Detection of [bla.sub.IMP] gene encoding metallo-beta-lactamase resistance among clinical isolates of Pseudomonas aeruginosa.
In the past few decades, antibiotic resistance ratio has been increased drastically, particularly among non-fermenting bacteria, which became a substantial challenge to treat. 
Among the various nosocomial pathogens, Pseudomonas aeruginosa a soil inhabitant, and human saprophyte is well-known opportunistic human pathogen. It causes various life-threatening infections such as ventilator-associated pneumonia, surgical site, and urinary tract infections in patients from intensive care units.  Infections become severe when associated with immunosuppressive states mainly diabetes, carcinomas and also in burns injury and cystic fibrosis. Major risk factors are prolonged hospitalization, ventilation, exposure to inadequate antimicrobial therapy and immunocompromised state to get this infection.  In recent decades, P. aeruginosa has emerged as a multidrug-resistant bacteria by acquiring intrinsic resistance to a number of antimicrobial agents. Multidrug-resistant status in-turn leads to increased hospital expenditure, prolonged hospitalization, narrowing of therapeutic options, cross infection thus, and landing to increased mortality and morbidity finally. This bacteria uses distinctive resistant mechanisms to all the available antibiotics, which include metallo-[beta]-lactamases (MBL) production, extended spectrum [beta]-lactamase production, AmpC production, decreased permeability, altered penicillin binding proteins and rarely, and overexpression of efflux pumps.  As carbapenems are the considered to be the potent antimicrobial agent against multidrug-resistant P. aeruginosa (MDRPA), this bacterium has developed resistance even against this group of drugs by producing MBLs (carbapenemase).  Imipenem and meropenem in carbapenems have gained increased therapeutic access in many hospital settings against MDRPA. However, as this pathogen has gained already resistance even to these available drugs, identification of nosocomial strains capable of producing MBL has aroused more interest and importance in current status.  Acquired MBLs includes the VIM and IMP enzymes, of which there are several variants of the original VIM-1 and IMP-1 MBLs as well as the SPM-1, GIM-1, NDM-1, AIM-1, and SIM-1 enzymes.  The VIM and IMP enzymes are by far the most common MBLs found in carbapenem-resistant bacteria, including carbapenem-resistant P. aeruginosa.  Thus, this study was conducted to know the presence of IMP gene among our P. aeruginosa isolates.
MATERIALS AND METHODS
A total of 20 of nonrepetitive clinical isolates of P. aeruginosa were collected from Saveetha Medical College, Thandalam. They were processed for a battery of standard biochemical tests and confirmed. Isolates were preserved in semi-solid trypticase soy broth stock and stored at 4[degrees]C until further use.
Antibiotic Susceptibility Testing
Antibiotic susceptibility testing was determined for this isolates to routinely used antibiotics such as to piperacillintazobactam, cefotaxime, ceftazidime, tetracycline, cotrimoxazole, aztreonam, gentamicin, and imipenem by Kirby Bauer disc diffusion method as per CLSI guideline. 
Detection of [bla.sub.IMP] Gene In Pseudomonas Aeruginosa
P. aeruginosa isolates were detected for the presence of [bla.sub.IMP] gene by polymerase chain reaction (PCR) analysis. Detection of the gene was carried out using primer [Table 1].
Bacterial DNA was extracted by boiling lysis method. 1 [micro]L of DNA extract was used as template for PCR reaction. The reaction mixture contained 2 mM of Mg[cl.sub.2] 0.2 mM dNTP mix and 1[micro]M of [bla.sub.IMP] gene with 0.5U of Taq polymerase (New England Biolabs) in a 1 x PCR buffered reaction. A positive control of P. aeruginosa with [bla.sub.VIM] gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 98[degrees]C for 6 min and 30 cycles for 40 s, 57[degrees]C for 30 s, and 76[degrees]C for 30 s followed by a final extension of 5 min at 74[degrees]C. PCR products were resolved in 2% agarose gel. A 100 bp ladder was including in all the gel analysis. 
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, and 3/20 (15%) from pus [Figure 1].
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, aztreonam, and gentamicin isolates showed complete resistance 20/20 (100%) [Table 2].
17/20 (85%) clinical isolate of P. aeruginosa was found to possess [bla.sub.IMP] gene. L3-100bp ladder, L4- [bla.sub.IMP] gene positive [Figure 2].
Multidrug-resistant P. aeruginosa is a major cause of hospital acquired infections and known to cause a wide spectrum of life-threatening diseases. These organisms are resistant to almost all commonly available antibiotics with limited treatment options. Several mechanisms such as carbapenemase production, oprD mutation, AmpC, and efflux pumps overexpression are involved in carbapenems resistance among P. aeruginosa strains. However, the emergence of carbapenem resistant P. aeruginosa isolates has become a serious clinical concern because of its intrinsic and acquired resistance mechanisms, limiting the treatment option.
Study conducted by Ramakrishnan et al. from Puducherry reported that MDRPA isolates showed the highest resistance to carbapenems such as meropenem (84%) and imipenem (40%), which were found to be the precious weapon against MDRPA infections and this is an alarming sign. All the isolates showed 100% sensitive to polymyxin B and colistin.  In this study, we have observed complete resistance to all the antibiotics tested except imipenem which showed 85% isolates were resistance, which is in concordance with other earlier studies, however, we did not include colistin in our study. A report by Aliskan et al, with 1071 MDRPA, reported resistance to imipenem (22.5%) and meropenem (31%).  Deepak et al. during 2009-2010 with 193 P. aeruginosa reported resistance to imipenem (3.7%), which is less compared with the present study.  A study conducted by Manoharan and his colleagues in 2010, found 30% of their isolates were found to harbor IMP gene by PCR,  we also seen similar kind of result as we observed 85% of positivity of having this gene. They have already reported novel IMP type of MBL producing Pseudomonas spp. detected in India during 2006 in which strains were clustered in 33 ribotypes with clones found in multiple hospitals. 
Figure 1: Sample wise distribution of clinical isolates of Pseudomonas aeruginosa sputum 45% blood 25% urine 15% Dpus 15% Note: Table made from pie chart.
It seems that chronic underlying conditions, prolonged period of intensive care unit stay and the use of invasive techniques and devices predispose patients to infection with these resistant isolates. However, to derive a conclusion, a more number of isolates are recommended and even other types of genes are also screened. [15,16] This indicates the important role of clinical microbiology laboratories to distinguish MBL-producing P. aeruginosa from strains with other mechanisms responsible for carbapenem resistance.
Therefore, in our study, we observed increased percentage of IMP mediated carbapenem resistance in our isolates, which indicates that this gene may be found in most of the P. aeruginosa carbapenem isolates. The early detection of MBL-producing P. aeruginosa may help in appropriate antimicrobial therapy and avoid the development and dissemination of these multidrug-resistant strains.
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How to cite this article: Raj V, Pa G, Jain AR. Detection of [bla.sub.IMP] gene encoding metallo-beta-lactamase resistance among clinical isolates of Pseudomonas aeruginosa. Natl J Physiol Pharm Pharmacol 2018;8(6):775-778.
Source of Support: Nil, Conflict of Interest: None declared.
Varshan Raj (1), Gopinath Pa (1), Ashish R Jain (2)
(1) Department of Microbiology, Saveetha Dental College and Hospital, Saveetha University, Chennai, Tamil Nadu, India, (2) Department of Prosthodontics, Saveetha Dental College and Hospital, Saveetha University, Chennai, Tamil Nadu, India
Correspondence to: Ashish R Jain, E-mail: firstname.lastname@example.org
Access this article online
Received: November 23, 2017; Accepted: January 09, 2018
Table 1: Primer detail of [bla.sub.IMP] gene Primer Primer sequence Product size [bla.sub.IMP] 5'-GTT TGG CCG CAT TTC CA AC-3' 393 bp 5'-AAT GCG GAG CAC AAG GAT AG-3' Table 2: Results of antibiotic susceptibility pattern of P aeruginosa Antibiotics Sensitivity Intermediate Resistant (20) (%) (20) (%) (20) (%) Piperacillin-Tazobactam 0 (0) 0 (0) 20 (100) Cefotaxime 0 (0) 0 (0) 20 (100) Ceftazidime 0 (0) 0 (0) 20 (100) Tetracycline 0 (0) 0 (0) 20 (100) Cotrimoxazole 0 (0) 0 (0) 20 (100) Aztreonam 0 (0) 0 (0) 20 (100) Gentamicin 0 (0) 0 (0) 20 (100) Imipenem 2 (10) 1 (5) 17 (85) P. aeruginosa: Pseudomonas aeruginosa
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Raj, Varshan; Pa, Gopinath; Jain, Ashish R.|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Jun 1, 2018|
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