Introduction

Antibiotic resistance in Pseudomonas aeruginosa, one of the most common pathogens in complicated urinary tract infections (UTIs), has spread in the Asia-Pacific region.1, 2, 3 The overexpression of efflux pumps was reported to contribute to multi-drug resistance in P. aeruginosa.4 Most Gram-negative bacteria have the genes for efflux pumps belonging to the resistance nodulation division family,5, 6 and several homologous resistance nodulation cell division-type pumps are representative in P. aeruginosa such that MexAB-OprM (coding gene: mexA, mexB and oprM), MexCD-OprJ (mexC, mexD and oprJ), MexEF-OprN (mexE, mexF and oprN), MexXY(-OprA) (mexX, mexY and oprA) and some P. aeruginosa strains lost oprA gene.7 MexAB-OprM contributes to antibiotic resistance to β-lactams such as cephalosporines or penicillins, macrolides, chloramphenicol, tetracycline and fluoroquinolones.8, 9, 10 MexCD-OprJ contributes to antibiotic resistance to macrolides, tetracyclines, fluoroquinolones and some β-lactams including cefepime.9, 11, 12 MexEF-OprN contributes to antibiotic resistance to fluoroquinolones, chloramphenicol and trimethoprim.13 MexXY-(OprA) contributes to antibiotic resistance to aminoglycosides, cefepime, ciprofloxacin and levofloxacin (LVFX).14

On the other hand, Sacha et al. stated that among the strains belonging to different clones isolated from ICUs, different levels of activity of the MexAB-OprM pump were observed. It can be assumed that this mechanism is also responsible for resistance to multiple classes of antibiotics (for example, ciprofloxacin and meropenem) in the population of P. aeruginosa strains colonizing hospital environments.15 Moreover, Ozer et al. reported in their intensive care units patients 86% of the isolates were determined to carry one and more resistance genes. The significant relationship between the resistance to cefepime, piperacilline/tazobactam and the mexC, that between the resistance to mezlocillin, piperacilline/tazobactam, ceftazidime, cefepime and ampC, and that between the resistance to ciprofloxacin, norfloxacin and oprJ, oprN and nfxB were identified.16

We demonstrated that two or more mutations in the quinolone resistance determining the region of gyrA and parC had a significant relationship with LVFX resistance of UTI-causing P. aeruginosa in the previous study.17

In this study, we investigated the correlation between antibiotic susceptibilities and expression of the efflux pump genes in P. aeruginosa in UTI patients and which efflux pump gene related to antibiotic resistance, mutations of quinolone resistance genes and the patients’ backgrounds, such as the presence of urinary tract underlying disease (complicated UTI) or diabetes mellitus (DM).

Materials and methods

Bacterial isolates

Urine cultures were obtained from patients with UTI treated at three hospitals (Kobe University Hospital, Miki City Hospital and Akashi Municipal Hospital) in Hyogo prefecture in Japan. Post-treatment isolates and other repeat isolates from the same patients were excluded from this study. A total of 105 cases of P. aeruginosa strains from UTI patients were investigated. The study obtained Institutional Review Board approval.

Susceptibility testing

Susceptibility testing was performed by measuring MIC for nine antibiotics: piperacillin, ceftazidime, cefozopran, aztreonam (AZT), imipenem, gentamicin, tobramycin, amikacin and LVFX, and the definition of susceptible and their ranges for concentrations tested were referred according to Clinical Laboratory Standards Institute guideline M07-A8 (Table M100-S19), using Frozen plates (Eiken Chemical Co. Ltd, Tokyo, Japan).18 The bacterial isolates were cultured as shown in the previous literature and on heart infusion agar plates at 37 °C for 22 h, prepared as 1 ml of 1.0 McFarland standard and then diluted in 9 ml of sterile saline and inoculated into Frozen plates for 20 h. MICs of each antibiotics were analyzed by IA 01 MIK mk II plate reader (Eiken Chemical Co. Ltd). P. aeruginosa PAO1 was used as a quality control.17

RNA extraction and quantification of RNA expression using quantitative reverse-transcription PCR

Taqman Reverse Transcription Reagents (Applied Biosystems, Carlsbad, CA, USA) were used for the synthesis of complementary DNA for all samples. Complementary DNA synthesis was performed for the amplification as follows: 48 °C for 30 min, 95 °C for 10 min and 50 cycle of 95 °C for 15 s and 62 °C for 1 min. One microliter of complementary DNA was used as the template in quantitative reverse-transcription PCR amplification with designed primers (Table 1) as described in previous studies.13, 19, 20 rpsL was used as housekeeping gene to calculate the relative level of expression of each gene.2 Quantitative reverse-transcription PCR was performed in MyiQ real-time PCR systems (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendation. The protocol was as follows: initial denaturation at 95 °C for 10 min, 45 cycles of amplification; denaturation at 95 °C for 15 s; annealing at 55 °C for 5 s, and extension at 72 °C for 10 s (mexB, mexY and rpsL) and at 95 °C for 15 min, 40 cycles of amplification; denaturation at 95 °C for 20 s; annealing at 60 °C for 20 s and extension at 72 °C for 30 s (mexC and mexE). Each sample was assayed in triplicate on a 96-well plate.

Table 1 Primer sequences used in this study
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The relative expression level of efflux pump genes was calculated by the level of relative quantification in each efflux pump gene divided by that of rpsL and then the expression rate of each efflux pump gene was defined as the relative expression level of efflux pump gene in each isolate divided into greater than equal to twofold gene expression compared with that of the control (P. aeruginosa PAO1).21, 22

Association of increased expression of efflux pump genes with risk factors, each antibiotic resistance and mutations of quinolone resistance-related genes

We analyzed the association of expression of mexB, mexC, mexE and mexY with risk factors, the MICs of each antibiotics and the presence of mutations of fluoroquinolone resistance-related genes (gyrA and parC).13

We set as the potential risk factors for P. aeruginosa antibiotic resistance such as gender (male), complicated UTI, DM and previous use of LVFX. Complicated UTI is defined as UTIs in which the patients had urinary tract underlying disease such as urinary tract stones, benign prostate hyperplasia or neurogenic bladder. Antibiotic resistance or the four efflux pump gene expressions tested and the mutations of fluoroquinolone resistance genes with the presence of these potential risk factors were investigated for potential correlations. We also compared the increased expression of efflux pump genes with antibiotic resistance. In addition, we confirmed the associations between increased expression of efflux pump genes and mutations of quinolone resistance-related genes.

Statistical analysis

Statistical analyses were performed by univariate and multivariate logistic regression using PASW Statistics 17.0 software packages (for Windows, SPSS Inc., Chicago, IL, USA) with P<0.05 considered to indicate statistical significance. Multivariable logistic regression was used to calculate odds ratios and 95% confidence intervals after controlling simultaneously for potential confounders.

Results

Patient's characteristics, sequence and efflux pump genes expressions

Patients’ backgrounds are shown in Table 2. As Table 2 shows, 38.1% of all cases had complicated UTI and 18.1% of the patients had DM. The increased expressed strains were seen in 28 (26.7%) in mexB, 12 (11.4%) in mexC, 44 (41.9%) in mexE and 40 (38.1%) in mexY, suggesting that mexE and mexY tended to be increasingly expressed in our studies strains.

Table 2 Patient's characteristics of urinary tract infection
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Susceptibility to each antibiotics

In 105 P. aeruginosa isolated from UTI patients, 9 (8.6%), 9 (8.6%), 9 (8.6%), 28 (26.7%), 12 (11.4%), 21 (20.0%), 7 (6.7%), 10 (9.5%) and 23 (21.9%) isolates were resistant to piperacillin, ceftazidime, cefozopran, AZT, imipenem, gentamicin, tobramycin, amikacin and LVFX, respectively (Table 3). That is, our studied strains tended to show comparatively higher resistant ratios especially in AZT, gentamicin and LVFX (MICs and efflux pump gene expressions are shown in a Supplementary Table).

Table 3 Susceptibilities of antimicrobial agents tested
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Correlation of increased expression of efflux pump genes with risk factors

We examined the association between the strains with increased expression of efflux pump genes and potential risk factors, and found an association between the overexpression of mexB and mexC and complicated UTI (P=0.001 and P=0.040, respectively), and overexpression of mexC and gene mutation (gyrA and parC) (P=0.033) by univariate analyses. In addition, we revealed a higher significant relationship between the overexpression of mexB and mexC, and complicated UTI (P<0.001 and P=0.032, respectively) by multivariate analyses. However, there were no significant associations between efflux pump gene expressions and gender (male), DM, previous use of LVFX and gene mutations (gyrA and parC) (Table 4). These findings suggest overexpression of mexB and/or mexC related to the complicated UTI.

Table 4 Correlation of overexpression of efflux pump genes with risk factors
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Association of increased expression of efflux pump genes with MICs of each antibiotics

We investigated the relationship between antibiotic MICs and efflux pump genes expressions. Our statistical data demonstrated a significant association between mexC and LVFX resistance (P=0.019) by univariate analyses. Multivariate analyses also showed a significant relationship between overexpression of mexC and LVFX resistance (Odds ratio=4.48, P=0.035) (Table 5), suggesting, taken together, that mexC correlated with quinolone (LVFX) resistances.

Table 5 Correlation of overexpression of efflux pump genes with antibiotics susceptibilities
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Association of increased MICs of each antibiotics and patients’ backgrounds such as complicated or uncomplicated UTI, DM and previous use of LVFX

We also examined the correlation between antibiotic MICs and patients’ backgrounds such as the classification of UTI, DM and previous use of LVFX. The results showed that previous use of LVFX significantly correlated with AZT and LVFX resistances (Table 6). As to AZT resistances and previous use of LVFX, it is not easy to find some reasons for this correlation; therefore, further research is necessary for this conclusion.

Table 6 Correlation of risk factors with antibiotics susceptibilities
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Discussion

Among the possible mechanisms for multi-drug resistance, efflux pump systems are located in bacterial membrane transporters, and increased levels of the efflux system expression are considered to lead to antibiotic resistance.23 Of the efflux pumps, the MexAB-OprM efflux pump is expressed continuously in susceptible strains, and an increase in gene transcription is apparently sufficient for further increase of the resistance level.4, 19, 20 The MexR protein is the transcriptional repressor of the mexA-mexB-oprM operon.12 On the other hand, MexCD-OprJ and MexEF-OprN were normally silent or weakly transcribed in susceptible strains.24 We revealed that increased expression of mexC leads to antibiotic resistance to LVFX, and MexCD-OprJ contributes to resistance to fluoroquinolones, often used in UTI treatments.17

Gorgani et al.25 discovered a mutation in codon 126 of the mexR regulatory gene changing amino acid Val to Glu that correlated with fluoroquinolone resistance. Moreover, it was suggested relationship between oxacilin resistance, and expression of mexR26 and mutation in mexR gene was seen in 64% of multi-drug resistance strains, which were resistant to at least three antibiotics out of ciprofloxacin, tobramycin, ceftazidime, or imipenem.27 Masuda et al. reported that MexAB-OprM is the primary system to extrude ofloxacin in the wild-type strain and that MexXY-OprM is a compensatory system to extrude ofloxacin in the mutant lacking MexAB,28 and mexE (MexEF-oprN) expression was reported to correlate to fluoroquinolone resistance.20 However, we were unable to detect that mexB, mexE and mexY were associated with the MIC of antibiotics including LVFX, except for mexC. As to the association of LVFX MIC and pump genes expressions, it was significant only in mexC differently from previous literatures. We speculate the reasons as follows: (1) the different materials and patients’ backgrounds may produce different results related to this association. (2) The susceptibilities to fluoroquinolones such as LVFX in P. aeruginosa have been changed from epoch to epoch and from region to region in the world. (3) The use of antibiotics was also varied from region to region including dose and duration. (4) Especially, in this study, our 105 P. aeruginosa strains had 80% of susceptible strains to LVFX and this may be higher than usual population in P. aeruginosa causing UTI.29

There are the reports regarding the risk factors for the expressions of efflux pump genes in Escherichia coli;25, 30 however, regarding P. aeruginosa, there were no reports of the risk factors for the expressions of efflux pump genes even though there are many reports of separate investigation of risk factors for antibiotic resistance.31 Our previous work using E. coli revealed that complicated UTI cases and female gender for LVFX resistance were significantly independent risk factors for transcriptional regulator gene marA expression.22 Our current study with P. aeruginosa showed that complicated UTI significantly correlated with high expressions of mexB and mexC, suggesting complicated UTI may have highly expressed efflux pump genes leading to comparatively higher antibiotic MICs. Our risk factors results by multivariate analyses showed that complicated UTI significantly associated with increased expressions of mexE and mexC. These findings clinically indicate that complicated UTI may possibly have some effects of efflux pump inhibitors for their inhibition or killing.

Previous reports have suggested that which mutations in gyrA and parC or efflux pump expressions have a main role for fluoroquinolone resistance remains contentious.28, 32 We previously showed that mutations in gyrA and/or parC correlated to fluoroquinolone resistance more than efflux pump gene expressions in E. coli causing UTI without their mutual relationships.28 Our multivariate analysis data showed no significant correlation of efflux pump gene expressions with mutations in gyrA and/or parC. Taken together, these facts suggest that gyrA and parC mutations and efflux pump system were involved in fluoroquinolone resistance through other independent mechanisms in both E. coli and P. aeruginosa.

We would like to emphasize the study limitations. First, the ratio of resistant strains was comparatively fewer than in other reports and this might result in a lower level of efflux pump gene-increased expression. Second, we lacked information of history of antibiotics other than LVFX and this point might affect the significant relationship between complicated UTI and efflux pump genes expressions such as mexB and mexC. These limitations may be overcome by further studies with more ratios of resistant strains.

In conclusion, our multivariate analyses data demonstrated that overexpression of mexC efflux pump genes can lead to LVFX resistance in clinically isolated P. aeruginosa from UTI patients and revealed a significant relationship between the mexB or mexC efflux pump genes and complicated UTI. These results contribute to our knowledge of the efflux pump system.