Issues and Trends in Collection Development for East Asia Legal Materials

The authors delineate the general policy and guidelines for developing foreign and transnational law collections in U.S. law libraries, and they analyze factors that shape East Asian collections, such as law libraries’ preservation and digitization efforts and their related cost-efficiency, and the availability and quality of English translations. The authors then discuss the main sources for Korean, Japanese, and Chinese law

Early Phylogenetic Diversification of SARS-CoV-2 : Determination of Variants and the Effect on Epidemiology, Immunology, and Diagnostics

The phylogenetic clustering of 95 SARS-CoV-2 sequences from the first 3 months of the pandemic reveals insights into the early evolution of the virus and gives first indications of how the variants are globally distributed. Variants might become a challenge in terms of diagnostics, immunology, and effectiveness of drugs. All available whole genome sequence data from the NCBI database (March 16, 2020) were phylogenetically analyzed, and gene prediction as well as analysis of selected variants were performed. Antigenic regions and the secondary protein structure were predicted for selected variants. While some clusters are presenting the same variant with 100% identical bases, other SARS-CoV-2 lineages show a beginning diversification and phylogenetic clustering due to base substitutions and deletions in the genomes. First molecular epidemiological investigations are possible with the results by adding metadata as travelling history to the presented data. The advantage of variants in source tracing can be a challenge in terms of virulence, immune response, and immunological memory. Variants of viruses often show differences in virulence or antigenicity. This must also be considered in decisions like herd immunity. Diagnostic methods might not work if the variations or deletions are in target regions for the detection of the pathogen. One base substitution was detected in a primer binding site

How to Show the Real Microbial Biodiversity? A Comparison of Seven DNA Extraction Methods for Bacterial Population Analyses in Matrices Containing Highly Charged Natural Nanoparticles

A DNA extraction that comprises the DNA of all available taxa in an ecosystem is an essential step in population analysis, especially for next generation sequencing applications. Many nanoparticles as well as naturally occurring clay minerals contain charged surfaces or edges that capture negatively charged DNA molecules after cell lysis within DNA extraction. Depending on the methodology of DNA extraction, this phenomenon causes a shift in detection of microbial taxa in ecosystems and a possible misinterpretation of microbial interactions. With the aim to describe microbial interactions and the bio-geo-chemical reactions during a clay alteration experiment, several methods for the detection of a high number of microbial taxa were examined in this study. Altogether, 13 different methods of commercially available DNA extraction kits provided by seven companies as well as the classical phenol-chloroform DNA extraction were compared. The amount and the quality of nucleic acid extracts were determined and compared to the amplifiable amount of DNA. The 16S rRNA gene fragments of several taxa were separated using denaturing gradient gel electrophoresis (DGGE) to determine the number of different species and sequenced to get the information about what kind of species the microbial population consists of. A total number of 13 species was detected in the system. Up to nine taxa could be detected with commercially available DNA extraction kits while phenol-chloroform extraction lead to three detected species. In this paper, we describe how to combine several DNA extraction methods for the investigation of microbial community structures in clay.BIOTON, German Federal Ministry of Education and Researc

Staphylococcus argenteus and Staphylococcus schweitzeri are cytotoxic to human cells in vitro due to high expression of alpha-hemolysin Hla

Staphylococcus argenteus and Staphylococcus schweitzeri are newly identified species of the S. aureus-related complex. S. argenteus, as occurring globally and showing significant prevalence and comparable infection and morbidity rates compared to S. aureus, is becoming clinically important. Whole genome sequencing has revealed the presence of several virulence genes but the molecular mechanisms of S. argenteus infection and virulence are largely unknown. Here, we studied the effect of a previously characterized clinical S. argenteus isolate on human cells in vitro. The clinical isolate, together with the S. argenteus type strain MSHR1132T and the S. schweitzeri type strain FSA084T, had a cytotoxic effect on the cells, which showed necrotic cell death after a few hours of treatment. The protein causing the cytotoxic effect was purified and identified by mass spectrometry as alpha-hemolysin, Hla, which is awell-known pore-forming toxin in S.aureus. The cytotoxic effect could be blocked with an antibody against Hla. S.argenteus showed 12-15 fold higher expression levels of hla at the RNA level and 4-6 fold higher expression levels at the protein level compared to S.aureus. The higher expression levels of hla were supported by higher RNA levels of the regulatory factors sarA and saeR. Also, the RNAIII component of the accessory gene regulator (agr) quorum sensing system was 8,000-10,000 fold higher in the S.argenteus isolates compared to S.aureus. This is the first study on the effect of S.argenteus on ahuman cell line and strengthens the idea of significant virulence of S.argenteus

GIFeGSH : A New Genomic Island Might Explain the Differences in Brucella Virulence

An imported dog was confirmed to be positive with canine brucellosis in Sweden in 2010. The whole genome of Brucella canis SVA10 was subjected to phage analysis (WGS-PA) and was assigned to the Asian B. canis cluster. Further analysis indicated that the genome of B. canis SVA10 is smaller compared to genomes of the same species. A 35,781 bp genomic island (GI) was found to be absent in strain SVA10 which was detected by read mapping the paired reads to the genome of B. canisATCC 23,365T. The lacking genes of genomic island GIFeGSH are mainly coding for iron uptake enzymes and parts of the glutathione pathway. A screening of all available whole genome sequences of Brucella strains confirmed that GIFeGSH is also missing in four more strains of B. canis but present in several strains of B. abortus, B. melitensis, B. suis, B. ovis, B. microti, B. pinnipedialis, and B. ceti. Parts of the GI were present, but scattered in two other B. canis strains. The aim of this study was to find differences in the genomes of Brucella which might explain former described differences in virulence. The analysis was extended to all available Brucella genomes after the detection of a genomic island in strain SVA10

Time to review the gold standard for genotyping vancomycin-resistant enterococci in epidemiology : Comparing whole-genome sequencing with PFGE and MLST in three suspected outbreaks in Sweden during 2013–2015

Vancomycin-resistant enterococci (VRE) are a challenge to the health-care system regarding transmission rate and treatment of infections. VRE outbreaks have to be controlled from the first cases which means that appropriate and sensitive genotyping methods are needed. The aim of this study was to investigate the applicability of whole genome sequencing based analysis compared to Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST) in epidemiological investigations as well as the development of a user friendly method for daily laboratory use. Out of 14,000 VRE - screening samples, a total of 60 isolates positive for either vanA or vanB gene were isolated of which 38 were from patients with epidemiological links from three suspected outbreaks at Uppsala University Hospital. The isolates were genotypically characterised with PFGE, MLST, and WGS based core genome Average Nucleotide Identity analysis (cgANI). PFGE was compared to WGS and MLST regarding reliability, resolution, and applicability capacity. The PFGE analysis of the 38 isolates confirmed the epidemiological investigation that three outbreaks had occurred but gave an unclear picture for the largest cluster. The WGS analysis could clearly distinguish six ANI clusters for those 38 isolates. As result of the comparison of the investigated methods, we recommend WGS-ANI analysis for epidemiological issues with VRE. The recommended threshold for Enterococcus faecium VRE outbreak strain delineation with core genome based ANI is 98.5%. All referred sequences of this study are available from the NCBI BioProject number PRJNA301929

Which methods are appropriate for the detection of Staphylococcus argenteus and is it worthwhile to distinguish S. argenteus from S. aureus?

Purpose: To further analyze a clinical isolate originally identified as methicillin-resistant Staphylococcus aureus (MRSA) using whole-genome sequencing and comparative genomics. Materials and methods: Classical diagnostic methods such as cultivation, biochemical tests, and PCR were supplemented with whole-genome sequencing and comparative genomics, to identify the isolate. Results: The isolate was phenotypically similar to MRSA. However, the presence of the nuc gene could not be confirmed using PCR, while it was positive for the mecA gene. Whole-genome sequencing correctly identified the isolate as Staphylococcus argenteus. The isolate possessed several resistance genes, such as mecA, blaZ (beta-lactam antibiotics) and dfrG (trimethoprim). The me gene differed from that of MRSA. Six phylogenetic distinct clusters were identified by average nucleotide identity (ANI) analysis of all available S. argenteus whole-genome sequences. Our isolate, RK308, clustered with those isolated in Europe and Asia. Conclusion: Due to the invasive potential, the multi-drug resistance and the similarity to MRSA, S. argenteus should be included in the MRSA screening. Due to the divergent genome compared to MRSA, new PCR approaches have to be developed to avoid an unnoticed spreading of S. argenteus

Differences in virulence gene expression between human blood and stool Campylobacter coli clade 1 ST828CC isolates

Background: Campylobacter colonise the gastrointestinal tract of warm-blooded animals and are major enteropathogens in humans. C. coli is less common than C. jejuni and accounts for about 10% of the total number of Campylobacter infections although the two species seem to share many virulence determinants. Campylobacter bacteraemia is rare, estimated to occur in less than 1% of the infections, and the exact mechanisms regulating the progression of the infection from the gastrointestinal tract to the blood stream are unclear. Here, we looked at the contribution of C. coli to Campylobacter infections and further compared various virulence traits in C. coli clade 1 blood and stool isolates. Results: We assessed the numbers of C. jejuni and C. coli among typed isolates in the PubMLST database and found that C. coli accounted for 25.9% of blood isolates, but only 8.9% of the stool isolates. Phylogenetic analysis of 128 C. coli clade 1 whole genome sequences deposited to NCBI revealed no specific clustering of the human blood, stool or animal isolates. Of the six C. coli isolates chosen for phenotypic analyses, stool isolates adhered significantly better to human HT-29 colon cancer cells than the blood isolates, while there was no difference in induced IL-8 levels between the isolates. Furthermore, the stool isolates had two-to fourfold higher RNA expression levels of the flpA, ciaB, iamA and cdt virulence genes than the blood isolates. Finally, we looked at the gene structure of the cdtA, B and C toxin genes and found numerous nucleotide additions and deletions disrupting the open reading frames. In contrast to 58% isolates of animal origin, only 38% and 32% of human blood and stool isolates, respectively, had all three cdt genes intact, a prerequisite to produce functional toxins. Conclusions: This study reveals interesting differences between C. coli clade 1 isolates of human and animal origin on one hand, and also between human blood and stool isolates, on the other. The results suggest that C. coli might downregulate and/or inactivate various virulence determinants as the isolates pass from the animal host to the human gastrointestinal tract and enter the human blood stream

Draft Genome Sequences of Brucella suis Biovar 4 Strain NCTC 10385, Brucella ceti Strain NCTC 12891T, Brucella inopinata Strain CAMP 6436T, and Brucella neotomae Strain ATCC 23459T

With the aim of developing quantitative PCR methods for the detection and differentiation of Brucella species, the genomes of Brucella ceti, Brucella inopinata, Brucella netotomae, and Brucella suis biovar 4 were sequenced and analyzed