Bacterial vaginosis (BV) is a very common gynaecologic condition affecting women of reproductive age worldwide. BV is characterized by a depletion of lactic acid-producing Lactobacillus species and an increase in strict and facultative anaerobic bacteria that develop a polymicrobial biofilm on the vaginal epithelium. Despite multiple decades over research, the etiology of this infection is still not clear. However, some BV-associated bacteria (BVAB) may play a key role in the development of this infection, namely Gardnerella species, Prevotella bivia, and Fannyhessea vaginae. In this work we aimed to characterize the growth of these three species in a rich medium and in a medium simulating vaginal tract secretions (mGTS). We first assessed planktonic growth in New York City (NYCIII) medium and mGTS and observed that the three species showed distinct capacities to grow in the two media. Surprisingly, despite the ability of all three species to grow in single-species in NYCIII, in a triple-species consortium, P. bivia was not able to increase its concentration after 48 h, as assessed by qPCR. Furthermore, when using the more restrictive mGTS media, G. vaginalis was the only BVAB able to grow in the triple-species consortia. Interestingly, we found that P. bivia growth in NYCIII was influenced by the cell-free supernatant (CFS) of F. vaginae and by the CFS of G. vaginalis in mGTS. This antimicrobial activity appears to happen due to the acidification of the media. Single- and triple-species biofilms were then formed, and the growth of each species was further quantified by qPCR. While G. vaginalis had a greater capacity to form biofilms in both media, F. vaginae and P. bivia biofilm growth was favored when cultured in rich media. Differences were also found in the structure of triple-species biofilms formed in both media, as assessed by confocal laser scanning microscopy. In conclusion, while all three species were able to grow in single-species biofilms in rich media, in mGTS the growth of G. vaginalis was essential for incorporation of the other species in the biofilm.

A vaginose bacteriana (VB) é uma infeção vaginal comum em mulheres em idade reprodutiva, caracterizada por um desequilíbrio na microbiota vaginal com um decréscimo do número de espécies de Lactobacillus e um crescimento excessivo de bactérias anaeróbias estritas e facultativas. Apesar de vários estudos demonstrarem uma elevada diversidade de espécies detetadas em casos de VB, a sua etiologia ainda não é clara. No entanto, uma característica bem conhecida da VB é a formação de um biofilme polimicrobiano no epitélio vaginal. Este estudo focou-se em três das espécies mais comumente associadas à VB, nomeadamente Gardnerella vaginalis, Fannyhessea vaginae, e Prevotella bivia, de forma a explorar a sua contribuição para o desenvolvimento do biofilme e interações microbianas que possam estar envolvidas na patogénese da VB. Primeiro, foram desenvolvidas duas sondas de ácido nucleico peptídico para a deteção específica de F. vaginae e P. bivia, e foi otimizado um protocolo multiplex que combina estas novas sondas com uma sonda de Gardnerella spp. anteriormente desenvolvida. As sondas permitiram a análise in vitro de biofilmes triplos e a deteção das espécies em amostras vaginais recolhidas de mulheres com VB. O crescimento destas três espécies foi avaliado num meio rico e num meio que simula as secreções do trato genital (mGTS). G. vaginalis mostrou a melhor capacidade de crescimento em ambos os meios, enquanto o crescimento de F. vaginae foi muito limitado em mGTS. Curiosamente, o crescimento de P. bivia foi afetado quando as três espécies cresceram juntas, tendo-se verificado que o sobrenadante de F. vaginae mostrou um efeito inibidor no crescimento de P. bivia em meio rico. O efeito do crescimento conjunto das três espécies foi depois avaliado por uma análise transcriptómica que comparou biofilmes simples e triplos em meio rico. Os resultados foram mais evidentes para G. vaginalis que mostrou vários processos metabólicos entre os genes subexpressos. Alterações transcriptómicas foram ainda estudadas quando os biofilmes triplos foram crescidos em mGTS, e um elevado número de genes diferencialmente expressos foi identificado para as três espécies. Alguns dos genes identificados na primeira análise não apresentaram alterações transcriptómicas significativas, o que sugere que estes genes podem ser relevantes para interações entre as três espécies. A expressão de quatro genes de G. vaginalis foi quantificada em amostras vaginais recolhidas de mulheres saudáveis e mulheres que desenvolveram VB, e os resultados mostraram o potencial destes genes para discriminar casos positivos e negativos de VB.

A vaginose bacteriana (VB) é a infeção vaginal mais comum em todo o mundo afetando principalmente mulheres em idade reprodutiva. Esta infeção é caracterizada pela redução do número de espécies de Lactobacillus, normalmente associadas a um estado saudável, e pelo aumento de bactérias anaeróbias que colonizam e formam um biofilme polimicrobiano no epitélio vaginal. A etiologia da VB e as interações microbianas entre espécies que levam ao desenvolvimento desta infeção não são ainda claras, no entanto, espécies de Gardnerella, Fannyhessea vaginae e Prevotella bivia estão associadas ao desenvolvimento da infeção e parecem ter um papel importante na colonização inicial do epitélio vaginal. O objetivo deste trabalho passou pela caracterização de biofilmes formados por estas três espécies bacterianas, quando crescidas em meio de cultura rico ou em meio simulando secreções vaginais (mSSV). Os resultados iniciais de crescimento de culturas plantónicas, demonstraram capacidades muito distintas no crescimento das três espécies, nas duas condições testadas. Em meios de cultura mais semelhantes ao ambiente vaginal (mSSV), P. bivia consegue prosperar enquanto que F. vaginae tem uma redução significativa da sua viabilidade. Curiosamente, G. vaginalis aparenta ter uma taxa de crescimento semelhante à sua taxa de morte, mantendo constante a biomassa composta por esta bactéria. Por outro lado, em meio nutritivo rico, todas as espécies apresentam crescimento significativo, embora em diferentes proporções. Quando analisado o crescimento em fenótipo de biofilmes, verificou-se que em mSSV, apenas G. vaginalis consegue crescer como biofilme de cultura pura. Já no meio nutritivo rico, todas as espécies testadas conseguem formar um biofilme. Curiosamente, quando incubadas as 3 espécies simultaneamente, o biofilme triplo formado em mSSV apresenta colónias de cada uma das 3 espécies, determinado por microscopia de fluorescência in situ. No entanto, a análise por microscopia confocal revela diferenças significativas nas estruturas dos biofilmes triplos, formados em cada um dos meios testados. Estes resultados confirmam que G. vaginalis tem uma grande adaptabilidade bioquímica, sendo essencial para a formação de biofilmes associados à VB quer em condições típicas de crescimento laboratorial (meios ricos), quer em condições que mimetizam a bioquímica vaginal. Futuramente, será realizada uma análise da expressão genética destes biofilmes por RNA-seq para perceber interações chave que possam levar ao desenvolvimento da infeção.

Bacterial vaginosis (BV), the most common vaginal infection worldwide, is characterized by the development of a polymicrobial biofilm on the vaginal epithelium. While Gardnerella spp. have been shown to have a prominent role in BV, little is known regarding how other species can influence BV development. Thus, we aimed to study the transcriptome of Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia, when growing in triple-species biofilms. Single and triple-species biofilms were formed in vitro, and RNA was extracted and sent for sequencing. cDNA libraries were prepared and sequenced. Quantitative PCR analysis (qPCR) was performed on the triple-species biofilms to evaluate the biofilm composition. The qPCR results revealed that the triple-species biofilms were mainly composed by G. vaginalis and P. bivia was the species with the lowest percentage. The RNA-sequencing analysis revealed a total of 432, 126, and 39 differentially expressed genes for G. vaginalis, F. vaginae, and P. bivia, respectively, when growing together. Gene ontology enrichment of G. vaginalis downregulated genes revealed several functions associated with metabolism, indicating a low metabolic activity of G. vaginalis when growing in polymicrobial biofilms. This work highlighted that the presence of 3 different BV-associated bacteria in the biofilm influenced each others transcriptome and provided insight into the molecular mechanisms that enhanced the virulence potential of polymicrobial consortia. These findings will contribute to understand the development of incident BV and the interactions occurring within the biofilm.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge and is often associated with other health consequences mainly in pregnant women. BV is described by an imbalance in the vaginal microbiota where strictly and facultative anaerobic bacteria outgrow the lactic acid- and hydrogen peroxide-producing Lactobacillus species. The species involved in BV are capable to grow and form a polymicrobial biofilm in the vaginal epithelium. The treatment of BV is usually performed using broad-spectrum antibiotics, including metronidazole and clindamycin. However, these conventional treatments are associated with high recurrence rates. The BV polymicrobial biofilm may have an important role on the treatment outcome and is accounted as one of the factors for treatment failure. Other possible reasons for treatment failure include the presence of species resistant to antibiotics or the chance of reinfection after treatment. Therefore, novel strategies to increase the rates of treatment have been studied namely the use of probiotics and prebiotics, acidifying agents, antiseptics, plant-based products, vaginal microbiota transplantation, and phage endolysins. Although some of them are still in an initial phase of development with very preliminary results, they show great perspectives for application. In this review, we aimed to study the role of the polymicrobial nature of BV in treatment failure and explore a few alternatives for treatment.

To study the influence of simulated in vivo conditions on the interactions of key BVAB, we analyzed the transcriptome of a triple-species biofilm composed of G. vaginalis, F. vaginae, and P. bivia, when biofilms were grown in a rich medium versus a medium mimicking genital tract secretions. Triple-species biofilms formed in mGTS were also analyzed by qPCR to determine each species percentage, revealing that the great majority of the biofilm was composed of G. vaginalis, followed by P. bivia, and F. vaginae, as shown in Supplementary Figure S1. Regarding RNA-seq data, the quality of the sequencing data was assessed by evaluating some of the summary parameters from the trimming and mapping steps as described at Supplementary Tables S3, S4, respectively. The number of sequencing reads ranged between 13137570 and 20716698. The results of mapping to the reference genome sequence revealed a percentage of mapping between 1.82% and 75.76% for the NYCIII medium and between 0.63% and 67.48% for the mGTS medium. Overall, the sequences from P. bivia were the ones with the lowest percentages of mapping. The PCA plots, depicted in Supplementary Figures S2S6, revealed differences among the triplicates analyzed for each condition. Triplicates of biofilms formed in mGTS were more similar than those formed in NYCIII medium for G. vaginalis and P. bivia. In contrast, more differences were found among the biofilm triplicates in the mGTS condition for F. vaginae. These variations are likely a result from biological heterogeneity found in biofilms (Sousa et al., 2014). However, a clear separation from the triplicates in NYCIII and mGTS was observed for all the species, indicating that the two conditions caused alterations in the transcriptome of the three species. The distribution of gene expression was evaluated in both conditions for each species, as shown in Supplementary Figure S7. The box plots, which display the distribution of the RPKM values, revealed a similar distribution of the data in NYCIII and mGTS for the three species. The scatter plots, which represent the correlation between RPKM values in the NYCIII and mGTS conditions, showed that more genes were upregulated in the mGTS condition for G. vaginalis and F. vaginae (more genes displayed above the line), whereas for P. bivia more genes were downregulated in the same condition (more genes displayed below the line). Finally, heat maps depicting the normalized gene expression pattern in the three species across conditions and triplicates showed a clear alteration in gene expression from NYCIII to mGTS condition (Supplementary Figures S8S12). For differential gene expression analysis, biofilms formed in the NYCIII medium were used as the control. A total of 1,315, 1,202, and 2,184 differentially expressed genes were found for G. vaginalis, F. vaginae, and P. bivia, respectively. MA and volcano plots (Figure 1) showed that the expression of the majority of the genes has a fold change between 32 and 32 (5 < Log2 > 5). Moreover, for F. vaginae and P. bivia, a wide range of FDR p-values was found in comparison with G. vaginalis.

A vaginose bacteriana (VB) é a infeção vaginal mais comum em mulheres em todo o mundo. A VB é caracterizada por um desequilíbrio na microbiota vaginal marcado por uma diminuição do número de espécies de Lactobacillus e um aumento do número de bactérias anaeróbias, que crescem num biofilme polimicrobiano. A formação de um biofilme polimicrobiano em casos de VB é uma das características mais associadas à VB, no entanto, o processo de formação desta comunidade microbiana e as consequências para a infeção são ainda largamente desconhecidos. Neste estudo, pretendemos analisar como 3 das espécies mais comummente associadas à VB, nomeadamente Gardnerella vaginalis, Fannyhessea vaginae e Prevotella bivia, adaptaram a sua fisiologia, quando crescidas num meio nutricionalmente rico [New York City (NYCIII)] ou num meio que simula as secreções do trato vaginal (mGTS). Para cumprir com este objetivo, foram formados os biofilmes contendo as 3 espécies nas duas condições distintas, tendo de seguida sido obtido o RNA destas culturas, e posteriormente sequenciado e analisado. A análise à composição dos biofilmes revelou que, em ambas as condições, os biofilmes eram maioritariamente compostos por G. vaginalis. Os resultados da sequenciação revelaram que, nos biofilmes formados em mGTS, foram identificados 927 genes sobre-expressos e 37 genes sub-expressos em G. vaginalis, 492 genes sobre-expressos e 104 genes sub-expressos em F. vaginae, e 166 genes sobre-expressos e 490 genes sub-expressos em P. bivia. Uma análise ontológica dos genes diferencialmente expressos mostrou que processos metabólicos e de biossíntese eram comuns entre os genes sobre-expressos em G. vaginalis, enquanto os genes sub-expressos estavam associados a transportadores de membrana. Para F. vaginae, transportadores transmembranares foram identificados entre os genes sobre-expressos, enquanto os genes sub-expressos estavam envolvidos em processos de metabolismo e celulares. Por fim, no caso de P. bivia, os genes sobreexpressos em mGTS estavam associados a processos transmembranares, enquanto os genes sub-expressos foram associados a processos metabólicos. Várias vias KEGG associadas a processos metabólicos foram também identificadas entre os genes subexpressos em F. vaginae e P. bivia. Este trabalho vem assim evidenciar a adaptação destas 3 espécies num meio que simula condições in vivo e que por isso pode contribuir para melhor compreensão das interações que ocorrem durante o desenvolvimento da VB.

Bacterial vaginosis (BV) is the most common vaginal infection affecting women worldwide. This infection is characterized by the loss of the dominant Lactobacillus community in the vaginal microbiota and an increase of anaerobic bacteria, that leads to the formation of a polymicrobial biofilm, mostly composed of Gardnerella spp. Treatment of BV is normally performed using broadspectrum antibiotics, such as metronidazole and clindamycin. However, the high levels of recurrence of infection after treatment cessation have led to a demand for new therapeutic alternatives. Thymbra capitata essential oils (EOs) are known to have a wide spectrum of biological properties, including antibacterial activity. Thus, herein, we characterized two EOs of T. capitata and tested their antimicrobial activity as well as some of their main components, aiming to assess possible synergistic effects. Our findings showed that carvacrol and -cymene established a strong synergistic antimicrobial effect against planktonic cultures of Gardnerella spp. On biofilm, carvacrol and linalool at sub-MIC concentrations proved more efficient in eliminating biofilm cells, while showing no cytotoxicity observed in a reconstituted human vaginal epithelium. The antibiofilm potential of the EOs and compounds was highlighted by the fact cells were not able to recover culturability after exposure to fresh medium.

Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and has been associated with serious health complications, mainly in pregnant women. It is characterized by a decrease in the number of Lactobacillus species in the healthy vaginal microbiota and an overgrowth of strict and facultative anaerobic bacteria that develop a polymicrobial biofilm. Despite over 60 years of research investigating BV, its etiology is not fully understood. Gardnerella spp. is a crucial microorganism that contributes to the formation of the biofilm and the development of BV, but the role of other BV-associated bacteria is not clear. Nevertheless, Fannyhessea vaginae (previously known as Atopobium vaginae) is a highly specific species for BV, and co-colonization with Gardnerella is thought to be a very specific diagnostic marker. The diagnosis of BV still presents some limitations, since currently used methods often fail to accurately detect BV. This work aims to develop a novel peptide nucleic acid (PNA) probe targeting F. vaginae. This probe was further validated in a multiplex assay, which included a Gardnerella-specific PNA probe, as a possible method for diagnosis of BV, and was compared with quantification by qPCR. The new PNA probe showed excellent sensitivity and specificity and could discriminate F. vaginae-Gardnerella biofilms, confirming the potential to be used for the detection of BV-associated pathogens.

Assessment of genomic DNA (gDNA) extraction efficiency is required for accurate bacterial quantification by qPCR. Exogenous DNA molecules are often added after bacterial cultures are lysed, but before DNA purification steps, to determine extraction efficiency. Herein we found that different exogenous DNA controls have different recovery rates, suggesting distinct DNA extraction efficiencies. Recovery rates are also affected by the gDNA extraction method being more affected in silica-based columns than in phenol-chloroform extraction. Overall, we determined that the use of long DNA fragments, such as gDNA, as exogenous controls have a higher recovery rate than use of smaller size DNA molecules.