Introduction
Pseudomonas aeruginosa is a Gram-negative bacterium with the ability to persist in both community and hospital settings. It is an important nosocomial pathogen with emerging resistance to many effective groups of antibiotics through both intrinsic and acquired resistance mechanisms. Carbapenems are the drug of choice for infections caused by Pseudomonas aeruginosa including other Gram-negative organisms.1 Global emergence of carbapenem resistance Pseudomonas aeruginosa (CRPA) including resistance to beta-lactams, Aminoglycosides, and Fluoroquinolones represents an extraordinary threat to public health. The major mechanisms of carbapenem resistance in Pseudomonas aeruginosa include carbapenem hydrolyzing enzymes i.e. carbapenemase, decrease outer membrane permeability and alteration penicillin-binding proteins.2 However, the clinical use of these antimicrobials is under threat with the emergence of carbapenemases, particularly Metallo- ß-lactamases (MBLs).2 MBL belongs to Ambler class B, and these enzymes can hydrolyze a wide variety of beta-lactam agents, such as penicillin, cephalosporins, and carbapenems. They require zinc for their catalytic activity and are inhibited by metal chelators, such as EDTA and thiol-based compounds.3 MBLs in P. aeruginosa were identified in 1991 in Japan4 and since then have been described from various parts of the world, including Asia, Europe, Australia, South America, and North America4, 5 whereas in India blaVIM, blaIMP and blaNDM genes are frequently encountered in P. aeruginosa.6
The genes responsible for the production of MBLs are typically part of an integron structure and are carried on transferrable plasmids but can also be part of the chromosome.7 Due to integron-associated gene cassettes, MBL producing P. aeruginosa isolates are often resistant to different groups of antimicrobial agents, which can be transferred to another Gram-negative bacteria.8
MBLs in Carbapenem-resistant Pseudomonas aeruginosa can be detected by different phenotypic methods and these methods are based on the ability of metal chelators to inhibit the activity of MBLs such as EDTA and thiol-based compounds.9 These include Combined Disk Test (CDT)9 using EDTA with imipenem (IPM), Modified Hodge test (MHT), MBL Epsilometer test (E-test) and EDTA disk potentiation test.10, 11 A PCR detection assay is considered as a gold standard method for the detection of MBL producers.12 Because of the increasing rate of resistance to the carbapenems, the treatment of infections produced by MBLs producing P. aeruginosa is becoming critical.
Hence the present study was undertaken to detect presence of MBLs among Carbapenem-Resistant Pseudomonas aeruginosa (CRPA) isolates by phenotypic methods like MHT and Combined Disk Test (CDT) by using Imipenem (IPM)- Ethylene diamine tetraacetic acid (EDTA) and to identify the MBL genes (blaVIM and blaIMP) coding for carbapenemase resistance by conventional PCR in a tertiary care hospital in Bengaluru.
Materials and Methods
This study was conducted over a period of one year from January 2018 to December 2018 at a tertiary hospital, BMCRI, Bengaluru. Ethical clearance was obtained from the institutional ethical committee. This study included 91 non-consecutive clinical samples from patients hospitalized for 48 hours and more, received for culture and sensitivity in the Department of Microbiology.
All the clinical specimens were subjected to direct microscopy, growth on culture media and series of tests for identification of P. aeruginosa. These isolates were subjected to antimicrobial susceptibility testing by Kirby-Bauer disk diffusion method according to CLSI guidelines.13 The antibiotics tested include amikacin (30µg), ciprofloxacin (5µg), ceftazidime (30µg), piperacillin-tazobactam (100/10µg), imipenem (10µg), meropenem (MEM-10µg), aztreonam (30 µg), colistin (10 µg) and polymyxin-B (300 units). All the disks were obtained commercially (Hi-Media Laboratories Limited. Mumbai, India). ATCC strain of Pseudomonas aeruginosa 27853 is used as control.
All isolates resistant to imipenem or meropenem or ceftazidime or any two of them were considered as probable MBL producer. All positive isolates were further tested by two phenotypic tests for the MBL detection, described as follows.
Imipenem- Ethylene diamine tetraacetic acid combined disc test
A lawn culture of test isolate was prepared. Allowed to dry for five minutes. Two imipenem (10 µg) discs, one with 0.5 M EDTA and other a plain imipenem disc, were placed on the surface of agar plates approximately 30mm apart. The plates were incubated overnight at 37oC for 16-18h. An increase in zone diameter of >7mm around the imipenem-EDTA disc in comparison to imipenem disk alone indicates the production of MBL.9
Modified Hodge Test (MHT)
A saline suspension of a 0.5 McFarland standard of E. coli ATCC 25922 was prepared and diluted 1:10 and lawn inoculated on Muller Hinton Agar (MHA). The plate was allowed to dry for 3-10 minutes. An imipenem (10 µg) disk was placed at the center and 3-5 colonies of test organisms were inoculated in a straight line drawn out from the edge of the disk. A known NDM positive strain was used as Positive control and incubated overnight at 350C for 20-24h. the presence of a distorted zone of inhibition or cloverleaf type of indentation at the intersection of the test organism and E. coli. within the zone of inhibition of the IPM disk was interpreted as a positive result.10, 11
PCR for carbapenem encoding gene
Isolates tested positive in the phenotypic test are subjected to conventional PCR for detection genes coding for MBLs. The DNA extraction was done by the crude method (boiling method). Freshly subcultured colonies were suspended in 50 µl of PCR grade water, heated to 990C for 10 minutes in a water bath and kept at room temperature for 5 minutes. The suspension is centrifuged at 14000 rpm for 1min at 40C, 5 µl of supernatant was used as the template for a 50 µl PCR reaction. PCR was performed by using previously designed primers (Sigma-Aldrich, Bengaluru) for blaVIM, and blaIMP (Table 1)14
Amplification was performed in 50 µl PCR mixture consisting of master mix (25 µl)- Taq DNA polymerase 2x master mix RED 1.5 mM MgCl2 (Synergy Scientific Service PVT. LTD), PCR grade water (18µl), Primer blaVIM, and blaIMP F’ and R’ (1µl each) and DNA (5µl).
DNA was amplified in a Master cycler Eppendorf under the following conditions (Table 1) and PCR products were kept at 40C. Known blaVIM, and blaIMP, producing laboratory strain Pseudomonas aeruginosa was used as the positive control. Pseudomonas aeruginosa ATCC 27853 reference strain was used as the MBLgene’s negative strain. Cycling conditions for MBL (blaVIM and blaIMP) gene were described (Table 2).12
The PCR products were analyzed by gel electrophoresis with 2% agarose gel in TAE (tris-acetate buffer) buffer with1.5µl ethidium bromide and were visualized and photographed under ultraviolet illumination.
Table 1
Primer sequence used in PCR and their product size as follows.14
Table 2
Cycling conditions for different gene identification12
Results
Among ninety-one CRPA isolates included in the study 70.3% were from the pus, 12.08% from blood and body fluids, 6.5% from urine, 3.3% from respiratory samples and 3.29% from others (Corneal and ENT). Distribution of isolates according to wards as follows, General surgery (42.8%), burns and plastic surgery (27.4%), ICUs (9.8%), orthopedics (4.3%), Nephro-urology (4.3%) and others -ENT and ophthalmic (2.19%).
The antimicrobial susceptibility testing of CRPA isolates showed resistance to other antimicrobials including ceftazidime and cefepime(95.6% each), ciprofloxacin and levofloxacin (91.2% each), piperacillin(94.5%), ticarcillin (96.7%), piperacillin-tazobactam(86.8%), amikacin(83.5%), gentamicin(86.8%), aztreonam(80.2%), whereas only 12.08% isolates were resistant to colistin (Figure 1).
Figure 1
Antimicrobial susceptibility test in Carbapenem resistant pseudomonas aeruginosa (CRPA) isolates. Antimicrobial susceptibility test in carbapenem resistant pseudomonas aeruginosa (CRPA) isolates
MBL phenotypic screening tests
Among the two phenotypic tests, Combined Disc Test using imipenem and EDTA was positive in 84 isolates and Modified Hodge Test was positive in only 12 isolates. In this study, CDT showed high sensitivity (92.3%) while MHT was least sensitive (13.18%) (Figure 2).
Discussion
Antimicrobial resistance is a significant route cause of healthcare-associated infections all over the world. Carbapenem has been used as the last choice of treatment of many Gram-negative organisms. In the current study, we have included carbapenem-resistant P. aeruginosa isolates. Carbapenem resistance among Gram-negative bacteria has been increased in recent years in the Indian subcontinent and MBL producing isolates have emerged worldwide and are associated with outbreaks in health care settings over the past few years.
In this study, most of the carbapenem-resistant Pseudomonas aeruginosa isolates were from pus (70.3%) and from general surgery ward (42.8%) and most of them were resistant to other class of antibiotics such as Anti-pseudomonal Penicillin’s, Aminoglycosides, Fluoroquinolones, Cephalosporins, and Aztreonam. This indicates the concomitant presence of other beta-lactamases. Polymyxins showed susceptibility of 87.9% (n=91) which was high compared to other classes of antibiotics and hence considered as a treatment option of CRPA isolates.
In this study, MBL production was screened by the Modified Hodge test and Combined Disk Test by using Imipenem-EDTA. Out of 91 isolates, MHT was positive in 84 isolates (92.3%) and MHT was positive in 12 isolates (13.18%). PCR detected MBL genes blaVIM/blaIMP in 20.2% (blaVIM-16 and blaIMP-1) among those positive isolates by phenotypic tests. This coincides with a high prevalence of blaVIM among Pseudomonas isolates, a study by Amudhan M et al.14 The remaining 71 isolates that were MBL phenotypic test positive, were negative for both MBL genes (blaVIM/blaIMP) suggesting the presence of other MBL genes such as SIM-1, GIM-1, NDM-1 or SPM-1.
Despite the good performance of inhibitor-based methods (Combined disk test using IMP-EDTA) for the detection of MBL, false-positive results were found, hence this is not considered as a specific test. False positives may be due to EDTA which acts on the membrane of a bacterial cell and increases cell permeability. MHT also showed very low sensitivity. Hence the results of the MBL phenotypic tests must be interpreted cautiously.
The overall blaVIM/blaIMP production among study isolate was 20.2% among them, blaVIM was found in 16 isolates whereas blaIMP was found in one isolate. In Asia MBL genes, blaVIM and blaIMP are prevalent and blaIMP is found in Japan, Korea, China, Taiwan and Iran.15, 16, 17
The prevalence of MBL genes in India ranges from 7-65% among P. aeruginosa. In a study from India showed, the rate of MBL production was 24.5% among 61 P. aeruginosa isolates and blaVIM was the most common. Another study from India also reported blaVIM -2 from P. aeruginosa.18 In a nationwide survey conducted to characterize 301 MBL producing Pseudomonas species in 10 medical centers from India, the MBL genes were detected in 18.9% isolates.19 There are fewer data available on the prevalence and distribution of MBL among Indian isolates.
In this study, MBL phenotypic tests were positive in 92.3% isolates and among them, 20.2% isolates were carrying blaVIM/blaIMP genes indicating carbapenemase production. So, early detection of MBL production in Carbapenem-resistant Pseudomonas aeruginosa will not only help in treating the infections caused by them adequately and also help in preventing the spread of multidrug resistance to other Gram-negative strains. Therefore, all clinical isolates that are resistant to carbapenem must be screened for MBL production by using simple phenotypic tests and confirmed by the MBL Epsiolometer test (E-test) or by PCR if possible.
To conclude, carbapenem resistance in P. aeruginosa is chiefly mediated by Metallo-beta-lactamase production. Among the two phenotypic tests performed in this study, CDT was more sensitive compared to the Modified Hodge Test in the screening of MBL production. Genotypically, the common MBL gene -found was blaVIM compared to blaIMP. Therefore, screening for MBL production in microbiology laboratories is crucial for optimal treatment of patients, particularly hospitalized patients and also to prevent the possible spread of resistance to other Gram-negative organisms because of their broad-spectrum drug resistance which creates a therapeutic challenge to clinicians. Finally, to understand the epidemiology, there is a need for genetic analysis and also typing of Metallo-ß-lactamase enzymes.
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