Antibacterial Activity of Solanum torvum Leaf Extract and Its Synergistic Effect with Oxacillin against Methicillin-Resistant Staphyloccoci Isolated from Dogs

Methicillin-resistant staphylococci (MRS) have been considered a veterinary and public health threat that needs to be addressed, as they are known to cause serious infections, with limited therapeutic options. Thus, in this study, we aimed to examine the potential antibacterial activity of the leaf extract of Solanum torvum against MRS isolated from clinically healthy dogs. In total, seven mecA-positive Staphylococcus isolates tested in this study were identified using 16S rRNA gene sequencing, and all of them were classified as multidrug-resistant using disk diffusion tests. According to gas chromatography-mass spectrometry analysis, the main phytochemical components found in the leaf extract were hexadecanoic acid and its ethyl ester and 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z). The minimum inhibitory concentration (MIC) breakpoints for the leaf extract against all tested isolates ranged from 2 to 16 mg/mL, while the MIC breakpoints for oxacillin were from 2 to 512 mg/L. Although varying effects were found, the positive effects of the leaf extract were most evident in combination with oxacillin. These results suggested that S. torvum leaf extract may complement classical antibiotics and may potentially drive the development of an effective therapeutic option for MRS.


Introduction
Although most staphylococci are commensal bacteria, some of them can cause serious infections of the skin and other tissues in both animals and humans. According to their ability to produce coagulase, staphylococci can be classified into the following two groups: coagulase-positive and coagulase-negative staphylococci. Coagulase-positive staphylococci, particularly Staphylococcus aureus and Staphylococcus pseudintermedius are commonly involved in infection in humans and companion animals, respectively, while coagulasenegative staphylococci (CoNS) are frequently related to nosocomial infection [1,2]. In the last few decades, the incidence of methicillin-resistant staphylococci (MRS) has seen a steady increase, which has led to it becoming a major concern worldwide. Since MRS can occur in companion animals and the transmission of MRS between animals and humans may potentially occur [3,4], MRS is considered to be a significant threat to public health. Methicillin resistance in staphylococci has mainly been associated with the mecA gene, which encodes a modified penicillin-binding protein 2a (PBP2a), causing a low binding affinity to β-lactam antibiotics [5]. In veterinary medicine, infections and colonization with MRS strains have been recognized as a serious problem because of their multidrug resistance and the possibility of transmission to humans. MRS strains are known to be resistant to several classes of antimicrobial agents, including β-lactam antibiotics, so therapeutic options are limited [6][7][8]. Therefore, traditional plant medicine may provide a potential alternative approach to treating and controlling the spread of MRS.
Solanum torvum (Swartz or turkey berry) is a small spiny tree widely distributed in India; China; Southeast Asian countries, including Thailand, Malaysia, and the Philippines; and tropical America. Different parts of this plant, particularly the fruits and leaves, produce antimicrobial effects and could therefore be used as medicinal plants [9][10][11]. Leaf extracts of S. torvum have been shown to effectively inhibit various pathogenic bacterial strains [12], although there is a lack of information regarding the antibacterial activity of S. torvum against antimicrobial-resistant bacteria. Due to the limited options for the treatment and control of MRS infection, the leaf extract of S. torvum is a potentially valuable candidate for the management of this public health threat.
In this study, we aimed to evaluate the antibacterial activity of the leaf extract of S. torvum against MRS isolated from clinically healthy dogs. The synergistic effects between the extract and oxacillin were also investigated.

Plant Preparation and Extraction
Leaves of S. torvum collected from the Nan province in the northern part of Thailand were used in this study. They were identified and authenticated at the Office of the Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Thailand (code: BKF 186114). After air-drying at room temperature, the sampled leaves were ground to a fine powder. Then, approximately 600 g of the leaf powder was extracted with 5400 mL of 95% ethanol, as described in a previous study [12].
The leaf extract was dissolved in dimethyl sulfoxide (DMSO) (Merck, Germany) to a final concentration of 50% (v/v) and filtered through No. 1 Whatman filter paper to obtain the crude extract used for the examination of antibacterial activity. The phytochemical components of the crude extract were analyzed using GC-MS with a 6980 GC system (Agilent Technologies; Santa Clara, CA, USA), as described previously [12].

Bacterial Strains and Identification
In total, seven mecA-positive Staphylococcus isolates collected from the head (n = 2), mouth (n = 3), and nasal cavity (n = 2) of six clinically healthy dogs were used in this study. These three sites of sample collection are considered to be areas of high-frequency contact between dogs and owners. Ethical approval for the animal research was obtained from Kasetsart University (ACKU 01557). The bacterial isolates were cultured on mannitol salt agar (Himedia, India) and presumptively identified as Staphylococcus spp. by Gram staining and catalase tests. After DNA extraction using an E.Z.N.A. ® Bacterial DNA kit (Omega Bio-Tek, Doraville, GA, USA) following the manufacturer's instructions, all isolates were definitely identified using 16S rRNA gene sequencing, as previously described [13], and the results were further analyzed using EzBioCloud (https://www.ezbiocloud.net/) (accessed on 13 May 2021) [14]. In addition, S. aureus ATCC 25923 was used as a quality control strain for antimicrobial susceptibility testing.
The detection of mecA was performed using PCR, as described previously [15], with slight modifications. Briefly, the primers used for the amplification of mecA were mecA-1 (5 -AAAATCGATGGTAAAGGTTGGC-3 ) and mecA-2 (5 -AGTTCTGCAGTACCGGATTT-GC-3 ), giving a product of 533 bp. The conditions for PCR amplification were as follows: 94 • C for 2 min, followed by 30 cycles of 94 • C for 15 s, 55 • C for 30 s, and 72 • C for 30 s, and a final extension at 72 • C for 10 min. The PCR products were analyzed using gel electrophoresis. After staining with SYBR ® green (Sigma Aldrich, MO, USA), the bands of the amplified products were visualized using a gel documentation system. Positive and negative controls were included in all reactions.

Microdilution Broth Susceptibility Assay
The determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of crude extracts of S. torvum leaves and oxacillin was performed using the microdilution broth method [16] with slight modifications. Twelve twofold serial dilutions of the extract (256-0.125 mg/mL) and oxacillin (0.512-0.005 mg/L) were prepared in a 96-well microplate. To each well, 100 µL of each dilution of the leaf extract or oxacillin and 100 µL of bacterial suspension in Mueller-Hinton broth (MHB) (Oxoid, Basingstoke, UK) with a concentration of 1 × 10 6 CFU/mL were added to produce a final bacterial number of approximately 1.5 × 10 5 CFU per well. Sterility control with only MHB medium and positive control with MHB medium and bacterial inoculum were included in the experiment. In addition, we added DMSO as a negative control at equal concentrations (12.5-0.05% v/v) to those used to dilute the leaf extract. The plates were incubated at 37 • C for 24 h and the bacterial viability was examined by adding p-iodonitrotetrazolium chloride (INT) (Sigma-Aldrich, St. Louis, MO, USA). After incubation at 37 • C for 30 min, the color alteration was observed in the wells containing viable bacteria. Then, the MIC values were determined. The MIC and the more concentrated test dilutions were inoculated into Mueller-Hinton agar (Oxoid, Basingstoke, UK); then, the MBC values were further determined after an overnight incubation at 37 • C. The assays were independently performed in triplicate.

Synergistic Interaction Analysis
To determine the potential synergistic antibacterial activity of the leaf extract of S. torvum and oxacillin, the checkerboard method was used [17]. Briefly, the leaf extract was diluted twofold in MHB along the vertical rows of the 96-well microplate, while oxacillin was cross-diluted horizontally by twofold serial dilution. Bacterial suspension was added into each well to produce a final concentration of 1.5 × 10 5 CFU/mL. The plates were incubated at 37 • C for 24 h. After adding INT and incubating at 37 • C for 30 min, the bacterial growth was assessed by observing the color of the solution. The tests were carried out in triplicate.

Statistical Analyses
All tests were independently performed in triplicate. The differences between the data were analyzed using one-way ANOVA with repeated measures in SPSS version 28.0.1.0 (142) (IBM, Armonk, NY, USA). The p-values were considered statistically significant at ≤0.05.

Resistance Profiles of Bacterial Strains
As shown in Table 1, seven mecA-positive Staphylococcus isolates were identified as Staphylococcus schleiferi subsp. schleiferi (n = 1), Staphylococcus epidermidis (n = 2), Staphylococcus intermedius (n = 3), and Staphylococcus pseudintermedius (n = 1), using 16S rDNA sequencing. These staphylococcal species are commensals on skin and mucous membranes and can cause infections in both humans and animals [19][20][21][22]. According to the results from disk diffusion tests, all isolates were found to be resistant to at least three classes of antimicrobial agents and were therefore considered to be multidrug-resistant (MDR) bacteria [23]. Their resistance profiles are presented in Table 1. Among the isolates used in this study, those isolated from the nasal cavities and mouths of dogs tended to be resistant to most antimicrobial agents tested. One isolate from the nasal cavities of dogs (DN41) and two isolates from the mouths of dogs (DN11 and DN40) which were identified as S. intermedius, as well as one S. pseudintermedius isolate (DN73) from a dog's mouth, were resistant to all tested antimicrobials. By D-test, two phenotypes were observed among the isolates tested in this study. S. schleiferi subsp. schleiferi (DN2) was erythromycin-sensitive and clindamycin-sensitive, indicating a susceptible phenotype, while the other six isolates showed resistance to both erythromycin and clindamycin, indicating a constitutive macrolide-lincosamide-streptogramin B (MLS B ) phenotype.
Although information on methicillin-resistant coagulase-negative staphylococci (MRCoNS) in pets is rare compared with that on methicillin-resistant S. aureus (MRSA), these results suggested that a considerable amount of antimicrobial resistance was present in CoNS and other commensal bacteria in healthy pets. Since a high prevalence of MRS has previously been reported in healthy dogs [24] and the transfer of methicillin-resistant S. pseudintermedius (MRSP) among pets, humans, and the environment has evidently occurred [3], people who have close contact with pets would be at increased risk of transmission of these MRS bacteria.
MRS (including MRCoNS) are potentially a neglected risk to public health. Therefore, the use of natural extracts or combinations of extracts and existing antimicrobial agents may provide effective alternative options for the treatment and prevention of MRS bacteria.

Phytochemical Components of Leaf Extract
An ethanol extraction of S. torvum leaves was analyzed using gas chromatographymass spectrometry (GC-MS). The phytochemical components of the extract and their potential biological activities are shown in Table 2. In total, 11 chemical compounds, including fatty acid ethyl esters, fatty acids, diterpenes, indoles, and alkanes, made up 60.12% of the total extract, as determined by GC fraction (Supplementary Figure S1). The major components were hexadecanoic acid, ethyl ester (11.13%), followed by hexadecanoic acid (8.63%) and 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z) (7.47%). These compounds have also been identified in extracts of the fruit of S. torvum [25] and other plants [26][27][28]. The common biological activities of the abundant compounds of S. torvum leaf extract are antibacterial and anti-inflammatory. Consistent with a previous study, hexadecanoic acid, ethyl ester, and 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z), were identified as major components of other leaf extracts which had potent activity against both Gram-positive and Gram-negative bacteria, as well as MRSA [28]. Hexadecanoic acid and its methyl and ethyl esters have previously been reported to exhibit antituberculotic activity in actinobacteria [29]. It is, therefore, suggested that the major phytochemical compounds (hexadecanoic acid, ethyl ester, hexadecanoic acid, 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z) found in our leaf extract play a crucial role in bacterial inhibition. The GC-MS results showed that neophytadiene was found twice, in peaks No. 4 and 10 ( Table 2, Supplementary Figure S1). However, due to their different retention times, these two peaks were possibly related compounds with slightly different chemical structures.

Minimum Inhibitory Concentration (MIC) and Synergistic Interaction Analysis
The results from MIC and checkerboard analysis are presented in Table 3. All isolates were phenotypically confirmed as MRS (MIC of oxacillin ≥ 0.5 mg/L) [16]. They had MIC and minimum bactericidal concentration (MBC) values ranging from 2 to 512 mg/L for oxacillin, with the MIC and MBC values of S. torvum leaf extract ranging from 2 to 16 mg/mL and 8 to 64 mg/mL, respectively. Three S. intermedius isolates-DN11, DN40, and DN41-which were resistant to all antimicrobial agents tested also showed high MIC values for oxacillin. However, the leaf extract exhibited effective antibacterial activity against these three isolates, with an MIC range of 2-4 mg/mL. Similarly, the leaf extract of S. torvum has been reported to be effective against several pathogenic bacteria [12] and mycotoxigenic fungi [39]. The combination of oxacillin and S. torvum leaf extract exhibited varying antibacterial effects against the MRS strains tested (Table 3 and Figure 1). Although no antagonism was detected, only limited effects were found for 2/7 isolates. Synergistic and partially synergistic effects were observed for 4/7 isolates, while an additive effect was noted in one isolate of S. intermedius. The positive interaction was observed against all staphylococcal species used in this study-S. schleiferi, S. epidermidis, S. intermedius, and S. pseudintermedius-indicating the potential of the use of S. torvum leaf extract in combination with common antibiotics to counteract MRS.
The cytotoxic effects of crude extract of S. torvum leaves were previously evaluated in vitro. Since the antimycobacterial activity of S. torvum leaves has been observed, the cytotoxic potential of S. torvum leaf extract was investigated against a human fetal lung fibroblast cell line [40]. Although the notable cytotoxicity of several compounds of S. torvum aerial parts against human cancer cell lines was identified [41,42], the S. torvum leaf extract was found to be safe in a human fetal lung fibroblast cell line, supporting the traditional use of these leaves to treat respiratory tract disorders [40].
Due to the limited therapeutic options available for the treatment of MRS infections, novel alternatives, particularly phytopharmaceuticals, have been attracting increasing interest. The antimicrobial activity of several natural product extracts, and the synergistic effects between the extracts and antimicrobial agents, have been investigated. The antibacterial activity of various leaf extracts and their synergy with antibiotics against MRSA have previously been observed [43,44]. The combination of methicillin or penicillin G and essential oil has recently been reported to be effective against MRSA [45]. In this study, we demonstrated the potent activity of S. torvum leaf extract against MRCoNS and MRSP. The major components, hexadecanoic acid and its ethyl esters, are likely to be the Antibiotics 2022, 11, 302 7 of 9 bioactive compounds detected in our crude extract. However, further study regarding the antibacterial activity, pharmacological properties, and cytotoxic effects of these purified compounds is required to elucidate the antibacterial mechanism and the possibility of their use in vivo.
three independent experiments.
The combination of oxacillin and S. torvum leaf extract exhibited varying antibacterial effects against the MRS strains tested (Table 3 and Figure 1). Although no antagonism was detected, only limited effects were found for 2/7 isolates. Synergistic and partially synergistic effects were observed for 4/7 isolates, while an additive effect was noted in one isolate of S. intermedius. The positive interaction was observed against all staphylococcal species used in this study-S. schleiferi, S. epidermidis, S. intermedius, and S. pseudintermediusindicating the potential of the use of S. torvum leaf extract in combination with common antibiotics to counteract MRS.

Conclusions
Methicillin-resistant staphylococci known to possess multidrug-resistant traits were isolated from healthy dogs. In this study, we investigated the antimicrobial activity of the crude extract of Solanum torvum leaves against the tested isolates and identified positive interactions between the extract and oxacillin. The results highlight S. torvum as a promising therapeutic option for methicillin-resistant staphylococci.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article.