Antibacterial Susceptibility of Plaque Bacteria MICHAEL G. NEWMAN*, CHARLES HULEM**, JUDITH COL(GATE**, and CARL AN SELMO * *
*U.C.L.A. School of Dentistry,
Section of Periodontics, Los Angeles, CA University, Lonig Beach, Department of 'Aicrobiology, Lonig Beach, CA 90840.
Selected anaerobic, capnophilic and ,fcultative bacteria isolated from patients with various forms of periodontal health and disease wvere tested for their susceptibility to antibiotics and antimicrobial agents. Specific bactericidal and minimum inhibitory concentrations were compared to disc zone diameters, thereby generating new standards for the potential selection of attimicrobial agents. J Dent Res 58(7):1722-1732, July 1979
Introduction. Periodontitis, the periodontal abscess and infection resulting from pulpal disease, can be initiated by many of the bacterial species colonizing the gingival and periodontal tissues. The empirical selection of an antibiotic is necessary when a patient first appears with acute infection. The selection of the most appropriate agent can be improved if there are criteria of susceptibility and resistance available to the dentist. There has been substantial research dealing with antimicrobial susceptibilities of medically important bacteria,5 ,3 6,37 but surprisingly there are few available data regarding the antibiotic or antimicrobial susceptibility of the bacterial strains which are implicated in dental diseases. The data which are available are either incomplete or based on older or nonreliable methods. Although the majority of available information has been based on the disc diffusion technique for antimicrobial susceptibility testing, it is only recently that attempts have been made to standardize the procedure and to correlate the results with the more accurate and sensitive dilution methods of
Received for publication March 3, 1978. Accepted for publication September 26, 1978. Supported by grants from the California Dental Association and Grant NIH-NIDR DE 0444-1.
90024; **Califbrnia State
testing. 17,36,38 In addition, some investigators have used standards for anaerobes that were originally developed for aerobic and facultative organisms. Growth rate of the bacteria. inoculum size, pH, medium, and carbon dioxide concentrations are some of the factors influenced by anaerobic conditions.17 A standardized dilution technique as well as a disc diffusion technique can be used to measure specific bactericidal and minimum inhibitory concentrations of anaerobes. Using a combination of these methods, new criteria of susceptibility and resistance can be generated.
Materials and methods. Bacterial cultures. Forty bacteria were obtained from patients with various forms of periodontal disease and from patients with periodontal health (Table 1). The organisms were: Actinomyces viscosus (4), Bac tero ides m elaninogen icus ss m elaninogenicus (2), Capnocytophaga2530'3133,34 species (Bacteroides ochraceus)(1 1), "corroding Bacteroides "i 3 (3), Eikenella corrodens (2), Fusobacterium nucleatum (4), Selenomonas sputigena (3) and Streptococcus sanguis (9). Bacteroides fragilis ss. ovatus (ATCC 8483) and Eikenella corrodens (Orange County Medical Center) were used as controls. All organisms except Capnocytophaga and "corroding Bacteroides" were classified according to established characteristics such as those listed in Bergey's Manual of Determinative Bacteriology, 8th edition ,1 0 Wadsworth Anaerobic Bacteriology Manual,38 and others.17'24 Capnocytophaga were characterized by criteria established by Savitt et al. 1975,3i Holt et al. 1975,2 5 Newman, et al. 1976,30 Socransky et al. 197934 and "corroding Bacteroides" were classified according to criteria by Crawford et al., 1 9715 3 Stock solutions. All antibiotics were
1722 Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
Vol. S8 No. 7
ANTIBACTERIAL SUSCEPTIBILITY OF PLAQUE BACTERIA
1723
TABLE 1
SOURCE AND DISTRIBUTION OF BACTERIAL ISOLATES Subgingival Isolates
ORGANISM
N umber Supraof gingival strains Plaque
Papillon Adoles- Perio- Lefevre Perio- Periocent dontal Aged SynHealthy* Healthy Abscess drome dontosis dontitis
4 Actinomyces viscosus 3 Bacteroides melaninogenicus ss melantino2 genicus 11 Capnocytophaga sp. 4 2 Eikenella corrodens 3 Corroding Bacteroides 3 4 Fusobacterium nucleatum 1 3 Selenomonas sputigena 9 1 Streptococcus sanguis 5 Control Organisms Bacteroides fragilis ss oratus ATCC 843 3 Eikenella corrodens. Orange County Medical Center
1 -) 2
51
5
-
2 1 3 2
1 I
*Plaque taken from an adult,older than 55 years,with the absence of periodontal disease.
dissolved in distilled water to achieve a final concentration of 100 pg/ml (units/ml for penicillin); filter sterilized; and stored frozen in 1 ml aliquots until used. The antibiotics used were: ampicillin (Bristol Laboratories), chloramphenicol (Sigma Chemical Company), clindamycin (Upjohn), erythromycin, penicillin and tetracycline (Sigma Chemical Company). The antimicrobial agents used were: metronidazole (Searle Company), alexidine 0.035% (Calgon Company), chlorhexidine 5% (Imperial Chemical Industries, Limited, Maclesfield, England), sodium fluoride 5.33% (Lorvic Company) and eugenol 100% (Merck Company). Filter paper discs. Antibiotic discs (Difco) were obtained in the following concentrations: ampicillin 10 pg, chloramphenicol 30 pg, clindamycin 2 pug, erythromycin 1 5 pg, penicillin 10 units, and tetracycline 30 pg. The metronidazole discs were prepared by adding 0.01 ml of the stock metronidazole solution to sterile 1/4 inch Bacto-Concentration Discs (Difco) with a micropipette, allowing them to dry, and then storing at 40C with a desicant until use, giving a final concentration of 10 pg per disc. The other antimicrobial agents were prepared in a similar manner, except 0.005 ml was used for chlorhexidine, eugenol, and
sodium fluoride and 0.01 ml of alexidine to give final disc concentrations of: chlorhexidine 0.025%, alexidine 0.00035%, sodium fluoride 0.02 7% and eugenol 0.5%. Broth dilution method. The medium employed was Trypticase-Soy Broth (with 0.25% dextrose) (BBL) which was supplemented with 5% Fildes Enrichment (BBL). Following a 1:50 dilution of the stock antibiotic or antimicrobial agent (this initial dilution was deleted for alexidine, eugenol and sodium fluoride), a two-fold serial dilution was made. An equal volume of a 1:100 dilution of culture grown anaerobically for 48 hours was then added. The tubes were incubated in anaerobic GasPak (BBL) jars, at 370C, for at least 48 hours or until the control tubes showed turbidity. The minimal inhibitory concentration (MIC) was determined as the lowest concentration of antibiotic showing no visible growth. Minimal bactericidal concentrations (MBC) were determined by streaking all tubes with no growth onto plates containing Trypticase Soy Agar with 5% Sheep's Blood (BBL) and incubated for 48 hours in GasPak jars at 370C. The MBC was determined as the lowest concentration of antibiotic resulting in no visible growth on these plates. Disc diffusion. Trypticase Soy Agar with
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NEJVMAN E T A L.
1 724
JDentResJuly 1979
5% Sheep's Blood (BBL) was used as the basal medium. The cultures were grown in supplemented trypticase soy broth in an anaerobic GasPak jar for at least 48 hours, or until turbid. These were then diluted to a No. 1 McFarland barium sulfate standard and swabbed over the entire surface of the plate. Antibiotic or antimicrobial discs were applied, and the plates were incubated for 48 hours at 370C in GasPak jars. The zones of inhibition were measured with a caliper, under reflected light. Regression curves. Regression curves were generated by plotting MICs against disc zone diameters. The locations of the straight lines on each graph were selected by a simple regression method,2 using zone diameter as the independent variable. The intercepts and slope of the lines were determined using a computer program (CHI) and a Control Data Cyber 173 computer. Determination of break points. Break points of "sensitive," "intermediate," and "resistant" were determined by the average MIC and achievable blood levels for each antibiotic.3 Ideally, the in vivo concentration of the antibiotic (at the site of infection) should be two (2) to four (4) times more than in vitro MIC.1'14 Organisms were considered sensitive if the achievable blood level of the antibiotic was 4 times the MIC; intermediate if it was 2-4 times the MIC; and resistant if it was less than 2 times the MIC. The antimicrobials tested are topical agents; therefore, determination of blood levels is not applicable. Antimicrobials were not graded as to sensitive or resistant, but rather according to their potential clinically achievable topical concentration (Table 2).
Results.
of two strains of "corroding Bacteroides" and B. fragilis, were susceptible to an average concentration of 0.86 ,ig/ml ampicillin (Table 3, Fig. 1). Chloramphenicol inhibited all organisms (average concentration 2.36 ,g/ml) but, because of achievable blood levels, B. fragilis, Capnocytophaga, and Eikenella corrodens were considered only intermediately susceptible (Table 3, Fig. 1). All organisms tested were highly susceptible to clindamycin, responding to an average concentration of 01.41 ig/ml (Table 3, Fig. 1). One strain of Selenomonas sputigena was resistant because it required a concentration of 10 ,g/ml. Only the control strain, B. fragilis, was resistant to penicillin. Most other strains were sensitive (average concentration 0.24 ,ig/ml) (Table 3, Fig. 2). All organisms, except one strain of Fusobacterium nucleatum, were inhibited by the highest concentration (10 ,ug/ml) of erythromycin tested (Table 3, Fig. 2). Because the maximum achievable blood level of erythromycin is 2 pg/ml, and since B. fragilis, Eikenella corrodens and Fusobacterium nuccleatum were susceptible only to an average concentration of 2.96 jzg/ml, these organisms were considered resistant. There were several strains not inhibited by the highest concentration (10 pg/ml) of metronidazole (Table 3, Fig. 2). These include Actinomyces viscosus (4 strains), Capnocytophaga (2 strains), Eikenella corrodens (2 strains), Selenomonas sputigena (2 strains), and Streptococcus sanguis (7 strains). Many other strains required relatively high concentrations (1.255.0 jig/ml) for inhibition. All Fusobacterium nucleatum strains were highly susceptible to a concentration of 0.005 -0.08 ,ug/ml but, because of the relatively high achievable blood level (2 - 20 ,g/ml), Actinomyces viscosus and Eikenella corrodens were con-
All organisms tested, with the exception TABLE 2 CLINICALLY ACHIEVABLE TOPICAL LEVELS OF ANTIMICROBIAL AGENTS
Mouthrinse
Gel
Agent Pulpal Pulpal Analgesic Analgesic
3 2 * 0.1-1.0% Chlorhexidine4''32' Chlorhxidin4, 6, 2 3, 2 7, 2 8,.2 Alexidine26'35 0.0 35-0.05%
Sodium Fluoride7'1 5,29 Eugenol8,2 1,22 *
0.2%
Dentifrice Cements
1%
0.5%
(0 .1-0 .2%) 40%
46%
3 7.5%
References Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
Vol. 58 No. 7
ANTIBACTERIAL SUSCEPTIBILITY OF PLAQUE BACTERIA
1 725
TABLE 3 SUSCEPTIBILITY OF ORAL BACTERIA TO ANTIBIOTICS
Antibiotic Ampicillin
Organism
Actinomyces viscosus B. fragilis B. melaninogenicus
Capnocytophaga
Chloramphenicol
Clindamycin
Erythromycin
Corroding Bacteroides E. corrodens Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus
Capnocytophaga Corroding Bacteroides E. corrodens
Metronidazole
Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus
Capnocytophaga Corroding Bacteroides
Penicillin
E. corrodens Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus
Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatum
Average MIC ,ug/ml
0.01 10.0 0.08 0.13 6.7 0.23 0.04 0.17 0.11 1.09 5.0 2.66 3.52 2.08 3.75 0.55 1.15 2.85 0.18 0.31 0.31 0.02 0.009 0.26 0.01 0.03 3.37 0.08 1.25
0.06 0.14 0.27 2.17 1.98 0.21 0.35 > 10.0 0.625 0.10 3.47 0.36 >10.0 0.04 0.04 8.95 0.096 >10.0 0.095 0.23 0.08 0.83 0.02
Average MBC ugjml
Average Zone Diameater mm
0.45 > 10.0 0.08 0.49 6.7 1.25 0.04 0.27 0.50 3.96 10.0 5.16 6.82 3.75 8.33 1.80 3.75 4.38 0.88 0.625 0.08 0.013 0.26 0.625 0.04 3.37 0.53 0.47 2.50 0.08 0.22 0.42 2.71 5.21 1.88 0.46 > 10.0 0.625 0.20 6.25 2.50 > 10.0 0.05 0.04 8.96 0.27 >10.0 0.095 0.70 0.08 3.75 0.02 (TABLE 3
Sa
47 8 63 49 21 33 51 41 39 42 29
x
54 39 43 37 52
x
38 32 36 22 55 54 39 24 55 30 32 43 34 40 40 39 18 40 42 32 7 54 65 32 58 7 70 67 9 45 7 49 39 36 24 49 is continued
Ib Rc x
x x
x x x x x x x x
x
x x x x x x x x x x x x x x x
x x x x x x x x x x x x x x x x
x x x
x x x x x on next
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page.)
J Dent Res July19 79 E T A L. NJE WIAlN
1726
(TABLE 3 continued) SUSCEPTlBILITY 01 ORAL BACTERIA TO ANTIBIOTICS
,ug/ml
Average MBC gin/ml
Average Zone Diameter mm
0.32 0.16 0.71 0.31 0.47 0.48 0.17 1.04 0.41 0.35 1.09
0.32 0.50 3.96 2.50 0.78 1.25 3.96 1.88 1.39 3.49 2.79
42 34 39 71 59 42 39 23 50 39 31
Average
MIC)
Organisnm
Antibiotic
Seleniomonas sputigena Streptococcus satnguis A ctinomnyces iscosus B. fragilis B. melaninogenicus Capnocy tophaga Corroding Bacteroides E. corrodens Fusobacterium ntuclea tutul Selenotinonias sputigena Streptococcus sanguiis
Tetracy cline
a -
sensitive, b
intermediate,
c
-
a/i
in
ss
lb RC
X x
x x x x x x x x x
resistant
sidered resistant, whereas Streptococcus sanguis was considered to be of intermediate susceptibility according to break points. Since tetracycline has a relatively high strains achievable blood level (8 Mg/ml), were susceptible (Table 3, Fig. 2). Alexidine was found to inhibit all of the test organisms at a concentration of 0.0021I% (Table 4). The zone diameters ranged from 7 mm to 15 mm around 0.00035% discs (Fig. 3). Bacteroides me/laninogenicuis ss muelaninogenicus were again found to be the most sensitive to this agent (0.000098%). All of the strains tested were susceptible to 0.0031% or less chlorhexidine and had zones of inhibition of 12 mm or larger around 0.025% discs (Table 4, Fig. 3). Bacteroides elaini(ogenicus ss melanitnogenicus were shown to be the most susceptible, while Bacteroides f'ragilis were shown to be the most resistant. Using the methods employed, a concentration of 0.33% sodium fluoride was necessary for inhibition of the 40 tested organisms (Table 4, Fig. 3). Zone diameters of 8 mm to 25 mm were observed around 0.0"7% discs. Bacteroides tnelanlinogeuicuts
Sa
melaninogen icus
were
found to be the organisms most sensitive (MBC 0.006%), followed closely by Fusobacterium nzucleatum species (MBC 0.01%) (Table 4). The gram-positive organisms A ctinomy ces i)iscosus and Streptococcus sanguis were shown to be more resistant than the gram-negative organisms. A visibility problem was present between the sodium fluoride and the trypticase soy broth. In
addition, a visibility problem was encountered when the trypticase soy broth was mixed with the sodium fluoride. The resultant dilutions were turbid before inoculation; therefore, only MBC's were determined. A concentration of 0.18% eugenol was found to inhibit all organisms tested, with the exception of one strain of Selenomonas sputigenza and one strain of ActinOomyces Viscosus, which required a 0.37% eugenol concentration (Fig. 3). Zone diailieters ranged greatly from 12 mm to 40 mm. As with the other three agents tested, Bactero'ides in elan inogenicus ss melaninogen icus were found to be the organisms most sensitive (0.023%). Selenomonas sputigena was the most resistant (0.1 6%).
Discussion. At the present time, there are four antibiotics generally used for the treatment of anaerobic infections: chloramphenicol, clindamycin, penicillin, and tetracycline.17 The results of this study indicate that the anaerobic isolates and the facultative organisms were extremely susceptible to most antibiotics tested. Tetracycline inhibited 98% of the organisms tested, whereas penicillin and ampicillin inhibited 93% of the isolates. Metronidazole inhibited 60% of the test organisms. The remaining 40% not inhibited by this drug were facultative bacteria. Erythromycin, while not as effective as the previously-mentioned antibiotics, was more effective than metronidazole. This can be accounted for since erythromycin
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ANTIBACTERIAL SUSCEPTIBILITY OF PLAQUE BACTERIA
Vol. 58 No. 7
has a wider spectrum affecting both aerobes and anaerobes. The empirical selection of an antimicrobial agent and dosage regimen is based on *
1 727
knowledge of the pathogens, clinical experience and pharmacology of the drug. The terms resistant and susceptible are categories designating the extremes of a spectrum.
ACTINONUYCES VISCOSUS
>10
I
El BACTEROIDES CORRODENS
BACTEROIDES FRAG LIS * BACTEROIDES MlELANINOGENICUS x CAPNOCYTOPHAGA A EIKENELLA CORRODENS FUSOBACTERIUM NUCLEATUAM A SELENONMONAS SPUTIGENA
10 - K
*
S
AMPICILLIN
2.5 1.25
STREPTOCOCCUS SANGUIS
-
E
N, 0.625 0.31
05
-
0
0.15
u
E x
0.08
c
0.04-
0
0
00
C
0.02
O
0.010.005 -,
0.0025
1 50
30
10
70
zone diameter in millimeters
>10
>10i
10-
10
A
x
at 0
2.5
2.5-
1.25 -
1.2S \-
E\
"
0
5
CLINDAMYCIN
s
0.625
1-
E
\
VI
0.625-
0.31
00
0.31
*0
05 o
0
uY
0.15-
C
0.15 -
0.08 \
0.04
-
0.040.02-
0.02
0.01
CHLORAMPHENICOL
0_
C
0.08
0.01-
-,
0.005
0.005
0.0025
0.0025
10
zone
30
50
70
diameter in millimeters
Fig. 1 -Regression
curves
10
zone
30
50
diameter in millimeters
for antibiotics and antimicrobials.
Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
70
1728
NEWMAN ETAL.
JDent Res July 1979
When information places a microorganism at one of the extremes, therapeutic responsiveness or unresponsiveness may be reasonably anticipated. A microorganism may
be considered susceptible to a particular agent if in vitro studies suggest that a patient infected by those microorganisms is likely to respond favorably to the drug when it is
>10 I 10
10
PENICILLIN 2.5 _
E
w
0.15
01 0
t
Isas
E
U.
-
0.825
2
0.31
0c
0 0.15
V
I 4ETRONIDAZOLE
E
E c
m
1.25
_
N, 0.625°
#
2.5
1.25
0.31
9
s
0.08
c
0.08
0.04
Y
0.04
0.02
0.02
0.01
0.01
0.005
0.005
0.0025
0.0025 30
1o
50
-
70
10
30
50
70
zone diameter in millimeters
zone diameter in millimeters
>10 {
10
"°11
I
10-
S.-.
5-
ERYTHROMYCIN
2.5 1.25 V
0.625-
2
0.31 -
E
,
8A
1.25
9D O
*
0.e25-
E 0
. X .1
0
a 0
01
-
0.04-]
L-
-
I-)
0.02 -
*0
0.15-
E
S^) A
O COO
a
0.31-
016
tJ
TETRACYCLINE
2.S -
c
0.08
-
0.04-
*
A x
_
0.02
0.01 -
-
0.01-
0.005 -
0.005
0.0025 -
0.0025
-r
- -T 30
10
zone
50
70
diameter in millimeters
Fig. 2-Regression
curves
10
zone
30
s0
diameter in millimeters
for antibiotics and antimicrobials.
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70
Vol. 58 No. 7
ANTIBACTERIAL SUSCEPTIBILITY OFPLAQUE BACTERIA
>0.018 I
1
>0.05
0.018
1 729
0.05
0.0087
0.025
ALEXIDINE
0.0043
0.0062
0.0021
0.0031
CHLORHEXIDINE
0.0010
>
0.0015
t
0.00054
c
0.00075
0
0.00027
0.00037
i
0.00013
U0.00018 0.000
.
Q
0\
*
-
0.000065 0.000034
0.000045
0.000017
0.000022
0.000008
0.000011
0.000004-
0.000005.-
-
I
10
30
zone
50
>501
zone
S0
70
diameter in millimeters
>2.66
sos
\
2.66 -
25-
1.33
-
12.5 -
0.66
-
6.25 -
0.33
-
EUGENOL
SODIUM FLUORIDE
0.16-
3.1-
0.08 -
1.5n-7r.U.n:> -
0.37
30
10
70
diameter in millimeters
o
-
0.04
-
0.02
-
M-
0.01-
0.18-
os
0.09 -
o
SW
0.045 -
0.0025
0.022-
o *
0.011-
o
X
V
10
-
0.0012 -
a
co
30
0.0006
50
curves
-
1
70
zone diameter in millimeters
Fig. 3-Regression
0
0.005 -
-
-I
10
zone
30
50
diameter in millimeters
for antibiotics and antimicrobials.
Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
70
1730
NEWAIAN ETAL.
J Deti t Res Julj) 1 9 79
given in dosages appropriate to the type of was found to inhibit the growth of all ormicroorganism and the type of infection. ganisms tested. This value of approximately A number of complicating variables-such 0.3%, which must be in contact with the as, trauma, injection of local anesthetic, dentogingival surface at all times in order to severe periodontal infection, systemic or inhibit plaque, is a concentration not achieved primary metabolic disease, radiation and in toothpastes. Yet the 0.1% to 0.2% submalignancy-must be considered before the optimal dosage in toothpaste has been response of an individual to chemotherapy shown to reduce caries 20% to 30%o.29 can be predicted. More information regard- Like that of chlorhexidine and alexidine, the ing the pharmacology and physiology of effectiveness of fluoride may be due in some these agents in the treatment of oral infec- part to its substantivity.29 tions is needed. Eugenol, a phenol derivative, is presently Chlorhexidine has been shown to be very used in a variety of dental therapeutics. effective in its bactericidal action in clinical Since it is a derivative of phenol, it has the supragingival plaque control, even thiough potential for antibacterial properties. The its use in the United States has been present study supports this claim, but not limited.16'19'27 In the in vitro tests per- necessarily as used in vivo. It has been formed in this study, the microorganisms shown in other studies20 that this activity isolated from supra- and subgingival plaque can be almost completely lost if an agar were found to be susceptible. medium that contains reducing agents is The MIC values, determined in this study chosen as a test vehicle. Since the present by the broth dilution method, were coinpar- study was carried out under anaerobic or able to those reported by others.18'23 reduced conditions, the activity on the agar These concentrations could be used to plates should have been decreased. This, determiine the amrount of agent which must however, was not the case, since eugenol be placed into a depot or slow release de- was shown to exhibit a high degree of vice so that these MIC levels will be present inhibition under anaerobic conditions (zone at all times, especially in subgingival areas. sizes 12 mm to 40 mm). A concentration of Loesche29 suggests that if these MIC values 0.37% was found to inhibit all organisms are used to determine the level of agent in tested. toothpastes, mouthwashes or gels, the The regression curves for all the antibioresults will inevitably be failures. In the case tics and antimicrobials tested showed a of dentifrices, one must bear in mind the positive correlation between the MIC and reactivity of the drug in question and its zone diameters. Examination of the regrescompatability with many conventional sion lines revealed that some organisms fell dentifrice ingredients. Chlorhexidine, for predominantly to the right of the regresexample, has reduced bactericidal effect sion line. This may be accounted for by the under acid conditions where surface ioni- fact that these strains had a slower growth zation of bacteria is suppressed. Other rate at 370C, thus producing larger zone factors are the length of contact the drug sizes. Reference to the regression lines has with the dental tissues, its substantivity shows that this phenomenon accounts for and its stability in the oral cavity. These the percentage of strains whose zone diadeterminations must be found by perform- meters do not correlate with their respective ing experiments which measure time expo- MIC's. sure of agent in relation to supragingival The results from this study provide preplaque inhibition.29 liminary data to clinicians to aid in the Alexidine possesses excellent bactericidal empirical selection of antibiotics and antipotential. It also appears to stain less.35 microbial agents used in the treatment of While still being evaluated for use in the dental infections. By adopting a standardized United States, alexidine can significantly method, similar to the method used in this reduce supragingival plaque and gingivitis in study, results may be compared from one clinical studies.12,26 In the present study, laboratory to the next. alexidine at a concentration of 0.0021% Acknowledgments-The authors wish to thank has been shown to inhibit all the surveyed David M. Carlberg for his assistance in preparing plaque bacteria. the computer program, and Dr. Vera Sutter and A concentration of 0.33% sodium fluoride Susan L. Newman, RDH for their valuable suggestions. Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
Vol. S8 No. 7
ANTIBACTERIAL SUSCEPTIBILITY OF PLAQUE BACTERIA
1 731
TABLE 4 SUSCEPTIBILITY O ORAL BACTERIA TO ANTIMICROBIAL AGENTS
Antibiotic
Organism
Chlorhexidine Actinomyces viscosus B. fragilis B. melaniniogeniicus Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatumni Selenomonas sputigena Streptococcuis sanguis A ctinom77yces viscosus Alexidine B. fragilis B. melaninogenicus Capnocytophaga Corroding Bacteroides E. corrodens
Ftusobacteriuni nucleatuim Selenomonias sputigena Streptococcus sanguis
Fluoride
Actinomn vces viscosus
Average Average MIC MBC % v/v % v/v 0.0007 0.003 1 0.00056 0.0023 0.0018 0.0023 0.0027 0.0017 0.0014 0.00042 0.001 0.000093 0.00079 0.0076 0.0045 0.0014 0.00045 0.00069 *
B. fragilis B. melaninogenicus
Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatun
Eugenol
Selenomonas sputigena Streptococcus saniguis A ctinomyces viscosus B. fragilis B. melaninogenicus Capniocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatunm Selenomonas sputigena Streptococcus sanguis
0.097 0.045 0.023 0.037 0.08 0.082 0.068 0.16 0.093
0.0011 0.003 1 0.00075 0.0035 0.0020 0.0044 0.0027 0.0017 0.0020 0.00042 0.001 0.00013 0.00097 0.0012 0.0015 0.0016 0.0014 0.00097 0.08 0.08 0.006 0.07 0.15 0.16 0.01 0.14 0.10 0.24 0.045 0.045 0.075 0.09 0.13 0.068 0.17 0.15
Average Zone Diameter MBC mm Clinically Achievable
20 16 27 18 17 20 17 23 20 10 7 12 8 8 10 7 9 9 11 15 23 14 21 16 22 19 12 17
20 37 34 37 26 23 27 18
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
*Impossible to read MIC-see text.
REFERENCES 1. BALLOWS, A.: Current Techniques for Antibiotic Susceptibility Testing. Charles Thomas, 1974. 2. BAILEY, N. 1. J.: Statistical Methods in Biology. The English Universities Press, Ltd., London, 1959. 3. BARKER, B. M.; and F. PRESCOT: Anti-
microbial Agents in Medicine. Blackwell Scientific Publications, London, 1973. 4. BASSIOUNY, M. A.; and A. A. GRANT: The toothbrush application of chlorhexidine. A clinical trial. Brit Dent J 139:8, 1972. 5. BAUER, A. W.; W. M. KIRBY; J. C. SHERRIS; and M. TURCK: Antibiotic susceptibility testing by single disc method. Am J Clin Path 45:993, 1966. 6. BERGSTROMN, J.; and B. HOLMBERG: The effect of chlorhexidine emulsion on
Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
1 732
7.
8.
9.
10.
11.
12.
J Den t Res Julj. 1 9 79
NEWAIANETAL.
plaque. An intraindividual study of local application. Scand J Dent Res 66:5, 1973. BIRKELAND, J. M.: Effect of fluoride on the amount of dental plaque in children. Scand JDent Res 80:82, 1972. BRAUER, G. M.; R. McLAUGHLIN; and E. HUGET: Aluminum oxide as a reinforcing agent for zinc oxide-eugenol o-ethoxybenzoic acid. J. Dent Res 47:622, 1968. BRINNER, W. W.; and J. A. WUNDER: Sensitivity of dog plaque microorganism to chlorhexidine during longitudinal studies. J Periodonit Res 12:135, 1977. BUCHANAN, R. E.; and N. E. GIBBONS: Bergey's Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Company, Baltimore, MD, 1974. BURNETT, G. W.; and H. W. SCHERP: Oral Microbiology and Infectious Disease, 3rd ed. The Williams and Wilkins Company, Baltimore, MD, 1968. CARLSON, H. C.; and C. K. PORTER: Inhibitory effect of a synthetic antibiotic mouthwash (QR-711) on dental plaque and gingivitis in young adults. JPeriodont 44:225,
1973. 13. CRAWFORD, A.; S. S. SOCRANSKY; and G. BRATTHALL: Predominant cultivable microbiota of advanced periodontitis. J Dent Res 54 Abst., 209, 1975. 14. DICKSON, D.: New procedure in microbiology. Path-Logics Quarterly 2:24, 1976. 15. ENGLANDER, H. R.; P. H. KEYES; M. GESTWICKI; and H. A. SULTZ: Clinical anticaries effect of replated topical sodium fluoride applications by mouthpieces. JADA 75 :638, 1967. 16. ERIKSEN, H. M.; and P. GJERMO: Incidence of stained tooth surfaces in students using chlorhexidine-containing dentifrices. ScandJDentRes 81:533, 1973. 17. FINEGOLD, S. M.: Anaerobic Bacteria in Human Disease. Academic Press, New York, 1977. 18. GJERMO, P.; K. BAASTAD; and G. ROLLA: The plaque inhibiting capacity of 11 antibacterial compounds. J Periodont Res 5:102, 1970. 19. GJERMO, P.; and G. ROLLA: The plaque inhibiting effect of chlorhexidine containing dentifrices. Scand J Dent Res 79:126, 1971. 20. GURNEY, B.F.: Chemotherapy in dental practice. Dental Digest 71:30, 1965. 21. GURNEY, B. F.: Chemotherapy in dental practice: Compound topical anesthetics. Dental Digest 74:129, 1968. 22. GURNEY, B. F.: Substituted phenols: Part one - Hydroxy phenols, guaiacol, eugenol. Denital Digest 78:260, 1972. 23. HANNESSEY, T. D.: Some antibacterial properties of chlorhexidine. J Periodont Res 8: Suppl., 12:61, 1973.
24. HOLDEMAN, L. V.; and W. E. C. MOORE: 4naerobe Laboratory Manual, 3rd ed. Virginia Polytechnic Institute and State University,
Blacksburg, VA, 1975. 25. HOLT, S. C.; J. L. SIMPSON; and E. R. LEADBETTER: Some characteristics of gliding bacteria isolated from human dental plaque. JDent Res 54 Abst., 1208, 1975. 26. LOBENE, R. R.; and P. M. SOPARKAR: The effect of an alexidine mouthwash on human plaque and gingivitis. JADA 87: 848, 1973. 27. LOE, H.; and C. R. SCHIOTT: The effect of mouthwashes and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. J Periodont Res 5:79, 1970. 28. LOE, H.; C. R. SCHIOTT; L. GLAVIND; and T. KARRING: Effects and side effects during two years' use of chlorhexidine in humans. J Dent Res 54:123, 1975. 29. LOESCHE, W. J.: Chemotherapy of dental plaque infections. Oral Sciences Rev 9:23, 1976. 30. NEWMAN, M. G.; S. S. SOCRANSKY; E. D. SAVITT; D. A. PROPAS; and A. CRAWFORD: Studies of the microbiology of periodontosis. J Periodont 47:483, 1976. 31. SAVITT, E. D.; S. S. SOCRANSKY; B. F. HAMMOND; and M. G. NEWMAN: Characterization of fusiform organisms isolated from periodontosis. J Dent Res 54:abst. 208, 1975. 32. SCHROEDER, H. E.: Formation and Inhibition of Dental Calculus. Hans Huber, Berne, Switzerland, 1969. 33. SOCRANSKY, S. S.: Microbiology of periodontal disease: Present status and future considerations. J Periodont 48:497, 1977. 34. SOCRANSKY, S. S.; S. C. HOLT; E. R. LEADBETTER; A. C. R. TANNER:, E. SAVITT; B. F. HAMMOND: Capnocytophaga: New genus of gram-negative gliding bacteria. III. Physiological characteristics. Arch Oral Biol, in press. 35. SPOLSKY, V.: Personal communication. 1977. 36. STALOM, D. R.; and C. ZHORNSBERRY: Broth-dilution method for determining the antibiotic susceptibility of anaerobic bacteria. Antiemicro Agent and Clhemother 7:15, 1975. 37. SUTTER, V. L.; U. U. KWAH; and S. M. FINEGOLD: Standardized antimicrobial disc susceptibility testing of anaerobic bacteria. A ppl Micr 23:268, 1972. 38. SUTTER, V. L.; V. L. VARGO; and S. M. F INEGOLD: Wadsworth Anaerobic Bacteriology Manual. UCLA Extension Division, University of California, Los Angeles, CA, 1975.
Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
*U.C.L.A. School of Dentistry,
Section of Periodontics, Los Angeles, CA University, Lonig Beach, Department of 'Aicrobiology, Lonig Beach, CA 90840.
Selected anaerobic, capnophilic and ,fcultative bacteria isolated from patients with various forms of periodontal health and disease wvere tested for their susceptibility to antibiotics and antimicrobial agents. Specific bactericidal and minimum inhibitory concentrations were compared to disc zone diameters, thereby generating new standards for the potential selection of attimicrobial agents. J Dent Res 58(7):1722-1732, July 1979
Introduction. Periodontitis, the periodontal abscess and infection resulting from pulpal disease, can be initiated by many of the bacterial species colonizing the gingival and periodontal tissues. The empirical selection of an antibiotic is necessary when a patient first appears with acute infection. The selection of the most appropriate agent can be improved if there are criteria of susceptibility and resistance available to the dentist. There has been substantial research dealing with antimicrobial susceptibilities of medically important bacteria,5 ,3 6,37 but surprisingly there are few available data regarding the antibiotic or antimicrobial susceptibility of the bacterial strains which are implicated in dental diseases. The data which are available are either incomplete or based on older or nonreliable methods. Although the majority of available information has been based on the disc diffusion technique for antimicrobial susceptibility testing, it is only recently that attempts have been made to standardize the procedure and to correlate the results with the more accurate and sensitive dilution methods of
Received for publication March 3, 1978. Accepted for publication September 26, 1978. Supported by grants from the California Dental Association and Grant NIH-NIDR DE 0444-1.
90024; **Califbrnia State
testing. 17,36,38 In addition, some investigators have used standards for anaerobes that were originally developed for aerobic and facultative organisms. Growth rate of the bacteria. inoculum size, pH, medium, and carbon dioxide concentrations are some of the factors influenced by anaerobic conditions.17 A standardized dilution technique as well as a disc diffusion technique can be used to measure specific bactericidal and minimum inhibitory concentrations of anaerobes. Using a combination of these methods, new criteria of susceptibility and resistance can be generated.
Materials and methods. Bacterial cultures. Forty bacteria were obtained from patients with various forms of periodontal disease and from patients with periodontal health (Table 1). The organisms were: Actinomyces viscosus (4), Bac tero ides m elaninogen icus ss m elaninogenicus (2), Capnocytophaga2530'3133,34 species (Bacteroides ochraceus)(1 1), "corroding Bacteroides "i 3 (3), Eikenella corrodens (2), Fusobacterium nucleatum (4), Selenomonas sputigena (3) and Streptococcus sanguis (9). Bacteroides fragilis ss. ovatus (ATCC 8483) and Eikenella corrodens (Orange County Medical Center) were used as controls. All organisms except Capnocytophaga and "corroding Bacteroides" were classified according to established characteristics such as those listed in Bergey's Manual of Determinative Bacteriology, 8th edition ,1 0 Wadsworth Anaerobic Bacteriology Manual,38 and others.17'24 Capnocytophaga were characterized by criteria established by Savitt et al. 1975,3i Holt et al. 1975,2 5 Newman, et al. 1976,30 Socransky et al. 197934 and "corroding Bacteroides" were classified according to criteria by Crawford et al., 1 9715 3 Stock solutions. All antibiotics were
1722 Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
Vol. S8 No. 7
ANTIBACTERIAL SUSCEPTIBILITY OF PLAQUE BACTERIA
1723
TABLE 1
SOURCE AND DISTRIBUTION OF BACTERIAL ISOLATES Subgingival Isolates
ORGANISM
N umber Supraof gingival strains Plaque
Papillon Adoles- Perio- Lefevre Perio- Periocent dontal Aged SynHealthy* Healthy Abscess drome dontosis dontitis
4 Actinomyces viscosus 3 Bacteroides melaninogenicus ss melantino2 genicus 11 Capnocytophaga sp. 4 2 Eikenella corrodens 3 Corroding Bacteroides 3 4 Fusobacterium nucleatum 1 3 Selenomonas sputigena 9 1 Streptococcus sanguis 5 Control Organisms Bacteroides fragilis ss oratus ATCC 843 3 Eikenella corrodens. Orange County Medical Center
1 -) 2
51
5
-
2 1 3 2
1 I
*Plaque taken from an adult,older than 55 years,with the absence of periodontal disease.
dissolved in distilled water to achieve a final concentration of 100 pg/ml (units/ml for penicillin); filter sterilized; and stored frozen in 1 ml aliquots until used. The antibiotics used were: ampicillin (Bristol Laboratories), chloramphenicol (Sigma Chemical Company), clindamycin (Upjohn), erythromycin, penicillin and tetracycline (Sigma Chemical Company). The antimicrobial agents used were: metronidazole (Searle Company), alexidine 0.035% (Calgon Company), chlorhexidine 5% (Imperial Chemical Industries, Limited, Maclesfield, England), sodium fluoride 5.33% (Lorvic Company) and eugenol 100% (Merck Company). Filter paper discs. Antibiotic discs (Difco) were obtained in the following concentrations: ampicillin 10 pg, chloramphenicol 30 pg, clindamycin 2 pug, erythromycin 1 5 pg, penicillin 10 units, and tetracycline 30 pg. The metronidazole discs were prepared by adding 0.01 ml of the stock metronidazole solution to sterile 1/4 inch Bacto-Concentration Discs (Difco) with a micropipette, allowing them to dry, and then storing at 40C with a desicant until use, giving a final concentration of 10 pg per disc. The other antimicrobial agents were prepared in a similar manner, except 0.005 ml was used for chlorhexidine, eugenol, and
sodium fluoride and 0.01 ml of alexidine to give final disc concentrations of: chlorhexidine 0.025%, alexidine 0.00035%, sodium fluoride 0.02 7% and eugenol 0.5%. Broth dilution method. The medium employed was Trypticase-Soy Broth (with 0.25% dextrose) (BBL) which was supplemented with 5% Fildes Enrichment (BBL). Following a 1:50 dilution of the stock antibiotic or antimicrobial agent (this initial dilution was deleted for alexidine, eugenol and sodium fluoride), a two-fold serial dilution was made. An equal volume of a 1:100 dilution of culture grown anaerobically for 48 hours was then added. The tubes were incubated in anaerobic GasPak (BBL) jars, at 370C, for at least 48 hours or until the control tubes showed turbidity. The minimal inhibitory concentration (MIC) was determined as the lowest concentration of antibiotic showing no visible growth. Minimal bactericidal concentrations (MBC) were determined by streaking all tubes with no growth onto plates containing Trypticase Soy Agar with 5% Sheep's Blood (BBL) and incubated for 48 hours in GasPak jars at 370C. The MBC was determined as the lowest concentration of antibiotic resulting in no visible growth on these plates. Disc diffusion. Trypticase Soy Agar with
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NEJVMAN E T A L.
1 724
JDentResJuly 1979
5% Sheep's Blood (BBL) was used as the basal medium. The cultures were grown in supplemented trypticase soy broth in an anaerobic GasPak jar for at least 48 hours, or until turbid. These were then diluted to a No. 1 McFarland barium sulfate standard and swabbed over the entire surface of the plate. Antibiotic or antimicrobial discs were applied, and the plates were incubated for 48 hours at 370C in GasPak jars. The zones of inhibition were measured with a caliper, under reflected light. Regression curves. Regression curves were generated by plotting MICs against disc zone diameters. The locations of the straight lines on each graph were selected by a simple regression method,2 using zone diameter as the independent variable. The intercepts and slope of the lines were determined using a computer program (CHI) and a Control Data Cyber 173 computer. Determination of break points. Break points of "sensitive," "intermediate," and "resistant" were determined by the average MIC and achievable blood levels for each antibiotic.3 Ideally, the in vivo concentration of the antibiotic (at the site of infection) should be two (2) to four (4) times more than in vitro MIC.1'14 Organisms were considered sensitive if the achievable blood level of the antibiotic was 4 times the MIC; intermediate if it was 2-4 times the MIC; and resistant if it was less than 2 times the MIC. The antimicrobials tested are topical agents; therefore, determination of blood levels is not applicable. Antimicrobials were not graded as to sensitive or resistant, but rather according to their potential clinically achievable topical concentration (Table 2).
Results.
of two strains of "corroding Bacteroides" and B. fragilis, were susceptible to an average concentration of 0.86 ,ig/ml ampicillin (Table 3, Fig. 1). Chloramphenicol inhibited all organisms (average concentration 2.36 ,g/ml) but, because of achievable blood levels, B. fragilis, Capnocytophaga, and Eikenella corrodens were considered only intermediately susceptible (Table 3, Fig. 1). All organisms tested were highly susceptible to clindamycin, responding to an average concentration of 01.41 ig/ml (Table 3, Fig. 1). One strain of Selenomonas sputigena was resistant because it required a concentration of 10 ,g/ml. Only the control strain, B. fragilis, was resistant to penicillin. Most other strains were sensitive (average concentration 0.24 ,ig/ml) (Table 3, Fig. 2). All organisms, except one strain of Fusobacterium nucleatum, were inhibited by the highest concentration (10 ,ug/ml) of erythromycin tested (Table 3, Fig. 2). Because the maximum achievable blood level of erythromycin is 2 pg/ml, and since B. fragilis, Eikenella corrodens and Fusobacterium nuccleatum were susceptible only to an average concentration of 2.96 jzg/ml, these organisms were considered resistant. There were several strains not inhibited by the highest concentration (10 pg/ml) of metronidazole (Table 3, Fig. 2). These include Actinomyces viscosus (4 strains), Capnocytophaga (2 strains), Eikenella corrodens (2 strains), Selenomonas sputigena (2 strains), and Streptococcus sanguis (7 strains). Many other strains required relatively high concentrations (1.255.0 jig/ml) for inhibition. All Fusobacterium nucleatum strains were highly susceptible to a concentration of 0.005 -0.08 ,ug/ml but, because of the relatively high achievable blood level (2 - 20 ,g/ml), Actinomyces viscosus and Eikenella corrodens were con-
All organisms tested, with the exception TABLE 2 CLINICALLY ACHIEVABLE TOPICAL LEVELS OF ANTIMICROBIAL AGENTS
Mouthrinse
Gel
Agent Pulpal Pulpal Analgesic Analgesic
3 2 * 0.1-1.0% Chlorhexidine4''32' Chlorhxidin4, 6, 2 3, 2 7, 2 8,.2 Alexidine26'35 0.0 35-0.05%
Sodium Fluoride7'1 5,29 Eugenol8,2 1,22 *
0.2%
Dentifrice Cements
1%
0.5%
(0 .1-0 .2%) 40%
46%
3 7.5%
References Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
Vol. 58 No. 7
ANTIBACTERIAL SUSCEPTIBILITY OF PLAQUE BACTERIA
1 725
TABLE 3 SUSCEPTIBILITY OF ORAL BACTERIA TO ANTIBIOTICS
Antibiotic Ampicillin
Organism
Actinomyces viscosus B. fragilis B. melaninogenicus
Capnocytophaga
Chloramphenicol
Clindamycin
Erythromycin
Corroding Bacteroides E. corrodens Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus
Capnocytophaga Corroding Bacteroides E. corrodens
Metronidazole
Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus
Capnocytophaga Corroding Bacteroides
Penicillin
E. corrodens Fusobacterium nucleatum Selenomonas sputigena Streptococcus sanguis Actinomyces viscosus B. fragilis B. melaninogenicus
Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatum
Average MIC ,ug/ml
0.01 10.0 0.08 0.13 6.7 0.23 0.04 0.17 0.11 1.09 5.0 2.66 3.52 2.08 3.75 0.55 1.15 2.85 0.18 0.31 0.31 0.02 0.009 0.26 0.01 0.03 3.37 0.08 1.25
0.06 0.14 0.27 2.17 1.98 0.21 0.35 > 10.0 0.625 0.10 3.47 0.36 >10.0 0.04 0.04 8.95 0.096 >10.0 0.095 0.23 0.08 0.83 0.02
Average MBC ugjml
Average Zone Diameater mm
0.45 > 10.0 0.08 0.49 6.7 1.25 0.04 0.27 0.50 3.96 10.0 5.16 6.82 3.75 8.33 1.80 3.75 4.38 0.88 0.625 0.08 0.013 0.26 0.625 0.04 3.37 0.53 0.47 2.50 0.08 0.22 0.42 2.71 5.21 1.88 0.46 > 10.0 0.625 0.20 6.25 2.50 > 10.0 0.05 0.04 8.96 0.27 >10.0 0.095 0.70 0.08 3.75 0.02 (TABLE 3
Sa
47 8 63 49 21 33 51 41 39 42 29
x
54 39 43 37 52
x
38 32 36 22 55 54 39 24 55 30 32 43 34 40 40 39 18 40 42 32 7 54 65 32 58 7 70 67 9 45 7 49 39 36 24 49 is continued
Ib Rc x
x x
x x x x x x x x
x
x x x x x x x x x x x x x x x
x x x x x x x x x x x x x x x x
x x x
x x x x x on next
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page.)
J Dent Res July19 79 E T A L. NJE WIAlN
1726
(TABLE 3 continued) SUSCEPTlBILITY 01 ORAL BACTERIA TO ANTIBIOTICS
,ug/ml
Average MBC gin/ml
Average Zone Diameter mm
0.32 0.16 0.71 0.31 0.47 0.48 0.17 1.04 0.41 0.35 1.09
0.32 0.50 3.96 2.50 0.78 1.25 3.96 1.88 1.39 3.49 2.79
42 34 39 71 59 42 39 23 50 39 31
Average
MIC)
Organisnm
Antibiotic
Seleniomonas sputigena Streptococcus satnguis A ctinomnyces iscosus B. fragilis B. melaninogenicus Capnocy tophaga Corroding Bacteroides E. corrodens Fusobacterium ntuclea tutul Selenotinonias sputigena Streptococcus sanguiis
Tetracy cline
a -
sensitive, b
intermediate,
c
-
a/i
in
ss
lb RC
X x
x x x x x x x x x
resistant
sidered resistant, whereas Streptococcus sanguis was considered to be of intermediate susceptibility according to break points. Since tetracycline has a relatively high strains achievable blood level (8 Mg/ml), were susceptible (Table 3, Fig. 2). Alexidine was found to inhibit all of the test organisms at a concentration of 0.0021I% (Table 4). The zone diameters ranged from 7 mm to 15 mm around 0.00035% discs (Fig. 3). Bacteroides me/laninogenicuis ss muelaninogenicus were again found to be the most sensitive to this agent (0.000098%). All of the strains tested were susceptible to 0.0031% or less chlorhexidine and had zones of inhibition of 12 mm or larger around 0.025% discs (Table 4, Fig. 3). Bacteroides elaini(ogenicus ss melanitnogenicus were shown to be the most susceptible, while Bacteroides f'ragilis were shown to be the most resistant. Using the methods employed, a concentration of 0.33% sodium fluoride was necessary for inhibition of the 40 tested organisms (Table 4, Fig. 3). Zone diameters of 8 mm to 25 mm were observed around 0.0"7% discs. Bacteroides tnelanlinogeuicuts
Sa
melaninogen icus
were
found to be the organisms most sensitive (MBC 0.006%), followed closely by Fusobacterium nzucleatum species (MBC 0.01%) (Table 4). The gram-positive organisms A ctinomy ces i)iscosus and Streptococcus sanguis were shown to be more resistant than the gram-negative organisms. A visibility problem was present between the sodium fluoride and the trypticase soy broth. In
addition, a visibility problem was encountered when the trypticase soy broth was mixed with the sodium fluoride. The resultant dilutions were turbid before inoculation; therefore, only MBC's were determined. A concentration of 0.18% eugenol was found to inhibit all organisms tested, with the exception of one strain of Selenomonas sputigenza and one strain of ActinOomyces Viscosus, which required a 0.37% eugenol concentration (Fig. 3). Zone diailieters ranged greatly from 12 mm to 40 mm. As with the other three agents tested, Bactero'ides in elan inogenicus ss melaninogen icus were found to be the organisms most sensitive (0.023%). Selenomonas sputigena was the most resistant (0.1 6%).
Discussion. At the present time, there are four antibiotics generally used for the treatment of anaerobic infections: chloramphenicol, clindamycin, penicillin, and tetracycline.17 The results of this study indicate that the anaerobic isolates and the facultative organisms were extremely susceptible to most antibiotics tested. Tetracycline inhibited 98% of the organisms tested, whereas penicillin and ampicillin inhibited 93% of the isolates. Metronidazole inhibited 60% of the test organisms. The remaining 40% not inhibited by this drug were facultative bacteria. Erythromycin, while not as effective as the previously-mentioned antibiotics, was more effective than metronidazole. This can be accounted for since erythromycin
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ANTIBACTERIAL SUSCEPTIBILITY OF PLAQUE BACTERIA
Vol. 58 No. 7
has a wider spectrum affecting both aerobes and anaerobes. The empirical selection of an antimicrobial agent and dosage regimen is based on *
1 727
knowledge of the pathogens, clinical experience and pharmacology of the drug. The terms resistant and susceptible are categories designating the extremes of a spectrum.
ACTINONUYCES VISCOSUS
>10
I
El BACTEROIDES CORRODENS
BACTEROIDES FRAG LIS * BACTEROIDES MlELANINOGENICUS x CAPNOCYTOPHAGA A EIKENELLA CORRODENS FUSOBACTERIUM NUCLEATUAM A SELENONMONAS SPUTIGENA
10 - K
*
S
AMPICILLIN
2.5 1.25
STREPTOCOCCUS SANGUIS
-
E
N, 0.625 0.31
05
-
0
0.15
u
E x
0.08
c
0.04-
0
0
00
C
0.02
O
0.010.005 -,
0.0025
1 50
30
10
70
zone diameter in millimeters
>10
>10i
10-
10
A
x
at 0
2.5
2.5-
1.25 -
1.2S \-
E\
"
0
5
CLINDAMYCIN
s
0.625
1-
E
\
VI
0.625-
0.31
00
0.31
*0
05 o
0
uY
0.15-
C
0.15 -
0.08 \
0.04
-
0.040.02-
0.02
0.01
CHLORAMPHENICOL
0_
C
0.08
0.01-
-,
0.005
0.005
0.0025
0.0025
10
zone
30
50
70
diameter in millimeters
Fig. 1 -Regression
curves
10
zone
30
50
diameter in millimeters
for antibiotics and antimicrobials.
Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
70
1728
NEWMAN ETAL.
JDent Res July 1979
When information places a microorganism at one of the extremes, therapeutic responsiveness or unresponsiveness may be reasonably anticipated. A microorganism may
be considered susceptible to a particular agent if in vitro studies suggest that a patient infected by those microorganisms is likely to respond favorably to the drug when it is
>10 I 10
10
PENICILLIN 2.5 _
E
w
0.15
01 0
t
Isas
E
U.
-
0.825
2
0.31
0c
0 0.15
V
I 4ETRONIDAZOLE
E
E c
m
1.25
_
N, 0.625°
#
2.5
1.25
0.31
9
s
0.08
c
0.08
0.04
Y
0.04
0.02
0.02
0.01
0.01
0.005
0.005
0.0025
0.0025 30
1o
50
-
70
10
30
50
70
zone diameter in millimeters
zone diameter in millimeters
>10 {
10
"°11
I
10-
S.-.
5-
ERYTHROMYCIN
2.5 1.25 V
0.625-
2
0.31 -
E
,
8A
1.25
9D O
*
0.e25-
E 0
. X .1
0
a 0
01
-
0.04-]
L-
-
I-)
0.02 -
*0
0.15-
E
S^) A
O COO
a
0.31-
016
tJ
TETRACYCLINE
2.S -
c
0.08
-
0.04-
*
A x
_
0.02
0.01 -
-
0.01-
0.005 -
0.005
0.0025 -
0.0025
-r
- -T 30
10
zone
50
70
diameter in millimeters
Fig. 2-Regression
curves
10
zone
30
s0
diameter in millimeters
for antibiotics and antimicrobials.
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70
Vol. 58 No. 7
ANTIBACTERIAL SUSCEPTIBILITY OFPLAQUE BACTERIA
>0.018 I
1
>0.05
0.018
1 729
0.05
0.0087
0.025
ALEXIDINE
0.0043
0.0062
0.0021
0.0031
CHLORHEXIDINE
0.0010
>
0.0015
t
0.00054
c
0.00075
0
0.00027
0.00037
i
0.00013
U0.00018 0.000
.
Q
0\
*
-
0.000065 0.000034
0.000045
0.000017
0.000022
0.000008
0.000011
0.000004-
0.000005.-
-
I
10
30
zone
50
>501
zone
S0
70
diameter in millimeters
>2.66
sos
\
2.66 -
25-
1.33
-
12.5 -
0.66
-
6.25 -
0.33
-
EUGENOL
SODIUM FLUORIDE
0.16-
3.1-
0.08 -
1.5n-7r.U.n:> -
0.37
30
10
70
diameter in millimeters
o
-
0.04
-
0.02
-
M-
0.01-
0.18-
os
0.09 -
o
SW
0.045 -
0.0025
0.022-
o *
0.011-
o
X
V
10
-
0.0012 -
a
co
30
0.0006
50
curves
-
1
70
zone diameter in millimeters
Fig. 3-Regression
0
0.005 -
-
-I
10
zone
30
50
diameter in millimeters
for antibiotics and antimicrobials.
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70
1730
NEWAIAN ETAL.
J Deti t Res Julj) 1 9 79
given in dosages appropriate to the type of was found to inhibit the growth of all ormicroorganism and the type of infection. ganisms tested. This value of approximately A number of complicating variables-such 0.3%, which must be in contact with the as, trauma, injection of local anesthetic, dentogingival surface at all times in order to severe periodontal infection, systemic or inhibit plaque, is a concentration not achieved primary metabolic disease, radiation and in toothpastes. Yet the 0.1% to 0.2% submalignancy-must be considered before the optimal dosage in toothpaste has been response of an individual to chemotherapy shown to reduce caries 20% to 30%o.29 can be predicted. More information regard- Like that of chlorhexidine and alexidine, the ing the pharmacology and physiology of effectiveness of fluoride may be due in some these agents in the treatment of oral infec- part to its substantivity.29 tions is needed. Eugenol, a phenol derivative, is presently Chlorhexidine has been shown to be very used in a variety of dental therapeutics. effective in its bactericidal action in clinical Since it is a derivative of phenol, it has the supragingival plaque control, even thiough potential for antibacterial properties. The its use in the United States has been present study supports this claim, but not limited.16'19'27 In the in vitro tests per- necessarily as used in vivo. It has been formed in this study, the microorganisms shown in other studies20 that this activity isolated from supra- and subgingival plaque can be almost completely lost if an agar were found to be susceptible. medium that contains reducing agents is The MIC values, determined in this study chosen as a test vehicle. Since the present by the broth dilution method, were coinpar- study was carried out under anaerobic or able to those reported by others.18'23 reduced conditions, the activity on the agar These concentrations could be used to plates should have been decreased. This, determiine the amrount of agent which must however, was not the case, since eugenol be placed into a depot or slow release de- was shown to exhibit a high degree of vice so that these MIC levels will be present inhibition under anaerobic conditions (zone at all times, especially in subgingival areas. sizes 12 mm to 40 mm). A concentration of Loesche29 suggests that if these MIC values 0.37% was found to inhibit all organisms are used to determine the level of agent in tested. toothpastes, mouthwashes or gels, the The regression curves for all the antibioresults will inevitably be failures. In the case tics and antimicrobials tested showed a of dentifrices, one must bear in mind the positive correlation between the MIC and reactivity of the drug in question and its zone diameters. Examination of the regrescompatability with many conventional sion lines revealed that some organisms fell dentifrice ingredients. Chlorhexidine, for predominantly to the right of the regresexample, has reduced bactericidal effect sion line. This may be accounted for by the under acid conditions where surface ioni- fact that these strains had a slower growth zation of bacteria is suppressed. Other rate at 370C, thus producing larger zone factors are the length of contact the drug sizes. Reference to the regression lines has with the dental tissues, its substantivity shows that this phenomenon accounts for and its stability in the oral cavity. These the percentage of strains whose zone diadeterminations must be found by perform- meters do not correlate with their respective ing experiments which measure time expo- MIC's. sure of agent in relation to supragingival The results from this study provide preplaque inhibition.29 liminary data to clinicians to aid in the Alexidine possesses excellent bactericidal empirical selection of antibiotics and antipotential. It also appears to stain less.35 microbial agents used in the treatment of While still being evaluated for use in the dental infections. By adopting a standardized United States, alexidine can significantly method, similar to the method used in this reduce supragingival plaque and gingivitis in study, results may be compared from one clinical studies.12,26 In the present study, laboratory to the next. alexidine at a concentration of 0.0021% Acknowledgments-The authors wish to thank has been shown to inhibit all the surveyed David M. Carlberg for his assistance in preparing plaque bacteria. the computer program, and Dr. Vera Sutter and A concentration of 0.33% sodium fluoride Susan L. Newman, RDH for their valuable suggestions. Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 18, 2015 For personal use only. No other uses without permission.
Vol. S8 No. 7
ANTIBACTERIAL SUSCEPTIBILITY OF PLAQUE BACTERIA
1 731
TABLE 4 SUSCEPTIBILITY O ORAL BACTERIA TO ANTIMICROBIAL AGENTS
Antibiotic
Organism
Chlorhexidine Actinomyces viscosus B. fragilis B. melaniniogeniicus Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatumni Selenomonas sputigena Streptococcuis sanguis A ctinom77yces viscosus Alexidine B. fragilis B. melaninogenicus Capnocytophaga Corroding Bacteroides E. corrodens
Ftusobacteriuni nucleatuim Selenomonias sputigena Streptococcus sanguis
Fluoride
Actinomn vces viscosus
Average Average MIC MBC % v/v % v/v 0.0007 0.003 1 0.00056 0.0023 0.0018 0.0023 0.0027 0.0017 0.0014 0.00042 0.001 0.000093 0.00079 0.0076 0.0045 0.0014 0.00045 0.00069 *
B. fragilis B. melaninogenicus
Capnocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatun
Eugenol
Selenomonas sputigena Streptococcus saniguis A ctinomyces viscosus B. fragilis B. melaninogenicus Capniocytophaga Corroding Bacteroides E. corrodens Fusobacterium nucleatunm Selenomonas sputigena Streptococcus sanguis
0.097 0.045 0.023 0.037 0.08 0.082 0.068 0.16 0.093
0.0011 0.003 1 0.00075 0.0035 0.0020 0.0044 0.0027 0.0017 0.0020 0.00042 0.001 0.00013 0.00097 0.0012 0.0015 0.0016 0.0014 0.00097 0.08 0.08 0.006 0.07 0.15 0.16 0.01 0.14 0.10 0.24 0.045 0.045 0.075 0.09 0.13 0.068 0.17 0.15
Average Zone Diameter MBC mm Clinically Achievable
20 16 27 18 17 20 17 23 20 10 7 12 8 8 10 7 9 9 11 15 23 14 21 16 22 19 12 17
20 37 34 37 26 23 27 18
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
*Impossible to read MIC-see text.
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