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

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Dr Archana Dambal

"Journal of clinical and diagnostic research is a welcome change in publishing practices. It aims to reach out to the grass-root level researchers who do not lack in experience, clinical material and ideas, but lack in their knowledge in English language and statistics. The journal achieves it's aim by supporting in these exact domains.
It also gives due credit to all research designs like descriptive and qualitative studies while many journals ignore these important study designs. The rigorous review process does not allow any compromise in quality
It is indexed in many indexing agencies and the articles are available under creative commons licence free of cost
The frequency of publication supports many aspiring authors from India and other countries.
It's wide scope welcomes articles across various specialities in medicine. In an era when there is an unscientific insistence on speciality specific research by regulatory bodies in medical education, JCDR supports collaborative research across specialities. I wish the publisher all the best in his future endeavors."



Dr. Archana Dambal
Department of General Medicine,
Belgaum Institute of Medical Sciences,Belgaum, Karnataka,INDIA,
On 30 Nov 2018




Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




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 Dematolgy,
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
Lucknow
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,
Muzaffarnagar.
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,
Bengaluru.
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
Consultant
(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)
E-mail: drrajendrak1@rediffmail.com
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.
E-mail: ravi.dr.shankar@gmail.com
On April 2011

Important Notice

Original article / research
Year : 2011 | Month : April | Volume : 5 | Issue : 2 | Page : 223 - 226

The Phenotypic Detection Of Carbapenemase In Meropenem Resistant Acinetobacter Calcoaceticus–Baumannii Complex In A Tertiary Care Hospital In South India

ANIL V KUMAR*, VISHNU S PILLAI**, KAVITHA R. DINESH***, SHAMSUL KARIM****

*MD, Clinical Assistant Professor, Microbiology, Amrita Institute of Medical Sciences, Ponekkara, Kochi, Kerala, India, Pin: 682041; **MSc, Amrita Institute of Medical Sciences, Ponekkara, Kochi, Kerala, India, Pin: 682041; *** MD, Clinical Professor, Microbiology, Amrita Institute of Medical Sciences, Ponekkara, Kochi, Kerala, India, Pin: 682041; ****MD, Professor& Head, Microbiology, Amrita Institute of Medical Sciences, Ponekkara, Kochi, Kerala, India, Pin: 682041

Correspondence Address :
Dr.V.Anil Kumar MD (Microbiology) Clinical Assistant Professor Department of Microbiology Amrita Institute of Medical Sciences Ponekara, Kochi,Kerala India: 682041. vanilkumar@aims.amrita.edu Office:0484 2801234 (Extn : 8010,8015] Mob: 09037401413


Abstract

Background and objectives: The predominant Acinetobacter spp. in clinical settings are the members of the Acinetobacter calcoaceticus– baumannii complex which are multi drug resistant and are responsible for causing outbreaks. Carbapenem resistance due to metallo-β-lactamase production in Acinetobacter spp. is on the rise. We investigated the production of carbapenemase among the meropenem resistant Acinetobacter spp. which were further screened for metallo-β-lacatmase production. The co-resistance to other classes of antibiotics was also investigated.
Materials and Methods: Forty five non duplicate consecutive meropenem resistant Acinetobacter calcoaceticus– baumannii complex were investigated for carbapenemase production by the modified Hodge test. The carbapenemase producing isolates were further screened for metallo-β-lacatmase production by the combined disc diffusion test by using imipenem with EDTA as the chelator. The co-resistance to other classes of antibiotics was also investigated to identify the multi drug resistant isolates.
Results: Of the 45 non duplicate consecutive meropenem resistant Acinetobacter calcoaceticus– baumannii complex which were screened, 95% (43/45) of them were multi drug resistant and 71% (32/45) were found to be carbapenemase producers by the modified Hodge test, of which 21% (7/32) were found to be metallo-β-lacatmase producers phenotypically by the combined-disk test.
Conclusion: Carbapenem resistance in Acinetobacter calcoaceticus– baumannii complex is very high and is predominantly due to carbapenemase production. However, metallo-β-lactamase production among these isolates is not very high but is gradually increasing. Only 21% of our isolates were metallo-β-lactamase phenotypes, thus suggesting that the production of carbapenem hydrolyzing oxacillinase is still the most common mechanism of resistance to carbapenems in this species.

Keywords

: metallo-β-lactamase; Acinetobacter spp.; carbapenems.

How to cite this article :

ANIL V KUMAR, VISHNU S PILLAI, KAVITHA R. DINESH, SHAMSUL KARIM. THE PHENOTYPIC DETECTION OF CARBAPENEMASE IN MEROPENEM RESISTANT ACINETOBACTER CALCOACETICUS–BAUMANNII COMPLEX IN A TERTIARY CARE HOSPITAL IN SOUTH INDIA. Journal of Clinical and Diagnostic Research [serial online] 2011 April [cited: 2019 Aug 21 ]; 5:223-226. Available from
http://www.jcdr.net/back_issues.asp?issn=0973-709x&year=2011&month=April&volume=5&issue=2&page=223-226&id=1128

Introduction
Acinetobacter spp. is considered as the most common oxidase negative non fermenting gram negative bacilli which have been encountered in the clinical laboratory. It is widely distributed in nature and in the hospital environment, thus causing opportunistic infections in debilitated patients, especially in intensive care units. (1) Acinetobacter spp. has been included the list of the six top priority dangerous drug resistant microbes, which has been released by the Infectious Disease Society of America. The predominant Acinetobacter spp. in clinical settings are members of the Acinetobacter calcoaceticus– baumannii complex (Acb complex), which are multi drug resistant (MDR), thus leaving carbapenems as the only therapeutic option¬. (2) The Acb complex can frequently cause outbreaks and are very versatile, surviving in the hospital environment for long periods, thereby posing a difficult challenge for infection control. The first known carbapenem resistant Acinetobacter spp. was isolated in 1983 in Scotland and the carbapenem hydrolyzing β-lactamase was designated as oxacillinase-23 (OXA-23). (3) Though OXA is the predominant carbapenemase which is responsible for carbapenem resistance, reports on the IMP- or VIM-class metallo-β-lactamase (MBL) producing Acinetobacter spp. are also on the rise. (4) We investigated the production of carbapenemase among the meropenem resistant strains of the Acinetobacter spp. which were further phenotypically screened for MBL production. The co- resistance to other classes of antibiotics was also investigated.

Material and Methods

Forty five consecutive, non duplicate, meropenem resistant isolates of the Acb complex which were isolated from the samples of patients who were admitted to our hospital from August 2009 to January 2010 were included in our study. Only the Acb complex isolates which were obtained as a predominant growth in the culture, with relevant clinical history, were included in our study.

There are 17 recognized species within the genus, Acinetobacter. The major genus characteristics include the inability to ferment glucose (non-fermenter), lack of oxidase production (oxidase negative), and non-motility. The specific phenotypic characteristics include the appearance as cocci or coccobacilli on gram staining, the ability to grow on MacConkey’s agar and the resistance to penicillin. Many of the species within this genus are difficult to separate reliably by phenotypic methods alone and frequently are placed into groups or complexes based on the biochemical test results. The predominant Acinetobacter spp. in clinical settings is identified as the Acb complex as all of them have the ability to oxidize glucose and are therefore described as the saccharolytic species of this genus. (2),(5) (6) Antibiotic susceptibility was assessed by the Kirby Bauer disc diffusion method. (7) The antibiotics which were included were ciprofloxacin (5µg), cefipime (30µg), ceftazidime (30µg) and gentamycin (10µg), piperacillin (100µg), amikacin (30µg), piperacillin /tazobactam (100/10µg), cefoperazone/sulbactam (75/30µg) and meropenem (10µg). Meropenem resistance was used as the indication for carbapenemase production. A total of 45 Acb complex isolates with a reduced susceptibility to meropenem were further screened for carbapenemase and MBL productions by the modified Hodge test (MHT) and the combined disc test (CDT) respectively.

The Clinical Laboratory Standard Institute (CLSI) has not yet included any standardized phenotypic detection method for screening MBL positive strains in the Acb complex, though it has included screening and confirmatory tests for suspected carbapenemase production in Enterobacteriaceae. (7)

The Modified Hodge test
Lee et. al had modified the Hodge test which was developed to detect penicillinase producing Neisseria gonorrhoea by substituting Escherichia coli ATCC 25922 for penicillin susceptible Staphylococcus aureus ATCC 25923, and a 10µg imipenem disc for a 10 U penicillin disk. (5) An overnight culture suspension of E.coli coli ATCC 25922 which was adjusted to one tenth turbidity of the McFarland no.0.5 tube was inoculated evenly on the surface of a Muller-Hinton agar plate containing 70g/ml of zinc sulfate. After a brief drying at room temperature, an imipenem disk was placed in the center of the plate. Meropenem resistant test strains from an overnight culture were streaked heavily from the edge of the disk to the periphery of the plate. The presence of a distorted or clover leaf shaped inhibition zone was interpreted as positive for carbapenemase producing isolates. (5)

The Combined disc test
The combined disc test was carried out for 32 MHT positive isolates. The test strain was inoculated on plates with Mueller Hinton agar as recommended by CLSI for antibiotic sensitivity testing by the disk diffusion method. The presence of MBL was determined by placing two imipenem disks on the inoculated plate, in which 10 l of 0.1 M EDTA (292µg) was added to one of the imipenem disks. After overnight incubation at 37C, the inhibition zones of imipenem and imipenem with EDTA were compared. A zone difference of >4mm between the imipenem and the imipenem-EDTA inhibition zones confirmed the isolate to be MBL positive. (4)

Results

Antibiotic susceptibility testing of the 45 non duplicate carbapenem resistant isolates of the Acb complex isolates by the Kirby-Bauer disk diffusion method showed that 95% (43/45) of them were multi drug resistant (MDR). Multi drug resistance is defined as the resistance to three or more classes of antibiotics, including aminoglycosides, antipseudomonal penicillins, carbapenems, cephalosporins and quinolones. Co-resistance to other classes of antibiotics is shown in (Table/Fig 1). The sample wise distribution of the 45 non duplicate carbapenem resistant isolates of the Acb complex is shown in (Table/Fig 2).Of the 45 isolates of the Acb complex which were screened, 71% (32) were found to be carbapenemase positive by MHT. These positive isolates were further tested by CDT Of the 45 isolates of the Acb complex which were screened, 71% (32) were found to be carbapenemase positive by MHT. These positive isolates were further tested by CDT and 21% (7/32) were found to be MBL producers phenotypically.

Discussion

Carbapenems are used as the last resort for treating MDR Gram-negative infections in any noscomial setting. Carbapenems have a broad spectrum activity and they are stable to hydrolysis by most of the β-lactamases, including the extended spectrum β lactamases (ESBLs) and the AmpC β-lactamases. However, there has been an alarming increase in the reports on carbapenem resistant Acinetobacter spp. over the last decade. (8)

Carbapenem resistance in the Acb complex is attributed to various causes such as the reduced expression of the outer membrane proteins (29 kDa, 33-36kDa), the modification of the penicillin binding proteins, the specific drug efflux and carbapenemase production. The carbapenemases which are found in A. baumannii belong to either the OXA class D family of serine-β-lactamases (OXA-23 to 27, 40, 51 ,64 to 71) or to the IMP/VIM class B family of MBLs (IMP-1, 2, 4 and 5 and VIM-1 and 2). (9) Carbapenem resistant Acb complex which produce carbapenemases are being increasingly isolated in recent times. (10) Recently, a new type of MBL called SIM-1 has also been reported. (11)

MBL producing isolates have the ability to rapidly disseminate within an institution and lead to poor outcomes when infection ensues; therefore, it is immensely important to detect MBL in laboratory settings to avoid therapeutic failure. (4) Unfortunately, CLSI has not yet documented any standardized phenotypic detection method for screening MBL positive strains in the Acb complex, though it has included screening and confirmatory tests for suspected carbapenemase production in Enterobacteriaceae. (7) However, MBL positive Acinetobacter spp. strains can phenotypically be detected by using 1) the Double disk synergy test (DDST) by using IPM (imipenem)-EDTA (ethylene diamine tetra acetic acid), IPM-SMA (sodium mercaptoacetic acid) and the CTZ (ceftazidime)-SMA combinations. (12) 2) the Modified Hodge test by using a 10 µg imipenem disk and zinc sulfate solution. (5) 3) the combined-disk test (CDT) by using the IPM-EDTA combination. (4) 4) the Etest by using the combinations of IPM-EDTA, IPM-MPA (mercaptopropionic acid) and CTZ-EDTA. (13) 5) the Agar dilution method to determine the MIC of the IPM-EDTA combination. (14) 6) the carbapenem hydrolysis assay by using crude cell sonicates with and without EDTA treatment at 300C by using a spectrophotometer. (15) Among these tests, we selected the MHT with zinc sulfate and the CDT methods for this study, as they were highly sensitive and specific, easy to carry out and the materials required were cost effective, non-toxic and were easily available and hence, they could be easily adapted in laboratories for routinely detecting MBL phenotypes.

Of the 45 carbapenem resistant isolates which were included in our study, only 32 isolates were found to produce the carbapenemase enzyme and the remaining 13 strains were found to be carbapenemase negative by MHT. They probably showed resistance to carbapenem due to the efflux mechanism. (16)

MHT is used as a screening method to detect carbapenemas producers and therefore, it gives a positive result with strains that produces carbapenemases like MBL, Klebsiella pneumonia carbapenemase (KPC) and OXA-type β lactamases. Carbapenem resistance can also occur as a result of the reduced expression of the outer membrane proteins combined with the AmpC β-lactamases and the modification of the penicillin binding proteins and the efflux mechanisms. (17) In our study, of the 32 modified Hodge test positive strains, only 7 isolates were found to be MBL phenotypes; the remaining 25 (78%) strains presumably had the oxacillinase enzyme, as the OXA-23 encoding gene was found to be highly disseminated in the Acb complex from the Asia-Pacific nations, including India. (18)

Lee et.al reported that only 66% of the 39 MBL producing isolates of Pseudomonas aeruginosa and Acinetobacter spp. gave positive results by the Hodge test. He also noted that 10 more isolates with equivocal results became positive when the 10 µg IPM disks were supplemented with 10 µl of 50 mM zinc sulfate. Alternatively, the addition of zinc sulfate to Mueller-Hinton agar to get a final concentration of 70µg/ml also improved the test performance. (12)

Koh et.al in 2006, found that 30.5% of the 114 isolates of imipenem resistant Acb complex isolates showed carbapenemase activity; of which 4 isolates carried blaIMP-4, 5 carried blaOXA-58 and 40 carried blaOXA- 23. All four strains which were positive for blaIMP-4 also had blaOXA-58, while one strain with blaOXA-23 also had blaOXA-58. (17)

Many Indian studies have documented the presence of MBLs in Pseudomonas aeruginosa (6); however, to our knowledge, only three studies have been carried out for the phenotypic detection of MBLs in A. baumannii in India. [6,19,20] Uma et.al in 2008, reported that 70% of the carbapenem resistant A. baumannii isolates were positive for MBL production by the EDTA-DDST, of which 42% harboured the blaIMP-1 gene, while none of them showed the presence of the blaVIM-2 gene. (6)

In our study, 71% (32/45) of the isolates of the Acb complex were found to be carbapenemase producers by the MHT. This was in concordance with the results which were obtained by Lee et.al(19) in Korea, where 73% (59/81) of the isolates were found to be carbapenemase positive by the MHT, but was in disagreement with the other Korean studies using the same method, reporting a 25% and 14% prevalence of carbapenemase among the Acb complex isolates. Only one study in India used the MHT to detect carbapenemase production in the Acb complex and reported a very low prevalence of 2.2%. (20)

The carbapenemase positive isolates from our study were further tested by the combined disk test (CDT) method and 21% were found to be MBL producers phenotypically. This was in concordance with the results obtained by Lee et.al in 2005 by using DDST with imipenem and EDTA disks, who reported a 25% MBL prevalence. (11) Similar results were also obtained by Franklin et.al by using CDT (16%) and by Lee et.al (2003) by using DDST (14%). [14, 12] Our results were also in disagreement with the results of two Indian studies on the detection of MBL by DDST; Uma et.al, 70% (39/55) and Gupta et.al, 7.5% (4/200). [6, 19]

However, a study on the mechanism of resistance to carbapenems in meropenem resistant Acinetobacter spp. isolates from clinical samples, which was conducted by Sinha et.al; 2006, found that none of the meropenem resistant isolates produced MBLs . (8)

The most active agent against the carbapenem resistant isolates in our study was cefoperazone/sulbactam, with a susceptibility rate of 49%. Yun-Song Yu et.al studied 45 carbapenem isolates and found that cefoperazone/sulbactam and ampicillin/sulbactam were the only active agents, with a susceptibility of 63% and 43.5% respectively. (2) This may be due to the unique activity of sulbactam against Acinetobacter spp. Sulbactam acts synergistically with cephalosporins in the treatment of infections caused by such isolates.

Conclusion

This study confirms that majority of the currently prevalent carbapenem resistant Acb complex are multidrug-resistant. Carbapenemase production appears to be the most common mechanism of carbapenem resistance by the phenotype screening method. Since among these isolates only 21% were of the MBL phenotype, the production of carbapenem hydrolyzing oxacillinase is the most likely mechanism of resistance. Based on our study, the modified Hodge test with zinc sulfate and the combined-disk test, which are simple and highly sensitive methods, can be used for the routine phenotypic detection of MBL production in any laboratory settings.

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