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225
Journal of Basic and Clinical Pharmacy
www.jbclinpharm.com
Emergence of Multidrug Resistant Extended-Spectrum
β-Lactamase Producing Eshcherichia coli Associated With Urinary
Tract Infections in Bangladesh
Rumana Mowla
1
, K.M. Al-Hasan Imam
1
, Muhammad Asaduzzaman
1*
, Nishat Nasrin
2
, Sheikh Zahir Raihan
1
, and
A.K. Azad Chowdhury
1
1
Deptartment of Clinical Pharmacy & Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh.
2
Deptartment of Pharmacy, East West University, Dhaka-1212, Bangladesh.
ABBREVIATIONS
AmC, ESBL, ESBLEC, MDR, MIC, USA, UTI
INTRODUCTION
U
rinary Tract Infection is the most commonly encountered infection
found in adult women in many parts of the world. Reports of high inci-
dents of community acquired urinary tract infections are available from
Asia Pacifi c, Denmark, Japan, India, Russia, and the USA [1-4]. In the USA
alone, every woman visits physician at least twice for the management of UTI [5].
In Bangladesh UTI is both community and hospital acquired. e disease has not
been yet occurred as outbreak but emerging resistance of pathogenic strains against
multidrug has imposed a threat of epidemiology all over the world. Strains highly
resistant to ciprofl oxacin were detected among commonly isolated gram-negative
urinary pathogens [6]. Transferable drug resistance is also been studied and found
common in organisms isolated from UTI patients in Bangladesh [7].
While
trimethoprim–sulfamethoxazole or trimethoprim alone had
been used
widely as empirical therapy for E. coli urinary tract
infection for a long period of
time, recent studies suggest alarming prevalence of multidrug resistance in E. coli
associated with UTIs. Very high percentages of multidrug resistant (including
fl uoroquinolones and cephalosporins) E. coli causing UTIs began to be reported
from earlier this decade from USA, Saudi Arabia, India, Japan , Nepal, China and
Brazil [8-14]. is made the treatment of UTIs even more complicated.
Until recently, most infections caused by ESBL-producing Escherichia coli
(ESBLEC) had been found to be nosocomial [15, 16]. e emergence of extend-
ed-spectrum β-lactamase (ESBL)-producing bacteria, particularly E. coli and
Klebsiella pneumoniae, is now a critical concern for the development of therapies
against bacterial infections. ESBL producing organisms exhibit co-resistance to
many other classes of antibiotics resulting in limitation of therapeutic option [17].
Emergence of ESBL producing E. coli in Bangladesh should be of no surprise as
signifi cant prevalence of ESBL producing multidrug resistant E. coli have been
reported recently in India and Pakistan [19-20]. Incidence of urinary infections
(UTIs) by ESBL-producing E. coli was found highest in India (60%), followed by
Hong Kong (48%) and Singapore (33%) [1].
Present study refl ects relatively high prevalence of multidrug resistant and
ESBL producing E. coli associated with UTIs in Bangladesh.
MATERIALS AND METHODS
Strains
irty six Eshcherichia coli strains were isolated from ‘clean-catch’’ midstream urine
and put into culture by the standard-loop semi-quantitative technique [18] from
UTI patients at the Sylhet Medical College, Sylhet, Bangladesh. e culture in
MacConkey agar plates were incubated aerobically at 37
0
C overnight (18- 24
hours). Any plate showing more than 100 colonies were considered as signifi -
cant. A single colony of confi rmed Eshcherichia coli was grown in trypticase soy
broth with 0.3% yeast extract and stored at -70
0
C after addition of 15% glycerol for
further use. e strains were identifi ed in the microbiology laboratory by standard
microbiological and biochemical methods [21].
Analytical Profile Index (API 20E)
e API 20E strips that consist of 20 microtubes containing dehydrated sub-
strates were used to confi rm the bacterial species [22]. For every strain, one specifi c
number was obtained by the change of colour of the strips upon addition of bacte-
rial suspension. e number was matched with the analytical profi le index.
Antibiogram
Bacterial susceptibility to antimicrobial agents was determined by using modifi ed
Kirby-Baur method or disk diff usion method as recommended by the National
Committee for Clinical Laboratory Standards [23] with commercial antimicro-
*Corresponding Author E-mail: azamanalo@yahoo.com
ABSTRACT
The incidence of infections due to extended-spectrum β-lactamase (ESBL)–producing Escherichia coli has been
i ncreased dramatically in recent years. Treatment is difficult because of frequent multidrug resistance. To identify the
sensitivity of commonly used antibiotics, 36 ESBL producing E. coli strains were isolated from young adult female pa-
tients in a govt. medical college hospital in Bangladesh. The samples were studied for antimicrobial sensitivity against
nine (9) commonly used antibiotics namely ampicillin (amp), trimethoprim-sulfomethoxazole (tms), tetracycline (tet),
ciprofloxacin (cip), mecillinum (mel), ceftriaxone (cef)
, nalidixic acid (nal), Azithromycin (azm) and Chloramphenicol
(chl) and the MIC values were determined by agar dilution method. Overall, 72% of the strains were multidrug resistant
(MDR) i.e. resistant to two or more drugs. Among 36 strains, 14 isolates were initially found to be resistant against third
generation cephalosporin, ceftriaxone. Those were subjected to the test for production of ESBL (Extended Spectrum
β-Lactamase) and 7 showed positive results.
received on 10-01-2011
accepted on 12-07-2011
available online 15-02-2012
www.jbclinpharm.com
KEY WORDS
ESBL, Escherichia coli, UTI, agar dilution,
multidrug resistance
Journal of Basic and Clinical Pharmacy
Rumana Mowla et al
www.jbclinpharm.comVol-003 Issue-001 February 2012
226
bial discs (Oxoid, Basingstoke, United Kingdom). e antibiotic discs used in this
study were ampicillin (10 μg), tetracycline (30μg), mecillinum (25μg), nalidixic
acid (30μg), sulfomethoxazole-trimethoprim (25μg), ciprofl oxacin (5μg), ceftri-
axone (30μg), azithromycin (15μg), and chloramphenicol (30μg). e diameters
of the zones of complete inhibition were measured to the nearest whole millimetre
by a ruler. e zone diameter for individual antimicrobial agents was then trans-
lated into susceptible, intermediate, moderately susceptible, or resistant categories
according to the interpretation table (supplied by the company, Oxoid limited,
England).
Determination of MIC
Minimum inhibitory concentration (MIC) values of fl uorooquinolone antibiotics
(ciprofl oxacin and nalidixic acid) were determined by agar dilution method [24] in
which the bacteria were allowed to grow on a series of agar plates containing dou-
bling dilution of antibiotics and the results were most often read after incubation
at 37°C for 20 hours. Young cultures of the representative strains were used in this
method where 10-15μl young culture was applied on the agar surface by means of
an inoculating device (micropipette). After inoculation, the plates were dried for 5
minutes in room temperature and then incubated at 37°C overnight. After incuba-
tion, the plates were observed for complete inhibition of bacterial growth.
Determination of production of ESBL
To test whether a β-lactam resistant organism produces ESBL or not, Disk ap-
proximation or double disk method was used as described earlier by Jarlier et.
al [25] by using multiple target disks and a clavulanic acid disk. Disk content
and disk placement followed a validated method [26]. e multiple disks used
in this process were disks containing the standard 30 μg of ceftazidime, aztre-
onam and ceftriaxone and the amoxicillin-clavulanic acid (AmC) disk containing
standard 10 μg of the later compound. Muller Hinton agar plates containing
the ceftriaxone resistant strains were prepared by the recommended method for
a standard disk diff usion susceptibility test. Disks containing the standard 30
μg of ceftazidime, aztreonam and ceftriaxone were placed 15 mm (edge to edge)
from an AmC disk. Disk placement was expedited by melting holes in the lid of
a petri dish and using the lid as a template to mark the bottom of the agar plate
for proper disk location. After 16 to 18 h of incubation, the plates were examined
and enhancement of the zone of inhibition between a beta-lactam disk and that
containing beta-lactamase inhibitor was decided to be indicative of the presence
of ESBL.
RESULTS
Analytical Profile Index (API20E)
e analytical profi le numbers of all 36 strains were matched with the analytical
profi le index supplied by the manufacturer and all the strain was confi rmed to be
E. coli.
Antibiotic Susceptibility Test
e antibiotic susceptibility test showed (Table 1) that 92% strains were re-
sistant to ampicillin (n=33), 52% of the isolates (n=19) were resistant against
sulfamethoxazole-trimethoprim, 50% (n=18) to tetracycline, 25% (n=9) to
chloramphenicol, 50% against azithromycin, 8.33% (n=3) isolates showed resist-
ance against to mecellinum. Among the strains, 72% were resistant to nalidixic
acid (n=26) and 50% (n=18) were resistant to Ciprofl oxacin. Lastly, 14 strains
(39%) were resistant to ceftriaxone. e antibiotic sensitivity pattern was such
that overall 72% of the strains were multidrug-resistant (MDR) i.e. resistant to
at least three drugs.
e antibiotic sensitivity was typed according to their resistance pattern,
namely Pattern 1 To Pattern 6 (Table 2)
All the strains were tested to determine the minimum inhibitory concentration
of fl uoroquinolone antibiotics – ciprofl oxacilin (cip) and nalidixic acid (nal) by
agar dilution method. e MIC values were determined on the basis of the recom-
mended break points for Enterobacteriacae [27] and were defi ned as the lowest con-
centration of antibiotic that completely inhibit the visible growth of test organism
at 37°C after 18 hours of incubation. With respect to MIC values, 50% (n=18) of
the strains were resistant to ciprofl oxacin (MIC value ranged from 16 to >128mg/
ml) and over 72% (n=26) were resistant to nalidixic acid (MIC value ranged from
512 to >1024mg/ml) (Table 3).
Test for production of ESBL
Among 36 strains of Escherichia coli, 14 isolates were initially found to be resistant
against third generation cephalosporin, i. e. ceftriaxone. ose 14 strains were sub-
jected to the test for production of ESBL and 50% (n = 7) showed positive results.
e presence of enhanced zone of inhibition between disks containing AmC (Amox-
yclavulanic acid) and β-lactams was taken as the indicator of producing ESBL.
Table 1: Antibiotic resistance of
Escherichia coli
(n=36).
Antibiotics used % of resistant strains
Ampicillin (Am) 92
Trimethoprim-sulfomethoxazole
(Sxt)
52
Ciprofl oxacin (Cip) 50
Nalidixic acid (Nal) 72
Mecillinum (Mel) 8
Azithromycin (Azm) 50
Chloramphenicol (C) 25
Tetracycline (Tc) 50
Ceftriaxone (CRO) 39
Table 2: Resistance pattern of the
E. coli
strains.
Pattern Resistance pattern % of strains (no.)
Pattern 1 Resistant to one drug 8 % (n=3)
Pattern 2 Resistant to two drugs 17% (n=6)
Pattern 3 Resistant to four drugs 11% (n= 4)
Pattern 4 Resistant to fi ve drugs 28% (n=10)
Pattern 5
Resistant to six or more
drugs
22% (n=8)
Pattern 6 Sensitive to all 2% (n=1)
Emergence of multidrug resistant
E. coli
in Bangladesh
Journal of Basic and Clinical Pharmacy
Vol-003 Issue-001 February 2012www.jbclinpharm.com
227
DISCUSSION
MDR (multi-drug resistant) pathogens pose a clear threat to public health. In the
present study, 36 Escherichia coli samples were collected from young females. We
found 72% of the strains to be resistant against two or more drugs. Resistance
to most commonly used antibiotic ampicillin was 92% (n=33). In addition, 50%
(n= 18) of the strains were resistant to ciprofl oxacin and 72% (n=26) strains were
resistant to nalidixic acid. Cephalosporin resistance was also found to be high. We
used ceftriaxone, a third generation cephalosporin and alarmingly the resistance
percentage was 39 (n=14). Extended spectrum β-lactamases are enzymes that
confer resistance to third generation cephalosporins and monolactams. A large
number of plasmid-transferable ESBLs capable of inactivating extended-spectrum
cephalosporins
have been discovered [15]. Production of ESBLs is one important
factor to study. As these enzymes are harbored by plasmids, the microorganisms
producing ESBLs not only confer resistance to cephalosporins but also can trans-
mit the resistance to other organisms. Again, another disastrous thing about ESBL
producing microorganism is that there is possibility of developing resistance to
other antimicrobial agents [25]. In the present study we used traditional double
disk approximation method [28] to detect the presence of ESBL and discovered
threatening results. Out of 14 ceftriaxone resistant isolates, 50% (n=7) were con-
fi rmed as ESBL producers. is fi nding clearly demonstrates the importance of us-
ing more modern broad spectrum antibiotics to treat this kind of infection. ese
enzymes are capable of hydrolyzing broad-spectrum cephalosporins and mono-
bactams but inactive against cephamycins and imipenem. So these new broader
classes of antibiotics in the treatment of UTIs should be introduced.
CONCLUDING REMARKS
UTIs are not very uncommon in Bangladesh and with this high level of antibiot-
ics it is likely that the disease could become more prevalent. Pathogens producing
Extended Spectrum β-lactamase limits the treatment option even further. More
surveillance work defi ning the types of ESBL should be conducted to develop em-
pirical treatment pattern for Eshcherichia coli mediated UTIs.
DISCLOSURES
ere are no confl icts of interest among the authors in relation to the manuscript.
GRANT SUPPORT
is work was neither funded nor supported by any grant. It was self-funded by
the authors.
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