Identification of interspecies interactions affecting Porphyromonas gingivalis virulence phenotypes

Figures & data

Fig. 1.  Effects of Porphyromonas gingivalis (Pg) on oral epithelial cells. (A) Pg W83, Streptococcus oralis 180B (So180B), or Pg+So180B were added to OKF6/TERT2 cells. Cocultures were allowed to incubate in 5% CO₂ at 37°C for 16 hr and then examined by blinded investigators. (B) OKF6/TERT-2 cells were grown to confluence and used in the wound-healing assay. A linear streak was made in the cell monolayer. Pg, So180B, or Pg+So180B were added to the cultures at MOI = 100. After incubation for 2 hrs, the plates were washed to remove bacteria and fresh media was added. The cultures were incubated for 16 hrs and read by blinded investigators.

Table 1. Inhibition of Porphyromonas gingivalis (Pg) cytotoxicity by clinical strains

		Inhibition of Pg cytotoxicity
Classification of clinical isolates	Number of strains	−	+	+ +
Actinobacteria (Gram+, high G + C ratio)
Actinomyces naeslundii	13	5	4	4
Actinomyces odontolyticus	2	1	0	1
Actinomyces viscosus	1	1	0	0
Propionibacterium acnes	3	0	2	1
Firmicutes (Gram+, low G + C ratio)
Bifidobacterium dentium	1	1	0	0
Gemella morbilorum	1	1	0	0
Lactobacillus rhamnosus	2	2	0	0
Staphylococcus pasteuri	2	2	0	0
Staphylococcus epidermidis	2	2	0	0
Streptococcus constellatus	3	2	0	1
Streptococcus cristatus	1	1	0	0
Streptococcus gordonii	2	2	0	0
Streptococcus intermedius	3	2	1	0
Streptococcus infantis	2	2	0	0
Streptococcus mitis	5	3	0	2
Streptococcus oligofermentans	1	1	0	0
Streptococcus oralis	13	12	0	1
Streptococcus parasanguinis	2	2	0	0
Streptococcus pneumoniae	3	3	0	0
Streptococcus sanguinis	17	16	0	1
Streptococcus vestibularis	3	3	0	0
Fusobacteria (Gram–)
Fusobacterium equinum	1	1	0	0
Fusobacterium periodonticum	1	1	0	0
Veillonella (Gram–)
Veillonella denticariosi	2	2	0	0
Veillonella dispar	2	1	0	1
Unidentified oral isolates	15	15	0	0
Total	103	84	7	12

Inhibition of Pg cytotoxicity was evaluated using an in vitro cell-rounding assay. OKF6/Tert-2 cells exposed for 16 hrs to Pg (MOI = 100) showed complete rounding and detachment upon microscopic observation. The effects of coincubation with isolates on Pg cytotoxicity were scored as follows: ‘–’ no visible inhibition (complete cell rounding and detachment), ‘ + ’ weak inhibition (partial cell rounding and detachment), and ‘ + +’ strong inhibition (minimal cell rounding and detachment). The table describes the number of isolates within each species of bacteria that inhibited P. gingivalis cytotoxicity. The assay was performed in duplicate and confirmed in at least two independent experiments. A total of 103 clinical strains were analyzed.

Table 2. Effects on Porphyromonas gingivalis (Pg) gingipain activity and production of acid and hydrogen peroxide by oral bacterial strain

	Percent change in gingipain activity				Production of
	Cocultured with Pg		Independent culture		Acid (pH)	H2O2 (ng/ml)
Clinical isolates	%	P value	%	P value
Actinomyces naeslundii 106	9	Ns	−24	Ns	6	476
A. naeslundii 109A	−12	Ns	−9	Ns	5.8	–
A. naeslundii^a 110A	14	Ns	−27	0.002	5.8	–
A. naeslundii 139A	0	Ns	17	Ns	5.8	–
A. naeslundii 141	2	Ns	−8	Ns	5.8	–
A. naeslundii 144A	7	Ns	14	Ns	5.8	–
A. naeslundii^a 164B	9	Ns	1	Ns	5.8	–
A. naeslundii^a 181A	4	Ns	−31	0.003	6.4	1,547
A. odontolyticus^a 178	13	Ns	−10	Ns	7	–
Propionibacterium acnes 115	4	Ns	−2	Ns	7	–
P. acnes 167	−2	Ns	−31	0.009	5.8	–
P. acnes 168	−28	0.001	−9	Ns	7	–
Streptococcus constellatus 165	−35	0.009	14	Ns	7	–
Streptococcus intermedius 173	5	Ns	7	Ns	5.5	–
Streptococcus mitis 166	−36	0.006	−35	0.002	6.2	759
Streptococcus mitis^a 185	10	Ns	−34	0.001	7	654
Streptococcus oralis 180B	4	Ns	−26	0.003	6.2	652
Streptococcus sanguinis 179	−55	0.005	−18	Ns	6.6	–
Veilonella dispar 103	−27	0.001	−8	Ns	6.2	–

Porphyromonas gingivalis (Pg) total gingipain activity was assayed by incubating Pg and each of 19 oral bacterial strains (identified in Table 1 as inhibitory to Pg cytotoxicity) with BANA substrate and observing the change in color upon the addition of Fast Blue BB detection solution. The oral bacteria were either cocultured with P. gingivalis for 1 day or cultured independently and mixed with Pg immediately prior to the assay. The OD_490nm of the reaction mixtures was measured and expressed as percent activity compared to Pg alone. Statistically significant (P>0.01) effects of clinical isolates on gingipain activity compared to Pg alone when analyzed using a paired t-test are indicated in bold type. Ns = nonsignificant. Acid production by inhibitory clinical isolates was assayed by measuring the pH of TS broth cultures after 48 hrs at 37°C. The presence of H₂O₂ in broth cultures after 48 hrs was measured using Hydrogen Peroxide Assay Kit as described in Methods. Data shown are the means of three experiments in triplicate. Samples below the level of detection of 15 ng/ml are marked as ‘–’.

^aLess than 98% 16S rRNA sequence similarity to the closest type strain.

Fig. 2.  Effect of oral bacterial isolates on Porphyromonas gingivalis (Pg) growth. Broth cultures of 19 clinical strains identified as inhibitory to Pg cytotoxicity were spotted on blood agar inoculated with a lawn of Pg and incubated anaerobically at 37°C for 48 hrs. The growth of Pg was inhibited near the colonies of 6 of 19 strains including (A) Streptococcus intermedius and Actinomyces naeslundii. (B) Growth inhibition was measured as the size of the clear zone (in mm) between the edge of the colony and the lawn of P. gingivalis. Bars represent the average of triplicate measurements confirmed in three independent experiments. Abbreviations used: An, Actinomyces naeslundii; Pa, Propionibacterium acnes; Si, Streptococcus intermedius; Smi, Streptococcus mitis; Sc, Streptococcus constellatus; So, Streptococcus oralis; Streptococcus sanguinis; Vd, Veillonella dispar. *less than 98% 16S rRNA sequence similarity to the closest type strain.