ANTIBIOTIC SUSCEPTIBILITY OF BACTERIAL STRAINS, WITH SPECIAL REFERENCE TO ESCHERICHIA COLI ISOLATED FROM URINARY TRACT INFECTIONS IN RURAL MAHARASHTRA

Objective: The objective of this study was to find out the etiology of urinary tract infections (UTIs) in patients attending B.K.L. Walawalkar Hospital, Dervan and to determine their antibiotic sensitivity pattern to currently used antimicrobial agents. Methods: A cross-sectional study was conducted in a rural hospital of Konkan Maharashtra, and urine samples were collected from 368 clinically suspected cases of UTIs using the mid-stream “clean catch” method and was tested and cultured using standard procedures. Antimicrobial susceptibility test (AST) was performed for the isolated pathogens according to the Clinical and Laboratory Standards Institute guidelines. Results: Escherichia coli (54.84%) was the most prevalent uropathogen. 76.47% of the isolated E. coli were found to be extended spectrum beta lactamase producers. A higher prevalence rate of resistance was seen among E. coli to the commonly prescribed antibiotic agents. 32 (94.11%) of 34 E. coli isolates recovered had multiple antibiotic resistance (MAR), with 16 isolates (50%) possessing MAR indices of 0.6. Conclusion: The study indicates the isolated microorganisms in UTI showed very high resistance to the commonly prescribed antimicrobial drugs. This suggests the monitoring and rational use of the antimicrobial agents.


INTRODUCTION
Urinary tract infections (UTIs) are some of the most common infections that occur in both male and female of all the ages, exceeded in frequency only by respiratory and gastrointestinal infections among ambulatory patients [1].
About 80-85% of community-acquired UTIs are caused by Escherichia coli and 5-10% by the Staphylococcus saprophyticus [2]. UTI cases are often treated empirically, and the antimicrobial resistance patterns of the urinary pathogens determine this empirical therapy [3]. However, uncontrolled antibiotic usage in large proportion has contributed to the emergence of microbial resistance to antibiotics. As a result, antimicrobial resistance is the more prevalent worldwide among urinary pathogens [4][5][6][7].
For this reason, having the knowledge of the changes in drug resistance patterns in specific geographical locations may help clinicians to choose the empirical antimicrobial treatment appropriately. The literature on the prevalence of UTI in this part of rural Maharashtra was scarce in recent decades; thereby, this study was conducted to find out the prevalence of UTI and to determine the antimicrobial susceptibility patterns of commonly used antibiotics.

METHODS
The study was performed on UTI cases attending B.K.L. Walawalkar Hospital, Dervan from July 2015 to December 2015 after ethical clearance from the Institution Review Board. A total of 368 clean catch midstream urine samples were collected in a wide mouth sterile container from the study subjects who had not taken antimicrobials in last 15 days. They were instructed to clean the area around the urethral opening with clean water, dry the area, and collect the urine with the labia held apart. Only one specimen per patient was accepted, and samples were processed within 1 hr of collection.
For culture, the urine sample (1 μl) was inoculated on Blood Agar and McConkey Agar plates, using a standard loop of internal diameter 1.34 mm (semi-quantitative method). The plates were read after 24 hrs of aerobic incubation at 37°C. They were further incubated for another 24 hrs before a negative report was issued. A specimen was considered positive for UTI if a single organism was cultured at a concentration of ≥10 5 cfu/ml [8]. No mixed infections were encountered.
The isolates were identified and their antibiotic susceptibility determined using the automated method for ID/antimicrobial susceptibility test using MicroScan (Siemens) rapid Gram-negative and Gram-positive panels. Intermediate isolates were counted as resistant to all the agents tested.
The results obtained were analyzed using descriptive statistics. Multiple antibiotic resistance (MAR) index is a tool to analyze health risk and is helpful to check the spread of bacterial resistance in a given population where there is resistance to more than three antibiotics [9]. It is calculated as the number of antibiotics to which test isolate displayed resistance divided by a total number of antibiotics to which the test organism has been evaluated for sensitivity.

RESULTS
A total of 368 urine samples were analyzed for culture and sensitivity during the study period, of which 60 (16.3%) had significant bacteriuria. The rate of positive culture was 63.33% for female subjects and 36.67% for male subjects.
The percentage frequency of MAR is shown in Table 3. In this study, we defined MAR as resistance to at least 3 antimicrobial agents [10]. 32 (94.11%) of 34 E. coli isolates re-covered had MAR, with 16 isolates (50%) possessing MAR indices of 0.6.

DISCUSSION
UTIs refer to the presence of microbial pathogens within the urinary tract and it is usually classified by the site of infection, if pathogen infects bladder (cystitis), if kidney (pyelonephritis), or if urine (bacteriuria). Bacteria that infect urinary tract may ascend toward the bladder causing cystitis, causing the peculiar symptoms of UTI (i.e., pain, frequency, and urgency). Bacteria may further ascend from bladder up to kidneys causing pyelonephritis, which may complicate later to irreversible kidney damage, renal failure, and death [11]. The etiology and antimicrobial susceptibility of UTI-causing bacteria's have been changing over years. Therefore, the overall, selecting empiric treatment for the patients with serious UTIs is difficult, necessitating the confirmation of etiologic by culture and susceptibility testing [12].
In this study, the isolation rate of bacteria from urine was 16.3% which is in line with studies done in Assefa et al. [13] and from Iran which had a rate of 13.2% [14]. The females were more susceptible to UTI than males, which is also similar to other studies [15][16][17]. Clinical UTIs are more common among females, with up to 60% of women having at least 1 episode in their lifetime [18]. The reason behind this high prevalence is close proximity of the urethral meatus to the anus, shorter urethra, sexual intercourse, incontinence, and bad toilet [19]. Considering the fact that most of infecting organisms are commensals of perianal and vaginal regions, emphasis on personal hygiene, especially in females may be important in reducing the incidence of UTI [20].
The results of our study show that among the heterogeneous causative organisms of UTI, Enterobacteriaceae are the predominant pathogens and E. coli is still the single most common uropathogen. This corresponds with the data obtained by other investigators [21][22][23]. Klebsiella pneumoniae, P. aeruginosa, Acinetobacter species, Citrobacter species, and P. mirabilis were isolated in small numbers while no Gram-positive organisms were isolated in this study. E. coli has the ability to colonize the urogenital mucosa with adhesins, pili, fimbriae, and P1 blood group phenotype receptor [24]. This probably explains its higher isolation from UTI.
Over the last decade, the treatment of choice for UTIs has changed owing to the rate of resistance and high level of therapeutic failure [25]. This study revealed a higher prevalence rate of resistance among E. coli to the commonly prescribed antibiotic agents such as ampicillin, amoxicillin -clavulanic acid, trimethoprim/sulfamethoxazole, norfloxacin, ciprofloxacin, and levofloxacin. The low susceptibility

Salunke and Gidamudi
(i.e., higher resistance) of the isolates to the common and cheap orally administered antibiotics is not surprising because these drugs are more commonly misused, thereby leading to the development of resistance, as previously reported [26]. Increased resistance in quinolones against E. coli may reflect the overuse of these drugs for the treatment of UTI [27]. Thus reducing the number of prescription for a particular antibiotic can lead to a decrease in resistance rates. Another factor could be the generalized use of fluoroquinolones in animals feed (especially in poultry), and the subsequent transmission of resistant to strains from animals to humans [28]. These findings are of great importance and imply that these antibiotics cannot be used as empirical therapy for UTI, particularly in the study area.
On the other hand, while no resistance was detected to tigecycline and fosfomycin, lower resistance was detected to nitrofurantoin, imipenem, and meropenem. The low resistance could be because they are not easily accessible and relatively expensive in price compared to others. Thus, these drugs could be considered as alternative options in the empirical treatment of UTIs. Resistance to nitrofurantoin among E. coli isolates from UTIs has remained low despite more than 50 year's widespread use of the drug. Reason for the lack of emerging resistance are not fully understood, but likely include restricting use to indication for urinary infection, limited systemic absorption, and the need for multiple genetic mutations for the bacteria to develop resistance [29].
The value of MAR index 0.2 differentiates the low and high risk. MAR index >0.2 implies that the strain of such bacteria originate from an environment where several antibiotics are used. The MAR indices of E. coli obtained in this study is a possible indication that a very large proportion of the bacterial isolates have been exposed to several antibiotics. In our study, 94.11% isolates of E. coli were MDR. This is quite high when compared to other studies [30]. Multi-resistance is usually related to production of ESBL, which in our study is very high as compared to other recently published data.

CONCLUSION
UTI is one of the common causes for seeking medical attention in the community and effective management of patients relays on the identification of the type of organisms that caused the disease and the selection of an effective antibiotic agent to the organism in question. This study provides valuable data to compare and monitor the status of antimicrobial resistance. Thus, fosfomycin and nitrofurantoin were found to be the most appropriate oral antibiotics and tigecycline, imipenem and meropenem were the most appropriate parenteral antibiotics, for the empirical therapy of UTIs.