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PubMed2004

Impairment of growth of Listeria monocytogenes in THP-1 macrophages by granulocyte macrophage colony-stimulating factor: release of tumor necrosis factor-alpha and nitric oxide.

BACKGROUND: Listeria monocytogenes tends to survive in phagocytes. Granulocyte macrophage colony-stimulating factor (GM-CSF) protects mice against L. monocytogenes infection, and mice knocked out for the GM-CSF gene are more susceptible to these infections. METHODS: THP-1 cells were used to characterize the GM-CSF receptor (binding isotherms; STAT5 phosphorylation), measure the intracellular growth of L. monocytogenes (5 h after phagocytosis), examine the influence of a 24-h incubation with GM-CSF before infection, measure the production of tumor necrosis factor (TNF)-alpha and the expression of nitric oxide synthase (iNOS), and evaluate the influence of anti-GM-CSF receptor (GM-CSFR alpha ) and anti-TNF-alpha antibodies and the addition of N(omega)-nitro-L-arginine methyl ester (L-NAME) and catalase. RESULTS: THP-1 cells display functional GM-CSFR alpha. GM-CSF impairs the intracellular growth of L. monocytogenes to approximately 65% of its value in unstimulated cells. This effect is abolished by anti-GM-CSFR alpha, anti-TNF-alpha antibodies, and catalase (and, to a lesser extent, by L-NAME). GM-CSF stimulates the release of TNF-alpha and the expression of iNOS. TNF-alpha added to unstimulated cells (even in large amounts) does not fully reproduce the impairment in the growth of L. monocytogenes caused by GM-CSF. CONCLUSIONS: GM-CSF impairs the intracellular growth of L. monocytogenes by a synergistic action of the GM-CSF-triggered release of autocrine TNF-alpha and hydrogen peroxide and the production of NO (associated with the stimulation of the expression of iNOS).

Blotting, Western↗

PubMed2000

Shelf life of ground poultry meat stored under modified atmosphere.

The shelf life of ground chicken and turkey meat packaged under a modified atmosphere containing O2 and a high level of CO2 (62% CO2, 8% O2, and 30% N2; gas-1), or a gas mixture without O2 (20% CO2 and 80% N2; gas-2) was evaluated for 20 d at 1 C. Meat packaged under gas-2 maintained a higher a* value (redness) throughout the experiment and generally had a more appealing color than the meat packaged using gas-1. Microbial populations were assessed after 8, 12, and 15 d of storage. Total aerobic mesophilic counts were higher in chicken meat than in turkey throughout storage. Coliforms and Escherichia coli counts were lower in meat packaged under gas-1. After 15 d of storage at 1 C, Brochothrix thermosphacta was detected only in ground chicken meat packaged using gas-2. Meat packaged under the gas mixtures tested had similar counts for presumptive pseudomonads, Staphylococcus aureus, and lactic acid bacteria. These results indicate that an appropriate gas mixture can maintain a desirable color in ground poultry meat but offers no guarantees with respect to the microbial profile of meat.

Animals↗

PubMed2002

Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment.

The majority (>99%) of microorganisms from the environment resist cultivation in the laboratory. Ribosomal RNA analysis suggests that uncultivated organisms are found in nearly every prokaryotic group, and several divisions have no known cultivable representatives. We designed a diffusion chamber that allowed the growth of previously uncultivated microorganisms in a simulated natural environment. Colonies of representative marine organisms were isolated in pure culture. These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms. This observation may help explain the nature of microbial uncultivability.

Bacteria↗

PubMed2000

Rapid virus production and removal as measured with fluorescently labeled viruses as tracers.

Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h(-1) and production rates in the dark ranging from 1.9 to 6.1% h(-1), corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.

Bacteria↗

PubMed2003

Lack of influence of body exhaust gowns on aerobic bacterial surface counts in a mixed-ventilation operating theatre. A study of 62 hip arthroplasties.

Aerobic bacterial surface contamination was studied with and without the use of body exhaust gowns in an operating room equipped with mixed/turbulent ventilation and separate operating and anaesthetic areas during 62 hip joint arthroplasties. In 31 operations conventional gowns were used, and 31 were performed with body exhaust gowns. Bacterial surface contamination was monitored in the operating and anaesthetic area using 9 cm diameter settle plates (1+1) and nitrocellulose membranes (2+2) transferred after sampling to nutrient pads. Compared with conventional clothing, the use of body exhaust gowns did not significantly reduce the microbial contamination (P=0.1-0.7). On the settle plates 1 m from the patient 279+/-326 cfu/m(2)/h were observed with conventional clothing compared with 142+/-227 cfu/m(2)/h with body exhaust gowns. The first membrane located on the patient in the sterile area detected 250+/-590 cfu/m(2)/h with conventional clothing and 210+/-320 cfu/m(2)/h with exhaust gowns. For the second membrane on the floor, the counts were 1790+/-2700 and 1590+/-1590 cfu/m(2)/h. For all operations the settle plates yielded 210+/-287 cfu/m(2)/h in the operating area and 720+/-564 cfu/m(2)/h in the anaesthetic area (P=0.01). Compared with the membrane placed on the anaesthetic equipment the counts on the membrane placed on patient were also significantly lower (P=0.01) while the membranes placed on the floor in each area showed no difference in counts. In conclusion, compared with conventional clothing, the use of body exhaust gowns could not be proven to provide more protection against microbial contamination. The low number of colony forming units found in the operating area was similar to that expected from an ultraclean laminar airflow unit, although achieved with a cheaper and more energy saving system.

Adult↗

PubMed1999

Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ.

We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial "black box" should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the alpha subdivision of the class Proteobacteria (alpha-Proteobacteria) and of the Cytophaga-Flavobacterium group obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined ( approximately 60% of the universal probe-labeled cells and approximately 50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the alpha-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the "dissection" of microbial communities by type and function.

Animals↗

PubMed2005

Comparison of microbial numbers in soils by using various culture media and temperatures.

The influence of different media and incubation temperatures on the quantification of microbial populations in sorghum, eucalyptus and forest soils was evaluated. Microbial growth was compared by using complex (tryptone soybean agar, TSA, casein-starch, CS, and Martin) and saline (Thorton, M3, Czapeck) media and incubation temperatures of 25 and 30 degrees C. Higher numbers of total bacterial and fungal colony-forming units (CFU) were observed in sorghum soils, and of spore-forming and Gram-negative bacteria in forest soils than other soils. Actinomycetes counts were highest in forest soil when using CS medium at 30 degrees C and in sorghum soil at 25 degrees C in M3 medium. Microorganism counts were dependent on the media and incubation temperatures. The counts at temperatures of 30 degrees C were significantly higher than at 25 degrees C. Microbial quantification was best when using TSA medium for total and spore-forming bacteria, Thorton for Gram-negative bacteria, M3 for actinomycetes, and Martin for fungi.

Bacteria↗

PubMed1993

Microbiological status of rabbit carcases in Egypt.

A total of 40 New Zealand white rabbits, 20 freshly slaughtered rabbits from an experimental farm and 20 processed rabbit carcases from grocery stores in Beni-Suef city, were examined bacteriologically. The mean values of aerobic plate counts at 37 degrees C and 1 degrees C, Enterobacteriaceae counts, Pseudomonas counts and Staphylococcus counts of freshly slaughtered rabbits were 10(4) +/- 2 x 10(3), 8 x 10(2) +/- 10(2), 6 x 10(2) +/- 10(2), 3 x 10(2) +/- 10(2), and 10(2) +/- 60 organisms per gram respectively, whereas the equivalent values were 8 x 10(5) +/- 3 x 10(4), 2 x 10(5) +/- 10(4), 4 x 10(4) +/- 8 x 10(3), 2 x 10(4) +/- 6 x 10(3), and 4 x 10(3) +/- 4 x 10(2) organisms per gram of processed rabbit carcases from grocery stores, respectively. The freshly slaughtered rabbits showed a low bacterial load compared with processed rabbit carcases from grocery stores. Escherichia coli and Listeria monocytogenes could be isolated from the rabbit carcases examined in addition to Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus from processed rabbit carcases. Yersinia enterocolitica could not be isolated from any sample examined. Suggested measures to reduce the microbial load in rabbit carcases are discussed.

Animals↗

PubMed1992

Subgingival temperature (III). Relation to microbial counts.

The present investigation examined the relationship of selected bacterial species and subgingival temperature. 35 subjects were measured at 6 sites per tooth for clinical parameters and subgingival temperature. Measurements were repeated for 21 subjects at 2 month intervals providing a total of 66 subjects visits. At each visit, subgingival plaque samples were taken from the mesial aspect of each tooth and anaerobically dispersed, diluted and plated on non-selective media. After anaerobic incubation, colonies were lifted to nylon filters and specific species detected using digoxigenin-labeled whole chromosomal DNA probes. Species enumerated were; A. actinomycetemcomitans serotypes a and b, B. forsythus, B. gingivalis, B. intermedius I and II, C. ochracea, F. nucleatum ss. vincentii, P. micros, S. intermedius, S. sanguis I and II, V. parvula and W. recta. Total viable counts and counts of Capnocytophaga sp. were determined directly from the primary isolation plates. A total of 1581 samples were evaluated. Subject visits with higher mean subgingival temperatures had significantly higher mean %s of B. intermedius I and P. micros, and lower mean %s of Capnocytophaga sp. Sites with higher subgingival temperatures had elevated proportions of B. intermedius I and II, A. actinomycetemcomitans serotype a and B. gingivalis more frequently than sites with lower temperatures, while Capnocytophaga sp. were elevated more often at cooler sites. 43 of the subject visits had follow up attachment level measurements at 2 months. The 1026 microbial samples and the subgingival temperature measurements from these visits were related to longitudinal attachment change.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed2006

Effectiveness of radiation processing in elimination of Salmonella typhimurium and Listeria monocytogenes from sprouts.

The effectiveness of radiation treatment in eliminating Salmonella Typhimurium and Listeria monocytogenes on laboratory inoculated ready-to-eat sprouts was studied. Decimal reduction doses (D10-values) for Salmonella Typhimurium and L. monocytogenes in dry seeds of mung (green gram), matki (dew gram), chana (chick pea), and vatana (garden pea) ranged from 0.189 to 0.303 kGy and 0.294 to 0.344 kGy, respectively. In sprouts made from these seeds, the D10-values ranged from 0.192 to 0.208 kGy for Salmonella Typhimurium and from 0.526 to 0.588 kGy for L. monocytogenes. Radiation treatment with a 2-kGy dose resulted in complete elimination of 10(4) CFU/g of Salmonella Typhimurium and 10(3) CFU/g of L. monocytogenes from all the four varieties of sprouts. No recovery of Salmonella Typhimurium and L. monocytogenes was observed in the radiation treated samples stored at 4 and 8 degrees C up to 12 days. Radiation treatment with 1 kGy and 2 kGy resulted in a reduction of aerobic plate counts and coliform counts by 2 and 4 log CFU/g, respectively; the yeast and mold counts and staphylococci counts decreased by 1 and 2 log CFU/g, respectively. However, during postirradiation storage at 4 and 8 degrees C, aerobic plate counts, coliform counts, yeast and mold counts, and staphylococci counts remained constant throughout the incubation period. This study demonstrates that a 2-kGy dose of irradiation could be an effective method of processing to ensure microbial safety of sprouts.

Colony Count, Microbial↗

PubMed2002

The degree of methylation influences the degradation of pectin in the intestinal tract of rats and in vitro.

We investigated the degradation, metabolism, fate, and selected effects of pectin in the intestinal tract of rats. Conventional and germfree rats were fed for 3 wk diets containing 6.5% pectin (degree of methylation 34.5, 70.8 and 92.6%, respectively) or pectin-free diets. Pectin passes the small intestine as a macromolecule. The molecular weight distribution of pectins isolated from intestinal contents of germfree rats were unaffected by diet. No or very little galacturonan was found in cecum, colon or feces of most of the conventional rats. In colon contents of some conventional rats, di- and trigalacturonic acid were present. Total anaerobic and Bacteroides counts were greater in groups fed pectin. The concentration of short-chain fatty acids (SCFA) was higher in cecum and feces in all pectin-fed groups. With increasing degree of methylation, the formation rate of SCFA decreased in the cecum of conventional rats. During in vitro fermentation of pectin with fecal flora from rats, unsaturated oligogalacturonic acids appeared as intermediate products. Low-methoxyl pectin was fermented faster than high-methoxyl pectins in vivo and in vitro. Pectin-fed rats had greater ileum, cecum and colon weights. We conclude that structural parameters of pectin influence its microbial degradation in the intestinal tract.

Animals↗

PubMed2002

Composition and physiological profiling of sprout-associated microbial communities.

The native microfloras of various types of sprouts (alfalfa, clover, sunflower, mung bean, and broccoli sprouts) were examined to assess the relative effects of sprout type and inoculum factors (i.e., sprout-growing facility, seed lot, and inoculation with sprout-derived inocula) on the microbial community structure of sprouts. Sprouts were sonicated for 7 min or hand shaken with glass beads for 2 min to recover native microfloras from the surface, and the resulting suspensions were diluted and plated. The culturable fraction was characterized by the density (log CFU/g), richness (e.g., number of types of bacteria), and diversity (e.g., microbial richness and evenness) of colonies on tryptic soy agar plates incubated for 48 h at 30 degrees C. The relative similarity between sprout-associated microbial communities was assessed with the use of community-level physiological profiles (CLPPs) based on patterns of utilization of 95 separate carbon sources. Aerobic plate counts of 7.96 +/- 0.91 log CFU/g of sprout tissue (fresh weight) were observed, with no statistically significant differences in microbial cell density, richness, or diversity due to sprout type, sprout-growing facility, or seed lot. CLPP analyses revealed that the microbial communities associated with alfalfa and clover sprouts are more similar than those associated with the other sprout types tested. Variability among sprout types was more extensive than any differences between microbial communities associated with alfalfa and clover sprouts from different sprout-growing facilities and seed lots. These results indicate that the subsequent testing of biocontrol agents should focus on similar organisms for alfalfa and clover, but alternative types may be most suitable for the other sprout types tested. The inoculation of alfalfa sprouts with communities derived from various sprout types had a significant, source-independent effect on microbial community structure, indicating that the process of inoculation alters the dynamics of community development regardless of the types of organisms involved.

Brassica↗

PubMed1999

Phenotypic changes in the lipopolysaccharide of Pseudomonas aeruginosa and Escherichia coli grown in milk-based enteral nutrition solutions.

Previous studies have shown enteral nutritional solutions (ENS) contaminated with large numbers of microorganisms from the environment or gastrointestinal (GI) tract of patients have caused respiratory infections, acute and chronic enteritis, and septicemia. The introduction of "closed" enteral feeding systems has been used to prevent contaminating organisms from entering enteral feeding systems in large numbers. However, there is some discussion as to whether this has been an effective measure in reducing ENS-related infections because there is anecdotal evidence to suggest that disease processes resulting from enteral feeding are still commonplace in the hospital and home. This is because there is very little information about the growth of microorganisms in ENS and whether growth in ENS may affect the virulence and pathogenicity of microorganisms. This study shows that Escherichia coli and Pseudomonas aeruginosa may grow at 25 degrees C from either high or low initial numbers to up to 9.2 log colony-forming units per mL in a range of milk-based ENS. However, these organisms did not grow in the fruit-based ENS. The effect on the lipopolysaccharide (LPS) of culturing E. coli and P. aeruginosa in milk-based ENS as opposed to standard laboratory media was examined using polyacrylamide gel electrophoresis. We found that there were significant qualitative changes in the phenotype of O-polysaccharide side chains of the LPS from these organisms. O-polysaccharide is known to mediate in the complement, antibiotic and bile resistance, and affect adherence. Therefore, changes in the virulence and pathogenicity of these microorganisms when cultured in ENS may be indicated. Thus, the study provides further evidence for reevaluating the microbiologic and immunologic effects of enteral feeding, especially on the microbial flora of the GI tract.

Animals↗

PubMed2003

Microbial contamination of artificially incubated Greater Rhea (Rhea americana) eggs.

1. This paper is a report of biological agents that contaminate Greater Rhea (Rhea americana) eggs during artificial incubation. 2. The cleanliness of eggs when collected, and the period of storage prior to incubation, were investigated to assess their effects on microbial contamination and hatchability. 3. A total of 14 bacteria and 4 fungi species were isolated within the egg in the laboratory. 4. Microbial contamination was higher (24%) in very dirty eggs than in eggs which were clean or dirty (16%). Hatching success was lower (30%) for very dirty eggs, compared with 42% for clean or dirty eggs. 5. The percentage of microbial contamination of stored eggs (10%) did not differ significantly from that of non-stored ones (5%). 6. The extreme lower and upper limits of infection rate estimated for artificially incubated Greater Rhea eggs were 4% and 40%, respectively, being higher than in poultry species. 7. It is concluded that collecting eggs soon after laying will reduce the risk of microbial contamination.

Animals↗

PubMed2005

Effect of heat, acidification, and chlorination on Salmonella enterica serovar typhimurium cells in a biofilm formed at the air-liquid interface.

Bacterial biofilms have great significance for public health, since biofilm-associated microorganisms exhibit dramatically decreased susceptibility to antimicrobial agents and treatments. To date most attention has focused on biofilms that arise from the colonization of solid-liquid or solid-air interfaces. It is of interest that colonization of the interface between air and liquid, which can be selectively advantageous for aerobic or facultative aerobic bacteria, has been rarely studied, although it may present a major problem in industrial aquatic systems. In this work we investigated the role of a biofilm at the interface between air and liquid (pellicle) in the susceptibility of Salmonella enterica serovar Typhimurium to stress conditions. For a control we used a mutant that had lost its ability to synthesize cellulose and thin aggregative fimbriae and thus did not produce the pellicle. Resistance of bacteria from the pellicle to heat, acidification, and chlorination was compared to resistance of planktonic cells from the logarithmic and stationary phases of growth. Pellicle cells were significantly more resistant to chlorination, and thus the surrounding matrix conferred protection against the reactive sodium hypochlorite. However, the stress management of pellicle cells in response to heat and low pH was not enhanced compared to that of stationary-phase cells. A long-period of incubation resulted in endogenous hydrolysis of the pellicle matrix. This phenomenon provides a potential new approach to combat microbial cells in biofilms.

Bacterial Adhesion↗

PubMed1996

Human oral microbial ecology and dental caries and periodontal diseases.

In the human oral cavity, which is an open growth system, bacteria must first adhere to a surface in order to be able to colonize. Ability to colonize a non-shedding tooth surface is necessary prior to any odontopathic or periodontopathic process. Complex microbe-host relationships occur and must be studied before the commensal-to-pathogenic nature of the human indigenous oral flora can be understood. Medical pathogens, if present in the appropriate host, always produce specific disease. Caries and periodontal diseases are conditional diseases, requiring numbers of certain indigenous species at various sites, particularly the tooth surface. In the case of caries, the condition is related to sugar consumption. Periodontal disease/s may require certain host and environmental conditions, such as local environment or nutritional factors in gingival crevicular fluids. Nonetheless, critical numbers of certain indigenous species must be present in order for these diseases to occur. The aim of this review is to understand the acquisition of the indigenous oral flora and the development of human dental plaque. The role of the salivary pellicle and adherence of indigenous bacteria to it are critical first steps in plaque development. Bacterial interactions with saliva, nutritional factors, growth factors, and microbial physiologic processes are all involved in the overall process of microbial colonization.

Bacteria↗

PubMed2006

Construction of a leftover bath water model for microbial testing.

In this study, in order to construct a model of leftover bath water, we analyzed one hundred samples of used bath water samples which were provided by twenty-eight volunteer families. It appeared that the number of detected bacteria from such bath water was correlated closely with the number of bathers. Moreover, the pH, acidity, chemical oxygen demand (COD), ion, protein content of the leftover bath water were measured. The number of bathers had no connection with the pH, acidity, COD, and ion content of the leftover bath water. However, the protein content of the bath water correlated with the number of detected bacteria. Based on these results, the model of leftover bath water was constructed. Achromobacter xylosoxidans, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa were incubated with the model bath water as indices of bath water contamination. The number of incubated viable cells in the model bath water increased with increasing concentrations of casamino acid. Consequently, it was suggested that varying the concentration of casamino acid based on family size or contamination would be necessary in the efficient use of the constructed model of leftover bath water for microbial testing.

Baths↗

PubMed2004

Menadione-catalyzed luminol chemiluminescence assay for the rapid detection of viable bacteria in foods under aerobic conditions.

A menadione-catalyzed luminol chemiluminescence assay was developed for the rapid detection and estimation of viable bacteria in foods. The principle of this assay is based on the extracellular menadione-catalyzed active oxygen spieces (O2- and H2O2) generated by the activity of NAD(P)H:menadione oxidoreductase in viable cells. This luminol chemiluminescence assay requires 10 min for the incubation of cells with menadione and then 2 s for the measurement of chemiluminescence intensity after an injection of luminol solution without the treatment of cell lysis. This method was evaluated using liquid food samples of milk, vegetable juice, green tea, and coffee spiked with Escherichia coli ATCC 25922. The study result revealed that E. coli contamination at 1 to 10 CFU/ml in these foods could be detected after incubation at 37 degrees C for 7 h in an enrichment medium; however, the green tea and coffee samples requires filtration. This method could be a useful tool for the rapid evaluation of microbial food contamination.

Colony Count, Microbial↗