Adherence Assay of Uropathogenic Escherichia coli In Vivo and In Vitro

Duk Yoon Kim; Je Chul Lee

doi:10.14777/uti.2017.12.03.122

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Kim and Lee: Adherence Assay of Uropathogenic Escherichia coli In Vivo and In Vitro

Original Article

Urogenit Tract Infect 2017;12(3):122-129.

Published online: 19 January 2017

DOI: https://doi.org/10.14777/uti.2017.12.03.122

Adherence Assay of Uropathogenic Escherichia coli In Vivo and In Vitro

Duk Yoon Kim, Je Chul Lee¹

Department of Urology, Catholic University of Daegu College of Medicine, Daegu

¹Department of Microbiology, Kyungpook National University, Daegu, Korea

Correspondence to: Duk Yoon Kim http://orcid.org/0000-0001-8341-3123 Department of Urology, Catholic University of Daegu College of Medicine, 33 Duryugongwon-ro 17-gil, Nam-gu, Daegu 42472, Korea Tel: +82-53-650-4663, Fax: +82-53-623-7507 E-mail: dykim@cu.ac.kr

Received 11 November 2017 Revised 12 December 2017 Accepted 12 December 2017

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose:

This study aims to investigate the specific pathogenic properties or virulent determinant characteristics of uropathogenic Escherichia coli (UPEC) as bacterial adherence in tissue culture cells in vitro and the pathogenicity in animal model in vivo.

Materials and Methods:

Thirty strains of E. coli were isolated from urine of patients with acute pyelonephritis. Four cell lines—HeLa cells, HEp-2 cells, A-498 cells, and J-82 cells—were used for bacterial adherence assay. Histologic examination and scanning electron microscopy examination of pyelonephritis or cystitis, which was caused by E. coli, in mice was performed.

Results:

Sixteen (53.3%) strains of E. coli appeared to adhere to at least one or more kinds of four cell lines, and seven strains were able to adhere to all four cell lines. All of the tested E. coli strains were adhered to the mouse bladder and kidneys. The number of bacteria colonized in the kidney was greater than that of bladders in the following 5 strains of E. coli: TME104, TME107, TME113, TME306, and TME119. There was no difference in the number of bacteria colonized in the bladder and kidneys in the aspects of adherence patterns to tissue culture cells.

Conclusions:

Although there was no best choice cell lines in the adherence assay to identify the adherence patterns, combined assays of in vitro cell culture and in vivo model of mouse urinary tract infection appeared to be efficient methods to investigate the role of bacterial adherence in the pathogenesis of UPEC.

Keywords: Urinary tract infections, Virulence factors, Escherichia coli

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Fig. 1.

Light micrographs showing 3 types of adherence pattern in HeLa, HEp-2, A-498, and J-82 cells. DA denotes a diffuse adherence to the TME104 strain on the dispersed surface of the cultured cells. LA represents a localized adherence of the TME303 strain to the localized areas of cultured cells and formed to particulate microcolonies. AA denotes an aggregative adherence of TME114 strain to the cultured cells. Note the characteristic aggregates of bacteria that showed “stacked-brick” appearance. They bound aggregately to both of cultured cells and bottom of cell culture dish (×500).

Fig. 2.

Inflammation of the mouse bladder (A) and kidney (B) caused by bacterial infections. H&E, ×100.

Fig. 3.

Scanning electron micrographs showing diffuse adherence (DA) of TME104, localized adherence (LA) of TME303, and aggregative adherence (AA) of TME114 Escherichia coli strains adhered to J-82 cells. Magnification, ×1,500.

Fig. 4.

Scanning electron micrographs showing TME116 (A, B), TME303 (C, D), and TME114 (E, F) Escherichia coli strains adhering to mouse urinary bladder. Magnification, A, C, E: ×2,200; B, D, F: ×15,000. DA: diffuse adherence, LA: localized adherence, AA: aggregative adherence, NA: non-adherence.

Table 1.

Adherence of Escherichia coli strains to the host cells

Strain no.	HeLa	HEp-2	A-498	J-82
TLE102	DA (54.5±14.8)	DA (82.0±7.4)	AA (68.2±5.3)	AA (45.3±9.6)
TLE103	NA	NA	LA (17.0±4.4)	LA (27.8±6.5)
TLE104	NA	NA	NA	LA (34.3±7.1)
TLE105	NA	NA	NA	LA (17.0±0.8)
TME102	LA (11.2±5.8)	LA (16.2±2.3)	NA	NA
TME103	NA	NA	DA (18.2±6.1)	DA (19.5±4.2)
TME104	DA (178.3±16.5)	DA (208.8±17.4)	DA (37.5±12.4)	DA (147.8±22.4)
TME107	LA (9.7±3.9)	LA (7.8±1.3)	NA	NA
TME108	DA (70.2±20.0)	DA (53.5±10.6)	AA (70.0±19.7)	DA (40.5±10.8)
TME113	LA (31.2±9.8)	LA (35.2±8.6)	DA (15.2±4.1)	DA (16.0±2.8)
TME114	AA (39.5±5.9)	AA (32.0±5.8)	AA (34.8±7.2)	AA (30.5±8.0)
TME116	DA (19.0±5.0)	DA (31.0±4.6)	DA (24.0±5.5)	DA (17.0±5.5)
TME303	LA (29.3±4.8)	LA (45.5±5.0)	LA (35.8±14.8)	LA (24.2±5.7)
TME304	NA	NA	LA (27.8±5.4)	LA (32.7±15.2)
TME306	AA (14.0±2.4)	AA (28.0±5.3)	NA	NA
TME307	AA (22.5±7.4)	AA (43.0±3.4)	ND	ND

The number of bacteria adhered to each host cells are presented as mean±standard error of the mean.

DA: diffuse adherence, AA: aggregative adherence, NA: non-adherence, LA: localized adherence, ND: almost all cultures cells were detached and lysed, and adherence pattern and degree of E. coli strains could not be determined.

Table 2.

In vivo adherence of Escherichia coli strains to mouse bladder and kidneys

Strain no.	Adherence pattern	No. of bacteria (/0.1 ml of tissue homogenate)^a)
Strain no.	Adherence pattern	Bladder	Kidneys
TME104	DA	5.5×10⁷	1.7×10⁸
TME108	DA	9.4×10⁴	3.5×10⁴
TME116	DA	2.1×10⁵	6.7×10⁴
TME107	LA	3.5×10⁴	9.5×10⁴
TME113	LA	3.2×10⁴	1.3×10⁵
TME303	LA	7.1×10⁶	2.2×10³
TME114	AA	7.4×10⁸	5.2×10⁸
TME306	AA	3.1×10⁵	3.3×10⁵
TME307	AA	1.2×10⁴	6.0×10²
TME115	NA	9.2×10⁵	7.2×10⁵
TME117	NA	5.1×10⁵	6.3×10³
TME119	NA	1.2×10⁴	6.1×10⁶

DA: diffuse adherence, LA: localized adherence, AA: aggregative adherence, NA: non-adherence.

^a) The numbers of bacteria adhered to bladder and kidneys were the mean numbers of double experiments.

Table 3.

Histological changes of mouse urinary tract after intravesical inoculation with Escherichia coli strains

Strain no.	Adherence pattern	No. of mice with
Strain no.	Adherence pattern	Cystitis & pyelitis	Only cystitis	Only pyelitis
TME116	DA	3/5	0/5	2/5
TME303	LA	2/3	1/3	0/3
TME114	AA	0/4	3/4	0/4
TME115	NA	0/3	1/3	1/3

DA: diffuse adherence, LA: localized adherence, AA: aggregative adherence, NA: non-adherence.

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