Doi:10.1016/s0378-1135(03)00061-0

Veterinary Microbiology 94 (2003) 39–45 Antimicrobial susceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, 2108 Tupper Hall, Davis, CA 95616, USA Received 6 September 2002; received in revised form 14 January 2003; accepted 24 January 2003 Abstract
Clostridium difficile and Clostridium perfringens are anaerobic, Gram-positive bacilli that are common causes of enteritis and enterotoxemias in both domestic animals and humans. Both or-ganisms have been associated with acute and chronic large and small bowel diarrhea, and acutehemorrhagic diarrheal syndrome in the dog. The objective of this study was to determine the in vitroantimicrobial susceptibilities of canine C. difficile and C. perfringens isolates in an effort to optimizeantimicrobial therapy for dogs with clostridial-associated diarrhea. The minimum inhibitory concen-trations (MIC) of antibiotics recommended for treating C. difficile (metronidazole, vancomycin) andC. perfringens-associated diarrhea in the dog (ampicillin, erythromycin, metronidazole, tetracycline,tylosin) were determined for 70 canine fecal C. difficile isolates and 131 C. perfringens isolates. AllC. difficile isolates tested had an MIC of ≤1 for both metronidazole and vancomycin. Ninety-five per-cent (124/131) of C. perfringens isolates tested had an MIC for ampicillin of ≤0.125 ␮g/ml. Two C.
perfringens isolates had an MIC of ≥256 ␮g/ml for both erythromycin and tylosin. A third C. perfrin-gens isolate had an MIC of 32 ␮g/ml for metronidazole. Based on the results of this study, ampicillin,erythromycin, metronidazole, and tylosin appear to be effective antibiotics for the treatment of C.
perfringens-associated diarrhea, although resistant strains do exist. However, because there is limitedinformation regarding breakpoints for veterinary anaerobes, and because intestinal concentrations arenot known, in vitro results should be interpreted with caution.
2003 Elsevier Science B.V. All rights reserved.
Keywords: Dog; Diarrhea; MIC; Drug resistance; Clostridium spp.
∗ Corresponding author. Tel.: +1-530-752-1387; fax: +1-530-752-9620.
E-mail address: slmarks@ucdavis.edu (S.L. Marks).
0378-1135/03/$ – see front matter 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1135(03)00061-0 S.L. Marks, E.J. Kather / Veterinary Microbiology 94 (2003) 39–45 1. Introduction
Clostridium difficile and Clostridium perfringens are spore-forming, anaerobic, Gram- positive bacilli that are common causes of enteritis and enterotoxemias in domestic animalsand humans Both organisms have been implicated as causes of canineacute and chronic large and small bowel diarrhea, as well as an acute hemorrhagic diarrhealsyndrome C. perfringens enterotoxin (CPE) is detected in 28–34% of diarrheic dogs and 5–14% ofnondiarrheic dogs, while C. difficile toxin A or B is detected in 13–21% of diarrheic dogs and2–7% of nondiarrheic dogs (of C. difficileor C. perfringens-associated diarrhea in the dog is typically made based on detection of tox-ins in fecal specimens in conjunction with clinical signs, and antimicrobial administrationis typically reserved for cases of severe diarrhea or for dogs with systemic manifestationsof disease. Antibiotics recommended for the treatment of C. difficile-associated diarrhea indogs include metronidazole, and to a lesser extent, vancomycin, whereas antibiotics rec-ommended for the treatment of C. perfringens-associated diarrhea include beta-lactams(ampicillin and amoxicillin), macrolides (erythromycin and tylosin), metronidazole, andtetracyclines However, the recommendationsfor treating clostridial-associated diarrhea in dogs have mostly been extrapolated from thehuman literature, as there is limited information concerning the in vitro or in vivo susceptibil-ities of these canine fecal isolates to these antibiotics. This information is particularly impor-tant in the face of increasing reports of resistance among anaerobic bacteria isolated fromboth humans and animals. Several studies have documented multiply antibiotic-resistantstrains of C. perfringens (and a recent study conducted at the University of California Davis Veterinary Medi-cal Teaching Hospital (VMTH) in horses with C. difficile-associated diarrhea reported that19% of the C. difficile isolates were resistant to metronidazole, one of the most commonlyadministered antibiotics used to kill this organism Because exposure toantibiotics at concentrations close to or below the MIC for a particular organism is one factorinvolved in the selection of resistant bacterial strains (administra-tion of antibiotics to treat the diseases associated with these organisms may in fact promoteresistant strains if the fecal concentrations fall below the MIC values for each antibiotic.
The objectives of this study were to determine the in vitro antimicrobial susceptibilities ofcanine C. difficile isolates to two antibiotics, and to determine the in vitro susceptibilitiesof canine C. perfringens isolates to five commonly utilized antibiotics to provide data onwhich to optimize antimicrobial therapy for dogs with clostridial-associated diarrhea.
2. Materials and methods
Seventy C. difficile isolates, each obtained from an individual dog (59 diarrheic dogs, 11 nondiarrheic dogs), and 131 C. perfringens isolates, each obtained from an individual S.L. Marks, E.J. Kather / Veterinary Microbiology 94 (2003) 39–45 dog (72 diarrheic dogs, 59 nondiarrheic dogs), were evaluated. All isolates were derivedfrom diarrheic and nondiarrheic dogs that presented to the University of California DavisVeterinary Medical Teaching Hospital between 1995 and 2001. Isolates were frozen at−80 ◦C in 20% skim milk until analyzed. Frozen isolates were thawed, plated onto reduced5% sheep blood agar (SBA) plates, and incubated at 37 ◦C in an anaerobic chamber (BactronIV, Sheldon Manufacturing Inc., Cornelius, OR). C. perfringens isolates were identifiedbased on gram-stain morphology, a double zone of hemolysis on SBA, and lecithinaseproduction on McClung’s egg yolk agar (EYA). C. difficile isolates were identified on thebasis of gram-stain, colony morphology and color on cycloserine–cefoxitin–fructose agar(CCFA), fluorescence, odor, and detection of l-proline aminopeptidase activity utilizing acommercially available kit (PRO Kit, Remel, Lenexa, KS).
Minimum inhibitory concentrations (MICs) for metronidazole and vancomycin were determined for all C. difficile isolates (n = 70) and MICs for ampicillin, erythromycin,metronidazole, tetracycline, and tylosin were determined for all C. perfringens isolates (n =131). All MIC values were determined using the National Committee for Clinical LaboratoryStandards Reference Agar Dilution (Brucella plates supplemented withhemin, Vitamin K1 and laked sheep blood, were prepared on the day of testing for eachantibiotic at the following antibiotic concentrations: 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64,128, and 256 ␮g/ml agar. Twenty-four hour cultures of each isolate were used to inoculatereduced brucella broth to match the turbidity of a 0.5 McFarland standard. Approximately1 ml of the inoculated broth was then transferred to a well of a Steer’s replicator seed blockand plates were inoculated from the lowest concentration to the highest. Control platescontaining no antibiotic were inoculated before and after the antibiotic plates as well asbetween each antibiotic series. Inoculated plates were incubated anaerobically at 37 ◦C andMICs were read at 24 and 48 h. For quality control, Staphylococcus aureus (ATCC 29213),Enterococcus faecalis (ATCC 29212), Bacteroides fragilis (ATCC 25285), Bacteroidesthetaiotamicron (ATCC 29741), and Eggerthella lenta (ATCC 43055) were included oneach plate. MIC values for each quality control organism were within reported ranges.
Each dog’s medical record was evaluated to determine the antibiotic history in the 6 months prior to collection of isolates. The Mann–Whitney test was used to compare theMIC values obtained for each isolate with antibiotic exposure within 6 months of fecalcollection for each dog. P-values <0.05 were considered statistically significant.
3. Results
All C. difficile isolates were susceptible to metronidazole and vancomycin at ≤1 ␮g/ml.
The MIC50 and MIC90 for metronidazole was 0.25 and 0.5 ␮g/ml, respectively. Both the S.L. Marks, E.J. Kather / Veterinary Microbiology 94 (2003) 39–45 Table 1MIC distribution of ampicillin, erythromycin, metronidazole, tetracycline, and tylosin for 131 canine Clostridiumperfringens isolates Number of C. perfringens isolates with MIC values (␮g/ml) MIC50 and MIC90 for vancomycin were 1 ␮g/ml. Antibiotic histories were available for 63of 70 dogs from which C. difficile was isolated. None of the dogs had received vancomycin6 months prior to fecal collection, and 9/63 dogs (14%) had received metronidazole within6 months of fecal collection.
The MIC values of each antibiotic for C. perfringens are summarized in five percent (124/131) of C. perfringens isolates tested had an MIC of ≤0.125 ␮g/ml forampicillin. The MIC90 for erythromycin and tylosin was 4 and 1 ␮g/ml, respectively. One C.
perfringens isolate had an MIC of ≥256 ␮g/ml for both erythromycin and tylosin, 2 ␮g/mlfor ampicillin, and 32 ␮g/ml for tetracycline. A second isolate had an MIC of >256 ␮g/mlfor erythromycin and 32 ␮g/ml for tylosin. The MIC90 for metronidazole was 2 ␮g/ml.
One isolate had an MIC of 64 ␮g/ml for metronidazole. The MIC90 for tetracycline was16 ␮g/ml. Antibiotic treatment histories of 126 of the 131 dogs from which C. perfringenswas isolated, were evaluated for determination of antibiotic administration in the 6 monthsprior to fecal collection. Ten dogs (8%) had received beta-lactam antibiotics within 6 monthsof fecal collection (five received amoxicillin/clavulanic acid, two received amoxicillin, andthree received ampicillin). The isolates obtained from these dogs were all susceptible toampicillin at 0.125 ␮g/ml. Three dogs had received tylosin prior to fecal collection and onedog had received erythromycin. One of the two C. perfringens isolates with a high MICfor both erythromycin and tylosin (≥256 ␮g/ml) was obtained from a dog that had receivedtylosin 2 months previously. The second C. perfringens isolate with a high MIC for themacrolides was obtained from a diarrheic dog with no history of macrolide antibiotic ad-ministration. Thirteen of the 126 dogs (10%) whose antibiotic histories could be determinedhad received metronidazole within 6 months of fecal collection. The isolates obtained fromthese dogs all had MICs of ≤2 ␮g/ml for metronidazole. Two of 126 dogs (1.6%) had re-ceived doxycycline within 6 months of fecal collection. The tetracycline MICs for thesetwo isolates were 0.125 and 8 ␮g/ml. Analysis of antibiotic exposure and in vitro suscep-tibilities for each antibiotic revealed no association between antibiotic exposure and anincreasing MIC.
S.L. Marks, E.J. Kather / Veterinary Microbiology 94 (2003) 39–45 4. Discussion
Because there is a paucity of information regarding breakpoints for anaerobic veteri- nary isolates to commonly utilized antibiotics, interpretation of in vitro MIC values isdifficult. However, some inferences can be made. The two C. perfringens isolates thathad an MIC for erythromycin of >256 ␮g/ml can be referred to as resistant based on pre-vious reported MICs of erythromycin-resistant C. perfringens strains (One of the two dogs from which a C. perfringens isolate witha high MIC for both erythromycin and tylosin (MIC > 256 ␮g/ml) was obtained, had re-ceived tylosin for a 1 month period at a very low dose (5 mg/kg twice daily), with treat-ment ending approximately 2 months before the fecal analysis was performed. In addition,the isolate obtained from this dog had an MIC of 2 ␮g/ml for ampicillin, and an MICof 32 ␮g/ml for tetracycline. Only one C. perfringens isolate, obtained from a dog withnosocomial diarrhea, had an MIC supportive of a resistant isolate (MIC of 64 ␮g/ml).
Metronidazole resistance among human and animal clinical C. perfringens isolates is rare,and to the authors’ knowledge, this is the first report of a metronidazole-resistant clini-cal C. perfringens isolate obtained from a dog. This same isolate also had a high MIC(64 ␮g/ml) for tetracycline. Furthermore, the dog had no history of antibiotic exposurewithin 6 months of fecal analysis. Although the majority of clostridial isolates evaluatedin this study were susceptible to the antibiotics tested, the high percentage of C. perfrin-gens isolates (21%) with an MIC of ≥16 ␮g/ml for tetracycline (the breakpoint for resis-tance for human anaerobes and veterinary isolates (andthe discovery of multiply-resistant C. perfringens strains emphasizes the importance ofsurveying antimicrobial susceptibility profiles of common bacterial pathogens. Based onthe results of this study, tetracycline is a poor antimicrobial choice for the treatment ofC. perfringens-associated diarrhea in the dog, due to the reduced susceptibility amongcanine C. perfringens isolates. Furthermore, it has recently been shown that exposure totetracycline concentrations below the MIC may actually induce conjugative transfer oftetracycline resistance plasmids from Bacteroides species, and it has been suggested thatgene transfer can occur not only within the Bacteroides genus but also between Bacteroidesspp. and Gram-positive bacteria (Administration of tetracycline,especially at a low dose, for treatment of C. perfringens-associated diarrhea could notonly select for resistant strains, but might potentially stimulate the transfer of tetracyclineresistance.
Because of the paucity of information pertaining to clostridial MIC breakpoints for specific antibiotics in dogs, determination of intrinsic resistance ascertained bygenetic analysis should provide additional clarification of these putative resistantisolates. These studies are currently underway to determine the presence of establishedtetracycline and macrolide resistance genes. In addition, fecal concentrations of ampi-cillin, metronidazole, and tylosin are currently being determined utilizing an HPLC method.
Determination of MIC values for C. perfringens and C. difficile used in conjunction withboth molecular resistance determinants and fecal antibiotic concentrations should pro-vide insight as to whether bactericidal intracolonic concentrations are being reached inthe lumen of the bowel, and may provide further information for antibioticdosing.
S.L. Marks, E.J. Kather / Veterinary Microbiology 94 (2003) 39–45 5. Conclusion
The results of this study emphasize the importance of careful selection of antimicrobials used to treat dogs with clostridial-associated diarrhea. For the treatment of C. perfringens-associated diarrhea, ampicillin, metronidazole, and tylosin appeared to be the most effectiveantibiotics, although it should be noted that resistant strains do exist. Metronidazole appearsto be an appropriate antibiotic for the treatment of canine C. difficile-associated diarrhea,due to the fact that all isolates were susceptible to ≤1 ␮g/ml of antibiotic.
Acknowledgements
This project was supported by a grant from the Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis, USA.
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