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Clostridium Hathewayi Strain from a Continuous-flow Exclusion Chemostat Culture Derived from the Cecal Contents of a Feral Pig

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Clostridium Hathewayi Strain from a Continuous-flow Exclusion Chemostat Culture Derived from the Cecal Contents of a Feral Pig ARTICLE IN PRESS Anaerobe 13 (2007) 153–160 www.elsevier.com/locate/anaerobe Veterinary anaerobes and diseases Characterization of an antibiotic resistant Clostridium hathewayi strain from a continuous-flow exclusion chemostat culture derived from the cecal contents of a feral pig N. Ramlachana, R.C. Andersona,Ã, K. Andrewsa, G. Labanb,c, D.J. Nisbeta aUnited States Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, Food & Feed Safety Research Unit, 2881 F&B Road, College Station, TX 77845, USA bDepartment of Forestry and Natural Resources, Pfender Hall, 715 W State Street Purdue University, West Lafayette, IN, USA cCenter for the Environment, 503 Northwestern Avenue, Purdue University, West Lafayette, IN 47907, USA Received 8 March 2007; accepted 14 March 2007 Available online 23 March 2007 Abstract The chemostat model has been an important tool in studying intestinal microflora. To date, several competitive exclusion products have been developed from such studies as prophylactic treatment against pathogenic bacteria. A continuous-flow chemostat model of a feral pig was developed using inocula from the cecal contents of a wild boar caught in East Texas. Several strains of antibiotic-sensitive bacteria were isolated including Bacteroides, Lactobacillus, Enterococcus and Clostridium sp. This study reports on the characterization of a multidrug-resistant Clostridum hathewayi strain that was isolated from this feral pig’s cecal contents maintained in a continuous-flow chemostat system showing high resistance to carbapenems and macrolides (including the growth promoter tylosin). Clostridium hathewayi has been documented to be pathogenic to both humans and animals. Feral pigs may be an important source of pathogenic and antibiotic resistant bacteria and may pose potential risk to domestic species. Further work is needed to elucidate the prevalence of these reservoirs and assess the contribution these may play in the spread of disease and resistance. Published by Elsevier Ltd. Keywords: Clostridium hathewayi; Antibiotic resistance; Feral pig; Chemostat 1. Introduction the basis of 16S rRNA sequencing isolated from the human gastrointestinal tract [7]. Clostridium hathewayi has also Clostridium sp. are anaerobic, Gram-positive bacilli that been identified as the cause of various infections in fatal are common causes of enteritis and enterotoxemias in both clinical cases, including sepsis [8–11]. domestic animals and humans [1]. Outbreaks of multidrug- Ampicillin, metronidazole, tetracycline, erythromycin, resistant Clostridium spp. have caused serious illness and and tylosin appear to be effective antibiotics for the even death in both humans and livestock animals world- treatment of Clostridium spp. -associated diarrhea in wide with Clostridium spp. accounting for about 1% of all domestic animals [1]. Tylosin is also a macrolide antibiotic significant blood culture isolates in bacteremia cases [2–5]. used as a growth promoter to prevent infections in young Many Clostridium species are pathogenic for humans and pigs [12]. Studies have shown transfer of macrolide animals, resulting in diseases such as tetanus, botulism, gas resistance elements occurring between several species of gangrene and pseudomembranous colitis due to the bacteria including Bacteroides, Enterococci, Lactobacilli production of potent extracellular toxins [6]. Clostridium and Clostridium in humans and animals [13–25]. Antibiotic hathewayi has recently been described as novel species on sensitivity to several feed-based antimicrobials, such as tylosin is of concern, as it has been proposed that the use of ÃCorresponding author. Tel.: +1 979 260 9317; fax: +1 979 260 9332. antibiotics for therapeutic and growth promotion in E-mail address: [email protected] (R.C. Anderson). livestock could lead to increasing resistance in bacteria of 1075-9964/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.anaerobe.2007.03.003 ARTICLE IN PRESS 154 N. Ramlachan et al. / Anaerobe 13 (2007) 153–160 human and animal origin to these drugs [26]. Competitive cultures achieved steady-state at a slightly lower pH exclusion (CE) cultures have been shown to inhibit the (between pH 5.7 and 6.0), than that of the RPCF which growth of pathogenic bacteria in chickens and swine stabilized between pH 5.9 and 6.1. [27–32]. Continuous-flow cultures have also been very useful as in vitro models of the ecology of intestinal flora 2.3. Bacterial isolation and biochemical characterization [33,34]. A recombined, porcine-derived, continuous-flow (RPCF) culture derived from a domestic pig has been After steady state conditions were achieved (approxi- shown to inhibit growth of pathogenic Salmonella enterica mately 7 Â 550 ml culture turnovers), 1 ml samples were serovars Typhimuirum, Choleraesius and Escherichia coli taken from the chemostats. Detection of anaerobic bacteria strains in vitro [35]. was performed in a Bactron IV Anaerobic/Environmental Limited information is available on the presence of Chamber (5% Co2,5%H2, 90% N2) (Sheldon Manufac- antibiotic resistant strains of bacteria in wild/feral pigs. turing Inc., Cornelius, OR) by serial dilution and plating There is a concern that transfer of multidrug-resistant onto Anaerobic Brucella Blood Agar (BRU) (Anaerobe genetic elements in bacteria occurs readily between the Systems, Morgan Hill, CA) and Bacteroides Bile Esculin environment, domestic animals and humans [36–43]. The Agar (BBE) (Anaerobe Systems, Morgan Hill, CA) for possibility that feral pigs may be an additional pool for detection of Bacteroides. Serial dilutions of feral pig culture antibiotic-resistant bacteria exists, since these are a source were plated onto CHROMagarTM E. coli (CHROMagar, for direct transmission of bacterial infections [44,45]. Paris, France) for detection of E. coli, brilliant green agar Evidence of antibiotic resistance has been seen in organi- (Oxoid, Hampshire, England) for detection of Salmonella, cally raised, feral swine or swine without exposure to Modified Charcoal Cefoperazone Deoxycholate Agar antibiotics in feed [36,46–48]. Recent studies show slow (mCCDA) (Oxoid, Hampshire, England) for detection of dissipation of antibiotics in soil and presence of antibiotic- Campylobacter and M Enterococcus agar (Becton Dick- resistant strains in the environment which may also inson and Company, Sparks, MD) for detection of increase exposure to domestic and feral animals Enteroccoci. Specific identification of select colonies of [43,49–51]. In our attempts to comparatively study transfer bacteria was performed according to manufacturer’s of antibiotic resistance in chemostat cultures derived from instructions using rapid ID 32 STREP, rapid 20E, 20NE, cecal contents from domestic and feral pigs, we isolated 20A, and rapid ID 32 A identification strips (bioMe´rieux, multidrug-resistant anaerobic bacteria from the gut con- Hazelwood, MO). Indole spot tests (Anaerobe Systems, tents of a feral pig. This study describes establishment of a Morgan Hill, CA), EtestsTM (AB Biodisk, Piscataway, NJ) continuous-flow culture derived from a feral pig obtained and gas chromatography were also used in this analysis. in rural East Texas and characterizes an endogenous Detection of b-lactamase production was performed using antibiotic resistant strain of C. hathewayi. BD BBL DrySlideTM NitrocefinTM (Becton Dickinson and Company, Sparks, MD) as per manufacturer’s directions. 2. Materials and methods 2.4. Antibiotic susceptibility testing 2.1. Inoculum The antimicrobial susceptibility profiles of the aerobic Inoculum was obtained from cecal contents harvested at isolates were determined by the broth microdilution method slaughter from an adolescent feral boar, approximately 6–9 with cation-adjusted Mueller–Hinton broth (BD Diagnostic months of age and weighing 80 kg. Because slaughter Systems [BDDS], Sparks, MD, USA), as described in the occurred in the evening (20:00 h), freshly collected cecal National Committee for Clinical Laboratory Standards contents were placed into 160 ml serum vials containing (NCCLS, Wayne, PA, USA) (now known as the Clinical approximately 32 ml anaerobic glycerol until completely and Laboratory Standards Institute [CLSI]) publication full and then immediately sealed and frozen within 1 h of M7-A6 [53]. Tests were interpreted at 18–24 h. The Etestss collection at À80 1C until inoculation into the chemostat. (AB Biodisk, Piscataway, NJ) were performed as described by the manufacturer with BRU; Etestss were interpreted at 2.2. Chemostat culture 24–48 h. Sensititre microdilution plates (Trek Diagnostics System, UK) were used according to CLSI guidelines to The continuous-flow feral pig culture was developed determine minimum inhibitory concentrations (MIC) using published protocols for establishment of the pre- of the following antibiotics at NCCLS breakpoints [54]: viously developed redefined porcine continuous-flow ampicillin, ceftiofur, chlortetracycline, clindamycin, dano- (RPCF) culture [29,30,35,52]. Cultures were maintained floxacin, enrofloxacin, erythromycin, florfenicol, gentamicin, in 550 ml of Viande–Levure medium as previously de- neomycin, oxytetracycline, penicillin, spectinomycin, sulpha- scribed [29] and continuously stirred at 100 rpm, at a flow choloropyridazine, sulphadimethoxime, sulphathiazole, tia- rate of 0.8 ml/min and a vessel turnover time of 24 h. Feral mulin, tilmicosin, trimethoprim/ sulphamethoxazole. The pig chemostat cultures were sparged with oxygen-free CO2 MIC breakpoint
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