CoChat
Sign Up
Cited by 140Reproductive tract infections researchProbiotics and Fermented FoodsGut microbiota and health

The DOI matches its Crossref record; 82 of the 82 references it cites were found in a registry or an index.

Lactobacillus crispatus inhibits growth of Gardnerella vaginalis and Neisseria gonorrhoeae on a porcine vaginal mucosa model

Open access
Breshears, Laura M., Edwards, Vonetta L., Ravel, Jacques, Peterson, Marnie L.·2015·BMC Microbiology
DOI 10.1186/s12866-015-0608-0PMC PMC4675025
Open this paper in CoChat — free accountOpen PDF

Abstract

BACKGROUND: The vaginal microbiota can impact the susceptibility of women to bacterial vaginosis (BV) and sexually transmitted infections (STIs). BV is characterized by depletion of Lactobacillus spp., an overgrowth of anaerobes (often dominated by Gardnerella vaginalis) and a pH > 4.5. BV is associated with an increased risk of acquiring STIs such as chlamydia and gonorrhea. While these associations have been identified, the molecular mechanism(s) driving the risk of infections are unknown. An ex vivo porcine vaginal mucosal model (PVM) was developed to explore the mechanistic role of Lactobacillus spp. in affecting colonization by G. vaginalis and Neisseria gonorrhoeae. RESULTS: The data presented here demonstrate that all organisms tested can colonize and grow on PVM to clinically relevant densities. Additionally, G. vaginalis and N. gonorrhoeae form biofilms on PVM. It was observed that lactic acid, acetic acid, and hydrochloric acid inhibit the growth of G. vaginalis on PVM in a pH-dependent manner. N. gonorrhoeae grows best in the presence of lactic acid at pH 5.5, but did not grow well at this pH in the presence of acetic acid. Finally, a clinical Lactobacillus crispatus isolate (24-9-7) produces lactic acid and inhibits growth of both G. vaginalis and N. gonorrhoeae on PVM. CONCLUSIONS: These data reveal differences in the effects of pH, various acids and L. crispatus on the growth of G. vaginalis and N. gonorrhoeae on a live vaginal mucosal surface. The PVM is a useful model for studying the interactions of commensal vaginal microbes with pathogens and the mechanisms of biofilm formation on the vaginal mucosa.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1186/s12866-015-0608-0

Cites

82 of 82 references verified0 citation errors · 0 not checkable

Statistics

Figures

Fig. 1 Growth of human clinical isolates on porcine vaginal mucosa (PVM). a PVM is obtained as large specimens (i) and 5 mm explants are trimmed (ii) and placed mucosal side up in transwells over liquid media (iii, iv). “Air” refers to either an aerobic or anaerobic environment depending on the experiment being performed. b L. crispatus, c G. vaginalis, and d N. gonorrhoeae were inoculated onto PVM explants at ~ 104 CFU/explant (dotted lines). Explants were processed for CFU/explant at each time point to evaluate bacterial growth. Data was log10 transformed and plotted on a log scale as the mean ± SD
Fig. 1 Growth of human clinical isolates on porcine vaginal mucosa (PVM). a PVM is obtained as large specimens (i) and 5 mm explants are trimmed (ii) and placed mucosal side up in transwells over liquid media (iii, iv). “Air” refers to either an aerobic or anaerobic environment depending on the experiment being performed. b L. crispatus, c G. vaginalis, and d N. gonorrhoeae were inoculated onto PVM explants at ~ 104 CFU/explant (dotted lines). Explants were processed for CFU/explant at each time point to evaluate bacterial growth. Data was log10 transformed and plotted on a log scale as the mean ± SDCC BY · PubMed Central
Fig. 2 G. vaginalis and N. gonorrhoeae form biofilm on PVM. Strains were inoculated onto PVM explants at ~ 104 CFU/explant and processed for microscopy at indicated times. The LIVE/DEAD stain allows for imaging of both the mucosal epithelium and bacteria. Green cells are alive while red cells are dead. a-d, i-l Uncolonized control (CNTL) tissue remains healthy throughout both experiments as evidenced by large green intact epithelial cells. e-h By 48 h post-colonization, G. vaginalis (GV) forms a patchy biofilm that persists and spreads over time (anaerobic growth). m-p In just 24 h N. gonorrhoeae (NG) forms a robust biofilm, which thickens and persists over time (aerobic growth). Epithelial cells that can be seen under and around the NG biofilm are alive as evidenced by their green staining, while those on GV-colonized explants are dead (large red cells under the green biofilm). Scale bars = 100 μm for all images
Fig. 2 G. vaginalis and N. gonorrhoeae form biofilm on PVM. Strains were inoculated onto PVM explants at ~ 104 CFU/explant and processed for microscopy at indicated times. The LIVE/DEAD stain allows for imaging of both the mucosal epithelium and bacteria. Green cells are alive while red cells are dead. a-d, i-l Uncolonized control (CNTL) tissue remains healthy throughout both experiments as evidenced by large green intact epithelial cells. e-h By 48 h post-colonization, G. vaginalis (GV) forms a patchy biofilm that persists and spreads over time (anaerobic growth). m-p In just 24 h N. gonorrhoeae (NG) forms a robust biofilm, which thickens and persists over time (aerobic growth). Epithelial cells that can be seen under and around the NG biofilm are alive as evidenced by their green staining, while those on GV-colonized explants are dead (large red cells under the green biofilm). Scale bars = 100 μm for all imagesCC BY · PubMed Central
Fig. 3 Various acids inhibit growth of G. vaginalis and N. gonorrhoeae on PVM. RPMI was pH-adjusted with lactic acid, acetic acid or hydrochloric acid (HCl) and placed under transwells containing PVM explants. Bacteria were enumerated 48 h post-colonization. L. crispatus and G. vaginalis were incubated anaerobically, while N. gonorrhoeae was incubated aerobically. a The low pH produced by each of the three acids has no effect on growth of L. crispatus. b G. vaginalis growth is inhibited at pH 4.0 in the presence of all three acids. c N. gonorrhoeae growth peaked at pH 5.5 in the presence of lactic acid and HCl, while growth was inhibited at pH 5.5 in the presence of acetic acid. Asterisks indicate significant difference from inoculum (dotted lines) (p < 0.0001)
Fig. 3 Various acids inhibit growth of G. vaginalis and N. gonorrhoeae on PVM. RPMI was pH-adjusted with lactic acid, acetic acid or hydrochloric acid (HCl) and placed under transwells containing PVM explants. Bacteria were enumerated 48 h post-colonization. L. crispatus and G. vaginalis were incubated anaerobically, while N. gonorrhoeae was incubated aerobically. a The low pH produced by each of the three acids has no effect on growth of L. crispatus. b G. vaginalis growth is inhibited at pH 4.0 in the presence of all three acids. c N. gonorrhoeae growth peaked at pH 5.5 in the presence of lactic acid and HCl, while growth was inhibited at pH 5.5 in the presence of acetic acid. Asterisks indicate significant difference from inoculum (dotted lines) (p < 0.0001)CC BY · PubMed Central
Fig. 4 Unbuffered RPMI does not support sufficient pH changes in the presence of L. crispatus on PVM. Unbuffered RPMI was placed under transwells and relevant explants were inoculated with L. crispatus. After 48 h, relevant explants were inoculated with G. vaginalis. Experiments were performed under anaerobic conditions and the pH of media below explants was monitored over time. a Media under uncolonized controls (CNTL) and those colonized with G. vaginalis (GV) alone reached pH 6.5 while those colonized with L. crispatus (LC) alone or co-colonized with both organisms (LC + GV) reached pH 6.0 (N = 4, n = 1). b The presence of L. crispatus had a statistically significant (asterisk, p < 0.016), yet minor effect on G. vaginalis growth. Dotted line represents GV inoculum
Fig. 4 Unbuffered RPMI does not support sufficient pH changes in the presence of L. crispatus on PVM. Unbuffered RPMI was placed under transwells and relevant explants were inoculated with L. crispatus. After 48 h, relevant explants were inoculated with G. vaginalis. Experiments were performed under anaerobic conditions and the pH of media below explants was monitored over time. a Media under uncolonized controls (CNTL) and those colonized with G. vaginalis (GV) alone reached pH 6.5 while those colonized with L. crispatus (LC) alone or co-colonized with both organisms (LC + GV) reached pH 6.0 (N = 4, n = 1). b The presence of L. crispatus had a statistically significant (asterisk, p < 0.016), yet minor effect on G. vaginalis growth. Dotted line represents GV inoculumCC BY · PubMed Central
Fig. 5 Conditioned media supports L. crispatus-induced pH changes, inhibiting growth of G. vaginalis on PVM. Media from overnight aerobic broth culture of L. crispatus was sterile filtered and mixed with unbuffered RPMI to achieve pH 5.5. This conditioned media (CM) was placed under transwells and relevant explants were inoculated with L. crispatus. After 48 h, relevant explants were inoculated with G. vaginalis. Experiments were performed under anaerobic conditions and the pH of media below explants was monitored over time. a Media under uncolonized controls (CNTL) and those colonized with G. vaginalis (GV) alone remained at pH 5.0 – 5.5 while those colonized with L. crispatus (LC) alone or co-colonized with both organisms (LC + GV) were reduced to pH 4.0 (N = 6, n = 1). b The presence of L. crispatus inhibited G. vaginalis growth. Dotted line represents GV inoculum. Asterisk indicates significant difference in growth between the two groups shown (p < 0.0001). c, d D- and L-lactate levels in the CM below transwells from experiment (a) were analyzed at 96 h. CM below explants colonized with L. crispatus (LC and LC + GV) showed a significant increase in lactic acid when compared with uncolonized controls (CM) and G. vaginalis (GV) alone (p < 0.0001). e A comparison of D- and L- lactate levels in CM below LC alone infections shows that D-lactate is produced at ~2X the level of L-lactate (p < 0.002). (C-E) Data shown are combined (N = 5, n = 1)
Fig. 5 Conditioned media supports L. crispatus-induced pH changes, inhibiting growth of G. vaginalis on PVM. Media from overnight aerobic broth culture of L. crispatus was sterile filtered and mixed with unbuffered RPMI to achieve pH 5.5. This conditioned media (CM) was placed under transwells and relevant explants were inoculated with L. crispatus. After 48 h, relevant explants were inoculated with G. vaginalis. Experiments were performed under anaerobic conditions and the pH of media below explants was monitored over time. a Media under uncolonized controls (CNTL) and those colonized with G. vaginalis (GV) alone remained at pH 5.0 – 5.5 while those colonized with L. crispatus (LC) alone or co-colonized with both organisms (LC + GV) were reduced to pH 4.0 (N = 6, n = 1). b The presence of L. crispatus inhibited G. vaginalis growth. Dotted line represents GV inoculum. Asterisk indicates significant difference in growth between the two groups shown (p < 0.0001). c, d D- and L-lactate levels in the CM below transwells from experiment (a) were analyzed at 96 h. CM below explants colonized with L. crispatus (LC and LC + GV) showed a significant increase in lactic acid when compared with uncolonized controls (CM) and G. vaginalis (GV) alone (p < 0.0001). e A comparison of D- and L- lactate levels in CM below LC alone infections shows that D-lactate is produced at ~2X the level of L-lactate (p < 0.002). (C-E) Data shown are combined (N = 5, n = 1)CC BY · PubMed Central
Fig. 6 L. crispatus inhibits growth of N. gonorrhoeae. a, b Unbuffered RPMI or (c) L. crispatus CM was adjusted to pH 5.5 and placed under transwells. Relevant explants were inoculated with L. crispatus. After 48 h, relevant explants were inoculated with N. gonorrhoeae. Experiments were performed under aerobic conditions and the pH of media below explants was monitored over time. a RPMI under uncolonized controls (CNTL) and those colonized with N. gonorrhoeae (NG) alone remained at pH 5.5 while RPMI under explants colonized with L. crispatus (LC) alone or co-colonized with both organisms (LC + NG) was reduced to pH 4.5 (N = 4, n = 1). b The presence of L. crispatus inhibited 48 h N. gonorrhoeae growth. Asterisk indicates significant difference in growth between the two groups shown (p < 0.002). c CM under CNTL or NG remains at pH 5.5 while CM under explants colonized with LC alone or co-colonized with both organisms (LC + NG) was reduced to pH 4.0 (N = 3, n = 1). d While N. gonorrhoeae did exhibit a growth defect over NYC + RPMI media (the background media for CM) it was completely killed by aerobic or anaerobic CM at pH 5.5, even in the absence of a L. crispatus co-infection. Asterisks indicate significant difference from inoculum (p < 0.0001). For (b) and (d), dotted lines represent NG inocula
Fig. 6 L. crispatus inhibits growth of N. gonorrhoeae. a, b Unbuffered RPMI or (c) L. crispatus CM was adjusted to pH 5.5 and placed under transwells. Relevant explants were inoculated with L. crispatus. After 48 h, relevant explants were inoculated with N. gonorrhoeae. Experiments were performed under aerobic conditions and the pH of media below explants was monitored over time. a RPMI under uncolonized controls (CNTL) and those colonized with N. gonorrhoeae (NG) alone remained at pH 5.5 while RPMI under explants colonized with L. crispatus (LC) alone or co-colonized with both organisms (LC + NG) was reduced to pH 4.5 (N = 4, n = 1). b The presence of L. crispatus inhibited 48 h N. gonorrhoeae growth. Asterisk indicates significant difference in growth between the two groups shown (p < 0.002). c CM under CNTL or NG remains at pH 5.5 while CM under explants colonized with LC alone or co-colonized with both organisms (LC + NG) was reduced to pH 4.0 (N = 3, n = 1). d While N. gonorrhoeae did exhibit a growth defect over NYC + RPMI media (the background media for CM) it was completely killed by aerobic or anaerobic CM at pH 5.5, even in the absence of a L. crispatus co-infection. Asterisks indicate significant difference from inoculum (p < 0.0001). For (b) and (d), dotted lines represent NG inoculaCC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 82 references

Cited by

Sorted by citations

See all 140

Related

Metadata

Authors
Breshears, L. M., Edwards, V. L., Ravel, J., & Peterson, M. L.
Affiliations
  • University of Minnesota (US)
  • University of Maryland, Baltimore (US)
Corresponding author
Peterson, Marnie L.
ORCID
Venue
BMC Microbiology
Publisher
Springer Science and Business Media LLC
Published
2015-12
Volume
15
Issue
1
Pages
276-276
ISSN
1471-2180 (ISSN-L)
DOI
10.1186/s12866-015-0608-0
PMC
PMC4675025
OpenAlex
W2192796897
Cited by
140
References
82
Citations by year
2026: 10 · 2025: 12 · 2024: 22 · 2023: 12 · 2022: 13 · 2021: 17 · 2020: 15 · 2019: 11 · 2018: 13 · 2017: 12 · 2016: 3
FWCI
7.86
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Microbiology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • University of Minnesota (22928)
  • National Institute of Allergy and Infectious Diseases (U19AI084044)
Topics
  • Life Sciences › Immunology and Microbiology › Microbiology › Reproductive tract infections research
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
Keywords
Lactobacillus crispatus, Gardnerella vaginalis, Neisseria gonorrhoeae, bacterial vaginosis, vaginal microbiota, biofilm formation, lactic acid, vaginal pH, sexually transmitted infections, vaginal colonization
MeSH terms
Animals, Antibiosis, Carboxylic Acids, Female, Gardnerella vaginalis, Humans, Hydrogen-Ion Concentration, Lactobacillus, Male, Mucous Membrane, Neisseria gonorrhoeae, Organ Culture Techniques, Swine, Vagina, Models, Animal

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review