Development of a Multiplex-PCR assay for the rapid identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus.

The presence of thermophilic bacilli in dairy products is indicator of poor hygiene. Their rapid detection and identification is fundamental to improve the industrial reactivity in the implementation of corrective and preventive actions. In this study a rapid and reliable identification of Geobacillus stearothermophilus and Anoxybacillus flavithermus was achieved by species-specific PCR assays. Two primer sets, targeting the ITS 16S-23S rRNA region and the rpoB gene sequence of the target species respectively, were employed. Species-specificity of both primer sets was evaluated by using 53 reference strains of DSMZ collection; among them, 13 species of the genus Geobacillus and 15 of the genus Anoxybacillus were represented. Moreover, 99 wild strains and 23 bulk cells collected from 24 infant formula powders gathered from several countries worldwide were included in the analyses. Both primer sets were highly specific and the expected PCR fragments were obtained only when DNA from G. stearothermophilus or A. flavithermus was used. After testing their specificity, they were combined in a Multiplex-PCR assay for the simultaneous identification of the two target species. The specificity of the Multiplex-PCR was evaluated by using both wild strains and bulk cells. Every analysis confirmed the reliable identification results provided by the single species-specific PCR methodology. The easiness, the rapidity (about 4 h from DNA isolation to results) and the reliability of the PCR procedures developed in this study highlight the advantage of their application for the specific detection and identification of the thermophilic species G. stearothermophilus and A. flavithermus.

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems.

Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life-threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 10(8) pfu ml(-1) of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (10(6) and 10(2) cfu ml(-1)). In contrast, broth inoculated with 10(4) phage and 10(2) bacteria per ml first showed normal bacterial growth until reaching a cell titre of 10(5) cfu ml(-1). Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml(-1). Phages could be produced with titres of 10(10) pfu ml(-1) in broth culture, but they were not stable upon freeze-drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.

Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection.

BACKGROUND: Enterobacter sakazakii is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. The current FDA detection method includes two enrichment steps, the subculturing of the second enrichment broth on a selective agar (VRBG), a further subculturing of selected grown colonies on TSA and the subsequent biochemical identification of yellow-pigmented colonies by API20E. However, there is a strong need for simplified methods for isolation and identification of E. sakazakii. In this study, two chromogenic media, which allow to indicate presumptive E. sakazakii colonies by the alpha glucosidase activity, as well as a newly developed 1,6-alpha-glucosidase based conventional PCR assay and a rRNA oligonucleotide probe based commercial test system for identification of presumptive E. sakazakii were evaluated on 98 target and non-target strains. The methods were compared with respect to specificity aspects. RESULTS: A total of 75 presumptive E. sakazakii and 23 non-target strains were analysed by using chromogenic media, alpha-glucosidase based PCR assay, and the VIT assay. For most presumptive E. sakazakii strains on the chromogenic media, the PCR and VIT assay confirmed the identification. However, for a number of presumptive E. sakazakii isolates from fruit powder, the alpha-glucosidase PCR and VIT assay did not correspond to the typical E. sakazakii colonies on DFI and ESIA. Further characterization by API32E identification, phylogenetic analysis of partial 16S rRNA sequences and ribotyping strongly suggested, that these strains did not belong to the species E. sakazakii. The newly developed alpha-glucosidase based PCR assay as well as the commercially available VIT Enterobacter sakazakii identification test showed an excellent correlation with the 16S rRNA data, and are thus well suited for identification of E. sakazakii. CONCLUSION: The results indicate that presumptive colonies on ESIA and DFI media need further species identification. Both evaluated molecular methods, the alpha-glucosidase PCR and the 16S RNA in situ hybridisation test (VIT), although based on completely different target regions and methodologies performed equally well in terms of specificity.

Molecular characterization of the alpha-glucosidase activity in Enterobacter sakazakii reveals the presence of a putative gene cluster for palatinose metabolism.

Enterobacter sakazakii is considered an opportunistic pathogen for premature infants and neonates. Although E. sakazakii has been isolated from various types of food, recontaminated dried infant formula has been epidemiologically identified as the major source of infection. Amongst others, alpha-glucosidase activity is one of the most important biochemical features, which differentiates E. sakazakii from other species in the family Enterobacteriaceae and has therefore been used as a selective marker in the development of differential media. However, it has been shown, that methods based on this biochemical feature are prone to producing false-positive results for presumptive E. sakazakii colonies due to the presence of this enzymatic activity in other species of the Enterobacteriaceae. Therefore, elucidation of the molecular basis responsible for the biochemical feature in E. sakazakii would provide novel targets suitable for the development of more specific and direct identification systems for this organism. By applying the bacterial artificial chromosome (BAC) approach, along with heterologous gene expression in Escherichia coli, the molecular basis of the alpha-glucosidase activity in E. sakazakii was characterized. Here we report the identification of two different alpha-glucosidase encoding genes. Homology searches of the deduced amino acid sequences revealed that the proteins belong to a cluster of gene products putatively responsible for the metabolism of isomaltulose (palatinose; 6-O-alpha-d-glucopyranosyl-d-fructose). The glycosyl-hydrolyzing activity of each protein was demonstrated by subcloning the respective open reading frames and screening of E. coli transformants for their ability to hydrolyze 4-methyl-umbelliferyl-alpha-d-glucoside. Analysis at the protein level revealed that both enzymes belong to the intracellular fraction of cell proteins. The presence of the postulated palatinose metabolism was proven by growth experiments using this sugar as a sole carbon source.

Biofilm formation, extracellular polysaccharide production, and cell-to-cell signaling in various Enterobacter sakazakii strains: aspects promoting environmental persistence.

Enterobacter sakazakii is considered an opportunistic pathogen and has been implicated in food-associated cases of meningitis or enteritis, especially in neonates and infants. The organism has been detected in various types of food and in food production units, but so far only powdered infant formula has been linked to outbreaks of disease. Survival and persistence in such environments requires the ability to adapt to high osmotic potentials and/or dry conditions. Fifty-six E. sakazakii strains were evaluated for several features important for persistence and survival: (i) biofilm formation and the putative production of cellulose as one of the components of the extracellular matrix, (ii) adherence to hydrophilic and hydrophobic surfaces, (iii) the production of extracellular polysaccharides, and (iv) the ability of E. sakazakii to produce cell-to-cell signaling molecules. Pellicle and flock formation was observed in 21 of the strains grown in Luria-Bertani broth and in 44 of the strains grown in brain heart infusion broth. Calcofluor-stained fibrils, observed microscopically in every (fragile or rigid) pellicle, suggested the presence of cellulose as an extracellular compound in this type of biofilm. Twelve isolates did not form any pellicle or flocks under either condition. Twenty-three of the isolates exhibited the potential to adhere to glass surfaces in shaken cultures, and 33 strains showed biofilm formation at the air-solid interface of polyvinyl chloride microtiter wells. Sixteen isolates adhered to both surfaces. Twenty-four of the isolates tested produced a milky, viscous mass, considered as extracellular polysaccharide. High-performance liquid chromatography analysis of the polysaccharide revealed the presence of glucose, galactose, fucose, and glucuronic acid. Thin-layer chromatography analyses performed on ethyl acetate extracts of cell-free supernatants of the 56 strains indicated the presence of two different types of acylated homoserine lactones (3-oxo-C6-HSL and 3-oxo-C8-HSL). These findings illustrate the ability of E. sakazakii to produce cell-to-cell signaling molecules.

Differentiation inside multicelled macroconidia of Fusarium culmorum during early germination.

Multicelled conidia are formed by many fungal species, but germination of these spores is scarcely studied. Here, the germination and the effects of antimicrobials on multicompartment macroconidia of Fusarium culmorum were investigated. Germ-tube formation was mostly from apical compartments. The intracellular pH (pH(in)) of the different individual cells of the macroconidia was monitored during germination. The pH(in) varied among different compartments and during different stages of germination. The internal pH was lowest in ungerminated cells and rose during germ-tube formation and was highest in new germ tubes. Antifungal compounds affect the pH(in) and differentiation of the conidia. The pH(in) inside the macroconidial compartments was lowered very fast in the presence of nystatin (1 and 4 microg/ml). At sublethal doses (0.3 microg/ml), the apical compartments were preferentially targeted showing lower pH(in) values. The reduced germination capacity of apical compartments under these conditions was compensated by an increased germination capacity of middle compartments.

Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress.

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

Assessment of the microbody luminal pH in the filamentous fungus Penicillium chrysogenum.

The enzymes of the penicillin biosynthetic pathway in Penicillium chrysogenum are located in different subcellular compartments. Consequently, penicillin pathway precursors and the biologically active penicillins have to cross one or more membranes. The final enzymatic step that is mediated by acyltransferase takes place in a microbody. The pH of the microbody lumen in penicillin producing cells has been determined with fluorescent probes and mutants of the green fluorescent protein and found to be slightly alkaline.