Combination of Whole Genome Sequencing and Metagenomics for Microbiological Diagnostics

Whole genome sequencing (WGS) provides the highest resolution for genome-based species identification and can provide insight into the antimicrobial resistance and virulence potential of a single microbiological isolate during the diagnostic process. In contrast, metagenomic sequencing allows the analysis of DNA segments from multiple microorganisms within a community, either using an amplicon- or shotgun-based approach. However, WGS and shotgun metagenomic data are rarely combined, although such an approach may generate additive or synergistic information, critical for, e.g., patient management, infection control, and pathogen surveillance. To produce a combined workflow with actionable outputs, we need to understand the pre-to-post analytical process of both technologies. This will require specific databases storing interlinked sequencing and metadata, and also involves customized bioinformatic analytical pipelines. This review article will provide an overview of the critical steps and potential clinical application of combining WGS and metagenomics together for microbiological diagnosis

Gut microbiota composition as a candidate risk factor for dimethyl fumarate-induced lymphopenia in multiple sclerosis

Mounting evidence points towards a pivotal role of gut microbiota in multiple sclerosis (MS) pathophysiology. Yet, whether disease-modifying treatments alter microbiota composition and whether microbiota shape treatment response and side-effects remain unclear. In this prospective observational pilot study, we assessed the effect of dimethyl fumarate (DMF) on gut microbiota and on host/microbial metabolomics in a cohort of 20 MS patients. Combining state-of-the-art microbial sequencing, metabolome mass spectrometry, and computational analysis, we identified longitudinal changes in gut microbiota composition under DMF-treatment and an increase in citric acid cycle metabolites. Notably, DMF-induced lymphopenia, a clinically relevant safety concern, was correlated with distinct baseline microbiome signatures in MS patients. We identified gastrointestinal microbiota as a key therapeutic target for metabolic properties of DMF. By characterizing gut microbial composition as a candidate risk factor for DMF-induced lymphopenia, we provide novel insights into the role of microbiota in mediating clinical side-effects

Diagnostic challenges within the Bacillus cereus-group: finding the beast without teeth

The Bacillus cereus-group (B. cereus sensu lato) includes common, usually avirulent species, often considered contaminants of patient samples in routine microbiological diagnostics, as well as the highly virulent B. anthracis. Here we describe 16 isolates from 15 patients, identified as B. cereus-group using a MALDI-TOF MS standard database. Whole genome sequencing (WGS) analysis identified five of the isolates as B. anthracis species not carrying the typical virulence plasmids pXO1 and pXO2, four isolates as B. paranthracis, three as B. cereus sensu stricto, two as B. thuringiensis, one as B. mobilis, and one isolate represents a previously undefined species of Bacillus (B. basilensis sp. nov.). More detailed analysis using alternative MALDI-TOF MS databases, biochemical phenotyping, and diagnostic PCRs, gave further conflicting species results. These cases highlight the difficulties in identifying avirulent B. anthracis within the B. cereus-group using standard methods. WGS and alternative MALDI-TOF MS databases offer more accurate species identification, but so far are not routinely applied. We discuss the diagnostic resolution and discrepancies of various identification methods

Sodium benzoate induced developmental defects, oxidative stress and anxiety-like behaviour in zebrafish larva

Sodium benzoate (SB) is a common food preservative. Its FDA described safety limit is 1000 ppm. Lately, increased use of SB has prompted investigations regarding its effects on biological systems. Data regarding toxicity of SB is divergent and controversial with studies reporting both harmful and beneficial effects. Therefore, we did a systematic dose dependent toxicity study of SB using zebrafish vertebrate animal model. We also investigated oxidative stress and anxiety-like behaviour in zebrafish larva treated with SB. Our results indicate that SB induced developmental (delayed hatching), morphological (pericardial edema, yolk sac edema and tail bending), biochemical (oxidative stress) and behavioural (anxiety-like behaviour) abnormalities in developing zebrafish larva. LC50 of SB induced toxicity was approximately 400 ppm after 48 h of SB exposure. Our study strongly supports its harmful effects on vertebrates at increasing doses. Thus, we suggest caution in the excessive use of this preservative in processed and convenience foods

ZebraPace: An Open-Source Method for Cardiac-Rhythm Estimation in Untethered Zebrafish Larvae

For the assessment of cardiac function, heartbeat represents one key parameter. Current methods of heartbeat measurements in the zebrafish larvae usually require larval immobilization, fluorescent transgenic strains and a confocal microscope, costly commercial software for analysis, or strong programming skills if the software is open-source. Here, we present a simple yet powerful method of heartbeat analysis using untethered, unlabeled zebrafish larva using ImageJ (open-source software), which does not require programming skills. We named it as ZebraPace for Zebrafish Precise Algorithm for Cardiac-rhythm Estimation. ZebraPace works directly with AVI videos and requires no image processing steps. ZebraPace uses pixel intensity change in a grayscale video to count the number of beats. We have validated the ZebraPace method by pharmacological alterations of the heartbeat in zebrafish larvae of 48 and 72 hpf stages. We have also determined beat-to-beat interval, which relates to rhythmicity of heartbeat. The results obtained by using ZebraPace corroborates well with the heartbeat values previously reported for similarly aged larvae as determined by using specialized software. We believe that the ZebraPace method is simple, cost-effective, and easy to grasp as it involves fewer steps. It not only reduces the manual workload but also eliminates sample preparation time and researcher subjectivity