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.

Microsatellite Analysis of Perch (Perca fluviatilis) and its Genetic Authentication of Geographical Localization.

European perch (Perca fluviatilis) is an economically important freshwater species in Europe. In Switzerland, where the demand largely exceeds the production coming from Swiss lakes, nearly 90% of the requirements come from importation with the majority of perch originating from Estonia and Russia. The price of perch fillet varies considerably depending on the origin. Therefore traceability in the fish food sector plays an increasingly important role for consumer protection. Currently the traceability of perch can be assessed through chemical isotopic analysis. The 180/160 isotopic abundance ratio is used as geographical traceability marker, but several aspects affect the accuracy of the method, i.e. the distinct geographical area ratio differs only very slightly with overlapping standard deviation, the need for a large amount of fish material requires the mix of many fillets, the impossibility of analyzing processed matrix, the comparison of the ratio with the ratio of a sample of the presumed originating water makes the analyses more complicated. New application of DNA markers for the traceability of food products plays an increasingly important role for consumer protection. Microsatellites, which are short tandemly repetitive DNA sequences, are genetic markers of choice for traceability because of their abundance and high polymorphism. Moreover, fluorescent labelling and capillary electrophoresis separation increase efficiency and precision of genotyping microsatellites. The method can also be efficiently applied in processed food products where other methods have limited applications. In this study, we tested the efficiency of three polymorphic microsatellites and their combinations for their ability to correctly assign or exclude 195 reference perch to their origin population. Using the maximum likelihood and Bayesian methods computed by the software GeneClass2, the three loci microsatellite were optimized and allowed the correct assignation of all but two Swiss perch (60/62) into Swiss population. The markers also exclude 132/133 imported fish from the Swiss population with a match probability of more than 95%. The number of markers required for correct assignation differs from species to species, and depends on many factors such as genetic diversity and population structure. For perch populations, the results showed that only three polymorphic microsatellite markers are required to perform a reliable attribution or exclusion of a perch to the Swiss population with more than 98% correct assignations.