Phage Activity Against Planktonic and Biofilm Staphylococcus aureus Periprosthetic Joint Infection Isolates

We recently reported the successful treatment of a case of periprosthetic joint infection (PJI) with phage. Phage activity against bacteria causing PJI has not been systematically evaluated. Here we examined the in vitro activity of seven lytic phages against 122 clinical isolates of Staphylococcus aureus recovered between April 1999 and February 2018 from subjects with PJI. Phages were assessed against planktonic and biofilm phenotypes. Activity of individual phages was demonstrated against up to 73% of bacterial isolates in the planktonic state and up to 100% of biofilms formed by isolates that were planktonically phage-susceptible. Susceptibility to phage was not correlated with small colony variant status. These results demonstrate that phages can infect S. aureus causing PJI in both planktonic and biofilm phenotypes, and thus are worthy of investigation as an alternative or addition to antibiotics in this setting.

Investigation of phage and molasses interactions for the biocontrol of E. coli O157:H7

Nowadays, resistance in pathogens against antibiotics is one of the most critical health-threatening problems in the world. Therefore, finding new treatment methods to be used as an alternative to antibiotics has become a priority for researchers. Like phages, certain products containing antimicrobial components such as molasses are widely used to eliminate resistant bacteria. Molasses has a strong antimicrobial effect on the bacterial cell, and this effect is thought to be due to the breakdown of the cytoplasmic cell membrane and cell proteins of the polyphenols in molasses. In the present study, phages-molasses interactions were investigated to examine the effects arising from concomitant use. It was found that molasses samples increased the size of phage plaques by up to 3-fold, and MIC and 1/2×MIC concentration of molasses increased the burst size of phages. Although no synergistic effect was found between the phage and the molasses, the antimicrobial activities of the components and the effect of molasses on phage activity were demonstrated.

Engineering a dynamic, controllable infectivity switch in bacteriophage T7

Transcriptional repressors play an important role in regulating phage genomes. Here, we examined how synthetic regulation based on repressors can be to create a dynamic, controllable infectivity switch in bacteriophage T7. We engineered T7 by replacing a large region of the early phage genome with combinations of ligand-responsive promoters and ribosome binding sites (RBS) designed to control the phage RNA polymerase. Phages with the engineered switch showed virulence comparable to wildtype when not repressed, indicating the phage can be engineered without a loss of fitness. When repressed, the most effective switch used a TetR promoter and a weak RBS, resulting in a two-fold increase in latent period (time to lyse host) and change in phage titer. Further, phage activity could be tuned by varying inducer concentrations. Our study provides a proof of concept for a simple circuit for user control over phage infectivity.

In Vitro Newly Isolated Environmental Phage Activity against Biofilms Preformed by Pseudomonas aeruginosa from Patients with Cystic Fibrosis

As disease worsens in patients with cystic fibrosis (CF), Pseudomonas aeruginosa (PA) colonizes the lungs, causing pulmonary failure and mortality. Progressively, PA forms typical biofilms, and antibiotic treatments determine multidrug-resistant (MDR) PA strains. To advance new therapies against MDR PA, research has reappraised bacteriophages (phages), viruses naturally infecting bacteria. Because few in vitro studies have tested phages on CF PA biofilms, general reliability remains unclear. This study aimed to test in vitro newly isolated environmental phage activity against PA isolates from patients with CF at Bambino Gesù Children’s Hospital (OBG), Rome, Italy. After testing in vitro phage activities, we combined phages with amikacin, meropenem, and tobramycin against CF PA pre-formed biofilms. We also investigated new emerging morphotypes and bacterial regrowth. We obtained 22 newly isolated phages from various environments, including OBG. In about 94% of 32 CF PA isolates tested, these phages showed in vitro PA lysis. Despite poor efficacy against chronic CF PA, five selected-lytic-phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) showed wide host activity. The Φ4_ZP1-meropenem and Φ14_OBG-tobramycin combinations significantly reduced CF PA biofilms (p < 0.001). To advance potential combined phage-antibiotic therapy, we envisage further in vitro test combinations with newly isolated phages, including those from hospital environments, against CF PA biofilms from early and chronic infections.

Viral satellites exploit phage proteins to escape degradation of the bacterial host chromosome

SummaryPhage defense systems are often found on mobile genetic elements (MGEs), where they constitutively defend against invaders or are induced to respond to new assaults. Some MGEs, the phage satellites, exploit phages for their own transmission after induction, reducing phage production and protecting their hosts in the process. One such satellite inVibrio cholerae, PLE, is triggered by the lytic phage ICP1 to excise from the chromosome, replicate, and transduce to neighboring cells, completely sabotaging phage production. Here, we found that ICP1 has evolved to possess one of two syntenic loci encoding an SF1B-type helicase, either of which PLE can exploit to directly drive PLE replication. Further, loss of PLE mobilization limits anti-phage activity due to phage-mediated degradation of the bacterial genome. Our work provides insight into the unique challenges imposed on the parasites of lytic phages and underscores the adaptions of these satellites to their ever-evolving target phage.

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: Insights from a natural arms-race

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defense against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not equally interfere with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses.

Rapid and Accurate Detection of Bacteriophage Activity againstEscherichia coliO157:H7 by Propidium Monoazide Real-Time PCR

Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol ofE. colirequire several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intactE. colicells that survive phage exposure.Escherichia coliO157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cellsE. coliO157:H7. Compared to PMA-qPCR, direct plating overestimated (P< 0.01) phage efficacy as cell surface-attached phage particles lysedE. coliO157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol ofE. coliO157:H7.