Introduction and persistence of tularemia in Bulgaria

Figures & data

Fig. 1 The old and new tularemia foci in Bulgaria. A. The genetic distance between strains L2 and 81 (orange dots) comprised five SNPs in 33 years. B. The genetic distance between strain 94 from USSR and strain 19c from Srebarna (yellow dots) comprised two SNPs in 5 years. C. There was an accumulation of >20 SNPs between the Srebarna 19 from the old focus and L1, MN, and MMM in the new focus (red dots). D. The first Bulgarian genome in the B.4 basal clade was represented by strain B1 isolated in Breznik in 1999 (black dot).

Table 1 Francisella tularensis strains investigated in the study

Original ID	Collection ID	Whole genome	Source	Year of isolation	Place of isolation	Fig. 3 Basal clade (subclade)
L1^a,b	FDC179	Yes	Hare	1998	Slivnitsa	B.12 (B.77)
Dm1^a	FDC180	Yes	Tick, Dermacentor marginatus	1998	Slivnitsa	B.12 (B.34/35)^c
Dm2^a	FDC181	Yes	Tick, D. marginatus	1998	Slivnitsa	B.12 (B.34/35)^c
B1^a	FDC182	Yes	Tick, Ixodes ricinus, collected from goat	1999	Breznik	B.4 (B.78)
MMM^a	FDC183	Yes	Common vole	2003	Meshtitsa	B.12 (B.77)
MN^a	FDC184	Yes	Human	2003	Meshtitsa	B.12 (B.77)
L2^a,b	FDC185	Yes	Hare	2005	Prosena	B.12 (B.34/35)^c
Srebarna 19^a	FDC186	Yes	Muskrat	1962	Srebarna	B.12 (B.77)
Srebarna 5^a		No		1962	Srebarna	B.12 (B.23)^d
Srebarna 56^a		No		1962	Srebarna	B.12 (B.23)^d
19c^e	FSC930	Yes	Muskrat	1961	Srebarna^f	B.12 (B.76)
81^e	FSC937	Yes	Water	1962	Srebarna^f	B.12 (B.34/B.35)
94 or N94	FSC931	Yes	Water rat	1956	USSR	B.12 (B.76)

^aThe strains are from the strain collection of the Military Medical Academy (MMA), Sofia, Bulgaria. The strains Srebarna 5, Srebarna 19, and Srebarna 56 were recovered by an MMA team (Gotev/Kupenov) and originate from the Bulgarian Type Culture Collection (BTCC) in Sofia; ^bNote that the strain L1 is not identical to a strain also named L1 in the reference (16); ^cB.34/35 should be read as that the strain showed the derived SNP state for B.34 and the ancestral state for B.35; ^dNo further subtyping with B.77 was performed on these two strains due to insufficient amount of DNA (Fig. 3); ^eThe strains 19c, 81, and 94 are part of a strain collection maintained at the Military Institute of Hygiene and Epidemiology (MIHE) in Warsaw, Poland, and originate from a strain collection at the former Institute of Marine Medicine in Gdansk, Poland (21). Strains 19c and 94 originated from the Medical Academy, Moscow, USSR and strain 81 originated from Centre of Epidemiology and Microbiology in Prague, former Czechoslovakia; ^fThe original documentation reads Bulgaria (21). No other outbreaks than the Srebarna outbreak nor other areas of strains isolation are described in the literature, institutional records, or known by personal communication to our knowledge (Marinov, personal communication).

Table 2 Genome data for 10 Bulgarian strains and one archive strain sequenced in this study

Original ID	Total number of bases (>1,000 bp)	Total number of contigs (>1,000 bp)	N50^a	Number of reads/pairs	Coverage	Sample accession^b	Sample provider^c
L1	1,886,108	95	28469	26,517,196	2812	SAMN03773843	MMA
Dm1	1,883,038	91	28802	25,066,628	2662	SAMN03773844	MMA
Dm2	1,881,120	93	28426	8,579,883	912	SAMN03773845	MMA
B1	1,897,672	93	28339	22,520,234	2373	SAMN03773846	MMA
MMM	1,875,643	95	28022	8,821,904	941	SAMN03773847	MMA
MN	1,884,503	94	28467	34,247,320	3635	SAMN03773848	MMA
L2	1,889,172	93	28557	28,616,993	3030	SAMN03773849	MMA
Srebarna 19	1,881,962	94	28470	6,532,837	694	SAMN03773850	MMA
19c	1,847,256	97	28076	18,541,564	2007	SAMN03774233	MIHE
81	1,832,473	93	29527	24,573,197	2682	SAMN03774238	MIHE
94 or N94	1,818,365	97	27934	7,428,933	817	SAMN03774234	MIHE

^aN50 is the length of the smallest contig in the set that contains the fewest (largest) contigs whose combined length represents at least 50% of the assembly; ^bNCBI BioProject PRJNA285143; ^cMMA=Military Medical Academy, Sofia, Bulgaria; MIHE=Military Institute of Hygiene and Epidemiology, Pulawy, Poland.

Table 3 Four new assays B.76−B.79 for Bulgarian clades. Existing assays using modified primer sequences from previous publications

canSNP	SNP state	Position SCHU S4^a	Primer^b	Primer sequence 5’–3’
First order of discrimination
B.16^c	T	608,245	D	gcgggcagggcggcATGCTAGCAAATTACCATCAAAAcT
	G		A	ATGCTAGCAAATTACCATCAAAAtG
			C	AACTCTTCTCGCCATCAACTTCTAT
B.4^d	T	823,672	D	cggggcggggcggggGACTTTTGGGGTTTTTGCTGgT
	A		A	GACTTTTGGGGTTTTTGCTGgA
			C	GCGATCACCAAAACTACCYATAACCACACTG
B.6^d	A	713,647	D	ggggcggggcggggTGTTATTGAATGTAAGGAAAATGATTCcA
	G		A	tttttttttttttttttttTGTTATTGAATGTAAGGAAAATGATTCaG
			C	GAAATAGTAAGTCCAAACGCATGAAATA
B.19^c	A	1,373,999	D	gcgggcTTGCTACTGATGGTTTAAgTA
	C		A	gcgggcagggcggcTTGCTACTGATGGTTTAAaTC
			C	CAATACGTCACTTATGCAGTGAT
Second order of discrimination
B.10^d	G	387,537	D	cggggcggggcggggGCCATCACTAGTAAATACCACATTAAaC
	A		A	TGCCATCACTAGTAAATACCACATTAAgT
			C	TTGTAATATTAGCTMGAAAAGTAGATGAA
B.11^d	A	1,282,029	D	cggggcggggcggggTGTGATCAAGGGAAATGCTCAgA
	G		A	TGTGATCAAGGGAAATGCTCAtG
			C	TATTCTGTTATATTGTTGCGCATAGGCATAC
B.20^c	G	1,396,082	D	gcgggcagggcggcTCTGATGAAGAATATCTTACtG
	A	1,789,417	A	gcgggcTCTGATGAAGAATATCTTACcA
			C	ATTATGGCAAAACTATACCTT
B.23^c	T	253,120	D	gcgggcTTACTACAAATTCGCCTCTgAT
	G		A	gcgggcagggcggcTTACTACAAATTCGCCTCTtAG
			C	AGCAAAAGAGCTTACTAAACAATTTGA
Third order of discrimination
B.34^e	A	766,614	D	ggggcggggcggggcTAGCGAGCATTATTTGCTGGgTT
	G		A	GTAGCGAGCATTATTTGCTGGtTC
			C	ATAAAACTATAAATTTACATAAAATGAAAACTTCTC
B.76	T	1,509,966	D	ccccgccccgccccgATATCCGAGCATATTCCTAGaT
	C		A	ATATCCGAGCATATTCCTAGtC
			C	ATGGAGGTAGAGAGTATCTTATAG
B.77	A	1,802,833	D	cggggcggggcggggTGTTGAAAAGCCAAAAGTTcT
	G		A	ATGTTGAAAAGCCAAAAGTTtC
			C	GTAAATGCTGCTGCCATC
B.78	C	1,634,094	D^f	cggggcggggcggggcAGTGTGCGTGGTTTCTTgCG
	T		A	GGAAGTGTGCGTGGTTTCTTtCA
			C	AAAGGTACAGGGAATAATACATCAT
B.79	A	1,661,376	D	cggggcggggcggggACCTGGGATTCTTGGcGT
	G		A	gcgggcATACCTGGGATTCTTGGtGC
			C	ggggcggggGAATGGCTGCATATAATATGATAT

^aSCHU S4 has GenBank accession no. AJ749949.2; ^bD=Derived SNP state primer, A=Ancestral SNP state primer, C=Common primer; ^cSvensson et al. (6). A real-time PCR array for hierarchical identification of Francisella isolates. B.19 was used for identifying branch B.12 (Fig. 2 and Fig. 3); ^dVogler et al. (5). Phylogeography of Francisella tularensis: Global expansion of a highly fit clone; ^eGyuranecz et al. (20) Phylogeography of Francisella tularensis subsp. holarctica, Europe; ^fPrimer generates a product with double melt peaks.

Fig. 2 Phylogeny. Whole genome phylogeny of F. tularensis subsp. holarctica based on SNPs in an alignment of nine published genomes (strain names are indicated) showing the placement of the Bulgarian strains in a global phylogenetic framework. Bulgarian outbreak strains and the non-outbreak strain 94 sequenced in this study are indicated in bold text. B.4, B.6, B.12, and B.16 indicate the four major canSNP clades of the subspecies. B.20 and B.23 indicate two B.12 subclades that included Bulgarian strains The tree was mid-point rooted in FSC022. The total length of the root branch was 3,317 base differences.

Fig. 3 The new Bulgarian genomes placed in the phylogenetic framework of canSNPs. Four assays B.76 − B.79 were developed in this study to target subclades, where strains from Bulgaria were members. B.4, B.12, B.23, B.24, B.34, and B.35 have been previously published (5, 6) (20). Alternative canSNPs (B.17, B.19, and B.42) are indicated in gray below the first published canSNP in indicated branch (6, 19). s=the numbers of canSNPs on the indicated branch.

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