Frequency and Sensitivity of Extended Spectrum Beta-Lactamase Positive Organisms in a Secondary and Tertiary Level Hospital Network in Dhaka

Background: Extended spectrum β-lactamase (ESBL) positive organisms are now a global health concern including in Bangladesh. These are associated with treatment failure, increased morbidity and mortality and increased health care costs. In this study, frequency of ESBL positive organisms in some health care centres in Dhaka city has been observed and their current status of antibiogram has also been observed. Objective: To observe the current status of antibiogram of ESBL positive organisms. Materials and Methods: This cross-sectional study was done in the Department of Microbiology, Bangladesh Institute of Health Sciences (BIHS) General Hospital, Dhaka, Bangladesh from March, 2012 to February, 2013. Only E. coli and Klebsiella spp. from pus and urine specimens were included in this study. Isolation, identification and antibiotic sensitivity of the organisms were done by standard procedures. Results: Organisms (Escherichia coli and Klebsiella spp.) isolated from urine and pus collected from different sites of 472 subjects were studied. Predominant organisms were Escherichia coli (82.8%) and remaining 17.2% were Klebsiella spp. ESBL positive organisms were higher in Escherichia coli (54.5%) than in Klebsiella spp. (44.4%) and higher in pus (77.0%) than in urine (49.1%) isolates. Imipenem is the most effective drug for treating ESBL positive organisms followed by colistin, tigecycline and piperacillin/tazobactam. Conclusion: Imipenem, colistin, tigecycline and piperacillin/tazobactam drugs should be kept reserved and used only when other effective drugs are not available so that emergence of resistance against these drugs is deferred. While reporting the culture and sensitivity tests, the ESBL positive organisms should be pointed out with comment like this – “The organisms are ESBL positive and resistant to penicillins, cephalosporins and monobactams”.


Frequency and Sensitivity of Extended Spectrum Beta-Lactamase
Positive Organisms in a Secondary and Tertiary Level Hospital Network in Dhaka USA in late 1988. 4,5In Bangladesh, it was reported first in 2001. 6ESBLs are enzymes that mediate resistance to β-lactam antibiotics, viz., penicillins, cephalosporins and monobactams, but do not affect cephamycins or carbapenems.Because of greatly extended substrate range, these enzymes are called extended spectrum β-lactamases. 2ESBL enzymes are most commonly produced by Escherichia coli (E.coli) and Klebsiella spp.and to some extent by other members of Enterobacteriaceae and Pseudomonadaceae. 2,7BL organisms have continued to increase in varieties (more than 400 variants detected) 8 and prevalence and are now a global health concern. 4,9,10The ESBL organisms have implications for clinicians and patients because these are associated with treatment failure, increased morbidity and mortality, poor outcomes, increased length of stay (LOS) in hospital and health care costs. 9So, it is important to treat it properly as soon as it is diagnosed.Aim of this study was to observe the current status of antibiogram of ESBL positive organisms and their prevalence in and around Dhaka city.

Materials and Methods
This cross-sectional study was done in the Department of Microbiology, Bangladesh Institute of Health Sciences (BIHS) General Hospital, Dhaka, Bangladesh from March, 2012 to February, 2013.Specimens were collected from inpatient and outpateint departments of BIHS General Hospital and nine more hospitals/centres of Dhaka city affiliated to BIHS General Hospital.
Organisms isolated from urine and pus of 472 subjects were studied.There were 411 urine and 61 pus specimens.Pus was collected from diabetic foot lesions, post surgical infected wounds and traumatic wounds.Only E. coli and Klebsiella spp.were included in this study.][13][14] For detection of ESBL positive organisms, screening test 15 was done along with routine sensitivity test.
Positive screening tests were confirmed later by Phenotypic Confirmatory Test (PCT). 13

Screening test
Disks of cefotaxime (CTX), ceftazidime (CAZ) and amoxycillin/clavulanic acid (AMC) were placed in a line, placing AMC in the centre and other two on either side about 30 mm apart from centre to centre.Widening of cephalosporin's inhibition zone adjacent to the disk containing AMC was regarded as ESBL positive. 14,15,

Isolation of ESBL positive organisms in BIHS General
Hospital and different hospitals/centres located at different sites of Dhaka city has been shown in  In this study, 472 specimens were tested, of which 249 (52.8%) were found positive for ESBL organisms.This is consistent with findings of Ahmed et al (53.4%, n=221) 1 and Sashirekha (48.9%, n=225) 16 .Findings of Dalela (61.6%, n=219) 17 , Oliveira et el (61.1%, n=90) 18 and Yasmin 19 (71.3%, n=300) were slightly higher; the findings of Rahman et al 6 was slightly lower (41.4%,n=157), but that of Hansen et al 7 (2.4%, n=14674) was very low.It is to be noted that in countries where there is good infection control practices and antimicrobial stewardship, the prevalence of ESBL +ve organisms is lower than other countries as was evidenced in a study in 2009 where global prevalence rate was found as follows: Latin America -44%, Asia / Pacific -22.4%, Europe-13.3%and North America-7.5%. 20However, prevalence of ESBL positive organisms is increasing throughout the world. 4edominating organism in our study was E. coli (82.8%, n=472) and the rest (17.2%) was Klebsiella spp.; 54.5% of E. coli (n=391) and 44.4% of Klebsiella spp.(n=81) were ESBL positive.These findings are consistent with the study of Rahman et al 6 and Sashirekha. 16In the study of Rahman et al 6 predominating organism was E. coli (54.4%, n=241) of which 43.2% were ESBL positive and 45.6% were K. pneumoniae of which 39.5% were ESBL positive.
In the study of Sashirekha 16 predominating organism was E. coli (28%, n=325) of which 52.8% were ESBL positive and 25.2% were K. pneumoniae of which 45.1% were ESBL positive.In the study of Dalela 17 73.5% of E. coli and 58.1% of K. pneumoniae were ESBL positive.In contrast, study of Olveira et al 18 showed K. pneumoniae (71.1%, n=90) as predominating organism followed by E. coli (24.4%); 71.9% of K. pneumoniae and 36.4% of E. coli were ESBL positive.Study of Yasmin 19 in Bangladesh also showed different scenario where predominating organism was E. coli (52%, n=300) followed by Proteus spp.(18.3%) and Klebsiella spp.(15%).In her study, Klebsiella spp.(80%) topped the list as ESBL producers followed by Proteus spp.(72%) and E. coli (67.3%).ESBL producing organism is now a global threat 1 and its treatment has become difficult because of drug resistance over a wide spectrum of β-lactam and non βlactam drugs.In this study, in case of most antibiotics the difference of sensitivity between ESBL positive and ESBL negative organisms was statistically significant.
All ESBL positive organisms (100%) were found resistant to cephalosporins (except cephamycins as discussed below) and monobactams while all (100%) ESBL negative organisms were sensitive to these drugs.Other authors showed varying degrees of sensitivity of ESBL producers to cephalosporins. 1,16,17It is to be noted that ESBL producers should be taken as resistant to all cephalosporins and monobactams even if these show sensitivity against some of the members.
Cefepime is considered as a therapeutic option by some authors, but clinical data show high failure rate. 20Some patients with ESBL positive organisms were treated successfully with cephalosporins where organisms appeared sensitive in disc diffusion methods and had very low MIC. 1,16,17,21,22However, MIC is not done routinely in our department except in cases of multidrug resistance when higher than MIC level of antibiotic is targeted.
Currently, carbapenems are the most effective antibiotic for treatment of infections due to ESBL producing organisms as the outcome is better than treatment with other antibiotics. 4,18,22,23This has been true in this study also where all (100%, n=391) E coli strains, both ESBL positive and negative, were sensitive to imipenem.This is consistent with the findings of Sasirekha (n=225) 16 , Yasmin (n=201) 19 and Paterson 23 who also found 100% of isolates sensitive to imipenem.Dalela (n=215) 17 found 98.5% (n=135) of ESBL producer and 94% (n=219) of non-ESBL producers sensitive to imipenem and Ahmed et al 1 found 98.3% of ESBL producer E. coli sensitive to imipenem.However, 5.6% of ESBL positive Klebsiella spp. in our study showed resistance to imipenem which is similar to the study of Hawser et al 24 where 10% of ESBL positive Klebsiella spp.were resistant to imipenem; but it contrasts with the study of Sasirekha 16 , Yasmin 19 and Paterson 23 who found all ESBL producing Klebsiella spp.sensitive to imipenem.Imipenem resistance of ESBL positive Klebsiella spp. is increasing all over the world day by day. 22 this study all isolates except ESBL positive Klebsiella spp.were found sensitive to tigecycline; 80.6% of ESBL +ve Klebsiella spp.were sensitive to tigecycline.In other studies, it was found that tigecycline had excellent in vitro sensitivity but data reflecting clinical outcome is lacking. 20,23,25It was also reported that there was increased mortality rate in tigecycline-treated patients than in patients treated by other antibiotics. 20FDA warned health professionals and their medical care organizations about the increased risk of death when intravenous tigecycline is used.They suggested to reserve tigecycline for use only in situations when alternative treatments are not suitable. 26out 94% of ESBL positive E. coli were found sensitive to colistin in this study.All ESBL negative E. coli and all Klebsiella spp.were sensitive to colistin.Other studies also demonstrated its efficacy in treating multidrug resistant organisms including ESBL producers. 20,23Although recent studies have shown that it has acceptable effectiveness and has been used to treat infections due to multiresistant Gram-negative organisms, its use should be reserved as a last resort mainly for ESBL producing bacteria that are also resistant to aminoglycosides and carbapenems. 25 this study, <20% ESBL producers and >80% non-ESBL producers (both E. coli and Klebsiella) were sensitive to amoxycillin/clavulanic acid.In the study of Dalela  [20][21][22][23] The cephamycins (e.g.cefoxitin) which are also 2 nd generation cephalosporins demonstrate markedly greater resistance to microbial degradation by βlactamases than is normally displayed by the cephalosporins. 22In our study, 85.9% of ESBL positive and 100% of ESBL negative E. coli and 86.1% of ESBL positive and 93.3% of ESBL negative Klebsiella were sensitive to cefoxitin.However, clinical data regarding its use is scarce and clinical failure has been documented due to co-resistance. 4,20 16 .
While reporting the culture and sensitivity tests, the ESBL positive organisms should be pointed out with comment like this -"The organisms are ESBL positive and resistant to penicillins, cephalosporins and monobactams".In this study imipenem was found as the most effective drug for treatment of ESBL positive as well as ESBL negative organisms followed by colistin, tigecycline, piperacillin/tazobactam.However, these drugs should be kept reserved and used only when other sensitive drugs are not available so that emergence of resistance against these drugs is deferred.While selecting antibiotic against ESBL positive organisms, it should be remembered that this group of organisms are relatively less sensitive than ESBL negative organisms.Also in cases of ESBL positive organisms, it seems prudent to avoid β-lactam antibiotics as far as possible.

Table II :
Frequency of ESBL positive Esch. coli and Klebseilla spp.
in different specimens (N= 472) BIHSGH, Bangladesh Institute of Health Sciences General Hospital

Table I :
Distribution of ESBL positive organisms in different sites of Dhaka city organisms in Banani (32.4%), the area where most affluent people live, apparently gives a good relationship of economic status, improved environmental sanitation and infection control.

Table III :
Sensitivity pattern of ESBL positive and ESBL negative E. coli and Klebsiella spp. to different antibiotics (except cephalosporins) (N=472) Rate of ESBL positive E. coli and Klebsiella spp. was 81.1% and 70.8% respectively.This is consistent with the study of Yasmin 19 and Dalela 17 except that the rate of ESBL positive E. coli in the study of Yasmin 19 (50%, n=42) and rate of ESBL positive Klebsiella spp. in the study of Dalela (50%, n=20) 17 were lower.
*Urine isolates only; In urine isolates, ESBL +ve E coli 183, ESBL -ve E coli 171 and ESBL +ve Klebsiella spp.19,ESBL -ve Klebsiella spp.38 17, 15.6% of ESBL producers and 23.8% of non- 20,23 ESBL negative Klebsiella spp.were sensitive to this drug.In the study of Ahmed et al 1 34.8% of ESBL positive and 21.5% of ESBL negative E. coli were sensitive to gentamicin.However, aminoglycosides may be potentially useful in the treatment of complicated urinary tract infections due to ESBL-producing organisms; but these are not recommended as routine monotherapy for severe infections at other sites.20,23Nitrofurantoinwas used in case of urine isolates only; both ESBL positive and ESBL negative E. coli were found almost equally sensitive (~85%).But Klebsiella spp.showed different figure -31.6% of ESBL positive Klebsiella spp.and 65.8% of ESBL negative strains were sensitive to nitrofurantoin. Or findings are almost similar to that of Yasmin 19 in case of E. coli (about 96% sensitive) but differed in case of Klebsiella spp.(about 95% sensitive).Ahmed et al 1 found 54% cases sensitive in case of both ESBL positive and negative E. coli.Sasirekha 16 found about 40% cases sensitive in case of both ESBL producer and non-producer organisms.Sensitivity of ESBL positive organisms to other antibiotics was very low (doxycycline 29.1% and zero, cotrimoxazole 24.9% and 19.4% and ciprofloxacin 11.3% and 16.7% for E.coli and Klebsiella spp.respectively). But sitivity of ESBL negative strains were significantly higher than ESBL positive organisms (p<0.05).These findings are consistent with that of Yasmin 19 , Dalela 17 , Ahmed et al 1 and Sasirekha In our study, 77.5% of ESBL positive E. coli and 55.6% of ESBL positive Klebsiella spp.were sensitive to gentamicin whereas 94.4% of ESBL negative E. coli and 97.