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451
INDIAN JOURNAL OF
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All India Institute of Medical Sciences
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Nizam’s Institute of Medical Sciences
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Professor
Department of Microbiology
Kasturba Medical College
Manipal - 576 119, India
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Medical College and Research Institute, Kadhirkamam,
Puducherry - 605 009, India
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Indian Journal of Medical Microbiology
vol. 25, No. 4
INDIAN JOURNAL
OF MEDICAL MICROBIOLOGY
(Publication of Indian Association of Medical Microbiologists)
ISSN 0255-0857
Volume 25
Number 4
October-December, 2007
CONTENTS
Page No.
Guest Editorial
The Need for Control of Viral Illnesses in India: A Call for Action
C Lahariya, UK Baveja
.......309
Review Article
Immunobiology of Human ImmunodeÞciency Virus Infection
P Tripathi, S Agrawal
.......311
Special Articles
Serum Levels of Bcl-2 and Cellular Oxidative Stress in Patients with Viral Hepatitis
HG Osman, OM Gabr, S Lotfy, S Gabr
.......323
Rapid IdentiÞcation of Non-sporing Anaerobes using Nuclear Magnetic Resonance Spectroscopy
and an IdentiÞcation Strategy
.......330
S Menon, R Bharadwaj, AS Chowdhary, DV Kaundinya, DA Palande
Original Articles
Species Distribution and Physiological Characterization of Acinetobacter Genospecies from
Healthy Human Skin of Tribal Population in India
SP Yavankar, KR Pardesi, BA Chopade
Extended-spectrum Beta-lactamases in Ceftazidime-resistant Escherichia coli and Klebsiella
pneumoniae Isolates in Turkish Hospitals
S Hoşoğlu, S Gündeş, F Kolaylõ, A Karadenizli, K Demirdağ, M Günaydõn, M Altõndis, R Çaylan,
H Ucmak
Typhoid Myopathy or Typhoid Hepatitis: A Matter of Debate
M Mirsadraee, A Shirdel, F Roknee
.......336
.......346
.......351
Correlation Between in Vitro Susceptibility and Treatment Outcome with Azithromycin in Gonorrhoea:
A Prospective Study
.......354
P Khaki, P Bhalla, A Sharma, V Kumar
Comparison of Radiorespirometric Buddemeyer Assay with ATP Assay and Mouse Foot Pad Test in
Detecting Viable Mycobacterium leprae from Clinical Samples
.......358
VP Agrawal, VP Shetty
Detection of Mycoplasma Species in Cell Culture by PCR And RFLP Based Method: Effect of
BM-cyclin to Cure Infections
V Gopalkrishna, H Verma, NS Kumbhar, RS Tomar, PR Patil
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.......364
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453
Virulence Factors and Drug Resistance in Escherichia coli Isolated from Extraintestinal Infections .......369
S Sharma, GK Bhat, S Shenoy
Antimicrobial Susceptibility Testing of Helicobacter pylori to Selected Agents by Agar Dilution
Method in Shiraz-iran
J Kohanteb, A Bazargani, M Saberi-Firoozi, A Mobasser
Outbreak of Acute Viral Hepatitis due to Hepatitis E virus in Hyderabad
P Sarguna, A Rao, KN Sudha Ramana
.......374
.......378
A Comparative Study for the Detection of Mycobacteria by BACTEC MGIT 960, Lowenstein Jensen
Media and Direct AFB Smear Examination
.......383
S Rishi, P Sinha, B Malhotra, N Pal
Cytokine Levels in Patients with Brucellosis and their Relations with the Treatment
H Akbulut, I Celik, A Akbulut
.......387
Brief Communications
Rapid Detection of Non-enterobacteriaceae Directly from Positive Blood Culture using Fluorescent
In Situ Hybridization
.......391
EH Wong, G Subramaniam, P Navaratnam, SD Sekaran
Latex Particle Agglutination Test as an Adjunct to the Diagnosis of Bacterial Meningitis
K Surinder, K Bineeta, M Megha
.......395
Helminthic Infestation in Children of Kupwara District: A Prospective Study
SA Wani, F Ahmad, SA Zargar, BA Fomda, Z Ahmad, P Ahmad
.......398
Clinical and Mycological ProÞle of Cryptococcosis in a Tertiary Care Hospital
MR Capoor, D Nair, M Deb, B Gupta, P Aggarwal
.......401
Candida spp. other than Candida albicans: A Major Cause of Fungaemia in a Tertiary Care Centre .......405
S Shivaprakasha, K Radhakrishnan, PMS Karim
Case Reports
Enterobacter sakazakii in Infants: Novel Phenomenon in India
P Ray, A Das, V Gautam, N Jain, A Narang, M Sharma
.......408
Ocular Toxocariasis in a Child: A Case Report from Kashmir, North India
BA Fomda, Z Ahmad, NN Khan, S Tanveer, SA Wani
.......411
Cutaneous Actinomycosis: A Rare Case
SC Metgud, H Sumati, P Sheetal
.......413
Fatal Haemophagocytic Syndrome and Hepatitis Associated with Visceral Leishmaniasis
P Mathur, JC Samantaray, P Samanta
.......416
A Rare Case of Mucormycosis of Median Sternotomy Wound Caused by Rhizopus arrhizus
R Chawla, S Sehgal, S Ravindra Kumar, B Mishra
.......419
Mycobacterium fortuitum Keratitis
C Sanghvi
.......422
Correspondence
Prevention of Parent-to-Child Transmission of HIV: An Experience in Rural Population
N Nagdeo, VR Thombare
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.......425
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Indian Journal of Medical Microbiology
vol. 25, No. 4
Combining Vital Staining with Fast Plaque: TB Assay
D Rawat, MR Capoor, A Hasan, D Nair, M Deb, P Aggarwal
.......426
Disseminated Histoplasmosis
PK Maiti, MS Mathews
.......427
Authors’ Reply
RS Bharadwaj
.......428
Microwave Disinfection of Gauze Contaminated with Bacteria and Fungi
VH Cardoso, DL Gonçalves, E Angioletto, F Dal-Pizzol, EL Streck
.......428
Endoscope Reprocessing: Stand up and Take Notice!
A Das, P Ray, M Sharma
.......429
Prevalence of Toxoplasma gondii Infection amongst Pregnant Women in Assam, India
BJ Borkakoty, AK Borthakur, M Gohain
.......431
Evaluation of Glucose-Methylene-Blue-Mueller-Hinton Agar for E-Test Minimum Inhibitory
Concentration Determination in Candida spp.
MR Capoor, D Rawat, D Nair, M Deb, P Aggarwal
.......432
Resurgence of Diphtheria in the Vaccination Era
N Khan, J Shastri, U Aigal, B Doctor
.......434
A Report of Pseudomonas aeruginosa Antibiotic Resistance from a Multicenter Study in Iran
MA Boroumand, P Esfahanifard, S Saadat, M Sheihkvatan, S Hekmatyazdi, M Saremi, L Nazemi
.......435
Trends of Antibiotic Resistance in Salmonella enterica Serovar Typhi Isolated from Hospitalized
Patients from 1997 to 2004 in Lagos, Nigeria
KO Akinyemi, AO Coker
.......436
Book Review
Hospital-Acquired Infections: Power Strategies for Clinical Practice
Reba Kanungo
.......438
Title Index, 2007
Author Index, 2007
Scientific Reviewers, 2007
.......440
.......442
.......446
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2007 Microbiology, (2007) 25(4): 369-73
Indian
Journal of Medical
369
Original Article
VIRULENCE FACTORS AND DRUG RESISTANCE IN ESCHERICHIA COLI
ISOLATED FROM EXTRAINTESTINAL INFECTIONS
S Sharma, *GK Bhat, S Shenoy
Abstract
Purpose: To determine the virulence factors produced by Escherichia coli isolated from extraintestinal infections, to
study the drug resistance pattern in E. coli with special reference to extended spectrum β-lactamase (ESBL) and to
evaluate screening methods for ESBL. Methods: A total of 152 isolates of E. coli from various extraintestinal infections
were screened for virulence factors such as haemolysin, surface hydrophobicity, serum resistance and protease. All the
isolates were also studied for antibiotic susceptibility pattern using modiÞed Kirby Bauer disk diffusion method. ESBL
production was screened by standard disk diffusion method and conÞrmed using phenotypic conÞrmatory method.
Results: Among 152 isolates, 36 (23.7%) were haemolytic, 42 (27.6%) were hydrophobic, 132 (86.8%) were serum
resistant and only four were positive for protease. Multiple virulence factor were observed in 67 (44%) of isolates.
Seventy-nine (51.4%) isolates produced ESBL. ESBL producing isolates showed multidrug resistance. There was a
signiÞcant association (P < 0.001) between multiple virulence factors and ESBL production by extraintestinal E. coli.
Conclusions: The present study shows the expression of virulence factors and multidrug resistance in E. coli isolated
from various extraintestinal infections. The study also shows that appropriate methods of detecting drug resistance and
ESBL production are required for the judicious use of antibiotics in managing these infections.
Key words: Drug resistance, Escherichia coli, haemolysin, serum resistance, surface hydrophobicity
Escherichia coli is one of the commensals in the human
intestinal tract. As a commensal, it contributes to the
maintenance of health of a person. However, E. coli, when
enters into unnatural sites, can cause variety of infectious
diseases such as urinary tract infections, wound infections,
bacteraemia, meningitis and other soft tissue infections.1
The ability of E. coli to cause extraintestinal infections
depends largely on several virulence factors, which help in
the survival of E. coli under adverse conditions present in
those sites. The virulence of individual strains in a given
infection is determined by the presence and actual expression
of the virulence genes present in them, and also by the
environmental conditions in the host.2
The treatment of E. coli infections is increasingly
becoming difÞcult because of the multidrug resistance
exhibited by the organism. Extended spectrum β-lactamase
(ESBL) producing organisms pose a major problem for
clinical therapeutics. The incidence of ESBL producing
strains of E. coli among clinical isolates has been steadily
increasing over the past few years resulting in limitation of
therapeutic options.3
The knowledge of drug resistance pattern in a geographical
area and the formulation of an appropriate hospital antibiotic
policy will go a long way in the control of these infections.
Therefore, it is necessary to know the antibiotic susceptibility
pattern of pathogenic E. coli to select the correct antibiotic(s)
for proper treatment of infections caused by it. The objectives
of the present study were to demonstrate the virulence factors
and drug resistance in E. coli isolated from extraintestinal
infections and to evaluate screening procedures for ESBL.
Materials and Methods
Clinical isolates
A total of 152 isolates of E. coli from extraintestinal
infections obtained from January 2004 to June 2005 were
included in the study. The study population included
hospitalised patients of all age groups at Government Wenlock
Hospital, Government Lady Goschen Hospital, KMC Hospital
Attavar and KMC Hospital Ambedkar Circle, Mangalore.
Specimens collected were pus, exudates, clean catch
midstream urine, urine from indwelling catheter, blood
and CSF using standard sterile procedures. The samples
were processed immediately using standard procedures.
The isolates were identiÞed based on colony morphology
on blood agar, MacConkey’s agar, Gram staining and by
standard biochemical tests.4
Detection of virulence factors
*Corresponding author (email: <[email protected]>)
Department of Microbiology, Kasturba Medical College,
Mangalore - 575 001, Karnataka, India
Received: 26-04-06
Accepted: 10-05-07
Cell surface hydrophobicity: The cell surface
hydrophobicity of E. coli was determined by salt aggregation
test (SAT).5,6 One loopful (10 µL) of bacterial suspension
made in phosphate buffer was mixed with equal volume
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Indian Journal of Medical Microbiology
of ammonium sulphate solution of different molarity, i.e.,
from 0.3125 M through 5.0 M, on a glass slide and observed
for 1 min while rotating. The highest dilution of ammonium
sulphate solution giving visible clumping of bacteria was
scored as the salt aggregation test (SAT) value. Strains
showing aggregation in 0.002 M phosphate buffer alone (pH
6.8) were considered auto aggregative. E. coli strains that
had SAT value ≤1.25 M were considered hydrophobic.5,6
Haemolysin production: Plate haemolysis test was done
for the detection of α-haemolysin produced by E. coli.5 The
bacteria were inoculated onto 5% sheep blood agar and
incubated overnight at 35 °C. Haemolysin production was
detected by the presence of a zone of complete lysis of the
erythrocytes around the colony and clearing of the medium.
Serum resistance: Serum resistance was studied using
fresh culture of the isolates.5,6 Overnight cultures of E. coli,
grown at 37 °C on blood agar, were harvested and the cells
were suspended in Hank’s balanced salt solution (HBSS).
Bacterial suspension (0.05 mL) was incubated with serum
(0.05 mL) at 37 °C for 180 min. Ten microlitres of samples
were withdrawn and spread on blood agar plates which
were then incubated at 37 °C for 18 h and the viable count
was determined. Resistance of bacteria to serum bactericidal
activity was expressed as the percentage of bacteria
surviving after 180 min of incubation with serum, in relation
to the original count. Bacteria were termed serum sensitive,
if viable count dropped to 1% of initial value and resistant, if
>90% of organisms survived after 180 min.
Gelatinase test: Gelatinase production was tested using
gelatin agar.7 The plate was inoculated with test organism
and incubated at 37 °C for 24 h. The plate was then ßooded
with mercuric chloride solution. Development of opacity
in the medium and zone of clearing around colonies were
considered positive for gelatinase.
Test for extended spectrum β-lactamase production
Screening by standard disk diffusion method: Screening for
ESBL production was done according to criteria recommended
by NCCLS.8 Two discs, ceftazidime (30 µg) and cefotaxime
(30 µg), were used for in vitro sensitivity testing by Kirby-Bauer
disk diffusion method. Zone diameters were read using NCCLS
vol. 25, No. 4
criteria. An inhibition zone of ≤ 22 mm for ceftazidime and ≤
27 mm for cefotaxime indicated a probable ESBL producing
strain requiring phenotypic conÞrmatory testing.
Phenotypic conÞrmatory methods: Disk diffusion
method was used to conÞrm ESBL production by E. coli
strains.9 Ceftazidime (30 µg) vs. ceftazidime/clavulanic acid
(30/10 µg) and cefotaxime (30 µg) vs. cefotaxime/clavulanic
acid (30/10 µg) were placed onto Mueller Hinton agar plate
lawned with the test organisms and incubated overnight at
35 °C. Regardless of zone diameters, a ≥ 5 mm increase in a
zone diameter of an antimicrobial agent tested in combination
with clavulanic acid vs. its zone size when tested alone,
indicated ESBL production.
Antibiotic susceptibility testing: The antibiotic
susceptibility testing was done using modiÞed Kirby-Bauer
disk diffusion method. The antibiotic disks (Hi Media,
Mumbai) used were ampicillin (10 µg), amikacin (30 µg),
co-trimoxazole (25 µg), cefotaxime (30 µg), ciproßoxacin
(5 µg), gentamicin (30 µg), netlimicin (30 µg).
After 18 hours of incubation at 37 °C, the diameter of
the zone of inhibition was measured using a millimetre
scale around each antimicrobial disk on the undersurface of
the plate. The zone size around each antimicrobial disk was
interpreted as sensitive, intermediate or resistant according
to NCCLS criteria.10
Results
A total of 152 strains of E. coli were isolated from
specimens collected from extraintestinal infections. Out of
these, 62 (40.8%) were from pus, 60 (39.5%) from urine,
14 (9.2%) from blood, 8 (5.3%) from ascites, 7 (4.6%) from
sputum and 1 (0.7%) from CSF (Table 1).
Virulence factors such as haemolysin, surface
hydrophobicity, serum resistance and protease were studied
for all the isolates. The most common virulence factor
identiÞed was serum resistance in 132 (86.8%) isolates.
Haemolysin was produced by 36 (23.7%) isolates, 42 strains
(27.6%) were hydrophobic and only 4 (6.9%) produced
protease. Multiple virulence factors were observed among
67 (44.0%) isolates of E. coli.
Table 1: Virulence factors in extraintestinal E. coli
Nature of specimen
Pus
Urine
Blood
Ascites
Sputum
CSF
Number of isolates
62
60
14
8
7
1
Haemolysin positive
10 (16.1)
15 (25.0)
9 (64.2)
2 (25.0)
0
0
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Number (%) of E. coli
Hydrophobic
Serum resistant
16 (25.8)
48 (77.4)
20 (33.4)
52 (86.7)
3 (21.4)
12 (85.7)
2 (25.0)
5 (62.5)
0
4 (57.1)
1
1
Protease positive
0
4 (6.9)
0
0
0
0
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Sharma et al - Virulence and Drug Resistance in Extraintestinal E. coli
The analysis of drug resistance pattern showed that
among 152 isolates of E. coli, maximum numbers (76.9%)
were resistant to ampicillin and lowest to netillin (42.8%).
ESBL production was detected in 79 (51.4%) of isolates.
The maximum number of ESBL producing E. coli (66.1%)
was isolated from pus.
Out of the 75 isolates resistant to cefotaxime, 70 (93.4%)
were ESBL producers whereas 9 (11.7%) isolates sensitive to
cefotaxime were positive for ESBL production. On the other
hand, of the 85 isolates, which were positive for ESBL by
screening test, 79 (92.9%) were positive by conÞrmatory test
for ESBL, whereas all the 67 isolates negative by screening
were also negative by conÞrmatory test.
The ESBL producing isolates were also studied for the
presence of co-resistance to other antibiotics. Among the 79
ESBL producers, all were resistant to ampicillin followed
by 89.8% to ciproßoxacin, 64.5% to cotrimoxazole, 88.6%
to cefotaxime, 65.8% to gentamicin, 46.8 to amikacin and
37.9% to netillin.
All the ESBL producing isolates were studied for the
presence of multiple virulence factors (Table 2). Application
of Gaussian’s formula showed that, there was signiÞcant
association (P < 0.001) between multiple virulence factors
and ESBL production in extraintestinal E. coli. Haemolysin
production was predominantly correlated with MDR/ESBL
production. As the number of virulence factors increased, the
rate of ESBL production in E. coli decreased.
Discussion
Virulence factors enable E. coli to colonise selectively the
mucosal uro-epithelium, evoke an inßammatory reaction and
eventually proceed from lower urinary tract to renal cavities
and tissue invasion. The capacity of E. coli to produce many
virulence factors contributes to its pathogenicity. E. coli
is able to cause a variety of infections such as urinary tract
infection, soft tissue infections, bacteraemia and neonatal
meningitis. These virulence factors enable some members of
the normal ßora to elicit an infection by overcoming the host
defence mechanisms.11
Haemolysin production is associated with pathogenicity
of E. coli, especially the more severe forms of infection.12 In
the present study, a few strains of E. coli (23.7%) produced
haemolysin. Haemolysin production was more common
among the isolates from blood. Haemolysin production
Table 2: Presence of multiple virulence factors in
ESBL producing isolates of E. coli
Multiple virulence factors
+
−
Total
ESBL+
21
58
79
ESBL−
46
27
73
371
as a virulence factor by urinary isolates of E. coli has been
shown by previous workers.6,12 It has been suggested that
colonization with haemolytic strains of E. coli is more likely
to develop into urinary tract infections. Haemolysis, though
not essential for establishment of acute pyelonephritis, may
contribute to tissue injury, survival in renal parenchyma
and entry into blood stream. The higher rate of haemolysin
producing strains isolated from blood may indicate its
importance in the invasive strains.
Surface hydrophobicity is another important virulence
factor of E. coli that causes extraintestinal infections. In
the present study, 27.6% of the strains were hydrophobic
and compared to other specimens, more isolates from urine
(33.4%) were hydrophobic. This is consistent with the
results of previous studies.6,13 The high hydrophobicity of
the bacterial cell surface promotes their adherence to various
surfaces like mucosal epithelial cells.
Serum resistance is the property by which the bacteria
resist killing by normal human serum due to the lytic
action of complement system. In the present study, 86.8%
of the isolates were resistant to serum bactericidal activity,
majority of which were isolated from urine (86.7%) and
blood (77.4%). A previous study showed serum resistance in
32.7% of E. coli isolated from urine.6 In another study, 68%
of the urinary isolates were resistant to serum bactericidal
activity which is comparable to our results.5 The rate of
serum resistant strains of E. coli isolated from blood in the
present study is consistent with a previous study.12
Isolates from patients with pyelonephritis, cystitis and
bacteraemia are typically serum resistant whereas patients with
asymptomatic bacteriuria have serum sensitive strains.12 The
serum resistant gram negative bacteria possess a signiÞcant
survival advantage in the blood during bacteraemia. There is
a strong correlation between serum resistance and the ability
of a variety of gram negative bacteria to invade and survive in
human bloodstream. A previous study has shown that serum
resistance is important in the pathogenesis of symptomatic
UTI, regardless of the severity.12
The present study also revealed expression of multiple
virulence factors by extraintestinal E. coli. Most of the
haemolysin producing isolates were also hydrophobic and
serum resistant. This is consistent with the Þndings of a
previous study.14 A combination of all three virulence factors
such as haemolysin, surface hydrophobicity and serum
resistance was present in 11.2% of the isolates. A previous
study indicated that although virulence of an organism
cannot be accurately predicted on the basis of its measurable
virulence factor phenotype, the presence of multiple virulence
factors does increase the virulence of organisms.12 The
virulence factors function additively or synergistically in
overcoming normal host defences. The strains with a more
extensive complement of virulence factors are more effective
pathogens, and the compromising host conditions decrease
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Indian Journal of Medical Microbiology
the need for multiple virulence factors in strains causing
serious infections.
Antibiotic susceptibility pattern was studied for all
isolates of E. coli. Resistance was observed to commonly
used antibiotics such as ampicillin, ciproßoxacin, cotrimoxazole, cefotaxime, gentamicin, amikacin and netillin.
The greater prevalence of resistance to common antibiotics
has also been reported by other workers.15,16 The presence
of multidrug resistance may be related to the dissemination
of antibiotic resistance among hospital isolates of E. coli.
Among aminoglycosides, netillin was found to have an edge
over gentamicin and amikacin. Similar observations have
been made by a previous group of workers.15 Maximum
number of isolates (76.9%) were resistant to ampicillin and
the lowest (42.8%) to netillin. These results are consistent
with the previous studies on drug resistance in E. coli. 17,18
We observed a high rate of ESBL production by E.
coli which may be due to the selective pressure imposed
by extensive use of antimicrobials. The indiscriminate
use of cephalosporins is responsible for the high rate of
selection of ESBL producing microorganisms. These
results are consistent with previous studies from India.9,19
For predicting ESBL production, it is important to mention
that for screening test, negative results are a better guide
than positive results. Therefore, conÞrmation of all positive
results by screening should be done to prevent unnecessary
avoidance of conventional β-lactams. These results are
consistent with the other studies on ESBL detection.9,20
In the present study, we found that a majority (68.7%) of
ESBL negative strains of E. coli produced multiple virulence
factors whereas most of the ESBL producers (68.2%) did
not produce multiple virulence factors. These results support
the hypothesis that although virulence factors and antibiotic
resistance may confer increased Þtness for extraintestinal
infections in humans, they may do so via mutually exclusive
pathways and in distinct populations.21 A rise in the number
of virulence factors was associated with a decrease in the rate
of ESBL production. A robust virulence factor repertoire may
be essential for a pathogen to overcome intact host defences,
whereas it may be unnecessary in a compromised host, where
antibiotic resistance may provide a substantial advantage to the
survival of the pathogen. Some strains sensitive to cefotaxime
were positive for ESBL. The false susceptibility observed
could be due to inoculum effect.22 Since ESBL production is
usually plasmid mediated, it is possible for one specimen to
contain both ESBL producing and non-producing cells.
The present study has shown the capacity of E. coli to
adapt and survive in different tissues by producing virulent
factors and developing drug resistance. The expression of
virulence factor(s) may depend on the need and it varies in
different kinds of infections. Drug resistance is on the rise
among E. coli strains that cause human infections. Proper
selection of antibiotics for treatment depends on the results
vol. 25, No. 4
of antibiotic sensitivity test. Therefore, the correct detection
of drug resistant bacteria is important. Judicious use of
antibiotics and good antibiotic policy are needed to limit the
emergence and spread of antibiotic resistance in bacteria.
Acknowledgements
The authors are grateful to the Manipal Academy of Higher
Education (MAHE) for providing the research fund to conduct the
study.
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Source of Support: Manipal Academy of Higher Education.
Conßict of Interest: None declared.