An Experimental Study On The Release Of Endotoxin From Gram Negative Bacteria Induced By Different Antibiotics

Objective: To study the release of LPS from Gram-negative bacteria (GNB) exposed to varying concentrations of antibiotics, and the relationship between LPS release and morphological changes of bacteria.Methods : Selected the common clinical GNB infection of E.coli and Acinetobacter baumannii, bacteria were exposed to cefoperazone sulbactam (CPZ/SBT), levofloxacin(LVFX), and amikacin(AMK) at 0.5MIC, 2MIC, 5MIC and 20MIC. Samples(1ml)were obtained at 0h, 2h, 4h, 6h, 8h after bacteria exposing to antibiotics. LPS release was measured by Tachypleus amebocyte lysate Test( TAL Test ). The culture without antibiotic acted as a control, broth without drug and bacteria was a blank tube. Collected 0.5MIC culture medium at different times, centrifuged to take sediment, Gram stained and the morphology of bacteria was evaluated by light microscope. Acinetobacter baumannii isolated on Mueller-Hinton agar were exposed to antibiotics for 8h and their morphology was examined by scanning electron microscope.Results:1 Different types antibiotics effect on bacteria LPSWhen Acinetobacter baumannii were treated with three types of antibiotics at 0.5MIC, LPS release is: LVFX> CPZ /SBT> AMK; at 2MIC, 5MIC, 20MIC, LPS release is: CPZ / SBT> LVFX> AMK.2 Different concentrations of antibiotics effect on bacteria LPSWhen bacteria were treated with CPZ / SBT and LVFX, at 0.5MIC induced more LPS release than those at 5MIC and 20MIC.When Acinetobacter baumannii were treated with AMK, at 0.5MIC medium LPS release were induced; When E.coli were treated with AMK, low LPS release were induced. 3 Incubation time of antibiotics effect on bacteria LPSWith the prolongation of incubation time, antibiotics induced the bacteria LPS release gradually increased.4 Different strains effect on bacteria LPSControl group: With the prolongation of incubation time, both bacteria LPS release gradually increased. At each time point (after 2h) E. coli released LPS higher than A. baumannii, there is a significant difference between E. coli and A. baumannii (P <0.05).Antibiotic group: LPS released from GNB exposed to varying concentrations of antibiotics were different.5 The morphological changes of 0.5MIC culture mediumAcinetobacter baumannii: In CPZ / SBT group, the morphology of bacteria had no changes at 6h under light microscope, however, the cell elongation, distortion and the middle of depression were seen at 8h under scanning electron microscope. In LVFX group, it changed into filamentous form at 6h under light microscope. In AMK group, either light microscope or scanning electron microscope morphological changes were not observed.E. coli: Both CPZ / SBT group and the LVFX group could observe changes in bacterial biomass of filamentous at 2h under the light microscope. AMK group at 2h it induced cell lysis.Conclusions:1 When three kinds of antibiotics acted on A.baumannii and E. coli , CPZ/SBT and LVFX induced more LPS release than AMK. Therefore, the clinical anti-infective therapy should be considered seriously, trying to find the best antibiotics which have more anti-bacterial effects and inducing less LPS release.2 CPZ/SBT, LVFX and AMK could induce A. baumannii and E. coli to release different levels of LPS. LPS released from bacteria was related to types of antibiotics, concentrations and time of incubation.3 CPZ/SBT, LVFX and AMK could induce A. baumannii and E. coli to have morphological changes. It maybe one of the mechanisms of which induced bacteria to release different levels of LPS.