Neighbour-Joining phylogenetic tree based on divergences of ribosomal 5.8S-ITS sequences.

<p>The alignment of ribosomal 5.8S-ITS sequences from 10 isolates and 10 reference strains of <i>Aspergillus</i> spp. was performed using the CLUSTALΩ program. Nucleotide divergences were estimated according to the Jukes–Cantor model. Node numbers represent the frequency (proportion) with which a cluster appears in 1000 bootstrap runs.</p

Alignment of the ribosomal 5.8S-ITS amplicon sequences of representative <i>Aspergilli</i> isolates.

<p>A part of the amplicon sequences (nucleotides 101 to 200) alignment is presented. Restriction sites are presented as bold-underlined, and variable nucleotides are highlighted. The extra <i>Hha</i>I restriction site present in the Ai-1 sequence is also shown.</p

5.8S-ITS RFLP DNA fragment sizes profiles.

<p>Individual isolates and reference strain isolates from grapes are clustered according to their RFLP profile deduced after sequencing of the 5.8S-ITS amplicon. Fragment sizes generated respect to the restriction endonuclease site position in the sequence, are given in bp.</p

<i>Aspergillus</i> section <i>Nigri</i> strains distribution.

<p><i>A</i>. section <i>Nigri</i> distribution in total mycoflora (bars) and different species distribution within the <i>A</i>. section <i>Nigri</i> group (percentages inside bars) for each sampled prefecture. The percentages underneath prefectures' names refer to distribution and incidence of <i>A</i>. section <i>Nigri</i>.</p

Identification, origin and OTA production of <i>A</i>. section <i>Nigri</i> isolates.

<p>Species identification and origin of <i>A</i>. section <i>Nigri</i> spp. isolated from the present study and their ochratoxigenic potential assayed by HPLC and ELISA methods.</p><p>Limit of Quantification (LOQ) 2 ng OTA g⁻¹ CYA and Limit of Detection (LOD) 1 ng OTA g⁻¹ CYA.</p

Restriction digestion patterns of sequenced ribosomal 5.8S-ITS region amplicons.

<p>Restriction digestion patterns (designated as <b>A</b>, <b>B</b>, <b>C</b> and <b>D</b>) of five sequenced ribosomal 5.8S-ITS DNA amplicons from five different <i>Aspergilli</i> grape isolates (presented as isolate designations), after digestion with the restriction endonucleases <b><i>Hha</i></b><b>I</b>, <b><i>Hinf</i></b><b>I</b> and <b><i>Rsa</i></b><b>I</b>. Each isolate is a representative of the five different <i>Aspergillus</i> species characterized in this study. <b>L</b>: DNA ladder.</p

Correlation of HPLC and ELISA values for OTA determination.

<p>Linear correlation and <i>R²</i> for the two methods assayed for OTA quantification (ELISA & HPLC).</p

Ribosomal 5.8S-ITS region restriction digestion patterns of <i>Aspergillus</i> grape isolates.

<p>Restriction digestion patterns (designated as <b>A</b>, <b>B</b> and <b>C</b>) of ribosomal 5.8S-ITS DNA amplicons from various <i>Aspergilli</i> grape isolates (presented as isolate designations), after digestion with the restriction endonucleases <b><i>Hha</i></b><b>I</b> and <b><i>Hinf</i></b><b>I</b>. 50 bp <b>(</b><b><i>l</i></b><b>)</b> and 100 bp <b>(L)</b> DNA ladders are also shown.</p

Investigating the Effect of Different Treatments with Lactic Acid Bacteria on the Fate of <i>Listeria monocytogenes</i> and <i>Staphylococcus aureus</i> Infection in <i>Galleria mellonella</i> Larvae

<div><p>The use of <i>Galleria mellonella</i> as a model host to elucidate microbial pathogenesis and search for novel drugs and therapies has been well appreciated over the past years. However, the effect of microorganisms with functional appeal in the specific host remains scarce. The present study investigates the effect of treatment with selected lactic acid bacteria (LAB) with probiotic potential, as potential protective agents by using live or heat-killed cells at 6 and 24 h prior to infection with <i>Listeria monocytogenes</i> and <i>Staphylococcus aureus</i> or as potential therapeutic agents by using cell-free supernatants (CFS) after infection with the same pathogens. The employed LAB strains were <i>Lactobacillus pentosus</i> B281 and <i>Lactobacillus plantarum</i> B282 (isolated from table olive fermentations) along with <i>Lactobacillus rhamnosus</i> GG (inhabitant of human intestinal tract). Kaplan-Meier survival curves were plotted while the pathogen’s persistence in the larval hemolymph was determined by microbiological analysis. It was observed that the time (6 or 24 h) and type (live or heat-killed cells) of challenge period with LAB prior to infection greatly affected the survival of infected larvae. The highest decrease of <i>L</i>. <i>monocytogenes</i> population in the hemolymph was observed in groups challenged for 6 h with heat-killed cells by an average of 1.8 log units compared to non challenged larvae for strains B281 (<i>p</i> 0.0322), B282 (<i>p</i> 0.0325), and LGG (<i>p</i> 0.0356). In the case of <i>S</i>. <i>aureus</i> infection, the population of the pathogen decreased in the hemolymph by 1 log units at 8 h post infection in the groups challenged for 6 h with heat-killed cells of strains B281 (<i>p</i> 0.0161) and B282 (<i>p</i> 0.0096) and by 1.8 log units in groups challenged with heat-killed cells of LGG strain (<i>p</i> 0.0175). Further use of CFS of each LAB strain did not result in any significant prolonged survival but interestingly it resulted in pronounced decrease of <i>L</i>. <i>monocytogenes</i> in the hemolymph at 24 h and 48 h after infection by more than 1 log unit (<i>p</i> < 0.05) depending on the strain. The results of the present work support the broader use of <i>G</i>. <i>mellonella</i> larvae as a low cost <i>in vivo</i> tool for screening for probiotic properties.</p></div

Population dynamics of <i>L</i>. <i>monocytogenes</i> in the host’s hemolymph.

<p>Larvae were injected with live (A,C) or heat-killed (B,D) cells of <i>L</i>. <i>pentosus</i> B281 (white bars), <i>L</i>. <i>plantarum</i> B282 (dotted bars) and <i>L</i>. <i>rhamnosus</i> LGG (grey bars). Infection with the pathogen took place at 6 (A, B) and 24 h (C, D) post LAB administration. Black bars correspond to the control group. Bars with asterisks show groups with significant differences in comparison with the control group with a <i>p</i>-value ≤ 0.05. A total of 30–35 larvae were used for each treatment in order to determine the population of the pathogen in the hemolymph.</p