Introduction for detection of ESBLs like phenotypic confirmatory disc



With the advancement in medical practice,
new problems are surfacing, one of most important and common being emergence of
resistance in Gram negative bacteria towards the ? lactams due to ? lactamases
enzyme. These enzymes inactivate beta lactam ring containing antibiotics, which
in the course of time has evolved to extended spectrum ?-lactamases (ESBL), which confers bacteria enhance ability to be resistant
against wide variety of new beta-lactams1 including the III
generation cephalosporins, and aztreonam (but not the cephamycins or
carbapenems). The ESBLs hydrolysis the antibiotics, but are inhibited by ?-lactamase inhibitors such as clavulanic acid.2

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ESBLs are a cause
of major challenge in therapeutic treatment in hospitals3 due to
increase in expenditure of money on ineffective antibiotics which further
develops resistance in bacteria making patient more susceptible to infection
and colonization by them.4


ESBL strains remain undetected as they are difficult
to detect by routine susceptibility testing methods and may show false
susceptibility to antibiotics by Kirby- Bauer disc diffusion methods5.
Identification of all ESBLs producing organisms in clinical Microbiology
laboratory is a major challenge because of
inoculum effect and substrate specificity1.


ESBL detection is important as knowledge about its
prevalence is helpful to formulate infection control measures and to prevent
their spread6. There are different methods for detection of ESBLs
like phenotypic confirmatory disc diffusion test (PCDDT), Modified
Double Disc Synergy Test (MDDST), Indirect Modified Three Dimensional Test
(IMTDT), etc.


Review of literature


There is high
prevalence of ESBLs in clinical isolates. Sharma
M. et al reported frequency of 52.49% in gram negative isolates at NIMS
University, Jaipur4,while Hima Bindu M et al, reported ESBLs frequency
of 58.8% in isolates7 at Mallareddy Institute of Medical Sciences,
India. Where else study by Bajpai T et al, determined that among gram negative
isolate 36.8% were found to be ESBL producers8.


Indirect modified
3-dimensional enzyme extract test for detection of ESBLs is sensitive and gives
rapid result. Modi D et al, reported sensitivity of IMTDT is 98% to 100% and
found it to be most sensitive test among other available tests such as double
disc synergy test and modified direct three dimensional test.

Khodare A et al,
study showed 76% strains gave positive result with IMTDT, and was found to be
better than phenotypic confirmatory disc diffusion test (PCDDT) for detection
of ESBLs although it was a little labour intensive and may be technically
challenging. 10.


study by Shaikh el at IMTDT was found to be superior method than Modified Double
Disc Synergy Test (MDDST), PCDDT and DDST for detection of production of ESBL
alone or in presence of other ?- lactamases like AmpC. PCDDT& DDST should
be used in the isolates which produce only ESBL but are not useful for
detection of ESBL in isolates who also produces other ?-lactamases like AmpC



With the advent
of ESBLs which are reducing the treatment options, it is necessary to detect
these with a reasonable accuracy so that appropriate treatment may be initiated
in the patients. Hence this study is planned with the following objective:


1. To detect ESBL
producing strains using the indirect modified three dimensional test.

2. To study the
utility of IMTDT in detecting ESBLs in tertiary care hospital




The study will
be carried out in the Department of Microbiology of a tertiary care hospital.


Study Type: Cross-sectional
prospective study.

Study Time: Two months.

Type: All samples received for
culture and sensitivity in the Microbiology Laboratory

Sample Size:
30 strains



All samples received for culture and
sensitivity in the Microbiology Laboratory will be inoculated on routine media
like blood agar and Mac Conkey agar; in case of urine it will be inoculated on
cysteine lactose electrolyte deficient medium (CLED). After 24 hours of
incubation, the bacterial isolates, if any, will be identified by standard
biochemical tests12 and antibiotic sensitivity will be performed by
Kirby Bauer method as per CLSI guidelines. Also ESBL screening will be done by
standard disc diffusion using cefpodoxime, cefotaxime, ceftriaxone and
ceftazidime with zones of inhibition