The emergence of acquired high-level resistance among Enterococcus species has renewed interest in mechanisms of resistance to glycopeptide antibiotics in gram-positive bacteria. In Enterococcus faecalis and Enterococcus faecium, resistance is encoded by the van gene cluster and is due to the production of a peptidoglycan precursor terminating in D-alanyl-D-lactate, to which vancomycin does not bind. MostLeuconostoc and many Lactobacillus species are intrinsically resistant to high levels of glycopeptide antibiotics, but the mechanism of resistance has not been elucidated. To determine whether the mechanisms of resistance are similar in intrinsically resistant bacteria, cytoplasmic pepti4oglycan precursors were isolated from Leuconostoc mesenteroides and Lactobacilus casei and analyzed by mass spectrometry, revealing structuresGlycopeptide antibiotics such as vancomycin act by binding to the D-alanyl-D-alanine terminus of stem pentapeptides present in bacterial peptidoglycan (16,21). It had been widely assumed that this terminus was ubiquitous among eubacterial species producing peptidoglycan and that vancomycin resistance was therefore unlikely to emerge in the absence of a barrier to vancomycin binding such as the outer membrane of gram-negative bacteria. However, high-level transmissible resistance to glycopeptides has recently been recognized among several gram-positive species, particularly enterococci, in which the incidence of resistance has been rising among clinical isolates (2).Most Leuconostoc and Pediococcus and some Lactobacillus species have been described as being intrinsically resistant to glycopeptides (9,15,17,20), but the mechanism of resistance has not been systematically investigated, perhaps because these organisms were considered to be of little clinical significance. However, the widespread use of vancomycin has led to more frequent recognition of these species as opportunistic pathogens (9,20 depsipeptide (D-alanyl-D-hydroxy acid) which is added in place of D-alanyl-D-alanine to the UDP-muramyl-tripeptide precursor, resulting in a peptidoglycan precursor that is not bound by vancomycin (6). This hypothesis was supported by the observation that the MIC for resistant enterococci was diminished when tested with high concentrations of D-amino acids that can be incorporated into pentapeptide and that are bound by vancomycin to a greater extent than D-2-hydroxy acids (23). Subsequently, the cytoplasmic peptidoglycan precursor in resistant E. faecium and E. faecalis was identified as UDP-

Vancomycin resistance plasmids in enterococci carry the genes vanH and vanA, which encode enzymes catalyzing, respectively, the reduction of 2-keto acids to 2-D-hydroxy acids and the addition of D-hydroxy acids to D-alanine. It has therefore been postulated that resistant cells produce peptidoglycan precursors that terminate in the depsipeptide D-alanine-2-D-hydroxy acid rather than the dipeptide D-alanine-D-alanine, thus preventing vancomycin binding (M. Arthur, C. Molinas, T.

The electrochemical oxidation pathway of uric acid was determined by on-line electrochemistry/thermospray/tandem mass spectrometry. Intermediates and products formed as a result of electrooxidation were monitored as the electrode potential was varied. Several reaction intermediates have been identified and characterized by tandem mass spectrometry. The tandem mass spectrometric results provide convincing evidence that the primary intermediate produced during the electrooxidation of uric acid has a quinonoid diimine structure. The results indicate that once formed via electrooxidation, the primary intermediate can follow three distinct reaction pathways to produce the identified final products. The final electrochemical oxidation products observed in these studies were urea, CO2, alloxan, alloxan monohydrate, allantoin, 5-hydroxyhydantoin-5-carboxamide, and parabanic acid. The solution reactions that follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe. In particular, it was found that at different tip temperatures either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur. Most importantly, the results show that the on-line combination of electrochemistry with thermospray/tandem mass spectrometry provides otherwise difficult to obtain information about redox and associated chemical reactions of biological molecules such as the structure of reaction intermediates and products, as well as providing insight into reaction pathways.

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