Genome-Wide Single Nucleotide Polymorphism Typing Method for Identification of Bacillus anthracis Species and Strains among B. cereus Group Species ▿ †

As an issue of biosecurity, species-specific genetic markers have been well characterized. However, Bacillus anthracis strain-specific information is currently not sufficient for traceability to identify the origin of the strain. By using genome-wide screening using short read mapping, we identified strain-specific single nucleotide polymorphisms (SNPs) among B. anthracis strains including Japanese isolates, and we further developed a simplified 80-tag SNP typing method for the primary investigation of traceability. These 80-tag SNPs were selected from 2,965 SNPs on the chromosome and the pXO1 and pXO2 plasmids from a total of 19 B. anthracis strains, including the available genome sequences of 17 strains in the GenBank database and 2 Japanese isolates that were sequenced in this study. Phylogenetic analysis based on 80-tag SNP typing showed a higher resolution power to discriminate 12 Japanese isolates rather than the 25 loci identified by multiple-locus variable-number tandem-repeat analysis (MLVA). In addition, the 80-tag PCR testing enabled the discrimination of B. anthracis from other B. cereus group species, helping to identify whether a suspected sample originates from the intentional release of a bioterrorism agent or environmental contamination with a virulent agent. In conclusion, 80-tag SNP typing can be a rapid and sufficient test for the primary investigation of strain origin. Subsequent whole-genome sequencing will reveal apparent strain-specific genetic markers for traceability of strains following an anthrax outbreak

Genomewide Screening for Novel Genetic Variations Associated with Ciprofloxacin Resistance in Bacillus anthracis▿ †

Fluoroquinolone (FQ) resistance of Bacillus anthracis is a serious concern in the fields of biodefense and bioterrorism since FQs are very effective antibiotics and are recommended as first-line treatment against this lethal bacterium. In this study, we obtained 2 strains of B. anthracis showing resistance or intermediate resistance to ciprofloxacin (CIP) by a stepwise selection procedure with increasing CIP concentrations. Fifteen genetic variations were identified between the parental and CIP-resistant strains by next-generation sequencing. Nonsynonymous mutations in the quinolone resistance-determining region (QRDR) of type II DNA topoisomerase were identified in the resistant strain but not in the intermediate-resistant strain. The GBAA0834 (TetR-type transcriptional regulator) locus was also revealed to be a novel “mutation hot spot” that leads to the increased expression of multidrug efflux systems for CIP resistance. As an initial step of CIP resistance in B. anthracis, such disruptive mutations of GBAA0834 appear to be more easily acquired than those in an essential gene, such as that encoding type II DNA topoisomerase. Such an intermediate-resistant phenotype could increase a cell population under CIP-selective pressure and might promote the emergence of highly resistant isolates. Our findings reveal, in addition to QRDR, crucial genetic targets for the investigation of intermediate resistance of B. anthracis to FQs

Molecular Epidemiological Study of Bacillus anthracis Isolated in Mongolia by Multiple-Locus Variable-Number Tandem-Repeat Analysis for 8 Loci (MLVA-8)

SUMMARY: The incidence of anthrax, which is caused by Bacillus anthracis, in the human and animal population of Mongolia has increased recently, and control of this infection is a nationwide concern. In this study, 29 isolates obtained from animals and various regions in Mongolia from 2001 to 2007 were analyzed by performing multiple-locus variable-number tandem-repeat analysis for 8 loci (MLVA-8) to understand the genetic relationship between the Mongolian B. anthracis isolates. We found that all the Mongolian isolates can be classified into A3 cluster along with the Japanese and the Chinese B. anthracis isolates. Our data revealed that MLVA-8 is useful for studying the molecular epidemiology of the Mongolian B. anthracis isolates and would help characterize B. anthracis infections in Mongolia

Epidemiological and comparative genomic analysis of Bacillus anthracis isolated from northern Vietnam.

To understand the epidemiological and genetic background of anthrax cases occurring in Vietnam from 2011 to 2015, we surveilled and genetically analyzed Bacillus anthracis isolated in the north of the country. Epidemiological surveillance showed that most human cutaneous anthrax cases occurred in association with animal dissection. Whole-genome sequences were obtained from six B. anthracis strains from human patients with cutaneous anthrax in the endemic area. Comparative genomic analysis showed that the genetic homogeneity among Vietnamese B. anthracis strains was very high. All Vietnamese B. anthracis strains belonged to the canSNP lineage of A.Br.011/009, which mostly consists of strains of the trans-Eurasian (TEA) group, including the most closely related strain, Carbosap. To clarify the genetic diversity of Vietnamese strains and strains belonging to A.Br.011/009 and A.Br.008/011 canSNP lineages, we applied a reference genome-based single-nucleotide polymorphism (SNP) and gene-by-gene genomic analysis (whole-genome MLST) strategy. The phylogeny from core genome SNPs revealed that the Vietnamese strains were positioned close to each other; moreover, several SNPs specific to Vietnamese B. anthracis were identified. Whole-genome MLST analysis revealed the differences in the number of SNPs between Vietnamese strains, which could enable discrimination at the strain level

Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

<div><p>LC16m8 (m8), a highly attenuated vaccinia virus (VAC) strain, was developed as a smallpox vaccine, and its safety and immunogenicity have been confirmed. Here, we aimed to develop a system that recovers infectious m8 from a bacterial artificial chromosome (BAC) that retains the full-length viral genomic DNA (m8-BAC system). The infectious virus was successfully recovered from a VAC-BAC plasmid, named pLC16m8-BAC. Furthermore, the bacterial replicon-free virus was generated by intramolecular homologous recombination and was successfully recovered from a modified VAC-BAC plasmid, named pLC16m8.8S-BAC. Also, the growth of the recovered virus was indistinguishable from that of authentic m8. The full genome sequence of the plasmid, which harbors identical inverted terminal repeats (ITR) to that of authentic m8, was determined by long-read next-generation sequencing (NGS). The ITR contains x 18 to 32 of the 70 and x 30 to 45 of 54 base pair tandem repeats, and the number of tandem repeats was different between the ITR left and right. Since the virus recovered from pLC16m8.8S-BAC was expected to retain the identical viral genome to that of m8, including the ITR, a reference-based alignment following a short-read NGS was performed to validate the sequence of the recovered virus. Based on the pattern of coverage depth in the ITR, no remarkable differences were observed between the virus and m8, and the other region was confirmed to be identical as well. In summary, this new system can recover the virus, which is geno- and phenotypically indistinguishable from authentic m8.</p></div

Identity of the ITR region in authentic m8 and 8.8S-clone4, a clone that was plaque-purified from EGFP-negative and bacterial replicon-free vLC16m8.8S-BAC.

<p>The coverage depth computed by the alignment of short-reads to the sequence of the left ITR region, which was extracted from the consensus sequence of pLC16m8.8S-BAC is shown (A and D). The coverage depth in the ITR left region from the terminus (position 1) to the C23L gene of m8 or 8.8S-clone 4 was plotted as either a black or a red line. The mean of the coverage depth by each of the regions (i.e., 70 base pair repeat, non-repeated region II to 125 base pairs, 54 base pair repeat, non-repeated region III, and C23L) was plotted (B, C, E, and F). One-way ANOVA with Bonferroni's multiple-comparison test was used to determine the level of statistical significance. The effect size (r), which indicates the magnitude of the mean difference between the regions, was also calculated. The mean of coverage depth at the 70 base pairs repeats were calculated from position 474 to 1423 to exclude the effect of low coverage depth artificially occurred at the terminus of the reference sequence. The number of 54 base pair tandem repeats in the reference sequence was artificially modified from x 41 (A-C) to x 23 (D-E). “n.s.” indicates "not significant”.</p

Number of 54 bp or 70 bp repeats in the ITR left or right.

<p>Number of 54 bp or 70 bp repeats in the ITR left or right.</p

Restriction fragment analysis of BAC plasmids.

<p>Prediction of NcoI digested fragment patterns based on the sequences of pLC16m8-BAC and the derivatives were shown (A). Purified BAC plasmids, pLC16m8-BAC (m8-BAC), pLC16m8.8S/8.OS-BAC (8.8S or 8.OS) and pLC16m8.8/8.O (8.8/8.O) were digested with NcoI and separated on a 0.75% agarose gel (B). Color of the gel was inverted. Sizes of a molecular weight marker are shown as marker, and the sizes are given. Changes in the restriction pattern due to the presence of EGFP, mini-F cassettes or rear part of B5R are indicated with a red arrow.</p

Coverage depth through the ITR left to the right region flanked by the C23L gene of pLC16m8.8S-BAC computed by HGAP (black line) or by Canu (red line).

<p>Coverage depth through the ITR left to the right region flanked by the C23L gene of pLC16m8.8S-BAC computed by HGAP (black line) or by Canu (red line).</p