Abstract: Bacterial toxin of Porphyromonas asaccharolytica strains obtained from adult periodontitis were analysed using mouse model. Results from subcutaneous injection with whole bacterial cells of 108 and 1010 cells mL-1 failed to induce any infection. However when bacterial concentration of 1012 cells mL-1 was used, all P. asaccharolytica strains were able to induced the development of localized lesion on balb/c mice. In addition, infected mice appeared cachectic with ruffled hair. Thus, infection with the latter concentration was pathogenic and was dose dependent. The toxin was also found to be heat labile for all strains. Therefore, we conclude that the extracellularly toxin produced by P. asaccharolytica of our local isolates which were obtained from adult periodontitis, is virulent and heat labile.
INTRODUCTION
Inflammatory reaction due to response to the penetration of bacterial products at the subgingival areas in the periodontal pockets of teeth leads to periodontal disease. Infected individuals may suffer tooth loss and require life-long review. As a result of various improved methods for isolation and classification of oral microorganisms, more groups of bacteria have been shown to be associated with periodontal disease. Microorganisms that are commonly associated with periodontitis are Porphyromonas species, Prevotella sp., Bacteroides sp. Fusobacterium sp. and Actinobacillus sp. (Dahlèn and Leonhardt, 2006).
Porphyromonas gingivalis is a black-pigmented gram-negative anaerobe and has been found to have virulence factors that can directly affect the periodontium (Fletcher et al., 1994, 1995). Another species in the same genus of Porphyromonas is Porphyromonas asaccharolytica. This species can be isolated from a variety of clinical specimens but it is commonly found in urogenital and intestinal tracts (Jousimies-Somer et al., 2003). P. asaccharolytica showed black pigmented colonies on blood agar and is a Gram negative rod anaerobic bacteria. It is highly resistant to beta lactams antibiotics (Morizano et al., 2005). P. asaccharolytica has also been reported to have collagenase and trypsin-like activities which is similar to the properties of P. gingivalis (http://www.gideononlin.com). However, until to date the contribution and pathogenesis of P. asaccharolytica in oral diseases, especially periodontitis is still remain unknown. Thus, the study was undertaken with the aim of identifying P. asaccharolytica virulent toxin to analyze its possible contribution to pathogenicity in vitro.
MATERIALS AND METHODS
Bacterial Strains and Culture Conditions
All Porphyromonas asaccharolytica strains were of previous
research collection obtained in year 2005 in University of Malaya, Kuala
Lumpur, Malaysia. These samples were collected from deep periodontal pocket
of 5 mm or more from adult periodontitis patients. All strains were revived
from -80°C storage and thawed to room temperature before being grown
on enriched tryptic soy agar supplemented with 5 μl mL-1
hemin, 0.5 μl mL-1 menadione and 5% defibrinated blood.
As Porphyromonas assacharolytica is anaerobic organism incubation
was carried out in anaerobic jars placed in the 37°C incubator for
up to 14 days.
Porphyromonas gingivalis ATCC 33277 was used as the positive control whereas PBS (phosphate buffered saline) and saline were used as negative controls in all studies.
Toxin Pathogenicity Analysis
Porphyromonas asaccharolytica isolates were tested for
invasiveness in a mouse model as previously described by Neiders et
al. (1989). Strains were grown for 18 h using media and anaerobic
environment as described above. Then, the cells were centrifuged, washed
twice in sterile phosphate buffered saline (0.147 M NaCl, 0.01 M sodium
phosphate) and counted in a Petroff-Hausser chamber. Mice were challenged
with subcutaneous injections of 0.1 mL of 108, 1010
and 1012 bacterial suspensions at 2 sites on the dorsal surface.
The presence, size, consistency and location of each ulceration lesions
were evaluated. Mice were also examined daily to assess their general
health status. A number of 6 mice were used for every strain tested, in
order to confirm the accuracy including the reproducibility of the experiment.
Preparation of Unsonicated Bacterial Suspension
All strains were grown overnight at 37°C for 48 h in pre-reduced
enriched trypticase yeast extract broth supplemented with 5 μl mL-1
hemin and 0.5 μl mL-1 menadione. Incubation was carried
out under standard anaerobic condition (80%N2, 10%H2,
10%CO2). The cells were sedimented by centrifugation at 12000
rpm for 20 min at 4°C, after which 0.1, 0.2 and 0.3 mL of the unsonicated
cell-free filtrate supernatant prepared from each of these isolates were
used to inject the mice subcutaneously at 2 dorsal sites of the balb/c
mice. Positive and negative controls were always used in the mouse pathogenicity
study to ensure results accuracy and to avoid biases throughout the experiments.
Preparation of Sonicated Bacterial Suspension
All strains were grown overnight at 37°C for 48 h in pre-reduced
enriched trypticase yeast extract broth supplemented with 5 μl mL-1
hemin and 0.5 μl mL-1 menadione. Incubation was carried
out under standard anaerobic condition (80%N2, 10%H2,
10%CO2).
For preparation of sonicated bacterial suspension, the bacterial cells were washed and resuspended in 1.0 mL 0.85% normal saline and sonicated for 30 sec. The sonication was repeated 3 times after which the suspension was centrifuged and the sonicated pellet was discarded. The supernatant fluid was filtered through a 0.22 μm membrane and retained. 0.1, 0.2 and 0.3 mL of the sonicated cell-free filtrate supernatant prepared from each of these isolates were used to inject the mice subcutaneously at 2 sites about 1 cm lateral on the dorsal surface of the balb/c mice.
Temperature Lability of Porphyromonas asacharolytica Toxin
Bacterial cell-free supernatants were heated to 37 and 60°C for
60 and 15 min, respectively. Toxic activity was then tested as described
above, with 0.2 mL filtrates injected subcutaneously at two dorsal sites
of the balb/c mice after the heat treatment.
RESULTS AND DISCUSSION
Porphyromonas asaccharolytica labelled as Pa1, Pa2, Pa3 and Pa4
were used to study the bacterial toxin pathogenicity, which cause skin
lesion in mice. This study was carried out to determine
| Table 1: | Virulence in Porphyromonas asaccharolytica bacterial cells via mice injection at 108, 1010 and 1012 cells mL-1 on day 7 |
| Positive control: ATCC 33277, Negative controls: Saline | |
toxin production among clinical isolates via mice injection of Porphyromonas asaccharolytica. Six mice were used for every set of injection for each bacterial strain tested. The toxicity activity from bacterial cell culture was observed as abscesses on the skin of the injected mice. Development and severity of lesion or abscesses were observed and recorded. There was no death occurred after the injection. However, all mice produced localized lesion/abscesses and showed signs of mild to moderate cachexia with ruffled hair on day 3 onwards after getting the injection. It has been shown in this study that the response to develop skin lesion on balb/c mice is similar with previous reports of Van Steenbergen et al. (1992) on Porphyromonas gingivalis strains and Himratul-Aznita et al. (2005) on Prevotella intermedia strains obtained from deep periodontal pockets.
The subcutaneous injection with 108 and 1010 cells mL-1 of Porphyromonas assacharolytica and Porphyromonas gingivalis ATCC 33277 failed to produce any skin lesion. However when 1012 cells mL-1 cultures used, lesions developed about 3-5 days after infection (Table 1), with varied localised lesion areas, measuring about 3-5 mm in diameter occurred for all mice. Similar results have been shown in another finding of Himratul-Aznita et al. (2005) on Prevotella intermedia that 1012 cells mL-1 concentration of bacterial suspension was required to induce infection in mice.
Bacterial cell-free supernatant of sonicated and unsonicated cells were
also injected subcutaneously in mice to determine the toxicity effect.
It was found that the sonicated and unsonicated cell-free filtrate supernatant
prepared from each of these isolates developed lesion when 0.1, 0.2 and
0.3 mL supernatant were used. Thus, suggesting that the isolates produced
extracellular toxigenic compounds. For both sonicated and unsonicated
cell-free filtrates, all mice produced localized lesions or abscesses
at the site of injection starting from day 7 onwards and appeared to be
cachectic. The destruction of tissue observed in the study may be correlated
to the destruction of the periodontal tissues in individual with periodontitis
(Van Steenbergen et al., 1982). However, injection with 0.1 mL
cell-free filtrate is found to be too little to cause severe skin lesions
compared to 0.2 and 0.3 mL filtrate used. In contrast, the use of 0.2
and 0.3 mL cell-free filtrates produced big lesion areas (Table
2) with higher level of severity for all mice. Both sonicated and
unsonicated cell-free extracts from all strains used in the study showed
a relative increment of volume used to the sizes of abscesses produced.
However the distribution of toxigenicity varied among the isolates. Therefore,
it is clearly shown in present study that this toxin is produced extracellularly
by this bacterial strain. In addition, it
| Table 2: | Toxigenicity of Porphyromonas asaccharolytica isolates from unsonicated and sonicated cell-free filtrates via mice injection |
| Positive control: ATCC 33277, Negative controls: Saline | |
was found that periodontopathic bacteria is dose dependent in causing skin lesion on balb/c mice and is in agreement with those of other scientist elsewhere (Van Steenbergen et al., 1982; Himratul-Aznita et al., 2005).
The toxicity activity effect of the toxin towards heat treatment was studied at 2 different temperatures (37°C heated for 60 min and 60°C heated for 15 min). The results showed that the toxin activity of Porphyromonas asaccharolytica cell filtrates were stable when heated at 37°C for 60 min. However, the toxicity activity was terminated when the filtrates were heated at 60°C after 15 min, as none of the injected mice developed skin lesion. Similar result has also been obtained in another study on Prevotella intermedia toxin which was found to be stable at 37°C (Himratul-Aznita and Ansary, 2006).
CONCLUSION
Therefore, P. asaccharolytica isolates from adult periodontitis in our Malaysian sample have toxic activity and the toxin is heat labile, which functioned optimally at 37°C.