Changes in the Dependence of Pseudomonas aeruginosa O Serogroup Strains and Their Resistance to Antibiotics in a University Hospital During a 5-year Period

The aim of our study was to determine the changes in antibiotic resistance and O serogroup dependence of P. aeruginosa strains isolated from lower respiratory tract specimens of patients in 2003 and 2008; the patients were treated in intensive care units of the biggest treatment facility in Lithuania (Hospital of Lithuanian University of Health Sciences, HLUHS, former Hospital of Kaunas University of Medicine) Material and Methods. The study included 90 P. aeruginosa strains serotyped in 2003 and 101 P. aeruginosa strains serotyped in 2008, which were randomly selected. The resistance of P. aeruginosa strains was determined by the disc diffusion method based on the standard guidelines. The sizes of inhibition zones were interpreted according to the National Committee for Clinical Laboratory Standards (M2-A6). Isolates were serotyped using sera with specific antibodies against the O antigens of P. aeruginosa (Bio-Rad, France). Results. Comparison of changes in the distribution of P. aeruginosa serogroups in 2003 and 2008 showed that P. aeruginosa strains of serogroups O:1, O:2, and O:3 were more prevalent in 2003 as compared with 2008 (23.3%, n=21; 27.8%, n=25; 12.2%, n=11 vs. 9.9%, n=10; 10.9%, n=11; 4.0%, n=4, P<0.05). P. aeruginosa strains of serogroups O:6 and O:11 were isolated more frequently in 2008 than 2003 (26.7%, n=27; 34.7%, n=35 vs. 4.4%, n=4; 10.0%, n=9, P<0.001). The results showed that 18 of the 90 P. aeruginosa strains in 2003 and 25 of the 101 P. aeruginosa strains in 2008 were resistant to three or more antibiotics tested, i.e., they were multidrug-resistant. Analysis of the distribution of serogroups among these P. aeruginosa strains isolated in 2003 and 2008 revealed a significantly higher frequency of O:11 serogroup than other serogroups. Meanwhile, in the group of nonmultidrug-resistant P. aeruginosa strains, P. aeruginosa O:11 serogroup strains were identified less frequently and accounted only for 2.8% (n=2, P<0.001) of the isolates in 2003 and 27.6% (n=21, P<0.01) in 2008. Conclusions. During the 5-year period, the isolation rate of P. aeruginosa strains belonging to serogroup O:11 increased. P. aeruginosa strains isolated in 2003 and 2008 belonging to serogroup O:11 were more frequently multidrug resistant. The increasing resistance of P. aeruginosa to reserve antibiotics of carbapenem group was observed.


Introduction
Pseudomonas aeruginosa (P. aeruginosa) strains have an innate characteristic of the resistance to antibiotics and capability to rapidly acquire the resistance to antibiotics with antipseudomonal activity. Due to these reasons, P. aeruginosa remains an important nosocomial pathogen and still causes considerable problems in most countries including Lithuania (1)(2)(3)(4). It has been demonstrated that resistance patterns of P. aeruginosa strains to an-tibiotics differ depending on lipopolysaccharide structure of the bacterial outer membrane, which is part of the bacterial cell wall and O serogroup (according to O antigen), to which they belong (5,6). However, an important role in the development of the resistance to antibiotics among P. aeruginosa strains is played by antibiotic policy and prescribing strategies in hospitals and within a country. There is a large body of evidence that frequent usage of antibiotics can cause alterations in P. aeruginosa strains and can drive transformation to strains having more pathogenicity factors and strains more resistant to antibiotics (7,8). Due to the effect of antibiotics, resistant strains of P. aeruginosa emerge. Therefore, the antibiotic resistance of P. aeruginosa strains, belonging to the same serogroup, isolated from the specimens of patients treated in different clinical settings can differ as different antibiotics and their dosages are administered. In Poland, P. aeruginosa strains resistant to aminoglycosides and beta-lactam antibiotics were serotyped as O:12 more frequently (6); in Greece, P. aeruginosa strains resistant to antibiotics belonged to O:11 serogroup more frequently (9). The fi ndings of our previous studies showed that P. aeruginosa strains resistant to most antipseudomonal antibiotics (multidrug resistant, MDR) belonged to O:E serogroup (O:11) more frequently (10). It is important to monitor the changes in the serogroup dependence and antibiotic resistance of P. aeruginosa strains in a larger treatment facility over a period of several years since knowledge of these changes can contribute to a successful choice of empirical treatment for critically ill patients. Our study aimed at determining the changes in antibiotic resistance and O serogroup dependence of P. aeruginosa strains isolated from lower respiratory tract specimens of patients in 2003 and 2008 treated in intensive care units of the biggest treatment facility in Lithuania (Hospital of Lithuanian University of Health Sciences, HLUHS).

Material and Methods
P. aeruginosa Strains. 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 in the intensive care units (central, neurosurgery, and cardiac surgery). The strains were identifi ed according to the standard methodology. The study included 90 P. aeruginosa strains serotyped in 2003 and 101 P. aeruginosa strains serotyped in 2008, which were randomly selected; one patient, one strain. The molecular fi ngerprinting was not performed, and copy strains were not excluded. Although only noncopy strains were included in this study, it cannot be excluded at this stage that clonal strains bearing certain serotypes spread locally. Appropriate studies to test the clonal diversity of P. aeruginosa strains are needed.
Susceptibility Testing. The resistance of P. aeruginosa strains was determined by the disc diffusion method based on the standard guidelines (11). The sizes of inhibition zones were interpreted according to the National Committee for Clinical Laboratory Standards (M 2 -A 6 ). Standard P. aeruginosa ATCC 27853 strain was used as a control strain.
Serotyping. Isolates were serotyped using sera with specifi c antibodies against O antigens of P. aeruginosa (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. At the same time, serogroups were identifi ed using the agglutination method according to the methodology approved by the manufacturer of specifi c antibodies (Bio-Rad, France). At the beginning, a bacterial suspension was prepared: no later than after 24 hours, the isolated P. aeruginosa colony grown on the nonselective growth medium was diluted with sterile physiological saline. One drop of a polyvalent serum (pooled conjugate for several specifi c P. aeruginosa serogroups) was mixed with 10 μL of the bacterial suspension solution on a glass slide; by rocking the slide gently in a rotary motion, no later than after 5 minutes, it was observed if agglutination occurred. If agglutination was observed during the testing with a polyvalent serum, further testing was carried out by employing particular specifi c sera belonging to that group of a polyvalent serum. Physiological saline without serum was used as a negative control. If agglutination occurred later than after 5 minutes or bacterial suspension agglutinated with more than one specifi c serum, the testing was repeated by heating the suspension for 30 minutes at 120°C before agglutination. Statistical Analysis. Statistical analysis was conducted using the SPSS (Statistical Package for Social Sciences, Microsoft Inc., USA) software, version 10.0 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.       Table 4).

Distribution of O Serogroups
Analysis of changes in the resistance of P. aeruginosa strains to beta-lactam antibiotics in 2003 and 2008 revealed that there was an increase in the rates of the resistance to ceftazidime (from 5.6% to 13.9%, P<0.05), imipenem (from 7.8% to 39.6%, P<0.001), and meropenem (from 16.7% to 28.7%, P<0.05) dur-ing the 5-year period. However, there were no changes in the resistance of P. aeruginosa strains to piperacillin and piperacillin/tazobactam (Fig. 1).
While evaluating the resistance of P. aeruginosa strains to antibiotics of aminoglycoside group, no changes were observed during the 5-year period. However, the resistance of P. aeruginosa strains to ciprofl oxacin increased from 15.6% in 2003 to 39.6% in 2008 (P<0.001) (Fig. 2).

Discussion
The increasing resistance of P. aeruginosa strains to antibiotics has become a recognized problem not only in most European countries, but also across the world; it has also been linked to the frequent usage of antibiotics (12)(13)(14)(15)(16). Bacteria have an innate characteristic enabling them to exchange diverse mobile genetic elements, which are responsible for the resistance to antibiotics, and this leads to the emergence of multidrug-resistant strains spreading in a hospital environment (17,18). The increasing resistance of P. aeruginosa strains even to reserve antibiotics -carbapenems -causes consider-    able problems in choosing the empirical treatment of infections caused by P. aeruginosa (18,19). It has been proved that pathogenicity factors are found more frequently in P. aeruginosa strains with the outer membrane formed from O antigen-containing lipopolysaccharides (20,21 Although the present study shows that the resistance to ceftazidime increased to 13.9% during the 5-year period, it is still lower than in some other centers reaching even 48.9% (23).

Conclusions
During the 5-year period, the isolation rate of P. aeruginosa strains belonging to O:11 serogroup increased. P. aeruginosa strains both isolated in 2003 and 2008 belonging to O:11 serogroup were more frequently MDR. The increasing resistance of P. aeruginosa to reserve antibiotics of carbapenem group was observed.

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