title: Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus.
abstract: BACKGROUND
  Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin.
  
  
  RESULTS
  The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 μg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 μg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI) <0.5. The killing curve assay had confirmed that the viability of ARSA was dramatically reduced from 5 x 10(5) cfu/ml to 10(3) cfu/ml within 6 h after exposure to SSE (2 mg/ml) plus ampicillin (0.15 μg/ml) combination. Electron microscopic study clearly revealed that these ARSA cells treated with this combination caused marked morphological damage, peptidoglycan and cytoplasmic membrane damage, and average cell areas significant smaller than control. Obviously, Immunofluorescence staining and confocal microscopic images confirmed that the peptidoglycan of these cells were undoubtedly disrupted by this combination. Furthermore, the CM permeability of ARSA was also increased by this combination. Enzyme assay demonstrated that SSE had an inhibitory activity against β-lactamase in concentrations manner.
  
  
  CONCLUSIONS
  So, these findings provide evidence that SSE has the high potential to reverse bacterial resistance to originate traditional drug susceptibility of it and may relate to three modes of actions of SSE: (1) inhibits peptidoglycan synthesis, resulting in morphological damage, (2) inhibits β-lactamases activity, and (3) increases CM permeability. It is widely recognized that many types of drugs are derived from alkaloids. So, this SSE offers the prominent potential to develop a novel adjunct phytopharmaceutical to ampicillin for the treatment of ARSA. Further active ingredients study, toxicity of it, and the synergistic effect on blood and tissue should be performed and confirmed in an animal test or in humans.
authors.affiliation: School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 3000, Thailand. teethaisong.y@gmail.com.
authors.forename: Yothin
authors.initials: Y
authors.lastname: Teethaisong
authors.affiliation: School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 3000, Thailand. autarkool@hotmail.com.
authors.forename: Nongluk
authors.initials: N
authors.lastname: Autarkool
authors.affiliation: School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 3000, Thailand. kittipot_rs68@hotmail.com.
authors.forename: Kittipot
authors.initials: K
authors.lastname: Sirichaiwetchakoon
authors.affiliation: School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 3000, Thailand. pongrit@gmail.com.
authors.forename: Pongrit
authors.initials: P
authors.lastname: Krubphachaya
authors.affiliation: School of Physiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 3000, Thailand. sajeera@sut.ac.th.
authors.forename: Sajeera
authors.initials: S
authors.lastname: Kupittayanant
authors.affiliation: School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 3000, Thailand. griang@sut.ac.th.
authors.forename: Griangsak
authors.initials: G
authors.lastname: Eumkeb
mesh_terms: D000667:Ampicillin / Q000494:pharmacology*; D000668:Ampicillin Resistance; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D004338:Drug Combinations; D004357:Drug Synergism; D057075:Enzyme Assays; D008826:Microbial Sensitivity Tests; D046529:Microscopy, Electron, Transmission; D010936:Plant Extracts / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D031607:Stephania / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D004338:Drug Combinations; D010936:Plant Extracts; D000667:Ampicillin