Prevalence and antibiotic susceptibility pattern of pathogens from urinary tract infections (UTI) in a private diagnostic laboratory in Bangladesh

Urinary tract infection (UTI) is one of the commonest infections encountered by clinicians and despite the widespread availability of antimicrobial agents UTI has become difficult to treat because of appearance pathogens with increasing resistance to antimicrobial agents. The aim and objectives of this study were to determine the pathogens causing UTI and to determine the antibiotic sensitivity status among these isolates in a diagnostic laboratory in Dhaka city. A laboratory based cross sectional survey was conducted in Popular Diagnostic Centre Ltd. Dhanmondi, Dhaka-1205, Bangladesh from July 2016 to December 2016. A total of 553 freshly voided midstream urine samples (10-20 ml) were collected in a wide mouth sterile container from patients and processed in microbiology laboratory to isolate pathogens and antibiotic susceptibility test using standard procedure. Among 553 urine sample, the culture positivity in urine samples was found to be 158 (28.57%) of which 39 (24.70%) were isolated from male patients and 119 (75.30%) from female patients. Escherichia coli (43.67%) were found to be the predominant pathogen followed by Staphylococcus spp. (16.45%), Enterococcus spp. (13.39%), Klebsiella spp. (13.29%), Candida spp. (5.70%), Acinetobacter spp. (4.43%), Psudomonas spp. (3.80%) and Proteus spp. (1.27%). Carbapenem group (Imipenem, Meropenem) were the most effective antibiotic with resistance between 0 and 5.1% of the gram negative isolates and Linezolid and Vancomycin was most effective in gram positive isolates. Nitrofurantoin was most effective both gram negative and gram positive isolates. This study finding showed That Escherichia. coli isolates were the predominant pathogens and showed increasing pattern to the commonly prescribed drugs in private practice that in turn leaves the clinicians with very few alternative options in drug for the treatment of UTIs.


Introduction
Urinary tract infection (UTI) is the common bacterial infectious disease in community practice with high rate of morbidity and financial cost all over the world. Worldwide, about 150 million people are suffering from UTI/year, costing 6 billion US dollar ( Gonzalez et al., 1999). Nearly about 10% of people will experience in UTI during lifetime (Hoberman et al., 1997). UTIs refer to the presence of microbial pathogens within the urinary tract and it is usually classified by the site of infection of urinary bladder (cystitis), kidney (pyelonephritis) or urine (bacteriuria). Almost 95% of cases of UTI are caused by bacteria that typically multiply at the opening of the urethra and travel up to the bladder. A limited predictable spectrum of organism is responsible for urinary tract infection. Most of the UTI are caused by gram negative bacteria like Esherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Proteus spp., Acinetobacter spp., Serratia and Morganella margani. UTI also caused by Gram positive bacteria like Enterococcus spp., Staphylococcus especially coagulase negative Staphylococci and Streptococcus agalactiae (Shaaban et al., 2012). UTI is much more common in Women than in men due to anatomical and physiological reason; by virtue of its position urogenital tract is more vulnerable to bacterial infection caused by internal and external flora. Studies from India, Navaneeth et al., 2002, Bangladesh, Iqbal et al., 1997 andNepal, Srinivassa et al., 199 have reported an increased resistance of the urinary pathogens to commonly used antibiotics. The aim of this study was to determine microbial etiologic agents responsible for urinary tract infection and to evaluate them in vitro susceptibility pattern to commonly used antimicrobial agents in a private practice set up of Dhaka city. This study is important for clinicians in order to facilitate the effective treatment and management of patients with symptoms of urinary tract infection.

Study design
A prospective cross-sectional study was conducted in the dept. of Microbiology, Popular Diagnostic Centre Ltd. Dhaka, Bangladesh. Total 553 samples were collected during this study period from July 2016 -Dec 2016.

Inclusion/Exclusion criteria
Study population consisted of all the patients who visited the concerned laboratory for urine culture examination.

Sample collection and processing
Study population were instructed on how to collect clean-catch mid-stream urine into sterile container after carefully cleaning the genitalia, especially around the opening the urethra. Samples were collected wide mouthed, leak proof container supplied by the laboratory and brought to the laboratory as early as possible, usually within 1 hour after collection.

Microscopy
The urine samples were mixed thoroughly, centrifuged and examined microscopically for wet mount preparation.

Culture
A calibrated sterile Nicrome wire loop for the semi-quantitative method was used for the plating. It has a 4 mm diameter to deliver 0.01 ml. A loopful of the well mixed urine sample was inoculated on Hi-Chrome (Himedia, India), MacConkey and Blood agar media (Mast diagnostic, UK) and then incubated at 37 0 C aerobically for 24 hrs and for 48 hrs in negative cases. They were then examined for bacterial growth. A significant bacterial count was taken as any count equal to or in excess of 100,000 CFU/ml. A less than 100 CFU/ml was interpreted as negative.

Cultural observation
Color, size, and colony morphology are observed from the incubated plates.

Microscopic examination of pathogens
Slides were prepared from each different colonies observed on the plates and gram staining was performed. The results such as the gram positive or gram negative, shape of the bacteria are observed from the examinations.

Biochemical examination
The selected colonies based on the cultural, microscopic and microbiological examinations, were subjected to biochemical examination (starch hydrolysis, lipid hydrolysis, casein hydrolysis, triple sugar iron agar test, oxidase test, catalase test, nitrate reduction test, indole production test, methyl red test, voges-proskauer test, citrate utilization test, urease test) for confirmation of the pathogens (Cheesbrough, 2006).

Statistical analysis
The statistical analysis of data was performed using Microsoft Excell-2011 version. Discrete values were expressed as percentage. Descriptive statistics were used to summarize patient characteristics and the prevalence of antimicrobial sensitivity.

Results
Out of 553 urine samples, the culture positive (≥10 5 cfu/ml) was found 158 (28.57%) of which 39 (24.70%) were isolated from male patients and 119 (75.30%) from female patients and other 395 (71.43%) sample were negative or normal flora (Table 1). Most of the patients were in age group of 21 to 40 years, which were 38 (17.92%) cases positive; among them 34 (98.12%) were female and 4 (1.88%) cases were male. In the age group of less than 20 years' female is also predominant than male which is 16 (61.54%) cases and 10 (38.46%) cases respectively. Overall female was more common than male, shown in Table 2.  (Table 3).

Discussion
This study provides valuable data to isolate and identify the pathogen which cause urinary tract infection and monitor the status of antimicrobial sensitivity among uropathogens to improve efficient empirical treatment. In addition to that the susceptibility pattern of these bacteria is very important to avoid development of drug resistant (Behzadi et al., 2010). In our study isolation & identification of uropathogens were performed and 158(28.57%) urine sample showed significant growth of bacteria so, remaining majority 395 (71.43%) of the cases showed either insignificant bacteriuria or no growth with urine from the suspected cases of UTI. This study resembles to the study conducted by Yusuf et al., 2015 andBarber et al., 2013 and they showed 28% significant bacterial growth. The reason of low growth rate may be due to irrational use of antibiotic which is available in the local market in this country and these are given without prior culture and antibiotic sensitivity pattern. In addition to that, incomplete doses are another factor. Prior antibiotic therapy before sending urine sample for culture and sensitivity and other clinical conditions like non-gonococcal urethritis could be the factors responsible for insignificant bacteriuria or no growth. (Barber et al., 2013).
In this study showed the prevalence was high in females than males for each isolates 75.31% and 24.68% respectively, so we can say strictly female suffers more than male from UTI. Other studies also showed that urinary tract infection was more common females. Akhter et al., 2012 andYusuf et al., 2015 was found 71.6 % and 66.6% respectively. It is well established that female is more commonly infected with UTI than male due to anatomical position of urethra, influence of hormone and pregnancy (Jones et al., 1999) The international studies have shown that UTIs in women are very common; therefore, one in five adult women experience UTI in her life and it is extremely common, clinically apparent, worldwide patient problem.  Oluremi et al., 2011 and also in study conducted by Chakupurakal et al., 2010 where predominant organism was E. coli and in number of reports worldwide where the particular organisms is identified as most common uropathogens. The dominance of E. coli is followed by Staphylococcus spp. 16.45% in this study resembles to the study done by Jha et al., 2005 which was the second most organisms causing UTI. In sectional study by University of Florida USA of a group of patient, 81 patients met the inclusion criteria of this study of these 81 patients 89% had UTI E. coli (Mcloughlin et al., 2003). Antibiotic susceptibility test reveals that higher percentage of susceptibility for Imipenem & Meropenem 96%, followed by Nitrofurantoin (88%), Amikacin (88%), Netilmicin (88%), shown in Table 4. This study resembles to the study conducted by Farjana et al., 2009 where higher percentage of susceptibility was seen for amikacin (88%). Second is the nitrofurantoin which considered as an appropriate agent for first line treatment of community acquired UTIs. Ampicillin was found to show the higher resistance rate (98%) which resembles to the study conducted by Nerukar et al., 2012 which show that isolates of most of the species exhibited a high rate of resistance to ampicillin. Resistance to antibiotics develops due to its frequent misuse. (Nerukar et al., 2012) According to the above result, among antibiotics used for susceptibility test for gram negative bacteria, amikacin, netilmicin, and nitrofurantoin was found to be the most effective antibiotics followed by gentamicin, cefoxitin, cefepime and ceftazidime for the isolates respectively were sensitive. In present study, E. coli and Klebsiella have maximum sensitive to nitrofurantoin. Almost all gram negative organisms are very less sensitive to cephlosporin and flouroquinolones groups, Pseudomonas species was found resistant against all commonly used and maximum sensitive was imepenem, meropenem. The results of present study show higher rate of resistance in a private diagnostic laboratory, which may be a result of the irrational use of antibiotics (Dund et al., 2015).
Staphylococcus & Enterococcus spp. was sensitive to Linezolid (100%), Vancomycin (94%). This is comparable with works of Dund et al., 2015. The antimicrobial susceptibility pattern confirms that most of the urinary isolates in our environment are resistant to the commonly used antibiotic including the cephalosporin and fluoroquinolones.
In particular, the high resistance of the gram negative isolates to the fluoroquinolones is worrisome as these are reserve drugs for treating resistance infections (Aderounmu et al., 2006)

Conclusions
A drug resistance among bacterial pathogen is an evolving process, regular surveillance and monitoring is necessary to provide physicians knowledge on the updated and most effective empirical treatment of UTIs. Periodic reassessment of in vitro susceptibility pattern of urinary pathogens to serve as a guide for antibiotic therapy since these organism exhibit resistances to first-line drugs used for UTI. In order to prevent resistance to antibiotics, the use of antibiotics should be kept under supervision, should be given in appropriate doses for an appropriate period of time.
In the present study, community-acquired UTI and nosocomial UTI were not been distinguished. This was the main limitation of the study.

Conflict of interest
None to declare.