Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation

The use of pathogen-free plant material is the main strategy for controlling bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. However, detection and isolation of this pathogen from seeds before field or greenhouse cultivation is difficult when the bacterium is at low concentration and associated microbiota are present. Immunomagnetic separation (IMS), based on the use of immunomagnetic beads (IMBs) coated with specific antibodies, was used to capture C. michiganensis subsp. michiganensis cells, allowing removal of non-target bacteria from samples before plating on non-selective medium. Different concentrations of IMBs and of two antisera were tested, showing that IMS with 10(6)IMBs/ml coated with a polyclonal antiserum at 1/3200 dilution recovered more than 50% of target cells from initial inocula of 10(3) to 10(0)CFU/ml. Threshold detection was lower than 10CFU/ml even in seed extracts containing seed debris and high populations of non-target bacteria. The IMS permitted C. michiganensis subsp. michiganensis isolation from naturally infected seeds with higher sensitivity and faster than direct isolation on the semiselective medium currently used and could become a simple viable system for routinely testing tomato seed lots in phytosanitary diagnostic laboratories.     

A magnetoelastic resonance biosensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium

Mass-sensitive, magnetoelastic resonance sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied, time varying, magnetic field. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that measurement is wireless and remote. A biosensor for the detection of Salmonella typhimurium was constructed by immobilizing a polyclonal antibody (the bio-molecular recognition element) onto the surface of a magnetostrictive platform. The biosensor was then exposed to solutions containing S. typhimurium bacteria. Binding between the antibody and antigen (bacteria) occurred and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Sensors with different physical dimensions were exposed to different concentrations of S. typhimurium ranging from 10(2) to 10(9)CFU/ml. Detection limits of 5x10(3) CFU/ml, 10(5) CFU/ml and 10(7) CFU/ml were obtained for sensors with the size of 2 mmx0.4 mmx15 microm, 5 mmx1 mmx15 microm and 25 mmx5 mmx15 microm, respectively. Good agreement between the measured number of bound bacterial cells (as measured by scanning electron microscopy (SEM)) and frequency shifts was obtained.     

Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride

In a biomass assay based on adenosine 5(')-triphosphate (ATP) bioluminescence, extracellular ATP is removed; then intracellular ATP is extracted from the microorganism by an ATP extractant and subsequently reacted with luciferase. To provide a highly sensitive assay, the concentration of benzalkonium chloride (BAC) in the ATP extractant was optimized by using a mutant luciferase resistant to BAC. The use of 0.2% BAC, which was acceptable for the luciferase, simultaneously achieved the maximum extraction of intracellular ATP from microorganisms and the inactivation of the ATP-eliminating enzymes for removal of extracellular ATP. The detection limit (blank+3 SD) for ATP was 1.8x10(-14)M (1.8x10(-18)mol/assay) in the presence of the ATP extractant with coefficients of variation of 0.7 to 6.3%. The reagent system coupled with the ATP-eliminating enzymes allowed for the detection of 93 colony-forming units (CFU)/ml of Escherichia coli ATCC 25922, 170CFU/ml of Pseudomonas aeruginosa ATCC 27853, 170CFU/ml of Proteus mirabilis ATCC 29906, 68CFU/ml of Staphylococcus aureus ATCC 25923, and 7.7CFU/ml of Bacillus subtilis ATCC 6051. The yeast cell of Saccharomyces cerevisiae IFO 10217 could be detected at 1CFU/ml. With 54 kinds of microorganisms, the average ATP extraction efficiency compared to the trichloroacetic acid extraction method was 81.0% in 24 strains among gram-negative bacteria, 99.4% in 13 strains among gram-positive bacteria, and 97.0% in 17 strains among yeast. The ATP contents of the gram-negative bacteria, gram-positive bacteria, and yeasts ranged from 0.40 to 2.70x10(-18)mol/CFU (mean=1.5x10(-18)mol/CFU), from 0.41 to 16.7x10(-18)mol/CFU (mean=5.5x10(-18)mol/CFU), and from 0.714 to 54.6x10(-16)mol/CFU (mean=8.00x10(-16)mol/CFU), respectively.     

Quantitative use of fluorescent in situ hybridization to examine relationships between mycolic acid-containing actinomycetes and foaming in activated sludge plants

The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid-containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods (P < 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m(3) completely mixed activated sludge plant. Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata. We tentatively estimated that the threshold foaming concentration of mycolata was about 2 x 10(6) cells ml(-1) or 4 x 10(12) cells m(-2). We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.     

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.     

Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

Washing bacteria by centrifugation through a water-immiscible layer of silicones

Hurwitz, Charles (Veterans Administration Hospital, Albany, N.Y.), Catherine B. Braun, and Richard A. Peabody. Washing bacteria by centrifugation through a water-immiscible layer of silicones. J. Bacteriol. 90:1692-1695. 1965.-A method is described which enables the separation of cells from aqueous suspension without altering the internal aqueous environment of the cells. The method consists of centrifuging the cells from the aqueous environment through a more dense, immiscible solvent consisting of a mixture of two silicones. A thin film of the aqueous environment equal to 7 x 10(-13) ml per cell remains with the bacteria during the separation procedure. The method by which this volume was determined is described. The procedure itself has no measurable effect on viability or permeability of the cells and permits recovery of about 90% of the cells from the aqueous environment. With this method, it has been found that the intracellular water volume of Escherichia coli ML 35 accessible to sucrose or inositol is 1.96 x 10(-12) ml +/- 6%, or 85% of the total volume of the cell determined by visual measurement.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Evaluation of different growth media for the recovery of the species of Alicyclobacillus

AIMS: Five different isolation media, namely potato dextrose agar (PDA), orange serum agar (OSA), K agar, yeast-starch-glucose agar and Bacillus acidocaldarius medium were evaluated for the recovery of Alicyclobacillus spp. from inoculated diluted and undiluted fruit-juice concentrates. METHODS AND RESULTS: Plates of PDA (pH 3.7), spread with vegetative cells (3.9 x 10(6) CFU ml(-1)) of Alicyclobacillus acidoterrestris from single-strength pear juice, recovered 2.9 x 10(6 )CFU ml(-1) after 5 days at 50 degrees C (74% recovery). The recovery of endospores from single-strength pear juice, after a heat treatment at 80 degrees C for 10 min, was higher on spread plates of OSA (pH 5.5) at 50 degrees C for 5 days (97% recovery). CONCLUSIONS: PDA (pH 3.7) and OSA (pH 5.5) at 50 degrees C for 3-5 days recovered the highest numbers of vegative cells and endospores of Alicyclobacillus spp. from sterilized fruit juices and concentrates. SIGNIFICANCE AND IMPACT OF THE STUDY: The most appropriate synthetic media for the recovery of Alicyclobacillus species from inoculated fruit juices and concentrates are shown.     

A comparison of sensitivity between direct plate culture, immunomagnetic separation and polymerase chain reaction for the isolation of enterohemorrhagic Escherichia coli O157]

Sensitivities of direct plate culture (DPC) method, immunomagnetic separation (IMS) method, and polymerase chain reaction (PCR) assay for successful detection Escherichia coli O157 in the food samples were compared. Three lots of minced beef and three lots of radish sprout, both of which were commercially retailed, were enriched with non-selective broth media at 36 degrees C for 6 h. After enrichment, the cultures of the minced beef and those of the radish sprout were found to have background microflora at ca.10(5)-10(7) CFU/ml and ca.10(8) CFU/ml, respectively. The cultures were then experimentally inoculated with E. coli O157 strains at various final concentrations ranging from ca.10 to 10(7) CFU/ml. The samples thus prepared were subjected to the above three methods to evaluate their detection limits. For the samples of minced beef, the detection limits of the DPC method was 10(2) CFU/ml whilst that of the IMS method was ca.10 CFU/ml. For the samples of radish sprout, the detection limits of the DPC method, the IMS method, and the PCR assay were ca.10(4) CFU/ml, ca.10(2) CFU/ml, and ca.10(6) CFU/ml, respectively. There results strongly suggest that the IMS method is most sensitive method for the detection of O157 from food samples among the methods currently available.     

Detection of low levels of Listeria monocytogenes cells by using a fiber-optic immunosensor

Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 x 10(3) CFU/ml for a pure culture of L. monocytogenes grown at 37 degrees C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10 degrees C with 3.5% NaCl, the detection threshold was 4.1 x 10(4) or 2.8 x 10(7) CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.     

Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice

Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1). They were still 50-80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1). The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1). Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.     

Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis

The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6+/-0.2%) and sucrose the highest (6.4+/-1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1 x 10(7) to 5 x 10(10) CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium. The highest freeze-drying survival values, 15-25%, were obtained for initial cell concentrations between 1 x 10(9) and 1 x 10(10) CFU/ml. For cell concentrations outside this window more than 10 times lower survival values were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26+/-6%.     

Manganese Oxidation by Bacterial Isolates from the Indian Ridge System

The abundance and activity of culturable manganese-oxidizing bacteria were assessed from near-bottom water samples of the tectonically active Carlsberg Ridge. Retrievable counts as colony forming units (CFU) on dilute nutrient agar medium (dilNA = 2 gm l⁻¹ nutrient broth+2% agar) and on dilNA supplemented with 1, 2 and 3 mM MnCl₂·4H₂O were in the order of 10⁶ CFU l⁻¹. Retrievability of heterotrophs ranged from non-detectable levels (ND) to 2.82 × 10⁶ CFU l⁻¹. The retrievable counts on Mn amended dilNA ranged from ND to 3.21× 10⁶, 1.47 × 10⁶ and 1.45 × 10⁶ CFU l⁻¹ on 1, 2 and 3 mM, respectively. About 87% of the Mn tolerant isolates (n = 39) showed taxonomic affinities to Pseudomonas I and II sp. Two representative strains CR35 and CR48 (CR–Carlsberg Ridge) isolated on manganese-supplemented media were tested for their ability to tolerate a range of Mn amendments from 1 nM to 100 mM in terms of growth and respiration. CR35 represents 66% of the total CFU (3.04 × 10⁶ CFU l⁻¹), while CR48 represented only 6% of the total CFU (1.05 × 10⁶ CFU l⁻¹). The colonies of these two isolates were dark brown in color suggesting precipitation of Mn as oxide. Tests for the effect on growth and respiration were conducted in media simulating heterotrophic (amended with 0.01% glucose) and lithotrophic (unamended) conditions. Maximum stimulation in growth and respiration of CR35 occurred at 100 μM Mn both in unamended and amended media. At levels of Mn greater than 100 μM the counts decreased steadily. Total respiring cells of CR48 were stimulated to a maximum at 1 μM Mn in unamended medium and 1 nM in amended medium. Total cells counts for the same decreased beyond 100 μM Mn in unamended and 1 nM in amended medium. The isolates were tested for their ability to oxidize Mn ammendments from 1 μM to 10 mM Mn. At the end of a 76-day incubation period, there was evidence of manganese oxide precipitation at high Mn concentrations (≥1 mM) as a dark brown coloration on the sides of culture tubes. Highest Mn oxidation rates were observed at 10 mM Mn(II) concentration with CR35 oxidizing 27 and 25 μM Mn day⁻¹ in unamended and amended condition, respectively. CR48 oxidized Mn at the rate of 26 μM Mn day⁻¹ in unamended medium and 35 μM Mn day⁻¹ in amended medium. Scanning electron microscope (SEM) observations of both isolates revealed free-living cells in clustered matrices ≈2 μm diameter. Energy dispersive spectrum of the cell matrix of CR35 cultured in 1 mM Mn detected 30% Mn, while the cell aggregates of CR48 harbored 7–10% Mn. The relatively high specific activity of these mixotrophic bacteria under relatively oligotrophic conditions suggests that they may be responsible for scavenging dissolved Mn from the Carlsberg Ridge waters and could potentially participate in oxidation.     

Lateral diffusion of the secretory component (SC) in the basolateral membrane of the human colon carcinoma cell line HT29 assessed with fluorescence recovery after photobleaching

The lateral diffusion of the secretory component (SC), acting as a receptor for dimeric IgA in the basolateral side of intestinal epithelial cells, was studied in the human colonic carcinoma cell line HT29. The HT29 cells were grown in Dulbecco's modified Eagle's medium in which galactose had been substituted for glucose to promote development of small intestine-like cells, with a distinct separation of the basolateral side from the apical surface. The SC was stained with rhodamine-labeled polyclonal anti-human SC rabbit antibodies (Ig) or Fab fragments, and the lateral mobility was assessed with the fluorescence recovery after photobleaching technique. The average lateral diffusion was consistent with a diffusion constant of 7.7 +/- 2.0 (mean value +/- SD; n = 29) and 7.1 +/- 2.3 (n = 30) x 10(-10) cm2s-1 for Ig-and Fab-labeled receptors, respectively, which is slower than lipid diffusion but is similar to that found for other membrane receptors. The corresponding values for the fraction of mobile receptors were 66 +/- 13% and 71 +/- 12%, respectively. Cells were labeled from the top of the culture plate, and cells adjacent to a mechanically made rift or a natural opening in the cell monolayer were labeled more strongly, confirming the microscope-based impression that the basolateral surface primarily harboured the SC receptor.     

Persistence and multiplication of obligate anaerobe bacteria in amebae under aerobic conditions

After co-cultivation of Mobiluncus curtisii, an obligate non-sporeforming anaerobe, with free living amebae from the Acanthamoeba spp. under aerobic conditions, internalization, multiplication and persistence of bacterial cells were established for at least 4-6 weeks. Under the same conditions and media without viable amebae, the cells of M. curtisii did not replicate and died in 4-7 days. The infection of amebae occurred with 10 to 100 bacteria per ml of co-cultivation media. In 7-14 days the amount of bacterial cells increased to 1x10(5)-1x10(6) CFU/mL. Electron microscopic examinations revealed bacteria within vacuoles in the amebae and intracellular replication. These results suggest a previously undescribed mechanism for spread, replication and persistence of obligately anaerobe bacteria in the environment and new possible sources, reservoirs and transfer mechanisms of infections caused by obligate anaerobe bacteria.     

Lactobacillus rhamnosus strain GG reduces aflatoxin B1 transport, metabolism, and toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B1 (AFB1) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB1 availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB1 levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB1 transport from the apical to the basolateral chamber was reduced from 11.1%+/-1.9% to 6.4%+/-2.5% (P=0.019) and to 3.3%+/-1.8% (P=0.002) within the first hour in monolayers coincubated with GG (1x10(10) and 5x10(10) CFU/ml, respectively). GG (1x10(10) and 5x10(10) CFU/ml) bound 40.1%+/-8.3% and 61.0%+/-6.0% of added AFB1 after 1 h, respectively. AFB1 caused significant reductions of 30.1% (P=0.01), 49.4% (P=0.004), and 64.4% (P<0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1x10(10) CFU/ml GG after 24 h protected against AFB1-induced reductions in transepithelial resistance at both 24 h (P=0.002) and 48 h (P=0.04). DNA fragmentation was apparent in cells treated only with AFB1 cells but not in cells coincubated with either 1x10(10) or 5x10(10) CFU/ml GG. GG reduced AFB1 uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.     

Rapid and sensitive biosensor for Salmonella

The rapid and sensitive detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated. The binding of bacteria to the surface changed the crystal resonance parameters; these were quantified by the output voltage of the sensor instrumentation. The sensor had a lower detection limit of a few hundred cells/ml, and a response time of < 100 s over the range of 10(2)-10(10) cells/ml. The sensor response was linear between bacterial concentrations of 10(2)-10(7) cells/ml, with a sensitivity of 18 mV/decade. The binding of bacteria was specific with two binding sites needed to bind a single cell. The sensors preserve approximately 75% of their sensitivity over a period of 32 days.