Jin-Hyung Lee

Indole as an intercellular signal in microbial communities

Bacteria can utilize signal molecules to coordinate their behavior to survive in dynamic multispecies communities. Indole is widespread in the natural environment, as a variety of both Gram-positive and Gram-negative bacteria (to date, 85 species) produce large quantities of indole. Although it has been known for over 100 years that many bacteria produce indole, the real biological roles of this molecule are only now beginning to be unveiled. As an intercellular signal molecule, indole controls diverse aspects of bacterial physiology, such as spore formation, plasmid stability, drug resistance, biofilm formation, and virulence in indole-producing bacteria. In contrast, many non-indole-producing bacteria, plants and animals produce diverse oxygenases which may interfere with indole signaling. It appears indole plays an important role in bacterial physiology, ecological balance, and possibly human health. Here we discuss our current knowledge and perspectives on indole signaling.

3-Indolylacetonitrile Decreases Escherichia coli O157:H7 Biofilm Formation and Pseudomonas aeruginosa Virulence

Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish Pseudomonas aeruginosa virulence. However, indole and bacterial indole derivatives are unstable in the microbial community because they are quickly degraded by diverse bacterial oxygenases. Hence, this work sought to identify novel, non-toxic, stable and potent indole derivatives from plant sources for inhibiting the biofilm formation of E. coli O157:H7 and P. aeruginosa. Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit the biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting its growth. IAN more effectively inhibited biofilms than indole for the two pathogenic bacteria. Additionally, IAN decreased the production of virulence factors including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), pyocyanin and pyoverdine in P. aeruginosa. DNA microarray analysis indicated that IAN repressed genes involved in curli formation and glycerol metabolism, whereas IAN induced indole-related genes and prophage genes in E. coli O157:H7. It appeared that IAN inhibited the biofilm formation of E. coli by reducing curli formation and inducing indole production. Also, corroborating phenotypic results of P. aeruginosa, whole-transcriptomic data showed that IAN repressed virulence-related genes and motility-related genes, while IAN induced several small molecule transport genes. Furthermore, unlike bacterial indole derivatives, plant-originated IAN was stable in the presence of either E. coli or P. aeruginosa. Additionally, indole-3-carboxyaldehyde was another natural biofilm inhibitor for both E. coli and P. aeruginosa.

Roles of Indole as an Interspecies and Interkingdom Signaling Molecule

A number of bacteria, and some plants, produce large quantities of indole, which is widespread in animal intestinal tracts and in the rhizosphere. Indole, as an interspecies and interkingdom signaling molecule, plays important roles in bacterial pathogenesis and eukaryotic immunity. Furthermore, indole and its derivatives are viewed as potential antivirulence compounds against antibiotic-resistant pathogens because of their ability to inhibit quorum sensing and virulence factor production. Indole modulates oxidative stress, intestinal inflammation, and hormone secretion in animals, and it controls plant defense systems and growth. Insects and nematodes can recognize indole, which controls some of their behavior. This review presents current knowledge regarding indole and its derivatives, their biotechnological applications and their role in prokaryotic and eukaryotic systems.

Cinnamon bark oil and its components inhibit biofilm formation and toxin production.

The long-term usage of antibiotics has resulted in the evolution of multidrug resistant bacteria, and pathogenic biofilms contribute to reduced susceptibility to antibiotics. In this study, 83 essential oils were initially screened for biofilm inhibition against Pseudomonas aeruginosa. Cinnamon bark oil and its main constituent cinnamaldehyde at 0.05% (v/v) markedly inhibited P. aeruginosa biofilm formation. Furthermore, cinnamon bark oil and eugenol decreased the production of pyocyanin and 2-heptyl-3-hydroxy-4(1H)-quinolone, the swarming motility, and the hemolytic activity of P. aeruginosa. Also, cinnamon bark oil, cinnamaldehyde, and eugenol at 0.01% (v/v) significantly decreased biofilm formation of enterohemorrhagic Escherichia coli O157:H7 (EHEC). Transcriptional analysis showed that cinnamon bark oil down-regulated curli genes and Shiga-like toxin gene stx2 in EHEC. In addition, biodegradable poly(lactic-co-glycolic acid) film incorporating biofilm inhibitors was fabricated and shown to provide efficient biofilm control on solid surfaces. This is the first report that cinnamon bark oil and its components, cinnamaldehyde and eugenol, reduce the production of pyocyanin and PQS, the swarming motility, and the hemolytic activity of P. aeruginosa, and inhibit EHEC biofilm formation.

ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production

The opportunistic pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilms of this bacterium are much more resistant to antibiotics than planktonic cells. Thirty-six metal ions have been investigated to identify antivirulence and antibiofilm metal ions. Zinc ions and ZnO nanoparticles were found to markedly inhibit biofilm formation and the production of pyocyanin, Pseudomonas quinolone signal (PQS), pyochelin, and hemolytic activity of P. aeruginosa without affecting the growth of planktonic cells. Transcriptome analyses showed that ZnO nanoparticles induce the zinc cation efflux pump czc operon and several important transcriptional regulators (porin gene opdT and type III repressor ptrA), but repress the pyocyanin-related phz operon, which explains observed phenotypic changes. A mutant study showed that the effects of ZnO nanoparticles on the control of pyocyanin production and biofilm formation require the czc regulator CzcR. In addition, ZnO nanoparticles markedly increased the cellular hydrophilicity of P. aeruginosa cells. Our results support that ZnO nanoparticles are potential antivirulence materials against recalcitrant P. aeruginosa infections and possibly other important pathogens.

Anti-biofilm activities of quercetin and tannic acid against Staphylococcus aureus

Staphylococcus aureus is a leading cause of nosocomial infections because of its resistance to diverse antibiotics. The formation of a biofilm is one of the mechanisms of drug resistance in S. aureus. The anti-biofilm abilities of 498 plant extracts against S. aureus were examined. Seventy-two plant extracts belonging to 59 genera and 38 families were found to significantly inhibit the formation of biofilms of S. aureus without affecting the growth of planktonic cells. The most active extract, from Alnus japonica, inhibited the formation of biofilms by three S. aureus strains by >70% at 20 μg ml−1. Transcriptional analyses showed that extract of A. japonica repressed the intercellular adhesion genes icaA and icaD most markedly. Quercetin and tannic acid are major anti-biofilm compounds in the extract of A. japonica. Additionally, the extract of A. japonica and its component compound quercetin, reduced hemolysis by S. aureus. This phenomenon was not observed in the treatment with tannic acid. This study suggests that various plant extracts, such as quercetin and tannic acid, could be used to inhibit the formation of recalcitrant biofilms of S. aureus.

Environmental factors affecting indole production in Escherichia coli

A variety of both Gram-positive and Gram-negative bacteria produce large quantities of indole as an intercellular signal in microbial communities. Biosynthesis of indole is well-studied, and while carbon sources and amino acids are important environmental cues for indole production in Escherichia coli, other environmental factors affecting indole production for this strain are less clear. This study demonstrates that the environmental cue pH is an important factor for indole production that further controls biofilm formation of E. coli. Moreover, E. coli produced a higher level of extracellular indole in the presence of the antibiotics ampicillin and kanamycin, and the increased indole enhanced cell survival during antibiotic stress. Additionally, we found here that temperature is another important factor for indole production; E. coli produces and accumulates a large amount of indole at 50 °C, even at low cell densities. Overall, our results suggest that indole is a stable biological compound, and E. coli may utilize indole to protect itself against other microorganisms.

Electrochemically active biofilm assisted synthesis of Ag@CeO2 nanocomposites for antimicrobial activity, photocatalysis and photoelectrodes

Ag@CeO2 nanocomposites were synthesized by a biogenic and green approach using electrochemically active biofilms (EABs) as a reducing tool. The as-synthesized Ag@CeO2 nanocomposites were characterized and used in antimicrobial, visible light photocatalytic and photoelectrode studies. The Ag@CeO2 nanocomposites showed effective and efficient bactericidal activities and survival test against Escherichia coli O157:H7, and Pseudomonas aeruginosa. The as-synthesized Ag@CeO2 nanocomposites also exhibited enhanced visible light photocatalytic degradation of 4-nitrophenol and methylene blue than pure CeO2. A photocatalytic investigation showed that the Ag@CeO2 nanocomposites possessed excellent visible light photocatalytic activities compared to pure CeO2. Electrochemical impedance spectroscopy and photocurrent measurements showed that the as-synthesized Ag@CeO2 nanocomposites exhibited excellent and enhanced responses to visible light irradiation. These results suggest that the AgNPs anchored at CeO2 induced visible light photoactivity by decreasing the recombination of photogenerated electrons and holes, and extending the response of pure CeO2 to visible light. Overall, as-synthesized Ag@CeO2 nanocomposites are smart materials that can be used for a range of applications, such as antimicrobial activity, visible light photocatalysis and photoelectrode.

Low concentrations of honey reduce biofilm formation, quorum sensing, and virulence in Escherichia coli O157:H7

Bacterial biofilms are associated with persistent infections due to their high resistance to antimicrobial agents. Hence, controlling pathogenic biofilm formation is important in bacteria-related diseases. Honey, at a low concentration of 0.5% (v/v), significantly reduced biofilm formation in enterohemorrhagic Escherichia coli O157:H7 without inhibiting the growth of planktonic cells. Conversely, this concentration did not inhibit commensal E. coli K-12 biofilm formation. Transcriptome analyses showed that honey significantly repressed curli genes (csgBAC), quorum sensing genes (AI-2 importer and indole biosynthesis), and virulence genes (LEE genes). Glucose and fructose in the honeys were found to be key components in reducing biofilm formation by E. coli O157:H7 through the suppression of curli production and AI-2 import. Furthermore, honey, glucose and fructose decreased the colonization of E. coli O157:H7 cells on human HT-29 epithelial cells. These results suggest that low concentrations of honey, such as in honeyed water, can be a practical means for reducing the colonization and virulence of pathogenic E. coli O157:H7.

Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7.

E. coli O157:H7 is the most common cause of hemorrhagic colitis, and no effective therapy exists for E. coli O157:H7 infection. Biofilm formation is closely related to E. coli O157:H7 infection and constitutes a mechanism of antimicrobial resistance. Hence, the antibiofilm or antivirulence approach provides an alternative to antibiotic strategies. Coumarin and its derivatives have a broad range of biological effects, and in this study, the antibiofilm activities of nine coumarins were investigated against E. coli O157:H7. Coumarin or umbelliferone at 50μg/ml was found to inhibit biofilm E. coli O157:H7 formation by more than 80% without affecting bacterial growth. Transcriptional analysis showed that coumarins repressed curli genes and motility genes in E. coli O157:H7, and these findings were in-line with observed reductions in fimbriae production, swarming motility, and biofilm formation. In addition, esculetin repressed Shiga-like toxin gene stx2 in E. coli O157:H7 and attenuated its virulence in vivo in the nematode Caenorhabditis elegans. These findings show that coumarins have potential use in antivirulence strategies against persistent E. coli O157:H7 infection.