AMR occurs when a microorganism is able to survive in the presence of antibiotics at a concentration that would normally inhibit their growth (1). Antimicrobial resistant (AMR) bacteria are able to survive and thrive through natural selection (2). Multidrug resistance (MDR) refers to bacteria that are resistant to at least three classes of antibiotics. It arises from the presence of resistance-associated genes in the bacterial genome (3). Currently, an estimated 700,000 fatalities are attributed to (AMR) per year. By 2050, AMR could lead to about 10 million deaths per year, as well as a 2% to 3.5% loss in the gross domestic product and global social costs of up to 100 trillion USD (4).

A primary issue with the introduction of a new antibiotic is whether antimicrobial resistance (AMR) to it will eventually emerge (5). Multidrug resistance has been detected among gram-positive pathogens, with penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium, being of particular concern (6). Among gram-negative bacteria, resistance to third-generation cephalosporins followed by fluoroquinolones, carbapenems, and currently colistin among Enterobacteriaceae poses a global threat that has resulted in a large increase in mortality and treatment costs and changed the guidelines for the treatment and control of infection (7).

The major driver of resistance evolution is the overuse of antibiotics fostered by factors such as inadequate regulations and misuse, lack of awareness about proper practices and consequent unjustified or unskilled use of antibiotics, the use of antibiotics as growth promotors in poultry and livestock, unrestricted access to antibiotics (8). Antimicrobial resistance is a natural phenomenon that occurs over time and is usually due to genetic changes in an organism. Antimicrobial-resistant organisms are found in humans and all their living environments (animals, plants, water and soil) and can spread from human to human or through zoonotic transmission from animal(or animal products) to humans (9).

Resistance may be caused by one or more of the following mechanisms (2). Enzymatic inactivation of the antimicrobial compound as the case with beta-lactamases (10). Reducing the antimicrobial effect by modifying the metabolic pathways alters bacterial cell walls making antimicrobial agents lose their binding ability to the bacterial target (11). Modifying the antimicrobial targets includes overamplifying the target or altering the permeability of the cell membrane by either decreased influx (porin loss) or increased efflux (efflux pumps) leading to a reduction in the accumulation of antimicrobial agents inside the cell (12). Another mechanism by which bacteria might develop antimicrobial resistance is by acquiring efflux pumps that extrude the antibacterial agent from the cell before reaching the target site (11). However, antimicrobial resistance may be intrinsic or acquired; it can develop through the mutation of existing genes (13, 14), or through the transfer of genes from other species or strains (15, 16).

Strategies employed to overcome AMR include reducing the extensive use of antimicrobials, collecting and analysis of data, avoiding the overuse of antimicrobials in farm animals, and developing novel treatment approaches (17, 18). The development of novel nanoscale antimicrobial agents/nanocomposites has been reported on different microorganisms (18). The limitations, and/or health risks associated with these nano-sized particles need to be taken into consideration (19).

Antimicrobial peptides (AMPs) have been widely tested in the fight against AMR bacterial infections (20, 21). However, overuse of AMPs may result in more resistant forms of bacteria resulting in deadly infections (11). Another potential alternative is bacteriophages; these are bacterial viruses that act as pathogens against bacteria. They are abile to specifically attack and kill only their host bacterial cells (22). Their limitations include the development of antibodies after repeated treatment, rapid inactivation of phages by the spleen, endotoxin contamination of the therapeutic phage preparations from bacterial debris, limited host range, regulation, and bacterial resistance to phages (11). Plant-based therapeutic agents evolved as a therapeutic alternative due to the emergence of AMR infections and the growth of scientific knowledge about herbal medicines as a promising complementary treatment (23).

Antibodies, mostly produced in mammals, provide a useful alternative in the treatment of bacterial infections either directly by targeting bacterial surfaces or indirectly by neutralizing bacterial toxins and the virulence factors that are responsible for infection (24, 25). However, several challenges face the production of IgG antibodies in mammals including the weak immune responses of the antigens used, the pain and distress caused to animals by immunization, blood sample collection, and ultimately sacrifice (26) and the cost of the production, poor shelf life, and the scale-up required for the large-scale production (24, 27).

The search for a more efficient and economical approach for the production of antibodies without the harm caused to the animals has led to a growing interest in egg yolk antibodies (IgY) (28). IgY is an isotype of immunoglobulin found in birds. Large-scale production of antigen-specific IgY can be obtained from eggs laid by immunizing hens with the specific antigens (27). Passive immunization using IgY is a promising alternative approach to combat the emergence of new and current drug-resistant pathogens (29) The yield of IgY antibodies produced in eggs is 18 times superior to the amount produced in rabbits (30) thus reducing the number of animals needed and pain caused by blood collection and sacrifice (31). An extra benefit that IgYs provide is their high content of sialic acid (32) reported to increase the half-life of the drug (33) leading to the increased shelf life of the IgY antibodies. IgY antibodies retain activity through different manufacturing steps and dried IgY batches can keep their biological activity over several years (34, 35).

IgY antibodies are reported as potent preventive and/or therapeutic agents against several viruses such as Influenza A (36, 37), Rotavirus (38), Dengue (39), Zika (40), Ebola (41) and as we reported previously against MERS-CoV (42, 43) and SARS-COV-2 (44). IgYs were also tested for their anti-parasitic activities against Trypanosoma cruzi (45, 46), Cryptosporidium parvum (47), Eimeria (48), and Candida albicans as a fungal infection (49).

According to the WHO, there are 12 bacterial priority pathogens for which novel antibiotics are urgently needed (50). IgY antibodies showed activity against most of these pathogens. The aim of this review is to highlight the potential role that specific IgYs can play in the immunotherapeutic prevention and treatment of these antimicrobial-resistant pathogens.

IgYs are safer than IgGs as they do not bind to human Fc receptors or fix mammalian complement components; hence they do not induce dangerous immune responses (51). Hakalehto et al., 2021 reported that IgY antibodies are one of the safest possible therapeutic agents (52). IgY consumed orally is Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (53). Additionally, IgY antibodies have been used orally to treat pulmonary Pseudomonas aeruginosa-infected patients with no negative complications for up to 10 years (54).

Topical applications of the IgY antibodies (55) were reported against S. mutans as a gel or powder in a rat dental caries model and showed inhibition of S. mutans (56, 57). Patients who used toothpaste containing anti-gingivitis IgY showed significant differences in bleeding on probing and gingival index (58, 59). Short-term (three weeks) use of IgY mouth spray resulted in a significant decrease in S. mutans in dental plaque, and low levels of S. mutans were detected for at least 5 weeks after withdrawal of IgY (60).

Nasal delivery is superior in many cases to systemic delivery due to its non-invasive nature, fast onset of action, and low side effects due to targeted delivery (61–63). Anti-P. aeruginosa IgY were reported to inhibit murine pneumonia when administered intranasally (64). The protective effect and safety of intranasally administered anti-SARS-CoV-2 IgY antibodies were confirmed in a mouse model, with no adverse effects observed (65). Other reports showed that the superaficial application of anti-SARS-CoV-2 IgY would not be expected to elicit antibody-dependent enhancement of infection due to its topical application (53).

Systemic administration of IgY has not been clinically evaluated. IgY-based antivenom was given parenterally and showed complete protection in animal models of lethal venomous bites and stings (66–68). Moreover, intraperitoneal administration of polyvalent-specific anti-Zika virus IgY in a mouse model did not induce antibody-dependent enhancement (ADE) and did not display any side effects (40). More in depth studies on the safety and efficacy of IgY systemic delivery are needed before clinical use (69).

The concept of passive immunization describes the administration of specific antibodies obtained from an immunized donor in a prophylactic or therapeutic setting. In general, passive immunization is a naturally occurring means of transferring immunity from a mother to an offspring, such as through the immunoglobulins contained in breastmilk in mammals and the transfer of IgY antibodies through the egg yolk in chickens (70). The concept of passive immunization has drawn increasing interest in the past several years owing to the increase in antibiotic-resistant pathogens. In general, the IgY technology can be used to develop highly specific antibodies against a vast variety of antigens including bacteria, viruses, and even bacterial enzymes such as beta-lactamase, which is able to inactivate antibiotics. These antibodies are seen as a novel approach to targeting antibiotic-resistant bacteria through passive immunization. The administration of developed and isolated IgYs can provide rapid protection against diseases that are currently unresponsive to antibiotic therapies, including among immunocompromised patients for whom conventional treatment or vaccinations are not effective. The commercial availability of reagents that are specifically designed and developed for use in egg yolk antibody isolation and characterization has increased the amount of research in this area, with the term IgY being internationally recognized in research and industry (71). One of the humane advantages of IgY use is the ability to extract the antibodies from the eggs and not from blood, which makes it more favorable for animal safety and care (72). In the 1990s, several reports investigated the various aspects of IgY technology and applications (73). A particular advantage of IgY-based diagnosis and treatment is that the phylogenetic distance between mammals and birds enables the generation of IgY antibodies against conserved mammalian or pathogen proteins (74). This phylogenetic distance results in no recognition of mammalian Fc receptors and does not trigger the mammalian complement activation in vitro or in vivo (75, 76). IgY provide an added environmental advantage by using nontoxic techniques for purification (77). Precipitation techniques using water dilution and low pH-induced precipitation, as well as polyethylene glycol-, dextran sulfate- and xanthan gum-induced precipitations (78) or NaCl extraction (79) have been employed for IgY purification.

IgY antibodies have a large number of advantages over mammalian IgG antibodies such as cost-effectiveness, the short time needed for preparation and production, the wide range of potential pathogen targets, convenience in handling and storage, and the high yield of the target IgYs (53, 80). A hen can be considered a small “factory” for antibody production, as one hen can produce more than 22.5 g of total IgY per year of which 2% to 10% is composed of target-specific antibodies (81). This quantity is the equivalent of the IgG antibody production of 4.3 rabbits over the course of a year; further, this large amount of IgY can be harvested without killing the hen (29). IgY is more resistant to proteolysis than its mammalian IgG counterparts (82), and it has been found to retain 40% of its activity after incubation with trypsin or chymotrypsin for 8 hours (83). Moreover, owing to the phylogenetic distance between birds and mammals, immunizing laying hens twice with the specific antigen is enough to produce a humoral immune response that leads to the transfer of large amounts of specific IgY antibodies to the eggs for several months (53). IgYs are safer than IgGs as they do not bind human Fc receptors or fix mammalian complement components; hence, they do not initiate potentially dangerous immune responses (51). Owing to the lack of a hinge region between the two “arms” of the antibody molecule, the IgY molecular structure is more rigid than that of IgGs and thus somewhat stronger (84, 85).

The use of polyclonal IgY against infectious diseases minimizes the risk of developing AMR. Since the antibodies are directed to various antigens of the same microorganism, this lowers the chance of developing resistance to all of these antigens at the same time because they require multiple genes for synthesis (86). Therefore, specific IgY antibodies are promising alternatives for use as antimicrobials in human and veterinary health to combat the emergence of resistant bacteria (28). The use of IgY is environmentally friendly and elicits no undesirable side effects, disease resistance, or toxic residues (87). IgY-based therapy does not cause disruption of the host flora because the treatments target specific disease-causing pathogens (88).

The choice of a specific antigenic target depends on the characteristics of the pathogen and the therapeutic strategy itself. For example, IgY antibodies can target different factors important for the survival of bacteria such as enzymes, toxins, colonization factors, flagella, and mucosal receptors (89, 90). The actual mode of action of passive immunization includes the agglutination of bacteria, inhibition of bacterial adhesion, suppression of virulence factors, toxin neutralization, opsonization and enzyme inactivation (81).

The antigenicity of specific targets that are used to immunize the chickens can be influenced by the immunogen itself, the type of adjuvant used for immunization, route of antigen delivery, frequency of administration, and general avian properties (breed, commensal bacterial footprint, age, egg lying capacity) (91).

Four different target strategies of passive immunization with IgY antibodies can be distinguished and are further displayed in ( Figure 1 ).

IgYs have been developed against crucial enzymes such as urease, which is expressed by bacteria such as Helicobacter pylori for survival in harsh conditions (low pH in the gastric mucosa), and beta-lactamases (92), which enable bacteria to degrade antibiotics and survive exposure to them. IgYs inhibit the enzymatic function (urease or beta-lactamase) through blockage of the active site, which prevents the substrate from binding to the enzyme and neutralizes the ability of bacteria to survive adverse conditions.

Research shows that IgYs generated against UreC, one of the subunits of urease, resulted in the prevention and even eradication of antibiotic-resistant H. pylori infections, which cause gastritis and gastric ulcers leading to gastric cancer (93). The addition of IgYs to yogurt as a functional food against H. pylori, for example, makes the usage of IgYs attractive (94).

Further research conducted by LeClaire et al. and Trott et al. (95, 96) also showed promising results regarding the therapeutic usage of IgYs generated against bacterial enzymes/toxins. These researchers reported that IgYs specific to enterotoxin B and botulinum type A neutralize their activity and can therefore prevent and treat infections due to S. aureus and Clostridium spp., respectively.

In addition, Hirai et al. (97) passively immunized mice with three different types of IgYs specific to Vibrio cholerae (anti-01, O139, and anti-cholera toxin B), which effectively prevented cholera infection in the mice.

Several studies have shown that it is possible to generate IgYs that specifically target pathogens directly. These IgYs act by blocking the adhesion/virulence molecules of the bacteria or by coating the whole bacterial cell wall, which suppresses the biological functions of the pathogen itself. This method was adapted by Nilsson et al. (98) who conducted a 12-year study on prophylactic oral IgY treatment against Pseudomonas aeruginosa in 17 patients and reported the prevention of bacterial colonization in most cases. These results clearly indicated the potential of IgYs as a preventative option for respiratory infections. IgY in this treatment is reported to form an antibody barrier preventing P. aeruginosa from entering the lungs through the nose/oropharynx and binding to the epithelial surface of the mucosa. Gargling with IgY at night keeps active concentrations of Anti-Pseudomonas IgY in saliva and oropharyngeal mucosa till the next morning (98).

Furthermore, Dapunt et al. (99) generated IgYs to target quorum-sensing molecules of Staphylococcus spp. associated with implant infections. In particular, the adhesion molecule AtlE (S. epidermidis), which is a member of the autolysin family and mediates the attachment to the implant surface, was identified as a target of interest. Autolysins themselves are a group of enzymes that catalyze the degradation of the bacterial cell wall at specific sites. Besides the quorum-sensing molecules, the bacterial heat shock protein GroEL was also investigated in the context of biofilm formation. GroEL is a highly conserved protein that is homologous to the human heat shock protein 60 (HSP-60) and is essential for protein folding. It was previously shown that bacteria are not able to survive without GroEL, which makes it a perfect candidate as a potential target. Immune cells are able to recognize GroEL, which induces several bactericidal strategies. So far, two scientific reports have mentioned GroEL in relation to IgY and as a potential target. Hermans et al. (100) focused on Campylobacter jejuni in broiler chickens and used whole cell lysate to immunize chickens for the generation of IgY antibodies specific against the pathogen. GroEL was identified via HPLC-MS/MS in the whole cell lysate, and it was predicted that the polyclonal IgYs would also contain an antibody portion against GroEL. Therefore, there is no clear indication of the impact of GroEL-specific IgYs on the growth of C. jejuni. Dapunt et al. (101) have also investigated GroEL-specific IgYs against S. epidermis in biofilm formation. Unfortunately, the impact was only minimal and further research is needed.

Successful generation of the monoclonal IgY or IgY fragments in the last few years has increased the functional use of IgY fragments, such as single chain (scFv) (190), chimeric (191), and humanized IgY (192). Monoclonal IgY antibodies combines the benefits of avian IgY antibodies and the features of monoclonal antibodies (193). They have the potential for use as therapeutics in both veterinary and human applications, immunological detection and diagnosis, and for screening and validating biomarkers (193).

Phage display production of IgY monoclonal antibody is more likely to generate a robust immune response against various highly conserved mammalian protein molecules (194). Monoclonal IgY antibodies conjugated with phthalocyanine—a synthetic photosensitizing dye used in near-infrared phototherapy—were recently used against Candida albicans and provided highly effective and specific success in an in vivo skin infection model with no damage to the healthy epithelium (195). In another study, hens were immunized with canine parvovirus VP2 (CPV-VP2) virus-like particles (VLP) and the specific IgY-scFv were generated using the T7 phage display technique (196). Transgenic IgY antibodies containing the bird constant regions and the human variable regions allow the use of the highly specific IgY antibodies against mammalian conserved proteins (197). So far one clinical trial is reported on the parenteral administration of monoclonal IgY product Sym021 (trial ID: NCT03311412) against human programmed cell death protein 1 (PD1) with promising inhibitory binding to the target protein (198).

The vast number of research studies in the past few years has led to an increase in the number of registered products for therapy and diagnosis together with an increase in the number of patents filed and clinical trials registered. For human use several IgY products are registered in the market such as IgY Max (against 26 human-relevant bacteria), Ig-Guard Helico, GastimunHP, and Ovalgen^® HP (Helicobacter pylori) (69). For veterinary use, about 56 products are reported at various stages of evaluation, including products in the market such as Ig-Guard Calf, Ig Lock Calves, Globigen^® Dia Stop, and IgY DNT (for calf diarrhea); PG-002 (for cow mastitis); Ig-Guard Swine, Ig Lock Pig (for swine diarrhea), Ig-Guard Puppy, ParvoONE^®, Ig Lock Canine, GastroMate^®, and Guardizen (for pets, especially in canines); Ig-Guard Duck, Ig-Guard Poultry, BIOAb DHV-IgY (for poultry) (69)

The market value of IgY polyclonal antibodies is estimated to be USD 14.2 million by 2027. However, the total antibody market share is only 0.24% (199). About 95% of IgY antibody productions are polyclonal antibodies with future prospects of IgY fragments and monoclonal IgYs (69). Several of the production companies now available provide custom antibody production services (53, 200). As the concept of using IgY antibodies in therapy is a new approach for alternative treatments, the USFDA has enforced strict regulations for the parenteral administration products (193, 201) which mandates more studies on the safety and efficacy of this route of administration. The documentation in these studies is an essential part of the process as well as the utility of standard processes such as good manufacturing practice (GMP) conditions, and immunization of specific- pathogen-free (SPF) birds (69).

The susceptibility of IgY to proteolysis is one of the limitations to the oral use of IgY for passive immunotherapy. Although IgY antibodies are resistant to inactivation by the gastric proteolytic enzymes trypsin and chymotrypsin, it is degraded by pepsin (82). To overcome this obstacle, microencapsulation is found to be an effective approach to protect IgY from gastric inactivation (202).

The lack of standardization in the experimental animals (i.e., specific-pathogen-free birds) for the production and extraction and purification procedures of IgY antibodies is one of the major difficulties facing the progress in product licensing so far as well as the consensus on regulation and approval of IgY-based health products (53). More safety studies are needed to evaluate their safety for use as human and veterinary therapeutics. Research is also needed to develop more industrial scale standardized extraction and purification methods to fit the needs of clinical applications (69).

MDR pathogens are a growing threat to human health and welfare. The problem requires more research into innovative, and effective approaches including immunotherapies. Economically one of the main advantages of avian immunoglobulins is their cost-effective production, with the benefit of upscaling in the poultry industry, which may allow low-income countries to easily adopt technological capacities in their health systems. Overall, the use and application of IgY antibodies will emerge as an alternative to antibiotics and will help in the design of novel, safe and effective biologicals for the treatment of various MDR pathogens.

SE-K, Conceptualization, Supervision, Writing – original draft, Writing – review & editing; AA, Conceptualization, Supervision, Writing – original draft, Writing – review & editing. CO, Conceptualization, Supervision, Writing – original draft, Writing – review & editing. MT, Writing, original draft, Writing – review & editing. SE Writing, original draft, Writing – review & editing. AZ, Validation, Supervision, Writing – original draft, Writing – review & editing. EA, Conceptualization, Supervision, Writing – original draft, Writing – review & editing. All authors contributed to the article and approved the submitted version.