Acriflavine resistance in the clinical meticillin-resistant Staphylococcus aureus isolate KT24 was found not to be mediated by multidrug efflux pumps encoded by qacA/B, smr, qacE, qacG, qacH, qacJ or norA. Early uptake and accumulation of ethidium bromide in MRSA KT24 was significantly lower than that in a susceptible strain, although the efflux rates were similar. Therefore, a permeability barrier in MRSA KT24 may be the conceivable mechanism of acriflavine resistance. Interestingly, it was found that MRSA KT24 had a significantly thickened cell wall, and that cellwall thickness increased gradually during bacterial growth. In contrast, cell size and surface area in MRSA KT24 were not different from those in the susceptible strain. Moreover, MRSA KT24 exposure to sub-MIC concentrations of acriflavine resulted in a thicker cell wall. These results indicate that cell-wall thickness may be responsible for acriflavine resistance in S. aureus.

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.Scanning electron microscopy (SEM) has been used widely in environmental microbiology for characterization of the surface structure of biomaterials, as well as measurement of biological responses to biomaterials, including cell attachment and changes in morphology (1,37,38,42). SEM is very useful for defining the number and distribution of microorganisms adherent to surfaces. Size and some basic morphometric parameters were identified through the application of image analysis techniques to SEM (26,39). One limitation of SEM in environmental microbiology is that it cannot provide phylogenetic or genetic information about target microbes. When target microbes are observed by SEM, they must be distinguished from nontarget cells by morphological characteristics. However, microorganisms in natural samples, as well as pure culture, are morphologically highly diversified. Immunogold labeling marks cellular components (13,21,36). Antibodylabeled colloidal gold particles react to surface antigen and allow observation of specific microorganisms under SEM.In contrast, molecular studies based on rRNA analysis have led to an understanding of the microbial diversity and community composition of aquatic environments. For reliable quantitative characterization of community structure, in situ hybridization (ISH) with rRNA-targeted fluorescent oligonucleotide probes (FISH) is an increasingly popular method. FISH permits the identification and quantification of individual cells and has demonstrated great power in the analysis of bacterial community composition in several environments (2,4,15).In order to detect, identify, and enumerate target microbes by electron microscopy ...

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Aims:  To develop a scanning electron microscopic approach using in situ hybridization (SEM–ISH) for gaining both genetic and morphological information about target bacteria. Methods and Results:  Target cells were hybridized with DNA‐targeted polynucleotide probes, and a tyramide signal amplification system was used to increase the sensitivity. The protocol of SEM–ISH enabled to detect low copy number target DNA sequences in individual cells. Conclusions:  SEM–ISH allowed the in situ detection of bacteria carrying a specific gene. Significance and Impact of the Study:  Combining morphological study with SEM and ISH techniques appears to be a valuable tool to understand the spatial distribution of target cells in complex microbial communities on various materials.