Image_1_Antimicrobial activity of bovine lactoferrin against Gardnerella species clinical isolates.TIF

Gardnerella species play a key role in the development and recurrence of Bacterial Vaginosis (BV), a common imbalance of the vaginal microbiota. Because of the high rates of BV recurrence reported after treatment with standard of care antibiotics, as well as the emergence of antibiotic-resistant BV, the development of alternative treatment approaches is needed. Bovine lactoferrin, a well studied iron-binding glycoprotein with selective antimicrobial activity, may ameliorate vaginal dysbiosis either alone or in combination with antibiotics. The present study evaluated the antimicrobial resistance/susceptibility profile of seventy-one presumptive G. vaginalis clinical isolates to metronidazole and clindamycin. In addition, the in vitro antimicrobial activity of Metrodora Therapeutics bovine Lactoferrin (MTbLF) against the tested clinical isolates, both alone and in combination with metronidazole and clindamycin, was in depth evaluated using defined-iron culture conditions. All 71 presumptive G. vaginalis clinical isolates exhibited resistance to metronidazole, with MIC values greater than 256 μg/ml. Different susceptibility profiles were detected for clindamycin. In detail, the vast majority of the tested strains (45%), exhibiting MIC lower than 2 μg/ml, were considered sensitive; 18 strains (25%) with MIC higher or equal to 8 μg/ml, were classified as resistant, whereas the remaining 21 (30%) were classified as intermediate. MTbLF was tested in culture medium at different concentrations (32, 16, 8, 4, 2, 1, and 0.5 mg/ml) showing ability to inhibit the growth of the tested presumptive G. vaginalis clinical isolates, including those metronidazole-resistant, in a dose-dependent and not in a strain-dependent manner. MTbLF, at concentrations ranging from 32 to 8 mg/ml, exerted a statistically different antimicrobial activity compared with lower concentrations (4, 2, 1, and 0.5 mg/ml). A synergistic effect between MTbLF (8 and 4 mg/ml) and clindamycin was revealed for all the tested strains. When tested in the absence of other sources of iron, MTbLF did not support the growth of the tested presumptive G. vaginalis clinical isolates. Bovine lactoferrin may be a potential candidate to treat Gardnerella species infection.</p

Image_1_Fermentation of Nocellara Etnea Table Olives by Functional Starter Cultures at Different Low Salt Concentrations.TIF

<p>Nocellara Etnea is one of the main Sicilian cultivars traditionally used to produce both olive oil and naturally fermented table olives. In the present study, the effect of different salt concentrations on physico-chemical, microbiological, sensorial, and volatile organic compounds (VOCs) formation was evaluated in order to obtain functional Nocellara Etnea table olives. The experimental design consisted of 8 treatments as follow: fermentations at 4, 5, 6, and 8% of salt with (E1-E4 samples) and without (C1-C4 samples) the addition of starters. All the trials were carried out at room temperature (18 ± 2°C) and monitored for an overall period of 120 d. In addition, the persistence of the potential probiotic Lactobacillus paracasei N24 at the end of the process was investigated. Microbiological data revealed the dominance of lactic acid bacteria (LAB), starting from the 7th d of fermentation, and the reduction of yeasts and enterobacteria in the final product inoculated with starters. VOCs profile highlighted a high amount of aldehydes at the beginning of fermentation, which significantly decreased through the process and a concomitant increase of alcohols, acids, esters, and phenols. In particular, esters showed an occurrence percentage higher in experimental samples rather than in control ones, contributing to more pleasant flavors. Moreover, acetic acid, ethanol, and phenols, which often generate off-flavors, were negatively correlated with mesophilic bacteria and LAB. It is interesting to note that salt content did not affect the performances of starter cultures and slightly influenced the metabolome of table olives. Sensory data demonstrated significant differences among samples registering the highest overall acceptability in the experimental sample at 5% of NaCl. The persistence of the L. paracasei N24 strain in experimental samples, at the end of the process, revealed its promising perspectives as starter culture for the production of functional table olives with reduced salt content.</p

Image_2_Antimicrobial activity of bovine lactoferrin against Gardnerella species clinical isolates.JPEG

Gardnerella species play a key role in the development and recurrence of Bacterial Vaginosis (BV), a common imbalance of the vaginal microbiota. Because of the high rates of BV recurrence reported after treatment with standard of care antibiotics, as well as the emergence of antibiotic-resistant BV, the development of alternative treatment approaches is needed. Bovine lactoferrin, a well studied iron-binding glycoprotein with selective antimicrobial activity, may ameliorate vaginal dysbiosis either alone or in combination with antibiotics. The present study evaluated the antimicrobial resistance/susceptibility profile of seventy-one presumptive G. vaginalis clinical isolates to metronidazole and clindamycin. In addition, the in vitro antimicrobial activity of Metrodora Therapeutics bovine Lactoferrin (MTbLF) against the tested clinical isolates, both alone and in combination with metronidazole and clindamycin, was in depth evaluated using defined-iron culture conditions. All 71 presumptive G. vaginalis clinical isolates exhibited resistance to metronidazole, with MIC values greater than 256 μg/ml. Different susceptibility profiles were detected for clindamycin. In detail, the vast majority of the tested strains (45%), exhibiting MIC lower than 2 μg/ml, were considered sensitive; 18 strains (25%) with MIC higher or equal to 8 μg/ml, were classified as resistant, whereas the remaining 21 (30%) were classified as intermediate. MTbLF was tested in culture medium at different concentrations (32, 16, 8, 4, 2, 1, and 0.5 mg/ml) showing ability to inhibit the growth of the tested presumptive G. vaginalis clinical isolates, including those metronidazole-resistant, in a dose-dependent and not in a strain-dependent manner. MTbLF, at concentrations ranging from 32 to 8 mg/ml, exerted a statistically different antimicrobial activity compared with lower concentrations (4, 2, 1, and 0.5 mg/ml). A synergistic effect between MTbLF (8 and 4 mg/ml) and clindamycin was revealed for all the tested strains. When tested in the absence of other sources of iron, MTbLF did not support the growth of the tested presumptive G. vaginalis clinical isolates. Bovine lactoferrin may be a potential candidate to treat Gardnerella species infection.</p

Image_3_Antimicrobial activity of bovine lactoferrin against Gardnerella species clinical isolates.JPEG

Gardnerella species play a key role in the development and recurrence of Bacterial Vaginosis (BV), a common imbalance of the vaginal microbiota. Because of the high rates of BV recurrence reported after treatment with standard of care antibiotics, as well as the emergence of antibiotic-resistant BV, the development of alternative treatment approaches is needed. Bovine lactoferrin, a well studied iron-binding glycoprotein with selective antimicrobial activity, may ameliorate vaginal dysbiosis either alone or in combination with antibiotics. The present study evaluated the antimicrobial resistance/susceptibility profile of seventy-one presumptive G. vaginalis clinical isolates to metronidazole and clindamycin. In addition, the in vitro antimicrobial activity of Metrodora Therapeutics bovine Lactoferrin (MTbLF) against the tested clinical isolates, both alone and in combination with metronidazole and clindamycin, was in depth evaluated using defined-iron culture conditions. All 71 presumptive G. vaginalis clinical isolates exhibited resistance to metronidazole, with MIC values greater than 256 μg/ml. Different susceptibility profiles were detected for clindamycin. In detail, the vast majority of the tested strains (45%), exhibiting MIC lower than 2 μg/ml, were considered sensitive; 18 strains (25%) with MIC higher or equal to 8 μg/ml, were classified as resistant, whereas the remaining 21 (30%) were classified as intermediate. MTbLF was tested in culture medium at different concentrations (32, 16, 8, 4, 2, 1, and 0.5 mg/ml) showing ability to inhibit the growth of the tested presumptive G. vaginalis clinical isolates, including those metronidazole-resistant, in a dose-dependent and not in a strain-dependent manner. MTbLF, at concentrations ranging from 32 to 8 mg/ml, exerted a statistically different antimicrobial activity compared with lower concentrations (4, 2, 1, and 0.5 mg/ml). A synergistic effect between MTbLF (8 and 4 mg/ml) and clindamycin was revealed for all the tested strains. When tested in the absence of other sources of iron, MTbLF did not support the growth of the tested presumptive G. vaginalis clinical isolates. Bovine lactoferrin may be a potential candidate to treat Gardnerella species infection.</p

API 50CH fermentative profile of <i>L. rhamnosus</i> strains.

<p>Fermentation ability is indicated in black for positive, grey for partially positive and white for negative. Strains are organized according to their genetic relatedness as defined in the hierarchical clustering and coloured according to their respective niche/origin (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-g001" target="_blank">Figure 1</a>). Carbohydrates of interest are marked by a red asterisk. Black arrows show fermentative profile shifts among <i>L. rhamnosus</i> strains.</p

Analysis of genome diversity in <i>L. rhamnosus</i> by mapped SOLiD sequencing.

<p>The 100 <i>L. rhamnosus</i> strains were clustered using hierarchical clustering <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen.1003683-Sturn1" target="_blank">[78]</a> based on their relative shared gene content with <i>L. rhamnosus</i> GG. Strain names were colour-coded as follows: green for dairy isolates, purple for intestinal isolates, orange for oral isolates, magenta for vaginal isolates and blue for clinical/other isolates. Four main groups or clusters were highlighted and numbered. The <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-g001" target="_blank">Figure 1</a> also shows the 17 variable chromosomal regions identified in GG, as further detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-t001" target="_blank">Table 1</a>. Each row corresponds to one strain, and each column shows the genes in these variable regions, colour-coded as follows: blue for present and yellow for absent.</p

CRISPR spacer oligotyping and CRISPR-associated protein diversity in <i>L. rhamnosus</i> species.

<p>Panel (A) illustrates the genetic organization of the CRISPR system and its associated genes in <i>L. rhamnosus</i> GG. Panel (B) shows the conservation (blue), the partial conservation (grey) or the absence (yellow) of <i>L. rhamnosus</i> GG spacers. The presence (green) or the absence (red) of the <i>cas</i> genes is also indicated in Panel (C). Strains are organized according to their genetic relatedness defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-g001" target="_blank">Figure 1</a>.</p

Mucus adhesion and SpaCBA pili gene diversity among <i>L. rhamnosus</i>.

<p>Panel (A) shows the genotype and phenotype of all strains. Based on our genomic analysis, pilin and sortase genes were assigned as present (green) or divergent (red). Sequences of corresponding genes were further analyzed using blastx. The sequence identity was shown by an upper triangle superposed to the SOLiD genomic data, where the colour gradient corresponds to the identity percentage to GG pili genes. We also indicated if the strains were tested by immunoblotting analysis (DB), electron microscopy (EM) or <i>in vitro</i> competitive binding assay (AB). Green is for pili positive and red for pili negative. Panel (B) shows the human mucus binding ability (%) of all <i>L. rhamnosus</i> isolates ranked from the lowest to the highest mucus binder.</p

Features of the variable chromosomal regions found in <i>L. rhamnosus</i>.

<p>Features of the variable chromosomal regions found in <i>L. rhamnosus</i>.</p

Pilosotype distribution in our <i>L. rhamnosus</i> collection.

<p>The table describes the niches or isolation sources, the number of strains per group and their pilosotype, <i>i.e.</i> the presence of an intact and functional SpaCBA pili cluster as determined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003683#pgen-1003683-g006" target="_blank">Figure 6</a>. Probiotic strains GG, VIFIT, IDOF, AK-RO and CO-RO were classified as intestinal isolates. The group ‘Others’ contained strains of unspecified origins (clinical specimens) or from minor isolation source (<i>n</i><2), <i>i.e.</i> hip punction or pus.</p