Bacillus cereus in free-stall bedding.

To increase the understanding of how different factors affect the bacterial growth in deep sawdust beds for dairy cattle, the microbiological status of Bacillus cereus and coliforms in deep sawdust-bedded free stalls was investigated over two 14-d periods on one farm. High counts of B. cereus and coliforms were found in the entire beds. On average, 4.1 log(10) B. cereus spores, 5.5 log(10) B. cereus, and 6.7 log(10) coliforms per gram of bedding could be found in the upper layers of the sawdust likely to be in contact with the cows' udders. The highest counts of B. cereus spores, B. cereus, and coliforms were found in the bedding before fresh bedding was added, and the lowest immediately afterwards. Different factors of importance for the growth of B. cereus in the bedding material were explored in laboratory tests. These were found to be the type of bedding, pH, and the type and availability of nutrients. Alternative bedding material such as peat and mixtures of peat and sawdust inhibited the bacterial growth of B. cereus. The extent of growth of B. cereus in the sawdust was increased in a dose-dependent manner by the availability of feces. Urine added to different bedding material raised the pH and also led to bacterial growth of B. cereus in the peat. In sawdust, a dry matter content greater than 70% was needed to lower the water activity to 0.95, which is needed to inhibit the growth of B. cereus. In an attempt to reduce the bacterial growth of B. cereus and coliforms in deep sawdust beds on the farm, the effect of giving bedding daily or a full replacement of the beds was studied. The spore count of B. cereus in the back part of the free stalls before fresh bedding was added was 0.9 log units lower in stalls given daily bedding than in stalls given bedding twice weekly. No effect on coliform counts was found. Replacement of the entire sawdust bedding had an effect for a short period, but by 1 to 2 mo after replacement, the counts of B. cereus spores in the beds had increased about 2 log units and were as high as they were before bed replacement. Therefore, free-stall management could, to a limited extent, reduce the content of B. cereus spores in the beds by daily bedding and entire bed replacement.

Bacillus cereus spores during housing of dairy cows: factors affecting contamination of raw milk.

The contamination of raw milk with Bacillus cereus spores was studied during the indoor confinement of dairy cattle. The occurrence of spores in fresh and used bedding material, air samples, feed, feces, and the rinse water from milking equipment was compared with the spore level in bulk tank milk on 2 farms, one of which had 2 different housing systems. A less extensive study was carried out on an additional 5 farms. High spore concentrations of >100 spores/L in the raw milk were found on 4 of the farms. The number of spores found in the feed, feces, and air was too small to be of importance for milk contamination. Elevated spore contents in the rinse water from the milking equipment (up to 322 spores/L) were observed and large numbers of spores were found in the used bedding material, especially in free stalls with >5 cm deep sawdust beds. At most, 87,000 spores/g were found in used sawdust bedding. A positive correlation was found between the spore content in used bedding material and milk (r = 0.72). Comparison of the genetic fingerprints obtained by the random amplified polymorphic DNA PCR of isolates of B. cereus from the different sources indicated that used bedding material was the major source of contamination. A separate feeding experiment in which cows were experimentally fed B. cereus spores showed a positive relationship between the number of spores in the feed and feces and in the feces and milk (r = 0.78). The results showed that contaminated feed could be a significant source of spore contamination of raw milk if the number of spores excreted in the feces exceeded 100,000/g.

Effect of different premilking manual teat-cleaning methods on bacterial spores in milk.

Different teat-cleaning methods were evaluated to determine their effect on the presence of spores from anaerobic bacterial spore-formers in the milk. Artificial contamination was used to achieve uniform contamination of teats to reduce the number of cows and samples needed in the experiments and still obtain adequate power to detect differences among tested methods. Teats were contaminated experimentally with a large amount of Clostridium tyrobutyricum spores in a manure-water slurry. Various types of dry and moistened towels and different combinations of methods using soap or 2 types of towels, together with cleaning times of 10 or 20 s, were compared in 2 Latin square-designed experiments with 7 cows, 7 treatments, and 4 replications in each experiment. In comparison with control (no cleaning and no forestripping), cleaning teats with dry paper towels for 10 s reduced concentration of spores in milk by 45 to 50%. A 50 to 74% reduction was achieved using different types of moist towels for 10 s. Methods using 2 towels, soap, or a longer cleaning time reduced bacterial contamination by 85 to 91%. The most effective methods in reducing milk spore content (96% reduction) were use of a moist washable towel with or without soap followed by drying with a dry paper towel, for a total time of 20 s per cow. One of the best cleaning methods was studied in an additional experiment to determine the effect of different teat contamination mixtures. The Latin square-designed experiment with 8 cows, 8 treatments, and 2 replications showed that cleaning was independent of the tested contamination matrix (manure, soil, or sawdust), type of spores (Cl. tyrobutyricum and Bacillus cereus), or degree of contamination (manure or extra manure).

Contamination of pasteurized milk by Bacillus cereus in the filling machine.

The contamination of pasteurized milk by Bacillus cereus during the filling process was studied in two dairy plants. Samples of pasteurized milk were taken at four different sites along the production line. The samples were stored at 7 degrees C for 7 d, or at 10 degrees C for 5 d, before plate counting and random selection of B. cereus isolates. Isolates of B. cereus were typed by the polymerase chain reaction (PCR)-based method randomly amplified polymorphic DNA (RAPD). Samples taken at three different sites between the pasteurizer and the filling machine were all holding similar low concentrations of B. cereus, while an increase of the B. cereus count was seen in the consumer packages. More B. cereus of different RAPD types was growing in the consumer packages than in samples taken just before the filling machine. Several RAPD types found in the consumer packages were not detected in the samples taken just before the filling machine.

Comparison between automatic ribotyping and random amplified polymorphic DNA analysis of Bacillus cereus isolates from the dairy industry.

Discrimination by automatic ribotyping and random amplified polymorphic DNA PCR, RAPD, was compared for 40 different B. cereus dairy isolates, 4 different B. mycoides isolates and 6 culture collection strains. RAPD-PCR has previously shown to be useful for tracing contamination routes of B. cereus to milk. Automatic ribotyping using EcoRI and PvuII separated the B. cereus and B. mycoides isolates/strains into 36 different ribotypes. RAPD-typing with primers generated 40 different RAPD-profiles. However, 17 isolates clustered into eight groups, irrespective of the primer and restriction enzyme used, and in all but one case, the isolates with the same pattern were isolated from the same dairy. Automatic ribotyping proved to be a useful, standardized and quick method to discriminate between B. cereus strains, only slightly less discriminatory than RAPD-typing.

Bacillus cereus spores in raw milk: factors affecting the contamination of milk during the grazing period.

Psychrotrophic Bacillus cereus is a limiting factor for the shelf-life of pasteurized milk, particularly during the grazing season. Potential sources of contamination and factors that might affect the spore content of milk were studied in detail for a group of eight cows during three 2-wk study periods from June to September over 2 yr. The spore content of milk was strongly associated with the degree of contamination of the teats with soil. High water content of soil, low evaporation of water and dirty access alloys were the most important factors correlating with high spore concentrations. The spore content of soil varied from < 50 to 380,000/g, depending on time and sampling site. The milking equipment did not contribute significantly to the contamination. The spore contents in air during milking (< 100 cfu/m3) and in feed (silage, hay, fresh grass, and concentrates) were too low to be of importance for contamination. The spore content in dung was also low. Further support that soil was the major contamination source was found by comparison of genetic fingerprints by random amplified polymorphic DNA polymerase chain reaction of isolates of B. cereus from soil and milk and by teat cleansing experiments, which resulted in reduced contamination levels in milk.

A RAPD-PCR method for large-scale typing of Bacillus cereus.

A robust RAPD-PCR procedure for large-scale typing of Bacillus cereus was developed. It is based on a simple DNA preparation, involving only freezing and boiling of cells in water with active carbon. By using a computerized system for data collection and processing, an efficient system for handling RAPD patterns for large-scale investigations was achieved. The procedure was highly discriminatory for Bacillus cereus strains and was found to give reproducible classification of RAPD fingerprints for five reference strains.

Toxin production by Bacillus cereus dairy isolates in milk at low temperatures.

A total of 136 strains of Bacillus cereus isolated from milk and cream were evaluated for toxin production based on HeLa S3, Vero, and human embryonic lung (HEL) cell cytotoxicity in vitro. HEL cell monolayers were more susceptible than the other two cell lines. The percentage of isolates exhibiting HEL cytotoxicity was similar (43.0 and 48.4%) when the strains were grown in brain heart infusion broth containing 0.1% glucose (BHIG) at 7 and 24 h, respectively, at 30 degrees C. In milk, only 21.8% of isolates showed HEL cytotoxicity at 7 h, and the number increased significantly to 73.2% at 24 h at 30 degrees C. Further, 102 toxin-positive isolates were acclimatized to grow at 8 degrees C in milk. Ninety-four (92.2%) of the strains produced HEL cytotoxicity of various degrees with no strict correlation to bacterial cell numbers and also elicited vascular permeability reaction in rabbit skin. Under aerated growth conditions (agitation, 200 rpm) B. cereus elicited cytotoxicity in BHIG and in milk at temperatures of 30, 15, and 8 degrees C. However, in nonaerated (stagnant) cultures toxin production was diminished (BHIG) or completely lost (milk) at all temperatures. Toxin production at 8 degrees C was evaluated in two different types of commercial cardboard milk packages by inoculation with a potent toxigenic dairy isolate. No detectable HEL cytotoxicity was observed in milk in any of the packages either at stagnant conditions or during mechanical shaking. However, the same strain produced cytotoxin in whipped cream at 8 degrees C.

Membrane potential, lipid regulation and adenylate energy charge in acyl chain modified Acholeplasma laidlawii.

In Acholeplasma laidlawii variations induced in the transmembrane electrical potential have been shown to affect the membrane lipid composition. Particularly the molar ratio between the predominant glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, decreases upon hyperpolarization and increases upon depolarization (Clementz et al. (1986) Biochemistry 25, 823-830). Upon variation of the degree of membrane fatty acyl chain unsaturation, known to affect the passive permeability for a number of small molecules, there was no significant correlation between acyl chain composition and the magnitude of the electrical potential. Hyperpolarization by valinomycin decreased the glucolipid ratio for all kinds of membranes, but the size of the decrease was not correlated to the acyl chain composition. However, a clear relationship, independent of acyl chain composition, was found between the extent of hyperpolarization and the size of the decrease in the glucolipid ratio. The adenylate energy charge value (Ec) of the cells was affected by the acyl chain composition, although not exclusively by the proportion of unsaturation. Furthermore, a larger hyperpolarization upon valinomycin addition was accompanied by a stronger reduction in Ec.

Adsorption of mycoplasmavirus MV-L2 to Acholeplasma laidlawii: effects of changes in the acyl-chain composition of membrane lipids.

The enveloped mycoplasmavirus MV-L2 and its host Acholeplasma laidlawii JA1 were used to study the ways in which changes in the membrane lipid bilayer affect virus adsorption. The physical state of the membranes was altered by (i) using viruses and bacteria with different membrane lipid acyl-chain compositions, (ii) using incorporation of cholesterol, and (iii) changing the temperature. Adsorption of viruses was strongly dependent on the acyl-chain composition of the virus and the host. Adsorption to homologous hosts was poor, whereas adsorption to hosts with highly different membrane lipid acyl-chain composition was much stronger. We found a heterogeneity within virus populations produced from hosts with different acyl-chain compositions. In a given virus population, various subpopulations differing in acyl-chain composition were found that differed in their ability to adsorb to cells with a specific acyl-chain composition. The adsorption rate increased slightly when cholesterol was present in the viral membranes but decreased considerably when cholesterol was present in the bacterial membranes. The rate of adsorption was temperature dependent with an increase in adsorption rate above 20 degrees C (for hosts with equal amounts of palmitoyl and oleoyl acyl chains). MV-L2 did not adsorb to the persistently L2-infected strain JA1(2R) but adsorbed very well to the virus-resistant strain A(EF22). The physicochemical properties of the lipid matrix of both virus and host are obviously important factors in the adsorption process.

Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy.

The extraction of proteins and membrane lipids from biological materials during embedding procedures for electron microscopy carried out at temperatures down to 223 K was studied. Glutaraldehyde-fixed cells of Acholeplasma laidlawii mainly served as test material. More than 99% of the protein and 88% of the lipid of these cells were retained after dehydration with ethanol or acetone between 277 and 223 K and infiltration with methacrylate at 223 K. When methanol was used for dehydration, only 54% of the lipid was retained. The amount of extracted lipid was essentially independent of the ratio between volume of extraction liquid and amount of material subjected to extraction. The cytoplasmic membrane of sectioned Acholeplasma-cells dehydrated and infiltrated as described above appeared more diffuse than that of cells fixed with glutaraldehyde and osmium tetroxide in epoxy resin at room temperature. Glutaraldehyde-fixed erythrocyte ghosts retained 85% of their phospholipid content when dehydrated with ethanol between 277 and 223 K and infiltrated with methacrylate at 223 K. Spinach chloroplasts and thylakoid vesicles retained 61% and 35%, respectively, of their chlorophyll content.

Transmembrane electrical potential affects the lipid composition of Acholeplasma laidlawii.

In membranes of Acholeplasma laidlawii, lipid composition is regulated as a function of several stimuli affecting the volume and length of the hydrocarbon chains and the hydrocarbon-water interfacial area. This regulation is vizualized as changes in the relative amounts of the major polar lipids monoglucosyl diglyceride and diglucosyl diglyceride. These lipids form reversed hexagonal and lamellar phases with water, respectively. However, mixtures of the two lipids, in the molar proportions found in the A. laidlawii membrane, form a lamellar phase. By adjustment of the glycolipid ratio as a response to environmental stimuli, a certain stability of the lamellar membrane is maintained. In growing cells with oleoyl membrane lipids, a transmembrane electrical potential of approximately -50 mV (inside negative), but no transmembrane pH difference, was found. Addition of the K+ ionophore valinomycin caused a rapid and dose-dependent hyperpolarization remaining for at least 7 h. Simultaneously, a rapid and lasting metabolic decrease in the ratio monoglucosyl diglyceride/diglucosyl diglyceride occurred. The increase in potential and the decrease in the lipid ratio were both reversed in a dose-dependent manner by extracellular KCl. Likewise, the lipophilic cation tetraphenylphosphonium caused a dose-dependent decrease in membrane potential and an increase in the monoglucosyl diglyceride/diglucosyl diglyceride ratio, respectively. The ionophores monensin and particularly nigericin had similar but less pronounced effects on the potential and lipid ratios as valinomycin. The uncoupler carbonyl cyanide m-chlorophenylhydrazone had no effect on cell growth, membrane potential, or lipid regulation at 10 microM. These dissimilar structures and the low concentrations used make a direct disturbance of drug molecules on lipid packing in membranes less likely.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2.

In a previous report it was shown that the replacement of native erythrocyte phosphatidylcholine (PC) with different PC species which have defined acyl chain compositions can lead to morphological changes (Kuypers, F.A., W. Berendsen, B. Roelofsen, J. A. F. Op den Kamp, and L.L.M. van Deenen, 1984, J. Cell Biol., 99:2260-2267). It was proposed that differences in molecular shape between the introduced PC species and normal erythrocyte PC caused the membrane to bend outwards or inwards, depending on the shape of the PC exchanged. To support this proposal, two requirements would have to be fulfilled: the exchange reaction would take place only with the outer lipid monolayer of the erythrocyte, and the extent of lipid transbilayer movement would be restricted. If this theory is correct, any treatment causing unilateral changes in lipid molecular shape should lead to predictable morphological changes. Since this hypothesis is a refinement of the coupled bilayer hypothesis, but so far lacks experimental support, we have sought other means to change lipid molecular shape unilaterally. Shape changes of human erythrocytes were induced by the replacement of native PC by various PC species using a phosphatidylcholine-specific transfer protein: by hydrolysis of phospholipids in intact cells using sphingomyelinase C or phospholipase A2, and by the combination of both procedures. The morphological changes were predictable; additive when both treatments were applied, and explicable on the basis of the geometry of the lipid molecules involved. The results strongly support the notion that lipid molecular shape affects erythrocyte morphology.

Involvement of surface potential in regulation of polar membrane lipids in Acholeplasma laidlawii.

Upon induced variation of membrane lipid acyl chain unsaturation in Acholeplasma laidlawii, the cells strongly change in a characteristic manner the proportions of individual (charged and noncharged) polar lipids synthesized. Monolayer analysis of polar lipid extracts revealed different mean lateral molecular areas but similar surface charge densities. Microelectrophoresis of these lipids indicated an almost constant lipid membrane zeta-potential of about -35 mV. Simulation by the Gouy-Chapman-Stern (GCS) relations verified that the zeta (surface)-potentials remain constant. Exposing cells to increasing concentration of Na+ yielded a substantial increase in amounts of charged lipids synthesized. In model systems consisting of mixtures of A. laidlawii phosphatidylglycerol (anionic) and glucolipid (diglucosyldiglyceride, noncharged) microelectrophoresis showed; (i) increasing PG amounts resulted in an increased-, and (ii) increasing Na+ concentration resulted in a decreased zeta-potential, respectively, (iii) at physiological ionic strength and lipid surface charge densities the zeta-potential was approximately -35 mV, and (iv) simulation according to the GCS theory yielded an acceptable fit with experimental data. This behavior of the phosphatidylglycerol/diglucosyldiglyceride mixtures is very similar to that of phosphatidylserine/phosphatidylcholine mixtures. It is concluded that the changes in lipid surface charge densities (and surface potential) in A. laidlawii membranes brought by variation in lateral areas of lipid acyl chains and the concentration-dependent quenching of lipid charge by Na+, is compensated for by the cellular regulation of charged lipid amounts thereby maintaining a constant lipid surface potential.

A comparative fluorescence polarization study of cis-parinaroyl-phosphatidylcholine and diphenylhexatriene in membranes containing different amounts of cholesterol.

The steady state fluorescence anisotropy (rs) of 1-acyl-2-cis parinaroyl phosphatidylcholine (PnPC) was compared with that of diphenylhexatriene (DPH) in a variety of model- and biological membrane systems. The fluorescence anisotropy of both probes responded similarly to temperature changes and variations in the acyl chain composition in phosphatidylcholine (PC) liposomes. The presence of proteins and cholesterol increased rs for both DPH and PnPC in the biological membranes as compared to the isolated polar membrane lipids. Comparison of DPH and PnPC in dipalmitoyl-PC-liposomes with and without 50 mol% cholesterol, showed at temperatures above the phase transition of pure dipalmitoyl-PC the presence of cholesterol increased the rs-value for DPH strongly, whereas the rs-value for PnPC was much less affected. In the cholesterol-rich erythrocyte membrane as well as in microsomes from Morris hepatoma 7787, which have an increased cholesterol content as compared to normal rat liver microsomes, the rs of DPH was higher than that of PnPC. No large differences between the rs-values of both probes were evident in the normal cholesterol-poor rat liver microsomes. These effects are discussed in terms of structural differences between the probes and variation of cholesterol content. Alterations in the fatty acid composition of PC present in human erythrocyte membranes were introduced with the aid of a PC-specific transfer protein. Fluorescence anisotropy values of both probes hardly changed upon enrichment of the red cell membrane with either dipalmitoyl PC or 1-palmitoyl-2-arachidonyl PC.

Chemical composition and ultrastructure of Mycoplasma hominis.

Mycoplasma hominis belongs to the family Mycoplasmataceae, which includes the smallest free-living organisms known to exist. Despite the small size of the organism, its chemical composition and cell structure are almost as complex as in other bacteria. The cytoplasm contains typical 70S bacterial ribosomes and a circular double-stranded DNA molecule. Both of these structures have been well characterized. Like other mycoplasmas, M. hominis lacks a cell wall. The cytoplasmic membrane is the outer boundary of the cell. The membrane lipid and protein composition of M. hominis has been established. The presence of various high-molecular-weight proteins at the extracellular side of the membrane in different strains is probably important in terms of the immunogenic heterogeneity of M. hominis. Furthermore, sugar-containing structures at the outside of the membrane may be important in the interaction between M. hominis and the mucosal surfaces of its human hosts. However, the information available on the cell structure of M. hominis is still inadequate for an exact definition of the relation between host and parasite.

Tetracycline resistance in Mycoplasma hominis.

The composition of the cytoplasmic membrane of a clinical isolate of Mycoplasma hominis that was resistant to tetracycline (minimal inhibitory concentration, 30 micrograms/ml) was compared with that of a susceptible strain (minimal inhibitory concentration, less than 1 microgram/ml). Neither differences in passive permeability of the lipid portion of the membrane to the drug nor modulation of active transport by membrane lipids explained the resistance to tetracycline. Polyacrylamide gel electrophoresis revealed several differences between the membrane protein composition of the two strains. One 25,000-dalton protein was prominent in the membrane of the resistant strain but almost nonexistent in that of the sensitive strain. Therefore, the resistance may have been correlated with differences in protein composition. Preliminary evidence suggested that the resistance trait was plasmid-mediated.

Lipid phase structure in the regulation of lipid composition in Acholeplasma laidlawii membranes.

In Acholeplasma laidlawii membranes the ratio between the dominating lipids, monoglucosyldiglyceride (MGDG) and diglucosyldiglyceride (DGDG), depends on temperature, configuration of incorporated fatty acids, and membrane cholesterol content, which affect the molecular geometry of the lipids. MGDG and DGDG have wedge- and rod-like molecular shapes, respectively, that are modifiable. The packing constraints of lipids in amphiphilic aggregates, i.e., the area of the hydrocarbon-water interface and the volume and length of the hydrocarbon chains, are important in determining the aggregate structure. Pure MGDG forms a reversed hexagonal- (HII) phase structure with different acyl chain contents, while DGDG forms a lamellar phase. Depending on the unsaturated acyl chain content in the lipids, an in vitro mixture of MGDG and DGDG forms lamellar or cubic phases at physiologic temperatures. A high degree of cis-unsaturation, large amounts of MGDG and high temperatures favor formation of the cubic phase. Addition of cholesterol corresponding to the maximal amount incorporable into A. laidlawii induces a transition from a lamellar or a cubic phase to a reversed hexagonal phase. Lipid mixtures containing only unsaturated acyl chains are more sensitive to the bilayer-destabilizing effect of cholesterol than are mixtures with equal amounts of saturated and unsaturated acyl chains. The lamellar phase is the only one compatible with a functional biological membrane. Consequently, the balance between lipids that form lamellar and other mesophase structures must keep within certain limits. The cubic and reversed hexagonal structures were discovered under conditions not existing in the living Acholeplasma cell. Thus, the response of A. laidlawii lipid metabolism to external and internal stimuli can be predicted on the basis of molecular shapes and is necessary to the maintenance of optimal membranes stability. The reduced capacity of Acholeplasma membranes to incorporate cholesterol is a consequence of this regulation.