Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

Users Online : 29276

AbstractMaterial and MethodsResultsDiscussionConclusionAcknowledgementReferencesTable and FiguresDOI and Others
Article in PDF How to Cite Citation Manager Readers' Comments (0) Audio Visual Article Statistics Link to PUBMED Print this Article Send to a Friend
Advertisers Access Statistics Resources

Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"

Dr Mohan Z Mani,
Professor & Head,
Department of Dermatolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018

Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."

Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018

Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."

Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018

Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."

Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
On Sep 2018

Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."

Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata

Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
On Aug 2018

Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".

Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
On Aug 2018

Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".

Dr. Mamta Gupta
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018

Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.

Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."

Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
On May 11,2011

Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."

Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
On April 2011

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.

Dr. Anuradha
On Jan 2020

Important Notice

Original article / research
Year : 2012 | Month : September | Volume : 6 | Issue : 7 | Page : 1196 - 1199 Full Version

The Prospective Evaluation of Four Convenient Methods for Detecting MBLs in the Clinical Isolates

Published: September 1, 2012 | DOI:
Mridula Raj Prakash, Veena.M, Archana Sharma, Basavaraj.K.N, Viswanath .G

1. Post graduate student, Department of Microbiology, 2. Associate Professor, Department of Microbiology, 3. Post graduate, Department of Microbiology, 4. Associate Professor, Department of Microbiology, 5. Professor & Head, Department of Microbiology, J.J.M. Medical College Davangere-577004, Karnataka India.

Correspondence Address :
Dr. Veena. M,
Associate Professor, Department of Microbiology,
J.J.M. Medical College, Davangere,
Karnataka 577004 India.
Phone: 9480599771


Background: The emergence and the spread of metallo-beta-lactamases (MBLs) in non fermenters like Pseudomonas aeruginosa and the Acinetobacter spp has become a therapeutic challenge. In this study, the phenotypic confirmation of the MBL production was done by different methods.

Method: The isolates were screened for metallo-beta-lactamase production by using imipenem. All the imipenem resistant isolates were further subjected to a phenotypic confirmation by the combined disk method, the double disk synergy test and the modified Hodge test. The results were analyzed and tabulated.

Results: Of the total 200 isolates, 124(62%) were of P. aeruginosa and 76(38%) were of the Acinetobacter spp. Out of the 200 isolates, 28 (19(68%)- P. aeruginosa and 9(32%)- Acinetobacter isolates were found to be MBL producers by the combined disk test which used imipenem and EDTA , by the double disk synergy test and by the modified Hodge test. But the combined disk test which used ceftazidime detected only 25 out of the 28 MBLs.

Conclusion: The results of our study showed that the combined disk test which used imipenem, the double disk synergy test and the modified Hodge test, all had equal sensitivity. But when the ease of the performance and the interpretation of the results were considered cost effectively, the combined disk test was found to be a more feasible option than the other tests. Overall, these tests can be done easily as they are less laborious and are cost – effective. Hence, the routine testing of the metallo beta-lactamase producers is of great value, in order to take measures to advocate a proper antibiotic policy and also to take effective steps for controlling their spread worldwide.


MBLs, Pseudomonas aeruginosa, Imipenem-EDTA combined disk test, Double disk synergy test, Modified Hodge test.

The mechanisms of bacterial resistance are complex, varied and not completely understood (1). In the past two decades, the clinical microbiologists have been finding it difficult to test the antibiotics against many of the common pathogens.The reason being the emergence of the increasing resistance to the betalactam group of drugs which include the carbapenems, apart from the indiscriminate use of antibiotics. The carbapenems (imipenem, ertapenem and meropenem) are the mainstay of treatment for serious infections which are caused by non fermenting bacilli like Pseudomonas aeruginosa, Acinetobacter spp, etc. The carbapenemases are diverse enzymes that vary in their abilities in hydrolyzing carbapenems and other betalactams. Hence, their detection is a crucial issue because they often show an extensive and sometimes a total antibiotic resistance. The more resistant organisms like the strains of Pseudomonas and Acinetobacter spp. have acquired the resistance from the Enterobacteriaceae (2). The plasmid mediated carbapenemases pose more danger than the chromosomal mediated carbapenemases. The carbapenemases belong to the molecular classes, A, B and D. The class B enzymes (Bush group 3) are metallo betalactamases (MBLs). The MBLs hydrolyze almost all the betalactam antibiotics. The MBLs typically hydrolyze carbapenem efficiently, but theySectionare inhibited by chelating agents such as EDTA.

The MBL activity can be detected by both phenotypic and genotypic methods. Different studies have used different methods according their feasibility. Though the molecular methods are highly sensitive and specific, their use is limited only to the research laboratories. The phenotypic methods being simple, sensitive, economical and reliable, they can be routinely performed in microbiological laboratories. Several phenotypic methods are available for the detection of the MBLs which are produced by bacteria. Most of these methods are based on the ability of the metal chelator (EDTA) and the thiol based compounds to inhibit the enzyme activities (3). The spread of the MBL genes from Pseudomonas aeruginosa to Enterobacteriacea is posing a difficulty in selecting the antibiotics for controlling serious infections like septicaemia, pneumonia, etc,. Thus, in order to control the spread of resistance, the detection of MBL is of prime importance. This study was aimed at detecting the MBL producing Pseudomonas aeruginosa as well as members of the Enterobacteriaceae family.

Material and Methods

The study period and the clinical samples - A total of 200 samples (from the patients who were admitted to the Bapuji and the Chigateri Government Hospital, Davangere) which included blood, urine,pus , wound swabs , suction tips, catheter tips and other body fluids were included in this study. This study was carried out over a period of 11 months (from Jan 2011 to Nov 2011).The samples were processed as per the standard microbiological procedures (1). Pseudomonas aeruginosa was identified, based on its colony morphology on blood agar and Mac Conkey’s agar, the oxidase test , its pigment production and polymyxin B (300 units) on the Muller Hinton agar (MHA). Antimicrobial susceptibility testing- Antibiotic susceptibility testing was performed on Mueller Hinton Agar (Hi Media) by the Kirby- Bauer disk diffusion method according to the CLSI guidelines (2006). The antibiotic disks for the studies were procured from Hi Media, Mumbai, India. The drug disks which were tested in our study were, (in micrograms)- Gentamicin (10), Amikacin (30), Ciprofloxacin (30), Ceftazidime (30), Ceftriaxone (30), Imipenem (10), Polymyxin B (300), Piperacillin-Tazobactum (100/10), Cefotaxime (30) and Cefepime (30). The isolates which showed a zone diameter of ≤13mm were considered as resistant, those which showed a zone diameter of 14-15mm were considered as intermediate and those which showed a zone diameter of ≥16mm were considered to be sensitive to imipenem. The isolates which showed resistance to imipenem were further subjected to the MBL phenotypic confirmation.

Pseudomonas aeruginosa ATCC 27853 and E. coli ATCC 25922, were used as the control strains, which were obtained from HI-Media, Mumbai. The different phenotypic confirmatory methods which were used were as follows: 1. The Imipenem –EDTA combined disk method – A 10μg Imipenem disk was placed on a Mueller Hinton agar plate at a distance of 20mm from an Imipenem-EDTA disk, on a lawn culture of an imipenm resistant isolate. The plates were incubated overnight at 370 C and the zone of inhibition of the imipenem and the imipenem -EDTA disks were compared on the next day. If the increase in the inhibition zone with the Imipenem-EDTA disk was ≥ 7mm than the imipenem disk alone, it was considered to be MBL positive. 2. The Ceftazidime - EDTA combined disk method - Two 30μg ceftazidime disks were placed on a Muller Hinton agar plate, on which the lawn culture of an imipenem resistant isolate was made. 10μl of EDTA solution was added to one of them, to obtain the desired concentration of 750μg. The plates were incubated overnight at 370C and the zone of inhibition of the ceftazidime and the ceftazidime EDTA disks were compared on the next day. If the increase in the inhibition zone with the ceftazidime-EDTA disk was ≥ 7 mm than the ceftazidime disk alone, it was considered to be MBL positive.

3. The Imipenem- EDTA double disk synergy test (DDST) – A 10 μg imipenem disk was placed at 20 mm centre to centre from a blank disk which contained 10ul of 0.5 molar EDTA (750 μg). After an overnight incubation at 370C, the enhancement of the zone of inhibition around the imipenem EDTA disk in comparison with the zone of inhibition on the far side of the drug was interpreted as positive for MBL production. 4. The Modified Hodge test - E. coli ATCC25922 was inoculated on to a MHA plate as per the CLSI guidelines. The test organisms were heavily streaked from the centre to the periphery of the plate. The plate was allowed to stand for 15 minutes at room temperature. A 10 μg imipenem disk was placed at the centre of the plateand it was incubated overnight. The presence of a distorted zone of inhibition was interpreted as a positive result.


Out of the 200 samples which were collected, 112(56%) were from males and 88(44%) were from females. Of the 200 samples, 124(62%) were of Pseudomonas and 76(38%) were of Acinetobacter. 36(18%) of them showed resistance to Imipenem (screen positives). But when they were subjected to various phenotypical confirmatory methods, we found that a total of 28 (78%) were MBL producers. This result indicated that 14% of all the isolates (200) were MBL positives. The details of the organisms and the MBL production and the methods which were used have been shown in the tables below (Table/Fig 1), (Table/Fig 2), (Table/Fig 3) and (Table/Fig 4).


Pseudomonas aeruginosa which produces metallo-β- lactamases (MBLs) was first reported from Japan in 1991 (4), andit has spread worldwide since then. Apart from P.aerugenosa, other bacteria like Serratia, Klebsiella pneumonia , Escherichia coli, Enterobacter aerogens, E.cloacae, Citrobacter freundii, Proteus vulgaris, P.putida, Acinetobacter and Alcaligenes xylosoxidans were also shown to produce MBLs (5). The introduction of carbapenems for treating serious bacterial infections which were caused by the β- lactam resistant bacteria was a great leap in the antibiotic history (6). But certain organisms have evolved in such a way that the effect of these carbapenems no longer seem to be very promising. Two types of carbapenem hydrolyzing enzymes have been described –the serine enzymes which have a serine moiety at the active site and Metallo-β-Lactamases (MBLs) which require divalent cations like zinc as a cofactor for their enzymatic activity (7),(8),(9),(10). The serine carbapenemases are the derivatives of the class A or the class D enzymes and they usually mediate carbapenem resistance in Enterobacteriaceae or in the Acinetobacter spp (11). Despite the high avidity of these organisms for the carbapenems, they do not always mediate high-level resistance and not all are inhibited by clavulinic acid (12). The classification scheme which was proposed by Ambler in 1980 was modified, depending on the functional properties of the carbapenems, by Bush in 1989.This has been a widely accepted referencing system for the β-lactamases .This was mainly based on their Imipenem hydrolysis, their sensitivity to EDTA and their lack of inhibition by the serine β-lactamase inhibitors. This scheme was further updated in 1994 and 1997 to accomodate the increasing new enzymes which were continually being discovered (13),(9),(14). Class B1, B2 and B3 were the chromosomally mediated MBLs, whereas the transferable MBLs included IMP, VIM, GIM and SPM-1 (15). However, these enzymes are universally inhibited by EDTA and other chelating agents of the divalent cations (14). Several non-molecular methods have been studied for the identification of the carbapenemases and in many cases, their detection is based on the use of specific inhibitors. Hence, the MBL detection is based on its dependence on zinc and on the use of inhibitors/chelating agents such as EDTA (15),(16).

Since there are no standardized detection methods, many researchers have used many different methods to detect and to confirm the MBL production. In our study, we used the combined disk test, the double disk synergy test and the modified Hodge test. In the present study, out of 124 P. aeruginosa isolates, 19(15.32%) and out of 76 Acinetobacter isolates, 9(11.84%) were found to be MBL positives. Hence, out of 200 isolates, a total of 28(14%) were MBL positive. The details have been shown in (Table/Fig 3). The Combined Disk Test (CDT) which used ceftazidime (CAZ) and EDTA detected 17(60.7%) out of 19 isolates of Pseudomonas and 8(28.5%) out of 9 isolates of Acinetobacter. But on using imipenem and the I- EDTA combined disk test as was described by Yong et al., (5) all the 28(14%) isolates were found to be MBL producers. This result was similar to that of the study which was done by Deeba Bashir et al., where they found 12% of the P. aeruginosa isolates to be MBL producers by CDT (17). But in another study which was done by Horeish Saderi et al,. 39.06% of MBLs were observed. In a study which was done by Noyal et al,. 6.5% of the Acinetobacter isolates were found to be MBL producers . The DDST in our study also confirmed all the 28 isolates to be MBL producers. When they were subjected to the modified Hodge test again, all the 28 isolates were found to be1198confirmed MBL positives. Three different methods (CDT, DDST, MHT) gave consistent results, whereas a slightly different result was given by CDT which used ceftazidime. The reason could be that the MBL producing organisms had other ceftazidime resistance mechanisms. Such strains will not show MBL production (18). Many, including Arakawa et al,. have suggested that the combined disk test was superior to the double disk synergy test. The major drawback of DDST was the subjective interpretation of the results at times (19). But our study showed both of them to be equally effective in detecting the MBLs. It has been suggested that the selection of the optimal MBL screening method be based not only on the bacterial species, but also on the strains which are collected and the local prevalence of the MBL producers (20),(21). When all the 28 isolates were tested for their susceptibility patterns by using the Mueller Hinton Agar, both Pseudomonas and Acinetobacter showed 94%, and 96% resistance to the fluoroquinolones respectively and 92% and 100% resistance to the aminoglycosides. An increased level of resistance was also seen by Prajapathi et al,. (22). In our present study, we also noted that the isolates showed 78% resistance to cefepime and 61% resistance to piperacillin-tazobactum. A similar pattern was observed by Behera et al,. With the MBLs showing an increased resistance pattern to a variety of commonly used and effective drugs, one is left with a very minimal choice of drugs for the treatment of the severe infections which are caused by these organisms.


To conclude, the results of our study showed that the combined disk test and the double disk synergy test were equally effective and that they were at par with the modified Hodge test which was commonly used as a final confirmatory test, apart from the E-test and molecular based methods. Many studies have proved that the MBL E-test and the combined disk test had equal sensitivity. As the E-test is highly expensive, CDT, which is rather economical and easy to do, can be routinely implemented. DDST and MHT can also be used to corroborate the results .Our study results give a warning regarding the serious scenario of the MBLs. This also points towards the implementation of a strict intra-institutional antibiotic policy and infection control measures to limit the spread of these MBLs globally.


We thank Dr.Viswanath.G , Professor and Head , Dept of Microbiology for his constant guidance and support to carry out this study in the department.


Winn WC, Allen SD, Janda WM, Koneman EW, Procop GW, Schreckenberger C, Woods GL-Koneman’s Color Atlas and Textbook of Diagnostic Microbiology.,6th ed Philadelphia, USA: Lippincott Williams and Wilkins ;2006; 959-60.
Thomson KS.The extended-spectrum-β-lactamases, AmpC, and the carbapenemase issues. J Clin Microbiol 2010 ;Commentary:1019-25.
Saderi H, Karimi Z, Owlia P, Bahar MA, Rad SM BA. The phenotypic detection of metallo-beta-lactamase producing Pseudomonas aeruginosa strains which were isolated from burns patients. Iranian Journal of Pathology 2008;3(1), 20-24.
Watanabe M, Iyobe S, Inoue M, Mitsuhashi S. The transferable imipenem resistance in Psedomonas aeruginosa. Antimicrob Agents Chemother 1991; 35:147-51.
Yong D, Lee K ,Yum JH, Shin HB, Rossolini GM, Chong Y. Theimipenem-EDTA disk method for the differentiation of the metallo- β-lactamase producing clinical isolates of Pseudomonas spp and Acinetobacter spp. J Clin Microbiol 2002; 40:3798-801.
Kahan FM, Kropp H , Sundelof JG , Brinbaum J. Thienamycin: the development of imipenem-cilastin. J Antimicrob Agents Chemother 1983;12(Suppl D):S1-35.
Bush K. The β-Lactamases of increasing clinical importance. Curr. Pharm.Des 1999;. 5:839-45.
Bush K. The metallo-β-lactamases: a class apart. Clin. Infect. Dis. 1998;27: S48-53.
Bush K. New β-lactamases in the gram-negative bacteria: their diversity and impact on the antimicrobial therapy. Clin. Infect. Dis. 2001; 32:1085-89.
Bush K, Jacoby GA, .Medeiros AA. A functional classification scheme for β-lactamases and its correlation with the molecular structure. Antimicrobe. Agents Chemother. 1995; 39:1211-33.
Nordman P, Mariotte S, Naas T, Labia R, Nicholas MH. The biochemical properties of a carbapenem –hydrolysing β-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli .Antimicrob. Agents Chemother. 1993;37:939-46.
Nordman P, Poirel L. The emerging cabapenemases in the gramnegative aerobes. Clin. Microbiol. Infect. 2002; 8:321-31.
Bush K. Classification of the β-lactamases –group -2c ,group-2d , group- 2e, andgroup-4. Antimicrobe. Agent Chemother. 1989; 33:271-76.
Rasmussen B, Bush K. The carbapenem-hydrolysing β-lactamases. Antimicrob. Agents Chemother. 1997; 41:223-32.
Queenan AM, Bush K. Carbapenemases: the versatile β-lactamases. Clin Microbiol Rev 2007; 20:440-58.
Franklin C, Liolis L, Peleg AY. The phenotypic detection of carbapenem susceptible, metal-β-lactamase-producing, gram-negative bacilli in the clinical laboratory . J Clin Microbiol 2006;44:3139-44
Bashir D, Thokar MA, Fomda BA, Bashir G, Zahoor D, Ahmad S et al., The detection of metallo-β-lactamase (MBL) Producing Pseudomonas aeruginosa at a tertiary care hospital in Kashmir. Afr. J. Microbiol. Res. 5(2): 164-72.
Behera B, Mathur P, Das A, Kapil A, Sharma V. An evaluation of four different phenotypic techniques for the detection of metallo-β- lactamase producing Pseudomonas aeruginosa. Indian J. Med Microbiol. 2008;26:233-37.
Arakawa Y, Shibata N, Shibayama K, Kurokawa H, Yagi T, Fujiwarw H et al., A convenient test for screening the metallo-β-lactamase producing gram negative bacteria by using thiol compounds. J Clin Microbiol 2000;40:2755-59.
Lee K, Yong D, Yum JH, Lim YS, Bolmstrom A, Qwarnstrom A, Karlsson A, Chong Y. Evaluation of the E test MBL for the detection of the blaIMP-1 and the blaVIM-2 allele-positive clinical isolates of the Pseudomonas spp and the Acinetobacter spp. J. Clin. Microbiol. 2005; 43:942–44.
Pica˜o RC, Andrade SS, Nicoletti AG, Campana EH, Moraes G C,Mendes RE, Gales AC. Metallo-_-lactamase detection: a comparative evaluation of the double-disk synergy versus the combined disk tests for IMP-, GIM-, SIM- and SPM-, or the VIM-producing isolates. J. Clin. Mic Robiol. 2008; 46:2028–37.
Prajapati SB, Vegad MM, Mehta SJ, Kikani KM, Kamothi MN. Pandya J.M. An evaluation of two different phenotypic methods for the detection of metallo-β-lactamase producing Pseudomonas isolates J. Cell Tissue Research 2011;11:2601-60.

DOI and Others

ID: JCDR/2012/4148:2469

Date of Submission: Feb 19, 2012
Date of Peer Review: Mar 13, 2012
Date of Acceptance: Jul 27, 2012
Date of Publishing: Sep 30, 2012

JCDR is now Monthly and more widely Indexed .
  • Emerging Sources Citation Index (Web of Science, thomsonreuters)
  • Index Copernicus ICV 2017: 134.54
  • Academic Search Complete Database
  • Directory of Open Access Journals (DOAJ)
  • Embase
  • EBSCOhost
  • Google Scholar
  • HINARI Access to Research in Health Programme
  • Indian Science Abstracts (ISA)
  • Journal seek Database
  • Google
  • Popline (reproductive health literature)