antibiotics Article Characterization of Two New Multidrug-Resistant Strains of Mycobacterium smegmatis: Tools for Routine In Vitro Screening of Novel Anti-Mycobacterial Agents Patrick K. Arthur 1,* , Vincent Amarh 1 , Precious Cramer 1, Gloria B. Arkaifie 1, Ethel J. S. Blessie 1, Mohammed-Sherrif Fuseini 1, Isaac Carilo 1, Rebecca Yeboah 1, Leonard Asare 1 and Brian D. Robertson 2 1 West African Center for Cell Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology, University of Ghana, P. O. Box LG 54, Accra, Ghana; vincentamarh02@gmail.com (V.A.); samalin01@gmail.com (P.C.); gloria.arkaifie@gmail.com (G.B.A.); jeblessie@gmail.com (E.J.S.B.); msherrif04@gmail.com (M.-S.F.); isaaccarilo@gmail.com (I.C.); rebecca.yeboah23@gmail.com (R.Y.); asaleonne@gmail.com (L.A.) 2 Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK; b.robertson@imperial.ac.uk * Correspondence: parthur@ug.edu.gh  Received: 13 November 2018; Accepted: 7 December 2018; Published: 2 January 2019  Abstract: Mycobacterium tuberculosis is a pathogen of global public health concern. This threat is exacerbated by the emergence of multidrug-resistant and extremely-drug-resistant strains of the pathogen. We have obtained two distinct clones of multidrug-resistant Mycobacterium smegmatis after gradual exposure of Mycobacterium smegmatis mc2 155 to increasing concentrations of erythromycin. The resulting resistant strains of Mycobacterium smegmatis exhibited robust viability in the presence of high concentrations of erythromycin and were found to be resistant to a wide range of other antimicrobials. They also displayed a unique growth phenotype in comparison to the parental drug-susceptible Mycobacterium smegmatis mc2 155, and a distinct colony morphology in the presence of cholesterol. We propose that these two multidrug-resistant clones of Mycobacterium smegmatis could be used as model organisms at the inceptive phase of routine in vitro screening of novel antimicrobial agents targeted against multidrug-resistant Mycobacterial tuberculosis. Keywords: antibiotics; antimicrobial resistance; tuberculosis; mycobacteria 1. Introduction Tuberculosis is one of the most successful human infections with an extensive global coverage [1]. It is caused by members of the Mycobacterium tuberculosis complex, following inhalation of aerosols containing the tubercle bacilli [2]. By estimation, one-third of the world’s population has latent tuberculosis infection and this represents a considerable reservoir from which future tuberculosis cases can emanate [3]. Currently, tuberculosis is a major cause of morbidity and death in low- and middle-income countries. In the clinical setting, drug-resistant Mycobacterium tuberculosis is a serious and persistent threat to global health [4]. Drug resistance in Mycobacterium tuberculosis is largely associated with genetic mutations [5,6]. Resistance to at least rifampicin and isoniazid, also termed multidrug-resistant (MDR) tuberculosis, is on the rise. Over 490,000 MDR tuberculosis cases are recorded yearly, resulting in over 130,000 deaths [7]. Though host genetic factors may play a role, insufficient or incomplete treatment is Antibiotics 2019, 8, 4; doi:10.3390/antibiotics8010004 www.mdpi.com/journal/antibiotics Antibiotics 2019, 8, 4 2 of 14 the most important determinant of the development of MDR tuberculosis [8]. In the global setting, MDR tuberculosis accounts for 3.8% of new cases of the disease and presents in 20% of patients with a history of tuberculosis treatment [9]. Though a number of therapeutic strategies have been employed to treat and eradicate the disease, the emergence of drug-resistant strains has attracted worldwide concern. These strategies include the development of new drugs that target new bacterial proteins, cellular structures, and processes. Routine in vitro screening for novel antimycobacterial compounds would be facilitated by the availability of avirulent mycobacterial strains that are easy to grow and handle. Even though H37Ra is an avirulent strain of Mycobacterium tuberculosis, derived from the virulent H37 parent strain, its slow growth rate hinders the rapid initial screening of antimicrobials to identify candidates that exhibit antimycobacterial activity [10]. An alternative avirulent mycobacterial strain which could be used as a surrogate for Mycobacterium tuberculosis is the Mycobacterium smegmatis mc2 155 strain; Mycobacterium smegmatis is suitable for rapid bioassays, which can be performed in less sophisticated biosafety cabinets. Notably, the diarylquinoline TMC207, which is an anti-tubercular drug, was identified using Mycobacterium smegmatis as a model organism [11]. This study set out to develop extensively-drug-resistant Mycobacterium smegmatis strains to provide a model which could be useful at the initial phase of screening for new antibiotics to combat drug-resistant infections. Continuous exposure of microbial populations to antibiotics, as employed in this study, is considered the most relevant factor that influences the evolution of drug-resistant strains [12]. Continuous exposure to antibiotics promotes gradual acquisition of resistance by subpopulations of the mycobacterium, leaving their antibiotic susceptible counterparts extinct [13,14]. Interactions among other antibiotics also influence the extent of sensitivity of a microbe to a particular antibiotic. Antibiotic resistance has been shown to impose metabolic and fitness costs that reduces the growth of resistant strains [15,16]. Resistance to an antibiotic has been found to either promote cross-resistance or sensitivity to other antibiotics [17,18]. In the present study, drug-susceptible populations of Mycobacterium smegmatis mc2 155 were driven, under in vitro conditions, to evolve into two distinct erythromycin-resistant strains. The study demonstrates the unique phenotypes exhibited by the two erythromycin-resistant strains, in comparison to the original mycobacterium strain from which they were derived. Collectively, we provide additional data which corroborate our current understanding of the emergence and characteristic phenotypes of drug-resistant strains of mycobacterium following continuous exposure to a drug. The two strains will also serve as valuable drug screening tools for the development of the next generation of anti-mycobacterial agents. 2. Materials and Method 2.1. Bacterial Strains, Media Preparation, and Antibiotic Treatments Mycobacterium smegmatis mc2 155 was used as a drug-susceptible strain for all experiments. The MDR Mycobacterium smegmatis strains (referred to as erythromycin-resistant Mycobacterium smegmatis A and B) were generated in this study, under in vitro conditions, and were used as drug-resistant strains. Middlebrook 7H10 agar base (Sigma Aldrich) and Middlebrook 7H9 broth base (Sigma Aldrich) were used for preparing agar plates and liquid broth, respectively, according to the manufacturer’s instructions. The M7H10 agar base was supplemented with 0.085% NaCl and 0.5% dextrose and the M7H9 broth base was supplemented with 0.085% NaCl, 0.44% glycerol, and 0.25% Tween 80. All commercially available antibiotics that were used for this study are listed in Tables S2 and S3. The amount or concentration of each antibiotic used for specific assays are indicated in the text of the results. 2.2. Drug Susceptibility Assay Prior to a drug susceptibility assay, mycobacterial cells were streaked onto fresh M7H10 agar plates and incubated at 30 ◦C [19] for 48 h. The cells that grew on the agar plates were confirmed to Antibiotics 2019, 8, 4 3 of 14 be mycobacteria, via acid-fast staining, and were used for the inoculation of 50 mL of M7H9 broth base. The inoculated broths were incubated at 30 ◦C for 24 h with shaking (160 rpm). The bacterial cultures that were obtained were diluted to an optical density at 600 nm (OD600) of 0.7 in 50 mL of freshly prepared M7H9 broth base and incubated at 30 ◦C for 24 h with shaking. The 24 h, day two cultures were diluted to OD600 of 0.7 and were used for the uniform inoculation (spreading) of M7H10 agar plates. Paper discs containing the indicated amount of antibiotic were placed on the inoculated plates and the plates were incubated at 30 ◦C for 48 h. The zone of inhibition (in mm) around each antibiotic disc was measured and used to ascertain the susceptibility profile of the organism to the panel of antibiotics. 2.3. Testing Cell Viability via Spot Test Assay The 24-h, day two cultures were diluted to OD600 of 0.7 in 1 mL of M7H9 broth base containing the indicated concentration of erythromycin. The control for the diluted samples, indicated as untreated, did not contain erythromycin. The diluted mycobacterial cultures (± erythromycin) were incubated at 30 ◦C for 0 h, 3 h, and 6 h, and an aliquot of 5 µL was spotted on M7H10 agar plates after the indicated duration of incubation. The M7H10 agar plates containing the spots of the bacterial culture were incubated at 30 ◦C for 48 h. 2.4. Growth Profile of Mycobacterial Cells The 24-h, day two cultures were diluted to OD600 of 0.01 in 50 mL of M7H9 broth base. The diluted cultures were incubated at 30 ◦C, with shaking, and the OD600 were measured periodically at the indicated time intervals. 2.5. Colony Morphology Assay The 24-h, day two cultures were diluted to OD600 of 0.7 and aliquots of 5 µL were spotted on M7H10 agar plates containing 100 mM of cholesterol. The corresponding control M710 agar plates did not contain cholesterol. For each M7H10 agar plate, 5 µL of the diluted bacterial cultures were spotted at four positions, which were separated approximately 20 mm from each other. The agar plates containing the spots of the bacterial cultures were incubated at 30 ◦C for 48 h. Colonies that grew on the agar plates were confirmed to be mycobacterial cells by acid-fast staining. 2.6. Staining of Mycobacterial Cells A colony of cells that had grown on M7H10 agar plates were spread uniformly on microscope slides. Gram staining and acid-fast staining were performed to confirm that the cells were mycobacteria. Following the Gram staining and acid-fast staining, the cells were visualized using a Bresser Science MPO 401 light microscope equipped with a 100× objective lens. Images were acquired using a camera that was in-built within the microscope. 3. Results 3.1. Exposure of Mycobacteria to Increasing Antibiotic Concentrations Drives Evolution of Drug Resistance Starting with a single colony of Mycobacterium smegmatis mc2 155, drug-resistant populations of the bacteria were allowed to evolve by systematically exposing the cells to increasing concentrations of either erythromycin, streptomycin, or tetracycline (Figure 1). Prior to each repetition (R), the minimal inhibitory concentrations (MICs) of erythromycin, streptomycin, and tetracycline were determined for the distinct colonies that had grown either within or around the zones of inhibition of these antibiotics (Figure 1). The MIC determination was used to ascertain the extent of the antibiotic resistance acquired by these distinct colonies of Mycobacterium smegmatis. The cells gradually adapted to the systematic exposure to increasing concentrations of each antibiotic, revealing gradual acquisition of resistance (Table 1). Measurement of OD600 at the end of the 2nd, 4th, and 5th steps also confirmed Antibiotics 2019, 8, 4 4 of 14 Antibiotics 2018, 7, x FOR PEER REVIEW 4 of 15 thiAsnotibbisoteicrsv 2a0t1i8o, n7, x( TFaObRl ePESE1R) R. EAVtIEtWhe end of the 5th step, colonies of Mycobacterium smegmatis w4e orfe 15n ot destreecptetodmwyictihni nort hteetrzaocyncelionfe idnihsicbsi t(iToanbloen 1)t.h He oMw7eHve1r0, adgisatirnpctl actoelsoncoienst aoifn Minygcoebiatchteriustmre spmtoegmmyactiisn or tewtsretarceyp cdtloeintmeecytdceidsnc wsor(i tTthaeibtnrl aetch1ye)c .zliHonneoe wds ieosvfc esinr (,hTdiaibsbittlieino 1cnt) .oc Hfo leorwnytiehevsreormf, Mdyicsyitncion dbcaitsc ctseor loiounnm itehsem oMefg 7MmHay1tci0os bawagceatrerer ipudlmaet esscm.t eTgdhmewastieit sh in thmewyzecoroneb edasceotefrciitnaehld i cbweitlilitsoh niwno etfhreer yzcothhnarreoasmc oteyf rciiniznheidb iistacisos noe ronyft ehreryoMtmhr7yoHcmi1ny0-rcaeingsi asdrtiaspnclsta toMensy .tchToehb aeMcste7eHrmiu1ym0c oasgbmarec gtpemlraaitaeilsc.. eTllhwsewose e re chcaomrloayncctoieebrsai,z ciesteodrliaaatsle decr ebylyltsh srpwormerayed ciicnhg-ar raea s1ci0tse−t6ar idnziteludMt iyaocsno beoarfcy ttthehreri uoemryytshmcrineog-mrmeysacitsiinsta.-rnTetws iMostaycnocotlo bManciyteecsro,ibuiasmcot elasrmitueemdg mbsmyateisgsp.m rTeatawidso i ng a c1uc0olt−luo6rnedi,e isl,hu iotsiwoolneadteo df ibftfhyee rsepenrrtey astudhsirncoegmp aty i1bc0iil−ni6t -idreeisls utitosit oaan np otafnM tehyle co oefbr tyahtcihtrertreoiemunmy ccoisnm-rmeegsemirscatiatainsl ta cMnutlyitbcuoiorbetai,cctsse hr(iFouiwmgu esrdme ed2gamif,bfae)t.ri se nt suTschcueelstpeut riebtw,i lsoiht ioedwsisettodin adctip ffacenlroeennletos sf utwhsceierrtpee teifbnuirlcittohimesr mtcoeh arc rpiaaaclntaernli tzoiefb dtiho itarisct ese(nrFy ictgohumrorme 2eyarcc,ibina)-l.r aeTnshitseitbsaienott iwcMso (yFdcioigsbutaicrntece r2tiauc,lmbo) n. es wsemTrheegefmsuear ttithsw eAor acdnhidastr iBan ccattne drci lzwoenedersea scwoenerrtyient hufruaolrlmtyh eymrc aincinh-traaerisanicestdtea rnoiznte MdM y7aHcso 1be0ar caytgtehrairuo pmmlaystcmeisne (-gwrmeisatihtsiotsaunAtt a anMntiydbcioBbtaiacnste)d rfiouwrm e re cothnsmeti nenguemxatal lt6yi sm mAoa naintnhtdsa ,iB np earidnodro tnwo Metrhe7e H csou1nb0tsianegquauarellpnytl amatseassian(ytwas.ii nt hedo uotna Mnt7ibHio1t0i casg)afro pr ltahteesn (ewxitt6homuot nanthtisb,iportiicosr) tfoort he subthsee qnueexnt 6t amssoanyths.s, prior to the subsequent assays. Figure 1. Generating multidrug-resistant (MDR) clones of Mycobacterium smegmatis, in vitro. 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AMllinythococebualcgattaeerrdiup malag tsaemrs epwglmaetareeti. si nAthclula tbt hgaerte wadg awatri 3tph0lian◦tC ethsf eow zreo4rn8ee hsi n.ocDfu iinbshacrtieebdtiet ioactno l3wo0ne ir°eCs r oefo-firnM o4yc8uc ohlab. ateDcdtie sricniruteomt efrs emcsoehlgo Mmnia7etHsi s9ot fh at grbeMrwoythcwo fbiotahrc itdnereituhemrem zsimonneagetimsooantf iosin ft hthaibet igmtiroeinwim wueirtmhei rnien t-hhiniebo iztcounrlyae stce odnf iicnehtnoitbrfairtteiioshn swM (eM7rHeI C9resb-)i rnooft chtuhfeloasrtee dceo itlneotrnomi efirsne fsaohtri o Mena7coHhf 9t he maibnrtiiombtihuo mtfiocr.i nTdheitebe dritmiosricnyreacteoio ncnoc eloonfn ttriheaest imwoneinsrei(m MaulsImCo sii)nohcfiubtlihatoetesryed cinotlnoc nefrinetsrhaf toMiron7eHsa c(9hM baIrCnotsih)b ociof tntihctae. isTnehi nceogld oainsci eirnsec tfreoerca oseelaodcnh i es wceoarnectiaeblnisotortaiictni. oTnch uoelf ad atinescdtirbeiintoett oicco flfroenrs ihreespM we7teiHtrieo9 nabl osrof tihneoc ceounnltatirateid np iirnnogtcoea dfnrueirsneh c( rM5e ta7ismHe9ed sb)c.r oDntuhcr ecinotgnr tatahtiineo irnegpo eaftnait ninotincbrsei o(aRtsie)c,d f or retphceo tnciotcienoncnternoatftritaohtnieo onefn oatfni rateinbptiiorbtoiiocte fidco rut hraetp( we5taittisim oande sdo)ef. dtDh tueo r eMint7giHrteh9 p ebrrooectpehde wtuitraieso (ni5ns ct(rimRea)es,set)hd. e Dsycusortniencmge antthtireca rtleiloyp ne(2toi×tf,i o4an×s,t 8i(b×Ri,) o, tic th1a6tht×ew, caosnndac dend3t2er×da)t itopnrM iof7r aHnt9oti btirhoetti hc dtwhisacts widnaicfsfr uaedsaidsoend satoys sMatey7m.H aT9t hbicera oltilhny iwt(i2a×ls ,icn4oc×nrec,ea8ns×etrd,a 1stiy6o×sntes,m aonafdt ics3at2lrl×eyp )(t2op×mr,i 4oy×rc,i tn8o,× t, he deisr1cy6t×dh,i rfofaumnsdyio cni3n2a,× as)sn adpy r.tieoTtrh aectoyin clitithniael (cd1o×isn)c,c aednditfrefaudts ition es aoachsfs sfatryree.s phTt Mohme7 Hyinc9ii ntbi,aroel trhyc,ot hwnrceoermnet 3rya0ct iiμnog,n/asμ nLdo,f 6 te0st trμraegcp/yμtocLlmi, nayencid(n1 ,× ), ad30edr eμydtght/μoroLem,a cryehcsifpnre,e castnhivdMe tle7ytH.r Za9c1yb, crZoli2tnh,e a, (nw1d×e )rZ,e a33d r0deµepdgre /tsoµe nLeta, c6trh0i pµfrlgiecs/ahµt eLM m,7aHenad9s ub3rr0eomµthge,/ nwµtseL ro,efr e3es0ap cμehgc zt/iμovLnee,l y 6o.0fZ μi1ng,h/Zμib2Li,t,a iaonndd Z 3 re(pi3nr0 em sμemgn/)μt. tLr,i preliscpaetcetimveelays. uZr1e,m Ze2n, tasnodf Zea3c rhepzorenseenotf tirniphliibciattieo nm(einasmurmem).ents of each zone of inhibition (in mm). 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ZoZnoense osfo IfnIhnihbiibtitoionn (m(mmm)) SSttrreeppttoommyycicnin( µ(µg/µL) Erythromycin (µg/µL) Tetracycline (µg/µL) ReRpepetitions (R) g/µL) Erythromycin (µg/µL) Tetracycline (µg/µL) etitions (R) 0.10.1 1 1 66 3300 115500 0.01. 1 1 1 6 6 30 30 15105 0 0.10 .1 1 1 6 6 30 30150 150 - 0 6 12 15 22 0 0 0 11.5 19.5 8 9.5 18 22 27.5 - 1 0 0 6 0 1120 1152 1722 0 0 0 11.5 19.5 8 9.51 0 0 10 12 17..55 0 0 0 0 0 0 6 6 131.53 .5 0 0 8 8 11 18 11 15.5 22 19 27.515.5 19 2 2 0 0 0 0 99 88 1133 0 0 0 0 0 0 6 6 101 0 0 0 0 0 8.5 8.5 11 1114.5 14.5 3 3 0 0 0 0 00 0 1188 0 0 0 0 0 0 16.156 .5 242.54 .5 101 0 6 6 10 10 13 13 15 15 4 4 0 0 0 0 00 0 1177 0 0 0 0 0 0 10 10 171.57 .5 N/NA/ A N/AN /AN/AN /AN/AN /AN/A N/A 5 5 N/NA/A N/NA/A NN/A/A NN//AA NN//AA 0 0 0 0 0 0 6 6 131 3 N/NA/ A N/AN /AN/AN /AN/AN /AN/A N/A NN/A/ Adedneontoetse snnoot taavvaailialabbllee.. 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T. hTehsees oebosbesrevravtiaotniosn asgraeger eweitwh iath praepvrioeuvsio rueps orertp ort thtahtatin inddiciacateteddt thhaatt aa ssiinnggllee aannttiibbiioottiicc ttrreeaattmmeennt tmmaayy sesleelcetc ftofro rrerseisstiasntacne caegaaginasitn ostthoetrh merumltiuplltei pdlreudgsr ugs vivaiaa as esteto of fm meecchhaanniissmmss ((ccoollllaatteerraall rreessiissttaannccee) )oorr aa ssiningglel emmecehcahnainsmism (cr(ocrsos-srse-sriesstaisntcaen)c [e2)0[].2 0W].hWat hisa t is more striking about this data is that the acquisition of broad-spectrum resistance cuts across all mcolaresssetsr iokfi nangtaibbiooutitcsth. Tishdisa otabsisertvhaattiothne isa cthqeu ifsiristito tno obfe brreopaodrt-esdp efcotrr ummycroebsaiscttaenricae wcuhtesrea cerxopsossaulrlec ltaos ses ofonaen tainbtiiobtiioctsi.c hTahsi sleodb tsoe rthvea tdioevneilsopthmeenfitr sotf teoxtbreemreep dorrutge drefsoisrtamncyec o(MbaDcRte)r. iTahwush, etrheeseex tpwoos usrtreatinoso ne ancotinbsitoittuicteh aa svalleudabtole tthoeol dine vtheelo cpomnteinnuteodf seexatrrcehm foerd nreuwg arnetsiibsitoatnicc eca(nMdiDdaRt)e.sT. hus, these two strains co nstitute a valuable tool in the c ontinued search for new antibiotic candidates. 3.2. MDR Mycobacterium Smegmatis is Viable in the Presence of Higher Concentrations of Erythromycin A spot test assay was used to validate the extent of resistance of the two clones of erythromycin-resistant Mycobacterium smegmatis (clones A and B) relative to the Mycobacterium smegmatis mc2 155 strain. These three mycobacterial strains were exposed to increasing concentrations Antibiotics 2018, 7, x FOR PEER REVIEW 6 of 15 3.2. MDR Mycobacterium Smegmatis is Viable in the Presence of Higher Concentrations of Erythromycin AntibioAtic ssp20o1t9 t,e8s, t4 assay was used to validate the extent of resistance of the two clones of erythrom6yocfin14- resistant Mycobacterium smegmatis (clones A and B) relative to the Mycobacterium smegmatis mc2 155 strain. These three mycobacterial strains were exposed to increasing concentrations of erythromycin ofofre r0y thh, r3o mh,y cainndf o6r h0,h p, 3rihor, atnod s6poht,tpinrigo rthtoe scpeolltst inogn tMhe7cHel1l0s oangaMr 7pHla1t0esa.g Tarhpe laerteyst.hTrohme eyrcyitnh-rroesmisytcainnt- rMesyicsotabnacttMeriyucmob samcteegrimumatissm stergaminasti ws setrrea ivnisabwlee raet vailal bolfe thaet athllroefe tthime teh preoeinttism (e0 pho, 3in hts, a(0ndh ,63 hh o,fa enxdp6oshuoref etox peorsyutrherotomeyrcyitnh)r, oamnydc inth),ea nvdiatbhileitvyi awbialist yrwobaussrto baugsatinasgta itnhset thloewlo wanadn dhihgihg hccoonncceennttrraattioionnss ooff eerryytthhrroomyycciinn uusseeddf ofor rt htehea sasassya(yF i(gFuigreu3re). 3O).n Othne tchoen tcroanrytr, athrye,M thyec oMbayctceorbiaucmtesrmiuemgm samtiesgmmact2is1 5m5cs2t r1a5in5 wstraasinv iwabalse vaitatbhlee alot wthceo lnocwe nctornactieonntsraotifoenrsy tohf reormytyhcrionm, wychiner, ewashegrreoaws tghrowwatshu wndase tuenctdeedteacttethde aht itghhe choignhce nctornactieonntrsaotifotnhse aonft ibthioet iacn(Ftiibgiuortiec3 a(F,big).uNreo ta3ba,lby,).g rNowottahbwlya, sgurnodwetthe ctwedasf ournthdeetMecytecodb afcoterr ituhme sMmyecgombaatcitsermiucm2 1 5sm5 setgrmaiantifso lmlocw2 i1n5g56 shtroafienx pfoolsluorweitnogt h6e lho wofc oenxcpeonsturartei otnos tohfee rlyotwhr ocmonycceinnt(rFaitgiuornes3 oc)f. Terhyetsheroobmsyercvina ti(oFnigsudreem 3ocn).s trTahteesteh aotbthseertvwatoioenrys thdreomoyncsintr-artees istthaantt tshtrea itnws oe xehribyittharodmisyticninct-rveisaibstilaintyt psthraeninost yepxheibinit tah edipstrienscetn vcieaboifliteyr ypthhernomotyypcien ,ini nthceo pmrpesaerniscoen oft oertyhtehrMomycyocbianc,t einri ucmomspmaergimsoant istom thce2 1M55ycpoabarectnetrisutrma isnm. egmatis mc2 155 parent strain. Figure 3. Cont. Antibiotics 2019, 8, 4 7 of 14 Antibiotics 2018, 7, x FOR PEER REVIEW 7 of 15 FFigiguurree3 3. .V Viaiabbiliiltiytya assssaayyo offt htheed drruugg-s-suusscceepptitbiblelea annddM MDDRRM Myyccoobbaaccteterriuiumms smmeeggmmaatitsiss strtraaininssf ofolllolowwiningg eexxppoossuurreet otol olowwa annddh higighhc oconncecenntrtaratitoionnsso offe eryryththrorommyyccinin. .O Ovveerrnnigighhttc cuultltuurreesso offt htheessees strtraaininssw weerree either untreated or exposed to low (0.39 µg/mL; 0.78 µg/mL; 1.56 µg/mL; 3.1 µg/mL) or high either untreated or exposed to low (0.39 μg/mL; 0.78 μg/mL; 1.56 μg/mL; 3.1 μg/mL) or high (100 (100 µg/mL; 200 µg/mL; 800 µg/mL) concentrations of erythromycin for either 0 h (a), 3 h (b), μg/mL; 200 μg/mL; 800 μg/mL) concentrations of erythromycin for either 0 h (a), 3 h (b), or 6 h (c). An or 6 h (c). An aliquot of 5 µL of the untreated and treated cultures were spotted onto M7H10 aliquot of 5 μL of the untreated◦ and treated cultures were spotted onto M7H10 agar plates and agar plates and incubated at 30 C for 48 h. For each treatment condition, the three spots on the incubated at 30 °C for 48 h. For each treatment condition, the three spots on the M7H10 agar plate M7H10 agar plate represent biological replicates. Ery M. smeg A and Ery M. smeg B represent the two represent biological replicates. Ery M. smeg A and Ery M. smeg B represent the two erythromycin- erythromycin-resistant clones derived from the Mycobacterium smegmatis mc2 155 strain. resistant clones derived from the Mycobacterium smegmatis mc2 155 strain. 3.3. MICs of Selected Antibiotics against MDR Mycobacterium Smegmatis 3.3. MICs of Selected Antibiotics against MDR Mycobacterium Smegmatis The MICs of thirteen commercially-available antibiotics were determined for the Mycobacterium smegmTahteis MmIcC2s1 o5f5 tshtirrateinena ncodmthmeetrwcioallMy-DavRasiltarbalien sa.nFtiobrioetaiccsh woregrea ndiestmer,mthineeMd IfCoro tfhaen Maynctoibbaioctteirciuomf insmteergemstawtisa smdce2t e1r5m5 isnterdaibny apnlda ctihneg tfwouor MdisDcRs, csotrnatianisn.i nFgord eecarceha soirnggacnoisnmce,n tthraet iMonICs o offt haen aanntitbibioiotitcic, aotf pionstietrioesnts wdaisst adnettefrrommineeadc hbyo tphlearcoinngt hfoeuarg dairspcsla, tceo.nAtallinoifntgh edethcrreeeasminygc ocobnaccteenrtiaral tsitornaisn osfw theere arnetsiibsitoatnict , toatt hpeoshiitgiohnesst dcoisntacennt tfrraotimon eoafchP yortahzeinr aomni dthee( 1a6g0aµr gp)laatned. wAlelr eofs uthscee pthtirbelee mtoytchoeblaocwteersitacl osntrcaeinntsr awtioenre orfemsisotxainflto txoa tchine (h0i.g2h5eµsgt ;cToanbcelen2tr)a. tTiohne otwf PoyMraDziRnasmtraidines (w16e0r eμrge)s aisntadn wt teoret hseuhsciegphteisbtlec oton ctehnet rlaotwioenst ocfoinsocenniatrzaitdio, net hofa mbouxitfolol,xlaincienz o(0li.d25, aμngd; Teraybtlhe r2o)m. Tyhcein t,wwoh MileDtRh estMrayincosb waceterrei uremsissmtaengtm toat tishem hci2g1h5e5st sctroanicnenwtraastisouns coefp tiisbolenitaozitdh,e eltohwamesbt uctoonl,c elnintreaztoiolind,o fanthde seeryatnhtriobmioyticcisn., TwhheilMe ItCheo fMvyacnocboamcteyrciuinm asnmdegsmtraetpist ommcy2 c1i5n5 wstraasin8 0waµsg sufosrcetphteibMle DtoR thsetr laoiwnse;stt hcoenMceynctorbaaticotenr ioufm thsemsee gamntaitbiisotmiccs2. T1h5e5 MstIrCai nof wvaasncsoumscyecpitnib alnedt osttrheeptloomwyecsitnc ownacse 8n0tr μatgi ofnors tohfet hMeDseRa sntrtiabiniost;i cthseu Mseydcofboarcttehreiuamss samy.egImnatetirse smticn2g 1l5y5, thsteratwino wMaDs Rssutsrcaeinpstiwbleer etosu stcheep tliobwleetsot 2c0oµngceonftaramtipoincsil lionf anthdeasem oaxnitciiblliiont,icesv eunstehdo ufgohr otuhre eaasrlsiaeyr. oIbnsteerrvesattiinognlsy,i ntdheic atwteod MthaDtRt hsetsreaiMnsD wResretr asiunsscewpetirbelere tsois t2a0n μt tgo o4f0 aµmgpoicfielliitnh earnodf athmeosexiacinlltiinb,i oetvicesn (Fthigouurgeh2 obu).rM eaorrleioerv eorb, scelorvnaetBioonfst ihnedMicDatReds ttrhaaint twheasses uMscDeRp tsibtrlaeitnos 1w5eµrge arensdis3t0anµtg too f4t0e tμragc oyfc leinitehearn dof cthhleosrea manpthibeinoitciocsl, (rFeisgpuercet i2vbe)l.y M; aonreoobvseerrv, caltoionne tBh aotf wthaes MinDcoRn sstirsateinn twwaist hsuthsceedpatitbalesh toow 15n μing Faingdu r3e02 μbg. Woef tpetrroapcoyscelidneth aantdt hchelpolraacmempheennticoof ld, rifefsepreencttivcelalyss; easn oofbasnertvibaitoiotinc tdhiasct swoans ianncoangsairstpelnatt ewdituhr tihneg dthaeta teshstofworn tihne Fdigruugres 2ubs.c eWpeti bpirloitpyopseadtt etrhnat( Fthigeu prleac2eBm),eanstc oofm dpifafreerdentto ctlhaesspelsa ocfe manetnibtiooftidc idscisscosf otnh eansa amgaer apnltaibtei odtuicrifnogr tthhee tMesItC fodr ettheer mdriunga tsiounsc(eTpatbibleili2ty), pwaattsertnh e(Fbiagsuirsef o2Br )t,h aes acboomvpea-mreedn ttoio tnheed pldaecveimateinotn os.f Tdhiescas notifb tiohtei csaamssea yanfotirbmioattici sfotrh ethset aMndICar ddewteirdmeliynautisoend (fToarbtlhee 2d),e twerams itnhaet iobnasiosf ftohre tahnet iabbiootviec- smusecnetpiotinbeildit ydpevaitatetironnosf. bTahcet earniatlibisiootlaict eass.sTahy ufosrcmroasts -iisn ttheera scttaionndabredtw weiednedlyi fufesreedn tfoclra tshsee sdoeftearnmtibinioattiicosn moifg thhteb aenitnibfliuoetincc sinugsctehpetirbeisliisttya npcaettperronfi olef sboafcttehreiaMl iDsoRlaMte.ss. mTehgumsa ctirsosstsr-ainintesrtaocatiosnu bb-ecotwlleecetnio dnifoffertehnet scellaescsteesd ocfo amnmtibeirocitaiclsa mntiigbhiott bices .influencing the resistance profiles of the MDR M. smegmatis strains to a sub-collection of the selected commercial antibiotics. Antibiotics 2019, 8, 4 8 of 14 Table 2. Zones of inhibition. Zones of Inhibition (mm) Antibiotic (×) Antibiotic Concentration M. smeg mc2 155 Ery M. smeg A Ery M. smeg B 0.5× 0 12 10 Amp (40 1× 6 11 14µg) 2× 9 17 14 4× 18 21 20 0.5× 6 12 11 Amx (40 g) 1× 9 18 17µ 2× 13 18 19 4× 10 18 20 0.5× 13 0 0 Van (40 g) 1× 20 0 0µ 2× 20 7 9 4× 21 9 10 0.5× 8 0 0 Inh (10 g) 1× 29 0 0µ 2× 37 0 0 4× 40 0 0 0.5× 15 0 0 Emb (10 g) 1× 36 0 0µ 2× 46 0 0 4× 51 0 0 0.5× 0 0 0 Pzd (40 1× 0 0 0µg) 2× 0 0 0 4× 0 0 0 0.5× 30 10 10 Moxi (0.5 g) 1× 36 18 12µ 2× 40 22 20 4× 47 29 24 0.5× 0 18 12 Rif (10 g) 1× 8 20 16µ 2× 8 23 19 4× 11 25 21 0.5× 0 0 0 Lin (5 g) 1× 7 0 0µ 2× 8 0 0 4× 10 0 0 0.5× 46 20 7 Tet (30 g) 1× 40 20 9µ 2× 42 23 14 4× 54 27 18 0.5× 14 9 0 Chlo (30 g) 1× 18 9 7µ 2× 22 15 10 4× 31 22 13 0.5× 8 0 0 Ery (40 µg) 1× 10 0 0 2× 10 0 0 4× 15 0 0 0.5× 18 0 0 Strep (20 µg) 1× 20 0 0 2× 31 6 6 4× 38 8 9 The full definitions of the antibiotics are as follows: Ampicillin (Amp), Amoxicillin (Amx), Vancomycin (Van), Isoniazid (Inh), Ethambutol (Emb), Pyrazinamide (Pzd), Moxifloxacin (Moxi), Rifampicin (Rif), Linezolid (Lin), Tetracycline (Tet), Chloramphenicol (Chlo), Erythromycin (Ery), Streptoimycin (Strep). 3.4. Effect of Neighboring Antibiotic Discs on the Antimycobacterial Activity of Selected Antibiotics Using the disc diffusion assay, a number of antibiotics are usually placed at unique positions on an agar plate that has been inoculated with the strain of interest during the test for drug susceptibility Antibiotics 2018, 7, x FOR PEER REVIEW 9 of 15 AntibioUtiscsin20g1 9th, 8e, 4disc diffusion assay, a number of antibiotics are usually placed at unique position9 so fo1n4 an agar plate that has been inoculated with the strain of interest during the test for drug susceptibility ppaatttteerrnnss ((FFiigguurree 44)).. WWee iinnvveessttiiggaatteedd tthhee eeffffeecctt ooff nneeiigghhbboorriinngg aannttiibbiioottiicc ddiissccss oonn tthhee aannttiimmyyccoobbaacctteerriiaall aaccttiivviittyyo foaf mapmicpililicnil,lainm, oaxmiciollxinic, illilnienz, olliidn,etzeotrlaidcy, ctlientera, cchylcolriname, pchhenloicroalm, eprhytehnriocmoly, cienr,yatnhdrosmtreypctionm, yacnind. sUtrlteipmtaotmelyy,citnh.i sUexltpimeraimteelyn,t athl aisp pexropaecrhimweanstaaln taicpippraoteadcht owparso vaindteiciinpfaotremda ttioo nproonvsipdeec iifincfonremigahtiboonr ionng sapneticbifiioct incedigihscbsorthinagt aenitthibeiroetinc hdainsccse dthoart erietdhuerc eednhtahneceadn toimr yrecdoubcaecdte rthiael aancttiimviytycoobfacthteersieal saecleticvtietdy oanf ttihbeisoeti scedleiscctes.d antibiotic discs. FFiigguurree 44.. SScchheemmaatitcic rereppreresesenntatatitoionn ofo fthteh eararrarnagnegmemenetn ot fo afnatnibtiibotioict idcisdciss cosno angaagr aprlaptleast.e As. tAottaol toafl nofinnetieneente deinffedriefnfetr ceonmt mcoemrcmiale arcnitailbiaontitcisb iwoteirces pwlaecreed aptl aucneidquaet puonsiitqiounes poons tihtiroene sMo7nH1th0 raegearM p7laHte1s0, waghairchp lwate 2 erse, iwnohcicuhlatweder ewiitnho ccuullatuterdes woift heitchuelrt uMreyscoobfacetietrhieurmM smyceogbmacatteisr iummc2 s1m5e5g mora teisrymthcrom15y5cionr- reersyitshtaronmt Mycyicno-braecstiesrtiaunmt sMmyecgombaactitse rAiu. mThsem aemgmouatnist (Ain. Tμhge) oafm eaocuhn ste(lienctµegd) aonftiebaicohtics-ecloenctteadinianngt idbiisoct iics- icnodnitcaainteindg ind iTscabislein Sd2i caantedd tihneT aambloeuSn2ta onfd etahceha amnotiubniot toicf e(ainc hμagn) tuibsieodti cas(i na µnge)iguhsbeodra fsoar ntheeig shebloerctfeodr athnetisbeiloetcicte ids ianndtiibcaiotetidc iins iTnadbiclea tSe3d. in Table S3. The erythromycin-resistant M. smegmatis A strain was resistant to ampicillin, amoxicillin, The erythromycin-resistant M. smegmatis A strain was resistant to ampicillin, amoxicillin, and cahnldorcahmloprhamenpichoeln iwcohlewn heeanche aocfh tohfetshee sseelseeclteecdt edanatnibtiiobtiioctsi cws wereer esusurrrorouunnddeded bbyy tthhee nneeiigghhbboorriinngg aannttiibbiioottiicc ddiissccss iinnddiiccaatteedd iinn TTaabbllee 33.. IInntteerreessttiinnggllyy,, tthhee ssaammee ccoonncceennttrraattiioonn ooff tthheessee sseelleecctteedd aannttiibbiioottiiccss ((aammppiicciilllliinn,, aammooxxiicciilllliinn,, aanndd cchhlloorraammpphheenniiccooll)) eexxhhiibbiitteedd aannttiimmyyccoobbaacctteerriiaall aaccttiivviittyy aaggaaiinnsstt tthhee ssaammee rreessiissttaanntt ssttrraaiinn iinn tthhee aabbsseennccee oof fththe eneniegihgbhobroirnign ganatnibtiiboitoict idcidsciss.c Ts.hTe hoeppopospiotes ietfefeecftf ewctasw daestedcetteedc tfeodr sfotrrepsttroemptyocminy acginaiangsta itnhset rtehseisrteasnits tsatnratinst rwaihnenw dheisncsd oisfc rsifoafmrpifiacmin,p iiscoinn,iaiszoidn,i aaznidd ,aamnpdicaimllinp icwilelirne wuseerde uass endeaigshnbeoigrsh.b Tohrse.sTe hoebsseeorvbasetirovnast iwonesrew neoret nuontiquuneiq tuoe etroyethryrothmroymciny-criens-irsetsainstta Mnt. Msm. semgmegamtisa tAis Astrsatirna; inth; et h 2 Me .M s.msemgmegamtiast ims mc c 155 strain was highly susceptible to linezolid in the presence of2 155 strain was highly susceptible to linezolid in the presence of nneeiigghhbboorriinngg aannttiibbiioottiicc ddiissccss,, bbuuttw waassr eresissitsatannt tt otot hthees asmame ec ocnocnecnetnratrtaiotinono folfi nleinzeozliodliidn itnh ethaeb saebnsceencoef onfe ignheibgohrbinogrinangt iabniotitbiciodtiiscc2 sd.isCcosl. leCcotilvleeclyti,vtehleys,e tohbesseer voabtsioernvsadtieomnso ndsetrmatoentshtarattteh ethraest istthaen creespisrotafinlcees pofroMfi.lesms eogfm Mati.s smmcegm15a5tias nmd cthe corresponding MDR strains are influenced b2 155 and the corresponding MDR strains ayrec rionsfslu-ienntecreadc tbioyn corfotshse- idnitffeerraecntitocnla osfs eths eo fdaifnfteirbeinotti cclsa.sTshesis osfa amnetidbiaottaiscest. dTehmiso snasmtrea tdeastaasseutb dtleempohnesntoratytepsi cad siuffbetrleen pcheebneotwtyepeinc dthifefetwreoncMe DbeRtwstereanin tsh. e two MDR strains. Table 3. Selected antibiotics. Table 3. Selected antibiotics. Selected Selected Antibiotics in the Presence of Neighbors Selected Antibiotics alone SeleAcntteidb iotics SelecteMd. Asmnetigbmioct2ic1s5 i5n the PreseEnrcye Mof. NsmeeigghAbors M. smSeeglected Neighbors mc2 15 A5 ntibEriyotMic.ss amleognAe Neighbors Antibiotics M. smeg mc2 155 Ery M. smeg A M. smeg mc2 155 Ery M. smeg A Amp N/A 0 N/A 11 Strep, Inh, Moxi AmpA mx N/A N/A 0 0 N/NA/A 18 11 LiSnt,rPezpd,, ICnyhs, Moxi Amx Lin N/A 50 0 N/A N0 /A N/A18 Van,LPiznd, ,PAzmd,x Cys Lin Tet 50 N/A N/A 7 N/A0 20N/A RiVf,aInh, ,PEzrdy, Amx Tet Chlo N/A N/A 7 0 NN/A/A 9 20 Emb,RPizf,d I,nChy,s Ery Chlo Ery N/A 20 0 N/A 1N0 /A N/A9 Tet, InhStrep 48 14 20 0 Rif,EImnhb , ,, M A P oxi mzdp, Cys Ery 20 N/A 10 N/A Tet, Inh, Moxi StNarse upmbers represent zones of inhibition (mm) of the selected antibiotics. The amount of antibiotics (µg) used for thesa y are indicated48in Tables S2 and S3. 14 20 0 Rif, Inh, Amp Antibiotics 2019, 8, 4 10 of 14 3.5. Evolution of the MDR Phenotype Affects the Growth Profile of Mycobacterium Smegmatis Antibiotics 2018, 7, x FOR PEER REVIEW 10 of 15 The growth profiles of the two clones of erythromycin-resistant Mycobacterium smegmatis and the Numbers represent zones of inhibition (mm) of the selected antibiotics. The amount of antibiotics (μg) Mycobacterium smeugsemd faotr itshem 2 asscay a1r5e 5indsitcartaedin in wTabelrese Si2n anvde Ss3t. igated in order to ascertain the effect of the MDR phenotype on growth rate. Single colonies from these three mycobacterial strains were initially grown 3.5. Evolution of the MDR Phenotype Affects the Growth Profile of Mycobacterium Smegmatis in M7H9 broth for 24 h at 30 ◦C with shaking. These mycobacterial cultures were diluted to an OD600 of The growth profiles of the two clones of erythromycin-resistant Mycobacterium smegmatis and 0.01 and the grtohew Mthycorbaactteerwiuma ssmmegomnatiist omrce2 d15f5o srtra6i0n whe(rFe iignvuersteig5atae)d. iTn horedetrw too acscleorntaeins tohef eefrfeyctt horf othme ycin-resistant MycobacteriumMsDmRe pghmenaottiyspes ohno gwroewdth aratree. lSaintgivle ecolylonriaesp firdomg throeswe tthhreer amtyecodbaucrteirniagl strhaiensi nwietriea inl i1tia2llyh compared to the Mycobactergiuromwns min eMg7mHa9t bisromth cfo2r 1245 5h astt 3r0a i°nC .wIintht eshraeksitnign. gThlyes,et hmeycloabtatceterrisatl rcauiltnurrees cwuerpee drialutteedd tfor om the initial an OD600 of 0.01 and the growth rate was monitored for 60 h (Figure 5a). The two clones of lag phase of gerroytwhrtohmyacfinte-rres1is2tanht Mayncdobasctheroiuwm esmdegamartaisp sihdowgerdo aw retlhativrealtye r,apaitdt agrionwinthg raaten dOuriDng6 0th0e of 3.2 by 48 h (Figure 5a). Onintithiael 1c2o hn tcroamrpya,retdh eto etrhye tMhyrcoobmacytecriiunm- rsmesegismtaatins tmsct2 r1a5i5n sstrasihn.o Iwnteerdestaingrelyl,a tthiev lealttyers sltorawine r growth rate after the initialre1cu2pher.atTedh firsomp athtet einritnialo lafgg prhoawse tohf gwrowasth caoftenrfi 12r mh aendd sfhoolwloedw ai rnapgidm greoawsthu rate, attaining an OD600 of 3.2 by 48 h (Figure 5a). On the contrary, the erythromycin-resistant strarinesm sheonwtedo fa OD600 at 12-h intervals for 96rehlat(ivFeilgy uslroew5erb g)r,oiwlltuh sratrtea atfitnerg thteh iantititahl e12a hc. qTuhiiss pitaitotenrn oof gMroDwtRh wpahse cnonoftirympeed fcoalluowseinsg a concomitant alteration in thmeegasruorewmtehnt pofr OoDfi6l0e0 aot f12M-h yinctoerbvaaclst eforri u96m h s(Fmigeugrme 5abt)i, sil.lustrating that the acquisition of MDR phenotype causes a concomitant alteration in the growth profile of Mycobacterium smegmatis. Figure 5. GrowFtighurper o5.fi Glersowotfh thpreofdilersu gof- stuhes cderpugt-isbulseceapntibdleM anDdR MMDRy cMobyacocbtaecrteiruiumm ssmmeeggmmataist isstrsatirnas.i ns. Overnight Overnight cultures of the three strains were diluted to an optical density at 600 nm (OD600) of 0.01 and cultures of the tthhe rgereowstthr araitne swwas emroenditoirleudt efodr 6t0o ha (na) oorp 9t6i cha (bl )d. Feonr s(ait),y thaet O6D00600 wnmas m(OeaDsur6e0d0 )evoefry0 3. 0h1 foar nd the growth rate was monit1o2r he,d fofllowr e6d0 bhy a(na )inoterrm96itthent( b12)-.h Fmoeras(uar)e,mtehnet. OFoDr (6b0),0 thwe aOsDm600 weas umreeasdurevd evryery3 1h2 hf oforr 12 h, followed by an intermitt9e6n ht. E1r2y- Mh.m smeega sAu arnedm Ereyn Mt.. sFmoegr B( bre)p,rtehseentO thDe two ewryathsrommeyacisnu-rreesidstaenvt celroyne1s 2dehrivfeodr fr9o6mh . Ery M. smeg the Mycobacterium smegmatis mc2 155 strain. 600 A and Ery M. smeg B represent the two erythromycin-resistant clones derived from the Mycobacterium smegmatis mc2 155 strain. 3.6. Cholesterol Enhances the Growth of Colonies of Mycobacterium Smegmatis It has been reported that Mycobacterium tuberculosis utilizes the cholesterol of its host organism, for virulence and invasion of macrophages, in a mechanism dependent on the modulation of lysosomal calcium levels [21,22]. Cholesterol metabolism in M. tuberculosis is facilitated by the presence of an operon, which consist of genes encoding acrylamine N-acetyltransferase and other associated proteins [23]. Proteins associated with cholesterol metabolism are usually conserved in M. tuberculosis and M. smegmatis and represent a possible target for novel anti-tubercular drugs [24,25]. Even though M. smegmatis is avirulent and does not persist in macrophages, the presence of these conserved proteins makes M. smegmatis a suitable laboratory model for studying the phenotypic effect of cholesterol on Antibiotics 2018, 7, x FOR PEER REVIEW 12 of 15 According to the WHO, a strain of Mycobacterium tuberculosis is classified as MDR if it is resistant Antibiotics 2019, 8, 4 11 of 14 to at least the two most potent anti-TB drugs (rifampicin and isoniazid). Extremely-drug-resistant TB strains exhibit additional resistance to isoniazid, a fluoroquinolone, and one of the second-line anti- mycobTaBc tderruiag.sH (keanncaem, wyceini,n avmesitkiagcaitne, danthde ceafpferecotmofyccihno).l eTshteer otwl oo ncltohneems oofr pthheo elorygtyhroofmMyycicno-braecstisetraiunmt smegmMatyiscombacc2te1r5iu5m, a ssmweeglml aatsist hgeenereyrathterdo mbyyc itnh-irse ssitsutdany t wsterarein sb.oth susceptible to rifampicin and a Cfoluloonroieqsuionfoltohnee M(myocxoibflaocxtearciiunm), bsumt ewgmeraet risesmistca2nt1 t5o5 issotrnaiainzidw. eTrheusd, ettheec tderduga sresbirsotaandc,e flpurofffyil-el iokfe structuthree seorynthtrhoemcyhcoinle-rsetseirsotal-ncto MntyacionbiancgterMiu7mH s1m0egamgaatrisp wlaates s,nowt heenrteiraeslyt hceoncsoinstternotl w(niothc hthoel eWsteHrOol ) criteria for the classification of MDR phenotypes of mycobacterium. The susceptibilities to agar pmlaotxeisflsohxoacwine dantdh setreexpptoemcteydcinc owloerney mpahrkeendoltyy rpeedu(Fciegdu irne th6eas)e. nTehwe McoDloRn sitersaionfs,t hwehitcwh osocmloenwehsaot f erythropmlacyec itnh-ermes isbteatnwteMeny ctohbea cctlearsiusimcasl mMegDmRa taisnwd eXreDmR omdyecsotlbyaectnelrairagl esdtroainnsth. eNcohnoeltehsetleersosl,- ctohne taMinDinRg agar pplahteencootymppea erxehdibtoitetdh ebcyo tlhoen itewsoo nditshtienccto ncltornoelsa goaf rerpyltahterosm(Fyicgiun-rrees6ias)t.anVti sMuaycliozbaatcitoerniuomf isnmdeigvmidautisa l cells wmitahkinest htehsee ocorgloanniiesms r eav emaloerde tshuaittaabllleo fmthoedtehl reoergmanyicsomb afcotre rtihale stirdaeinntsiftiecsatteiodnp oofs itniovveeflo rbaiocaidct-ifvaes t stainincgom(FpigouunredsS 1a)g. aMinosrt eMovDeRr,-TinBd iavnidd uXaDl Rce-TllBs ,f raosm cocmolpoanrieeds otof tthhee Myyccoobbaacctteerriiuum ssmeeggmaattisis mcc2 2115555 strainshtraadina. Dslueen dtoe trhaen sdigrnoifdic-alinkte gmenoertipch doislopgaryit(yF bigetuwree6nb M). yEcvobeancttehriouumg thubtehrecuelroysitsh arnodm Mycyicnob-raectseirsituamn t strainssmweegrmeaatlis,o throe dM-lDikRe sitnrasihnas poef ,ththee lyattwere rweoruelda tbive emlyorsem uaslelefurlt ahsa mn othde lM orygcaonbiascmtesr iautm thsem inegitmiaal tpishmasce2 155 stroafi nro.uImtinpeo crhteamntilcya,lt lhiberaprrye secrneceenoinfgc phroolecesdteurroelsh. Faudtunroe dsteutdecietsa bwleille bffee dcetdoincattheed mto othrpe hgoenloogmyico afnthde individpuroatlecoemllisc( Fchigaurarcete6rbi)z.aHtioen coef, ctheoslee sMteDroRl esntrhaainsc etso thuenrsapvreela tdhien gmoefcthhaenicsomlosn oief sroesfiMstaynccoeb.a cNteorviueml smegmcaotmispaocurnodsss tthhaet aargea srelseuctrefdac aes, pooftewnthiaicl hanwti-eTBp odsrtuuglsa dteurminigg hsctrebeenianng eosf sthene tcihaelmfeicaatlu lriberfaoryr utissisnuge invasiothnesbey MMDyRc oMbaycctoebraiactletruiubmer csumleogsmisaitfist hsterapinhse nmoutsytp uenidsecrogno seexrtvenesdivaec rcohsasraMctyerciozbaatciotenr ituom vaslpid.ate their efficacy. FigureF6ig. uErfefe c6t. oEffcfehcotl eosft ecrhoolloenstethroelm oonr ptheo lmogoyrpohfoclolgoyn ioefs acnoldoniniedsi vainddu ailncdeilvlsidoufatlh ecedllrsu go-fs uthsec edprtuibgl-e and MDsuRscMepytciboblea ctaenrdiu mMsDmReg mMayticsobsatrcateirnisu.m( a)smCeoglmonaytism sotrpaihnosl.o g(ay) oCf tohloenthy reme oMrpyhcobloacgtye rioufm tshmee gtmhraeteis strainsMiyncotbhaectearbiusmen scmeegamnadtisp srtersaeinsc ein othfec ahbosleensctee raonld. Oprveeserncige hotf cchuoltleusrteesrool.f Otvheerntihgrhete cuslttruarienss owf tehree diluted to an OD600 of 0.7, and aliquots of 5 µL were spotted at four positions on M7H10 agar plates containing 100 mM cholesterol, as well as M7H10 agar plates that did not contain cholesterol (control). The plates were incubated for 48 h at 30 ◦C. (b) Visualization of individual cells from the mycobacterial colonies on the M7H10 agar plates, shown in (a). Ery M. smeg A and Ery M. smeg B represent the two erythromycin-resistant clones derived from the Mycobacterium smegmatis mc2 155 strain. Antibiotics 2019, 8, 4 12 of 14 4. Discussion Mycobacterium smegmatis has been reported as a suitable surrogate for Mycobacterium tuberculosis during screening for novel anti-tuberculosis (anti-TB) drugs [26]. However, the emergence of MDR-tuberculosis (MDR-TB) and extensively-drug-resistant tuberculosis (XDR-TB) restrict the suitability of the drug-susceptible Mycobacterium smegmatis for the development of novel drugs against resistant TB strains. In the clinical settings, drug-resistant strains of TB emerge following the exposure of the drug-susceptible strain to mono-drug treatment conditions [27]. This strategy was explored in the present study for isolating two unique MDR strains of Mycobacterium smegmatis, under in vitro conditions. We have demonstrated that the drug-susceptible Mycobacterium smegmatis mc2 155 strain is capable of evolving into distinct clones of MDR strains, following gradual exposure to increasing concentrations of erythromycin. The MDR Mycobacterium smegmatis (referred to as erythromycin-resistant Mycobacterium smegmatis A and B) were robust and viable in the presence of high concentrations of erythromycin. Interestingly, the MDR phenotype affected the growth profile of Mycobacterium smegmatis. This observation is not entirely surprising, as the ability of a drug-susceptible mycobacterium to adapt to increasing concentrations of an antibiotic has been linked to compensatory adaptations, which impacts fitness and metabolic costs [28]. The strategy of switching between broth and plate cultures and the use of antibiotic discs was critical in the isolation of these unique strains. Antibiotics such as ampicillin and amoxicillin, which did not exhibit activity against the MDR Mycobacterium smegmatis strain, gained their activity in the presence of neighboring antibiotics, and vice versa. These observations illustrate the effect of antibiotic cross-interaction on the dynamic profile of the MDR phenotype. Even though combination chemotherapy has been well established as a robust strategy for eradicating MDR strains [29], our data also suggest that it is capable of suppressing or eliminating the activity of a useful antibiotic. Collectively, these provide insights into the intricate dynamics of drug resistance profiles that are exhibited by pathogenic bacteria, in general. According to the WHO, a strain of Mycobacterium tuberculosis is classified as MDR if it is resistant to at least the two most potent anti-TB drugs (rifampicin and isoniazid). Extremely-drug-resistant TB strains exhibit additional resistance to isoniazid, a fluoroquinolone, and one of the second-line anti-TB drugs (kanamycin, amikacin, and capreomycin). The two clones of the erythromycin-resistant Mycobacterium smegmatis generated by this study were both susceptible to rifampicin and a fluoroquinolone (moxifloxacin), but were resistant to isoniazid. Thus, the drug resistance profile of the erythromycin-resistant Mycobacterium smegmatis was not entirely consistent with the WHO criteria for the classification of MDR phenotypes of mycobacterium. The susceptibilities to moxifloxacin and streptomycin were markedly reduced in these new MDR strains, which somewhat place them between the classical MDR and XDR mycobacterial strains. Nonetheless, the MDR phenotype exhibited by the two distinct clones of erythromycin-resistant Mycobacterium smegmatis makes the organism a more suitable model organism for the identification of novel bioactive compounds against MDR-TB and XDR-TB, as compared to the Mycobacterium smegmatis mc2 155 strain. Due to the significant genetic disparity between Mycobacterium tuberculosis and Mycobacterium smegmatis, the MDR strains of the latter would be more useful as model organisms at the initial phase of routine chemical library screening procedures. Future studies will be dedicated to the genomic and proteomic characterization of these MDR strains to unravel the mechanisms of resistance. Novel compounds that are selected as potential anti-TB drugs during screening of the chemical library using these MDR Mycobacterium smegmatis strains must undergo extensive characterization to validate their efficacy. 5. Conclusions Continuous exposure of Mycobacterium smegmatis mc2 155 to erythromycin generated two clones that were extremely resistant to isoniazid, ethambutol and linezolid, while exhibiting reduced susceptibility to other notable anti-TB drugs. The fast growth phenotype of M. smegmatis was maintained in these MDR strains, thereby consolidating their suitability as model organisms for rapid and routine in vitro screening of novel drugs targeted against MDR Mycobacterium tuberculosis. Antibiotics 2019, 8, 4 13 of 14 Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/8/1/4/s1, Figure S1: Acid-fast staining of the drug-susceptible and MDR Mycobacterium smegmatis strains following exposure to cholesterol. Scale bar is 5 µm; Table S1: Optical Density of M. smegmatis as a measure of cell survival and proliferation during isolation of the MDR strains; Table S2: Amount of antibiotic in the paper discs used for the disc diffusion assay; Table S3: Amount of antibiotic in the paper discs used as neighbours for the selected antibiotics. Author Contributions: Conceptualization, P.K.A.; Data curation, P.K.A., P.G., G.B.A., E.J.S.B., M.-S.N.F., I.C. and R.Y.; Formal analysis, P.K.A.; Funding acquisition, P.K.A.; Investigation, P.K.A., V.A., P.G., G.B.A., E.J.S.B., I.C., R.Y. and L.A.; Methodology, P.K.A., V.A., P.G., G.B.A. and E.J.S.B.; Project administration, P.K.A.; Resources, P.K.A.; Supervision, P.K.A.; Validation, P.K.A.; Writing—original draft, P.K.A., V.A., M.-S.N.F. and I.C.; Writing—review & editing, P.K.A., V.A. and B.D.R. Funding: P.K.A. was supported by equipment grant (P/12/08960 in May 2012) from DAAD (Deutscher Akademischer Austausch Dienst—German Academic Exchange Service) and also funds from a World Bank African Centers of Excellence grant (ACE02-WACCBIP: Awandare) and a DELTAS Africa grant (DEL-15-007: Awandare). V.A., Ethel J. S. Blessie, I.C., R.Y., and L.A. were supported by a WACCBIP–World Bank ACE PhD fellowship (ACE02-WACCBIP: Awandare). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency (NEPAD Agency), with funding from the Wellcome Trust [107755/Z/15/Z: Awandare) and the UK government. The views expressed in this publication are those of the author(s) and not necessarily those of AAS, NEPAD Agency, Wellcome Trust, or the UK government. P.G. and Mohammed-Sherrif Fuseini were supported by the Department of Biochemistry, Cell and Molecular Biology, University of Ghana as National Service Personnel. P.K.A. and G.B.A. were supported by a grant from the Grand Challenges Canada (grant number: GCC 0237-01). Acknowledgments: The authors acknowledge the assistance of Dr. Michael Zimmermann of the ETH-Zurich for the supply of the wild type Mycobacterium smegmatis mc2 155 strains. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Kirschner, D.E.; Young, D.; Flynn, J.L. Tuberculosis: Global approaches to a global disease. Curr. Opin. Biotechnol. 2010, 21, 524–531. [CrossRef] [PubMed] 2. Forrellad, M.A.; Klepp, L.I.; Gioffré, A.; Sabio y Garcia, J.; Morbidoni, H.R.; Santangelo, M.d.L.P.; Cataldi, A.A.; Bigi, F. Virulence factors of the Mycobacterium tuberculosis complex. Virulence 2013, 4, 3–66. [CrossRef] [PubMed] 3. Borkowska, D.I.; Napiorkowska, A.M.; Brzezińska, S.A.; Kozińska, M.; Zabost, A.T.; Augustynowicz-Kopeć, E. From Latent Tuberculosis Infection to Tuberculosis. News in Diagnostics (QuantiFERON-Plus). Pol. J. Microbiol. 2017, 66, 5–8. [CrossRef] [PubMed] 4. Pai, M.; Nicol, M.P.; Boehme, C.C. Tuberculosis Diagnostics: State of the Art and Future Directions. Microbiol. Spectr. 2016, 4. [CrossRef] 5. McGrath, M.; Gey van Pittius, N.C.; van Helden, P.D.; Warren, R.M.; Warner, D.F. Mutation rate and the emergence of drug resistance in Mycobacterium tuberculosis. J. Antimicrob. Chemother. 2014, 69, 292–302. [CrossRef] [PubMed] 6. Tadesse, M.; Aragaw, D.; Dimah, B.; Efa, F.; Abdella, K.; Kebede, W.; Abdissa, K.; Abebe, G. Drug resistance-conferring mutations in Mycobacterium tuberculosis from pulmonary tuberculosis patients in Southwest Ethiopia. Int. J. Mycobacteriol. 2016, 5, 185–191. [CrossRef] [PubMed] 7. Yew, W.W.; Lange, C.; Leung, C.C. Treatment of tuberculosis: Update 2010. Eur. Respir. J. 2011, 37, 441–462. [CrossRef] [PubMed] 8. Sharma, S.K.; Mohan, A. Multidrug-resistant tuberculosis. Indian J. Med. Res. 2004, 120, 354–376. [CrossRef] 9. World Health Organization (WHO). Global Tuberculosis Report 2013; WHO: Geneva, Switzerland, 2013. 10. Forbes, L.; Ebsworth-Mojica, K.; DiDone, L.; Li, S.G.; Freundlich, J.S.; Connell, N.; Dunman, P.M.; Krysan, D.J. A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis. PLoS ONE 2015, 10, e0129234. [CrossRef] 11. Andries, K.; Verhasselt, P.; Guillemont, J.; Gohlmann, H.W.; Neefs, J.M.; Winkler, H.; Van Gestel, J.; Timmerman, P.; Zhu, M.; Lee, E.; et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 2005, 307, 223–227. [CrossRef] 12. Canton, R.; Morosini, M.I. Emergence and spread of antibiotic resistance following exposure to antibiotics. FEMS Microbiol. Rev. 2011, 35, 977–991. [CrossRef] [PubMed] Antibiotics 2019, 8, 4 14 of 14 13. Levin-Reisman, I.; Ronin, I.; Gefen, O.; Braniss, I.; Shoresh, N.; Balaban, N.Q. Antibiotic tolerance facilitates the evolution of resistance. Science 2017, 355, 826–830. [CrossRef] [PubMed] 14. Baquero, F.; Lemonnier, M. Generational coexistence and ancestor’s inhibition in bacterial populations. FEMS Microbiol. Rev. 2009, 33, 958–967. [CrossRef] [PubMed] 15. Händel, N.; Schuurmans, J.M.; Brul, S.; ter Kuile, B.H. Compensation of the metabolic costs of antibiotic resistance by physiological adaptation in Escherichia coli. Antimicrob. Agents Chemother. 2013. [CrossRef] [PubMed] 16. Zampieri, M.; Enke, T.; Chubukov, V.; Ricci, V.; Piddock, L.; Sauer, U. Metabolic constraints on the evolution of antibiotic resistance. Mol. Syst. Biol. 2017, 13, 917. [CrossRef] [PubMed] 17. Obolski, U.; Stein, G.Y.; Hadany, L. Antibiotic restriction might facilitate the emergence of multi-drug resistance. PLoS Comput. Biol. 2015, 11, e1004340. [CrossRef] [PubMed] 18. Imamovic, L.; Sommer, M.O. Use of collateral sensitivity networks to design drug cycling protocols that avoid resistance development. Sci. Transl. Med. 2013, 5, 204ra132. [CrossRef] [PubMed] 19. Noens, E.E.; Williams, C.; Anandhakrishnan, M.; Poulsen, C.; Ehebauer, M.T.; Wilmanns, M. Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1DeltaC expression strain. BMC Biotechnol. 2011, 11, 27. [CrossRef] 20. Love, W.J.; Zawack, K.A.; Booth, J.G.; Grhn, Y.T.; Lanzas, C. Markov Networks of Collateral Resistance: National Antimicrobial Resistance Monitoring System Surveillance Results from Escherichia coli Isolates, 2004–2012. PLoS Comput. Biol. 2016, 12, e1005160. [CrossRef] 21. Ouellet, H.; Johnston, J.B.; de Montellano, P.R. Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis. Trends Microbiol. 2011, 19, 530–539. [CrossRef] 22. Pandey, A.K.; Sassetti, C.M. Mycobacterial persistence requires the utilization of host cholesterol. Proc. Natl. Acad. Sci. USA 2008, 105, 4376–4380. [CrossRef] [PubMed] 23. Ryan, A.; Polycarpou, E.; Lack, N.A.; Evangelopoulos, D.; Sieg, C.; Halman, A.; Bhakta, S.; Eleftheriadou, O.; McHugh, T.D.; Keany, S.; et al. Investigation of the mycobacterial enzyme HsaD as a potential novel target for anti-tubercular agents using a fragment-based drug design approach. Br. J. Pharmacol. 2017, 174, 2209–2224. [CrossRef] [PubMed] 24. Evangelopoulos, D.; Gupta, A.; Lack, N.A.; Maitra, A.; ten Bokum, A.M.; Kendall, S.; Sim, E.; Bhakta, S. Characterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis. Tuberculosis 2014, 94, 664–671. [CrossRef] [PubMed] 25. Bhakta, S.; Besra, G.S.; Upton, A.M.; Parish, T.; Sholto-Douglas-Vernon, C.; Gibson, K.J.; Knutton, S.; Gordon, S.; DaSilva, R.P.; Anderton, M.C.; et al. Arylamine N-acetyltransferase is required for synthesis of mycolic acids and complex lipids in Mycobacterium bovis BCG and represents a novel drug target. J. Exp. Med. 2004, 199, 1191–1199. [CrossRef] [PubMed] 26. Chaturvedi, V.; Dwivedi, N.; Tripathi, R.P.; Sinha, S. Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis. J. Gen. Appl. Microbiol. 2007, 53, 333–337. [CrossRef] [PubMed] 27. Kerantzas, C.A.; Jacobs, W.R., Jr. Origins of Combination Therapy for Tuberculosis: Lessons for Future Antimicrobial Development and Application. mBio 2017, 8. [CrossRef] [PubMed] 28. Vogwill, T.; MacLean, R.C. The genetic basis of the fitness costs of antimicrobial resistance: A meta-analysis approach. Evol. Appl. 2015, 8, 284–295. [CrossRef] [PubMed] 29. Blumberg, H.M.; Burman, W.J.; Chaisson, R.E.; Daley, C.L.; Etkind, S.C.; Friedman, L.N.; Fujiwara, P.; Grzemska, M.; Hopewell, P.C.; Iseman, M.D.; et al. American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: Treatment of tuberculosis. Am. J. Respir. Crit Care Med. 2003, 167, 603–662. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).