ABSTRACT
Staphylococcus aureus is a human pathogen with many infections originating on mucosal surfaces. One common group of S. aureus is the USA200 (CC30) clonal group, which produces toxic shock syndrome toxin-1 (TSST-1). Many USA200 infections occur on mucosal surfaces, particularly in the vagina and gastrointestinal tract. This allows these organisms to cause cases of menstrual TSS and enterocolitis. The current study examined the ability of two lactobacilli, Lactobacillus acidophilus strain LA-14 and Lacticaseibacillus rhamnosus strain HN001, for their ability to inhibit the growth of TSST-1 positive S. aureus, the production of TSST-1, and the ability of TSST-1 to induce pro-inflammatory chemokines from human vaginal epithelial cells (HVECs). In competition growth experiments, L. rhamnosus did not affect the growth of TSS S. aureus but did inhibit the production of TSST-1; this effect was partially due to acidification of the growth medium. L. acidophilus was both bactericidal and prevented the production of TSST-1 by S. aureus. This effect appeared to be partially due to acidification of the growth medium, production of H2O2, and production of other antibacterial molecules. When both organisms were incubated with S. aureus, the effect of L. acidophilus LA-14 dominated. In in vitro experiments with HVECs, neither lactobacillus induced significant production of the chemokine interleukin-8, whereas TSST-1 did induce production of the chemokine. When the lactobacilli were incubated with HVECs in the presence of TSST-1, the lactobacilli reduced chemokine production. These data suggest that these two bacteria in probiotics could reduce the incidence of menstrual and enterocolitis-associated TSS.
IMPORTANCE Toxic shock syndrome (TSS) Staphylococcus aureus commonly colonize mucosal surfaces, giving them the ability to cause TSS through the action of TSS toxin-1 (TSST-1). This study examined the ability of two probiotic lactobacilli to inhibit S. aureus growth and TSST-1 production, and the reduction of pro-inflammatory chemokine production by TSST-1. Lacticaseibacillus rhamnosus strain HN001 inhibited TSST-1 production due to acid production but did not affect S. aureus growth. Lactobacillus acidophilus strain LA-14 was bactericidal against S. aureus, partially due to acid and H2O2 production, and consequently also inhibited TSST-1 production. Neither lactobacillus induced the production of pro-inflammatory chemokines by human vaginal epithelial cells, and both inhibited chemokine production by TSST-1. These data suggest that the two probiotics could reduce the incidence of mucosa-associated TSS, including menstrual TSS and cases originating as enterocolitis.
INTRODUCTION
Staphylococcus aureus is a ubiquitous pathogen that most often originates from mucosal surfaces but can also cause infections across skin barriers (1, 2). The CDC classifies S. aureus strains as USA100 to -1100 based on pulsed-field gel electrophoresis (3). Among these clonal groups is the USA200 (CC30) clonal group. USA200 strains of S. aureus, present on mucosal surfaces, have unique properties compared to other clonal groups. USA200 S. aureus produces the superantigen toxic shock syndrome toxin-1 (TSST-1), produces β-cytotoxin (the hot-cold hemolysin), and has greatly reduced alpha-toxin production compared to other clonal groups (2, 4–6). This combination of virulence factors appears to restrict the group to mucosal surfaces or damaged skin (2, 5, 6).
TSST-1 is the cause of 100% of menstrual TSS cases from human vaginal USA200 S. aureus (2, 7–9). Additionally, a few cases of enterocolitis are associated with intestinal infection with TSST-1 USA200 S. aureus (10, 11). The most common origin site of these organisms, both in the vagina and intestinal tract, is the anterior nares (1, 12).
Various lactobacilli and related bacteria are the dominant microbiome organisms in both the vagina and intestinal tract (13–15). For example, women are typically colonized with 5 × 107 vaginal lactobacilli per tampon, and these organisms persist throughout the menstrual cycle (13), even during menstruation, when pathogens such as TSS S. aureus may grow to even higher numbers, reaching even 1011 per tampon (16).
In our prior studies, we have observed that some women have only lactobacilli present in the vagina (17). These women do not appear able to be colonized with potential pathogens on this mucosal surface. These observations suggest that it may be possible to colonize women on mucosal surfaces with probiotic lactobacilli to reduce the presence of TSS S. aureus. Two such probiotic lactobacilli are Lacticaseibacillus rhamnosus strain HN001 and Lactobacillus acidophilus strain LA-14. Furthermore, the combination of these probiotic strains has shown beneficial effects on vaginal health, particularly in women with dysbiotic vaginal microbiota, in randomized placebo-controlled clinical trials (18, 19). However, the effects of these probiotics on TSS-associated S. aureus or TSST-1 production are unclear.
This study was undertaken to determine the in vitro effects of both L. rhamnosus and Lactobacillus acidophilus, both separately and when combined, on TSS S. aureus, its production of TSST-1, and the ability of TSST-1 to induce pro-inflammatory chemokine production by human vaginal epithelial cells (HVECs). L. rhamnosus HN001 prevented TSST-1 production, partially through acid production, but did not inhibit growth of TSS S. aureus. In contrast, L. acidophilus LA-14 both killed TSS S. aureus and simultaneously prevented TSST-1 production, partially through acid and H2O2 production. When it was incubated together with TSS S. aureus, the effect of L. acidophilus dominated. L. acidophilus did not kill L. rhamnosus. Finally, neither lactobacillus alone induced interleukin-8 (IL-8) chemokine production by HVECs, or induced only low-level production, but both lactobacilli inhibited IL-8 production by HVECs, as induced by TSST-1.
RESULTS
Growth of L. rhamnosus HN001, L. acidophilus LA-14, and S. aureus.
In our first set of studies, the stationary phase of all three organisms was determined after growth in Todd Hewitt (TH) medium with shaking (150 rpm) at 37°C. The inoculum size was estimated to be 107 CFU/mL and then verified by serial dilution plate counts. After 48 h, L. rhamnosus had grown to approximately 109 CFU/mL. The colonies, as grown on chocolate agar, were approximately 2 mm in diameter after 48 h incubation in a 5% CO2 incubator. In contrast, L. acidophilus grew to only 2 × 108 CFU/mL after the 48-h growth period. When plated on chocolate agar, the L. acidophilus colonies remained as pinpoints, even after incubation for up to 1 week in the presence of 5% CO2. These stationary phases agreed with those provided by the commercial source of the lactobacilli.
The stationary phase of USA200 (CC30) TSS S. aureus MN8 (20) was determined to be approximately 7 × 109 CFU/mL after 24 h culture in Todd Hewitt broth at 37°C with 150-rpm shaking. The starting inoculum was 1.2 × 107 CFU/mL, the approximate average CFU/mL found in the vagina during menstruation. The colonies grew on sheep blood agar plates as relatively non-hemolytic colonies about 2 mm in size, as expected for this organism. Three additional strains of USA200 (CC30) S. aureus strains were cultured similarly to MN8 and their stationary phases were determined. CDC587 (7) and Harrisburg (7, 21), from menstrual TSS cases, and a clinical isolate from enterocolitis/TSS (10, 11) had stationary phases of 5.3 × 109, 7.8 × 109, and 9.8 × 109 CFU/mL, respectively. Their appearances on blood agar plates were similar to that of strain MN8.
L. acidophilus LA-14 and L. rhamnosus HN001 growth kinetics.
Initially, the growth of the two lactobacillus strains was determined after 24 h of incubation at 37°C with shaking at 150 rpm, as incubated separately and when mixed at various starting inocula (Fig. 1A to C). The 24-h time point was chosen as the end of experiment because all four S. aureus strains, used in subsequent experiments, achieved their stationary phases before 24 h. L. acidophilus at 109 CFU/mL dropped to 108 CFU/mL, its usual stationary phase (Fig. 1A). L. acidophilus at 108 CFU/mL remained at 108 CFU/mL. L. acidophilus at starting inocula of 107 and 106 CFU did not reach the expected stationary phase by 24 h…
