Introduction:
With the emergence of carbapenemases, particularly Ambler class B metallo-ß-lactamases (MBLs) the utility of carbapenems is under threat. Such enzymes have emerged in many geographical locations1 and often confer high-level resistance to all ß-lactams except aztreonam. Carbapenems have been the drugs of choice for treatment of infections caused by MDR because of its broad spectrum activity.2 Metallo-ß-lactamase was first detected in Bacillus cereus in 1960s and was chromosomal in location. Since then, MBL encoding genes have been reported all over the world among fermenters and non fermenters gram negative bacilli.3 The MBL producing strains are frequently resistant to aminoglycosides and fluoroquinolones but remain susceptible to polymyxins. Unlike carbapenem resistance due to several other mechanisms, the resistance due to MBL and other carbapenemase production has a potential for rapid dissemination, as it is often plasmid mediated.1 MBLs spread easily and cause nosocomial infections and outbreaks. Such infections mainly concern patients admitted to Intensive Care Units with several co-morbidities and a history of prolonged administration of antibiotics. There was no standard guideline for screening of Carbapenemases though lot of authors have described various phenotypic methods for its detection. PCR gives reliable specific and sensitive result for gene coding for carbapenem resistance4, but due to cost and labour constrain this method is of limited practical use for daily application. A simple and inexpensive testing method for screening of carbapenemase producers is the need of hour. Therefore this study was undertaken to detect MBLs in clinical isolates by a low cost, convenient and sensitive procedure.
Materials and Methods:
This was a cross sectional study conducted in the Department of Microbiology, for a period of 24 months. Total 989 patients received mechanical ventilator support during the study who were admitted in the ICUs; Respiratory Intensive Care Unit(RICU), Surgical Intensive Care Unit (SICU), Medical Intensive Care Unit (MICU)and Intensive Cardiac Care Unit (ICCU). Among them 669 patients were on ventilator for more than 48 hours. Out of 669 patients, 100 Ventilator Associated Pneumonia (VAP) patients were included in the present study who satisfied the Clinical Pulmonary Infection Score (CPIS) >6.5,6 Samples of these patients were sent to the microbiology laboratory for routine culture identification and sensitivity testing. The isolates were identified based on standard bacteriological techniques7,8 and were screened for meropenem resistance by Kirby-Bauer disk diffusion method according to CLSI guidelines.9
Phenotypic detection of Metallo-betalactamase was done by performing three methods:
1. Modified Hodge Test (MHT)
2. Combined disc test (CDT: Meropenem and Meropenem+EDTA)
3. Double Disc Synergy Test (DDST: Meropenem and EDTA)
Modified Hodge Test10-14
A 0.5 McFarland matched suspension of E.coli ATCC 25922 was diluted 1:10. From this diluted suspension lawn culture was done on Muller Hinton agar (MHA) plate with sterile cotton swabs. The plate was allowed to dry for 3-4 minutes at room temperature. A 10 mcg meropenem disc (BD Company) was placed at the centre and test organism was streaked in a straight line from edge of the disc. Plate was incubated at 37ºc for 24 hours in ambient air. The presence of clover leaf type of indentation at the intersection of the test organism and ATCC E.coli 25922, within the zone of inhibition of meropenem susceptibility disc was interpreted as positive result as per CLSI guideline 2013.(Figure 1)
 |
Figure1: Modified Hodge Test - Positive |
Combined Disc Test (CDT)11,13
0.5 M EDTA solution was prepared by dissolving 18.61 gram of disodium EDTA.2H2O in 100 ml of distil water and its pH was adjusted to 8.0 by using NaOH. The mixture was then sterilized by autoclaving. 10µl EDTA solution was poured on meropenem disk to obtain a desired concentration of 1900 µg per disc. Two to three identical colonies of test organism were inoculated in to nutrient broth and incubated at 37ºc for 4-6 hours then turbidity was adjusted to 0.5 Mcfarland. Lawn culture of this suspension of test organism was done on Muller Hinton Agar (MHA) plate with a sterile cotton swab. One 10µg meropenem disk was placed on MHA plate. EDTA impregnated meropenem disc was also placed on the same MHA plate at the distance of 20-25 mm from centre to centre. The plate was incubated at 37ºc for 16-18 hours. An increase in zone size of atleast 7mm around the meropenem –EDTA disc compared to meropenem without EDTA was recorded as an MBL producing strain.(Figure 2)
 |
Figure 2: Positive CDT Test |
Double Disc Synergy Test (DDST)12,13
A Lawn culture was done on to Muller Hinton Agar (MHA) plate from 0.5 McFarland matched suspension of Test organism by using sterile cotton swab. A meropenem disc (10µg) and one sterile blank disc (6mm size) was placed on Muller Hinton Agar plate (MHA) plate at a distance of 20 mm from centre to centre. 10µl of 0.5 M EDTA solution was put on to sterile blank disc to obtain 1900µg/disc concentration. Plate was kept for incubation at 35ºc at ambient air for 24 hours. Enhancement of zone of inhibition in the area between Meropenem and EDTA disc in comparison with the zone of inhibition on the far side of the drug (meropenem) was interpreted as a positive result.(Figure 3)
 |
Figure 3: Positive DDST Test |
Statistics: No statistical method was required for screening method
Results
Out of 126 gram negative bacterial isolates, 80 isolates (63.49%) showed resistance to carbapenem group of drugs (meropenem & imipenem). Among them maximum resistance was shown by Acinetobacter baumanii (90.32%), followed by Klebseilla pneumoniae (45.7%), Pseudomonas aeruginosa (26%).(Table 1)
| Table 1: Susceptibility pattern of gram negative bacilli to carbapenems |
Organisms |
Isolates number |
Carbapenem resistance (%) |
Carbapenem sensitive (%) |
Acinetobacter baumanii |
62 |
56 (90.32%) |
06 (9.68%) |
Klebseilla pneumoniae |
35 |
16 (45.7%) |
19 (54.3%) |
Pseudomonas aeruginosa |
23 |
06 (26%) |
17 (74%) |
Escherichia coli |
01 |
00 (00%) |
01 (100%) |
Klebseilla oxytoca |
02 |
01 (50%) |
01 (50%) |
Serratia marcesence |
01 |
00 (00%) |
01 (100%) |
Citrobacter species |
02 |
01 (50%) |
01 (50%) |
Total no |
126 |
80 (63.49%) |
46 (36.51%) |
Out of 80 isolates, 66 were positive for MBL by MHT, 64 by CDT and 61 were detected positive for MBL by DDST (As in Graph 1). These three methods were done for detection of metallo-betalactamases (MBL) & it was found that Modified Hodge Test (MHT) & Combined Disc Test (CDT) are better method of choice for MBL detection as compared to DDST. In the present study, most of the carbapenem resistant A.baumanii isolates were MBL producer. Among A.baumanii 49 isolates were MBL positive by MHT test, 47 isolates by CDT test and 45 isolates by DDST. While in case of K.pneumoniae; 10 isolates were MBL positive by MHT test and by CDT test where as 9 were positive by DDST (Table 2). All the carbapenem resistant isolates of P.aeruginosa and K.oxytoca were detected MBL positive by all three methods.
| Table 2: Results of modified Hodge test, combined disk test and EDTA disc synergy test |
Bacteria
|
Number of meropenem resistant isolates |
No. of positives (%) |
Modified Hodge test |
Combined disc test |
EDTA disc synergy test |
A.baumannii |
56 |
49 (87.5%) |
47 (84%) |
45 (80.3%) |
K.pneumoniae |
16 |
10 (62.5%) |
10 (62.5%) |
9(56.25%) |
P.aeruginosa |
06 |
6 (100%) |
6 (100%) |
6 (100%) |
K.oxytoca |
01 |
1 (100%) |
1(100%) |
1 (100%) |
Citrobacter spp. |
01 |
0 |
0 |
0 |
 |
Graph 1: MBL detection by different phenotypic methods |
Discussion
Since discovery of Penicillin antibioticsopened a new door in field of medicine, ß lactam antibiotics such as carbapenems are one of the potent drugs to treat infection by MDR. Prevalence of carbapenemase among gram negative bacilli varies greatly from country to country and among different institutions within the country. In the present study out of 80 isolates, 82.5%, 80%, 76.25% were positive for MBL by MHT, CDT and DDST respectively. Our findings are concordant with Behra B et al, who found 76.19% isolates positive for MBL by CDT and 57.14% isolates positive for MBL by DDST.15 Similar results were reported by Amudhan S M et al, who found 97.4% isolates positive for MBL by MHT and 79.31% isolates positive for MBL by CDT.16 In other studies done by Franklin et al17 showed 79% positive isolates by DDST and 100% by CDT. In the present study maximum MBL production was seen in A.baumannii by all three methods. In other study done by Manoharan18 et al showed 65% prevalence of MBL in P.aeruginosa while in Acinetobacter spp, the MBLs prevalence was found to be 50% by Manikal et al19study. In other study done by Enas Ghoneim et al20 MBL was produced by 61.1% of Pseudomonas isolates and 80% of Acinetobacter isolates.
Carbapenem hydrolyzing enzymes are most commonly seen in non-fermenter gram negative organisms. In the recent years there is an increasing incidence of these enzymes in enterobacteriaceae family as well. Intravenous colistin combined with rifampin and imipenem was recommended for the treatment of carbapenem-resistant isolates lacking MBLs, whereas the combination of colistin and rifampicin (with or without tigecycline) was recommended for treatment of MBL producing carbapenem-resistant isolates.20,21
Conclusion
High rate of MBL producing gram negative bacteria in this study emphasizes on the need for active surveillance in the microbiology laboratories for the detection of these resistant strains and also stresses on the judicious use of carbapenems to prevent the spread of resistance. MHT and CDT were found equally efficient method to detect MBL followed by DDST. Main disadvantage in case of Double Disk Synergy Test was subjective interpretation of result. Maximum MBL production was detected in Acinetobacter baumanii and Pseudomonas aeruginosa. There is need of simple and accurate test for MBL detection to prevent spreading of infection with nosocomial strain in hospital settings. E-test and PCR are other method for MBL detection but due to their high cost, not feasible in routine laboratory practice. Hence CDT and MHT can be used as alternative method for testing in laboratory to monitor the emergence of MBLs which are economical and easy to perform. Controversies exist regarding the choice of optimal laboratory method. Microbiology laboratories must be prepared to screen for MBL-producing isolates by a low cost, convenient and sensitive procedure.
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