Molecular Diversity of Bacillus anthracis in Italy

We used multiple-locus variable-number tandem repeat analysis(MLVA) to type 64 Bacillus anthracis isolates from outbreaksthat have occurred during the past 40 years in Italy. MLVA ofthe 64 isolates revealed 10 unique genotypes; 9 of these genotypesand the majority of isolates (63/64) belonged to the previouslydescribed genetic cluster A1.a. Within the A1.a isolates, twopreviously described genotypes (G1 and G3), which differ bya single mutation in the pX01 locus, account for the majorityof isolates in the country (53/63). The low diversity of B.anthracis genotypes in Italy suggests a single, dominant historicalintroduction, followed by limited localized differentiation.

INTRODUCTION

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Introduction

Bacillus anthracis, the causative agent of anthrax, is a geneticallyhomogeneous and recently emerged pathogen, which complicatesefforts to subtype it (7, 8, 11). In 1995, Henderson et al.detected the first molecular difference among B. anthracis strains,and in the following year Andersen et al. demonstrated thatthe variation was due to a 12-nucleotide variable-number tandemrepeat (VNTR), named vrrA (1, 4). The molecular basis for thevariation was due to differences in the number of repeated units,and five different allelic forms could be detected (6). An expansionon this strategy that further advanced the subtyping of B. anthracisinvolved the analysis of multiple VNTR loci (8). Multiple-locusVNTR analysis (MLVA) typing systems use PCR amplification andfragment sizing to detect length polymorphisms in several VNTRregions (8, 10). MLVA has proven to be a powerful tool for distinguishingamong B. anthracis isolates and has been applied to examinationof bioterrorism events as well as naturally occurring anthraxoutbreaks (5, 8, 9, 10, 13, 14). Keim et al. in 2000 used MLVAto examine a worldwide collection of B. anthracis isolates anddescribed 89 unique genotypes (8). Further studies have usedMLVA to examine regional patterns of anthrax diversity; theseinclude studies of the genetic diversity of B. anthracis isolatesin France and Poland (2, 3, 13, 14).

In Italy, anthrax is generally a sporadic disease that occurs,with a few exceptions, in the central and southern regions andin the major islands, where it almost exclusively affects animalsat pasture. Since the late 1950s, the Italian anthrax epidemicsituation has improved as a result of vaccination campaignswith an attenuated live vaccine, composed of spores in 1% saponin(Carbosap). This nationwide control system also includes diseaseoutbreak surveillance, and over the past 5 years, the AnthraxReference Institute of Italy has been recovering B. anthracisisolates from national outbreaks, including archived specimensfrom historical outbreaks. As a result of this collection effort,we have recovered 64 isolates of B. anthracis that are archivedas spores at the maximum security laboratory of the AnthraxReference Institute of Italy. However, the genetic compositionof B. anthracis in Italy has not been adequately studied. Analysisof the genetic similarity or differences would lead to an understandingof the disease origins and transmission patterns. In this studywe used MLVA to genotype the B. anthracis isolates from Italianoutbreaks in order to examine relationships among these outbreaksand to understand the diversity of B. anthracis isolates inItaly as it relates on a regional and a global scale.

MATERIALS AND METHODS

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Materials and Methods

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

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

MLVA PCR. For the MLVA test, we utilized fluorescently labeled primersthat 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 reservedfor molecular weight size standards (ROX). Primers amplifyingVNTRs that exhibit overlapping allele size ranges are labeledwith different fluorophores to ensure that the VNTRs can beexamined in a single electrophoretic injection. The final volume(50 µl) of reaction mixture contained 200 µM deoxynucleosidetriphosphates (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.), 50mM KCl (Perkin-Elmer), 1.5 mM MgCl₂ (Perkin-Elmer), 1.25 U ofTaq DNA polymerase gold (Perkin-Elmer), and 5.0 µl ofextracted DNA. The PCR samples were amplified in a MastercyclerPersonal (Eppendorf AG, Hamburg, Germany) as follows: 95°Cfor 10 min, followed by 30 cycles of 92°C for 30 s, annealingfor 30 s (57°C for VrrA, VrrB₁, VrrB₂, and VrrC₁; 60°Cfor VrrC₂; 55°C for CG3, pX01, and pX02), and extensionat 72°C for 30 s. To ensure that the loci were amplified,20.0 µl of each amplified sample was subjected to electrophoresisin a 2.0% agarose gel with 0.5 µg/ml of ethidium bromideat 100 V for 120 min. The amplified DNA bands were visualizedupon UV light exposure (Eagle Eye II; Stratagene, La Jolla,Calif.).

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

Data analysis. UPGMA (unweighted-pair group method using arithmetic averages)cluster analysis was performed on genotype scores from a combineddata set of the Italian isolates and a panel of 89 diverse genotypesas reported by Keim et al. in 2000 (8). To ensure comparabilityof genotype scores between the 89 diverse genotypes and Italianisolates, a subset of isolates was genotyped at both the KeimGenetics Laboratory (Northern Arizona University) and the AnthraxReference Institute of Italy (Foggia, Italy), and raw VNTR sizeswere compared and normalized.

RESULTS

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Results

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

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TABLE 1. Characteristics of Bacillus anthracis isolates in this study

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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; Table1). UPGMA cluster analysis grouped 63/64 isolates with the previouslydescribed A1.a cluster (8), with only a single isolate associatedwith the A3.b cluster (Table 1). Four of the genotypes wereexact matches to previously described genotypes, including thetwo 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 previouslydescribed genotypes (G_K); those that did not were assigned onlyItalian genotype designations (G_I). For two samples, assigninggenotypes was difficult due to missing data. One A1.a isolateis missing the pXO2 plasmid marker (G_I H), whereas the singleA3.b representative (G_I J) is missing both the pXO2 and pXO1markers. Therefore, these samples could match several previouslydefined genotypes in these groups or could represent novel genotypes.

A mutational-step model attempts to describe genetic relationshipsbased on the minimum number of mutations needed to create theobserved diversity in the Italian B. anthracis genotypes (Fig.1A). The A3.b G_I genotype J, which is multiple mutational stepsdifferent from the A1.a genotypes and which lacks data for theplasmid markers, is not included in this model. The most commongenotypes (G_I genotypes A and F, G_K genotypes 1 and 3) differby only a single mutation in the pXO1 marker locus. With theexception of G_I genotypes D and E, there is only a single mutationalstep separating the rarer genotypes from either of the two dominantgenotypes. In this study, with one exception (G_I J), the B.anthracis isolates are closely genetically related within theA1.a cluster.

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

DISCUSSION

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Discussion

Worldwide, B. anthracis exhibits a clonal population structurewith ties among geographically distant regions (8, 10). A worldwidedistribution is particularly true for the A1.a group, whichhas been highly successful. The major Italian genotypes arepart of this very fit strain complex, which has succeeded inbecoming ecologically established in many regions. This resultis in contrast to that for adjacent southern France, where B2genotypes have become established and are the most common subtype(2).

The close similarity of the B. anthracis isolates is consistentwith a single introduction of the pathogen into Italy. Onlya few postulated mutations are needed to explain most of thegenotypes observed. Of the 10 genotypes observed in this study,the vast majority of samples (53/64) belonged to genotype A(G_K 1) or F (G_K 3), which differ only by a single 3-bp mutationin the pXO1 plasmid marker (Table 1; Fig. 1). Based on thesedata, it could be supposed that the B. anthracis strains responsiblefor anthrax outbreaks in Italy are the result of evolution froma recent, common ancestral strain. These genotyping markersare highly mutable loci, and the few mutational events thatseparate the isolates could have occurred in Italy after theintroduction of this strain. A single origin would be true formost of the recent anthrax outbreaks but not for genotype J,which likely was independently introduced into Sardinia, Italy.The center of diversity for Italian B. anthracis is in Puglia,where five different genotypes are observed. Other regions,notably Sardinia, are more homogeneous. It appears that theB. anthracis populations in Puglia are older and have had timeto generate more diverse genotypes that were then dispersedto other sites. Dispersal can be associated with populationbottlenecks that eliminate diversity due to founder effectsin the new locations. Alternatively, the low sample sizes fromother regions could result in underestimation of their diversity.

This study establishes a genetic landscape for Italy that canbe used for future epidemiological and forensic analyses. Aswas shown in the U.S. anthrax letter attacks, strain identitycan differentiate between a natural outbreak and one associatedwith bioterrorism (5, 10, 12). We would expect to see genotypesA and F associated with future Italian anthrax outbreaks causedby current ecological foci. In contrast, association with anexotic B. anthracis strain would point to a new introductionvia commodities or even the deliberate release of this pathogen.Strain identity is useful in these situations only if the naturalpopulation is characterized and its strain genotype compositionknown.

ACKNOWLEDGMENTS

We thank Antonio Battisti of the Istituto Zooprofilattico Sperimentaleof Lazio and Toscana, Lucio Cirelli of the Istituto ZooprofilatticoSperimentale of Venezie, and Vincenzo Di Marco of the IstitutoZooprofilattico Sperimentale of Sicilia for the B. anthracisstrains collected in their regions.

FOOTNOTES

* Corresponding author. Mailing address: Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata—Anthrax Reference Institute of Italy, Foggia 71100, Italy. Phone: 39.0881.786330. Fax: 39.0881.786362. E-mail: a.fasanella{at}izsfg.it.

REFERENCES

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