Modeling the rate of attachment of Listeria monocytogenes, Pantoea agglomerans, and Pseudomonas fluorescens to, and the probability of their detachment from, potato tissue at 10 degrees C

The rate of attachment of bacteria to, and their subsequent detachment from, the cut surface of raw potato tissue was measured and modeled by using mathematical approaches that allowed detailed objective comparisons of adhesion processes under different conditions. Attachment was rapid and reached equilibrium after contact for 60 min. A new method to measure the probability of detachment was developed and modeled, revealing that the probability of detachment for Pseudomonas fluorescens remained unchanged for contact times between less than 5 s and 60 min. Listeria monocytogenes, however, was more easily removed initially, with the probability of detachment decreasing over the first 2 min of contact but remaining constant and equivalent to that for Pseudomonas fluorescens thereafter. For all of the bacteria tested, the number of bacteria attached after 2 min of contact was proportional to the inoculum concentration raised to the power of 0.79.     

The Effect of Phylogenetically Different Bacteria on the Fitness of Pseudomonas fluorescens in Sand Microcosms

In most environments many microorganisms live in close vicinity and can interact in various ways. Recent studies suggest that bacteria are able to sense and respond to the presence of neighbouring bacteria in the environment and alter their response accordingly. This ability might be an important strategy in complex habitats such as soils, with great implications for shaping the microbial community structure. Here, we used a sand microcosm approach to investigate how Pseudomonas fluorescens Pf0-1 responds to the presence of monocultures or mixtures of two phylogenetically different bacteria, a Gram-negative (Pedobacter sp. V48) and a Gram-positive (Bacillus sp. V102) under two nutrient conditions. Results revealed that under both nutrient poor and nutrient rich conditions confrontation with the Gram-positive Bacillus sp. V102 strain led to significant lower cell numbers of Pseudomonas fluorescens Pf0-1, whereas confrontation with the Gram-negative Pedobacter sp. V48 strain did not affect the growth of Pseudomonas fluorescens Pf0-1. However, when Pseudomonas fluorescens Pf0-1 was confronted with the mixture of both strains, no significant effect on the growth of Pseudomonas fluorescens Pf0-1 was observed. Quantitative real-time PCR data showed up-regulation of genes involved in the production of a broad-spectrum antibiotic in Pseudomonas fluorescens Pf0-1 when confronted with Pedobacter sp. V48, but not in the presence of Bacillus sp. V102. The results provide evidence that the performance of bacteria in soil depends strongly on the identity of neighbouring bacteria and that inter-specific interactions are an important factor in determining microbial community structure.     

Biofilms of nitrile-hydrolyzing bacteria: Dynamics of growth,resistance to toxic substances,and biotechnological potential

Monospecies and mixed bacterial biofilms of Rhodococcus ruber gt1, Pseudomonas fluorescens C2, Alcaligenes faecalis 2, and Rh. erythropolis 11-2 were obtained during growth in presence of the carriers. The transformation of aliphatic and aromatic nitriles by the biofilms of nitrile-hydrolyzing bacteria, as well as the growth dynamics of Rh. ruber gt1 and P. fluorescens C2 biofilms and their resistance to toxic substrates and products of nitrile hydrolysis, were studied. It was shown that the P. fluorescens C2 biofilm mass and total ATP content reached the maxima after 1 day of growth, whereas Rh. ruber gt1 reached them after 3–4 days of cultivation. The biofilms of Rh. ruber gt1 and P. fluorescens C2 were more resistant to the effects of high concentrations of acrylamide and acrylonitrile and had a greater adaptive capacity than planktonic cells.     

Pseudomonas fluorescens can induce and divert the human β-defensin-2 secretion in intestinal epithelial cells to enhance its virulence

The effect of intestinal molecules produced by the host on the virulence of Pseudomonas fluorescens is poorly documented. In the present work, we evaluated the secretion of human β-defensin-2 (hBD-2) by enterocytes after infection with P. fluorescens (a species previously suggested to be involved in inflammatory bowel disease) and investigated the effect of this host-defense peptide on the bacterial virulence. The results showed that P. fluorescens can induce hBD-2 production in Caco-2/TC7 cells via P38 and ERK MAPK-dependent pathways. Surprisingly, the exposure of P. fluorescens to low doses of the antimicrobial peptide was found to enhance its cytotoxic and proinflammatory effects suggesting a potential feedback mechanism in the dialog between bacteria and the host.     

Influence of nanophase titania topography on bacterial attachment and metabolism

Surfaces with nanophase compared to conventional (or nanometer smooth) topographies are known to have different properties of area, charge, and reactivity. Previously published research indicates that the attachment of certain bacteria (such as Pseudomonas fluorescens 5RL) is higher on surfaces with nanophase compared to conventional topographies, however, their effect on bacterial metabolism is unclear. Results presented here show that the adhesion of Pseudomonas fluorescens 5RL and Pseudomonas putida TVA8 was higher on nanophase than conventional titania. Importantly, in terms of metabolism, bacteria attached to the nanophase surfaces had higher bioluminescence rates than on the conventional surfaces under all nutrient conditions. Thus, the results from this study show greater select bacterial metabolism on nanometer than conventional topographies, critical results with strong consequences for the design of improved biosensors for bacteria detection.     

Construction and Analysis of Fractional Multifactorial Designs To Study Attachment Strength and Transfer of Listeria monocytogenes from Pure or Mixed Biofilms after Contact with a Solid Model Food

The aim of this study was to establish which of seven factors influence the adhesion strength and hence bacterial transfer between biofilms containing Listeria monocytogenes (pure and two-species biofilms) and tryptone soya agar (TSA) as a solid organic surface. The two-species biofilms were made of L. monocytogenes and one of the following species of bacteria: the nonpathogenic organisms Kocuria varians, Pseudomonas fluorescens, and Staphylococcus sciuri and CCL 63, an unidentified gram-negative bacterium isolated from the processing plant environment. We used biofilms prepared under conditions simulating open surfaces in meat-processing sites. The biofilm's adhesion strength and population were evaluated by making 12 contacts on a given whole biofilm (4.5 cm²), using a new slice of a sterilized TSA cylinder for each contact, and plotting the logarithm CFU · cm⁻² detached by each contact against the contact number. Three types of detachment kinetics were observed: biphasic kinetics, where the first slope may be either positive or negative, and monophasic kinetics. The bacteria that resisted a chlorinated alkaline product and a glutaraldehyde- and quaternary ammonium-based disinfectant had greater adhesion strengths than those determined for untreated biofilms. One of the four non-Listeria strains studied, Kocuria varians CCL 56, favored both the attachment and detachment of L. monocytogenes. The stainless steel had smaller bacterial populations than polymer materials, and non-Listeria bacteria adhered to it less strongly. Our results helped to evaluate measures aimed at controlling the immediate risk, linked to the presence of a large number of CFU in a foodstuff, and the delayed risk, linked to the persistence of L. monocytogenes and the occurrence of slightly contaminated foods that may become dangerous if L. monocytogenes multiplies during storage. Cleaning and disinfection reduce the immediate risk, while reducing the delayed risk should be achieved by lowering the adhesion strength, which the sanitizers used here cannot do at low concentrations.     

Distribution of a Take-All Suppressive Strain of Pseudomonas fluorescens on Seminal Roots of Winter Wheat

An antibiotic-resistant strain of Pseudomonas fluorescens, that suppresses take-all of wheat, was used to study the distribution of the bacteria on seminal roots of wheat after being introduced onto seeds. Cells of P. fluorescens were isolated from the entire length of the root, and density of the introduced bacteria declined with the distance from the base of the root. Maximum populations of 10⁵ to 10⁶ CFU and 10³ to 10⁵ CFU per cm of root were detected on sections of roots near the seed and root tip, respectively. The introduced bacteria competed well with indigenous bacteria, comprising at least 25% of the fluorescent pseudomonads detected by plate counts for 48 days after planting.     

Evaluation of a liquid formulation of Pseudomonas fluorescens against Fusarium oxysporum f. sp. cubense and Helicotylenchus multicinctus in banana plantation

Plant growth promoting rhizobacteria (PGPR) are eco-friendly alternatives to chemical fungicides to manage plant diseases. We evaluated the efficacy of a Pseudomonas fluorescens formulation against Fusarium oxysporum f. sp. cubense and Helicotylenchus multicinctus at multiple banana plantations. Three field trials were conducted to assess the wilt incidence and the populations of nematode and bacteria in the soil treated with a liquid formulation of P. fluorescens at 2.0, 3.0 and 4.0 l ha^?1 using drip irrigation system at 60, 120, 180 and 240 days after planting. The results showed that the treatment at 4.0 l ha^?1 reduced the wilt incidence by 60 %. It also reduced the overall population of H. multicinctus by 41.3–89.0 % in the treated fields. The presence of P. fluorescens in the treated soil was 5.6 × 10⁶ cfu g^?1 of soil at the time of harvest. The treatment of biocontrol agent P. fluorescens also resulted in an overall yield increase in banana production by 36.6–46.5 % compared to the control.     

Heat-stable proteases of psychrotrophic bacteria isolated from cooled raw milk

Summary 143 psychrotrophic bacteria were isolated and identified from 53 cooled raw bulk milk samples. The most encountered isolates were Pseudomonas fluorescens and P. fluorescens-like organisms, Achromobacter xylosoxidans, Acinetobacter lwoffii, Aeromonas hydrophila and enterobacteria. The strongest activity was displayed by the pseudomonads, especially the P. fluorescens-like organisms. All the excreted proteases examined were heat-stable: the best inactivation was obtained after a heat-treatment of 1 h at 55°C and 2 min at 140°C. At temperatures of 80, 90 and 100°C often increasing heat-resistance was observed.     

Dexamethasone,a Specific PLA2 Inhibitor,Allows Nonpathogenic Pseudomonas fluorescens to Induce Virulence Against Spodoptera exigua

Phospholipase A₂ (PLA₂) catalyzes phospholipids at sn-2 position to release arachidonic acid (20:4n-6). The arachidonic acid is further oxidized to form different eicosanoids, which play biological mediators to express cellular or humoral immune reactions in response to pathogen infection. Xeno-rahbdus and Photorhabdus, the symbiotic bacteria of entomopathogenic nematodes, have been known to inhibit PLA₂ to express their pathogenicity. This research aimed to test a hypothesis that other entomopathogenic bacteria also inhibit PLA₂ to express their pathogenicity in Spodopera exigua. Two bacterial species of Enterococcus faecalis and Pseudomonas fluorescens presumably different in ento-mopathogenicity were analyzed in their PLA₂ inhibitory activities. A pathogenic E. faecalis induced significantly immunodepression of S. exigua by inhibiting PLA₂ activity because the bacteria-infected S. exigua recovered immune reactions after the addition of arachidonic acid. However, the nonpathogenic P. fluorescens did not induce immunodepression because the addition of arachidonic acid to P. fluorescens-infected S. exigua did not further increase immune capacities while dexamethasone, a PLA₂ inhibitor, could decrease the immune activities. Injection of E. faecalis along with 10 μg of dexamethasone significantly increased pathogenicity in comparison with the bacteria alone. Moreover, the addition of dexamethasone transformed nonpathogenic P. fluorescens into pathogenic bacterium. This study suggests an evidence that PLA₂ is an inhibitory target even for entomopathogenic bacteria not related with entomopathogenic nematodes, and that the inhibition of PLA₂ determines the bacterial virulence in S. exigua.     

Chromosomal insertion of the entire Escherichia coli lactose operon,into two strains of Pseudomonas,using a modified mini-Tn5 delivery system

A 12-kb PstI fragment including the entire E. coli lactose operon (lacIPOZYA) was inserted in one copy into the chromosome of Pseudomonas putida, Pseudomonas fluorescens and an E. coli strain with lac⁻ phenotype. This was made possible by improvements of an already existing mini-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on the chromosome of the recipient bacteria in single copies. This has resulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites needed in the mini-Tn5 delivery system; (b) the generation of E. coli nonlysogenic strains expressing the π protein thus being capable of maintaining and delivering R6K-based mini-Tn5 vectors to other E. coli strains; (c) the successful insertion of the E. coli lactose operon into the P. fluorescens chromosome giving P. fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the assumption that the mini-Tn5 delivery system always creates one-copy inserts. These improvements allow insertion of large DNA fragments encoding highly expressed proteins into the chromosome of a large variety of Gram-negative bacteria including E. coli.     

Bacterial biosynthesis of 1-aminocyclopropane-1-caboxylate (ACC) deaminase,a useful trait to elongation and endophytic colonization of the roots of rice under constant flooded conditions

This study was conducted to investigate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Pseudomonas fluorescens strain REN₁ and its ability to reduce ethylene levels produced during stress, endophytically colonize and promote the elongation of the roots of rice seedlings under gnotobiotic conditions. We isolated 80 bacteria from inside roots of rice plants grown in the farmers’ fields in Guilan, Iran. All of the isolates were characterized for plant growth promoting (PGP) traits. In addition, the colonization assay of these isolates on rice seedlings was carried out to screen for competent endophytes. The best bacterial isolate, based on ACC deaminase production, was identified and used for further study. 16S rDNA sequence analysis revealed that the endophyte was closely related to Pseudomonas fluorescens. The results of this study showed ACC deaminase containing P. fluorescens REN1 increased in vitro root elongation and endophytically colonized the root of rice seedlings significantly, as compared to control under constant flooded conditions. The trait of low amount of indole-3-acetic acid (IAA) production (<15 μg mL⁻¹) and the high production of ACC deaminase by bacteria may be main factors in colonizing rice seedling roots compared to other PGP traits (siderophore production and phosphate solubilization) in this study. Endophytic IAA and ACC deaminase-producing bacteria may be preferential selections by rice seedlings. Therefore, it may be suggested that the utilization of ACC as a nutrient gives the isolates advantages in more endophytic colonization and increase of root length of rice seedlings.     

In Situ Monitoring of the Nascent Pseudomonas fluorescens Biofilm Response to Variations in the Dissolved Organic Carbon Level in Low-Nutrient Water by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy

Drinking water quality management requires early warning tools which enable water supply companies to detect quickly and to forecast degradation of the microbial quality of drinking water during its transport throughout distribution systems. This study evaluated the feasibility of assessing, in real time, drinking water biostability by monitoring in situ the evolution of the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) fingerprint of a nascent reference biofilm exposed to water being tested. For this purpose, the responses of nascent Pseudomonas fluorescens biofilms to variations in the dissolved organic carbon (DOC) level in tap water were monitored in situ and in real time by ATR-FTIR spectroscopy. Nascent P. fluorescens biofilms consisting of a monolayer of bacteria were formed on the germanium crystal of an ATR flowthrough cell by pumping bacterial suspensions in Luria-Bertani (LB) medium through the cell. Then they were exposed to a continuous flow of dechlorinated sterile tap water supplemented with appropriate amounts of sterile LB medium to obtain DOC concentrations ranging from 1.5 to 11.8 mg/liter. The time evolution of infrared bands related to proteins, polysaccharides, and nucleic acids clearly showed that changes in the DOC concentration resulted in changes in the nascent biofilm ATR-FTIR fingerprint within 2 h after exposure of the biofilm to the water being tested. The initial bacterial attachment, biofilm detachment, and regrowth kinetics determined from changes in the areas of bands associated with proteins and polysaccharides were directly dependent on the DOC level. Furthermore, they were consistent with bacterial adhesion or growth kinetic models and extracellular polymeric substance overproduction or starvation-dependent detachment mechanisms.     

Fine-scale recombination and adaptive radiation could be linked

The difficult reconstruction of the evolutionary history of the major surface protein gene oprF highlighted an adaptive radiation in the Pseudomonas fluorescens group. The recent work of Hao (2013) showed that partial recombination events in oprF gene occurred specifically in a P. fluorescens lineage under ecological niche segregation. So, I suggest that identification of lineage-specific fine-scale recombination may be a way to detect putative adaptive radiation in bacteria.     

Influence of Solid Surface, Adhesive Ability, and Inoculum Size on Bacterial Colonization in Microcosm Studies

Microcosm studies were performed to evaluate the effect of solid surfaces, bacterial adhesive ability, and inoculum size on colonization success and persistence of Pseudomonas fluorescens and Xanthomonas maltophilia, each with a Tn5 insertion that conferred resistance to kanamycin and streptomycin. Two types of microcosms were used: (i) a simple system that was colonized by Aeromonas hydrophila and a coryneform and (ii) a complex system produced from lake water enrichment cultures. Simple microcosms contained 100 ml of peptone- and yeast extract-supplemented artificial lake water or 60 ml of peptone- and yeast extract-supplemented artificial lake water with 70 g of 3-mm glass beads. Complex microcosms contained 100 ml of lake water with no nutrient additions or 100 ml of lake water with 70 g of glass beads. The microcosms were incubated for 35 days at 20°C. In lake water enrichment microcosms, the presence of beads increased the abilities of P. fluorescens or X. maltophilia to colonize, but their numbers decreased with time in microcosms both with and without beads. The adhesiveness of the bacteria, measured in an in vitro assay, did not relate to colonization success. In simple microcosms, the inoculum size (10, 10², or 10³) of P. fluorescens did not influence colonization success. However, in complex microcosms, an inoculum of 10³ cells was insufficient to ensure colonization by P. fluorescens, while 10⁶ cells resulted in colonization of liquid and beads. Simple microcosm studies, utilizing only a few species, were poor models for complex natural systems. In complex enrichment systems, colonization of surfaces resulted in higher numbers of organisms but did not noticeably promote persistence. Adhesiveness of a particular organism may be a relatively minor factor influencing its ability to colonize solid surfaces in complex natural environments.     

6:2 Fluorotelomer alcohol (6:2 FTOH) biodegradation by multiple microbial species under different physiological conditions

Factors affecting microbial aerobic biodegradation of 6:2 fluorotelomer alcohol [6:2 FTOH, F(CF₂)₆CH₂CH₂OH] were investigated using three alkane-degrading bacteria (Mycobacterium vaccae JOB5, Pseudomonas oleovorans, and Pseudomonas butanovora) and one fluoroacetate-degrading bacterium (Pseudomonas fluorescens DSM 8341). In the presence of formate (an external reducing energy source), P. fluorescens DSM 8341 produced perfluorobutanoic acid by removing three –CF₂– groups from 6:2 FTOH. Only P. fluorescens DSM 8341 transformed 5:3 acid to 4:3 acid and perfluoropentanoic acid. However, formate showed no effects on the degradation rates, patterns, or transformation products of 6:2 FTOH by M. vaccae JOB5. When dicyclopropylketone (an alkane hydroxylase inducer) or formate was added, P. oleovorans rapidly degraded 6:2 FTOH and produced PFPeA. In the presence of lactate, P. butanovora degraded 6:2 FTOH slowly but produced diverse metabolites. Our results demonstrate that the extent and mechanisms of 6:2 FTOH biotransformation are affected by strain types, enzyme inducers, and levels of reducing energy.     

No apparent costs for facultative antibiotic production by the soil bacterium Pseudomonas fluorescens Pf0-1

Background

Many soil-inhabiting bacteria are known to produce secondary metabolites that can suppress microorganisms competing for the same resources. The production of antimicrobial compounds is expected to incur fitness costs for the producing bacteria. Such costs form the basis for models on the co-existence of antibiotic-producing and non-antibiotic producing strains. However, so far studies quantifying the costs of antibiotic production by bacteria are scarce. The current study reports on possible costs, for antibiotic production by Pseudomonas fluorescens Pf0-1, a soil bacterium that is induced to produce a broad-spectrum antibiotic when it is confronted with non-related bacterial competitors or supernatants of their cultures.

Methodology and Principal Findings

We measured the possible cost of antibiotic production for Pseudomonas fluorescens Pf0-1 by monitoring changes in growth rate with and without induction of antibiotic production by supernatant of a bacterial competitor, namely Pedobacter sp.. Experiments were performed in liquid as well as on semi-solid media under nutrient-limited conditions that are expected to most clearly reveal fitness costs. Our results did not reveal any significant costs for production of antibiotics by Pseudomonas fluorescens Pf0-1. Comparison of growth rates of the antibiotic-producing wild-type cells with those of non-antibiotic producing mutants did not reveal costs of antibiotic production either.

Significance

Based on our findings we propose that the facultative production of antibiotics might not be selected to mitigate metabolic costs, but instead might be advantageous because it limits the risk of competitors evolving resistance, or even the risk of competitors feeding on the compounds produced.     

Natural Transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in Soil

Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria, Agrobacterium tumefaciens and Pseudomonas fluorescens, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover, P. fluorescens produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested. A. tumefaciens could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.     

Characterization of new bacterial biocontrol agents Acinetobacter,Bacillus, Pantoea and Pseudomonas spp. mediating grapevine resistance against Botrytis cinerea

A collection of 282 bacterial isolates from the rhizosphere and different organs of healthy field-grown grapevine plants was obtained and screened for their ability to protect grapevine leaves against Botrytis cinerea, the causal agent of gray mold. Twenty-six strains effectively controlled B. cinerea infections on leaves. After phenotypic and molecular analysis, seven strains were identified as Pseudomonas fluorescens PTA-268 and PTA-CT2, Bacillus subtilis PTA-271, Pantoea agglomerans PTA-AF1 and PTA-AF2, and Acinetobacter lwoffii PTA-113 and PTA-152. In vitro antifungal experiments showed that from these seven strains, only PTA-AF1 and PTA-CT2 exhibited a direct antagonism against B. cinerea. Furthermore, the biocontrol activity of the seven bacteria was associated with differential induction of defense-related responses lipoxygenase, phenylalanine ammonia-lyase and chitinase in grapevine leaves. Our results show that the selected bacteria can efficiently protect grapevine leaves against gray mold disease through an induction of plant resistance and in some cases by an additional antagonistic activity.     

Catalase and Superoxide Dismutase of Root-Colonizing Saprophytic Fluorescent Pseudomonads

Root-colonizing, saprophytic fluorescent pseudomonads of the Pseudomonas putida-P. fluorescens group express similar levels of catalase and superoxide dismutase activities during growth on a sucrose- and amino acid-rich medium. Increased specific activities of catalase but not superoxide dismutase were observed during growth of these bacteria on components washed from root surfaces. The specific activities of both enzymes were also regulated during contact of these bacteria with intact bean roots. Increased superoxide dismutase and decreased catalase activities were observed rapidly, by 10 min upon inoculation of cells onto intact bean roots. Catalase specific activity increased with time to peak at 12 h before declining. By 48 h, the cells displayed this low catalase but maintained high superoxide dismutase specific activities. Catalase with a low specific activity and a high superoxide dismutase activity also were present in extracts of cells obtained from 7-day-old roots colonized from inoculum applied to seed. This specific activity of superoxide dismutase of root-contacted cells was about fourfold-higher in comparison to cells grown on rich medium, whereas the specific activity for catalase was reduced about fivefold. A single catalase isozyme, isozyme A, and one isozyme of superoxide dismutase, isozyme 1, were detected during growth of the bacteria on root surface components and during exposure of cells to intact bean roots for 1 h. An additional catalase, isozyme B, was detected from bacteria after exposure to the intact bean roots for 12 h. Catalase isozyme A and superoxide dismutase isozyme 1 were located in the cytoplasm and catalase band B was located in the membrane of P. putida.