Anaerobe 10 (2004) 47–50

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doi:10.1016/j.an

Veterinary anaerobes and diseases

Antimicrobial susceptibility of Clostridium difficile isolated fromneonatal pigs with enteritis

Karen W. Posta, J. Glenn Songerb,*aRollins Animal Disease Diagnostic Laboratory, North Carolina Department of Agriculture and Consumer Services, Raleigh, NC 27601, USA

bDepartment 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 totetracycline varied widely, with MIC50 and MIC90 of 8 and 32mg/mL, respectively. The MICs90 for tiamulin (8 mg/mL) andvirginiamycin (16 mg/mL) suggest moderate susceptibility. Bacitracin and ceftiofur (MICs90X256 mg/mL) have little activity againstC. 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 pseudomembranouscolitis and antibiotic-associated diarrhea in humans,horses, and laboratory rodents [1–9]. It has recentlybeen recognized as a significant etiologic agent ofenteritis in suckling swine [10,11]. Sows may serve as asource 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 vanco-mycin and 90% are susceptible to tetracycline anderythromycin; minimum inhibitory concentrations(MIC) for clindamycin and fluoroquinolones are vari-able, and most are insusceptible to cephalosporins[13,14]. Resistance to bacitracin has been reported[15,16]. The drugs of choice for treatment of humaninfections are vancomycin and metronidazole [17].Knowledge of antimicrobial susceptibility of animalstrains of C. difficile is limited. Equine isolates have low

ng author. Tel.: +1-520-621-2962; fax: +1-520-621-

ss: gsonger@u.arizona.edu (J.G. Songer).

front matter r 2004 Elsevier Ltd. All rights reserved.

aerobe.2004.01.003

MICs for chloramphenicol, rifampin, vancomycin, andmetronidazole and a high MIC for bacitracin [2].Antimicrobial susceptibility of porcine C. difficile

strains has not previously been investigated, so we usedan agar dilution method [18] to examine 80 recentisolates from pigs. Agents were selected on the basis ofpreviously demonstrated in vitro activity against anae-robes and label claims which might allow their use tolimit fecal shedding of C. difficile by sows or to treatinfected piglets.

2. Materials and methods

2.1. Isolates and strains

Eighty C. difficile isolates were obtained fromneonatal piglets with enteritis. Specimens of feces orcolonic contents were subjected to bacteriologic cultureon C. difficile selective medium [CDSA, brain–heartinfusion agar (Difco) supplemented with 5% defibri-nated bovine blood, 0.1% taurocholate, and 250 mgcycloserine and 8 mg cefoxitin per mL]. Plates wereincubated in an anaerobic atmosphere for 48 h at 37�Cand presumptive identification of C. difficile was madeon the basis of colony morphology on CDSA and

ARTICLE IN PRESSK.W. Post, J.G. Songer / Anaerobe 10 (2004) 47–5048

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 werestored in 25% glycerol at �80�C until tested.Control strains were Bacteroides fragilis ATCC 25285

and Eggerthella lenta ATCC 43055. Current NationalCommittee for Clinical Laboratory Standards (NCCLS)recommendations do not include clostridia among theorganisms for quality-control of antimicrobial suscept-ibility tests, but we included C. difficile ATCC 9689 andC. perfringens ATCC 13124 on each plate to evaluatethe internal reproducibility of the method.

2.2. Determination of MICs

Susceptibilities were determined by the reference agardilution procedure recommended by NCCLS [18]. Serialtwo-fold dilutions of bacitracin, erythromycin (Sigma-Aldrich, St. Louis, MO), tetracycline HCl (US Pharma-copeia, Rockville, MD), ceftiofur (Pharmacia AnimalHealth, Kalamazoo, MI), tiamulin (Boehringer Ingel-heim 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, withvitamin K (1 mg/mL), hemin (5 mg/mL), and laked sheepblood (5%), at final concentrations of 0.125–256 mg/mL.There are no current NCCLS quality control ranges foractivity of macrolide antibiotics against clostridia, but aprevious NCCLS document [19] listed the MIC ofclindamycin for C. perfringens ATCC 13124. Thus,clindamycin was used at the same range of concentra-tions (0.125–256 mg/mL) for quality control of tests ontilmicosin and tylosin.Prior to antimicrobial susceptibility testing, isolates

were subcultured twice on prereduced Brucella agarsupplemented with bovine blood (5%), hemin (5 mg/mL), and vitamin K1 (mg/mL). Colonies were suspendedin trypticase soy broth to an optical density equivalentto that of a McFarland 0.5 standard. Plates wereinoculated with a Steer’s replicator which delivered1–2 mL per spot. Plates were incubated at 37�C in anatmosphere of 5% H2:5% CO2:90% N2, and wereexamined after 48 h. The lowest dilution of antimicro-bial which affected growth on the test plate (ascompared to control plates containing no antimicrobial)constituted the MIC. MIC50 and MIC90 were theconcentrations at which 50% and 90%, respectively, ofstrains were inhibited.

3. Results and discussion

Others have reported in vitro anti-clostridial activityof tylosin [20], and our findings concur; MICs50 for

erythromycin, tilmicosin, and tylosin were relatively low(0.25–0.50 mg/mL). However, MICs90 (64 orX256mg/mL) suggest that a proportion of isolates will notbe susceptible in vivo. Human isolates of C. difficile alsoexhibit this bimodal pattern of susceptibility to ery-thromycin [12,14] and it seems likely that this is due tothe non-uniform distribution of a resistance element inthe population. C. difficile from humans vary widely insusceptibility to tetracycline [12,14], and our findingswere similar, with MIC50 and MIC90 of 8 and 32 mg/mL,respectively. Tiamulin, a pleuromutilin derivative usedin the treatment of animal infections with Gram-positiveand Gram-negative bacteria, is active against anaerobicbacteria [21]. Our MIC90 for tiamulin (8 mg/mL) suggestsmoderate susceptibility, as did that for virginiamycin(16 mg/mL).Bacitracin had little activity against C. difficile

(MICX256 mg/mL), in keeping with results of MICtesting of human and equine strains [2,16]. Thepreviously reported high MICs for cephalosporins[13,14] mirror our findings with ceftiofur(MIC90X256 mg/mL, Table 1), a third generationcephalosporin used exclusively in veterinary medicine.Results for the reference antimicrobial, clindamycin,were within acceptable limits [19] and MICs for otherquality control strains were within acceptable limits[18,22].Antimicrobials such as bacitracin, tetracycline, tia-

mulin, tilmicosin, tylosin, and virginiamycin are admi-nistered to swine in feed. Intestinal levels ofantimicrobials administered by this route are difficultto measure, so the MIC may not reliably predict in vivoefficacy. However, negative clinical outcome might beexpected for those agents with a MIC higher than feedlevels. Furthermore, parenteral administration may notyield gut concentrations of the agent which areequivalent to the achievable serum concentrations whichhave traditionally been used to interpret antimicrobialsusceptibility. Drugs administered parenterally (ceftio-fur, erythromycin, tetracycline and tylosin) and havinghigher MICs would not be predicted to be as efficaciousas those with lower MICs.Based upon low MIC values, tiamulin and virginia-

mycin might be useful in decreasing fecal shedding of C.

difficile by sows, while bacitracin may not be efficaciousin this regard. Fifty percent of strains examined had lowMICs for erythromycin, tetracycline, and tylosin, sothese agents may be useful for treatment of infectedpiglets. The MIC90 for ceftiofur (>256mg/mL) suggeststhat it might not be efficacious in the treatment ofneonatal enteritis due to C. difficile.Antimicrobial susceptibility testing of veterinary

anaerobes and veterinary-exclusive agents is affectedby general lack of standardization; antimicrobial agentshave not been evaluated using NCCLS-approvedsusceptibility testing methods and there are no

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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 >256 >256 >256

Ceftiofur 256–>256 256 >256

Erythromycin 0.125–256 0.5 256

Tetracycline 0.125–128 8 32

Tiamulin 0.125–256 4 8

Tilmicosin 0.125–256 0.5 >256

Tylosin 0.125–128 0.25 64

Virginiamycin 0.125–16 0.25 2

aMIC50 and MIC90 concentrations at which the growth of 50% and 90%, respectively, of the isolates is inhibited.

K.W. Post, J.G. Songer / Anaerobe 10 (2004) 47–50 49

interpretive criteria available for veterinary anaerobicpathogens against these drugs and there are nointerpretive criteria to apply to results of testing ofveterinary anaerobic pathogens. In many cases, MICshave not been determined by any method.In addition, it is difficult to interpret MIC informa-

tion on enteric pathogens. The generally accepted rulebases efficacy upon achievement in vivo of a peakconcentration of four times the MIC; intestinal con-centrations are unknown for many antimicrobials,making it difficult to draw valid conclusions aboutsusceptibility or resistance. Thus, application of in vitrosusceptibility information must be strongly influencedby the resulting clinical response, since therapy basedsolely upon MICs may not translate to in vivo efficacy.Clinical studies will be needed to establish field efficacyof these agents. Continued efforts are needed to developstandardized methods for testing veterinary antimicro-bials and enteric pathogens.

Acknowledgements

This work was supported in part by funds from theNational Pork Board, Alpharma, and Novartis AnimalVaccines.

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