Introduction

Nosocomial lower respiratory tract infection (LRTI) is the most frequent hospital acquired infection. It is the most common cause of death among nosocomial infections and is the primary cause of death in intensive care units (ICUs).1 Pseudomonas aeruginosa is an important pathogen of nosocomial LRTI especially in ICUs and is commonly resistant to many antibiotics.2 Multidrug-resistant (MDR) P. aeruginosa (resistant to at least three of the following antimicrobials: ceftazidime, imipenem, gentamicin and ciprofloxacin) are often isolated from nosocomial infections in ICUs.2 MDR is often related to the specific efflux pumps and porins in P. aeruginosa strains.3, 4 And four efflux pumps, all of the Resistance Nodule Cell Division Family (RND) type, have been described as MexAB–OprM, MexCD–OprJ, MexEF–OprN and MexXY–OprM, and an outer membrane porin (OprD). Three genes encoding these pumps are arranged as operons. The first gene encoding a membrane fusion protein that is associated with the cytoplasmic membrane (MexA, MexC, MexE and MexX). The second gene encodes the transporter (MexB, MexD, MexF and MexY) thought to export the substrate across the inner membrane. The third gene encodes an outer membrane protein (OprM, OprJ and OprN) that facilitates passage of the substrate across the outer membrane.5 In many ICUs, MDR P. aeruginosa isolates represent a major therapeutic problem. Therefore, understanding the mechanisms of resistance and developing therapy alternatives for these isolates is very important.

The aim of this study was to determine the antimicrobial resistance rates and the resistance genes of P. aeruginosa strains isolated from the patients who had LRTI in ICU.

Materials and methods

The strains were collected during the period February 2007–April 2009 from the patients who acquired LRTIs in ICU of Mustafa Kemal University Hospital. This study was approved by the Local Ethical Committee and was carried out in Mustafa Kemal University, School of Medicine, Department of Medical Microbiology.

Bacterial strains

Fifty P. aeruginosa strains isolated from the lower respiratory tract specimens of the patients who acquired LRTIs in ICU were included in this study. P. aeruginosa strains (fifty) were isolated from tracheal aspirate (27), bronchoalveolar lavage (14) and sputum (9). Isolates were identified as P. aeruginosa based on colony morphology, odor, Gram staining, production of blue-green pigment on Mueller Hinton agar, reactions (k/k) on triple sugar iron agar slants, positive oxidase reaction.5 The species identification was confirmed with the Vitek 2 compact system (bioMérieux, Marcy l'Etoile, France) as required.

Antibiotic susceptibility testing

The isolates were evaluated for their susceptibilities to mezlocillin (75 μg), piperacillin/tazobactam (100/10 μg), ceftazidime (30 μg), cefepime (30 μg), imipenem (10 μg), meropenem (10 μg), gentamicin (10 μg), amikacin (30 μg), tobramicin (10 μg), ciprofloxacin (5 μg), and norfloxacin (10 μg; Oxoid, Basingstoke, UK) by the disk diffusion method, and evaluated according to Clinical and Laboratory Standards Institute.6

Determination of the resistance genes by PCR

Bacterial DNA was extracted from the strains using the method of Chen and Kuo7 with some modifications. The primers of resistance genes were selected from a research article of Dumas et al.,8 shown in Table 1. Multiplex PCR assay was carried out for the detection of antibiotic-resistance genes in a thermal cycler (Bioder/Thermal Blocks xp cycler, Tokyo Japan). The primers were selected according to their base pair. There were four primer groups including; mexE (114 bp), mexR (150 bp), mexT (216 bp), mexA (316 bp) as the first, oprD (156 bp), oprD (232 bp) and oprJ (305 bp) as the second, mexC (164 bp), mexC (344 bp) and ampC (218 bp) as the third, and nfxB (164 bp), oprN (235 bp) and mexX (326 bp) as the fourth group.

Table 1 Primer sequences used in the study
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The PCR amplification was carried out in a total volume of 25 μl reaction mixture. PCR amplification was performed as follows: The reaction mixture consisted of 2.5 ml of 10 × reaction buffer without MgCl2 (Promega, Madison, WI, USA); 200 μM of each deoxynucleoside triphosphate (ABgene, Epsom, UK), 2 mM MgCl2; 0.4 μM of primers and ∼10 ng of template DNA, and brought up to a 25 μl final volume with distilled water. Reactions were started at 94 °C for 4 min and placed on ice, and 1 U of Taq polymerase (Fermentas, Hanover, MD, USA) was added. The amplification process was started with an initial denaturation step (94 °C, 1 min). Each cycle consists of three steps (denaturation, annealing and extension). PCR reaction consisted of 35 cycles of amplification for only mexA, mexT, mexE and mexR genes. The other PCR reaction consisted of 30 cycles of amplification. Amplification consisted of denaturation at 94 °C for 1 min, annealing at 57 °C for 45 s and DNA chain extension at 72 °C for 45 s. And a final extension cycle was performed at 72 °C for 10 min. After the amplification of antibiotic-resistance genes, 10 μl volumes of PCR samples were mixed with 3 μL of loading buffer (10% (w/v), ficoll 400; 10 mmol l−1 Tris-HCl, pH 7.5; 50 mmol l−1 EDTA; 0.25% bromophenol blue). The PCR products were analyzed in a 2% (w/v) agarose gel in 1 × TAE buffer (40 mmol l−1 Tris-acetate, 1 mmol l−1 EDTA). Ethidium bromide (0.5 μg ml−1 TAE)-stained DNA amplicons were visualized using a gel imaging system (Wealtec, Dolphin-View, NV, USA). To determine the expected bp lengths, DNA marker with defined molecular weights in the range 100–2000 were used.

Statistical methods

Analysis was performed using Statistical Package for Social Sciences version 13.0 (SSPS Inc, Chicago, IL, USA). Comparison for categorical variables was calculated using χ2 test. A P-value <0.05 was considered statistically significant.

Results

The highest resistance rate was found against to mezlocillin (50%), followed by norfloxacin (48%), ciprofloxacin (46%), meropenem (40%; Table 2). We measured gene expression of seven mex efflux pumps, the chromosomal ampC β-lactamase, the porin oprD, oprJ, oprN and nfxB in clinical isolates. Expression of mexA, mexE, mexR, mexT genes in group one, oprD and oprJ genes in group two, ampC and mexC genes in group three and nfxB, oprN and mexX genes in group four is shown in Figures 1, 2, 3, 4, respectively.

Table 2 The resistance rates of strains by the disk diffusion method
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Figure 1
figure 1

Multiplex PCR amplification products showing expression of the mexA, mexT, mexR and mexE genes of P. aeruginosa. A 100-bp DNA size ladder is shown; 100 bp DNA size ladder includes fragments of 3000, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp.

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Figure 2
figure 2

Multiplex PCR amplification products showing expression of the oprJ and oprD genes of P. aeruginosa. A 100-bp DNA size ladder is shown.

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Figure 3
figure 3

Multiplex PCR amplification products showing expression of the mexC and ampC genes of P. aeruginosa. A 100-bp DNA size ladder is shown.

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Figure 4
figure 4

Multiplex PCR amplification products showing expression of the mexX, oprN, nfxB genes of P. aeruginosa. A 100-bp DNA size ladder is shown.

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Seven of 50 P. aeruginosa strains had none of these resistance genes. Fourty-three isolates (86%) were determined to be positive for one and more resistance genes. Only four isolates were found to be positive for one resistance gene. The presence of the resistance genes by multiplex PCR is shown in Table 3.

Table 3 The presence of antibiotic resistance genes in Pseudomonas aeruginosa by multiplex PCR method
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Number of the resistant isolates to cefepime and piperacilline/tazobactam carrying mexC gene were found to be 12 (24%; P=0.048) and 15 (30%; P=0.025), respectively; and the number of the resistant isolates to mezlocillin, piperacilline/tazobactam, ceftazidime and cefepime-carrying ampC gene were found to be 16 (32%; P=0.002), 15 (30%; P=0.002), 12 (24%; P=0.035) and 12 (24%; P=0.008), respectively. The isolates carrying nfxB, oprN and oprJ genes were found to be more resistant to norfloxacin and ciprofloxacin (P<0.05). The relationship between antibiotic resistance and the presence of the resistance genes is shown in Table 4.

Table 4 Relationship between antibiotic resistance and the presence of the resistance genes
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Discussion

P. aeruginosa is an important pathogen associated with serious nosocomial infections. In 2003, P. aeruginosa was reported to be the most commonly isolated Gram-negative bacteria (18.1%) for nosocomial pneumonia in the United States.9 Inside and outside ICUs, MDR-P. aeruginosa strains has becoming an increasingly reported problem.10 The ICU isolates gained significant resistance to the antibiotics used for the treatment of the life-threatening infections in ICUs.10 Increasing resistance rates to the antibiotics in P. aeruginosa strains were reported by several studies during the last years.10, 11, 12, 13

In this study, 50 P. aeruginosa isolates from the patients with LRTIs in ICU were investigated for 13 genes, mostly for efflux proteins leading to antimicrobial resistance. To our knowledge, although there are studies investigating the resistance genes from Turkey,14, 15 there aren’t any studies investigating a large number of resistance genes in P. aeruginosa strains isolated from nosocomial LRTIs. The results of the study have shown antimicrobial resistance rates of the isolates were found high, and 86% of them were determined to carry at least one resistance gene. P. aeruginosa exhibited the highest rates of resistance to mezlocillin, with resistance to norfloxacin and ciprofloxacin ranging from 46 to 50%. In the National Surveillance Program in USA, it was reported that antimicrobial resistance was highest for the beta-lactams and ciprofloxacin.13 Our findings support the results of that study. In our study, the resistance rates of P. aeruginosa were higher than the resistance rates in multicenter study in Spain12 and lower than the resistance rates in the study from Bulgaria.11

Beta lactams in combination with aminoglycosides are commonly used as antipseudomonal agents because they may exhibit synergy with aminoglycosides.16 In the current study, the highest resistance rate of the isolates were determined against mezlocillin (50%). The resistance rates against other beta lactams; ceftazidime, piperacilline/tazobactam and cefepime were determined to be 30, 24, 18 and 16%, respectively. These resistance rates were lower than the study performed in an another university hospital in Turkey.17 Antipseudomonal beta lactam antibiotics (piperaciline, cefepime and meropenem) are among mexCD-OprJ's substrates.18 We found that the isolates carrying mexC gene were more resistant to cefepime and piperacilline/tazobactam.

Beta lactam antibiotics (piperacilline, ceftazidime, cefepime aztreonam and meropenem) are substrats of mexAB-OprM.19 Also, no significant relation was determined between mexA gene and ceftazidime, cefepime and piperacilline/tazobactam resistance. Piperacilline, cefepime, ceftazidime, meropenem, imipenem are substrats of mexXY-OprM efflux system.18 Furthermore, no significant relation between mexX gene and resistance against these antibiotics was found.

MexR negatively regulates mexAB-oprM efflux system.20 However, no relation was found between mexR gene and resistance against ceftazidime, cefepime and meropenem in our study.

AmpC gene causes production of chromosomal beta lactamase. The overproduction of AmpC beta lactamases can result in resistance to nearly all beta-lactam antibiotics except the carbapenems.21 We found that isolates carrying ampC gene were more resistant to mezlocillin, piperacilline/tazobactam, ceftazidime and cefepime.

mexT negatively regulates mexAB-oprM efflux system and oprD.22 We didn’t find a significant relation between mexT gene and ceftazidime, cefepime, piperacilline/tazobactam, imipenem and meropenem (the substrates of mexAB-oprM efflux system and oprD).

Carbapenems are one of the most active groups of beta lactam antibiotics against P. aeruginosa. The outer membrane protein OprD allows entry of carbapenems, and its reduced expression is frequently noted in carbapenem-resistant isolates.23 In this study, no relation was found between the persistence of the oprD gene and susceptibility of carbapenems. Outer membrane proteins; oprJ and oprN are related to multidrug resistance.23 No relation between the antibiotics (cefepime, imipenem and meropenem) that were investigated in the other studies and these genes (oprJ and oprN) was determined in this study.

Carbapenem remains as an important agent for the therapy of serious infections secondary to MDR P. aeruginosa. The development of carbapenem resistance severely compromises effective therapeutic options. In the absence of carbapenem-hydrolyzing enzymes, the mechanism leading to carbapenem resistance is usually multifactorial. We determined that the isolates that were resistant to carbapenems were also resistant to other beta lactam antibiotics in this study. Only one isolate was resistant to carbapenems and didn’t show cross-resistance to other beta lactams so it can be imipenem-resistant P. aeruginosa mutant.24

We investigated the relation between mexA, mexC, mexX, mexE genes and quinolones because they are substrates of four efflux system but we didn’t find. We also found no relation between mexR gene that negatively regulates mexAB-oprM, and mexT gene that positively regulates mexCD-oprJ and the resistance against the antibiotics.

The isolates carrying oprJ and oprN, which cause multidrug resistance, and nfxB gene were determined to be more resistant to ciprofloxacin and norfloxacin.

Aminoglycosides are frequently used in pseudomonal infections.16 In our study, the aminoglycoside resistance rates in P. aeruginosa were lower than that in the study from Korea.25 Aminoglycoside resistance arises more frequently via enzymatic modification of the aminoglycosides, and less frequently via mexXY-oprM efflux systems.16 So no relation was found between the presence of mexX gene and aminoglycosides (amikacin, gentamicin and tobramycin).

Conclusion

These data showed that antimicrobial resistance rates of the isolates were high and the highest resistance was against mezlocillin. Most of the isolates were determined to carry one and more resistance genes. NfxB gene was most often seen in the genes that were investigated.

There were strains that were susceptible to most of the antibiotics although they contained large number of antibiotic resistance genes. These strains have very high chance of developing resistance during treatment. And also it should be remembered that the mechanism leading to antimicrobial resistance is usually multifactorial. For this reason, rather than investigating the susceptibility to antimicrobials by phenotypic methods, investigating genotypically the antimicrobial-resistance genes is more meaningful. Understanding the prevalence and mechanism of antimicrobial resistance may help to select empirical therapy for nosocomial LRTIs due to P. aeruginosa in our ICU.