Spread of Plasmids Carrying Multiple GES Variants

Abstract

Five GES-producing Enterobacteriaceae isolates that displayed an extended-spectrum β-lactamase (ESBL) phenotype harbored two GES variants: GES-7 ESBL and GES-6 carbapenemase. In all isolates, the two GES alleles were located on the same integron that was inserted into an 80-kb IncM1 self-conjugative plasmid. Whole-genome sequencing suggested in vivo horizontal gene transfer of the plasmid along with clonal diffusion of Enterobacter cloacae. To our knowledge, this is the first description in Europe of clustered Enterobacteriaceae isolates carrying two GES β-lactamases, of which one has extended activity toward carbapenems.

TEXT

GES-type extended-spectrum β-lactamases (ESBLs) are increasingly reported in Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae (1, 2, 3). More than 27 GES variants have now been identified throughout the world (3). One feature of these enzymes is that they may modify their spectrum of hydrolysis by point mutations. GES-2 was the first described example of an ESBL that extended its spectrum of activity against carbapenems through a single point mutation (4). To date, at least 12 variants (GES-2, -4, -5, -6, -13, -14, -15, -16, -18, -20, -21, and -24) possess a substitution at position 170 and are theoretically able to hydrolyze carbapenems. Some variants also have the ability to hydrolyze cefoxitin (GES-4, GES-5, GES-6, and GES-11) and/or aztreonam (GES-9 and GES-14) (3). The bla_GES genes have been essentially described as gene cassettes associated with class 1 or class 3 integrons on plasmids with different types of replicases (3, 5).

Here, we describe enterobacterial isolates recovered from a Belgian hospital that harbored two variants of bla_GES genes that were inserted back to back in the same integron, one coding for an ESBL and the second one coding for a carbapenemase.

In May 2007, an ESBL-producing Enterobacter cloacae isolate (EB-1) was isolated from the stool sample of a two-year-old girl who was hospitalized in the pediatric surgical ward of a Belgian hospital for a follow-up after a second liver transplantation. This patient underwent a first transplantation in Israel in January 2006 and was transferred to Belgium for a second transplantation. During initial hospitalization, all of the stool samples obtained for screening of ESBL carriage remained negative. The child left the hospital in May 2006 and did not travel until rehospitalization in May 2007. One week after the first case, an ESBL-producing Citrobacter youngae isolate (EB-2), displaying a similar resistance pattern, was cultured from the urine sample of a 1-year-old girl who was hospitalized for acute pyelonephritis in the same ward. A third child, a 3-year-old boy transferred from Macedonia for a liver transplant in November 2007, was negative for ESBL carriage at admission. He was found to be colonized with ESBL-producing Enterobacteriaceae in December 2007, first with an E. cloacae isolate (EB-3) and then with another E. cloacae isolate (EB-4) and a K. pneumoniae isolate (EB-5).

Antibiograms performed by disk diffusion and interpreted according to EUCAST guidelines (http://www.eucast.org) revealed that all isolates (EB-1 to EB-5) were resistant to penicillins, cephalothin, and ceftazidime but remained susceptible to cefotaxime, cefepime, aztreonam, and imipenem. Moreover, synergy images were demonstrated between clavulanate and third/fourth-generation cephalosporins, suggesting the presence of an ESBL. Etests revealed that the MICs for aztreonam were in the intermediate range and that the MICs of carbapenems were in the susceptible range (imipenem, MICs ≤ 0.5 μg/μl; ertapenem, MICs ≤ 0.047 μg/μl; and meropenem, MICs ≤ 0.047 μg/μl). In addition, the five isolates were resistant to all aminoglycosides, except gentamicin, and to co-trimoxazole but remained susceptible to fluoroquinolones, tigecycline, and colistin (data not shown).

Whole-cell DNA extraction and ESBL- and carbapenemase-specific PCR, as previously described (6), revealed that the five isolates possessed only the bla_GES gene. However, sequencing of the PCR products on the two strands using an ABI 3100 automated Sanger sequencer (Applied Biosystems, Les Ulis, France) revealed a double peak (A or G) at position 508 in all five isolates. At this position, an AGC codon codes for Ser at Ambler's position 170 and corresponds to the GES-6 variant whereas a GGC codon codes for Gly and corresponds to a GES-7 variant.

Kieser's extraction of natural plasmids revealed that all of the isolates possessed at least a ca. 80-kb plasmid that could be transferred by conjugation into E. coli as previously described (6, 7). Transconjugants expressed resistance patterns similar to those of the parental strains. PCR experiments confirmed the presence of the bla_GES gene in the transconjugants, and sequencing of the PCR products revealed the same double peak at position 508, suggesting the presence of two copies of the bla_GES gene on the same plasmid.

The entire genomes of E. cloacae EB-1, EB-3, and EB-4 and of K. pneumoniae EB-5 along with the natural plasmids pEB-1 and pEB-2 extracted from E. coli transconjugants were sequenced using the Nextera XT v3 kit (Illumina, San Diego, CA, USA) according to the manufacturer's recommendations and then run on MiSeq (Illumina) to generate paired-end 300-bp reads (6, 8). De novo assembly was performed by CLC Genomics Workbench v7.0.4 (Qiagen, Hilden, Germany). The acquired antimicrobial resistance genes and multilocus sequence types (MLSTs) were identified by uploading assembled genomes to the ResFinder server v2.1 (http://cge.cbs.dtu.dk/services/ResFinder-2.1/) and MLST 1.8 (https://cge.cbs.dtu.dk/services/MLST/), respectively. The genome was annotated using the RAST server (9).

Plasmid pEB-1 was constructed and confirmed the presence of two copies of the bla_GES gene, bla_GES-6 and bla_GES-7 (Fig. 1). The two bla_GES genes were embedded together in a gene cassette array of a class 1 integron, along with four other gene cassettes, aacA4, smr2, dfrA1, and aadA1. The 3′ conserved sequence was composed by the qacE and sul1 genes as frequently described (10). The gene aacA4 encodes a 6′-N-aminoglycoside acetyltransferase [AAC(6′)-I] conferring resistance to amikacin, tobramycin, and netilmicin but not to gentamicin, and dfrA1 and sul1 are responsible for trimethoprim and sulfonamide resistances, respectively, which is consistent with the observed phenotype of all isolates.

FIG 1.

Schematic representation of plasmid pEB-1 carrying bla_GES-6 and bla_GES-7 genes and comparison with four other IncL/M plasmids harboring various β-lactamases, pACM-1 (GenBank accession number KJ541681), pEL60 (GenBank accession number AY422214), pNDM-OM (GenBank accession number JX988621), and pOXA-48a (GenBank accession number JN626286). White boxes indicate plasmid scaffold regions that are common to all of the plasmids or are of unknown function. Resistance genes are indicated by gray boxes, except for the β-lactamase genes, which are indicated by black boxes. Transposon-related genes (tnpA, tnpR, and tnpM), insertion sequences, and integrase genes are indicated by hatched boxes. Replicase genes are indicated by boxes with vertical lines. Genes encoding mobilization and partition systems are indicated by dotted boxes.

Plasmid pEB-1 was 78,907 bp in size and belonged to the IncM1 subgroup according to the IncRNA classification scheme, with 100%, 98%, and 98% IncRNA nucleotide sequence identity with pACM1, R69, and pFOX-7a, respectively (11–14). This plasmid contained open reading frames (ORFs) including genes involved in replication, mobilization, partitioning, and conjugation. Overall, the backbone of pEB-1 was similar to that of the IncL/M family plasmids described previously, e.g., pEL60 or pNDM-OM (Fig. 1), with the exception of the integration site of the resistance gene array (13).

Sequences of the other natural plasmids revealed a high degree of identity, except for pEB-4, which has another mercury operon inserted, while the rest of the plasmid was entirely identical. Semiautomated repetitive extragenic palindromic PCR (rep-PCR) typing (DiversiLab; bioMérieux) of the E. cloacae isolates (EB-1, EB-3, and EB-4) revealed that EB-1 and EB-3 were related (more than 95% identity) whereas EB-4 was unrelated to the two other strains (Table 1). These results were confirmed by MLST using the next-generation sequencing (NGS) results of each entire genome (Table 1). Thus, our results strongly suggest clonal spread of E. cloacae along with plasmid diffusion.

TABLE 1.

Clinical data of the patients with GES-producing Enterobacteriaceae

Patient	Country of origin	Age (yr)	Sex	Date of hospitalization (mo/yr)	Underlying disease	Infection	Previous antimicrobial treatment(s)	Isolate	Date of isolation of GES isolate (mo/yr)	Site of isolation	Plasmid	rep-PCRa	MLSTb
A	Israel	2	F	05/2007	Liver transplantation	Pseudomonas aeruginosa pneumonia	Co-trimoxazole ceftazidime, colistin	Enterobacter cloacae EB-1	05/2007	Stool	pEB-1	A	ST-346
B	Belgium	1	F	04/2007	Congenital gastroesophageal reflux	Urinary tract infection (acute pyelonephritis)	Cefuroxime + amikacin	Citrobacter youngae EB-2	05/2007	Urine	pEB-2 = pEB-1c		ND
C	Macedonia	3	M	11/2007	Liver transplantation	Fever of unknown origin post liver graft	Ampicillin, temocillin, co-trimoxazole, fluconazole	Enterobacter cloacae EB-3	12/2007	Stool	pEB-3 = pEB-1c	A	ST-346
								Enterobacter cloacae EB-4	01/2008	Stool	pEB-4 = pEB-1 ins Merd	B	ST-68
								Klebsiella pneumoniae EB-5	01/2008	Stool	pEB-5 = pEB-1Δ152bpe		ST-318

^aRep-PCR results were obtained for the three E. cloacae isolates with the semiautomated DiversiLab system (bioMérieux).

^bND, not done. Since isolate did not regrow, however, the plasmid sequence was obtained from an E. coli transformant.

^cThese plasmids are 100% identical to pEB-1.

^dpEB-1 ins Mer is 100% identical to pEB-1, except for the mercury transposon that was replaced with that of pSF088-1.

^epEB-1Δ152bp is 100% identical to pEB-1, except for a 152-bp deletion between nucleotide positions 8823 and 8975 compared to pEB-1.

GES variants have already been described in association with other β-lactamases, such as carbapenemases like VIM, IMP, or OXA-23, especially in P. aeruginosa and in A. baumannii isolates (15–17). GES-1 and GES-5 were the two first GES variants identified in an epidemic P. aeruginosa clone ST235 in Spain (18). Another study highlighted the presence of GES-19 and GES-20 in K. pneumoniae and E. coli isolates from Mexico (19).

Molecular techniques such as PCR are not sufficient to identify GES carbapenemases, as sequencing of the entire gene is necessary. Only biochemical methods that address carbapenem hydrolysis may differentiate ESBL from carbapenemase variants (20). However, several studies report false-negative results from detection tests with GES-5- and GES-6-producing isolates (20–22).

To the best of our knowledge, this is the first description in Europe of clustered Enterobacteriaceae isolates carrying two GES β-lactamases, with one an ESBL and the other one a carbapenemase.

Nucleotide sequence accession number.

The nucleotide sequence of the pEB-1 plasmid has been deposited in GenBank under accession number KX230795.

Funding Statement

This work, including the efforts of Thierry Naas, was funded by University Paris 11 (EA7361), by LabEx LERMIT (ANR-10-LABX-33), and by the European Commission (EC) (MAGIC-BULLET FP7/HEALTH-F3-2001-27823). This work, including the efforts of Youri Glupczynski, was funded by Fondation Mont-Godinne.