Campylobacter sputorum subsp. bovis subsp. nov., isolated from cattle, and an emended description of Campylobacter sputorum

Abstract

Six urease-negative Campylobacter strains were isolated from cattle faeces over a 19-month period from 2009 to 2010. These strains were initially identified as Campylobacter sputorum by 16S rRNA gene and atpA typing. Initial studies characterizing these strains by multilocus sequence typing and genome sequencing further supported their classification as C. sputorum but indicated that these strains form a divergent clade within the species. A polyphasic study was undertaken here to clarify their taxonomic position. Phylogenetic analyses were performed based on 16S rRNA gene sequences and the concatenated sequences of 330 core genes, with the latter analysis also placing the six strains into a clade distinct from the three C. sputorum biovars. Pairwise digital DNA–DNA hybridization values identified these strains as C. sputorum, and the pairwise average nucleotide identity values were consistent with those observed between current Campylobacter subspecies pairs. Standard phenotypic testing was also performed. All strains are microaerobic, anaerobic, motile, Gram-negative and oxidase- and catalase-positive; cells are curved rods or spirals. Strains can be distinguished from the C. sputorum biovars by the presence of alkaline phosphatase activity and triphenyltetrazolium chloride reduction and absence of nitrate reduction. The data presented here show that these strains represent a novel subspecies within C. sputorum, for which the name C. sputorum subsp. bovis subsp. nov. (type strain RM8705^T=LMG 32300^T=CCUG 75470^T) is proposed.

Data Summary

One supplementary figure and four supplementary tables are provided in the online version of this article. All supplementary data are available through Figshare at https://doi.org/10.6084/m9.figshare.27105994.

Introduction

Campylobacter sputorum is a non-thermotolerant Campylobacter that has been recovered from cattle [1,2], sheep [3,4], swine [5] and dogs [6]. Although strains of this species have been isolated from humans [2,7, 8], its pathogenicity is presently undetermined. The taxonomic evolution of C. sputorum is long and complex. Originally isolated in 1914 from a human patient with bronchitis [9], it was named ‘Vibrio sputorum’ by Prévot [10] and subsequently characterized as a catalase-negative anaerobe [11]. The related species ‘Vibrio bubulus’ [12] and ‘Vibrio faecalis’ [3] were also described, with the latter being distinguishable from the other two by the presence of catalase activity. Loesche et al. [13] demonstrated that ‘V. sputorum’ and ‘V. bubulus’ were microaerophiles and proposed that these two taxa were subspecies of the same species; ‘V. sputorum var. sputorum‘ and ‘V. sputorum var. bubulus’ could be differentiated based on tolerance to both 3.5% (w/v) NaCl and 1% (w/v) bile. These two taxa were assigned to the genus Campylobacter by Véron and Chatelain in 1973 [14] and a third C. sputorum subspecies, C. sputorum subsp. mucosalis, was proposed by Lawson et al. [15,16]. C. sputorum was reorganized in 1985 by Roop et al. [17,18], who demonstrated that subspp. sputorum and bubulus could not be distinguished by DNA homology and that subsp. mucosalis had no significant DNA homology to the other two C. sputorum subspecies. This created the novel species Campylobacter mucosalis and reclassified the remaining two C. sputorum subspecies as C. sputorum biovars. ‘V. faecalis’ (later ‘Campylobacter faecalis’) was added as a third C. sputorum biovar [17]. The current taxonomic composition of C. sputorum was proposed in 1998 by On et al. [2], who determined that the salt and bile tolerance tests used to distinguish bvs. sputorum and bubulus were unreliable. These two biovars were combined into bv. sputorum, and a third biovar, the catalase-negative, urease-positive C. sputorum bv. paraureolyticus, was added. The three C. sputorum biovars (i.e., bvs. sputorum, faecalis and paraureolyticus) could be readily distinguished based on catalase and urease activities [2].

Six Campylobacter strains were recovered from cattle faeces in Monterey County, California, from March 2009 to October 2010 (Table S1, available in the online Supplementary Material). Initially known as C. sputorum by 16S rRNA gene sequencing [19], these strains were catalase-positive and urease-negative, the phenotype associated with C. sputorum bv. faecalis. However, additional characterization using a novel C. sputorum multilocus sequence typing (MLST) method demonstrated that the strain set comprised four sequence types that were distinct from those of C. sputorum bv. faecalis [19]; phylogenetic analysis also placed these sequence types into a clade well separated from the clade comprising the three C. sputorum biovars [19]. Four of the six cattle strains were typed using atpA gene sequencing and also shown to compose a clade separate from the three C. sputorum biovars but sister to C. sputorum bv. paraureolyticus, thus confirming the MLST results [20]. A complete, gap-free genome of a representative cattle-associated strain, RM8705^T, was constructed and compared to the complete genomes of C. sputorum bv. sputorum RM3237, C. sputorum bv. faecalis LMG 8532 and C. sputorum bv. paraureolyticus LMG 17589 [21]. The four genomes are relatively colinear, with a high percentage of shared gene content. Nevertheless, while average nucleotide identity (ANI) values ranged between 98 and 99% among the three established biovars, pairwise values between these biovars and strain RM8705^T were 94–95% [21]. Additionally, digital DNA–DNA hybridization (dDDH) values were 92–95% between the three biovars but 82–87% when these biovars were compared to strain RM8705^T [21]. These dDDH values were all above the 70% threshold recommended to define novel species [22]. However, the ANI values were slightly lower than the proposed 95% value recommended for species delineation [23,24] but similar to ANI values observed between other Campylobacter subspecies pairs. This suggested that strain RM8705^T and perhaps the other five cattle-associated C. sputorum strains were members of a novel C. sputorum subspecies [21]. We present here a polyphasic study that provides further evidence that the six cattle-associated strains represent a novel C. sputorum subspecies.

Genomic analysis and taxonomic placement

The five remaining cattle-associated C. sputorum strains were sequenced here to draft level using Illumina MiSeq sequencing, as described by Miller et al. [25]. The genome metrics are presented in Table S1. Proteins predicted to be encoded by the draft genomes were identified by GeneMark [26]. The draft proteomes were compared to the proteomes of C. sputorum bv. sputorum LMG 7795^T, C. sputorum bv. sputorum RM3237, C. sputorum bv. faecalis LMG 8532, C. sputorum bv. paraureolyticus LMG 17589 and C. sputorum strain RM8705^T by pairwise BLASTP analysis. A high conservation in gene content was observed among the six cattle-associated strains: 96.8% of the genes identified in strain RM8705^T were also identified in each of the other five strains. Additionally, loci described as absent in strain RM8705^T [21], when strain RM8705^T was compared to the C. sputorum bv. strains, are also missing from the other five cattle-associated strains. These include a ttrACBSR tetrathionate reductase locus, the ceuBCDE-exbBD-tonB ferric enterobactin transporter locus and a 15-gene locus putatively encoding an allophanate hydrolase, radical S-adenosyl methionine-associated transferases, membrane proteins and rhodanese and Fido/AMPylation domain-containing proteins. As expected, all six genomes contain the katA catalase gene and do not encode any urease locus proteins. The 16S rRNA genes of these six strains are 1742 bp, which are longer than most 16S rRNA genes typically observed in Campylobacter but consistent with those reported previously in C. sputorum [2,27, 28]. These larger 16S rRNA genes are the result of structured intervening sequences (IVSs) in helix 10 of the 5′ major domain. Such IVSs are also found at the same position within the 16S rRNA genes of some strains in other Campylobacter species, e.g., Campylobacter curvus and Campylobacter helveticus [29,30].

16S rRNA gene and core gene sequence phylogenetic analyses were performed using the six cattle-associated C. sputorum strains and the full set of Campylobacter type strains. 16S rRNA genes were aligned using Clustal X. The nucleotide sequences of 330 core genes were extracted from the C. sputorum and Campylobacter type strain genomes and aligned individually using muscle [31] in Geneious (ver. 2024.0.5); within Geneious, the 330 core gene alignments were concatenated alphabetically by gene name into a single alignment. The core genes used and their cognate locus tags are listed in Table S2. Phylogenetic trees were constructed within mega version 6.06 [32] using the neighbour-joining method [33], the Kimura 2-parameter distance estimation method [34] and 1000 bootstrap replicates. The 16S rRNA gene sequences of the cattle-associated C. sputorum strains are nearly identical (~98% similarity; data not shown) to those of the three C. sputorum biovars (Fig. 1). With the exception of one single-nucleotide polymorphism (SNP), the observed differences between the C. sputorum 16S rRNA gene sequences reside in the IVSs. Analysis of the C. sputorum IVSs shows that those from the six cattle-associated strains are identical and that multiple SNPs can be observed when these IVSs are compared to those of the C. sputorum biovars (Fig. S1A). These sequence differences are also reflected in the predicted two-dimensional structures of the IVSs (Fig. S1B–D). Therefore, although the C. sputorum 16S rRNA genes are nearly indistinguishable phylogenetically, they can be sorted into two discrete groups on the basis of the sequence and structure of the 16S rRNA gene internal spacer. Core gene phylogeny demonstrates that the six novel strains can be clearly distinguished from the other Campylobacter species and form a discrete sister clade to the three C. sputorum biovars (Fig. 2). These data are consistent with previous studies using MLST or atpA typing, in which these strains are also composed of clades similar to but distinct from C. sputorum.

Fig. 1. 16S rRNA gene phylogenetic tree showing the position of C. sputorum subsp. bovis subsp. nov. within the genus Campylobacter. Included in the tree are the 16S rRNA gene sequences of the six subsp. bovis strains and Campylobacter type strain 16S rRNA gene sequences. Bootstrap values of ≥75%, generated from 1000 replicates, are shown at the nodes. GenBank accession numbers (in parentheses) are provided for each strain. The Campylobacter magnus type strain contains two different 16S rRNA gene sequences and both are included in the tree. The Helicobacter pylori type strain was used to root the tree. The scale bar represents nucleotide sequence divergence.

Fig. 2. Core genome phylogeny of C. sputorum subsp. bovis subsp. nov. within the genus Campylobacter. Phylogeny is based on the concatenated nucleotide sequence alignment of 330 core genes and compares the six subsp. bovis strains, the three C. sputorum subsp. sputorum subsp. nov. biovars and Campylobacter type strains. Bootstrap values of >75%, generated from 1000 replicates, are shown at the nodes. The Helicobacter pylori type strain was used to root the tree. The scale bar represents nucleotide sequence divergence.

These phylogenetic analyses further indicated that the six cattle-associated strains are either C. sputorum or highly related to C. sputorum. To clarify the taxonomic position of the six strains, ANI [23] and dDDH [35] analyses were performed. A dDDH value of 70% for species delineation is approximately equivalent to an ANI value of 95% [23,24]. dDDH analyses were performed using the Genome-to-Genome Distance Calculator (GGDC ver. 3.0; https://ggdc.dsmz.de/ggdc.php# [36,37]); GGDC formula 3 was used for all calculations, as recommended by the latest minimal standards for Campylobacter [38]. Pairwise dDDH values between each of the six strains and the C. sputorum biovars are >70% (81.2–86.7%; Table 1), indicating the placement of all six cattle strains within C. sputorum. ANI analyses were performed here using JSpecies (ver. 1.2.1) [24]. Pairwise ANI values between the six strains and the three C. sputorum biovars ranged from 94.5 to 95.5% (Table 1), which is either slightly below or slightly above the species delineation threshold. However, within Campylobacter, pairwise ANI values between the six established subspecies pairs range from 91.9 to 96.1% (Table S3A). The pairwise ANI values between the six strains and the three C. sputorum biovars fall within this range. Moreover, the relatively low dDDH values described above are also consistent with the dDDH values observed between current Campylobacter subspecies (Table S3A).

Table 1. Pairwise dDDH and ANI values between C. sputorum subsp. bovis subsp. nov. and related Campylobacter taxa.

dDDH	RM8705T		RM11259		RM11302		RM12321		RM12327		RM13538
C. sputorum bv. paraureolyticus LMG 17589	86.7		86.6		85.2		86.5		85.6		86.4
C. sputorum bv. faecalis LMG 8532	85.2		85.3		83.9		85.1		84.7		85.1
C. sputorum bv. sputorum ATCC 35980T	82.4		83.4		81.2		82.5		82.2		82.4
C. ureolyticus LMG 6451T	14.6		14.4		14.5		14.4		14.5		14.5
C. blaseri LMG 30333T	14.3		14.2		14.2		14.2		14.3		14.2
C. corcagiensis LMG 27932T	14.3		14.3		14.2		14.2		14.3		14.2
C. portucalensis LMG 31504T	14.3		14.4		14.2		14.3		14.3		14.3
ANI	1	2	3	4	5	6	7	8	9	10	11	12
C. sputorum bovis RM8705T	–	100	100	99.8	99.8	99.7	94.6	95.4	94.9	70.6	70.3	70.3
C. sputorum bovis RM12321	100	–	100	99.8	99.8	99.7	94.6	95.4	95.0	70.8	70.3	70.3
C. sputorum bovis RM13538	100	100	–	99.9	99.8	99.7	94.7	95.5	95.0	70.7	70.4	70.2
C. sputorum bovis RM12327	99.8	99.8	99.9	–	99.8	99.8	94.6	95.3	94.9	70.9	70.3	70.2
C. sputorum bovis RM11259	99.8	99.8	99.8	99.8	–	99.7	94.7	95.3	95.0	70.9	70.3	70.3
C. sputorum bovis RM11302	99.7	99.7	99.7	99.8	99.7	–	94.6	95.3	95.0	70.7	70.3	70.1
C. sputorum sputorum ATCC 35980T	94.5	94.6	94.5	94.5	94.6	94.5	–	97.8	98.6	71.2	70.4	70.4
C. sputorum paraureolyticus LMG 17589	95.3	95.3	95.3	95.3	95.3	95.3	97.9	–	97.9	70.6	70.4	70.2
C. sputorum faecalis LMG 8532	94.7	94.8	94.8	94.9	94.8	94.8	98.6	97.9	–	70.7	70.5	70.3
C. portucalensis LMG 31504T	70.7	70.7	70.7	70.9	70.9	70.7	70.9	70.4	70.4	–	73.3	71.7
C. ureolyticus LMG 6451T	70.5	70.5	70.4	70.3	70.5	70.5	70.4	70.5	70.5	73.6	–	72.1
C. blaseri LMG 30333T	70.4	70.4	70.4	70.3	70.4	70.4	70.4	70.3	70.4	72.0	72.1	–

Pairwise dDDH values between the C. sputorum subsp. bovis type strain and other Campylobacter taxa >14% and pairwise ANI values between C. sputorum subsp. bovis and other Campylobacter taxa >70% are shown.

Therefore, these results suggest that the six strains recovered from cattle form a novel C. sputorum subspecies, for which we propose the name C. sputorum subsp. bovis subsp. nov. The creation of this subspecies would move the current C. sputorum bv. strains into the novel subspecies C. sputorum subsp. sputorum subsp. nov., with C. sputorum subsp. sputorum bv. sputorum strain LMG 7795^T as the type strain of the subspecies. While not a requirement for defining novel subspecies, it is also noteworthy that there is a consistent difference in the G+C content between the two proposed subspecies, with subsp. bovis values ranging between 29.19 and 29.26 mol% and subsp. sputorum values ranging between 29.63 and 29.72 mol% (Table S3B). Although these G+C content data may change with the addition of new genomic data, it provides further supporting evidence of the taxonomic separation of the two proposed subspecies.

Morphology and phenotypic characterization

Cells from these strains are motile with a cellular morphology typical of other Campylobacter species, i.e., a mixture of curved rods and spiral cells (Fig. 3). The six C. sputorum subsp. bovis subsp. nov. strains were characterized using the standard phenotypic tests defined in the minimal standards for Campylobacter [38]. Strains were tested for: growth at 30, 37 and 42 °C under microaerobic conditions; growth at 37 °C under aerobic and anaerobic conditions; motility; selenite, indoxyl acetate and triphenyltetrazolium chloride (TTC) reduction; oxidase, catalase, alkaline phosphatase, hippuricase, urease and nitrate reductase activity; growth on modified charcoal–cefoperazone–deoxycholate agar (mCCDA) plates or media amended with 2% (w/v) NaCl, 1% (w/v) glycine or 0.04% (w/v) TTC; α-haemolysis on anaerobe basal agar (ABA; Oxoid) amended with 5% lysed horse blood (Innovative Research, Novi, MI) [ABA with blood (ABA-B)]; H₂S production on triple sugar iron (TSI) agar; and resistance to 30 mg l⁻¹ nalidixic acid or 30 mg l⁻¹ cephalothin. All tests were performed in triplicate using appropriate positive and negative controls [39]. The results of these phenotypic tests are presented in Table 2. All strains: could grow under microaerobic conditions at 37 and 42 °C or under anaerobic conditions at 37 °C, but not aerobically or at 30 °C microaerobically; were motile; were oxidase-, catalase- and alkaline phosphatase-positive and urease- and hippuricase-negative; did not reduce nitrate but could reduce both selenite and TTC; produced H₂S on TSI agar; did not hydrolyse indoxyl acetate; could grow on media supplemented with 2% (w/v) NaCl or 1% (w/v) glycine and weakly on media supplemented with 0.04% (w/v) TTC and were resistant to both 30 mg l⁻¹ nalidixic acid and 30 mg l⁻¹ cephalothin.

Fig. 3. Scanning electron micrograph images of C. sputorum subsp. bovis subsp. nov. strains (a) RM8705^T (=LMG 32300^T) and (b) RM11302 (=LMG 32301) at ×10 000 magnification. Sample preparation and microscopy were performed as described by Miller et al. [39].

Table 2. Phenotypic characteristics of C. sputorum subsp. bovis subsp. nov. and related Campylobacter species.

	1	2	3	4	5	6	7	8	9	10
Motility	+	+	+	+	−	−	−	−	−	−
Temperature (atmosphere)
37 °C (aerobic)	−	−	−	−	−	−	−	−	−	−
30 °C (microaerobic)	−	M	M	M	+	+	+	−	U	+
37 °C (microaerobic)	+	+	+	+	+	+	+	+	+	+
42 °C (microaerobic)	+	V	V	V	+	+	−	−	+	V
37 °C (anaerobic)	+	+	+	+	+	+	+	+	w	+
Oxidase	+	+	+	+	+	+	+	+	+	+
Catalase	+	−	+	−	+	+	+	−	−	F
Urease	−	−	−	+	+	+	−	−	−	+
Alkaline phosphatase	+	−	−	−	+	+	−	−	U	−
Hippuricase	−	−	−	−	−	−	+	−	−	−
Indoxyl acetate hydrolysis	−	−	−	−	+	V	−	−	−	F
Reduction:
Nitrate	−	+	M	+	+	M	+	V	−	+
Selenite	+	V	V	V	U	U	−	−	U	−
TTC	+	−	−	−	U	U	−	−	U	−
H2S production on TSI	+	+	+	+	+	+	−	−	−	−
α−Haemolysis	−	+	+	+	−	−	−	−	−	V
Growth on:
2% (w/v) NaCl	+	+	+	+	U	+	+	U	−	+
1% (w/v) glycine	+	+	+	+	w	+	+	+	V	+
0.04% (w/v) TTC	w	−	−	−	U	−	U	−	−	−
mCCDA	V	M	M	M	−	U	+	U	U	V
Resistance to:
Nalidixic acid (30 mg l−1)	R	V	V	V	S	R	R	V	U	S
Cephalothin (30 mg l−1)	R	S	S	S	S	S	S	S	U	S

Species: 1, C. sputorum subsp. bovis subsp. nov. (n = 6); 2, C. sputorum bv. sputorum; 3, C. sputorum bv. faecalis; 4, C. sputorum bv. paraureolyticus; 5, Campylobacter blaseri; 6, Campylobacter corcagiensis; 7, Campylobacter geochelonis; 8, Campylobacter hominis; 9, Campylobacter portucalensis and 10, Campylobacter ureolyticus. Positive strains: + (95−100%), M (70−95%), V (30−70%), F (10−30%), − (0−10%); w, weak growth; for antibiotic resistance, S/V/R indicates sensitive, variable or resistant, respectively; U, unknown/data not available. The complete comparison within the genus Campylobacter is shown in Table S4. Data for columns 2−10 are derived from the original species descriptions and/or On et al. [38].

Strains from both proposed subspecies share multiple phenotypic features that together distinguish them from the other Campylobacter taxa (Tables 2 and S4). Phenotypic profiles unique to each subspecies were also observed. As shown here and as described previously [21], the C. sputorum subsp. bovis strains are uniformly catalase-positive and urease-negative, a phenotype proposed by On et al. [2] to classify such strains within C. sputorum as bv. faecalis. However, the C. sputorum subsp. bovis strains do not reduce nitrate and demonstrate alkaline phosphatase activity, reduction of TTC and resistance to cephalothin (Table 2) and are thus clearly distinct from C. sputorum subsp. sputorum. The absence of nitrate reduction is noteworthy since all six subsp. bovis strains contain complete, untruncated nitrate reductase (napAGHBFLD) loci. However, alignment of the C. sputorum nitrate reductase proteins identified multiple amino acid substitutions within the subsp. bovis proteins when they were compared to those of subsp. sputorum (data not shown). One or more of these substitutions, as well as possible point mutations in the upstream promoter region, may potentially lead to a loss of nitrate reductase activity. Further work will be necessary to clarify the absence of nitrate reduction in C. sputorum subsp. bovis.

Emended description of Campylobacter sputorum (Prévot 1940) Véron and Chatelain 1973 (Approved Lists 1980)

The species description is as previously described by On et al. [2], with the following emendations. Some strains demonstrate alkaline phosphatase activity, reduce TTC, do not reduce nitrate and grow weakly on blood agar amended with 0.04% TTC. Strains of C. sputorum may be assigned to C. sputorum subsp. sputorum or C. sputorum subsp. bovis according to their alkaline phosphatase activity and reduction of TTC. C. sputorum subsp. sputorum strains do not demonstrate alkaline phosphatase activity and do not reduce TTC, while C. sputorum subsp. bovis strains demonstrate alkaline phosphatase activity and reduce TTC. C. sputorum subsp. sputorum strains may be further assigned to one of three biovars based on their catalase and urease activities, as previously described [2].

Description of Campylobacter sputorum subsp. sputorum subsp. nov.

Campylobacter sputorum subsp. sputorum (spu.to’rum. L. neut. n. sputum, spit, sputum; L. gen. pl. n. sputorum, of sputa). Strains conform to the description of C. sputorum, as previously described by On et al. [2].

Description of Campylobacter sputorum subsp. bovis subsp. nov.

Campylobacter sputorum subsp. bovis (bo’vis. L. gen. n. bovis, of a cow, of bovine). Gram-negative cells are motile with a curved or spiral morphology. After 72 h culture at 37 °C under microaerobic conditions on ABA-B, colonies glisten, are opaque, convex and circular with entire margins and are 1–2 mm in diameter. Growth occurs on ABA-B at both 37 and 42 °C under microaerobic conditions and at 37 °C under anaerobic conditions. No growth on ABA-B at 30 °C under microaerobic conditions or at any temperature under aerobic conditions. All strains have oxidase, catalase and alkaline phosphatase activities but no urease activity. Strains do not hydrolyse indoxyl acetate or hippurate or reduce nitrate. All strains reduce selenite and TTC. Produces H₂S on TSI agar. Growth is supported on ABA-B with a final NaCl concentration of 2% (w/v) and on ABA-B amended with 1% (w/v) glycine. Strains demonstrate weak growth on ABA-B amended with 0.04% TTC. Strains are resistant to 30 mg l⁻¹ cephalothin and 30 mg l⁻¹ nalidixic acid. Pathogenicity is unknown. Six strains were isolated from cattle. The G+C content of its DNA is 29.2–29.3 mol%. The type strain is RM8705^T (=LMG 32300^T = CCUG 75470^T), recovered in 2009 from cow faeces in California. Accession numbers for the 16S rRNA gene and genome sequence of the type strain are MW157372 and CP019685, respectively.

Abbreviations

ABA

anaerobe basal agar

ABA-B

anaerobe basal agar with blood

ANI

average nucleotide identity

dDDH

digital DNA–DNA hybridization

GGDC

Genome-to-Genome Distance Calculator

IVS

intervening sequence

mCCDA

modified charcoal–cefoperazone–deoxycholate agar

MLST

multilocus sequence typing

TSI

triple sugar iron

TTC

triphenyltetrazolium chloride