Molecular Genotyping of Bacillus anthracis Strains from Georgia and Northeastern Part of Turkey

Research Article

J Bacteriol Mycol. 2017; 4(3): 1053.

Molecular Genotyping of Bacillus anthracis Strains from Georgia and Northeastern Part of Turkey

Khmaladze E¹, Su W², Zghenti E¹, Buyuk F³, Sahin M³, Nicolich MP2,4, Baillie L5, Obiso R6 and Kotorashvili A¹*

¹Lugar Center for Public Health Research at the National Center for Disease Control, Tbilisi, Georgia

²Walter Reed Army Institute of Research, USA

³Kafkas University, Turkey

4US Army Medical Research Unit, Georgia

5Cardiff University, Cardiff, Wales, UK

6Avila Scientific, USA

*Corresponding author: Adam Kotorashvili, Lugar Center for Public Health Research at the National Center for Disease Control, Tbilisi, Georgia

Received: July 28, 2017; Accepted: August 30, 2017; Published: September 06, 2017


Bacillus anthracis is the causal agent of anthrax and has a history of use as a biological weapon. Anthrax cases occur worldwide and the disease is endemic in certain regions. Here we describe a study of the genetic diversity of B. anthracis strains in two endemic areas: The country of Georgia and the Kars region of Turkey. Thirty Turkish isolates and thirty Georgian isolates were subjected to Single Nucleotide Polymorphism (SNP) sub typing, followed by higher-resolution genotyping using 25-loci variable-number tandem repeat analysis (MLVA-25). Canonical SNP typing indicated that Turkish strains belonged to both the A.Br.003 linage and the Australian 94 lineage. In light of a recent analysis that placed the majority of Georgian B. anthracis isolates in one phylogenetic group, we screened the Turkish strains using a previously developed Georgian SNP panel. Minimal diversity was observed among the Kars strains within the Georgian SNP lineage: all 30 of these strains grouped with A.Br.026, ten strains were derived from A.Br.028, and only two isolates belonged to A.Br.029. According to the results of MLVA-25 genotyping, all 30 Turkish strains belong to two clusters. Cluster A is more diverse than cluster B. Our results suggest that B. anthracis strains originating from Georgia and the northeastern part of Turkey are genetically interrelated, which could be explained by the geographic proximity of the countries.

Keywords: B. anthracis; Especially Dangerous pathogens; MLVA and SNP genotyping


The etiologic agent of anthrax, Bacillus anthracis, is a monomorphic member of a highly diverse group of endosporeforming bacteria. There are at least 51 known Bacillus species and many more of uncertain taxonomic status [1]. B. anthracis spores are typically found in soil and may be spread through contaminated dust, water, and plant and animal materials. The toxins produced by vegetative B. anthracis dictate its virulence and differ from the toxins produced by other Bacillus species.

Although anthrax is primarily a disease of herbivores, humans may contract anthrax directly or indirectly from animals [2]. The most common form of human anthrax, cutaneous anthrax, accounts for 95 to 99% of human cases worldwide and usually results from handling contaminated animal products. Infection occurs through a break in the skin and results in lesions on exposed regions of the body. After an incubation period of 2 to 3 days, a small papule appears, vesicles develop in a ring around the papule, and the papule subsequently ulcerates, dries, and blackens to form a distinctiveeschar. Less than 20% of untreated cases of cutaneous anthrax are fatal. In fatal cases, generalized symptoms may be mild (e.g., malaise and a slight fever) or absent before the sudden onset of acute illness, which is characterized by dyspnea, cyanosis, severe pyrexia, and disorientation followed by circulatory failure, shock, coma, and death in quick succession [3]. Concomitant with the severe signs of illness, the number of B. anthracis in the blood increases rapidly and reaches a maximum concentration during the last few hours of life.

Two other forms of human anthrax have been described. Gastrointestinal anthrax is caused by the consumption of contaminated animal products, and pulmonary anthrax occurs when B. anthracis spores are inhaled. Although rare, these forms of anthrax are much more severe than cutaneous anthrax because they are more likely to result in the rapid dissemination of bacteria to regional lymph nodes and the development of fatal septicemia.

Anthrax vaccines are available for animals and humans, but the disease remains endemic in many countries, particularly those without effective vaccination policies. Bacillus anthracis is extremely difficult to eradicate from endemic areas because its spores remain viable in soil for many years, and because bacterial persistence is not dependent on animal reservoirs [4].

In Georgia, anthrax is classified as endemic and has persisted for centuries [5]. During 2000 – 2012, there were 592 reported cases of human cutaneous anthrax in Georgia. 299 cases (51%) were classified as rural, 103 (17%) were peri-urban and 190 (32%) were urban [6]. Recent evidence suggests an increase in the incidence rate of infection in Georgia and in neighboring countries including thehyperendemic regions in Turkey [7].

Although rare in large parts of the world, B. anthracis infection presents a significant medical problem in the Kars region of Turkey, where human infection occurs amongst local farmers who live near their animals. From 1995 to 2005 there were 2,415 human cases of anthrax in Turkey of which 19.7% occurred in the area around Kars [8].

There have been relatively few studies to characterize the strains of B.anthracis circulating Turkey [9,10], but an in-depth understanding of Turkish B. anthracis population is necessary to effectively identify strains and trace them to their origin. In addition, a more complete understanding of antigenic differences among Turkish strains could contribute to improved vaccine intervention strategies to curtail natural or weaponized B. anthracis outbreaks.

Studies carried out in Turkey and Georgia have sought to clarify genetic relationships among B. anthracis strains circulating in the region. In 2006, Merabishvili et al. used eight-loci variable-number tandem repeat analysis (MLVA-8) to determine the subtypes of 18 Georgian field-isolated and five B. anthracis vaccine strains (former Soviet Union (FSU) vaccines administered to livestock throughout the FSU). They found that these strains fell within the A.3.a subgroup (previously defined by Keim et al.) in two genotype clades shared with regional Turkish isolates [5,11]. Similarly, Durmaz et al. studied 251 B.anthracis strains isolated from human, animal, and environmental samples collected throughout Turkey and found a total of 12 distinct MLVA-25 A.3.a subtypes [11]. Ortatatli et al. examined the genetic diversity of 55 B. anthracis isolates from 16 distinct regions of Turkey [12] and identified three geographically related subgroups circulating in three distinct regions; genotype dispersal patterns were indicative of trans-boundary contamination from livestock. Khmaladze et al. screened multiple Georgian strains using 26 canonical single nucleotide polymorphism (can SNPs) assays, which placed these strains into eight newly identified groups within the A.Br.013/015 lineage [15]. Canonical SNP analysis is a phylogenetic approach used to identify SNPs that efficiently partition bacterial strains in genetic groups consistent with their recognized population structure.

Here we describe the use of can SNP analysis and MLVA to determine the subtypes of B. anthracis strains from Georgia and northeastern Turkey. Comparative analysis was conducted to get insight into the regional phylogenetic placement of the Georgian and Turkish strains, provide new insight on the evolutionary history, regional settlement and differentiation of B. anthracis strains of Caucasus region.

Materials and Methods

Bacillus anthracis strain culture and inactivation

In total, 60 B. anthracis samples were studied: 30 samples were provided by Kafkas University in Kars, Turkey and 30 samples were provided by the National Center for Disease Control and Public Health in Tbilisi, Georgia. Bacillus anthracis isolates from pure cultures grown on 5% Sheep Blood Agar (SBA) plates (Eliava Media Production, Georgia) were incubated at 37°C for 24hours. Several loops of culture were transferred to 1.5-mL micro centrifuge tubes and heat-inactivated in an autoclave at 121°C for 20 minutes [13].

DNA isolation and sterility testing

Sterile genomic DNA was extracted using QIAamp DNA Mini Kits (Qiagen, USA) according to the manufacturer’s instructions. Purified DNA was divided into 100μL aliquots and stored at -20°C pending analysis. DNA concentrations were measured using a NanoDrop 2000 (Thermo Fisher Scientific, USA).

We determined sample sterility by pipetting 5% of the final volume of the DNA and incubating at 37°C in the same growth media used in bacterial culturing. To confirm sterility, at day +3 and day +7, 5μL of isolated DNA was placed on 5% SBA and incubated at 37°C. If no growth was observed after 72hours at either time point, then the preparation was considered sterile. Primary and secondary containers were decontaminated with 1% sodium hypochlorite for 30 minutes, and stored at -20°C. After surface decontamination, sterile samples could be handled under biosafety level-1 containment.

SNP analysis

To conduct can SNP analysis of B. anthracis strains, a specific SNP melt analysis of mismatch amplification mutation assay (melt- MAMA) was used [14]. Primer concentrations were adjusted to 100pmol/μL in Tris-EDTA buffer. Each primer was diluted to 10pmol/μL with distilled water to create a working stock.

Synthetic, allele-specific, positive control templates were created by conventional PCR. Primer mixes contained 10pmol/μL of ancestral allele primer (SA), derived-allele primer (SD) and reverse primer (SC). Each 40-μL, single-primer-set PCR reaction contained 1μL of primer mix; 36μL Platinum PCR SuperMix and 2.0μL genomic DNA (> 5ng/ μL). Conventional PCR products were verified by electrophoresis on 2% agarose gel in 1X Tris-acetate-EDTA buffer at 10V/cm for two hours. Original PCR products amplified using SD-SC and SASC primers were diluted 10,000X for use as synthetic allele-specific positive control templates for determination of melting temperature (Tm) for both SD and SA with SC. Real-Time PCR amplification followed with melt analysis was then performed using genomic DNA and the primer mixes on CFX 96 Real-Time PCR detection system (Bio-Rad). Each 10-μL PCR reaction contained of 1μL primer mix for SD, SA, and SC (10pmol/μL); 5μL of 2 X SYBR Green master mixes; 1μL of genomic DNA (10ng/μL); and 3μLddH2O. The Tm from each sample was compared to the appropriate Tm reference table to determine the SNP base call.

MLVA-25 analysis

Forward and reverse primers were combined and diluted to 5μM or 10μM to create working primer stocks. The DNA was amplified in four multiplex PCR reactions (multiplex A, B, C and D). The PCR master mix was prepared with 7.55μL molecular-grade water (ddH2O), 1.5μL 10X PCR buffer, 2.25μL primers in total, 1.5μL 50mMMgCl2, 1μL dNTPs (2.5mM each), and 0.2μL Platinum Taq DNA Polymerase.

Each PCR contained 14μL of master mix and 1μL of sample DNA; 1μL of ddH2O served as the negative control. After heat-denaturing the DNA for 5minutes at 95°C, PCR reactions were performed with the following cycling conditions: 38 cycles of 30 sec at 95°C, 30 sec at 55°C and 1 min at 72°C. 7min at 72°C, final extension 5min at 72°C and 4°C hold.

After amplification, 2μL of each PCR reaction was diluted 100- fold in 198μL of ddH2O. A denaturation solution/sizing standard solution was prepared from 18.7μL of HiDi Formamide and 0.3μL of 1200LIZ size standard; 19μL of the resulting solution and 1μL of the diluted multiplex samples were added to the wells of an ABI platform-compatible plate, e.g., MicroAmp Optical 96-well Reaction Plate (life technologies). Samples were denatured in a GeneAmp PCR System 9700 (Applied Biosystems) for five minutes at 95°C and then placed on ice for three to five minutes. Reactions were run on an ABI 3130xl instrument (Thermo Fisher Scientific) and fragment analysis was performed with GeneScan and GeneMapper software packages (Applied Biosystems). GeneMapper software was used to analyze electropherograms and score VNTR sizes.


Canonical SNP typing of B. anthracis strains from Turkey and Georgia revealed that Turkish strains belonged to B. anthracis group A.Br.003 and the Australian 94 lineages. The lineages of the Georgian isolates used in the study are shown Table 1. The lineages of Turkish isolates used in the study are shown in Table 2. The SNPs are defined by their positions in the B. anthracis genome as shown in these two tables. Given the results of our recent study, which indicated that the majority of Georgian B. anthracis isolates belong to the same phylogenetic group, the Turkish strains were screened against the Georgian SNP panels as described by Khmaladze et al. [15]. Some diversity was observed among the Turkish strains within the Georgian SNP lineage: all 30 of these strains grouped with A.Br.026, ten strains were derived from A.Br.028, and only two isolates belonged to A.Br.029. Figure 1 is a dendrogram depicting the results of MLVA- 25 analysis of B. anthracis specimens from Kars region of Turkey. Figure 2 is a comparison of MLVA-25 data for B. anthracis specimens derived from Georgia and Kars region of Turkey. According to the results of MLVA-25 genotyping, all 30 Turkish strains belong to two clusters. Cluster A is more diverse than cluster B, which could be explained by the sample size and location.