Characterization of Two New Multidrug-Resistant Strains of Mycobacterium Smegmatis: Tools for Routine in Vitro Screening of Novel Anti-Mycobacterial Agents

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; [email protected] (V.A.); [email protected] (P.C.); gloria.arkaiﬁ[email protected] (G.B.A.); [email protected] (E.J.S.B.); [email protected] (M.-S.F.); [email protected] (I.C.); [email protected] (R.Y.); [email protected] (L.A.) 2 Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK; [email protected] * Correspondence: [email protected]

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 (Figure1). 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 (Figure1). 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 (Table1). Measurement of OD 600 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 thisAntibiotics observation 2018, 7, x (Table FOR PEER S1). REVIEW At the end of the 5th step, colonies of Mycobacterium smegmatis were4 of 15 not detectedstreptomycin within orthe tetracycline zone of inhibitiondiscs (Table on 1). the However, M7H10 agardistinct plates colonies containing of Mycobacterium either streptomycin smegmatis or tetracyclinewerestreptomycin detected discs withinor (Table tetracycline the1). zones However, discs of inhibition (Table distinct 1). coloniesof However, erythromycin of Mycobacteriumdistinct discs colonies on the smegmatis ofM7H10 Mycobacterium agarwere plates. detected smegmatis These within themycobacterialwere zones detected of inhibition cellswithin were of the erythromycin zonescharacterized of inhibition discs as onerythromycin-resistant of theerythromycin M7H10 agar discs plates. onMycobacterium the These M7H10 mycobacterial agarsmegmatis plates. cells. TheseTwo were characterizedcolonies,mycobacterial isolated as erythromycin-resistantcells by spreadingwere characterized a 10−6 dilutionMycobacterium as erythromycin-resistantof the erythromycin-resistant smegmatis. Two Mycobacterium colonies, Mycobacterium isolated smegmatis bysmegmatis spreading. Two aculture, 10colonies,−6 dilution showed isolatedof different by the spreading erythromycin-resistant susceptibilities a 10−6 dilution to a panel ofMycobacterium the of erythromycin-resistant thirteen commercial smegmatis antibiotics culture,Mycobacterium showed (Figure smegmatis different2a,b). susceptibilitiesTheseculture, two showed distinct to adifferent panel clones of susceptibilities thirteenwere further commercial tocharacterized a panel antibiotics of thirteen as erythromycin-resistant (Figure commercial2a,b). Theseantibiotics two Mycobacterium (Figure distinct 2a,b). clones weresmegmatisThese further two A and characterizeddistinct B and clones were as continuallywere erythromycin-resistant further maintained characterized on MycobacteriumM7H10 as erythromycin-resistant agar plates smegmatis (withoutA antibiotics) andMycobacterium B and for were continuallythesmegmatis next 6 months, maintainedA and B prior and on towere M7H10the continuallysubsequent agar plates maintainedassays. (without on antibiotics) M7H10 agar for plates the next (without 6 months, antibiotics) prior tofor the the next 6 months, prior to the subsequent assays. subsequent assays.

Figure 1. Generating multidrug-resistant (MDR) clones of Mycobacterium smegmatis, in vitro. Single Figure 1. Generating multidrug-resistant (MDR) clones of Mycobacterium smegmatis, in vitro. Single coloniesFigure 1.of Generatingthe Mycobacterium multidrug-resi smegmatisstant mc 2 (MDR)155 strain clones were of transferred Mycobacterium into asmegmatis M7H9 broth,, in vitro. containing Single 2 coloniesa coloniessublethal of of the concentration theMycobacterium Mycobacterium of an smegmatis smegmatisantibioticmc (streptomc2 155 mycin,strain were wereerythromycin, transferred transferred or into te intotracycline), a aM7H9 M7H9 broth, and broth, incubated containing containing aat sublethala 30sublethal °C for concentration 24concentration h with shaking. of of an an An antibiotic antibiotic aliquot (streptomycin,(strepto of eachmycin, broth cultureerythromycin, erythromycin, was spread, or or te tetracycline),tracycline), uniformly, andonto and incubated M7H10 incubated ◦ atagarat 30 30 C plates°C for for 24 24and h h with withtwo shaking. shaking.paper AndiscsAn aliquotaliquot containing of each th broth e same culture culture amount was was spread, spread,of antibiotic uniformly, uniformly, (streptomycin, onto onto M7H10 M7H10 agarerythromycin,agar plates plates and and twoor tetracycline) papertwo paper discs containingwerediscs placedcontaining the sameapproximately th amounte same of amount30mm antibiotic apart of (streptomycin, antibioticon the corresponding(streptomycin, erythromycin, orinoculatederythromycin, tetracycline) agar were orplate. tetracycline) placed All the approximately agar were plates placed were 30 mmapproximately incubated apart on at the 3030mm corresponding°C for apart 48 h.on Discrete inoculatedthe corresponding colonies agar of plate. ◦ AllMycobacteriuminoculated the agar plates agar smegmatis plate. were incubated Althatl the grew agar atwithin 30platesC the for were zones 48 h.incubated of Discrete inhibiti onat colonies 30were °C re-inoculated offorMycobacterium 48 h. Discrete into fresh smegmatiscolonies M7H9 ofthat grewbrothMycobacterium within for determination the zonessmegmatis of of inhibition that the grewminimum werewithin re-inoculatedinhibitory the zones concentrations of intoinhibiti freshon were M7H9 (MIC re-inoculateds) broth of these for determinationcolonies into fresh for M7H9each of the minimumantibiotic.broth for inhibitory Thedetermination discrete concentrations colonies of the miniwere (MICs)mum also inoculatinhibitory of theseed coloniesconcentrationsinto fresh for M7H9 each (MIC broth antibiotic.s) ofcontaining these The colonies discretean increased for colonies each wereconcentrationantibiotic. also inoculated The of discrete antibiotic into colonies freshfor repetition M7H9 were also broth of theinoculat containingentireed procedure into an fresh increased (5 M7H9 times). broth concentration During containing the repetitions of an antibiotic increased (R), for repetitiontheconcentration concentration of the of entire ofantibiotic antibiotic procedure for that repetition (5was times). added of the During to entiM7H9re the procedure broth repetitions was (5increased times). (R), the Duringsyst concentrationematically the repetitions (2×, of 4×, antibiotic 8×,(R), that16×,the was concentrationand added 32×) toprior M7H9 of antibioticto broththe discthat was was increaseddiffusion added systematicallyassay.to M7H9 The broth initial was (2× ,increasedconcentrations 4×, 8×, 16syst×ematically, andof streptomycin, 32× )(2×, prior 4×, to8×, the discerythromycin,16×, diffusion and 32×) assay. and prior tetracycline The to initial the (1×), concentrationsdisc addeddiffusion to each ofassay. streptomycin,fresh The M7H9 initial broth, erythromycin, concentrations were 30 μg/ andμ L,of 60 tetracyclinestreptomycin, μg/μL, and (1 × ), added30erythromycin, μg/ toμL, each respectively. fresh and M7H9 tetracycline Z1, broth, Z2, and (1×), were Z3 added 30 representµg/ toµ L,each 60triplica µfreshg/µte L,M7H9 measurements and broth, 30 µg/ wereµL, of respectively. 30each μg/ zoneμL, 60of Z1, μinhibitiong/ Z2,μL, andand Z3 represent(in30 mm). μg/μ triplicateL, respectively. measurements Z1, Z2, and of Z3 each represent zone of triplica inhibitionte measurements (in mm). of each zone of inhibition (in mm).

Figure 2. Cont. Antibiotics 2019, 8, 4 5 of 14 Antibiotics 2018, 7, x FOR PEER REVIEW 5 of 15

FigureFigure 2. 2. IsolationIsolation of of two two clones clones of ofMDR MDR MycobacteriumMycobacterium smegmatis smegmatis. (a) Schematic.(a) Schematic representation representation of the of theprocedure procedure used used for forisolating isolating the the two two clones clones of oferythromycin-resistant erythromycin-resistant MycobacteriumMycobacterium smegmatis smegmatis. . FollowingFollowing thethe 5th repeat for for ge generatingnerating resistant resistant clones clones of Mycobacterium of Mycobacterium smegmatis smegmatis, in vitro,, in vitro(R5),, a (R5), a10-fold 10-fold serial serial dilution dilution of of the the bacterial bacterial culture culture was was performed. performed. The The10−6 culture 10−6 culture dilution dilution was spread wasspread on ona M7H10 a M7H10 agar agar plate, plate, from from which which two two distinct distinct colonies colonies of acid-fast of acid-fast bacilli bacilli were were isolated isolated andand identified identiﬁed asas erythromycin-resistant erythromycin-resistant MycobacteriumMycobacterium smegmatis smegmatis A andA and B. (b B.) Drug (b) Drug susceptibility susceptibility pattern pattern of the two of the twoclones clones of the of the erythromycin-resistant erythromycin-resistant MycobacteriumMycobacterium smegmatis smegmatis. The. Thedefinitions deﬁnitions of the of theabbreviated abbreviated commercialcommercial antibiotics antibiotics andand their co correspondingrresponding amounts amounts in in the the discs discs are are indicated indicated in inTable Table S2.S2.

TableTable 1. 1. ZoneZone of of inhibition. inhibition.

ZonesZones of ofInhibition Inhibition (mm) (mm) Streptomycin (µg/µL) Erythromycin (µg/µL) Tetracycline (µg/µL) Repetitions (R) Streptomycin (µg/µL) Erythromycin (µg/µL) Tetracycline (µg/µL) Repetitions (R) 0.1 1 6 30 150 0.1 1 6 30 150 0.1 1 6 30 150 0.1 1 6 30 150 0.1 1 6 30 150 0.1 1 6 30 150 - 0 6 12 15 22 0 0 0 11.5 19.5 8 9.5 18 22 27.5 - 0 6 12 15 22 0 0 0 11.5 19.5 8 9.5 18 22 27.5 1 0 0 10 12 17.5 0 0 0 6 13.5 0 8 11 15.5 19 1 0 0 10 12 17.5 0 0 0 6 13.5 0 8 11 15.5 19 22 0 0 00 99 88 13 13 0 00 00 06 610 10 0 00 08.5 8.511 1114.5 14.5 33 0 0 00 00 00 18 18 0 00 00 0 16.516.5 24.5 24.5 1010 6 610 1013 1315 15 44 0 0 0 0 0 0 0 0 17 17 0 0 0 00 0 10 10 17.517.5 N/AN/A N/A N/A N/A N/A N/A N/AN/A N/A 55 N/A N/A N/AN/A N/AN/A N/AN/A N/A N/A 0 0 0 00 0 6 6 13 13 N/A N/A N/A N/A N/A N/A N/A N/AN/A N/A N/AN/A denotes denotes not not available. available.

TheTheMycobacterium Mycobacterium smegmatis mcmc2 2155155 was was susceptible susceptible to totwelve twelve out out of ofthe the thirteen thirteen panel panel of of commercialcommercial antibiotics,antibiotics, while while the the two two clones clones of erythromycin-resistant of erythromycin-resistant MycobacteriumMycobacterium smegmatis smegmatis (A (Aand and B) Bwere) were both both susceptible susceptible to only to rifampicin, only rifampicin, moxifloxacin, moxiﬂoxacin, and streptomycin and streptomycin (Figure 2b). Notably, (Figure2 b). Notably,clone A, clone but A,not but clone not cloneB, of B,the of erythromycin-resistant the erythromycin-resistant MycobacteriumMycobacterium smegmatis smegmatis strainstrain was wasalso also susceptiblesusceptible toto tetracyclinetetracycline and chloramphenicol. Henc Hence,e, the the distinct distinct antibiotic antibiotic susceptibility susceptibility pattern pattern forfor each each erythromycin-resistant erythromycin-resistant strain was was the the basi basiss for for the the characterization characterization of erythromycin-resistant of erythromycin-resistant MycobacteriumMycobacterium smegmatis smegmatis asas either clone A A or or clone clone B. B. These These ob observationsservations agree agree with with a previous a previous report report that indicated that a single antibiotic treatment may select for resistance against other multiple drugs that indicated that a single antibiotic treatment may select for resistance against other multiple drugs via a set of mechanisms (collateral resistance) or a single mechanism (cross-resistance) [20]. What is via a set of mechanisms (collateral resistance) or a single mechanism (cross-resistance) [20]. What is more striking about this data is that the acquisition of broad-spectrum resistance cuts across all more striking about this data is that the acquisition of broad-spectrum resistance cuts across all classes classes of antibiotics. This observation is the first to be reported for mycobacteria where exposure to of antibiotics. This observation is the ﬁrst to be reported for mycobacteria where exposure to one one antibiotic has led to the development of extreme drug resistance (MDR). Thus, these two strains antibioticconstitute has a valuable led to thetool developmentin the continued of search extreme for drugnew antibiotic resistance candidates. (MDR). Thus, these two strains constitute a valuable tool in the continued 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

AntibioticsA spot2019 test, 8, 4 assay was used to validate the extent of resistance of the two clones of erythromycin-6 of 14 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 offor erythromycin 0 h, 3 h, and for 6 h, 0 h, prior 3 h, andto spotting 6 h, prior the to spottingcells on theM7H10 cells onagar M7H10 plates. agar The plates. erythromycin-resistant The erythromycin- resistantMycobacteriumMycobacterium smegmatis smegmatis strains werestrains viable were at viableall of the at allthree of thetime three points time (0 pointsh, 3 h, and (0 h, 63 h h, of and exposure 6 h of exposureto erythromycin), to erythromycin), and the andviability the viability was robust was robust against against the thelow low and and high high concentrations concentrations of 2 erythromycin usedused for for the the assay assay (Figure (Figure3). On3). On the contrary,the contrary, the Mycobacterium the Mycobacterium smegmatis smegmatismc 155 mc strain2 155 wasstrain viable was viable at the at low the concentrations low concentrations of erythromycin, of erythromycin, whereas whereas growth growth was undetected was undetected at the at high the concentrationshigh concentrations of the antibioticof the antibiotic (Figure3 a,b).(Figure Notably, 3a,b). growth Notably, was growth undetected was for undetected the Mycobacterium for the 2 smegmatisMycobacteriummc 155 smegmatis strain following mc2 155 6 strain h of exposure following to the 6 lowh of concentrations exposure to the of erythromycin low concentrations (Figure3 c).of Theseerythromycin observations (Figure demonstrate 3c). These that observations the two erythromycin-resistant demonstrate that the strains two exhibit erythromycin-resistant a distinct viability 2 phenotypestrains exhibit in thea distinct presence viability of erythromycin, phenotype in in th comparisone presence of to erythromycin, the Mycobacterium in comparison smegmatis tomc the 155Mycobacterium parent strain. smegmatis mc2 155 parent strain.

Figure 3. Cont. Antibiotics 2019, 8, 4 7 of 14 Antibiotics 2018, 7, x FOR PEER REVIEW 7 of 15

Figure 3. Viability assay of the drug-susceptible and MDR Mycobacterium smegmatis strains following Figure 3. Viability assay of the drug-susceptible and MDR Mycobacterium smegmatis strains following exposure to low and high concentrations of erythromycin. Overnight cultures of these strains were exposure to low and high concentrations of erythromycin. Overnight cultures of these strains were 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 smegmatisThe MICsmc2 155 of thirteen strain and commercially-availa the two MDR strains.ble antibiotics For each were organism, determined the MIC for ofthe an Mycobacterium antibiotic of interestsmegmatis was mc determined2 155 strain by and placing the two four MDR discs, containingstrains. For decreasing each organism, concentrations the MIC ofof the an antibiotic,antibiotic atof positionsinterest was distant determined from each by other placing onthe four agar discs, plate. cont Allaining of the decreasing three mycobacterial concentrations strains of werethe antibiotic, resistant toat thepositions highest distant concentration from each of Pyrazinamide other on the (160 agarµ g)plate. and wereAll of susceptible the three mycobacterial to the lowest concentration strains were ofresistant moxifloxacin to the (0.25highestµg; concentration Table2). The twoof Pyrazinamide MDR strains (160 were μ resistantg) and were to the susceptible highest concentration to the lowest ofconcentration isoniazid, ethambutol, of moxifloxacin linezolid, (0.25 andμg; Table erythromycin, 2). The two while MDR the strainsMycobacterium were resistant smegmatis to themc highest2 155 strainconcentration was susceptible of isoniazid, to the ethambutol, lowest concentration linezolid, ofand these erythromycin, antibiotics. Thewhile MIC the of Mycobacterium vancomycin andsmegmatis streptomycin mc2 155 wasstrain 80 wasµg susceptible for the MDR to the strains; lowest the concentrationMycobacterium of these smegmatis antibiotics.mc2 The155 MIC strain of wasvancomycin susceptible and to streptomycin the lowest concentrations was 80 μg for the of theseMDR antibioticsstrains; the used Mycobacterium for the assay. smegmatis Interestingly, mc2 155 thestrain two was MDR susceptible strains were to susceptible the lowest to 20concentratµg of ampicillinions of andthese amoxicillin, antibiotics even used though for ourthe earlierassay. observationsInterestingly, indicated the two MDR that these strains MDR were strains susceptible were resistant to 20 μ tog of 40 ampicillinµg of either and of amoxicillin, these antibiotics even (Figurethough2 b).our Moreover, earlier observations clone B of the indicated MDR strain that wasthese susceptible MDR strains to 15 wereµg andresistant 30 µg to of 40 tetracycline μg of either and of chloramphenicol,these antibiotics (Figure respectively; 2b). Moreover, an observation clone thatB of wasthe MDR inconsistent strain was with susceptible the data shown to 15 μ ing Figureand 302 μb.g Weof tetracycline proposed that and the chloramphenicol, placement of different respectively; classes an ofobservation antibiotic that discs was on inconsistent an agar plate with during the data the testshown for thein Figure drug susceptibility 2b. We proposed pattern that (Figure the placement2B), as compared of different to classes the placement of antibiotic of discs discs of on the an same agar antibioticplate during for the test MIC for determination the drug susceptibility (Table2), waspattern the (Figure basis for 2B), the as above-mentioned compared to the placement deviations. of Thediscs antibiotic of the same assay antibiotic format isfor the the standard MIC determi widelynation used (Table for the 2), determination was the basis of for the the antibiotic above- susceptibilitymentioned deviations. pattern of bacterialThe antibiotic isolates. assay Thus format cross-interaction is the standard between widely different used for classes the determination of antibiotics mightof the beantibiotic influencing susceptibility the resistance pattern profiles of bacteria of the MDRl isolates.M. smegmatis Thus cross-interactionstrains to a sub-collection between different of the selectedclasses of commercial antibiotics antibiotics. might be 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 1× 6 11 14 Amp (40 µg) 2× 9 17 14 4× 18 21 20 0.5× 6 12 11 1× 9 18 17 Amx (40 µg) 2× 13 18 19 4× 10 18 20 0.5× 13 0 0 1× 20 0 0 Van (40 µg) 2× 20 7 9 4× 21 9 10 0.5× 8 0 0 1× 29 0 0 Inh (10 µg) 2× 37 0 0 4× 40 0 0 0.5× 15 0 0 1× 36 0 0 Emb (10 µg) 2× 46 0 0 4× 51 0 0 0.5× 0 0 0 1× 0 0 0 Pzd (40 µg) 2× 0 0 0 4× 0 0 0 0.5× 30 10 10 1× 36 18 12 Moxi (0.5 µg) 2× 40 22 20 4× 47 29 24 0.5× 0 18 12 1× 8 20 16 Rif (10 µg) 2× 8 23 19 4× 11 25 21 0.5× 0 0 0 1× 7 0 0 Lin (5 µg) 2× 8 0 0 4× 10 0 0 0.5× 46 20 7 1× 40 20 9 Tet (30 µg) 2× 42 23 14 4× 54 27 18 0.5× 14 9 0 1× 18 9 7 Chlo (30 µg) 2× 22 15 10 4× 31 22 13 0.5× 8 0 0 1× 10 0 0 Ery (40 µg) 2× 10 0 0 4× 15 0 0 0.5× 18 0 0 1× 20 0 0 Strep (20 µg) 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

AntibioticsUsing2019 the, 8, 4disc diffusion assay, a number of antibiotics are usually placed at unique positions9 of on 14 an agar plate that has been inoculated with the strain of interest during the test for drug susceptibility patterns (Figure 44).). WeWe investigated investigated the the effect effect of of ne neighboringighboring antibiotic antibiotic discs discs on on the the antimycobacterial antimycobacterial activity ofof ampicillin, ampicillin, amoxicillin, amoxicillin, linezolid, linezolid, tetracycline, tetracycline, chloramphenicol, chloramphenicol, erythromycin, erythromycin, and streptomycin. and streptomycin.Ultimately, this Ultimately, experimental this approach experimental was anticipated approach to was provide anticipated information to provide on speciﬁc information neighboring on specificantibiotic neighboring discs that antibiotic either enhanced discs that or either reduced enhanced the antimycobacterial or reduced the antimycobacterial activity of these selectedactivity ofantibiotic these selected discs. antibiotic discs.

Figure 4. SchematicSchematic representation representation of of the the arrangement arrangement of of antibiotic antibiotic discs discs on on ag agarar plates. plates. A total A total of nineteenof nineteen different different commercial commercial antibi antibioticsotics were placed were at placed unique at posi uniquetions positionson three M7H10 on three agar M7H10 plates, Mycobacterium smegmatis 2 whichagar plates, were inoculated which were with inoculated cultures withof either cultures Mycobacterium of either smegmatis mc2 155 or erythromycin-mc 155 or erythromycin-resistant Mycobacterium smegmatis A. The amount (in µg) of each selected antibiotic- resistant Mycobacterium smegmatis A. The amount (in μg) of each selected antibiotic-containing disc is containing disc is indicated in Table S2 and the amount of each antibiotic (in µg) used as a neighbor for indicated in Table S2 and the amount of each antibiotic (in μg) used as a neighbor for the selected the selected antibiotic is indicated in Table S3. antibiotic is indicated 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 and chloramphenicol when each of these selected antibiotics were surrounded by the neighboring chloramphenicol when each of these selected antibiotics were surrounded by the neighboring antibiotic discs indicated in Table3. Interestingly, the same concentration of these selected antibiotics antibiotic discs indicated in Table 3. Interestingly, the same concentration of these selected antibiotics (ampicillin, amoxicillin, and chloramphenicol) exhibited antimycobacterial activity against the same (ampicillin, amoxicillin, and chloramphenicol) exhibited antimycobacterial activity against the same resistant strain in the absence of the neighboring antibiotic discs. The opposite effect was detected resistant strain in the absence of the neighboring antibiotic discs. The opposite effect was detected for for streptomycin against the resistant strain when discs of rifampicin, isoniazid, and ampicillin streptomycin against the resistant strain when discs of rifampicin, isoniazid, and ampicillin were were used as neighbors. These observations were not unique to erythromycin-resistant M. smegmatis used as neighbors. These observations were not unique to erythromycin-resistant M. smegmatis A A strain; the M. smegmatis mc2 155 strain was highly susceptible to linezolid in the presence of strain; the M. smegmatis mc2 155 strain was highly susceptible to linezolid in the presence of neighboring antibiotic discs, but was resistant to the same concentration of linezolid in the absence of neighboring antibiotic discs, but was resistant to the same concentration of linezolid in the absence neighboring antibiotic discs. Collectively, these observations demonstrate that the resistance proﬁles of neighboring antibiotic discs. Collectively, these observations demonstrate that the resistance of M. smegmatis mc2 155 and the corresponding MDR strains are inﬂuenced by cross-interaction of the profiles of M. smegmatis mc2 155 and the corresponding MDR strains are influenced by cross- different classes of antibiotics. This same dataset demonstrates a subtle phenotypic difference between interaction of the different classes of antibiotics. This same dataset demonstrates a subtle phenotypic the two MDR strains. difference between the two MDR strains. Table 3. Selected antibiotics. Table 3. Selected antibiotics. Selected Selected Antibiotics in the Presence of Neighbors Selected Antibiotics alone Selected Selected Antibiotics in the Presence of Neighbors Selected Antibiotics alone Neighbors Antibiotics M. smeg mc2 155 Ery M. smeg A M. smeg mc2 155 Ery M. smeg A 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 AmpAmx N/A N/A 0 0 N/A N/A 18 11 Lin, Strep, Pzd, Inh, Cys Moxi Amx Lin N/A 50 0 N/A N/A0 N/A 18 Van, Lin, Pzd, Pzd, Amx Cys Lin Tet50 N/A N/A 7 N/A 0 20 N/A Rif,Van, Inh, Pzd, Ery Amx Tet Chlo N/A N/A 7 0 N/A N/A 9 20 Emb, Rif, Pzd, Inh, Cys Ery Ery 20 N/A 10 N/A Tet, Inh, Moxi ChloStrep N/A 48 0 14 20 N/A 0 9 Rif, Emb, Inh, AmpPzd, Cys Ery 20 N/A 10 N/A Tet, Inh, Moxi Numbers represent zones of inhibition (mm) of the selected antibiotics. The amount of antibiotics (µg) used for the Strep 48 14 20 0 Rif, Inh, Amp assay are indicated in Tables S2 and S3. 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 represent2 zones of inhibition (mm) of the selected antibiotics. The amount of antibiotics (μg) Mycobacterium smegmatisused for themc assay are155 indicated strain in wereTables S2 investigated and S3. 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 growththe Mycobacterium rate was smegmatis monitored mc2 155 for strain 60 were h (Figure investigated5a). Thein order two to ascertain clones ofthe erythromycin-resistanteffect of the MycobacteriumMDR smegmatis phenotypeshowed on growth arate. relatively Single colonies rapid from growth these three rate mycobacterial during strains the initial were initially 12 h compared to the Mycobacteriumgrown smegmatis in M7H9 brothmc for2 15524 h at strain. 30 °C with Interestingly, shaking. These the mycobacterial latter strain cultures recuperated were diluted to from 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 growtherythromycin-resistant after 12 h Mycobacterium and showed smegmatis a rapid showed growth a relatively rate, rapid attaining growth rate an during OD600 the of 3.2 by 48 h (Figure5a). Oninitial the contrary,12 h compared the to erythromycin-resistant the Mycobacterium smegmatis mc strains2 155 strain. showed Interestingly, a relatively the latter slower strain growth rate recuperated from the initial lag phase of growth after 12 h and showed a rapid growth rate, attaining after the initial 12 h. This pattern of growth was confirmed following measurement of OD at 12-h an OD600 of 3.2 by 48 h (Figure 5a). On the contrary, the erythromycin-resistant strains showed a 600 intervals for 96relatively h (Figure slower5b), growth illustrating rate after the that initial the 12 acquisition h. This pattern of of MDRgrowth phenotypewas confirmed causesfollowing a concomitant alteration in themeasurement growth profileof OD600 at of 12-hMycobacterium intervals for 96 h smegmatis(Figure 5b), illustrating. that the acquisition of MDR phenotype causes a concomitant alteration in the growth profile of Mycobacterium smegmatis.

Figure 5. GrowthFigure proﬁles 5. Growth of theprofiles drug-susceptible of the drug-susceptible and MDRand MDRMycobacterium Mycobacterium smegmatissmegmatis strains.strains. Overnight Overnight cultures of the three strains were diluted to an optical density at 600 nm (OD600) of 0.01 and cultures of thethe three growth strains rate was were monitored diluted for 60 to han (a) opticalor 96 h (b). density For (a), the at OD 600600 was nm measured (OD600 )every of 0.01 3 h for and the growth rate was monitored12 h, followed for 60 by h an (a )intermi or 96ttent h ( b12-h). Formeasurement. (a), the ODFor (600b), thewas OD measured600 was measured every every 3 12 h h for for 12 h, followed 96 h. Ery M. smeg A and Ery M. smeg B represent the two erythromycin-resistant clones derived from by an intermittent 12-h measurement. For (b), the OD600 was measured every 12 h for 96 h. Ery M. smeg the Mycobacterium smegmatis mc2 155 strain. 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- mycobacteria.TB drugs Hence,(kanamycin, we investigated amikacin, and the capreomycin). effect of cholesterol The two on clones the morphology of the erythromycin-resistant of Mycobacterium smegmatisMycobacteriummc2 155, assmegmatis well as the generated erythromycin-resistant by this study were strains. both susceptible to rifampicin and a Coloniesfluoroquinolone of the Mycobacterium(moxifloxacin), but smegmatis were resistantmc2 155 to isoniazid. strain were Thus, detected the drug as resistance broad, ﬂuffy-likeprofile of the erythromycin-resistant Mycobacterium smegmatis was not entirely consistent with the WHO structures on the cholesterol-containing M7H10 agar plates, whereas the control (no cholesterol) criteria for the classification of MDR phenotypes of mycobacterium. The susceptibilities to agar plates showed the expected colony phenotype (Figure6a). The colonies of the two clones of moxifloxacin and streptomycin were markedly reduced in these new MDR strains, which somewhat erythromycin-resistant Mycobacterium smegmatis were modestly enlarged on the cholesterol-containing place them between the classical MDR and XDR mycobacterial strains. Nonetheless, the MDR agar platephenotype compared exhibited to the by colonies the two on distinct the control clones agar of erythromycin-resistant plates (Figure6a). Visualization Mycobacterium of individual smegmatis cells withinmakes these the organism colonies revealed a more thatsuitable all of model the three organism mycobacterial for the strainsidentification tested positiveof novel for bioactive acid-fast 2 stainingcompounds (Figure S1). against Moreover, MDR-TB individual and XDR-TB, cells from as compared colonies ofto thethe MycobacteriumMycobacterium smegmatis smegmatis mcmc2 155155 strainstrain. had a Due slender to the and significant rod-like genetic morphology disparity (Figure between6b). Mycobacterium Even though tuberculosis the erythromycin-resistant and Mycobacterium strainssmegmatis were also, the rod-like MDR strains in shape, of the they latter were would relatively be more smaller usefulthan as model the Mycobacterium organisms at the smegmatis initial phasemc2 155 strain.of routine Importantly, chemical thelibrary presence screening of cholesterol procedures. had Future no detectablestudies will effectbe dedicated on the morphologyto the genomic of and the individualproteomic cells (Figurecharacterization6b). Hence, of cholesterolthese MDR enhances strains to the unravel spreading the mechanisms of the colonies of resistance. of Mycobacterium Novel smegmatiscompoundsacross that the are agar selected surface, as potential of which anti-TB we postulate drugs during might screening be an essentialof the chemical feature library for using tissue invasionthese by MDRMycobacterial Mycobacterium tuberculosis smegmatisif the strains phenotype must undergo is conserved extensive across characterizationMycobacterium to validatesp. their efficacy.

FigureFigure 6. Effect 6. ofEffect cholesterol of cholesterol on the morphologyon the morphology of colonies of andcolonies individual and individual cells of the cells drug-susceptible of the drug- and MDRsusceptibleMycobacterium and MDR smegmatis Mycobacteriumstrains. ( a)smegmatis Colony morphology strains. (a) ofColony the three morphologyMycobacterium of the smegmatis three strainsMycobacterium in the absence smegmatis and presence strains in ofthe cholesterol. absence and Overnightpresence of cholesterol. cultures of Overnight the three cultures strains of were the

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/).