Humulus lupulus aqueous extract and hydrolate as a potential ingredient for cosmetics: chemical characterization and in vitro antimicrobial, cytotoxicity, antioxidant and anti-inflammatory assessment.

Humulus lupulus extracts have in their composition different molecules, such as polyphenols, α-acids, ß-acids, and hydrocarbons, which contribute to the plant's medicinal properties. These molecules are associated with antimicrobial, antioxidant and anti-inflammatory activities. OBJECTIVE: This work focuses on the evaluation of H. lupulus biological activities, with the aim of evaluating its potential for inclusion in cosmetic formulations. METHODS: Two distinct aqueous extracts and two hydrolates obtained via hydrodistillation were evaluated. These include the flower parts (FE, FH) and the mix of aboveground parts (ME, MH). The chemical profiles for both aqueous extracts and hydrolates were identified by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Antimicrobial, antioxidant, cytotoxicity, and anti-inflammatory activity were tested in vitro using standard methods. RESULTS: Rutin was the major compound found in FE (40.041 µg mg-1 of extract) and ME (2.909 µg mg-1 of extract), while humulenol II was the most abundant compound in hydrolates (FH: 20.83%; MH: 46.80%). Furthermore, FE was able to inhibit the growth of Staphylococcus aureus and Staphylococcus epidermis with MIC values of 50% and 25% (v/v), respectively. FH showed the same effect in Staphylococcus aureus (50% v/v). FH evidenced poor antioxidant potential in DPPH scavenging test and demonstrated significant antioxidant and anti-inflammatory effects by reducing (***p < 0.001) intracellular reactive oxygen species (ROS), NO (nitric oxide) levels (***p < 0.001) and cyclooxygenase-2 (COX-2) protein expression (***p < 0.001) in lipopolysaccharide (LPS)-stimulated macrophages. Nevertheless, it is important to note that FH exhibited cytotoxicity at high concentrations in 3T3 fibroblasts and RAW 264.7 macrophages. CONCLUSION: The studied H. lupulus aqueous extracts and hydrolates revealed that FH stands out as the most promising bioactive source for cosmetic formulations. However, future research addressing antimicrobial activity is necessary to confirm its potential incorporation into dermatological and cosmetic formulations.

Study of Ecological Relationship of Yeast Species with Candida albicans in the Context of Vulvovaginal Infections.

The role of the fungal community, the mycobiota, in the health of the vagina is currently an important area of research. The emergence of new sequencing technologies and advances in bioinformatics made possible the discovery of novel fungi inhabiting this niche. Candida spp. constitutes the most important group of opportunistic pathogenic fungi, being the most prevalent fungal species in vulvovaginal infections. However, fungi such as Rhodotorula spp., Naganishia spp. and Malassezia spp. have emerged as potential pathogens in this niche, and therefore it is clinically relevant to understand their ecological interaction with Candida spp. The main aim of this study was to evaluate the impact of yeasts on Candida albicans' pathogenicity, focusing on in-vitro growth, and biofilm formation at different times of co-culture and germ tube formation. The assays were performed with isolated species or with co-cultures of C. albicans (ATCC10231) with one other yeast species: Rhodotorula mucilaginosa (DSM13621), Malassezia furfur (DSM6170) or Naganishia albida (DSM70215). The results showed that M. furfur creates a symbiotic relationship with C. albicans, enhancing the growth rate of the co-culture (149.69%), and of germ tube formation of C. albicans (119.8%) and inducing a higher amount of biofilm biomass of the co-culture, both when mixed (154.1%) and preformed (166.8%). As for the yeasts R. mucilaginosa and N. albida, the relationship is antagonistic (with a significant decrease in all assays), thus possibly repressing the mixture's pathogenicity. These results shed light on the complex interactions between yeasts in the vaginal mycobiome.

The Role of Yeasts in Human Health: A Review.

The microbiome consists mostly of bacteria, but new evidence and developments in sequencing methods have shown that fungi play an important role in human health and in the stability of the microbiota. Scientific knowledge about the role of commensal fungi in intestinal, oral, vaginal and cutaneous communities has been increasing; however, more studies are still needed to better understand their action in these niches. To date, fungal research focuses primarily on opportunistic diseases caused by fungal species, leaving unclear the possible role of fungi as an integral part of the microbiota. Although they are much less abundant than bacteria, fungi such as species belonging to the genus Candida, Malassezia, Rhodotorula and Cryptococcus are some of the yeasts that have been in the focus of the scientific community because they inhabit various niches. In this review, we have summarized the current information about the yeasts that inhabit the human body, including some of the diseases that they can cause when the microbiota becomes unstable.

Uncovering the Yeast Diversity in the Female Genital Tract: An Exploration of Spatial Distribution and Antifungal Resistance.

Candida albicans is the leading cause of vulvovaginal yeast infections; however, other species are becoming relevant in this niche. The spatial distribution of these fungi in the female genital tract remains poorly understood. In this study, swab samples were collected from 33 patients, first from the anterior vulva and then from the upper third and right lateral wall of the vagina: 16 were with symptoms of vulvovaginal candidiasis and 17 were without characteristic symptoms; furthermore, the genus and species of each isolate were identified. In vitro susceptibility testing for fluconazole and clotrimazole was performed for all isolates. Candida albicans was the most common species (63.6%), followed by Rhodotorula spp. (51.5%), and then Candida parapsilosis (15.2%). Rhodotorula spp. and C. parapsilosis were more commonly associated with colonization, and C. albicans with infection. Rhodotorula spp. isolates presented a low susceptibility to fluconazole, with the MIC ranging from 32 to >64 µg/mL. Differences in susceptibility to fluconazole and clotrimazole between the pairs of vaginal and vulvar isolates were found for Candida albicans, Rhodotorula spp., and Nakaseomyces glabratus. The results suggest that different niches may impact the susceptibility profiles of the isolates, as well as their different clinical behaviors.

Thymus mastichina (L.) L. and Cistus ladanifer L. for skin application: chemical characterization and in vitro bioactivity assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus mastichina (L.) L. (TM) and Cistus ladanifer L. (CL) are two Portuguese autochthonous species with traditional skin application in folk medicine. TM is majorly known for its antiseptic and wound healing properties, as an external anti-inflammatory agent and for its application in folk cosmetics and hygiene products. Its use in acne vulgaris has also been reported. CL is traditionally used in remedies for wounds, ulcers and other skin ailments such as psoriasis and eczema. Its application has been found useful due to its anti-inflammatory, astringent, wound healing and antiseptic properties. AIM OF THE STUDY: With this work, we aimed to investigate relevant bioactivities related with the traditional application of TM and CL essential oils (EOs) and hydrolates (by-products of EO production) in skin ailments. Specifically their in vitro antioxidant, anti-inflammatory, cytotoxic, wound healing and antimicrobial properties were evaluated. The chemical composition of both EOs and respective hydrolates was also characterized. MATERIALS AND METHODS: Chemical characterization of EOs and hydrolates was performed by GC-FID and GC-MS. Cellular biocompatibility was evaluated using the MTT assay in macrophages (RAW 264.7) and fibroblasts (L929) cell lines. Anti-inflammatory activity was investigated by studying nitric oxide (NO) production by macrophages with Griess reagent. Wound healing potential was evaluated with the scratch-wound assay. The antioxidant potential was studied by the DPPH scavenging method. Antimicrobial activity was evaluated by broth microdilution assay against relevant microbial strains and skin pathogens, namely Staphylococcus aureus, Staphylococcus epidermidis, Cutibacterium acnes, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Aspergillus brasiliensis. RESULTS: The major compounds present in TM and CL EOs were 1,8-cineole and α-pinene, respectively. 1,8-cineole and E-pinocarveol were the major compounds in the correspondent hydrolates. CL EO presented the highest anti-inflammatory potential [EC50 = 0.002% (v/v)], still with significant cytotoxicity [IC50 = 0.012% (v/v)]. TM preparations presented anti-inflammatory potential, also presenting higher biocompatibility. The same profile was present on fibroblasts regarding biocompatibility of the tested preparations. CL EO and hydrolate increased fibroblasts' migration by 155.7% and 148.4%, respectively. TM hydrolate presented a milder activity than CL hydrolate, but wound healing potential was still present, increasing cell migration by 125.1%. All preparations presented poor antioxidant capacity. CL EO presented higher antimicrobial activity, with MICs ranging from 0.06% (v/v) to 2% (v/v), against different microorganisms. CONCLUSIONS: Anti-inflammatory and skin repairing potential were present for CL preparations. TM hydrolate presented an interesting biocompatible profile on both cell lines, also presenting anti-inflammatory potential. Furthermore, EOs from both species presented antimicrobial activity against a panel of different microorganisms. These in vitro bioactivities support some of their traditional skin applications, specifically regarding their antiseptic, wound healing and anti-inflammatory uses.

Vulvovaginal Candida albicans Clinical Isolates' Resistance to Phagocytosis In-Vitro

Previous studies have revealed that Candida albicans isolates involved in chronic vulvovaginal candidosis (cVVC) phenotypically express less virulent traits than clinical isolates involved in sporadic infections. In this study, we aimed to further explore this finding by studying the behaviour of those same clinical isolates in in-vitro models of infection. Eighteen clinical Candida albicans isolates were collected from women suffering sporadic (eight isolates) or chronic infections (ten isolates). Adhesion to HeLa cells (human cervical cancer epithelial cell line) and resistance to phagocytosis by RAW 264.7 cells (murine macrophages cell line) were tested in-vitro. In addition, phenotypic expression of virulence factors related with either adhesion or resistance to phagocytosis was tested in-vitro. Results indicated that yeast isolates involved in sporadic infection adhered in a higher proportion of HeLa cells than those of chronic infections, which was related with their ability to produce biofilm (p < 0.05). The ability to evade phagocytosis was related to an elevated production of proteases (p < 0.05) by chronic isolates, while sporadic isolates' resistance to phagocytosis was related to a higher hydrophobicity of cell walls (p < 0.05). We conclude that the evasion of macrophage-mediated phagocytosis related to the production of proteases might be an important factor involved in the recurrence of vulvovaginal candidosis infection.

Women's preferences and acceptance for different drug delivery routes and products.

To use or not to use, that is the first decision to take regarding a drug product. This mandatory step for adherence dictates product efficacy. The determinants for such decision do not only rely on the priority of the therapeutic or preventive strategy, but are related to a complex network of perceptions, preferences, personal and cultural backgrounds, and results from previous experiences. Women's preferences for dosage forms and even for drug delivery routes have been mainly studied in the fields of contraception and HIV prevention (and their related multipurpose approaches). Much less attention has been devoted to other therapeutic or preventive strategies. In a time when patient-centred approaches and shared decisions are increasingly valued, considering women's preferences and their main determinants is essential for product development and selection. Such products will be more likely to be chosen and used as intended, increasing efficacy, and reducing the overall costs related with these treatments. This knowledge shall be integrated in early stages of product development. This article reviews the state of the art related with women's preferences and acceptance for different dosage forms and drug delivery routes involved in women's health. The methodologies used for collecting these data and their major drawbacks are discussed. Results obtained from acceptability studies and the main determinants for selection of preventive and treatment drug products are discussed as tools for new developments in the field.

Development of a new multiplex PCR to detect prevalent species of house dust mites in house dust.

Dermatophagoides pteronyssinus and Dermatophagoides farinae are the most common House Dust Mite (HDM) species in home environments worldwide and responsible for HDM allergy. Since the prevalence of HDM-related clinical conditions is linked to exposure to the mite itself, the detection of HDM in the human households gains importance. We aimed to develop a fast and accessible multiplex PCR to detect and distinguish two relevant HDM species in house dust. New primers were designed, and sensitivity analysis was performed. Sequencing of PCR products was also performed to confirm the method's specificity. The limit of detection of the multiplex PCR for both species was as low as 30 pg µL-1. The application of the multiplex PCR to dust samples also resulted in the identification of both species with high sensitivity. The protocol required small amount of template, reagents and a short reaction time thus presenting an alternative to classically used techniques for HDM identification.

Chemical characterization and bioactive potential of Thymus×citriodorus (Pers.) Schreb. preparations for anti-acne applications: Antimicrobial, anti-biofilm, anti-inflammatory and safety profiles.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus × citriodorus (Pers.) Schreb. is an interspecific hybrid between Thymus pulegioides and Thymus vulgaris, known for its pharmacological activities as diaphoretic, deodorant, antiseptic and disinfectant, the last mostly related with its antimicrobial activity. The folk use of other extracts, as hydrolates, have also been disseminated, as regulators of oily skin with anti-acne effect. AIM OF THE STUDY: We aimed to evaluate the anti-acne potential of two Thymus x citriodorus (TC) preparations, the essential oil (EO) and the hydrolate, to be used as active ingredients for skin applications. Specifically, we intend to validate their anti-acne potential by describing their activity on acne related bacteria, bacterial virulence, anti-oxidant and anti-inflammatory potential, and biocompatibility on inflammatory cells. Additionally, we aimed to report their ecotoxicity under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), thus focusing not only on the consumer, but also on environmental safety assessment. MATERIALS AND METHODS: Minimum inhibitory concentration (MIC) against C. acnes, S. aureus and S. epidermidis was evaluated. Minimum lethal concentration (MLC) was also determined. The effect on C. acnes biofilm formation and disruption was evaluated with crystal violet staining. Anti-inflammatory activity was investigated on LPS-stimulated mouse macrophages (RAW 264.7), by studying nitric oxide (NO) production (Griess reagent) and cellular biocompatibility through MTT assay. In-vitro NO and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging potential were also evaluated. The ecotoxicity was evaluated using Daphnia magna acute toxicity assays. RESULTS: EO presented direct antimicrobial activity, with visual MICs ranging from 0.06% for S. epidermidis and C. acnes to 0.125% for S. aureus. MLCs were higher than the obtained MICs. Hydrolate revealed visual MIC only for C. acnes. TC essential oil was effective in preventing biofilm formation and disrupting preformed biofilms even at sub-inhibitory concentrations. Hydrolate showed a more modest anti-biofilm effect. Regarding anti-inflammatory activity, TC hydrolate has a higher cellular biocompatibility. Still, both plant preparations were able to inhibit at least 50% of NO production at non-cytotoxic concentrations. Both EO and hydrolate have poor anti-oxidant activities. Regarding the ecotoxicity, TC essential oil was classified under acute 3 category, while the hydrolate has proved to be nontoxic, in accordance to the GHS. CONCLUSIONS: These results support the anti-acne value of different TC preparations for different applications. TC hydrolate by presenting higher biocompatibility, anti-inflammatory potential and the ability to modulate C. acnes virulence, can be advantageous in a product for everyday application. On the other hand, EO by presenting a marked antimicrobial, anti-biofilm and anti-inflammatory activities, still with some cytotoxicity, may be better suited for application in acute flare-ups, for short treatment periods.

Allergic vulvovaginitis: a systematic literature review.

PURPOSE: Despite the vaginal mucosa is able to respond to allergenic stimuli, vaginal allergic responses have been under investigated in clinical practice. Thus, we aimed to identify the most frequent etiological agents responsible for vulvovaginal allergies, the prevalent signs/symptoms, and the diagnostic tests applied in this clinical condition. METHODS: Literature search was performed on PubMed, Scopus, Scielo, Web of Science, and EMBASE. The study protocol was registered on PROSPERO (CRD42020167238). Studies were divided in two groups depending on allergen exposure route. Due to a significant number of studies correlating allergy to Candida infection, subgroup analysis was included. RESULTS: In direct exposure cases, Human Seminal Plasma was the most prevalent allergen, sensitizing 73% of affected women. These women presented localized swelling and burning as prevalent symptoms, affecting 42/68 and 36/68 women, respectively. Cutaneous Prick tests were applied in 58/68 women, either alone or combined with IgE measurements. Regarding cases of indirect/unidentified exposure, house dust mites was the most prevalent allergen (54%), followed by pollen (44%). Predominant symptoms were vulvar pruritus and burning, affecting 67/98 and 52/98 women. Skin prick test was the most prevalent diagnostic method used among different studies. Hypersensitivity toward Candida antigen was present in only half (163/323) of women presenting concomitant allergy and Candida infection. CONCLUSION: From the two types of allergen exposure that can cause vulvovaginal allergic responses, direct contact of the antigen with the vulva and/or vagina was the most prevalent. Still, allergens can also sensitize the vaginal mucosa secondarily to other exposure route, specifically aeroallergens.

Virulence Factors as Promoters of Chronic Vulvovaginal Candidosis: A Review.

INTRODUCTION: The vast majority of the species of the genus Candida spp. is commensal in humans; however, some are opportunistic pathogens that can cause infection, called candidosis. Among the different types of candidosis, we highlight the vulvovaginal (VVC) which can occur in two main clinical variants: chronic (cVVC) and episodic or sporadic. The incidence of cVVC has been worrying the scientific community, promoting the research on genotypic and phenotypic causes of its occurrence. We summarize important findings on factors that favor chronic vulvovaginal candidosis with respect to molecular epidemiology and the expression of various virulence factors, while clarifying the terminology involving these infections. AIM AND METHODOLOGY: The aim of this review was to gather research that linked virulence factors to VVC and its persistence and recurrence, using two databases (Pubmed and Google Scholar). Predisposing factors in women for the occurrence of cVVC and some studies that refer new preventive and alternative therapies were also included, where appropriate. RESULTS AND DISCUSSION: Several studies have been shedding light on the increasing number of persistence and recurrences of VVC. The expression of virulence factors has been related to both chronic forms of VVC and antifungal resistance. Other studies report mutations occurring in the genome of Candida spp. during the infection phase which may be important indications for new therapies. The introduction of preventive therapies and new therapies has revealed great importance and is also highlighted here.

Evaluation of overtime phenotypic variation of yeasts in chronic vulvovaginal candidosis cases.

Chronic vulvovaginal candidosis results either from reinfection or from the ability of Candida spp. to persist in the vulva and/or vagina. Persistence is usually associated with increased antifungal (mainly azoles) resistance rates, which can explain treatment failure, and/or increased expression of virulence factors by Candida spp. The aim of this study was to assess the mechanisms leading to Candida spp persistence, by studying sequential isolates from women with chronic vulvovaginal candidosis, focusing on strains genotypes, azole resistance, and ability to form biofilms along the period of clinical evaluation. The strains were identified at species level by automated analysis of biochemical profiles and molecular typing evaluated by polymorphic DNA analysis. The capacity to form biofilm was assessed with a microtiter plate assay. Fluconazole susceptibility was determined by the microdilution broth assay at both pH 7 (following the recommended guideline) and pH 4.5 (as representative of vaginal pH). We studied samples from 17 clinically recurrent cases. In 53% of the chronic cases there were two or more isolates that had a phylogenetic relationship while the remaining (47%) were caused by different species. In those cases where related strains were involved in recurrence, we verified an increase in MIC at pH 7 and also an increased capacity to form biofilms over time. Significant correlation between these two parameters was observed only in cases caused by C. glabrata, evidencing the importance of these two factors to enhance persistence in the vaginal mucosa for this particular species.

Chronic vulvovaginal candidosis (VVC) affects millions of women worldwide. We found that persistence of the same Candida strain on the vaginal mucosa does not account for the great majority of VVC cases. Moreover, modulation of biofilm formation and azole resistance overtime was investigated.

Dequalinium Chloride Effectively Disrupts Bacterial Vaginosis (BV) Gardnerella spp. Biofilms.

Bacterial vaginosis (BV) is the most frequent vaginal infection worldwide. It is caused by the overgrowth of anaerobic vaginal pathogens such as Gardnerella spp. BV has been associated with the occurrence of dense multispecies biofilms on the vaginal mucosa. Treatment of biofilm-associated infections such as BV is challenging. In this study, we have tested the role of a quaternary ammonium compound, dequalinium chloride (DQC), in the eradication of Gardnerella spp. biofilms. The effects of the test substance on the biomass and the metabolic activity of the biofilm of Gardnerella spp. were assessed in vitro using a microtiter plate assay. In addition, the effect of DQC on the Gardnerella spp. biofilm was further assessed by using scanning electron microscopy and confocal laser scanning microscopy. The results showed that DQC was particularly effective in the destruction of BV-associated Gardnerella spp. biotypes, impacting both their biomass and metabolic activity. In addition, the disruption of biofilm architecture was evident and was probably caused by multiple mechanisms of action. We conclude that DQC is an antibiofilm agent and is able to efficiently destroy Gardnerella spp. BV-associated biofilms. Therefore, it is a valid option for BV therapy and has the potential to prevent BV recurrences.

Species Distribution and Antifungal Susceptibility Profiles of Isolates from Women with Nonrecurrent and Recurrent Vulvovaginal Candidiasis.

Recurrent vulvovaginal candidiasis (RVVC) is caused by Candida spp., a vaginal colonizer. Despite the clinical importance of RVVC, little is known regarding the characteristics of the disease in Portugal. Thirty-six clinical cases were analyzed, comprising 93 yeast vulvovaginal isolates obtained from women attending a gynecologic consultation at a private clinic. Of these, 18 women were diagnosed with RVVC, while other 18 women had a sporadic episode of infection (nonrecurrent vulvovaginal candidiasis [NR-VVC]). Species identification was performed with CHROMagar chromogenic medium and by analysis of biochemical profiles. In addition, antifungal susceptibility testing for two azole compounds was performed by broth microdilution. We found that Candida albicans was isolated from both NR-VVC and RVVC cases, being highly predominant; C. glabrata and C. tropicalis were also isolated. Resistance to at least one antifungal was detected in up to 65% of the isolates, and resistance to both antifungals reached a frequency of 25%. Moreover, azole-resistant isolates were distributed among all species identified. We conclude that in the studied group of patients, C. albicans is in fact the major player both in NR-VVC and in RVVC, C. glabrata being more frequently associated with recurrence (p < 0.05). In addition, we found a high proportion of azole-resistant strains.

Sodium bicarbonate gels: a new promising strategy for the treatment of vulvovaginal candidosis.

Vulvovaginal candidosis (VVC), caused mainly by the yeast Candida albicans, is the second most prevalent vaginal infection. It has been found to have a large impact on women's quality of life, self-esteem and routines. The prevalence of recurrent vulvovaginal candidosis (RVVC) remains high so the development of alternative treatments is needed. The main objective of this study was to develop and characterize sodium bicarbonate gels to treat VVC. We described key formulation characteristics and analyzed their influence on in vitro performance evaluations. The potential to inhibit Candida albicans's growth, the pH, osmolality, viscosity and rheological performance in contact with vaginal fluid simulant and the bioadhesion's profile (using a vaginal ex vivo porcine model) were studied for all formulations. Among the formulations, formulation C (5% sodium bicarbonate, 1% carbomer and 94% water) was the most effective in inhibiting the C. albicans' growth. This gel presented the same potential (the same MIC 2.5%) to inhibit other etiological agents of VVC (C. glabrata, C. krusei, C. tropicalis and C. parapsilosis) for all species tested. Additionally, sensorial characteristics of gel C were in accord with users' preferences. This gel exhibited physicochemical characteristics acceptable for short term treatments, suggesting good overall performance for the treatment of VVC. Furthermore, Gel C was biocompatible with the HeLa cell line (MTT assay) and was classified as a non-severe irritant in the HET-CAM assay (irritation score 4 ± 1). Overall, gel C was deemed the best performing of the set tested, and suitable for further development.

Recurrent vulvovaginal Candida spp isolates phenotypically express less virulence traits.

OBJECTIVE: Vulvovaginal candidosis (VVC) is a condition that impacts the quality of life of women worldwide. At least 5-8% of all VVC cases re-occur. Recurrent vulvovaginal candidosis (RVVC) can be defined as the occurrence of a VVC episode at least four times per year. The reasons for recurrence to occur are poorly understood. This work aims to identify key phenotypic traits associated with RVVC Candida spp. isolates that might be used to plan strategies to control RVVC. METHODS: The capacity to form biofilms (with the microtitration plate assay), to develop germinative tube in the presence of fetal bovine serum and to produce phospholipase (in the egg-yolk plate assay) was assessed for a collection of Candida spp. isolates obtained from 17 women diagnosed with RVVC and 16 women with non-recurrent VVC (VVC). The differences obtained regarding the proportion of isolates expressing each virulence factor was assessed by statistical analysis (χ2). RESULTS: We found that C. albicans isolates had a higher ability to form germinative tubes than RVVC isolates (29% vs 4%, p < 0.05). In addition, the ability of Candida spp. isolates to form biofilm (63% vs 51%) and to produce phospholipase (13% vs 11%) was also higher, though not statistically different (p > 0.05). CONCLUSIONS: We conclude that biofilm formation and phenotypic-switching associated with germinative tube production are particularly important C. albicans virulence factors for acute, sporadic VVC cases.

Semen supports growth of Candida albicans: A putative risk factor for recurrence of vulvovaginal infections?

AIM: Vulvovaginal candidosis (VVC) is the second most common vaginal infection (20-25%), and about 90% of all VVC cases are caused by Candida albicans. Unprotected sexual intercourse has been implicated as one of the risk factors that lead to an outbreak of VVC. To further investigate the relevance of this particular risk factor, in this study, we aim to evaluate the effect of human semen in the promotion of the growth of C. albicans. METHODS: The disposable amount of 41 samples of semen obtained from infertility patients were included in this study, with informed consent. The spermogram and physical characteristics of the samples were performed at the Unit; this information was provided with the anonymity of the samples. Samples were inoculated with a calibrated suspension of C. albicans ATCC 10231 in culture media. After the incubation time, C. albicans CFU/mL was determined. RESULTS: We found that semen allowed the growth of C. albicans (4.30 ± 1.00 CFU/mL), but not as much as the culture medium (9.45 ± 1.90 CFU/mL). Interestingly, we found that the increase in viscosity impaired significantly C. albicans growth. In addition, in what respects to the rate of multiplication of C. albicans in semen, we observed two different trends. However, we found no relation between these and the physical characteristics of the semen samples in which these behaviors were differently observed. CONCLUSION: Semen has the ability to sustain C. albicans growth, but further studies are needed to elucidate its role in VVC.

Development and validation of a new one step Multiplex-PCR assay for the detection of ten Lactobacillus species.

Lactobacillus sp. are well-known colonizers of human mucosa and frequently used as probiotics. Accurate species identification is crucial both for fundamental studies and biotechnology applications; however, it has been thus far challenging. The aim of this work was to develop a one-step multiplex-PCR assay for detection of ten Lactobacillus species (L. jensenii, L. fermentum, L. acidophilus, L. crispatus, L. reuteri, L. iners, L. casei, L. gasseri, L. plantarum, L. rhamnosus) directly in complex bacterial genomic DNA. A multiplex-PCR assay was optimized based on Box-Behnken experimental design, which showed to be efficient for optimization of all crucial reaction components. Nineteen Lactobacillus strains, including six collection strains and thirteen human isolates were used in order to verify the specificity and sensitivity of the assay. In addition, a set of PCR adjuvants was introduced to remove non-specific amplifications and enhance reaction yield. Among them, Triton™ X-100, Tween® 20, BSA, and dithiothreitol showed beneficial effects when compared with other adjuvants. The application of the developed method to samples that resulted from the mixing of DNA from the ten strains, resulted in amplicons of the expected sizes (from about 100 to 1000 bp). The detection limit was 1.25 ng/µl for all species with the exception of L. gasseri (0.31 ng/µl). In order to confirm the method applicability on human samples, ten vaginal fluids were enrolled in this study showing that the method can be successfully used on these biological materials. The proposed multiplex-PCR assay was shown to be selective, sensitive and efficient for detection of ten Lactobacillus species directly in human vaginal samples. This method provides a cost-effective and accessible methodology applicable to the detection of Lactobacillus species to different environments. At the same time, this approach represents a considerable improvement over other PCR-based approaches for identification of these species.

Evolutionary Origin of the Staphylococcal Cassette Chromosome mec (SCCmec).

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the ß-lactam resistance gene mecA However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCCmec, we sequenced the genomes of 106 isolates, comprising S. sciuri (n = 76), Staphylococcus vitulinus (n = 18), and Staphylococcus fleurettii (n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCCmec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCCmec: S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCCmec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus.

Evidence for the evolutionary steps leading to mecA-mediated ß-lactam resistance in staphylococci.

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA-an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all ß-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the ß-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to ß-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of ß-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics.