Evaluation Of In Vitro And In Vivo Antimicrobial Activity Of Pheromonicin-SA

Staphylococcus aureus infections are a major cause of morbidity and mortality in community and hospital populations. The emergence of routine antibiotic-resistant strains of S. aureus have been representing an enormous threat to public health. Therefore, there is an urgent demand of identify new types of antibacterial agents.Colicins are bacteriocins secreted by E.coli. They have several modes of lethal action, including inhibition of protein and DNA synthesis and cellular deenergization. Colicin la, a 626-amino-acid protein, belongs to ion-channel forming colicins and kills E.coli and related cells by deenergization mode. However, colicin la can not be directly used as new antibiotic because it only recognizes receptors in the outer and inner membranes of E.coli and related bacteria.Therefore, if we want to reform colicin la to be a new antibacterial agent, an "inducer" must be finded to taget it to other types of bacteria. Pheromons are chemical signals secreted by bacteria to communicate with the other bacteria of the same species in order to synchronize their behavior. It has the ability of autoinducing to find the receptors in the membrane. For instance,agrD, the pheromone of Staphylococcos aureus , can be induced to near the cell and specificly bind to the receptor-histidine kinase (agrC) in the cell membrane, trigger a standard two-component signal-transduction pathway and regulate the expression of virulence factors and surface proteins . By using agrD as a potential candidate of colicin la' "inducer", Qiu et al constructed the fusion protein pheromonicin-SA by linking the C-terminal of colicin la with the eight-amino-acid pheromone, agrDl. According to Qiu's study, ph-SA presented relatively strong antimicrobial activity against MR.SA(methicillin-resistant staphylococcus aureus) and MSSA( methicillin-sensitive staphylococcus aureus). At this present study, the antimicrobial activity of Ph-SA against MRSA and some other bacteria were evaluated in vitro and in vivo.Methods: To test the minimal inhibitory concentration of ph-SA against 69 strains pathogens by using agar disk dilution assay proposed by the National Committee for Clinical Laboratory Standards (NCCLS) and compare the antimicrobial activity with that of cefazolin, oxacillin, vancomycin and other antibacterial agents. And in vivo activity of Ph-SA was tested via intraperitoneal infected mice with intravenous drug treatment.Results: In vitro data showed the MICS50 (minimal inhibitory concentration of fifty-percent bacteria) of ph-SA against MRSA, MSSA , MESE( methicillin- resistant staphylococcus epidermidis), MSSE {methicillin-sensitive staphylococcus epidermidis), were 8, 0.5, 2, lmg/L respectively. And the MICS50 against E.coli, Pseudomonas aeruginosa, Enterobacter cloacae, Acinetobacter barmannii were 16, 32, 64, 16mg/L respectively. By comparison with the results of vacomycin, the bactericidalactivity of Ph-SA against MRSA was approximately 12 times greater, on a molar basis, than that of vancomycin.At present in vivo study, all mice in the Oxacillin (9.75mg/kg) and untreated groups died in less than 3d. Seventy percent of Vancomycin (5.20mg/kg) -treated mice died the 8-d experimental period , while only twenty percent of Ph-SA (4.23mg/kg)-treated mice died in this period. Therefore, according to the same evaluation procedure as we done in in vitro study, the bactericidal activity of Ph-SA was approximately 59 times greater than that of vancomycin.Implication of results: Ph-SA presented much stronger bactericidal activity for MRSA ,on a molar basis, than that of any other routine antibiotics used in present in vitro and in vivo studis., and may be of value as new antibiotic against MRSA.