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Biodegradation of diesel oil by cold-adapted microorganisms in presence of sodium dodecyl sulfate.

The effect of different concentrations of the anionic surfactant sodium dodecyl sulfate (SDS) on biodegradation of diesel oil was assessed during 32 days at 10 degrees C, under simulated environmental conditions, in liquid culture and in an alpine soil. Low SDS concentrations (50-100 mg l-1) significantly enhanced oil biodegradation by a psychrotrophic inoculum in liquid culture, whereas higher SDS concentrations (500-1000 mg l-1) inhibited hydrocarbon biodegradation. Oil biodegradation by the indigenous microorganisms in soil was inhibited at all SDS concentrations tested. The surfactant itself was rapidly biodegraded both in liquid culture and in soil.

Arthrobacter↗

Rapid and cost-effective multiparameter toxicity tests for soil microorganisms.

Three biochemical parameters, DNA quantification in soil samples and two enzymatic activities, beta-galactosidase and dehydrogenase have been assessed as potential end-points for the use in cost-effective toxicity tests on soil microorganisms. The assessment included the development of a classical dose-response 24-h assay and the incorporation of measurements of the effects on microbial activities in soil column leaching studies and multispecies miniaturised terrestrial systems (MTS). Four different chemicals, copper, a new herbicide, thiabendazole and fenthion were studied. A rapid fluorescence DNA quantification technique did not produce adequate responses. The efforts to quantify DNA after extraction and clean-up procedures failed due to the presence of humic acids. From the protocol of the technique one could see that the technical procedure is time-consuming and expensive and, for this reason, not suitable for use as a parameter in rapid and cost-effective tests. However, the enzymatic activities showed their potential as toxicity end-points. Copper produced a concentration/response inhibition of beta-galactosidase and dehydrogenase with EC50 values of 78.39 and 24.77 mg Cu/kg soil, respectively. In the soil column study, these endpoints allowed the measurement of the microbial activities through the column. The effects of the new herbicide on beta-galactosidase and dehydrogenase activities were statistically significant for the highest application dose (40 g/ha). Thiabendazole affected the microbial activity when mixed within the soil, but no effects were observed when this fungicide was applied on the soil surface. Fenthion produced effects when applied either in the soil or on the soil surface. These results can be explained by the low mobility of thiabendazole. The results show the capabilities of these biochemical parameters to be included as endpoints in cost-effective bioassays.

Biological Assay↗

Metal ion transport in eukaryotic microorganisms: insights from Saccharomyces cerevisiae.

Metal ions such as iron, copper, manganese, and zinc are essential nutrients for all eukaryotic microorganisms. Therefore, these organisms possess efficient uptake mechanisms to obtain these nutrients from their extracellular environment. Metal ions must also be transported into intracellular organelles where they function as catalytic and structural cofactors for compartmentalized enzymes. Thus, intracellular transport mechanisms are also present. When present in high levels, metal ions can also be toxic, so their uptake and intracellular transport is tightly regulated at both transcriptional and post-transcriptional levels to limit metal ion overaccumulation and facilitate storage and sequestration. Remarkable molecular insight into these processes has come from recent studies of the yeast Saccharomyces cerevisiae. This organism, which is the primary subject of this chapter, serves as a useful paradigm to understand metal ion metabolism in other eukaryotic microbes.

Biological Transport↗

Correlation between thermal sensitivity and microorganisms isolated from deep carious dentin.

The correlation between thermal sensitivity and the microorganisms present in 29 deep carious lesions was studied. The numbers of lactobacilli and total Gram-positive rods in the carious lesions were found to be negatively related to the length of pain triggered by cold and heat stimulants. The presence of Gram-positive cocci and non-black-pigmented Bacteroides were positively associated with both cold and heat sensitivities. Black-pigmented Bacteroides, Streptococcus mutans, and total anaerobic colony counts were positively related to the heat sensitivity. Recovery of Fusobacterium nucleatum, Actinomyces viscosus, and enterics on the selective plates was associated with cold sensitivity. Total counts of Gram-positive cocci and Gram-negative rods on the anaerobic nonselective medium were positively related to the cold sensitivity. It appeared that teeth with low numbers of lactobacilli in the carious lesions usually responded to thermal tests with longer duration of pain. Conversely, teeth with high numbers of lactobacilli in the carious lesions usually responded with shorter duration of pain.

Bacteroides↗

Identification of an epitope derived from human proteolipid protein that can induce autoreactive CD8+ cytotoxic T lymphocytes restricted by HLA-A3: evidence for cross-reactivity with an environmental microorganism.

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is in part due to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. In this study, we have identified a peptide sequence derived from the CNS-specific myelin protein proteolipid protein (PLP) which could bind to HLA-A3 and induce a HLA-A3-restricted CD8+ CTL response from HLA-A3+ donors. These PLP peptide-specific CTL could lyse HLA-A3+ target cells pulsed with a homologous peptide derived from the CRM1 protein of Saccharomyces cerevisae. These findings demonstrate the immunogenic potential of a PLP-derived peptide for generation of autoreactive HLA-A3-restricted CD8+ CTL, and further show that these CTL can be activated by a peptide derived from a common environmental microorganism.

Adult↗

Characterization of a heme c nitrite reductase from a non-ammonifying microorganism, Desulfovibrio vulgaris Hildenborough.

A cytochrome c nitrite reductase (NiR) was purified for the first time from a microorganism not capable of growing on nitrate, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It was isolated from the membranes as a large heterooligomeric complex of 760 kDa, containing two cytochrome c subunits of 56 and 18 kDa. This complex has nitrite and sulfite reductase activities of 685 micromol NH(4)(+)/min/mg and 1.0 micromol H(2)/min/mg. The enzyme was studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies. The overall redox behavior was determined through a visible redox titration. The data were analyzed with a set of four redox transitions, with an E(0)' of +160 mV (12% of total absorption), -5 mV (38% of total absorption), -110 mV (38% of total absorption) and -210 mV (12% of total absorption) at pH 7.6. The EPR spectra of oxidized and partially reduced NiR show a complex pattern, indicative of multiple heme-heme magnetic interactions. It was found that D. vulgaris Hildenborough is not capable of using nitrite as a terminal electron acceptor. These results indicate that in this organism the NiR is not involved in the dissimilative reduction of nitrite, as is the case with the other similar enzymes isolated so far. The possible role of this enzyme in the detoxification of nitrite and/or in the reduction of sulfite is discussed.

Amino Acid Sequence↗

Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms.

As a technique allowing simultaneous visualization, identification, enumeration and localization of individual microbial cells, fluorescence in situ hybridization (FISH) is useful for many applications in all fields of microbiology. FISH not only allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms, and can therefore help in understanding complex microbial communities. In this review, methodological aspects, as well as problems and pitfalls of FISH are discussed in an examination of past, present and future applications.

Animals↗

Combination of ATP-bioluminescence and PNA probes allows rapid total counts and identification of specific microorganisms in mixed populations.

We have combined ATP-dependent bioluminescence with a novel chemiluminescent in situ hybridization (CISH) method using peroxidase-labeled peptide nucleic acid (PNA) probes targeting species-specific rRNA sequences to provide total counts and subsequent identification of specific microorganisms. Both methods are applied to the same membrane filter following a short incubation time and both methods provide results in the form of spots of light that are captured by the MicroStar detection system. Each spot of light represents individual micro-colonies detected by either ATP bioluminescence or PNA CISH. This new concept is particularly intended for in process and quality control of non-sterile products to rapidly provide total counts as well as presence/absence of specific indicators and/or pathogens in non-sterile, filterable samples.

Adenosine Triphosphate↗

Identification of medically relevant microorganisms by vibrational spectroscopy.

In the recent years, vibrational spectroscopies (infrared and Raman spectroscopy) have been developed for all sorts of analyses in microbiology. Important features of these methods are the relative ease with which measurements can be performed. Furthermore, in order to obtain infrared or Raman spectra, there is only a limited amount of sample handling involved without the need for expensive chemicals, labels or dyes. Here, we review the potential application of vibrational spectroscopies for the use in medical microbiology. After describing some of the basics of the techniques, considerations on reproducibility and standardisation are presented. Finally, the use of infrared and Raman spectroscopy for the (rapid) identification of medically relevant microorganisms is discussed. It can be concluded that vibrational spectroscopies show high potential as novel methods in medical microbiology.

Bacterial Infections↗

Optimization of the growth conditions of the extremely thermophilic microorganisms Thermococcus celer and Pyrococcus woesei.

Growth medium components and cultivation conditions for the extremely thermophilic Archaea Thermococcus celer and Pyrococcus woesei were optimized. A culture media based in marine water was formulated. Both Archaea demonstrated to be strictly anaerobic with optimal growth temperature of 85 degrees and 95 degrees C, respectively. Sodium sulfide, but not cysteine, was used as a sulfur and reductive capacity source. It was observed that hydrogen sulfide could be replaced by 30 microM titanium (III) nitrile acetate. The addition of elemental S(o) enhanced growth of both microorganisms, with T. celer far more sensitive than P. woesei to the absence of S(o). P. woesei utilized maltose as a carbon source, while T. celer was able to use only peptides from yeast extract, peptone and tryptone as its carbon source. Optimum carbon source concentrations were 1.25 g/L for T. celer and 5 g/L for P. woesei. Although both Archaea required peptides as a nitrogen source, the addition of ammonia chloride to a nitrogen-limited media did not stimulate growth, which suggests that neither Archaea appear to metabolize ammonia. The growth of P. woesei, but not T. celer, was stimulated considerably in the presence of iron. Co, Ni, Zn, Mo. Mn and Mg were essential trace elements needed for optimal growth of both bacteria.

Bacteriological Techniques↗

Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The Safety in Biotechnology Working Party of the European Federation of Biotechnology.

Risk assessment for the deliberate release of microorganisms into the environment is traditionally carried out on a case-by-case basis. In a similar approach to that used when assessing human pathogenicity, we propose an alternative approach by introducing risk classes to facilitate or complement this type of risk assessment. These consider several sets of scenarios that address the different values that need to be protected. Examples of this approach include risk-class definitions for soil fertility and biodiversity.

Bacteria↗

Assessment of viability of microorganisms employing fluorescence techniques.

Viability assessment of microorganisms is relevant for a wide variety of applications in industry, including evaluation of inactivation treatments and quality assessment of starter cultures for beer, wine, and yoghurt production. Usually, the ability of microbial cells to reproduce is considered as the benchmark method for determination of cell viability, and this is most commonly determined by the plate count method. The time needed to form visible colonies, however, is relatively long. Therefore, there is an increasing interest in rapid methods which exploit criteria other than reproduction. In this review the applications of fluorescent probes for, e.g., determination of membrane integrity, enzyme activities, respiration, membrane potential and intracellular pH, are discussed in detail.

Bacteria↗

A novel area of predictive modelling: describing the functionality of beneficial microorganisms in foods.

Predictive microbiology generally focuses on the potential outgrowth of spoilage bacteria and foodborne pathogens in foods. Little attention has been paid to the biokinetics of beneficial foodgrade microorganisms, such as lactic acid bacteria. The latter is commonly used in the food fermentation industry, mainly for the in situ production of the antimicrobial lactic acid to extend the shelf life of the food. Furthermore, many strains show additional industrial potential as novel starter cultures since they produce functional metabolites, such as bacteriocins and exopolysaccharides. The production of these functional metabolites has been demonstrated during in vitro experiments, but in many cases these novel starter cultures seem to be less efficient when applied in a food system. A modelling approach may contribute to a better understanding of the tight relation between the food environment and bacterial functionality. Primary modelling can be applied to fit the experimental data concerning cell growth, sugar metabolism, and the production of functional metabolites for a given set of environmental conditions. This led to conclusions concerning the growth-associated production of bacteriocin and exopolysaccharides, the inactivation of these molecules when cell growth levels off, and a minimum cell concentration to trigger on bacteriocin production. Examples deal with the production of the bacteriocin sakacin K by the natural fermented sausage isolate Lactobacillus sakei CTC 494, and the production of heteropolysaccharides by the yoghurt starter culture Streptococcus thermophilus LY03. Secondary modelling of biokinetic parameters quantifies the production of bacteriocin and exopolysaccharides in function of environmental factors. As an example, the specific bacteriocin production by Lb. sakei CTC 494 decreases with increasing sodium chloride concentrations. Furthermore, since the assessment of functionality is frequently hampered by the nature of the food system, mathematical modelling techniques may help to predict the functional behaviour of novel lactic acid bacteria starter cultures in a food matrix, and hence quantify in situ production. For example, a model may simulate cell growth and exopolysaccharide production of S. thermophilus LY03 in a milk environment, where direct measurements are difficult to perform.

Bacteriocins↗

Osmoregulation and its importance to food-borne microorganisms.

The control of water activity has been used as a means of preserving foods for thousands of years. This preservation strategy presents food-borne microorganisms with serious problems, many of which relate to the management of water flow. Although the specific details of how each organism deals with these problems are different, several common themes have emerged. Bacteria induce specific responses. both physiological and genetic, to respond to either the loss or the gain of water, triggered by changes in the osmolarity of the environment. Many of the key systems have now been identified and the mechanisms of their regulation are beginning to be understood. Here we review recent developments in the field of bacterial osmoregulation with emphasis on key food-borne genera.

Bacterial Physiological Phenomena↗

Combining nonthermal technologies to control foodborne microorganisms.

Novel nonthermal processes, such as high hydrostatic pressure (HHP), pulsed electric fields (PEFs), ionizing radiation and ultrasonication, are able to inactivate microorganisms at ambient or sublethal temperatures. Many of these processes require very high treatment intensities, however, to achieve adequate microbial destruction in low-acid foods. Combining nonthermal processes with conventional preservation methods enhances their antimicrobial effect so that lower process intensities can be used. Combining two or more nonthermal processes can also enhance microbial inactivation and allow the use of lower individual treatment intensities. For conventional preservation treatments, optimal microbial control is achieved through the hurdle concept, with synergistic effects resulting from different components of the microbial cell being targeted simultaneously. The mechanisms of inactivation by nonthermal processes are still unclear; thus, the bases of synergistic combinations remain speculative. This paper reviews literature on the antimicrobial efficiencies of nonthermal processes combined with conventional and novel nonthermal technologies. Where possible, the proposed mechanisms of synergy is mentioned.

Biotechnology↗

Adherence to stainless steel by foodborne microorganisms during growth in model food systems.

Biofilm formation on stainless steel by Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7, Pseudomonas fragi and Pseudomonas fluorescens during growth in model food systems was studied. Test growth media included tryptic soy broth (TSB), diluted TSB (dTSB), 1% reconstituted skim milk (RSM) and diluted meat juice (DMJ). Adherent cells were stained with acridine orange and enumerated using epifluorescent microscopy and computerized image analysis. Cells were observed on the stainless steel surface after 1 h in all of the media. However, the increases in the number of adherent cells over time was seen only with S. typhimurium in DMJ, E. coli O157:H7 in TSB, dTSB and DMJ, P. fragi in RSM and P. fluorescens in RSM. The medium which produced the highest observed level of adherent cells was different for each microorganism.

Bacteria↗

Method for the rapid quantitative detection of lipolytic activity among food fermenting microorganisms.

A standard method for the detection of free fatty acids (FFAs) in milk was modified and applied to the measurement of the lipolytic activity of microorganisms in a model system containing either homogenised pork or beef fat tissue. The increase in FFAs was measured colorimetrically using palmitic acid as a standard. Among the strains tested, two strains of Staphylococcus xylosus and one strain of Staphylococcus carnosus were found to display lipolytic activity. For all strains, a higher increase in FFA was observed in broth supplemented with pork fat than with beef fat. All three strains displayed lipolytic activity when tested on tributyrin agar plates.

Fatty Acids, Nonesterified↗

Amino acid decarboxylase capability of microorganisms isolated in Spanish fermented meat products.

Enterobacteria, lactic acid bacteria (LAB) and Gram-positive cocci were isolated from Spanish meat products. The most frequent species in the meat products studied were identified as Lactobacillus sake, Lactobacillus plantarum and Lactobacillus curvatus from De Man-Rogosa-Sharpe agar; Staphylococcus xylosus, Staphylococcus saprophyticus and Micrococcus varians from mannitol salt phenol-red agar; and Hafnia alvei, Escherichia coli, Pseudomonas fluorescens, Enterobacter amnigenes and Enterobacter aerogenes from violet red bile dextrose agar. The amino acid decarboxylase activity of the microorganisms isolated was assayed. Enterobacteria had higher amino acid decarboxylase activity than the other groups. LAB did not show any significant amino acid decarboxylase capability in this study.

Amino Acids↗