Visualizing Bacterial Motility Based on a Color Reaction.

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Anti-quorum sensing activity of Forsythia suspense extract against Chromobacterium violaceum by targeting CviR receptor.

The increasing incidence of antimicrobial-resistant bacterial pathogens has focused researchers on quorum sensing inhibition strategies instead of those conventional approaches to fight bacterial infections. Anti-quorum sensing (QS) activity of aqueous extract from Forsythia suspense (FSE) was assessed, and its potential QS inhibition mechanisms were also analyzed. The minimal inhibitory concentration (MIC) of FSE to Chromobacterium violaceum 12472 is 0.5 mg mL-1. Inhibition of QS-regulated violacein production and biofilm formation in C. violaceum 12472 by FSE occurred in a concentration-dependent manner at sub-MIC, with > 70.12 and > 85.31% inhibition at 0.25 mg mL-1, respectively. N-Acyl homoserine lactones (AHLs) extracted from cultures of C. violaceum 31532 grown in the presence of FSE could not change the violacein production in C. violaceum 026, which indicated that FSE did not inhibit AHL synthesis. We also found that FSE cannot degrade AHLs. Finally, in silico molecular docking was conducted. The computed binding energy data suggested that components of F. suspense have a tendency to inhibit CviR with varying binding affinities and the energy score of Pinoresinol (- 26.02 kcal/mol) is higher than that of C6-HSL (- 16.09 kcal mol-1). We concluded that FSE acts as an antagonist of bacterial quorum sensing by competing with AHL receptor binding site.

Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp.

Quorum quenching (QQ) is a promising alternative infection-control strategy to antibiotics that controls quorum-regulated virulence without killing the pathogens. Aeromonas hydrophila is an opportunistic gram-negative pathogen living in freshwater and marine environments. A. hydrophila possesses an N-acyl homoserine lactone (AHL)-based quorum-sensing (QS) system that regulates virulence, so quorum signal-inactivation (i.e., QQ) may represent a new way to combat A. hydrophila infection. In this study, an AHL lactonase gene, aiiA was cloned from Bacillus sp. strain QSI-1 and expressed in Escherichia coli strain BL21(DE3). The A. hydrophila hexanoyl homoserine lactone (C6-HSL) QS signal molecule was degraded by AiiAQSI-1, which resulted in a decrease of bacterial swimming motility, reduction of extracellular protease and hemolysin virulence factors, and inhibited the biofilm formation of A. hydrophila YJ-1 in a microtiter assay. In cell culture studies, AiiAQSI-1 decreased the ability of A. hydrophila adherence to and internalization by Epithelioma papulosum cyprini (EPC) cells. During in vivo studies, oral administration of AiiAQSI-1 via feed supplementation attenuated A. hydrophila infection in Crucian Carp. Results from this work indicate that feed supplementation with AiiAQSI-1 protein has potential to control A. hydrophila aquaculture disease via QQ.

Isolation, Identification of Carotenoid-Producing Rhodotorula sp. from Marine Environment and Optimization for Carotenoid Production.

Carotenoids are natural pigments found in plants and microorganisms. These important nutrients play significant roles in animal health. In contrast to plant production, the advantages of microbial fermentation of carotenoids are the lower media costs, fast growth rate of microorganisms, and the ease of culture condition control. In this study, a colony of red pigment-producing yeast, Rhodotorula sp. RY1801, was isolated from the sediment of marine environment with the potential to produce carotenoids. Optimization of carotenoid production in Rhodotorula sp. RY1801 was also discussed. The optimum conditions found for carotenoid production were as follows: temperature, 28 °C; pH 5.0; carbon source, 10 g/L glucose, nitrogen source, 10 g/L yeast extract, maximum concentration of 987 µg/L of total carotenoids was obtained. The results of this study show that the isolated yeast strain Rhodotorula sp. RY1801 can potentially be used in future as a promising microorganism for the commercial production of carotenoids.

Tea polyphenols as an antivirulence compound Disrupt Quorum-Sensing Regulated Pathogenicity of Pseudomonas aeruginosa.

Green tea, a water extract of non-fermented leaves of Camellia sinensis L., is one of the nonalcoholic beverages in China. It is becoming increasingly popular worldwide, because of its refreshing, mild stimulant and medicinal properties. Here we examined the quorum sensing inhibitory potentials of tea polyphenols (TP) as antivirulence compounds both in vitro and in vivo. Biosensor assay data suggested minimum inhibitory concentrations (MICs) of TP against selected pathogens were 6.25 ~ 12.5 mg/mL. At sub-MIC, TP can specifically inhibit the production of violacein in Chromobacterium violaceum 12472 with almost 98% reduction at 3.125 mg/mL without affecting its growth rate. Moreover, TP exhibited inhibitory effects on virulence phenotypes regulated by QS in Pseudomonas aeruginosa. The total proteolytic activity, elastase, swarming motility and biofilm formation were reduced in a concentration-dependent manner. In vivo, TP treatment resulted in the reduction of P. aeruginosa pathogenicity in Caenorhabditis elegans. When its concentration was 3.125 mg/mL, the survival rate reached 63.3%. In the excision wound infection model, the wound contraction percentage in treatment groups was relatively increased and the colony-forming units (CFU) in the wound area were significantly decreased. These results suggested that TP could be developed as a novel non-antibiotic QS inhibitor without killing the bacteria but as an antivirulence compound to control bacterial infection.

Quorum quenching bacteria Bacillus sp. QSI-1 protect zebrafish (Danio rerio) from Aeromonas hydrophila infection.

Quorum Sensing (QS) is a bacterial regulatory mechanism, which is responsible for controlling the expression of various biological macromolecules such as the virulence factors in a cell density-dependent manner. Disruption of the QS system of pathogens has been proposed as a new anti-infective strategy. Biodegradation of AHLs proves to be an efficient way to interrupt QS, since AHLs are the main family of QS autoinducers used in Gram negative bacteria. In this study, the effect of Bacillus sp. QSI-1 as an efficient quorum quencher on virulence factors production and biofilm formation of fish pathogen Aeromonas hydrophila was investigated. QSI-1 reduced the accumulation of AHLs but did not affect the growth of A. hydrophila YJ-1 when cocultured. In the result, the supernatant of QSI-1 showed significant inhibition of protease production (83.9%), hemolytic activity (77.6%) and biofilm formation (77.3%) in YJ-1. In biocontrol experiment, QSI-1 significantly reduced the pathogenicity of A. hydrophila strain YJ-1 in zebrafish (Danio rerio). The fish fed with QSI-1 was observed to have a relative percentage survival of 80.8%. Our results indicate that AHLs degrading bacteria should be considered as an alternative for antibiotics in aquaculture for the biocontrol of bacterial fish diseases.

Azithromycin Reduces the Production of α-hemolysin and Biofilm Formation in Staphylococcus aureus.

Staphylococcus aureus causes a broad range of life-threatening diseases in humans. This bacterium produces a large number of extracellular virulence factors that are closely associated with specific diseases which are controlled by quorum sensing. In this study, we show that azithromycin was active against methicillin-resistant Staphylococcus aureus (MRSA) strains with MICs ranged from 32 to 64 µg/mL. Azithromycin at subinhibitory concentration, markedly reduced the production of α-hemolysin at (1/16MIC, 1/8MIC) and biofilm formation at (1/16MIC, 1/8MIC), respectively. The results indicated that sub-inhibitory concentrations of azithromycin decreased the production of α-hemolysin and biofilm formation in MRSA in a dose-dependent manner. Therefore, azithromycin may be useful in the treatment of α-hemolysin producing and biofilm formation MRSA infections.

Effect of Traditional Chinese Herbal Medicine with Antiquorum Sensing Activity on Pseudomonas aeruginosa.

Traditional Chinese herbal medicines (TCHMs) were tested for their ability of antiquorum sensing. Water extracts of Rhubarb, Fructus gardeniae, and Andrographis paniculata show antiquorumsensing activity when using Chromobacterium violaceum CV12472 as reporter; the sub-MIC concentrations of these TCHMs were tested against AHL-dependent phenotypic expressions of PAO1. Results showed significant reduction in pyocyanin pigment, protease, elastase production, and biofilm formation in PAO1 without inhibiting the bacterial growth, revealing that the QSI by the extracts is not related to static or killing effects on the bacteria. The results indicate a potential modulation of bacterial cell-cell communication, P. aeruginosa biofilm, and virulence factors by traditional Chinese herbal medicine. This study introduces not only a new mode of action for traditional Chinese herbal medicines, but also a potential new therapeutic direction for the treatment of bacterial infections, which have QSI activity and might be important in reducing virulence and pathogenicity of pathogenic bacteria.

Production of N-acyl Homoserine Lactones and Virulence Factors of Waterborne Aeromonas hydrophila.

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules and some virulence factors, including hemolysins, proteases, extracellular nucleases production and cytotoxicity by waterborne Aeromonas hydrophila. A total of 24 strains isolated from fresh-water or diseased fish were used in the study. The majority A.hydrophila strains produce two AHL molecules (21/24), one is N-butanoyl homoserine lactone (BHL), and the other is N-hexanoyl homoserine lactone (HHL) according to thin-layer chromatography analysis. Among the virulence factors tested, more than 83 % of the isolates produced ß haemolysin when inoculated on sheep blood agar, only 50 % of the isolates displayed DNase activity, 75 % of the isolates shown proteolytic activity on skimmed milk plate, and cytotoxic activity was detected in 20 of 24 of the isolates. The strains producing AHLs possessed one or more virulence factors. In conclusion, the production of quorum sensing signal molecules is common among the strains that we examined, and there seems to some relationships between quorum sensing signal production and virulence factors in A. hydrophila.

[Generation of Aeromonas hydrophila ghosts and their evaluation as oral vaccine candidates in Carassius auratus gibelio].

The bacterial ghost (BG) system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures. They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria, and can induce humoral and cellular immune response, including mucosal immune responses. Plasmid pElysis consisting E gene was transformed into AhJ-1. Through shifting the culture temperature from 28 degrees C to 42 degrees C, A. hydrophila J-1 (pElysis) was induced to lyse and the OD600 value of culture media was measured every 15 minutes during the induction. The lysed bacteria were observed by scanning electron microscopy (SEM). The A. hydrophila ghosts (AHG) used as oral vaccine were also investigated. The OD600 value of A. hydrophila J-1(pElysis) began to decline after 30 min of induction, and after 75 min of induction, the OD600 value decline speed become slowly. The efficiency of ghost induction in non-lyophilized A. hydrophila was 99.99%, 16 hours post induced, no live bacteria can be detected in culture. Scanning electron microscopy observation proved that most lysed bacteria were emptied. Fish vaccination experiments shows that the antibody evoked highest degree after 5 weeks by oral administration of bacterial ghost vaccine and the agglutination antibody titer reached 2(7) and continued two weeks, while the agglutination antibody titer of formalin killed vaccine only reached 26 and only maintained one week. After challenged with the parent strain J-1, the survival rate of bacterial ghost vaccinated fish was higher than the control group and formalin killed vaccine group, the relative percent survival (RPS) was 78.95% (16/20), but the RPS of formalin killed vaccine group was 57.9% (12/20). This suggests that the bacterial ghost vaccine has higher potential to induce protective adaptive immunity than normal vaccine.