Effect of Phosphate Limited Growth on Drug Resistance of Pseudomonas Aeruginosa

Abstract

The nutritional requirements of P. aeruginosa NCTC 6750 were defined using a chemically defined medium (C.D.M.) buffered with 3-N-morpholinopropane sulphonic acid (MOPS). Utilisation of MOPS as a source of sulphur by strain 6750 necessitated medium reformulation and the use of acid ammonium phosphate buffer when sulphate requirements were investigated. The C.D.M. was used to study the response to Polymyxin B (PB) and EDTA of batch and chemostat cultured cells grown in media containing graded amounts of phosphate. The response of batch grown cells to PB was dependent upon the medium phosphate content and the drug concentration used. Cells treated with 10 i.u. m1-1 PB showed increasing sensitivity as the medium phosphate concentration increased to a certain level above which the cells were apparently more resistant to the drug. Evidence suggested this resistance was in part due to cell envelope components released into the medium interacting with PB. Phosphate limitation of growth determined resistance. Phosphate or carbon limited chemostat grown cells showed increasing sensitivity to PB and EDTA as the dilution rate was increased. Phosphate limited chemostat cells, at D = 0.17 bro! were more sensitive to PB as the growth medium phosphate was increased, except when viable count was studied, when increasing resistance was seen at higher phosphate levels. Batch and chemostat_(D = 0.17 hr-1 ) grown cells showed increasing sensitivity to 785yg m1-1 EDTA as the growth medium phosphate content increased over the range tested. Inner and outer membrane preparations from batch and chemostat cells grown in various levels of phosphate were chemically analysed. Quantitative changes in outer membrane lipopolysaccharide, divalent cations and lipids and inner membrane lipids were detected. Phosphate depletion resulted in replacement of phospholipids by fatty acids and neutral lipids. Qualitative changes in outer membrane proteins were also noted. Some of these changes were possibly responsible for the altered sensitivity to PB and EDTA.