Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2؉ -dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap R 1, from the model bacterium Bacillus subtilis 168. Dap R 1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap R 1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap R 1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB, the stringent response gene relA, and the phosphatidylglycerol synthase gene pgsA. Genetic reconstruction studies indicated that the pgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca 2؉ -DAP complex.Daptomycin (DAP) is a cyclic lipopeptide antibiotic used to treat complicated skin and skin structure infections caused by Staphylococcus aureus or enterococci. In addition, it has been approved to treat S. aureus-induced bacteremia and infective endocarditis (21), and animal model studies suggest that it may be a useful alternative for treatment of inhalational anthrax (26). The mechanism of action involves the calcium-dependent insertion of DAP into the bacterial membrane, followed by depolarization of the membrane and extrusion of potassium ions, leading to arrest of macromolecular synthesis and to cell death (49, 51).The introduction of new antibacterial compounds seems to be followed inevitably by the emergence of resistant isolates. It is estimated that over 1 million patients have been treated with DAP (J. Silverman, personal communication). According to the SENTRY Antimicrobial Surveillance Program in the United States for the years 2002 to 2010, 99.9% of methicillin-resistant S. aureus (MRSA) isolates treated with DAP had an MIC of 1.0 g/ml or lower, with only a slight increase of MIC over time (47; http://www.gp-pathogens.com/data/default.cfm).Previous studies to define mechanisms of resistance to DAP were performed on clinical isolates and by in vitro selection (22,31). After serial passages with increasing DAP concentrations, Friedman et al. characterized three...

Entomopathogenic fungi (EPFs) play an important role for regulating insect pest populations, as they exist in many different ecosystems. Within these fungi, Beauveria and Metarhizium spp. genera include species that are the most commercially important. The aim of this study was to determine the diversity and distribution of Beauveria and Metarhizium spp. in walnut fields of Kırşehir, Turkey, and to evaluate their pathogenicity against Cydia pomonella (L.) (Lepidoptera: Tortricidae). To perform this, 90 soil samples were collected from walnut fields where Beauveria and Metarhizium spp. were isolated from these soils, using selective media. The isolated 40 fungi were characterized based on their morphological and molecular characteristics including Bloc and β-tubulin gene sequences. Also, eight selected fungi were tested against C. pomonella larvae under laboratory conditions. The fungal isolates were identified as Beauveria pseudobassiana (15), B. bassiana (12), Metarhizium robertsii (11), and M. brunneum (13). M. brunneum ELA-38 caused 83% mortality within 2 weeks after application of 1 × 10 8 conidia/ml. Consequently, Beauveria and Metarhizium spp. are the common component of the soils collected from walnut fields and some of fungi obtained from this work might be beneficial in the future biological control programs of C. pomonella.

Plagiodera versicolora (Laicharting, 1781) (Coleoptera: Chrysomelidae) is an important forest pest which damages many trees such as willow, poplar, and hazelnut. In order to find new microbes that can be utilized as a possible microbial control agent against this pest, we investigated the culturable bacterial flora of it and tested the isolated bacteria against P. versicolora larvae and adults. We were able to isolate nine bacteria from larvae and adults. The isolates were characterized using a combination of morphological, biochemical, and physiological methods. Additionally, we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results. Based on characterization studies, the isolates were identified as Staphylococcus sp. Pv1, Rahnella sp. Pv2, Rahnella sp. Pv3, Rahnella sp. Pv4, Rahnella sp. Pv5, Pantoea agglomerans Pv6, Staphylococcus sp. Pv7, Micrococcus luteus Pv8, and Rahnella sp. Pv9. The highest insecticidal activity against larvae and adults was obtained from M. luteus Pv8 with 50 and 40 % mortalities within 10 days after treatment, respectively. Extracellular enzyme activity of the bacterial isolates such as amylase, proteinase, lipase, cellulose, and chitinase was also determined. Consequently, our results show that M. luteus Pv8 might be a good candidate as a possible microbial control agent against P. versicolora and were discussed with respect to biocontrol potential of the bacterial isolates.