Biology subjects

Hynes, A. P.

Publications and source records attributed to Hynes, A. P..

4 recordsLinked to original sources

bioRxiv2024

Temperate phages increase antibiotic effectiveness in a Caenorhabditis elegans infection model

The bactericidal nature of obligately lytic bacterial viruses (phages) is of increasing interest for the treatment of drug-resistant bacterial infections, either administered alone or in combination with antibiotics. In contrast, temperate phages are largely ignored in a therapeutic context due to their ability to lie dormant within the bacterial host. However, these phages often undergo a lytic cycle. Furthermore, even in their dormant state they can be a considerable burden to the bacterium - most famously by their ability to switch to lytic replication in response to environmental triggers, such as antibiotics, that stress the bacterial host. Recent reports of antibiotics synergizing with temperate phages in vitro, termed "temperate phage antibiotic synergy" (tPAS), present a potentially scalable opportunity to make use of these abundant entities for the treatment of bacterial infections. Here we employ Caenorhabditis elegans as a robust in vivo animal model for testing the efficacy of temperate phages as adjuvants to antibiotics. In vivo, temperate phage Hali alone results in 60% dormancy in the bacterial survivors. However, the antibiotic ciprofloxacin can abolish dormancy of temperate phage Hali - infecting a ciprofloxacin resistant Pseudomonas aeruginosa clinical strain. The phage Hali-ciprofloxacin pairing increased the lifespan of P. aeruginosa infected worms to that of the uninfected control, at doses where neither the phage nor the antibiotic had any effect alone. Complete rescue was also observed in worms infected with a phage-carrying strain treated with the otherwise ineffective antibiotic, supporting that the phage - even in its dormant form - can greatly enhance antibiotic effectiveness. This illustrates potential "accidental" phage therapy when antibiotics are prescribed. Our work establishes C. elegans as a suitable model for studying the in vivo efficacy of tPAS and is the first in vivo demonstration of this synergy, greatly expanding the therapeutic potential of temperate phages.

microbiology↗

bioRxiv2024

Temperate phage-antibiotic synergy is widespread, but varies by phage, host, and antibiotic pairing

With a decline in antibiotic effectiveness, there is a renewed interest in bacteriophage (phage) therapy. Phages are bacterial-specific viruses that can be used alone or with antibiotics to reduce bacterial load. Most phages are unsuitable for therapy because they are temperate and can integrate into the host genome, forming a lysogen which is protected from subsequent phage infections. However, integrated phages can be awakened by stressors such as antibiotics. This interaction was previously reported to result in a potent synergy between antibiotic classes and a model E. coli temperate phage, which can readily eradicate the bacterium at sub-lethal concentrations of antibiotics, despite the poor effectiveness of the phage alone. Here we explore the generalizability of this synergy to a clinically relevant pathogen: Pseudomonas aeruginosa. Thirty-six temperate phages isolated from clinical strains were screened for synergy with six antibiotics (ciprofloxacin, levofloxacin, meropenem, piperacillin, tobramycin, polymyxin B), using checkerboard assays. Interestingly, our screen identified phages that can synergize with each antibiotic, despite their widely differing targets - however, these are highly phage-antibiotic and phage-host pairing specific. Screening the strongest pairings across multiple clinical strains reveal that these phages can reduce the antibiotic minimum inhibitory concentration up to 32-fold, even in a resistant isolate, functionally re-sensitizing the bacterium to the antibiotic. When meropenem and tobramycin were effective synergistic agents, they did not reduce the frequency of lysogens, suggesting a mechanism of action independent of the temperate nature of the phages. In contrast, ciprofloxacin and piperacillin were able to reduce the frequency of lysogeny, the former by inducing phages - as previously reported in E. coli. Curiously, synergy with piperacillin reduced the frequency of lysogeny, but not by inducing the phages, and therefore likely acts by biasing the phage away from lysogeny in the initial infection. Overall, our findings indicate that temperate phages can act as adjuvants to antibiotics in clinically relevant pathogens, even in the presence of antibiotic resistance, thereby drastically expanding their therapeutic potential.

microbiology↗

bioRxiv2024

Micro-plaque assays: A high-throughput method to detect, isolate, and characterize bacteriophages

The gold standard for the isolation and characterization of bacteriophages (phages), the plaque assay, has remained almost unchanged for over 100 years. The need for improvements to its scalability has been driven home by successes with personalized phage therapy requiring large phage libraries and rapid sensitivity testing. Using a robotic pinning platform, we miniaturized plaque assays from bacterial lawns to micro-colonies from 100 nl of inoculant, increasing throughput by >1000 fold without compromising sensitivity. A comparable manual workflow with one quarter the throughput maintained the same sensitivity. These micro-plaque assays can replace plaque assays as a new gold standard in phage biology. As proof of principle, we used our technique to isolate and de-replicate 21 unique Pseudomonas aeruginosa phages from a single environmental sample. We then demonstrated - using the same assay - that of 17 multi-drug resistant clinical P. aeruginosa strains, 15 were susceptible to infection by one or more of the 21 phages tested. Our method allows rapid isolation and de-replication of phages, as well as enabling screening of large phage libraries against bacterial isolates of interest.

microbiology↗

bioRxiv2024

CRISPR adaptation in Streptococcus thermophilus can be driven by phage environmental DNA

The CRISPR-Cas system is a bacterial adaptative immune system which protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers -- fragments of the invading nucleic acids -- into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, phages quickly take over the metabolism of the bacteria, leaving little time for the bacteria to acquire new spacers, transcribe them and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases eDNA which could be involved in the development of CRISPR immunity. Using S. thermophilus and phages 2972 and 858 as a model for CRISPR immunity, we show that eDNA is crucial to the development of optimal CRISPR immunity, as generation of phage-immune bacterial colonies decrease with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since its addition increases the generation of phage-immune colonies. We also show that the effect of eDNA is phage-specific, sequence specific and can even be traced to a region of the genome covering the early-expressed genes which differ between phages 2972 and 858. However, we also show that eDNA is not used as a source of genetic information for spacer acquisition. This suggests that the effect of eDNA involves a new mechanism of phage resistance. Moreover, the effect of eDNA is highly dependent on environmental conditions as variation in media suppliers are sufficient to interfere with this effect. These results link environmental conditions, specifically eDNA, to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect the presence of phages before infection.

microbiology↗