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Phage-Host Interaction: an Ecological Perspective. Sandra Chibani-Chennoufi, 2004. Hypermutation and the Preexistence of Antibiotic-Resistant Pseudomonas aeruginosa Mutants: Implications for Susceptibility Testing and Treatment of Chronic Infections. Antonio Oliver, 2004.Whether or not resistant mutants will be present before the start of antibiotic treatment of an initially susceptible population of bacteria depends on the size of the infecting population, the rate of mutation to resistance, and the amount of time that the population has been maintained . In the present investigation, we argue that for the treatment of chronic infections caused by hypermutable Pseudomonas aeruginosa of the sort frequently found in cystic fibrosis patients, mutants resistant to all single antipseudomonal drugs will almost invariably be present in a high proportion at the onset of treatment, and consequently, these strains should be considered resistant to all agents when they are used as monotherapy . Using a construct of P . aeruginosa strain PAO1 with a mutS deletion (strain PAO  mutS), we show that when in vitro populations of less than 5 x 104 seemingly susceptible hypermutable bacteria are confronted with any of 11 antipseudomonal agents, mutants for which the MICs and the minimum bactericidal concentrations are in the range of clinical resistance will almost invariably ascend to dominance within 24 to 36 h . This does not occur for PAO1 without the mutS deletion . The results of our detailed analysis of this evolution of acquired resistance to two of these antibiotics, imipenem and ciprofloxacin, indicate that although the rates of mutation to resistance in PAOmutS are on the order of 1 x 10–6 per generation, resistant mutants are very likely to either be present in cultures of between 2 x 104 and 4 x 104 bacteria or arise after the bacterial populations are confronted with antibiotics . We also demonstrate with in vitro experiments that the problem of acquired resistance to treatment with single antibiotics can be thwarted by combination therapy with pairs of antibiotics of different classes with synergistic activities . We discuss the clinical implications of our analysis of these observations .
Construction, Characterization, and Use of Two Listeria monocytogenes Site-Specific Phage Integration Vectors. Peter Lauer, 2002.Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes . The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion . pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L . monocytogenes, forms stable, single-copy integrants at a frequency of  10-4 per donor cell, and can be used in the L . monocytogenes 1/2 and 4b serogroups . Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed . pPL1 was used to introduce the hly and actA genes at comK-attBB' in deletion strains derived from 10403S and SLCC-5764 . These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays . Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay . Additionally, the PSA phage attachment site on the L . monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site . The listeriophage PSA integrates in the 3' end of an arginine tRNA gene . There are 17 bp of DNA identity between the bacterial and phage attachment sites . The PSA prophage DNA sequence reconstitutes a complete tRNAArg gene . The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains .
Characterization of a New LexA Binding Motif in the Marine Magnetotactic Bacterium Strain MC-1. Antonio R. Fernández de Henestrosa, 2003.MC-1 is a marine, magnetotactic bacterium that is phylogenetically associated with the alpha subclass of the Proteobacteria and is the first and only magnetotactic coccus isolated in pure culture to date . By using a TBLASTN search, a lexA gene was identified in the published genome of MC-1; it was subsequently cloned, and the protein was purified to >90% purity . Results from reverse transcription-PCR analysis revealed that the MC-1 lexA gene comprises a single transcriptional unit with two open reading frames encoding proteins of unknown function and with a rumA-like gene, a homologue of the Escherichia coli umuD gene . Mobility shift assays revealed that this LexA protein specifically binds both to its own promoter and to that of the umuDC operon . However, MC-1 LexA does not bind to the promoter regions of other genes, such as recA and uvrA, that have been previously reported to be regulated by LexA in bacterial species belonging to the alpha subclass of the Proteobacteria . Site-directed mutagenesis of both the lexA and umuDC operator regions demonstrated that the sequence CCTN10AGG is the specific target motif for the MC-1 LexA protein . Detection of Prochlorothrix in Brackish Waters by Specific Amplification of pcb Genes. Ulrike Geiß, 2003.Prochlorothrix hollandica is the only filamentous chlorophyll b (Chlb)-containing oxyphotobacterium that has been found in freshwater habitats to date . Chlb serves as a light-harvesting pigment which is bound to special binding proteins (Pcb) . Even though Prochlorothrix was initially characterized as a highly salt-sensitive species, we detected it in a brackish water environment that is characterized by salinities of up to 12 practical salinity units . Using PCR and reverse transcription, we amplified pcb gene fragments of phytoplankton samples taken along a salinity gradient in the eutrophic Darss-Zingst estuary (southern Baltic Sea) . After sequencing, high levels of homology to the pcbB and pcbC genes of P . hollandica were found . Furthermore, autofluorescence of Prochlorothrix-like filaments that indicated that Chlb was present was detected in enrichment cultures prepared from the estuarine phytoplankton . The detection of Chlb-containing filaments, as well as the pcb and 16S ribosomal DNA sequences, suggests that Prochlorothrix is an indigenous genus in the Darss-Zingst estuary and may also inhabit many other brackish water environments . The potential of using pcb gene detection to differentiate Prochlorothrix from morphologically indistinguishable species belonging to the genera Pseudanabaena and Planktothrix (Oscillatoria) in phytoplankton analyses is discussed .
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