Screening and quantification of anti-quorum sensing and antibiofilm activity of Actinomycetes isolates against food spoilage biofilm-forming bacteria.

BACKGROUND: Biofilms can form in many industries, one of them is the food industry. The formation of biofilms in this industry could cause immense economic losses and endanger public health. Biofilms formation is mainly triggered by quorum sensing. Therefore, inhibition of quorum sensing could be an innovative approach to inhibit the formation of biofilms. One way to inhibit quorum sensing is by using anti-quorum sensing compounds. Actinomycetes are a group of bacteria that is acknowledged to produce these compounds. RESULTS: There were eight crude extracts of Actinomycetes isolates that showed promising anti-quorum sensing activity against Chromobacterium violaceum. The concentration of the crude extracts was 20 mg/mL. All the crude extracts showed no antibacterial activity against food spoilage bacteria, except for crude extracts of isolate 18 PM that showed antibacterial activity against Bacillus subtilis. They also showed various antibiofilm activity, both inhibition and destruction. The highest inhibition and destruction activity sequentially was done by crude extracts of isolate 12 AC with 89.60% against Bacillus cereus and crude extracts of isolate SW03 with 93.06% against Shewanella putrefaciens. CONCLUSIONS: Actinomycetes isolates that isolated from different regions in Indonesia can be used as potential candidates to overcome biofilms formed by food spoilage bacteria using their ability to produce anti-quorum sensing compounds.

Antibacterial Activity and Mechanism of Linalool against Shewanella putrefaciens.

The demand for reduced chemical preservative usage is currently growing, and natural preservatives are being developed to protect seafood. With its excellent antibacterial properties, linalool has been utilized widely in industries. However, its antibacterial mechanisms remain poorly studied. Here, untargeted metabolomics was applied to explore the mechanism of Shewanella putrefaciens cells treated with linalool. Results showed that linalool exhibited remarkable antibacterial activity against S. putrefaciens, with 1.5 µL/mL minimum inhibitory concentration (MIC). The growth of S. putrefaciens was suppressed completely at 1/2 MIC and 1 MIC levels. Linalool treatment reduced the membrane potential (MP); caused the leakage of alkaline phosphatase (AKP); and released the DNA, RNA, and proteins of S. putrefaciens, thus destroying the cell structure and expelling the cytoplasmic content. A total of 170 differential metabolites (DMs) were screened using metabolomics analysis, among which 81 species were upregulated and 89 species were downregulated after linalool treatment. These DMs are closely related to the tricarboxylic acid (TCA) cycle, glycolysis, amino acid metabolism, pantothenate and CoA biosynthesis, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism. In addition, linalool substantially affected the activity of key enzymes, such as succinate dehydrogenase (SDH), pyruvate kinase (PK), ATPase, and respiratory chain dehydrogenase. The results provided some insights into the antibacterial mechanism of linalool against S. putrefaciens and are important for the development and application of linalool in seafood preservation.

The applications of Lactobacillus plantarum-derived extracellular vesicles as a novel natural antibacterial agent for improving quality and safety in tuna fish.

Bacteria release membrane vesicles into the extracellular environment but which activity is unclear. We investigated the applications of extracellular vesicles (EVs) isolated from probiotic Lactobacillus plantarum to protect tuna fish against spoilage and quality loss in this study. A significant difference was found in EVs size obtained from L. plantarum after 8, 24, and 48 hr incubation. The L. plantarum-derived EVs were collected and used to confirm the anti-bacterial activity versus Shewanella putrefaciens. Finally, the tuna fish was stored at 4 °C for 5 days after coating with EVs or sodium erythorbate, and the quality indexes were assayed. Results indicated that EVs markedly inhibited oxidation reaction, total volatile base nitrogen (TVBN), peroxide value (PV), malondialdehyde (MDA), and bacteria levels. These results finding out that EVs from L. plantarum may have potential for application in food storage technology. Overall, we indicated this new material may be developed as an anti-bacterial agent for prolonging the shelf life of tuna fish.

Extraction of specific egg yolk antibodies and application in chitosan coating: effect on microbial and sensory properties of rainbow trout fillet during chilled storage.

BACKGROUND: This study investigated the effect of chitosan coating enriched with extracted egg yolk antibodies on microbial and sensory quality of rainbow trout fillet during refrigeration (4 ± 1 °C). Firstly, bacterial antigen suspensions (total psychrophilic bacteria, Pseudomonas fluorescens and Shewanella putrefaciens) were injected into the breast muscles of chickens. Eggs of immunized chickens were then collected to isolate immunoglobulin from egg yolks (IgY). Fresh fish fillets were coated by chitosan solution containing different types of IgY separately, at two concentrations (60 and 90 mg mL-1 ), refrigerated for 16 days and analysed for total viable count, psychrotrophic, Pseudomonas spp., P. fluorescens and S. putrefaciens count as well as sensorial properties. RESULTS: The tested microbial values increased in all samples during the storage period; however, CH + IgY treated samples could significantly retard microbial growth compared to control. The shelf life of CH + IgY-P samples was extended for about 4 days, while it was extended for about 8 days in CH + IgY-S and CH + IgY-T samples, when they were compared to control (p < 0.05). Higher scores for sensory attributes were also observed in CH + IgY treated samples, especially in CH + IgY-S samples until the end of storage period. CONCLUSIONS: Accordingly, use of chitosan coating containing IgY increases the microbial and sensory quality of fish flesh at 4 °C. Therefore, given the consumer interest in natural additives, chitosan coating containing IgY can be a promising candidate. © 2018 Society of Chemical Industry.

Shewanella putrefaciens CN32 outer membrane cytochromes MtrC and UndA reduce electron shuttles to produce electricity in microbial fuel cells.

The extracellular electron transfer (EET) process of Shewanella species is believed to be indispensable for their anaerobic respiration with an electrode. However, the function of outer membrane c-type cytochromes (OM c-Cyts, the primary components of the EET pathway) is still controversial. In this study, we investigated the effect of two OM c-Cyts (MtrC and UndA) of Shewanella putrefaciens CN32 with respect to electricity production and anodic EET efficiency. Deletion of the mtrC gene severely prolonged the microbial fuel cell (MFC) start-up time and decreased electricity production due to depressed flavin-mediated electron transfer, whereas deletion of the undA gene did not have a significant impact. Strikingly, the depression of EET by the deletion of mtrC could be partially relieved by acclimation, which might be due to an increase in the transmembrane transport of electron shuttles and/or the activation of other redox proteins. These results suggested that MtrC may be the primary reductase of flavins to ensure fast indirect EET, which plays a crucial role in MFC electricity generation.

Antimicrobial peptide AMPNT-6 from Bacillus subtilis inhibits biofilm formation by Shewanella putrefaciens and disrupts its preformed biofilms on both abiotic and shrimp shell surfaces.

Shewanella putrefaciens biofilm formation is of great concern for the shrimp industry because it adheres easily to food and food-contact surfaces and is a source of persistent and unseen contamination that causes shrimp spoilage and economic losses to the shrimp industry. Different concentrations of an antimicrobial lipopeptide, the fermentation product of Bacillus subtilis, AMPNT-6, were tested for the ability to reduce adhesion and disrupt S. putrefaciens preformed biofilms on two different contact surfaces (shrimp shell, stainless steel sheet). AMPNT-6 displayed a marked dose- and time-dependent anti-adhesive effect>biofilm removal. 3MIC AMPNT-6 was able both to remove biofilm and prevent bacteria from forming biofilm in a 96-well polystyrene microplate used as the model surface. 2MIC AMPNT-6 prevented bacteria from adhering to the microplate surface to form biofilm for 3h and removed already existing biofilm within 24h. Secretion of extracellular polymeric substances incubated in LB broth for 24h by S. putrefaciens was minimal at 3× MIC AMPNT-6. Scanning electron microscopy showed that damage to S. putrefaciens bacteria by AMPNT-6 possibly contributed to the non-adherence to the surfaces. Disruption of the mature biofilm structure by AMPNT-6 contributed to biofilm removal. It is concluded that AMPNT-6 can be used effectively to prevent attachment and also detach S. putrefaciens biofilms from shrimp shells, stainless steel sheets and polystyrene surfaces.

Continuous bioelectricity generation through treatment of Victoria blue R: A novel microbial fuel cell operation.

A novel two-chamber microbial fuel cell (MFC) operation with a continuous anaerobic-aerobic decolorization system was developed to improve the degradation of the triphenylmethane dye, Victoria blue R (VBR). In addition, bioelectricity was generated during the VBR degradation process, and the operation parameters were optimized. The results indicated that the VBR removal efficiency and electricity generation were affected by the VBR concentration, liquid retention time (LRT), external resistance, gas retention time (GRT), and shock loading. The optimal operation parameters were as follows: VBR concentration, 600 mg L-1; LRT, 24 h; external resistance, 3300 Ω; and GRT, 60 s. Under these operating conditions, the VBR removal efficiency, COD removal efficiency, and power density were 98.2% ± 0.3%, 97.6% ± 0.5%, and 30.6 ± 0.4 mW m-2, respectively. According to our review of the relevant literature, this is the first paper to analyze the electrical characteristics of a continuous two-chamber MFC operation and demonstrate the feasibility of the simultaneous electricity generation and decolorization of VBR.

Facile Fabrication of Graphene-Containing Foam as a High-Performance Anode for Microbial Fuel Cells.

Facile fabrication of novel three-dimensional anode materials to increase the bacterial loading capacity and improve substrate transport in microbial fuel cells (MFCs) is of great interest and importance. Herein, a novel graphene-containing foam (GCF) was fabricated easily by freeze-drying and pyrolysis of a graphene oxide-agarose gel. Owing to the involvement of graphene and stainless-steel mesh in the GCF, the GCF shows high electrical conductivity, enabling the GCF to be a conductive electrode for MFC applications. With the aid of agarose, the GCF electrode possesses a supermacroporous structure with pore sizes ranging from 100-200 µm and a high surface area, which greatly increase the bacterial loading capacity. Cell viability measurements indicate that the GCF possesses excellent biocompatibility. The MFC, equipped with a 0.4 mm-thick GCF anode, shows a maximum area power density of 786 mW m(-2) , which is 4.1 times that of a MFC equipped with a commercial carbon cloth anode. The simple fabrication route in combination with the outstanding electrochemical performance of the GCF indicates a promising anode for MFC applications.

Cytometric patterns reveal growth states of Shewanella putrefaciens.

Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species.

Inference of interactions in cyanobacterial-heterotrophic co-cultures via transcriptome sequencing.

We used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella putrefaciens W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph-heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic-heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.

In vivo study of spoilage bacteria on polyphenoloxidase activity and melanosis of modified atmosphere packaged Pacific white shrimp.

This study investigated the effect of the three spoilage bacteria (Carnobacterium maltaromaticum, Shewanella putrefaciens and Aeromonas salmonicida) on the development of melanosis by inoculating the bacteria on modified atmosphere packaged shrimp. The three bacteria, which inoculated at about 5 log cfu/g, proliferated to a maximum level of 7.49, 6.86 and 6.89 log cfu/g, respectively at the end of storage. In regards to the effect of bacteria on melanosis, it was found that C. maltaromaticum did not display a significant effect on PPO activity and melanosis, and A. salmonicida showed an inhibitory effect on PPO activity with an activity value of less than 1.5 Units/ml. However, the PPO activity of shrimp inoculated S. putrefaciens was about one time higher than other samples and greater melanosis was displayed in the first 48 h. As melanosis can cause sensory quality loss, the growth of S. putrefaciens should be limited.

Characterization of antioxidant-antibacterial quince seed mucilage films containing thyme essential oil.

In this study thyme essential oil (TEO) concentrations ranging from 0% to 2.0%, incorporated in quince seed mucilage (QSM) film were used. Antibacterial activity, physical, mechanical, barrier and antioxidant properties of QSM films were evaluated. The antimicrobial activity of the QSM films incorporated with thyme essential oil was screened against 11 important food-related bacterial strains by agar disc-diffusion assay. Films containing 1% of thyme essential oil were effective against all test microorganisms and exhibited a strong inhibitory effect on the growth of Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus. QSM films exhibited some antioxidant activity, which was significantly improved by the addition of the essential oil. A reduction of the glass transition temperature, as determined by differential scanning calorimetry (DSC), was caused by addition of thyme essential oil into the QSM films. Scanning electron microscopy was carried out to explain structure-property relationships. Incorporating thyme essential oil into edible QSM films provides a novel way to improve the safety and shelf life of ready-to-eat foods.

Uranium removal from a contaminated effluent using a combined microbial and nanoparticle system.

Reduction of soluble uranium(VI) to insoluble uranium(IV) for remediating a uranium-contaminated effluent (EF-03) was examined using a biotic and abiotic integrated system. Shewanella putrefaciens was first used and reduced U(VI) in a synthetic medium but not in the EF-03 effluent sample. Subsequently the growth of autochthonous microorganisms was stimulated with lactate. When lactate was supported on active carbon 77% U(VI) was removed in 4 days. Separately, iron nanoparticles that were 50 nm in diameter reduced U(VI) by 60% in 4 hours. The efficiency of uranium(VI) removal was improved to 96% in 30 min by using a system consisting of lactate and iron nanoparticles immobilized on active carbon. Lactate also stimulated the growth of potential uranium-reducing microorganisms in the EF-03 sample. This system can be efficiently used for the bioremediation of uranium-contaminated effluents.

Modeling antimicrobial activity of lipopeptides from Bacillus amyloliquefaciens ES-2 against Shewanella putrefaciens in shrimp meat using a response surface method.

Bacillus amyloliquefaciens ES-2 can produce antimicrobial lipopeptides, including surfactin and fengycin. In this study, the model of antimicrobial activity against Shewanella putrefaciens in shrimp meat by antimicrobial lipopeptides from B. amyloliquefaciens ES-2 was researched by response surface methodology. The results showed that S. putrefaciens had high sensitivity to antimicrobial lipopeptides, which had a MIC of 0.6 mg/ml. A quadratic mathematical model representative of the action of antimicrobial lipopeptides on S. putrefaciens in shrimp meat was developed as a function of concentration, time, and temperature. A reduction of S. putrefaciens cells of over 2 log cycles could be realized when the temperature was below 5.4°C, the time was over 6 h, and the concentration of the lipopeptides was over 0.3 mg/g.

Shewanella putrefaciens, a major microbial species related to tetrodotoxin (TTX)-accumulation of puffer fish Lagocephalus lunaris.

AIMS: To investigate the major micro-organisms, which were isolated from internal organs, related to tetrodotoxin (TTX)-accumulation of puffer fish Lagocephalus lunaris. METHODS AND RESULTS: Puffer fish Lagocephalus lunaris around Chang Island in the Gulf of Thailand were collected to examine TTX-accumulation and microbial load in internal organs. The nine predominant micro-organisms isolated from the internal organs were determined TTX and studied in relation to the TTX-accumulation of the puffer fish. Shewanella putrefaciens, a predominant bacterium and related to the TTX-accumulation of the puffer fish, was examined growth and TTX-production after culture in modified Zobell medium. The results revealed that the average TTX-accumulation of the puffer fish directly varied to the bacterium load periodically, year-round. Furthermore, it coincided with the growth and the TTX-production of the bacterium, which grew slowly but produced high TTX at low temperature. CONCLUSIONS: Shewanella putrefaciens was a major bacterium relating to TTX-accumulation of puffer fish L. lunaris. It resulted in high TTX-accumulation of the puffer fish at low temperatures of seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: Temperature affected growth and TTX-production of S. putrefaciens that resulted in TTX accumulated in puffer fish L. lunaris.

Effects of specific egg yolk antibody (IgY) on the quality and shelf life of refrigerated Paralichthys olivaceus.

BACKGROUND: The spoilage of fishery food has been attributed to limited types of microorganisms called specific spoilage organisms (SSO). Unlike traditional food-preserving techniques which usually exploit broad-spectrum antimicrobial agents, here, based on the specific antimicrobial activity of egg yolk antibodies (IgY) against two SSO in refrigerated fish (Shewanella putrefaciens and Pseudomonas fluorescens), a novel strategy for fish preservation was suggested and evaluated. RESULTS: During storage of Paralichthys olivaceus fillets at 4 ± 1 °C, the bacteria growth (including total microorganisms and the two SSO) in test groups was significantly inhibited in comparison to that of controls (P < 0.05). This antibacterial activity of the specific IgY was also confirmed by chemical analysis (pH, total volatile base nitrogen and 2-thiobarbituric acid value) and sensory evaluation, and the shelf life of samples was extended approximately from 9 days to 12-15 days in the presence of the specific IgY. CONCLUSION: These results indicated a significant antimicrobial activity of the anti-SSO IgY for refrigerated fish products, which allowed us to suggest its potential as a bio-preservative for seafood.

Effect of gutting on microbial loads, sensory properties, and volatile and biogenic amine contents of European hake (Merluccius merluccius var. mediterraneus) stored in ice.

The effect of gutting on sensory, microbiological, and chemical properties of European hake (Merluccius merluccius var. mediterraneus) stored in ice was studied. Gutting of hake noticeably affected the development of gram-negative bacteria: counts of Enterobacteriaceae, Shewanella putrefaciens, and Pseudomonas throughout ice storage were higher in gutted than in ungutted samples. These differences in microbial loads were also reflected in the lower sensory scores of both raw and cooked hake, in the quicker trimethylamine accumulation, and in the higher contents of putrescine, cadaverine, tyramine, and histamine found in gutted hake. All of the fish quality indicators studied showed that gutting made hake more susceptible to spoilage during ice storage and decreased its shelf life by 4 days.

Studies of iron-uptake mechanisms in two bacterial species of the shewanella genus adapted to middle-range (Shewanella putrefaciens) or antarctic (Shewanella gelidimarina) temperatures.

Iron(III)-uptake mechanisms in bacteria indigenous to the Antarctic, which is the most Fe-deficient continent on Earth, have not been extensively studied. The cold-adapted, Antarctic bacterium, Shewanella gelidimarina, does not produce detectable levels of the siderophore, putrebactin, in the supernatant of Fe(III)-deprived cultures. This is distinct from the putrebactin-producing bacterium from the same genus, Shewanella putrefaciens, which is adapted to middle-range temperatures. The production of putrebactin by S. putrefaciens is optimal, when the pH value of the medium is 7.0. According to the strong positive response from whole cells in the Chrome Azurol S (CAS) agar diffusion assay, Shewanella gelidimarina appears to produce cell-associated siderophores. In the RP-HPLC trace of an Fe(III)-loaded extract from the cell-associated components of S. gelidimarina cultured in media with [Fe(III)] ca. 0 microM, a peak appears at [MeCN] ca. 77%, which decreases in intensity in a parallel experiment in which [Fe(III)] ca. 5 microM, and is barely detectable in Fe(III)-replete media ([Fe(III)] ca. 20 microM). The Fe(III)-dependence of this peak suggests that the attendant species, which is significantly more hydrophobic than putrebactin (RP-HPLC elution: [MeCN] ca. 14%), is associated with Fe(III)-management in S. gelidimarina. This study highlights the diversity in Fe(III)-uptake mechanisms in Shewanella species adapted to different environmental and thermal niches.

Inhibition of NO3- and NO2- reduction by microbial Fe(III) reduction: evidence of a reaction between NO2- and cell surface-bound Fe2+.

A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO(3)(-) and NO(2)(-) (NO(x)(-)) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe(2+) and NO(2)(-). This coating could then inhibit reduction of NO(x)(-) by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of approximately 3 mM NO(3)(-) reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe(2+) and NO(2)(-), demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe(2+) onto Paracoccus denitrificans inhibited NO(x)(-) reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO(2)(-) was chemically reduced to N(2)O by goethite or cell-sorbed Fe(2+), but not at appreciable rates by aqueous Fe(2+). Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe(2+) and NO(2)(-). The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.

Cultivation of microorganisms in the cultural medium made from squid internal organs and accumulation of polyunsaturated fatty acids in the cells.

The disposal and more efficient utilization of marine wastes is becoming increasingly serious. A culture media for microorganisms has been prepared from squid internal organs that are rich in polyunsaturated fatty acids (PUFAs). Both freshwater and marine bacteria grew well in this medium and some bacteria accumulated PUFAs in their lipids, suggesting uptake of exogenous PUFAs. Higher PUFA accumulations were observed in Escherichia coli mutant cells defective either in unsaturated fatty acid biosynthesis or fatty acid degradation, or both, compared to those without these mutations. Therefore, PUFA accumulation in cells can be improved by genetic modification of fatty acid metabolism in the bacteria. Squid internal organs would be a good source of medium, not only for marine bacteria but also for freshwater bacteria, and that this process may be advantageous to make efficient use of the fishery wastes and to produce PUFA-containing microbial cells and lipids.