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Year : 2014  |  Volume : 57  |  Issue : 4  |  Page : 579-582
Molecular characterization of metallo β-lactamase producing multidrug resistant Pseudomonas aeruginosa from various clinical samples

1 Department of Clinical Microbiology, Pondicherry Institute of Medical Sciences, Puducherry, India
2 Department of Biotechnology, Pondicherry University, Puducherry, India

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Date of Web Publication11-Oct-2014


Introduction: Pseudomonas aeruginosa is a potent opportunistic nosocomial human pathogen among Gram-negative bacteria causing various life-threatening infections in patients from Intensive Care Units. This bacterium has become resistant to almost all commonly available antibiotics with limited treatment options. Multi drug resistant P. aeruginosa (MDRPA) is a major cause of concern among hospital acquired infections. It uses distinctive resistant mechanisms virtually to all the available antibiotics such as Metallo β-lactamases (MBL) production, extended spectrum β-lactamase production (ESBL), up regulation of efflux systems related genes and decreased outer membrane permeability. This study was carried out to find one the predominant resistance mechanisms among MDRPA and the prevalence of corresponding resistance genes. Materials and Methods: MDRPA isolates collected from various clinical samples for a period of 1-year (November 2009-Octo ber 2010) were included to detect the predominant mechanism of resistance using phenotypic and molecular methods. Molecular characterization of all these isolates was done by polymerase chain reaction (PCR) for the presence of blaVIM-2, blaIMP-1, blaOXA-23, and blaNDM-1 genes with specific primers. Results: Among 75 MDRPA isolates 84% (63) were MBL producers. Molecular characterization studied by PCR showed the presence of blaVIM-2 gene in 13% of MBL producers. Conclusion: The prevalence of MBLs has been increasing worldwide, particularly among P. aeruginosa, leading to severe limitations in the therapeutic options for the management. Thus, proper resistance screening measures and appropriate antibiotic policy can be strictly adopted by all the healthcare facility providers to overcome these superbugs.

Keywords: Pseudomonas aeruginosa , metallo β-lactamase genes, blaVIM-2

How to cite this article:
Ramakrishnan K, Rajagopalan S, Nair S, Kenchappa P, Chandrakesan SD. Molecular characterization of metallo β-lactamase producing multidrug resistant Pseudomonas aeruginosa from various clinical samples . Indian J Pathol Microbiol 2014;57:579-82

How to cite this URL:
Ramakrishnan K, Rajagopalan S, Nair S, Kenchappa P, Chandrakesan SD. Molecular characterization of metallo β-lactamase producing multidrug resistant Pseudomonas aeruginosa from various clinical samples . Indian J Pathol Microbiol [serial online] 2014 [cited 2023 Nov 30];57:579-82. Available from:

   Introduction Top

In the last few decades, antimicrobial resistance ratio has been increased drastically, especially among nonfermenting bacteria, which became a substantial challenge to treat. [3] Among the various nosocomial pathogens, Pseudomonas aeruginosa a soil inhabitant, and human saprophyte is well-known opportunistic human pathogen. It causes life-threatening ventilator associated pneumonia, surgical site, and urinary tract infections in patients from Intensive Care Units. [1] Infections are more severe when associated with immunosuppressive states mainly diabetes, carcinomas and also in case of burns injury and cystic fibrosis. Major risk factors being, prolonged hospitalization, ventilation, exposure to inadequate antimicrobial therapy and immunocompromised state. [2]

With improvements in antibiotic therapy and diagnosis, in recent decades P. aeruginosa has emerged as a multidrug resistant superbugs by acquiring intrinsic resistance to a number of antimicrobial agents. [2] Multi drug resistance in-turn leads to increased hospital expenditure, prolonged hospitalization, narrowing of therapeutic options, cross infection thus, landing to increased mortality and morbidity finally. This organism uses distinctive resistant mechanisms virtually to all the available antibiotics, which include metallo β-lactamases (MBL) production, extended spectrum β-lactamase production, AmpC production, decreased permeability, altered penicillin binding proteins and rarely, overexpression of efflux pumps. [4]

As carbapenems are the potent antimicrobial weapon against multi drug resistant P. aeruginosa (MDRPA), this bacterium has developed resistance even against this group of drugs by producing MBLs (carbapenemase). [5] Imipenem and meropenem among carbapenems have gained increased therapeutic access in many medical centers 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 the recent years. [6]

Carbapenemases are Class B MBLs; IMP, VIM or Class D oxacillinases (OXA 23-OXA 27) (carbapenem-hydrolyzing Class D β-lactamases or Class A clavulanic acid inhibitory enzymes (SME, NMC, IMI, KPC). Class A β-lactamases with activity again carbapenems, are uncommon and divided into five groups (GES, IMI, KPC, NMC-A and SME). ESBLs and carbapenemases are typically encoded by plasmid or transposon-borne genes, often on integron, Which are genetic elements capable of capturing and subsequently mobilizing resistance genes, although some β-lactamase genes are associated with novel mobile insertion sequences termed insertion sequence common region elements. [6] Acquired MBLs includes the VIM and IMP enzymes, of which there are numerous 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. [7],[8] The VIM and IMP enzymes are by far the most common MBLs found in carbapenem-resistant bacteria, including carbapenem-resistant P. aeruginosa. [9] Thus, this study was conducted to know the prevalence of MBL producing multidrug resistant P. aeruginosa and the molecular characterization of prevalent genes present in them in order to improve the therapeutic options and to decrease the morbidity and mortality.

   Materials and methods Top

Study design

Between November 2009 and October 2010 (1-year), a prospective study was conducted with various clinical samples in the Department of Clinical Microbiology from a tertiary care hospital. A total of 226 P. aeruginosa was isolated, out of these, 75 were multi drug resistant (resistant to three or more groups of antibiotics by in vitro Kirby Bauer disc diffusion method). [10] Inclusion criteria: All clinical isolates of P. aeruginosa from inpatients, which were found to be multidrug resistant (total of 75 MDRPA) were included in this study for further characterization. Repeated isolates from the same patients were excluded.

Cultures identification and characterization

Antibiotic susceptibility pattern for all these isolates were done by Kirby Bauer disc diffusion method. Isolates were confirmed for MBL production by EDTA double disc synergy test [10] and Molecular characterization for these isolates were done by polymerase chain reaction (PCR) for the presence of VIM 2, IMP 1, OXA 23 and NDM 1 genes. ATCC P. aeruginosa 27853 was used as a control strain for all the procedures.

Polymerase chain reaction amplification of resistance genes

The genomic DNA was extracted from all the 75 MDRPA isolates followed by agarose gel electrophoresis. The isolated DNA was used as a template for amplification of specific genes described below. PCR amplification was done with specific gene primers and checked for the presence of the corresponding gene responsible for MBL production such as blaVIM-2, blaIMP-1, blaOXA-23, and blaNDM-1 genes. The PCR amplification was performed with Eppendorf Master cycler.

Amplification of the resistant genes were carried out with the following reaction mixture composition (10 μl): DNA template (50 ng), 1 μl each of deoxynucleotide triphosphates (2.5 μM), Taq buffer (10), forward and reverse primers (2.5 μM) and 1 U of Taq DNA polymerase (Merck Biosciences, Darmstadt, Germany) [Table 1]. All PCR amplifications were performed using thermal cycler (Veriti Thermal cycler, Applied Biosystems, USA.) using the following conditions for 30 cycles: 94 °C for 5 min, 94 °C for 45 s, annealing at 54 °C for 30 s and extension at 72 °C for 45 s. The PCR products were analyzed on 1% agarose gel, stained with ethidium bromide, and the amplicons were purified using HiPura PCR product purification kit (Himedia, Mumbai, India) and sent for custom sequencing (Macrogen Inc., Seoul, Korea).
Table 1: List of primers used

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Statistical analysis

All the data were entered in Microsoft Excel sheet and the results were analyzed by SPSS software. (IBM, USA)

   Results Top

Out of 75 MDRPA, carbapenemase resistance was documented among 84% (63) toward meropenem and 40% (30) toward imipenem, respectively. All these 75 isolates showed 100% susceptibility toward polymxin B and colistin by Kirby Bauer disc diffusion method. The minimum inhibitory concentration (MIC) of meropenem for resistant strains ranged from 8 μg/ml to >64 μg/ml. (Break point MIC-≤4 μg/ml to ≤16 μg/ml). A total of 84% (63) MBL production was observed.

Among the MBL producers, 13.33% (10) of MDRPA showed presence of VIM 2 gene [Figure 1] and only one (1.33%) was positive for IMP 1 gene [Figure 2]. Finally, none of the 75 MDRPA have produced OXA 23 and NDM genes in our study.
Figure 1: Polymerase chain reaction amplification of blaVIM-2

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Figure 2: Polymerase chain reaction amplification of blaIMP-1

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   Discussion Top

Multi drug 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.

In the present study, MDRPA isolates showed the highest resistance to carbapenems like 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 2008, a study by Alis‚ kan et al. with 1071 MDRPA, reported resistance to imipenem (22.5%) and meropenem (31%). [11] Deepak et al. during 2009 to 2010 with 193 P. aeruginosa reported resistance to imipenem (3.7%), which is less compared with the present study. [12] Minimum Inhibitory concentration of meropenem ranged from 8 μg/ml to >64 μg/ml, which is comparable to other studies. About 63.33% of these MDRPA isolates showed higher MIC to meropenem. Shashikala et al. with carbapenem resistant P. aeruginosa had reported MIC ranging from 8 μg/ml to 64 μg/ml. [13] In a study by Fernαndez et al. higher MIC of 128 μg/L for meropenem got documented. [14] All these resistance ranging pattern is mostly directly dependent on various factors, which mainly includes the antibiotic policy in practice in the respective healthcare setups.

As MBLs production is the major mechanism of resistance among MDRPA, Navaneeth et al. reported 12% MBL production in P. aeruginosa. [15] Another recent study by Varaiya et al. showed 20.8% of MDRPA were found to be MBL producers. [16] Upadhyay et al. reported 46.6% of MBL production among MDRPA strains. [17] Another study by Jayakumar et al. reported 54.5% MBL producers. [18] In our present study, we reported a very high prevalence of 84% MBL producers. This is less comparable with other studies. Remaining MDRPA strains may be harboring some other resistance mechanism like, ESBL production, AmpC production, biofilm formation or through various virulence factors.

In P. aeruginosa number of different β-lactamases has been described including MBL, ESBL and OXA production. This present study investigated the predominant β-latamase coding genes such as, VIM-2, IMP-1, OXA-23 and NDM-1 through PCR. Among MBL producing isolates in our study, the presence of VIM-2 gene is predominant when compared with IMP-1 MBL gene. Surprisingly, none of our isolates were positive for OXA-23 and NDM genes. The presence of VIM-2 gene appears to be more prevalent in our setup, wherein 13% of isolates were positive for VIM-2 MBL.

In 2007, the first case of MBL gene blaVIM-2 was reported in a strain isolated in India. This VIM-2, which is present on integron had its ancestral Class I integron documented in United States and Russia. [1],[19],[20],[21] This Class I integron having 3' conserved sequence have arisen from an ancestral integron predating the formation of 3' conserved, which was found in United States and Russia. The present study documented occurrence of blaVIM-2 among its collection, wherein 10 isolates of its collection were positive for blaVIM-2 . Interestingly, the DNA sequence analysis of all our VIM-2 showed 100% identity with the sequence of global genotypes retrieved from the GenBank public database. This finding suggests successful global dissemination of VIM-2 resistant gene that is of great concern. VIM-2 gene was found to be more prevalent among MDRPA in our setup as revealed by PCR method.

Fortunately in our setup we encountered very less prevalence of resistance genes among our P. aeruginosa isolates when compared to rest of the world, wherein, high incidence of MBL have been reported. From the results obtained through our investigation, it can be concluded that VIM-2 was the most frequently isolated β-lactamase genes among the P. aeruginosa isolated from Pondicherry. The sequencing results further confirmed, there is less variance among our β-lactamase genes when compared to global genotypes.

The present investigation also shows that, multidrug resistant P. aeruginosa is still a gigantic problem in the hospital setup. MDRPA infections are likely to affect critically ill patients who require prolonged hospitalization. Infections with MDRPA are also associated with adverse clinical outcome. Strict isolation of patients infected with MDRPA and judicial use of antibiotics should be emphasized in order to prevent the spread of MDRPA infections. Further, more clinical studies are needed to identify risk factors for MDRPA development and to determine the economic impact of these infections, as well as to determine the most efficacious antimicrobial regimens and duration of therapy to maximize the outcome of MDRPA infections.

   Conclusion Top

The present study gives the alarming sign toward the high prevalence of carbapenem resistant nonfermenting pathogens. Thus, this calls for stringent preventive measures, which includes strict infection control practices and judicious use of antibiotics with implementation of antibiotic policy. These kind of important measures might overcome the challenge of high mortality posed by MDRPA and other nonfermenting bacterial pathogens.

   Acknowledgment Top

We acknowledge our Professor and Head, Department of Microbiology, Dr. Mary V. Jesudasan for her guidance.

   References Top

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Correspondence Address:
Kalaivani Ramakrishnan
No.37, Marie Street, Bahour, Puducherry - 607 402
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.142670

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