Zinc is an essential element for all living cells. Recent studies have shown that the ZnuABC zinc uptake system significantly contributes to the ability of several pathogens to multiply in the infected host and cause disease, suggesting that zinc is scarcely available within different tissues of the host. To better understand the role of zinc in bacterial pathogenicity, we have undertaken a functional characterization of the role of the ZnuABC-mediated zinc uptake pathway in enterohemorrhagic Escherichia coli O157:H7.

Every year, the pharmaceutical and food industries produce over 1000 tons of essential oils (EOs) exploitable in different fields as the development of eco-friendly and safe antimicrobial inhibitors. In this work we investigated the potential of some EOs, namely Cinnamomum verum, Cymbopogon martini, Cymbopogoncitratus and Cymbopogon flexuosus, on the growth, biofilm formation and gene expression in four strains of enterohemorrhagic Escherichia coli O157:H7. All EOs were analyzed by gas chromatography-mass spectrometry (GC-MS). The antimicrobial activity was performed by using dilutions of EOs ranging from 0.001 to 1.2% (v/v). Subinhibitory doses were used for biofilm inhibition assay. The expression profiles were obtained by RT-PCR. E. coli O157:H7 virulence was evaluated in vivo in the nematode Caenorhabditis elegans. All EOs showed minimal inhibitory concentrations (MICs) ranging from 0.0075 to 0.3% (v/v). Cinnamomum verum bark EO had the best activity (MIC of 0.0075% (v/v) in all strains) while the C. verum leaf EO had an intermediate efficacy with MIC of 0.175% (v/v) in almost all strains. The Cymbopogon spp. showed the more variable MICs (ranging from 0.075 to 0.3% (v/v)) depending on the strain used. Transcriptional analysis showed that C. martini EO repressed several genes involved in biofilm formation, virulence, zinc homeostasis and encoding some membrane proteins. All EOs affected zinc homeostasis, reducing ykgM and zinT expression, and reduced the ability of E. coli O157:H7 to infect the nematode C. elegans. In conclusion, we demonstrated that these EOs, affecting E. coli O157:H7 infectivity, have a great potential to be used against infections caused by microorganisms.

Background: Highly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated. Results: We report that strains deleted of one or more sodC genes are less resistant than the wild type strain to a challenge with hydrogen peroxide, thus confirming their involvement in the bacterial antioxidant apparatus. To understand if the different sodC genes have truly overlapping functions, we have carried out a comparison of the functional, structural and regulatory properties of the various E. coli O157:H7 SodC enzymes. We have found that the chromosomal and prophagic sodC genes are differentially regulated in vitro. sodC is exclusively expressed in aerobic cultures grown to the stationary phase. In contrast, sodC-F1 and sodC-F2 are expressed also in the logarithmic phase and in anaerobic cultures. Moreover, the abundance of SodC-F1/SodC-F2 increases with respect to that of SodC in bacteria recovered from infected Caco-2 cells, suggesting higher expression/stability of SodC-F1/SodC-F2 in intracellular environments. This observation correlates with the properties of the proteins. In fact, monomeric SodC and dimeric SodC-F1/SodC-F2 are characterized by sharp differences in catalytic activity, metal affinity, protease resistance and stability. Conclusion: Our data show that the chromosomal and bacteriophage-associated E. coli O157:H7 sodC genes have different regulatory properties and encode for proteins with distinct structural/functional features, suggesting that they likely play distinctive roles in bacterial protection from reactive oxygen species. In particular, dimeric SodC-F1 and SodC-F2 possess physico-chemical properties which make these enzymes more suitable than SodC to resist the harsh environmental conditions which are encountered by bacteria within the infected host.

New strategies are needed to fight the emergence of multidrug-resistant bacteria caused by an overuse of antibiotics in medical and veterinary fields. Due to the importance of biofilms in clinical infections, antibiofilm peptides have a great potential to treat infections. In recent years, an increased interest has emerged in antimicrobial peptides (AMPs). One of the richest sources of AMPs is represented by amphibian skin. In the present work, we investigated the effects of two peptides derived from the frog skin AMP esculentin-1, namely, Esc(1-21) and Esc(1-18), on the growth, biofilm formation, and gene expression of the non-pathogenic Escherichia coli strain K12 and of enterohemorrhagic E. coli O157:H7. Both peptides showed minimal bactericidal concentrations ranging from 4 to 8 µM for Esc(1-21) and from 32 to 64 µM for Esc(1-18). They also, at sub-MIC doses, reduced the formation of biofilm, as supported by both microbiological assays and scanning electron microscopy, while they displayed no marked activity against the planktonic form of the bacteria. Transcriptional analysis in E. coli O157:H7 showed that both AMPs induced the expression of several genes involved in the regulation of formation and dispersal of biofilm, as well as in the stress response. In conclusion, we demonstrated that these AMPs affect E. coli O157:H7 growth and biofilm formation, thus suggesting a great potential to be developed as novel therapeutics against infections caused by bacterial biofilms.

The absence of effective therapy against Escherichia coli O157:H7 infections has led to the need to develop new antimicrobial agents. As the use of synergistic combinations of natural antimicrobial compounds is growing as a new weapon in the fight against multidrug-resistant bacteria, here, we have tested new synergistic combinations of natural agents. Notably, we investigated a possible synergistic effect of combinations of essential oils and natural peptides to counteract the formation of biofilm. We chose three essential oils (i.e., Cymbopogon citratus, C. flexuosus and C. martinii) and one peptide already studied in our previous works. We determined the fractional inhibitory concentration (FIC) by analyzing the combination of the peptide derived from esculentin-1a, Esc(1–21), with the three essential oils. We also studied the effects of combinations by time–kill curves, scanning electron microscopy on biofilm and Sytox Green on cell membrane permeability. Finally, we analyzed the expression of different genes implicated in motility, biofilm formation and stress responses. The results showed a different pattern of gene expression in bacteria treated with the mixtures compared to those treated with the peptide or the single C. citratus essential oil. In conclusion, we demonstrated that the three essential oils used in combination with the peptide showed synergy against the E. coli O157:H7, proving attractive as an alternative strategy against E. coli pathogen infections.