One of the most important mechanisms of microbial resistance to β-lactam antibiotics (penicillins, cephalosporins, monobactams, and carbapenems) is hydrolysis by β-lactamases. Genes

Simple screening tests for detection of carbapenemases in clinical isolates of nonfermentative Gram-negative bacteria

M.J.C. Noyal, G.A. Menezes, B.N. Harish, S. Sujatha & S.C. Parija

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & ResearchPuducherry, India

Received April 10, 2008

Background & objectives: The production of carbapenemases is an important mechanism responsible for the carbapenem resistance. A simple and inexpensive testing method for screening of carbapenemase producers is essential. A prospective study was undertaken to detect metallo-β-lactamases (MBLs) and AmpC β-lactamases in nonfermentative Gram negative bacteria and to evaluate the various methods for detection of carbapenemases and MBLs.

Methods: A total of 100 Acinetobacter spp. (78 A. baumannii and 22 A. baumannii and 22 A. baumannii A. lwoffi i) and 140 Pseudomonas spp. (103 P. aeruginosa and 37 other Pseudomonas spp.) were screened for meropenem resistance by Kirby-Bauer disc diffusion method. Modifi ed Hodge test, EDTA disk synergy (EDS) test and AmpC disk test were used for the detection of carbapenemases, MBLs and AmpC β-lactamases, respectively.

Results: Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffi i, 32 (31.1%) P. aeruginosa and 7 (18.9%) Pseudomonas spp. were resistant to meropenem. Among the 32 meropenem resistant P. aeruginosa, 15 (46.9%) were AmpC β-lactamase producers, 16 (50.0%) MBL producers by EDS test, but only 9 (28.1%) found positive for carbapenemases by modifi ed Hodge test. Among the 46 meropenem resistant A. baumannii, 31 (67.4%) were AmpC β-lactamase producers, 3 (6.5%) MBL producers, but only 1 (14.3%) was positive for carbapenemases by modifi ed Hodge test. One P. aeruginosa was positive for carbapenemase by modifi ed Hodge test, but was negative for MBL and AmpC β-lactamase.

Interpretation & conclusions: MBL production is an important mechanism of carbapenem resistance among Pseudomonas species but not among Acinetobacter species. EDS is more sensitive for detection Acinetobacter species. EDS is more sensitive for detection Acinetobacterof MBLs than modifi ed Hodge test. Both EDTA-meropenem and EDTA-ceftazidime combination must be used to detect all the MBL producers. Carbapenemases other than MBL may also be responsible for carbapenem resistance. AmpC β-lactamase is also a contributory factor for carbapenem resistance among the isolates in the hospital.

Key words AmpC β - lactamases - AmpC disk test - EDTA disk synergy test - metallo-β-lactamases - modifi ed Hodge test - nonfermentative bacteria

coding for β-lactamase enzymes mutate continuously in response to the heavy pressure of antibiotic use leading to development of newer β-lactamases with a broad spectrum of activity. Among the β-lactamases,

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Indian J Med Res 129, June 2009, pp 707-712

the carbapenemases especially transferrable metallo-β-lactamases (MBLs) are the most feared because of their ability to hydrolyze virtually all drugs in that class, including the carbapenems. There are several mechanisms for carbapenem resistance such as the lack of drug penetration due to mutation in porins, loss of certain outer membrane proteins and effl ux mechanisms1.

In addition to their resistance to all β-lactams, the MBL producing strains are frequently resistant to aminoglycosides and fl uoroquinolones2. However, these usually remain susceptible to polymyxins2. 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 mediated2.Consequently, the rapid detection of carbapenemase production is necessary to initiate effective infection control measures to prevent their dissemination.

Modifi ed Hodge test is a phenotypic method for detection of carbapenemases3. Various methods like, EDTA disk synergy (EDS) test3, MBL E-test1, EDTA-based microbiological assay4 are used for detection of MBLs. But EDS test is a relatively simple and sensitive method for MBL detection. There are no established phenotypic methods for detection of specifi c serine carbapenemases. AmpC disk test5 and three-dimensional extract method6 are the commonly used tests for detection of AmpC β-lactamases.

Although different phenotypic methods have been described, for a long time there was no standard guideline for screening of carbapenemases. Recently, CLSI has recommended modifi ed Hodge test for detection of carbapenemases activity in Enterobacteriaceae, but not in non-fermenters7. Detection of genes coding for carbapenem resistance by PCR, usually give reliable and satisfactory results8, but this method is of limited practical use for daily application in clinical laboratories because of the cost. Thus, a simple and inexpensive testing method for screening of carbapenemase producers is necessary. Therefore this study was undertaken to detect MBLs and AmpC β-lactamases in clinical isolates of nonfermentative Gram-negative bacteria. Clinical usefulness of the various methods for detection of carbapenemases and MBLs as a screening test was also evaluated.

Material & Methods

A prospective study was conducted over a period of nine months (June 2007 to February 2008) at the Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry. A total of 100 Acinetobacter species and 140 Pseudomonas species were included in this study. These organisms were isolated from specimens like sputum, tracheal aspirate, pus, urine, blood, pleural fl uid and ascitic fl uid of patients admitted to different wards, which were sent to the microbiology laboratory for routine culture identifi cation and sensitivity testing.

The Acinetobacter species and Acinetobacter species and Acinetobacter Pseudomonas species were identifi ed based on standard bacteriological techniques9,10. All these isolates were screened for meropenem resistance by Kirby-Bauer disk diffusion method according to CLSI guidelines11.

Modifi ed Hodge test: The meropenem resistant strains were subjected to modifi ed Hodge test for detection of carbapenemases3. An overnight culture suspension of Escherichia coli ATCC 25922 adjusted to 0.5 McFarland standard12 was inoculated using a sterile cotton swab on the surface of a Mueller-Hinton agar (MHA) (HI-MEDIA, Mumbai, India). After drying, 10 µg meropenem disk (HI-MEDIA, Mumbai, India) was placed at the center of the plate and the test strain was streaked from the edge of the disk to the periphery of the plate in four different directions. The plate was incubated overnight at 37oC. The presence of a ‘cloverleaf shaped’ zone of inhibition due to carbapenemase production by the test strain was considered as positive (Fig. 1).

EDTA disk synergy (EDS) test: EDTA disk synergy (EDS) test was done with simultaneous testing of two different β-lactams (meropenem and ceftazidime), for

Fig. 1. Modifi ed Hodge test. Positive strain shows a ‘cloverleaf shaped’ zone of inhibition due to carbapenemase production, while the negative strain shows an undistorted zone of inhibition.

708 INDIAN J MED RES, JUNE 2009

detection of metallo-β-lactamases in the meropenem resistant isolates3.

A 0.5 M EDTA solution was prepared by dissolving 186.1 g of disodium EDTA. 2H

2O (REACHEM,

Chennai, India) in 1,000 ml of distilled water. The pH was adjusted to 8.0 by using NaOH (HI-MEDIA, Mumbai, India) and was sterilized by autoclaving13.

An overnight liquid culture of the test isolate was adjusted to a turbidity of 0.5 McFarland standard12

and spread on the surface of a MHA plate. A 10 µg meropenem disk or 30 µg ceftazidime disk (HI-MEDIA, Mumbai, India) was placed on the agar. A blank disk (6 mm in diameter, Whatmann fi lter paper no. 1) was kept on the inner surface of the lid of the MHA plate and 10 µl of 0.5 M EDTA is added to it. This EDTA disk was then transferred to the surface of the agar and was kept 10 mm edge-to-edge apart from the meropenem or ceftazidime disk. After incubating overnight at 37oC, the presence of an expanded growth inhibition zone between the two disks was interpreted as positive for MBL production (Fig. 2).

Amp C disk testAmp C disk testAmp C d : AmpC disk test was also done for the meropenem resistant strains for detection of AmpC β-lactamases5. On a MHA plate, lawn culture of E. coli ATCC 25922 was made from an overnight culture coli ATCC 25922 was made from an overnight culture colisuspension adjusted to 0.5 McFarland standard12. A 30 µg cefoxitin disk was kept on the surface of the agar. A blank disk (6 mm in diameter, Whatmann fi lter paper no. 1) was moistened with sterile saline and inoculated with a few colonies of the test strain. The inoculated disk was then placed beside the cefoxitin disk almost touching it. The plate was incubated overnight at 37oC. A fl attening or indentation of the cefoxitin inhibition zone in the vicinity of the disk with test strain was interpreted as positive for the production of AmpC β-lactamase. An undistorted zone was considered as negative (Fig. 3).

Results

Of the 100 clinical isolates of Acinetobacterspecies, 78 were A. baumannii, while 22 were A. lwoffi i. Among the 140 Pseudomonas isolates screened, 103 were P. aeruginosa, while the remaining 37 wereother Pseudomonas spp.

Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffi i, 32 (31.1%) P. aeruginosa and 7 (18.9%) Pseudomonasspp. were found resistant to meropenem.

Among the 32 meropenem resistant P. aeruginosa, 15 (46.9%) were AmpC β-lactamase producers, 16 (50.0%) were MBL producers by EDTA disk synergy test, but only 9 (28.1%) were positive for carbapenemases by modifi ed Hodge test (Table). Two isolates were positive for both MBL and AmpC β-lactamase. One was positive for carbapenemase by modifi ed Hodge test, but was negative for MBL and AmpC β-lactamase by EDTA disk synergy test and AmpC disk test respectively. Of the 16 MBL producers, 8 were detected by simultaneously testing with both meropenem and ceftazidime in EDS, 6 were detected only using EDTA-ceftazidime combination and 2 were positive by EDTA-meropenem combination alone.

Of the 7 meropenem resistant Pseudomonas spp., 4 (57.1%) were AmpC β-lactamase producers, 2 (28.6%) were MBL producers by EDTA disk synergy test, but only 1 (14.3%) was positive for carbapenemases by modifi ed Hodge test (Table). Of the 2 MBL producers, one was detected by simultaneously testing with both meropenem and ceftazidime in EDS, while the other was detected only using EDTA-ceftazidime combination.

Fig. 2. EDTA disc synergy test. Positive strain shows a synergistic zone of inhibition between ceftazidime or meropenem disc and EDTA disc, while the negative strain shows no synergistic zone of inhibition.

Fig. 3. AmpC disc test. Indentation of the cefoxitin zone of inhibition is seen in the vicinity of the disk with positive strain, while there is an undistorted zone of inhibition near the negative strain.

NOYAL et al: DETECTION OF CARBAPENEMASES IN GNBS 709

Among the 46 meropenem resistant A. baumannii, 31 (67.4%) were AmpC β-lactamase producers, 3 (6.5%) were MBL producers, but only 1 (14.3%) was positive for carbapenemases by modifi ed Hodge test (Table). Among the 3 MBL producers, one was detected by simultaneously testing with both meropenem and ceftazidime in EDS and 2 were detected only using EDTA-ceftazidime combination.

Of the 7 meropenem resistant A. lwoffi i, 4 (57.1%) were AmpC β-lactamase producers, but none were positive for MBL and other carbapenemases (Table).

Discussion

Carbapenems have a broad spectrum of antibacterial activity, and these are resistant to hydrolysis by most β-lactamases including extended spectrum β-lactamases (ESBL) and AmpC β-lactamases14. These are often used as a last resort in infections due to multidrug-resistant Gram-negative bacilli. However there is an alarming increase in reports of carbapenem resistance in Acinetobacter species and Acinetobacter species and Acinetobacter P. aeruginosa15. The fi rst metallo-β-lactamase-producing P. aeruginosa strain was isolated in Japan in 198816. For many years, these MBL producing isolates were restricted to Japan, but now it has disseminated worldwide17-19. In India, MBL producing P. aeruginosa was fi rst reported in 200220.

In our study we report a high prevalence of meropenem resistance among A. baumannii. In a prospective study with 150 Acinetobacter species Acinetobacter species Acinetobacterconducted at Bangalore, India, 14.0 per cent resistance to carbapenems was reported21. Surveillance in Brooklyn, New York, revealed that approximately 2 of every 3 isolates of Acinetobacter species were Acinetobacter species were Acinetobacterresistant to carbapenem22. In an Australian study with ninety A. baumannii isolates recovered from blood, 64 per cent resistance to meropenem was noted23. Though our result was contradictory to the relatively lesser resistance rates among Acinetobacter species Acinetobacter species Acinetobacter

documented in India, it was similar to the New York and Australian studies, which also have reported high prevalence of carbapenem resistance among Acinetobacter species. High antibiotic pressure due Acinetobacter species. High antibiotic pressure due Acinetobacterto indiscriminate use of carbapenems in this part of the world could have resulted in the increase in carbapenem resistant Acinetobacter spp. Acinetobacter spp. Acinetobacter

Among the P. aeruginosa 31.1 per cent meropenem resistance was documented in this study. A 5 year longitudinal study involving many centers from Latin America has reported that, P. aeruginosa resistance to carbapenems has risen to 40.0 per cent 24. In a study done at a tertiary care hospital in Puducherry (India), with 266 isolates of P. aeruginosa, 10.9 per cent resistance to carbapenems was reported25. Though the carbapenems resistance among P. aeruginosa in our study was lesser than the Latin American surveillance study24, it was higher than the resistance documented in 2006 from this part of the world25. These fi ndings clearly show a rising trend in the carbapenem resistance among the nonfermenters.

We also found that 50 per cent of the carbapenem resistance among P. aeruginosa was attributable to the production of MBLs. This was surprisingly higher than the documented report in Korea, where 11.4 per cent of imipenem-resistant P. aeruginosa isolates produced MBLs26. But another Indian study has reported MBL production among 75 per cent of the imipenem resistant Pseudomonas species27. Among the meropenem resistant A. baumannii, 6.5 per cent were MBL producers, which was relatively less compared to 14.2 per cent imipenem-resistance attributable to the production of MBLs reported in Korea26. In an Indian study on meropenem resistant Acinetobacter species Acinetobacter species Acinetobacternone of the isolates produced MBLs21. Consequently in the Indian scenario production of MBL may not play a major role in carbapenem resistance among Acinetobacter species.Acinetobacter species.Acinetobacter

EDTA disk synergy test detected MBL production in additional 9 Pseudomonas species and 2 Acinetobacter species, which were missed by the Acinetobacter species, which were missed by the Acinetobactermodifi ed Hodge test. Based on these fi ndings, EDTA disk synergy test seems to be a better method for MBL detection then modifi ed Hodge test. Though the reason for the difference in the performance of these two tests is not clear, similar results have been observed in other studies3,27. In the EDS test, EDTA-ceftazidime combination detected additional MBL producers which were not identifi ed by EDTA-meropenem

Table. Results of modifi ed Hodge test, EDTA disc synergy test & AmpC disc test

Bacteria No. of meropenem

resistant isolates

No. of positives (%)

Modifi ed Hodge

test

EDTA disc synergy

test

AmpC disc test

P. aeruginosa 32 9 (28.1) 16 (50.0) 15 (46.9)Pseudomonas spp. 7 1 (14.3) 2 (28.6) 4 (57.1)A. baumannii 46 1 (2.2) 3 (6.5) 31 (67.4)A. lwoffi i 7 0 (0) 0 (0) 4 (57.1)

710 INDIAN J MED RES, JUNE 2009

combination. The reason for the increased sensitivity of EDTA-ceftazidime combination is the ability of ceftazidime to produce a marked inhibitory effect with EDTA. Therefore, ceftazidime appears to be the better substrate for EDS. Similar results were observed in a study done at the National Institute of Infectious Diseases, Tokyo, Japan28.

Two MBL producing P. aeruginosa were detected only by EDTA-meropenem combination, but not by EDTA-ceftazidime combination. A synergistic zone of inhibition which must have been normally present between EDTA and ceftazidime due to MBL production was not observed with these two isolates. These two MBL producers were also simultaneously producing AmpC β-lactamase. Hence it could be proposed that the synergistic zone of inhibition was masked by the resistance to ceftazidime conferred by the AmpC β-lactamase, which is independent of zinc ions for its action. Based on this study it is clear that both EDTA-meropenem and EDTA-ceftazidime combination must be used simultaneously to detect all the MBL producers, which may otherwise be missed by using either of this combination alone.

One of the meropenem resistant P. aeruginosa was positive for carbapenemase by modifi ed Hodge test, but was negative for MBL and AmpC β-lactamase. This may be because of the production of carbapenemase other than MBL, which is not dependent on zinc ion for its action. Accordingly, apart from MBL, other classes of carbapenemases (class A or D) can also be responsible for meropenem resistance in P. aeruginosa29.

AmpC β-lactamase was produced by 46.9 and 67.4 per cent of the meropenem resistant P. aeruginosaand A. baumannii respectively. Therefore, AmpC β-lactamase could be an important contributory factor for meropenem resistance among the isolates in our hospital similar to other studies22,30,31. This seems to be more likely with A. baumannii, most of which in our study, despite being meropenem resistant, did not show production of MBL or other carbapenemases.

The detection of MBL and other carbapenemases is of utmost importance in deciding the most appropriate therapeutic regimen for treatment of the carbapenem resistant nonfermenters. In several studies, 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 isolates30,31. Moreover, studies have shown that effl ux pumps mainly affect meropenem, while specifi c β-lactamases (carbapenemases) hydrolyze imipenem more effi ciently30. So, detection of carbapenemases is necessary for administration of appropriate therapy.

The drawbacks in our study are the relatively small sample size and failure to evaluate the clinical usefulness of detection of carbapenemases. Our study was restricted mainly to detection of carbapenemases and the comparison of the effi cacy of different techniques for detection of carbapenemases. Further studies are needed to evaluate the clinical usefulness of detection of carbapenemases.

In conclusion, MBL production is an important mechanism of carbapenem resistance among Pseudomonas species, while it may not play a major role in carbapenem resistance among Acinetobacterspecies. Modifi ed Hodge test may not be a useful screening test for carbapenemases as many MBL producing isolates were not detected by this test. EDS is a more sensitive method for detection of MBLs. Ceftazidime appears to be the better substrate for EDS compared to meropenem, but both meropenem and ceftazidime must be used simultaneously to detect all the MBL producers. Carbapenemases other than MBL may also be responsible for carbapenem resistance in these nonfermenters. AmpC β-lactamase is also a contributory factor for carbapenem resistance among the isolates in the hospital.

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Reprint requests: Dr B.N. Harish, Professor, Department of Microbiology, Jawaharlal Institute of PostgraduateMedical Education & Research, Puducherry 605 006, Indiae-mail: drbnharish@yahoo.com

712 INDIAN J MED RES, JUNE 2009