Leptotrichia hongkongensis sp. nov., a novel Leptotrichia species with the oral cavity as its natural reservoir

Abstract

A straight, non-sporulating, Gram-variable bacillus (HKU24(T)) was recovered from the blood culture of a patient with metastatic breast carcinoma. After repeated subculturing in BACTEC Plus Anaerobic/F blood culture broth, HKU24(T) grew on brucella agar as non-hemolytic, pinpoint colonies after 96 h of incubation at 37 degrees C in an anaerobic environment and aerobic environment with 5% CO2. Growth was enhanced with a streak of Staphylococcus aureus. HKU24(T) was non-motile and catalase-negative, but positive for alkaline phosphatase, beta-glucosidase, and alpha-glucosidase. It hydrolyzed phenylphosphonate and reduced resazurin. 16S rRNA, groEL, gyrB, recA, and rpoB sequencing showed that HKU24(T) occupies a distinct phylogenetic position among the Leptotrichia species, being most closely related to Leptotrichia trevisanii. Using HKU24(T) groEL, gyrB, recA, and rpoB gene-specific primers, fragments of these genes were amplified from one of 20 oral specimens. Based on phenotypic and genotypic characteristics, we propose a new species, Leptotrichia hongkongensis sp. nov., to describe this bacterium.

Fig. 1

Gram smear and scanning electron micrograph of HKU24^T. (a) Gram smear showing a straight, non-sporulating, Gram-variable bacillus; (b) Scanning electron micrograph showing that the bacterium is aflagellate and straight Cells vary in length from 5.86 to 11.94 µm and diameter from 0.50 to 0.74 µm (mean=8.92 µm×0.62 µm, n=20)

Fig. 1

Gram smear and scanning electron micrograph of HKU24^T. (a) Gram smear showing a straight, non-sporulating, Gram-variable bacillus; (b) Scanning electron micrograph showing that the bacterium is aflagellate and straight Cells vary in length from 5.86 to 11.94 µm and diameter from 0.50 to 0.74 µm (mean=8.92 µm×0.62 µm, n=20)

Fig. 2

Phylogenetic relationship of 16S rRNA between the L. hongkongensis HKU24^T and other Leptotrichia species. The tree was inferred from 16S rRNA data by the neighbor-joining method and rooted using the 16S rRNA gene sequence of Fusobacterium nucleatum (FJ471645). Bootstrap values were calculated from 1000 trees. The scale bar indicates the estimated number of substitutions per 100 bases. Names and accession numbers are given as cited in the GenBank database

Fig. 3

Phylogenetic relationships of groEL (a), gyrB (b), recA (c), and rpoB (d) genes between the L. hongkongensis HKU24^T and other Leptotrichia species. The trees were constructed by the neighbor-joining method and bootstrap values were calculated from 1000 trees. The corresponding nucleotide sequences of Escherichia coli (NC_013364) were used as outgroups. The scale bar indicates the estimated number of substitutions per 50 bases. All names and accession numbers are given as cited in the GenBank database

Fig. 3

Phylogenetic relationships of groEL (a), gyrB (b), recA (c), and rpoB (d) genes between the L. hongkongensis HKU24^T and other Leptotrichia species. The trees were constructed by the neighbor-joining method and bootstrap values were calculated from 1000 trees. The corresponding nucleotide sequences of Escherichia coli (NC_013364) were used as outgroups. The scale bar indicates the estimated number of substitutions per 50 bases. All names and accession numbers are given as cited in the GenBank database

Fig. 3

Phylogenetic relationships of groEL (a), gyrB (b), recA (c), and rpoB (d) genes between the L. hongkongensis HKU24^T and other Leptotrichia species. The trees were constructed by the neighbor-joining method and bootstrap values were calculated from 1000 trees. The corresponding nucleotide sequences of Escherichia coli (NC_013364) were used as outgroups. The scale bar indicates the estimated number of substitutions per 50 bases. All names and accession numbers are given as cited in the GenBank database

Fig. 3

Phylogenetic relationships of groEL (a), gyrB (b), recA (c), and rpoB (d) genes between the L. hongkongensis HKU24^T and other Leptotrichia species. The trees were constructed by the neighbor-joining method and bootstrap values were calculated from 1000 trees. The corresponding nucleotide sequences of Escherichia coli (NC_013364) were used as outgroups. The scale bar indicates the estimated number of substitutions per 50 bases. All names and accession numbers are given as cited in the GenBank database

Fig. 4

Phylogenetic relationships of groEL (a), gyrB (b), recA (c), and rpoB (d) genes between the L. hongkongensis amplified from the oral specimen of the healthy volunteer in the present study and other Leptotrichia species. The trees were constructed by the neighbor-joining method and bootstrap values were calculated from 1000 trees. The corresponding nucleotide sequences of Escherichia coli (NC_013364) were used as outgroups. The scale bar indicates the estimated number of substitutions per 20 or 50 bases as indicated. All names and accession numbers are given as cited in the GenBank database

Fig. 4

Phylogenetic relationships of groEL (a), gyrB (b), recA (c), and rpoB (d) genes between the L. hongkongensis amplified from the oral specimen of the healthy volunteer in the present study and other Leptotrichia species. The trees were constructed by the neighbor-joining method and bootstrap values were calculated from 1000 trees. The corresponding nucleotide sequences of Escherichia coli (NC_013364) were used as outgroups. The scale bar indicates the estimated number of substitutions per 20 or 50 bases as indicated. All names and accession numbers are given as cited in the GenBank database

Fig. 4

Phylogenetic relationships of groEL (a), gyrB (b), recA (c), and rpoB (d) genes between the L. hongkongensis amplified from the oral specimen of the healthy volunteer in the present study and other Leptotrichia species. The trees were constructed by the neighbor-joining method and bootstrap values were calculated from 1000 trees. The corresponding nucleotide sequences of Escherichia coli (NC_013364) were used as outgroups. The scale bar indicates the estimated number of substitutions per 20 or 50 bases as indicated. All names and accession numbers are given as cited in the GenBank database

Fig. 4

Phylogenetic relationships of groEL (a), gyrB (b), recA (c), and rpoB (d) genes between the L. hongkongensis amplified from the oral specimen of the healthy volunteer in the present study and other Leptotrichia species. The trees were constructed by the neighbor-joining method and bootstrap values were calculated from 1000 trees. The corresponding nucleotide sequences of Escherichia coli (NC_013364) were used as outgroups. The scale bar indicates the estimated number of substitutions per 20 or 50 bases as indicated. All names and accession numbers are given as cited in the GenBank database