Potential antimicrobial properties of the Ulva lactuca extract against methicillin-resistant Staphylococcus aureus-infected wounds: A review

Methicillin-resistant Staphylococcus aureus (MRSA), currently a major problem in hospitals worldwide, is one of the most common causes of nosocomial disease through surgical wound infection. MRSA-infected wounds have very low recovery rates and have become more problematic as some antibiotics are not effective against MRSA. Several antimicrobial and anti-inflammatory agents of green algae (Ulva lactuca) in the form of alkaloids, triterpenoids, steroids, saponins, and flavonoids have the potential to accelerate the wound healing process following MRSA wound infection. Various active compounds contained in the U. lactuca extract are thought to have multiple antibacterial and anti-inflammatory properties that can overcome the MRSA antimicrobial resistance and accelerate tissue growth in the wound healing process. This review aims to describe the potential of Ulva lactuca extract against MRSA-infected wound healing.


Introduction
Nosocomial infections are infections acquired from the hospital during medical treatment. About 15% of the hospitalized patients acquire nosocomial infections [1]. Worldwide statistics show that nosocomial infections occur in 7% of all disease incidences in developed countries and in 10% of disease incidences in developing countries. Surgical site infection (SSI) is considered one of the most common routes of nosocomial disease [2]. Nosocomial pathogens include bacteria, viruses, and fungal parasites. Staphylococcus aureus is often considered an agent of nosocomial infection [3]. S. aureus, from which some strains have evolved into Methicillin-resistant S. aureus (MRSA), is a bacteria that are resistant to various kinds of antibiotics; it is also known as a superbug (multi-resistant bacteria) [4]. MRSA is currently recognized as a significant problem in hospitals worldwide [3,5]. S. aureus resistant strains can interfere with the wound healing process, which causes prolonged wound healing, and lead to increased mortality and morbidity [6][7][8]. Until now, there is no anti-MRSA agent that is accepted globally.
Ulva lactuca, also known as green algae (sea lettuce), is classified as macroalgae in the phylum Chlorophyta [9]. U. lactuca contains secondary metabolites such as alkaloids, triterpenoids, steroids, saponins, phenolic compounds, and flavonoids [10,11]; these active compounds have the potential to accelerate wound healing of nosocomial wound infections due to their antibacterial, anti-inflammatory, antioxidant, and anticoagulant activities [12]. With various active compounds in the U. lactuca extract, it is thought to be effective against multi-resistant bacteria. This extract also acts through multiple antimicrobial mechanisms to penetrate bacterial resistance barriers [13].
Information on the potential of U. lactuca as an antibacterial and anti-inflammatory agent in wound healing is still limited. This review addresses what is known about the potency of U. lactuca as a source of anti-MRSA agents.

SSI and Inflammation
SSI increases morbidity and mortality, thereby increasing health cost burdens and lowering patients' quality of life, as reported in European countries [14]. As summarized by Mawalla et al. [5], SSI rates have been reported to range from 2.5% to 41.9% globally.
According to the European Center for Disease Prevention and Control, SSIs are divided into superficial, deep, and organ/cavity infections (Table-1) [4,5,15]. Superficial infection indicates local wound symptoms, such as sero-sanguineous-to-purulent discharge from the wound or accumulation of pus associated with swelling, redness, heat, and pain. Deep infection is related to symptoms such as systemic sepsis, including fever, tachypnea, and left shift leukocytosis. Radiological or ultrasound examination is required to determine organ/ cavity infections [5,15,16]. A prospective cross-sectional study has been reported by Mawalla et al. [ [5].
The most common infectious agents found in surgical wounds include S. aureus, coagulase-negative Staphylococci, Enterococcus species, and Escherichia coli. At present, there has also been an increase in the incidence of SSI caused by antibiotic-resistant pathogens, such as MRSA [15]. Mawalla et al. [5] reported that S. aureus was the predominant (28.6%) SSI organism, as 18.8% of the SSI cases reported in major surgery patients were infected with MRSA.
Inflammation is a defense mechanism against pathogens and other causes of tissue damage [17]. The molecular mechanisms of inflammation are fairly complex and begin with the recognition of specific molecular patterns associated with cellular infections or injury. The entire inflammatory response is mediated by several key regulators involved in the selective expression of pro-inflammatory molecules [18]. This complex response involves leukocytes, such as macrophages, neutrophils, and lymphocytes, which are also known as inflammatory cells [17]. Various chemical mediators from the circulatory system, inflammatory cells, and injured tissues actively contribute to and adjust the inflammatory response. The chemical mediators released include (1) vasoactive amines (e.g., histamine and serotonin), (2) peptides (e.g., bradykinin), and (3) eicosanoids (e.g., thromboxane, leukotrienes, and prostaglandins) [19].
The damage-associated molecular patterns and pathogen-associated molecular patterns of invading bacteria, fungi, or viruses are the main mediators of cell injury and microbial invasion, respectively. The molecular patterns are detected by receptors called pattern recognition receptors (PRRs). PRRs, which give rise to direct immune response, are constitutively expressed on host macrophages, monocytes, dendritic cells, neutrophils, and epithelial cells. The first inflammatory leukocytes to be recruited to the wound site are neutrophils. Under the influence of chemokines, there is an increase in the expression of adhesion molecules (ICAM, VCAM1, and e-selectin) on vascular endothelial cells, leading to the adherence of neutrophils to the vessel wall, followed by extravasation and migration along the chemokine gradient. This neutrophil migration is accompanied by phagocytosis, killing of ingested bacteria, release of antimicrobial cationic proteinases and peptides, and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, which maintain the inflammatory state [20].

Staphylococcus aureus and Multi-resistant Superbug
S, aureus is an invasive bacterium that can infect both healthy and unhealthy individuals in hospitals and through infected communities. S. aureus is also well adapted to human hosts. It can cause bacteremia, septicemia, soft-tissue infections, endocarditis, osteomyelitis, pneumonia, and meningitis [21]. S. aureus infection causes significant morbidity in some infected patients and a mortality rate that may approach 40% [22].
The nasal carriage of multi-resistant S. aureus seems to be the origin for the development of staphylococcal infections in patients with post-surgical wounds [21][22][23][24]. S. aureus is commonly adherent to the skin of atopic-dermatitis patients, and prolonged infection may exacerbate the clinical condition [25,26]. Staphylococcal enterotoxin Y may accelerate localized inflammation through skin-resident T-cell activation, thereby facilitating the pathogenesis of S. aureus infection in patients with disrupted epithelial barriers [26]. The persistent carriage of strains harboring these virulence determinants may increase the risk for subsequent invasive infections in carriers [21][22][23]25,26].
S. aureus expresses a variety of virulence factors, including toxins (hemolysin and leukocidins), surface immune-evasive factors (capsules and protein A), and enzymes that promote tissue invasion (hyaluronidase enzymes) [24]. Microorganisms that can develop resistance to antimicrobials are called superbugs [4]. Antimicrobial resistance (AMR) occurs when microorganisms (such as bacteria, fungi, viruses, and parasites) adapt when exposed to antimicrobial drugs (such as antibiotic, antifungal, antiviral, antimalarial, and anthelmintic drugs). "Superbug" is a term used to describe the evolution of bacterial species resistant to antibiotics. Antibiotic resistance by superbugs causes economic losses by increasing the duration of infections, increasing the cost of treatment, and decreasing the success of surgical treatment due to nosocomial infections [27]. AMR threatens the effective prevention and treatment of a growing range of infections caused by bacteria, parasites, viruses, and fungi. As  [15] a result, drugs become ineffective and the infections remain in the body, thereby increasing the risk of transmission to others [4].

Resistance Mechanism
The use of large quantities of antimicrobials over a long period of time can provide selection pressures that support the evolution of resistant strains. Resistant strains can spread horizontally and cause the spread of the virus in the environment [27][28][29]. From an evolutionary perspective, bacteria use two main genetic strategies to adapt to antibiotic attack: (1) Gene mutations that are often associated with the mechanism of action of the antibiotic compounds and (2) acquisition of foreign DNA coding for determinants of resistance through horizontal gene transfer (HGT) [27,29].
Gene mutations can accelerate the resistance to antimicrobial molecules. Once resistant mutants emerge, antibiotics eliminate the susceptible population, and resistant bacteria predominate. In general, mutations resulting in AMR alter the action of antibiotics through one of the following mechanisms: (1) Modification of the antimicrobial target by decreasing bacterial affinity for the drug, (2) reducing drug uptake, (3) activating the excretion mechanism to excrete harmful molecules, or (4) global change in important metabolic pathways through modulation of regulatory networks. Therefore, raising microbial resistance due to mutational changes can be varied and complex [27,29].
HGT is one of the most important drivers of bacterial evolution and is often responsible for developing AMR. Classically, bacteria acquire external genetic material through three main strategies, (1) transformation (fusion of bare DNA), (2) transduction, and (3) conjugation. The emergence of resistance in a hospital setting often involves conjugation, a highly efficient gene transfer method that involves cell-to-cell contact, and is most likely to occur at high levels in the human gastrointestinal tract under antibiotic treatment [27,29].
The main antibiotic resistance mechanisms in superbugs can be briefly described as follows: (1) Production of hydrolysis products such as β-lactams by various β-lactamase enzymes (Class A to D β-lactamases), (2) changes in penicillin-binding proteins to prevent β-lactam action, (3) changes in the structure and amount of porin protein, resulting in decreased permeability to antibiotics through the outer membrane of bacterial cells, and (4) efflux pump activity, which further reduces the antibiotic concentration in bacterial cells [28,29].

MRSA Infection
Resistance to S. aureus first emerged in the 1940s, mediated by the bacterial β-lactamase gene [24]. Hospital-acquired infections are still a major cause of morbidity and mortality worldwide. MRSA has been identified as one of the most frequently infecting nosocomial pathogens, accounting for about 25% of over 2 million nosocomial infections ranging from mild skin infections to septicemia [30,31]. Vancomycin is the last choice of antibiotics that can inhibit the growth of MRSA bacteria. The emergence of resistance to vancomycin is the most feared genetic adaptation in S. aureus till date [24]. Bacteria that are completely resistant to vancomycin are also considered as superbugs [28,32].
In general, risk factors that predispose patients to postsurgical MRSA infections include: (1) Duration of surgery or anesthesia, which may explain the increased risk of infection associated with simultaneous bilateral orthopedic procedures, and (2) surgical implant placement, in which the surgical plate acts as a nidus for infection and a site for biofilm formation, which can help bacteria avoid the immune system and antibiotics [16].
The body's main defense against S. aureus infection is the neutrophil response. When S. aureus enters the skin, neutrophils and macrophages migrate to the infection site. S. aureus avoids this response and increases the number of skin and soft-tissue infections in many ways, including blocking leukocyte chemotaxis, confiscating host antibodies, hiding from detection through polysaccharide capsules or biofilm formation, and fighting damage after ingestion by phagocytes. Several virulence factors appear to contribute to infection, including Panton-Valentine leukocidin (PVL), α-hemolysin (also called alpha-toxin), phenol-soluble modulin (PSM), arginine catabolic mobile element (ACME), and regulatory loci referred to as Agr. PVL causes lysis of white blood cells. In a meta-analysis of several studies, the presence of PVL was clearly associated with abscesses and furuncles at a ratio of 10.5 (95% CI, 7.4-14.9). α-hemolysin is a toxin that can form pores in various human cells and cause cell lysis. In skin infections, α-hemolysin also contributes to keratinocyte penetration. PSM and its proteolytic products facilitate MRSA colonization and lyse human cells, including neutrophils and erythrocytes. The speG gene in the ACME locus also increases resistance to polyamines produced by the skin, thus leading to the possibility of a selective advantage during colonization and bacterial infection of the skin. The Agr locus regulates the release of virulence factors such as PVL, α-hemolysin, and ACME from MRSA bacteria [30][31][32][33].
In a study conducted by El-Gayar et al. [34], macroscopic and microscopic results were obtained for wounds on the skin of rats infected with MRSA. Biopsies were taken from rats of MRSA-infected and uninfected group immediately after fasting for different time intervals, fixed in 10% formalin salt solution for 24 h, and stained with hematoxylin and eosin for examination under a microscope. Macroscopically, wounds without MRSA infection did not show any pus, whereas wounds with MRSA infection appeared wet (oozing) and pus formation began after 5-11 days. Microscopically, wounds without MRSA infection showed no histopathological changes, whereas wounds with MRSA infection showed necrosis of the epidermis and dermis on day 3 and ulceration of the epidermis and dermis on day 9.

Benefits of Green Algae (U. lactuca)
U. lactuca is a green algae, commonly known as sea lettuce, belongs to the phylum Chlorophyta. This sessile algae is able to grow and float freely [35,36]. U. lactuca reproduces through sexual reproduction, and also through thallus fragmentation, although rare. U. lactuca is a polymorphic species with a morphology that depends on salinity or symbiosis with bacteria [9]. U. lactuca is considered a widespread therapeutic herbal source for some oriental remedies of some noxious diseases [36,37].

Antibacterial Activity of U. lactuca Against MRSA
The U. lactuca extract has been tested in vitro against several Gram-positive and Gram-negative bacteria as well as some fungi [35,38,52,54]. According to Abdel-Khaliq et al. [55], U. lactuca showed an average inhibition zone of 22.0±0.8 mm against Gram-positive bacteria S. aureus, which is higher than that showed by Ulva intestinalis (20.1±0.4 mm). The inhibition zone of U. lactuca was almost equivalent to the antibiotic ampicillin, which showed an inhibition zone of 28.3±0.1 mm.
Kolanjinathan and Stella [52] reported that the U. lactuca methanol extract (5.0 mg/mL) showed an average inhibition zone of 13±0.6 mm against the Grampositive bacilli S. aureus, with a minimum inhibitory concentration (MIC) value of 2.5 mg/mL. Talreja [35] evaluated the methanolic extract of U. lactuca against resistant strains of human pathogenic bacteria species. Lauric, palmitic, linolenic, oleic, stearic, and myristic acids are known to be potential antibacterial and antifungal agents [56], and these compounds in the methanolic extract of U. lactuca may confer their antimicrobial activity against resistant strains [35]. The crude extract of U. lactuca also showed significantly stronger antimicrobial activity (zone of inhibition, 8.2 mm) against MRSA infections than that shown by the standard drug streptomycin (4.6 mm). Thus, crude antimicrobial activity was significant in the case of MRSA [35,57].
Kim et al. [58] reported that the U. lactuca ethyl-ether extract was able to suppress bacteria, including MRSA with MIC MRSA CCARM3561 (12.5 μg/mL), MRSA CCARM3115 (12.5 μg/mL), and MRSA CCARM3089 (50 μg/mL). Another study also showed that there is a significant antibacterial activity increase from U. lactuca specifically against MRSA when they are harvested at lunar Phase III [59].

Potential of U. lactuca as an Antiinflammatory Agent in Wound Healing
The inflammatory response is an essential aspect of the tissue response to harmful infections. A prolonged inflammatory process leads to chronic disease, which can eventually lead to tissue damage [17]. Several compounds isolated from marine algae possess varied chemical structures that have potent anti-inflammatory and antioxidant activities [37,[60][61][62][63]. These marine algae compounds may affect multiple targets in the immune and inflammatory systems that influence disease progression [43]. U. lactuca contains active compounds such as saponins, terpenoids, and flavonoids that function as anti-inflammatory agents [38][39][40]64].
Mezdour et al. [43] reported that the U. lactuca extract that was subcutaneously injected into rats with leg edema produced a significant anti-inflammatory effect (48-51%) at 3 h after treatment, equivalent to the positive control diclofenac-sodium (52% anti-inflammatory effect). The U. lactuca lectin and

Pharmacological activity References
Saponin Accelerates the wound healing process and inhibits inflammatory reactions in wounds during the initial phase [38] Steroid Antimicrobial agent [39] Triterpenoid As an anti-tumor, antibacterial, anti-inflammatory, and anti-hyperglycemic [40] Alkaloid Inhibits the growth of Grampositive bacteria [41] Flavonoid Has antioxidant properties that can reduce cell necrosis and wound tissue damage due to oxidation by repairing damaged blood vessels and reducing lipid peroxidation, has antibacterial activity by inhibiting bacterial growth and is able to maintain wound contraction and increase the rate of epithelialization [13,41] Tannin Antimicrobial agent [39] Fenol Antioxidants [42] U. lactuca=Ulva lactuca Available at www.veterinaryworld.org/Vol.14/May-2021/8.pdf polysaccharide extracts exerted anti-inflammatory activities by suppressing the LPS-stimulated production of TNF-α and NO in the RAW 264.7 cells treated with LPS [43,65]. Awad [66] reported the topical anti-inflammatory activity of U. lactuca using mouse ear edema as an experimental model. U. lactuca was shown to contain the anti-inflammatory compound 3-O-β-D glucopyranosyl-stigmasta-5,25-dien. Devaki et al. [67] also showed that sulfated polysaccharides from U. lactuca could prevent the oxidative stress induced by D-galactosamine intoxication.
A similar study was also conducted by de Araújo et al. [68], who showed that the U. lactuca extract has an anti-vascular anti-inflammatory effect through the targeting of bradykinin, which is one of the chemical factors that stimulate the inflammatory process. The anti-inflammatory activity of the U. lactuca extract was shown to accelerate the wound healing process, as evidenced in the study of Premarathna et al. [69]. These authors reported that after 7 days of treatment with the U. lactuca extract, rats with excised wounds showed 61.67% wound healing compared with the control group without treatment (52.76%). Based on this description, U. lactuca has potential in accelerated wound healing because of its activity as an anti-inflammatory agent.

Conclusion
MRSA is the primary strain of antimicrobial-resistant bacteria in hospitals. It is the leading cause of nosocomial infection and secondary infection in postoperated patients. As of yet, there is no anti-MRSA agent that is accepted globally. U. lactuca contains active compounds that have the potential to accelerate wound healing of nosocomial wound infections due to its antibacterial, anti-inflammatory, antioxidant,

Efficacy Treatment Method Result References
Anti-inflammatory Acute inflammatory leg edema (Wistar rats) The extract was injected into the legs; Foot thickness was measured before and after injection at 0, 1, 3, and 6 h U. lactuca extract had a significant antiinflammatory effect (48%-51%) at 3 h after treatment compared to diclofenac-sodium as a positive control showing an antiinflammatory effect of 52% [43] Anti-inflammatory antioxidants Rheumatism (Wistar rats) Extract was given orally; for 1, 2, and 3 weeks ankle circumference was measured Treatment of rheumatic rats with U. lactuca extract induced significant improvement at 1 st and 2 nd weeks. U. lactuca extract had strong anti-inflammatory effects and antioxidant properties [44] Anti-bacteria -The extracts were tested on an antibiotic disc and the inhibition zone diameter was measured Antibacterial activity revealed against pathogens S. aureus, K. pneumonia, P. vulgaris and B. subtilis [45] Anti-biofilm - S. aureus=Staphylococcus aureus, U. lactuca=Ulva lactuca and anticoagulant activities. With various active compounds contained in the U. lactuca extract, it may demonstrate efficacy against multi-resistant bacteria. However, information on the potential of U. lactuca as an antibacterial and anti-inflammatory agent in wound healing is still limited. This review describes the potential of U. lactuca as a source of anti-MRSA agent that might be used to treat common SSIs.

Authors' Contributions
SIOS conceived, wrote, and revised the manuscript. NFA, LM, and DU wrote, prepared, and revised the manuscript. All authors read and approved the final manuscript.