Isolation of Shewanella putrefaciens GRD 03 from Fish and Explication of Biofilm Adherence Potency on Different Substrates

Foodborne pathogens are the main threat and cause of food poisoning. The majority of food infections have been related to the biofilm formation of foodborne pathogens in the food industry. Shewanella putrefaciens (KX355803, GRD 03), a Gram-negative pathogen isolated from mackerel fish, was identified and recognized as a food spoilage bacterium and a strong biofilm producer. The adhesion or attachment ability of Shewanella putrefaciens was determined on steel, plastic, glass, PVC and wood. NB (Nutrient broth), LB (Luria-Bertani broth), TSB (Tryptic soy broth) and BHI (Brain heart infusion broth) were enriched with glucose and shows optimum for bacterial adhesion. In the microtiter plate method (MTP), the strong attachment was observed at 48 and 72 hours of incubation and significant differences were obtained at p < 0.05. As the incubation period increases, the OD value (Optical density) of samples also increase. Biofilm formation is the major cause cross-contamination, and shows resistance to certain disinfectants, which leads to environmental stress tolerance. This study suggested with optimum biofilm production of isolate from fish by using glucose enriched media on different substrates, also comparing different growth media provide a detailed idea about biofilm-forming ability at different incubation time intervals.

Comparative Proteomics Reveals the Spoilage-Related Factors of Shewanella putrefaciens Under Refrigerated Condition

Shewanella putrefaciens is a microorganism with strong spoilage potential for aquatic products. This study aimed to investigate the potential spoilage factors of S. putrefaciens by comparative proteomic analysis. The spoilage potential of two strains of S. putrefaciens (00A and 00B) isolated from chilled spoiled bigeye tuna was investigated. The results of total volatile basic nitrogen (TVB-N), trimethylamine (TMA) in fish inoculated with S. putrefaciens, extracellular protease activity of S. putrefaciens, and degradation of fish proteins indicated that the spoilage potential of S. putrefaciens 00A was much higher than that of 00B. Fish proteins are usually degraded by spoilage microorganism proteases into small molecular peptides and amino acids, which are subsequently degraded into spoilage metabolites in bacterial cells, leading to deterioration of fish quality. Thus, proteomic analysis of the extracellular and intracellular proteins of 00A vs. 00B was performed. The results indicated that the intracellular differentially expressed protein (IDEP) contained 243 upregulated proteins and 308 downregulated proteins, while 78 upregulated proteins and 4 downregulated proteins were found in the extracellular differentially expressed protein (EDEP). GO annotation revealed that IDEP and EDEP were mainly involved in cellular and metabolic processes. KEGG annotation results showed that the upregulated proteins in IDEP were mainly involved in sulfur metabolism, amino acid metabolism, and aminoacyl-tRNA biosynthesis, while downregulated proteins were related to propanoate metabolism. In contrast, EDEP of KEGG annotation was mainly involved in ribosomes, quorum sensing, and carbohydrate metabolism. Proteins associated with spoilage containing sulfur metabolism (sulfite reductase, sulfate adenylyltransferase, adenylyl-sulfate kinase), amino acid metabolism (biosynthetic arginine decarboxylase, histidine ammonia-lyase), trimethylamine metabolism (trimethylamine-N-oxide reductase), and extracellular proteins (ATP-dependent Clp protease proteolytic subunit) were identified as upregulated. These proteins may play a key role in the spoilage potential of S. putrefaciens. These findings would contribute to the identification of key spoilage factors and understanding of the spoilage mechanism of microorganisms.

Microbiologically influenced corrosion inhibition of carbon steel via biomineralization induced by Shewanella putrefaciens

Microbiologically influenced corrosion inhibition (MICI) of Q235 carbon steel by biomineralization was investigated via a combination of surface analysis, electrochemistry, and scanning electrochemical microscopy (SECM). The results showed that Shewanella putrefaciens used the cell walls as the nucleation sites to induce the formation of a protective biomineralized layers which contained calcite and extracellular polymeric substances on the steel surface. The potentiodynamic polarization results demonstrated that the corrosion current density (icorr value) of the biomineralized steel surface was 0.38 μA cm−2, which was less than one-tenth that of the blank steel in a sterile medium (4.86 μA cm−2) after 14 days. The biomineralized layers presented wear resistance and could self-repair after undergoing mechanical damage under microbial conditions as verified by morphological and SECM observations. This work reveals that microbial-induced carbonate biomineralization, as a MICI approach, may be considered as a reliable, low-cost, environmentally friendly corrosion inhibition strategy.

Comparison of Physicochemical Changes and Water Migration of Acinetobacter johnsonii, Shewanella putrefaciens, and Cocultures From Spoiled Bigeye Tuna (Thunnus obesus) During Cold Storage

This study investigates the physicochemical changes and water migration of Acinetobacter johnsonii (A), Shewanella putrefaciens (S), and cocultured A. johnsonii and S. putrefaciens (AS) inoculated into bigeye tuna during cold storage. The physicochemical indexes [fluorescence ratio (FR), total volatile base nitrogen (TVB-N), thiobarbituric acid (TBA), trimethylamine (TMA), peroxide value (POV), and pH] of bigeye tuna increased cold storage. A significant decrease in trapped water was found in the AS samples, and direct monitoring of the water dynamics was provided by low-field nuclear magnetic resonance. Samples inoculated with A. johnsonii and S. putrefaciens also induced the degradation of myofibrillar proteins and weakness of some Z-lines and M-lines. Higher values of physicochemical indexes and water dynamics were shown in the coculture of S. putrefaciens and A. johnsonii than in the other groups. Therefore, this paper reveals that the coculture of A. johnsonii and S. putrefaciens resulted in a bigeye tuna that was more easily spoiled when compared to the single culture. This study provides insight into the spoilage potential of A. johnsonii and S. putrefaciens during cold storage, which further assists in the application of appropriate technologies to keep the freshness of aquatic foods.

Molecular cloning and characterization of NAD+ dependent isocitrate dehydrogenase enzyme from Shewanella putrefaciens

Isocitrate dehydrogenase (IDH) is a fundamental enzyme for carbon metabolism in the Krebs cycle. This enzyme is required for oxidation-reduction reactions in both eukaryotic and prokaryotic cells and plays a critical role in their growth and pathogenesis. In this study, we cloned the gene encoding NAD+ dependent isocitrate dehydrogenase from Shewanella putrefaciens. The expression of recombinant protein was induced with 0.5 mM of IPTG. His-tagged IDH overexpressed in E. coli was purified and characterized. The expressed IDH enzyme was purified in an active soluble form. The molecular weight of the enzyme was confirmed with Western blotting. High sequence homology was observed with IDH sequences of other Shewanella strains and remarkable sequence homology was found with other bacteria reported in the database. The open reading frame of the gene encoding IDH of S. putrefaciens was 1069 bp in length, which codes a polypeptide composed of 356 amino acids and with a 38 KDa molecular weight. The optimum enzyme activity was obtained at pH 8. We cloned, purified and characterized Shewanella putrefaciens isocitrate dehydrogenase enzyme (SpIDH). The recombinant enzyme demonstrated a specific activity of Vmax 4.6±0.3 μM/min and Km 334 μM using NAD+ as a cofactor.