Introduction to Antibacterial Therapeutics (Antibiotics) MEDCH 561P

Introduction to Antibacterial Therapeutics (antibiotics) MEDCH 561P

Penicillium chrysogenum + Staph. Aureus

March 28, 2016 Kelly Lee, Ph.D. H-172J

[email protected] http://botit.botany.wisc.edu/toms_fungi/ nov2003.html MedChem561P

• Antibacterials: mechanisms of action and resistance • Cell-wall synthesis inhibitors: • Beta-lactams, vancomycin, etc. • Antibiotics for anaerobic infections • Protein synthesis inhibitors: • Macrolides, tetracyclines, aminoglycosides • Sulfa drugs, nitrofurantoin • New antibiotics and combination therapies • Antiviral drugs: influenza, HIV, herpesvirus, HCV • Therapeutic and clinical perspective from Prof. Doug Black: Fridays 12:30-1:30 MedChem561P

• Catalyst site where slides, notes, exam keys, etc will be posted: https://catalyst.uw.edu/workspace/kklee/53461/ ! • Course materials including schedule, syllabus, project/paper instructions, general course info can also be found at: http://courses.washington.edu/medch561/NEW/index.html Miracle drugs

Can take a potentially fatal condition and CURE an individual Much of modern medical care relies upon the ability to effectively treat microbial infections

Surgery Organ transplants Catheterization Joint replacement Cancer treatment AIDS Wounds Birth and delivery Chronic infections Much of modern medical care relies upon the ability to effectively treat microbial infections

• For example, hip and knee replacements (~800,000 per year in US): • Antibiotic prophylaxis is commonly used to prevent hospital acquired infections • Currently, infection rates ~0.5-2% • Without antibiotics, the likely postoperative infection rate would be 40-50%, and ~30% of those infections would likely be fatal

R. Smith, J. Coast, “The true cost of antimicrobial resistance”, BMJ 2013, vol 346 Impact on health care: big business

• In 2004, total global trade in antibiotics > $27 billion • Beta-lactams accounted for ~45% • 6 antibiotics topped $1 billion in sales: • ceftriaxone (beta-lactam) • amoxicillin/clavulanate (beta-lactam+beta-lactamase inhibitor) • azithromycin (macrolide) • clarithromycin (macrolide) • ciprofloxacin (fluoroquinolone) • levafloxacin (fluoroquinolone)

Taylor JB and Triggle DJ (eds), Comprehensive Medicinal Chemistry II, vol7! Walsh CT, Antibiotics: Actions, origins, resistance, (2003) ASM Press, Washington DC, USA! Coates A, et al., Nature Rev. Drug Discovery (2002) 1:895-910 Impact on health care

• Antibiotics make up a substantial fraction of prescriptions: • ~42% of patients admitted to hospitals receive antibiotics • ~50% of drug prescription costs go to antibiotics, where prescription costs amount to 10-15% of total health care costs ! ! • Places where use is most intense lead to greatest resistance. Most likely place for an individual to acquire an antibiotic-resistant infection is the ICU ! ! • Frequently prescribed unnecessarily. U. Michigan Health System study: In 2002, 41 million antibiotic prescriptions for people suffering from colds (viral), more than one-third of patients who saw a doc about a cold (Feb 24, 2003 edition of Archives of Internal Medicine) The problem we face Incidence of bacterial resistant isolates

New FDA-approved antibiotics New FDA-approved Infectious Disease 1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 2008-2011 Society of America Life in a pre-antimicrobial world

Tuberculosis (Mycobacterium tuberculosis) Typhus (Rickettsia prowazekii) Scarlet Fever (Streptococcus pyogenes) Pneumonia (Streptococcus pneumonia, Haemophilus influenzae) Whooping cough (Bordetella pertussis) Cholera (Vibrio cholerae) Diphtheria (Corynebacterium diphtheriae)

• Mortality due to infectious disease was ~20x higher in 1900 than now • Before antibiotics Streptococcus pyogenes responsible for ~50% of infant mortality • S. pyogenes also frequently caused death in infected burn wounds • Staphylococcus aureus fatal in nearly 80% of infected wounds • Prior to antibiotics, wound infection killed more soldiers than weapons in warfare 9th century medicine for a stye (typically caused by Staph. aureus)

Take cropleek and garlic, of both equal quantities, pound them well together, take wine and bullocks’ gall, of both equal quantities, mix with the leek, put this then into a brazen vessel, let it stand nine days in the brass vessel, wring out through a cloth and clear it well, put it into a horn, and about night time apply it with a feather to the eye; the best leechdom. Bald's Leechbook Old English, 9th century C.E. How to cure MRSA according to 9th-century! Downloaded from Anglo Saxon medical knowledge RESEARCH ARTICLE crossmark

A 1,000-Year-Old Antimicrobial Remedy with Antistaphylococcal mbio.asm.org Activity on September 3, 2015 - Published by Freya Harrison,a Aled E. L. Roberts,a Rebecca Gabrilska,b Kendra P. Rumbaugh,b Christina Lee,c Stephen P. Digglea Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, University Park, Nottingham, United Kingdoma; Department of Surgery, Texas Tech University Health Sciences Center, School of Medicine, Lubbock, Texas, USAb; School of English and Centre for the Study of the Viking Age, University of Nottingham, University Park, Nottingham, United Kingdomc

ABSTRACT Plant-derived compounds and other natural substances are a rich potential source of compounds that kill or attenu- ate pathogens that are resistant to current antibiotics. Medieval societies used a range of these natural substances to treat conditions clearly recognizable to the modern eye as microbial infections, and there has been much debate over the likely efficacy of these treatments. Our interdisciplinary team, comprising researchers from both sciences and humanities, identified and reconstructed a potential remedy for Staphylococcus aureus infection from a 10th century Anglo-Saxon leechbook. The remedy re- peatedly killed established S. aureus biofilms in an in vitro model of soft tissue infection and killed methicillin-resistant S. aureus (MRSA) in a mouse chronic wound model. While the remedy contained several ingredients that are individually known to mbio.asm.org have some antibacterial activity, full efficacy required the combined action of several ingredients, highlighting the scholarship of premodern doctors and the potential of ancient texts as a source of new antimicrobial agents. IMPORTANCE While the antibiotic potential of some materials used in historical medicine has been demonstrated, empirical tests of entire remedies are scarce. This is an important omission, because the efficacy of “ancientbiotics” could rely on the combined activity of their various ingredients. This would lead us to underestimate their efficacy and, by extension, the scholarship of premodern doctors. It could also help us to understand why some natural compounds that show antibacterial promise in the laboratory fail to yield positive results in clinical trials. We have reconstructed a 1,000-year-old remedy which kills the bacteria it was designed to treat and have shown that this activity relies on the combined activity of several antimicrobial ingredients. Our results highlight (i) the scholarship and rational methodology of premodern medicalHarrison professionals et al., mBio and (ii) (2015) the untapped 6(4):e01129-15 potential of premodern remedies for yielding novel therapeutics at a time when new antibiotics are desperately needed.

Received 7 July 2015 Accepted 14 July 2015 Published 11 August 2015 Citation Harrison F, Roberts AEL, Gabrilska R, Rumbaugh KP, Lee C, Diggle SP. 2015. A 1,000-year-old antimicrobial remedy with antistaphylococcal activity. mBio 6(4):e01129- 15. doi:10.1128/mBio.01129-15. Editor Dianne K. Newman, California Institute of Technology/HHMI Copyright © 2015 Harrison et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Freya Harrison, [email protected], or Stephen P. Diggle, [email protected].

ntibiotic resistance is a clear and present danger to human Bald’s Leechbook (7) (Materials and Methods) is an English Ahealth, and there are worryingly few new antibiotics in the medical text from the Anglo-Saxon period. The manuscript was developmental pipeline (1). A rich source of new antimicrobials written in the 10th century and contains remedies for various potentially resides in medieval and early modern medical texts: ailments, including clearly recognizable microbial infections. Me- microbial infection has been a constant presence throughout hu- dieval medicine has generally been dismissed as backwards or su- man history, and manuscript evidence shows that early modern perstitious, but recent scholars have suggested that among the and premodern societies used a range of natural compounds to remedies there may be methods and recipes that indicate a more treat conditions that are clearly recognizable as microbial infec- factual application (8–10). One of Bald’s remedies, a salve for a tions. Some substances used in premodern treatments for infec- “wen” or lump in the eye (Fig. 1), is particularly interesting to the tion (e.g., extracts from garlic [2–5]) show antimicrobial or modern microbiologist. It aims to treat a condition caused by virulence-reducing effects under certain conditions, but experi- bacterial infection and contains ingredients with the potential for ments that test the antibiotic activities of entire historical remedies antimicrobial activity. The recipe instructs the reader to crush are few and far between. This is an important omission, because garlic and a second Allium species (whose translation into modern the efﬁcacy of these “ancientbiotics” could rely on the combined English is ambiguous), combine these with wine and oxgall (bo- activity of their various ingredients. This would lead us to under- vine bile), and leave the mixture to stand in a brass or bronze vessel estimate their efﬁcacy and, by extension, the scholarship of pre- for 9 days and nights. modern doctors. It could also help to explain why the in vitro The “wen” is most likely a sty: an infection of an eyelash follicle. antibacterial activity of individual compounds (e.g., garlic [6]) Today, most styes are caused by the Gram-positive bacterium does not always reliably translate into in vivo antimicrobial poten- Staphylococcus aureus (11), which also causes other severe and tial. persistent infections of various tissues. The spread of methicillin-

July/August 2015 Volume 6 Issue 4 e01129-15 ® mbio.asm.org 1 Selective Toxicity

Antimicrobial: Microbial secondary metabolites or synthetic compounds that in small doses inhibit the growth and survival of microorganisms without serious toxicity to the host (us) ! Antibiotic: Natural product subset of antimicrobials We are >90% bacterial

For our 10 trillion cells, ~100 trillion bacterial cells make their home in and on us. For our 10 trillion cells, ~100 trillion1,000-5,000 bacterial species cells make their home in and on us. Targeting pathogenic bacteria with antibacterials will impact our normal flora. Normal ﬂora Ways commensal bacteria impact our health

Organs and internal tissues are normally sterile. Commensal bacteria do colonize “exterior” including skin, gut, respiratory tract, mouth, eyes, urogenital tract, etc.

• In many cases the flora-host interaction is mutualistic. Commensal bacteria provide: • Aid in digestion of food and production of vitamins, link to obesity • Processing of nutrients and drugs in our gut • Overall metabolite profile (metabolome) of host with natural bacterial flora is significantly different from those that are germ-free (tested in animal models) • Prevent establishment of pathogenic competitors • Immunity • Imbalance can impact asthma, atherosclerosis, allergies, obesity, cancer, … • Is the impact on flora transient or long-lasting? Some evidence suggests the affect can persist and lead to long-term health consequences. Growing appreciation of enterotypes! Native flora is specific to an individual, impact of treatment may differ significantly

“Enterotypes of the human gut microbiome,” Arumugam et al., Nature (2011) 472:7343

• Appears human microbiome of the gut may be categorizable into 3 main “enterotypes”, each dominated by a main genus • Not related to nation, ethnicity, gender, or age • May be linked to long-term diet • There may be a link between the enterotype found in an individual and susceptibility to disorders/diseases

© 2013 Nature America, Inc. All rights reserved. © 2013 Nature America, Inc. All rights reserved. (myocardial infarction, strokeordeath),butonlyamongsubjects withconcurrentlyhighTMAOlevels.Chronic dietary ing microbiota choline metabolism of and dietary ated during cooking ingestion, such salt content as high or heterocyclic compounds gener alternative disease-promoting exposures that accompany meat dietary CVDandbetween meat consumption has stimulated investigation of not enough sufficiently high to be account association for observed the picion that cholesterol the and saturated fat content of red meat may in mice.Omnivoroushumansubjectsproduced moreTMAOthandidvegansorvegetariansfollowingingestionof Received 7December2012;accepted27February2013;publishedonlineApril California, USA.CorrespondenceshouldbeaddressedtoS.L.H.( ( Cleveland, Ohio,USA. 8 1 cholesterol in meat CVD risk, presumably owing to large the content of saturated fats and levelThe of high meat consumption developed world in the is to linked microbiota ofdietary metabolized toaproatherogenicspecies,trimethylamine- Intestinal microbiotametabolismofcholine andphosphatidylcholineproducestrimethylamine(TMA),whichisfurther Koeth A Robert a nutrient inred meat, promotes atherosclerosis Intestinal microbiota metabolism of NATURE MEDICINE Earl BBrittEarl linked tolinked increased meat consumption are responsible and CVD, prompting suggestion the that other environmental exposures cohort studies showed saturated no association dietary between fat intake ance recently, complex phenotypes such disease as obesity and insulin resist immune function,bioactivation of nutrients and vitamins, and, more diet-host interaction with reference to red meat consumption. participationthe of commensal intestinal microbiota the inmodifying Philadelphia, Pennsylvania,USA. both plasmaTMAOconcentrationanddietary status.Plasma through amicrobiota-dependentmechanism. Thepresenceofspecificbacterialtaxainhumanfeceswasassociatedwith Cleveland, Ohio,USA. California, USA. Hongzhe Li Section onLipidSciences,Wake ForestSchoolofMedicine,Winston-Salem, NorthCarolina,USA. Intestinal microbiota may thuscontributetothewell-established linkbetweenhighlevelsofredmeatconsumption andCVDrisk. microbiota, dietarysupplementation withTMAOoreithercarnitine orcholinereduced atherosclerosis, butthis didnotoccurifintestinalmicrobiotawas concurrentlysuppressed.Inmicewithan intactintestinal supplementation inmice alteredcecalmicrobialcomposition,markedly enhancedsynthesisofTMAandTMAO, andincreased W HWilson Tang (myocardial infarction,strokeordeath),butonlyamongsubjectswithconcurrentlyhighTMAOlevels.Chronicdietary microbiota ofdietary ing microbiota choline metabolism of and dietary ated during cooking ingestion, such salt content as high or heterocyclic compounds gener alternative disease-promoting exposures that accompany meat dietary CVDandbetween meat consumption has stimulated investigation of not enough sufficiently high to be account association for observed the picion that cholesterol the and saturated fat content of red meat may in mice.OmnivoroushumansubjectsproducedmoreTMAOthandidvegansorvegetariansfollowingingestionof Received 7December2012;accepted27February2013;publishedonline 7 April2013; California, USA.CorrespondenceshouldbeaddressedtoS.L.H.( ( Cleveland, Ohio,USA. both plasmaTMAOconcentrationanddietarystatus.Plasma through amicrobiota-dependentmechanism.Thepresenceofspecificbacterialtaxainhumanfeceswasassociatedwith a nutrient inred meat, promotes atherosclerosis 8 1 cholesterol inmeat CVD risk, presumably owing to large the content of saturated fats and levelThe of high meat consumption developed world inthe is to linked metabolized toaproatherogenicspecies,trimethylamine- Intestinal microbiotametabolismofcholineandphosphatidylcholineproducestrimethylamine(TMA),whichisfurther Koeth A Robert Intestinal microbiota metabolism of NATURE MEDICINE linked tolinked increased meat consumption are responsible and CVD, prompting suggestion the that other environmental exposures cohort studies showed saturated no association dietary between fat intake ance recently, complex phenotypes such disease as obesity and insulin resist immune function,bioactivation of nutrients and vitamins, and, more diet-host interaction with reference to red meat consumption. participationthe of commensal intestinal microbiota the inmodifying Cleveland, Ohio,USA. Philadelphia, Pennsylvania,USA. Earl BBrittEarl California, USA. Intestinal microbiotamaythuscontributetothewell-establishedlinkbetweenhighlevelsofredmeatconsumptionandCVDrisk. microbiota, dietarysupplementationwithTMAOoreithercarnitinecholinereduced atherosclerosis, butthisdidnotoccurifintestinalmicrobiotawasconcurrentlysuppressed.Inmicewithanintact supplementation inmicealteredcecalmicrobialcomposition,markedlyenhancedsynthesisofTMAandTMAO,increased Section onLipidSciences,Wake ForestSchoolofMedicine,Winston-Salem, NorthCarolina,USA. Hongzhe Li W HWilson Tang n Department ofMedicine,PerelmanSchoolMedicineattheUniversityPennsylvania,Philadelphia, USA. Department ofCellular&MolecularMedicine,ClevelandClinic,Cleveland,Ohio,USA. n Department ofMedicine,PerelmanSchoolMedicineattheUniversityPennsylvania,Philadelphia,USA. Department ofCellular&MolecularMedicine,ClevelandClinic,Cleveland,Ohio,USA. =2,595)predictedincreasedrisksforboth prevalentcardiovasculardisease(CVD)andincidentmajoradversecardiacevents The microbiota of humans to linked intestinal has been health, =2,595)predictedincreasedrisksforbothprevalentcardiovasculardisease(CVD)andincidentmajoradversecardiacevents The microbiota of humans to linked intestinal has been health, 6–8 6–8 . We recently reported apathway humans in both and micelink . We recently reported apathway humans inboth and micelink 4 1,2 6 Department ofMathematics,ClevelandStateUniversity, Cleveland,Ohio,USA. , Gary DWu, Gary , Xiaoming Fu 1,2 6 3 4,5 1,2 Department ofMicrobiology, CenterforClinicalEpidemiologyandBiostatistics,PerelmanSchoolofMedicineattheUniversityPennsylvania, Department ofMedicine,DivisionCardiology, DavidGeffenSchoolofMedicine,UniversityCalifornia–LosAngeles,Los 4 1,2 ADVANCE ONLINEPUBLICATION 6 . However, a recent meta-analysis of prospective Department ofMathematics,ClevelandStateUniversity, Cleveland,Ohio,USA. . To our knowledge, no studies have yet explored 1,2,5 , Zeneng Wang , Gary DWu, Gary L -carnitine, atrimethylamineabundantinred meat,alsoproducesTMAOandacceleratesatherosclerosis , Xiaoming Fu , Frederic DBushman 1,2 6 3 4,5 1,2 7 Department ofMicrobiology, CenterforClinicalEpidemiologyandBiostatistics,PerelmanSchoolofMedicineattheUniversityPennsylvania, Department ofMedicine,DivisionCardiology, DavidGeffenSchoolofMedicine,UniversityCalifornia–LosAngeles,Los Division ofGastroenterology, PerelmanSchoolofMedicineattheUniversityPennsylvania,Philadelphia,USA. ADVANCE ONLINEPUBLICATION . However, arecent meta-analysis of prospective 7 . To our knowledge, no studies have yet explored 1,2,5 , James DLewis , Zeneng Wang L 1,2 -carnitine, atrimethylamineabundantinredmeat,alsoproducesTMAOandacceleratesatherosclerosis , Yuping Wu , Frederic DBushman 1,2 7 Division ofGastroenterology, PerelmanSchoolofMedicineattheUniversityPennsylvania,Philadelphia,USA. , Bruce SLevison , Bruce 7 , James DLewis phosphatidylcholine 1,2 6,8 3 4

. In sus the fact, , Lin Li , Lin , Yuping Wu 5 , Manya Warrier, [email protected] , Aldons JLusis 1,2 N 1,2 , Bruce SLevison , Bruce -oxide (TMAO).We demonstrateherethatmetabolism byintestinal 1,2 , Jonathan DSmith L -carnitine levelsinsubjectsundergoingcardiac evaluation - - - - ). , Jennifer ABuffa

phosphatidylcholine a trimethylamine structure similar to that of choline ( to TMAO formation, have clarified. not fully been tional mechanisms, microbial and contribute specific which species tolinked development the of accelerated atherosclerosis through addi to CVD pathogenesis as TMA ( bylized gut microbiota to produce an intermediate compound known pound and of part group head the of phosphatidylcholine, is metabo to enhanced “forward cholesterol transport.” macrophage scavenger receptors and thereby potentially contribute atherosclerosis. TMAO proposed has been to induce upregulation of generatealso TMAO from gut microbiota and promote accelerated nutrientsother dietary possessing atrimethylamine structure may ated with cardiovascularfindings raise possibility risks. These the that monooxygenases to form TMAO, is which proatherogenic and associ and serves an essential intransporting function and fatty serves acids into the vores, itendogenously is also producedmammalsin from lysine ingestionAlthough dietary is amajor source of 2 l 3 6,8 3 9 Center forCardiovascularDiagnostics&Prevention,ClevelandClinic, -carnitine is an abundant nutrient in red meat and contains & Stanley LHazen &Stanley 4 , JMark Brown

. In sus the fact, doi:10.1038/nm.314 , Lin Li , Lin 5 , Manya Warrier, [email protected] , Aldons JLusis Fig. 1 5 Department ofCardiovascularMedicine,ClevelandClinic, 10 Children’s HospitalOaklandResearchInstitute,Oakland, a l ). TMA is rapidly oxidized by further hepatic flavin N 1,2,5 1,2 1,2 -carnitine, -oxide (TMAO).We demonstrateherethatmetabolismbyintestinal , Elin Org , Elin in vivo 9 1,2 , Joseph ADiDonato , Jonathan DSmith . Choline, atrimethylamine-containing com 5 9 L , Ronald MKrauss -carnitine levelsinsubjectsundergoingcardiacevaluation - - - - ). , Jennifer ABuffa 1,2,5 reversecholesteroltransport. a trimethylamine structure similar to that of choline ( to TMAO formation, have clarified. not fully been tional mechanisms, microbial and contribute specific which species tolinked development the of accelerated atherosclerosis through addi to CVDpathogenesis to enhanced “forward cholesterol transport.” macrophage scavenger receptors and thereby potentially contribute atherosclerosis. TMAO proposed has been to induce upregulation of generatealso TMAO from gut microbiota and promote accelerated nutrientsother dietary possessing atrimethylamine structure may ated with cardiovascularfindings raise possibility risks. These the that monooxygenases to form TMAO, is which proatherogenic and associ as TMA ( bylized gut microbiota to produce an intermediate compound known pound and of part group head the of phosphatidylcholine, is metabo Although dietary ingestionAlthough dietary is amajor source of and serves an essential intransporting function and fatty serves acids into the vores, itendogenously is also producedmammalsin from lysine 3 9 , Brendan TSheehy Department ofPathology, 2 l 3 9 Center forCardiovascularDiagnostics&Prevention,ClevelandClinic, -carnitine is an abundant nutrient inred meat and contains & Stanley LHazen &Stanley , JMark Brown doi:10.1038/nm.314 Fig. 1

10 5 10 Department ofCardiovascularMedicine,ClevelandClinic, 1,2 10 . Whether TMAO is l -carnitine inomni , ARTICLES Children’s HospitalOaklandResearchInstitute,Oakland, L , Jun Chen -carnitine a l ). TMA is rapidly oxidized by further hepatic flavin L

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mitochondrial compartment10,11. l-Carnitine ingestion and sup- measuring endogenous l-carnitine concentrations in all subsequent plementation in industrialized societies have markedly increased12. investigations (Supplementary Figs. 1–3). Whether there are potential health risks associated with the rapidly growing practice of consuming l-carnitine supplements has not Human gut microbiota is required to form TMAO from L-carnitine been evaluated. The participation of gut microbiota in TMAO production from Herein we examine the gut microbiota–dependent metabolism of dietary l-carnitine in humans has not previously been shown. In l-carnitine to produce TMAO in both rodents and humans (omnivores initial subjects (omnivores), we developed an l-carnitine challenge and vegans or vegetarians). Using isotope tracer studies in humans, test in which the subjects were fed a large amount of l-carnitine clinical studies to examine the effects on cardiovascular disease risk, (an 8-ounce sirloin steak, corresponding to an estimated 180 mg and animal models including germ-free mice, we demonstrate a role of l-carnitine)13–15, together with a capsule containing 250 mg for gut microbiota metabolism of l-carnitine in atherosclerosis patho- of a heavy isotope–labeled l-carnitine (synthetic d3-(methyl)-l- genesis. We show that TMAO, and its dietary precursors choline and carnitine). At visit 1 post-prandial increases in plasma d3-TMAO carnitine, suppress reverse cholesterol transport (RCT) through gut and d3- l-carnitine concentrations were readily detected, and 24-h microbiota–dependent mechanisms in vivo. Finally, we define micro- urine collections also revealed the presence of d3-TMAO (Fig. 1b–e bial taxa in feces of humans whose proportions are associated with and Supplementary Figs. 4 and 5). Figure 1 and Supplementary both dietary carnitine ingestion and plasma TMAO concentrations. Figure 4 show tracings from a representative omnivorous subject, of We also show microbial compositional changes in mice associated five studied with sequential serial blood draws after carnitine chal- with chronic carnitine ingestion and a consequent marked enhance- lenge. In most subjects examined, despite clear increases in plasma ment in TMAO synthetic capacity in vivo. d3-carnitine and d3-TMAO concentrations over time (Fig. 1e), post- prandial changes in endogenous (unlabeled) carnitine and TMAO RESULTS concentrations were modest (Supplementary Fig. 5), consistent with Metabolomic studies link L-carnitine with CVD total body pools of carnitine and TMAO that are relatively very large Given the similarity in structure between l-carnitine and choline in relation to the amounts of carnitine ingested and TMAO produced (Fig. 1a), we hypothesized that dietary l-carnitine in humans, like from the carnitine challenge. choline and phosphatidylcholine, might be metabolized to pro- To examine the potential contribution of gut microbiota to duce TMA and TMAO in a gut microbiota–dependent fashion TMAO formation from dietary l-carnitine, we placed the five and be associated with atherosclerosis risk. To test this hypothesis, volunteers studied above on oral broad-spectrum antibiotics we initially examined data from our recently published unbiased to suppress intestinal microbiota for a week and then performed small-molecule metabolomics analyses of plasma analytes and a second l-carnitine challenge (visit 2). We noted near complete CVD risks9. suppression of detectable endogenous TMAO in both plasma An analyte with identical molecular weight and retention time to and urine after a week-long treatment with the antibiotics (visit 2) l-carnitine was not in the top tier of analytes that met the stringent P value cutoff for association with CVD. However, a hypothesis-driven a examination of the data using less stringent criteria (no adjustment for

Nature America, Inc. All rights reserved. Inc. Nature America, multiple testing) revealed an analyte with the appropriate molecular FMO Carnitine Gut flora Atherosclerosis weight and retention time for l-carnitine that was associated with cardiovascular event risk (P = 0.04) (Supplementary Table 1). In further TMA TMAO © 201 3 studies we were able to confirm the identity of the plasma analyte as Choline l-carnitine and develop a quantitative stable-isotope-dilution liquid b Visit 1 Visit 2 Visit 3 chromatography tandem mass spectrometry (LC-MS/MS) method for Steak Gut flora Steak Reacquisition Steak + + + d3-carnitine suppression d3-carnitine of gut flora d3-carnitine Figure 1 TMAO production from L-carnitine is a microbiota-dependent c 100 TMAO 100 TMAO 100 TMAO process in humans. (a) Structure of carnitine and scheme of carnitine m/z = m/z = m/z = and choline metabolism to TMAO. L-Carnitine and choline are both 76 58 76 58 76 58 dietary trimethylamines that can be metabolized by microbiota to TMA. 50 50 50 TMA is then further oxidized to TMAO by flavin monooxygenases (FMOs). Intensity (%) (b) Scheme of the human L-carnitine challenge test. After a 12-h 0 0 0 overnight fast, subjects received a capsule of d3-(methyl)-carnitine 0510 0510 0510 (250 mg) alone, or in some cases (as in data for the subject shown) also an 8-ounce steak (estimated 180 mg L-carnitine), whereupon serial d 100 d3-TMAO 100 d3-TMAO 100 d3-TMAO m/z = m/z = m/z = plasma and 24-h urine samples were obtained for TMA and TMAO 79 61 79 61 79 61 analyses (visit 1). After a weeklong regimen of oral broad-spectrum Knock50 down gut flora with50 antibiotics and TMAO50 is not produced antibiotics to suppress the intestinal microbiota, the challenge was • repeated (visit 2), and then again a final third time after a q3-week period Intensity (%) 0 0 0 to permit repopulation of intestinal microbiota (visit 3). (c,d) LC-MS/MS 0510 0510 0510 chromatograms of plasma TMAO (c) and d3-TMAO (d) in an omnivorous Time (min) Time (min) Time (min) subject using specific precursor l product ion transitions indicated at e t = 8 h for each visit. (e) Stable-isotope-dilution LC-MS/MS time course 2.50 2.50 12.00 d3-TMAO 2.50 measurements of d3-labeled TMAO and carnitine in plasma collected from d3-TMAO 1.25 sequential venous blood draws at the indicated time points. Data shown in 1.25 d3-carnitine 1.25 d3-carnitine c–e are from a representative female omnivorous subject who underwent d3-TMAO Plasma ( M) 0 0 0 d3-carnitine carnitine challenge. Data are organized vertically to correspond with the 01224 01224 01224 visit schedule indicated in b. Time (h) Time (h) Time (h)

2 ADVANCE ONLINE PUBLICATION NATURE MEDICINE © 2013 Nature America, Inc. All rights reserved. or vegetarian( grouped bydietarystatusaseithervegan Supplementary Methods on taxonomic operationalunits(OTUs,plotted (plotted on percentiles. ( and whiskersrepresentthe10th90th 50th (middlelines)and75thpercentiles, enterotype. Boxesrepresentthe25th, subjects withacharacterizedgutmicrobiome enterotype 2( TMAO plasmaconcentrationsassociate with ( d3-TMAO formation, consistent with challenge plasma and samples urine again showed TMAO and mean nonparametric test.Datapointsrepresent the curve(AUC)ofgroupsusingWilcoxon shown isforthecomparisonofarea under Vegans andvegetariansproduceless TMAO from humans is dependent on intestinal microbiota. data show that TMAO production from dietary discontinuation of antibiotics (visit and 3).Baseline post- Fig. 5 concomitant steakconsumption. The carnitine (250mg)challenge without after (visit 2) ant was little change overall inthe course time before (visit 1)versus ( and unlabeled Supplementary Fig.4 microbiota inTMAO formation from after carnitine challenge, consistent with an obligatory role for gut post-prandialin all plasma samples or samples 24-hurine examined tually no detectable formation of either native or TMAO d3-labeled and vegetarians( concentrations inmaleandfemale vegans and 90thpercentiles. ( percentiles and whiskersrepresent the 10th Boxes represent the 25th, 50th, and 75th vegetarians ( d3-TMAO from male and female vegans and fasting plasma concentrations ofTMAOand TMAO andd3-TMAOwerequantifiedafter frequently consumesredmeat.Plasma a representativefemaleomnivorewho 8-ounce sirloinsteakand,forcomparison, of 250mgd3-(methyl)-carnitineandan challenge consistingofco-administration from amalevegansubjectinthecarnitine expressed as means±s.e.m.( (Cr) toadjustforurinary dilution.Dataare reported asratiowith urinarycreatinine collection ( expressed asmeans±s.e.m.forbothTMAOconcentration( L ingested Figure 2 NATURE MEDICINE ( ingestion was variable among individuals. A The capacity to produce TMAO (native and after d3-labeled) with plasmaTMAOconcentrations.( and fecalmicrobiotacompositionassociates n -carnitine challenge ( Fig. 1b Fig. 1b =5)participatingin ad3-(methyl)- y axes),determinedasdescribedin o ). We rechallenged weeks same the after subjects several s.e.m.of

– – L The formationofTMAOfrom e -carnitine isnegligibleinvegans, e and and b and Supplementary Figs. and 4 x ). UrineTMAOandd3-TMAO axes)andtheproportionof n f Prevotella ) PlasmaTMAOconcentrations = 26) and omnivores ( n =23)oromnivore( l Supplementary Fig. 5 -carnitine after the n n =5)andomnivores =5pergroup.( i biotic treatment (Fig. 1 ADVANCE ONLINEPUBLICATION a d ). In contrast, d3- we both detected ) andina24-hurine ) inmaleandfemale . Subjectswere ) Plasmad3-TMAO c ) Baseline e n a ) Baseline n =30). , P l = 51). b • value

-carnitine challenge, and there ) Data ). Moreover, vir we observed carnitine dose Vegetarians and Vegans didnotform TMAO evengiven samesteakand l

and -carnitine ( intestinal recolonization P valueshownisforcomparisonsbetweendietarygroupsusingarobustHotelling e 5 and and ). Collectively,). these post hoc c a –4 –4 f Plasma TMAO (µM) Plasma (µM) 0 4 8 0 4 6 Supplementary Supplementary Proportion OTUs (×10 ) Proportion OTUs (×10 ) 20 40 0 2 4 L

0 -carnitine l . . 3.6 2.7 1.8 Fig. 1b vegetarian P <0.05 . . 3.6 2.7 1.8 -carnitine in 0 (n =26) P =0.13 Peptostreptococcaceae Vegan/ P <0.05 vegetarian nutritional nutritional (n =23) Vegan/ incertae sedisXII x l l l Omnivore axis)andtheproportionofOTUs( -carnitine -carnitine incertae sedis Clostridiales -carnitine -carnitine TMAO (µM) TMAO (µM) – TMAO Time (h) e Omnivore and and (n =51) 12 Omnivore (n =30) Vegan -

vegans and vegetarians ( vegetarians and vegans ( plasma levelsof challenge (Fig. 2a capacity to generate TMAO or d3-TMAO inplasma after carnitine the nominal plasmaand TMAO urine levels at baseline, andno virtually d3-(methyl)-carnitine consumption) ( consentedwho to carnitine the challenge (including steakand both same the prospectively, we examined TMAO and d3-TMAO production after to generate TMAO from habitsdietary (red meat consumption) may influence capacity the that volunteers the survey completed suggested that antecedent 24-h urine collection sample collection 24-h urine ( tions sequential plasma inboth measurements ( omnivore showed increases inTMAO and d3-TMAO concentra venison,(beef, lamb, mutton, duck or pork). Post-prandially, the self-reported frequent consumption (near daily) dietary of red meat comparison are data from asingle representative omnivore with Supplementary Fig. 6 15 30 0 2 4 To and confirm extendfindings, we these examined additional 0 24 . . 3.6 2.7 1.8 . . 3.6 2.7 1.8 P =0.13 P <0.05 d

Clostridiaceae Plasma d3-TMAO (µM) Fusibacterium 15 30 TMAO (µM) 0 TMAO (µM) l

-carnitine challenge, first inalong-term (>5years) vegan Plasma (µM) 0.125 0.250 0 0 0 l -carnitine compared to omnivorous the subject , y b axis). Time (h) Vegan/vegetarian ). The vegan had lower subject also fasting Omnivore d3-TMAO 12 50 25 ). 40 20

0 0 P <0.05

Time (h) n (n =5) . . 3.6 2.7 1.8 . . 3.6 2.7 1.8

= 23) and =23) omnivorous ( subjects Omnivore

Peptostreptococcaceae 122

= 5) = n P <0.05 ( -carnitine (data not shown). To test this

Vegan

Fig. 2 Lachnospira TMAO (µM) TMAO (µM) 24 b P <0.05 ). In contrast, vegan the showed 4 Fig. 2 e Plasma TMAO (µM) 0 4 8 12 24 15 30 b 0 0 Enterotype 1 Bacteroides d3-TMAO/Cr TMAO/Cr a . . 3.6 2.7 1.8 . . 3.6 2.7 1.8 (n =49) ). Also shown). Also for P <0.05 (mmol/mol) (mmol/mol) T 25 50 ARTICLES 2 0 1 2 0 Fig. 2 test.Dataare Sporobacter TMAO (µM) TMAO (µM) Clostridium ea Omnivore Vegan ea Omnivore Vegan Urine d3-TMAO Urine TMAO a Enterotype 2 ) and ina P =0.10 Prevotella P <0.05 (n =4) n =51). 3 -

© 2013 Nature America, Inc. All rights reserved. on taxonomic operational units(OTUs,plotted or vegetarian ( grouped by dietarystatus as eithervegan Supplementary Methods (plotted on percentiles. ( and whiskersrepresentthe10th 90th 50th (middlelines)and75thpercentiles, enterotype. Boxesrepresentthe25th, subjects withacharacterizedgutmicrobiome enterotype 2( TMAO plasmaconcentrationsassociate with mean nonparametric test.Datapointsrepresent the curve(AUC)ofgroupsusingWilcoxon shown isforthecomparisonofareaunder concomitant steakconsumption.The carnitine (250mg)challengewithout ( d3-TMAO formation, consistent with challenge plasma and samples urine again showed TMAO and discontinuation of antibiotics (visit and 3). Baseline post- Fig. 5 ( Vegans andvegetariansproducelessTMAOfrom humans is dependent on intestinal microbiota. data show that TMAO production from dietary after (visit 2) ant was little change overall inthe course time before (visit 1)versus microbiota inTMAO formation from after carnitine challenge, consistent with an obligatory role for gut post-prandialin all plasma samples or samples 24-hurine examined tually no detectable formation of either native or TMAO d3-labeled and vegetarians( concentrations inmaleandfemalevegans and 90thpercentiles.( percentiles andwhiskersrepresentthe10th Boxes representthe25th,50th,and75th vegetarians ( d3-TMAO frommaleandfemalevegans fasting plasmaconcentrationsofTMAOand and unlabeled Supplementary Fig.4 TMAO andd3-TMAOwerequantifiedafter frequently consumesredmeat.Plasma a representativefemaleomnivorewho 8-ounce sirloinsteakand,forcomparison, of 250mgd3-(methyl)-carnitineandan challenge consistingofco-administration from amalevegansubjectinthecarnitine expressed asmeans±s.e.m.( (Cr) toadjustforurinarydilution.Dataare reported asratiowithurinarycreatinine collection ( expressed as means ± s.e.m. for both TMAO concentration ( ( L ingested Figure 2 NATURE MEDICINE with plasmaTMAOconcentrations.( and fecalmicrobiotacompositionassociates ingestion was variable among individuals. A The capacity to produce TMAO (native and after d3-labeled) n -carnitine challenge( Fig. 1b Fig. 1b =5)participatinginad3-(methyl)- y axes),determined asdescribedin o ). We rechallenged weeks same the after subjects several s.e.m.of

– – L The formationofTMAOfrom e -carnitine isnegligibleinvegans, e and and b and Supplementary Figs. and 4 x ). UrineTMAOandd3-TMAO axes)andtheproportion of n f Prevotella ) PlasmaTMAOconcentrations =26)andomnivores( n =23) oromnivore ( l Supplementary Fig. 5 -carnitine after the n n =5)andomnivores =5pergroup.( i biotic treatment (Fig. 1 ADVANCE ONLINEPUBLICATION a d ). In contrast, d3- we both detected ) andina24-hurine ) inmaleandfemale . Subjects were ) Plasmad3-TMAO c ) Baseline e n a ) Baseline n =30). , P l =51). b value

-carnitine challenge, and there

and -carnitine ( intestinal recolonization P or vegetarian ( grouped by dietarystatusaseither vegan Supplementary Methods on taxonomic operational units(OTUs,plotted (plotted on percentiles. ( and whiskersrepresentthe10th90th 50th (middlelines)and75thpercentiles, enterotype. Boxesrepresentthe25th, subjects withacharacterizedgutmicrobiome enterotype 2( TMAO plasmaconcentrationsassociate with ( d3-TMAO formation, consistent with challenge plasma and samples urine again showed TMAO and mean nonparametric test.Datapointsrepresent the curve(AUC)ofgroupsusingWilcoxon shown isforthecomparisonofareaunder Vegans andvegetariansproducelessTMAOfrom humans is dependent on intestinal microbiota. data show that TMAO production from dietary discontinuation of antibiotics (visit and 3).Baseline post- Fig. 5 concomitant steakconsumption.The carnitine (250mg)challengewithout after (visit 2) ant was little change overall inthe course time before (visit 1)versus ( and unlabeled Supplementary Fig.4 microbiota inTMAO formation from after carnitine challenge, consistent with an obligatory role for gut post-prandialin all plasma samples or samples 24-hurine examined tually no detectable formation of either native or TMAO d3-labeled and vegetarians( concentrations inmaleandfemalevegans and 90thpercentiles.( percentiles andwhiskersrepresentthe10th Boxes representthe25th,50th,and75th vegetarians ( d3-TMAO frommaleandfemalevegans fasting plasmaconcentrationsofTMAOand TMAO andd3-TMAOwerequantifiedafter frequently consumesredmeat.Plasma a representativefemaleomnivorewho 8-ounce sirloinsteakand,forcomparison, of 250mgd3-(methyl)-carnitineandan challenge consistingofco-administration from amalevegansubjectinthecarnitine expressed as means ± s.e.m. for both TMAO concentration ( expressed asmeans±s.e.m.( (Cr) toadjustforurinarydilution.Dataare reported asratiowithurinarycreatinine collection ( NATURE MEDICINE ( L ingested Figure 2 with plasmaTMAOconcentrations.( and fecalmicrobiotacompositionassociates ingestion was variable among individuals. A The capacity to produce TMAO (native and after d3-labeled) n -carnitine challenge( Fig. 1b Fig. 1b value shownis forcomparisons between dietary groupsusing arobust Hotelling =5)participatinginad3-(methyl)- e y 5 axes),determinedas describedin and and ). Collectively,). these o post hoc ). We rechallenged weeks same the after subjects several s.e.m.of c a

– – f L The formationofTMAOfrom e –4 –4 -carnitine isnegligibleinvegans, e Plasma TMAO (µM) Plasma (µM) 0 4 8 0 4 6 Supplementary Supplementary

and and Proportion OTUs ( 10 ) Proportion OTUs ( 10 ) × × b and Supplementary Figs. and 4 x 20 40 0 2 4 L

). UrineTMAOandd3-TMAO axes)andtheproportion of n 0 -carnitine l f . . 3.6 2.7 1.8 Fig. 1b vegetarian Prevotella ) PlasmaTMAOconcentrations =26)andomnivores( P <0.05 n . . 3.6 2.7 1.8 -carnitine in 0 (n =26) P =0.13 Peptostreptococcaceae Vegan/ P <0.05 =23) oromnivore ( vegetarian l Supplementary Fig. 5 nutritional nutritional (n =23) -carnitine after the n Vegan/ incertae sedisXII x n l l l Omnivore =5)andomnivores axis) and the proportion of OTUs ( =5pergroup.( -carnitine -carnitine incertae sedis i Clostridiales -carnitine -carnitine TMAO (µM) biotic treatment (Fig. 1 TMAO (µM) ADVANCE ONLINEPUBLICATION a – d TMAO ). In contrast, d3- we both detected ) andina24-hurine Time (h) ) inmaleandfemale e . Subjects were ) Plasmad3-TMAO Omnivore and and (n =51) 12 Omnivore (n =30) Vegan - c

) Baseline vegans and vegetarians ( vegetarians and vegans ( d3-(methyl)-carnitine consumption) ( consentedwho to carnitine the challenge (including steakand both same the prospectively, we examined TMAO and d3-TMAO production after plasma levelsof challenge (Fig. 2a capacity to generate TMAO or d3-TMAO inplasma after carnitine the nominal plasmaand TMAO urine levels at baseline, andno virtually to generate TMAO from habitsdietary (red meat consumption) may influence capacity the that volunteers the survey completed suggested that antecedent 24-h urine collection sample collection 24-h urine ( tions sequential plasma inboth measurements ( omnivore showed increases inTMAO and d3-TMAO concentra venison,(beef, lamb, mutton, duck or pork). Post-prandially, the self-reported frequent consumption (near daily) dietary of red meat comparison are data from asingle representative omnivore with e • • n Supplementary Fig. 6 a ) Baseline n =30). 15 30 0 2 4 , To and confirm extendfindings, we these examined additional P l 0 =51). b 24 Enterotype dependenceon TMAO production Veg vsomnivoreshavedifferent bugs:long-term dietaryinfluenceonbugs . . 3.6 2.7 1.8 value

-carnitine challenge, and there ) Data . . 3.6 2.7 1.8 ). Moreover, vir we observed P =0.13 P <0.05 d l

and Clostridiaceae -carnitine ( Plasma d3-TMAO (µM) Fusibacterium intestinal recolonization P 15 30 TMAO (µM) 0 value shownis forcomparisons between dietary groups usinga robust Hotelling TMAO (µM) l e 5 -carnitine challenge, first in along-term (>5years) vegan Plasma (µM) 0.125 0.250 and and 0 ). Collectively,). these post hoc c a 0 –4 –4 f Plasma TMAO (µM) Plasma (µM) 0 4 8 0 4 6 Supplementary Supplementary Proportion OTUs (×10 ) Proportion OTUs (×10 ) 20 40 0 2 4 L

0 l 0 -carnitine l . . 3.6 2.7 1.8 Fig. 1b vegetarian P <0.05 -carnitine compared to omnivorous the subject . . 3.6 2.7 1.8 -carnitine in 0 (n =26) P =0.13 , Peptostreptococcaceae Vegan/ y P <0.05 vegetarian b nutritional nutritional axis). (n =23) Vegan/ Time (h) incertae sedisXII x Vegan/vegetarian l l ). The vegan had lower subject also fasting l Omnivore axis) and the proportion of OTUs ( -carnitine -carnitine incertae sedis Clostridiales -carnitine -carnitine TMAO (µM) Omnivore d3-TMAO 12 25 50 ). TMAO (µM) 20 40 0 0 P <0.05 – Time (h) n TMAO (n =5) Time (h) . . 3.6 2.7 1.8 . . 3.6 2.7 1.8 e l = 23) and =23) omnivorous ( subjects Omnivore Omnivore

Peptostreptococcaceae and and 122

= 5) = n P <0.05 ( (n =51) -carnitine (data not shown). To test this 12 Omnivore (n =30) Vegan Vegan Fig. 2 - Lachnospira

TMAO (µM) TMAO (µM) vegans and vegetarians ( vegetarians and vegans ( d3-(methyl)-carnitine consumption) ( consentedwho to carnitine the challenge (including steakand both same the prospectively, we examined TMAO and d3-TMAO production after to generate TMAO from habitsdietary (red meat consumption) may influence capacity the that volunteers the survey completed suggested that antecedent plasma levelsof challenge (Fig. 2a capacity to generate TMAO or d3-TMAO inplasma after carnitine the nominal plasmaand TMAO urine levels at baseline, andno virtually 24-h urine collection sample collection 24-h urine ( tions sequential plasma inboth measurements ( omnivore showed increases inTMAO and d3-TMAO concentra venison,(beef, lamb, mutton, duck or pork). Post-prandially, the self-reported frequent consumption (near daily) dietary of red meat comparison are data from asingle representative omnivore with Supplementary Fig. 6 24 15 30 0 2 4 b To and confirm extendfindings, we these examined additional 0 P <0.05 24 . . 3.6 2.7 1.8 ). In contrast, vegan the showed . . 3.6 2.7 1.8 4 P =0.13 P <0.05 d

Clostridiaceae Plasma d3-TMAO (µM) Fusibacterium Fig. 2 15 30 e TMAO (µM) 0 TMAO (µM) l Plasma TMAO (µM) -carnitine challenge, first inalong-term (>5 years) vegan Plasma (µM) 0.125 0.250 0 0 4 8 12 24 15 30 b 0 0 0 Enterotype 1 Bacteroides d3-TMAO/Cr TMAO/Cr a . . 3.6 2.7 1.8 . . 3.6 2.7 1.8 (n =49) ). Also shown). Also for P <0.05 0 l (mmol/mol) (mmol/mol) T 25 50 ARTICLES -carnitine compared to omnivorous the subject 2 0 0 1 2 Fig. 2 , y b test. Dataare axis). Time (h) Vegan/vegetarian ). The vegan had lower subject also fasting Sporobacter TMAO (µM) TMAO (µM) Clostridium Omnivore d3-TMAO 12 ea Omnivore Vegan ea Omnivore Vegan 25 50 Urine TMAO Urine d3-TMAO ). 20 40 0 0 P <0.05 Time (h) n (n =5) . . 3.6 2.7 1.8 . . 3.6 2.7 1.8 l

a = 23) and =23) omnivorous ( subjects Omnivore

Peptostreptococcaceae 122

= 5) = n Enterotype 2 ( P <0.05 -carnitine (data not shown). To test this ) and ina P =0.10 Prevotella P <0.05 Vegan (n =4) Fig. 2 n Lachnospira TMAO (µM) TMAO (µM) =51). 24 b P <0.05 ). In contrast, vegan the showed 4 3 -

Fig. 2 e Plasma TMAO (µM) 0 4 8 12 24 15 30 b 0 0 Enterotype 1 Bacteroides d3-TMAO/Cr TMAO/Cr a . . 3.6 2.7 1.8 3.6 2.7 1.8 (n =49) ). Also shown). Also for P <0.05 (mmol/mol) (mmol/mol) T 25 50 ARTICLES 2 0 0 1 2 Fig. 2 test. Dataare Sporobacter TMAO (µM) TMAO (µM) Clostridium ea Omnivore Vegan ea Omnivore Vegan Urine TMAO Urine d3-TMAO a Enterotype 2 ) and ina P =0.10 Prevotella P <0.05 (n =4) n =51). 3 -

© 2013 Nature America, Inc. All rights reserved. We next investigated dietary whether age) collection) resultsinadult female(>8weeks of (+ ABS). Alsoshownare RCT (72-hstool of microbiota using cocktail of antibiotics root atherosclerotic plaque quantification revealed chow dietversus same the dietsupplemented with chow versusdiet supplemented with either Data areexpressedasmeans±s.e.m. involved inbileacidsynthesisor transport. levels (to supplemented with TMAO.( female (>8 weeksofage) killing. ( plasma recovered frommiceatthetimeof stable-isotope-dilution LC-MS/MSanalysisof TMAO concentrationsasdetermined using the OnlineMethods.( and lesionareawasquantifiedas describedin time ofweaning(4weeksage) beforekilling, of agewerestartedontheindicated dietsatthe 6 6 factors ( after adjustments for traditional CVD risk an independent predictor of MACE, even carnitine concentration quartile) (4th was stroke and revascularization). Elevated composite of death, myocardial infarction, for major adverse cardiac events (MACE: of carnitine and incident (3-year) risk fastingbetween plasma concentrations byrevealed L Figure 5 A ( ship carnitine between and MACE risk was completely abolished medications and estimated renal significant the function), relation example, extent of CAD, fraction, ejection might known be at of time presentation (for andlarger a number of comorbidities that for plasma both TMAO concentration Dietary of carnitine with cardiovascular risks. rather than carnitine, driver of is primary the association the incident cardiovascular results risks, these suggest that TMAO, tions of associated to carnitine prevalent with be both seem and trations ( among subjects with concurrent those plasma high TMAO concen in Cox regression models after multivariate adjustment, but only carnitine concentration and incident cardiovascular event risks Apoe (counterstained withhematoxylin)of19-week-old (a) RepresentativeoilredO–stainedaorticroots transport inamicrobiotadependentfashion. atherosclerosis andinhibitsreversecholesterol lesion area. (b) Quantificationofmouseaorticrootplaque (ABS) asdescribedintheOnlineMethods. in thepresenceversusabsenceofantibiotics formation. We fed extentthe of atherosclerosis presence inthe or absence of TMAO (Kruskal-Wallis -carnitine or choline, as well as after suppression Fig. 4 We examined relationship the also RTICLES Apoe −/ femalemiceontheindicateddiets e d) RCT(72-hstool collection)inadult Dietary L ). Notably, asignificant association we between observed Actb Fig. −/ -carnitine promotesmicrobiota-dependentatherosclerosis P Apoe

miceonnormal chowversusdiet <0.001)( diagnostic cardiac catheterization ) ofmouselivercandidate genes 4e −/ L ). After further adjustment). After further -carnitine accelerates P femalemiceat19weeks <0.001)(Fig. 4 Apoe c) Carnitine,TMAand Fig. 4 Apoe − e,f) RelativemRNA / − micefrom of time the weaning anormal −/ f ). Thus, although plasma concentra miceon normal d ).

-carnitine has an impact on

• with greaterplasmalevelsofcarnitine(andmore sodueto TMAO) Greater levelsofcoronaryarterialdiseaseandmajor cardiaceventsassociated c a f d l Carnitine (M) -carnitine. Aortic Relative Liver 200 RCT (%) 100

expression 0 1.5 0.5 1.0 Stool 2 0 1 approximately approximately 0 (n =9) Chow (n =43) Chow +ABS Chow P <0.01 (n =16)( hwTMAO Chow P <0.05 Chow P <0.05 P =0.22 Carnitine Oatp1 =9 (n=11) (n=9) (n =11) P <0.05 antn hln Chow Choline Carnitine 28% (n =27)( n =16) P <0.01 250 m + ABS 250 m Chow - - -

P <0.01 31% n =21) =1)(n=16) (n =16) arm of study the ( the increase the inatheroscleroticplaque burden with dietary l concentrations ( microbiota showed marked reductions inplasma TMA and TMAO in miceplacedon an oral antibiotic cocktail to suppress intestinal compared to normal chow–fed mice( a doubling burden of disease in ( capacity of microbiota to catabolize inmicesupplementedhigher with in Supplementary Fig. 12 to show of evidence steatosis ( and histological analyses oflivers from anygroup of failedmice the lipoprotein, or glucose insulin levels; moreover, biochemical both absence inthe ofoccurred proatherogenic changes inplasma lipid, hwTMAO Chow Carnitine P -carnitine–dependent increase inatherosclerosis ( + ABS P <0.05 <0.05)( Plasma concentrations of carnitine were significantly higher + ABSstool Oatp4 l 2 0 1 -carnitine–fed micecompared to normal chow–fed controls =1)(n=16) (n=12) (n =30) P =0.34 TMA ( M) 120 Carnitine +ABS 60 0 P =0.39 antn Choline Carnitine Carnitine Fig. 5 =9 (n=11) (n=9) (n=11) (n =9) Chow (n =16)( hwTMAO Chow P <0.01 ADVANCE ONLINEPUBLICATION P =0.77 Bsep Fig. 5 P <0.05 antn Chow Carnitine c Fig. 5 n =16) ). Plasma carnitine concentrations were even 250 m 250 m 1.5 0.5 1.0 Stool c ). P <0.01 0 ) and complete inhibition of dietary the + ABS c ), presumably as aresult of reduced the P =0.31 (n =21)( hwTMAO Chow =1)(n=16) (n =16) hwTMAO Chow b P <0.05 2 P <0.01 Carnitine Aortic lesion (m ) + ABS Mrp2 2.5 10 5.0 10 Supplementary Tables 3 n =29) 35% l -carnitine supplemented mice 0 TMAO ( M) 5 5 l l 200 100 e -carnitine antibiotic inthe -carnitine. However, as the =9 (n=11)( (n =9) Chow 0 Fig. 5a Relative expression 1.5 0.5 1.0 Liver (n =16)( hwTMAO Chow 1.8-fold 0 P <0.01 =9 (n=11) (n=9) (n=11) (n =9) Chow P =0.97 =1)(n=16) (n =16) hwTMAO Chow Ephx1 P <0.01 antn Chow Carnitine P <0.05 Cyp7a1 P =0.89

, antn Chow Carnitine n =16) P <0.01 b NATURE MEDICINE ). Parallel Fig. 5b + ABS P <0.01 =9 (n=10) n =9) P <0.01 + ABS (n =15)( =1)(n=16) (n =16) hwTMAO Chow hwTMAO Chow P =0.84 P <0.01 l Cyp27a1 P <0.05 and P <0.05 ). Ofnote, -carnitine Ntcp Carnitine Carnitine

+ ABS + ABS studies 4 n =16) and and

© 2013 Nature America, Inc. All rights reserved. • • • (myocardial infarction,strokeordeath),butonlyamongsubjectswithconcurrentlyhigh TMAOlevels.Chronicdietary ing microbiota choline metabolism of and dietary ated during cooking ingestion, such salt content as high or heterocyclic compounds gener alternative disease-promoting exposures that accompany meat dietary CVDandbetween meat consumption has stimulated investigation of not enough sufficiently high to be account association for observed the picion that cholesterol the and saturated fat content of red meat may in mice.OmnivoroushumansubjectsproducedmoreTMAOthandidvegansorvegetarians followingingestionof Received 7December2012;accepted27February2013; publishedonline7April2013; California, USA.Correspondenceshouldbeaddressed to S.L.H.( ( Cleveland, Ohio,USA. microbiota ofdietary metabolized toaproatherogenicspecies,trimethylamine- Intestinal microbiotametabolismofcholineandphosphatidylcholineproducestrimethylamine (TMA),whichisfurther Koeth A Robert a nutrient inred meat, promotes atherosclerosis Intestinal microbiota metabolism of NATURE MEDICINE 8 1 cholesterol inmeat CVD risk, presumably owing to large the content of saturated fats and levelThe of high meat consumption developed world inthe is to linked Earl BBrittEarl linked tolinked increased meat consumption are responsible and CVD, prompting suggestion the that other environmental exposures cohort studies showed saturated no association dietary between fat intake ance recently, complex phenotypes such disease as obesity and insulin resist immune function,bioactivation of nutrients and vitamins, and, more diet-host interaction with reference to red meat consumption. participationthe of commensal intestinal microbiota the inmodifying Philadelphia, Pennsylvania,USA. both plasmaTMAOconcentrationanddietarystatus.Plasma through amicrobiota-dependentmechanism.Thepresenceofspecificbacterialtaxa in humanfeceswasassociatedwith Cleveland, Ohio,USA. California, USA. Hongzhe Li Section onLipidSciences,Wake ForestSchoolofMedicine,Winston-Salem, NorthCarolina,USA. Intestinal microbiotamaythuscontributeto thewell-establishedlinkbetweenhighlevelsofredmeatconsumptionandCVDrisk . microbiota, dietarysupplementationwithTMAO oreithercarnitinecholinereduced atherosclerosis, butthisdidnotoccurifintestinalmicrobiotawasconcurrentlysuppressed. Inmicewithanintactintestinal supplementation inmicealteredcecalmicrobialcomposition,markedlyenhancedsynthesis ofTMAandTMAO,increased W HWilson Tang n Department ofMedicine,PerelmanSchoolMedicine attheUniversityofPennsylvania,Philadelphia,USA. Department ofCellular&Molecular Medicine,ClevelandClinic,Cleveland,Ohio,USA. =2,595)predictedincreasedrisksforbothprevalentcardiovasculardisease(CVD)and incidentmajoradversecardiacevents The microbiota of humans to linked intestinal has been health, physiology such ascoronaryfunction Antibiotics can alterthenatureofmetabolites anddirectlyimpact downstream Different enterotypesproducedifferent metabolicprofiles Gut floraplaysanimportant roleinmetabolizing,processingwhatweingest 6–8 . We recently reported apathway humans inboth and micelink 4 1,2 6 Department ofMathematics,ClevelandStateUniversity, Cleveland,Ohio,USA. , Gary DWu, Gary , Xiaoming Fu 1,2 6 3 4,5 1,2 Department ofMicrobiology, Centerfor Clinical EpidemiologyandBiostatistics,PerelmanSchoolofMedicineattheUniversityPennsylvania, Department ofMedicine,DivisionCardiology, DavidGeffenSchoolofMedicine,UniversityCalifornia–LosAngeles,Los ADVANCE ONLINEPUBLICATION . However, arecent meta-analysis of prospective . To our knowledge, no studies have yet explored 1,2,5 , Zeneng Wang L -carnitine, atrimethylamineabundantinredmeat,alsoproducesTMAOandaccelerates atherosclerosis , Frederic DBushman 7 Division ofGastroenterology, PerelmanSchool ofMedicineattheUniversityPennsylvania,Philadelphia,USA. 7 , James DLewis 1,2 , Yuping Wu 1,2 , Bruce SLevison , Bruce phosphatidylcholine 6,8 3 4

. In sus the fact, , Lin Li , Lin 5 , Manya Warrier, [email protected] , Aldons JLusis N 1,2 -oxide (TMAO).We demonstrateherethatmetabolismbyintestinal 1,2 , Jonathan DSmith L -carnitine levelsinsubjectsundergoingcardiacevaluation - - - - ). , Jennifer ABuffa a trimethylamine structure similar to that of choline ( to TMAO formation, have clarified. not fully been tional mechanisms, microbial and contribute specific which species tolinked development the of accelerated atherosclerosis through addi to CVDpathogenesis as TMA ( bylized gut microbiota to produce an intermediate compound known pound and of part group head the of phosphatidylcholine, is metabo to enhanced “forward cholesterol transport.” macrophage scavenger receptors and thereby potentially contribute atherosclerosis. TMAO proposed has been to induce upregulation of generatealso TMAO from gut microbiota and promote accelerated nutrientsother dietary possessing atrimethylamine structure may ated with cardiovascularfindings raise possibility risks. These the that monooxygenases to form TMAO, is which proatherogenic and associ and serves an essential intransporting function and fatty serves acids into the vores, itendogenously is also producedmammalsin from lysine ingestionAlthough dietary is amajor source of 2 l 3 9 Center forCardiovascularDiagnostics &Prevention,ClevelandClinic, -carnitine is an abundant nutrient inred meat and contains & Stanley LHazen &Stanley , JMark Brown doi:10.1038/nm.314 Fig. 1 5 Department ofCardiovascularMedicine,ClevelandClinic, 10 Children’s HospitalOaklandResearchInstitute,Oakland, a l ). TMA is rapidly oxidized by further hepatic flavin 1,2,5 1,2 -carnitine, , Elin Org , Elin in vivo 9 , Joseph ADiDonato . Choline, atrimethylamine-containing com 5 9 , Ronald MKrauss 1,2,5 reversecholesteroltransport. 3 9 , Brendan TSheehy Department ofPathology,

10 10 1,2 . Whether TMAO is l -carnitine inomni , ARTICLES L , Jun Chen -carnitine L

-carnitine 1 ,

Fig. 1

6 , a ). ). 1 - - - - - Normal ﬂora Sources of pathogenic bacterial infections

Organs and internal tissues are normally sterile. Commensal bacteria do colonize “exterior” including skin, gut, respiratory tract, mouth, eyes, urogenital tract, etc.

• Some of the commensal bacteria may become pathogens if they gain access to normally sterile internal sites through wounds, medical device insertion, etc. OPPORTUNISTIC PATHOGENS. ! • Compromised immune systems also can create opportunities for pathogenesis. ! • Some pathogens are extrinsic and not related to our commensal flora. Some non-commensal pathogens

• Mycobacterium tuberculosis (“acid-fast”; waxy mycolic acid cell surface) • Acinetobacter baumannii (Gram-, highly resistant to most antimicrobials; significant source of hospital-acquired infections) • Stenotrophomonas maltophilia (Gram-, naturally resistant to broad spectrum antimicrobials; medical devices) • Burkholderia cepacia (Gram-; naturally resistant to many antimicrobials including polymyxin; transmissible, pathogenic; cystic fibrosis) • Neisseria gonorrhoeae: gonorrhea • Treponema pallidum: syphilis • Chlamydia trachomatis: chlamydia • Bacillus anthracis (Gram+; environmental exposure, bioweapon): anthrax • Salmonella typhi: typhoid fever Impact of antibiotics on our microbial flora

• The antimicrobials are not so speciﬁcally targeted that they knock out only the primary pathogen, they can act potently against other species of the ﬂora.

• Can compromise the balanced bacterial ecology, especially of the gut • e.g. leading to diarrhea (antibiotic associated diarrhea, AAD), C. difﬁcile overgrowth

• The flora can also be reservoirs for transferable resistance factors (R-factors). • R-factors can be detected even during the course of the therapy, and persist for years after antibiotic therapy Bacteria involved in common infections Common infections •Sinusitus • 30-40% Streptococcus pneumoniae (“S. pneumo.”) • 20% Haemophilus influenzae (“H. flu.”) • 20% Moraxella catarrhalis (“M. cat.”) ! •Acute otitis media (middle ear infection) • By age 3, up to 70% of children have experienced at least 1 infection • 40% S. pneumoniae; 30% penicillin-resistant (PRSP) • 30% H. influenzae; 30% produce beta-lactamases • 15-25% M. catarrhalis; 90-95% of these isolates produce beta-lactamases ! •Community-acquired pneumonia • 50-80% S. pneumonia • 2-18% H. influenzae • 2-5% S. aureus • ~5% anaerobes • ~5% other Gram- ! •By comparison: Hospital-acquired pneumonia • Pseudomonas aeruginosa • Staph. aureus • Klebsiella pneumoniae • Enterobacteriaceae (Enterobacter, E. coli, Proteus, Serratia marcescens, A. baumanii) Common infections •Urinary tract infections • Community acquired UTI: • 80% E. coli • 10% Staphylococcus saprophyticus • Nosocomial UTI: • Klebsiella • Proteus • Enterobacter • Pseudomonas Respiratory, sinus and ear infections: S. pneumoniae

• Streptococcus pneumoniae: Gram+ • Pneumonia (500,000 cases/yr); S. pneumo the most common cause • ~40,000 cases/yr of invasive pneumococcal infection • 28% resistant to at least one antibiotic • 11% resistant to 3 or more antibiotics • Sinusitis and otitis media (7,000,000 cases/yr) • Sepsis (55,000 cases/yr) • Meningitis (6,000 cases/yr) • Penicillins front-line drugs, but now 30% is • penicillin resistant (PRSP); multi-drug resistance • also observed • Vaccine helping to reduce prevalence • of antibiotic resistance Respiratory, sinus and ear infections: H. inﬂuenzae

• Haemophilus influenzae: Gram-, facultative anaerobe • Opportunistic commensal bacteria • Pneumonia • Sinusitis • Otitis media (middle ear infection) • Vaccine (HiB conjugate vaccine) is available • and has reduced frequency of invasive infections • relating to encapsulated serotype B • 30% beta-lactamase producing • Some show modified PBPs conferring • penicillin resistance, but cephalosporins may • be effective, as well as macrolides, • fluoroquinolones Respiratory, sinus and ear infections: M. catarrhalis •Moraxella catarrhalis: Gram-, aerobic, commensal found in upper resp. tract •Carriage in children up to 75%, in adults only 1-3%, esp in fall and winter •Emerged as pathogen, esp for children, adults w/ COPD, immune compromised •Otitis media (middle ear infection) •Pneumonia •Bronchitis •Sinusitis •Meningitis, sepsis more rare •Lower respiratory tract infections •COPD patients •Pneumonia in elderly •Hospital outbreaks Strep. pyogenes • Strep. pyogenes: Gram+; “group A beta-hemolytic streptococcus (GAS) • Sometimes part of flora, nonpathogenic, asymptomatic • Skin and wound infections • 10,000,000 cases/yr in USA infection: impetigo, cellulitis • 4500 cases/yr invasive infection: necrotizing fasciitis • Strep throat • Scarlet fever • Streptococcal toxic shock (TSS): reaction to • toxin • Acute rheumatic fever; autoimmune reaction • triggered by Strep. pyogenes • Penicillin is the drug of choice, very little • resistance has emerged; for those penicillin • allergic, macrolides, clindamycin Strep. pyogenes

• Strep. pyogenes: Gram+; “group A beta-hemolytic streptococcus (GAS) • 4500 cases/yr invasive infection: necrotizing fasciitis • Penicillin is the drug of choice, very little resistance has emerged. So why is necrotizing fasciitis so hard to treat? Staphylococcus aureus ๏ Gram+, facultative anaerobe ๏ Often found on skin and respiratory tract without causing illness ๏ Typical infections: ๏ Wound ๏ Cellulitis ๏ Sinusitis ๏ Pneumonia ๏ Food poisoning ๏ Bacteremia (sepsis) ๏ Bone (osteomyelitis) ๏ Meningitis Janice H. Carr, C.D.C ๏ Endocarditis ๏ Toxic shock syndrome (TSS)

Staph skin infection Nosocomial infections and pathogens • ~10% infection rate (btxn 48h of admission to 30d after discharge); ~90,000 deaths/yr • Tend to exhibit greater frequencies and complexities of antimicrobial resistance • Often related to insertion devices ! •Types of infections: • Urinary tract infections (30%) • Hospital-aquired pneumonia and respiratory infections (22%) • Surgery-related (14%) • Skin and mucosa (7%) • Bacteremia 6% ! •Pathogens: • Pseuodomonas aeruginosa • Klebsiella pneumoniae • Staph. aureus • Enterococcus faecalis • Enterococcus faecium • Serratia marcescens • Proteus mirabilis • Enterobacter • Citrobacter • Stenotrophomonas maltophila • Acinetobacter baumanii • Burkholderia cepacia Nosocomial pathogens: example P. aeruginosa • Pseudomonas aeruginosa: Gram-, aerobic/facultative anaerobe, opportunist • Minimal nutrient requirements, capable of colonizing wide range of environments • Frequent colonizer of medical device surfaces, e.g. catheters, ventilators • Burn and wound infections • UTI • Gastrointestinal • Bone and joint • Bacteremia (blood infection) • Respiratory infections, cystic fibrosis • 10% of hospital-acquired infections • Significant antimicrobial resistance • Biofilm formation • Low cellular permeability to antibiotics • Efflux pumps; multi-drug efflux pumps transport across BOTH membranes Enterococci: E. faecalis and E. faecium • Gram+ facultative anaerobes • Part of commensal gut flora • Major therapeutic challenges due to intrinsic and acquired resistance • Gut infection, bacteremia, endocarditis, UTI, surgical wounds • Significant antimicrobial resistance • Significant penicillin and other beta-lactam resistance • For example, vancomycin resistance emerged in Enterococci • Passed through plasmid conjugation to Staph. giving rise to VRSA

CDC Clostridium difﬁcile (C. diff.)

healthy colon Pseudomembranous colitis • Gram+, anaerobe; spore (infective state) forming • While some normally carry the bacteria, most are exposed to it in health care settings; ingested from contaminated surfaces, contact • Spores are resistant to antimicrobial therapy, can lead to relapse • Gains a foothold when other gut ﬂora are wiped out or imbalanced. • Produces enterotoxin (toxin A) and cytotoxin (toxin B) that damage host cells • ~14,000 deaths/year in US • AAD, fever, abdominal pain • Pseudomembranous colitis: a severe infection of the colon Where do antibacterials (antibiotics) come from? What Fleming saw Penicillin History

๏ 1928 Alexander Fleming observed Penicillium notatum (now classiﬁed P. chrysogenum) killing staphylococci

๏ 1940 Florey, Chain, Heatley led industrialization of penicillin, increased yields 100-fold

๏ By 1942, the ﬁrst patient was treated for septicemia, using up half of the entire world’s supply of penicillin

๏ By 1943, only enough penicillin available for treatment of ~100 patients. It could be repuriﬁed from urine and readministered.

๏ 1943 Mary Hunt, a lab worker in Peoria, Illinois, found a moldy cantaloupe at a market sporting a strain of P. chrysogenum that combined with improvements in fermentation recipes, increased yields so dramatically that 2.3 million doses were available by D-Day, 1944. Where do antimicrobials come from?

ANTIBIOTICS:! ANTIBACTERIALS:! Natural products of! Synthetic products from microbial warfare chemical screens

Penicillium chrysogenum

Arsphenamine (Salvarsan, compound 606) Streptomyces 1910 used to treat syphilis the ﬁrst chemotherapeutic drug

Prontosil, a sulfa drug (1930s) by Domagk at Bayer; useful for Streptococci www.flickr.com/photos/ ajc1/2902232380/ Where do antimicrobials come from?

ANTIBIOTICS:! ANTIBACTERIALS:! Natural products of! Synthetic products from microbial warfare chemical screens

• Beta-lactams: • Sulfa drugs (sulfonamides) • Penicillins • Quinolones • Cephalosporins • Linezolid (Zyvox) • Carbapenems • Monobactams • Aminoglycosides • Macrolides • Tetracyclines • Daptomycin (Cubicin) • Vancomycin • Chloramphenicol • Bacitracin • Phosphomycin • Polymyxin Actinobacteria: especially genus Streptomyces • Approximately two-thirds of the antibiotics (natural products) in use are from Streptomyces, including:

Erythromycin (Saccharopolyspora erythrea)! Production of pigmented • secondary metabolites by Neomycin (S. fradiae)! Streptomyces colonies. (a) • Typical colonial • Streptomycin (S. griseus)! morphologies of Streptomyces isolated from • Tetracycline (S. rimosus)! the soil. Colonies often excrete colored pigments, • Vancomycin (S. orientalis)! providing a visual recording of secondary metabolite • Daptomycin (S. roseosporus)! biosynthesis. The chemically diverse compounds • Rifamycin (Amycolatopsis rifamycinica/S. mediterranei)! represent a vast array of bioactive compounds that • Chloramphenicol (S. venezuelae)! often have pharmaceutical Puromycin (S. alboniger)! applications. (b) A • panoramic view of Lincomycin (S. lincolnensis)! Streptomyces coelicolor • colonial morphology. Both • Cefoxitin (S. lactamdurans)

Thompson CJ et al. (2002) Genome Biology 3:reviews1020 Semi-synthetic derivatives

• Medicinal chemistry starting from the natural products to produce derivatives with greater potency, broader antimicrobial spectrum, lower toxicity, and improved pharmacokinetic profile, and to overcome resistance • 1950s and ‘60s much work along these lines, especially for the beta- lactams (penicillins, cephalosporins, etc).

Penicillin G (natural product) General concepts for antibacterial drugs Therapeutic Control of Infection

Bacteriostatic: some antimicrobials do not necessarily kill the bacteria • Break the logarithmic growth phase, allowing the immune system to deal with the infection. Tend to involve inhibition of protein synthesis. • Examples: Tetracyclines, Sulfonamides, Chloramphenicol, Macrolides, Lincosamides ! Bactericidal: kill the bacterium • Examples: Beta-lactams (penicillins, cephalosporins, carbapenems), Glycopeptides (vancomycin), Aminoglycosides, Fluoroquinolones, Metronidazole • Weaken the cell wall, leading to lysis (e.g. penicillins) • Disrupt DNA replication (quinolones: DNA gyrase and topoisomerase IV) • Disrupt RNA synthesis (rifampin: RNA polymerase) • Disrupt protein synthesis (some however are bacteriostatic) • Some drugs that are bacteriostatic at lower concentrations can be -cidal at higher concentrations MIC: Minimum Inhibitory Concentration! MBC: Minimum Bactericidal Concentration

MIC Minimum Inhibitory Concentration: lowest concentration of drug that gives no visible growth after 24h incubation [drug]

medium drug+

medium drug-

Minimum Bactericidal Concentration: concentration of drug that gives no visible MBC growth even in the absence of drug

partially KILLED not inhibited inhibited A Kirby-Bauer disc test

disc saturated plated with with antibiotic Staph. Aureus

Don Stanlons; phil.cdc.gov Bioavailability

• Before the anti-microbials such as penicillin, arsenicals and sulfa drugs, both with some associated toxicities, topical antiseptics and disinfectants were the only tools available for treating infection. • Penicillin and beta-lactams in particular provided low host toxicity, high potency that could get to the site of infection and permeate it. • The drug must get to its target: • Tissue penetration • Penetrate biofilms • Bacterial cell penetration to bind to the target • Attain adequate concentrations to occupy a sufficient number of target active sites to produce desired effect, but without toxicity to host • Must remain bound for sufficient time to inhibit the biological/metabolic process that will lead to bacterial cell death Narrow vs broad spectrum antimicrobial

• Generally would like to use as narrow spectrum of antibiotic as possible so as to reduce impact on non-pathogenic flora and so as to maintain balance, prevent overgrowth • Broader spectrum increases risk of antibiotic-associated diarrhea (AAD) • Potentially provides opportunity for Clostridium difficile or Candida yeast overgrowth • But often we do not know the bacterial target, or may have a super- infection involving multiple species • Empiric therapy with broad spectrum • Identify pathogen • Switch to narrower spectrum Good things to know

• Role of antibiotics in modern medical care • Role of bacterial flora in host physiology; Impact of drugs on bacterial flora • Origin of the bacteria that cause infections: commensal vs non-commensal • Where do antibacterial drugs come from? • Frequently encountered bacteria associated with common infections • Conceptual points: • Selective toxicity • Bactericidal vs bacteriostatic • Minimum inhibitory concentration (MIC) • Minimum bactericidal concentration (MBC) • Bioavailability of antibacterials • Spectrum of coverage (broad vs narrow) Mechanisms of action of antibacterial therapeutics MEDCH 561P

E. Coli by David Goodsell E. coli a gram negative bacterium! by David Goodsell E. coli growing under nutrient-rich conditions

Brett Finlay, HHMI ribosomes peptidoglycan cell wall

metabolism

DNA replication machinery RNA synthesis DavidK. Lee Goodsell ([email protected])([email protected]) Targets are unique to bacteria, not found in humans

Murray PR, Rosenthal KS, Pfaller MA, (2009) Medical Microbiology 6th Edition Natural product antibiotics and their derivatives

Murray PR, Rosenthal KS, Pfaller MA, (2009) Medical Microbiology 6th Edition Synthetic antimicrobial agents

Murray PR, Rosenthal KS, Pfaller MA, (2009) Medical Microbiology 6th Edition Mechanisms of action: broad array of drug classes

penicillin G

tetracycline ciproﬂoxacin

prontosil

vancomycin streptomycin fosfomycin Narrow or broad spectrum

๏ Differences among bacterial species mean a drug will only be active against certain types of bugs ๏ Narrow vs. broad spectrum ๏ Gram+ vs Gram- ๏ Target expressed? ๏ Details of target enzyme structure ๏ Differences in resistance mechanisms Things to know

Drug’s mechanism of action

Spectrum of activity

Resistance

Adverse effects

Uses Disruption of the cell wall and its synthesis

cell membrane (outer): polymixin peptidoglycan cell wall: beta-lactams vancomycin cycloserine cell membrane (inner): fosfomycin daptomycin bacitracin

DavidK. Lee Goodsell ([email protected])([email protected]) Gram-positive vs. Gram-negative bacteria

Gram-positive Gram-negative

outer membrane membrane inner membrane peptidoglycan peptidoglycan

cytoplasm cytoplasm

lipopolysaccharide (endotoxin) periplasmic space

Purple crystal violet stain washes away; Retains the purple crystal violet stain to see bacteria, stain with a second dye Gram-positive bacteria secreted beta-lactamases proteins, glycoproteins

peptidoglycan

teichoic acid PBP cellular membrane

efﬂux pump ๏ Relatively simple cell wall ๏ Single membrane ๏ Thick (20-80nm) peptidoglycan layer, can be up to 40 layers thick ๏ High internal osmolarity ๏ Less developed biosynthetic capability ๏ Lysozyme, a protein in our innate immune defense, digests peptidoglycan; found in tears, mucus, saliva Gram-negative bacteria

porin lipopolysaccharide

outer membrane

peptidoglycan beta-lactamases periplasmic space PBP

inner membrane efﬂux pump Gram-negative bacteria

๏ Complex cell wall ๏ Outer and inner membranes ๏ Thin (7-8nm) peptidoglycan layer only 1 or 2 layers in thickness ๏ Periplasmic space separating the two membrane barriers ๏ Porin channels in outer membrane can restrict uptake of drug ๏ Low internal osmolarity ๏ Highly developed synthetic capability ๏ Highly adaptive Outer membrane can drastically limit drug uptake: Porins

porin lipopolysaccharide

outer membrane

peptidoglycan

beta-lactamases periplasmic space PBP

inner membrane efﬂux pump ๏ Large, bulky drugs (e.g. vancomycin), >700 Daltons excluded ๏ Apolar compounds tend to be excluded ๏ Smaller, polar compounds may cross outer membrane via porins

Pages JM et al., “The Porin and the Permeating Antibiotic: a selective diffusion barrier in Gram-negative Bacteria” (2008) Nature Reviews Microbiology 6: 893-903 Outer membrane can drastically limit drug uptake: Porins penicillin G ampicillin porin

beta-lactamases PBP

๏ Large, bulky drugs (e.g. vancomycin), >700 Daltons excluded ๏ Apolar compounds tend to be excluded ๏ Smaller, polar compounds may cross outer membrane via porins

Pages JM et al., “The Porin and the Permeating Antibiotic: a selective diffusion barrier in Gram-negative Bacteria” (2008) Nature Reviews Microbiology 6: 893-903 In general, the additional, outer membrane of Gram- bacteria make them considerably harder to treat than Gram+ bacteria, and intrinsically more resistant to some drug classes Cell wall synthesis inhibitors target the peptidoglycan cell wall, its biosynthesis and maintenance.! Generally bactericidal. This is your bacteria

Brett Finlay, HHMI This is your bacteria on drugs

Brett Finlay, HHMI E. coli treated with ampicillin (a beta-lactam drug)

Brett Finlay, HHMI Peptidoglycan networks

lysozyme cuts the 1,4-beta NAG/NAM linkage

๏ Note, our cells do not make peptidoglycan, thus its synthesis in bacteria can be inhibited without affecting related processes in us. Selective toxicity. Intracellular, transport, and extracellular stages of biosynthesis

cycloserine bacitracin

beta-lactams

vancomycin

5 5

5 Protein synthesis inhibitors target the bacterial ribosomes, shutting down protein translation and elongation.! Generally bacteriostatic. Inhibition of bacterial protein synthesis

bacterial ribosome: tetracyclines macrolides aminoglycosides chloramphenicol lincosamides

DavidK. Lee Goodsell ([email protected])([email protected]) Inhibition of bacterial protein synthesis

30s 50s

70s bacterial ribosome ๏ The 70s (30s+50s) bacterial ribosomes are sufﬁciently different from eukaryotic 80s ribosomes (40s+60s subunits) that antimicrobials act selectively PDB coordinate-based illustrations by David Goodsell Inhibition of bacterial protein synthesis

A: aminoacyl-tRNA large subunit (50s): P: peptidyl-tRNA peptide chain synthesis E: free, exiting tRNA P A E

small subunit (30s): mRNA codon, anticodon pairing

•Ribosome: a protein/RNA ribozyme, functions like a machine with moving parts and substrates including mRNA, tRNA, amino acids, co- factors, the nascent polypeptide 2wdk+2wdl PDB coordinate-based illustrations by David Goodsell Inhibition of bacterial protein synthesis

๏ Ribosome: a protein/RNA ribozyme, functions like a machine with moving parts and substrates including mRNA, tRNA, amino acids, co- factors, the nascent polypeptide wikimedia commons Antimicrobials directed against protein synthesis bind to the rRNA

tetracycline

chloramphenicol

30s subunit 50s subunit PDB:1hnw PDB:1nji

PDB coordinate-based illustrations by David Goodsell Inhibition of bacterial protein synthesis 30s 50s

macrolides aminoglycosides chloramphenicol tetracyclines lincosamides streptogramins linezolid

ﬁgure by Stephen Douthwaite, University of Southern Denmark Inhibition of bacterial protein synthesis

๏ MACROLIDES: Bind to 50s subunit: ๏ Induce premature dissociation of peptidyl-tRNA from ribosome, hence premature termination ๏ Prevent addition of residues onto nascent polypeptide by blocking A to P translocation ! ๏ TETRACYCLINES: Bind to 30s subunit: ๏ Prevent aminoacyl-tRNA binding, hence peptide elongation ! ๏ AMINOGLYCOSIDES: Bind to 30s subunit: ๏ Prevent tRNA movement from A to P site ๏ Induce errors into “proofreading” and induce premature release of nonsense peptides

ﬁgure by Stephen Douthwaite, University of Southern Denmark DNA replication and RNA synthesis:! nucleic acid synthesis inhibitors

RNA polymerase rifamycins

DNA topoisomerases DavidK. Lee Goodsell ([email protected])([email protected]ﬂuoroquinolones) Interfere with DNA replication: quinolones/ﬂuoroquinolones

DNA gyrase

quinolone nucleus

๏ During DNA replication, helicases and DNA polymerase introduce supercoiling into DNA, gyrase, a topoisomerase, relieves that strain, allowing replication to proceed. ๏ Block topoisomerases II (gyrase in Gram-) and IV (in Gram+): inhibits control of DNA supercoiling and hence gene regulation and nucleoid (chromosome) packaging in the cell ๏ Pass through porins; active against both Gram+ and Gram- ๏ Bactericidal ๏ E.g. ciproﬂoxacin, levaﬂoxacin rendering of topoisomerase II by D. Goodsell

Interfere with DNA replication: quinolones/ﬂuoroquinolones

g i i i F iF

S.M. Vos et al., Nature Reviews Molecular Cell Biology 2011, 12:827 However, there also is mounting evidence that type IA protein (BLM), leads to enhanced formation of ultrafine topoisomerases, particularly topo III, can participate DNA tangles, termed microbridges, between condensed directly in hemicatenane resolution before forks con- chromosomes106. The natural fusion of a RecQ-like SF2 verge. For example, in bacteria, temperature-sensitive helicase domain with a type IA topoisomerase module topo IV-encoding alleles can be rescued by overexpres - from reverse gyrase — which likewise can resolve hemi- sion of topo III102. Topo III, which decatenates single- catenanes13,107,108 — further highlights the frequent need stranded DNA in vitro, has been shown to collaborate for pairing RecQ helicase and type IA topoisomerase with RecQ family DNA helicases to disentangle hemi- activities in the cell. catenated structures (see Supplementary information S1 (table)); in this partnership, the helicase may help gener- Topoisomerases in replication termination. With respect ate single-stranded DNA to aid topo III function. The to replication termination, topoisomerases have been functions of these two enzyme classes are entwined, as found to play a part in at least two instances. In bacteri a, evidenced by the fact that they frequently form stable the completion of DNA synthesis can be assisted by or colocalized complexes along with auxiliary single- chromosomally encoded termination regions (Ter sites), stranded-DNA-binding proteins such as Ssb (in bacteria) which bind dedicated factors that s help arrest replicative or one or more replication protein A (RPA)-like factors helicases. In E. coli, the absence of topo IA diminishes (that is, RecQ-mediated genome instability protein 1 the ability of the bacterium’s cognate termination factor, (RMI1), present in all eukaryotes, and RMI2, a second terminus site-binding protein (Tus), to block progression protein present in metazoans)9,103–105. Consistent with of the replicative helicase, DnaB109. This effect may arise the collaboration between these proteins being part of as a consequence of the increased levels of negative super- a functional ‘resolvosome’, ablation of the RecQ family coiling resulting from the absence of topo IA: increased partner of topo III in eukaryotes,In bacteria, Bloom’s a need forsyndrome compaction supercoiling has been invoked can stimulate remains DNA an unwinding open question, by DnaB but the and mechanism almost cer- as one of the principal reasons why chromosomal DNA tainly relies on proteinaceous factors. Overall, the role of is negatively supercoiled and why gyrase, which adds supercoiling in DNA compaction, and how topoisomer- | DECEMBER 2011 | VOLUME 12 those supercoils to DNA, is so ubiquitous throughout ases collaborate to define the superhelical ‘set point’ of the 53,55 © 2011the bacterialMacmillan Publishersdomain Limited.. More All recentrights reserved studies, however, chromosome, is not fully understood. suggest that there are additional layers of complexity to Topoisomerases play another part in chromosome Superhelical densities this view. For example, two closely related bacterial organ- compaction by working with large condensation machin- Measurements of the over- or isms, Escherichia coli and Salmonella enterica (specifically, eries (see Supplementary information S1 (table)) — prin- under-twistedness of DNA, S. enterica subsp. enterica serovar Typhimurium), have cipally a group of ATP-binding-cassette ATPases (ABC generally expressed as the about 90% sequence identity across homologous genes56 ATPases) known as structural maintenance of chromo- ratio by which the twist of the superhelical densities 63–67 supercoiled state differs from but have highly dissimilar , with E. coli somes (SMC) proteins . SMCs, and their paralogous that of the relaxed state. DNA being substantially more underwound than that of counterparts RAD50 (in eukaryotes) and SbcC and RecF S. enterica57. Gyrase itself is not always necessary for DNA (in bacteria), are conserved factors involved in multiple ATP-binding-cassette packaging, as the lone type IIA topoisomerase present in aspects of chromosome cohesion and condensation, and ATPases the hyperthermophilic bacterium Aquifex aeolicus is not a DNA repair68. SMCs and type IIA topoisomerases indi- (ABC ATPases). A family of proteins that include gyrase, as might be predicted on the basis of its sequence rectly colocalize as part of the protein network that helps membrane-bound similarity with other gyrase amino-terminal domains, stabilize long-range contacts between chromosomal seg- transporters, DNA repair but is rather a topo IV58. However, many thermophiles ments67,69. SMCs, or their affiliated accessory factors, have factors and structural also possess reverse gyrase59, which introduces positive also been reported to directly associate with both topo II maintenance of chromosomes (SMC) proteins. ABC ATPases supercoils that may aid compaction as well as counter- and topo IV. For example, the D. melanogaster subunit 60,61 possess a conserved ATPase act thermal denaturation . Although the steady-state Barren (an orthologue of condensin H (also known as site that is often formed at superhelical density of DNA in most bacteria and archaea condensin complex subunit 2)) co-immunoprecipiates dimer interfaces. ATP binding has not been measured directly, multiple findings suggest with topo II in vitro66, and a dimerization region of results in conformational that many species, particularly those that are adapted the E. coli SMC homologue, MukB, interacts with the changes (often dimerization) that affect the associated to growth at high temperatures, have relaxed chromo- carboxy-terminal domain of the ParC subunit of topo 62 64,65 partner proteins and somes . How such organisms are able to sufficiently com- IV . Interestingly, both interactions appear to potenti- substrates. pact their chromosomes in the absence of supercoiling ate the relaxation of negatively supercoiled DNA by the

๏ The quinolones block the religation step. The nuclease domain still functions properly. ๏ The DNA ends up getting fragmented, thus killing the bacterium. target RNA synthesis: rifamycins

RNA polymerase by D. Goodsell rifampin

๏ From Actinobacteria Amycolatopsis mediteranie ๏ Binds to bacterial RNA polymerase, inhibit RNA synthesis by blocking chain elongation, blocks mRNA transcription ๏ Bactericidal ๏ Treatment of mycobacteria infection (TB, leprosy), some Gram+ ๏ Some activity against HIV’s reverse transcriptase (not clinically tested) Folic acid synthesis inhibitors

๏ Inhibition of folate synthesis in bacteria: ๏ Sulfa drugs (sulfonamides): an “antimetabolite” that inhibits dihydropteroate synthase by competitive binding with p-aminobenzoic acid (PABA) ๏ Folate is a critical for DNA synthesis ๏ Bacteria make their own folate, we do not synthesize our own folic acid, our cells import it ๏ Prontosil, the original sulfonamide drug (actually a prodrug) ๏ Trimethoprim/Sulfamethoxazole (TMP-SMX) synergistic

Dihydropteroate! diphosphate! + PABA Targets are unique to bacteria, not found in humans

Murray PR, Rosenthal KS, Pfaller MA, (2009) Medical Microbiology 6th Edition