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The antibiotic sensitivity test in dental practice
The antibiotic sensitivity test in dental practice
T
he selection of an ant,ibiotic becomes a serious matter in cases of acute oral infection caused by a mutant organism that is resistant to penicillin or other antibiotics. The question of which antibiotic is to be used to overcome the resistant strain is easily resolved through the use of the microbial sensitivity test. By exposing pure cultures of organisms isolated from the infected area to commercially prepared paper discs impregnated with antibiotics of specific dosage, one can determine the antibiotics to which t,he organism is sensitive or resistant. Most drug-resistant organisms develop as the result of genetic mutation independent of antibiotic use.l However, inadequate dosage, administration by an improper route, and prolonged duration of drug administration encourage the evolution of resistant strains. Initially, the mutant strain is slightly resistant to the particular antibiotic, but when treatment is inadequate subsequent generations that are more resistant develop, and in time a truly resistant strain of organism en1erges.l Clinical studies show that in most cases, selection of a specific antibiotic for an oral infection on the basis of the microbial sensitivity test findings results in the successful treatment of acute symptoms. The test also has the advantage of being carried out quickly and easily. In the original method suggested by Bondi and associates,’ antibiotic discs were placed on blood agar plates streaked directly with clinical material, and organism susceptibility was determined in 24 hours, at the same time that the organisms were being isolated and identified. This method has several disadvantages : 1. There must be a heavy growth and an even distribution of colonies around the discs in order to obtain significant zones of inhibition. 2. A plate which has a good distribution of colony growth for isolation and identification of the microorganism may be poor for “Career
Resident
in Periodontia,
Veterans
Administration
Hospital,
New York,
N. Y.
Volume Number
22 5
,4ntibiotic
sensitivity
test
683
determining the antibiotic sensitivity of the organism by zones of inhibition. 8. Four or rnorc antibiotics are often tested on a single plate, but a direct. smear may not, yield an cvcn enough spread of the colonies bc4ng tested to come in contact, with each disc. Therefore, it is suggested that the organism first be isolated and that a pure culture be obtained from the isolated colonies on the original plate.” In cases of a grave infectious process:, however, a direct smear is plated and the results are interpreted while the results obtained from pure cultures are being await,ed. N?henever a patient presclnts scutc symptorns, an antibiotic (for example, t~rtmcyclinc, crythrompcin, or chloramphenicol) should be administered immediately, since the sensitivity test does require some time. In addition, rnost pathogenic organisms involved in acute oral infections arc susceptible to the antibiotics mentioned.4 The putposc of t,he microbial sensitivity test is to provide an important safety factor, since the organism may prove to be resistant to the antibiotic administered. In a critical case, valuable tirne can be saved by the use of this test. METHOD
OF CULTURING
In cases of acute oral infection, purulcnt material is obtained either by opening directly into the pulp chamber under aseptic conditions or by aspirating pus from the surrounding soft-tissue swelling. When material is to be obtained directly from the canal, a rubber dam is applied to isolate the tooth and then the crown is swabbed with %ephira.n to prevent contamination of the specimen. An opening is then cut into the pulp chamber with a sterile bur. A sterile paper point is ins&cd into the canal and allowed to remain in place for one minute. The point is then withdrawn and placed in a culture tube” by an aseptic technique. Soft,-tissue swellings should be dried, isolated with cotton rolls, and swabbed with tincture of Merthiolate. The necdlr point of a sterile aspirating syringe is then insert4 into the swcllin, n and purulent, material is withdrawn. The plunger of t,he syringe is withdrawn, and the specimen is picked up with a sterile swab. The swab is then placed in the culture tube by an aseptic technique. The t,ube is incubated for 2-l hours. acrobic*ally, at 37’ C”. After 24 hours, a srnear of the culture is gram stain4 unlrss the> tube is perfectly clear. If organisms a~‘(: seen in the smear, t\vo blood agar platrst shollld be streaked with broth from the original cwlture." C)nc plate is incubattd ac~robic*ally and the other in an anaerobic jar for 24 hours. lf a has mol*(~than one type of organism colony, catel: diRcrcnt t-p<’ &>1’4(in,v i< picked up, J’iaWd in an individual tube, and incubated, and then tht- I~r*c?tl~c.tttlt;riltill~ tltcs 1~111~’ c~ultrlle is streaked on indisc& are now placed dividual blood plates. A nlm~bcr of different antibiotic (hvenly on the agar by Itlwcin, 0 the dispenser over the plate (Fig. 1’) and pressing plilt?
O.S., O.M. & O.P. November, 1966
Fig. 1. Difco
tlisc dispenser
placed over agar plate.
the plunger handle of the disc dispenser (Fig. 2).* Discs may be placed also by an individual disc dispenser (Fig. 3). The discs are then gently pressed to the agar with sterile tweezers to assure contact of the discs against the agar surface. The plates are incubated overnight at 37” C. before reading and can be incubated in the atmosphere, anaerobically, or under increased carbon dioxide tension, depending on the type of organism. The organism’s susceptibility to each antibiotic being tested is determined by the zone of growth inhibition around the disc (F’ig. 4). The diameter of the zone where the organism has failed to develop any visible growth indicates the organism’s relative susceptibility to a particular antibiotic3 Some organisms will show a profuse growth around the disc, indicating a synergistic effect or a possible antibiotic dependency. A number of variables enter into the complex reaetions in\-olved in the antibiotic diff&ion disc technique, including the molecular weight of the a,ntibiotic, the presence of sufficient sodium chloride in the medium, and t,he size of the inoculum. In general, however, the following conclusions can be derived from the test when it is accurately performed.3 A zone of inhibition greater t,han 15 mm. usually means that the organism is susceptible to the particular a.ntibiotic being tested. A 10 to 15 mm. zone indicates that the organism is moderately resistant, and a zone of less than 10 mm. means that the organism is resistant except to polymyxin, bacitracin, kanamycin and ristoeetin, for which any zone indicates sensitivity. “Dispens-O-Disc
dispenser,
Difco
Laboratories,
Detroit,
Mich.
Volume Numlw-
25 5
Fig. 2. Stroke OII! 0 ru1ture gate.
A4ntibiotic
of dispenser
handle
automatically
deposits
dformiy
sensitidy
test
spaced criwlv
of
685
cliws
A susceptible organism will usually be inhibited by the blood lc~l OF an alttihiotic, following administ.ration of t.he usual therapeutic dose.” A moderatc~l~ resistant organism will require an increased dose, and a resistant strain will not respond. r~g;irdless of the dose or the route of administration. In vitro sensitivity tests are essentially artiticia~l, since they cannot include the part. that normal body defense mechanisms play in combating an infectious process. Therefore, the disc technique is useful primarily as a qua,litativc test, designed to determine whether an organism is suscept,ihle or resistant to an antimirivhjal ny.cnt.3 SUMMARY
The use of pa~)er discs for routine determination of the in vitro susceptibilit) of microorganisms to antimicrobial agents is a universally accepted procedure.
686
OS., O.M. & O.P. November, 1966
Larato
Fil/.
3. Di sposable
Fig. 4. Blood discs.
disc dispenser.
agar plate
showing
A single
disc is dispensed
different.
zones of inhibition
each time trig
:ger is pressczd.
around individual
antibiotic
Volume Number
2% 5
.4 ,dihiotic
semiticity
test
607
The microbial sensitirit,y test, may be utilized whew\-er an acute dental infection is being treated or a resistant strain of organism is suspected during antibiotic therapy. The dentist, need only obtain a sample of csudate or purulent material from t.he infected area. and send it. in a culture tube t,o a diagnostic laborat.ory for determination of the organism type and its antibiotic sensit,ivity. From the resulk of the test, the antibiotic of choice can be administered to combat the infection and prcvcnt the development of resistant organism strains. REFERENCES
1. Zubrom, H. J., Rpat,z, S. S., and Kline, H. N.: Antibiotics in Oral Surgery, D. Clin. North America, p. 680, November, 1959. 2. Rondi, A., Jr., Kpsulding, E:. H., Smith, 1). E.. and Dietz, C!. C.: Routine Method for Rapid 1)etermination of Susceptibility to Penicillin and Other Antibiotics, Am. J. M. SC. 213: 22;, 1947. 3. Ralley, W. R., and Scott, E. G.: Diagnostic Microl)iology, St, Louis, 1962, The (1. V. Mosby Company, pp. 119-122, 249-262. cd. 1, Pl~iladclphia, 1955, Lea Sr Febiger, pp. 4. Clark, H. FL, Jr.: Practical Oral Surgery, 318-322.
T
he selection of an ant,ibiotic becomes a serious matter in cases of acute oral infection caused by a mutant organism that is resistant to penicillin or other antibiotics. The question of which antibiotic is to be used to overcome the resistant strain is easily resolved through the use of the microbial sensitivity test. By exposing pure cultures of organisms isolated from the infected area to commercially prepared paper discs impregnated with antibiotics of specific dosage, one can determine the antibiotics to which t,he organism is sensitive or resistant. Most drug-resistant organisms develop as the result of genetic mutation independent of antibiotic use.l However, inadequate dosage, administration by an improper route, and prolonged duration of drug administration encourage the evolution of resistant strains. Initially, the mutant strain is slightly resistant to the particular antibiotic, but when treatment is inadequate subsequent generations that are more resistant develop, and in time a truly resistant strain of organism en1erges.l Clinical studies show that in most cases, selection of a specific antibiotic for an oral infection on the basis of the microbial sensitivity test findings results in the successful treatment of acute symptoms. The test also has the advantage of being carried out quickly and easily. In the original method suggested by Bondi and associates,’ antibiotic discs were placed on blood agar plates streaked directly with clinical material, and organism susceptibility was determined in 24 hours, at the same time that the organisms were being isolated and identified. This method has several disadvantages : 1. There must be a heavy growth and an even distribution of colonies around the discs in order to obtain significant zones of inhibition. 2. A plate which has a good distribution of colony growth for isolation and identification of the microorganism may be poor for “Career
Resident
in Periodontia,
Veterans
Administration
Hospital,
New York,
N. Y.
Volume Number
22 5
,4ntibiotic
sensitivity
test
683
determining the antibiotic sensitivity of the organism by zones of inhibition. 8. Four or rnorc antibiotics are often tested on a single plate, but a direct. smear may not, yield an cvcn enough spread of the colonies bc4ng tested to come in contact, with each disc. Therefore, it is suggested that the organism first be isolated and that a pure culture be obtained from the isolated colonies on the original plate.” In cases of a grave infectious process:, however, a direct smear is plated and the results are interpreted while the results obtained from pure cultures are being await,ed. N?henever a patient presclnts scutc symptorns, an antibiotic (for example, t~rtmcyclinc, crythrompcin, or chloramphenicol) should be administered immediately, since the sensitivity test does require some time. In addition, rnost pathogenic organisms involved in acute oral infections arc susceptible to the antibiotics mentioned.4 The putposc of t,he microbial sensitivity test is to provide an important safety factor, since the organism may prove to be resistant to the antibiotic administered. In a critical case, valuable tirne can be saved by the use of this test. METHOD
OF CULTURING
In cases of acute oral infection, purulcnt material is obtained either by opening directly into the pulp chamber under aseptic conditions or by aspirating pus from the surrounding soft-tissue swelling. When material is to be obtained directly from the canal, a rubber dam is applied to isolate the tooth and then the crown is swabbed with %ephira.n to prevent contamination of the specimen. An opening is then cut into the pulp chamber with a sterile bur. A sterile paper point is ins&cd into the canal and allowed to remain in place for one minute. The point is then withdrawn and placed in a culture tube” by an aseptic technique. Soft,-tissue swellings should be dried, isolated with cotton rolls, and swabbed with tincture of Merthiolate. The necdlr point of a sterile aspirating syringe is then insert4 into the swcllin, n and purulent, material is withdrawn. The plunger of t,he syringe is withdrawn, and the specimen is picked up with a sterile swab. The swab is then placed in the culture tube by an aseptic technique. The t,ube is incubated for 2-l hours. acrobic*ally, at 37’ C”. After 24 hours, a srnear of the culture is gram stain4 unlrss the> tube is perfectly clear. If organisms a~‘(: seen in the smear, t\vo blood agar platrst shollld be streaked with broth from the original cwlture." C)nc plate is incubattd ac~robic*ally and the other in an anaerobic jar for 24 hours. lf a has mol*(~than one type of organism colony, catel: diRcrcnt t-p<’ &>1’4(in,v i< picked up, J’iaWd in an individual tube, and incubated, and then tht- I~r*c?tl~c.tttlt;riltill~ tltcs 1~111~’ c~ultrlle is streaked on indisc& are now placed dividual blood plates. A nlm~bcr of different antibiotic (hvenly on the agar by Itlwcin, 0 the dispenser over the plate (Fig. 1’) and pressing plilt?
O.S., O.M. & O.P. November, 1966
Fig. 1. Difco
tlisc dispenser
placed over agar plate.
the plunger handle of the disc dispenser (Fig. 2).* Discs may be placed also by an individual disc dispenser (Fig. 3). The discs are then gently pressed to the agar with sterile tweezers to assure contact of the discs against the agar surface. The plates are incubated overnight at 37” C. before reading and can be incubated in the atmosphere, anaerobically, or under increased carbon dioxide tension, depending on the type of organism. The organism’s susceptibility to each antibiotic being tested is determined by the zone of growth inhibition around the disc (F’ig. 4). The diameter of the zone where the organism has failed to develop any visible growth indicates the organism’s relative susceptibility to a particular antibiotic3 Some organisms will show a profuse growth around the disc, indicating a synergistic effect or a possible antibiotic dependency. A number of variables enter into the complex reaetions in\-olved in the antibiotic diff&ion disc technique, including the molecular weight of the a,ntibiotic, the presence of sufficient sodium chloride in the medium, and t,he size of the inoculum. In general, however, the following conclusions can be derived from the test when it is accurately performed.3 A zone of inhibition greater t,han 15 mm. usually means that the organism is susceptible to the particular a.ntibiotic being tested. A 10 to 15 mm. zone indicates that the organism is moderately resistant, and a zone of less than 10 mm. means that the organism is resistant except to polymyxin, bacitracin, kanamycin and ristoeetin, for which any zone indicates sensitivity. “Dispens-O-Disc
dispenser,
Difco
Laboratories,
Detroit,
Mich.
Volume Numlw-
25 5
Fig. 2. Stroke OII! 0 ru1ture gate.
A4ntibiotic
of dispenser
handle
automatically
deposits
dformiy
sensitidy
test
spaced criwlv
of
685
cliws
A susceptible organism will usually be inhibited by the blood lc~l OF an alttihiotic, following administ.ration of t.he usual therapeutic dose.” A moderatc~l~ resistant organism will require an increased dose, and a resistant strain will not respond. r~g;irdless of the dose or the route of administration. In vitro sensitivity tests are essentially artiticia~l, since they cannot include the part. that normal body defense mechanisms play in combating an infectious process. Therefore, the disc technique is useful primarily as a qua,litativc test, designed to determine whether an organism is suscept,ihle or resistant to an antimirivhjal ny.cnt.3 SUMMARY
The use of pa~)er discs for routine determination of the in vitro susceptibilit) of microorganisms to antimicrobial agents is a universally accepted procedure.
686
OS., O.M. & O.P. November, 1966
Larato
Fil/.
3. Di sposable
Fig. 4. Blood discs.
disc dispenser.
agar plate
showing
A single
disc is dispensed
different.
zones of inhibition
each time trig
:ger is pressczd.
around individual
antibiotic
Volume Number
2% 5
.4 ,dihiotic
semiticity
test
607
The microbial sensitirit,y test, may be utilized whew\-er an acute dental infection is being treated or a resistant strain of organism is suspected during antibiotic therapy. The dentist, need only obtain a sample of csudate or purulent material from t.he infected area. and send it. in a culture tube t,o a diagnostic laborat.ory for determination of the organism type and its antibiotic sensit,ivity. From the resulk of the test, the antibiotic of choice can be administered to combat the infection and prcvcnt the development of resistant organism strains. REFERENCES
1. Zubrom, H. J., Rpat,z, S. S., and Kline, H. N.: Antibiotics in Oral Surgery, D. Clin. North America, p. 680, November, 1959. 2. Rondi, A., Jr., Kpsulding, E:. H., Smith, 1). E.. and Dietz, C!. C.: Routine Method for Rapid 1)etermination of Susceptibility to Penicillin and Other Antibiotics, Am. J. M. SC. 213: 22;, 1947. 3. Ralley, W. R., and Scott, E. G.: Diagnostic Microl)iology, St, Louis, 1962, The (1. V. Mosby Company, pp. 119-122, 249-262. cd. 1, Pl~iladclphia, 1955, Lea Sr Febiger, pp. 4. Clark, H. FL, Jr.: Practical Oral Surgery, 318-322.