Alternative Anti-Infective Treatments to Traditional Antibiotherapy against Staphylococcal Veterinary Pathogens

The genus Staphylococcus encompasses many species that may be pathogenic to both humans and farm animals. These bacteria have the potential to acquire multiple resistant traits to the antimicrobials currently used in the veterinary or medical settings. These pathogens may commonly cause zoonoses, and the infections they cause are becoming difficult to treat due to antimicrobial resistance. Therefore, the development of novel alternative treatments to traditional antibiotherapy has gained interest in recent years. Here, we reviewed the most promising therapeutic strategies developed to control staphylococcal infections in the veterinary field to overcome antibiotic resistance.


Introduction
Over the past few years, the one health concept has increased significantly in importance to understand the evolution of antimicrobial resistance. This concept has evolved from one medicine to one health, and it refers to the increasing number of pathogenic bacteria affecting both animals and humans [1]. Indeed, the number of epizootic diseases and zoonoses is increasing. Thus, animal and human health should be treated jointly to avoid the spread of antimicrobial resistance.
Members of the genus Staphylococcus are common causative agents of both human and animal infections [2] (Table 1). Moreover, staphylococcal infections are related to the development and spread of antimicrobial resistance. Indeed, Methicillin-Resistant Staphylococcus aureus (MRSA) is one of the most important multidrug-resistant pathogens in humans and animals [3]. Thus, the use of antibiotics against staphylococcal infections in animals can trigger the emergence of antimicrobial-resistant strains in humans [4]. Besides, the number of pan-susceptible staphylococci is decreasing substantially, and low-pathogenic Staphylococcus (coagulase-negative strains) may be the reservoirs of resistant genes [2,5]. Consequently, it is estimated that the number of resistant strains will increase substantially in the near future. Thus, the development of new anti-infective strategies to improve animal care is becoming urgent.
Staphylococcal infection in animals is caused by different species of Staphylococcus that may affect a variety of domestic and livestock animals. Staphylococcus spp. are part of the normal cutaneous and mucosal microbiota of mammals and birds [2]. Most animals are colonized by one or more Staphylococcus spp. during their lives and in different body parts [6,7]. There are Table 1. List of pathogenic staphylococcal species and the diseases they may cause in humans and animals.

Treatments Based on Feed Supplements
Different treatments based on natural compounds are available to control staphylococcal mastitis, one of the leading causes of disease in dairy animals [13]. However, the use of antibiotics is still the most common treatment, which has led to the emergence of Livestock Associated MRSA (LA-MRSA) [14].
A promising solution to this problem is based on the use of antimicrobial peptides produced by generally recognized as safe (GRAS) bacteria [15][16][17]. Nisin is a bacteriocin produced by Lactococcus lactis with antimicrobial activity against several animal pathogens, including S. aureus [13,18] ( Table 2). Moreover, bacteriocins can inhibit S. aureus' biofilm formation, especially relevant to host colonization [18,19].

S. aureus
Ex-vivo Curative [40,41] Several natural variants of nisin are produced by Lactococcus lactis and different species of Streptococcus [42], whose biomedical application has been extensively studied. Nisin A was the first discovered bacteriocin, but new nisin variants have been found [42]. Despite the fact that the antimicrobial effect of nisin variants has been demonstrated [13,[18][19][20][21], very few scientific studies have focused on their possible use against subclinical mastitis [43]. The same applies to plantaricin NC8 αβ, a bacteriocin produced by Lactobacillus plantarum with great heat and pH stability [44].
Moreover, Bacillus thuringiensis is a very well-known producer of bacteriocins with antimicrobial activity against several S. aureus strains, morricin 269 being the most active bacteriocin against this pathogen. In addition, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524 also show a certain degree of activity against S. aureus [13,21].
Other very promising bacteriocins are colicins and pyocins, which are small proteins (~100 amino acids) used by bacteria for intraspecies competition [15]. Colicins are produced by Escherichia coli and pyocins by Pseudomonas aeruginosa.
Similarly, cyclotides are plant-derived peptides with an antibacterial activity produced by a large variety of species [48][49][50]. Their antimicrobial activity against staphylococci was demonstrated 20 years ago [51]. Two different cyclotides (Table 2), cycloviolacin 2 and kalata B2, have been used to treat cellular infection caused by S. aureus in RAW 264.7 monocytes and mice, without producing any cytotoxicity against host cells [22].
In parallel, the use of probiotics and prebiotics in animal healthcare has increased during the last few years (Table 2). Probiotics are living organisms recognized as safe that could be used as part of the animal diet (see Section 3.2), whereas prebiotics are molecules that elicit the growth of natural gut microbiota. Both pro-and prebiotics are natural strategies implemented to fight multidrug-resistant bacteria in animal feeds [52,53].
Prebiotics could be used to elicit the growth of natural commensal bacteria that may exclude staphylococci colonization of animals (Table 2). However, the use of prebiotics in animal feed has led to a variable efficacy, and not always resulted in changes in the natural gut microbiota [35]. The most promising prebiotics for animal healthcare are nondigestible oligosaccharides (NDOs) such as galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), cello-oligosaccharides (COS) or mannan-oligosaccharides (MOS), among others, which stimulate the growth of beneficial commensal bacteria in the animal intestine [23,35,54,55]. In particular, NDOs have been tested in pigs, ruminants, and poultry, where they elicit the growth of Bifidobacteria and Lactobacilli and increase the host immunological defenses [23]. Similarly, the plant-derived inulin or anthocyanins elicit the proliferation of probiotic bacteria, which potentially leads to the exclusion of S. aureus in the host [25]. In particular, inulin could be used as a feed additive in poultry to prevent S. aureus' colonization [23,24,54,56]. However, an inulin-based feed may generate intestinal damage in the gilthead seabream [57]. Therefore, the use of prebiotic should be evaluated individually to exclude any adverse effects in the host.
Feed supplements could also be based on inorganic molecules with antimicrobial activity, such as zeolites (e.g., clinoptilolite) with antimicrobial activity against S. aureus [26]. Other compounds that could be an excellent solution to prevent staphylococcal infections and bacterial resistance are phenolic compounds such as resveratrol or dihydroquercetin [27][28][29] (Table 2). In particular, resveratrol is an efflux pump inhibitor that increases the efficacy of other antimicrobials [27]. However, resveratrol is an antioxidant compound [58], and therefore, its use in combination with oxidative stress-generating antimicrobials could decrease the efficacy of the anti-staphylococcal therapy [59].

Phage Therapy
Bacteriophages, also known informally as phages, are viruses that infect bacteria. Bacteriophage-based therapies are a century-old approach to control bacterial infections [60,61]. However, their clinical use was discarded after the discovery of antibiotics due to their high strain specificity, low stability, and the rapid development of bacterial resistance to specific bacteriophage-based therapies. Antimicrobial resistance has renewed the interest in developing treatments against multidrug-resistant bacterial infections based on bacteriophages [60,61]. Indeed, phages do have several advantages over traditional antimicrobial treatments.
In particular, phages are highly specific, and therefore, bacteriophage-based therapies do not affect the normal flora nor eukaryotic cells. Besides, these are low dosage treatments, they rapidly proliferate inside the host bacteria, and they could be obtained from the natural environment, which makes them a perfect therapy against antimicrobial-resistant infections in animals [60,61]. Moreover, unlike antibiotics, bacteriophages can control putative reinfections, and they may mutate alongside their hosts to prevent the apparition of resistant bacteria [62].
However, the use of bacteriophages requires a detailed study of their interaction with targeted bacteria due to their strain specificity [53]. Therefore, monophage therapy must always be preceded by an in vitro assay to study the efficacy of the selected bacteriophages against the specific bacterial strain causing the disease [60]. Moreover, some bacteriophages may even introduce or activate virulence genes into the bacterial genome [53].
Despite these negative aspects, bacteriophages have already been used against staphylococci in animals, frequently in phage cocktails [61,63,64]. Interestingly, the bacteriophage cocktails formulated against the dog pathogen Staphylococcus pseudintermedius have shown activity against other pathogen species of Staphylococcus, including S. epidermidis, S. haemolyticus, and S. saprophyticus [65,66], suggesting that many bacteriophage-based therapies could be repurposed against different staphylococcal infections. In addition, the lytic bacteriophage phiSA012 and its endolysin Lys-phiSA012 ( Table 2) have different veterinary Staphylococcus sp. targets [30], which may pave the way to develop pan-staphylococcal phage-based therapies.

Competitive Exclusion of Pathogens
Probiotics can inhibit colonization of the gut by pathogens by direct competition or by the production of toxic substances (i.e., bacteriocins) [67][68][69][70]. For instance, natural skin microbiota can inhibit the colonization of the host by staphylococci. Indeed, the external application of beneficial bacteria is essential for preventing and treating staphylococcal skin infections in atopic dermatitis' patients [71]. Besides, probiotics reduce the concentration of methicillin-resistant S. aureus in farm animals [72]. Many strains of the genera Lactobacillus and Lactococcus can produce bacteriocins that are active against staphylococci [34,35,42,73] (Table 2). In addition, many of these lactic acid bacteria are resistant to antibiotics that could be used in combination with these probiotics and enhance their antimicrobial activity [34].
Moreover, the list of bacteria and fungi that could be used as animal probiotics is increasing. Lactobacillus, Lactococcus, and Bifidobacterium are the most commonly used, but different species of Enterococcus, Leuconostoc, Pediococcus, Propionibacterium, Streptococcus, Bacillus, Saccharomyces, Kluyveromyces, and Aspergillus have shown very promising probiotic assets [35,36]. Interestingly, it has been discovered that a Bacillus sp. probiotic strain produces a lipopeptide capable of inhibiting the quorum-sensing signaling system of S. aureus, which abolishes the pathogen's colonization of the human gastrointestinal tract [74]. Similar strategies based on quorum-sensing blockers may be used in animal health in the near future.
However, the precise mechanism of action of many of these probiotics is still not well understood. Also, the significant dose of probiotics required to cause an effect on the host, combined with their poor mucosal colonization, have hampered the development of animal feed supplements based on this strategy [67,69,75]. Therefore, more research must be done before extending the use of probiotics in animal feed. Importantly, gut microbiome changes must be thoroughly analyzed after antibiotic treatments because some changes in their composition could alter the beneficial effects of probiotics [67,68].
An interesting alternative to probiotics has recently emerged in the form of predatory bacteria against several important human pathogens [76] ( Table 2). Many of the Bdellovibrio and Micavibrio species described are common Gram-negative predatory bacteria. However, they can also disrupt biofilms created by S. aureus, which facilitates the antimicrobial response of macrophages against this pathogen and liberates free amino acids in the process [77,78]. Other predatory bacteria, such as those belonging to the genus Herpetosiphon, produce secondary metabolites with antimicrobial effects and show prey activity against S. aureus, S. epidermidis, and S. saprophyticus [31]. Finally, some predatory bacteria, such as Myxococcus xanthus, may secrete outer membrane vesicles (OMV) that carry antimicrobial compounds and proteins active against S. aureus and S. epidermidis [31][32][33].

Host-Directed Therapies against Staphylococcus spp. Infections
Host-directed therapies (HDT) against Staphylococcus species are still poorly studied in farm animals. However, the host response during staphylococcal infection is well known in some animal models (in particular rabbits and cattle), and this knowledge could pave the way to develop novel host-targeted therapies [10,79].
For example, dairy cows suffer a reduction in immune function during lactation, with a subsequent predisposition to infectious disease [37,80]. This immunosuppression could be reversed to avoid staphylococcal infections with pegbovigrastim, a recombinant DNA-derived bovine granulocyte colony-stimulating factor (G-CSF) analog (Table 2). G-CSF-based treatments increase the number of neutrophils with bactericidal activity [37,[80][81][82].
On the other hand, diet and nutritional supplements based on garlic have many advantages for animal health, particularly in poultry [83]. Some secondary metabolites present in garlic enhance the humoral immunity in chickens and elicit a direct antimicrobial effect against S. aureus by generating oxidative stress [38,58] (i.e., allicin; Table 2) [84,85]. In addition, different probiotics elicit a potent cellular and humoral immune response against staphylococci by increasing the production of IgG, IgM, and IgA [86,87].

Immunotherapies
Immunotherapies are well-established treatments in cancer therapy that are now considered a very promising strategy to control infections caused by multidrug-resistant bacteria [88]. In particular, immunotherapies are based on the use of antibodies that may interfere with the pathogenicity of the bacteria [89].
For example, monoclonal antibodies targeting bacterial membrane proteins called adhesins are efficient treatments against S. aureus infections by inhibiting the pathogen's adhesion to host cells and tissues and its subsequent colonization [12]. Another important target of monoclonal antibodies could be specific membrane proteins of eukaryotic cells that act as receptors of toxins produced by the pathogen during infection. These toxins may generate necrosis in the infected tissues to release the intracellular contents of host cells and increase the availability of nutrients and growth factors in the interstitial fluid [90].
However, the current high cost of immunotherapies is still a critical factor that hampers their application in farm animals, despite its up-and-coming applications. Consequently, immunotherapy has only been applied in small animals such as cats or dogs [91,92], and it is unclear if this strategy could be eventually applied in animal production at a reasonable cost.

Small-Interference RNAs (siRNAs)
Another strategy to fight against multidrug-resistant staphylococci infections in animals could be based on the use of siRNAs. Similarly to some immunotherapies, siRNAs could be used to silence the expression of specific virulence factors in the pathogen by targeting their messenger RNAs [87,88]. Importantly, siRNAs can be synthetically designed to match the sequence of any gene, and therefore, this is a very versatile strategy to combat multidrug-resistant bacteria. siRNAs-based therapies have been proposed as a putative solution against MRSA in humans by targeting the coagulase expressed by the pathogen [39]. However, other targets will have to be validated to control infections caused by coagulase-negative staphylococci. Besides, little is known about the applicability of this strategy to control staphylococcal infections in animals. Synthetic siRNAs have been designed to silence targets of viruses that cause diseases in cats [93]. Interestingly, a combination of different siRNAs may show additive or synergistic interactions [93].
However, the efficient delivery of siRNAs to their targeting cell is still one of the significant limitations of this strategy because siRNAs could be degraded by extracellular nucleases present in host tissues [94,95]. Therefore, new strategies have been developed to improve siRNAs' delivery, including different conjugation methods with immunoproteins, peptide ligands, or aptamers [95][96][97]. Besides, siRNAs could also be encapsulated in gold or silver-based nanoparticles, liposomes, or cyclodextrin complexes that may protect their cargo against nuclease attacks [95][96][97].

Nanoparticles
Metal nanoparticles have recently emerged as another alternative strategy for treating bacterial infections. In particular, zinc oxide nanoparticles are considered a safe and low-cost therapy against many human infections [40,98] (Table 2). Interestingly, nanoparticles have shown antimicrobial activity against intracellular pathogens because they may stimulate the biosynthesis of Reactive Oxygen Species (ROS) within subcellular compartments of the infected host cell, causing oxidative stress and damage to bacterial membranes [41,58]. Zinc oxide nanoparticles have broad-spectrum antimicrobial activity, and they may be useful in treating MRSA infections [40,41]. Additionally, the combination of pancreatin and zinc oxide nanoparticles showed synergy against MRSA by simultaneously affecting multiple virulence factors, biofilm formation, and pathogen growth [40]. Consequently, the use of zinc oxide nanoparticles against infectious diseases is now widely accepted in animal production [99,100].
However, the extensive use of heavy metal nanoparticles could also risk the co-selection of heavy metal resistance genes and antimicrobial resistance genes [101,102], which may aggravate the problem of antimicrobial resistance in the long term. In addition, the release of zinc to the environment, a highly contaminating heavy metal, is considered a significant limitation of this strategy.

Conclusions
The one-health concept is of major importance for the rational design of strategies centered on controlling the spread of antimicrobial resistance. It is becoming clear that the abuse of antimicrobials in animal production results in an increase of multidrug-resistant infections in humans. Any alternative to using antimicrobials in the control of animal infections will have a significant impact on human health. Here, we have summarized the most promising anti-infective therapeutic strategies to treat staphylococcal infections that could be used in animal health as an alternative to antibiotics. Many of the treatments covered in this article are currently considered effective, safe, and low-cost treatments, and may reduce the use of antimicrobials in animal production in the long term. In particular, the use of phage cocktails or probiotics may be a natural and inexpensive solution to the problem of antimicrobial resistance in animal health. However, further research is needed to implement these anti-infective strategies in animal production at the industrial scale.