Reversible monensin adaptation in Enterococcus faecium, Enterococcus faecalis and Clostridium perfringens of cattle origin: potential impact on human food safety.

OBJECTIVES: To determine the stability/reversibility and mechanism of monensin adaptation in monensin-treated cattle isolates compared with reference bacterial isolates, exposed in vitro to high monensin concentrations. METHODS: We evaluated the potential for cattle-origin strains of Clostridium perfringens, Enterococcus faecium and Enterococcus faecalis exposed to monensin in vivo (in vivo monensin-exposed isolates) to maintain or achieve the ability to grow in the presence of high monensin concentrations (in vitro monensin-adapted isolates). Twenty-one consecutive subcultures of the in vitro monensin-adapted strains were performed, and monensin MICs were determined for the 3rd, 7th, 14th and 21st subcultures (subcultured isolates). SDS-PAGE and transmission electron microscopy (TEM) were used to determine protein expression and visualize extracellular morphology changes. RESULTS: Monensin-non-exposed isolates did not display monensin adaptation during in vitro monensin exposure. In contrast, in vivo monensin-exposed isolates displayed monensin adaptation enabling growth at 32× MIC. Upon consecutive subculturing, monensin MICs returned to baseline, or one dilution above, for the monensin-adapted strains. SDS-PAGE identified overexpression of a 14 kDa protein (C. perfringens) and a 20.5 kDa protein (E. faecium and E. faecalis) in the monensin-adapted isolates. TEM demonstrated that in vitro monensin-adapted strains had a significantly thicker cell wall or glycocalyx compared with in vivo monensin-exposed or subcultured isolates. CONCLUSIONS: In vivo monensin-exposed isolates of C. perfringens, E. faecium and E. faecalis have the ability to grow in the presence of high monensin concentrations in vitro. This is associated with an increased thickening of the cell wall or glycocalyx that is reversible upon serial passage, suggesting a phenotypically expressed, but not genetically stable, trait.

Pan-European monitoring of susceptibility to human-use antimicrobial agents in enteric bacteria isolated from healthy food-producing animals.

OBJECTIVES: To determine the antimicrobial susceptibility of Escherichia coli, Salmonella, Campylobacter and Enterococcus from cattle, pigs and chickens across the European Union (EU) using uniform methodology. METHODS: Intestinal samples (1624) were taken at slaughter across five EU countries. Bacteria were isolated in national laboratories, whilst MICs were determined in a central laboratory for key antimicrobials used in human medicine. Clinical resistance was based on CLSI breakpoints and decreased susceptibility based on European Food Safety Authority (EFSA)/EUCAST epidemiological cut-off values. RESULTS: Isolation rates were high for E. coli (n=1540), low for Salmonella (n=201) and intermediate for Campylobacter (n=940) and Enterococcus (n=786). For E. coli and Salmonella, clinical resistance to newer compounds (cefepime, cefotaxime and ciprofloxacin) was absent or low, but decreased susceptibility was apparent, particularly in chicken strains. Resistance to older compounds (except gentamicin) was variable and higher. Colistin resistance was absent for E. coli, but apparent for Salmonella. For Campylobacter jejuni, ciprofloxacin resistance was markedly prevalent for chickens, whereas clinical resistance and decreased susceptibility to erythromycin was absent or very low. For Campylobacter coli, resistance was notably higher. None of the Enterococcus faecium strains was resistant to linezolid, but some were resistant to ampicillin or vancomycin. Resistance to quinupristin/dalfopristin was frequent. CONCLUSIONS: Resistance patterns varied widely depending on bacterial species, antibiotics, hosts and region. Resistance varied among countries, particularly for older antimicrobials, but clinical resistance to newer antibiotics used to treat foodborne disease in humans was generally very low. In the absence of resistance to newer compounds in E. coli and Salmonella, the apparent decreased susceptibility should be monitored.

Relationship of in vitro minimum inhibitory concentrations of tilmicosin against Mannheimia haemolytica and Pasteurella multocida and in vivo tilmicosin treatment outcome among calves with signs of bovine respiratory disease.

OBJECTIVE: To determine associations between in vitro minimum inhibitory concentrations (MICs) of tilmicosin against Mannheimia haemolytica and Pasteurella multocida and in vivo tilmicosin treatment outcome among calves with clinical signs of bovine respiratory disease (BRD). DESIGN: Observational, retrospective, cohort study. ANIMALS: 976 feeder calves with clinical signs of BRD enrolled in 16 randomized clinical trials. PROCEDURES: Records of clinical trials from October 26, 1996, to November 15, 2004, were searched to identify calves with BRD from which a single isolate of M haemolytica or P multocida was identified via culture of deep nasal swab samples prior to treatment with tilmicosin (10 mg/kg [4.5 mg/lb], SC) and for which MICs of tilmicosin against the isolate were determined. The MICs of tilmicosin against recovered isolates and response to tilmicosin treatment were evaluated. RESULTS: Tilmicosin resistance among M haemolytica and P multocida isolates was uncommon (6/745 [0.8%] and 16/231 [6.9%], respectively). Treatment outcome, defined as success or failure after tilmicosin treatment, did not vary with the MIC of tilmicosin against recovered isolates. The proportion of treatment failures attributed to M haemolytica isolates categorized as resistant (MIC of tilmicosin, ≥ 32 µg/mL) or not susceptible (MIC of tilmicosin, ≥ 16 µg/mL), was 0.2% and 0.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Recovery of tilmicosin-resistant M haemolytica or P multocida isolates was rare, and no association was detected between MIC of tilmicosin and treatment response.

Overview of the animal health drug development and registration process: an industry perspective.

Products for animal health commercialization follow a structured progression from initial concept through to regulatory approval. Typically, products are developed for use in either food animals or companion animals. These can be for the intention of disease intervention, productivity enhancement or improvement in a quality of life capacity. The animal health industry is a regulated industry, meaning that a government agency is responsible for oversight of products, both pre- and post-approval. There are three primary US government agencies that ensure quality, safety and effectiveness for the approval of new products and post-marketing compliance.

The interface between veterinary and human antibiotic use.

The identification and early development of novel antimicrobial agents for use in veterinary medicine is subject to many of the same business and technical challenges as those found in antimicrobial agent use for human infectious disease. However, as awareness that some of the antimicrobial classes used in veterinary medicine are the same as used in human medicine, concern by multiple stakeholders has increased that this nonhuman use might be contributing to the problem of antimicrobial resistance to pathogens in humans, particularly with regard to food-borne diseases, such as salmonellosis and campylobacteriosis. Consequently, the interface between veterinary and human antibiotic use and resistance, especially with respect to human microbial food safety, has begun to redirect the industry pipeline of novel antimicrobial agents to be commercialized for use in veterinary medicine.

Macrolide-resistant Campylobacter: the meat of the matter.

The use of macrolide antibiotics in food animals has the potential to select for macrolide-resistant strains of resident bacterial flora. This may include the animal pathogens that are the intended targets of macrolide antibiotic intervention and Campylobacter, common inhabitants of the intestinal tract of food animals that are zoonotic pathogens in man. Such Campylobacter strains are not only resistant to the macrolide antibiotics used in food animals, e.g. tylosin, tilmicosin and tulathromycin, but to the macrolide antibiotics used in human medicine, e.g. erythromycin, azithromycin and clarithromycin, as well. Retail meat is a possible source of Campylobacter and persons consuming the meat derived from macrolide-treated food animals could acquire infections due to macrolide-resistant strains of this organism. Erythromycin is sometimes used to treat human cases of campylobacteriosis and those infected with animal-derived macrolide-resistant Campylobacter may not respond to treatment. The actual risk to human health from the use of macrolide antibiotics in food animals has been difficult to determine because of a lack of information about the macrolide-resistant Campylobacter found on the farm and in the clinic. Recently, however, a plethora of new information has become available on this topic. This review discusses what is currently known about the selection of macrolide-resistant Campylobacter in food animals, the prevalence of macrolide-resistant Campylobacter on retail meat, the prevalence of animal-derived macrolide-resistant Campylobacter in the clinic and the human health consequences associated with macrolide-resistant Campylobacter infection. This work will emphasize the comprehensive body of data generated in Denmark and the US as part of government-sponsored research studies over the last 10 years. These scientific findings may allow informed decisions to be made in the future about how macrolide antibiotics should be used in food animals while still safeguarding human health.

Animal health pharmaceutical industry.

The animal health pharmaceutical industry has proactively reported on the volumes of member company antimicrobial active ingredients sold in the U.S. At the individual company level, reporting of finished product distribution data to the FDA is a regulatory requirement, with applications to surveillance and pharmacovigilance. An accounting of product manufactured is done for purposes of good business practices, as well as marketing analyses. Additional applications of antimicrobial usage data might include use in risk assessments, such as for the FDA's Center for Veterinary Medicine Guidance for Industry #152 for the evaluation of the microbiological safety of antimicrobials intended for use in food animals. Compilation of national usage data will be a complex undertaking, hindered by issues such as confidentiality, auditing, field use practice variations, population dynamics (e.g. disease incidence, market conditions for poultry and livestock production), and generic usage. The amounts or volumes in pounds should be considered relative to the large number of animals under husbandry in the United States. Large volumes might seem impressive unless put into proper context. Until such time as a clearly defined application of national usage data is agreed, it is recommended that local usage programs will provide more useful information to perpetuate prudent antimicrobial use in animals.

Minimum inhibitory concentration breakpoints and disk diffusion inhibitory zone interpretive criteria for tilmicosin susceptibility testing against Pasteurella multocida and Actinobacillus pleuropneumoniae associated with porcine respiratory disease.

Tilmicosin is a novel macrolide antibiotic developed for exclusive use in veterinary medicine. Tilmicosin has been approved as a feed premix to control porcine respiratory disease associated with Pasteurella multocida and Actinobacillus pleuropneumoniae. The development of antimicrobial susceptibility testing guidelines for tilmicosin was predicated on the relationship of clinical efficacy studies that demonstrated a favorable therapeutic outcome, on pharmacokinetic data, and on in vitro test data, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The approved breakpoints for the minimum inhibitory concentration dilution testing for both species are resistant, > or = 32 microg/ml, and susceptible, < or = 16 microg/ml. The zone of inhibition interpretive criteria for disk diffusion testing with a 15-microg tilmicosin disk are resistant, < or = 10 mm, and susceptible, > or = 11 mm.

Prevalence of a novel capsule-associated lipoprotein among pasteurellaceae pathogenic in animals.

Capsular serotype A strains of Pasteurella multocida of avian origin express a 40-kDa lipoprotein (Plp-40) thought to attach the extracellular polysaccharide to the cell surface. The objective of the present study was to assess the prevalence of Plp-40 in P. multocida strains of disparate serotypes and host origins, as well as other pathogenic members of the family Pasteurellaceae. Exponential-phase reference and clinical isolates were radiolabeled with [3H]-palmitate, lysed to obtain whole-cell protein fractions, and analyzed using SDS-PAGE and fluorography to assess lipoprotein content. The ability to produce Plp-40 was found to be conserved among certain P. multocida reference and clinical strains of different host origins including avian, human, porcine, bovine, feline, canine, ovine, and cervine, but not rabbit. Production of a 40-kDa lipoprotein was exhibited by all clinical isolates of Pasteurella aerogenes, Pasteurella pneumotropica, Actinobacillus suis, Actinobacillus suis-like organism, and Actinobacillus pleuropneumoniae examined, but not Pasteurella (Mannheimia) haemolytica, Actinobacillus lignieresii, or Haemophilus spp. These data suggest that, while not all Pasteurellaceae are able to produce a 40-kDa lipoprotein under the present experimental conditions, expression is somewhat conserved among diverse isolates of disparate host origins.