Porphyromonas: A neglected potential key genus in human microbiomes.

Anaerobes form a large part of microbial communities, and have begun to be specifically studied in both healthy and pathologic contexts. Porphyromonas is one of the top ten anaerobic taxa in the microbiome (anaerobiome) in healthy subjects. However, to date, most studies focused on the deleterious role of P. gingivalis, the most widely described species. Interestingly, targeted metagenomics reveals Porphyromonas other than gingivalis (POTG), highlighting other species such as P. catoniae or P. pasteri as potential biomarkers in disease progression or pathogen colonization susceptibility. From the sparse data, it appears that the Porphyromonas genus may also be a relevant target of investigation in several pulmonary diseases. Moreover, deciphering cutaneous, gastric and oral microbiomes hint that Porphyromonas may be a genus of interest in non-pulmonary diseases. This review aims to summarize the major data on POTG and to report their impact on the various human microbiomes in different clinical states.

In Vitro Activities of Pexiganan and 10 Comparator Antimicrobials against 502 Anaerobic Isolates Recovered from Skin and Skin Structure Infections.

Pexiganan, a cationic peptide, exhibited a broad range of anti-anaerobic antimicrobial activity. The MIC90s of studied isolates were as follows: Bacteroides fragilis, 16 µg/ml; other B. fragilis group spp., 4 µg/ml; Prevotella and Fusobacterium spp., 32 µg/ml; Porphyromonas spp., 64 µg/ml; Propionibacterium acnes, 4 µg/ml; Eggerthella lenta and Peptostreptococcus anaerobius, 32 µg/ml; other Gram-positive rods and cocci, 4 µg/ml; Clostridium perfringens, 128 µg/ml; and other clostridia, 256 µg/ml. Pexiganan cream shows potential as adjunctive therapy for skin and skin structure infections (SSSIs) involving anaerobes.

A reproducible microcosm biofilm model of subgingival microbial communities.

OBJECTIVE: To develop a reproducible subgingival microcosm biofilm model. MATERIAL AND METHODS: Subgingival plaque samples were collected from four deep pockets (probing pocket depth ≥6 mm) in each of seven patients with periodontitis and from shallow pockets (probing pocket depth ≤3 mm) in two periodontally healthy donors. An active attachment model and a peptone medium (Thompson et. al., Appl Environ Microbiol 2015;81:8307-8314) supplemented with 30% serum was used. Biofilms were harvested at 2 and 4 weeks. DNA of dead cells was blocked for amplification by propidium monoazide treatment. Composition was analyzed using 16S rRNA gene amplicon pyrosequencing. Similarities between the biofilm samples were assessed by non-metric multidimensional scaling using the Bray-Curtis similarity index and similarity percentage analysis. Data from duplicate experiments, different biofilm sources and different biofilm age were compared. RESULTS: The non-metric multidimensional scaling revealed a strong clustering by the inoculum source, the donor and their periodontal status. Statistically significant differences were found between the sources of inoculum (P=.0001) and biofilm age (P=.0016). Furthermore, periodontitis biofilms (P) were distinct in composition from health-derived biofilms (H) by genera: Porphyromonas (P=19%; H=0%), Filifactor (P=10%; H=0%), Anaeroglobus (P=3%; H=0%), Phocaeicola (P=1.5%; H=0%), Parvimonas (P=19%; H=14%), Fusobacterium (P=2%; H=26%), Peptostreptococcus (P=20%; H=30%), Veillonella (P=7%; H=8%) and 57 other genera. Similarity distances (Bray-Curtis) (mean 0.73, SD 0.15) and the Shannon diversity index (mean 2, SD 0.2) revealed no differences between duplicate experiments (P=.121). CONCLUSION: This biofilm model allows reproducible production of complex subgingival microbial communities.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages.

Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 µM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 µM (p<0.05) and 250 µM (p<0.01). The POH increased ROS production at both 10 µM and 100 µM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 µM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 µM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Impact of the CFTR-potentiator ivacaftor on airway microbiota in cystic fibrosis patients carrying a G551D mutation.

BACKGROUND: Airway microbiota composition has been clearly correlated with many pulmonary diseases, and notably with cystic fibrosis (CF), an autosomal genetic disorder caused by mutation in the CF transmembrane conductance regulator (CFTR). Recently, a new molecule, ivacaftor, has been shown to re-establish the functionality of the G551D-mutated CFTR, allowing significant improvement in lung function. OBJECTIVE AND METHODS: The purpose of this study was to follow the evolution of the airway microbiota in CF patients treated with ivacaftor, using quantitative PCR and pyrosequencing of 16S rRNA amplicons, in order to identify quantitative and qualitative changes in bacterial communities. Three G551D children were followed up longitudinally over a mean period of more than one year covering several months before and after initiation of ivacaftor treatment. RESULTS: 129 operational taxonomy units (OTUs), representing 64 genera, were identified. There was no significant difference in total bacterial load before and after treatment. Comparison of global community composition found no significant changes in microbiota. Two OTUs, however, showed contrasting dynamics: after initiation of ivacaftor, the relative abundance of the anaerobe Porphyromonas 1 increased (p<0.01) and that of Streptococcus 1 (S. mitis group) decreased (p<0.05), possibly in relation to the anti-Gram-positive properties of ivacaftor. The anaerobe Prevotella 2 correlated positively with the pulmonary function test FEV-1 (r=0.73, p<0.05). The study confirmed the presumed positive role of anaerobes in lung function. CONCLUSION: Several airway microbiota components, notably anaerobes (obligate or facultative anaerobes), could be valuable biomarkers of lung function improvement under ivacaftor, and could shed light on the pathophysiology of lung disease in CF patients.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

The efficacy of tinidazole in naturally occurring periodontitis in dogs: bacteriological and clinical results.

The efficacy of tinidazole in addition to a single course of scaling was studied in 14 dogs with periodontitis. Three test teeth, two with periodontitis and one with healthy periodontium, were selected per dog. Subgingival bacterial samples were taken, and clinical examination was carried out at each of four visits (0, 14, 90 and 180 days). The bacterial samples were cultured anaerobically using selective and nonselective media. All teeth in the dentition, except one diseased test tooth with periodontitis were scaled at the first visit and tinidazole or placebo administered twice a day for 7 days. The mean probing depth of the diseased test pockets was significantly more reduced in tinidazole+scaling (T1S1) than scaling alone (T0S1) group at all visits compared to baseline values. The most significant decreases of bacterial counts in T1 or T0 groups were found in agar corroding Gram-negative rods in both diseased and healthy T1S1 pockets and in slimy Gram-negative rods in diseased T1S0 pockets between visits 1 and 2. The intergroup comparisons showed that Gram-positive cocci decreased significantly more in S1 pockets as well as sporeforming Gram-positive rods in diseased S0 pockets of T1 than T0 group. The highest number of isolates was found in the group of pigmented Gram-negative anaerobe rods, mainly Porphyromonas spp. The percentage of Porphyromonas gingivalis-like isolates decreased to zero and Porphyromonas endodontalis-like isolates increased in all test teeth of T1 group at 14 days. It is concluded that tinidazole has good efficacy against P. gingivalis-like bacteria which seem to be periodontal pathogens in dogs.

Associations amongst three feline Porphyromonas species from the gingival margin of cats during periodontal health and disease.

Digoxigenin labelled whole chromosomal DNA probes directed against three feline members of the genus Porphyromonas (P. gingivalis VPB 3492, P. circumdentaria NCTC 12469T and P. salivosa VPB 3313) were used to identify and quantify organisms in samples taken from the gingival margins of 40 domestic cats with different grades of periodontal disease. At the right upper canine tooth, the grade of periodontal disease ranged from 0 to 5 and the cfu of facultative/obligate anaerobes ranged from 5.5 x 10(4) to 2.0 x 10(6)). In 38 of the 40 cats, at least one of the three Porphyromonas species was isolated and regression analysis showed that the cfu of total Porphyromonas sp. was a highly significant indicator of the grade of periodontal disease (p < 0.001, R2 0.510). Feline P. gingivalis was isolated from 37 of the 40 cats and regression analysis showed that it was a highly significant predictor of the grade of periodontal disease (p < 0.001, R2 0.561). The cfu of P. salivosa was a significant predictor of the grade of periodontal disease (p < 0.001, R2 0.286) and regression analysis showed that there was a significant positive relationship between cfu of P. circumdentaria and grade of periodontal disease (p = 0.018, R2 0.116). The periodontal grades at the right upper third premolar tooth ranged from 0 to 6. The cfu of facultative/obligate anaerobes isolated ranged from 1.2 x 10(5) to 7.9 x 10(6), and regression analysis showed that cfu was a significant predictor of periodontal grade (p < 0.001, R2 0.378). The cfu of total Porphyromonas species ranged from 1.2 x 10(4) to 1.7 x 10(6) and regression analysis of the cfu against the grade of periodontal disease showed a highly significant association (p < 0.001, R2 0.633). The cfu of P. gingivalis ranged from 0 to 1.1 x 10(6) and regression analysis of the cfu of P. gingivalis against the grade of periodontal disease showed a highly significant association (p < 0.001, R2 0.439). The cfu of P. salivosa was a significant predictor of the grade of periodontal disease (p < 0.001, R2 0.479) and the same association was found between cfu of P. circumdentaria and grade of periodontal disease (p = 0.002, R2 0.204). This study has established Porphyromonas as anumerically significant and highly prevalent genus in feline periodontal disease.

Effects of combined topical metronidazole and mechanical treatment on the subgingival flora in deep periodontal pockets in cuspids and bicuspids.

The Effect on the subgingival microflora of a single topical administration of a 95% collagen and 5% metronidazole device in combination with debridement was investigated in 30 adult periodontitis patients in comparison with mechanical treatment alone. For each patient, plaque samples from test and control sites in cuspids and bicuspids were collected for culture and enumeration of total anaerobically cultivable bacteria (TA), black-pigmented anaerobes (BPA), and Actinobacillus actinomycetemcomitans (Aa). Spirochetes and fusiforms were quantified by direct microscopic examination after Giemsa staining. A decrease was observed for all parameters, and a significant difference in comparison with the control group was found for fusiforms. After treatment, a lower number of Aa positive sites were observed in the test group (13/25). These results show that a single application of topical metronidazole seems to be effective as adjunctive antimicrobial treatment in adult periodontitis.

Microbiota associated with experimental peri-implantitis and periodontitis in adult Macaca mulatta monkeys.

This study examines the microbiota associated with the progression of experimental peri-implantitis and periodontitis induced concurrently in partially edentulous adult monkeys. Root-form and plate-form implants with fixed prosthesis in place for at least 12 months and their corresponding opposite molar teeth were ligated for 6 months. The microbiota in plaque around these ligated dental implants and molars were studied at 0, 1, 2, 3, and 6 months post-ligation. Plaque samples were analyzed by dark-field microscopy and selective and non-selective culture. Putative periodontal pathogens were detected as a major component of the microbiota cultured from plaque samples obtained from experimental peri-implantitis sites. Overall, the types and relative proportions of putative periodontal pathogens in plaque associated with ligature-induced peri-implantitis and ligature-induced periodontitis were similar. Only levels of anaerobic Actinomyces and spirochetes were significantly different between both sites. Spirochete levels were significantly higher at peri-implantitis sites when compared with levels at periodontitis sites after 6 months, and spirochete levels increased significantly between 0 and 6 months post-ligation at implant sites. Levels of spirochetes correlated significantly with probing depth and bone loss at peri-implantitis sites. Overall, Actinomyces levels were higher at periodontitis sites. Porphyromonas species were not detected continuously as part of the peri-implantitis microbiota. In conclusion, this study finds that the microbiota associated with the progression of experimental peri-implantitis and periodontitis occurring concurrently in partially edentulous mouths are similar.

Development of an anaerobic bacterial leakage model.

The majority of bacteria associated with infections of endodontic origin are strict anaerobes. The purpose of this study was to develop an endodontic microleakage model using strict anaerobic bacteria in a two-chamber system. Nine species of anaerobic bacteria were tested for viability and detection by either turbidity or color change of the broth. A survey of pH chromogenic substrates revealed that bromcresol purple (pH 5.2 = yellow, pH 6.8 = purple) could be used as a chromogenic indicator to detect the growth of anaerobic bacteria. Peptone-yeast extract-glucose broth (PYG) and brain heart infusion broth (BHI) were each used alone and with bromcresol purple (bpPYG, bpBHI) in this study. Fusobacterium nucleatum and F. necrophorum were viable in all four media for > 2 wk and produced both turbidity and a color change after only 1 day of incubation. Veillonella parvula in either bpBHI or BHI and Peptostreptococcus anaerobius in either bpPYG or BHI were viable for > 2 wk and showed a color change or turbidity after 1 or 2 days. The results indicate that leakage of strict anaerobes may be evaluated in a two-chamber system.

Viable bacteria in root dentinal tubules of teeth with apical periodontitis.

Two sets of teeth with apical periodontitis were collected at different geographic locations to study the identity of bacteria left in the root dentinal tubules. Root dentin of 20 of these teeth was cultured from three locations between pulp and cementum (A, B, and C). In addition dentin from eight teeth was examined histologically. Using the culturing technique bacteria were found in 77% of the dentin samples from set 1 (Amsterdam) and in 87.5% of the dentin samples from set 2 (Glasgow). At greater distance, in layer C, from the pulp bacteria were found in 62% (13 of 21) of the dentin samples. Twenty-three percent (3 of 13) of set 1 and 25% (2 of 8) of set 2 contained >50,000 colony-forming units/mg of dentin in layer C. In layers closer to the pulp higher numbers of anaerobic bacteria and gram-positive rods were found, as well as a larger number of bacterial species. Histological sections showed bacterial penetration in dentinal tubules in 5 of 8 teeth. In the other three teeth where the colony-forming units/mg recovered was <10,000, no histological signs of tubule penetration was seen. It seems clear that, in more than half of the infected roots, bacteria are present in the deep dentin close to the cementum and that anaerobic culturing of dentin is more sensitive than histology to detect these bacteria.

Experimental root canal infections in conventional and germ-free mice.

A small animal model was evaluated to study the interrelationships between microorganisms after their implantation in root canals (inferior central incisors) using germ-free (GF) and conventional (CV) mice. The selected microorganisms were: Porphyromonas endodontalis (ATCC 35406), Eubacterium lentum (ATCC 25559), Peptostreptococcus anaerobius (ATCC 27337), Fusobacterium nucleatum (ATCC 10953), Escherichia coli (ATCC 25922), and Enterococcus faecalis (ATCC 4083). Only P. anaerobius, E. coli, and E. faecalis, respectively, were able to colonize when inoculated alone into the root canal of both CV and GF mice. E. lentum, when inoculated alone colonized only in CV animals. P. endodontalis and F. nucleatum were unable to colonize in CV and GF animals after single inoculation. It is concluded that the experimental animal model presented herein is valuable for ecological studies of root canal infections and that only some strict anaerobic bacteria are able to colonize mice root canals when inoculated by themselves alone in pure culture.

The effects of plant extracts on microbial community structure in a rumen-simulating continuous-culture system as revealed by molecular profiling.

An in vitro study in dual-flow continuous-culture fermentors was conducted with two different concentrations of monensin, cinnamaldehyde or garlic extract added to 1:1 forage-to-concentrate diet in order to determine their effects on selected rumen bacterial populations. Samples were subjected to total DNA extraction, restriction analysis of PCR amplified parts of 16S rRNA genes (ARDRA) and subsequent analysis of the restriction profiles by lab-on-chip technology with the Agilent's Bioanalyser 2100. Eub338-BacPre primer pair was used to select for the bacteria from the genera Bacteroides, Porphyromonas and Prevotella, especially the latter representing the dominant Gram-negative bacterial population in the rumen. Preliminary results of HaeIII restriction analysis show that the effects of monensin, cinnamaldehyde and garlic extract on the BacPre targeted ruminal bacteria are somewhat different in regard to targeted populations and to the nature of the effect. Garlic extract was found to trigger the most intensive changes in the structure of the BacPre targeted population. Comparison of the in silico restriction analysis of BacPre sequences deposited in different DNA databanks and of the results of performed amplified ribosomal DNA restriction analysis showed differences between the predicted and obtained HaeIII restriction profiles, and suggested the presence of novel, still unknown Prevotella populations in studied samples.

Oral microbiota and implant type membranes.

Candida albicans (Ca), Staphylococcus aureus (Sa), Streptococcus sanguis (Ss), Actinomyces naeslundii (An), Actinomyces odontolyticus (Ao), Porphyromona spp (P spp), Candida glabrata (Cg), Candida krusei (Ck), and Rhodotorula spp (R spp) were tested with equal pieces of biodegradable membranes. Membranes pretreated with saliva or clorhexidine and nontreated control membranes were tested in three different culture media containing 0.1 mL homologous suspension for each strain under study. Incubation was performed at 37 degrees C for 48 hours for aerobiosis and for five days for anaerobiosis. Macroscopy and microscopy were carried out. Membranes were removed, washed, and resuspended. Samples were sonicated, and the supernatant was disseminated on brain heart infusion broth or blood agar. Incubation was repeated, colony-forming unit counts were performed, and statistical analysis was carried out using analysis of variance transforming results to Log10 (x + 1), the highest interaction level was used to calculate standard error. Orthogonal contrast was used to compare the different microorganisms under study. Highest adhesion was found with Ca, Cg, Ck, Sa, and Ss. A sufficient quantity of Actinomyces could not be recovered from the membranes. Results with P spp were poor, confirming lower gram-negative adhesion. Replicate flasks with Ss and Ca were cultivated. Membranes were removed after washing and subjected to scanning electron microscopy, as were untreated control pieces. A cavelike surface was observed. Streptococcus sanguis adhering to the membranes showed extracellular projections. Candida and gram-positive cocci showed great recovery capacity.

Effects of root planing on the distribution of microorganisms adult periodontitis sites.

The aims of this study were to identify the microbial species present in subgingival plaque in patients with adult periodontitis, and to investigate the effects of root planing upon the microbial flora for 12 months following root planing. Microorganisms from samples of subgingival plaque obtained from adult periodontitis sites in 15 patients were cultured, both aerobically and anaerobically, on a variety of media and identified. Prevotella intermedia and Porphyromonas asaccharolytica were the most frequently identified species before treatment. Bacteroides ureolyticus, the Prevotella veroralis/buccalis complex and other microorganisms including Capnocytophaga ochracea, species of streptococci, staphylococci and diphtheroids were also frequently isolated. Root planing improved periodontal health, and was accompanied by reductions in the frequencies with which P. intermedia, P. asaccharolytica, the P. veroralis/buccalis complex and C. ochracea were isolated. Regular scaling and oral hygiene reviews maintained the clinical improvements and balance in favour of beneficial microbial species during the study.

Evaluation of a monovalent companion animal periodontal disease vaccine in an experimental mouse periodontitis model.

Periodontal disease in companion animals is clinically similar to that of human periodontal disease. Despite the usage of veterinary procedures and antibiotic therapy, the disease still remains as one of the most highly prevalent disorders seen by veterinarians. The goal of this study was to evaluate the immunogenic properties and vaccine performance of a monovalent canine periodontal disease vaccine in the mouse oral challenge model of periodontitis. Mice vaccinated subcutaneously with inactivated, whole-cell bacterin preparations of Porphyromonas gulae displayed both high titers of anti-P. gulae specific antibodies and significantly reduced alveolar bone loss in response to homologous, heterologous, and cross-species challenge. Based on the results of these studies, a periodontal disease vaccine may be a useful tool in preventing the progression of periodontitis in animals.

Growth characteristics and a novel method for identification (the WEE-TAB system) of Porphyromonas species isolated from infected dog and cat bite wounds in humans.

Thirty-nine clinical isolates of Porphyromonas species recovered from infected cat and dog bite wounds in humans and eight American Type Culture Collection and National Collection of Type Cultures type strains were characterized by using the API ZYM system, the RapID ANA II system, and conventional biochemical methods. Growth characteristics on various agar media were compared. All strains grew on brucella blood agar supplemented with vitamin K1 and hemin and on brucella laked blood agar supplemented with vitamin K1 and hemin. In contrast, only 34% of strains grew on unsupplemented brucella blood agar, 62% grew on Columbia blood agar, and 70% grew on tryptic soy blood agar (the last three media did not contain vitamin K1 or hemin). The ability of the single-tube, triple-substrate WEE-TAB system to detect the preformed enzymes N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase, beta-D-galactosidase, alpha-fucosidase, trypsin-like activity, and chymotrypsin was evaluated. The WEE-TAB test results were easy to interpret; the WEE-TAB tests were more sensitive than the comparable tests with the API ZYM and RapID ANA II systems for the detection of alpha-D-galactosidase, beta-D-galactosidase, trypsin, and chymotrypsin, and the WEE-TAB tests accurately identified Porphyromonas species.

Porphyromonas uenonis sp. nov., a pathogen for humans distinct from P. asaccharolytica and P. endodontalis.

Three Porphyromonas species (Porphyromonas asaccharolytica, P. endodontalis, and the novel species that is the subject of the present report, P. uenonis) are very much alike in terms of biochemical characteristics, such as enzyme profiles and cellular fatty acid contents. P. asaccharolytica is distinguished from the other two species by virtue of production of alpha-fucosidase and glyoxylic acid positivity. The novel species is difficult to differentiate from P. endodontalis phenotypically and was designated a P. endodontalis-like organism for some time. However, P. endodontalis is recovered almost exclusively from oral sources and also grows poorly on Biolog Universal Agar, both characteristics that are in contrast to those of the other two organisms. Furthermore, P. uenonis is glycerol positive in the Biolog AN Microplate system. Both P. asaccharolytica and P. uenonis are positive by 13 other tests in the Biolog system, whereas P. endodontalis is negative by all of these tests. P. asaccharolytica grew well in both solid and liquid media without supplementation with 5% horse serum, whereas the other two species grew poorly without supplementation. Sequencing of 16S rRNA revealed about 10% divergence between the novel species and P. endodontalis but less than 2% sequence difference between the novel species and P. asaccharolytica. Subsequent DNA-DNA hybridization studies documented that the novel organism was indeed distinct from P. asaccharolytica. We propose the name Porphyromonas uenonis for the novel species. We have recovered P. uenonis from four clinical infections in adults, all likely of intestinal origin, and from the feces of six children.

Growth stimulation of Porphyromonas endodontalis by hemoglobin and protoporphyrin IX.

Porphyromonas endodontalis, like other Porphyromonas species, has a complex set of nutritional requirements. In addition to being an obligate anaerobe, the bacterium must be grown in a complex medium consisting of amino acids, reducing agents and heme compounds. P. endodontalis accumulates high concentrations of heme pigments to the extent that colonies appear black on blood agar. This accumulation of heme and the need for these compounds has been characterized as iron requirements by these species. However, in our studies, P. endodontalis demonstrated growth dependence on hemoglobin or protoporphyrin IX but not on free iron. Iron added to other heme compounds actually decreased growth stimulation by porphyrin-containing compounds. P. endodontalis actively transported free iron, but this process did not appear to be critical for growth. The maximum stimulation of growth by protoporphyrin IX, under conditions of iron deprivation, suggests that P. endodontalis requires the porphyrin moiety as a growth factor.