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Fusobacterium nucleatum is one of the most frequently found anaerobic species in the oral cavity in early childhood (11). It is also commonly found in various infections in oral and nonoral sites. Pediatric infections in which F. nucleatum is involved are located mainly in the upper respiratory tract and the head and neck (5), suggesting an oral source. F. nucleatum is a heterogeneous bacterial group; its division into several subspecies has been made on the basis of electrophoretic patterns of enzyme mobilities or whole-cell proteins and on the basis of DNA-DNA homology (6, 7). However, F. nucleatum subspecies cannot be separated from each other by conventional biochemical testing alone. Differences in pathogenic potential among F. nucleatum subspecies have been reported (8, 27), indicating that the various subspecies may exhibit differences in such characteristics as -lactamase production. Except for one report of a -lactamase-producing Fusobacterium nucleatum subsp. polymorphum strain from the blood of a seriously ill patient (9), no data on -lactamase production by different F. nucleatum subspecies exist. The first reports of penicillin resistance due to -lactamase production by F. nucleatum were published in the mid-1980s (1, 3, 24). The frequency of -lactamase production by fusobacteria seems to be increasing (2). We have observed surprisingly high frequencies of -lactamase production by several anaerobic, gram-negative species in oral sites, especially by pigmented Prevotella spp., in infants and young children (13, 14, 19). In the present study, our aim was to examine -lactamase production among heterogeneous oral F. nucleatum populations isolated from young, healthy children. Secondly, by using cellular fatty acid (CFA) analysis for subspecies identification, we tried to determine if -lactamase production is characteristic only of a certain subspecies. Finally, the activity of potential alternative antimicrobials for F. nucleatum was determined.

Altogether, 123 F. nucleatum isolates originated from young, healthy children (11) from whom at least three simultaneous oral isolates were available. The children (10 boys and 10 girls ranging in age from 2 to 3.4 years) had not received systemic antimicrobial treatment within at least 1 month preceding the specimen collection (Table 1). The clinical F. nucleatum isolates, with various colony morphologies, were indole positive and lipase negative, produced butyric acid as a major metabolic end product, and did not convert lactate to propionate. F. nucleatum subsp. polymorphum ATCC 10953^T, Fusobacterium nucleatum subsp. nucleatum ATCC 25586^T, Fusobacterium nucleatum subsp. vincentii ATCC 49256^T, Fusobacterium nucleatum subsp. fusiforme NCTC 11326^T, Fusobacterium nucleatum subsp. animalis NCTC 12276^T, Fusobacterium periodonticum ATCC 33693^T (a closely related strain) were used as reference strains. The bacterial isolates were maintained in vials containing 20% sterilized skim milk at 70°C until further testing. An automatic CFA analysis, based on capillary column gas-liquid chromatography designed by the Microbial Identification System (MIS) (MIDI, Newark, N.J.) and with the Moore Broth Library database (versions 3.8 and 3.9) as a reference, was used as previously described (15) to presumptively identify the clinical F. nucleatum isolates to the subspecies level. A dendrogram (cluster analysis) was constructed for -lactamase-positive F. nucleatum isolates and all reference strains.

View this table: [in this window] [in a new window]   TABLE 1.   Characteristics of 20 children and the distribution of -lactamase-producing F. nucleatum subspecies among their oral F. nucleatum populationsa

-Lactamase production of all 123 clinical F. nucleatum isolates was examined by the qualitative chromogenic cephalosporin disk (AB Biodisk, Solna, Sweden) test (20). In vitro antimicrobial susceptibility to penicillin G, amoxicillin-clavulanic acid, tetracycline hydrochloride, metronidazole, trovafloxacin, and azithromycin was examined for all -lactamase-positive isolates and the corresponding number of -lactamase-negative isolates by using the National Committee for Clinical Laboratory Standards (NCCLS)-approved agar dilution method (18). MICs were determined in parallel on brucella blood agar and on fastidious anaerobe agar (FAA; Lab M Ltd., Bury, England), both supplemented with sheep blood, hemin, and vitamin K₁ (21). To aid the endpoint reading, a viability indicator dye, triphenyltetrazolium chloride (TTC) (21), was used for F. nucleatum isolates with hazy growth.

All F. nucleatum isolates produced major amounts of C_14:0, C_16:0, and C_16:1-cis-9. The identification provided by MIS was used for the presumptive identification of the clinical F. nucleatum isolates to the subspecies level. A dendrogram was constructed for the -lactamase-positive F. nucleatum isolates and all reference strains (Fig. 1). Most of the isolates clustered together with the indicated F. nucleatum type strains, but some clinical isolates formed a subcluster that did not concisely conform to the pattern of any type strain. For the latter isolates, similarity indices were less than 0.3 (the highest possible match is 1.0) and/or the differences between the primary and secondary identification choices by MIS were less than 0.1. 

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Dendrogram of -lactamase-producing F. nucleatum isolates and the type strains F. nucleatum subsp. polymorphum ATCC 10953T, F. nucleatum subsp. nucleatum ATCC 25586T, F. nucleatum subsp. vincentii ATCC 49256T, F. nucleatum subsp. fusiforme NCTC 11326T, F. nucleatum subsp. animalis NCTC 12276T, and F. periodonticum ATCC 33693T, generated by cluster analysis of CFA profiles.

Ten children (50%) harbored a total of 25 -lactamase-positive F. nucleatum isolates. Both -lactamase-positive and -lactamase-negative F. nucleatum strains were simultaneously isolated from 9 of 10 children. According to MIS, 16 of the -lactamase-positive isolates were identified as F. nucleatum subsp. polymorphum, 7 isolates were identified as F. nucleatum subsp. nucleatum, and 2 isolates were identified as F. nucleatum supsp. vincentii. The distribution of the -lactamase-producing subspecies within the group of children studied is seen in Table 1. Activities of several antimicrobials against -lactamase-positive F. nucleatum isolates showed similar patterns on both agar media; the MICs determined on brucella agar and FAA agreed with each other within 1 log₂ dilution. However, -lactamase-negative isolates frequently exhibited poor growth on brucella plates. Table 2 presents the in vitro activities of penicillin G, amoxicillin-clavulanic acid, tetracycline hydrochloride, metronidazole, trovafloxacin, and azithromycin against 25 -lactamase-producing and 26 non--lactamase-producing F. nucleatum isolates. MICs for the -lactamase-positive isolates ranged from intermediate susceptibility (one isolate; MICs of 1 and 2 µg/ml on FAA and brucella agar, respectively) to high resistance to penicillin G (MIC of 256 µg/ml). Except for two isolates from the same child for which the MIC was 1 µg/ml, -lactamase-negative isolates exhibited high susceptibility to penicillin G (MIC  0.03 µg/ml). Amoxicillin-clavulanic acid, tetracycline hydrochloride, metronidazole, and trovafloxacin had good activity against all F. nucleatum isolates. Azithromycin also proved to be effective against F. nucleatum, as only one strain for which MICs were 2 and 4 µg/ml on brucella agar and on FAA, respectively, was found.

View this table: [in this window] [in a new window]   TABLE 2.   In vitro activities of antimicrobial agents against -lactamase-producing and non--lactamase-producing F. nucleatum isolates

A surprisingly high frequency of -lactamase production by oral F. nucleatum was found in these young, healthy children, as half of them harbored -lactamase-producing isolates. No differences between children with and without -lactamase-positive F. nucleatum isolates with respect to gender, age, or preceding antimicrobial treatment were observed. -Lactamase production coincided well with penicillin resistance; the MICs of penicillin G on brucella agar varied from 2 to 256 µg/ml for -lactamase-positive isolates compared with the low MICs of penicillin G for -lactamase-negative isolates. Notably, as both -lactamase-producing and non--lactamase-producing variants can be simultaneously present in the mouth, several isolates per sample should be tested to determine the true rate of -lactamase production within a bacterial species. As with our previous experience with pigmented Prevotella species (13, 14), the multiple isolate testing may partly explain the high frequency of -lactamase production by F. nucleatum in young children observed in the present study.

CFA analysis under standardized conditions has proved to be a useful tool for the taxonomic characterization of anaerobic gram-negative bacilli (16). Tunér et al. (23) have successfully used CFA analysis to separate different Fusobacterium species. However, in differentiating F. nucleatum subspecies, an improved and expanded database that recognizes the National Collection of Type Cultures strains of F. nucleatum is needed. Another problem arises from the vast heterogeneity among F. nucleatum populations. The reported overlapping of subspecies (7) has caused uncertainty about whether three or four human subspecies exist. More recently, even the validity of the division of F. nucleatum into subspecies has been questioned (17). In the present study, CFA analysis was performed to presumptively identify the -lactamase-positive F. nucleatum isolates to the subspecies level. The majority of these isolates were identified as F. nucleatum subsp. polymorphum; however, some were also identified as F. nucleatum subsp. nucleatum and F. nucleatum subsp. vincentii, which in most cases clustered together with indicated type strains. Thus, -lactamase production seems not to be confined to one subspecies only but is shared by several F. nucleatum subspecies.

Special efforts are needed to test fusobacteria for their antimicrobial susceptibilities. Even by using an NCCLS-approved agar dilution method (18) that allows the addition of blood to the culture medium as an appropriate growth supplement for anaerobic bacteria, the problem of poor or hazy growth arises. "Tailing" of growth occurs due to cell wall-defective variants of Fusobacterium species (10). To solve the problem with exact end-point determination, we used TTC, a viability indicator dye, as an indicator to recognize the demarcation zone of viable growth. TTC has been successfully used to minimize inconsistency in interpreting endpoints for Bilophila wadsworthia (22), a fastidious anaerobic species with tendency to hazy growth on antimicrobial-containing media, a phenomenon identical to that seen with fusobacteria. Brazier et al. (4) compared different culture media for their capacity to support the growth of fusobacteria and found that the enriched culture medium designed especially for anaerobes, FAA, promoted the growth of fusobacteria and reduced their "tailing." In the present study, we accordingly determined the susceptibilities in tandem by using the NCCLS-recommended supplemented brucella agar and FAA. Both culture media were associated with nearly identical MICs for -lactamase-positive F. nucleatum isolates. However, we were repeatedly confronted with difficulties in determining MICs due to the poor growth of -lactamase-negative isolates on brucella agar. According to the previous (4) as well as present results, FAA favors the growth of fusobacteria and, conceivably, promotes the susceptibility testing of F. nucleatum.

In a recent study (14), the penicillin breakpoint of 0.5 µg/ml precisely separated -lactamase-producing Prevotella melaninogenica isolates from non--lactamase-producing isolates; the observation is in accordance with the latest breakpoint determination by NCCLS (18). In the present study, the lowest MIC measured on FAA was 1 µg/ml for -lactamase-producing F. nucleatum, but the MICs for two -lactamase-negative F. nucleatum isolates from one child (probably the same strain) were also 1 µg/ml. The MICs of amoxicillin-clavulanic acid, tetracycline hydrochloride, metronidazole, and trovafloxacin were unambiguously below the current susceptible breakpoints approved by NCCLS (18). Trovafloxacin, a novel fluoroquinolone, has shown promising in vitro activity against anaerobes (25, 26). In the present study, the MICs of trovafloxacin and also of metronidazole were similar to MICs for 28 F. nucleatum strains reported by Wexler et al. (26). Although no NCCLS breakpoint for azithromycin has been approved for anaerobes, the low MICs seen in this study indicate that azithromycin has better activity than other macrolides against F. nucleatum.

The clinical significance of fusobacteria in pediatric infections has recently been pointed out by Brook (5). In fact, F. nucleatum was the Fusobacterium species most frequently isolated from these infections. According to our preliminary experience, F. nucleatum might play a role in the pathogenesis of acute otitis media in infancy, as it was the anaerobic species most frequently isolated from the nasopharynx during bouts of ear infection (12). As seen in the present study, penicillin resistance due to -lactamase production by oral F. nucleatum occurs frequently in childhood. This phenomenon is not confined to F. nucleatum subsp. polymorphum but seems to be a characteristic of several other F. nucleatum subspecies. As beta-lactams are among the antimicrobials most commonly used in bacterial pediatric infections, routine -lactamase testing of multiple isolates from such infections could be of benefit.

In conclusion, the reported high resistance rates among oral anaerobic species in childhood can have a significant impact on the treatment practices and outcomes of pediatric infections that originate in the oral cavity.