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

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.The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have evolved in prokaryotes to protect against phage attack and undesired plasmid replication by targeting foreign DNA or RNA (1–3). These systems target nucleic acids, based on short DNA sequences, called spacers, that exist between repeats in the CRISPR array. Transcribed spacers guide Cas proteins to homologous sequences within the foreign nucleic acid, called protospacers, which are subsequently cleaved. The CRISPR-Cas systems have revolutionized molecular biology by providing efficient tools to precisely engineer genomes and manipulate gene expression in various organisms (4–10). CRISPR-Cas systems have also recently been used to phenotypically correct genetic diseases in live animals (11), and their utility is being explored for various therapeutic approaches in mammals. Nevertheless, only limited studies have shown the use of CRISPR-Cas systems to target antibiotic resistance genes or a specific population of virulent bacterial strains (12–17).Two recent elegant studies demonstrated that phage-transferable CRISPR-Cas systems are capable of specifically killing pathogens or resensitizing them to antibiotics (16, 17). These studies, and another study (13), also showed that the transferred CRISPR-Cas system is capable of eliminating specific bacterial populations. Furthermore, they demonstrated that the system might be used against pathogens to effectively treat infected animals. Consequently, it was suggested that the system could be used as a potent antimicrobial agent. Nevertheless, although the results of these studies highlight the potential of a phage-transferable CRISPR-Cas system, the concept of using the system as a direct antimicrobial is similar to conventional phage therapy, which currently faces various obstacles (18). One major obstacle is phage administration into infected tissues; this stems from the phages’ immunogenicity and relative large size compared with antibiotics. One may argue that it would be more efficient to directly kill a pathogen by a lytic phage if it were possible to deliver the CRISPR-Cas–encoding cassette into this pathogen by a phage. Moreover, using the proposed systems in infected patients to resensitize pathogens to antibiotics while antibiotics counterselect for these sensitized pathogens would most likely fail due to escape mutants that are selected by the antibiotics.Here we demonstrate a strategy to counteract the emerging threat of antibiotic-resistant bacteria that evades the above shortcomings. Instead of directly killing the pathogens, we propose to sensitize the pathogens on surfaces or in the human skin flora while concomitantly enriching for these sensitized populations. Patients infected by these antibiotic-sensitive bacteria would thus be treatable by traditional antibiotics. In this strategy, the CRISPR-Cas system is used to destroy specific DNAs that confer antibiotic resistance and to concurrently confer a selective advantage to antibiotic-sensitive bacteria by virtue of resistance to lytic phages. The selective advantage enables to efficiently displace populations of nonsensitized bacteria by killing them with lytic phages. In contrast to conventional phage therapy, this approach does not require administration of phages into the host’s tissues. In addition, it does not aim to directly kill treated bacteria but rather to sensitize them to antibiotics and to kill the nonsensitized bacteria. Therefore, there is no counterselection against the sensitization. The strategy relies on CRISPR spacers that can be rationally designed to target any DNA sequence, including those that encode resistance genes and lytic phages. It thus allows genetically linking a trait that is beneficial to the bacteria (i.e., spacers protecting from lytic phage) with a trait that reverses drug resistance (i.e., spacers targeting resistance genes). The genetic linkage enables selecting antibiotic-sensitized bacterial population by using lytic phages. The integrated construct is designed not only to actively eradicate existing resistance genes but also to eliminate horizontal transfer of these genes between bacteria. Extended use of this technology should thus reduce drug-resistant populations of pathogens on major sources of contamination. Consequently, well-established antibiotics for which resistance currently exists could once again be effective.