Molecular Diversity of Bacillus anthracis in Italy

B. anthracis isolates.We have analyzed a collection of 64 B. anthracis isolates that are representative samples from outbreaks occurring in Italy during the past 40 years. B. anthracis isolates were collected from the following regions: Puglia, Campania, Basilicata, Calabria, Tuscany, Sicily, Sardinia, Lazio, and Veneto.

DNA preparation.Each B. anthracis strain was streaked onto tryptose agar plates and then incubated at +37°C for 24 h. The single colonies were suspended in 1 ml of TE (Tris-HCl [pH 8], 1.0 mM EDTA) and incubated at +98°C for 20 min. After centrifugation at 15,000 × g for 1 min, the supernatant was collected and filtered using 0.22-μm filters.

MLVA PCR.For the MLVA test, we utilized fluorescently labeled primers that flank the eight VNTR regions (vrrA, vrrB₁, vrrB₂, vrrC₁, vrrC₂, CG3, pXO1, pXO2) as described by Keim et al. in 2000 (8). Three different dyes are used for the reaction primers (6-carboxyfluorescein, VIC, and NED), while one is reserved for molecular weight size standards (ROX). Primers amplifying VNTRs that exhibit overlapping allele size ranges are labeled with different fluorophores to ensure that the VNTRs can be examined in a single electrophoretic injection. The final volume (50 μl) of reaction mixture contained 200 μM deoxynucleoside triphosphates (Amersham Pharmacia Biotech, Piscataway, N.J.), 0.2 μM primers (Applied Biosystems, Foster City, Calif.), 10 mM Tris-HCl, pH 8.3 (Perkin-Elmer, Wellesley, Mass.), 50 mM KCl (Perkin-Elmer), 1.5 mM MgCl₂ (Perkin-Elmer), 1.25 U of Taq DNA polymerase gold (Perkin-Elmer), and 5.0 μl of extracted DNA. The PCR samples were amplified in a Mastercycler Personal (Eppendorf AG, Hamburg, Germany) as follows: 95°C for 10 min, followed by 30 cycles of 92°C for 30 s, annealing for 30 s (57°C for VrrA, VrrB₁, VrrB₂, and VrrC₁; 60°C for VrrC₂; 55°C for CG3, pX01, and pX02), and extension at 72°C for 30 s. To ensure that the loci were amplified, 20.0 μl of each amplified sample was subjected to electrophoresis in a 2.0% agarose gel with 0.5 μg/ml of ethidium bromide at 100 V for 120 min. The amplified DNA bands were visualized upon UV light exposure (Eagle Eye II; Stratagene, La Jolla, Calif.).

Automated genotype analysis.The amplified MLVA PCR products were subjected to capillary electrophoresis in polyacrylamide gels under denaturing conditions on an ABI Prism 310 automated DNA sequencer (Applied Biosystems) and were analyzed using Genescan and Genotyper software (Applied Biosystems).

Data analysis.UPGMA (unweighted-pair group method using arithmetic averages) cluster analysis was performed on genotype scores from a combined data set of the Italian isolates and a panel of 89 diverse genotypes as reported by Keim et al. in 2000 (8). To ensure comparability of genotype scores between the 89 diverse genotypes and Italian isolates, a subset of isolates was genotyped at both the Keim Genetics Laboratory (Northern Arizona University) and the Anthrax Reference Institute of Italy (Foggia, Italy), and raw VNTR sizes were compared and normalized.

Geographical distribution and sources of Italian isolates.While anthrax occurs in much of Italy, the majority of anthrax outbreaks occurred in central Italy, southern Italy, and major islands (Table 1; Fig. 1B). The samples from this study are representative of anthrax activity for the past 40 years.

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FIG. 1.

Genetic and geographic relationships among Italian B. anthracis isolates. (A) Mutational-step model for isolate genetic relationships. Shown is a network of genotypes with the minimum mutational changes needed to convert one genotype into another. Note that genotype designations are consistent with those in Table 1. The G_I designation is given first, and G_K designation is given in parentheses following G_I if an equivalent genotype was described by Keim et al. in 2000 (8). Connecting lines are labeled with the genetic locus and the number of repeat units that differ between individual genotypes. (B) Geographic distribution of B. anthracis isolates. Genotype distribution and frequency are indicated by colors corresponding to designations in panel A. Pie chart sizes are scaled categorically according to the number of isolates (in three categories: 1 to 5, 6 to 10, and >10 isolates). Note that the two isolates with missing data (plasmid markers) were not included in either panel.

Genetic diversity of B. anthracis in Italy.While MLVA of 64 B. anthracis isolates revealed 10 unique genotypes, these were very closely related genetically (Fig. 1A; Table 1). UPGMA cluster analysis grouped 63/64 isolates with the previously described A1.a cluster (8), with only a single isolate associated with the A3.b cluster (Table 1). Four of the genotypes were exact matches to previously described genotypes, including the two dominant Italian genotypes (G_I) A (n = 28) and F (n = 25), which match the Keim et al. 2000 genotypes (G_K) 1 and 3, respectively (Fig. 1; Table 1). Not all of the Italian isolates matched previously described genotypes (G_K); those that did not were assigned only Italian genotype designations (G_I). For two samples, assigning genotypes was difficult due to missing data. One A1.a isolate is missing the pXO2 plasmid marker (G_I H), whereas the single A3.b representative (G_I J) is missing both the pXO2 and pXO1 markers. Therefore, these samples could match several previously defined genotypes in these groups or could represent novel genotypes.

A mutational-step model attempts to describe genetic relationships based on the minimum number of mutations needed to create the observed diversity in the Italian B. anthracis genotypes (Fig. 1A). The A3.b G_I genotype J, which is multiple mutational steps different from the A1.a genotypes and which lacks data for the plasmid markers, is not included in this model. The most common genotypes (G_I genotypes A and F, G_K genotypes 1 and 3) differ by only a single mutation in the pXO1 marker locus. With the exception of G_I genotypes D and E, there is only a single mutational step separating the rarer genotypes from either of the two dominant genotypes. In this study, with one exception (G_I J), the B. anthracis isolates are closely genetically related within the A1.a cluster.

Different Italian regions have distinctive genotype compositions and population structures (Fig. 1B). The A genotype (G_K 1) dominates the peninsular provinces of Puglia, Basilicata, and Calabria. This genotype is not found in Sardinia or Sicily, where the F genotype (G_K 3) is dominant. The close genetic relationship between these genotypes argues for a historical relationship, but the spatial differences indicate that recent anthrax outbreaks are now independent between the regions.