pathogens Conference Report
Cytoprotective Effect of Lactobacillus crispatus CTV-05 against Uropathogenic E. coli Daniel S. C. Butler 1 , Aurelio Silvestroni 2 and Ann E. Stapleton 2, * 1 2
*
Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, 221 00 Lund, Sweden;
[email protected] Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98195-5852, USA;
[email protected] Correspondence:
[email protected]; Tel.: +1-206-616-4121
Academic Editor: Lawrence S. Young Received: 12 November 2015; Accepted: 29 February 2016; Published: 8 March 2016
Abstract: The vaginal flora consists of a subset of different lactic acid producing bacteria, typically creating a hostile environment for infecting pathogens. However, the flora can easily be disrupted, creating a favorable milieu for uropathogenic Escherichia coli (UPEC), making it possible to further infect the urinary system via the urethra. Probiotic use of different lactobacilli to restore the normal flora of the vagina has been proposed as a potential prophylactic treatment against urinary tract infections. This project evaluated the protective- and anti-inflammatory roles of the probiotic Lactobacillus crispatus strain CTV-05 in an in vitro system. The inflammatory response and the cytotoxic effect were studied by Enzyme-linked immunosorbent assays and by trypan blue exclusion of cells inoculated with L. crispatus CTV-05 and comparing it to non-infected controls and UPEC infected cells. L. crispatus CTV-05 showed no cytotoxicity to vaginal epithelial cells compared to non-infected controls and provided significant protection against UPEC infection (p < 0.05). Further more, L. crispatus CTV-05 did not create a pro-inflammatory response in vitro, with no significant increase of IL-1β or IL-6. These results demonstrate the protective effect of using L. crispatus CTV-05 as a probiotic treatment to reduce the risk of recurrent urinary tract infections. Keywords: urinary tract infection; Lactobacillus crispatus; innate immunity; probiotics
1. Introduction Recurrent acute uncomplicated cystitis (AUC) is a common infection, most often caused by Escherichia coli strains known as uropathogenic E. coli (UPEC) originating in the rectal microbiota. Women are far more affected than men. Half of the female population in the US will have at least one episode of acute cystitis during their lifespan, and around a third of these women will develop recurrent UTIs, resulting in increased morbidity and health care expenditures [1,2] A strong link between vaginal health and UTIs has been described and may serve as a basis for the skewed gender distribution of AUC [3–5]. The healthy vagina is colonized by different lactic acid producing bacteria (LAB), most commonly strains of Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus iners [6–8]. The vaginal flora is easily disrupted; several studies have indicated that frequent sexual activity [9], the use of spermicide contraceptives [10], and certain hygiene regimens [11] allow different pathogens to colonize vaginal epithelial cells (VECs) and establish a potential reservoir for future UTIs. After an episode of AUC, the healthy, protective vaginal microbiota may take weeks to re-establish and an increased risk of new vaginal colonization accompanies this period, increasing the risk for recurrent UTI [12]. This reservoir of bacteria may further invade the urethra, facilitating the bacterial spread and infection of the urothelium of the bladder or the renal pelvis, thus precipitating clinical disease [13,14].
Pathogens 2016, 5, 27; doi:10.3390/pathogens5010027
www.mdpi.com/journal/pathogens
Pathogens 2016, 5, 27
2 of 12
Various normally resident Lactobacillus spp. have been postulated to protect the vagina from invading pathogens by means of mechanisms including the acidification of the vagina creating an unfavorable milieu for invading pathogens. In addition, Lactobacillus adhesion to the vaginal epithelium and the resulting competitive exclusion of adhering UPEC strains has been proposed as a mechanism for both resident and probiotic Lactobacilli to impair UPEC colonization and infection. Several successful studies have been performed using Lactobacilli to re-establish the normal vaginal microbiota, establishing the efficacy of prophylactic lactobacilli in replenishing the vaginal flora. In addition, this approach might avoid the negative effects of antibiotics, some of which alter the vaginal microbiota [15–18]. The severity of UTI reflects the magnitude and functionality of the innate immune response to infection. Adhering UPEC bacteria stimulate a cytokine response in uroepithelial and vaginal cells [19,20]. IL-1β and IL-6 concentrations are elevated in the urine of patients with UTI following infection. In acute pyelonephritis, blood cytokine levels are augmented and play a role in the generation of fever and the acute phase response [21]. The use of probiotics needs to be further explored as a potential prophylactic and therapeutic measure against uropathogens with increasing resistance to antibiotics. In this study we used an immortalized primary cultured vaginal epithelial cell (VEC) system [22] as an in vitro model of the protective effects of the probiotic L. crispatus strain CTV-05. We evaluated the effects on UPEC cytotoxicity, adherence, and pro-inflammatory response activation, quantifying pro- and anti-inflammatory cytokines regulated by UPEC infection. 2. Results and Discussion 2.1. Results 2.1.1. L. crispatus CTV-05 Reduces the Toxicity of UPEC Strains to Vaginal Epithelial Cells To exclude cytotoxicity of L. crispatus CTV-05, a cell viability assay was performed using VECs immortalized with the amphotropic retrovirus vector, LXSN16E6E7 (gift from D. Galloway, Seattle, WA, USA) containing the E6 and E7 genes of HPV 16 with the neomycin resistance gene. The loss of cell viability after in vitro infection with L. crispatus CTV-05 was quantified by trypan blue exclusion compared to non-infected cells and bacterial numbers were determined by viable counts of the supernatants, 3, 8, and 24 h after incubation with the VECs. L. crispatus CTV-05 did not alter cell viability (108 cfu/mL), mean viability 87.8% ˘ 5.1%, 24 h, p = 0.15 compared to non-stimulated VECs (Figure 1a,b). In contrast, the UPEC strains E. coli R45 and E. coli J96 were cytotoxic for the VECs. This effect was dose-dependent for cells infected with 105 , 104 and 103 cfu/mL. To avoid excessive cytotoxicity, 103 cfu/mL was selected for further studies. The average viability of non-stimulated VECs showed no significant difference between the three time-points analyzed. To address if Lactobacilli may protect against the cytotoxicity of the UPEC strains, VECs were pre-inoculated with 108 cfu/mL of L. crispatus CTV-05 for 1 h prior to UPEC infection and cell death was measured by trypan blue exclusion, as described above. Pre-inoculation of L. crispatus CTV-05 yielded significant differences between cells exposed to L. crispatus CTV-05 before inoculation with E. coli J96 and VECs only infected with E. coli J96 (p < 0.001 after 8 h), The same effects were seen with E. coli R45, where pre-inoculation with L. crispatus CTV-05 for one hour yielded a significant difference compared to E. coli R45 stimulated cells (p < 0.001 after 8 h) (Figure 2a).
Pathogens 2016, 5, 27 Pathogens 2016, 5, 27 Pathogens 2016, 5, 27
3 of 12 3 of 12 3 of 12
Figure crispatusCTV-05 CTV-05 is epithelial cellscells compared to non-infected control control Figure 1. 1. L.L.crispatus is non-toxic non-toxictotovaginal vaginal epithelial compared to non-infected cells and UPECinfected infected cells. cells. Vaginal cells were inoculated with with the probiotic L. crispatus cells and UPEC Vaginalepithelial epithelial cells were inoculated the probiotic L. crispatus 8 cfu/mL) Figure 1. L. crispatus CTV-05 iscystitis non-toxic to vaginal epithelial cells compared to non-infectedstrain control 3 3cfu/mL), 8 cfu/mL) cfu/mL), the isolate E. E. colicoli R45 (10(10 or theorpyelonephritis strain CTV-05 (10 strain CTV-05 (10 the cystitis isolate R45 the pyelonephritis strain 3 cfu/mL) and infected Vaginal epithelial cells were inoculated with the of probiotic L. crispatus E. cells coli J96 (103UPEC for 3,cells. 8 or 24 h. (A) Graphs illustrate the average percentage viable cells after E. coli J96 (10 cfu/mL) for 3, 8 or 24 h. (A) Graphs illustrate the average percentage of viable cells 8 cfu/mL) or the pyelonephritis strain cystitis coliright, R45 8(10 strain CTV-05 (103 cfu/mL), infection for the three differentthe time pointsisolate (3 h toE.the h in the middle, and 24 h to the left) after infection for the three different time points (3 h to the right, 8 h in the middle, and 24 h to the E. colitrypan J96 (103 cfu/mL) for 3, 8 or 24 h. (A) Graphs illustratewas the average percentage viable cells after using blue exclusion. Statistical significance calculated using ofpaired t-test. left) using trypan blue exclusion. Statistical significance was calculated using paired p < 0.01; forpthe three different time points (3 h to right, 8 effect h in the middle, andCTV-05, 24 t-test. h to E. the** left) ** pinfection < 0.01; *** < 0.001; (B) Line graph illustrating thethe cytotoxic of L. crispatus coli *** p
