Prevalence and Antimicrobial Susceptibilities of Bacteria Isolated from Circumcised and Non-Circumcised Women with Urinary Tract Infections in Different Gynecological Clinics in Khartoum Locality, Sudan

Urinary tract infections (UTIs) are one of the diseases that are widely spreading among women. A number of factors contribute to UTIs, including circumcision, which narrows the opening of the urinary system. This cross-sectional study was conducted from April 2019 to February 2021 to detect the frequency of antimicrobial-resistant bacteria isolated from circumcised women attending two Clinics, for Gyncology in Khartoum locality. Conventional methods were used for isolation, identification, and antimicrobial susceptibility testing. A total of 80 midstream urine samples (n = 80) were collected from all female eligible volunteers, of which 40 had been circumcised and 40 had not. The study investigated 80 females aged 7-70 years, with a mean of 29.3 + 13.1 SD. There were 16/40 (40%) circumcised women who were married and 23/40 (60%) single, whereas for non-circumcised women there were 7/40 (17.5%) married and 33/40 (82.5%) single. Among the circumcised patients, 34/40 (85%) had growths compared to 6/40 (15%) of the non-circumcised participants, and UTIs were significantly associated with circumcision (P=0.001). Circumcised females had a 32 times higher odd ratio (O.R) of UTIs than non-circumcised females. Escherichia coli was the most predominant isolate among circumcised and non-circumcised women (15(37.5%)). The isolated bacteria in circumcised women were moderately sensitive to Augmentin 22/34 (67.7%) and Gentamycin 20/34 (58.8%) compared to other antimicrobial agents; Ciprofloxacin 16/34(47.1%), Cefuroxime 12/34(35.3) and Amoxycillin 10/34 (29.4%) while all Gram negative rods were highly resistant to Nalidixic acid (100.0%). In contrast to non-circumised women; all isolated bacteria were highly sensitive to Gentamicin 6/6 (100.0%) and Cefuroxime 5/6(83.3%), and moderate sensitive to Augmentin 4/6 (66.7%) and Ciprofloxacin 4/6(66.7%). Also all isolated were highly resistant to Nalidixic acid (100.0%) and Amoxycillin 1/6 (16.7%). UTIs and antimicrobial-resistant bacteria were more prevalent among circumcised women than non-circumcised women. E.coli was the most prevalent bacteria among circumcised and non-circumcised women.

Urinary tract infections (UTIs) can affect any part of the urinary system, and women are more likely than men to develop UTIs 1 . Generally, UTIs occur when bacteria enter the urinary tract through the urethra and begin to multiply in the bladder. Despite the urinary system's designed defenses, these can sometimes fail. When that happens, bacteria may take hold and grow into a full-blown infection in the urinary tract. Women experience more than one infection during their lifetimes 2 . The anatomy of the female urethra is of particular importance to the pathogenesis of UTIs. The female urethra is relatively short compared to the male urethra, and it is situated close to the warm, moist, per rectal region, which is highly populated by microorganisms 1 .
Other risk factors include: sexual activity, certain type of birth control and menopause (after menopause a decline in circulating estrogen causes change in the urinary tract that make women more vulnerable to infection) 2 , urinary tract abnormalities (babies born with urinary tract abnormalities), blocking in the urinary tract; as kidney stone, catheterize (people who can't urinate on their own and use a tube to urinate), urinary procedure (which can be recent urinary surgery or an exam of the urinary tract that involves medical instrument) 3 . Female genital mutilation (FGM) also known as female genital cutting or female circumcision that don't allow the urine to leave completely or cause urine to back up in the urethra have an increased risk of UTIs and menstrual problem 4 . At least 200 million girls and women alive today have undergone FGM 7 living in 30 countries in Africa, the Middle East and Asia where FGM is practiced 8 . The most serious type of FGM is type three that create covering seal and small opening is left for urine and menstrual blood to escape 4 . FGM is thought to be a tribal tradition or an Islamic imperative, and several studies have reported the reasons that a girl might undergo FGM include providing her with an honorable social life, preserving her virginity, and allowing her to become a mature woman for a safe marriage 5,6 The overall prevalence of urological complications in women with genital mutilation is 20%. Recurrent urinary tract infections, lower urinary tract symptoms, urinary retention, urogenital fistulas, meatus stenosis, urethral stones, and megaurethra are the reported ones 9 . All those risk factors can lead us to recurrent infection and may result in antibiotic resistant bacteria. Also geographic variation in etiologic agents of UTIs and their resistance patterns in antibiotics 10,11 UTIs is the second most common infection presenting in community practice which associated with elevated antibiotic resistance in Sudan 12 . Female genital mutilation may be increasing UTIs 5 .
To our knowledge, there are few studies concerning the consequences of circumcision on the urinary system and its risk for increasing antibiotic-resistant bacteria associated with UTIs. So, the ultimate goal of this study is to provide information about this problem which may highlight circumcision and its association with antibiotic-resistant bacteria causing UTIs.

Methods
This cross-sectional, case-control, hospital-based study was conducted in two clinics in Khartoum locality. The study was carried out between July 2019 and March 2021. The study included 80 Sudanese women (n=80) with genital mutilation (n=40 case groups) and 40 non-genital mutilated women (n=40 control groups); all of them had UTI symptoms including (pyuria, loin pain, burning micturition, frequency, urinary retention and urgency of urination); and with different ages.

ethical considerations
Ethical approval No. (MLS-RB-07-3-18) to conduct this study was obtained from the Scientific Research Committee, Collage of Medical Laboratory Science, Sudan University of Science and Technology and Dan and Nobatia Clinics in Khartoum Locality. Informed consent was obtained from participants before collection of the urine samples.

data collection
Data were collected using direct interview the patients, which provided information conceding each case examined.

Laboratory processing specimen collection and processing
The participants collected mid-stream urine specimens in a sterile wide-mouth container after they were informed of the collection procedure, and all specimens were processed within one hour of collection.

Isolation
Aseptically, and with a 0.005 calibrated loop, a loop full of well-mixed urine was incubated aerobically at 37°C overnight on blood and Cysteine Lactose Electrolyte Deficient (CLED) agar plates.

Identification of the isolated bacteria
Identification of the isolated bacteria was done by colonial morphology after assessment of significant bacteriuria (>10 5 CFU/1ml of urine), Gram's stain and conventional biochemical tests.

Biochemical tests for identification of Grams negative rods
Grams By using a sterile wire loop, 3-5 wellisolated, purified and fresh colonies of similar appearance of the tested organism were touched and emulsified in 3-4 ml of sterile physiological saline. The turbidity of the suspension was matched to the standard turbidity (0.5% McFarland standard) on good light. Sterile swab was used to inoculate a plate of Mueller Hinton agar. Excess fluid was removed by pressing and rotating the swab against the side of the tube above the level of the suspension. Streaked the swab evenly over the surface of the medium in three directions, rotated the plate approximately 60 to ensure even distribution. With the petri dish lid in place, allowed about 3-5minutes (no longer than 15 minutes) for the surface of the agar to dry. By using a sterile forceps, the appropriate antimicrobial discs was placed, evenly distributed on the inoculated plate. After overnight incubation, the plates was examined to ensure the growth was confluent and by a ruler the diameter of each zone of inhibition was measured in mm. The zone sizes of each antimicrobial disc was interpreted by interpretative chart, according to CLSI guidelines 16 .

statistical analysis
The obtained data from this study was analyzed using statistical package of the social science (SPSS) version 20.0. Frequencies were expressed in form of table and Chi-Square test was used to determine the significant differences at p-value d"0.05.

ResuLts
A total of 80 urine specimens had been collected as follow: 40 were circumcised and other 40 were not, and their ages ranged from 7 to 70 years old with a mean age of 28.8± 13.8 S.D. About 16/40 (40%) of circumcised women were married and 23/40 (57%) were single, while among non-circumcised group there were 7/40 (17%) married and 33/40 (82%) single as in table 1. The growth rate was 34/40 (85%) among circumcised females and 6/40 (15%) among non-circumcised participants and there was high statistical difference between circumcision and UTIs (P.value=0.001) and circumcised women were 32 times exposed to risk of UTIs than non-circumcised one as showed in table (2). The isolated bacteria were 24/34 (70.6%) G-ve rods and 10/34 (29.4%) G+ve cocci among circumcised women, while all isolated bacteria were G-ve rods 6/6 (100%) and no isolated G+ve cocci among non-circumcised one as displayed in table (3).
Among the isolated bacteria; Escherichia coli was the most predominant isolate among circumcised and non-circumcised women. Circumcised women were 5, 8, 2, and 3 times more susceptible to urinary tract infection with Escherichia coli, K.pneumoniae, Portus mirabilis, and Pseudomonas aeruginosa, respectively than non-circumcised ones. There was a high association between the isolated organism and circumcised women (P. value=0.001) as illustrated in table (4). The all isolated bacteria from circumcised women were highly sensitive to Gentamycin (7/12 (58.0%)) compared to other antimicrobial agents and in non-circumcised was 100.0% for Gentamycin and Augmentin. Also all isolated G-ve rods in both groups were totally resistant to Nalidixic acid as displayed in table (5) and (6).

dIscussIon
In this study, the growth rate was 34 (85%) among circumcised women rather than non-circumcised 6 (15%). This result almost confirms the theory of Elduma when he mentioned UTIs as one of the circumcision complications in Sudan (2018) 5 . In the current study, the circumcised women were more likely to have and develop UTIs (85%) than the non-circumcised group (15%). This result was similar to that obtained by Amin et al. (2013) 17 in Egypt which reported highly significant different types of UTIs by 86.6% of the study group (251 circumcised women participate in the study).
Gram negative rods isolated more frequently than G+ve cocci in this study, 24/34 (70.6%) and 10/34 (29.4%), respectively in circumcised women, Also in non-circumcised women Gram negative rods also grew more frequently than G+ve cocci in 6/40 (15%) of the cases, but there were no isolated G+ve cocci among circumcised women. This was harmonized to that reported in Sudan by Saeed who found 45(65%) Gram negative bacteria more than Gram positive 24(35%) from 69 of significant growth urine sample 12 .
The present study found the commonest frequent was E. coli 12(35.3%), (followed by En. faecalis 8(23.5%) and K. pneumoniae 7(20.6%) among circumcised women, Moreover, while E. coli 3(50%) was the most prevalent among noncircumcised women. It was close to the study carried out by Theodore 18 in Nigeria who found 141 out of 181 urine specimens and E.coli was 47 (33.3%) followed by Klebsiella species 28 (19.9%) and 5(3%) En. faecalis. Also among noncircumcised females E.coli was more predominant than others.
The isolated bacteria from circumcised women were highly resistant to a number of the antimicrobial agents used, including: Nalidixic acid (100%), Amoxycillin (70.5%), and cefuroxime (64.7%). This was in agreement with a study conducted by Ahmed and colleagues 19 in Sudan, in which E.coli showed high resistance to amoxicillin, but low resistance to nalidixic acid. In non-circumcised women, (83%) were resistant to Nalidixic acid and Amoxycillin, whereas (0%) were resistant to cefuroxime.