The agar diffusion assay is one method for quantifying the ability of antibiotics to inhibit bacterial growth. Interpretation of results from this assay relies on model-dependent analysis, which is based on the assumption that antibiotics diffuse freely in the solid nutrient medium. In many cases, this assumption may be incorrect, which leads to significant deviations of the predicted behaviour from the experiment and to inaccurate assessment of bacterial susceptibility to antibiotics. We sought a theoretical description of the agar diffusion assay that takes into consideration loss of antibiotic during diffusion and provides higher accuracy of the MIC determined from the assay. We propose a new theoretical framework for analysis of agar diffusion assays. MIC was determined by this technique for a number of antibiotics and analysis was carried out using both the existing free diffusion and the new dissipative diffusion models. A theory for analysis of antibiotic diffusion in solid media is described, in which we consider possible interactions of the test antibiotic with the solid medium or partial antibiotic inactivation during diffusion. This is particularly relevant to the analysis of diffusion of hydrophobic or amphipathic compounds. The model is based on a generalized diffusion equation, which includes the existing theory as a special case and contains an additional, dissipative term. Analysis of agar diffusion experiments using the new model allows significantly more accurate interpretation of experimental results and determination of MICs. The model has more general validity and is applicable to analysis of other dissipative processes, for example to antigen diffusion and to calculations of substrate load in affinity purification.

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1–3)-β-Glcp-(1–5)-β-Galf-(1–6)-α-Glcp-(1–4)-β-Galp-(1–4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

The increasing resistance of human pathogens to conventional antibiotics presents a growing threat to the chemotherapeutic management of infectious diseases. The lanthionine antibiotics, still unused as therapeutic agents, have recently attracted significant scientific interest as models for targeting and management of bacterial infections. We investigated the action of one member of this class, subtilin, which permeabilizes lipid membranes in a lipid II-dependent manner and binds bactoprenyl pyrophosphate, akin to nisin. The role the C and N termini play in target recognition was investigated in vivo and in vitro by using the natural N-terminally succinylated subtilin as well as enzymatically truncated subtilin variants. Fluorescence dequenching experiments show that subtilin induces leakage in membranes in a lipid II-dependent manner and that N-succinylated subtilin is roughly 75-fold less active. Solid-state nuclear magnetic resonance was used to show that subtilin forms complexes with membrane isoprenyl pyrophosphates. Activity assays in vivo show that the N terminus of subtilin plays a critical role in its activity. Succinylation of the N terminus resulted in a 20-fold decrease in its activity, whereas deletion of N-terminal Trp abolished activity altogether.

Nisin is a small cationic lanthionine antibiotic produced byLactococcus lactis. During its antimicrobial action, it targets intermediates in the bacterial cell-wall biosynthesis, lipid II, and undecaprenyl pyrophosphate. Here, we report results from electron microscopic investigations of the effects of lethal nisin doses onBacillus subtiliscell morphology. Bacterial membranes were permeabilized shortly afterB. subtiliswas incubated with nisin, but this did not lead to immediate cell death. Cell division, as well as other life functions, persisted over at least half an hour after nisin was added. Slower bacterial elongation, consistent with cell envelope inhibition and accelerated division, resulted in cell-length reduction. Abnormal morphogenesis near the division site suggests this to be the primary site of nisin action. Morphological changes are characteristic of deregulation of a filamentous cell envelope protein, Mbl, and the division-inhibiting Min system. We propose a previously undescribed model, in which the lethal action of nisin againstB. subtilisstarts with membrane permeabilization and is followed by accelerated cell division, cell envelope inhibition, and aberrant cell morphogenesis.

The spread of infectious diseases and the increase in antibiotic resistance represent a life‐threatening global development that calls for new approaches to control microorganisms. Of all potential targets, the essential and unique pathway of bacterial cell wall synthesis, targeted by the first known antibiotic penicillin, remains a perfect candidate for the development of new antibiotics. Here we show that the lantibi‐otic nisin exercises its antibacterial action by targeting peptidoglycan intermediates’ extracellular pyrophosphate, unique to bacterial cell wall precursors. We show that nisin sequesters cell wall precursors found in the outer leaflet of bacterial plasma membranes, Lipid II and undecaprenyl pyrophosphate, into stable complexes. We propose a model of antibacterial action for nisin in which the terminal amino group of Ile1 targets the pyrophosphate groups of the bacterial cell wall precursors, where it docks via a hydrogen bond. The pyrophosphate moiety, a highly conserved chemical group different from the L‐Lys‐D‐Ala‐D‐Ala docking motif for vancomycin, has no biochemical analogs with comparable properties and is unlikely to be susceptible to bacterial adaptations akin to those responsible for resistance to penicillins and vancomycin.—Bonev, B. B., Breukink, E., Swiezewska, E., de Kruijff, B., Watts, A. Targeting extracellular pyrophosphates underpins the high selectivity of nisin. FASEB J. 18, 1862‐1869 (2004)