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

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Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
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On Sep 2018




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"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."



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




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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.
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Dr. Mamta Gupta
Consultant
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Aug 2018




Dr. Rajendra Kumar Ghritlaharey

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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.
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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 : 2012 | Month : May | Volume : 6 | Issue : 4 | Page : 660 - 663

The Simultaneous Detection of the ESBL and the AmpC b-Lactamases in Gram Negative Bacilli

Avinash Laghaw e, Neelam Jaitly, Vilas Thombare

1. M.D.(Microbiology) Assist. Professor, Dept. of Microbiology, Sri Aurobindo Inst. Of Medical Sciences, Indore, M.P. , India. 2. M.D.(Microbiology) Professor, Dept. of Microbiology, NKP Salve Inst. of Medical Sciences and Research Centre, Nagpur, MH, India. 3. M.D. (Microbiology) Professor and HOD, Dept. of Microbiology, NKP Salve Inst. of Medical Sciences and Research Centre, Nagpur, MH, India.

Correspondence Address :
Avinash Laghawe
Asst. Professor, Dept. of Microbiology,
SAIMS Medical College, Sanwer Road, Indore,
Madhya Pradesh, India - 453111
Phone: 09713357133
E-mail: lavinash@rediffmail.com; dravinash73@gmail.com

Abstract

Purpose: The purpose of this study was to detect the prevalence of the ESBLs and the AmpC β-lactamases in gram-negative bacilli.

Methods: The detection of ESBLs was done by using various third generation cephalosporins (3GC), along with imipenem, aztreonam, cefoxitin and ceftazidime + clavulanic acid. The criteria which were used for the identification of ESBL – were resistance to 3GC, sensitivity to cefoxitin and increase in the zone size by 5 mm or more of ceftazidime + clavulanic acid as compared to the ceftazidime zone size. The organisms which were resistant to cefoxitin were tested for the presence of AmpC by the AmpC disc test.

Results: A total of 432 isolates were isolated from 414 samples. Out of these 432 isolates, 85(19.67%) were (64 pure and 21 mixed) ESBL producers, 69(15.97%) were (48 pure and 21 mixed) AmpC β-lactamase producers and 299(69.22%) isolates didn’t show any evidence of the production of β-lactamases. 21(4.86%) isolates were positive for both ESBLs and AmpC. Out of the 64 pure ESBL producers, 55 (85.94%) were from indoor patients and 9(14.06%) were from outdoor patients. Out of the 48 pure AmpC producers, 42(87.5%) were from indoor patients and 6(12.5%) were from outdoor patients. All (21) the mixed beta lactamase producers (ESBLs + AmpC) were from indoor patients.

Conclusion: Regular monitoring of the incidence of the β-lactamase production by the organisms is necessary. As the β-lactamase producing organisms are also present in the outdoor patients, they also should be screened for the production of β-lactamases. The detection of ESBLs and AmpC beta lactamases by this method is simple and any microbiology laboratory can do it along with the routine antibiotic susceptibility testing.

Keywords

ESBL, AmpC, Clavulanic acid, Cefoxitin, Third generation cephalosporins

How to cite this article :

Avinash Laghaw e, Neelam Jaitly, Vilas Thombare. THE SIMULTANEOUS DETECTION OF THE ESBL AND THE AMPC B-LACTAMASES IN GRAM NEGATIVE BACILLI. Journal of Clinical and Diagnostic Research [serial online] 2012 May [cited: 2019 Nov 15 ]; 6:660-663. Available from
http://www.jcdr.net/back_issues.asp?issn=0973-709x&year=2012&month=May&volume=6&issue=4&page=660-663&id=2123

INTRODUCTION
At least a quarter of all the illnesses for which patients consult their doctors are infective. Gram-negative bacteria may exhibit a reduced susceptibility to the β-lactam antibiotics by a number of mechanisms which include reduced outer membrane permeability, target site modification, and efflux of β-lactams out of the cell. However, by far, the most common mechanism of resistance is the enzymatic inactivation of the β-lactams by a β-lactamase (1). The extensive use of the third generation cephalosporins (3GC) like cefotaxime, ceftriaxone and ceftazidime has led to the evolution of newer β-lactamases such as the Extended Spectrum Beta Lactamases (ESBLs). ESBLs are Plasmid-mediated enzymes that hydrolyze the oxyimino β-lactams (3GC) and the monobactams (aztreonam) but have no effect on the cephamycins (cefoxitin, cefotitan) and the carbapenems (Imipenem). Being plasmid mediated, they can be easily transferred from one organism to another. Based on their physical properties, more than 300 ESBLs have been identified and they have been found to be inhibited by clavulanic acid, sulbactam and tazobactam. This is the property which helps in their detection in the laboratory. ESBLs are more prevalent in the Klebsiella species and in E. coli, but they may also be found in other genera such as Citrobacter, Serratia, Proteus, Salmonella, Enterobacter, Pseudomonas, etc (2).

The expression of the AmpC β-Lactamases can be generated by chromosomal or plasmid genes. The plasmid mediated AmpC β-lactamases are thought to have originated from the chromosomes of several Enterobacteriaceae species and they are rarely inducible. Unlike ESBLs, the plasmid encoded cephalosporinases are active against the cephamycins and they are not inhibited by clavulanic acid. The plasmid mediated AmpC β-lactamases were first reported in 1988. The number of infections which are caused by the AmpC producing organisms is increasing. Distinguishing between the AmpC and the ESBL producing organisms has epidemiological significance and it may have a therapeutic importance as well (3). The AmpC producing organisms can act as hidden reservoirs for ESBLs. Enterobacteriaceae which produce both AmpC and ESBLs have been increasingly reported worldwide. Also, the high-level expression of the AmpC β-lactamases may mask the recognition of the ESBLs (4). With this background, the current study was undertaken to detect the prevalence of ESBLs and AmpC β-lactamases in gram-negative bacilli, as not many such studies have been done on this topic, especially in central India.

Material and Methods

All the gram-negative bacilli which were isolated from various clinical samples from hospitalized patients as well as from patients who attended the out patients departments were collected and identified by standard biochemical reactions. Anti-microbial susceptibility testing was performed by the Kirby Bauer disc diffusion method for cefotaxime, ceftriaxone, ceftazidime, ceftazidime + clavulanic acid, imipenem, cefoxitin and aztreonam. The disc placement was designed in such a fashion so as to assess the ESBLs and the Amp C enzymes [Table/Fig-1].

Imipenem, an inducer, was placed in the centre and on either side of it, at a 15 mm distance, ceftazidime and cefotaxime (indicators of induction) were placed. The ceftazidime + clavulanic acid disc was placed 15-20 mm apart from the ceftazidime disc. In addition, another inducer, cefoxitin, was placed 15 mm apart from cefotaxime (indicator). The cefoxitin disc was placed opposite to that of ceftazidime + clavulanic acid to avoid any effect of the inducible β-lactamase on the zone of inhibition of ceftazidime + clavulanic acid. Aztreonam was placed in between ceftazidime + clavulanic acid and cefotaxime, while ceftriaxone was placed in between cefoxitin and ceftazidime. All the discs were placed at a distance of 15-20 mm from each other (5).

Interpretation (5)
An isolate was suspected to be an ESBL producer if it had the zone sizes for the cephalosporins like cefotaxime (30μg) ≤ 27 mm, ceftazidime (30μg) ≤ 22 mm, ceftriaxone (30μg) ≤ 25 mm and aztreonam (30μg) ≤ 27 mm. The criteria which were used for deciding whether an organism was an ESBL producer (Table/Fig 2):1) Zone diameter for various 3GCs as has been mentioned above. 2) Susceptibility to cefoxitin 3) Increase in the zone size with the addition of an inhibitor, by ≥ 5 mm. The criteria which were used for suspecting whether an organism was an AmpC producer [Tabe/Fig-3]: 1) Zone diameter for various 3GCs as has mentioned above. 2) Resistance to cefoxitin 3) No increase in the zone size with the addition of an inhibitor, by ≥ 5 mm

CONFERMATION OF THE AMPC b-LACTAMASES [Table/Fig-4] (6)
The isolates which showed blunting of the ceftazidime or the cefotaxime zone of inhibition which was adjacent to the inducer (imipenem or cefoxitin) or those which showed a reduced susceptibility to either of the above test drugs (ceftazidime and cefotaxime) and cefoxitin were considered as “screen positive” and they were selected for the confirmation of the AmpC β-lactamases. A lawn culture of E.coli ATCC 25922 was prepared on a Mueller- Hinton agar plate. A sterile disk (6mm) was moistened with sterile saline (20μl) and it was inoculated with several colonies of the test organism. The inoculated disk was then placed besides a cefoxitin disk (almost touching) on the inoculated plate. The plate was incubated overnight at 350C. A positive test appeared as a flattening or an indentation of the cefoxitin inhibition zone in the vicinity of the test disk. A negative test produced a undistorted zone.

Results

This study was conducted from February 2005 to July 2007 in the Department of Microbiology, NKP Salve Institute of Medical Sciences and Research Center and Lata Mangeshkar Hospital, Digdoh, Nagpur, India. A total of 432 isolates were isolated from 414 samples. Out of the 414 samples, 392 were mono microbial and 22 were poly microbial. The maximum number [154(35.65%)] of isolates were of E.coli, followed by those of Pseudomonas aeruginosa [113(26.16%)] and Klebsiella pneumoniae [111(25.69%)]. Other isolates were those of Enterobacter cloacae [19(4.4%)], Proteus mirabilis [14(3.24%)], non-fermenters [9(2.08%)], Citrobacter freundii and Serratia marcescens [4(0.93%)]. Of the 432 isolates, 137 were found to be resistant to cefoxitin and to one or more of the third generation cephalosporins. When the AmpC disk test was applied, 69 isolates were found to be positive for the AmpC β-lactamases.

Out of the 432 isolates, [Table/Fig-5] 85(19.67%) were ESBL producers, 69(15.97%) were AmpC β-lactamase producers and 299(69.22%) isolates didn’t show any evidence of the production of β-lactamases. 21(4.86%) isolates were positive for both ESBLs and AmpC.

ESBL production was observed in 23.38% of isolated E.coli, 19.82% of the Klebsiella pneumoniae isolates and in 62.5% of the Citrobacter freundii isolates. Out of the 23.38% ESBL producing E. coli, 20.78% produced pure ESBLs and 2.6% produced mixed ESBLs i.e. ESBLs + AmpC. Out of the 19.82% ESBL producing Klebsiella pneumoniae, 14.41% produced pure ESBLs and 5.41% produced mixed ESBLs i.e. ESBLs + AmpC. The AmpC production was predominant in Citrobacter freundii (62.5%), followed by Enterobacter cloacae (52.63%) and the non-fermenters (44.44%).∗ A mixed β-lactamase (ESBL and AmpC) production was observed in 4 - E.coli, 4 - Ps. aeruginosa, 6 - Klebsiella pneumoniae, 2 - Enterobacter cloacae, 2 - Proteus mirabilis, and 3-Citrobacter freundii isolates.

All the β-lactamase producing organisms were susceptible to Imipenem. Out of the 64 pure ESBL producers, 55 (85.94%) were from indoor patients and 9(14.06%) were from outdoor patients. Out of the48 pure AmpC producers, 42(87.5%) were from indoor patients and 6(12.5%) were from outdoor patients. All (21) the mixed beta lactamase (ESBL + AmpC) producers were from indoor patients.

Discussion

The knowledge on the extent of the ESBL mediated resistance appears to be limited due to the inability of the standardized methods of susceptibility testing or the commercially available systems to detect this resistance (7). The emergence and the spread of the ESBL producing strains have led to questions regarding the optimal therapy for infections which are caused by the ESBL producing strains (8). The confirmation of the ESBL production by clavulanic acid inhibition can be difficult in some strains, not only because the activity of the β-lactamase varies with different substrates, but also because the organism may contain additional resistance mechanisms that can mask the presence of the ESBL activity (9).

The AmpC β-lactamases are cephalosporinases which belong to the molecular class C, as was classified by Ambler and group I under a classification scheme of Bush and Jacoby et al. These are clinically significant as they may confer resistance to a wide variety of β-lactam drugs which include α-methoxy-β-lactams, narrow, expanded and broad-spectrum cephalosporins, aztreonam and most significantly, the β-lactam plus the β-lactamase inhibitor combinations (10). The AmpC producing organisms can act as a hidden reservoir for the ESBLs. Enterobacteriaceae which produce both AmpC and ESBLs have been increasingly reported worldwide. Also, the high-level expression of the AmpC β-lactamases may mask the recognition of the ESBLs (4).

In our study, 19.67% were ESBL producers as against Emery et al’s (11) and Spanu et al’s (12) findings which reported ESBL prevalence in 1.2% and 6.3% isolates respectively. Stratchounski et al (13) and Sanguinetti et al (14) had reported in their studies that 66.7% and 62.55% were ESBL producers. The studies which were carried out by Cormican et al (7), D’Agata E. et al (15), Navon et al (16), Tankhiwale et al (17) and Wang et al (18) reported isolation rates of 17.33%, 13.1%, 19%, 19.35% and 16.67% respectively. This also proved that the prevalence of ESBLs among the clinical isolates varied from country to country and institution to institution within the country. In our study, AmpC production was seen in 15.97% isolates as against Ratna AK et al’s (19) findings (3.3%) and Rodrigues et al’s (5) findings (66.43%). Moland et al (20) and Sanguinetti et al (14) found the prevalence of the AmpC production to be 10.67% and 15.1% respectively.

Though the presence of the beta lactamases in the indoor patients was significantly high as compared to that in the outdoor patients, beta lactamases are also found in community acquired infections.

Conclusion

Regular monitoring of the incidence of the β-lactamase production by the organisms is necessary. Along with the detection of ESBL, it is necessary to detect AmpC β-lactamases, as they can act as hidden reservoirs for the ESBLs. Enterobacteriaceae which produce both AmpC and ESBLs have been increasingly reported worldwide. Also, the high-level expression of the AmpC β-lactamases may mask the recognition of the ESBLs.

To prevent the spread of the β-lactamase producing strains, hospitals must have functional hospital infection control committee with an appropriate hospital antibiotic policy, with regular updates. As the β-lactamase producing organisms are also present in the outdoor patients, they also should be screened for the presence of β-lactamases. Also, attempts should be made to prevent the dissemination of the β-lactamases in the community. The carbapenems should be kept as reserve drugs and they should be used only in patients who have infections which are caused by multi-drug resistant strains, especially the strains which produce extended-spectrum and AmpC β-lactamases. The detection of ESBLs and AmpC beta lactamases by this method is simple and any microbiology laboratory can do it along with the routine antibiotic susceptibility testing.

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