Evaluation of subgingival bacteria in the dog and susceptibility to commonly used antibiotics
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Evaluation of subgingival bacteria in the dog and susceptibility to commonly used antibiotics



Resistência bacteriana: o uso indiscriminado de antimicrobianos indicados para tratamento de afecções orais tem sido discutido como fonte importante de resistência bacteriana. ...

Resistência bacteriana: o uso indiscriminado de antimicrobianos indicados para tratamento de afecções orais tem sido discutido como fonte importante de resistência bacteriana.
Este estudo apresenta cepas resistentes e discute novas formas de aplicação dos medicamentos.



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Evaluation of subgingival bacteria in the dog and susceptibility to commonly used antibiotics Evaluation of subgingival bacteria in the dog and susceptibility to commonly used antibiotics Document Transcript

  • Evaluation of Subgingival Bacteria in the Dog andSusceptibility to Commonly Used AntibioticsMirko Radice, DVM; Piera Anna Martino, DBSc, PhD; Alexander M. Reiter, Dip! Tzt, Dr med vet progression from a healthy periodontium to gingivitis and Summary: periodontitis, there is a shift from a gram-positive oriented, aerobic facultative flora to a predominantly gram-negative, The aim of the present investigation was to exaluate the anaerobic flora." Periodontopathogens are bacteria that cause subgingival aerobic and anaerobic flora of 13 dogs with gingivitis and periodontitis. A catalase-positive form ofthe gram periodontal disease and the susceptibility of these bacteria negative Porphyromonas gingivaUs is considered to be the key to antibiotics currently approved in Italy for ti-eatment of periodontopathogen in cats and dogs--- and is recognized as P canine infections. Of the anaerobic bacteria, Bacteroides giilae.- Other canine and feline Porphyromonas organisms fragilis was most frequently isolated, followed by include P. assacharolytica, P. cangingivalis, P canoris, P. cansulci, Peptostreptococciis + Porphyromonas gingivaUs and P. endodontalis, P. circumdentaria, P. crevioricanis, P. salivosa.. P. Prevotella intermedia. Ofthe aerobic bacteria, a-hemolytic denticanis, and P. gingivicanis."-" Additional black-pigmented Streptococcus was most frequently isolated, ofien associated anaerobic bacteria associated with periodonta! disease include with Escherichia coli or Pctsteurella multocida. Resistance PreOtella intennedia-- and Bacteroides spp.""-" Pathogen- of anaerobic and aerobic bacteria to various antibiotics was related oral spirochetes also are considered to play an important generally high. Anaerobic bacteria appeared to be role in periodontal disease, but cultivation studies in cats and dogs susceptible to amoxicillin + clavulanic acid, doxycycline, have only been reported sparsely.*"-^ and erythromycin; aerobic bacteria appeared to be Previous studies showed that amoxicillin + clavulanic acid susceptible to amoxicillin + clavulanic acid, erythromycin, and clindamycin had high in vitro susceptibility against anaerobes gentamycin, and sulfa-trimethoprim. Bacteroides fragilis and enrofloxacin high in vitiv susceptibility against aerobes from was resistant to all of the antibiotics tested. The emerging subgingival plaque samples in cats and dogs," Although worldwide problem of bacterial resistance to antibiotics periodontal disease is caused by bacteria, antibiotic therapy should resulting from overuse and misuse of antibiotics is not be the primary treatment strategy,*^-" Unfortunately, there is a discussed J Vet Dent 23 (4); 219 - 224,2006 tendency among veterinarians to use antibiotics as part of the management of any animal with periodontal disease or other oral condition. Resistance of plaque bacteria to antibiotics has clearly been demonstrated in humans,**^ and a similar pattern of bacterialIntroduction resistance development may be present in cats and dogs. Periodontal disease is an infectious condition of the toothsupporting tissues (gingiva, periodontal ligament,, alveolar bone, The aim of the present investigation was to evaluate theand cementum) and is considered to be the most common disease subgingival flora (aerobic and anaerobic bacteria) of dogs within companion animals. The accumulation of plaque on tooth periodontal disease and the susceptibility of these bacteria tosurfaces is responsible for tiie development of gingivitis and antibiotics currently approved in Italy for treatment of canineperiodontitis. infections. Furthermore, the emerging problem of bacterial Gingivitis is inflammation ofthe gingiva and is reversible, if resistance to antibiotics in human and veterinary medicine isplaque is removed by home or professional oral hygiene reviewed.procedures.^ In addition to toxins and tissue-destructive enzymesproduced by periodontopathogenic bacteria, the hosts response to Materials and Methodsplaque leads to the release of agents from damaged neutrophils Thirteen chent-owned dogs with various degrees ofthat can cause injury to the bodys own tissues. hiflammation periodontal disease (ranging from gingivitis to periodontitis asmay spread along the periodontal space and ultimately progresses assessed by means of periodontal probing) were included in thisto periodontitis, which is diagnosed as loss of attachment study. There were three Yorkshire terriers, one German shepherd,(gingival recession, résorption of alveolar bone, and formation of one poodle, and eight mixed-breed dogs. Two dogs receivedperiodontal pockets). The periapical region ofthe tooth root may sporadic oral hygiene at home, A professional scaling andbecome affected, leading to retrograde pulpal infection. Thus, polishing had been performed on all dogs 6-months prior toendodontic disease can occur as a result of severe periodontal sample collection. Antibiotics had been given to most ofthe dogsdisease. Eventually the tooth becomes mobile and is lost due to in the past for conditions other than periodontal disease. Nospontaneous exfoliation or professional extraction. attempt was made to assess details of antibiotic history because Gingivitis and periodontitis are referred to as bacterial owners were not able to verify names of antibiotics used, datesinfections, but several hundred bacterial species have been and routes of administration, duration of therapies, etc. However,identified to date in normal and diseased mouths of cats and dogs must not have received antibiotic therapy for at least 2-weeks With maturation of plaque in subgingival areas and before sample collection to be included in the study. J,VET,DENT Vol. 23 No. 4 December 2006 219
  • Sample collection was performed under general anesthesia at the detachment of microbial cells. Two dilutions (Logio) werethe right maxillary canine tooth (104) and the right maxillary made for all samples that were plated on Tryptic Soy Agar platesfourth premolar tooth (108) since both teeth had been excluded witb 5 % sheep blood for aerobic bacteria and on Bnicella Agar^from prior periodontai probing, A sterile endodontic paper point for anaerobic bacteria. The plates were incubated aerobically forwas inserted into the depth of the gingival sulcus or periodontai 24 to 48-hours at 37°C, and anaerobicalV for 48 to 72-hours atpocket at buccal aspects of the teeth. The paper point was 37°C.removed after a few seconds and placed into tubes containing a The anaerobic flora was identified by growth on Bnicellatransport liquid media (thioglycollate broth)- for growth of Agaf, a medium containing Vit K and haemin. Grams staining,aerobic and anaerobic bacteria. The samples were immediately Schaifer & Fultons staining for spores, and API System 20Atransported to the reference laboratory and were vortexed to allow were used as biochemical reference methods. Aerobic bacteria were identified by their macroscopic {e.g.., morphology of colonies, presence of hemolysis) and microscopic Table 1 {using Gram-staining) characteristics; moreover, biochemical Signalment of the 13 dogs enrolled in the study. tests were performed using macro- or micrometbods*^. For the identification oí Streptococcus strains, the presence of hemolysis Variable n % (a partial or ß total) and the growth on Mitis SalivariusAgar^, a Male 11 84.6 medium for tbe isolation and identification of the streptococci of Sex the oral cavity, were evaluated. For identification oí Pasteurella Female 2 15.4 multocida, the lack of growth on Mac Conkey Aga) was < 5 years 3 23.1 evaluated. Age 5-10 years 7 53.8 Evaluation of microbial sensitivity/resistance to antibiotics was performed using the Kirby-Bauer reference method or the > 10 years 3 23.1 agar disk diffusion test. A bacterial suspension, performed in <12kg 8 61.5 saline buffer (0.9 % NaCl), was delivered onto a Mueller-Hinton Weight 12+kg 5 38.5 plate, and then the disks containing different antibiotic molecules were placed on the plate. After incubation at 37°C under aerobic Mixed 10 76.9 Diet or anaerobic atmosphere for 24 to 48-hours, the susceptibility of Dry 3 23.1 each microorganism was recorded to the following antibiotics: amikacin, amoxicillin + clavulanic acid, doxycycline, erythromycin, gentamycin, kanamycin, metronidazole (only for anaerobes), and sulfa-trimethoprim.^ Table 2 Isolation of anaerobic bacteria. Results Results of variables of signalment (sex, age, weight, and diet) Anaerobic bacteria Percent (%) of the 13 dogs enrolled in the study are reported in Table 1. The Peplostreptococcus + Porphyromonas majority of dogs were male (84.6 %), 5 to 10-years of age (53.8 gingivaiis 30.8 %), < 12.0 kg (61.5 %), and eating a mixed (soft and dry) diet Bacteroides fragilis 46.1 (76.9 %). Prevotella Intermedia 23.1 100 Of the anaerobic bacteria (Table 2), Bacteroides fi-agilis was most frequently isolated from subgingival samples, followed by Peptostreptococcus + Porphyromonas gingivaiis, and Prevotella Anaerobic bacteria intermedia. Of the aerobic bacteria (Table 3), a-haemolytic Streptococcus was most frequently isolated, often associated with Peptostreptococcus + Escherichia coli or Pasteurella multocida. Porphyromonas gingivaiis Susceptibility of anaerobic and aerobic bacteria to various antibiotics is shown in Tables 4-8. Resistance of isolated bacteria to tested antibiotics was generally high. Anaerobic bacteria appeared to be susceptible to amoxicillin + clavulanic acid, doxycycline, and erythromycin, while aerobic bacteria appeared to be susceptible to amoxicillin + clavulanic acid, erythromycin, gentamycin, and sulfa-trimetboprim. Bacteroides fragilis was Prevotella intermedia resistant to all of the antibiotics tested. Bacteroides fragilis Discussion The aim of the present investigation was to evaluate the subgingival aerobic and anaerobic bacterial flora of 13 dogs with220 J.VET.DENT Vol. 23 No, 4 December 2006
  • periodontal disease and the susceptibility of these bacteria to veterinarians, and diagnostic uncertainty. Several recent studiesantibiotics currently approved in Italy for treatment of canine showed that pediatricians prescribe antibiotics significantly moreinfections. Except for the high prevalence of Bacteroidesß-agilis, often, if they perceive parents expect them, and significantly lessthe predominant subgingival flora obtained in this study confirms often, if they feel parents do not expect them."^** The best way toresults reported in previous studies."•"•"•^•-"-- Of the anaerobic combat this situation is to educate patients/patient owners andbacteria, Bacteroides fragilis was most frequently isolated, doctors/veterinarians to decrease both demand and over-followed by Peptostreptococcus + Porphyromonas gingivalis and prescribing. Unfortunately, there is a tendency to use antibiotics asPrevotella intermedia. The reason for the unusually high part of the management of any animal with periodontal disease orprevalence oí Bacteroides fi-agilis is not clear. Differing results other oral condition, although there is no apparent justification forbetween isolation studies may be due to differences in study this practice. Similar to dogs in the present study, resistance ofmethodology, including sample population utilized and isolation plaque bacteria to antibiotics has clearly been demonstrated intechniques applied. Of the aerobic bacteria, a-haemolyticStreptococcus was most frequently isolated, often associated withEscherichia coli or Pasteurella nndtocida. Table 3 The fast growth rate, high concentration of cells, genetic isolation of aerobic bacteria.processes of mutation and selection, and ability to exchange genesaccount for the extraordinary adaptation and evolution of Aerobic bacteria Percent {%)bacteria." For these reasons bacterial resistance to antibiotics may a-hemolytic Sireptococcus + E. coli 38take place very rapidly in evolutionary time. Risk factors a-hemolytic Streptococcus + P. fvultocida 38responsible for the emergence and spread of resistant bacteria a-hemoiytic Streptococcus + E. coli + P. muitocida 8include: (1) antibiotic use; (2) reservoirs for resistance; (3) a-hemolytic Streptococcus + E. colimedical advances; and (4) societal changes."" Antibiotics make + S. intermedius 8conditions favorable for overgrowth of some bacteria, including a-hemolytic Streptococcusthose that possess mechanisms of drug resistance. If a resistant S, intermedius + P. muitocida 8organism is present, antibiotics will create selective pressure 100favoring the growth ofthat organism. A number of studies havedemonstrated conclusively that the development of bacterial Aerobic bacteria a-hemolytic Streptococcusresistance to antibiotics is correlated with the level of antibiotic + E. coli -h P. muttocidause."^* Antibiotic resistance of nosocomial pathogens in hospitals, a-fiemolytic Streptococcusnursing homes, day-care centers, and animal facilities is increased + E. cell + S. intermediusby the transfer of individuals already colonized by resistant a-hemolytic Streptccoccusorganisms from one location to another. Progress in the treatment a-iiemolytic + S. intermedius Streptococcus + P. multocidaof many diseases has led to an increased life span of humans and E. coiianimals. Consequently, with advanced age, chronic disease orimmunosuppression, individuals can be more susceptible tobacterial infections, resulting in greater use of antibiotics.Worldwide spread of bacterial resistance to antibiotics hasoccurred due to the increased mobility of todays society.-"- Pet animal numbers have substantially increased in modem a-hemolytic Streptococcus + P. muitocidasociety, and attention is increasingly devoted to pet welfare.Antibiotics are frequently used in small animal practice, withheavy use of broad-spectrum agents such as amoxicillin +clavulanic acid, cephalosporins, and ñuoroquinoIones. The Table 4practice of antibiotic overuse and misuse in cats and dogs hascontributed to the development of Staphylococcus spp., Antibiotic activity versus Prevotella intermedia.Escherichia coli and various other bacteria that are resistant toantibiotics.""^^ The role of pets in the dissemination of bacterial Antibiotic Sensitivity (%) Resistance (%)resistance to antibiotics has been given relatively little attention Amikacin - 100when compared with that of food animals, and a marked contrast Amoxicillin-^CIavuianic acid 66.7 33,3is evident between the current policies on antibiotic usage in Doxycycline 33.3 66.7companion and food animals. However, the possible transfer of Erythromycin 66.7 33.3resistant bacteria from cats and dogs to humans has recently been Gentamycin - 100acknowledged as a potential threat to public health.""-^* Kanamycin - 100 One of the biggest problems is inappropriate prescribing of Metronidazole - 100antibiotics. There are many reasons for this, including demand Suifa-trimethoprim 100 -from patients/patient owners, time pressure on physicians/ J,VET,DENT, Vol. 23 No, 4 December 2006 221
  • Table 5 Table 6 Antibiotic activity versus Bacteroides fragilis. Antibiofic activify versus Porphyromonas gingivalis Peptostreptococcus. Antibiotic Sensitivity (%) Resistance (%) Antibiotic Sensitivity (%) Resistance (%) Amikacin - 100 Amikacin 25 75 Amoxicillin+Clavulanic acid - 100 Amoxicillin+Clavulanic acid 75 25 Doxycycline - 100 Doxycycline 100 - Erythromycin - 100 Erythromycin 25 75 Genlamycin - 100 Gentamycin 25 75 Kanamycin - 100 Kanamycin 25 75 Metronidazole - 100 Metronidazole 25 75 Sulfa-trimethoprim - 100 Sulfa-trimethoprim - 100 Table 7 Table 8 Antibiotic activity versus a-haemolytic Streptococcus -f £ Antibiofic activity versus a-haemolyflc Streptococcus + P. coli. multccida. Antibiotic Sensitivity (%) Resistance (%) Antibiotic Sensitivity (%) Resistance (%} Amikacin 20 80 Amikacin - 100 Amoxicillin+Clavulanic acid 40 60 Amoxicillin+Clavulanic acid - 100 Doxycycline 20 80 Doxycycline - 100 Erythrcmycin 20 80 Erythromycin 40 60 Gentamycin 80 20 Gentamycin - 100 Kanamycin 20 80 Kanamycin 100 Sulfa-trimethoprim 60 40 Sulfa-trimethoprim - 100humans." It is therefore imperative to review periodontaltreatment strategies and determine whether systemic antibiotics Table 9have a role to play in the management of periodontal disease. If accumulation of plaqne is prevented, periodontal disease American Veterinary Dental College (AVDC) Positiondoes not develop." Although this condition is caused by bacteria, Statement on fhe Use of Antibiotics in Veterinary Dentistry.antibiotic therapy is not considered the primary treatmentstrategy."^" Instead, treatment of periodontal disease should be The AVDC endorses the use of systemic antibiotics indirected at mechanical removal or reduction of plaque and veterinary dentistry for treatment of some infectiouscalculus accumulation, suppression of the tissue-destructive conditions of the orai cavity Alttiough cuiture andeffects of the inflainmatory response, surgical management of susceptibility testing is rarely performed on individual patients that have an infection extending from/to the oralperiodonfal pockets, extraction of more severely affected teeth, cavity, ttie selection of an appropriate antibiotic should beand thorough debridement of extraction sites. A controlled- based on pubiisfied data regarding susceptibility testingrelease local antibiotic delivery system, reaching of the spectra of known oral pathogens. Patients that areperiodontopathogens deep within periodontal pockets, has been scheduled for an oral procedure may benefit from pre- treatment with an appropriate antibiotic to improve thedescribed in dogs.^" Professional supra- and subgingival scaling, health of infected oral tissues. Baoteremia is a recognizedfollowed by daily tooth brushing, is the gold standard" for seguela to dental scaling and other oral procedures.prevention of periodontal disease. Home oral hygiene may be Healthy animais are able to overoome this bacteremiaenhanced by offering products that support dietary abrasion or without the use of systemic antibiotics. However, use of achemically suppress plaque and calculus accumulation.™ systemically administered antibiotic is recommended to reduce bacteremia for animals that are immune Bacteremia secondary to periodontal disease occui"s daily in compromised, have underlying systemic disease (suchpatients with periodontal disease, and it is normally rapidly as clinically-evident cardiac, hepatic, and renal diseases) and/or when severe oral infection is present. Antibioticscleared hy the reticulo-endothelial system in the healthy patient." should never be considered a monotherapy for treatmentTherefore, for the great majority of otherwise healthy cats and of oral infections, and should not be used as preve •••/edogs presenting with periodontal disease and other oral management of oral conditions. Adopted by the Bo.conditions, systemic antibiotics are not indicated." Bacteremia Directors. April 2005can be prevented or reduced in severity by rinsing the oral cavity222 J.VETDENT Vol. 23 No. 4 December 2006
  • di cani con parodontopatie e ioro antibiotioo-sensibilità. Tesi di laurea, Reiatore Dott.ssawith dilute chlorhexidine gluconate (0.12 %) prior to PA, Marlino, 2003 Facoitâ di Medicina Velennaria, Miianocommencing the oral procedure. Perioperative systemic 4, Courant PR, Saxe SR, Nasii L, Roddy S, Sulcuiar bacteria in the beagle dog,antibiotics are indicated in: ( 1 ) debilitated and Periodontics 1968: 6: 250-252.lnununocompromised patients; (2) patients suffering from organ 5. Harvey CE, Thornsberry C, et al. Subgingivai bacteria-comparison of culture results mdisease, endocrine disorders, cardiovascular disease, and severe dogs and cats with gingivitis. J Vet Dent 1995; 12:147-150.local and/or systemic infections; and (3) patients having 6, Hennet PR, Harvey CE Aerobes in periodontal disease in the dog: a review. J Vel Dentpermanent implants and transplants. Unless there is a well- 1991; 8: 9-11,founded positive reason for their administration, systemic 7. Hennet PR, Harvey CE. Anaerobes in periodontai disease in the dog. A Review. J Vet Denf 1991:8: 18-21.antibiotics should not be used. Although there is a position statement on the use of 8 Hennel PR, Harvey CE. Spirochetes m periodontal disease in the dog a review J Vet DenM991:8: 16-17.antibiotics in veterinary dentistry provided by the American 9. isogai E, isogai H, etal, Orel flora of mongrei and beagie dogs with periodontai diseaseVeterinaiy Dental College (Table 9),"- compulsory guidelines for Nippon JuigakuZasshi ^äSQ.b^: 110-118,prudent prescription patterns and use of antibiotics in small 10. Isogai H, Isogai E, ef al. Detection of serum antibodies of oral Porphyrornonasanimals with periodonta] disease or other oral conditions, which (Bacteroides) asaccharolyticus in dogs: relationship to periodontal disease. Nippon JuigakuZasshn9e9: 5V. 1239-1241.describe the minimum requirements to be followed byveterinarians, are not available. Key elements of these guidelines 11. isogai HY, Kosako Y, et al. 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