Influence of sub-inhibitory concentrations of antimicrobial agents on biofilm formation in indwelling medical devices

Biofilms of Staphyloccocus epidermidis and Candida spp. are two of the most frequent factors of infection and pathogenesis associated to the use of indwelling medical devices. Several strategies have been proposed and/or developed to prevent infection. The aim of this study was to compare the effect of sub-inhibitory concentrations of anti-microbial agents on biofilm formation. Biofilms of three strains of S. epidermidis and two of both Candida albicans and Candida dubliniensis where formed in the presence of three antibiotics and two antifungal agents respectively. Based in the control samples, the percentage of biofilm formation inhibition by the different agents was determined and compared. The results showed that the influence of the antibacterial and antifungal agents tested is strain dependent, with the effect of the different agents also varying among strains, even if they have the same mechanism of action.Fundação para a Ciência e a Tecnologia (FCT)

Biofilms of nosocomial microorganisms

The majority of nosocomial infections associated with the use of indwelling devices are caused by microorganisms of the genera Staphylococcus and Candida, being S. epidermidis and Candida albicans the predominant species. These species are able to adhere to abiotic and biotic surfaces and form biofilms (cellular communities embedded in a polymeric matrix) that are inherently tolerant to antimicrobial agents and host defences. Our work is focused on the mechanisms of biofilm formation on biomaterials and human tissue and on the virulence factors triggered by the microbial sessile mode of life. Particularly, we are interested on the physico-chemical mechanisms that rule microbial adhesion and the identification of the proteins involved in the adhesion process. We also study biofilm structure through CSLM and SEM observations and the composition of the biofilm-matrices [1]. As far as virulence is concerned we evaluate the antimicrobial tolerance of biofilm cells of different clinical isolates and the extent of expression of virulent molecules such as proteinases, lipases and intercellular-adhesion-polysaccharides. We are also studying the interaction of the biofilm cells with the immune system through in vitro and in vivo assays [2]. Finally, new antimicrobial agents such as bacteriophages and quorum sensing molecules are being tested against bacteria and yeast biofilms, respectively

Competitive initial adhesion between Lactobacillus spp. and Gardnerella vaginalis strains against vaginal epithelium

Bacterial vaginosis (BV) is a common disorder in women of reproductive age. BV is characterized by the replacement of vaginal lactobacilli, such as Lactobacillus crispatus, by predominantly anaerobic microorganisms. However, Lactobacillus iners is frequently found in the BV. Gardnerella vaginalis, commonly associated with BV, can also be present in 50-95% of BV patients and in 20-30% of healthy women. The capacity of G. vaginalis to form biofilms on the vaginal epithelium has recently been demonstrated. Our goal was to study the colonization of endogenous vaginal microflora from Lactobacillus spp. and G. vaginalis 5-1 (isolated from a healthy woman) and G. vaginalis 101 (isolated from a BV patient), at different initial concentrations and consequently to analyze the competition and interaction during the primary step of biofilm formation: initial adhesion. ME-180 and HeLa epithelial cell monolayers were challenged with the two G. vaginalis strains with different adhesion conditions. For the competition assays, cultures of Lactobacillus casei, Lactobacillus crispatus or Lactobacillus iners were mixed G. vaginalis strains at different concentrations and allowed to adhere to the two cell lines for 30 minutes. To analyse interference of lactobacilli in G. vaginalis initial adhesion, different lactobacilli concentrations were allowed to adhere to the cell monolayer for 4 hours and then G. vaginalis strains at different concentrations were added and allowed to adhere for 30 minutes. These adhesion times were previously optimized. All adhesion assays were quantified by fluorescence microscopy, using DAPI for total cell count and PNA-FISH probe for G. vaginalis quantification. Our results showed that G. vaginalis 101 (pathogenic strain) had a greater adhesion capacity than G. vaginalis 5-1 in all cases tested. Also, L. casei was the least adherent of the all lactobacilli used in this study. L. casei was included in this study as a non-sense control, since this lactobacilli strain is not a common colonizer of the vagina epithelium. L. crispatus showed decreased adherence to epithelial cells in the presence of G. vaginalis 101. In contrast, adherence of L. iners did not decrease in presence of G. vaginalis 101. Our study suggests that adherence of the G. vaginalis to epithelial cells is a critical step during the stage of vaginal colonization. It was found that adherence of Lactobacillus spp to epithelial cells was influenced by a specific G. vaginalis strains. These studies help to provide insight into the clinical situation in which indigenous vaginal lactobacilli can interfere with G. vaginalis presence

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.This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008) and in part by funds from the National Institutes of Health (P60-MD002256). AM acknowledges the FCT individual fellowship SFRH/BD/62375/2009)

Extracellular matrix in Staphylococcus epidermidis biofilms: a consequence of bacterial production or cell wall degradation?

Staphylococcus epidermidis is a leading pathogen accounting for nosocomial infections. The ability to form biofilms is considered the major virulence factor of this bacterium. The hallmark of this type of infection is the presence of an extracellular polymeric matrix that, in the case of S. epidermidis biofilms, is mainly constituted by an N-acetylglucosamine polymer. We have identified a subpopulation of bacteria that we believe to be the responsible for the extracellular matrix accumulation in S. epidermidis biofilms as they have comparative significant higher amount of surface N-acetylglucosamine. Flow cytometric evaluation of cell wall permeability and transmission electronic microscopy are highly suggestive of primary wall degradation in these bacteria. In overall, these results suggest that the extracellular matrix in S. epidermidis biofilms is a consequence of the degradation of the bacteria cell wall and that propide iodium should be used with care when used as a marker for bacteria dead in biofilms

Vaginal sheets with Thymbra capitata essential oil for the treatment of bacterial vaginosis: design, characterization and in vitro evaluation of efficacy and safety

We aimed to incorporate Thymbra capitata essential oil (TCEO), a potent antimicrobial natural product against bacterial vaginosis (BV)-related bacteria, in a suitable drug delivery system. We used vaginal sheets as dosage form to promote immediate relief of the typical abundant vaginal discharge with unpleasant odour. Excipients were selected to promote the healthy vaginal environment reestablishment and bioadhesion of formulations, while the TCEO acts directly on BV pathogens. We characterized vaginal sheets with TCEO in regard to technological characterization, predictable in vivo performance, in vitro efficacy and safety. Vaginal sheet D.O (acid lactic buffer, gelatine, glycerine, chitosan coated with TCEO 1% w/w) presented a higher buffer capacity and ability to absorb vaginal fluid simulant (VFS) among all vaginal sheets with EO, showing one of the most promising bioadhesive profiles, an excellent flexibility and structure that allow it to be easily rolled for application. Vaginal sheet D.O with 0.32 µL/mL TCEO was able to significantly reduce the bacterial load of all in vitro tested Gardnerella species. Although vaginal sheet D.O presented toxicity at some concentrations, this product was developed for a short time period of treatment, so this toxicity can probably be limited or even reversed when the treatment ends.This work supported by the Portuguese Foundation for Science and Technology within the research project PTDC/BIA-MIC/28271/2017 under the scope of COMPETE 2020 (POCI-01- 0145-FEDER-028271) including an individual scholarship and general funding. This work was also developed within the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES.info:eu-repo/semantics/publishedVersio

Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urothelium

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This work investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4 and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45 % reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium adhered bacteria

Staphylococcus epidermidis glucose uptake in biofilm versus planktonic cells

The aim of this work was to compare the glucose uptake of biofilms formed by four different Staphylococcus epidermidis strains as well as to compare between sessile and planktonic cells of the same strain. Biofilm cells showed a lower level of glucose uptake compared to planktonic cells. Moreover, glucose uptake by cells in the sessile form was strongly influenced by biofilm composition. Therefore, this work helps to confirm the phenotypic variability of S. epidermidis strains and the different behaviour patterns between sessile and free cells.Fundação para a Ciência e a Tecnologia (FCT) - POCTI/ESP/42688/2001; SFRH/BD/19265/2004

Genome size variation and polyploidy incidence in the alpine flora from Spain

The interest to study genome evolution, in particular genome size variation and polyploid incidence, has increased in recent years. Still, only a few studies have been focused at a community level. Of particular interest are high mountain species, because of the high frequency of narrow endemics and their higher susceptibility to extinction due to the effects of climate change. In the present study we explored genome size variation and polyploidy incidence in the entomophilous plant communities of two distinct mountain ranges, the Sierra Nevada and Picos de Europa National Parks. For that, chromosome numbers and DNA ploidies were assessed through a review of the literature, and the genome size and incidence of polyploidy in 39 taxa from several key genera were estimated using flow cytometry. In this study, first genome size estimations are given for 32 taxa. The majority of the analyzed taxa presented very small to small genome sizes (2C ≤ 7.0 pg), with no differences being detected between genome size and geographic origin and distribution ranges. A low incidence of polyploid taxa was observed (23.3%), with polyploids being more common in Picos de Europa than in Sierra Nevada. Most taxa inferred as polyploids were high altitude plants, but no clear pattern between polyploidy incidence and endemic status was observed. The obtained results are discussed within the context of angiosperm’s genome size variation and of polyploidy incidence in alpine and arctic flora, contributing to the scientific knowledge of these natural communities of great biological importance.This work was partially supported by the Organismo Autónomo de Parques Nacionales (014/2009 - POLPINE - Interacciones entre polinizadores y plantas alpinas: conservación de la biodiversidad en áreas protegidas de alta montaña). R. Torices was partially supported by a postdoctoral fellowship from Spanish Minister of Education (BVA 2010-0375)

Front Matter

The interest to study genome evolution, in particular genome size variation and polyploid incidence, has increased in recent years. Still, only a few studies have been focused at a community level. Of particular interest are high mountain species, because of the high frequency of narrow endemics and their higher susceptibility to extinction due to the effects of climate change. In the present study we explored genome size variation and polyploidy incidence in the entomophilous plant communities of two distinct mountain ranges, the Sierra Nevada and Picos de Europa National Parks. For that, chromosome numbers and DNA ploidies were assessed through a review of the literature, and the genome size and incidence of polyploidy in 39 taxa from several key genera were estimated using flow cytometry. In this study, first genome size estimations are given for 32 taxa. The majority of the analyzed taxa presented very small to small genome sizes (2C ≤ 7.0 pg), with no differences being detected between genome size and geographic origin and distribution ranges. A low incidence of polyploid taxa was observed (23.3%), with polyploids being more common in Picos de Europa than in Sierra Nevada. Most taxa inferred as polyploids were high altitude plants, but no clear pattern between polyploidy incidence and endemic status was observed. The obtained results are discussed within the context of angiosperm’s genome size variation and of polyploidy incidence in alpine and arctic flora, contributing to the scientific knowledge of these natural communities of great biological importance.El interés en el estudio de la evolución del genoma, especialmente de la variación en tamaño y de la incidencia de poliploidía, se ha incrementado en los últimos años. Sin embargo, sólo unos pocos estudios se han centrado en el nivel de comunidades. Las especies de alta montaña son especialmente interesantes debido a la alta frecuencia de especies endémicas y a que son consideradas muy susceptibles a la extinción por los efectos del cambio climático. En el presente estudio, exploramos la variación en el tamaño genómico y la incidencia de poliploidía en las comunidades de plantas entomófilas de alta montaña de dos macizos montañosos: el Parque Nacional de Sierra Nevada y el Parque Nacional de Picos de Europa. Para ello, se evaluó el número de cromosomas y el nivel de ploidía por medio de una revisión bibliográfica, mientras que el tamaño genómico y la incidencia de poliploidía se estimaron en 39 taxones de varios géneros usando citometría de flujo. En este estudio, se proporcionan las primeras estimaciones del tamaño genómico para 32 taxones. La mayoría de los taxones analizados presentaron tamaños genómicos pequeños o muy pequeños (2C ≤ 7.0 pg), sin mostrar diferencias en el tamaño genómico asociadas a su origen geográfico o rango de distribución. Se observó una baja incidencia de taxones poliploides (23.3%), siendo éstos más comunes entre las plantas de Picos de Europa que entre las de Sierra Nevada. La mayor parte de los taxones considerados como poliploides fueron plantas restringidas a las montañas, sin embargo no se observó un patrón claro entre la incidencia de poliploidía y el grado de endemismo. Los resultados obtenidos son discutidos dentro del contexto de variación en el tamaño del genoma y de la incidencia de poliploidía en las floras árticas y alpinas, contribuyendo al conocimiento científico de estas comunidades naturales de gran importancia biológica