OprD Expression and Imipenem Resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that produces highly resistant to antibiotic treatment. Here we show a presence of correlation between oprD expression and imipenem resistance. Minimal inhibitory concentration (MIC) for planktonic cells of P. aeruginosa was measured using E test. The results revealed the presence of oprD expression in 10 strains of P. aeruginosa isolates from patients with cystic fibrosis in order to evaluate their impact on imipenem susceptibility profiles. We investigated the relation between oprD gene expression and imipenem susceptibility profile showed isolates. Surprisingly, the imipenem-susceptible (P8) has low oprD expression that appeared in imipenem-susceptible, indicated a correlation of other mechanisms. This work forms a basis for future studies revealing the mechanisms of imipenem resistance in P. aeruginosa.


INTRODUCTION
Pseudomonas aeruginosa is an important opportunistic human pathogen that can cause life-threatening infections especially in cystic fibrosis (CF) patients and individuals with a compromised immune system.This environmental bacterium is willingly able to survive both as free swimming planktonic form and in surface-associated communities known as biofilms.Although there are several antimicrobials (carbapenems, cefepime, ceftazidime, tobramycin and amikacin) that continue to be effective against P. aeruginosa, in the last few years the bacterium's increasing resistance to many others has been reported (Sanchez-Romero et al, 2007;Ruiz-Martinez et al, 2011), Carbapenems, particularly imipenen, are suitable alternative in treating multi drug resistant P. aeruginosa, yet the emergence and spread of carbapenem resistant strains have compromised the progress of therapeutic and control efforts (Riera et al, 2011).
OprD porin of Pseudomonas aeruginosa facilitates the uptake across the outer membrane of basic amino acids, small peptides that contain these amino acids, and their structural analogue imipenem.Indeed, prolonged imipenem treatment of patients with P. aeruginosa infections leads to imipenem resistant mutants that either lack OprD due to an oprD gene mutation (Lynch et al., 1987) or have strongly reduced OprD levels due to an nfxC-type mutation (mexT) which suppresses oprD expression at the same time as upregulation of the mexEF-oprN multidrug efflux operon (Fukuda et al., 1995;Kohler et al., 1997).Inactivating mutations in OprD have been documented to confer resistance to imipenem and to a lesser extent to meropenem and doripenem (Sanbongi et al., 2009).
The goal of the present study was to analyze the expression of OprD in a number of clinical isolates of P. aeruginosa with different imipenem susceptibility profiles, ranging from susceptible to intermediately susceptible, and how much this oprD expression gene would affect the imipenem resistance.

MATERIALS AND METHODS
Strains and media.The strains used in this study were routinely cultured on lysogeny broth (LB) medium, which was solidified with 1.5% agar when necessary, bacterial strains and susceptibility testing P. aeruginosa were isolated from sputum samples of different cystic fibrosis patients.

Determination of Imipenem Minimal Inhibitory Concentration (MIC)
The E-test method was used for MIC determination according to the manufactures instructions.In brief, bacterial suspensions were prepared from fresh colonies, the concentration adjusted to 0.5 McFarland turbidity, each isolate was uniformly spread on the surface of a Mueller Hinton agar (MHA) plate, and an imipenem E-test strip (from 0.002 to 32 µg/mL; bioMérieux, France) placed on the surface of agar plate.After overnight incubation at 37°C, MIC was been determined and categorized as sensitive (≤ 2 µg/ml), intermediate (4 µg/ml) or resistant (≥8 µg/ml) according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2013).In select cases, we also determined the MIC by a microdilution assay in microtiter plates to confirm the E-test findings (Andrews, 2001;Wiegand et al, 2008).Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control strains for E test and microdilution assay, respectively.

Modified Hodge Test (MHT)
Modified Hodge test was adopted for the detection of carbapenemases following the procedure described by Anderson et al. (2007) and Noyal et al. (2009).In brief; 0.5 McFarland dilution of the E. coli ATCC 25922 was prepared in 5 ml of saline.1:10 dilution was prepared by adding 0.5 ml of overnight culture to 4.5 ml of saline.Thereafter, a lawn of the 1:10 dilution of E. coli ATCC 25922 was streaked to a MHA.Afterward, 10 µg Imipenem disc was placed in the center of the test area.In a straight line, the test organism was streaked from the edge of the disc to the edge of the plate.Up to four organisms can be tested on the same plate with one drug, and then the plate was incubated overnight at 37ºC in ambient air for 16-24 hours.After 16-24 hours of incubation, the plate was examined for a clover leaf-type indentation at the intersection of the test organism and the E. coli 25922, within the zone of inhibition of the carbapenem susceptibility disk.MHT positive test has a clover leaf-like indentation of the E. coli 25922 growing along the test organism growth streak within the disk diffusion zone.MHT negative test has no growth of the E. coli 25922 along the test organism growth streak within the disc diffusion.

qRT-PCR
Strains were diluted from LB-grown overnight cultures 1:100 into M63 minimal medium supplemented with glucose, MgSO 4 , and CAA and grown to an optical density at 600 nm (OD 600 ) of 0.6.RNA was isolated, and cDNA was prepared as previously described (Kuchma et al., 2005).Quantitative reverse transcription-PCR (qRT-PCR) was performed using an ABI 7500 Fast System and analyzed using ABI Fast System software version 1.4.Expression levels were quantified in picograms of input cDNA using a standard curve method for absolute quantification, and these values were normalized to rplU expression.Each experiment was done with three replicates per sample.The primers used are oprD-RT Forward CCGCAGGTAGCACTCAGTTCG and oprD-RT Reverse GTAGTTGCGGAGCAGCAGGTC.

Statistical analysis
Data are presented as mean ± standard deviation.ANOVA test, correlation coefficient (r) and LSD 0.05 were employed for data analysis using Microsoft EXCELL 2010 application

RESULT AND DISCUSSION
Imipenem susceptibility and carbapenemase detection Among fifty eight P. aeruginosa isolates evaluated with E test, forty seven (81.03%) isolates were susceptible, two (3.45%) isolates were intermediate, and nine (15.52%) isolates were resistant to imipenem.
All these 9 isolates were previously isolated from patients with cystic fibrosis.No carbapenemase activity was found among the intermediate and resistant isolates, as it is confirmed by the Hodge test.A positive strain would develop a 'cloverleaf shaped' (figure 1) zone of inhibition due to carbapenemase production, while our strains showed negative results (undistorted zone of inhibition).These results suggested that the imipenem resistance is due to oprD malfunction rather than carbapenemase.Carbapenems are not prone to inactivation by extended spectrum ß-lactamases and penetrate across the outer membrane of P. aeruginosa through a porin OprD, which allows selective penetration of basic amino acids, small peptides containing these amino acids, and carbapenems, their structural analogs.Prolonged treatment of P. aeruginosa-infected patients with imipenem has often allowed for the emergence of imipenem-resistant mutants.These resistant strains have either lost OprD or have strongly reduced OprD levels due to an nfxC type of quinolone-resistant mutation (mexT) which represses oprD expression and activates the mexEF-oprN multidrug efflux operon (Yoneyama and Nakae, 1993).Ochs et al. (1999) reported in their study that the possible mechanisms by which resistance to imipenem emerged in 17 imipenem-resistant P. aeruginosa clinical isolates, related to the loss of OprD was the predominant reason of imipenem resistance, OprD loss was caused by a chromosomal oprD mutation.

Analysis of oprD expression
The relationship between oprD expression and imipenem resistance were assessed in some imipenem resistant clinical strains.As it is illustrated in table 1, all resistant isolates showed low oprD expression values by comparison with the resistance isolates., LSD = 0.132 The results showed all the resistance isolates (P1, P2, P3, P4, P5, P6) have low oprD expression; whereas the sensitive isolates (P7, P9) developed high oprD expression.Interestingly, the low oprD expression that appeared in imipenem-susceptible (P8) and intermediate susceptible isolates (P10) indicated a correlation of other mechanisms.Less commonly, there is a mutation or deletions within mexT convert inactive MexT into an active form.Somehow, mutations occur in mexS located upstream of mexT, lead to accumulate various metabolites that serve as effectors molecules for MexT, which, in turn or in both cases, the expression of mexEF-oprN occurs at high level, alongside with a decline in the expression of oprD which is inadequate to elaborate quantities of OprD in the outer membrane sufficient for normal cellular function (Fukuda et al., 1995;Köhler et al., 1997).Wolter et al. (2009) also demonstrated down-regulation in the production of the carbapenem channel OprD despite carbapenem hyper susceptibility.These isolates had decreased expression of the mexAB-oprM pump involved with intrinsic antibiotic resistance but over expressed the mexCD-oprJ and mexEF-oprN efflux systems normally associated with acquired resistance.Once again this might mean that there are other routes for carbapenems entrance.
We concluded that oprD expression correlated with imipenem resistance in these clinical isolates (r = 0.8).Loss of oprD is one of the most important mechanisms of resistance to imipenem in P. aeruginosa.Multiple studies have evaluated the importance of oprD mutation in clinical isolates of P. aeruginosa resistant to carbapenems.Always authors demonstrated a correlation between the levels of expression of oprD and the degrees of susceptibility to imipenem (Dib et al., 1995;Ocampo-Sosa et al., 2012;Lee et al., 2012).In this step, we aimed to gain an insight into the relationship between oprD expression and imipenem susceptibility profiles in imipenem-susceptible -intermediate and resistance clinical strains of P. aeruginosa.Our selection of these isolates was based on their imipenem susceptibility profiles, including organisms with a broad range of susceptibility: susceptible (MICs ≤ 2 µg/ml), intermediately susceptible (MIC = 4 µg/ml) or resistance (MICs ≥ 8 µg/ml) to imipenem.
In a conclusion, the results revealed a good correlation between oprD expression and imipenem resistance; however, there are other mechanisms that might be related to low oprD expression in sensitive isolates.

Figure 1 :
Figure 1: Modified Hodge test, the negative strain shows an undistorted zone of inhibition.