Design, synthesis, and discovery of novel oxindoles bearing 3-heterocycles as species-specific and combinatorial agents in eradicating Staphylococcus species

A series of new functionalized 3-indolylindolin-2-ones, 3-(1-methylpyrrol-2-yl)indolin-2-ones, and 3-(thiophen-2-yl)indolin-2-ones were synthesized by using novel indium (III)-catalysed reaction of various 3-diazoindolin-2-ones with indoles, 1-methylpyrrole, or thiophene via one-pot procedure. The newly synthesized compounds were characterized and screened for their in vitro antibacterial activity against various Staphylococcus species, including methicillin-resistant Staphylococcus aureus. results revealed that five compounds KS15, KS16, KS17, KS19, and KS20 exhibited potent and specific antibacterial activity against Staphylococcus species albeit inactive against Gram-negative bacteria. Especially, compounds exhibited superior antibacterial potency against Staphylococcus epidermidis compared to the reference drug streptomycin. The most potential compound KS16 also increased the susceptibility of Staphylococcus aureus to ciprofloxacin, gentamicin, kanamycin, and streptomycin. Among them, KS16 was found to be a synergistic compound with gentamicin and kanamycin. Furthermore, the cellular level of autolysin protein was increased from the KS16-treated Staphylococcus aureus cells. Finally, in vitro CCK-8 assays showed that KS16 exhibited no cytotoxicity at the minimum inhibitory concentrations used for killing Staphylococcus species. From all our results, novel oxindole compounds directly have lethal action or boost existing antibiotic power with the reduction of doses and toxicity in the treatment of multidrug-resistant Staphylococcus species.

particular, S. aureus and S. epidermidis, methicillin-resistant strains, are now common to hospitals and have spread in a pandemic fashion within the community, producing community-associated methicillin resistant Staphylococcus species 12,13 . Many different approaches have been tried to prevent or eradicate the infection of Staphylococcus species. One approach is that the development of promising vaccines against S. aureus species. However, there is no S. aureus vaccine on the market. There are main reasons why S. aureus vaccine development has made it to challenge 14 . First, the immune responses caused by S. aureus infection has not been clearly understood. Second, S. aureus also expresses a large array of virulence factors, resulting any one may not prove effective. Therefore, current trials are focused on multiple antigen preparations. Recently, it was hypothesized that the master mechanism of S. aureus counteracts may hinder the immunological responses, resulting in failure of target-oriented vaccine development 15 . Therefore, the understanding of such mechanisms are the key success for a developing vaccine for the future, but it is still remaining as a challenge. In other aspects, new potential anti-Staphylococcus compounds with their plausible action mechanism [16][17][18][19] and therapeutics 19,20 have been discovered in recent years, but there is still a high demand for highly potent compounds to specifically target AMR Staphylococcus species among bacterial mixtures. Biofilm, a surface-associated bacterial community surrounded by a self-produced extracellular matrix [21][22][23][24][25] associated with resistance to antimicrobial agents 26,27 . Recently, new materials such as dihydrazone analogues against biofilm forming Gram-negative 28 and benzodioxane midst piperazine decorated chitosan silver nanoparticles (BP*C@AgNPs) was controlled the MRSA biofilm 29 , still indole and its derivatives have been regarded as interesting heterocyclic antibacterial molecules against AMR bacteria owing to their promising potential to inhibit the formation of biofilm. In addition, the modification of indoles with diverse functional groups would change the efficiency of biofilm inhibition and the antibacterial specificity. For example, C-5/C-7-hydroxylated indoles were more efficient with respect to biofilm inhibition in Escherichia coli (E. coli) 30,31 . Moreover, various halogenated indoles, especially 5-iodoindole derivatives, potently inhibit biofilm formation of both E. coli and S. aureus 32 . Alternatively, oxindoles, which are structurally similar to indoles, have diverse pharmacological profiles [33][34][35] and are the most recent synthetic class of new antibacterial agents. For example, a series of oxindole-based 3(Z)-{4- [4-(arylsulfonyl)piperazin-1-ylbenzylidene]-1 ,3-dihydro-2H-indol-2-one derivatives exhibited significant antibacterial activity against S. aureus, Streptococcus pyrogenes, E. coli, and Pseudomonas (P.) aeruginosa 36 . Moreover, several pharmacophores combined with a single heterocyclic indole or oxindole molecule can result to an enhanced antibacterial activity. For example, novel heterocyclic spiro-oxindole derivatives were active against E. coli and S. aureus, with minimum inhibitory concentrations (MIC) of 500 μg equivalent to that of the standard drug, streptomycin 37 . One study reported that a combination of spiro-oxindole, 2-amino-4H-pyran, and 1,2,3-triazoles in a single matrix showed good Gram-positive antibacterial activity against S. aureus (MIC = 32-256 μg/mL); however, none of the compounds affected Gram-negative bacteria (E. coli and P. aeruginosa) 38 . In addition, indole derivatives containing heterocyclic moieties synthesized by using 3-chloro-1H-indole-2-carbaldehyde have shown excellent antibacterial activity 39 . Based on these previous reports, the antibacterial activity of oxindole derivatives has been observed to enhance and alter for specificity through different functionalization and structural modifications. Therefore, it is of great interest to synthesize and identify highly efficient and selective oxindoles bearing pharmacologically active heterocycles at the 3-position, such as pyrrole, thiophene, and indole moieties, that are probable novel antibacterial agents against health-threatening Staphylococcus species.
Based on the above considerations, we have designed and synthesized several different C-3-functionalized oxindoles bearing indole, pyrrole, and thiophene moieties by a novel indium(III)-catalysed reaction (Fig. 1). In addition, we also screened the synthetic compounds for their anti-Staphylococcus activity and synergistic action www.nature.com/scientificreports www.nature.com/scientificreports/ to existing antibiotics. A preliminary studies of structure-activity relationship (SAR) for the synthesized compounds have also been performed.

Materials.
All materials were obtained from commercial suppliers and were used without further purification.

Synthesis of 3-(1H-indol-3-yl)indolin-2-ones KS1 to KS12.
A reaction mixture of the corresponding 3-diazoindolin-2-ones (1, 0.5 mmol) and indoles (2, 0.55 mmol) in 1,2-dichloroethane was stirred at 50 °C for 6 h in the presence of 10 mol% In(OTf) 3 (Fig. 2). After completion of the reaction, as indicated by TLC, the reaction mixture was evaporated in a rotary evaporator and the residue was purified by silica gel column chromatography using hexane/ethyl acetate as the eluent to afford the desired products KS1 to KS12.

Characterization
All melting point (MP) was obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Switzerland). Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, USA). 1 H NMR and 13 C NMR spectra were recorded on Bruker ARX-400, 400 MHz or Bruker ARX-600, 600 MHz spectrometers (Bruker Bioscience, USA) with TMS as an internal standard. Reaction time of the products were monitored by TLC on FLUKA silica gel aluminium cards (0.2 mm thickness) with fluorescent indicator at 254 nm. Column chromatography was run on silica gel. The purity of all the synthesized compounds, determined by their 1 H and 13 C NMR spectra, was observed to be 95-99% (see Supporting Information, SI1 for details). In addition, the purity of compound KS16 was determined by Gas Chromatography (GC) and was found to be 99% (see SI2 for details).               www.nature.com/scientificreports www.nature.com/scientificreports/   www.nature.com/scientificreports www.nature.com/scientificreports/     www.nature.com/scientificreports www.nature.com/scientificreports/ Screening of active compounds with anti-S. aureus activity. The screening for compounds expressing anti-S. aureus activity was performed by using a disc diffusion method 40 . For all bacterial strains, overnight culture grown in MHB (400 μL) was inoculated into 20 mL of Difco Muller Hinton Agar (MHA; BD, USA) and solidified on petri dishes with a diameter of 90 mm (SPL, Korea). Sterile filter discs (6 mm in diameter; Advantec MFS, Taiwan) were placed on the surface of inoculated agar plates. Compounds, to a final amount of 20 μg, were impregnated by dropping 10 μL of 2 mg/mL stock solution to the sterile filter discs. The plates were then incubated at 37 °C for 24 h, and antibacterial activity was evaluated through the measurement of the diameter of the inhibition zone (mm) by using a transparent ruler. Each compound was analysed a minimum of three times and one representative image was presented.

Antibacterial Activity studies
Validation of MRSA strains. MRSA strains were purchased from CCARM (Culture Collection of Antimicrobial Resistant Microbes; www.ccarm.or.kr). The verification of strains was performed in two independent experiments. First, assay plates for both six individual MRSA strains and S. aureus (ATCC 25923) were prepared as described in disc diffusion method and the susceptibility to oxacillin was determined by using an E-test Oxacillin (BioMérieux, France). Second, S. aureus strains were subjected to PCR amplification for the detection of mecA gene in accordance with a previous report 41 . The PCR protocol was performed in a total 20 μL volume using 2x Quick Taq mix (Toyobo, Japan). The PCR program was conducted with initial denaturation at 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 30 s, and ended with a final extension at 68 °C for 5 min. The PCR product was electrophoresed on 2% agarose gel and imaged with an E-Graph Gel Documentation System (Atto, Japan). Synthesized primers mecA-F (5′-GTAGAAATGACTGAACGTCCGATGA -3′) and mecA-R (5′-CCAATTCCACATTGTTTCGGTCTAA-3′) were used in the amplification, which produced a PCR amplicon of 310 bp in size.

Determination of MIC.
Microbroth dilution MIC testings were performed 42 . Briefly, fresh overnight cultured cells were grown until absorbance at 600 nm (A 600 ) value of 0.5 was reached and the 500-fold diluted cells  www.nature.com/scientificreports www.nature.com/scientificreports/ in accordance with the manufacturer's instructions. Therefore, the final concentrations of the working MIC plates were diluted to one-fourth of that in the original EUST plates.
Determination of synergistic action of compounds to antibiotics. The fractional inhibitory concentration indices (FICIs) of the designed combinations against S. aureus were determined by using checkerboard www.nature.com/scientificreports www.nature.com/scientificreports/ assays, as previously described 43 under the same conditions used to screening the antibiotics to evaluate the combinatorial activity between antibiotics and selected compounds.

Characterization of proteins affected by KS16. Total cellular proteins extracted from ATCC25923 cells
(1 × 10 9 ) treated with different concentrations of KS16 in 96-well plates were resolved in 12% TGX strain-free gels (Bio-Rad), followed by imaging with ChemiDoc-MP (Bio-Rad). Amounts of protein bands migrated on the gel was analyzed with Image Lab Software (v. 5.2.1; Bio-Rad, USA). For peptide mass fingerprinting (PMF), gels were stained with Brilliant Blue R (Sigma-Aldrich, USA) and interesting protein bands were excised from the gel, followed by subjecting them to in-gel trypsin digestion as described 44 . Then, the in-gel digested sample was subjected to MALDI-TOF MS (Microflex LRF 20, Bruker, USA) as previously described 45 . Spectra were acquired from 300 shots per spectrum in the m/z range of 600-3,000 and calibrated by two-point internal calibration using trypsin auto-digestion peaks (m/z = 842.5099 and 2211.1046). The processing and peak picking of the spectra were automatically carried out using Flex analysis software v3.0. Threshold used for peak-picking was as follows: 500 for minimum resolution of monoisotopic mass and 5 for S/N. The MS/MS database search was conducted in NCBInr and Swiss-databases using the online MS/MS ion search software, Mascot (http://www.matrixscience. com/) with subsequent parameters: trypsin as the cleaving enzyme, a maximum of one missed cleavage, iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of ±0.1 Da. PMF acceptance criteria is probability scoring. Cytotoxicity assay. The cytotoxicity assay was performed as described in a previous report 46

Screening of oxindole derivatives as anti-S. aureus agents. The newly synthesized heterocyclic
indolin-2-ones were next evaluated for their antibacterial activity against S. aureus by using the disc diffusion method. The anti-S. aureus activity for the 20 individual compounds (KS1 to KS20) evaluated showed that 14 chemicals produced a clear zone of inhibition against S. aureus (Figs 4, 5 and Table 1). Among them, five compounds (KS15, KS16, KS17, KS19, and KS20) that produced a clear zone of >10 mm were selected as the target compounds (Figs 4, 5A and Table 1). To demonstrate the enhanced antibacterial activity of the synthesized compounds, we have evaluated the discrete antibacterial activity of the starting materials (1-methylpyrrole, oxindole, or thiophene) used for the synthesis of the active compounds under equal amounts (20 μg). However, none of these substrates resulted in any clear zone of inhibition against S. aureus at 20 μg ( Fig. 5B and Table 1). To compare the effectiveness of the target compounds to that of the nuclear compounds, different amounts (between 20 and 1,000 μg) of 1-methylpyrrole, oxindole, and thiophene were evaluated for the production of a clear zone. We found that only oxindole at 1,000 μg produced a turbid zone (Fig. 5C), whereas other compounds did not exhibit such inhibitory activity (Fig. 5D,E). These results indicated that the newly synthesized chemical compounds were at least > 50-fold more active than the starting materials. Moreover, the compounds showed the comparable activity to streptomycin, while slightly less active to chloramphenicol, erythromycin, and methicillin (Table 1 & Fig. 5F). From a structural perspective (Fig. 3), five active compounds (KS15, KS16, KS17, KS19, and KS20) have common structural features: C-4/C-5-halogenated indolin-2-ones bearing 5-membered heterocycles, 1-methylpyrrol-2-yl or thiophen-2-yl group, at the 3-position. Meanwhile, 3-indolylindolin-2-ones KS1-KS12 were relatively inactive, which indicated that the nature of the functionalization at 3-position was a key component in the regulation of function. Specifically, active KS15-KS17 were either brominated or chlorinated at the 4-or 5-position on the benzene ring of the indolin-2-one moiety, whereas inactive KS13-KS14 were either non-halogenated at 4-or 5-position or methoxy-substituted at 5-position. Hence, halogenation at the 5-position of the indolin-2-one scaffold appears to be an important factor of the activity. This structural effect on the enhanced antibacterial activity was further observed for compounds KS19 and KS20 bearing -Br or -Cl group at the 5-position.
In general, the SAR results indicated that heterocyclic indolin-2-ones KS13-KS20 bearing 1-methylpyrrol-2-yl or thiophen-2-yl group at the 3-position on the indolin-2-one moiety and the halogenation at the 4-or 5-position on the benzene ring of the oxindole moiety significantly influenced their anti-S. aureus activities.
Determination of MIC of compounds against S. aureus. We further evaluated the anti-S. aureus activity of the target compounds by determining the MIC for different concentrations of KS15, KS16, KS17, KS19, and KS20 in comparison to chloramphenicol, methicillin, and streptomycin as control antibiotics. We found that the MIC values for KS15, KS16, KS17, KS19, and KS20 were 6. 38, 6.40, 8.72, 9.07, and 8.87 μg/mL, respectively (Fig. 6A,B), whereas the MIC value for chloramphenicol, methicillin, and streptomycin, was 4.60, 4.07, and 1.73 μg/mL, respectively. Owing to the lower MIC value of KS15 and KS16 compared with KS17, the position www.nature.com/scientificreports www.nature.com/scientificreports/ of the halogen at the 4-position of oxindoles with 1-methylpyrrole or thiophene at 3-position appeared to be the most important moieties for enhancing anti-S. aureus activity. Moreover, our newly synthesized compounds were comparable with commercial antibiotics, which suggested that our compounds are potentially good candidate for antibiotics.

Specificity of compounds against Staphylococcus species.
We speculated whether the active compounds selectively inhibited the growth of S. aureus. To answer this question, we determined the MIC of the compounds against subspecies of Staphylococcus such as S. epidermidis (ATCC 12228) and S. saprophyticus (ATCC 15305) and compared them with that of chloramphenicol, erythromycin, methicillin, and streptomycin. From the data, we found that all five compounds (KS15, KS16, KS17, KS19, and KS20) were active against tested species (Fig. 7A,B) with equivalent or higher efficacy with S. epidermidis and S. saprophyticus, respectively, than S. aureus (Figs 6 and 7A,B). Meanwhile, commercial antibiotics showed different activity among species. For example, chloramphenicol and methicillin, were found to be active against both S. saprophyticus and S. epidermidis, while streptomycin was inactive against S. epidermidis (MIC > 20 μg/mL) (Fig. 7A). Therefore, the experimental results suggest that the heterocyclic oxindole derivatives express a broader and specific range of antibacterial activity against members of the common colonization species of S. aureus than the control antibiotic, streptomycin.
We further tested whether new compounds are effective against two Gram-negative bacteria. First, E. coli strain (ATCC 25922) was used to examine the effect of compounds on the inhibition of bacterial growth. As shown in Fig. 7C (left), all compounds were inactive (MIC > 20 μg/mL) as methicillin (MIC > 100 μg/mL) against E. coli cells, while chloramphenicol and streptomycin showed inhibitory effects. Second, we measured MIC values against P. aeruginosa, a common interactor with S. aureus in chronic wounds and other types of infections [47][48][49][50][51][52][53] . As shown in Fig. 7C (right), the MIC values of all compounds against P. aeruginosa (ATCC 27853) were >20 μg/ mL, as seen for E. coli, whereas streptomycin showed inhibitory effects only at 20 μg/mL, but this concentration was regarded as non-effective considering that <10 μg/mL of compounds was effective against Staphylococcus species (Figs 6B and 7B). Moreover, chloramphenicol and methicillin showed almost no activity to P. aeruginosa (MIC > 100 μg/mL; Fig. 7C, right). All the above data showed that compounds (KS15, KS16, KS17, KS19, and KS20) are specific to Staphylococcus species and indicated that such compounds could be utilized for the eradication of Staphylococcus species as narrow spectrum antibiotics in infections co-colonized with P. aeruginosa or other Gram-negative bacteria.
Activity of compounds against MRSA strains. We expected that the active compounds would be also effective for the inhibition of clinically-isolated MRSA strains. MRSA strains to be tested were confirmed by PCR for the amplification of the mecA gene 41 , a defining standard in the determination of the resistance of S. aureus to methicillin including MIC determination. The data showed that all six MRSA strains tested were much less sensitive than MSSA against oxacillin (Fig. 8A) and produced a mecA PCR product of 533 bp in size (Fig. 8B). Furthermore, we have determined the MIC for five active compounds (KS15, KS16, KS17, KS19, and KS20) against MRSA strains. The results showed that MIC values for the compounds against all MRSA strains were between 4.5 and 9.7 μg/mL (Fig. 8C,D), which were similar or slightly lower than that of ATCC 25923 (MSSA). Interestingly, KS15 and KS16 showed stronger activity against MRSA than against MSSA, which suggested that KS15 and KS16 might be potential compounds for the eradication of MRSA strains which may have been due to the unique functionalization in comparison to other evaluated compounds. The results can provide further insights on how to alter the antibacterial activity of the heterocyclic-bearing oxindole derivatives with specific functionalization at different positions. Therefore, our compounds may be alternative antibiotics for the specific eradication of both methicillin-susceptible and methicillin-resistant Staphylococcus species.
Synergistic action of KS16 with aminoglycosides antibiotics. The five compounds (KS15, KS16, KS17, KS19, and KS20) showed comparable activity to S. aureus species with a commercial antibiotic, streptomycin ( Fig. 4 & Table 1). Moreover, above compounds showed specific activity to Staphylococcus species unlike streptomycin (Figs 6 and 7). This indicates that newly synthesized compounds could directly use as specific anti-S. aureus agents. To understand their plausible mechanisms, we approached to identify synergistic antibiotics that is increased their activities by new active compounds assuming that the antibiotics and compounds inhibit the same pathway for the killing of S. aureus. To this end, we selected KS16, one of the most active compounds in this study, and evaluated its combinatorial action with existing antibiotics planted in a pre-made Sensititre Gram-positive MIC plate (Fig. 9A) by determining MIC values of with or without 2.5 μg/mL of KS16, the concentration at which no killing of S. aureus was found (Fig. 6). Results showed that the addition of KS16 to the antibiotics (ciprofloxacin, gentamicin, kanamycin, and streptomycin) increased the susceptibility to S. aureus compared with the control sample (Fig. 9B,C; Table 2). Addition to antibiotics seeded on the pre-made Sensititre Plate, we separately evaluated the effect of KS16 to the susceptibility to methicillin, an ineffective antibiotic to MRSAs. Neither the increase in MIC values of methicillin itself (2.0 μg/mL) nor the synergistic action by KS16 was detected (Fig. 9D). Therefore, KS16 may not expand the arsenal of therapeutic agents for eradicating MRSAs. Interestingly, all selected antibiotics, except ciprofloxacin, a fluoroquinolone class broad-spectrum antibiotic, are known to work as protein synthesis inhibitors targeting the bacterial ribosome. Among them, the gentamicin and kanamycin susceptibility to S. aureus was increased to more than 4-fold. To evaluate the effect of potency of the combination of KS16 and gentamicin or kanamycin in comparison to their individual activities, synergy testing by checkerboard assay was performed and found that combinations of KS16 with gentamicin or kanamycin expressed FICI of 0.5 (Fig. 9D), an expected value as synergistic drug combination.
www.nature.com/scientificreports www.nature.com/scientificreports/ Increased cellular level of autolysin by KS16. Checkerboard assays indicate that KS16 is a compound for the synergistic combination with gentamicin and kanamycin by sharing the part of cellular targets or pathways for the antibacterial action by gentamicin and kanamycin. Second possibility is that KS16 increases the permeability for the entry into ribosome targeting antibiotics as reported on the combination of gentamicin and vancomycin although the exact mechanism of synergistic combination has not been verified 54 . In opposite to broad-spectrum activity of gentamicin and kanamycin to multiple species 55 , KS16 may target gene product(s) only presented within S. aureus to function as both antibacterial and synergistic agent with high specificity.
Because KS16 showed the synergy to antibiotics modulating protein synthesis (Fig. 9) we assumed that the protein composition in S. aureus cells is changed by KS16. To verify this hypothesis, total proteins from S. aureus cultures treated with KS16 of sublethal concentrations (0 to 5 μg/mL) were compared to that of control sample. Interestingly, we found that a protein band migrating ~150 kDa (band A) on SDS-PAGE gel was increased to 4.72-fold by KS16 with concentration-dependent manner (Fig. 10A). To characterize the protein MALDI-TOF/peptide mass fingerprinting (PMF) was further performed (Fig. 10B). By Mascot and Swiss-Prot analysis of trypsin-treated fragments the protein from band A was denoted as mannosyl-glycoprotein endo-β-N-acetylglucosamidase (EC 3.2.1.96), also known as autolysin (Atl) by sequence similarity (Fig. 10C). The Atl is a bifunctional protein originally produced as a 138-kDa precursor form and processed to generate mature 62-kDa amidase (AM) and 51-kDa glucosamidase (GM) domains by sequencially [56][57][58] .
In functional aspects, Atl is known as a peptidoglycan hydrolase involved in bacterial cell wall degradation through trimming of peptidoglycan and the separation of the daughter cells after division 59,60 . Moreover, the protein is involved in the initial perforation of the cell wall during the autolysis of penicillin-treated S. aureus 61 . Therefore, our results suggest that KS16 seems to function as an anti-S. aureus by activating the degradation of peptidoglycan layers of target cells and/or perforation of the cell wall. However, future studies are required to understand how KS16 showed the synergistic activity to antibiotics related to protein synthesis and how KS16 selectively affected to the viability of Staphylococcus species. For this purpose, profiling analyses of both total RNAs and proteins by high-throughput techniques such as RNA-sequencing and proteomics are required. Such studies will broaden the usage of heterocyclic oxindole compounds as new lead molecules with greater activity and specificity for the eradication of Staphylococcus species.

Cytotoxicity of KS16.
For the evaluation of cytotoxicity of KS16, human skin fibroblast cell (CRL 2097) was selected in the aspect that one of colonization sites for S. aureus is skin tissues 62 . The cytotoxicity of KS16 with a range of increasing concentrations (0 to 20 μg/mL) after 24 and 48 h of incubation was determined using the CCK-8 assay. Data showed that KS16 at MIC level (4.5 to 6.4 μg/mL) against Staphylococcus species including MRSAs did not exhibit any cytotoxicity (Fig. 11). Moreover, even at 20 μg/mL, which is 3.1-to 4.4-fold of MIC value did not exhibit significant cytotoxicity. This indicates that KS16 could lead to possible non-toxic anti-MRSA application for skin infection.

Conclusion
In summary, we have designed and synthesized diverse oxindoles bearing 3-heterocycles by indium (III)-catalysed reaction of 3-diazoindolin-2-ones and indoles, 1-methylpyrrole, or thiophene. Among the synthesized compounds KS15, KS16, KS17, KS19, and KS20 were the most active compounds with high specificity against various Staphylococcus species including MRSA with equivalent or superior activity to streptomycin. The study on synergistic activity of KS16 with existing antibiotics indicates that KS16 increased the susceptibility of S. aureus to ciprofloxacin, gentamicin, kanamycin, and streptomycin. Among them, gentamicin and kanamycin exhibited a synergistic combination with KS16 to S. aureus. Moreover, the KS16 increased the cellular level of autolysin (Atl) protein and expressed the antibacterial activity. Finally, the treatment of KS16 to skin fibroblast cells at the MIC levels used for killing Staphylococcus species was non-toxic. All our findings suggest that our newly developed heterocyclic oxindoles are valuable lead antibacterial agents in the eradication of multidrug-resistant Staphylococcus species.