Impact of Shiga-toxin encoding gene transduction from O80:H2 Shiga toxigenic Escherichia coli (STEC) on non-STEC strains

Abstract

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens that cause human diseases ranging from diarrhea to life-threatening complications including hemolytic–uremic syndrome. Virulence of STEC strains and their ability to cause severe diseases are associated with the activity of prophage-encoded Shiga toxins (Stxs). The first objective of this work was to isolate and characterize the Stx2d phage from STEC O80:H2 and to study the transfer of this phage in non-STEC strains. The second objective was to assess the survival of Galleria mellonella larvae inoculated with these transduced strains. Firstly, one bacteriophage isolated from a STEC O80:H2 strain was used to infect six non-STEC strains, resulting in the conversion of three strains. Then, stability assays were performed, showing that this phage was stable in the new STEC strains after three successive subculturing steps, as confirmed by a combination of short and long read genome sequencing approaches. This phage, vB_EcoS_ULI-O80_Stx2d, is resistant to moderate temperature and pH. It belongs to a currently unclassified genus and family within the Caudoviricetes class, shares 98% identity with Stx2_112808 phage and encodes several proteins involved in the lysogenic cycle. The yecE gene was identified at the insertion site. Finally, G. mellonella experiments showed that the transduced strains caused significantly higher mortality rates than the corresponding non-STEC strains. In conclusion, this study showed that stx2d gene from O80:H2 E. coli can be transferred to non-STEC strains and contributes to their virulence.

Virulence of Shigatoxigenic and Enteropathogenic Escherichia coli O80:H2 in Galleria mellonella Larvae: Comparison of the Roles of the pS88 Plasmids and STX2d Phage

“…The remaining two AE-STEC O80:H2 were identified by PCR, as previously described [ 21 ], and were genome sequenced for confirmation (see Section 2.2 ). One of the AE-STEC O80:H2 (EH3320/SES3090) had been previously tested in larvae of G. mellonella [ 30 ] and was the O80:H2 positive control.…”

“…The pS88 plasmid conjugation strategy was based on the presence of the ColV-encoding cva operon on the pS88 plasmid of bovine AE-STEC and EPEC O80:H2 [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. The recipient strain was the E. coli K12 DH10B strain harboring the recombinant plasmid pAuto-ColV-Switch1.0 carrying a synthetic ColV-encoding locus (received from Syngulon Company, Seraing, Belgium; ).…”

“…Thirty larvae per strain divided into three groups of 10 larvae (Animal Confort, Loncin, Belgium) were inoculated in the last proleg, as previously described [ 30 ], with 10 μL of each of the tenfold dilutions of the bacterial culture corresponding to 10 to 10 6 CFU using an automatic injector (ColeParmer, Vernon Hills, IL, USA). In addition, 30 larvae were injected with 10 μL of PBS as a control group.…”

“…Besides their low cost and the possibility of testing multiple groups of larvae in a short time, G. mellonella larvae possess an innate immune system similar to mammals and can be maintained at 37 °C, like the bacterial pathogens of mammals [ 25 , 29 ]. So, it was recently observed that the STX2d phage and the type 3 secretion system (T3SS) contribute to the virulence of AE-STEC O80:H2 and EPEC O127:H6, respectively, in G. mellonella larvae [ 25 , 30 , 31 ], while the contribution of the T3SS to the virulence of AE-STEC O157:H7 is less clear [ 32 , 33 ]. However, no study has been performed yet to assess the role of the pS88 plasmids of AE-STEC and EPEC O80:H2.…”

Horizontal Gene Transfer of Antibiotic Resistance Genes in Biofilms

“…Transduction experiments have commonly been performed using planktonic cultures with characterized environmental, clinical, or laboratory strains [230][231][232][233]. To the best of our knowledge, only one study has demonstrated phage-mediated gene transfer through lysogenic bacteriophages in biofilms of potentially pathogenic E. coli [234].…”