Bacteriological profile, antimicrobial susceptibility, and factors associated with urinary tract infection in pregnant women

Introduction: Urinary tract infection (UTI) is a common bacterial complication in pregnancy. The study aimed to estimate the prevalence, risk factors, and bacterial etiology of UTI during pregnancy and determine the efficacy of antimicrobial drugs in treating UTIs. Methodology: Urine specimens and clinical data were collected from pregnant women who attended primary health centers in Erbil, Iraq. All specimens were cultured on appropriate media and identified by standard microbiological methods. The pregnant women were grouped into symptomatic UTI group, asymptomatic bacteriuria group, and the control group. The agar dilution method was used to determine antimicrobial susceptibility. Results: Among the 5,042 pregnant women included in this study, significant bacteriuria was found in 625 (12.40%) of the cases, and 198 (31.68%) had symptomatic UTI, of which 43.59% were diagnosed during the third trimester. Out of the 643 bacteria isolated, 33.28% were symptomatic UTI, of which 43.59% developed during the third trimester. There was a significant difference in the bacterial etiology between symptomatic UTI and asymptomatic bacteriuria ( p = 0.002), as well as between cystitis and pyelonephritis ( p = 0.017). The most common bacterial species isolated was Escherichia coli , which was susceptible to fosfomycin (100%), meropenem (99.45%), and nitrofurantoin (97.8%). Conclusions: Pregnant women are more likely to develop UTI in the third trimester. Escherichia coli is the predominant pathogen. The study suggests the use of fosfomycin, meropenem, and nitrofurantoin for the treatment of UTI. No Gram-positive isolates were resistant to daptomycin.


Introduction
Urinary tract infection (UTI) is a common and serious bacterial infection in pregnant women worldwide, leading to costly medical complications [1].Morphological and physiological changes in the genitourinary tract during pregnancy increase the incidence of UTIs [2].About 40-50% of women experience UTI during their lifetime, and its incidence has significantly increased globally each year [3,4].Infection of the urinary tract is caused by different types of microorganisms [5], which depend on where the infection develops along the urinary tract during pregnancy [6].UTIs have been classified as symptomatic UTIs or asymptomatic bacteriuria (ASB).ASB is defined as the presence of significant bacteria (i.e., the presence of ≥ 10 5 bacteria/mL of urine) in the absence of symptoms of UTI [7][8][9].
Screening and treatment of ASB are recommended in pregnant women because if left untreated, it will lead to symptomatic UTI and cause serious risks for both the mother and the fetus [10].UTIs have been associated with neonatal sepsis and an increased risk of stillbirth.Thus, treatment is important for the mother and child [11,12].Furthermore, symptomatic UTIs are classified as cystitis and pyelonephritis involving the bladder and kidneys, respectively.Treatment of cystitis and pyelonephritis requires attention to the growing antimicrobial resistance [13].The patterns of antimicrobial resistance in a wide variety of uropathogenic bacteria can vary over a short period [14], and resistance of uropathogenic bacteria is increasing globally, mainly against commonly used antimicrobials [15].Therefore, knowledge about antimicrobial resistance patterns is required when selecting antimicrobial agents [16].Incorrect UTI diagnosis and treatment can result in newborn complications [17].
Appropriate studies involving the treatment of UTIs are required to avoid life-threatening illness and morbidity related to UTI complications in pregnant women [18].However, in many developing countries, like Iraq, routine urine culture test for pregnant women is not performed, and antimicrobials are usually prescribed empirically without laboratory urine culture results.Furthermore, current awareness about bacteria causing UTIs and their antibiotic resistance is essential for ensuring successful therapy through periodic evaluation of the antibacterial activity.The goal of this study was to assess the etiologic and antibiotic susceptibility patterns of bacteria isolated from pregnant women with UTIs, and investigate whether UTIs are associated with the third trimester.

Study design and patient population
A cross-sectional study was conducted on 5,042 pregnant women attending primary care health centers in Erbil, the capital of Iraq's Kurdistan Region, who were assessed for UTI from October 2018 to February 2022.Exclusion criteria included pregnant women who were below the age of 18 years; refused to participate in the study; were diagnosed with hypertension and/or diabetes mellitus; received antimicrobial treatment within two weeks; had recent hospitalization, catheterization, surgery, or urethral instrumentation in the previous two weeks; were diagnosed with COVID-19; had urologic abnormalities or nephrolithiasis; and had a known serum creatinine level of more than 2.2 mg/d.

Ethical approval
Ethics committee approval was obtained from the Medical Research Ethics Committee of Hawler Medical University, Erbil, Kurdistan Region, Iraq.All pregnant women provided verbal informed consent for participation in the study before specimen collection and authorized the use of their clinical data for subsequent publication.All identifying information about the women was kept confidential.

Data collection
Demographic data, including age, educational level, parity (number of live births), trimester, and medical history, were obtained from the pregnant women.The body mass index (BMI) was calculated by measuring height and weight.

Urine collection and analysis
Clean-catch midstream urine was collected from pregnant women and then cultured.The bacterial culture was performed by streaking 1 µL of urine on MacConkey agar and 5% blood agar plates (Lab M, Lancashire, UK) with a calibrated loop.The plates were incubated at 37 °C for 18-24 hours under aerobic conditions [19].Plates with mixed cultures were subcultured to obtain a pure bacterial culture.Gram staining was performed to identify whether the bacteria were Gram-negative or Gram-positive [20].The isolated bacteria were identified to genus or species level using the analytical profile index (API) system.The results were interpreted according to the guidelines of the Infectious Diseases Society of America as follows [10]: (1) Sterile urine: negative urine culture (control group).
(2) Contaminated specimen: urine specimen containing more than two species.
(3) Insignificant bacteriuria: clean-catch urine containing < 10 5 colony-forming units (CFU) of bacteria per mL.(4) Significant bacteriuria: growth of bacteria at ≥ 10 5 CFU/mL in urine culture, which was sub-classified into two groups [21]: a. ASB: significant bacteriuria in the absence of signs or symptoms attributable to an UTI.b.Symptomatic UTI: presence of significant bacteriuria with the clinical symptoms of UTI.Urine specimens that produced insignificant bacteriuria or that were contaminated were excluded from the study.

Identification with API systems
The API system is a method used to identify bacteria based on their biochemical characteristics [22].The isolated bacterial species were identified using the API system with the API 20E kit for Gram-negative bacteria, the API Staph kit for staphylococci, and the API 20 Strep kit for streptococci and enterococci (bioMérieux, Marcy l'Etoile, France).The tests were performed according to the manufacturer's instructions.

Antimicrobial susceptibility testing
The agar dilution method was performed according to the guidelines of the Clinical Laboratory Standard Institute (CLSI) document M07-A10 to determine antimicrobial susceptibility [23].Mueller-Hinton agar (Lab M, Lancashire, UK) plates were prepared with a doubling concentration of the antimicrobial.The antimicrobials used for Gram-negative and Grampositive bacteria were ampicillin (AMP), amoxicillin (AMX), cephalexin (LEX), cefuroxime (CXM), ceftriaxone (CRO), cefepime (FEP), meropenem (MEM), azithromycin (AZM), nitrofurantoin (NIT), and fosfomycin (FOF) (Sigma-Aldrich, St. Louis, MO, USA).In addition, daptomycin (DAP) (Sigma-Aldrich) was used for Gram-positive bacteria.The bacterial suspensions of each bacteria strain were prepared and adjusted to a turbidity equivalent to 0.5 McFarland standard turbidity, and then the bacteria were inoculated on these plates with approximately 10 4 CFU per spot of 5-8 mm in diameter, and the plates were incubated aerobically at 35 ± 2 °C for 16-20 hours.The plates were examined for bacterial growth to determine the minimum inhibitory concentration (MIC), which is the lowest concentration of the antimicrobial that inhibited the growth of the isolate when compared to the growth of the control.The MIC value was compared with the CLSI susceptibility breakpoints [24], and the percentage of antimicrobial susceptibility was determined based on the CLSI breakpoints.

Data analysis
The data were coded and analyzed using SPSS version 25.0 (SPSS, Chicago, IL).The Chi-square test is used to compare categorical variables.A p value less than 0.05 was considered a significant association between the variables.
Table 1 shows the distribution and comparison of the characteristics of symptomatic UTI with ASB and  control group.During the third trimester, 43.59% of pregnant women had symptomatic UTIs.Statistical differences in age and trimester were observed between symptomatic UTI and the other two groups (ASB and control).The differences in parity (p = 0.003) and BMI (p = 0.014) were also significant between the symptomatic UTI group and the control group.

Discussion
Women are predisposed to ASB and symptomatic UTI or pyelonephritis during pregnancy, which can cause significant maternal and fetal morbidity [25].This study discovered a significant number of ASBs and symptomatic UTIs, which is consistent with previous research [25,26].ASB predisposes to the development of UTIs [27] and increases the risk of acquiring UTIs due to hormonal and anatomical changes during pregnancy [28].Furthermore,  pyelonephritis is most prevalent in late pregnancy [29].When UTIs were detected and treated early, the complications were reduced [30].The study also concluded that the majority of UTIs developed during the third trimester.This finding corresponds with another study [31].UTIs cause adverse pregnancy outcomes, and the identification of uropathogenic bacteria may contribute to the early treatment of pregnant women.Undiagnosed and/or untreated ASB in the first and second trimesters causes cystitis and pyelonephritis in the third trimester.If ASB is left untreated, it may be associated with acute cystitis and pyelonephritis [32].This emphasizes the significance of screening for ASB and treating symptomatic UTIs with appropriate antimicrobial therapy to reduce the incidence of pyelonephritis and UTI complications during pregnancy [33].
The prevalence of symptomatic UTIs in pregnancy was affected by age, educational level, parity, and BMI compared to ASB or control group.UTIs and ASBs were common in pregnant women of all ages.But some studies have suggested that older age may be associated with a higher risk of bacteriuria and UTIs during pregnancy [34,35], which agrees with our results.The effect of educational level on UTIs in pregnant women is not well established in the current study.This might be attributed to increased awareness about UTI symptoms and prevention measures, as well as high adherence to treatment and follow-up at different educational levels.However, lower levels of education have been associated with a higher prevalence of ASB in other studies [36,37].The effect of nulliparity on UTIs in pregnant women is not well established.However, the prevalence of UTIs in nulliparous women  was higher than in ASB and the control group in this study, which may be because the nulliparous women were screened and treated for ASB.Moreover, some studies reported a higher prevalence of UTIs associated with nulliparity [38,39].Elevated BMI was associated with UTIs, and this result is consistent with other studies [40].
In this study, the most common bacteria isolated from symptomatic UTI and ASB were E. coli, which has been reported in other geographical areas with resistance to various antimicrobial agents [41].Antimicrobial resistance in uropathogenic bacteria is increasing worldwide, especially to the commonly used antimicrobial agents [42].UTIs caused by E. coli pose a therapeutic challenge and are associated with an increased risk of serious complications for the mother and fetus during pregnancy [43].Antimicrobial resistance of E. coli has become an alarming problem in both developed and developing countries because the resistance is increasing faster than the development of antimicrobial agents [44].Fosfomycin affected all strains of E. coli and the majority of the isolated species, but E. coli was most likely to be susceptible to fosfomycin, according to the CANWARD surveillance study [45].
Most of the isolates in the current study were susceptible to nitrofurantoin, which is one of the most prescribed antibiotics [46,47].Therefore, during the first trimester, nitrofurantoin can be used for the prevention and treatment of UTIs.The American College of Obstetricians and Gynecologists recommended that prescribing nitrofurantoin in the first trimester is still appropriate when no other alternative antimicrobial therapies are available [48].About twothirds of the uropathogens were susceptible to cephalexin in this study, and it has been recommended as a first-line treatment for UTI in pregnancy by the National Institute for Health and Care Excellence [49].However, approximately one-third of isolated bacteria were susceptible to ampicillin, which reduced their overall susceptibility to the commonly used form of the antimicrobial agent.The study provides a choice for treatment of UTIs during pregnancy when microbiological results are unavailable, especially in developing countries.A limitation of this study was the lack of testing for extended-spectrum beta-lactamase (ESBL)-producing bacteria.

Conclusions
A significant number of UTIs were reported in pregnant women, and the likelihood was higher during the third trimester.The most common bacterial isolate from UTI and ASB was E. coli, and fosfomycin was effective against all strains of this bacterium.The most effective treatment for this infection was meropenem, followed by nitrofurantoin, ceftriaxone, cefepime, and fosfomycin.There was no Gram-positive bacterial resistance to daptomycin.Thus, the efficiency of the antimicrobial drug contributed to the successful treatment of UTIs during pregnancy.

Figure 1 .
Figure 1.Urine culture results of 5,042 pregnant women.

Table 1 .
Comparison of the characteristics of symptomatic UTI in pregnant women with asymptomatic bacteriuria and control group.Comparison of symptomatic UTI with asymptomatic bacteriuria using Chi-square test; b Comparison of symptomatic UTI with control group using Chi-square test; UTI: urinary tract infection; BMI: body mass index. a

Table 3 .
Distribution of uropathogenic bacteria in cystitis and pyelonephritis.