Detection of blaPER-1 & blaOxa10 among imipenem resistant isolates of Pseudomonas aeruginosa isolated from burn patients hospitalized in Shiraz Burn Hospital.

BACKGROUND AND OBJECTIVES: Pseudomonas aeruginosa is one of the most important Gram negative opportunistic bacteria which causes infection among burn patients. Resistance to the antibiotics in this group of bacteria is increased due to the activity of extended spectrum β-lactamase (ESBLs) genes. In the current study, we investigated the prevalence of two genes (blaPER-1 & blaOxa10 ) related β-lactamase genes among imipenem resistance clinical isolates of P. aeruginosa in hospitalized patients.
MATERIALS AND METHODS: From May 2010 to March 2011, 270 P. aeruginosa isolated from hospitalized burned patients' wounds in Shiraz Burn Hospital, were tested for Imipenem resistance by disk diffusion method. Presence of ESBLs exo-enzyme, blaPER-1 and blaOxa10 genes were also evaluated in the resistant isolate.
RESULTS: 210 (77.7%) of 270 P. aeruginosa isolates were resistant to imipenem. blaPER-1 and blaOxa10 were detected among 168 (80.0%) of imipenem resistant isolates. Furthermore, 160 (76.2%) of them had blaOxa10 gene and 84 (40.0%) of them had blaPER-1 while 63 (30.0%) resistant isolates contained both genes simultaneously.
CONCLUSION: This study showed a high prevalence of blaPER-1 and blaOxa10 genes in hospitalized burn patients in south west of Iran. Therefore, it's highly recommended to perform such tests routinely to evaluate the resistance pattern in order to better antibiotic selection in the burned patients.

INTRODUCTION

Pseudomonas aeruginosa is a Gram negative opportunistic human pathogen, which causes various acute and chronic nosocomial infections such as pneumonia, urinary tract and wound infections in immunocompromised hosts, particularly in burn hospitalized patients (1).

These infections are responsible for significant human mortality, morbidity, prolonged hospital stays and increased health care costs (2,3). This organism possesses different factors that promote adherence to host cells and mucosal tissues, damage host tissue, elicit inflammation and disrupt defense mechanisms. This conditions aggravate in burn patients due to impairment of the skin barrier in burn patients and frequent scrubbing, debridement and manipulation of the burn site (1). Resistance of P. aeruginosa to a wide spectrum of antibiotics has become a major clinical concern worldwide (1, 2). The extended spectrum β-lactamases (ESBLs), may lead P. aeruginosa to be resistant to β-lactam antibiotics, including penicillins, cephalosporins and monobactams (4,5).

OXA and PER can be mentioned as the two of important β-lactamase enzymes, in P. aeruginosa, (3, 4). bal was the first group of this gene reported from France in 1991 in a single P. aeruginosa isolate from a Turkish patient (3). P. aeruginosa strains contacting bal are highly resistant to β-lactamase, and have strong hydrolytic activity against cephalosporins but can not hydrolyze carbapenems and cephamycins (5). Another group of such enzymes which has high incidence in Enterobacteriaceae especially in P. aeruginosa is OXA. Owing to hydrolytic activity OXA-10 (a class D β-lactamase) is responsible for a high resistance to amino-group antibiotics, carboxy-penicillins, ureido-penicillins and cephalosporins in P. aeruginosa isolates (6).

Due to importance of the carbapenems in resistance infections management, and increasing of the imipenem resistance ESBL P. aeruginosa strains, finding the true frequency of such enzymes is mandatory. The purpose of the present study was to investigate the prevalence of these two β-lactamase genes (bla and bla) in imipenem resistant clinical isolates of P. aeruginosa in hospitalized patients in a main burn center of southwest of Iran.

MATERIALS AND METHODS

Bacterial Isolation.

The study included 270 P. aeruginosa isolates that were recovered consecutively from clinical sites of separate patients’ wounds hospitalized in Shiraz Burn Hospital (the main burn center in southwest of Iran) from May 2010 to March 2011. Collected strains were assessed with routine microbiology methods like Gram stain, pigment production on Muller-Hinton agar media, Oxidase test and non-fermentative result in TSI media. Then, PCR based assay was performed by specific primers for 16s rDNA (7) to confirm presence of P. aeruginosa. Confirmed strains were stored at −200C in Trypticase Soy Broth containing 10% glycerol.

Antimicrobial susceptibility testing.

Isolated strains were tested for their resistance to imipenem using the disc diffusion method (DD), according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (8). Resistance to imipenem, were also evaluated for ESBL production using the combination disc diffusion method (CDD), recommended by the CLSI guidelines (9). In this step, antibiotic disks (aztreonam (/30μg), ceftazidime (/30μg) cephotaxime (/30μg)) were placed around a clavulanic acid (15μg) disk on 10 cm Mueller-Hinton agar plates inoculated with 0.5 McFarland suspensions of the isolates, with 30 mm distance between each disk. After the incubation time (18 hours/37°C) inhibition zone diameters were measured to the minimum distance. Difference of 5mm ≥ in the zone between each disk with clavulanic acid disk compare to another side of the disk showed that the strain is ESBL positive. For ESBL negative control, P. aeruginosa (ATCC 27853) was used.

Detection of bla and bla PCR.

Imipenem-Resistant P. aeruginosa strains were refreshed in Muller-Hinton broth for about 4 hours and their DNA were extracted with Accuprep® Genomic DNA Extraction Kit (Bioneer-USA) according to the manufacture protocol. To evaluate the presence of bla, bla genes in these strains, the PCR was performed with the specific primers for these regions according to the Table 1.

Table 1.

Primer seq. target genes, product size and PCR protocol

Bacteria Gene Primer	Seq. (5/-3/direction)	cycle profile (35X)	Product size
P. aeruginosa
blaOXA-10 F	TAT CGC GTG TCT TTC GAG TA	610C/1min- 720/2min- 950/1min	760 bp
blaOXA-10 R	TTA GCC ACC AAT GAT GCC C
blaPER-1 F	ATG AAT GTC ATT ATA AAA GCT	520C/1min- 720/2min- 950/1min	927 bp
blaPER-1 R	TA ATT TGG GCT TAG G

RESULTS

In this cross-sectional of 270 isolates of P. aeruginosa, 210 (77.7%) were resistant to the imipenem. According to the results of CDD screen, 168(80.0%) isolates were ESBL producing strains. 160 (76.2%) of them contain bla and 84 (40%) of resistant isolates contain bla related genes, and 63(30.0%) of them contain both genes. 148(92.5%) of 160 bla containing isolates, and all of bla containing isolates produced ESBL (Figs. 1–2).

Fig. 1

PCR amplification of bla and bla genes. 1, 3, 7, 15: No. of positive samples for per-1 (927bp) - 11, 25, 42, 57: No. of positive samples for bla (760 bp)

La: DNA ladder, NC: negative control, C+: Positive control.

Fig. 2

Number of Imipenem resistant isolates, ESBL producing isolates in Imipenem Resistance’s, number of bla and bla related genes.

DISCUSSION

Burn injury is a major public health problem in many countries, and requires immediate specialized care in order to minimize mortality and morbidity (10,11). It is estimated that 75% of all deaths following burn injury are related to infection (12). The infection in such patients is difficult to control due to the presence of dead burn eschar, and moist environment, that act as a good growth medium for microbes. Prolonged hospital stay and invasive diagnostic and therapeutic procedures (13, 14).

P. aeruginosa, known as major colonizer of the burn wound, is able to accumulate different resistance and virulence factors, thrives on moist burn wound surface and survives well in the hospital environment, once it is established (14). Burn hospitals often harbor multidrug-resistant P. aeruginosa that can serve as the source of infection (2). Previous studies in Iran confirmed resistance to many antibiotics used routinely for treatment of burn wounds infected by P. aeruginosa. Hadadi et al. showed that P. aeruginosa isolates were resistance to ceftizoxime (99%), ceftazidime (59.6%), ticarcilin (50%), ceftriaxone (44.3%), and cefoperazone (37.5%) (15). According to a study conducted in Shiraz Burn hospital in 2006 by Japoni A et al. almost all P. aeruginosa isolated from burn patients were resistant to all tested anti-Pseudomonal antibiotics except carbapenems (meropenem and imipenem) (16).

Carbapenems are useful in treatment of some cases of multi-drug resistant strains of P. aeruginosa (16). The resistance of P. aeruginosa was 48% against imipenem in a study conducted by Singh et al. in Korea in 2001 (17). In another study in Iran in 2009 Shahcheraghi et al. reported 75% resistance for imipenem in P. aeruginosa isolated from nosocomial sources (12). In the current study 210 (77.7%) of 270 isolated P. aeruginosa, were resistant to imipenem.

It seems that most of this multidrug resistance reflects the accumulation of multiple mutations and acquirement of many resistance genes (2). Different studies evaluated the P. aeruginosa resistant strains in different world centers specially in ICU & Burn wards so far (5,16,18), which showed different pattern for the resistance of this bacteria to the different antibiotics and the frequency of the important β-lactamases (such as bla and bla) among resistant isolates. According to the results of CDD screening in our study, 168 (80.0%) isolates from 210 Imipenem resistant isolates were ESBL producing strains. Mirsalehian et al. highlighted that 39.41% of the P. aeruginosa strains isolated from hospitalized burn patients in Tehran were ESBL producers (16).

For many years, PER β-lactamases were thought to be significant only in Turkey (PER-1) and Argentina (PER-2) (5, 18, 19). Since 1995, PER-1 producing organisms have been disseminating in Italy (20, 21), France (21), Spain (22), Romania (23), Korea (24), Japan (26), and China (27). Bacteria with the OXA (a class D β-lactamase) have evolved to destroy β-lactam antibiotics, and presented high levels of resistance to a broad spectrum of β-lactam antibiotics (28, 29).

Mirsalehian et al. in 2010 stated that 74.62% and 49.25% of the isolated P. aeruginosa strains from hospitalized burn patients in Tehran contain bla and bla gene, respectively (17). In addition, Vahaboglu et al. detected PER-1-type b-lactamases in 11% (40/367) of P. aeruginosa strains (30). While in our study, 160 (76.2%) of the resistant isolates contain bla and 84 (40%) of resistant isolates contain bla related genes, and 63 (30.0%) of them contains both related genes simultaneously. 148 (92.5%) of 160 bla containing isolates, and all of bla containing isolates were ESBL producing.

The results of our study showed a high prevalence of the imipenem resistant strains in burn patients, which is an alarming sign and should be taken into the consideration, because increasing of the antimicrobial resistant bacteria isolated from burn patients is an important issue (13).

The accumulation of multiple mutations and acquirement of resistance genes is believed as one of the most important reasons for the multidrug resistance (2). Hence, it is very important to set up a strict and logical infection control program to detect the source of infection, bacteriological profile, antibiogram of burn wound isolates and find the responsible genes for the antimicrobial resistant in order to decrease the incidence of nosocomial infections in hospitalized burn patients and help the clinicians to better drug selection for this kind of patients.