Bacteriophage therapy as a treatment strategy for orthopaedic-device-related infections: where do we stand?

Antibiotic resistance represents a key challenge of the 21st century. Since the pipeline of new antibiotics in development is limited, the introduction of alternative antimicrobial strategies is urgently required. Bacteriophage therapy, the use of bacterial viruses to selectively kill bacterial pathogens, is re-emerging as a potential strategy to tackle difficult-to-treat and multidrug-resistant pathogens. The last decade has seen a surge in scientific investigation into bacteriophage therapy, including targeting orthopaedic-device-related infections (ODRIs) in several successful case studies. However, pharmacological data, knowledge on the interplay with the immune system and, especially in ODRIs, the optimal local application strategy and treatment outcomes remain scarce. The present review reports the state-of-the-art in bacteriophage therapy in ODRIs and addresses the hurdles in establishing bacteriophage therapy under good clinical practice guidelines. These hurdles include a lack of data concerning bacteriophage production, processing, administration and dosing, as well as follow-up clinical monitoring reports. To overcome these challenges, an integrated clinical approach is required, supported by comprehensive legislature to enable expansive and correctly implemented clinical trials.

[Beyond antibiotic therapy - Future antiinfective strategies - Update 2017]. / "Beyond antibiotic therapy" ­ Zukünftige antiinfektiöse Strategien ­ Update 2017.

BACKGROUND: The key elements in the therapy of surgical site infections (SSI) are surgical debridement and local and systemic antibiotic therapy; however, due to increasing antibiotic resistance, the development of additional therapeutic measures is of great interest for future trauma and orthopedic surgery. METHOD: Against the background of our own experimental and clinical experiences and on the basis of the current literature, possible future anti-infective strategies were elaborated. RESULTS/CONCLUSIONS: Bacteriophages were discovered and clinically implemented approximately one century ago and have been used in Western Europe for about one decade. They are currently used mainly in patients with burn injuries. It is likely that bacteriophages will become of great importance in view of the increasing antibiotic multi-drug resistance; however, they will probably not entirely replace antibiotic drugs. A combined use of bacteriophages and antibiotics is likely to be a more reasonable efficient therapy. In addition, the clinical importance of antimicrobial peptides (AMP) also increases. Up to now the possible use of AMPs is still experimental; however, individual AMPs are already established in the routine therapy (e. g. colistin). Further diagnostic and therapeutic measures may include photodynamic therapy, ultraviolet (UV) light application and differentiated genome analysis as well as the individual metabolism situation (metabolomics) of the pathogen cell and the patient tissue.

Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates.

Recent years have seen renewed interest in phage therapy--the use of viruses to specifically kill disease-causing bacteria--because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre-adapted all phage strains against all bacterial strains and then compared the efficacy of pre-adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre-adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.

Cleanrooms and tissue banking how happy I could be with either GMP or GTP?

The regulatory framework of tissue banking introduces a number of requirements for monitoring cleanrooms for processing tissue or cell grafts. Although a number of requirements were clearly defined, some requirements are open for interpretation. This study aims to contribute to the interpretation of GMP or GTP guidelines for tissue banking. Based on the experience of the participating centers, the results of the monitoring program were evaluated to determine the feasibility of a cleanroom in tissue banking and the monitoring program. Also the microbial efficacy of a laminar airflow cabinet and an incubator in a cleanroom environment was evaluated. This study indicated that a monitoring program of a cleanroom at rest in combination with (final) product testing is a feasible approach. Although no statistical significance (0.90 < p < 0.95) was found there is a strong indication that a Grade D environment is not the ideal background environment for a Grade A obtained through a laminar airflow cabinet. The microbial contamination of an incubator in a cleanroom is limited but requires closed containers for tissue and cell products.

Personalized bacteriophage therapy to treat pandrug-resistant spinal Pseudomonas aeruginosa infection.

Bone and joint infections (BJI) are one of the most difficult-to-treat bacterial infection, especially in the era of antimicrobial resistance. Lytic bacteriophages (phages for short) are natural viruses that can selectively target and kill bacteria. They are considered to have a high therapeutic potential for the treatment of severe bacterial infections and especially BJI, as they also target biofilms. Here we report on the management of a patient with a pandrug-resistant Pseudomonas aeruginosa spinal abscess who was treated with surgery and a personalized combination of phage therapy that was added to antibiotics. As the infecting P. aeruginosa strain was resistant to the phages developed by private companies that were contacted, we set up a unique European academic collaboration to find, produce and administer a personalized phage cocktail to the patient in due time. After two surgeries, despite bacterial persistence with expression of small colony variants, the patient healed with local and intravenous injections of purified phages as adjuvant therapy.

Efficacy assessment of PEV2 phage on Galleria mellonella larvae infected with a Pseudomonas aeruginosa dog otitis isolate.

Pseudomonas (P.) aeruginosa is the most frequently isolated Gram-negative bacteria in dog otitis. Antimicrobial resistance is particularly prevalent in P. aeruginosa and phage therapy represents a promising alternative therapeutic strategy. The aim of this study was to assess the efficacy of the PEV2 phage against a clinical P. aeruginosa isolate from a canine otitis using a Galleria (G.) mellonella larvae model. The genomic DNA of PAV237 P. aeruginosa isolate was sequenced and analysed. In a first main experiment, the efficacy of PEV2 phage against PAV237 was assessed at different multiplicities of infection (MOI) (50,000, 5000, 500, 50) by analyzing the larvae survival rate during 4 days. In a second experiment, the bacterial and phage titer evolutions were assessed depending on two MOIs (50,000, 5000). No significant survival increase was observed with PEV2 therapy in the infected larvae groups. The generated Kaplan-Meier curves showed that the rate of alive larvae was significantly higher in the non-infected larvae compared to the infected-treated ones irrespective of phage MOIs. An increase of the phage titer was observed at 24 and 48 h post-inoculation (HPI) with both MOIs and the P. aeruginosa titers were lower with MOI 50,000 and 5000 compared to the infectivity control at 24 and 48 HPI. Even if an ineffectiveness of the PEV2 phage was observed on the larvae survival, PEV2 is active against P. aeruginosa in this model and PEV2 replication is correlated with a lower bacterial proliferation in the phage treated larvae.

Access to bacteriophage therapy: discouraging experiences from the human cell and tissue legal framework.

Cultures of human epithelial cells (keratinocytes) are used as an additional surgical tool to treat critically burnt patients. Initially, the production environment of keratinocyte grafts was regulated exclusively by national regulations. In 2004, the European Tissues and Cells Directive 2004/23/EC (transposed into Belgian Law) imposed requirements that resulted in increased production costs and no significant increase in quality and/or safety. In 2007, Europe published Regulation (EC) No. 1394/2007 on Advanced Therapy Medicinal Products. Overnight, cultured keratinocytes became (arguably) 'Advanced' Therapy Medicinal Products to be produced as human medicinal products. The practical impact of these amendments was (and still is) considerable. A similar development appears imminent in bacteriophage therapy. Bacteriophages are bacterial viruses that can be used for tackling the problem of bacterial resistance development to antibiotics. Therapeutic natural bacteriophages have been in clinical use for almost 100 years. Regulators today are framing the (re-)introduction of (natural) bacteriophage therapy into 'modern western' medicine as biological medicinal products, also subject to stringent regulatory medicinal products requirements. In this paper, we look back on a century of bacteriophage therapy to make the case that therapeutic natural bacteriophages should not be classified under the medicinal product regulatory frames as they exist today. It is our call to authorities to not repeat the mistake of the past.

HIV-1 detection by nested PCR and viral culture in fresh or cryopreserved postmortem skin: potential implications for skin handling and allografting.

AIMS: To date, the risk relating to the handling or allografting of human immunodeficiency virus type 1 (HIV-1) infected postmortem skin remains hypothetical. While blood screening for HIV antibodies is still the key safety procedure to detect HIV infected cadavers, false negative results are a concern. Conversely, false positive results may hamper the collection of skin allografts. Accordingly, viral culture was used to clarify skin infectivity and the nested polymerase chain reaction (PCR) was used to assess the reliability of skin PCR testing. METHODS: Viral culture and nested PCR performed with gag and pol specific primers were investigated in cadaveric skin and blood from 12 HIV-1 infected patients. Samples were collected repeatedly between one and five days in seven patients. In most cases, analyses were performed on triplicate skin samples: fresh (n = 26); cryopreserved in 5% dimethylsulphoxide (n = 21), or cryopreserved in 30% glycerol (n = 26). RESULTS: HIV was isolated in two of 26 cultures of fresh skin specimens (8%), seven of 47 cryopreserved skin specimens (15%), and eight of 26 blood specimens (31%). The nested PCR detected HIV-1 in all skin samples (n = 73), regardless of the postmortem interval or cryopreservation. In blood, a positive signal was found in eight of 12 patients but two of them had discordant results on successive samples. CONCLUSIONS: These data suggest that nested PCR on postmortem skin samples can detect HIV more reliably than on blood. They also demonstrate the potential viral infectivity of fresh or stored skin postmortem samples in HIV infected patients. They underscore the need for caution during the handling of skin tissue from HIV infected cadavers and confirm the potential risk related to accidental allografting of HIV contaminated skin.

HIV transmission by transplantation of allograft skin: a review of the literature.

The fear of human immunodeficiency virus (HIV) transmission by means of allograft skin has led to a cautious approach to allograft donor selection. However, no irrefutable diagnostic test exists to determine the possible presence of HIV at the time of donation. In order to find ways of improving HIV donor screening practices for skin banks, we review the presence of HIV in human skin, explore the possible transmission of HIV by transplantation of human allograft skin, and discuss the reliability of existing HIV tests. The use of the polymerase chain reaction (PCR) as a sensitive detection system for HIV infection of skin biopsies, in combination with conventional routine HIV blood screening tests; could lower the risk of transmitting HIV to severely burned patients.

Analysis of epidemic Pseudomonas aeruginosa isolates by isoelectric focusing of pyoverdine and RAPD-PCR: modern tools for an integrated anti-nosocomial infection strategy in burn wound centres.

The Gram-negative bacterium Pseudomonas aeruginosa is an important life-threatening nosocomial pathogen in burn units. In this study we analysed epidemic P. aeruginosa isolates from patients and their hospital environment using two new molecular techniques in order to establish strain relatedness for epidemiological purposes. One technique was pyoverdine typing by isoelectric focusing (PVD-IEF) and the other was a genomic PCR-based fingerprinting technique called random amplification of polymorphic DNA actually referred to as RAPD-PCR. The described short epidemic (6 weeks) included 37 consecutive isolates from 9 different patients as well as two environmental isolates recovered, at the same time, from one of the hydrotherapy facilities. Only two of the three known pyoverdine types of P. aeruginosa could be found. Type I was absent while type II represented 49 per cent and type III, 51 per cent of the isolates. The two consecutive isolates from the environment were both of type III. The RAPD-PCR fingerprinting discriminated four patterns. Profile 1 represented 60 per cent; profile 2, 34 per cent; and profiles 3 and 4 only 3 per cent of the isolates respectively. The environmental isolates also had a RAPD-PCR 1 profile, arguing for the hydrotherapy facility as a possible contamination source. Prompt measures could prevent an outbreak. The study demonstrates the applicability of the techniques in a routine microbiology lab as well as their usefulness, in combination with other techniques, in the fight against nosocomial infections, which are so critical in burn units. Both techniques showed undoubtable evidence of the occurrence of polymicrobial infection of individual patients by P. aeruginosa species. Meanwhile pyoverdine typing by IEF seems suited to studying more profoundly the role of pyoverdines in burns.

Effectiveness of bacteriophages in the sputum of cystic fibrosis patients.

Bacteriophages have been shown to be effective for treating acute infections of the respiratory tract caused by antibiotic-resistant bacteria in animal models, but no evidence has yet been presented of their activity against pathogens in complex biological samples from chronically infected patients. We assessed the efficacy of a cocktail of ten bacteriophages infecting Pseudomonas aeruginosa following its addition to 58 sputum samples from cystic fibrosis (CF) patients collected at three different hospitals. Ten samples that did not contain P. aeruginosa were not analysed further. In the remaining 48 samples, the addition of bacteriophages led to a significant decrease in the levels of P. aeruginosa strains, as shown by comparison with controls, taking two variables (time and bacteriophages) into account (p = 0.024). In 45.8% of these samples, this decrease was accompanied by an increase in the number of bacteriophages. We also tested each of the ten bacteriophages individually against 20 colonies from each of these 48 samples and detected bacteriophage-susceptible bacteria in 64.6% of the samples. An analysis of the clinical data revealed no correlation between patient age, sex, duration of P. aeruginosa colonization, antibiotic treatment, FEV1 (forced expiratory volume in the first second) and the efficacy of bacteriophages. The demonstration that bacteriophages infect their bacterial hosts in the sputum environment, regardless of the clinical characteristics of the patients, represents a major step towards the development of bacteriophage therapy to treat chronic lung infections.

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

Seoul hantavirus in Europe: first demonstration of the virus genome in wild Rattus norvegicus captured in France.

Although rats (Rattus rattus or Rattus norvegicus) worldwide have been found to carry Seoul hantavirus, there are at present only a very few reports of confirmed human Seoul hantavirus infections outside Asia, where the virus, in certain areas, is responsible for approximately 25% of the human hantavirus infections. In Europe, no confirmed human infections outside laboratories have been described, and although rats occasionally have been found to be antibody positive, the viral genome has not been demonstrated in these animals. The present report describes the first confirmed finding of Seoul hantavirus in R. norvegicus captured in Europe.

Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid 'real-time' polymerase chain reaction.

STATEMENT OF FINDINGS: We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.