Browsing by Author "Kullin, Brian"

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    Characterisation of vaginal Prevotella strains from a cohort of South African adolescent girls and young women with and without bacterial women with and without bacterial vaginosis
    (2024) Welp, Kirsten; Kullin, Brian; Paul , Lynthia; Passmore, Jo-Ann
    A common condition in reproductive age women, bacterial vaginosis (BV), is characterised by a dysbiosis in the vaginal microbiome, and frequently associated with abundance of the genus Prevotella. Prevalence of BV is particularly high in sub-Saharan Africa and comprehensive characterisation of South African BV-associated bacteria is vital for BV management in the region. However, to date, most research has focussed on BV-associated Prevotella species from the global North. A total of 69 Prevotella isolates were purified from vaginal samples from the UChoose cohort study, which was designed to assess the effect of hormonal contraceptives on HIV risk in adolescent girls and young women (16-19 years old). Isolate genus was confirmed by Prevotella genus-specific PCR for all isolates. A subset was speciated by 16S rRNA gene sequencing to be P. bivia (n=36) and a putative P. melaninogenica (n=1) and were carried forward for further phenotypic characterisation, including antimicrobial resistance testing by Etest methodology, and quantification of biofilm formation by crystal violet staining. South African Prevotella isolates displayed 2.7% and 8.3% resistance to metronidazole and clindamycin, respectively, with higher rates of resistance against non-BV antibiotics (19.44%-38.89%). Several strains exhibited co- resistance to two (12/36), three (6/36) or four (2/36) antibiotics. Biofilm formation was significantly greater than the P. bivia ATCC 29303T type-strain in over a quarter of strains (p>0.05), with some intra-sample diversity in biofilm-forming ability. Additionally, 36 strains (35 P. bivia and 1 P. melaninogenica) were whole genome sequenced using Illumina MiSeq technology. Genomes of these isolates, and of published sequences on the NCBI RefSeq database, were annotated and screened for antimicrobial resistance and virulence genes, as well as for the presence of prophages or anti-phage defence mechanisms. Phylogenetic analysis by cpn60 gene sequencing and core genome alignment revealed that intra-sample diversity was generally low, but at least one sample had two distinct strains at the same time point. The putative P. melaninogenica isolate was most closely related to another vaginal Prevotella spp., but both were potential novel species based on average nucleotide identity score (<95%). Isolation location (South African versus international) was not reflected in the phylogenetic trees and there were no differential genes associated with isolate origin. Antimicrobial resistance genes relevant to metronidazole (nimK) and clindamycin resistance (ermF) were identified in 2.27% (1/44) and 29.09% (4/44) strains, respectively. The variants of the β-lactamase gene, cfxA, were differentially associated with resistance to a β-lactam antibiotic (p=0.000212). Lipopolysaccharide (LPS) production was assessed in a subset of Prevotella strains using a commercially available isolation and quantification kit. LPS production was 24-fold higher in the P. melaninogenica-related strain, compared to five P. bivia isolates tested (p<0.05), and was structurally different to P. bivia LPS as observed by SDS-PAGE. Overall, isolates, added to a Prevotella biorepository, were phenotypically and genotypically characterised, which will enable regionally relevant BV research into future intervention and treatment plans.
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    Characterising vaginal Lactobacillus strains from young South African women with persistently optimal vaginal microbiota – Developing the framework for an African vaginal probiotic product development platform for reproductive health
    (2024) Chicken, Anika; Kullin, Brian; Passmore, Jo-Ann; Happel, Anna-Ursula
    Background: Bacterial vaginosis (BV) is a genital condition with a high prevalence in sub-Saharan Africa, which can cause adverse sexual and reproductive health outcomes. Treatment with antibiotics such as metronidazole results in only a brief resolution of the vaginal dysbiosis present in BV and the recurrence rates are high. Probiotics have been explored as a possible adjunctive treatment option, but most commercially available formulations do not contain vaginally-derived strains and there is limited evidence for their efficacy in local populations. Therefore, there is an urgent need for the development of vaginal probiotics tailored towards sub-Saharan African women containing strains with useful probiotic characteristics that have the potential to persistently colonise the female genital tract. Aim: This project aimed to isolate and characterise Lactobacillus strains from young South African women with a stable optimal vaginal microbiota for consideration as candidate strains in a vaginal probiotic product. Methods: Samples from a previously completed study cohort where hormonal contraceptive preferences were examined in adolescent and young women in Cape Town (UChoose) were used for this study. Samples were collected from healthy, HIV uninfected young women (aged 15-19) at three timepoints, 16 weeks apart. To identify women with a stable and optimal vaginal microbiota over time, vaginal microbial communities were classified using VALENCIA. Lactobacillus spp. were selectively isolated from participants with longitudinally stable Lactobacillus crispatus- dominant communities and assayed for their ability to inhibit Prevotella bivia, a BV-associated organism using a soft agar overlay method. Candidates with strong inhibition potential against P. bivia were characterised further fortheir inhibition potential against regionally relevant BV-associated organismsisolated from women within the same study cohort, as well astheir growth at physiologically relevant pH (pH 4, 4.5 and 5), their ability to produce lactic acid and finally their resistance to commonly used BV and urinary tract infection antibiotics. Additionally, whole genome sequencing (WGS) data were available for three strains and these were assessed to determine additional safety characteristics of the probiotic candidates. The strains were screened for antimicrobial resistance genes using several online databases (ResFinder, ARG-ANNOT, CARD, NCBI AMRFinderPlus and VFDB), the presence of integrated prophages using VirSorter and phage defence mechanisms using the DefenseFinder tool. Lastly, putative bacteriocins were identified using BAGEL4. Results: The most common community state type (CST) identified across all participants and visits was the highly diverse CST IV, which is dominated by a variety of anaerobes and shows a strong association with BV. In total 3 participants had an optimal, L. crispatus-dominant vaginal microbiota (CST I) across all three study visits and did not use any antibiotics during the study. Samples from these participants yielded a combined 337 isolates. Isolates displayed a wide variety in inhibition potential against P. bivia ATCC 29303T . The isolates selected for further characterisation were all identified as L. crispatus using 16S rRNA sequencing. More than half of the selected isolates inhibited local Prevotella strains more than they inhibited the P. bivia ATCC 29303T type strain. All L. crispatusisolates produced some of each lactic acid isomer, with the top three L-lactate producers all isolated from the same participant (UC101). No correlation was found between the amount of lactate produced and antimicrobial activity against Prevotella strains suggesting that the inhibition was not lactate dependent. All isolates were resistant to metronidazole, but susceptible to other BV and urinary tract infection treatment antibiotics indicating that co administration with metronidazole is a possibility for these candidate probiotic strains. Of the three genomes analysed, two of the isolates harboured plasmids. No previously characterised antimicrobial resistance determinants were identified. Each of the three strains harboured five putative bacteriocins, which may play a role in activity against competing bacterial strains. Additionally, 12 putative prophages were identified, with none found on the plasmid. Overall, seven different potential phage defence mechanisms were identified on the bacterial chromosomes. Conclusion: The proportion of women with longitudinally stable, optimal vaginal communities in our setting was relatively low. However, targeting samples provided by these women for the isolation of potential probiotic bacteria yielded a large number of isolates with inhibitory activity against a BV-associated pathogen. Phenotypic variation among isolates illustrates the importance of screening multiple strains of the same species per sample. The WGS screen did not identify any virulence or antimicrobial resistance genes suggesting that the probiotic candidates are safe to use. The putative bacteriocins found in the isolates could be targeted for future research. This pilot study provides proof-of-principle for a pipeline that will screen L. crispatus strains with probiotic potential from African women to treat BV and improve reproductive health in Africa.
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    Fermented foods production using isolated Lactobacillaceae species for the improvement of vaginal health: the case of mageu and yoghurt
    (2022) Hartzenberg-Aeroe, Ellen; Fagan-Endres, Marijke; Kullin, Brian
    Bacterial vaginosis (BV) is the most prevalent vaginal dysbiosis affecting women's reproductive health. The condition is characterised by the disruption of a Lactobacillus spp.-dominated microbiota that is associated with positive health outcomes and a shift to a microbial community that consists of diverse obligately anaerobic bacteria. Currently, antibiotics are used to treat BV and other vaginal infections. However, post-therapy recurrence and reinfection rates are high, suggesting the need for adjunctive therapy. Probiotic supplements can be used in conjunction with antibiotic treatment to re-establish the optimal vaginal microbial community. Of the probiotic products available in South Africa, only a limited number are specifically for vaginal health and none contain probiotic microorganisms found in the female genital tract. Additionally, the healthy vaginal microbiota differ with geography. Therefore, there is a need for an affordable probiotic product targeted for South African women. In this dissertation, mageu and yoghurt are investigated for their potential as probiotic delivery vehicles. Previously isolated Lactobacillaceae strains from the genital tracts of healthy South African women are tested for their ability to ferment maize meal to mageu and milk to yoghurt, both as pure cultures and supplemented with traditionally-used microorganisms: Saccharomyces cerevisiae for mageu and Streptococcus thermophilus for yoghurt. Protocols were developed for the production of mageu and yoghurt with the bacterial strains. During production, fermentation was monitored by measuring pH until the end points of 3.5 and 4.5 were reached for mageu and yoghurt, respectively. After fermentation, the products were analysed by measuring titratable acidity, lactic acid and ethanol concentrations, total solids content, viable cell counts, qualitative analysis and shelf-life. Four mageu samples were then analysed for their consumer acceptability by an untrained consumer panel. The Lactobacillaceae isolates, both as pure strains and supplemented with S. cerevisiae, were able to ferment maize meal to mageu, producing a product with a final pH of around 3.5, which qualitatively resembled mageu. The presence of pH-lowering metabolites including lactic acid and other acids was also measured using titratable acidity. The addition of S. cerevisiae significantly reduced the fermentation time and increased the titratable acidity of all mixed culture samples compared to the pure culture mageu samples. All mageu samples produced using the Lactobacillaceae isolates met the ethanol (maximum 2.5 g/L) and total solids content (minimum 8% (m/m)) production requirements set by the South African National Standards. After one week at room temperature or refrigerated, none of the mageu samples had pH values below 3, which is acceptable for mageu. However, the mixed culture mageu samples saw a larger drop in pH over the week than the pure culture samples. The sensory analysis investigated the consumer acceptability of four probiotic mageu samples, a positive control produced with flour as the inoculum source, and a commercial sample. The commercial sample was generally preferred, and was scored highest by the consumer panel, while the other samples received mixed results. The spread of results was attributed to the influence of individual preference due to the small cohort size. The pure Lactobacillaceae strains were unable to ferment milk to yoghurt. By hour 12, the pH values were still between 6.32 and 6.37, and curdling of the product had occurred by hour 24. However, when supplemented with S. thermophilus, the mixed cultures were able to produce a product with a final pH of around 4.5 that qualitatively resembled traditional yoghurt. However, it is not known if the improved fermentation was due to S. thermophilus activity alone. The titratable acidity results also confirmed that pH-lowering metabolites were produced and all yoghurt samples had titratable acidity values greater than 0.6% (m/m), the minimum requirement for yoghurt. Little difference was observed between the samples for the qualitative assessment; however, the positive control had a thicker consistency than the probiotic samples. These results show that the Lactobacillaceae isolates when supplemented with traditional fermentation microorganisms are able to produce mageu and yoghurt. During this project protocols for mageu and yoghurt production using the Lactobacillaceae isolates specific to South African women were designed. This project serves as the first step towards investigating the use of fermented foods as affordable probiotic delivery vehicles for the improvement of vaginal health in South African women.
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    The effect of bacterial vaginosis on HIV infection
    (2024) Abrahams, Bianca; Kullin, Brian
    Human Immunodeficiency Virus-1 (HIV) and Bacterial Vaginosis (BV) are both among the most common diseases affecting young women in Sub-Saharan Africa. BV is characterised by dysbiosis in the female reproductive tract (FRT) when optimal Lactobacillus spp. such as L. crispatus, are displaced by anaerobes such as Gardnerella spp., consistently isolated from the FRT of BV-positive women. Gardnerella spp. produce a number of important virulence factors such as vaginolysin (VLY) and sialidase and are known to initiate the formation of biofilms and hence, contribute to the pathology associated with BV. As many studies have suggested that BV increases susceptibility to HIV infection, it stands to reason that Gardnerella spp. might either indirectly, (initiate the onset of BV and subsequent immune responses) or directly (via its virulence factors) play a role in enhancing HIV acquisition. This study investigated whether VLY, sialidase and biofilm formation played a role in HIV infection. Recently, it was discovered that Gardnerella spp. comprises G. leopoldii, G. vaginalis, G. piotii and G. swidsinskii as well as nine other genome species which vary in virulence potential. When we compared twenty strains isolated from BV-positive women belonging to G. vaginalis (n = 16), G. piotii (n = 2) and G. swidsinskii (n = 2), we found differences in the presence, expression, and activity of VLY and sialidase as well as biofilm-forming capacity between the strains, suggesting a wide range in virulence. However, there was no overt association between HIV infection and Gardnerella virulence factors. Gardnerella sialidase consists of three isoforms: NanH1, NanH2 and NanH3 and as the latter is responsible for the sialidase activity in the FRT, nanH3 was cloned, expressed in E.coli and purified by His-Tag affinity chromatography. We found that purified, recombinant NanH3 increased HIV infection in vitro, most likely by removing the sialic acid moieties on the surface of host cells, reducing the negative repulsive force between the viral Envelope and cell membrane. This may then facilitate virus accumulation at the cell surface, favouring attachment to CD4 and/or its co-receptors and thereby enhance HIV infection. Interestingly, only the two G. piotii strains expressed NanH3, suggesting that perhaps only some species of Gardnerella may play a role in enhancing HIV infection. This has important implications for diagnosis and treatment as BV-positive women shown to carry G. piotii strains, might benefit from preexposure prophylaxis with antiretroviral drugs.