Resistance of Spheroplasts and Whole Cells of Pseudomonas cepacia to Polymyxin B

Abstract

Sucrose-lysozyme spheroplasts were prepared from two strains ofPseudomonas cepacia and tested for susceptibility to polymyxin B and benzalkonium chloride. Spheroplasts were more susceptible than whole cells to benzalkonium chloride but not to polymyxin B. Disruption of the outer membrane layer was not by itself sufficient to render P. cepacia susceptible to polymyxin B. Pseuldomonas cepacia, originally described as a plant pathogen (2), has been implicated in nosocomial infections with increasing frequency (3,6 (EDTA) at a concentration of 15 mg, contained in 0.24 ml of a neutralized 6.25% (wt/vol) solution, and 300 ug of lysozyme (0.10 ml of a 0.3% solution) were added. Phase microscopy indicated the presence of 99.9 to 100% spheroplasts in all preparations. Control cells were washed with distilled water and resuspended in fresh medium prior to susceptibility testing. The susceptibility of cells and spheroplasts to PMB and BC was determined as follows. Samples (3 ml) of whole-cell or spheroplast suspensions were added to 7-ml portions of medium containing appropriate concentrations of the test compound. P. cepacia was tested in semidefined medium; E. coli was tested in nutrient broth. Assay tubes for Tris-EDTA-lysozyme spheroplasts were osmotically stabilized with 0.25 M sucrose. The number of viable whole cells and spheroplasts was determined by plate counts immediately after contact (0.5 min) with the antimicrobial agents and after 60 min of incubation at room temperature. Serial dilutions were made into tubes containing 0.01 M MgCl2, 0.22% lecithin, 1.55% polysorbate 80, and 0.25 M sucrose. Samples were plated on Trypticase soy agar with lecithin and polysorbate 80 (BBL) and incubated at 30°C (P. cepacia) or 37°C (E. coli) for 48 h. Controls were plated in the same manner immediately prior to the addition of the antimicrobial agents.

Characterization of Lipopolysaccharides of Polymyxin‐Resistant and Polymyxin‐Sensitive Klebsiella pneumoniae O3

“…Our results invite comparison to those published by Cox and Wilkinson [31] on the LPS of Pseudomonas cepacia. This species is known for its resistance to many antibacterial agents, including polymyxin [37]. In the analysis of P cepacia LPS, only two phosphates were found, yielding "P-NMR diester signals at -1.5 ppm and -2.0 ppm.…”

Mechanisms of Antimicrobial Peptide Action and Resistance

“…For example, in a variety of studies, Serratia, Proteus, and Providencia species often prove to be refractory to inhibition or killing by cationic peptides (Viljanen and Vaara, 1984). Burkholderia (formerly Pseudomonas) species also exhibit exceptionally broad resistance to antimicrobial peptides in vitro (Manniello et al, 1978). These examples illustrate the likelihood that certain microbial pathogens are inherently more resistant to antimicrobial peptides due to stable structural or functional properties or pathogenesis strategies.…”

Fosmidomycin Decreases Membrane Hopanoids and Potentiates the Effects of Colistin on Burkholderia multivorans Clinical Isolates

“…Polymyxin therapy is not considered an option for CF patients with Bcc infections because of the high constitutive polymyxin resistance of these bacteria. The intrinsic resistance of Bcc bacteria to polymyxins is multifaceted (12) but is due primarily to unique features of the cell membrane (12,13). Polymyxin B binds poorly to its lipopolysaccharide (LPS) target sites on intact Bcc cells (14).…”