Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis

Abstract

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.

Fig 1. Hypothesis on an active life cycle of B. anthracis in soil environments.

Soil surrounding the carcass at the lower part of the figure harbors a very high spore burden. The left part of the panel predicts massive soil proliferation of B. anthracis (hypothesis). In this case of an active near-surface life cycle, local accumulation of spores is suggested to be due to repeated rounds of germination, replication and sporulation in the near-surface soil environment. During genome amplification random mutations occur resulting in derived genotypes compared to the genotypes of the initial spore population within the carcass. The right part of the panel depicts events if there was no soil-borne life cycle of the pathogen (competing hypothesis). Inert spores are supposed to accumulate in rainwater depressions. Genotypes differing from the original animal-infecting population cannot be observed in near-surface isolates.

Fig 2. Location and details of sampling sites in southern Italy.

Panel A indicates the locations of burial sites in Southern Italy (left) and positions of burial sites A, B and C at Pollino National Park (right). Panel B shows burial site C after sampling at positions 1, 2 and 3 (holes, approx. 50 cm apart).

Fig 3. Temperature-dissociation (melt) curve derivatives of HRM-SNP.

The relative fluorescence signal is plotted against the melting temperature (°C) for each SNP. Curves showing ancestral alleles (same base as predominant Pollino allele) are displayed in green. Curves indicating derived alleles (variant allele at the SNP-position) are displayed in red. The respective bases at the SNP position (“G” or “A”) are indicated for each allele group. Inconsistent curves (“?”) are displayed in blue. Negative controls (“NC”) are displayed in light-blue.

Fig 4. Modified (synthesis) model for B. anthracis genotype dynamics in soil of Pollino burial sites.

Soil surrounding the carcass at the lower part of the figure harbors a very high endospore burden. Genetically diverse genotypes, which can be found near-carcass and near-surface, are the result of either multiple genotype infection or mutations during the course of host infection. Endospores reach the surface and accumulate via physical diffusion. Sporadic germination in soil or possibly in transient vectors, replication and sporulation under favorable conditions can lead to genetically diverse genotypes that can be found in near-surface soil.