Antimicrobial activity of intraurethrally administered probiotic Lactobacillus casei in a murine model of Escherichia coli urinary tract infection

Abstract

The antimicrobial activity of the intraurethrally administered probiotic Lactobacillus casei strain Shirota against Escherichia coli in a murine urinary tract infection (UTI) model was examined. UTI was induced by intraurethral administration of Escherichia coli strain HU-1 (a clinical isolate from a UTI patient, positive for type 1 and P fimbriae), at a dose of 1 x 10(6) to 2 x 10(6) CFU in 20 microl of saline, into a C3H/HeN mouse bladder which had been traumatized with 0.1 N HCl followed immediately by neutralization with 0.1 N NaOH 24 h before the challenge infection. Chronic infection with the pathogen at 10(6) CFU in the urinary tract (bladder and kidneys) was maintained for more than 3 weeks after the challenge, and the number of polymorphonuclear leukocytes and myeloperoxidase activity in the urine were markedly elevated during the infection period. A single administration of L. casei Shirota at a dose of 10(8) CFU 24 h before the challenge infection dramatically inhibited E. coli growth and inflammatory responses in the urinary tract. Multiple daily treatments with L. casei Shirota during the postinfection period also showed antimicrobial activity in this UTI model. A heat-killed preparation of L. casei Shirota exerted significant antimicrobial effects not only with a single pretreatment (100 microg/mouse) but also with multiple daily treatments during the postinfection period. The other Lactobacillus strains tested, i.e., L. fermentum ATCC 14931(T), L. jensenii ATCC 25258(T), L. plantarum ATCC 14917(T), and L. reuteri JCM 1112(T), had no significant antimicrobial activity. Taken together, these results suggest that the probiotic L. casei strain Shirota is a potent therapeutic agent for UTI.

FIG. 1

A murine chronic UTI model. C3H/HeN mice were divided into two groups. Traumatization of the bladder was performed as described in Materials and Methods for one group (●), and another group (control) (○) was mock treated with saline. E. coli strain HU-1 at a dose of 2 × 10⁶ CFU was infused into the bladders of anesthetized mice. The counts of viable bacteria in the bladder (A) and kidneys (B) were determined on days 1, 4, 7, 10, 14, and 24 after the challenge. Results are expressed as the means and SDs for six mice. Significant differences between the untreated controls and the treated group: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

FIG. 2

Preventive effect of L. casei Shirota against chronic UTI. (A through C) Either L. casei Shirota at a dose of 1.2 × 10⁸ CFU in 20 μl of saline (○) or saline alone (control) (●) was infused into the bladder 15 min after traumatization, and E. coli strain HU-1 at a dose of 1.8 × 10⁶ CFU was infused into the bladder 24 h later. On days 0 (just after infection), 1, 4, and 7 after the challenge infection, eight mice per period were dissected for bacteriological determination in the bladder (A) and kidneys (B). (A and B) changes in viable E. coli counts, (C) changes in viable L. casei Shirota counts in the bladder (◊) and kidneys (▵). (D) Mice pretreated with saline (solid bars) or L. casei Shirota at a dose of 10⁸ CFU 24 h earlier (open bars) were infected intravesically with E. coli strain HU-1 (2.1 × 10⁶ CFU), HU-2 (2.5 × 10⁶ CFU), or RI-1 (2.2 × 10⁶ CFU) and dissected for bacterial examination 24 h after infection. Results are expressed as the means and SDs for eight mice. Plus symbols and minus symbols in parentheses in panel D indicate whether or not the strain expresses type 1 fimbriae (first symbol) and P fimbriae (second symbol). Significant differences between untreated controls and the treated group: ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 3

Adhesion of lactobacillus strains to MBT-2 cell cultures, presented as the number of bacteria bound per 100 MBT-2 cells versus the concentration of bacteria added (CFU per milliliter). Representative results from two separate experiments are shown. Symbols: ○, L. casei Shirota; ●, L. fermentum ATCC 14931^T; ▵, L. jensenii ATCC 25258^T; ▴, L. plantarum ATCC 14917^T; □, L. reuteri JCM 1112^T.

FIG. 4

Effects of L. casei Shirota on histopathological changes in the urinary tract during E. coli infection. Mice were challenged intravesically with E. coli strain HU-1 at a dose of 2 × 10⁶ CFU and were dissected for histopathological examination of the bladder 4 days later. Either saline (A and B), L. casei Shirota at a dose of 1.6 × 10⁸ CFU (C and D), or L. fermentum ATCC 14931^T at a dose of 1.0 × 10⁸ CFU (E and F) was infused into the bladder 24 h before the challenge infection. Panels A, C, and E were stained with hematoxylin and eosin. Magnification, ×108. Panels B, D, and F were Gram stained. Magnification, ×432.

FIG. 5

Inhibition of infection-induced inflammatory responses by L. casei Shirota. Mice pretreated intravesically with saline (●) or L. casei Shirota at a dose of 1.6 × 10⁸ CFU (○) were infected intravesically with E. coli strain HU-1 at an inoculum of 2 × 10⁶ CFU 24 h later. Urination was obtained immediately after the challenge infection and on days 1, 4, and 7 from nine mice in each group per period. The number of leukocytes was counted (A), and MPO activity in the urine was determined (B). Results are expressed as the means and SDs for three samples (from nine mice). Δ, normal control. Significant differences between untreated controls and the treated group: ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 6

Antimicrobial activity of an HK preparation of L. casei Shirota. (A) Preventive activity. E. coli strain HU-1 at an inoculum of 1.8 × 10⁶ CFU was infused 24 h after treatment with HK L. casei Shirota (100 μg/mouse), and viable E. coli counts were determined on days 0 (immediately after the challenge), 1, 4, and 7. (B) Therapeutic activity of HK L. casei Shirota with postinfection administration. Mice that had been infected intravesically with E. coli strain HU-1 at an inoculum of 2.0 × 10⁶ CFU received 7 daily intravesical infusions of saline or HK L. casei Shirota at a dose of 100 μg/mouse starting on day 7 after the infection, and the mice were dissected for bacteriological examination on days 7 (controls only), 8, 11, and 14 after the challenge infection. Symbols: ●, saline-treated control, ○, group treated with HK L. casei Shirota. Results are expressed as the means and SDs for eight mice. ∗, P < 0.05 for differences between untreated controls, and treatment groups.