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

doi:10.5493/wjem.v16.i2.119306

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World Journal of Experimental Medicine

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2220-315x

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

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Copyright: ©Author(s) 2026. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial (CC BY-NC 4.0) license. No commercial re-use. See permissions. Published by Baishideng Publishing Group Inc.

World J Exp Med. Jun 20, 2026; 16(2): 119306
Published online Jun 20, 2026. doi: 10.5493/wjem.v16.i2.119306

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

Swathi Pandey, Arul Senghor Kadalangudi Aravaanan, Emmanuel Bhaskar, Santhi Silambanan

Swathi Pandey, Emmanuel Bhaskar, Department of General Medicine, Sri Ramachandra Institute of Higher Education and Research, Chennai 600116, Tamil Nadu, India

Arul Senghor Kadalangudi Aravaanan, Department of Biochemistry, SRM Institute of Science and Technology, Kattangulathur, Chennai 603203, Tamil Nadu, India

Santhi Silambanan, Department of Biochemistry, Sri Ramachandra Institute of Higher Education and Research, Chennai 600116, Tamil Nadu, India

Co-corresponding authors: Emmanuel Bhaskar and Santhi Silambanan.

Author contributions: Swathi P, Bhaskar E, Aravaanan ASK, and Silambanan S designed the research study, analyzed the data, and wrote the manuscript; Swathi P and Silambanan S contributed analytic tools; Bhaskar E and Silambanan S have played important and indispensable roles in the manuscript preparation as the co-corresponding authors; all authors have read and approved the final manuscript.

Supported by Founder Chancellor Shri. N.P.V. Ramasamy Udayar Research Fellowship, SRIHER, Chennai, No. U022300967.

Institutional review board 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).

Clinical trial registration statement: The study does not require registration as it does not meet the definition of a clinical trial.

Informed consent 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.

Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.

CONSORT 2010 statement: The authors have read the CONSORT 2010 Statement, and the manuscript was prepared and revised according to the CONSORT 2010 Statement.

Data sharing statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Corresponding author: Santhi Silambanan, MD, Professor, Department of Biochemistry, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai 600116, Tamil Nadu, India. santhisilambanan@sriramachandra.edu.in

Received: January 26, 2026
Revised: February 13, 2026
Accepted: April 2, 2026
Published online: June 20, 2026
Processing time: 140 Days and 1.3 Hours

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.

Keywords: 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.