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

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Dr Bhanu K Bhakhri

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Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




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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
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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."



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Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
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Lucknow
On Sep 2018




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On Aug 2018




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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.
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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 : 2010 | Month : August | Volume : 4 | Issue : 4 | Page : 2679 - 2684

Existence of Metallo beta lactamases in carbapenem susceptible gram negative bacilli: a cause for concern

G RENU*, T RAJEEV**, S SMITA**

*MD, Assistant Professor, Department of Microbiology, Institute of Human Behaviour and Allied Sciences, Delhi-95. **MD, Professor, Department of Microbiology, Institute of Human Behaviour and Allied Sciences, Delhi-95. ***MD, Senior Resident, Department of Microbiology, Institute of Human Behaviour and Allied Sciences, Delhi-95.

Correspondence Address :
Dr Renu Goyal, Department of Microbiology, Institute of Human Behaviour and Allied Sciences, Dilshad Garden, Delhi 110095, INDIA, Phone numbers: 9868396833, E-mail address renugoyal_123@yahoo.co.in

Abstract

Background: Metallo--lactamases (MBLs) have been increasingly recognized among imipenem resistant isolates, but they also appear to exist in imipenem susceptible isolates. These undetected hidden MBLs in sensitive isolates can spread unnoticed in hospitals if such isolates are reported susceptible to carbepenem without screening for the presence of MBLs. The laboratory detection of such isolates is crucial as they pose a serious therapeutic challenge.
Aims: The aim of our study was to detect MBLs in both the imipenem resistant and sensitive isolates by using combination of available phenotypic methods.
Settings and design: This was a hospital based prospective study which was carried out in a tertiary neuropsychiatric centre from April 2008 to April 2009.
Methods and Material: A total of 130 gram negative isolates (50 imipenem sensitive, 30 imipenem intermediate and 50 imipenem resistant by the Kirby Bauer disc diffusion method) were tested for the presence of MBLs by the double disk synergy test (imipenem, EDTA 750g/ml), the combined disk test (imipenem, imipenem + EDTA 750g/ml) and the MBL E test strip.
Statistical analysis: Descriptive statistics was used and the percentages of MBLs carrying imipenem resistant and sensitive isolates were calculated.
Results: MBLs were detected in 11 (20%) imipenem sensitive isolates. Out of these 11 imipenem sensitive MBL producing strains, 8 were from the imipenem sensitive category and 3 were from the intermediate category. These 3 isolates in the intermediate category had MIC for imipenem in the sensitive range (MIC ≤4 ug/ml) by E test method. All the MBL carrying imipenem sensitive isolates had zone diameters in between 16 to 22 mm by the Kirby Baeur disc diffusion method. Among the imipenem resistant isolates, 38 isolates were MBL producers (32 from the imipenem resistant category and 6 from the intermediate category).
Conclusions: This study reports the existence of MBLs in carbapenem susceptible organisms and proposes that gram negative bacterial isolates having an imipenem zone diameter ≤22mm by the disk diffusion method should be routinely screened for presence of MBLs.

Keywords

Metallo--lactamases (MBLs), antibiotic resistance, imipenem, Double disk synergy test (DDST)

INTRODUCTION
Till the emergence of the carbapenemases, carbapenems were the drugs of choice for penicillin resistant or cephalosporin resistant gram negative bacilli; as these were stable to hydrolysis by most β-lactamases (extended-spectrum and AmpC -lactamases).(1),(2) These carbapenemases are most often metallo--lactamases (MBLs) which are capable of hydrolyzing not only carbapenems, but also all -lactam antibiotics except aztreonam.(1),(2),(3) MBLs are resistant to classical β-lactamase inhibitors, but are susceptible to EDTA and thiol-based compounds.(4)(5),(6),(7) IMP-1 MBL was first reported from Japan, from Serratia marcescens and Pseudomonas aeruginosa, after which several variants of MBLs like IMP 1 (IMP-2 to 9), VIM, SPM and GIM have been detected and characterized worldwide. (1),(2),(6),(10)Although scanty data is available on the overall prevalence of these enzymes among clinical isolates, a particular concern is that acquired MBL genes are located on integron structures that reside on mobile genetic elements such as plasmids or transposons and can widely disseminate in hospitals.(8)
These MBLs, as thought earlier, are just not restricted to the carbapenem resistant strains, but some recent reports have argued about their presence in carbapenem susceptible organisms also.(11),(12) As seen with extended spectrum beta lactamases (ESBLs) and AmpC type lactamases with cephalosporins, MBL carrying organisms can appear to be susceptible to carbapenems by current clinical and laboratory standard guidelines.(13) These carbapenem susceptible organisms with hidden MBL genes can spread unnoticed in hospitals if such isolates are reported as sensitive without screening for the presence of MBLs. The treatment of these organisms pose a serious therapeutic challenge as these strains are most often resistant to multiple drugs and can spread unnoticed in hospitals along with other hospital related organisms.(1),(2) The laboratory detection of carbapenem susceptible MBL carrying organisms is of significant clinical importance in order to stop their uncontrolled spread.
The present study was undertaken to detect metallo- β- lactamases in carbapenem resistant and susceptible isolates by the double-disk synergy test (DDST), combined-disk test (CDT) and an MBL Etest strip

Material and Methods

Bacterial strains: A total of 50 consecutive, non repeat, IMP-resistant (Zone diameter ≤13), 30 intermediate (Zone diameter 14-15mm) and 50 IMP sensitive (zone diameter ≥16) gram-negative bacterial isolates obtained from various clinical specimens were included in the study.(13) All the isolates were characterized up to the species level by using standard microbiological techniques.(14) A list of the bacterial strains which were tested and their source of isolation is shown in Table 1.
Antimicrobial susceptibility testing was done for all the bacterial isolates by using commercially available disks (Himedia, Mumbai, India) in accordance with the CLSI guidelines.(13) The antibiotics which were tested were piperacillin 100 g (PIP), ceftazidime 30 g (CAZ), imipenem 10 g (IPM), ciprofloxacin 5g (CIP), gentamicin 10 g (GEN) amikacin 30 g (AK) and aztreonam 30 g (ATM). The quality control strains which were used were Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.
The Phenotypic MBL detection method: All the isolates were screened for the presence of MBLs by the double-disk synergy test (DDST), the combined-disk test (CDT), and the MBL E test strip (AB BioDisk Company, Sweden).
The DDST and the Combined Disk Test: A 0.5 M EDTA solution was prepared by dissolving 186.1 gm of EDTA. 2H2O in 1000 ml of distilled water and its pH was adjusted to 8.0 by using NaOH. This mixture was sterilized by autoclaving.(7)
For the combined disk test, two 10 g IMP discs were placed on the surface of an agar plate and 5l EDTA solution was added to one of them to obtain a concentration of 750 g. The inhibition zones of IMP and IMP-EDTA were compared after 16-18 hours of incubation in air at 350C. An increase in zone size to ≥7 mm was taken as positive.(7),(12)
For the disk synergy test, an IMP disk was placed near a blank filter paper disk at a centre to centre distance of 10 to 25 mm. 5 l of 0.5 M EDTA was applied to the blank disk (750 g). After incubation for 16-18 hours, the presence of an enlarged zone of inhibition was interpreted as EDTA synergy test positive.(5) For the detection of MBLs in IMP susceptible isolates, IMP and a blank disk were kept at a distance of 25 mm.
The MBL Etest procedure: MBL E test strips with IMP (4 to 256 g/ml) and IMPE (1 to 64 g/ml) were applied on Mueller-Hinton agar and were incubated for 16 to 20 hrs at 35C. A reduction of IMP MIC 3 twofold dilutions in the presence of EDTA was interpreted as being suggestive of metallo--lactamase production. Equally, the presence of a "phantom" zone between the two gradient sections or deformation of the IP ellipses was also indicative of the presence of metallo--lactamases.(15)
The MBL E test strip was also used to detect MICs for IMP simultaneously with MBL ase detection. The MICs were interpreted as resistant, intermediate and sensitive as per the CLSI guidelines (Resistant: MIC 16 ug/ml, sensitive: MIC 4< ug/ml).(13)
Ethics: All the procedures were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975 that was revised in 2000.

Statistical analysis:
Descriptive statistics was used and the percentages of MBLs carrying imipenem resistant and sensitive isolates were calculated

Results

All the isolates which were included in the study were multi drug resistant, with resistance to 4 or more drugs as shown in Table 2.
Table 3 depicts the MBL detection in different gram negative bacteria. Of the 50 IMP resistant isolates (MIC≥16 ug/ml), 32 isolates were detected as MBL producers by all three methods. Individually, 42 isolates were found to be positive by DDST, 32 by CDT and 32 by the MBL E test strip. Of the 42 DDST positive isolates, 32 isolates gave increase in the zone diameter of ≥7mm (CDT positive), while 4 isolates gave a zone diameter between 4 - 6 mm and 6 gave less than 4 mm.
Among 50 IMP sensitive isolates (MIC ≤4 ug/ml), 16 isolates had zone diameters varying from 16-20 mm (group A) and 14 isolates had zone diameters varying from 21-22 mm (group B), while 20 isolates had zone diameters ≥ 23 mm (group C). A total of 6 isolates from Group A, 2 isolates from Group B and none from Group C were detected as MBL producers by all the three methods. However, 8 isolates from Group A, 4 isolates from Group B and none from Group C were detected as MBL producers by the double disk synergy test. All the isolates which were positive for MBL by CDT were also positive by the MBL E test strip.
Of the 30 IMP intermediate isolates, 13 isolates were found to be MBL producers by the combined use of all the three methods. 15 isolates were found to be MBL producers by the DDST method. In the MBL E test strip method, the MICs of 6 isolates were found to be in the sensitive range, 16 were in the intermediate range and 10 were in the resistant range. Of these, 3 from the sensitive category, 4 from the intermediate category and 6 from the resistant category were found to be MBL producers.
Thus, a total of 11 imipenem sensitive isolates (8 from the sensitive and 3 from the intermediate categories) and 38 imipenem resistant isolates (32 from the resistant and 6 from the intermediate categories) were found to be MBL positive by all the three methods.

Discussion

The increasing prevalence of MBL-producing gram-negative bacilli in many geographical regions and their propensity to rapidly disseminate within an institution, makes it essential to detect MBL- producing isolates by simple and rapid phenotypic methods.(1),(2) Unfortunately, MBL production is just not limited to carbapenem resistant strains, but has also been demonstrated in some carbapenem susceptible isolates.(11),(12) These organisms carrying hidden MBL genes, may spread unnoticed and may lead to untoward infection control problems.(3) Screening of only carbapenem-resistant organisms is insufficient and screening of all the IMP susceptible isolates for MBL creates unnecessary work with a lower yield. Hence, some criteria is needed to select out IMP susceptible isolates for MBL screening, as has been suggested by some other workers.(11)
In the present study, all the isolates which were screened were multidrug resistant (resistant to 4 or more classes of antimicrobials), as shown in Table 2. 68% of the IMP resistant isolates were susceptible to aztreonam, a profile which was compatible with MBL production. However 32 % of IMP resistant isolates revealed full or intermediate resistance to aztreonam, suggesting the co-existence of another mechanism of resistance among these isolates, most importantly ESBL or the AmpC-type -lactamases. Similar findings were also reported by Franklin et al who found that 37% of the MBL-carrying isolates were resistant to aztreonam.(11)
The inhibition of enzyme activity by EDTA and thiol compounds was an important characteristic which was used to distinguish MBLs from other -lactamases, but MBL detection was difficult among imipenem susceptible isolates by using IMP, as MBLs are inhibited by low concentrations of IPM.(11),(12) CAZ has been recommended by many authors to screen MBL producers, but the MBL-producing strains may also have another CAZ resistance mechanism, thus having a chance to result in false positive results.(6),(11),(16) Hence, we detected MBL in both IMP resistant and susceptible isolates by using IMP EDTA.
In the present study, we utilized the CDT (IMP and IMP EDTA), DDST (IMP and EDTA) and MBL Etest strip methods in an attempt to detect such challenging organisms. 2-mercaptopropionic acid and 1, 10-phenanthroline were not used to inhibit MBLs, as these are toxic for routine handling and required special precautions.(11)
In our study, 20% of the MBL carrying isolates were found to be susceptible to IMP as against a very high rate varying from 30-88%, which was reported by other workers.(11),(12) There was 100% concordance between the CDT and MBL E test strips. DDST detected more number of isolates as MBL producers, as even a slight increase in the synergistic zone was taken as positive. This could have been due to a zone difference of 7 mm which was taken as positive by the IPM-EDTA method as compared with using IPM alone in the CDT method. Had a cut off of 4mm been chosen for MBL detection, as done by some workers, some additional isolates would have been screened to be positive for MBLs by CDT.(11) However, all the isolates with an increase in the zone diameter of less than 7mm were screened to be negative for MBLs by the MBL Etest strip method, suggesting that increase in the zone diameter of 7 mm is an acceptable cut off with 750 g EDTA.
In this study, 6 MBLs carrying IMP sensitive isolates had a zone diameter in between 16-20 mm and 2 isolates had a zone diameter between 21-22 mm. No IMP sensitive isolate with a zone diameter 23 mm was detected as an MBL producer, thus indicating that all the isolates with a zone diameter ≤22 mm should be routinely screened for MBL production.
Thirty eight (63%) IMP R isolates were detected as MBL producers by all the three methods. Among other IMP resistant isolates, carbapenem resistance may be due to other mechanisms of resistance such as decreased permeability of the outer membrane and/or active efflux, which is possibly associated to the overproduction of the endogenous class C -lactamases.(16),(17)
Several phenotypic methods have been evaluated by various workers by using different combinations of antibiotic disks along with different concentrations of EDTA and different inoculum sizes of the test strain.[5-7] No single method has been proven as an ideal method for MBL detection in all the isolates and so, we used the three most user friendly techniques for MBL detection. Our results demonstrated that the combined-disk test was a better method for screening purposes as it was simple to perform and the materials used were cheap, nontoxic, easily accessible and allowed for the objective interpretation of results. Moreover, this method was quite good in detecting carbapenem-susceptible MBL-producing isolates, a phenotype that is being described with increasing frequency. Therefore, it is recommended that MBL detection must be done in all diagnostic laboratories on a daily basis to prevent the emergence and spread of this worrying resistance mechanism.

Key Message

Metallo--lactamases (MBLs) are not only restricted to the carbapenem resistant strains, but are also present in carbapenem susceptible organisms.
These carbapenem susceptible organisms with hidden MBL genes can spread unnoticed in hospitals if such isolates are reported as sensitive without screening for the presence of MBLs.
The laboratory detection of MBL carrying organisms is of significant clinical importance to stop their uncontrolled spread and the emergence of antimicrobial resistance.

References

1.
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