Magistral Phage Preparations: Is This the Model for Everyone?

Phage therapy is increasingly put forward as a promising additional tool to help curb the global antimicrobial resistance crisis. However, industrially manufactured phage medicinal products are currently not available on the European Union and United States markets. In addition, it is expected that the business purpose-driven phage products that are supposed to be marketed in the future would mainly target commercially viable bacterial species and clinical indications, using fixed phage cocktails. hospitals or phage therapy centers aiming to help all patients with difficult-to-treat infections urgently need adequate phage preparations. We believe that national solutions based on the magistral preparation of personalized (preadapted) phage products by hospital and academic facilities could bring an immediate solution and could complement future industrially manufactured products. Moreover, these unlicensed phage preparations are presumed to be more efficient and to elicit less bacterial phage resistance issues than fixed phage cocktails, claims that need to be scientifically substantiated as soon as possible. Just like Belgium, other (European) countries could develop a magistral phage preparation framework that would exist next to the conventional medicinal product development and licensing pathways. However, it is important that the current producers of personalized phage products are provided with pragmatic quality and safety assurance requirements, which are preferably standardized (at least at the European level), and are tiered based on benefit-risk assessments at the individual patient level. Pro bono phage therapy providers should be supported and not stopped by the imposition of industry standards such as Good Manufacturing Practice requirements. Keywords: antimicrobial resistance; antibiotic resistance; bacterial infection; bacteriophage therapy; magistral preparation.

Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity.

Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung.

Preanalytical variables influencing the interpretation and reporting of biological tests on blood samples of living and deceased donors for human body materials.

With the present paper, the Working Group on Cells, Tissues and Organs and other experts of the Superior Health Council of Belgium aimed to provide stakeholders in material of human origin with advice on critical aspects of serological and nucleic acid test (NAT) testing, to improve virological safety of cell- and tissue and organ donation. The current paper focusses on a number of preanalytical variables which can be critical for any medical biology examination: (1) sampling related variables (type of samples, collection of the samples, volume of the sample, choice of specific tubes, identification of tubes), (2) variables related to transport, storage and processing of blood samples (transport, centrifugation and haemolysis, storage before and after centrifugation, use of serum versus plasma), (3) variables related to dilution (haemodilution, pooling of samples), and (4) test dependent variables (available tests and validation). Depending on the type of donor (deceased donor (heart-beating or non-heart beating) versus living donor (allogeneic, related, autologous), and the type of donated human material (cells, tissue or organs) additional factors can play a role: pre- and post-mortem sampling, conditions of sampling (e.g. morgue), haemodilution, possibility of retesting.

Correction to: Biological tests carried out on serum/plasma samples from donors of human body material for transplantation: Belgian experience and practical recommendations.

The article Biological tests carried out on serum/plasma samples from donors of human body material for transplantation: Belgian experience and practical recommendations.

Biological tests carried out on serum/plasma samples from donors of human body material for transplantation: Belgian experience and practical recommendations.

This paper on the biological tests carried out on serum/plasma samples from donors of human body material (HBM) is the result of a project of the working Group of Superior Health Council of Belgium formed with experts in the field of HBM and infectious serology. Indeed, uncertainty about the interpretation of biological test results currently leads to the sometimes unjustified cancelling of planned donations or the rejection of harvested HBM, whilst more sophisticated diagnostic algorithms would still allow the use of organs or HBM that would otherwise have been rejected. NAT tests will not be discussed in this publication. In the first part some general aspects as the need for a formal agreement between the Tissue Establishment l and the laboratory responsible for the biological testing, but also some specifications regarding testing material, the choice of additional biological tests, and some general aspects concerning interpretation and reporting are discussed. In a second part, detailed information and recommendations concerning the interpretation are presented for each of the mandatory tests (human immunodeficiency virus, hepatitis B virus, hepatitis C virus and syphilis) is presented. A number of not mandatory, but regularly used optional serological tests (e.g. for the detection of antibodies to Toxoplasma gondii, Epstein-Barr virus, human T cell leukemia virus and cytomegalovirus) are also extensively discussed. Although the project was meant to provide clarification and recommendations concerning the Belgian legislation, the majority of recommendations are also applicable to testing of donors of tissues and cells in other (European) countries.

Antimicrobial resistance and genomic rep-PCR fingerprints of Pseudomonas aeruginosa strains from animals on the background of the global population structure.

BACKGROUND: Pseudomonas aeruginosa is an important human opportunistic pathogen responsible for fatal nosocomial infections worldwide, and has emerged as a relevant animal pathogen. Treatment options are dramatically decreasing, due to antimicrobial resistance and the microorganism's large versatile genome. Antimicrobial resistance profiles, serotype frequency and genomic profile of unrelated P. aeruginosa isolates of veterinary origin (n = 73), including domesticated, farm, zoo and wild animals mainly from Portugal were studied. The genomic profile, determined by DiversiLab system (Rep-PCR-based technique), was compared with the P. aeruginosa global population structure to evaluate their relatedness. RESULTS: Around 40% of the isolates expressed serotypes O6 (20.5%) and O1 (17.8%). A total of 46.6% of isolates was susceptible to all antimicrobials tested. Isolates obtained from most animals were non-multidrug resistant (86.3%), whereas 11% were multidrug resistant, MDR (non-susceptible to at least one agent in ≥ three antimicrobial categories), and 2.7% extensively drug resistant, XDR (non-susceptible to at least one agent in all but two or fewer antimicrobial categories). Resistance percentages were as follows: amikacin (0.0%), aztreonam (41.1%), cefepime (9.6%), ceftazidime (2.7%), ciprofloxacin (15.1%), colistin (0.0%), gentamicin (12.3%), imipenem (1.4%), meropenem (1.4%), piperacillin + tazobactam (12.3%), ticarcillin (16.4%), ticarcillin + clavulanic acid (17.8%), and tobramycin (1.4%). Animal isolates form a population with a non-clonal epidemic structure indistinguishable from the global P. aeruginosa population structure, where no specific 'animal clonal lineage' was detected. CONCLUSIONS: Serotypes O6 and O1 were the most frequent. Serotype frequency and antimicrobial resistance patterns found in P. aeruginosa from animals were as expected for this species. This study confirms earlier results that P. aeruginosa has a non-clonal population structure, and shows that P. aeruginosa population from animals is homogeneously scattered and indistinguishable from the global population structure.

Recellularizing of human acellular dermal matrices imaged by high-definition optical coherence tomography.

High-definition optical coherence tomography (HD-OCT) permits real-time 3D imaging of the impact of selected agents on human skin allografts. The real-time 3D HD-OCT assessment of (i) the impact on morphological and cellular characteristics of the processing of human acellular dermal matrices (HADMs) and (ii) repopulation of HADMs in vitro by human fibroblasts and remodelling of the extracellular matrix by these cells. Four different skin decellularization methods, Dispase II/Triton X-100, Dispase II/SDS (sodium dodecyl sulphate), NaCl/Triton X-100 and NaCl/SDS, were analysed by HD-OCT. HD-OCT features of epidermal removal, dermo-epidermal junction (DEJ) integrity, cellularity and dermal architecture were correlated with reflectance confocal microscopy (RCM), histopathology and immunohistochemistry. Human adult dermal fibroblasts were in vitro seeded on the NaCl/Triton X-100 processed HADMs, cultured up to 19 days and evaluated by HD-OCT in comparison with MTT proliferation test and histology. Epidermis was effectively removed by all treatments. DEJ was best preserved after NaCl/Triton X-100 treatment. Dispase II/SDS treatment seemed to remove all cellular debris in comparison with NaCl/Triton X-100 but disturbed the DEJ severely. The dermal micro-architectural structure and vascular spaces of (sub)papillary dermis were best preserved with the NaCl/Triton X-100. The impact on the 3D structure and vascular holes was detrimental with Dispase II/SDS. Elastic fibre fragmentation was only observed after Dispase II incubation. HD-OCT showed that NaCl/Triton X-100 processed matrices permitted in vitro repopulation by human dermal fibroblasts (confirmed by MTT test and histology) and underwent remodelling upon increasing incubation time. Care must be taken in choosing the appropriate processing steps to maintain selected properties of the extracellular matrix in HADMs. Processing HADMs with NaCl/Triton X-100 permits in vitro the proliferation and remodelling activity of human dermal fibroblasts. HD-OCT provides unique real-time and non-invasive 3D imaging of tissue-engineered skin constructs and complementary morphological and cytological information.

Quality and safety requirements for sustainable phage therapy products.

The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.

Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa.

Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (∼ 5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to >4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections.

Real-time three-dimensional imaging of epidermal splitting and removal by high-definition optical coherence tomography.

While real-time 3-D evaluation of human skin constructs is needed, only 2-D non-invasive imaging techniques are available. The aim of this paper is to evaluate the potential of high-definition optical coherence tomography (HD-OCT) for real-time 3-D assessment of the epidermal splitting and decellularization. Human skin samples were incubated with four different agents: Dispase II, NaCl 1 M, sodium dodecyl sulphate (SDS) and Triton X-100. Epidermal splitting, dermo-epidermal junction, acellularity and 3-D architecture of dermal matrices were evaluated by High-definition optical coherence tomography before and after incubation. Real-time 3-D HD-OCT assessment was compared with 2-D en face assessment by reflectance confocal microscopy (RCM). (Immuno) histopathology was used as control. HD-OCT imaging allowed real-time 3-D visualization of the impact of selected agents on epidermal splitting, dermo-epidermal junction, dermal architecture, vascular spaces and cellularity. RCM has a better resolution (1 µm) than HD-OCT (3 µm), permitting differentiation of different collagen fibres, but HD-OCT imaging has deeper penetration (570 µm) than RCM imaging (200 µm). Dispase II and NaCl treatments were found to be equally efficient in the removal of the epidermis from human split-thickness skin allografts. However, a different epidermal splitting level at the dermo-epidermal junction could be observed and confirmed by immunolabelling of collagen type IV and type VII. Epidermal splitting occurred at the level of the lamina densa with dispase II and above the lamina densa (in the lamina lucida) with NaCl. The 3-D architecture of dermal papillae and dermis was more affected by Dispase II on HD-OCT which corresponded with histopathologic (orcein staining) fragmentation of elastic fibres. With SDS treatment, the epidermal removal was incomplete as remnants of the epidermal basal cell layer remained attached to the basement membrane on the dermis. With Triton X-100 treatment, the epidermis was not removed. In conclusion, HD-OCT imaging permits real-time 3-D visualization of the impact of selected agents on human skin allografts.

Guidelines to Compose an Ideal Bacteriophage Cocktail.

Properly designed bacteriophage therapeutics are the cornerstone for a successful outcome of bacteriophage therapy. Here we present an overview of the different strategies and steps that can be taken to develop a bacteriophage cocktail that complies with relevant quality and safety requirements. It is based on empirical bacteriophage therapy knowledge from over a century of experience, more recently performed studies, and emerging technologies. We emphasize the selection of adequate bacteriophages and describe a modified Appelmans' method to improve the overall performance of therapeutic bacteriophages individually and collectively in the cocktail. We present two versions of the method, which differ from each other by the employed techniques to evaluate phage activity and synergy: photometric assessment of bacterial growth versus measurement of bacterial respiration via the Omnilog® system.

Burn or trauma scoring: experience of the burn unit of the Queen Astrid Military Hospital during the terror attacks on 22 March 2016.

PURPOSE: On 22 March 2016, the burn unit (BU) of Queen Astrid Military Hospital assessed a surge in severely injured victims from terror attacks at the national airport and Maalbeek subway station according to the damage control resuscitation (DCR) and damage control surgery (DCS) principles. This study delves into its approach to identify a suitable triage scoring system and to determine if a BU can serve as buffer capacity for mass casualty incidents (MCIs). METHODS: The study reviewed retrospectively the origin of explosion, demographic data, sustained injuries, performed surgery, and length of stay of all admitted patients. Trauma scores (Injury Severity Score (ISS) and New Injury Severity Score (NISS)) and triage scores (Revised Trauma Score (RTS), New Trauma Score (NTS), and Trauma Score Injury Severity Score (TRISS)), were compared to burn mortality scores (Osler updated Baux Score and Tobiasen's Abbreviated Burn Severity Index (ABSI)). RESULTS: Of the 23 casualties admitted to the BU, the scores calculated on average 3.5 indications for a level 1 trauma center (ISS 4, NISS 6, RTS 0, T-NTS 4). Nevertheless, no deaths occurred during admission or the 1-year follow-up. CONCLUSION: MCIs create chaos and a high demand for care. Avoiding bottlenecks and adhering to the DCR/DCS principles are necessary to deliver the best care to the largest number of people. This study indicates that a BU can serve as buffer capacity for MCIs. Nevertheless, its integration into the medical resilience plan depends on accurate scoring, comprehensive care availability, and understanding of the DCR/DCS concept. NTS for triage seems the best fit for scoring polytrauma referrals to a BU during MCIs.

Bacteriophage Production in Compliance with Regulatory Requirements.

In this chapter, we discuss production requirements for therapeutic bacteriophage preparations. We review the current regulatory expectancies and focus on pragmatic production processes, implementing relevant controls to ensure the quality, safety, and efficacy of the final products. The information disclosed in this chapter can also serve as a basis for discussions with competent authorities regarding the implementation of expedited bacteriophage product development and licensing pathways, taking into account some peculiarities of bacteriophages (as compared to conventional medicines), such as their specificity for, and co-evolution with, their bacterial hosts. To maximize the potential of bacteriophages as natural controllers of bacterial populations, the implemented regulatory frameworks and manufacturing processes should not only cater to defined bacteriophage products. But, they should also facilitate personalized approaches in which bacteriophages are selected ad hoc and even trained to target the patient's infecting bacterial strain(s), whether or not in combination with other antimicrobials such as antibiotics.

Use of the Naturally Occurring Bacteriophage Grouping Model for the Design of Potent Therapeutic Cocktails.

The specificity of phages and their ability to evolve and overcome bacterial resistance make them potentially useful as adjuncts in the treatment of antibiotic-resistant bacterial infections. The goal of this study was to mimic a natural grouping of phages of interest and to evaluate the nature of their proliferation dynamics with bacteria. We have, for the first time, transferred naturally occurring phage groups directly from their sources of isolation to in vitro and identified 13 P. aeruginosa and 11 K. pneumoniae phages of 18 different genera, whose host range was grouped as 1.2-17%, 28-48% and 60-87%, using a large collection of P. aeruginosa (n = 102) and K. pneumoniae (n = 155) strains carrying different virulence factors and phage binding receptors. We introduced the interpretation model curve for phage liquid culturing, which allows easy and quick analysis of bacterial and phage co-proliferation and growth of phage-resistant mutants (PRM) based on qualitative and partially quantitative evaluations. We assayed phage lytic activities both individually and in 14 different cocktails on planktonic bacterial cultures, including three resistotypes of P. aeruginosa (PAO1, PA14 and PA7) and seven K. pneumoniae strains of different capsular serotypes. Based on the results, the natural phage cocktails designed and tested in this study largely performed well and inhibited PRM growth either synergistically or in proto-cooperation. This study contributes to the knowledge of phage behavior in cocktails and the formulation of therapeutic phage preparations. The paper also provides a detailed description of the methods of working with phages.

Bacteriophages as potential antibiotic potentiators in cystic fibrosis: A new model to study the combination of antibiotics with a bacteriophage cocktail targeting dual species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

OBJECTIVES: Staphylococcus aureus and Pseudomonas aeruginosa co-infections in patients with cystic fibrosis (CF) are associated with disease severity. Their treatment is complicated by biofilm formation in the sticky mucus obstructing the airways. We investigated the activity of phages-antibiotics combinations using a dual species biofilm (P. aeruginosa/S. aureus) formed in artificial sputum medium. METHODS: Biofilmswere incubated with broad-spectrum antibiotics (meropenem, ceftazidime, ciprofloxacin, tobramycin) combined with a cocktail of two (bacterio)phages (PSP3 and ISP) proven active via spot tests and double agar on P. aeruginosa PAO1 and S. aureus ATCC 25923. RESULTS: At the highest tested concentrations (100 x MIC), antibiotics alone caused a 20-50% reduction in biomass and reduced S. aureus and P. aeruginosa CFU of 2.3 to 2.8 and 2.1 to 3.6 log10, respectively. Phages alone reduced biofilm biomass by 23% and reduced P. aeruginosa CFU of 2.1 log10, but did not affect S. aureus viability. Phages enhanced antibiotic effects on biomass and exhibited additive effects with antibiotics against P. aeruginosa, but not against S. aureus. Following inhibition of bacterial respiration by phages in planktonic cultures rationalised these observations by demonstrating that PSP3 was effective at multiplicities of infection (MOI) as low as 10-4 plaque forming units (PFU)/CFU on P. aeruginosa, but ISP, at higher MOI (> 0.1) against S. aureus. CONCLUSION: Pre-screening inhibition of bacterial respiration by phages may assist in selecting those showing activity at sufficiently low titers to showcase anti-biofilm activity in this complex but clinically-relevant in vitro model of biofilm.

Case report: Local bacteriophage therapy for fracture-related infection with polymicrobial multi-resistant bacteria: hydrogel application and postoperative phage analysis through metagenomic sequencing.

Fracture-related infections can be challenging, particularly with concomitant severe bone defects and multi-resistant microorganisms. We present a case of a 42-year-old patient with a fracture-related infection following a war injury from a gunshot, resulting in a 12-cm subtrochanteric segmental bone defect and the detection of four different multi-resistant Gram-negative bacteria. Due to antibiotic drug resistance, treatment with bacteriophages was considered. Phage susceptibility testing revealed the activity of a commercially available bacteriophage cocktail (Intesti bacteriophage, Eliava Institute, Tbilisi, Georgia). This phage cocktail was included in a modified two-stage Masquelet technique. During the first intervention, the bone was debrided and samples for microbiological and phage testing were harvested. The indwelling intramedullary rod was removed, and the bone defect was filled with a PMMA spacer loaded with colistin and the bone stabilized with a plate. During the second procedure, the PMMA spacer was removed and a silver-coated angular stable plate was implanted. The bone defect was filled with a fibular autograft and allograft cancellous bone chips. At the end of the procedure, the Intesti bacteriophage cocktail was injected into a DAC hydrogel and this bacteriophage hydrogel composite was then put onto the angular stable plate. Postoperatively the wound fluid was collected over 72 h, and high-throughput metagenomic sequencing was performed. This showed a time-dependent release of the bacteriophages in the wound fluid, with a relatively high concentration after 12 h, decreasing to DNA copies of 0 after 72 h. Furthermore, we have assessed the release of phages from DAC gel and the effect of DAC gel on the phages in vitro. The results showed a stable and rapid release of phages from the DAC gel (~1×103 PFU/mL). The clinical course of the patient showed no relapse of the infection with good bone consolidation of the bone defect after 1 year without the need for any surgical revision. To the best of our knowledge, this is the first case that shows the detection of bacteriophage DNA copies by high-throughput metagenomics sequencing in a patient with a complex fracture-related infection. Successful treatment of this case encourages further investigation of bacteriophage therapy in patients with complex bone and joint infections.

Bacteriophages for the treatment of pseudomonas-infected vascular prosthesis.

We present a case report detailing therapeutic application of two lytic antipseudomonal bacteriophages to treat a chronic relapsing Pseudomonas aeruginosa infection of a prosthetic aortic graft. As there are currently no Danish laboratories offering phages for clinical therapy, and this case, to our knowledge represents the first applied phage therapy in Denmark, the practical and regulatory aspects of offering this treatment option in Denmark is briefly reviewed along with the clinical case.

Exploiting phage-antibiotic synergies to disrupt Pseudomonas aeruginosa PAO1 biofilms in the context of orthopedic infections.

IMPORTANCE: Biofilm-related infections are among the most difficult-to-treat infections in all fields of medicine due to their antibiotic tolerance and persistent character. In the field of orthopedics, these biofilms often lead to therapeutic failure of medical implantable devices and urgently need novel treatment strategies. This forthcoming article aims to explore the dynamic interplay between newly isolated bacteriophages and routinely used antibiotics and clearly indicates synergetic patterns when used as a dual treatment modality. Biofilms were drastically more reduced when both active agents were combined, thereby providing additional evidence that phage-antibiotic combinations lead to synergism and could potentially improve clinical outcome for affected patients.

Improving phage therapy by evasion of phage resistance mechanisms.

Antibiotic failure is one of the most worrisome threats to global health. Among the new therapeutic efforts that are being explored, the use of bacteriophages (viruses that kill bacteria), also known as 'phages', is being extensively studied as a strategy to target bacterial pathogens. However, one of the main drawbacks of phage therapy is the plethora of defence mechanisms that bacteria use to defend themselves against phages. This review aims to summarize the therapeutic approaches that are being evaluated to overcome the bacterial defence systems, including the most innovative therapeutic approaches applied: circumvention of phage receptor mutations; modification of prophages; targeting of CRISPR-Cas systems and the biofilm matrix; engineering of safer and more efficacious phages; and inhibition of the anti-persister strategies used by bacteria.