1. Introduction

Infectious diseases have affected the health of humans in many aspects and always remained one of the major causes of mortality and morbidity [1]. Amongst different microbial pathogens, Pseudomonas aeruginosa has been declared as one of the major cause of infectious agents belonging to the group of gram negative bacteria. It’s an alarming threat to the hospitalized and immunosuppressed patients and has the ability to persist in different environmental conditions [2]. According to Center of Disease Control and Prevention (CDC), P. aeruginosa has been ranked as the fourth most commonly isolated nosocomial pathogen and its multidrug resistance poses a challenge for its treatment [3]. The emergence of imipenem resistance in P. aeruginosa has become a severe threat, especially in poor and developing countries due to the limitation of sources including Pakistan. The epidemiological data available in Pakistan indicates that the P. aeruginosa is one of the frequent pathogen cause infections in hospitalized patients. Other reports in Pakistan reported that in clinical isolates of P. aeruginosa an increase in percent resistance against imipenem was observed [4, 5].

Carbapenems and fluoroquinolones are potent and first line of therapy against P. aeruginosa. But now in some reported cases, P. aeruginosa has developed resistance against fluoroquinolones [6]. Carbapenems are the second choice of treatment and have been developed from thienamycin, a naturally derived product of Streptomyces cattleya. They inhibit L-D-transpeptidases during the cell wall synthesis. Among carbapenems known; imipenem is more important and effective antibiotic. It is the first-line of therapy against pseudomonal infections. It can bind strongly with penicillin binding proteins (PBPs), especially with PBP-2, PBP-1a and PBP-1b [7].

Resistance against imipenem knows as Imipenem Resistant P. aeruginosa (IRPA). There are two mechanisms by which P. aeruginosa has developed resistance against imipenem: by decreased drug permeability through the outer membrane proteins known as OprD and carbapenem hydrolyzing β-lactamase (metallo-β-lactamase). The resistance may be either by one or both of the two mechanisms [8]. The second major cause other is the metallo-β-lactamase (MBL) production. Metallo-β- Lactamase (MBL), according to the Ambler's classification is class B. It’s a Zn²⁺ dependent carbapenem, active site of it have zinc ions for nucleophilic attack by zinc-bound water hydroxide. It is resistant to clavulanic acid but can be inhibited by EDTA (Ethylene-Diamine-Tetra-Acetic Acid), Tetramethylethylenediamine and other chelating agents [9].

MBL was first detected in Bacillus cereus, in 1960s and they have intrinsic chromosome resistance against β-lactam antibiotics. Approximately 9 different types of MBL have been identified in gram negatives but most common and important types of acquired MBLs include IMP (Imipenem Carbapenemase) (Kyorin Health Science MBL 1) is rare [10, 11]. In 1991, first plasmid-mediated MBL gene, IMP-1 was identified in P. aeruginosa from Japan [4], then subsequently other IMP-type of MBLs were detected in other Eastern countries, including Europe, Canada and Brazil. Analysis of gene bla_IMP showed the features of different class integrons. Integrons are DNA-based structures that carry the genes of antibiotic resistance, known as gene cassettes and most transferable MBLs are encoded by type 1 and 3 integrons [12, 13].

Objectives of Study: Main objective of this research is to investigate the prevalence of Imipenem Resistant P. aeruginosa (IRPA) and detection of bla_IMP gene in the confirmed cases of MBL strains.

2. Material and Methods

2.1. Study and Location

Our study was carried out in the Government College University, Lahore and King Edward Medical College Lahore.

2.2. Bacterial Isolation and Identification

Two Hundred laboratory samples were collected and inoculated on MacConkey and Blood agar for the isolation of P. aeruginosa. Initial identification was done phenotypicaly including API 20NE galleries (BioMerieux, France).

2.2.1. Antibiotic Sensitivity Test and Detection of Imipenem Resistance

Antimicrobial sensitivity testing was performed by according to CLSI recommendation 2012, in order to investigate the presence of imipenem resistance in P. aeruginosa.

2.2.2. Detection of MBL

IRPA are MBL producers. Confirmation of metallo-β- lactamase production was done by imipenem-EDTA combined disk diffusion method. MBL need Zn²⁺ at its active site for activation. Whereas, EDTA as a chelating agent to inactivate the MBL by capturing the zinc ions. EDTA solution of 0.5 M (750 µg EDTA) concentration was prepared in this method by adding 186.1 g of di-sodium EDTA. 2H₂0 (Sigma) in 1 liter distilled water. NaOH was added to adjust the pH of solution 8.0. This solution was sterilized by autoclaving. On the dried plate of Mueller-Hinton agar two imipenem (10 µg) disks (Oxoid, UK) were placed. Out of two imipenem disks, on one disk 4 µl volume of 0.5 M EDTA was added. Plates were incubated for 18-20 hours at 35°C. EDTA causes an increase in the zone of inhibition from 8-15 mm (imipenem plus EDTA) were regarded as MBL producers, whereas, an increase in the zone of inhibition from 0-5 mm was regarded as MBL non-producers [14] (as shown in the Fig. 1).Positive and negative controls as P. aeruginosa (MBL positive) and P. aeruginosa (MBL negative) respectively were also included.

2.2.3. Ribotyping

All the imipenem resistant MBL positive P. aeruginosa isolates were re-confirmed by ribotyping of 16S rRNA by polymerase chain reaction (PCR) and amplified product was isolated on 1% agarose gel electrophoresis. The forward and reverse primers are utilized for PCR reaction was as follows [15]:

Forward Primer: 5' -AGA GTT TGA TCC TGG CTC AG -3'

Reverse Primer: 5' –TAC GGT TAC CTT GTT ACG ACT T- 3'

The correct size of end product and for the separation of mixed population of DNA was determined by running the product on 1% agarose gel.

2.2.4. Phylogenetic Analysis

Phylogenetic trees, based on homology search results through BLAST [16] were constructed taking the 16S rRNA sequences of each of IRPA sequence as query. As the 16S RNA is very much conserved in all of the bacteria, therefore, during BLAST search the option of ‘similar sequences’ was chosen instead that of ‘dissimilar sequences’ so as search may include more diverse sequences to find the Phylogenetic relationship of 16S RNA sequence of IRPA isolates of this study. Moreover, 16S ribosomal RNA sequences database was selected for the homology search through BLAST at NCBI. The Phylogenetic trees of relationship were through neighbor joining method using percent identity through Jalview [17].

2.2.5. Detection of bla_IMP Gene

For the detection of bla_IMP gene_, PCR and 1% agarose gel electrophoresis was performed on MBL producing isolates of P. aeruginosa. The primers utilized for PCR reaction were as follows [18].

bla_IMP Forward Primer: 5' - GAA GGY GTT TAT GTT CAT AC-3'

bla_IMP Reverse Primer: 5' - GTA MGT TTC AAG AGT GAT GC - 3'

The correct size of end product and for the separation of mixed population of DNA was determined by running the product on 1% agarose gel.

2.2.6. ATCC Strain

ATCC 27853 Pseudomonas aeruginosa and were included to monitor quality control.

2.2.7. Statistical Analysis

Data was analyzed by using computer software program SPSS 19.0. The values are expressed in percentages. Chi square test was used for the determination of the statistical significance (p-value).

3. Results and Discussion

In the present study, we have collected 17 different categories of clinical samples (Table 1). The percentage of the indoor patients 70.5% (n=141), having P. aeruginosa, were observed to be much higher compared to the outdoor patients 29.5% (n=59). These findings are similar to the study of Prashant et al., in 2011 [19]. According to them, indoor patients, with P. aeruginosa, were 84.92% compared to the outdoor patients (15.07%). These results correspond to the fact that duration of hospital stay has been related with incidence and prevalence of P. aeruginosa infections.

Table 1. List of Collected Clinical Samples having P. aeruginosa infection

Some P. aeruginosa isolates were found as either a single pathogen 40% (n=80) or in the combination with other organisms (polymicrobial) 60% (n=120). Some isolates have produced a blue-green pigment (pyocyanin) 72% (n=144). Finlayson in 2011 concluded that production of pigment significantly associated with the multidrug resistance and also the virulence in P. aeruginosa [20]. MBL production also affected by pigment production; according the statistical analysis; p-value (probability significance) was found less than the targeted value (p<0.05) as p=0.021 (Table 2).

Table 2. Association of Pigment Production with MBL Production

Resistance against imipenem was found in 28% (n=56) IRPA isolates whereas 72% (n=144) were sensitive (Fig. 1). Different percentages of imipenem resistance were found in different regions of world. In 2010, Akhtar et al. observed 26.1% IRPA in Pakistan [21]. Prashant et al. in 2011 had found 12.69% IRPA in India [19]. In 2012, Fatimah et al., have found imipenem resistance in 24% isolates of P. aeruginosa in Karachi, Pakistan [22], Zoghlami et al. observed the imipenem resistance in 37.1% isolates of P. aeruginosa in Tunisia [2] and Dong et al., observed 15% IRPA in Taiwan [23]. In 2013, Morrow et al. observed 78.1% IRPA in USA [24]. The data shows an increase in percentage of resistance in P. aeruginosa which is an alarming threat.

Figure 1. Resistance Pattern of 200 isolates of P. aeruginosa against various antimicrobials (%age)

Main culprit for the imipenem resistance is the production of Metallo-β-lactamase (MBL). MBL was detected in 87.5% (n=49) of IRPA isolates (n=56). Prashant e al., 2011 in India, determined the MBL in 62.5% isolates of imipenem resistant P. aeruginosa [19]. Vahdani et al. in 2012 have observed 38% MBL positivity in burns, Iran [25].

Figure 2. MBL Detection by Imipenem-EDTA Disc diffusion Method

Re-confirmation of all the MBL positive P. aeruginosa isolates was done by ribotyping for further bla_IMP gene detection. After ribotyping of 16S rRNA, a product gene around 500bp was produced and all were found to be P. aeruginosa confirmed by gene sequencing of the end product. Sequencing of 16S rRNA of all IRPA isolates resulted in three different lengths of sequences. Of the total 49 IRPA isolates 23 (47%) specimen were with a gene length of 1461 bp whereas, 9 were with a sequence length of 1456 bp and remaining 7 isolates resulted in a length of 1326 bp. Homology search results and phylogenetic analysis revealed that all IRPA isolates of the present study were very much related to P. aeruginosa PAO1 strain (Figure 3).

Figure 3. Phylogenetic Tree

The confirmed isolates of MBL positive P. aeruginosa were evaluated by PCR to detect bla_IMP gene, end product of 230 bp was produced (Figure 4) which was compared with 1Kb DNA Ladder. This gene product was much smaller than our desired product of 587 bp of bla_IMP gene. It confirmed that bla_IMP gene was not found in any of the isolate resistant to imipenem.

Figure 4. 1Kb DNA Ladder, 2-4: Test isolates for blaIMP detection

Imipenem resistance either by decreased drug permeability (OprD) or MBL production both can be present simultaneously or individually [8]. Amongst 9 different categories of bla genes bla_IMP, bla_VIM, bla_SIM, bla_NDM, and bla_KHM-1 were found common and important ones [10]. bla_IMP was not detected in the present study; the reasons behind it may be the decreased drug permeability or deficiency of OprD or any type of MBL gene production. Data regarding investigation on MBL gene detection in Pakistani is limited and only one study reported was on bla_IMP-13 and OprD in P. aeruginosa, in Karachi [26].

The current study is in line with Mohammad et al. in 2008 in Iran; they have detected 23 imipenem resistant isolates of P. aeruginosa in Iranian burn center of level I, but did not found the bla_IMP gene in their hospital settings [15]. Khosravi et al. in 2011 (Malaysia) have collected 90 IRPA isolates, 32 were MBL positive whereas bla_IMP was found in 14, bla_VIM in 18, bla_IMP-7 in 12, bla_IMP-4 in 2, bla_VIM-2 in 17 and bla_VIM-11 in 1 isolates of MBL producers in IRPA isolates [27] . In 2011, Mereuta et al. observed bla_VIM in MBL isolates of IRPA but absence of bla_IMP in Lasi [28]. Jacome et al. (2012) in Brazil, did not found the blaIMP or blaVIM genes but blaSMP-1 was detected from 44.8% of MBL producers [29]. In the end, blaIMP gene has not been found in our isolated strains of MBL producing P. aeruginosa. Reason behind it might be the production other bla genes or OprD deficiency.

4. Conclusions

Overall, 28% IRPA has been observed in this study which is an important issue regarding the treatment of infection. Drugs including Tazocin and Sulzone have been found to be the most effective antimicrobials against IRPA. However no bla_IMP gene has been found in our hospital settings but still scope of detection for the presence of MBL gene in Pakistani IRPA isolates is much high.

ACKNOWLEDGEMENTS

We are grateful to kind Edward Medical College, Mue Hospital Lahore for providing us the clinical materials for analysis and with special thanks to the University of Punjab Lahore, for the use of their research facilities and to all the participants.