Reference ID: 2013-01436

Title of the study: Study of drug resistance and -lactamase production in clinical isolates
of E coli and Klebsiella spp.

1. Introduction
Members of the family Enterobacteriaceae are inhabitants of human intestine and are
among the most common human pathogens causing urinary tract infection, blood stream
infections, pneumonias, intra-abdominal infections, gastroenteritis, etc. E. coli and Klebsiella
spp. are the most common isolates among them. They are the source of community and hospitalacquired infections.
The injudicious, empirical use of antibiotics has contributed to the emergence of
drug resistance. Antibiotic resistance is a significant problem not only among nosocomial bacterial
isolates, but also in isolates from community-acquired infections, where it has traditionally been
absent.
Due to the life-threatening infections caused by E. coli and Klebsiella spp, their
resistance to antibiotics is a major cause of concern. They have the propensity to spread
genetic material through horizontal gene transfer, mediated mostly by plasmids and transposons.
There may be various ways by which E. coli and Klebsiella spp. acquire resistance, but production
of extended- spectrum beta lactamase (ESBL) is more important. Carbapenem drugs are currently
considered as the best treatment for these ESBL-producing E. coli and Klebsiella spp. But, there
are few studies, which indicate emergence of resistance against carbapenems (Kumarasamy et al
2010).
1

Carbapenem resistance among clinical isolates of Enterobacteriaceae, especially E

coli and K pneumoniae, is largely attributed to metallo--lactamase (MBL).

Currently, the

spread of carbapenem resistant bacteria is a serious issue. This emerging trend of resistance may
lead to disastrous consequences, as in the years to come, no antibiotics may remain
effective. This may lead to high mortality and morbidity in patients. Steps need to be taken at
many levels. One important step could be the regular monitoring of these organisms for drug
resistance. Antibiotic policy of the particular hospital should be based on antibiotic sensitivity
profile of microorganisms. Hence, we are trying to analyze antibiotic sensitivity pattern of E.
coli and Klebsiella spp. of bacteria
Nagpur, India.

at

Indira Gandhi Government Medical

College,

2. Review of literature
E coli is the commonest cause of urinary tract infections in both community and
nosocomial settings (Mohanty et al 2003).
E coli was first identified by the German paediatrician Theodore Escherich during
his studies of intestinal flora of infants. He described the organism in 1885 as Bacterium coli
commune (Escherich 1885) and established in pathogenic properties in extraintestinal infections.
The name Bacterium coli was widely used until 1919, when Castellani and Chalmers defined the
genus Escherichia and established the type species E. coli (Castellani et al 1919).
E. coli is gram-negative, rod-shaped, non-spore forming bacteria, usually motile by
peritrichous flagella. They are facultative anaerobe and gas is usually produced from fermentable
carbohydrates. Most strains of this species promptly ferment lactose or give a positive Onitrophenyl--D-galactopyranoside reaction. They are methyl red positive and Voges-Prokauer
negative. They produce indole, fail to hydrolyse urea or to grow in Mollers KCN broth. H 2S
production is not detectable on triple sugar iron (TSI) agar or Kliglers iron agar (KIA),
phenylalanine is not deaminated, gelatin is not liquefied and gluconate is not oxidized. Most strains
decarboxylate lysine and utilize sodium acetate, but they do not grow on Simmons citrate agar
(Cheasty et al 2005).

K pneumoniae is a Gram-negative, non-motile, encapsulated, lactose fermenting,

facultative anaerobic, rod shaped bacterium. K pneumoniae is lactose fermenting, H2S and indolenegative, has a positive voges-proskauer reaction, is capable of growth in KCN and use citrate as a
sole carbon source, and is incapable of growth at 10C.

Klebsiella is gram-negative bacteria that can cause different types of healthcareassociated infections, including pneumonia, bloodstream infections, wound or surgical site
infections, and meningitis (Wikipedia).
Klebsiella bacteria have developed antimicrobial resistance, most recently to the
class of antibiotics known as carbapenems. Klebsiella bacteria are normally found in the human
intestines (where they do not cause disease). They are also found in human stool (feces). In
healthcare settings, Klebsiella infections commonly occur among sick patients who are receiving
treatment for other conditions. Patients whose care requires devices like ventilators (breathing
machines) or intravenous (vein) catheters, and patients who are taking long courses of certain
antibiotics are most at risk for Klebsiella infections. Healthy people usually do not get Klebsiella
infections.

Antibiotic Resistance
The injudicious, empirical use of antibiotics has contributed to the emergence of
resistant bacteria. Antibiotic resistance is a significant problem not only among nosocomial
bacterial isolates, where it has traditionally been recognized, but also in CA, where it has
traditionally been absent.
The likelihood of multi-drug resistance is much greater in patients with nosocomial
infections. In recent years, bacterial resistance to different antibiotics has risen dramatically leaving
physicians with few therapeutic options. Extended spectrum - lactamases (ESBL) producing
organisms have become common hospital problems. Since these rates of resistance to antibiotics
differ from region to region, in making an appropriate choice of empiric or definitive therapy, it is
useful to avail of the information on prevailing levels of antimicrobial resistance among common
pathogens.

 -lactamase production is the most common mechanism of -lactam drug resistance

in gram-negative bacteria (Black 2005). Enterobacteriaceae producing both ESBLs and AmpC lactamases have been increasingly reported worldwide. Since AmpC producing organisms can act
as hidden reservoirs for ESBLs, it is important for clinical microbiology laboratories to be able to
detect ESBL production in these organisms on a routine basis. Until recently, carbapenems were
the choice for the therapeutic management of multidrug-resistant gram-negative bacterial
infections. Currently, the spread of carbapenem-resistant bacteria has caused grave concern due to
the limited choice in antibiotics for treating infections caused by them. The resistance to
carbapenems due to MBLs in enterobacteriaceae is increasingly recognized. For this reason,
aggressive surveillance of MBL producers were extremely important.
Various mechanisms of drug resistance in gram-negative bacilli include ESBL,
AmpC -lactamase production, efflux mechanisms and porin deficiency. lactamases continue to
be the leading cause of resistance to -lactam antibiotics in gram negative bacteria. Amongst the
mechanisms of resistance to third generation cephalosporins, production of ESBLs and AmpC lactamases are the most common. Since both ESBL and AmpC -lactamases are encoded on
plasmids and confer a selective advantage to strains harbouring these in a hospital setting. It is
important to know the occurrence of ESBL and AmpC producing strains as well as their antibiotic
susceptibilities to newer agents to guide empirical therapy for various infections (Taneja et al
2008).
ESBL are the enzymes that hydrolyse and cause resistance to oxyminocephalosporins and aztreonam. Production of these enzymes is either chromosomally mediated or
plasmid mediated. Point amino acid substitution of the classical plasmid mediated -lactamases
like TEM-1, TEM-2 and SHV-1 increases the spectrum of activity from earlier generation rd

lactams to 3

generation cephalosporins and monobactams. However, they retain their stability

against cephamycins and carbapenems and are inhibited to an extent by -lactamase inhibitors such

as clavulanic acid. Being plasmid mediated these enzymes spread fast amongst various bacteria and
are important by infection control, clinical and therapeutic implication (Rodrigues et al 2004).
As per CLSI 2012 guidelines, when using the new interpretive criteria, routine
ESBL testing is no longer necessary before reporting results (i.e., it is no longer necessary to edit
results for cephalosporins, aztreonam, or penicillins to resistant). However, ESBL testing may still
be useful for epidemiological or infection control purposes (Manual 2011). ESBL production can
be detected by double disk synergy test (DDST) using amoxiclav and cefotaxime (AMC-CTX)
(Pitout et al 2003, Shobha et al 2007, Shobha et al 2009) and piperacillin-tazobactum and cefepime
(PIT-CPM) (Pitout et al 2003, Shobha et al 2007, Shobha et al 2009, Rodrigues et al 2004). The
inhibitor-based confirmatory test approach is most promising for isolates that do not coproduce an
inhibitor-resistant -lactamase like AmpC (Khan et al 2009). Since high-level expression of AmpC
-lactamases may mask recognition of ESBLs, therefore the unique combination of cefepime and
piperacillin-tazobactam can be used to detect ESBLs (Manual 2011). High-level expression of
AmpC production has minimal effect on activity of cefepime; making this drug is more reliable for
ESBL detection in presence of AmpC (Rodrigues et al 2004).
AmpC -lactamases are clinically important because they confer resistance to
narrow-,

expanded-,

and

broad-spectrum

cephalosporins,

-lactam--lactamase

inhibitor

combinations and aztreonam. The chromosomally mediated -lactamase production is mainly

through expression of AmpC gene which is either constitutive or inducible. Plasmid-encoded
AmpC have been found around the world in nosocomial and non-nosocomial isolates and have
been linked to treatment failure (Ruppe et al 2006, Jacoby 2009). In most genera of the family
enterobacteriaceae, AmpC is inducible (Philippon et al 2002). Unlike chromosome-mediated
AmpC, plasmid-encoded AmpC enzymes are almost always expressed constitutively (Bush et al
1995). Plasmid mediated inducible -lactamases are extremely rare.

Enzyme extraction methods have traditionally been cited as the optimum phenotypic
detection method for AmpC activity. However, these are labour-intensive and not suitable for
routine clinical use (Tan et al 2009). Therefore, disk based methods are preferred. Cefoxitin and
imipenem are strong inducers of AmpC -lactamases but are much more stable for hydrolysis.
Whereas cefotaxime, ceftriaxone, ceftazidime, cefepime, cefuroxime, piperacillin, and aztreonam
are weak inducers and weak substrates but can be hydrolysed if enough enzyme is made (Jacoby
2009). Further, AmpC -lactamases are poorly inhibited by clavulanic acid; however, they are
inhibited by cloxacillin. Other inhibitors of the AmpC enzyme are also well described and which
include boronic acid compounds and novel inhibitors such as Syn2190 (Tan et al 2009). Cloxacillin
based methods such as cloxacillin combined disk diffusion (Tan et al 2009) and double disk
synergy test (Ruppe et al 2006) can detect both inducible and non-inducible AmpC -lactamase.
Carbapenemases are classified into class A, B and D. Class B carbapenemases
consist of MBL only. As per CLSI 2012 guidelines, institutional infection control procedures or
epidemiological

investigations

may

require

identification

of

carbapenemase-producing

enterobacteriaceae. Carbapenemase-producing isolates usually test intermediate or resistant to one

or more carbapenems using the current interpretive criteria and test resistant to one or more agents
in cephalosporin subclass III (e.g., cefoperazone, cefotaxime, ceftazidime, ceftizoxime, and
ceftriaxone). Therefore, testing could be limited to isolates with these characteristics. CLSI 2012
guidelines recommended modified Hodge test for carbapenemase production amongst
enterobacteriaceae isolates. Further they added that for isolates with positive modified Hodge test,
minimum inhibitory concentration (MIC) test should be performed before reporting any
carbapenem results, since carbapenem MIC interpretations are solely based on the MIC and should
not be changed regardless of the MHT result. Although CLSI 2012 has not recommended MHT for
gram negative non-fermentative bacilli (Noyal et all 2009) used it for the carbapenemase detection
in gram-negative non-fermentative bacilli.

Since MBL can hydrolyse a very wide range of broad-spectrum -lactams, MBLproducing gram-negative bacteria usually demonstrate consistent resistance to a variety of broadspectrum -lactams, including oxyiminocephalosporins, cephamycins and carbapenems which are
the last resort for the control of infections caused by gram negative bacteria. Thus, MBL-producing
gram-negative bacteria have been recognized to be among the most important nosocomial
pathogens. With the worldwide increase in the occurrence, types and rate of dissemination of MBL,
early detection is critical. The benefits of such include timely implementation of strict infection
control practices as well as clinical guidance regarding the potential risks for therapeutic failure.
MBLs are inhibited by chelating agents such as EDTA. Addition of zinc has been known to increase
the activity of metallo beta lactamases. Therefore, methods using these substances can be used to
detect MBL production.

3. Aims and Objectives

1. To study the antimicrobial susceptibility in clinical isolates of E. coli and Klebsiella spp.
2. To study the -lactamase production in the clinical isolates of E. coli and Klebsiella spp.

10

4. Materials and Methods

The study was conducted at Department of Microbiology, Indira Gandhi
th

Government Medical College, and Nagpur from 1st May 2013- 30 June 2013.Various samples like
pus, urine, CSF, sputum, blood etc. were collected and processed by standard microbiological
techniques. All the samples were cultured on blood and McConkey media. The isolates of E. coli
and Klebsiella spp. were identified by colony characteristics, morphology and biochemical
reactions (Ballows et al 1991).

Identification of E. coli (Crichton 2007)

E. coli is a gram negative, straight, short, slender, sluggishly motile bacillus. After
o
1824 hours of incubation at 37 C, E. coli forms colonies as follows:
1. Blood agar - large (2-3 mm), circular, low convex and non-pigmented.
2. McConkey medium: Large, lactose fermenting, moist.
3. Biochemical reactions: Catalase positive; oxidase negative; lactose, glucose and mannitol
fermented with production of acid and gas; sucrose not fermented; indole positive; methyl red
positive; citrate negative; urease negative; H2S not produced.

Identification of Klebsiella spp.

Klebsiella is shorter and thicker gram negative, non-motile, capsulated bacilli.
The colony characteristics are:
1. Blood agar - luxuriant, greyish-white, convex and mucoid
2. McConkey medium lactose fermenting, mucoid.
3. Biochemical reactions: Catalase positive; oxidase negative; lactose, glucose, mannitol and
sucrose fermented with production of acid and gas; indole negative; citrate positive; urease

positive; H2S not produced.

Antibiotic sensitivity test Antimicrobial susceptibility testing was performed by Kirby Bauer method
(Bauer et al 1966) as per the CLSI guidelines (2012).
Medium used was Muller Hinton agar (MHA). The inoculum turbidity was
adjusted to 0.5 McFarland standard. Commercially available disks (Hi media pvt ltd.) of 6 mm
diameter with recommended potency were used. MHA plates were inoculated by streaking evenly
the swab three times. The diameters of the zone of inhibition including antibiotic disk
were measured. Interpretation i.e. susceptible, intermediate and resistant was done.

Methods of beta-lactamases detection (Manual 2011)

1.

ESBL detection

i. Initial screening and phenotypic confirmatory test (CLSI 2012): A lawn culture of 0.5
McFarland inoculum of the test strain was exposed to a disk of ceftazidime. After incubation at
o
37 C for 16-18 hours, the zone diameter 22 mm indicates ESBL production. The lawn culture
of the test strain was done on MHA. Disks of ceftazidime (30 g) and ceftazidime-clavulanic
o
acid (30/10 g) were placed on it. After incubation at 37 C for 16-18 hour, a 5-mm increase in
a zone diameter for ceftazidime-clavulanic acid versus its zone when tested alone indicates
ESBL production.
ii. Double disk synergy test (DDST) using amoxyclav and cefotaxime and piperacillintazobactum and cefepime: MHA plates were swabbed to form a lawn culture with 0.5 McFarland
standard inoculum of the test strain. On the MHA plate, a disk of cefotaxime (30 g) was
placed 20 mm apart, centre-to-centre, from amoxiclav (20/10 g) disk whereas piperacillin10

tazobactam (100/10 g) disk was placed 25 mm apart, centre-to-centre from cefepime (30 g)
o
disk. Plates were incubated at 37 C overnight and were examined for enhancement of zone
inhibition of cefotaxime and cefepime at the side facing amoxiclav and piperacillin-tazobactam
disk respectively. Organisms that will show a clear extension of inhibition zone towards the disk
of amoxiclav and piperacillin-tazobactam were considered ESBL positive.

2.

AmpC -lactamase detection

i. Screening test: A lawn culture of 0.5 McFarland inoculum of the test strain was exposed
o
to a disk of cefoxitin (30 g). After overnight incubation at 37 C, the zone diameter 18 mm
was suspected as AmpC producer.
ii. Cefoxitin-cefotaxime disk antagonism test: A lawn culture of 0.5 McFarland inoculum of the
test strain was exposed to a disk of cefotaxime (30 g) and cefoxitin (30 g) placed at a distance
of 1.5 cm from edge to edge. After overnight incubation, there was flattening of radius of
zone of inhibition produced by cefotaxime on the side nearest the cefoxitin disk in case of AmpC
-lactamase producer organisms.
iii. Ceftazidime-imipenem

antagonism

test:

MHA

plate

was

inoculated

with

0.5

McFarland inoculum of the test organism. Ceftazidime (30 g) and imipenem (10 g) disks were
o
placed 20 mm apart from centre to centre. The plate were incubated overnight at 37 C. Isolates
showing blunting of ceftazidime zone of inhibition adjacent to imipenem disk were confirmed
positive for inducible AmpC production.

3.

MBL detection
It was tested by performing initial screening test followed by combined disk test.

i. Screening test: in the test, lawn culture of 0.5 McFarland inoculum of the test strain was
exposed to a disc of meropenem (10 g). After overnight incubation, the zone diameter around 1621mm indicated carbapenemase MBL production.
ii. Combined disk test: - In this test, the lawn culture of 0.5 McFarland inoculum of the test strain
was exposed to a disk of imipenem (10 g) and imipenem-EDTA (10/750 g). The difference of
7 mm in zones of inhibitions of two disks will indicate MBL production.

5. Observations and Results

The study was conducted in Department of Microbiology, Indira Gandhi
st

th

Government Medical College, Nagpur during 1 May 2013 to 30 June 2013.

A total of 60 strains of Klebsiella and 40 strains of E coli isolated from
samples collected from different infections were included in the study. All the 60 strains of
Klebsiella were K pneumoniae.
Table 1 shows various infections from which K pneumoniae and E coli
strains were isolated.
Table 1. Different infections caused by K pneumoniae and E coli
Infection

No. of K pneumoniae isolates (%)

No. of E coli isolates (%)

20 (33.3)

3 (7.5)

Urinary tract infection (UTI)

15 (25.0)

25 (62.5)

Bacteremia

15 (25.0)

8 (20.0)

Wound Infection

10 (16.7)

4 (10.0)

Total

60 (100)

40 (100)

Lower respiratory tract

infection (LRTI)

Table 1 shows that K pneumoniae and E coli were causative agents of lower
respiratory tract infection, urinary tract infection, bacteremia and wound infection. Further K
pneumoniae was more common in LTRI as compared to E coli and E coli was more common in
UTI.
Table 2 shows age and sex distribution of cases of K pneumoniae and E coli
infections.
1
4

Table 2 shows that K pneumoniae infections were more common in >60 (40%)
followed by 21-30 (20%) and 0-10 (16.6%) age groups. E coli infections were more common in
21-30 (37.5%) followed by 31-40 (25%) age groups. K pneumoniae infections were more
common in males and E coli infections were more common in females.

Table 2. Age and sex distribution of cases in K pneumoniae and E coli infections
K pneumoniae infection

E coli infection

Age (years)
Male (%)

Female (%)

Total

Male (%)

Female (%)

Total

0-10

6 (18.8)

4 (14.3)

10 (16.7)

2 (12.5)

1 (4.2)

3 (7.5)

11-20

2 (6.3)

1 (3.6)

3 (5.0)

1 (6.3)

1 (4.2)

2 (5.0)

21-30

4 (12.5)

8 (28.6)

12 (20.0)

3 (18.8)

12 (50)

15 (37.5)

31-40

2 (6.3)

2 (7.1)

4 (6.7)

4 (25)

6 (25)

10 (25.0)

41-50

2 (6.3)

1 (3.6)

3 (5.0)

1 (6.3)

1 (4.2)

2 (5.0)

51-60

2 (6.3)

2 (7.1)

4 (6.7)

1 (6.3)

1 (4.2)

2 (5.0)

>60

14 (23.3)

10 (35.7)

24(40)

4 (25)

2 (8.3)

6 (15)

Total

32

28

60

16

24

40

Table 3 shows Antimicrobial resistance amongst E coli and K pneumonia

isolates. K pneumoniae isolates were completely resistant to aminopenicillin (100%). However,
they showed variable resistance to cephalosporins (11.6% - 30%), tetracycline (60.0%), norfloxacin
(50.0%), cotrimaxazole (46.6%). E coli showed 20% resistance to aminopenicillin and 25%
st

nd

resistance to 1 and 2 generation cephalosporins. All K pneumoniae and E coli isolates were

Table 3. Antimicrobial resistance amongst E coli and K pneumonia isolates

E coli

K pneumoniae

n = 40 (%)

n = 60 (%)

Norfloxacin*

16 (40.0)

30 (50.0)

Cotrimoxazole*

14 (35.0)

28 (46.6)

Carbenicillin*

4 (10.0)

6 (10.0)

Ampicillin

8 (20.0)

60 (100)

Amoxyclav

8 (20.0)

42 (70.0)

Cephalothin

10 (25.0)

18 (30.0)

Cefuroxime

10 (25.0)

18 (30.0)

Cefoxitin

2 (5.0)

2 (3.3)

Cephotaxime

2 (5.0)

7 (11.6)

Cefipime

2 (5.0)

7 (11.6)

Piperacillin

4 (10.0)

7 (11.6)

Piperacillin Tazobactum

Imipenem

4 (10.0)

6 (10.0)

Toberamycin

4 (10.0)

6 (10.0)

Netillin

1 (2.5)

4 (6.6)

20 (50.0)

36 (60.0)

Drugs
Nitrofurantoin*

Gentamycin
Amikacin

Tetracycline

10

*- Urinary antibiotics though useful in UTI only, the isolates from all the infections were
tested for susceptibility to them.

susceptible to piperacillin tazobactum, imipenem and amikacin. Among aminoglycosides, amikacin

showed best susceptibility in our study.

-LACTAMASE PRODUCTION
In present study, ESBL production amongst enterobacteriaceae isolates showing positive screening
was tested by
1. Phenotypic confirmatory test
2. Double disk synergy test
Table 4 shows the comparison of different methods of ESBL production amongst
enterobacteriaceae isolates showing positive screening test.

Table 4. Comparison of different methods of ESBL production testing amongst

enterobacteriaceae isolates (n = 100)
Phenotypic confirmatory

Double disk synergy

Method
(CAZ CAC)

AMC - CTX

PIT - CPM

6 (6.0)

4 (4.0)

5 (5.0)

No. of strains
showing ESBL
production (%)
CAZ Ceftazidime, CAC - Ceftazidime clavulanic acid, AMC Amoxyclav, CTX Cefotaxime,
PIT Piperacillin-tazobactam, Cefepime - CPM
17

Table 4 shows that amongst 100 enterobacteriaceae isolates, ESBL production was
maximally (6.0%) detected by phenotypic confirmatory method (CAZ-CAC). Double disk synergy
test was able to detect ESBL production in 4.0% isolates by using AMC-CTX and in 5.0% isolates
by using PIT-CPM. All isolates detected as ESBL positive by double disk synergy test by using
either of the methods have shown ESBL production by phenotypic confirmatory method also.
In present study, AmpC production was tested amongst enterobacteriaceae isolates
showing positive screening. It was tested by

Cefoxitin-cefotaxime disk antagonism test

Ceftazidime-imipenem antagonism test

Table 5 shows the comparison of tests of AmpC production amongst
enterobacteriaceae isolates.

Table 5. Comparison of different tests for AmpC production amongst enterobacteriaceae

isolates (n = 100)
Tests for AmpC production
Cefoxitin-cefotaxime disk
antagonism

Ceftazidime-imipenem
antagonism

No. of strains showing

2 (2.0)

2 (2.0)

AmpC production (%)

Table 5 shows that amongst 100 enterobacteriaceae isolates, AmpC production was
equally detected by both ceftazidime-imipenem disk antagonism test and cefoxitin-cefotaxime disk
antagonism test (2.0%). The isolates detected positive for AmpC production by cefoxitin-

cefotaxime disk antagonism had shown AmpC production by ceftazidime-imipenem disk

antagonism test too.
Table 6 shows -lactamase production amongst enterobacteriaceae isolates in
present study.
Table 6. -lactamase production amongst enterobacteriaceae isolates (n = 100)

Enterobacteriaciae isolates

ESBL (%)

AmpC (%)

MBL (%)

Total

E coli (n=40)

2(5.0)

2(5.0)

K pneumoniae (n=60)

4(6.6)

2(3.3)

6(10)

6(6)

2(2)

8(8)

Total (n=100)

Table 6 shows that ESBL production was more (6.6%) in K pneumoniae as

compared to E coli (5%). AmpC production was seen in K pneumoniae only (3.3%) and not in E
coli. None of the strains were positive for MBL. Thus, total B lactamase production was 8% in E
coli and K pneumoniae isolates in our study.

6. Discussion
The study was conducted in Department of Microbiology, Indira Gandhi
st

th

Government Medical College, and Nagpur during 1 May 2013 to 30 June 2013.
A total of 60 strains of Klebsiella and 40 strains of E coli isolated from samples
collected from different infections were included in the study. All the 60 strains of Klebsiella were
K pneumoniae.
It was found out that K pneumoniae was more common in lower respiratory tract
infections (33.3%). K pneumoniae is a primary pathogen capable of causing pneumonia in other
wise, healthy people. However most infections are acquired in the hospital and occur in those who
are debilitated by various underlying conditions (Jarvis et al 1985).
E coli was more common in urinary tract infections (62.5%) as compared to K
pneumoniae. E coli is the leading cause of both community acquired and nosocomial urinary tract
infections (Donnenberg 2005).

Other infections caused by K pneumoniae and E coli include

bacteremia and wound infection (Table 1).

When compared with the age aspect, it was found out that K pneumoniae infections
were more common in extremes of ages (16.6% and 40%) in which risk of infection is higher.
Elderly patients are more prone to infections due to age-related decrease in immunity and also due
to rapidly fatal underlying illness (Guillermo et al 2013).
E coli infections were found to be more common in 21-30 and 31-40 age groups
(37.5% and 25% resp.). The infections were again found to be more common in females. The
infections in this age group were mostly urinary tract infection. Women are more susceptible to
urinary tract infections than men, and their infections tend to recur. One reason is that the urethra
(the tube that carries urine away from the bladder) is shorter in women than in men. Frequent

sexual intercourse also increases a womans risk of developing UTIs. Contraceptive spermicides
and diaphragm use are other risk factors (Urinary Tract Infections, Web).
It was observed that (Table 3) resistance to routinely prescribed urinary antibiotic
such as norfloxacin, cotrimoxazole and carbenicillin has been introduced in commonest etiological
agents of UTI i.e E coli and K pneumoniae. The unfrequently used urinary antibiotic nitrofurantoin
is spared from the development of the resistance in the isolates in our study. K pneumoniae showed
complete resistance to aminopenicillin and E coli showed only 20% resistance to the drug. The
resistance to different cephalosporins in both genera varied from 5% to 30% (Table 3). The
resistance to cefamycin was 3.3% and 5% in E coli and K pneumoniae respectively. Isolates of
neither of the genera showed resistance against piperacillin and tazobactum, imipenem. Amongst
aminoglycosides amikacin showed complete sensitivity. The resistance to other aminoglycosides
varied from 2.5% to 10%. Overall K pneumoniae isolates showed more resistance as compared to E
coli.
-lactamase production is the most common mechanism of -lactum drug resistance
in gram-negative bacteria (Black et al 2005). Amongst different -lactamases, MBL production
was not observed in our study. Since AmpC-producing organisms can act as hidden reservoirs for
ESBLs, it is important for clinical microbiology laboratories to be able to detect ESBL production
in these organisms on a routine basis (Pitout et al 2003). Until recently, carbapenems were the
choice for the therapeutic management of multidrug-resistant gram-negative bacterial infections.
Currently, the spread of carbapenem-resistant bacteria has caused grave concern due to the limited
choice in antibiotics for treating infections caused by them (Walsh 2010). The resistance to
carbapenems due to metallo--lactamases (MBLs) in enterobacteriaceae is increasingly recognized
(Tato et al 2010). For this reason, aggressive surveillance of MBL producers were extremely
important (Shibata et al 2003).

In present study, ESBL production was detected amongst enterobacteriaceae isolates

(Table 4) by using different methods. Amongst 100 enterobacteriaceae isolates, ESBL production
was maximally (6.0%) detected by phenotypic confirmatory method (CAZ-CAC). Whereas double
disk synergy test was able to detect ESBL production in 4.0% isolates by using AMC-CTX and in
5.0% isolates by using PIT-CPM. All isolates detected as ESBL positive by double disk synergy
test by using either of the methods have shown ESBL production by phenotypic confirmatory
method also. As compared to double disk synergy test by any method, phenotypic confirmatory
method suggested by CLSI guidelines for ESBL production detected ESBL production in more
number of the isolates (Khan et al 2008) reported that amongst the 90 isolates tested, double disk
synergy test detected ESBLs in 40 isolates, whereas, phenotypic confirmatory test detected ESBLs
in 39 isolates.
As per CLSI 2012 guidelines, when using the current interpretive criteria, routine
ESBL testing is no longer necessary before reporting results (i.e., it is no longer necessary to edit
results for cephalosporins, aztreonam, or penicillins to resistant). However, ESBL testing may still
be useful for epidemiological or infection control purposes (CLSI 2012). In the present study, we
have followed CLSI 2012 guidelines for ESBL testing by using initial screening and phenotypic
confirmatory test but additionally we have performed double disk synergy test (DDST) using
amoxiclav and cefotaxime (AMC-CTX) and piperacillin-tazobactum and cefepime (PIT-CPM).
The inhibitor-based confirmatory test approach is most promising for isolates that do not coproduce
an inhibitor-resistant -lactamase like AmpC. Since high level expression of AmpC -lactamases
may mask recognition of ESBLs, therefore in present study, the unique combination of cefepime
and piperacillin-tazobactam was used to detect ESBLs. High level expression of AmpC production
has minimal effect on activity of cefepime; making this drug more reliable for ESBL detection in
presence of AmpC. Tazobactam and sulbactam are much less likely to induce AmpC -lactamases
22

and are, therefore, preferable inhibitors for ESBL detection (Khan et al 2008). In the present study,
the coproduction of ESBL and AmpC was not detected. Hence, superiority of this test was not
completely revealed. Some authors advocate inclusion of cefepime in differentiation of ESBL
versus AmpC. However, it is important to remember that cefepime has low MIC to ESBLs,
because it is a zwitterion and enters the periplasmic space efficiently. High inoculum testing
generally uncovers the intrinsic activity of ESBL against cefepime. Clinical laboratories are still
not fully aware of the importance of ESBL and AmpC. They need to develop quick screening
methods to assess the mechanism of -lactam resistance in the isolates so that appropriate
medication can be given (Rodrigues et al 2004).
In present study, AmpC production was tested amongst enterobacteriaceae isolates
by using different tests (Table 5). Amongst 100 enterobacteriaceae isolates, AmpC production was
equally (2.0%) detected by ceftazidime-imipenem disk antagonism test and cefoxitin-cefotaxime
disk antagonism test. The isolates detected positive for AmpC production by cefoxitin-cefotaxime
disk antagonism had shown AmpC production by ceftazidime-imipenem disk antagonism test too.
CLSI 2012 guidelines have not mentioned any criteria for AmpC detection. In
present study, we had performed different tests to detect AmpC production amongst
enterobacteriaceae isolates. AmpC -lactamases are clinically important because they confer
resistance to narrow-, expanded-, and broad-spectrum cephalosporins, -lactam--lactamase
inhibitor combinations and aztreonam. Clinical microbiology laboratories should be able to detect
bacterial strains producing AmpC enzymes, since these strains may appear susceptible to a
particular -lactam antibiotic in vitro, but show no clinical response when used to treat serious
infections (Cantarelli et al 2007). The chromosomally mediated -lactamase production is mainly
through expression of AmpC gene that is either constitutive (non-inducible) or inducible. Enzyme
extraction methods have traditionally been cited as the optimum phenotypic detection method for
23

AmpC activity. However, these are labour-intensive and not suitable for routine clinical use.
Therefore, in present study, we have used disk-based methods to detect the production of AmpC lactamases. AmpC -lactamases production was detected by using the disks of cefoxitincefotaxime and ceftazidime-imipenem. Cefoxitin and imipenem are strong inducers of AmpC lactamases but are much more stable for hydrolysis. Whereas cefotaxime, ceftriaxone, ceftazidime,
cefepime, cefuroxime, piperacillin and aztreonam are weak inducers and weak substrates but can
be hydrolysed if enough enzyme is made (Jacoby 2009).
In present study, ESBL and AmpC production was tested amongst E coli and K
pneumoniae isolates. All the strains were screening test negative for MBL hence none are MBL
producer. Co-production of ESBL and AmpC was not observed. In present study, ESBL production
was seen in 5% of E coli strains and 6.6% of K pneumoniae isolates making 6% in
enterobacteriaceae strains studied (Table 6). AmpC production was seen in 2% of K pneumoniae
isolates and not seen in E coli isolates. Thus in total 8% of the E coli and K pneumoniae isolates
showed beta-lactamase production. Taneja et al (2008) reported 36.5% ESBL production with the
highest ESBL positivity in klebsiella (51.2%) followed by that in E. coli (40.2%). Gupta et al
(2012) in their study on nosocomial isolates of K. pneumoniae reported 32% AmpC production.

7. Conclusion

K pneumoniae was more common in lower respiratory tract infections (33.3%) and E coli was
more common in urinary tract infections (62.5%).

K pneumoniae infections were more common in extremes of ages in which risk of infection is
higher.

E coli infections were found to be more common in 21-30 and 31-40 age groups (37.5% and
25% resp.). The infections were again found to be more common in females.

Resistance to routinely prescribed urinary antibiotic such as norfloxacin, cotrimoxazole and

carbenicillin has been introduced in commonest etiological agents of UTI i.e E coli and K
pneumoniae. The unfrequently used urinary antibiotic nitrofurantoin is spared from the
development of the resistance in the isolates.

K pneumoniae showed complete resistance to aminopenicillin and E coli showed only 20%
resistance to the drug.

The resistance to different cephalosporins in both genera varied from 5% to 30%. The
resistance to cefamycin was 3.3% and 5% in E coli and K pneumoniae respectively.

Isolates of neither of the genera showed resistance against piperacillin and tazobactum,
imipenem.

Amongst aminoglycosides amikacin showed complete sensitivity. The resistance to other

aminoglycosides varied from 2.5% to 10%.

Overall drug resistance was more common in K pneumoniae isolates as compared to E coli.

Amongst different -lactamases, MBL production was not observed in our study.

ESBL production was maximally (6.0%) detected by phenotypic confirmatory method (CAZCAC). Whereas double disk synergy test was able to detect ESBL production in 4.0% isolates
by using AMC-CTX and in 5.0% isolates by using PIT-CPM.

AmpC production was equally (2.0%) detected by ceftazidime-imipenem disk antagonism test
and cefoxitin-cefotaxime disk antagonism test.

Co-production of ESBL and AmpC was not observed.

ESBL production was seen in 5% of E coli strains and 6.6% of K pneumoniae isolates making
6% in enterobacteriaceae strains studied. AmpC production was seen in 2% of K pneumoniae
isolates and not seen in E coli isolates. Thus in total 8% of the E coli and K pneumoniae
isolates showed beta-lactamase production.

Introduction of MBL or carbapenemase production in enterobacteriaceae is a matter of great

concern. Luckily the strains in our setup are spared till date. The widespread use of
antimicrobials in community either due to self-medication or incorrect prescription is the
possible factor fuelling the emergence of resistant strains. Therefore, careful monitoring of drug
resistance and -lactamase especially carbapenemase is necessary. It is a high time for
microbiology laboratories to introduce -lactamase testing routinely for the knowledge of their
prevalence and for the measures to be taken to control their spread. Timely knowledge of
aetiology and antimicrobial resistance pattern of bacterial isolates can help in rational use of
antibiotics and control of drug resistance.

8. Summary

The study was conducted at Department of Microbiology, Indira Gandhi

th

Government Medical College, and Nagpur from 1st May 2013- 30 June 2013.
The aims and objectives were to study the antimicrobial susceptibility and the lactamase production in the clinical isolates of E. coli and Klebsiella spp.
A total of 60 strains of Klebsiella and 40 strains of E coli isolated from samples
collected from different infections were included in the study. All the 60 strains of Klebsiella were
K pneumoniae. The isolates were subjected to antibiotic susceptibility test by Kirby Bauer method
as per the CLSI guidelines. Different beta-lactamase production i.e ESBL, AmpC, MBL was
studied. Extended spectrum -lactamase production amongst enterobacteriaceae isolates showing
positive screening was tested by phenotypic confirmatory test and double disk synergy test using
cefotaxime-amoxiclav and piperacillin-tazobactam - cefepime disks. , AmpC production was tested
amongst enterobacteriaceae isolates showing positive screening. It was tested by cefoxitincefotaxime disk antagonism test and ceftazidime-imipenem antagonism test.
K pneumoniae and E coli were causative agents of lower respiratory tract infection,
urinary tract infection, bacteremia and wound infection.

Further K pneumoniae was more

common in LTRI as compared to E coli and E coli was more common in UTI.
K pneumoniae infections were more common in >60 (40%) followed by 21-30 (20%) and
0-10 (16.6%) age groups. E coli infections were more common in 21-30 (37.5%) followed by 3140 (25%) age groups. K pneumoniae infections were more common in males and E coli
infections were more common in females.

K pneumoniae isolates were completely resistant to aminopenicillin (100%).

However, they showed variable resistance to cephalosporins (11.6% - 30%), tetracycline (60.0%),
norfloxacin (50.0%), cotrimaxazole (46.6%). E coli showed 20% resistance to aminopenicillin and
st

nd

25% resistance to 1 and 2 generation cephalosporins. All K pneumoniae and E coli isolates were
susceptible to piperacillin tazobactum, imipenem and amikacin. Among aminoglycosides, amikacin
showed best susceptibility in our study.
Amongst 100 enterobacteriaceae isolates, ESBL production was maximally (6.0%)
detected by phenotypic confirmatory method (CAZ-CAC). Double disk synergy test was able to
detect ESBL production in 4.0% isolates by using AMC-CTX and in 5.0% isolates by using PITCPM. All isolates detected as ESBL positive by double disk synergy test by using either of the
methods have shown ESBL production by phenotypic confirmatory method also.
Amongst 100 enterobacteriaceae isolates, AmpC production was equally detected
by both ceftazidime-imipenem disk antagonism test and cefoxitin-cefotaxime disk antagonism test
(2.0%).
-lactamase production was observed in enterobacteriaceae isolates. ESBL was
observed in 6 (6.0%) isolates and AmpC production in 2 (2.0%) isolates. None of the strains were
positive for MBL. ESBL production was seen in 5% of E coli strains and 6.6% of K pneumoniae
isolates making 6% in enterobacteriaceae strains studied. AmpC production was seen in 2% of K
pneumoniae isolates and not seen in E coli isolates. Thus in total 8% of the E coli and K
pneumoniae isolates showed beta-lactamase production.

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