Authors: Meseret Geremew, Destaw Kebede, Abebe Fenta, Gebreselassie Demeke, Milkiyas Toru
Categories: Research, Antimicrobial susceptibility, Asymptomatic bacteriuria, Risk factors
Source: BMC Microbiology
Authors: Meseret Geremew, Destaw Kebede, Abebe Fenta, Gebreselassie Demeke, Milkiyas Toru
In pregnant women, asymptomatic bacteriuria is a public health problem. Untreated asymptomatic bacteriuria is the primary cause of cystitis and pyelonephritis, which increase prenatal mortality and morbidity. Limited research has been conducted on asymptomatic bacteriuria in our study setting. Thus, this study aimed to assess antimicrobial susceptibility and risk factors of asymptomatic bacteriuria among pregnant women who attended antenatal care at public health facilities in Debre Markos town, Northwest Ethiopia.
Using a consecutive convenience sampling technique, an institutionally based cross-sectional study was carried out in Debre Markos town's health facilities between May 1, 2023, and July 30, 2023. Pretested structured questionnaires were used to collect clinical and sociodemographic data. Additionally, a sterile container was used to collect the midstream urine sample, which was then inoculated onto sheep blood agar, MacConkey agar (MAC), and cysteine-lactose-electrolyte-deficient agar (CLED). Gram stain, biochemical tests, and colony morphology were used to identify and characterize the isolates. On Mueller–Hinton agar (MHA), the disk diffusion method was used to determine the antimicrobial susceptibility pattern. EpiData 3.0 was used to enter the data. Finally, bivariable and multivariable logistic regression was performed via SPSS version 23 to identify related factors, with a P value < 0.05 being deemed statistically significant.
The overall prevalence of asymptomatic significant bacteriuria (ASB) among study participants was 30/185 (16.2%; 95% CI 10.8–21.6). Escherichia coli (E. coli) 13/30 (43.3%) is the predominant isolates, and followed by Staphylococcus aureus (S. aureus) accounting for 10/30 (33.3%), Coagulase-negative Staphylococcus (CoNS) 6/30 (20.0%), and Pseudomonas aeruginosa (P. aeruginosa) 2/30 (3.3%) decreasingly. Six (100%) CoNS isolates were susceptible to norfloxacin, trimethoprim-sulfamethoxazole, tetracycline, and nitrofurantoin. About 11/13 (84.6%) of E. coli was susceptible to ceftriaxone, imipenem, gentamicin, and norfloxacin. In contrast, E. coli was resistant to cefuroxime 6/13 (54.5%) and amoxicillin-clavulanic acid 9/13 (69.2%). Despite this, only a few number of P. aeruginosa strains were isolated, and were 100% susceptible to amoxicillin-clavulanic acid, ciprofloxacin, meropenem, and norfloxacin. Asymptomatic bacteriuria was significantly associated with a history of UTI (AOR = 7.281; 95% CI: 2.53–20.96; P = 0.001), catheterization (AOR = 6.985; 95% CI: 1.61–28.91; P = 0.007), diabetes mellitus (AOR = 5.009; 95% CI: 1.55–16.19; P = 0.007), and vaginal douching (AOR = 3.125; 95% CI: 1.10–8.92; P = 0.033) by considering P < 0.05 as significant association.
ASB prevalence was 16.2%; E. coli was the predominant isolate; the MDR rate was 40%. Diabetes, vaginal douching, catheterization, and prior UTI were associated with ASB. Routine urine culture should be considered for targeted screening and culture-guided therapy for high-risk groups. Further studies are needed to evaluate prophylaxis and treatment outcomes.
Asymptomatic significant bacteriuria (ASB) during pregnancy is defined as the presence of bacteria in the urine with a quantitative count of 10^5^ colony-forming units/ml without signs or symptoms of urinary tract infection [1]. One of the most prevalent issues that pregnant women face is urinary tract infections (UTIs), which are caused by bacteria and can result in serious consequences due to the growth and presence of microorganisms in the urinary tract [2]. For mechanical reasons, hormonal changes, decreased immunity, urinary stasis, and the reflux of urine from the bladder into the ureters, pregnant women are more likely to experience asymptomatic bacteriuria [3]. As a result, 10–20% of women will experience a UTI, which increases the risk of maternal and fetal morbidity [4]. About 21.2% of cases at Adigrat Hospital in Ethiopia are asymptomatic bacteriuria, making it one of the primary risk factors for urinary tract infections during pregnancy [5]. In various patient populations, the prevalence of asymptomatic UTIs ranges from 2 to 15% [6].
Numerous factors, including maternal age, location, marital status, educational attainment, monthly family income, and occupation, can be linked to ASB. Additionally, medical and obstetric factors such as anemia, HIV status, prior history of UTI, history of catheterization, parity, and gestational age are linked to asymptomatic UTIs in pregnant women [5, 7–13]. Preterm birth, low birth weight, preeclamptic toxemia, anemia, and other issues can result from asymptomatic urinary tract infections during pregnancy [14].
Ethiopia is among the countries where antimicrobial resistance in bacteriuria is increasing. Certain bacteria are pathogenic and can develop resistance to antibiotics [15]. Improper antibiotic utilization during pregnancy also can cause antimicrobial resistance [15–17]. However, the rates of antibiotic resistance vary by region and are empirically correlated with both antimicrobial usage and abuse [18]. Such empirical treatment causes bacterial resistance to routinely prescribed antibiotics through a variety of processes, such as the formation of the extended-spectrum β-lactamase enzyme, which hydrolyzes the β-lactam ring of antimicrobials [18, 19].
In Ethiopia, research efforts have focused on examining the bacterial profile and antimicrobial susceptibility patterns of asymptomatic UTIs in pregnant women across various regions and hospitals [5, 20–28]. If untreated, asymptomatic bacteriuria in pregnant womensignificantly increases the risks for serious maternal and fetal complications, including kidney infections (pyelonephritis), premature birth, low birth weight, preeclampsia, anemia, and even fetal death [29]. However, there is a significant data gap regarding asymptomatic UTIs and infections caused by extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing uropathogens, particularly in our study area. Thus, this study aimed to assess antimicrobial susceptibility and risk factors of asymptomatic bacteriuria among pregnant women who attended antenatal care at public health facilities in Debre Markos town, Northwest Ethiopia.
The public health facilities in Debre Markos Town, Northwestern Ethiopia, served as the study site. Debre Markos town is located 265 km from Bahir Dar, the capital of the Amhara regional state, and 299 km from Addis Ababa, the capital of Ethiopia. According to the 2022 population projection in the Amhara National Regional State Health Bureau and the Central Statistical Agency of Ethiopia (CSA), approximately 71,079 women live among 140,699 people in this town [30]. There are four health centers and one government hospital in Debre Markos town. Therefore, from May 1, 2023, to July 30, 2023, a cross-sectional study was carried out at five health facilities, namely, Debre Markos Comprehensive Specialized Hospital (DMCSH), Debre Markos Health Center (DMHC), Hidassie Health Center (HHC), Gozamen (GHC), and Wuseta Health Center (WHC).
The source population was all pregnant women who went to public health facilities in Debre Markos town for antenatal care.
The study included all pregnant women with asymptomatic UTIs who got ANC services at Debre Markos town's public health facilities during the study period. However, this study excluded pregnant women who were extremely unwell, had a history of antibiotic therapy during the previous two weeks, or had signs of a UTI.
Using the single population proportion calculation, the sample size was determined by taking the 14% prevalence from a previous study in Ethiopia [31], assuming a 95% confidence level (Z = Zα/2 = 1.96), and a margin of error (d = 5% = 0.05). The sample size was then calculated as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ N=\frac{{\left(\frac{za}{2}\right)}^{2}*p\left(1-P\right)}{{d}^{2}}=\frac{{\left(1.96\right)}^{2}\times 0.14(1-0.14)}{(0.05{)}^{2}}=185
Thus, the final 185 minimum sample size was proportionally allocated for the five health facilities on the basis of the number of pregnant women attending such facilities in the previous year. We reviewed the number of last year's attendees from May to July pregnant women who attended Debre Markos Comprehensive Specialized Hospital (DMCSH) (415), Debre Markos Health Center (312), Hidassie Health Center (208), Gozamen (267), and Wuseta Health Center (158), out of the total 1360 ANC attendees. Accordingly, the number of study participants enrolled at DMCSH was (415 ÷ 1360) × 185 = 57, Debre Markos Health Center (312 ÷ 1360) × 185 = 43, Hidassie Health Center (208 ÷ 1360) × 185 = 28, Gozamen Health Center (267 ÷ 1360) × 185 = 36, and Wuseta Health Center (158 ÷ 1360) × 185 = 21. Therefore, participants were enrolled consecutively using convenience sampling until the sample size was reached. ### Operational definition Asymptomatic the presence of significant bacteriuria (≥10^5^ cfu/ml) in urine culture without urinary tract infection symptoms or indicatorsHistory of UTI: any history of urinary tract infection that has been identified by a medical professionalMultidrug bacteria that exhibit concurrent resistance to two or more antimicrobials from distinct chemical groupsBacteriuria: bacterial infections in the urinary system that result in the presence of bacteria in the urineFrequent vaginal douching once per week or more that disrupts normal vaginal flora ### Data collection and processing The lead investigator and a trained BSc midwife collected the data. Therefore, a structured, pretested Amharic version of the questionnaire was used to collect sociodemographic data through in-person interviews with study participants or their parents immediately after consent or assent was obtained. Additionally, the questionnaire included demographic information (age, sex, etc.) and other crucial clinical data (patient history), and related data were collected by data collectors. Before performing an interview, study participants were adequately informed about the purpose of the research at each data collection location. ### Urine specimen collection and transportation Upon sociodemographic data collection, all study participants were oriented and instructed to collect clean-catch midstream urine after perineal cleaning by using a sterile, screw-capped, wide-mouth container with a secure lid and leak-resistant containers. Proper cleaning instructions were followed to prevent possible false positives. The collected urine samples were immediately brought to the DMCSH Microbiology laboratory for bacteriological analysis within two hours after collection. Urine samples were stored in the refrigerator for culture by placing a tightly sealed container in the fridge at about 2–8 °C for up to 24 h to slow bacterial growth; if we can't refrigerate, get it to the laboratory within 1–2 h, as room temperature allows bacteria to multiply quickly, affecting results. ### Bacterial isolation The collected urine samples were inoculated into Cystine-Lysine-Electrolyte-Deficient medium (CLED) (Oxoid, England) via a calibrated wire loop (0.001 mL). After incubation at 37 °C for 24 h, colonies were counted to assess significant growth. Colony counts yielding bacterial growth of > 10^5^ CFU/mL were regarded as significant bacteriuria (SBU). Colonies of SBU from CLED were also subcultured into MacConkey (MAC) (Oxoid, Ltd., England) and blood agar (BA) (Oxoid, Ltd., England) and incubated at 37 °C for 24–48 h. In accordance with the Clinical and Laboratory Standard Institute (CLSI) M100:2022 standard guidelines [32], bacteria were identified on the basis of colony characteristics, Gram reactions, and a panel of biochemical tests. The gram-negative bacteria were identified by indole and H₂S production in Kligler's iron agar (KIA), citrate utilization, urease tests, motility tests, lysine decarboxylase, and oxidase tests (Oxoid). A catalase test was performed to differentiate *Staphylococcus* from *Streptococcus,* and a coagulase test was used to differentiate *Staphylococcus aureus* from other coagulase-negative *Staphylococci* (CoNS) [33]. A repeat test was done for mixed growths (multiple organisms) by collecting a new sample using correct collection technique to minimize contamination and confirm a true infection or if the sample was contaminated. Plating in duplicates/triplicates like serial dilutions was used for estimating bacterial concentration expressed as colony-forming units per milliliter, accordingly. ### Antibiotic susceptibility testing The antimicrobial susceptibilities of the bacterial isolates were determined according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI,M100, 2022) guidelines [32], which used the Kirby–Bauer disc diffusion method on Mueller–Hinton Agar (MHA) (Oxoid, Ltd., England) for the following antimicrobial Meropenem (MEM, 10 µg), Ceftazidime (CAZ, 30 µg), penicillin (PEN, 10 µg), nitrofurantoin (F, 30 µg), gentamycin (GEN, 10 µg), ampicillin (AMP, 5 µ), amoxicillin-clauvlanic acid (AMC, 30 µg), ciprofloxacin (CIP, 5 µg), trimethoprim-sulphamethaxzole (SXT, 25 µg), Norfloxacin (NOR, 10 µg), Ceftriaxone (CRO, 30 µg), cefotaxime (CTX, 30 µg), imipenem (IMP, 30 µg), cefpodoxime (CPD, 30 µg), cefuroxime (CXM, 30 µg) and nalidixic acid (NA, 30 μg) for gram-negative bacteria, whereas nitrofurantoin (F, 30 µg), gentamicin (GEN, 10 µg), ciprofloxacin(CIP,), tetracycline (TET, 30 µg), trimethoprim/sulfamethoxazole (SXT, 25 µg), norfloxacin (NOR, 10 µg), and penicillin (PEN, 10 µg) for gram positive isolates. A loop full of bacterial colonies was taken from a culture and transferred to a tube containing 5 ml of phosphate-buffered saline and mixed gently until it formed a homogenous suspension. The turbidity of the suspension was adjusted to the turbidity of the 0.5 McFarland standard in a tube, and the mixture was swabbed on Mueller–Hinton medium. After the antibiotic discs were subsequently placed on MHA and incubated at 37 °C for 16–18 h, the zones of inhibition were measured, and the results were interpreted as susceptible, intermediate, and resistant on the basis of the CLSI M100, 2022 guidelines [32]. ### Extended-spectrum beta-lactamase detection The widths of the zones of inhibition created by ceftriaxone (30 µg), ceftazidime (30 µg), cefotaxime (30 µg), or cefpodoxime (30 µg) on Mueller–Hinton media (Conda Ltd.) were used for the initial screening criteria for ESBL in accordance with the CLSI M100:2022. Ceftriaxone (< 25 mm), ceftazidime (30 µg) ≤ 22 mm, cefotaxime 27 mm, and cefpodoxime (10 µg) < 17 mm were the breakpoints that were suggestive of suspicion for ESBL formation. Following this, the combined disk test was used to confirm the phenotypic detection of ESBL generation in accordance with CLSI M100, 2022 standards [32]. Drug susceptibility testing was used to distribute the organism to be evaluated on a Mueller–Hinton agar plate. For phenotypic confirmation of the existence of ESBLs, ceftazidime (30 µg) disks and cefotaxime (30 µg) disks were employed both alone and in combination with clavulanic acid (30 µg/10 µg). A ≥ 5 mm increase in the zone of inhibition diameter for either of the cephalosporin-clavulanate disk combinations compared with the zone diameter of the corresponding cephalosporin disk was deemed positive following a 24-h incubation period at 37 °C, and the isolate was interpreted as an ESBL producer [32]. ### Detection of carbapenemase-producing bacteria One microliter loopful of Enterobacteriaceae or ten microliter loopfuls of *P. aeruginosa* from blood agar were emulsified in two milliliters of tryptosoya broth (TSB) as part of the modified carbapenem inactivation method (mCIm). To detect carbapenemase-producing bacteria, a meropenem disk was submerged in the suspension and incubated at 35 °C for at least 4 h, resulting in antibiotic inactivation by bacterial carbapenemase. *E. coli* ATCC 25922 serves as a susceptible indicator strain, revealing if a test isolate produces carbapenemase, breaks down the meropenem in the disk, leading to a smaller zone of inhibition (positive) or larger zone (negative) on an agar plate. The direct colony suspension method was used to create a 0.5 McFarland standard solution of *E. coli* ATCC 25922 in saline*. E. coli* ATCC 25922 was inoculated into a Mueller–Hinton Agar (MHA) plate via the standard disk diffusion method. After being removed from the trypto-soya broth (TSB), the meropenem disk was placed on an MHA plate that had already been inoculated with the indicator strain of *E. coli* ATCC 25922. The plates were incubated at 35 °C for 18 to 24 h. Then, a zone of inhibition larger than or equal to 19 mm was regarded as carbapenemase negative, whereas colonies inside a 16–18 mm zone or an inhibition zone width of 6–15 mm were regarded as positive results [32]. ### Quality control To check for word meaning discrepancies, the structured questionnaires were first written in English, translated into Amharic, and then translated back into English. One BSc midwife received training on how to gather clinical and sociodemographic data for one day, and approximately eight (5%) of the structured questionnaires were pretested at Lumame Primary Hospital. Data completeness was checked for its quality. Following standard operating procedures, all media were autoclaved aseptically in accordance with the manufacturer's instructions. A 5% batch of culture media was incubated for 24 h at 35–37 °C to ensure sterility. All materials, tools, and processes were suitably regulated according to the pre-analytical and post-analytical phases of quality assurance that are integrated into the microbiological laboratory's standard operating procedures (SOPs). Furthermore, reference strains of *S. aureus* (ATCC 25923), *P. aeruginosa* ATCC 27853e, and *E. coli* ATCC 25922 were used to evaluate the performance of the culture media. A quality control strain of *E. coli* (ATCC 25922), *P. aeruginosa* (ATCC 278530), or *S. aureus* (ATCC 25923) was inoculated as part of the quality control of the disc diffusion technique. After the discs were correctly positioned and incubated for 24 h at 37 °C, the zone of inhibition was measured and compared with the standard reference. Various quality control strains, including *S. aureus*–ATCC 33592, *Staphylococcus epidermidis* (*S. epidermidis)* ATCC 12228 or ATCC 14990, *E. coli* ATCC 25922, *Proteus mirabilis* (*P. mirabilis)* NCTC 10975, and *Klebsiella pneumonia* (*K. pneumonia)* ATCC 13883, were also used to verify the quality control of each biochemical test. Gram-staining techniques were examined using several quality control strains of *E. coli* and *S. aureus*. To evaluate the validity of the test process, quality control strains were also examined for growth, susceptibility pattern, Gram stain, and biochemical testing in parallel with urine samples. The expiration date of each reagent, supply, and antimicrobial disc was checked. ### Data analysis Version 23 of the statistical software for social sciences (SPSS) was used to analyze the data after they were entered into EpiData version 3.1. The prevalence of ASB and antibiotic susceptibility profiles were ascertained via descriptive statistics. To determine the factors linked to asymptomatic UTIs in pregnant women, as defined by a *p-*value < 0.05 indicating a statistically significant association, multivariable logistic regression was performed by including variables with p < 0.25 in the bivariable logistic regression. In our study, the p-value of the Hosmer–Lemeshow test was 0.78 (approaches 1), which is greater than 0.05 and showed a good fitness of the logistic regression model. Therefore, a good Hosmer–Lemeshow P-value did not preclude the existence of multicollinearity among variables. Since the variance inflation factors (VIFs) were 4 in this study, there is a moderate correlation but no need to take corrective measures. ### Ethical considerations The College of Medicine and Health Sciences Ethical Review Board at Debre Markos University provided ethics approval (Ref. No.: HSC/RCS/131/11/12). The Amhara Public Health Institute (APHI) provided a supportive letter, and the Debre Markos City Zonal Health Department provided a written permission letter to collect the data in health facilities. Written informed consent was obtained from participants and assent from parents/guardians of participants whose age was less than 18 years in accordance with the Declaration of Helsinki. Participation in the study was completely voluntary-based. Coding was used to protect participants’ privacy. Significant urine culture results were reported to the physician for further management. ## Results ### Sociodemographic characteristics of the study participants The study was conducted by recruiting 185 study participants. Approximately 103 out of 185 (55.7%) of the study participants fell within the age range of 25 to 34 years. The mean age of participants was 26.9 years, with a standard deviation of 4.9. The majority of the study participants, 145/185 (78.4%), 148/185 (80.0%), and 125/185 (67.6%), were urban residents, married, and housewives, respectively (Table 1).Table 1Sociodemographic characteristics of the study participants attending public health facilities in Debre Markos town from May 1, 2023, to July 30, 2023VariablesCategoriesFrequencyPercent (%)Age15–2425–34 > 35621032033.555.710.8ResidenceUrbanRural1454078.421.6Marital statusMarriedSingleDivorcedWidowed1481712880.09.26.54.3Education levelCannot read and writeCan read and writePrimary school (1–8)Secondary school (9–12)College/University361928495319.510.315.126.528.6OccupationHouse wifeMerchantStudentEmployee1253791467.620.04.97.6 ### Clinical and behavioral characteristics of the study participants The majority of the study participants, with clinical features, had never had a previous UTI history 151/185 (81.6%), had never undergone catheterization 172/185 (93.0%), and had diabetes mellitus 163/185 (88.1%). Similarly, 147/185 (79.5%) of the study participants said that they engage in frequent vaginal douching, 171/185 (92.4%) holding urine for long period, and 144/185 (7.8%) did not regularly urinate following sexual activity (Table 2).Table 2Clinical and behavioral characteristics of study participants attending at Debre Markos town public health facilities from May 01, 2023 to July 30, 2023VariablesCategoriesFrequencyPercent (%)Previous history of UTIYesNo3415118.481.6Catheterization historyYesNo131727.093.0Gestational periods1–3 month4–6 month7–9 month43776523.241.635.1Diabetes MellitusYesNo2216311.988.1History of chronic diseaseYesNo4314223.276.8Drinking 2 L of water per dayYesNo879847.053.0Holding urineYesNo141717.692.4Bladder emptying before bedYesNo1711492.47.6Vaginal douchingYesNo1473879.520.5Use of contraceptiveYesNo1642188.611.4Regular urination after sexual intercourseYesNo4114422.277.8GravidityPrimi gravidiaMulti gravidia929349.750.3 ### Prevalence of asymptomatic significant bacteriuria in health facilities The overall prevalence of asymptomatic significant bacteriuria (ASB) was 30/185 (16.2%) with a 95% Confidence interval (CI) that ranges from 10.8% to 21.6%. Among the total 30 isolates from 185 urine samples collected from health facilities, 11/30 (36.7%), 7/30 (23.3%), 5/30 (16.7%), 3/30 (10.0%), and 4/30 (13.3%) were culture-positive samples from DMCSH, DMHC, GHC, WHC, and HHC, respectively (Fig. 1).Fig. 1Prevalence of significant bacteriuria among asymptomatic pregnant women in selected public health facilities in Debre Markos town from May 01, 2023 to July 30, 2023 ### Bacterial profiles of asymptomatic bacteriuria among pregnant women Four distinct bacterial species were isolated from a midstream urine sample for this investigation. Among the thirty bacterial uropathogens that were identified, 16/30 (53.3%) were gram-positive, and 14/30 (46.6%) were gram-negative. The most predominant bacterial isolate was *E. coli* 13/30 (43.3%) from gram-negative bacteria, followed by *S. aureus* 10/30 (33.3%) and CoNS 6/30 (20.0%) from gram-positive bacteria (Fig. 2).Fig. 2Bacterial urophatogens isolated from urine culture from asymptomatic pregnant women attending Debre Markos town public health facilities from May 01, 2023 to July 30, 2023 ### Antimicrobial susceptibility pattern of gram-positive isolates In accordance with the antibiotic profile of the gram-positive isolates, the antibiotics ciprofloxacin 8/10(80%), gentamycin 8/10(80%), norfloxacin 9/10 (90%), trimethoprim-sulfamethoxazole 9/10 (90%), and nitrofurantoin 10/10 (100%) were susceptible to *S. aureus*. Conversely, *S. aureus* has been shown to be 10/10 (100%) penicillin-resistant. Furthermore, all Coagulase-negative Staphylococcus 6/6 (100%) were susceptible to norfloxacin, tetracycline, and trimethoprim-sulfamethoxazole. Even so, most of the 5/6 (83.3%) CoNS were penicillin-resistant (Table 3).Table 3Antimicrobial susceptible pattern of Gram-positive bacteria isolated from urine culture of asymptomatic pregnant women at Debre Markos town public health facilities from May 01, 2023 to July 30, 2023BacteriapatternFGENCIPTESXTNORPN(%)N(%)N(%)N(%)N(%)N(%)N(%)*S. aureus*S10(100)8(80)8 (80)8(80)9(90)9(90)0(0)R0(0)2(20)1(10)2(20)1(10)1(10)10(100)I0(0)0(0)1(10)0(0)0(0)0(0)0(0)*CoNS*S6(100)1(16.7)5(83.3)6(100)6(100)6(100)1(6.3)R0(0)5(83.3)1(16.7)0(0)0(0)0(0)5(93.7)I0(0)0(0)0 (0)0(0)0(0)0(0)0(0)*F* nitrofurantoin, *GEN* gentamicin, *CIP* ciprofloxacin, *TE* tetracycline, *SXT* trimethoprim/sulfamethoxazole, *NOR* norfloxacin, *P* penicillin, *I* intermediate, *S* sensitive, *R* resistant ### Antimicrobial susceptibility pattern of gram-negative-negative isolates The antibiotic profile of the gram-negative isolates revealed that 11/13 (84.6%) *E. coli* were susceptible to ceftriaxone, imipenem, gentamicin, norfloxacin, and nitrofurantoin, whereas 12/13 (92.3%) were susceptible to meropenem and nitrofurantoin. Additionally, *E. coli* were susceptible to ceftazidime, ampicillin, cefotaxime, ciprofloxacin, and cefpodoxime with 10/13 (76.9%), 9/13 (69.2%), 9/13 (69.2%), 10/13 (76.9%), and 9/13 (69.2%), respectively. In contrast, 6/13 (54.5%) cefuroxime and 9/13 (67.2%) amoxicillin-clavulanic acid resistance were reported in *E. coli*. Despite the small number of *P. aeruginosa* isolates, it was 1/1 (100%) susceptible to ciprofloxacin, meropenem, and norfloxacin. Nevertheless, it exhibited 1/1 (100%) resistance to tazobactam, piperacillin, ceftaxidime, cefpodoxime, and gentamicin (Table 4).Table 4Antimicrobial susceptible pattern of gram-negative bacteria isolated from urine culture of asymptomatic pregnant women at Debre Markos town public health facilities from May 01, 2023 to July 30, 2023Antibiotics*E. coli**P. aeruginosa*SN (%)RN (%)IN (%)SN (%)RN (%)NOR11(84.6)2(15.4)0(0)1(100)0(0)GEN11(84.6)2(15.4)0(0)0(0)1(100)IMP11(84.6)2(15.4)0(0)0(0)1(100)SXT11(84.6)2(15.4%)0(0%)NANACTR11(84.6)2(15.4)0(0)--NAL6(46.2)6(46.2)1(7.6)NANACAZ10(76.9)1(7.7)2(15.4)0(0)1(100)TE7(53.8)6(46.2)0(0)NANAAMP9(69.2)3(23.1)1(7.7)NANAMEM12(92.3)1(7.7)0(0)1(100)0(0)CTX9(69.2)2(15.4)2(15,4)--F12(92.3)1(7.7)0(0)NANACIP10(76.9)2(15.4)1(7.7)1(100)0(0)CXM5(45.5)6(54.5)2(15.4)--CPD9(69.2)2(15.4)2(15.4)0(0)1(100)AMC4(30.8)9(69.2)0(0)--TZPNANANA0(0)1(100)*N* number, *F* nitrofurantoin, *GEN* gentamicine, *CIP* ciprofloxacin, *TE* tetracycline, *SXT* trimethoprim/sulfamethoxazole, *NOR* norfloxacin, *P* penicillin, *AMP* ampecillin, *MEM* Meropenem, *NAL* nalidicik acid, *AMC* amoxicillin-clavulanic acid, *IMP* impemenem, *CAZ* ceftaxidime, *CXM* cefuroxime, *CTX* cefotaxime, *CRO* ceftriaxone, *TZP* tazobactam pepracillin, *CPD* cefpodoxime, *NA* not applicable ### The multidrug resistance pattern of the isolates Twelve (40%) of the identified bacterial uropathogens had multidrug resistance. About 10 (71.4%) and 2 (12.5%) of these were gram-positive and gram-negative bacteria, respectively. Two out of ten (20.0%) *S. aureus* isolates and nine out of thirteen (69.2%) *E. coli* isolates were primarily multidrug resistant (MDR) (Table 5).Table 5Multi-drug resistance pattern of bacterial isolates from asymptomatic pregnant women at Debre Markos town public health facilities from May 01, 2023 to July 30, 2023**Antimicrobial resistance pattern****bacteria isolate**Total (%)R0R1R2R3R4 ≥ R5MDRGram-negative14(46.6)0(0)1(7.1)3(21.4)5(35.7)4(28.6)1(7.1)10(71.4)*E.coli*13(43.3)0(0)1(7.7)3(23.0)5(38.4)3(23.0)1(7.7)9(69.2)*P. aeruginosa*1(3.3)0(0)0(0)0(0)0(0)1(100)0(0)1(100)Gram positive16(53.3)0(0)7(43.8)7(43.8)1(6.3)1(6.3)0(0)2(12.5)*S. aureus*10(33.3)0(0)6(60.0)2(20)1(10)1(10)0(0)2(20.)*CoNS*6(20)0(0)`1(16.6)5(83.3)0(0)0(0)0(0)0(0)**Total**30(100)0(0)8(26.6)10(33.3)6(20)5(16.7)1(3.3)12(40)Key: *R0 *No antibiotic resistance, *R1* Resistance to one drug, *R2 *Resistance to two drugs, *R3 *Resistance to three drugs, *R4 *Resistance to four drugs, Resistance to five and more than five drugs, *MDR *Resistance more than two class of drugs ### ESBL and carbapenemase-producing gram-negative bacteria Among the 14/30 (46.7%) isolated gram-negative bacteria that were evaluated for the generation of ESBL, 3/14 (21.4%) produced ESBL, with 2/3 (66.7%) and 1/3 (33.3%) of these being *E. coli* and *P. aeruginosa*, respectively. Among 2/14 (14.3%) carbapenemase-producing gram-negative isolates, 1/2 (50.0%) *E. coli* and 1/2 (50.0%) *P. aeruginosa were reported*. However, 1/2 (50.0%) of *E. coli* were both ESBL and carbapenemase-producing isolates *in our study* (Fig. 3).Fig. 3ESBL and carbapenemase producing Gram-negative bacteria isolated from urine culture of asymptomatic pregnant women at Debre Markos town public health facilities from May 01, 2023 to July 30, 2023 For the ESBL- and/or carbapenemase-producing isolates, antibiotic susceptibility tests were conducted for meropenem, imipenem, ceftazidime, cefotaxime, ceftriaxone, and cefpodoxime. As such, ESBL-producing *E. coli* was susceptible to cefpodoxime, accounting for 2/2 (100%), and meropenem, 1/2 (50.0%). In contrast, it was resistant to imipenem, ceftazidime, and meropenem, accounting for 1/2 (50.0%), 2/2 (100%), and 1/2 (50.0%), respectively. Moreover, cefpodoxime and cefotaxime were also 2/2 (100%) intermediates. *P. aeruginosa* was the another ESBL-producing isolate that was 1/1 (100%) susceptible to cefpodoxime and ceftriaxone, but 1/1 (100%) was resistant to imipenem, meropenem, and ceftazidime. Among carbapenemase-producing isolates, *E. coli* 1/1 (100%) was susceptible to cefpodoxime. However, it was 1/1 (100%) resistant to imipenem, meropenem, and ceftazidime. Similarly, carbapenemase-producing *P. aeruginosa* 1/1 (100%) was susceptible to cefpodoxime, and 1/1 (100%) was resistant to ceftazidime, imipenem, meropenem, and ceftriaxone (Table 6). Table 6Antimicrobial susceptible pattern of ESBL and Carbapenemase producing isolates among pregnant women at Health facilities in Debre Markos city from May 01, 2023 to July 30, 2023AntibioticsESBL producer (*N* = 3)Carbapenemase producing(*N* = 2)*E. coli* (*N* = 2)*P. aerugnosa* (*N* = 1)*E. coli* (*N* = 1)*P. aerugnosa* (*N* = 1)SN (%)RN (%)IN (%)SN (%)RN (%)IN (%)SN (%)RN (%)IN (%)SN (%)RN (%)CAZ0(0)2(100)0(0)0(0)1(100)0(0)0(0)1(100)0(0)0(0)1(100)MEM1(50.0)1(50.0)0(0)0(0)1(100)0(0)0(0)1(100)0(0)0(0)1(100)CTR0(0)0(0)2(100)1(100)0(0)0(0)0(0)0(0)1(100)0(0)1(100)CTX0(0)0(0)2(100)0(0)0(0)0(0)0(0)0(0)1(100)0(0)0(0)CPD2(100)0(0)0(0)1(100)0(0)0(0)1(100)0(0)0(0)1(100)0(0)*N* number, *S* sensitive, *R* resistance, *I* intermidiate, *MEM* Meropenem, *IMP* impemenem, *CAZ* ceftazidime, *CTX* cefotaxime, *CTR* ceftriaxone, *CPD* cephodoxime ### Factors associated with asymptomatic bacteriuria Prior UTI history, catheterization history, diabetes mellitus, chronic disease, urine holding, emptying the bladder before bed, vaginal douching, and other variables with a P value less than < 0.25 in the bivariable logistic regression analysis were entered into the multivariable logistic regression analysis. Parity was excluded from the final model because of its multicollinearity effect. We did a model diagnostic test using the Hosmer–Lemeshow test for goodness-of-fit statistic that had a significance value of > 0.05, and this model has adequately described the data.Thus, residence, presence of chronic disease, urine holding, and empty bladder before bedding were not associated with asymptomatic UTIs. Whereas a history of catheterization (AOR = 6.985; 95% CI = 1.61–28.91; *p* = 0.007), vaginal douching (AOR = 3.125; 95% CI: 1.10–8.92; *p* = 0.033), diabetes mellitus (AOR = 5.009; 95% CI = 1.55–16.19; *p* = 0.007), and a history of UTI (AOR = 7.281; 95% CI: 2.53–20.96; *p* = 0.001) were significantly associated with asymptomatic UTIs in pregnant women (Table 7). Compared with those without a history of UTIs, pregnant women with a history of UTIs had a 7.281-fold increased risk of having a history of UTIs. Furthermore, asymptomatic UTIs were 6.985 times more common in women who underwent catheterization than in those who did not. Additionally, compared with women without diabetes mellitus, pregnant women with diabetes mellitus had a 5.009-fold increased risk of developing an asymptomatic UTI. Accordingly, the risk of developing a UTI during pregnancy was 3.125 times greater for women who engaged in vaginal douching than for those who did not (Table 7).Table 7Bivariable and Multivariable Logistic Regression Analysis of Factors Associated with Asymptomatic UTI among Pregnant Women attending Debre Markos Town Public Health Facilities from May 01, 2023 to July 30, 2023VariablesCategoriesBacteriuriaCOR (95%CI) *P* valueAOR (95% CI)*P*valueNoN (%)YesN(%)ResidenceUrbanRural124(85.5)31 (77.5)21(14,5)9(22.5)11.714(0.72–4.11)0.227*15.314(0.57–6.23)0.084Previous UTI historyNoYes138(91.4)17(50.0)13(8.6)17(50.0)110.615(4.40–25.61)0.001*17.281(2.53–20.96)0.001**History of catheterizationNoYes151(87.8)4(30.8)21(12.2)9(69.2)116.179(4.58–57.22)0.001*16.985(1.61–28.91)0.007**Diabetes mellitusNoYes142(87.1)13(59.1)21(12.9)9(40.9)14.681(1.78–12.29) 0.002*15.009(1.55–16.19)0.007**Presence of Chronic diseaseNoYes128(90.1)27(62.8)14(9.9)16(37.2)15.418(2.37–12.41) 0.001*19.213(0.95–10.11)0.354Urine holdingNoYes8(57.1)147(86.0)6(42.9)24(1.0)14.594(1.47–14.41) 0.009*14.544(0.98–20.91)0.053Empty bladder before beddingNoYes8(57.1)147(86.0)6(42.9)24(14.0)4.594(1.47–14.41) 0.009*14.567(0.89–10.51)10.457Vaginal douchingNoYes26(68.4)129(87.8)12(31.6)18(12.2)13.308(1.42–7.61) 0.005*3.125(1.10–8.92)10.033***COR* crude odd ratio, *AOR* Adjusted odd ratio, *CI* confidence interval, *UTI* urinary tract infection, * = variables *p* value < 0.25 and entered to multivariable logistic regression, ** = variable that *p* value < 0.05 and significantly associated with UTI in multivariable logistic regression ## Discussion This study assessed the bacterial profile, antibiotic susceptibility pattern, and associated risk factors for asymptomatic UTIs among pregnant women visiting public health institutions in Debre Markos, Ethiopia. In the present study, the prevalence of significant bacteriuria was 16.2%. This finding was in line with the study findings from Bangladesh, 16.5% [34], and Madda Walabu, Ethiopia, 16% [35]. However, it was lower than the prevalence of ASB reported from India (25.3%) [36], Nepal (42%) [37], Ghana (33.3%) [38], and Hawasa, Ethiopia (58.1%) [39]. In addition, the findings of the present study are higher than the prevalence of AUTIs reported in Saudi Arabia (3.42%) [40], Cameroon (7.8%) [41], Uganda (3.75%) [42], and Nairobi, Kenya (4.3%) [43]. This variation might be due to methodological differences in specimen collection, laboratory thresholds, and trimester distribution, in addition to sample size, geographical variations, social behaviors of the population, and health education practices. In this study, 53.3% of the gram-positive bacteria were identified, which was higher than the percentage of gram-negative bacteria identified (46.6%). The gram-positive bacteria identified in this study are in line with those reported from Dessie, Ethiopia (63.8%) [9], and Hawassa, Ethiopia (51%) [44]. *E. coli* was the most prevalent (43.3%) bacteria isolated in this study. Similar findings were reported at 44.26%, 44.5%, 62%, 68%, and 57.1% in Saudi Arabia [40], Jordan University Hospital [43], Nellore [45], Addis Ababa, the capital city of Ethiopia [46], and Felege Hiwot in Bahir Dar [47], respectively. This could be due to virulence factors harbored by the bacteria to colonize the urinary epithelium and the difficulty of maintaining personal hygiene during pregnancy and fecal flora contamination. However, *S. aureus* (33.3%) was the second predominant bacteria in the current study. This finding was comparable with those of studies conducted in Uganda [42], Addis Ababa, Ethiopia [46], Ambo Hospital, the Ambo Health Center, and the Awaro Health Center [48]. In this study, the highest resistance was shown to be to amoxicillin-clavulanic acid (69.2%) among the gram-negative bacteria. This could be due to overuse of the drug for many years. On the other hand, a higher rate of susceptibility was observed against nitrofurantoin (92.3%), ceftazidime (76.9%), norfloxacin (84.6%), meropenem (92.3%), and gentamicin (84.6%), which is an indication of the possible use of these drugs as empiric therapy, particularly in the study area. Similar findings have been reported in previous studies performed in Ethiopia, such as Bale [13], Hawassa [39], and Dessie [49]. This might be due to medications that are thought to be alternative for treating UTIs, which may be the reason for this low degree of resistance. Every gram-positive isolate demonstrated susceptibility to nitrofurantoin (100.0%), norfloxacin (100.0%), trimethoprim sulfamethoxazole (90%), and gentamicin (84.6%). These results were also consistent with those of other studies conducted in different parts of Ethiopia, including Bale [13], Hawassa [39], and Addis Ababa [27]. Conversely, penicillin was comparatively ineffective against gram-positive bacteria (100%). This may be because PBP modification results in resistance to the majority of beta-lactam antibiotics. Significantly fewer MDR bacteria (40%) were found in our investigation than those reported from Dessie, Ethiopia (72.4%) [9]. Addis Ababa, Ethiopia (57.1%) [46], and Iraq (100%) [50]. As a result, the causes may include using antibiotics precisely as directed, administering them appropriately in empirical treatments, and never taking an antibiotic that has been recommended for someone else. These actions can help reduce the prevalence of resistant organisms in the population. Extended-spectrum beta-lactamase production was detected in 21.4% of the gram-negative isolates in this study. A similar result was reported in Iraq (21.1%) [51]. A result was higher than that of a study published in Dessie, Ethiopia (15.8%) [49]. In contrast, it was less common than studies from Nigeria (36.1%) [52] and Addis Ababa, Ethiopia (50%) [53]. This could be caused by variations in the circulating strains and virulence characteristics of organisms across different regions and nations. Like the research reported in Iraq [51] and Addis Ababa in Ethiopia [53], approximately 66.7% of the *E. coli* in our study produced ESBL. *E. coli* isolates that were ESBL positive in our study presented high levels of resistance to 100% ceftazidime and intermediate levels of resistance to cefotaxime and ceftriaxone, but they were 100% sensitive to CPD, which is comparable to the results reported by Iraq [51] and Ethiopia, such as Dessie [49] and Addis Ababa [53]. However, a 14.3% production of carbapenemase was noted in our study. The carbapenemase output was higher than the values of 4.6%, 2.73%, and 15.12% reported from Algeria [54]. Referral Hospitals of Ethiopia [55] and Ethiopia [56], respectively. The degradation of antibiotics, the inhibition of antibiotic penetration into bacterial cells, pore protein mutation or deletion, excessive efflux pump activation, and PBP modification could all be contributing factors. Conversely, it was less than the 36.14% reported in Qurrayat, Saudi Arabia [57]. The right use of antibiotics, less frequent use of antibiotics, and differences in laboratory test methods could be the causes of this variation. One (100%) resistance to imipenem and meropenem was demonstrated by *P. aeruginosa* and *E. coli* isolates among carbapenemase producers. Antimicrobial stewardship can help reduce resistance by optimizing drug selection, which can include de-escalating therapy, discontinuing therapy, and using local resistance patterns to guide empirical choices. To align with antimicrobial stewardship principles, susceptibility suggests nitrofurantoin may remain effective; treatment decisions should consider pregnancy trimester and clinical guidelines. Pregnant women who had a history of previous UTIs were 7.28 times more likely to develop ASB than those without any history of UTI in this study. A similar finding was reported by Dessie [9]. Addis Ababa, Ethiopia [46]; Somaliland [58]; Saudi Arabia [40]; and Dhaka, Bangladesh [34], in which previous UTIs are significantly associated with asymptomatic UTIs among pregnant women. This connection might be explained by the presence of antibiotic-resistant bacteria from prior infections. Furthermore, compared with pregnant women without a history of catheterization, those with a history of catheterization had a sevenfold increased risk of developing ASB. This was consistent with research conducted in Somaliland [58], Addis Ababa, Ethiopia [46], and Dessie, Ethiopia [9]. This could be due to the introduction of bacteria to the sterile area during catheterization, and repeated, prolonged catheterization could create an environment that encourages the colonization of bacteria. Pregnant women with diabetes mellitus had a fivefold greater risk of developing ASB in the current study than did those without a history of diabetes mellitus. This conclusion was corroborated by comparable research conducted in Dhaka, Bangladesh [34]. This might be because diabetes lowers both local and systemic immunity. The risk of developing ASB was 3.308 times greater for pregnant women who practiced vaginal douching than for those who did not. Similar results were also reported in Harare, Ethiopia [59], and Cairo, Egypt [60]. This might be because frequent vaginal douching can irritate the urethral lining, and washing the genitalia from back to front can allow anal or vaginal bacteria to move into the urethra, which can result in a UTI. ### Limitations of the study This study has lacked minimum inhibition concentration (MIC) or E tests for vancomycin resistance determination in addition to selection bias as a limitation. A small sample size with only 30 isolates altogether and very few isolates per species limits the validity of the results to represent the spectrum of organisms and the prevalent antibiotic susceptibility profiles, non-speciation of the CoNS study population from a specific area, and hence may not reflect epidemiology in other settings. ## Conclusion ASB prevalence was 16.2%; E. coli was the predominant isolate; the MDR rate was 40%. Diabetes, vaginal douching, catheterization, and prior UTI were associated with ASB. Routine urine culture should be considered for targeted screening and culture-guided therapy for high-risk groups. Further studies are needed to evaluate prophylaxis and treatment outcomes.