Newer Bacteriologic Considerations of Dental Infections as Factors in Systemic Diseases

There are wide differences of opinion as to the significance of dental infection in systematic disease and ordinary bacteriologic methods are of little assistance. Our researches during the past 17 years have thrown considerable light on the bacteriology and pathology of this relationship and suggest explanations for the usual failure to secure desired results from application of the focal infection hypothesis. We will present an outline of our findings and will discuss certain technical details which were found necessary for best diagnostic and therapeutic results.

First of all, we must correct certain misunderstandings concerning the mechanism of focal infection. The usual concept is that of an isolated mass of infected tissue which gives off bacterial bodies to the blood and lymph streams, which in turn carry them to the rest of the body, where they set up secondary foci. These secondary foci are kept inflamed by constant or frequent showers of bacteria. If elimination of the suspected “primary” focus does not cure the disease, other foci are sought. Then, if they cannot be found or if eliminating them does not help and vaccine treatment gives no relief, it is considered proof that foci of infection had no bearing on the disease. Such an assumption, however, does not seem justified in the light of recently discovered facts.

The conception just outlined takes into consideration only one phase of focal infection. Many inflamed tissues undoubtedly are true foci of infection, i.e., masses of infected tissue but the transport of bacteria from primary to secondary foci is effectively checked by the bactericidal power of the blood and the phagocytic action of the reticuloendothelial system. Thus, there is little likelihood that they are constantly fed by bacteria from primary foci. It is possible that blood-borne bacteria might be deposited in distant tissues (i.e., secondary foci), particularly after trauma or acute infection and that some force, possibly a circulating toxin, activates the infection after it has once been established in the secondary focus. However, many “foci” cannot be explained on this basis.

The evidence offered by the tissue reactions in certain inflammatory diseases, e.g., of the eye, joints, gall bladder and intestine, from which no bacteria can be cultivated, suggests that an entirely different mechanism may be responsible for this type of “focal” inflammation. It is possible that in such instances the inflammation in the secondary “foci” might be caused by factors other than irritation from the presence or multiplication of bacterial bodies. It is our belief that among these factors may be (1) the interaction of sensitized tissues with toxin or antigen which has been brought from a distant focus or from outside the body, (2) a mechanism resulting from autonomic unbalance, such as occurs in certain intestinal diseases associated with vagotonia, or (3) endocrine or nutritional disturbances (some of which are now recognized as a possible result of gastrointestinal infection). Therefore, elimination of the secondary focal symptoms following extraction of teeth in such cases cannot be caused by elimination of infection in the secondary foci resulting from improved immunity because no infection existed in such foci. It is more likely that the symptomatic improvement is caused by the fact that a major source of toxins has been eliminated and not enough toxin then reaches the circulation from other sources to produce symptoms associated with secondary “foci.”

Streptococcus viridans is unquestionably the common denominator of chronic diseases associated with focal infection because pathogenic types can be recovered in every case. However, since bacteria cannot be found in many diseased tissues, the factor causing pathologic changes must be something more than just the bacterial bodies. This factor probably is a group of metabolites or “toxins”, capable of producing toxic symptoms in persons whose tissues have become sensitized to them. Thus, a concept of focal infection must take into consideration such toxigenic bacteria and their “toxins.”

Another factor causing confusion is improper use of the term “primary focus”. Our investigations suggest that the membranes lining the oral cavity, particularly those lining the pharynx and nasopharynx and possibly the gum margins, are the primary sources (i.e., the primary foci) of pathogenic streptococci. Although such infected areas may be the primary foci they may not be the major foci (i.e., the foci harboring most of the toxigenic streptococci and giving them off to the circulation). Thus, an infected tooth, residual area or periodontal pocket may be the major focus but the pharyngeal or post-nasal membranes are more likely to be the primary foci because organisms must first gain a foothold in some exposed tissue. Our experiments indicate that this occurs in early infancy in the majority of such infected persons.

There may be several foci of equal significance, all of importance as sources of infection. However, the infection is not usually confined to isolated foci but continues as diffuse inflammations of the contiguous membranes, frequently extending to the gastrointestinal membranes also. Quite often there may be more absorption of toxins from such diffusely inflamed areas than from isolated foci. Hence, elimination of obvious focal areas may not suffice to eliminate symptoms because the bulk of the toxins may originate in these diffusely inflamed membranes. Since such areas may persist after apparently adequate focal surgery, further methods must be found to deal with them. Obviously, surgery cannot be used. General measures, such as physiotherapy, heliotherapy, diathermy, change of climate, rest and improved diet are often inadequate to relieve the symptoms of infection. Hence, the use of vaccines. These have, however, been disappointing in many cases. Our experiments suggest several possible explanations.

Usual methods for selecting bacteria are inadequate. They lack specificity.¹ Moreover, even though a major focus may yield an apparently pure culture of a certain bacterium, the cultural methods may not be suitable for producing a vaccine in a form which,. when injected into the patient, will stimulate a high titer of specific antibodies. Let us examine some of these findings more closely.

A most important bacteriological fact, neglected by almost every worker in this field, is that the pathogenic properties of a culture must be known before it is possible to determine its significance. The fact that a streptococcus can be recovered from a tooth, residual area or periodontal pocket, or that the bacteriologist finds a “pure culture of Streptococcus veridans” has no significance at all because the culture may be non-pathogenic and thus has only feeble power of producing disease or of stimulating the production of antibodies. Further, although the growth may appear to be a pure culture on superficial examination, more careful study of such cultures frequently demonstrates that they are mixtures of types known to bacteriologists as “variants” or “dissociants”, i.e., cells directly descended from a common parent strain but differing from each other in important properties. Our researches indicate that such differences in properties are often associated with differences in pathogenicity. During the growth of such a dissociating culture there is often a change in the numerical relationships among the different types of cells as a result of uneven multiplication and of differences in dissociating power of different cells in the culture (Fig. 1). Thus, if non-pathogenic variants should overgrow the pathogenic variants of a pathogenic culture, the resulting culture will then be non-pathogenic. This dissociative tendency can be minimized by growing the culture in a more suitable medium under reduced oxygen tension and by securing the final growth as quickly as possible.

Fig. 1–Diagrams illustrating the changes that take place when streptococcal cells divide, giving rise to daughter cells.

+ Pathogenic cell.

± Degenerate pathogenic cell.

0 Non-pathogenic cell.

With Streptococcus viridans and non-hemolytic streptococci known to bacteriologists as A_ and r_hemolytic streptococci, respectively, the power to produce disease is associated with the production of weak toxins which affect sensitized tissues. Therefore, the inflammations produced by them are usually low grade and almost always chronic.

On the other hand, the toxins produced by hemolytic streptococci (known to bacteriologists as B_hemolytic streptococci, the cause of such acute conditions as septic sore throat, scarlet fever, puerperal fever, rheumatic fever and erysipelas) are much more potent. However, these B-hemolytic streptococci are not associated with dental infections. They have been reported in the literature as having been recovered from such sources but their presence in these cultures may have been the result of faulty technic.

Because it is the toxic metabolites which cause damage to dental tissues and injure tissues in other parts of the body, knowledge of the extent of toxin production (i.e., toxigenicity) by a streptococcal culture becomes of vital importance to the dentist and physician. We have developed simple methods for isolating these toxigenic strains and for differentiating them from non-toxigenic variants or strains, such as comprise the normal flora of the oral cavity. Differentiation is based upon the fact that toxigenic strains or variants are more resistant to injurious agents, such as antiseptics and the bactericidal factor in fresh blood, than are non-toxigenic strains or variants.

Strictly speaking, such resistant cultures are only probably pathogenic. It is difficult at the present time to prove that they are pathogenic for the host from whom they were isolated. However, this assumption is quite reasonable because strains from pathologic lesions in man produce pathogenic effects in animals,² and are related to other findings in diseases of man.^1,3,4,5 Moreover, pathogenic cultures are more likely to produce infection than are non-pathogenic bacteria. The number of pathogenic streptococci in a particular culture may have some significance because organisms present in small numbers are more likely to be contaminants.

Because the oral cavity is probably the portal of entry of streptococci, unexposed oral foci and foci in other parts of the body are more likely to be secondary to the exposed oral foci. This does not mean that strains from teeth, residual areas or perodontal pockets may not be more important that those recovered from the pharynx and tonsils but it does mean that pathogenic strains recovered from teeth, residual areas and periodontal pockets frequently are similar to those found, e.g., in the pharynx. However, it is wise to include streptococci from infected dental areas when making vaccines because they may be better antigens than those recovered elsewhere.

Our studies suggest further that periodontal pockets frequently contain a higher proportion of pathogenic streptococci than do any other part of the body (Tables 1 and 2).

Pathogenic staphylococci are found in about 60 per cent of chronic invalids, chiefly in the nasal cavity. While not directly related in many cases6 they may, however, enhance the pathogenicity of streptococci.

Culture of Bacteria From Suspected Teeth

It would seem superfluous to describe details of extraction of teeth for bacteriological study but we have seen so many teeth obviously contaminated by bacteria from the mouth that we are describing the method which we have found successful in securing teeth with a minimum of contamination.

1. Remove gross food particles from all teeth, particularly from the one to be extracted.
2. Spray the mouth with a suitable antiseptic.
3. Dry the area around the tooth with sterile cotton.
4. Swab the area around the tooth with tincture of iodine.
5. Have all instruments sterile.
6. Use a lip retractor and tongue depressor to keep the tooth from coming in contact with soft tissues.
7. Extract the tooth and be careful that it does not come in contact with anything except sterile instruments.
8. Place the tooth in sterile gauze.
9. Do not drop the tooth into a liquid medium. Any liquid will spread bacteria from one part of the tooth to other parts.

The problem of proper handling of the tooth for culture should not be dismissed lightly. The “broth” culture medium, as ordinarily prepared, contains only “peptone” and meat extract, which are unsatisfactory for adequate culture of streptococci and certainly do not permit production of much toxin. Our researches³ show that best results are obtainable by using brain heart infusion (available in dehydrated form from the Difco Laboratories of Detroit). Even with a suitable culture medium the entire tooth should not be dropped into a liquid culture medium, because any rapidly growing organism will overgrow streptococci and because it is better to make a culture of a particular part of the tooth. A glass container is unsatisfactory because the tooth rolls around in it and any contaminating organism will be transported to other parts of the tooth.

We have found pieces of folded gauze most satisfactory. While it tends to dry the tooth, the moist tissue around the tooth preserves streptococci from the deleterious effects of oxygen and drying, even though this moist covering should itself become dry. By folding the gauze so that it forms a pocket open at one side and by placing this in a paper envelope with the open side facing the opening of the envelope, the closed (but not sealed) envelope can be placed in an outer envelope and sterilized by dry heat. When ready for use the inner envelope is opened cautiously, the gauze pocket opened with sterile pliers and the tooth dropped into it. Then the envelope is sealed and sent to the laboratory.

Bacteriologic Study of the Extracted Tooth, Residual Area or Periodontal Pocket

We shall describe certain bacteriological details for the benefit of those who might try these methods. The first problem involves selection of material for culture. Streptococci on the outside of the root apex are more likely to be those causing periapical infection. A swab of the socket often provides excellent organisms. On the other hand, streptococci recovered after probing periodontal pockets with a platinum loop or capillary tube represent those responsible for the production of periodontal intoxication. The periapical culture can be studied by smearing the apex on the surface of a blood agar plate, either directly or after soaking the apex in a suitable fluid, such as brain heart infusion.

For culture of the root canal, it is best to put the dried remainder of the tooth into many thicknesses of sterile gauze and smash it on a sterile anvil. Scrapings of the canal can then be cultured as just described.

Platinum loop probings or suction capillary fluid of periodontal pockets can be seeded in brain heart infusion or plated directly on blood agar.

To prevent ruining blood agar cultures when a “spreader” (e.g., Proteus) is present we have found the addition of 0.01 per cent sodium azide, as recommended by Snyder and Lichstein,⁷ satisfactory in most cases. Bacto tryptose agar (Difco Laboratories) to which is added crystal violet 1:500,000 was found to be a superior base medium for blood agar, although it is unsatisfactory for differentiating types of hemolysis. It gives much larger colonies and permits better differentiation of colonies of streptococci. Incubation should be exactly 20 hours, not “overnight.”

The next step is the selection of colonies from these initial cultures. This involves certain bacteriologic principles which are not widely appreciated. First, during multiplication of ^A_ and ^r_hemolytic streptococci, there is usually a dissociation of the culture, with the result that daughter races (variants or dissociants) may be lacking in certain properties possessed by the parent strain. Dissociation involving loss of pathogenic properties is of particular importance in preparing vaccines because, while pathogenic variants make excellent antigens, provoking considerable stimulation of antibodies, non-pathogenic variants are poor antigens, having only feeble power of stimulating antibody production.

Second, when a culture of streptococci is transplanted to brain heart infusion or other liquid medium and incubated at 37° C., it begins to multiply and reaches its maximum growth in from 8 to 16 hours, depending upon the number of times it is shaken and upon the characteristics of the culture. During the phase of vigorous visible growth (the logarithmic phase), pathogenic streptococci are highly pathogenic and make excellent antigens. As they approach maximum turbidity they begin to lose vigor and soon afterward they decline in physiologic, biochemical immunologic and pathogenic activity. Therefore, cultures should be studied for pathogenicity during the phase of vigorous visible growth, and further changes in the culture should then be prevented by the addition of an antiseptic to the washed culture.

Based on these facts, the following plan is suggested for studying streptococci from teeth, residual areas and periodontal pockets. The swab, loop or apex, or a measured amount of the brain heart infusion suspension is plated on blood agar, spread by means of a sterile glass spreader and incubated exactly 20 hours. If such a procedure should make the end of incubation occur during the night the inoculated plates can be left in an anaerobic jar in a cool place for a few hours before being put in the incubator. After incubation, colonies representing each of the different types are cut from the medium by a spear-headed inoculating needle and each one is put into a separate tube of brain heart infusion. We prefer Pyrex titer test tubes because the culture can be grown and then centrifuged in them, thus avoiding transfer to a separate centrifuge tube. The tubes of brain heart infusion are kept cool until late afternoon and then put in the incubator equipped with a time switch to start it about 8 pm. The tubes are examined at 9 am. or earlier and the turbid ones are then removed, tested for resistance, and centrifuged. The supernatant is decanted and 1 per cent phenol is added to the sediment. The tubes of brain heart infusion which did not show sufficient growth are shaken and returned to the incubator for another 2 hours, when they are examined again and the turbid ones removed and tested. This process of shaking and selecting the turbid cultures is repeated every 2 hours until 12 noon, after which time there is little likelihood of further growth.

Resistance Tests of Streptococci

It has been shown^2,4,5 that resistance of streptococci to appropriate time/dilutions of hexylresorcinol is parallel with certain pathogenic properties of the cultures. It should be pointed out, however, that the cultures are most pathogenic and most resistant during the phase of vigorous visible growth and may lose these properties after they have reached maximum turbidity.

The resistance tests^1,8,9 consist of adding 1 loopful of the growth in brain heart infusion to 0.5 cc. of 1:125,000 hexylresorcinol. A loopful of this mixture is streaked on blood agar to serve as a control of the number of viable organisms. The hexylresorcinol mixture is incubated exactly 2 hours and another loopful of it is streaked on the blood agar plate alongside the control. After 18 to 20 hours incubation of the blood agar plates the two streaks are compared. If the second streak shows as much growth as the control, the resistance is considered 8+ and the strain is probably highly pathogenic. If the second streak shows less growth than the control the strain has little significance and should be discarded.

In cases where the growth in brain heart infusion does not reach maximum turbidity, 1 drop of the culture should be mixed with 1.0 cc. of the 1:125,000 hexylresorcinol.

Summary

It is probable that several mechanisms are involved in the pathogenesis of focal infection of dental origin.

In addition to the type in which bacteria are distributed from primary to secondary foci and the secondary foci are activated by frequent showers of bacteria from the primary focus, there appear to be types caused by the interaction of toxic antigens with sensitized tissues, by a disturbed autonomic balance, and possibly by other factors.

Since the toxic action of streptococcal metabolites appears to play a major role in most of these mechanisms, study of the toxigenic power of streptococci associated with focal infection assumes a new significance.

Methods are described by means of which it is possible to differentiate toxigenic from non-toxigenic streptococci. The significance of microbic dissociation and other factors affecting the pathogenic properties of these streptococci is discussed.

A method is described for the bacteriological examination of extracted teeth, residual areas and periodontal pockets which embodies these principles. Such a combination of methods provides a better estimate of the probable significance of dental and periodontal infections than is possible by methods in common use.

The differentiation of toxigenic streptococci is of further value in the preparation of vaccines because toxigenic strains provide better stimulation of antibodies than do nontoxigenic strains.

References Cited:

1. Chapman, G. H. Berens, C., Lieb, C. W., Rawls, W. B., and Stiles, M. H. “Examination of cultures from persons suspected of having chronic infection.” Am. Jour. Clin. Path., 9: 491-503 (July) 1939.
2. Chapman, G. H., Berens, C., and Nilson, E. L. “Studies of streptococci, III. Preliminary attempts to correlate resistance to chemicals, etc. with pathogenic effects.” Jour. Bact. 32: 339-346 (April) 1936.
3. Chapman, G. H., Stiles, M. H., and Berens, C. “The isolation and in vitro testing of pathogenic types of non-exotoxic streptococci.” Am. Jour. Clin. Path., Tech. Suppl., 3: 20-27 (January) 1939.
4. Rawls, W. B., and Chapman, G. H. “Experimental arthritis in rabbits. Comparison of the arthritis-producing ability of inagglutinable streptococci which resist the ‘bactericidal’ action of fresh, diluted, defibrinated guinea pig blood and those which are agglutinable but sensitive to the ‘bactericidal’ agent.” Jour. Lab. & Clin. Med., 21: 49-64 (October) 1935.
5. Chapman, G. H., and Lieb, C. M. “Bacteriology of the intestinal tract in certain diseases. II. The possible inhibition of colon bacilli by pathogenic streptococci and staphylococci.” Rev. Gastroent. 5: 234-240 (September) 1938.
6. Stiles, M. H., and Chapman, G. H. “Probable pathogenic streptococci and staphylococci in chronic low grade illness.” Arch. Otolaryngol. 31: 458-466 (March) 1940.
7. Snyder, M. L., and Lichstein, H. C. “Sodium azide as an inhibiting substance for Gram-negative bacteria.” Jour. Infect. Dis. 67: 113-115 (September-October) 1940.
8. Chapman, G. H., and Rawls, W. B. “Studies of streptococci. I. Qualitative differences in resistance to various agents.” Jour. Bact., 31: 323-331 (April) 1936.
9. Chapman, G. H., and Curcio, L. “Studies of streptococci. II. Quantitative differences in resistance to sodium bicarbonate and hexylresorcinol.” Joul. Bact., 31: 333-337 (April) 1936.