Muropeptide signature of inhibitors of protein synthesis correlates with ß-lactam synergism against methicillin-resistant Staphylococcus aureus.

Quinupristin/dalfopristin (Q/D) and ß-lactams interact positively against methicillin-resistant Staphylococcus aureus (MRSA). The effect extends to other inhibitors of protein synthesis, but not to inhibitors of polynucleotide synthesis or assembly, or to Q/D plus non-ß-lactam cell wall inhibitors. Moreover, electron microscopy studies have correlated this effect with a thickened cell wall. In this study, we sought to determine whether inhibitors of protein synthesis might produce a specific peptidoglycan muropeptide signature that would correlate with their positive ß-lactam interaction. The muropeptides of six S. aureus isolates (three methicillin-susceptible and three MRSA) were analysed using high-performance liquid chromatography and mass spectrometry. Exposure to 0.25× the minimum inhibitory concentration of inhibitors of protein synthesis consistently produced three main alterations irrespective of methicillin resistance: (i) an increase in peak 12 (a cyclic dimer of glycine-containing disaccharide-tetrapeptide); (ii) an increase in poorly resolved late-eluting materials; and (iii) a decrease in peak 1 (a disaccharide-pentapeptide). Eventually, the rate of autolysis was also decreased, supporting the structural alteration of the peptidoglycan. Other drug classes did not produce these anomalies. An increase in peak 12 was also observed in staphylococci treated with fosfomycin, which decreases expression of the native penicillin-binding protein (PBP) 2 and 4. Parallel blockage of normal PBPs with ß-lactams abolished the anomalies, indicating that they resulted from altered function of native PBPs. This underlines the potential of inhibiting both protein synthesis and transpeptidation simultaneously and suggests that such a drug combination strategy might be efficaciously exploited.

Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin.

The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4'R, 2''R, 4''R (pyochelin I) and 4'R, 2''S, 4''R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.

The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus.

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.