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

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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

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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.
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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.
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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 ones 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 journalsNo manuscriptsNo 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 : 2009 | Month : February | Volume : 3 | Issue : 1 | Page : 1307 - 1312

Extended Spectrum Beta-Lactamases (ESBL) In Gram Negative Bacilli At A Tertiary Care Hospital

SHOBHA K.L*,RAMACHANDRA L**,RAO G*** ,MAJUMDER S****, RAO S P*****.

*Prof,Dept of Microbiology,Melaka Manipal Medical College,**Prof.,Deptof Surgery Kasturba Medical College,Manipal,***Sr.lecturer,Dept.of Microbiology,Melaka ManipalMedicalCollege,Manipal,****Post graduatestudent,*****Prof.Dept.of Microbiology Kasturba MedicalCollege,Manipal

Correspondence Address :
Dr Shobha K.L,Professor,Dept.of Microbiology, Melaka Manipal Medical College,Manipal,Udupi,Karnataka(India),Pin:576104,Ph:0820– 2922519/2922570 Email:shobhamicro@yahoo.com

Abstract

Extended spectrum beta lactamases (ESBL) hydrolyse expanded spectrum cephalosporins like ceftazidime and cephotaxime ,which are used in the treatment of Pseudomonas and other gram negative bacteria. ESBL producing bacteria may not be detectable by the routine disk diffusion susceptibility test, which leads to inappropriate use of antibiotics and treatment failure. Not much information on ESBL producing organisms causing infection is available from the south west coastal region. An effort was therefore made to study the ESBL producing gram negative organisms by the phenotypic confirmatory test and the novel fashion method. Antibiotic susceptibility pattern of ESBL organisms were also analysed.
Methods:160 clinical strains were included in the study. Strains were obtained from adult patients who were either admitted to or attended the outpatient departments of medicine, surgery, obstetrics and gynaecology in a tertiary care hospital .The study was conducted from June 2005 to December 2005. Informed consent was taken from the patients for collecting the samples. The samples were processed for the identification of organisms and were screened for ESBLs. The isolates were also tested for antimicrobial susceptibility by the Kirby-Bauer disc diffusion technique, using Muller Hinton agar. Screening for ESBL was done as per the guidelines recommended by CLSI . Organisms were further tested for confirmation of ESBL production by the phenotypic confirmatory test and by the Novel fashion .
Results And Conclusion:Out of the total 160 strains, the common organisms isolated were Klebsiella pneumoniae with 73 strains (45.62%), followed by Escherichia coli with 63 strains (39.37%) and Pseudomonas spp with 14 strains (8.75%), respectively. ESBL positive strains detected by the screening test for Klebsiella pneumoniae were 20(27.39%), for Escherichia coli were 16 (25.39%) and for Pseudomonas species were 03(21.42%), respectively. ESBL positive organisms were also found to remain positive by the Phenotypic confirmatory test, when combinations of Cefotaxime against amoxicillin /clavulanic acid and Cefipime against piperacillin/tazobactum were used. The novel fashion method showed that ESBL and de-repressed mutants in E.coli were 29(46.03%), only de-repressed mutants strains were 15 (23.80%) and inducible Amp C gene producers were 03(4.76%) . Among 48(65.75%) strains, Klebsiella pneumoniae showed ESBL and de-repressed mutants , de-repressed mutants alone in 08(10.95%) strains and inducible Amp C mutants in 02(2.73%). The antimicrobial susceptibility test showed that ESBL organisms were resistant to gentamicin and trimethoprim / sulphamethoxazole, but all were susceptible to imipenem, We conclude that clinical laboratories should develop quick screening methods to assess the different mechanisms of ESBL production, so that the patients can be treated with appropriate antibiotics .

Keywords

ESBL, Gram negative bacilli, novel fashion method

Introduction
The widespread use of antibiotics in hospitals has led to the emergence of multi-drug resistant organisms like Klebsiella spp, Pseudomonas spp , Escherichia coli and Enterobacter spp .Over the last few years, numerous outbreaks of infections with organisms producing extended spectrum beta lactamase (ESBLs), has been observed worldwide. The advent of ESBL producers has posed a great threat to the use of antibiotics like cephalosporins .There are indications that poor outcome occurred when patients with serious infections caused by ESBL producing organisms were treated with antibiotics to which the organisms were resistant .The aim of the present study was to study the prevalence of ESBL production among gram negative organisms by the different mechanisms and to analyse the antibiotic susceptibility of ESBL producing organisms.

Material and Methods

A total number of 160 clinical strains were included in the study .The strains were obtained from patients admitted to or those who attended the outpatient departments of medicine, surgery , obstetrics and gynecology at a tertiary care hospital. Out of the 160 patients, 87 were males and 73 were female patients in the age group of 18 years to 70 years. No specific criteria were followed in the selection of males and females. The study was conducted during the period from June 2005 to December 2005. Informed consent was taken from the patients before collecting the samples, which included urine, pus and sputum. The samples were processed for the identification of organisms according to W.winn et al (1) and were later screened for ESBLs. Screening for ESBL (2) was done according to the guidelines recommended by the Clinical Laboratory Standards Institute (CLSI). Control strains, Escherichia coli ATCC 25922 (Beta – Lactamase negative) and Klebsiella pneumoniae ATCC 700603(ESBL positive) were used for quality control. The screening test for ESBL was considered positive when an inhibition zone of <27 mm for cefotaxime and an inhibition zone of <22 mm for ceftazidime was indicated. ESBL producing organisms were further confirmed by the phenotypic confirmatory test .The method followed for the phenotypic confirmatory test was the antibiotic disc test, An amoxicillin - clavulanic acid (20/10 µg ) disc was placed in the centre of the Muller Hinton culture plate, and a disc of cefotaxime was placed at a distance of 20 mm from the amoxicillin - clavulanic acid disc. The third antibiotic disc, aztreonam (30 µg) was placed at a distance of 25 mm from the amoxicillin-clavulanic acid disc and another antibiotic disc ceftazidime (30 µg) was placed at a distance of 30mm from the amoxicillin-clavulanic disc. In the same culture medium, a piperacillin – tazobactum (100 µg/10 µg) disc was placed at a distance of 25 mm from the cefipime disc, and on the opposite side, a tazobactum (110 µg) disc was placed at a distance of 25 mm from the cefipime disc. All the afore said antibiotic discs were purchased from Hi-Media, ,Mumbai, India . The plates were incubated at 370C overnight, and were examined for formation of a synergistic zone or enhancement of zone of inhibition by the cefotaxime and cefipime disc at the side facing the amoxicillin-clavulanic acid (20/10 µg) disc and the piperacillin-tazobactum disc (100/10 µg) . The organisms that showed a clear extension of zone of inhibition towards the amoxicillin-clavulanic acid and the piperacillin-tazobactum discs were considered to be ESBL strains. Apart from the above procedure, a different method called the Novel fashion method for detection of ESBL (3) , was also studied for ESBL strains. The disc placements in the novel fashion method were as shown in the (Table/Fig 1).

The ceftazidime and ceftazidime-clavulanic acid discs were kept 15-20 mm apart from each other .Imipenem, an inducer, was placed in the centre. On either side of the imipenem discs, at a distance of 15 mm,the ceftazidime and cefotaxime discs were placed (indicators of induction). Another inducer, cefoxitin was placed at 15mm from cefotaxime (indicator). Cefoxitin discs were placed opposite the ceftazidime - clavulanic acid discs to avoid any effect of inducible beta -lactamase on the zone of inhibition of the latter. The remaining discs were placed as showed in (Table/Fig 1). The criteria used in the Novel fashion method (3) for ESBL producer detection was, the resistance of the strain to 3rd generation cephalosporins, susceptibility of the strains to cefoxitin and the tendency of the strains to show increase of zone size by 5mm with the addition of an inhibitor. Inducible Amp C lactamase production was considered when there was a blunting of the zone towards the inducer, when there was no increase in zone size with the addition of an inhibitor and when the strain was susceptible to cefipime. De-repressed mutants were considered when the strain was resistant to cefotaxime and cefoxitin, when it showed blunting of the zone towards the inducer and when it showed no increase in zone size with the addition of an inhibitor. The strain was considered to have multiple mechanisms of resistance to cefoxitin, showed blunting of the zone towards the inducer and showed increase of zone size by 5mm with the addition of an inhibitor. The isolates were also tested for antimicrobial susceptibility by the Kirby-Bauer disc diffusion technique, using Muller Hinton agar (4) . Muller Hinton agar was purchased from Hi-media, Mumbai, India. In the Kirby-Bauer disc diffusion method , test strains were pre-incubated in peptone water at 370C at an optical density of 0.5Mc Farland standard .This suspension was used to inoculate the strains on to the Muller Hinton agar plate by swabbing them with a sterile cotton swab. The different antibiotic discs used for the disc diffusion test were amikacin(30µg), gentamicin (10µg), ciprofloxacin (5µg), trimethoprim/sulphamethoxazole(TSX) (1.25/23.75µg), netilmicin((30µg), cefotaxime(30µg) ,ceftazidime (30µg) and imipenem (110µg). The antibiotic discs were purchased from Hi-media, Mumbai, India .The plates were incubated at 370C overnight, a zone of inhibition was measured for each antibiotic and it was recorded as sensitive or resistant.

Results

73 strains of Klebsiella pneumoniae , 63 strains of Escherichia coli, 14 strains of Pseudomonas spp , 04 strains of Enterobacter spp and 06 strains of Acinetobacter spp were isolated from 160 strains. ESBL strains which tested positive by the screening test were 20(27.39%) Klebsiella pneumoniae, 16(25.2%) Escherichia coli, 03(21.42%) Pseudomonas spp, 01(25%) Enterobacter spp and 01(17%) Acinetobacter spp. In the phenotypic confirmatory test, when the combinations of cefotaxime against amoxicillin /clavulanic acid and cefipime against piperacillin/tazobactum was used, the results showed that the strains which were positive by the screening test were also positive by phenotypic confirmatory test . The novel fashion method showed that ESBL and de-repressed mutants in E.coli were 29(46.03%), only de-repressed mutants strains were 15 (23.80%) and inducible Amp C gene producers were 03(4.76%). Klebsiella pneumoniae spp showed 48(65.75%) strains of ESBL and de-repressed mutants 08(10.95%) strains of de-repressed mutants alone and 02(2.73%) of inducible Amp C mutants (Table/Fig 2). Different mechanisms of Beta –Lactamase production were identified by the novel fashion method. Antibacterial susceptibility test to various antibiotics, indicated that 17 (85%) ESBL producing Klebsiella pneumoniae organisms were sensitive to amikacin and all ESBL strains were sensitive to imipenem (Table/Fig 3).

Discussion

The number of strains which tested positive by the screening test, were also found to be positive by the phenotypic confirmatory test. The percentage of ESBL producers detected by the phenotypic confirmatory method for Klebsiella pneumoniae strains were 20/73 (27.39%), for Escherichia coli were 16/63 (25.39%), for Pseudomonas spp were 3/14 (21.42%) , for Enterobacter spp were 1/4(25%) and for Acinetobacter spp were 1/6 (17%). These results were in concordance with the study conducted by C.Rodriques (3), David L Peterson (7) and S.Babypadmini et al (8) . The novel fashion method of study indicated that ESBL organisms like E.coli, Pseudomonas spp and Klebsiella pneumoniae produced various different mechanisms for the production of multiple β-Lactamases. . Our study was in concordance with the study conducted by C.Rodrigues et al (3). The percentage of plain ESBL producers among Ecoli was 16(25.3%) and among Klebsiella pneumoniae were less frequent (15%), when compared to studies conducted by A Varaiya et al (9), and Pseudomonas spp had fewer plain ESBL producers when compared to studies conducted by C Rodrigues et al (3).The percentages of acinetobacter spp and enterobacter spp were 01(16.66%) and 01(25%), respectively, and the percentages of those producing plain ESBL was less, when compared to Mahua et al (10) A similar study conducted by Giuseppe Celenza (11) from a Bolivian hospital, showed a higher percentage (34%) of ESBL production. It could be because only a few strains of enterobacter spp and acinetobacter spp were isolated at our centre. De-repressed mutants in our study were 29(46.03%) for Ecoli and 02(14.28%) for Pseudomonas, which was less when compared to studies conducted by Rodrigues et al (3) . However, in our study, 48(65.75%) klebsiella pneumoniae, 03(50%) acinetobacter spp and 02(50%) enterobacter spp were both ESBL producers with derepressed mutants, which was high when compared to studies conducted at Mumbai (3). Pseudomonas spp 01(7.14%) and acinetobacter spp 02(33.33%) were detected to be de-repressed mutants, which was less when compared to studies conducted at Mumbai (3). All the four organisms tested, namely Escherichia coli 3/63(4.76%), Pseudomonas spp 10/14(71.42%) , Klebsiella pneumoniae 2/73(2.75%) and acinetobacter spp 1/6(16.66%), were detected to produce inducible Amp C lactamase, except Enterobacter spp. The percentage of inducible Amp C lactamase production was higher when compared to studies done at Mumbai. At Mumbai, only 26.5% Pseudomonas spp showed inducible Amp C lactamase production. When the phenotypic confirmatory method was compared to the novel fashion method, the latter was found to be better than the former, because it assesses ESBL producers, de-repressed mutants, inducible AmpC lactamase production and multiple mechanisms in a single culture plate and it was also easy to perform. The antimicrobial susceptibility test showed that strains were resistant to gentamicin, netilmicin, ciprofloxacin and trimethoprim/sulphamethoxazole.

Susceptibility to imipenem and amikacin was found to be 14(100%) in Pseudomonas spp , 63(100%) in E.coli 63, 17(85%) in Klebsiella pneumoniae and the susceptibility to netilmicin for Pseudomons spp was 14 (100%) strains, for E.coli it was 15( 93.75% ) and for Klebsiella pneumoniae it was 17 (85%), but all ESBL producers were found to be susceptible to imipenem and amikacin. However, amikacin and carbapenems are usually used only as the reserve drugs . A similar study conducted by Hanstia JB et al (5) and Abigal S Mathai et al (6) showed 100% susceptibility to amikacin and imipenem. The marked increase in β-Lactamase production, including the high level constitutive ESBL producers (de-repressed mutants), have left us with few alternatives in combating serious infections.

Conclusion

Clinical laboratories must be aware of the importance of ESBL and plasmid mediated AmpC Beta- Lactamase production. Although CLSI recommendations exist, they are limited to ESBL producers of Ecoli and Klebsiella spp . No recommendations exist for ESBL detection and reporting for other organisms, or for the detection of Amp C Beta- Lactamases .Clinical laboratories need to develop quick screening methods to assess the mechanisms of Beta- Lactamase resistance in their isolates, so that appropriate antibiotics can be given to patients. Screening methods of ESBL with recommended zone size should be immediately applied to suggest the presence of an ESBL. From the study, it was apparent that various different mechanisms exist for production of multiple Beta-Lactamases, especially in places where newer Beta-Lactams were being routinely prescribed. The marked increase in Beta-Lactamase production and the high level constitutive producers (de-repressed mutants) with ESBL leave us with few alternatives in combating serious infections. Good infection control practice and careful introspection while prescribing Beta- Lactam drugs, with the background of high risk category in acquiring infections with ESBL producing organisms, have to be considered for good anti-microbial stewardship in hospitals. The battle between the antibiotics and bacteria possessing Beta- Lactamase, is far from over .Unfortunately, as new antibiotics are introduced that are capable of resisting the effects of bacterial Beta Lactamases, bacteria develop novel ways of overcoming the new antibiotics. A small number of new Beta-Lactamase antibiotics and Beta-Lactam inhibitors are undergoing development at the present time(12). It is unclear whether we will be able to keep pace with bacterial genetic changes and be able to effectively treat gram negative infections in future.

Key Message

ESBL by phenotypic confirmatory test, novel fashion method

References

1.
. WinnW.Jr, Allen S, Janda W et al.Koneman’s color atlas and textbook of diagnostic microbiology.6th edn. 2006 Philadelphia USA.
2.
. Amitha Jain, Indranil Roy, Mahendra KGupta ,Mala Kumar and S.K. Agarwal .Prevalence of ESBL producing gram negative bacteria in septicaemic neonates in tertiary care hospital .J Medl Microbiology 2003; 52: 421- 25 .
3.
. C.Rodrigues,P.Joshi ,SH Jain ,M Aiphons ,R Radhakrishna,A Mehata .2004. Detection of β lactamases in nosocomial gram negative clinical isolates. JCM. 22(4):247-50.
4.
. Clinical and Laboratory Standards Institute, 2006. Performance standards for antimicrobial susceptibility testing; 16th informational supplement. CLSI document M100-S16. Clinical and Laboratory Standards Institute, Wayne, PA.
5.
. Hanstia JB ,Agarwal V,Patha KAA,Saoji AM.ESBL mediated resistance to 3rd generation cephalosporins in Klebsiella pneumonia ,Nagpur Central India.Indian Journal of Medical Research.1997;105:158- 61.
6.
. Abigal S Mathai ,E Jesudasan MV,Jhon TJ: Ceftazidim resistance among Klebsiella pneumoniae in South India.Indian Journal Of Medical research .1995:102:53-88
7.
. David L Peterson MD. β lactamase inhibitor and new β lactamase :JCM.1999; 85-87
8.
. S Babypadmini, B Appalaraju:Extended spectrum –lactamases in urinary isolates of Escherichia coil and Klebsiella pneumoniae-Prevalence and susceptibility pattern in a tertiary care hospital:IJMM. 2004;22:3:172 - 74.
9.
. A Varaiya ,J Dogra ,M Kulkarni ,PBhalekar :Extended spectrum beta lactamase(ESBL) producing Escherichia coli and Klebsiella pneumoniae in diabetic foot infection:IJMM. 2008; 26:3:281- 82.
10.
. Mahua Sinha, H Srinivasa and R Macaden:ESBL-Production of Acinetobacter species :IJMR. 2007;126:63 – 67.
11.
. Giuseppe Celenza, Cristina Pellegrini, Marisa Caccamo ,Bernardella Segatore ,Gianfranco Amicosante and Mariagrazia Perilli :Spread of bla CTX- Mtype and bla PER-2 β- Lactamase genes in clinical isolates from Bolivian hospitals. Journal of Antimicrobial Chemotherapy.2006;57(5):975 – 78.
12.
. K.Bush:Beta-lactamase inhibitors from laboratory to clinic.Clin Microbiol Rev. 1988;1(1):109 – 23.

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