Resolvin D1 and D2 reduce SARS-CoV-2-induced inflammatory responses in cystic fibrosis macrophages.

An excessive, non-resolving inflammatory response underlies severe COVID-19 that may have fatal outcomes. Therefore, the investigation of endogenous pathways leading to resolution of inflammation is of interest to uncover strategies for mitigating inflammation in people with SARS-CoV-2 infection. This becomes particularly urgent in individuals with preexisting pathologies characterized by chronic respiratory inflammation and prone to bacterial infection, such as cystic fibrosis (CF). Here, we analyzed the immune responses to SARS-CoV-2 virion spike 1 glycoprotein (S1) of macrophages (MΦ) from volunteers with and without CF and tested the efficacy of resolvins (Rv) D1 and D2 in regulating the inflammatory and antimicrobial functions of MΦ exposed to S1. S1 significantly increased chemokine release, including interleukin (IL)-8, in CF and non-CF MΦ, while it enhanced IL-6 and tumor necrosis factor (TNF)-α in non-CF MΦ, but not in CF cells. S1 also triggered the biosynthesis of RvD1 and modulated microRNAs miR-16, miR-29a, and miR-103, known to control the inflammatory responses. RvD1 and RvD2 treatment abated S1-induced inflammatory responses in CF and non-CF MΦ, significantly reducing the release of select chemokines and cytokines including IL-8 and TNF-α. RvD1 and RvD2 both restored the expression of miR-16 and miR-29a, while selectively increasing miR-223 and miR-125a, which are involved in NF-κB activation and MΦ inflammatory polarization. During Pseudomonas aeruginosa infection, S1 stimulated the MΦ phagocytic activity that was further enhanced by RvD1 and RvD2. These results provide a map of molecular responses to SARS-CoV-2 in MΦ, key determinants of COVID-19-related inflammation, unveiling some peculiarity in the response of cells from individuals with CF. They also demonstrate beneficial, regulatory actions of RvD1 and RvD2 on SARS-CoV-2-induced inflammation.

High mortality by nosocomial infections caused by carbapenem-resistant P. aeruginosa in a referral hospital in Brazil: facing the perfect storm.

Introduction. Carbapenem-resistant Pseudomonas aeruginosa is responsible for increased patient mortality.Gap Statement. Five and 30 day in-hospital all-cause mortality in patients with P. aeruginosa infections were assessed, followed by evaluations concerning potential correlations between the type III secretion system (TTSS) genotype and the production of metallo-ß-lactamase (MBL).Methodology. This assessment comprised a retrospective cohort study including consecutive patients with carbapenem-resistant infections hospitalized in Brazil from January 2009 to June 2019. PCR analyses were performed to determine the presence of TTSS-encoding genes and MBL genes.Results. The 30-day and 5-day mortality rates for 262 patients were 36.6 and 17.9 %, respectively. The unadjusted survival probabilities for up to 5 days were 70.55 % for patients presenting exoU-positive isolates and 86 % for those presenting exo-negative isolates. The use of urinary catheters, as well as the presence of comorbidity conditions, secondary bacteremia related to the respiratory tract, were independently associated with death at 5 and 30 days. The exoS gene was detected in 64.8 % of the isolates, the presence of the exoT and exoY genes varied and exoU genes occurred in 19.3 % of the isolates. The exoU genotype was significantly more frequent among multiresistant strains. MBL genes were not detected in 92 % of the isolates.Conclusions. Inappropriate therapy is a crucial factor regarding the worse prognosis among patients with infections caused by multiresistant P. aeruginosa, especially those who died within 5 days of diagnosis, regardless of the genotype associated with TTSS virulence.

Susceptibility of Pseudomonas aeruginosa veterinary isolates to Pbunavirus PB1-like phages.

In recent years, antimicrobial-resistant Pseudomonas aeruginosa strains have increased in the veterinary field. Therefore, phage therapy has received significant attention as an approach for overcoming antimicrobial resistance. In this context, we isolated and characterized four Pseudomonas bacteriophages. Phylogenetic analysis showed that the isolated phages are novel Myoviridae Pbunavirus PB1-like phages with ØR12 belonging to a different clade compared with the other three. These phages had distinct lytic activity against 22 P. aeruginosa veterinary isolates. The phage cocktail composed from the PB1-like phages clearly inhibited the occurrence of the phage-resistant variant, suggesting that these phages could be useful in phage therapy.

Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections.

Pf bacteriophage are temperate phages that infect the bacterium Pseudomonas aeruginosa, a major cause of chronic lung infections in cystic fibrosis (CF) and other settings. Pf and other temperate phages have evolved complex, mutualistic relationships with their bacterial hosts that impact both bacterial phenotypes and chronic infection. We and others have reported that Pf phages are a virulence factor that promote the pathogenesis of P. aeruginosa infections in animal models and are associated with worse skin and lung infections in humans. Here we review the biology of Pf phage and what is known about its contributions to pathogenesis and clinical disease. First, we review the structure, genetics, and epidemiology of Pf phage. Next, we address the diverse and surprising ways that Pf phages contribute to P. aeruginosa phenotypes including effects on biofilm formation, antibiotic resistance, and motility. Then, we cover data indicating that Pf phages suppress mammalian immunity at sites of bacterial infection. Finally, we discuss recent literature implicating Pf in chronic P. aeruginosa infections in CF and other settings. Together, these reports suggest that Pf bacteriophage have direct effects on P. aeruginosa infections and that temperate phages are an exciting frontier in microbiology, immunology, and human health.

Co-infection of H9N2 influenza virus and Pseudomonas aeruginosa contributes to the development of hemorrhagic pneumonia in mink.

Influenza A virus (IAV) and bacteria co-infection can influence the host clinical conditions. Both H9N2 IAV and Pseudomonas aeruginosa (P. aeruginosa) are potential pathogens of respiratory diseases in mink. In this study, to clarify the effects of H9N2 IAV and P. aeruginosa co-infections on hemorrhagic pneumonia in mink, we carried out to establish the mink models of the two-pathogen co-infections in different orders. Compared with the single infections with H9N2 IAV or P. aeruginosa, the mink co-infected with H9N2 IAV and P. aeruginosa showed severe respiratory diseases, and exacerbated histopathological lesions and more obvious apoptosis in the lung tissues. H9N2 IAV shedding and viral loads in the lungs of the mink co-infected with H9N2 IAV and P. aeruginosa were higher than those in the mink with single H9N2 IAV infection. Furthermore, the clearance of P. aeruginosa in the co-infected mink lungs was delayed. In addition, the anti-H9N2 antibody titers in mink with P. aeruginosa co-infection following H9N2 IAV infection were significantly higher than those of the other groups. This implied that H9N2 IAV and P. aeruginosa co-infection contributed to the development of hemorrhagic pneumonia in mink, and that P. aeruginosa should play a major role in the disease. The exact interaction mechanism among H9N2 IAV, P. aeruginosa and the host needs to be further investigated.

Resistance Evolution against Phage Combinations Depends on the Timing and Order of Exposure.

Phage therapy is a promising alternative to chemotherapeutic antibiotics for the treatment of bacterial infections. However, despite recent clinical uses of combinations of phages to treat multidrug-resistant infections, a mechanistic understanding of how bacteria evolve resistance against multiple phages is lacking, limiting our ability to deploy phage combinations optimally. Here, we show, using Pseudomonas aeruginosa and pairs of phages targeting shared or distinct surface receptors, that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance. Whereas sequential exposure allowed bacteria to acquire multiple resistance mutations effective against both phages, this evolutionary trajectory was prevented by simultaneous exposure, resulting in quantitatively weaker resistance. The order of phage exposure determined the fitness costs of sequential resistance, such that certain sequential orders imposed much higher fitness costs than the same phage pair in the reverse order. Together, these data suggest that phage combinations can be optimized to limit the strength of evolved resistances while maximizing their associated fitness costs to promote the long-term efficacy of phage therapy.IMPORTANCE Globally rising rates of antibiotic resistance have renewed interest in phage therapy where combinations of phages have been successfully used to treat multidrug-resistant infections. To optimize phage therapy, we first need to understand how bacteria evolve resistance against combinations of multiple phages. Here, we use simple laboratory experiments and genome sequencing to show that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance evolution in the opportunistic pathogen Pseudomonas aeruginosa These findings suggest that phage combinations can be optimized to limit the emergence and persistence of resistance, thereby promoting the long-term usefulness of phage therapy.

Identification of a lytic Pseudomonas aeruginosa phage depolymerase and its anti-biofilm effect and bactericidal contribution to serum.

Pseudomonas aeruginosa (P. aeruginosa) infection has imposed a great threat to patients with cystic fibrosis. With the emergence of multidrug-resistant P. aeruginosa, developing an alternative anti-microbial strategy is indispensable and more urgent than ever. In this study, a lytic P. aeruginosa phage was isolated from the sewage of a hospital, and one protein was predicted as the depolymerase-like protein by genomic sequence analysis, it includes two catalytic regions, the Pectate lyase_3 super family and Glycosyl hydrolase_28 super family. Further analysis demonstrated that recombinant depolymerase-like protein degraded P. aeruginosa exopolysaccharide and enhanced bactericidal activity mediated by serum in vitro. Additionally, this protein disrupted host bacterial biofilms. All of these results showed that the phage-derived depolymerase-like protein has the potential to be developed into an anti-microbial agent that targets P. aeruginosa.

Mechanistic research holds promise for bacterial vaccines and phage therapies for Pseudomonas aeruginosa.

Vaccines for Pseudomonas aeruginosa have been of longstanding interest to immunologists, bacteriologists, and clinicians, due to the widespread prevalence of hospital-acquired infection. As P. aeruginosa becomes increasingly antibiotic resistant, there is a dire need for novel treatments and preventive vaccines. Despite intense efforts, there currently remains no vaccine on the market to combat this dangerous pathogen. This article summarizes current and past vaccines under development that target various constituents of P. aeruginosa. Targeting lipopolysaccharides and O-antigens have shown some promise in preventing infection. Recombinant flagella and pili that target TLR5 have been utilized to combat P. aeruginosa by blocking its motility and adhesion. The type 3 secretion system components, such as needle-like structure PcrV or exotoxin PopB, are also potential vaccine targets. Outer membrane proteins including OprF and OprI are newer representatives of vaccine candidates. Live attenuated vaccines are a focal point in this review, and are also considered for novel vaccines. In addition, phage therapy is revived as an effective option for treating refractory infections after failure with antibiotic treatment. Many of the aforementioned vaccines act on a single target, thus lacking a broad range of protection. Recent studies have shown that mixtures of vaccines and combination approaches may significantly augment immunogenicity, thereby increasing their preventive and therapeutic potential.

Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model.

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.

Screening for Growth-Inhibitory ORFans in Pseudomonas aeruginosa-Infecting Bacteriophages.

Like all viruses, bacteriophages heavily depend on their host's physiology for reproduction. Therefore, phages have evolved numerous proteins that influence the host metabolism to facilitate the infection process. Some of these proteins strongly perturb the host cell, ultimately leading to cell death. These growth-inhibitory phage proteins presumably target key metabolic processes, which may provide a basis for innovative phage-derived antibacterials. Unfortunately, most of these proteins are the so-called ORFans, since they have no known function or sequence homology to any other gene. We here describe a screening method for the identification of growth-inhibitory ORFans of bacteriophages infecting gram-negative bacteria (e.g., Pseudomonas aeruginosa), using the pUC18-mini-Tn7T-Lac vector system, which allows for stable single-copy integration of the phage ORFans in the Pseudomonas genome under the control of an IPTG-inducible promoter. Furthermore, we describe a method to examine the effect of the phage proteins in different hosts, using different vector copy numbers. Finally, we explain how to investigate the effect of ORFan expression on the host morphology using time-lapse microscopy.

Exploitation of Drosophila Infection Models to Evaluate Antibacterial Efficacy of Phages.

Nonmammalian infection models have been exploited to understand the various aspects of host-pathogen interactions and also provided innovative research platforms for identification of virulence factors, screening for antimicrobial hits, and evaluation of antimicroial efficacy. Here we describe a relatively straightforward protocol to assess the antibacterial efficacy of bacteriophages (phages) toward the opportunistic human pathogen, Pseudomonas aeruginosa, based on the systemic infection model using the fruit fly, Drosophila melanogaster. Since phages, unlike antibacterial chemicals, can be easily and sensitively enumerated by simple assays, it is also possible to address the pharmacokinetic properties of administered phages even in this small-scale infection model.

BrlR from Pseudomonas aeruginosa is a receptor for both cyclic di-GMP and pyocyanin.

The virulence factor pyocyanin and the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP) play key roles in regulating biofilm formation and multi-drug efflux pump expression in Pseudomonas aeruginosa. However, the crosstalk between these two signaling pathways remains unclear. Here we show that BrlR (PA4878), previously identified as a c-di-GMP responsive transcriptional regulator, acts also as a receptor for pyocyanin. Crystal structures of free BrlR and c-di-GMP-bound BrlR reveal that the DNA-binding domain of BrlR contains two separate c-di-GMP binding sites, both of which are involved in promoting brlR expression. In addition, we identify a pyocyanin-binding site on the C-terminal multidrug-binding domain based on the structure of the BrlR-C domain in complex with a pyocyanin analog. Biochemical analysis indicates that pyocyanin enhances BrlR-DNA binding and brlR expression in a concentration-dependent manner.

Recovery and Characterization of Bacteria Resisting Infection by Lytic Bacteriophage.

Bacteria and bacteriophages coexist and coevolve, bacteriophages being obligatory predators exerting an evolutionary pressure on their prey. Mechanisms in action vary depending on the bacterial genomic content and on the regulation of the bacteriophage cycle. To assess the multiplicity of bacterial genes involved in resistance as well as the changes in the bacteriophage interactions with the bacteria, it is necessary to isolate and investigate large numbers of independent resistant variants. Here we describe protocols that have been applied to the study of Pseudomonas aeruginosa and four of its virulent bacteriophages belonging to the Podoviridae and Myoviridae bacteriophage families. Mutations are identified using whole genome sequencing of resistant variants. Phenotypic analyses are performed to describe the changes conferred by the mutations.

Activity of Bacteriophages in Removing Biofilms of Pseudomonas aeruginosa Isolates from Chronic Rhinosinusitis Patients.

Introduction:Pseudomonas aeruginosa infections are prevalent amongst chronic rhinosinusitis (CRS) sufferers. Many P. aeruginosa strains form biofilms, leading to treatment failure. Lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death. Aim: To assess the activity of a phage cocktail in eradicating biofilms of ex vivo P.aeruginosa isolates from CRS patients. Methods: P. aeruginosa isolates from CRS patients with and without cystic fibrosis (CF) across three continents were multi-locus sequence typed and tested for antibiotic resistance. Biofilms grown in vitro were treated with a cocktail of four phages (CT-PA). Biofilm biomass was measured after 24 and 48 h, using a crystal violet assay. Phage titrations were performed to confirm replication of the phages. A linear mixed effects model was applied to assess the effects of treatment, time, CF status, and multidrug resistance on the biomass of the biofilm. Results: The isolates included 44 strain types. CT-PA treatment significantly reduced biofilm biomass at both 24 and 48 h post-treatment (p < 0.0001), regardless of CF status or antibiotic resistance. Biomass was decreased by a median of 76% at 48 h. Decrease in biofilm was accompanied by a rise in phage titres for all except one strain. Conclusion: A single dose of phages is able to significantly reduce biofilms formed in vitro by a range of P.aeruginosa isolates from CRS patients. This represents an exciting potential and novel targeted treatment for P. aeruginosa biofilm infections and multidrug resistant bacteria.

Quorum sensing controls the Pseudomonas aeruginosa CRISPR-Cas adaptive immune system.

CRISPR-Cas are prokaryotic adaptive immune systems that provide protection against bacteriophage (phage) and other parasites. Little is known about how CRISPR-Cas systems are regulated, preventing prediction of phage dynamics in nature and manipulation of phage resistance in clinical settings. Here, we show that the bacterium Pseudomonas aeruginosa PA14 uses the cell-cell communication process, called quorum sensing, to activate cas gene expression, to increase CRISPR-Cas targeting of foreign DNA, and to promote CRISPR adaptation, all at high cell density. This regulatory mechanism ensures maximum CRISPR-Cas function when bacterial populations are at highest risk for phage infection. We demonstrate that CRISPR-Cas activity and acquisition of resistance can be modulated by administration of pro- and antiquorum-sensing compounds. We propose that quorum-sensing inhibitors could be used to suppress the CRISPR-Cas adaptive immune system to enhance medical applications, including phage therapies.

Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo.

Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

Characterizing Non-Tuberculous Mycobacteria Infection in Bronchiectasis.

Chronic airway infection is a key aspect of the pathogenesis of bronchiectasis. A growing interest has been raised on non-tuberculous mycobacteria (NTM) infection. We aimed at describing the clinical characteristics, diagnostic process, therapeutic options and outcomes of bronchiectasis patients with pulmonary NTM (pNTM) disease. This was a prospective, observational study enrolling 261 adult bronchiectasis patients during the stable state at the San Gerardo Hospital, Monza, Italy, from 2012 to 2015. Three groups were identified: pNTM disease; chronic P. aeruginosa infection; chronic infection due to bacteria other than P. aeruginosa. NTM were isolated in 32 (12%) patients, and among them, a diagnosis of pNTM disease was reached in 23 cases. When compared to chronic P. aeruginosa infection, patients with pNTM were more likely to have cylindrical bronchiectasis and a "tree-in-bud" pattern, a history of weight loss, a lower disease severity and a lower number of pulmonary exacerbations. Among pNTM patients who started treatment, 68% showed a radiological improvement, and 37% achieved culture conversion without recurrence, while 21% showed NTM isolation recurrence. NTM isolation seems to be a frequent event in bronchiectasis patients, and few parameters might help to suspect NTM infection. Treatment indications and monitoring still remain an important area for future research.

Production of Inhalation Phage Powders Using Spray Freeze Drying and Spray Drying Techniques for Treatment of Respiratory Infections.

PURPOSE: The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders. METHOD: A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1 = 60:20:20 and F2 = 40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed. RESULTS: A significant titer loss (~2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1 = 13.1 ± 1.7 × 10(4) pfu and SD-F2 = 11.0 ± 1.4 × 10(4) pfu) than from their counterpart SFD formulations (SFD-F1 = 8.3 ± 1.8 × 10(4) pfu and SFD-F2 = 2.1 ± 0.3 × 10(4) pfu). CONCLUSION: Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.

Bacteriophage can lyse antibiotic-resistant Pseudomonas aeruginosa isolated from canine diseases.

Pseudomonas aeruginosa is a pathogen frequently identified as the cause of diverse infections or chronic disease. This microbe has natural resistance to several kinds of antibiotics, because of the species' outer membrane, efflux pumps and growth as a biofilm. This bacterium can acquire increased resistance with specific point mutations. Bacteriophage (phage), however, can lyse these bacteria. Therefore, in the present study, we assessed the host range of phages isolates and their ability to lyse antibiotic-resistant P. aeruginosa. Present phages could lyse many strains of P. aeruginosa (28/39), including strains with high resistance to fluoroquinolones (4/6). In conclusion, application of phages for antibiotic-resistant bacteria is greatly effective. To avoid pervasive antibiotic-resistant bacteria, further development of phage usage for disease treatment is required.

Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion.

To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS) and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.