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The mechanisms responsible for the increasing prevalence of colistin-only-sensitive (COS) Pseudomonas aeruginosa isolates in a Spanish hospital were investigated. Pulsed-field gel electrophoresis revealed that 24 (50%) of the studied isolates belonged to the same clone, identified as the internationally spread sequence type 235 (ST235) through multilocus sequence typing. In addition to several mutational resistance mechanisms, an integron containing seven resistance determinants was detected. Remarkably, the extended-spectrum β-lactamase GES-1 and its Gly170Ser carbapenem-hydrolyzing derivative GES-5 were first documented to be encoded in a single integron. This work is the first to describe GES enzymes in Spain and adds them to the growing list of β-lactamases of concern (PER, VIM, and OXA) detected in ST235 clone isolates.
The growing threat of antimicrobial resistance in Pseudomonas aeruginosa is driven by the extraordinary capacity of this microorganism for developing resistance to almost any available antibiotic by the selection of mutations in chromosomal genes, in conjunction with the emergence and dissemination of transferable resistance determinants of high concern during the last decade (8, 10, 11, 14-16). Class B carbapenemases (or metallo-β-lactamases [MBLs]) and extended-spectrum β-lactamases (ESBLs), frequently encoded on integrons along with aminoglycoside-modifying enzymes, are likely the transferable resistance determinants currently having the highest impact on antimicrobial therapy in hospitals worldwide (2, 26, 30). The simultaneous detection of MBLs and ESBLs in the same clone (32) and the recent emergence of ESBLs with carbapenem-hydrolyzing activity, such as KPC and GES enzymes, add further complexity and concern (7, 23, 28).
The objective of this study was to determine the underlying mechanisms responsible for the increasing prevalence of P. aeruginosa clinical isolates showing resistance to all antipseudomonal agents except colistin (colistin-only-sensitive [COS] isolates) in a Spanish hospital (Hospital Universitario 12 de Octubre, Madrid).
A retrospective study of all patients colonized/infected by P. aeruginosa isolates resistant to penicillins, cephalosporins, and carbapenems during 2007 and 2008 was conducted. Identification and susceptibility testing were performed using semiautomatic microdilution Wider panels (Soria Melguizo, Spain) (3). The antibiotics tested were piperacillin-tazobactam (PTZ), ceftazidime (CAZ), cefepime (FEP), aztreonam (ATM), imipenem (IMP), meropenem (MER), ciprofloxacin (CIP), gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and colistin. Breakpoints were applied according to Clinical and Laboratory Standards Institute (CLSI) recommendations (4). Additionally, MICs for the same antibiotics were determined by Etest (AB Biodisk, Solna, Sweden) in selected strains.
During the 2-year study, a total of 1,182 patients were colonized/infected with P. aeruginosa, and 56 (4.7%) of them met the inclusion criteria. The proportion of resistant isolates in the second year of the study was significantly higher than that in the first year, 7.1% (41/580) versus 2.5% (15/602) (P < 0.001), respectively. β-Lactam-resistant isolates frequently showed coresistance to non-β-lactam antibiotics, and up to 65% of them were COS.
Genotyping, through pulsed-field gel electrophoresis (PFGE), revealed that 24 (50%) isolates were part of the same clone (A). The remaining 24 isolates showed a high clonal diversity (21 different PFGE patterns). Multilocus sequence typing (MLST) analysis was performed, following previously established protocols (6), in three isolates belonging to clone A, identified as sequence type 235 (ST235) according to the MLST database (http://pubmlst.org/paeruginosa).
Almost all (22/24) clone A isolates showed a COS phenotype characterized by resistance to PTZ, CAZ, FEP, IMP, MER, CIP, GEN, TOB, and AMK; only two clone A isolates maintained susceptibility against TOB and/or AMK. On the other hand, the susceptibility patterns of non-clone A isolates were much more variable.
The presence of MBLs was explored through phenotypic (Etest MBL) and genotypic (PCR and sequencing) approaches. While all clone A isolates were negative for MBL production, blaVIM-2 was detected in 9 of the 24 non-clone A isolates, each of them belonging to a different PFGE clonal type. Except for ATM (five/nine susceptible), VIM-2-producing isolates showed a conserved resistance profile that included all antibiotics except colistin (COS).
Clone A persisted at least from March 2007 to December 2008. There were no differences between clone A and non-clone A isolates in the temporal distribution. The comparison of clinical variables of patients infected/colonized by clone A or non-clone A isolates is shown in Table Table1.1. The analysis revealed a strong association with admission in the hematology department (33.3% versus 4.2%, P = 0.023) and, remarkably, higher mortality during hospitalization (41.7% versus 12.5%, P = 0.023) (Table (Table1).1). Nevertheless, further studies are needed in order to elucidate whether infection by this clone is an independent risk factor for poor prognosis. Patients with MBL-producing P. aeruginosa isolates were hospitalized in different departments, including surgery (three patients), internal medicine (three), pulmonology (two), and intensive care (one).
A deep analysis of the mutational and horizontally acquired resistance mechanisms was performed in three representative isolates from clone A. The mutational resistance mechanisms detected are summarized in Table Table2.2. The relative mRNA levels of the genes encoding the major P. aeruginosa efflux pumps (mexB, mexD, and mexY) and the chromosomal cephalosporinase (ampC) were determined by real-time PCR as previously described (13, 19). The presence of mutations in oprD, gyrA, gyrB, parC, and parE was investigated by PCR amplification and sequencing, following previously described protocols (1, 10). No significant changes were found in the expression of mexD or ampC genes with respect to that in PAO1, and the slightly increased expression of mexB and mexY suggests a limited impact (if any) on the resistance phenotype. On the other hand, high-level fluoroquinolone resistance was produced by GyrA Thr83Ile and ParC Ser87Leu mutations, while carbapenem resistance was, at least partially, a consequence of the disruption of oprD at nucleotide 629 by the insertion sequence IS1001.
Through isolectric focusing, performed as described elsewhere (12), two β-lactamase bands, of pIs 8.4 and 6, were detected. The identification of the involved β-lactamases was attempted through PCR amplification of genes encoding VIM-, IMP-, PER-, CTX-M-, SHV-, TEM-, OXA-, and PSE-type enzymes, using previously described primers and conditions (5, 10, 20). While the pI 8.4 band corresponded to OXA-2, the other β-lactamase could not be identified with the primers used. Therefore, cloning of the corresponding bla gene was attempted. An approximately 10-kb DNA fragment, obtained through BamHI digestion of genomic DNA from isolate PA-A1, was cloned into pUCP24 (31) to yield plasmid pA1, which was electroporated into PAO1, following previously described procedures (27). To explore the phenotypes of the cloned resistance determinants, MIC determinations were performed and the results are shown in Table Table2.2. Interestingly, the cloned fragment conferred resistance to aminoglycosides and all tested β-lactams, including piperacillin, PTZ, CAZ, FEP, ATM, IMP (intermediate resistance), and MER. The MICs of IMP and MER were not modified in the presence of clavulanic acid (4 μg/ml).
Sequencing of the cloned DNA fragment revealed the presence of a complete integron structure, represented in Fig. Fig.1.1. The integron contained up to seven resistance determinants, including three genes encoding aminoglycoside-modifying enzymes and three β-lactamases. The genes encoding aminoglycoside-modifying enzymes were aacA4, aadA1, and a previously undescribed aac variant designated aac(6′)-33, showing 92% identity to aac(6′)-I30 (17). One of the β-lactamase genes coded for the already-detected narrow-spectrum OXA-2, but remarkably, the other two coded for the ESBLs GES-1 and GES-5. PCR analysis confirmed that the structure of the integron was conserved among the three isolates of clone A studied.
The potential plasmid location of the integron was initially evaluated through transformation assays. For this purpose, plasmid DNA (Genopure plasmid maxikit [Roche Diagnostics, Mannheim, Germany]) was introduced by electroporation into PAO1, and potential transformants were selected in 16 μg/ml CAZ or 200 μg/ml carbenicillin-LB agar plates. Since several attempts at electroporation failed, Southern blot hybridization with blaGES PCR probes was performed with chromosomal and plasmid DNA, using the ECL kit (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Results showed hybridization with chromosomal but not plasmid DNA (not shown). Therefore, the blaGES integron was apparently located in the chromosome of clone A, in contrast to other previous works in which a plasmid location was revealed (22, 23, 25).
Particularly noteworthy is the first detection of the ESBL GES-1 and its Gly170Ser weakly clavulanate-inhibited carbapenem-hydrolyzing derivative GES-5 (29) simultaneously encoded in the same integron. A close analysis of the nucleotide sequences suggested that blaGES-5 could have originated through a tandem duplication of blaGES-1 occurring between two 5-nucleotide (ACAAA) direct repeats (located at the end of the attI region [upstream of blaGES-1] and at the beginning of the aacA4 cassette [downstream of blaGES-1]), followed by the selection of the Gly170Ser mutation and the acquisition of the last cassette of the integron [aac(6′)-33] (Fig. (Fig.1).1). Although the alternative explanation (independent acquisition of the two blaGES cassettes) cannot be completely ruled out, the presence of the ACAAA sequence between blaGES-1 and blaGES-5 supports the former but not the latter hypothesis. Interestingly, Picão et al. (21) have recently reported the independent detection of blaGES-1 or blaGES-5 in a P. aeruginosa clone from Brazil, possibly representing a further example of in vivo evolution of GES-1 to GES-5.
GES-1 was first isolated from a Klebsiella pneumoniae strain recovered from a patient hospitalized in French Guiana (GES stands for Guiana ESBL) in 1998 (22). Several variants have been identified afterwards, some of them, such as GES-2, GES-4, GES-5, or GES-6, characterized by their extended spectrum toward carbapenems (24, 26). These enzymes have been detected in Enterobacteriaceae (K. pneumoniae, Enterobacter cloacae, or Escherichia coli) and P. aeruginosa, with a growing geographical dissemination (including South Africa, South Korea, France, Greece, Canada, China, and Brazil) in the last few years (18, 21, 25, 26). This is the first report of this type of β-lactamase in Spain.
MLST analysis identified clone A as ST235. Interestingly, this clone, belonging to the BG11 complex, has been recently found to be very widespread, since it has been detected in Turkey, Greece, Italy, Hungary, Poland, Sweden, and Russia (9). Remarkably, ST235 has been previously associated with PER, OXA, and VIM enzymes; our work adds GES ESBLs to this growing and significant list.
Indeed, this work emphasizes the increasing global threat of COS P. aeruginosa. Certainly, a deep understanding of the genetic mechanisms involved and their horizontal and longitudinal dissemination, on both global and local scales, is critical for implementing strategies for the prevention, detection, and control of nosocomial infections by COS P. aeruginosa strains.
The nucleotide sequences described in this work have been deposited in the GenBank database under the accession number GQ337064.
This work was supported by the Ministerio de Educación y Ciencia of Spain, Instituto de Salud Carlos III, through the Spanish Network for the Research in Infectious Diseases (REIPI C03/14 and RD06/0008) and SAF2006-8154.
Published ahead of print on 8 September 2009.