Whole Genome Sequencing for Studying Bacillus anthracis from an Outbreak in the Abruzzo Region of Italy

Abstract

Anthrax is a serious infectious disease caused by the gram-positive and spore-forming bacterium Bacillus anthracis. In Italy, anthrax is an endemic disease with sporadic cases each year and few outbreaks, especially in Southern Italy. However, new foci have been discovered in zones without previous history of anthrax. During summer 2016, an outbreak of anthrax caused the death of four goats in the Abruzzo region, where the disease had not been reported before. In order to investigate the outbreak, we sequenced one strain and compared it to 19 Italian B. anthracis genomes. Furthermore, we downloaded 71 whole genome sequences representing the global distribution of canonical SNP lineages and used them to verify the phylogenetic positioning. To this end, we analyzed and compared the genome sequences using canonical SNPs and the whole genome SNP-based analysis. Our results demonstrate that the outbreak strain belonged to the Trans-Eurasian (TEA) group A.Br.011/009, which is the predominant clade in Central-Southern Italy. In conclusion, the high genomic relatedness of the Italian TEA strains suggests their evolution from a common ancestor, while the spread is supposedly driven by trade as well as human and transhumance activities. Here, we demonstrated the capabilities of whole genome sequencing (WGS), which can be used as a tool for outbreak analyses and surveillance activities.

A Whole-Genome-Based Gene-by-Gene Typing System for Standardized High-Resolution Strain Typing of Bacillus anthracis

“…The high virulence potential of this pathogen, its worldwide and unknown dispersal of spore-contaminated fields (“champs maudit”) in the environment but also its potential use as an agent for bioweapons motivate in-depth genotyping for routine surveillance and outbreak investigation. As such, whole-genome SNP analysis has been applied by many laboratories for detailed subtyping of B. anthracis ( 5 , 15 ). However, the use of custom software and in-house workflows for SNP analysis affects the reproducibility and standardization of SNP typing approaches.…”

“…However, MLVA is laborious and prone to homoplasy problems ( 5 ). Today, whole-genome sequencing (WGS) is the method of choice to identify genome-wide SNPs in B. anthracis , which allows the definition of new canonical genetic lineages ( 7 , 15 ), but also greatly enhances the resolution of phylogenetic analyses in outbreak settings. Core-genome-based multilocus sequence typing (cgMLST) is a genome-wide typing system for a high-resolution clustering that indexes strain genotyping results into allelic numbers that can be accessed via a central database.…”

Genetic Diversity of Australian Bacillus anthracis Isolates Revealed by Multiple-Locus Variable-Number Tandem Repeat Analysis

“…Whole genome sequencing is a powerful alternative method to MLVA, with the versatility to look at disease outbreak origins, introductions, and routine surveillance to support epidemiological investigations. This technology is becoming standard practice for examining the genetic diversity of clonal bacterial species such as B. anthracis [ 32 , 33 ]. Rapid classification frameworks made available by researchers such as Bruce et al [ 32 ] offer a simplified and robust tool for the analysis of B. anthracis evolution and classification.…”

“…Rapid classification frameworks made available by researchers such as Bruce et al [ 32 ] offer a simplified and robust tool for the analysis of B. anthracis evolution and classification. Owing to the expansion of available genome sequence data and the comparison of B. anthracis strains worldwide [ 32 , 33 , 34 , 35 , 36 , 37 ], this approach offers greater insight into the global placement of the Australian strains and may provide a deeper understanding of the genomic diversity at a local level.…”

Identification of the molecular characteristics of Bacillus anthracis (1982-2020) isolates in East Indonesia using multilocus variable-number tandem repeat analysis

“…A genetic diversity study of B. anthracis has been performed worldwide using several techniques, such as amplified fragment length polymorphism [ 6 ], multilocus variable-number tandem repeat (VNTR) analysis (MLVA) [ 7 - 22 ], single-nucleotide polymorphism (SNP) [ 1 , 2 , 6 , 7 , 12 , 14 - 19 , 23 - 27 ], and whole-genome sequencing (WGS) [ 1 , 2 , 6 , 7 , 22 , 23 , 28 ]. MLVA detects VNTRs through polymerase chain reaction (PCR) based on target genes, which usually contain low-frequency mutations in the bacterial genome [ 29 - 31 ].…”