Integrative omics analysis of Pseudomonas aeruginosa virus PA5oct highlights the molecular complexity of jumbo phages

Pseudomonas virus vB_PaeM_PA5oct is proposed as a model jumbo bacteriophage to investigate phage-bacteria interactions and is a candidate for phage therapy applications. Combining hybrid sequencing, RNA-Seq and mass spectrometry allowed us to accurately annotate its 286,783 bp genome with 461 coding regions including four non-coding RNAs (ncRNAs) and 93 virion-associated proteins. PA5oct relies on the host RNA polymerase for the infection cycle and RNA-Seq revealed a gradual take-over of the total cell transcriptome from 21% in early infection to 93% in late infection. PA5oct is not organized into strictly contiguous regions of temporal transcription, but some genomic regions transcribed in early, middle and late phases of infection can be discriminated. Interestingly, we observe regions showing limited transcription activity throughout the infection cycle. We show that PA5oct upregulates specific bacterial operons during infection including operons pncA-pncB1-nadE involved in NAD biosynthesis, psl for exopolysaccharide biosynthesis and nap for periplasmic nitrate reductase production. We also observe a downregulation of T4P gene products suggesting mechanisms of superinfection exclusion. We used the proteome of PA5oct to position our isolate amongst other phages using a gene-sharing network. This integrative omics study illustrates the molecular diversity of jumbo viruses and raises new questions towards cellular regulation and phage-encoded hijacking mechanisms

Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their efficacy against Pseudomonas aeruginosa biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants

A proposed integrated approach for the preclinical evaluation of phage therapy in Pseudomonas infections

Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications

Treating Bacterial Infections with Bacteriophage-Based Enzybiotics: In Vitro, In Vivo and Clinical Application

Over the past few decades, we have witnessed a surge around the world in the emergence of antibiotic-resistant bacteria. This global health threat arose mainly due to the overuse and misuse of antibiotics as well as a relative lack of new drug classes in development pipelines. Innovative antibacterial therapeutics and strategies are, therefore, in grave need. For the last twenty years, antimicrobial enzymes encoded by bacteriophages, viruses that can lyse and kill bacteria, have gained tremendous interest. There are two classes of these phage-derived enzymes, referred to also as enzybiotics: peptidoglycan hydrolases (lysins), which degrade the bacterial peptidoglycan layer, and polysaccharide depolymerases, which target extracellular or surface polysaccharides, i.e., bacterial capsules, slime layers, biofilm matrix, or lipopolysaccharides. Their features include distinctive modes of action, high efficiency, pathogen specificity, diversity in structure and activity, low possibility of bacterial resistance development, and no observed cross-resistance with currently used antibiotics. Additionally, and unlike antibiotics, enzybiotics can target metabolically inactive persister cells. These phage-derived enzymes have been tested in various animal models to combat both Gram-positive and Gram-negative bacteria, and in recent years peptidoglycan hydrolases have entered clinical trials. Here, we review the testing and clinical use of these enzymes

Friends or Foes? Rapid Determination of Dissimilar Colistin and Ciprofloxacin Antagonism of Pseudomonas aeruginosa Phages

Phage therapy is a century-old technique employing viruses (phages) to treat bacterial infections, and in the clinic it is often used in combination with antibiotics. Antibiotics, however, interfere with critical bacterial metabolic activities that can be required by phages. Explicit testing of antibiotic antagonism of phage infection activities, though, is not a common feature of phage therapy studies. Here we use optical density-based ‘lysis-profile’ assays to assess the impact of two antibiotics, colistin and ciprofloxacin, on the bactericidal, bacteriolytic, and new-virion-production activities of three Pseudomonas aeruginosa phages. Though phages and antibiotics in combination are more potent in killing P. aeruginosa than either acting alone, colistin nevertheless substantially interferes with phage bacteriolytic and virion-production activities even at its minimum inhibitory concentration (1× MIC). Ciprofloxacin, by contrast, has little anti-phage impact at 1× or 3× MIC. We corroborate these results with more traditional measures, particularly colony-forming units, plaque-forming units, and one-step growth experiments. Our results suggest that ciprofloxacin could be useful as a concurrent phage therapy co-treatment especially when phage replication is required for treatment success. Lysis-profile assays also appear to be useful, fast, and high-throughput means of assessing antibiotic antagonism of phage infection activities

Characterization of lytic bacteriophages infecting Pseudomonas aeruginosa and their peptidoglycan and exopolysaccharide degrading enzymes

Antibiotic resistance of bacterial pathogens is an emerging problem worldwide. As multidrug resistant organisms fail to respond to antimicrobial therapy, infections become more severe and cause more complications which in turn leads to longer illnesses (Gould, 2006; Haecker, 2009; Hübner et al., 2012). The aerobic Gram-negative bacterium Pseudomonas aeruginosa is one of the most important and dangerous microbes inhabiting the hospital environment. It has the ability to adapt to and thrive in many ecological niches, from water and soil environments to plant and animal tissues. P. aeruginosa also possesses a wide array of virulence factors, that do not only cause extensive tissue damage, but also interfere with the human immune system (Dzierżanowska, 2008). As an opportunistic pathogen it is particularly known for causing endogenous infections in immune-deficient individuals (AIDS, cystic fibrosis, cancer) and presents resistance to many antibiotics (Cornelis et al., 2008). During chronic infections populations of P. aeruginosa undergo characteristics evolutionary adaptations, including reduction of virulence factors production and transition from planktonic form to biofilm-associated lifestyle. The biofilm play an important role in evolution of high-level antibiotic resistance and protects the embedded bacteria from antimicrobial agents applied from without. Moreover, during prolonged infections coexistence of divergent phenotypic lineages of Pseudomonas within patients was observed, which makes accurate diagnosis and treatment even more challenging (Winstanley et al., 2016). For these reason, new antibacterial therapies are urgently needed. With hurdles in the antibiotic research and development pipelines, especially against Gram-negative pathogens, the scientific community is exploring the development of alternative forms of antimicrobial therapies. The idea of using bacteriophages as natural parasites of bacteria is well known. Bacteriophages have co-evolved intimately with their host for three billion years, and therefore highly efficient antibacterial mechanisms have emerged, granting them unique advantages over classical antibiotics to kill bacteria. Indeed, phage cocktails are commonly applied as alternative or as supportive treatments simultaneously with antibiotics in Eastern Europe, as part of standard care. Positive results are routinely obtained with the eradication of Escherichia, Pseudomonas, Proteus, Klebsiella and Staphylococcus clinical strains from various kinds of purulent wounds (Slopek et al., 1981; Wright et al., 2009; Maura and Debarbieux, 2011). These viruses developed also the ability to tunnel through bacterial biofilms by employment of specific enzymes, which degrade the bacterial exopolysaccharides (EPS), one of the main component of the biofilm matrix. Such phages can be identified by the appearance of a halo zone around the phage plaques, which results from the enzymatic degradation of bacterial EPS without phage infection (Azeredo et al., 2008). Furthermore, bacteriophages can kill bacteria with peptidoglycan degrading enzymes, endolysins and virion-associated peptidoglycan hydrolases (VAPGH), that disrupt specific bonds in the peptidoglycan, a major component of bacterial cell wall. The use of bacteriophages and their recombinantly manufactured proteins offers a great opportunity to bypass antibiotic therapy hurdles. This dissertation specifically focuses on bacteriophages and their two types of enzymes: the polysaccharide depolymerases and peptidoglycan degrading enzymes, endolysins and VAPGHs, active against P. aeruginosa. In first part of this study we focused on characterization of four newly isolated bacteriophages KT28, KTN6, KTN4 and PA5oct, lytic towards P. aeruginosa. Their basic biology was evaluated, including morphology, host surface receptors, host range, stability and infection process. All phages were stable in a range of temperatures, pH and in presence of chloroform. KT28, KTN6 and KTN4 had relatively broad host range, in contrast to PA5oct. Host cell receptors were different for each type of phage, and included LPS, type IV pili and flagella. Furthermore, the phage genomes were sequenced and analyzed in depth. Unwanted genes or mobile elements were not found, which supports obligatory lytic nature of these bacteriophages. Functional genome analysis was supported with ESI-MS/MS analysis of phage proteome and RNA seq. With the use of comparative genomics, the protein-sharing network was constructed, that revealed phage evolution and homology. KTN6 and KT28 belong to Pbunavirus, KTN4 is a Phikzvirus and PA5oct is a unique giant virus. Finally phage antibacterial activity was evaluated in vitro using a novel Airway Surface Liquid model on non-CF and CF epithelial cells lines, in an effort to mimic in vivo conditions of the respiratory tract, developed at the laboratory of Prof. B. Harvey (Department of Molecular Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin, Ireland). Phages KTN4 and PA5oct presented substantial antibacterial activity, reducing bacterial load from 2.5 to 7 logs, depending on the P. aeruginosa strain used. Second part of this dissertation focuses on phage-encoded PG degrading enzymes (KT28 gp49, KT28 gp41 and its domain, KTN6 gp46, PA5oct gp214, PA5oct gp250, KTN4 gp48) and polysaccharide depolymerases (LKA1 gp49, LUZ7 gp56 and their domains). Their secondary and tertiary structure was analyzed in depth based on available homology and crystal structures. The corresponding recombinant proteins have been produced and their bactericidal activity and biofilm eradication potential was evaluated. Among the PG degrading enzymes included in this study, the strongest antibacterial activity presented KTN6 gp46, a globular endolysin. Research of polysaccharide depolymerases was complicated, time consuming and required the greatest effort to obtain bioactive, recombinant and purified protein. LKA1 gp49 and its domain reduced EPS slime surrounding bacterial cell. In the future, their biofilm degradation activity should be further evaluated with more specific and quantitative biofilm assays. This research presents the potential of bacteriophages and their enzymes, which can be considered for therapy or industrial purposes. However, further research is required to analyze e.g. antibacterial activity in details, stability, dose, toxicity or structure. Furthermore, in vivo animal studies would show the true power of this alternative therapy.nrpages: 304status: publishe

Phage Therapy in the 21st Century: Is There Modern, Clinical Evidence of Phage-Mediated Efficacy?

Many bacteriophages are obligate killers of bacteria. That this property could be medically useful was first recognized over one hundred years ago, with 2021 being the 100-year anniversary of the first clinical phage therapy publication. Here we consider modern use of phages in clinical settings. Our aim is to answer one question: do phages serve as effective anti-bacterial infection agents when used clinically? An important emphasis of our analyses is on whether phage therapy-associated anti-bacterial infection efficacy can be reasonably distinguished from that associated with often coadministered antibiotics. We find that about half of 70 human phage treatment reports—published in English thus far in the 2000s—are suggestive of phage-mediated anti-bacterial infection efficacy. Two of these are randomized, double-blinded, infection-treatment studies while 14 of those studies, in our opinion, provide superior evidence of a phage role in observed treatment successes. Roughly three-quarters of these potentially phage-mediated outcomes are based on microbiological as well as clinical results, with the rest based on clinical success. Since many of these phage treatments are of infections for which antibiotic therapy had not been successful, their collective effectiveness is suggestive of a valid utility in employing phages to treat otherwise difficult-to-cure bacterial infections

Phage Cocktail Development for Bacteriophage Therapy: Toward Improving Spectrum of Activity Breadth and Depth

Phage therapy is the use of bacterial viruses as antibacterial agents. A primary consideration for commercial development of phages for phage therapy is the number of different bacterial strains that are successfully targeted, as this defines the breadth of a phage cocktail’s spectrum of activity. Alternatively, phage cocktails may be used to reduce the potential for bacteria to evolve phage resistance. This, as we consider here, is in part a function of a cocktail’s ‘depth’ of activity. Improved cocktail depth is achieved through inclusion of at least two phages able to infect a single bacterial strain, especially two phages against which bacterial mutation to cross resistance is relatively rare. Here, we consider the breadth of activity of phage cocktails while taking both depth of activity and bacterial mutation to cross resistance into account. This is done by building on familiar algorithms normally used for determination solely of phage cocktail breadth of activity. We show in particular how phage cocktails for phage therapy may be rationally designed toward enhancing the number of bacteria impacted while also reducing the potential for a subset of those bacteria to evolve phage resistance, all as based on previously determined phage properties