Ana Filipa Frutuoso Mendes Henriques

Fluorescence in situ hybridization method using peptide nucleic acid probes for rapid detection of Lactobacillus and Gardnerella spp.

BackgroundBacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples.ResultsTherefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH.ConclusionsThis quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.

Isolation and characterization of bacteriophages for avian pathogenic E. coli strains

Aims:  To isolate and characterize bacteriophages, and to evaluate its lytic performance against avian pathogenic Escherichia coli (APEC) strains with high patterns of antibiotic resistance, in order to select phages for a therapeutic product to treat colibacillosis in chickens.

Reciprocal Interference between Lactobacillus spp. and Gardnerella vaginalis on Initial Adherence to Epithelial Cells

Bacterial vaginosis (BV) is the most common vaginal disorder in women of child-bearing age. It is widely accepted that the microbial switch from normal microflora to the flora commonly associated with BV is characterized by a decrease in vaginal colonization by specific Lactobacillus species together with an increase of G. vaginalis and other anaerobes. However, the order of events leading to the development of BV remains poorly characterized and it is unclear whether the decrease in lactobacilli is a cause or a consequence of the increase in the population density of anaerobes. Our goal was to characterize the interaction between two Gardnerella vaginalis strains, one of which was isolated from a healthy woman (strain 5-1) and the other from a woman diagnosed with BV (strain 101), and vaginal lactobacilli on the adherence to cervical epithelial cells. In order to simulate the transition from vaginal health to BV, the lactobacilli were cultured with the epithelial cells first, and then the G. vaginalis strain was introduced. We quantified the inhibition of G. vaginalis adherence by the lactobacilli and displacement of adherent lactobacilli by G. vaginalis. Our results confirmed that pathogenic G vaginalis 101 had a higher capacity for adhesion to the cervical epithelial cells than strain 5-1. Interestingly, strain 101 displaced L. crispatus but not L. iners whereas strain 5-1 had less of an effect and did not affect the two species differently. Furthermore, L. iners actually enhanced adhesion of strain 101 but not of strain 5-1. These results suggest that BV-causing G. vaginalis and L. iners do not interfere with one another, which may help to explain previous reports that women who are colonized with L. iners are more likely to develop BV.

Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.

In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp.

BackgroundBacterial vaginosis (BV) is a common pathology of women in reproductive age that can lead to serious health complications, and is associated with shifts in the normal microflora from predominance of Lactobacillus spp. to a proliferation of other anaerobes such as G. vaginalis and A vaginae, which can be detected by PCR. The optimal PCR pathogen detection assay relies mainly on the specificity and sensitivity of the primers used.FindingsHere we demonstrate that in silico analytical testing of primer specificity is not a synonym to in vitro analytical specificity by testing a range of published and newly designed primers with both techniques for the detection of BV-associated microorganisms.ConclusionsBy testing primer in vitro specificity with a sufficient range of bacterial strains, we were able to design primers with higher specificity and sensitivity. Also by comparing the results obtained for the newly designed primers with other previously published primers, we confirmed that in silico analysis is not sufficient to predict in vitro specificity. As such care must be taken when choosing the primers for a detection assay.

Synergistic Antimicrobial Interaction between Honey and Phage against Escherichia coli Biofilms

Chronic wounds afford a hostile environment of damaged tissues that allow bacterial proliferation and further wound colonization. Escherichia coli is among the most common colonizers of infected wounds and it is a prolific biofilm former. Living in biofilm communities, cells are protected, become more difficult to control and eradicate, and less susceptible to antibiotic therapy. This work presents insights into the proceedings triggering E. coli biofilm control with phage, honey, and their combination, achieved through standard antimicrobial activity assays, zeta potential and flow cytometry studies and further visual insights sought by scanning electron microscopy and transmission electron microscopy. Two Portuguese honeys (PF2 and U3) with different floral origin and an E. coli-specific phage (EC3a), possessing depolymerase activity, were tested against 24- and 48-h-old biofilms. Synergic and additive effects were perceived in some phage–honey experiments. Combined therapy prompted similar phenomena in biofilm cells, visualized by electron microscopy, as the individual treatments. Honey caused minor membrane perturbations to complete collapse and consequent discharge of cytoplasmic content, and phage completely destroyed cells leaving only vesicle-like structures and debris. Our experiments show that the addition of phage to low honey concentrations is advantageous, and that even fourfold diluted honey combined with phage, presents no loss of antibacterial activity toward E. coli. Portuguese honeys possess excellent antibiofilm activity and may be potential alternative therapeutic agents in biofilm-related wound infection. Furthermore, to our knowledge this is the first study that assessed the impacts of phage–honey combinations in bacterial cells. The synergistic effect obtained was shown to be promising, since the antiviral effect of honey limits the emergence of phage resistant phenotypes.

Prevalence of Gardnerella vaginalis and Atopobium vaginae in Portuguese women and association with risk factors for bacterial vaginosis

Bacterial vaginosis is the most common clinical condition among women of reproductive age. It is characterized by a shift in normal vaginal flora and the overgrowth of anaerobicmicroorganisms [1]. Bacterial vaginosis is a risk factor for the development of pregnancy complications and sexually transmitted diseases [2]. Initially, it was described as a sexually transmitted infection caused by Gardnerella vaginalis [3] but subsequent studies revealed that other microorganisms are associated with bacterial vaginosis [1], forming multispecies biofilms comprising mainly G. vaginalis and Atopobium vaginae [4]. There are no Portuguese studies related to vaginal colonization by bacterial vaginosis-associated microorganisms; thus, the aim of the present study was to determine the prevalence of bacterial vaginosis and of G. vaginalis and A. vaginae in Portuguese women and to correlate the presence of G. vaginalis and A. vaginae with risk factors for bacterial vaginosis. Portuguese women were invited to participate in the study by completing an anonymous questionnaire assessing their behavioral risk factors and by collecting a self-sample of vaginal exudates. Study approval was provided by the institutional review board of the University of Minho, Braga, Portugal; all participants providedwritten informed consent. Previously describedmolecularmethods [5] were used to detectG. vaginalis and A. vaginae in the collected samples. Statistical analyses

Doctor's perception on bacterial vaginosis in Portugal: prevalence, diagnostic methods and choice of treatment

Bacterial vaginosis (BV) is one of the most common vaginal disorders in women of reproductive age and has been linked to increased risks of preterm labour in pregnant women, HIV and postoperative infections and inflammatory pelvic disease.1 Studies performed in different countries around the world showed that the prevalence of BV varies with geographical location, socioeconomic status and race.1 Data regarding the prevalence of BV in Portugal are almost non-existent, being limited to a paper …

Synergistic antimicrobial interaction of honey and bacteriophage in Escherichia coli biofilms

Acknowledgements Wound colonization by biofilms-forming bacteria is one of the main obstacles to the treatment of chronic wounds, causing a number of biological and financial problems. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymeric matrix, adhered to inert or living surfaces, blocking antibiotics and patient’s immune cells from reaching bacteria. Bacterio(phages), viruses infecting exclusively bacteria, and honey are being considered as valuable alternatives to treat a variety of infections (Table 1). Phages are harmless to mammalian cells, specific for target bacteria therefore not affecting commensal microflora, have the ability to selfreplicate as long as the host is present, and are effective against antibiotic resistant bacteria. Honey is a complex substance with broad spectrum antimicrobial activity, essentially attributed to the high sugar content, low pH, the presence of hydrogen peroxide and methylglyoxal that reacts with important biological molecules (RNA, DNA and proteins). Honey had yet the potential to promote tissue regeneration, cicatrization, decrease inflammation, improving wound healing. In this work the combination of those two antimicrobial agents was considered in the control E. coli biofilms.