Secondary metabolites profoundly affect microbial physiology, metabolism and stress responses. Increasing evidence suggests that these molecules can modulate microbial susceptibility to commonly used antibiotics; however, secondary metabolites are typically excluded from standard antimicrobial susceptibility assays. This may in part account for why infections by diverse opportunistic bacteria that produce secondary metabolites often exhibit discrepancies between clinical antimicrobial susceptibility testing results and clinical treatment outcomes. In this Review, we explore which types of secondary metabolite alter antimicrobial susceptibility, as well as how and why this phenomenon occurs. We discuss examples of molecules that opportunistic and enteric pathogens either generate themselves or are exposed to from their neighbours, and the nuanced impacts these molecules can have on tolerance and resistance to certain antibiotics.
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Maplestone, R. A., Stone, M. J. & Williams, D. H. The evolutionary role of secondary metabolites — a review. Gene 115, 151–157 (1992).
Demain, A. L. & Fang, A. The natural functions of secondary metabolites. Adv. Biochem. Eng. Biotechnol. 69, 1–39 (2000).
Keller, N. P., Turner, G. & Bennett, J. W. Fungal secondary metabolism–from biochemistry to genomics. Nat. Rev. Microbiol. 3, 937–947 (2005).
Tyc, O., Song, C., Dickschat, J. S., Vos, M. & Garbeva, P. The ecological role of volatile and soluble secondary metabolites produced by soil bacteria. Trends Microbiol. 25, 280–292 (2017).
Wang, S. & Lu, Z. in Biocommunication of Archaea (ed. Witzany, G.) 235–239 (Springer, 2017).
Price-Whelan, A., Dietrich, L. E. P. & Newman, D. K. Rethinking “secondary” metabolism: physiological roles for phenazine antibiotics. Nat. Chem. Biol. 2, 71–78 (2006).
Davies, J. Specialized microbial metabolites: functions and origins. J. Antibiot. 66, 361–364 (2013).
Haslam, E. Secondary metabolism — fact and fiction. Nat. Prod. Rep. 3, 217 (1986).
Dietrich, L. E., Price-Whelan, A., Petersen, A., Whiteley, M. & Newman, D. K. The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol. Microbiol. 61, 1308–1321 (2006).
Glasser, N. R., Kern, S. E. & Newman, D. K. Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force. Mol. Microbiol. 92, 399–412 (2014).
Wang, Y. et al. Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition. J. Bacteriol. 193, 3606–3617 (2011).
McRose, D. L. & Newman, D. K. Redox-active antibiotics enhance phosphorus bioavailability. Science 371, 1033–1037 (2021).
Ling, L. L. et al. A new antibiotic kills pathogens without detectable resistance. Nature 517, 455–459 (2015).
Kester, J. C. & Fortune, S. M. Persisters and beyond: mechanisms of phenotypic drug resistance and drug tolerance in bacteria. Crit. Rev. Biochem. Mol. Biol. 49, 91–101 (2014).
Brauner, A., Fridman, O., Gefen, O. & Balaban, N. Q. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat. Rev. Microbiol. 14, 320–330 (2016).
Balaban, N. Q. et al. Definitions and guidelines for research on antibiotic persistence. Nat. Rev. Microbiol. 17, 441–448 (2019).
Piddock, L. J. V. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 19, 382–402 (2006).
Li, X.-Z., Plésiat, P. & Nikaido, H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin. Microbiol. Rev. 28, 337–418 (2015).
Piddock, L. J. V. Multidrug-resistance efflux pumps – not just for resistance. Nat. Rev. Microbiol. 4, 629–636 (2006).
Martinez, J. L. et al. Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems. FEMS Microbiol. Rev. 33, 430–449 (2009).
Mousa, J. J. & Bruner, S. D. Structural and mechanistic diversity of multidrug transporters. Nat. Prod. Rep. 33, 1255–1267 (2016).
Du, D. et al. Multidrug efflux pumps: structure, function and regulation. Nat. Rev. Microbiol. 16, 523–539 (2018).
Anes, J., McCusker, M. P., Fanning, S. & Martins, M. The ins and outs of RND efflux pumps in Escherichia coli. Front. Microbiol. 6, 587 (2015).
Li, X.-Z. & Nikaido, H. in Efflux-Mediated Antimicrobial Resistance in Bacteria (eds Li, X.-Z., Elkins, C. A. & Zgurskaya, H. I.) 219–259 (Springer, 2016).
Ruiz, C. & Levy, S. B. Regulation of acrAB expression by cellular metabolites in Escherichia coli. J. Antimicrob. Chemother. 69, 390–399 (2014). This work identifies endogenous cellular metabolites that induce expression of a major multidrug efflux pump in E. coli.
Chubiz, L. M. & Rao, C. V. Aromatic acid metabolites of Escherichia coli K-12 can induce the marRAB operon. J. Bacteriol. 192, 4786–4789 (2010).
Hirakawa, H., Inazumi, Y., Masaki, T., Hirata, T. & Yamaguchi, A. Indole induces the expression of multidrug exporter genes in Escherichia coli. Mol. Microbiol. 55, 1113–1126 (2005).
Nishino, K., Honda, T. & Yamaguchi, A. Genome-wide analyses of Escherichia coli gene expression responsive to the BaeSR two-component regulatory system. J. Bacteriol. 187, 1763–1772 (2005).
Nikaido, E. et al. Effects of indole on drug resistance and virulence of Salmonella enterica serovar Typhimurium revealed by genome-wide analyses. Gut Pathog. 4, 5 (2012).
Nishino, K., Nikaido, E. & Yamaguchi, A. Regulation of multidrug efflux systems involved in multidrug and metal resistance of Salmonella enterica serovar Typhimurium. J. Bacteriol. 189, 9066–9075 (2007).
Lee, H. H., Molla, M. N., Cantor, C. R. & Collins, J. J. Bacterial charity work leads to population-wide resistance. Nature 467, 82–85 (2010). This work demonstrates that the production of indole by highly antibiotic-resistant mutants of E. coli increases the antibiotic tolerance and resistance of less-resistant strains, thus establishing a precedent for the role of a secondary metabolite in mediating the overall antibiotic susceptibility of a bacterial population.
Paździor, E., Pękala-Safińska, A. & Wasyl, D. Phenotypic diversity and potential virulence factors of the Shewanella putrefaciens group isolated from freshwater fish. J. Vet. Res. 63, 321–332 (2019).
Shyu, J. B. H., Lies, D. P. & Newman, D. K. Protective role of tolC in efflux of the electron shuttle anthraquinone-2,6-disulfonate. J. Bacteriol. 184, 1806–1810 (2002).
Sakhtah, H. et al. The Pseudomonas aeruginosa efflux pump MexGHI–OpmD transports a natural phenazine that controls gene expression and biofilm development. Proc. Natl Acad. Sci. USA 113, E3538–E3547 (2016).
Meirelles, L. A. & Newman, D. K. Both toxic and beneficial effects of pyocyanin contribute to the lifecycle of Pseudomonas aeruginosa. Mol. Microbiol. 110, 995–1010 (2018).
Meirelles, L. A., Perry, E. K., Bergkessel, M. & Newman, D. K. Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics. PLoS Biol. 19, e3001093 (2021). This work shows that a toxic secondary metabolite can increase tolerance to fluoroquinolones in strains of P. aeruginosa and other opportunistic pathogens, and can also promote the establishment of spontaneous antibiotic-resistant mutants in populations of these bacteria.
Lau, G. W., Hassett, D. J., Ran, H. & Kong, F. The role of pyocyanin in Pseudomonas aeruginosa infection. Trends Mol. Med. 10, 599–606 (2004).
Mavrodi, D. V., Blankenfeldt, W. & Thomashow, L. S. Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. Annu. Rev. Phytopathol. 44, 417–445 (2006).
Llanes, C. et al. Role of the MexEF-OprN efflux system in low-level resistance of Pseudomonas aeruginosa to ciprofloxacin. Antimicrob. Agents Chemother. 55, 5676–5684 (2011).
Richardot, C. et al. Amino acid substitutions account for most MexS alterations in clinical nfxC mutants of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 60, 2302–2310 (2016).
Lomovskaya, O. & Bostian, K. A. Practical applications and feasibility of efflux pump inhibitors in the clinic — a vision for applied use. Biochem. Pharmacol. 71, 910–918 (2006).
Jamshidi, S., Sutton, J. M. & Rahman, K. M. Computational study reveals the molecular mechanism of the interaction between the efflux inhibitor PAβN and the AdeB transporter from Acinetobacter baumannii. ACS Omega 2, 3002–3016 (2017).
Wolloscheck, D., Krishnamoorthy, G., Nguyen, J. & Zgurskaya, H. I. Kinetic control of quorum sensing in Pseudomonas aeruginosa by multidrug efflux pumps. ACS Infect. Dis. 4, 185–195 (2018).
Schiessl, K. T. et al. Phenazine production promotes antibiotic tolerance and metabolic heterogeneity in Pseudomonas aeruginosa biofilms. Nat. Commun. 10, 762 (2019). This study reveals that phenazine production alters both the metabolic profile of biofilms and their tolerance to different classes of clinical antibiotics, suggesting that beyond induction of specific cellular defences, secondary metabolites can also affect antibiotic susceptibility via indirect mechanisms.
Zhu, K., Chen, S., Sysoeva, T. A. & You, L. Universal antibiotic tolerance arising from antibiotic-triggered accumulation of pyocyanin in Pseudomonas aeruginosa. PLoS Biol. 17, e3000573 (2019). In this work, the authors report that sublethal antibiotic treatment can trigger PYO production in P. aeruginosa, and that PYO enables multiple bacterial species to grow to higher cell densities in the presence of diverse clinical antibiotics.
Lister, P. D., Wolter, D. J. & Hanson, N. D. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin. Microbiol. Rev. 22, 582–610 (2009).
Yonehara, R., Yamashita, E. & Nakagawa, A. Crystal structures of OprN and OprJ, outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa. Proteins 84, 759–769 (2016).
Glavier, M. et al. Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA–OprM chaperone-like complex. Nat. Commun. 11, 4948 (2020).
Clarke-Pearson, M. F. & Brady, S. F. Paerucumarin, a new metabolite produced by the pvc gene cluster from Pseudomonas aeruginosa. J. Bacteriol. 190, 6927–6930 (2008).
Iftikhar, A. et al. Mutation in pvcABCD operon of Pseudomonas aeruginosa modulates MexEF–OprN efflux system and hence resistance to chloramphenicol and ciprofloxacin. Microb. Pathog. 149, 104491 (2020).
Sokol, P. A., Lewis, C. J. & Dennis, J. J. Isolation of a novel siderophore from Pseudomonas cepacia. J. Med. Microbiol. 36, 184–189 (1992).
Darling, P., Chan, M., Cox, A. D. & Sokol, P. A. Siderophore production by cystic fibrosis isolates of Burkholderia cepacia. Infect. Immun. 66, 874–877 (1998).
Visca, P., Ciervo, A., Sanfilippo, V. & Orsi, N. Iron-regulated salicylate synthesis by Pseudomonas spp. J. Gen. Microbiol. 139, 1995–2001 (1993).
Bakker, P. A. H. M., Ran, L. & Mercado-Blanco, J. Rhizobacterial salicylate production provokes headaches! Plant. Soil. 382, 1–16 (2014).
Nair, B. M., Cheung, K.-J., Griffith, A. & Burns, J. L. Salicylate induces an antibiotic efflux pump in Burkholderia cepacia complex genomovar III (B. cenocepacia). J. Clin. Invest. 113, 464–473 (2004).
Cohen, S. P., Levy, S. B., Foulds, J. & Rosner, J. L. Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway. J. Bacteriol. 175, 7856–7862 (1993).
Brochado, A. R. et al. Species-specific activity of antibacterial drug combinations. Nature 559, 259–263 (2018).
Burkhead, K. D., Schisler, D. A. & Slininger, P. J. Pyrrolnitrin production by biological control agent Pseudomonas cepacia B37w in culture and in colonized wounds of potatoes. Appl. Environ. Microbiol. 60, 2031–2039 (1994).
Jeong, Y. et al. Toxoflavin produced by Burkholderia glumae causing rice grain rot is responsible for inducing bacterial wilt in many field crops. Plant. Dis. 87, 890–895 (2003).
Depoorter, E. et al. Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers. Appl. Microbiol. Biotechnol. 100, 5215–5229 (2016). This review catalogues the toxic secondary metabolites known to be produced by Burkholderia species and describes what is known about their regulation, thus serving as a useful resource for identifying endogenous compounds that might affect antibiotic susceptibility in this family of opportunistic pathogens.
Depoorter, E., De Canck, E., Coenye, T. & Vandamme, P. Burkholderia bacteria produce multiple potentially novel molecules that inhibit carbapenem-resistant Gram-negative bacterial pathogens. Antibiotics (Basel) 10, 147 (2021).
Lipuma, J. J. The changing microbial epidemiology in cystic fibrosis. Clin. Microbiol. Rev. 23, 299–323 (2010).
Jones, C. et al. Kill and cure: genomic phylogeny and bioactivity of Burkholderia gladioli bacteria capable of pathogenic and beneficial lifestyles. Microb. Genom. 7, mgen.0.000515 (2021).
Stern, K. G. Oxidation–reduction potentials of toxoflavin. Biochem. J. 29, 500–508 (1935).
Latuasan, H. E. & Berends, W. On the origin of the toxicity of toxoflavin. Biochim. Biophys. Acta 52, 502–508 (1961).
Gencheva, R., Cheng, Q. & Arnér, E. S. J. Efficient selenocysteine-dependent reduction of toxoflavin by mammalian thioredoxin reductase. Biochim. Biophys. Acta Gen. Subj. 1862, 2511–2517 (2018).
Li, X., Li, Y., Wang, R., Wang, Q. & Lu, L. Toxoflavin produced by Burkholderia gladioli from Lycoris aurea is a new broad-spectrum fungicide. Appl. Environ. Microbiol. 85, e00106-19 (2019).
Kim, J. et al. Quorum sensing and the LysR-type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae. Mol. Microbiol. 54, 921–934 (2004).
Tahlan, K. et al. Initiation of actinorhodin export in Streptomyces coelicolor. Mol. Microbiol. 63, 951–961 (2007).
Willems, A. R. et al. Crystal structures of the Streptomyces coelicolor TetR-like protein ActR alone and in complex with actinorhodin or the actinorhodin biosynthetic precursor (S)-DNPA. J. Mol. Biol. 376, 1377–1387 (2008).
Kohanski, M. A., Dwyer, D. J., Hayete, B., Lawrence, C. A. & Collins, J. J. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130, 797–810 (2007).
Keren, I., Wu, Y., Inocencio, J., Mulcahy, L. R. & Lewis, K. Killing by bactericidal antibiotics does not depend on reactive oxygen species. Science 339, 1213–1216 (2013).
Liu, Y. & Imlay, J. A. Cell death from antibiotics without the involvement of reactive oxygen species. Science 339, 1210–1213 (2013).
Dwyer, D. J., Collins, J. J. & Walker, G. C. Unraveling the physiological complexities of antibiotic lethality. Annu. Rev. Pharmacol. Toxicol. 55, 313–332 (2015). This work comprehensively discusses the evidence that oxidative stress contributes to antibiotic lethality.
Zuccato, E. et al. Role of bile acids and metabolic activity of colonic bacteria in increased risk of colon cancer after cholecystectomy. Dig. Dis. Sci. 38, 514–519 (1993).
Karlin, D. A., Mastromarino, A. J., Jones, R. D., Stroehlein, J. R. & Lorentz, O. Fecal skatole and indole and breath methane and hydrogen in patients with large bowel polyps or cancer. J. Cancer Res. Clin. Oncol. 109, 135–141 (1985).
Vega, N. M., Allison, K. R., Khalil, A. S. & Collins, J. J. Signaling-mediated bacterial persister formation. Nat. Chem. Biol. 8, 431–433 (2012).
Shen, X., Lind, J. & Merenyi, G. One-electron oxidation of indoles and acid-base properties of the indolyl radicals. J. Phys. Chem. 91, 4403–4406 (1987).
Garbe, T. R., Kobayashi, M. & Yukawa, H. Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity. Arch. Microbiol. 173, 78–82 (2000).
Perry, E. K. & Newman, D. K. The transcription factors ActR and SoxR differentially affect the phenazine tolerance of Agrobacterium tumefaciens. Mol. Microbiol. 112, 199–218 (2019).
Voggu, L. et al. Microevolution of cytochrome bd oxidase in staphylococci and its implication in resistance to respiratory toxins released by Pseudomonas. J. Bacteriol. 188, 8079–8086 (2006).
Hassett, D. J., Charniga, L., Bean, K., Ohman, D. E. & Cohen, M. S. Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase. Infect. Immun. 60, 328–336 (1992).
Priuska, E. M. & Schacht, J. Formation of free radicals by gentamicin and iron and evidence for an iron/gentamicin complex. Biochem. Pharmacol. 50, 1749–1752 (1995).
Prayle, A., Watson, A., Fortnum, H. & Smyth, A. Side effects of aminoglycosides on the kidney, ear and balance in cystic fibrosis. Thorax 65, 654–658 (2010).
Mosel, M., Li, L., Drlica, K. & Zhao, X. Superoxide-mediated protection of Escherichia coli from antimicrobials. Antimicrob. Agents Chemother. 57, 5755–5759 (2013).
Alexander, H. K. & MacLean, R. C. Stochastic bacterial population dynamics restrict the establishment of antibiotic resistance from single cells. Proc. Natl Acad. Sci. USA 117, 19455–19464 (2020).
Dwyer, D. J. et al. Antibiotics induce redox-related physiological alterations as part of their lethality. Proc. Natl Acad. Sci. USA 111, E2100–E2109 (2014).
Belenky, P. et al. Bactericidal antibiotics induce toxic metabolic perturbations that lead to cellular damage. Cell Rep. 13, 968–980 (2015).
Saini, V. et al. Ergothioneine maintains redox and bioenergetic homeostasis essential for drug susceptibility and virulence of Mycobacterium tuberculosis. Cell Rep. 14, 572–585 (2016). This work indicates that a metabolite involved in redox homeostasis has a key role in mediating antibiotic resistance and tolerance in M. tuberculosis, suggesting that such metabolites might also contribute to these phenotypes in other bacteria.
Hall, J. W., Yang, J., Guo, H. & Ji, Y. The Staphylococcus aureus AirSR two-component system mediates reactive oxygen species resistance via transcriptional regulation of staphyloxanthin production. Infect. Immun. 85, e00838-16 (2017).
Clauditz, A., Resch, A., Wieland, K.-P., Peschel, A. & Götz, F. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infect. Immun. 74, 4950–4953 (2006).
Liu, G. Y. et al. Sword and shield: linked group B streptococcal beta-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense. Proc. Natl Acad. Sci. USA 101, 14491–14496 (2004).
Wu, C. et al. Genomic island TnSmu2 of Streptococcus mutans harbors a nonribosomal peptide synthetase-polyketide synthase gene cluster responsible for the biosynthesis of pigments involved in oxygen and H2O2 tolerance. Appl. Environ. Microbiol. 76, 5815–5826 (2010).
Edge, R. & Truscott, T. G. Singlet oxygen and free radical reactions of retinoids and carotenoids–a review. Antioxidants (Basel) 7, 5 (2018).
Young, A. J. & Lowe, G. L. Carotenoids–antioxidant properties. Antioxidants (Basel) 7, 28 (2018).
Hong, Y., Zeng, J., Wang, X., Drlica, K. & Zhao, X. Post-stress bacterial cell death mediated by reactive oxygen species. Proc. Natl Acad. Sci. USA 116, 10064–10071 (2019).
Shatalin, K., Shatalina, E., Mironov, A. & Nudler, E. H2S: a universal defense against antibiotics in bacteria. Science 334, 986–990 (2011).
Tkachenko, A. G. & Fedotova, M. V. Dependence of protective functions of Escherichia coli polyamines on strength of stress caused by superoxide radicals. Biochemistry. 72, 109–116 (2007).
El-Halfawy, O. M. & Valvano, M. A. Putrescine reduces antibiotic-induced oxidative stress as a mechanism of modulation of antibiotic resistance in Burkholderia cenocepacia. Antimicrob. Agents Chemother. 58, 4162–4171 (2014).
El-Halfawy, O. M. & Valvano, M. A. Chemical communication of antibiotic resistance by a highly resistant subpopulation of bacterial cells. PLoS ONE 8, e68874 (2013).
Tkachenko, A. G., Akhova, A. V., Shumkov, M. S. & Nesterova, L. Y. Polyamines reduce oxidative stress in Escherichia coli cells exposed to bactericidal antibiotics. Res. Microbiol. 163, 83–91 (2012).
Kreamer, N. N., Costa, F. & Newman, D. K. The ferrous iron-responsive BqsRS two-component system activates genes that promote cationic stress tolerance. mBio 6, e02549 (2015).
Häussler, S. & Becker, T. The pseudomonas quinolone signal (PQS) balances life and death in Pseudomonas aeruginosa populations. PLoS Pathog. 4, e1000166 (2008).
Bredenbruch, F., Geffers, R., Nimtz, M., Buer, J. & Häussler, S. The Pseudomonas aeruginosa quinolone signal (PQS) has an iron-chelating activity. Environ. Microbiol. 8, 1318–1329 (2006).
Nguyen, D. et al. Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science 334, 982–986 (2011).
Han, M.-L. et al. Comparative metabolomics and transcriptomics reveal multiple pathways associated with polymyxin killing in Pseudomonas aeruginosa. mSystems 4, e00149-18 (2019).
Sampson, T. R. et al. Rapid killing of Acinetobacter baumannii by polymyxins is mediated by a hydroxyl radical death pathway. Antimicrob. Agents Chemother. 56, 5642–5649 (2012).
Trimble, M. J., Mlynárčik, P., Kolář, M. & Hancock, R. E. W. Polymyxin: alternative mechanisms of action and resistance. Cold Spring Harb. Perspect. Med. 6, a025288 (2016).
Bottery, M. J., Pitchford, J. W. & Friman, V.-P. Ecology and evolution of antimicrobial resistance in bacterial communities. ISME J. 15, 939–948 (2021).
Welp, A. L. & Bomberger, J. M. Bacterial community interactions during chronic respiratory disease. Front. Cell Infect. Microbiol. 10, 213 (2020).
Vega, N. M., Allison, K. R., Samuels, A. N., Klempner, M. S. & Collins, J. J. Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance. Proc. Natl Acad. Sci. USA 110, 14420–14425 (2013). This work reveals how a secondary metabolite that induces antibiotic tolerance can work across bacterial species.
Kim, J., Shin, B., Park, C. & Park, W. Indole-induced activities of β-lactamase and efflux pump confer ampicillin resistance in Pseudomonas putida KT2440. Front. Microbiol. 8, 433 (2017).
Bhargava, N., Sharma, P. & Capalash, N. Pyocyanin stimulates quorum sensing-mediated tolerance to oxidative stress and increases persister cell populations in Acinetobacter baumannii. Infect. Immun. 82, 3417–3425 (2014).
Heindorf, M., Kadari, M., Heider, C., Skiebe, E. & Wilharm, G. Impact of Acinetobacter baumannii superoxide dismutase on motility, virulence, oxidative stress resistance and susceptibility to antibiotics. PLoS ONE 9, e101033 (2014).
Bandara, H. M. H. N. et al. Fluconazole resistance in Candida albicans is induced by Pseudomonas aeruginosa quorum sensing. Sci. Rep. 10, 7769 (2020).
Chmiel, J. F. et al. Antibiotic management of lung infections in cystic fibrosis. I. The microbiome, methicillin-resistant Staphylococcus aureus, Gram-negative bacteria, and multiple infections. Ann. Am. Thorac. Soc. 11, 1120–1129 (2014).
Schwab, U. et al. Localization of Burkholderia cepacia complex bacteria in cystic fibrosis lungs and interactions with Pseudomonas aeruginosa in hypoxic mucus. Infect. Immun. 82, 4729–4745 (2014).
Wood, K. B. & Cluzel, P. Trade-offs between drug toxicity and benefit in the multi-antibiotic resistance system underlie optimal growth of E. coli. BMC Syst. Biol. 6, 48 (2012).
Yung, D. B. Y., Sircombe, K. J. & Pletzer, D. Friends or enemies? The complicated relationship between Pseudomonas aeruginosa and Staphylococcus aureus. Mol. Microbiol. 116, 1–15 (2021).
Camus, L., Briaud, P., Vandenesch, F. & Moreau, K. How bacterial adaptation to cystic fibrosis environment shapes interactions between Pseudomonas aeruginosa and Staphylococcus aureus. Front. Microbiol. 12, 617784 (2021).
Briaud, P. et al. Impact of coexistence phenotype between Staphylococcus aureus and Pseudomonas aeruginosa isolates on clinical outcomes among cystic fibrosis patients. Front. Cell Infect. Microbiol. 10, 266 (2020).
Radlinski, L. et al. Pseudomonas aeruginosa exoproducts determine antibiotic efficacy against Staphylococcus aureus. PLoS Biol. 15, e2003981 (2017). In this work, the authors screen clinical isolates of P. aeruginosa for effects on the antibiotic susceptibility of S. aureus and find that the interactions are strain specific and complex, highlighting the challenges of understanding how secondary metabolites affect polymicrobial infections.
Noto, M. J., Burns, W. J., Beavers, W. N. & Skaar, E. P. Mechanisms of pyocyanin toxicity and genetic determinants of resistance in Staphylococcus aureus. J. Bacteriol. 199, e00221-17 (2017).
Orazi, G., Ruoff, K. L. & O’Toole, G. A. Pseudomonas aeruginosa increases the sensitivity of biofilm-grown Staphylococcus aureus to membrane-targeting antiseptics and antibiotics. mBio 10, e01501-19 (2019).
Orazi, G. & O’Toole, G. A. Pseudomonas aeruginosa alters Staphylococcus aureus sensitivity to vancomycin in a biofilm model of cystic fibrosis infection. mBio 8, e00873-17 (2017).
Biswas, L., Biswas, R., Schlag, M., Bertram, R. & Götz, F. Small-colony variant selection as a survival strategy for Staphylococcus aureus in the presence of Pseudomonas aeruginosa. Appl. Environ. Microbiol. 75, 6910–6912 (2009).
McNamara, P. J. & Proctor, R. A. Staphylococcus aureus small colony variants, electron transport and persistent infections. Int. J. Antimicrob. Agents 14, 117–122 (2000).
Wilson, R. et al. Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium. Infect. Immun. 56, 2515–2517 (1988).
Cruickshank, C. N. & Lowbury, E. J. The effect of pyocyanin on human skin cells and leucocytes. Br. J. Exp. Pathol. 34, 583–587 (1953).
Levin-Reisman, I., Brauner, A., Ronin, I. & Balaban, N. Q. Epistasis between antibiotic tolerance, persistence, and resistance mutations. Proc. Natl Acad. Sci. USA 116, 14734–14739 (2019).
Santi, I., Manfredi, P., Maffei, E., Egli, A. & Jenal, U. Evolution of antibiotic tolerance shapes resistance development in chronic Pseudomonas aeruginosa infections. mBio 12, e03482-20 (2021). In this work, the authors analyse the microevolution of P. aeruginosa within patients with chronic infection and find that antibiotic tolerance promotes the evolution of antibiotic resistance, validating earlier in vitro studies that showed a link between tolerance and resistance.
Windels, E. M. et al. Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates. ISME J. 13, 1239–1251 (2019).
Levin-Reisman, I. et al. Antibiotic tolerance facilitates the evolution of resistance. Science 355, 826–830 (2017).
Liu, J., Gefen, O., Ronin, I., Bar-Meir, M. & Balaban, N. Q. Effect of tolerance on the evolution of antibiotic resistance under drug combinations. Science 367, 200–204 (2020).
Martina, P. et al. Hypermutation in Burkholderia cepacia complex is mediated by DNA mismatch repair inactivation and is highly prevalent in cystic fibrosis chronic respiratory infection. Int. J. Med. Microbiol. 304, 1182–1191 (2014).
Ryan, R. P. et al. The versatility and adaptation of bacteria from the genus Stenotrophomonas. Nat. Rev. Microbiol. 7, 514–525 (2009).
Walterson, A. M. & Stavrinides, J. Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiol. Rev. 39, 968–984 (2015).
Smith, D. D. N., Kirzinger, M. W. B. & Stavrinides, J. Draft genome sequence of the antibiotic-producing cystic fibrosis isolate Pantoea agglomerans Tx10. Genome Announc. 1, e00904-13 (2013).
König, S., Vogel, H.-J., Harms, H. & Worrich, A. Physical, chemical and biological effects on soil bacterial dynamics in microscale models. Front. Ecol. Evol. 8, 53 (2020).
Severi, E. & Thomas, G. H. Antibiotic export: transporters involved in the final step of natural product production. Microbiology 165, 805–818 (2019).
Martín, J. F., Casqueiro, J. & Liras, P. Secretion systems for secondary metabolites: how producer cells send out messages of intercellular communication. Curr. Opin. Microbiol. 8, 282–293 (2005).
Crits-Christoph, A., Bhattacharya, N., Olm, M. R., Song, Y. S. & Banfield, J. F. Transporter genes in biosynthetic gene clusters predict metabolite characteristics and siderophore activity. Genome Res. 31, 239–250 (2020).
Glasser, N. R., Saunders, S. H. & Newman, D. K. The colorful world of extracellular electron shuttles. Annu. Rev. Microbiol. 71, 731–751 (2017).
Yan, J. & Bassler, B. L. Surviving as a community: antibiotic tolerance and persistence in bacterial biofilms. Cell Host Microbe 26, 15–21 (2019).
Ersoy, S. C. et al. Correcting a fundamental flaw in the paradigm for antimicrobial susceptibility testing. EBioMedicine 20, 173–181 (2017).
Song, Y. et al. Inhibition of staphyloxanthin virulence factor biosynthesis in Staphylococcus aureus: in vitro, in vivo, and crystallographic results. J. Med. Chem. 52, 3869–3880 (2009).
Liu, C.-I. et al. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence. Science 319, 1391–1394 (2008).
Costa, K. C., Glasser, N. R., Conway, S. J. & Newman, D. K. Pyocyanin degradation by a tautomerizing demethylase inhibits Pseudomonas aeruginosa biofilms. Science 355, 170–173 (2017).
VanDrisse, C. M., Lipsh-Sokolik, R., Khersonsky, O., Fleishman, S. J. & Newman, D. K. Computationally designed pyocyanin demethylase acts synergistically with tobramycin to kill recalcitrant Pseudomonas aeruginosa biofilms. Proc. Natl Acad. Sci. USA 118, e2022012118 (2021). This works reveals that degradation of a secondary metabolite that promotes antibiotic tolerance in biofilms can increase antibiotic lethality, suggesting that targeting such secondary metabolites may be a viable approach to potentiating exisiting clinical antibiotics.
Liu, G. Y. & Nizet, V. Color me bad: microbial pigments as virulence factors. Trends Microbiol. 17, 406–413 (2009).
Shatalin, K. et al. Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance. Science 372, 1169–1175 (2021). In this work, the authors demonstrate that antibiotic efficacy is increased both in vitro and in a mouse infection model by inhibiting the production of a bacterial metabolite previously implicated in intrinsic antibiotic tolerance and resistance.
Abbott, I. J. & Peleg, A. Y. Stenotrophomonas, Achromobacter, and nonmelioid Burkholderia species: antimicrobial resistance and therapeutic strategies. Semin. Respir. Crit. Care Med. 36, 99–110 (2015).
Hazan, R. et al. Auto poisoning of the respiratory chain by a quorum-sensing-regulated molecule favors biofilm formation and antibiotic tolerance. Curr. Biol. 26, 195–206 (2016).
Shukla, P. et al. “On demand” redox buffering by H2S contributes to antibiotic resistance revealed by a bacteria-specific H2S donor. Chem. Sci. 8, 4967–4972 (2017).
Anderson, R. J. & Newman, M. S. The chemistry of the lipids of tubercle bacilli. J. Bio. Chem. 103, 405–412 (1933).
Gardner, P. R. Superoxide production by the mycobacterial and pseudomonad quinoid pigments phthiocol and pyocyanine in human lung cells. Arch. Biochem. Biophys. 333, 267–274 (1996).
Giddens, S. R., Feng, Y. & Mahanty, H. K. Characterization of a novel phenazine antibiotic gene cluster in Erwinia herbicola Eh1087. Mol. Microbiol. 45, 769–783 (2002).
Giddens, S. R. & Bean, D. C. Investigations into the in vitro antimicrobial activity and mode of action of the phenazine antibiotic d-alanylgriseoluteic acid. Int. J. Antimicrob. Agents 29, 93–97 (2007).
Krishnamurthi, V. S., Buckley, P. J. & Duerre, J. A. Pigment formation from l-tryptophan by a particulate fraction from an Achromobacter species. Arch. Biochem. Biophys. 130, 636–645 (1969).
Wells, J. M., Cole, R. J. & Kirksey, J. W. Emodin, a toxic metabolite of Aspergillus wentii isolated from weevil-damaged chestnuts. Appl. Microbiol. 30, 26–28 (1975).
Lim, F. Y. et al. Genome-based cluster deletion reveals an endocrocin biosynthetic pathway in Aspergillus fumigatus. Appl. Environ. Microbiol. 78, 4117–4125 (2012).
Imlay, J. A. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat. Rev. Microbiol. 11, 443–454 (2013).
Dietrich, L. E. P., Teal, T. K., Price-Whelan, A. & Newman, D. K. Redox-active antibiotics control gene expression and community behavior in divergent bacteria. Science 321, 1203–1206 (2008).
Gu, M. & Imlay, J. A. The SoxRS response of Escherichia coli is directly activated by redox-cycling drugs rather than by superoxide. Mol. Microbiol. 79, 1136–1150 (2011).
Singh, A. K., Shin, J.-H., Lee, K.-L., Imlay, J. A. & Roe, J.-H. Comparative study of SoxR activation by redox-active compounds. Mol. Microbiol. 90, 983–996 (2013).
Tanabe, M. et al. The multidrug resistance efflux complex, EmrAB from Escherichia coli forms a dimer in vitro. Biochem. Biophys. Res. Commun. 380, 338–342 (2009).
Lu, S. & Zgurskaya, H. I. Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter. Mol. Microbiol. 86, 1132–1143 (2012).
Wei, Q. et al. Global regulation of gene expression by OxyR in an important human opportunistic pathogen. Nucleic Acids Res. 40, 4320–4333 (2012).
Ochsner, U. A., Vasil, M. L., Alsabbagh, E., Parvatiyar, K. & Hassett, D. J. Role of the Pseudomonas aeruginosa oxyR–recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB–ankB, ahpB, and ahpC–ahpF. J. Bacteriol. 182, 4533–4544 (2000).
Romsang, A., Dubbs, J. M. & Mongkolsuk, S. in Stress and Environmental Regulation Of Gene Expression and Adaptation in Bacteria (ed. de Bruijn, F. J.) 1090–1102 (John Wiley & Sons, 2016).
Blin, K. et al. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 47, W81–W87 (2019).
Liu, J. et al. Metabolic co-dependence gives rise to collective oscillations within biofilms. Nature 523, 550–554 (2015).
Liu, J. et al. Coupling between distant biofilms and emergence of nutrient time-sharing. Science 356, 638–642 (2017).
Spero, M. A. & Newman, D. K. Chlorate specifically targets oxidant-starved, antibiotic-tolerant populations of Pseudomonas aeruginosa biofilms. mBio 9, e01400-18 (2018).
Saunders, S. H. et al. Extracellular DNA promotes efficient extracellular electron transfer by pyocyanin in Pseudomonas aeruginosa biofilms. Cell 182, 919–932.e19 (2020).
Rosche, W. A. & Foster, P. L. Determining mutation rates in bacterial populations. Methods 20, 4–17 (2000).
Zheng, Q. A new practical guide to the Luria–Delbrück protocol. Mutat. Res. 781, 7–13 (2015).
Somayaji, R. et al. Antimicrobial susceptibility testing (AST) and associated clinical outcomes in individuals with cystic fibrosis: a systematic review. J. Cyst. Fibros. 18, 236–243 (2019).
Hurley, M. N., Ariff, A. H. A., Bertenshaw, C., Bhatt, J. & Smyth, A. R. Results of antibiotic susceptibility testing do not influence clinical outcome in children with cystic fibrosis. J. Cyst. Fibros. 11, 288–292 (2012).
Jorgensen, J. H. & Ferraro, M. J. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin. Infect. Dis. 49, 1749–1755 (2009).
Brown, M. R. Nutrient depletion and antibiotic susceptibility. J. Antimicrob. Chemother. 3, 198–201 (1977).
Work in the corresponding author’s laboratory was supported by grants to D.K.N. from the NIH (1R01AI127850-01A1, 1R01HL152190-01) and the Doren Family Foundation. E.K.P. was supported by a National Science Foundation Graduate Research Fellowship under Grant No. DGE-1745301.
The authors declare no competing interests.
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The ability to survive transient antibiotic exposure.
The ability to grow in the presence of antibiotics at a given concentration.
- Antibiotic resilience
The ability of a bacterial population to be refractory to antibiotic treatment via tolerance and/or resistance.
- Efflux pumps
Membrane-associated transport proteins that are responsible for the extrusion of various compounds out of the cell.
A subpopulation of bacteria that is killed by a given antibiotic at a much slower rate than the rest of the population, in a manner that is non-heritable.
- Antioxidant activity
The ability to neutralize highly reactive free radicals.
- Pro-oxidant activity
The ability to induce oxidative stress.
- Polymicrobial infection
An infection that is caused by more than one species of microorganism.
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Perry, E.K., Meirelles, L.A. & Newman, D.K. From the soil to the clinic: the impact of microbial secondary metabolites on antibiotic tolerance and resistance. Nat Rev Microbiol (2021). https://doi.org/10.1038/s41579-021-00620-w