Porphyromonas bennonis sp. nov., isolated from human clinical specimens

Members of the genus Porphyromonas are indigenous bacterial flora in the oral cavity of humans and animals; many species are also found in the urogenital and intestinal tracts. Some species are considered true pathogens and are associated with human or animal infections. Porphyromonas species have been isolated from oral infections and from many infections throughout the body. Porphyromonas species of animal origin have been encountered in humans with animal-bite infections. During our investigation of the bacteriology of human wound infections and abscesses (Finegold et al., 2008), a novel anaerobic, non-spore-forming, Gram-negative bacillus related distantly to Porphyromonas somerae (Summanen et al., 2005) was frequently isolated. Comparative 16S rRNA gene sequencing studies demonstrated that the 14 novel strains were genotypically homogeneous, clustering within the phylogeny of the genus Porphyromonas. In this polyphasic taxonomic study, we describe the genotypic and phenotypic characterization of this organism, which represents a novel species of the genus Porphyromonas.

Fourteen strains isolated at the VA Wadsworth Medical Center were included in this study (Table 1⇓). All strains were characterized by using 16S rRNA gene sequencing and conventional biochemical methods, unless otherwise indicated. P. somerae ATCC BAA-1230^T was included in the biochemical and molecular characterizations. The strains were maintained at −70 °C in double-strength skimmed milk before characterization. The strains were grown at 37 °C on Brucella blood agar (Anaerobe Systems) or CDC blood agar (Becton Dickinson) under anaerobic conditions (N₂ : CO₂ : H₂, 90 : 5 : 5). All tests were performed on 24–72 h pure cultures, unless otherwise indicated.

The strains were characterized by using conventional biochemical tests (Durmaz et al., 1995; Jousimies-Somer, 1995; Jousimies-Somer et al., 2002; Holdeman et al., 1977), including the assessment of susceptibility to special potency identification discs, the fermentation of pre-reduced, anerobically sterilized biochemicals (Anaerobe Systems), GLC of metabolic end products, API ZYM and Rapid ID 32A (bioMérieux) and Rosco Diagnostic Tablets (Rosco). Pigment production was tested on laked blood agar (Anaerobe Systems). The production of β-lactamase was assessed by using nitrocefin-impregnated discs (Cefinase; Becton Dickinson). In all tests with pre-reduced, anerobically sterilized biochemicals, supplementation with haemin (0.001 %) and rabbit serum (1 %) was used to obtain growth. For cellular fatty acid analyses, the cells were grown on Brucella blood agar for 48 h. Growth was collected in 0.7 % (w/v) MgSO₄ and harvested by centrifugation (5000 g for 20 min); the pellets were processed immediately or kept frozen at −20 °C. Methyl ester extraction was carried out according to the manufacturer's procedure (MIDI; Microbial ID) and the analysis was performed by using a Microbial Identification System (model 5898A; Hewlett Packard) gas chromatograph, with software from MIDI. The corresponding library (anaerobe, version Moore 5.0) was used in successive analyses. All biochemical tests were performed in duplicate. The KOH string test was performed as described by Halebian et al. (1981).

Genomic DNA was extracted and purified from bacterial cells in the mid-exponential growth phase, using a QIAamp DNA Mini kit (Qiagen). 16S rRNA gene fragments were amplified by means of a PCR with universal primers. The PCR products were purified by using a QIAquick gel extraction kit (Qiagen). The purified PCR products were sequenced directly with BigDye Terminator cycle sequencing kits (Biotech Diagnostics) on an ABI 3100 Avant Genetic System (Applied Biosystems). The sequencing data were analysed by comparing the consensus sequences with sequences obtained from GenBank, using the Ribosomal Database Project (rdp-II) (Maidak et al., 2001) and blast software (Benson et al., 1999): similarity (%) with respect to other sequences was determined. Closely related sequences were retrieved from GenBank and were aligned with the newly determined sequences by using the program clustal w (Thompson et al., 1994). The phylogenetic tree was constructed by using the clustal w method, with the SeqBuilder and MegAlign program modules of lasergene 7 (DNASTAR); bootstrap analysis was also performed with MegAlign. The G+C content (mol%) of the DNA was determined by using HPLC according to the method of Mesbah et al. (1989).

The unidentified isolates were recovered from clinical infections as a part of the infecting flora together with other anaerobes or aerobes, generally at high counts (>10⁵ c.f.u. ml⁻¹) (Table 1⇑). The mean number of accompanying aerobes for each c.f.u. of the unidentified isolates was 1.8 and that for anaerobes was 4.1. Colonies grown on anaerobic blood agar plates for 48–72 h were small (0.2–0.5 mm in diameter), circular, entire, low convex, opaque and slightly β-haemolytic. At an early stage of growth, the colonies did not show pigmentation or fluorescence under long-wave UV light (366 nm); the colour of colonies ranged from grey to cream. After more prolonged incubation (≥10 days), a light beige to tan pigment developed; fluorescence remained negative. On Brucella blood agar, cells were generally Gram-negative, 0.7–1.0×1.0–6.0 μm in size and pleomorphic, occasionally with long filaments and swellings. Occasionally, cells appeared Gram-positive in young cultures; however, the string test was positive, indicating that they were Gram-negative. The novel isolates were all resistant to colistin and kanamycin special potency identification discs, and nine of the 14 strains tested were sensitive to the vancomycin disc. All of the isolates studied were lipase-, indole- and nitrate-negative and sensitive to bile. Catalase results were variable, with six of the 14 strains registering as catalase-positive. All strains hydrolysed gelatin. All were asaccharolytic and produced acetic acid and succinic acid, and occasionally trace amounts of iso-valeric acid and lactic acid, as metabolic end products. All strains were positive for alkaline phosphatase, esterase (weakly positive), leucine arylamidase, chymotrypsin (weakly positive), acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, α-glucosidase and N-acetyl-β-glucosaminidase, but were negative for cystine arylaminidase, trypsin, α-galactosidase, β-glucuronidase, β-glucosidase, α-mannosidase and α-fucosidase (in the API ZYM system). Esterase lipase, lipase and valine arylamidase activities were variable in the API ZYM test. Rosco diagnostic tablets indicated that the strains were positive for α-glucosidase, N-acetyl-β-glucosaminidase and ONPG, but were negative for α-fucosidase, trypsin, urease and aesculin. In the Rapid ID 32A system, β-galactosidase, β-galactosidase phosphate, α-glucosidase, N-acetyl-β-glucosaminidase, alkaline phosphatase, leucyl glycine arylamidase and alanine arylamidase were positive, whereas urease, arginine dihydrolase, α-galactosidase, β-glucosidase, α-arabinosidase, β-glucuronidase, mannosidase and raffinose fermentation, α-fucosidase and pyroglutamic acid arylamidase gave negative results. Other reactions with Rapid ID 32A were variably negative or weakly positive. β-Lactamase was produced by two of 13 strains (15 %). The main cellular fatty acids detected in the novel organism belonged to summed feature 5 (C_15 : 0 DMA and/or C_14 : 0 3-OH, which could not be separated by the method used; 23–40 % of the total fatty acids). Other cellular fatty acids (detected at levels in the range 9–15 % of total fatty acids) included C_12 : 0, C_14 : 0 and C_16 : 0. Cluster analysis of the principal components showed a Euclidian distance of <10 between the unidentified strains, thus indicating that they belong to the same species. Biochemical tests that are useful for differentiating this novel organism from other species and genera are listed in Table 2⇓. The key characteristics that serve to differentiate this novel organism from other Porphyromonas species are the negative α-fucosidase results, the positive N-acetyl-β-glucosaminidase and β-galactosidase results and the production of acetic and succinic acids as metabolic end products. In addition, the fact that the novel organism produces only weakly pigmented, small colonies on blood agar is also useful for differentiation (except with respect to Porphyromonas catoniae, which is non-pigmented; Willems & Collins, 1995).

In this polyphasic taxonomic study, the unidentified strains were related closely to each other genetically, as demonstrated by the 16S rRNA gene sequence similarities (99–100 %). Phylogenetic analysis demonstrated that the novel organism represents a distinct line of descent within the genus Porphyromonas, as exemplified by strain WAL 1926^T (Fig. 1⇓). The mean interspecies similarity within the Porphyromonas cluster is approximately 87 % (Paster et al., 1994); in GenBank, the closest match to the novel organism was P. somerae WAL 6690^T, at 89–90 % sequence similarity. There is no precise correlation between 16S rRNA gene sequence divergence (%) and species delineation, but it is generally recognized that divergence values of 3 % or more are significant (Stackebrandt & Goebel, 1994). Support for the distinctiveness of the unknown bacterium was also very evident from the phenotypic analyses.

Whilst the data from the phylogenetic analysis were consistent with the view that the unknown organism belongs to the genus Porphyromonas, several of the organism's phenotypic characteristics did not conform to the description of the genus (Willems & Collins, 1995). Rather like the members of the genus Porphyromonas, most of the novel isolates were sensitive to the special potency disc containing vancomycin. In addition, like most other human Porphyromonas species, the unknown organism was asaccharolytic. The organism produced very slightly pigmented colonies on laked blood agar only after prolonged incubation, whereas pigment production in most Porphyromonas species is evident after 2–4 days. Unlike most Porphyromonas species, which produce complex metabolic end-product profiles that include acetic, propionic, isobutyric, butyric and isovaleric acids, the novel organism produced moderate amounts of acetic and succinic acids, occasionally with trace amounts of iso-valeric acid and lactic acid. The cellular fatty acid profile of the novel organism is unlike that of any other Porphyromonas species, in that predominating cellular fatty acids described for the latter are iso-C_15 : 0, iso-C_17 : 0 3-OH and anteiso-C_15 : 0 (Brondz et al., 1989; Shah & Collins, 1988; Brondz & Olsen, 1991; Könönen et al., 1996; Summanen et al., 2005), whereas the main cellular fatty acid(s) in the former was C_15 : 0 DMA and/or C_14 : 0 3-OH (23–40 % of total fatty acids). In the case of iso-C_15 : 0, iso-C_17 : 0 3-OH and anteiso-C_15 : 0, the levels detected were very low (<1 %) or non-existent. Furthermore, the DNA G+C content of the unknown organism (58 mol%) is somewhat higher than those of recognized Porphyromonas species (44–55 mol%).

With an accurate identification scheme, this hitherto-unidentified organism may prove to be more common in clinical specimens than has been appreciated. During our comprehensive microbiological evaluation of infections of skin and soft tissue (Finegold et al., 2008), the novel organism was the most frequently isolated anaerobic, Gram-negative rod: 5 % (19 of 394) of specimens yielded this organism. Even though the novel organism was also isolated from areas above the diaphragm, we found that the isolates were predominantly recovered from patients with chronic infections of skin or soft tissue in the perirectal/buttock/groin regions (63 %). The most common predisposing condition in the patients with skin and soft-tissue infections was diabetes mellitus. There were several relatively serious infections in which the unidentified organism was part of the infecting flora at high counts (Table 1⇑). The full extent of the virulence of this newly identified organism remains to be determined, as it was always isolated from mixed culture. It is notable that 15 % of the novel strains were β-lactamase producers; in the genus Porphyromonas, this is a trait found only in P. somerae (Summanen et al., 2005), Porphyromonas uenonis (Finegold et al., 2004) and perhaps also P. catoniae (Könönen et al., 1996). In mixed infections, β-lactamase-producing species may protect other penicillin-susceptible bacteria by releasing free β-lactamase into their immediate environment (Brook, 2004).

Our studies indicate that this novel organism isolated from human sources clusters phylogenetically within the genus Porphyromonas, but is phenotypically and genetically different from recognized Porphyromonas species. We therefore propose the name Porphyromonas bennonis sp. nov. for this novel species.