Persister Cells Andauthor Antimicrobial Toleranceby

Bucharest, 20-22 Feb 2019

Persister cells andauthor antimicrobial toleranceby

Anca Streinu-eLibraryCercel, MD, PhD Assoc. Prof. Carol Davila University of Medicine and Pharmacy, Bucharest, Romania National Institute for Infectious Diseases “Prof. Dr. Matei Balș”, Bucharest, Romania ESCMID © Disclosure slide for speaker at EUCIC Local module for Infection Prevention and Control

Disclosure of speaker’s interests author (Potential) conflict of interest None to declare by Potentially relevant company relationships in connection with event 1 None to declare

• 2 Sponsorship or research fundingeLibrary

• Fee or other (financial) payment3 None to declare • Shareholder4 5 • Other relationship,ESCMID i.e. … © Persister cells and antimicrobial tolerance

• Bacterial populations produce persister cells that neither grow nor die in the presence of bactericidal agents, and thus exhibitauthor multidrug tolerance (MDT).1 by

• The mechanism of MDT and the nature of persisters, which were discovered in 1944, have remained elusive1

• Scientific literature presents persisterseLibraryas largely responsible for the recalcitrance of infections caused by bacterial biofilms.

• The majority of infections in the developed world are caused by biofilms, which sparked a renewed interest in persisters1 ESCMID

1Lewis K. Biochemistry (Mosc©). 2005;70:267 -74. 2Roberts ME, et al. Microbiology. 2005 Jan;151(Pt 1):75-80. Persister cells and antimicrobial tolerance

author •Although we use the term persister, an alternative and possibly synonymous term is phenotypicby variant.

•Phenotypic variants are cells that have spontaneously switched from the wild-type state into a variant state in which the cell exhibits alteredeLibrary phenotypic properties.

•These properties may include enhanced antimicrobial tolerance (Drenkard & Ausubel, 2002; Balaban et al., 2004). ESCMID © Roberts ME, et al. Microbiology. 2005 Jan;151(Pt 1):75-80. author by

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life cycle of biofilms (open source: MicrobeWiki) Persister cells

• A method to isolate persister cells was developed (Lewisauthor K1); a gene expression profile of Escherichia coli persisters was obtained. • The profile indicated an elevated expression ofby toxin -antitoxin modules and other genes that can block important cellular functions such as translation. • Bactericidal antibiotics kill cells by corrupting the target function (i.e., translation). • aminoglycosides interrupt translation,eLibrary producing toxic peptides. • Inhibition of translation leads to a shutdown of other cellular functions as well, preventing antibiotics from corrupting their targets, which will give rise to tolerant persister cells. ESCMID Ashby MJ et al. J Antimicrob©Chemother. 33:443–452. Balaban NQ, et al. Science 305:1622-5. Persister cells

• Overproduction of chromosomally-encoded "toxins"author such as RelE, an inhibitor of translation, or HipA, causes a rapid increase in persisters. • Deletion of the hipBA module produces a sharpby decrease in persisters in both stationary and biofilm cells.

• HipA is the first validated persister/MDT gene. • The function of "toxins" is the exacteLibrary opposite of the term, namely, to protect the cell from lethal damage. • It appears that stochastic fluctuations in the levels of MDT proteins lead to formation of rare persister cells. • Persisters are essentially altruistic cells that forfeit propagation in order to ensure survivalESCMID of kin cells in the presence of lethal factors. Ashby MJ et al. J Antimicrob©Chemother. 33:443–452. Balaban NQ, et al. Science 305:1622-5. Bacterial populations author • Produce a small number of dormant persister cells that exhibit multidrug tolerance. by • All resistance mechanisms do essentially the same thing: prevent the antibiotic from hitting a target.

• By contrast, tolerance apparentlyeLibrary works by shutting down the targets. • Bactericidal antibiotics kill bacteria by corrupting their targets, rather than merely inhibiting them.

• Shutting down the targets then protects from killing. ESCMID © Lewis K. Curr Top Microbiol Immunol. 2008;322:107-31. Biofilms

• The number of persisters in a growing population of bacteria rises at mid- log and reaches a maximum of approximately 1% at stationaryauthor state. • Slow-growing biofilms produce substantial numbers of persisters. • A biofilm has the ability to: by • limit access of the immune system components • sustain an antibiotic attack (persisters)

• And is responsible for the recalcitrance of the infections in vivo and for their relapsing nature. eLibrary

• Isolation of Escherichia coli persisters by lysing a population or by sorting GFP- expressing cells with diminished translation allowed to obtain a gene expression profile. • The profile indicated downregulated biosynthetic pathways (i.e., dormant nature), and overexpressionESCMID of toxin/antitoxin (TA) modules. © Lewis K. Curr Top Microbiol Immunol. 2008;322:107-31. Translation – persister formation

• Stochastic overexpression of toxins that inhibit essentialauthor functions such as translation may contribute to persister formation.by • Ectopic expression of: RelE, MazF, and HipA toxins produced multidrug tolerant cells. • Also glpD and plsB were identified as potential persister genes by overexpression cloning of a genomic library and selection for antibiotic tolerance. eLibrary • Yeast Candida albicans forms recalcitrant biofilm infections that are tolerant to antifungals, similarly to bacterial biofilms. • C. albicans biofilms produce multidrug tolerant persisters that are not mutants, but rather phenotypic variants of the wild type. ESCMID Ashby MJ et al. J Antimicrob©Chemother. 33:443–452. Balaban NQ, et al. Science 305:1622-5. New treatment approaches author • Unlike bacterial persisters, C. albicans persisters were only observed in a biofilm, not in a planktonic stationary population.by

• Identification of persister genes opens the way to a new design of anti- biofilm therapy. • Combination of a conventional antibiotic with a compound inhibiting persister formation or maintenanceeLibrary may produce an effective therapeutic. • Other approaches to the problem include sterile-surface materials, prodrug antibiotics, and cyclic application of conventional antimicrobials ESCMID © Proctor RA, et al. Clin Infect Dis 1998;27,:419-22. SCVs • The presence of persister cells and small-colony variants (SCVs) has been associated with enhanced antibiotic resistance of many organisms in biofilms author • Singh R, et al. studied dose-dependent killing ofby biofilms and planktonic cells with five antibiotics: • oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin • Biofilms were treated with each antibiotic at a concentration of 0-100 microg/mL at 37 °C for 48 h. eLibrary • The killing of biofilm cells by all of the antibiotics showed the presence of persister cells – most cells in the population died, leaving a fraction that persisted, even at higher concentrations of the antibiotics. • These persisters represented a transient resistant phenotype and reverted to a killing curve resembling that of the wild-type parent upon re- exposure to the antibiotics.ESCMID Singh R, et al. J Med Microbiol.© 2009;58(Pt 8):1067-73. SCVs

• SCVs were observed in biofilms only after treatment withauthor ciprofloxacin, and these SCVs were of a transient nature. by • The treatment of planktonic cells with oxacillin, cefotaxime, ciprofloxacin and vancomycin killed the entire population and no persisters were detected. • Transient SCVs, observed in planktonic cells following exposure to these antibiotics, were killed at higher antibiotic concentrations. • The treatment of planktonic cells eLibrarywith amikacin yielded a small subpopulation of survivors that included persisters (at numbers significantly lower than for the biofilms) and highly resistant, stable SCVs with an increased biofilm-forming capacity in comparison with the wild-type parent. • Thus the high resistance of S. aureus biofilms to multiple unrelated antibiotics is largelyESCMID dependent on the presence of persister cells. Singh R, et al. J Med Microbiol.© 2009;58(Pt 8):1067-73. Biofilms author • In biofilms we identify a large number of persisters compared to planktonic cultures by • In planktonic cultures there are rarely persisters or they are found in a small number. • SCVs, although not specifically associated with S. aureus biofilms, have an increased biofilm-forming capacity and this may explain the frequent isolation of SCVs from biofilm-associatedeLibrary infections. • The intrinsic resistance of these variants may contribute to the enhanced antibiotic resistance of the biofilms formed.

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ESCMID © Bacteria

• Bacteria can transmit memory across generations soauthor their descendants will know how to attach themselves to surfaces and form biofilm communities by

• Researchers’ findings shed light on cystic fibrosis-related bacteria’s early steps in biofilm formation. • These pathogens cause persistent airway infections in people with cystic fibrosis. eLibrary • Multigenerational memory and adaptive adhesion in early bacterial biofilm communities

• Resistance mechanism: multifactorial • AB resistance is heritable • Biofilms are perfectESCMID to develop resistant subpopulations

Calvin K. et al. PNAS. 2018;115:4471© -6. Bacteria ……. author • The collective virulence of bacteria is increased by intercellular communication – quorum sensing by

• Many of the virulence factors are related to either: • direct tissue and cell destruction or • up regulation of the inflammatory response causing indirect tissue destruction eLibrary

• To conclude……..ESCMID © Antimicrobial tolerance Difference: Imipenem Subinhibitory concentrations of AB colistin Q-S no bacterial extra cellular DNA Metabolic rate -> low O2 level author by

planktonic 1 day 3 day 7 day Biofilm tolerance Colistin acts on the bacterial

membrane; effective at low O2 Imipenem eLibrary concentrations (the inner-part). colistin Aminoglycosides,

Antibiotic concentration fluorquinolones, betalactams kill the outer-part. ESCMID planktonic© 1 day 3 day 7 day Tolerance is different author by

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Anwar et al. J Antimicrob© Chemother 1989;24:647-55. Tackling antimicrobial tolerance author Quorum sensing mutants are more fragile than the wild type. More vulnerable to shear forces and phagocytosisby.

Phagocytosis Shear forces eLibrary Biofilm

ESCMID Antibiotics © author by

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The tolerance to antibiotics is part of the biofilm definition ESCMID © author Models for studying biofilmby and assessing anti biofilm activity

Anca StreinueLibrary-Cercel, MD, PhD Assoc. Prof. Carol Davila University of Medicine and Pharmacy, Bucharest, Romania National Institute for Infectious Diseases “Prof. Dr. Matei Balș”, Bucharest, Romania

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© https://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancac3/2017/ancac3.2017 de Anda J, et al. ACS Nano 2017;11:9340-51. .11.issue-9/acsnano.7b04738/20170920/pdfimages_v02/master.img-000.jpg • This experimental technique is capable of reconstructing 3D phenomena near a surface with 5 ms time resolution.1,2 As a test case, they used a type of “4D” computational microscopy to study flagellum-driven bacterial motility near surfaces. author • Two types of unanticipated flagellum-driven surfaceby motions:1,3,4 1. vertical-spinning bacteria with unanchored poles that exhibit diffusive motion on the surface 2. horizontal bacteria that are driven by the flagellum to execute purely superdiffusive motion on a surface. These fundamental flagellum-basedeLibrary motility mechanisms are reminiscent of various TFP-based movements (such as “walking” and “crawling”) that enforce diffusive or superdiffusive motility outcomes.2

ESCMID 1de Anda J, et al. ACS Nano 2017;11:9340-51. 2Conrad JC, et al. Biophys J. 2011;100:1608-16. 3Darnton NC, et al. J Bacteriol©. 2007;189:1756 -64. 4Marquet P, et al. Opt Lett. 2005;30:468-70. author •Start from patients: patient’s needs by •Infection: identify bacteria… •Persister cells •Biofilm eLibrary •Need help

ESCMID © CDC biofilm reactor author • BioSurfaceTechnologies’ Biofilm Reactors provide easy-to-sample biofilm growth systems by • Aids in the evaluation of antimicrobial agents, surface treatments, and biofilm processes • These systems allow multi-sample opportunities and repeatable data. eLibrary • ASTM Standard Methods are available for most biofilm reactors.

ESCMID © Azeredo J, et al. Crit Rev Microbiol. 2016;43:313-51. Microscopy flow cells author • Reusable and autoclavable devices designed for monitoring and evaluating biofilm processes using microscopyby and image analysis. • All flow cells are compatible with confocal, fluorescent, and reflected light microscopy.

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ESCMID © Azeredo J, et al. Crit Rev Microbiol. 2016;43:313-51. Test coupons and surfaces author • Many variations of test surfaces can be used for biofilm reactors and flow cells. by • These surfaces (coupons) come in more than 40 different materials across several different configurations.

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ESCMID © Azeredo J, et al. Crit Rev Microbiol. 2016;43:313-51. Continuous flow systems/flow chambers author • Biofilms grow on glass supplied with a continuous flow carbon content media by • Enable structural analysis on biofilm grow by CLSM

• Expensive and low reproducible rates; good for modelling urinary infections eLibrary

ESCMID © https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcTzLSU1mjXJgKXwlidyuN7LPYD6D3a0GN9yj4nYHsjDLO4vGqEh Silicon media

• Mimics multispecies biofilm growth author by

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ESCMID © https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcSdPcE7x4IvIahyRzXTLtaGMsdiKaj19tb-yXZ-lxgKKDK-RfNB Microtiter plate assay

• Shows a representative result for biofilm formation assays performedauthor for Pseudomonas aeruginosa, Pseudomonas fluorescens and Staphylococcus aureus. (A) side view of the well with a biofilm of P. aeruginosa (8 hrsby, 37 °C). (B) side view of the well with a biofilm of P. fluorescens (6 hrs, 30°C). (C) top-down view of the biofilm formed by S. aureus in a flat-bottom microtiter plate (two wells, 24 hrs, 37°C). P. aeruginosa and P. fluorescens are both motile organisms and form a biofilm at the air- liquid interface. S. aureus is non-motile andeLibrary forms a biofilm on the bottom of the well.

ESCMID https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/bin/jove© -47-2437.mov More linked to the clinical part author • Sepsis model: ex vivo whole blood sepsis model (Aarag et al, FEMS Immunology Med Microbiol 2011) by • Synthetic CF sputum media/artificial sputum media (Palmerv et al, J Bact 2007) • Nose: artificial nose secretion (Krismer et al, PLOS Pathogens 2014) • Chronic wound: artificial wound secretion (Kucera et al, J Microbiol Methods 2014, Werthrn et al, APMISeLibrary 2010)

ESCMID © Critical review on biofilm methods author • November 2016 Critical Reviews in Microbiology 43:313-51. • DOI: 10.1080/1040841X.2016.1208146 by

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ESCMID © To sum up author • Methods for in vitro biofilm analysis are available • All have advantages or disadvantages that needby to be considered • The inability to resemble in vivo infection condition • Future models to incorporate: • Growth media mimic infection patterns • Ways to study non-adherent eLibrarybacteria aggregates • Host immune response • … ESCMID © author Models for studying biofilmby and assessing anti biofilm activity