APPLICATION OF BACTERIOPHAGE THERAPY IN FARM ANIMALS TO REDUCE ANTIBIOTIC USE
by-DR. RAJESH KUMAR SINGH, (LIVESTOCK & POULTRY CONSULTANT), JAMSHEDPUR, JHARKHAND,INDIA 9431309542, rajeshsinghvet@gmail.com
Bacteriophages (BPs) are viruses that can infect and kill bacteria without any negative effect on human or animal cells. For this reason, it is supposed that they can be used, alone or in combination with antibiotics, to treat bacterial infections.
Bacteriophage (often abbreviated to ‘phage’) are viruses which infect and kill bacteria. The idea of using them to treat infections (aka ‘phage therapy’) is not new, and predates the discovery of antibiotics by approximately 10 years. Early in the 20th century, Félix d’Hérelle, who co-discovered bacteriophage, conducted several phage therapy experiments in animals and humans, successfully treating bacterial dysentery, bubonic plague, cholera and Salmonella infections among others. Phage therapy fell out of favour in the West during the 1930s and 1940s, due partly to a poor understanding of bacteriophage biology, and also the rise of antibiotics, which was seen as more promising. The resurgent interest in phage in the animal husbandry in recent years is chiefly a consequence of AMR.
Bacteriophages as potential new therapeutics to replace or supplement antibiotics.
Over recent decades, a growing body of literature has validated the use of bacteriophages for therapy and prophylaxis in the war against drug-resistant bacteria. Today, much more is known about bacteriophages than in the 1930s when phage therapy first appeared and began to spread to many countries. With rapid dissemination of multi-drug-resistant bacterial pathogens, the interest in alternative remedies to antibiotics, including bacteriophage treatments, is gaining new ground. Based on recent experience and current results of bacteriophage applications against bacterial infections in countries where this alternative therapy is approved, many scientists and companies have come to believe that the use of phages for treating and preventing bacterial diseases will be successful.
Since the discovery of bacteriophages during 1915-1917 (see Phage History), they have been used to prevent and treat various bacterial infections. Although “phage therapy” has been historically associated with the use of bacteriophages in human medicine, phages also have been extensively used in veterinary medicine1. The first-known therapeutic use of phages in veterinary medicine is associated with Felix d’Herelle (who was also the first to use phages to treat human infections, see Human Therapeutics), the co-discoverer of bacteriophages, who examined their efficacy – in France, during 1919 – in preventing and treating Salmonella infections in chickens. Phages effectively reduced chicken mortality, which prompted other investigators to examine their possible usefulness in preventing and treating other naturally-occurring and experimental bacterial infections in animals. In that regard, phages have been reported to be a safe and effective preventive/treatment modality against numerous bacterial infections of animals. Some examples would include:
• Salmonella infections in poultry
• Escherichia coli infections in mice, poultry, calves, piglets, and lambs
• Clostridium difficile infections in hamsters
• Acinetobacter baumanii infections in mice
• Pseudomonas aeruginosa infections in mice
• Staphylococcus aureus infections in mice and cows
Why phage?————
Phage have many potential advantages as antimicrobials. They are self-replicating and self-limiting; multiplying only when susceptible bacteria are available. Unlike antibiotics, they are specific to a genus, species or strain of bacteria and can precisely target bacterial subpopulations. This reduces the possibility of imbalances of microbiota (dysbiosis) which have been linked to disorders such as inflammatory bowel disease, coeliac disease, asthma and metabolic syndrome. They can be prepared inexpensively, and locally, which facilitates their use in underserved populations (e.g. in developing countries). They can also be targeted towards receptors on the bacterial cell surface which are involved in virulence. The loss or modification of these receptors by the bacterium often leads to attenuated virulence.
History of phage therapy:
Phage therapy was first introduced by the French-Canadian scientist Felix d’Herelle, who co-discovered bacteriophages around 1910. Bacteriophages are a class of viruses that specifically target and kill bacteria. The bacteriophage firstbinds to specific surface receptors on the host bacteria before injecting its genetic material and hijacking the bacterial cell machinery. This process ultimately causes the bacteria to burst, killing the bacteria and releasing a multitude of newly formed phages to target and kill additional bacteria. d’Herelle later used these phages tosuccessfully treat dysentery in a number of patients, providing proof of principle that bacteriophages could therapeutically treat bacterial infections.
Applications of phage in livestock—————-
Interest in phage therapy in livestock was rekindled during the 1980s with some exquisitely designed experiments by H. Williams Smith. This work, initially performed in mice, then in cattle, demonstrated that a single dose of phage was as effective as multiple doses of antibiotics when treating systemic Escherichia coli infections. Subsequent experiments demonstrated that phage can be used both prophylactically and therapeutically to treat intestinal and systemic diseases in a range of livestock, including chickens, pigs, cattle, sheep and fish.
Phage therapy in livestock has been largely directed towards foodborne zoonoses, chiefly those caused by Campylobacter, Salmonella, E. coli and Listeria. The phage used for this purpose are often characterised as exclusively lytic, i.e. their infection cycle does not involve integration in the bacterium’s chromosome (a property of ‘temperate’ bacteriophage). The use of lytic phage arguably improves the predictability of phage therapy, and also reduces the possibility of phage-mediated transfer of DNA between bacteria. More recently, it has become commonplace to sequence and annotate the genomes of candidate therapeutic phage to ensure they do not carry potentially harmful genes associated with virulence or AMR.
Phage treatment of systemic infections in animals is usually by intravenous or intramuscular injections whereas gastrointestinal infection or colonisation is usually treated by phage supplementation of feed or water. Experimental phage therapy trials typically report a 90 to 99% reduction in the targeted bacterial population, and sometimes much greater. However, the phage do not usually eliminate their targets. Once the bacterial population falls below a critical level (sometimes called the ‘phage proliferation threshold’) it becomes less likely that the phage will come into direct contact with the bacteria and initiate an infection. This scenario is perhaps more likely in the guts of animals than other environments because phage may attach to particles of food or non-host ‘decoy’ bacteria, which will further reduce the available pool of phage. In addition, the host bacteria are not evenly distributed throughout the animal gut, but exist in localised populations and particular environmental niches which may not always be accessible to the phage.
A 99% reduction in bacterial pathogens has been shown to significantly improve clinical outcomes in many experimental livestock infections, including cattle, pigs and chickens, and is comparable to reductions seen with therapeutic levels of antibiotics such as enrofloxacin in poultry. Selectively reducing bacterial populations to this degree may also result in appreciable public health benefits. For example, some estimates suggest a 99% reduction in the level of Campylobacter on retail chickens would reduce the number of human campylobacteriosis cases by more than 30-fold.
Limitations and barriers—————
Despite impressive results in experimental infections, there are some important limitations on the useful scope of phage therapy. Phage are unlikely to be effective for the treatment of tuberculosis, brucellosis or other diseases involving intracellular pathogens. The specificity of phage also makes them less useful in the treatment of polymicrobial infections. The emergence of phage-resistant pathogens is also a concern, but can be addressed by using ‘cocktails’ of phage, targeting different receptors. In addition to these technical limitations, there are practical considerations, such as where phage fit into national and international regulatory frameworks which are not designed to deal with ‘living’ antibiotics. Also, like antibiotics, there is no financial incentive for pharmaceutical companies to develop phage-based therapeutic products. Finally, there are concerns about the public acceptability of food, or food animals, deliberately treated with viruses.
Future prospects————
The rise of antimicrobial resistance has forced us to reappraise phage therapy as an alternative or adjunct to antibiotic treatments. While phage are unlikely to be a panacea, they clearly have significant potential to control bacterial infections which may no longer be amenable to antibiotic chemotherapy. As such, phage therapy promises to be a valuable tool in the fight against antimicrobial resistance.
The goal of sustainable animal husbandry is to implement practices that will attain healthy disease-free animals, provide safe food for a growing global population, and minimize the impact of animal husbandry practices on the environment . Conversely, animal husbandry practices are impacted by economic and disease pressures, consumer preferences, geographic location, weather conditions, and government regulations. Following the Second World War, antibiotics have been incorporated into animal husbandry. The overuse in medicine and animal husbandry has contributed to the rise of worldwide antimicrobial resistant (AMR) bacteria.
Most antibiotics are non-specific, acting not only against the target pathogen, but also against other bacteria naturally present in the environment or plant and animal microflora. Drug-resistant infections result in millions of people being affected from drug-resistant bacteria each year, with an estimated 700,000 deaths worldwide each year, a number that could increase to 10 million by 2050 if the drug resistance trend continues . Imprudent use of antimicrobials in animal husbandry may result in reduced efficacy of antibiotics due to facilitated emergence of antibiotic resistant human pathogens, increased human morbidity and mortality, increased healthcare costs, and increased potential for carriage and dissemination of pathogens. Together with consumers’ calls for antibiotic-free products, popularity of organic products and the removal of antibiotics for veterinary use in certain jurisdictions have led to the search for alternatives. Use of phages, which infect and destroy bacteria, could significantly reduce the environmental impact of antibiotic use in veterinary , while potentially increasing profitability by lowering loss or animal mortality in early stages of the breeding process.
Bacteriophages in Food Animal Production———–
By volume, the vast majority of antibiotics consumed worldwide are for veterinary purposes, predominantly in intensive and large-scale animal production systems, such as dairy, livestock, poultry, and aquaculture . Animal husbandry practices widely use antibiotics therapeutically to treat infectious diseases, as well as non-therapeutically to prevent the spread of disease (prophylaxis) and to promote growth. Controversy, however, surrounds the widespread use of antibiotics for animal production, as their overuse and possible misuse is driving antibiotic microbial resistance. For instance, the practice of prolonged exposure to sub-therapeutic antibiotic doses, the context in which prophylactic and growth-promoting antibiotics are administered, exerts an inestimable amount of selective pressure toward the emergence of AMR . Furthermore, AMR bacteria and AMR genes of animal origin can then be transmitted to humans through environmental contamination, food distribution, or direct contact with farm animals . Intensive animal production systems necessitate antibiotics to keep animals healthy and maintain productivity, and with rising incomes in transitioning countries expected to boost antibiotic consumption by 67% by 2030 , this presents a major health risk to humans and animals.
The World Health Organization, the European Commission, the Centers for Disease Control and Prevention, and Health Canada, to name a few, all support immediate antimicrobial stewardship in animal food production, aimed primarily at reducing or eliminating the nontherapeutic use of medically important antibiotics. Eliminating prophylactic antimicrobials outright may not be feasible in intensive animal production systems due to increasing worldwide demand for protein, the potential compromise in animal welfare and health, and in human health and food safety. Phages instead of antibiotics are a promising option in food animal production to maintain animal health and limit the transfer of AMR and zoonotic pathogens that may be harmful to consumers. So the on application of phages as alternatives to antibiotic growth promoters, prophylaxis, and zoonotic pathogen animal decolonization at the farm level has become an important tool .
Phages as Growth Promoters—————-
Antibiotics in subtherapeutic doses have played important roles in the promotion of growth, enhancement of feed efficiency and improvement of the quality of animal products . To combat the increased rate of mortality and morbidity due to reduction of in-feed antibiotics, phages have been proposed as the replacement, particularly in the early stages when vaccination is not possible and the maintenance of the bacterial ecosystem is crucial . The distinction between growth promotion and prevention or treatment of diseases is subtle and further work is needed to see if phages do offer growth promotion effects other than simply reducing disease incidence.
Clostridium perfringens is a major problem for the poultry industry, resulting in both clinical and subclinical infections. A cocktail of five phages could effectively control necrotic enteritis in chicken broilers and thus improve feed conversion ratios and weight gain . This efficacy was independent of whether the phages were administered in feed or in drinking water. Dietary supplementation with phages has also been shown to improve on growth performance in pigs . Feed supplemented with a commercial phage product, which contained a mixture of phages targeting several pathogens, including Salmonella spp., Escherichia coli, Staphylococcus aureus, and C. perfringens, improved different aspects of grower pig’s performance, such as average daily feed intake .
For dairy herds, mastitis is the most important disease worldwide . S. aureus, one of the etiological agents for mastitis, which has a propensity to recur chronically, causes a potentially fatal inflammatory response in gland tissues
Phages have the potential to be a viable and eco-friendly alternative to antibiotics in aquaculture. Aquaculture is the fastest growing food production sector, providing over fifty percent of the world’s supply of fish and seafood. Antibiotics in feed are commonly used as prophylactics to decrease the corresponding heavy economic losses due to bacterial diseases worldwide.
Phages that Combat Zoonotic Pathogens———-
Phages offer a non-antibiotic method to improve food safety as a preharvest intervention to reduce zoonotic pathogens from the food supply. For instance, contaminated poultry, pork, beef, and fish have led to food poisoning and food-related disease. Often, food-borne pathogen contamination of meat products occurs during processing when carcasses are exposed to infected animal faeces. Campylobacteriosis caused by Campylobacter jejuni, is the most frequent food-borne human enteritis in developed countries, the major source being tainted poultry meat.
Salmonellosis is another common cause of gastroenteritis in humans. Pigs can become colonized with Salmonella spp. from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Use of a mixture of three different Salmonella-specific phages to reduce S. enteritidis colonization in the ceca of laying hens resulted in a significant decrease in bacterial prevalence of incidence of up to 80% .
pplication of phages in biocontrol and therapeutic design————
Phage therapies are also an effective tool in eliminating bacterial infections in various species of animals. Bacteriophages have also proven successful in treating diseases in poultry. One of the objectives of phage therapy in animals is to assess the suitability of bacterial viruses for control of pathogens having an important influence on animal productivity and health. Phages used in treatment have been effective in preventing infections and in treatment of colibacteriosis in poultry . Positive results, with a high success rate in eliminating pathogens, have also been obtained in combating infections induced by various Salmonellaserotypes in gamefowl, such as Enteritidis and Typhimurium , as well as campylobacteriosis in poultry, particularly infections induced by Campylobacter jejuni and C. coli . The effectiveness of phage therapy has also been confirmed in infections of broiler chickens by anaerobic Clostridium perfringens during the course of necrotic enteritis.
The advantages of phage therapy over antibiotics:———-
Phage therapy provides a number of advantages compared to therapy with antibiotic compounds. These advantages are presented below.
Bacteriophages are easy to identify & isolate:
Bacteriophages are ubiquitous in nature, making them fairly simple to identify and isolate. While bacteria can mutate and adapt to resist antimicrobials such as antibiotic compounds, bacteriophages can also mutate and adapt to overcome these bacterial resistances. This means that additional bacteriophages can be screened and isolated to respond to resistant bacterial strains.
Bacteriophages are highly specific:
Another benefit of phage therapy is that bacteriophages are highly specific for their target bacteria. It has been more widely appreciated in recent years that usage of broad spectrum antibiotics can have off-target effects on patients,disrupting their healthy microbiota and exacerbating disease. The use of bacteriophages targeted towards a specific species of bacteria greatly minimizes the chance of off-target effects on the microbiome or on the human patient themselves, as bacteriophages do not directly affect human cells.
Bacteriophages can kill antibiotic-resistant bacteria:
Finally, bacteriophages may be effective in a variety of situations where antibiotics are generally ineffective, such as treating antibiotic-resistant bacteria or bacteria within a biofilm. The mechanism by which phages infect and kill bacteria is often completely different from the mechanism of antibiotic resistance, meaning antibiotic-resistant bacteria may still be sensitive to phages. Bacterial biofilms, which have emerged as a major public health problem, are generally resistant to antibiotics, which are unable to penetrate the matrix of the biofilm to kill bacteria. In contrast, bacteriophages may be more effective at killing bacterial biofilms than antibiotics, making them a viable alternative for this purpose. Bacteriophages may even be effective as additives to minimize food contamination and reduce the amount of antibiotics given to farm animals, one of the drivers of antibiotic resistance.
What are the challenges faced by phage therapy?———-
While the use of bacteriophages as a therapeutic for drug-resistant bacterial infections is promising, widespread use is still in early stages of development and faces some questions concerning indirect effects of the bacteriophage on human patients.
Release of byproducts:
As mentioned above, bacteriophages inject their genetic material into the bacteria and use it to make more copies of the phage, ultimately killing the bacteria. While bacteriophages do not directly affect human cells, there are concerns that byproducts of the infection process, such as bacterial toxins or either bacterial or viral products released when the bacteria burst, may affect human cells. Existing data suggests that treatment of bacteria with bacteriophages does not increase the release of bacterial products that elicit an immune response in humans,suggesting that phage therapy does not result in more harmful byproducts than treatment with antibiotics.
Transfer of resistant genes to naïve bacteria:
Another potential concern with phage therapy is that some bacteriophages are capable of integrating into the bacterial genome, potentially resulting in theincorporation of antibiotic resistance genes or other virulence factors into the viral genome and the transfer to naïve bacteria. Verification that bacteriophages used for phage therapy do not integrate into the bacterial genome is therefore an important regulatory check before they are incorporated into a bacteriophage cocktail.
Complying with regulatory standards:
The most pressing hurdle for phage therapy is passing clinical trials under modern regulatory standards. In order for therapeutics to be approved for use, their makeup and stability must be clearly defined. This process was initially designed for chemical compounds, and can be difficult with phages, especially because phage therapy often uses multiple phage isolates in a cocktail for a single treatment, all of which must be assessed. Resistance is usually minimized by changing the components of the cocktail over time, but adding new phages increases the regulatory burden. While the number of phages can be reduced to minimize the complexity of documentation, this eliminates one of the major benefits of phage therapy: the diversity of the cocktail minimizes the possibility of bacterial resistance.
Bacteriophage specificity:
The specificity of bacteriophages for their targets has also emerged as a potential complication. While the specificity of phages for their target minimizes the risk of off-target effects, it also means that more time is needed to know exactly what strain of bacteria an infected patient has, and whether a specific cocktail of phages will be effective. This may take time that is not available in a critical-care situation. Additionally, the PhagoBurn study found complications in enrolling participants into the trial, as patients were often infected with multiple bacterial species, while the study was trying to show validation of phages specifically targeted to one type of bacteria. This resulted in getting much fewer participants than required for approval, and ultimately led to one of the trials being canceled.
Conclusions:
The increasingly observed acquisition of antibiotic resistance by bacteria necessitates new strategies for combating drug-resistant bacteria. The results of research on bacteriophages, indicating that they can be an alternative means of eliminating pathogens posing a threat to humans and animals, justify its continuation, particularly in view of increasing drug-resistance in bacteria and restrictions on the use of antibiotics. The development of adequate phage preparations may in the future prove to be one of the most effective methods for fighting bacteria that are pathogenic for humans and animals, and will also make it possible to obtain products that are safe and free of antibiotics.
