Secondary selective enrichment of salmonellae from naturally contaminated specimens by using a selective motility system.

A selective motility medium was used as a secondary selective enrichment medium to examine specimens naturally contaminated with salmonellae. The medium, incubated at 37 degrees C, was inoculated from either selenite brilliant green sulfa enrichment broth or Müller-Kauffman tetrathionate broth, both of which had been incubated at 42 degrees C. The use of the selective motility medium resulted in an increase in the number of positive specimens from 65 and 74% to 80 and 82%, when inoculated at 24 and 48 h, respectively, from tetrathionate broth. Tetrathionate broth, when used singly, was significantly better than selenite brilliant green sulfa broth, which detected 55% of positive specimens at both 24 and 48 h. The use of the selective motility medium of Harper and Shortridge (J. Hyg. 67: 181--186, 1969) for the further examination of specimens culturally negative on primary selective enrichment is advocated.     

Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated retail turkey meat products

AIMS: The aim of this study was to compare the real-time iQ-Check Salmonella kit (Bio-Rad) with the immunocapture assay RapidCheck Salmonella method, and a conventional culture method (FSIS, USDA) in detecting Salmonella in naturally contaminated turkey meat products. This study was also designed to determine if a selective enrichment step might improve the real-time detection of Salmonella. METHODS AND RESULTS: Using the culture method, Salmonella was recovered from 49 out of 99 retail turkey meat samples collected. RapidCheck failed to detect 11 Salmonella samples that were positive by the culture method. The iQ-Check real-time PCR also failed to detect three samples that were positive by the culture method. However, when carried out after a selective enrichment step, the iQ-Check real-time PCR detected all 49 Salmonella samples recovered by the culture method. The iQ-Check real-time PCR detected the presence of Salmonella in some samples that were not recovered by the culture method. CONCLUSIONS: Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated turkey meat samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The iQ-Check Salmonella real-time PCR can be used as a rapid method to monitor Salmonella in turkey meat, together with conventional culture methodology.     

Effects of enrichment media and incubation conditions on isolating salmonellae from ground-meat filtrate.

Forty-eight combinations of enrichment media, secondary enrichment, incubation times and temperatures, and atmospheres were examined for their efficacy in recovering different serovars of Salmonella that had been inoculated into ground-meat extract. Variations included three selective-enrichment media, two (37 and 43 degrees C) incubation temperatures, two (24 and 48 h) incubation times, two (aerobic and anaerobic) incubation atmospheres, and secondary enrichment to two of the selective-enrichment media. The ratio of Salmonella to other microorganisms was 10: greater than 1,000,000. One-hundred and twenty-four tests were conducted for each enrichment under each condition of incubation. None of the methods recovered Salmonella in more than 60% of the trials. Salmonella typhimurium was recovered most frequently of the serovars tested; S. abortusovis was recovered least frequently. There was considerable variation in the results obtained by the different methods, but there was a statistically significant advantage in the 43 degrees C incubation temperature. Secondary enrichment in tetrathionate broth showed a statistically significant advantage over secondary enrichment in selenite broth. Secondary enrichment into a different medium from the primary enrichment also was advantageous.     

Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media.

Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.     

Isolation and enumeration of Campylobacter jejuni from poultry products by a selective enrichment method.

A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delayed secondary enrichment for the isolation of salmonellae from broiler chickens and their environment.

Specimens collected from six broiler flocks were cultured for salmonellae by three methods. (i) For direct enrichment, the specimen was homogenized, and 1 ml of the homogenate was inoculated into tetrathionate-brillant green broth; (ii) for preenrichment, liquid specimens and homogenates were incubated at 37 degrees C, and on the next day 1 ml was inoculated into tetrathionate-brillant green broth; and (iii) for delayed secondary enrichment, incubated preenrichment cultures were held at room temperature for 7 to 10 days and then subcultured to fresh tetrathionate-brilliant green broth. All tetrathionate-brilliant green broth cultures were incubated at 42 degrees C for 24 to 48 h before plating. Significantly more isolations of salmonellae were obtained by delayed secondary enrichment than by direct enrichment or preenrichment. Salmonellae were isolated from 417 of 2,283 (18.3%) samples of litter, intestinal contents, and feces cultured by all three methods. Of these positive specimens, direct enrichment detected 208 (49.9%), preenrichment detected 282 (67.6%), and delayed secondary enrichment detected 373 (89.4%). Of 896 specimens of swabs and rinse fluids that were cultured by preenrichment and delayed secondary enrichment, 259 (28.9%) yielded salmonellae. Delayed secondary enrichment detected 254 (98.1%) of these, and preenrichment detected 147 (56.8%). A total of 23 serotypes of salmonellae were identified. The greater effectiveness of delayed secondary enrichment for the isolation of salmonellae was not likely due to the selection of certain serotypes or to an increased inhibition of competing flora.     

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group.

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.     

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group.

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.     

Recovery of Campylobacter jejuni and Campylobacter coli from inoculated foods by selective enrichment.

A direct enrichment procedure was developed to selectively recover small numbers of Campylobacter jejuni, C. coli, and nalidixic acid-resistant thermophilic Campylobacter from foods. The procedure includes an enrichment medium composed of brucella broth, 7% lysed horse blood, 0.3% sodium succinate, 0.01% cysteine hydrochloride, vancomycin (15 micrograms/ml), trimethoprim (5 micrograms/ml), polymyxin B (20 IU/ml), and cycloheximide (50 micrograms/ml) that is inoculated with 10 or 25 g of food and incubated with agitation under microaerophilic conditions at 42 degrees C for 16 to 18 h. After incubation, the medium is plated directly onto Campy-BAP agar plates (M. J. Blaser et al., Ann. Intern. Med. 91:179-185, 1979), and resulting colonies that resemble Campylobacter are identified by conventional tests. The foods evaluated included raw milk, hamburger, and chicken skin which had aerobic plate counts of 10(5) to 10(9) bacteria/g. The procedure was effective in recovering as few as 0.1 cell of Campylobacter per g of food. Of the 50 isolates of Campylobacter evaluated, all were recovered from raw milk and hamburger at a level of 1 to 4 cells/g, and 41 and 40 isolaes were recovered from the hamburger and milk, respectively, at 0.1 to 0.4 cell/g. The enrichment was least effective for recovering campylobacters from chicken skin, as 7 and 26 of 50 isolates were not recovered at 1 to 4 and 0.1 to 0.4 cell/g, respectively. This new procedure is more rapid, direct, and effective than other enrichment or direct plating procedures for recovering small numbers of campylobacters from foods.     

Isolation and enumeration of Campylobacter jejuni from poultry products by a selective enrichment method

A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns

Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented.     

Pasteurization of naturally contaminated water with solar energy.

A solar box cooker (SBC) was constructed with a cooking area deep enough to hold several 3.7-liter jugs of water, and this was used to investigate the potential of using solar energy to pasteurize naturally contaminated water. When river water was heated either in the SBC or on a hot plate, coliform bacteria were inactivated at temperatures of 60 degrees C or greater. Heating water in an SBC to at least 65 degrees C ensures that the water will be above the milk pasteurization temperature of 62.8 degrees C for at least an hour, which appears sufficient to pasteurize contaminated water. On clear or partly cloudy days, with the SBC facing magnetic south in Sacramento, bottom water temperatures of at least 65 degrees C could be obtained in 11.1 liters of water during the 6 weeks on either side of the summer solstice, in 7.4 liters of water from mid-March through mid-September, and in 3.7 liters of water an additional 2 to 3 weeks at the beginning and end of the solar season. Periodic repositioning of the SBC towards the sun, adjusting the back reflective lid, and preheating water in a simple reflective device increased final water temperatures. Simultaneous cooking and heating water to pasteurizing temperatures was possible. Additional uses of the SBC to pasteurize soil and to decontaminate hospital materials before disposal in remote areas are suggested.     

Highly selective enrichment of phosphorylated peptides using titanium dioxide

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).     

Influence on motility of Escherichia coli and Salmonella typhimurium by a naturally occurring conjugative plasmid.

In a collection of 45 R-plasmids, one was found to be associated with loss of motility of its Escherichia coli K-12 and Salmonella typhimurium host bacteria when tested in conventional motility agar. Genetic experiments, as well as analyses of deoxyribonucleic acid, showed that inhibition of motility was caused by a conjugative plasmid that was separate from the R-plasmid. This second plasmid, named pUM5, was fi- and mediated the same type of sex pilus (F-like) as the accompanying R-plasmid but lacked resistance determinants. Preliminary studies indicated that bacterial cells carrying the motility inhibition plasmid pUM5 were still equipped with flagella. The mechanism by which flagellar action is disturbed by the plasmid is presently not known.     

Glycine-containing selective medium for isolation of Legionellaceae from environmental specimens.

Glycine, at a final concentration of 0.3%, has been shown to be an excellent selective agent for the isolation of Legionellaceae. Stock cultures of Legionella pneumophila were not inhibited on buffered charcoal-yeast extract agar containing the amino acid. Among the other Legionellaceae tested, only one of two strains of L. dumoffii and two of six strains of L. micdadei were appreciably inhibited. This medium permitted the isolation of L. pneumophila from environmental specimens with marked inhibition of many non-Legionellaceae bacteria. The selectivity of the medium was subsequently improved by the incorporation of vancomycin (5 microgram/ml) and polymyxin B (100 U/ml). This selective medium, glycine-vancomycin-polymyxin B agar, should facilitate the recovery of Legionellaceae from environmental sources.     

Highly selective carbamate-based butyrylcholinesterase inhibitors derived from a naturally occurring pyranoisoflavone

This current study described the design and synthesis of a series of derivatives based on a natural pyranoisaflavone, which was obtained from the seeds of Millettia pachycarpa and displayed attractive BChE inhibition and high selectivity in our previous study. The inhibitory potential of all derivatives against two cholinesterases was evaluated. Only a few compounds demonstrated AChE inhibitory activity at the tested concentrations, while 26 compounds showed significant inhibition on BChE (the IC₅₀ values varied from 9.34 μM to 0.093 μM), most of them presented promising selectivity to ward BChE. Prediction of ADME properties for 7 most active compounds was performed. Among them, 9g (IC₅₀ = 222 nM) and 9h (IC₅₀ = 93 nM) were found to be the most potent BChE inhibitors with excellent selectivity over AChE (SI ratio = 1339 and 836, respectively). The kinetic analysis demonstrated both of them acted as mixed-type BChE inhibitors, while the molecular docking results indicated that they interacted with both residues in the catalytic active site. A cytotoxicity test on PC12 cells showed that both 9g and 9h had a therapeutic safety range similar to tacrine. Overall, the results indicate that 9h could be a good candidate of BChE inhibitors.