1. Introduction

Urinary tract infections (UTIs) are among the most common bacterial infections worldwide and represent a major public health concern, affecting millions of individuals each year, particularly women. UTIs are generally classified as uncomplicated or complicated depending on host characteristics and underlying urinary tract abnormalities. The increasing prevalence of antimicrobial-resistant uropathogens has become a significant challenge for effective clinical management and has contributed to higher healthcare costs, prolonged hospitalization, and increased morbidity [1].

The pathogenesis of UTIs is closely associated with bacterial virulence factors that facilitate colonization, invasion, persistence, and evasion of host immune defenses. These virulence determinants enable uropathogens to adhere to the urothelial surface, establish infection, and promote recurrent disease [2]. Biofilm formation is considered one of the most important pathogenic mechanisms because bacteria embedded within the extracellular polymeric matrix exhibit enhanced resistance to host immune responses and antimicrobial agents, resulting in persistent and recurrent infections [3,4].

The rapid emergence of antimicrobial resistance has become one of the greatest threats to global public health. Inappropriate antibiotic use, inadequate antimicrobial stewardship, widespread use of broad-spectrum antibiotics, globalization, and the extensive application of antibiotics in agriculture and animal production have accelerated the dissemination of resistant microorganisms [5]. Bacterial isolates are commonly classified as multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan drug-resistant (PDR). According to internationally accepted definitions, MDR organisms exhibit acquired non-susceptibility to at least one antimicrobial agent in three or more antimicrobial classes, XDR organisms remain susceptible to only one or two antimicrobial categories, whereas PDR organisms are resistant to all available antimicrobial agents [6].

The increasing prevalence of MDR, XDR, and PDR uropathogens has stimulated interest in alternative antimicrobial strategies. Among these, bacteriocins have attracted considerable attention because they are ribosomally synthesized antimicrobial peptides produced by bacteria that exhibit potent bactericidal activity against closely related or pathogenic bacterial species [7]. Unlike many conventional antibiotics, bacteriocins possess multiple mechanisms of action, are generally regarded as safe for human use, and are readily degraded by gastrointestinal proteases, thereby reducing toxicity and adverse effects [7].

Biofilm-associated infections remain particularly difficult to eradicate because bacterial cells within biofilms demonstrate substantially reduced susceptibility to antimicrobial therapy. Consequently, conventional antibiotic monotherapy is often insufficient for successful treatment. Current antibiofilm approaches include disruption of the extracellular polymeric matrix, inhibition of bacterial adhesion, use of antimicrobial biomaterials, small-molecule inhibitors, and naturally derived antimicrobial compounds, including bacteriocins [8].

Given the growing burden of antimicrobial resistance and biofilm-associated UTIs, the present study aimed to characterize antimicrobial resistance patterns and biofilm formation among bacterial isolates recovered from women with urinary tract infections, evaluate the antibacterial and antibiofilm activities of bacteriocin produced by Staphylococcus epidermidis, investigate its synergistic activity with conventional antibiotics, and determine the genetic relationships among resistant uropathogens using molecular and cluster analyses.

2. Materials and Methods

2.1 Study Design and Sample Collection

A laboratory-based cross-sectional study was conducted between October 2024 and January 2025. A total of 100 bacterial isolates were recovered from midstream urine specimens collected from women clinically diagnosed with urinary tract infections attending Nwa Laboratory, Shar Hospital, Asia Hospital, and Smart Tower Hospital in Sulaymaniyah City, Iraq. Demographic and clinical information was obtained using a structured questionnaire that included patient identification code, age, place of residence, educational level, marital status, symptomatic or asymptomatic presentation, history of recurrent UTIs, underlying diseases, and previous antimicrobial use.

2.2 Isolation and Identification of Uropathogens

Urine specimens were cultured on blood agar, MacConkey agar, and nutrient agar media and incubated aerobically at 37°C for 24 hours. Colony morphology was evaluated, and bacterial growth was quantified. Significant bacteriuria was defined as a bacterial count of ≥10⁵ colony-forming units (CFU)/mL [9].

Pure bacterial isolates were identified using conventional morphological, microscopic, biochemical, and physiological methods. Species confirmation and antimicrobial susceptibility testing were subsequently performed using the VITEK® 2 Compact System (bioMérieux, France).

2.3 Isolation of Staphylococcus epidermidis

Clinical isolates of Staphylococcus epidermidis were cultured on blood agar and MacConkey agar and incubated aerobically at 37°C for 48–72 hours. Identification was confirmed using Gram staining, biochemical testing, microscopic examination, and the VITEK® 2 Compact identification system.

2.4 Antimicrobial Susceptibility Testing

Antimicrobial susceptibility profiles were determined using the VITEK® 2 Compact system according to the manufacturer's recommendations. Bacterial isolates were categorized as multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan drug-resistant (PDR) according to internationally accepted definitions [6].

2.5 Biofilm Formation Assays

Biofilm production was evaluated using two complementary methods: the Congo red agar (CRA) assay and the tissue culture plate (TCP) method.

Tissue Culture Plate Method

The tissue culture plate assay was performed as previously described by O'Toole [10]. Briefly, overnight bacterial cultures were adjusted to the 0.5 McFarland standard, diluted in fresh brain-heart infusion (BHI) broth, and inoculated into sterile 96-well microplates. Following incubation at 37°C for 48 hours, wells were washed, stained with 0.1% crystal violet, and air-dried before biofilm biomass was quantified spectrophotometrically.

Congo Red Agar Method

Biofilm production was also assessed on Congo red agar containing brain-heart infusion broth, sucrose, Congo red dye, and agar. Plates were incubated at 37°C for 24 hours. Black colonies were interpreted as biofilm producers, whereas red colonies were considered non-biofilm producers [11].

2.6 Extraction of Bacteriocin

Crude bacteriocin produced by Staphylococcus epidermidis was obtained by culturing the organism in brain-heart infusion broth under anaerobic conditions at 37°C for 24 hours. Cultures were centrifuged at 6000 rpm for 10 minutes at 4°C, and the cell-free supernatant was sterilized through a 0.22-µm membrane filter. The pH was adjusted to 7.0 using 1 M NaOH to eliminate the antimicrobial effects of organic acids [12].

2.7 Evaluation of Antibacterial Activity

The antibacterial activity of crude bacteriocin was evaluated using agar well diffusion and cup assay methods. Mueller–Hinton agar plates inoculated with standardized bacterial suspensions received 150 µL of bacteriocin extract per well, followed by incubation at 37°C for 24 hours. Antibacterial activity was expressed as the diameter of inhibition zones (mm) surrounding each well [13,14].

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using serial two-fold dilution according to Clinical and Laboratory Standards Institute (CLSI, 2024) recommendations [15].

2.8 Antibiofilm Activity

The antibiofilm activity of crude bacteriocin was evaluated using a modified 96-well microtiter plate assay. Biofilms were allowed to develop for 24 hours at 37°C, after which bacteriocin extract was added to each well. Following incubation, residual biofilm biomass was quantified using crystal violet staining and optical density measurements at 550 nm. The percentage reduction in biofilm formation was calculated using the previously described equation [16].

2.9 Synergistic Activity Between Bacteriocin and Antibiotics

The synergistic antibacterial activity of bacteriocin in combination with conventional antibiotics was evaluated using the disk diffusion method. For Gram-positive bacteria, bacteriocin was combined with benzylpenicillin, levofloxacin, and gentamicin, whereas ceftriaxone, ciprofloxacin, and trimethoprim/sulfamethoxazole were evaluated against Gram-negative isolates. Inhibition zones were measured after overnight incubation at 37°C [17].

2.10 Detection of the Bacteriocin Gene

The bacteriocin gene was amplified by polymerase chain reaction (PCR) using previously published primers targeting a 179-bp fragment [18]. PCR amplification consisted of an initial denaturation at 95°C for 10 minutes, followed by 32 cycles of denaturation (95°C for 30 s), annealing (57°C for 30 s), extension (72°C for 10 s), and a final extension at 72°C for 5 minutes. PCR products were separated on 1.5% agarose gel electrophoresis and visualized under ultraviolet illumination.

2.11 Statistical Analysis

Statistical analyses were performed using IBM SPSS Statistics version 27.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA). Hierarchical cluster analysis was performed using PRIMER-E version 7 to evaluate relationships among bacterial isolates based on antimicrobial resistance patterns, biofilm-forming capacity, and susceptibility to bacteriocin. Statistical significance was defined as P < 0.0

3. Results

3.1 Demographic Characteristics and Distribution of Uropathogens

A total of 100 bacterial isolates were recovered from urine specimens collected from women diagnosed with urinary tract infections (UTIs) attending five healthcare facilities in Sulaymaniyah City. Most isolates originated from Nwa Laboratory (40%), followed by Asia Hospital (25%), Shar Hospital (20%), Smart Tower Hospital (10%), and the Teaching Hospital (5%). All participants were female, with 90% being married and 10% unmarried. Previous antibiotic exposure was reported by 65% of the participants, while 49% had diabetes mellitus, 39% had recurrent UTIs, and 30% had a history of hospitalization (Table 1).

The majority of patients (62%) were ≥50 years of age, followed by those aged 40–49 years (14%), 30–39 years (12%), 20–29 years (7%), and ≤19 years (5%) (Figure 1).

Among the isolated uropathogens, Gram-negative bacteria predominated (89%), whereas Gram-positive bacteria accounted for only 11% of all isolates. Escherichia coli was the most frequently isolated pathogen (76%), followed by Klebsiella pneumoniae (9%), Staphylococcus haemolyticus (6%), Klebsiella oxytoca (4%), Staphylococcus aureus (2%), Staphylococcus hominis (2%), and Staphylococcus sciuri (1%) (Figure 2).

3.2 Antimicrobial Susceptibility Profiles

Antimicrobial susceptibility testing performed using the VITEK® 2 Compact system demonstrated substantial variability in resistance patterns among the isolated uropathogens. Gram-negative isolates exhibited the highest resistance rates to ciprofloxacin, trimethoprim/sulfamethoxazole, and third-generation cephalosporins, whereas resistance to carbapenems remained relatively uncommon. Among Gram-positive isolates, resistance was highest against tetracycline, benzylpenicillin, and oxacillin, while glycopeptides retained comparatively good activity (Tables 3 and 4).

Overall, antimicrobial susceptibility testing identified multiple multidrug-resistant phenotypes, including MDR, XDR, and PDR isolates among both Gram-negative and Gram-positive bacteria.

3.3 Detection of the Bacteriocin Gene

A bacteriocin-producing strain of Staphylococcus epidermidis was isolated from the normal skin flora of healthy outpatients. PCR amplification successfully detected the bacteriocin gene, producing the expected 179-bp amplicon, thereby confirming the presence of the target gene (Figure 4).

3.4 Antibacterial Activity of Bacteriocin

The crude bacteriocin demonstrated broad-spectrum antibacterial activity against 96% of the tested uropathogens. MIC values ranged from 10–20 µg/mL for most Gram-positive bacteria and 20–160 µg/mL for Gram-negative isolates. Klebsiella oxytoca showed complete resistance to bacteriocin, with no detectable MIC or MBC values (Table 5).

The antibacterial activity evaluated by agar-well diffusion and cup-assay methods demonstrated significantly greater inhibition against Gram-positive bacteria than Gram-negative bacteria. Using the agar-well diffusion method, inhibition zones ranged from 17–23 mm for Gram-positive isolates and 11–15 mm for Gram-negative isolates. Similar findings were observed using the cup-assay method, although inhibition zones were consistently smaller (12–16 mm and 7–11 mm, respectively). No inhibitory activity was observed against Klebsiella oxytoca. Overall, Gram-positive bacteria required lower bacteriocin concentrations for growth inhibition than Gram-negative bacteria (Table 6; Figure 5).

3.5 Synergistic Activity Between Bacteriocin and Conventional Antibiotics

The combination of bacteriocin with conventional antimicrobial agents significantly enhanced antibacterial activity compared with bacteriocin alone. Among Gram-negative isolates, the greatest synergistic effect was observed when bacteriocin was combined with trimethoprim/sulfamethoxazole, producing larger inhibition zones than combinations with ceftriaxone or ciprofloxacin (Table 8).

Similarly, combinations of bacteriocin with benzylpenicillin, gentamicin, and levofloxacin significantly increased inhibition zones against Gram-positive bacteria, particularly among MDR and XDR Staphylococcus isolates (Table 9). These findings indicate that bacteriocin can potentiate the activity of conventional antibiotics against resistant uropathogens.

3.6 Biofilm Formation

Biofilm production was evaluated using both the tissue culture plate (TCP) and Congo red agar (CRA) methods. The TCP assay identified 72% of isolates as moderate or strong biofilm producers, whereas the CRA method identified 70% as biofilm producers, indicating that the TCP method was slightly more sensitive for biofilm detection.

All Gram-positive isolates (100%) produced biofilms, whereas 61 of 89 Gram-negative isolates (68.5%) exhibited moderate or strong biofilm-forming ability (Figure 6).

3.7 Antibiofilm Activity of Bacteriocin

Treatment with crude bacteriocin significantly reduced biofilm formation in most uropathogenic isolates. The antibiofilm activity was particularly pronounced among Gram-positive bacteria, with biofilm reduction ranging from 57.6% to 91.5%. Among Gram-negative bacteria, biofilm reduction ranged from 35.1% to 79.7%.

Notably, strong and moderate biofilm-producing isolates were frequently converted to weak biofilm producers following bacteriocin treatment. In contrast, Klebsiella oxytoca remained unaffected, exhibiting neither antibacterial nor antibiofilm susceptibility (Table 10; Figure 7).

3.8 Cluster Analysis of Uropathogenic Isolates

Hierarchical cluster analysis using PRIMER-E version 7 grouped the 100 bacterial isolates into three major clades (A–C) according to their antimicrobial resistance profiles, bacteriocin susceptibility, and biofilm-forming characteristics (Figure 8).

Clade A consisted predominantly of MDR Escherichia coli isolates recovered mainly from married women aged ≥50 years. Most isolates exhibited strong biofilm production before treatment, which was markedly reduced following bacteriocin exposure.

Clade B comprised mainly Klebsiella pneumoniae and Klebsiella oxytoca isolates, with MDR and XDR phenotypes predominating. This group demonstrated the lowest susceptibility to bacteriocin, and K. oxytoca showed complete resistance with persistent biofilm formation after treatment.

Clade C contained predominantly Staphylococcus species together with several highly resistant E. coli isolates. This clade exhibited the highest prevalence of XDR and PDR phenotypes. Although bacteriocin produced the largest antibacterial inhibition zones in this group, its antibiofilm activity varied considerably among isolates, indicating differences in biofilm susceptibility despite strong antibacterial effects.

Overall, the cluster analysis demonstrated a close association between antimicrobial resistance, biofilm-forming capacity, and bacteriocin susceptibility, highlighting the potential utility of bacteriocins as alternative therapeutic agents against resistant uropathogens.

Table 1. Demographic and clinical characteristics of women with urinary tract infections (N = 100)

Table 2. Distribution of uropathogenic bacterial isolates (N = 100)

Summary by Gram stain

Table 3. Overall antimicrobial resistance among Gram-negative uropathogens

Table 4. Overall antimicrobial resistance among Gram-positive uropathogens

Table 5. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of bacteriocin

Table 6. Antibacterial activity of bacteriocin against uropathogens

Table 7. Biofilm production determined by two detection methods

Table 8. Antibiofilm activity of bacteriocin

Table 9. Summary of synergistic activity between bacteriocin and antibiotics

Table 10. Characteristics of bacterial clusters identified by PRIMER-E analysis

4. Discussion

Urinary tract infections (UTIs) remain among the most common bacterial infections worldwide, and the increasing prevalence of antimicrobial-resistant uropathogens has become a major therapeutic challenge. In the present study, Escherichia coli was identified as the predominant causative pathogen, accounting for 76% of all isolates, followed by Klebsiella pneumoniae and coagulase-negative Staphylococcus species. These findings are consistent with previous epidemiological studies demonstrating that E. coli is responsible for approximately 70–80% of community-acquired UTIs because of its ability to adhere to uroepithelial cells, produce multiple virulence factors, and establish persistent infections [1,2]. Similar findings have been reported in Iraq, where E. coli represented 74.4% of bacterial isolates recovered from women with UTIs [3].

A major finding of this study was the high prevalence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan drug-resistant (PDR) phenotypes among the recovered uropathogens. The emergence of these resistant organisms considerably limits available treatment options and emphasizes the urgent need for alternative antimicrobial agents. The observed resistance patterns are likely attributable to inappropriate antibiotic use, repeated antimicrobial exposure, and the widespread dissemination of resistance genes among clinical bacterial populations, findings that are consistent with recent global reports on antimicrobial resistance [4,5].

Biofilm formation represented another important characteristic of the isolated uropathogens. Using both the tissue culture plate and Congo red agar methods, approximately 70% of isolates were identified as moderate or strong biofilm producers, with the tissue culture plate assay demonstrating slightly greater sensitivity. These findings agree with previous studies from India, which reported biofilm formation rates ranging from 60% to 67% among urinary isolates [6,7]. The high prevalence of biofilm-producing bacteria observed in the present study may explain the persistence and recurrence of UTIs because biofilms provide a protective environment that enhances bacterial survival, facilitates horizontal transfer of resistance genes, and markedly reduces susceptibility to antimicrobial therapy [8].

One of the most important findings of this study was the potent antibacterial activity of bacteriocin produced by Staphylococcus epidermidis. The crude bacteriocin inhibited the growth of 96% of the tested uropathogens and demonstrated broad-spectrum activity against both Gram-positive and Gram-negative bacteria. Gram-positive isolates were generally more susceptible than Gram-negative isolates, likely because the outer membrane of Gram-negative bacteria acts as an additional permeability barrier that limits bacteriocin penetration. These findings are consistent with previous reports demonstrating that bacteriocin-producing staphylococci naturally colonize the human skin microbiome and produce antimicrobial peptides capable of inhibiting clinically important pathogens [9].

In addition to its antibacterial activity, bacteriocin exhibited remarkable antibiofilm effects. Treatment with crude bacteriocin significantly reduced biofilm biomass in most MDR, XDR, and PDR isolates, converting many strong biofilm producers into weak producers. These findings support previous evidence indicating that staphylococcal bacteriocins interfere with biofilm maturation and disrupt established biofilm architecture through mechanisms distinct from those of conventional antibiotics [10]. Such activity is particularly valuable because biofilm-associated infections are notoriously difficult to eradicate using standard antimicrobial therapy alone.

The synergistic activity observed between bacteriocin and conventional antibiotics further strengthens its therapeutic potential. The combination of bacteriocin with trimethoprim/sulfamethoxazole against Gram-negative bacteria and benzylpenicillin against Gram-positive bacteria produced significantly larger inhibition zones than either agent alone. These findings suggest that bacteriocins may enhance antibiotic penetration or increase bacterial membrane permeability, thereby improving antimicrobial efficacy against resistant pathogens. Combination therapy may therefore represent a promising strategy for reducing antibiotic dosage, minimizing toxicity, and slowing the development of antimicrobial resistance [11].

Hierarchical cluster analysis further demonstrated a close relationship between antimicrobial resistance, biofilm-forming ability, and bacteriocin susceptibility. MDR and XDR Escherichia coli and Klebsiella pneumoniae isolates tended to cluster together and were characterized by strong biofilm formation and reduced susceptibility to bacteriocin, whereas Staphylococcus isolates generally exhibited greater antibacterial susceptibility despite possessing robust biofilm-forming capabilities. These findings highlight the phenotypic diversity of clinical uropathogens and emphasize the importance of considering both antimicrobial resistance and virulence characteristics when evaluating alternative therapeutic strategies.

Although the findings are encouraging, several limitations should be acknowledged. The study was conducted at a single geographical location using a relatively limited number of isolates, and only crude bacteriocin preparations were evaluated. Purification and biochemical characterization of the bacteriocin were not performed, and its molecular mechanism of action was not investigated. Therefore, future multicenter studies should evaluate purified bacteriocins, determine their structural characteristics, investigate their mechanisms of antibacterial and antibiofilm activity, and assess their efficacy and safety in in vivo infection models before clinical application.

5. Conclusion

The present study demonstrated that Escherichia coli remains the predominant uropathogen among women with urinary tract infections and that a substantial proportion of isolates exhibit multidrug-resistant phenotypes and strong biofilm-forming capacity, both of which contribute to treatment failure and recurrent infection. Crude bacteriocin produced by Staphylococcus epidermidis exhibited potent antibacterial activity against the majority of clinical uropathogens, significantly inhibited biofilm formation, and enhanced the activity of conventional antibiotics through synergistic interactions. These findings suggest that bacteriocins represent promising alternative or adjunctive antimicrobial agents for the management of MDR, XDR, and biofilm-producing urinary pathogens.

Further studies should focus on purification and structural characterization of bacteriocins, elucidation of their molecular mechanisms of action, optimization of combination therapies with conventional antibiotics, and evaluation of their efficacy and safety in animal models and clinical trials. Such investigations may facilitate the development of bacteriocin-based therapeutics as an innovative approach to combating antimicrobial-resistant urinary tract infections.