Ultrasound-assisted fermentation of Porphyra yezoensis sauce at different growth stages using Lactiplantibacillus plantarum: Metabolic response and biological activity.

This study first employed ultrasonic-assisted fermentation of seaweed foot material with Lactiplantibacillus plantarum to produce Porphyra yezoensis sauce. The aim was to examine L. plantarum's growth and metabolism of nutritional components at different growth stages under low- (133.99 W/L) and high-ultrasonic power densities (169.17 W/L). After 24-h fermentation, L. plantarum exhibited a 21.32 % increase in the sonicated P. yezoensis sauce at 133.99 W/L and the logarithmic growth phase compared to that at 169.17 W/L. In addition, compared to the non-sonicated sauce, total phenolic and flavonoid contents increased by around 58 % and 27 % in sonicated sauce at 133.99 W/L, reaching 92.38 mg GEA/g DW and 111.08 mg RE/g DW, respectively. Principal Component Analysis (PCA) of the evaluation criteria for different fermentation stages under 133.99 W/L power ultrasonication revealed that the P. yezoensis sauce generated more phenolic compounds and exhibited stronger antioxidant capabilities in the sonicated sample at the logarithmic phase of L. plantarum. Compared to the traditional treated P. yezoensis sauce, the content of free amino acids was significantly increased in sonicated sauce, especially for logarithmic phase. Finally, GC-IMS analysis demonstrated that the ultrasonication at logarithmic phase released more volatile compounds compared to the non-sonicated sauce. This led to a reduction in the fishy odour of the Porphyra yezoensis sauce and an improved release of favourable flavour compounds.

The antibacterial activity of plantaricin GZ1-27 against MRSA and its bio-preservative effect on chilled pork in combination with chitosan.

Methicillin-resistant Staphylococcus aureus (MRSA) poses great threats to human health. In this research, we found that the newly discovered bacteriocin plantaricin GZ1-27 could efficiently inhibit the MRSA, with a MIC of 32 µg/mL. Comprehensive investigations were carried out by analysis of K+ leakage, propidium iodide assay, and cell ultra-structure analysis with scanning electron microscopy and transmission electron microscopy. The results consistently showed that plantaricin GZ1-27 could increase the permeability of the membrane and impair its integrity, which induced the collapse of the cell structure and thus led to cell death. Furthermore, by sensory evaluation and biochemical analysis, it was found that plantaricin GZ1-27 combined with chitosan could significantly improve the preservation of pork when applied to the surface of meat slices. Overall, our research clearly showed the anti-MRSA bactericidal mechanism of plantaricin GZ1-27, which, together with its preliminary application trials in pork, suggested plantaricin GZ1-27 could be a potential anti-MRSA agent and is promising to be applied in pork preservation to extend the shelf life.

Genome Mining, Heterologous Expression, Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost-Associated Bacillus subtilis fmb60.

Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D-F (1-3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D-F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS+ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated.

Efficacy of Individual Bacteriophages Does Not Predict Efficacy of Bacteriophage Cocktails for Control of Escherichia coli O157.

Effectiveness of bacteriophages AKFV33 (Tequintavirus, T5) and AHP24 (Rogunavirus, T1), wV7 (Tequatrovirus, T4), and AHP24S (Vequintavirus, rV5), as well as 11 cocktails of combinations of the four phages, were evaluated in vitro for biocontrol of six common phage types of Escherichia coli O157 (human and bovine origins) at different multiplicities of infection (MOIs; 0.01-1,000), temperatures (37 or 22°C), and exposure times (10-22 h). Phage efficacy against O157 was highest at MOI 1,000 (P < 0.001) and after 14-18 h of exposure at 22°C (P < 0.001). The activity of individual phages against O157 did not predict the activity of a cocktail of these phages even at the same temperature and MOI. Combinations of phages were neutral (no better or worse than the most effective constituent phages acting alone), displayed facilitation (greater efficacy than the most effective constituent phages acting alone), or antagonistic (lower efficacy than the most effective constituent phages acting alone). Across MOIs, temperatures, exposure time, and O157 strains, a cocktail of T1, T4, and rV5 was most effective (P < 0.05) against O157, although T1 and rV5 were less effective (P < 0.001) than other individual phages. T5 was the most effective individual phages (P < 0.05), but was antagonistic to other phages, particularly rV5 and T4 + rV5. Interactions among phages were influenced by phage genera and phage combination, O157 strains, MOIs, incubation temperatures, and times. Based on this study, future development of phage cocktails should, as a minimum, include confirmation of a lack of antagonism among constituent phages and preferably confirmation of facilitation or synergistic effects.

Acetate and auto-inducing peptide are independent triggers of quorum sensing in Lactobacillus plantarum.

The synthesis of plantaricin in Lactobacillus plantarum is regulated by quorum sensing. However, the nature of the extra-cytoplasmic (EC) sensing domain of the histidine kinase (PlnB1) and the ability to recognize the auto-inducing peptide PlnA1 is not known. We demonstrate the key motif Ile-Ser-Met-Leu of auto-inducing peptide PlnA1 binds to the hydrophobic region Phe-Ala-Ser-Gln-Phe of EC loop 2 of PlnB1 via hydrophobic interactions and hydrogen bonding. Moreover, we identify a new inducer, acetate, that regulates the synthesis of plantaricin by binding to a positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1 via electrostatic interaction. The side chain of Phe143 on loop 4 determined the specificity and affinity of PlnB1 to recognize acetate. PlnA1 activates quorum sensing in log phase growth and acetate in stationary phase to maintain the synthesis of plantaricin under conditions of reduced growth. Acetate activation of PlnB was also evident in four types of PlnB present in different Lb. plantarum strains. Finally, we proposed a model to explain the developmental regulation of plantaricin synthesis by PlnA and acetate. These results have potential applications in improving food fermentation and bacteriocin production.

Bacteriophage biocontrol of Shiga toxigenic Escherichia coli (STEC) O145 biofilms on stainless steel reduces the contamination of beef.

Shiga toxigenic Escherichia coli (STEC) can form biofilms and frequently cause serious foodborne illnesses. A strain of STEC O145:H25 (EC19990166) known to be a strong biofilm former was used to evaluate the efficacy of bacteriophage AZO145A against biofilms formed on stainless steel (SS) coupons. Exposure of STEC O145:H25 to phage AZO145A (1010 PFU/mL) for 2 h resulted in a 4.0 log10 reduction (P < 0.01) of planktonic cells grown in M9 broth at 24 °C for 24 h, while reductions were 2.0 log10 CFU/mL if these cells were grown for 48 h or 72 h prior to phage treatment. STEC O145 biofilms formed on SS coupons for 24, 48 and 72 h were reduced (P < 0.01) 2.9, 1.9 and 1.9 log10 CFU/coupon by phages. STEC O145 cells in biofilms were readily transferred from the surface of the SS coupon to beef (3.6 log10 CFU/coupon) even with as little as 10 s of contact with the meat surface. However, transfer of STEC O145 cells from biofilms that formed on SS coupons for 48 h to beef was reduced (P < 0.01) by 3.1 log10 CFU by phage (2 × 1010 PFU/mL) at 24 °C. Scanning electron microscopy revealed that bacterial cells within indentations on the surface of SS coupons were reduced by phage. These results suggest that bacteriophage AZO145A could be effective in reducing the viability of biofilm-adherent STEC O145 on stainless steel in food industry environments.

Transcriptomic and proteomic profiling response of methicillin-resistant Staphylococcus aureus (MRSA) to a novel bacteriocin, plantaricin GZ1-27 and its inhibition of biofilm formation.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a worrisome superbug, due to its wide distribution and multidrug resistance. To characterize effects of a newly identified plantaricin GZ1-27 on MRSA, transcriptomic and proteomic profiling of MRSA strain ATCC43300 was performed in response to sub-MIC (16 µg/mL) plantaricin GZ1-27 stress. In total, 1090 differentially expressed genes (padj < 0.05) and 418 differentially expressed proteins (fold change > 1.2, p < 0.05) were identified. Centralized protein expression clusters were predicted in biological functions (biofilm formation, DNA replication and repair, and heat-shock) and metabolic pathways (purine metabolism, amino acid metabolism, and biosynthesis of secondary metabolites). Moreover, a capacity of inhibition MRSA biofilm formation and killing biofilm cells were verified using crystal violet staining, scanning electron microscopy, and confocal laser-scanning microscopy. These findings yielded comprehensive new data regarding responses induced by plantaricin and could inform evidence-based methods to mitigate MRSA biofilm formation.

Effect of Plantaricin 163 in Combination with Thymol and Surfactin on Crucian Carp (Carassius auratus).

The use of natural preservatives has attracted considerable attention owing to their generally safe and environmentally friendly properties. In this study, we investigated the effects of the preservative A1, composed of plantaricin 163, thymol, and surfactin, on bacterial communities and storage quality of refrigerated crucian carp. A total of 522 operational taxonomic units belonging to 20 phyla and 272 genera were identified by high-throughput sequencing, showing a comprehensive coverage of bacterial composition of crucian carp. In untreated samples after spoilage, Brochothrix was the predominant genus, followed by Aeromonas and Pseudomonas. After treatment with A1, the growth of these spoilage bacteria was significantly inhibited according to high-throughput sequencing and plate counts, and Lactococcus became the most abundant organism at the end of storage. Meanwhile, compared with control samples, the shelf life of A1-treated samples extended from 3 to 12 days on the basis of the sensory evaluation and the total viable counts. Furthermore, the total volatile basic nitrogen, thiobarbituric acid, and pH values for A1-treated samples were significantly lower than that of control samples. The results indicate that preservative A1 has potential commercial application in the preservation of refrigerated crucian carp.

Purification, Characterization, and Mode of Action of Plantaricin GZ1-27, a Novel Bacteriocin against Bacillus cereus.

Bacillus cereus is an opportunistic pathogen that causes foodborne diseases. We isolated a novel bacteriocin, designated plantaricin GZ1-27, and elucidated its mode of action against B. cereus. Plantaricin GZ1-27 was purified using ammonium sulfate precipitation, gel-filtration chromatography, and RP-HPLC. MALDI-TOF/MS revealed that its molecular mass was 975 Da, and Q-TOF-MS/MS analysis predicted the amino acid sequence as VSGPAGPPGTH. Plantaricin GZ1-27 showed thermostability and pH stability. The antibacterial mechanism was investigated using flow cytometry, confocal laser-scanning microscopy, scanning and transmission electron microscopy, and RT-PCR, which revealed that GZ1-27 increased cell membrane permeability, triggered K+ leakage and pore formation, damaged cell membrane integrity, altered cell morphology and intracellular organization, and reduced the expression of genes related to cytotoxin production, peptidoglycan synthesis, and cell division. These results suggest that plantaricin GZ1-27 effectively inhibits B. cereus at both the cellular and the molecular levels and is a potential natural food preservative targeting B. cereus.

Identification of novel virulence-related genes in Aeromonas hydrophila by screening transposon mutants in a Tetrahymena infection model.

Outbreaks of motile Aeromonad septicemia (MAS) in fish caused by sequence type (ST) 251 Aeromonas hydrophila have become a prominent problem for the aquaculture industry. The pathogenesis of A. hydrophila is very complicated, and some virulence factors remain to be identified. In this study, to identify novel virulence-related factors, ST251 A. hydrophila strain NJ-35 was used as the parental strain to construct a mutant library comprising 1030 mutant strains by transposon insertion mutagenesis. Subsequently, 33 virulence-attenuated transposon insertion mutants were identified using Tetrahymena and zebrafish as model hosts in sequence. Thermal asymmetric interlaced (Tail)-PCR and Southern blot analysis identified 21 single transposon insertion sites. Seven of the insertion sites are located in non-coding regions, whereas the other 14 insertion sites are located in genes, including aroA, rmlA, rtxA, chiA and plc. All insertion mutants exhibited attenuated virulence in Tetrahymena and zebrafish. Furthermore, the relationship of two genes, chiA and trkH, to virulence was confirmed by gene inactivation and subsequent restoration assays. This study provides new information about the genetic determinants of A. hydrophila pathogenicity and validates the Aeromonas-Tetrahymena co-culture model for high-throughput screening of A. hydrophila virulence factors.

Catecholamine-Stimulated Growth of Aeromonas hydrophila Requires the TonB2 Energy Transduction System but Is Independent of the Amonabactin Siderophore.

The growth-stimulating effects of catecholamine stress hormones have been demonstrated in many pathogens. However, catecholamine-induced growth and its underlying mechanisms remain poorly understood in Aeromonas hydrophila. The present study sought to demonstrate that norepinephrine (NE), epinephrine (Epi), dopamine (Dopa), and L-dopa stimulate the growth of A. hydrophila in iron-restricted media containing serum. NE exhibited the strongest growth stimulation, which could be blocked by adrenergic antagonists. Furthermore, it was demonstrated that NE could sequester iron from transferrin, thereby providing a more accessible iron source for utilization by A. hydrophila. The deletion of the amoA gene associated with amonabactin synthesis revealed that the amonabactin siderophore is not required for NE-stimulated growth. However, the deletion of the TonB2 energy transduction system resulted in the loss of growth promotion by NE, indicating that a specific TonB-dependent outer membrane receptor might be involved in the transport of iron from transferrin. Collectively, our data show that catecholamine sensing promotes the growth of A. hydrophila in a manner that is dependent on the TonB2 energy transduction system.

Identification and Characterization of an Aeromonas hydrophila Oligopeptidase Gene pepF Negatively Related to Biofilm Formation.

Bacterial biofilms are involved in adaptation to complex environments and are responsible for persistent bacterial infections. Biofilm formation is a highly complex process during which multifarious genes work together regularly. In this study, we screened the EZ-Tn5 transposon mutant library to identify genes involved in biofilm formation of Aeromonas hydrophila. A total of 24 biofilm-associated genes were identified, the majority of which encoded proteins related to cell structure, transcription and translation, gene regulation, growth and metabolism. The mutant strain TM90, in which a gene encoding oligopeptidase F (pepF) was disturbed, showed significant upregulation of biofilm formation compared to the parental strain. The TM90 colony phenotype was smaller, more transparent, and splendent. The adhesive ability of TM90 to HEp-2 cells was significantly increased compared with the parental strain. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the enhanced-biofilm mutant TM90 was highly attenuated relative to the wild-type strain. In conclusion, the pepF gene is demonstrated for the first time to be a negative factor for biofilm formation and is involved in A. hydrophila pathogenicity.

Identification of Aeromonas hydrophila Genes Preferentially Expressed after Phagocytosis by Tetrahymena and Involvement of Methionine Sulfoxide Reductases.

Free-living protozoa affect the survival and virulence evolution of pathogens in the environment. In this study, we explored the fate of Aeromonas hydrophila when co-cultured with the bacteriovorous ciliate Tetrahymena thermophila and investigated bacterial gene expression associated with the co-culture. Virulent A. hydrophila strains were found to have ability to evade digestion in the vacuoles of this protozoan. In A. hydrophila, a total of 116 genes were identified as up-regulated following co-culture with T. thermophila by selective capture of transcribed sequences (SCOTS) and comparative dot-blot analysis. A large proportion of these genes (42/116) play a role in metabolism, and some of the genes have previously been characterized as required for bacterial survival and replication within macrophages. Then, we inactivated the genes encoding methionine sulfoxide reductases, msrA, and msrB, in A. hydrophila. Compared to the wild-type, the mutants ΔmsrA and ΔmsrAB displayed significantly reduced resistance to predation by T. thermophila, and 50% lethal dose (LD50) determinations in zebrafish demonstrated that both mutants were highly attenuated. This study forms a solid foundation for the study of mechanisms and implications of bacterial defenses.