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Background: Infections are a major cause of morbidity in burn patients. We aimed to investigate the epidemiology and antibiotic susceptibility of blood stream infections in order to gain a better understanding of their role and burden in our Burn Wound Center. Methods: This retrospective epidemiological investigation analyzed data derived from medical files of patients admitted to our Burn Wound Center having had at least one positive blood culture between 1 January and 31 December 2018. We focused on the prevalence of causative agents in blood stream infections in function of the time after injury and on their drug sensitivity. Results: Among the 363 patients admitted to our Burn Wound Center during the study period, 29 had at least one episode of blood stream infection. Gram-negative organisms accounted for 56,36% of the pathogens in blood stream infections, Gram-positives for 38,17%, and yeasts for 5,45%. Pseudomonas aeruginosa was the most common bacterium (20%), followed by Staphylococcus epidermidis (16.36%), Escherichia coli and Klebsiella pneumoniae (9,09% each). A third of the Gram-negative isolates were multidrug resistant. Gram-positive cocci were isolated from blood cultures at a median of 9 days after the injury, earlier than Gram-negative rods (median 15 days). The main sources of blood stream infections were the burn wounds, followed by infected catheters. Conclusions: Multidrug resistant bacteria must be considered when selecting empirical antibiotic therapy in septic burn patients. In our center, we need to update our antibiotic guidelines, to review the hospital infection control measures and to introduce routine typing technology.

With rising antibiotic resistance, there has been increasing interest in treating pathogenic bacteria with bacteriophages (phage therapy). One limitation of phage therapy is the ease at which bacteria can evolve resistance. Negative effects of resistance may be mitigated when resistance results in reduced bacterial growth and virulence, or when phage coevolve to overcome resistance. Resistance evolution and its consequences are contingent on the bacteria-phage combination and their environmental context, making therapeutic outcomes hard to predict. One solution might be to conduct “in vitro evolutionary simulations” using bacteria-phage combinations from the therapeutic context. Overall, our aim was to investigate parallels between in vitro experiments and in vivo dynamics in a human participant. Evolutionary dynamics were similar, with high levels of resistance evolving quickly with limited evidence of phage evolution. Resistant bacteria – evolved in vitro and in vivo - had lower virulence. In vivo, this was linked to lower growth rates of resistant isolates, whereas in vitro phage resistant isolates evolved greater biofilm production. Population sequencing suggests resistance resulted from selection on de novo mutations rather than sorting of existing variants. These results highlight the speed at which phage resistance can evolve in vivo, and in vitro experiments may give useful insights for clinical evolutionary outcomes.

In Belgium, the incorporation of phages into magistral preparations for human application has been permitted since 2018. The stability of such preparations is of high importance to guarantee quality and efficacy throughout treatments. We evaluated the ability to preserve infectivity of four different phages active against three different bacterial species in five different buffer and infusion solutions commonly used in medicine and biotechnological manufacturing processes, at two different concentrations (9 and 7 log pfu/mL), stored at 4 °C. DPBS without Ca2+ and Mg2+ was found to be the best option, compared to the other solutions. Suspensions with phage concentrations of 7 log pfu/mL were unsuited as their activity dropped below the effective therapeutic dose (6–9 log pfu/mL), even after one week of storage at 4 °C. Strong variability between phages was observed, with Acinetobacter baumannii phage Acibel004 being stable in four out of five different solutions. We also studied the long term storage of lyophilized staphylococcal phage ISP, and found that the titer could be preserved during a period of almost 8 years when sucrose and trehalose were used as stabilizers. After rehydration of the lyophilized ISP phage in saline, the phage solutions remained stable at 4 °C during a period of 126 days.

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., Author summary In many countries, Burkholderia multivorans is the most prevalent species within the Burkholderia cepacia complex (Bcc) found infecting the lungs of patients with cystic fibrosis (CF). Its positive identification is of immediate concern to the health of the patient as it is notoriously hard to eradicate using antibiotics and can cause necrosis of the lung tissues (cepacia syndrome). Infection control measures reduced the prevalence of B. cenocepacia in CF wards, but patients continue to acquire infections by B. multivorans from environmental sources. In most reported cases, the infecting strains are unique except in rare cases in which cross-infection is observed between patients. We report here an endemic strain of B. multivorans with sequence type ST-742 that has been infecting multiple patients, without evidence for cross-infection. We investigated the epidemiology and genomics of this ST-742 strain and show that it is microdiverse, as isolates between-patients exhibit numerous genomic differences, at scales that have not been observed previously when looking at evolutionary trajectories within-patients. Additionally, we found that the specific genomic background of a given strain may dictate the strategy of adaptation within the CF lung.

Background The Democratic Republic of the Congo (DRC) has one of the highest neonatal death rates (between 14% and 28%) in the world. In the DRC, neonatal sepsis causes 15.6% of this mortality, but data on the bacterial etiology and associated drug susceptibility are lacking. Methods Hemocultures of 150 neonates with possible early-onset neonatal sepsis (pEOS) were obtained at the Hôpital Provincial Général de Référence de Bukavu (Bukavu, DRC). The newborns with pEOS received an empirical first-line antimicrobial treatment (ampicillin, cefotaxime, and gentamicin) based on the synopsis of international guidelines for the management of EOS that are in line with World Health Organization (WHO) recommendations. Isolates were identified using matrix-assisted laser desorption/ ionization time-of-flight mass spectrophotometry. Antibiotic resistance was assessed using the disk diffusion method. Results Fifty strains were obtained from 48 patients and identified. The 3 most prevalent species were Enterobacter cloacae complex (42%), Klebsiella pneumoniae (18%), and Serratia marcescens (12%). Enterobacter cloacae isolates were resistant to all first-line antibiotics. All K. pneumoniae and S. marcescens isolates were resistant to ampicillin, and the majority of the K. pneumoniae and half of the S. marcescens isolates were resistant to both cefotaxime and gentamicin. All E. cloacae complex strains, 89% of K. pneumoniae, and half of S. marcescens had an extended-spectrum ß-lactamase phenotype. Conclusions The most prevalent pathogens causing EOS in Bukavu were E. cloacae complex, K. pneumoniae, and S. marcescens. Most of these isolates were resistant to the WHO-recommended antibiotics.

Non-typhoidal Salmonella present a major threat to animal and human health as food-borne infectious agents. We characterized 91 bacterial isolates from Armenia and Georgia in detail, using a suite of assays including conventional microbiological methods, determining antimicrobial susceptibility profiles, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, serotyping (using the White-Kauffmann-Le Minor scheme) and genotyping (repetitive element sequence-based PCR (rep-PCR)). No less than 61.5% of the isolates were shown to be multidrug-resistant. A new antimicrobial treatment strategy is urgently needed. Phage therapy, the therapeutic use of (bacterio-) phages, the bacterial viruses, to treat bacterial infections, is increasingly put forward as an additional tool for combatting antibiotic resistant infections. Therefore, we used this representative set of well-characterized Salmonella isolates to analyze the therapeutic potential of eleven single phages and selected phage cocktails from the bacteriophage collection of the Eliava Institute (Georgia). All isolates were shown to be susceptible to at least one of the tested phage clones or their combinations. In addition, genome sequencing of these phages revealed them as members of existing phage genera (Felixounavirus, Seunavirus, Viunavirus and Tequintavirus) and did not show genome-based counter indications towards their applicability against non-typhoidal Salmonella in a phage therapy or in an agro-food setting. ispartof: VIRUSES-BASEL vol:12 issue:12 ispartof: location:Switzerland status: published

BackgroundBacterial vaginosis (BV) is the most common gynecological condition in women of reproductive age and associated with adverse pregnancy outcomes. In the Democratic Republic of the Congo (DRC), neonatal mortality rate is as high as 2.8 percent with preterm birth (PTB) and low birth weight (LBW) as leading causes. Because no studies have addressed BV in DRC, we aimed to investigate the prevalence of BV, the risk factors and the association between BV and adverse pregnancy outcomes in a population of pregnant women from Bukavu, DRC.MethodsA total of 533 pregnant women in the second trimester of pregnancy were recruited in the Provincial Reference Hospital of Bukavu, DRC, between January and October 2017, and followed until delivery. Clinical and sociodemographic data of mother and newborn, and data on (vaginal) hygiene practices, sexual behavior and reproductive history were collected. BV was diagnosed by Nugent scoring of Gram-stained vaginal smears. Two multivariate regression models were built to identify risk factors for BV and to investigate BV as a risk factor for adverse pregnancy outcomes.ResultsThe prevalence of BV was 26.3% and approximately half of the women with BV were asymptomatic. Independent risk factors for BV were the use of alternatives to water for intravaginal washing, concurrent partners, unemployed status, the presence of vaginalCandidaand clay consumption. BV was independently associated with both LBW and PTB of an infant with LBW.ConclusionThe prevalence of BV in Bukavu is high but in line with the global average. BV was associated with adverse pregnancy outcomes in our study population. Hence, research on modifiable risk factor-based interventions to reduce the prevalence of BV, and on screening/treatment of BV during antenatal care should be explored to reduce neonatal mortality and morbidity.

Dissemination of antibiotic resistance is a major concern, especially in aquatic environments, where pollution contributes for resistant bacteria selection. These strains may have serious health implications, especially for endangered species, including the sea turtles' hawksbill Eretmochelys imbricata and green turtles Chelonia mydas. We aimed to evaluate the presence of antibiotic resistant pseudomonads in wild sea turtles from Principe Island, Sao Tome and Principe, Guinea Gulf. Isolates were obtained from oral and cloacal swabs of free-living turtles by conventional techniques. Pseudomonads screening was performed by multiplex-PCR (oprl/oprL) and biochemical identification and antibiotic resistance profiling were achieved using Vitek2. All pseudomonad isolates were genotyped by Rep-PCR. Thirteen isolates were oprl-positive and classified as pseudomonads, eight from the genus Pseudomonas with the species P. aeruginosa, P. stutzeri, and P. mendocina, and five co-isolated Alcaligenes faecalis. The P. aeruginosa isolate was also oprL-positive. Regarding isolates susceptibility profile, 38.5% were susceptible to all antibiotics tested, and multidrug resistant (MDR) strains were not identified. DNA fingerprinting did not show any specific clonal-cluster similarity. Data on the worldwide incidence of antibiotic resistance among wildlife is still very scarce, especially concerning remote tropical areas. Since Pseudomonas genus has emerged as a group of increasingly reported opportunistic microorganisms in human and veterinary medicine with high resistance levels, it could be used as a tool for environmental resistance surveillance, particularly considering their ubiquity. Oceandrio de Lisboa, Portugal Marine Turtle Conservation Act - U.S. Fish & Wildlife Service grant Interdisciplinary Research Centre for Animal Health, Faculty of Veterinary Medicine, University of Lisbon (FMV/UL) [UID/CVT/00276/2013] Laboratory of Molecular and Cellular Technology, Queen Astrid Military Hospital, Brussels, Belgium info:eu-repo/semantics/publishedVersion

To determine phage titers accurately, reproducibly and in a non-laborious and cost-effective manner, we describe the development of a qPCR platform for molecular quantification of five phages present in bacteriophage cocktail 2 (BFC2). We compared the performance of this molecular approach, with regard to quantification and reproducibility, with the standard culture-based double agar overlay method (DAO). We demonstrated that quantification of each of the five phages in BFC2 was possible by means of qPCR, without prior DNA extraction, but yields were significantly higher in comparison to DAO. Although DAO is assumed to provide an indication of the number of infective phage particles, whereas qPCR only provides information on the number of phage genomes, the difference in yield (qPCR/DAO ratio) was observed to be phage-dependent and appeared rather constant for all phages when analyzing different (freshly prepared) stocks of these phages. While DAO is necessary to determine sensitivity of clinical strains against phages in clinical applications, qPCR might be a valid alternative for rapid and reproducible quantification of freshly prepared stocks, after initial establishment of a correction factor towards DAO.

Bacteriophages (phages for short) are increasingly put forward as potential (additional) tools in the fight against the worldwide antibiotic crisis. However, the setup of clinical trials, which are necessary to demonstrate the efficacy of phages and to optimize their clinical application protocols, and consequently to their implementation in the clinic is still hindered by the lack of an adapted regulatory framework. Although some (local) progresses have been made in recent years, no real paradigm shift has occurred. This chapter discusses the current status in Europe with regard to the two most pertinent barriers to phage therapy: the regulatory and intellectual property protection issues.

Ten Belgian travelers returned from Mali with a Schistosoma haematobium-Schistosoma bovis hybrid infection, confirmed by DNA sequencing from eggs. Clinical symptoms and laboratory findings resembled those of classic acute schistosomiasis, but the detected eggs were morphologically unusual.

This multidisciplinary expert panel opinion on bacteriophage therapy has been written in the context of a society that is confronted with an ever increasing number of antibiotic resistant bacteria. To avoid the return to a pre-antibiotic era, alternative treatments are urgently needed. The authors aim to contribute to the opinion formation of relevant stakeholders on how to potentially develop an infrastructure and legislation that paves the way for the acceptance and re-implementation of bacteriophage therapy.;

Recently, aTyphi isolate producing CTX-M-15 extended spectrum β-lactamase (ESBL) and with decreased ciprofloxacin susceptibility was isolated in the Democratic Republic of the Congo. We have selected bacteriophages that show strong lytic activity against this isolate and have potential for phage-based treatment of. Typhi, andin general. ispartof: Viruses vol:10 issue:4 ispartof: location:Switzerland status: published

Since time immemorial, phages—the viral parasites of bacteria—have been protecting Earth’s biosphere against bacterial overgrowth. Today, phages could help address the antibiotic resistance crisis that affects all of society. The greatest hurdle to the introduction of phage therapy in Western medicine is the lack of an appropriate legal and regulatory framework. Belgium is now implementing a pragmatic phage therapy framework that centers on the magistral preparation (compounding pharmacy in the US) of tailor-made phage medicines.

The emergence of antibiotic-resistant bacteria and decrease in the discovery rate of novel antibiotics takes mankind back to the ‘pre-antibiotic era' and search for alternative treatments. Bacteriophages have been one of promising alternative agents which can be utilised for medicinal and biological control purposes in agriculture and related fields. The idea to treat bacterial infections with phages came out of the pioneering work of Félix d‘Hérelle but this was overshadowed by the success of antibiotics. Recent renewed interest in phage therapy is dictated by its advantages most importantly by their specificity against the bacterial targets. This prevents complications such as antibiotic-induced dysbiosis and secondary infections. This article is compiled by the participants of the Expert Round Table conference ‘Bacteriophages as tools for therapy, prophylaxis and diagnostics' (19–21 October 2015) at the Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia. The first paper from the Round Table was published in the Biotechnology Journal1. This In Focus article expands from this paper and includes recent developments reported since then by the Expert Round Table participants, including the implementation of the Nagoya Protocol for the applications of bacteriophages.

Bacteriophages could be used along with burn wound care products to enhance antimicrobial pressure during treatment. However, some of the components of the topical antimicrobials that are traditionally used for the prevention and treatment of burn wound infection might affect the activity of phages. Therefore, it is imperative to determine the counteraction of therapeutic phage preparations by burn wound care products before application in patients. Five phages, representatives of two morphological families (Myoviridae and Podoviridae) and active against 3 common bacterial burn wound pathogens (Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus) were tested against 13 different products commonly used in the treatment of burn wounds. The inactivation of the phages was quite variable for different phages and different products. Majority of the anti-infective products affected phage activity negatively either immediately or in the course of time, although impact was not always significant. Products with high acidity had the most adverse effect on phages. Our findings demonstrate that during combined treatment the choice of phages and wound care products must be carefully defined in advance.

International audience; 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.

Lectin-like bacteriocins of the LlpA family, originally identified in plant-associated bacteria, are narrow-spectrum antibacterial proteins composed of two tandemly organized monocot mannose-binding lectin (MMBL) domains. The LlpA-like bacteriocin of Pseudomonas aeruginosa C1433, pyocin L1, lacks any similarity to known P. aeruginosa bacteriocins. The initial interaction of pyocin L1 with target cells is mediated by binding to D-rhamnose, present in the common polysaccharide antigen of lipopolysaccharides but the actual cytotoxic mechanism is unknown. In this study, we characterized the activity range of pyocin L1 and two additional L pyocins revealed by genome mining, representing two highly diverged LlpA groups in P. aeruginosa. The recombinant proteins exhibit species-specific antagonistic activities down to nanomolar concentrations against clinical and environmental P. aeruginosa strains, including several multidrug-resistant isolates. The overlap in target strain spectrum between two close homologues of the pyocin L1 group is only minimal, contrasting with the considerable spectral redundancy of LlpA proteins reported for other Pseudomonas species. No correlation was found between L pyocin susceptibility and phylogenetic relatedness of P. aeruginosa isolates. Sensitive strains were retrieved in 13 out of 15 O serotypes tested, excluding the possibility that the highly variable and immunogenic O serotype antigen of the lipopolysaccharide coating would represent a dominant susceptibility-discriminating factor. ispartof: MicrobiologyOpen vol:3 issue:6 pages:875-884 ispartof: location:England status: published

Bacteriophages could help address the antibiotic resistance crisis that impacts health systems all over the world. In 2011, the European Commission formally confirmed that phage products used as therapeutics are medicinal products and thus manufacturers need to navigate the extremely arduous and enormously expensive medicine development and marketing pathway. However, up until now, not one therapeutic phage product has made it to the European market, and yet clinicians are under increasing pressure to use phages in the treatment of multidrug-resistant bacterial infections. While a handful of small European enterprises are struggling to squeeze therapeutic phage products through the conventional and centralised European medicinal products funnel, some clinicians and academics are exploring (European) national solutions to accelerate the availability of phages for the treatment of an increasing number of desperate patients. This mini-review summarises the actual status and perspectives of clinical phage application in Europe.

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.

The transplantation of conventional human cell and tissue grafts, such as heart valve replacements and skin for severely burnt patients, has saved many lives over the last decades. The late eighties saw the emergence of tissue engineering with the focus on the development of biological substitutes that restore or improve tissue function. In the nineties, at the height of the tissue engineering hype, industry incited policymakers to create a European regulatory environment, which would facilitate the emergence of a strong single market for tissue engineered products and their starting materials (human cells and tissues). In this paper we analyze the elaboration process of this new European Union (EU) human cell and tissue product regulatory regime—i.e. the EU Cell and Tissue Directives (EUCTDs) and the Advanced Therapy Medicinal Product (ATMP) Regulation and evaluate its impact on Member States’ health care systems. We demonstrate that the successful lobbying on key areas of regulatory and policy processes by industry, in congruence with Europe’s risk aversion and urge to promote growth and jobs, led to excessively business oriented legislation. Expensive industry oriented requirements were introduced and contentious social and ethical issues were excluded. We found indications that this new EU safety and health legislation will adversely impact Member States’ health care systems; since 30 December 2012 (the end of the ATMP transitional period) there is a clear threat to the sustainability of some lifesaving and established ATMPs that were provided by public health institutions and small and medium-sized enterprises under the frame of the EUCTDs. In the light of the current economic crisis it is not clear how social security systems will cope with the inflation of costs associated with this new regulatory regime and how priorities will be set with regard to reimbursement decisions. We argue that the ATMP Regulation should urgently be revised to focus on delivering affordable therapies to all who are in need of them and this without necessarily going to the market. The most rapid and elegant way to achieve this would be for the European Commission to publish an interpretative document on “placing on the market of ATMPs,” which keeps tailor-made and niche ATMPs outside of the scope of the medicinal product regulation.

With the growing global antimicrobial resistance crisis, there is a critical need for alternatives to conventional antibiotics, especially in developing countries. Virulent bacteriophages (phages) represent a viable antibacterial technology that could be particularly beneficial, since phages are active against antimicrobial-resistant bacteria, easy to isolate from contaminated environments, and relatively inexpensive to produce. We discuss here examples of infectious diseases that significantly affect developing countries, phage applications that could be especially impactful in those settings, and special considerations for implementing phages in the developing world.

On 8 June, 2015, London, a workshop on bacteriophage therapy was organized by the European Medicines Agency (EMA). About 60 delegates from various stakeholders (participants belonged to public bodies such as WHO, ECDC, WAAAR, national health agencies from different European countries, politicians, journalists, clinicians, researchers, including six members of P.H.A.G.E., as well as private companies.) were convened to participate, with the workshop broadcast on the EMA website. Why did EMA organize a workshop on bacteriophage therapy? The answer is the emergency to find solutions to overcome the global antibiotic crisis and the unfortunately corresponding lack of significant interest in bacteriophage therapy. The past two decades, public health agencies reported on the dramatic increase of drug-resistant pathogens, a worrisome situation leading the World Health Organization (WHO) to declare a new ‘preantibiotic era’ in its 2014 surveillance report (http://www.who.int). In the USA, based on data collected in 2009, over 20 000 annual deaths were ascribed to the lack of effective antibiotics. Comparably in Europe, we estimate that more than 100 000 people worldwide die annually. The situation gets worse: a recent study mentions that in France about 13 000 people died in 2012 (http://www.invs.sante.fr). Worldwide, the estimation is rising to 700 000 deaths annually, and therefore G7 Health Ministries recently called for a global ‘One Health’ approach (https://www.g7germany.de). Future predictions are alarming, with 10 million people estimated to die from resistant microorganisms in 2050 if nothing changes (Carlet 2015). Bacteriophage therapy is nearly 100 years old and was first introduced in France and Belgium a decade before the discovery of penicillin by A. Fleming. After an initial worldwide interest (including from the …

Increases in antibiotic minimum inhibitory concentrations (MICs) for Pseudomonas aeruginosa during treatment are commonly observed but their relationship to efflux overexpression remains poorly documented. In this study, pairs of first [at time of diagnosis (D0)] and last [during treatment (DL)] P. aeruginosa isolates were obtained from patients treated for suspicion of nosocomial pneumonia. Pair clonality was determined by repetitive extragenic palindromic PCR. Overexpression of mexA and mexX was assessed by real-time PCR, and expression of mexC and mexE was assessed by PCR. Antibiotics received by patients before and during treatment were determined from clinical charts. For D0 isolates, 24% were from patients without antibiotics for 1 month and 64% were negative for mexA/mexX overexpression and mexC/mexE expression. For DL isolates, approximately one-half of the patients had received piperacillin/tazobactam, amikacin, meropenem and/or cefepime, and 17% had received ciprofloxacin (alone or in combination); 38% did not show changes in expression of the four genes, whereas 38% showed increased expression for one gene (mainly mexA or mexX), 19% for two genes (mainly mexA and mexX) and 5% for three or four genes. Isolates overexpressing mexA or mexX had median MICs above EUCAST clinical resistance breakpoints for ciprofloxacin, cefepime and meropenem, or for ciprofloxacin, amikacin, cefepime and meropenem, respectively. mexA or mexX overexpression was statistically significantly associated with patients’ exposure to ciprofloxacin and meropenem or cefepime and meropenem, respectively. Overexpression of genes encoding antibiotic transporters in P. aeruginosa during treatment is frequent and is associated with increases in MICs above EUCAST clinical susceptibility breakpoints.

Viable donor skin is still considered the gold standard for the temporary covering of burns. Since 1985, the Brussels military skin bank supplies cryopreserved viable cadaveric skin for therapeutic use. Unfortunately, viable skin can not be sterilised, which increases the risk of disease transmission. On the other hand, every effort should be made to ensure that the largest possible part of the donated skin is processed into high-performance grafts. Cryopreserved skin allografts that fail bacterial or fungal screening are reworked into 'sterile' non-viable glycerolised skin allografts. The transposition of the European Human Cell and Tissue Directives into Belgian Law has prompted us to install a pragmatic microbiological screening and acceptance procedure, which is based on 14 day enrichment broth cultures of finished product samples and treats the complex issues of 'acceptable bioburden' and 'absence of objectionable organisms'. In this paper we evaluate this procedure applied on 148 skin donations. An incubation time of 14 days allowed for the detection of an additional 16.9% (25/148) of contaminated skin compared to our classic 3 day incubation protocol and consequently increased the share of non-viable glycerolised skin with 8.4%. Importantly, 24% of these slow-growing microorganisms were considered to be potentially pathogenic. In addition, we raise the issue of 'representative sampling' of heterogeneously contaminated skin. In summary, we feel that our present microbiological testing and acceptance procedure assures adequate patient safety and skin availability. The question remains, however, whether the supposed increased safety of our skin grafts outweighs the reduced overall clinical performance and the increase in work load and costs.

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts.

This perspective paper follows up on earlier communications on bacteriophage therapy that we wrote as a multidisciplinary and intercontinental expert-panel when we first met at a bacteriophage conference hosted by the Eliava Institute in Tbilisi, Georgia in 2015. In the context of a society that is confronted with an ever-increasing number of antibiotic-resistant bacteria, we build on the previously made recommendations and specifically address how the Nagoya Protocol might impact the further development of bacteriophage therapy. By reviewing a number of recently conducted case studies with bacteriophages involving patients with bacterial infections that could no longer be successfully treated by regular antibiotic therapy, we again stress the urgency and significance of the development of international guidelines and frameworks that might facilitate the legal and effective application of bacteriophage therapy by physicians and the receiving patients. Additionally, we list and comment on several recently started and ongoing clinical studies, including highly desired double-blind placebo-controlled randomized clinical trials. We conclude with an outlook on how recently developed DNA editing technologies are expected to further control and enhance the efficient application of bacteriophages.

A collection of 15 newly isolated (bacterio)phages infecting the opportunistic pathogen Pseudomonas aeruginosa was established to investigate their global diversity and potential in phage therapy. These phages were sampled in 14 different countries traversing four continents, from both natural environments and hospital sewage. They all display unique DNA and protein profiles and cluster morphologically into six groups within the three major families of the Caudovirales. Extensive host range studies on a library of 122 AFLP-genotyped clinical P. aeruginosa strains (of which 49 were newly isolated at the University Hospital of Leuven, Belgium) showed that the phages lysed 87% of the strains. Infection analysis of outer membrane mutants identified 10 phages as type IV pili-dependent. More detailed information about the evolutionary relatedness of the phages was gathered by de novo peptide sequencing of major virion proteins using tandem Matrix-Assisted Laser Desorption/Ionization Time of Flight technology. Applying this technique for the first time to viruses, seven groups of closely related phages were identified without the need of prior knowledge of genome content and/or electron microscopic imaging. This study demonstrates both the epidemic population structure of P. aeruginosa and the global spread of P. aeruginosa phage species, and points at the resistance of two clinically predominant, widespread P. aeruginosa strains against phage attack.

The treatment of infectious diseases with antibiotics is becoming increasingly challenging. Very few new antimicrobials are in the pharmaceutical industry pipeline. One of the potential alternatives for antibiotics is phage therapy. Major obstacles for the clinical application of bacteriophages are a false perception of viruses as ‘enemies of life’ and the lack of a specific frame for phage therapy in the current Medicinal Product Regulation. Short-term borderline solutions under the responsibility of a Medical Ethical Committee and/or under the umbrella of the Declaration of Helsinki are emerging. As a long-term solution, however, we suggest the creation of a specific section for phage therapy under the Advanced Therapy Medicinal Product Regulation.

Type III secretion system (T3SS) in Pseudomonas aeruginosa is associated with poor clinical outcome in acute infections. T3SS allows for injection of bacterial exotoxins (e.g. ExoU or ExoS) into the host cell, causing cytotoxicity. It also activates the cytosolic NLRC4 inflammasome, activating caspase-1, inducing cytotoxicity and release of mature IL-1β, which impairs bacterial clearance. In addition, flagellum-mediated motility has been suggested to also modulate inflammasome response and IL-1β release. Yet the capacity of clinical isolates to induce IL-1β release and its relation with cytotoxicity have never been investigated. Using 20 clinical isolates from acute infections with variable T3SS expression levels and human monocytes, our aim was to correlate IL-1β release with toxin expression, flagellar motility and cytotoxicity. ExoU-producing isolates caused massive cell death but minimal release of IL-1β, while those expressing T3SS but not ExoU (i.e. expressing ExoS or no toxins) induced caspase-1 activation and IL-1β release, the level of which was correlated with cytotoxicity. Both effects were prevented by a specific caspase-1 inhibitor. Flagellar motility was not correlated with cytotoxicity or IL-1β release. No apoptosis was detected. Thus, T3SS cytotoxicity is accompanied by a modification in cytokine balance for P. aeruginosa clinical isolates that do not express ExoU.

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 allowsa 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., SCOPUS: re.j, info:eu-repo/semantics/published

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.

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.

Chronic Pseudomonas aeruginosa infections are the main cause of morbidity among patients with cystic fibrosis (CF) due to persistent lung inflammation caused by interaction between this bacterium and the immune system. Longitudinal studies of clonally related isolates of a dominant CF clone have indicated that genome reduction frequently occurs during adaptation of P. aeruginosa in the CF lung. In this study, we have evaluated the P. aeruginosa population structure of patients attending the Universitair Ziekenhuis Brussel (UZ Brussel) CF reference center using a combination of genotyping methods. Although the UZ Brussel P. aeruginosa CF population is characterized by the absence of a dominant CF clone, some potential interpatient transmissions could be detected. Interestingly, one of these clones showed deletion of the alternative type I ferripyoverdine receptor gene fpvB . Furthermore, we found that several other TonB-dependent receptors are deleted as well. The genome of one potentially transmissible CF clone was sequenced, revealing large deleted regions including all type III secretion system genes and several virulence genes. Remarkably, a large number of deleted genes are shared between the P. aeruginosa CF clone described in this study and isolates belonging to the dominant Copenhagen CF DK2 clone, suggesting parallel evolution.

The excessive and improper use of antibiotics has led to an increasing incidence of bacterial resistance. In Europe the yearly number of infections caused by multidrug resistant bacteria is more than 400.000, each year resulting in 25.000 attributable deaths. Few new antibiotics are in the pipeline of the pharmaceutical industry. Early in the 20th century, bacteriophages were described as entities that can control bacterial populations. Although bacteriophage therapy was developed and practiced in Europe and the former Soviet republics, the use of bacteriophages in clinical setting was neglected in Western Europe since the introduction of traditional antibiotics. Given the worldwide antibiotic crisis there is now a growing interest in making bacteriophage therapy available for use in modern western medicine. Despite the growing interest, access to bacteriophage therapy remains highly problematic. In this paper, we argue that the current state of affairs is morally unacceptable and that all stakeholders (pharmaceutical industry, competent authorities, lawmakers, regulators, and politicians) have the moral duty and the shared responsibility towards making bacteriophage therapy urgently available for all patients in need.

There is a distinct difference between tissue and organ donation, although it is usually not well perceived by the public. Solid organs (e.g. kidney, liver and heart) can be taken only from donors who are brain-dead and on life support or immediately after irreversible cardio-respiratory arrest. Organs have to be transported quickly from donor to recipient and are not, or only slightly, processed. Their procurement is generally controlled by surgeons in transplant hospitals and allocation is usually coordinated by national or regional organizations. In contrast, human cells and tissues (e.g. bone, skin and heart valves) may come from live organ donors but more usually come from deceased donors in hospitals, morgues or even funeral homes. These cells and tissues are often transformed and stored, sometimes for years, in “tissue establishments” from which they can be distributed across the world. Tissue brokers, processors and distributors steer the allocation of the resulting human cells, tissues, cellular and tissue-based products.

The Cram-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 knownpyoverdine 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.