Bacterial Biofilm Inhibition: A Focused Review on Recent Therapeutic Strategies for Combating the Biofilm Mediated Infections.

Biofilm formation is a major concern in various sectors and cause severe problems to public health, medicine, and industry. Bacterial biofilm formation is a major persistent threat, as it increases morbidity and mortality, thereby imposing heavy economic pressure on the healthcare sector. Bacterial biofilms also strengthen biofouling, affecting shipping functions, and the offshore industries in their natural environment. Besides, they accomplish harsh roles in the corrosion of pipelines in industries. At biofilm state, bacterial pathogens are significantly resistant to external attack like antibiotics, chemicals, disinfectants, etc. Within a cell, they are insensitive to drugs and host immune responses. The development of intact biofilms is very critical for the spreading and persistence of bacterial infections in the host. Further, bacteria form biofilms on every probable substratum, and their infections have been found in plants, livestock, and humans. The advent of novel strategies for treating and preventing biofilm formation has gained a great deal of attention. To prevent the development of resistant mutants, a feasible technique that may target adhesive properties without affecting the bacterial vitality is needed. This stimulated research is a rapidly growing field for applicable control measures to prevent biofilm formation. Therefore, this review discusses the current understanding of antibiotic resistance mechanisms in bacterial biofilm and intensely emphasized the novel therapeutic strategies for combating biofilm mediated infections. The forthcoming experimental studies will focus on these recent therapeutic strategies that may lead to the development of effective biofilm inhibitors than conventional treatments.

Anti-quorum Sensing and Protective Efficacies of Naringin Against Aeromonas hydrophila Infection in Danio rerio.

It is now well known that the quorum sensing (QS) mechanism coordinates the production of several virulence factors and biofilm formation in most pathogenic microorganisms. Aeromonas hydrophila is a prime pathogen responsible for frequent outbreaks in aquaculture settings. Recent studies have also continuously reported that A. hydrophila regulates virulence factor production and biofilm formation through the QS system. In addition to the presence of antibiotic resistance genes, biofilm-mediated antibiotic resistance increases the severity of A. hydrophila infections. To control the bacterial pathogenesis and subsequent infections, targeting the QS mechanism has become one of the best alternative methods. Though very few compounds were identified as QS inhibitors against A. hydrophila, to date, the screening and identification of new and effective natural QS inhibitors is a dire necessity to control the infectious A. hydrophila. The present study endorses naringin (NA) as an anti-QS and anti-infective agent against A. hydrophila. Initially, the NA showed a concentration-dependent biofilm reduction against A. hydrophila. Furthermore, the results of microscopic analyses and quantitative virulence assays displayed the promise of NA as a potential anti-QS agent. Subsequently, the downregulation of ahh1, aerA, lip and ahyB validate the interference of NA in virulence gene expression. Furthermore, the in vivo assays were carried out in zebrafish model system to evaluate the anti-infective potential of NA. The outcome of the immersion challenge assay showed that the recovery rate of the zebrafish has substantially increased upon treatment with NA. Furthermore, the quantification of the bacterial load upon NA treatment showed a decreased level of bacterial counts in zebrafish when compared to the untreated control. Moreover, the NA treatment averts the pathogen-induced histoarchitecture damages in vital organs of zebrafish, compared to their respective controls. The current study has thus analyzed the anti-QS and anti-infective capabilities of NA and could be employed to formulate effective treatment measures against A. hydrophila infections.

In vitro and in vivo effect of 2,6-Di-tert-butyl-4-methylphenol as an antibiofilm agent against quorum sensing mediated biofilm formation of Vibrio spp.

This study unveils the in vitro and in vivo antibiofilm potential of 2,6-Di-tert-butyl-4-methylphenol (DTBMP) from Chroococcus turgidus against Vibrio spp. In the preliminary study, cell free culture supernatant (CFCS) of C. turgidus inhibited the violacein production in biomarker strain Chromobacterium violaceum and its mutant strain CV026 in a dose dependent manner. The effective biofilm inhibitory concentration (BIC) of pure compound DTBMP from C. turgidus was identified as 250 µg/ml concentration in tested Vibrio species. Furthermore, DTBMP proved to effectively inhibit the bioluminescence production in V. harveyi and other biofilm related virulence traits such as exopolysaccharides (EPS) production, hydrophobicity index, swimming and swarming motility at its BIC concentration in three major pathogenic vibrios: V. harveyi, V. parahaemolyticus and V. vulnificus. The antibiofilm potential of DTBMP was validated through light, confocal laser scanning and scanning electron microscopic analyses. In addition, the non-bactericidal effect of DTBMP was determined through growth curve and 2,3-bis (2-methyloxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Real-time PCR studies revealed the down-regulation of master quorum sensing (QS) regulator genes of V. harveyi such as luxR, luxS, luxP, luxQ and luxO on treatment with DTBMP. In vivo results confirmed that DTBMP augmented the survival rate of Litopenaeus vannamei larvae up to 75, 88 and 66% upon infection with V. harveyi, V. parahaemolyticus and V. vulnificus, respectively. The results of this study ascertain the promising effects of DTBMP as an antibiofilm agent, which could be positively explored to treat biofilm-associated vibrios infections in aquaculture.

Rapid biosynthesized AgNPs from Gelidiella acerosa aqueous extract mitigates quorum sensing mediated biofilm formation of Vibrio species-an in vitro and in vivo approach.

The present study explores the non-bactericidal anti-virulence efficacy of green synthesized silver nanoparticles (AgNPs) from Gelidiella acerosa against multi-drug resistant Vibrio spp. Spectral characterization of AgNPs was performed through UV-Visible, FT-IR, and energy-dispersive spectroscopic techniques followed by X-ray crystallography and zeta potential analysis. Further, the structural characterization was done by electron and atomic force microscopic techniques. AgNPs profoundly quelled the quorum sensing mediated violacein production in Chromobacterium violaceum and CV026. Characterized AgNPs at 100 µg mL-1 concentrations depicted a phenomenal anti-biofilm efficacy against Vibrio parahaemolyticus (71%) and Vibrio vulnificus (83%) biofilms, which was further confirmed through light, confocal, and scanning electron microscopic analyses. In vitro bioassays revealed the remarkable inhibitory values of AgNPs, by inhibiting the exopolysaccharide production, hydrophobicity, and motility. In vivo studies using Artemia franciscana larvae also confirmed the anti-infective proficiency, as the AgNPs effectively reduced the bacterial colonization and enhanced the survival rate of larvae up to 100% without any toxicity effect. Graphical abstract Rapid biosynthesized AgNPs from Gelidiella acerosa quench quorum sensing controlled virulence traits in vibrios.

In vitro and in vivo exploration of palmitic acid from Synechococcus elongatus as an antibiofilm agent on the survival of Artemia franciscana against virulent vibrios.

Biofilm formation of Vibrio spp. has been demonstrated as a potentially important mechanism contributing antibiotic treatment failure in aquaculture. In the present study, the effect of palmitic acid (PA) identified from Synechococcus elongatus was assessed for the inhibition of quorum sensing (QS) regulated biofilm formation in aquatic bacterial pathogens. The biofilm inhibitory concentration (BIC) of PA against Vibrio spp. was found to be 100µgml-1. In this concentration, PA exhibited a significant inhibition in biofilm biomass of Vibrio harveyi MTCC 3438, V. parahaemolyticus ATCC 17802, V. vulnificus MTCC 1145 and V. alginolyticus ATCC 17749 without hindering their planktonic growth. Also, PA displayed gradual decrease in bioluminescence production of V. harveyi. The results of extracellular polymeric substances quantification, microbial adhesion to hydrocarbons and Fourier transform infrared spectroscopic (FT-IR) analyses suggested that PA positively interferes with the initial adhesion stages of biofilm formation. In addition, confocal and scanning electron microscopic analysis substantiates the antibiofilm efficacy of the PA. The transcriptomic analysis revealed the down-regulation of QS mediated response regulator genes expression in V. harveyi. Concomitantly, PA reduced the intestinal colonization of vibrios in brine shrimp larvae and thereby attenuates the biofilm assemblage and its associated virulence. In vivo studies using brine shrimp larvae manifested the reduction in adherence and virulence, which prompts further investigation about the potential of PA for the treatment of vibriosis.

In vitro antibiofilm efficacy of Piper betle against quorum sensing mediated biofilm formation of luminescent Vibrio harveyi.

Vibrio harveyi is a potent biofilm former, which confers resistance to multiple antimicrobials, disinfectants, chemicals and biocides. The prevalence of biofilm mediated antibiotic resistance among aquatic bacterial pathogens stresses the search for novel alternative approach to treat vibriosis in aquaculture. Exploring suitable therapeutics from natural resources could be a novel area of research. Therefore, this work was executed to evaluate the inhibitory effect of Piper betle ethyl acetate extract (PBE) on bioluminescence production and biofilm formation of V. harveyi. Minimal inhibitory concentration (MIC) of PBE against planktonic V. harveyi was found to be 1600 µg ml-1; furthermore, PBE inhibited the quorum sensing (QS) mediated bioluminescence production and biofilm formation in V. harveyi upto 98 and 74% respectively, at its sub-MIC concentration of 400 µg ml-1 without affecting their cell viability. Similar results were obtained for exopolysaccharides production and swimming motility related to biofilm formation of V. harveyi, where PBE reduced EPS production upto 64%. Light and confocal laser scanning microscopic analyses further confirmed that the PBE effectively prevented the initial attachment as well as microcolonies formation of V. harveyi biofilm, when compared to their untreated controls. This study demonstrates the promising antibiofilm activity of PBE and confirms the ethnopharmacological potential of this plant against V. harveyi infections.

In vitro and in vivo efficacy of rosmarinic acid on quorum sensing mediated biofilm formation and virulence factor production in Aeromonas hydrophila.

Rosmarinic acid (RA) was assessed for its quorum sensing inhibitory (QSI) potential against Aeromonas hydrophila strains AH 1, AH 12 and MTCC 1739. The pathogenic strains of A. hydrophila were isolated from infected zebrafish and identified through biochemical analysis and amplification of a species-specific gene (rpsL). The biofilm inhibitory concentration (BIC) of RA against A. hydrophila strains was found to be 750 µg ml-1. At this concentration, RA reduced the QS mediated hemolysin, lipase and elastase production in A. hydrophila. In FT-IR analysis, RA treated A. hydrophila cells showed a reduction in cellular components. Gene expression analysis confirmed the down-regulation of virulence genes such as ahh1, aerA, lip and ahyB. A. hydrophila infected zebrafish upon treatment with RA showed increased survival rates. Thus, the present study demonstrates the use of RA as a plausible phytotherapeutic compound to control QS mediated biofilm formation and virulence factor production in A. hydrophila.