Intereactions between doripenem and clavulanate ­ Application of minimal inhibitory concentration analysis and cytometry flow for bactericidal studies

Background: In view of the current low efficacy of bacterial infection treatment the common trend towards searching for antibiotic systems exhibiting synergistic action is well justified. Among carbapenem analogues a particularly interesting option is provided by combinations of clavulanic acid with meropenem, which have proven to be especially effective. Results: Determination of the minimal inhibitory concentration (MIC) along with the method based on flow cytometry constitutes an important tool in the identification of bacterial sensitivity to active substances. Within this study the inhibitory effect of doripenem, clavulanic acid and the doripenem-clavulanate acid system was analyzed in relation to such bacteria as Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Clostridium butyricum and Clostridium pasteurianum, Acinetobacter baumannii, Enterobacter aerogenes. The lowest MIC, amounting to 0.03 µg/mL, was observed for the doripenem-clavulanate acid system in the case of E. coli ATCC 25922. In turn, the lowest MIC for doripenem applied alone was recorded for K. pneumoniae ATCC 31488, for which it was 0.1 µg/mL. The strain which proved to be most resistant both to doripenem and the doripenem-clavulanate acid system, was A. baumannii, with MIC of 32 µg/mL (clinical isolate) and 16 µg/mL (reference strain). Cytometric analysis for P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 showed changes in cells following exposure to limiting concentrations of the active substance. Conclusions: Analysis of MIC supplies important information concerning microbial sensitivity to active substances, mainly in terms of limiting concentrations causing mortality or vitality of the tested species, which is essential when selecting appropriate antibiotic therapy.

A Comparative Analysis of Monofunctional Biosynthetic Peptidoglycan Transglycosylase (MBPT) from Pathogenic and Non-pathogenic Bacteria

Monofunctional biosynthetic peptidoglycan transglycosylase (MBPT) catalyzes the formation of the glycan chain in bacterial cell walls from peptidoglycan subunits: N-acetylglucosamine (NAG) and acetylmuramic acid (NAM). Bifunctional glycosyltransferases such as the penicillin binding protein (PBP) have peptidoglycan glycosyltransferase (PGT) on their C terminal end which links together the peptidoglycan subunits while transpeptidase (TP) on the N terminal end cross-links the peptide moieties on the NAM monosaccharide of the peptide subunits to create the bacterial cell wall. The singular function of MBPT resembles the C terminal end of PBP as it too contains and utilizes a similar PGT domain. In this article we analyzed the infectious and non infectious protein sequences of MBPT from 31 different strains of bacteria using a variety of bioinformatic tools. Motif analysis, dot-plot comparison, and phylogenetic analysis identified a number of significant differences between infectious and non-infectious protein sequences. In this paper we have made an attempt to explain, analyze and discuss these differences from an evolutionary perspective. The results of our sequence analysis may open the door for utilizing MBPT as a new target to fight a variety of infectious bacteria.