In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli.

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic Escherichia coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse precolonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study suggests that commensal E. coli engineered to deliver payloads that block essential virulence determinants can be developed as a new means to prevent and potentially treat infections including those due to antibiotic resistant microbes.

In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli.

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality.

Frequency of deployed emergency donor panel use prior to implementation of the low titre group O whole blood program.

INTRODUCTION: The US military has frequently used a 'walking blood bank', formally known as an 'emergency donor panel' (EDP) to obtain warm fresh whole blood (WFWB) which is then immediately transfused into the casualty. We describe the frequency of EDP activation by the US military. METHODS: We analysed data from 2007 to 2015 within the Department of Defense Trauma Registry for US, Coalition and US contractor casualties that received at least 1 unit of blood product within the first 24 hours and described the frequency of WFWB use. RESULTS: There were 3474 casualties that met inclusion, of which, 290 casualties (8%) required activation of the EDP. The highest proportion of EDP events was in 2014, whereas the highest number of EDP events was in 2011. Median injury severity scores were higher in the recipients, compared with non-EDP recipients (29 vs 20), as were proportions with serious injuries to the abdomen (43% vs 19%) and extremities (77% vs 65%). The median number of units of all blood products, except for packed red blood cells, was higher for WFWB recipients. Of the WFWB recipients, the median was 5 units (IQR 2-10) with a maximum documented 144 units. There were four documented cases of EDP recipients receiving >100 units of WFWB with only one surviving to hospital discharge. During the study period, there were a total of 3102 (3%) units of WFWB transfused among a total of 104 288 total units. CONCLUSIONS: We found nearly 1 in 11 casualties who received blood required activation of the EDP. Blood from the EDP accounted for 3% of all units transfused. These findings will enable future mission planning and medical training, especially for units with smaller, limited blood supplies. The lessons learned here can also enable mass casualty planning in civilian settings.