Biological characteristics and anti-biofilm activity of a lytic phage against vancomycin-resistant Enterococcus faecium

Background and Objectives: An important leading cause of the emergence of vancomycin-resistant enterococci, especially Enterococcus faecium, is the inefficiency of antibiotics in the elimination of drug-resistant pathogens. Consequently, the need for alternative treatments is more necessary than ever. Materials and Methods: A highly effective bacteriophage against vancomycin-resistant E. faecium called vB-EfmS-S2 was isolated from hospital sewage. The biological properties of phage S2 and its effect on biofilm structures were determined. Results: Phage S2 was specifically capable of lysing a wide range of clinical E. faecium isolates. According to Electron mi- croscopy observations, the phage S2 belonged to the Siphoviridea family. Suitable pH spectra for phage survival was 5-11, at which the phage showed 100% activity. The optimal temperature for phage growth was 30-45°C, with the highest growth at 37°C. Based on one-step growth curve results, the latent period of phage S2 was 14 min with a burst size of 200 PFU/ml. The phage S2 was also able to tolerate bile at concentrations of 1 and 2% and required Mg2+ for an effective infection cycle. Biofilms were significantly inhibited and disrupted in the presence of the phage. Conclusion: According to the results, phage S2 could potentially be an alternative for the elimination and control of vancomycin-resistant E. faecium biofilm.

Genome-scale top-down reduction of phages to generate viable minimal phage genomes

Reduction of tailed-phage genomes to generate viable minimal genome phages is important for expanding our understanding of phage biology, providing insights for phage synthetic biology. Many efforts have been made to minimize living cells, but such work remains a challenge for phages due to the extraordinary genomic diversity and lack of genome-scale editing techniques. Here, we developed a CRISPR/Cas9-based iterative phage genome reduction (CiPGr) approach to detect the nonessential gene set of phages and minimize phage genomes. By CiPGr, inactivated genes accumulated on the phage genome, and mutant progeny with robust growth gradually arose, eventually becoming predominant in the populations. CiPGr was applied to four distinct tailed phages (model phages T7 and T4; wild-type phages seszw and selz), resulting in mutants of these phages with deletion of 8–20% (3.3–33 kbp) sequences, and leading to minimal genomes. Metagenomic sequencing of the mutant phage populations generated showed that 46.7 to 65.4% of genes of these phages were removed. Loss of some genes (39.6%-50%) in the removable gene sets was likely severely detrimental to phage growth. This made the corresponding mutant progenies recede in the populations, leading to the failure of detection of these genes in the genomes of the isolated mutants. In summary, our results for these four distinct tailed phages demonstrated that CiPGr is a generic yet effective approach suitable for use in novel phages without prior knowledge.

Isolation and characterization Phage UPM1705 against multi-drug resistant K. pneumoniae 1705

The rise in in the number of drug-resistant bacteria that can resist almost all kinds of antibiotics is due to the overuse of these antibiotics (e.g., carbapenems). Thus, there is a need to find an alternative to antibiotic treatment such as the use of phages. In this study, phage UPM1705 was isolated from a polluted lake which can lyse its host Klebsiella pneumoniae ATCC BAA-1705. Based on morphological appearance from transmission electron microscopy, UPM1705 belongs to Caudovirales (Myoviridae). UPM1705 can reach a titer of 107 PFU/ml based on the double-layer method. It has a burst size of 298 PFU/bacteria cell and a latent period of 80 min, a rise period of 75 min, and adsorption time of 20 min based on a one-step growth curve assay using an MOI of 0.02. It was stable from 4°C to 80°C and retained its functionality at pH between 4 to 11, with pH of 7 being the optimum pH for the phage growth. The efficiency of UPM1705 was tested via a turbidity assay at MOI of 0.02, 0.2, and 2. UPM1705 was able to clear the turbidity of the host bacteria culture at all of these three MOIs. Thus, UPM1705 has the potential to be used for phage therapy.

Phage Cocktails can Prevent the Evolution of Phage-Resistant Enterococcus

ABSTRACTAntibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy is often deployed as a cocktail of phages, but there is little understanding of how to most effectively combine phages. Here we utilized a collection of 20 Enterococcus phages to test principles of phage cocktail design and determine the phenotypic effects of evolving phage resistance in Enterococcus isolates that were susceptible or resistant to antibiotics (e.g., Vancomycin Resistant Enterococcus (VRE)). We tested the ability of each phage to clear Enterococcus host cultures and prevent the emergence of phage resistant Enterococcus. We found that some phages which were ineffective individually were effective at clearing the bacterial culture when used in cocktails. To understand the dynamics within phage cocktails, we used qPCR to track which phages increased in abundance in each cocktail, and saw dynamics ranging from one dominant phage to even phage growth. Further, we isolated several phage-resistant mutants to test for altered Vancomycin sensitivity. We found that mutants tended to have no change or slightly increased resistance to Vancomycin. By demonstrating the efficacy of phage cocktails in suppressing growth of antibiotic susceptible and VRE clinical isolates when exposed to phages, this work will help to inform cocktail design for future phage therapy applications.IMPORTANCEAntibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy in the form of cocktails is often suggested, with similar goals as the combination therapy that has been successful in the treatment of HIV infection, but there is little understanding about how to combine phages most effectively. Here we utilized a collection of 20 Enterococcus phages to test whether several phage cocktails could prevent the host from evolving resistance to therapy and to determine whether evolving resistance to phages affected host susceptibility to antibiotics. We showed that cocktails of two or three unrelated phages often prevented the growth of phage-resistant mutants, when the same phages applied individually were not able to.

Population dynamics of decision making in temperate bacteriophages

Due to their ability to choose between lysis and lysogeny, temperate bacteriophages represent a classic model system to study the molecular basis of decision making. The coinfection of individual bacteria by multiple, genetically identical phages is known to alter the infection outcome and favor lysogeny over lytic development. However, it is not clear what role the ability of individual phages to sense and respond to coinfections plays in the phage-host infection dynamics at the population level. To address this question, we developed a full-stochastic model to capture the interaction dynamics between billions of bacteria and phages with single-cell and -phage resolution. While, at the level of individual bacteria, the probability of coinfections depends mainly on the phage concentration at the time of infection, the average number of coinfections at the population level is primarily determined by the relative growth rate of phage. Because the maximum attainable phage growth rate is constrained by basic life history parameters, the average number of coinfections has an upper bound of around two. However, for a broad range of conditions, the average number of coinfections stays well below this value. Consequently, we find that coinfections provide only very limited information to individual phages about the state of the infection at the population level. Nevertheless, this information can still provide a strong competitive advantage for phages that base fate decisions on the number of coinfections.

Escherichia coli and Salmonella Enteritidis dual-species biofilms: interspecies interactions and antibiofilm efficacy of phages

Escherichia coli and Salmonella Enteritidis are foodborne pathogens forming challenging biofilms that contribute to their virulence, antimicrobial resistance, and survival on surfaces. Interspecies interactions occur between species in mixed biofilms promoting different outcomes to each species. Here we describe the interactions between E. coli and S. Enteritidis strains, and their control using specific phages. Single-species biofilms presented more cells compared to dual-species biofilms. The spatial organization of strains, observed by confocal microscopy, revealed similar arrangements in both single- and dual-species biofilms. The EPS matrix composition, assessed by Fourier-transform infrared spectroscopy, disclosed that the spectra extracted from the different dual-species biofilms can either be a combination of both species EPS matrix components or that the EPS matrix of one species predominates. Phages damaged more the single-species biofilms than the mixed biofilms, showing also that the killing efficacy was greatly dependent on the phage growth characteristics, bacterial growth parameters, and bacterial spatial distribution in biofilms. This combination of methodologies provides new knowledge of species-species and phage-host interactions in biofilms of these two major foodborne pathogens.

Promises and Pitfalls of In Vivo Evolution to Improve Phage Therapy

Phage therapy is the use of bacterial viruses (phages) to treat bacterial infections, a medical intervention long abandoned in the West but now experiencing a revival. Currently, therapeutic phages are often chosen based on limited criteria, sometimes merely an ability to plate on the pathogenic bacterium. Better treatment might result from an informed choice of phages. Here we consider whether phages used to treat the bacterial infection in a patient may specifically evolve to improve treatment on that patient or benefit subsequent patients. With mathematical and computational models, we explore in vivo evolution for four phage properties expected to influence therapeutic success: generalized phage growth, phage decay rate, excreted enzymes to degrade protective bacterial layers, and growth on resistant bacteria. Within-host phage evolution is strongly aligned with treatment success for phage decay rate but only partially aligned for phage growth rate and growth on resistant bacteria. Excreted enzymes are mostly not selected for treatment success. Even when evolution and treatment success are aligned, evolution may not be rapid enough to keep pace with bacterial evolution for maximum benefit. An informed use of phages is invariably superior to naive reliance on within-host evolution.

Evolution of Superinfection Immunity in Cluster A Mycobacteriophages

ABSTRACTTemperate phages encode an immunity system to control lytic gene expression during lysogeny. This gene regulatory circuit consists of multiple interacting genetic elements, and although it is essential for controlling phage growth, it is subject to conflicting evolutionary pressures. During superinfection of a lysogen, the prophage’s circuit interacts with the superinfecting phage’s circuit and prevents lytic growth if the two circuits are closely related. The circuitry is advantageous since it provides the prophage with a defense mechanism, but the circuitry is also disadvantageous since it limits the phage’s host range during superinfection. Evolutionarily related phages have divergent, orthogonal immunity systems that no longer interact and are heteroimmune, but we do not understand how immunity systems evolve new specificities. Here, we use a group of Cluster A mycobacteriophages that exhibit a spectrum of genetic diversity to examine how immunity system evolution impacts superinfection immunity. We show that phages with mesotypic (i.e., genetically related but distinct) immunity systems exhibit asymmetric and incomplete superinfection phenotypes. They form complex immunity networks instead of well-defined immunity groups, and mutations conferring escape (i.e., virulence) from homotypic or mesotypic immunity have various escape specificities. Thus, virulence and the evolution of new immune specificities are shaped by interactions with homotypic and mesotypic immunity systems.IMPORTANCEMany aspects regarding superinfection, immunity, virulence, and the evolution of immune specificities are poorly understood due to the lack of large collections of isolated and sequenced phages with a spectrum of genetic diversity. Using a genetically diverse collection of Cluster A phages, we show that the classical and relatively straightforward patterns of homoimmunity, heteroimmunity, and virulence result from interactions between homotypic and heterotypic phages at the extreme edges of an evolutionary continuum of immune specificities. Genetic interactions between mesotypic phages result in more complex mesoimmunity phenotypes and virulence profiles. These results highlight that the evolution of immune specificities can be shaped by homotypic and mesotypic interactions and may be more dynamic than previously considered.