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Pharmacodynamic Functions: a Multiparameter Approach to the Design of Antibiotic Treatment Regimens. Roland R. Regoes, 2004.There is a complex quantitative relationship between the concentrations of antibiotics and the growth and death rates of bacteria . Despite this complexity, in most cases only a single pharmacodynamic parameter, the MIC of the drug, is employed for the rational development of antibiotic treatment regimens . In this report, we use a mathematical model based on a Hill function—which we call the pharmacodynamic function and which is related to previously published Emax models—to describe the relationship between the bacterial net growth rates and the concentrations of antibiotics of five different classes: ampicillin, ciprofloxacin, tetracycline, streptomycin, and rifampin . Using Escherichia coli O18:K1:H7, we illustrate how precise estimates of the four parameters of the pharmacodynamic function can be obtained from in vitro time-kill data . We show that, in addition to their respective MICs, these antibiotics differ in the values of the other pharmacodynamic parameters . Using a computer simulation of antibiotic treatment in vivo, we demonstrate that, as a consequence of differences in pharmacodynamic parameters, such as the steepness of the Hill function and the minimum bacterial net growth rate attained at high antibiotic concentrations, there can be profound differences in the microbiological efficacy of antibiotics with identical MICs . We discuss the clinical implications and limitations of these results . Effect of Pyocyanin on a Crude-Oil-Degrading Microbial Community. R. Sean Norman, 2004.Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments . In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P . aeruginosa and is a redox-active compound capable of inhibiting microbial growth . To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 µM pyocyanin and microbial community structure and oil degradation were monitored for 50 days . Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures . Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively . Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture . Pyocyanin also affected the overall degradation of the crude oil . At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C29 and C30 hopanes . This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community . Involvement of the PrrB/PrrA Two-Component System in Nitrite Respiration in Rhodobacter sphaeroides 2.4.3: Evidence for Transcriptional Regulation. William P. Laratta, 2002.Rhodobacter sphaeroides strain 2.4.3 is capable of diverse metabolic lifestyles, including denitrification . The regulation of many Rhodobacter genes involved in redox processes is controlled, in part, by the PrrBA two-component sensor-regulator system, where PrrB serves as the sensor kinase and PrrA is the response regulator . Four strains of 2.4.3 carrying mutations within the prrB gene were isolated in a screen for mutants unable to grow anaerobically on medium containing nitrite . Studies revealed that the expression of nirK, the structural gene encoding nitrite reductase, in these strains was significantly decreased compared to its expression in 2.4.3 . Disruption of prrA also eliminated the ability to grow both photosynthetically and anaerobically in the dark on nitrite-amended medium . Complementation with prrA restored the wild-type phenotype . The PrrA strain exhibited a severe decrease in both nitrite reductase activity and expression of a nirK-lacZ fusion . Nitrite reductase activity in the PrrA strain could be restored to wild-type levels by using nirK expressed from a heterologous promoter, suggesting that the loss of nitrite reductase activity in the PrrA and PrrB mutants was not due to problems with enzyme assembly or the supply of reductant . Inactivation of prrA had no effect on the expression of the gene encoding NnrR, a transcriptional activator required for the expression of nirK . Inactivation of ccoN, part of the cbb3-type cytochrome oxidase shown to regulate the kinase activity of PrrB, also caused a significant decrease in both nirK expression and Nir activity . This was unexpected, since PrrA-P accumulates in the ccoN strain . Together, these results demonstrate that PrrBA plays an essential role in the regulation of nirK . Molecular Analysis of Transport and Oligomerization of the Yersinia enterocolitica Adhesin YadA. Andreas Roggenkamp, 2003.The Yersinia adhesin YadA is the prototype of a novel class of bacterial adhesins which form oligomeric lollipop-like structures and are anchored in the outer membrane by the C terminus . For YadA, six different regions (R) or domains (D) are predicted from the amino acid sequence: the N-terminal leader sequence, head-D, neck-D, stalk-D, linking-R, and a C-terminal transmembrane region consisting of four ß-strands . To identify structural and functional features of these domains, we performed in-frame deletion mutagenesis and constructed N-terminally tagged YadA variants . Diverse YadA variants were analyzed for outer membrane localization, surface exposure, oligomerization adhesion properties, and ability to protect against complement-mediated lysis . We demonstrated that (i) the C-terminal region (amino acids [aa] 353 to 422) is sufficient for outer membrane insertion and formation of trimers in the outer membrane; (ii) the head, neck, and stalk domains (aa 26 to 330) are surface exposed, forming a passenger domain; and (iii) the linking region (aa 331 to 369) is responsible for outer membrane translocation of the passenger domain . Thus, YadA meets all the criteria of an autotransporter . The same may be true for all other members of the YadA family, forming a subfamily of surface-attached oligomeric autotransporters . Moreover, in-frame truncation mutagenesis suggested that the head and neck domains together form the YadA-binding module which is located on the top of the stalk . However, the YadA-binding module did not confer serum resistance . Mutants lacking the head and neck domain were resistant to complement-mediated lysis . In-frame truncation of the stalk domain did not result in significant attenuation of the mutant in an orogastric mouse infection model . Structure of Haloacetate-Catabolic IncP-1ß Plasmid pUO1 and Genetic Mobility of Its Residing Haloacetate-Catabolic Transposon. Masahiro Sota, 2003.The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury . The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1ß plasmid R751 . Comparison of pUO1 with three other IncP-1ß plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids . Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition . Osmosensitivity Associated with Insertions in argP (iciA) or glnE in Glutamate Synthase-Deficient Mutants of Escherichia coli. Madhusudan R. Nandineni, 2004.An ampicillin enrichment strategy following transposon insertion mutagenesis was employed to obtain NaCl-sensitive mutants of a gltBD (glutamate synthase [GOGAT]-deficient) strain of Escherichia coli . It was reasoned that the gltBD mutation would sensitize the parental strain even to small perturbations affecting osmotolerance . Insertions conferring an osmosensitive phenotype were identified in the proU, argP (formerly iciA), and glnE genes encoding a glycine betaine/proline transporter, a LysR-type transcriptional regulator, and the adenylyltransferase for glutamine synthetase, respectively . The gltBD+ derivatives of the strains were not osmosensitive . The argP mutation, but not the glnE mutation, was associated with reduced glutamate dehydrogenase activity and a concomitant NH4+ assimilation defect in the gltBD strain . Supplementation of the medium with lysine or a lysine-containing dipeptide phenocopied the argP null mutation for both osmosensitivity and NH4+ assimilation deficiency in a gltBD background, and a dominant gain-of-function mutation in argP was associated with suppression of these lysine inhibitory effects . Osmosensitivity in the gltBD strains, elicited either by lysine supplementation or by introduction of the argP or glnE mutations (but not proU mutations), was also correlated with a reduction in cytoplasmic glutamate pools in cultures grown at elevated osmolarity . We propose that an inability to accumulate intracellular glutamate at high osmolarity underlies the osmosensitive phenotype of both the argP gltBD and glnE gltBD mutants, the former because of a reduction in the capacity for NH4+ assimilation into glutamate and the latter because of increased channeling of glutamate into glutamine .
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