Phylogenetic and comparative genomics of the family Leptotrichiaceae and introduction of a novel fingerprinting MLVA for Streptobacillus moniliformis.

BACKGROUND: The Leptotrichiaceae are a family of fairly unnoticed bacteria containing both microbiota on mucous membranes as well as significant pathogens such as Streptobacillus moniliformis, the causative organism of streptobacillary rat bite fever. Comprehensive genomic studies in members of this family have so far not been carried out. We aimed to analyze 47 genomes from 20 different member species to illuminate phylogenetic aspects, as well as genomic and discriminatory properties.
RESULTS: Our data provide a novel and reliable basis of support for previously established phylogeny from this group and give a deeper insight into characteristics of genome structure and gene functions. Full genome analyses revealed that most S. moniliformis strains under study form a heterogeneous population without any significant clustering. Analysis of infra-species variability for this highly pathogenic rat bite fever organism led to the detection of three specific variable number tandem analysis loci with high discriminatory power.
CONCLUSIONS: This highly useful and economical tool can be directly employed in clinical samples without laborious prior cultivation. Our and prospective case-specific data can now easily be compared by using a newly established MLVA database in order to gain a better insight into the epidemiology of this presumably under-reported zoonosis.

Background

The Leptotrichiaceae are a family of underexplored and rarely isolated microorganisms within the phylum Fusobacteria containing both species known from certain pathologies as well as colonising members of the resident microbiota. Many if not all species of the Leptotrichiaceae inhabit the oral cavities, gastrointestinal or urogenital tracts of humans and animals [1-3]. One of the reasons they are rarely encountered is the obligate anaerobic or capnophilic growth dependence of these fastidious bacteria and the usual presence of a high number of concomitant microorganisms. Some members of this family are well known pathogens, such as Streptobacillus (S.) moniliformis, one of the two causative organisms of the bacterial zoonosis rat bite fever [4]. Recently, a number of novel species have been described, most of which could be attributed to clinical disease [5-8]. It can also be concluded from numerous phylotypes, Leptotrichiaceae normally colonize mucous membranes [9-15], but when introduced into new tissue or host sites they are also able to shift their pathogenic potential and cause severe and even life-threatening disease. With increasing availability of next generation sequencing a number of single genomes have been published [6, 16–20]. However, almost no comprehensive genomic studies including these microorganisms have been completed, nor have virulence properties been identified in these species. Phylogenetic studies and identifications within the phylum Fusobacteria have been carried out and based on single or multiple gene sequences such as 16S rRNA, 16S–23S rRNA internal transcribed spacer, gyrB, groEL, recA, rpoB, conserved indels and genes for group-specific proteins, 43-kDa outer membrane protein and zinc protease [18, 21–30]. In an attempt to characterize different members of this phylum Gupta & Seti proposed various conserved signature indels (CSIs) in amino acid sequences for the Leptotrichiaceae from which three CSIs were found to be specific for this family [31]. On the other hand, no detailed phylogenetic and comparative genome studies dedicated to Leptotrichiaceae have been published up to now. Furthermore, and due to a general paucity of strains and attempts to differentiate members from the same species there is currently no tool available to type isolates in order to prove transmission chains. Our data, presented here, were derived from 46 complete genomes from 20 different taxa of the family Leptotrichiaceae aiming to provide the first such comparative analysis. Our study results confirm the picture of earlier phylogenies from this group that are now based on a larger scale of orthologous genes. We give a surveying insight into the investigated genomes, thereby also including recently described species from this family. With a novel approach it was, furthermore, possible to accurately and unequivocally type isolates of S. moniliformis based on three variable number tandem repeat (VNTR) sequences. With this, we are presenting a culture-independent, species-specific fingerprinting tool in order to type the most important causative organism of rat bite fever for the first time.

Results

Accession numbers

The GenBank/EMBL/DDBJ accession numbers for the genome sequences used in this study are summarized in Table 1.

Table 1

Strains as well as origins, clinical symptoms and host species of the Leptotrichiaceae members used in this study

Strain no.	Strain designation	Species	Year of isolation	Host	Clinic/sample	Country	Strain reference	Genome reference	Accession number
1	DSM 12112T (=ATCC 14647T)	Streptobacillus moniliformis	1925	Human	Rat bite fever	France	[4]	[16]	CP001779.1CP001780.1
2	CIP 55-48	Streptobacillus moniliformis	1947	Mouse	Lymph adenitis	UK	n. d. a.	this study	LWQV00000000
3	ATCC 27747	Streptobacillus moniliformis	1964	Turkey	Septic arthritis	USA	[51]	this study	LWQW00000000
4	NCTC 10773	Streptobacillus moniliformis	1971	Human	Blood culture	UK	n. d. a.	this study	LYRU00000000
5	NCTC 11194	Streptobacillus moniliformis	1977	Human	Rat bite fever	UK	n. d. a.	this study	LWQX00000000
6	IPDH 144/80	Streptobacillus moniliformis	1980	Turkey	Septic arthritis	Germany	n. d. a.	this study	LWQY00000000
7	CIP 81-99	Streptobacillus moniliformis	1981	Human	Blood culture (wild rat bite)	France	n. d. a.	this study	LWSZ00000000
8	AHL 370-4	Streptobacillus moniliformis	1982	Mouse	Ear infection	Australia	n. d. a.	this study	LWTA00000000
9	NCTC 11941	Streptobacillus moniliformis	1983	Human	Haverhill fever	UK	n. d. a.	this study	LXKD00000000
10	IPDH 109/83	Streptobacillus moniliformis	1983	Turkey	Septic arthritis	Germany	n. d. a.	this study	LWTB00000000
11	ATCC 49567	Streptobacillus moniliformis	1989	Mouse	Lymph adenitis	Germany	[52]	this study	LWTC00000000
12	Kun 3 (RIVM)	Streptobacillus moniliformis	1991	Rat	Healthy	The Netherlands	[53]	this study	LWTD00000000
13	ATCC 49940	Streptobacillus moniliformis	1992	Rat	Otitis media	Germany	[54]	this study	LWTE00000000
14	B10/15	Streptobacillus moniliformis	Unknown	Wild rat	Unknown	The Netherlands	n. d. a.	this study	LWTF00000000
15	A378/1	Streptobacillus moniliformis	1995	Wild rat	Vaginal swab	Germany	DKFZ strain collection	this study	LWTG00000000
16	VA11257/2007	Streptobacillus moniliformis	2007	Human (farmer)	Rat bite fever, endocarditis	Germany	[55]	this study	LWTI00000000
17	VK105/14	Streptobacillus moniliformis	2008	Domestic rat	Abscess	Germany	TiHo strain collection	this study	LWTJ00000000
18	B5/1	Streptobacillus moniliformis	2009	Laboratory mouse	After rat bite	Germany	DKFZ strain collection	this study	LXKJ00000000
19	Marseille	Streptobacillus moniliformis	2009	Rat	Rat bite fever	La Réunion	[56]	this study	LXKI00000000
20	IKC1	Streptobacillus moniliformis	n. d. a.	Rat	Oral swab	Japan	[39]	this study	LXKH00000000
21	IKC5	Streptobacillus moniliformis	n. d. a.	Rat	Oral swab	Japan	[39]	this study	LXKG00000000
22	IKB1	Streptobacillus moniliformis	n. d. a.	Rat	Oral swab	Japan	[39]	this study	LXKF00000000
23	TSD4	Streptobacillus moniliformis	n. d. a.	Rat	Oral swab	Japan	[39]	this study	LXKE00000000
24	131000547T (DSM 29248T)	Streptobacillus felis	2013	Cat	Pneumonia	Germany	[5, 7]	[18]	LOHX00000000
25	DSM 26322T (HKU33T)	Streptobacillus hongkongensis	2014	Human	Abscess	Hong Kong	[8]	[18]	LOHY0000000
26	AHL 370-1T	Streptobacillus notomytis	1979	Spinifex hopping mouse	Sepicaemia, cultured from liver tissue	Australia	[57]	[6]	SAMN04038436
27	KWG2	Streptobacillus notomytis	n. d. a.	Rat (Rattus rattus)	Oral swab	Japan	[39]	this study	SAMN04099645
28	KWG24	Streptobacillus notomytis	n. d. a.	Rat (Rattus rattus)	Oral swab	Japan	[39]	this study	SAMN04099670
29	OGS16T	Streptobacillus ratti	n. d. a.	Rat (Rattus rattus)	Oral swab	Japan	[39]	[18]	SAMN04099675
30	CCUG 41628T	Sneathia sanguinegens	1999	Human	Blood	Sweden	[58, 59]	[38]	LOQF00000000
31	Sn35	“Sneathia amnii”	n. d. a.	Human	Vaginal microbiota	n. d. a.	[19]	[19]	NZ_CP011280
32	NCTC 11300T (ATCC 33386T)	Sebaldella termitidis	1962	Termite	Intestine	n. d. a.	[60]	[17]	CP001739
33	DSM 1135 (C-1013-b)	Leptotrichia buccalis	2009	Human	Supragingival calculus	USA	n. d. a.	n. d. a.	CP001685
34	DSM 19756 (LB 57)	Leptotrichia goodfellowii	2013	Human	Prosthetic aortic valve	Germany	n. d. a.	n. d. a.	NZ_AZXW00000000
35	F0264	Leptotrichia goodfellowii	n. d. a.	Human	Oral cavity	n. d. a.	n. d. a.	n. d. a.	NZ_ADAD00000000
36	F0254	Leptotrichia hofstadii	n. d. a.	n. d. a.	n. d. a.	n. d. a.	n. d. a.	n. d. a.	NZ_ACVB00000000
37	DSM 19757	Leptotrichia shahii	2013	Human	Gingivitis	Norway	n. d. a.	n. d. a.	NZ_ARDD00000000
38	DSM 19758	Leptotrichia wadei	2004	Human	Saliva	Norway	[2]	n. d. a.	NZ_ARDS00000000
39	F0279	Leptotrichia wadei	n. d. a.	Human	Subgingival plaque	n. d. a.	n. d. a.	n. d. a.	NZ_AWVM00000000
40	Str. W10393	Leptotrichia sp. oral taxon 212	2015	Human	Oral microbiome project	n. d. a.	n. d. a.	n. d. a.	CP012410
41	Str. W9775	Leptotrichia sp. oral taxon 215	2015	Human	Oral microbiome project	n. d. a.	n. d. a.	n. d. a.	NZ_AWVR00000000
42	Str. F0581	Leptotrichia sp. oral taxon 225	2015	Human	Oral microbiome project	n. d. a.	n. d. a.	n. d. a.	NZ_AWVS00000000
43	Str. F0557	Leptotrichia sp. oral taxon 879	2015	Human	Oral microbiome project	n. d. a.	n. d. a.	n. d. a.	NZ_AWVL00000000
44	CCUG 39713T	Caviibacter abscessus	1998	Guinea pig	Cervical abscess	Sweden	n. d. a.	[38]	LOQG00000000
45	1510011837	Caviibacter abscessus	2015	Guinea pig	Cervical abscess	Germany	[38]	[38]	LOQH00000000
46	AVG2115T	Oceanivirga salmonicida	1992	Atlantic salmon	Septicaemia	Ireland	[32, 61]	[37]	LOQI00000000
47	ATCC 25586	Fusobacterium nucleatum subsp. nucleatum	n. d. a.	Human	Cervico-facial lesion	n. d. a.	n. d. a.	[62]	AE009951

type strain, n. d. a. no data available, ATCC American Type Culture Collection, Rockville, USA, NCTC National Collection of Type Cultures, London, UK, CIP Collection Institut Pasteur, Paris, France, IPDH Institute for Poultry Diseases, Hannover, Germany, RIVM Rijksinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands, AHL Animal Health Laboratory, South Perth, Australia, ZfV Zentralinstitut für Versuchstierzucht, Hannover, Germany, DKFZ Deutsches Krebsforschungszentrum, Heidelberg, Germany, TiHo Tierärztliche Hochschule Hannover, Germany, RBF rat bite fever

Phylogenetic analysis based on orthologous genes

To determine the phylogeny within the genus Streptobacillus we aligned the allelic variations of 281 orthologous genes from 29 strains of S. moniliformis, S. ratti, S. notomytis, S. felis and S. hongkongensis which resulted in 57,841 single nucleotide polymorphisms (SNPs). From these SNPs we inferred a maximum likelihood phylogeny showing the distance between the different species within this genus (Fig. 1). To zoom deeper into the phylogeny of the S. moniliformis group we repeated this analyses with 775 orthologous genes present in 23 S. moniliformis strains which resulted in 5,211 SNPs. These SNPs were also used to construct a maximum likelihood phylogeny (Fig. 2).

Fig. 1

Maximum likelihood phylogenetic tree of the genus Streptobacillus (strains 1–29 according to Table 1). The tree is based on 281 orthologous genes including 57,841 SNPs

Fig. 2

Unrooted maximum likelihood phylogenetic tree of 23 Streptobacillus moniliformis strains from this study. The tree is based on 775 orthologous genes including 5,211 SNPs

As shown in the tree, most S. moniliformis strains used for this study are unrelated and form a heterogeneous population without any significant clustering. Solely strains A378/1 and B5/1 that both originate from the same source but without a common epidemiological background were phylogenetically indistinguishable.

Analysis of genomes and protein functions

The genome size in members of the Leptotrichiaceae varies between 1.22 and 4.42 Mbp with Caviibacter (C.) abscessus and Sebaldella (Se.) termitidis being the smallest und largest genomes, respectively. Generally, and with the exception of Sebaldella termitidis, genomes are smaller than 2.45 Mbp. The genera Caviibacter and Sneathia (Sn.) are comparable with respect to genome size (1.22–1.34 Mbp) as are the genera Streptobacillus and Oceanivirga (O.) (1.38–1.90 Mbp). Members of the genus Leptotrichia (L.) are the second largest group with 2.31–2.47 Mbp. A general overview on the genomes of all strains under study is depicted in Table 2. A similar order can be observed with respect to coding DNA sequences (CDS), i.e., C. abscessus and Sneathia spp. possess 1212–1282 CDS, followed by Streptobacillus spp. and O. salmonicida (1293–1679), Leptotrichia spp. (1930–2365) and Sebaldella termitidis (4083). The average percentage of CDS within the whole genome displays a graded distribution within the family: a highly coding group consisting of the genera Caviibacter, Oceanivirga and Sneathia (89–93 %), an intermediate Streptobacillus spp. group (87 %) and a group containing the genera Leptotrichia and Sebaldella (84 %) with lower coding density. Nevertheless, intra-genus variability can be considerably high, the former results can inevitably also be shown for the average gene densities and the average intergenic regions (in parentheses average genes/Mbp; number of intergenic nt): O. salmonicida (1056; 79), C. abscessus (996; 76), Sneathia spp. (989; 84), Streptobacillus spp. (987; 115), Leptotrichia spp. (967; 144) and Sebaldella (936; 149). An organization of the genomes under study into clusters of orthologous groups (COGs) is depicted in Additional files 1 and 2 and shows, however, high intra-species as well as inter-species variations. On a generic level, gene contents of COG classes J, L, D and F are inversely correlated with increasing genome size, whereas COG classes K, N, T and Q are positively correlated (see Additional files 1 and 2).

Table 2

Analysis of genome data as well as predictions of coding regions of the Leptotrichiaceae members used in this study

Strain no.	Organism	Approx. genome size (nt)	CDSa	rRNA	tRNAb	% GCc	Total DNA coding regions (nt)	Total non-coding regions (nt)	Coding genome space (%)	Average gene density (genes/Mbp)	Average inter-genic region (nt)
1	S. moniliformis	1673280	1568	16	39	26.3	1556870	116410	93	937	74
2	S. moniliformis	1678906	1658	12	37	26.1	1508835	170071	89	988	103
3	S. moniliformis	1684459	1591	14	35	26.1	1486041	198418	87	945	125
4	S. moniliformis	1897024	2244	9	43	28.9	1651665	245359	85	1183	109
5	S. moniliformis	1712153	1764	3	38	26.1	1542831	169322	89	1030	96
6	S. moniliformis	1668382	1615	13	36	26.1	1484745	183637	88	968	114
7	S. moniliformis	1686977	1543	12	35	26.4	1449924	237053	84	915	154
8	S. moniliformis	1598404	1608	14	38	25.9	1470174	128230	91	1006	80
9	S. moniliformis	1689124	1675	4	36	26.1	1399686	289438	79	992	173
10	S. moniliformis	1756513	1765	14	37	26.1	1559103	197410	87	1005	112
11	S. moniliformis	1763717	1621	9	35	26.1	1488168	275549	81	919	170
12	S. moniliformis	1518628	1540	12	33	25.9	1442043	76585	95	1014	50
13	S. moniliformis	1689360	1765	5	36	26.1	1526748	162612	89	1045	92
14	S. moniliformis	1674237	1597	13	37	26.2	1477515	196722	87	954	123
15	S. moniliformis	1667701	1692	14	36	26.0	1518810	148891	90	1015	88
16	S. moniliformis	1690579	1538	16	37	26.1	1468143	222436	85	910	145
17	S. moniliformis	1608659	1507	22	34	26.2	1433763	174896	88	937	116
18	S. moniliformis	1497161	1644	8	36	25.8	1322022	175139	87	1098	107
19	S. moniliformis	1696954	1774	5	38	26.1	1521612	175342	88	1045	99
20	S. moniliformis	1696554	1688	17	37	26.0	1509528	187026	88	995	111
21	S. moniliformis	1792325	1664	16	42	26.2	1550631	241694	84	928	145
22	S. moniliformis	1759287	1737	13	43	25.9	1566621	192666	88	987	111
23	S. moniliformis	1608076	1559	10	35	26.0	1445580	162496	89	969	104
24	S. felis	1610666	1754	3	37	26.4	1450014	160652	89	1089	92
25	S. hongkongensis	1543001	1485	14	35	26.1	1324059	218942	83	962	147
26	S. notomytis	1762984	1773	9	43	28.1	1511157	251827	83	1006	142
27	S. notomytis	1426245	1349	8	40	26.4	1257996	168249	87	946	125
28	S. notomytis	1384502	1341	19	39	26.3	1256817	127685	90	969	95
29	S. ratti	1499353	1411	11	39	25.9	1318767	180586	86	941	128
30	Sneathia sanguinegens	1300753	1329	2	34	26.7	1214541	86212	93	1022	65
31	“Sn. amnii”	1339284	1282		34	28.3	1207722	131562	89	957	103
32	Sebaldella termitidis	4418842	4135	13	40	33.5	3802074	616768	84	936	149
33	Leptotrichia buccalis	2465610	2299	15	46	29.6	2062809	402801	80	932	175
34	L. goodfellowii	2281162	2241	7	39	31.6	2045213	235949	88	982	105
35	L. goodfellowii	2287284	2373	3	39	31.5	2055020	232264	89	1037	98
36	L. hofstadii	2453253	2720	13	47	30.8	2059248	394005	81	1109	145
37	L. shahii	2144606	1969	10	41	29.5	1812950	331656	82	918	168
38	L. wadei	2316529	2139	11	42	29.3	1973929	342600	83	923	160
39	L. wadei	2353455	2212	3	27	29.2	2008568	344887	83	940	156
40	Leptotrichia sp. oral taxon 212	2444904	2231	14	43	31.4	2146482	298422	86	936	130
41	Leptotrichia sp. oral taxon 215	2308492	2195	3	34	31.4	2039067	269425	87	951	123
42	Leptotrichia sp. oral taxon 225	2400083	2306	3	24	29.6	2061283	338800	84	961	147
43	Leptotrichia sp. oral taxon 879	2415750	2361	4	25	29.6	2026284	389466	81	977	165
44	C. abscessus	1219935	1198			26.5	1131456	88479	92	982	74
45	C. abscessus	1304155	1316	4	35	26.4	1201320	102835	91	1009	78
46	O. salmonicida	1769081	1869	2	38	25.4	1621182	147899	91	1056	79
47	Fusobacterium nucleatum (outgroup)	2174500	2022	15	47	27.2	1937724	236776	88	930	117

aCDS: DNA coding sequences; btRNA: transfer ribonucleic acid; cGC: guanine-cytosine content

Multiple-Locus Variable number tandem repeat Analysis (MLVA)

In silico VNTR analysis

Under default conditions, 127 repeats were identified by the tandem repeat finder. For further analysis, the three most variable VNTRs were identified according to the degree of variability of allele types identified by alignment analysis (Table 3). These three allelic loci were only present in S. moniliformis and thus proved to be specific for this microorganism (all other members of the Leptotrichiaceae were negative). The combination of the three loci yielded a high discriminatory index (0.94296 DI; Table 4).

Table 3

Streptobacillus moniliformis specific Variable Number of Tandem Repeat (VNTR) primer sequences used in this study

Primer ID	VNTR positiona	Repeat size in nt (identity in %)	Sequence (5–3)	PCR product size (bp)
VNTR_Sm1	1576120 - 1576145	3 (100)	TCA TTT ACT CAC CCT AGT AGT GGT	210
			CCA GTT GAA TAT AAG CTT GCT ATG G
VNTR_Sm2	1182890 - 1182907	6 (100)	TGG AAC TGT TTG TTG AGT ATT TCC A	298
			AGG GAC AGA TGT TCA ATT TGT GTA
VNTR_Sm3	284997 - 285268	36 (91)	TAC GCT GTA GGG TTG AAC GG	830
			ACA GTT TGA GCA CGT CTT AAT CC

Primers were designed with Geneious (v. 8.1.3; Biomatters, Auckland, NZ) [43] and to be complementary to VNTR flanking regions that were conserved among genomes; aaccording to the S. moniliformis DSM 12112T genome (CP001779.1)

Table 4

VNTR allele types of the Streptobacillus moniliformis strains used in this study

Isolate ID	VNTR_Sm1a	VNTR_Sm2	VNTR_Sm3b	Allele code
DSM 12112 T	9	3	16	LHL1
CIP 55-48	7	3	16	LHL10
ATCC 27747	10	3	16	LHL4
NCTC 10773	8	4	17	LHL15
NCTC 11194	6	3	17	LHL16
IPDH 144/80	6	3	16	LHL5
CIP 81-99	7	3	16	LHL10
AHL 370-4	7	2	15	LHL3
NCTC 11941	6	3	18	LHL11
IPDH 109/83	6	3	16	LHL5
ATCC 49567	6	3	16	LHL5
Kun 3 (RIVM)	6	3	18	LHL11
ATCC 49940	6	3	14	LHL6
B10/15	6	4	15	LHL7
A378/1	8	5	16	LHL2
VA11257/2007	6	3	16	LHL5
VK105/14	8	3	16	LHL13
B5/1	8	5	16	LHL2
Marseille	6	4	14	LHL14
IKC1	6	3	15	LHL8
IKC5	5	3	15	LHL9
IKB1	6	3	16	LHL5
TSD4	11	3	18	LHL12
A40-13 c	11	2	17	LHL17

Bold rows represent strains used for a PCR-based validation of in silico identified VNTR allele types (underlined alleles were not found in silico and only identified after PCR amplification); a in order to fit requirements of the database, the repeat copy numbers at locus VNTR_Sm1 have been rounded up to receive integer values (e.g., 9 instead of 8.7); bwhile the repeat copy numbers at locus VNTR_Sm3 have been rounded up to the next half-value and doubled to receive integer values (e.g., 15 instead of 7.2); T: type strain; cstrain was only used for validation (no complete genome available)

PCR-based validation of in silico results

The absence of the calculated VNTR loci could also be proven by polymerase chain reaction (PCR) in all Leptotrichiaceae members other than S. moniliformis (data not shown). Contrarily, each of the ten S. moniliformis strains exhibited a specific band corresponding to their predicted tandem repeats pattern. Analysis of the sequenced PCR products confirmed the allele type allocation determined in silico (Table 4). VNTR_Sm1 alleles of two isolates, which were not found in silico, were successfully assigned (Table 4). Re-calculation revealed a DI of 0.9529 after including these two isolates, as well as one isolate for which no genome data was available. In order to facilitate comparisons of results in future studies, every genotype (from the allele types of the three loci) was expressed as a specific allele combination resulting in a specific allele code (Table 4). An online database dedicated to MLVA results of S. moniliformis has been established on the webserver of University Paris-Saclay, Orsay, France (http://microbesgenotyping.i2bc.paris-saclay.fr/databases/public) which is open to future entries and strain comparisons.

Discussion

Members of the Leptotrichiaceae are rarely encountered microorganisms, a phenomenon that seems to be highly dependent on difficulties with cultivation. With the availability of molecular methods in this field the number of findings and frequencies has significantly increased [10–15, 32–36]. On the other hand, we still need deeper insight into the genomes of this group. In particular, the mechanisms involved in pathogenesis and virulence of pathogenic species are completely unexplored. We have undertaken a first step into this direction by analysing a broad spatio-temporal collection of strains, thereby including especially species with regular evidence for pathologies. Firstly, the large dataset from this study has been utilized for the confirmation of our phylogenetic picture from earlier studies [18, 30, 37, 38]. An intra-genus phylogeny that was based on 775 orthologous genes revealed a very similar picture to previous studies involving only four selected functional genes (Figs. 1 and 2). Conversely and in contrast to almost identical average nucleotide identity (ANI) values [30], full genome analyses revealed a high level of heterogeneity for all but two strains (no. 15 and 18) of S. moniliformis without any significant clustering. This is, albeit, not surprising, because the present study included a large spatio-temporal collection of 23 S. moniliformis strains that have been isolated over a period of 90 years from at least five different host species and from almost all subcontinents. We were also able to display the three predicted Leptotrichiaceae specific CSIs of MreB/MrI (2 aa deletion), AlaS and RecA (5 and 2 aa insertions, respectively) in all of our genomes as well as in the recently described members of the family (data not shown) [31].

Genome size dependent gene content has been described and could also be confirmed for the genomes from this study [19]. With increasing genome size gene contents of COG classes J, L, D and F involved in DNA replication, cell cycle regulation and protein translation are inversely correlated, whereas COG classes K, N, T and Q involved in transcription, signal transduction, cell motility and the biochemistry of secondary metabolites are positively correlated (see Additional files 1 and 2). This makes sense when essential gene functions are preserved in smaller genomes and less important gene functions which are dispensable or can be ‘outsourced’ to the host, are lost [19]. On first impression the group of S. moniliformis strains is highly similar as can be concluded from related morphological and phenotypical properties and also from their high intra-species ANI of 98.5–99.3 % (cf. Table S2 in [30]). Based on data from this study very similar COG classes were also observed within this group (see Additional files 1 and 2), but differences in coding densities suggested, on the other hand, remarkable discrepancies. Fuelled by the idea that these discrepancies could, furthermore, be utilized with respect to epidemiology, we have developed a specific MLVA typing scheme for the major pathogen from this group, S. moniliformis, and the causative organism of rat bite fever. This scheme proved to be sufficient in unequivocally typing all 23 S. moniliformis strains under study plus one additional isolate with high discriminatory power (0.9529 DI). Interestingly, only four allele codes (genotypes; LHL2, LHL5, LHL10 and LHL11) were found more than once among isolates (Table 4). At least for LHL2 isolates, a connection could be pursued in that both isolates have been stored in the same strain collection, although a direct transmission could not be proven. To check the clonality of isolates belonging to these four genotypes we have investigated further loci with high discriminatory potential, i.e., the clustered regularly interspaced short palindromic repeats (CRISPR) region known to occur in S. moniliformis (http://crispr.u-psud.fr/cgi-bin/crispr/SpecieProperties.cgi?Taxon_id=519441). In contrast to all other allele codes (LHL5, LHL10, LHL11), both strains (no. 15 and 18) belonging to the allele code LHL2 indeed shared an identical CRISPR region, thereby pointing towards a clonal relation of these two isolates (data not shown) as could also be concluded from the phylogenetic tree (Fig. 2). Due to its length of up to approximately 3,000 nucleotides and its high level of heterogeneity the CRISPR region seems, on the other hand, presently not very well suited as a direct typing tool, but could be useful in certain situations to confirm or negate clonality of strains. A second advantage of the MLVA method described in this study is that it can effectively be pursued directly from the original matrix (e.g., a mouth microbiota swab and a clinical sample) without prior cultivation of the organism, which offers the possibility to better understand transmission chains in the future. This seems to be especially relevant since established PCR assays are not species specific, but limited to genus level specificity [37, 39, 40]. The majority of diagnoses of rat bite fever cases in the recently published literature relies only on partial 16S rRNA gene sequence analysis that may – in the light of very similar novel Streptobacillus spp. that also colonize rats – be quite uncertain for proper pathogen identification [41]. Hopefully, the newly established MLVA database will help to clarify regional infectious clusters and confirm transmission of certain lineages.

Conclusion

We have undertaken a first analysis of Leptotrichiaceae genomes using a large spatiotemporal collection of strains also including novel members of this group. Our dataset unveiled a first insight into characteristics founding a stable phylogeny, genome structure and COG classes. Beside apparent intra-species similarities we have detected also genetic heterogeneities that provided a basis for fingerprinting the most relevant pathogen from this group, the rat bite fever organism, S. moniliformis. This highly useful and economical tool can be directly used from clinical samples without ambitious prior cultivation and with high discriminatory power. Our data form the basis for a newly established MLVA database that provides the opportunity to store and compare isolate-specific information in future cases with this neglected zoonosis.

Methods

Generation of genomic data

Twenty-two strains of S. moniliformis were sequenced in this study, ten strains were taken from previous publications of our group and 15 strains were descended from other projects (Table 1). Genomic DNA was extracted from a 72 h bacterial culture with a commercial kit according to the manufacturer’s instructions (MasterPure™ Complete DNA and RNA Purification Kit, Epicentre, distributed by Biozym Scientific, Hessisch Oldendorf, Germany). Whole genome sequencing of the strains was performed on an Illumina MiSeq with v3 chemistry resulting in 300 bp paired end reads and a coverage of greater than 90×. Quality trimming and de novo assembly was performed with CLC Genomics Workbench, Version 7.5 (CLC Bio, Aarhus, Denmark). For automatic annotation we used the RAST Server: Rapid Annotations using Subsystems Technology [42]. Data from further relevant reference genomes from the Leptotrichiaceae were also utilized and obtained from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). Sequence analyses and genome calculations as well as oligonucleotide primer generation were carried out with Geneious (v. 8.1.3; Biomatters, Auckland, NZ) [43]. Table 1 depicts the set of strains and reference genomes used for this study.

The determination of the maximum common genome (MCG) alignment was done comprising those genes present in all genomes considered for comparison [44]. Based on the parameters sequence similarity (minimum 70 %) and coverage (minimum 90 %) the genes were clustered and those genes that were present in each genome, fulfilling the threshold parameters were defined as MCG. This resulted in 281 orthologous genes for the comparison of 29 strains of S. moniliformis, S. ratti, S. notomytis, S. felis and S. hongkongensis and in 775 orthologous genes for the comparison within 23 strains of S. moniliformis only.

The following extraction of the allelic variants of these genes from all genomes was performed by a blast based approach after which they were aligned individually for each gene and concatenated which resulted in an alignment of 219,961 bp for the 29 strains and of 546,508 bp for the 23 S. moniliformis strains [45].

This alignment was used to generate a phylogenetic tree with randomized axelerated maximum likelihood (RAxML) 8.1 [46] using a General Time Reversible model and gamma correction for among site rate variation.

Genes were predicted with Prodigal [47] and assigned to COGs with the NCBI’s Conserved Domain Database [48].

The complete genome sequence of the S. moniliformis type strain DSM12112T (accession number CP001779.1) was used to search for potential VNTRs using a tandem repeat finder web tool (http://tandem.bu.edu/trf/trf.basic.submit.html). We focused our search on repeats that were characterized by high purity, large size, and/or large number of repeat copies [49]. Repeats of interest were aligned against a set of available genomes depicted in Table 1 using Geneious and allele types were determined as shown in repeat copy numbers. The DI was calculated for a combination of three most variable VNTRs using an online discriminatory power calculator (http://insilico.ehu.es/mini_tools/discriminatory_power/).

Ten S. moniliformis strains (strain nos. 1, 2, 3, 12, 14, 15, 21, 22 and 23 according to Table 1 plus strain A40-13 for which complete genomic data were not available) as well as all accessible members of the Leptotrichiaceae other than S. moniliformis were used for validation. DNA was extracted from respective isolates (2–3 colonies) by boiling in 100 μL distilled water for 20 min (min.) followed by centrifugation at 20,817 × g for 5 min. The 20 μL final PCR reaction contained 10 μL of Hotstar Taq MasterMix (Qiagen, Hilden, Germany), 1 μL of each forward and reverse primer (10 pmol/μL) (TIB MOLBIOL, Berlin, Germany) (Table 3), 6 μL DNase free PCR grade water (Qiagen), and 2 μL of the extracted DNA. PCR conditions were as following: 1× (95 °C, 15 min), 40x (94 °C, 30 s; 58 °C, 30 s; 72 °C, 30 s), 1× (72 °C, 10 min). PCR products were stained with ethidium bromide in a 2 % agarose gel (100 V for 1.5 h) and then analyzed with a gel documentation system (BioDoc-It, UVP, UK). The PCR amplicons were purified using MicroElute DNA Cycle-Pure Kit (OMEGA bio-tek, Norcross, USA) and sequenced at Seqlab-Microsynth laboratories (Göttingen, Germany). All sequences were analyzed by tandem repeat finder web tool and/or BLASTN 2.3.1+ [50] hosted by NCBI website and compared to the in silico results.