Natalia Corbalan

<p>One hundred µL of serial dilutions (from 10<sup>−3</sup> to 10<sup>−8</sup>) from a stationary phase culture were plated in the specified solid medium (M9 agar, M9A or LB agar, LBA) and incubated overnight. The type of growth was observed: L, lawn; C, colony, NG, no growth.</p><p><sup>a</sup>+ Fe, indicate medium supplementation with 100 µM FeCl<sub>3.</sub></p>b<p>+ ASC indicate medium supplementation with 1 mM ascorbic acid.</p>c<p>+CAS indicate medium supplementation with 1% casamino acid.</p

The ability of siderophores to play roles beyond iron acquisition has been recently proven for many of them and evidence continues to grow. An earlier work showed that the siderophore enterobactin is able to increase copper toxicity by reducing Cu2+ to Cu+, a form of copper that is more toxic to cells. Copper toxicity is multifaceted. It involves the formation of reactive oxygen species (ROS), mismetallation of enzymes and possibly other mechanisms. Given that we previously reported on the capacity of enterobactin to alleviate oxidative stress caused by various stressors other than copper, we considered the possibility that the siderophore could play a dual role regarding copper toxicity. In this work, we show a bimodal effect of enterobactin on copper toxicity (protective and harmful) which depends on the siderophore concentration. We found that the absence of enterobactin rendered Escherichia coli cells more sensitive to copper, due to the reduced ability of those cells to cope wi...

<p>A) Liquid aerated minimal M9 medium cultures of wild-type strain (blue squares), <i>entE</i> strain (green circles) and <i>entE</i> strain in the same media but supplemented with 100 µM FeCl<sub>3</sub> (red triangles). Growth (OD<sub>600</sub>) was determined at the indicated times. B) Lawn growth of wt and <i>entE E. coli</i> strains on M9A. A stationary phase culture of <i>entE E. coli</i> strain was serially diluted (10<sup>−1</sup> to 10<sup>−4</sup>) and an aliquot of these dilutions was applied on M9A or M9A supplemented with 100 µM FeCl<sub>3</sub>. As control, the same dilutions of a wt strain overnight culture were applied on M9A medium. Lawn growth was compared at 8 hours of incubation. C) Colony growth of wt and <i>entE E. coli</i> on LBA. A stationary phase culture of <i>entE E. coli</i> strain was serially diluted and an aliquot of dilutions 10<sup>−6</sup> to 10<sup>−8</sup> were applied on LBA or LBA supplemented with 100 µM FeCl<sub>3</sub>. As control, the same dilutions of an overnight culture of the wt strain were applied on LBA medium. After overnight incubation, colonies sizes were compared. D) Activity of the <i>rhyB</i> promoter estimated by β-galactosidase activity as an indirect measure of the intracellular iron content (The higher the promoter expression, the lower the iron content <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084734#pone.0084734-Ma2" target="_blank">[48]</a>). Both wild-type strain and <i>entE</i> mutant respond to iron addition. The plasmid pALM23 carries the <i>ryhB- lacZ</i> fusion.</p

<p>Quantitation of ROS levels was done using the DCFA-DA probe. Fluorescence intensities are relative to that of the control. Control: wild-type strain grown in M9 medium; <i>entE</i> M9: indicates cells grown in M9 medium, <i>entE</i> LB: indicates cells grown in LB medium, <i>entE</i> M9 1% cas: indicates cells grown in M9 medium supplemented with 1% casamino acids. Error bars = SD, n = 3.</p

<p>A) Type of growth of <i>E. coli fepD</i> and <i>fepG</i> mutants streaked in M9A and incubated overnight in aerobic conditions. B) Levels of reactive oxygen species in <i>E. coli</i> wild-type and <i>fepD</i>, <i>fepG, entS</i> and <i>entE</i> mutants grown in M9 medium. Quantitation of ROS levels was done using the DCFA-DA probe. Fluorescence intensities are relative to that of the control. Control: wt grown in M9 medium. Error bars = SD, n = 3.</p

<p>In plates in which no growth was obtained after overnight incubation (10<sup>−5</sup> dilutions), 1 µL of 1 µM enterobactin (A), 5 µL of 1 mM ascorbic acid (B), 5 µL of 2% casamino acids (C), 10 µL of 1 mM FeCl<sub>3</sub> (D) or 10 µL of 1 mM sodium citrate (E) were spotted. After a second overnight incubation, a size gradient of colonies was clearly observed around the spots containing enterobactin (A), ascorbic acid (B) and casamino acids (C). However, no growth was observed with FeCl<sub>3</sub> (D) or citrate (E).</p

The use of bacteriocins is a promising alternative to improve food security through the biocontrol of food pathogens and spoilage microorganisms. Gram-negative produced microcin J25(G12Y), known as (MccJ25(G12Y)) is a variant of the well-studied and characterized antimicrobial peptide, microcin J25 (MccJ25). In the present work, we explored the activity of this microcin against Gram-negative bacteria linked to foodborne diseases. We evaluated the in vitro antimicrobial activity of MccJ25(G12Y) in solid medium against a collection of pathogenic and food-altering strains and studied its activity and stability in meat and dairy food systems. We show that MccJ25(G12Y) exhibited the same in vitro antimicrobial spectrum as its parental microcin (MccJ25) against different Gram-negative foodborne pathogens and spoilage strains. We highlight that low concentrations of MccJ25(G12Y) between 0.45 and 29.4 μM were able to inhibit a substantial number of pathogens, including Salmonella, Escherichia, Shigella and Enterobacter genus. We also demonstrate the antimicrobial effectiveness of the peptide against Escherichia coli O157:H7 NCTC 12900, Enterobacter cloacae CECT 194, and Salmonella enterica CECT 4396 in fish and beef burgers and yogurt. MccJ25(G12Y) was added or not to food matrices inoculated with the foodborne pathogens at 105 CFU/g or mL. Afterward, food products were stored at 4 °C and selective media for the specific enumeration were used to determine the antimicrobial susceptibility of each pathogen to MccJ25(G12Y). The viability of the three pathogens was significantly reduced in the different food biological environments. In yogurt, the peptide decreased E. coli numbers on day 5 by about 4 log 10 CFU/mL as compared to non-treated samples. For S. enterica and E. cloacae no viable cells were detected at the end of the treatment. Adding MccJ25(G12Y) to fish burgers decreased E. cloacae numbers during storage 2 log10 CFU/g on the first day, reaching a difference of about 5 log 10 CFU/g after 10 days compared to non-treated control. Finally, the peptide decreased E. coli O157:H7 numbers on the beef burgers samples during storage on day 10 by about 3 log 10 CFU/g as compared to non-treated samples. The stability analysis demonstrated that MccJ25(G12Y) is capable of remaining active in these food matrices for a considerable time during the storage at refrigeration temperatures. These results reinforce the studies on the potential applicability of this microcin as a biopreservative in the food industry.

Background Microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide produced by Escherichia coli (E. coli). MccJ25 enters into the sensitive E. coli strains by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD and SbmA. The resistance of Salmonella enterica serovar Typhimurium (S. Typhimurium) to MccJ25 is attributed to the inability of its FhuA protein to incorporate the antibiotic into the cell. Results In this work we demonstrate that S. Typhimurium becomes notably susceptible to MccJ25 when replicating within macrophages. In order to determine the possible cause of this phenomenon, we studied the sensitivity of S. Typhimurium to MccJ25 at conditions resembling those of the internal macrophage environment, such as low pH, low magnesium and iron deprivation. We observed that the strain was only sensitive to the antibiotic at low pH, leading us to attribute the bacterial sensitization to this condition. A MccJ25-resistant E. coli strain in which...

Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, antibacterial peptide produced by Escherichia coli . MccJ25 inhibits RNA polymerase and the membrane respiratory chain. MccJ25 uptake into E. coli -sensitive strains is mediated by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. This peptide is active on some E. coli , Salmonella , and Shigella species strains, while other Gram-negative bacteria, such as clinical isolates of Enterobacter cloacae , Citrobacter freundii , Klebsiella pneumoniae , Pseudomonas aeruginosa , Acinetobacter baumannii , Moraxella catarrhalis , and Salmonella enterica serovar Typhimurium, are completely resistant. In the present work, we demonstrated that the membrane-permeabilizing peptide (KFF) 3 K made some resistant strains sensitive to MccJ25, among them S. Typhimurium, where the antibiotic inhibits in vitro cell growth and bacterial replication within macrophages. The results demonstrate that the membr...