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Bacterial isolates and antimicrobial susceptibilities from odontogenic abscesses in rabbits: 48 cases
  1. Sara Gardhouse, DVM1,
  2. David Sanchez-Migallon Guzman, LV, MS, DECZM (Avian, Small Mammal), DACZM2,
  3. Joanne Paul-Murphy, DVM, DACZM, DACAW2,
  4. Barbara A Byrne, DVM, PhD, DACVIM, DACVM3 and
  5. Michelle G Hawkins, VMD, DABVP (Avian)2
  1. 1 School of Veterinary Medicine, University of California Davis, Veterinary Medical Teaching Hospital, Davis, California, USA
  2. 2 Departments of Medicine and Epidemiology, School of Veterinary Medicine, UC Davis Veterinary Medical Teaching Hospital, University of California, Davis, California, USA
  3. 3 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, UC Davis Veterinary Medical Teaching Hospital, University of California, Davis, California, USA
  1. E-mail for correspondence; guzman{at}ucdavis.edu

Abstract

The medical record database of a veterinary teaching hospital was searched from 2000 through 2014 for records of client-owned rabbits with positive cultures from odontogenic abscesses. Data reviewed included sex, age, abscess location, sampling technique, previous antimicrobial treatments, microorganisms identified and antibiotic susceptibility of aerobic bacteria. Forty-eight client owned rabbits with one or more odontogenic abscesses and culture results were evaluated. One hundred and eighty-five isolates (52 aerobic, 133 anaerobic) were identified from 61 positive cultures from odontogenic abscesses. The most common aerobic bacterial isolates were Pseudomonas aeruginosa (14/52), Pasteurella species (10/52), Streptococcus species (8/52) and Staphylococcus species (7/52). The most common anaerobic bacterial isolates were Fusobacterium species (36/133), Peptostreptococcus species (27/133) and Bacteroides species (27/133). Mixed aerobic and anaerobic isolates were detected in 73.3 per cent of cultures and 50.8 per cent of the samples had greater than three isolates. P aeruginosa was susceptible to amikacin and gentamicin, had variable resistance to enrofloxacin, and resistance to chloramphenicol. Pasteurella species, Streptococcus species and Staphylococcus species isolates were susceptible to most antibiotics tested. The bacterial isolates from rabbit odontogenic abscesses are a mixture of aerobic and anaerobic Gram-positive and Gram-negative organisms and without culture and susceptibility testing, it is difficult to predict appropriate antimicrobial treatment.

  • rabbit
  • oryctolagus cuniculus
  • odontogenic
  • culture
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Introduction

Acquired dental disease is a common reason for rabbits to be examined by veterinarians.1 Frequent features of the earliest stage of the disease include elongation of the crowns of the teeth, curving of the premolars and molars and widened interdental spaces.1 Further progression of the disease includes development of dental spurs that can lead to lingual and buccal mucosal ulcerations.1 One of the most common complications of acquired dental disease is the formation of odontogenic abscesses, often with subsequent osteomyelitis.1 However, despite the commonality of this disease condition, the underlying pathophysiology is not completely understood.2

Obligate anaerobic bacteria have an important role in odontogenic infections in rabbits and are often found in mixed infections with aerobes.3 Diagnostic sample collection of the abscessed material presents a challenge, as poor sampling technique can result in isolation of normal gingival flora; however, if the sample has been collected from inside the abscess or tissue, then the bacterial isolate is likely significant.3 Additionally, with poor sampling technique, the practice of coprophagy can result in the isolation of non-oral organisms that are of little significance.4 For example, isolation of some Enterobacteriaceae and Bacteroides fragilis is common and can represent fecal organisms.4

Identification of common bacterial species associated with rabbit odontogenic abscesses is of great value to optimise early treatment decisions when isolates and susceptibilities are not yet available.5 There are two previous studies that have evaluated the causative organisms and the susceptibilities associated with odontogenic abscesses.3 6 In the study by Tyrrell and others,3 12 rabbits were evaluated, with the most common isolates being Fusobacterium nucleatum, Streptococcus intermedius and Peptostreptococcus micros.3 The most effective antibiotics from that study were chloramphenicol, clindamycin, penicillin, ceftriaxone and cefazolin.3 Resistance to azithromycin, ciprofloxacin and trimethoprim-sulfamethoxazole was common.3 In the case series by Taylor and others,6 13 cases were included. The most common anaerobic isolates included Prevotella melaninogenicus, B fragilis, Bacteroides species and the most common aerobic isolates were Actinomyces species, Proteus vulgaris, Pasteurella species, Streptococcus species and Escherichia coli.6 Susceptibility results were not reported in this study.6

The goal of the current study was to describe the aerobic and anaerobic bacterial isolates and antimicrobial susceptibilities of aerobic organisms found in odontogenic abscesses in rabbits.

Materials and methods

Case selection

Medical records of all rabbits with cultures from odontogenic abscesses examined at a teaching hospital between 2000 and 2014 were reviewed retrospectively. An electronic search was performed for any rabbits that had positive culture results in the time period with inclusion criteria for abscesses of odontogenic origin. Each of the retrieved medical records were reviewed by one investigator (SG) to verify that the rabbit had evidence of a maxillary or mandibular abscess associated with the teeth, evidence of purulent material in the mouth or further diagnostic testing such as advanced imaging to confirm or identify the presence of an odontogenic abscess. For each rabbit, information extracted from the record included sex, age and previous antibiotic use.

Sample collection

Sample collection methods for the cultures varied, ranging from swabbing of the exudate inside the abscess to submission of the abscessed tooth, bone or capsule. The culture swabs were immediately transferred to the diagnostic laboratory for the teaching hospital, for aerobic and anaerobic bacterial isolation and identification and for antimicrobial susceptibility testing for aerobic isolates.

Bacterial isolation and identification

To identify aerobic bacteria, samples were inoculated on to five per cent defibrinated sheep blood, Rose with five per cent sheep blood and MacConkey agars (Hardy Diagnostics, Santa Maria, California, USA). Inoculated plates were incubated aerobically at 35°C with five per cent carbon dioxide (CO2) and examined daily for five days for bacterial growth. Bacteria were identified using Gram stain, catalase and spot tests, tubed media and identification strips (API, BioMerieux, Durham, North Carolina, USA).

To isolate and identify anaerobic bacteria, samples were inoculated on to prereduced Brucella blood and phenylethyl alcohol blood agars and incubated under 90 per cent nitrogen gas (N2), 5 per cent hydrogen gas (H2) and 5 per cent CO2 in the presence of a palladium catalyst (Anaerobe Systems, Morgan Hill, California, USA). Colonies were identified based on Gram stain characteristics, susceptibility to antibiotic-containing disks (colistin, kanamycin and vancomycin) and various spot tests (eg, nitrate reduction, indole), reactions on egg yolk agar and production of acid from various sugars.

Antimicrobial susceptibility testing

Susceptibility testing to determine minimum inhibitory concentrations was performed on aerobic isolates against 15 antimicrobial agents using the broth microdilution technique according to guidelines described by the Clinical and Laboratory Standards Institute (Sensititre, Thermo Fisher Scientific, Waltham, Massachusetts, USA).7 ,8 Susceptibility testing was carried out as previously described.9 Streptococci and Pasteurella species were inoculated into cation-adjusted Mueller-Hinton broth containing laked horse blood before inoculation of Sensititre plates, sealed with perforated seals and incubated with 5 per cent CO2.

Since rabbit-specific criteria for determining susceptibility based on minimum inhibitory concentration values do not exist, criteria for interpretations of minimum inhibitory concentrations were based on Clinical and Laboratory Standards Institute guidelines for animals, and when not available for animals, criteria for human isolates were used.7 ,8

Statistical analysis

The results were reported as Clopper-Pearson binomial proportions with exact 95 per cent confidence intervals (CIs) using EpiTools software (http://epitools.ausvet.com.au/content.php?page=CIProportion).

Results

A total of 185 isolates (52 aerobic, 133 anaerobic) were isolated from 61 positive cultures from 48 odontogenic abscesses over the 14-year time period. The data set included 32 male and 16 female rabbits. The median age was 3 years (range 0.5–7 years). At the time of culture collection, 82 per cent (95% CI=70.0 to 90.6 per cent; 50/61) of rabbits were being treated with antibiotics, with the most common antibiotics being enrofloxacin 32.8 per cent (95 per cent CI=21.3 to 46.0 per cent; 20/61), penicillin 24.6 per cent (95 per cent CI=14.5 to 37.3 per cent; 15/61) and chloramphenicol 14.8 per cent (95 per cent CI=7.0 to 26.2 per cent; 9/61). The total number of culture samples (n=61) includes 13 rabbits that were sampled more than once due to recurrent or new abscesses. One of the 61 samples was submitted for aerobic culture alone, and 60 samples were submitted for both aerobic and anaerobic testing. Of the samples submitted for both aerobic and anaerobic culture, 6.7 per cent (95 per cent CI=1.8 to 16.2 per cent; 4/60) detected anaerobic isolates with no aerobes detected, 20.0 per cent (95 per cent CI=10.8 to 32.3 per cent; 12/60) detected aerobic isolates alone with no anaerobes detected and, 73.3 per cent (95 per cent CI=60.3 to 83.9 per cent; 44/60) cultures demonstrated mixed aerobic and anaerobic isolates. Thirty of the 61 samples had one to three isolates. Greater than three isolates were detected in 31 out of 61 samples, with the maximum number of isolates being 10.

The most frequently isolated aerobic bacteria were Pseudomonas aeruginosa, 26.9 per cent (95 per cent CI=15.6 to 41 per cent; 14/52), Pasteurella species 19.2 per cent (95 per cent CI=9.6 to 32.5 per cent; 10/52), Streptococcus species 15.4 per cent (95 per cent CI=6.9 to 28.1 per cent; 8/52) and Staphylococcus species 13.5 per cent (95 per cent CI=5.6 to 25.8 per cent; 7/52). The most common anaerobic isolates were Fusobacterium species 27.1 per cent (95 per cent CI=19.7 to 35.5 per cent; 36/133), Peptostreptococcus species 20.3 per cent (95 per cent CI=13.8 to 28.1 per cent; 27/133) and Bacteroides species 20.3 per cent (95 per cent CI=13.8 to 28.1 per cent; 27/133). The aerobic and anaerobic bacterial isolates are presented in Table 1 and Table 2. There were five cultures of abscesses of odontogenic origin that were negative for any growth. Of these rabbits, two out of five were receiving antimicrobials at the time of culture collection.

TABLE 1:

Aerobic bacterial isolates (n=52) from odontogenic abscesses from 48 domestic rabbits.

TABLE 2:

Anaerobic bacterial isolates (n=133) from odontogenic abscesses from 48 domestic rabbits.

The aerobic isolates were evaluated for susceptibility against commonly used antimicrobials. Susceptibility results are presented in Table 3. In all cases in this study, P aeruginosa isolates were susceptible to amikacin and gentamicin, had variable resistance to enrofloxacin (8/14 susceptible), and were resistant to chloramphenicol. Pasteurella species in this study demonstrated susceptibility to most antibiotics tested, including trimethoprim-sulfamethoxazole, aminopenicillins, amikacin and gentamicin. Streptococcus species isolates were susceptible to most antibiotics tested except aminoglycosides. None of the Staphylococcus species was methicillin resistant and the great majority (25/28) demonstrated susceptibility to the aminoglycosides, chloramphenicol and trimethoprim-sulfamethoxazole. The remainder of the isolates were represented by Enterococcus species, which was most susceptible to chloramphenicol.

TABLE 3:

In vitro susceptibilities of aerobic bacteria isolated (n=52) from 48 domestic rabbit odontogenic abscesses.

Anaerobic susceptibilities were not evaluated in this study because of the difficulty in performing them, the labour intensive nature, and the associated high cost.10 Additionally, anaerobic susceptibilities are not routinely performed at this institution.

Discussion

This study analysed results from 61 odontogenic abscesses in rabbits and found that rabbit odontogenic infections are often mixed aerobic and anaerobic infections with multiple isolates, highlighting the importance of a multidrug treatment protocol. The antibiotic treatment selections should be changed if necessary when microbiology results and antibiotic susceptibilities are available if empirical selection was not appropriate.

Treatment of odontogenic abscesses in rabbits is thought to require multimodal therapy with surgical intervention in conjunction with administration of appropriate antimicrobial agents.5 Although no studies exist to demonstrate that odontogenic abscesses require antimicrobial therapy, antimicrobial therapy is an important adjunct to treatment in many species.11 The antimicrobials used should be selected on the basis of culture and susceptibility testing of the purulent material, abscessed tooth, bone or abscess capsule.3 5 Additionally, treatment is often selected based on susceptibilities reported in the scientific literature, since most laboratories do not routinely perform susceptibility testing on anaerobic isolates.10 Although controversial to culture the purulent material, in the study by Tyrrell and others,3 comparison of capsule and purulent material culture demonstrated approximately one-half of the cultures of purulent material yielded a greater variety or quantity of organisms than cultures of the margin. In addition, poor sampling technique can result in isolation of normal gingival flora or fecal organisms due to the practice of coprophagy.3 4 However, if sample collection is appropriate, isolation of oral or gastrointestinal flora may be clinically significant as the starting point of an opportunistic infection.11

The most frequently identified aerobic isolate in this study was P aeruginosa (26.9 per cent), an opportunistic Gram-negative, aerobic, coccobacillus.12 P aeruginosa is a ubiquitous organism that is present in many diverse environmental settings and has been isolated from many living sources.12 Pseudomonas species have been reported to commonly infect other body systems of rabbits such as the respiratory system13 and can pose a serious therapeutic challenge due to its ability to develop resistance to multiple classes of antimicrobial agents, even during the course of a single treatment.9 Although this study was not designed to examine the development of resistance by Pseudomonas, the high degree of resistance of these organisms might explain some of the difficulty in successfully treating these abscesses. In this study, P aeruginosa was susceptible to amikacin and gentamicin, uncommonly used antibiotics in rabbits and exhibited variable resistance to enrofloxacin (6/14 resistant), and resistance to chloramphenicol. The resistance of Pseudomonas species to chloramphenicol was expected and has been demonstrated previously.14 Chloramphenicol and enrofloxacin are common treatments employed for odontogenic abscesses in rabbits.5 In the previous studies of odontogenic abscesses in rabbits, Pseudomonas was not identified.3 6 In the current study, most rabbits were on antimicrobial therapy at the time of culture, whereas only 2/12 rabbits were receiving antimicrobial therapy in the previous study by Tyrrell and others.3 Other notable differences in comparison to the study by Tyrrell and others include the percutaneous collection of all abscesses in the previous study and the smaller sample set compared with the current study.3

Pasteurella species (19.2 per cent) were common aerobic isolates from odontogenic abscesses in this study. Pasteurella species are Gram-negative facultative anaerobic organisms that cause significant disease in rabbits, with a wide array of clinical signs, including purulent nasal discharge, pneumonia, otitis media, pyometra, orchitis, abscesses, oculoconjunctivitis and septicaemia.15 Strains of Pasteurella species vary in their pathogenicity.13 They are considered part of the normal oral flora in many species, including dogs and cats.16 To our knowledge, the normal oral flora of rabbits has not been reported. Pasteurella species in this study were susceptible to most antibiotics tested, including trimethoprim-sulfamethoxazole, aminopenicillins, fluoroquinolones, amikacin and gentamicin. Given the commonality of this pathogen in rabbits and its role in multiple disease processes, its isolation in this study was not unexpected. However, in the two previous studies of odontogenic abscesses in rabbits, Pasteurella species was not isolated3 and was only identified in 1/13 rabbits in the other.6 Based on the findings from this large-scale study, susceptibility of Pasteurella species should be considered when treating odontogenic abscesses.

Streptococci were the third most common aerobic bacterial isolates found in 15.4 per cent of the odontogenic abscesses from rabbits in this study. Streptococcus species are Gram-positive, facultative anaerobic bacteria with variable pathogenicity, depending on the susceptibility of the host and the bacterial virulence.17 In this study, Streptococcus species isolates were susceptible to most antibiotics tested, indicating that most antimicrobial options would be appropriate to target this isolate in odontogenic abscesses.

Staphylococcus was also a common finding representing 13.5 per cent of the aerobic bacterial isolates, being susceptible to a large number of the antibiotics assessed. Other aerobic organisms were also isolated in a smaller number, and some such as Enterococcus have not previously been reported.

The most common anaerobic bacteria isolated in this study were Fusobacterium species, representing 27 per cent of the anaerobic bacterial isolates, followed by Peptostreptococcus species and Bacteroides species representing 20.3 per cent of the anaerobic isolates each. Fusobacterium species and Peptostreptococcus species were also the most common anaerobic isolates in the study by Tyrrell and others.3 Isolation of B fragilis was common, representing 33 per cent of the Bacteroides species in this study and could represent contamination from oral flora and coprophagy if poor sampling technique was used, or could represent an inciting cause of the abscess from oral flora when proper sampling technique was used.4 Bacteroides was a common isolate in this study, but was not noted in the study by Tyrrell and others3; however, it has been noted as a common isolate in another study of odontogenic abscesses in rabbits.6 Prevotella species represented 9.8 per cent of the isolates, followed by a number of other isolates represented in only a few cases. Anaerobic susceptibilities are rarely performed in most microbiology laboratories and hence, anaerobic spectrum antimicrobials are usually selected empirically.10 In addition to the high expense of anaerobic susceptibility testing, the results can be challenging to interpret due to the variety of susceptibility testing methodology used, the selective antibiotic pressures associated with antimicrobial usage and the lack of uniformity among laboratories of the appropriate interpretive breakpoints.18 19 However, with increasing antimicrobial bacterial resistance among anaerobic bacteria, susceptibility testing is becoming more readily available and commonly performed.10

Options for treatment of anaerobic bacteria with antimicrobials in rabbits are limited and each must be used with extreme caution and monitoring for gastrointestinal signs of dysbiosis. Possible options for systemic treatment of anaerobes in rabbits include the use of metronidazole, chloramphenicol or parenteral penicillin G.3 An additional antimicrobial to consider is azithromycin, as it demonstrated some efficacy in one study.3 Some antibiotics administered orally (eg, aminopenicillins, clindamycin, erythromycin) will result in dysbiosis and enterotoxemia.1 Chloramphenicol poses a low risk for serious enteropathies in hind-gut fermenting species, but does pose a human health risk.20 It has a wide spectrum of activity against many Gram-positive aerobic organisms including streptococci and staphylococci, Gram-negative aerobes and anaerobes including Clostridium species, Bacteroides species (including B fragilis), Fusobacterium species and Veillonella spp.20 Penicillins are usually bactericidal and effective against a wide variety of bacteria including beta-haemolytic streptococci, many Gram-positive anaerobes, spirochetes and Gram-negative aerobic bacilli depending on the class of penicillin.21 Due to the common presence of beta-lactamases in B fragilis isolates, penicillin may not be the drug of choice for infections with this agent.22 Metronidazole can also be used cautiously in rabbits and is reported to have activity against most obligate anaerobes including Bacteroides species (including B fragilis), Fusobacterium species, Veillonella species, Clostridium species, Peptococcus species and Peptostreptococcus species23 Given the propensity of rabbit odontogenic abscesses to result in secondary osteomyelitis, the penetration of antimicrobials into the bone should be considered.6 Penicillins are efficacious for the treatment of osteomyelitis in humans when given for a prolonged period of time.24 In a rat model, both metronidazole and chloramphenicol showed penetration into long and flat bones in amounts sufficient to exceed the minimal inhibitory concentrations for most anaerobic bacteria; however, the bone/serum ratio for metronidazole was lower than that for chloramphenicol.25 Alternatively, local treatment with antibiotic impregnated gauze or antimicrobial beads has been reported to provide long-term resolution of odontogenic abscesses in rabbits.5 6 These options also allow the use of alternative antimicrobial options, such as clindamycin, which are not safe to administer orally in rabbits.5 6

Five of the cultures of abscesses of odontogenic origin did not demonstrate growth of any bacteria. Only two of these rabbits were receiving antimicrobials at the time of culture collection. A review of the medical records did not demonstrate any differences with sample collection in these rabbits compared with the other rabbits with positive culture results in this study. In the human literature, culture negative abscesses are commonly seen in clinical practice.26 Considerations for negative culture results despite presence of an abscess or infection include lack of sensitivity to detect all infecting bacteria, sterile abscess formation, hidden bacteria within macrophages, organism levels below the limit of detection and use of inappropriate media or incubation temperatures.26 It is unknown in the case of these rabbits, the underlying reason why culture did not yield a positive result, but it is likely multifactorial.

Significant challenges are faced with treatment of odontogenic abscesses in rabbits.1 Although this study focuses on medical treatment of these odontogenic abscesses, medical therapy as the sole treatment is generally inadequate; however, it is an important adjunct to therapy.1 Surgical intervention and pain management is almost always necessary for treatment, in conjunction with the appropriate antimicrobial use.1 However, complete removal of rabbit odontogenic abscesses is technically difficult and can result in damage to surrounding facial structures.1 Passive drainage and flushing of rabbit abscesses is often ineffective due to the thick, caseous nature of the exudate.1 A number of surgical treatment options have been described for the treatment of abscesses of odontogenic origin in rabbits.5 Various options include opening the capsule, excising the entire abscess capsule with primary closure or marsupialisation1 27 packing the surgical site with polymethylmethacrylate beads,28 or antimicrobial impregnated gauze,6 calcium hydroxide, honey or sugar solution29 or bioactive ceramics1 have all been reported with various success.

Limitations of this study include the accuracy of information available in the record, which is inherent in any retrospective study. Additionally, since cultures were performed over a 14-year period by numerous clinicians, sample collection was not consistent. Poor sampling technique can contribute to isolation of normal gingival flora or a negative culture.3 Future prospective studies with a standardised collection technique and location may yield different results. Susceptibility testing also carries limitations as not all antibiotics that are routinely used in rabbits were tested in the susceptibility panel (ie, azithromycin, marbofloxacin). As well, some of the antibiotics in the susceptibility panel are not routinely used in rabbits and would require further safety and efficacy evaluation before attempting parenteral use, such as amoxicillin/clavulanic acid, ampicillin, cefazolin, imipenem, oxacillin and ticarcillin/clavulanic acid. Another limitation and struggle faced by most veterinarians treating odontogenic abscesses, and encountered in this study, is that most rabbits were already on antibiotics at the time of culture collection. With empirical selection of antimicrobials, or the potential for polymicrobial therapy to have already been initiated in these rabbits, concerns for the selection of resistant strains also arise. Additionally, obtaining cultures before antibiotic use improves the chances of isolating the offending microorganism, thus improving patient care. Obtaining cultures after antimicrobial therapy has been initiated can result in inconclusive results because organisms that would normally be detected may not necessarily grow after exposure to an antibiotic agent. Studies in humans have demonstrated that long term antimicrobial use can result in the persistence of antibiotic resistance genes, further complicating interpretation of culture results.30 Additionally, without anaerobic susceptibilities, selection of antimicrobials is still empirical in these cases for the anaerobic isolates. Lastly, and an important consideration that represents a limitation of this study and limitation of treatment of rabbit odontogenic abscesses is the production of biofilm by many of these bacteria. The production of biofilm has the potential to interfere with the success of antimicrobial therapy.31 Many of the agents cultured in this study, including Streptococcus species, Pseudomonas species, Staphylococcus species and Pasteurella species are recognised as biofilm-producing bacteria, which plays a significant role in the pathogenesis and virulence of the bacteria.31 32 The biofilm can also interfere with accurate determination of MIC data for the bacteria and response to therapy.33

This study determined that organisms isolated from rabbit odontogenic abscesses in this population consist of a mixture and variable number of aerobic and anaerobic Gram-positive and Gram-negative organisms, including most often P aeruginosa, Pasteurella species, Fusobacterium species and Peptostreptococcus species. It is important to direct antimicrobial therapy against these organisms when treating odontogenic abscesses in rabbits as a part of the multimodal treatment recommended for this clinical presentation. Anaerobes present the biggest challenge because susceptibility results are usually not known. Culture and susceptibility testing of any abscess is recommended to provide optimal antimicrobial treatment whenever possible, and this should always be combined with other appropriate therapies including surgical intervention and pain management.

References

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Footnotes

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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