Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

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A SARS-CoV-2 variant B1.1.7 containing a mutation Δ69/70 has spread rapidly in the UK and shows an identifiable profile in ThermoFisher TaqPath RTqPCR (S-gene target failure; SGTF). We analysed recent test data for trends and significance. Linked Ct values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1x10 7 and 1x10 8. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in humans and animals. Non-O157:H7 EHEC strains contain the gene efa-1 (referred to in previous publications as efa1), which influences adherence to cultured epithelial cells. An almost identical gene in enteropathogenic E. coli (lifA) mediates the inhibition of lymphocyte proliferation and proinflammatory cytokine synthesis. We have shown previously that significantly lower numbers of EHEC O5 and O111 efa-1 mutants are shed in feces following experimental infection in calves and that these mutants exhibit reduced adherence to intestinal epithelia compared with isogenic wild-type strains. E. coli O157:H7 strains lack efa-1 but encode a homolog on the pO157 plasmid (toxB/l7095) and contain a truncated version of the efa-1 gene (efa-1/z4332 in O island 122 of the EDL933 chromosome). Here we report that E. coli O157:H7 toxB and efa-1 single and double mutants exhibit reduced adherence to cultured epithelial cells and show reduced expression and secretion of proteins encoded by the locus of enterocyte effacement (LEE), which plays a key role in the host-cell interactions of EHEC. The activity of LEE1, LEE4, and LEE5 promoters was not significantly altered in E. coli O157:H7 strains harboring toxB or efa-1 mutations, indicating that the effect on the expression of LEE-encoded secreted proteins occurs at a posttranscriptional level. Despite affecting type III secretion, mutation of toxB and efa-1 did not significantly affect the course of fecal shedding of E. coli O157:H7 following experimental inoculation of 10-to 14-day-old calves or 6-week-old sheep. Mutation of tir caused a significant reduction in fecal shedding of E. coli O157:H7 in calves, indicating that the formation of AE lesions is important for colonization of the bovine intestine.Enterohemorrhagic Escherichia coli (EHEC) strains are zoonotic enteric pathogens of worldwide importance (39). In humans, infection by some EHEC serotypes may cause diarrhea, which may be complicated by hemorrhagic colitis and severe systemic sequelae, including hemolytic-uremic syndrome (45). EHEC strains are closely related to enteropathogenic E. coli (EPEC) strains, which are a leading cause of infantile diarrhea in developing countries, and have many of the EPEC genes implicated in virulence (13,51).Ruminants are an important reservoir of EHEC (15, 44), and direct or indirect contact with ruminant feces is the leading antecedent to EHEC infection in humans (17,30,43). Natural and experimental infections of calves or sheep with EHEC result in efficient colonization of the intestinal tract, and large numbers of bacteria are shed in the feces for several weeks (3, 5-9, 18, 59, 67, 68). Strategies to reduce the prevalence of EHEC in ruminants offer the potential to lower the incidence of human infections (58). However, little is currently known about the mechanisms underlying intestinal colonization of cattle and sheep by EHE...

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SummaryType III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

The prebiotic Bimuno® is a mixture containing galactooligosaccharide, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41171 in the presence of lactose. Previous studies have implicated prebiotics in reducing infections by enteric pathogens, thus it was hypothesized that Bimuno® may confer some protection in the murine host from Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. In this study, infection caused by S. Typhimurium SL1344nalr in the presence or absence of Bimuno® was assessed using tissue culture assays, a murine ligated ileal gut loop model and a murine oral challenge model. In tissue culture adherence and invasion assays with HT-29-16E cells, the presence of ∼2 mM Bimuno® significantly reduced the invasion of S. Typhimurium SL1344nalr (P<0.0001). In the murine ligated ileal gut loops, the presence of Bimuno® prevented colonization and the associated pathology of S. Typhimurium. In the BALB/c mouse model, the oral delivery of Bimuno® prior to challenge with S. Typhimurium resulted in significant reductions in colonization in the five organs sampled, with highly significant reductions being observed in the spleen at 72 and 96 h post-challenge (P=0.0002, <0.0001, respectively). Collectively, the results indicate that Bimuno® significantly reduced the colonization and pathology associated with S. Typhimurium infection in a murine model system, possibly by reducing the invasion of the pathogen into host cells.

Intimin facilitates intestinal colonization by enterohemorrhagic Escherichia coli O157:H7; however, the importance of intimin binding to its translocated receptor (Tir) as opposed to cellular coreceptors is unknown. The intimin-Tir interaction is needed for optimal actin assembly under adherent bacteria in vitro, a process which requires the Tir-cytoskeleton coupling protein (TccP/EspF U ) in E. coli O157:H7. Here we report that E. coli O157:H7 tir mutants are at least as attenuated as isogenic eae mutants in calves and lambs, implying that the role of intimin in the colonization of reservoir hosts can be explained largely by its binding to Tir. Mutation of tccP uncoupled actin assembly from the intimin-Tir-mediated adherence of E. coli O157:H7 in vitro but did not impair intestinal colonization in calves and lambs, implying that pedestal formation may not be necessary for persistence. However, an E. coli O157:H7 tccP mutant induced typical attaching and effacing lesions in a bovine ligated ileal loop model of infection, suggesting that TccP-independent mechanisms of actin assembly may operate in vivo.