Characterisation of vaginal Lactobacillus isolates from South African women towards the development of a biotherapeutic to optimise the vaginal microbiome

This study characterizes vaginal *Lactobacillus* isolates from South African women and identifies *L. crispatus* strains as the most promising candidates for a tailored live biotherapeutic due to their superior acidification, low inflammatory response, and population-specific genomic features.

The Gist

Imagine your vagina is a bustling, healthy city. In this city, the "good guys" are a specific type of bacteria called Lactobacillus. They act like the city's police force and sanitation crew, keeping the streets clean, the pH balanced (not too acidic, not too alkaline), and keeping the "bad guys" (harmful bacteria that cause infections) out.

However, in many parts of Africa, this city is often under siege. A condition called Bacterial Vaginosis (BV) happens when the good bacteria leave or die off, and the bad bacteria take over. This isn't just uncomfortable; it's dangerous. It makes the city walls (the vaginal tissue) inflamed and weak, making it much easier for HIV to break in and infect the residents.

The Problem:
Doctors have tried to fix this city by using antibiotics (like a heavy-handed bulldozer) to knock down the bad bacteria. But the problem is, the bulldozer often knocks down the good guys too, and the bad bacteria come back stronger than before. We need a better solution: a way to bring in a new, super-strong police force that can stay and protect the city long-term.

The Mission:
This paper is about a team of scientists in South Africa who went on a "recruitment drive." They wanted to find the ultimate superhero bacteria from local women to create a new kind of medicine called a Live Biotherapeutic. Think of this as a "probiotic on steroids"—a pill or gel containing live, super-charged bacteria designed to repopulate the city and keep it safe.

The Search:
The scientists collected samples from 25 healthy women in South Africa. They found 181 different strains of good bacteria. But they didn't just pick any; they needed the best of the best. They put these bacteria through a series of "training camps" (lab tests) to see which ones had the right skills:

1. The Acid Test: Good bacteria produce lactic acid, which is like a natural shield that kills bad invaders. The scientists checked which bacteria produced the most acid.
2. The Peacekeeper Test: Sometimes, even good bacteria can accidentally cause a riot (inflammation). The scientists tested if the bacteria made the city walls angry (inflammatory) or calm. They wanted the ones that kept the peace.
3. The Genome Scan: They looked at the bacteria's "ID cards" (DNA) to see if they were truly local heroes.

The Results:
The clear winner was a specific type of bacteria called Lactobacillus crispatus.

• The Acid King: These bacteria were the best at making lactic acid, creating a strong shield against HIV and other infections.
• The Calmest: Unlike some other bacteria that caused a bit of a fuss (inflammation), L. crispatus kept the city walls calm and healthy.
• The Local Heroes: When the scientists looked at the DNA, they found that the South African L. crispatus were like a close-knit family. They were very similar to each other but quite different from bacteria found in the US or Europe.

Why This Matters (The "Local vs. Global" Analogy):
Imagine you are trying to plant a forest. If you plant trees from a tropical rainforest in a snowy tundra, they won't survive. Similarly, bacteria that work well for women in the US or Europe might not be able to survive or thrive in the unique environment of South African women.

This study proves that you need local heroes to protect local cities. The South African bacteria found here are genetically distinct and perfectly adapted to the local environment. Using bacteria from other parts of the world might be like trying to fit a square peg in a round hole.

The Future:
The scientists found that these local L. crispatus bacteria are safe (they don't carry dangerous drug-resistance genes) and they have special weapons (called bacteriocins) to fight off bad bacteria.

In a nutshell:
This paper is a blueprint for building a custom-made "bodyguard" for women in South Africa. By using the best local bacteria, scientists hope to create a treatment that stops BV, calms inflammation, and ultimately helps prevent HIV, giving women a stronger, more natural defense system. It's about giving the city its own local police force, trained and ready to protect the people who live there.

Technical Summary

1. Problem Statement

• HIV Risk in Sub-Saharan Africa: Adolescent girls and young women in sub-Saharan Africa face a disproportionately high risk of HIV infection. A primary biological risk factor is Bacterial Vaginosis (BV), a condition characterized by a loss of optimal Lactobacillus species and an overgrowth of anaerobic bacteria.
• Limitations of Current Treatments: Standard antibiotic treatments for BV have high recurrence rates. Furthermore, the efficacy of topical HIV pre-exposure prophylaxis (PrEP) is reduced in women with BV, and adherence to oral PrEP remains poor in this demographic.
• The Biotherapeutic Gap: While live biotherapeutics (probiotics) containing Lactobacillus species show promise for treating BV and reducing HIV risk, existing candidates are largely derived from Western populations. There is a critical lack of well-characterized Lactobacillus isolates from African women, despite evidence suggesting that vaginal microbiota composition and strain-level adaptations vary significantly by geography and ethnicity.

2. Methodology

The study employed a multi-stage approach involving clinical sampling, phenotypic characterization, and genomic analysis:

• Cohort and Sampling:

  • Source: 25 BV-negative (Nugent score 0–3) South African women (aged 16–32) from the MISC study in Cape Town.
  • Isolation: 181 Lactobacillus isolates were cultured from cervicovaginal secretions. Species identified included L. crispatus (72), L. reuteri (30), L. jensenii (24), L. gasseri (23), L. vaginalis (22), L. johnsonii (7), L. salivarius (2), and L. coleohominis (1).
  • Selection: 50 morphologically distinct isolates from 18 women were selected for detailed evaluation. Notably, 9 isolates came from women with concurrent sexually transmitted infections (STIs) to assess if STI status altered isolate characteristics.

• Phenotypic Characterization:

  • Metabolite Production: Quantification of D-lactate, L-lactate, total lactate, and culture pH after 24-hour anaerobic incubation. Lactic acid concentrations were calculated using the Henderson-Hasselbalch equation.
  • Inflammatory Response: Co-culture of isolates with VK2/E6E7 vaginal epithelial cells. Cytokine levels (IL-6, IL-8, IL-1α, IL-1β, IL-1RA, CCL2, CCL3, CCL4, CCL5) were measured via Luminex. Gardnerella vaginalis served as a positive control for inflammation. Cell viability was assessed via Trypan blue exclusion.
  • Ranking System: Isolates were scored (1–4) based on D-lactate, L-lactate, pH (lower is better), anti-inflammatory IL-1RA (higher is better), and inflammatory cytokines (lower is better). Inflammatory scores were doubled to prioritize low inflammation.

• Genomic Analysis (Whole Genome Sequencing - WGS):

  • Selection: 10 L. crispatus isolates (3 top-performing + 7 varying performers) were sequenced using PacBio HiFi reads.
  • Bioinformatics: Assemblies were screened for Antimicrobial Resistance (AMR) genes (ResFinder, CARD), plasmids (PlasmidFinder), bacteriocins (BAGEL4), and prophages (PHASTEST, checkV, Pharokka).
  • Phylogenetics: Maximum likelihood phylogeny was constructed to compare South African isolates against global reference strains (US, Brazil, Europe, HMP) and ATCC 33820.

3. Key Results

• Metabolic Performance:

  • Lactic Acid Production: L. crispatus isolates produced the highest levels of D-lactate, L-lactate, and total lactate, resulting in the most significant culture acidification (lowest pH).
  • Correlation: Total lactate production was strongly inversely correlated with culture pH ($R = -0.80$).
  • Species Comparison: L. crispatus outperformed L. jensenii, L. gasseri, and L. vaginalis in acidification and total lactate production.

• Immunomodulation:

  • Inflammation: Most Lactobacillus isolates induced lower inflammatory cytokine responses (IL-6, IL-8, IL-1α, etc.) compared to G. vaginalis.
  • Anti-inflammatory Activity: L. crispatus strains elicited the lowest levels of pro-inflammatory cytokines and the highest levels of the anti-inflammatory cytokine IL-1RA.
  • STI Impact: No significant differences were found in the overall ranking or inflammatory profiles between isolates from STI-positive and STI-negative women, though STI-positive isolates showed a trend toward lower L-lactate production.

• Genomic Findings:

  • Phylogeny: The 10 sequenced L. crispatus isolates formed a distinct clade, clustering more closely with each other than with isolates from the US, Brazil, Europe, or the ATCC reference strain. This suggests population-specific adaptation.
  • Safety Profile: No antimicrobial resistance (AMR) genes, plasmids, or chromosomal mutations conferring resistance were detected.
  • Functional Genes:
    • Bacteriocins: All isolates harbored putative bacteriocin genes, including penocin A, enterolysin A, and helveticin J. Seven of ten isolates contained linear azol(in)e-containing peptides (LAPs).
    • Prophages: Intact putative prophages were identified in all isolates. While most were similar to known Lactobacillus phages, some showed limited similarity to reference databases, indicating novel sequences.
    • Enzymes: L-lactate and D-lactate dehydrogenase genes were highly conserved (99.7–100% identity), despite phenotypic variations in lactate production.

• Top Candidates:

  • L. crispatus dominated the top rankings. The top 10 isolates included 8 L. crispatus strains.
  • Top Performer: Isolate PID182 3a (L. crispatus) achieved the highest total score (22/24), characterized by high D/L-lactate, low pH, and low inflammation.

4. Significance and Contributions

• Population-Specific Biotherapeutics: The study provides strong evidence that vaginal Lactobacillus strains are geographically distinct. The South African isolates form a unique phylogenetic cluster, supporting the hypothesis that live biotherapeutics must be tailored to the specific population they intend to treat to ensure effective colonization.
• Identification of Candidates: The study identifies specific L. crispatus strains from South African women that possess the ideal "biotherapeutic signature": high lactic acid production (crucial for HIV inactivation and pH control), low inflammatory potential, and the presence of bacteriocins (for pathogen inhibition).
• Safety Validation: The absence of detectable AMR genes and plasmids in the sequenced isolates addresses a critical safety concern for clinical development.
• Future Directions: The identification of intact prophages highlights a need for further functional characterization to ensure they are not inducible or virulence-enhancing before clinical trials. The study lays the groundwork for developing a region-specific live biotherapeutic product to treat BV and reduce HIV acquisition risk in African women.

Conclusion

This research successfully characterizes a novel panel of vaginal Lactobacillus isolates from South Africa, identifying L. crispatus as the superior candidate for live biotherapeutic development. The findings underscore the necessity of using locally derived strains to address the specific microbiome and HIV risks of African women, moving beyond the reliance on Western-derived probiotic strains.