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Influence of vaginal microbiota on toxic shock syndrome toxin-1 production by 1
Staphylococcus aureus. 2
3
Roderick MacPhee (1,2), Wayne L. Miller (1), Greg Gloor (3), John K. McCormick 4
(1,2), Jeremy Burton (1,2) and Gregor Reid (1,2) 5
6
1. Canadian Research & Development Centre for Probiotics, Lawson Health Research 7
Institute, 268 Grosvenor Street, London, Ontario, N6A 4V2, Canada. 8
2. Department of Microbiology & Immunology, The University of Western Ontario, 9
London, Ontario, N6A 5C1, Canada. 10
3. Department of Biochemistry, The University of Western Ontario, London, Ontario, 11
N6A 5C1, Canada. 12
* Address for Correspondence: Gregor Reid, Canadian Research & Development 13
Centre for Probiotics, Lawson Health Research Institute, 268 Grosvenor St., N6A 4V2, 14
London, Ontario, Canada. E-mail: [email protected]. Tel: 519-646-6100 x 65256 Fax: 15
519-646-6031 16
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Copyright © 2013, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02908-12 AEM Accepts, published online ahead of print on 11 January 2013
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Abstract 32
Menstrual toxic shock syndrome (TSS) is a serious illness that afflicts women of 33
pre-menopausal age worldwide, and arises from vaginal infection by Staphylococcus 34
aureus and concurrent production of toxic shock syndrome toxin-1 (TSST-1). Studies 35
have illustrated the capacity of lactobacilli to reduce S. aureus virulence, including 36
TSST-1-suppression. We hypothesized that an aberrant microbiota characteristic of 37
pathogenic bacteria would induce an increased production of TSST-1 and that this might 38
represent a risk factor for the development of TSS. A S. aureus TSST-1 reporter strain 39
was grown in the presence of vaginal swab contents collected from women with a 40
clinically healthy vaginal status, an intermediate status, and those diagnosed with 41
bacterial vaginosis (BV). Bacterial supernatant-challenge assays were also performed to 42
test the effects of aerobic vaginitis (AV)-associated pathogens towards TSST-1 43
production. While clinical samples from healthy and BV women suppressed toxin 44
production, in vitro studies demonstrated that Streptococcus agalactiae and 45
Enterococcus spp. significantly induced TSST-1 production, while some Lactobacillus 46
spp. suppressed it. The findings suggest that women colonized by S. aureus and with 47
aerobic vaginitis (AV), but not BV, may be more susceptible to menstrual-TSS and 48
would most benefit from prophylactic treatment. 49
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Introduction 53
Toxic shock syndrome (TSS) is a systemic illness characterized by extensive T-54
cell proliferation throughout the body. This leads to systemic inflammation and 55
concurrent health problems, including rash formation, multiple organ failure and 56
potentially death. Menstrual-TSS is a form of the disease prominent in pre-menopausal 57
women, afflicting approximately 1 in every 100,000 women in the Western world (1, 2). 58
This condition arises from vaginal infection by Staphylococcus aureus and subsequent 59
production of toxic shock syndrome toxin-1 (TSST-1), a superantigen thought to be 60
unique with the capability of crossing the vaginal epithelial layer (3, 4). It is estimated 61
that 70% of women in North America use tampons (5), and although the rates of 62
menstrual-TSS have dropped significantly since the early 1980’s, it remains crucial to 63
better understand the pathogenesis of this condition to prevent its re-emergence. 64
Production of TSST-1, encoded by the tst gene, is regulated in large part by the 65
agr system of S. aureus (6), a well-characterized quorum sensing system (7) that 66
regulates many secreted and surface expressed virulence factors (8). Activation of this 67
system is regulated by several factors, including the SarA protein (9, 10), RAP/RIP 68
proteins (11) and alternative sigma-factor B (σB ) (12). Much research has been placed 69
on investigating the nature of TSST-1 production in response to environmental cues that 70
may be present in the vagina. For instance, Yarwood and Schlievert (13) revealed that 71
elevated oxygen levels in the presence of carbon dioxide are necessary for TSST-1 72
production. It is also well established that a neutral pH of 6.5-7.0, which is found in 73
aberrant vaginal conditions, is optimal for production of this toxin, in contrast to a healthy 74
vaginal pH of 4.5 (14, 15). It is therefore readily apparent that the vaginal environment, 75
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characteristic of fluctuating gas levels and pH, could influence the degree to which 76
colonized S. aureus produce TSST-1. However, little research has been conducted to 77
evaluate the putative role of the residing vaginal microbiota, which arguably shapes this 78
environment as a whole. 79
The vaginal microbiota consists largely of Lactobacillus species, which produce 80
lactic acid and other antimicrobial metabolites that prevents the overgrowth of 81
opportunistic pathogens (16). These bacteria have been shown to inhibit the growth of 82
several urogenital pathogens, including S. aureus (17, 18). Furthermore, the vaginal 83
probiotic strain L. reuteri RC-14 has been shown to produce cyclic dipeptides capable of 84
suppressing TSST-1 production in S. aureus (19), suggesting that indigenous vaginal 85
lactobacilli may protect the host from menstrual-TSS progression. 86
Changes in the vaginal environment render the vaginal microbiota susceptible to 87
constant fluctuations in its members. This occasionally leads to depletion in the 88
lactobacilli population and a subsequent increase in pathogenic bacteria, resulting in 89
bacterial vaginosis (BV) or aerobic vaginitis (AV). BV is characterized by the overgrowth 90
of strict anaerobic bacteria including Gardnerella vaginalis, Atopobium vaginae, 91
Prevotella bivia and Leptotrichia amnionii (20, 21), and has been associated with 92
increased susceptibility to sexually transmitted infections, preterm labour (22) and pelvic 93
inflammatory disease (23, 24). In contrast, AV is characterized by the overgrowth of 94
enteric aerobic and facultative anaerobic commensals such as Escherichia coli, 95
Streptococcus agalactiae, Enterococcus faecalis and S. aureus (25) and is an 96
inflammatory condition that can often lead to pregnancy complications (26-29). 97
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The aim of the present study is to investigate a potential relationship between the 98
vaginal microbiota and TSST-1 production by S. aureus, and thus the susceptibility to 99
menstrual-TSS. We hypothesize that an aberrant microbiota that alters the vaginal pH 100
and environment will lead to increased production of TSST-1, and thus might represent 101
a risk factor for the development of TSS. In contrast, we believe that TSST-1 production 102
will be suppressed in a healthy vaginal environment dominated by Lactobacillus 103
species. If this link between the vaginal microbiota and TSST-1 production is 104
established, it may be feasible to control toxin production of S. aureus by altering the 105
vaginal microbiota through the application of exogenous bacteria. 106
107
Materials and Methods 108
Clinical Study 109
Details of the clinical study were reviewed and approved by the Health Sciences 110
Research Ethics Board at the University of Western Ontario. Participants were provided 111
with a package detailing all relevant information of the study, including an in-depth 112
explanation of the clinical procedure, and signed a consent form prior to sample 113
collection. 114
Recruitment of pre-menopausal women between the ages of 18-40 years with BV 115
as well as healthy controls took place in London, Ontario and was based on a selective 116
criteria designed to ensure that the vaginal samples reflected a representative 117
microbiota of the general female pre-menopausal population. Recruitment posters were 118
placed in various locations in London, Ontario, including The University of Western 119
Ontario and local hospitals and medical clinics. The poster emphasized a target 120
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audience of premenopausal women with a suspected history of BV. Participants were 121
excluded if they had reached menopause, had a urogenital infection other than BV in 122
the past 6 months, were pregnant, had a history of gonorrhoea, chlamydia, estrogen-123
dependent neoplasia, abnormal renal function or pyelonephritis, were taking prednisone, 124
immune-suppressive agents or antimicrobial medication, or had undiagnosed abnormal 125
vaginal bleeding. Participants were asked to refrain from oral or vaginal intercourse and 126
consuming probiotic supplements or foods for 48 hours prior to the clinical visit. No 127
participants were menstruating at time of the clinical visit. At the conclusion of the study, 128
a total of 34 participants were recruited: 11 healthy, 10 intermediate and 13 BV, with a 129
mean age of 24.6 years. None of the subjects were diagnosed with AV nor had a 130
microbiome indicative of this condition. 131
Weekly clinics were held at the Victoria Family Medical Centre (London, Ontario) 132
over a 6-month period. A total of 7 samples were collected from each participant at a 133
single time point: one pHem-alert applicator (Gynex) was used to detect the vaginal pH; 134
three CultureSwab polyester-tipped swabs (BD Biosciences) were used for bacterial 135
isolation (for diagnosis of BV by Nugent score, for bacterial DNA and RNA collection); 136
two ESwabs (Copan) were processed for culturing the organisms; and one cytobrush 137
(Cooper Surgical) was processed for vaginal epithelial cell RNA. Swabs were inserted 138
approximately 5 cm into the vaginal canal, pressed against the wall and rotated 4 times. 139
The microbial status was assessed through the Nugent scoring method (30). A 140
CultureSwab polyester-tipped swab (BD Biosciences) was pressed against the vaginal 141
wall and smeared onto a glass microscope slide, heat-fixed and Gram-stained. Four 142
fields of view were chosen at random at a magnification of 1,000 X, and bacterial 143
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numbers from each were recorded. A participant was diagnosed as having BV if the 144
Nugent score was 7-10 and if they had a vaginal pH greater than 4.5. All participants 145
with a healthy Nugent score of 0-3 had a pH that was equal to or less than 4.5. 146
Sequencing by Illumina 147
Bacteria were collected on a CultureSwab polyester-tipped swab (BD 148
Biosciences) which was immediately placed in 700 µL RNAprotect (Qiagen). The tube 149
was vortexed for 5 sec, swab discarded and the solution stored at -20°C. DNA isolation 150
was performed using the InstaGene matrix (Bio-Rad) DNA preparation kit according to 151
the manufacturers’ instructions. The collected DNA was stored at -20°C and was used 152
as DNA template for subsequent PCR reactions. 153
PCR was carried out in 50 µL reactions consisting of 40 µL master mix and 10 µL 154
template (or 10 µL Milli-Q H20 [Millipore] for negative control). The master mix consisted 155
of 1X PCR buffer (Invitrogen), 1.7 mM MgCl2, 210 µM dNTPs, 640 nM forward and 156
reverse primer, 0.05 U/µL Platinum Taq Polymerase (Invitrogen) and Milli-Q H20 157
[Millipore]. Amplification was done using eubacterial primers flanking the V6 region of 158
the 16S rRNA gene: V6-F (5’-CAACGCGARGAACCTTACC -3’) and V6-R (5’-159
ACAACACGAGCTGACGAC -3’). The PCR reactions were carried out in a Mastercycler 160
(Eppendorf) under the following annealing temperature touchdown program: 94°C for 2 161
min, followed by 10 cycles of 94°C for 45 sec, 61°C-51°C for 45 sec (each cycle 162
dropping by 1°C), 72°C for 45 sec, and then 15 cycles of 94°C for 45 sec, 51°C for 45 163
sec, 72°C for 45 sec, and ending with a final elongation step of 72°C for 2 min. PCR 164
products (5 µL) were mixed with 1 µL 6X Loading Dye and loaded onto a 1.5% agarose 165
gel. The gel was then viewed under UV light in an AlphaImager (Alpha Innotech 166
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Corporation). The ImageJ program was used to quantify the brightness of each DNA 167
band so that equal amounts of each sample were added to the subsequent Illumina 168
reaction. DNA samples were then sent for Illumina sequencing at The Next-Generation 169
Sequencing Facility in The Centre for Applied Genomics at the Hospital for Sick 170
Children in Toronto, Canada. Data analysis of the sequencing reads was performed 171
according to our colleagues’ protocol (31). 172
Luminescence Assay 173
Vaginal samples were collected from participants using the ESwab (Conan) swab 174
and transport system containing 1 mL liquid Amies medium. Following sample 175
collection, the ESwab was submerged into the Amies medium for 2 hours at room 176
temperature followed by a 10 sec vortex at high speed. The swab was removed and 177
discarded. The ESwab solution was aliquoted 2 x 350 µL and centrifuged at 10,000 rpm 178
for 5 min. The supernatants were transferred to a fresh tube and stored at -80°C, which 179
was later used for the in vitro luminescence assay. 180
A culture of a reporter strain of S. aureus MN8, containing the cloning vector 181
pAmilux with an incorporated promoter for tst, was grown in brain heart infusion (BHI) 182
media with 10 µg/µL chloramphenicol overnight at 37°C with a shaking speed of 250 183
rpm (19, 32). The culture was diluted to an OD600 of 0.02 with a) supernatant from the 184
clinical sample, b) Amies transport medium or c) BHI, of which the latter two acted as 185
controls. Next, 200 µL of the subculture was grown for 16 hr in a Fluoroskan Ascent FL 186
luminometer (Thermo) where luminescence production was detected from the activated 187
tst promoter every 30 min. Another 200 µL of the same subculture was grown in a 188
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Bioscreen C Reader (MTX Lab Systems) at 37°C with continuous shaking for 24 hr to 189
monitor growth of the bacteria. 190
Bacterial Cultures and Conditions for Supernatant Challenge Assay 191
All bacterial strains used in the supernatant challenge assay, along with respective 192
growth conditions, are listed in Table 1. These bacteria were obtained from either ATCC 193
or previous clinical isolations. The bacterial strains were divided into 3 groups for 194
analysis: healthy, AV-associated and BV-associated bacteria. The S. aureus MN8 strain 195
was selected as a prototype of menstrual-TSS strains, as it has been isolated from a 196
pre-menopausal woman suffering from the condition (33). All aerobic bacteria were 197
cultured in shaking conditions for 16 hr at 37°C and then sub-cultured to an OD600 of 198
0.02 using their growth media. The sub-cultures were grown for 12 hr. The lactobacilli 199
were grown in anaerobic conditions using the GasPak EZ system (Becton-Dickenson) 200
for 24 hr at 37°C and sub-cultured for an additional 24 hr, starting at an OD600 of 0.02. 201
The strict anaerobes, Atopobium vaginae and Prevotella bivia were cultured and sub-202
cultured in a Forma anaerobic chamber (Model 1025, Thermo) for 48 hr at 37°C to allow 203
for growth into stationary phase. Sub-cultures underwent centrifugation at 3500 x g for 204
10 min at 4°C and the supernatants were transferred to a fresh tube. The pH of the 205
supernatants were determined and adjusted to that of the S. aureus MN8 supernatant 206
(pH 5.95), sterilized via filtration using 0.2 µm filters, transferred to a clean tube and 207
frozen at -20°C for no longer than 1 week. Next, S. aureus MN8 cultures were grown for 208
16 hr and sub-cultured starting at an initial OD600 of 0.02 in half BHI and half 209
supernatant of the challenger bacteria. The sub-cultures were grown for 16 hr at 37°C in 210
shaking conditions and were then centrifuged at 3500xg for 10 min at 4°C. Sub-cultures 211
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were also grown in a Multiskan Ascent Plate Reader (Thermo-Scientific) to detect OD600 212
values to ensure similar S. aureus growth across the various conditions. The 213
supernatants were subsequently used for real-time PCR. 214
Quantitative Real-Time PCR 215
A culture of S. aureus MN8 challenged with supernatants from the other bacteria (as 216
described previously) was grown from an initial OD600 of 0.02 for 16 hr. Ten millilitres of 217
RNAprotect (Qiagen) was added to 5 mL culture, and the solution was vortexed and 218
incubated for 10 min at room temperature, then centrifuged at 6000 x g for 20 min at 219
4°C. The supernatant was then discarded and the pellet stored at -80°C. To isolate 220
bacterial RNA, the pellet was suspended in 5 mL lysis solution (10 mM Tris-HCl, 1 mM 221
EDTA [pH 8.0] and 50 µg/mL lysostaphin) and vortexed. The solution was incubated for 222
10 min at 37°C then centrifuged at 6000 x g for 20 min. Isolation of RNA from the pellet 223
was performed using TRIzol (Invitrogen) in accordance with the manufacturers’ 224
instructions. 225
Contaminants were removed from the RNA samples using the RNeasy Mini Kit 226
(Qiagen). RNA products were viewed via electrophoresis in a 1% agarose gel using 1X 227
TBE, stained with ethidium bromide and viewed under UV light in an AlphaImager 228
(Alpha Innotech Corporation). The RNA concentration and quality in the samples were 229
then determined through a biophotometer (Eppendorf) (RNA quality cut-off: 260/280 ≥ 230
1.8; 260/230 ≥ 1.6). Five hundred nanograms of RNA from each sample was used as a 231
template for reverse transcription PCR. Conversion to cDNA was done using the 232
Multiscribe Reverse Transcriptase mix (Invitrogen). The reactions were carried out in a 233
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Mastercycler (Eppendorf) with the following program: initial step of 25°C for 10 min, 234
followed by 37°C for 120 min, and a final step of 85°C for 5 min. 235
The cDNA samples were used as templates for real-time PCR reactions. Primers 236
included tst-f (5’- CTGATGCTGCCATCTGTGTT -3’) and tst-r (5’- 237
GTAAGCCCTTTGTTGCTTGC -3’) for expression of the tst gene, and rpoB-f (5’- 238
TCCTGTTGAACGCGCATGTAA -3’) and rpoB-r (5’-GCTGGTATGGCTCGTGATGGTA-239
3’) for expression of the rpoB housekeeping gene. The iQ SYBR Green Supermix (Bio-240
Rad) was used to carry out 20 µL reactions, which were composed of the following: 10 241
µL 2X iQ SYBR Green Supermix, 1 µL of each primer at 10 µM, 7 µL RNase-free H2O 242
and 1 µL cDNA template. Reactions were run in a Rotor –Gene 6000 thermocycler 243
(Corbett) under the following program: an initial melting ramp from 72°C to 95°C, 244
followed by 40 cycles of 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec, ending 245
with a hold at 95°C for 10 min. Data was analyzed using the Rotor-Gene 6000 Series 246
Software 1.7 (Corbett). Expression of tst by S. aureus in each condition was compared 247
to tst expression when S. aureus was grown in media alone. For the purpose of 248
comparison, the control expression was set at 100%. Overall, the supernatant challenge 249
experiment was performed three times, with each run having three technical replicates 250
starting from the reverse transcription stage. 251
A series of standards were created to quantify gene expression during data 252
analysis. A sub-culture of S. aureus MN8 was grown for 12 hr, and total DNA isolated 253
using InstaGene matrix (Bio-Rad). PCR was carried out in 50 µL reactions consisting of 254
the following components: 10X PCR buffer, 1.7 mM MgCl2, 210 mM dNTPs mix, 640 255
nM of each primer (tst and rpoB primer pairs used for real-time PCR), 5U Platinum Taq 256
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DNA Polymerase (Invitrogen), 5 µL template and Milli-Q H20 [Millipore] to reach 50 µL. 257
Amplification was performed in a Mastercycler (Eppendorf) using the following program: 258
94°C for 1 min, followed by 30 cycles of 94°C for 45 sec, 60°C for 45 sec and 72°C for 259
45 sec, and a final elongation step of 72°C for 2 min. Products were viewed via 260
electrophoresis in a 1% agarose gel using 1X TBE, stained with ethidium bromide and 261
viewed under UV light in an AlphaImager (Alpha Innotech Corporation). Concentration 262
of DNA in each sample was determined through spectrophotometry, and a series of 263
standards were made ranging from 5000 ng/µL to 5 x 10-9 ng/µL. These standards were 264
used as templates in the real-time PCR reactions to construct standard curves with 265
known concentrations of DNA. 266
Statistical Analysis 267
Statistical analysis was performed on the real-time PCR data using the GraphPad 268
Prism® 4 program. Gene expression of the various conditions was compared to the 269
control using one-way ANOVA analysis with a Dunnett’s multiple comparison test. 270
Changes in gene expression were considered significant for p values <0.05. The 271
standard error of the mean (SEM) for each condition tested was displayed on the bar-272
plots. 273
Results 274
Vaginal microbiota profile of pre-menopausal women 275
A clinical study was undertaken to collect vaginal samples and determine the 276
microbiomes of women with and without BV, for subsequent comparison of their effects 277
on TSST-1 production in S. aureus. The vaginal microbiota of participants were 278
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determined through 16S rRNA sequencing by Illumina, and bacterial DNA from 21 279
participants was successfully isolated and amplified for sequencing (9 healthy, 5 280
intermediate and 7 BV). The vaginal microbiota of healthy versus BV participants were 281
very distinct. The predominant species associated with healthy and intermediate 282
samples was L. crispatus (50.7% and 41.6%, respectively) followed by L. iners (27.4% 283
and 20.5%, respectively), with an average species abundance of 3.6 species per 284
sample (Figure 1). Other bacterial species, including those associated with BV, were 285
present in relatively low abundance in these subjects. Three outliers were present 286
among these samples, having diversities of 11, 12 and 17 species per sample. One 287
intermediate sample had no L. iners or L. crispatus (at ≥1% abundance), but instead 288
consisted of L. jensenii, L. gasseri/johnsonii and G. vaginalis of nearly equal 289
abundances. Participants assigned to the BV group, however, had a predominant 290
Gardnerella vaginalis population (average abundance of 49.7%), along with Leptotrichia 291
amnionii (3.9%) and Atopobium vaginae (3.7%). The BV samples had a higher bacterial 292
species diversity than healthy and intermediate, with an average of 10.5 species per 293
sample. 294
Expression of tst in S. aureus in women with and without bacterial vaginosis 295
In order to investigate the ability of the different vaginal environments to influence 296
the production of TSST-1, vaginal samples collected from non-pregnant, pre-297
menopausal subjects in London, Ontario were tested against cultures of the reporter 298
strain S. aureus MN8/pAmilux-Ptst to monitor activity of the tst promoter (reflecting tst 299
expression). Samples from all three vaginal states (healthy, intermediate and BV) were 300
tested in this luminescence assay, and following 16 hours of growth, expression of tst in 301
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S. aureus was found to be completely suppressed from all 3 sample categories, as 302
illustrated in Figure 2A. However, samples from two healthy participants were found to 303
have no effect and only some inhibition on tst expression (Figure 2B and 2C, 304
respectively). Total protein levels were determined via a bicinchoninic acid (BCA) assay 305
in these two samples, as well as in three samples demonstrating strong inhibition (data 306
not shown). Results showed similar protein levels in all the samples, regardless of anti-307
tst activity, suggesting that the lack of suppression observed in these samples were 308
likely not due to the absence of host proteins in these two samples. 309
TSST-1 production in response to bacteria associated with BV, AV and a healthy 310
microbiota 311
In order to investigate whether vaginal bacterial species have an influence on TSST-312
1 production, cultures of S. aureus MN8 were challenged with supernatants of AV-313
associated, BV- associated and healthy-associated bacteria, and tst expression was 314
monitored by real-time PCR. These bacterial strains were obtained through commercial 315
means or previous clinical isolations. The probiotic strain L. reuteri RC-14 was also 316
included in the experiment, as this strain has been shown to suppress TSST-1 317
production (12). Growth of S. aureus in these conditions was monitored and grew to 318
similar OD600 values (data not shown). Bacteria used to challenge S. aureus were 319
separated into 3 groups for data analysis: lactobacilli, AV-associated and BV-320
associated. Several Lactobacillus species were found to significantly decrease tst 321
expression (Figure 3A). L. johnsonii, L. jensenii and L. gasseri decreased tst expression 322
by 54%, 59% and 49%, respectively (p<0.05). The probiotic strains L. rhamnosus GR-1 323
and L. reuteri RC-14 decreased expression by 57% and 72%, respectively (p<0.05). 324
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Interestingly, L. iners and L. crispatus (the most common vaginal residents) did not 325
significantly decrease tst expression. 326
In general, the AV-associated bacteria increased tst expression. Secreted 327
products from S. agalactiae significantly increased tst expression 3.7-fold relative to S. 328
aureus grown in BHI (p<0.05) (Figure 3B). E. faecalis and E. faecium increased tst 329
expression 1.7- and 1.9-fold, respectively, while E. coli did not affect expression. 330
However, these latter changes in gene expression were not statistically significantly 331
when compared to the control of S. aureus grown in BHI. Interestingly, we acquired 332
preliminary data showing that S. aureus challenged with a combination of S. agalactiae 333
and L. reuteri RC-14 supernatants, as well as with S. agalactiae and L. jensenii 334
supernatants, expressed basal levels of the tst gene, suggesting a dampening effect 335
from the lactobacilli (data not shown). 336
Finally, the BV-associated organisms slightly decreased tst expression. In 337
particular, G. vaginalis, A. vaginalis and P. bivia decreased expression by 15%, 16% 338
and 27%, respectively (the latter of which was deemed statistically significant) (Figure 339
3C), when compared to tst expression S. aureus grown in half BHI and half VDMP. 340
Discussion 341
To our knowledge, this study is the first report of indigenous vaginal bacteria 342
altering TSST-1 production in S. aureus. In particular, several species of Lactobacillus 343
significantly suppressed tst expression, including L. gasseri and L. jensenii, which are 344
common vaginal residents, and L. johnsonii which is a gut commensal occasionally 345
found in the vagina. Surprisingly, however, L. iners and L. crispatus did not demonstrate 346
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tst suppression. We confirmed anti-TSST-1 activity from the vaginal probiotic strain L. 347
reuteri RC-14, but also that L. rhamnosus GR-1, another vaginal probiotic strain, was 348
capable of suppressing this toxin. 349
An interesting discovery was that aerobic bacteria induced tst expression. During 350
menses, increased oxygen levels and a neutral pH would increase the chances of 351
TSST-1 production (13-15), and the presence of neighboring aerobic bacteria may be 352
enough for S. aureus to secrete a threshold level of the toxin, leading to disease 353
progression. The finding that S. agalactiae (group B Streptococcus: GBS) significantly 354
induced expression by more than 3-fold was particularly interesting considering the 355
seemingly-symbiotic relationship between this species and S. aureus. Multiple studies 356
have found that S. agalactiae inhibits Lactobacillus spp. and G. vaginalis without 357
inhibiting S. aureus (34-36) and that S. aureus colonization was significantly associated 358
with colonization by S. agalactiae in a population of pregnant women (37). Our findings 359
thus illustrate another way in which GBS could promote the virulence of S. aureus. In 360
the context of menstrual-TSS, women with AV who are colonized predominantly with 361
these two species might be at a higher risk of developing the disease; elevated oxygen 362
levels in the vagina would presumably induce TSST-1 production in S. aureus (13), and 363
the presence of GBS may further perpetuate toxin production. If this relationship were to 364
hold true in suitable animal studies, the application to human of certain Lactobacillus 365
species intra-vaginally may be worth considering since they can displace S. aureus and 366
S. agalactiae from vaginal epithelial cells (38). 367
A number of Lactobacillus species tested (L. jensenii, L. gasseri, L. johnsonii) 368
suppressed tst expression in the supernatant challenge assay, in addition to the vaginal 369
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probiotic strain L. reuteri RC-14 which is known to confer this effect (19). Screening 370
these supernatants for the cyclic dipeptides linked to L. reuteri RC-14 is underway to 371
determine whether this or another substance is responsible for the tst-suppression. 372
Surprisingly, L. rhamnosus GR-1, another vaginal probiotic strain, also suppressed the 373
toxin. A study by Laughton et al. (2006) found that this strain, in contrast to L. reuteri 374
RC-14, was not able to suppress the RNAIII-initiating P3 promoter in S. aureus Newman 375
(39). This suggests that the inhibitory effect on tst seen by L. rhamnosus GR-1 was not 376
due to inhibition of agr but perhaps from altered SarA activity, as this regulator has been 377
shown to bind and activate the tst promoter directly (10). Lastly, L. iners and L. 378
crispatus, the predominant vaginal residents of healthy women, did not demonstrate 379
anti-tst activity. Considering the rare nature of menstrual-TSS, this suggests that other 380
factors are at play in preventing S. aureus in the vagina from secreting this toxin, such 381
as vaginal metabolites from the host. Nevetheless, this finding will aid in identifying 382
putative tst-suppressing factor(s) secreted by the other Lactobacillus members by 383
comparing their secretory products under the experimental conditions. 384
The complete content of vaginal swabs from women of a healthy, BV and 385
intermediate status were introduced to cultures of the reporter strain S. aureus 386
MN8/pAmilux-Ptst. Monitoring the luminescence produced from this strain allowed us to 387
monitor tst promoter activity in real time during the growth of S. aureus. It was 388
discovered that samples from all three vaginal states were able to significantly suppress 389
tst expression. However, the microbiota-sequencing data showed that these samples 390
had high abundances of L. iners, L. crispatus and G. vaginalis¸ all of which failed to 391
suppress tst in the challenge assay of the present study. This supports the idea that the 392
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majority of pre-menopausal women have host-derived factors that prevents the 393
development of menstrual-TSS. Identifying host-derived vaginal metabolites shared 394
among both healthy and BV women will be important for shedding light on putative 395
candidate compounds to test. It is important to note that two samples from the healthy 396
group failed to show any suppression, which illustrates that there may be a sub-397
population of women who would benefit from TSST-1-suppressive agents more so than 398
the general population. Interestingly, these were the same healthy samples to show 399
increased bacterial diversity, and although no unique bacteria were identified within the 400
samples, this suggests that an altered vaginal environment may contribute to increased 401
menstrual-TSS susceptibility. The suppression of tst expression in response to BV 402
samples was surprising, as these samples had pH’s greater than 4.5, with some as high 403
as 6.0, which is more conducive to TSST-1 production (14). However, the effect of pH 404
would likely be minimized as the transport medium used to preserve contents of the 405
swabs contained various buffering agents. Levels of oxygen and carbon dioxide would 406
not likely contribute to suppression, as all samples were exposed to atmospheric 407
conditions during collection and preparation. 408
The vaginal microbiota profiles of women recruited here were similar to those 409
seen in other studies, in that L. iners, L. crispatus and G. vaginalis made up the core 410
members of the microbiota (21, 40). However, the microbiota of the subjects varied from 411
person to person, regardless of health status. For instance, 4 of the 5 intermediate 412
samples consisted primarily of L. crispatus and L. iners, while the other was composed 413
of L. jensenii, L. gasseri/johnsonii and G. vaginalis. As well, although healthy vaginal 414
samples typically contain less diverse populations compared to BV, very complex 415
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populations were seen in two healthy samples (17 and 12 species per sample) and one 416
intermediate sample (11 species per sample) compared to the other samples (average 417
of 3.6 species per sample). Members of these populations included Streptococcus and 418
Enterococcus spp. Therefore, healthy women into which S. aureus colonize may already 419
be colonized with a diverse population of bacteria, some of which may release 420
compounds that induce TSST-1 production. 421
Overall, this study has shown that the vaginal environment of women with BV and 422
with a healthy background is able to suppress TSST-1, which is consistent with the rare 423
occurrence of menstrual-TSS. However, samples from a few subjects of the clinical 424
study failed to suppress the toxin, which illustrates a sub-population of women who may 425
be more susceptible to developing this condition. Identifying the factor(s) responsible for 426
the observed suppression will help identify a target population of pre-menopausal 427
women who would benefit most from prophylactic options, such as the application of 428
exogenous lactobacilli. Perhaps the biggest finding of this study was that aerobic 429
bacteria, including S. agalactiae, E. faecalis and E. faecium, had a TSST-1-inducing 430
effect on S. aureus. This suggests that women with AV or a complex aerobic microbiota, 431
which is occasionally seen during menses, may be at an increased risk of menstrual-432
TSS and would benefit most from a vaginally-applied probiotic.433
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48(5):1812-9. 547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
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569
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Table 1. Bacterial strains used for supernatant challenge assay. 573
Bacteria Source Growth Media/Conditions Staphylococcus aureus MN8 Clinical Isolate BHI/aerobic
Escherichia coli J96 Clinical Isolate BHI/aerobic Streptococcus agalactiae ATCC
13813 ATCC BHI/aerobic
Enterococcus faecalis ATCC 33186
ATCC BHI/aerobic
Enterococcus faecium ATCC 19434
ATCC BHI/aerobic
Atopobium vaginae BAA-55 Reid Culture
Collection
Vaginally-Defined Medium + 0.5% Proteose Peptone (VDMP)/anaerobic
Prevotella bivia 29303 Reid Culture Collection
VDMP/anaerobic
Gardnerella vaginalis Clinical Isolate VDMP/anaerobic Lactobacillus crispatus ATCC
33820 ATCC de Man, Rogosa and Sharpe
(MRS)/anaerobic Lactobacillus jensenii ATCC
25258 ATCC MRS/anaerobic
Lactobacillus johnsonii DSM20553
DSM MRS/anaerobic
Lactobacillus gasseri ATCC 33323
ATCC MRS/anaerobic
Lactobacillus reuteri RC-14 Clinical Isolate MRS/anaerobic Lactobacillus rhamnosus GR-1 Clinical Isolate MRS/anaerobic
Lactobacillus iners AB-1 Clinical Isolate MRS/anaerobic 574
575
576
577
578
579
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580
581
Figure 1. Vaginal microbiota of 21 subjects recruited from the clinical study, as 582
determined through 16S rRNA sequencing by Illumina. Vaginal state determined 583
via Nugent Scoring; score of 1-3 is healthy, 4-6 is intermediate, and 7-10 is BV. 584
Each column represents a participant (marked by their corresponding ID 585
number), with each coloured bar representing a type of bacteria. 586
587
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588
Figure 2. Luminescence activity of S. aureus MN8 tst gene in response to 589
vaginal swab contents from women of varying vaginal health (healthy, 590
intermediate and BV). (A) Vaginal samples from all 3 health groups were 591
capable of suppressing tst expression, as shown by three representative 592
samples. (B, C) Two healthy clinical samples failed to suppress tst expression. 593
S=Staphylococcus aureus MN8; #=Participant ID number; TM=Transport 594
Medium, the medium in which the swabs were preserved. 595
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596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
Figure 3. Fold change in gene expression of tst in S. aureus MN8 in response to 618
supernatants from lactobacilli (A), AV-associated (B) and BV-associated (C) bacteria, as 619
detected by real-time PCR. * indicates p<0.05. 620
621
Media
E. coli
E. faecalis
E. faecium
S. agalactiae
0
50
100
150
200
250
300
350
400
450
500
550*
Re
lativ
e ts
t Ex
pre
ssi
on
Media
L. rhamnosus G
R-1
L. reuteri RC-14
L. johnsonii
L. jensenii
L. gasseri
L. crispatus
L. iners
0
10
20
30
40
50
60
70
80
90
100
110
120
Re
lativ
e ts
t Ex
pre
ss
ion
Media
G. vaginalis
A. vaginalis
P. bivia
0
10
20
30
40
50
60
70
80
90
100
*
Source of Supernatant
Re
lati
ve
tst E
xp
res
sio
n
A
B
C
*
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