Abstract

BackgroundAntibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity.ResultsCells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the β-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after β-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different β-lactams and vancomycin, confirming the applicability of the methodology.ConclusionThe technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis.

Highlights

  • Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice

  • A background of extracellular microgranular-fibrilar material released by the bacteria was observed with a density dependent on the efficacy of the antibiotic, being especially intense in susceptible strains exposed to relative high doses

  • We have adapted our methodology for assessment of susceptibility or resistance to quinolones [14,15,16] to develop a simple and rapid procedure to determine susceptibility or resistance to antibiotics that act at the cell wall

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Summary

Introduction

Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. Peptidoglycan or murein is an important component of bacterial cell wall. This forms an enormous network of interlinked chains of alternating subunits of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Short stem peptides that are attached to NAM are cross-linked to stem peptides from nearby muropeptide strands. There, the penicillin-binding proteins (PBPs) or DD-peptidases catalyze the formation of glycosidic linkages between two muropeptide units producing linear glycan chains and the formation of the peptide bonds between adjacent murein strands, i.e

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