Bacteriophage-based decontamination to control environmental colonization by Staphylococcus capitis in neonatal intensive care units: An in vitro proof-of-concept.

Introduction: In neonatal intensive care units (NICUs), the standard chemical-based disinfection procedures do not allow a complete eradication of pathogens from environmental surfaces. In particular, the clone Staphylococcus capitis NRCS-A, a significant pathogen in neonates, was shown to colonize neonatal incubators. The aim of this study was to evaluate the in vitro effect of a bacteriophage cocktail on NRCS-A eradication. Methods: Three bacteriophages were isolated, genetically characterized and assessed for their host range using a collection of representative clinical strains (n=31) belonging to the clone NRCS-A. The efficacy of a cocktail including these three bacteriophages to eradicate the reference strain S. capitis NRCS-A CR01 was determined in comparison or in combination with the chemical disinfectant Surfanios Premium on either dry inoculum or biofilm-embedded bacteria. The emergence of bacterial resistance against the bacteriophages alone or in cocktail was evaluated by growth kinetics. Results: The three bacteriophages belonged to two families and genera, namely Herelleviridae/Kayvirus for V1SC01 and V1SC04 and Rountreeviridae/Andhravirus for V1SC05. They were active against 17, 25 and 16 of the 31 tested strains respectively. Bacteriophage cocktails decreased the bacterial inoculum of both dry spots and biofilms, with a dose dependent effect. The sequential treatment with bacteriophages then Surfanios Premium did not show enhanced efficacy. No bacterial resistance was observed when using the bacteriophage cocktail. Discussion: This study established a proof-of-concept for the use of bacteriophages to fight against S. capitis NRCS-A. Further investigations are needed using a larger bacterial collection and in real-life conditions before being able to use such technology in NICUs.

Phage Therapy against Staphylococcus aureus: Selection and Optimization of Production Protocols of Novel Broad-Spectrum Silviavirus Phages.

Background: Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus. Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 1011 PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages. Conclusions: In sum, we propose a quality-by-design approach for the amplification of broad-spectrum anti-S. aureus phages, facilitating the subsequent steps of the manufacturing process; namely, purification and quality control.

Paving the way for phage therapy using novel drug delivery approaches.

Bacterial resistance against antibiotics is an emergent medical issue. The development of novel therapeutic approaches is urgently needed and, in this context, bacteriophages represent a promising strategy to fight multi resistant bacteria. However, for some applications, bacteriophages cannot be used without an appropriate drug delivery system which increases their stability or provides an adequate targeting to the site of infection. This review summarizes the main application routes for bacteriophages and presents the new delivery approaches designed to increase phage's activity. Clinical successes of these formulations are also highlighted. Globally, this work paves the way for the design and optimization of nano and micro delivery systems for phage therapy.

Past and Future of Phage Therapy and Phage-Derived Proteins in Patients with Bone and Joint Infection.

Phage-derived therapies comprise phage therapy and the use of phage-derived proteins as anti-bacterial therapy. Bacteriophages are natural viruses that target specific bacteria. They were proposed to be used to treat bacterial infections in the 1920s, before the discovery and widespread over-commercialized use of antibiotics. Phage therapy was totally abandoned in Western countries, whereas it is still used in Poland, Georgia and Russia. We review here the history of phage therapy by focusing on bone and joint infection, and on the development of phage therapy in France in this indication. We discuss the rationale of its use in bacterial infection and show the feasibility of phage therapy in the 2020s, based on several patients with complex bone and joint infection who recently received phages as compassionate therapy. Although the status of phage therapy remains to be clarified by health care authorities, obtaining pharmaceutical-grade therapeutic phages (i.e., following good manufacturing practice guidelines or being "GMP-like") targeting bacterial species of concern is essential. Moreover, multidisciplinary clinical expertise has to determine what could be the relevant indications to perform clinical trials. Finally "phage therapy 2.0" has to integrate the following steps: (i) follow the status of phage therapy, that is not settled and defined; (ii) develop in each country a close relationship with the national health care authority; (iii) develop industrial-academic partnerships; (iv) create academic reference centers; (v) identify relevant clinical indications; (vi) use GMP/GMP-like phages with guaranteed quality bioproduction; (vii) start as salvage therapy; (vii) combine with antibiotics and adequate surgery; and (viii) perform clinical trials, to finally (ix) demonstrate in which clinical settings phage therapy provides benefit. Phage-derived proteins such as peptidoglycan hydrolases, polysaccharide depolymerases or lysins are enzymes that also have anti-biofilm activity. In contrast to phages, their development has to follow the classical process of medicinal products. Phage therapy and phage-derived products also have a huge potential to treat biofilm-associated bacterial diseases, and this is of crucial importance in the worldwide spread of antimicrobial resistance.

Evaluation of the Activity of a Combination of Three Bacteriophages Alone or in Association with Antibiotics on Staphylococcus aureus Embedded in Biofilm or Internalized in Osteoblasts.

Staphylococcus aureus is responsible for difficult-to-treat bone and joint infections (BJIs). This is related to its ability to form biofilm and to be internalized and persist inside osteoblasts. Recently, bacteriophage therapy has emerged as a promising option to improve treatment of such infections, but data on its activity against the specific bacterial lifestyles presented above remain scarce. We evaluated the activity of a combination of three bacteriophages, recently used for compassionate treatment in France, against S. aureus HG001 in a model of staphylococcal biofilm and a model of osteoblasts infection, alone or in association with vancomycin or rifampin. The activity of bacteriophages against biofilm-embedded S. aureus was dose dependent. In addition, synergistic effects were observed when bacteriophages were combined with antibiotics used at the lowest concentrations. Phage penetration into osteoblasts was observed only when the cells were infected, suggesting a S. aureus-dependent Trojan horse mechanism for internalization. The intracellular bacterial count of bacteria in infected osteoblasts treated with bacteriophages as well as with vancomycin was significantly higher than in cells treated with lysostaphin, used as a control condition, owing to the absence of intracellular activity and the rapid killing of bacteria released after the death of infected cells. These results suggest that bacteriophages are both inactive in the intracellular compartment after being internalized in infected osteoblasts and present a delayed killing effect on bacteria released after cell lysis into the extracellular compartment, which avoids preventing them from infecting other osteoblasts. The combination of bacteriophages tested was highly active against S. aureus embedded in biofilm but showed no activity against intracellular bacteria in the cell model used.

Salvage Debridement, Antibiotics and Implant Retention ("DAIR") With Local Injection of a Selected Cocktail of Bacteriophages: Is It an Option for an Elderly Patient With Relapsing Staphylococcus aureus Prosthetic-Joint Infection?

Local injection of a bacteriophages mix during debridement, antibiotics and implant retention ("DAIR") was performed to treat a relapsing Staphylococcus aureus chronic prosthetic joint infection (PJI). This salvage treatment was safe and associated with a clinical success. Scientific evaluation of the potential clinical benefit of bacteriophages as antibiofilm treatment in PJI is now feasible and required.

Cytosolic phospholipase A2α enhances mouse mortality induced by Pseudomonas aeruginosa pulmonary infection via interleukin 6.

Pseudomonas aeruginosa pulmonary infection is a leading cause of death in numerous diseases such as cystic fibrosis (CF). The host cytosolic phospholipase A2α (cPLA2α) releases lipid mediators that play an important role in the pathogenesis of diseases, but its role in lung injury induced by P. aeruginosa infection is still obscure. Using an animal model of P. aeruginosa lung infection, we showed that the CHA strain of P. aeruginosa was more potent than the PAK strain in inducing mouse mortality and lung injury, and that both mouse mortality and lung injury were reduced in cPLA2α(-/-) mice as compared to cPLA2α(+/+) mice. This was accompanied by decreased levels of IL6 but not other inflammatory cytokines (IL1ß, KC and TNFα) in the bronchoalveolar lavage fluids (BALFs) of cPLA2α(-/-) mice. Given that CFTR(-/-) mice exhibit increased cPLA2α activation in the lung, the role of cPLA2α was further examined in this lung infection model. Compared to littermates, P. aeruginosa infection caused increased mortality in CFTR(-/-) mice with high IL6 levels in BALFs, which was attenuated by pharmacological inhibition of cPLA2α. In addition, compared to IL6(-/-) mice, an enhanced mortality was also observed in P. aeruginosa infected IL6(+/+) mice. Since alveolar macrophages (AMs) are the primary inflammatory cytokine source in the lung, murine AMs cell line (MH-S) were used to investigate the signalling pathways involved in this process. Incubation of MH-S cells with P. aeruginosa induced IL6 production, which was mediated by MAPKs ERK/p38 and was abolished by cPLA2α inhibitors. Furthermore, among cPLA2 downstream signalling pathways, only 15-lipoxygenase (15-LOX) and cyclooxygenase-2 (COX-2) were proven to participate in this P. aeruginosa-induced IL6 expression. Based on all these observations, we conclude that cPLA2α enhances P. aeruginosa-induced animal lethality in part via IL6 induction and that MAPKs ERK/p38, 15-LOX and COX-2 signalling pathways were involved in this process.

Toll-like receptor 9 deficiency protects mice against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. While a number of studies have demonstrated the roles of TLR2, TLR4 and TLR5 in host defense againt P. aeruginosa infection, the implication of TLR9 in this process has been overlooked. Here, we show that P. aeruginosa DNA stimulates the inflammatory response through TLR9 pathway in both a cell line and primary alveolar macrophages (AMs). This activation requires asparagine endopeptidase- and endosomal acidification. Interestingly, TLR9-/- mice resisted to lethal lung infection by P. aeruginosa, compared to WT C57BL/6 mice. The resistance of TLR9-/- mice to P. aeruginosa infection was associated with: (i) a higher ability of TLR9-/- AMs to kill P. aeruginosa; (ii) a rapid increase in the pro-inflammatory cytokines such as TNFα, IL-1ß and IL-6 production; and (iii) an increase in nitric oxide (NO) production and inductible NO synthase expression in AMs. In addition, inhibition of both IL-1ß and NO production resulted in a significant decrease of P. aeruginosa clearance by AMs. Altogether these results indicate that TLR9 plays a detrimental role in pulmonary host defense toward P. aeruginosa by reducing the AMs clearance activity and production of IL-1ß and NO necessary for bacteria killing.

Non-invasive in vivo study of the Trypanosoma vivax infectious process consolidates the brain commitment in late infections.

Trypanosoma vivax, one of the leading parasites responsible for Animal African Trypanosomosis (Nagana), is generally cyclically transmitted by Glossina spp. but in areas devoid of the tsetse flies in Africa or in Latin American countries is mechanically transmitted across vertebrate hosts by other haematophagous insects, including tabanids. We followed on from our recent studies on the maintenance of this parasite in vivo and in vitro, and its genetic manipulation, by constructing a West African IL1392 T. vivax strain that stably expresses firefly luciferase and is fully virulent for immunocompetent mice. We report here on a study where murine infection with this strain was monitored in vivo using a non-invasive method. Study findings fully support the use of this strain in the assessment of parasite dynamics in vivo since a strong correlation was found between whole body light emission measured over the course of the infection and parasitemia determined microscopically. In addition, parasitemia and survival rates were very similar for mice infected by the intraperitoneal and sub-cutaneous routes, except for a longer prepatent period following sub-cutaneous inoculation with the parasite. Our results clearly show that when administered by the subcutaneous route, the parasite is retained few days in the skin close to the inoculation site where it multiplies before passing into the bloodstream. Ex vivo bioluminescence analyses of organs isolated from infected mice corroborated our previous histopathological observations with parasite infiltration into spleen, liver and lungs. Finally, our study reinforces previous observations on the presence of the parasite in the central nervous system and consequently the brain commitment in the very late phases of the experimental infection.

A crucial role of Flagellin in the induction of airway mucus production by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis.

Genetic engineering of Trypanosoma (Dutonella) vivax and in vitro differentiation under axenic conditions.

Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents.

Trypanosoma vivax infections: pushing ahead with mouse models for the study of Nagana. I. Parasitological, hematological and pathological parameters.

African trypanosomiasis is a severe parasitic disease that affects both humans and livestock. Several different species may cause animal trypanosomosis and although Trypanosoma vivax (sub-genus Duttonella) is currently responsible for the vast majority of debilitating cases causing great economic hardship in West Africa and South America, little is known about its biology and interaction with its hosts. Relatively speaking, T. vivax has been more than neglected despite an urgent need to develop efficient control strategies. Some pioneering rodent models were developed to circumvent the difficulties of working with livestock, but disappointedly were for the most part discontinued decades ago. To gain more insight into the biology of T. vivax, its interactions with the host and consequently its pathogenesis, we have developed a number of reproducible murine models using a parasite isolate that is infectious for rodents. Firstly, we analyzed the parasitical characteristics of the infection using inbred and outbred mouse strains to compare the impact of host genetic background on the infection and on survival rates. Hematological studies showed that the infection gave rise to severe anemia, and histopathological investigations in various organs showed multifocal inflammatory infiltrates associated with extramedullary hematopoiesis in the liver, and cerebral edema. The models developed are consistent with field observations and pave the way for subsequent in-depth studies into the pathogenesis of T. vivax - trypanosomosis.