Joana Isabel Reis Castro
@uminho.pt
Centre of Biological Engineering
University of Minho
Joana Castro graduated in Biomedical Engineering by Minho University (December 2012). She performed her master thesis in a Bacterial Vaginosis project. After concluding her master thesis, she became a research fellow in the same project. In 2014, she was awarded for a PhD Grant by Fundação para a Ciência e a Tecnologia and enrolled a Doctoral Program in Biomedical Sciences, from Abel Salazar Institute Sciences (ICBAS). During her PhD, Joana explored the phenotypical differences between Gardnerella vaginalis isolates from healthy women or women with BV, in order to better understand the etiology of BV. She performed the work in collaboration with University of Minho and Virginia Commonwealth University, Richmond, USA. She finished her PhD on 28th November 2018. In addition, she became a member to Agência de Avaliação e Acreditação do Ensino Superior (A3ES), participating in diverse audits.
EDUCATION
Ph.D. in Biomedical Sciences, Instituto de Ciências Biomédicas Abel Salazar, University of Porto
MSc in Biomedical Engineering, University of Minho
RESEARCH INTERESTS
Bacterial biofilms; Bacterial Vaginosis; Antimicrobial resistance; Virulence; Gardnerella; Gene expression; Medicinal Plants;
Scopus Publications
Scholar Citations
Scholar h-index
Scholar i10-index
Scopus Publications
Joana Castro, Ricardo Oliveira, Liliana Fernandes, Isabel Carvalho, Hugo Oliveira, Erik Brinks, Gyu-Sung Cho, Charles Franz, Carina Almeida, Sónia Silva,et al.
Elsevier BV
Diana Vilas Boas, Joana Castro, Daniela Araújo, Franklin L. Nóbrega, Charles W. Keevil, Nuno F. Azevedo, Maria João Vieira, and Carina Almeida
MDPI AG
Flagellum-mediated motility has been suggested to contribute to virulence by allowing bacteria to colonize and spread to new surfaces. In Salmonella enterica and Escherichia coli species, mutants affected by their flagellar motility have shown a reduced ability to form biofilms. While it is known that some species might act as co-aggregation factors for bacterial adhesion, studies of food-related biofilms have been limited to single-species biofilms and short biofilm formation periods. To assess the contribution of flagella and flagellum-based motility to adhesion and biofilm formation, two Salmonella and E. coli mutants with different flagellar phenotypes were produced: the fliC mutants, which do not produce flagella, and the motAB mutants, which are non-motile. The ability of wild-type and mutant strains to form biofilms was compared, and their relative fitness was determined in two-species biofilms with other foodborne pathogens. Our results showed a defective and significant behavior of E. coli in initial surface colonization (p < 0.05), which delayed single-species biofilm formation. Salmonella mutants were not affected by the ability to form biofilm (p > 0.05). Regarding the effect of motility/flagellum absence on bacterial fitness, none of the mutant strains seems to have their relative fitness affected in the presence of a competing species. Although the absence of motility may eventually delay initial colonization, this study suggests that motility is not essential for biofilm formation and does not have a strong impact on bacteria’s fitness when a competing species is present.
D. Araújo, J. Castro, F. Matos, R. Oliveira, C. Ramos, C. Almeida, and S. Silva
Elsevier BV
Maria Margarida Barros, Joana Castro, Daniela Araújo, Ana Maria Campos, Ricardo Oliveira, Sónia Silva, Divanildo Outor-Monteiro, and Carina Almeida
MDPI AG
Swine pathogenic infection caused by Escherichia coli, known as swine colibacillosis, represents an epidemiological challenge not only for animal husbandry but also for health authorities. To note, virulent E. coli strains might be transmitted, and also cause disease, in humans. In the last decades, diverse successful multidrug-resistant strains have been detected, mainly due to the growing selective pressure of antibiotic use, in which animal practices have played a relevant role. In fact, according to the different features and particular virulence factor combination, there are four different pathotypes of E. coli that can cause illness in swine: enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC) that comprises edema disease E. coli (EDEC) and enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and extraintestinal pathogenic E. coli (ExPEC). Nevertheless, the most relevant pathotype in a colibacillosis scenario is ETEC, responsible for neonatal and postweaning diarrhea (PWD), in which some ETEC strains present enhanced fitness and pathogenicity. To explore the distribution of pathogenic ETEC in swine farms and their diversity, resistance, and virulence profiles, this review summarizes the most relevant works on these subjects over the past 10 years and discusses the importance of these bacteria as zoonotic agents.
Alice Ferreira, Daniela Silva, Carina Almeida, Maria Elisa Rodrigues, Sónia Silva, Joana Castro, Dalila Mil-Homens, Isidro García-Meniño, Azucena Mora, Mariana Henriques,et al.
Springer Science and Business Media LLC
AbstractEnterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs’ viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.
Lúcia G. V. Sousa, Joana Castro, Carlos Cavaleiro, Lígia Salgueiro, Mariana Tomás, Rita Palmeira-Oliveira, José Martinez-Oliveira, and Nuno Cerca
Springer Science and Business Media LLC
AbstractBacterial 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 broad-spectrum 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 ρ-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.
Ricardo Oliveira, Joana Castro, Sónia Silva, Hugo Oliveira, Maria José Saavedra, Nuno Filipe Azevedo, and Carina Almeida
MDPI AG
While antibiotic resistance is rising to dangerously high levels, resistance mechanisms are spreading globally among diverse bacterial species. The emergence of antibiotic-resistant Klebsiella pneumoniae, mainly due to the production of antibiotic-inactivating enzymes, is currently responsible for most treatment failures, threatening the effectiveness of classes of antibiotics used for decades. This study assessed the presence of genetic determinants of β-lactam resistance in 102 multi-drug resistant (MDR) K. pneumoniae isolates from patients admitted to two central hospitals in northern Portugal from 2010 to 2020. Antimicrobial susceptibility testing revealed a high rate (>90%) of resistance to most β-lactam antibiotics, except for carbapenems and cephamycins, which showed antimicrobial susceptibility rates in the range of 23.5–34.3% and 40.2–68.6%, respectively. A diverse pool of β-lactam resistance genetic determinants, including carbapenemases- (i.e., blaKPC-like and blaOXA-48-like), extended-spectrum β-lactamases (ESBL; i.e., blaTEM-like, blaCTX-M-like and blaSHV-like), and AmpC β-lactamases-coding genes (i.e., blaCMY-2-like and blaDHA-like) were found in most K. pneumoniae isolates. blaKPC-like (72.5%) and ESBL genes (37.3–74.5%) were the most detected, with approximately 80% of K. pneumoniae isolates presenting two or more resistance genes. As the optimal treatment of β-lactamase-producing K. pneumoniae infections remains problematic, the high co-occurrence of multiple β-lactam resistance genes must be seen as a serious warning of the problem of antimicrobial resistance.
Aliona S. Rosca, Joana Castro, Ângela França, Mario Vaneechoutte, and Nuno Cerca
Springer Science and Business Media LLC
Bacterial vaginosis (BV) is one of the most common bacterial vaginal infections worldwide. Despite its high prevalence, BV etiology is still unknown. Nevertheless, a hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp. and other anaerobic species, of which co-colonization with Fannyhessea vaginae is considered an important diagnostic marker. We previously developed an in vitro biofilm model wherein Gardnerella was first allowed to establish an early biofilm that served as a scaffold for other species to adhere to. To better understand ecological interactions between BV-associated bacteria, we compared triple-species biofilms formed using two distinct models: a pre-conditioned (wherein Gardnerella vaginalis formed the early biofilm) model and a competitive (wherein all three bacteria were co-incubated together) model. Interestingly, synergistic growth interactions were more significant in the competitive model. Furthermore, the biofilm structure and species-specific distribution, as assessed by confocal laser scanning microscopy and using peptide nucleic acid fluorescence in situ hybridization method, revealed two very different triple-species morphotypes, suggesting that different interactions occur in the different models. Interestingly, independent of the model or triple-species consortium tested, we observed that G. vaginalis represented most of the biofilm bacterial composition, further highlighting the relevance of this taxon in BV.
Joana Castro, Maria Margarida Barros, Daniela Araújo, Ana Maria Campos, Ricardo Oliveira, Sónia Silva, and Carina Almeida
Frontiers Media SA
Enteric colibacillosis is a common disease in nursing and weanling pigs. It is caused by the colonization of the small intestine by enterotoxigenic strains of Escherichia coli (ETEC) that make use of specific fimbria or pili to adhere to the absorptive epithelial cells of the jejunum and ileum. Once attached, and when both the immunological systems and the gut microbiota are poorly developed, ETEC produce one or more enterotoxins that can have local and, further on, systemic effects. These enterotoxins cause fluid and electrolytes to be secreted into the intestinal lumen of animals, which results in diarrhea, dehydration, and acidosis. From the diversity of control strategies, antibiotics and zinc oxide are the ones that have contributed more significantly to mitigating post-weaning diarrhea (PWD) economic losses. However, concerns about antibiotic resistance determined the restriction on the use of critically important antimicrobials in food-producing animals and the prohibition of their use as growth promoters. As such, it is important now to begin the transition from these preventive/control measures to other, more sustainable, approaches. This review provides a quick synopsis of the currently approved and available therapies for PWD treatment while presenting an overview of novel antimicrobial strategies that are being explored for the control and treatment of this infection, including, prebiotics, probiotics, synbiotics, organic acids, bacteriophages, spray-dried plasma, antibodies, phytogenic substances, antisense oligonucleotides, and aptamers.
Hanna Fokt, Sara Cleto, Hugo Oliveira, Daniela Araújo, Joana Castro, Nuno Cerca, Maria João Vieira, and Carina Almeida
MDPI AG
Escherichia coli is a highly versatile bacterium ranging from commensal to intestinal pathogen, and is an important foodborne pathogen. E. coli species are able to prosper in multispecies biofilms and secrete bacteriocins that are only toxic to species/strains closely related to the producer strain. In this study, 20 distinct E. coli strains were characterized for several properties that confer competitive advantages against closer microorganisms by assessing the biofilm-forming capacity, the production of antimicrobial molecules, and the production of siderophores. Furthermore, primer sets for E. coli bacteriocins–colicins were designed and genes were amplified, allowing us to observe that colicins were widely distributed among the pathogenic E. coli strains. Their production in the planktonic phase or single-species biofilms was uncommon. Only two E. coli strains out of nine biofilm-forming were able to inhibit the growth of other E. coli strains. There is evidence of larger amounts of colicin being produced in the late stages of E. coli biofilm growth. The decrease in bacterial biomass after 12 h of incubation indicates active type I colicin production, whose release normally requires E. coli cell lysis. Almost all E. coli strains were siderophore-producing, which may be related to the resistance to colicin as these two molecules may use the same transporter system. Moreover, E. coli CECT 504 was able to coexist with Salmonella enterica in dual-species biofilms, but Shigella dysenteriae was selectively excluded, correlating with high expression levels of colicin (E, B, and M) genes observed by real-time PCR.
Aliona S Rosca, Joana Castro, Lúcia G V Sousa, Angela França, Mario Vaneechoutte, and Nuno Cerca
Oxford University Press (OUP)
Abstract Background Bacterial vaginosis (BV), the most common cause of vaginal discharge, is characterized by the presence of a polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp., but also other anaerobic species. Interactions between bacteria in multi-species biofilms are likely to contribute to increased virulence and to enhanced antimicrobial tolerance observed in vivo. However, functional studies addressing this question are lacking. Objectives To gain insights into the role that interactions between BV-associated species in multi-species BV biofilms might have on antimicrobial tolerance, single- and triple-species biofilms formed by Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae and Peptostreptococcus anaerobius were characterized, before and after metronidazole or clindamycin treatment. Methods Total biofilm biomass, total cells and cfu counts prior to and after antibiotic treatment were first determined. In addition, bacterial populations in the triple-species biofilms were also quantified by quantitative PCR (qPCR) and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH). Results Despite the effect observed in single-species biofilms, neither metronidazole nor clindamycin was effective in reducing triple-species biofilm biomass. Similar results were obtained when evaluating the number of total or culturable cells. Interestingly, despite differences between strain susceptibilities to antibiotics, the composition of the triple-species biofilms was not strongly affected by antibiotics. Conclusions Taken together, these results strengthen the idea that, when co-incubated, bacteria can interact synergistically, leading to increased tolerance to antimicrobial therapy, which helps explain the observed clinically high BV recurrence rates.
Aliona S. Rosca, Joana Castro, Lúcia G. V. Sousa, Angela França, Carlos Cavaleiro, Lígia Salgueiro, and Nuno Cerca
Frontiers Media SA
Bacterial vaginosis (BV) is associated with serious gynaecologic and obstetric complications. The hallmark of BV is the presence of a polymicrobial biofilm on the vaginal epithelium, but BV aetiology is still a matter of debate. We have previously developed an in vitro biofilm model that included three BV-associated species, but, up to now, no studies are available whereby more bacterial species are grown together to better mimic the in vivo situation. Herein, we characterized the first polymicrobial BV biofilm consisting of six cultivable BV-associated species by using both in vitro and ex vivo vaginal tissue models. Both models revealed that the six species were able to incorporate the polymicrobial biofilm, at different bacterial concentrations. As it has been thought that this polymicrobial biofilm may increase the survival of BV-associated species when exposed to antibiotics, we also assessed if the Thymbra capitata essential oil (EO), which has recently been shown to be highly bactericidal against several Gardnerella species, could maintain its anti-biofilm activity against this polymicrobial biofilm. Under our experimental conditions, T. capitata EO exhibited a high antibacterial effect against polymicrobial biofilms, in both tested models, with a significant reduction in the biofilm biomass and the number of culturable cells. Overall, this study shows that six BV-associated species can grow together and form a biofilm both in vitro and when using an ex vivo model. Moreover, the data obtained herein should be considered in further applications of T. capitata EO as an antimicrobial agent fighting BV.
Joana Castro, Lúcia G. V. Sousa, Ângela França, Lenka Podpera Tisakova, Lorenzo Corsini, and Nuno Cerca
MDPI AG
Bacterial vaginosis (BV) is the most frequent vaginal infection in women of reproductive age. It is caused by the overgrowth of anaerobic vaginal pathogens, such as Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia, which are vaginal pathogens detected during the early stages of incident BV and have been found to form multi-species biofilms. Treatment of biofilm-associated infections, such as BV, is challenging. In this study, we tested the role of an investigational engineered phage endolysin, PM-477, in the eradication of dual-species biofilms composed of G. vaginalis–F. vaginae or G. vaginalis–P. bivia. Single-species biofilms formed by these species were also analysed as controls. The effect of PM-477 on biomass and culturability of single- and dual-species biofilms was assessed in vitro using a microtiter plate assay, epifluorescence microscopy, confocal laser scanning microscopy, and quantitative PCR. The results showed that PM-477 was particularly effective in the disruption and reduction of culturability of G. vaginalis biofilms. In dual-species biofilms, PM-477 exhibited lower efficiency but was still able to selectively and significantly eliminate G. vaginalis. Since polymicrobial interactions have been shown to strongly affect the activity of various antibiotics, the activity of PM-477 in dual-species biofilms is a potentially promising result that should be further explored, aiming to completely eradicate multi-species biofilms associated with BV.
Joana Castro, Ângela Lima, Lúcia G. V. Sousa, Aliona S. Rosca, Christina A. Muzny, and Nuno Cerca
Frontiers Media SA
Bacterial Vaginosis (BV) involves the presence of a multi-species biofilm adhered to vaginal epithelial cells, but its in-depth study has been limited due to the complexity of the bacterial community, which makes the design of in vitro models challenging. Perhaps the most common experimental technique to quantify biofilms is the crystal violet (CV) staining method. Despite its widespread utilization, the CV method is not without flaws. While biofilm CV quantification within the same strain in different conditions is normally accepted, assessing multi-species biofilms formation by CV staining might provide significant bias. For BV research, determining possible synergism or antagonism between species is a fundamental step for assessing the roles of individual species in BV development. Herein, we provide our perspective on how CV fails to properly quantify an in vitro triple-species biofilm composed of Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, and Prevotella bivia, three common BV-associated bacteria thought to play key roles in incident BV pathogenesis. We compared the CV method with total colony forming units (CFU) and fluorescence microscopy cell count methods. Not surprisingly, when comparing single-species biofilms, the relationship between biofilm biomass, total number of cells, and total cultivable cells was very different between each tested method, and also varied with the time of incubation. Thus, despite its wide utilization for single-species biofilm quantification, the CV method should not be considered for accurate quantification of multi-species biofilms in BV pathogenesis research.
Lúcia G. V. Sousa, Joana Castro, Angela França, Carina Almeida, Christina A. Muzny, and Nuno Cerca
Frontiers Media SA
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.
Joana Castro, Aliona S. Rosca, Christina A. Muzny, and Nuno Cerca
MDPI AG
Bacterial vaginosis (BV) is associated with a highly structured polymicrobial biofilm on the vaginal epithelium where Gardnerella species presumably play a pivotal role. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia are vaginal pathogens detected during the early stages of incident BV. Herein, we aimed to analyze the impact of A. vaginae and P. bivia on a pre-established G. vaginalis biofilm using a novel in vitro triple-species biofilm model. Total biofilm biomass was determined by the crystal violet method. We also discriminated the bacterial populations in the biofilm and in its planktonic fraction by using PNA FISH. We further analyzed the influence of A. vaginae and P. bivia on the expression of key virulence genes of G. vaginalis by quantitative PCR. In our tested conditions, A. vaginae and P. bivia were able to incorporate into pre-established G. vaginalis biofilms but did not induce an increase in total biofilm biomass, when compared with 48-h G. vaginalis biofilms. However, they were able to significantly influence the expression of HMPREF0424_0821, a gene suggested to be associated with biofilm maintenance in G. vaginalis. This study suggests that microbial relationships between co-infecting bacteria can deeply affect the G. vaginalis biofilm, a crucial marker of BV.
Joana Castro, Aliona S. Rosca, Piet Cools, Mario Vaneechoutte, and Nuno Cerca
Frontiers Media SA
Bacterial vaginosis (BV) is the most common vaginal infection among women of reproductive age. A hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, presumably initiated by facultative anaerobes of the genus Gardnerella, which then becomes a scaffold for other species to adhere to. One of the species often found incorporated in Gardnerella mediated biofilms is Atopobium vaginae. Interestingly, A. vaginae is very rarely found without the presence of Gardnerella. However, not much is known regarding the interactions between A. vaginae and Gardnerella species. This study assessed biological interactions between Gardnerella vaginalis and A. vaginae. In our in vitro model, by using specific Gardnerella and A. vaginae Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH) probes, we confirmed that A. vaginae was able to incorporate a pre-formed G. vaginalis biofilm, accounting for up to 20% of the total number of biofilm cells. However, our findings showed that almost 92% of A. vaginae cells lost viability after 48 h of mono-species planktonic growth, but were able to maintain viability when co-cultured with Gardnerella or after pre-conditioning with cell-free supernatant of Gardnerella cultures. While the in vitro conditions are very different from the in vivo microenvironment, this study contributes to a better understanding of why A. vaginae vaginal colonization rarely occurs in the absence of Gardnerella. Overall, this highlights the importance of microbial interactions between BV-associated bacteria and demands more studies focused on the polymicrobial bacterial communities found in BV.
Joana Castro, Kimberly K. Jefferson, and Nuno Cerca
Elsevier BV
Gardnerella vaginalis has been considered a pivotal player in the progression of bacterial vaginosis (BV), a condition associated with serious health complications. However, G. vaginalis is also commonly found in asymptomatic or BV-negative women. This has generated interest in the question of whether genetic differences among isolates might distinguish pathogenic from commensal isolates. G. vaginalis was the only recognized species in its genus for four decades, but recently an emended description of G. vaginalis and descriptions of three new species - Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii - have been proposed. This review provides background on the heterogeneity and diversity within the genus Gardnerella, highlighting the main features that distinguish species and clades, and how these features may impact BV development.
Aliona S. Rosca, Joana Castro, and Nuno Cerca
PeerJ
Background Bacterial vaginosis (BV) is one of the most common vaginal infections worldwide. It is associated with the presence of a dense polymicrobial biofilm on the vaginal epithelium, formed mainly by Gardnerella species. The biofilm also contains other anaerobic species, but little is known about their role in BV development. Aim To evaluate the influence of different culture media on the planktonic and biofilm growth of six cultivable anaerobes frequently associated with BV, namely Gardnerella sp., Atopobium vaginae, Lactobacillus iners, Mobiluncus curtisii, Peptostreptococcus anaerobius and Prevotella bivia. Methods A total of nine different culture media compositions, including commercially available and chemically defined media simulating genital tract secretions, were tested in this study. Planktonic cultures and biofilms were grown under anaerobic conditions (10% carbon dioxide, 10% helium and 80% nitrogen). Planktonic growth was assessed by optical density measurements, and biofilm formation was quantified by crystal violet staining. Results Significant planktonic growth was observed for Gardnerella sp., A. vaginae and L. iners in New York City III broth, with or without ascorbic acid supplementation. Biofilm quantification showed high in vitro biofilm growth for Gardnerella sp., P. anaerobius and P. bivia in almost all culture media excluding Brucella broth. Contrary, only New York City III broth was able to promote biofilm formation for A. vaginae, L. iners and M. curtisii. Conclusions Our data demonstrate that New York City III broth relative to the other tested media is the most conducive for future studies addressing polymicrobial biofilms development as this culture medium allowed the formation of significant levels of single-species biofilms.
Aliona S Rosca, Joana Castro, Lúcia G V Sousa, and Nuno Cerca
Oxford University Press (OUP)
Abstract The human vagina is a dynamic ecosystem in which homeostasis depends on mutually beneficial interactions between the host and their microorganisms. However, the vaginal ecosystem can be thrown off balance by a wide variety of factors. Bacterial vaginosis (BV) is the most common vaginal infection in women of childbearing age, but its etiology is not yet fully understood, with different controversial theories being raised over the years. What is generally accepted is that BV is often characterized by a shift in the composition of the normal vaginal microbiota, from a Lactobacillus species dominated microbiota to a mixture of anaerobic and facultative anaerobic bacteria. During BV, a polymicrobial biofilm develops in the vaginal microenvironment, being mainly composed of Gardnerella species. The interactions between vaginal microorganisms are thought to play a pivotal role in the shift from health to disease and might also increase the risk of sexually transmitted infections acquisition. Here we review the current knowledge regarding the specific interactions that occur in the vaginal niche and discuss mechanisms by which these interactions might be mediated. Furthermore, we discuss the importance of novel strategies to fight chronic vaginal infections.
Joana Castro, Daniela Machado, and Nuno Cerca
Springer Science and Business Media LLC
Bacterial vaginosis (BV) is characterized by a highly structured polymicrobial biofilm, which is strongly adhered to the vaginal epithelium and primarily consists of the bacterium Gardnerella vaginalis. However, despite the presence of other BV-associated bacteria, little is known regarding the impact of other species on BV development. To gain insight into BV progress, we analyzed the ecological interactions between G. vaginalis and 15 BV-associated microorganisms using a dual-species biofilm model. Bacterial populations were quantified using a validated peptide nucleic acid fluorescence in situ hybridization approach. Furthermore, biofilm structure was analyzed by confocal laser scanning microscopy. In addition, bacterial coaggregation ability was determined as well as the expression of key virulence genes. Remarkably, our results revealed distinct biofilm structures between each bacterial consortium, leading to at least three unique dual-species biofilm morphotypes. Furthermore, our transcriptomic findings seem to indicate that Enterococcus faecalis and Actinomyces neuii had a higher impact on the enhancement of G. vaginalis virulence, while the other tested species had a lower or no impact on G. vaginalis virulence. This study casts a new light on how BV-associated species can modulate the virulence aspects of G. vaginalis, contributing to a better understanding of the development of BV-associated biofilms.
Joana Castro, Kimberly K Jefferson, and Nuno Cerca
Oxford University Press (OUP)
Mucosal surfaces of the female reproductive tract contain a variety of antimicrobial components that provide the first line of defense against bacteria involved in the development of bacterial vaginosis (BV). Microbiological analysis of BV has shown Gardnerella vaginalis to be a prominent species in BV development. However, G. vaginalis colonization does not always lead to BV. Over the last decade, phenotypic and genotypic studies have demonstrated the existence of strain variants. Therefore, this study aimed to investigate if the major components of the vaginal immune response, specifically lysozyme, lactoferrin and β-defensin 2, differently affected virulence traits of G. vaginalis strains isolated from healthy women or from women with BV. Gardnerella vaginalis strains were first genotyped by the clade classification system and then phenotypically characterized. Our results revealed that key differences in initial adhesion existed among the isolates but that these differences could not be predicted using the clade-genotyping approach. Importantly, we found that growth, initial adhesion and biofilm formation were strongly affected by lysozymes, but at similar levels in both groups, suggesting that the response to host immune components is not a distinguishing characteristic of isolates from women with BV versus those from healthy women.
Joana Castro, Ana Paula Martins, Maria Elisa Rodrigues, and Nuno Cerca
Elsevier BV
Using a chemically-defined medium simulating genital tract secretions, we have shown that pre-adhering Lactobacillus crispatus to Hela epithelial cells reduced cytotoxicity caused by Gardnerella vaginalis. This effect was associated to the expression of vaginolysin and was specific to L. crispatus interference, as other vaginal facultative anaerobes had no protective effect.
Joana Castro, Angela França, Katie R. Bradwell, Myrna G. Serrano, Kimberly K. Jefferson, and Nuno Cerca
Springer Science and Business Media LLC
Bacterial vaginosis is the most common gynecological disorder affecting women of reproductive age. Bacterial vaginosis is frequently associated with the development of a Gardnerella vaginalis biofilm. Recent data indicates that G. vaginalis biofilms are more tolerant to antibiotics and are able to incorporate other bacterial vaginosis -associated species, yielding a multi-species biofilm. However, despite its apparent role in bacterial vaginosis, little is known regarding the molecular determinants involved in biofilm formation by G. vaginalis. To gain insight into the role of G. vaginalis in the pathogenesis of bacterial vaginosis, we carried out comparative transcriptomic analysis between planktonic and biofilm phenotypes, using RNA-sequencing. Significant differences were found in the expression levels of 815 genes. A detailed analysis of the results obtained was performed based on direct and functional gene interactions. Similar to other bacterial species, expression of genes involved in antimicrobial resistance were elevated in biofilm cells. In addition, our data indicate that G. vaginalis biofilms assume a characteristic response to stress and starvation conditions. The abundance of transcripts encoding proteins involved in glucose and carbon metabolism was reduced in biofilms. Surprisingly, transcript levels of vaginolysin were reduced in biofilms relative to planktonic cultures. Overall, our data revealed that gene-regulated processes in G. vaginalis biofilms resulted in a protected form of bacterial growth, characterized by low metabolic activity. This phenotype may contribute towards the chronic and recurrent nature of bacterial vaginosis. This suggests that G. vaginalis is capable of drastically adjusting its phenotype through an extensive change of gene expression.Vaginal infection: Gene activity protects biofilmsAltered gene activity may promote chronic and recurrent infections in the most common gynecological disorder in women of reproductive age. Bacterial vaginosis (BV) frequently involves the formation of biofilms by Gardnerella vaginalis bacteria. Nuno Cerca and colleagues at the University of Minho in Portugal, with co-workers elsewhere in Portugal and in the USA, studied changes in gene activity in BV biofilms. Their results linked differences in the activity of 815 genes to an ability of the bacteria to enter a protected form of growth when producing biofilms. The differences included increased activity of specific genes associated with antibiotic resistance. This insight into the bacterial ability to undergo drastic changes in gene activity could guide work to understand chronic and recurrent BV. It may also lead to new ways to treat this common and troublesome condition.
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16| Castro J, Jefferson KK, Cerca N (2019). Genetic Heterogeneity and Taxonomic Diversity among Gardnerella Species. Trends in Microbiology, In press, doi: 10.1016/j.. Impact factor (2018): 11.974; Q1
15| Castro J #, Rosca AS#, Sousa LGV, Cerca N (2019). Gardnerella and vaginal health: the truth is out there. FEMS Microbiology Reviews, In press, doi: 10.1093/femsre/fuz027. #Both authors contributed equally to this work. Impact factor: 11.524 (2018); Q1
14| Castro J, Jefferson KK, Cerca N (2018). Innate immune components affect growth and virulence traits of bacterial vaginosis-associated and non-BV associated Gardnerella vaginalis strains similarly. Pathogens and Disease. Accepted. Impact factor: 2.335 (2016); Q1 (Medicine-miscellaneous)
13| Castro J, Machado D, Cerca N (2018). Unveiling the role of Gardnerella vaginalis in polymicrobial Bacterial Vaginosis biofilms: the impact of other vaginal pathogens living as neighbors. The ISME Journal. Accepted. Impact factor: 9.520 (2017); Q1
12| Castro J #, Martins AP #, Rodrigues ME, Cerca N (2018). Lactobacillus crispatus represses vaginolysin expression by BV associated Gardnerella vaginalis and reduces cell cytotoxicity. Anaerobe 50, 60-63 (doi: 10.1016/j. #Both authors contributed equally to this work. 5-Year Impact factor: 2.943; Q2
11| Machado D, Castro J, Martinez-de-Oliveira J, Nogueira-Silva C, Cerca N (2017). Prevalence of bacterial vaginosis in Portuguese pregnant women and vaginal colonization by Gardnerella vaginalis. PeerJ 5, e3750 (doi: 10.7717/. Impact factor: 2.118 (2017); Q1
10| Castro J, França A, Bradwell KR, Serrano MG, Jefferson KK, Cerca N (2017). Comparative transcriptomic analysis of Gardnerella vaginalis biofilms versus planktonic cultures using RNA-seq. NPJ Biofilms Microbiomes 3, 3 (doi: 10.1038/s41522-017-0012-7). 2-year Impact factor: 4.128; Q1
9| Machado D, Castro J, Palmeira-de-Oliveira A, Martinez-de-Oliveira J, Cerca N. (2016). Bacterial vaginosis biofilms: challenges to current therapies and emerging solutions. Frontiers in Microbiology 6, 1528 (doi: 10.3389/. Impact factor: 4.019; Q1
8| Castro J, Machado D, Cerca N. (2016). Escherichia coli and Enterococcus faecalis are able to incorporate and enhance a pre-formed Gardnerella vaginalis biofilm. Pathogens and Disease 74 (doi: 10.1093/femspd/ftw007). Impact factor: 2.335 (2016); Q1 (Medicine-miscellaneous)
7| Castro J, Cerca N (2015). BV and non-BV associated Gardnerella vaginalis establish similar synergistic interactions with other BV-associated microorganisms in dual-species biofilms. Anaerobe 36, 56-9 (doi: 10.1016/j.. 5-Year Impact factor: 2.943; Q2
6| Castro J, Alves P, Sousa C, Cereija TB, França A, Jefferson KK, Cerca N (2015). Using an in-vitro biofilm model to assess the virulence potential of bacterial vaginosis or non-bacterial vaginosis Gardnerella vaginalis isolates. Scientific Reports 5:11640 (doi: 10.1038/srep11640).5-year Impact factor: 4.609; Q1
5| Machado A, Castro J, Cereja T, Almeida C and Cerca N (2015). Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method. PeerJ 3:e780 (doi: 10.7717/. Impact factor: 2.118 (2017); Q1
4| Ojala T, Kankainen M, Castro J, Cerca N, Edelman S, Westerlund-Wikstrom B, Paulin L, Holm, L, Auvinen P (2014). Comparative genomics of Lactobacillus crispatus suggests novel mechanisms for the competitive exclusion of Gardnerella vaginalis. BMC Genomics 15, 1070 (doi: 10.1186/1471-2164-15-1070). Impact factor: 3.729 (2016); Q1
3| Alves P #, Castro J #, Sousa C, Cereija TB, Cerca N (2014). Gardnerella vaginalis outcompetes 29 other bacterial species isolated from patients with bacterial vaginosis, using in an in vitro biofilm formation model. Journal of Infectious Diseases 210, 593-596 (doi: 10.1093/infdis/jiu131) #Both authors contributed equally to this work. 5-year Impact factor: 5.345; Q1
2| Cereja T, Castro J, Alves P and Cerca N (2013). Influence of anaerobic conditions on vaginal microbiome recovery from Bacterial Vaginosis patients. Sexually Transmitted Infections, 89:307 (doi:10.1136/sextrans-2013-051100). Impact factor: 3.346; Q1
1| Castro J, Henriques A, Machado A, Henriques M, Jefferson KK and Cerca N. (2013). Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells. International Journal of Medical Science, 10(9):1193-8 (doi: 10.7150/. Impact factor: 2.399; Q1
GRANT DETAILS
FCT Grant SFRH/BD/93963/2013 – Determining the pathogenic potential of commensal and clinical Gardnerella vaginalis isolates