Prevalence and characterization of the seven major serotypes of Shiga toxin-producing Escherichia coli (STEC) in veal calves slaughtered in France.

Shiga toxin (Stx)-producing Escherichia coli (STEC) belonging to the "top 7″ serotypes (i.e. O157:H7, O26:H11, O45:H2, O103:H2, O111:H8, O121:H19 and O145:H28) are considered as the main pathogenic enterohemorrhagic E. coli (EHEC). As ruminants, including calves, are a reservoir of pathogenic STEC, we investigated the prevalence, major virulence genes and genetic relatedness of top7 STEC in veal calves slaughtered in France, through the analysis of 500 fecal samples collected over one year. Thirty top7 STEC isolates were recovered from 28 calves. The two serotypes O103:H2 and O26:H11 accounted for 73% of STEC strains, followed by O145:H28 and O157:H7. STEC super-shedding levels were identified for two calves carrying STEC O103:H2 and O157:H7, respectively. Thirty-nine atypical enteropathogenic E. coli (aEPEC) were also recovered from calves. Overall, a prevalence of 5.6% top7 STEC-positive calves was found, thus higher than that previously determined for the French slaughtered adult cattle (1.8%), confirming the impact of animals age on STEC carriage. Most top7 STEC strains carried the stx1a subtype suggesting a low pathogenicity for humans. Seasonal variation in STEC carriage was also observed, with two peaks of higher prevalence during spring and fall. Genetic similarity of top7 STEC isolates was found for calves originating from the same fattening facilities, reflecting STEC circulation between animals kept in groups. This study indicates that veal calves grown for meat production are at higher risk of shedding top7 STEC compared to adult cattle. They thus represent ideal targets for the implementation of farm interventions aimed at reducing STEC burden in cattle and the food chain.

Surface Engineering of Top7 to Facilitate Structure Determination.

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular ß-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular ß-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

Changes in STEC and bacterial communities during enrichment of manufacturing beef in selective and non-selective media.

Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from manufacturing beef is challenging and it may be affected by microbial changes during enrichment. This study was designed to understand population changes during enrichment of beef from an integrated (Samples A and B) and a fragmented (Samples C and D) abattoir. The samples were enriched in buffered peptone water (BPW), Assurance GDS MPX top 7 STEC mEHEC®, BAX® E. coli O157:H7 MP and PDX-STEC media then were processed for 16 S rRNA sequencing. Escherichia dominated Sample B enrichment broths regardless of the media used (71.6-97.9%) but only in mEHEC broth (79.6%) of Sample A. Escherichia was dominant in Sample C in mEHEC (95.2%) and PDX-STEC (99.2%) broths but less in BPW (58.5%) and MP (64.9%) broths. In Sample D, Clostridium dominated in mEHEC (65.5%), MP (80.2%) and PDX-STEC (90.6%) broths. O157 STEC was isolated from Sample C only. The study suggested that MP may not be as effective as mEHEC and PDX-STEC and that Clostridium could interfere with enrichment of Escherichia. Understanding the ecological changes during enrichment provides meaningful insight to optimising the enrichment protocol for STEC and subsequently enhance the efficiency of STEC detection.

Added Value of Genomic Surveillance of Virulence Factors in Shiga Toxin-Producing Escherichia coli in New South Wales, Australia.

The disease caused by Shiga toxin-producing Escherichia coli (STEC) remains a significant public health challenge globally, but the incidence of human STEC infections in Australia remains relatively low. This study examined the virulence characteristics and diversity of STEC isolates in the state of New South Wales between December 2017 and May 2020. Utilisation of both whole and core genome multi-locus sequence typing (MLST) allowed for the inference of genomic diversity and detection of isolates that were likely to be epidemiologically linked. The most common STEC serotype and stx subtype detected in this study were O157:H7 and stx 1a, respectively. A genomic scan of other virulence factors present in STEC suggested interplay between iron uptake system and virulence factors that mediate either iron release or countermeasures against host defence that could result in a reduction of stx 1a expression. This reduced expression of the dominant stx genotype could contribute to the reduced incidence of STEC-related illness in Australia. Genomic surveillance of STEC becomes an important part of public health response and ongoing interrogation of virulence factors in STEC offers additional insights for the public health risk assessment.

Multiplex PCR Assays for the Detection of One Hundred and Thirty Seven Serogroups of Shiga Toxin-Producing Escherichia coli Associated With Cattle.

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called "non-top-7" have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7-12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.

The influence of biotinylation on the ability of a computer designed protein to detect B-cells producing anti-HIV-1 2F5 antibodies.

Antibodies against the HIV-1 2F5 epitope are known as one of the most powerful and broadly protective anti-HIV antibodies. Therefore, vaccine strategies that include the 2F5 epitope in their formulation require a robust method to detect specific anti-2F5 antibody production by B cells. Towards this goal, we have biotinylated a previously reported computer-designed protein carrying the HIV-1 2F5 epitope aiming the further development of a platform to detect human B-cells expressing anti-2F5 antibodies through flow cytometry. Biophysical and immunological properties of our devised protein were characterized by computer simulation and experimental methods. Biotinylation did not affect folding and improved protein stability and solubility. The biotinylated protein exhibited similar binding affinity trends compared to its unbiotinylated counterpart and was recognized by anti-HIV-1 2F5 antibodies expressed on the surface of patient-derived peripheral blood mononuclear cells. Moreover, we present a high affinity marker for the identification of epitope-specific B cells that can be used to measure the efficacy of vaccine strategies based on the HIV-1 envelope protein.

Designing cooperativity into the designed protein Top7.

The topology of the designed protein Top7 is not found in natural proteins. Top7 shows signatures of non-cooperative folding in both experimental studies and computer simulations. In particular, molecular dynamics of coarse-grained structure-based models of Top7 show a well-populated C-terminal folding-intermediate. Since most similarly sized globular proteins are cooperative folders, the non-natural topology of Top7 has been suggested as a reason for its non-cooperative folding. Here, we computationally examine the folding of Top7 with the intent of making it cooperative. We find that its folding cooperativity can be increased in two ways: (a) Optimization of packing interactions in the N-terminal half of the protein enables further folding of the C-terminal intermediate. (b) Reduction in the packing density of the C-terminal region destabilizes the intermediate. In practice, these strategies are implemented in our Top7 model through modifications to the contact-map. These modifications do not alter the topology of Top7 but result in cooperative folding. Amino-acid mutations that mimic these modifications also lead to a significant increase in folding cooperativity. Finally, we devise a method to randomize the sizes of amino-acids within the same topology, and confirm that the structure of Top7 makes its folding sensitive to packing interactions. In contrast, the ribosomal protein S6, which has secondary structure similar to Top7, has folding which is much less sensitive to packing perturbations. Thus, it should be possible to make a sequence fold cooperatively to the structure of Top7, but to do so its side-chain packing needs to be carefully designed.

Folding of Top7 in unbiased all-atom Monte Carlo simulations.

For computational studies of protein folding, proteins with both helical and ß-sheet secondary structure elements are very challenging, as they expose subtle biases of the physical models. Here, we present reproducible folding of a 92 residue α/ß protein (residues 3-94 of Top7, PDB ID: 1QYS) in computer simulations starting from random initial conformations using a transferable physical model which has been previously shown to describe the folding and thermodynamic properties of about 20 other smaller proteins of different folds. Top7 is a de novo designed protein with two α-helices and a five stranded ß-sheet. Experimentally, it is known to be unusually stable for its size, and its folding transition distinctly deviates from the two-state behavior commonly seen in natural single domain proteins. In our all-atom implicit solvent parallel tempering Monte Carlo simulations, Top7 shows a rapid transition to a group of states with high native-like secondary structure, and a much slower subsequent transition to the native state with a root mean square deviation of about 3.5 Å from the experimentally determined structure. Consistent with experiments, we find Top7 to be thermally extremely stable, although the simulations also find a large number of very stable non-native states with high native-like secondary structure.