Porphyromonas bronchialis sp. nov. Isolated from Intraoperative Bronchial Fluids of a Patient with Non-Small Cell Lung Cancer

Abstract

Porphyromonas strains, including Porphyromonas-like strains, have been isolated from oral and various other systemic infections. The characterization of such strains is a crucial issue, because such information contributes to both the taxonomy of anaerobic bacteria and the clinical aspects of infectious diseases. We previously isolated four Porphyromonas-like strains from intraoperative bronchial fluids of a patient with non-small cell lung cancer. This study aimed to characterize the genetic, biochemical and chemotaxonomic aspects of these isolates. Each strain only grew under anaerobic conditions and their colony morphology was convex, 0.1-1.0 mm in diameter, light gray, and slightly glistening colony, with no black or brown pigmentation on blood agar plates after five-day incubation. The pigmentation was helpful to differentiate the isolates from other Porphyromonas, as most of Porphyromonas species show the pigmentation. In the 16S rRNA gene phylogenetic analysis (98% sequence identity of isolates indicates the same species), the four isolates were closely related to one another (99.7-100.0%), but not related to Porphyromonas (P.) catoniae, the closest species (96.9%). In addition, the DNA-DNA hybridization data revealed less than 16% similarity values between a representative isolate and the P. catoniae, indicating that the strains were genetically independent. Biochemically, the isolates could be differentiated from closely related species, i.e., P. catoniae, P. gingivalis, P. gulae, and P. pogonae, with trypsin activity (negative only in the isolates) and leucine arylamidase activity (positive only in the isolates). We therefore propose a new species to include these isolates: Porphyromonas bronchialis sp. nov.

Introduction

Numerous bacterial strains of Gram-negative, non-spore-forming, rod-shaped, obligate anaerobic bacteria, belonging to the genus Porphyromonas, have been isolated from oral infections and many other infections throughout the body, e.g., abscesses in the buttock and groin areas, soft tissue infections, amniotic fluid, umbilical cord, pelvic abscesses, vulvovaginitis, and other infected tissues (Shah and Collins 1988; Krieg 2011; Kawamura et al. 2015). Within the genus Porphyromonas, 16 species are currently recognized, i.e., Porphyromonas asaccharolyticus, Porphyromonas bennonis, Porphyromonas cangingivalis, Porphyromonas canoris, Porphyromonas catoniae, Porphyromonas circumdentaria, Porphyromonas crevioricanis, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gingivicanis, Porphyromonas gulae, Porphyromonas levii, Porphyromonas macacae, Porphyromonas pogonae, Porphyromonas somerae and Porphyromonas uenosis.

Taxonomy within the genus Porphyromonas has progressed in these days; however, many strains remain uncharacterized (Fournier et al. 2001; Bemis et al. 2011). The characterization of such not-yet-identified strains, e.g., how different from established Porphyromonas species on the genetical, biochemical, and chemotaxonomical basis, is essential for the completion of Porphyromonas taxonomy, and thus attracting attentions of researchers. Furthermore, information on such not-yet-identified strains has been, in general, thought to contribute greatly to the clinical aspects of infectious diseases.

Porphyromonas-like strains have been isolated from oral cavities, urogenital system, and intestinal tracts of their hosts (Willems and Collins 1995; Fournier et al. 2001; Finegold et al. 2004; Hardham et al. 2005; Summanen et al. 2005, 2009; Mikkelsen et al. 2008; Bemis et al. 2011; Filioussis et al. 2015; Kawamura et al. 2015). Usually, most of the uncharacterized strains are known to be unavailable between different laboratories. Recently, as one of the means of obtaining uncharacterized Porphyromonas-like strains, we quantified and identified bacteria in intraoperative bronchial fluids of nine elderly patients using a bronchoscopic micro-sampling probe and a well maintained anaerobic glove box system, and successfully isolated 79 predominant facultative and obligate anaerobes (Hasegawa et al. 2014). Among the 79 strains, there were four Porphyromonas-like organisms; however, to date, their phenotypic and genetic characteristics remain to be fully defined. Thus, the purpose of the present study was to characterize the genetic, biochemical, and chemotaxonomic aspects of the four strains. In this paper, we report the results of the phenotypic analysis, 16S rRNA gene phylogenetic analysis, DNA-DNA hybridization, G + C content and cellular fatty acid analysis, in order to determine the taxonomic position of the four strains.

Materials and Methods

Bacterial strains and growth conditions

Four isolates of a new candidate species were used in the present study; these four Porphyromonas isolates, PAGU 1600^T to 1603 (PAGU, School of Pharmacy, Aichi Gakuin University), were also numbered, Tohoku 18-2 to 18-5, respectively (Table 1). They were isolated from intraoperative bronchial fluids of a patient (male, aged 74 years) with non-small cell lung cancer, i.e., adenocarcinoma in the left upper lobe and squamous cell carcinoma in the left lower lobe, at Tohoku University Hospital, Sendai, Japan, utilizing a bronchoscopic micro-sampling probe and an anaerobic glove box system (model AZ-Hard, containing 80% N₂, 10% H₂, and 10% CO₂; Hirasawa, Tokyo, Japan). Informed consent was obtained from the patient, and this study was approved by the Research Ethics Committee of Tohoku University Graduate School of Dentistry, Sendai, Japan, as described previously (Hasegawa et al. 2014). The following type strains were also used in this study; Porphyromonas gingivalis PAGU 1218^T (= JCM, Japan Collection of Microorganisms 12257^T), Porphyromonas asaccharolyticus PAGU 1229 ^T (= JCM 6326^T), Porphyromonas catoniae PAGU 1627^T (= JCM 13863^T), and Porphyromonas gulae PAGU 1794^T (= JCM 13865^T). All strains were grown on Brucella Blood Agar with Hemin and Vitamin K (Difco) plates, at 37°C under an anaerobic atmosphere consisting of 80% N₂, 10% CO₂ and 10% H₂, generated using an Anoxomat Mark II anaerobic system (Mart Microbiology, Drachten, Netherlands). Black or brown pigmentation on the agar plates after 5 to 14 days incubation at 37°C was checked by the visual inspection.

Table 1.

Strains used in the present study.

Phenotypic analysis

The biochemical traits of each of these isolates were characterized using API-ZYM and Rapid ID32A systems (Sysmex bioMérieux Co., Ltd., Tokyo, Japan), according to the manufacturer’s instructions. Catalase activity was monitored by adding a drop of 3% hydrogen peroxide to a microbial smear. Fermentation tests for each sugar were performed using Fastidious Anaerobe Broth (FAB; Lab M Ltd., Lancashire, UK) supplemented with 1% glucose, lactose, or maltose. After 3 days of incubation at 37°C under anaerobic conditions, pH of the medium was measured. A pH value lower than 5.5 was considered to indicate a positive fermentation result, pH of 5.5-6.0 was considered to be weakly positive for fermentation, and a pH value higher than 6.0 indicated no fermentation (Holdeman et al. 1977). Volatile fatty acids (C₂-C₆), acetoin, diacetyl alcohols (C₂-C₅), and free acids or methyl derivatives of lactic, succinic, phenyl acetic, phenyl propionic, and formic acids, produced in peptone-yeast extract-glucose broth (PYG), were assayed by gas chromatography, as described previously (Hoshino et al. 1978; Hoshino and Sato 1986).

16S rRNA gene phylogenetic analysis

Phylogenetic analysis was carried out using 16S rRNA gene sequences, as described previously (Kawamura et al. 2009). Briefly, the 16S rRNA gene (> 1,300 bases) sequences for each isolate were determined using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Tokyo, Japan) and the 3130 Genetic Analyzer (Applied Biosystems). Other sequences used for alignment and calculating the identity levels were obtained from the DNA Data Bank of Japan (DDBJ) database (http://www.ddbj.nig.ac.jp/). Multiple sequence alignments of DNA sequences were carried out using Clustal X software (http://www.clustal.org/). Phylogenetic distances were estimated using the neighbor-joining method, and the phylogenetic tree was drawn using TreeView (http://www.ddbj.nig.ac.jp/search/help/tv-setup.html) and NJPlot (http://www.softpedia.com/get/Science-CAD/NJPlot.shtml) software.

DNA-DNA hybridization and determination of G + C content

Quantitative microplate DNA-DNA hybridization was carried out, as described previously (Kawamura et al. 1999). Hybridization experiments were carried out at 34°C (optimal conditions) or 44°C (stringent conditions) in 2× SSC and 50% formamide. The mol% G + C contents of the DNA were determined by high-performance liquid chromatography as described previously (Kawamura et al. 1998).

Cellular fatty acid analysis

Cellular fatty acids of each isolate were liberated with 10% KOH/methanol at 100°C for 30 min, followed by extraction with n-hexane. After methylation with 10% trimethylsilyldiazomethane in n-hexane (Nacalai Tesque Inc., Kyoto, Japan), methyl ester derivatives of fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS) (Li et al. 2003; Tomida et al. 2011; Kawamura et al. 2012).

Scanning electron microscopy

Bacterial cell shape was observed by scanning electron microscopy, as described previously (Kawamura et al. 2015). Briefly, bacterial colonies were suspended in phosphate-buffered saline, washed three times, and smeared onto a cover glass. Bacteria were fixed with 0.1 M sodium cacodylate containing 2.5% (v/v) glutaraldehyde for 1 h at 4°C. After washing with 0.1 M sodium cacodylate, samples were dehydrated in a graded series of ethanol solutions ranging from 50% (v/v) ethanol in distilled water to 100% ethanol. All samples were coated with 5 nm of platinum by magnetron sputtering and examined by scanning electron microscopy (JXA-8530FA; JOEL, Tokyo, Japan).

Results and Discussion

The genetic, biochemical, and chemotaxonomic aspects of the four unidentified strictly anaerobic strains, isolated from intraoperative bronchial fluids of a patient with pulmonary carcinoma, were characterized in this study.

Genetic analysis

The phylogenetic analysis of 16S rRNA sequences demonstrated that the four isolates belong within the genus Porphyromonas. It is generally accepted that a 98% 16S rRNA sequence identity is an indication that isolates are members of the same species (Stackebrandt and Goebel 1994). In this study, these four isolates were closely related to one another (99.7-100.0% sequence identity), but not related to Porphyromonas (P.) catoniae PAGU 1600^T, the closest species (96.9% sequence identity, Fig. 1). In addition, in the whole-genome DNA-DNA hybridization studies, a threshold of 70% or greater similarity indicates a relationship at the species level (Wayne et al. 1987). In our study, the DNA-DNA hybridization data revealed less than 16% DNA-DNA similarity values between a representative isolate PAGU 1600^T and the P. catoniae type strain (Table 2), indicating that the strains were genetically independent. Our hybridization data appear to be reliable, as the similarity values between P. gingivalis and P. gulae observed in our experiments (42.2-55.6%, Table 2) were in almost the same range as those (53-65%) reported by Kato et al. (1997). Based on these data, we concluded that the Porphyromonas isolates from intraoperative bronchial fluids fell into a single taxon, but did not belong to any established species of the genus.

The G + C mol% of a representative isolate PAGU 1600^T was 56.50 ± 0.67 mol%, which were clearly distinct from other non-pigmented Porphyromonas species, i.e., P. catoniae (49 mol%; Willems and Collins 1995), P. bennonis (58 mol%; Summanen et al. 2009), and P. pogonae (43.00 ± 0.62 mol%; Kawamura et al. 2015).

Fig. 1.

Phylogenetic tree, based on 16S rRNA gene sequences, showing the position of Porphyromonas bronchialis within the genus Porphyromonas. The numbers at the branching points are bootstrap percentages (based on 1,000 replications). Bacteroides fragilis DSM 2151^T is used as an out group. Scale bar represents one inferred nucleotide substitution per 100 nucleotides. GenBank/DDBJ accession numbers are given in parenthesis, and type strains are indicated as ^T (superscript notations). In addition to PAGU and JCM, the following acronyms of Culture Collections are used in Fig. 1.

NCTC; National Collection of Type Cultures, Central Public Laboratory Service, London, UK.

ATCC; American Type Culture Collection, Manassas, VA.

VPB; Veterinary Pathology and Bacteriology Collection, University of Sydney, New South Wales, Australia.

NUM; Nihon University School of Dentistry at Matsudo, Chiba, Japan.

WAL; Wadsworth Anaerobe Laboratory, Wadsworth Hospital Center, Los Angeles, CA.

DSM; DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

Table 2.

Whol-genome DNA-DNA hybridization assay between P. bronchialis and closely related species of the genus Porphyromonas.

DNA-DNA hybridization was carried out using the photobiotin microplate method. Values are percentage results from hybridization at 34°C (optimal conditions) and 44°C (stringent conditions). Data represent means ± standard deviation of three experiments.

Cellular fatty acid and fermentation end product analyses

Fatty acid analysis revealed that 3OH-iso-C17:0 (18.8-20.4%) predominated, followed by anteiso-C15:0, C16:0, and C18:0 in almost equal amounts (11.1-12.2%, 9.5-10.6%, and 9.6-11.2%, respectively), which could be separated from other non-pigmented Porphyromonas species, i.e., P. catoniae (C15:0 iso, C15:0 antiiso; Willems and Collins 1995; Kawamura et al. 2015), P. bennonis (C15:0 DMA, 3OH-C14:0; Summanen et al. 2009; Kawamura et al. 2015), and P. pogonae (C15:0 iso, C15:0 anteiso, 3OH-C17:0 iso; Kawamura et al. 2015) (Table 3). Fermentation end product data showed that propionic acid was the most abundant product (3.94-13.10 mM), followed by acetic acid (1.84-5.26 mM), which differed from other non-pigmented Porphyromonas species, i.e., P. catoniae (propionic and succinic acids, followed by acetic, iso-valeric and lactic acids; Willems and Collins 1995; Kawamura et al. 2015), P. bennonis (acetic and succinic acids; Summanen et al. 2009; Kawamura et al. 2015), and P. pogonae (succinic, propionic and acetic acids, followed by iso-valeric and lactic acids; Kawamura et al. 2015) (Table 3).

Table 3.

Differential characteristics of P. bronchialis sp. nov. and closely related species of the genus Porphyromonas.

^aData of P. catoniae, P. gingivalis, P. gulae and P. pogonae were taken from Krieg (2011), Fournier et al. (2001), Kawamura et al. (2015) and our own results.

+: more than 90% of the strains are positive, d: 89-11% of the strains are positive (and their actual numbers of positive/tested strains were described in parentheses), −: less than 10% of the strains are positive, w: weakly positive reaction.

^bNegative but weakly positive after incubation for more than 10 to 14 days.

^cReaction in the API ZYM system and/or Rapid ID32A system (bioMérieux).

^dReaction in the Fastidious Anaerobe Broth (Lab M Ltd.).

^eReaction in the Rapid ID32A system (bioMérieux).

^fA, acetic acid; P, propionic acid; IB, isobutyric acid; B, butyric acid; IV, isovaleric acid; L, lactic acid; S, succinic acid; PA, phenylacetic acid. Capital letters represent an amount of metabolic product equal to or greater than 10 mM from peptone-yeast extract-glucose (PYG); small letters represent an amount of metabolic product less than 10 mM.

^gData are not available for the present.

Colony and cell morphology, and phenotypic analysis

All four isolates showed very similar colony morphology; convex, 0.1-1.0 mm in diameter, light gray, slightly glistening colony, and did not show black or brown pigmentation (Table 3) on Brucella Blood Agar with Hemin and Vitamin K (Difco) plate after five days incubation under anaerobic condition at 37°C, which were distinct from closely related species of pigmented Porphyromonas, such as P. gingivalis (Krieg 2011) and P. gulae (Fournier et al. 2001). In addition, no pigment production was observed in the four isolates even after incubation for more than 10 to 14 days, which differed from non-pigmented Porphyromonas species, P. bennonis (Summanen et al. 2009; Kawamura et al. 2015) and P. pogonae (Kawamura et al. 2015). Colonies did not show red fluorescence under UV (366 nm) light, similar to P. catoniae, P. gingivalis, and P. gulae, but in contrast to P. pogonae (Table 3). Catalase tests were negative for all isolates, similar to P. catoniae and P. gingivalis, but in contrast to P. gulae and P. pogonae (Table 3). Cells from each isolate appeared microscopically as small, approximately 0.5-0.8 μm × 0.8-2.5 μm, Gram-negative rods, and coccobacilli.

The isolates could be differentiated from closely related species, such as P. catoniae, P. gingivalis, P. gulae, and P. pogonae by measuring trypsin activity (negative only in the isolates, but positive in the other four species) and leucine arylamidase activity (positive only in the isolates, but negative in the other four species) (Table 3). In addition to the pigmentation and catalase activity (described above), α-fucosidase activity, and fermentation tests for glucose, lactose, maltose, mannose, and raffinose as substrates were also helpful to differentiate the isolates from other members of the genus Porphyromonas (Table 3).

Clinical implication and summary

It has been suggested that silent aspiration of bacteria occurs in elderly patients with pulmonary carcinoma and that the risk of pneumonia in these patients is high (Hasegawa et al. 2014); however, neither postoperative pneumonia nor carcinoma recurrence was observed in any patients in that study, whose intraoperative bronchial fluids were the source of the isolates. Thus, the pathological characteristics of this species of Porphyromonas (such as on pulmonary carcinoma as well as aspiration pneumonia) are unknown, and further study is necessary.

All four isolates collected from human bronchial fluids are novel members of the genus Porphyromonas, and here, we propose the name Porphyromonas bronchialis sp. nov. The summary of the taxonomic properties of the species is described as follows.

Description of Porphyromonas bronchialis sp. nov.

Porphyromonas bronchialis (L. pl. n. bronchia, the bronchial tubes; L. fem. suff.-alis, suffix used with the sense of pertaining to; N.L. fem. adj. bronchialis, pertaining to the bronchi, coming from the bronchi [bronchial fluids]).

The description below is based on our own observations.

Cells are Gram-negative, non-spore-forming rods, 0.5-0.8 μm × 0.8-2.5 μm in size. They grow under anaerobic conditions only.

Convex, 0.1-1.0 mm in diameter, light gray, and slightly glistening colonies appeared on Brucella Blood Agar with Hemin and Vitamin K (Difco) plates after 5 days of incubation under anaerobic conditions at 37°C. The differential characteristics of this organism and related species are described as follows (and Table 3). Black or brown pigmentation was not observed. No fluorescence was observed under long-wave UV light. The organism is catalase-, indole-, urease-, and nitrate-negative. Positive reactions were exhibited for α-fucosidase, β-galactosidase, N-acetyl-β-glucosaminidase, leucine arylamidase, and chymotrypsin (weak positive) and fermentation tests for raffinose and mannose (1/2 strains). The organism is negative for other tests that were run on the API-ZYM and Rapid-ID32A systems.

The metabolic end products identified by gas chromatography were propionic acid followed by acetic acid. The major fatty acids detected in the strains were 3OH-iso-C17:0, anteiso-C15:0, C16:0, and C18:0. The G + C mol% content was 56.50 ± 0.67%.

We propose that this type strain be designated PAGU 1600^T. The organism was isolated from human bronchial fluids.

Acknowledgments

This study was supported in part by Grants-in-Aid for Scientific Research (20591661, 24390511, 25462945, 25460177, 25463237, 25670777, 25861785, and 26462869) from the Japan Society for the Promotion of Science, Tokyo, Japan. We gratefully acknowledge Drs. Y. Yoshida and Y. Morita for their helpful assistance with this study, and Drs. G. Mayanagi, H. Ohshima and J. Washio for their invaluable comments on this manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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