A proposed scheme for the monitoring of antibiotic resistance in veterinary pathogens of food animals in the UK.

BACKGROUND: Antibiotic resistance in bacteria is a global threat to both animal and public health, and detecting its occurrence is an important component of control strategies. Monitoring programmes for antibiotic resistance are currently in place in food-producing animals in the European Union covering the zoonotic bacteria Salmonella enterica, Campylobacter coli and Campylobacter jejuni and the indicator bacteria Escherichia coli, Enterococcus faecalis and Enterococcus faecium. However, there is no equivalent pan-European statutory monitoring programme covering the antibiotic susceptibility of veterinary bacterial pathogens in food animals. This paper considers that issue and aims to facilitate and stimulate further discussion. METHODS: Recommendations, proposed by the authors from the scientific literature and following expert discussion at international meetings, are presented for monitoring the susceptibility of key veterinary pathogens. RESULTS: The selected veterinary pathogens comprise Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, E. coli, Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Staphylococcus aureus and Streptococcus spp. from the major food animal species cattle, pigs and poultry. The organisms are tested using harmonised panels of antibiotics over specified dilution ranges in a broth microdilution method. CONCLUSION: The selected antibiotics and their respective dilution ranges are presented together with the underlying rationale for inclusion; the ranges chosen are suitable for incorporation into three microtitre plates, with each organism tested using a single plate. The recommendations are being implemented in 2020 in the UK for monitoring of the susceptibility of veterinary bacterial pathogens.

Human Health Risk Assessment (HHRA) for environmental development and transfer of antibiotic resistance.

BACKGROUND: Only recently has the environment been clearly implicated in the risk of antibiotic resistance to clinical outcome, but to date there have been few documented approaches to formally assess these risks. OBJECTIVE: We examined possible approaches and sought to identify research needs to enable human health risk assessments (HHRA) that focus on the role of the environment in the failure of antibiotic treatment caused by antibiotic-resistant pathogens. METHODS: The authors participated in a workshop held 4-8 March 2012 in Québec, Canada, to define the scope and objectives of an environmental assessment of antibiotic-resistance risks to human health. We focused on key elements of environmental-resistance-development "hot spots," exposure assessment (unrelated to food), and dose response to characterize risks that may improve antibiotic-resistance management options. DISCUSSION: Various novel aspects to traditional risk assessments were identified to enable an assessment of environmental antibiotic resistance. These include a) accounting for an added selective pressure on the environmental resistome that, over time, allows for development of antibiotic-resistant bacteria (ARB); b) identifying and describing rates of horizontal gene transfer (HGT) in the relevant environmental "hot spot" compartments; and c) modifying traditional dose-response approaches to address doses of ARB for various health outcomes and pathways. CONCLUSIONS: We propose that environmental aspects of antibiotic-resistance development be included in the processes of any HHRA addressing ARB. Because of limited available data, a multicriteria decision analysis approach would be a useful way to undertake an HHRA of environmental antibiotic resistance that informs risk managers.

A Clostridium difficile gene encoding flagellin.

Six strains of Clostridium difficile examined by electron microscopy were found to carry flagella. The flagella of these strains were extracted and the N-terminal sequences of the flagellin proteins were determined. Four of the strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins. Using degenerate primers based on the N-terminal sequence and the conserved C-terminal sequence of several flagellins, the gene encoding the flagellum subunit (fliC) was isolated and sequenced from two virulent strains. The two gene sequences exhibited 91% inter-strain identity. The gene consists of 870 nt encoding a protein of 290 amino acids with an estimated molecular mass of 31 kDa, while the extracted flagellin has an apparent molecular mass of 39 kDa on SDS-PAGE. The FliC protein displays a high degree of identity in the N- and C-terminal amino acids whereas the central region is variable. A second ORF is present downstream of fliC displaying homology to glycosyltransferases. The fliC gene was expressed in fusion with glutathione S-transferase, purified and a polyclonal monospecific antiserum was obtained. Flagella of C. difficile do not play a role in adherence, since the antiserum raised against the purified protein did not inhibit adherence to cultured cells. PCR-RFLP analysis of amplified flagellin gene products and Southern analysis revealed inter-strain heterogeneity; this could be useful for epidemiological and phylogenetic studies of this organism.