The Hydrophobic Stabilization of Pseudomonas aeruginosa Bacteriophage F8 and the Influence of Modified Bacteriophage Preparation on Biofilm Degradation.

The bacteriophage F8 belongs to the Myoviridae group of phages and is a pathogen of Pseudomonas aeruginosa. Since Pseudomonas aeruginosa is a multidrug-resistant opportunistic bacterium and can cause serious challenges for health services, studying the potential use of phages against them is a promising approach. Pseudomonas aeruginosa can be found on medical devices because bacteria can attach to surfaces and develop biofilms, which are difficult to eradicate because of their high resistance to environmental conditions and antimicrobial therapeutics. Phage therapy is becoming promising as an alternative for the treatment of antibiotic-resistant infections, but there is still a lack of standardized protocols approved by health organizations for possible use in the clinic. In our research, we focused on the potential use of 1-octanol, which was previously used by our team to develop a method for phage purification from bacterial lysate. 1-octanol is a fatty alcohol that is mostly used in the cosmetics industry, and its advantage is that it is approved by the FDA as a food additive. In this paper, we studied the protective properties of the addition of 1-octanol for storing phage liquid preparations. We demonstrated the stabilization effect of 1-octanol addition on F8 bacteriophage preparation during storage under various conditions. Interestingly, more effective biofilm reduction was observed after treatment with the purified bacteriophage and with 1-octanol addition compared to crude lysate.

Phage-mediated resolution of genetic conflict alters the evolutionary trajectory of Pseudomonas aeruginosa lysogens.

The opportunistic human pathogen Pseudomonas aeruginosa is naturally infected by a large class of temperate, transposable, Mu-like phages. We examined the genotypic and phenotypic diversity of P. aeruginosa PA14 lysogen populations as they resolve clustered regularly interspaced short palindromic repeat (CRISPR) autoimmunity, mediated by an imperfect CRISPR match to the Mu-like DMS3 prophage. After 12 days of evolution, we measured a decrease in spontaneous induction in both exponential and stationary phase growth. Co-existing variation in spontaneous induction rates in the exponential phase depended on the way the coexisting strains resolved genetic conflict. Multiple mutational modes to resolve genetic conflict between host and phage resulted in coexistence in evolved populations of single lysogens that maintained CRISPR immunity to other phages and polylysogens that lost immunity completely. This work highlights a new dimension of the role of lysogenic phages in the evolution of their hosts.IMPORTANCEThe chronic opportunistic multi-drug-resistant pathogen Pseudomonas aeruginosa is persistently infected by temperate phages. We assess the contribution of temperate phage infection to the evolution of the clinically relevant strain UCBPP-PA14. We found that a low level of clustered regularly interspaced short palindromic repeat (CRISPR)-mediated self-targeting resulted in polylysogeny evolution and large genome rearrangements in lysogens; we also found extensive diversification in CRISPR spacers and cas genes. These genomic modifications resulted in decreased spontaneous induction in both exponential and stationary phase growth, increasing lysogen fitness. This work shows the importance of considering latent phage infection in characterizing the evolution of bacterial populations.

Capsid structure of bacteriophage ΦKZ provides insights into assembly and stabilization of jumbo phages.

Jumbo phages are a group of tailed bacteriophages with large genomes and capsids. As a prototype of jumbo phage, ΦKZ infects Pseudomonas aeruginosa, a multi-drug-resistant (MDR) opportunistic pathogen leading to acute or chronic infection in immunocompromised individuals. It holds potential to be used as an antimicrobial agent and as a model for uncovering basic phage biology. Although previous low-resolution structural studies have indicated that jumbo phages may have more complicated capsid structures than smaller phages such as HK97, the detailed structures and the assembly mechanism of their capsids remain largely unknown. Here, we report a 3.5-Å-resolution cryo-EM structure of the ΦKZ capsid. The structure unveiled ten minor capsid proteins, with some decorating the outer surface of the capsid and the others forming a complex network attached to the capsid's inner surface. This network seems to play roles in driving capsid assembly and capsid stabilization. Similar mechanisms of capsid assembly and stabilization are probably employed by many other jumbo viruses.

Bacterial homologs of innate eukaryotic antiviral defenses with anti-phage activity highlight shared evolutionary roots of viral defenses.

Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes.

Control of lysogeny and antiphage defense by a prophage-encoded kinase-phosphatase module.

The filamentous 'Pf' bacteriophages of Pseudomonas aeruginosa play roles in biofilm formation and virulence, but mechanisms governing Pf prophage activation in biofilms are unclear. Here, we identify a prophage regulatory module, KKP (kinase-kinase-phosphatase), that controls virion production of co-resident Pf prophages and mediates host defense against diverse lytic phages. KKP consists of Ser/Thr kinases PfkA and PfkB, and phosphatase PfpC. The kinases have multiple host targets, one of which is MvaU, a host nucleoid-binding protein and known prophage-silencing factor. Characterization of KKP deletion and overexpression strains with transcriptional, protein-level and prophage-based approaches indicates that shifts in the balance between kinase and phosphatase activities regulate phage production by controlling MvaU phosphorylation. In addition, KKP acts as a tripartite toxin-antitoxin system that provides defense against some lytic phages. A conserved lytic phage replication protein inhibits the KKP phosphatase PfpC, stimulating toxic kinase activity and blocking lytic phage production. Thus, KKP represents a phosphorylation-based mechanism for prophage regulation and antiphage defense. The conservation of KKP gene clusters in >1000 diverse temperate prophages suggests that integrated control of temperate and lytic phage infection by KKP-like regulatory modules may play a widespread role in shaping host cell physiology.

Phages produce persisters.

Arguably, the greatest threat to bacteria is phages. It is often assumed that those bacteria that escape phage infection have mutated or utilized phage-defence systems; however, another possibility is that a subpopulation forms the dormant persister state in a manner similar to that demonstrated for bacterial cells undergoing nutritive, oxidative, and antibiotic stress. Persister cells do not undergo mutation and survive lethal conditions by ceasing growth transiently. Slower growth and dormancy play a key physiological role as they allow host phage defence systems more time to clear the phage infection. Here, we investigated how bacteria survive lytic phage infection by isolating surviving cells from the plaques of T2, T4, and lambda (cI mutant) virulent phages and sequencing their genomes. We found that bacteria in plaques can escape phage attack both by mutation (i.e. become resistant) and without mutation (i.e. become persistent). Specifically, whereas T4-resistant and lambda-resistant bacteria with over a 100,000-fold less sensitivity were isolated from plaques with obvious genetic mutations (e.g. causing mucoidy), cells were also found after T2 infection that undergo no significant mutation, retain wild-type phage sensitivity, and survive lethal doses of antibiotics. Corroborating this, adding T2 phage to persister cells resulted in 137,000-fold more survival compared to that of addition to exponentially growing cells. Furthermore, our results seem general in that phage treatments with Klebsiella pneumonia and Pseudomonas aeruginosa also generated persister cells. Hence, along with resistant strains, bacteria also form persister cells during phage infection.

Evaluation of MDR-specific phage Pɸ-Mi-Pa loaded mucoadhesive electrospun nanofibrous scaffolds against drug-resistant Pseudomonas aeruginosa- induced wound infections in an animal model.

Given the escalating prevalence of drug-resistant wounds, there is a justified imperative to explore innovative and more efficacious therapies that diverge from conventional, ineffective wound healing approaches. This research has introduced a strategy to address multi-drug resistant (MDR) Pseudomonas aeruginosa infections in a chronic wound model, employing MDR-specific phage Pɸ-Mi-Pa loaded onto mucoadhesive electrospun scaffolds. A cocktail of three isolates of P. aeruginosa-specific lytic phages, Pɸ-Mi-Pa 51, Pɸ-Mi-Pa 120, and Pɸ-Mi-Pa 133 were incorporated into varying ratios of fabricated PCL-PVP polymer. These formulations were assessed for their therapeutic efficacy in achieving bacterial clearance in P. aeruginosa-induced wound infections. The study encompassed biological characterization through in vivo wound healing assessments, histology, and histomorphometry. Additionally, morphological, mechanical, and chemical analyses were conducted on the fabricated PCL-PVP electrospun nanofibrous scaffolds. Three clonal differences of the MDR P. aeruginosa-specific phages (Pɸ-Mi-Pa 51, Pɸ-Mi-Pa 120, and Pɸ-Mi-Pa 133) produced lytic activity and were seen to produce distinct and clear zones of inhibition against MDR P. aeruginosa strains Pa 051, Pa 120 and Pa 133 respectively. The average porosity of the nanofibrous scaffolds PB 1, PB 2, PB 3, and PB 4 were 12.2 ± 0.3 %, 22.1 ± 0.7 %, 31.1 ± 2.4 %, 28.0 ± 0.8 % respectively. In vitro cumulative release of MDR-specific phage Pɸ-Mi-Pa from the mucoadhesive electrospun nanofibrous scaffolds was found to be 70.91 % ± 1.02 % after 12 h of incubation after an initial release of 42.8 % ± 3.01 % after 1 h. Results from the in vivo wound healing study revealed a substantial reduction in wound size, with formulations PB 2 and PB 3 exhibiting the most significant reduction in wound size, demonstrating statistically significant results on day 5 (100 % ± 31.4 %). These findings underscore the potential of bacteriophage-loaded electrospun PCL-PVP nanofibrous scaffolds for treating drug-resistant wounds, generating tissue substitutes, and overcoming certain limitations associated with conventional wound care matrices.

An intron endonuclease facilitates interference competition between coinfecting viruses.

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ. We show that gp210 targets a specific sequence in ΦKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions.

Screening of the PA14NR Transposon Mutant Library Identifies Genes Involved in Resistance to Bacteriophage Infection in Pseudomomas aeruginosa.

Multidrug-resistant P. aeruginosa infections pose a serious public health threat due to the rise in antimicrobial resistance. Phage therapy has emerged as a promising alternative. However, P. aeruginosa has evolved various mechanisms to thwart phage attacks, making it crucial to decipher these resistance mechanisms to develop effective therapeutic strategies. In this study, we conducted a forward-genetic screen of the P. aeruginosa PA14 non-redundant transposon library (PA14NR) to identify dominant-negative mutants displaying phage-resistant phenotypes. Our screening process revealed 78 mutants capable of thriving in the presence of phages, with 23 of them carrying insertions in genes associated with membrane composition. Six mutants exhibited total resistance to phage infection. Transposon insertions were found in genes known to be linked to phage-resistance such as galU and a glycosyl transferase gene, as well as novel genes such as mexB, lasB, and two hypothetical proteins. Functional experiments demonstrated that these genes played pivotal roles in phage adsorption and biofilm formation, indicating that altering the bacterial membrane composition commonly leads to phage resistance in P. aeruginosa. Importantly, these mutants displayed phenotypic trade-offs, as their resistance to phages inversely affected antibiotic resistance and hindered biofilm formation, shedding light on the complex interplay between phage susceptibility and bacterial fitness. This study highlights the potential of transposon mutant libraries and forward-genetic screens in identifying key genes involved in phage-host interactions and resistance mechanisms. These findings support the development of innovative strategies for combating antibiotic-resistant pathogens.

Combinations of Bacteriophage Are Efficacious against Multidrug-Resistant Pseudomonas aeruginosa and Enhance Sensitivity to Carbapenem Antibiotics.

The Gram-negative ESKAPE bacterium Pseudomonas aeruginosa has become a pathogen of serious concern due its extensive multi-drug resistance (MDR) profile, widespread incidences of hospital-acquired infections throughout the United States, and high occurrence in wound infections suffered by warfighters serving abroad. Bacteriophage (phage) therapy has received renewed attention as an alternative therapeutic option against recalcitrant bacterial infections, both as multi-phage cocktails and in combination with antibiotics as synergistic pairings. Environmental screening and phage enrichment has yielded three lytic viruses capable of infecting the MDR P. aeruginosa strain PAO1. Co-administration of each phage with the carbapenem antibiotics ertapenem, imipenem, and meropenem generated enhanced overall killing of bacteria beyond either phage or drug treatments alone. A combination cocktail of all three phages was completely inhibitory to growth, even without antibiotics. The same 3× phage cocktail also disrupted PAO1 biofilms, reducing biomass by over 75% compared to untreated biofilms. Further, the phage cocktail demonstrated broad efficacy as well, capable of infecting 33 out of 100 diverse clinical isolate strains of P. aeruginosa. Together, these results indicate a promising approach for designing layered medical countermeasures to potentiate antibiotic activity and possibly overcome resistance against recalcitrant, MDR bacteria such as P. aeruginosa. Combination therapy, either by synergistic phage-antibiotic pairings, or by phage cocktails, presents a means of controlling mutations that can allow for bacteria to gain a competitive edge.

Phage-Mediated Digestive Decolonization in a Gut-On-A-Chip Model: A Tale of Gut-Specific Bacterial Prosperity.

Infections due to antimicrobial-resistant bacteria have become a major threat to global health. Some patients may carry resistant bacteria in their gut microbiota. Specific risk factors may trigger the conversion of these carriages into infections in hospitalized patients. Preventively eradicating these carriages has been postulated as a promising preventive intervention. However, previous attempts at such eradication using oral antibiotics or probiotics have led to discouraging results. Phage therapy, the therapeutic use of bacteriophage viruses, might represent a worthy alternative in this context. Taking inspiration from this clinical challenge, we built Gut-On-A-Chip (GOAC) models, which are tridimensional cell culture models mimicking a simplified gut section. These were used to better understand bacterial dynamics under phage pressure using two relevant species: Pseudomonas aeruginosa and Escherichia coli. Model mucus secretion was documented by ELISA assays. Bacterial dynamics assays were performed in GOAC triplicates monitored for 72 h under numerous conditions, such as pre-, per-, or post-bacterial timing of phage introduction, punctual versus continuous phage administration, and phage expression of mucus-binding properties. The potential genomic basis of bacterial phage resistance acquired in the model was investigated by variant sequencing. The bacterial "escape growth" rates under phage pressure were compared to static in vitro conditions. Our results suggest that there is specific bacterial prosperity in this model compared to other in vitro conditions. In E. coli assays, the introduction of a phage harboring unique mucus-binding properties could not shift this balance of power, contradicting previous findings in an in vivo mouse model and highlighting the key differences between these models. Genomic modifications were correlated with bacterial phage resistance acquisition in some but not all instances, suggesting that alternate ways are needed to evade phage predation, which warrants further investigation.

Pseudomonas aeruginosa Bacteriophages and Their Clinical Applications.

Antimicrobial resistance poses a serious risk to contemporary healthcare since it reduces the number of bacterial illnesses that may be treated with antibiotics, particularly for patients with long-term conditions like cystic fibrosis (CF). People with a genetic predisposition to CF often have recurrent bacterial infections in their lungs due to a buildup of sticky mucus, necessitating long-term antibiotic treatment. Pseudomonas aeruginosa infections are a major cause of CF lung illness, and P. aeruginosa airway isolates are frequently resistant to many antibiotics. Bacteriophages (also known as phages), viruses that infect bacteria, are a viable substitute for antimicrobials to treat P. aeruginosa infections in individuals with CF. Here, we reviewed the utilization of P. aeruginosa bacteriophages both in vivo and in vitro, as well as in the treatment of illnesses and diseases, and the outcomes of the latter.

Phage Therapy in a Burn Patient Colonized with Extensively Drug-Resistant Pseudomonas aeruginosa Responsible for Relapsing Ventilator-Associated Pneumonia and Bacteriemia.

Pseudomonas aeruginosa is one of the main causes of healthcare-associated infection in Europe that increases patient morbidity and mortality. Multi-resistant pathogens are a major public health issue in burn centers. Mortality increases when the initial antibiotic treatment is inappropriate, especially if the patient is infected with P. aeruginosa strains that are resistant to many antibiotics. Phage therapy is an emerging option to treat severe P. aeruginosa infections. It involves using natural viruses called bacteriophages, which have the ability to infect, replicate, and, theoretically, destroy the P. aeruginosa population in an infected patient. We report here the case of a severely burned patient who experienced relapsing ventilator-associated pneumonia associated with skin graft infection and bacteremia due to extensively drug-resistant P. aeruginosa. The patient was successfully treated with personalized nebulized and intravenous phage therapy in combination with immunostimulation (interferon-γ) and last-resort antimicrobial therapy (imipenem-relebactam).

Bacteriophages isolated from mouse feces attenuates pneumonia mice caused by Pseudomonas aeruginosa.

BACKGROUND: Most of the current bacteriophages (phages) are mostly isolated from environments. However, phages isolated from feces might be more specific to the bacteria that are harmful to the host. Meanwhile, some phages from the environment might affect non-pathogenic bacteria for the host. METHODS: Here, bacteriophages isolated from mouse feces were intratracheally (IT) or intravenously (IV) administered in pneumonia mice caused by Pseudomonas aeruginosa at 2 hours post-intratracheal bacterial administration. As such, the mice with phage treatment, using either IT or IV administration, demonstrated less severe pneumonia as indicated by mortality, serum cytokines, bacteremia, bacterial abundance in bronchoalveolar lavage fluid (BALF), and neutrophil extracellular traps (NETs) in lung tissue (immunofluorescence of neutrophil elastase and myeloperoxidase). RESULTS: Interestingly, the abundance of phages in BALF from the IT and IV injections was similar, supporting a flexible route of phage administration. With the incubation of bacteria with neutrophils, the presence of bacteriophages significantly improved bactericidal activity, but not NETs formation, with the elevated supernatant IL-6 and TNF-α, but not IL-1ß. In conclusion, our findings suggest that bacteriophages against Pseudomonas aeruginosa can be discovered from feces of the host. CONCLUSIONS: The phages attenuate pneumonia partly through an enhanced neutrophil bactericidal activity, but not via inducing NETs formation. The isolation of phages from the infected hosts themselves might be practically useful for future treatment. More studies are warranted.

High throughput platform technology for rapid target identification in personalized phage therapy.

As bacteriophages continue to gain regulatory approval for personalized human therapy against antibiotic-resistant infections, there is a need for transformative technologies for rapid target identification through multiple, large, decentralized therapeutic phages biobanks. Here, we design a high throughput phage screening platform comprised of a portable library of individual shelf-stable, ready-to-use phages, in all-inclusive solid tablets. Each tablet encapsulates one phage along with luciferin and luciferase enzyme stabilized in a sugar matrix comprised of pullulan and trehalose capable of directly detecting phage-mediated adenosine triphosphate (ATP) release through ATP bioluminescence reaction upon bacterial cell burst. The tablet composition also enhances desiccation tolerance of all components, which should allow easier and cheaper international transportation of phages and as a result, increased accessibility to therapeutic phages. We demonstrate high throughput screening by identifying target phages for select multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli, and Staphylococcus aureus with targets identified within 30-120 min.

Structure of the Bacteriophage PhiKZ Non-virion RNA Polymerase Transcribing from its Promoter p119L.

Bacteriophage ΦKZ (PhiKZ) is the founding member of a family of giant bacterial viruses. It has potential as a therapeutic as its host, Pseudomonas aeruginosa, kills tens of thousands of people worldwide each year. ΦKZ infection is independent of the host transcriptional apparatus; the virus forms a "nucleus", producing a proteinaceous barrier around the ΦKZ genome that excludes the host immune systems. It expresses its own non-canonical multi-subunit non-virion RNA polymerase (nvRNAP), which is imported into its "nucleus" to transcribe viral genes. The ΦKZ nvRNAP is formed by four polypeptides representing homologues of the eubacterial ß/ß' subunits, and a fifth that is likely to have evolved from an ancestral homologue to σ-factor. We have resolved the structure of the ΦKZ nvRNAP initiating transcription from its cognate promoter, p119L, including previously disordered regions. Our results shed light on the similarities and differences between ΦKZ nvRNAP mechanisms of transcription and those of canonical eubacterial RNAPs and the related non-canonical nvRNAP of bacteriophage AR9.

A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant Gram-negative pathogens.

Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.

Safety and tolerability of bronchoscopic and nebulised administration of bacteriophage.

INTRODUCTION: Pseudomonas aeruginosa is an organism well known for causing significant morbidity and mortality in people living with chronic lung conditions such as cystic fibrosis. We describe the safety, tolerability, and potential efficacy of bronchoscopic and nebulised bacteriophage administration, offering insights into a potential breakthrough for the treatment of chronic infections particularly in children and adolescents. METHOD: A 12-year-old female (F12) and a 17-year-old male (M17), both diagnosed with cystic fibrosis and chronic P. aeruginosa lung infection, underwent bacteriophage treatment (BT). The administration involved bronchoscopic instillation and subsequent nebulisation. This was performed concurrently with intravenous antibiotics and regular physiotherapy delivered in an in-patient setting for 14 days. Microbiological, clinical, and lung function assessments were conducted to assess this treatment modality. RESULTS: No adverse events (fever, localised reaction, wheeze or bronchospasm) occurred during BT. F12 demonstrated a 4% increase, while M17 showed a 5% improvement in FEV1% from their best FEV1% over the past three years following BT. A 12% (F12) and an 8% (M17) improvement from baseline FEV1% was observed. For F12 P. aeruginosa was not isolated from her sputum despite 12 previous hospitalisations for intravenous antibiotics. CONCLUSION: Bronchoscopic and nebulised routes of bacteriophage administration were well-tolerated in these two adolescents. This early report underscores the potential of this treatment modality and encourages clinicians and researchers to actively explore this innovative approach.

The uncharacterized PA3040-3042 operon is part of the cell envelope stress response and a tobramycin resistance determinant in a clinical isolate of Pseudomonas aeruginosa.

Bacteriophages (hereafter "phages") are ubiquitous predators of bacteria in the natural world, but interest is growing in their development into antibacterial therapy as complement or replacement for antibiotics. However, bacteria have evolved a huge variety of antiphage defense systems allowing them to resist phage lysis to a greater or lesser extent. In addition to dedicated phage defense systems, some aspects of the general stress response also impact phage susceptibility, but the details of this are not well known. In order to elucidate these factors in the opportunistic pathogen Pseudomonas aeruginosa, we used the laboratory-conditioned strain PAO1 as host for phage infection experiments as it is naturally poor in dedicated phage defense systems. Screening by transposon insertion sequencing indicated that the uncharacterized operon PA3040-PA3042 was potentially associated with resistance to lytic phages. However, we found that its primary role appeared to be in regulating biofilm formation, particularly in a clinical isolate of P. aeruginosa in which it also altered tobramycin resistance. Its expression was highly growth-phase dependent and responsive to phage infection and cell envelope stress. Our results suggest that this operon may be a cryptic but important locus for P. aeruginosa stress tolerance. IMPORTANCE: An important category of bacterial stress response systems is bacteriophage defense, where systems are triggered by bacteriophage infection and activate a response which may either destroy the phage genome or destroy the infected cell so that the rest of the population survives. In some bacteria, the cell envelope stress response is activated by bacteriophage infection, but it is unknown whether this contributes to the survival of the infection. We have found that a conserved uncharacterized operon (PA3040-PA3042) of the cell envelope stress regulon in Pseudomonas aeruginosa, which has very few dedicated phage defense systems, responds to phage infection and stationary phase as well as envelope stress and is important for growth and biofilm formation in a clinical isolate of P. aeruginosa, even in the absence of phages. As homologs of these genes are found in other bacteria, they may be a novel component of the general stress response.

Bacteriophages as potential antibiotic potentiators in cystic fibrosis: A new model to study the combination of antibiotics with a bacteriophage cocktail targeting dual species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

OBJECTIVES: Staphylococcus aureus and Pseudomonas aeruginosa co-infections in patients with cystic fibrosis (CF) are associated with disease severity. Their treatment is complicated by biofilm formation in the sticky mucus obstructing the airways. We investigated the activity of phages-antibiotics combinations using a dual species biofilm (P. aeruginosa/S. aureus) formed in artificial sputum medium. METHODS: Biofilmswere incubated with broad-spectrum antibiotics (meropenem, ceftazidime, ciprofloxacin, tobramycin) combined with a cocktail of two (bacterio)phages (PSP3 and ISP) proven active via spot tests and double agar on P. aeruginosa PAO1 and S. aureus ATCC 25923. RESULTS: At the highest tested concentrations (100 x MIC), antibiotics alone caused a 20-50% reduction in biomass and reduced S. aureus and P. aeruginosa CFU of 2.3 to 2.8 and 2.1 to 3.6 log10, respectively. Phages alone reduced biofilm biomass by 23% and reduced P. aeruginosa CFU of 2.1 log10, but did not affect S. aureus viability. Phages enhanced antibiotic effects on biomass and exhibited additive effects with antibiotics against P. aeruginosa, but not against S. aureus. Following inhibition of bacterial respiration by phages in planktonic cultures rationalised these observations by demonstrating that PSP3 was effective at multiplicities of infection (MOI) as low as 10-4 plaque forming units (PFU)/CFU on P. aeruginosa, but ISP, at higher MOI (> 0.1) against S. aureus. CONCLUSION: Pre-screening inhibition of bacterial respiration by phages may assist in selecting those showing activity at sufficiently low titers to showcase anti-biofilm activity in this complex but clinically-relevant in vitro model of biofilm.