Effects of Terminalia Citrina Roxb. alcoholic extract on extended spectrum beta-lactamase producing Escherichia Coli

Abstract

Extended spectrum beta-lactamase producing Escherichia coli is a problem in hospitalized patients worldwide and are increasingly associated with community acquired infection. The most frequent coresistances found in ESBL-producing organisms are broad spectrum beta-lactam, third generation cephalosporins, aminoglycosides and fluoroquinolones. Combination therapy is the other choice in the treatment of ESBLproducing E.coli infections with the aim of decreasing the emergence of resistance strains and increasing bacterial killing. Therefore, the purpose of the present study is to determine the susceptibility of alcoholic extract of T. citina ROXB, ampicillin and norfloxacin against 30 E.coli ( 29 ESBL-producing,1 non- ESBL-producing) strains. The MIC90 values were 10 mg/ml, >256 μg/ml and 64 μg/ml, respectively. All of 30 strains were beta-lactamase positive by Nitrocefin base test. The high ampicillin resistance were detected (MIC range 256- >256 μg/ml). There were 18 strains of ESBL –producing E.coli (60%), which were resistant to both ampicillin and norfloxacin. Checkerboard method served to determine the activity of extract in combination with ampcillin and norfloxacin. Combination of the extract with ampicillin showed indifference against 30 E.coli strains while norfloxacin plus the extract against 18 strains of ESBL –producing E.coli (resistance to both ampicillin and norfloxacin) showed synergistic effect in 11 strains (61.11%), partial synergistic effect in 6 strains (33.33%) and additive effect in 1 strain (5.55%). In the Time kill study using 18 ESBL-producing E.coli strains, extract alone (½ MIC, 1 MIC) showed bacteriostatic activity (90% killing), whereas norfloxacin alone (½ MIC) showed bactericidal activity (99.9% killing) in 2 strains at 8 hour of growth. However, the regrowth were observed in 14, 16 and 12 strains at 24 hour by the extract alone or norfloxacin alone. When the concentration of the extract was increased from ½ MIC to 1 MIC and combined with norfloxacin, the bactericidal rate was faster and the number of strains killed was increased. For example, in strain no. U16 had the bactericidal activity was seen at 2 hour instead of 4 hour when using the conbination of the extract 1 MIC plus norfloxacin. At 24 hour of growth, the bacteriostatic and bactericidal activity (90-99.9% killing ) were observed in only 4 strains [BA24=164.75 ±30.69 log CFU/ml·h] (1/2 MIC of the extract plus norfloxacin), while conbined 1 MIC of the extract plus norfloxacin showed the bacteriostatic and bactericidal activity (90-99.9% killing) against 16 strains [BA24=175.78±30.41 log CFU/ml·h]. The results obtained suggested that antibacterial activity of the combination extract plus norfloxacin were higher than the antibacterial activity of each drug. It is concluded that the combination of extract plus norfloxacin could be promising alternatives in the treatment of infections due to ESBL-producing E.coli that were resistant to ampicillin and norfloxacin.