ABSTRACT- Purpose: Multidrug resistant organisms are on rise. Various enzymes present in the organisms are
responsible for this resistance. Detection of these enzymes become challenging if organisms harbor multiple enzymes.
This study was done to find the prevalence of various enzymes at our tertiary care hospital.
Materials and methods: Extended spectrum beta lactamases (ESBL) detection was done by screening method followed
by two phenotypic confirmatory methods (double disc synergy and disc potentiation method). Carbapenems (imipenem,
meropenem) resistant strain were analyzed for metallo beta lactamases (MBL) and carbapenemases (KPC) using
combined disc test and modified Hodge test. Amp C detection was done by using cefoxitin disc on heavy lawn of E. coli
ATCC 25922. Distortion of the zone size on the streaked line of test was taken as positive for Amp C.
Results: 87.15% were screened positive for ESBL and confirmed cases were 36.80%. Carbapenem resistant was 31.86%,
MBL was 7.52%, KPC was 0.82 %, Amp C in 0.23%.
Conclusions: There is high prevalence of ESBL. Detection of these enzymes is important in routine diagnostics for
treatment. Co-expression of multiple enzymes was detected in this study. Judicious and rational use of antibiotics is
required which might lead to decrease in emergence of resistance. Also knowledge of the prevalence of these enzymes
helps in empirical antibiotic therapy and in infection control purpose.
Key-words- Multidrug resistant, ESBL, MBL, KPC, Amp C
Emergence of multidrug resistance among the pathogens is
on rise and it is posing a serious threat to the management
of infections in a hospital care. Initially the most frequently
used antimicrobials for empirical therapy were beta
lactams. Bacteria produce Beta lactamases which is
responsible for resistance to Beta lactam antibiotics. The
first plasmid mediated beta lactamases were TEM-1
(Temoniera-1) and SHV-1 (sulfhydryl “variable”) reported
in 1965 from Escherichia coli and Klebsiella pneumoniae
. The introduction of third generation cephalosporins in
early 1980s particularly ceftazidime and aztreonam after
cefotaxime has accelerated the evolution of ESBL
worldwide roughly at the same time and the first report of
plasmid encoded beta lactamase capable of hydrolyzing the
extended spectrum cephalosporin was published in 1983
from Germany [1-3]. These ESBLs are derived from
mutation in older beta lactamases like (TEM-1, TEM2 and
SHV-1) and are resistant to third generation cephalosporins
(3GCs) and monobactams but are sensitive to cephamycins
and carbapenems. They are inhibited by beta lactamase
inhibitor combinations (BLI). ESBLs are encoded by
transferable conjugative plasmids which are responsible for
dissemination of resistance to other bacteria in the hospital
and in community .
Amp C beta lactamases were first discovered in 1970.
These organisms are resistant to penicillins, cephalosporins,
monobactums, BL/BLI, cephamycins. These are usually
sensitive to carbapenems, floroquinolones .
The first carbapenemases was identified in 1993.Since then
a large number of carbapenemases have been identified,
most of them belong to Ambler class A, B, D beta
lactamases. True carbapenemases hydrolyse most beta
lactams, including carbapenems .
KPC-producing Enterobacteriaceae were first reported in a
clinical specimen from a patient in North Carolina in
MATERIALS AND METHODS
A retrospective study was done for a period of 30 months
(April 2012 to September 2014) to analyze various
enzymes namely ESBL, Amp C, MBL in a tertiary care
hospital. Isolates were obtained from various samples
submitted to our lab: urine, respiratory sample, blood,
sterile body fluid (CSF, pleural fluid), pus, high vaginal
swab. For statistical analysis location was categorized into
four groups namely OPD; representing people from
community, Emergency; representing admissions from
other healthcare area, ICU; representing all critical care
areas and wards with stabilized and not serious patients.
The study was conducted on non duplicate isolates of
E.coli, K. pneumoneae, K. oxytoca. Bacterial identification
was performed by Vitek2C (Biomereux). For ESBL
screening test ceftazidime disc and phenotypic
confirmatory was done using two methods- double disc
synergy (ceftazidime, cefotaxime, cefpodoxime, ceftiaxone,
amoxiclavulinic acid, Oxoid) (Fig.1) and disc potentiation
(ceftazidime clavulanic acid, cefotaxime clavulanic acid
combination) (Fig 2). All the isolates resistant to
ceftazidime were taken as screening test positive and
strains were considered as ESBL positive if either
phenotypic confirmatory test was positive . Carbapenems
(Imipenem and Meropenem) resistant strains were analysed
for metallo beta lactamases (MBL) (Fig. 3) and
carbapenemases (KPC) (Fig. 4) using combined disc test
using EDTA, Modified Hodge test [7-10]. As there are
presently no CLSI or approved criteria for Amp C detection
it was performed as a heavy inoculums streaked radially
from the cefoxitin disc on the agar surface already streaked
with E. coli ATCC 25922.Distortion of the zone size was
taken positive (Fig 5) . Quality control used is ATCC
K. pneumoneae 700603.
Fig. 1: Phenotypic Confirmation Test by Double Disc
Synergy Test of Screening Positive Isolate
Fig. 2: Phenotypic Confirmatory Test by Disc
Potentiation Method showing Zone Size of >5mm in the
Disc with Ceftazidime and Clavulanic acid as
Compared to Ceftazidime
Fig. 3: Detection of Mbl (>7mm Augmentation) by
Combined Disc Test
Fig. 4: Isolate Showing Carbapenamase (KPC)
Enzyme –Modified Hodge Test
Fig. 5: Detection of Amp C. Isolate showing
Distortion of Zone Size along the Streaked Line
A total of 2584 isolates of E. coli
were obtained from April 2012 to September 2014. ESBL
positive strains obtained were 951(36.80%), whereas
87.15% (2252/2584) were screening test positive. Amp C
was detected in 0.23% (4/1738). Carbapenem resistance
was seen in 31.86% (824/2584), MBL 7.52%
(91/1210).KPC 0.82% (14/1711).
Prevalence of various enzymes present in different location
is shown in Table 1-4.
Table 1: Prevalence of Resistant Enzymes in OPD
Table 2: Prevalence of Resistant Enzymes in Emergency
||No. of Isolates
test ) ||893 ||666 ||74.58%|
|ESBL (Confirmed) ||893 ||365 ||40.87%|
(Not Confirmed) ||893 ||301 ||33.71%|
|Amp C ||658 ||2 ||0.30%|
|KPC ||648 ||3 ||0.46%|
|MBL ||466 ||15 ||3.22%|
|893 ||108 ||12.09%|
Table 3: Prevalence of Resistant Enzymes in ICU
||No of Isolates
test ) ||472 ||439 ||93.01%|
|ESBL (Confirmed) ||472 ||247 ||52.33%|
(Not Confirmed) ||472 ||192 ||40.68%|
|Amp C ||294 ||1 ||0.34%|
|KPC ||288 ||4 ||1.39%|
|MBL ||201 ||8 ||3.98%|
|472 ||141 ||29.87%|
Table 4: Prevalence of Resistant Enzymes in Ward
||No of Isolates
test ) ||812 ||786 ||96.80%|
|ESBL (Confirmed) ||812 ||233 ||28.69%|
(Not Confirmed) ||812 ||553 ||68.10%|
|Amp C ||526 ||1 ||0.19%|
|KPC ||517 ||6 ||1.16%|
|MBL ||363 ||47 ||12.95%|
|812 ||425 ||52.34%|
(Screening test ) ||407 ||361 ||88.70%|
|ESBL (Confirmed) ||407 ||106 ||26.04%|
(Not Confirmed) ||407 ||255|| 62.65%|
|Amp C ||260 ||0 ||0.00%|
|KPC ||258 ||1 ||0.39%|
|MBL ||181 ||21 ||11.60%|
|407 ||150 ||36.86%|
It was found that resistance to ceftazidime was maximum
in ICU (96.80%) as shown in Table 3 and least in wards
accounting for 26.04% (Table 4). Carbepenem resistance
and MBL detection was seen in 52.34% and 12.95%
respectively in ICU (Table 3) which was quite high when
compared to other areas (Table1&Table 2). Detection of
other enzymes Amp C, KPC remained low in all the areas.
All the confirmed ESBL were uniformly sensitive to
carbapenems. Not confirmed isolates were found resistant
to various agents including aminoglycosides, carbapenems,
floroquinolones. Isolates with carbapenem resistance and
harbouring other enzymes were sensitive only to
polymixins and tigecycline. An isolate was defined as
multidrug resistant organism when found resist to BL,
BL/BLI, carbapenems at our institution for infection
This study demonstrates the prevalence of resistant enzyme
expression in a tertiary care hospital. Enzyme detection is
generally not performed in most of the laboratories due to
lack of knowledge, lack of facilities to conduct or lack of
resources which can lead to therapeutic failure. ESBL
producing Enterobacteriaceae are resistant to
cephalosporins, aztreonam and monobactam while
resistance to co-trimoxazole and aminoglycosides is
frequently co-transferred on the same plasmid. Many ESBL
producing organisms express Amp C beta-lactamases thus
conferring resistance to cephalosporins in the 7
alpha-methoxy cephalosporins , oxyimino group, and are
poorly inhibited by clavulanic acid. Carbepenems are given
for the treatment of infections caused by ESBL
producing organisms. With the emergence of
carbapenemases and its spread from pseudomonas to
Enterobacteraceae, resistance to Carbapenems has been
Co-expression of multiple ESBL enzymes (CTX-M, TEM,
SHV) and at the same time multiple enzymes (AmpC,
ESBL, MBL, KPC) are known to occur in a single isolate.
These enzymes if present cannot be identified phenotypically,
thus making impossible for any lab to identify. In
such cases isolates are screening test positive but fail
confirmatory tests. These isolates are found resistant to
multiple antibiotics including Carbapenems [13-15]
In India, the prevalence rate of ESBL varies in different
institutions from 28% to 84% 
and in our hospital it is
36.80% which is similar to a study conducted by Bhutada
. In a study done by Grover N et al the prevalence
was 40.07% and Amp C was 14.8% 
. In a study done by
Basavaraj MC the prevalence was 32.1% 
. In a study by
Wattal et al the prevalence of ESBL in E coli increased to
. In a study conducted in Mysore the rate was 43%
The prevalence of plasmid mediated Amp C varies widely
in different parts of the world from 2% to 46%. In Indian
studies, the prevalence of Amp C ranged from 8% to 47%
. In our study Amp C detected very low 0.23%.Various
studies have demonstrated the prevalence as
3.3%,14.8%,15.97 % respectively [4,22-23]
The prevalence of carbapenem resistance among isolates
reported to the National Healthcare Safety Network
(NHSN) in 2006–2007, was up to 4.0% of Escherichia
and 10.8% of K. pneumoniae
isolates that were
associated with certain device-related infections 
. In a
study done by Datta P et al the prevalence of carbapenem
resistance was 7.87% and MBL was 5.75% 
. A study
reported high prevalence of resistance to carbapenems
ranging from 13 to 51% in E. coli
and Klebsiella spp.
ICUs and wards from a tertiary care hospital in Delhi 
Gupta E et al. 
also reported high prevalence of
resistance varying from 17 to 22% to various carbapenems
. In a study
carbapenemases detection was15% and 0.5% by combined
disc and modified hodge test 
. In our study
carbapenemases 0.82% and 7.52% by MHT and CDT
respectively. In a study done by Wadekar M and Bhutada
KH et al 
the pevalance of MBL was 18% and 9.48%
. The prevalence of KPC in a study by Sarita N et
al was found to be 16.6% 
In the present study we found various enzymes prevalent in
our set up. Routine screening and confirmatory test should
be performed so that appropriate therapy can be chosen for
management of patients and containment of infections.
Resistant enzymes trend and patterns in different location is
important for empirical therapy, epidemiological and
infection control purpose. If screening is positive and
confirmatory method is negative then possibility of
organisms harbouring other enzymes (other than detected)
or multiple enzymes should be considered which is more
prevalent in critical care areas due to selection pressure.
The most active antibacterial agents against Carbapenemases
producing with either KPCs or MBLs are colistin,
Detection, and confirmation of the presence of various
enzymes, is important for surveillance, infection control
and treatment purpose and to avoid inadvertent use of
antibiotics. Challenges are there in detection of enzymes if
multiple enzymes are present in an isolate making it
multidrug resistant. Molecular methods are there for
identification of various enzymes but they are costly and
cost effectiveness should always be kept in mind in
treatment of patient.
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