Prevalence and Sensitivity of Bacterial Urinary Tract Infection among Adult Diabetic Patients in Misan Province, iraq

Urinary tract infections and their complications causing serious health problems especially in diabetic patients in Misan province, it is essential to determine the causative agent for appropriate treatment of this disease. One hundred and fifty urine samples were taken from adult patients with diabetes. The microbial growth appeared in 106 (70.6%) samples. Axenic culture was 83% versus 17% mixed growth. UTI prevalence was 65% with females comparing 35% of males and it was 56.6% in age group 35-49 years. Furthermore, UTIs were more incident in patients without antibiotics use and diabetic period 5-10 years (82% and 64%) respectively. Escherichia coli was identified as the most common causative agent of UTIs (52.9%), followed by Klebsiella pneumoniae and Enterococcus faecalis (9,1% and 6.6%, respectively). Streptococcus agalactiae and Klebsiella aerugenes were (4.2%, for each). Moreover, Pseudomonas aeruginosa, Klebsiella oxytoca and Staphylococcus haemolyticus were 2.48% for each. Additionally, the frequency of Proteus mirablis, Staphylococcus aureus, Streptococcus pyogenes, Serratia marcessens, Staphylococcus epidermidis and Staphylococcus warnerii was 1.65% for each, while Acinetobacter baumani and Bacillus subtilis were 0.82% for each. Most bacterial isolates had a high sensitivity to imipenem (78.8%) followed by amikacin (61.9%), but low sensitivity to ceftriaxone, tetracycline and Co-trimaxazole (36.4%, 29.7% and 26.3%, respectively) whereas, highly resistance to ampiclox and nitrofurantoin (98.3% and 87.3%, respectively). High rate of multidrug resistance observed among bacterial isolates.


INTRODUCTION
Urinary tract infections (UTIs) are one of the most common infectious diseases encountered in medical practice affecting humans of all ages 1 . Globally, UTIs incidence was estimated about 150 million persons per year 2 .
Diabetes mellitus is a group of chronic metabolic disorders characterized by increased blood glucose level resulting from defects in secretion, action of insulin or both 3 . The chronic hyperglycemia is associated with long term damage, dysfunction and failure of different organs especially the eyes, urinary system, nerves and cardiovascular system 3 . Over time, diabetic patients may develop cystopathy, nephropathy and renal papillary necrosis complications that predispose them to UTIs 4 . Higher severity of UTI can be cause many complications, ranging from dysuria to pyelonephritis 5 . Moreover, diabetic patients encounter further urinary urgency and incontinence during night 6 . Further more, those patients frequently suffer from bacterial cystitis with higher prevalence in diabetic women including higher prevalence of both asymptomatic bacteriuria and symptomatic UTI added to recurrent complications comparing with healthy women 7,8 .
In diabetic and non-diabetic patients, about 80% of UTIs cases were caused by gramnegative bacteria mainly Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Proteus spp., while only 15% were caused by gram-positive including Enterococcus spp., Staphylococcus spp. and Streptococcus spp. [9][10][11] . Susceptibility to antibiotics is usually variable among species and strains, therefore, the resistance to different antibiotics making it difficult to treat in some infections 12 . The widespread and indiscriminate use of broad-spectrum antibiotics led to the emergence of multi-drug resistant bacteria 13 .
Briefly, this study was designed to determine the prevalence of causative microbial agents of urinary tract infections in diabetic adult patients and their susceptibility to antibiotics.

Study design
A descriptive study was conducted at the Microbiology Laboratory, Department of Clinical Laboratories Sciences, College of Pharmacy, University of Misan, Misan city, South of Iraq over the period from May to August in 2018. This study was approved by Ethical Committees of College of Pharmacy of Misan University with written consent withdrawn from all the patients.

Sample collection
One hundred and fifty midstream urine samples were obtained from 150 diabetic adult outpatients between 20-64 years old attending to the Microbiological Examinations Unit at Al-Sadar Teaching Hospital in Misan city with written consent. The urine samples were collected by sterilized disposable containers (BIOZEK, Netherlands) after necessary precautions 14 .
Samples were brought to the laboratory within one hour. Questionnaires had completed covering the information pertaining to sex, age, period of diabetes and antibiotics used.

Urine culture
A 0.1 ml of urine sample was inoculated separately onto Blood agar (LAB, UK) and MacConkey agar (LAB, UK) plates, and incubated aerobically at 37°C for 24 h. The grown colonies were calculated colony forming unit (CFU) per ml. The plates containing more than 10 CFU/ml were selected as a significant growth, then gram stain for initial identification 15 . All grown colonies from primary cultures were subcultured onto Nutrient agar (LAB, UK) plates for the following confirm assays.
The confirm diagnosis for bacterial isolates was accomplished by analytical profile index (API) system (API 20E, API 20 STREP and API STAPH) add to motility, catalase, coagulase and oxidase tests described by Cheesbrough 12 .

Statistical analysis
Statistical analysis was achieved with the Chi-square test by Statistical Package for Social Sciences (SPSS) version 18. P-value less than 0.05 was considered as statistically significant and P-value less than 0.01 considered as highly significant.

Prevalence of UTIs in diabetic patients according to some factors
A total of 150 urine samples enrolled in this study, 84(56%) were of females and 66(44%) were of males. Out of 106 positive cultures, the prevalence of UTIs was high in females 69 (65%) than males with a significant difference at P≤0.01. Furthermore, UTIs were more incidence in patients without antibiotics use and period of diabetes 5-10 years (82% and 64%, respectively), while it was 56.6% in age group 35-49 years without signification, as in Table 3.
A total of 121 microbial isolates were obtained, Escherichia coli was the most common causative agent of UTIs with 64 isolates, followed by Klebsiella pneumonia, and Enterococcus faecalis (11 and 8, respectively

DISCUSSION
The present study showed that axenic culture was 83% versus to mixed growth, this result in agreement with Abd Al Abbas and Jasim 17 founding 82% of samples gave axenic growth. Axenic refer to pure colonies from the primary culture, as in Table 1. Furthermore, this study showed that a large proportion of uropathogens were gram-negative comparing to gram-positive isolates as in Table 2. This result is consistent with previous studies 10,18,19 . Table (3) shows the prevalence of UTI according to some factors. The results revealed a high prevalence of UTI in females (65%), in agreement with other previously reported studies [9][10][11]19 . Females are more prone to have UTI than males because the urethra is shorter and closer to the anus adding to a sexual intercourse 20 . Nevertheless, this study showed that the occurrence of UTI in diabetes was more frequently in the age group (35-49 years), followed by the age group(50-64 years) in agreement with study done by Adeyeba et al. 21 . Generally, engagement in sexual activity and increasing age of diabetics make them vulnerable to urinary tract infection 22 . Moreover, UTI occurrence was more frequent in patients without antibiotics use and diabetic period (5-10 years). Indiscriminate and uncontrolled use of antibiotics for long period with chronic glucosuria encouraging the microbial growth and thereby increase the risk of UTIs 23,24 .
The present study showed that E. coli was the most common bacteria with 64 isolates (52.9%), followed by Klebsiella spp. 19(15.7%) isolates then E. faecalis 8(6.6%) isolates and S. agalactiae 5(4.1%) isolates. Several studies reported that E. coli and Klebsiella spp. were the most frequent uropathogens in diabetic patients, while the other species were different 10,18,19,25 . Relatively, UTIs occur when gastro-intestinal bacteria can be entering to the urethra and then colonization, once these bacteria gain access to the urinary bladder they may multiply and transmitting up through ureters to kidneys 14 . E. coli is normally habitat in the intestinal tract, it is most predominant bacterium causing UTI in both diabetic and non-diabetic patients 11,25,26 . E. coli have numerous virulence factors which enable to colonize the urethra such as P-fimbria to bind the epithelium, other factors contribute in its pathogenicity like α and β-hemolysins, colicins and cytotoxic necrotizing factor 27 . Klebsiella spp. are opportunistic pathogens and they are the etiological agents for urinary tract infection in both community-acquired and hospital-acquired infections. Capsule production, siderophore activity and biofilm formation are an important virulence factors in pathogenesis of Klebsiella 28 . Enterococcus faecalis is also opportunistic bacterium and it's the causative agent for UTI in the general population 26 . This bacterium has many virulence factors playing an important role in its pathogenicity such as aggregation substance, Enterococal surface protein and hemolysins 29 . On the other hand, S. agalactiae has been isolated from patient with poorly glucosuria 10 . Although, the bacterium form a part of urogenital flora, but became opportunistic, particularly in patient with poor immunity.
Table (4) showed that the uropathogenic bacteria were highly sensitive to imipenem (78.8%), followed by Amikacin (61.9%). These results are consistent with other previous studies 18,19,25 . In contrast, all bacterial species appeared a complete resistance to ampiclox (100%) with the exception of S. agalactiae (60%). This is due to the acquisition of mec A gene by plasmid transferring 30,31 . On the other hand, many bacterial species appeared resistant to three or more antibiotics which may be due to plasmid carrying drug resistant genes, a biofilm formation inside the urinary bladder increases the chance of multi-drug resistance bacteria to colonize the urinary tract and thus to infection 32,33 . Furthermore, MDR efflux -system contributes to the emergence of multi-drug resistance strains, especially in gram -negative bacteria 34,35 . Additionally, the exposure of bacterial pathogens to high concentration of antibiotics for long times creates severe selection pressure leading to emergence of resistance 36 .