Extracellular matrix affects mature biofilm and stress resistance of psychrotrophic spoilage Pseudomonas at cold temperature.

Psychrotrophic Pseudomonas as the dominant spoilage bacteria, have biofilm forming ability, increasing persistence and contamination in the chilled food. Biofilm formation of spoilage Pseudomonas at cold temperature was documented, however, role of extracellular matrix in mature biofilm and stress resistance of psychrotrophic Pseudomonas are much less abundant. The aim of this study was to investigate the biofilm forming characteristics of three spoilers P. fluorescens PF07, P. lundensis PL28, and P. psychrophile PP26 at 25 °C, 15 °C and 4 °C, and to explore their stress resistance to chemical and thermal treatments of mature biofilms. The results showed that biofilm biomass of three Pseudomonas at 4 °C was significantly higher than that at 15 °C and 25 °C. The secretion of extracellular polymeric substances (EPS) greatly increased in those Pseudomonas under low temperature, of which extracellular protein constituted about 71.03%-77.44%. Compared to 25 °C, the mature biofilms were observed to more aggregation and thicker spatial structure at 4 °C ranging from 42.7 to 54.6 µm, in contrast to 25.0-29.8 µm at 25 °C, especially strain PF07. These Pseudomonas biofilms switched into moderate hydrophobicity, and their swarming and swimming were significantly inhibited at low temperature. Furthermore, the resistance to NaClO and heating at 65 °C apparently enhanced for mature biofilm formed at 4 °C, indicating the difference in EPS matrix production influenced the stress resistance of biofilm. In addition, three strains contained alg and psl operons for exopolysaccharide biosynthesis, and biofilm related genes of algK, pslA, rpoS, and luxR were significantly up-regulated, while flgA gene was down-regulated at 4 °C compared to 25 °C, consistent with the above phenotype changes. Thus, the dramatic increase of mature biofilm and their stress resistance in psychrotrophic Pseudomonas were associated with large secretion and protection of extracellular matrix under low temperature, which provide a theoretical basis for subsequent biofilm control during cold chain.

Synergism With ε-Polylysine Hydrochloride and Cinnamon Essential Oil Against Dual-Species Biofilms of Listeria monocytogenes and Pseudomonas lundensis.

Various pathogenic and spoilage bacteria frequently coexist in meat processing environments and can form multispecies biofilms, causing significant health and economic issues. Despite the prevalence and coexistence, only less is known about possible interactions between Listeria monocytogenes (LM) and spoilers like Pseudomonas species, and their community-wide resistance against natural preservatives. This study evaluates the interactions between mono- or dual-species biofilms formed by LM and Pseudomonas lundensis (PL), as well as the sensitivity of these bacteria in dual-species biofilms to ε-polylysine hydrochloride (ε-PLH) alone or combined with cinnamon essential oil (CEO). The results showed that the biofilm cell density of P. lundensis in dual species was higher (p < 0.05) than LM, constituting about 85% of the total population. More biofilms and exopolysaccharide both in mono- or dual species of the two psychrotrophic strains were greatly produced at 15°C than at 30°C. The biomass, biovolume, and thickness of dual-species biofilms were significantly lower than single PL biofilm when tested using crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy, indicating the competitive interactions between them prevail. Additionally, ε-PLH significantly reduced the biofilm development as mono- and dual species in a concentration-dependent manner, especially single LM biofilm, which was consistent with the decrease in autoinducer-2 (AI-2) activity. LM as dual-species biofilms exhibited lower sensitivity to ε-PLH than its mono-biofilm probably due to protective effect conferred by PL. ε-PLH in combination with CEO, at the maximum sublethal concentrations (MSCs), showed enhanced inhibitory activity against dual-species biofilm formation, as evidenced by thin spare spatial structures and reduced AI-2 activity. In addition, the preformed dual biofilms were dramatically eradicated following treatment with ε-PLH combined with CEO at higher than minimum inhibitory concentration in comparison with either of the compounds used alone, indicating the synergistic antibiofilm of the two preservatives. This study reveals the competitive interactions between the two strains in dual-species biofilms, in which the dominant PL significantly contributed toward the tolerance of LM to ε-PLH, and the use of combined preservatives shows it is an effective strategy to control the multispecies biofilms in meat processing.

Interactions between fish isolates Pseudomonas fluorescens and Staphylococcus aureus in dual-species biofilms and sensitivity to carvacrol.

Biofilm formation is a frequent source of contamination of food products, which results in significant economic losses through microbial spoilage and poses serious health concerns. Little is known about the fate of Staphylococcus aureus in the dual-species biofilms with Pseudomonas fluorescens an important spoiler commonly found in aquatic products. This study evaluates the interactions between mono- or dual-species biofilms formed by P. fluorescens and S. aureus, as well as the sensitivity of the two tested strains to carvacrol. The biofilm cell population, expolysaccharide production, biofilm structures of P. fluorescens as mono- and dual-species with S. aureus at ratios of 1:1 and 1:0.01 were investigated with different concentrations of carvacrol (0, 0.4, 0.8 and 1.6 mM) in fish juice at 30 °C. The results show that the biofilm cell population of S. aureus in the dual-species was significantly lower (p < 0.05) than that in the mono-species, compared to no difference for P. fluorescens. In the co-culture the dominance of P. fluorescens inhibited the growing population of S. aureus in both planktonic and biofilm cells, however, two strains were stimulated to produce the large expolysaccharides and coaggregation, forming the complex spatial multibiofilm structures. The large increase in the dual-species biofilms was positively correlated with high quorum sensing autoinductor-2 (AI-2), and exogenous 4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor, DPD), rather than C4-HSL, greatly stimulated the dual-species biofilm formation. In addition, carvacrol significantly reduced the tested biofilms and expolysaccharide secretion without affecting cell viability in a concentration-dependent manner, especially for S. aureus. Furthermore, the two strains as the dual-species biofilms exhibited lower sensitivity to carvacrol than the mono-culture, regardless of the level of inoculum of S. aureus, which was consistent with the decrease of AI-2 activity. The present study highlights that the interactions between P. fluorescens and S. aureus in dual-species biofilms promoted the large production of expolysaccharides and complex biofilm structures modulated by AI-2 signal, which results in the community-level resistance to carvacrol.