Virulence Gene Sequencing Highlights Similarities and Differences in Sequences in Listeria monocytogenes Serotype 1/2a and 4b Strains of Clinical and Food Origin From 3 Different Geographic Locations

peer-reviewedThe Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018.01103/full#supplementary-materialThe prfA-virulence gene cluster (pVGC) is the main pathogenicity island in Listeria monocytogenes, comprising the prfA, plcA, hly, mpl, actA, and plcB genes. In this study, the pVGC of 36 L. monocytogenes isolates with respect to different serotypes (1/2a or 4b), geographical origin (Australia, Greece or Ireland) and isolation source (food-associated or clinical) was characterized. The most conserved genes were prfA and hly, with the lowest nucleotide diversity (π) among all genes (P < 0.05), and the lowest number of alleles, substitutions and non-synonymous substitutions for prfA. Conversely, the most diverse gene was actA, which presented the highest number of alleles (n = 20) and showed the highest nucleotide diversity. Grouping by serotype had a significantly lower π value (P < 0.0001) compared to isolation source or geographical origin, suggesting a distinct and well-defined unit compared to other groupings. Among all tested genes, only hly and mpl were those with lower nucleotide diversity in 1/2a serotype than 4b serotype, reflecting a high within-1/2a serotype divergence compared to 4b serotype. Geographical divergence was noted with respect to the hly gene, where serotype 4b Irish strains were distinct from Greek and Australian strains. Australian strains showed less diversity in plcB and mpl relative to Irish or Greek strains. Notable differences regarding sequence mutations were identified between food-associated and clinical isolates in prfA, actA, and plcB sequences. Overall, these results indicate that virulence genes follow different evolutionary pathways, which are affected by a strain's origin and serotype and may influence virulence and/or epidemiological dominance of certain subgroups.This study was supported by the 7th Framework Programme projects PROMISE, contract number 265877

Inhibition of Listeria monocytogenes Growth, Adherence and Invasion in Caco-2 Cells by Potential Probiotic Lactic Acid Bacteria Isolated from Fecal Samples of Healthy Neonates

Lactic acid bacteria (LAB) isolated from healthy humans may prove an effective tool against pathogen growth, adherence and invasion in intestinal epithelial cells. This study aimed to evaluate the antilisterial properties of LAB isolated from fecal samples of healthy neonates. Forty-five LAB strains were tested for their antimicrobial activity against ten Listeria monocytogenes strains with spot-on-lawn and agar-well diffusion assays, and ten lactobacilli strains were further assessed for their inhibitory effect against adherence and invasion of Caco-2 cells by L. monocytogenes EGDe. Inhibition was estimated in competition, exclusion or displacement assays, where lactobacilli and L. monocytogenes were added to Caco-2 monolayers simultaneously or 1 h apart from each other. Inhibition of L. monocytogenes growth was only displayed with the spot-on-lawn assay; cell-free supernatants of lactobacilli were not effective against the pathogen. Lactobacillus (L.) paragasseri LDD-C1 and L. crispatus LCR-A21 were able to adhere to Caco-2 cells at significantly higher levels than the reference strain L. rhamnosus GG. The adherence of L. monocytogenes to Caco-2 cells was reduced by 20.8% to 62.1% and invasion by 33.5% to 63.1% during competition, which was more effective compared to the exclusion and displacement assays. These findings demonstrate that lactobacilli isolated from neonatal feces could be considered a good candidate against L. monocytogenes

Inhibition of <i>Listeria monocytogenes</i> Growth, Adherence and Invasion in Caco-2 Cells by Potential Probiotic Lactic Acid Bacteria Isolated from Fecal Samples of Healthy Neonates

Lactic acid bacteria (LAB) isolated from healthy humans may prove an effective tool against pathogen growth, adherence and invasion in intestinal epithelial cells. This study aimed to evaluate the antilisterial properties of LAB isolated from fecal samples of healthy neonates. Forty-five LAB strains were tested for their antimicrobial activity against ten Listeria monocytogenes strains with spot-on-lawn and agar-well diffusion assays, and ten lactobacilli strains were further assessed for their inhibitory effect against adherence and invasion of Caco-2 cells by L. monocytogenes EGDe. Inhibition was estimated in competition, exclusion or displacement assays, where lactobacilli and L. monocytogenes were added to Caco-2 monolayers simultaneously or 1 h apart from each other. Inhibition of L. monocytogenes growth was only displayed with the spot-on-lawn assay; cell-free supernatants of lactobacilli were not effective against the pathogen. Lactobacillus (L.) paragasseri LDD-C1 and L. crispatus LCR-A21 were able to adhere to Caco-2 cells at significantly higher levels than the reference strain L. rhamnosus GG. The adherence of L. monocytogenes to Caco-2 cells was reduced by 20.8% to 62.1% and invasion by 33.5% to 63.1% during competition, which was more effective compared to the exclusion and displacement assays. These findings demonstrate that lactobacilli isolated from neonatal feces could be considered a good candidate against L. monocytogenes

Estimates of Weibull model for inactivation curves during acid challenge, using HCl, for 6 h at 25°C.

<p>Estimates of Weibull model for inactivation curves during acid challenge, using HCl, for 6 h at 25°C.</p

Parameter estimates of Weibull model for inactivation curves during osmotic stress, in TSB containing 20% (w/v) NaCl, at 25°C.

<p>Parameter estimates of Weibull model for inactivation curves during osmotic stress, in TSB containing 20% (w/v) NaCl, at 25°C.</p

<i>L</i>. <i>monocytogenes</i> heat response after habituation on fresh produce.

<p>Survival (%) of <i>L</i>. <i>monocytogenes</i> population was determined after exposure to heat stress 60°C for 150 s. Cell cultures were previously habituated on lettuce, tomatoes, or in TSB, at 5°C for 24 h and 5 days, and in TSB for 18 h at 30°C (control). Values represent the mean of two independent experiments, with 3 technical replicates each (n = 6).</p

Growth of <i>L</i>. <i>monocytogenes</i> and total microbiota on fresh produce.

<p>Size of populations (log CFU/g) of <i>L</i>. <i>monocytogenes</i> and total microbiota on inoculated lettuce and tomatoes were determined following 24-h and 5-day storage at 5°C. Bars represent mean populations ± standard error mean of eight independent experiments with three technical replicates each (n = 24). Significant differences (<i>P</i> < 0.05) between 24-h and 5-day stored samples are shown as (*).</p

Parameter estimates of Weibull model for inactivation curves during heat stress, carried out in TSB at 60°C, for 150 s.

<p>Parameter estimates of Weibull model for inactivation curves during heat stress, carried out in TSB at 60°C, for 150 s.</p

<i>L</i>. <i>monocytogenes</i> acid stress survival after habituation on fresh produce.

<p>Survival (%) of <i>L</i>. <i>monocytogenes</i> population was determined after exposure to lactic acid (A-B); acetic acid (C-D); HCl at pH 3.5 (E-F); and HCl at pH 1.5 (G-H), for 6 h. Cell cultures were previously habituated on lettuce, tomatoes, or in TSB, at 5°C for 24 h and 5 days, and in TSB for 18 h at 30°C (control). Values represent the mean of two independent experiments, with 3 technical replicates each (n = 6).</p