Detection of intracellular bacterial communities and biofilms in urinary tract infections with Escherichia coli using staining protocols

Abstract

BACKGROUND

Urinary tract infections (UTIs) are prevalent worldwide, and Escherichia coli(E. coli) is the most common causative agent. The ability of the bacteria to form intracellular bacterial communities (IBCs) and biofilm is a major reason for UTIs. Studies have indicated that the persistence of uropathogenic E. coli as IBCs and biofilms has been implicated in UTIs. However, IBCs are not routinely identified by standard diagnostic methods.

AIM

To compare the various staining techniques for the detection of IBCs in urine samples from E. coli culture-positive UTI patients with the biofilm-forming capability of the isolates.

METHODS

The study included 73 patients with E. coli culture-confirmed UTI. Before antibiotic treatment midstream urine sample was collected, and the sediment was obtained by centrifugation. The samples were visualized using Sternheimer-Malbin, Wright-Giemsa, Safranin, and immunofluorescence staining to detect IBCs. Formation of biofilms was analyzed by the tube method. Descriptive statistics were used.

RESULTS

E. coli clusters were seen by light microscopy using various stains. However, immunofluorescence staining showed a better picture in the form of bright intracellular signals, which indicate bacterial aggregates. Biofilm assay showed an association with intracellular colonization.

CONCLUSION

The various staining techniques help in the identification of uropathogenic E. coli as IBCs inside superficial epithelial cells. These bacteria are also capable of forming biofilms, which resists action of antibiotics. Thus, IBCs and biofilms are rich reservoirs of organisms in the urinary bladder, paving the way for chronic treatment-resistant UTIs. This study requires further larger studies to substantiate these findings.

Key Words: Urinary tract infections; Intracellular bacterial communities; Biofilm formation; Sustainable Development Goals-3; Escherichia coli

Core Tip: Escherichia coli urinary tract infections are persistent through the formation of intracellular bacterial communities (IBCs) and biofilms, which are not detected by conventional diagnostic techniques. This work was designed to compare Sternheimer-Malbin, Wright-Giemsa, Safranin, and immunofluorescent staining methods for the detection of IBCs in urinary sediments. Compared with Wright-Giemsa and Sternheimer-Malbin, immunofluorescence was proven to have a higher detection rate for diagnosing IBCs. The results underscore the importance of novel diagnostic procedures and therapeutic strategies directed not only towards intracellular but also biofilm-embedded bacteria for the successful treatment of urinary tract infections.

INTRODUCTION

People of all ages, genders, and demographics are susceptible to urinary tract infections (UTIs), making them one of the most prevalent bacterial infections across the world. UTIs are the major causes for frequent hospital visits, laboratory tests to confirm the infection, and further initiation of multiple therapeutic regimens. Young- and middle-aged women are at higher risk of developing UTI, due to variations in anatomical structure, hormones, and sexual activity[1]. In addition, UTI is also prevalent in elderly individuals, diabetics, patients on in-dwelling catheters, and immunocompromised individuals. Patients with UTI present with dysuria, hesitancy, fever, malaise, and frequency of urination. If left untreated, patients may develop spread of infections to the upper urinary system, resulting in pyelonephritis or sepsis, repeated infections, and resistance to antibiotic treatment[2]. The economic burden is also high due to chronicity. It affects individuals and society in the form of repeated visits to hospitals, frequent laboratory tests, administration of various therapeutic modalities, and loss of productivity[3]. This study aimed to analyze intracellular bacterial communities (IBCs) and biofilm formation in E. coli culture-positive UTI patients.

Uropathogenic Escherichia coli and its virulence factors

The most common pathogen of UTIs is Escherichia coli (E. coli). This is found in 80%-90% of community-acquired and nosocomial infections[2,4]. The uropathogenic E. coli (UPEC) strains have a rich and wide range of virulence factors that allow them to colonize, invade, and survive in the urinary tract[5]. Compared to commensal strains of the gastrointestinal tract, UPEC possesses specific virulence factors that are unique to the pathogen. The adhesins, such as type 1 and P fimbriae, help the bacteria invade the epithelial cells in the urinary bladder, and also, they resist being flushed during urination. UPEC also synthesizes toxins that could damage host tissues, produces siderophores to scavenge iron, and expresses immune-evasive proteins, thereby contributing to persistence within the urinary tract of the host[6]. UPEC persists in the urinary tract by alternating between free-floating (planktonic) growth, intracellular localization, and biofilm formation, despite adequate host defenses and antimicrobial treatments. These adaptive features are the reasons for the infection to recur, even with appropriate management. Unlike extracellular bacteria that can be detected by standard urine culture, bacteria like UPEC adopt an intracellular or biofilm-associated form, which creates a challenging situation for diagnosis and management[7].

IBCs as reservoirs of bacteria in UTI

Among the most remarkable adaptations of UPEC to survive in the urinary tract is their ability to invade epithelial cells in the superficial layer of the urinary bladder and establish the IBCs. The microorganism initially takes the shape of a rod, which tends to adhere to epithelial cells. Then, with the help of fimbriae and other adhesins, it starts invaginating the epithelial cells. After reaching the intracellular environment of epithelial cells, it multiplies using the intracellular milieu. Now the bacteria transform into a dense, clustered, and spherical form called IBCs. After reaching the maximum replication, and also during the routine turnover, the epithelial cells open up to shed the bacteria in urine. This facilitates the release of numerous UPEC into the external environment in a filamentous form. New UPEC revert to their original rod-shaped form, seeking new epithelia in the urinary tract to adhere to, thus leading to a constant invasion of the urinary system, and the cycle repeats (Figure 1)[8]. These IBCs are biofilm-like formations that are found inside the cytoplasm of urothelial cells[9].

Figure 1 The morphological cycle of intracellular bacterial communities[8] (Supplementary material). IBC: Intracellular bacterial community; UPEC: Uropathogenic Escherichia coli.

The intracellular environment provides several benefits to UPEC. The bacteria proliferate without any restriction until the infected cells are expelled directly or burst open to release the stored organisms into the lumen of the bladder. Obtaining the intracellular niche offers protection against host immune responses as well as the therapeutic effects of antibiotics[10]. In a few patients who are found to have IBCs in their urine samples, the culture samples show few colony counts, and they are falsely negative for the organism[8]. The infected urothelial cells are excreted in urine, and thus, there is a strong opportunity to observe the IBCs in urine sediments[11]. Since the existence of IBCs is strongly and closely linked to infection persistence, it has emerged as an interesting field of study.

Biofilm and treatment resistance

Along with intracellular colonization, biofilm formation is also another important survival technique of UPEC. Research explains that biofilms are organized communities of microbes that are surrounded by an extracellular matrix that facilitates their adhesion to surfaces, nutrient exchanges, and increased resistance to environmental stresses[12]. The biofilms may develop on the wall of the bladder and are more predominant on the indwelling catheters[13]. Catheter-induced UTI is hard to control, as the exposed surfaces are constantly covered with biofilm. E. coli is a particularly hard-to-eradicate biofilm-forming strain, whose biofilm environment reduces the penetration of antibiotics and thus develops tolerance[14]. Biofilm-forming UPEC strains are also linked to chronic infections even without catheterization[15]. Laboratory techniques, like the tube method, offer a viable means to assess the biofilm-forming ability of clinical isolates, and their usage has expanded the understanding of the persistence of bacteria.

Limitations for IBCs detection

IBCs are not easily identified in routine clinical practice, which is a major limitation. Standard methods can identify causative pathogens and antibiotic sensitivity, but cannot confirm intracellular colonization. To counter this, a more direct method is provided by microscopy-based techniques coupled with appropriate staining procedures. Classical stains, including Sternheimer-Malbin, Wright-Giemsa, and Safranin, give morphological images of bacteria in the exfoliated cells, whereas the immunofluorescence staining has a higher detection rate due to the stringent use of appropriate antibodies. Very few studies have explored the use of various staining methods for the detection of IBCs in clinical samples. The benefits and limitations need to be understood to introduce them into routine practice. These laboratory techniques have distinct advantages; however, the diagnostic ability of the various methods using clinical urine samples has not been thoroughly analyzed.

According to earlier studies, while intracellular protection of E. coli is offered by IBCs within host cells, biofilms offer extracellular protection[16]. Research has indicated that these microbial strains that can generate IBCs might also be in a better position to generate biofilms, but the degree of this association is not well comprehended[17]. Researching the association of the two survival mechanisms may help us to understand why certain patients are more susceptible to the occurrence of the disease, and why the traditional treatment procedures are not effective in all clinical encounters.

Rationale of the study

There is a need for effective methods to identify intracellular bacterial colonization, given the clinical significance of chronic UTIs, especially in the vulnerable population. The relative analysis of staining methods, including classic stains and other sophisticated immunofluorescence methods, can provide a good understanding of the diagnostic potential of microscopic methods. Simultaneously investigating the biofilm-forming potential of clinical isolates and their association with IBCs may provide further insight into the pathogenesis of UPEC.

Summary of research gaps

Limited studies are available on the effectiveness of various staining protocols for the detection of IBCs in clinical urine samples. Similarly, although biofilm formation has been extensively studied, few studies are available on its association with IBC. The existence of these gaps provides justification to conduct a study that assesses the effectiveness of the staining procedures, analyzes the biofilm formation, and explores the potential relationships between the two.

Aim: To compare the various staining techniques for the detection of IBCs in urine samples from E. coli culture-positive UTI patients with the biofilm-forming capability of the isolates.

Objectives: To compare the IBCs visualized by the four staining techniques in E. coli positive urine samples. To score the biofilm-forming ability of E. coli isolates from culture-positive urine samples. To compare the biofilm-forming ability and immunofluorescence staining techniques of E. coli culture-positive urine samples.

Null hypothesis: Identifying IBCs using staining techniques and the biofilm-forming tube methods is not superior to traditional urine culture in E. coli-positive urine samples.

Alternate hypothesis: Identifying IBCs using staining techniques and the biofilm-forming tube method is superior to traditional urine culture in E. coli-positive urine samples.

MATERIALS AND METHODS

Study design

The prospective study was conducted at Sri Ramachandra Institute of Higher Education and Research (SRIHER), Chennai. The culture-positive UTI patients were recruited from the Department of General Medicine. The laboratory work was conducted collaboratively in the Departments of Biochemistry, Pathology and Microbiology. The patients were recruited between July 2025 and December 2025.

Study participants and their selection criteria

The study included 73 patients of both genders with E. coli culture-confirmed UTI. The sample size was calculated using Buderer’s method. The calculation assumed an expected sensitivity of 80%, specificity of 80%, disease prevalence of 15%, an absolute precision of ± 20%, and a confidence level of 90%. Based on these parameters, the estimated sample size was 73[18].

The inclusion criteria: Were patients aged 18-70 years of both genders. They should present with at least two clinical features of UTI, such as dysuria, fever, or frequency of urination. The included participants showed a colony count ≥ 10⁵ CFU/mL in their urine culture samples.

The exclusion criteria: Were presence of polymicrobial growth in urine, patients with any indwelling urinary tract device (catheter, ureteral stent), pregnant women, menstruating women, patients with hyperthyroidism, inflammatory bowel disease, acute coronary syndrome, acute ischemic stroke, acute respiratory failure, acute bronchospasm, and anaphylaxis, and patients on immunomodulating drugs, and steroids within 6 months. The study excluded patients older than 70 years to minimize confounding factors from age-related immunological changes and the high rate of comorbidities.

Data collection

Personal data, disease history, temperature, clinical features, and drug susceptibility of the participants were collected, along with relevant laboratory investigations.

Sample collection

Before starting antibiotic treatment, midstream urine samples were collected under complete aseptic conditions from patients in a sterile container. All urine samples were subjected to complete urine analysis and urine culture. Among them, the samples with E. coli culture growth ≥ 10⁵ were selected as participants.

Ethics statement

Data from all the participants were collected only after written informed consent was obtained. Personal data will not be revealed at any time to maintain participants’ confidentiality. Ethics approval was obtained from the Institutional Ethics Committee (IEC), SRIHER, Porur, Chennai (IEC-NI/24/APR/93/63, dated 24-06-2024).

Analytical methodology

Sample preparation and storage were done in the Department of Biochemistry. Light microscopy was used to detect IBCs in urine samples. Staining and immunofluorescence methodology were performed in the Department of Pathology and Sri Ramachandra Innovation and Incubation Centre, SRIHER, Chennai, respectively. The test-tube method to detect biofilm formation was evaluated in the Department of Microbiology, SRIHER.

Preparation of a urine sample for analysis

Within an hour of the urine collection, the samples were centrifuged for 10 minutes at 2000 rpm. The sediment was fixed with 10% formalin and analyzed to identify IBCs. Formalin-fixed sediments were stained with Sternheimer-Malbin, Wright-Giemsa, Safranin stains, and immunofluorescence microscopy to identify IBCs.

Sternheimer-Malbin staining

Formalin-fixed urinary sediment was mixed with an equal volume of Sternheimer-Malbin stain (1 drop each) (Globe Scientific, Mahwah, NJ, United States) and air-dried for 2 minutes to 6 minutes at room temperature. After placing the coverslip on the stained sample, it was allowed to dry for 10-20 minutes. The slides were viewed under 100 × oil immersion to identify rod-shaped gram-negative bacteria in the exfoliated urothelial cells[19].

Wright-Giemsa and Safranin staining

Formalin-fixed samples were placed on two clean glass slides and spread evenly using a sterile inoculating loop. The smears were allowed to air-dry and then heat-fixed. One slide was stained with Wright-Giemsa stain (Abcam, Cambridge, United Kingdom) and another with Safranin stain (Abcam, Cambridge, United Kingdom), and both were examined under light microscopy. This method was used to observe the morphology of the bacteria at 100 × oil immersion.

Immunofluorescence

In light microscopy, the light passes through the sample over the field of view. But it is not suitable for thicker solutions. In a confocal microscope, the illumination and detection optics are focused on the diffraction-limited (thicker) spot in the sample. This gives the advantage of constructing a three-dimensional image of the structure within the cells[11,20]. According to Robino et al[21], confocal microscopy shows bacterial collections in the cytoplasm of uroplakin cells in patients with UTIs.

Confocal microscopy was used to perform immunofluorescence staining using goat polyclonal fluorescein isothiocyanate anti-E. coli-E. coli K antibody (Abcam, Cambridge, United Kingdom, catalog No. ab20856), and rabbit polyclonal uroplakin III (Abcam, Cambridge, United Kingdom, No. ab137967). Formalin-fixed sediment was washed with phosphate buffer saline (PBS) (Thermo Fisher Scientific, Waltham, MA, United States, catalog No. 10010023), and the slide was blocked in 1% bovine serum albumin (Sigma-Aldrich, Missouri, United States, catalog No. A3858) and permeabilized with 0.3% Triton X-100 (SRL, Mumbai, India, catalog No. 64518) for one hour at room temperature. Then incubated for one hour with goat anti-E. coli (1/50) and rabbit anti-uroplakin III (1/50) primary antibodies in a dark room. Then, the samples were washed three times in PBS (5 minutes each) and incubated with Alexa Fluor 647 conjugated donkey anti-rabbit immunoglobulin G secondary antibodies (1/400) (Thermo Fisher Scientific, Waltham, MA, United States, catalog No. A21207) for 30 minutes. The slides were then stained with Thiazole Red (TOPRO-3) (Thermo Fisher Scientific, Waltham, MA, United States, catalog No. R37170) and mounted with fluoromount G (Thermo Fisher Scientific, Waltham, MA, United States, catalog No. 00-4958-02). Immunofluorescence is able to locate the bacteria, whether they were intracellular or extracellular. IBCs were identified based on established morphological criteria described in previous studies[21,22].

Biofilm detection by the tube method

Confirmed E. coli culture plates were used to assess the biofilm formation. Biofilm production was evaluated by 0.1% crystal violet staining intensity and classified as weak, moderate, or strong, as described by Christensen et al[23]. Trypticase soy broth was used as a nutrient medium for bacterial growth. A loopful of isolate from the culture plate was inoculated in a test tube containing 10 mL of trypticase soy broth supplemented with 1% glucose. The tubes were incubated at 37 °C for 24 hours. The broth was discarded the next day. The tube was washed with PBS (pH: 7.2) and dried at room temperature for 60 minutes. Later, the tubes were stained with 0.1% crystal violet, which was bound to bacterial cell walls, and the tubes were allowed to dry for 60 minutes. Excess stain was removed with deionized water, and the tubes were allowed to dry in an inverted position for 60 minutes. Based on the intensity of the stain, which was visible on the walls and bottom of the tubes, staining was scored as strong: Thick, and dense film, moderate: Visible but less dense film, and weak/absent: No film or just a ring at the liquid interface.

Statistical analysis

The data obtained from various IBC analyses and biofilm formation methods were qualitative or semi-quantitative in nature. The data expressions were mostly descriptive. Hence, frequency and percentage were used to compile the data. The data were only from culture-confirmed E. coli UTIs; culture-negative controls were not included in the study. Hence, formal diagnostic accuracy measures such as sensitivity, specificity, and receiver operating characteristic (ROC) curve analysis could not be calculated. The absence of a positive control and formal inferential statistical analysis (including sensitivity, specificity, and ROC curve evaluation) limits definitive conclusions regarding diagnostic performance.

RESULTS

A total of 73 urine samples from patients with E. coli culture-confirmed UTI were examined for the presence of IBCs using 4 different stains: Sternheimer-Malbin, Wright Giemsa, Safranin, and immunofluorescence. The demographic details are given in Table 1. Female participants were around 60%; the mean age was lower in females compared to males (Table 1). The detection rate of each method varied, and immunofluorescence showed the highest positivity in IBC identification. Biofilm assay was also performed on the corresponding E. coli isolates and associated with the immunofluorescence results. The number of IBC-positive samples detected by each method is summarized in Table 2.

Table 1 Demographic characteristics of the study participants, n (%).

Variables	Overall (n = 73)
Age (range) years
Male	24-70
Female	23-70
Gender
Male	29 (39.8)
Female	44 (60.2)
Number of UTI episodes in the past
NIL	73 (100)
> 1	0 (0)
Urine culture identification for E. coli
Positive	73 (100)
Negative	0 (0)

UTI: Urinary tract infection; NIL: Nothing; E. coli: Escherichia coli.

Table 2 Performance evaluation of staining protocols for intracellular bacterial community detection (n = 73), n (%).

Method	Number of samples with IBCs	Interpretation
Sternheimer-Malbin stain	15 (20.6)	Highlighted the urine sediment morphology, but the visualization of the intracellular bacteria against the background was vague
Wright-Giemsa stain	19 (26.0)	Better than Sternheimer-Malbin and Safranin stains for visualization of intracellular clusters
Safranin stain	11 (16.4)	Weak staining for intracellular bacteria; lowest sensitivity among the stains
Immunofluorescence (anti-E. coli)	27 (37.0)	Highest sensitivity due to specific antigen targeting

IBC: Intracellular bacterial community; E. coli: Escherichia coli.

Sternheimer-Malbin staining of IBCs

Out of 73 samples, the Sternheimer-Malbin stain identified IBC-like features in 15 samples (20.6%). Sternheimer-Malbin staining improved the differentiation of urinary sediments and showed the exfoliated epithelial cells with bacterial inclusions. Rod-shaped bacilli were seen in urothelial cells, much better than Safranin staining. The stain also made it easier to see white blood cells and casts, helping avoid misinterpreting the background debris (Figure 2). This method was found useful in identifying IBCs, but the poor contrast between the bacterial clusters and host cellular components reduced the detection rate.

Figure 2  Sternheimer-Malbin staining shows bacteria as purple rod-shaped bacilli present within the urothelial cells (arrow).

Wright-Giemsa staining of IBCs

The Wright-Giemsa stain detected IBCs in 19/73 samples (26.0%), indicating better detection compared to Sternheimer-Malbin and Safranin stains. Wright-Giemsa staining showed bacterial clusters in exfoliated urothelial cells present in urine sediment (Figure 3A). Host nuclei and cytoplasm were well differentiated, and dense, dark-blue aggregates were observed within the cytoplasm that were consistent with IBCs. In some sections, elongated filamentous bacterial organisms (Figure 3B) were also found, which were indicative of maturing IBCs. Some background staining and overlapping occurred in a limited number of fields.

Figure 3 Wright-Giemsa stain. A: Dense dark-blue bacterial cluster; B: Filamentous bacterial forms (indicated by arrows), highlighting elongated morphology suggestive of intracellular persistence.

Safranin staining of IBCs

Safranin staining showed the presence of intracellular bacterial inclusions in 12/73 samples (16.4%). However, contrast was less distinctive between the bacterial clusters and host cell structures (Figure 4) than with the Wright-Giemsa staining. This made it less reliable to differentiate intracellular from extracellular organisms.

Figure 4  Safranin staining showing pink granules of bacterial colonies (arrow) observed within exfoliated urothelial cells.

Identification of IBCs by immunofluorescence microscopy

IBC positivity was operationally defined as the localized presence of thick, discrete bacterial aggregates in the cytoplasm of exfoliated urothelial cells, easily distinguishable from extracellular bacteria or background debris. Co-localization of E. coli-specific fluorescence with uroplakin in urothelial cells was used to identify IBCs by immunofluorescence microscopy. Immunofluorescence is advantageous over the other three staining techniques since its detection capacity is higher, as well as a clear distinction of the location of bacteria within uroplakins. The Immunofluorescence microscopy method detected IBCs in 27/73 samples (37.0%) and proved to be the most effective method. Red fluorescence was used to visualize urothelial cells (Figure 5A), while bright green fluorescent signals were consistent with bacterial aggregates within the cells (Figure 5B). The merged image confirmed the intracellular localization of E. coli clusters (Figure 5C). Compared with other staining techniques evaluated in this study, immunofluorescence demonstrated a higher detection frequency of IBCs. IBC positive samples are further classified as shown in Table 3 and Figure 6.

Figure 5 Immunofluorescence microscopy showing intracellular Escherichia coli within urothelial cells. A: Red fluorescence indicating urothelial cell staining; B: Green fluorescence representing Escherichia coli-specific antibody labeling of bacterial aggregates; C: Merged image showing intracellular co-localization of red and green signals (orange), confirming intracellular bacterial communities.

Figure 6 Scoring of intracellular bacterial communities within uroplakin cells by the four staining methods. IBC: Intracellular bacterial community.

Table 3 Scoring of intracellular bacterial communities within uroplakin cells by the four staining methods.

Scoring1	Sternheimer-Malbin stain (n = 15)	Wright-Giemsa stain (n = 19)	Safranin stain (n = 11)	Immunofluorescence staining (n = 27)
Occasional	2	3	1	2
Few	4	1	2	9
Mild	1	2	2	1
Moderate	1	3	1	2
Plenty	7	10	5	13

¹Based on the number of intracellular bacterial communities within uroplakin cells, scoring was defined as: Occasional: < 5, few: 6-10, mild: 11-30, moderate: 31-60, plenty: > 61.

Biofilm formation assay - the tube method

The biofilm-forming ability of 73 E. coli isolates was tested by the semi-quantitative tube method, and the results are depicted in Table 4. After crystal violet staining, tubes had variable densities of biofilm attached to the walls and bases (Figure 7). In 38 out of 73 isolates (52.05%), there were thick linings of violet color inside the tubes, while 35/73 isolates (47.9%) exhibited poor or the absence of biofilm. These results suggested that a significant percentage of the clinical isolates possessed a strong ability to form biofilms, further supporting the ability of clinical isolates to persist and be resistant to management.

Figure 7  Test tube method showing biofilm formation from strong to weak.

Table 4 Analysis of Biofilm formation by Escherichia coli urinary tract infection patients.

	Strong/moderate biofilm	Weak/no biofilm
IBC positive (n = 27)	21	6
IBC negative (n = 46)	17	29

IBC: Intracellular bacterial community.

Based on the intensity of the stain, which was visible on the walls and bottom of the tubes, staining was scored from 3 to 1. Score 3 (Strong): Thick, and dense film, score 2 (moderate): Visible but less dense film, and score 1 (weak/absent): Just a ring at the liquid interface or absence of film. This scoring system is a semi-quantitative assessment, as adapted from a previously described tube-based biofilm assay[23]. By assigning the scores, the degree of variability between the isolates could be captured in a more consistent way, enabling meaningful comparisons. Such grading is important because the strength of biofilm is directly related to the persistence of bacteria, tolerance to antibiotics, and poor clinical outcomes in UTIs.

Association of IBCs with biofilm formation

When the presence of IBCs was compared to the biofilm-forming ability of the corresponding E. coli isolates, a significant trend was observed. Among 27 IBC-positive samples, 21 (77.8%) isolates showed moderate to high biofilm formation, while 29 out of 46 isolates (63.04%) from IBC-negative samples showed weak or no biofilm formation. These findings suggested a possible association between intracellular colonization of bacterial clusters and their biofilm-forming ability, and both mechanisms play crucial roles in the continuous colonization of the urinary tract.

DISCUSSION

UTIs continue to be a major health concern worldwide. The current study evaluated the sensitivity of IBC detection in E. coli culture-positive urine specimens by various staining techniques and determined their biofilm-forming capacity. The results indicated that IBCs are detectable in the exfoliated urothelial cells in urine. The immunofluorescence microscopy provided the best method, compared to the standard staining techniques. In the present study, the data obtained from various IBC analyses and biofilm formation methods were qualitative or semi-quantitative in nature. The data expressions were mostly descriptive. Hence, frequency and percentage were used to compile the data.

The IBC detection rate in the current study was 37.0% by immunofluorescence, which is consistent with previous studies that have shown IBC prevalence ranging from 30% to 40% in symptomatic UTI patients. This is similar to the findings of Rosen et al[11], who observed IBCs by confocal microscopy in 50% of urine specimens from women with uncomplicated cystitis. According to Robino et al[7], intracellular bacteria are detected by confocal microscopy in 36.8% of pediatric urine samples, reinforcing therefore that this phenomenon occurs in real clinical practice among different age groups of patients. In the present study, immunofluorescence microscopy allowed clear visualization of bacterial aggregates within urothelial cells through specific antibody-based labeling. This approach helps to obviate many of the interpretative difficulties encountered with conventional staining, as cellular debris, crystalline material, or extracellular bacteria could be mistaken for true IBCs. The better detection of immunofluorescence coincides with the findings of Kwak et al[9], who pointed out that sensitive methods to detect intracellular bacteria are crucial because standard culture methods may be falsely negative in certain circumstances. Since these results are consistent across studies, it is increasingly evident that intracellular colonization is a replicable aspect of UTIs, thus underscoring its major concern[7,11].

Sternheimer-Malbin staining, which identified IBC-like features in 20.6% of cases, was shown to be useful in differentiating bacteria from cellular debris, especially in specimens containing large amounts of inflammatory exudate. In our study, although sensitivities differed significantly, the contrast of the bacteria within cells was problematic with all the staining techniques. The Wright-Giemsa stain resulted in optimal contrast between bacterial aggregates and host cell features, enabling visualization of a high-density intracellular cluster (26.0%). This efficacy may be due to the differential affinities of the Romanowsky type dyes for bacterial nucleic acids and cytoplasmic material, leading to distinct coloration of bacterial structure. Safranin itself appeared to be least supportive of differentiation between intracellular bacteria from adjacent cellular structures, with a relatively low detection in 16.4% cases (Table 2). The fact that safranin works sub-optimally is possibly due to its application as a counter-stain rather than a primary method for visualization of bacteria, which may be present in complex biological matrices[24].

According to the biofilm assay, a large portion of E. coli isolates (52.05%) displayed moderate to strong capacity for forming biofilms. And most importantly, isolates from IBC-positive samples were more inclined to develop robust biofilm in 77.8% of the cases, which was significantly higher than that for IBC-negative patients (36.9%) (Table 4). This relationship indicated that the intracellular invasive and IBC-forming molecular machinery may utilize some of the same pathways for biofilm formation, also[25]. Adhesive structures, quorum sensing, and the capacity to synthesize components of the extracellular matrix are required by the two phenotypes of bacterial persistence, through two complementary mechanisms: IBCs offering intracellular sanctuaries shielded from extracellular immune responses and antibiotics, whereas biofilms form external protected niches on mucosal surfaces and indwelling devices[5].

As stated by Cangui-Panchi et al[16], IBCs and biofilms are adaptive bacterial responses to a hostile host environment, allowing them to avoid immune clearance and antibiotic treatment. In the same way, Tenke et al[17] reported that biofilm-producing bacteria developed increased resistance to antibiotics, and their minimum inhibitory concentrations often exceeded achievable in vivo concentrations, including in urine. The current results are in alignment with the early studies, that some IBC-forming clinical isolates from UTI patients are also strong biofilm producers, indicating a broad spectrum of virulence among certain uropathogenic strains[17].

The finding of filamentous bacterial forms in some specimens is another clinically relevant observation. Filamentous phenotype has been linked to late-stage IBC progression, and it is a survival strategy of the bacterium to evade immune killing. These long forms are too big to be effectively phagocytosed by the neutrophils, and can potentially promote attachment of bacteria to urothelial cells[25]. There have been reports that the IBC growth resembled the filamentous phenotype, which would be indicative of cellular stress caused by antibiotics and nutritional limitations due to high bacterial load[11,26].

The results of this study emphasized the importance of identifying IBCs and biofilms and that they are associated with survival strategies of UPEC. This information facilitates the derivation of combination therapy, which targets intracellular and extracellular bacterial populations[14,15]. The study highlighted the clinical significance of IBCs in chronic UTIs and demonstrated applications of various staining techniques. Immunofluorescence showed a higher detection rate compared to other stains. In resource-limited settings, the ease of Wright-Giemsa and Sternheimer-Malbin staining techniques may be potential screening tools. A two-tiered diagnostic approach can be employed, with conventional staining as a screening tool followed by immunofluorescence for diagnosis.

Clinically, our study highlighted the need for therapeutic strategies targeting intracellular reservoirs, in addition to biofilm structures, to decrease the recurrence and increase the treatment success. The significance of IBCs and biofilms in the pathogenesis of UTIs aligns with the Sustainable Development Goals. In alignment with this, the study contributes to Sustainable Development Goals-3 (Good health and wellbeing) as it targets chronic UTIs and antimicrobial resistance through improved diagnostics. Consequently, critical breakthroughs may enhance the diagnosis and treatment of UTIs through the advent of highly sensitive laboratory methods, and thus introduce specific treatment to the affected individuals, which would discourage further advancement of the disease.

According to the literature, urine culture is considered to be the method of choice in identifying the causative microorganism in UTIs[27]. However, it cannot be used for identifying IBCs. Hence, staining methods may offer the advantage of circumventing the limitations of cultures. Among the staining methods used for identifying IBCs, Sternheimer-Malbin and Safranin stains have limited specificity, hence may be used for screening purposes. Wright-Giemsa staining is better at giving cellular details, but effective clarity is low. Immunofluorescence staining seems to deliver the specificity and sensitivity compared to other staining techniques because it offers the morphological advantage. But the limitation with immunofluorescence is technical expertise and infrastructure expertise facilities.

Limitations

There are a few limitations in sampling and techniques. Due to the heterogeneity in IBC prevalence across the patient groups or infection severity, the sample size may be insufficient to address all the characteristics of IBCs. Moreover, the research was limited to samples with only E. coli infection. Since IBCs may be transiently shed in the urine, single-sample analysis may result in false-negative findings. The absence of a positive control and formal inferential statistical analysis (including sensitivity, specificity, and ROC curve evaluation) limits definitive conclusions regarding diagnostic performance. In immunofluorescence, Z-stack/orthogonal imaging of confocal microscopy was not performed. Additionally, variability in semi-quantitative scoring may introduce subjective bias. The impact of IBC on clinical progression and treatment outcomes requires further studies.

Future research directions

Since IBCs may be transiently shed in the urine, multiple-sample analysis may reduce or remove false-negative findings. Future multicenter studies with larger sample sizes, longitudinal follow-up, antimicrobial susceptibility profiling, a standardized scoring system, and comprehensive statistical validation are required to establish the diagnostic utility of these staining techniques. Use of positive and negative controls may enhance the credibility of the data obtained. Formal inferential statistical analysis (including sensitivity, specificity, and ROC curve evaluation) could enhance conclusions regarding diagnostic performance. In immunofluorescence, technical performance regarding three-dimensional visualization can be incorporated. Additionally, variability in semi-quantitative scoring may be reduced with stringent scoring methods. The impact of IBC on clinical progression and treatment outcomes will be studied in further research.

CONCLUSION

The current study revealed that IBCs are detected in exfoliated urothelial cells, and the immunofluorescent test had a high detection rate of 37.0%, while Wright-Giemsa (26.0%) and Sternheimer-Malbin (20.6%) are appropriate as screening tests. Safranin had a poor detection rate (16.4%) and showed supportive findings. Biofilm test showed that a large proportion of E. coli isolates were either moderate or strong biofilm producers. Crucially, isolates associated with IBC-positive samples were also more likely to exhibit good biofilm formation, which could be linked to intracellular evasion and extracellular persistence.

ACKNOWLEDGEMENTS

The authors would like to express their sincere gratitude to the management of SRIHER, Chennai.