1. Background
Klebsiella pneumoniae is a ubiquitous encapsulated bacterium found in diverse environments, including soil, water, and medical surfaces. It commonly colonizes human mucosal sites, particularly the oropharynx and gastrointestinal tract, from which it can disseminate to other tissues (1). Klebsiella pneumoniae is a significant opportunistic pathogen responsible for serious clinical conditions, such as liver abscesses, meningitis, and urinary tract infections (UTIs) (2). Although newborns, older adults, and immunocompromised individuals are at the highest risk, community-acquired infections are increasingly reported (3). Klebsiella pneumoniae is recognized as a major cause of Gram-negative infections, including septicemia, endocarditis, pneumonia, and surgical site infections, and is characterized by increasing rates of antimicrobial resistance (4-7). Biofilm formation by K. pneumoniae was first reported in the late 1980s and was later confirmed in vivo. Approximately 40% of isolates form biofilms, increasing to 63% in catheterized patients, and many ventilator-associated pneumonia strains also demonstrate this ability. Biofilms increase stress tolerance and antibiotic resistance, thereby enhancing bacterial virulence (8, 9).
Bacteria communicate through quorum sensing (QS), a regulatory system based on the production and detection of extracellular signaling molecules known as autoinducers (AIs) (10). The LuxS enzyme facilitates the synthesis of AI-2 from S-ribosylhomocysteine (11). AI-2 signaling regulates the expression of numerous genes and influences key physiological processes, such as virulence, protein secretion, extracellular polysaccharide production, iron uptake, motility, and biofilm formation (12). LuxS strongly affects the bacterial phenotype and contributes to K. pneumoniae pathogenicity through several mechanisms (13). LuxS has been implicated in the modulation of host immune responses, biofilm formation, and the regulation of capsular polysaccharide (14). Previous studies have demonstrated that LuxS-mediated AI-2 quorum sensing influences biofilm architecture and the expression of outer-membrane-associated genes in K. pneumoniae. Subsequent evidence indicates that LuxS-dependent virulence effects are strain-specific and growth phase-dependent, particularly among isolates with different mucoid phenotypes (15).
Although the molecular role of LuxS in quorum sensing, virulence modulation, and biofilm formation in K. pneumoniae has been extensively studied, few studies have reported the distribution of the luxS gene among clinical urinary isolates and its possible association with biofilm-forming ability. Furthermore, epidemiological evidence on this topic in Iran remains limited. Therefore, this study was conducted to determine the prevalence of the luxS gene among urinary Klebsiella isolates and to assess its association with biofilm formation and antimicrobial resistance patterns, thereby providing clinically relevant information on the role of LuxS in UTIs.
2. Objectives
The present study aimed to determine the prevalence of the luxS gene in clinical urinary isolates of K. pneumoniae and to assess the association of the luxS gene with biofilm formation and antimicrobial resistance patterns, thereby providing clinically relevant insights into the molecular determinants of K. pneumoniae UTIs.
3. Methods
3.1. Bacterial Isolates
This cross-sectional study was conducted from September 2022 to March 2023 at the reference laboratory of Kermanshah University of Medical Sciences, Iran. The study used anonymized urinary isolates routinely received by the microbiology laboratory for diagnostic purposes. Clinical samples were cultured on MacConkey and blood agar and incubated at 37 °C for 24 hours. Colonies were identified by microscopy, biochemical tests, and IMViC. Confirmed isolates were stored at -70 °C in trypticase soy broth containing 18% glycerol for further analysis. All eligible Klebsiella isolates recovered from urine specimens during the study period were consecutively included. Accordingly, all available isolates meeting the inclusion criteria were included in the study population, and the sample size was determined by the total number of eligible isolates collected rather than by a predefined target sample size.
3.2. Antimicrobial Susceptibility Testing
Antibiotic susceptibility was assessed using the disk diffusion method on Mueller-Hinton agar. The antibiotics tested were amikacin, ampicillin, sulfamethoxazole-trimethoprim, ceftazidime, cefazolin, gentamicin, nalidixic acid, and nitrofurantoin. Results were interpreted according to CLSI guidelines (16). Escherichia coli ATCC 25922 and K. pneumoniae ATCC 9997 were used as quality-control strains.
3.3. Biofilm Assay
Biofilm formation was evaluated using the microtiter plate assay. Briefly, bacterial suspensions were inoculated into sterile 96-well microplates and incubated under appropriate conditions for biofilm development. The wells were gently washed to remove planktonic cells, and the adherent biofilm was fixed and stained with crystal violet. The bound dye was then solubilized, and optical density (OD) was measured using a microplate reader. Based on the OD values, isolates were classified as non-biofilm producers or as weak, moderate, or strong biofilm producers according to established criteria described in previous studies (17-19).
3.4. Genomic DNA Preparation
DNA was extracted using the boiling method (20). DNA absorbance and concentration at 260/280 nm were measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). The DNA templates were stored at -20 °C until further analysis.
3.5. PCR Amplification of the luxS Gene
Polymerase chain reaction (PCR) using gene-specific primers (Table 1) was performed to detect the luxS gene. The PCR was performed in a final volume of 25 µL containing 15 µL Taq premix, 2 µL template DNA, 1 µL forward primer, 1 µL reverse primer, and 6 µL sterile distilled water. Amplification began with initial denaturation at 95 °C for 3 minutes, followed by 35 cycles of denaturation, annealing at 53 °C for 1 minute, and extension. A final extension at 72 °C for 5 minutes was performed to complete DNA synthesis. The amplified PCR products were separated by electrophoresis on a 1% agarose gel and visualized under UV illumination. Klebsiella pneumoniae ATCC 9997 was included as a control strain to verify amplification accuracy.
3.6. Statistical Analysis
Statistical analysis was performed using SPSS version 19 (Chicago, IL, USA). Chi-square tests were used to evaluate associations between categorical variables, such as luxS gene presence and biofilm intensity. A P value < 0.05 was considered statistically significant.
4. Results
4.1. Bacterial Identification and Demographic Features
A total of 51 urinary isolates were obtained and examined. Of these, 6 (11.8%) isolates were recovered from male patients and 45 (88.2%) from female patients. The mean age of the patients was 40.12 years. Using conventional culture methods and IMViC biochemical testing, 32 isolates (62.75%) were identified as K. pneumoniae, whereas 19 isolates (37.25%) were identified as K. oxytoca. These results indicate that K. pneumoniae was the predominant species among the urinary isolates.
4.2. Antimicrobial Susceptibility Pattern
Antimicrobial susceptibility testing showed that the isolates exhibited distinct resistance profiles. The highest resistance rates were observed for ampicillin (88.2%) and gentamicin (86.2%). In contrast, the highest susceptibility rate was observed for amikacin (74.5%) (Table 2).
Table 2.
Antimicrobial Susceptibility Profile of Klebsiella pneumoniae Isolates Obtained from Urine Samples a
| Antibiotics | Susceptibility | Intermediate | Resistant |
|---|---|---|---|
| AM | 6 (11.8) | - | 45 (88.2) |
| CAZ | 16 (31.4) | 22 (43.1) | 13 (25.5) |
| NA | 16 (31.4) | 23 (45) | 12 (23.5) |
| GM | 44 (86.2) | 4 (7.8) | 3 (6) |
| FM | 32 (62.74) | 7 (13.72) | 12 (23.5) |
| AN | 38 (74.5) | 8 (15.68) | 5 (9.8) |
| SXT | 35 (68.6) | 4 (7.8) | 12 (23.5) |
| CZ | 15 (29.4) | 7 (13.7) | 29 (56.9) |
a Values are expressed as No. (%). Abbreviations: AM, ampicillin; CAZ, ceftazidime; NA, nalidixic acid; GM, gentamicin; FM, nitrofurantoin; AN, amikacin; SXT, sulfamethoxazole/trimethoprim; CZ, cefazolin.
4.3. Biofilm Formation
Biofilm-forming capacity was assessed for all isolates. A considerable proportion of the isolates was able to produce biofilm. The proportions of strong, moderate, and weak biofilm producers were significantly associated with one another (chi-square = 10.83, df = 2, P = 0.0044), indicating variation in biofilm-forming potential among the isolates. Biofilm formation indicates an increased ability of the isolates to persist in the urinary tract and to withstand antimicrobial therapy (Table 3).
Table 3.
Association Between Biofilm-Forming Capacity and Presence of the luxS Gene Among Urinary Klebsiella pneumoniae Isolates a
| Biofilm strength | luxS+ | luxS- | Total |
|---|---|---|---|
| Weak | 1 | 5 | 6 |
| Moderate | 9 | 0 | 9 |
| Strong | 22 | 14 | 36 |
| Total | 32 | 19 | 51 |
a Values are expressed as number. Chi-square test = 10.83; df = 2; P = 0.0044.
4.4. Detection of the luxS Gene
To determine the distribution of the luxS gene, PCR was performed on 51 Klebsiella strains isolated from urine cultures. The results showed that 32 isolates (62.7%) harbored the gene (Figure 1), indicating a substantial frequency within the tested population. Furthermore, the data demonstrated a clear correlation between the presence of luxS and biofilm-forming capacity. Overall, these clinical isolates exhibited substantial luxS carriage, marked biofilm development, and antibiotic resistance.
Figure 1.
Agarose gel electrophoresis of PCR products obtained for detection of the luxS gene in Klebsiella pneumoniae isolates. Lane M: 100-bp DNA ladder; lane 1: negative control; lane 2: positive control; lanes 3 and 4: positive isolates (475 bp).
5. Discussion
The increasing incidence of antibiotic-resistant Klebsiella species worldwide represents a serious public health threat (22). Biofilm formation further increases the pathogenic potential of these organisms by protecting bacterial cells from host immune responses and reducing antimicrobial penetration. In the present study, we evaluated antimicrobial resistance patterns, biofilm-forming ability, and the presence of the luxS gene among urinary Klebsiella isolates. Among the 51 isolates examined, most were obtained from female patients (88.2%), consistent with previous reports indicating a higher prevalence of UTIs among women (23). This increased susceptibility is attributed to the shorter female urethra and its close anatomical relationship with the perianal area, which facilitates colonization of the urinary tract by Gram-negative bacteria (24). Female sex remains a major epidemiological risk factor for UTIs.
Antimicrobial susceptibility testing revealed high resistance rates to ampicillin (88.2%) and cefazolin (56.9%). Resistance to the other tested antibiotics varied but remained clinically significant. These findings are consistent with previous reports indicating high resistance levels among Klebsiella isolates from UTIs (23, 25-26). Differences among studies are likely related to geographic location, clinical specimen type, antibiotic-prescribing guidelines, and local hygiene practices. In the current study, most isolates were strong biofilm producers (70.5%), whereas fewer were moderate (17.6%) or weak (11.7%) biofilm producers. These results are similar to the findings of Vuotto et al., who reported a predominance of strong biofilm-producing urinary K. pneumoniae isolates (22). Other studies have confirmed the ability of clinical Klebsiella strains to form biofilms, although prevalence rates vary by methodology and sample type (25, 27). Biofilm formation is a key factor in catheter- and device-related infections and a major contributor to chronicity and treatment failure (28).
In addition to its general role as a virulence factor, biofilm formation in K. pneumoniae is a complex, highly regulated process influenced by multiple regulatory systems, including quorum sensing. Quorum sensing enables bacterial populations to coordinate gene expression in response to cell density through the production and detection of signaling molecules. Among these systems, the LuxS-mediated signaling pathway is particularly important because it is responsible for the synthesis of autoinducer-2 (AI-2), a universal signaling molecule involved in interspecies bacterial communication (21, 29). In the present study, the luxS gene was detected in 62.7% of the isolates, and the results suggested an association between luxS gene carriage and biofilm-forming capacity. These findings support the hypothesis that LuxS-mediated quorum sensing may contribute to the regulation of biofilm formation in clinical Klebsiella isolates. Previous studies have shown that disruption of the luxS gene can significantly affect biofilm architecture, bacterial adhesion, and community behavior, indicating that the LuxS/AI-2 system plays an important role in biofilm development (30, 31).
The AI-2 signaling molecule produced by LuxS participates in the regulation of genes involved in adhesion, extracellular matrix production, motility, and stress responses, all of which are critical for the initiation and maturation of bacterial biofilms (32, 33). In K. pneumoniae, quorum sensing has also been associated with increased virulence, enhanced persistence in host tissues, and improved adaptation to hostile environmental conditions. Recent investigations focusing specifically on K. pneumoniae have further highlighted the importance of the LuxS/AI-2 system in biofilm regulation and pathogenicity. Experimental studies have demonstrated that alteration or deletion of luxS affects biofilm biomass, outer membrane protein expression, and the structural stability of biofilm communities (34, 35). Moreover, the LuxS/AI-2 signaling pathway has been implicated in facilitating horizontal gene transfer and plasmid dissemination, potentially contributing to the spread of antimicrobial resistance among clinical isolates (15, 36). Additional reports indicate that quorum sensing may modulate virulence-associated traits, including capsule production and stress adaptation, thereby enhancing bacterial persistence in host tissues (37, 38).
Overall, the findings of the present study indicate that biofilm formation and the presence of the luxS gene are closely associated virulence characteristics in urinary Klebsiella isolates. This relationship may partly explain infection persistence and the reduced effectiveness of antimicrobial therapy in biofilm-associated UTIs. Therefore, understanding the molecular mechanisms underlying quorum sensing and biofilm regulation may contribute to the development of novel therapeutic strategies targeting bacterial communication systems in addition to conventional antibiotic therapy.
5.1. Conclusions
The present study demonstrated frequent strong biofilm-forming ability and a high prevalence of the luxS gene in urinary Klebsiella isolates, especially K. pneumoniae. The observed association between luxS carriage and biofilm formation suggests that quorum sensing may play an important role in the pathogenicity and persistence of these isolates in UTIs. Furthermore, the high level of antimicrobial resistance observed, particularly to ampicillin and cefazolin, underscores the therapeutic challenges posed by these organisms. Overall, the results highlight the clinical importance of biofilm formation and luxS-dependent quorum sensing in Klebsiella infections. Improved understanding of these mechanisms could lead to new strategies for preventing and treating UTIs associated with biofilms and drug resistance.
