- Volume 8, Issue 11, 2022

Whole-genome sequencing is widely used to better understand the transmission dynamics, the evolution and the emergence of new variants of viral pathogens. This can bring crucial information to stakeholders for disease management. Unfortunately, aquatic virus genomes are usually difficult to characterize because most of these viruses cannot be easily propagated in vitro. Developing methodologies for routine genome sequencing of aquatic viruses is timely given the ongoing threat of disease emergence. This is particularly true for pathogenic viruses infecting species of commercial interest that are widely exchanged between production basins or countries. For example, the ostreid herpesvirus type 1 (OsHV-1) is a Herpesvirus widely associated with mass mortality events of juvenile Pacific oyster Crassostrea gigas. Genomes of Herpesviruses are large and complex with long direct and inverted terminal repeats. In addition, OsHV-1 is unculturable. It therefore accumulates several features that make its genome sequencing and assembly challenging. To overcome these difficulties, we developed a tangential flow filtration (TFF) method to enrich OsHV-1 infective particles from infected host tissues. This virus purification allowed us to extract high molecular weight and high-quality viral DNA that was subjected to Illumina short-read and Nanopore long-read sequencing. Dedicated bioinformatic pipelines were developed to assemble complete OsHV-1 genomes with reads from both sequencing technologies. Nanopore sequencing allowed characterization of new structural variations and major viral isomers while having 99,98 % of nucleotide identity with the Illumina assembled genome. Our study shows that TFF-based purification method, coupled with Nanopore sequencing, is a promising approach to enable in field sequencing of unculturable aquatic DNA virus.

Shiga toxin-producing Escherichia coli (STEC) are a cause of severe human illness and are frequently associated with haemolytic uraemic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause human disease. In addition to the Shiga-toxin (stx genes), many additional factors have been reported, such as intimin (eae gene), which is clearly an aggravating factor for developing HUS. Current STEC detection methods classically rely on real-time PCR (qPCR) to detect the presence of the key virulence markers (stx and eae). Although qPCR gives an insight into the presence of these virulence markers, it is not appropriate for confirming their presence in the same strain. Therefore, isolation steps are necessary to confirm STEC viability and characterize STEC genomes. While STEC isolation is laborious and time-consuming, metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Recently, short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics for identifying eae-positive STEC strains using raw cow's milk as a causative matrix for STEC food-borne outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing and generating an easy-to-use STEC characterization pipeline, the direct identification of an eae-positive STEC strain was successful after enrichment of artificially contaminated raw cow's milk samples at a contamination level as low as 5 c.f.u. ml−1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk in combination with a complete STEC analysis pipeline from long-read sequencing metagenomics data. This study shows the potential of the innovative methodology for characterizing STEC strains from complex matrices. Further developments will nonetheless be necessary for this method to be applied in STEC surveillance.

Dinoflagellates of the genus Alexandrium are responsible for harmful algal blooms and produce paralytic shellfish toxins (PSTs). Their very large and complex genomes make it challenging to identify the genes responsible for toxin synthesis. A family-based genomic association study was developed to determine the inheritance of toxin production in Alexandrium minutum and identify genomic regions linked to this production. We show that the ability to produce toxins is inheritable in a Mendelian way, while the heritability of the toxin profile is more complex. We developed the first dinoflagellate genetic linkage map. Using this map, several major results were obtained: 1. A genomic region related to the ability to produce toxins was identified. 2. This region does not contain any polymorphic sxt genes, known to be involved in toxin production in cyanobacteria. 3. The sxt genes, known to be present in a single cluster in cyanobacteria, are scattered on different linkage groups in A. minutum. 4. The expression of two sxt genes not assigned to any linkage group, sxtI and sxtG, may be regulated by the genomic region related to the ability to produce toxins. Our results provide new insights into the organization of toxicity-related genes in A. minutum, suggesting a dissociated genetic mechanism for the production of the different analogues and the ability to produce toxins. However, most of the newly identified genes remain unannotated. This study therefore proposes new candidate genes to be further explored to understand how dinoflagellates synthesize their toxins.

Gene transfer agents (GTAs) are phage-like particles that transfer pieces of cellular genomic DNA to other cells. Homologues of the Rhodobacter capsulatus GTA (RcGTA) structural genes are widely distributed in the Alphaproteobacteria and particularly well conserved in the order Rhodobacterales. Possible reasons for their widespread conservation are still being discussed. It has been suggested that these alphaproteobacterial elements originate from a prophage that was present in an ancestral bacterium and subsequently evolved into a GTA that is now widely maintained in extant descendant lineages. Here, we analysed genomic properties that might relate to the conservation of these alphaproteobacterial GTAs. This revealed that the chromosomal locations of the GTA gene clusters are biased. They primarily occur on the leading strand of DNA replication, at large distances from long repetitive elements, and thus are in regions of lower plasticity, and in areas of extreme GC skew, which also accumulate core genes. These extreme GC skew regions arise from the preferential use of codons with an excess of G over C, a distinct phenomenon from the elevated GC content that has previously been found to be associated with GTA genes. The observed properties, along with their high level of conservation, show that GTA genes share multiple features with core genes in the examined lineages of the Alphaproteobacteria.

Enterococcus faecium is a lactic acid bacterium that confers beneficial health effects in humans. However, lately, a number of E. faecium strains have been linked to the spread of nosocomial infections in the hospital environment. Therefore, any potential commercial usage of E. faecium isolates should be preceded by an assessment of infection risk. In the current study, the genomes of two novel E. faecium strains Am1 (larval isolate) and Bee9 (adult bee isolate) isolated from the gut of Apis mellifera L. (honeybee) were sequenced to allow evaluation of their safety. In particular, their genomes were screened for antibiotic-resistance and virulence genes. In addition, their potential to spread resistance in the environment was evaluated. The analysis revealed that Am1 and Bee9 possess 2832 and 2844 protein-encoding genes, respectively. In each case, the genome size was 2.7 Mb with a G+C content of 37.9 mol%. Comparative analysis with probiotic, non-pathogenic and pathogenic enterococci revealed that there are variations between the two bee E. faecium isolates and pathogenic genomes. They were, however, closely linked to the probiotic comparison strains. Phenotypically, the Am1 and Bee9 strains were susceptible to most antibiotics tested, but showed intermediate sensitivity towards erythromycin, linezolid and trimethoprim/sulfamethoxazole. Notably, no genes associated with antibiotic resistance in clinical isolates (e.g. vancomycin resistance: vanA, vanB, vanS, vanX and vanY) were present. In addition, the insertion sequences (IS16, ISEfa11 and ISEfa5), acting as molecular pathogenicity markers in clinically relevant E. faecium strains, were also absent. Moreover, the analysis revealed the absence of three key pathogenicity-associated genes (acm, sgrA, ecbA) in the Am1 and Bee9 strains that are found in the prominent clinical isolates DO, V1836, Aus0004 and Aus0085. Overall, the findings of this investigation suggest that the E. faecium isolates from the bee gut have not suffered any recent clinically relevant antibiotic exposure. It also suggests that E. faecium Am1 and Bee9 are safe potential probiotic strains, because they lack the phenotypic and genetic features associated with strains eliciting nosocomial infections.

Most bacteria can form biofilms, which typically have a life cycle from cells initially attaching to a surface before aggregation and growth produces biomass and an extracellular matrix before finally cells disperse. To maximize fitness at each stage of this life cycle and given the different events taking place within a biofilm, temporal regulation of gene expression is essential. We recently described the genes required for optimal fitness over time during biofilm formation in Escherichia coli using a massively parallel transposon mutagenesis approach called TraDIS-Xpress. We have now repeated this study in Salmonella enterica serovar Typhimurium to determine the similarities and differences in biofilm formation through time between these species. A core set of pathways involved in biofilm formation in both species included matrix production, nucleotide biosynthesis, flagella assembly and LPS biosynthesis. We also identified several differences between the species, including a divergent impact of the antitoxin TomB on biofilm formation in each species. We observed deletion of tomB to be detrimental throughout the development of the E. coli biofilms but increased biofilm biomass in S. Typhimurium. We also found a more pronounced role for genes involved in respiration, specifically the electron transport chain, on the fitness of mature biofilms in S. Typhimurium than in E. coli and this was linked to matrix production. This work deepens understanding of the core requirements for biofilm formation in the Enterobacteriaceae whilst also identifying some genes with specialised roles in biofilm formation in each species.

  Streptococcus suis   is a leading cause of bacterial meningitis in South-East Asia, with frequent zoonotic transfer to humans associated with close contact with pigs. A small number of invasive lineages are responsible for endemic infection in the swine industry, causing considerable global economic losses. A lack of surveillance and a rising trend in clinical treatment failure has raised concerns of growing antimicrobial resistance (AMR) among invasive   S. suis  . Gene flow between healthy and disease isolates is poorly understood and, in this study, we sample and sequence a collection of isolates predominantly from healthy pigs in Chiang Mai province, Northern Thailand. Pangenome characterization identified extensive genetic diversity and frequent AMR carriage in isolates from healthy pigs. Multiple AMR genes were identified, conferring resistance to aminoglycosides, lincosamides, tetracycline and macrolides. All isolates were non-susceptible to three or more different antimicrobial classes, and 75 % of non-serotype 2 isolates were non-susceptible to six or more classes (compared to 37.5 % of serotype 2 isolates). AMR genes were found on integrative and conjugative elements previously observed in other species, suggesting a mobile gene pool that can be accessed by invasive disease isolates. This article contains data hosted by Microreact.

Antimicrobial resistance (AMR) has become a critical threat to public health worldwide. The use of antimicrobials in food and livestock agriculture, including the production of poultry, is thought to contribute to the dissemination of antibiotic resistant bacteria (ARB) and the genes and plasmids that confer the resistant phenotype (ARG). However, the relative contribution of each of these processes to the emergence of resistant pathogens in poultry production and their potential role in the transmission of resistant pathogens in human infections, requires a deeper understanding of the dynamics of ARB and ARG in food production and the factors involved in the increased risk of transmission.

Salmonella enterica serovar Typhimurium (S. Typhimurium) comprises a group of closely related human and animal pathogens that account for a large proportion of all Salmonella infections globally. The epidemiological record of S. Typhimurium in Europe is characterized by successive waves of dominant clones, each prevailing for approximately 10–15 years before replacement. Succession of epidemic clones may represent a moving target for interventions aimed at controlling the spread and impact of this pathogen on human and animal health. Here, we investigate the relationship of phage sensitivity and population structure of S. Typhimurium using data from the Anderson phage typing scheme. We observed greater resistance to phage predation of epidemic clones circulating in livestock over the past decades compared to variants with a restricted host range implicating increased resistance to phage in the emergence of epidemic clones of particular importance to human health. Emergence of monophasic S. Typhimurium ST34, the most recent dominant multidrug-resistant clone, was accompanied by increased resistance to phage predation during clonal expansion, in part by the acquisition of the mTmII prophage that may have contributed to the fitness of the strains that replaced ancestors lacking this prophage.

The   Escherichia coli   species contains a diverse set of sequence types and there remain important questions regarding differences in genetic content within this population that need to be addressed. Pangenomes are useful vehicles for studying gene content within sequence types. Here, we analyse 21   E. coli   sequence type pangenomes using comparative pangenomics to identify variance in both pangenome structure and content. We present functional breakdowns of sequence type core genomes and identify sequence types that are enriched in metabolism, transcription and cell membrane biogenesis genes. We also uncover metabolism genes that have variable core classification, depending on which allele is present. Our comparative pangenomics approach allows for detailed exploration of sequence type pangenomes within the context of the species. We show that ongoing gene gain and loss in the   E. coli   pangenome is sequence type-specific, which may be a consequence of distinct sequence type-specific evolutionary drivers.

Lambdoid (or Lambda-like) phages are a group of related temperate phages that can infect Escherichia coli and other gut bacteria. A key characteristic of these phages is their mosaic genome structure, which served as the basis for the ‘modular genome hypothesis’. Accordingly, lambdoid phages evolve by transferring genomic regions, each of which constitutes a functional unit. Nevertheless, it is unknown which genes are preferentially transferred together and what drives such co-transfer events. Here we aim to characterize genome modularity by studying co-transfer of genes among 95 distantly related lambdoid (pro-)phages. Based on gene content, we observed that the genomes cluster into 12 groups, which are characterized by a highly similar gene content within the groups and highly divergent gene content across groups. Highly similar proteins can occur in genomes of different groups, indicating that they have been transferred. About 26 % of homologous protein clusters in the four known operons (i.e. the early left, early right, immunity and late operon) engage in gene transfer, which affects all operons to a similar extent. We identified pairs of genes that are frequently co-transferred and observed that these pairs tend to be near one another on the genome. We find that frequently co-transferred genes are involved in related functions and highlight interesting examples involving structural proteins, the cI repressor and Cro regulator, proteins interacting with DNA, and membrane-interacting proteins. We conclude that epistatic effects, where the functioning of one protein depends on the presence of another, play an important role in the evolution of the modular structure of these genomes.