Whole-genome analysis of Haemophilus influenzae strains isolated from persons with cystic fibrosis.

Introduction. Haemophilus influenzae is a commensal of the respiratory tract that is frequently present in cystic fibrosis (CF) patients and may cause infection. Antibiotic resistance is well described for CF strains, and virulence factors have been proposed.Hypothesis/Gap. The genetic diversity of H. influenzae strains present in the lungs of persons with CF is largely unknown despite the fact that this organism is considered to be a pathogen in this condition. The aim was to establish the genetic diversity and susceptibility of H. influenzae strains from persons with CF, and to screen the whole genomes of these strains for the presence of antibiotic resistance determinants and proposed virulence factors.Methods. A total of 67 strains, recovered from respiratory samples from persons with CF from the UK (n=1), Poland (n=2), Spain (n=24) and the Netherlands (n=40), were subjected to whole-genome sequencing using Illumina technology and tested for antibiotic susceptibility. Forty-nine of these strains (one per different sequence type) were analysed for encoded virulence factors and resistance determinants.Results. The 67 strains represented 49 different sequence types. Susceptibility testing showed that all strains were susceptible to aztreonam, ciprofloxacin, imipenem and tetracycline. Susceptibility to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, cefuroxime, cefixime, ceftriaxone, cefepime, meropenem, clarithromycin, co-trimoxazole and levofloxacin ranged from 70.2-98.5%. Only 6/49 strains (12.2%) harboured acquired resistance genes. Mutations associated with a ß-lactamase-negative ampicillin-resistant phenotype were present in four strains (8.2 %). The potential virulence factors, urease, haemoglobin- and haptoglobin-binding protein/carbamate kinase, and OmpP5 (OmpA), were encoded in more than half of the strains. The genes for HMW1, HMW2, H. influenzae adhesin, a IgA-specific serine endopeptidase autotransporter precursor, a TonB-dependent siderophore, an ABC-transporter ATP-binding protein, a methyltransferase, a BolA-family transcriptional regulator, glycosyltransferase Lic2B, a helix-turn-helix protein, an aspartate semialdehyde dehydrogenase and another glycosyltransferase were present in less than half of the strains.Conclusion. The H. influenzae strains showed limited levels of resistance, with the highest being against co-trimoxazole. Sequences encoding a carbamate kinase and a haemoglobin- and haemoglobin-haptoglobin-binding-like protein, a glycosyl transferase and an urease may aid the colonization of the CF lung. The adhesins and other identified putative virulence factors did not seem to be necessary for colonization.

Taxonomic position, antibiotic resistance and virulence factors of clinical Achromobacter isolates.

The role of Achromobacter species in lung disease remains unclear. The aim of this study was to characterize Achromobacter isolated from persons with cystic fibrosis and from other clinical samples. Whole genome sequences from 101 Achromobacter isolates were determined (81 from patients with cystic fibrosis and 20 from other patients) and analysed. Taxonomic analysis showed nine species including two putative novel species. Thirty-five novel sequence types were present. The most active agent was co-trimoxazole followed by imipenem, but Minimal Inhibitory Concentrations (MICs) were high. Acquired antibiotic resistance genes were rare. Their presence did not correlate with minimal inhibitory concentrations suggesting that other mechanisms are involved. Genes for proposed virulence factors were present in only some isolates. Two putative novel species were identified. The putative virulence properties of Achromobacter involved in infections are variable. Despite the high MICs, acquired resistance genes are uncommon.

Taxonomic position, antibiotic resistance and virulence factor production by Stenotrophomonas isolates from patients with cystic fibrosis and other chronic respiratory infections.

BACKGROUND: The potential pathogenic role of Stenotrophomonas maltophilia in lung disease and in particular in cystic fibrosis is unclear. To develop further understanding of the biology of this taxa, the taxonomic position, antibiotic resistance and virulence factors of S. maltophilia isolates from patients with chronic lung disease were studied. RESULTS: A total of 111 isolates recovered between 2003 and 2016 from respiratory samples from patients in five different countries were included. Based on a cut-off of 95%, analysis of average nucleotide identity by BLAST (ANIb) showed that the 111 isolates identified as S. maltophilia by Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) belonged to S. maltophilia (n = 65), S. pavanii (n = 6) and 13 putative novel species (n = 40), which each included 1-5 isolates; these groupings coincided with the results of the 16S rDNA analysis, and the L1 and L2 ß-lactamase Neighbor-Joining phylogeny. Chromosomally encoded aminoglycoside resistance was identified in all S. maltophilia and S. pavani isolates, while acquired antibiotic resistance genes were present in only a few isolates. Nevertheless, phenotypic resistance levels against commonly used antibiotics, determined by standard broth microbroth dilution, were high. Although putative virulence genes were present in all isolates, the percentage of positive isolates varied. The Xps II secretion system responsible for the secretion of the StmPr1-3 proteases was mainly limited to isolates identified as S. maltophilia based on ANIb, but no correlation with phenotypic expression of protease activity was found. The RPF two-component quorum sensing system involved in virulence and antibiotic resistance expression has two main variants with one variant lacking 190 amino acids in the sensing region. CONCLUSIONS: The putative novel Stenotrophomonas species recovered from patient samples and identified by MALDI-TOF/MS as S. maltophilia, differed from S. maltophilia in resistance and virulence genes, and therefore possibly in pathogenicity. Revision of the Stenotrophomonas taxonomy is needed in order to reliably identify strains within the genus and elucidate the role of the different species in disease.

Draft genome sequence of the strain 16-537536, isolated from a patient with bronchiectasis and its relationship to the Pseudomonas koreensis group of the Pseudomonas fluorescens complex.

OBJECTIVE: The Pseudomonas koreensis group bacteria are usually found in soil and are associated with plants. Currently they are poorly described. Here we report on the whole genome sequence of a bacterial isolate from a patient with bronchiectasis that was first identified as P. koreensis, and on its position in the P. koreensis group. RESULTS: Strain 16-537536 was isolated from a patient with bronchiectasis from Spain and initially identified by MALDI-TOF as P. koreensis, a member of the Pseudomonas fluorescens complex. However, the average nucleotide identity analysis (ANIb) and whole genome alignments of the draft genome sequence of this strain showed it to be a member of the P. koreensis group of the P. fluorescens complex, but belonging to an undescribed species. In addition, based on ANIb analysis, the P. koreensis group contains several other unnamed species. Several genes for putative virulence factors were identified. The only antibiotic resistance gene present in strain 16-537536 was a class C ß-lactamase. The correct identification of bacterial species from patients is of utmost importance in order to understand their pathogenesis and to track the potential spread of pathogens between patients. Whole genome sequence data should be included for the description of new species.

Whole-genome analysis of Pandoraea species strains from cystic fibrosis patients.

Aim: Genetic characterization of Pandoraea strains recovered from cystic fibrosis patients. Materials & methods: The whole-genome sequence of 12 Pandoraea strains was determined using Illumina technology. The position of the strains within the genus Pandoraea was analyzed using selected partial gene sequences, core genome multi-locus sequence typing and average nucleotide identity analysis. Furthermore, the sequences were annotated. Results: The results show that some strains previously identified as Pandoraea pnomenusa, Pandoraea sputorum, Pandoraea oxalativorans and Pandoraea pulmonicola belong to novel species. The strains did not harbor acquired antibiotic resistance genes but encoded an OXA-type ß-lactamase. Conclusion: The taxonomy of the genus Pandoraea needs to be revised.

Therapy and Outcome of Staphylococcus aureus Infections of Intracorporeal Ventricular Assist Devices.

Infection of the driveline or pump pocket is a common complication in patients with ventricular assist devices (VADs) and Staphylococcus aureus is the main pathogen causing such infections. Limited evidence is currently available to guide the choice of antibiotic therapy and the duration of treatment in these patients. Patients at the University Medical Center Utrecht who developed a VAD-related S. aureus infection between 2007 and 2016 were retrospectively assessed. Blood culture isolates were typed by whole genome sequencing to differentiate between relapses and reinfections, and to determine whether antibiotic therapy had led to acquisition of resistance mutations. Twenty-eight patients had S. aureus VAD infections. Ten of these patients also suffered S. aureus bacteremia. Discontinuation of antibiotic therapy was followed by relapse in 50% of the patients without prior S. aureus bacteremia and in 80% of patients with bacteremia. Oral cephalexin could ultimately suppress the infection for the duration of follow-up in 8/8 patients without S. aureus bacteremia and in 3/6 patients with S. aureus bacteremia. Clindamycin failed as suppressive therapy in 4/4 patients. Cephalexin appears an adequate choice for antibiotic suppression of VAD infections with methicillin-susceptible S. aureus. In patients without systemic symptoms, it may be justified to attempt to stop therapy after treatment of the acute infection, but antibiotic suppression until heart transplant seems indicated in patients with S. aureus bacteremia.

Th1-directing adjuvants increase the immunogenicity of oligosaccharide-protein conjugate vaccines related to Streptococcus pneumoniae type 3.

Oligosaccharide (OS)-protein conjugates are promising candidate vaccines against encapsulated bacteria, such as Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae. Although the effects of several variables such as OS chain length and protein carrier have been studied, little is known about the influence of adjuvants on the immunogenicity of OS-protein conjugates. In this study, a minimal protective trisaccharide epitope of Streptococcus pneumoniae type 3 conjugated to the cross-reacting material of diphtheria toxin was used for immunization of BALB/c mice in the presence of different adjuvants. Subsequently, half of the mice received a booster immunization with conjugate alone. Independent of the use and type of adjuvant, all mice produced long-lasting anti-polysaccharide type 3 (PS3) antibody levels, which provided full protection against challenge with pneumococcal type 3 bacteria. All adjuvants tested increased the anti-PS3 antibody levels and opsonic capacities as measured by an enzyme-linked immunosorbent assay and an in vitro phagocytosis assay. The use of QuilA or a combination of the adjuvants CpG and dimethyl dioctadecyl ammonium bromide resulted in the highest phagocytic capacities and the highest levels of Th1-related immunoglobulin G (IgG) subclasses. Phagocytic capacity correlated strongly with Th1-associated IgG2a and IgG2b levels, to a lesser extent with Th2-associated IgG1 levels, and weakly with thiocyanate elution as a measure of avidity. Thus, the improved immunogenicity of OS-protein conjugates was most pronounced for Th1-directing adjuvants.