and 67% of the P. nigrescens isolates were susceptible to tetracycline. A higher ... Systemic or topical antibiotics have been employed as an adjunct in treat-.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2001, p. 3253–3255 0066-4804/01/$04.00⫹0 DOI: 10.1128/AAC.45.11.3253–3255.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 11
Susceptibilities of Eikenella corrodens, Prevotella intermedia, and Prevotella nigrescens Clinical Isolates to Amoxicillin and Tetracycline N. LUONG, J. TSAI,
AND
C. CHEN*
Division of Primary Oral Health Care, School of Dentistry, University of Southern California, Los Angeles, California 90089 Received 28 September 2000/Returned for modification 28 February 2001/Accepted 16 July 2001
The AB Biodisk Etest showed that 106 (100%) and 98 (92%) isolates of Eikenella corrodens were susceptible to amoxicillin and tetracycline, respectively. Twenty-three (68%) Prevotella intermedia isolates and 14 (67%) Prevotella nigrescens isolates were susceptible to amoxicillin. Seventy-nine percent of the P. intermedia isolates and 67% of the P. nigrescens isolates were susceptible to tetracycline. A higher percentage of -lactamaseproducing isolates of P. intermedia and P. nigrescens were identified with selective agar containing amoxicillin than with nonselective agar. (supra- and subgingival plaque samples, saliva, and mucosal surfaces) of periodontally healthy subjects (9 isolates), adult periodontitis patients (45 isolates), and localized juvenile periodontitis patients (37 isolates). The remaining E. corrodens isolates included 14 nonoral isolates (9 isolates from blood, 4 isolates from nonoral abscesses, and 1 isolate from a bite wound) and 1 isolate from sputum. Sixty-five P. intermedia oral isolates and 33 P. nigrescens oral isolates were examined in this study. The isolates were recovered from the primary cultures of the subgingival plaque of periodontitis patients over 1 year from 1999 to 2000. The isolates can be divided into three categories based on the culture medium used in primary cultures; nonselective brucella blood agar (34 P. intermedia isolates and 21 P. nigrescens isolates), brucella blood agar containing 1 g of amoxicillin per ml (21 P. intermedia isolates and 9 P. nigrescens isolates), and brucella blood agar containing 1 g of tetracycline per ml (10 P. intermedia isolates and 3 P. nigrescens isolates). The species identities of the P. intermedia and P. nigrescens isolates were confirmed by 16S rRNA-based PCR detection methods as described previously (1). The susceptibilities of E. corrodens, P. intermedia, and P. nigrescens to amoxicillin and tetracycline were determined by the Etest method (AB Biodisk, Piscataway, N.J.). E. corrodens was grown on brucella blood agar plates for 3 days in 5% CO2 at 37°C and washed off the plates with Todd-Hewitt broth. P. intermedia and P. nigrescens were grown on brucella blood agar plates anaerobically at 37°C for 3 to 5 days and washed off the plates with dilution broth consisting of sodium chloride (5,000 g/ml), Thiotone peptone (2,500 g/ml), and tryptose (2,500 g/ml). The E. corrodens, P. intermedia, and P. nigrescens bacterial suspensions were adjusted to a turbidity of a 1.0 McFarland standard and inoculated onto brucella blood agar plates with sterile cotton swabs. The Etest strips (AB Biodisk) were placed onto the agar surface. The plates were cultured for 3 days, and the MICs were determined according to the manufacturer’s guidelines. Selected P. intermedia and P. nigrescens isolates were further subjected to a -lactamase test with Cefinase -lactamase disc (Becton Dickenson Microbiology Sys-
Periodontitis is a bacterial inflammatory disease characterized by the destruction of connective tissues, including alveolar bone, and it may eventually lead to tooth loss. Systemic or topical antibiotics have been employed as an adjunct in treating periodontal disease (10). A majority of the practicing periodontists in the United States prescribe antibiotics following periodontal surgery. Penicillin and tetracycline are among the most frequently prescribed antibiotics (10). Eikenella corrodens, Prevotella intermedia, and Prevotella nigrescens have been implicated as the causative agents of periodontitis (4). E. corrodens is a gram-negative facultative anaerobic rod that is occasionally found in high numbers in people with periodontal disease (3). This organism has also been identified either as the sole pathogen or as part of mixed microflora in various extraoral sites (2, 3). P. intermedia and P. nigrescens are part of a heterogeneous group of gram-negative obligate anaerobic bacteria formerly designated as black-pigmented Bacteroides (7–9). The two species are phenotypically difficult to distinguish and are commonly identified as the P. intermedia/ nigrescens group in primary culture. However, more recently developed 16S rRNA-based PCR methods can rapidly distinguish between these two microbial species (1, 11). The aims of this study were to examine the susceptibilities to amoxicillin and tetracycline among E. corrodens, P. intermedia, and P. nigrescens clinical isolates. We also evaluated the use of agar containing either amoxicillin or tetracycline in primary culture to help identify resistant isolates of P. intermedia and P. nigrescens. The incidence of -lactamase production was determined among P. intermedia and P. nigrescens isolates. The presence or absence of plasmid was also examined for P. intermedia and P. nigrescens isolates with resistance to antibiotics. One hundred six E. corrodens clinical isolates were examined. The isolates were collected between 1985 and 1999. Ninety-one of these isolates originated from various intraoral sites
* Corresponding author. Mailing address: DEN 4335, USC School of Dentistry, Division of Primary Oral Health Care, Los Angeles, CA 90089. Phone: (213) 740-1075. Fax: (213) 740-2194. E-mail: ccchen @hsc.usc.edu. 3253
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NOTES
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Antimicrobial susceptibilities of E. corrodens, P. intermedia, and P. nigrescens
Species (no. of isolates)
Medium used in primary culture
E. corrodens (106) P. intermedia (34)
—e Nonselective blood agar Nonselective blood agar Blood agar with amoxicillin Blood agar with amoxicillin Blood agar with tetracycline Blood agar with tetracycline
P. nigrescens (21) P. intermedia (21) P. nigrescens (9) P. nigrescens (3) P. intermedia (10)
Amoxicillin MIC (g/ml)a Range
0.03–0.75 0.016–8f ⬍0.016–8j 0.25–12f j
0.5–3
⬍0.016–2 0.016–8
Tetracycline MIC (g/ml)a
50%
90%
Susceptibility ratesb,c,d
0.25 0.016
0.5 1.5
100, 0, 0 68, 0, 32g
0.12–8 0.016–24h
1 0.25
2 12
92, 7, 1 79, 15, 6i
2
67, 0, 33k
0.023–24
0.125
12
67, 29, 5
4
0, 0, 100g
0.047–16
1.5
12
76, 14, 10
1
3
k
0, 0, 100
0.094–16
6
16
44, 44, 12
1.5
2
33, 33, 33
6
8
0, 100, 0
6
12
⬍0.016 2
0.38
6
40, 0, 60
Range
50%
90%
Susceptibility ratesb,c,d
6–8 h
1.5–16
40, 50, 10i
a
50% and 90%, MICs at which 50 and 90% of the isolates tested, respectively, are inhibited. Data represent the percentage of isolates with a MIC corresponding to the interpretive categories of susceptible, intermediate, and resistant, respectively. The interpretive categories for E. corrodens were adopted from the interpretive standards of ampicillin and tetracycline susceptibilities for Haemophilus spp. outlined by the National Committee for Clinical Laboratory Standards: MICs of ⱕ1, 2, and ⱖ4 g of ampicillin per ml and ⱕ2, 4, and ⱖ8 g of tetracycline per ml. d The interpretive categories for P. intermedia and P. nigrescens were adopted from the interpretive standards of ampicillin and tetracycline susceptibilities for anaerobic bacteria outlined by the National Committee for Clinical Laboratory Standards: MICs of ⱕ0.5, 1, and ⱖ2 g of ampicillin per ml. All -lactamase-positive isolates were considered resistant, and MICs of tetracycline were ⱕ4, 8, and ⱖ16 g/ml. e —, most of the E. corrodens isolates were recovered in primary cultures on blood agar plates with 1 g of clindamycin per ml. f,h,j Statistically significant difference between selective and nonselective medium at P ⬍ 0.01 by Mann-Whitney test. g,k P ⬍ 0.0001 by chi-square test between selective and nonselective medium. i P ⬍ 0.05 by chi-square test between selective and nonselective medium. b c
tems, Cockeysville, Md.). The presence of plasmid in P. intermedia and P. nigrescens isolates was evaluated by plasmid extraction with the QIAprep Miniprep kit (Qiagen, Inc., Valencia, Calif.) according to the manufacturer’s recommendation. Table 1 shows antimicrobial susceptibilities of E. corrodens, P. intermedia, and P. nigrescens. Because no interpretive criteria exist for amoxicillin or for E. corrodens, we adopted the interpretive categories of ampicillin and tetracycline susceptibilities for Haemophilus influenzae as described by the National Committee for Clinical Laboratory Standards (6). The interpretive categories of ampicillin and tetracycline susceptibilities for anaerobic bacteria outlined by the National Committee for Clinical Laboratory Standards (5) were used for P. intermedia and P. nigrescens. For all 106 E. corrodens isolates, the MICs of amoxicillin were 0.75 g/ml or less, and the isolates were categorized as susceptible (MIC of ⱕ1 g/ml) (Table 1). The susceptibility rates (number of isolates) of E. corrodens to tetracycline, categorized as susceptible, intermediate, and resistant (MICs of 2, 4, and 8 g/ml, respectively), were 98 (92%), 7 (7%), and 1 (1%), respectively. Our findings were in agreement with those of several studies showing sensitivity of E. corrodens to amoxicillin (2) and also indicated that the occurrence of tetracycline resistance among E. corrodens isolates is rare. Table 1 also shows the susceptibilities of P. intermedia and P. nigrescens isolates recovered from nonselective brucella blood agar (34 P. intermedia and 21 P. nigrescens isolates), selective brucella blood agar with amoxicillin (21 P. intermedia and 9 P. nigrescens isolates), and selective brucella blood agar with tetracycline (10 P. intermedia and 3 P. nigrescens isolates). Onethird of these nonselective P. intermedia and P. nigrescens isolates were resistant to amoxicillin (MIC of ⱖ2 g/ml, or -lactamase positive), and the remaining two-thirds were sus-
ceptible (MIC of ⱕ0.5 g/ml). The MICs of amoxicillin for P. intermedia (21 isolates) and P. nigrescens (9 isolates) recovered from selective agars containing amoxicillin were significantly higher than those for the corresponding species recovered from nonselective plates (P ⬍ 0.01; Mann-Whitney test). Of isolates recovered from nonselective agar, 27 of the 34 (79%) P. intermedia isolates and 14 of the 21 (67%) P. nigrescens isolates were susceptible to tetracycline (MIC of ⱕ4 g/ml). Five (15%) P. intermedia and six (29%) P. nigrescens isolates were of intermediate susceptibility to tetracycline (MIC ⫽ 8 g/ml). Resistance was found in the remaining two (6%) P. intermedia isolates and one (5%) P. nigrescens isolate (MIC of ⱖ16 g/ml). The MICs of tetracycline for P. intermedia (10 isolates) recovered from selective agar containing tetracycline were significantly higher than those for nonselective P. intermedia isolates (P ⬍ 0.01; Mann-Whitney test). The use of selective agar containing amoxicillin increased the detection rates of -lactamase among P. intermedia and P. nigrescens isolates from approximately one-third with nonselective agar (11 of 34 [32%] P. intermedia and 7 of 21 [33%] P. nigrescens isolates) to 100% with selective agar (21 of 21 P. intermedia and 9 of 9 P. nigrescens isolates) containing 1 g of amoxicillin per ml. Thirty-one P. intermedia isolates and 12 P. nigrescens isolates recovered from selective agar plates with amoxicillin or tetracycline were examined for the presence of plasmid. None of the isolates examined revealed a plasmid. In summary, the present study showed that 100% and 92% of the E. corrodens isolates were susceptible to amoxicillin and tetracycline, respectively. Of P. intermedia and P. nigrescens isolates recovered from nonselective agar, 67 to 68% were susceptible to amoxicillin, and 79% of P. intermedia isolates and 67% of P. nigrescens isolates were susceptible to tetracycline. Approximately one-third of the P. intermedia and P. nigrescens isolates recovered from nonselective agar plates
VOL. 45, 2001
were -lactamase positive. A higher percentage of -lactamase-producing isolates of P. intermedia and P. nigrescens were identified with selective agar containing amoxicillin than with nonselective agar. We thank Hannele Jousimies-Somer of the National Public Health Institute, Helsinki, Finland, for critical review of the manuscript. This work was supported in part by grant no. ROI DE 12212 from the National Institute of Dental and Craniofacial Research. REFERENCES 1. Ashimoto, A., C. Chen, I. Bakker, and J. Slots. 1996. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol. Immunol. 11:266–273. 2. Chen, C., M. L. Towns, and E. J. C. Goldstein. 1999. Eikenella corrodens. Williams and Wilkins, Baltimore, MD. 3. Chen, C., and M. E. Wilson. 1992. Eikenella corrodens in human oral and non-oral infections: a review. J. Periodontol. 63:941–953. 4. Haffajee, A. D., and S. S. Socransky. 1994. Microbial etiological agents of destructive periodontal diseases. Periodontology 2000 5:78–111.
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5. National Committee for Clinical Laboratory Standards. 1997. Methods for antimicrobial susceptibility testing of anaerobic bacteria, 4th ed. Approved standard. NCCLS document M11–A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. 6. National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard. NCCLS document M7–A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. 7. Paster, B. J., F. E. Dewhirst, I. Olsen, and G. J. Fraser. 1994. Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria. J. Bacteriol. 176:725–732. 8. Shah, H. N., and D. M. Collins. 1990. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int. J. Syst. Bacteriol. 40:205–208. 9. Shah, H. N., and S. E. Gharbia. 1992. Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov. Int. J. Syst. Bacteriol. 42:542–546. 10. Slots, J., and T. E. Rams. 1990. Antibiotics in periodontal therapy: advantages and disadvantages. Oral Microbiol. Immunol. 17:479–493. 11. Umeda, M., A. Contreras, C. Chen, I. Bakker, and J. Slots. 1998. The utility of whole saliva to detect the oral presence of periodontopathic bacteria. J. Periodontol. 69:828–833.
