Direct Quantitative Evaluation of the Effects of Biocides on Pseudomonas fluorescens in Various Nutrient Media

A method for quantitative evaluation of the effects of biocides is presented. The method was tested in experiments with Pseudomonas fluorescens grown on various agar nutrient media. The effective concentrations of biocides that decreased the maximum specific rate of the colony biomass growth ("_m) were called S (suppressing) concentrations, and concentrations that decreased the number of colony-forming units (CFU) were taken as L (sublethal) concentrations. The efficiency of the reported approach was demonstrated in experiments with three biocides tested in four nutrient media. It was found that the biocide sensitivity of Pseudomonas fluorescens varied by a factor of 30, depending on the amount and type of the nutrient substrate.     

Biocidal activity of formaldehyde and nonformaldehyde biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in pure and mixed suspensions in synthetic metalworking fluid and saline

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.     

Biocontrol strain Pseudomonas fluorescens WCS365 inhibits germination of Fusarium oxysporum spores in tomato root exudate as well as subsequent formation of new spores

Fusarium oxysporum f.sp. radicis-licopersici (Forl) is a soilborne pathogenic fungus which can cause tomato foot and root rot (TFRR). Tomato root exudate is a good source of nutrients for both Forl and the TFRR-suppressing biocontrol bacterium Pseudomonas fluorescens strain WCS365. Incubation of Forl microconidia in tomato root exudate stimulates their germination. This phenomenon is observed, to a lesser extent, upon incubation in plant nutrient solution supplemented with citrate or glucose, the major organic acid and sugar components, respectively, of tomato root exudate. Here we show that induction of germination of microconidia is significantly reduced in the presence of P. fluorescens WCS365 in all tested media. Scanning electron microscopy revealed that P. fluorescens WCS365 colonizes developing hyphae. Efficient colonization correlates with low nutrient availability. Eventually, new microconidia are formed. The presence of P. fluorescens WCS365 reduces the number of newly formed microconidia. This reduction does not depend on physical contact between bacteria and hyphae. We discuss that the ability of P. fluorescens WCS365 to slow down the processes of microconidia germination and development of new microconidia of the phytopathogen, and therefore the ability to reduce fungal dissemination, is likely to contribute to the biocontrol efficacy of this strain.     

Organic acid exudation and pH changes by Gordonia sp. and Pseudomonas fluorescens grown with P adsorbed to goethite

The actinomycete Gordonia sp. and the bacterium Pseudomonas fluorescens Pf-5 were grown in liquid media (pH 6.5) with phosphate adsorbed to the Fe-oxide/hydroxide goethite (Goe-P) and with soluble phosphate (0.1 mM or 1.0 mM P as KH2PO4). The two isolates showed distinct differences in their physiology. The pH of the medium was increased by Gordonia sp. by 1.1-1.7 units while it was decreased by P. fluorescens by 1.4-2.4 units. In all treatments the concentration of organic acids in the media with Gordonia sp. was up to 10 times lower (0.4-10.9 micromol L(-1)) than in media with P. fluorescens (33.4-84.4 micromol L(-1)). Gordonia sp. produced five different organic acids in varying amounts depending on P source and time. In contrast, P. fluorescens exuded mainly citrate and only small amounts of two to three other organic acids irrespective of P source or time.     

Determining the sensitivity of Pseudomonas to chemotherapeutic preparations by the micromethod in liquid synthetic medium

Ever increasing interest is being displayed lately to simple, economic and standard systems for assay of antibiotic sensitivity of microbes with microtechniques in nutrient media requiring no raw materials in short supply. For determining sensitivity of Pseudomonas spp. to chemotherapeutics a liquid synthetic medium balanced by its cationic composition and containing no competing agents of sulfanylamides was used. Three procedures were comparatively estimated: the method of serial dilutions in the liquid medium with using immunological trays, the method of serial dilutions in agar and the diffusion test. In the estimation 185 strains of various Pseudomonas species were used: P. aeruginosa, P. cepacia, P. fluorescens, P. stutzeri, P. putida and P. pseudomallei. The method using the liquid synthetic medium and trays provided more precise interpretation of the results of the assay of the Pseudomonas spp. sensitivity to aminoglycosides, tetracycline, polymyxin and sulfamonomethoxine that the routine procedures. It showed some other advantages such as simplicity, low cost, low medium requirement and glassware economy. The application of the method allowed to exclude the use of expensive imported nutrient media in assay of sulfanylamide sensitivity.     

Proposal for a method to estimate nutrient shock effects in bacteria

ABSTRACT: BACKGROUND: Plating methods are still the golden standard in microbiology; however, some studies have shown that these techniques can underestimate the microbial concentrations and diversity. A nutrient shock is one of the mechanisms proposed to explain this phenomenon. In this study, a tentative method to assess nutrient shock effects was tested. FINDINGS: To estimate the extent of nutrient shock effects, two strains isolated from tap water (Sphingomonas capsulata and Methylobacterium sp.) and two culture collection strains (E. coli CECT 434 and Pseudomonas fluorescens ATCC 13525) were exposed both to low and high nutrient conditions for different times and then placed in low nutrient medium (R2A) and rich nutrient medium (TSA). The average improvement (A.I.) of recovery between R2A and TSA for the different times was calculated to more simply assess the difference obtained in culturability between each medium. As expected, A.I. was higher when cells were plated after the exposition to water than when they were recovered from high-nutrient medium showing the existence of a nutrient shock for the diverse bacteria used. S. capsulata was the species most affected by this phenomenon. CONCLUSIONS: This work provides a method to consistently determine the extent of nutrient shock effects on different microorganisms and hence quantify the ability of each species to deal with sudden increases in substrate concentration.     

Adaptation of Pseudomonas fluorescens to the plant rhizosphere

Saprophytic Pseudomonas are common root-colonizing bacteria that can improve plant health. Efficient exploitation of these bacteria in agriculture requires knowledge of traits that enhance ecological performance in the rhizosphere. Here, I describe the development and application of a promoter-trapping technology (IVET) that enables the isolation of Pseudomonas fluorescens genes that show elevated levels of expression in the rhizosphere. Using IVET, 20 P. fluorescens genes were identified that are induced during rhizosphere colonization, and their patterns of expression were analysed in laboratory media and in the rhizosphere. Fourteen genes showed significant homology to sequences in GenBank that are involved in nutrient acquisition, stress response, or secretion; six showed no homology. Seven of the rhizosphere-induced ( rhi ) genes have homology to known non- Pseudomonas genes. One of the rhi genes ( hrcC ) is a component of a type III secretion pathway, not previously known in non-parasitic bacteria. Together, these genes provide a view of the rhizosphere environment as perceived by a rhizosphere colonist, and suggest that the nature of the association between P. fluorescens and the plant root may be more complex and intimate than previously thought.     

Factors Affecting the Antimicrobial Activity of Vitamin K5

Pure cultures of Escherichia coli, Bacillus subtilis, Proteus vulgaris, Staphylococcus aureus, and Pseudomonas fluorescens were used in this investigation. The bactericidal concentrations of vitamin K(5) required for E. coli, B. subtilis, P. vulgaris, S. aureus, and P. fluorescens; the effect of an absence of oxygen; the effect of contact time with E. coli and S. aureus; and the effect of initial counts per milliliter of E. coli were studied. The bactericidal concentrations ranged from 60 ppm of K(5) for S. aureus to 220 ppm for E. coli, with an initial count of 160,000 to 200,000 cells per milliliter and a contact time of 12 hr in nutrient broth. The gram-positive bacteria tested were more susceptible to the antimicrobial activity of vitamin K(5) than the gram-negative bacteria. In the studies conducted under nitrogen atmosphere, the per cent inhibition showed an inverse relationship to the bactericidal concentrations required for complete inhibition in studies conducted under air atmosphere. This finding suggested that there might be different factors responsible for inhibition depending on the species of bacteria being tested, and it also might help explain the difference in concentrations necessary for inhibition. Cells of E. coli and S. aureus were not inhibited immediately on coming into contact with vitamin K(5); 50% inhibition occurred after 25 and 32 min, respectively. A rapid inhibition rate was maintained until approximately 90% inhibition occurred, after whch a rapid decrease in the rate was noted.     

Plasmid Stability in Pseudomonas fluorescens in the Rhizosphere

Plasmids belonging to various incompatibility (Inc) groups were introduced into the efficiently root-colonizing strain Pseudomonas fluorescens WCS365, and their stabilities in complex and minimal media and in the rhizospheres of tomato, wheat, and potato plants grown under gnotobiotic conditions without selection pressure were tested. The IncP plasmid was found to be highly unstable under all conditions tested, whereas the IncQ and IncW plasmids showed intermediate stabilities and the plasmids pVSP41 and pWTT2081, for which the Inc group is unknown, both containing the origin of replication (rep) and stability (sta) regions of the Pseudomonas aeruginosa pVS1 replicon, were stably maintained under all conditions tested. Growth experiments in which cells of strain WCS365 carrying the plasmid pWTT2081 were grown in the presence of WCS365 without the plasmid showed that the presence of pWTT2081 acts as a burden. We conclude that pVSP41 and pWTT2081 are valuable as stable vectors for the functional analysis of genes involved in root colonization, provided that control cells carry the empty vector.     

Effect of temperature and plasmid carriage on nonculturability in organisms targeted for release

Abstract: The ability to track genetically modified bacteria released into the environment is essential for assessing their persistence and dispersal. Some bacteria can enter a 'viable but nonculturable' (VBNC) state in which the cells remain viable while losing the ability to grow on routine culture media. Thus, VBNC cells are not detectable by standard plating methods. In order to determine what conditions, if any, induce this state in Pseudomonas fluorescens, Pseudomonas syringae , and Escherichia coli , cells were 'marked' with lux genes, either chromosomally or on one of two different plasmids. Variations in temperature, but not nutrient or NaCl concentrations, affected culturability of these strains and induced the VBNC state. The temperature which induced the VBNC state in the two pseudomonads depended on whether or not the cell carried one of the two lux -marked plasmids. This effect was shown not to be due to the presence of the lux genes, as their removal from the plasmid had no effect on entry into the VBNC state. Instead, the effect appeared to depend on the location of the plasmid DNA, as a strain of P. fluorescens with the same plasmid integrated into the chromosome behaved identically to the parent strain. The fact that plasmids may have such a dramatic effect on culturability has significant implications for the monitoring of genetically modified bacteria intended for environmental release.     

Quantitative determination of infinite inhibition concentrations of antimicrobial agents.

We developed a method to determine the infinite inhibition concentrations (IICs) of antimicrobial agents. This method was based on finding the relative effectiveness of an inhibitor at various concentrations. Benzoic acid and parabens were tested on Saccharomyces bayanus, Hansenula sp., and Pseudomonas fluorescens. The relative effectiveness values of these compounds were established. A plot of the inhibitor concentration versus the reciprocal of relative effectiveness was linear. The chi-axis intercept was the concentration of the inhibitor which gave infinite microbial inhibition. For S. bayanus the IICs were 330, 930, 480, and 220 ppm (330, 930, 480, and 220 ml/liter) for benzoic acid and methyl-, ethyl-, and propylparabens, respectively. For Hansenula sp. the IIC was 180 ppm for benzoic acid. For P. fluorescens the IICs were 1,310, 960, and 670 ppm for methyl-, ethyl-, and propylparabens, respectively. Our results indicated that the IIC is affected by the growth medium. The advantages and applications of this method are discussed.     

Quantitative determination of infinite inhibition concentrations of antimicrobial agents

We developed a method to determine the infinite inhibition concentrations (IICs) of antimicrobial agents. This method was based on finding the relative effectiveness of an inhibitor at various concentrations. Benzoic acid and parabens were tested on Saccharomyces bayanus, Hansenula sp., and Pseudomonas fluorescens. The relative effectiveness values of these compounds were established. A plot of the inhibitor concentration versus the reciprocal of relative effectiveness was linear. The chi-axis intercept was the concentration of the inhibitor which gave infinite microbial inhibition. For S. bayanus the IICs were 330, 930, 480, and 220 ppm (330, 930, 480, and 220 ml/liter) for benzoic acid and methyl-, ethyl-, and propylparabens, respectively. For Hansenula sp. the IIC was 180 ppm for benzoic acid. For P. fluorescens the IICs were 1,310, 960, and 670 ppm for methyl-, ethyl-, and propylparabens, respectively. Our results indicated that the IIC is affected by the growth medium. The advantages and applications of this method are discussed.     

Effect of N concentration and N source on root colonization by Pseudomonas fluorescens 2-79RLI

Wheat (Triticum aestivum L.) was grown in nutrient solution with low or high N supply (NH₄NO₃ as N source). To further evaluate the influence of N form and its interaction with the nutrient solution pH, wheat plants were grown with NH ₄ ⁺ or NO ₃ ^- either in an conventional nutrient solution or in a nutrient solution in which the pH was maintained at pH 6.5 using a pH-stat system. The nutrient solution was inoculated with Pseudomonas fluorescens 2-79RLI, a genetically modified bacterium that contains lux genes activated by a ribosomal promoter. Cell numbers and physiological status of P. fluorescens 2-79RLI (length of the lag phase of bioluminescence) in the rhizosphere were determined at the root tip and in the lateral root zone. Nitrogen deficiency decreased both plant growth and root colonization by P. fluorescens 2-79RLI at the root tip while it had no effect on root colonization in the lateral root zone. The physiological status of P. fluorescens 2-79RLI was not affected by nitrogen deficiency. Ammonium nutrition increased root colonization by P. fluorescens 2-79RLI at the root tip and in the lateral root zone when the pH of the nutrient solution was allowed to change according to the N form provided. Under these conditions, the physiological status of P. fluorescens 2-79RLI was higher in the lateral root zone than at the root tip. In contrast, N source had no effect on root colonization or physiological status of P. fluorescens 2-79RLI in the nutrient solution maintained at pH 6.5. It is concluded that the stimulation of root colonization by NH ₄ ⁺ in the nutrient solution, not maintained at a constant pH, may be due to increased leakage of solutes into the rhizosphere as a result of impaired exudate retention by high H⁺ concentration in the rhizosphere or the apoplast. This revised version was published online in June 2006 with corrections to the Cover Date.     

Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates).

The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK 13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.     

Molecular analysis of the Pseudomonas aeruginosa regulatory genes ptxR and ptxS.

We have previously described two Pseudomonas aeruginosa genes, ptxR, which enhances toxA and pvc (the pyoverdine chromophore operon) expression, and ptxS, the first gene of the kgu operon for the utilization of 2-ketogluconate by P. aeruginosa. ptxS interferes with the effect of ptxR on toxA expression. In this study, we have utilized DNA hybridization experiments to determine the presence of ptxR and ptxS homologous sequences in several gram-negative bacteria. ptxR homologous sequences were detected in P. aeruginosa strains only, while ptxS homologous sequences were detected in P. aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens. Using Northern blot hybridization experiments and a ptxS-lacZ fusion plasmid, we have shown that P. aeruginosa ptxR and ptxS are expressed in P. putida and P. fluorescens. Additional Northern blot hybridization experiments confirmed that ptxS is transcribed in P. putida and P. fluorescens strains that carried no plasmid. The presence of a PtxS homologue in these strains was examined by DNA-gel shift experiments. Specific gel shift bands were detected when the lysates of P. aeruginosa, P. putida, and P. fluorescens were incubated with the ptxS operator site as probe. kgu-hybridizing sequences were detected in P. putida and P. fluorescens. These results suggest that (i) ptxR is present in P. aeruginosa, while ptxS is present in P. aeruginosa, P. putida, and P. fluorescens; (ii) both ptxR and ptxS are expressed in P. putida and P fluorescens; and (iii) a PtxS homologue may exist in P. putida and P. fluorescens.     

Comparative adherence to human A549 cells, plant fibronectin-like protein, and polystyrene surfaces of four Pseudomonas fluorescens strains from different ecological origin

The main objective of this study was to compare the adherence properties of four Pseudomonas fluorescens isolates from different ecological niches (human tissue, rhizosphere, drinking water, and cow milk). The substrates used to test P. fluorescens adherence were as follows: cultured human respiratory epithelial cells A549, immobilized plant fibronectin-like protein, and polystyrene. For all the experiments, bacteria were grown at 27 degrees C. The adherence assay to human cells was performed at 37 degrees C, whereas adherence to fibronectin and polystyrene was done at 27 degrees C. The four strains tested adhered to A549 cells but showed different adherence patterns. At 3 h, the milk isolate showed an aggregative adherence phenotype, whereas the three other isolates showed a diffuse adherence pattern. With a longer incubation time of 24 h, the aggregative pattern of the milk isolate disappeared, the adherence of the clinical strain increased, the adherence of the water isolate decreased, and morphological changes in A549 cells were observed with the clinical, water, and soil isolates. The four strains tested formed biofilms on polystyrene dishes. The clinical and milk isolates were the more efficient colonizers of polystyrene surfaces and also the more adherent to immobilized plant fibronectin-like protein. There was no relation between bacterial surface hydrophobicity and P. fluorescens adherence to the substrates tested. The main conclusions of these results are that P. fluorescens is an adherent bacterium, that no clear correlation exists between adherence and ecological habitat, and that P. fluorescens can adhere well to substrates not present in its natural environment.     

Isolation and growth of a Pseudomonas species that utilizes cyanide as a source of nitrogen

A simple method of isolating bacteria that utilize cyanide as a source of nitrogen for growth has been developed. This involved supplying hydrogen cyanide as a vapour to glucose-containing minimal-salts agar plates. The bacteria isolated were Gram-negative, oxidase-positive rods producing a fluorescent green pigment and were tentatively identified as strains of Pseudomonas fluorescens. Three organisms were studied further and shown to be P. fluorescens biotype II. One of these (NCIB 11764) was grown in a glucose-containing fed-batch culture with either NH4Cl or KCN as the limiting nutrient. Cyanide-grown bacteria produced stoichiometric amounts of ammonia from cyanide when pulsed with cyanide under aerobic conditions. Stimulation of oxygen uptake was seen on addition of cyanide to suspensions of cyanide-grown but not ammonia-grown bacteria.     

Specific ion concentration as a factor in barotolerant protein synthesis in bacteria.

The degree of barotolerance exhibited by Pseudomonas fluorescens and Pseudomonas bathycetes in vitro polyphenylalanine-synthesizing systems can be modified by altering the concentrations of specific ions in the reaction mixture. Hybrid-protein-synthesizing systems, utilizing all the possible S-100 supernatant fluid and ribosome combinations from Escherichia coli, P. fluorescens, and P. bathycetes, were tested for barotolerance under conditions of low (16 mM Mg2+ plus 0 mM Na+) and high (150 mM Na+ plus 60 mM Mg2+) ion concentrations. The results reveal that barotolerant synthesis is a characteristic determined by the origin of the ribosome. Systems utilizing E. coli ribosomes are barosensitive at both low and high ion concentrations, P. fluorescens ribosomes barotolerant under both conditions, and P. bathycetes ribosomes barosensitive at low and barotolerant at high ion concentrations. Therefore, certain concentrations of specific ions will increase barotolerance, but only if the ribosomes are capable of functioning at high pressures.     

The effect of fifteen biocides on formaldehyde-resistant strains ofPseudomonas aeruginosa

Summary Evaluation of formaldehyde and fifteen biocides in formaldehyde sensitive (S) and resistant (R) strains ofPseudomonas aeruginosa revealed a pattern of response that allowed a comparison of the mode of action of these biocides. The response of these strains to the various biocides, as well as the induction of transient resistance or cross-resistance in the (S) strain, allowed a grouping of biocides based on this pattern of response. Group 1 biocides acted in a manner indistinguishable from formaldehyde for both the (S) and (R) strains. Group 2 biocides were not effective against either the (S) or (R) strains at concentrations calculated to release equimolar concentrations of formaldehyde. However, treatment of the (S) strain with formaldehyde or Group 2 biocides resulted in the development of cross-resistance. Group 3 biocides were equally effective against the (S) and (R) strain, but the (S) strain survivors of treatment with Group 3 biocides were resistant to formaldehyde. Group 4 biocides (controls) had no presumed connection to formaldehyde mode of action. These four groupings, based on pattern of response, also resulted in groupings of biocides based on chemical structure.     

Use of in vivo expression technology to identify genes important in growth and survival of Pseudomonas fluorescens Pf0-1 in soil: discovery of expressed sequences with novel genetic organization

Studies were undertaken to determine the genetic needs for the survival of Pseudomonas fluorescens Pf0-1, a gram-negative soil bacterium potentially important for biocontrol and bioremediation, in soil. In vivo expression technology (IVET) identified 22 genes with elevated expression in soil relative to laboratory media. Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation. Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci. Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in laboratory media. Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional laboratory approaches.