Nitric Oxide Induced stx2 Expression Is Inhibited by the Nitric Oxide Reductase, NorV, in a Clade 8 Escherichia coli O157:H7 Outbreak Strain

Escherichia coli O157:H7 pathogenesis is due to Shiga toxin (Stx) production, though variation in virulence has been observed. Clade 8 strains, for instance, were shown to overproduce Stx and were more common among hemolytic uremic syndrome cases. One candidate gene, norV, which encodes a nitric oxide (NO) reductase found in a clade 8 O157:H7 outbreak strain (TW14359), was thought to impact virulence. Hence, we screened for norV in 303 O157 isolates representing multiple clades, examined stx2 expression following NO exposure in TW14359 for comparison to an isogenic mutant (ΔnorV), and evaluated survival in THP-1 derived macrophages. norV was intact in strains representing clades 6–9, whereas a 204 bp deletion was found in clades 2 and 3. During anaerobic growth, NO induced stx2 expression in TW14359. A similar increase in stx2 expression was observed for the ΔnorV mutant in anaerobiosis, though it was not impaired in its ability to survive within macrophages relative to TW14359. Altogether, these data suggest that NO enhances virulence by inducing Stx2 production in TW14359, and that toxin production is inhibited by NorV encoded by a gene found in most clade 8 strains. The mechanism linked to these responses, however, remains unclear and likely varies across genotypes.

Inhibitory Effect of Sodium Citrate, Sodium Nitrite and Cinnamaldehyde on Biofilm-forming Escherichia coli O157:H7

The development of biofilms by the foodborne pathogens attached to surfaces in the food processing environments results in the deterioration of products, persistence of pathogenic bacteria and transmission of food-associated diseases. In addition, biofilms are more resistant to antimicrobials than their planktonic counterparts which make their elimination from food and the food processing facilities a great challenge. This study aim was to determine the inhibitory effect of food additives on biofilm forming Escherichia coli O157:H7. The isolate obtained was subjected to Gram’s staining and various biochemical identifications and later confirmed by latex agglutination test. Biofilm formation potential was done on Congo red media and the confirmed biofilm former was subjected to biofilm formation at 10℃ and 37℃ for 168hrs. Antimicrobial susceptibility testing, MIC, MBC, and antibiofilm effect was determined following CLSI 2017 guideline. The highest zone of growth inhibition of 31 mm was exhibited by cinnamaldehyde, sodium nitrite with 26 mm and sodium citrate with 13 mm. The MIC 2.5 mg/mL was recorded for sodium citrate, 0.25 mg/mL for sodium nitrite and 0.125 μl/mL for cinnamaldehyde. Strong biofilm was formed at 37 ℃ with 7.82 x 109 CFU/mL viable cells at 168hrs while 6.79 x 109 CFU/mL were obtained at 10 ℃. All the three additives showed antibiofilm effect (at 10℃ and 37℃), cinnamaldehyde exhibited 70%-90.1%, sodium nitrite; 70%-88.2% inhibition and sodium nitrite; 75%-88% inhibition respectively. This study showed that sodium citrate, sodium nitrite and cinnamaldehyde exerted strong antimicrobial and antibiofilm properties indicating their potential as good preservatives.

The Structure and Function of Modular Escherichia coli O157:H7 Bacteriophage FTBEc1 endolysin, LysT84: Defining a New Endolysin Catalytic Subfamily

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level—key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1–4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.