Showing papers in "Journal of Rapid Methods and Automation in Microbiology in 2002"
TL;DR: The results support previous work which showed a similar small rIMB deviation possibly due to the hydro-dynamic behavior of the IMBs in relatively dilute buffer suspensions and argues that IMB-based cell capture is almost exclusively a function of mass transport.
Abstract: In this manuscript we present a modified kinetic model for pathogen capture efficiency (E) variation with mixing time (Tmix) which is applicable for most combinations of immuno-magnetic bead (IMB)-target organism concentrations (e.g., [IMB] ˜ 8 × 106 IMB mL-1; Salmonella enterica serovar Enteritidis, [SE] ˜ 102-108 CFU mL-1). We found that as the ratio of [IMB] to [SE] was decreased from more than 105 to ca. 10-1, the average number of cells captured per IMB SE complex increased from ca. 1 to 4 ± 1. Concurrently, using a modified kinetic model for fitting E variation with Tmix, the maximum possible level of capture (§) was observed to decline from ˜ 100% (§˜ 1; [IMB] > > [SE]) to 23% (§˜ 0.23; [IMB]:[SE] ˜ 0.1). Using a purely kinetic approach we also found that an empirical mass transport term (yobs= (3.1 ± 1.2) × 10-9 mL IMB-1 min 1; averaged across all [SE] tested: [IMB]:[SE] ˜ 200000, 200, 3, and 0.1) did not differ much from yobs reported in earlier work (3.35 ± 0.2 × 10-9 mL min-1 IMB-1) at extremely low [SE] (e.g., ± 100 CFU mL-1). Upon inserting the classically-derived equation for γ into the kinetic model and solving for the effective IMB radius (rIMB), we found that the rIMB was 2.3 ± 0.16 μ (§ S; averaged across all [IMB]:[SE]) which was only 0.5–0.9 μ greater than the rIMB reported by the manufacturer (1.4 ± 0.2 μ). These results support previous work which showed a similar small rIMB deviation possibly due to the hydro-dynamic behavior of the IMBs in relatively dilute buffer suspensions ([IMB ˜ 8 × 106 IMB mL-1) and argues that IMB-based cell capture is almost exclusively a function of mass transport.
20 citations
TL;DR: It was determined that once the pathogen had been positively identified, it could then be recovered by culturing the cells captured on the waveguide used in the biosensor assay, which significantly improved the speed of the confirmation of the presence of thePathogen and of the isolation of the Pathogen, from 4 days using conventional methods to one day using the bios sensor assay.
Abstract: Consumption of ground beef has been implicated in the majority of outbreaks of disease from Escherichia coli O157:H7. There is a great need for rapid, sensitive, and specific microbial detection and isolation methods for the detection and recovery of pathogens such as E. coli O157:H7. Evanescent wave, fiber optic biosensors are an innovative, cutting-edge technology with the potential to meet such a need. Food pathogens such as E. coli O157:H7 can be detected in minutes using the biosensor assay rather than days using conventional methods. The biosensor assay is sensitive and presently has the ability to directly detect 102 CFU of E. coli O157:H7 in E. coli O157:H7-seeded PBS or ground beef. In this study, it was determined that once the pathogen had been positively identified, it could then be recovered by culturing the cells captured on the waveguide used in the biosensor assay. E. coli O157:H7-seeded PBS or ground beef samples were assayed using an evanescent wave, fiber optic biosensor. After a positive identification on the biosensor, the waveguide was added to modified LB medium supplemented with 10 μg/mL acriflavin (mLB) and incubated at 42C for a minimum of 4 h followed by selection on Rainbow O157 agar incubated 16 to 18 h at 37C. E. coli O157:H7 was confirmed using agglutination with E. coli O157:H7 antiserum. It was possible to recover E. coli O157:H7 colonies in E. coli O157:H7-seeded PBS or ground beef when 1 mL samples containing at least 2.0 × 103 CFU of E. coli O157:H7 had been injected into the biosensor. It took only 24 h to detect the target pathogen, recover the pathogen from the sample, and grow isolated colonies. This total time included sample preparation, detection, enrichment culture, and growth of cells to produce isolated colonies on a selective and differential medium. The identification of isolated colonies was then quickly confirmed using slide agglutination with the appropriate antiserum. This method significantly improved the speed of the confirmation of the presence of the pathogen and of the isolation of the pathogen, from 4 days using conventional methods to one day using the biosensor assay.
14 citations
TL;DR: It was found that the capture increased as the total mass of SAMB increased and greater capture with SAMB of a higher density was observed with the same bead mass, in agreement with the predictions from the hydrodynamic properties of tested SAMB.
Abstract: Two types of commercially available, streptavidin-coated magnetic beads (SAMB) were labeled with the same biotinylated anti E. coli O157 antibodies. Labeled SAMB and other necessary antibody derivatives needed for Light-addressable Potentiometric Sensor (LAPS) detection were used to capture E. coli O157:H7 in solution. The LAPS signal intensity was then used to determine the overall bacterial capture efficiency. The static and hydrodynamic properties of the beads were then used to correlate with determined efficiencies. It was found that the capture increased as the total mass of SAMB increased. Also, greater capture with SAMB of a higher density was observed with the same bead mass. In addition, the SAMB of higher density also required less time of rocking to reach a maximum capture. These observations are in agreement with the predictions from the hydrodynamic properties of tested SAMB.
13 citations
TL;DR: In this article, the authors compared the performance of two spin-column methods, namely, DNeasy Tissue and QIAamp Stool Kit, for DNA extraction from swine fecal samples and a bacterial culture extracted by a conventional phenol-chloroform extraction method.
Abstract: Application of reliable, rapid and sensitive methods to laboratory diagnosis of zoonotic infections continues to challenge microbiological laboratories. The recovery of DNA from a swine fecal sample and a bacterial culture extracted by a conventional phenol-chloroform extraction method was compared to a rapid silica-membrane spin-column method (DNeasy Tissue or QIAamp Stool Kit, QIAGEN GmbH). The two spin column methods yielded 3.5 and 2.7 μg of DNA, respectively, when the elution volume was 200 μL, compared to 1.3 and 1.5 μg of DNA, respectively, with the phenol-chloroform method.
In addition, the detection range of λ-DNA of a spectrophotometric and a fluorometric (PicoGreen) method was compared. The PicoGreen showed a quantification limit of 1 ng/mL, consistent triplicate measurements, and finally a linear relationship between the concentrations of DNA standards and the fluorescence readings (R2= 0.99 and R2= 1.00).
In conclusion, silica-membrane columns can provide a more convenient and less hazardous alternative to the conventional phenol-based method. The results have implication for further improvement of sensitive amplification methods for laboratory diagnosis.
13 citations
TL;DR: The transformed E. coli methionine auxotroph strain was transformed with a plasmid containing genes encoding ampicillin resistance and bioluminescence in order to develop a microbiological technique for methamphetamineionine quantitation.
Abstract: Methionine is one of the essential and first limiting amino acids in animal nutrition. In this study, an Escherichia coli methionine auxotroph bacterial strain that exhibits a linear growth response to methionine concentrations was transformed with a plasmid containing genes encoding ampicillin resistance and bioluminescence in order to develop a microbiological technique for methionine quantitation. Transformants were selected based on antibiotic resistance and plasmid containing candidates were confirmed by restriction enzyme digestion and gel electrophoresis. To confirm the bioluminescent phenotype, video imaging of the strain using long exposure photography yielded colonies exhibiting bioluminescence. The strain was also tested in the presence of ampicillin supplemented media with increasing methionine concentrations and growth response (measured as optical density, OD), growth rates and methionine affinities were compared before and after transformation. Although the transformed E. coli methionine auxotroph exhibited somewhat different growth kinetic responses than the nontransformed strain, the standard curves used for estimating methionine concentrations were not different. Based on the results in this study the transformed bioluminescent strain could be used as an OD-based assay if bioluminescence equipment and materials are not available.
10 citations
TL;DR: Methionine microtiter standard curves yielded linear growth responses to increasing concentrations of methionine in the range of 0 to 26.8 μM, which has potential as a rapid in vitro assay method for quantifying methamphetamineionine.
Abstract: A rapid microtiter methionine assay was developed using a methionine auxotroph E. coli strain. The bacterial strain was first grown on rich media to promote extensive bacterial growth and the cells were depleted to exhaust all endogenous methionine. After depletion, cells were transferred to minimal media with increasing concentrations of methionine and microtiter plates were incubated at 37C for 6 h. Methionine microtiter standard curves yielded linear growth responses to increasing concentrations of methionine in the range of 0 to 26.8 μM. Addition of different antibiotic and antifungal agents to the media did not significantly alter the linear growth response observed in the microtiter assay. This microtiter plate E. coli methionine assay has potential as a rapid in vitro assay method for quantifying methionine.
9 citations
TL;DR: BHI media proved to be the most consistent negative control for evaluating hilA response to poultry house water by yielding the lowest overall Miller unit values compared to other laboratory control media.
Abstract: Salmonella is regarded as a primary foodborne pathogen commonly found in the gastrointestinal tract and internal organs of poultry. However, Salmonella is also capable of surviving in fresh water supplies. In this study, our efforts were to determine which media controls of varying nutrient availability would allow for accurately assessing the potential of a poultry house water source to influence virulence gene expression in Salmonella. The cages of the sampled layer house were divided into four separate quadrants. Virulence expression was measured using a β-galactosidase assay on a hilA:lacZY fusion strain of S. Typhimurium and calculated as Miller units to adjust for bacterial cell concentration measured as optical density. Virulence assays were performed with brain heart infusion broth (BHI) serving as the primary overall control which yielded values ranging from approximately 60–100 Miller units. Results showed that standing water obtained from quadrant 2 yielded a significantly different (P<0.05) virulence responses compared to other standing water samples from other quadrants. However, no significant difference was observed between the quadrants for the fresh water samples. BHI media proved to be the most consistent negative control for evaluating hilA response to poultry house water by yielding the lowest overall Miller unit values compared to other laboratory control media.
9 citations
TL;DR: The objective of this study was to examine the feasibility of using impedance technology as an alternative method for the enumeration of clostridial spores and found that data derived were statistically comparable to those obtained using the reference method.
Abstract: Late spoilage of cheese is due to gas formation during lactic acid fermentation by spore-forming, gram-positive, anaerobic clostridia of the species Ciostridium tyrobutyricum, Clostridium butyricum and Clostridium sporogenes. Since small numbers of such clostridial spores readily cause considerable losses in cheese production, spore numbers of fewer than 100 spores/liter must be determined reliably. Until recently, the only reliable method available was the time-consuming (7 days) and cumbersome Most Probable Number Method (MPN). The objective of this study was to examine the feasibility of using impedance technology as an alternative method for the enumeration of clostridial spores. Three to fifteen replicates of 7.5–12.0 mL samples were tested using an impedimetric method with and without the addition of Oxyrase to generate anaerobic conditions within the impedance measurement cells. Results were obtained in less than 48 h. Data derived from the rapid impedance method were statistically comparable to those obtained using the reference method (MPN).
7 citations
TL;DR: There was a wide range of recoveries ranging from 0–100% when the cellulose acetate filter was used to detect phages in bioaerosols within and around broiler houses, and further studies are needed to accurately determine the collection efficiencies of viruses.
Abstract: Air quality within and around confined animal housing operations is important from both occupational exposure and environmental quality perspectives. Appropriate sampling equipment should be available so that bioaerosols are adequately characterized in terms of their component microbial populations. In this study the efficacy of a commercially available impaction-based bioaerosol sampler (SAS-100) was evaluated in terms of its ability to detect male-specific coliphages within and around poultry broiler houses. In addition to the manufacturer recommended agar medium, cellulose and cellulose-acetate filter media were also used as the collection surface. The agar medium and the cellulose ester filters provided very high recoveries of phages as compared to the cellulose filter (P<0.05). There was a wide range of recoveries ranging from 0–100% when the cellulose acetate filter was used to detect phages in bioaerosols within and around broiler houses. The results suggest that the sampler is capable of concentrating male-specific coliphages from bioaerosols. However, further studies are still needed to accurately determine the collection efficiencies of viruses.
6 citations
TL;DR: It appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.
Abstract: A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E coli strain Bacterial cells were grown in minimal media containing a concentration range of 0 to 268 μM of either L-methionine or MHA as ALIMET® Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 013) and the maximum growth rate estimates were also similar (P > 034) Methionine and MHA standard curves yielded linear responses (R2= 096) to increasing concentrations of the respective substrate Based on these results it appears that the E coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®
6 citations
TL;DR: A PCR method is developed and optimized which targets the tor A gene sequence that encodes TMAO reductase and could characterize fish freshness in the laboratory.
Abstract: Objective criteria used to assess the fish freshness in the laboratory are currently inadequate. During contamination of muscle tissue, bacteria reduce trimethylamine oxide (TMAO) to trimethylamine (TMA) in the absence of oxygen. Based on this reaction, we envisaged a new approach and have developed and optimized a PCR method which targets the tor A gene sequence that encodes TMAO reductase. We applied this method to two fish species (Whiting (Merlangus merlangus) and Pouting (Gadus luscus)) during monitoring of spoilage, in parallel with assay of TVBN and enumeration of total aerobic flora. The PCR results tally with the chemical and microbiological findings. Used in quantitative PCR, this method could characterize fish freshness.
TL;DR: Comparisons of the transposon footprints from the pools of S. typhimurium Tn5 mutants before and after exposure to desiccation revealed mutants that were not recovered after the selection were rapidly identified that would be easily isolated for further genetic analysis.
Abstract: Salmonella spp. are one of the foodborne pathogens that can be isolated in the environments of poultry houses and desiccation is a potential stress condition that can influence the survival of Salmonella spp. in this environment. In order to investigate the desiccation survival mechanism of Salmonella spp. the genome of S. typhimurium ATCC 14028 was screened for the genes potentially required for survival during desiccation using a novel method based on Tn5 mutagenesis previously developed in our laboratory. This method, termed transposon footprinting, simultaneously amplifies the Tn5-flanking sequences in a complex pool of the Tn5 mutants. As the length of the amplified DNA fragment should be unique for each distinct Tn5 mutant, the polymerase chain reaction (PCR) products separated on an agarose gel generate transposon footprints with each band in the footprint representing the corresponding Tn5 mutant. By comparing the transposon footprints from the pools of S. typhimurium Tn5 mutants before and after exposure to desiccation, Tn5 mutants that were not recovered after the selection were rapidly identified that would be easily isolated for further genetic analysis.
TL;DR: In this paper, the authors show that MS-dependent pathogen losses are related to changes in the buoyancy of the IMB·S.E. complex, and that these apparent losses in pathogen activity are not due to the injury or decease of IMB-bound bacteria inasmuch as numerous MS steps, accomplished without changing the supernatant, result in no significant diminution in bound Salmonellae.
Abstract: One question associated with the immuno-magnetic bead (IMB) protocol for pathogen isolation and subsequent concentration is why a significant number of captured cells are lost during each magnetic separation step (MS). For instance, we observe an average IMB-Salmonella concentration decrease of 4–7% per MS even at extremely low Salmonella Enteritidis cell densities ([S.E.] > 100 mL-1; [IMB]:[S.E.] 1). Such a change in [IMB·S.E.] per separation step could be significant since, in practice, more washing/rinsing steps are necessary when isolating organisms from environmental samples. These apparent losses in pathogen activity are not due to the injury or decease of IMB-bound bacteria inasmuch as numerous MS steps, accomplished without changing the supernatant, result in no significant diminution in bound Salmonellae. In this manuscript we show that [IMB·S.E.] losses monotonically increase from ca. 5 to 15% per MS with [S.E.]:[IMB]. These observations argue that MS-dependent pathogen losses are related to changes in the buoyancy of the IMB·S.E. complex since, at elevated pathogen levels ([S.E.]:[IMB]> > 1), we observe as many as 4–6 Salmonella cells binding per IMB which could decrease the IMB·S.E. complex's density by about 8% or 0.1 g cm-3.
TL;DR: The PCR-based fluorescence method could detect the target bacteria in minutes after PCR amplification compared to hours by gel electrophoresis and also could be done at an earlier time during PCR amplification.
Abstract: A DNA binding fluorescence method based on polymerase chain reaction (PCR) products was evaluated for rapid detection of Salmonella Typhimurium in poultry products. Wash water samples of chicken carcasses and ground turkey were inoculated with S. Typhimurium to obtain final concentrations of 10° - 105 CFU/mL. One mL of each sample was used to get the DNA template and 5 μL of the sample template was added into 25 μL of SYBR Green PCR Master Mix and two specific Salmonella ompC gene primers. The negative control was the same except 5 μL of each wash solution was added instead of 5 μL sample template. The reaction was carried out in a thermocycler. Finally, the fluorescence signal of each PCR product was measured using a fluorometer. The PCR products were also confirmed by ethidium bromide agarose gel, and the DNA concentrations of the PCR products were measured by a filter fluorescence photometer. The results showed that when bacterial cells increased from 0 to 2 CFU/mL, the fluorescence signal increased significantly. The PCR-based fluorescence method could detect the target bacteria in minutes after PCR amplification compared to hours by gel electrophoresis and also could be done at an earlier time during PCR amplification. The detection limit of this method for S. Typhimurium in the poultry samples was 2 CFU/mL without any enrichment.
TL;DR: The use of SFP in the FDT system provides an efficient, easy-to-use method that is less expensive than the conventional method to detect and enumerate C. perfringens in foods.
Abstract: This study compared the ability of Clostridium perfringens to recover in Fung's Double Tube (FDT) system using either tryptose-sulfite-cycloserine without egg yolk (TSC) or Shahidi-Ferguson-perfringens (SFP) agar both in three typical meat products and peptone water. A three-strain cocktail of C. perfringens spores (ATCC 10388, NCTC 8238, and NCTC 8239) was inoculated in each food system at three inoculum levels: 102, 104, and 106 cfu/g or mL. FDT system consists of a smaller glass test tube inserted into a larger glass test tube in which sample and agar have been poured. By inserting the inner tube, the agar squeezes between the exterior wall of the small tube and the interior wall of the larger tube to form a thin agar layer, which creates an anaerobic environment. Tubes are then incubated in aerobic incubators. There were no significant differences in enumeration of C. perfringens between the two media in any of the meat products and peptone water at any inoculation level. Thus, the use of SFP, which is less expensive and easier to prepare than TSC, is recommended as an alternative agar for analyses of C. perfringens in FDT system. Compared to the petri dish method, the FDT method generates greater anaerobiosis, is easier to prepare, more compact, and eliminates the need for an overlay and an anerobic chamber or jar. Combined together, the use of SFP in the FDT system provides an efficient, easy-to-use method that is less expensive than the conventional method to detect and enumerate C. perfringens in foods.
TL;DR: The constructed mutant could grow in the absence of lysine supplementation although its growth rate was significantly (P < 0.05) depressed when compared to the parent strain.
Abstract: The objective of this study was to construct an insertion mutation in the lysA gene of Escherichia coli and investigate the effect of the insertion mutation on the growth rate response. The lysA gene encodes the last enzyme in the lysine biosynthetic pathway in most bacteria. A suicide plasmid pXL1 carrying a segment of the lysA sequence was transformed into E. coli SM10 that is lysogenic for λ pir. The plasmid was extracted from the transformant and confirmed to contain the segment of lysA by restriction enzyme digestion. After this confirmation, pXL1 was transferred by conjugation to an E. coli strain that cannot support replication of the plasmid. Maintenance of the selectable drug marker on the plasmid requires that the plasmid integrate into the chromosome at the lysA locus. The constructed mutant could grow in the absence of lysine supplementation although its growth rate was significantly (P < 0.05) depressed when compared to the parent strain.
TL;DR: A new rapid test platform for the direct detection of E. coli O157:H7 in stool samples of infected patients was compared with the current standard methods, and the new test, DIAPRO FAST-Q®, used biochip ICEflo ®technology that provided results within 20 min.
Abstract: A new rapid test platform for the direct detection of E. coli O157:H7 in stool samples of infected patients was compared with the current standard methods. The new test, DIAPRO FAST-Q®, used biochip ICEflo ®technology that provided results within 20 min. Twenty-one stool samples from patients infected with E. coli O157:H7 or of unknown status were studied. Using the DIAPRO FAST-Q® method, within 20 min, we confirmed positive results for the seven known E. coli O157:H7 samples. While the standard culture method gave rise to only four positives in the remaining 14 unknown samples, the DIAPRO FAST-Q® detected E. coli O157:H7 in eight of these samples, which was confirmed subsequently by broth enrichment culture method.
TL;DR: The method sensitivity has significantly improved comparing with the visual immunoassay assessment method when detecting the presence of this bacterium in spiked beef samples and a possible new application for the CCD based light scattering instrument is demonstrated.
Abstract: A sensitive and easy-to-perform instrumentational method for the detection of Escherichia coli O157 in raw minced beef is described. The detection is based on a light scattering immunoassay and a charge-coupled device (CCD) direct readout spectrometer measuring the scattered light spectral signals at an optimized angle of 20° to the axis of transmitted light. Using latex particles coated with antibodies for E. coli O157, the method sensitivity has significantly improved comparing with the visual immunoassay assessment method when detecting the presence of this bacterium in spiked beef samples. The method is capable of detecting E. coli O157 at the level of 103 cfu mL-1 after 6 h of incubation of the spiked samples. This study has demonstrated a faster technique (within 8 h) for the detection of E. coli O157 in raw beef and a possible new application for the CCD based light scattering instrument.