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Antimicrobial susceptibility of Clostridium difficile isolated from neonatal pigs with enteritis
ARTICLE IN PRESS
Anaerobe 10 (2004) 47–50
Veterinary anaerobes and diseases
Antimicrobial susceptibility of Clostridium difficile isolated from neonatal pigs with enteritis Karen W. Posta, J. Glenn Songerb,* a
Rollins Animal Disease Diagnostic Laboratory, North Carolina Department of Agriculture and Consumer Services, Raleigh, NC 27601, USA b Department of Veterinary Science and Microbiology, The University of Arizona, Tucson, AZ 857212, USA Received 21 July 2003; received in revised form 2 January 2004; accepted 20 January 2004
Abstract The minimum inhibitory concentration (MIC) of eight antimicrobial agents was determined by the agar dilution method for 80 isolates of Clostridium difficile from neonatal pigs with enteritis. MICs50 for erythromycin, tilmicosin, and tylosin were relatively low (0.25–0.50 mg/mL), but MICs90 (64 or X256 mg/mL) suggest in vivo resistance of a proportion of isolates. Susceptibility to tetracycline varied widely, with MIC50 and MIC90 of 8 and 32 mg/mL, respectively. The MICs90 for tiamulin (8 mg/mL) and virginiamycin (16 mg/mL) suggest moderate susceptibility. Bacitracin and ceftiofur (MICs90X256 mg/mL) have little activity against C. difficile. Tiamulin and virginiamycin may decrease fecal shedding of C. difficile by sows, and erythromycin, tetracycline, and tylosin may be useful for treatment of infected piglets. r 2004 Elsevier Ltd. All rights reserved. Keywords: Antimicrobial susceptibility; Porcine enteric disease; Clostridium difficile; Neonatal enteritis
1. Introduction Clostridium difficile is a cause of pseudomembranous colitis and antibiotic-associated diarrhea in humans, horses, and laboratory rodents [1–9]. It has recently been recognized as a significant etiologic agent of enteritis in suckling swine [10,11]. Sows may serve as a source of infection for their offspring. Human strains of C. difficile have a wide range of susceptibility to erythromycin and tetracycline [12]. Nearly all human isolates are susceptible to ampicillin, penicillin G, metronidazole, carbapenems, and vancomycin and 90% are susceptible to tetracycline and erythromycin; minimum inhibitory concentrations (MIC) for clindamycin and fluoroquinolones are variable, and most are insusceptible to cephalosporins [13,14]. Resistance to bacitracin has been reported [15,16]. The drugs of choice for treatment of human infections are vancomycin and metronidazole [17]. Knowledge of antimicrobial susceptibility of animal strains of C. difficile is limited. Equine isolates have low
*Corresponding author. Tel.: +1-520-621-2962; fax: +1-520-6216366. E-mail address: [email protected] (J.G. Songer). 1075-9964/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2004.01.003
MICs for chloramphenicol, rifampin, vancomycin, and metronidazole and a high MIC for bacitracin [2]. Antimicrobial susceptibility of porcine C. difficile strains has not previously been investigated, so we used an agar dilution method [18] to examine 80 recent isolates from pigs. Agents were selected on the basis of previously demonstrated in vitro activity against anaerobes and label claims which might allow their use to limit fecal shedding of C. difficile by sows or to treat infected piglets.
2. Materials and methods 2.1. Isolates and strains Eighty C. difficile isolates were obtained from neonatal piglets with enteritis. Specimens of feces or colonic contents were subjected to bacteriologic culture on C. difficile selective medium [CDSA, brain–heart infusion agar (Difco) supplemented with 5% defibrinated bovine blood, 0.1% taurocholate, and 250 mg cycloserine and 8 mg cefoxitin per mL]. Plates were incubated in an anaerobic atmosphere for 48 h at 37 C and presumptive identification of C. difficile was made on the basis of colony morphology on CDSA and
ARTICLE IN PRESS 48
K.W. Post, J.G. Songer / Anaerobe 10 (2004) 47–50
cefoxitin cycloserine fructose agar (CCFA, Difco), Gram stain, odor, and production of l-aminopeptidase. Identification was confirmed by a rapid test system (RapID ANAII, Remel, Inc., Lenexa, KS). Isolates were stored in 25% glycerol at 80 C until tested. Control strains were Bacteroides fragilis ATCC 25285 and Eggerthella lenta ATCC 43055. Current National Committee for Clinical Laboratory Standards (NCCLS) recommendations do not include clostridia among the organisms for quality-control of antimicrobial susceptibility tests, but we included C. difficile ATCC 9689 and C. perfringens ATCC 13124 on each plate to evaluate the internal reproducibility of the method. 2.2. Determination of MICs Susceptibilities were determined by the reference agar dilution procedure recommended by NCCLS [18]. Serial two-fold dilutions of bacitracin, erythromycin (SigmaAldrich, St. Louis, MO), tetracycline HCl (US Pharmacopeia, Rockville, MD), ceftiofur (Pharmacia Animal Health, Kalamazoo, MI), tiamulin (Boehringer Ingelheim Vetmedica, St. Joseph, MO) tilmicosin (Eli Lilly, Indianapolis, IN), tylosin tartrate (Sigma-Aldrich, St. Louis, MO) and virginiamycin (Phibro Animal Health, Fairfield, NJ) were incorporated into Brucella agar, with vitamin K (1 mg/mL), hemin (5 mg/mL), and laked sheep blood (5%), at final concentrations of 0.125–256 mg/mL. There are no current NCCLS quality control ranges for activity of macrolide antibiotics against clostridia, but a previous NCCLS document [19] listed the MIC of clindamycin for C. perfringens ATCC 13124. Thus, clindamycin was used at the same range of concentrations (0.125–256 mg/mL) for quality control of tests on tilmicosin and tylosin. Prior to antimicrobial susceptibility testing, isolates were subcultured twice on prereduced Brucella agar supplemented with bovine blood (5%), hemin (5 mg/ mL), and vitamin K1 (mg/mL). Colonies were suspended in trypticase soy broth to an optical density equivalent to that of a McFarland 0.5 standard. Plates were inoculated with a Steer’s replicator which delivered 1–2 mL per spot. Plates were incubated at 37 C in an atmosphere of 5% H2:5% CO2:90% N2, and were examined after 48 h. The lowest dilution of antimicrobial which affected growth on the test plate (as compared to control plates containing no antimicrobial) constituted the MIC. MIC50 and MIC90 were the concentrations at which 50% and 90%, respectively, of strains were inhibited.
3. Results and discussion Others have reported in vitro anti-clostridial activity of tylosin [20], and our findings concur; MICs50 for
erythromycin, tilmicosin, and tylosin were relatively low (0.25–0.50 mg/mL). However, MICs90 (64 orX256 mg/mL) suggest that a proportion of isolates will not be susceptible in vivo. Human isolates of C. difficile also exhibit this bimodal pattern of susceptibility to erythromycin [12,14] and it seems likely that this is due to the non-uniform distribution of a resistance element in the population. C. difficile from humans vary widely in susceptibility to tetracycline [12,14], and our findings were similar, with MIC50 and MIC90 of 8 and 32 mg/mL, respectively. Tiamulin, a pleuromutilin derivative used in the treatment of animal infections with Gram-positive and Gram-negative bacteria, is active against anaerobic bacteria [21]. Our MIC90 for tiamulin (8 mg/mL) suggests moderate susceptibility, as did that for virginiamycin (16 mg/mL). Bacitracin had little activity against C. difficile (MICX256 mg/mL), in keeping with results of MIC testing of human and equine strains [2,16]. The previously reported high MICs for cephalosporins [13,14] mirror our findings with ceftiofur (MIC90X256 mg/mL, Table 1), a third generation cephalosporin used exclusively in veterinary medicine. Results for the reference antimicrobial, clindamycin, were within acceptable limits [19] and MICs for other quality control strains were within acceptable limits [18,22]. Antimicrobials such as bacitracin, tetracycline, tiamulin, tilmicosin, tylosin, and virginiamycin are administered to swine in feed. Intestinal levels of antimicrobials administered by this route are difficult to measure, so the MIC may not reliably predict in vivo efficacy. However, negative clinical outcome might be expected for those agents with a MIC higher than feed levels. Furthermore, parenteral administration may not yield gut concentrations of the agent which are equivalent to the achievable serum concentrations which have traditionally been used to interpret antimicrobial susceptibility. Drugs administered parenterally (ceftiofur, erythromycin, tetracycline and tylosin) and having higher MICs would not be predicted to be as efficacious as those with lower MICs. Based upon low MIC values, tiamulin and virginiamycin might be useful in decreasing fecal shedding of C. difficile by sows, while bacitracin may not be efficacious in this regard. Fifty percent of strains examined had low MICs for erythromycin, tetracycline, and tylosin, so these agents may be useful for treatment of infected piglets. The MIC90 for ceftiofur (>256 mg/mL) suggests that it might not be efficacious in the treatment of neonatal enteritis due to C. difficile. Antimicrobial susceptibility testing of veterinary anaerobes and veterinary-exclusive agents is affected by general lack of standardization; antimicrobial agents have not been evaluated using NCCLS-approved susceptibility testing methods and there are no
ARTICLE IN PRESS K.W. Post, J.G. Songer / Anaerobe 10 (2004) 47–50
49
Table 1 Antimicrobial susceptibilities of 80 C. difficile isolates from neonatal pigs with enteritis Antimicrobial
Range (mg/mL)
MIC50 (mg/mL)a
MIC90 (mg/mL)a
Bacitracin Ceftiofur Erythromycin Tetracycline Tiamulin Tilmicosin Tylosin Virginiamycin
>256 256–>256 0.125–256 0.125–128 0.125–256 0.125–256 0.125–128 0.125–16
>256 256 0.5 8 4 0.5 0.25 0.25
>256 >256 256 32 8 >256 64 2
a
MIC50 and MIC90 concentrations at which the growth of 50% and 90%, respectively, of the isolates is inhibited.
interpretive criteria available for veterinary anaerobic pathogens against these drugs and there are no interpretive criteria to apply to results of testing of veterinary anaerobic pathogens. In many cases, MICs have not been determined by any method. In addition, it is difficult to interpret MIC information on enteric pathogens. The generally accepted rule bases efficacy upon achievement in vivo of a peak concentration of four times the MIC; intestinal concentrations are unknown for many antimicrobials, making it difficult to draw valid conclusions about susceptibility or resistance. Thus, application of in vitro susceptibility information must be strongly influenced by the resulting clinical response, since therapy based solely upon MICs may not translate to in vivo efficacy. Clinical studies will be needed to establish field efficacy of these agents. Continued efforts are needed to develop standardized methods for testing veterinary antimicrobials and enteric pathogens.
Acknowledgements This work was supported in part by funds from the National Pork Board, Alpharma, and Novartis Animal Vaccines.
References [1] Blankenship-Paris TL, Walton BJ, Hayes YO. Clostridium difficile infection in hamsters fed an atherogenic diet. Vet Pathol 1995;32:269–73. [2] Jang SS, Hansen HM, Breher JE. Antimicrobial susceptibilities of equine isolates of Clostridium difficile and molecular characterization of metronidazole-resistant strains. Clin Infect Dis 1997;25(Suppl 2):S266–7. [3] Jones RL, Adney WS, Alexander AF. Hemorrhagic necrotizing enterocolitis associated with Clostridium difficile infection in four foals. J Am Vet Med Assoc 1988;193:76–9. [4] Knoop FC, Owens M, Crocker IC. Clostridium difficile: clinical disease and diagnosis. Clin Microbiol Rev 1993;6:251–65.
[5] Lowe BR, Fox J, Bartlett JG. Clostridium difficile-associated cecitis in guinea pigs exposed to penicillin. Am J Vet Res 1980;41:1277–9. [6] Lyerly DM, Krivan H, Wilkins TD. Clostridium difficile: its disease and toxins. Clin Microbiol Rev 1988;1:1–18. [7] Madewell BR, Tang YJ, Jang SS. Apparent outbreaks of Clostridium difficile-associated diarrhea in a veterinary medical teaching hospital. J Vet Diagn Invest 1995;7:343–6. [8] Perrin J, Cosmetatos I, Gallusser A. Clostridium difficile with typholcolitis in an adult horse. J Vet Diagn Invest 1993;5:99–101. [9] Rehg JE, Lu YS. Clostridium difficile typhlitis in hamsters not associated with antibiotic therapy. J Am Vet Med Assoc 1982;181:1422–3. [10] Songer JG, Post KW, Larson DJ. Infection of neonatal swine with Clostridium difficile. Swine Health Prod 2000;8:185–9. [11] Waters EH, Orr JP, Clark EG, Schaufele CM. Typhlocolitis caused by Clostridium difficile in suckling piglets. J Vet Diagn Invest 1998;10:104–8. [12] Delmee M, Avesani V. Correlation between serogroup and susceptibility to chloroamphenicol, clindamycin, erythromycin, rifampin and tetracycline. J Antimicrob Chemother 1988;22: 325–31. [13] Chow AW, Cheng N, Bartlett KH. In vitro susceptibility of Clostridium difficile to new b-lactam and quinolone antibiotics. Antimicrob Agents Chemother 1985;28:842–4. [14] Dzink J, Bartlett JG. In vitro susceptibility of Clostridium difficile isolates from patient with antibiotic-associated diarrhea or colitis. Antimicrob Agents Chemother 1980;17:695–8. [15] Bacon AE, McGrath S, Fekety R, Holloway WJ. In vitro synergy studies with Clostridium difficile. Antimicrob Agents Chemother 1991;35:582–3. [16] Citron DM, Warren YA, Tyrrell KL. Comparative in vitro activity of ramoplanin, teicoplanin, vancomycin, linezolid, bacitracin, and four other antimicrobials against intestinal anaerobic bacteria. 41st Interscience Conference on Antimicrobial Chemotherapy, 2001, p. 193. [17] Barbut F, Decre D, Burghoffer B, Lesage D, Delisle F, Lalande V, Delmee M, Avesani V, Sano N, Coudert C, Petit J-C. Antimicrobial susceptibilities and serogroups of clinical strains of Clostridium difficile isolated in France in 1991 and 1997. J Clin Microbiol 1999;43:2607–11. [18] National Committee for Clinical Laboratory Standards. Methods for antimicrobial susceptibility testing of anaerobic bacteria, 5th ed. Approved standard M11-A5. Wayne, PA: National Committee for Clinical Laboratory Standards; 2001. [19] National Committee for Clinical Laboratory Standards. Methods for antimicrobial susceptibility testing of anaerobic bacteria, 2nd ed. Approved standard M11-A2. Villanova, PA: National Committee for Clinical Laboratory Standards; 1990.
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[20] Dutta GN, Devriese LA, van Assche PF. Susceptibility of clostridia from farm animals to 21 antimicrobial agents including some used for growth promotion. J Antimicrob Chemother 1983;12:347–56. [21] Werner G, Laber G, Schutze E. In vitro activity of tiamulin (81.723 hfu) a new pleuromutilin derivative against clinically significant anaerobes. J Antibiot 1978;30:756–60.
[22] Benning VR, Mathers JJ. Comparison of agar dilution and broth microdilution methods of anaerobic antimicrobial susceptibility testing using several veterinary antibiotics against Clostridium perfringens strains originating from porcine and avian sources. Anaerobe 1999;5:561–9.
Anaerobe 10 (2004) 47–50
Veterinary anaerobes and diseases
Antimicrobial susceptibility of Clostridium difficile isolated from neonatal pigs with enteritis Karen W. Posta, J. Glenn Songerb,* a
Rollins Animal Disease Diagnostic Laboratory, North Carolina Department of Agriculture and Consumer Services, Raleigh, NC 27601, USA b Department of Veterinary Science and Microbiology, The University of Arizona, Tucson, AZ 857212, USA Received 21 July 2003; received in revised form 2 January 2004; accepted 20 January 2004
Abstract The minimum inhibitory concentration (MIC) of eight antimicrobial agents was determined by the agar dilution method for 80 isolates of Clostridium difficile from neonatal pigs with enteritis. MICs50 for erythromycin, tilmicosin, and tylosin were relatively low (0.25–0.50 mg/mL), but MICs90 (64 or X256 mg/mL) suggest in vivo resistance of a proportion of isolates. Susceptibility to tetracycline varied widely, with MIC50 and MIC90 of 8 and 32 mg/mL, respectively. The MICs90 for tiamulin (8 mg/mL) and virginiamycin (16 mg/mL) suggest moderate susceptibility. Bacitracin and ceftiofur (MICs90X256 mg/mL) have little activity against C. difficile. Tiamulin and virginiamycin may decrease fecal shedding of C. difficile by sows, and erythromycin, tetracycline, and tylosin may be useful for treatment of infected piglets. r 2004 Elsevier Ltd. All rights reserved. Keywords: Antimicrobial susceptibility; Porcine enteric disease; Clostridium difficile; Neonatal enteritis
1. Introduction Clostridium difficile is a cause of pseudomembranous colitis and antibiotic-associated diarrhea in humans, horses, and laboratory rodents [1–9]. It has recently been recognized as a significant etiologic agent of enteritis in suckling swine [10,11]. Sows may serve as a source of infection for their offspring. Human strains of C. difficile have a wide range of susceptibility to erythromycin and tetracycline [12]. Nearly all human isolates are susceptible to ampicillin, penicillin G, metronidazole, carbapenems, and vancomycin and 90% are susceptible to tetracycline and erythromycin; minimum inhibitory concentrations (MIC) for clindamycin and fluoroquinolones are variable, and most are insusceptible to cephalosporins [13,14]. Resistance to bacitracin has been reported [15,16]. The drugs of choice for treatment of human infections are vancomycin and metronidazole [17]. Knowledge of antimicrobial susceptibility of animal strains of C. difficile is limited. Equine isolates have low
*Corresponding author. Tel.: +1-520-621-2962; fax: +1-520-6216366. E-mail address: [email protected] (J.G. Songer). 1075-9964/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2004.01.003
MICs for chloramphenicol, rifampin, vancomycin, and metronidazole and a high MIC for bacitracin [2]. Antimicrobial susceptibility of porcine C. difficile strains has not previously been investigated, so we used an agar dilution method [18] to examine 80 recent isolates from pigs. Agents were selected on the basis of previously demonstrated in vitro activity against anaerobes and label claims which might allow their use to limit fecal shedding of C. difficile by sows or to treat infected piglets.
2. Materials and methods 2.1. Isolates and strains Eighty C. difficile isolates were obtained from neonatal piglets with enteritis. Specimens of feces or colonic contents were subjected to bacteriologic culture on C. difficile selective medium [CDSA, brain–heart infusion agar (Difco) supplemented with 5% defibrinated bovine blood, 0.1% taurocholate, and 250 mg cycloserine and 8 mg cefoxitin per mL]. Plates were incubated in an anaerobic atmosphere for 48 h at 37 C and presumptive identification of C. difficile was made on the basis of colony morphology on CDSA and
ARTICLE IN PRESS 48
K.W. Post, J.G. Songer / Anaerobe 10 (2004) 47–50
cefoxitin cycloserine fructose agar (CCFA, Difco), Gram stain, odor, and production of l-aminopeptidase. Identification was confirmed by a rapid test system (RapID ANAII, Remel, Inc., Lenexa, KS). Isolates were stored in 25% glycerol at 80 C until tested. Control strains were Bacteroides fragilis ATCC 25285 and Eggerthella lenta ATCC 43055. Current National Committee for Clinical Laboratory Standards (NCCLS) recommendations do not include clostridia among the organisms for quality-control of antimicrobial susceptibility tests, but we included C. difficile ATCC 9689 and C. perfringens ATCC 13124 on each plate to evaluate the internal reproducibility of the method. 2.2. Determination of MICs Susceptibilities were determined by the reference agar dilution procedure recommended by NCCLS [18]. Serial two-fold dilutions of bacitracin, erythromycin (SigmaAldrich, St. Louis, MO), tetracycline HCl (US Pharmacopeia, Rockville, MD), ceftiofur (Pharmacia Animal Health, Kalamazoo, MI), tiamulin (Boehringer Ingelheim Vetmedica, St. Joseph, MO) tilmicosin (Eli Lilly, Indianapolis, IN), tylosin tartrate (Sigma-Aldrich, St. Louis, MO) and virginiamycin (Phibro Animal Health, Fairfield, NJ) were incorporated into Brucella agar, with vitamin K (1 mg/mL), hemin (5 mg/mL), and laked sheep blood (5%), at final concentrations of 0.125–256 mg/mL. There are no current NCCLS quality control ranges for activity of macrolide antibiotics against clostridia, but a previous NCCLS document [19] listed the MIC of clindamycin for C. perfringens ATCC 13124. Thus, clindamycin was used at the same range of concentrations (0.125–256 mg/mL) for quality control of tests on tilmicosin and tylosin. Prior to antimicrobial susceptibility testing, isolates were subcultured twice on prereduced Brucella agar supplemented with bovine blood (5%), hemin (5 mg/ mL), and vitamin K1 (mg/mL). Colonies were suspended in trypticase soy broth to an optical density equivalent to that of a McFarland 0.5 standard. Plates were inoculated with a Steer’s replicator which delivered 1–2 mL per spot. Plates were incubated at 37 C in an atmosphere of 5% H2:5% CO2:90% N2, and were examined after 48 h. The lowest dilution of antimicrobial which affected growth on the test plate (as compared to control plates containing no antimicrobial) constituted the MIC. MIC50 and MIC90 were the concentrations at which 50% and 90%, respectively, of strains were inhibited.
3. Results and discussion Others have reported in vitro anti-clostridial activity of tylosin [20], and our findings concur; MICs50 for
erythromycin, tilmicosin, and tylosin were relatively low (0.25–0.50 mg/mL). However, MICs90 (64 orX256 mg/mL) suggest that a proportion of isolates will not be susceptible in vivo. Human isolates of C. difficile also exhibit this bimodal pattern of susceptibility to erythromycin [12,14] and it seems likely that this is due to the non-uniform distribution of a resistance element in the population. C. difficile from humans vary widely in susceptibility to tetracycline [12,14], and our findings were similar, with MIC50 and MIC90 of 8 and 32 mg/mL, respectively. Tiamulin, a pleuromutilin derivative used in the treatment of animal infections with Gram-positive and Gram-negative bacteria, is active against anaerobic bacteria [21]. Our MIC90 for tiamulin (8 mg/mL) suggests moderate susceptibility, as did that for virginiamycin (16 mg/mL). Bacitracin had little activity against C. difficile (MICX256 mg/mL), in keeping with results of MIC testing of human and equine strains [2,16]. The previously reported high MICs for cephalosporins [13,14] mirror our findings with ceftiofur (MIC90X256 mg/mL, Table 1), a third generation cephalosporin used exclusively in veterinary medicine. Results for the reference antimicrobial, clindamycin, were within acceptable limits [19] and MICs for other quality control strains were within acceptable limits [18,22]. Antimicrobials such as bacitracin, tetracycline, tiamulin, tilmicosin, tylosin, and virginiamycin are administered to swine in feed. Intestinal levels of antimicrobials administered by this route are difficult to measure, so the MIC may not reliably predict in vivo efficacy. However, negative clinical outcome might be expected for those agents with a MIC higher than feed levels. Furthermore, parenteral administration may not yield gut concentrations of the agent which are equivalent to the achievable serum concentrations which have traditionally been used to interpret antimicrobial susceptibility. Drugs administered parenterally (ceftiofur, erythromycin, tetracycline and tylosin) and having higher MICs would not be predicted to be as efficacious as those with lower MICs. Based upon low MIC values, tiamulin and virginiamycin might be useful in decreasing fecal shedding of C. difficile by sows, while bacitracin may not be efficacious in this regard. Fifty percent of strains examined had low MICs for erythromycin, tetracycline, and tylosin, so these agents may be useful for treatment of infected piglets. The MIC90 for ceftiofur (>256 mg/mL) suggests that it might not be efficacious in the treatment of neonatal enteritis due to C. difficile. Antimicrobial susceptibility testing of veterinary anaerobes and veterinary-exclusive agents is affected by general lack of standardization; antimicrobial agents have not been evaluated using NCCLS-approved susceptibility testing methods and there are no
ARTICLE IN PRESS K.W. Post, J.G. Songer / Anaerobe 10 (2004) 47–50
49
Table 1 Antimicrobial susceptibilities of 80 C. difficile isolates from neonatal pigs with enteritis Antimicrobial
Range (mg/mL)
MIC50 (mg/mL)a
MIC90 (mg/mL)a
Bacitracin Ceftiofur Erythromycin Tetracycline Tiamulin Tilmicosin Tylosin Virginiamycin
>256 256–>256 0.125–256 0.125–128 0.125–256 0.125–256 0.125–128 0.125–16
>256 256 0.5 8 4 0.5 0.25 0.25
>256 >256 256 32 8 >256 64 2
a
MIC50 and MIC90 concentrations at which the growth of 50% and 90%, respectively, of the isolates is inhibited.
interpretive criteria available for veterinary anaerobic pathogens against these drugs and there are no interpretive criteria to apply to results of testing of veterinary anaerobic pathogens. In many cases, MICs have not been determined by any method. In addition, it is difficult to interpret MIC information on enteric pathogens. The generally accepted rule bases efficacy upon achievement in vivo of a peak concentration of four times the MIC; intestinal concentrations are unknown for many antimicrobials, making it difficult to draw valid conclusions about susceptibility or resistance. Thus, application of in vitro susceptibility information must be strongly influenced by the resulting clinical response, since therapy based solely upon MICs may not translate to in vivo efficacy. Clinical studies will be needed to establish field efficacy of these agents. Continued efforts are needed to develop standardized methods for testing veterinary antimicrobials and enteric pathogens.
Acknowledgements This work was supported in part by funds from the National Pork Board, Alpharma, and Novartis Animal Vaccines.
References [1] Blankenship-Paris TL, Walton BJ, Hayes YO. Clostridium difficile infection in hamsters fed an atherogenic diet. Vet Pathol 1995;32:269–73. [2] Jang SS, Hansen HM, Breher JE. Antimicrobial susceptibilities of equine isolates of Clostridium difficile and molecular characterization of metronidazole-resistant strains. Clin Infect Dis 1997;25(Suppl 2):S266–7. [3] Jones RL, Adney WS, Alexander AF. Hemorrhagic necrotizing enterocolitis associated with Clostridium difficile infection in four foals. J Am Vet Med Assoc 1988;193:76–9. [4] Knoop FC, Owens M, Crocker IC. Clostridium difficile: clinical disease and diagnosis. Clin Microbiol Rev 1993;6:251–65.
[5] Lowe BR, Fox J, Bartlett JG. Clostridium difficile-associated cecitis in guinea pigs exposed to penicillin. Am J Vet Res 1980;41:1277–9. [6] Lyerly DM, Krivan H, Wilkins TD. Clostridium difficile: its disease and toxins. Clin Microbiol Rev 1988;1:1–18. [7] Madewell BR, Tang YJ, Jang SS. Apparent outbreaks of Clostridium difficile-associated diarrhea in a veterinary medical teaching hospital. J Vet Diagn Invest 1995;7:343–6. [8] Perrin J, Cosmetatos I, Gallusser A. Clostridium difficile with typholcolitis in an adult horse. J Vet Diagn Invest 1993;5:99–101. [9] Rehg JE, Lu YS. Clostridium difficile typhlitis in hamsters not associated with antibiotic therapy. J Am Vet Med Assoc 1982;181:1422–3. [10] Songer JG, Post KW, Larson DJ. Infection of neonatal swine with Clostridium difficile. Swine Health Prod 2000;8:185–9. [11] Waters EH, Orr JP, Clark EG, Schaufele CM. Typhlocolitis caused by Clostridium difficile in suckling piglets. J Vet Diagn Invest 1998;10:104–8. [12] Delmee M, Avesani V. Correlation between serogroup and susceptibility to chloroamphenicol, clindamycin, erythromycin, rifampin and tetracycline. J Antimicrob Chemother 1988;22: 325–31. [13] Chow AW, Cheng N, Bartlett KH. In vitro susceptibility of Clostridium difficile to new b-lactam and quinolone antibiotics. Antimicrob Agents Chemother 1985;28:842–4. [14] Dzink J, Bartlett JG. In vitro susceptibility of Clostridium difficile isolates from patient with antibiotic-associated diarrhea or colitis. Antimicrob Agents Chemother 1980;17:695–8. [15] Bacon AE, McGrath S, Fekety R, Holloway WJ. In vitro synergy studies with Clostridium difficile. Antimicrob Agents Chemother 1991;35:582–3. [16] Citron DM, Warren YA, Tyrrell KL. Comparative in vitro activity of ramoplanin, teicoplanin, vancomycin, linezolid, bacitracin, and four other antimicrobials against intestinal anaerobic bacteria. 41st Interscience Conference on Antimicrobial Chemotherapy, 2001, p. 193. [17] Barbut F, Decre D, Burghoffer B, Lesage D, Delisle F, Lalande V, Delmee M, Avesani V, Sano N, Coudert C, Petit J-C. Antimicrobial susceptibilities and serogroups of clinical strains of Clostridium difficile isolated in France in 1991 and 1997. J Clin Microbiol 1999;43:2607–11. [18] National Committee for Clinical Laboratory Standards. Methods for antimicrobial susceptibility testing of anaerobic bacteria, 5th ed. Approved standard M11-A5. Wayne, PA: National Committee for Clinical Laboratory Standards; 2001. [19] National Committee for Clinical Laboratory Standards. Methods for antimicrobial susceptibility testing of anaerobic bacteria, 2nd ed. Approved standard M11-A2. Villanova, PA: National Committee for Clinical Laboratory Standards; 1990.
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[20] Dutta GN, Devriese LA, van Assche PF. Susceptibility of clostridia from farm animals to 21 antimicrobial agents including some used for growth promotion. J Antimicrob Chemother 1983;12:347–56. [21] Werner G, Laber G, Schutze E. In vitro activity of tiamulin (81.723 hfu) a new pleuromutilin derivative against clinically significant anaerobes. J Antibiot 1978;30:756–60.
[22] Benning VR, Mathers JJ. Comparison of agar dilution and broth microdilution methods of anaerobic antimicrobial susceptibility testing using several veterinary antibiotics against Clostridium perfringens strains originating from porcine and avian sources. Anaerobe 1999;5:561–9.