Bacteriophages, viruses that only kill specific bacteria, are receiving substantial attention as nontraditional antibacterial agents that may help alleviate the growing antibiotic resistance problem in medicine. We describe the design and preclinical development of AB-SA01, a fixed-composition bacteriophage product intended to treat Staphylococcus aureus infections. AB-SA01 contains three naturally occurring, obligately lytic myoviruses related to Staphylococcus phage K. AB-SA01 component phages have been sequenced and contain no identifiable bacterial virulence or antibiotic resistance genes. In vitro, AB-SA01 killed 94.5% of 401 clinical Staphylococcus aureus isolates, including methicillin-resistant and vancomycin-intermediate ones for a total of 95% of the 205 known multidrug-resistant isolates. The spontaneous frequency of resistance to AB-SA01 was ≤3 × 10−9, and resistance emerging to one component phage could be complemented by the activity of another component phage. In both neutropenic and immunocompetent mouse models of acute pneumonia, AB-SA01 reduced lung S. aureus populations equivalently to vancomycin. Overall, the inherent characteristics of AB-SA01 component phages meet regulatory and generally accepted criteria for human use, and the preclinical data presented here have supported production under good manufacturing practices and phase 1 clinical studies with AB-SA01.

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Numerous high-value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin-arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed-batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial-type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA-GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co-expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over-expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed-batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h(-1), the cultures reached OD(600) values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L(-1) culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec-dependent export approaches.

Filamentous bacteriophages and their derivatives are showing great promise as a whole new class of industrial agents, such as biologically based nano-materials and viral vectors. This raises challenges for their large-scale manufacture, principally due to the lack of bioprocessing knowledge. This article addresses what will be a potentially important option in the primary purification of the bacteriophages. Polyethylene glycol (PEG)-salt dual precipitants, calcium ions, spermidine, and isoelectric precipitation were first examined for their potential suitability for bacteriophage concentration under both pure and broth conditions. Successful precipitants were further studied on the basis of their selective purification ability from DNA and protein contaminants in a clarified broth system. Both PEG-based and isoelectric precipitations resulted in bacteriophage purity improvements, and PEG-based precipitations offered the highest selectivities. This work shows that precipitation of bacteriophages can be an effective primary purification step in a large-scale bioprocess.