ISPpu22, a novel insertion sequence in the oprD porin gene of a carbapenem-resistant Pseudomonas aeruginosa isolate from a burn patient in Tehran, Iran.

BACKGROUND AND OBJECTIVES: The oprD mutation and AmpC overproduction are the main mechanisms of intrinsic resistance to carbapenems such as imipenem and meropenem in Pseudomonas aeruginosa.
MATERIALS AND METHODS: In this study, we investigated intrinsic resistance to carbapenems including mutation of oprD and AmpC overproduction in a carbapenem-resistant P. aeruginosa isolated from a burn patient by phenotypic and molecular methods.
RESULTS: In our study, the carbapenem-resistant P. aeruginosa isolate was resistant to imipenem, meropenem, cefepime, gentamicin, ceftriaxone, carbenicillin, aztreonam and ciprofloxacin but was susceptible to ceftazidime and polymyxin B. The minimum inhibitory concentrations (MICs) against imipenem, meropenem and ceftazidime were 64 μg/ml, 16 μg/ml and 2μg/ml, respectively. The isolate was ESBLs and AmpC overproducer. No carbapenemase activity was detected by Modified Hodge test (MHT). This isolate was carrying only bla OXA-10 . PCR amplification and sequencing of oprD performed on isolate resulted in PCR product of 2647bp. Sequence analysis of the 2647bp product revealed insertion of a sequence of 1232 bp at position 8 in coding region of oprD.
CONCLUSION: According to the results of this study, oprD mutation and AmpC overproduction can cause the main mechanism of resistance of P. aeruginosa to carbapenems.

INTRODUCTION

Pseudomonas aeruginosa is a ubiquitous organism present in many diverse environmental settings and it is an important cause of bacterial infections associated with hospitalization (1). It presents a serious therapeutic challenge for treatment of related infections because the majority of isolates show a high level of intrinsic resistance to antimicrobial drugs and has potential to acquire resistance (2,3). Carbapenems such as imipenem and meropenem are the major antibiotics used for treatment of P. aeruginosa infections (2,3). In recent years, carbapenem resistance has developed among P. aeruginosa worldwide (2–4). Several mechanism including loss of the outer membrane OprD porin, efflux-pumps over-expression and production of β-lactamases are involved in resistance to carbapenems. However, oprD mutation and AmpC overproduction are the main mechanisms of intrinsic resistance to carbapenem. Loss of the outer membrane OprD porin causes blocking of entrance of carbapenems particularly imipenem into the cell (5,6). One of the factors that causes loss of the OprD is the disruption of oprD by insertion sequence (IS) elements (7). Also, the mutation of oprD usually couples with AmpC over-expression and thus leads to increased resistance to imipenem (8,9). In this study, we report a novel insertion sequence in the oprD porin gene of a carbapenem-resistant P. aeruginosa isolate from a burn patient in Tehran, Iran.

MATERIALS AND METHODS

The carbapenem-resistant P. aeruginosa strain was isolated in 2011 from a wound sample of a female burn patient who was admitted to Shahid Motahari Hospital of Tehran University of Medical Sciences. The identification of isolate was performed by standard biochemical tests (10). The antibiotic resistance pattern of isolate was determined using disk diffusion method according to the CLSI guidelines (11). The following antibiotics (MAST, UK) were tested: imipenem (10 μg), meropenem (10 μg), polymyxin B (300 Unit), ceftazidime (30 μg), cefepime (30 μg), gentamicin (10 μg), amikacin (10 μg) ceftriaxone (30 μg), carbenicillin (30 μg), aztreonam (30 μg) and ciprofloxacin (5 μg). The isolate was also screened for production of carbapenemase using the Modified Hodge test (11). The isolate screened for extended spectrum β-lactamases (ESBLs) production using combined double-disk test, double-disk synergy and β-lactamase disk test (9,11). The β-lactamase disk test is based on use of Tris-EDTA (TE) buffer to permeabilize a bacterial cell and release of β-lactamases into the external environment. In brief, the surface of a Mueller-Hinton agar plate was inoculated with a lawn of the cefotaxime (CTX) susceptible (P. aeruginosa ATCC 27853) according to the standard disk diffusion method. A CTX (30μg) disk was placed on the bacterial lawn on the surface of the Mueller-Hinton agar and flanked by two blank disks, each containing 20 μl of a 1:1 mixture of saline and 100X Tris-EDTA solution. Colonies of the test strain and control strains were applied to blank disks (Fig. 1). Flattening or indentation of the growth inhibition zone of the CTX disk at the side of blank disks containing the test strain indicated the release of β-lactamase. The minimum inhibitory concentrations (MICs) of imipenem (IMI), meropenem (MEM) and ceftazidime (CAZ) were determined in micro broth dilution assay. Susceptibility breakpoints were defined according to CLSI recommendations (11). AmpC overproduction was confirmed according to the method of Rodríguez-Martínez et al. (12). Furthermore, the presence of carbapenemase genes (blaVIM, blaIMP, blaGIM, blaAIM, blaSPM, blaNDM, blaSIM, blaOXA-48, blaKPC and blaGES) and ESBLs genes (blaTEM, blaSHV, bla-PER, blaPSE, blaCTX-M and blaOXA-10) were investigated by PCR method as previously described (4). PCR amplification and sequencing of oprD and the ISPpu22 was performed using primers oprD-F-1-5′-CGCCGACAAGAAGAACTAGC-3′ and oprD- R-5′-GTCGATTACAGGATCGACAG-3′ (9) and with additional primer designed in this study (ISPpu22- F-5′- G GTAG CCGTATACACC TCCG -3′, oprD-F-2-5′-TCGATGCCTTCGGCTACCT-3′)

Fig. 1.

β-lactamase Disk Test. A, B; Negative controls (P. aeruginosa ATCC 27853 and E. coli ATCC 25922), C; Positive control, D; Clinical isolate and E; Lawn culture (P. aeruginosa ATCC 27853).

RESULTS

The carbapenem-resistant P. aeruginosa isolate was resistant to imipenem, meropenem, cefepime, gentamicin, ceftriaxone, carbenicillin, aztreonam and ciprofloxacin but was susceptible to ceftazidime and polymyxin B using disk diffusion method. MICs revealed a high resistance to imipenem (MICs=64 μg/ml). MICs against meropenem and ceftazidime were 16 and 2μg/ml, respectively. This strain was AmpC overproducer. Addition of cloxacillin reduced the MICs from 64 to 16μg/ml for imipenem. No carbapenemase activity was detected in MHT. Diameter of inhibition zone around the antibiotics in combined disk test and double-disk synergy was zero without detection of ESBLs by these tests but β-lactamase disk test was positive for this isolate (Fig. 1). PCR results were negative for blaTEM, blaSHV, blaPER, blaPSE, blaCTX-M, blaOXA-48, blaKPC, blaGES, blaVIM, blaIMP, blaGIM, blaAIM, blaSPM, blaNDM and blaSIM genes. This isolate was positive only for blaOXA-10. PCR amplification and sequencing of oprD performed on strain resulted in PCR product of 2647bp instead of 1413 bp by oprD-F-1 and oprD-R primers (Fig. 2). Sequence analysis of the 2647bp product revealed insertion of a sequence of 1232 bp at position 8 in coding region of oprD. This IS that had 97% identity with IS3 family transposed from P. aeruginosa PA96 (GenBank accession no. CP007224). This IS named ISPpu22, belonging to the IS3 family according to the IS finder database nomenclature (. The full sequence of the oprD gene interrupted by this novel IS element has been deposited in the Gen-Bank database with accession no. KJ825703.

Fig. 2.

PCR product of oprD, Lane M: DNA size marker, Lane 1:PAO1, Lane 2: Clinical isolate

DISCUSSION

In this study, the isolate was positive for β-lactamase by β-lactamase disk test method. This result shows a probable activation of one of the efflux pumps system and impermeability, or both, against beta-lactam antibiotics which causes falsely negative results in β-lactamase detections in combined disk method and double-disk synergy (4,8). This investigation showed that, in the absence of carbapenemases, the oprD mutation and AmpC over expression can cause resistance to carbapenems, however, oprD mutation can cause decreased meropenem susceptibility and AmpC over expression not effective on meropenem (5,6, 13). Literature revealed various routes, including deletion or insertion of sequences, can cause of oprD inactivation. Recently, IS elements disrupting of oprD has been reported in France, USA, China, Korea and Spain among resistant carbapenem P. aeruginosa isolates (7,12). In this study AmpC-β-lactamase caused a reduction in susceptibility to imipenem but not to meropenem. These data suggest that AmpC may play an important role in the level of intrinsic susceptibility of P. aeruginosa to imipenem. Quale, Rodríguez and Lee et al, reported that AmpC-β-lactamase causes an increase of MIC to carbapenems in clinical P. aeroginosa isolates (3,12–15). Therefore, mutation of oprD and AmpC overproduction are the main mechanisms of carbapenem resistance in the absence of acquired carbapenemases such as metallo-beta-lactamases.

CONCLUSION

According to the results in our study, intrinsic resistance mechanism such as oprD mutation and AmpC overproduction are the main mechanism of resistance of P. aeruginosa to carbapenems.