Prevalence and Molecular Epidemiology of Imipenem-Resistant Pseudomonas aeruginosa Carrying Metallo-Beta-Lactamases from Two Central Hospitals in Portugal

Introduction

Carbapenems, a class of beta-lactams, are the most potent antimicrobial agents for the treatment of Pseudomonas aeruginosa infections. ⁴ Since their introduction in the clinical use, strains expressing metallo-beta-lactamases (MBLs) have been reported from around the world and in the last decade have emerged particularly in P. aeruginosa. This pathogen is one of the most prevalent nosocomial infection agents, mainly associated to septicemias and respiratory infections in immunocompromised patients, and is sometimes responsible for hospital outbreaks associated with high mortality. ¹⁰

MBLs hydrolyze all beta-lactams, but monobactams. ⁴ Several MBLs have been reported in P. aeruginosa, including the IMP and VIM families, SPM-1, GIM-1, SIM-1, AIM-1, KHM-1, NDM-1, and DIM-1, where in particular, VIM-2 has emerged as a dominant MBL variant worldwide. ⁴ A first description of VIM-2 (2002) and further studies dealing with its spread have demonstrated that VIM-2 is the only MBL in Portugal.^8,11,12 VIM genes (bla_VIM) are often encoded on mobile gene cassettes inserted in class 1 integrons, promoting their spread.^2,4

The aim of this work was to document the occurrence of MBLs, as well as integrons, in imipenem (IP)-resistant P. aeruginosa isolates (IRPA) from two central hospitals in central Portugal, to understand if intra- or interhospital clonal spread has occurred and to optimize the therapeutics in patients carrying P. aeruginosa MBL producers.

Materials and Methods

Results

The 91 P. aeruginosa isolates collected from H1 were distributed in HP, PH, and in eight different wards from CH: HP (5.8%), PH (11.3%), internal medicine (16.8%), surgery (14.6%), neurology (13.5%), pneumology (12.5%), neurosurgery (7.9%), infectious diseases (6.9%), emergency room (5.9%), and intensive care unit (ICU) (4.8%). In H2, the 31 isolates were collected from 9 wards: surgery (19.3%), liver transplant unit (16.1%), internal medicine (9.6%), neurotraumatology (12.9%), ICU (12.9%), orthopedic (9.7%), cardiology (6.5%), infectious diseases (6.5%), and emergency room (6.5%). Regarding clinical specimens, 44.3% isolates were from bronchial secretions, 27% from urine, 17.2% from wounds, 4.1% from blood, and 7.4% from other origins.

Susceptibility to beta-lactams of all IRPA was 79.1% for CAZ, 76.9% for CEP, 70.3% for PIP, 34.1% for AZT, and 23.1% for MP. The aminoglycoside AMK showed good activity (96.7%), but the fluoroquinolone CIP had a diminished activity (25.3%).

The combined double-disk test screened for 50 MBL-positive results: 32 from H1 and 18 from H2. An 815 bp amplicon, corresponding to bla_VIM, was detected in 25 of those 50 MBL: 12 from H1 and 13 from H2. Obtained amplicons were interpreted by Blast analysis and all regarded a VIM-2-type gene. No amplification was detected with other MBL primers. Overall, 20.5% of the isolates presented VIM-2. MICs of beta-lactams were determined on those isolates showing that 84% were resistant to MP, 72% to CAZ, 28% to PIP, and 16% to AZT (Table 1).

Class 1 integrons were detected in 10 of the 25 VIM-2 isolates (40%): four from H1, where two had 3,000 bp integrons, one isolate presented two integrons, one with 3,000 bp and another with 1,000 bp, and the fourth isolate had a 2,000 bp integron. In H2 isolates, two of the six isolates presented a single integron of 3,000 bp. The other four isolates had two integrons, one with 3,000 bp and the other with 1,000 bp. Only two integrons, with 3,000 bp, contained the VIM-2 gene, one from H1 and another from H2. When their sequences were compared by the megablast alignment tool, we observed that the integrons were different. Other integrons, without VIM-2, from H2 were similar (100% identical) with each other, and contained aminoglycoside resistance genes (aacA4 and aacA7). H1 integrons were different from each other and from those of H2. These integrons also contained aacA4 and aacA7 genes.

RAPD analysis of the isolates harboring VIM-2 showed 14 different patterns (table 1). A to G profiles were attributed to isolates from H2 and were spread through eight different wards. Pattern A was represented by six isolates, collected from three wards, and these isolates harbored all the class 1 integrons identified in H2. H to N profiles belonged to H1 and were distributed through six wards from CH, PH, and HP. Five of the 12 H1 VIM-2 producing isolates were collected from two wards of PH and presented the same profile H. Three of the four integrons observed in H1, belonged to this profile. L, M, and N profiles were obtained from different urine isolates of the CH Internal Medicine ward, showing the great variability between strains in the same ward. The M isolate contained the other integron detected in H1.

Discussion

P. aeruginosa has clearly emerged as a leading nosocomial pathogen, because of its ubiquitous nature and its ability to acquire resistance to several antimicrobial agents under drug exposure. Carbapenems, mainly IP and MP, are good agents to treat infections caused by multidrug resistance (MDR) P. aeruginosa, however, the prevalence of carbapenem-resistant P. aeruginosa has been increasing over the years due to the loss of OprD porin, AmpC overexpression, efflux pumps, and also by MBL production. ¹⁸

In the present study, the overall MBL prevalence in IRPA was relatively higher (20.5%) than in those reported in Italy (12.7%), Germany (11.7%), and Spain (6.8%),^13,14,16 which could be associated to isolates coming from patients with invasive devices, complicated infections, and long-term hospitalization. A recent study from Netherlands stated higher values of VIM-2 producers (33%), ¹⁷ and an older work from Greece pointed 62% VIM producers in nine hospitals. ⁶ In our study, VIM-2 IRPA isolates were highly resistant to MP (84%), but AZT and PIP displayed some activity (16% and 28%, respectively). Spanish, Greek, and Italian studies evidenced higher rates of antibiotic resistance, showing a MDR pattern, including CAZ, CEP, PIP/tazobactam, gentamicin, tobramycin, and quinolone resistance.^9,13–15 In Europe, bla_VIM-type determinants are more prevalent and widespread in P. aeruginosa than bla_IMP type. In the present study, molecular analysis only detected bla_VIM-2, the only MBL observed until now in Portugal, as well as the studies from Germany, Netherlands, and Greece.^6,16,17 IMP has only been detected in P. aeruginosa in Italy, France, and Spain.^7,13,14 These great differences in MBL prevalence, drug susceptibility patterns, and MBL content between different European countries are probably due to different antibiotherapy policies.

The MBL phenotypic screening yielded 50% of false-positive results since from 50 phenotypic-positive isolates, only 25 demonstrated to harbor a MBL gene, VIM-2. In Valenza's study, the EDTA disk test was able to discriminate between all MBL-positive and MBL-negative by using a breakpoint >=14 mm. ¹⁶ This could be a remark to be applied in future studies to diminish false-positive results. However, PCR failure in enzyme variants or newer MBLs may also have occurred in this study, for which, the observed differences between genotypic and phenotypic MBL detection might be due, in part, to PCR constraints and not only to EDTA-testing failure.

The presence of class 1 integrons in VIM-2 P. aeruginosa isolates was observed in 40% of all VIM-2 isolates, which suggests that horizontal gene transfer may play a role in the dissemination of MBL producing P. aeruginosa in these settings. Indeed, two integrons contained the MBL gene, one from each hospital. Association of MBL genes with class 1 integrons was also demonstrated in Germany and Italy.^14,16

RAPD analysis of VIM-2 isolates showed 14 patterns scattered in both hospitals. Although the number of studied isolates was limited, the results suggest that there is no interhospital dissemination, but intrahospital spread was observed. This clonal diversity was also seen in a French survey, where 137 isolates were clustered in 113 Pulsed-Field-Gel-Electrophoresis patterns and in most cases, the clonally related isolates were recovered from the same hospital. ⁷ A Belgium study, however, documented an interhospital epidemic spread, ⁵ as well as in Italy, Spain, Greece, and Netherlands, where the same cluster was observed in different hospitals wards.^9,14,15,17

Pattern A was the most prevalent in the 25 detected VIM-2 P. aeruginosa isolates, represented by six isolates collected from three different wards of H2. The other patterns were from different wards and the majority was represented by a single isolate, suggesting their possible source in the patients themselves and not in the hospital environment. ⁶ The five VIM-2 isolates obtained in PH, from different wards, presented the same profile H indicating the selection of a unique clone in this hospital, as stated in an Italian sudy. ¹⁴ Three of these isolates presented a 3,000 bp integron, therefore, horizontal gene transfer may be important in this setting and the hospital personnel should be aware to avoid cross contamination.^4,14 L, M, and N profiles were obtained from urine isolates from the Internal Medicine ward, reflecting the variability between isolates, and reducing the possibility of cluster dissemination in this ward. ⁶

Emergence of MBLs producing bacilli resistant to carbapenems is an increasing clinical problem. Moreover, they are associated to a MDR phenotype that not only includes carbapenems, but also last generation cephalosporins and ureidopenicilins. ⁴ In this study, we demonstrated that the presence of VIM-2 is a reality in our hospitals, with P. aeruginosa carrying bla_VIM-2 typed into 14 different patterns detected in different wards of nearby hospitals, suggesting that the prevalence of carbapenemase-encoding genes was mainly due to gene spread than to clonal dissemination. These results reinforce the need of screening all carbapenem-resistant isolates for MBL production to better address the prevalence and dissemination of these enzymes. We suggest the use of enhanced phenotypic tools, as proposed by Valenza's work regarding the combined double-disk test interpretation. ¹⁶ This might be useful to improve antibiotherapy regimens and policies of infection control in Portuguese and other European hospitals, to prevent the wider spread of this worrisome resistance determinant.