Prudent use and regulatory guidelines for veterinary antibiotics-politics or science?

Regulatory guidelines are in place across the world to ensure that approval of antibiotics is consistent with current scientific understanding of quality, efficacy and safety including minimizing the risk of the development of antibiotic resistance. We suggest the regulatory process is fit for purpose and does indeed approve products that are safe for use with regard to development of antibiotic resistance. However, we maintain that in order to preserve the longevity of antibiotics, treatment should be based on an established diagnosis and normally only antibiotics authorized for the diagnosed indication and indicated bacteria are used. Furthermore, susceptibility testing should be carried out whenever possible. Despite a general acceptance that antibiotic resistance is a significant issue, antibiotics can still receive a marketing authorization without a sponsor having to generate a clinical breakpoint. The consequence of this is that for many antibiotics we have no measure of what is resistant and what is susceptible at the approved dose. We argue that the time is right for all approvals of new or existing antibiotics to have independently agreed clinical breakpoints, as part of the regulatory process, without which talk of resistance is somewhat meaningless. This is relevant not only for novel antibiotics but also for generic compounds.

Workshop report: the 2012 antimicrobial agents in veterinary medicine: exploring the consequences of antimicrobial drug use: a 3-D approach.

Antimicrobial resistance is a global challenge that impacts both human and veterinary health care. The resilience of microbes is reflected in their ability to adapt and survive in spite of our best efforts to constrain their infectious capabilities. As science advances, many of the mechanisms for microbial survival and resistance element transfer have been identified. During the 2012 meeting of Antimicrobial Agents in Veterinary Medicine (AAVM), experts provided insights on such issues as use vs. resistance, the available tools for supporting appropriate drug use, the importance of meeting the therapeutic needs within the domestic animal health care, and the requirements associated with food safety and food security. This report aims to provide a summary of the presentations and discussions occurring during the 2012 AAVM with the goal of stimulating future discussions and enhancing the opportunity to establish creative and sustainable solutions that will guarantee the availability of an effective therapeutic arsenal for veterinary species.

Susceptibility testing methods, resistance and breakpoints: what do these terms really mean?

The Clinical and Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing can be considered the major international contributors to antimicrobial susceptibility testing. In this review, the author considers the differences between the respective organisations, examines the terminology used in antimicrobial susceptibility testing and argues for an urgent need to harmonise these definitions. While this may seem somewhat surprising, the terminology used to define resistance does differ. In this context, attention is given to the trend for 'resistance' to be defined by the epidemiological cut-off value, rather than by the long-established clinical breakpoint. The author goes on to discuss susceptibility testing methodologies and present an approach to setting clinical breakpoints.

Surveillance and monitoring of antimicrobial resistance and antibiotic consumption in humans and animals.

Surveillance and monitoring studies of antimicrobial resistance in bacteria of human and animal origin and antimicrobial consumption in humans and animals have been conducted in various countries throughout the world. In the veterinary field, in particular, programmes have been installed which target bacteria of zoonotic, foodborne and/or veterinary relevance. Each year, the European Surveillance of Veterinary Antimicrobial Consumption project summarises and evaluates antimicrobial consumption in ambulatory and hospital care in many European countries. In contrast, antimicrobial consumption data in veterinary medicine are available from only a few countries and the type of information that is collected or reported varies. To address this challenge, the European Surveillance of Veterinary Antimicrobial Consumption project was launched by the European Medicines Agency in September 2009 and has just published its first report. This comparison of the different studies for surveillance and monitoring of antimicrobial resistance and antimicrobial consumption in humans and animals shows the need to improve harmonisation.

Susceptibility of coagulase-negative staphylococci to a kanamycin and cefalexin combination.

A combination of kanamycin and cefalexin was licensed in Europe in 2008 to treat bovine clinical mastitis. Preliminary broth and disk clinical breakpoints for this antibiotic combination have been proposed for Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis, and Escherichia coli. This study indicates that these proposed breakpoints also hold for coagulase-negative staphylococci (CNS), a group of bacteria frequently isolated in milk samples from cows with clinical mastitis. The data show that clinical bovine mastitis isolates of CNS from Europe have a high degree of susceptibility to the kanamycin/cefalexin combination, with minimal resistance to either agent alone. The use of the available kanamycin and cefalexin combination disk for testing the susceptibility of bovine mastitis isolates of Staph. aureus, Strep. uberis, Strep. dysgalactiae, and E. coli is also reliable for use in the testing of CNS, as disk results correlated with broth minimum inhibitory concentrations. The study reports, for the first time, the approved Clinical Laboratory Standards Institute quality control ranges for the kanamycin/cefalexin combination and wild-type cutoff values for major bacterial pathogens implicated in bovine mastitis.

Fluoroquinolone resistance of Escherichia coli and Salmonella from healthy livestock and poultry in the EU.

The potential for transmission of antibiotic-resistant enteric zoonotic bacteria from animals to humans has been a public health concern for several decades. Bacteria carrying antibiotic resistance genes found in the intestinal tract of food animals can contaminate carcasses and may lead to food-borne disease in humans that may not respond to antibiotic treatment. It is consequently important to monitor changes in antimicrobial susceptibility of zoonotic and commensal organism; in this context, there are a number of veterinary monitoring programmes that collect bacteria in food-producing animals at slaughter and determine their susceptibility against antibiotics relevant for human medicine. The data generated are part of the risk analysis for potential food-borne transmission of resistance. There has been much debate about the use of fluoroquinolones in veterinary medicine, and so, this review will consider the fluoroquinolone data from two surveys and compare them to national surveillance programmes. At the outset, it must be pointed out that there is, however, a lack of agreement between several programmes on what is meant by the term 'fluoroquinolone resistance' through use of different definitions of resistance and different resistance breakpoints. An additional aim of this paper is to clarify some of those definitions. Despite the debate about the contribution of antibiotic use in veterinary medicine to the overall resistance development in human pathogens, the data suggest that clinical resistance to fluoroquinolones in Escherichia coli and nontyphoidal Salmonella is generally uncommon, except for a few countries. Ongoing surveillance will continue to monitor the situation and identify whether this situation changes within the respective animal populations. For the benefit of both the epidemiologist and the clinician, it would be strongly advantageous that national monitoring surveys report both percentages of clinical resistance and decreased susceptibility.

Impact of antimicrobial residues on gut communities: are the new regulations effective?

Veterinary medicines are subject to a rigorous evaluation with regard to safety, efficacy and quality before they are licensed. For drugs used in food producing animals, it is necessary to establish what is referred to as the acceptable daily intake (ADI), this is defined as an estimate of the amount of a substance, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable risk to human health. It is necessary to determine a toxicological, pharmacological and microbiological ADI. This article describes a recently harmonized guideline that outlines the process for determining the need for a microbiological ADI and discusses the test systems that take into account the complexity of the human intestinal flora. The described process is used to address the effects of antimicrobial drug residues on human intestinal flora for regulatory purposes. The guideline does not recommend any one particular system for use in regulatory decision making but provides recommendations for a harmonized approach to establish a microbiological ADI and offers test options rather than specifying a testing regimen. The process and the challenges of this new guideline are discussed.

Do bacteria need to be regulated?

Additives for use in animal nutrition are regulated under Regulation (EC) No. 1831/2003. The scope of this paper addresses the specific microbiological issues relevant to a microbial feed additive, containing a Bacillus spp. and uses as an example a product with the trade name, Calsporin. Bacillus subtilis C-3102 is the active ingredient in Calsporin and is added to animal feed to favourably affect animal production and performance (growth and feed efficiency), by modulating the gastrointestinal flora. It is not the purpose of this review to present the raw data for Calsporin but rather to use Calsporin as an example of the type of data required by the European regulatory authorities. At the time of preparation of this manuscript Calsporin has yet to be reviewed by the authorities. The regulatory system under the auspices of the EFSA FEEDAP Panel is clearly attempting to move in line with development of scientific opinion and is to be applauded for such efforts. Bacteria do need to be regulated, and the regulations clearly provide adequate and appropriate protection to human health and to environmental considerations.

Influence of sample preparation technique on two-dimensional gel electrophoresis of proteins from Porphyromonas gingivalis.

Sample preparation methods were compared for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of cellular proteins from the proteolytic bacterium Porphyromonas gingivalis. Standard solubilization buffer yielded poorly resolved protein spots, but pre-treatment of cells with trichloroacetic acid or inclusion of the protease inhibitor TLCK during solubilization improved definition and separation. The latter approach allowed reliable detection of a 55 kDa immunodominant surface antigen by Western immunoblotting. Further improvements in resolution occurred when SDS was included in the sample preparation. Thus, controlling proteolysis and optimizing protein solubilization were essential for reproducible separations and maximal protein recovery during 2D-PAGE of P. gingivalis.

Harmonic generation in "non-linear" biological systems.

We describe a high performance, low frequency Fourier Transform based spectrum analyser, with design features particularly suited to the harmonic analysis of the non-linear amplitude transfer functions of various biological systems. A previous published method, using general purpose systems to produce reference and signal plus reference "power" spectra, was susceptible to ambiguous interpretation of results. Unlike that design, the present spectrometer derives quantitative complex voltage (amplitude and phase) harmonics of sampled voltage data, maintaining potentially important information. Based on the use of a basic IBM or compatible PC, it features high sensitivity, high speed data processing and large dynamic range, is compact, easy to use, flexible in operation and low in cost. Its precision current signal source eliminates harmonics generated at the drive electrodes from the measurement. Use of the instrument leads us to conclude that, contrary to previous assertions, no repeatable dielectric or conductive non-linearity is exhibited in the bulk cell suspensions tested under the field and frequency conditions reported.

Harmonic 'signatures' of microorganisms.

The frequency/amplitude effect of various microorganisms exposed to periodic (time varying) electric fields, when proximate to immersed electrodes, has been studied using a novel analytical instrument. The harmonic distribution, in complex signals caused by cells exposed to harmonic free waveforms and occupying part of the electrode/suspension interface volume, was shown to be almost entirely due to the change in the standing interfacial transfer function by the (dielectrically nonlinear) presence of cells. Thus, the characteristic interfacial non-linearity is viewed as variable, being uniquely modulated by the presence of particular cells in the interfacial region. Little can be attributed to bulk (far field) effects. The tendency for subtle (characteristic) signal distortion to occur as a function of particulate (cell or molecular) occupancy of the near electrode interfacial region under controlled current conditions leads to the method of sample characterisation by harmonic (Fourier) analysis. We report here, as a sequel to our original studies (Hutchings et al., 1993; Hutchings and Blake-Coleman, 1993), preliminary results of the harmonic analysis of microbial suspensions under controlled signal conditions using a three-electrode configuration. These data provide three-dimensional graphical representations producing harmonic 'surfaces' for various microorganisms. Thus, cell type differences are characterised by their 'harmonic signature'. The visual distinction provided by these 'surface' forming three-dimensional plots is striking and gives a convincing impression of the ability to identify and enumerate specific microorganisms by acquisition of cell-modulated electrode interfacial Fourier spectra.

Mode of action of GR69153, a novel catechol-substituted cephalosporin, and its interaction with the tonB-dependent iron transport system.

GR69153 is a novel cephalosporin incorporating a catechol-substituted 7-aminothiazolyl-oxime. The antibiotic is actively transported into gram-negative cells via iron-regulated outer membrane proteins regulated by the tonB product. This transport enhances bactericidal activity most significantly at low concentrations, essentially removing the permeability barrier for antibiotic uptake.

Cephalexin in ponies: a preliminary investigation.

The administration of a single dose of the antibacterial agent cephalexin intramuscularly to six ponies at a dose rate of 7 mg/kg was well tolerated. No reactions at the injection site were apparent. It was absorbed rapidly and reached a mean peak plasma concentration of 6.77 micrograms/ml after a mean of 1.41 hours; plasma concentrations above 2.0 and 0.5 micrograms/ml were maintained for 3.8 and 9.8 hours, respectively.

Model simulation of a single oral dose of cefuroxime axetil and the related in-vitro kill kinetics against four bacterial species.

An in-vitro model was shown to be capable of simulating a cefuroxime serum profile equivalent to that observed in human volunteer studies, following a single dose of 250 mg cefuroxime axetil. The model was used to carry out kill kinetic studies and showed cefuroxime to lyse the four bacterial test strains, time of onset of lysis being related to the sensitivity of the respective organisms. The more sensitive Staphylococcus aureus and Haemophilus influenzae strains were subject to a higher absolute kill and showed no regrowth over the duration of the simulated serum profile. In contrast, Proteus mirabilis and Escherichia coli showed regrowth after 4 and 5 h respectively. The kill kinetic profiles of the respective organisms are discussed in relation to the pharmacokinetic analysis of the cefuroxime serum profile.

Kill kinetics of the cephalosporin antibiotics cephalexin and cefuroxime against bacteria of veterinary importance.

Kill kinetic studies for two cephalosporin antibiotics, cephalexin and cefuroxime were carried out against veterinary strains of Escherichia coli, Pasteurella multocida, Streptococcus suis, Erysipelothrix rhusiopathiae and a laboratory culture of Staphylococcus aureus. In more than 90 per cent of cases a kill of more than 99 per cent was achieved within four hours of antibiotic treatment at concentrations of 2 or 4 micrograms/ml. Although cefuroxime was effective at lower concentrations than cephalexin the rates of kill of the two antibiotics were comparable. The results are discussed in relation to in vivo dosage regimens.