Transmission Electron Microscopic Morphological Study and Flow Cytometric Viability Assessment ofAcinetobacter baumanniiSusceptible toMusca domesticacecropin

Multidrug-resistant (MDR)Acinetobacter baumanniiinfections are difficult to treat owing to the extremely limited armamentarium. Expectations about antimicrobial peptides' use as new powerful antibacterial agents have been raised on the basis of their unique mechanism of action.Musca domesticacecropin (Mdc), a novel antimicrobial peptide from the larvae of Housefly (Musca domestica), has potently active against Gram-positive and Gram-negative bacteria standard strain. Here we evaluated the antibacterial activity of Mdc against clinical isolates of MDR-A. baumanniiand elucidate the related antibacterial mechanisms. The minimal inhibitory concentration (MIC) of Mdc was 4 μg/mL. Bactericidal kinetics of Mdc revealed rapid killing ofA. baumannii(30 min). Flow cytometry using viability stain demonstrated that Mdc causesA. baumanniimembrane permeabilization in a concentration- and time-dependent process, which correlates with the bactericidal action. Moreover, transmission electron microscopic (TEM) examination showed that Mdc is capable of disrupting the membrane of bacterial cells, resulting in efflux of essential cytoplasmic components. Overall, Mdc could be a promising antibacterial agent for MDR-A. baumanniiinfections.

Secondary metabolite production of Epicoccum sp. isolated from lignite

We have extracted secondary metabolites from fossil fungus Epicoccum sp. isolated from lignite and characterized changes in production on various growth media. Chromatographic separation yielded more than 7 fractions that were characterized spectroscopically. One secondary metabolite showed antimicrobial activity. Peaks in the NMR spectra of isolated fractions differ from compounds described in literature that were isolated from recent strains of Epicoccum. The remaining water phase after the extraction was shown to have protein staining properties and could be also used for fluorescent viability stain of yeast cells.

Acid Tolerance of Biofilm Cells of Streptococcus mutans

ABSTRACT Streptococcus mutans, a member of the dental plaque community, has been shown to be involved in the carious process. Cells of S. mutans induce an acid tolerance response (ATR) when exposed to sublethal pH values that enhances their survival at a lower pH. Mature biofilm cells are more resistant to acid stress than planktonic cells. We were interested in studying the acid tolerance and ATR-inducing ability of newly adhered biofilm cells of S. mutans. All experiments were carried out using flow-cell systems, with acid tolerance tested by exposing 3-h biofilm cells to pH 3.0 for 2 h and counting the number of survivors by plating on blood agar. Acid adaptability experiments were conducted by exposing biofilm cells to pH 5.5 for 3 h and then lowering the pH to 3.5 for 30 min. The viability of the cells was assessed by staining the cells with LIVE/DEAD BacLight viability stain. Three-hour biofilm cells of three different strains of S. mutans were between 820- and 70,000-fold more acid tolerant than corresponding planktonic cells. These strains also induced an ATR that enhanced the viability at pH 3.5. The presence of fluoride (0.5 M) inhibited the induction of an ATR, with 77% fewer viable cells at pH 3.5 as a consequence. Our data suggest that adhesion to a surface is an important step in the development of acid tolerance in biofilm cells and that different strains of S. mutans possess different degrees of acid tolerance and ability to induce an ATR.

Persistence of Streptococcus pyogenes in Stationary-Phase Cultures

ABSTRACT In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 103 CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.

An investigation of the thermal death kinetics ofMycobacterium paratuberculosisduring pasteurisation

Mycobacterium paratuberculosis is the known cause of Johne's disease in cattle and has been implicated as a cause of Crohn's disease in humans. Concern has been expressed that the organism, which is excreted in milk and faeces of infected cattle, may be transmitted via pasteurised milk. This is largely from work that has shown an unacceptable risk of survival of the organism when it is present in raw milk at numbers exceeding 102cfu/ml. Three possible reasons for this apparent heat resistance were investigated viz. use of a milk heating menstruum, presence of a heat resistant sub–population, and the tendency of the organism to form clumps. Heat resistance studies using a combined acid–fast/viability stain, and a comparison of the relative heat sensitivities of clumped and de-clumped M. paratuberculosis cells provided circumstantial evidence that it is the organism's tendency to form clumps which confers the apparent heat resistance. This work casts doubt on the efficacy of current commercial pasteurisation heat treatments (72°C/15s) for the inactivation of M. paratuberculosis. This would be of concern if a link between M. paratuberculosis and Crohn's disease is eventually established.