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Esp-Independent Biofilm Formation by Enterococcus faecalis. Christopher J. Kristich, 2004.Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro . In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices . However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown . Recent work has suggested that a specific cell surface protein (Esp) of E . faecalis is critical for biofilm formation by this organism . However, in the same study, esp-deficient strains of E . faecalis were found to be capable of biofilm formation . To test the hypothesis that Esp is dispensable for biofilm formation by E . faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains . Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence . Using scanning electron microscopy to evaluate biofilms of E . faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels . Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions . Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E . faecalis . In summary, E . faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein . A Chimeric Peptide Composed of a Dermaseptin Derivative and an RNA III-Inhibiting Peptide Prevents Graft-Associated Infections by Antibiotic-Resistant Staphylococci. Naomi Balaban, 2004.Staphylococcal bacteria are a prevalent cause of infections associated with foreign bodies and indwelling medical devices . Bacteria are capable of escaping antibiotic treatment through encapsulation into biofilms . RNA III-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal biofilm formation by obstructing quorum-sensing mechanisms . K4-S4(1-13)a is a 13-residue dermaseptin derivative (DD13) believed to kill bacteria via membrane disruption . We tested each of these peptides as well as a hybrid construct, DD13-RIP, for their ability to inhibit bacterial proliferation and suppress quorum sensing in vitro and for their efficacy in preventing staphylococcal infection in a rat graft infection model with methicillin-resistant Staphylococcus aureus (MRSA) or S . epidermidis (MRSE) . In vitro, proliferation assays demonstrated that RIP had no inhibitory effect, while DD13-RIP and DD13 were equally effective, and that the chimeric peptide but not DD13 was slightly more effective than RIP in inhibiting RNA III synthesis, a regulatory RNA molecule important for staphylococcal pathogenesis . In vivo, the three peptides reduced graft-associated bacterial load in a dose-dependent manner, but the hybrid peptide was most potent in totally preventing staphylococcal infections at the lowest dose . In addition, each of the peptides acted synergistically with antibiotics . The data indicate that RIP and DD13 act in synergy by attacking bacteria simultaneously by two different mechanisms . Such a chimeric peptide may be useful for coating medical devices to prevent drug-resistant staphylococcal infections . Elevated Levels of Ketopantoate Hydroxymethyltransferase (PanB) Lead to a Physiologically Significant Coenzyme A Elevation in Salmonella enterica Serovar Typhimurium. Aileen Rubio, 2002.Pantothenate is the product of the ATP-dependent condensation of pantoate and ß-alanine and is a direct precursor of coenzyme A . A connection exists between pantothenate biosynthesis and thiamine biosynthesis in Salmonella enterica serovar Typhimurium since derivatives of a purF mutant that can grow (on glucose medium) in the absence of thiamine excrete pantothenate . We show here that the causative mutation in three such mutants was the addition of a CG base pair upstream of the panB gene . This base addition brings the spacing between the -10 and -35 hexamers of the promoter to a consensus spacing of 17 bp and results in increased transcription of the pan operon . Furthermore, overexpression of PanB caused by this mutation, or by other means, was necessary and sufficient to increase pantothenate production and allow PurF-independent thiamine synthesis on glucose medium . Mechanism of Pseudomonas aeruginosa RhlR Transcriptional Regulation of the rhlAB Promoter. Gerardo Medina, 2003.Pseudomonas aeruginosa contains two transcription regulators (LasR and RhlR) that, when complexed with their specific autoinducers (3-oxo-dodecanoyl-homoserine lactone and butanoyl-homoserine lactone, respectively) activate transcription of different virulence-associated traits . We studied the RhlR-dependent transcriptional regulation of the rhlAB operon encoding rhamnosyltransferase 1, an enzyme involved in the synthesis of the surfactant monorhamnolipid, and showed that RhlR binds to a specific sequence in the rhlAB regulatory region, both in the presence and in the absence of its autoinducer . Our data suggest that in the former case it activates transcription, whereas in the latter it acts as a transcriptional repressor of this promoter . RhlR seems to repress the transcription of other quorum-sensing-regulated genes; thus, RhlR repressor activity might be of importance in the finely regulated expression of P . aeruginosa virulence-associated traits .
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