Evaluation of WGS performance for bacterial pathogen characterization with the Illumina technology optimized for time-critical situations

Whole genome sequencing (WGS) has become the reference standard for bacterial outbreak investigation and pathogen typing, providing a resolution unattainable with conventional molecular methods. Data generated with Illumina sequencers can however only be analysed after the sequencing run has finished, thereby losing valuable time during emergency situations. We evaluated both the effect of decreasing overall run time, and also a protocol to transfer and convert intermediary files generated by Illumina sequencers enabling real-time data analysis for multiple samples part of the same ongoing sequencing run, as soon as the forward reads have been sequenced. To facilitate implementation for laboratories operating under strict quality systems, extensive validation of several bioinformatics assays (16S rRNA species confirmation, gene detection against virulence factor and antimicrobial resistance databases, SNP-based antimicrobial resistance detection, serotype determination, and core genome multilocus sequence typing) for three bacterial pathogens (Mycobacterium tuberculosis, Neisseria meningitidis, and Shiga-toxin producing Escherichia coli) was performed by evaluating performance in function of the two most critical sequencing parameters, i.e. read length and coverage. For the majority of evaluated bioinformatics assays, actionable results could be obtained between 14 and 22 h of sequencing, decreasing the overall sequencing-to- results time by more than half. This study aids in reducing the turn-around time of WGS analysis by facilitating a faster response in time-critical scenarios and provides recommendations for time-optimized WGS with respect to required read length and coverage to achieve a minimum level of performance for the considered bioinformatics assay(s), which can also be used to maximize the cost-effectiveness of routine surveillance sequencing when response time is not essential.The Belgian Federal Public Service of Health, Food Chain Safety and Environment and Sciensano RP-PJ - Belgium.https://www.microbiologyresearch.org/content/journal/mgenam2022Genetic

Whole-genome sequencing-based antimicrobial resistance characterization and phylogenomic investigation of 19 multidrug-resistant and extended-spectrum beta-lactamase-positive Escherichia coli strains collected from hospital patients in Benin in 2019

The increasing worldwide prevalence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli constitutes a serious threat to global public health. Surgical site infections are associated with high morbidity and mortality rates in developing countries, fueled by the limited availability of effective antibiotics. We used whole-genome sequencing (WGS) to evaluate antimicrobial resistance and the phylogenomic relationships of 19 ESBL-positive E. coli isolates collected from surgical site infections in patients across public hospitals in Benin in 2019. Isolates were identified by MALDI-TOF mass spectrometry and phenotypically tested for susceptibility to 16 antibiotics. Core-genome multi-locus sequence typing and single-nucleotide polymorphism-based phylogenomic methods were used to investigate the relatedness between samples. The broader phylogenetic context was characterized through the inclusion of publicly available genome data. Among the 19 isolates, 13 different sequence types (STs) were observed, including ST131 (n = 2), ST38 (n = 2), ST410 (n = 2), ST405 (n = 2), ST617 (n = 2), and ST1193 (n = 2). The blaCTX-M-15 gene encoding ESBL resistance was found in 15 isolates (78.9%), as well as other genes associated with ESBL, such as blaOXA-1 (n = 14) and blaTEM-1 (n = 9). Additionally, we frequently observed genes encoding resistance against aminoglycosides [aac-(6')-Ib-cr, n = 14], quinolones (qnrS1, n = 4), tetracyclines [tet(B), n = 14], sulfonamides (sul2, n = 14), and trimethoprim (dfrA17, n = 13). Nonsynonymous chromosomal mutations in the housekeeping genes parC and gyrA associated with resistance to fluoroquinolones were also detected in multiple isolates. Although the phylogenomic investigation did not reveal evidence of hospital-acquired transmissions, we observed two very similar strains collected from patients in different hospitals. By characterizing a set of multidrug-resistant isolates collected from a largely unexplored environment, this study highlights the added value for WGS as an effective early warning system for emerging pathogens and antimicrobial resistance.The ARES (Académie de la Recherche pour l’Enseignement Supérieur), Belgium.http://www.frontiersin.org/Microbiologyam2022Genetic

Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin‑producing Escherichia coli isolates

Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized and harmonized workflows, e.g. for DNA extraction, are required to ensure robust and exchangeable WGS data. Data sharing between (inter)national laboratories is essential to support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial DNA preparation kits for their potential influence on: (i) DNA quality for Nextera XT library preparation; (ii) MiSeq sequencing (data quality, read mapping against plasmid and chromosome references); and (iii) WGS data analysis, i.e. isolate characterization (serotyping, virulence and antimicrobial resistance genotyping) and phylogenetic relatedness (core genome multilocus sequence typing and single nucleotide polymorphism analysis). Shiga toxin-producing Escherichia coli (STEC) was selected as a case study. Overall, data quality and inferred phylogenetic relationships between isolates were not affected by the DNA extraction kit choice, irrespective of the presence of confounding factors such as EDTA in DNA solution buffers. Nevertheless, completeness of STEC characterization was, although not substantially, influenced by the plasmid extraction performance of the kits, especially when using Nextera XT library preparation. This study contributes to addressing the WGS challenges of standardizing protocols to support data portability and to enable full exploitation of its potential.The data generated and analyzed during the current study are available in the NCBI SRA repository (https:// www.ncbi.nlm.nih.gov/sra) under accession number PRJNA574887 (in-house sequenced data) and its accession numbers are listed in Supplementary Table S11 online.Sciensano, Belgium and the Belgian Federal Public Service of Health, Food Chain Safety and Environment.http://www.nature.com/srepam2021Genetic

Phylogenomic investigation of increasing fluoroquinolone resistance among Belgian cases of Shigellosis between 2013 and 2018 indicates both travel-related imports and domestic circulation

Shigellosis is an acute enteric infection caused mainly by the species Shigella flexneri and Shigella sonnei. Since surveillance of these pathogens indicated an increase in ciprofloxacin-resistant samples collected in Belgium between 2013 and 2018, a subset of 148 samples was analyzed with whole genome sequencing (WGS) to investigate their dispersion and underlying genomic features associated with ciprofloxacin resistance. A comparison between observed phenotypes and WGS-based resistance prediction to ciprofloxacin revealed perfect correspondence for all samples. Core genome multi-locus sequence typing and single nucleotide polymorphism-typing were used for phylogenomic investigation to characterize the spread of these infections within Belgium, supplemented with data from international reference collections to place the Belgian isolates within their global context. For S. flexneri, substantial diversity was observed with ciprofloxacin-resistant isolates assigned to several phylogenetic groups. Besides travel-related imports, several clusters of highly similar Belgian isolates could not be linked directly to international travel suggesting the presence of domestically circulating strains. For S. sonnei, Belgian isolates were all limited to lineage III, and could often be traced back to travel to countries in Asia and Africa, sometimes followed by domestic circulation. For both species, several clusters of isolates obtained exclusively from male patients were observed. Additionally, we illustrated the limitations of conventional serotyping of S. flexneri, which was impacted by serotype switching. This study contributes to a better understanding of the spread of shigellosis within Belgium and internationally, and highlights the added value of WGS for the surveillance of this pathogen.SUPPLEMENTARY MATERIAL : FIGURE S1: Occurrence of gyrA p83, gyrA p87 and parC p80 mutations associated with ciprofloxacin resistance in the background collection. FIGURE S2: Network representation of minimum spanning trees for S. sonnei (A) and S. flexneri (B), TABLE S1: Accession number and metadata for the Belgian samples, TABLE S2: Read trimming statistics, TABLE S3: Assembly and coverage statistics, TABLE S4: Percentage of cgMLST alleles identified, TABLE S5: In silico determined serotypes for all S. flexneri samples, TABLE S6: Detected AMR genes for all samples, TABLE S7: Detected AMR point mutations for all samples, TABLE S8: Detected qnr genes, TABLE S9: Classification of contigs carrying qnr genes, TABLE S10: Lineage and PG classification, and ciprofloxacin resistance for the Belgian samples and background collection.The NRC is partially supported by the Belgian Ministry of Social Affairs through a fund within the Health Insurance System.https://www.mdpi.com/journal/microorganismsam2022Genetic

A shotgun metagenomics approach to detect and characterize unauthorized genetically modified microorganisms in microbial fermentation products

The presence of a genetically modified microorganism (GMM) or its DNA, often harboring antimicrobial resistance (AMR) genes, in microbial fermentation products on the market is prohibited by European regulations. GMMs are currently screened for through qPCR assays targeting AMR genes and vectors, and then confirmed by targeting known specific GM constructs/events. However, when the GMM was not previously characterized and an isolate cannot be obtained, its presence cannot be proven. We present a metagenomics approach capable of delivering the proof of presence of a GMM in a microbial fermentation product, with characterization based on the detection of AMR genes and vectors, species and unnatural associations in the GMM genome. In our proof-of-concept study, this approach was performed on a case with a previously isolated and sequenced GMM, an unresolved case for which no isolate was obtained, and a non-GMM-contaminated sample, all representative for the possible scenarios to occur in routine setting. Both short and long read sequencing were used. This workflow paves the way for a strategy to detect and characterize unknown GMMs by enforcement laboratories

Application of a strain-level shotgun metagenomics approach on food samples : resolution of the source of a Salmonella food-borne outbreak

Food-borne outbreak investigation currently relies on the time-consuming and challenging bacterial isolation from food, to be able to link food-derived strains to more easily obtained isolates from infected people. When no food isolate can be obtained, the source of the outbreak cannot be unambiguously determined. Shotgun metagenomics approaches applied to the food samples could circumvent this need for isolation from the suspected source, but require downstream strain-level data analysis to be able to accurately link to the human isolate. Until now, this approach has not yet been applied outside research settings to analyse real food-borne outbreak samples. In September 2019, a Salmonella outbreak occurred in a hotel school in Bruges, Belgium, affecting over 200 students and teachers. Following standard procedures, the Belgian National Reference Center for human salmonellosis and the National Reference Laboratory for Salmonella in food and feed used conventional analysis based on isolation, serotyping and MLVA (multilocus variable number tandem repeat analysis) comparison, followed by whole-genome sequencing, to confirm the source of the contamination over 2 weeks after receipt of the sample, which was freshly prepared tartar sauce in a meal cooked at the school. Our team used this outbreak as a case study to deliver a proof of concept for a short-read strain-level shotgun metagenomics approach for source tracking. We received two suspect food samples: the full meal and some freshly made tartar sauce served with this meal, requiring the use of raw eggs. After analysis, we could prove, without isolation, that Salmonella was present in both samples, and we obtained an inferred genome of a Salmonella enterica subsp. enterica serovar Enteritidis that could be linked back to the human isolates of the outbreak in a phylogenetic tree. These metagenomics-derived outbreak strains were separated from sporadic cases as well as from another outbreak circulating in Europe at the same time period. This is, to our knowledge, the first Salmonella food-borne outbreak investigation uniquely linking the food source using a metagenomics approach and this in a fast time frame.The Belgian Federal Public Service of Health, Food Chain Safety and Environment and Sciensano.https://www.microbiologyresearch.org/content/journal/mgenam2022Genetic

Time series modelling for wastewater-based epidemiology of COVID-19 : a nationwide study in 40 wastewater treatment plants of Belgium, February 2021 to June 2022

Background: Wastewater-based epidemiology (WBE) has been implemented to monitor surges of COVID-19. Yet, multiple factors impede the usefulness of WBE and quantitative adjustment may be required.Aim: We aimed to model the relationship between WBE data and incident COVID-19 cases, while adjusting for confounders and autocorrelation.Methods: This nationwide WBE study includes data from 40 wastewater treatment plants (WWTPs) in Belgium (02/2021-06/2022). We applied ARIMA-based modelling to assess the effect of daily flow rate, pepper mild mottle virus (PMMoV) concentration, a measure of human faeces in wastewater, and variants (alpha, delta, and omicron strains) on SARS-CoV-2 RNA levels in wastewater. Secondly, adjusted WBE metrics at different lag times were used to predict incident COVID-19 cases. Model selection was based on AICc minimization.Results: In 33/40 WWTPs, RNA levels were best explained by incident cases, flow rate, and PMMoV. Flow rate and PMMoV were associated with-13.0 % (95 % prediction interval:-26.1 to +0.2 %) and +13.0 % (95 % prediction interval: +5.1 to +21.0 %) change in RNA levels per SD increase, respectively. In 38/40 WWTPs, variants did not explain variability in RNA levels independent of cases. Furthermore, our study shows that RNA levels can lead incident cases by at least one week in 15/40 WWTPs. The median population size of leading WWTPs was 85.1 % larger than that of non-leading WWTPs. In 17/40 WWTPs, however, RNA levels did not lead or explain incident cases in addition to autocorrelation.Conclusion: This study provides quantitative insights into key determinants of WBE, including the effects of wastewater flow rate, PMMoV, and variants. Substantial inter-WWTP variability was observed in terms of explaining incident cases. These findings are of practical importance to WBE practitioners and show that the early-warning potential of WBE is WWTP-specific and needs validation