Background Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus , Lactobacillus helveticus and Lactobacillus delbrueckii . Results Two NWCs from Swiss Gruyère producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats. Conclusions Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities. Electronic supplementary material The online version of this article (10.1186/s12866-019-1500-0) contains supplementary material, which is available to authorized users.
Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus—to our knowledge—identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.
The dairy industry suffers massive economic losses due to staphylococcal mastitis in cattle. The Staphaureux latex agglutination test (Oxoid, Basel, Switzerland) was reported to lead to negative results in 54% of bovine Staphylococcus aureus strains, and latex-negative strains are thought to be less virulent than Staphaurex latex-positive strains. However, comparative information on virulence and resistance profiles of these 2 groups of Staph. aureus is scarce. Our objective was to associate the latex agglutination phenotype of Staph. aureus strains isolated from bovine mastitis milk with data on clonal complexes, virulence genes, and antibiotic resistance to (1) determine the virulence profiles of the Staphaureux test positive and Staphaurex test negative groups, and (2) provide data needed to improve treatment of bovine mastitis and to identify potential vaccine targets. Seventy-eight Staph. aureus strains isolated from 78 cows on 57 Swiss farms were characterized. Latex agglutination was tested by Staphaureux kit, and resistance profiles were generated by disk diffusion. A DNA microarray was used to assign clonal complexes (CC) and to determine virulence and resistance gene profiles. By the Staphaureux test, 49% of the isolates were latex-positive and 51% were latex-negative. All latex-negative strains were assigned to CC151, whereas latex-positive strains were assigned to various clonal complexes, including CC97 (n=16), CC8 (n=10), CC479 (n=5), CC20 (n=4), CC7 (n=1), CC9 (n=1), and CC45 (n=1). Although the latexnegative isolates were susceptible to all antimicrobial agents tested, 24% of latex-positive isolates were classified as intermediate with regard to cefalexin-kanamycin and 13% were resistant to both ampicillin and penicillin. Microarray profiles of latex-negative isolates were highly similar, but differed largely from those of latex-positive isolates. Although the latex-negative group lacked several enterotoxin genes and sak, it exhibited significantly higher prevalence rates of genes encoding enterotoxin C, toxic shock syndrome toxin, and leukocidins (lukM/lukF-P83, lukD). Our findings suggest that latex-negative isolates represent a group of closely related strains with specific resistance and virulence gene patterns. The dairy industry suffers from massive economic losses due to staphylococcal mastitis in cattle. 39Staphaureux latex agglutination test was reported to lead to negative results in 54% of bovine S. 40 aureus strains and latex-negative strains were hypothesized to be less virulent than Staphaurex 41 test positive strains. However, comparative information on virulence and resistance profiles of 42 these two groups of S. aureus is scarce. Our objective was to associate the latex agglutination 43 phenotype of S. aureus strains isolated from bovine mastitis milk with data on clonal complexes, 44virulence genes, and antibiotic resistance in order to 1) determine the virulence profiles of the 45Staphaureux test positive and Staphaurex test negative groups, and 2) provide data neede...
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