TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.The bacterium Erwinia chrysanthemi is a gram-negative broad-host-range phytopathogen causing soft-rot disease. Production of plant cell wall-degrading enzymes and avirulence proteins by the phytopathogen contributes to the destruction of plant structural barriers and evasion of certain host defense responses, respectively (4, 9, 10, 21, 37). Nutrient acquisition in E. chrysanthemi from degraded plant cell-wall pectin is facilitated by transport systems for pectin-derived oligomers and monomers (18,20,22,46). Secretion of pathogenesis-related proteins across the bacterial cytoplasmic and outer membranes requires several export systems that have been extensively studied. Pectin-degrading enzymes are secreted by a type IIdependent mechanism (encoded by the out genes), and secretion of avirulence proteins into the host is mediated by a type III-dependent process (encoded by the hrp genes) (9,33,35). Finally, secretion of metalloproteases by a sec-independent type I mechanism (prt genes) in E. chrysanthemi has also been demonstrated (11,25,26).TolC is an important though low-abundance protein in the outer membrane of gram-negative bacteria (17). The crystal structure of this protein has recently been determined (24). The protein exists functionally as a trimer forming a -barrel with a large internal diameter, facilitating movement of both large and small molecules through the outer membrane (2). TolC functions as a component of multidrug resistance (MDR) efflux systems in the removal of a broad range of toxic chemicals from the cell (16, 41). In Escherichia coli, type I-dependent secretion of certain virulence-associated proteins also requires TolC (48). The role of TolC in virulence and survival strategies of the pathogenic enteric bacteria E. coli (49), Vibrio cholerae (5), Salmonella enterica serovar Enteritidis (40), and Serratia marcescens (6) has been established. While the existence and ...
OBJECTIVE: To establish reliability and validity of real-time fluorescent PCR for early detection of bacterial invasion of the amniotic cavity. METHODS: Amniotic fluid samples from 40 patients undergoing mid-trimester genetic amniocentesis were incubated for 6 h at 37 degrees C and were cultured on media specific for group B streptococcus (GBS) and E. coli. Concurrently, samples were analyzed with real-time fluorescent PCR (Roche LightCycler) using DNA primers and probes designed to detect the CAMP factor encoding cfb gene and uidA gene of GBS and E. coli, respectively. For positive control and to simulate amniotic fluid colonization, 104 cfu/ml of GBS and E. coli were inoculated on sterile amniotic fluid and incubated for 6 h. Bacterial genomic DNA for the two organisms was extracted and purified via the two-step precipitation method using a commercial kit. The real-time PCR assays were also tested against 25 non-GBS and non-E. coli bacterial species. The lower limit of detection for each pathogen was established using serial dilution of bacterial genomic DNA. RESULTS: All patient samples were negative for evidence of GBS and E. coli with both culture and real-time PCR methods. Amniotic fluid samples inoculated with GBS and E. coli were positive with real-time PCR whereas the 25 bacterial species other than GBS or E. coli tested negative with the assay. Average total sample processing time including the pre-enrichment step was 7 h 40 min. The average cost for DNA extraction and PCR testing was 8.50 dollars per test. CONCLUSION: Real-time fluorescent PCR is a valid and reliable method for detection of specific pathogens in amniotic fluid. This technique is sensitive for low inoculation levels. Real-time fluorescent PCR has potential to impact clinical management as a rapid, reliable detection method for GBS and E. coli in chorioamnionitis.
Clostridium perfringens is one of the etiologic agents of gas gangrene that can occur when a wound is contaminated with soil. Type A C. perfringens can cause foodborne and nonfoodborne gastrointestinal illnesses due to an enterotoxin (CPE) produced by some strains during sporulation. We developed a quantitative real-time PCR assay based on fluorescence resonance energy transfer hybridization chemistry that targets the C. perfringens-specific phospholipase C (plc) gene and the enterotoxigenic gene (cpe) with the LightCycler and the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.). The assay can detect as few as 20 copies of target sequences per PCR. The total assay time, from extraction to PCR analysis, is 90 min. This assay is rapid, sensitive, and specific and will allow direct detection of C. perfringens in water, food, and stool samples. It should prove helpful in investigating foodborne illnesses due to C. perfringens and can be used as a tool to ensure the safety of food and water supplies.
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