P. aeruginosa is the leading cause of nosocomial infections in immuno-compromised patients in hospitals. The frequency of P. aeruginosa infections is high in burn patients due to easier access of pathogens [1]. Emergence of antibiotics resistance in P. aeruginosa is a major public health concern because the resistant organisms produce a wide range of beta lactamases. Production of extended spectrum beta lactamases (ESBLs) is the principle mechanism involved in coping with antibiotics by P. aeruginosa [2]. The recent increase in ESBLs production by P. aeruginosa is alarming in developing countries due to non-judicious use/abuse of antibiotics. This practice engenders new modes of resistance while augmenting the prevalence of drug-resistant bacteria [3-5]. Among the ESBLs, TEM-1 beta-lactamase was first identified in E. coli and later-on in P. aeruginosa [6,7]. TEM-1 is the most commonly encountered beta-lactamase in Gram-negative bacteria. Up to 90 % of ampicillin resistance in E. coli is due to the production of beta-lactamase by TEM-1 gene. TEM-2, the first derivative of TEM-1, has a single amino acid substitution from the original beta-lactamase [8,9]. Characterization of beta-lactamases in drug-resistant P. aeruginosa will be crucial in the study of mechanisms of molecular pathogenesis and virulence.

The present study was undertaken to screen and identify TEM beta lactamases among clinical isolates of P. aeruginosa in Pakistan. Moreover, antibiotic susceptibility was performed in TEM producing isolates.

The study was conducted at the Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat, Pakistan. Samples totalling 102 were collected from a tertiary care hospital, Peshawar, Khyber Pakhtunkhwa, Pakistan and aseptically transported to the research laboratory.

Pure culture of P. aeruginosa isolates

In our previous study, a total of 102 isolates were identified as P. aeruginosa on the basis of phenotypic and species specific PCR [10]. Pure cultures of P. aeruginosa were processed for this study. All the isolates were re-confirmed by biochemical assays using standard protocols.

Molecular detection of TEM beta-lactamase in P. aeruginosa

TEM gene amplification was checked in all 102 isolates of P. aeruginosa. Primers used were as follows: TEM forward 5׳-TCGGGGAAATGTGCG-3׳ and TEM reverse 5׳-TGCTTAATCAGTGA GGCACC-3׳primers [8]. Ready PCR mixture contained 10 mM Tris HCl, 50 mM KCl, 1.5 mM MgCl₂, 0.5 mM DDT, glycerol, Gelatin, NP-40, 0.05 mM Tween-20, 0.2 mM dNTPs and Taq DNA polymerase. 1 µL genomic DNA was used for every reaction in a total volume of 25 µL. The following PCR conditions were used for TEM gene amplification of P. aeruginosa: initial denaturation at 95 ^oC for 5min followed by 35 cycles, 94 ^oC for 1 min, 58 ^oC for 1 min, 72 ^oC for 1 min and final extension for 7 min. The amplified product was separated on agarose gel and visualized under UV trans-illuminator.

Sequencing of amplified TEM gene and bioinformatics analysis

Amplified PCR product was sequenced using forward primer for the TEM gene. Sequencing reactions were performed by Macrogen (Seoul) Korea. Online available Bioedit software was used for sequence analysis. Amino acid sequence of TEM was analysed on translation tool (www.expasy.org/) and was matched with all previously reported TEM genes [11].

Antibiotic susceptibility assay of TEM-116 beta-lacatamase-producing P. aeruginosa

Antibiotic sensitivity pattern of TEM-116 beta-lactamase producing P. aeruginosa isolates were determined by modified Kirby-Bauer method [12]. A 0.5 McFarland dilution of refreshed bacterial isolate was inoculated on Mueller Hinton agar (MHA). Antibiotic discs (Oxoid, UK) including amoxycillin/clavulanic acid (AMC), aztreonam (ATM), ceftazidime (CAZ), cefepime (FEP), imipenem (IPM)  ceftriaxone (CRO), amikacin (AK), doxycycline (DO), gentamicin (CN), ciprofloxacin (CIP), norfloxacin (NOR), levofloxacin (LEV), sulphamethoxazole/trimethoprim (SXT), sulzone (SCF) and chloramphenicol (C)  were placed on MHA plates and then incubated overnight. After incubation, zones of inhibition were measured and interpreted in accordance with Clinical Laboratory Standard Institute (CLSI) guidelines [13].

E-test

Quantitative minimum inhibitory concentrations (MICs) of the selected antibiotics (including ciprofloxacin, ceftriaxone, gentamicin and amikacin) against TEM 116 beta-lacatamse- producing P. aeruginosa were evaluated by E-test in accordance with manufacturer’s instructions (AB Biodisk, Solna, Sweden).

Among the 102 P. aeruginosa isolates, the amplified product of 972 bp corresponding to TEM gene size was found in 23 isolates (22.5 %) ().

TEM amplified gene product was processed for sequencing. When nucleotide sequence was translated into amino acids, it showed 100 % amino acid sequence homology with TEM 116. In chromatogram the nucleotides at position c.250G>A and c.551C>T, refer to the substituted nucleotides and p.Val84Ile and p.Ala184Val refer to the amino acids substitutions at these two positions in comparison to the original TEM 1 gene sequence (). BLAST analysis of the translated amino acids sequence with TEM 1, showed two mutations that include replacement of valine by isoleucine at position 84, and replacement of alanine by valine at position 184 (). The two mutations at positions 84 and 184 confirmed the presence of TEM-116 when used online tool (http://www.laced.uni-stuttgart.de).

After analysis of sequence homology, the TEM-116 beta-lactamase producing isolates were processed for antibiotic susceptibility pattern. The TEM-116 isolates showed 100 % resistance (n = 7) to sulphamethoxazole/trimethoprim, amoxycillin/clavulanic acid and doxycycline. However they showed increased sensitivity to levofloxacin, norfloxacin and amikacin. One isolate, PA11 showed resistance to all the antibiotics used in the study ().

MICs were determined to assess the quantitative inhibitory concentration of commonly used antibiotics against TEM producing P. aeruginosa. It was found that MIC of ceftriaxone was >512 µg/ml against three isolates (PA5, PA9 and PA11) while PA11 isolate was found to be the most resistant among all TEM-116 producing isolates ().

Resistance to beta-lactam antibiotics in communities and hospitals has created a major health problem leading to increased mortality, morbidity and high health care expenses.

Production of extended spectrum beta-lactamases (ESBLs) in bacteria is the principle mechanism of resistance against beta-lactam antibiotics. Among ESBLs, TEM beta-lactamase family has variable diversity, prevalence and global distribution [11]. In TEM family, TEM-116 was first reported among Enterobacteriaceae in Korea [14] and in P. fluorescens [15]. One study has documented co-production of TEM-116 and SHV-2a in P. aeruginosa isolates in France [16].In the present study, TEM-116 showed two point mutations which cause amino acid substitution from valine to isoleucine at position 84 and alanine to valine at position 184. Similar mutations in TEM 116 were also reported in P. fluorescens [15].

When antibiotic susceptibility pattern was checked, it was found that TEM 116-producing P. aeruginosa isolates exhibited maximum resistance to sulphamethoxazole/trimethoprim, amoxycillin/clavulanic acid and doxycycline. In several studies, ESBLs and TEM 116 isolates were highly resistant to commonly available antibiotics [16-18]. These reports are   in line with our findings. Moreover TEM-116 producers in the current study exhibited sensitivities to levofloxacin, norfloxacin and amikacin, which indicate effective bacteriostatic activity against the TEM-116 isolates. When bacteriostatic activities of selected antibiotics including ciprofloxacin, ceftriaxone, gentamicin and amikacin were checked, they showed similar variable activities as reported earlier [17].

Among these isolates (PA11) showed resistance to all the antibiotics used in the study. The possible reason might be the presence of full spectrum of beta lactamases and other virulence factors in this isolate (PA11).

In the present study, increased antibiotic resistance was reported in the TEM-116 P. aeruginosa isolates from Pakistan. These findings are considered beneficial in understanding the mechanism(s) involved in development of antibiotic resistance in P. aeruginosa.