Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria

Abstract

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.

Keywords: CRISPR-Cas; ex vivo treatment; lysogenization; positive selection.

Fig. 1.

Schematics of the lysogenizing phages. The CRISPR-associated genes cas3, cse1, cse2, cas7, cas5, and cas6e (blue) were inserted in place of nucleotides at position 19,014–27,480 of the λ chromosome (National Center for Biotechnology Information Reference Sequence: NC_001416.1) yielding the control lysogenizing phage λ_cas (Lower). The λ_cas-CRISPR phage (Upper) encodes, in addition to the cas genes, a CRISPR array with spacers targeting the genes ndm-1 (N₁, N₂, N₃) and ctx-M-15 (C₁, C₂, C₃). P_T7, T7 promoter.

Fig. 2.

Lysogenization effect on transformation of antibiotic resistance plasmids. E. coli K-12 were lysogenized with λ_cas (light gray bars) or λ_cas-CRISPR (dark gray bars). These lysogens were transformed with a control (pVEC), ndm-1 (pNDM), or ctx-M-15 (pCTX) encoding plasmids and plated on agar plates supplemented with streptomycin. Bars represent average and SD of the number of CFUs per microgram plasmid counted after plating serial dilutions of the cultures in three independent experiments.

Fig. 3.

Lysogenization effect on protection against lytic phages. E. coli K-12 were lysogenized with λ_cas (light gray bars) or λ_cas-CRISPR (dark gray bars). These lysogens were infected with a control T7-gp8 lacking targeted protospacers, or with T7 phages encoding two protospacers from ndm-1 (T7-N₁N₂) or two protospacers from ctx-M-15 (T7-C₂C₁) or one spacer from each gene (T7-N₁C₁ and T7-C₂N₂). Bars represent average and SD of the number of plaque-forming units (PFUs) per milliliter counted after plating serial dilutions of the phages in three independent experiments.

Fig. 4.

Enrichment of antibiotic-sensitized bacteria by lytic phages. (A) Schematics of the procedure to enrich for antibiotic-sensitized bacteria. A bacterial culture is mixed with lysogenizing phages, resulting in both lysogens and nonlysogens in the culture. Lysogens are both antibiotic sensitized and phage resistant as the CRISPR-Cas system degrades the antibiotic resistance-conferring plasmid and the lytic-phage chromosome. The treated culture is inoculated on agar-containing lytic phages that selectively kill the nonlysogens and thus enrich for antibiotic-sensitized bacteria. (B) Enrichment of phage-resistant E. coli. E. coli K-12 harboring a control (pVEC), ndm-1 (pNDM), ctx-M-15 (pCTX), or ndm-1 + ctx-M-15 (pNDM*/pCTX) encoding plasmids were treated with λ_cas (light gray bars) or λ_cas-CRISPR (dark gray bars) and plated on T7-N₁C₁–coated plates as shown in the scheme presented in A. Bars represent average and SD of the number of surviving CFUs per milliliter counted in three independent experiments. (C) Enrichment of antibiotic-sensitive E. coli. Surviving colonies (20–48 CFUs) from each culture described in B were inoculated on plates having or lacking streptomycin or gentamicin. Bars represent percentage and SD from three independent experiments of streptomycin- or gentamicin-sensitive bacteria scored as CFU unable to grow on plates with streptomycin or gentamicin out of the total number of CFU able to grow on plates lacking these antibiotics.