The antimicrobial effects of the alginate oligomer OligoG CF-5/20 are independent of direct bacterial cell membrane disruption

Concerns about acquisition of antibiotic resistance have led to increasing demand for new antimicrobial therapies. OligoG CF-5/20 is an alginate oligosaccharide previously shown to have antimicrobial and antibiotic potentiating activity. We investigated the structural modification of the bacterial cell wall by OligoG CF-5/20 and its effect on membrane permeability. Binding of OligoG CF-5/20 to the bacterial cell surface was demonstrated in Gram-negative bacteria. Permeability assays revealed that OligoG CF-5/20 had virtually no membrane-perturbing effects. Lipopolysaccharide (LPS) surface charge and aggregation were unaltered in the presence of OligoG CF-5/20. Small angle neutron scattering and circular dichroism spectroscopy showed no substantial change to the structure of LPS in the presence of OligoG CF-5/20, however, isothermal titration calorimetry demonstrated a weak calcium-mediated interaction. Metabolomic analysis confirmed no change in cellular metabolic response to a range of osmolytes when treated with OligoG CF-5/20. This data shows that, although weak interactions occur between LPS and OligoG CF-5/20 in the presence of calcium, the antimicrobial effects of OligoG CF-5/20 are not related to the induction of structural alterations in the LPS or cell permeability. These results suggest a novel mechanism of action that may avoid the common route in acquisition of resistance via LPS structural modification

Reaching the Hard-to-Reach: A Probability Sampling Method for Assessing Prevalence of Driving under the Influence after Drinking in Alcohol Outlets

Drinking alcoholic beverages in places such as bars and clubs may be associated with harmful consequences such as violence and impaired driving. However, methods for obtaining probabilistic samples of drivers who drink at these places remain a challenge – since there is no a priori information on this mobile population – and must be continually improved. This paper describes the procedures adopted in the selection of a population-based sample of drivers who drank at alcohol selling outlets in Porto Alegre, Brazil, which we used to estimate the prevalence of intention to drive under the influence of alcohol. The sampling strategy comprises a stratified three-stage cluster sampling: 1) census enumeration areas (CEA) were stratified by alcohol outlets (AO) density and sampled with probability proportional to the number of AOs in each CEA; 2) combinations of outlets and shifts (COS) were stratified by prevalence of alcohol-related traffic crashes and sampled with probability proportional to their squared duration in hours; and, 3) drivers who drank at the selected COS were stratified by their intention to drive and sampled using inverse sampling. Sample weights were calibrated using a post-stratification estimator. 3,118 individuals were approached and 683 drivers interviewed, leading to an estimate that 56.3% (SE = 3,5%) of the drivers intended to drive after drinking in less than one hour after the interview. Prevalence was also estimated by sex and broad age groups. The combined use of stratification and inverse sampling enabled a good trade-off between resource and time allocation, while preserving the ability to generalize the findings. The current strategy can be viewed as a step forward in the efforts to improve surveys and estimation for hard-to-reach, mobile populations

Nanoscale visualization and characterization of Myxococcus xanthus cells with atomic force microscopy

Multicellular microbial communities are the predominant form of existence for microorganisms in nature. As one of the most primitive social organisms, Myxococcus xanthus has been an ideal model bacterium for studying intercellular interaction and multicellular organization. Through previous genetic and EM studies, various extracellular appendages and matrix components have been found to be involved in the social behavior of M. xanthus, but none of them was directly visualized and analyzed under native conditions. Here, we used atomic force microscopy (AFM) imaging and in vivo force spectroscopy to characterize these cellular structures under native conditions. AFM imaging revealed morphological details on the extracellular ultrastructures at an unprecedented resolution, and in vivo force spectroscopy of live cells in fluid allowed us to nanomechanically characterize extracellular polymeric substances. The findings provide the basis for AFM as a useful tool for investigating microbial-surface ultrastructures and nanomechanical properties under native conditions

Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy

During the past decades, several methods (e.g., electron microscopy, flow chamber experiments, surface chemical analysis, surface charge and surface hydrophobicity measurements) have been developed to investigate the mechanisms controlling the adhesion of microbial cells to other cells and to various other substrates. However, none of the traditional approaches are capable of looking at adhesion forces at the single-cell level. In recent years, atomic force microscopy (AFM) has been instrumental in measuring the forces driving microbial adhesion on a single-cell basis. The method, known as single-cell force spectroscopy (SCSCFS), consists of immobilizing a single living cell on an AFM cantilever and measuring the interaction forces between the cellular probe and a solid substrate or another cell. Here we present SCSCFS protocols that we have developed for quantifying the cell adhesion forces of medically important microbes. Although we focus mainly on the probiotic bacterium Lactobacillus plantarum, we also show that our procedures are applicable to pathogens, such as the bacterium Staphylococcus epidermidis and the yeast Candida albicans. For well-trained microscopists, the entire protocol can be mastered in 1 week