Molecular Detection of extended spectrum Beta-lactamases in Clinical isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa producing extended spectrum beta lactamases (esBl) is a major concern in the hospital settings. it is usually reported in Enterobacteriaceae and is less frequently observed in P. aeruginosa. there is no recommended test for esBl detection in P.aeruginosa. therefore, we determined the occurrence of esBl in clinical isolates of P.aeruginosa by both phenotypic and genotypic methods. Antimicrobial susceptibility tests were done on two hundred and thirteen isolates of P. aeruginosa . Phenotypic detection of esBl was performed using combined disk method and esBl encoding genes such as blaVEB, blaPER, blaPSE, blaGES, blaTEM, blaSHV, blaCTX-M, blaBEL, blaOXA1, blaOXA10, blaOXA2 were studied by simplex PCR. Of the 213 isolates, 85 were identified as resistant to ceftazidime and 27/85 isolates were confirmed to be esBl producers by phenotypic method. the presence of genes encoding esBls comprising of blaTEM (n=44) , blaOXA-10 (n=19) isolates, blaOXA-1 (n=5), blaOXA-2 (n=3) were found. All OXA gene positive isolates exhibited the esBl phenotype. the blaGES gene were identified in 4/85 (5%) isolates. this study shows the prevalence of esBl among clinical isolates of P.aeruginosa and in particular, the presence of Ges β lactamases.


iNtRODuCtiON
Pseudomonas aeruginosa is a multidrug resistant bacterium which has intrinsic resistant mechanisms and is responsible for hospital acquired infections. The production of betalactamases is the most common mechanism of bacterial antimicrobial resistance, which is primarily mediated by plasmids. 1 Extended spectrum beta lactamases (ESBL) are a group of β lactamases which hydrolyze penicillins and cephalosporins including oxyimino b-lactamases and aztreonam, but are inhibited by β-lactamase inhibitors. Co-resistance to many additional antibiotic classes is common in organisms that produce ESBLs, thereby limiting therapeutic options. Penicillinases belonging to the molecular class A serine β lactamases (CTX-M families, TEM and SHV) and class D (OXA-type) β lactamases are the commonly reported β-lactamases; VEB, PSE, CARB, PER and GES are less frequently reported. 2 In addition, Insertion sequence (IS) elements are closely linked to blaESBL genes. Insertion Sequence 26 (IS26) is located upstream of bla TEM gene and is frequently reported to reside in a resistance plasmid which has the feature of transposition and target site duplication (TSD). 3 IS26 is significant in the acquisition and dissemination of antibiotic resistance gene. The overexpression of inducible chromosomal AmpC β-lactamases in P. aeruginosa confers resistance to broad spectrum antibiotics which may be difficult to detect by phenotypic methods. 4 Increased membrane permeability and the existence of several efflux systems adds to the difficulties in detection of antibiotic resistance. There is no reliable test for phenotypic detection of ESBLs in clinical isolates of P. aeruginosa. Molecular methods are used to detect antibiotic resistance genes for monitoring the emergence of drug resistance in the clinical setting. The detection of these genes could help to establish standards for hospital infection control measures. Hence the study was aimed to detect ESBL production by both phenotypic and genotypic methods.

MethODOlOGy
During the period of one year (2013-2014), 213 Pseudomonas aeruginosa clinical isolates were collected from two tertiary care hospitals in South India. The ethical clearance was obtained from the institutional human ethics committee (IHEC No.: UM/IHEC/02-2014-I). Kirby Bauer disc diffusion method was used to evaluate all clinical isolates for antibiotic susceptibility. 5 The following antibiotics (HiMedia Labs Mumbai, India) were used: piperacillin (100 μg), piperacillin/ tazobactum (100μg/10 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), ofloxacin (5 µg), aztreonam (30 μg), cefepime (30 μg), ceftazidime (30 μg), amikacin (30 μg) gentamicin (10 μg), imipenem (10 μg) and meropenem (10 μg). Clinical and Laboratory Standards Institute (CLSI) 2013 guidelines were used for interpretation. 6 For the phenotypic detection of ESBL, combined disk method was used. Briefly, the test isolate was swabbed on Mueller Hinton agar plates after adjusting the opacity to 0.5 McFarland standard. Ceftazidime (30 μg) and ceftazidime/ clavulanic acid (30/10 μg) discs was kept adjacent to each other at a distance of 20mm. After incubation at 37°C for 24 hrs, a zone size greater than 5mm with the ceftazidime/clavulanic acid disc when compared to the ceftazidime disc was considered as positive for ESBL production. 7 The identification of P. aeruginosa by biochemical tests was confirmed with PCR. The boiling lysis method was used to extract DNA. An overnight culture was centrifuged at 10,000rpm for 10 minutes and to the supernatant 300μl of nuclease free water was added, boiled at 100°C for 10 minutes and stored at -20°C for at least 6 hours. The supernatant was collected after centrifugation at 10,000 rpm for 10 minutes and stored at 20°C. For PCR amplification, 2μl was used as a template and kept at -20°C for further use. 8 PCR was performed for the confirmation of species using a species-specific primer which targets the 16S rRNA gene (Primer F: GGGGGATCTTCGGACCTCA and R: TCCTTAGAGTGCCCACCCG) and the amplicon size was 956bp. 9 The presence of ESBL-encoding genes was determined using primer specific for bla VEB , bla PER , bla PSE , bla GES , bla TEM , bla SHV , bla CTX-M , bla BEL , bla OXA1 , bla OXA10 , bla OXA2 by simplex PCR (Table 1). 2,10-11 For bla TEM positive isolates, IS26 transposon was detected by PCR.
Of the 213 isolates, 85(40%) isolates were found to be resistant to ceftazidime and 27 (32%) isolates were confirmed as ESBL producing strains by the combined disk method. The remaining 58 isolates (68%) were resistant to the clavulanate combination, and this may be due to the overproduction of Amp C β-lactamases. All the tested isolates were confirmed as P. aeruginosa by species specific 16S rRNA PCR (Figure 1). PCR detected bla TEM in 44/85(52%) isolates and bla SHV gene was not identified in any of the isolates tested. Forty-four bla TEM positive isolates were further tested for IS26 transposon gene by PCR. Of the 44 isolates, 37 (84%) were positive for the IS26 transposase gene indicating their possible role in acquisition and mobilization of bla TEM (Figure 2).
Among 85 clinical isolates, bla OXA-10 was detected in 19(23%) isolates; bla OXA-1 , bla OXA-2 , were found in 5(6%) and 3(4%) isolates respectively (Table 2, Figure 3). All bla OXA gene positive isolates exhibited the ESBL phenotype. bla VEB and bla PER types were found to be the most common ESBL in P. aeruginosa in several countries, whereas in this study all the isolates were negative for bla VEB and blaPER. The bla GES gene were identified in 4/82 (5%) isolates; two isolates showed resistant to both carbapenems tested; one isolate was susceptible to both the carbapenems, and another was resistant to meropenem and susceptible to imipenem. This shows varying extended activity of GES enzymes in hydrolyzing the carbapenem. All 4 bla GES positive isolates were sequenced and submitted to GenBank and accession numbers were obtained (Table 3). Among the Enterobacteriaceae, the most prevalent enzymes are the CTX-M group of enzymes; however, they were absent in this study.
Other genes frequently seen in Pseudomonas such as bla VEB , bla PER and the minor ESBLs such as bla PSE and bla BEL were not found in any of the tested isolates.

DisCussiON
Phenotypic detection of ESBL in P. aeruginosa is difficult due to the presence of various resistance mechanisms such as over expression of inducible chromosomal AmpC β-lactamase and greater degree of impermeability or efflux-mediated resistance. 12 Current ESBL detection methods employed for Enterobacteriaceae are not recommended to be used for P. aeruginosa. Since there were no CLSI guidelines for P. aeruginosa in the year that this study was conducted, the interpretation criteria for Enterobacteriaceae was followed. Resistance to the clavulanate combination was observed in 58% of isolates and this may be due to the coexistence of ampC production or the presence of inhibitor resistant ESBL variants. The level of AmpC production may obstruct or even obscure the phenotypic detection. In ESBL-producing P. aeruginosa isolates, the spread of metallo beta lactamases may be another reason for the non-detection by phenotypic tests. 4 PCR cannot differentiate the broad spectrum and extended spectrum variants of TEM    β-lactamases. Previously, there have not been many reports of bla TEM and bla SHV genes in P. aeruginosa, due to the high prevalence of oxacillinase and carbenicillinase genes, upregulation of chromosome-encoded cephalosporinase and the rarity of narrow-spectrum TEM type enzymes. 2 In this study, bla TEM was the most prevalent followed by bla OXA-10 whereas another study showed that the most prevalent ESBL gene was bla VEB followed by bla TEM , bla GES and bla SHV . 13 These may vary in different geographical regions.
Our study result shows that bla TEM was found in 52% of isolates and bla SHV was not detected in any of the isolates. The study further demonstrated the presence of IS26 association with bla TEM gene. Horizontal gene transfer mechanisms may have played a role in the dissemination of this kind of enzymes from the Enterobacteriaceae family. The selective pressure of antibiotics on the bacteria could have modified the resistant mechanisms. 14 CTX-M variant was previously one of the most frequently reported enzymes in Enterobacteriaceae; later it was reported in P. aeruginosa. 15 Dissemination of the gene may be due to the insertion sequence ISEcp1 which is frequently observed upstream of the bla CTX-M genes. However, in this study, none of the isolates exhibited bla CTX-M which differed from previous findings. [16][17][18][19] Molecular class D β-lactamases mainly OXA group exhibit greater diversity in their enzymatic activities and are the most frequent ESBLs found in Pseudomonas sp. The present study reports the presence of bla OXA-10 , bla OXA-1 and bla OXA-2 in 19 isolates, 5 isolates and 3 isolates respectively. OXA-10 and to a lesser extent, OXA-2 are the origin of the majority of OXA-type ESBLs. 20 OXA 10 and OXA 2 are classified as narrow-spectrum Class D β-lactamases; however, when expressed in A. baumannii, they can show carbapenemase activity. 21 In the present study, bla GES was identified in four isolates which showed both the ESBL phenotype and carbapenemase activities. This could be a stage in the transition between ESBLs and carbapenem-hydrolyzing enzymes of class A. GES has previously been observed in different places in India. 22 The high frequency of ESBLs in the present study indicates the necessity for standardization of phenotypic method of detection and increasing its sensitivity. Further, PCR is essential for the characterization of ESBLs and monitoring the dissemination of resistant genes. Although the prevalence of ESBLs has been documented in numerous studies from around the world, geographical and institutional differences in their prevalence have been observed.

CONClusiON
ESBL-producing P. aeruginosa strains are reported world-wide. Use of molecular methods for the detection of resistant genes is important to study their prevalence.