1. The Microbiology of Bad Breath and Periodontitis
By Kate Egge

2. Overview Background information about bad breath and Periodontitis
Discuss 3 bacteria that cause oral malodor
Methods for detecting bacteria and?or Bad breath:
PCR, organoleptic, VSC concentrations, and BANA tests
Research: that indicates bacteria cause bad breath
Research: that indicates that bacteria may cause periodontitis
Conclusions
References
Questions

3. Bad Breath
Americans spent 625 million dollars on breath fresheners in 2000.
85-95% of cases originate in the mouth
Microorganisms on the posterior dorsal side of tongue is the main source of bad breath
Area is poorly cleansed by saliva and contains invaginations where bacteria can hide and putrefy postnasal drip

4. Periodontitis
Some bad breath is associated with periodontal disease and destruction of gums
Periodontitis is a common, progressive disease that affects the supporting structures of the teeth, causing loss of attachment to the bone and often resulting in tooth loss

5. Bad breath Bad breath primarily result of microbial metabolism.
Organisms feed off of glycoproteins and release offensive chemical odors
Oral gram-negative organisms such as spirochetes and some bacteroides have the capacity to produce oral malodor through putrification of sulfur containing protein substrates such as cysteine and methionine
Chemistry

6. Important microorganisms
Key anaerobic organisms Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus

7. Phylogenetic tree of major lineages of Bacteria based on 16s ribosomal RNA sequence comparisons

8. Treptonema denticola Gram negative, anaerobic spirochete
A lot of size variation among the spirochetes
Form a major phylogenetic lineage of Bacteria
Protoplasmic cylinder
Flagella emerge from each pole and are located in the periplasm of the cell (endoflagella)
Endoflagella and protoplasmic cylinder are surrounded by a flexible membrane called the outer sheath

9. Porphyromonas gingivalis and Bacteroides forsythus
Member of genus Bacteroides
Gram negative obligate anaerobe
Ferments sugars to primarily acetate and succinate
Bacteroides species dominant bacteria in large intestine, found 10^10 – 10^11 cells per gram of human feces

10. Porphyromonas gingivalis
Several clinical studies show that P. gingivalis is a factor in periodontitis
Study Journal of Clinical Microbiology compared the prevalence of P. gingivalis in a group with periodontitis to a different group that was periodontally healthy
Results:

11. Porphyromonas gingivalis
P. gingivalis was detected in only 25% of the healthy subjects but was detected in 79% of the periodontitis group
The odds ratio for being infected with P. gingivalis was 11.2 times greater in the periodontits group than in the healthy group.

12. Porphyromonas gingivalis and PCR
PCR assays were used for detection of P.gingivalis
Target sequence was the ribosomal DNA spacer region between 16S and 23S ribosomal genes
DNA fragments were separated by agarose gel electrophoresis

13. Detecting Porphyromonas gingivalis

14. Methods for detecting oral malodor
Organoleptic measurements:
Scale 0=no odor up to 5=foul odor
Tongue malodor was assessed by scraping the posterior tongue dorsum with gauze and immediately smelling it’s odor.
Malodor in dental sites was assessed by pushing waxed dental floss between teeth and immediately smelling and recording the odor based on the organoleptic scale

15. Methods for detecting oral malodor continued…
High VSC concentrations indicate oral malodor
Volatile Sulfur Compounds (VSC) are measured with a portable sulfide monitor
VSC monitor contains a pump which sucks air from a plastic straw placed inside the subjects mouth
The mouth air is passed through an electrolytic sensor which measures the concentration of volatile sulfurs
VSC levels are strongly associated with organoleptic scores

16. Methods for detecting oral malodor continued
BANA test
Test kit for bacterial trypsin like activity based on the hydrolysis of benzoyl-DL-arginine-naphthylamide (BANA) used to asses the presence of one or more of
T. denticola , P. gingivalis , B. forsythus
These bacteria are capable of hydrolyzing the synthetic trypsin substrate BANA- an activity not identified in other oral microbial species
Conditions favoring a Positive BANA test on the tongue dorsum and tooth sites were associated with oral malodor production

17. Relationship of Oral Malodor to Periodontitis
Species identified by BANA tests have been tentatively implicated as putative periodontal pathogens
However, some studies have found that species Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus identified by BANA tests do not cause periodontitis.
When peridontitis was present 87.5% of tooth sites and 92.5% of tongue sites were BANA (+)
In healthy individuals 74.4% of tooth sites and 93% of tongue sites were BANA (+)

18. Relationship of Oral Malodor to Periodontitis continued
Oral malodor is not directly associated with presence of periodontitis
Substantial oral malodor may be present in individuals without periodontitis

19. Conclusions
Gram negative anaerobic oral bacteria (Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus ) cause bad breath
Key anaerobic organisms Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus degrade glycoproteins proteins and release foul smelling chemicals
Strong evidence indicates that these bacteria cause bad breath
Treponema denticola: Anaerobic spirochete, Protoplasmic cylinder and endoflagella
Porphyromonas gingivalis, and Bacteroides forsythus are both members of the genus Bacteroides

20. Conclusions continued
Bacteria on the posterior dorsal side of tongue is the main source of bad breath
One study using PCR found that P.gingivalis was 11x more likely to be found in individuals that had periodontitis- P. gingivalis may be a periodontal pathogen
However, research using BANA tests found that the presence of T. denticola, P. gingivalis, and B. forsythus was NOT associated with periodontitis
Oral malodor is not directly associated with the presence of periodontitis

21. References:
Rosenberg, Mel. Clinical Assessment of Bad Breath: Current Concepts. Journal of the American Dental Association 1996; 127:
Griffen A, Becker M, Lyons S. Prevalence of Porphyromonas gingivalis and Periodontal Health Status. Journal of Clinical Microbiology 1998; 36(11) :
Madigan M, Martinko J, Parker J. Biology of Microorganisms. Ninth edition
A, Bosy, G.V. Kulkarni, M. Rosenberg and C.A.G. McCulloch. Relationship of oral malodor to periodontitis: Evidence of independence in discrete subpopulations. Journal of Periodontology 1994; 65:37-46.
Rosenberg Mel. The science of Bad Breath. Scientific American 2002;
Bruce P,Boches S, Galvin J. Bacterial Diversity in human subgingival plaque. Journal of Bacteriology 2001;183(12):
De Boever E, Uzeda M, Loesche W. Relationship between volatile sulfur compounds,Bana-Hydrolyzing bacteria and gingival health in patients with and without complaints of oral malodor. Journal of Clinical Dentistry 1994; 4(4):