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Real-Time Monitoring of Intracellular Staphylococcus aureus Replication. S. N. A. Qazi, 2004.A high-throughput system to rapidly assess the intracellular replication of Staphylococcus aureus has been developed utilizing S . aureus transformed with a dual gfp-luxABCDE reporter operonunder the control of a growth-dependent promoter . Replication of tagged bacteria internalized into bovine mammary epithelial cells [MAC-T] could be measured by monitoring fluorescence and bioluminescence from the reporter operon following removal of extracellular bacteria from the plates . Bacterial replicationinside cells was confirmed by a novel ex vivo time-lapse confocal microscopic method . This assay of bacterial replication wasused to evaluate the efficacy of antibiotics which are commonlyused to treat staphylococcal infections . Not all antibioticstested were able to prevent intracellular replication of S.aureus and some were ineffective at preventing replication ofintracellular bacteria at concentrations above the MIC determinedfor bacteria in broth culture . Comparison of the fluorescenceand bioluminescence signals from the bacteria enabled effectson protein synthesis and metabolism to be discriminated andgave information on the entry of compounds into the eukaryoticcell, even if bacterial replication was not prevented . Elevatedresistance of S . aureus to antibiotics inside host cells increasesthe likelihood of selecting S . aureus strains which are resistantto commonly used antimicrobial agents within the intracellularniche . The approach presented directly assesses intracellularefficacy of antibiotics and provides an evidence-based approachto antibiotic selection for prescribing physicians and medicalmicrobiologists. Combination Therapy Counteracts the Enhanced Transmission of Drug-Resistant Malaria Parasites to Mosquitoes. Rachel L. Hallett, 2004.Malaria parasites carrying genes conferring resistance to antimalarials are thought to have a selective advantage which leads to higher rates of transmissibility from the drug-treated host . This is a likely mechanism for the increasing prevalence of parasites with resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine in sub-Saharan Africa . Combination therapy is the key strategy being implemented to reduce the impact of resistance, but its effect on the transmission of genetically resistant parasites from treated patients to mosquito vectors has not been measured directly . In a trial comparing CQ monotherapy to the combination CQ plus artesunate (AS) in Gambian children with uncomplicated falciparum malaria, we measured transmissibility by feeding Anopheles gambiae mosquitoes with blood from 43 gametocyte-positive patients through a membrane . In the CQ-treated group, gametocytes from patients carrying parasites with the CQ resistance-associated allele pfcrt-76T prior to treatment produced infected mosquitoes with 38 times higher Plasmodium falciparum oocyst burdens than mosquitoes fed on gametocytes from patients infected with sensitive parasites (P < 0.001) . Gametocytes from parasites carrying the resistance-associated allele pfmdr1-86Y produced 14-fold higher oocyst burdens than gametocytes from patients infected with sensitive parasites (P = 0.011) . However, parasites carrying either of these resistance-associated alleles pretreatment were not associated with higher mosquito oocyst burdens in the CQ-AS-treated group . Thus, combination therapy overcomes the transmission advantage enjoyed by drug-resistant parasites . Plasmid-Encoded Multidrug Efflux Pump Conferring Resistance to Olaquindox in Escherichia coli. Lars Hestbjerg Hansen, 2004.We report here the first gene-encoded resistance mechanism to the swine growth enhancer olaquindox . The genetic elements involved in resistance to olaquindox were subcloned and sequenced from a conjugative plasmid isolated from Escherichia coli . The subcloned fragment contained two open reading frames, oqxA and oqxB, that are homologous to several resistance-nodulation-cell-division family efflux systems from different species . The putative protein sequences were aligned to both experimentally verified and putative efflux pumps . We show that oqxA and oqxB are expressed in E . coli . Plasmids containing the oqxAB genes yielded high (>128 µg/ml) resistance to olaquindox in E . coli, whereas strains containing the control plasmid showed low resistance to the drug (8 µg/ml) . The oqxAB-encoded pump also conferred high (>64 µg/ml) resistance to chloramphenicol . We demonstrate that the subcloned fragment conferred H+-dependent ethidium efflux abilities to E . coli strain N43 . In addition, we show that the efflux system is dependent on the host TolC outer membrane protein when expressed in E . coli . Anaerobic Degradation of Flavonoids by Clostridium orbiscindens. Lilian Schoefer, 2003.An anaerobic, quercetin-degrading bacterium was isolated from human feces and identified as Clostridium orbiscindens by comparative 16S rRNA gene sequence analysis . The organism was tested for its ability to transform several flavonoids . The isolated C . orbiscindens strain converted quercetin and taxifolin to 3,4-dihydroxyphenylacetic acid; luteolin and eriodictyol to 3-(3,4-dihydroxyphenyl)propionic acid; and apigenin, naringenin, and phloretin to 3-(4-hydroxyphenyl)propionic acid, respectively . Genistein and daidzein were not utilized . The glycosidic bonds of luteolin-3-glucoside, luteolin-5-glucoside, naringenin-7-neohesperidoside (naringin), quercetin-3-glucoside, quercetin-3-rutinoside (rutin), and phloretin-2'-glucoside were not cleaved . Based on the intermediates and products detected, pathways for the degradation of the flavonol quercetin and the flavones apigenin and luteolin are proposed . To investigate the numerical importance of C . orbiscindens in the human intestinal tract, a species-specific oligonucleotide probe was designed and tested for its specificity . Application of the probe to fecal samples from 10 human subjects proved the presence of C . orbiscindens in 8 out of the 10 samples tested . The numbers ranged from 1.87 x 108 to 2.50 x 109 cells g of fecal dry mass-1, corresponding to a mean count of 4.40 x 108 cells g of dry feces-1 .
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