Detection of bla_TEM-1 gene for ESBL Production among Clinical isolates of Pseudomonas aeruginosa

 

Varshitha A¹ , Dr. Gopinath P²

¹BDS 2 year, Saveetha Dental College, Saveetha University, Chennai

²Senior lecturer, Department of Microbiology, Saveetha University, Chennai

 

ABSTRACT:

The occurrence of multi drug resistant P. aeruginosastrains is growing in the world, limiting the therapeutic options. Conventionally it was believed that ESBLs are derivatives of TEM and SHV parent enzymes but recently in Europe it was proved that ESBLs can be of non-TEM, non-SHV lineage. In this study we have taken 20 clinical isolates of P. aeruginosa and were subjected to antibiotic sensitivity pattern followed by the detection of bla_TEM-1 gene by PCR. Higher degree of resistance to routinely used antibiotics is reported, 15% of them were found to have this gene by PCR. Proper detection of desired panel of genes is required to emphasize the resistance mechanism being operated in these pathogens for better outcome.

 

 

 

 

INTRODUCTION

Pseudomonas aeruginosa is ubiquitous in nature and it is found as a commensal in the skin and gastrointestinal tract of humans. It causes opportunistic infections in immune-compromised patients, not commonly seen in immune-competent patients. ^[1]It also causes serious nosocomial infections such as ventilator associated pneumonia and various sepsis syndromes because it thrives on moist surfaces of the medical equipment like catheter, causing cross infections in clinic and hospitals. ^[2] It typically affects the airways, urinary tract, burns, wounds, gastrointestinal system and also causes blood infections. The most prominent attributes is the resistance of this bacteria to multiple clinically important antibiotics like third generation cephalosporins (imipenem and aztreonam) and extended –spectrum cephalosporins (cefotaxime, ceftriaxone, ceftazidime).^[3] Production of extended-spectrum β-lactamases (ESBLs) can be the cause for resistance of P. aeruginosa strains to the broad-spectrum cephalosporins.

 

The occurrence of multi drug resistant P. aeruginosa strains is growing in the world and limiting the therapeutic options. ^[4,5]  Conventionally it was believed that ESBLs are derivatives of TEM and SHV parent enzymes but recently in Europe it was proved that ESBLs can be of non-TEM, non-SHV lineage. ^[6] ESBLs are undergoing continuous mutations leading to the progression of new enzymes. More than 300 different ESBL variants, based on their amino acid sequences have been grouped into nine different structural and evolutionary families, TEM is one of the major type.^[7]

 

MATERIALS AND METHODS:

Bacterial Isolates:

A total of 20 of non-repetitive clinical isolates of Pseudomonas aeruginosa were collected from Saveetha Medical College, Thandalam. They were processed for a battery of standard bio chemical tests and confirmed. Isolates were preserved in semi-solid trypticase soy broth stock and stored at 4^oC until further use.

 

Antibiotic Susceptibility Testing:

Antibiotic susceptibility testing was determined for this isolates to routinely used antibiotics such as to piperacillin-tazobactam, cefotaxime, ceftazidime, tetra-cyclin, cotrimoxazole, aztreonam, gentamicin and imipenem by Kirby Bauer disc diffusion method as per CLSI guideline. ^[8][9]

Detection of bla_TEM-1 gene in P. Aeruginosa:

Pseudomonas aeruginosa isolates were detected for the presence of bla_TEM-1 gene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 2. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture contained 2mM of Mgcl₂0.2mM dNTP mix and 0.8µM of bla_TEM-1 gene with IU of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of P. aeruginosa with bla_TEM-1 gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 96^oC for 5 minutes and 30 cycles for 30s, 56^oC for 30s and 74^o C for 30s followed by a final extension of 5 min at 72^oC.PCR products were resolved in 1.5% agarose gel. A 100bp ladder was including in all the gel analysis.^[10]

 

Table 1: Primer detail of bla_TEM-1 gene

Primer	Primer sequence	Product size
BlaTEM-1	TGGATCTCAACAGCGGTA TTTATCCGCCTCCATCCA	893 bp

 

RESULTS:

Sample wise distribution of clinical isolates of P. aeruginosa of the 20 clinical isolates of P. aeruginosa, 9/20 (45%) isolates were from sputum, 5/20 (25%) from blood, 3/20 (15%) from urine, 3/20 (15%) from pus.

 

Results of Antibiotic Susceptibility Testing:

In our isolates, we have observed that an increased percentage of isolates have shown to be resistant to most of the routinely used antibiotics. Only 2/20 (10%) isolates showed sensitivity to imipenem. Other than that, for all other antibiotics such as piperacillin-tazobactam, cefotaxime, ceftazidime, tetracycline, cotrimoxazole, aztrionum, gentamicin isolates showed complete resistance 20/20 (100%). The detailed resistant pattern of P. aeruginosa isolates were showed in table1.

 

Table2: Results of antibiotic susceptibility pattern of P. aeruginosa

 

Result of bla_TEM-1gene in P. aeruginosa:

3/20 (15%) clinical isolate of P. aeruginosa was found to possess bla_TEM-1gene.

 

 

     L1       L2      L3       L4     L5

Figure 2: Representative gel picture showing bla_TEM-1 gene

 

L3- 100bp ladder; L1, L4, L5- 693bp bla_TEM-1 gene

 

DISCUSSION:

Pseudomonas aeruginosa are the commonly isolated organisms in health care settings. They cause nosocomial infections in hospitalized patients and immune-compromised patients, because of their ability to live in moist environment. Previously, the species were considered as common contaminants and later the organisms were frequently isolated in many nosocomial infections. MDR organisms are common and known to produce ESBLs, MBL, AmpC, aminoglycoside modifying enzymes to persist the bacteria resistant to all classes of antibiotics. Increasing incidence of these organisms may occur in many nosocomial outbreak infections such as ventilated associated pneumonia, bacteremia, surgical site infection etc. Study conducted by George and coworkers in 2015 reported 21.8% of positivity of having bla_TEM-1 gene and 2.7% of SHV and 91.8%  of CTX-M by PCR analysis.^[11] In our study only 15% of P. aeruginosa isolates were positive for blaTEM-1 gene, However, we did not detect the other genes that encode for ESBLs. In a recent study in north-western India by Kaur and Aggarwal,^[12]45.8% isolates were found to be ESBL producers. The multiplex PCR for TEM, SHV and CTX-M showed 59.32% of P.aureginosapossessed the CTX-M genes. These results are not in concordance with the previously existing reports as we have seen only few clinical isolates.

 

CONCLUSION:

We have observed a high degree of multiple drug resistance among our P. aeruginosa isolates. But only 15% of them were harboring this gene. This indicates that apart from this gene there several other genes that encodes for ESBLs such as CTX-M, SHV etc may be the reason for showing the resistance. To rule out this more number of isolateshave to be included with a battery of other genes to be screened to get the actual outcome.

 

ACKNOWLEDGMENT:

We thank Dr. Kalyani, Professor and Head of the Department of Microbiology, Saveetha Dental College, Chennai for kindly providing the clinical isolates to carry out our research work.

 

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Accepted on 27.07.2016           © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(10):1623-1625.