Carbapenem-resistant Pseudomonas aeruginosa strains from a Spanish hospital: Characterization of metallo-beta-lactamases, porin OprD and integrons

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Abstract

Molecular typing and mechanisms of carbapenem resistance such as alterations in porin OprD and presence of metallo-beta-lactamases (MBLs), as well as integrons have been studied in a collection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Spanish hospital. One hundred and twenty-three CRPA isolates were recovered from different samples of 80 patients. Clonal relationship among CRPA was analyzed by SpeI-PFGE. Susceptibility testing to 11 antibiotics and MBL phenotype was determined by microdilution, IP/IPI E-test and double disc method. The oprD gene was studied by PCR and sequencing, and mutations were determined comparing with P. aeruginosa PAO1 sequence. Characterization of MBLs, and class 1 and 2 integrons were studied by PCR and sequencing. SDS-PAGE analysis of outer membrane proteins of selected strains was performed.

Seventy-four-per-cent of patients with CRPA were hospitalised in the ICU setting and 50% had long hospitalization stays. Sixty-four different PFGE patterns were detected, and 87 CRPA strains were further analyzed. MBL phenotype was detected in 43 of 87 strains (49.4%), which contained blaVIM-2 gene inside class 1 integrons. VIM-2-producing strains belonged to lineages ST175, ST235, and ST973. A great diversity of nucleotide insertions, deletions, and mutations in oprD gene, and the presence of a new insertion sequence (ISPa45) truncating oprD were identified among CRPA strains. Class 1 integrons were detected in 75% of CRPA strains, blaVIM-2 and the new arrangement aac(3)-Ia+ISPa34+aadA1 (named as In661) being the most frequent gene-cassette arrays detected. Other gene cassettes detected in integrons were: aadB, aadA6, aadA7, aac(6′)-Ib′, and blaOXA-46.

Introduction

Pseudomonas aeruginosa is a common opportunistic and nosocomial pathogen that causes severe infections with a high mortality rate, especially in immunocompromised patients or those with underlying diseases (Poole, 2011). Multidrug resistant (MDR) P. aeruginosa isolates, resistant to almost all beta-lactams, aminoglycosides and quinolones, often ascribed to epidemic clones (ST235 or ST111), have been detected in hospitals worldwide, mainly within intensive care units (ICU) (Woodford et al., 2011, Paterson, 2006). The increasing prevalence of MDR P. aeruginosa isolates is a global health problem, because of the limitation in clinical treatment options.

Nowadays, intensive clinical use of carbapenems has caused the presence of carbapenem heteroresistant P. aeruginosa subpopulations (Pournaras et al., 2007, Oikonomou et al., 2011) and an increase in carbapenem resistance by acquisition of different mechanisms, such as hyperproduction of chromosomal AmpC beta-lactamase, overexpression of efflux systems, alteration or lack of outer membrane proteins (such as porin OprD), and production of carbapenemases (Lister et al., 2009).

Porin OprD is a 443 amino acid protein composed by a 16 strand transmembrane beta-barrel structure connected with 7 short periplasmic turn sequences and 8 loops on the external surface (Huang et al., 1995, Ochs et al., 2000). The P. aeruginosa protein OprD is a substrate-specific porin that facilitates the diffusion of basic amino acids, small peptides and carbapenems into the cell. The alterations or loss of OprD significantly decreases the susceptibility to available carbapenems in P. aeruginosa (Lister et al., 2009).

The most significant carbapenemases acquired by P. aeruginosa are the zinc-dependent metallo-beta-lactamases (MBLs) that are capable of hydrolyzing imipenem, and extended-spectrum cephalosporins, but not aztreonam, and are inhibited by the EDTA chelator. The most important types of MBLs for epidemiological dissemination and clinical relevance are: IMP, VIM, SPM and currently NDM types (Cornaglia et al., 2011, Poole, 2011). Nevertheless, the MBLs most frequently detected worldwide in P. aeruginosa are IMP and VIM types that have been reported within mobilizable elements such as integrons (Partridge et al., 2009, Walsh et al., 2005). Integrons are genetic elements able to capture, integrate and express gene cassettes. The presence of genes encoding VIM enzymes, especially VIM-2, included inside integron structures has been reported in clinical MDR P. aeruginosa isolates, however the prevalence reported in Spanish hospitals is still low (Gutiérrez et al., 2007).

In this study, a collection of carbapenem-resistant P. aeruginosa (CRPA) isolates from a Spanish hospital has been studied in order to determine their molecular typing, and to characterize the carbapenemase genes and integrons, as well as the alterations in porin OprD.

Section snippets

Bacterial isolates

One hundred and twenty-three CRPA isolates were recovered from 80 patients in the Hospital Clínico Universitario Lozano Blesa (Zaragoza, Spain) during June 2008 to October 2010. These CRPA isolates were recovered from different types of samples [surgical wound, 33; urine, 28; bronchial aspirate, 22; skin lesion, 13; blood, 9; abscess, 5; tracheal aspirate, 4; sputum, 3; peritoneal fluid, 3, and others, 3].

Molecular typing

The clonal relationship among CRPA isolates was evaluated by PFGE using SpeI enzyme for

Clinical and epidemiological data

The characteristics of the 80 patients (55 males and 25 females) who carried the 123 CRPA isolates are shown in Table 1. The average age of patients was 64 years. Most of patients with CRPA isolates (74%) were hospitalised in the ICU setting, and 50% of total patients were hospitalised over 30 days (Table 1).

Beta-lactams, fluoroquinolones, aminoglycosides and colistin were the antibiotics most commonly used in the previous 30 days of CRPA recovery (Table 1). Beta-lactams were used in 65

Characterization of protein OprD

The oprD gene was amplified in 82 of 87 CRPA strains, and their deduced OprD amino acid sequences (443 amino acids) were compared with the OprD sequence of P. aeruginosa PAO1. Nucleotide insertions, deletions, mutations and the presence of insertion sequences truncating the oprD gene were obtained among the strains. Table 2 shows the high polymorphism detected in OprD amino acid sequence of the 82 CRPA strains, being higher among the non-MBL-producing strains.

The most frequent amino acid

Discussion

The prevalence of CRPA isolates has increased in Spain from 13.8% in 2008 to 17.8% in 2010 (http://ecdc.europa.eu/en/activities/surveillance/EARS-Net/database/Pages/database.as). However, the CRPA frequency in the studied hospital was 12.8% in the period 2008–2010, being lower than the upward trend (23%) observed in a Spanish multicenter study (Peña et al., 2012). The high percentage of co-resistance to carbapenems and aminoglycosides is relevant among the studied CRPA, highlighting the

Acknowledgments

We thank Patricia Siguier for analysing the ISPa45 sequence (http://www-is.biotoul.fr/).

V. Estepa has a pre-doctoral fellowship from the Universidad de La Rioja, Spain (grant number FPI-UR-09/16599009). M. de Toro has a pre-doctoral fellowship from the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, Spain (grant number FI08/00506). S. Somalo has a contract associated with project SAF2009-08570. This work was partially supported by Instituto de Salud Carlos III of Spain (FIS

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