Commercially available anti-
Staphylococcus aureus is a colonizing microorganism of the nasal region of both humans and animals and represents an important opportunistic pathogen. The acquisition of the mecA and mecC genes by S. aureus led to the emergence of methicillin resistance (MRSA), becoming a public health problem in both human and animal areas. In addition to resistance to β-lactam antibiotics, MRSA strains have multidrug resistance to antimicrobials, significantly limiting therapeutic options, making it crucial to have effective alternatives for treating staphylococcal infections. In this context, the use of lytic bacteriophages, which are viruses that infect and lyse bacteria, as well as the use of their by-products, such as endolysins, has shown potential in the control of S. aureus, including MRSA. Due to the specificity of bacteriophages to infect particular prokaryotic hosts, these viruses represent an antibacterial resource for the control of public health relevant microorganisms, especially antibiotic-resistant bacteria.
- Methicillin resistant Staphylococcus aureus
- phage therapy
- phage by-products
1.1 The role of
S. aureusin human and animals
Among the different relevant bacterial genus in Veterinary and Human Medicine,
Historically, the first publications related to the human carriage of
, emerged in mid-1940s  and showed the relevance of the bacteria in the human infections. On the other side, the approach to this theme in the vet sphere was only evidenced from the year 2000. Regardless,
1.2 Infections related to
S. aureusand Methicilli Resistant S. aureus(MRSA)
1.3 Perspectives to MRSA infections treatment
Alternatively, with the development of the new antibiotics to supplant the resistance, there is the possibility of using viral agents to control unwanted bacteria. Viruses termed “bacteriophages” or “phages” are the most abundant agents in the environment and are host-specific, i.e., they infect only prokaryotes that have their own specific receptors for their adsorption. The absence of such receptors makes phage binding to the target cell as well as subsequent infection impossible, characterizing the specificity of these viruses [16, 17]. Phages are easily recovered from soil, sewage, and feces and their numbers are about 3 to 10 times higher than bacterial counts even though variations exist between ecosystems [18, 19]. Like other viruses, bacteriophages are obligate intracellular, and are characterized according to the replication cycle exhibited after infection of the bacterial host. The cycle can be lytic or lysogenic, but only phages that exclusively perform the lytic cycle are of interest for use as therapeutic agents, since they will promote cell lysis at the end of the cycle .
2. Methicillin resistant
2.1 What is MRSA?
2.2 Laboratory detection of MRSA
Phenotypic tests for laboratory identification of
2.3 MRSA colonization and MRSA infection
Historically MRSA was described in humans in 1961 , while MRSA colonization and infection in animals was first reported in 1972 in asymptomatic dogs in Nigeria and a case of bovine mastitis in Belgium . Around 25–30% of the human population is asymptomatically colonized by
It is now well established that MRSA isolates are often non-susceptible to different classes of antibiotics and are considered multidrug-resistant (MDR) when resistance is observed for at least three different classes of antimicrobials . The great adaptability of this pathogen is due to its expressive genetic plasticity, in which approximately 25% of the
When human MRSA infections persist, worsen, or recur despite surgical treatment, additional use of systemic antibiotic therapy is required . Different clinical treatment options are available to combat MRSA infections, including vancomycin. Although this drug is the main therapeutic option, there are several limitations in its use, such as the achievement of optimal serum concentration, long-term treatment, renal toxicity, and restricted route of administration (intravenous) . In the veterinary field, there is no effective therapy to treat MRSA infections, so prevention and control measures are critical to contain the further spread of MRSA . While this challenge remains unresolved, successful treatment of infections may require the development of new antibiotics and the use of bacteriophages and phage-derived lytic proteins  as alternative therapeutic resources.
2.4 Bacteriophages as anti-MRSA agents
With the emergence of MRSA, staphylococcal infections have become difficult to control. MRSA is typically resistant to beta-lactams and can even present resistance to other antimicrobials , thus requiring new therapeutic alternatives. In this sense, phage therapy resurfaces as a promising tool for the control of unwanted bacteria, since it consists of the use of viruses, called bacteriophages, capable of infecting and killing prokaryotes without harming human or animal cells.
2.4.1 What are bacteriophages?
Bacteriophages, also known as phages, are viruses that infect and lyse prokaryotes. They are considered the most numerous infectious entities on the planet, being found in different environmental matrices, such as sewage, water, soil, among others . Phages have been proposed as an alternative resource to the problem of resistant bacteria since they infect bacterial cells and, at the end of their reproduction cycle, promote the lysis of the host bacterium [18, 32]. After their discovery in 1917, phages were successfully used for the treatment of several bacterial infections . However, the advent of antibiotics and their industrial-scale production, coupled with the lack of adequate studies and the poor understanding of phage biology at the time, resulted in the abandonment of studies related to these viruses as therapeutic agents in most institutions. A few places followed up on these studies, such as Eastern Europe, mainly Russia, Georgia, and Poland. Truly, the production and use of phages for prophylaxis and therapy never stopped in the last two countries mentioned . From these countries emerged the main research in the phage therapy field.
Subsequently, the indiscriminate use of antibiotics enabled progressive bacterial resistance, leading to the resumption of studies with phages. Thus, bacteriophages and their products, such as enzymes released at the end of their replication cycle, were once again considered as therapeutic agents . Phage therapy is the use of bacteriophages to eliminate bacterial pathogens, and fortunately, innovative research techniques have made several advances in the field possible. One of the most important discoveries has been the distinction between the replication cycles carried out by phages. The replication of these viruses occurs mainly through two cycles: the lysogenic and the lytic.
2.4.2 Phage replication: Lytic and lysogenic cycles
Regardless of the type of cycle (lytic or lysogenic) performed by the bacteriophage, the replication process will begin by the adsorption of the virus to receptors on the surface of the host cell wall. During the infection of Gram-positive bacteria, as is the case of
The lysogenic cycle is characterized by phages that are able to infect and integrate their genetic material into the DNA of the bacteria, thus forming a prophage. The ability to integrate its genetic material with the bacteria is due to the presence of genes that encode the integrase protein, an enzyme that mediates the recombination between the phage’s DNA and that of the host . Subsequently, proteins are produced that induce viral latency, implying a pause in the transcription of gene products, allowing the prophage to exist with the bacteria for several bacterial generations without major consequences. Furthermore, the prophage induces immunity in the bacteria against infection via new phages. Bacteriophages that exhibit this type of replication cycle are not suitable in the context of phage therapy, since at the end of the viral cycle the death of the bacteria will not necessarily occur. In addition, bacteriophages that perform the lysogenic cycle may be responsible for producing toxic substances and carrying resistance genes , implying benefits for the bacteria.
On the other hand, in the lytic cycle there is no integration of the phage genetic material to the prokaryote DNA. At the end of this viral replication cycle, when the new virions are already formed and ready to be released, there is the production of enzymes capable of lysing the bacteria cell wall, inducing bacterial rupture and death for the release of new virions. Therefore, phages whose replication cycle is lytic are the most suitable for use in phage therapy, precisely because they cause bacterial lysis . The schematic representation of the lytic and lysogenic cycles in
2.4.3 History of phage therapy in
The attempt to use phages for the treatment of infections caused by
Studies with phages for the control of staphylococcal infections were continued in some regions of the world. In the United States (1952), a laboratory (Delmont Laboratories) licensed, for human use, a bacterial lysate produced from the infection of bacteriophages in two virulent strains of
Because of the resistance of
2.4.4 Commercial phage products anti-staphylococcal
Commercial products containing phages or enzymes produced by them are manufactured and available in some countries, mainly in Russia and Georgia, but also in Canada, the Netherlands and the Czech Republic. The following table (e.g., Table 1) gathers different commercial phage products, the target bacteria of each product, their main uses and the manufacturer [47, 48, 49, 50].
|Product name||Active against||Informations/use||Manufacturer|
|Complex Pyo bacteriophage||Mix of sterile lysate phages.|
Used for the treatment of diseases of the eyes/ear/nose, throat, infections of respiratory tract, lungs, surgical sites, urogenital, enteric, septic diseases. operational and newly infected wounds, for the prevention of hospital-acquired infections.
|Fersisi bacteriophage||Sterile filtrate of phage lysates.|
Used for the treatment of otolaryngological diseases; infections of skin, urogenital, gynecologic, enteric, pyo-inflammatory disease in children (including newborns).
|Eliava BioPreparations (Georgia)|
|Gladskin Acne, Gladskin Eczema, Gladskin Rosacea, Gladskin Shaving Irritation||Endolysin XZ.700. Used for the treatment of skin disorders (acne, eczema, rosacea, psoriasis).||Micreos (Netherlands)|
|Intesti bacteriophage||Mix of sterile filtrates of phage lysates.|
Used for the treatment of enteric infections.
|Eliava BioPreparations (Georgia)|
|Intesti- bacteriophage||Mixture of sterile filtrates of phage lysates.|
Used for the treatment of bacterial dysentery, dyspepsia, disbacteriosis, enterocolitis, colitis, salmonellosis.
|Pyophage||Mix of sterile lysate phages.|
Used for the treatment of infections of upper respiratory tract,
dermatological, surgical site, ocular urogenital, gastrointestinal, purulent septic infections in children, for prevention of post- operational complications and hospital infections.
|Eliava BioPreparations (Georgia)|
|Pyofag® polyvalent bacteriophage||Solution in vial with bacteriophages.|
Used for the treatment of pyoinflammatory diseases of ears, throat, nose, oral cavity, eyes, surgical infections, burn wounds; urogenital, gynecologic, and enteric infections.
|Pharmex Group LLC (Ukraine) for NeoProbioCare Inc. (Canada)|
|Stafal ®||Polyvalent bacteriophages of the family ||Bohemia Pharmaceuticals (Czech Republic)|
|Staphefekt TM||Endolysin XZ.700. Used for treatment of inflammatory skin conditions such as eczema, acne, rosacea, psoriasis.||Micreos (Netherlands)|
In recent years, different studies involving commercial phage products with anti-staphylococcal activity have been undertaken. Most of them were related to
Some commercial products with the same name, but produced by different manufacturers, are proposed for the control of
In a recent study, the action of Stafal® (a preparation with polyvalent bacteriophages active
Commercially, the recombinant endolysins Staphefekt SA.100 and Staphefekt XDR.300 (Micreos Human Health BV, Netherlands) which act on
Other commercially available products are: Bronchophage, Otophage, Phagodent, Phagoderm, Phagogyn, Phagovet, Vetagyn (Micromir, Russia); ENKO bacteriophage, SES Bacteriophage, Staphylococcal bacteriophage (Eliava BioPreparations, Georgia); Dysentery bacteriophage,
2.4.5 Non-commercial anti-S. aureus bacteriophages
Fortunately, since the year 2000, different studies have contributed to a better understanding of phages as anti-
A cocktail containing two bacteriophages, designated K and 44AHJD, was tested against clinical isolates of
The Clinical Trials platform, a database of clinical studies conducted worldwide, reports the existence of eight studies related to the use of bacteriophages against
2.4.6 Advantages and challenges of phage therapy
Among the principal attractive aspects of phage therapy, the main ones are: i) high specificity of the virus for the bacteria providing freedom from side effects on cells that are not targeted by the therapy; ii) activity against different bacteria, including multidrug resistant bacteria; iii) reduced treatment costs compared to antibiotic therapy; iv) prevention to the growth of secondary pathogens; v) ability to degrade bacterial biofilm by lysing bacteria; vi) high body distribution and vii) high efficacy compared to antimicrobials . On the other hand, there are some limitations to the use of phages in therapy, among them: i) the possibility of antibody production by the immune system; ii) the difficulty of measuring the application dose; iii) the possibility of gene transfer among pathogens through phages, which may be responsible for passing pathogenic determinants and virulence factors, resulting in a possible resistance of bacteria; iv) the ability of bacteria to develop resistance against bacteriophages; v) elucidation of the correct route of administration and treatment time and vi) accurate and rapid diagnosis of the microorganism that is provoking the illness .
Fortunately, for all the limitations previously indicated, there are already studies that aim to circumvent these problems. For example, viral genome sequencing avoids the use of phages that are lysogenic or contain toxic and resistant genes. Along with this is the progressive search for new phages to be used if antibodies are produced by the immune system, or to replace phages for which the bacteria have become resistant. In addition, it is already known that viruses can mutate and adapt to resistance mechanisms created by bacteria. In other words, after the creation of barriers that make it impossible for the phage to replicate in the bacteria, changes occur in the viruses that allow their replication cycle to continue, even with the presence of the bacterial adaptations . Further in this context, the use of new diagnostic resources allows the rapid differentiation of the disease-causing bacteria, in addition to the use of cocktails with different phages for the same bacterium, enhancing even more the specificity and avoiding the manifestation of resistance [32, 66].
MRSA represents a global threat due to its progressive resistance to antimicrobials, as well as the future prospect of no effective antibiotics. The use of lytic bacteriophages and their by-products are promising alternatives for bacterial control, since they infect and lyse the pathogen without the inconvenience of side effects, as well as contributing to lower consumption of antimicrobials, reflecting in the reduction of antibiotic resistance rates. The study of phages has always occurred in countries such as Georgia and Russia, where phage-based commercial products are relevant antibacterial alternatives. Although different
The authors would like to thank Ana Carolina Klein for the graphic art.