1 Anaerobe Reference Laboratory, Department of Microbiology and 3 Department of Vaccines, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki; 4 Department of Education, University of Helsinki, PO Box 39, FIN-00014 Helsinki, Finland; 2 Department of Surgical Sciences, Kuwait University, PO Box 24923, Safat 13110, Kuwait
Received 13 May 2002; returned 2 September 2002; revised 24 September 2002; accepted 26 September 2002
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Abstract |
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Keywords: Fusobacterium nucleatum, in vitro susceptibility, MIC, penicillin resistance, ß-lactamase production
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Introduction |
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Fusobacterium nucleatum is a strictly anaerobic, Gram-negative organism, which naturally colonizes the oral cavity in early childhood.4 Generally, fusobacteria seen in paediatric infections include F. nucleatum, Fusobacterium necrophorum, Fusobacterium gonidiaformans, Fusobacterium naviforme, Fusobacterium mortiferum and Fusobacterium varium.6 Of these, F. nucleatum is most often reported to produce ß-lactamase, which is known to be a penicillinase.7 F. nucleatum is a heterogeneous species, and at least three subspecies seem to produce ß-lactamase.2,8 Among the oral commensal flora, both ß-lactamase-producing and -non-producing variants of F. nucleatum can be present simultaneously.2 Several subspecies and strains can colonize the oral cavity, therefore as many isolates as possible per sample should be examined to demonstrate the true rate of resistance among this heterogeneous species.
Strain turnover in the developing anaerobic commensal flora occurs frequently.9 In addition, selection of bacterial populations after antimicrobial exposure has been demonstrated.10 Increased contact with other children in day care, age under 2 years and having older siblings have been shown to be predictive risk factors for resistant respiratory pathogens.11,12 Children are one of the major consumers of antimicrobial agents, due to the frequent occurrence and treatments of acute otitis media.13,14 Our previous study3 of ß-lactamase production among oral Gram-negative anaerobes during the first year of life, which included some part of this study material, raised the question of the level of penicillin resistance.
F. nucleatum was chosen as a representative for the present, more detailed study where the aims were to longitudinally examine the emergence of ß-lactamase-producing strains in the oral cavity and to determine the level of penicillin resistance of these isolates collected from saliva of infants during their first 2 years of life. The positive findings during the follow-up period were compared with infants age, day care attendance, sibling history and antibiotic exposure.
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Materials and methods |
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At baseline, 50 healthy 2-month-old infants were chosen for a detailed bacteriological investigation of the composition of early oral microflora.4 The infants, constituting a subpopulation of the Finnish Otitis Media (FinOM) Cohort Study, were followed up to 2 years of age in a study clinic at scheduled visits. In addition, if an infant became sick between these visits, the infection was diagnosed and treated in the same clinic.15 The present study consists of 44 infants who attended at least four of the five scheduled visits. Information about their siblings, day care attendance and usage of antimicrobial agents was included in the statistical analysis.
During the scheduled visits at 2, 6, 12, 18 and 24 months of age, unstimulated saliva samples were collected and cultured as previously described in detail.4 A minimum of five F. nucleatum isolates per infant on each sampling occasion was the target to be isolated and, if available, additional isolates were collected. The identification of F. nucleatum was mainly based on anaerobic growth, cell morphology in Grams stain (spindle-shaped Gram-negative bacillus), special potency antimicrobial disc profiles (susceptible to kanamycin and colistin, and resistant to vancomycin) and positive indole but negative lipase reactions.16 The isolates were stored at 70°C in vials containing 20% sterilized skimmed milk until further testing.
Testing of the ß-lactamase activity
A total of 1492 F. nucleatum isolates were revived from frozen stocks and tested for their ß-lactamase production using a chromogenic cephalosporin test.17 Results were read after 15, 30 and 60 min.
Susceptibility testing
In vitro antimicrobial susceptibility to penicillin for the ß-lactamase-positive isolates was determined as recommended by the NCCLS agar dilution method.18 MICs were determined in parallel on brucella agar and on fastidious anaerobe agar (FAA; Lab M Ltd, Bury, UK), both supplemented with 5% sheep blood, haemin and vitamin K1.16 Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, Eggerthella lenta ATCC 25559, F. nucleatum subsp. fusiforme NCTC 11326, F. nucleatum subsp. nucleatum ATCC 25586, F. nucleatum subsp. polymorphum ATCC 10953, F. nucleatum subsp. vincentii ATCC 49256 and Fusobacterium periodonticum ATCC 33693 were used as reference strains. Incubations in anaerobic jars filled with a gas mixture (80% N2, 10% H2, 10% CO2) were carried out at 36°C for 2 days.
Statistics
Statistical analysis of the distributions in the data and the associations between penicillin-resistant isolates and infants age, day care attendance, number of siblings and their ear infections, and exposure to antimicrobial agents was performed by SPSS Windows Version 10. The initial screening was conducted using frequencies, cross-tabulation and correlation procedures. The data matrix consisted of 44 cases. The main variable was the resistance in each of the time points (in form yes/no) and the frequency was positively skewed. It was winsorized (the tail was cut) as necessary for the statistical procedures. The rest of the variables described the stationary background variables of the family and the changing conditions in the family (such as sibling infections or day care status) between the study points. All the procedures were bivariate in their nature. Associations were examined using both parametric and distribution-free techniques. The association reported is based on the odds ratio technique (bivariate binary logistic regression). The analyses were carried out so that time point data were stacked to form a matrix with n = 216 (missing values lower than the theoretical 5 x 44). This approach was used to give more power to the analysis.
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Results |
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The overall prevalence of F. nucleatum in infants mouths increased from 11% to 93% during the follow-up period (Table 1). Less than five isolates per infant were available as follows: 5/5 infants at 2 months of age, 13/23 at 6 months, 13/38 infants at 12 months, 5/41 at 18 months and 1/40 at 24 months. In 90% of the cases, multiple F. nucleatum isolates per infant were available for ß-lactamase testing (Table 1).
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The prevalence of infants harbouring ß-lactamase-producing F. nucleatum isolates increased with age, from 1/44 (2%) at 2 months up to 21/43 (49%) at 24 months (Table 1). Of the 1492 isolates examined, 276 (18%) were ß-lactamase producers. In 30 infants ß-lactamase-producing isolates were found at least once during the study period. In 96% of these cases ß-lactamase-negative F. nucleatum isolates were found simultaneously (data not shown). In 13 infants ß-lactamase-producing isolates were found once, in 10 infants twice and in seven infants on three different sampling occasions. When ß-lactamase-producing F. nucleatum were found during the follow-up, in 13/17 (76%) of the cases they were in successive samplings.
MICs for ß-lactamase-producing F. nucleatum
The distribution of the 276 ß-lactamase-producing F. nucleatum isolates (including multiple ß-lactamase-producing isolates available from most study infants) into four different categories according to their MIC of penicillin is presented in Table 2. Only 2% of the isolates clustered near the NCCLS susceptibility breakpoint of 0.5 mg/L.18 Most of the ß-lactamase-producing isolates (80%) were clearly resistant to penicillin G (MIC 8 mg/L). For all ß-lactamase-producing isolates MIC50 was 16 mg/L and MIC90 64 mg/L. The MICs determined on brucella agar and FAA agreed with each other.
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Twenty-four of the 44 infants had one or more siblings. Nine infants had siblings of school age, nine had siblings who attended day care and 15 had siblings under school age who stayed at home. Fourteen infants had siblings with ear infections during the study period. Fifteen infants attended day care some time during the follow-up. No correlation (irrespective of siblings age grouping <5 or 5 years) between these factors and the incidence of penicillin-resistant F. nucleatum isolates was found.
Use of antimicrobial agents
During the follow-up, 36 infants (82%) had received 141 courses of antimicrobial agents. The main indication for the use of antimicrobials was an upper respiratory tract infection (mainly acute otitis media), which was diagnosed 87 times. For 14 antimicrobial courses the diagnosis remained unknown. ß-Lactams were the most commonly prescribed antimicrobial courses: 81 amoxicillin courses, nine co-amoxiclav courses, four cephalosporin courses and one course of penicillin V. In addition, three azithromycin courses were used and, usually as a secondary course after initial ß-lactam therapy, sulphonamides combined with trimethoprim were used 43 times. Eight infants received no antibiotics during the follow-up period. Antimicrobials were consumed on 29 occasions over a 30 day period prior to the study sampling. Taking this fact separately, no statistically significant association could be shown between the consumption of antimicrobial agents just before the sampling and the presence of penicillin-resistant isolates.
One antimicrobial agent was sufficient to treat the infection in 53 cases (61%), but two or more courses were needed in 34 cases (39%). Additionally, 12 infants had been exposed to antibiotics via their mothers over a 6 month period before the scheduled visit at 2 months. The logistic regression analysis showed a significant association (P = 0.044) between the amount of consumed antimicrobial agents (both ß-lactams and sulphonamides) during the first year of life and the presence of resistant F. nucleatum at 12 months of age. With a 95% confidence interval, the odds ratio was 1.50 (1.002.24).
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Discussion |
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Fusobacteria are fastidious and a special effort is needed to test their antimicrobial susceptibilities. Tailing of growth often occurs due to cell-wall-defective variants of Fusobacterium species.19 In the present study, dealing with ß-lactamase-producing F. nucleatum strains, tailing or poor growth did not appear. Brazier et al.20 found that the enriched culture medium FAA, designed especially for fastidious anaerobes, promoted the growth of fusobacteria and, in addition, reduced their tailing. We determined the susceptibility of ß-lactamase-producing F. nucleatum to penicillin in parallel on supplemented brucella agar and FAA, and the growth on both media agreed with each other. The susceptibility breakpoint of 0.5 mg/L separated ß-lactamase-producing F. nucleatum isolates from ß-lactamase-non-producing-isolates, which is in accordance with the breakpoint determination by the NCCLS.18
The significance of ß-lactamase production present within the normal flora is unknown. However, F. nucleatum, a component of the mouths commensal flora,21 is one of the potential anaerobic participants in mixed infections of the upper respiratory tract and in other sites of the body in children.6,2224 Anaerobes seem to be absent in the nasopharynx during health,25 but F. nucleatum and the Prevotella melaninogenica group of organisms can be frequently isolated from the nasopharynx during acute otitis media.26 These findings may indicate an oral source for nasopharyngeal anaerobes. Interestingly, nasopharyngeal F. nucleatum isolated during acute otitis media episodes may produce ß-lactamase.27 Previously, Brook & Gober23 have reported ß-lactamase-producing F. nucleatum isolates from nasopharyngeal samples.
Multiple F. nucleatum isolates per subject were tested for their ß-lactamase production in a recent cross-sectional study2 on different F. nucleatum subspecies present in the oral cavity. The test detected penicillin-resistant F. nucleatum strains in 50% of the examined healthy children. This finding in another group of Finnish children aged 23.4 years is in line with the present results. These prevalence rates for oral F. nucleatum isolated from young children are higher than reported in previous studies on clinical specimens.20,2830 In an earlier study by Könönen et al.1 great strain variation with different susceptibilities to penicillin was demonstrated within the populations of another heterogeneous commensal of the mouth, P. melaninogenica. Therefore, multiple isolates, whenever available, should be tested to demonstrate the true rate of penicillin resistance among oral commensal populations.
Tunér et al.7 found that 20% of the ß-lactamase produced by F. nucleatum is extracellular. In the case the enzyme is secreted into tissues and MICs of penicillin are as high as demonstrated in the present study, even non-pathogenic strains, with the ability to produce ß-lactamases, may be of concern when protecting otherwise penicillin-susceptible pathogens present in the same microenvironment.31 It is also noteworthy that resistant strains present in the commensal flora offer a reservoir of resistance genes potentially transferable to pathogens.32,33 On the other hand, one mechanism behind the significance of anaerobic bacteria in colonization resistance34 may be the capability of some strains to produce ß-lactamases. It is possible that ß-lactamase-producing strains of F. nucleatum, among other ß-lactamase-producing oral anaerobes,1,3 could block the overgrowth of harmful microorganisms and maintain the equilibrium in the oral commensal flora despite occasional courses of ß-lactams. Indeed, among intestinal anaerobic microflora derepressed mutants capable of enhanced ß-lactamase production are selected as a result of ß-lactam administration.35
Among intestinal bacteria, several research groups have demonstrated increasing frequencies of antibiotic resistance in healthy, hospitalized and community subjects of different age groups.3638 Although induction of ß-lactamase production by anaerobic species may occur,39 the selection of ß-lactamase-producing subgroups among anaerobic bacterial populations most probably explains the decreased susceptibility during and after antibiotic administration. In the present study, recurrent courses of antibiotics partly explain the high incidence of ß-lactamase-producing F. nucleatum. However, some examined infants without any history of the use of antimicrobial agents harboured ß-lactamase-producing F. nucleatum strains, indicating the possibility of transmission between individuals (mother, siblings) as a source of penicillin-resistant strains. We probably failed to demonstrate the possible transmission due to the limited number of subjects in the present study population.
In conclusion, the carriage rates of penicillin-resistant F. nucleatum strains increased with age and usage of antimicrobial agents during the first year of life. To demonstrate the plausible route from the mouth to nasopharynx, some of the oral F. nucleatum isolates included in the present study and nasopharyngeal F. nucleatum isolates, when available, from the infants with acute otitis media episodes were compared at clonal level.40 Such a bacterial translocation seems to occur frequently, indicating the involvement of oral anaerobic commensals, in some instances ß-lactamase-producing strains,27 in biofilms of the upper respiratory tract during infection.
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Acknowledgements |
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Footnotes |
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References |
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2
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40 . Haraldsson, G., Jousimies-Somer, H., Nyfors, S., Holbrook, W. P. & Könönen, E. (2002). Salivary Fusobacterium nucleatum as a potential source of nasopharyngeal F. nucleatum during acute otitis media in infancy. In Program and Abstracts of the Eightieth International Association for Dental Research, San Diego, CA, USA, 2002. Abstract 84, p. A-39. International Association for Dental Research, Alexandria, VA, USA.