of antimicrobial resistance in bacteria isolated from food animals to antimicrobial growth promoters and related therapeutic agents in Denmark. APMIS 106: 606-622, 1998. This study was conducted to describe the occurrence of acquired resistance to antimicrobials used for growth promotion among bacteria isolated from swine, cattle and poultry in Denmark. Resistance to structurally related therapeutic agents was also examined. Three categories of bacteria were tested: 1) indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium), 2) zoonotic bacteria (Campylobacter, Salmonella, Yersinia enterocolitica), and 3) animal pathogens (E. coli, Staphylococcus aureus, coagulase-negative staphylococci (CNS), Staphylococcus hyicus, Actinobacillus pleuropneumoniae). All antimicrobials used as growth promoters in Denmark and some structurally related therapeutic agents (in brackets) were included: Avilamycin, avoparcin (vancomycin), bacitracin, carbadox, flavomycin, monensin, olaquindox, salinomycin, spiramycin (erythromycin, lincomycin), tylosin (erythromycin, lincomycin), and virginiamycin (pristinamycin). Bacterial species intrinsically resistant to an antimicrobial were not tested towards that antimicrobial. Breakpoints for growth promoters were established by population distribution of the bacteria tested. A total of 2,372 bacterial isolates collected during October 1995 to September 1996 were included in the study. Acquired resistance to all currently used growth promoting antimicrobials was found. A frequent occurrence of resistance were observed to avilamycin, avoparcin, bacitracin, flavomycin, spiramycin, tylosin and virginiamycin, whereas resistance to carbadox, monensin, olaquindox and salinomycin was less frequent. The occurrence of resistance varied by animal origin and bacterial species. The highest levels of resistance was observed among enterococci, whereas less resistance was observed among zoonotic bacteria and bacteria pathogenic to animals. The association between the occurrence of resistance and the consumption of the antimicrobial is discussed. The results show the present level of resistance to growth promoters in bacteria from food animals in Denmark. They will form the baseline for comparison with future prospective studies, thereby enabling the determination of trends over time.

Resistance to antimicrobial agents used for animal therapy in pathogenic-, zoonotic-and indicator bacteria isolated from different food animals in Denmark: a baseline study for the Danish Integrated Antimicrobial Resistance Monitoring Programme (DANMAP). APMIS 106: 745-770, 1998. This study describes the establishment and first results of a continuous surveillance system of antimicrobial resistance among bacteria isolated from pigs, cattle and broilers in Denmark. The three categories of bacteria tested were: 1) indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium), 2) zoonotic bacteria (Campylobacter coli/jejuni, Salmonella enterica, Yersinia enterocolitica), and 3) animal pathogens (E. coli, Staphylococcus aureus, coagulase-negative staphylococci (CNS), Staphylococcus hyicus, Actinobacillus pleuropneumoniae). A total of 3304 bacterial isolates collected from October 1995 through December 1996 were tested for susceptibility to all major classes of antimicrobial agents used for therapy in Denmark. Bacterial species intrinsically resistant to an antimicrobial were not tested towards that antimicrobial. Acquired resistance to all antimicrobials was found. The occurrence of resistance varied by animal origin and bacterial species. In general, resistance was observed more frequently among isolates from pigs than from cattle and broilers. The association between the occurrence of resistance and the consumption of the antimicrobial is discussed, as is the occurrence of resistance in other countries. The results of this study show the present level of resistance to antimicrobial agents among a number of bacterial species isolated from food animals in Denmark. Thus, the baseline for comparison with future prospective studies has been established, enabling the determination of trends over time.

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Two different genotypic methods, colony hybridization and polymerase chain reaction (PCR), were applied to detect enterotoxin, verotoxin and fimbrial genes in 708 Escherichia coli (E. coli) strains from piglets with diarrhoea. The results were compared with those obtained by phenotypic methods. D N A fragments specific for each of the enterotoxins LT, ST, and ST,, the verotoxins VT,, VT, and VT,,, and for each of the fimbrial genes K88 (F4), K99 (F5), 987P (F6), F41 and F107, respectively, were used as probes in a colony hybridization assay of the E. coli strains. A PCR assay was used as genotypic test for the verotoxin gene. An Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was performed to test for the presence of K88 and K99 fimbriae, and a Vero cell test was performed to test for verotoxin production. Toxin detection kits were applied to detect the E. coli heat-labile enterotoxin (LT) and the heat-stable (ST,) enterotoxin. The correlation between the results obtained by genotypic and phenotypic methods was 97.7-100 %. The prevalence of the different fimbrial and toxin genes in E. coli strains from piglets with neonatal and postweaning diarrhoea were recorded. The verotoxin and the fimbrial F107 genes were found to be more frequent in postweaning E. coli strains than in neonatal E. coli strains. IntroductionEnterotoxigenic Escherichia coli (E. coli) (ETEC) is an important cause of diarrhoea in piglets. The diarrhoea occurs just after birth, in 1-7 day-old piglets, as neonatal diarrhoea. Postweaning diarrhoea occurs just after weaning in 22-49 days old piglets. Recognized virulence factors of the enterotoxigenic E. coli are the fimbrial adhesins K88, K99, 987P and F41 and the enterotoxins ST,, ST, and LT. Due to the lack of suitable methods a complete survey of virulence factors of E. coli strains pathogenic for piglets has not been carried out in Denmark before this study.The plasmid borne genes encoding ST,, STb and LT have been cloned and sequenced 50 OJENIYI, AHRENS and MEYLING (1981), the K99 fimbrial gene by ROOSENDAAL et al. (1984), the F41 fimbrial gene by FIDOCK et al. (1989) and the 987P fimbrial gene by DE GRAFF & KLAASEN (1987). The F107 fimbrial gene has been sequenced by IMBERECHTS et al. (1992). This information made it possible to synthesize oligonucleotides which, after labelling, may be used directly to identify fimbrial or toxin genes in a colony hybridization test, or used as primers for the amplification of a specific region of the toxin gene using the polymerase chain reaction (PCR) technique. These methods are readily applicable to large numbers of strains in contrast t o the classical methods such as agglutination tests (for fimbriae), infant mouse test (for ST, toxin), ligated swine intestine (for STb) and cell culture tests (for LT toxin), all of which are unsuitable for large scale studies.In the present study, specific DNA probes were constructed by the PCR technique and used in colony hybridization assays t o identify fimbrial and toxin genes of E. coli iso...

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