SASP: targeted delivery to Gram-negative pathogens

Abstract

Background: Gram-negative bacteria are responsible for significant morbidity and mortality worldwide. Multi-drug resistance emergence has rendered many therapies ineffective. New therapies are urgently required to widen treatment options. SASPject technology delivers small acid-soluble spore protein (SASP) genes to target bacteria using modified bacteriophage vectors, resulting in rapid killing. SASP is a unique antibacterial protein that non-specifically binds bacterial DNA and halts DNA replication and gene expression. In this study we present the first data for a Gram-negative targeted SASPject vector (PT3.1) which shows in vitro activity against Escherichia coli (EC) and Pseudomonas aeruginosa (PA). Methods: We evaluated efficacy of SASPject PT3.1 vs. EC (N=5) & PA (N=5) using a microtitre tray fixed duration (3 h) kill method. Log-phase cultures (1x105 cfu/mL) were prepared in supplemented (MgSO4 & CaCl2, 5 mM; glucose, 0.1% w/v) LB broth (LBC) & exposed to 2x108 plaque forming units (pfu)/mL of PT3.1. PT3.1 antimicrobial activity was determined using agar-based culture following incubation (37oC). Additionally, rate of PT3.1 kill was determined using a kill-curve technique; a selection of EC & PA strains from the fixed duration kill study were evaluated & bacterial viable counts determined over 3 h on LBC agar. Results: SASPject PT3.1 elicited good antimicrobial activity vs. EC & PA evaluated in this study; the median reduction in viable counts for PT3.1-treated cultures in the fixed 3 h kill was 99.1%. Kill curve data suggested rapid EC & PA killing; viable counts (log10 cfu/mL range) of PT3.1-treated cultures were 2.36->4.01, 3.04-4.06, and 3.40-4.16 lower than corresponding controls after 1, 2, and 3 h respectively. Conclusions: 1. SASPject PT3.1 demonstrated good antimicrobial activity vs. EC & PA evaluated in this study in a fixed 3 h exposure period 2. PT3.1 was bactericidal (≥3 log10 cfu/mL decline) vs. 3 of 4 isolates (1EC & 2PA) evaluated in time-kill curves after 1 h and against all isolates after 2 h 3. Further evaluations of Gram-negative SASPject phage are warranted