneumoniae isolates, and 2/404 Escherichia coli isolates; no GES-positive (0/32) E. cloacae isolates were identified (Table 1). For GES-type-positive isolates, antibiotic susceptibility testing was carried out by broth microdilution, following CLSI recommendations (2). All isolates turned out to be resistant to ceftazidime, cefotaxime, piperacillin, and ciprofloxacin. Three isolates turned out to be resistant to gentamicin and two to imipenem and meropenem (Table 1). Genomic DNA was analyzed (4, 11), and it revealed a nongenetic relationship between the GES-positive isolates (data not shown).

Table 1.

Molecular characteristics of GES-positive isolates and transconjugants^a

Strainb	Isolation date (mo/day/yr)	Hospitalc	Ward	Origin	Plasmid size(s) (kb)	Incompatibility group	PMQR gene	ESBL gene type(s)		MIC (mg/ml)
								Non-GES	GES	CAZ	CAZ/CLA	CTX	PIP	PIP/TAZ	CPO	IMP	IMP/CLA	MER	Gm
K01201	11/27/2006	1	Hematology	Blood	220, 140, 90, 40	NA	qnrA1/qnrS1/aac (6′)-Ib-cr	SHV-11	19, 20	256	128	>256	128	128	>32	1	0.25	0.125	8
TK01201	Not applicable	Not applicable	Not applicable	Not applicable	40	FIIs	qnrA1/aac(6′)-Ib-cr	Neg	19, 20	256	4	64	64	16	<0.002	1	0.25	0.125	2
K01239	10/10/2008	1	ICU	Wound	190, 160, 40	NA	Neg	SHV-11	19, 20	>256	>256	128	128	128	>32	32	16	8	16
TK01239	Not applicable	Not applicable	Not applicable	Not applicable	40	FIIs	Neg	Neg	19, 20	256	4	64	256	16	<0.002	0.5	0.125	0.03	2
K01256	09/22/2006	1	OR	Wound	190, 120, 110, 40	NA	Neg	SHV-26	19, 20	>256	>256	256	512	512	32	16	16	32	4
TK01256	Not applicable	Not applicable	Not applicable	Not applicable	40	FIIs	Neg	SHV-26	19, 20	>256	16	64	128	16	<0.002	0.5	0.125	0.03	2
K01295	04/01/2009	1	OR	Catheter	200, 100, 40	NA	qnrA1	SHV-1	19, 20	256	8	32	128	256	>32	1	0.25	0.125	8
TK01295	Not applicable	Not applicable	Not applicable	Not applicable	40	FIIs	qnrA1	Neg	19, 20	256	8	64	256	128	<0.002	0.5	0.125	0.03	2
K06220	09/14/10	2	ICU	Urine	180, 100, 50, 40	NA	qnrS1/aac(6′)-Ib-cr	SHV-5	19, 20	64	16	64	512	256	>32	1	0.5	0.5	>64
TK06220	Not applicable	Not applicable	Not applicable	Not applicable	50, 40	FIIsd	aac(6′)-Ib-cr	SHV-5	19, 20	64	4	32	128	8	<0.002	1	0.25	0.03	2
E01298	06/20/2009	1	Nephrology	Urine	110, 40	NA	aac(6′)-Ib-cr	CTX-M-15	19, 20	>256	8	>256	>512	64	>32	0.5	0.25	0.5	4
TE01298	Not applicable	Not applicable	Not applicable	Not applicable	40	FIIs	aac(6′)-Ib-cr	CTXM-15	19, 20	256	8	64	64	4	<0.002	0.5	0.125	0.03	2
E09280	03/10/2009	3	Hematology	Blood	190, 40	NA	aac(6′)-Ib-cr	Neg	19, 20	>256	16	128	32	8	>32	1	0.125	0.125	2
TE09280	Not applicable	Not applicable	Not applicable	Not applicable	40	FIIs	aac(6′)-Ib-cr	Neg	19, 20	256	8	64	32	8	<0.002	1	0.125	0.125	2
Kx09201	09/26/2007	3	Hematology	Blood	220, 190, 100, 80	NA	qnrB2	SHV-12/CTX-M-15	1	64	0.5	>256	>512	8	32	0.25	0.0625	0.015	64
TKx09201	Not applicable	Not applicable	Not applicable	Not applicable	80	FIIs	qnrB2	SHV-12/CTXM-15	1	32	1	>256	>512	8	<0.002	0.25	0.0625	0.015	32
E. coli J53-2	Not applicable	Not applicable	Not applicable	Not applicable	Not applicable	NA	Not applicable	Not applicable	Not applicable	0.5	0.25	0.0625	8	8	<0.002	0.125	0.0625	0.015	0.5

^aCAZ, ceftazidime; CLA, clavulanic acid; CTX, cefotaxime; PIP, piperacillin; TAZ, tazobactam; CPO, ciprofloxacin; IMP, imipenem; MER, meropenem; Gm, gentamicin; ICU, intensive care ward; NA, not analyzed; Neg, negative; OR, operating room.

^bK, K. pneumoniae; E, E. coli; Kx, K. oxytoca; T, transconjugant.

^cHospitals: 1, Hospital Civil de Guadalajara (HCG); 2, Hospital Universitario (CRCEI); 3, Instituto Nacional de Cancerología (INCan).

^dAdditional incompatibility group identified in transconjugants FII_y, FII_k, and IncR (see text).

GES-1 was identified in the K. oxytoca isolate (Kx09201). Whereas all K. pneumoniae and E. coli isolates contained ESBL GES-19 and carbapenemase GES-20, these proteins differed from GES-11 and GES-5 β-lactamases by the replacement of Ala by Gly at Ambler position 17 (leader peptide). The mating experiments (7, 9) showed that both GES-19 and GES-20 genes were transferred onto a 40-kb conjugative plasmid from K. pneumoniae and E. coli isolates and that GES-1 was transferred onto an 80-kb plasmid from K. oxytoca (Table 1). Plasmids were digested with XhoI and EcoRI restriction enzymes. The fingerprinting showed identical patterns among the 40-kb plasmids (data not shown). These data are in accordance with those corresponding to the FII_s incompatibility group identified in the plasmids. Moreover, members of incompatibility groups FII_y and FII_k and IncR were also identified in transconjugant TK06220, which contains an additional 50-kb plasmid (Table 1). The plasmid incompatibility groups were identified using recent PCR-based replicon typing (3, 13).

All GES-type alleles have been mainly described in class 1 integrons (15). The class 1 integron structure that encoded the GES-type alleles was determined using a PCR strategy with generic primers (1, 5, 14); in addition, GES-243F (5′-TGTGTTGTCGCCCATCTCCG-3′) and GES-104R (5′-ATGATCGTCGAATGGTCTCC-3′) were used to amplify the intergenic region between the two GES-type genes. All transconjugants harboring the 40-kb plasmid contained the class 1 integron with the following array: aacA42, bla_GES-19, and bla_GES-20 (tandem duplication) and aacA4′, bla_OXA-2, qacH4, and aadA1b (named In724). The nucleotide analysis showed the following characteristics. The bla_GES-19 gene is not followed by any attC recombination site; instead, there is a “TAAAACAAAGTTAG” fragment (2795 to 2908) that is a duplication at the end of the attI1 (1141 to 1154) region. This In724 class 1 integron is very similar to the one located on the Pseudomonas aeruginosa chromosome (In647) previously described (12). Interestingly, the intergenic region between the GES-type tandem duplications in both class 1 integrons is the same fragment (with the exception of a deleted A) which separates the two bla_GES genes in In647; this situation supports the idea that the In724 integron derives from In647 by variations in the bla_GES alleles (GES-19 and GES-20). Most likely, this duplication occurred via an insertion sequence (IS)-mediated event (8). Therefore, the plasmid-located integron facilitates the dissemination of integrons of these classes. On the other hand, the GES-1 gene encoding the class 1 integron showed the following structure: aacA4, bla_GES-1, qacF5, aacA4-18, and ΔaadA1, corresponding to a new class 1 structure called In725.

The GES-positive isolates were also screened for plasmid-mediated quinolone resistance (PMQR), as well as for SHV, CTX-M, and TLA-1 ESBL genes by the use of the respective primers (10). Our study showed that 6/8 GES-positive isolates contained at least one PMQR gene (Table 1). The GES alleles coexist with qnrA1, qnrB2, qnrS1, and aac(6′)-Ib-cr determinants. On the other hand, the bla_SHV-5, bla_SHV-12, and bla_CTX-M-15 genes coexist with the qnrB2, qnrS1, and aac(6′)-Ib-cr determinants.

The transconjugants encoding the SHV- and CTX-M-type ESBLs (TK01256, TK06220, TE01298, and TKx09201) showed a high drug MIC value (≥256) with respect to ceftazidime (TK01256 and TE01298) and cefotaxime (TKx09201). In terms of imipenem, the transconjugants carrying the GES-20 allele displayed a 1-to->3-fold MIC increase with respect to E. coli J53-2. These multiple-ESBL-containing isolates could be playing an important role in terms of cephalosporin resistance, and they might limit the therapeutic options when combined with PMQR genes.

Nucleotide sequence accession numbers.

The nucleotide sequence data reported in this paper appear in the GenBank/EMBL nucleotide database under accession numbers JN596279 (In725) and JN596280 (In724).