Application of Phage Therapy in a Case of a Chronic Hip-Prosthetic Joint Infection due to Pseudomonas aeruginosa: An Italian Real-Life Experience and In Vitro Analysis

Background: Prosthetic joint infection (PJI) caused by Pseudomonas aeruginosa represents a severe complication in orthopedic surgery. We report the case of a patient with chronic PJI from P. aeruginosa successfully treated with personalized phage therapy (PT) in combination with meropenem. Methods: A 62-year-old woman was affected by a chronic right hip prosthesis infection caused by P. aeruginosa since 2016 . The patient was treated with phage Pa53 (I day 10 mL q8h, then 5 mL q8h via joint drainage for 2 weeks) in association with meropenem (2gr q12h iv) after a surgical procedure. A 2-year clinical follow up was performed. An in vitro bactericidal assay of the phage alone and in combination with meropenem against a 24-hour-old biofilm of bacterial isolate was also carried out. Results: No severe adverse events were observed during PT. Two years after suspension, there were no clinical signs of infection relapse, and a marked leukocyte scan showed no pathological uptake areas. In vitro studies showed that the minimum biofilm eradicating concentration of meropenem was 8 µg/mL. No biofilm eradication was observed at 24 hours incubation with phages alone (108 plaque-forming units [PFU]/mL). However, the addition of meropenem at suberadicating concentration (1 µg/mL) to phages at lower titer (103 PFU/mL) resulted in a synergistic eradication after 24 hours of incubation. Conclusions: Personalized PT, in combination with meropenem, was found to be safe and effective in eradicating P. aeruginosa infection. These data encourage the development of personalized clinical studies aimed at evaluating the efficacy of PT as an adjunct to antibiotic therapy for chronic persistent infections

Mobile Regulatory Cassettes Mediate Modular Shuffling in T4-Type Phage Genomes

Coliphage phi1, which was isolated for phage therapy in the Republic of Georgia, is closely related to the T-like myovirus RB49. The ∼275 open reading frames encoded by each phage have an average level of amino acid identity of 95.8%. RB49 lacks 7 phi1 genes while 10 phi1 genes are missing from RB49. Most of these unique genes encode functions without known homologs. Many of the insertion, deletion, and replacement events that distinguish the two phages are in the hyperplastic regions (HPRs) of their genomes. The HPRs are rich in both nonessential genes and small regulatory cassettes (promoterearly stem-loops [PeSLs]) composed of strong σ70-like promoters and stem-loop structures, which are effective transcription terminators. Modular shuffling mediated by recombination between PeSLs has caused much of the sequence divergence between RB49 and phi1. We show that exchanges between nearby PeSLs can also create small circular DNAs that are apparently encapsidated by the virus. Such PeSL “mini-circles” may be important vectors for horizontal gene transfer

POTENTIAL USE OF PHAGES AS SANITIZING AGENTS TO REDUCE HOSPITAL PATHOGENS ON HARD SURFACES

Introduction: Hospital-acquired infections (HAI) can be transmitted by pathogens persistently contaminating hospital surfaces,1 often multidrug-resistant (MDR), and not efficiently controlled by conventional sanitation protocols, which indeed contribute to selection of drug-resistant strains.2 Due to the selective killing of specific bacteria, bacteriophages have been repeatedly suggested as decontaminating agents.3,4 This work was aimed to assess phage usability as sanitizing agents in routine hospital sanitation. Materials & Methods: Phage activity was assessed in vitro and in situ, in aqueous buffer or probiotic eco-sustainable detergents,5 on glass, plastic or ceramic surfaces artificially contaminated by S. aureus, E. coli and P. aeruginosa. Both ATCC strains and wild-type MDR hospital isolates were used, at a density consistent with what detected on hospital surfaces. Results: Phage application significantly reduced (up to 90%) all tested bacteria on all treated surfaces. Notably, phages suspended in probiotic detergents not only retained their full activity, but resulted even more effective especially at later times. Conclusions: Results suggest that phages might be successfully included in probiotic detergents currently used for hospital sanitation, potentially resulting in innovative products highly effective in the safe elimination of MDR nosocomial pathogens from the hospital environment. References: 1. Otter JA, Yezli S, French GL. The role played by contaminated surfaces in the transmission of nosocomial pathogens. Infect Control Hosp Epidemiol 2011; 32(7): 687-99. 2. Wand ME, Bock LJ, Bonney LC, Sutton JM. Mechanisms of Increased Resistance to Chlorhexidine and Cross-Resistance to Colistin following Exposure of Klebsiella pneumoniae Clinical Isolates to Chlorhexidine. Antimicrob Agents Chemother 2017; 61(1). 3. Jensen KC, Hair BB, Wienclaw TM, et al. Isolation and Host Range of Bacteriophage with Lytic Activity against Methicillin-Resistant Staphylococcus aureus and Potential Use as a Fomite Decontaminant. PLoS One 2015; 10(7): e0131714. 4. Tomat D, Quiberoni A, Mercanti D, Balague C. Hard surfaces decontamination of enteropathogenic and Shiga toxin-producing Escherichia coli using bacteriophages. Food Res Int 2014; 57: 123-9. 5. Caselli E, D'Accolti M, Vandini A, et al. Impact of a Probiotic-Based Cleaning Intervention on the Microbiota Ecosystem of the Hospital Surfaces: Focus on the Resistome Remodulation. PLoS One 2016; 11(2): e0148857

Susceptibilities of Multidrug-Resistant Pathogens Responsible for Complicated Skin and Soft Tissue Infections to Standard Bacteriophage Cocktails

Skin and soft tissue infections (SSTIs) may represent a wide clinical spectrum from cellulitis to high mortality associated necrotizing fasciitis. Limitations in therapy due to the multiple drug resistance, leads to increase in the morbidity and mortality rates, especially in complicated SSTIs such as diabetic foot, decubitus, and surgical wound infections. Therefore, alternative treatment strategies other than antibiotics are needed in appropriate clinical conditions. "Bacteriophage therapy", which is an old method and has been used as part of standard treatment in some countries such as Georgia and Russia, has again become popular worldwide. The aim of this study was to investigate the in vitro susceptibilities of multidrug-resistant (MDR) pathogens isolated from patients with complicated SSTIs, against standard bacteriophage (phage) cocktails. Six different ready-made phage preparations [Pyophage, Intestiphage, ENKO, SES, Fersisi and Staphylococcal Bacteriophage (Sb)] used in this study have been provided by G. Eliava Institute, Georgia. Because of the absence of ready-made phage preparations for Acinetobacter baumannii and Klebsiella pneumoniae, Phi 1-Phi 7 and Phi KL1-Phi KL3 phages were used provided from the same institute's phage library, respectively. Isolation and identification of the pathogens from abscess and wound samples of patients with SSTIs were performed by conventional methods and automatized VITEK (R)-2 (bioMerieux, ABD) system. Antimicrobial susceptibility testing was conducted complying CLSI standards' and the bacteria that were resistant to at least two different antibiotic groups were considered as MDR. Accordingly, a total of 33 isolates, nine of them were E.coli (8 ESBL and 1 ESBL + carbapenemase positive); nine were MDR P.aeruginosa; nine were MDR A.baumannii; three were methicillin-resistant Staphylococcus aureus (MRSA) and three were K.pneumoniae (1 ESBL, 1 carbapenemase and 1 ESBL + carbapenemase positive) were included in the study. The phage susceptibilities of the pathogens were performed by using spot test. In the study, 29 (87.9%) out of 33 MDR pathogens were found to be susceptible to at least one of the tested phage/phage preparations. All MRSA (3/3) strains were susceptible to ENKO, SES, Fersisi and Sb phage cocktails, while all A.baumannii isolates (9/9) were susceptible to Phi 5 and Phi 7 phages. However, two E.coli, one K. pneumoniae and one P.aeruginosa strains were resistant to the all phage preparations tested. Although the clinical use of phages has not been approved yet, except a few Eastern European countries, this study exhibits the potential use of the topical bacteriophage therapy in the treatment of complicated SSTIs caused by MDR pathogens with limited treatment options, such as diabetic foot, decubitus, and surgical wound infections

Centennial celebration of the bacteriophage research

International audienceThe year 2017 marked the 100th anniversary of the publication of Félix d’Herelle entitled “On an invisible microbe antagonistic toward dysenteric bacilli” in which he coined the term “bacteriophage [...

Phage Therapy in a 16-Year-Old Boy with Netherton Syndrome

Netherton syndrome (NS) is a rare autosomal recessive disorder, characterized by a classical triad of clinical features, including congenital ichthyosiform erythroderma, trichorrhexis invaginata, and atopic diathesis coupled with frequent bacterial infections (1). The genetic basis for the disease has been recently identified with mutations in gene SPINK5, which is involved in the regulation of formation of skin barriers. We report on a 16-year-old male with all the typical manifestations of NS, including atopic diathesis and ongoing serious staphylococcal infections and allergy to multiple antibiotics whose family sought help at the Eliava Phage Therapy Center when all other treatment options were failing. Treatment with several antistaphylococcal bacteriophage preparations led to significant improvement within 7 days and very substantial changes in his symptoms and quality of life after treatment for 6 months, including return visits to the Eliava Phage Therapy Center after 3 and 6 months of ongoing use of phage at home

Bacteriophage effectiveness in hard surfaces decontamination

Objectives: Persistently contaminated hospital surfaces represent a reservoir of infectious pathogens, hence contributing to the development of healthcare associated infections (HAI), which are often sustained by antibiotic-resistant pathogens. So far, decontamination of hospital surfaces was performed by conventional disinfectants, that cannot prevent recontamination phenomena, which occur in about 30 minutes. Previous studies suggested that specific bacteriophage mixtures could be used to reduce bacterial viability on food, thus the present study was aimed to determine the effect of phage treatment as decontaminating agents on hard surfaces, specifically targeting hospital pathogens. Methods: Both Gram positive and Gram negative bacteria were included in the study, focusing on strains most frequently detected on hospital surfaces, based on previous observations [Caselli E. et al., PLoS One 2016]. Bacterial strains were applied on sterile ceramic tiles and plastic surfaces, at a concentration similar to what detected on hospital surfaces. Namely, cultures of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Proteus mirabilis (ATCC 29906) were separately seeded on surfaces at 102 or 103 CFU per 24 cm2 (corresponding to 4,000 or 40,000 CFU/m2), and dried. Phage mixtures (Staphylococcal phage and Pyophage; Eliava Institute) were then applied uniformly on surfaces after dilution in saline at 1, 10, 100 and 1000 multiplicity of infection (m.o.i.; phage PFU/bacterial CFU ratio), and incubated at room temperature for 0.5, 1, 3, 6 and 24 hours. Bacterial survival was determined by standard CFU count after applying surfaces contact Rodac plates containing the selective media for the analyzed bacterial strain. All assays were also repeated by diluting phage preparations in conventional detergents at work dilution, to ascertain their usability during the usual sanitation procedures. Results: Both used phage mixtures reduced bacterial CFU on artificially contaminated surfaces 70-90%, depending on the used m.o.i., after 1 hour of incubation, compared to what detected on control surfaces (treated with phage diluent only). After 6 hours, almost no survivors were detected, and the results were maintained in the subsequent 24 hours. Phages activity was dose-dependent, but no statistically significant differences were observed between 100 and 1,000 m.o.i.. The results were similar for all the tested bacteria. Notably, phages retained their 100% activity when diluted in detergents commonly used for hospital surface sanitation. Conclusions: Our data indicate that bacteriophages are active in decontaminating in vitro dry hard surfaces, acting against pathogens levels similar to those detected on field on hospital surfaces. They are active within 1 hour at room temperature, and maintain their full activity when suspended in conventional detergents at work dilution. These features render phage mixtures suitable for use as sanitizing agents, and, especially in consideration of the high proportion of antibiotic-resistant isolates on hospital surfaces, open the way to the development of innovative products for the effective elimination of nosocomial pathogens on field