Characteristics of Carbapenem-Resistant Pseudomonas aeruginosa Strains in Patients with Ventilator-Associated Pneumonia in Intensive Care Units

Astra Vitkauskienė1, Erika Skrodenienė1, Asta Dambrauskienė1, Giedrė Bakšytė2, Andrius Macas3, Raimundas Sakalauskas4 1Department of Laboratory Medicine, Medical Academy, Lithuanian University of Health Sciences, 2Department of Cardiology, Medical Academy, Lithuanian University of Health Sciences, 3Department of Anesthesiology, Medical Academy, Lithuanian University of Health Sciences, 4Department of Pulmonology and Immunology, Medical Academy, Lithuanian University of Health Sciences, Lithuania


Introduction
It has been proved that early appropriate antimicrobial therapy in patients with nosocomial infections may have a major impact on mortality, length of stay, emergence of resistant strains, and overall health care costs. Treatment of serious infections in an intensive care unit requires an empirical strategy providing broad-spectrum coverage to a wide range of suspected pathogens, such as Pseudomonas aeruginosa (P. aeruginosa) (1). Infections with multidrug-resistant P. aeruginosa has been recognized as a growing problem in clinical settings, and the occurrence of infections due to strains that are resistant to almost all commercially available antibacterial drugs has become a rather common event. The carbapenems (meropenem [MEM] and imipenem [IMP]/cilastatin) represent a realistic option for initial empirical therapy in many serious nosocomial infections because of their broad spectrum of activity and the continued susceptibility of diffi cultto-treat and antibiotic-resistant pathogens to these agents (2). However, resistance to carbapenem is being observed more frequently among non-fermenting bacteria, such as P. aeruginosa and Acinetobacter spp. Data from many European centers show an increasing resistance of P. aeruginosa strains to carbapenems, conditioned by beta-lactamase synthesis (3)(4)(5). Increasing resistance to carbapenems mediated by metallo-β-lactamase (MBL) is a cause for concern because MBL-producing P. aeruginosa strains have been reported to be important causes of nosocomial infections, and it adversely affects clinical outcomes and adds to treatment costs (6). Other mechanisms of the resistance of P. aeruginosa strains to carbapenems, such as the production of AmpC, extended-spectrum β-lactamases, or Toho-1-type β-lactamases, can be involved as well, but the overall rates of morbidity and mortality among patients infected with MBL-producing strains are high (7). Early detection of MBL-producing strains is important for clinicians for the selection of appropriate antimicrobial agents. During the last years, the prevalence of carbapenem-resistant P. aeruginosa strains in the tertiary hospital of our university has increased from 10% to 40% (data not published). Such a high rate prompted us to study the characteristics of carbapenem-resistant P. aeruginosa strains and 5-year changes in resistance at our hospital and to present the guidelines in the future in order to decrease such a resistance.

Material and Methods
Patients and Bacterial Strains. This study was carried out at the 2000-bed tertiary-care teaching Hospital of the Lithuanian University of Health Sciences (HLUHS). According to the data of the Laboratory of Microbiology at the HLUHS, a total of 145 P. aeruginosa strains in 2003 and 151 in 2008 were isolated from lower respiratory tract specimens (bronchial or bronchoalveolar lavage) of patients treated for ventilator-associated pneumonia in the intensive care units. The study included 90 P. aeruginosa strains serotyped in 2003 and 101 P. aeruginosa strains serotyped in 2008, which were randomly selected. P. aeruginosa strains were identifi ed according to the standard methodology described previously (8).
Antimicrobial Susceptibility, Interpretive Criteria, and Detection of Metallo-β-Lactamase. All P. aeruginosa strains were screened for resistance to carbapenems (IMP and MEM) by the routine disk diffusion test with antibiotic-containing disks (BBL, USA) according to the Clinical and Laboratory Standards Institute (CLSI) recommendations (9 To investigate the resistance level of MBL-producing and non-MBL-producing carbapenem-resistant strains to antibiotics, the MICs of ciprofl oxacin, amikacin, and piperacillin/tazobactam against P. aeruginosa strains of both groups were determined. The MIC for each bacterial strain was interpreted according to the CLSI criteria. Standard P. aeruginosa ATCC 27853 strain was used as a control strain. Serogroup Detection. To identify serogroups, sera containing specifi c antibodies against O group antigens of P. aeruginosa were used (Bio-Rad, France). Serogroups were denominated by Arabic numerals from O:1 to O:16 according to the classifi cation of the ICSB Subcommittee on Pseudomonas and Related Organisms and identifi ed using the agglutination method according to the methodology approved by the manufacturer of specifi c antibodies (Bio-Rad, France).
Statistical Analysis. Statistical analysis was conducted using the SPSS (Statistical Package for Social Sciences, Microsoft Inc., USA) software, version 12.0 for Windows. While analyzing differences in frequency, nonparametric statistical criterion χ 2 and Fisher exact test were used. The differences between the groups were considered signifi cant if P was <0.05.

Results
In 2003, of the 90 clinical P. aeruginosa isolates included in this study, 19 (21.1%) were resistant or intermediate resistant to carbapenems (IMP or/and MEM). In 2008, there were more carbapenem-resistant or intermediate resistant P. aeruginosa strains as compared with 2003 (41.6%, 42/101; P=0.002). Comparison of the changes in resistance of P. aeruginosa strains to carbapenems within the 5-year period revealed that the level of resistance to IMP increased, but that to MEM did not change. In 2003, 53.3% of P. aeruginosa strains were found to be highly resistant to IMP, while in 2008, this percentage increased to 87.8% (P=0.01) (Fig.).
All P. aeruginosa strains that were found to be resistant to at least one of carbapenems using the disk diffusion test were screened for MBL activity. In 2008, a considerable increase in the proportion of carbapenem-resistant MBL-producing P. aeruginosa strains was observed as compared with 2003 (61.9%, 26/42, and 15.8%, 3/19, respectively; P<0.001).
The percentages of carbapenem-resistant P. aeruginosa strains resistant to ciprofl oxacin and gentamicin were signifi cantly greater than those of carbapenemsensitive P. aeruginosa strains both in 2003 and 2008.

Discussion
The emergence of resistance to carbapenems in P. aeruginosa is a serious concern. The prolonged use of imipenem/meropenem for the treatment of nosocomial infections can favor the development of resistance to other antibiotics (10). The increasing prevalence of strains possessing resistance to carbapenems still remains a major issue in the management of hospitalized patients. In this study, a high rate of increased resistance among P. aeruginosa strains isolated from respiratory tract specimens of patients treated at the ICUs was documented. Susceptibility testing revealed that carbapenem-resistant P. aeruginosa strains were highly resistant to other antibiotics: ciprofl oxacin, ceftazidime, piperacillin, and gentamicin. Trouillet et al. have reported that ventilator-acquired pneumonia (VAP) episodes due to piperacillin-resistant strains were more frequently associated with prior fl uoroquinolone administration than were VAP episodes due to piperacillinsusceptible strains. The results of this study highlighted the major role of fl uoroquinolones in the emergence of multidrug-resistant P. aeruginosa responsible for ventilator-associated pneumonia (11). In contrast, a study by Mueller et al. demonstrated that the use of fl uoroquinolones was not a risk factor for nosocomial colonization or infection with P. aeruginosa isolates resistant to both fl uoroquinolones and imipenem (12). Our fi ndings suggest that the resistance level of P. aeruginosa to imipenem increased during the 5-year period. Our previous studies that compared the data on defi ned daily doses per every 100 occupied bed-days during the period 2004-2007 revealed a signifi cant increase in meropenem use, but a decrease in ofl oxacin use (13). Other studies have shown that prolonged exposure to carbapenems and colistin independently predicted pandrugresistant P. aeruginosa pneumonia (14). Many factors play a role in the acquisition of multidrug-resistant (MDR) P. aeruginosa; previous exposure to quinolones and carbapenems is also recognized as an important contributor to the acquisition of MDR P. aeruginosa (15).
In the present study, 29 P. aeruginosa isolates were found to produce MBLs. MBL-producing P. aeruginosa strains were resistant to multiple antibiotics belonging to different structural families, and this makes our study more relevant from a clinical point of view. The majority of MBL-producing clinical P. aeruginosa isolates in our study were resistant to ciprofl oxacin. It was determined that a greater percentage of non-MBL-producing strains had low MICs against ciprofl oxacin and amikacin as compared with MBLproducing strains.
Our results in this study remind us that MBLproducing P. aeruginosa has become a serious clinical and therapeutic problem in our hospital. Other studies demonstrated that almost all MBL-producing organisms were highly resistant to extendedspectrum cephalosporins and beta-lactamase inhibitor combinations (16).
The present study has demonstrated a considerable increase in the percentage of P. aeruginosa strains belonging O:11 serogroup in our hospital in 2008. Our previous study revealed that there were no imipenemresistant strains among P. aeruginosa strains belonging to the O:11 serogroup in 2003, while in 2008, even 47.5% of imipenem-resistant strains were assigned to the O:11 serogroup (17). The data reported by Patzer and Dzierzanowska showed that in Poland, P. aeruginosa strains resistant to aminoglycosides and beta-lactams more often belonged to the O:12 serogroup (18), while in Greece, P. aeruginosa strains of the O:11 serogroup were more frequently resistant to different antibiotics (19). Increasing resistance rates of P. aeruginosa to carbapenems in our hospital are associated not only with an increase in carbapenem use, but also with the spread of MDR O:11 serogroup P. aeruginosa strains.

Conclusions
The results of our study emphasize the need to restrict the spread of O:11 serogroup Pseudomonas aeruginosa strains and usage of carbapenems to treat infections with Pseudomonas aeruginosa in the intensive care units of our hospital.

Statement of Conflict of Interest
The authors state no confl ict of interest.