Among the most common bacterial infections are Urinary Tract Infections (UTIs), which pose a major public health challenge, affecting individuals of all ages [1] . Uropathogens such as Klebsiella spp., Escherichia coli, Acinetobacter spp., Staphylococcus spp., and other pathogenic bacteria are frequent causes of nosocomial infections, including UTIs, which contribute significantly to mortality in clinical settings [2] - [6] . UTIs are the leading cause of morbidity in the general population and are the second most common infectious disease after respiratory infections [7] . Each year, approximately 150 million people worldwide are diagnosed with UTIs, resulting in a global economic burden exceeding US$6 billion [8] . Historically, UTIs have been recognized for millennia. As early as the 16th century BC, they were described in the Ebers Papyrus, one of the oldest surviving medical texts. The Egyptians attributed UTIs to heat in the bladder and treated them with herbal decoctions, bloodletting, and rest (Bent et al., 2002) [9] . Effective treatments for UTIs only emerged in the 1930s with the discovery of antibiotics.

Despite advances in diagnostics and the widespread use of antibiotic therapy, antimicrobial resistance (AMR) in uropathogens remains a significant concern [10] . UTIs, caused by the colonization of the urinary tract by infectious agents, lead to an inflammatory response in the urothelium. Among the pathogens involved, bacteria of the Klebsiella and Acinetobacter genera play a predominant role [11] .

The diagnosis of UTIs is based on clinical symptoms, as well as the detection and identification of microorganisms in urine [9] . UTIs are prevalent in both community and hospital settings and affect people across all demographics [12] . However, women are four times more likely than men to develop UTIs due to anatomical differences [13] .

Currently, particularly in low-income countries, the standard method for diagnosing UTIs remains the Cytobacteriological Examination of Urine (CEBU) through urine culture [14] . Urine culture provides valuable information regarding the presence or absence of a uropathogen, facilitates identification of the causative organism, and in some cases, determines antibiotic susceptibility or resistance. However, the main limitation of this method is the time required to obtain results, which typically takes 2 to 3 days [14] . To alleviate patient symptoms, empirical antibiotic therapy is often initiated while awaiting these results. This practice, however, can contribute to the overuse of antibiotics.

The development of a rapid diagnostic system based on molecular biology, such as automated urine analysis, could provide same-day results, detecting the presence or absence of significant bacteriuria, identifying the pathogen, and determining genetic resistance. Such a system could also reduce the need for urine culture in many negative cases, preventing unnecessary antibiotic prescriptions for patients who do not require them.

In this context, molecular characterization using the Polymerase Chain Reaction (PCR) has emerged as a vital innovation for the rapid and precise identification of pathogens in urinary tract infections. Multiplex real-time PCR allows for the specific detection and amplification of DNA fragments unique to targeted bacteria, offering a more sensitive and specific method compared to traditional urine culture. Urine culture often struggles to differentiate between Klebsiella species (K. pneumoniae and K. oxytoca) and is unable to detect Acinetobacter spp. [15] .

Given the limitations of traditional urine culture, particularly its lengthy turnaround time and difficulty in distinguishing certain uropathogens like Klebsiella spp. and Acinetobacter spp., there is a pressing need for faster, more accurate diagnostic methods. The emergence of molecular techniques, such as multiplex real-time PCR, offers a solution by enabling rapid identification of pathogens and their genetic resistance profiles within the same day. This advancement can significantly reduce unnecessary antibiotic use and improve patient outcomes by offering precise diagnoses in a timely manner. The objectives of this study were: (i) to evaluate, using urine culture, the prevalence rate of urinary tract infections (UTIs) and the resistance rate, and (ii) to identify Klebsiella spp. and Acinetobacter spp. in urine through a molecular approach using multiplex real-time PCR.

In this study, we evaluated the prevalence of uropathogens, their resistance to antimicrobials, and the potential for diagnosing pathogens that are difficult to identify using conventional bacterial culture, through multiplex real-time PCR.

Uropathogens Identified by Urine Cultures

Among the 209 patients who underwent urine culture, 23.44% (49/209) were diagnosed with a urinary tract infection (UTI). This rate is comparable to the findings in Burkina Faso (24.07%) [16] , in Bamako (21.9%) [17] , in Ghana (22.1%) [18] , and in Gabon (29.2 %) [8] . However, it is higher than the 18.5% reported in N’Djamena, Chad (18.5%) [19] ; but lower than the 59.8% found in Cameroun [20] . The prevalence of uropathogenic bacteria varies across regions and continents [21] , highlighting the need to understand local epidemiology and its changes over time to select effective first-line antibiotic therapies tailored to each area [8] .

In this study, the most prevalent uropathogens, in decreasing order, were: Escherichia coli (12.92%), Klebsiella pneumoniae (4.31%), Enterobacter cloacae (1.44%), and Staphylococcus aureus (0.96%). The prevalence of Escherichia coli in this study is lower than that reported in Ouagadougou (32.76%) [22] and (35.5%) [23] . Similarly, the prevalence of Klebsiella pneumoniae is lower than that found in Ouagadougou (22%) [23] and (10.45%) [22] and in Mbarara, in Uganda (12.57%) [24] . Variations in prevalence rates could be attributed to differences in study periods, sampling locations, and identification techniques.

Given that UTIs typically spread through an ascending route and that E. coli is a natural component of the digestive flora, this likely explains its strong predominance in urins samples [25] . As several authors have indicated, among the enterobacteria isolated in UTIs, the genus Escherichia is most involved, followed by Klebsiella [26] - [28] .

Moreover, this study revealed that UTIs were more prevalent in women (13.40%) compared to men (7.18%), which is consistent with findings from other studies [24] [26] [29] . Factors such as environmental pressure, seasonal variation [24] , insufficient hygiene or the anatomical configuration of the female urinary tract could contribute to this difference. However, genetically, women are more resistant to bacterial infections than men due to the “Multiple Copies in T-cell Lymphoma-1” (MCTS1) gene, located at the Xq24 locus on the X chromosome. This gene regulates Interferon gamma (IFN-γ) and the “Janus Kinase 2” (JAK2) gene, which activate immune responses to infections. Since women have two X chromosomes (XX) and men have one (XY), a mutation in the MCTS1 gene on the X chromosome in men could make them more susceptible to infections [30] . This genetic difference may explain why certain infectious diseases, such as COVID-19 and AIDS, have higher mortality rates in men than in women [31] [32] .

Antibiotic resistance

In this study, Klebsiella pneumoniae exhibited 100% resistance to Amoxicillin (AML) and Amoxicillin/Clavulanic Acid (AMC), while Escherichia coli showed 96.2% and 65.4% resistance to AML and AMC, respectively. However, Klebsiella pneumoniae remained 87.5% sensitive to Imipenem (IPM) and 77.8% sensitive to Gentamicin (GM). Numerous studies have confirmed the high resistance of uropathogens to antibiotics in Africa [33] - [35] . Furthermore, Klebsiella spp. resistance to Ceftriaxone (60.0%), Imipenem (26.7%) and Cefoxitin (26.7%) in this study is comparable to that found in Uganda (72.7%), 31.8% and 27.3% respectively [24] .

However, the primary risk factor for antibiotic resistance is repeated prior exposure to the same antibiotic [8] . The overuse or misuse of an antibiotic, or a class of antibiotics, fosters the development of bacterial resistance, which can extend to other antibiotic families through the cross-transmission of mobile genetic elements carrying antibiotic resistance genes [36] [37] . Since UTIs often arise from the ascending contamination of the perineal flora, which reflects the digestive flora, this selection pressure has significant clinical implications [8] .

Although urine culture is currently considered the “gold standard” in bacteriology, it has several limitations. One major drawback is the time required cultures take at least two days to yield results, including sensitivity profiles. This delay in treatment increases the risk of complications, such as upper urinary tract infections (e.g., pyelonephritis), especially when early intervention with empirical antibiotics could have mitigated the symptoms [38] . Additionally, certain rare pathogenic species are difficult to identify using conventional bacterial culture techniques. In contrast, molecular diagnostic methods offer a solution to these identification challenges.

Identification of Uropathogens by Multiplex Real-Time PCR

Multiplex real-time PCR analysis of three uropathogens yielded an overall positivity prevalence rate of 23.38% (18/77), with prevalence rates of Klebsiella pneumoniae at 10.39% (8/77), Klebsiella oxytoca at 7.79% (6/77), and Acinetobacter spp. at 7.79% (6/77). Among the 18 positive cases, the majority were female, with a prevalence of 12.99%, compared to 10.39% in males (P = 0.462). Additionally, the multiplex real-time PCR identified one co-infection involving Klebsiella pneumoniae and Acinetobacter spp.

Comparison Between the Diagnosis of Uropathogens by Bacterial Culture and the Multiplex PCR Technique

According to Sacace Biotechnologies, multiplex real-time PCR offers 100% sensitivity [79.41 - 100] and 100% specificity [98.71 - 100] [39] . The results of this study demonstrated the effectiveness and reliability of multiplex real-time PCR by confirming the positivity of all fifteen (15) Klebsiella spp. samples identified by urine culture, including nine (09) samples of Klebsiella pneumoniae and six (06) of Klebsiella oxytoca, which were further identified by PCR.

Although urine culture is currently considered the gold standard for diagnosing bacterial infections, it faces challenges such as high vulnerability to contamination, with an average contamination rate of 15% in many laboratories [40] .

Contamination, often occurring during the pre-analytical phase of urine culture, can be minimized with proper urine collection, storage, and transport methods [41] . False-positive results due to contamination can lead to inappropriate or unnecessary treatments, increasing bacterial resistance, compromising patient outcomes, and escalating healthcare costs [38] . Furthermore, the time-consuming nature of culture techniques, often requiring several days for results, has driven the search for faster diagnostic methods like multiplex real-time PCR [42] . Multiplex real-time PCR-based bacterial diagnostics represent a significant advancement in DNA-based laboratory technologies and have become widely available in clinical settings. The use of multiplex PCR, which employs multiple primers to simultaneously detect multiple targets, has reduced both the cost and time of analysis, which explains its growing adoption in clinical medicine [43] . According to Xu et al. (2021), PCR not only shows higher detection rates of single pathogens compared to urine culture [44] [45] , but it is also highly effective at detecting multiple pathogens in urine samples [46] such as Klebsiella oxytoca and Acinetobacter spp., which were identified in this study—pathogens that standard urine cultures consistently failed to detect.

Bacteriological culture, first pioneered in the 19th century by Louis Pasteur, revolutionized medical diagnostics. However, with ongoing advances in medical technology, newer diagnostic tools with greater sensitivity and specificity—such as multiplex real-time PCR, expanded quantitative urine culture (EQUC), and next-generation sequencing (NGS)—now offer more comprehensive pathogen analyses in significantly less time. This study, for instance, demonstrated that molecular characterization of Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. Using multiplex real-time PCR was completed in less than 24 hours, whereas traditional urine culture required two to three days. The ability to automate urine PCR testing, combined with its accuracy and rapid results, makes multiplex real-time PCR a powerful addition to the diagnostic tools available for managing urinary tract infections in modern medicine.

The authors thank Saint Camille Hospital of Ouagadougou (HOSCO) and the Centre de Recherche Biomoléculaire Pietro Annigoni (CERBA) for facilitating data collection.

JS, HT, TMZ, DO, CN, TS, designed the study.

JS, TMZ, DO, TS, FWD, AKO, LT, RAO, wrote the manuscript.

HT, JS, DO, TS, CN, AKO, NB, ATY, PB collected the data.

JS, HA, AKO, computerized the data.

HT, DO, TS, AKO, LZ, JS, TMZ, EJTV performed statistical analysis.

JS, TMZ, DO, TS, DO, FWD, AKO, LT, LZ, RAO, revised and edited the manuscript.

The HOSCO/CERBA Institutional Ethics Committee approved this study in Deliberation No. 2023-02-05 of 15 April 18, 2023.The study was conducted according to the declaration of Helsinki.