Search

The technical, biological, and inter-center reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) typing data has not yet been explored. The aim of this study is to compare typing data from multiple centers employing bioinformatics using bacterial strains from two past outbreaks and non-related strains.Participants received twelve extended spectrum betalactamase-producing E. coli isolates and followed the same standard operating procedure (SOP) including a full-protein extraction protocol. All laboratories provided visually read spectra via flexAnalysis (Bruker, Germany). Raw data from each laboratory allowed calculating the technical and biological reproducibility between centers using BioNumerics (Applied Maths NV, Belgium).Technical and biological reproducibility ranged between 96.8-99.4% and 47.6-94.4%, respectively. The inter-center reproducibility showed a comparable clustering among identical isolates. Principal component analysis indicated a higher tendency to cluster within the same center. Therefore, we used a discriminant analysis, which completely separated the clusters. Next, we defined a reference center and performed a statistical analysis to identify specific peaks to identify the outbreak clusters. Finally, we used a classifier algorithm and a linear support vector machine on the determined peaks as classifier. A validation showed that within the set of the reference center, the identification of the cluster was 100% correct with a large contrast between the score with the correct cluster and the next best scoring cluster.Based on the sufficient technical and biological reproducibility of MALDI-TOF MS based spectra, detection of specific clusters is possible from spectra obtained from different centers. However, we believe that a shared SOP and a bioinformatics approach are required to make the analysis robust and reliable.

The global widespread use of antimicrobials and accompanying increase in resistant bacterial strains is of major public health concern. Wastewater systems and wastewater treatment plants are considered a niche for antibiotic resistance genes (ARGs), with diverse microbial communities facilitating ARG transfer via mobile genetic element (MGE). In contrast to hospital sewage, wastewater from other health care facilities is still poorly investigated. At the instance of a nursing home located in south-west Germany, in the present study, shotgun metagenomics was used to investigate the impact on wastewater of samples collected up- and down-stream in different seasons. Microbial composition, ARGs and MGEs were analyzed using different annotation approaches with various databases, including Antibiotic Resistance Ontologies (ARO), integrons and plasmids. Our analysis identified seasonal differences in microbial communities and abundance of ARG and MGE between samples from different seasons. However, no obvious differences were detected between up- and downstream samples. The results suggest that, in contrast to hospitals, sewage from the nursing home does not have a major impact on ARG or MGE in wastewater, presumably due to much less intense antimicrobial usage. Possible limitations of metagenomic studies using high-throughput sequencing for detection of genes that seemingly confer antibiotic resistance are discussed.

The clinical course of Staphylococcus aureus bacteremia varies extensively. We sought to determine the relationship between genetic characteristics of the infecting pathogen and clinical outcomes in an exploratory study. In two study centers, 317 blood culture isolates were analyzed by DNA microarray and spa genotyping. By uni- and multivariate regression analyses associations of genotype data with 30-day all-cause mortality, severe sepsis/septic shock, disseminated disease, endocarditis, and osteoarticular infection were investigated. Univariate analysis showed significant association between S. aureus genes/gene-clusters or clonal complexes and clinical endpoints. For example CC15 was associated with 30-day mortality and CC22 with osteoarticular infection. In multivariate analysis methicillin resistance (mecA, OR 4.8 [1.43-16.06]) and the beta-lactamase-gene (bla, OR 3.12 [1.17-8.30]) remained independently associated with 30-day mortality. The presence of genes for enterotoxins (sed/sej/ser) was associated with endocarditis (OR 5.11 [1.14-18.62]). Host factors such as McCabe classification (OR 4.52 [2.09-9.79] for mortality), age (OR 1.06 [1.03-1.10] per year), and community-acquisition (OR 3.40 [1.31-8.81]) had a major influence on disease severity, dissemination and mortality. Individual genotypes and clonal complexes of S. aureus can only partially explain clinical features and outcomes of S. aureus bacteremia. Genotype-phenotype association studies need to include adjustments for host factors like age, comorbidity and community-acquisition.

Residual microorganisms and/or re-infections are a major cause for root canal therapy failure. Understanding of the bacterial content could improve treatment protocols. Fifty samples from 25 symptomatic and 25 asymptomatic previously root-filled teeth were collected from Sudanese patients with periradicular lesions. Amplified 16S rRNA gene (V1-V2) variable regions were subjected to pyrosequencing (FLX 454) to determine the bacterial profile. Obtained quality-controlled sequences from forty samples were classified into 741 operational taxonomic units (OTUs) at 3% dissimilarity, 525 at 5% dissimilarity and 297 at 10% dissimilarity, approximately corresponding to species-, genus- and class levels. The most abundant phyla were: Firmicutes (29.9%), Proteobacteria (26.1%), Actinobacteria (22.72%), Bacteroidetes (13.31%) and Fusobacteria (4.55%). Symptomatic patients had more Firmicutes and Fusobacteria than asymptomatic patients, while asymptomatic patients showed more Proteobacteria and Actinobacteria. Interaction of disease status and age was observed by two-way ANOSIM. Canonical correspondence analysis for age, tooth restoration and disease status showed a correlation of disease status with the composition and prevalence of different members of the microbial community. The pyrosequencing analysis revealed a distinctly higher diversity of the microbiota compared to earlier reports. The comparison of symptomatic and asymptomatic patients showed a clear association of the composition of the bacterial community with the presence and absence of symptoms in conjunction with the patients' age.

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements.

Gaseous and semi-volatile organic compounds emitted by the transport sector contribute to air pollution and have adverse effects on human health. To reduce harmful effects to the environment as well as to humans, renewable and sustainable bio-hybrid fuels are explored and investigated in the cluster of excellence "The Fuel Science Center" at RWTH Aachen University. However, data on the effects of bio-hybrid fuels on human health is scarce, leaving a data gap regarding their hazard potential. To help close this data gap, this study investigates potential toxic effects of a Ketone-Ester-Alcohol-Alkane (KEAA) fuel blend on A549 human lung cells. Experiments were performed using a commercially available air-liquid interface exposure system which was optimized beforehand. Then, cells were exposed at the air-liquid interface to 50–2000 ppm C3.7 of gaseous KEAA for 1 h. After a 24 h recovery period in the incubator, cells treated with 500 ppm C3.7 KEAA showed significant lower metabolic activity and cells treated with 50, 250, 500 and 1000 ppm C3.7 KEAA showed significant higher cytotoxicity compared to controls. Our data support the international occupational exposure limits of the single KEAA constituents. This finding applies only to the exposure scenario tested in this study and is difficult to extrapolate to the complex in vivo situation. [ABSTRACT FROM AUTHOR]