Ijodontostomat.com

Int. J. Odontostomat.,
7(2):193-198, 2013.
Antimicrobial Susceptibility to b-lactams and
Metronidazole of Microorganisms Isolated
from Chronic and Aggressive Periodontitis
Susceptibilidad Antimicrobiana a b-lactámicos y Metronidazol de
Microorganismos Aislados de Periodontitis Crónica y Agresiva
Elerson Gaetti-Jardim Júnior* & Christiane Marie Schweitzer**
GAETTI-JARDIM JÚNIOR, E. & SCHEITZER, C. M. Antimicrobial susceptibility to b-lactams and metronidazole of
microorganisms isolated from chronic and aggressive periodontitis. Int. J. Odontostomat., 7(2):193-198, 2013.
ABSTRACT: The objective of this study was to evaluate the microbial susceptibility to ß-lactams and metronidazole,
and evaluate the production of ß-lactamases by microorganisms isolated from patients with chronic or aggressive periodontitis.
The samples were obtained from 50 patients with periodontitis and microorganisms were isolated onto selective andnonselective culture media, identified by biochemical methods and tested for susceptibility to antimicrobial agents (amoxicillin,amoxicillin/clavulanate, cefoxitin, imipenem, metronidazole, penicillin G). The isolates were resistant to at least 1 mg/ml ofany drug tested were evaluated to verify the production of ß-lactamases by the method of double layer (or biological) andchromogenic cephalosporin using nitrocefin. The results evidenced resistance to amoxicillin and penicillin G, while thesusceptibility to association amoxicillin/clavulanate, imipenem and cefoxitin was widely disseminated among the organisms.
Resistance to these drugs showed a clear correlation with the production of ß-lactamase in the majority of microbial groups.
KEYWORDS: periodontitis, antimicrobials, b-lactams, bacteria, metronidazole.
INTRODUCTION
Periodontal diseases represent serious health positive and Gram-negative bacteria (López et al., problems and affect all ethnic groups, regardless gender and socio-economic conditions of the population. They result from the loss of equilibrium Antimicrobials and antibiotics used during between the immune response and virulence factors treatment of patients unable to maintain oral hygiene, of the resident microbiota. The control of the oral biofilm mentally or physically handicapped, suffering from is a prerequisite to the health maintenance in the dental and/or surgical trauma, and with refractory, periodontium (Eick et al., 2011). In general, aggressive or advanced periodontitis have been antimicrobial drugs are not indicated as monotherapy prescribed, but most of these compounds have several for patients with periodontitis, but some aggressive side effects. Moreover, the widespread use of forms of periodontitis may benefit with adjunctive use antimicrobial agents against oral anaerobes has been of systemic antimicrobials (Ahuja et al., 2012).
associated with a significant increase of the antimicrobial resistance, particularly against b-lactams Several microorganisms have been implicated (Senhorinho et al., 2012). In addition, most of as pathogens in periodontitis, such as Porphyromonas microorganisms recovered from periodontal sites are gingivalis, Prevotella intermedia, P. nigrescens, strict anaerobes and culture and susceptibility tests of Tannerella forsythia, spirochetes, Aggregatibacter anaerobes are not frequently performed in Brazil, what constitutes a serious problem since clinicians have to nucleatum, frequently associated with other Gram- choose antimicrobials just using international literature.
* Department of Pathology and Clinical Propaedeutics. School of Dentistry of Araçatuba, UNESP-Univ Estadual Paulista, Araçatuba, SP, Brazil** Department of Mathematics, Faculty of Engineering of Ilha Solteira, UNESP-Univ Estadual Paulista, Ilha Solteira, SP, Brazil, Grants: Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP. Process number: 2007/51016-3.
GAETTI-JARDIM JÚNIOR, E. & SCHEITZER, C. M. Antimicrobial susceptibility to b-lactams and metronidazole of microorganisms isolated from chronic and aggressive periodontitis.
Int. J. Odontostomat., 7(2):193-198, 2013.
Thus, the aim of this study was to evaluate the A total of 187 isolates of strict and facultative susceptibility of microorganisms isolated from chronic anaerobic bacteria were submitted to the susceptibility and aggressive periodontitis to b-lactams and tests. Susceptibility to antimicrobial agents was metronidazole, and evaluate the production of b- evaluated on 21 isolates of Aggregatibacter actinomycetemcomitans, 11 isolates of Bacteroides sp., 13 isolates of Eubacterium sp., 3 isolates of E.
lentum, 4 isolates of Fusobacterium sp., 33 isolates MATERIAL AND METHOD
of F. nucleatum, 5 F. periodonticum, 12 Parvimonasmicra, 9 Peptostreptococcus sp., 4 isolates of P.
anaerobius, 10 Porphyromonas sp., 23 Patients. This study was approved by the Ethics
Porphyromonas gingivalis, 5 Prevotella sp., 16 P. in- Committee in Research of the School of Dentistry of termedia, 7 P. nigrescens, and 11 isolates of Araçatuba. The population of this study consisted of 50 patients with clinical and radiographic characteristics of chronic periodontitis, seen at the School of Dentistry Antimicrobial susceptibility tests. The minimal
of Araçatuba and private clinics for initial trial for inhibitory concentrations were determined by agar treatment. Patient ages ranged from 26 to 59 years dilution method using Wilkins-Chalgren agar (mean 43.3). Additionally, 8 patients with clinical and supplemented with horse blood, hemin (1 mg/mL), radiographic features of aggressive periodontitis, aged and menadione (5 mg/mL). The bacterial inoculum from 4.5 to 13 years were included. Medical data was standardized in 105 cells/spot and transferred revealed that all patients were in good general health.
to the Petri plates containing the antimicrobials and The diagnostic and evaluation of the periodontal sta- the control plates (without drugs), using a Steer`s tus of the patients were carried out by specialists in replicator (Cefar Ltda, São Paulo, Brazil). The minimum inhibitory concentration (MIC) was defined as the smallest concentration of the drug that During the first visit, written informed consent was completely inhibited the bacterial growth. The obtained from each patient or their responsible before following antimicrobial drugs were tested: amoxicillin enrolment in the study. Patients who had received (Bayer S.A., São Paulo, Brazil), amoxicillin/ antibiotics 6 months before the initial clinical trial were clavulanate (Smithkline Beecham Brasil Ltda, São excluded. The clinical samples were collected from the Paulo, Brazil), cefoxitin (Merck Sharp & Dohme, São 3 deepest non-contiguous periodontal sites (probing Paulo, Brazil), imipenem (Merck Sharp & Dohme, São depth ≥ 5 mm) presenting clinical characteristics of Paulo, Brazil), benzylpenicillin (Fontoura-Wyeth S.A., inflammation and loss of attachment. Sample collection São Paulo, Brazil) and metronidazole (Laboratórios was performed by mean of sterile paper points, which Pfizer Ltda, Guarulhos, Brazil). The breakpoints used were introduced into periodontal pockets and remained for interpretation were those recommended by the there for 30s. After this procedure, the paper points were Clinical and Laboratory Standards Institute (2007).
pooled and transferred to transport medium VMGA III The test and control dishes were incubated (Gaetti-Jardim et al., 2012).
anaerobically (90% N2 + 10% CO2) at 37ºC, for 48 Microbial isolation and identification. In the
laboratory, aliquots of VMGA III were submitted to se- F. nucleatum ATCC 10953, F. nucleatum ATCC rial 10 fold dilutions in VMG I and were plated on 25586, E. lentum ATCC 43055, B. fragilis ATCC fastidious anaerobic agar (FAA) supplemented with 23745, E. coli ATCC 25922, S. aureus ATCC 29213, yeast extract (0.5%), hemin (1 mg/mL), menadione (5 P. aeruginosa ATCC 27853, and E. faecalis ATCC mg/mL) and 5% horse blood, tryptic soy serum 29212 were used as reference strains for quality con- bacitracin vancomycin agar (TSBVA), and incubated in anaerobiosis (90% N2 + 10% CO ), at 37ºC, for 14 and 3 days, respectively. Pure cultures were obtained Detection of b-lactamases. All the microorganisms
and the isolates were subjected to microbial speciation.
able to resistant to at least 1,0 mg/mL of the tested b- Initially, the isolates were subjected to Gram staining, lactms were submitted to assays to evaluate the evaluation colony morphology on blood agar plates, production of b-lactamases, using biological and identification by biochemical tests and amplification of chromogenic cephalosporin methods (Gaetti-Jardim DNA by PCR (Gaetti-Jardim et al.).
et al., 2007). The tested strains included 9 Prevotella GAETTI-JARDIM JÚNIOR, E. & SCHEITZER, C. M. Antimicrobial susceptibility to b-lactams and metronidazole of microorganisms isolated from chronic and aggressive periodontitis.
Int. J. Odontostomat., 7(2):193-198, 2013.
spp., 2 Peptostreptococcus spp., 12 fusobacteria, and DISCUSSION
2 Bacteroides sp. A chromogenic cephalosporin b- lactamase assay using Cefinase disks was performed The effectiveness of the antimicrobials in the according to the manufacturer’s instructions.
treatment of mixed anaerobic infections of the head and neck is undermined by empirical selection of drugs In the biological method, 20 µL of cultures of and the problem of self-medication. In addition, the resistant isolate were plated on the surface of microorganisms producing b-lactamases are frequently Wilkins-Chalgren agar, containing 1,0 µg/mL of the detected in dental biofilm from childhood to adulthood tested b-lactam (benzylpenicillin or cefoxitin). These particularly in infectious processes (Fosse et al., 2002; plates were then incubated anaerobically at 37°C for 48-72 h. After incubation, the cultures were exposed to chloroform fumes for 20 min and then covered with The strict anaerobes play a major role in the 3 mL of semi-solid brain heart infusion (BHI) agar pathogenesis of chronic or aggressive periodontitis and previously inoculated with 106 cells of Streptococcus the systemic use of antimicrobial agents may improve pyogenes FOA-94F14, sensitive to all tested b- the treatment of such infections when associated to lactams in a concentration of ≤ 0.06 mg/mL. The Petri local procedures and modification of oral hygiene dishes were then incubated under aerobiosis for 24 standards. Moreover, periodontitis is a reservoir of h at 37°C. After incubation, the presence of this halo opportunistic pathogens and their eradication depends of growth was indicative of the degradation of the b- on use of antimicrobials, particularly b-lactams (Ahuja lactam. Bacteroides fragilis ATCC 43858 was used et al.), while metronidazole is the frequently used in as the positive control of b-lactamase production.
the therapy of anaerobic mixed infections, specially due to its action on Gram-negative anaerobes associatedwith oral infections, and its association with amoxicillin has been evaluated in the treatment of aggressive periodontitis (Casarin et al., 2012).
It was not verified any difference in the Most studies that deal with the emergence of susceptibility patterns of the isolates recovered from antimicrobial resistance of oral bacteria have been chronic or aggressive periodontitis and the results done in the developed countries and little information are presented regardless the source of the clinical is available from south hemisphere (Gaetti-Jardim samples, in Tables I and II. All strains tested were Júnior et al., 2007). In addition, antimicrobial resistance susceptible to amoxicillin/clavulanate and imipenem, has expanded considerably, which has rendered but variable levels of resistance to amoxicillin, traditional prescription habits critical in the absence of benzylpenicillin, and cefoxitin were detected.
laboratorial tests (Baumgartner & Xia, 2003). This fact Resistance to b-lactams was frequently detected in is further aggravated when one considers the relevance Gram-negative strict anaerobes, particularly in the of the phenomenon of self-medication, which limits the genera Fusobacterium, Prevotella and Bacteroides, efficacy of treatment and can make prescribing a although some anaerobic cocci also evidenced difficult task, as it modifies the local patterns of actinomycetemcomitans (9.5%); in addition, 19% of The resistance to amoxicillin and benzylpenicillin was similar to previously reported in the literature from Peptostreptococcus spp. presented intermediate developing countries (Brescó-Salinas et al., 2006; susceptibility to this antimicrobial agent.
Gaetti-Jardim Júnior et al., 2007) and evidenced that most of resistant isolates belonged to a peculiar group Out of 25 bacterial isolates resistant or of Gram Clinical and Laboratory Standards Institute presenting intermediate susceptibility to to b-lactams, Clinical -negative strict anaerobes, as also reported to 20 (80%) were b-lactamase producers (10.7% of the bacteria isolated from endodontic infections (Gaetti- isolated bacteria). The production of these hydrolytic Jardim Júnior et al., 2007). The bacteria that accounted enzymes seems to be the major mechanism of for the significantly increased proportion of resistance resistance to b-lactams, excluding Bacteroides sp.
to amoxicillin and penicillin G in clinical samples were and peptostreptococci, where b-lactamases were not Gram-negative rods, susceptible to metronidazole. The results of Table II suggest that b-lactamases from GAETTI-JARDIM JÚNIOR, E. & SCHEITZER, C. M. Antimicrobial susceptibility to b-lactams and metronidazole of microorganisms isolated from chronic and aggressive periodontitis.
Int. J. Odontostomat., 7(2):193-198, 2013.
Table I. Susceptibility to antimicrobials of 187 clinical isolates recovered from chronic and aggressive periodontitis.
1 Peptostreptococcus spp. (25) GAETTI-JARDIM JÚNIOR, E. & SCHEITZER, C. M. Antimicrobial susceptibility to b-lactams and metronidazole of microorganisms isolated from chronic and aggressive periodontitis.
Int. J. Odontostomat., 7(2):193-198, 2013.
Table II. Production of b-lactamases by oral microorganisms isolated GAETTI-JARDIM JÚNIOR, E. & SCHEITZER, C.
from chronic or aggressive periodontitis.
M. Susceptibilidad antimicrobiana a b-lactámicos y
*Taxon (N)
Production of β−lactamases
metronidazol de microorganismos aislados de periodontitis crónica y agresiva. Int. J.
Odontostomat., 7(2):193-198, 2013.
RESUMEN: El objetivo fue evaluar la suscep-
tibilidad a ß-lactámicos y metronidazol, y evaluar la producción de ß-lactamasas por microorganismos aislados de pacientes con periodontitis crónica y agre-sivo. Las muestras fueron obtenido de 50 pacientes *Isolates presenting resistance or intermediate susceptibility to the tested b- con periodontitis y microorganismos aislados en me- dios de cultivo selectivos y no selectivos, identifica-dos por métodos bioquímicos y probados a la sus- anaerobic Gram-negative bacteria are active on penicillins and ceptibilidad a los antimicrobianos (amoxicilina, cephalosporins, as also described by Wybo et al. (2007) and amoxicilina/clavulanato, cefoxitina, imipenem, Gaetti-Jardim Júnior et al. (2007).
metronidazol, penicilina G). Los aislados fueron re- sistentes a por lo menos 1 mg/ml de cualquier dro-gas analizadas fueron evaluados para verificar la pro- The association of amoxicillin/clavulanate and imipenem ducción de ß-lactamasas por el método de doble capa were the most effective b-lactams against all tested isolates, (o biológico) y nitrocefina. Los resultados mostraron including amoxicillin, penicillin G and metronidazole resistant resistencia a amoxicilina y penicilina G, mientras la microorganisms. This effectiveness is related to the action of susceptibilidad a la asociación amoxicilina/ clavulanate in inhibiting most b-lactamases of oral clavulanate, cefoxitina y imipenem fue ampliamente microorganisms (Gaetti-Jardim Júnior et al., 2007) and the difundido entre los microrganismos. Resistencia a stability of carbapenems against most frequent b-lactamases.
estas drogas mostraron una clara correlación con la Although imipenem-hydrolyzing class D b-lactamase has been producción de ß-lactamasas en la mayoría de gru- identified, the present investigation evidenced that all b- lactamase producers were highly susceptible to this PALABRAS CLAVE: periodontitis,
carbapenem. However, due to its effectiveness on antimicrobianos, b-lactámicos, bacteria,
pseudomonads and other resistant microorganisms, the use metronidazol.
of carbapenems should be restrict to severe infections, particularly life-threatening diseases, to avoid disseminationof resistance (Gaetti-Jardim Júnior et al., 2007).
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