The control of E. coli activity from forming biofilm and persister cells is an essential factor in both the health and food industries. The efficacy of antimicrobial treatment is often limited due to their low penetrability as biofilm formation protect cells within from physical or chemical threats. Among other factors, osmotic stress has shown to have a high capacity to enhance the antimicrobial activities against various pathogens. Thus, this study aimed to test the hypothesis that the antimicrobial activity of cineole (CN) could be enhanced under osmotic stress to inhibit biofilm and persister cells. Time-kill analysis revealed that CN under NaCl-induced osmotic stress (CN-S) had better inhibitory effect on E. coli biofilm. 5% CN-S altered the integrity, hydration, motilities and exopolysaccharide production of E. coli cells. Also, the outer membrane permeability, surface roughness and hydrophobicity which determine initial cell adhesion, aggregation and colony assembly were significantly perturbed. Furthermore, the expression levels of virulence genes stx1, stx2, eae, flhD, and the TA system antitoxin genes mazE, hipB were downregulated. When applied to cucumber, the rate of increase in internalized bacterial cells significantly reduced after storage at 4 °C for 48 h. Thus, the results suggested that the application of osmotic stress could minimize the working concentration of antimicrobials in real food systems, which could be helpful in counteracting the growing concern of microbial resistance.

Microbial communities enmeshed in a matrix of macromolecules, termed as biofilms, are the natural setting of bacteria. Exopolysaccharide is a critical matrix component of biofilms. Here, we focus on biofilm matrix exopolysaccharides in Pseudomonas aeruginosa. This opportunistic pathogen can adapt to a wide range of environments and can form biofilms or aggregates in a variety of surfaces or environments, such as the lungs of people with cystic fibrosis, catheters, wounds, and contact lenses. The ability to synthesize multiple exopolysaccharides is one of the advantages that facilitate bacterial survival in different environments. P. aeruginosa can produce several exopolysaccharides, including alginate, Psl, Pel, and lipopolysaccharide. In this review, we highlight the roles of each exopolysaccharide in P. aeruginosa biofilm development and how bacteria coordinate the biosynthesis of multiple exopolysaccharides and bacterial motility. In addition, we present advances in antibiofilm strategies targeting matrix exopolysaccharides, with a focus on glycoside hydrolases.

Tetragenococcus halophilus, a halophilic lactic acid bacterium (LAB), plays an important role in the production of high-salt fermented foods. Generally, formation of biofilm benefits the fitness of cells when faced with competitive and increasingly hostile fermented environments. In this work, the biofilm-forming capacity of T. halophilus was investigated. The results showed that the optimal conditions for biofilm formation by T. halophilus were at 3-9% salt content, 0-6% ethanol content, pH 7.0, 30°C, and on the surface of stainless steel. Confocal laser scanning microscopy (CLSM) analysis presented a dense and flat biofilm with a thickness of about 24 μm, and higher amounts of live cells were located near the surface of biofilm and more dead cells located at the bottom. Proteins, polysaccharides, extracellular-DNA (eDNA), and humic-like substances were all proved to take part in biofilm formation. Higher basic surface charge, greater hydrophilicity, and lower intracellular lactate dehydrogenase (LDH) activities were detected in T. halophilus grown in biofilms. Atomic force microscopy (AFM) imaging revealed that biofilm cultures of T. halophilus had stronger surface adhesion forces than planktonic cells. Cells in biofilm exhibited higher cell viability under acid stress, ethanol stress, heat stress, and oxidative stress. In addition, T. halophilus biofilms exhibited aggregation activity and anti-biofilm activity against Staphylococcus aureus and Salmonella Typhimurium. Results presented in the study may contribute to enhancing stress tolerance of T. halophilus and utilize their antagonistic activities against foodborne pathogens during the production of fermented foods.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm.

OBJECTIVE: Bacterial biofilms on the surface of prostheses are becoming a rising concern in managing prosthetic joint infections. The inherent resistant features of biofilms render traditional antimicrobial therapy unproductive and revision surgery outcomes uncertain. This situation has prompted the exploration of novel antimicrobial strategies. The synergy of ultrasound microbubbles and vancomycin has been proposed as an efficient alternative for biofilm eradication. The purpose of this study was to evaluate the anti-biofilm effect of stimulated phase-shift acoustic nanodroplets (NDs) combined with vancomycin.
METHODS: We fabricated lipid phase-shift NDs with a core of liquid perfluoropentane. A new phase change mode for NDs incorporating an initial unfocused low-intensity pulsed ultrasound for 5 minutes and a subsequent incubation at 37°C into a 24-hour duration was developed. Methicillin-resistant Staphylococcus aureus (MRSA) biofilms were incubated with vancomycin and NDs under the hybrid stimulation. Biofilm morphology following treatment was determined using confocal laser scanning microscopy and scanning electron microscopy. Resazurin assay was used to quantify bactericidal efficacy against MRSA biofilm bacteria.
RESULTS: NDs treated sequentially with ultrasound and heating at 37°C achieved gradual and substantial ND vaporization and cavitation in a successive process. NDs after stimulation were capable of generating stronger destruction on biofilm structure which was best characterized by residual circular arc margins and more dead bacteria. Furthermore, NDs combined with vancomycin contributed to significantly decreasing the metabolic activity of bacteria in MRSA biofilms (P<0.05).
CONCLUSIONS: Phase-shift acoustic NDs could exert a significant bactericidal effect against MRSA biofilms through a new stimulation mode. Acoustic NDs present advantages over microbubbles for biofilm damage. This anti-biofilm strategy could be used either alone or as an enhancer of traditional antibiotics in the control of prosthetic joint infections.

Quorum sensing (QS) is a cell-to-cell communication system based on the exchange of small intercellular signal molecules, such as N-Acyl homoserine lactones (AHLs), which act as cell-density mediators of QS gene expression, and are highly variable both in types and amounts in most Gram-negative Proteobacteria. Understanding the regulation of AHLs may contribute to the elucidation of cell density-dependent phenomena, such as biofilm formation. Vibrio alginolyticus is among the most frequently observed marine opportunistic Vibrio pathogens. However, AHL production of this species and its effects on biofilm formation remain to be understood. Here, our study reported the diverse AHL profiles of 47 marine-isolated V. alginolyticus strains and the effects of exogenous 3-oxo-C10-HSL on biofilm formation under different temperature conditions (16°C and 28°C). A total of 11 detected AHLs were produced by the isolates, of which 3-OH-C4-HSL, 3-oxo-C10-HSL and 3-oxo-C14-HSL comprised the largest proportions. We also observed that moderate levels of exogenous 3-oxo-C10-HSL (10 and 20 μM) could induce or enhance biofilm formation and alter its structure, while high levels (40 and 100 μM) did not significantly improve and even inhibited biofilm formation in V. alginolyticus. Further, regulation by exogenous 3-oxo-C10-HSL was both concentration- and temperature-dependent in V. alginolyticus.