Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors

Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternative antimicrobials. However, phage-mediated killing is often limited by bacterial resistance development. Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using phage-based companion diagnostics, we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials

Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥10$^{3}$ CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application

Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course.

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 and autoimmune disorders, but they target different chemokines than those in COVID-19. Finally, monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. One-Sentence Summary Naturally arising anti-chemokine antibodies associate with favorable COVID-19 and are predictive of lack of long COVID

Autoantibodies against chemokines post-SARS-CoV-2 infection correlate with disease course

Infection with severe acute respiratory syndrome coronavirus 2 associates with diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse coronavirus disease 2019 (COVID-19) outcomes. Here we discovered that antibodies against specific chemokines were omnipresent post-COVID-19, were associated with favorable disease outcome and negatively correlated with the development of long COVID at 1 yr post-infection. Chemokine antibodies were also present in HIV-1 infection and autoimmune disorders, but they targeted different chemokines compared with COVID-19. Monoclonal antibodies derived from COVID-19 convalescents that bound to the chemokine N-loop impaired cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising chemokine antibodies may modulate the inflammatory response and thus bear therapeutic potential

Autoantibodies against chemokines post-SARS-CoV-2 infection correlate with disease course

Infection with severe acute respiratory syndrome coronavirus 2 associates with diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse coronavirus disease 2019 (COVID-19) outcomes. Here we discovered that antibodies against specific chemokines were omnipresent post-COVID-19, were associated with favorable disease outcome and negatively correlated with the development of long COVID at 1 yr post-infection. Chemokine antibodies were also present in HIV-1 infection and autoimmune disorders, but they targeted different chemokines compared with COVID-19. Monoclonal antibodies derived from COVID-19 convalescents that bound to the chemokine N-loop impaired cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising chemokine antibodies may modulate the inflammatory response and thus bear therapeutic potential

Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors

Abstract Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternative antimicrobials. However, phage-mediated killing is often limited by bacterial resistance development. Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using phage-based companion diagnostics, we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials

The Impact of Free Cell Area in Asymptomatic Patients Undergoing CAS: Results from a Multicentre Registry

Objective: The stent cell design has been advocated as a crucial factor that may impact on reducing cerebrovascular ischaemic events during carotid artery stenting (CAS). New generation dual layer stents (DLSs) seem to perform better than the previous generations but long term data are lacking. The present multi- centre study investigated the association between stent cell design and stroke prevention in asymptomatic patients undergoing CAS. Methods: From January 2006 to January 2022, 1 118 transfemoral CAS procedures were planned in 1 069 patients (males 803, 71.8%) with an asymptomatic severe carotid artery stenosis in six tertiary referral centres. Patients were divided in three groups considering the stent design: open cell (OC), closed cell (CC), and dual layer stents (DLS). Pre-operative data included demographics, comor- bidities, ipsilateral and contralateral carotid artery stenosis degree, occlusion, or previous surgical treatment of the contralateral ca- rotid bifurcation. Post-operative data included technical success (intention to treat basis), access site and systemic complications, and therapy at discharge. Thirty day outcomes were stroke, death (all cause mortality), stroke/death, and myocardial infarction (MI) rates. Follow up outcomes were evaluated at one, 2.5, and five years. Primary follow up outcomes included overall survival, stroke (overall and ipsilateral), and stroke related mortality estimations. Secondary follow up outcomes were restenosis and carotid re- intervention estimations. Data were analysed using the chi squared test and Fisher’s exact test while follow up outcomes were investigated with life table KaplaneMeier curves. The log rank test was used to determine differences between the groups and uni- variate analysis was employed to identify the association between risk factors and stroke rates. A p value < .05 was considered to be statistically significant. Results: In six patients technical success was not achieved (0.5%); thus, 1 112 patients were finally analysed (OC 48.6%, CC 30%, DLS 17.9%); 49 procedures were bilateral (4.4%). Mean age was 73.3  8.2 years (OC 72.9  7.6; CC 73.6  8.4, DLS 74.6  8.2). Thirty day outcomes were stroke rate 2.3% (OC 1.1%, CC 3.4%, DLS 3.8%; p 1⁄4 .018), mortality rate 0.6% (OC 0.9%, CC 0.3%, DLS 0.5%; p 1⁄4 .687), stroke/death rate 2.9% (OC 2.0%, CC 3.7%, DLS 4.2%; p 1⁄4 .151), MI rate 0.2% (OC 0.2%, CC 0.3%, DLS 0.0%; p 1⁄4 .742). A total of 959CAS procedures were followed up with a mean of 50.1  38.8 months (OC 68.8  40.6; CC 43.3  30.1, DLS 17.6  14.5). At one, 2.5, and five years of follow up overall survival was 96%, 91%, 79.2% (OC 96.1%, 92.4%, 81%; CC 96.2%, 89.1%, 77.2%, DLS 95%, 90.1%, 73.1; log rank 2.1, p 1⁄4 .336), overall stroke rate: 0.7%, 1.6%, 1.6% (OC: none, 0.5%, 0.5%; CC: 2%, 3.9%, 3.9%, DLS: none; log- rank 9.7, p 1⁄4 0.008) and 0.3%, 1%, 1.2% ipsilateral (OC: none, 0.3%, 0.3%; CC: 1%, 2.5%, 3.1%, DLS: none; log-rank 8.4, p 1⁄4 0.015), stroke-related mortality rate: 0.2%, 0.7%, 1.2% (OC: none, 0.3%, 0.6%; CC: 0.7%, 1.6%, 2.5%, DLS: none; log-rank 2.5, p 1⁄4 0.283). Restenosis rate was 1.5%, 2.8%, 5.4% (OC 1.7%, 2.7%, 5.5%; CC 2.1%, 3%, 5.8%; DLS: none, 1.5% 3.8%; log rank 0.82, p 1⁄4 .661) and carotid re-intervention rate was 0.9%, 1.9%, 2.9% (OC 1.4%, 2.1%, 3.5%; CC 0.6%, 2%, 2.6%; DLS none; log rank 2.4, p 1⁄4 .296) at one, 2.5, and five years, respectively. The risk factor analysis shows a higher significant incidence of dyslipidaemia (p 1⁄4 .0001), hypertension (p 1⁄4 .0001), smoking habit (p 1⁄4 .001), and a severe degree of stenosis (p 1⁄4 .001) in the CC and DLS groups. These data may explain the 30 day stroke rates in these groups being these stents selected to treat a high risk population. The 2.5 and five year KaplaneMeier curves showed higher incidence of overall (p 1⁄4 .008) and ipsilateral (p 1⁄4 .015) stroke rates in the CC group. The univariate analysis demonstrated that ipsilateral stroke rate was influenced by age (p 1⁄4 .005) and peripheral artery dis- ease (p 1⁄4 .015). Conclusion: In this study, CC stents and DLS showed higher rate of post-operative stroke probably because they were used to treat a risky population. DLS performed better than OC and CC stents in terms of five year stroke prevention

Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine

Abstract The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥103 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application

Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 and autoimmune disorders, but they target different chemokines than those in COVID-19. Finally, monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. One-sentence summary: Naturally arising anti-chemokine antibodies associate with favorable COVID-19 and are predictive of lack of long COVID

A national cross-sectional survey on time-trends for endovascular repair of genetically-triggered aortic disease and connective tissue disorders over two decades

Background: By this survey, we aim to gain national-based information regarding trends in endovascular repair (ER) for the treatment of aortic disease in patients with genetically-triggered aortic disease (GTAD) and connective tissue disorder (CTD) over the last two decades. Methods: All Italian vascular surgery centers (N.=80) were invited to participate in an anonymous electronic cross-sectional survey on ER for GTAD/CTD. Results: Overall, 29 institutions completed the survey, thereby yielding a 36% response rate. The percentage of responding institutions rises to 64% if only regional hubs were considered (23/36). The median number of index procedures per center was 6.2, and a steady increase in the overall number of interventions over time was also noted. Most patients were males (73%) with a median age of 48 years. The most common endovascular procedure was TEVAR (N.=101), followed by F/BEVAR (N.=43) and EVAR (N.=37). The overall technical success rate was 83.4% while major adverse events and mortality at thirty days were reported at 18.2% and 9.9%, respectively. An additional 5.0% mortality rate was noted for an overall one-year mortality of 14.9%, while 3.7% of all treated patients were diagnosed with a type 1 endoleak. Conclusions: This national cross-sectional survey, investigating trends in ER of GTADs and CTDs over two decades, highlights a consistent increase in the use of endovascular techniques for their treatment. Early mortality was acceptably low, yet influenced by the urgency of presentation. At one-year follow-up, a 5% additional death rate was noted, and the reintervention rate remained below one in ten