Survival of free and alginate-encapsulated Pseudomonas aeruginosa UG2Lr in soil treated with disinfectants

Pseudomonas aeruginosa UG2Lr, a rifampicin-resistant strain possessing the luxAB on a chromosomal Tn5 insert, was inoculated into soil microcosms as either free cells or encapsulated in dry alginate beads. A 100-fold increase in cell number g^-1 dry soil was observed in microcosms inoculated with alginate-encapsulated UG2Lr after 3 weeks incubation at 22°C compared to microcosms inoculated with free cells. After 98 d, microcosms inoculated with free UG2Lr cells contained 10⁴ cfu g^-1 dry soil compared to 10⁷ cfu g^-1 dry soil in microcosms inoculated with alginate-encapsulated UG2Lr cells. The effects of disinfectants on both the free and alginate-encapsulated UG2Lr cells were also examined. 1·0% (w/g dry soil) calcium hypochlorite, formaldehyde and Spectrum Clear Bath, were added to microcosms each week for 4 weeks. Formaldehyde killed both free and alginate-encapsulated UG2Lr cells within 14 d after only two amendments. Calcium hypochlorite reduced free UG2Lr cell numbers 10-fold 2 d after initial application; however, the introduced population recovered and was unaffected by subsequent treatments at 7, 14 and 21 d. Alginate-encapsulated UG2Lr cells were not affected by calcium hypochlorite treatment. Spectrum Clear Bath did not kill either free or alginate-encapsulated UG2Lr cells in soil. Alginate encapsulation improved survival of introduced bacteria in soil except in the presence of formaldehyde. Killing genetically-engineered bacteria in soil may be difficult unless a powerful disinfectant such as formaldehyde is used or the genetically-engineered micro-organism is allowed to become non-viable over time.     

A biological containment system for Rhizobium meliloti based on the use of recombination-deficient (recA−) strains

Abstract Using a newly developed integration vector, the Escherichia coli gusA gene conferring GUS-activity or the firefly ( Photinus pyralis ) luc gene mediating bioluminescence were integrated into a non-essential site of the chromosome of Rhizobium meliloti 2011. The integration of the constitutively expressed marker genes into the chosen site per se did not affect the strains' ability to perform homologous recombination, its growth characteristics or its symbiotic nitrogen fixation. Comparative microcosm analyses between the bioluminescent, recombination-proficient (RecA⁺) R. meliloti strain L33 whose construction is reported in this paper, and its previously described recombination-deficient (RecA⁻) isogenic counterpart L1 indicate that RecA⁻ strains of Rhizobium are safe hosts for deliberate release experiments.     

Drought tolerance by lentil rhizobia (Rhizobium leguminosarum) from arid and semiarid areas of Pakistan

Ten strains of lentil rhizobia (Rhizobium leguminosarum ) were evaluated for drought tolerance by exposing them to soil moisture potentials of −0·03, −1·0 and −1·5 MPa. Water availability, rhizobial strain and time of exposure to drought had a significant ( P ≤ 0·001) effect on the number of surviving rhizobia g⁻¹ of soil. Highest cell counts were observed at −0·03 MPa, followed by soil maintained at −1·0 and −1·5 MPa. Five strains originating from saline areas showed significantly ( P ≤ 0·05) better survival under low water potential after 35 days. Two strains exhibited greatest survival under low water potential and produced viable cell counts of more than 10⁷ rhizobia g⁻¹ of soil. These strains could probably be used successfully as inoculants for lentil production in arid and semi-arid environments.     

Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples

Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 10², by PCR amplification; 10³, by whole cell luminescence; and 10³−10⁴, by measurement of luminescence in cell extracts.     

Survival and infectivity of a Rhizobium meliloti strain maintained in water and buffer suspensions

Rhizobium meliloti B323 cells were suspended in deionized water, phosphate buffer pH 6.5 and 5.5 and these buffers supplemented with Ca²⁺, Mg²⁺ (1 mmol/l) and Fe³⁺ (0.1 mmol/l). Initial cell count was 1.10⁸ cells/ml. The viable count of rhizobia suspended in buffer at pH 6.5, with and without salts, remained constant or even increased during storage. Cells suspended in buffer at pH 5.5 with salts, decreased in numbers in the first 5 months, then, until the 10th month, the count remained at 10⁵ cells/ml. Rhizobia suspended in buffer at pH 5.5 and deionized water decreased in viability almost to zero by the 10th month. In those suspensions where viability was maintained, the symbiotic infectivity of cells was also maintained as compared with a control performed with fresh cultured rhizobia. In suspensions in deionized water and buffer at pH 5.5 where the viability diminished during the experiment, the rhizobia lost their ability to infect roots immediately after inoculation but maintained their capacity to form late nodules on the hosts.     

An Ecological Study of Marked Rhizobium trifolii Strains on the Host Plant Trifolium repens var. Huai in an Acidic Peat and a Neutral Mineral Soil

An ecological study of the nodulation of Trifolium repens var. grassland Huai by genetically marked Rhizobium trifolii was carried out in two Irish soils, a neutral mineral and an acidic peat. An indigenous population of 2 x 10⁴ R. trifolii /g was found in the mineral soil. In the peat soil, 4 x 10¹ R. trifolii /g was found in the uninoculated peat. This number increased to 4.5 x 10⁵ R. trifolii /g, however, eight weeks after the peat soil was neutralized, supplemented with nutrients and sown with uninoculated clover seed. Indigenous R. trifolii strains from the mineral soil were effective whereas strains from the peat soil were ineffective on the host plant T. repens under plant room conditions. The introduced strains were inoculated on to clover seed at the rate of 1 x 10⁵ R. trifolii /seed. In the mineral soil, the introduced inoculum failed to establish at any period during the growing season. In the peat soil, the percentage establishment of the introduced inoculum varied from 40-50% of nodules selected eight weeks after sowing to 70-90% of nodules selected at the end of the growing season.     

The effect of moisture potential on growth and survival of root nodule bacteria in peat culture and on seed

Peat from three sources was dried, milled and packed separately in polyethylene bags and sterilized by irradiation. The carrier was impregnated with broth cultures of either Rhizobium leguminosarum bv. trifolii strain WU95, Bradyrhizobium japonicum strain CB1809 or B. lupini strain WU425 and sterile water to provide five moisture potentials in the range > - 1 × 10⁴ - 1 × 10⁶ Pa. The packets were stored at 26°C under conditions which restricted moisture loss. Numbers of root nodule bacteria were counted at intervals up to 12 weeks. No single moisture potential was optimum for all strains in all carriers because of a significant ( P < 0.05) interaction between moisture potential × strain × carrier × time. Where direct comparisons could be made, all strains survived best at - 1 × 10⁴ and/or −3.2 × 10⁴ Pa. Seeds of Trifolium subterraneum and polypropylene beads (used to avoid seed coat toxicity), were inoculated with WU95 prepared in two sources of peat and at each of the above moisture potentials and stored at 15°C. Seed coat toxicity significantly effected the log death rate ( k ) of WU95 on subterraneum clover seed for the period 0–0.25 d ( k 1.796) compared with k - 0.399 for polypropylene beads. In the first 24 h moisture did not affect survival but by 28 d rhizobia grown in Badenoch peat survived best at −3.2 × 10⁴ Pa. In Millicent peat, survival was equally as good at −3.2 × 10⁴ and −1 × 10⁴ Pa.     

Plasmid transfer in natural soil: a case by case study with nitrogen-fixing Enterobacter

Abstract In strains of nitrogen-fixing Enterobacter agglomerans , isolated from the rhizosphere of cereals, the nif genes are located on large plasmids. Plasmid pEA9 (200 kb) is self-transmissible between closely related strains. To collect data on possible uncontrolled gene spread, for planned releases of such bacteria, plasmid pEA9 was labelled with transposons (Tn 1725 and Tn 5 ) and used in mating experiments between homologous Enterobacter strains with soil as substrate. The soil was from a plot into which an actual release was being planned. In the majority of experiments it was not sterilized.
Survival and plasmid transfer is described, as are variations in temperature, time, moisture, pH and soil packing. Further experiments were with or without added energy sources, and with or without plant roots. Under standard conditions (22°C, pH 5.2, 15.5% moisture, loose soil, 2 × 10⁷ inoculated donor and recipient cells each per g soil, 3 days incubation) sterilized soil gave low rates of plasmid transfer (10⁻⁶ per donor) but non-sterilized soil gave none. Adding Luria broth or sucrose to non-sterilized soil elicited strong cell propagation, together with plasmid transfer (optimum after incubation for 1 day: 10⁻⁴ exconjugants per donor). No transfer could be registered in the presence of wheat seedling roots for periods up to 5 weeks.     

Characterization of sulfate-reducing bacteria isolated from Senegal ricefields

Abstract Sulfate-reducing bacteria were enumerated in soils and water samples from Senegal ricefields using lactate and sulfate as substrates. When rice plants were severely injured by sulfides, maximum densities ranged from 10⁷ to 10⁹ bacteria g⁻¹ of dry spermosphere or rhizosphere soil. Seven non-sporulating, mesophilic strains were isolated. The strains had motile curved cells and stained Gram-negative. Lactate, pyruvate, H₂+ CO₂, malate, fumarate, or ethanol could serve as electron donors. Organic acids were incompletely oxidized to acetate. Alcohols were degraded to the corresponding fatty acids. Sulfate, sulfite, or thiosulfate could serve as electron acceptors and were reduced to sulfide. Vitamins, yeast extract, Biotrypcase, or additional NaCl were not required for growth. On the basis of morphological and physiological properties, and the G + C mol % of the DNA, six isolates were identified as Desulfovibrio vulgaris and one as Desulfovibrio desulfuricans . The comparison of their main physiological properties with the physico-chemical properties of sampling sites indicated that they were better adapted to conditions prevailing in the rice rhizosphere than to those prevailing in the bulk of soil.     

Sulphate-reducing bacteria in bovine faeces

Sulphate-reducing bacteria (SRB) were found in all of 200 bovine faeces examined. The number of SRB in bovine faeces ranged from 5 times 10² to 6 times 10⁸ bacteria g^-1. Of 50 isolates identified, all were assigned to the genus Desulfovibrio .     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Electrotransformation of meat lactobacilli. Effect of several parameters on their efficiency of transformation

Intact cells of several lactobacilli isolated from Spanish dry fermented sausages ( Lactobacillus curvatus, Lact. sake, Lact. plantarum and Lact. bavaricus ) were transformed by electroporation. With pNZ12 as a vector, transformation efficiencies of 2.4 times 10⁵, 3.8 times 10³ and 8.8 times 10² transformants μg^-1 DNA were observed for Lact. curvatus CTC435, Lact. sake CTC335 and Lact. bavaricus CTC232, respectively.
Effects of variation in experimental parameters on transformation efficiency were evaluated. Strains, vectors and buffers were the determinant parameters. The growth phase of the culture, cell concentration, voltage, use of cell wall weakening agents and the purity of the vector influenced the transformation efficiency in most strains.     

A determination of protective microhabitats for bacteria introduced into soil

Abstract Survival studies with rhizobia introduced into loamy sand showed that a kaolinite amendment of the soil improved the survival of Rhizobium , and that bentonite had a very strong positive effect on rhizobial survival. The survival level was significantly higher in soil amended with 10% than with 5% bentonite. The amount of water present in the bentonite amended soil had a significant influence on rhizobial survival; in drier soil, survival levels were highest. For the loamy sand, the loamy sand amended with 5 and 10% bentonite or with 10% kaolinite, the number of rhizobial cells surviving on day 57 after introducing 2.5–5.0×10⁷ cells g⁻¹ dry soil could be described using the distribution of pores from three size classes in a mathematical relationship. Pores with necks < 3 μm and between 3 and 6 μm positively affected the survival of introduced rhizobia whereas pores with necks > 6 μm had a negative effect.     

The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) to assess microbial populations on food contact surfaces

Two rapid methods, direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) on swab resuspension fluids, were compared with the traditional total viable count (TVC) on swab resuspension fluids for their ability to enumerate surface populations of attached bacteria. The degree of error in estimating surface populations was shown to be significantly less with DEM than DEFT followed by TVC. DEM estimated populations in the range 3 times 10³ to 5 times 10⁷ colonies/cm² whilst DEFT enumerated populations above 3 times 10⁴ colonies/cm² and TVC above 3 times 10⁵ colonies/cm² (as measured by DEM). Swabbing was shown to remove a constant proportion of organisms from the surface populations tested, although below 3 times 10⁵ colonies/cm² most of the organisms remained in the cotton matrix and were difficult to resuspend. DEFT was more able to enumerate swab resuspension fluids obtained from surface populations below 3 times 10⁵ colonies/cm² than was TVC.     

Plasmid and chromosomally encoded luminescence marker systems for detection of Pseudomonas fluorescens in soil

Luminescent strains of Pseudomonas fluorescens 10586 were constructed in which luciferase production was constitutive by introduction of Vibrio fischeri luxABE genes on the chromosome and on a multicopy plasmid. Light production in liquid batch culture was directly proportional to biomass concentration during exponential growth and enabled detection by luminometry of 1.7 × 10³ and 8.9 × 10⁴ cells/ml for the plasmid and chromosomally marked strains, respectively. Luminescent colonies of both strains were detectable by eye, enabling viable cell enumeration on solid media against a background of non-luminescent strains. Following inoculation into sterile and non-sterile soil lower levels of detection were increased but detection of 8.1–59 × 10³and 2.2–30 × 10³ cells per g of soil was possible for plasmid and chromosomally marked strains. Maximum specific growth rate in liquid culture was unaffected by introduction of lux marker genes on the chromosome, but was reduced in the plasmid marked strain. The chromosomally encoded marker was stable in both liquid culture and in soil, but the plasmid was unstable during continuous subculturing in liquid medium and during growth in soil. The chromosomally encoded luminescence-marker system therefore provides a convenient, non-extractive technique for quantification of genetically modified soil microbial inocula.     

Isolation and identification of Burkholderia pseudomallei from soil using selective culture techniques and the polymerase chain reaction

An environmental soil survey to detect Burkholderia pseudomallei was performed during the dry and wet seasons in Darwin, Northern Territory, Australia. Soil was sampled at regular intervals during a 15-month period at different depths from areas which were representative of the local, soil environment. Selective culture techniques using Ashdown's and Galimand and Dodin's methods and the polymerase chain reaction (PCR) using specific 16S rRNA primers were used to detect and identify the organism and determine its distribution within the soil stratum over the change in seasons. Results showed that Ashdown's method gave higher isolation rates in the dry season, and Galimand and Dodin's method gave higher isolation rates during the wet season. PCR of the soil enrichment proved to be a more sensitive method than culture and was also a useful confirmatory test in determining the identification of isolates where biochemical tests gave inconsistent results. The PCR primers were specific and able to detect 10¹ cfu g^-1 soil and 10⁴ cfu g^-1 of soil using Ashdown's enrichment broth and Galimand and Dodin's broth, respectively. Overall the isolation of B. pseudomallei was greatest during the dry season and at the higher and lower soil depths, which is contradictory to epidemiological evidence that melioidosis occurs primarily during the wet season among patients exposed to contaminated surface soil and water.     

Application of subtraction hybridization for the development of a Rhizobium leguminosarum biovar phaseoli and Rhizobium tropici group-specific DNA probe

Abstract A combined subtraction hybridization and polymerase chain reaction/amplification technique was used to develop a DNA probe which was specific for the Rhizobium leguminosarum biovar phaseoli and the Rhizobium tropici group. Total genomic DNA preparations from Rhizobium leguminosarum biovar viciae, Rhizobium leguminosarum biovar trifolii, Rhizobium sp., Agrobacterium tumefaciens, Rhizobium fredii, Bradyrhizobium japonicum, Bradyrhizobium ssp. and Rhizobium meliloti were pooled and used as subtracter DNA against total genomic DNA from the Rhizobium leguminosarum biovar phaseolo strain KIM5s. Only one round of subtraction hybridization at 65°C was necessary to remove all cross-hybridizing sequences. Dot blot hybridizations with total genomic DNA of the eight subtracter organisms and 29 bacteria of different groups confirmed the high specificity of the isolated DNA sequences. Dot blot hybridizations and total genomic DNA from ten different R. Leguminosarum biovar phaseoli and R. tropici strains resulted in strong hybridization signals for all strains tested. The DNA probe for the R. tropici and R. leguminosarum biovar phaseoli group was used for dot blot hybridization with DNA extracts from three tropical and one boreal soil. When correlated with data from Most Probable Number analyses the probe was capable of detecting as low as 3 × 10⁴ homologous indigenous rhizobia per g soil. The technique offers great benefits for the development of DNA probes for monitoring bacterial populations in environmental samples.     

Effect of Saccharomyces boulardii against experimental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice

A.C.P. RODRIGUES, R.M. NARDI, E.A. BAMBIRRA, E.C. VIEIRA AND J.R. NICOLI. 1996. Saccharomyces boulardii was shown to be capable of inhibiting multiplication of enteropathogenic bacteria in vitro and is currently used for its anti-diarrhoea properties. We studied the capacity of this yeast to antagonize Salmonella typhimurium and Shigella flexneri in the intestinal tract of conventional or gnotobiotic NMRI mice. Conventional animals were given daily 10 mg doses of S. boulardii , whereas germ-free animals were given a single 10 mg dose. Both groups were challenged orally 5 d later with the pathogenic bacteria (10⁸ or 10² viable cells, respectively). Control groups were treated with saline instead of S. boulardii. Mortality and/or histopathological data showed a protective effect against the pathogenic bacteria in yeast-treated mice. Saccharomyces boulardii colonized the digestive tract of gnotobiotic mice and the number of viable cells ranged around 10¹⁰ g^-1 of faeces. In experimental and control gnotobiotic animals, Salm. typhimurium and Sh. flexneri became rapidly established at a level of about 10¹⁰ viable cells g^-1 of faeces and remained at high levels until the animals died or were sacrificed. The protection against Salm. typhimurium and Sh. flexneri obtained in conventional and/or gnotobiotic mice previously associated with S. boulardii is not due to the reduction of the bacterial populations in the intestines.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

Dominance of Lactobacillus plantarum strains in grass silage as demonstrated by a novel competition assay

An assay was developed for assessing the competitive ability of potential Lactobacillus plantarum silage inoculants. This assay was based on the ability of the test inoculant to outcompete a standard strain ( Lact. plantarum DCU101) co-inoculated at the same rate of 5 × 10⁵ colony forming units g^-1 of grass. Total populations of Lact. plantarum were enumerated with a selective medium and Lact. plantarum DCU101 was identified with a strain-specific DNA probe. The DNA probe was based on a small (2.2 kb), cryptic, indigenous plasmid which was cloned into pAT153, a multicopy cloning vector. Seven Lact. plantarum strains, six of which were isolated from well-preserved grass silages, were used to inoculate laboratory scale silos, and variation in strain dominance was monitored over the 14 d ensilage period.