Noncontiguous finished genome sequence and description of Bartonella mastomydis sp. nov.

Bartonella mastomydis sp. nov. strain 008 is the type strain of B. mastomydis sp. nov., a new species within the genus Bartonella. This strain was isolated from Mastomys erythroleucus rodents trapped in the Sine-Saloum region of Senegal. Here we describe the features of this organism, together with the complete genome sequence and its annotation. The 2 044 960 bp long genomes with 38.44% G + C content contains 1674 protein-coding and 42 RNA genes, including three rRNA genes.

Introduction

Just over a century ago, the first historical record of the emerging Bartonella genus was made during World War I, when a million frontline troops were shown to be plagued by a disease later known as trench fever. This was caused by the louse-borne bacterium now known as Bartonella quintana [1]. Bartonella are small facultative intracellular, vector-transmitted, Gram-negative, haemotropic bacilli, classified within the class of α-proteobacteria [2]. The genus was significantly expanded after Brenner et al. proposed the unification of genera Bartonella and Rochalimaea in 1993 and Birtles et al. unified Bartonella and Grahamella genera in 1995 [3]. The Bartonellaceae family (Gieszczykiewicz 1939) [4] contains 35 species and three subspecies (http://www.bacterio.net/) as of 1 August 2017 [5]. Bartonellae usually exist in two specific habitats: the gut of the obligately blood-sucking arthropod vector and the bloodstream of the mammalian host [1]. Among the 38 recognized Bartonella species, 17 have been described as pathogenic in humans [6]. In humans, Bartonella bacteria are among the most described as being associated with endocarditis or cardiopathy. In animal hosts, a wide array of clinical syndromes, as well as asymptomatic infection and endocarditis, have been described [6], [7], [8].

New species and subspecies are constantly being proposed. Candidate species belonging to the genus Bartonella from a wide range of animal reservoirs have been described but not yet assigned new species designations [1]. Parasitism by bartonellae is widespread among small mammals. Potentially new Bartonella species infecting bat communities were reported in Madagascar [9], Kenya [10], Puerto Rico [11] and French Guiana [12]. Rodents and insectivores were showed to maintain bartonellae infections. Additionally, a large number of partially characterized Bartonella have been isolated from rodents in Southeast Asia [13], South Africa [14], [15], Europe, North and South America [16], Nigeria [17], the Republic of Congo and Tanzania [16]. In Senegal, West Africa, using the criteria proposed by La Scola et al. [18] based on the multilocus sequence analyses of four genes and the intergenic spacer (ITS) as a tool to the description of bartonellae, three new bartonellae were isolated and described: Bartonella senegalensis, Bartonella massiliensis from the soft tick Ornithodoros sonrai [13] and Bartonella davoustii from cattle [19].

We sought to describe an additional Bartonella species isolated from small mammals in the region of Sine-Saloum, in western Senegal [20]. In this rural region, the biotype is favourable to the spread of commensal mammals harbouring pathogenic microorganisms and is often found in close contact with humans. This situation increases the risk of human and animal transmission of infectious disease from rodent-associated tick-borne pathogens. This work describes the genome sequence of the proposed candidate Bartonella mastomydis strain 008 isolated from Mastomys erythroleucus using a polyphasic approach combining matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and genomic properties, as well as next-generation sequencing technology to complete description of a potentially new species [21].

Here we present the summary classification and a set of features for B. mastomydis sp. nov. strain 008 together with the description of the complete genomic sequences and annotation. These characteristics support the definition of the species B. mastomydis.

Samples and bacterial culture

In February 2013, rodents and insectivores were captured alive at two sites (Dielmo and Ndiop) using wire mesh traps baited with peanut butter or onions. Our aim was to investigate the presence of Bartonella spp. in commensal rodents in Sine-Saloum, Senegal. In this region, rodents and rodent-associated soft ticks are respectively the reservoirs and vectors of relapsing fever caused by Borrelia crocidurae. Trapped rodents and insectivores were anesthetized and necropsied in sterile conditions. Sampled blood was inoculated on homemade Columbia agar plates supplemented with 5% sheep's blood. The results of this study have been reported elsewhere [20]. In total, within a 6-day period, 119 small mammals were captured: 116 rodents and three shrews (Crocidura cf. olivieri). Rodents were identified morphologically as follows: five Arvicanthis niloticus, 56 Gerbilliscus gambianus, 49 M. erythroleucus, five Mus musculus and one Praomys daltoni. Thirty isolates of Bartonella spp. were recovered from the rodent bloodstreams. None of those isolated belonged to previously described Bartonella species (Table 1).

Table 1

Classification and general features of Bartonella massiliensis strain 008.

MIGS ID	Property	Term	Evidence codea
	Current classification	Domain Bacteria	TAS [42]
		Phylum Proteobacteria	TAS [43]
		Class Alphaproteobacteria	TAS [44]
		Order Rhizobiales	TAS [45], [46]
		Family Bartonellaceae	TAS [4], [22]
		Genus Bartonella	TAS [3], [4], [22], [47]
		Species Bartonella mastomydis	IDA
		Type strain 008	IDA
	Gram stain	Negative	IDA
	Cell shape	Rod	IDA
	Motility	Nonmotile	IDA
	Sporulation	Nonsporulating	IDA
	Temperature range	Mesophilic	IDA
	Optimum temperature	37°C	IDA
MIGS-22	Oxygen requirement	Aerobic	IDA
	Carbon source	Unknown	IDA
	Energy source	Unknown	IDA
MIGS-6	Habitat	Mastomys erythroleucus bloodstream	IDA
MIGS-15	Biotic relationship	Facultative intracellular	IDA
	Pathogenicity	Unknown	IDA
	Biosafety level	3	IDA
MIGS-14	Isolation	M. erythroleucus	IDA
MIGS-4	Geographic location	Senegal	IDA
MIGS-5	Sample collection	February 2013	IDA
MIGS-4.2	Latitude	14°03′N	IDA
MIGS-4.3	Longitude	15°31′W	IDA
MIGS-4.4	Altitude	8 m	IDA

MIGS, Minimum Information About a Genome Sequence.

Evidence codes are as follows: IDA, inferred from direct assay; TAS, traceable author statement (i.e. a direct report exists in the literature); NAS, nontraceable author statement (i.e. not directly observed for the living, isolated sample, but based on a generally accepted property for the species or anecdotal evidence). These evidence codes are from the Gene Ontology project (http://www.geneontology.org/GO.evidence.shtml). If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or by an expert or reputable institution mentioned in the acknowledgements.

Classification and features

The gltA, rpoB, 16S rRNA and ftsZ genes as well as the ITS have been amplified and sequenced from recovered Bartonella isolates [18], [22], [23], [24], [25]. Bartonella mastomydis (21 isolates) recovered only from M. erythroleucus was obtained following the fifth to tenth incubations at 37°C in a 5% CO2-enriched atmosphere on Columbia agar plates supplemented with 5% sheep's blood. Other morphologically and genetically indistinguishable strains were isolated from M. erythroleucus. The 21 isolates of B. mastomydis are almost genetically identical; however, strains type 008, 025, 086 and 202 showed different nucleotide identity. The identities between them are as follows: 100% for the rrs gene, 99% for the rpoB gene and 98% to 99% for the ftsZ and gltA genes. The sequence of the ITS of strain 008 present 94% to 99% identity with strains 025, 086 and 202, as presented by a 23 bp deletion and a 4 bp insertion compared to the other strains. This study focused on the taxonomic description and identification of strain 008.

Strain 008 exhibits the following nucleotide sequence similarities for the rrs gene (KY555064): 99% with Bartonella tribocorum str. BM1374166 (HG969192), Bartonella grahamii str. as4aup (CP001562), Bartonella vinsonii subsp. arupensis str. OK 94-513 (NR_104902) and subsp. berkhoffii (CP003124), Bartonella elizabethae str. F9251 (NR_025889), Bartonella henselae str. Houston-1 (NR_074335) and finally Bartonella quintana str. Toulouse (BX897700). For the ITS (KY555067), 95% similarity was observed with B. elizabethae (L35103). For the gltA gene (KY555066), 97% similarity was observed with B. elizabethae (Z70009), 94% with B. tribocorum str. BM1374166 (HG969192), B. grahamii str. as4aup (CP001562) and Bartonella queenslandensis str. AUST/NH12 (EU111798). For the ftsZ gene (KY555065), 98% of similarity was observed with B. elizabethae (AF467760), 96% with B. tribocorum str. BM1374166 (HG969192), B. grahamii str. as4aup (CP001562) and B. queenslandensis str. AUST/NH12 (EU111798). For the rpoB gene (KY555068), 99% similarity was observed with multiple uncultured Bartonella amplified from small mammals from Ethiopia [26], Benin [27], Congo and Tanzania [16] and Nepal [28]. The closest recognized species was B. elizabethae (AF165992) at 98% homology (Fig. 1).

Fig. 1

Evolutionary history of sequenced samples was inferred using maximum likelihood method implemented in MEGA7 [40] and based on concatenated gltA, rpoB, 16S RNA and ftsZ (total length of 2731 bp) sequences. Sequences of gltA, rpoB,16S RNA and ftsZ genes used for comparison were obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/). Sequences were aligned using BioEdit [41]. Firstly, for each gene individually, sequences we used for comparison were first aligned using CLUSTALW. All positions containing gaps and missing data were eliminated manually; then each alignment was concatenated and second alignment was performed. Evolutionary history was inferred by maximum likelihood method based on Hasegawa-Kishino-Yano model. Percentage of trees in which associated taxa clustered together is shown next to branches. Initial tree for heuristic search was obtained automatically by applying the Neighbor Joining and BioNJ algorithms to matrix of pairwise distances estimated using maximum composite likelihood (MCL) approach and then selecting topology with superior log likelihood value. Discrete gamma distribution was used to model evolutionary rate differences among sites (two categories (+G, parameter = 0.2144)). Tree is drawn to scale, with branch lengths measured in number of substitutions per site. Statistical support for internal branches of trees was evaluated by bootstrapping with 1000 iterations. Analysis involved 39 nucleotide sequences.

MALDI-TOF MS protein analysis was carried out as previously described [21]. Five isolated colonies of strain 008 were deposited as individual spots on the MALDI target plate. Each smear was overlaid with 2 μL of matrix solution (a saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50% acetonitrile and 2.5% trifluoroacetic acid, and allowed to dry for 5 minutes. Measurements were performed with a Microflex spectrometer (Bruker Daltonics, Bremen, Germany). The five 008 spectra were imported into MALDI BioTyper software version 2.0 (Bruker) and were analysed by standard pattern matching (with default parameter settings) against the main spectra of 4613 bacteria in the BioTyper database and the 25 Bartonella species in our own database. The identification method included the m/z from 3000 to 15 000 Da. For every spectrum, a maximum of 100 peaks was considered and compared with the spectra in the database. A score of below 1.7 meant identification was not possible. For strain 008, the scores obtained were always below 1.5, suggesting that our isolate was not a member of a known species. We added the spectrum from strain 008 to the database (Fig. 2). A gel view comparing the spectrum of strain 008 with those of other Bartonella species is shown in Fig. 3.

Fig. 2

Reference mass spectrum from Bartonella mastomydis strain 008. Spectra from 12 individual colonies were compared and reference spectrum was generated.

Fig. 3

Gel view comparing Bartonella mastomydis strain 008 spectra with other members of Bartonella genus. Gel view displays raw spectra of loaded spectrum files arranged in pseudo–gel-like look. X-axis records m/z value. Left y-axis displays running spectrum number originating from subsequent spectra loading. Peak intensity is expressed by greyscale scheme code. Colour bar and right y-axis indicate relation between colour in which peak is displayed and peak intensity in arbitrary units. Displayed species are indicated at left.

Biochemical characterization and antibiotic susceptibility

Different growth temperatures (32, 37, 42°C) were tested. Growth occurred only at 37°C in 5% CO2. Colonies were grey, opaque and 0.5 mm to 1 mm in diameter on blood-enriched Columbia agar. A motility test was negative. Cells grown on agar were Gram negative and had a mean length and width of 1369.8 ± 423.8 nm and 530.9 ± 105.8 nm respectively by electron microscopy (Fig. 4). No flagella or pili were observed. Strain 008 exhibited neither catalase nor oxidase activity. Biochemical characteristics were assessed using API 50 CH (bioMérieux, Marcy l’Etoile, France), API ZYM (bioMérieux) and API Coryne (bioMérieux); none of the available biochemical tests was positive. Similar profiles were previously observed for B. senegalensis [29]. Bartonella mastomydis is sensitive to amoxicillin, amoxicillin/clavulanic acid, oxacillin, imipenem, rifampicin, nitrofurantoin, doxycycline, linezolid, tobramycin, gentamycin, trimethoprim/sulfamethoxazole, fosfomycin and ciprofloxacin. Bartonella mastomydis is resistant to metronidazole and colistin.

Fig. 4

Transmission electron microscopy of Bartonella mastomydis strain 008 using Tecnai G20 device (FEI Company, Limeil-Brévannes, France) at operating voltage of 200 keV. Scale bar = 200 nm.

Genome sequencing information

Genome project history

The organism was selected for sequencing on the basis of the similarity of its 16S rRNA, ITS, ftsZ, gltA and rpoB to other members of the genus Bartonella. Nucleotide sequence similarities for these genes suggested that strain 008 represents a new species in the genus Bartonella. A summary of the project information is shown in Table 2. The GenBank accession number is G + CA_900185775, and the entry consists of 12 scaffolds (>1500 bp). Table 2 shows the project information and its association with Minimum Information About a Genome Sequence (MIGS) version 2.0 compliance.

Table 2

Project information

MIGS ID	Property	Term
MIGS-31	Finishing quality	High-quality draft
MIGS-28	Libraries used	One paired-end 3 kb library
MIGS-29	Sequencing platforms	454 GS FLX Titanium
MIGS-31.2	Fold coverage	30×
MIGS-30	Assemblers	Newbler version 2.5.3
MIGS-12	Gene calling method	Prodigal
	GenBank ID	GCA_900185775
MIGS-13	Project relevance	Biodiversity of Bartonella spp. in rodents from Senegal

MIGS, Minimum Information About a Genome Sequence.

Genome sequencing and assembly

Bartonella mastomydis sp. nov. strain 008 (DSM 28002; CSUR B643) was grown on 5% sheep's blood–enriched Columbia agar at 37°C in a 5% CO2 atmosphere. Genomic DNA of B. mastomydis sp. nov. strain 008 was extracted in two steps. A mechanical treatment was first performed by acid-washed glass beads (G4649-500g; MilliporeSigma, St Louis, MO, USA) using a FastPrep BIO 101 instrument (Qbiogene, Strasbourg, France) at maximum speed (6.5 m/s) for 90 s. Then after 2 hours' lysozyme incubation at 37°C, DNA was extracted on the EZ1 biorobot (Qiagen, Hilden, Germany) with the EZ1 DNA tissue kit. The elution volume was 50 μL. Genomic DNA was quantified by a Qubit assay with the High Sensitivity Kit (Life Technologies, Carlsbad, CA, USA) to 66 ng/μL. Genomic DNA was sequenced on the MiSeq Technology device (Illumina, San Diego, CA, USA) with the mate-pair strategy. The genomic DNA was barcoded to be mixed with 11 other projects with the Nextera Mate Pair sample prep kit (Illumina).

The mate-pair library was prepared with 1.5 μg of genomic DNA using the Nextera mate-pair Illumina guide. The genomic DNA sample was simultaneously fragmented and tagged with a mate-pair junction adapter. The profile of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) with a DNA 7500 labchip. The optimal size of obtained fragments was 7.77 kb. No size selection was performed, and 600 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with Optima on a bimodal curve at 593 and 1377 bp on a Covaris (Woburn, MA, USA) S2 device in T6 tubes. The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent), and the final concentration library was measured at 49.16 nmol/L. The libraries were normalized at 2 nM, pooled with 11 other projects, denatured and diluted at 15 pM. Automated cluster generation and 2 × 250 bp sequencing runs were performed in a 39-hour run.

Total information of 7.2 Gb was obtained from a 765K/mm2 cluster density, with a cluster passing quality control filters of 94.7% (14 162 000 passed filter clusters). Within this run, the index representation for B. mastomydis was determined to 12.30%. The 1 742 441 paired end reads were filtered according to the read qualities.

Genome assembly

The genome's assembly was performed with a pipeline that enabled creation of an assembly with different software programs (Velvet [30], Spades [31] and Soap Denovo [32]), on trimmed (MiSeq and Trimmomatic [33]) or untrimmed data (only MiSeq). For each of the six assemblies performed, GapCloser [32] was used to reduce gaps. Then contamination with Phage Phix was identified (BLASTN against Phage Phix174 DNA sequence) and eliminated. Finally, scaffolds under 800 bp were removed, and scaffolds with a depth value lower than 25% of the mean depth were removed (identified as possible contaminants). The best assembly was selected by using different criteria (number of scaffolds, N50, number of N characters).

Genome annotation

Open reading frames (ORFs) were predicted using Prodigal [34] with default parameters, but the predicted ORFs were excluded if they spanned a sequencing gap region (contained N). The predicted bacterial protein sequences were searched against the Clusters of Orthologous Groups (COGs) database using BLASTP (E value of 1e-03, coverage 0.7 and identity percentage 30%). If no hit was found, it searches against the NR database using BLASTP (E value of 1e-03, coverage 0.7 and identity percentage of 30%). If the sequence length was <80 aa, we used an E value of 1e-05. The tRNAScanSE [35] tool was used to find transfer RNA genes, whereas ribosomal RNA genes were found by using RNAmmer [36]. Lipoprotein signal peptides and the number of transmembrane helices were predicted using Phobius [37]. ORFans were identified if not all of the BLASTP performed gave positive results (E value smaller than 1e-03 for ORFs with sequence size >80 aa or E value smaller than 1e-05 for ORFs with sequence <80 aa). Such parameter thresholds have already been used in previous work to define ORFans.

Genome properties

The genome is 2 044 960 bp long with 38.44% G + C content. It is composed of 12 scaffolds (composed of 14 contigs) (Fig. 5). Of the 1716 predicted genes, 1674 were protein-coding genes and 42 were RNAs (one gene is 5S rRNA, one gene is 16S rRNA, one gene is 23S rRNA, 39 genes are tRNA genes). A total of 1212 genes (72.4%) were assigned as putative function (by COGs or by NR BLAST). Fifty-six genes were identified as ORFans (3.35%). The remaining 338 genes were annotated as hypothetical proteins (20.19%). The distribution of genes into COGs functional categories is presented in Table 3. The propriety and statistics of the genome are summarized in Table 3, Table 4. The most predicted functional genes are associated with translation (9.38%), followed by those involved in the basic biological functions, such as amino acid transport and metabolism (6.33%), energy production and conversion (4.42%) and carbohydrate transport and metabolism (3.35%) (Table 4).

Fig. 5

Graphical circular map of chromosome. From outside to centre: genes on forward strand coloured by COGs categories (only genes assigned to COGs), genes on reverse strand coloured by COGs categories (only gene assigned to COGs), RNA genes (tRNAs green, rRNAs red), G + C content and G + C skew. COGs, Clusters of Orthologous Groups database.

Table 3

Number of gene associated with 25 general COGs functional categories

Code	Value	% of total	Description
J	157	9.38	Translation
A	0	0	RNA processing and modification
K	58	3.46	Transcription
L	73	4.36	Replication, recombination and repair
B	0	0	Chromatin structure and dynamics
D	17	1.02	Cell cycle control, mitosis and meiosis
Y	0	0	Nuclear structure
V	21	1.25	Defense mechanisms
T	37	2.21	Signal transduction mechanisms
M	74	4.42	Cell wall/membrane biogenesis
N	4	0.24	Cell motility
Z	0	0	Cytoskeleton
W	0	0	Extracellular structures
U	42	2.51	Intracellular trafficking and secretion
O	74	4.42	Posttranslational modification, protein turnover, chaperones
X	25	1.49	Mobilome: prophages, transposons
C	74	4.42	Energy production and conversion
G	56	3.35	Carbohydrate transport and metabolism
E	106	6.33	Amino acid transport and metabolism
F	47	2.81	Nucleotide transport and metabolism
H	63	3.76	Coenzyme transport and metabolism
I	44	2.63	Lipid transport and metabolism
P	57	3.41	Inorganic ion transport and metabolism
Q	15	0.89	Secondary metabolites biosynthesis, transport and catabolism
R	74	4.42	General function prediction only
S	68	4.06	Function unknown
—	603	36.02	Not in COGs

COGs, Clusters of Orthologous Groups database.

Table 4

Nucleotide content and gene count levels of genome

Attribute	Genome (total)
	Value	% of Totala
Size (bp)	2 044 960	100
G + C content (bp)	785 960	38.44
Coding region	1 555 569	76.07
Total gene	1716	100
RNA genes	42	2.45
Protein-coding genes	1674	100
Protein assigned to COGs	1071	63.99
Protein with peptide signals	263	15.71
Genes with transmembrane helices	372	22.22

COGs, Clusters of Orthologous Groups database.

Total is based on either the size of the genome in base pairs of the total of protein-coding genes in the annotated genome.

Insights from genome sequence

The draft genome sequence of B. mastomydis is smaller than those of Bartonella rattaustraliani, Bartonella florencae, B. queenslandensis and B. tribocorum (2045, 2158, 2054, 2378 and 2631 Mb respectively), but larger than those of B. elizabethae and B. vinsonii subsp. berkhoffii (1964 and 1803 Mb respectively). The G + C content of B. mastomydis is smaller than those of B. rattaustraliani, B. vinsonii subsp. berkhoffii, B. florencae and B. tribocorum (38.44, 38.8, 38.83, 38.45 and 38.81% respectively), but larger than those of B. elizabethae and B. queenslandensis (38.32 and 38.38% respectively). The protein-coding gene content of B. mastomydis is smaller than those of B. rattaustraliani, B. florencae, B. queenslandensis and B. tribocorum (1674, 1943, 1886, 2466 and 2295 respectively), but larger than those of B. elizabethae and B. vinsonii subsp. berkhoffii (1663 and 1434 respectively). Similarly, the gene content of B. mastomydis (1674) is smaller than those of B. rattaustraliani, B. florencae, B. queenslandensis and B. tribocorum (1943, 1886, 2466 and 2295 respectively), but larger than those of B. elizabethae and B. vinsonii subsp. berkhoffii (1663 and 1434 respectively). The COGs category gene distribution is not similar. B. mastomydis has fewer COGs category genes belonging to transcription (n = 58) than B. tribocorum (73). Bartonella mastomydis has also fewer genes belonging to the replication, recombination and repair COGs category (73) than B. rattaustraliani (108), B. queenslandensis (100) and B. tribocorum (95). Finally, B. mastomydis has also fewer genes belonging to mobilome: prophages, transposons COGs category (25) than B. tribocorum, B. rattaustraliani, B. queenslandensis, B. vinsonii subsp. berkhoffii and B. florencae (125, 56, 50, 45 and 43 respectively) (Fig. 6). Among species with standing in nomenclature, average genomic identity of orthologous gene sequences (AGIOS) values ranged from 0.96 between B. mastomydis and B. elizabethae to 0.66 between B. vinsonii subsp. berkhoffii and B. rattaustraliani, B. queenslandensis, B. elizabethae, B. mastomydis, B. rattaustraliani, B. tribocorum, B. florencae and B. tribocorum (Table 5). To evaluate the genomic similarity among the strains, we determined two parameters, digital DNA-DNA hybridization, which exhibits high correlation with DNA-DNA hybridization (DDH) [38] and AGIOS [39], which was designed to be independent of DDH (Table 6).

Fig. 6

Distribution of functional classes of predicted genes according to clusters of orthologous groups of proteins.

Table 5

Numbers of orthologous protein shared between Bartonella genomes (upper right)a

	B. vinsonii subsp. berkhoffii	B. rattaustraliani	B. florencae	B. tribocorum	B. queenslandensis	B. elizabethae	B. mastomydis
B. vinsonii subsp. berkhoffii	1434	1115	1121	1154	1043	1143	1144
B. rattaustraliani	0.66	1943	1134	1164	1057	1148	1154
B. florencae	0.67	0.83	1886	1210	1081	1201	1201
B. tribocorum	0.80	0.66	0.66	2295	1136	1257	1258
B. queenslandensis	0.66	0.82	0.83	0.70	2466	1114	1115
B. elizabethae	0.66	0.82	0.84	0.70	0.90	1663	1264
B. mastomydis	0.66	0.82	0.84	0.70	0.90	0.96	1674

Average percentage similarity of nucleotides corresponding to orthologous protein shared between genomes (lower left) and numbers of proteins per genome (bold).

Table 6

Pairwise comparison of Bartonella mastomydis with six other species using GGDC, formula 2 (DDH estimates based on identities/HSP length)a

	B. vinsonii subsp. berkhoffii	B. rattaustraliani	B. florencae	B. tribocorum	B. queenslandensis	B. elizabethae	B. mastomydis
B. vinsonii subsp. berkhoffii	100% ± 00	25.8% ± 2.45	27.1% ± 2.45	25.8% ± 2.4	25.9% ± 2.4	25.6% ± 2.4	25.5% ± 2.4
B. rattaustraliani		100% ± 00	25.5% ± 2.4	25.1% ± 2.4	27.5% ± 2.45	24.4% ± 2.4	24.2% ± 2.4
B. florencae			100% ± 00	26.7% ± 2.4	26.3% ± 2.45	26.8% ± 2.4	26.7% ± 2.4
B. tribocorum				100% ± 00	42% ± 2.55	37.3% ± 2.45	36.8% ± 2.5
B. queenslandensis					100% ± 00	37.6% ± 2.45	37.3% ± 2.5
B. elizabethae						100% ± 00	60.3% ± 2.8
B. mastomydis							100% ± 00

DDH, DNA-DNA hybridization; GGDC, Genome-to-Genome Distance Calculator; HSP, high-scoring segment pairs.

Confidence intervals indicate inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets (which are always limited in size). These results are in accordance with phylogenomic analyses as well as the GGDC results.

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Bartonella mastomydis sp. nov. that contains the strain 008. This bacterial strain has been isolated from M. erythroleucus blood samples from animals trapped in the Sine-Saloum region of Senegal.

Description of Bartonella mastomydis sp. nov. strain 008

Bartonella mastomydis (mas.to'my.dis, N.L. gen. n., mastomydis, ‘of Mastomys,’ isolated from Mastomys erythroleucus) is a nonmotile, Gram-negative rod. Growth is only obtained at 37°C. Colonies are opaque, grey and 0.5 to 1 mm in diameter on blood-enriched Columbia agar. Cells are rod shaped without flagella or pili. Length and width are 1369.8 ± 423.8 nm and 530.9 ± 105.8 nm respectively. Bartonella mastomydis strain 008 exhibits neither biochemical nor enzymatic activities. The type strain 008 is sensitive to rifampicin, amoxicillin, amoxicillin/clavulanic acid, oxacillin, nitrofurantoin, doxycycline, linezolid, tobramycin, gentamycin, imipenem, trimethoprim/sulfamethoxazole, fosfomycin and ciprofloxacin and resistant to metronidazole and colistin. The G + C content of the genome is 38.44%. The 16S rRNA gene sequence and whole-genome shotgun sequence of strain 008 are deposited in GenBank under accession numbers KY555064 and GCA_900185775 respectively. The type strain 008 (CSUR B643, DSM2802) was isolated from the rodent Mastomys erythroleucus trapped in the region of Sine-Saloum, Senegal.