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

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

Evaluation of the Bruker® MBT Sepsityper IVD module for the identification of polymicrobial blood cultures with MALDI-TOF MS.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) considerably reduces timeframe required from initial blood culture positivity towards complete bacterial identification. However, rapid identification of polymicrobial blood cultures remains challenging. We evaluated the performances of the Bruker® MBT Sepsityper IVD module on MALDI-TOF MS for the direct identification of polymicrobial blood culture bottles. This module has the ability to give a strong indication that a sample contains a mixture of organisms and to identify two of them. Blood culture bottles considered as polymicrobial using routine subculture were collected and processed using the Sepsityper kit. MALDI-TOF MS identification was performed using the MBT Compass IVD software including the Sepsityper module. From 143 polymicrobial blood culture bottles tested, 34.3% (49/143) were completely identified by the module. Both microorganisms were more easily detected by the module in samples containing two pathogens than in samples containing two contaminants (36.8% vs 29.4%). Additionally, in more than half of the samples, the module detected 1 of the different microorganisms contained in the same vial. In these cases, with a pathogen and contaminant in the same sample, the module detected the pathogen in more than 80%. The Sepsityper module identified 14 microorganisms which were not recovered by conventional culture methods. The Bruker® MBT Sepsityper IVD module contributed to a valuable identification of polymicrobial blood cultures in more than a third of all cases. Conventional culture methods are still required to complete the results and to carry on susceptibility testing.

Molecular typing and antifungal susceptibility of Exophiala isolates from patients with cystic fibrosis.

The black yeast Exophiala dermatitidis is a frequent agent of colonization of the lungs of patients with cystic fibrosis (CF). A total of 71 clinical isolates of Exophiala from 13 patients were identified at the species level by sequencing the internal transcribed spacer (ITS) regions 1 and 2 of the rDNA genes and typed by random amplification of polymorphic DNA (RAPD), using two different primers, BG-2 and ERIC-1. In vitro susceptibility of these isolates to some systemic antifungal drugs was investigated using the CLSI method. Almost all the isolates were identified as E. dermatitidis, but long-term colonization with the closely related species E. phaeomuriformis was observed in one patient. No clustering was found according to the geographical origin of the isolates, the isolation date or the antifungal susceptibility. Variations were seen in the susceptibility of studied isolates to antifungals but most of them exhibited low susceptibility to amphotericin B and although some patients were successively colonized by two distinct genotypes, most of the isolates were distributed in patient-specific clusters. This phenomenon may be due to genomic variations of E. dermatitidis in the lung environment of CF patients. These results are typical of colonization of the airways of patients by a poorly distributed environmental fungus, which occupies particular reservoirs that need to be defined.

Exophiala (Wangiella) dermatitidis and cystic fibrosis - Prevalence and risk factors.

The objective of this prospective study was to assess the prevalence of Exophiala dermatitidis in respiratory secretions of patients with cystic fibrosis (CF) and to identify risk factors for its presence. The results of all cultures performed over a 2-year period in non lung-transplant patients in our CF clinic were included in the study. Samples consisted of sputum (whenever possible) or deep pharyngeal aspirate after a session of physiotherapy. Specimens were inoculated onto Sabouraud gentamicin-chloramphenicol agar (SGCA) medium (Becton-Dickinson) and incubated at 35°C for 2 days and then at ambient temperature (15-25°C) for 3 weeks. The whole study group included 154 patients (mean age ± SD: 18.5 y ± 11.69). E. dermatitidis was isolated from 58 specimens (2.8%) of nine patients (5.8%) out of total of 2065 cultures prepared during the study period. All E. dermatitidis culture-positive patients were pancreatic insufficient and ≥12 y of age. Almost all (8/9) were homozygous for the F508 del mutation. Aspergillus fumigatus colonization and genotype seemed to be predisposing factors. No other significant characteristic was identified in this group, either in terms of predominant bacterial pathogen or treatment. A distinct comparative study performed over 3 months in our laboratory revealed that the use of SGCA yielded identical isolation rates of E. dermatitidis as erythritol-chloramphenicol agar (ECA).

Continuous intravenous infusion of a low-molecular-weight heparin during allogenic haematopoietic stem-cell transplantation.

Low-molecular-weight heparins are routinely administered once or twice daily by subcutaneous injection. With the exception of patients on haemodialysis or presenting with unstable angina or flat Q-wave myocardial infarction, in which short-term intravascular administration is recommended, little information is available regarding the efficacy of continuous intravenous administration of low-molecular-weight heparins. We report the case of a 50-year-old patient who underwent an allogenic haematopoietic stem-cell transplantation for acute myeloid leukaemia. Prior to transplantation, the patient was on long-term oral anticoagulant (acenocoumarol) following the placement of a mechanical aortic valve. Acenocoumarol was stopped and low-molecular-weight heparin (nadroparin calcium) was administered intravenously through a continuous infusion pump (30 000 anti-Xa U/day) starting from day 0 until day 23 after transplantation. The patient was prophylactically transfused with platelets when the daily platelet count fell below 50 x 10 l. Repeated blood measurements showed that a therapeutic level of anti-Xa activity was achieved and maintained at a fairly constant level. No haemorrhagic or thrombotic complications occurred. This observation suggests that intravenous continuous infusion of low-molecular-weight heparin may be an alternative to subcutaneous injections in selected patients who need anticoagulation.