The emergence of antibiotic resistance has necessitated new therapeutic approaches to combat recalcitrant bacterial infections. Persister cells, often found in biofilms, are metabolically dormant, and thus, are highly tolerant to all traditional antibiotics and represent a major drug resistance mechanism. In the present study, 36 diverse indole derivatives were investigated with the aim of identifying novel compounds that inhibit persisters and biofilm formation by Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. 5-Iodoindole and other halogenated indoles, 4-fluoroindole, 7-chloroindole, and 7-bromoindole, eradicated persister formation by E. coli and S. aureus, and 5-iodoindole most potently inhibited biofilm formation by the two bacteria. Unlike other antibiotics, 5-iodoindole did not induce persister cell formation, and 5-iodoindole inhibited the production of the immune-evasive carotenoid staphyloxanthin in S. aureus; hence, 5-iodoindole diminished the production of virulence factors in this strain. These results demonstrate halogenated indoles are potentially useful for controlling bacterial antibiotic resistance.

Background: Bacteria use diverse signaling molecules to ensure the survival of the species in environmental niches. A variety of both Gram-positive and Gram-negative bacteria produce large quantities of indole that functions as an intercellular signal controlling diverse aspects of bacterial physiology. Results: In this study, we sought a novel role of indole in a Gram-positive bacteria Paenibacillus alvei that can produce extracellular indole at a concentration of up to 300 μM in the stationary phase in Luria-Bertani medium. Unlike previous studies, our data show that the production of indole in P. alvei is strictly controlled by catabolite repression since the addition of glucose and glycerol completely turns off the indole production. The addition of exogenous indole markedly inhibits the heat resistance of P. alvei without affecting cell growth. Observation of cell morphology with electron microscopy shows that indole inhibits the development of spore coats and cortex in P. alvei. As a result of the immature spore formation of P. alvei, indole also decreases P. alvei survival when exposed to antibiotics, low pH, and ethanol. Additionally, indole derivatives also influence the heat resistance; for example, a plant auxin, 3-indolylacetonitrile dramatically (2900-fold) decreased the heat resistance of P. alvei, while another auxin 3-indoleacetic acid had a less significant influence on the heat resistance of P. alvei. Conclusions: Together, our results demonstrate that indole and plant auxin 3-indolylacetonitrile inhibit spore maturation of P. alvei and that 3-indolylacetonitrile presents an opportunity for the control of heat and antimicrobial resistant spores of Gram-positive bacteria.

Acinetobacter baumannii is a nosocomial pathogen, and its biofilms are tolerant to desiccation, nutrient starvation, and antimicrobial treatment on biotic and abiotic surfaces, tissues, and medical devices. Biofilm formation by A. baumannii is triggered by a quorum sensing cascade, and we hypothesized that fatty acids might inhibit its biofilm formation by interfering with quorum sensing. Initially, we investigated the antibiofilm activities of 24 fatty acids against A. baumannii ATCC 17978 and two clinical isolates. Among these fatty acids, two unsaturated fatty acids, nervonic and oleic acid, at 20 μg/mL significantly inhibited A. baumannii biofilm formation without affecting its planktonic cell growth (MICs were >500 μg/mL) and markedly decreased the motility of A. baumannii but had no toxic effect on the nematode Caenorhabditis elegans. Interestingly, molecular dynamic simulations showed that both fatty acids bind to the quorum sensing acyl homoserine lactone synthase (AbaI), and decent conformational stabilities of interactions between the fatty acids and AbaI were exhibited. Our results demonstrate that nervonic and oleic acid inhibit biofilm formation by A. baumannii strains and may be used as lead molecules for the control of persistent A. baumannii infections.

Skin microbiota, such as acne-related Cutibacterium acnes, Staphylococcus aureus, and fungal Candida albicans, can form polymicrobial biofilms with greater antimicrobial tolerance to traditional antimicrobial agents and host immune systems. In this study, the phytopigment shikonin was investigated against single-species and multispecies biofilms under aerobic and anaerobic conditions. Minimum inhibitory concentrations of shikonin were 10 µg/mL against C. acnes, S. aureus, and C. albicans, and at 1–5 µg/mL, shikonin efficiently inhibited single biofilm formation and multispecies biofilm development by these three microbes. Shikonin increased porphyrin production in C. acnes, inhibited cell aggregation and hyphal formation by C. albicans, decreased lipase production, and increased hydrophilicity in S. aureus. In addition, shikonin at 5 or 10 µg/mL repressed the transcription of various biofilm-related genes and virulence-related genes in C. acnes and downregulated the gene expression levels of the quorum-sensing agrA and RNAIII, α-hemolysin hla, and nuclease nuc1 in S. aureus, supporting biofilm inhibition. In addition, shikonin prevented multispecies biofilm development on porcine skin, and the antimicrobial efficacy of shikonin was recapitulated in a mouse infection model, in which it promoted skin regeneration. The study shows that shikonin inhibits multispecies biofilm development by acne-related skin microbes and might be useful for controlling bacterial infections.

Acne vulgaris is a common chronic inflammatory skin disease involving Cutibacterium acnes with other skin commensals such as Staphylococcus aureus and Candida albicans in the anaerobic and lipid-rich conditions of pilosebaceous units. These microbes readily form multispecies biofilms that are tolerant of traditional antibiotics as well as host immune systems. The phytopigment alizarin was previously found to prevent biofilm formation by S. aureus and C. albicans strains under aerobic conditions. Hence, we hypothesized that alizarin might control C. acnes and multispecies biofilm development. We found that under anaerobic conditions, alizarin efficiently inhibited single biofilm formation and multispecies biofilm development by C. acnes, S. aureus, and C. albicans without inhibiting planktonic cell growth. Alizarin increased the hydrophilicities of S. aureus and C. albicans cells, decreased lipase production by S. aureus, diminished agglutination by C. acnes, and inhibited the aggregation of C. albicans cells. Furthermore, the co-administration of alizarin and antibiotics enhanced the antibiofilm efficacies of alizarin against C. acnes. A transcriptomic study showed that alizarin repressed the transcriptions of various biofilm-related genes such as lipase, hyaluronate lyase, adhesin/invasion-related, and virulence-related genes of C. acnes. Furthermore, alizarin at 100 µg/mL prevented C. acnes biofilm development on porcine skin. Our results show that alizarin inhibits multispecies biofilm development by acne-causing microbes and suggest it might be a useful agent for treating or preventing C. acnes-causing skin diseases.