Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018.

In Italy anthrax is an endemic disease, with a few outbreaks occurring almost every year. We surveyed 234 B. anthracis strains from animals (n = 196), humans (n = 3) and the environment (n = 35) isolated during Italian outbreaks in the years 1972-2018. Despite the considerable genetic homogeneity of B. anthracis, the strains were effectively differentiated using canonical single nucleotide polymorphisms (CanSNPs) assay and multiple-locus variable-number tandem repeat analysis (MLVA). The phylogenetic identity was determined through the characterization of 14 CanSNPs. In addition, a subsequent 31-loci MLVA assay was also used to further discriminate B. anthracis genotypes into subgroups. The analysis of 14 CanSNPs allowed for the identification of four main lineages: A.Br.011/009, A.Br.008/011 (respectively belonging to A.Br.008/009 sublineage, also known Trans-Eurasian or TEA group), A.Br.005/006 and B.Br.CNEVA. A.Br.011/009, the most common subgroup of lineage A, is the major genotype of B. anthracis in Italy. The MLVA analysis revealed the presence of 55 different genotypes in Italy. Most of the genotypes are genetically very similar, supporting the hypothesis that all strains evolved from a local common ancestral strain, except for two genotypes representing the branch A.Br.005/006 and B.Br.CNEVA. The genotyping analysis applied in this study remains a very valuable tool for studying the diversity, evolution, and molecular epidemiology of B. anthracis.

Introduction

Anthrax is a non-contagious zoonotic disease affecting a broad range of animal species including humans. Bacillus anthracis, the etiological agent of anthrax, forms highly resistant spores that can to persist in the environment for several decades [1]. Domestic and wild ruminants are species most susceptible to anthrax [2]. Animals are infected during grazing in areas contaminated with anthrax spores, while humans can contract the disease by contact with anthrax-infected animals or anthrax-contaminated animal products. Most frequently this involves employment in specific high risk occupation; such a farmer, butcher, tanner, wool carder, shearer and veterinarian. Exposure most commonly occurs during the skinning and butchering of cattle that are sick or dead because of anthrax [3]. Three forms of anthrax occur in humans, depending on exposure type: cutaneous (which is usually non-fatal), gastrointestinal, and inhalational, both of which can be potentially fatal [4]. Recently, a fourth form of the disease was reported in drug users who inject drugs contaminated with anthrax spores [5]. Further, since it is relatively easy and inexpensive to obtain B. anthracis, the bacterium is one of the preferred pathogenic agents for use as a bacteriological weapon in bio-terrorist attacks [6]. In Italy, anthrax is typically a sporadic disease, particularly occurring during the summer (with a few exceptions) in the central and southern regions, and in the major islands, where it almost exclusively affects animals at pasture [7]. Between 1972 and 2018, approximately 200 outbreaks of animal anthrax were recorded (unpublished data). Very rarely, anthrax infection takes the form of an epidemic-like disease, characterized by outbreaks in limited areas involving a great number of animals. In Italy, two major epidemic-like anthrax outbreaks have been reported: one during the summer of 2004 in Basilicata, and one during the summer of 2011, in an area between Basilicata and Campania [8, 9]. Molecular tools, such as the canonical SNPs assay (CanSNPs) and multiple-locus variable-number tandem repeat analysis (MLVA) are highly effective for differentiating B. anthracis strains. The overall goal of this study was to utilize SNP analysis to establish the phylogenetic relationship between the B. anthracis strains examined, and further discriminate them in the context of the MLVA assay, in order to examine correlations among the B. anthracis isolates associated with the Italian anthrax outbreaks and to assess genetic diversity at regional and broader scales.

Materials and methods

Ethics statement

The animal and environmental strains used in the current study were isolated at the Anthrax Reference Institute of Italy (Ce.R.N.A.), a public laboratory mandated by the Italian Ministry of Health to confirm diagnosis of all animal anthrax cases in Italy. During outbreaks, samples were collected by the veterinary services of the Ministry of Health. Specific permission for soil sampling was not required. B. anthracis DNAs from anthrax human cutaneous cases were also included in the current study; two came from the “San Carlo” Hospital, Department of Infectious Disease, Potenza, Italy, and one from the “L. Spallanzani” National Institute for Infectious Disease, Rome, Italy [10].

Bacterial strains

A collection of 234 B. anthracis strains, including 196 strains isolated from animal and 35 from the environment, isolated during Italian anthrax outbreaks in the years 1972–2018, were analyzed in the current study (Table 1). Furthermore, 3 B. anthracis DNAs from anthrax human cutaneous cases were also analyzed.

Table 1

Overview of Bacillus anthracis isolates from the years 1972–2018 analyzed in the current study.

Sample type	Source	No. of isolates	Regions
Environmental samples	Water	3	Tuscany
	Soil	32	Basilicata, Tuscany
Animal samples	Bovine	101	Basilicata, Campania, Lazio, Apulia, Sardinia, Sicily, Tuscany, Umbria, Veneto, Lombardy
	Caprine	20	Abruzzo, Basilicata, Calabria, Campania, Apulia, Sardinia, Trentino
	Deer	7	Basilicata
	Equine	12	Basilicata, Campania, Apulia
	Ovine	53	Basilicata, Campania, Lazio, Apulia, Sicily
	Swine	3	Basilicata
Human samples (DNAs)	Human	3	Basilicata, Lazio

DNA extraction

B. anthracis strains were seeded on 5% sheep blood agar plates and then incubated at +37°C for 24 h. Bacterial DNA was extracted using the DNAeasy Blood and Tissue kit (Qiagen, Hilden, Germany), following the protocol for Gram-positive bacteria. All manipulations of B. anthracis strains were performed in a biosafety level 3 laboratory at the Experimental Zooprophylactic Institute of Apulia and Basilicata Regions in a class II type A 2 biosafety cabinet.

Real-time polymerase chain reaction (PCR) assay

Molecular identification of B. anthracis was performed using qualitative real-time PCR. The method is based on the amplification of specific DNA sequences using three pairs of specific primers [11] as follows: R1/R2 primers, specific for the BA813 gene located on the B. anthracis chromosome; PAG 23/24 primers, specific for the protective antigen (PA) gene located on the virulence plasmid pXO1; and CAP 57/58 primers, specific for the capsule (CAP) gene located on the virulence plasmid pXO2. Each 20 μl reaction mixture contained 1x Sso Advanced TM SYBR® Green Supermix (BIORAD), 300 nM each forward and reverse primer, and approximately 10 ng DNA template. The amplification was performed using the CFX Connect Real Time PCR Detection System (BIORAD). A melting curve was generated at 0.5°C increments between 65°C and 95°C, and was analyzed by CFX Manager TM Software, Version 3.0 (BIORAD).

CanSNP analysis

CanSNP profiles were obtained using 13 allelic discrimination assays involving specific oligonucleotides and probes, as described by Van Ert et al. [12]. Each 10 μl reaction mixture contained 1x TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA), 250 nM probe, 600 nM each of forward and reverse primer, and approximately 10 ng DNA template. For all assays, the thermal cycling parameters used were as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Endpoint fluorescent data were acquired by using the ABI 7900HT instrument. The CanSNPs data were compared with the data for 12 recognized sublineage or subgroups. The 14th SNP was detected using a High Resolution Melting (HRM) assay for a specific A.Br.011 CanSNP [13,14]. Position 2,552,486, based on the Ames Ancestor genome (NC_007530.2), was analyzed. Amplification was performed using the CFX Connect Real Time PCR Detection System (BIORAD) and Precision Melt Supermix (BIORAD). The reaction mixture contained 0.2 μM of each primer and 1x Precision Melt Supermix (BIORAD) in a 20 μl final volume. The following cycling parameters were used: 2 min at 95°C, were followed by 35 cycles of 10 s at 95°C and 30 s at 60°C. The samples were then heated to 95°C for 30 s, cooled down to 60°C over 1 min, and then heated from 65°C to 95°C at a rate of 0.5°C/s. High-resolution melting data were analyzed using Precision Melt Analysis Software (BIORAD).

31-loci MLVA analysis

For the 31-marker MLVA, 5' fluorescently labeled oligonucleotides (6-FAM, VIC, NED and PET), specifically selected for variable number tandem repeats (VNTR) analysis were used. Twenty-seven chromosomal VNTR loci (vrrA, vrrB1, cg3, vrrB2, vntr19, vrrC1, vrrC2, vntr32, vntr12, vntr35, vntr23, bams03, bams05, bams13, bams15, bams21, bams22, bams23, bams24, bams25, bams28, bams30, bams31, bams34, bams44, bams51 and bams53) and four plasmid loci (vntr16, vntr17, pxO1 and pxO2) [12, 15–18] were analyzed. The MLVA assay involved preparation of two singleplex and nine multiplex reactions, in a final volume of 15 μl. Each reaction mixture contained the following: 1x PCR reaction buffer (Qiagen, Hilden, Germany); 3 mM MgCl2, 0.2 mM for each dNTP; 1 U Hot Star Plus Taq DNA polymerase (Qiagen, Hilden, Germany), the appropriate concentration of each primer (as described in Table 2); and approximately 10 ng DNA template.

Table 2

Primer concentration for each set of marker in PCR reactions of MLVA analysis.

PCR Reactions	Primers conc. [μM]
Singleplex 1	vrrC1 [0.2 μM]
Singleplex 2	vrrC2 [0.2 μM]
Multiplex 1	vrrA, vrrB1 [0.2 μM]; CG3 [0.4 μM]
Multiplex 2	vrrB2 [0.25 μM]; pXO1 [0.3 μM]; pXO2 [0.1 μM]
Multiplex 3	vntr12 [0.25 μM]; vntr19 [0.2 μμM]; vntr35 [0.2 μM]
Multiplex 4	vntr16 [0.25 μM]; vntr23 [0.2 μM]
Multiplex 5	vntr17 [0.1 μM]; vntr32 [0.4 μM]
Multiplex 6	bams03 [0.8 μM]; bams05 [0.2 μM]; bams15, bams44 [0.5 μM]
Multiplex 7	bams21 [0.4 μM]; bams24, bams25 [0.3 μM]; bams34 [0.2 μM]
Multiplex 8	bams13 [0.3 μM]; bams28 [0.15 μM]; bams31, bams53 [0.6 μM]
Multiplex 9	bams22, bams51 [0.3 μM]; bams23 [0.2 μM]; bams30 [0.6 μM]

The following PCR cycling program was used for the two singleplex reactions and for multiplex reactions 1 and 2: 5 min at 95°C; followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C, with a final step of 5 min at 72°C. The following amplification program was used for multiplex reactions 3: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 54°C, 45 s at 72°C, and 5 min at 72°C. The following amplification program was used for multiplex reaction 4: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 45 s at 56°C, 1 min at 72°C, and 5 min at 72°. The following amplification program was used for multiplex reaction 5: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 45 s at 59°, 1 min at 72°C, and 5 min at 72°C. The following amplification program was used for multiplex reactions 6 to 9: 5 min at 94°C, followed by 35 cycles of 1 min at 94°C, 90 s at 60°, 90 s at 72°C, and 15 min at 72°C.

Automated genotype analysis

The MLVA PCR products were diluted 1:80 and analyzed by capillary electrophoresis using the ABI Prism 3130 genetic analyzer (Applied Biosystems) and 0.25 μl GeneScan 1200, and were sized by using Gene Mapper 4.0 (Applied Biosystems Inc.). Assignment of the sizes and corresponding repeating unit numbers for each locus was performed using the following strains as references: Ames Ancestor (NCBI Reference Sequence: NC_007530.2, chromosome), pXO1 (NCBI Reference Sequence: NC_007322.2, plasmid), and pXO2 (NCBI Reference Sequence: NC_007323.2, plasmid). Data were analyzed using conventional values proposed in the updated version of the 2016 Bacillus anthracis MLVA database, available at MLVAbank (http://mlva.u-psud.fr/). A phylogram was derived by clustering with the unweighted pair group method with arithmetic means (UPGMA), using ‘categorical’ character table values. All markers were given equal weight, irrespective of the number of repeats.

The discriminatory ability of the MLVA technique was determined by calculating the discriminatory index (D) for 234 typed strains. The discriminatory power of each VNTR was estimated by the number of alleles detected and the allele diversity using BioNumerics 7.6 software (Applied Maths, Belgium) [19].

Results

Real Time PCR, CanSNPs and MLVA analysis of anthrax strains

All the analyzed strains tested positive after the PCR amplification of chromosomal, plasmid pXO1 (toxins coding) and pXO2 (capsule formation) targets. The analysis of 13 classical CanSNPs revealed that 231 analyzed strains belonged to the sublineage A.Br. 008/009, also known as Trans-Eurasian (TEA) group. The TEA group was established in southern and eastern Europe and represents the dominant subgroup in Italy, Bulgaria, Hungary and Albania [7, 12, 20–22]. The analysis of an additional 14th CanSNP (A.Br.011), recently allowed for the differentiation of the A.Br.008/009 group into 2 subgroups. Accordingly, the analysis of the 14th CanSNP in the current study revealed that 207 of the 231 strains belonged to the main sub-lineage A.Br.011/009, while 24 strains belonged to the sublineage A.Br.008/011. All but one strain belonging to the latter sublineage were isolated in Sicily; one strain was isolated in Umbria. Further, one strain isolated in Veneto belonged to the main lineage A, sublineage A.Br.005/006, while two other strains, one from Veneto and one from Trentino, belonged to the main lineage B, sublineage B.Br.CNEVA.

MLVA based on the analysis of 31 VNTRs revealed 55 different genotypes, as shown in S1 Table, distributed in the Italian regions named GT-1 to GT-55, accordingly (Fig 1). The GT-14 genotype was the most common and was represented by 34 strains, mostly from Basilicata, Apulia, and Calabria. The GT-31 genotype was represented by 19 isolates: 16 from Tuscany, two from Apulia and one from Sardinia. The GT-26 and GT-27 genotypes were only isolated in the Basilicata and Campania regions. Other genotypes were characteristic for single regions, as showed in Table 3.

Fig 1

The geographical distribution of 55 Bacillus anthracis genotypes in Italy.

Image modified from the “Map of Italy”; “World of Maps” Public Domain ().

Table 3

Distribution of Bacillus anthracis CanSNPs and genotypes isolated in Italy in the years 1972–2018.

Number of isolates	Regions	CanSNPs sublineage	Genotype
1	Apulia	A.Br. 011/009	MLVA31-1
1	Apulia	A.Br. 011/009	MLVA31-2
1	Apulia	A.Br. 011/009	MLVA31-3
3	Campania	A.Br. 011/009	MLVA31-4
1	Sardinia	A.Br. 011/009	MLVA31-5
3	Sardinia	A.Br. 011/009	MLVA31-6
2	Apulia	A.Br. 011/009	MLVA31-7
1	Umbria	A.Br. 008/011	MLVA31-8
14	Tuscany	A.Br. 011/009	MLVA31-9
3	Sicily	A.Br. 011/009	MLVA31-10
1	Tuscany	A.Br. 011/009	MLVA31-11
3	Sicily	A.Br. 011/009	MLVA31-12
1	Lombardy	A.Br. 011/009	MLVA31-13
34	Basilicata/Apulia/Calabria	A.Br. 011/009	MLVA31-14
1	Apulia	A.Br. 011/009	MLVA31-15
2	Apulia	A.Br. 011/009	MLVA31-16
1	Apulia	A.Br. 011/009	MLVA31-17
1	Basilicata	A.Br. 011/009	MLVA31-18
1	Apulia	A.Br. 011/009	MLVA31-19
1	Apulia	A.Br. 011/009	MLVA31-20
1	Apulia	A.Br. 011/009	MLVA31-21
1	Apulia	A.Br. 011/009	MLVA31-22
1	Apulia	A.Br. 011/009	MLVA31-23
57	Basilicata	A.Br. 011/009	MLVA31-24
3	Basilicata	A.Br. 011/009	MLVA31-25
3	Campania/Basilicata	A.Br. 011/009	MLVA31-26
9	Campania/Basilicata	A.Br. 011/009	MLVA31-27
5	Basilicata	A.Br. 011/009	MLVA31-28
1	Apulia	A.Br. 011/009	MLVA31-29
1	Sardinia	A.Br. 011/009	MLVA31-30
19	Tuscany/Apulia/Sardinia	A.Br. 011/009	MLVA31-31
1	Apulia	A.Br. 011/009	MLVA31-32
1	Apulia	A.Br. 011/009	MLVA31-33
5	Apulia	A.Br. 011/009	MLVA31-34
6	Apulia	A.Br. 011/009	MLVA31-35
2	Apulia	A.Br. 011/009	MLVA31-36
1	Apulia	A.Br. 011/009	MLVA31-37
1	Lazio	A.Br. 011/009	MLVA31-38
1	Lazio	A.Br. 011/009	MLVA31-39
1	Tuscany	A.Br. 011/009	MLVA31-40
1	Apulia	A.Br. 011/009	MLVA31-41
1	Apulia	A.Br. 011/009	MLVA31-42
1	Campania	A.Br. 011/009	MLVA31-43
1	Abruzzo	A.Br. 011/009	MLVA31-44
2	Lazio	A.Br. 011/009	MLVA31-45
1	Lazio	A.Br. 011/009	MLVA31-46
5	Lazio	A.Br. 011/009	MLVA31-47
3	Sicily	A.Br. 008/011	MLVA31-48
1	Sicily	A.Br. 008/011	MLVA31-49
2	Sicily	A.Br. 008/011	MLVA31-50
9	Sicily	A.Br. 008/011	MLVA31-51
7	Sicily	A.Br. 008/011	MLVA31-52
1	Sicily	A.Br. 008/011	MLVA31-53
1	Veneto	A.Br. 005/006	MLVA31-54
2	Trentino/Veneto	B.Br. CNEVA	MLVA31-55

The number of different alleles ranged from 1 for bams21 and bams25 to 10 for bams15. Highest allelic diversities measured by Shannon Diversity Index (0.40632) was observed for the locus bams15 (Table 4). The relationship among the strains based on MLVA results is represented in Fig 2.

Table 4

Shannon Diversity Index and allele numbers of MLVA markers with respect to the collection investigated.

Locus	No. alleles	Diversity Index (Shannon)
vrrA	4	0.172297
vrrB1	2	0.021373
vrrB2	3	0.073064
vrrC1	2	0.021373
vrrC2	2	0.082347
CG3	2	0.02979
pXO1aat	4	0.344872
pXO2at	4	0.118086
vntr32	3	0.033334
bams03	2	0.021373
bams05	5	0.08735
bams13	5	0.1482
bams15	10	0.40632
bams21	1	0
bams22	3	0.09788
bams23	4	0.06145
bams24	4	0.208345
bams25	1	0
bams28	2	0.23682
bams30	6	0.11232
bams31	7	0.224167
bams34	3	0.030103
bams44	2	0.147596
bams51	5	0.183046
bams53	3	0.021602
vntr12	4	0.08852
vntr16	5	0.219688
vntr17	4	0.215683
vntr19	2	0.234608
vntr23	2	0.0708
vntr35	2	0.159057

Fig 2

A UPGMA phylogram of MLVA profiles.

The phylogram was built using BioNumerics 7.6 software (Applied Maths, Belgium). The visualization and the annotation of the genetic distances were performed using the web-based tool Interactive Tree of Life (iTOL). Circling the phylogram from the external to internal region are: genotype number, sublineage, species, year, regions (differently colored) of isolation and identification number of each analyzed strain.

Discussion

Bacillus anthracis is clonal in nature and often exhibits a high degree of genetic homogeneity due to the fact that is has a single stranded chromosome and reproduces asexually. This characteristic has traditionally made the discrimination of isolates for epidemiological purposes difficult. Furthermore the high survivability of spores in the soils, allowed B. anthracis to reproduce a relatively limited number of times during its evolution [23]. The 31-loci MLVA analysis carried out on 234 B. anthracis strains, isolated in Italy during the years 1972–2018, revealed the circulation of 55 B. anthracis genotypes. The performed CanSNPs analysis placed 53 of the 55 identified genotypes in a common cluster (TEA). The analysis of the classical 13 CanSNPs revealed that most of the analyzed strains (98%) belonged to the sublineage A.Br.008/009 (the TEA group), which is the most common group in Europe and Asia [15]. However, except for the genotypes of strains isolated in Umbria and some others isolated in Sicily belonging to sublineage A.Br.008/011, all strains belonged to the sublineage A.Br.011/009. Interestingly, genotype GT-54 isolated in Veneto was very different from the other characteristic Italian strains. CanSNPs analysis confirmed this observation placing this genotype in the branch A.Br.005/006. This branch is generally present in the central-southern Africa, although it was also identified in Europe [12, 24].Furthermore, genotype GT-55; B.Br.CNEVA, isolated in Veneto and Trentino is highly differentiated from most other Italian strains examined here. This genotype is widespread in Europe and found in France, Switzerland and Germany [12, 25, 26]. In Italy, the population of B. anthracis is mainly divided into two sublineages: A.Br.011/009, definitely the most common and A.Br.008/011 present only in Umbria and Sicily. Both these sublineages belong to the large TEA group (Fig 2). The TEA group A.Br.008/009 contains a B. anthracis subpopulation that is well adapted to the northern hemisphere and predominant in Europe, Russia, Kazakhstan, Caucasus and western China [12, 27]. It has also been detected in Africa [18, 28]. This group is thought to have given rise to the western north American sublineage (A.Br.WNA), which is dominant in central Canada and much of the western USA. The presence of strains belonging to sublineages A.Br.008/011 and A.Br.011/009 might represent an effect of evolution on a common ancestral strain at the territorial level. In particular, A.Br.008/011 represents a rare and deep branching sublineage, also observed in Bulgaria, France and Turkey [29]. The spread of the TEA group to Europe and Asia is postulated to be linked to animal handling along the ancient East-West commercial routes of the Silk Road [30]. In the current study, strains belonging to the B.Br.CNEVA lineage were isolated in a relatively small area of north-eastern Italy. The relatively low diversity between the two strains demonstrated in the current study is consistent with a single introduction event of the B.Br.CNEVA lineage into the country, followed by ecological establishment and progressive in situ differentiation around the Italian Alps area [21]. Consistent with this hypothesis, the Italian strains form a cluster that is distinct from the other European B.Br.CNEVA strains. Identification of one A.Br.005/006 strain in Italy could be associated with the trade exchanges dating back when city states competed for trade and commerce throughout the Mediterranean [7]. This subgroup is well represented in Africa, but rare in Europe [12]. It is therefore quite surprising that past importations of ill or dying animals or spore-infected items from Africa, the Middle East, or even Asia, did not have a greater impact on the genetic structure of B. anthracis in the region. The higher variety of B. anthracis genotypes identified in southern Italy relative to genotypes from other Italian regions may be explained by the differences in the breeding systems between northern and southern Italy. In southern Italy, many livestock farmers use extensive farming methods, which increases the chances of grazer exposure to historical spore sites and deposits. The possibility of exposure is lower in northern Italy because most livestock farmers use intensive breeding systems. Another observation from the current study was that the neighboring regions share just a few genotypes. In particular, the GT-24 genotype was present in Apulia, Basilicata and Calabria; the GT-26 and GT-27 genotypes were identified in Basilicata and Campania; and the GT-55 genotype was identified in Veneto and Trentino. Noteworthy and difficult to explain is the dislocation of genotype GT-31, identified in Apulia, Tuscany and Sardinia. These are not neighboring regions; on the contrary, they are quite far from one another. Also in this national scenario one of the explanations could be the trade of animals or animal products within the country over the years. Nevertheless, since most genotypes are exclusive to each region, it appears that Italian B. anthracis strains may be autochthonous for a single territory. Interestingly, genotypes exclusive to specific regions were detected especially in Sicily and Sardinia, probably because of low animal movements between these islands and the rest of Italy. The analysis of chromosomal and plasmid hypervariable regions using such methods as MLVA constitutes a valuable approach for studying the diversity, evolution and molecular epidemiology of B. anthracis. Therefore, MLVA is a valid method that enables the understanding of the distribution of B. anthracis within a country.

Allele distribution of the 55 genotypes identified using 31 VNTR analysis.

(XLSX)

Click here for additional data file.

21 Sep 2019

PONE-D-19-23489

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for an interesting paper. The description of the genotypic distribution of B. anthracis isolates across Italy adds to the body of work which highlights the diversity of this pathogen internationally.

That said, please be careful of your repetition of the word "results" in regards to canSNP lineages. I have made some editorial suggestions in comment boxes. Also, please check your references, especially with regards to the PCR's used. You referred to real time PCR and then referenced Fasanella et al. which describes conventional PCR. You must credit the designers of the primers used in your methodology references. You did not refer to Patra and Ramisse for the pag, lef, cap and BA813 primers. You also did not list which primers exactly were used. If indeed you did use qPCR in diagnostics this study, please state the chemistry employed; if not...adjust the method description accordingly.

Would it be possible to add more detail surrounding the history of the isolates? Your references were only in relation to the epizootics involving tabanids/horseflies. More context would help flesh out your discussion and provide more evidence for solid conclusions. As it stands, you cannot conclude a common ancestor of strains using a limited panel of canSNP's and MLVA. If there is whole genome data to support this, you should refer to it. Your speculation around the introduction of the A.Br.005/006 also needs stronger evidence before you can call it a viable hypothesis. Otherwise, just call it speculation.

Reviewer #2: The manuscript is sound, although they need to drop the term phylogenetic tree from their UPGMA analysis. Not much analysis in this paper overall, mostly descriptive in scope.

Really needs some serious grammatical work.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PONE-D-19-23489_reviewer (2).pdf

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Submitted filename: PLOS_review_SB_Sep_18.docx

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7 Nov 2019

Dear Editorial Office,

we made and submitted the corrections required by the Journal and by the reviewers. The manuscript was revised by a professional English proofreading service as the revievers asked.

Thank you for the precious suggestions and observations.

We submitted the revised manuscript following PLOS ONE's style requirements and we replaced the overlapping text with previous publications as you asked.

The human samples we used in this study are just 3 DNAs coming from people affected by cutaneous anthrax for genotyping analyses. We received them in anonymous form. We described the origin in the manuscript.

Reviewer 1: We answered the questions directly in the pdf file he sent and in a separate word file.

However we changed the reference he asked and we better described the primers used in the manuscript. We disecribed the Real Time PCR we used with Sybr Green technology.

Most of the isolates we tested are all the isolates we collected all over the years during the Italian anthrax outbreaks in animals. The environmental samples were taken from soils and water following anthrax outbreaks. The references you are referring to are related to the description of the epidemic-like anthrax outbreaks occurred in Italy in Basilicata and Campania. They were not inserted for a description of all the samples we analyzed.

As suggested we reworded the sentence with this hypothesis and deleted part of It.

Reviewer 2: The term "phylogenetic tree" was replaced by the term "phylogram" and diversity index analysis was later performed in the manuscript.

The manuscript was revised by a professional English proofreading service for grammar.

Submitted filename: PONE-D-19-23489_reviewer (2) reply.pdf

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4 Dec 2019

PONE-D-19-23489R1

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018.

PLOS ONE

Dear Dr. Galante,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewers note edits and minor corrections still necessary, but otherwise, a much improved paper. Please address these final comments.

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We look forward to receiving your revised manuscript.

Kind regards,

Wendy C. Turner

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The document still requires a bit of editorial correction, although vastly improved from the previous round.

I still have issue with some of the conclusions made in the discussion portion of the paper. While it is true that the trade history of Italy has been extensive over the centuries, conclusions regarding the genotypes of this study (limited to 1972-2018) cannot be compared to the middle ages unless you have solid data to support it. By the logic of your hypothesis, all isolates can be linked to trade with southern Africa. The time scales with which such genotypic distribution could have occured cannot be determined using MLVA (which inherently includes homoplasy/homoplasticity) and a limited panel of SNPs. Only whole genome sequencing and comparative genomics which includes diverse lineages can provide enough support for such a narrative. There is enough data to recommend this paper without the inclusion of such wild conclusions on the origins of these isolates.

As to the inclusion of the Simpson's index: In my exprerience, the Shanon diversity better represents allelic diversity in VNTR loci. It will also make your work comparable to other papers.

It reads much better overall and the methods are also much improved.

Reviewer #2: Overall this MS has improved greatly from the previous version, although there is still extensive editing to be done in terms of grammar. I have tried to address all of these issues below but have likely still missed some. I would suggest the authors use the free program/web app Grammarly to specifically address the issue of the overuse of commas throughout. With the incorporation of the edits below I think this manuscript would be suitable for publication in PLOS One.

Line 51: remove s at the end of “occupations”

Line 53: add s at the end of “human”.

Line 55: change “a fourth disease form” to “a fourth form of the disease”

Line 57: change “for use as bacteriological” to “for use as a bacteriological”

Line 66: remove comma

Line 75: remove comma after “laboratory”

Line 130: Remove comma after “analysis” and replace “we used” with “were used”.

Line 139: Table 2: Make sure all rows are equally spaced (rows 6 through 9 are spaced farther apart then the rest)

Lines 157-158: The authors should state what the reference numbers refer to (ncbi or ena, and what specific database)

Line 159: Change “Phylogram”, to “A phylogram”.

Line 171: Why does sentence end mid-page?

Line 172: Remove comma

Line 173: Remove “or”

Line 174: Remove comma after Europe

Line 175: Remove both commas surrounding “14th”

Line 176: change “to” to “the”

Line 183: remove comma after “VNTRs”

Line 184: remove comma after “regions”

Line 198: Table 4: Change “Simpson’s Index of Diversity” to “Simpson's Diversity Index” and throughout.

Line 202: Remove extreme spacing between words

Line 207: Change “Around the phylogram are shown, from the external part to the internal part” to “Circling the phylogram from the external to internal region are”

Line 214: replace comma with “and”

Line 215: change “purpose” to “purposes”

Line 216-217: I’m not sure this sentence is true, and it is also grammatically incorrect. Does the high survivability of spores lead to genetic homogeneity? I don’t think so, I would argue that genetic homogeneity in this species is due to the fact that it has a single stranded chromosome and reproduces asexually. Persistence of spores in the environment complicate this but is not the defining feature that dictates homogeneity.

Line 217-219: This sentence should be reworded to reduce the number of commas and read more coherently.

Line 221: Remove comma

Line 225: Remove comma after “GT-54” and “Veneto”

Line 226: Remove comma after “observation”

Line 228: Remove comma after “GT-55”

Line 229: Remove comma after “Trentino”

Line 228-229: This sentence is confusing because your allocating a genotype to a genotype. I would reword this to something like: “Furthermore, genotype GT-55; B.Br.CNEVA, isolated in Veneto and Trentino is highly differentiated from most other Italian strains examined here.

Line 230: change “branch” to “genotype”, remove “mainly” and change “in particular” to “and found in”

Line 236: Change “this group gave rise” to “this group is thought to have given rise” and cite the paper that originally presented this hypothesis.

Line 238: Change “an effect of evolution of a common ancestral strain at territorial level” to “an effect of evolution on a common ancestral strain at the territorial level”

Line 240, change “as well as” to “and”

Line 241: change “seems” to “is postulated”

Line 247: add the word “strains” after B.Br.CNEVA

Line 248: remove the word “the”

Line 254: remove comma after “regions” and change “can” to “may”

Line 256: change “increasing the chances” to “which increases the chances”

Line 257: Change “this” to “the”

Line 263: Change “This” to “these”

Line 264: change space in front of period

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Spencer A Bruce

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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Submitted filename: PONE-D-19-23489_Reviewer1_02122019.pdf

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23 Dec 2019

Dear Editorial Office,

we made and submitted the corrections required by the second revision of the Journal and of the reviewers.

The corrections are reported in the attached files "response to reviewers" and "revised manuscript with track changes".

Thank you for the further precious suggestions and observations.

Submitted filename: RESPONSE TO REVIEWERS.docx

Click here for additional data file.

2 Jan 2020

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018.

PONE-D-19-23489R2

Dear Dr. Galante,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Wendy C. Turner

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

6 Jan 2020

PONE-D-19-23489R2

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018.

Dear Dr. Galante:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Wendy C. Turner

Academic Editor

PLOS ONE