The role of microorganisms in the production of oral malodor

The role of microorganisms in the production of oral malodor Thomas P. McNamara, Ph.D., Joseph F. Alexander, AS., Morris Plains, N. J. DEPARTMENT

OF DENTAL

SCIENCE,

WARNER-LAMBERT

B.S., and Mary Lee, RESEARCH

INSTITUTE

An in vitro method has been established for studying the role microorganisms play in the formation of oral malodor. Using this procedure, the formation of putrid odors was correlated with a shift in flora from a predominantly grampositive flora to one having an unusually high number of gram-negative anaerobes. Some of the conditions contributing to this shift in flora are the stagnation of saliva, a depletion of available carbohydrates, and a rise in pH. On the basis of these findings, it is suggested that intrinsic oral malodor is readily produced by microorganisms in localized intraoral areas that favor such environmental conditions.

E

arlier work by Allen1 Prinz,2 and Sulser and associates3 established that a major source of offensive breath odors originates from stagnation of saliva and the presence of food debris. Using stimulated whole saliva as the odor-producing medium, a number of chemical changes in saliva have been recorded.4p s These investigators demonstrated that protein decomposition occurred in incubated whole saliva, with the resultant production of ammonia, sulfides, indole, and lactic acid. Further investigations into the role of saliva have attempted to identify the bacterial organisms involved in the formation of malodorous compounds associated with bad breath. Berg and Fosdick6 studied the effect of seventeen different oral microorganisms on the rate of putrefaction of saliva and salivary protein. Recognizing that despite the differences that did exist between their in vitro study and those conditions resident in the oral cavity, the investigators reported that practically all of the microorganisms studied could play a role in putrefaction of salivary proteins, with the mixed microflora producing putrefaction more rapidly than any single species. In another in vitro experiment Shiota and Kunkel? also concluded that no single organism could produce a malodor identical to that found in vivo, Importantly, both of the latter studies established that microorganisms in the presence of saliva are involved in the production of malodor. 41

42

McNamara,

Alexawh-,

awl

Lee

A number of invrstigators have attc~lupted lo identfy the itr viva conditions cbontributing to the formation of oral nlalotlor. I
AND

METHODS

In all of the in vitro experiments, odor was formed either by incubating whole saliva at 3’7OC. for 4 to 24 hours to simulate stagnation or by similarly incubating broth inoculated with saliva or stock cultures of selected organisms. The resultant odor was characterized by smelling to determine whether the typical putrid odor commonly associated with intrinsic oral malodor was produced. There was no attempt in these experiments to quantitate the strength of the odor. All judgments were on an all-or-none basis and aimed at determining qualitatively whether or not the characteristic putrid odor was present. Saliva

Twenty healthy adult human subjects chewed a 10 by 10 cm. piece of Parafilm “M” and expectorated 20 ml. of stimulated saliva into a sterile glassstoppered vial during a 20-minute collection period. The individual saliva samples were pooled prior to use in all investigations. Sterile

saliva

Fresh whole saliva was collected as above and filtered through a Millipore HA filter (0.45 micron). Broth

medium

Various types of medium were evaluated to determine their suitability as substrates for the in vitro production of odor. The following medium was found to be satisfactory for this purpose : 18 Gm. Brain heart infusion broth Difco #0037-01 Fluid thioglycollate Difco #0256-02-g 14 Gm. Yeast extract Difco #012’7-02-6 5 Gm. Distilled water 1,000 ml. Carbohydrate

stock

solution

Glucose was chosen for testing the effect of carbohydrate on odor production. A 5 per cent aqueous solution of glucose was sterilized by filtration through a

Volume 34 Number 1

Microorganisms and oral malodor

43

Millipore filter and stored at 4O C. until use. Varying quantities of the stock solution were added to the broth medium which, in turn, was inoculated with 0.1 ml. of pooled saliva and incubated at 37’ C. for 24 hours. Following incubation the broth tubes were evaluated organoleptically for odor. Gram-stained smears were also prepared from the broths to determine the ratio of gram-positive to gram-negative organisms. Buffer

McIlvaine’s standard buffer solutions were used to buffer the broth medium at pH 6.5 and pH 7.5. The buffers were sterilized by filtration through a Millipore filter and stored aseptically. The pH of the buffer solutions was checked with a Beckman pH meter, and the buffers were added to the broth medium in those experiments carried out to determine the relationship between pH and odor production. These broths were inoculated with 0.1 ml. of saliva and incubated at 37’ C. for 24 hours, after which they were evaluated for the presence of odor. Following this initial 24-hour incubation period, the growth in each of these buffered broth media was used to cross-inoculate fresh broth buffered at pH 6.5 and pH 7.2. These latter broths were incubated for an additional 24-hour period at 37O C. and evaluated for odor formation. Oral

microorganisms

The following microorganisms representative of the normal oral flora were studied to determine their capacity for producing putrid odors and were carried as stock cultures on either brain heart infusion agar (Difco) or broth medium : Agar: Streptococcus salivarius S. pyogenes

S. faecalis S. mitis

Lactobacillus

acidophilus L. casei Staphylococcus aureus Streptococcus pneumoniae Candida albicans Klebsiella pneumoniae Broth medium: Leptotrichia sp. Fusobacterium polymorphum Veillonella alcalescens Bacteroides fundiliformis Prior to evaluation, a 24-hour broth culture of each organism was used to prepare a suspension adjusted to a 50 per cent transmission f 2 per cent using a Bausch and Lomb Spectronic 20 at a wavelength of 465 nm. One tenth milliliter of a 1 A,000 dilution of this suspension was used to inoculate the broth medium. When saliva served as the inoculum for the broth, 0.1 ml. was used.

44

McNamara,

Alexalzder,

I. Odor-producing

Table

Odor-producing Sterile Broth Broth Sterile

Stagnant

saliva medium medium saliva

medium filtrate filtrate

and. Lee

characteristics

Oral July,

of salivary

components odor

InoculzLm None Sterile saliva filtrate Organisms recovered Organisms recovered

from from

Surg. 1972

saliva saliva

production

Negative Negative: Positive Positive

saliva

Separate tenfold serial dilutions were carried out to determine the total number of viable organisms present in fresh and stagnated saliva. The dilutions were prepared by adding a 1.0 ml. aliquot from the 20 ml. fresh pooled saliva sample to 9.0 ml. of broth medium and continuing such tenfold dilutions through a total of ten tubes. All tubes were incubated at 37O C. for 48 hours. A 10 ml. aliquot of the remainder of the original pooled saliva sample was then incubated for 24 hours at 37O C. to simulate stagnation as might be found in the oral cavity. After incubation, a 1.0 ml. aliquot of the stagnated saliva was also diluted in the broth medium in the same manner as the fresh saliva and incubated at 37O C. for 48 hours. Following incubation, all broth tubes were checked macroscopically for evidence of growth. The total number of organisms present in the fresh and the stagnated saliva samples was determined by multiplying the last tube showing growth in each of the respective series by its corresponding dilution factor. Each incubated tube of broth medium used for determining the number of organisms present was evaluated for odor formation. RESULTS

The data in Table I show a positive cause-and-effect relationship between the presence of salivary microorganisms and the production of malodor. The bacteriafree samples did not produce any malodor that would be considered similar to in vivo intrinsic oral malodor. Of the fourteen species of oral organisms used as inoculum for the broth medium, the nine gram-positive organisms did not produce any putrid odors. The remaining five test cultures (four gram-negative and one gram-variable) did produce unpleasant odors. These results are summarized in Table II. When the total number of viable organisms in freshly collected saliva was compared with those present in saliva incubated at 3i’O C., the results (Table III) show that all of the samples contain similar concentrations of organisms, that is, about lo8 organisms per milliliter. It should be noted, however, that putrid malodor was detected in the broth-dilution series prepared from the stagnant saliva samples at much greater dilutions than in the broth-dilution series prepared from fresh saliva samples. The data in Table IV show that the ratio of gram-positive to gram-negative organisms present in fresh saliva upon stagnation undergoes a dramatic shift in favor of the growth of the latter organisms. This shift is accompanied by the appearance of putrid odor. The malodors produced by stagnated saliva samples and by broth inoculated with saliva were indistinguishable.

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34 1

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45

II. The odor-producing characteristics of selected salivary organisms when used as inoculum for broth medium and incubated at 37O Cr. for 24 hours

Table

Organism

odor

Streptooocczls salivarizcs S. pyogenes S. faecalis S. mitis Laotobacillzcs acidophilus L. casei Staphylococcus aureus Diploooccus pneumoniae Can&da albicans Leptotrichia sp. Fusobacteriwm polymorphzlm. Veillonella aloalescens Bacteroides fundiliformis Elehsiella pnezlmoniae

None None None None None None None None None Unpleasant Unpleasant Unpleasant Unpleasant Unpleasant

Table

Ill. Determination

Gram

produced

character

Positive Positive Positive Positive Positive Positive Positive Positive Positive Variable Negative Negative Negative Negative

(putrid) (putrid) (putrid) (putrid) (putrid)

of the number of organisms and malodor present in

fresh and stagnant saliva Saliva

sample

Fresh Serial broth dilzction SO-1 10-z 10-a 10-b 10-S 10-e 10-T 10-S 10-g

Bacterial

growth

Stagnant Pzltrid

odor

+ + + +

Bacterial

growth

P&rid

odor

+ + f + f + +

+ = present. - = absent.

Freshly collected whole human saliva has a slightly acid pH and a rather pleasant sweet scent, but when it is incubated it gradually becomes alkaline and takes on a distinctive putrid malodor. This same finding is noted when 0.1 ml. of saliva was used to inoculate the specially prepared buffered broth. Following a 24-hour incubation period, the broth buffered at pH 6.5 produced no odor while the broth buffered at pH 7.5 produced a typical malodor. When material from each of these tubes was used to cross-inoculate separate broth tubes at pH 6.5 and pH 7.2, a typical malodor was produced only in those tubes at pH 7.2, regardless of the inoculum used. The effect of adding various concentrations of glucose to saliva samples is shown in Table V. The results show that a concentration of 0.02 M glucose suppresses odor formation while maintaining the balance of gram-positive to gram-negative organisms. With a glucose level of 0.002 M, the ratio of grampositive to gram-negative organisms begins to shift and favors the growth of the gram-negative types, with the concomitant formation of odor. The control sample without added glucose contained a 40 :60 ratio of gram-positive to gram-negative

Table IV. The Gram character, fresh and stagnated saliva

morphologic

Fresh iv0

I

Per

0a0r

@am-positive Gram-positive Gram-positive Gram-negative Gram-negative

cocci filaments rods cocci filaments

type, and presenc:c of malodor of

Stugnant

Gram-positive Gram-positive Grxmpositive Gram-negative Gram-negat,ive

Table V. The effect of glucose on odor formation to gram-negative organisms in stagnated saliva Glucose 0.0 Control 0.002 M 0.02 M - No odor. ++ Typical

Pev

im~a0~0~

cent

45 20 20 10 5

odor formation ++ ++ -

cocci filaments rods cocci filaments

cent 25 10 10 25 00

and the ratio of gram-positive Oral flora Gram-positiz;e/Gram-aegative 40/60 55/45 75/25

malodor.

organisms and pr0ducea malodor similar to that recorded for the saliva sample incubated with 0.002 M glucose added. DISCUSSION The results of this study confirm the findings of Berg and FosdickC and Shiota and Kunkel’ in clearly indicating that the capacity for producing oral malodor resides in the oral microbial flora. When a bacteria-free saliva filtrate is incubated alone or added to the broth medium, no malodor is produced. Conversely, when the organisms filtered from saliva were either added to the sterile saliva filtrate or added to the broth medium, malodor was produced (Table I). While microorganisms are essential for the production of intrinsic oral odor, not all resident species have this odor-producing capability. When whole saliva is allowed to stagnate for 24 hours at 37O C., the formation of odor is coincident with an increase by three orders of magnitude of odor-generating organisms ovei the number initially present in fresh saliva. These data (Tables II, III, and IV) indicate that a change has occurred with reference to the gram-staining character of the organisms that are present and that the capability for producing oral malodor resides with the gram-negative organisms. Accordingly, the factors involved in the production of malodor from incubated saliva would tend to confirm Everett’s* clinical findings of objectionable breath odor associated with stagnation of saliva accompanied by a concomitant rise in the total number of gramnegative filamentous organisms. There are several areas in the oral cavity where saliva readily stagnates-the gingival crevice, interdental spaces, dental plaque, tonsillar folds, and papillary crypts of the tongue. The technique of incubating saliva in the present study

Volume 34 Number 1

Microorganisms

and oral malodor

47

approximates the in vivo saliva stagnation process and has resulted in favoring the growth of gram-negative organisms with a concurrent production of putrid odor (typical intrinsic oral malodor) , Gibbons and colleaguesl” have studied the microbiota of samples of dental plaque, debris taken from the gingival crevice area, and swabbings made from the tongue, cheek, and saliva and have reported on the distribution in these various areas of the gram-positive organism, Streptococcus saliva&s, and the gram-negative organism, Bacteroides melanogenicus. Where little or no saliva stagnation would be expected, the gram-positive organism was found to be 20 to 100 times more numerous than the gram-negative organism. Conversely, in areas, such as in the gingival crevice, where one would expect saliva stagnation to occur, the gram-negative organism was ten times more numerous than the gram-positive organism. Although the aforementioned in vivo study did not involve any measurement of odor, the findings on the relative number of gram-positive and gram-negative indicator organisms as related to areas of saliva stagnation parallel the results obtained in the present study. It is not surprising to find that bacterial populations vary within different microenvironments in the mouth. In fact, different micro-environments most likely determine the composition of the microflora resident within them. On the basis of these data, it is proposed that intrinsic oral malodor is readily produced by microorganisms in localized areas within the mouth where saliva stagnation commonly occurs; such malodor most likely does not occur in areas of copious flow of saliva. Saliva stagnation, however, is not the only factor involved in oral odor production. As noted from the results of the present study, such subtle differences as a change to an acid pH or the presence of small amounts of glucose can affect the metabolism of the oral flora in a way that results in the formation of nonodorous end products. An acid pH prevents the formation of odorous metabolic end products by inactivating the enzymes required for the putrefaction of amino acids. The principal effect of glucose on odor-generating organisms is to alter their metabolism of tryptophane and other amino acidsI which inhibits the formation of odorous end products. Additional factors that may have a role in intrinsic odor formation are currently being investigated. It is, of course, recognized that not all malodors emanating from the mouth are caused by microorganisms. Common examples of nonmicrobial-induced odors would include the extrinsic odor of onion, alcohol, or garlic. “Stomach odors” would not be considered an important contributing cause of oral odor, since the esophagus is normally a collapsed tube and only during eructation would odiferous gases be expelled into the mouth from the stomach, Other physiologic factors involved in oral odor production are increased incidence with age,l* the “hunger state, “I3 and the time of day.l” The results of the present study strongly suggest that intrinsic malodor produced locally in the oral cavity is derived from the metabolism of some organism(s) resident in the oral flora. Toothbrushing as a general hygienic measureI and the use of an antiseptic rinse I6 to reduce the number of oral organisms are broad-spectrum measures effective in reducing locally produced oral malodor. Future research efforts should be directed toward identifying and selectively controlling the specific organisms(s) responsible for odor formation.

1. An in vitro method simulating in viva conditions has been established to assess the causes of intrinsic oral malodor (organolepticallg offensive breath odor). 2. These studies confirm the findings of previous investigations that bacteria are essential to the production of intrinsic oral malodor. 3. Gram-negative microorganisms have been identified as those chiefly responsible for the production of intrinsic oral malodor. 4. Saliva that is allowed to incubate (stagnate) results in a shift in the ratio of gram-positive to gram-negative organisms and favors the growth of the latter. 5. A slightly alkaline pH (7.2) favors malodor production, while a slightly acid pH (6.5) has the opposite effect. 6. The addition of glucose to the salivary flora inhibits the production of intrinsic oral malodor. The authors wish to thank aration of the manuscript.

Hazen

J. Baron,

D.D.S.,

Ph.D.,

for

his

assistance

in the

prep-

REFERENCES

i, 3: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Allen, H. J.: Fetor in the Breath, Welch’s Monthly 1: 257261, 1896. Prinz. H.: Offensive Breath, Its Causes and Prevention, Dent. Cosmos 72: 700-707. 1930. Sulser, G. F., Lesney, T. A:, and Fosdick, L. S.: The Reduction of Breath and Mouth Odors by Brushing the Teeth, J. Dent. Res. 19: 173-177, 1940. Cary, J. E.: The Development of Alkali Within Saliva and Its R.elation to Dental Caries, Aust. J. Dent. 50: 4-9, 1946. Rae, J. J., and Clegg, C. T.: Lactic Acid Production in Saliva, J. Dent. Res. 35: 612. 614, 1956. Berg, M., and Fosdick, L. S.: Studies in Periodontal Disease. II. Putrefactive Organisms in the Mouth, J. Dent. Res. 25: 73-81, 1946. Shiota, T., and Kunkel, M. F.: In Vitro Chemical and Bacterial Changes in Saliva, J. Dent. Res.37: 780-7&X7? 1958. Everett, M. R.: Medical Biochemistry, ed. 2, New York, 1949, Paul B. Hoeber, Inc., Medical Students Series, p. 155. Fox, N., and Kesel, R. G.: Hyperplastic Sinopharyngostomatitis, Arch. Otolargngol. 42: 368-371. 1945. Gibbons, RI J. Kapsimalis, B., and Socransky, 8. S.: The Source of Salivary Bacteria, Arch. Oral Bioi. 9: 101-103, 1964. Bovd. W.. and Liehstein, H.: The Effect of Carbohvdrate on the Trvotouhanase Activitv “I 1 of Bacteria, J. Bacteriol’69: 584-589, 1955. Sulser, G. F., Brening, K. H., and Fosdick, L. S.: Some Conditions That Affect the Odor Concentration of Breath, J. Dent. Res. 18: 355-359, 1939. Best! C. H., and Taylor, N. B.: The Physiological Basis of Medical Practice, ed. 3, Baltimore, 1943, Williams & Wilkins Company, p. 828. Massler, M., Emslie, R. D., and Bolden, T. E.: Fetor Ex Ore, ORAI, SURG. 4: 110-125,

__--.

lRR1

J. Am. Dent. Assoc. 55: 37-46, 15. Hine,, M. K.: Halitosis, P. P., and Read, R. R.: Halitosis: Variations 16. Morris? Intensity Resulting From Prophylaxis and Antisepsis, Reprint requests to: Dr. T. F. McNamara Department of Dental Science Warner-Lambert Research Institute 170 Tabor Rd. Morris Plains, N. J. 07950

1957. in Mouth J. Dent.

and Total Breath Odor Res. 28: 324-333, 1949.