Antimicrobial resistance in livestock: advances and alternatives to antibiotics

Antibiotic microbial resistance, as reported by the American Medical Association, is considered to be one of the greatest threats to human health. Advances in biotechnology have demonstrated that the efficacy of antibiotics can be restored by the use of antibiotic-peptide conjugates. Recently, several highly effective and alternative means of treating and controlling disease caused by microorganisms have been developed. This includes the use of a new gene editing technology (CRISPR/Cas9), genetically modified bacteriophages, engineered peptides, nanoantibiotics, improved vaccines, highly effective chicken and plant immunoglobulins, and Eubiotics. Improved DNA technology has greatly facilitated the selection of livestock that have genetic resistance to pathogenic microorganisms. Scientific breakthroughs in disease control will be able to safely overcome the problem of antibiotic resistance. More research, development, and evaluation, worldwide, is required. Antibiotic microbial resistance is considered to be one of the greatest threats to human health. In the United States, more than 2 million people are infected with antibiotic resistant bacteria annually, with 23,000 deaths as a direct result (Hampton, 2013). The O’Neil commission reviewed means to counteract the global threat of antibiotic resistance (O’Neil, 2016). It predicted that by 2050, 10 million deaths world-wide will be attributable to antimicrobial resistance. In addition to increased resistance to existing agents, there is a lack of new antibiotics in development. The commission made the following recommendations to reduce the consumption of antibiotics. Implement a massive global public campaign to improve global awareness of antimicrobial resistance. Improve hygiene and prevent the spread of infection. Reduce unnecessary use of antimicrobials in agriculture and their dissemination into the environment. Improve global surveillance of drug resistance and antimicrobial consumption in humans and animals. Promotes new, rapid diagnostics to cut unnecessary use of antibiotics. Promote development and use of vaccines and alternatives. A committee of the European Medicines Agency and the European Food Safety Authority outlined measures that could be implemented to reduce the use of antimicrobial agents in animal husbandry in the European Union, and its resulting impact on food safety (EMA and EFSA, 2017). The recommended options (non-prioritized) included the following: Develop national strategies for monitoring antimicrobial use and AMR development. Establish national targets for antimicrobial use reduction. Use of on-farm health plans. Increase the responsibility of veterinarians for prescribing antimicrobials. Increase the availability of rapid and reliable diagnostics. Improve husbandry and management procedures for disease prevention and control. Rethink livestock production systems to reduce inherent disease risk. Possible recommended alternatives to antibiotic use include probiotics and prebiotics, competitive exclusion, bacteriophages, immunomodulators, organic acids, and teat sealants. One Health is a “collaborative effort of multiple disciplines -working locally, nationally, and globally – to obtain optional health for people, animals and our environment.” Antimicrobial resistance is one of the most important issues that epitomizes the principles of One Health. Integrated approaches to reduce selection pressure and disrupt antimicrobial resistance transmission cycles on a global scale must be sought that are founded not only on sound One Health principles, but also based on economic evidence and on principles of social equity and global access to effective healthcare for people and their animals. An international agreement would help ensure the global coordination needed to accomplish these aims (Robinson et al., 2016). The goal of the current review is to provide an overview of recent advances and alternatives to the use of antibiotics by the animals feed industry. Currently used antibiotics tend to be broad spectrum, leading to in-discriminable killing of beneficial commensal bacteria and the evolution of drug resistance. CRISPR/Cas systems have been successfully used to targeted virulence factors and antibiotic resistance genes in bacteria and, as such, constitute an appealing option for the development of programmable and sequence specific antimicrobials (Bikard and Barrangou, 2017). CRISPR systems are a crucial component of the immune system of simple organisms. They are able to cut up any viral DNA sequences resulting from a viral attack. CRISPR technology is considered to be the discovery of the century in biotechnology, permitting a whole new field of gene-editing for therapeutic purposes including the development of engineered antimicrobials (Figure 1). The technology can be used to create antimicrobials whose spectrum of activity is chosen by design and to efficiently kill a target bacterial population when delivered by phage capsids both in vitro and in vivo. In addition, resistant bacteria can be re-sensitized to an antibiotic. In order to bring these strategies to the clinic, specific therapeutic approaches will have to be established. The unique advantage of CRISPR-based antimicrobials over all other strategies is their ability to kill bacteria based on their genetic sequence. This should prove advantageous in cases where it is desirable to eliminate only a select group of bacteria within a species, something that would be hard to achieve with incumbent strategies. CRISPR-based approaches will also address two grand challenges currently associated with antibiotics, namely (1) to prevent the indiscriminate eradication of intestinal bacteria that might be beneficial and (2) to lessen the selective pressure for resistance by allowing the non-target population to thrive and occupy the ecological niche. CRISPR-based technologies will open new avenues to control the composition of microbial communities rather than the traditional use of broad-spectrum antibiotics. Owing to the modularity and simplicity of CRISPR/Cas engineering, libraries of multiplexed RNA-guided nucleases can be rapidly constructed to simultaneously target antibiotic resistance and virulence determinants and to modulate the composition of complex microbial communities. This technology will reinvigorate the pipeline for new antimicrobials (Bikard and Barrangou, 2017; Kim et al., 2017). Gene silencing and editing with CRISPR. Guided RNA directs molecular machinery to cut both strands of the targeted DNA. During gene silencing, the DNA is broken and the gene is inactivated. For gene editing, a repair template with a specified sequence is added and incorporated into the DNA. The targeted DNA is now altered to carry this new sequence. (Data from Pak, 2014) Antibiotic conjugates are increasing used as a targeted therapy for the treatment or prevention of several bacterial diseases (Cal et al., 2017). Antibiotics in their natural form have limitations associated with bioavailability, toxicity, and biodistribution, as well as efficacy. A problem with the use of antibiotic therapy is often related to the inability to target specific moieties. Due to these limitations, and since free antibiotics have fast and short-acting effects, several daily high doses are required to maintain therapeutic concentrations at specific locations. This, in turn, can result in concomitant damage of commensal microbiota. The possibility of using conjugates to avoid antibiotic resistant pathways has the potential to revitalize antibiotics that have lost effectiveness against resistant bacteria. Conjugation of antibiotics provides a novel means for the delivery of antibiotic to any specific tissue in the body. Bacteriophages (phages for short) are viruses that can affect and kill bacteria. Bacteriophages infect bacterial cells with high specify, and in the case of lytic phages, they disrupt and lyse their host cells, resulting in cell death (Figure 2). Tail penetration through cell walls drives insertion of phage DNA into the cytoplasm of the host. Once inside the cell, specific enzymes encoded by the phage genome are synthesized to divert the host cell’s DNA and protein synthesis toward the generation of new phage particles. At a precise time at the end of the phage cycle, phage-encoded holins form pores in the cell membrane resulting in rapid cell destruction. Lytic phages have the ability to replicate exponentially and can rapidly eliminate bacteria regardless of their antibiotic resistance profiles. An overview of a phage attack on a bacterium and sites (red) of bacterial defense systems against phage attack. CRISPR gene-editing system can target each resistant site resulting in the ability of phage to kill bacteria. (Data from Seed (2015).) Virulent phages are very appealing candidates for use as biotherapeutic agents for treatment of acute infection in animals caused by both Gram-positive and Gram-negative bacteria (Endersen et al., 2014). Orally administered phages generally reduce the intestinal pathogen concentration or cause mortality. Some of the problems previously associated with the use of phages in animals are that they have a narrow range of hosts resulting in a limitation for their use for broad spectrum protection, administered phages may induce an immune response in the animal body, bacteria may become resistant to phages, and, finally, phages are not stable at the low pH of the stomach (pH ~2) but are almost completely stable at the pH of the large intestine (around pH 6.8) (Zhang et al., 2015). Colom et al. (2015) showed that encapsulation of phages in liposomes resulted in significantly longer periods of phage retention (several days longer) in the cecum of chicken. Park et al. (2017) has recently developed a genetic engineered phage-based delivery system as an antimicrobial against Staphylococcus aureus. They were able to overcome the current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of virulence genes. This same system can be adapted for use with other important pathogens. Recent research developments will allow the field to continue to address concerns regarding phage therapies and finally unlock their significant potential as antimicrobial agents. Additional research must be carried out to demonstrate the health/safety concerns of engineered phages including their ability to vector antimicrobial resistance. Antimicrobial peptides are promising next generation antibiotics that hold great potential for combating bacterial resistance. Antimicrobial peptides are small amphipathic peptides (29 to 42 amino acids) that are cationic (positively charged) and have direct and indirect antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and viruses (Li et al., 2017; Pachón-Ibáñez et al., 2017). They induce rapid killing and display a lower propensity to develop resistance than do conventional antibiotics. Despite significant progress in the past 30 yr, no peptide antibiotic has reached the clinic yet. Unfortunately, some disadvantages including stability, susceptibility to proteolysis, low activity under physiological conditions, and high cost of production must be circumvented before these peptides will reach the market place. Lam et al. (2016) synthesized a new class of antimicrobial agents, termed “structurally nanoengineered antimicrobial peptide polymers.” They exhibit sub-micromolar activity against all Gram-negative bacteria tested, while demonstrating low toxicity. Overall, structurally nanoengineered antimicrobial peptide polymers show great promise as low-cost and effective antimicrobial agents. They may be effective in combating the growing threat of resistant Gram-negative bacteria. Further research must be carried out to confirm the safety and efficacy of engineered antimicrobial peptides. Antimicrobial (host defense) peptides and their synthetic mimics have emerged as promising candidates for the killing of pathogenic bacteria. Cationic charge and amphiphilicity were identified as the two key antibiotic traits that help many antimicrobial peptides disrupt bacterial membranes via synergistic hydrophobic and charge interactions. Direct use of antimicrobial peptides is hindered by their expense, toxicity, and limited tissue distribution. Since the activity of antimicrobial peptides relies on their overall physicochemical property rather than their specific composition, much interest is put on developing synthetic peptides. A central dichotomy of synthetic antimicrobial peptides persists in that their hydrophobicity, which may be critical for antimicrobial activity, may also cause toxicity to mammalian cells. The prevalent wisdom on developing membrane active antimicrobials is to seek a delicate cationic–hydrophobic balance. Jiang et al. (2017) studied the antibiotic role of nanostructures by designing spherical and rod-like polymer molecular brushes that mimic the two basic structural motifs of the bacteriophage tail. The synthetically produced tail brushes were involved in the binding of the particle to bacterial cells. They demonstrated that, while the individual polymer molecular brushes are hydrophilic and a weak antimicrobial, amphiphilicity is not a required antibiotic trait once nanostructures come into play. The nanostructured polymeric molecular brushes induced pore formation (lethality) in bacterial but not in mammalian membranes. The sizes and shapes of the nanostructures further helped to define the antibiotic activity and selectivity of the polymer molecular brushes against of bacteria. 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