Antimicrobial Resistance in Bacteria of Animal Origin

The introduction of antimicrobial drugs into agriculture shortly after the Second World War caused a revolution in the treatment of many diseases of food animals. A broad overview of key features of the history of antimicrobial drug usage in food animals is given, which traces developments from the preantibiotic era to the present day, where there are highly arguable fears that we are moving into the "postantibiotic" era. The majority of antimicrobial drugs used in animals belong to a small number of major classes, and only one new class of antimicrobial drugs, fluoroquinolones has been introduced for food animal use in the last 30 years. Improvements in understanding of the microbiology of infectious diseases acquired from animals were less important, but also critical, forces in the reexamination of antimicrobial usage in agriculture. Ready access to the ‘’miracle drugs’’ by medicine and agriculture, coupled with the ability of chemists to alter existing drugs or develop new ones to counter resistance, led to the expectation that the preantibiotic era was to become a folk memory marked only on mossy gravestones. The new technologies of recombinant DNA, diagnostic DNA microarrays, genomics, proteomics, and combinatorial chemistry hold considerable promise for the development of new, possibly pathogen-targeted, antimicrobial drugs.

The combination of molecular characterization of resistant strains with precise identification of the antibiotic resistance gene(s) or mutation(s) and the genetic elements involved in the dissemination of these genes is an effective approach in the control of the spread of antibiotic resistance. Molecular methods also help to determine the location of the gene and to differentiate between horizontal gene transfer and clonal spread. This chapter describes polymerase chain reaction (PCR) and microarray analysis in detail, and specifies applications of these methods in the investigation of antibiotic-resistant strains. Numerous PCR assays for the detection of antibiotic resistance genes have been developed and the development of microarrays for the simultaneous detection of a large number of these genes and the genetic elements involved in their dissemination is in progress. The implementation of molecular methods in routine analysis can be achieved only when it is supported by the proper validation of the methods and the availability of the necessary controls, reference strains, and educated personnel. The molecular characterization of antibiotic-resistant strains can help to identify atypical resistant strains, describe new outbreak strains at an early stage, elucidate the epidemiology of resistant strains at a genotypic level, and explain the processes leading to the selection of resistant and virulent strains. In addition, molecular methods can allow proper risk assessment with respect to the use of antimicrobial substances. If the transcriptional and translational expression of antibiotic resistance genes can be better understood, molecular methods may replace phenotypic measurements.

Nowadays, antimicrobial agents are among the most frequently used therapeutics in human and veterinary medicine. Resistance to antimicrobial agents can be subdivided into two basic types of resistance, intrinsic resistance and acquired resistance. Resistance to ß-lactam antibiotics is mainly due to inactivation by ß-lactamases and decreased ability to bind to penicillin-binding proteins (PBPs) in both gram-positive and gram-negative bacteria, but may also be based on decreased uptake of ß-lactams due to permeability barriers or increased efflux via multidrug transporter systems. Different types of multidrug transporters mediating resistance to tetracycline in addition to resistance to a number of structurally unrelated compounds are described, for instance, in Escherichia coli, Salmonella, and P. aeruginosa. Plasmids, genomic islands, transposons, gene cassettes, and integrons are spread vertically during the division of the host cell, but can also be transferred horizontally between bacteria of the same or different species and genera via transduction, conjugation and mobilization, or transformation. The development of antimicrobial resistance—by either mutations, generation of new resistance genes, or acquisition of resistance genes already present in other bacteria—is a complex process that involves different mechanisms. Due to the usage of all types of antimicrobial substances for selection of resistant bacteria, prudent use of the antimicrobial agents is strongly recommended in both human and veterinary medicine, as well as in food animal production, to retain the efficacy of antimicrobial agents for the control of bacterial infections in animals.

Disinfectants are used extensively in animal husbandry, hospital and food industry. Particularly important in the food industry is the inactivation of bacteria attached to surfaces and at low temperatures, both conditions where bacterial tolerance to disinfectants can be enhanced. Concerns about possible antibiotic and disinfectant cross-resistance are discussed in “Potential for Selection of Resistant Strains”. Gram-negative bacteria are generally less susceptible to disinfectants than gram-positive bacteria, presumably due to the reduced permeability of the double membrane. Mutants of various bacterial species (S.enteric serovar Typhimurium, E.coli, and S.aureus) with reduced susceptibility to triclosan can be selected in vitro after exposure to sublethal concentrations of the compound. The inherent resistance of gram-negative bacteria to antibacterial agents and disinfectants is often attributed to poor permeability of the cell to these agents. The MICs of disinfectants for most bacteria are normally greatly below the concentrations used in practice. A recent study demonstrated differences between the hand flora isolated from “homemakers” and intensive care nurses. A key difference between the two groups was the increased hand hygiene practiced by the nurses, and it is possible that disinfectant exposure has contributed to the differences in flora observed in this study. Cleaning prior to disinfection is important in order to remove organic material and other contaminants that might interfere with disinfectant activity.

This chapter focuses on the prevalence of antimicrobial resistance phenotypes among Escherichia coli isolates associated with enteric and systemic colibacillosis in minor species, cattle, pigs, and poultry, as well as bovine mastitis and uterine and urinary tract infections in companion animals. Antimicrobial resistance phenotypes among avian pathogenic E. coli recovered from diseased turkeys are similar to those reported from chickens. Fluoroquinolones are highly efficacious antimicrobial agents commonly used in human medicine. This class of drugs has also been approved for certain bacterial diseases in animals, including acute bovine respiratory disease and avian colibacillosis. Resistance to narrow-spectrum cephalosporins and tetracyclines among E. coli isolates may be due to their use for intramammary treatment for clinical mastitis in dairy cows. Treatment in grower-finisher rations was significantly associated with resistance to ampicillin, spectinomycin, sulfisoxazole, and tetracycline. Nineteen percent of swine E. coli isolates were also shown to possess class 1 integrons bearing gene cassettes conferring resistance primarily to trimethoprim and streptomycin. Plasmids containing the oqxAB genes yielded high resistance to olaquindox as well as chloramphenicol in E. coli. Diarrhea associated with E. coli infection is responsible for high rates of morbidity and mortality in goat kids and lambs. Antimicrobials are essential tools of disease management regimens in food and companion animals worldwide. The most commonly used antimicrobial drugs in food and companion animals are from five major classes: β-lactams, tetracyclines, aminogylcosides, macrolides, and sulfonamides.

This chapter reviews the summarized data on susceptibility patterns and prevalence and epidemiology of resistance for the most clinically relevant staphylococcal and streptococcal pathogens in animals. The chapter focuses mainly on resistance in Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus intermedius. The first isolation of methicillin resistant Staphylococcus aureus (MRSA) from a veterinary specimen was reported from bovine mastitis in Belgium by Devriese et al. in 1972. The presence of other resistance genes and mechanisms has not been examined in streptococci of animal origin. Antimicrobial agents are most often used to control infections caused by these two groups of bacteria. The continuous monitoring of staphylococci and streptococci from animal infections is an essential prerequisite for early detection of new trends in the development of resistance to antimicrobials commonly used to control these infections. However, standardized procedures need to be made available for unambiguous species identification and for in vitro susceptibility testing, and such procedures also need to be applied by researchers in this field to make the results of susceptibility testing more comparable. More knowledge about the ability of certain subtypes to acquire resistance, together with the development of means to control such clones, could perhaps provide new means of controlling the increase in resistance.

The main species that cause infections in animals belongs to the genera Mycoplasma and Ureaplasma. In veterinary medicine the most commonly used antimicrobial agents are fluoroquinolones, macrolides, tetracyclines, and tiamulin. Susceptibility testing for Mycoplasma can, as for all other bacterial species, be performed by agar or broth dilution or disk diffusion. Susceptibility testing of mycoplasmas requires special media because of their slow growth and fastidious medium requirements. Worldwide, there have been very few reports on the antimicrobial susceptibility of the different Mycoplasma species. M. synoviae is intrinsically resistant to erythromycin and flumequin. Most studied strains are susceptible to josamycin, spiramycin, lincomycin, tylosin, tilmicosin, tiamulin, danofloxacin, and enrofloxacin. The genetic background for resistance has been studied only with mycoplasmas causing infections in humans. A high frequency of tetracycline resistance has been reported in several Mycoplasma species. Quinolones interfere with DNA replication by binding to DNA gyrase or topoisomerase IV. Resistance to quinolones is caused mainly by mutations in the genes encoding DNA gyrase or topoisomerase IV. Mutations at positions 83, 87, and 119 in gyrA have been frequently found to be associated with quinolone resistance in several gram-negative bacterial species, whereas changes at positions 81 and 84 seem to be infrequently found. There are only a very limited number of reports on the antimicrobial susceptibility of the different Mycoplasma species pathogenic for animals, and there is an obvious need for standardization of methods and interpretation.

This chapter provides a review of prevalence and trends of resistance in Campylobacter jejuni and Campylobacter coli isolated from humans in different parts of the world and a more thorough description of the mechanisms of resistance, origin, spread, and clinical consequences of resistance. Aminoglycosides exhibit rapid and significant bactericidal effects in vitro and should initially be included for the treatment of Campylobacter bacteremia in patients who appear very ill. The only mechanism of chloramphenicol resistance identified in Campylobacter occurs through modification of chloramphenicol by chloramphenicol acetyltransferase, which prevents its binding to the ribosome. The majority of contacts between Tet(O) and the ribosome are mediated by the rRNA, and one interaction with ribosomal protein S12. Most of the antimicrobials used in veterinary medicine are tetracyclines and macrolides, which result in high and continuous selective pressure for the animal-colonizing bacteria, ultimately resulting in the acquisition of antimicrobial resistance genes. Investigation into the mechanisms of action of antimicrobials, as well as the transfer of resistance determinants, is necessary to gain effective control of antimicrobial resistance. Epidemiological and microbiological studies show that poultry is the most important source for quinolone-susceptible and quinolone-resistant Campylobacter infections in humans. Trends over time for macrolide resistance show stable low rates in most countries, and macrolides should remain the drug class of choice for C. jejuni and C. coli enteritis.

This chapter focuses on the presence of virulence and antimicrobial resistance genes in enterococci of animal origin and the possible spread of resistance between the animal and human reservoir, probably through the food chain. Some species, such as Enterococcus gallinarum and E. casseliflavus, are motile. At least 23 distinct Enterococcus species are recognized and new species continue to be identified. The defined species have been separated into five groups on the basis of acid formation in mannitol and sorbose broths and hydrolysis of arginine. Identification of the different Enterococcus species can be done by conventional physiological tests, by commercial systems, or by molecular methods. The major natural habitat of organisms appears to be the gastrointestinal tract of animals and humans, where they make up a significant portion of the normal aerobic gut flora. Some Enterococcus species are host specific, while others are more broadly distributed. The emergence of antimicrobial resistance represents the greatest threat to the treatment of human enterococcal infections. The chapter also focuses on the antimicrobial resistance selected for animals that can be transferred to humans, causing treatment failures. Tetracycline resistance is probably the most common resistance phenotype in enterococci from food. Enterococci have an ability to become resistant to antimicrobials. The role of enterococci in disease raises valid concerns regarding their safety for use in foods or as probiotics. If Enterococcus strains are selected for use as starter or probiotic cultures, ideally such strains should harbor no virulence determinants and should be susceptible to clinically relevant antibiotics.

The origin of acquired antimicrobial resistance is in general believed to be adoption of resistance genes from antimicrobial-producing organisms and/or nucleotide changes in housekeeping genes. This chapter provides a brief overview of the putative origins of some of the most important antimicrobial resistance determinants and some of the genetic mechanisms. The actual origin of antimicrobial resistance genes is unknown, but environmental microbes, including the natural producers of antimicrobial substances, are believed to be important primary sources. The ß-lactamases are believed to have evolved from enzymes involved in cell wall biosynthesis. The main mechanism of resistance to aminoglycosides is enzymatic inactivation. Comparison of the DNA and deduced amino acid sequence of the erm genes from macrolide producing organisms and macrolide-resistant bacteria strongly suggests that the origin of macrolide resistance is macrolide-synthesizing organisms. Resistance to tetracyclines is mediated either by genes encoding ribosomal protection proteins (RPPs) or efflux pumps. Food animals and pets produce large amounts of waste products, which may contain many pathogenic bacteria, antimicrobial-resistant bacteria, and resistance genes. Human waste contains large amounts of antimicrobial-resistant bacteria and resistance genes. Such waste is normally treated to avoid transmission of pathogenic bacteria.

This chapter evaluates the usefulness and limitations of the various sources of input data, discusses how usage should be expressed in order to comply with the various purposes of monitoring, and presents examples on the application of data obtained through various monitoring programs. Data on antimicrobial drug usage collected from veterinarians and farmers may give information about dosage schemes and usage per species, per age group, by indication, and at herd level. Data on antimicrobial drug usage could also be collected by the use of questionnaires to veterinarians or farmers. Ideally, all antimicrobial ingredients and classes of antimicrobials used in animals should be included in a monitoring program on antimicrobial drug usage. Conversion of aggregate usage data to defined daily dose (DDD) may thereby create over- or underestimates of usage. The prescribed daily doses (PDDs) for veterinary antimicrobials may be calculated from prescriptions, questionnaires, or information obtained from veterinarians’ invoices. Usage of antimicrobials in selected food producing animal species should, if possible, be calculated to express doses per number of animal species and per age group, e.g., weaning pigs. In order to examine the selective pressure exerted by the various antimicrobial growth promoters (AGPs) or coccidiostat feed additives (CFAs), the proportional use of the different feed additives may be expressed as amounts of feed (in weight) containing an AGP or a CFA per 1,000 animals per time period.

The purpose of risk assessment is to help a manager better understand the risks (and opportunities) being faced and to evaluate the options available for their control. The chapter describes some antimicrobial resistance risk assessments that have been completed. The Food and Drug Administration (FDA) wanted to produce a model that was based on reliable data. The FDA deemed that the risk assessment had quantitatively demonstrated that the resistance in question presented a risk to human health, and subsequently moved to withdraw use of the antimicrobial. The major strengths of the model were its mathematical simplicity, its reliance on mostly federally collected data, its very limited set of assumptions, and the ease with which it could be updated as new data became available. The data problems underlined the difficulties of evaluating even relatively simple model parameters for antimicrobial risk assessments. The FDA’s quantitative risk analysis model was successful in that it provided robust information from which the FDA was able to make an important decision. A key to the successful involvement of risk assessment in decision making is the neutrality of the risk assessor. There is, unfortunately, a growing trend in published antimicrobial resistance risk assessments that are clearly selective about their sources or manipulate a risk assessment model to produce the desired answer. The FDA guidance takes an essentially qualitative approach to evaluating drugs for approval and continued use.