Emerging resistance to carbapenem among Gram-negative bacteria in a tertiary care hospital

The emergence of bacterial antibiotic resistance is a cardinal concern in the health care system. The spread of resistance in Enterobacteriaceae and nonfermenters to the currently available drugs make the treatment of serious nosocomial infections troublesome. The purpose of the study is to ind out the carbapenem resistance among Gram-negative bacilli in a tertiary care hospital. Antibiotic susceptibility pattern of 1913 aerobic Gram-negative bacilli isolated from clinical samples wasmade for a period of 6months. All the isolates were tested for susceptibility to antibiotics by the Kirby-Bauer disc diffusion technique according to CLSI guidelines. Carbapenemase production was conirmed by theModi iedHodge Test (MHT).Minimum Inhibitory Concentration (MIC) by Epsilometer (E) testwas performed (for Imipenem andMeropenem) for carbapenem-resistant strains. A total of 1731 clinical samples, 1913Gramnegative bacilli were isolated. 1476 (77.1%) were Enterobacteriaceae and 433 (22.6%)werenon-fermenters. 54were carbapenemase-producingGramnegative bacilli. Meropenem E test was done for carbapenemase-producing Gram-negative bacilli. Theminimum inhibitory concentration forMeropenem ranged from 0.002μg/ml to 32μg/ml. To overcome the problem of emerging resistance, combined interaction and cooperation of microbiologists, clinicians and the infection control team is needed.


INTRODUCTION
One of the major burdens in the health care system is the emergence of antibiotic resistance. The spread of resistance among Enterobacteriaceae and non-fermenters to the currently available drugs makes the treatment of severe nosocomial infec-tions complicated (Paterson, 2006). Carbapenemase has high hydrolytic activity against penicillins, cephalosporins and carbapenems (John and Balagurunathan, 2011). Acquired resistance has been reported in E.coli, Klebsiella pneumoniae, Pseudomonas spp, Acinetobacter spp and various other non-fermenter Gram-negative bacilli (Zavascki et al., 2013).
Carbapenemase gene detection by a molecular method is the gold standard but is available in only a few reference laboratories and phenotypic tests have therefore been developed. The Modiied Hodge Test (MHT) is the phenotypic test recommended by the Clinical and Laboratory Standards Institute (CLSI, 2013) for carbapenemase screening method (Birgy et al., 2012). The purpose of this study was to determine the carbapenem resistance among Gram-negative bacilli isolated from clinical samples and to con irm the carbapenemase pro-duction by Modi ied Hodge Test (MHT) and Minimum Inhibitory Concentration (MIC) by E test for carbapenem-resistant strains.

MATERIALS AND METHODS
The study was performed in a tertiary care hospital in Tamil Nadu for a period of 6 months. 1913 aerobic gram-negative bacilli were isolated in the clinical microbiology laboratory from various samples. The study was reviewed and approved by the Institutional Ethical Committee.

Modi ied Hodge Test (MHT)
The imipenem resistant strains were subjected to the Modi ied Hodge test for the detection of carbapenemases. A 0.5 McFarland dilution of the Escherichia coli ATCC 25922 was inoculated by lawn culture on the surface of Mueller-Hinton agar (MHA). After drying, 10µg imipenem disc was placed at the center of the plate and the 0.5 McFarland dilution of the test strain was streaked from the edge of the disc to the periphery of the plate in four different directions. The plates were incubated overnight at 37 • C. The presence of a 'cloverleaf shaped' zone of inhibition due to the production of carbapenemase production by the test strain was considered as positive (Chande et al., 2013).

Minimum Inhibitory Concentration (MIC) by E psilometer test
MIC to Imipenem and Meropenem of isolates which are positive for carbapenemase activity were tested by using E strips and inhibition ellipses around the strip were measured (Nair, 2013).

RESULTS AND DISCUSSION
In the present study, an attempt was made to know the rate of carbapenemase-producing aerobic Gramnegative bacilli and to know their antibiogram in a tertiary care hospital. A total number of Gramnegative bacilli isolated from 1731 clinical samples during the study period of 6 months was 1913. Out of the 1731 clinical samples, 1088 (62.8%) isolates from urine samples, 517 (29.9%) isolates from exudates samples, 92 (5.3%) isolates from respiratory samples and 34 (2%) isolates were from blood samples. Among these 1913, Escherichia coli was the commonest followed by Klebsiella spp and Pseudomonas spp (Figure 1). In this study, it was found that about 77.1% of Gram-negative bacterial isolates belonged to Enterobacteriaceae, which was almost similar to that of the study done in Pakistan (Saghir et al., 2009) with 63%.  Figure 3).
Among the 54 clinical samples from which carbapenem-resistant organisms were isolated,   In our study, among the carbapenemase producers, Pseudomonas spp (37%) was the predominant one

Figure 4: Minimum Inhibitory Concentration (MIC) of Meropenem by E test
followed by Acinetobacter spp (33.3%), as shown in Table 1. But in a study for simple screening for carbapenemase, 25.64% of Pseudomonas spp were found to be carbapenemase producers (Noyal et al., 2009). The percentage of carbapenem resistance from different literatures is outlined below in Table 3.
Minimum inhibitory concentration for Meropenem ranged from 0.002µg/ml to 32µg/ml (Figure 4). Meropenem E test was done for all the 54 carbapenemases producing Gram-negative bacilli [ Table 2]. The MIC of Meropenem for resistant strains of Pseudomonas spp and Acinetobacter spp is > 16µg/ml. The MIC value of meropenem for resistant strains of members of the Enterobacteriaceae is >4µg/ml. The strains of Acinetobacter spp with MIC of 0.75µg/ml for meropenem were sensitive to aminoglycosides, nitrofurantoin and combination of beta-lactamase inhibitors with cephalosporins. These strains were resistant to third-generation cephalosporins and imipenem only.
All Meropenem resistant isolates were resistant to Imipenem, but not all Imipenem resistant organisms were resistant to Meropenem. Some of the imipenem resistant strains of Acinetobacter spp showed MIC < 4µg/ml (sensitive). This could be because the mechanisms of resistance to these two drugs are different (Quale et al., 2003). In another study, among the 21 carbapenemases producing Klebsiella pneumoniae, 7 strains were found to have 0.5µg/ml MIC for meropenem, which is sensitive (Chande et al., 2013). In a similar study, out of 36 multidrug-resistant Acinetobacter spp, 5 strains that were resistant to meropenem by the disk diffusion method were found to have MICs to meropenem in the sensitive zone (John and Balagurunathan, 2011).

CONCLUSION
In this study, the Modi ied Hodge Test and test for Minimum Inhibitory Concentration were used for detecting carbapenemase production and con irming resistance to carbapenem. Modi ied Hodge Test proved to be a good screening test for the detection of carbapenemase production as the results obtained with this study correlates with other studies. To overcome the problem of emerging resistance, combined interaction and cooperation of microbiologists, clinicians and infection control teams are needed.