Uropathogenic Analysis and Commonly Used Drug Sensitivity Patterns of the Pathogens in Dhaka City, Bangladesh

Aims: Human urinary tract infections (UTI) are very common in Bangladesh. The objectives of the current study are to identify the uropathogenic agents infecting males and females of different age groups, and commonly used drugs sensitivity profiles. Study Design: A total of 980 urine samples from both sex and different age groups, were collected for the study. Chemical analyses of the samples were done by Dipstick method. Cultural, microscopic and biochemical analyses were done to identify the isolates. Finally antibiotic sensitivity was tested against conventionally used antibiotics. and May following published procedures. Results: Only 29.6% of the urine samples yielded positive culture. UTI showed more prevalence among female than male. Female belonging to the age group of 41-50 years were detected as high risk groups for UTI. Most common Gram negative isolates were Escherichia coli , Klebsiella sp., Candida sp., Pseudomonas sp., Proteus sp., and Acinetobacter baumannii . Gram positive bacteria comprised Enterococcus sp. and non-hemolytic Streptococci. Like most of the previous reports, E. coli was predominant, which is also corroborated in this study. However, the sensitivity pattern of the organisms differed from the previous studies. Conclusion: The isolates were found resistant to most common oral antibiotics used, such as cotrimoxazole, nitrofurantoin, and nalidixic acid. This finding, however, need further work to validate reliability.


INTRODUCTION
Urinary tract infections (UTI) are common and frequently encounter serious morbidity that badly affects its levy not only to all human population but also results in increasing antibiotic resistance due to persistence and unprofessional conduct of the ailment. Even today UTI is one of the most important causes of morbidity and mortality in the developing countries like Bangladesh and even in developed countries [1]. This may be attributed to lack of proper research, faulty diagnostic procedures, abuse of chemotherapeutic agents of the people and little or no preventive measures. The alarming phenomenon is that UTI does not restrict itself to the urinary tract only rather it can spread. UTI usually cause inflammation of the affected tissues of the urethra (urethritis) and urinary bladder. The most significant danger from lower urinary tract infections is that they can affect the kidney (causing pyelonephritis) and develop bladder infections subsequently [2]. Bacteria carried by blood stream can also infect the kidney and the infections can be very difficult to eradicate, are often chronic, and lead to marked damage of the kidney. Death promptly follows kidney failure unless the patient is lucky enough to be able to use artificial kidneys, or perhaps to receive a kidney transplant. UTI present as the clinical syndromes of acute, uncomplicated, urinary infection, including acute non obstructive pyelonephritis, complicated urinary tract infection, asymptomatic bacteriuria, and in men, bacterial prostatitis. Severe or life-threatening infection usually occurs with complicated urinary infection, which occurs in men and women with functional or structural abnormalities of the urinary tract. Obstruction or mucosal traumas are the most common precipitating events for urosepsis. Although 20% to 30% of women with acute non obstructive pyelonephritis or men with acute bacterial prostatitis have bacteremia, these syndromes seldom progress to severe sepsis or shock [3].
Any individual may be susceptible to UTI, however, the prevalence of infection differs with age, sex and certain predisposing factors such as diabetes, pregnancy, impaired voiding of the bladders etc [4]. The incidence of infection is greater in females than in males with two exceptions, infants and the catheter related infections [5,6]. Women are likely to get UTI frequently. The incidence of UTI is greater in women (20%) because of the anatomical predisposition or urothelial mucosa adherence to the mucopolysaccharide lining or other host factors. In children approximately 5% of girls and 1% of boys have a UTI by 11 years of age, in the neonates is 0.01-1% and can also be as high as 10% in low birth weight and preterm babies [7]. UTI is considered significant and requires treatment when more than 10 5 microorganisms per ml of urine are present in a properly collected sample [8]. Uropathogenic E. coli causes 90% of the UTIs in anatomically-normal, unobstructed urinary tracts [9]. After E. coli, the most common UTIs pathogens include Staphylococcus saprophyticus, Enterococcus spp., Pseudomonas aeruginosa, Candida spp., Klebsiella pneumonia, Proteus spp. and Enterobacter spp. Group B streptococci are rare pathogens in UTIs in young healthy women [10]. Interestingly, the pathogens traditionally associated with UTI are known to change many of their features, particularly due to their antimicrobial resistance patterns [11]. Though antibiotics are the mainstay treatment for all UTIs, the increasing trend of resistance in bacterial pathogens is of worldwide concern that can vary according to geographical and regional locations [12].
Since the initiation of antimicrobial therapy in UTI is empirical, a huge need demand for antimicrobial resistance exists at local, national and international levels [13]. Knowledge on the antimicrobial resistance patterns of common uropathogens and the subsequent treatment are thus required to minimize urinary diseases [14]. The current study aimed to identify the uropathogenic agents of UTI in males and females of different age groups, their sensitivity and resistance patterns against locally available antibiotics frequently prescribed by the physicians in order to find suitable antimicrobial agents to treat UTI.

Sample Collection
Urine samples were collected aseptically from out-patient department (OPD) and in-patient department (IPD) patients of all age groups including age 0 to 90 from United Hospitals Ltd, Gulshan, Dhaka, Bangladesh, having clinical symptoms of microbial infection. The study was conducted over a period of five months from January 1, 2012 to May 31, 2012 at the hospital setting. A total of 980 clinical isolates were tested from OPD and IPD patients.

Chemical Analysis
Chemical analysis of the urine specimens were done using COMBINA 11S dipstick. The analysis was done immediately within one hour of specimen collection. The specimens were collected from the midstream flow of the first morning urine. Specimens were thoroughly mixed by inversion several times and no centrifugation was done before analysis.

Microscopic Analysis
12 mL of each specimen was centrifuged at 1500 rpm for 5 minutes. After discarding the supernatant leaving only around 1 mL of concentrated specimen pellet. The pellet was mixed well and 20 µL of the suspension was placed over glass slide with cover slip on top for microscopic observation. Average number of casts were scanned and counted at low power field (LPF). RBCs, WBCs, crystals, yeast, bacteria, epithelial cells, mucus, and other formed elements were also scanned and counted at high power field (HPF).

Microbiological Analysis
10 µL of the specimens were directly streaked on blood agar and macConkey agar plates and incubated for 24-48 hours at 37ºC. After incubation the colony characteristics were observed and recorded. Gram staining was also done for all the colonies obtained on blood agar and macConkey agar plates [15].

Biochemical Analysis
Microgen GnA+B-ID system containing two separate micro well test strips GnA and GnB, was used for biochemical analysis of the isolates following their instruction manual. Each Micro well test strip contains 12 standardized biochemical substrates which have been selected on the basis of extensive computer analysis. The dehydrated substrates in each well are reconstituted with a saline suspension of the organism to be identified. If the individual substrates are metabolized by the organisms, a color change occurs during incubation or after addition of the specific reagents. The permutation of metabolized substrates was interpreted using the Microgen Identification System Software (MID60) to identify the test organism.

RESULTS AND DISCUSSION
Identification of the etiological agents and their susceptibility to antimicrobial agents is very important for choosing proper drug to treat the patient in their early stage of UTI. It is therefore, recommended that routine uropathological analysis and antibiotic sensitivity test of mid stream urine samples of the patients be carried out before the treatment of UTI [17].
A total number of 980 specimens were collected and analyzed for uropathogenic analysis. 288 specimens (29.4%) were positive for growth of urinary pathogen, 60 specimens (6.1%) showed growth of non pathogenic organisms and 632 specimens (64.5%) showed no apparent growth.
A total of 288 patients of UTI of either sex with the respective ratio of 27:73 (male: female) between age group of 1-90 years was studied (Fig. 1). Schaeffer and Dielubanzan [18] and Ahmed et al. [19] also found similar results in their study. Among the 288 UTI patients, 185 patients were from In Patient Department (IPD) and 103 patients were from Out Patient Department (OPD). Fig. 1 showed the number of UTI suspected cases according to different age group where higher UTI suspected age groups were from 41-50 for female and 0-10 and 11-20 age groups for male. However, maximum UTI patients were from age group of 30-35 years according to Ahmed et al. [19]. For male and female UTI patients, age group of 11-20 and 41-50 were found to be high risk group, respectively (Fig. 1). For females, after the age of 40, they became prone to UTI.

Fig. 1. Gender wise UTI patients (%) by age group
The current study indicated that E. coli is the primary etiological agent of UTI (Fig. 2). Besides, Klebsiella spp. and Candida spp. were also predominant among the UTI patients. Acinetobacter baumanni was detected from male patients mostly (80%), whereas E. coli and Morganella morgannii were detected mostly from female (80%) patients (Fig. 3). Other than A. baumanni; Enterococcus sp., Candida spp. and Klebsiella sp. were predominant causal agent of UTI among male patients. Klebsiella sp., Pseudomonas, Proteus and others were predominant for female UTI (Fig. 3), which is similar to previous findings [18].

Fig. 2. Etiological agents of UTI
The distribution of abnormal findings such as presence of pus cell, RBC, albumin, glucose, nitrite and ketone bodies were analyzed and the findings are presented in Fig. 4. Nitrite, pus cells, RBC, ketone bodies and albumin were found predominantly among female patients. Glucose was reported equally for patients of either sex (Fig. 4). Pus cells were found in urines of all UTI patients no matter what pathogen was involved (Fig. 5). Patients infected with Enterococcus sp. showed highest pus cells as well as albumin in urine (Fig. 5). As Enterococcus spp. are frequently encountered uropathogens in complicated UTIs [20], pyuria and albuminuria might have a relationship with that. However, patients infected with other uropathogens also have higher pus and albumin in urine [20], which indicated that these two abnormalities are very common in UTI.

CONCLUSION
This study intended to ascertain the existing situation of UTIs and drug resistance among different age groups of patients in Dhaka City. The study concluded that the incidence of UTI is higher in females and infection is higher among the patients of 41-50 age group of females and 11-20 age group of males as compared to other groups. However, UTI can affect anyone at any age. UTI is mainly caused by Gram negative organisms whereas Gram positives can also cause the infection. The study also revealed that patients of UTI cases were associated with abnormal count of pus cells, RBC, albumin, glucose, nitrite and ketone bodies. Most of the isolates were found resistant against commonly used antibiotics such as Cotrimoxazole, Nitrofurantion, Nalidixic acid. E. coli were found sensitive to Amikacin, Colistin and Imipenem ranging from 80-90%, followed by Netilmycin and Meropenem from 70-80%. Further research is needed to better understand the real situation of UTI and treatment efficacy in Dhaka City.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.