Isolation and antibiotic susceptibility of E. coli from urinary tract infections in a tertiary care hospital

Objective: The study was conducted to isolate and determine the antibiotic resistance in E. coli from urinary tract infections in a tertiary care hospital, Lahore. Methods: Urine samples (n=500) were collected from patients with signs and symptoms of Urinary tract infections. Bacteria were isolated and identified by conventional biochemical profile. Antibiotic resistance pattern of E. coli against different antibiotic was determined by Kirby-Baur method. Results: Bacterial etiological agent was isolated from 402 samples with highest prevalence of E. coli (321, 80%) followed by Staphylococcus aureus (9.4%), Proteus species (5.4%) and Pseudomonas species (5.2%). The E. coli were highly resistant to penicillin (100%), amoxicillin (100%) and cefotaxime (89.7%), followed by intermediate level of resistance to ceftazidime (73.8%), cephradine (73.8%), tetracycline (69.4%), doxycycline (66.6%), augmentin (62.6%), gentamycin (59.8%), cefuroxime (58.2%), ciprofloxacin (54.2%), cefaclor (50%), aztreonam (44.8%), ceftriaxone (43.3%), imipenem (43.3%), and low level of resistance to streptomycin (30%), kanamycin (19.9%), tazocin (14%), amikacin (12.7%) and lowest to norfloxacin (11.2%). Out of 321 E. coli isolates, 261 (81%) were declared as multiple drug resistant and 5 (1.5%) were extensive drug resistant. Conclusion: It is concluded that most of the urinary tract infections in human are caused by multiple drug resistant E. coli.


INTRODUCTION
Urinary tract infections (UTIs) are serious health affecting problems worldwide. 1 E. coli, E. faecalis, K. pneumoniae, S. marcescens, P. aeruginosa, S. saprophyticus, S. aureus and Proteus mirabilis are most common bacteria causing UTIs in human beings. [2][3][4] The E. coli accounts for approximately 85% of community acquired UTIs and 50% of hospital acquired UTIs. 5 Different factors like age, gender, immunosuppression and urological instruments may affect prevalence of UTIs. 6 Catheter-associated UTIs are one of the most dangerous health risks contributing 34% of all health care associated infections. 7 The emergence of extended-spectrum beta-lactamases has threatened the empirical use of cephalosporins and ciprofloxacin. 8,9 Microorganisms use various mechanisms to develop drug resistance, such as recombination of foreign DNA in bacterial chromosome, horizontal gene transfer and alteration in genetic material. 10 Resistance pattern of microorganisms vary from country to country, state to state, large hospital to small hospital and hospital to community. In Pakistan, the problem of antibiotic resistance is compounding because of overuse and misuse of antibiotics. 6,11 There is no systematic national surveillance of antibiotic resistance and insufficient data is available to quantify the problem. 12 Detection of UTI causing pathogens and resistance of these pathogens to commonly prescribed antibiotics in clinical set ups is essential and helpful in improving the efficacy of empirical treatment. 13 Objective of the present study was to highlight the bacterial etiology of UTIs and determination of resistance pattern of E. coli isolates.

METHODS
The observational and prospective study was conducted at Mayo Hospital Lahore, which is one of the oldest and biggest hospitals in Punjab.

Sample collection and Isolation of Bacteria:
Urine samples (n=500) were collected from patients in different wards (n=400) and outpatient department (n=100) from Mayo Hospital, Lahore. Samples were centrifuged and sediments were cultured primarily on blood agar and macConkey's agar by spread plate technique. Bacterial colonies having different morphology were selected, purified and identified by their biochemical profiles. Multiple drug resistance: Antibiotic sensitivity pattern of E. coli isolates was determined on Muller Hinton agar plates by Kirby-Bauer disc diffusion. 14 Isolates were declared as sensitive or resistant on the basis of zone of inhibition following the criteria of Clinical Laboratory standards Institute.

RESULTS
Bacterial etiology of Urinary tract infections (UTIs) in patients admitted in or visiting Mayo hospital, Lahore as out patient was determined. Resistance pattern of Escherichia coli against a number of antibiotics was also checked. Bacteria were successfully isolated from 402/500 samples. Rate of isolation of bacterial etiological agent from female samples (87.5%) was not-significantly higher as compared to male (71.3%) patients. Out of 402 bacterial isolates from patients, rate of E. coli (321, 80%) isolation was highest followed by Staphylococcus aureus (38 9.4%), Proteus species (22 5.4%) and Pseudomonas spp (21 5.2%). E. coli exhibited

DISCUSSION
UTIs are caused by microbial invasion and subsequent multiplication in urinary tract. 15 Eighty percent of the patients with UTI had bacterial etiology in this study. Although the infection rate was higher in female (87.5%) patients as compared to male (71.3%), it was not-significant, which is in accordance with finding of Shah et al. 16 Rate of bacterial isolation was highest in elderly patients (>50 years), which is in accordance with Iqbal et al. 6 E. coli was observed as the most common etiologic agent of UTI, which is also in accordance with previous studies. 1,17,18 Antibiotics are amongst the most important achievements of the twentieth century, used to kill or inhibit the growth of microorganisms. Antibiotic resistance in E. coli isolated from UTIs is increasing day by day, making it a major public health problem. So it is very important to determine the antibiotic resistance patterns in E. coli isolates for proper and accurate prescriptions.
UTIs caused by antibiotic resistant and multiple drug resistant bacteria have been increased in recent times. Complications in UTIs have increased because of the prevalence of extended spectrum beta-lactamases (ESBL) producing bacterial pathogens which are also causing many management and epidemiological issues. There were times almost a decade ago, when most of the ESBLs producing organisms were Klebsiella spp. and mostly were nosocomial. But in recent times the problem has been compounded by the prevalence of ESBL and MDR E. coli as well. Most of the ESBL E. coli are resistant to a wide range of beta lactams including cephalosporins, penicillins and piperacillin/ tazobactam, and non beta lactams including fluoroquinolones, trimethoprim and gentamycin. One of the major reasons for this high resistance can be co-expressed resistance mechanisms in the species of different pathogens isolated from patients of urinary tract infections admitted to different wards of Mayo hospital Lahore, Pakistan. In the present study we analyzed their antibiotic sensitivity pattern was determined by Kirby Bauer technique.
In present study all E. coli species(n=321) were resistant to penicillin and amoxicillin indicating a cautious use of these antibiotics for the treatment of urinary tract infections. In different parts of the world, resistance of E. coli to penicillins group of antibiotics have been on higher side and is increasing day by day, but there are only few reports which indicates 100% resistance to penicillins 19 Resistance to the combination of amoxicillin and a beta lactam inhibitor (augmentin 62.6%) was also on the higher side. Similar kinds of results, where beta lactam inhibitors increase the efficiency of penicillin group of antibiotic against E. coli, have been reported in previous studies. 20 Resistance to other beta lactam antibiotics including cefotaxime (89.7%), ceftazidime (73.8%), cephradin (73.8%), cefuroxime (58.26), cefaclor (50%), Ceftrioxone (43.3%) was also very high rendering many of these inefficient for empirical prescription of these antibiotics to treat UTIs. Previous studies in Pakistan have also shown very high antibiotic resistance in E. coli against cephalosporins and penicillins. 21 Generally, in developing countries like Pakistan, penicillines and cephalosporins are not active against the UTI infections and our results suggest that these antibiotics should not be used in the treatment of UTIs. Inefficiency of penicillins and cephalosporins in this study does not indicate that these antibiotics are not in use in any part of world to treat UTIs caused by E. coli. In some of the recent reports a higher number of E. coli was found sensitive to penicillins or cephalosporins from European countries. 22 A decade before, these antibiotics were active against E. coli even in Pakistan. 20 In this study, the resistance of E. coli against aztreonam and imipenem was 44.8% and 43.3%, respectively, which is higher than previous studies. 23,24 Higher resistance in E. coli against carbapenams indicates that these may have been misused and overused in health care set ups. Tazocin, a combination of piperacillin and beta lactamases inhibitor tazobactam, showed best results, for which resistance in E. coli was only 14% suggesting that this antibiotic can still be used for the treatment of UTIs. 25 Although, tetracycline group of antibiotics are not used now a days for human infectious agents, E. coli were highly resistant to tetracycline (69.4%) and doxycycline (67.6). In the present study variable resistance patterns were found for the aminoglycosides. E. coli were highly resistant to gentamycin, while low level of resistance was for kanamycin (19.9%), and amikacin (12.7%).
Quinolones, especially ciprofloxacin have been used for E. coli infections in recent past. In the present study however E. coli were highly resistant to ciprofloaxacin (54.2%), which is consistent with the previous reports. 26 Other fluoroquinolones such as norfloxacin (11.2% resistance) and levofloaxacin (all sensitive) were found efficient for the E. coli. Other studies from the different parts of the world also show that quinolones are still active against UTI infections. 26 Multiple drug resistance (MDR) and extensive drug resistance (XDR) was also determined in this study. MDR is described as resistant to at least one member from three different antibiotic classes being used for the treatment of E. coli, while extensive drug resistance (XDR) is described as resistance to at least one member of all but two antibiotic classes. MDR and XDR E. coli in this study were 81% and 8.7% respectively. The antibiotics active against the XDR were amikacin and norfloxacin generally. It is concluded that higher level of antibiotic resistance, MDR and XDR is present in E. coli. To treat the UTIs caused by E. coli combination therapy especially amikacin and ciprofloaxacin may provide better results. Antibiotic resistance in E. coli isolated from UTIs insinuates for its close monitoring and prescription of antibiotics after the culture sensitivity tests.