Microbial composition and dynamics in environmental samples from a ready-to-eat food production facility with a long-term colonization of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen of significant concern for the food industry due to its remarkable ability to persist through safety control efforts, posing a subsequent health threat to consumers. Understanding the microbial communities coexisting with L. monocytogenes in food processing environments provides insights into its persistence mechanisms. We investigated the microbial communities on non-food contact surfaces in a facility producing ready-to-eat foods, known to harbour a ST121 L. monocytogenes strain over multiple years. A 10-week sampling period was coordinated with the company and public health authorities. Metagenomic analysis revealed a stable microbial composition dominated by Pseudomonas fluorescens. While highly related populations were present in high-care production zones, distinctive taxa characteristic of specific areas were observed (e.g., Sphingomonas aerolata). Although Listeria spp. were not detected in metagenomes, they were detected in cultured samples, suggesting low relative abundance in factory settings. The findings suggest that a stable resident microbiota, with distinct adaptations to different areas within the factory, was selected for by their collective ability to survive control efforts in this environment. Listeria spp. was a member of this microbial community, albeit at low abundance, and may likewise benefit from the mutualism of the overall microbial community.

Rhizobacterial community and growth-promotion trait characteristics of Zea mays L. inoculated with Pseudomonas fluorescens UM270 in three different soils.

There is an increasing demand for bioinoculants based on plant growth-promoting rhizobacteria (PGPR) for use in agricultural ecosystems. However, there are still concerns and limited data on their reproducibility in different soil types and their effects on endemic rhizosphere communities. Therefore, this study explored the effects of inoculating the PGPR, Pseudomonas fluorescens strain UM270, on maize growth (Zea mays L.) and its associated rhizosphere bacteriome by sequencing the 16S ribosomal genes under greenhouse conditions. The results showed that inoculation with PGPR P. fluorescens UM270 improved shoot and root dry weights, chlorophyll concentration, and total biomass in the three soil types evaluated (clay, sandy-loam, and loam) compared to those of the controls. Bacterial community analysis of the three soil types revealed that maize plants inoculated with the UM270 strain showed a significant increase in Proteobacteria and Acidobacteria populations, whereas Actinobacteria and Bacteroidetes decreased. Shannon, Pielou, and Faith alpha-biodiversity indices did not reveal significant differences between treatments. Beta diversity revealed a bacterial community differential structure in each soil type, with some variation among treatments. Finally, some bacterial groups were found to co-occur and co-exclude with respect to UM270 inoculation. Considered together, these results show that PGPR P. fluorescens UM270 increases maize plant growth and has an important effect on the resident rhizobacterial communities of each soil type, making it a potential agricultural biofertilizer.

Antagonistic effect of Bacillus and Pseudomonas combinations against Fusarium oxysporum and their effect on disease resistance and growth promotion in watermelon.

AIMS: We aimed to develop an effective bacterial combination that can combat Fusarium oxysporum infection in watermelon using in vitro and pot experiments. METHODS AND RESULTS: In total, 53 strains of Bacillus and 4 strains of Pseudomonas were screened. Pseudomonas strains P3 and P4 and Bacillus strains XY-2-3, XY-13, and GJ-1-15 exhibited good antagonistic effects against F. oxysporum. P3 and P4 were identified as Pseudomonas chlororaphis and Pseudomonas fluorescens, respectively. XY-2-3 and GJ-1-15 were identified as B. velezensis, and XY-13 was identified as Bacillus amyloliquefaciens. The three Bacillus strains were antifungal, promoted the growth of watermelon seedlings and had genes to synthesize antagonistic metabolites such as bacilysin, surfactin, yndj, fengycin, iturin, and bacillomycin D. Combinations of Bacillus and Pseudomonas strains, namely, XY-2-3 + P4, GJ-1-15 + P4, XY-13 + P3, and XY-13 + P4, exhibited a good compatibility. These four combinations exhibited antagonistic effects against 11 pathogenic fungi, including various strains of F. oxysporum, Fusarium solani, and Rhizoctonia. Inoculation of these bacterial combinations significantly reduced the incidence of Fusarium wilt in watermelon, promoted plant growth, and improved soil nutrient availability. XY-13 + P4 was the most effective combination against Fusarium wilt in watermelon with the inhibition rate of 78.17%. The number of leaves; aboveground fresh and dry weights; chlorophyll, soil total nitrogen, and soil available phosphorus content increased by 26.8%, 72.12%, 60.47%, 16.97%, 20.16%, and 16.50%, respectively, after XY-13 + P4 inoculation compared with the uninoculated control. Moreover, total root length, root surface area, and root volume of watermelon seedlings were the highest after XY-13 + P3 inoculation, exhibiting increases by 265.83%, 316.79%, and 390.99%, respectively, compared with the uninoculated control. CONCLUSIONS: XY-13 + P4 was the best bacterial combination for controlling Fusarium wilt in watermelon, promoting the growth of watermelon seedlings, and improving soil nutrient availability.

Lytic bacteriophages UFJF_PfDIW6 and UFJF_PfSW6 prevent Pseudomonas fluorescens growth in vitro and the proteolytic-caused spoilage of raw milk during chilled storage.

In this study, P. fluorescens-infecting phages were isolated, characterized, and evaluated to their potential to control the bacterial counts and, consequently, the proteolytic spoilage of raw milk during cold storage. The UFJF_PfDIW6 and UFJF_PfSW6 phages showed titers of 9.7 and 7.6 log PFU/ml; latent period of 115 and 25 min, and burst size of 145 and 25 PFU/infected cell, respectively. They also were highly specific to the host bacterium, morphologically classified as the Podoviridae family, stable at pH 5 to 11 and were not inactivated at 63 °C or 72 °C for 30 min. These phages found to be effective against P. fluorescens, reducing bacterial count throughout the entire exponential growth phase in broth formulated with milk at both 4 °C and 10 °C. This effect on bacteria growth led to inhibition by at least 2 days in proteases production, delaying the degradation of milk proteins. When applied together in raw milk stored at 4 °C, they reduced the total bacteria, psychrotrophic, and Pseudomonas by 3 log CFU/ml. This study's findings indicate that these phages have a great potential to prevent the growth of Pseudomonas and, consequently, to retard proteolytic spoilage of raw milk during chilled storage.

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.

Differential protein expression during growth on model and commercial mixtures of naphthenic acids in Pseudomonas fluorescens Pf-5.

Naphthenic acids (NAs) are carboxylic acids with the formula (Cn H2n+Z O2 ) and are among the most toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf-5 on a model NA (4'-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs, including several transporters (e.g., ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid, and amino acid metabolism were also overexpressed, including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.

Influence of molecular weight fractionation on the antimicrobial and anticancer properties of a fucoidan rich-extract from the macroalgae Fucus vesiculosus.

The objective of this study was to investigate the antimicrobial and anticancer properties of a fucoidan extract and subsequent fractions isolated from the macroalgae Fucus vesiculosus. The fractions obtained (>300 kDa, <300 kDa, <100 kDa, <50 kDa and <10 kDa) could inhibit the growth of B. subtilis, E. coli, L. innocua and P. fluorescens when assayed at concentrations between 12,500 and 25,000 ppm. The bacterial growth was monitored by optical density (OD) measurements (600 nm, 24 h) at 30 °C or 37 °C, depending upon on the strain used. The extracted fractions were also tested for cytotoxicity against brain glioblastoma cancer cells using the Alamar Blue assay for 24 h, 48 h and 6 days. The >300 kDa fraction presented the lowest IC50 values (0.052% - 24 h; 0.032% - 6 days). The potential bioactivity of fucoidan as an antimicrobial and anticancer agent was demonstrated in this study. Hence, the related mechanisms of action should be explored in a near future.

Survivability and controlled release of alginate-microencapsulated Pseudomonas fluorescens VUPF506 and their effects on biocontrol of Rhizoctonia solani on potato.

Preserving the efficacy of plant probiotic bacteria in soil is a major challenge to the biological control of plant diseases. The microencapsulation technique is an important step in preserving the viability and activity of probiotics in adverse environmental conditions. The main objective of this study was to choose an appropriate coating for probiotic encapsulation. For this purpose, the survivability and controlled release of Pseudomonas fluorescens VUPF506 encapsulated with alginate (Alg) combined with whey protein concentrate (WPC), carboxymethyl cellulose (CMC), and peanut butter (PB) were evaluated. Moreover, the encapsulated cells were evaluated to control for Rhizoctonia solani in potato plants under in vivo conditions. The results showed that all tested wall material maintained more than 80% of the bacterial cells. The Alg-WPC microcapsules provided a better controlled release over two months. Interestingly, the greenhouse experiment also revealed that the treatment of potato plants with Alg-WPC microcapsules was the most effective treatment, suppressing 90% of the pathogen. The results showed that Alg-WPC is the most promising combination to improve the survivability of P. fluorescens VUPF506. Moreover, it can be used as a fertilizer due to its content of valuable amino acids.

Investigation of presence of endofungal bacteria in Rhizopus spp. isolated from the different food samples.

Rhizopus species are opportunistic pathogens and cause infections which lead to deaths in individuals with the weakened immune system. Some strains of Rhizopus species have been detected to have a symbiotic relationship with bacteria. The toxicity of the Rhizopus species is important. Because strains harbouring endofungal bacteria are able to produce secondary metabolites and if endofungal bacteria are released from mycelium, serious problems can occur. We aimed to investigate the presence of endofungal bacteria in Rhizopus species isolated from food samples. Rhizopus species were isolated from different food samples. The presence of endofungal bacteria in the Rhizopus isolates was investigated. Rhizopus strains containing the endofungal bacteria were identified through phenotypic and genotypic methods. Universal primers amplifying bacterial 16S rRNA region were used to amplify 1.2-1.5-kb fragment from fungal metagenomic DNA. Sequence analysis of PCR products amplified from fungal metagenomic DNA was made. Fluorescence microscopy and scanning electron microscopy were used to visualize the presence of endofungal bacteria in fungal hyphae. According to our results, the Rhizopus strains is associated with Serratia marcescens, Pseudomonas fluorescens and Klebsiella pneumoniae. Until now there is no evidence that Pseudomonas fluorescens and Klebsiella pneumoniae were identified as endofungal. These species are opportunistic pathogen dangerous for humans. It is important for humans not only the presence of the fungi but also the presence of the endofungal bacteria in foods. Our work is important because it draws attention to the presence of endofungal bacteria in foods. Because there is danger releasing of a bacterium from the mycelium, it is likely to face sepsis or serious problems.

Antimicrobial effect and mechanism of non-antibiotic alkyl gallates against Pseudomonas fluorescens on the surface of Russian sturgeon (Acipenser gueldenstaedti).

Since Pseudomonas fluorescens is the main microorganism causing severe spoilage in refrigerated aquatic products, the searching for non-antibiotic antibacterial agents effective against it continues to receive increasing interest. This study aimed to investigate the antibacterial effects and mechanisms of alkyl gallic esters against P. fluorescens isolated from the Russian sturgeon (Acipenser gueldenstaedti), as well as the effectiveness in combination with chitosan films on the preservation of sturgeon meats at 4 °C. Our data shows that the alkyl chain length plays a significant role in eliciting their antibacterial activities and octyl gallate (GAC8) exhibited an outstanding inhibitory efficacy. GAC8 can rapidly enter into the membrane lipid bilayer portion to disorder the membrane, and further inhibit the growth of the P. fluorescens through interfering both tricarboxylic acid cycle related to energy supply and amino acid metabolism associated with cell membranes, suppressing oxygen consumption and disturbing the respiration chain. Moreover, the alteration in membrane fatty acids indicated that GAC8 could disrupt the composition of cell membrane fatty acids, rendering the bacteria more sensitive to the antibacterial. The SEM results also substantiate the damage of the structure of the bacterial membrane caused by GAC8. Additionally, the edible chitosan-based films incorporated with GAC8 showed the enhanced antibacterial efficacy to remarkably extend the shelf life of Russian sturgeon. Overall, our findings not only provide new insight into the mode of action of GAC8 against P. fluorescens but also demonstrate composite films containing GAC8, as a kind of safe and antibacterial material, have a great promise for application in food preservations.

Modulation of Siderophore Production by Pseudomonas fluorescens Through the Manipulation of the Culture Medium Composition.

Pseudomonas fluorescens has the ability to produce the siderophore pyoverdine, a biotechnologically significant iron chelator, which has a wide range of potential applications, such as in agriculture (iron fertilizers) and medicine (development of antibiotics). The present work aimed to evaluate the influence of culture medium composition on the production of siderophores by P. fluorescens DSM 50090, an industrial relevant strain. It was found that the bacterium grown in minimal medium succinate (MMS) had a higher siderophore production than in King B medium. The replacement of succinate by glycerol or dextrose, in minimal medium, originated lower siderophore production. The increase of succinate concentration, the addition of amino acids or the reduction of phosphate in the culture medium did not improve siderophore production by P. fluorescens. The results obtained strongly suggest that (i) MMS is more appropriate than King B for large-scale production of siderophores; (ii) the modification of the culture medium composition, particularly the type of carbon source, influences the level of siderophore secreted; (iii) the production of siderophore by P. fluorescens seems to be a tightly regulated process; once a maximum siderophore concentration has been reached in the culture medium, the bacterium seems to be unable to produce more compound.

Biocontrol of Rhizoctonia solani (Kühn) and Fusarium solani (Marti) causing damping-off disease in tomato with Azotobacter chroococcum and Pseudomonas fluorescens.

BACKGROUND AND OBJECTIVE: The Damping-off disease is one of the most reasons for low productively of tomato in the world, especially in Iraq. In the current study, two types of bacteria (Azotobacter chroococcum and Pseudomonas fluorescens) were used to evaluate their efficacy in inhibiting the growth of pathogenic fungi Rhizoctonia solani and Fusarium solani and protecting the seeds of tomato and increasing their germination percentage. MATERIALS AND METHODS: Dual culture technique and Food poisoning technique were used to study the effect of bacteria on the growth of fungi understudy, and study the effect of bacterial filtrates on germination of tomato seeds. RESULTS: A. chroococcum showed the strongest antagonistic activity followed by P. fluorescens with the percentage of inhibition ranging between 72.9-77.1 and 69.5-70.3% for R. solani and F. solani respectively after 7 days of incubation. The effect of A. chroococcum and P. fluorescens filtrates were increased and also increased the inhibition of growth of fungi understudy, A. chroococcum filtrate also showed the strongest inhibitory effect followed by P. fluorescens with the percentage of inhibition ranging between 86.0-87.0 and 83.0-83.5% for R. solani and F. solani respectively at 20% concentration of filtrate. The percentage of seeds germination reached 90% in the treatment of A. chroococcum filtrate and 80% in the treatment of P. fluorescens filtrate. CONCLUSION: It can be concluded that the filtrates of A. chroococcum and P. fluorescens have antifungal properties against R. solani and F. solani and provided a high protection and increasing tomato seeds germination percentage.

Pseudomonas fluorescens: A Bioaugmentation Strategy for Oil-Contaminated and Nutrient-Poor Soil.

Bioremediation technology is one of the most profitable and sustainable strategies for remediating soils contaminated with hydrocarbons. This study focuses on assessing the influence of biostimulation and bioaugmentation with Pseudomonas fluorescens to contribute to the removal of total petroleum hydrocarbons (TPHs) of a soil. Laboratory studies were carried out (measurements of emitted CO2, surface tension, and residual TPH) to select the best bioaugmentation and biostimulation treatment. The sources of C, N, and P were glucose-yeast extract, NH4Cl-NaNO3, and K2HPO4-K3PO4, respectively. The effect of culture conditions on the reduction of TPH and respiratory activity was evaluated through a factorial design, 23, in a solid culture system. After 80 days of incubation, it was observed that treatments of yeast extract-NH4Cl-K2HPO4 (Y4) and glucose-NaNO3-K3PO4 (Y5) presented a higher level of TPH removal (20.91% and 20.00% degradation of TPH, respectively). Biostimulation favors the production of biosurfactants, indirectly measured by the change in surface tension in the soil extracts. The treatments Y4 and Y5 showed a lower change value of the surface tension (23.15 and 23.30 mN·m-1 at 25 °C). A positive correlation was determined between the change in surface tension and the removal of TPH; hence there was a contribution of the biosurfactants produced to the removal of hydrocarbons.

Interactions between functionalised silica nanoparticles and Pseudomonas fluorescens biofilm matrix: A focus on the protein corona.

Biofilms are microbial communities embedded in an extracellular polymeric matrix and display an enhanced tolerance to the action of antimicrobials. The emergence of novel functionalised nanoparticles is considered a promising avenue for the development of biofilm-specific antimicrobial technologies. However, there is a gap in the understanding of interactions between nanoparticles and the biofilm matrix. Particularly, questions are raised on how nanoparticle charge and surface groups play a role in aggregation when in contact with biofilm components. Herein we present the synthesis of four types of silica nanoparticles and undertake an analysis of their interactions with Pseudomonas fluorescens biofilm matrix. The effect of the biofilm matrix components on the charge and aggregation of the nanoparticles was assessed. Additionally, the study focused on the role of matrix proteins, with the in-depth characterisation of the protein corona of each nanoparticle by Liquid Chromatography with Tandem Mass Spectrometry experiments. The protein corona composition is dependent on the nanoparticle type; non-functionalised nanoparticles show less protein selectivity, whereas carboxylate-functionalised nanoparticles prefer proteins with a higher isoelectric point. These outcomes provide insights into the field of biofilm-nanoparticle interactions that can be valuable for the design of new nano-based targeting systems in future anti-biofilm applications.

Pleiotropic effects of RsmA and RsmE proteins in Pseudomonas fluorescens 2P24.

BACKGROUND: Pseudomonas fluorescens 2P24 is a rhizosphere bacterium that produces 2,4-diacetyphloroglucinol (2,4-DAPG) as the decisive secondary metabolite to suppress soilborne plant diseases. The biosynthesis of 2,4-DAPG is strictly regulated by the RsmA family proteins RsmA and RsmE. However, mutation of both of rsmA and rsmE genes results in reduced bacterial growth. RESULTS: In this study, we showed that overproduction of 2,4-DAPG in the rsmA rsmE double mutant influenced the growth of strain 2P24. This delay of growth could be partially reversal when the phlD gene was deleted or overexpression of the phlG gene encoding the 2,4-DAPG hydrolase in the rsmA rsmE double mutant. RNA-seq analysis of the rsmA rsmE double mutant revealed that a substantial portion of the P. fluorescens genome was regulated by RsmA family proteins. These genes are involved in the regulation of 2,4-DAPG production, cell motility, carbon metabolism, and type six secretion system. CONCLUSIONS: These results suggest that RsmA and RsmE are the important regulators of genes involved in the plant-associated strain 2P24 ecologic fitness and operate a sophisticated mechanism for fine-tuning the concentration of 2,4-DAPG in the cells.

Three biofilm types produced by a model pseudomonad are differentiated by structural characteristics and fitness advantage.

Model bacterial biofilm systems suggest that bacteria produce one type of biofilm, which is then modified by environmental and physiological factors, although the diversification of developing populations might result in the appearance of adaptive mutants producing altered structures with improved fitness advantage. Here we compare the air-liquid (A-L) interface viscous mass (VM) biofilm produced by Pseudomonas fluorescens SBW25 and the wrinkly spreader (WS) and complementary biofilm-forming strain (CBFS) biofilm types produced by adaptive SBW25 mutants in order to better understand the link between these physical structures and the fitness advantage they provide in experimental microcosms. WS, CBFS and VM biofilms can be differentiated by strength, attachment levels and rheology, as well as by strain characteristics associated with biofilm formation. Competitive fitness assays demonstrate that they provide similar advantages under static growth conditions but respond differently to increasing levels of physical disturbance. Pairwise competitions between biofilms suggest that these strains must be competing for at least two growth-limiting resources at the A-L interface, most probably O2 and nutrients, although VM and CBFS cells located lower down in the liquid column might provide an additional fitness advantage through the colonization of a less competitive zone below the biofilm. Our comparison of different SBW25 biofilm types illustrates more generally how varied biofilm characteristics and fitness advantage could become among adaptive mutants arising from an ancestral biofilm-forming strain and raises the question of how significant these changes might be in a range of medical, biotechnological and industrial contexts where diversification and change may be problematic.

Plant Extracts Containing Saponins Affects the Stability and Biological Activity of Hempseed Oil Emulsion System.

In this study, two saponins-rich plant extracts, viz. Saponaria officinalis and Quillaja saponaria, were used as surfactants in an oil-in-water (O/W) emulsion based on hempseed oil (HSO). This study focused on a low oil phase content of 2% v/v HSO to investigate stable emulsion systems under minimum oil phase conditions. Emulsion stability was characterized by the emulsification index (EI), centrifugation tests, droplet size distribution as well as microscopic imaging. The smallest droplets recorded by dynamic light scattering (droplets size v. number), one day after the preparation of the emulsion, were around 50-120 nm depending the on use of Saponaria and Quillaja as a surfactant and corresponding to critical micelle concentration (CMC) in the range 0-2 g/L. The surface and interfacial tension of the emulsion components were studied as well. The effect of emulsions on environmental bacteria strains was also investigated. It was observed that emulsions with Saponaria officinalis extract exhibited slight toxic activity (the cell metabolic activity reduced to 80%), in contrast to Quillaja emulsion, which induced Pseudomonas fluorescens ATCC 17400 growth. The highest-stability samples were those with doubled CMC concentration. The presented results demonstrate a possible use of oil emulsions based on plant extract rich in saponins for the food industry, biomedical and cosmetics applications, and nanoemulsion preparations.

Stress response and survival of Salmonella Enteritidis in single and dual species biofilms with Pseudomonas fluorescens following repeated exposure to quaternary ammonium compounds.

Biofilms formed on food contact surfaces are frequently exposed to disinfectants at different concentrations. This study was designed to evaluate how S. Enteritidis in single species and dual species biofilms with P. fluorescens respond to quaternary ammonium compounds (QAC) residues on food contact surfaces. The 48 h-biofilms of S. Enteritidis and P. fluorescens in single/dual species were continuously exposed to 20 ppm QAC for 5 days, followed by QAC challenge at 200 ppm and 100 ppm for attached and detached cells, respectively. Biofilm structures were observed by confocal laser scanning microscopy (CLSM) and extracellular polymeric substances (EPS)-related gene expression was also evaluated. Results showed that QAC stress led to one log lower cell counts of S. Enteritidis and P. fluorescens single species biofilms. More cellulose observed by CLSM images and increased transcript levels of cellulose-related genes (csgD, bcsA and ardA) of S. Enteritidis were induced by QAC stress. Nevertheless, high percentage of membrane damaged cells in QAC pre-exposed biofilms might contribute to the increased sensitivity of S. Enteritidis in both attached and detached cells. Previous QAC exposure did not influence S. Enteritidis viable cell counts in dual specie biofilms, in which S. Enteritidis showed strong resistance to QAC with <2 log CFU/cm2 reductions. Decreased transcript levels of cellulose-related genes were observed of S. Enteritidis in dual species biofilms, but EPS-related gene expression of P. fluorescens was not affected by single/duals species. The dual species biofilm matrix which has big microcolonies extruding from bottom layers with great amounts of polysaccharides mainly produced by P. fluorescens could possibly protect S. Enteritidis against disinfection. Enhanced survival of S. Enteritidis in dual species biofilms was also found when they were detached from the coupons. Overall, our findings highlight that although repeated exposures to low dose of QAC sensitized S. Enteritidis, the presence of P. fluorescens in dual species biofilms could enhance QAC resistance of S. Enteritidis, probably contributing to survival of S. Enteritidis in food processing plants.

Growth Kinetics and Spoilage Potential of Co-culturing Acinetobacter johnsonii and Pseudomonas fluorescens from Bigeye Tuna (Thunnus obesus) During Refrigerated Storage.

A. johnsonii and P. fluorescens are the well-known specific spoilage organisms in aquatic products and the study of the interactions between A. johnsonii and P. fluorescens are limited. This study aims to evaluate the growth kinetics, spoilage potential and interactions of A. johnsonii and P. fluorescens isolated from spoiled bigeye tuna (Thunnus obesus) by inoculating into sterile fish slices and stored at 4 °C for 6 days. The growth kinetics of A. johnsonii and P. fluorescens were fitted with Baranyi and Roberts model. The chemical indexes (total volatile base nitrogen (TVB-N), trimethylamine (TMA), pH, proteolytic activity and protein content) of each inoculated block of bigeye tuna were increased during refrigerated storage. Moreover, the higher contents of chemical indexes were observed in co-culture with A. johnsonii and P. fluorescens compared with single culture of A. johnsonii and P. fluorescens. In addition, atomic force microscopy (AFM) observation of co-culturing A. johnsonii and P. fluorescens inoculation into sterile fish slices revealed damage of myofibrillar protein structures and the protein degradation. Based on these parameters, a rapid method to evaluate spoilage potential of A. johnsonii and P. fluorescens was positively correlated with TVB-N value, TMA value and pH value (P < 0.05) by the correlation coefficient. Consequently, spoilage potential of microorganisms became stronger evaluated in a mixed culture than single culture. This paper provides insight for a detection method of interactions of A. johnsonii and P. fluorescens during refrigerated storage.

Bioinoculants play a significant role in shaping the rhizospheric microbial community: a field study with Cajanus cajan.

The present study is an attempt to understand the impact of bioinoculants, Azotobacter chroococcum (A), Bacillus megaterium (B), Pseudomonas fluorescens (P), on (a) soil and plant nutrient status, (b) total resident and active bacterial communities, and (c) genes and transcripts involved in nitrogen cycle, during cultivation of Cajanus cajan. In terms of available macro- and micro-nutrients, triple inoculation of the bioinoculants (ABP) competed well with chemical fertilizer (CF). Their 'non-target' effects were assessed in terms of the abundance and activity of the resident bacterial community by employing denaturing gradient gel electrophoresis (DGGE). The resident bacterial community (16S rRNA gene) was stable, while the active fraction (16S rRNA transcripts) was influenced (in terms of abundance) by the treatments. Quantification of the genes and transcripts involved in N cycle by qPCR revealed an increase in the transcripts of nifH in the soil treated with ABP over CF, with an enhancement of 3.36- and 1.57- fold at flowering and maturity stages of plant growth, respectively. The bioinoculants shaped the resident microflora towards a more beneficial community, which helped in increasing soil N turnover and hence, soil fertility as a whole.