Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 3, 2011, pp. 275-279

Indian Journal of Medical Microbiology, Vol. 29, No. 3, July-September, 2011, pp. 275-279

Original Article

Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae

S Rai¹, V Manchanda², NP Singh¹, IR Kaur¹

¹ Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Dilshad Garden, Delhi - 110 095, India
² Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya, Geeta Colony, Delhi - 110 031, India
Correspondence Address: S Rai, Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Dilshad Garden, Delhi - 110 095, India, sumit_rai@yahoo.com

Date of Submission: 19-Jun-2011 
Date of Acceptance: 28-Jun-2011

Code Number: mb11066

PMID: 21860109

DOI: 10.4103/0255-0857.83912

Abstract

Keywords: Enterobacteriaceae, modified Hodge test, metallo beta lactamases

Introduction

Rising resistance to reserved antimicrobials in the family Enterobacteriaceae has increased therapeutic challenges. Among the most notorious mechanisms is production of carbapenemases. These enzymes are usually present in integrons on plasmids and pose a serious threat of massive dissemination among the gram negative fraternity. First isolation of carbapenemase enzyme in Enterobacteriaceae dates back in 1991 when the metallo beta lactamase (MBL) gene bla_IMP-1 was discovered in Serratia marsascens from Japan. ^[1] This was followed by identification of bla_VIM-1 and bla_KPC in Greece ^[2] and United States. ^[3] In 2009, Clinical and Laboratory Standards Institute (CLSI) incorporated the modified Hodge test (MHT) for the detection of carbapenemases especially Klebsiella pneumoniae carbapenemases (KPC′s) from members of family Enterobacteriaceae. ^[4] Despite having a sensitivity and specificity of >90%, interpretation of MHT is often difficult due to possible bacteriocin production by the some test isolates, as the indicator strain ATCC 25922 does grow up to the edge of the test strain. ^[4] Alternatively, a similar phenomenon has been observed due to Amp C hyper-production or porin loss. ^[5] Moreover, there are many isolates that have a derepressed carbapenemase enzyme but are sensitive to carbepenems in vitro according to CLSI breakpoints. The MHT also does not indicate if the carbapenemases are dependent on zinc and enzyme activity of derepressed zinc-dependent carbapenemases may be missed. ^[6] Recently, the novel gene encoding the metallo-enzyme, the New Delhi Metallo-Beta Lactamase-1 (NDM-1) has been identified from an isolate of Klebsiella pneumoniae from a Swedish patient of Indian origin. ^[7] This would mean that we must incorporate effective screening procedures for such isolates.

The present study focused on phenotypic characterization of carbapenem-resistant isolates from family Enterobacteriaceae was performed to evaluate if they produced zinc-dependent carbapenemases, using multiple phenotypic methods.

Materials and Methods

This was an observational study conducted in the Departments of Microbiology of two tertiary level health care and teaching facilities in East Delhi, India. The study period was from July to November 2010.

Bacterial isolates

A total of 7342 specimens of urine and blood were received for culture in the bacteriology laboratory from the patients visiting and or admitted to the hospitals from June to November 2010 (6 months). From these specimens, 1477 continuous non-repeat isolates of family Enterobacteriaceae isolated were screened. Among these bacterial strains, 102 non-repeat isolates that were resistant to meropenem (10 μg) (Oxoid, Basingstoke, UK) according to CLSI breakpoints were included in the present study. These isolates were multi-drug resistant (resistant to carbapenems, third and fourth generation cephalosporins, aminoglycosides and fluorquinolones).^[4] Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as control strains. Identification and minimum inhibitory concentrations were determined using Vitek2compact (BioMerieux).

Modified Hodge test (MHT)

The test was performed as suggested by Lee et al. ^[8],[9] Briefly, a 1/10 dilution of 0.5 McFarland turbidity standard of fresh overnight growth of the indicator organism Escherichia coli ATCC 25922 was inoculated on Mueller Hinton Agar (MHA) (HiMedia Laboratories Pvt. Ltd., India). Two points were marked approximately 3 cm apart in the centre of the plate. One of the points was marked ′Zn′ and the other point left unmarked and an imipenem disc (10 μg) (Oxoid, Basingstoke, UK) was placed at the unmarked point. The test strains were heavily streaked from the edge of the disc to periphery. Three strains were inoculated per plate [Figure - 1]. The re-modified Hodge test was also done on the same plate.

Re-modified Hodge test

Addition of zinc has been known to increase the activity of metallo beta lactamases. ^[9] On the same plate as mentioned above, on the second point marked ′Zn′, another Imipenem disc was placed and the same test strains were heavily streaked in an approximately equal amount as the mirror images of the strains previously inoculated from the unmarked point. A total of 10 μl of 50 mM (140 μg per disc) of zinc sulphate (Rankem Chemicals, India) solution was added to this second disc. This was done so as to have a better comparison of the effect of zinc on the same plate and to avoid plate to plate variation. The plates were incubated overnight. In the case of MBL production a positive test was interpreted as an inward indentation of the growth of the indicator strain along the streak line of the test strain. This was read as negative, equivocal, positive and strongly positive [Figure - 1].

EDTA double disc synergy test

The imipenem-EDTA double disc synergy test has been described by Lee et al^[9] and Arakawa et al. ^[10] The synergy effect of imipenem (10 μg) and imipenem with Zn discs was observed with EDTA. EDTA discs (6 mm) were prepared by incorporating 10 μl of 0.5 mM solution of EDTA on each disc (equivalent to 750 μg per disc). The test organisms were adjusted to a 0.5 McFarland turbidity standard and swabbed on MHA plates. The EDTA disc was placed 20 mm apart edge to edge from the imipenem discs and the plates were incubated overnight. A zone of synergy between the antibiotic discs and EDTA was taken as a positive result [Figure - 2].

Combined disc test or disc enhancement test

The combined disc test or the disc enhancement test was performed as described by Yong et al^[11] with minor modifications. Briefly, three 10 μg imipenem discs were placed on a plate inoculated with 0.5 McFarland turbidity standard of fresh overnight growth of the test organism. To one of the imipenem discs, 10 μl of 0.5M EDTA (750 mg of disodium salt, dehydrate per disc) solution was added. To the second disc 10 μl of 50 mM (140 μg per disc) of zinc sulphate (Rankem Chemicals, India) solution was added and the third imipenem disc was placed unchanged. The inhibition zones of the imipenem, imipenem/EDTA and imipenem/zinc discs were compared after overnight incubation at 37 ^o C. A zone diameter difference between the imipenem and imipenem/EDTA discs of >5 mm or a decrease of 3 mm in the imipenem/zinc disc compared with Imipenem zone was interpreted as a positive result for MBL production. There was no deterioration of the imipenem disc on addition of Zn or EDTA as these combinations were checked using ATCC 25922 E. coli control.

Results

Among the 102 Meropenem resistant Enterobacteriaceae isolates, 91 (89%) were from urine and 11 (11%) were isolated from blood samples. These isolates included clinical strains of E.coli (51), Klebsiella spp. (33), Citrobacter spp. (8), Enterobacter spp. (7) and Proteus spp. (3). Eighteen isolates were obtained from patients visiting outpatient departments, while 74 and four isolates were from in-patients and ICUs respectively. The location of remaining six patients could not be identified. Among all these isolates 63 (61.7%) were imipenem sensitive while 39 were resistant [Table - 1]. Besides these antimicrobials, all the strains were resistant to piperacillin + tazobactam, ampicillin + sulbactam, amikacin, norfloxacin, nitrofurantoin, tobramycin, cefotaxime, amoxicillin + clavulanate (Oxoid, Basingstoke, UK). Proteus spp being naturally resistant to polymyxins, all other isolates sensitive to polymxin B and colistin. The MHT detected 92 (90.2%) isolates as carbapenemase producers while the re-modified Hodge test was positive for 98 isolates. Among those found negative for MHT (n=10), metallo beta lactamases (MBLs) activity was demonstrated using the EDTA disc synergy test and combined disc test in eight and nine isolates respectively.

Of the eighteen OPD patients, 14 were positive for carbapenemase activity by MHT. The remaining four MHT negative isolates were positive by the disc synergy test and combined disc test methods. Among the 78 hospitalized patients (both in patients and ICU patients), 72 were positive by MHT, while enzyme activity for the MHT negative isolates was picked up in at least four of the six isolates.

Among the imipenem sensitive isolates (n=63) the MHT picked up 57 (90.4%) isolates with carbapenemase activity while the disc synergy test and combined disc test picked up at least four of the six isolates missed by the MHT. The combined disc test picked up one extra isolate (5). Among the 39 Imipenem resistant isolates, the MHT picked 35 (90%) isolates. The disc synergy test and combined disc test picked up carbapenemase activity in all four isolates missed by the MHT. However the overall positivity for either a disc synergy test or a combined disc test was less than MHT in the MHT positive isolates [Table - 1].

Of all the 102 isolates, 99 isolates were positive for either the MHT or the re-modified Hodge test. Seven isolates which were negative by MHT were positive by the re-modified Hodge test and 39 isolates had an enhanced indentation in the re-modified Hodge test compared with the MHT. That is there was an enhancement of indentation demonstrating zinc dependence in a total of 46 isolates. In the remaining 52 isolates there was no further change or enhanced indentation. One isolate was positive by MHT but negative by the re-modified Hodge test. Remaining three isolates produced a possible bacteriocin that inhibited the growth of the indicator strain [Figure - 3].

Discussion

Spread of resistance by mobile genetic elements in health care settings has been well known. We have enough published data from India that indicate that a selection pressure due to frequent use of carbapenems has caused an increase in carbapenem-resistant strains. ^[12]

During the 6-month study period, 102 out of 1477 (7%) Enterobacteriaceae isolates were found to be MBL producers. The present study represents the prevalence of metallo beta lactamases in Enterobacteriaceae in the two hospitals of East Delhi and also an improved ability of their detection using multiple methods. We suggest that besides the MHT at least one more test must be included for carbapenemase/MBL screening for all isolates of family Enterobacteriaceae irrespective of their sensitivity to carbapenems. In the present study, the combined disc test fare slightly better than the disc synergy test. Reports for carbapenem susceptibility must be provided only after a strain has been screened by one of these methods besides the MHT as isolates showing sensitive breakpoint zones for Imipenem may be carbapenemase/MBL producers. Use of more than one test besides MHT may also be augmented by the fact that some strains produced bacteriocin like substances that rendered the MHT non-interpretable. Besides, some strains of Proteus pose difficulty in interpretation of MHT due to swarming [Figure - 4]. Enhancement of indentation of the indicator strain in the re-modified Hodge test was observed in 46 isolates demonstrating definite zinc dependence; however validation studies need to be done for the re-modified Hodge test using genetically characterized strains.

Considering the fact that the novel carbapenemase gene bla_NDM-1 found in India is located on a very mobile genetic element and the pattern of spread is more complex and more unpredictable than the genes encoding other carbapenemases, ^[13] their timely detection and control must be made an important practice guideline. Multiple studies have suggested Colistin and Tigecycline as the only treatment options. However both of these drugs are not the treatment option for Proteus spp as they are inherently resistant to them. This happens to be one particular concern in the present study where the presence of three Proteus isolates all of which were Zn-dependent carbapenemase producers but their carbapenemase activity was non-interpretable in the MHT. Tigecycline sensitivity was not performed. However experience dictates that these treatment options would also be short lived. ^[13] Resistance has already developed to Tigecycline in Klebsiella as evident by two studies ^[14],[15] and in our institutions (unpublished data). The call of the hour appears to be detection and containment and not mere treatment. Multiple reviews and guidelines have been published for detection and surveillance of carbapenemase producing Enterobacteriaceae. ^{[16],[17],[18]}

There has been outrage and concern regarding the origin of the recently described New Delhi Metallobetalactamase (NDM′s) bearing superbug. ^[19] There has also been a proposal to rename NDM as PCM (Plasmid encoding Carbapenemase-resistant Metallobetalactamase). ^[20] It is best for the scientific fraternity to find solutions for the detection and control of these NDMs rather than allegations and counter allegations. ^[21] The MHT has been routinely incorporated as a screening test for carbapenemase production. However, we suggest supplementing MHT either with the re-modified Hodge test, disc synergy test or combined disc test or all the methods, at least for all isolates negative or non-interpretable by MHT so as to identify most isolates with carbapenemase activity by phenotypic tests.

Acknowledgement

The authors would like to thank Mrs. Mercy Austin for laboratory and logistical assistance.

References

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2.	Miriagou V, Tzelepi E, Gianneli D, Tzouvelekis LS. Escherichia coli with a self-transferable, multiresistant plasmid coding for metallo-beta-lactamase VIM-1. Antimicrob Agents Chemother 2003;47:395-7.  Back to cited text no. 2
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18.	Stuart JC, Van-Hall MA. Guideline for Phenotypic Screening and Confirmation of Carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents 2010;36:201-10.  Back to cited text no. 18
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20.	Singh AR. Science, Name Giving and Names Calling: Change NDM - 1 to PCM. Mens Sana Monographs 2011;9:294-319.  Back to cited text no. 20
21.	Nataraj G. New Delhi metallo beta lactamase: What is in a name? J Post Grad Med 2010;56:251-2.  Back to cited text no. 21

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