Pilot study on selective antimicrobial effect of a halitosis mouthrinse: monospecies and saliva-derived microbiome in an in vitro model system

Figures & data

Table 1. Species and strains and their growth conditions

Species	ATCC number	Medium	Growth conditions
Fusobacterium periodonticum	33693	Columbia broth, Columbia blood agar	anaerobic
Fusobacterium nucleatum	23726	Columbia broth, Columbia blood agar	anaerobic
Tannerella forsythia	43037	SHI medium liquid, agar	anaerobic
Prevotella intermedia	49046	TSB supplemented broth, agar	anaerobic
Veillonella atypica	17744	BHI+0.06% lactic acid (broth, agar)	anaerobic
Porphyromonas gingivalis	33277	Columbia broth, Columbia blood agar	anaerobic
Streptococcus sanguinis	10556	BHI broth, agar	microaerophilic
Streptococcus gordonii	10558	BHI broth, agar	microaerophilic
Streptococcus mutans	UA140	BHI broth, agar	microaerophilic
Candida albicans	SN152	TSB broth, agar	aerobic

Anaerobic: 0% O₂, 5% CO₂, 5% H₂; Microaerophilic: 2.6% O_2, 5% CO₂; Aerobic: 21%O₂, 0.04% CO₂, 0.9% Ar, 78% N₂. BHI: brain heart infusion; TSB: tryptic soy broth; TSB supplemented: tryptic soy broth supplemented with yeast extract, hemin, vitamine K, L-cysteine; SHI: medium developed in Dr. W Shi’s laboratory [24].

Table 2. Activity of fresh breath rinse against common oral species in monoculture

Associated conditions	Strain	FBR exposure (min)	% Reduction	Listerine exposure (min)	% Reduction
Halitosis and periodontitis [7,9–12]	Fusobacterium periodonticum	2	82.0 (± 2.2)	2	22.2 (± 2.0)
		5	97.7 (± 1.1)	5	100.0 (± 0.9)
		10	99.9 (± 0.1)	10	-
		20	100.0 (± 0.4)	20	-
	Fusobacterium nucleatum	2	95.6 (± 14.0)	2	13.5 (± 2.0)
		5	99.5 (± 3.3)	5	100.0 (± 1.9)
		10	100.0 (± 0.2)	10	-
		20	-	20	-
	Tannerella forsythia	2	61.7 (± 1.0)	2	12.9 (± 1.0)
		5	86.5 (± 7.0)	5	100.0 (± 0.0)
		10	88.0 (± 4.0)	10	-
		20	95.7 (± 1.0)	20	-
Halitosis [9,10,13,14]	Prevotella intermedia	2	96.5 (± 3.0)	2	20.2 (± 3.7)
		5	100 (± 2.5)	5	100.0 (± 0.0)
		10	-	10	-
		20	-	20	-
	Veillonella atypica	2	24.7 (± 1.9)	2	11.6(± 1.9)
		5	36.5 (± 6.0)	5	100.0 (± 0.0)
		10	40.0 (± 1.0	10	-
		20	99.9 (± 4.3)	20	-
Periodontitis [14,15]	Porphyromonas gingivalis	no effect		2	23.7 (± 9.3)
				5	100.0 (± 0.0)
				10	-
				20	-
Commensal [32,33]	Streptococcus sanguinis	no effect		2	6.1 (± 2.9)
				5	100.0 (± 0.0)
				10	-
				20	-
	Streptococcus gordonii	no effect		2	12.4 (± 4.0)
				5	100.0 (± 0.0)
				10	-
				20	-
Caries [41]	Streptococcus mutans	no effect		2	5.2 (± 3.0)
				5	100.0 (± 0.0)
				10	-
				20	-
Stomatitis [42]	Candida albicans	no effect		2	12.3 (± 3.9)
				5	100.0 (± 0.0)
				10	-
				20	-

Figure 1. The test mouthrinse antimicrobial activity against common oral species in monoculture. Single-species cultures of Fusobacterium periodonticum, F. nucleatum, Prevotella intermedia, Tannerella forsythia, Veillonella atypica, Porphyromonas gingivalis, Streptococcus gordonii, S. sanguinis, S. mutans, and Candida albicans were exposed to the test mouthrinse (FBR, red symbol and line) or phosphate-buffered saline solution (PBS, blue symbol and line) for the indicated amount of time, and viability was assessed by CFUs. The curves represent the means and standard deviation of duplicated samples from two independent experiments. Differences in significance between groups were analyzed using two-way anova; *p < 0.05, **p < 0.005, ***p < 0.0005

Table 3. Decrease in genus relative abundance after 5 and 30 min exposure to FBR compared to saline control

% Relative abundance after 5 min exposure
	O-Mix			H1			H2			H3
	PBS	FBR	↓	PBS	FBR	↓	PBS	FBR	↓	PBS	FBR	↓
Fusobacterium	7.38	8.27		0.02	0.01	54.9	11.51	0.68	94.1	15.93	10.69	32.9
Parvimonas	16.66	19.50		0.01	0.01	46.5	8.00	11.01		0.18	0.18	3.3
Peptostreptococcus	4.45	5.22		13.99	1.97	85.9	8.76	0.69	92.1	0.01	0.01
Porphyromonas	0.25	0.81		0.00	0.00		1.47	2.38		3.88	4.34
Prevotella	8.10	0.10	98.7	20.38	0.24	98.8	10.71	3.78	64.7	16.74	11.44	31.7
Pseudomonas	8.18	0.00	100.0	0.00	0.00		0.00	0.00		0.00	0.00
Solobacterium	4.53	2.83	37.5	0.13	0.00	98.2	0.69	0.14	79.1	0.05	0.03	47.6
Stomatobaculum	9.05	10.79		3.33	0.06	98.2	4.16	0.81	80.5	0.05	0.01	72.7
% Relative abundance after 30 min exposure
	O-Mix			H1			H2			H3
	PBS	FBR	↓	PBS	FBR	↓	PBS	FBR	↓	PBS	FBR	↓
Fusobacterium	7.03	0.68	90.3	0.02	0.00	77.8	11.20	0.01	99.9	13.15	0.04	99.7
Parvimonas	22.71	1.56	93.1	0.01	0.01	9.1	7.50	9.97		0.21	0.01	96.8
Peptostreptococcus	4.19	1.11	73.4	12.56	0.44	96.5	10.53	0.01	99.9	0.01	0.01	43.8
Porphyromonas	0.24	0.05	79.1	0.00	0.00		1.68	0.09	94.8	4.10	0.68	83.4
Prevotella	7.40	0.04	99.5	25.39	0.05	99.8	9.07	0.10	98.9	16.30	7.69	52.8
Pseudomonas	4.71	0.01	99.9	0.00	0.00		0.00	0.00		0.00	0.00
Solobacterium	4.53	0.05	98.9	0.12	0.00	98.5	0.46	0.00	99.4	0.00	0.00
Stomatobaculum	9.37	0.31	96.7	3.17	0.02	99.41	4.36	0.24	94.6	0.07	0.00	97.5

PBS – phosphate-buffered saline solution; FBR – TheraBreath Fresh Breath Rinse.

↓ – % of decrease.

Figure 2. Microbial composition of saliva-derived cultures. Saliva was first cultured in SHI media and subsequently exposed to the test mouthrinse (FBR), phosphate-buffered saline solution (PBS), or Listerine with aliquots collected 5 min and 30 min after treatment. Aliquots were pelleted and then re-cultured in SHI media overnight before extracting DNA and performing 16S sequencing. The relative abundance (percentage) of the genus-level taxonomic results are shown in the stacked bar graphs. (a) O-Mix; pooled saliva from healthy volunteers was used to generate a healthy community culture. (b-d) H1, H2, and H3 refer to saliva-generated cultures with saliva from individuals with self-reported halitosis

Figure 3. Alpha diversity analysis of saliva-derived microbial communities. The alpha diversity of saliva microbial communities upon treatment with the test mouthrinse (FBR), phosphate-buffered saline solution (PBS), or Listerine (5 min and 30 min after treatment) was analyzed using Shannon index, and the respective rarefaction curves are shown. (a) O-Mix; mixed saliva from healthy volunteers was used to generate a healthy community culture. (b-d) H1, H2, and H3 refer to saliva-generated cultures with saliva from individuals with self-reported halitosis

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