Isolation and characterization of novel Staphylococcus aureus bacteriophage Hesat from dairy origin.

A novel temperate phage, named Hesat, was isolated by the incubation of a dairy strain of Staphylococcus aureus belonging to spa-type t127 with either bovine or ovine milk. Hesat represents a new species of temperate phage within the Phietavirus genus of the Azeredovirinae subfamily. Its genome has a length of 43,129 bp and a GC content of 35.11% and contains 75 predicted ORFs, some of which linked to virulence. This includes (i) a pathogenicity island (SaPln2), homologous to the type II toxin-antitoxin system PemK/MazF family toxin; (ii) a DUF3113 protein (gp30) that is putatively involved in the derepression of the global repressor Stl; and (iii) a cluster coding for a PVL. Genomic analysis of the host strain indicates Hesat is a resident prophage. Interestingly, its induction was obtained by exposing the bacterium to milk, while the conventional mitomycin C-based approach failed. The host range of phage Hesat appears to be broad, as it was able to lyse 24 out of 30 tested S. aureus isolates. Furthermore, when tested at high titer (108 PFU/ml), Hesat phage was also able to lyse a Staphylococcus muscae isolate, a coagulase-negative staphylococcal strain. KEY POINTS: • A new phage species was isolated from a Staphylococcus aureus bovine strain. • Pathogenicity island and PVL genes are encoded within phage genome. • The phage is active against most of S. aureus strains from both animal and human origins.

Discovery of 2-phenylquinazolines as potent Mycobacterium avium efflux pump inhibitors able to synergize with clarithromycin against clinical isolate.

Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop drug resistance against the most common antibiotics used to treat NTM infections. The overexpression of efflux pumps (EPs) was demonstrated to be a key mechanism of clarithromycin (CLA) resistance in NTM. Therefore, in this work, 24 compounds from an in-house library, characterized by chemical diversity, were tested as potential NTM EP inhibitors (EPIs) against Mycobacterium smegmatis mc2 155 and M. avium clinical isolates. Based on the acquired results, 12 novel analogs of the best derivatives 1b and 7b were designed and synthesized to improve the NTM EP inhibition activity. Among the second set of compounds, 13b emerged as the most potent NTM EPI. At a concentration of 4 µg/mL, it reduced the CLA minimum inhibitory concentration by 16-fold against the clinical isolate M. avium 2373 overexpressing EPs as primary mechanism of CLA resistance.

Biosensing systems for the detection and quantification of methane gas.

Climate change due to the continuous increase in the release of green-house gasses associated with anthropogenic activity has made a significant impact on the sustainability of life on our planet. Methane (CH4) is a green-house gas whose concentrations in the atmosphere are on the rise. CH4 measurement is important for both the environment and the safety at the industrial and household level. Methanotrophs are distinguished for their unique characteristic of using CH4 as the sole source of carbon and energy, due to the presence of the methane monooxygenases that oxidize CH4 under ambient temperature conditions. This has attracted interest in the use of methanotrophs in biotechnological applications as well as in the development of biosensing systems for CH4 quantification and monitoring. Biosensing systems using methanotrophs rely on the use of whole microbial cells that oxidize CH4 in presence of O2, so that the CH4 concentration is determined in an indirect manner by measuring the decrease of O2 level in the system. Although several biological properties of methanotrophic microorganisms still need to be characterized, different studies have demonstrated the feasibility of the use of methanotrophs in CH4 measurement. This review summarizes the contributions in methane biosensing systems and presents a prospective of the valid use of methanotrophs in this field. KEY POINTS: • Methanotroph environmental relevance in methane oxidation • Methanotroph biotechnological application in the field of biosensing • Methane monooxygenase as a feasible biorecognition element in biosensors.

Advancing bacteriophages as a treatment of antibiotic-resistant bacterial pulmonary infections.

PURPOSE OF REVIEW: The current article summarizes the recent advances in the use of bacteriophages to treat pulmonary infections, particularly those caused by Gram-negative drug-resistant bacteria, including Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and Burkholderia species. It provides an updated overview of the current available evidence, with a summary of published clinical cases, case series and clinical trials currently underway.Recent finding Personalized treatment with bacteriophages is still in its infancy in Europe and the USA, despite extensive experience in Eastern countries. However, more patients are expected to be treated with clinical trials in progress and others planned. SUMMARY: Despite very promising initial results and the confirmation of phage safety, there are still many ethical and practical implications to be considered, from the necessary regulatory approval to optimization of dose and route of administration, to developing strategies to tackle bacterial resistance. Patients with cystic fibrosis are a group where phage therapy, if successful, could have a major impact.

Phage-Based Control of Methicillin Resistant Staphylococcus aureus in a Galleria mellonella Model of Implant-Associated Infection.

Staphylococcus aureus implant-associated infections are difficult to treat because of the ability of bacteria to form biofilm on medical devices. Here, the efficacy of Sb-1 to control or prevent S. aureus colonization on medical foreign bodies was investigated in a Galleria mellonella larval infection model. For colonization control assays, sterile K-wires were implanted into larva prolegs. After 2 days, larvae were infected with methicillin-resistant S. aureus ATCC 43300 and incubated at 37 °C for a further 2 days, when treatments with either daptomycin (4 mg/kg), Sb-1 (107 PFUs) or a combination of them (3 x/day) were started. For biofilm prevention assays, larvae were pre-treated with either vancomycin (10 mg/kg) or Sb-1 (107 PFUs) before the S. aureus infection. In both experimental settings, K-wires were explanted for colony counting two days after treatment. In comparison to the untreated control, more than a 4 log10 CFU and 1 log10 CFU reduction was observed on K-wires recovered from larvae treated with the Sb-1/daptomycin combination and with their singular administration, respectively. Moreover, pre-infection treatment with Sb-1 was found to prevent K-wire colonization, similarly to vancomycin. Taken together, the obtained results demonstrated the strong potential of the Sb-1 antibiotic combinatory administration or the Sb-1 pretreatment to control or prevent S. aureus-associated implant infections.

Anti-Staphylococcal Activity of the Auranofin Analogue Bearing Acetylcysteine in Place of the Thiosugar: An Experimental and Theoretical Investigation.

Auranofin (AF, hereafter) is an orally administered chrysotherapeutic agent approved for the treatment of rheumatoid arthritis that is being repurposed for various indications including bacterial infections. Its likely mode of action involves the impairment of the TrxR system through the binding of the pharmacophoric cation [AuPEt3]+. Accordingly, a reliable strategy to expand the medicinal profile of AF is the replacement of the thiosugar moiety with different ligands. Herein, we aimed to prepare the AF analogue bearing the acetylcysteine ligand (AF-AcCys, hereafter) and characterize its anti-staphylococcal activity. Biological studies revealed that AF-AcCys retains an antibacterial effect superimposable with that of AF against Staphylococcus aureus, whereas it is about 20 times less effective against Staphylococcus epidermidis. Bioinorganic studies confirmed that upon incubation with human serum albumin, AF-AcCys, similarly to AF, induced protein metalation through the [AuPEt3]+ fragment. Additionally, AF-AcCys appeared capable of binding the dodecapeptide Ac-SGGDILQSGCUG-NH2, corresponding to the tryptic C-terminal fragment (488-499) of hTrxR. To shed light on the pharmacological differences between AF and AF-AcCys, we carried out a comparative experimental stability study and a theoretical estimation of bond dissociation energies, unveiling the higher strength of the Au-S bond in AF-AcCys. From the results, it emerged that the lower lipophilicity of AF-AcCys with respect to AF could be a key feature for its different antibacterial activity. The differences and similarities between AF and AF-AcCys are discussed, alongside the opportunities and consequences that chemical structure modifications imply.

In vitro antimicrobial activity against Abiotrophia defectiva and Granulicatella elegans biofilms.

OBJECTIVES: To determine the efficacy of different antibiotics (alone or in combination) against Abiotrophia defectiva and Granulicatella elegans biofilms and to investigate the anti-biofilm activity of gentamicin alone versus blood culture isolates from both species. METHODS: The activity of benzylpenicillin, clindamycin, daptomycin, fosfomycin, gentamicin, levofloxacin and rifampicin against 24-hour-old biofilms of A. defectiva and G. elegans was investigated in vitro by conventional microbiological methods and isothermal microcalorimetry. RESULTS: For planktonic bacteria, the MIC values of tested antibiotics ranged from 0.016 to 64 mg/L, as determined by microcalorimetry. Higher antibiotic concentrations, ranging from 1 to >1024 mg/L, were needed to produce an effect on biofilm bacteria. Gentamicin was an exception as it was active at 1 mg/L against both planktonic and biofilm G. elegans. A synergistic effect was observed when daptomycin was combined with benzylpenicillin, gentamicin or rifampicin against A. defectiva biofilms and when gentamicin was combined with rifampicin or levofloxacin against G. elegans biofilms. A. defectiva clinical isolates displayed greater variability in gentamicin susceptibility as compared with G. elegans strains. CONCLUSIONS: Antimicrobial susceptibility profiles vary widely between Abiotrophia and Granulicatella biofilms, and synergistic effects of the tested antibiotics were heterogeneous. The clinical relevance of these in vitro observations needs to be confirmed in experimental in vivo conditions and human trials, before guidelines for the treatment of A. defectiva and G. elegans infections are established. This study suggests the benefit of further clinical exploration of antibiotic combinations with anti-biofilm effect.

Real-Time Antimicrobial Susceptibility Assay of Planktonic and Biofilm Bacteria by Isothermal Microcalorimetry.

Most antimicrobials currently used in the clinical practice are tested as growth inhibitors against free-floating microorganisms in a liquid suspension, rather than against sessile cells constituting biofilms. Hence, reliable, fast, and reproducible methods for assessing biofilm susceptibility to antimicrobials are strongly needed. Isothermal microcalorimetry (IMC) is a nondestructive sensitive technique that allows for the real-time monitoring of microbial viability in the presence or absence of antimicrobial compounds. Therefore, the efficacy of specific antimicrobials, alone or in combination, may be promptly validated supporting the development of new drugs and avoiding the administration of ineffective therapies. Furthermore, the susceptibility of both planktonic and biofilm cells to antimicrobials can be conveniently assessed without the need for elaborated staining procedures and under nontoxic working conditions. Quantitative data regarding the antimicrobial effect against different strains might be collected by monitoring the microbial cell replication, and, more importantly, a dose-dependent activity can be efficiently detected by measuring the delay and decrease in the heat flow peak of the treated samples. A limitation of IMC for anti-biofilm susceptibility test is the inability to directly quantify the non-replicating cells in the biofilm or the total biomass. However, as IMC is a nondestructive method, the samples can be also analyzed by using different techniques, acquiring more information complementary to calorimetric data. IMC finds application also for the investigation of antibiotic eluting kinetics from different biomaterials, as well as for studying bacteriophages activity against planktonic and biofilm bacteria. Thus, the wide applicability of this ultra-sensitive and automated technique provides a further advance in the field of clinical microbiology and biomedical sciences.

Detection of Biofilms in Biopsies from Chronic Rhinosinusitis Patients: In Vitro Biofilm Forming Ability and Antimicrobial Susceptibility Testing in Biofilm Mode of Growth of Isolated Bacteria.

Chronic rhinosinusitis (CRS) is the most common illness among chronic disorders that remains poorly understood from a pathogenic standpoint and has a significant impact on patient quality of life, as well as healthcare costs. Despite being widespread, little is known about the etiology of the CRS. Recent evidence, showing the presence of biofilms within the paranasal sinuses, suggests a role for biofilm in the pathogenesis. To elucidate the role of biofilm in the pathogenesis of CRS, we assessed the presence of biofilm at the infection site and the ability of the aerobic flora isolated from CRS patients to form biofilm in vitro. For selected bacterial strains the susceptibility profiles to antibiotics in biofilm condition was also evaluated.Staphylococci represented the majority of the isolates obtained from the infection site, with S. epidermidis being the most frequently isolated species. Other isolates were represented by Enterobacteriaceae or by species present in the oral flora. Confocal laser scanning microscopy (CLSM) of the mucosal biopsies taken from patients with CRS revealed the presence of biofilm in the majority of the samples. Strains isolated from the specific infection site of the CRS patients were able to form biofilm in vitro at moderate or high levels, when tested in optimized conditions. No biofilm was observed by CLSM in the biopsies from control patients, although the same biopsies were positive for staphylococci in microbiological culture analysis. Drug-susceptibility tests demonstrated that the susceptibility profile of planktonic bacteria differs from that of sessile bacteria in biofilms.

Synergistic antibiotic activity against planktonic and biofilm-embedded Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis.

OBJECTIVES: To determine the antimicrobial activity against streptococcal biofilm in species mostly isolated from implant-associated infections and examine the effect of enzyme treatment of biofilm on the antimicrobial activity of different antibiotics. METHODS: The activities of fosfomycin, rifampicin, benzylpenicillin, daptomycin, gentamicin, levofloxacin, proteinase K and their combinations on planktonic and/or biofilm-embedded standard laboratory strains of Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis were investigated in vitro by standard methods and isothermal microcalorimetry. RESULTS: MIC values obtained for the tested antimicrobials against planktonic bacteria ranged from 0.016 to 128 mg/L for the three species tested. Higher antibiotic concentrations were usually required to reduce biofilm in comparison with planktonic bacteria, with the exception of gentamicin, for which similar concentrations (4-16 mg/L) exerted an effect on both planktonic and biofilm cells. A synergistic effect against the streptococcal biofilm of the three species was observed when gentamicin was combined with benzylpenicillin or with rifampicin. Moreover, antibiotic concentrations comparable to the MIC observed against planktonic cells induced a strong reduction of viable bacteria in proteinase K pre-treated biofilm. CONCLUSIONS: This study shows that the combination of gentamicin with either benzylpenicillin or rifampicin exerts a synergistic effect against biofilms produced by the tested streptococci strains in vitro. Our results also suggest that coupling a dispersal agent with conventional antibiotics may facilitate their access to the bacteria within the biofilm. In vivo and clinical studies are needed in order to confirm whether such a strategy may be effective in the treatment of implant-associated infections caused by streptococci.

Diffusion Tensor Analysis by Two-Dimensional Pair Correlation of Fluorescence Fluctuations in Cells.

In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF yields an accurate description of the preferential diffusive routes. Furthermore, we combine this analysis with the image-derived mean-square displacement approach and gain information on the average nanoscopic molecular displacements in different directions. Through these quantities, we build a fluorescence-fluctuation-based diffusion tensor that contains information on speed and directionality of the local dynamical processes. Contrary to classical fluorescence correlation spectroscopy and related methods, this combined approach can distinguish between isotropic and anisotropic local diffusion. We argue that the measurement of this iMSD tensor will contribute to advance our understanding of the role played by the intracellular environment in the regulation of molecular diffusion at the nanoscale.

Anti-biofilm properties of the antimicrobial peptide temporin 1Tb and its ability, in combination with EDTA, to eradicate Staphylococcus epidermidis biofilms on silicone catheters.

In search of new antimicrobials with anti-biofilm potential, in the present study activity of the frog-skin derived antimicrobial peptide temporin 1Tb (TB) against Staphylococcus epidermidis biofilms was investigated. A striking ability of TB to kill both forming and mature S. epidermidis biofilms was observed, especially when the peptide was combined with cysteine or EDTA, respectively. Kinetics studies demonstrated that the combination TB/EDTA was active against mature biofilms already after 2-4-h exposure. A double 4-h exposure of biofilms to TB/EDTA further increased the therapeutic potential of the same combination. Of note, TB/EDTA was able to eradicate S. epidermidis biofilms formed in vitro on silicone catheters. At eradicating concentrations, TB/EDTA did not cause hemolysis of human erythrocytes. The results shed light on the anti-biofilm properties of TB and suggest a possible application of the peptide in the lock therapy of catheters infected with S. epidermidis.

BaAMPs: the database of biofilm-active antimicrobial peptides.

Antimicrobial peptides (AMPs) are increasingly being considered as novel agents against biofilms. The development of AMP-based anti-biofilm strategies strongly relies on the design of sequences optimized to target specific features of sessile bacterial/fungal communities. Although several AMP databases have been created and successfully exploited for AMP design, all of these use data collected on peptides tested against planktonic microorganisms. Here, an open-access, manually curated database of AMPs specifically assayed against microbial biofilms (BaAMPs) is presented for the first time. In collecting relevant data from the literature an effort was made to define a minimal standard set of essential information including, for each AMP, the microbial species and biofilm conditions against which it was tested, and the specific assay and peptide concentration used. The availability of these data in an organized framework will benefit anti-biofilm research and support the design of novel molecules active against biofilm. BaAMPs is accessible at http://www.baamps.it.

Antimicrobial peptides design by evolutionary multiobjective optimization.

Antimicrobial peptides (AMPs) are an abundant and wide class of molecules produced by many tissues and cell types in a variety of mammals, plant and animal species. Linear alpha-helical antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement in innate defense. Recently, AMPs have received increasing attention as potential therapeutic agents, owing to their broad activity spectrum and their reduced tendency to induce resistance. The introduction of non-natural amino acids will be a key requisite in order to contrast host resistance and increase compound's life. In this work, the possibility to design novel AMP sequences with non-natural amino acids was achieved through a flexible computational approach, based on chemophysical profiles of peptide sequences. Quantitative structure-activity relationship (QSAR) descriptors were employed to code each peptide and train two statistical models in order to account for structural and functional properties of alpha-helical amphipathic AMPs. These models were then used as fitness functions for a multi-objective evolutional algorithm, together with a set of constraints for the design of a series of candidate AMPs. Two ab-initio natural peptides were synthesized and experimentally validated for antimicrobial activity, together with a series of control peptides. Furthermore, a well-known Cecropin-Mellitin alpha helical antimicrobial hybrid (CM18) was optimized by shortening its amino acid sequence while maintaining its activity and a peptide with non-natural amino acids was designed and tested, demonstrating the higher activity achievable with artificial residues.

Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis.

Staphylococcus epidermidis plays a major role in biofilm-related medical device infections. Herein the anti-biofilm activity of the human liver-derived antimicrobial peptide hepcidin 20 (hep20) was evaluated against polysaccharide intercellular adhesin (PIA)-positive and PIA-negative clinical isolates of S. epidermidis. Hep20 markedly inhibited biofilm formation and bacterial cell metabolism of PIA-positive and PIA-negative strains, but the decrease in biofilm biomass only partially correlated with a decrease in viable bacteria. Confocal microscope images revealed that, in the presence of hep20, both PIA-positive and PIA-negative strains formed biofilms with altered architectures and reduced amounts of extracellular matrix. Co-incubation of hep20 with vancomycin produced no synergistic effect, evaluated as number of viable cells, both in preventing biofilm formation and in treating preformed biofilms. In contrast, biofilms obtained in the presence of hep20, and then exposed to vancomycin, displayed an increased susceptibility to vancomycin. These results suggest that hep20 may inhibit the production/accumulation of biofilm extracellular matrix.

Evaluation of antibiofilm activity of cefiderocol alone and in combination with imipenem against Pseudomonas aeruginosa.

OBJECTIVES: The main aim of this study was to evaluate the antibiofilm activity of cefiderocol alone and in combination with imipenem vs. sessile cells of Pseudomonas aeruginosa, assessing a potential synergistic bactericidal effect. METHODS: Ten P. aeruginosa clinical isolates from infected implants and bloodstream were included in the study. Cefiderocol was tested alone and in combination with imipenem on 24-h-old P. aeruginosa biofilm formed on porous glass beads. For each antibiotic formulation, minimum bactericidal biofilm concentration (MBBC), defined as the lowest concentration that determined a reduction of at least 3 log10 CFU/mL compared with the untreated control, was evaluated. Scanning electron microscopy (SEM) was used to investigate the biofilm of P. aeruginosa treated with cefiderocol, imipenem, or their combination. RESULTS: Cefiderocol and imipenem were tested alone on P. aeruginosa biofilm and a reasonable reduction in the number of viable cells was observed, especially at high drug concentrations tested. The synergistic effect of cefiderocol in combination with imipenem was evaluated for five selected isolates. Cotreatment with the two drugs led to a remarkable reduction of cell viability by resulting in synergistic bactericidal activity in all tested strains and in synergistic eradicating activity in only one isolate. SEM analysis revealed that, in cefiderocol-treated biofilm, bacterial cells became more elongated than in the untreated control, forming filaments in which bacterial division seems to be inhibited. CONCLUSIONS: Cefiderocol exhibited an encouraging antibiofilm activity against tested strains, representing a valid option for the treatment of P. aeruginosa biofilm-associated infections, especially when administered in combination with imipenem.

Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation.

The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1-ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA(5), eccD(5), rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD(5) ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD(5) ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.

Isolation, Characterization, Genome Analysis and Host Resistance Development of Two Novel Lastavirus Phages Active against Pandrug-Resistant Klebsiella pneumoniae.

Klebsiella pneumoniae is a global health threat and bacteriophages are a potential solution in combating pandrug-resistant K. pneumoniae infections. Two lytic phages, LASTA and SJM3, active against several pandrug-resistant, nosocomial strains of K. pneumoniae were isolated and characterized. Their host range is narrow and latent period is particularly long; however, their lysogenic nature was refuted using both bioinformatic and experimental approaches. Genome sequence analysis clustered them with only two other phages into the new genus Lastavirus. Genomes of LASTA and SJM3 differ in only 13 base pairs, mainly located in tail fiber genes. Individual phages, as well as their cocktail, demonstrated significant bacterial reduction capacity in a time-dependent manner, yielding up to 4 log reduction against planktonic, and up to 2.59 log on biofilm-embedded, cells. Bacteria emerging from the contact with the phages developed resistance and achieved numbers comparable to the growth control after 24 h. The resistance to the phage seems to be of a transient nature and varies significantly between the two phages, as resistance to LASTA remained constant while resensitization to SJM3 was more prominent. Albeit with very few differences, SJM3 performed better than LASTA overall; however, more investigation is needed in order to consider them for therapeutic application.

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.