Exploring potential of vaginal Lactobacillus isolates from South African women for enhancing treatment for bacterial vaginosis

Antibiotics continue to be the standard-of-care for bacterial vaginosis (BV), although recurrence rates are high. Vaginal probiotics may improve durability of BV treatment, although few probiotics for vaginal health contain Lactobacillus spp. that commonly colonize the lower female genital tract. Characteristics of vaginal Lactobacillus strains from South African women were evaluated for their probiotic potential in vitro compared to strains from commercial vaginal products, including growth at varying pHs, ability to lower pH, produce D-/L-lactate and H2O2, influence growth of BV-associated Gardnerella vaginalis and Prevotella bivia, adherence to cervical cells and susceptibility to antibiotics. Fifty-seven Lactobacillus strains were purified from cervico-vaginal fluid, including L. crispatus, L. jensenii, L. gasseri, L. mucosae, and L. vaginalis. L crispatus strains grew better at pHs below 4.5 and lowered pH more effectively than other strains. Production of D-/L-lactate and H2O2 varied between Lactobacillus species and strains. Lactobacillus strains generally inhibited P. bivia more uniformly than G. vaginalis isolates. All vaginal Lactobacillus isolates were resistant to metronidazole while susceptibility to clindamycin varied. Furthermore, vaginal Lactobacillus strains tended to be broadly susceptible to penicillin, amoxicillin, rifampicin and rifabutin. Whole-genome-sequencing of five of the best-performing vaginal Lactobacillus strains confirmed their likely safety, due to antimicrobial resistance elements being largely absent, while putative intact prophages were present in the genomes of two of the five strains. Overall, vaginal Lactobacillus strains largely performed better in these in vitro assays than probiotic strains currently used in probiotics for vaginal health. Including the best-performing vaginal Lactobacillus isolates in a region-specific probiotic for vaginal health may result in improved BV treatment options.


Introduction
Maintenance of vaginal health is important in protecting women from adverse urogenital and reproductive health outcomes [1]. Optimally, the lower female genital tract (FGT) has a pH <4.5 [2,3] and a Lactobacillus-dominated microbiota [4,5]. However, the lower FGT microbiota frequently shifts to a non-optimal state that is characterised by a depletion of Lactobacillus spp. and high relative abundance of a diverse array of anaerobic bacteria, coinciding with elevation in vaginal pH �4.5-referred to as bacterial vaginosis (BV) [1]. BV can lead to severe reproductive complications [6][7][8], such as an increase in the risk for acquiring and transmitting sexually-transmitted infections (STIs) including human immunodeficiency virus (HIV) [9][10][11]. The current clinical standard of care (SOC) for BV is either oral or vaginal metronidazole or clindamycin [12]. However, antibiotic treatment of BV only results in a short-term cure as the recurrence rates are high, with~50% of women recurring within six months [13,14]. As a result of this, several clinical studies evaluated Lactobacillus-containing probiotics as an adjunct to BV treatment and have shown largely beneficial although heterogeneous outcomes [14][15][16][17]. It is therefore crucial to develop more effective probiotic treatment strategies for BV. Few probiotics are explicitly marketed for vaginal health internationally and in South Africa [18], and only few of these contain Lactobacillus spp. commonly found in FGTs of women with optimal microbiota. Bacterial strains should fulfil specific biological criteria if their intended purpose is to be developed into a probiotic to improve FGT health-collectively referred to as the preferred product profile (PPP). In this study, we evaluated a range of PPP characteristics that should be considered in the development of vaginal probiotics. These included: (1) originating from the FGT, as vaginal Lactobacillus spp. are highly adapted for this specialized niche [19]; (2) ability to adhere well to FGT cells, as adherent isolates are more likely to remain locally [20][21][22]; (3) inhibiting the growth of BV-associated species, including G. vaginalis and P. bivia, [23][24][25] (4) tolerating low pH associated with vaginal health [26][27][28]; (5) lowering in vitro culture pH conditions, thereby inhibiting viral and bacterial pathogens [2,13,29,30]; (6) producing L-and D-lactate, which are involved in viral and bacterial pathogen inhibition [30][31][32]; and (7) tolerating antibiotics used to treat BV if administered in conjunction SOC [33][34][35]. All these PPP characteristics call for the use of bacterial strains adapted to the particular conditions of the FGT, which will mostly be met in Lactobacillus spp. originating from the FGT of generally healthy women. Thus, this study aimed to isolate and evaluate key characteristics of vaginal Lactobacillus strains from South African women to select the top performing strains for the development of a probiotic for vaginal health. Furthermore, the characteristics of vaginal strains were compared to those of commercially available probiotic strains currently used in probiotic formulations for vaginal health in South Africa and internationally.

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Comparing probiotic potential of vaginal Lactobacillus isolates Ability to lower culture pH All vaginal Lactobacillus strains tended to have similar kinetics in their ability to lower pH under anaerobic conditions, with the lowest pH reached being pH 3.7, while none of the probiotic isolates lowered the pH <4.2 (S1 Fig). Overall, L. crispatus lowered the pH significantly more than L. gasseri, L. mucosae, L. vaginalis and any of the probiotic strains at 48 hours, after adjusting for multiple comparisons (Fig 2). Growth of individual isolates at pH 6.0 did not significantly predict their ability to lower culture pH at 48 hours (Spearman rho = -0.17, p = 0.165), indicating that differences in growth between strains did not account for differences in their ability to lower pH.

L-and D-lactate production
L-and D-lactate are considered important metabolic by-products of Lactobacillus strain metabolism that have anti-viral and anti-bacterial activity, respectively [30][31][32]. Therefore, the ability of vaginal Lactobacillus strains (46/57, due to assay limitations) to produce L-and Dlactate were evaluated under anaerobic conditions (Fig 3A and 3B). Vaginal Lactobacillus spp.
tended to produce lower amounts of L-than D-lactate (median 10.6 ng/μL [IQR 5.6-19.4] vs. 35.8 ng/μL [30.4-41.9], p<0.0001), although all appeared to produce both isomers. Some vaginal strains produced high levels of L-but almost no D-lactate (such as L. crispatus 95.34PA), while others produced large amounts of D-but little L-lactate (such as L. jensenii 96.45PA). Again, STI and BV status of the donor did not influence L-and D-lactate levels measured ( Fig  3A and 3B). Further, the majority of vaginal Lactobacillus isolates produced more of both isomers than their respective ATCC reference strains. With the exception of two probiotic L. acidophilus isolates (which produced similar levels of both isomers), all probiotic strains produced higher levels of L-than D-lactate, although concentrations of both were significantly lower than those of the vaginal strains (p<0.0001).
The ability of strains to produce L-and D-lactate did not correlate (Spearman rho = 0.00, p = 0.9592), neither did production of D-lactate (Spearman rho = 0.05, p = 0.7250) or L-lactate (Spearman rho = -0.23, p = 0.0838) with growth. There was also no correlation between ability of isolates to lower culture pH and their ability to produce L-or D-lactate (Spearman rho = 0.15, p = 0.2604 for L-lactate; rho = -0.02, p = 0.8960 for D-lactate), indicating that lactate was not the only cause of lowering culture pH.

H 2 O 2 production
Earlier studies suggested that H 2 O 2 was the most influential Lactobacillus spp.-produced metabolite to inhibit pathogens in the lower FGT, although these studies were conducted aerobically [39,40]. To mimic in vivo conditions in the lower FGT, all experiments in this study were performed under anaerobic conditions. All of the vaginal L. gasseri and L. vaginalis strains produced small quantities of H 2 O 2 (ranging from 0.5-15 μM), while some of the L. jensenii, L. crispatus, and L. mucosae strains did not produce any measurable H 2 O 2 at all ( Fig 3C). Of all previously evaluated in vitro characteristics, H 2 O 2 only correlated with L-lactate levels positively (Spearman rho = 0.61, p<0.0001), but not with D-lactate (Spearman rho = -0.20, p = 0.1504).

Ability to inhibit P. bivia and G. vaginalis strains
The ability of abiotic culture supernatants from a subset of vaginal Lactobacillus strains (25/57; selected based on performance in previous assays) to inhibit the growth of a panel of P. bivia and G. vaginalis strains was determined under anaerobic conditions (Fig 4). Most of the vaginal Lactobacillus isolates (besides L. gasseri 94.98PB, L. mucosae 86.30PA and 87.21PA) showed some degree of inhibition towards all clinical P. bivia strains, although the extent of inhibition differed by Lactobacillus and P. bivia strain (Fig 4). L. crispatus tended to inhibit P. bivia growth to a greater extent than other species, inhibiting all P. bivia strains > 90%. Vaginal Lactobacillus strains inhibited P. bivia similarly to their respective ATCC and the probiotic Lactobacillus strains (Fig 4).
In contrast, the inhibition of G. vaginalis strains was generally more variable and significantly lower (Fig 4). Only three vaginal Lactobacillus (L. crispatus 100.16A, L. jensenii 88.33PA and 92.27PA) and the L. crispatus ATCC strain inhibited the growth of the full panel of G.

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Comparing probiotic potential of vaginal Lactobacillus isolates

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Comparing probiotic potential of vaginal Lactobacillus isolates vaginalis strains. Inhibition appeared to be highly G. vaginalis strain-specific, as almost all Lactobacillus strains inhibited the vaginal G. vaginalis isolate 3H1 (Fig 4; bottom row), while only 9/25 inhibited the G. vaginalis ATCC strain (Fig 4; top row). Overall, L. crispatus strains tended to exhibit the strongest inhibitory activity against G. vaginalis, although this was not statistically significant. The ATCC reference Lactobacillus strains generally showed broader and better inhibitory activity against G. vaginalis than vaginal Lactobacillus isolates (Fig 4). Notably, the abilities of vaginal Lactobacillus strains to inhibit the pathogens tested were highly correlated (G. vaginalis and P. bivia: Spearman rho = 0.814, p<0.0001), indicating that individual Lactobacillus strains have a similar capacity to inhibit various BV-associated bacteria. Similarly to the previously assessed characteristics, BV/STI status of the donor did not influence inhibitory abilities.

Antibiotic susceptibility
Metronidazole and clindamycin are SOC for BV treatment and penicillin and amoxicillin are commonly used antibiotics. All vaginal Lactobacillus isolates (n = 57) were resistant to metronidazole, while the susceptibility to clindamycin varied within and between Lactobacillus spp. (Fig 5A). L. vaginalis and L. jensenii isolates were the most susceptible to clindamycin, while L. gasseri strains were the least. The inhibition zones of the probiotic strains were smaller than those of most clinical strains (with the exception of L. gasseri), indicating that these might be less susceptible to clindamycin than the majority of vaginal Lactobacillus strains.
All vaginal Lactobacillus isolates showed similar susceptibility to penicillin, with the exception of L. gasseri isolates, which tended to be less susceptible ( Fig 5A). Probiotic L. acidophilus strains were more susceptible to penicillin than most vaginal strains, while L. rhamnosus isolates tended to be similarly susceptible, and L. reuteri less. The inhibition zones for amoxicillin were highly comparable to those measured for penicillin, as was the intra-species variability ( Fig 5A). L. crispatus isolates were significantly more susceptible to amoxicillin than L. rhamnosus (adj. p = 0.0497), and L. mucosae than L. reuteri (adj. p = 0.0128) and L. rhamnosus (adj. p = 0.0033), indicating that L. reuteri and L. rhamnosus but not L. acidophilus strains may be less susceptible to amoxicillin than the vaginal isolates.
The rifamycins rifampicin and rifabutin are commonly used to treat tuberculosis (TB) and form part of the six-month antibiotic regimen administered to~500,000 people in South Africa who develop TB annually [41], and have previously been explored as a treatment option for BV [42,43]. All Lactobacillus isolates were highly susceptible to rifampicin (<12.5 μg/ml) and rifabutin (<10.0 μg/mL) and susceptibilities of the probiotic isolates were similar to the vaginal strains (Fig 5A).
Broader antibiotic resistance profiling to inhibitors of protein, cell wall/membrane and DNA synthesis was conducted on a subset of 14 vaginal and six probiotic Lactobacillus isolates ( Fig 5B). Vaginal L. gasseri strains appeared to be susceptible to a wider range of antibiotics than other Lactobacillus spp. L. crispatus strains showed exactly the same antibiotic susceptibility, while the intra-species variability was higher for other vaginal Lactobacillus spp. There were no clear differences between vaginal and probiotic Lactobacillus spp. in antibiotic susceptibility profiles. Based on the clinical MIC breakpoints defined by the EUCAST (2019), all vaginal isolates would be considered sensitive to ampicillin (MIC �4 μg/mL), and the majority to chloramphenicol (MIC �8μg/mL).

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Comparing probiotic potential of vaginal Lactobacillus isolates clindamycin (MIC �1 μg/mL), while all other isolates had MICs >1μg/mL. Since the breakpoint for clindamycin has been defined as >4μg/mL, accoding to the EUCAST 2019 guidelines, it was not possible to conclude with the current data whether these isolates are resistant. vaginalis (orange), and L. mucosae (grey) and probiotic (red) isolates to metronidazole (5 μg), clindamycin (2 μg), penicillin (2 μg) and amoxicillin (10 μg) was determined using disc diffusion assays. The susceptibility to rifampicin and rifabutin was determined using broth dilution assays. (B) For a subset of 20 Lactobacillus isolates, the MIC to antibiotics interfering with the protein synthesis (purple), cell wall and membrane (green) and DNA synthesis (red) was determined using Sensititre plates. Bar graphs show the median (IQR) for each species, while each line in the radar plot represent the susceptibility (log-transformed MICs) of individual isolates. https://doi.org/10.1371/journal.ppat.1008559.g005

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Comparing probiotic potential of vaginal Lactobacillus isolates
For a subset of vaginal Lactobacillus isolates that included three strains per vaginal Lactobacillus spp. and six probiotic strains, the effect on cervical epithelial Ca Ski cell viability and cytokine expression was determined (Fig 7). None of the vaginal strains decreased the viability of Ca Ski cells below 80%, as measured by trypan blue straining. After grouping the cytokines into their biological categories, all bacterial isolates up-regulated regulatory cytokines (including IL-1RA and IL-10) 1.5-fold (L. crispatus 100.16 a) to 10-fold (L. reuteri Z1006) compared to the control. In contrast, only five Lactobacillus strains (L. gasseri 100. 46

Assessment of overall PPP performance
To identify the most promising vaginal Lactobacillus strains for the development of a probiotic for vaginal health, a weighted scoring system was devised based on PPP characteristics,

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Comparing probiotic potential of vaginal Lactobacillus isolates including: their ability to grow well in vitro, lower pH, adhere to cervical epithelial cells, produce L-and D-lactate and H 2 O 2 , and inhibit growth of BV-associated species (G. vaginalis and P. bivia). Cytokine and antibiotic susceptibility profiles were not considered, as data were available only for a subset of vaginal strains. For this ranking, a total of 36/57 Lactobacillus strains (for which complete characteristic evaluations were available) were included. To allow objective comparisons, the performance of vaginal isolates was compared with their respective ATCC reference and the probiotic isolates. Because some of the characteristics included in the ranking correlated, a weighted scoring system was developed to account for co-linearity between characteristics (Fig 8A). Including all characteristics in the scoring matrix (model A; Fig 8B), L. mucosae 90.13PA, L. crispatus 70.6PA and 100.16A, L. vaginalis 80.23B, and L. jensenii 88.33PA and 92.27PA were the six highest scoring Lactobacillus strains, with similar points as the ATCC L. crispatus and ATCC L. gasseri strains, while all commercially available probiotics ranked lower. Further step-wise sensitivity analyses were performed to test the robustness of the selection. Model B: Because the concentrations of H 2 O 2 produced by Lactobacillus were all below the level found to be microbicidal [30], a ranking model was considered in which

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Comparing probiotic potential of vaginal Lactobacillus isolates H 2 O 2 was excluded, which did not change the ranking of the top six strains (Fig 8C). Model C: In addition to excluding H 2 O 2 production, the inhibitory effect on G. vaginalis and P. bivia were grouped to avoid skewing towards strains that performed generally well in the inhibition assays (Fig 8C), which resulted in two L. gasseri strains being included in the top-10 strains.
The sensitivity analysis suggested that the scoring system was quite robust and showed that isolates from both STI/BV-negative and women with STIs or BV were among the top-performing strains, indicating that even Lactobacillus strains that originate from women with BV or STIs can have promising probiotic characteristics. Although all commercially available probiotic strains were included in the scoring system, none scored in the top ten in any of the models considered.

WGS analysis of selected Lactobacillus strains
Prior to selecting vaginal Lactobacillus strains for the development of a probiotic, we selected well-performing strains from a range of Lactobacillus spp. for whole genome sequencing and genome analysis to assess the presence of antimicrobial resistance determinants, mobile elements and prophages, including L. mucosae 90.13PA (ranked consistently in the top 2, from BV/STI-positive women), L. crispatus 70.6PA (highest ranking L. cripsatus strain from BV/ STI-negative women) and L. crispatus 73.55a (highest ranking L. cripsatus strain from BV-positive women), L. gasseri 94.98PB (highest ranking L. gasseri strain, from BV/STI-negative women), and L. jensenii 95.1PA (from BV/STI-negative women).
The whole genomes of five selected strains were sequenced to a median depth of 117X (range 102-142X). Full assembly statistics are shown in S1 Table. Screening for the presence of well characterised antimicrobial resistance determinants revealed no matches to the Resfinder, CARD and AMRFinder databases for any of the assemblies. However, analysis of the RAST annotated assemblies revealed that all five strains harboured open reading frames (ORFs) with homology to the ldb0660 and copY-like genes in Lactobacillus delbrueckii subsp. bulgaricus that respectively encode a copper-translocating P-type ATPase and a negative transcriptional regulator, both of which play a role in copper homeostasis. In addition, three of the five strains (L. crispatus 70.6 PA, L. crispatus 73.55 a and L. mucosae 90.13 PA) harboured genes encoding putative TetM-type ribosomal protection proteins. Two strains (L. gasseri 94.98 PB and L. jensenii 95.1 PA) possessed potential beta-lactamase class A proteins, while L. crispatus 73.55 a harboured an ORF encoding a putative streptothricin acetyltransferase. The latter was flanked by genes encoding predicted mobile element-associated transposases. Finally, two classes of potential multidrug transporters were identified. All strains except L. gasseri 94.98 PB harboured genes encoding putative multidrug and toxin extrusion (MATE) family efflux pumps with homology to the MepA protein in Staphylococcus aureus and a further three strains (L. Putative intact prophages were present in the genomes of two of the five strains. L. crispatus 70.6 PA harboured a single predicted 43.9Kbp prophage, while L. mucosae 90.13 PA harboured three predicted prophages of 16.9Kbp, 37.5Kbp and 40.1Kbp, respectively. Complete CRISPR-Cas loci (i.e. one or more signature cas genes located alongside a CRISPR array) were identified in the genomes of three of the strains. L. crispatus 73.55 a harboured a type I-E locus with spacers showing matches to a lactococcal phage Tuc2009. L. mucosae 90.13 PA harboured two distinct loci encoding a type I-C and type III-A system, respectively. Spacers present in the type I-C locus showed homology to phage-like regions on the L. mucosae LM1 plasmid and the Streptococcus thermophilus phage Sfi19, while those in the type III-A locus showed

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Comparing probiotic potential of vaginal Lactobacillus isolates homology to several plasmids (L. mucosae LM1, C. botulinum pCBH, and an unnamed plasmid in Lactobacillus fermentum DR9) as well as the Streptococcus phage Javan623. L. jensenii 95.1 PA harboured a single type II-A locus with spacers showing homology to several phages (an enterococcal phage, EFRM31 and the Lactobacillus phages Lf1 and Lj928).

Discussion
A limited variety of probiotics for vaginal health are currently available internationally and in South Africa, and few of these contain FGT commensals [18]. Thus, there is an urgent need for the development of additional well-designed probiotics for vaginal health. In this study, we evaluated key probiotic characteristics of a large panel of vaginal Lactobacillus strains, including L. crispatus, L. gasseri, L. jensenii, L. vaginalis and L. mucosae, from South African women. L. iners was not included, since its role in FGT health and disease remains unclear [46]. Overall, the majority of vaginal Lactobacillus strains performed better according to the PPP criteria considered than the included currently available commercial probiotic strains.
All Lactobacillus strains grew optimally at pH 6.0, similar to what has been described previously [28], despite existing at pHs <4.5 in the FGT [36,47] and actively lowering culture pH. All vaginal L. crispatus strains lowered the pH in vitro to 3.7, while only some strains of other vaginal Lactobacillus spp. achieved pHs <4.0. In vivo, women with an L. crispatus-dominated vaginal microbiota have been shown to have a lower FGT pH than women with a L. gasseri-or L. jensenii-dominated microbiota [4], suggesting good alignment between these in vitro characteristics and in vivo phenotypes.
The ability to produce lactate varied between Lactobacillus spp. and strains, although vaginal L. jensenii strains produced the lowest L-lactate (close to detection limit) concentrations, which supports previous studies showing that L. jensenii strains commonly only produce Dlactic acid [48,49]. Hutt et al. (2016) also found that L. gasseri produced significantly more lactate than L. jensenii and L. crispatus, which agrees with our findings. No correlation between lactate production and culture pH was found, but it has been suggested that other acids, such as acetic acid, contribute to lowering pH [50]. Under anaerobic conditions, vaginal Lactobacillus strains produced <20μM H 2 O 2 . Considering that the microbicidal activity of H 2 O 2 has only been demonstrated at concentrations �1000 mM [30], H 2 O 2 production by vaginal Lactobacillus spp. seems to be biologically irrelevant [51].
Vaginal and probiotic Lactobacillus strains inhibited G. vaginalis growth in a highly strainspecific manner. This indicates that G. vaginalis genotype may influence susceptibility to Lactobacillus-mediated inhibition. Previous studies have shown that metronidazole-resistant G. vaginalis strains were less susceptible to inhibition by Lactobacillus spp. [23,52], and biofilmproducing G. vaginalis strains were more tolerant to lactic acid than non-biofilm producing strains [53]. As such, G. vaginalis strain 3H1, which was inhibited broadly and strongly, belongs to genotype B, is not resistant to metronidazole and a low biofilm producer, which might explain the high susceptibility to Lactobacillus-mediated inhibition. In contrast, G. vaginalis 3B1 that showed low levels of inhibition belongs to genotype C and is highly resistant to metronidazole, which might explain why it was less susceptible to inhibition by Lactobacillus spp.
Antibiotic susceptibility has an impact on whether the probiotic could be administered concurrently with SOC [54]. All vaginal Lactobacillus strains were resistant to metronidazole, as expected, and indeed, concentrations between 128 and 256 μg/ml of metronidazole have been shown to stimulate the growth of vaginal Lactobacillus spp. in vitro [55]. Few Lactobacillus strains were resistant to clindamycin, as described by others [56][57][58], indicating that these strains could not be administered concurrently with SOC clindamycin treatment. Of note, all

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Comparing probiotic potential of vaginal Lactobacillus isolates Lactobacillus strains were highly sensitive to rifamycins. Considering that these are administered to~500,000 people annually in South Africa for six months to treat TB, effects of longterm administration of rifamycins on the FGT microbiota should be evaluated.
It is also important that probiotic candidates do not cause pathology to the host, such as an increase in genital inflammation [44], especially in a setting with extremely high STI and HIV rates like in South Africa. Promisingly, we found that all vaginal Lactobacillus strains up-regulated the regulatory cytokine IL-1RA, which inhibits binding of pro-inflammatory cytokines like IL-1α and IL-1β to their cognate receptors [59]. Some of the Lactobacillus strains even increased IL-8 production, which is important to consider, as IL-8 has been found to decrease HIV-1 transcription in both lymphocytes and ectocervical tissue explants [60]. Only four strains induced the inflammatory cytokines TNF-α, IL-12(p70), IL-6 and IL-1β, while the remaining Lactobacillus strains downregulated these, which is again reassuring as genital inflammation has been linked to HIV and STI risk [61]. Other cytokines that have been related to the risk of HIV acquisition in South African women include MIP-1α, MIP-1β and IP-10 [62], and it is encouraging that all besides three Lactobacillus strains downregulated these. Together, these findings indicate that administration of most vaginal Lactobacillus strains evaluated here is likely to be considered safe in a population with high STI and HIV prevalence but this needs confirmation in preclinical animal models and small interventional trial in healthy humans.
Using a weighted PPP scoring system, the identified top-performing strains included only vaginal Lactobacillus strains, obtained both from BV/STI negative South African women as well as some from donors who had BV and/or STIs, while none of the commercially available strains ranked as well, suggesting that there is potential to improve currently available probiotic formulations for vaginal health. Surprisingly, we did not find that strains from BV and STI-negative women consistently performed better than those from BV/STI-positive women, indicating that the probiotic potential of vaginal Lactobacillus strains is not necessarily dependent on the vaginal health of the donor.
The WGS of five of the best-performing vaginal Lactobacillus strains further confirmed the likelihood of their safety, due to antimicrobial resistance elements being largely absent. L. crispatus 73.55a harboured a streptothricin acetyltransferase sequence flanked by mobile elements, which is concerning as this could be one potential mechanism leading to the transfer of streptothricin resistance [63]. Prophage sequences were also identified, in agreement with other studies that observed functional and non-functional integrated bacteriophages in vaginal Lactobacillus spp. isolates [64][65][66]. While the relevance of potentially functional prophages for vaginal health is still unclear, it may be necessary to ensure that these prophage sequences are non-functional prior to inclusion of the strains into a commercial probiotic.
Several limitations of our study need to be acknowledged. No information on the strains that were included in the commercial probiotics were made available by all manufacturers. Not all characteristics were determined for all 57 strains, but we selected strains for further characterisation based on evidence of promising PPP characteristics. We also did not evaluate the mechanisms of pathogen inhibition.
In summary, this study evaluated probiotic characteristics of numerous vaginal Lactobacillus strains from South African women. The probiotic potential of these isolates was found to be strain-specific, and vaginal Lactobacillus strains largely performed better than probiotic strains currently used in probiotics for vaginal health, available internationally and in South Africa. Some of the best-performing, sequenced vaginal Lactobacillus isolates are now being considered for the development of a probiotic for vaginal health. This probiotic will be vaginally administered because vaginal administration affects vaginal health more quickly than does oral administration and enhances the viability of the administered microorganism [67]. It remains to be decided whether this probiotic should be region-specific, and only available to Southern African women due to the origin of its Lactobacillus strains, or worldwide. Once its effect on BV cure and recurrence has been tested in South Africa and found to be effective, the probiotic should also be tested in non-African women to determine whether these Lactobacillus strains show a similarly beneficial effect in other populations.

Cohort from which vaginal Lactobacillus strains were isolated
Vaginal Lactobacillus isolates were derived from women (16)(17)(18)(19)(20)(21)(22) year old) enrolled in the Women's Initiative in Sexual Health (WISH) study [68], approved by the Human Research Ethics Committees of the University of Cape Town (UCT HREC #267/2013). Women who were �18 years old provided written informed consent and those <18 years provided written assent, and consent was obtained from their parent(s) or legal guardian(s). Eligibility criteria included being HIV negative, generally healthy, not pregnant or menstruating at the time of sampling. Participants were tested for BV (Nugent scoring) and STIs (including C. trachomatis, N. gonorrhoeae, T. vaginalis, Mycoplasma genitalium, HSV-2, Hemophilus ducreyi, Treponema pallidum and Lymphogranuloma venerum). The vaginal microbiota was characterised by 16s rRNA gene sequencing [36]. Cervicovaginal secretions were collected using menstrual cups, diluted 1:4 in PBS and stored with glycerol (20%v/v) at -80˚C for isolation of Lactobacillus strains.

Isolation of Lactobacillus strains
Cervicovaginal secretions or commercial probiotics dissolved in De-Man-Rogosa-Sharpe (MRS) were streaked onto MRS agar plates and incubated anaerobically (37˚C, 48 hours). Single colonies with distinct morphologies were picked and re-streaked until homogenous. Pure colonies were inoculated into MRS broth, grown anaerobically (37˚C, 48 hours), and stocks stored with glycerol (20% v/v) at -80˚C. Matrix Assisted Laser Desorption/Ionization time-offlight (MALDI-TOF, MALDI Biotyper, Bruker Daltonik, USA) was used to identify Lactobacillus spp. To determine bacterial concentrations at a standardized optical density (OD), overnight cultures were adjusted to OD 600nm 0.1±0.01, serial dilutions plated onto MRS agar plates that were incubated anaerobically (37˚C, 48 hours), colonies were counted, and CFU/mL calculated (S2 Fig).

Ability of Lactobacillus isolates to grow at low pH conditions
Cultures standardized to OD 600nm 0.1±0.01 in MRS broth adjusted to pH 3.5, 4.0, 4.5 or 6.0 using HCl were incubated in triplicate under anaerobic conditions in 96-well plates at 37˚C for 48 hours. OD 600nm was measured at 0, 3, 6, 12, 18, 24, 36 and 48 hours and area under the curve (AUC) was calculated for each isolate. Experiments were repeated three times.

Ability of Lactobacillus isolates to lower culture pH
Cultures adjusted to OD 600nm 0.1±0.01 in MRS broth were grown anaerobically at 37˚C, and culture supernatant pH measured at 3, 6, 12, 24 and 48 hours using a calibrated pH meter. Experiments were performed in duplicate.

Measurement of anaerobic lactate and H 2 O 2 production
Cultures adjusted to OD 600nm 0.1±0.01 were incubated anaerobically for 24 hours at 37˚C. Supernatants were filtered (0.2 μM) and concentrations of D-and L-lactate measured using colorimetric assays (Sigma-Aldrich, USA). H 2 O 2 concentrations were measured using a fluorometric kit (Sigma-Aldrich, USA), according to the manufacturer's instructions. Experiments were performed in duplicate.

Antimicrobial activity
G. vaginalis (ATCC 14018 and five vaginal isolates) and P. bivia (ATCC 29303 and five vaginal isolates) were grown in brain heart infusion (BHI) supplemented with 5% horse blood, standardised to OD 600nm 0.4±0.01, and 100 μL was added in triplicate to 96-well plates. Abiotic Lactobacillus-conditioned MRS (100 μL; same as used for lactate measurement) was added and incubated anaerobically at 37˚C for 48 hours. OD 600nm was measured at 0, 3, 6, 12, 18, 24, 36 and 48 hours. As control, growth of G. vaginalis and P. bivia strains in the presence of plain MRS, at the same ratio as abiotic culture supernatants, was measured. AUC for each growth curve was calculated and percentage of growth inhibition in the presence of Lactobacillus-conditioned MRS compared to the control calculated. Experiments were repeated three times.

Adhesion to human ectocervical epithelial cells
Ca Ski cells (ATCC CRL-1550) were grown to 80% confluence in 10% fetal calf serum (FCS) Dulbecco's Modified Eagle Medium (DMEM) in 24-well cell culture plates (37˚C, 5%CO 2 ). Lactobacillus isolates (4.2x10 6 CFUs) were added in triplicate and co-cultured for 3 hours at 37˚C (5%CO 2 ), after which unbound bacteria were gently removed by washing the cells three times with PBS. Cells and bound bacteria were detached using 0.1% Triton X-100, serially diluted, plated onto MRS agar plates, and incubated anaerobically at 37˚C for 48 hours. The percentage of adhered bacteria was calculated as the number of bound cells compared to total number of bacterial cells initially added. Experiments were repeated three times.

Impact on cervical cell viability and cytokine responses
Ca Ski cells were grown in 10% FCS DMEM to 80% confluence at 37˚C in 5% CO 2 . Lactobacillus strains (4.2x10 6 CFUs) were added in duplicate and incubated for 24 hours (37˚C, 5% CO 2 ). Ca Ski cells incubated without Lactobacillus strains were included as control. Supernatants were harvested and filtered (0.22 μm, Corning Costar Spin-X, Sigma-Aldrich, USA) to exclude bacterial cells. A Bio-Plex Pro Human Cytokine 27-Plex Luminex kit (Lot 64064139, Bio-Rad, USA) was used to measure concentrations of cytokines, chemokines and growth factors in the culture supernatant, according to the manufacturer's instructions. Data were acquired using a Bio-Plex Suspension Array Reader (Bio-Rad Laboratories Inc, USA), and a 5PL regression line was used to determine concentrations from standard curves. All values below the detection limit were recorded as half of the lowest measured concentration for each cytokine. Viability of Ca Ski cells after co-culture was evaluated using trypan blue staining (0.4%, Sigma-Aldrich, USA).

Whole genome sequencing
Genomic DNA was extracted from Lactobacillus isolates using the Quick-DNA Miniprep Kit (Zymo Research, USA) following the manufacturer's instructions after a pre-lysis step which consisted of resuspension of the cell pellet in 200 μl PrimeStore Molecular Transport Medium (Longhorn Vaccines-Diagnostics, USA) and storage at -80˚C for 24 hours. Libraries for sequencing were prepared using the Nextera DNA Flex kit (Illumina, USA), according to the manufacturer's instructions, and sequenced using the Illumina MiSeq Reagent Micro Kit v2 on an Illumina MiSeq instrument in a paired-end, dual indexed 2 x 151 cycle sequencing run. Draft de novo assemblies were prepared using a customised assembly pipeline. Briefly, raw reads were trimmed using Trimmomatic v0.3.9 [70] using a sliding window approach (Phred quality cut-off of 15 averaged across 4 bases), a leading/trailing quality cut-off of 3 and the removal of reads below 30bp. Trimmed reads were assembled using SPAdes v3.13.0 [71] using the '-careful' flag and omitting the initial read error correction step. To improve the draft assemblies, gap closure and contig extension were performed using GMcloser v1.6.2 [72]. The quality of the final assemblies for each strain was assessed using Quast v5.0.2 [73]. Rapid Annotation of microbial genomes using Subsystems Technology (RAST) was used to annotate the de novo assemblies [74].

PLOS PATHOGENS
Comparing probiotic potential of vaginal Lactobacillus isolates