Building a Morbidostat: an Automated Continuous-Culture Device For

Home , Erlenmeyer flask, Laboratory drying rack

© 2013 Nature America, Inc. All rights reserved. engineers familiar with basic microbiology. whole system can be built from readily available components within 2–3 weeks by biologists with some electronics experience or from bacterial cells suspended in the liquid culture. rate measurements are done using an optical detection system that is based on measuring the intensity of back-scattered light dynamically adjusts drug concentrations inside culture vials in order to maintain a constant drug-induced inhibition. predetermined drug environments, the morbidostat constantly measures the growth rates of evolving microbial populations and T We present a protocol for building and operating an automated fluidic system for continuous culture that we call the ‘morbidostat’. Published online 21 February 2013; doi:10.1038/nprot.2013.021 E.T. ( Colombia. Turkey. 1 intervals ( intervals ( volume fixed a in constantly challenged ( is population bacterial the that such adjusted dynamically is that concentration a at antibiotic an containing environment trolled the morbidostat, for studying evolution of drug resistance in a con different or drugs drug combinations in populations bacterial dynamics of to the quantify evolutionary mental systems become particularly important for studies that aim useful for the studying evolution drug resistanceof of growth inhibition on evolving bacterial populations are therefore automatically adjust drug concentrations to maintain a fixed level can that systems Experimental predicted. be cannot populations evolving complex on mutations emerging of effects phenotypic centrations are determined beforehand is almost impossible, as the out experiments using environmentscarrying in which drug con However, challenged. constantly is population bacterial evolving thus be able to keep increasing the drug concentration such that the multiple mutations,through accumulation sequential of we must levels resistance.of To be able to follow the evolution resistanceof drug is relieved and there is no additional pressure to evolve higher the by inhibition the population, the through sweep and emerge mutational few steps a conferring most resistance. at or Once such one single only mutations to limited generally are studies those concentration, drug fixed a to subjected typically are bacteria as emerge that mutants resistant the characterizing by and antibiotics by inhibited is to growth which culture in environment bacterial an a exposing by laboratory the in studied be lives of millions threatening and useless drugs available currently dering problem, ren health public important an is resistance Antibiotic I Erdal Toprak under dynamically sustained drug inhibition culture device for studying bacterial drug resistance Building a morbidostat: an automated continuous- Similar to a chemostat, in which cell cultures are ( Department of Systemsof Department Biology, Harvard Medical School, Boston, Massachusetts, USA. ∆ NTRO he he morbidostat is used to follow the evolution of microbial drug resistance in real time. Instead of exposing bacteria to We recently introduced an automated continuous culture device, V ) of fresh medium or fresh medium containing dissolved drugs. [email protected] 3 D Health Sciences and Technology Program, Harvard Medical School, Boston, Massachusetts, USA. 1– UCT 5 4 School of Engineering and Engineering School Appliedof Sciences, Harvard University, Cambridge, Massachusetts, USA. Correspondence should be addressed to . Evolution of resistance by spontaneous mutations can can mutations spontaneous by resistance . Evolution of ∆ t ) ) the culture a is fixeddiluted byof amount injection I ON 1 , 2 , Adrian Veres V ) with continuous stirring, and at fixed time time fixed at and stirring, continuous with ) Fig. 1 ) ) or R.K. ( ) 5 . The bacterial population is growing 3 , Sadik Yildiz [email protected] 8, 9 .

periodically diluted 2 7 , Juan M Pedraza . Such experi 5, T 6 he he morbidostat can additionally be used as a chemostat or a turbidostat. . However,. ). - - - - lation, the dilution rate of the morbidostat morbidostat the lation, of rate dilution the at a constant rate lower than the maximal growth rate of the popu tion and not by nutrient depletion, the evolutionary changes changes evolutionary drug the with are associated likely to be populations the in occurring depletion, nutrient by not and tion kept constant using a suction pump ( Throughout the entire experiment, the volumes of the cultures are ( drugs of the system is half of the growth rate of bacteria in the absence of V growing at a maximal rate of accordingly. rate Typically,growth the reduce to bacteria tion for rate of the bacteria forces the system to adjust the drug concentra dilution rate OD enough such that the population is never nutrient limited, typically ( injections medium fresh successive with dilution by reduced ally inside the culture vials increase with drug injections and are gradu concentrations Drug injected. is medium fresh cases, other all in ( rate dilution the than higher is only if the OD exceeds a threshold (OD fixed volume fresh medium or fresh medium plus drug (in either case, the same current OD of the culture, the morbidostat decides whether to add gray dots). Next, depending on the calculated growth rate and the (OD) measurements ( density optical lated on the of basis each period of concentration. drug Atend the adjusting byexternally controlled is rate growth its instead, limited; nutrient not is population the availability, in the morbidostat the cell density is kept low such that stat, in which the bacterial growth is inherently limited by nutrient chemo However,rate. a todilution this contrast matches in that value reach a must fixed.rate growth state,Insteady bacterial the 2 Fig. 1 Faculty of Engineering and Natural Engineering Faculty of Sciences, Sabanci University, Istanbul,

As the bacterial population growth is limited by drug inhibi drug by limited is growth population bacterial the As = , Remy Chait THR

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he he t 555 ) ) is b ------, © 2013 Nature America, Inc. All rights reserved. drug concentrations must be manually adjusted on a daily basis basis daily a on adjusted manually be must concentrations drug rate desired a at bacteria evolving of growth the bioreactor using drug-containing growth medium that will inhibit a in evolved be can bacteria Alternatively, populations. evolving in concentrations drug the adjusting dynamically by inhibition per hour, nearly continuous allowing growth rate maintenance of For ( period growth each for measurements rate growth precise perform can morbidostat the intervals, 1-s at continuously taken cally perturbed with high time resolution. As OD measurements are tion experiments in a dynamic environment that can be systemati evolu laboratory enabling of advantage the has morbidostat The other methods with Comparison toexperiments out be in carried a laboratory.biology of range diverse a enables morbidostat a Therefore,constructing sity, by making only simple modifications to the control algorithm. rate, or as a turbidostat, in which cells are grown at a constant den used as a chemostat, in which cells are grown at a constant growth staltic pumps with formulators. The morbidostat apparatus can be peri the replacing or optics detection density cell sensitive more sary, many other improvements to the system can be made by using neces control. If temperature with room environmental an in or capacity is easily increased if the setup is built in a larger incubator however, inches); 20 × 24 × this 24 dimensions: (inner incubator pendently controlled cultures in parallel in a mid-sized commercial inde 15 growing allows currently design transfer. Our liquid for assembly of a computer-controlled array of peristaltic pumps used bidostat culture vials, assembly of the optical detection system and 3 within weeks levels resistance steady high, attained cultures the of all studies, recent our in used drugs three increase all For observed. of is resistance rate in diminishing a until experiments morbidostat continue typically We mutations. resistance multiple mulating to population tion challenges the bacterial keep evolving by accu the system.the fixed rate of dilution inhibi continuous The drug to converges rate growth the and dose drug higher a using ited concentration is adjusted such is inhib that the again population drug the response in and drug, the of presence the to in faster grow starts it resistant, more becomes and evolves population the Once resistance. concentrations. TMP, trimethoprim. analyzing growth rates and corresponding drug inhibits growth by 50% (IC injections. ( (magenta) between two consecutive drug solution ( ( with drug solution and fresh medium, respectively. with magenta and green colors represent dilutions exponential growth functions (black lines). Markers populations are periodically calculated by fitting with gray dots. Growth rates ( growth in the morbidostat. OD values are shown of the morbidostat. ( bacterial populations. ( constant level of growth inhibition on evolving continuous-culture device that maintains a Figure 1 556 population evolving the of resistance the quantifying by d c protocol ) Final OD values at the end of each growth cycle. ) Growth rates (blue) and drug concentrations Construction of the involvesmorbidostat of Construction the assembly mor of

| ∆ VOL.8 NO.3VOL.8 t

| = The morbidostat is an automated

12 min, this results in five growth rate measurements measurements rate growth five in results this min, 12 e ) The drug concentration that 5 . | 2013 b ) Representative bacterial a ) The working algorithm | 50

natureprotocols ) is calculated by r ) of bacterial a c OD 10 10 Waste LED pump 6 . However, the . –2 –1 47 51 6 V .Unlike 55 Time (h)

∆ Detector t Control algorithm ). 59 ------Grow for

� t 63 drug levels, the morbidostat does not relax selection as the bacteria which selects just once for bacteria capable particular of surviving or selection on agar plates or liquid with fixed drug concentrations, assay diffusion disc conventionalthe such as techniques selection PEEK (polyether ether ketone; Fisher Scientific) tubing is is tubing Scientific) Fisher ketone; inserted into these openings for adding or removing ether (polyether PEEK excess liquid for keeping the culture volume constant. Autoclavable two separate drug solutions; filtered air intake; and removal of the medium; fresh of injection following: the for reserved are insert secure sealing of the bottle opening. The five openings on the Teflonthrough holes (Chemglass) and the open-top screw cap are used for fiveavailable,Teflon commercially nowwith but designed, insert the bottle for mixing the culture with a magnetic stirrer. into A dropped custom- is (Stirbars.com) bar stir magnetic A morbidostat. cells in the are for bacterial used growing screw (Chemglass) caps ( vials culture morbidostat 15 constructing with starts morbidostat the Construction of the morbidostat vials. the procedureof Overview cells. cancer in resistance drug of evolution the and communities microbial in interactions host-parasite yeast, and bacteria in response stress of evolution address that experiments adaptation long-term out carry to used be potentially could morbidostat the system the to modifications time-consuming any resistance. without that drug speculate bacterial We of evolution the studying to limited not are morbidostat the with done be can that experiments The Other applications potential multiple isolates resistance-conferring mutations that carry one mutation that will release the selection pressure, unlike clinical only develop to tend environments drug constant in selected are However,morbidostat. the comparedwith that mutants bacterial or medium havegrowth relative the main advantage of simplicity plates agar antibiotic-containing out using are that ments carried drug resistance in real time. In contrast, microbial selection experi in multistep increases makesto it follow possible evolution the of ( inhibition growth bacterial maintain to in drug and sensitivity adjusts the drug concentration accordingly resistant increasingly become Media pump Drug pum & 67 OD > ∆ OD >0 Fig. d p 47 THR 2 Yes ). Flat-bottom glass vials (Chemglass) with open-top open-top with (Chemglass) vials glass Flat-bottom ). No 51 Add Add media drug Time (h) 55 � � V V 59 63 b OD 7,9,1 67

0.10 0.15 0.17

0 1 2 3 4

µ ) ml g (

(TMP) (TMP) 52 0 . Instead, it follows the changes changes the . follows Instead, it –1 OD THR e The procedure for building Growth rate (1 h–1) r 10 0 0 –2 50% growth Fig. 1 Fig. ∆ inhibition OD Time (h) (TMP) 53 d IC ). This feature feature This ). r �

50 ( t liquids. High µ g ml 10 –1 � 0 V ) / V 5,1 1 54 . -

© 2013 Nature America, Inc. All rights reserved. evolved strains with or without drugs were very similar the to the the of verify sizes the size that found Forinstance,we effects. such of to of extent measurements control perform we cases, such in Furthermore, environment. nutrient-rich a in grow always cells in our measurements, the assaymorbidostat is such designed that effects these To vials. minimize culture the in lysates cell or cells filamentous long of because complications some have inherently temperature-controlled incubator. the entire ( holder tube array OD,to readings voltage converting for calibration a doing After (DAQ) acquisition data a using card (Measurement Computing). ( photodetector the across Shack; (Radio them feeding circuits the to connected and holder tube each to tered light. An LED light source and a photodetector are mounted scat of detection the maximize to angle 135° a at positioned are sources photodetectors. and for light LED openings These drilled ( rial mate Delrin black from machined are holders tube all,15 ets.In brack L using stirrer magnetic the on mounted and machined is ( Figure 3 and to read their ODs. A Plexiglas adaptor ( The tube holder array is used to continuously mix bacterial cultures tube holders, which sits on a stirrer magnetic ( 15-position array. holder tube the of Construction tube assembly can be sterilized by autoclaving when necessary. entire The Plastics). (Value connectors style thread Luer female vials are made using autoclavable silicone tubing culture (VWR) and and male/ pumps between connections PEEK tubing to the Teflon insert. All of the the upper face of the Teflon insert to fix the temperature–resistant silicone is applied to face of the Teflon insert. resistant silicone is applied to the entire upper volume of which is 12 ml. ( just touches the top of the culture in the vial, the the longer PEEK tubing is adjusted such that it ( insert assembled with PEEK tubing pieces. silicone tubing and Luer connector. ( assembly. ( Figure 2 LED holder. ( c d a ) Legs of the LED are soldered to wires. ( ) Assembled morbidostat vial. The height of Measuring the growth rate by directly using OD values may values OD using directly by rate growth the Measuring Supplementary Fig.1 Supplementary

| | Construction steps for tube holder assembly. ( Construction steps for morbidostat vial a ) PEEK tubing pieces. ( f ) LED and photodetector assemblies are inserted into the custom-machined Delrin tube holders. Fig. 4 b a ). A circuit the is voltagebuilt for measuring e ) High temperature– Fig. 4 Fig. ). Every tube holder has two openings openings two has holder tube ).Every Supplementary Fig. Supplementary 2 b ) Assembled c ) Teflon b ), and the voltages are recorded are voltages the and ), d

) Wire connections are insulated with heat-shrink insulators. ( c We construct an array of of We array an construct Supplementary Fig. 1 b a a ) Commercial LED holder. ( ) ) is placed in a d Fig. 3 ). ) - - - c

wild-type bacteria in the absence of drugs of absence the in bacteria wild-type on on a custom-made aluminum pump rack ( mounted are Solutions) (Clark pumps peristaltic 45 all, In tions. pump array that will be used to inject fresh medium and drug solu the of Construction pump array. causing drugs filamentation. or drugs bactericidal using when important especially are ments fromglycerol stock are added. These cultures are grown filled with 12 ml of growth medium, and then 10 Exponential growth rate measurements. are before performed each new long-term experiment. tests initial following the assembled, is apparatus entire the After Initial tests to assayoptimize the morbidostat Interface (GUI.m). MATLABcustom a with Usercontrolled is Graphical a code with safely, the system is sterilized and is ready for use. The entire system components work properly and are secure electrical and nections con tubing all that ensuring After (VWR). tubing silicone able autoclav with vials culture to connected are Pumps device. relay for pump Apparatus). the waste of pump The activation is also done the via waste a (Harvard vials as culture the in volumes used culture fixed maintaining is pump peristaltic 16-channel A pumps. these to connected are and bottles reservoir medium as Computing). Modified autoclavable screw cap glass bottles are used 24-channel electromechanical relay interface devices (Measurement 3 Fig. b ) The LED and the photodetectors are inserted into the LED holder. ). These pumps are activated via two computer-controlled computer-controlled two via activated are pumps These ). e d e ) Custom-machined LED housing is attached to the natureprotocols f The final step is to assemble the All of the culture vials are e

5 | Fig. VOL.8 NO.3VOL.8 . Such control experi control .Such 5 µ , protocol l of bacterial cells Supplementary Supplementary | 2013

overnight

557 - - - -

© 2013 Nature America, Inc. All rights reserved. stock. The morbidostat setup is operated using the chemostat chemostat the using operated is setup morbidostat The stock. ( ml 12 with filled are vials culture the tubing, peristaltic the of all initializing and system the of components medium-interfacing rate. dilution of Measurement medium used in the experiments. nentially. This range can vary depending on the strains and growth rate in the absence and drugs the of OD at which cells grow expo growth as such parameters experimental characterize to used are pieces are inserted through holes on a GL45 screw cap. High temperature–resistant silicone is applied to the entire upper face of ( the screw cap. ( Figure 5 all of the pumps are turned off. The resulting growth curves ( and the OD values are recorded throughout the while experiment are connected in parallel. ( connected to a 68- Figure 4 558 assembled medium reservoir. b b a V protocol b a ) Each photodetector is connected to a resistor in series. All photodetectors ) Two pieces of silicone tubing are attached to the pumps. ( ) of growth medium and 10 10 and medium growth of )

| VOL.8 NO.3VOL.8 6 6

V V | | Construction steps for pump array and medium reservoir assemblies. ( Circuit diagrams for the OD detection system. (

R = 68 Ω | Ω R = 100 k Detector-1

2013 Ω resistor in series. All LEDs are connected in parallel. LED-1 |

natureprotocols V

1 R = 68 Ω LED-2

b R = 100 kΩ Detector-2 µ After sterilizing all culture- and and culture- all sterilizing After l of bacterial cells from glycerol from cells bacterial of l R = 68 Ω V LED-3 2 a ) Each LED is c

) An assembled peristaltic pump with Luer connectors and silicone tubing. ( R = 100 kΩ Detector-15 R = 68 Ω c LED-15 Fig. V 15

6 ) - a ) A peristaltic pump is attached to the pump stand ( plates in which each consecutive well on a row is three-fold diluted, and the MIC is not known, we make a drug gradient on the 96-well multiple replicates is convenient. If the drug is used for the firstplates, 96-well require they volumeas less measurement and with time using prefer generally We availability. the on depending tubes, growth cell sterile or plates 96-well using done be can surements MIC of each drug is measured before the experiments. These mea (MIC). concentration inhibitory minimum the Measuring such parts as tubing pumps. and peristaltic commercial the of to inherent imperfections vesselsowing dostat necessary, as actual dilution rates can slightly vary between morbi is comparison Suchrate. dilution theoretical the with it compare and rate dilution data,chemostat the experimental the we extract formula: following the using calculated be can rate dilution theoretical The culture. the ( min 12 every ( min ~50 every double °C,cells 30 at amicase and glucose taining MG1655 exponential growth rate ( The algorithm such that fresh medium is periodically added to the vials. fresh medium containing drug solution to the bacterial cultures cultures bacterial the to solution drug containing medium fresh adds former the a is and that chemostat morbidostat the between difference operational main rate.The dilution fixed a at medium morbidostats and chemostats feed growing cell cultures with fresh inhibition. growth for concentrations drug of Estimation prepared according to the instructions provided by the suppliers. me MIC For necessary. is value MIC precise more a if used is surement, gradient, which covers mea theMICvalue found intheprevious drug linear a using measurement A MIC. the as assigned is OD) in increase no shows well (the survive cannot bacteria which at temperature. At the end of 24 h, the minimum drug concentration well and incubate the plate on a plate shaker for 24 h at the desired last well having no drugs. Later, we add roughly 1,000 cells to each with the first well having the strongest drug concentration and the r 0

dilution rate ( rate dilution

0.8 h 0.8 a surements, fresh drug solutions from powderarefromstocks solutions surements, drug fresh Escherichia coli Escherichia

− d

1 ). Under these settings, we dilute cultures by ~8% ~8% by cultures dilute we settings, these Under ). ∆ t ) by adding ~1 ml ( ml ~1 adding by ) r dilution r dilution cultures in M9 minimal medium con medium minimal M9 in cultures r ) of the system should be less than the the than less be should system the of ) 0 ) of ) the of cells. When we grow wild-type

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© 2013 Nature America, Inc. All rights reserved. • • • • • • Tube holder assembly array • • • • • • • • • • • • • • assembly Glass vial EQUIPMENT • • • • • • • • REAGENTS M drug-sensitive eral sev grow we cultures, the into added be to has drug much how Tois important. drugs toable properthe add of amount estimate rate is higher than the dilution rate of the system. Therefore, being growth the and threshold a exceeds culture the of OD the when shows the range in which the growth is exponential. overnight and OD values are recorded (gray line) every second. Red line is fitted for finding the calibration factor. ( created by cultures with known OD values are recorded. A line (red line) Figure 6 a O-rings for supporting tubes inside tubes theholder for supporting O-rings Custom-machined holder tube Delrin Machined cover Plexiglas ( Machined Lbrackets stirrer (Neutec, magnetic 15 position cat. no. VL -F203A0178) Machined ( sheet Plexiglas filterSyringe (0.2 filterSyringe (0.2 bar(Stirbars.com, stir Magnetic cat. no. SBM2003MIC) Female Luer cap(Value thread style Plastics, cat. no. FTLLP-6005) Male Luer cap(Value thread style Plastics, cat. no. MTLLP-6005) Permatex 81878no. Copper 101sensor safe Ultra hi-temp RTV silicone 24–400 open-top GPI cap(Chemglass, cat. no. CV-3750-0024) HMS-0909-151GC) no. cat. (Chemglass, cap GPI open-top 24–400 for insert Teflon vial,Flat-bottom glass 40ml(Chemglass, cat. no. CG-4902-08) Male Luer connector (Value Plastics, cat. no. MTLL004-6005) Female Luer connector (Value Plastics, cat. no. FTLL004-6005) Silicone tubing (1mminner diameter (i.d.), VWR, cat. no. 60985-708) PEEK cutter (Fisher Scientific, cat. no. 05-701-84) PEEK tubing (Fisher Scientific, cat. no. 05-701-6) Sterile water Ethanol Bleach Antibiotic(s) to tested be (Reagent Setup) pathogenic bacteria. for with experiments for microbiological practices. Note may permissions thatspecial required be rules thebiosafety following byresistant handled shouldbe strictly bacteria kept asglycerol stocks at subtilis Bacillus E. coli Amicase (Sigma-Aldrich, cat. no. 82514) Glucose (Sigma-Aldrich, cat. no. G7021) M9 minimalsalts, 5×(Sigma-Aldrich, cat. no. M6030)

ATER Optical density 0.2 0.4 0.6 0.8 1.0 0 0 , MG1655 wild-type laboratory strain. Other bacterial strains such strain. as strains Otherbacterial laboratory , MG1655wild-type I

| ALS Calibration of the detectors. ( 0.3 can also be grown inthemorbidostat. canalsobe are strains Bacterial 0.6 µ µ Volts m; VWR, cat. no. 514-0068) m; VWR, cat. no. 514-0066) E. coli E. 0.9

− Supplementary Fig.Supplementary 1 Supplementary Fig.Supplementary 1

80°C 1.2 cultures until they reach the threshold threshold the reach they until cultures !

CAUT 1.5 b a I Optical density ON ) The voltages (gray circles) 10 10 10 10 b –2 –1 ) An Both drug-sensitive Both anddrug- 0 1 5 0 E. coli ) ) culture is grown Time (h)

10 15 - (OD when when we use light-sensitive drugs. suppliers. For example, we drug solution aluminum wrap bottles with foil these solutions,lifetime of we follow provided strictly the byinstructions the to strains ensure that the the efficacy is of drugs not decreased. To extend the the MIC our of drug solutions on drug-sensitive a daily basis using wild-type Handling antibiotics REAGENT SETUP • • medium bottles reservoir Constructing • • • • • • Pump construction array • • • • • • • • • • • • • • • • • • If these If conditions are met, we the start long-term experiment. of ( nected to the peristaltic pumps and morbidostat algorithm is tested from stock B. All fresh medium and drug solution bottles are con injections making starts morbidostat the long, too takes A stock cells acquire resistance and inhibiting growth with injections from morbidostat tubes are initially made from stock A. However, if the stock B is 50× MIC (or 5× stock A). All the of drug into injections tion of stock A is typically 10× MIC and the drug concentration of medium are prepared (stock A and stock B). The concentra drug the solution drug that should be used, two solutions drug in fresh Trial run of the experiment. inhibited growth sufficiently. that drug solutions at concentrations ten times higher than the MIC depending on the drug type and concentration. We generally found concentrations. The growth rates of these cultures tend to decrease Supplementary Fig. 4 Medium storage bottle with GL45 screw cap, 1 liters (VWR, cat. no. 1395-1L) no.1395-5L) cat. (VWR, liters 5 cap, screw GL45 with bottle storage Medium Erlenmeyer flask,(VWR, 6liters cat. no. 1500–6000) Male Luer connector (Value Plastics, MTLL025-6005) Silicone tubing (0.125-inch i.d., VWR, cat. no. 89068-474) pump(Harvard 16-channel peristaltic Apparatus, cat. no. 73-3154) USB-ERB24) USB controller relay interface (Measurement device Computing, cat. no. Peristaltic Solutions, pumps(Clark cat. no. m451605) Spectrophotometer andcuvettes Serological pipettes tubes Eppendorf Wire (for zipties things) organizing insulation and/or heatshrink tape Electrical toolsSoldering Wire stripper software) Windows computer MATLAB with software (for 32-bit control installed Uninterruptable power supply (UPS; Amazon.com) DAQ card (Measurement Computing, cat. no. USB-1616FS) Temperature-controlled incubator (VWR, cat. no. 1535) High-power AC adaptor Shack, (Radio cat. no. 273-318) Various resistors Shack, (Radio 10–100k Resistor, 68 Colored (Amazon.com) wire stranded 22-gauge Machined housingfor LEDholder IR LEDemitter anddetector Shack, (Radio cat. no. 276-142) LED holder Shack, (Radio cat. no. 276-080) E. coli THR cells with one or two consecutive drug injections ( ) and then manually add 1 ml of drug solution with various Ω (Radio Shack, (Radio cat. no. 271-1106)

Throughout Throughout the experiments, morbidostat we measure ). We generally aim to slow down the growth natureprotocols After quantifying the concentration of Ω )

| VOL.8 NO.3VOL.8 protocol

| 2013 Fig. 1 |

559 b ). - - © 2013 Nature America, Inc. All rights reserved.  23| 22| 21| 20| 19| A 18| 17| 16| 15| 14| 13| Teflon insert ( 12| liquid levelinside the glass vial inorder toavoid contamination bydroplets.  11| inside the glassvial ( 10| 9| 8| 7| 6| 5| 4| vials. morbidostat 3| 2| piece using aPEEKcutter. These pieces willbeusedfor liquid injections ( 1| A PROCE 560 polarities. The length of the wires depends ontheir organization. protocol ssembling morbidostatvials ssembling ssembling tube holder array

CR CR

Attach syringe filters to the female Luer connectors reserved for air inlet, taking care to keep the vial side of the filters sterile. Solder four pieces of 22-gauge stranded wires to the legsof the LEDholder and photodetector ( Pushthe legsof the photodetector allthe wayinto the LEDholder from the small opening. Pushthe legsof the IRLEDallthe wayinto the LEDholder from the small opening ( Take twoLEDholders and discard the washers and the nuts thatcame withthem ( Mount the machined Plexiglas sheet onthe 15-position magnetic stirrer using Lbrackets. Autoclave allof the assembled vials at121°Cfor 20min. Transfer the assembledvial capstocleanglassvials, eachcontaining amagnetic stirbar. Closeallof the female Luerconnectors using male Luerthread stylecaps. Wait overnight until the silicone iscompletelydried. RepeatSteps1–12tomake atotalof 15culture vials. Applyhigh temperature–resistant silicone tothe entire upperface of the Teflon insert tofix the PEEKtubing to the Pushallof the other PEEKtubing pieces through the holes onthe Teflon insert ( Pushthe 5-inch (~12-cm)PEEKtubing through one of the holes until the tubing justmakes contact withthe water | VOL.8 NO.3VOL.8 Close the opening of the bottleusing a24–400open-topGPIcap. Close the opening of the vial withthe Teflon insert for 24–400open-topGPIcap. Fill a40-mlflat-bottomglassvial with12ml of water. Insert female Luerconnectors into the other end of the piece of silicone tubing ( Insert eachpiece of PEEKtubing into one piece of silicone tubing toadepth of ~0.5inches (~1cm). Cut fivepieces of silicone tubing (1mmi.d.), each4inches (~10cm)inlength. in culture excess of extraction for used be will This length. in cm) (~12 inches 5 tubing PEEK of piece one Cut Cut one piece of PEEKtubing 1.5inches (~4cm)inlength. Thiswillbeusedfor filtered airintake. Cut three pieces of PEEKtubing, each3inches (~8cm)inlength. Aftercutting these pieces, bevelone end of each I I T T D I I URE CAL CAL

STEP STEP Fig. 2 | 2013 The bevel-shapedend of the 3-inch (~8-cm)PEEKtubing should beatleast1 inch (~2.5cm)abovethe The useof wires withdifferent colors (i.e., red, green, blue, black)ishelpful tokeep track of devices and e |

). Fig. 2 natureprotocols c ). ● ●

T T IMI IMI N N G G ~24 h ~9hplusdryingovernight Fig. 2 a ). Fig. 2 Fig. 2 Fig. 3 Fig. 3 d b ). a ). ). b ). Fig. 3 c ).

© 2013 Nature America, Inc. All rights reserved. 40| 39| C polarity.  better than100. are median-filtered everysecond toeliminate noise. Typically, signal-to-noise ratio (mean/s.d.) inourvoltage readings is tions inincident light. The DAQ card weuse issettoacquire data atanacquisition rate of 500Hz,and the voltage readings 38| holder no. 2toterminal 1and soon.  37| DAQ card. Repeatthisfor all15tubeholders. 36|  35| port onthe right side of the incubator. 34| ( 33| measure the voltages across them. Therefore, keep the connections aslong asnecessary. resistances are generally ~100k  32| 31| (e.g., webcamormobile phone camera). supply. Setthe voltage to6V. Ensure thatallthe LEDsare emitting byusing adigital camera thatcandetect IR 30| 29| insulator. 28| avoid confusion and aid indebugging.  holders ( 27| 26| 25| 24| 41| S alibration alibration of the detectors

upplementary Fig.2 CR CR CR CR CR Turn onthe magnetic stirrer thatsits under the tubeholder array and setthe stirring speedto~200r.p.m. Add ~15mlof the diluted culture inaglassvial and drop amagnetic stir barinthe vial. Diluteanovernight bacterial culture to~OD Byusing the software provided bythe DAQ card supplier, ensure thatallof the photodetectors are responding tovaria Connect these pairsof wires tothe screw terminals of the DAQ card. Connect wires tobothsides of the photodetector resistors for measuring the voltage across them using amultichannel Connect allthe photodetectors toapowersupplyand setthe voltage to6V. Guide allthe wires coming from the tubeholder array tothe outside of the incubator using the 2-inch (~5-cm)-diameter Afterallthe connections are made, affixthe machined Plexiglas covertoprotect the wires from possibleliquid spills Byfollowing the circuit diagram givenin Byfollowing the circuit diagram givenin Byfollowing the circuit diagram givenin Mount allof the tubeholders tothe circular openings onthe Plexiglas sheet attached tothe magnetic stirrer. Connect a68- Placeallof the LEDand photodetector assemblies into the openings onthe sides of the machined Delrintube RepeatSteps20–25toprepare 15pairsof LEDand photodetector assemblies. Mount the LEDholder tothe machined housing for LEDholder ( Insulate allof the wire connections using electrical tapeorheat-shrink insulator ( I I I I I T T T T T I I I I I CAL CAL CAL CAL CAL Fig. 3

STEP STEP STEP STEP STEP f ). The most common reasons for having anonresponsive detector are broken connections orreversed The DAQ card has16screw terminals, numbered 0–15.Connect tubeholder no. 1toterminal 0,tube Allof the photodetectors should beconnected in parallel. The resistance of allresistors should bechosen such that1ODchange should correspond to2V. These At thispoint, there willbe15tubeholders and 60wires. Labeling the tubeholders and wires willhelp Ω resistor tothe wire from the LEDanode and insulate the connection using electrical tapeorheat-shrink ). ●

Ω T IMI and may differfor eachdetector. We prefer tokeep these resistors outside the incubator to N G ~2 h Figure 4 Figure 4 Figure 4

0.75inminimal growth medium. b a b , connect allof the LEDsinparallel and feedthem withapower , splice the wires coming from the short legof the photodetectors. , connect aresistor tothe other legof the photodetectors. Fig. 3 e ). Fig. 3 natureprotocols d ).

| VOL.8 NO.3VOL.8 protocol

| 2013

561 - © 2013 Nature America, Inc. All rights reserved. provided withthisprotocol ( configuration, weusethe 24th relay switch of the second relay box for controlling the suction pump.The MATLAB code following the instructions provided bythe pump supplier inorder toprotect the electronic pumpcircuits. Inourcurrent  56| modified ifthe connections are made differently. the drug Bpumpof culture 1.The MATLAB code (GUI.m)provided withthisprotocol ( relay switches. Forexample, relay switches nos. 1–3onthe relay box control the fresh medium pump,the drug Apumpand  55| box and 21relay switches from the second relay box.  ! 54| 53| tape orsimilarmaterial. Wearing electrician’s glovesisadvised. should betaken for safehandling of high voltage. Allof the electrical connections should besafelyinsulated withelectrical ! 52| the pumpsproperly.  51| 50| A ? the difference incalibration factors is~5%after1month of continuous use. a MATLAB command called‘robustfit’ for fitting aline ( 15 tubeholders and fitaline (OD 49| 48| the spectrophotometer. 47| 46| 45| and transfer ittoaspectrophotometer cuvette. 44| tube holders. 43| voltage value( 42| 562 protocol

ssembling ssembling the pump array CAUT CAUT TROU

CR CR CR CR

Use one of the remaining three unoccupied relay switches tocontrol the 16-channel peristaltic suction pump. Connect the second wire of the pumpstothe NO contacts onthe tworelay boxes. Connect allof the Ccontacts of the tworelay boxes together using insulated wires. Connect the Ccontact of relay switchno. 1tothe other poleof the AC powersource. Connect one of the twowires of eachpumptoone of the polesof the AC powersource. Attach allof the peristaltic pumpstothe pumpstand ( Assemble the pumpstand ( Afterfinishing voltage and OD recordings for all15cultures, plotvoltage valuesagainst corresponding ODvalues for all Repeatthe dilution and voltage recording process (Steps46and 47)until the ODdrops below0.03. Measure the voltage valuesfor the dilutedcellculture inalltubeholders and record the corresponding ODvalueusing Dilutethe cellculture inthe glassvial byadding 5mlof fresh medium and mixwell. Measure the ODof the removed culture using astandard spectrophotometer. Record the ODvalue. Aftercompleting the voltage recording for all15tubeholders, take out5mlof the culture using aserological pipette Move the culture vial tothe next tubeholder and record the median voltage asdescribed inStep42.Repeatthisfor all Putthe culture vial inthe firsttube holder and wait for 10 s. Record the voltage for 10sand calculatethe median | VOL.8 NO.3VOL.8 I I I I T T T T I I I I I I B ON ON CAL CAL CAL CAL LES Unplug the powersource before making the connections. The pumpsweuseworkwith110-VAC; therefore, one candirectly useapoweroutletonthe wall.Precautions

H STEP STEP STEP STEP OOT | S 2013 upplementary Fig.5 Turning this type of peristaltic pump on and off with an electronic control board should be done by strictly Inourcurrent configuration, the three pumpsfeeding aculture vial are controlled bythree neighboring Two relay boxes willhaveatotalof 48relay switches. We useallof the 24relay switches inthe first relay Forty-fivepumpsare necessary, asthree pumpsare reserved for each morbidostat culture vial. Labelall of I N G | natureprotocols S ● upplementary Data

S T upplementary Fig.3 IMI

calibration factor ×voltage N ). G ~24 h ) hastobemodified ifthe connection is made differently. ). Fig. 6 Fig. 5 ). Calibration factors may change overtime. Inourexperience,

+ a

offset) for finding the calibration factor. We typically use ). S upplementary Data ) must be

© 2013 Nature America, Inc. All rights reserved. 78| 77| 76| C  75| 74| 73| 72| tubing tothe measured length (1mmi.d.). 71| 70| the culture vials. 69| 16-channel pumpwilldraw liquid awayfrom the culture vials.  the male Luerconnectors onthe 16-channel suction pump. 68| on the exit portof the peristaltic pumps. 67| 66| number and the pumpnames (i.e., culture 1,medium pump:1M). All male Luerconnectors should beonthe same end of the bundle. Labeleverytubecarefully byincluding the culture 65| 64| pump) and cutthe silicone tubing (1mmi.d.) tothe appropriate length. 63| 62| 61| 60| 59| 58| 57| onstructing onstructing medium reservoir bottles

CR CR Cutapiece of silicone tubing (1mmi.d. )4feet(~1.2m)inlength. Cut15pieces of silicone tubing (1mmi.d.) 2feet(~60cm)inlength. Drill16holes of 2.5mmin diameter onGL45bottlecapsusing anelectrical drill. Putthe free end of the bundle into the Erlenmeyer flask.Sealthe mouth of the Erlenmeyer flask using Parafilm. Bundle allof the silicone tubestogether using plastic cableties orelectrical tape. Connect allof the silicone tubestothe 16-channel pumpusing Luerconnectors. Insert afemale Luerconnector toone end of allof the pieces of silicone tubing from Step71. Measure the distance betweenthe 16-channel pump and the mouth of the Erlenmeyer flask.Cut15pieces of silicone Placethe Erlenmeyer flaskinasafeplacethatissufficiently closeto the 16-channel pump. FillanErlenmeyer flaskwith1liter of pure bleach.Thiswillbeused for the collection of bacterial culture removed from Connect the lastfemale Luerconnector of the bundle thatwillbeusedtodraw excess volume from the culture vial to Connect the female Luerconnectors of the bundle (the ones reserved for liquid injections) tothe male Luerconnectors Connect the male Luerconnectors of the bundle tothe female Luerconnectors onthe morbidostat tubes. Group the four sections of tubing prepared for eachculture vial and bundle the tubestogether using electrical tape. Insert one male and one female Luerconnector tothe ends of eachsilicone tubing piece from Step63. Measure the distance betweenthe culture vials and the pumps(medium pump,drug Apump,drug B pumpand suction Placeallof the morbidostat culture vials inthe tubeholders. Insert amale Luerconnector tothe ends of eachpiece of silicone tubing installed inthe 16-channel suction pump. Insert amale Luerconnector toeachpiece of silicone tubing attached tothe pumps( Attach silicone tubing pieces tothe tubing connectors of eachpump( Cut90pieces of silicone tubing ~4cminlength (0.125-inch i.d.). Test allof the connections and ensure thatthe code controls the pumpsproperly ( I I T T I I CAL CAL

STEP STEP Emptythe flaskperiodically and always keep enough bleachtokillthe bacterial cells. Double-check the flowdirection of the suction pumpand properly connect the tubing such thatthe ●

T IMI N G ~6 h plus drying overnight Fig. 5 b ). S natureprotocols upplementary Fig.6 Fig. 5 c ).

| VOL.8 NO.3VOL.8 ). protocol | 2013

563 © 2013 Nature America, Inc. All rights reserved. and anopenflame, ifavailable.  100| 99| 98| 97| 96| 95| 94| 93|  92|  91| 90| 89| 88| S design. Smaller reservoirs are usedfor drug stocks;youmay need more drug solution reservoirs depending onthe experimental  keep the bottleside of the filtersterile. 87| 86| 85| 84| cap ( 83| 82| 81| the bottomsurface of the bottle. 80| 79| 564 protocol terilizing terilizing the tubing

CR CR CR CR

Insert a female Luer connector to the top ends of the pieces of silicone tubing pieces (these ends stay outside the bottle). Runallof the medium pumpsand the suction pump for 5min. Swapthe 70%(vol/vol)ethanol reservoir withthe sterile waterreservoir. Letthe ethanol stand in the systemfor 15minwiththe pumpsturned off. Runallof the medium pumps and the suction pumpfor 5min. Swapthe sterilewaterreservoir withthe 70%(vol/vol)ethanol reservoir. Runallof the medium pumps and the suction pumpfor 5min. Swapthe 10%(vol/vol)bleachreservoir withthe sterilewaterreservoir. Letthe bleachstand inthe systemfor 5minwiththe pumpsturned off. Runallof the medium pumpsand the suction pumpfor 5–10min. Spray 70%(vol/vol)ethanol onallthe Luerconnections. Connect the 10%(vol/vol)bleachreservoir tomedium pumpsusing Luerconnectors. Fillfour reservoirs with10%(vol/vol)bleach,70%ethanol, sterilewaterand growth medium, respectively. Replacethe male Luercapattached tothe silicone tubing reserved for airintake withasyringe filter, taking care to Autoclave the assembledreservoir at121°Cfor 20min. Closeallof the female Luerconnectors using male Luerthread stylecapstokeep the bottlesterile. Wait overnight until the silicone iscompletelydried ( Applyhigh temperature–resistant silicone tothe entire upperface of the GL45captofixthe silicone tubing tothe Pushthe 4-foot (~1.2-m)length of silicone tubing about1inch (~2.5cm)through the remaining hole onthe cap. Pushall15pieces of silicone tubing (2ft.inlength) through the holes onthe capsuch thatthe silicone tubing touches Closeabottletightly using the drilled GL45cap. | VOL.8 NO.3VOL.8 Fig. 5 Swapthe sterilewaterreservoir withthe growth medium reservoir. I I I I T T T T I I I I CAL CAL CAL CAL d

). STEP STEP STEP STEP | 2013 Bleachmay clogthe tubesifitstands for toolong. Closelywatchthe process and ensure thattubesand connections are not leaking. We recommend assembling atleasttworeservoirs of 5-litervolume and four reservoirs of 1-litervolume. To avoid contaminating the medium reservoir, thisstep hastobedone verycarefully using sterilegloves | natureprotocols ●

T IMI N G ~4 h Fig. 5 e ).

© 2013 Nature America, Inc. All rights reserved. is observed. 111| 110| anything goes wrong withaculture. stocks from eachday of the experiment iscrucial. Restartthe experiment using the glycerol stockfrom the previous day if  109| store itat 108| become visiblein2–3d.  growth medium. 107| ? solution. than stockB. Onthe next day, replace stockAwithBand replace stockBwiththe new more concentrated drug  injecting drug solution from stockB. tion from stockAwillbeadded. Ifthe morbidostat cannot inhibitgrowth byadding drug solution from stockA,itwillstart exceeds 0.03.Ifthe ODof aculture exceeds OD and feedthe culture withfresh medium every12min.No injections willbemade into avial until the ODinside the culture the experiment withcustomparameters thatyouspecify. The computerwillcontinuously record the ODvaluesof the cultures You canrunGUI.mfor basic pumpcontrols, automated calibration of the detectors and quick simulations, aswelltostart 106| inside them.  105| contamination inthe medium for atleast24h.  changes ingrowth rate. than stockA.Ifalternative media are used, the dilution rates inthe control code may need tobeadjustedaccommodate media are used. StockAhasadrug concentration thatistentimes higher thanMIC. StockBis5times more concentrated glucose. However, LBorother medium typescanbeused. Extra calibration of the detectors might beneeded ifdifferent  At thisstage, the ODof the culture isnot detectable. with 0.2%(wt/vol)amicase and 0.4%(wt/vol)glucose. Incubate the cellsfor 30mininaflaskat°Cusing ashaker. 104| R instead of the medium reservoir atStep100. 103| 102| and anopenflame, ifavailable.  Luer connectors attached tothe medium reservoir. 101|

unning unning the morbidostat assay TROU

CR CR CR CR CR CR CR Sealthe vials. Placeallof the vials inthe tubeholders. Next, runthe morbidostat software ( Fillallof the morbidostat vials with12mlof cellculture. Labelthe vials (culture no. 1,culture no. 2,etc.). Onthe first day of the experiment, add 100 RepeatSteps88–102for sterilizing the drug pumps. Drugpumpsare finally connected tostockAand stockB reservoirs Runallof the medium pumpsand the suction pumpfor 5mintoinitialize the tubing. Insert sterilesyringe filtersbetweenthe male Luerconnectors attached tothe peristaltic medium pumpsand female Continue the experiment until adiminishing return inthe rate of increase of resistance of the evolving populations Analyze the data onadaily basistocalculateIC Resume the experiment. Transfer 500 Pause the experiment after24hand transfer 500 I I I I I I I T T T T T T T I I I I I I I B CAL CAL CAL CAL CAL CAL CAL LES

−

H 80 °C.Eppendorf tubesmust beproperly labeled. STEP STEP STEP STEP STEP STEP STEP OOT Several problems, such ascontamination, may occurinmorbidostat experiments. Keeping the glycerol Switching tofresh vials every day isnecessary toavoid biofilm formation. Otherwise, bacterial biofilms Ifdrug solution isbeing added from stockB,prepare adrug solution thatisfivetimes more Use autoclavedsterileglassvials everytime. Glassvials are autoclavedwithone magnetic stirbar Growth medium should beprepared inadvance. Prepare the medium and ensure thatthere isno We did allexperiments withfiltered minimal M9 medium with0.2%(wt/vol)amicase and 0.4%(wt/vol) To avoid contaminating the medium reservoir, thisstephastobedone verycarefully using sterilegloves I µ N l of the cellstoasterile1.5-mlEppendorf tube, add 250 G ●

T IMI N G 2–4 h per day THR µ (currently 0.15)and the net growth of the culture ispositive, drug solu l of wild-type, drug-sensitive cellsinto 200mlof minimal M9medium 50 µ values( l of the cellstoanew sterile glassvial thatcontains ~12mlof Fig. 1 d ). µ l of sterile50%(wt/vol)glycerol and freeze/ natureprotocols S upplementary Data

| VOL.8 NO.3VOL.8 protocol

concentrated | 2013

565 - © 2013 Nature America, Inc. All rights reserved. GM081563-04 GM081563-04 and National Science Foundation grant no. DMS-074876-0. and J.P. were supported by US National Institutes of Health grant no. R01 E.T. is supported by a Marie Curie Career Integration grant (no. 303786). J.M.P. for Biodefense and Emerging Infectious Diseases grant no. AI057159 (to R.K.). R01 GM081617 (to R.K.), and by The New England Regional Center of Excellence This work was supported in part by US National Institutes of Health grant no. all members of the Kishony lab and Toprak lab for technical help and discussions. A is availableNote: information in the Supplementary in astepwisemanner. the assayissuccessfully carried outfor several weeks, drug resistance of bacterial populations increases either gradually or the bacterial growth. However, asthe bacteria evolveresistance, the number of drug injections noticeably increases. When In general, we adjust the parameters of the assay such that one or two injections of drug solution are adequate to slow down growth byfiftypercent (IC of drug resistance almost in real time. As illustrated in drug resistance inlessthan3weeks. One of the biggest advantages of the morbidostat isthe abilitytofollow the evolution In the morbidostat, for all of the antibiotic compounds wehaveusedsofar, drug-sensitive ANT Steps 104–111,running the morbidostat assay:2–4hdaily maintenance Steps 88–103,sterilizing the tubing: ~4h Steps 76–87,constructing medium reservoir bottles:~6hplusdrying overnight Steps 50–75,assembling the pumparray: ~24h(inexperienced usersmay need more time) Steps 39–49,calibration of the detectors: ~2h Steps 19–38,assembling tubeholder array: ~24h(inexperienced usersmay need more time) Steps 1–18,assembling morbidostat vials: ~9hplusdrying overnight ● T Troubleshooting advice canbefound in ? 566 a 106 49 S protocol ckno

TROU

tep b T

le le IMI | I VOL.8 NO.3VOL.8 C w I 1 1 le PATE N B | injections OD does not change after medium of antibiotics Bacteria do not grow after injections by antibiotics Bacterial growth is not inhibited readings Unexpected spikes in voltage Tubing is contaminated photo-detector is small Dynamic voltage range of a P dgm LES G

roblem Troubleshooting table. D ents H OOT RESULTS | 2013

The authors thank J. Horn, J. Marchionna, K. Reynolds and I N G | natureprotocols 50 ) canbecalculatedbydirectly analyzing the data acquired during the morbidostat experiment.

n l i T n able e

v e r s Biofilms on the inner wall of the cell vial Drug stock is too concentrated or old Drug stock is not concentrated enough Bacterial clumps in the liquid culture sufficiently sterile Working in conditions that are not amplification is not appropriate The resistor connected in series for P i o ossible reason 1 n

. o f

t h e

p a p e Figure 1b r . interests. CO J.M.P., J.P. and R.K. wrote the manuscript. analyzed the data. A.V. and S.Y. wrote the MATLAB code. E.T., A.V., S.Y., R.C., algorithm for the morbidostat. E.T., A.V. and R.K. performed the experiments and to the design of the setup. E.T., A.V. and R.K. developed the assay and the AUT – e M H , the amount of drug that is necessary to inhibit bacterial PET OR I

N CONTR G G FI NANC

IB UT

I I AL ONS I NTERESTS

E.T., A.V., S.Y., R.C., J.M.P., J.P. and R.K. contributed Dilute Dilute the drug stock from powder Make stronger fresh drug solutions experiment from the previous day Increase stirring speed or resume the all the tubing solution and sterile water. Autoclave Wash the tubing using 10% bleach different resistances Try a range of new resistors with P experiment from the previous day Replace the glass vial and resume the ossible solution

E. coli The authors declare no competing financial populations evolvedstrong

m / dynamically sustained drug selection. drug sustained dynamically USA Sci. prescriptions.and paradoxes hospitals: in resistance responses. and challenges resistance.multidrug Toprak, E. Toprak, antibiotic of epidemiology The B.R. Levin, & C.T.Bergstrom, M., Lipsitch, causes, worldwide:resistance Antibacterial B. Marshall, & S.B.Levy, back. fight bacteria The G. Taubes, antibacterial of mechanisms Molecular S.B.Levy, & M.N. Alekshun, r e p r i n t s /

97 i n et al. et d , 1938–1943 (2000). 1938–1943 , e x . h h Evolutionary paths to antibiotic resistance under resistanceantibiotic to paths Evolutionary t t t m p l : . / / Cell w w

w 128 . Nat. Med. Nat. n a t , 1037–1050 (2007). 1037–1050 , u r e . c o Science

m 10 / Nat. Genet. Nat. d , S122–S129 (2004). S122–S129 , o i f

i n 321 d e r , 356–361 (2008). 356–361 , / 1

0 44 . 1 , 101–105 (2012). 101–105 , Proc. Natl. Acad. Natl. Proc. 0 3 8 h / t n t p p : r / o / t w . 2 w 0 w 1 . 3 n . a 0

t 2 u 1 r . . e .

11. 10. 9. 8. 7. 6.

Science proteins. fitter to paths mutational few very only follow can evolution (1966). 493–496 method. disk single standardized a by testing USA Sci. Acad. resistance.of evolution for potential the modulateinteractions USA Sci. Acad. environments.multidrug in resistance of evolution Accelerated resistance.population-wide to leads Weinreich, D.M., Delaney, N.F., Depristo, M.A. & Hartl, D.L. Darwinian D.L. Hartl, & M.A. Depristo, Delaney,N.F., D.M., Weinreich, susceptibility Antibiotic M. Turck, & J.C. W.M.,Sherris,A.W.,Kirby,Bauer, Drug Kishony,R. & Jr. R.C. Moellering, R., P.J.,Chait, Yeh,J.B., Michel, Kishony,R. & D. Hartl, D., Damian, N., Shoresh, M., Hegreness, selection. W.Microbial Szybalski, V.& Bryson, work charity Bacterial J.J. Collins, & Cantor,C.R. M.N., Molla, H.H., Lee,

312 , 111–114 (2006). 111–114 , (2008). 105 , 13977–13981 (2008). 13977–13981 , natureprotocols Nature

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protocol 116 , 45–51 (1952). 45–51 , | 2013 Proc. Natl. Proc. Proc. Natl. Proc.

45 | ,

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