Study design, setting, and population
A cross-sectional study was conducted among gynecological cases admitted from September 16 to December 30, 2021 at Jimma University Medical Center, South West Ethiopia. The study area is located 348 km away from the capital city, Addis Ababa. The annual average admission of the centre is over 20,000 patients. The service delivery sites are the outpatient department, gynecology ward, maternity and labor ward, and operation rooms. All gynecological cases admitted to the gynecology ward during the study period were included in the study population, and those treated with antibiotics within 15 days of the study’s start date were excluded.
Sample size and sampling techniques
The sample size was calculated using a single population proportion formula \(n = \frac{{\left( {Z^{\frac{a}{2}} } \right)P\left( {1 – P} \right)}}{{d^{2} }}\), where n = number of sample size, Z is the statistics corresponding to a 95% level of confidence (1.96), d = margin of error, and P = is the assumed prevalence of uropathogenic bacteria among gynecologic cases (50%). The infection prevalence was assumed to be 50% because the current status of uropathogenic bacterial infection in the area is unknown. Therefore, the sample size was adjusted to 384 gynecological cases. A sequential sampling method was used to find study participants.
Data collection methods
Information on demographic variables was collected from each patients with gynecolological cases by a face-to-face interview using a structured questionnaire. Clinical data was gathered through a review of patients’ medical records and consultation with a gynecologist.
Specimen collection
After obtaining informed consent or assent from study subjects and/or parental/guardian, about 5–10 ml of urine was collected from patients by following aseptic technique and clean catch mid-stream urine (MSU) using a sterile screw-capped, wide-necked container. For those patients on a catheter, the sample was collected by aseptic techniques: cleansing the catheter port with alcohol and allowing drying time, and then aspirating the urine from the indwelling catheter with a sterile syringe. The container was labeled with the date, the name, and a code number. After collection, the specimen was immediately delivered to the microbiology laboratory for laboratory investigation. The specimens were processed within 2 h of collection. In the event of a delay in processing, the urine specimens were refrigerated at 4 °C until they were processed.
Culture and identification techniques
The collected urine sample was inoculated onto 5% Blood Agar, Mannitol Salt agar and MacConkey agar plates (Oxoid Ltd., Bashingstore Hampaire, UK) by streak plate methods following the standard microbiological techniques and procedures15. After incubating the plates aerobically at 37 °C for 24 h, they were inspected for the presence or absence of bacterial growth. If colonies were found, they were counted and multiplied by the reciprocal of the loop’s volume, or 1000. Counting colonies yielding bacterial growth of ≥ 105 cfu/ml with pure growth were considered as significant bacteriuria, and negative cultures contained no growth or mixed urogenital flora (more than two isolates). For cultures containing two types of colonies, sub-culture for further identification and antimicrobial susceptibility testing. For catheter collected urine samples, the presence of symptoms or signs compatible with UTI with no other identified source of infection along with ≥ 103 colony-forming units/ml and the presence of ≥ 105 cfu/ml in a single catheter urine specimen in a patient without symptoms compatible with UTI is recommended as representing significant bacteriuria16,17.
All positive urine cultures showing significant bacteriuria were further identified by their physical characteristics such as colony morphology, odor, swarming, and presence of hemolysis on their respective media, Gram-reaction, and pattern of biochemical reactions using the standard procedures15. The gram-negative rods were identified with the help of a series of biochemical tests, namely Citrate, Oxidase, Sulphur Indole Motility (SIM) media, Kligler’s Iron Agar (KIA), lysine decarboxylase, lactose fermentation, urea hydrolysis18. Gram-positive cocci were identified based on their Gram reaction, mannitol fermentation, catalase and coagulase tests15.
Antimicrobial susceptibility testing (AST)
The Kirby–Bauer disc diffusion method was employed for antibiotic susceptibility testing as recommended by Clinical Laboratory Standards Institute (CLSI) 2020 guidelines19. When a pure culture with significant bacteriuria was obtained, a loopful of bacteria was taken from a colony and transferred into a tube containing 5 ml of sterile normal saline (0.85% NaCl) and mixed gently until it formed a homogenous suspension. The turbidity of the suspension was adjusted to an optical density equivalent to 0.5 McFarland standards. The inoculated plates were left at room temperature to dry for 3–5 min while the Petridish lids were in place.
The following antimicrobial discs with their respective concentration were used for susceptibility testing and all the antimicrobials used for the study were obtained from Oxoid Ltd in the following concentrations: ciprofloxacin (CIP, 5 µg), trimethoprim-sulfamethoxazole (SXT, 1.25/23.75 µg), nalidixic acid (NA, 30 μg), meropenem (MEM, 10 μg ), cefotaxime (CTX, 30 µg), ceftazidime (CAZ, 30 µg), tetracycline (TTC, 30 µg) nitrofurantoin (F, 30 µg), norfloxacin (NOR, 10 µg), ceftriaxone (CRO, 30 µg), amoxicillin-clavulanic acid (AMC, 20/10 µg), ampicillin (AMP, 10 µg), gentamicin (CN, 10 µg), erythromycin (E, 15 µg), penicillin (PEN, 10 µg), clindamycin (DA, 10 µg) and chloramphenicol (C, 30 µg). Among these, nalidixic acid (NA), meropenem (MEM, 30 μg), cefotaxime (CTX, 30 µg), ceftazidime (CAZ, 30 µg), tetracycline (TTC, 30 µg) can be used only for gram-negative bacteria, whereas erythromycin (E, 15 µg), penicillin (PEN, 10 µg) and clindamycin (DA, 10 µg) can be used only for gram-positive bacteria; however, the rest of the antibiotics were used for both isolates. The diameter of the zone of inhibition around each disc was measured to the nearest whole millimeter (mm) by using a ruler, and the isolate was classified as sensitive, intermediate, or resistant according to the standardized table supplied by CLSI, 202019.
Detection of extended spectrum β-lactamase (ESBL)
ESBL-producing Escherichia coli and Klebsiella spp. were first screened for ESBL production by the phenotypic method and then confirmed by the phenotypic confirmatory test as per CLSI guidelines 202019.
Phenotypic screening for ESBL production
The ESBL screening test was performed by the standard disk diffusion method using ceftazidime (30 µg), cefotaxime (30 µg), and ceftriaxone (30 µg) (Oxoid, UK). More than one antibiotic disc was used for screening to improve the sensitivity of ESBL detection19. The three antibiotic discs were placed on Muller–Hinton agar and incubated at 37 °C for 18–24 h. These breakpoints for suspicion of ESBL production were: ≤ 25 mm for ceftriaxone (30 g), ≤ 22 mm for ceftazidime (30 g), and ≤ 27 mm for cefotaxime (30 g)19.
Phenotypic confirmation of ESBL producers
Confirmation of suspected ESBL producers was done by using the double-disk synergy (DDS) method. On Muller Hinton agar, an Amoxicillin/clavulanic acid (20/10 g) disc was placed in the center of the plate, and ceftazidime (30 g) and cefotaxime (30 g) discs were placed 15 mm apart, center to center. The plate was incubated at 37 °C for 18–24 h. A ≥ 5 mm increase in the diameter of the zone of inhibition for either of the cephalosporin-clavulanate disk combinations versus the zone diameter of the respective cephalosporin disk was considered positive, and the isolate was interpreted as an ESBL producer as recommended by CLSI guidelines 202019.
Data processing and analysis
Data were entered using EpiData version 3.1 and exported to SPSS version 25 software. Descriptive statistics were used to summarize socio-demographic data, bacterial profile and susceptibility patterns of isolates. Bivariate logistic regression was employed to look for associations between the outcome variable and each independent variable; and those variables significant at a P value of less than 0.25 in the bivariate regression were then selected for the multivariate analysis model. The corresponding variables with a P value ≤ 0.05 at a 95% confidence interval were then considered statistically significant.
Quality assurance
The legibility of the filled questionnaire and any labeling errors were confirmed immediately. Laboratory analyses were carried out using standard operating procedures. Culture media were tested for sterility and performance by incubating 5% of the batch at 35–37 °C overnight and observing the media for microbial growth. Those media which showed growth was discarded and replaced by a new sterile batch. Standard reference strains of Staphylococcus aureus (ATCC25923), E. coli (ATCC25922) and Pseudomonas aeruginosa (ATCC27853) were used during culture and antimicrobial susceptibility testing. Escherichia coli ATCC 25922 was used as an ESBL-negative whereas K. pneumoniae ATCC 700603 was used as an ESBL-positive reference strain19.
Ethical consideration
The study protocol was evaluated and approved by the Institutional Review Board of Jimma University (Ref.: IRB000144/2020), and ethical clearance was obtained. The support letter was obtained from Jimma University School of Medical Laboratory Science and then submitted to JMC, gynecology ward and all methods were performed in accordance with relevant guidelines and regulations. After adequately explaining the objectives and purpose of the study, written informed consent was obtained from all the study subjects and/ or assent were obtained from study subject less than 18 years and/or guardians before data and sample collection. All data obtained in the course of the study was kept confidential. Positive cases were referred to the attending clinician as soon as possible for their better management.
Ethics approval and consent to participate
Ethical permission for this study was approved by Jimma University, Research and Ethics Committee of the School of Medical Laboratory. All participants were voluntary and each supplied informed consent.