Investigation of Urine Samples for Isolation, Identification and Antibiotic Susceptibility Analysis of Bacterial Pathogens from Suspected Urinary Tract Infected Patients of Bangladesh

Urinary tract infection (UTI) is the most common community-acquired bacterial infection affecting people of all age groups and both sexes. Aims: This study was performed to isolate bacterial pathogens usually cause community-acquired uncomplicated UTI and to evaluate their sensitivity against 9 different antibiotics. and of Study: This study was conducted Department Microbiology, Jessore hundred fifty urine from Pathogenic were isolated and identified using conventional and biochemical methods. Kirby-Bauer disc diffusion method on Mueller Hinton agar media was used for the determination of sensitivity of the positive isolates to commonly prescribed antibiotics. Statistical Package for Social Sciences (SPSS) software, version 20 was used for statistical analysis. was the most predominant bacteria in both gender and different age groups. Cefixime (94.52%), Cephalexin (94.52%) and Azithromycin (93.15%) were the most effective drugs and Nalidixic acid (13.69%) was the least effective drug for the treatment of UTI.


INTRODUCTION
Urinary tract infection (UTI) is one of the most common life threatening and community acquired bacterial infection with a high rate of morbidity and financial cost [1][2][3]. UTI involves bacterial infections on one or more parts of urinary system and it usually occurs after bacteria overcome the natural host defence mechanism [4]. When infections occur in the lower urinary tract, it is known as a bladder infection or cystitis and infections in the upper urinary tract is known as kidney infection or pyelonephritis [5]. Cystitis is usually associated with the urgency of urination, dysuria, pyuria, irritation of urinary tract, discomfortable pressure, bloody urine which may have a strong smell and tiredness [6]. A kidney infection includes fever and flank pain as well as the symptoms of cystitis [5].
Though multiple species of microorganisms can be responsible for UTI, the vast majority of UTI is caused by Gram-negative E. coli, a common member of Enterobacteriaceae family, accounts for 75.0-90.0% of all UTI in both inpatients and outpatients [7]. Other members of the Enterobacteriaceae family that cause UTI are Pseudomonas species, Proteus species, Klebsiella species, and Citrobacter species. Among the Gram-positive organisms, the most common bacteria that frequently encountered are group B Streptococci, Staphylococcus aureus and Staphylococcus saprophyticus.
In case of uncomplicated urinary tract infection, nonresident infectious organisms invade structurally and functionally normal urinary tract and in complicated UTI, infections occur in patients with structurally and functionally abnormal urinary tract or both [8]. Nonresident infectious organisms include potential urinary pathogens from the bowel, or in some cases from the vagina of women as a result of sexual activity [8]. After colonization to the periurethral mucosa, organisms ascend to the bladder through the urethra and in some cases to the kidney through the ureter [8]. Antimicrobial secretions, polymorphonuclear cells, and Tamm-Horsfall glycoprotein of host inhibit bacterial adherence to the bladder mucosal wall [9]. But the uropathogens use different virulence factors and mechanisms to colonize and infect the urinary tract. In uncomplicated urinary tract infection, uropathogenic E. coli is the most common pathogens that have the enhanced virulence factor such as adhesins and fimbriae (pili), which help to bind to specific receptors of the uroepithelium [10].
Uncomplicated UTI is more commonly occurs in female because of the anatomical predisposition or urothelial mucosa adherence to the mucopolysaccharide lining or other host factors [11]. However, recent studies suggest that, uncomplicated UTI may also occur in men because of insertive anal intercourse or lack of circumcision or having sexual partner with vaginal colonization with uropathogenic microorganisms or lack of immunity [12][13][14]. Majority of UTIs don't cause any threat to the lives and irreparable damage. However, when the kidneys are involved, there is a risk of serious tissue damage with prevalence of bacteremia [15].
Clinical signs, symptoms and urinalysis results are important for the diagnosis of UTI. Results from urine culture play vital role in the diagnosis of UTI by determining the identity of infecting bacteria and their antimicrobial susceptibility [1,16]. Only a few studies have been documented about the susceptibility pattern of community-acquired UTI pathogens in different countries of the world [17][18][19]. However, there is no such information about the etiology and susceptibility patterns of community-acquired UTI pathogens in Jessore city of Bangladesh. Therefore, the present study was carried out to detect bacteria as etiological agents of community-acquired uncomplicated UTI and to determine their antimicrobial susceptibility pattern from urine samples of patients attending Jessore Medical College and Hospital of Bangladesh.

Study Area and Study Population
Present study was conducted between November, 2015 to February, 2016 in Department of Microbiology at Jessore Medical College and Hospital, Bangladesh. Urine samples were collected from 150 patients attending outpatients departments (OPDs) who were suspected of having UTI and referred to Jessore Medical College and Hospital for urine culture. Among the 150 urine samples, 75 samples were collected from female and another 75 samples were collected from male. Patients in the age range from 0 to 80 years were included in our study. The study was conducted after taking ethical approval from the hospital administrations.

Sample Processing
Samples for urine culture were processed within an hour of sample collection. A loopful urine sample was inoculated on Cystine-Lactose-Electrolyte Deficient (CLED) agar, blood agar and MacConkey agar by using sterile 4.0 mm platinum wired calibrated loop. The plates were incubated for 24 hours at 37°C in aerobic condition. After 24 hours, plates which showed no growth were further incubated for 24 hours at the same condition. When a single organism produced colony of ≥ 10 5 cfu/ml, then it was considered as positive sample for UTI [20].

Isolation and Identification of Bacterial Isolates
Bacteria were isolated and identified on the basis of standard culture and biochemical profiles of the isolates. Identification of Gram-negative bacteria were done by standard biochemical tests [21]. On the other hand, identification of Gram-positive microorganisms were done with the corresponding laboratory tests such as catalase, coagulase, CAMP test for Streptococcus species, mannitol test for Staphylococcus aureus [22]. The isolated strains were sub-cultured in nutrient agar slants and maintained as pure culture for further study.
Pure culture of bacteria was grown on nutrient agar media by streaking method. Then suspension was made in normal sterile saline by taking a single colony. The turbidity of the suspension was then adjusted to a McFarland 0.5 standard (3×10 8 cfu/ml). A sterile non toxic cotton swab was dipped into the standardized suspension and the swab was then streaked over the entire surface of the Mueller Hinton agar plates to obtain uniform inoculum. After 3 to 5 minutes, the discs were applied by using a sterile forcep and pressed down gently into the agar. Then the plates were incubated at 37°C for 24 hours. After 24 hours, the zones of inhibition were measured and interpreted by following the recommendations of the criteria of the Clinical and Laboratory Standards Institute (CLSI) [23]. E. coli (ATCC 25922), S. aureus (ATCC 25923), and P. aeruginosa (ATCC 27853) were used as quality control strains in our study.

Statistical Analysis
Chi square test was conducted to find out the significant difference between male and female patients in relation to the prevalence of isolated uropathogens. The frequency of the positive samples of each agent as a function of gender and age groups was also analyzed by Chi square test. At 95% level of confidence interval and a P value of <0.05 was considered as statistically significant.
The statistical analysis was performed by the Statistical Package for Social Sciences (SPSS) software, version 20.

RESULTS
Out of 150 urine samples, 73 (48.67%) samples were found positive for bacterial infection. The number of isolated Gram-negative bacilli was 55 (75.34%) and Gram-positive cocci was 18 (24.66%). From female, 62 (84.93%) positive isolates were identified and 11 (15.07%) positive isolates from male. Our study showed that prevalence of uropathogens was more common in females than males. However, the Chi square (߯2) test results showed that there was no significant variations (P > 0.05) between female and male in relation to the prevalence of the isolated bacterial pathogens at 95% confidence interval level (߯2 =2.544; degree of freedom = 5; P = 0.7698) ( Table 1).
E. coli was higher in percentage than other bacteria in both female and male. Patients within age group 11-50 years were found most susceptible for E. coli (Table 3). Klebsiella species, Pseudomonas species, and Proteus species were found more commonly in the age ranged between 21-40 years (Table 3). On the other hand, the distribution of Streptococcus species and Staphylococcus aureus were found to be the most frequent in age group 61-70 and 51-60 years respectively (Table 3). However, Data from statistical analysis showed that there were no significant variations in the prevalence of each isolated bacterial pathogens between the female and male of different age groups at 95% confidence interval level ( degree of freedom= 7, P > 0.05) ( Table 3). Proteus species was 100% susceptible to Cefixime, Cephalexin and Azithromycin and least susceptible to Ciprofloxacin (50%) and Gentamycin (50%). Also, Streptococcus species and Staphylococcus aureus were 100% susceptible to Cefixime and Azithromycin and 20% and 25% susceptible to Nalidixic acid respectively.

DISCUSSION
Urinary tract infection (UTI) is the most frequent community-acquired infection in the world. It is an extremely common cause of morbidity and mortality in developing countries like Bangladesh. This may be associated with lack of proper research, faulty diagnostic procedures, abuse of chemotherapeutic agents and little or no preventive measures. Generally, in almost all cases of UTI, antimicrobial treatment is started before the availability of results from microbiological tests. Therefore, it is important to obtain local and national data about the pathogens causing uncomplicated UTI and their antimicrobial susceptibility pattern. Results from region-specific surveillance studies provide additional information about causative agents and their antimicrobial susceptibility pattern and serve as a basis to develop national guidelines for the empirical treatment of UTI [24].
Detection of bacterial pathogens causing uncomplicated UTI and their antimicrobial susceptibility pattern has been demonstrated in this study. Our study showed that females (84.93%) are more prone to UTI than males (15.07%). Similar results were found in other studies performed in Turkey (82.3% female, 17.7% male), Kenya (64% female, 36% male), and Iran (76% female, 24% male) [24-28]. The incidence of urinary tract infections is far more frequent in women due to shorter and wider urethra, hormonal changes which affect the adherence of bacteria to mucosa, trauma of urethra during sexual intercourse. Besides, women lack the prostatic fluids which have bactericidal effects, usually secreted from male [28-29].
The results showed that the organisms isolated from urine samples were E. coli, Pseudomonas species, Klebsiella species, Proteus species, Streptococcus species and Staphylococcus aureus. Also, the most frequent causative agent of UTI was found to be E. coli (50.68%) in both sex groups. This result is consistent with other study conducted in Dhaka, the capital of Bangladesh where E. coli (59%) was found as the primary etiological agent of UTI [30]. Our report was higher than reports conducted in India and Southwest Ethiopia, where E. coli was 31.5% and 33.3% respectively. However, our report is lower than report carried out in Russia where E. coli (85.9%) was also found as predominant isolate [31-33].
Our research showed that Gram-negative bacilli (75.34%) are more commonly associated with UTI than Gram-positive cocci (24.66%). This data is consistent with other study conducted in Iran where prevalence of Gram-negative and Gram-positive bacteria was 85.6% and 9.3% respectively [20].
Antimicrobial susceptibility test inferred that in overall more than 80% of the uropathogens were susceptible to Cefixime, Cephalexin, Azithromycin as well as more than 50% were susceptible to Ciprofloxacin and Gentamycin ( This study has several inherent limitations. All of the urine samples in the study were collected from Jessore Medical College and Hospital, Jessore, Bangladesh. Samples from other hospitals located in Jessore city were not included in this study. The results obtained from this analysis might appear with some variation in comparison to results obtained from other regions of the country. The small sample size was a major constraint to convey meaningful outcomes by statistical analyses. Therefore, a prospective design of longitudinal study involving larger sample from different hospitals may provide more detail insight of bacterial pathogens causing UTI and their prevalence in different age groups of both male and female patients. The identification of differents bacterial pathogens was done based on various conventional biochemical tests. However, parallel molecular detection could provide extra strength in our findings.

CONCLUSION
Our study confirmed that E. coli, Pseudomonas species, Klebsiella species, Proteus species, Streptococcus species and Staphylococcus aureus are common pathogens usually associated with UTI. Results from different age groups of both genders revealed that female are more frequently infected with UTI than males. Cefixime, Cephalexin and Azithromycin could be of drug choice for community-acquired uncomplicated UTI and to control antimicrobial resistance, the physicians should prescribe antibiotics only after performing antibiogram.

CONSENT
All authors declare that written informed consent was obtained from the patients for publication of this case report and accompanying images.