RESUMEN
Contaminated beef and beef products remain a frequent vehicle for the transmission of Escherichia coli O157:H7. The current U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) regulatory testing for E. coli O157:H7 uses the method described in the USDA-FSIS Microbiology Laboratory Guidebook (MLG), chapter 5. At times, described presumptive test results are nonconfirmable, suggesting that recent PCR technological advancements and presumed enhanced sensitivity and specificity may offer beneficial changes. Here, we have evaluated the precision and sensitivity of a fluorescence resonance energy transfer-based real-time PCR assay called ECO for the detection of E. coli O157:H7. ECO detects the gene target specific to both E. coli O157:H7 and E. coli O157:non-H7 but distinguishes the two by using a melt curve analysis. A total of 3,113 O157:H7 and O157:non-H7 isolates were used to define this melting temperature-based criteria. The simulated comparative performance evaluation in the spiked beef samples indicated detection of 3 of 3 samples by ECO at <3.3 log CFU/mL, whereas MLG only detected 1 of 3 (<3.3 log CFU/mL). Using modified tryptic soy broth-enriched natural beef and veal product samples ( n = 452), the comparative sensitivity, specificity, false-positive rate, and false-negative rate against culture between MLG and ECO were 75 versus 92%, 91 versus 99%, 8.9 versus 0.77%, and 25 versus 8.3%, respectively. Positive predictive value, negative predictive value, and the overall accuracy were found to be 56 versus 94%, 96 versus 98%, and 88 versus 98%, for MLG and ECO, respectively. These data demonstrate that the ECO assay is comparable to MLG detection of E. coli O157:H7 and offers improved sensitivity.
Asunto(s)
Escherichia coli O157 , Transferencia Resonante de Energía de Fluorescencia/métodos , Contaminación de Alimentos/análisis , Productos de la Carne/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Bovinos , Recuento de Colonia Microbiana , Escherichia coli O157/aislamiento & purificación , Microbiología de Alimentos , Carne Roja/microbiologíaRESUMEN
The biodiversity of the bacterium Pseudomonas aeruginosa in an aquatic environment (the Woluwe River, Brussels, Belgium) was analysed. Surface water was sampled bimonthly over a 1-year period (2000-2001) at seven sites evenly dispersed over the river. Total bacterial counts were performed and P. aeruginosa strains were isolated on a selective medium. A weighed out sample of 100 randomly chosen presumptive P. aeruginosa isolates was further analysed. A set of data consisting of the nucleotide sequence of the oprL gene, a DNA-based fingerprint (amplified fragment length polymorphism, AFLP), serotype, pyoverdine type and antibiogram (MICs of 21 clinically relevant antibiotics) was assembled. These data were integrated with those previously obtained for 73 P. aeruginosa clinical and environmental isolates collected across the world. The combined results were analysed and compared using biological data analysis software. Our findings indicate a positive relationship between the extent of pollution and the prevalence of P. aeruginosa. Surprisingly, the Woluwe River P. aeruginosa community was almost as diverse as the global P. aeruginosa population. Indeed, the Woluwe River harboured members of nearly all successful clonal complexes. With the exception of one multidrug-resistant (MDR) strain, belonging to a ubiquitous and clinically relevant serotype O11 clone, antibiotic resistance levels were relatively low. These findings illustrate the significance of river water as a reservoir and source of distribution of potentially pathogenic P. aeruginosa strains and could have repercussions on antinosocomial infection strategies.
Asunto(s)
Biodiversidad , Pseudomonas aeruginosa/aislamiento & purificación , Ríos/microbiología , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Bélgica , Medios de Cultivo , Microbiología Ambiental , Proteínas de Escherichia coli , Humanos , Lipoproteínas/genética , Pruebas de Sensibilidad Microbiana , Oligopéptidos/metabolismo , Peptidoglicano/genética , Reacción en Cadena de la Polimerasa , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Análisis de Secuencia de ADNRESUMEN
Pseudomonas aeruginosa produces, under conditions of iron limitation, a high-affinity siderophore, pyoverdine (PVD), which is recognized at the level of the outer membrane by a specific TonB-dependent receptor, FpvA. So far, for P. aeruginosa, three different PVDs, differing in their peptide chain, have been described (types I-III), but only the FpvA receptor for type I is known. Two PVD-producing P. aeruginosa strains, one type II and one type III, were mutagenized by a mini-TnphoA3 transposon. In each case, one mutant unable to grow in the presence of the strong iron chelator ethylenediaminedihydroxyphenylacetic acid (EDDHA) and the cognate PVD was selected. The first mutant, which had an insertion in the pvdE gene, upstream of fpvA, was unable to take up type II PVD and showed resistance to pyocin S3, which is known to use type II FpvA as receptor. The second mutant was unable to take up type III PVD and had the transposon insertion in fpvA. Cosmid libraries of the respective type II and type III PVD wild-type strains were constructed and screened for clones restoring the capacity to grow in the presence of PVD. From the respective complementing genomic fragments, type II and type III fpvA sequences were determined. When in trans, type II and type III fpvA restored PVD production, uptake, growth in the presence of EDDHA and, in the case of type II fpvA, pyocin S3 sensitivity. Complementation of fpvA mutants obtained by allelic exchange was achieved by the presence of cognate fpvA in trans. All three receptors posses an N-terminal extension of about 70 amino acids, similar to FecA of Escherichia coli, but only FpvAI has a TAT export sequence at its N-terminal end.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Oligopéptidos , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/metabolismo , Receptores de Superficie Celular , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Medios de Cultivo , Fibrosis Quística/microbiología , Etilenodiaminas , Prueba de Complementación Genética , Humanos , Hierro/metabolismo , Datos de Secuencia Molecular , Mutación , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Piocinas/farmacología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Análisis de Secuencia de ADNRESUMEN
Pyoverdines, the main siderophores of fluorescent pseudomonads, contain a peptide moiety, different for each pyoverdine, and an identical chromophore. While it has been shown that non-ribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of the peptide chain of pyoverdines, this was not demonstrated for the biosynthesis of the chromo-phore part. We found that PvsA, from Pseudomonas fluorescens ATCC 17400, and PvdL (PA2424), from Pseudomonas aeruginosa are similar NRPSs and functional homologues, necessary for the production of pyoverdine. Transcriptional lacZ fusions showed that pvdL is co-transcribed with the upstream PA2425 gene, encoding a putative thioesterase, and is iron-regulated via PvdS. Similarly, RT-PCR analysis revealed that expression of pvsA is repressed by iron. Analysis of the adenylation domains of PvsA, PvdL and their homologues, revealed that their N-terminus starts with an acyl-CoA ligase module, followed by three amino acid activation domains. Computer modelling of these domains suggests that PvsA in P. fluorescens and PvdL in P. aeruginosa are orthologues involved in the biosynthesis of the pyoverdine chromophore.