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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 coevolves 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 how 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.

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.

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.

Correctly designed bacteriophage therapeutics are the cornerstone for a successful outcome of bacteriophage therapy. Here we overview strategies on how to choose bacteriophages and their bacterial hosts at different steps of a bacteriophage cocktail development in order to comply with all quality and safety requirements based on the already existing essentially empirical experience in bacteriophage therapy and current accomplishments in modern biomedical sciences. A modification of the classic Appelmans' method (1922) to assess stability of bacteriophage activity in liquid media is presented in order to improve the overall performance of therapeutic bacteriophages individually and collectively in the cocktail.

In this chapter we review bacteriophage production requirements to help institutions, which wish to manufacture bacteriophage products for human use in compliance with the applicable regulatory expectancies, defining production processes and implementing relevant controls ensuring 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 development of expedited bacteriophage product development and licensing pathways, including relevant and pragmatic requirements, and allowing for the full exploitation of bacteriophages as natural controllers of bacterial populations.

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.

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.

Multidrug resistant Acinetobacter baumannii and its closely related species A. pittii and A. nosocomialis, all members of the Acinetobacter calcoaceticus-baumannii (Acb) complex, are a major cause of hospital acquired infection. In the burn wound center of the Queen Astrid military hospital in Brussels, 48 patients were colonized or infected with Acb complex over a 52-month period. We report the molecular epidemiology of these organisms, their clinical impact and infection control measures taken. A representative set of 157 Acb complex isolates was analyzed using repetitive sequence-based PCR (rep-PCR) (DiversiLab) and a multiplex PCR targeting OXA-51-like and OXA-23-like genes. We identified 31 rep-PCR genotypes (strains). Representatives of each rep-type were identified to species by rpoB sequence analysis: 13 types to A. baumannii, 10 to A. pittii, and 3 to A. nosocomialis. It was assumed that isolates that belonged to the same rep-type also belonged to the same species. Thus, 83.4% of all isolates were identified to A. baumannii, 9.6% to A. pittii and 4.5% to A. nosocomialis. We observed 12 extensively drug resistant Acb strains (10 A. baumannii and 2 A. nosocomialis), all carbapenem-non-susceptible/colistin-susceptible and imported into the burn wound center through patients injured in North Africa. The two most prevalent rep-types 12 and 13 harbored an OXA-23-like gene. Multilocus sequence typing allocated them to clonal complex 1 corresponding to EU (international) clone I. Both strains caused consecutive outbreaks, interspersed with periods of apparent eradication. Patients infected with carbapenem resistant A. baumannii were successfully treated with colistin/rifampicin. Extensive infection control measures were required to eradicate the organisms. Acinetobacter infection and colonization was not associated with increased attributable mortality.

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.

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.

The worldwide emergence of antibiotic resistances and the drying up of the antibiotic pipeline have spurred a search for alternative or complementary antibacterial therapies. Bacteriophages are bacterial viruses that have been used for almost a century to combat bacterial infections, particularly in Poland and the former Soviet Union. The antibiotic crisis has triggered a renewed clinical and agricultural interest in bacteriophages. This, combined with new scientific insights, has pushed bacteriophages to the forefront of the search for new approaches to fighting bacterial infections. But before bacteriophage therapy can be introduced into clinical practice in the European Union, several challenges must be overcome. One of these is the conceptualization and classification of bacteriophage therapy itself and the extent to which it constitutes a human medicinal product regulated under the European Human Code for Medicines (Directive 2001/83/EC). Can therapeutic products containing natural bacteriophages be categorized under the current European regulatory framework, or should this framework be adapted? Various actors in the field have discussed the need for an adapted (or entirely new) regulatory framework for the reintroduction of bacteriophage therapy in Europe. This led to the identification of several characteristics specific to natural bacteriophages that should be taken into consideration by regulators when evaluating bacteriophage therapy. One important consideration is whether bacteriophage therapy development occurs on an industrial scale or a hospital-based, patient-specific scale. More suitable regulatory standards may create opportunities to improve insights into this promising therapeutic approach. In light of this, we argue for the creation of a new, dedicated European regulatory framework for bacteriophage therapy.

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.

The growing problem of antibiotic-resistant bacteria has re-kindled interest in phage-based therapies. Yet, the use of phages to treat life-threatening bacterial infections is held back by the lack of an appropriate regulatory framework for phage therapy. ispartof: EMBO reports vol:14 issue:11 pages:951-954 ispartof: location:England status: published

With this analysis we would like to raise some issues that emerge as a result of recent evolutions in the burgeoning field of human cells, tissues, and cellular and tissue-based product (HCT/P) transplantation, and this in the light of the current EU regulatory framework. This paper is intended as an open letter addressed to the EU policy makers, who will be charged with the review and revision of the current legislation. We propose some urgent corrections or additions to cope with the rapid advances in biomedical science, an extensive commercialization of HCT/Ps, and the growing expectation of the general public regarding the ethical use of altruistically donated cells and tissues. Without a sound wake-up call, the diverging interests of this newly established 'healthcare' industry and the wellbeing of humanity will likely lead to totally unacceptable situations, like some of which we are reporting here. © 2012 Springer Science+Business Media B.V., SCOPUS: ar.j, info:eu-repo/semantics/published

In 2011, a novel strain of O104:H4 Escherichia coli caused a serious outbreak of foodborne hemolytic uremic syndrome and bloody diarrhea in Germany. Antibiotics were of questionable use and 54 deaths occurred. Candidate therapeutic bacteriophages that efficiently lyse the E. coli O104:H4 outbreak strain could be selected rather easily from a phage bank or isolated from the environment. It is argued that phage therapy should be more considered as a potential armament against the growing threat of (resistant) bacterial infections. ispartof: PLoS One vol:7 issue:12 ispartof: location:United States status: published

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the 'Twort-like viruses'. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes. PC holds a postdoctoral fellowship of the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen' (FWO-Vlaanderen, Belgium). This study was performed by members of the research community 'PhageBiotics' and was funded by the Federal Public Service of Health, Food Chain Safety and Environment, Belgium (contract RT 08/6 PHAGE). DNA fingerprinting and hybridization experiments were supported by grant MED 19 of the Royal High Institute for Defence, Belgium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The present opinion is the result of discussions on the future of phage therapy (personalized or large-scale uniform therapy?) during the first International Congress on Viruses of Microbes, held at the Institut Pasteur in Paris on June 21–25, 2010.

SUMMARY: The open reading frame of the OprI lipoprotein gene from Pseudomonas aeruginosa was amplified by polymerase chain reaction (PCR) starting from purified DNA or colony lysates. A fragment of the expected size (249 bp) was detected in all P. aeruginosa strains from various clinical and geographical origins. The gene could only be amplified in pseudomonads of rRNA group I which are considered to be the authentic genus Pseudomonas. Digestions with HaeIII, PvuII and SphI of the amplified fragments demonstrated a sequence variation in the oprI gene. Colony, dot and Western blots with two monoclonal antibodies (mAbs) against the lipoprotein I confirmed our PCR results. These findings open interesting perspectives for the molecular taxonomy of the genus Pseudomonas and the development of diagnostic tools.

Phage therapy is the application of bacteria-specific viruses with the goal of reducing or eliminating pathogenic or nuisance bacteria. While phage therapy has become a broadly relevant technology, including veterinary, agricultural, and food microbiology applications, it is for the treatment or prevention of human infections that phage therapy first caught the world's imagination--see, especially, Arrowsmith by Sinclair Lewis (1925)--and which today is the primary motivator of the field. Nonetheless, though the first human phage therapy took place in the 1920s, by the 1940s the field, was in steep decline despite early promise. The causes were at least three-fold: insufficient understanding among researchers of basic phage biology; over exuberance, which led, along with ignorance, to carelessness; and the advent of antibiotics, an easier to handle as well as highly powerful category of antibacterials. The decline in phage therapy was neither uniform nor complete, especially in the former Soviet Republic of Georgia, where phage therapy traditions and practice continue to this day. In this review we strive toward three goals: 1. To provide an overview of the potential of phage therapy as a means of treating or preventing human diseases; 2. To explore the phage therapy state of the art as currently practiced by physicians in various pockets of phage therapy activity around the world, including in terms of potential commercialization; and 3. To avert a recapitulation of phage therapy's early decline by outlining good practices in phage therapy practice, experimentation, and, ultimately, commercialization.

Background Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. Results In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. Conclusion In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.

At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa "core lineage" and typically exhibited the exoS(+)/exoU(-) genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set.

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on successive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, phiKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee.

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or micro-arrays, while the introduction of closed-tube real-time polymerase chain reaction systems has resulted in the development of rapid microbial diagnostics with a reduced contamination risk. The use of these new molecular technologies is not restricted to detection and identification of microbial pathogens but also can be used for genotyping, allowing one to determine antibiotic resistance or to perform microbial fingerprinting.

Summary The biodiversity of the bacterium Pseudomonas aeruginosa in an aquatic environment (the Woluwe River, Brussels, Belgium) was analysed. Surface water was sampled bimonthly over a 1-year period (2000–2001) at seven sites evenly dispersed over the river. Total bacterial counts were performed and P. aeruginosa strains were isolated on a selective medium. A weighed out sample of 100 randomly chosen presumptive P. aeruginosa isolates was further analysed. A set of data consisting of the nucleotide sequence of the oprL gene, a DNA-based fingerprint (amplified fragment length polymorphism, AFLP), serotype, pyoverdine type and antibiogram (MICs of 21 clinically relevant antibiotics) was assembled. These data were integrated with those previously obtained for 73 P. aeruginosa clinical and environmental isolates collected across the world. The combined results were analysed and compared using biological data analysis software. Our findings indicate a positive relationship between the extent of pollution and the prevalence of P. aeruginosa. Surprisingly, the Woluwe River P. aeruginosa community was almost as diverse as the global P. aeruginosa population. Indeed, the Woluwe River harboured members of nearly all successful clonal complexes. With the exception of one multidrug-resistant (MDR) strain, belonging to a ubiquitous and clinically relevant serotype O11 clone, antibiotic resistance levels were relatively low. These findings illustrate the significance of river water as a reservoir and source of distribution of potentially pathogenic P. aeruginosa strains and could have repercussions on antinosocomial infection strategies.

The lungs of cystic fibrosis patients are frequently colonized by Pseudomonas aeruginosa, which produces high-affinity fluorescent peptidic siderophores, pyoverdines. Three pyoverdines which differ in their peptide chain and are easily differentiated by isoelectric focusing exist, only one being produced by a given strain. P. aeruginosa isolates from cystic fibrosis patients of a German hospital were analyzed by sequential, pulse-field gel electrophoresis (PFGE) and for pyoverdine production and type. Only producers of type I and type II pyoverdine were found. There was a perfect correlation between the type of pyoverdine produced and the clonality determined by PFGE. PFGE clone C, the most prevalent among cystic fibrosis patients, and found in an aquatic environment, produced type II pyoverdine. Pyoverdine-negative mutants seemed to increase as a function of the lung colonization time, but retained the capacity to take up pyoverdines. Most isolates that took up type II pyoverdine were also able to utilize type I pyoverdine as judged by growth stimulation experiments. No correlation was observed between the loss of pyoverdine production and mucoidy.

Vanadium is a metal that under physiological conditions can exist in two oxidation states, V(IV) (vanadyl ion) and V(V) (vanadate ion). Here, it was demonstrated that both ions can form complexes with siderophores. Pseudomonas aeruginosa produces two siderophores under iron-limiting conditions, pyoverdine (PVD) and pyochelin (PCH). Vanadyl sulfate, at a concentration of 1-2 mM, strongly inhibited growth of P. aeruginosa PAO1, especially under conditions of severe iron limitation imposed by the presence of non-utilizable Fe(III) chelators. PVD-deficient mutants were more sensitive to vanadium than the wild-type, but addition of PVD did not stimulate their growth. Conversely, PCH-negative mutants were more resistant to vanadium than the wild-type strain. Both siderophores could bind and form complexes with vanadium after incubation with vanadyl sulfate (1:1, in the case of PVD; 2:1, in the case of PCH). Although only one complex with PVD, V(IV)-PVD, was found, both V(IV)- and V(V)-PCH were detected. V-PCH, but not V-PVD, caused strong growth reduction, resulting in a prolonged lag phase. Exposure of PAO1 cells to vanadium induced resistance to the superoxide-generating compound paraquat, and conversely, exposure to paraquat increased resistance to V(IV). Superoxide dismutase (SOD) activity of cells grown in the presence of V(IV) was augmented by a factor of two. Mutants deficient in the production of Fe-SOD (SodB) were particularly sensitive to vanadium, whilst sodA mutants deficient for Mn-SOD were only marginally affected. In conclusion, it is suggested that V-PCH catalyses a Fenton-type reaction whereby the toxic superoxide anion O(2)- is generated, and that vanadium compromises PVD utilization.

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., Introduction: Early diagnosis of wound colonisation or prediction of wound sepsis provides an opportunity for therapeutic intervention. There is need for qualitative and quantitative tests that are more rapid than bacterial culture. Pseudomonas aeruginosa results in high morbidity and mortality rates, is inherently resistant to common antibiotics, and is increasingly being isolated as a nosocomial pathogen. We developed three PCR-based methods to detect and quantify P aeruginosa in wound biopsy samples: conventional PCR, enzyme-linked immunosorbent assay (ELISA)-PCR, and RTD-PCR with rapid thermal cycling (LightCycler™ technology), all based on the amplification of the outer membrane lipoprotein gene oprL. We compared the efficacy of these methods to bacterial culture by quantitatively measuring levels of P aeruginosa in serial dilutions, in reconstituted skin samples and 21 burn wound biopsy samples. Materials and methods: Serial 10-fold dilutions were made from an overnight P aeruginosa culture and plated out onto Luria-Bertani and cetrimide agar plates. The agar plates were incubated overnight at 37°C, and the colonies were counted in order to estimate the number of CFU per dilution tube. A sample was taken from each dilution tube as a template for the three PCR-based methods. Serial P aeruginosa dilutions (see above) were added to uninfected cadaveric skin. The reconstituted biopsy samples were homogenized using a tissue tearer and DNA was extracted using XTRAX DNA buffer. The DNA was resuspended in distilled water. A sample was taken as a template for the PCR-based methods. Twenty-one burn wound biopsy samples were taken from nine patients with suspected P aeruginosa burn wound infection. The biopsy samples were longitudinally divided into two pieces. From one piece, DNA was extracted (using XTRAX DNA buffer) and used as a template for PCR-based techniques (see above). The other piece was homogenized, in physiological water, using a tissue tearer. Serial 10-fold dilutions of the suspension were spread on Luria-Bertani and cetrimide agar plates. Colony counts were performed after overnight incubation at 37°C. The PCR mixture contained sterile distilled water, PCR buffer, deoxynucleotide mixture or digoxigenin labelling mix, MgCl2, diluted template, primers PAL1 and PAL2, and AmpliTaQ DNA polymerase. The amplification was performed in a GeneAmp® PCR System 2400. An aliquot of the reaction mixture was put on an agarose gel for electrophoresis and visualisation of the PCR product. An image of the gel was made using a digital camera. Image analysis software was used to calculate the band mass of the experimental bands. An aliquot of the digoxigenin labelling reaction was denatured and then hybridized with the biotinylated capture probe PrL. Some of the resultant solution was transferred to a well of a streptavidin-coated microtitre plate (MTP) and incubated for 3 h at 45°C. The solution was discarded. Peroxidase conjugated antidigoxigenin was added and the MTP was incubated for 30 min at 37°C. The solution was discarded and ABTS substrate was added. The MTP was incubated for 30 min at 37°C. Absorbance was read at 405 nm. The RTD-PCR mixture contained PCR grade sterile water, diluted template DNA, primers PAL1 and PAL2, 3' fluorescein (FL)-labelled probe oprL-FL, 5' LC Red 640-labelled and 3' phosphorylated probe oprL-LC, MgCl2, and LC DNA Master Hybridisation Probes, containing Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, and MgCl2. The amplification was performed in a LightCycler™. The fluorescence signal of LC Red 640 was measured during the annealing phase. The measured fluorescence data was processed with analysis software. Results and discussion: The three methods showed a good concordance with the culture results. Conventional PCR was at least 100 times less sensitive than bacterial culture and had a low dynamic range (2 logs). With a lower detection limit of 103 CFU/g tissue, ELISA-PCR was ten times more sensitive than conventional PCR. The dynamic range, however, did not increase. ELISA-PCR is very time consuming (8 h). The RTD-PCR produced a linear quantitative detection range of 7 logs with a lower detection limit of 103 CFU/g tissue. More important, however, was that the time from sample collection to result was less than 1 h. Two biopsy specimens scored significantly higher in ELISA-PCR and RTD-PCR than in bacterial culture. This could indicate that DNA from dead bacteria was amplified. One out of ten culture positive biopsy samples was found negative by all PCR-based methods. Topical antimicrobial agents possibly inhibited PCR. These results show that RTD-PCR has potential for the rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Further study is required to assess the reliability of this new technology, and its impact on patient outcome and hospital costs.

Since 1991, the skin bank of the Queen Astrid Military Hospital uses food-grade aluminum foil as a primary support for storing cryo preserved human donor skin (511 donors). The possible release of heavy metals into the cryo preservation media (30% (v/v) glycerol in physiological water) and the possible impact this release could have on the quality of the cryo preserved donor skin was evaluated. Aluminum was the principal detection target. Possible contaminants of the aluminum foil as such (arsenic, cadmium, chromium and lead) were also investigated. The evaluation was set up after a Belgian Competent Authority inspection remark. Aluminum was detected at a concentration of 1.4 mg/l, arsenic and lead were not detected, while cadmium and chromium were detected in trace element quantities. An histological analysis revealed no differences between cryo preserved and fresh donor skin. No adverse reactions in patients, related to the presence of aluminum or heavy metal traces, were reported since the introduction of the cryo preserved donor skin in our burn wound centre.

The detection of schistosomiasis cases among Belgian military personnel returning from a mission in the Democratic Republic of Congo (DRC) prompted a nested case-control study of all military personnel deployed in the DRC between 2005 and 2008 to identify all infections and to start appropriate treatment. Of 197 patients exposed at Lake Tanganyika in the Kalemie area of DRC, 49 (24.9%) were diagnosed with schistosomiasis. Swimming was significantly more frequent than wading in the seropositive group than in the seronegative group (88.9% vs. 73.6%; odds ratio [OR], 2.86; 95% confidence interval [CI], 0.97-9.01). Thirty-one of 49 patients (63.3%) were symptomatic; including skin problems in 34.7%, respiratory symptoms in 12.2%, fever in 14.3%, and 51.0% with gastrointestinal problems. Median eosinophil counts were significantly higher in seropositive patients (375 vs. 138 per tL; Wilcoxon rank sum test [Ws] = 10,559.00; p0.01; r = -0.49). In total, 20 (40.8%) of the 49 patients were treated for symptomatic infections and the remainder for asymptomatic schistosomiasis. Our study emphasizes the need for active systematic post-tropical screening in military personnel after deployment to Schistosoma-endemic regions of the world.