Bacteriophages can be used in various applications, from the classical approach as substitutes for antibiotics (phage therapy) to new biotechnological uses, i.e., as a protein delivery vehicle, a diagnostic tool for specific strains of bacteria (phage typing), or environmental bioremediation. The demand for bacteriophage production increases daily, and studies that improve these production processes are necessary. This study evaluated the production of a T4-like bacteriophage vB_EcoM-UFV09 (an E. coli-infecting phage with high potential for reducing environmental biofilms) in seven types of culture media (Luria–Bertani broth and the M9 minimal medium with six different carbon sources) employing four cultivation variables (temperature, incubation time, agitation, and multiplicity of infection). For this purpose, the rotatable central composite design (RCCD) methodology was used, combining and comparing all parameters to determine the ideal conditions for starting to scale up the production process. We used the RCCD to set up the experimental design by combining the cultivation parameters in a specific and systematic way. Despite the high number of conditions evaluated, the results showed that when specific conditions were utilized, viral production was effective even when using a minimal medium, such as M9/glucose, which is less expensive and can significantly reduce costs during large-scale phage production.

The design of proteins capable effectively binding to specific protein targets is crucial for developing therapies, diagnostics, and vaccine candidates for viral infections. Here, we introduce a complementarity-determining region (CDR) grafting approach for designing nanobodies (Nbs) that target specific epitopes, with the aid of computer simulation and machine learning. As a proof-of-concept, we designed, evaluated, and characterized a high-affinity Nb against the spike protein of SARS-CoV-2, the causative agent of the COVID-19 pandemic. The designed Nb, referred to as Nb Ab.2, was synthesized and displayed high-affinity for both the purified receptor-binding domain protein and to the virus-like particle, demonstrating affinities of 9 nM and 60 nM, respectively, as measured with microscale thermophoresis. Circular dichroism showed the designed protein’s structural integrity and its proper folding, whereas molecular dynamics simulations provided insights into the internal dynamics of Nb Ab.2. This study shows that our computational pipeline can be used to efficiently design high-affinity Nbs with diagnostic and prophylactic potential, which can be tailored to tackle different viral targets.

Proteus mirabilis is an opportunistic pathogen and is responsible for more than 40% of all cases of catheter-associated urinary tract infections (CAUTIs). Healthcare-associated infections have been aggravated by the constant emergence of antibiotic-resistant bacterial strains. Because of this, the use of phages to combat bacterial infections gained renewed interest. In this study, we describe the biological and genomic features of two P. mirabilis phages, named BigMira and MidiMira. These phages belong to the Acadevirus genus (family Autographiviridae). BigMira and MidiMira are highly similar, differing only in four missense mutations in their phage tail fiber. These mutations are sufficient to impact the phages’ depolymerase activity. Subsequently, the comparative genomic analysis of ten clinical P. mirabilis strains revealed differences in their antibiotic resistance profiles and lipopolysaccharide locus, with the latter potentially explaining the host range data of the phages. The massive presence of antimicrobial resistance genes, especially in the phages’ isolation strain P. mirabilis MCS, highlights the challenges in treating infections caused by multidrug-resistant bacteria. The findings reinforce BigMira and MidiMira phages as candidates for phage therapy purposes.