Systematic Evaluation of HILIC Stationary Phases for Global Metabolomics of Human Plasma.

Polar hydrophilic metabolites have been identified as important actors in many biochemical pathways. Despite continuous improvement and refinement of hydrophilic interaction liquid chromatography (HILIC) platforms, its application in global polar metabolomics has been underutilized. In this study, we aimed to systematically evaluate polar stationary phases for untargeted metabolomics by using HILIC columns (neutral and zwitterionic) that have been exploited widely in targeted approaches. To do so, high-resolution mass spectrometry was applied to thoroughly investigate selectivity, repeatability and matrix effect at three pH conditions for 9 classes of polar compounds using 54 authentic standards and plasma matrix. The column performance for utilization in untargeted metabolomics was assessed using plasma samples with diverse phenotypes. Our results indicate that the ZIC-c HILIC column operated at neutral pH exhibited several advantages, including superior performance for different classes of compounds, better isomer separation, repeatability and high metabolic coverage. Regardless of the column type, the retention of inorganic ions in plasma leads to extensive adduct formation and co-elution with analytes, which results in ion-suppression as part of the overall plasma matrix effect. In ZIC-c HILIC, the sodium chloride ion effect was particularly observed for amino acids and amine classes. Successful performance of HILIC for separation of plasma samples with different phenotypes highlights this mode of separation as a valuable approach in global profiling of plasma sample and discovering the metabolic changes associated with health and disease.

Towards Standards for Human Fecal Sample Preparation in Targeted and Untargeted LC-HRMS Studies.

Gut microbiota and their metabolic products are increasingly being recognized as important modulators of human health. The fecal metabolome provides a functional readout of the interactions between human metabolism and the gut microbiota in health and disease. Due to the high complexity of the fecal matrix, sample preparation often introduces technical variation, which must be minimized to accurately detect and quantify gut bacterial metabolites. Here, we tested six different representative extraction methods (single-phase and liquid-liquid extractions) and compared differences due to fecal amount, extraction solvent type and solvent pH. Our results indicate that a minimum fecal (wet) amount of 0.50 g is needed to accurately represent the complex texture of feces. The MTBE method (MTBE/methanol/water, 3.6/2.8/3.5, v/v/v) outperformed the other extraction methods, reflected by the highest extraction efficiency for 11 different classes of compounds, the highest number of extracted features (97% of the total identified features in different extracts), repeatability (CV < 35%) and extraction recovery (≥70%). Importantly, optimization of the solvent volume of each step to the initial dried fecal material (µL/mg feces) offers a major step towards standardization, which enables confident assessment of the contributions of gut bacterial metabolites to human health.

High diversity in SCCmec elements among multidrug-resistant Staphylococcus haemolyticus strains originating from paediatric patients; characterization of a new composite island.

PURPOSE: Staphylococcus haemolyticus has emerged as a highly antimicrobial-resistant healthcare-associated pathogen, in particular for patients admitted to neonatal intensive care. The objective of this study was to study the nature of SCCmec types among MDR-SH strains isolated from paediatric patients. METHODOLOGY: S. haemolyticus strains (n=60) were isolated from paediatric patients. Antibiotic resistance patterns were established using the disk agar diffusion and micro-broth dilution methods. SCCmec typing was performed using whole-genome sequencing (WGS) and an additional PCR analysis. RESULTS: All S. haemolyticus isolates demonstrated multidrug resistance. Using WGS, various novel mec types and combinations of SCCmec types were found, including a new composite island [SCCmec type V (Vd)+SCC cad/ars/cop] comprising 30 % of the strains. SCCmec type V was identified in 23 % of the isolates. A combination of the mecA gene enclosed by two copies of IS431 and absence of the mecRI and ccr genes was identified in 11 strains. In total, mecA regulatory genes were absent in all SH isolates used in this study. CONCLUSION: A high diversity of SCCmec elements with the prevalence of a new composite island was determined among MRSH strains. The structure of the composite island represented by MDR-SH strains in this study, in combination with the presence of a restriction-modification system type III, is described for the first time in this study. The presence of an 8 bp direct repeat (DR) and the sequences flanking the DR may support the integration of the mecA gene complex as a composite transposon (IS431-mecA-IS431) independently from recombinase genes.

High-Quality Genome Sequence of the Highly Resistant Bacterium Staphylococcus haemolyticus, Isolated from a Neonatal Bloodstream Infection.

Using Illumina HiSeq and PacBio technologies, we sequenced the genome of the multidrug-resistant bacterium Staphylococcus haemolyticus, originating from a bloodstream infection in a neonate. The sequence data can be used as an accurate reference sequence.