Linezolid resistance: detection of the cfr(B) gene in French clinical MRSA strains.

OBJECTIVES: To describe two linezolid-resistant MRSA strains carrying the cfr(B) gene detected in the French National Reference Centre for staphylococci. METHODS: Two linezolid-resistant MRSA strains isolated from cystic fibrosis patients in two different French hospitals in 2017 and 2019 were examined to explore the mechanisms of linezolid resistance. Antimicrobial susceptibility was tested using broth microdilution and gradient strips. The genetic determinants of linezolid resistance were assessed by a multiplex PCR targeting cfr/cfr(B), optrA and poxtA genes, by amplification and sequencing of individual 23S rRNA genes and by WGS using both Illumina and Nanopore technologies. RESULTS: The two MRSA strains were resistant to linezolid but susceptible to tedizolid, and PCR-positive for cfr/cfr(B). The WGS analysis indicated that they belonged to two different STs (ST8-MRSA-IV and ST5382-MRSA-IV) and that they both harboured the cfr(B) gene on the same 9.7 kb Tn6218-like chromosomal transposon, a finding only previously reported in Enterococcus sp. and Clostridioides difficile. CONCLUSIONS: To the best of our knowledge, this is the first description of the presence of cfr(B) in staphylococci, more specifically in linezolid-resistant MRSA strains. This finding illustrates the risk of horizontal intergenus transfer of oxazolidinone resistance genes in Staphylococcus aureus and highlights the need to monitor such emergence in this species.

Activity of Exebacase (CF-301) against Biofilms Formed by Staphylococcus epidermidis Strains Isolated from Prosthetic Joint Infections.

Staphylococcus epidermidis is one of the main pathogens responsible for bone and joint infections, especially those involving prosthetic materials, due to its ability to form biofilms. In these cases, biofilm formation, combined with increased antimicrobial resistance, often results in therapeutic failures. In this context, the development of innovative therapies active against S. epidermidis is a priority. The aim of this study was to evaluate the in vitro activity of the lysin exebacase (CF-301) against biofilms formed by 19 S. epidermidis clinical strains isolated from prosthetic joint infections (PJI). We determined the biomass and the remaining viable bacteria inside biofilms after 24 h of exposure to exebacase. Exebacase activity was compared to that of rifampicin, vancomycin, and daptomycin. The use of exebacase in addition to antibiotics was also assessed. Exebacase displayed (i) a significant anti-biomass activity on S. epidermidis biofilms at concentrations ≥5 mg/L (mean decrease up to 66% at 150 mg/L), (ii) significant bactericidal activity on biofilms at concentrations ≥50 mg/L (mean decrease up to 1.7 log CFU at 150 mg/L), (iii) synergistic effects when used in addition to rifampicin, vancomycin, or daptomycin. The extent of these activities varied by isolate. Exebacase can be considered a promising therapy in addition to rifampicin, vancomycin, or daptomycin in the context of PJI. Further in vitro studies are needed to understand its mechanism of action on S. epidermidis biofilms and in vivo investigations are required to confirm these data.

Clinical Metagenomic Sequencing for Species Identification and Antimicrobial Resistance Prediction in Orthopedic Device Infection.

Diagnosis of orthopedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful. Human DNA depletion may maximize the amount of microbial DNA sequence data available for analysis. Human DNA depletion by saponin was tested in 115 sonication fluid samples generated following revision arthroplasty surgery, comprising 67 where pathogens were detected by culture and 48 culture-negative samples. Metagenomic sequencing was performed on the Oxford Nanopore Technologies GridION platform. Filtering thresholds for detection of true species versus contamination or taxonomic misclassification were determined. Mobile and chromosomal genetic AMR determinants were identified in Staphylococcus aureus-positive samples. Of 114 samples generating sequence data, species-level positive percent agreement between metagenomic sequencing and culture was 50/65 (77%; 95% confidence interval [CI], 65 to 86%) and negative percent agreement was 103/114 (90%; 95% CI, 83 to 95%). Saponin treatment reduced the proportion of human bases sequenced in comparison to 5-µm filtration from a median (interquartile range [IQR]) of 98.1% (87.0% to 99.9%) to 11.9% (0.4% to 67.0%), improving reference genome coverage at a 10-fold depth from 18.7% (0.30% to 85.7%) to 84.3% (12.9% to 93.8%). Metagenomic sequencing predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient data were available for analysis. Metagenomic nanopore sequencing coupled with human DNA depletion has the potential to detect AMR in addition to species detection in orthopedic device-related infection. Further work is required to develop pathogen-agnostic human DNA depletion methods, improving AMR determinant detection and allowing its application to other infection types.

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.

Staphylococcus aureus: No ticket for the Paris 2024 Olympic Games!

Athletes are vulnerable to Staphylococcus aureus infections due to skin-to-skin contact and skin abrasions during training and competitions involving sharied sport equipment or toiletries, which promote the spread of the bacteria between athletes and within sport teams. This results not only in higher prevalence of S.aureus carriage among athletes compared to the general population, but also in outbreaks of infections, particularly skin infections, within sports teams. To limit the spread of S. aureus among athletes, a decolonization protocol can be applied when clustered cases of S. aureus infections occur, especially if Panton-Valentine leukocidin-producing strains are implicated. Finally, to avoid exposing athletes to S.aureus transmission/colonization, it is recommended to establish strict and clearly formulated individual and collective hygiene rules and to regularly disinfect shared sports equipment.

Case report: Continuous infusions of ceftazidime-avibactam and aztreonam in combination through elastomeric infusors for 12 weeks for the treatment of bone and joint infections due to metallo-ß-lactamase producing Enterobacterales.

Among carbapenem-resistant Enterobacterales, metallo-beta-lactamase producing strains represent a growing therapeutic challenge. While the association of aztreonam and ceftazidime-avibactam has been investigated in recent years for the treatment of infections involving these strains, little to no clinical data support the use of this association for the treatment of bone and joint infections. We report two cases of complex bone and joint infections involving metallo-beta-lactamase-producing Enterobacterales, successfully treated at our referral center with aztreonam and ceftazidime-avibactam for 12 weeks in continuous infusions through elastomeric infusors.

Sensitivity of the PBP2a SA Culture Colony Test on shortly incubated subcultures of methicillin-resistant staphylococci from positive blood cultures.

The sensitivity of an immunochromatographic assay for detecting methicillin resistance (PBP2a SA Culture Colony Test, Alere-Abbott) on shortly incubated subcultures of staphylococci in blood cultures was evaluated. The assay is highly sensitive for the detection of methicillin-resistant Staphylococcus aureus after 4 hour-subculture but requires 6 hour-incubation for methicillin-resistant coagulase-negative staphylococci.

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.

Bacteriophages Combined With Subtherapeutic Doses of Flucloxacillin Act Synergistically Against Staphylococcus aureus Experimental Infective Endocarditis.

Background The potential of phage therapy for the treatment of endovascular Staphylococcus aureus infections remains to be evaluated. Methods and Results The efficacy of a phage cocktail combining Herelleviridae phage vB_SauH_2002 and Podoviriae phage 66 was evaluated against a methicillin-sensitive S. aureus strain in vitro and in vivo in a rodent model of experimental endocarditis. Six hours after bacterial challenge, animals were treated with (1) the phage cocktail. (2) subtherapeutic flucloxacillin dosage, (3) combination of the phage cocktail and flucloxacillin, or (4) saline. Bacterial loads in cardiac vegetations at 30 hours were the primary outcome. Secondary outcomes were phage loads at 30 hours in cardiac vegetations, blood, spleen, liver, and kidneys. We evaluated phage resistance 30 hours post infection in vegetations of rats under combination treatment. In vitro, phages synergized against S. aureus planktonic cells and the cocktail synergized with flucloxacillin to eradicated biofilms. In infected animals, the phage cocktail achieved bacteriostatic effect. The addition of low-dose flucloxacillin elevated bacterial suppression (∆ of -5.25 log10 colony forming unit/g [CFU/g] versus treatment onset, P<0.0001) and synergism was confirmed (∆ of -2.15 log10 CFU/g versus low-dose flucloxacillin alone, P<0.01). Importantly, 9/12 rats given the combination treatment had sterile vegetations at 30 hours. In vivo phage replication was partially suppressed by the antibiotic and selection of resistance to the Podoviridae component of the phage cocktail occurred. Plasma-mediated inhibition of phage killing activity was observed in vitro. Conclusions Combining phages with a low-dose standard of care antibiotic represents a promising strategy for the treatment of S. aureus infective endocarditis.

[Viruses for health: Bacteriophages]. / Les virus au service de la santé : les bactériophages.

Bacteriophages are naturally occurring viruses that specifically target bacteria. They are widely distributed in the environment. The concept of phage therapy is to isolate phages, characterize them, cultivate them and then purify them to treat bacterial infections. There is currently a revival of phage therapy, and its implementation presupposes the availability of active phages of pharmaceutical quality. From a regulatory point of view, the status of phages is not yet clearly defined by the authorities. The availability of phages produced by the pharmaceutical industry and through academic development programs such as the PHAGEinLYON program represents a breakthrough in the development of phage therapy. Prosthetic joint infections and digestive diseases seem to be relevant indications, but preclinical studies and randomized clinical trials are now needed to be done.

Title: Les virus au service de la santé : les bactériophages. Abstract: Les bactériophages sont des virus naturels très répandus dans l'environnement qui ciblent spécifiquement les bactéries. Leur utilisation en médecine, connue sous le terme phagothérapie, consiste à les isoler, les caractériser, les cultiver, puis les purifier pour traiter des infections bactériennes. Il existe actuellement un renouveau pour la thérapie phagique, et sa mise en œuvre présuppose de disposer de phages actifs de qualité pharmaceutique. D'un point de vue réglementaire, le statut des phages n'est pas encore clairement défini par les autorités, mais la mise à disposition de phages produits par l'industrie pharmaceutique et les programmes de développement académiques, comme le programme PHAGEinLYON, constituent un tournant dans le déploiement de la phagothérapie.

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.

Experience With the Use of the MicroDTTect Device for the Diagnosis of Low-Grade Chronic Prosthetic Joint Infections in a Routine Setting.

Background: In prosthetic joint infections (PJIs), identification of the causative microorganisms is critical to successfully adapt and optimize treatment. However, microbiological diagnosis of PJIs remains a challenge notably because bacteria are embedded in biofilm adhered to the prosthetic material. Recently, dithiothreitol (DTT) treatment of prosthesis has been proposed as a new strategy to release bacteria from biofilm and to improve the yield of microbiological diagnosis. In this study, we evaluated the interest of a commercial device using DTT, the MicroDTTect system (Heraeus, Hanau, Germany), for the diagnosis of low-grade chronic PJIs, compared to the conventional culture of periprosthetic tissue (PPT) samples. Methods: Twenty patients undergoing a surgery procedure for removal of prosthetic material because of a suspicion of low-grade PJI without pre-operative microbiological documentation were included (NCT04371068). Bacteriological results using the fluid obtained after prosthesis treatment with the MicroDTTect system were compared to results obtained with conventional culture of PPT samples. Results: All the bacteria considered as responsible for PJIs recovered from culture of PPT samples were also detected using the MicroDTTect device. For one patient, an additional bacterial isolate (Staphylococcus haemolyticus) suspected to be involved in a polymicrobial PJI was identified using DTT treatment. Time to positivity of the cultures was also reduced using the MicroDTTect system, notably in case of Cutibacterium acnes infection. However, probable bacterial contaminants were found (MicroDTTect system, n = 5; PPT samples, n = 1). Conclusion: This study showed that DTT treatment of the prosthetic component using the MicroDTTect device could improve the microbiological diagnosis of low-grade PJIs.

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.

Case Report: Arthroscopic "Debridement Antibiotics and Implant Retention" With Local Injection of Personalized Phage Therapy to Salvage a Relapsing Pseudomonas Aeruginosa Prosthetic Knee Infection.

Bacteriophages are viruses that specifically target bacteria. They are considered to have a high potential in patients with prosthetic joint infection (PJI), as they have a synergistic anti-biofilm activity with antibiotics. We report here the case of an 88-year-old man (63 kg) with relapsing Pseudomonas aeruginosa prosthetic knee infection. The patient had severe alteration of the general status and was bedridden with congestive heart failure. As prosthesis explantation and/or exchange was not feasible, we proposed to this patient the use of phage therapy to try to control the disease in accordance with the local ethics committee and the French National Agency for Medicines and Health Products Safety (ANSM). Three phages, targeting P. aeruginosa, were selected based on their lytic activity on the patient's strain (phagogram). Hospital pharmacist mixed extemporaneously the active phages (initial concentration 1 ml of 1 × 1010 PFU/ml for each phage) to obtain a cocktail of phages in a suspension form (final dilution 1 × 109 PFU/ml for both phages). Conventional arthroscopy was performed and 30 cc of the magistral preparation was injected through the arthroscope (PhagoDAIR procedure). The patient received intravenous ceftazidime and then oral ciprofloxacin as suppressive antimicrobial therapy. Under this treatment, the patient rapidly improved with disappearance of signs of heart failure and pain of the left knee. During the follow-up of 1 year, the local status of the left knee was normal, and its motion and walking were unpainful. The present case suggests that the PhagoDAIR procedure by arthroscopy has the potential to be used as salvage therapy for patients with P. aeruginosa relapsing PJI, in combination with suppressive antimicrobial therapy. A Phase II clinical study deserves to be performed to confirm this hypothesis.

Arthroscopic "Debridement and Implant Retention" With Local Administration of Exebacase (Lysin CF-301) Followed by Suppressive Tedizolid as Salvage Therapy in Elderly Patients for Relapsing Multidrug-Resistant S. epidermidis Prosthetic Knee Infection.

Exebacase, a recombinantly produced lysin has recently (i) reported proof-of-concept data from a phase II study in S. aureus bacteremia and (ii) demonstrated antibiofilm activity in vitro against S. epidermidis. In patients with relapsing multidrug-resistant (MDR) S. epidermidis prosthetic knee infection (PKI), the only surgical option is prosthesis exchange. In elderly patients who have undergone several revisions, prosthesis explantation could be associated with definitive loss of function and mortality. In our BJI reference regional center, arthroscopic debridement and implant retention with local administration of exebacase (LysinDAIR) followed by suppressive tedizolid as salvage therapy is proposed for elderly patients with recurrent MDR S. epidermidis PKI with no therapeutic option or therapeutic dead end (for whom revision or transfemoral amputation is not feasible and no other oral option is available). Each use was decided in agreement with the French health authority and in accordance with the local ethics committee. A written consent was obtained for each patient. Exebacase (75 mg/mL; 30 mL) was administered directly into the joint during arthroscopy. Four patients (79-89 years old) were treated with the LysinDAIR procedure. All had several previous prosthetic knee revisions without prosthesis loosening. Three had relapsing PKI despite suppressive antibiotics following open DAIR. Two had clinical signs of septic arthritis; the two others had sinus tract. After the LysinDAIR procedure, no adverse events occurred during arthroscopy; all patients received daptomycin 8 mg/kg and linezolid 600 mg bid (4-6 weeks) as primary therapy, followed by tedizolid 200 mg/day as suppressive therapy. At 6 months, recurrence of the sinus tract occurred in the two patients with sinus tract at baseline. After >1 year follow up, the clinical outcome was favorable in the last two patients with total disappearance of clinical signs of septic arthritis even if microbiological persistence was detected in one of them. Exebacase has the potential to be used in patients with staphylococci PKI during arthroscopic DAIR as salvage therapy to improve the efficacy of suppressive antibiotics and to prevent major loss of function.