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Volatile Sulfur Compounds Produced by the Anaerobic Bacteria Porphyromonas spp. Isolated from the Oral Cavities of Dogs

A peer-reviewed version of this preprint was published in:
Veterinary Sciences 2023, 10(8), 503. https://doi.org/10.3390/vetsci10080503

Submitted:

04 July 2023

Posted:

05 July 2023

You are already at the latest version

Abstract
Porphyromonas spp. are oral anaerobic Gram-negative bacteria that form black-pigmented colonies on blood agar and produce volatile sulfur compounds (VSCs), such as hydrogen sulfide (H2S), methyl mercaptan (CH3SH), and dimethyl sulfide ((CH3)2S), which cause halitosis and the destruction of periodontal tissues. P. gulae is considered the main pathogen involved in periodontal disease in dogs. However, the characteristics of the VSCs produced by P. gulae are unknown. In the present study, VSCs were measured in 26 isolates of P. gulae and some isolates of the other Porphyromonas spp. obtained from the oral cavities of dogs with periodontal disease using an in vitro assay with an Oral ChromaTM gas chromatograph. The results demonstrated that P. gulae was able to produce large amounts of H2S and CH3SH, and the dominant one was CH3SH (CH3SH/H2S was approximately 2.2). Other Porphyromonas spp. that were also obtained from the oral cavities of dogs with periodontal disease indicated the similar levels of production of H2S and CH3SH to those of P. gulae. It is strongly suggested that the high levels of H2S and CH3SH in P. gulae and other Porphyromonas spp. contribute to halitosis and the destruction of periodontal tissues during the progression of periodontal disease in dogs.
Keywords: 
volatile sulfur compounds (VSCs)
;  
Porphyromonas gulae
;  
dog
;  
periodontal disease
;  
halitosis
Subject: 
Biology and Life Sciences  -   Animal Science, Veterinary Science and Zoology

1. Introduction

Volatile sulfur compounds (VSCs) are the main cause of halitosis (malodor) in humans and dogs with periodontal disease [1,2,3,4]. The major components of VSCs are hydrogen sulfide (H2S), methyl mercaptan (CH3SH), and dimethyl sulfide ((CH3)2S), which are derived from sulfur-containing amino acids (e.g., l-cysteine and l-methionine) [2,5]. Oral anaerobic Gram-negative bacteria that form black-pigmented colonies on blood agar, such as Porphyromonas spp., contribute significantly to the production of VSCs and are major pathogens in periodontal disease [2,4,6,7,8]. Simultaneously, it has also been suggested that, even at low levels, VSCs have cytotoxic potential for oral tissues and are associated with the progress of periodontal disease [5,9]. Specifically, the levels of VSC production are likely to reflect the pathogenicity of bacteria.
In dogs, periodontal disease is the most common disease, and a high prevalence (44–100%) has been reported [10]. Canine periodontal disease increases with age, but is predominant in toy breeds of dogs from the younger stages of their lives [10,11]. Recently, in Japan, elderly dogs and toy breeds of dogs have been growing in number. Halitosis is the first chance for owners to notice the presence of periodontal disease in their companions. However, due to owners’ frequent and close contact with dogs, halitosis has developed into a serious problem that causes anguish for owners.
The VSCs produced by human periodontal-disease-associated bacteria were assessed in vitro [12,13,14]. In dogs, in vivo analyses of VSCs were performed in clinical cases with or without periodontal disease [3,4]. However, there are no available reports regarding the in vitro assessment of VSC production via the periodontal-disease-associated Porphyromonas spp. in dogs. Therefore, the present study aimed to evaluate the characteristics of the VSCs produced by Porphyromonas spp. (Porphyromonas gulae, Porphyromonas macacae, Porphyromonas gingivalis, and Porphyromonas gingivicanis) isolated from canine oral cavities using an in vitro assay. Simultaneously, for comparison with Porphyromonas spp., we measured the VSCs produced by Bacteroides pyogenes, which is also an anaerobic Gram-negative bacterium, but which does not form black-pigmented colonies on blood agar; it was isolated from canine oral cavities containing normal flora or periodontal-disease-associated bacteria [15,16,17].

2. Materials and Methods

2.1. Ethics Statements and Sample Collection

The present study was approved by the ethics committees of two facilities: Veterinary Teaching Hospital of Kitasato University (approval number: 2022-8001) and Ito Animal Hospital (approval number: 1122-12-001). To detect the pathogenic microorganisms in dogs diagnosed with gingivitis while referring to the criteria provided in [18], the owners accepted and permitted the submission of oral swab samples from their animals after giving their informed consent.
The oral swab specimens were collected at the position of the gingival margin from the teeth of the canine to fourth premolars on the left side of the maxilla in 46 dogs without treatment with sedation and/or anesthesia by using a sterilized device (TX709A, Clean Foam® Series, Texwipe, Kernersville, NC 27284, USA).

2.2. Bacterial Culture and Isolation

The obtained swab samples were immediately inoculated on a non-selective anaerobic bacterial isolation agar plate that included hemin, vitamin K, l-arginine, and hemolytic rabbit blood (ABHK Agar Plate; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). The culture plates were incubated at 37 °C for 10 days under aerobic conditions using a commercial disposable self-contained anaerobic system (ANAEROMATE®-P “Nissui”, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). Then, black-pigmented bacterial colonies from 42 dogs and non-black-pigmented colonies from 4 other dogs were randomly selected and pure-cultured for 5 days according to the same procedure.

2.3. Bacterial Production of VSCs and Determination of VSCs

The concentrations of suspensions of pure-cultured bacterial colonies in axenic PBS were adjusted to have an optical density of 2.0 ± 0.1 at 600 nm by using SimpliNano (GE Health Care Ltd., Chicago, IL, USA). To determine the VSCs, the prepared 250 µL suspensions were placed in a KBM Anaerobic Test Tube Medium (Kohjin Bio Co., Ltd., Saitama, Japan) that contained 15 mL of liquid medium, which included enough nutrients for a wide range of anaerobic bacteria; this was incubated at 37 °C for 72 h under anaerobic conditions. Prior to the inoculation of the bacterial suspension into the culture bottle, a small hole was made in the screw cap of the bottle via puncturing and was sealed with a butyl rubber adhesive tape. After 72 h of incubation, the produced gas was collected from the upper layer of the air in the culture bottle using a disposable syringe with a 26G needle that was inserted through the small hole in the screw cap that was sealed with tape. The levels of VSCs (H2S, CH3SH, and (CH3)2S) were measured using an Oral ChromaTM gas chromatograph (CHM-2, FIS Inc., Hyogo, Japan). In addition, the ratio of H2S to CH3SH (CH3SH/H2S) was calculated.

2.4. Molecular Identification of Bacterial Species

For the molecular identification of the bacterial species, the 200 µL suspensions used for VSC determination were also used to extract the total bacterial DNA using the commercial kit (NucleoSpinl® DNA Stool, MACHEREY-NAGEL, GmbH & Co. KG, Düren, Germany) according to the manufacturer’s instructions. The obtained DNA specimens were stored at −20 °C prior to the analysis.
According to the attached manual, fast PCR targeting 16S rRNA genes was performed using a commercial kit (Bacterial 16S rDNA PCR Kit Fast (800), Takara Bio Inc., Shiga, Japan); this included the universal primer pairs (10F: GTTTGATCCTGGCTCA; 800R: TACCAGGGTATCTAATCC) in order to amplify regions of approximately 800 bp, a premix (the master mix was composed of modified Taq DNA polymerase and dNTPs), and a positive control. In brief, the total reaction volume was set to 25.0 µL, which included 12.5 µL of the master mix, 1.25 µL of each primer, 2.5 µL of the DNA template, and 7.5 µL of DNA-free water. The conditions of fast PCR were as follows: 25 cycles of 92 °C for 5 s, 50 °C for 1 s, and 68 °C for 8 s. The amplicons used in the PCR were electrophoresed on a 1.5% agarose gel and were stained (AtlasSight DNA Stain, Bioatlas, Tartu, Estonia) for the visualization of the bands. The expected size of the DNA fragments was confirmed via transillumination under UV light.
The PCR products were purified using a commercial kit (NucleoSpinl® Gel and PCR Clean-up, MACHEREY-NAGEL, GmbH and Co. KG, Düren, Germany). A sequencing analysis was executed in a commercial laboratory (FASMAC Co., Ltd., Atsugi, Kanagawa, Japan). Sequence alignment and compilation were performed using the MEGA 11.0.10 (https://www.megasoftware.net, accessed on 16 April 2023) program. To determine the bacterial species, the obtained DNA sequences were compared with GenBank references by using BLAST searches (http://www.ncbi.nlm.nih.gov/, accessed on 26 April 2023).

2.5. Data Analysis

The data were analyzed using “EZR” software (version 1.61). The measured values were expressed as the mean ± standard deviation. The Kruskal–Wallis test was used to compare the differences among groups, and the Steel–Dwass test was conducted to assess the differences between pairs of groups. Statistical differences were considered significant at p < 0.05.

3. Results

3.1. Molecular Identification of Bacteria

According to the molecular identification of the bacteria in the present study, among the 42 isolates of black-pigmented colony-forming bacteria were 26 isolates of P. gulae, 8 isolates of P. macacae, 4 isolates of P. gingivalis, and 4 isolates of P. gingivicanis. In addition, all four of the isolates of non-black-pigmented colony-forming bacteria were Bacteroides pyogenes (Table 1).

3.2. Volumes of VSCs Produced

The volumes of VSCs produced by the black-pigmented bacteria (Porphyromonas spp.) and non-black-pigmented bacteria (B. pyogenes) are shown in Table 2. Overall, the production of H2S and CH3SH in the Porphyromonas spp. group was revealed to have a higher tendency than that of B. pyogenes. Indeed, the volumes of H2S and CH3SH were significantly higher for P. gulae and P. macacae than for B. pyogenes. In addition, significant differences were observed between P. gulae and P. macacae regarding the levels of H2S and CH3SH. In other Porphyromonas spp., there were no statistical differences in H2S and CH3SH in comparison with B. pyogenes or among those species, which was due to the limited numbers that were investigated (e.g., there were only four isolates each of P. gingivalis, P. gingivicanis, and B. pyogenes) and the wide range of variation. The ratios of H2S to CH3SH (CH3SH/H2S) were approximately 2.2 for the Porphyromonas spp. group and 0.18 for B. pyogenes, and the ratios were significantly higher for P. gulae and P. macacae than for B. pyogenes. The volumes of (CH3)2S were irregular in all bacterial isolates, and many isolates were negative for the production of (CH3)2S (16/26 for P. gulae, 7/8 for P. macacae, 4/4 for P. gingivalis, 1/4 for P. gingivicanis, and 4/4 for B. pyogenes). No significant differences were demonstrated in the (CH3)2S levels among the investigated bacteria.

4. Discussion

The present study is the first report to demonstrate the characteristics of the VSCs produced by P. gulae, considered the main pathogen involved in periodontal disease in dogs [17,19], which was obtained from canine oral cavities. The results suggest that P. gulae has the potential to produce high levels of H2S and CH3SH, with CH3SH being dominant. As shown in the present study, the dominant gas was easy to determine due to the ratio of CH3SH/H2S. Although the examined numbers were insufficient, other Porphyromonas spp. (P. macacae, P. gingivalis, and P. gingivicanis) also indicated the same characteristics as those of P. gulae in terms of the VSCs that they produced. The characteristic of CH3SH-dominant VSC production was also reported in P. gingivalis in humans [12,13,14]. A clear difference was observed between Porphyromonas spp. and B. pyogenes. Regarding B. pyogenes, both H2S and CH3SH were produced at markedly low levels in comparison with Porphyromonas spp., and H2S was dominant in the VSCs, which was reflected in the low value of CH3SH/H2S. These differences in VSC levels and components between Porphyromonas spp. and B. pyogenes were easily calculable from the outset because these two bacteria belong to different genera. However, the enzyme l-cysteine desulfhydrase, which produces H2S from l-cysteine, was documented in both Porphyromonas spp. and Bacteroides spp. [2]. Additionally, l-methionine α-deamino-γ-mercaptomethane-lyase, which is an enzyme that produces CH3SH from l-methionine, was also demonstrated in both species of bacteria [2]. Therefore, it was suggested that the differences in the VSC levels and components between Porphyromonas spp. and Bacteroides spp. were derived from the levels of activity of these two enzymes. Despite being in the same genus, the VSCs produced by P. gingivalis and P. endodontalis had different components because P. endodontalis was shown to produce H2S-dominant VSCs in humans [12]. Due to the significant difference in the volumes of VSCs produced by P. gulae and P. macacae, it was also observed in the present study that the activities of l-cysteine desulfhydrase and l-methionine α-deamino-γ-mercaptomethane-lyase are likely to be characterized according to each species of bacteria.
Previous studies suggested that the main and important VSCs inducing halitosis are H2S and CH3SH, which are produced by some specific bacteria, such as P. gingivalis, Fusobacterium nucleatum, and Tannerella forsythia in humans [1,5,9]. The gases of H2S and CH3SH comprised approximately 90% of the VSCs in human breath [20], and some in vitro reports related to P. gingivalis in humans demonstrated a markedly low (almost negative) level of (CH3)2S production or even did not record it [12,13,14]. In addition, it is generally known that Gram-negative anaerobic bacteria predominantly produce H2S and CH3SH [1]. In the present study, although the details of the mechanism were unknown, the measured levels of (CH3)2S were unstable, and many samples revealed negative levels of (CH3)2S production. Thus, the importance of the H2S and CH3SH produced by periodontal-disease-associated bacteria was also confirmed in dogs.
The effects of VSCs are not only halitosis, but also toxicity, which is connected to the development of periodontal disease [5,9]. In practice, the VCSs of H2S and CH3SH play the main roles in the injuries in periodontal tissues. In particular, it was indicated that CH3SH had the greatest harmfulness [5]. Therefore, the CH3SH/H2S ratio has the potential to represent toxic activity in tissues and the characteristics of each periodontal bacterium. In the present study, because large amounts of dominant CH3SH and non-dominant H2S were demonstrated in P. gulae and other Porphyromonas spp. from canine cases with periodontal disease, it was suggested that these bacteria induce severe damage to the periodontal tissues in animals and contribute to the progress of periodontal disease. In contrast, low levels of H2S and CH3SH suggest the weak pathogenicity of B. pyogenes in comparison with that of Porphyromonas spp. However, we cannot neglect this status in B. pyogenes because VCSs can be toxic to tissues even at low levels and with short-term exposure [2,5]. The free thiols (–SH groups) of both H2S and CH3SH can react with DNA and proteins [2,5,9]. The thiols penetrate deeply into gingival tissues [5,9]. Previous research demonstrated that the mechanism of tissue damage by H2S and CH3SH was through the breaking of disulfide bonds in the proteins of the oral mucosa [5,9]. This is because disulfide bonds are critical for proteins’ structural integrity [5,9]. In addition, the inhibitory effect of CH3SH against proliferation and the apoptosis induced by H2S exposure were reported in oral epithelial cells [5,9]. The inhibited synthesis of collagen was shown with CH3SH exposure [5,9]. Apoptosis and damage to DNA strands via H2S were also demonstrated in human gingival fibroblasts [5,9]. Moreover, inflammatory reactions and immune responses, which included increased reactive oxygen species and inflammatory cytokine secretions, were initiated after damage to tissues and caused the degraded progress of periodontal disease [5,9]. Simultaneously, since VSCs can be transported to various organs of the body via the blood and inhibit the normal function and metabolic activity of tissues and organs, the development of systemic diseases is a concern [21]. Regarding (CH3)2S, this compound is basically inert because it contains no reactive thiols [5].

5. Conclusions

The present study characterized the VSCs produced by P. gulae originating from canine periodontal disease. The results demonstrated that P. gulae can produce large amounts of H2S and CH3SH. In particular, CH3SH was revealed to be dominant (the CH3SH/H2S ratio was approximately 2.2). Other Porphyromonas spp. also indicated levels of production of H2S and CH3SH that were similar to those of P. gulae. The high levels of H2S and CH3SH contribute to halitosis and the destruction of periodontal tissues after inflammatory reactions and immune responses in relation to the progression of periodontal disease in dogs. In addition, H2S and CH3SH have the potential to be transported into other tissues and organs, and then inhibit their functions, resulting in the risk of systemic disease. Appropriate oral care is required to prevent the toxic effects of VSCs in dogs with periodontal disease.

Author Contributions

Conceptualization, N.I. (Noriyuki Ito) and N.I. (Naoyuki Itoh); methodology, N.I. (Noriyuki Ito) and N.I. (Naoyuki Itoh); validation, N.I. (Noriyuki Ito), N.I. (Naoyuki Itoh), and S.K.; formal analysis, N.I. (Noriyuki Ito) and N.I. (Naoyuki Itoh); investigation, N.I. (Noriyuki Ito), N.I. (Naoyuki Itoh), and S.K.; resources, N.I. (Noriyuki Ito), N.I. (Naoyuki Itoh), and S.K.; data curation, N.I. (Noriyuki Ito) and N.I. (Naoyuki Itoh); writing—original draft preparation, N.I. (Noriyuki Ito) and N.I. (Naoyuki Itoh); writing—review and editing, N.I. (Noriyuki Ito), N.I. (Naoyuki Itoh), and S.K.; visualization, N.I. (Noriyuki Ito) and N.I. (Naoyuki Itoh); supervision, N.I. (Naoyuki Itoh); project administration, N.I. (Noriyuki Ito) and N.I. (Naoyuki Itoh). All authors read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The present study was approved by the ethical committees of two facilities: the Veterinary Teaching Hospital of Kitasato University (protocol code 2022-8001, on 11 October 2022) and Ito Animal Hospital (protocol code 1122-12-001, on 10 April 2022).

Data Availability Statement

The data presented in this study are included within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Table 1. Molecular identification of the bacteria used in the present study.
Table 1. Molecular identification of the bacteria used in the present study.
Bacterial Species Numbers Isolated Reference Isolates in GenBank Identity
Porphyromonas gulae (B)
1 JN713220 99.58%
1 JN713221 99.86%
1 JN713277 99.72%
1 KM461998 99.58%
9 KM462071 99.44–99.87%
7 KM462153 99.44–99.86%
3 LC749393 99.86–99.59%
2 LC749394 99.59%, 99.86%
1 LR134506 99.86%
Porphyromonas macacae (B)
2 AB547666 99.86%, 100%
6 KM461959 99.58–99.86%
Porphyromonas gingivalis (B)
1 CP024594 99.72%
1 CP024601 100%
2 CP025931 99.86%, 100%
Porphyromonas gingivicanis (B)
3 NR_104833 99.46–99.72%
1 JN713184 99.73%
Bacteroides pyogenes (NB)
1 HF558365 100%
1 JN713205 100%
1 MT271930 100%
1 NR_041280 100%
B: black-pigmented colony formed; NB: non-black-pigmented colony formed.
Table 2. The volumes of VSCs produced.
Table 2. The volumes of VSCs produced.
Bacterial Species Examined Numbers H2S (ppb) CH3SH (ppb) (CH3)2S (ppb) CH3SH/H2S
Black-pigmented colony formed
 Porphyromonas gulae 26 6275.6 ± 341.6 (a) 14304.1 ± 1119.5 (d) 64.2 ± 126.3 2.21 ± 0.15 (g)
 Porphyromonas macacae 8 5089.9 ± 973.3 (b) 12078.0 ± 1660.3 (e) 0.9 ± 2.5 2.40 ± 0.17 (h)
 Porphyromonas gingivalis 4 6044.8 ± 485.5 13471.5 ± 1578.5 0 ± 0 2.22 ± 0.14
 Porphyromonas gingivicanis 4 6384.9 ± 408.1 14292.3 ± 663.5 341.5 ± 327.1 2.24 ± 0.07
Non-black-pigmented colony formed
 Bacteroides pyogenes 4 2279.3 ± 1056.8 (c) 505.1 ± 901.4 (f) 0 ± 0 0.18 ± 0.32 (i)
Significant differences. (a) vs. (b): p < 0.01, (a) vs. (c): p < 0.05, (b) vs. (c): p < 0.05, (d) vs. (e): p < 0.05, (d) vs. (f): p < 0.05, (e) vs. (f): p < 0.05, (g) vs. (i): p < 0.05, (h) vs. (i): p < 0.05.
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