IADR Abstract Archives
Parabiotic Generation Using Ultrasound From L. Rhamnosus and L. Reuteri Probiotics
Objectives: Although probiotics’ use as alternative dental aids is increasing, the presence of live bacteria in these products has been a deterrent to their significant popularity in clinical practice. Administration of inactivated microorganisms, or parabiotics, has been tested in other fields to counteract this problem, but has only been sparingly tested in dentistry. This research aimed to assess the effects of ultrasound technology on commonly used probiotics L. rhamnosus and L. reuteri.
Methods: L. rhamnosus GG (ATCC) and L. reuteri DSM 17938 (Biogaia Protectis®) were cultured in MRS at 37oC in 5% CO2. Ultrasound treatment (UP400St, Hielscher) was carried out on 24-hour cultures (3 x 108 CFU/mL) on single species and 1:1 mixes at 240 W, 4.5 cm probe immersion depth (S24d3 sonotrode, Hielscher) for 3, 6, 8, 10 and 15 minutes.
Viability assays (colony counts, Baclight® and MTT) confirmed viability following treatment. Structural changes post-treatment were investigated through Scanning Electron microscopy (SEM) (Zeiss, 8 KV) following glutardehyde fixing, osmium tetroxide staining, ethanol dehydration and gold plating, while growth inhibition was assessed (spot test) against Streptococcus mutans (NCTC 10449 and ATCC 25175), Streptococcus gordonii (DL1 and ATCC 10558) and Streptococcus oralis (ATCC 35037). Sterile-filtered supernatants were also analysed (Bradford’s assay, Data-Independent Acquisition proteome analysis (BGI)).
Results: All probiotic groups lost viability after 15 minutes of treatment (p < 0.001). SEM showed progressive treatment time-dependent collapse of the bacterial membrane. L. rhamnosus products showed greatest inhibition, albeit with a treatment time-dependent decrease. Decreased viability, altered bacterial membrane and decreased inhibition with increased treatment time suggested probiotic product secretion, confirmed through supernatant analysis.
Conclusions: To our knowledge this is the first time parabiotics for oral health applications have been produced with the proposed methods. This research highlights ultrasound technology’s potential for translation for use on other probiotics for a wide range of medical applications.
Methods: L. rhamnosus GG (ATCC) and L. reuteri DSM 17938 (Biogaia Protectis®) were cultured in MRS at 37oC in 5% CO2. Ultrasound treatment (UP400St, Hielscher) was carried out on 24-hour cultures (3 x 108 CFU/mL) on single species and 1:1 mixes at 240 W, 4.5 cm probe immersion depth (S24d3 sonotrode, Hielscher) for 3, 6, 8, 10 and 15 minutes.
Viability assays (colony counts, Baclight® and MTT) confirmed viability following treatment. Structural changes post-treatment were investigated through Scanning Electron microscopy (SEM) (Zeiss, 8 KV) following glutardehyde fixing, osmium tetroxide staining, ethanol dehydration and gold plating, while growth inhibition was assessed (spot test) against Streptococcus mutans (NCTC 10449 and ATCC 25175), Streptococcus gordonii (DL1 and ATCC 10558) and Streptococcus oralis (ATCC 35037). Sterile-filtered supernatants were also analysed (Bradford’s assay, Data-Independent Acquisition proteome analysis (BGI)).
Results: All probiotic groups lost viability after 15 minutes of treatment (p < 0.001). SEM showed progressive treatment time-dependent collapse of the bacterial membrane. L. rhamnosus products showed greatest inhibition, albeit with a treatment time-dependent decrease. Decreased viability, altered bacterial membrane and decreased inhibition with increased treatment time suggested probiotic product secretion, confirmed through supernatant analysis.
Conclusions: To our knowledge this is the first time parabiotics for oral health applications have been produced with the proposed methods. This research highlights ultrasound technology’s potential for translation for use on other probiotics for a wide range of medical applications.