Staphylococcosis, usually known as Staphylococcus infection, is an important zoonotic disease primarily caused by Staphylococcus aureus. S. aureus is an important clinical pathogen and a common colonizing bacterium found on human skin and mucosal surfaces. It can cause a variety of infections at different sites in humans and animals, ranging from skin and soft tissue infections to serious life-threatening diseases such as osteomyelitis, endocarditis, and necrotizing pneumonia. Moreover, S. aureus can pollute food in various ways to produce toxins that cause food poisoning symptoms. Commonly affected foods include milk, raw meat, and frozen food products. In the United States, the number of food poisoning cases caused by S. aureus is estimated to be 241,000 per year (Scallan et al., 2011). Many such poisoning events have been reported in China each year (Yan et al., 2012; Lv et al., 2021).

Disinfection and hygienic food/feed management measures are the most important prevention and control strategies for S. aureus. S. aureus-infected patients and sick animals were first tested for drug sensitivity, and then treated with sensitive drugs. Beta-lactams and glycopeptide antibiotics are used to treat staphylococcosis. However, with the emergence of methicillin-resistant S. aureus (MRSA) and the introduction of strict laws on antibiotic usage in animals, an era characterized by the limited use of antibiotics is beginning in China. Many techniques and products replace antibiotics against Staphylococcus strains, such as antibacterial peptides, probiotics, prebiotics, enzyme preparations, and traditional Chinese medicine (David and Daum, 2017; Li et al., 2019). Plant extracts are becoming increasingly popular as alternatives to antibiotics, although they cannot completely replace antibiotics. Aqueous extracts of Galla chinensis can reduce biofilm formation in methicillin-resistant S. aureus and inhibit the invasive ability and pathogenicity of MRSA in vivo at a concentration of 31.25 μg/ml (Wu et al., 2019). Ginkgo biloba exocarp extracts at concentrations of 4–12 μg/ml were shown to inhibit S. aureus and MRSA biofilm formation in a dose-dependent manner and affected biofilm-associated gene expression (Wang et al., 2021). Moreover, herbal extracts and their active constituents can exert synergistic effects in combination with oxacillin or gentamicin against S. aureus (Kuok et al., 2017).

Portulaca oleracea L. is listed in the National Drug and Food Homologous Catalogue in China with the functions of clearing heat, removing toxins, cooling blood to stop bleeding, and treating dysentery (Zhou et al., 2015). It is mainly used to treat blood dysentery caused by heat toxins, eczema, inflammatory diseases, and redness of the skin. It is a medicine and food homologous drug with a good safety profile in humans. In recent years, many studies have been conducted on its chemical constituents, pharmacological effects, and molecular mechanisms (Rahimi et al., 2019). Two novel amide glycosides isolated from P. oleracea exhibit anticholinesterase and scavenging activities (Liu et al., 2021). P. oleracea leaf extract also displays antioxidant and anti-inflammatory activities against LPS-induced acute lung injury in a dose-dependent manner (Rahimi et al., 2019). Moreover, the P. oleracea extract show relatively potent anti-asthmatic effects owing to decreased nitric oxide production and increased antioxidant markers (Khazdair et al., 2019). Modern pharmacological studies on P. oleracea have demonstrated its gastroprotective and hepatoprotective activities in in vivo and in vitro models (Farkhondeh and Samarghandian, 2019). However, there have been few studies on antimicrobial activity against S. aureus. Two unsaturated fatty acids from P. oleracea act synergistically with erythromycin against MRSA in vitro, possibly by inhibiting methionine sulfoxide reductase (Fung et al., 2017). The carbon dioxide extraction of P. oleracea shows clinical effects against significant microorganisms, such as Escherichia coli and S. aureus (Tleubayeva et al., 2021). However, the chemical constituents that demonstrate antimicrobial activity against S. aureus remain unknown.

This study aimed to investigate the anti-Staphylococcus activity of an organic acid extract of P. oleracea in vitro and in vivo. First, the main chemical constituents of P. oleracea extract and the anti-Staphylococcus effects of the organic acid extract were evaluated using an in vitro model. Finally, organic acid extracts were used to cure skin wounds infected with S. aureus.

Staphylococcus aureus is an important zoonotic pathogen that not only causes local and systemic infections in humans but also induces dermatitis, mastitis, enteritis, and other infections in livestock and poultry (Zhou et al., 2018). The occurrence of methicillin-resistant or multidrug-resistant S. aureus strains makes the treatment of staphylococcal diseases more difficult. Moreover, the implementation of strict laws against antibiotic use in animals has complicated the husbandry of Staphylococcus-infected animals. However, many new methods could be applied to reduce antibiotic usage and kill S. aureus, such as herbal extracts, bacteriophages, acidifiers, and antimicrobial peptides. Aqueous extracts of P. oleracea have a slight inhibitory effect on E. coli (Okuda et al., 2021). However, the bacteriostatic mechanisms and active ingredients of P. oleracea remain unclear. The present study aimed to demonstrate that OAPO is the main active ingredient of P. oleracea against S. aureus through in vitro and in vivo experiments.

There have been many studies on the antimicrobial activity of P. oleracea against a broad spectrum of bacterial species (Soliman et al., 2017); however, the characteristics of antimicrobial activity determined by these studies were slightly different from those in ours. Tleubayeva et al. showed that the CO₂ extract of P. oleracea had the greatest bactericidal effectiveness against S. aureus at 250 μg/ml and against Escherichia coli, Bacillus subtilis, and Candida albicans at 500 μg/ml (Tleubayeva et al., 2021). Portulaca elatior leaf lectin also showed strong activity against some phytopathogenic bacteria with a MIC value of 0.185 μg/ml (da Silva et al., 2021). The methanol extracts of Portulaca quadrifida at 250 μg/ml and 200 μg/ml showed the most significant antimicrobial activity against all tested gram-positive and gram-negative bacteria (Desta and Cherie, 2018). However, the present study showed that the MIC of OAPO was 12.5 mg/ml, which is inconsistent with the results of the above studies. The sources of herbal medicine and the tested bacterial strains were very different, and these factors might be the main causes of the different MIC results. Moreover, some compounds identified from P. oleracea exhibited only weak dose-dependent inhibition against bacterial pathogens, whereas other compounds showed significant antibacterial activity against common enteropathogenic bacteria in vitro (Lei et al., 2015; Hu et al., 2021). Interestingly, two unsaturated fatty acids from P. oleracea acted synergistically with erythromycin in vitro against MRSA, possibly by inhibiting the multidrug efflux pump of the bacteria (Chan et al., 2015; Fung et al., 2017). In summary, the specific substances and/or chemicals that play an antibacterial role in P. oleracea are still unclear.

Portulaca oleracea has many constituents, including flavonoids, alkaloids, organic acids, terpenoids, polysaccharides, vitamins, sterols, and other compounds (Zhou et al., 2015). In our preliminary tests, the aqueous extract, flavonoids and polysaccharide extracts did not display antibacterial activity (data not shown), while the organic acids possessed certain Staphylococcus-killing properties. Organic acid extracts were prepared according to a previously reported method, with slight modifications (Zhang et al., 2020). HPLC-MS was used to analyze the chemical composition of OAPO, and many organic acids were identified in OAPO, such as α-eleostearic, palmitic, phloionolic, and stearidonic acids. Some organic acids, such as protocatechuic and caffeic acid, were not identified in OAPO by HPLC-MS. There are two important reasons for this: one is that P. oleracea contains too little protocatechuic and caffeic acids to be detected, and the other is that the mixture of OAPO affects the measurement accuracy of HPLC-MS. Nevertheless, an organic acid-enriched extract was obtained in this study, which proved that OAPO could kill S. aureus.

The integrity of the bacterial cell wall and membrane is vital for survival. Many antibiotics can inhibit bacterial growth and induce loss of cell homeostasis by altering or damaging the cell membrane structure (Turgis et al., 2009). Different degrees of OAPO-induced damage were observed in S. aureus using SEM, and the lysis effects of OAPO on the cell membrane were dose-dependent, which indicated that this damage in the cell wall and membrane was likely responsible for the observed growth inhibition and bactericidal effects. To further demonstrate the extent of OAPO-induced cell wall damage, the presence of AKP and nucleic acid substances in the supernatant of OAPO-treated S. aureus culture medium was used as an indicator of cell wall integrity. AKP is an enzyme located between the bacterial cell wall and cell membrane and, therefore, is undetectable when the bacterial structure is intact. Nevertheless, when the cell wall is damaged, AKP leaks out of the cell owing to an increased permeability (Tang et al., 2017). The present study showed that OAPO was more effective at elevating extracellular AKP levels at higher concentrations, which indicated that OAPO could increase the permeability of the cell wall, and damage to the integrity of the cell wall might be the main cause of AKP release into the supernatant. Moreover, nucleic acids are essential life substances in microorganisms and cannot permeate through the cell membrane during the normal growth of bacteria (Wu et al., 2016). The degree of leakage of nucleic acids from S. aureus cells treated with one-fold MIC and two-fold MIC of OAPO was significantly higher than that from untreated S. aureus, which suggested that OAPO might induce outer membrane damage in S. aureus, causing nucleic acids to leak out from the bacterial cells. Our results are consistent with those regarding the antibacterial activity of lactobionic acid against S. aureus by breaking down the structure of the bacterial cell wall and membrane (Cao et al., 2019).

Protein synthesis and energy metabolism are two other factors that affect bacterial survival. Soluble proteins are important osmotic pressure regulators of bacteria, which can improve the water retention capacity of bacteria and ensure their normal life activities (Nguyen et al., 2019). In this study, we determined the prevalence of intracellular biomacromolecules, such as total protein, ATPase, and glucose-6-phosphate dehydrogenase, in S. aureus treated with OAPO. The results of SDS-PAGE and BCA assays showed that the soluble protein levels of S. aureus were significantly elevated by OAPO, which indicated that interference with protein metabolism might not be the antibacterial mechanism of OAPO. Furthermore, OAPO did not affect intracellular ATPase and glucose-6-phosphate dehydrogenase content (data not shown), thereby indicating that OAPO could not inhibit ATPase activity and could not prevent ion transportation and nutrient absorption (Petrosyan et al., 2015).

To verify that OAPO has the same S. aureus-killing effects in vivo as it did in vitro, an animal model of wound infection with S. aureus mimicking clinical conditions was used in our study. The skin is the first line of defense against diseases, and skin wound infection is a useful animal model of S. aureus infection, characterized by a series of pathological changes, from local to systemic infection (Edwards and Harding, 2004; Gupta and Tyagi, 2021). Therefore, we used a skin wound infection model induced by S. aureus to evaluate the anti-infective effects of OAPO in vivo. The results demonstrate three aspects of the antibacterial activity of OAPO. First, OAPO could significantly promote skin wound healing compared with that by TMP-SMX; therefore, the skin histopathology of mice in the OAPO group was close to that of normal skin. It has long been reported that the crude extract of P. oleracea accelerates the wound healing process by decreasing the wound surface area and improving tensile strength (Rashed et al., 2003). Moreover, OAPO promotes wound healing better than homogenized plant material, indicating that organic acids might be the main active ingredients for improving wound injury. Second, the bacterial skin load in the OAPO group was significantly lower than that in the self-healing group, especially the bacterial load of the low-dosage group, which was significantly lower than that of the TMP-SMX group. Chan et al. reported that two fatty acids from P. oleracea exhibited synergistic effects with erythromycin in combating MRSA (Chan et al., 2015). Other organic acids from P. oleracea, such as protocatechuic and palmitic acids, also displayed effective bactericidal activity, which was in accordance with our results (Chao and Yin, 2009; Song et al., 2020). Third, the inflammatory responses induced by S. aureus were used to evaluate the anti-inflammatory effects of OAPO. The results demonstrated that OAPO exerted inhibitory effects on inflammatory cytokine expression, which was consistent with the results of previous studies on the anti-inflammatory activity of P. oleracea extracts (Rahimi et al., 2019; Moslemi et al., 2021).

To conclude, an extract of Portulaca oleracea was obtained in this study and it not only inhibits methicillin-resistant S. aureus activity in vitro but also inhibits S. aureus-induced skin damage in a mouse model. The chemical compositions of the extract contain lots of organic acids, which indicates that OAPO is the main active compositions against MRSA. However, the purity and toxicity of the extracted organic acids was unknown, and which organic acid has stronger antibacterial activity needs to be explored soon.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Animal Management and Ethics Committee of Guangxi University.

GL, HS, and CO conceived and designed this study. GL, AL, CZ, QZ, CY, HL, and HY participated in laboratory work. JM, ZZ, GL, and AL performed the data analysis and the writing of the manuscript. AL and CO participated in revising the manuscript critically. All authors contributed to the article and approved the submitted version.

This work was supported by scientific research startup funds of Guangxi University. We would like to thank Editage (www.editage.cn) for English language editing, Guangxi Science and Technology Base and Talent Special (2021AC19372) and TCM Industrial Pioneers (GuiNongKeMeng202211).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.