Microbiology of mandibular third molar pericoronitis: Incidence of -lactamase producing bacteria

Transcription

1 Microbiology of mandibular third molar pericoronitis: Incidence of -lactamase producing bacteria Jean-Louis Sixou, DDS, PhD, a Christophe Magaud, b Anne Jolivet-Gougeon, DPH, PhD, c Michel Cormier, DPH, PhD, d and Martine Bonnaure-Mallet, DDS, PhD, e Rennes, France UNIVERSITY OF RENNES 1 Objectives. The purpose of this study was to evaluate the predominant flora associated with pericoronitis in third molars and to investigate the presence of -lactamase producing strains. Study design.the third molars in 26 adults were evaluated by cultures with nonselective media and with selective media containing amoxicillin, pristinamycin, spiramycin, metronidazole, and spiramycin plus metronidazole. Results. In the majority of cases (19/26), the flora found in an anaerobic atmosphere predominated. Obligate anaerobes were present in 21 of the 26 samples. The bacteria most commonly detected were -hemolytic streptococci (26/26) and the genera Prevotella (15/26), Veillonella (15/26), Bacteroides (9/26), and Capnocytophaga (9/26). Amoxicillin and pristinamycin were the most active in reducing the anaerobic cultivable counts. -Lactamase producing strains were detected in 9 samples and were mostly bacteria of the genera Prevotella, Staphylococcus, and Bacteroides. Conclusion. These results highlight (1) the diversity of the microflora associated with pericoronitis and the importance of the anaerobic flora and (2) the existence of selection pressure related to the use of -lactams that may culminate in failure of prescribed penicillins. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:655-9) This research was funded by Fondation Langlois, Conseil Régional de Bretagne, and Laboratoire Aventis. This article was presented at the IADR Meeting (Continental European and Scandinavian Divisions) in Warsaw, Poland, August a Associate Professor and Head, Department of Pediatric Dentistry, Faculty of Dentistry, and Equipe de Biologie Buccale UPRES-EA 1256, University of Rennes 1. b Microbiologist, Equipe de Biologie Buccale UPRES-EA 1256, University of Rennes 1. c Associate Professor, Department of Bacteriology and Laboratory of Bacteriology UPRES-EA 1254, Faculty of Pharmacy, University of Rennes 1. d Professor and Head, Department of Bacteriology and Laboratory of Bacteriology UPRES-EA 1254, Faculty of Pharmacy, University of Rennes 1. e Professor and Head, Department of Oral Biology, Faculty of Dentistry, and Equipe de Biologie Buccale UPRES-EA 1256, University of Rennes 1. Received for publication Nov 27, 2002; returned for revision Feb 10, 2003; accepted for publication Mar 18, , Mosby, Inc. All rights reserved /2003/$ doi: /moe Pericoronitis is an inflammatory and infectious complication accompanying the clinical emergence of the third molars in the oral cavity. Severe complications accompanying the localized inflammation may range from major regional reactions, such as cellulitis and trismus, to general reactions, such as pain, fever and asthenia. The microbial flora that develop at the site of eruption are contained within a periodontal pocket-like crevice. The flora in this crevice cause pericoronitis and constitute a potential reservoir of microorganisms for the oropharyngeal sphere. 1-3 The identification of these flora and a knowledge of how susceptible they are to antibiotics are necessary to adapt the treatment. The flora have been described by photon microscopy as similar to those of acute necrotizing ulcerative gingivitis. 4,5 Studies by DNA probe 6 and culture on nonselective media 7,8 or selective media 2,9,10 have demonstrated a predominantly anaerobic flora comprising Campylobacter gracilis, Capnocytophaga species, Fusobacterium species, Micromonas (Peptostreptococcus) micros, Prevotella intermedia, Prevotella nigrescens, Selenomonas noxia, and Veillonella species. Streptococci, staphylococci, actinomycetes, and enterobacteria as well as protozoa and fungi have also been described. In view of the variety of clinical manifestations, treatment is limited to a local surgical procedure or involves empirical antibiotic treatment. There is no consensus rule for antibiotic treatment. Broad-spectrum -lactams are generally considered to be the primary choice of antibiotics in emergency cases, although the increasing frequency of -lactamase producing oral bacteria may result in prescription failure. Few data on the susceptibility to antibiotics of the flora associated with pericoronitis are available, particularly -lactamase producing bacteria. The aim of this study was to investigate the presence of -lactamase producing strains after evaluation of 655

2 656 Sixou et al ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY June 2003 the predominant flora of pericoronitis by using nonselective and selective media containing antibiotics. MATERIAL AND METHODS Twenty-six adults aged 18 to 30 years (10 men and 16 women, mean age years) without systemic disease, suffering from pericoronitis with acute pain and without antibiotic treatment in the previous 6 months, were included in this study. After a history taking, the clinical examination involved an investigation for general manifestations of the type of fever and asthenia and the evaluation of regional and local manifestations: lymphadenopathy, edema, trismus, presence of pus, and pain. The samples were taken after isolation from the concerned tooth by means of sterile cotton swabs and aspiration. The emerged surface of the third molar and the distal face of the second molar were cleaned. A tip of sterile paper (Mynol Fine; Block Professional Dental Products Company, Jersey City, NJ) was inserted into the pocket-like crevice for 30 seconds and then collected in 1.5 ml of prereduced transport medium (VMGA III) prepared according to Dahlén etal. 20 The samples were dispatched to the Rennes Oral Biology laboratory, where they were inoculated within 24 hours. After serial dilution, 100 L of each dilution were inoculated on Brucella agar (BBL, AES ; AES Laboratoires, Combourg, France) containing 5% sheep blood enriched with vitamin K1 (1 mg/l) and hemin (10 mg/l), and on Brucella agars supplemented with blood (5% vitamin K1 hemin) containing different antibiotics: amoxicillin (0.5 mg/l) (A1), amoxicillin (4 mg/l) (A2), pristinamycin (1mg/L) (P), spiramycin (1 mg/l) (S), metronidazole (4 mg/l) (M), and spiramycin metronidazole (1 mg/l 4 mg/l)(sm). Incubation was performed in an anaerobic atmosphere (80% N 2, 10% H 2, 10% CO 2 )at37 C. Duplicates of nonselective Brucella agar supplemented with blood were incubated in an aerobic atmosphere at 37 C. The dishes incubated in an anaerobic atmosphere were read after 3, 5, 7, 10, and 15 days of culture. The total flora incubated in an aerobic atmosphere were evaluated after 2 days of incubation at 37 C. The microorganisms were identified by standard methods 21 ;by their profiles in API ZYM, Rapid Id 32A, and Rapid ID 32 Strep strips (Biomerieux, Lyon, France); and by their fatty acid composition (gas chromatography by means of the SHERLOCK Microbial Identification System (MIDI Inc, Newark, Del). The organisms were classified as aerobic, microaerophilic, or anaerobic in accordance with the taxonomy established by Holt et al. 22 The presence of -lactamases in the strains isolated on amoxicillin-containing media was evaluated by using nitrocefin disks (BBL, Cefinase ; AES Laboratoires). The susceptibility of the -lactamase producing strains to amoxicillin was evaluated by the agar dilution method in accordance with the recommendations of the National Committee for Clinical and Laboratory Standards 23 and the French Microbiology Society. 24 RESULTS Population Clinically, 7 subjects exhibited suppuration only, 4 trismus, and 4 trismus and suppuration combined. The other 11 subjects exhibited inflammatory manifestations. Culture The mean values of the counts taken on dishes incubated in an anaerobic atmosphere (total anaerobic viable count [TAVC]) and in an aerobic atmosphere (AeroC) were respectively CFU/mL (TAVC) and CFU/mL (AeroC). In the majority of cases (19/26), the flora found in an anaerobic atmosphere were predominant (AeroC/TAVC 1). Identifications The main results are summarized in the Table. Apart from -hemolytic streptococci (26/26), the bacteria most commonly detected were obligate anaerobes (present in 21 of the 26 samples): the genus Prevotella (15/26), including black-pigmented Prevotella species (n 12) and nonpigmented Prevotella species (n 10); the genera Veillonella (n 15) and Bacteroides (n 9); and capnophilic bacteria, genera Capnocytophaga (n 9) and Campylobacter (n 7). Among the other nonobligate anaerobic microorganisms, the genera Staphylococcus and Gemella (8/26), members of the S anginosus group (belonging to the -hemolytic streptococci, 8/26) and the genus Actinomyces (7/26) were most commonly found. Three of the 4 Campylobacter rectus were found in patients with trismus. In 2 cases with suppuration, no obligate anaerobic bacterium was detected. No correlation was found between the presence of microorganisms and the sex of the subjects or the ingestion of estrogen-progestogen based drugs in female subjects. Effect of antibiotic-containing media The means of the ratios (count on selective medium/ total anaerobe count) were respectively (A1), (A2), (P), (S), (M), and (SM). No microorganisms were detected on an

3 ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 95, Number 6 Sixou et al 657 Table. Frequency of detection of different microorganisms Microorganisms * Frequency Microorganisms* Frequency Anaerobic gram-positive cocci Microaerophilic gram-negative rods Peptostreptococcus anaerobius 1 Campylobacter gracilis 2 Micromonas (Peptostreptococcus) micros 1 Campylobacter rectus 4 Anaerococcus (Peptostreptococcus) prevotii 1 Bacteroides ureolyticus 2 Unidentified gram-positive cocci 4 Aerobic gram-positive cocci Anaerobic gram-negative cocci Enterococcus spp 1 Veillonella spp 16 Gemella morbillorum 6 Anaerobic gram-positive rods Gemella haemolysans 5 Eggertella (Eubacterium) lentum 6 Gemella spp 2 Clostridium spp 3 Staphylococcus spp 8 Bifidobacterium spp 1 -haemolytic streptococci 26 Unidentified gram-positive rods 4 Streptococcus acidominimus 1 Anaerobic gram-negative rods Aerobic gram-positive rods Bacteroides ovatus 1 Actinomyces israelii 5 Bacteroides thetaiotaomicron 1 Actinomyces gerencsariae 1 Bacteroides vulgatus 1 Actinomyces odontolyticus 5 Bacteroides spp 2 Actinomyces viscosus 1 Fusobacterium necrogenes 1 Corynebacterium spp 1 Fusobacterium nucleatum 4 Lactobacillus acidophilus 1 Leptotrichia buccalis 2 Lactobacillus spp 1 Prevotella bivia disiens 2 Propionibacterium acnes 4 Prevotella bivia/tanerae/oeneca 2 Propionibacterium propionicus 2 Prevotella buccae 4 Aerobic gram-negative rods Prevotella buccalis 1 Capnocytophaga spp 9 Prevotella denticola 1 Enterobacterium 1 Prevotella intermedia/nigrescens/pallens 9 Hafnia alvei 1 Prevotella loescheii 2 Pasteurella multocida 2 Prevotella melaninogenica 7 Pseudomonas stutzeri 1 Prevotella oralis 2 Yeasts 5 Prevotella oris 4 Prevotella spp 2 Porphyromonas gingivalis 3 Unidentified black-pigmented bacteria 2 Unidentified gram-negative rods 4 spp, Species. * The microorganisms are classified according to Holt et al. 22 antibiotic-containing medium in 1 (A1), 5(A2), and 1 (P) case, respectively. -Lactamase producing strains Forty-four strains obtained from amoxicillincontaining media were tested for -lactamase production. Eighteen strains, obtained from 9 individuals, exhibited a positive response: bacteria of the genera Prevotella (n 9), Bacteroides (n 3), Staphylococcus (n 4), Capnocytophaga (n 1), and Fusobacterium (n 1). The resistance of these 18 strains to amoxicillin was confirmed by agar dilution method. DISCUSSION In this study, the benefit of deciding to use antibioticcontaining media was that resistant bacteria were selected and hence detected. The elimination of rapidly growing flora, particularly when predominant in the sample, also reveals bacteria that are present in small quantities and proportions, or whose growth is inhibited by the microorganisms eliminated by the antibiotics. The incorporation of inhibitory agents is a method commonly used in general microbiology and in oral microbiology to detect periodontal pathogens. The previous studies on pericoronitis flora in which selective media were used essentially sought to detect either periodontal and/or caries pathogens or, through standard selective media in microbiology, 1,9,10 particularly TSBV (tryptic soy agar containing bacitracin and vancomycin) medium, 25 Actinobacillus actinomycetemcomitans. The antibiotics used in this study are different from those used in the standard selective media. Their spectra of activity cover the majority of genera and species of the oral cavity. They correspond to those most regularly prescribed by French operators, 26,27 with the addition of pristinamycin, an antibiotic recom-

4 658 Sixou et al ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY June 2003 mended for patients at high risk of infection and allergic to amoxicillin. 14 The concentrations correspond to the critical concentrations defined by the Antibiotic Sensitivity Test Committee of the French Microbiology Society. 24 The 2 values for amoxicillin are those defined for gram-negative bacteria (0.5 mg/l) and grampositive bacteria (4 mg/l). The results obtained highlight the predominance of anaerobic flora in a heterogeneous and complex flora, as described previously. 7,8 The number of flora culturable in an anaerobic atmosphere is on average 3 times greater than that culturable in an aerobic atmosphere. The flora are composed of obligate anaerobic microorganisms (detected in 80.8% of samples), microaerophilic organisms, and facultative anaerobes. Apart from streptococci, the genera and species most commonly found are obligate anaerobes: Veillonella (61.5%), nonpigmented prevotellae, the Prevotella intermedia/nigrescens/pallens group, the Prevotella melaninogenica/denticola/loescheii group, Bacteroides (all 34.6%), or bacteria whose growth is preferentially anaerobic (Capnocytophaga, 34.6%, and even Campylobacter, 26.9%, and Actinomyces, 26.9%). Although these results demonstrate an anaerobic shift comparable to that found in the periodontal pockets, the composition of the flora shows a different profile. With the exception of the members of the P intermedia/nigrescens/pallens group (34.6%), periodontal pathogens were rarely detected (Porphyromonas gingivalis (11.5%), Campylobacter rectus (15.4%), Fusobacterium nucleatum (15.4%), Eggertella lentum (23,1%), and Micromonas micros (3.8%)). Actinobacillus actinomycetemcomitans and Bacteroides forsythus in particular were not found. Interestingly, most of the putative periodontal pathogens detected belong to Socransky s orange organisms 28 and have been previously shown to be predominant in pericoronitis. 6-8 Members of this complex are usually closely associated with one another, and treatment, including systematically administered metronidazole, has been shown to decrease their level and improve periodontal status. 28 -Hemolytic streptococci represent the type of bacteria most frequently found (100%). Although possibly associated with general conditions, particularly infectious endocarditis, 29 most of these microorganisms may potentially be considered as representatives of the normal flora of the oral cavity. Because the pseudopockets of the erupting teeth are open in the oral cavity and accessible to saliva, the microorganisms can infiltrate the superficial areas of the pseudopockets, which become reservoirs for -hemolytic streptococci. Among them, streptococci of the anginosus group (eg, milleri) have been regularly found (30.8%), as well as certain other gram-positive aerobic cocci: the genera Staphylococcus and Gemella. The members of these genera are known to be associated with local infections (abscesses) and systemic infectious diseases. Streptococci of the anginosus group have recently been described as possible major agents of pericoronitis. 10 Their frequency of detection ranges from 5% to 80% depending on the studies. 3,7-10 The frequency observed in our study (8/26, or 30.8%) corroborates these published results. The presence of gram-positive bacilli such as Actinomyces and Propionibacterium (isolated in 26.9% and 18.4% of samples, respectively) may be the sign of an inflammatory state comparable to that of gingivitis. These flora may also be compared with those found in cervicofacial actinomycosis, 21 in which obligate anaerobic bacteria and capnophilic bacteria are frequently found. These results suggest a substantial diversity in the pathologic phenomena associated with pericoronitis. -Lactamase producing strains were found in 34.6% of samples (9/26). They belonged to 5 bacterial genera: 3 obligate anaerobic genera (Prevotella, Bacteroides and Fusobacterium) and 2 aerobic genera (Staphylococcus and Capnocytophaga). Amoxicillin is a broadspectrum antibiotic that is active against both obligate anaerobic flora and facultative anaerobic flora. Its use causes the selection of natural or acquired resistants ( -lactamase producing strains). -Lactamase producing strains may be present in the oral cavity from a very early age 19,30 and may be associated with various types of oral infectious diseases. 12,15-18,31-33 Their frequency varies with the human population. 17 The results of this study highlight the existence of selection pressure related to the use of penicillins from a very early age and the risk of failure of antibiotic therapy with a -lactam when -lactamase producing strains are present. They demonstrate the benefit of antibiotics effective against obligate anaerobic flora and the risk of failure of a penicillin-based antibiotic in view of the constant increase in -lactamase producing strains. The authors wish to thank Hélène Pinsard-Solhi, Carine Desoindre, Xavier Moisan, Noël Grosset, and Céline Allaire for technical and editorial assistance. REFERENCES 1. Rajasuo A, Meurman JH, Murtomaa H, Torkko H. Effect of extraction of partly erupted third molars on subgingival microorganisms. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1992;74: Rajasuo A, Meurman JH, Murtomaa H. Periodontopathic bacteria and salivary microbes before and after extraction of partly erupted third molars. Scand J Dent Res 1993;101: Rajasuo A, Jousimies-Somer H, Savolainen S, Leppanen J, Murtomaa H, Meurman JH. Bacteriologic findings in tonsillitis and pericoronitis. Clin Infect Dis 1996;23:51-60.

5 ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 95, Number 6 Sixou et al Nitzan DW, Tal O, Sela MN, Shteyer A. Pericoronitis: a reappraisal of its clinical and microbiologic aspects. J Oral Maxillofac Surg 1985;43: Weinberg A, Nitzan DW, Shteyer A, Sela MN. Inflammatory cells and bacteria in pericoronal exudates from acute pericoronitis. Int J Oral Maxillofac Surg 1986;15: Blakey GH, White RP, Jr., Offenbacher S, Phillips C, Delano EO, Maynor G. Clinical/biological outcomes of treatment for pericoronitis. J Oral Maxillofac Surg 1996;54: Leung WK, Theilade E, Comfort MB, Lim PL. Microbiology of the pericoronal pouch in mandibular third molar pericoronitis. Oral Microbiol Immunol 1993;8: Wade WG, Gray AR, Absi EG, Barker GR. Predominant cultivable flora in pericoronitis. Oral Microbiol Immunol 1991;6: Mombelli A, Buser D, Lang NP, Berthold H. Suspected periodontopathogens in erupting third molar sites of periodontally healthy individuals. J Clin Periodontol 1990;17: Peltroche-Llacsahuanga H, Reichhart E, Schmitt W, Lutticken R, Haase G. Investigation of infectious organisms causing pericoronitis of the mandibular third molar. J Oral Maxillofac Surg 2000;58: Dahlén G. Microbiology and treatment of dental abscesses and periodontal-endodontic lesions. Periodontol ;28: Kuriyama T, Nakagawa K, Karasawa T, Saiki Y, Yamamoto E, Nakamura S. Past administration of beta-lactam antibiotics and increase in the emergence of beta-lactamase-producing bacteria in patients with orofacial odontogenic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000a;89: Sandor GK, Low DE, Judd PL, Davidson RJ. Antimicrobial treatment options in the management of odontogenic infections. J Can Dent Assoc 1998;64: Agence Française de Sécurité Sanitaire des Produits de Santé. Prescription des antibiotiques en odontologie et stomatologie. Recommandations et argumentaire. 2001: Fosse T, Madinier I, Hitzig C, Charbit Y. Prevalence of betalactamase-producing strains among 149 anaerobic Gram-negative rods isolated from periodontal pockets. Oral Microbiol Immunol 1999;14: Fosse T, Madinier I, Hannoun L, Giraud-Morin C, Hitzig C, Charbit Y, et al. High prevalence of cfxa beta-lactamase in aminopenicillin-resistant Prevotella strains isolated from periodontal pockets. Oral Microbiol Immunol 2002;17: Herrera D, van Winkelhoff AJ, Dellemijn-Kippuw N, Winkel EG, Sanz M. Beta-lactamase producing bacteria in the subgingival microflora of adult patients with periodontitis. A comparison between Spain and The Netherlands. J Clin Periodontol 2000;27: Kuriyama T, Karasawa T, Nakagawa K, Yamamoto E, Nakamura S. Incidence of beta-lactamase production and antimicrobial susceptibility of anaerobic Gram-negative rods isolated from pus specimens of orofacial odontogenic infections. Oral Microbiol Immunol 2001;16: Nyfors S, Kononen E, Takala A, Jousimies-Somer H. Betalactamase production by oral anaerobic gram-negative species in infants in relation to previous antimicrobial therapy. Antimicrob Agents Chemother 1999;43: Dahlén G, Pipattanagovit P, Rosling B, Moller AJ. A comparison of two transport media for saliva and subgingival samples. Oral Microbiol Immunol 1993;8: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 7th edition. Washington: ASM Press; p Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST, editors. Bergey s manual of determinative bacteriology. 9th edition. Philadelphia: Williams & Wilkins; p National Committee for Clinical and Laboratory Standards. Methods for antimicrobial susceptibility testing of anaerobic bacteria: approved standard. 5th edition. Wayne: NCCLS; p Société Française de microbiologie. Communiqué 2002 du Comité de l antibiogramme de la Société Française de Microbiologie. Paris: The Société; p Slots J. Selective medium for isolation of Actinobacillus actinomycetemcomitans. J Clin Microbiol 1982;15: Antibiotiques. Les prescriptions en question. La Lettre du Chirurgien-Dentiste 2002;6: Matysiak M, Gradelet J, Mignée MJ, Mabriez JC. Evaluation des prescriptions pharmacologiques des chirurgiens-dentistes de la circonscription de la CPAM de Grenoble. Revue Médicale de l Assurance Maladie 1997; Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL, Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25: Tomas Carmona I, Diz Dios P, Scully C. An update on the controversies in bacterial endocarditis of oral origin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93: Könönen E, Kanervo A, Takala A, Asikainen S, Jousimies- Somer H. Establishment of oral anaerobes during the first year of life. J Dent Res 1999;78: Kuriyama T, Karasawa T, Nakagawa K, Saiki Y, Yamamoto E, Nakamura S. Bacteriologic features and antimicrobial susceptibility in isolates from orofacial odontogenic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000b;90: Kuriyama T, Karasawa T, Nakagawa K, Yamamoto E, Nakamura S. Bacteriology and antimicrobial susceptibility of Grampositive cocci isolated from pus specimens of orofacial odontogenic infections. Oral Microbiol Immunol 2002;17: van Winkelhoff AJ, Winkel EG, Barendregt D, Dellemijn-Kippuw N, Stijne A, van der Velden U. Beta-lactamase producing bacteria in adult periodontitis. J Clin Periodontol 1997;24: Reprints requests: Dr Jean-Louis Sixou Faculty of Dentistry Equipe de Biologie Buccale UPRES-EA Place Pasteur Rennes, France jean-louis.sixou@univ-rennes1.fr AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics for the preceding 5 years are maintained and are available for purchase from the publisher, Mosby, until inventory is depleted. The following quantity discounts are available: 25% off on quantities of 12 to 23; one third off on quantities of 24 or more. Please write to Mosby, Subscription Customer Service, 6277 Sea Harbor Dr, Orlando, FL 32887, or call or for information on availability of particular issues and prices. If unavailable from the publisher, photocopies of complete issues are available from Bell & Howell Information and Learning, 300 N Zeeb Rd, Ann Arbor, MI ; (734) or (800)