Low-energy X-ray inactivation of Listeria monocytogenes in mono-/co-culture biofilms with Pseudomonas fluorescens on food contact surfaces.

This study assessed the inactivation kinetics of 150 keV low-energy X-ray on mono-/co-culture biofilms of Listeria monocytogenes and Pseudomonas fluorescens on three different food-contact-surfaces (polyethylene, acrylic, and stainless steel). The results indicated that the level of biofilm formation of mono-/co-cultures of L. monocytogenes and P. fluorescens was the highest on acrylic. The mono-culture L. monocytogenes biofilm cells exhibited higher resistance to the low-energy X-rays than the corresponding mono-culture P. fluorescens biofilm cells, regardless of surface types. Furthermore, co-culture had enhanced the resistance of both P. fluorescens and L. monocytogenes biofilm cells to the low-energy X-ray. Two kinetic models for the inactivation process were investigated, including (i) Linear model and (ii) Weibull model. Based on R2, RMSE and AIC analysis, the Weibull model was superior in fitting the inactivation curves of low-energy X-ray on L. monocytogenes in mono-/co-culture biofilms with P. fluorescens. For mono-culture biofilms, the irradiation achieved the tR1 value (derived from the Weibull model, i.e., the dose required for the first 1-log reduction) of 46.36-50.81 Gy for L. monocytogenes and the tR1 value of 25.61-31.33 Gy for P. fluorescens. For co-culture biofilms, higher tR1 values for L. monocytogenes (59.54-70.77 Gy) and P. fluorescens (32.73-45.13 Gy) were yielded than those for their individual counterparts in mono-culture biofilm.

Antibacterial effect and mechanisms of action of 460-470 nm light-emitting diode against Listeria monocytogenes and Pseudomonas fluorescens on the surface of packaged sliced cheese.

The aim of this study was to investigate the antibacterial effect of 460-470 nm light-emitting diode (LED460-470nm) illumination against pathogens and spoilage bacteria on the surface of agar media and packaged sliced cheese. LED460-470nm illumination highly inhibited the growth of Listeria monocytogenes and Pseudomonas fluorescens on agar media covered with oriented polypropylene (OPP) film (thickness, 0.03 mm). When sliced cheeses inoculated with L. monocytogenes or P. fluorescens and packaged with OPP film were illuminated by an LED460-470 nm at 4 or 25 °C, reduction levels of L. monocytogenes and P. fluorescens on packaged slice cheese were higher at 4 °C than at 25 °C. There were no significant differences in color between non-illuminated and illuminated sliced cheese after storage for 7 d at 4 °C. LED460-470 nm illumination at 4 °C for 4 d caused cellular injury of L. monocytogenes and P. fluorescens related to RNA, protein, and peptidoglycan metabolism, and a disruption of the cell membrane and loss of cytoplasmic components were observed from TEM results. These results suggest that LED460-470 nm illumination, in combination with refrigeration temperatures, may be applied to extend the shelf-life of packaged slice cheese and minimize the risk of foodborne disease, without causing color deterioration.

High intensity light pulses to reduce microbial load in fresh cheese.

The present study focused on the utilisation of High Intensity Light Pulses (HILP) treatment to preserve mozzarella cheese. First, the susceptibility of Pseudomonas fluorescens and Enterobacteriaceae to HILP (fluences from 0·39 to 28·0 J/cm2) in a transparent liquid was evaluated (in-vitro tests). Afterwards, the effects on inoculated mozzarella cheese were also assessed. Then untreated (Control) and HILP treated samples were packaged and stored at 10 °C for 2 weeks. Enterobacteriaceae, Pseudomonas spp. and pH were monitored during storage. In a transparent liquid (in-vitro tests) there was a significant microbial inactivation just with 2 s of treatment. On the inoculated cheese a relevant microbial reduction of about 1 log cycle was observed, according to the exposure to the treatments. For Pseudomonas spp. in particular, in the treated samples, the microbiological acceptability limit (106 cfu/g) was never reached after 2 weeks of refrigerated storage. To sum up, the efficacy of this treatment is very interesting because a microbial reduction was observed in treated samples. HILP treatment is able to control the microbial growth and may be considered a promising way to decontaminate the surface of mozzarella cheese.

Predictive modeling of Pseudomonas fluorescens growth under different temperature and pH values

Abstract Meat is one of the most perishable foods owing to its nutrient availability, high water activity, and pH around 5.6. These properties are highly conducive for microbial growth. Fresh meat, when exposed to oxygen, is subjected to the action of aerobic psychrotrophic, proteolytic, and lipolytic spoilage microorganisms, such as Pseudomonas spp. The spoilage results in the appearance of slime and off-flavor in food. In order to predict the growth of Pseudomonas fluorescens in fresh meat at different pH values, stored under refrigeration, and temperature abuse, microbial mathematical modeling was applied. The primary Baranyi and Roberts and the modified Gompertz models were fitted to the experimental data to obtain the growth parameters. The Ratkowsky extended model was used to determine the effect of pH and temperature on the growth parameter µmax. The program DMFit 3.0 was used for model adjustment and fitting. The experimental data showed good fit for both the models tested, and the primary and secondary models based on the Baranyi and Roberts models showed better validation. Thus, these models can be applied to predict the growth of P. fluorescens under the conditions tested.

Adaptive radiation of Pseudomonas fluorescens SBW25 in experimental microcosms provides an understanding of the evolutionary ecology and molecular biology of A-L interface biofilm formation.

Combined experimental evolutionary and molecular biology approaches have been used to investigate the adaptive radiation of Pseudomonas fluorescens SBW25 in static microcosms leading to the colonisation of the air-liquid interface by biofilm-forming mutants such as the Wrinkly Spreader (WS). In these microcosms, the ecosystem engineering of the early wild-type colonists establishes the niche space for subsequent WS evolution and colonisation. Random WS mutations occurring in the developing population that deregulate diguanylate cyclases and c-di-GMP homeostasis result in cellulose-based biofilms at the air-liquid interface. These structures allow Wrinkly Spreaders to intercept O2 diffusing into the liquid column and limit the growth of competitors lower down. As the biofilm matures, competition increasingly occurs between WS lineages, and niche divergence within the biofilm may support further diversification before system failure when the structure finally sinks. A combination of pleiotropic and epistasis effects, as well as secondary mutations, may explain variations in WS phenotype and fitness. Understanding how mutations subvert regulatory networks to express intrinsic genome potential and key innovations providing a selective advantage in novel environments is key to understanding the versatility of bacteria, and how selection and ecological opportunity can rapidly lead to substantive changes in phenotype and in community structure and function.

Predictive modeling of Pseudomonas fluorescens growth under different temperature and pH values.

Meat is one of the most perishable foods owing to its nutrient availability, high water activity, and pH around 5.6. These properties are highly conducive for microbial growth. Fresh meat, when exposed to oxygen, is subjected to the action of aerobic psychrotrophic, proteolytic, and lipolytic spoilage microorganisms, such as Pseudomonas spp. The spoilage results in the appearance of slime and off-flavor in food. In order to predict the growth of Pseudomonas fluorescens in fresh meat at different pH values, stored under refrigeration, and temperature abuse, microbial mathematical modeling was applied. The primary Baranyi and Roberts and the modified Gompertz models were fitted to the experimental data to obtain the growth parameters. The Ratkowsky extended model was used to determine the effect of pH and temperature on the growth parameter µmax. The program DMFit 3.0 was used for model adjustment and fitting. The experimental data showed good fit for both the models tested, and the primary and secondary models based on the Baranyi and Roberts models showed better validation. Thus, these models can be applied to predict the growth of P. fluorescens under the conditions tested.

Bacterial bioeffectors modify bioactive profile and increase isoflavone content in soybean sprouts (Glycine max var Osumi).

The effect of two bacterial strains to enhance bioactive contents (total phenolic compounds, total flavonoid compounds and isoflavones) and antioxidant activity on 3-day-old soybean sprouts were investigated. To identify bacterial determinants responsible for these effects, viable and UV-treated strains were delivered to wounded seeds at different concentration. Multivariate analysis performed with all the evaluated parameters indicated the different effectiveness of Stenotrophomonas maltophilia N5.18 and Pseudomonas fluorescens N21.4 based on different structural and metabolic determinants for each. N21.4 increased total phenolics and isoflavones from the genistein family, while N5.18 triggered biosynthesis of daidzein and genistein families coupled to a decrease in total phenolics, suggesting different molecular targets in the phenilpropanoid pathway. Only extracts from N5.18 treated seeds showed an improved antioxidant activity according to the ß-carotene bleaching prevention method. In summary, bioeffectors from both bacterial strains are effective tools to improve soybean sprouts quality; structural elicitors from N5.18 also enhanced antioxidant activity, being the best alternative for further development of a biotechnological procedure.

Physico-chemical factors affect chloramphenicol efflux and EmhABC efflux pump expression in Pseudomonas fluorescens cLP6a.

Protein synthesis inhibitors such as chloramphenicol and tetracycline may be inducers of efflux pumps such as MexY in Pseudomonas aeruginosa, complicating their use for the treatment of bacterial infections. We previously determined that chloramphenicol, a substrate of the EmhABC efflux pump in Pseudomonas fluorescens cLP6a, did not induce emhABC expression. In this study, we determined the effect of physico-chemical factors on chloramphenicol efflux by EmhABC, and the expression of emhABC. Efflux assays measuring accumulation of (14)C-chloramphenicol in cell pellets showed that chloramphenicol efflux is dependent on growth temperature, pH and concentration of Mg(2+). These physico-chemical factors modulated the efflux of chloramphenicol by 26 to >50%. All conditions tested that decreased the efflux of chloramphenicol unexpectedly induced transcription of emhABC efflux genes. EmhABC activity also effectively suppressed the deleterious effect of chloramphenicol on the cell membrane of strain cLP6a, which may explain why chloramphenicol is not an inducer of emhABC. Our results suggest that the detrimental effect of an antibiotic on cell membrane integrity and fatty acid composition may be the signal that induces emhABC expression, and that inducers of other bacterial efflux pumps may include environmental factors rather than their substrates per se.

Increase in oxidative stress at low temperature in an antarctic bacterium.

Association between cold stress and oxidative stress was demonstrated by measuring the activity of two antioxidant enzymes and the level of free radicals generated in two batches of cells of an Antarctic bacterium Pseudomonas fluorescens MTCC 667, grown at 22 and 4°C. Increase in oxidative stress in cells grown at low temperature was evidenced by increase in the activity of an enzyme and also in the amount of free radicals generated, in the cold-grown cells. The association between cold stress and oxidative stress demonstrated in this investigation bolsters the concept of interlinked stress response in bacteria.

Effectiveness of pulsed ultraviolet-light treatment for bacterial inactivation on agar surface and liquid medium.

In the present study, the efficiency of a broad-spectrum pulsed ultraviolet (UV)-light for the inactivation of Listeria monocytogenes Scott A, L. monocytogenes CNL 895807, and Pseudomonas fluorescens MF37 populations as agar seeded or suspended cells was investigated. The bacterial populations were treated by pulsed UV-light at different number of pulses (1 to 3), dose of energy (162, 243, or 324 J), and distance from the strobe (4, 9, or 12 cm). After pulsed UV-light treatment, the bacterial reduction was determined by standard plate count. The results showed that there was a significant reduction of population along with an increase of light energy and number of pulses. Decreasing the distance between the Petri dishes and the xenon lamp demonstrated an increase in bacterial reduction. Decontamination efficacy decreased significantly with the increase in level of contamination. This study demonstrates that pulsed UV-light can be used as an effective sterilizing method for the bacteria.

Role of RpoS in stress tolerance and environmental fitness of the phyllosphere bacterium Pseudomonas fluorescens strain 122.

Bacteria living epiphytically on aerial plant surfaces encounter severe and rapidly fluctuating environmental conditions, and their capacity to withstand environmental stress contributes to epiphytic fitness. The stationary phase sigma factor RpoS is a key determinant in stress response of gram-negative bacteria, including Pseudomonas spp. This study focused on the role of RpoS in stress response and epiphytic fitness of Pseudomonas fluorescens strain 122 on aerial plant surfaces. RpoS had a significant role in the response of the phyllosphere bacterium P. fluorescens 122 to stresses imposed by desiccation, UV irradiation, starvation, and an oxidative environment. While significant, the difference in stress response between an rpoS mutant and the parental strain was less for strain 122 than for the rhizosphere bacterium P. fluorescens Pf-5. No consistent influence of RpoS on epiphytic population size of strain 122 on pear or apple flowers or leaves was observed in field trials. These data may indicate that P. fluorescens occupies protected microsites on aerial plant surfaces where the bacteria escape exposure to environmental stress, or that redundant stress-response mechanisms are operating in this bacterium, thereby obscuring the role of RpoS in epiphytic fitness of the bacterium.

Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide.

AIMS: To evaluate the efficacy of ultraviolet (UV) light (254 nm) combined with hydrogen peroxide (H(2)O(2)) to inactivate bacteria on and within fresh produce. METHODS AND RESULTS: The produce was steep inoculated in bacterial cell suspension followed by vacuum infiltration. The inoculated samples were sprayed with H(2)O(2) under constant UV illumination. The log count reduction (LCR) of Salmonella on and within lettuce was dependent on the H(2)O(2) concentration, temperature and treatment time with UV intensity being less significant. By using the optimized parameters (1.5% H(2)O(2) at 50 degrees C, UV dose of 37.8 mJ cm(-2)), the surface Salmonella were reduced by 4.12 +/- 0.45 and internal counts by 2.84 +/- 0.34 log CFU, which was significantly higher compared with H(2)O(2) or UV alone. Higher LCR of Escherichia coli O157:H7, Pectobacterium carotovora, Pseudomonas fluorescens and Salmonella were achieved on leafy vegetables compared with produce, such as cauliflower. In all cases, the surface LCR were significantly higher compared with the samples treated with 200 ppm hypochlorite. UV-H(2)O(2)-treated lettuce did not develop brown discolouration during storage but growth of residual survivors occurred with samples held at 25 degrees C. CONCLUSIONS: UV-H(2)O(2) reduce the bacterial populations on and within fresh produce without affecting the shelf-life stability. SIGNIFICANCE OF THE STUDY: UV-H(2)O(2) represent an alternative to hypochlorite washes to decontaminate fresh produce.

The sigma factor RpoS is required for stress tolerance and environmental fitness of Pseudomonas fluorescens Pf-5.

Many micro-organisms exist in natural habitats that are subject to severe or dramatically fluctuating environmental conditions. Such is the case for bacteria inhabiting plant surfaces, where they are exposed to UV irradiation, oxygen radicals, and large fluctuations in temperature and moisture. This study focuses on the role of RpoS, a central regulator of stationary-phase gene expression in bacterial cells, in stress response and environmental fitness of Pseudomonas fluorescens Pf-5. Strain Pf-5 is a rhizosphere-inhabiting bacterium that suppresses plant diseases caused by several plant-pathogenic fungi and oomycetes. Previous studies demonstrated that rpoS was required for osmotic and oxidative stress resistance of Pf-5. The results of this study demonstrate a role for rpoS in tolerance of Pf-5 to freezing, starvation, UV irradiation and desiccation stress. In field studies, an rpoS mutant was compromised in rhizosphere colonization of plants in dry soil, whereas similar rhizosphere populations were established by Pf-5 and an rpoS mutant in well-irrigated soils. RpoS is a key determinant in stress response and environmental fitness of the rhizosphere bacterium P. fluorescens Pf-5.

The effect of electron beam irradiation on the survival of Salmonella enterica serovar typhimurium and psychrotrophic bacteria on raw chicken breasts stored at four degrees celsius for fourteen days.

The effect of high-energy electron beam irradiation on the survival of Salmonella enterica serovar Typhimurium and psychrotrophic bacteria on commercial chicken breast meat was evaluated. Fresh chicken breast meat was purchased from a local poultry processor, inoculated with 8 log10 cfu/mL Salmonella, packaged in Styrofoam trays and over wrapped with a polyvinyl chloride film, and subjected to 0, 1, 2, or 3 kGy of irradiation. The packaged samples were stored at 4 degrees C and analyzed for Salmonella Typhimurium and psychrotrophic organisms at 0, 2, 4, 6, 8, 10, 12, and 14 d of storage. Direct plating and enrichment methods were used for S. Typhimurium analyses. The direct plating method revealed a 4 log reduction in Salmonella for chicken breasts inoculated and treated with 1, 2, or 3 kGy of irradiation. Psychrotrophic counts were conducted at 7 degrees C for 10 d and 25 degrees C for 5 d to determine the effect of incubation methods on the recovery of psychrotrophic organisms. The enrichment method resulted in the repair of injured Salmonella cells and an elevated Salmonella Typhimurium count for all irradiation dosages when compared with data reported for the direct plating method. In general, psychrotrophic counts increased as storage time increased. However, psychrotrophic counts decreased (P < 0.05) as the irradiation dosage increased.

Effect of electrical charges and fields on injury and viability of airborne bacteria.

In this study, the effects of the electric charges and fields on the viability of airborne microorganisms were investigated. The electric charges of different magnitude and polarity were imparted on airborne microbial cells by a means of induction charging. The airborne microorganisms carrying different electric charge levels were then extracted by an electric mobility analyzer and collected using a microbial sampler. It was found that the viability of Pseudomonas fluorescens bacteria, used as a model for sensitive bacteria, carrying a net charge from 4100 negative to 30 positive elementary charges ranged between 40% and 60%; the viability of the cells carrying >2700 positive charges was below 1.5%. In contrast, the viability of the stress-resistant spores of Bacillus subtilis var. niger (used as simulant of anthrax-causing Bacillus anthracis spores when testing bioaerosol sensors in various studies), was not affected by the amount of electric charges on the spores. Because bacterial cells depend on their membrane potential for basic metabolic activities, drastic changes occurring in the membrane potential during aerosolization and the local electric fields induced by the imposed charges appeared to affect the sensitive cells' viability. These findings facilitate applications of electric charging for environmental control purposes involving sterilization of bacterial cells by imposing high electric charges on them. The findings from this study can also be used in the development of new bioaerosol sampling methods based on electrostatic principles.

[Some biological characteristics of genetically engineered insecticidal Pseudomonas fluorescens].

Plasmid stability, Antifungal activity, plant-colonizing ability, UV resistance and inseticidal activity in field were analysed for the engineered Pseudomonas fluorescens (Pf) strain IPP202. The results indicated that the recombinant plasmid in IPP202 was very stable after successive diluting culturing and after continuous culturing, There was no significant change in the properties beneficial to plants, such as antifungal activity and plant-colonizing ability as compared with the original strain P303. IPP202 was much more resistant to UV than Bt strain HD73. The control effect in field against cotton boll worm in field was close to that of a locally used Bt-chemical mixture in normal applied concentration. All the data indicated that the engineered Pf strain was a one with prosperous future after further study.

Lon protease influences antibiotic production and UV tolerance of Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5 is a soil bacterium that suppresses plant pathogens due in part to its production of the antibiotic pyoluteorin. Previous characterization of Pf-5 revealed three global regulators, including the stationary-phase sigma factor sigma(S) and the two-component regulators GacA and GacS, that influence both antibiotic production and stress response. In this report, we describe the serine protease Lon as a fourth global regulator influencing these phenotypes in Pf-5. lon mutants overproduced pyoluteorin, transcribed pyoluteorin biosynthesis genes at enhanced levels, and were more sensitive to UV exposure than Pf-5. The lon gene was preceded by sequences that resembled promoters recognized by the heat shock sigma factor sigma(32) (sigma(H)) of Escherichia coli, and Lon accumulation by Pf-5 increased after heat shock. Therefore, sigma(H) represents the third sigma factor (with sigma(S) and sigma(70)) implicated in the regulation of antibiotic production by P. fluorescens. Lon protein levels were similar in stationary-phase and exponentially growing cultures of Pf-5 and were not positively affected by the global regulator sigma(S) or GacS. The association of antibiotic production and stress response has practical implications for the success of disease suppression in the soil environment, where biological control organisms such as Pf-5 are likely to encounter environmental stresses.

Survival of Pseudomonas fluorescens and Salmonella typhimurium after electron beam and gamma irradiation of refrigerated beef.

The radurization effects of gamma ray and electron beam irradiation at 1.5 and 3.0 kGy on beef steaks inoculated with Salmonella Typhimurium and Pseudomonas fluorescens were investigated during 8 days of storage at 5 degrees C. Total bacterial counts and numbers of Salmonella Typhimurium and P. fluorescens were analyzed at 2-day intervals. Total bacterial counts of samples irradiated by both gamma rays and electron beam were significantly (P < 0.05) reduced by 3.8 to 5.3 log CFU/g. Salmonella Typhimurium was not detectable during the experimental period. P. fluorescens counts of beef samples irradiated by gamma rays at both 1.5 and 3.0 kGy were not detected; however, P. fluorescens in samples irradiated by electron beam at 1.5 and 3.0 kGy was recovered after 2 days, and bacterial counts reached 7.8 and 6.9 log CFU/g, respectively. Both gamma ray and electron beam irradiation reduced total bacterial counts initially, possibly extending shelf life. Irradiation was very effective in destroying Salmonella Typhimurium; however, P. fluorescens was not completely eliminated by electron beam irradiation. Consequently, gamma ray irradiation was more effective than electron beam irradiation in the destruction of P. fluorescens.

Inhibition of denitrification by ultraviolet radiation.

It has been shown that UV-A (lambda=320-400 nm) and UV-B (lambda=280-320 nm) inhibit photosynthesis, nitrogen fixation and nitrification. The purpose of this study was to determine the effects, if any, on denitrification in a microbial community inhabiting the intertidal. The community studied is the microbial mat consisting primarily of Lyngbya that inhabits the Pacific marine intertidal, Baja California, Mexico. Rates of denitrification were determined using the acetylene blockage technique. Pseudomonas fluorescens (ATCC #17400) was used as a control organism, and treated similarly to the mat samples. Samples were incubated either beneath a PAR transparent, UV opaque screen (OP3), or a mylar screen to block UV-B, or a UV transparent screen (UVT) for 2 to 3 hours. Sets of samples were also treated with nitrapyrin to inhibit nitrification, or DCMU to inhibit photosynthesis and treated similarly. Denitrification rates were greater in the UV protected samples than in the UV exposed samples the mat samples as well as for the Ps fluorescens cultures. Killed controls exhibited no activity. In the DCMU and nitrapyrin treated samples denitrification rates were the same as in the untreated samples. These data indicate that denitrification is directly inhibited by UV radiation.

Characterization of the recA gene from Pseudomonas fluorescens OE 28.3 and construction of a recA mutant.

The recA gene of Pseudomonas fluorescens OE 28.3 was isolated by complementation of the Fec- phenotype of recombinant lambda EMBL3 phages in a RecA- Escherichia coli strain. The subcloned recA restored resistance to UV and methyl methanesulphonate (MMS) exposure in recA mutants of E. coli. DNA sequence analysis showed that the coding region of the P. fluorescens gene, specifying a protein of 352 amino acid residues, was preceded by an SOS box highly similar to those of Pseudomonas aeruginosa and Azotobacter vinelandii. The deduced amino acid sequence displayed highest homology to the RecA proteins from P. aeruginosa (87.8% identity) and A. vinelandii (84.3% identity). In both the regulatory region and the structural gene, a relatively high degree of sequence divergence from the Pseudomonas cepacia gene was observed. A mutant of P. fluorescens was constructed by inserting a kanamycin resistance cassette into its recA gene. This mutant exhibited an increased sensitivity to UV irradiation and MMS, and was strongly impaired in homologous recombinational activity.