Abstract
While most bacteria involved in dentoalveolar infection are highly susceptible to penicillin, some Prevotella strains exhibit resistance to this agent through the production of β-lactamase. The production of β-lactamase by Prevotella spp. is in turn associated with the expression of the genes cfxA and cfxA2. The aim of the present study was to determine the prevalence of cfxA and cfxA2 in Prevotella strains by use of real-time PCR and to assess the performance of this molecular method for the direct detection of the genes in 87 clinical samples (pus and root canal exudates) from dentoalveolar infection. Production of β-lactamase by each isolate was determined using a nitrocefin disk. β-Lactamase production was seen in 31% of Prevotella isolates, while all isolates of other species were β-lactamase negative. The penicillin resistance of isolates strongly correlated with the production of β-lactamase. Real-time PCR was found to detect the cfxA and cfxA2 genes from at least five cells per reaction mixture (5 × 103 CFU/ml of pus). Using real-time PCR, the presence of cfxA and cfxA2 was evident for all 48 β-lactamase-positive Prevotella strains. In contrast, neither β-lactamase-negative Prevotella (n = 91) or non-Prevotella (n = 31) strains were positive for the genes. In this study, 31 of the 87 samples yielded β-lactamase-positive Prevotella results, and cfxA and cfxA2 were detected in all 31 samples. Of the 56 culture-negative samples, 8 (14%) were positive for cfxA and cfxA2 by the real-time PCR. This sensitive and specific molecular method offers a rapid clinical test for aiding in the selection of an appropriate antibiotic for treatment of dentoalveolar infection. Although penicillin remains largely effective in the treatment of dentoalveolar infection, β-lactamase-stable antibiotics should be considered in cases in which β-lactamase-positive Prevotella strains are involved.
Despite the improvement of dental health in developed countries, patients with dentoalveolar infections are still encountered. The majority of infections are associated with necrotic dental pulp tissue, although periodontal diseases and pericoronitis may also provide a source of infection (2). Drainage, which can be achieved by tooth extraction, surgical incision, or root canal treatment, is the most important factor in treatment of dentoalveolar infections (9, 11, 23, 24). In addition to drainage, systemic antibiotics can be prescribed to prevent the spread of infection and onset of serious complications (9, 11, 23, 24). Members of the penicillin group of antibiotics have long been the first-line treatment for dental infections because of their suitable antimicrobial spectrum, bactericidal activity, low incidence of adverse effects, and cost-effectiveness (1, 9, 11, 12, 23, 24).
The antimicrobial susceptibility of bacteria involved in the infection is a primary factor affecting the likely outcome of antibiotic therapy (9, 10, 24). The presence of penicillin-resistant bacteria is implicated as the cause of clinical failure of treatment in some cases of oral purulent infection (10). As a consequence, information determining whether penicillin-resistant bacteria are involved is important in predicting the effectiveness of treatment with penicillin for dentoalveolar infection. Although antibiotic sensitivity can be determined from standard cultural microbiological analysis, this generally takes several days due to the slow growth of fastidious anaerobic bacteria. Since infection can spread rapidly and cause severe complications such as sepsis and obstruction of the airway, such a delay can prove problematic and undesirable.
The introduction of PCR-based techniques has resulted in the development of tests that can detect specific pathogens and genes directly and rapidly from clinical samples. Indeed, conventional PCR has already become an important tool in clinical diagnostic and research laboratories. Recently, a more rapid, sensitive, and reproducible PCR has been described and is termed real-time PCR (18). This approach also allows the quantitative assessment of target nucleic acids.
Dentoalveolar infection usually involves bacteria residing in the oral cavity. In particular, strict anaerobes such as Peptostreptococcus, Prevotella, and Fusobacterium spp. and oral streptococci are the predominant isolates from the infection (9, 11-15, 23, 24). It has been demonstrated that the majority of these bacteria are highly susceptible to penicillin whereas some Prevotella strains have recently been reported as being resistant (9, 11-17, 24). Penicillin resistance of Prevotella strains is closely associated with the production of β-lactamase, which is an enzyme that degrades β-lactam agents (12-15, 24). β-Lactamase produced by Prevotella strains has the properties of class A-group 2e β-lactamases, which hydrolyze most penicillins and broad-spectrum cephalosporins but are inactive with respect to penicillin combined with clavulanic acid and imipenem (3). Although the genetic basis of β-lactamase-production by Prevotella strains has not been clarified completely, it has been demonstrated that the cfxA2 gene is associated with β-lactamase production (4, 6, 19). This gene shares 98% identity with cfxA, the structural gene of a β-lactamase produced by Bacteroides vulgatus (19).
The aim of this study was to determine the prevalence of cfxA and cfxA2 in Prevotella strains by use of real-time PCR and to assess the performance of this approach for direct gene detection in clinical samples from dentoalveolar infection.
MATERIALS AND METHODS
Clinical samples.
A total of 87 clinical samples were obtained from patients with dentoalveolar infection (53 males, 34 females; mean age, 48.1 years) attending the Oral and Maxillofacial Surgery Clinic of Kanazawa University Hospital between September 2001 and March 2005. Each of the samples obtained was recovered from a distinct and individual patient. A total of 72 pus samples (yellow thick pus, 50 samples; blood-like pus, 22 samples) were taken by aspiration from the abscess with a disposable syringe with an 18-gauge needle. Each sample was stored at −80°C prior to analysis by real-time PCR, with a portion immediately used for cultural bacterial examination. The remaining 15 samples were pus-like exudates from the root canal of the tooth involved. These samples were obtained using three sterilized paper points (size 25). Each sample held within the paper points was suspended in 100 μl of saline solution by vigorous vortexing for 20 s. A 20-μl volume of the suspension was cultured, and the remainder was stored at −80°C prior to DNA extraction.
Bacteriological culture examination.
Samples were inoculated aerobically and microaerophilically on brucella HK agar (Kyokuto, Tokyo, Japan) containing 5% (vol/vol) sheep blood for 48 h at 37°C. Culture of strict anaerobes was on brucella HK agar with 5% (vol/vol) sheep blood, which was incubated in an anaerobic atmosphere for up to 7 days at 37°C. Duplicate plates were prepared containing paromomycin (Pfizer, Tokyo, Japan) (75 mg/liter) and vancomycin (Shionogi, Osaka, Japan) (2.5 mg/liter) to selectively isolate strictly anaerobic gram-negative bacilli. Isolates were identified by conventional methods (20). Bacterial growth was recorded to determine the approximate number of bacteria in each sample. After identification, a colony of each isolate was stored at −80°C.
β-lactamase production and antimicrobial susceptibility.
A nitrocefin disk (Cefinase; Becton Dickinson, Cockeysville, Md.) was used to determine whether strains were positive for β-lactamase production (12-14). Susceptibility of 242 randomly chosen clinical isolates (Prevotella spp., 139 isolates; Fusobacterium, 33; Peptostreptococcus, 23; Campylobacter, 13; Porphyromonas, 12; Corynebacterium, 4; Veillonella, 7; Streptococcus, 5; Bacteroides, 3; Bifidobacterium, 1; Gemella, 1; Lactobacillus, 1) to penicillin G was determined by a disk diffusion method approved by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) (22). In addition, MICs of Prevotella isolates for amoxicillin (Astellas, Tokyo, Japan) and amoxicillin-clavulanate (GlaxoSmithKline, Middlesex, United Kingdom) were determined by the agar dilution method recommended by CLSI (21). Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741 were used as quality-control strains in each test. The resistance breakpoints for amoxicillin and amoxicillin-clavulanate were determined to be 2 and 16 μg/ml, respectively, based on the CLSI guidelines (21).
Extraction of DNA from bacteria.
Tested strains were subcultured on the brucella HK agar containing 5% (vol/vol) sheep blood for 48 h at 37°C, and resulting colonies were suspended in distilled water (100 μl) at a concentration equivalent to a MacFarland 1.5 standard. The suspension was heated to 96°C for 10 min and chilled to 4°C for 5 min, and the cell debris was removed by centrifugation. The supernatant was used as a template for PCR.
Extraction of DNA from clinical samples.
A 50-μl volume of pus was suspended in 50 μl of distilled water and treated as described above. For root canal exudates, the suspension (100 μl) was centrifuged at 12,000×g for 10 min and the supernatant (80 μl) was discarded. The concentrate (20 μl of the remainder) was then used for DNA extraction. This concentration procedure was also employed for blood-like pus samples.
Real-time PCR.
The primers and the TaqMan probe were designed using cfxA and cfxA2 gene sequences (GenBank accession no. U38243 and AF11810, respectively) and Primer Express software version 2 (Applied Biosystems, Foster, CA). The nucleotide sequence of the forward primer was 5′-GCGCAAATCCTCCTTTAACAA-3′ (KAG309), and the reverse primer sequence was 5′-ACCGCCACACCAATTTCG-3′ (KAG310). The sequence of the real-time PCR probe was 5′-TGATAGCATTTCTCAAATTGTCTCAGCTTGTCC-3′ (cfxA Taq Pr). The cfxA Taq Pr was 5′ end labeled with 6-carboxyfluoscein (FAM) as the reporter dye and 3′ end labeled with 6-carboxytetramethylrhodamine (TAMRA) as the quencher. The primers and probe were selected from a region with 100% nucleotide identity between cfxA and cfxA2. Although amino acid substitutions of cfxA and cfxA2 have been reported (6), the target regions for PCR amplification have not been found to differ in sequence. The real-time PCR was performed in a 25-μl final volume containing 12.5 μl of Premix Ex Taq (Perfect Real Time; Takara, Kyoto, Japan), 2 μl of DNA template, 0.5 μl of 10 μM KAG309 and KAG310 primers and 0.5 μl of 10 μM cfxA Taq Pr probe at a final concentration of 0.2 μM, 0.5 μl of ROX reference dye (50×), and 8.5 μl of distilled water. All reactions were run on an ABI Prism 7000 sequence detection system (Applied Biosystems) in triplicate with the following cycling parameters: 95°C for 10 s followed by 40 cycles of 95°C for 10 s and 60°C for 31 s.
Analytical specificity and sensitivity (detection limit).
Ten CfxA and CfxA2 β-lactamase-positive and 10 β-lactamase-negative Prevotella strains, which had been stored in our laboratory, were examined to confirm specificity of real-time PCR. β-Lactamase production by all these strains had previously been confirmed using the nitrocefin disk, antimicrobial susceptibility tests, and the presence of cfxA and cfxA2 by PCR as described previously (4) using primers 5′-GCAAGTGCAGTTTAAGATT-3′ and 5′-GCTTTAGTTTGCATTTTCATC-3′. In addition to these Prevotella strains, clinical isolates of Porphyromonas gingivalis (n = 12), Peptostreptococcus micros (n = 20), Fusobacterium nucleatum (n = 36), Campylobacter gracilis (n = 7), and Veillonella sp. (n = 6) were also examined. All these strains were β-lactamase negative.
Quantified dilutions of four β-lactamase-positive Prevotella strains (P. intermedia, 2 strains; P. melaninogenica, 1; P. buccae, 1) were prepared by measuring the number of CFU. A randomly selected pus sample was confirmed to be cfxA and cfxA2 negative and β-lactamase negative using the real-time PCR, conventional PCR (4), and culture examination. A 20-μl portion of each bacterial dilution was mixed with same volume of the pus. Bacterial DNA extraction was done as described above.
Ethics for study.
This study was approved by the Ethics Committee of Kanazawa University Graduate School of Medical Science.
RESULTS
Growth of bacteria from samples.
Bacterial growth was found in all clinical samples. Half of the thick pus samples, 4 of 22 blood-like pus samples, and 14 of 15 pus-like exudates from root canal yielded heavy bacterial growth under anaerobic conditions. The remaining samples produced limited bacterial growth.
Bacteriology, β-lactamase production, and penicillin disk susceptibility.
The results of cultural bacteriological examination are presented in Table 1. Prevotella, Peptostreptococcus, Fusobacterium, and Campylobacter spp. were predominantly isolated from the infections. Of the Prevotella isolates, 48 (31%) were found to be positive for β-lactamase; furthermore, there were no non-Prevotella β-lactamase-positive isolates detected. The penicillin disk susceptibility data was 100% concordant with the results of β-lactamase production testing (data not shown).
TABLE 1.
Identification and β-lactamase production by isolates from dentoalveolar infection
| Species | No. of β-lactamase-positive strains/total no. of isolates (%) |
|---|---|
| Prevotella intermedia-P. nigrescens | 12/39 (31) |
| Prevotella melaninogenica | 9/21 (43) |
| Prevotella denticola | 1/9 (11) |
| Prevotella loescheii | 5/23 (22) |
| Prevotella oralis | 8/26 (31) |
| Prevotella buccae | 8/24 (33) |
| Prevotella oris | 5/12 (42) |
| Prevotella bivia | 0/1 |
| Fusobacterium nucleatum/necrophorum | 0/66 |
| Peptostreptococcus micros | 0/55 |
| Peptostreptococcus prevotii | 0/4 |
| Peptostreptococcus anaerobius | 0/2 |
| Porphyromonas gingivalis | 0/16 |
| Porphyromonas endodontalis | 0/3 |
| Porphyromonas asaccharolytica | 0/3 |
| Campylobacter gracilis | 0/33 |
| Campylobacter rectus | 0/10 |
| Bacteroides capillosus | 0/7 |
| Gemella morbillorum | 0/3 |
| Propionibacterium acnes | 0/1 |
| Streptococcus sp. | 0/27 |
| Corynebacterium sp. | 0/14 |
| Veillonella sp. | 0/13 |
| Staphylococcus sp. | 0/4 |
| Lactobacillus sp. | 0/3 |
| Eubacterium sp. | 0/2 |
| Capnocytophaga sp. | 0/2 |
| Neisseria sp. | 0/2 |
| Actinomyces sp. | 0/1 |
| Bifidobacterium sp. | 0/1 |
| Unidentified anaerobic gram-negative bacilli | 0/12 |
| Unidentified aerobic gram-negative bacilli | 0/12 |
Analytical specificity and sensitivity of real-time PCR.
Real-time PCR confirmed the presence of the cfxA and cfxA2 genes for all 10 β-lactamase-positive and none of the 10 β-lactamase-negative Prevotella strains. This gene was not detected by real-time PCR in any strains of Porphyromonas, Peptostreptococcus, Fusobacterium, Campylobacter, or Veillonella. Using “spiked” pus, real-time PCR revealed a detection limit of 5 × 103 bacterial CFU/ml, corresponding to 5 CFU/reaction mixture, for three Prevotella strains tested. For one Prevotella strain, the detection limit was 1 ×103 bacterial CFU/ml of pus.
Prevalence of cfxA and cfxA2 in Prevotella species and the MIC for amoxicillin-clavulanate.
Although a total of 155 Prevotella isolates were recovered in this study, 16 strains that were stored by freezing were not subsequently recovered by culture for the PCR study. Therefore, to determine the prevalence of cfxA and cfxA2 in Prevotella isolates, a total of 139 clinical strains were examined. In this study, 48 (100%) of the β-lactamase-positive Prevotella strains were found to be positive for cfxA and cfxA2 by real-time PCR (Table 2). In contrast, all β-lactamase-negative strains were negative for the PCR. The β-lactamase-positive strains were highly susceptible to amoxicillin-clavulanate, although resistance to amoxicillin was recorded for all of the strains (Table 2). β-Lactamase-negative strain results showed low MICs for both amoxicillin and amoxicillin-clavulanate, with only one strain identified as having low-level resistance to amoxicillin (MIC, 2 μg/ml).
TABLE 2.
Prevalence of cfxA and cfxA2 and antimicrobial susceptibility in Prevotella strains isolated from dentoalveolar infections
| Isolate category | No. of tested strains | No. of cfxA- and cfxA2-positive strains | Amoxicillin
|
Amoxicillin-clavulanate
|
||||
|---|---|---|---|---|---|---|---|---|
| MIC50 (μg/ml) | MIC90 (μg/ml) | Resistance (%)a | MIC50 (μg/ml) | MIC90 (μg/ml) | Resistance (%)a | |||
| β-Lactamase positive | 48 | 48 | >64 | >64 | 100 | 2 | 4 | 0 |
| β-Lactamase negative | 91 | 0 | 0.06 | 0.25 | 1 | 0.06 | 0.25 | 0 |
Resistance rate. Breakpoints for amoxicillin and amoxicillin-clavulanate resistance results were 2 and 16 μg/ml, respectively.
Direct detection of cfxA and cfxA2 from clinical pus samples by real-time PCR.
Table 3 presents the agreement between cultural examination and PCR with respect to detection of β-lactamase-positive bacteria. Cultural bacteriological examination revealed that 31 (36%) of 87 clinical samples had β-lactamase-positive strains. Using the real-time PCR, cfxA and cfxA2 were detected for all these 31 β-lactamase-positive samples. Additionally, the real-time PCR also detected the resistance gene in 8 (14%) of the 56 samples deemed negative for β-lactamase by culture. Of these eight samples, six yielded β-lactamase-negative Prevotella results, and no Prevotella isolates were recovered from the remaining two samples.
TABLE 3.
cfxA and cfxA2 detection from clinical pus samples by real-time PCR
| Culture result | No. of samples | No. (%) of samples with indicated cfxA-cfxA2 result
|
|
|---|---|---|---|
| Positive | Negative | ||
| β-lactamase positive | 31 | 31 (100) | 0 |
| β-lactamase negative | 56 | 8 (14)a | 48 (86) |
Two of the eight samples did not yield Prevotella isolates by culture.
DISCUSSION
Due to improvement of culture and sampling techniques, recent studies demonstrate that dentoalveolar infections consist predominantly of strict anaerobes (9, 11-15, 23, 24). Our bacteriological investigation supports the results of these studies.
The nitrocefin method is capable of detecting β-lactamases produced by almost all bacterial species and has been commonly used in clinical laboratories (20). In this study, the detection of β-lactamase was limited to Prevotella strains (Table 1). Other studies have revealed that β-lactamase-positive strains of facultative anaerobic bacteria such as staphylococci and non-Prevotella anaerobes are also isolated from dental infections (4, 7, 16). However, even in these previous studies, the incidence of such bacteria and the prevalence of β-lactamase-positive strains in these species were very low. Consequently, with regard to β-lactamase production, Prevotella spp. would appear be the most important pathogen in dentoalveolar infection.
It has been reported that cfxA and cfxA2 occur in 100% of β-lactamase-positive Prevotella strains from American and Norwegian patients with periodontal disease (8). French investigators have also demonstrated a 100% prevalence of the resistance genes in β-lactamase-positive Prevotella strains (6). Our results are in good agreement with these studies and indicate that almost all β-lactamase-positive Prevotella strains are positive for cfxA and cfxA2. This would further suggest that detection of cfxA and cfxA2 by the molecular method is a useful indicator in determining the presence of β-lactamase-producing Prevotella strains. In addition, although β-lactamase-positive Prevotella strains are considered to produce CfxA and CfxA2 variants with minor nucleotide substitutions (6), the real-time PCR parameters, including the primers and probe employed in the present study, appeared to detect almost all types of cfxA and cfxA2 with high specificity and sensitivity.
There are various chromosome-encoding and plasmid-mediated genes associated with β-lactamase production (8). However, it has been demonstrated that no Prevotella strain harbors TEM, SHA, OHA, AmpC, and CF β-lactamase genes (8), and very few strains are positive for cepA and cblA (4). Together with these reports, our results showing cfxA and cfxA2 prevalence and drug MICs for Prevotella isolates support the idea that Prevotella β-lactamases are class A-group 2e β-lactamases (3) and that CfxA and CfxA2 are the most important and widely distributed β-lactamases produced by Prevotella strains.
In this study, PCR-positive results were obtained in 100% of clinical samples that were positive for β-lactamase by culture (Table 3). In contrast, 14% of the culture-negative samples were positive by PCR. If culture is considered the gold standard, it can be calculated that the diagnostic sensitivity and specificity of the real-time PCR are 100% and 86%, respectively, thus indicating the high performance of the real-time PCR for direct detection of β-lactamase-positive Prevotella strains from clinical samples. Nevertheless, a question about the significance of culture-negative PCR-positive samples and clinical relevance could be raised. As it is likely that real-time PCR is more sensitive for the detection of target bacteria than cultural examination, it is perhaps not surprising that the PCR detected the resistance genes from the samples containing low numbers of β-lactamase-positive Prevotella bacteria. It is also possible that the resistance genes would be detected by PCR from β-lactamase-positive Prevotella strains exhibiting highly fastidious growth on the culture media. In these circumstances, the improved sensitivity of the real-time PCR would be clinically advantageous.
In clinical laboratories, routine bacteriological investigation generally includes semiquantitation evaluating bacterial growth on agar. It has previously reported that multiple strains of the same species can be isolated from the same individual (5), and indeed, in this study, some clinical samples yielded both β-lactamase-positive and -negative strains of Prevotella species. It was therefore difficult to determine whether the cycle threshold (Ct) value accurately reflected the number of cfxA- and cfxA2-positive bacteria in sample. However, we confirmed that the Ct value was roughly correlated with the count of colonies in cfxA- and cfxA2-positive samples that yielded a single phenotype of suspected Prevotella colonies (data not shown). This suggests the applicability of real-time PCR for quantitative detection of β-lactamase-positive Prevotella strains in the clinical samples, although further study is necessary.
Penicillin, and in particular amoxicillin, has been the first-line antibiotic in the treatment of acute dentoalveolar infection (1, 9, 11, 23, 24). However, the incidence of penicillin resistance has brought into question the appropriateness of penicillin in the management of dental infections (24). In this study, only Prevotella strains exhibited resistance to penicillin, and the penicillin resistance was completely concordant with β-lactamase production, confirming the penicillin resistance mechanism of Prevotella spp. (9, 14, 24). These results support the continuance of penicillin use as the first-line antibiotic therapy and the proposal that Prevotella spp. remain highly significant pathogens with respect to treatment with penicillin (25).
The present report has highlighted the high performance of real-time PCR for the detection of cfxA and cfxA2 in clinical samples of dentoalveolar infection. This molecular method could provide a useful and rapid clinical test for aiding the selection of antibiotic for therapy. Some antibiotics, such as amoxicillin-clavulanate, cefmetazole, clindamycin, and metronidazole, have been demonstrated to be effective for treatment of β-lactamase-positive Prevotella infections (12, 14, 16, 17). These should be considered in circumstances where involvement of β-lactamase-positive Prevotella spp. is indicated. Since Prevotella spp. are also implicated in other oral and nonoral infections, such as periodontitis, osteomyelitis, sinusitis, and sepsis (4, 7, 23), this molecular diagnostic method may also prove of value in aiding treatment of these infections.
Acknowledgments
We thank E. Yamamoto (Kanazawa University) for his support. We also acknowledge Y. Ohta, T. Jyozen, M. Mori, S. Takai, H. Araki (Kanazawa University), and all technical staff members of Ishikawa Prefectural Institute of Public Health and Environmental Science for their kind cooperation.
This work was supported by a grant from the Japan Society for the Promotion of Science (Grant-in-aid for young scientists 177914443).
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