Sep 21, 2012 ... with cattle (078, 126, 033) were not clonal but differed in PFGE type, sporulation
.... PCR ribotyping was performed by primers and conditions described by Bidet
et al., (1999 ..... (FP7/2007-2013) under grant agreement no.
AEM Accepts, published online ahead of print on 21 September 2012 Appl. Environ. Microbiol. doi:10.1128/AEM.02185-12 Copyright © 2012, American Society for Microbiology. All Rights Reserved.
Multiclonal presence of Clostridium difficile PCR ribotypes 078, 126 and 033 within a
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single calf farm is associated with differences in antibiotic resistance and sporulation
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properties
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Brouwer 4, Adam P. Roberts4, Adriano O. Henriques3, and Maja Rupnik1, 5, 6
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7 8 9 10 11 12 13 14 15
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Institute of Public Health Maribor, Maribor, Slovenia;
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Department of Large Animal Internal Medicine, Faculty of Veterinary Medicine, Ghent
University, Salisburylaan 133, 9820 Merelbeke, Belgium 3
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da
República, Apartado 127, 2781-901 Oeiras, Portugal 4
Department of Microbial Diseases, UCL Eastman Dental Institute, University College
London, London, WC1X 8LD, United Kingdom 5 6
University of Maribor, Faculty of Medicine, Maribor, Slovenia Center of Excellence for integrated approaches in chemistry and biology of proteins,
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Ljubljana, Slovenia
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*Present address: F.Hoffmann-La Roche Ltd, Pharmaceuticals Division, PTDS - Bldg/Room 204/1.041, CH- 4070 Basel, Switzerland. Email:
[email protected]
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Corresponding author: Maja Rupnik;
[email protected]
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Running title: Multiclonality of animal associated C. difficile PCR ribotypes
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Keywords: Clostridium difficile, animals, sporulation, tetracycline determinants, PCR
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ribotyping
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Valerija Zidaric1, Bart Pardon2, Tiago dos Vultos3*, Piet Deprez2, Michael Sebastiaan Maria
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Abstract C. difficile strains were sampled periodically from fifty animals from a single veal calf farm
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over a period of 6 months. At arrival 10% of animals were C. difficile positive and the peak
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incidence was determined to be at the age of 18 days (16%). The prevalence then decreased and
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at slaughter C. difficile could not be isolated. Six different PCR ribotypes were detected and
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strains within a single PCR ribotype could be further differentiated by PFGE. The PCR ribotype
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diversity was high up to the animal age of 18 days, but at later sampling points PCR ribotype 078
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and the highly related PCR ribotype 126 predominated. Resistance to tetracycline, doxycycline
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and erythromycin was detected, while all strains were susceptible to amoxicillin and
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metronidazole. Multiple variations of the resistance gene tet(M) were present at the same
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sampling point and these changed over time. We have shown that PCR ribotypes often associated
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with cattle (078, 126, 033) were not clonal but differed in PFGE type, sporulation properties,
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antibiotic sensitivities and tetracycline resistance determinant suggesting that multiple strains of
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the same PCR ribotype infected the calves and calves were likely to be infected prior to arrival at
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the farm. Importantly, strains isolated at later time points were more likely to be resistant to
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tetracycline and erythromycin and showed higher early sporulation efficiencies when assayed in
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vitro, suggesting that these two properties converge to promote the persistence of C. difficile in
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the environment or the hosts.
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Introduction
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An increasing number of reports have been focused on the prevalence and shedding of C.
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difficile in domestic animals; especially pigs, cattle, other food animals and horses (1, 2, 11, 12,
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17, 18, 20, 28, 29, 32, 33, 34, 37, 44, 47). In cattle and pigs, PCR ribotype 078 (type 078) has
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frequently been identified as predominant (11, 12, 15, 16, 25, 44). More recently this type has 2
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also been considered as an important pathogen in humans and has been detected in different
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countries (5, 14, 21, 24, 36). In 2005, type 078 was the eleventh-most frequently found type in
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humans in Europe (4), becoming the third most frequent PCR ribotype in 2009 (5). Interestingly,
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human and pig type 078 isolates have been shown to be closely related (3, 24). Therefore, type
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078 has been speculated to be a new emerging strain associated with increased virulence and
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could be an example of C. difficile interspecies, foodborne transmission (38, 43, 45).
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Few longitudinal screenings on C. difficile in swine (20, 44), poultry (47) or cattle facilities
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have been published (11, 33). For cattle, one study followed the colonization and prevalence of
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tetracycline resistance in a single veal farm in Canada (11), while another study included older
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animals at a finishing facility and also looked at the prevalence of C. difficile, on-farm
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transmission and contamination at slaughter (33).
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The primary objective of this study was to assess the presence of certain genotypes of C.
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difficile in veal calves, and their prevalence at different time points in the production cycle.
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Additionally, the most prevalent PCR ribotypes 078 and 126 were characterized in terms of
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antibiotic resistance determinants and sporulation properties.
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Materials and methods
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Sampling. The veal production cohort in Belgium consisted of 340 Belgian Blue/ Holstein
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Friesian crossbreed veal calves. Calves arrived at the veal herd when they were 14 days old. The
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calves were housed within compartments of 112 animals on slatted floors. For the first 6 weeks
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the animals were housed individually and thereafter 5 or 6 animals were grouped per pen. The
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animals received a low iron milk powder diet, concentrates and roughage replacer twice daily.
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The calves received colistin (0.5g, bid, Promycin, VMD) and amoxicillin (1g, bid, Dokamox
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80%, Emdoka) for 10 days starting on day 7 after arrival. For reasons of diarrhea all animals 3
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received enrofloxacine for a single day (day 5 after arrival) (0.25g/calf sid, Baytril 2.5% oral
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solution, Bayer). As a metaphylactic treatment for respiratory disease doxycycline was
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administered (0.5g, bid, Doxyveto 50% pulvis, VMD) for 5 days and tylosin for 7 days (0.5g,
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bid, Tylan, Eli Lilli) (from day 13 after arrival). All individual treatments in the first two months
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after arrival were individually recorded by the owner (indication, drug, dose, route of
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administration).
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Sampling was carried out between February and August 2010 on six different occasions at the
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calf age of 14 (at arrival), 18, 25, 32, 46 days and just before slaughter (194 days). Within a
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single compartment, altogether 50 calves (arrived on the same day) were randomly selected and
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individually identified with ear tags. The rectal temperature was first recorded. As sampling was
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done shortly after the morning meal, most calves spontaneously defecated after rectal stimulation
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with the thermometer. The thermometer was disinfected with 97% alcohol and gloves were
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replaced after each calf. Only if an animal did not defecate spontaneously, the swab was
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introduced into the rectum and rotated. Rectal swabs were taken maximally in two animals per
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sampling point. Only at sampling point 4 13 of 50 animals (26%) were sampled by a rectal swab.
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Faecal samples were scored as normal (=firm), pasty diarrhea, watery diarrhea or clay-like faeces
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(sign of ruminal drinking). Samples were collected by submerging the swab in the middle of the
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faeces, and then placed into the recipient tube. During the shipping (taking 1-2-days), swabs were
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held at room temperature and were processed immediately upon arrival at the laboratory.
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C. difficile isolation. Swabs were put into enrichment broth CDA (C. difficile agar, without
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agar; Oxoid), supplemented with C. diffcile selective supplement (SR0096E; Oxoid), sporulation
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enhancers (0, 1% sodium choleate and 5mg/L of lysozyme), and incubated for 5-7 days.
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Subsequently, 500 µL of enriched culture was used for alcohol shock in a volume ratio of 1:1, for
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30 min at room temperature. Pellet was inoculated on commercial selective Clostridium difficile 4
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agar plates (CLO; bioMerieux) and incubated for three days. In several samples (n=11), 1-4
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colonies with typical C. difficile morphology were randomly collected from a single CLO plate
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and further subcultured on Columbia agar plates with 5% horse blood (COH; bioMerieux).
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Identification was confirmed by C. difficile specific PCR as described previously (47). Molecular typing of C. difficile strains. Toxinotyping was done by PCR amplification and
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restriction analysis of A3 and B1 fragments of tcdA and tcdB genes as previously described (35,
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www.mf.uni-mb.si/mikro/tox). The binary toxin gene was detected by partial amplification of
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cdtB (41).
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PCR ribotyping was performed by primers and conditions described by Bidet et al., (1999 (6)).
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Pulsed-field gel electrophoresis (PFGE) using restriction endonuclease SacII was used as
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described elsewhere (22). If the strain could not be typed by the standard protocol,
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electrophoresis was modified by addition of 180 µl of 1M urea into 2,4 L of electrophoresis
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buffer (13).
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Both, PCR ribotype and PFGE results were analyzed using the BioNumerics software
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(version 5.10 Applied Maths). To show similarity between PFGE patterns, dendograms were
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made by the unweighted-pair group method with arithmetic mean using Dice coefficients with
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position tolerance and optimization of 1,1%. Clusters with
