The Effectiveness of Streptococcus salivarius against the Otopathogens of Otitis Media

Transcription

1 The Effectiveness of Streptococcus salivarius against the Otopathogens of Otitis Media Dr. Robert C. Osgood Rochester Institute of Technology College of Health Sciences Biomedical Sciences Program

2 Definition: Biofilm Exopolysaccharide-encased thin layered 3D aggregates of microbial growth on living or non-living surfaces Additional Properties: Single species or multispecies Bacterial, Fungal or Algal Exopolysaccharide diversity

3 Our Group s Interest in Biofilms Dental Cavities Periodontal Disease Streptococcus mutans serotype k

4 Our interest in Biofilms Otitis Media Middle ear infection Otitis media (OM) is the 2nd most common disease of childhood, after upper respiratory infection (URI) OM is also the most common cause for childhood visits to a physician's office. Annual number of office visits is estimated to be about 16 million Total expenditures per year $2.5 million

5 Etiological Agents of Otitis Media Nontypeable Haemophilus influenzae Gram negative pleomorphic, coccibacillus, fastidious and facultative anaerobe Lacks the capsule that facilitates typing Streptococcus pneumoniae One of the top two isolates found in ear infection Gram positive diplococcus polysaccharide capsule Over 90 serotypes known *Fastidious, capnophilic - requires CO 2 for growth Moraxella catarrhalis fastidious, non-motile, gram negative diplococcus *microaerophilic common cause of otitis media and sinusitis

6 Oxygen Concentrations Under Microaerophilic and Anaerobic Conditions Oxygen Concentration CO2 Concentration Aerobic Atmospheric 5% Microaerophilic 10% 5% Anaerobic 0% ~10%

7 Insights from Early Investigations Effect of Oxygen on NTHi Biofilm Formation Effect of ph on NTHi Biofilm Formation

8 Crystal Violet Biofilm Assay V2 TC V2 LMEF A B C D E F G Prepare a 1/200 dilution of an overnight culture of organism 2. Add 200ul of diluted culture to wells of a 96 well plate 3. Incubate for 24 hours and add 50ul of % crystal violet to well and incubate for 20 min 4. Discard non-adherent cells of each well by sharp inverted ejection 5. Carefully wash wells with distilled water two times and allow to dry 6. Add 200ul of absolute ethanol to each well and mix gently while incubating at room temp H Controls

9 OD 595 OD 595 Crystal Violet Biofilm Assay Under Varying Oxygen Concentrations at ph V2 TC % CO2 Microaerophilic Anaerobic Condition Condition Condition V1 LMEF % CO2 Microaerophilic Anaerobic Conditions Conditions Conditions

10 SEM of NTHi Biofilm (86-028) Cross Sectional View

11 96 Well Biofilm Growth NTHi Isolate

12 NTHi Biofilm Close up #1 Color Inverted Image Color Inverted Image

13 The Eustachian Tube and Otitis Media

14 Biofilm Image of NTHi Strain (BLACK- WHITE IMAGE IN 96 Well Plate Well) Microaerophilic Conditions Anaerobic Conditions

15 This Investigation Focus on the ph of 8.0 Focus on the microaerophilic condition

16 Isolates Used in This Study Isolate ID Organism Comment V V6 Nontypeable Haemophilus influenzae Nontypeable Haemophilus influenzae 12 Month Colonization with AOM Long colonization Isolate 3 Streptococcus pneumoniae D39 Streptococcus pneumoniae Virulent serotype Moraxella catarrhalis Clinical Isolate

17 Is Our Chosen Probiotic Effective Against Our Organisms of Interest? The Chosen Probiotic: Streptococcus salivarius K12 BLIS o o o Produces bacteriocins o Substances produce by some bacteria (probiotics) that are effective in killing or inhibiting the growth of closely related species Specifically produces salivaricins o Salivaricin A, Salivaricin B, Salivarisin D Known to colonize the oral cavity of a small percentage of individuals Dr. Philip Wescombe, Scientist BLIS Technologies Ltd, New Zealand PO Box 5804 Moray Place, Dunedin, 9058 New Zealand

18 DISK METHOD Prepare and swab a 0.5% calcium carbonate and 1% sucrose supplemented Chocolate agar plate with a standardized amount of the test isolate Place sterile blank discs adequately spaced away from each other on the plate and aseptically add 100ul of a standardized concentration of Streptococcus salivarius K12 BLIS Place an extra sterile black disc on the plate and inoculate with sterile water to serve as a control Incubate duplicate plates at 37 C for 24 hours and evaluate for the production of inhibition zones

19 Disk Method Setup CONTROL: Add 100ul of Sterile broth to disk EXPERIMENTAL REPLICATES: Add 100ul of Streptococcus salivarius K12 BLIS to each disk of Salivaricins of Salivaricins

20 Disk Method S. salivarius k12 BLIS and NTHi V6 at ph-8.0 Aerobic Microaerophilic

21 Disk Method S. salivarius k12 BLIS and NTHi AOM at ph-8.0 Aerobic Microaerophilic

22 Antagonistic Activity of Streptococcus salivarius k12 BLIS against Streptococcus pneumoniae D39 using Disk Method at ph-8.0 Streptococcus pneumoniae is a capnophilic

23 Disk Method S. salivarius k12 BLIS and Moraxella catarrhalis at ph-8.0 Aerobic Microaerophilic

24 Confocal Microscopy

25 AOM1 ph 8.0 (NTHi--12 Hrs) 12 Hour Control 5X Mag Invitrogen s Live / Dead Stain Syto 9 and Propidium Iodide Green is LIVE and Red is DEAD

26 AOM ph 8.0 NTHi (24 Hrs) 10X 24 Hr Control Green is LIVE and Red is DEAD Invitrogen s Live / Dead Stain Syto 9 and Propidium Iodide

27 ua AOM ph 8.0 (NTHi - 36 Hrs) 36 Hr Control Green is LIVE and Red is DEAD Invitrogen s Live / Dead Stain Syto 9 and Propidium Iodide

28 A Few Conclusions 1. Streptococcus salivarius k12 BLIS shows promise in its ability to control NTHi, S. pneumoniae and Moraxella catarrhalis 2. Streptococcus salivarius k12 BLIS seems to exert its effect under aerobic or microaerophilic conditions which likely to exist in the ear during otitis media 3. The clinically significant ph of 8.0 does not seem to prevent the effect of Streptococcus salivarius k12 BLIS against 4 Assuming that recombinant production of the bacteriocins is possible, they do not harm host cells and a delivery mechanism across the tympanic membrane is identified, a new method of reducing episodes of otitis media may be obtained

29 Some Future Directions Clone the gene(s) and produce a recombinant form of the protein(s) that are responsible for the bactericidal effect Expand the testing of Streptococcus salivarius k12 BLIS against other pathogens) Evaluate the effect of Streptococcus salivarius k12 BLIS against purified endospores --Clostridium difficile Determine the cytopathic effect of Streptococcus salivarius k12 BLIS against middle ear epithelial cells and others

30 Acknowledgements Mike Pichichero, MD Director of RGH Research Institute Dr. Casey Legacy Pediatrics, RGH Research Institute Richard Hailstone, PhD Associate Professor, Rochester Institute of Technology Lab colleagues and Staff -- RGH Research Institute RIT Staff and Students

31 Thank You!!

32 Questions?

33 Additional Slides

34 Experimental Biofilm Assay Design Experimental AVE A B C D ph 3.5 ph 4.0 ph4.5 ph 5.0 ph 5.5 ph 6.0 ph 6.5 ph 7.0 ph 7.5 ph 8.0 ph 8.5 ph E F AVE Control Control G H Control AVE

35 OD (600 nm) OD (600 nm) OD (600 nm) Typical Results Microaerophilic ( RMEF AOM 1) Plat e 1 Plat e 2 Experimental Samples ph Plate 1 Plate 2 Positive Control ph Plate 1 Plate 2 Plate 3 Mean Negative Control ph

36 Are Alkaline Conditions Optimal for Biofilm Formation? Clinical ph Study Patient Initials Gender Age Sample ph 1 KF Male 10 Months BS Female 12 Months QNS 3 ZK Male 15 Months QNS 4 CR Male 15 Months DS Male 24 Months QNS 6 EB Female 24 Months GH Female 24 Months OF Male 48 Months ER Male 60 Months 8.5

37 Setting Up ph Volumes for Multichannel Pipetting Use a Pipette To add an equal Volume of Diluted Culture to each of the 12-Channels of the Sample Reservoir Pipette into 96-Well Plates ph 6 ph 7 ph 8

38 Incubate Under Appropriate Conditions Aerobic Incubator Microaerophilic Incubator Anaerobic Incubator

39 OD (600 nm) Typical Results Aerobic ( RMEF AOM 1) OD (600 nm) OD (600 nm) Plate 1 Plate 2 Plate 3 Mean Experimental Samples ph Plate 1 Plate 2 Positive Control ph Plate 1 Plate 2 Negative Control ph

40 OD (600 nm) OD (600 nm) OD (600 nm) Typical Results Microaerophilic ( RMEF AOM 1) Plat e 1 Plat e 2 Experimental Samples ph Plate 1 Plate 2 Positive Control ph Plate 1 Plate 2 Plate 3 Mean Negative Control ph

41 OD (600 nm) OD (600 nm) OD (600 nm) Typical Results Anaerobic ( RMEF AOM 1) Plate 1 Plate 2 Experimental Samples ph Plate 1 Plate 2 Positive Control ph Plate 1 Plate 2 Plate 3 Mean Negative Control ph