Short
communication
Selective
medium
for
culture
of
Mycoplasma
hyopneumoniae
Beth
S.
Cook
1,
Jessica
G.
Beddow,
Lucía
Manso-Silván
2,
Gareth
A.
Maglennon,
Andrew
N.
Rycroft
*
DepartmentofPathology&PathogenBiology,RoyalVeterinaryCollege,HawksheadLane,NorthMymms,AL97TA,UK
ARTICLE INFO
Articlehistory:
Received22April2016
Receivedinrevisedform27September2016 Accepted29September2016
Keywords:
Mycoplasmahyopneumoniae
Selectiveculture Enzooticpneumonia Pig
ABSTRACT
ThefastidiousporcinerespiratorypathogenMycoplasmahyopneumoniaehasprovendifficulttoculture sinceitwasfirstisolatedin1965.Areliablesolidmediumhasbeenparticularlychallenging.Moreover, clinicalandpathologicalsamplesoftencontainthefast-growingM.hyorhiniswhichcontaminatesand overgrowsM.hyopneumoniaeinprimaryculture.Theaimofthisstudywastooptimisetheculture mediumforrecoveryofM.hyopneumoniaeandtodeviseamediumforselectionofM.hyopneumoniae fromclinicalsamplesalsocontainingM.hyorhinis.ThesolidmediumdevisedbyNielsFriiswasimproved byuseofPurifiedagarandincorporationofDEAE-dextran.Additionofglucoseorneutralizationofacidity inliquidmediumwithNaOHdidnotimprovethefinalyieldofviableorganismsoralterthetimingof peakviability.AnalysisoftherelativesusceptibilityofM.hyopneumoniaeandM.hyorhinisstrainstofour antimicrobialsshowedthatM.hyopneumoniaeislesssusceptiblethanM.hyorhinistokanamycin.This wasconsistentinallUKandDanishstrainstested.Aconcentrationof2mg/mlofkanamycinselectively inhibitedthegrowthofallM.hyorhinistested,whileM.hyopneumoniaewasabletogrow.Thisformsthe basisofaneffectiveselectiveculturemediumforM.hyopneumoniae.
ã2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).
1.Introduction
Mycoplasma hyopneumoniaeistheprimarycauseof enzootic pneumonia(EP)inpigs.Thediseaseisofglobalsignificancebothin reducing growth efficiency and in promoting susceptibility to concurrentbacterial and viralinfections (Thacker et al.,1999). DiagnosisofEPisusuallybyacombinationofserologicaltests(Feld etal.,1992;Djordjevicetal.,1994),PCRofnasalsecretionorlung tissueatslaughter(Mattssonetal.,1995;Baumeisteretal.,1998; Stemke,1997),real-timePCR(Dubossonetal.,2004;Maroisetal., 2010)andisolationinculture(KobischandFriis,1996).However,
M.hyopneumoniaeisnotoriouslyfastidious(GoodwinandHurrell, 1970)andcultureremainschallengingandtimeconsuming.The mostwidelyusedliquidmediumforcultureofM.hyopneumoniae
wasdevelopedbyNielsFriis(1975).Homogenisedlungtissuewas seriallydilutedintubes ofthemediumandincubationledtoa gradualcolourchangeofthephenolredindicatorfrompinkto
yellow, without turbidity, over a period of 3–10days growth. However, individual mycoplasmas could only be purified by terminaldilutionintheliquidmedium.
AcommercialmediumisavailablefromMycoplasma Experi-ence1Ltd.U.K. (ME).Bothliquidand solidmediaareavailable. ThesesupportthegrowthofM.hyopneumoniaebutthe constit-uents of this commercial medium are not publicly available. SolidificationofFriismediumwithAgardidnotallowgrowthof colonies of M. hyopneumoniae, suggesting that the agar was inhibitorytogrowthorsequesteredanessentialnutrient.Theuse ofabetterdefinedsolidmediumwouldallowgreaterflexibilityfor research,suchastransformationofM.hyopneumoniae(Maglennon etal.,2013a).
Whenusedfordiagnosticpurposes,bothFriismediumandME media areeasily overgrown bythe faster-growing M.hyorhinis
which is sometimes present in pig lung lesions alongside
M. hyopneumoniae and frequently in apparently normal tissue (KobischandFriis,1996).TosuppressthegrowthofM.hyorhinis, Friisrecommendedtheuseof5%hyperimmuneanti-M.hyorhinis
rabbit serum and 500
m
g/ml cycloserine (Friis,1971).However, suchserumisnotalwaysavailable,isexpensive,andbatchesof serumvaryintheircapacitytosuppressM.hyorhinis.*Correspondingauthor.
E-mailaddress:arycroft@rvc.ac.uk(A.N. Rycroft).
1
Presentaddress:MSDAnimalHealth,MiltonKeynes,UK.
2
Presentaddress:INRA,UMR1309CMAEE,F-34398Montpellier,France.
http://dx.doi.org/10.1016/j.vetmic.2016.09.022
0378-1135/ã2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/). ContentslistsavailableatScienceDirect
Veterinary
Microbiology
Thepurposeofthisstudywasthereforetoimprovetheculture mediumfortransformationandmutagenesisofM.hyopneumoniae, allowingsinglecoloniestobereadilydistinguishedandselected, andtodeviseanimprovedselectionmediumforthesuppressionof
M.hyorhinis.
2.Materials&methods
2.1.Mycoplasmastrains
M.hyopneumoniaestrains277/94and325/95werethegiftof NielsFriis,DanishVeterinaryInstitute,Copenhagen.Otherstrains ofM.hyopneumoniaewereDanishfieldisolatesfromlesionsofEP, thegiftofDrBrankoKokotovic,DanishVeterinaryInstitute,orUK
fieldisolatesfromslaughterhouselesionsofEP.M.hyorhinisstrains wereUK fieldisolatesobtainedfrompiglungswithor without grosslesionsatpost-mortem.Theidentityofallorganismswas confirmedusingspecies-specificPCRamplifyinga regionof the conservedhypotheticalproteinmhp165fromM.hyopneumoniae
(Baumeisteretal.,1998)andthehighlyconserved16SrRNAregion of M. hyorhinis (Lin et al., 2006). The identity of all
M.hyopneumoniaestrainswassubsequentlyconfirmedbywhole genome sequencing and genome-wide analysis (J. Welch personalcommunication).
2.2.Liquidculturemedium
FriismediumwaspreparedlargelyasdescribedbyKobischand Friis (1996) with the following modifications: bacitracin and meticillinwere replaced withazlocillin and flucloxacillin(final concentration50
m
g/ml).To make500ml ofFriis medium,1.5g Brain Heart Infusion (BHI) (Difco) and 1.6g PPLO (Difco) was dissolved in365ml waterand sterilised byautoclaving.To this wereadded 18ml of yeastextract(prepared fromdried bakers yeast),12.5mlsterilesolutionA(160g/lNaCl,4g/l;8g/lKCl,2g/l MgSO47H2O,2g/lMgCl2.6H2O,3.7g/lCaCl22H2O),12.5mlsterilesolutionB(3.0g/lNa2HPO4.12H2O,1.2g/lKH2PO4),50mlpigserum
(Invitrogen)heat-treatedat 56C for20min,50mlheat-treated horseserum(Invitrogen),1mlphenolredsolution(0.6%in0.1M NaOH),25
m
lofeachof100mg/mlazlocillinandflucloxacillinand adjustedtopH7.4with1.0MNaOH.2.3.Solidculturemedium
Concentrated(2.8)Friismediumwaspreparedbytheaddition of88ml waterto4.3gBHI,4.6gPPLO sterilisedbyautoclaving, 51.4ml yeast extract, 35.7ml sterile solution A, 35.7ml sterile solutionB,143mlheat-treatedhorseserum,143mlheat-treated pig serum, 2.8ml 0.6% phenol red solution, azlocillin and
flucloxacillin(finalconcentration 140
m
g/ml), pH7.4. Friis agar waspreparedbymixingconcentratedFriismedium(2.8),mixed ataratioof35:65witheither1.8%agarNo.1(Oxoid,L11),Purified agar(Oxoid,L28)oragarose(Invitrogen).Whereindicated, DEAE-dextran (Sigma-Aldrich, Gillingham, UK) was added toagar at 0.1mg/ml.MycoplasmaExperience1solidmediumwasprepared accordingtothemanufacturer’sinstructions(Mycoplasma Expe-rience1Ltd,Reigate,UK).Cultureswereincubatedinahumidified incubatorat37Cin5%CO2.Charcoal-treated agar was prepared by incubating a 1.3% solutionofBacteriologicalagarwith2%w/vofactivatedcharcoal (Oxoid,Basingstoke,UK)for10minat55C,thecharcoalwasthen removedusingasieve.Followingsterilisation,eachagartypewas mixed withmedium supplement (Mycoplasma Experience Ltd, UK)orconcentratedFriismediumataratioof35:65(supplement: agar)andplatespoured.
2.4.Viabilitydetermination
Ten-fold serial dilutions in Hanks’ Balanced salts Solution (HBSS;LifeTechnologies)werespotted(10
m
lintriplicate)onto solid medium. Cultureswereincubatedfor 7days in5% CO2 at37C. Colonieswerecounted,fromthosedilutionsshowingthe largest number of separate colonies, using a dissecting micro-scope; the triplicate counts were combined and the viability calculated.Growthwasalsoestimatedfromthemetabolicactivity recordedasacolourshiftofthephenolredpHindicatorfrompH 7.4to6.8asdescribedbyFriis(1975).
2.5.Antibioticsusceptibilitytesting
Solutions of kanamycin (Sigma), tetracycline (Sigma) and puromycin(Fisher)werepreparedinde-ionisedwater. Chloram-phenicol (Sigma) was dissolved in ethanol at 10mg/ml final concentration.Allweresterilisedthrougha0.2
m
mfilter(Nalgene, UK).Afinalantibioticconcentrationrangewasachievedby two-foldserialdilutionsofeachantibioticinsterilewaterorethanolas appropriate.Antibioticswereincorporatedintosolidmediumover a range of concentrations 20–0.01m
g/ml (kanamycin), 4.0–0.03
m
g/ml (tetracycline), 20–0.06m
g/ml (chloramphenicol and puromycin)M.hyopneumoniaeandM.hyorhinisinoculawerepreparedin1x sterilephosphatebufferedsaline(BR0014G,Oxoid)asasuspension containingapproximately103CFU/ml.Asampleof10
m
lofeachstrainwas inoculated onto each platein triplicate. Plateswere incubatedat37Cwith5%CO2andobservedforgrowthofcolonies
betweenday2andday10.TheMICwasdeterminedasthehighest dilutionofantibioticgivinganabsenceofgrowthasdeterminedby theformationofmycoplasmacolonies.
3.Results
3.1.Therelationshipofviabilityandcolourshift
TheviabilityofM.hyopneumoniaetothecommonlyusedcolour shiftinliquidmediumwasdetermined(Fig.1).Culturesreacheda maximumviabilityof108.9atapproximately2.5daysincubation.
Thiscorrespondedtoacolourshiftbetween2(pH7.2)and3(pH 7.1).Afterthispointtherewasanexponentialdeclinereaching106
CFU/mlafter8days(beyondcolourshift4).
3.2.TheeffectofglucoseandNaOH
TheconcentrationofglucoseinFriismediumisapproximately 2mM:1mMisderivedfromthebrainheartinfusionbrothand another1mM from the dilutedserum. To investigate whether glucoseislimitingthegrowth,furtherglucosewassupplemented intotheliquidmediumtogive4mM(2)and8mM(4)glucose, andviablecountsperformedthroughthegrowthcycle.Thegrowth curvewas unchanged by the increased glucose concentrations (Fig.2).Friissuggestedthatthedeclineinviabilitywhenyellow colour shift was reached was due to the acidification of the medium.Ifso,thismightbepreventedbyadditionofalkalitothe mediumasthecolourchangesatthegrowthpeak(60h).Addition ofalkalitoneutralizethepHcausedamarginalimprovementin survival at 4–6days although this difference was not marked (Fig.2).
3.3.SolidifyingFriismedium
InordertouseFriismediumasthebaseforasolidmediumto cultureM.hyopneumoniae,differentagarpreparationsweremixed withFriisbaseandorMycoplasmaExperience1(ME)supplement and investigated for the ability to support the growth of M. hyopneumoniaecolonies.TheresultsareshowninTable1.
Friis medium solidified with thecommercially supplied ME agargrewcoloniesofM.hyopneumoniae277/94&325/95.When Agar no.1, used widely in the solidification of bacteriological culture medium, was mixed with either Friis medium or ME supplement there was no growth of M. hyopneumoniae. Pre-treatmentof theagarwithactivated charcoal,in anattemptto remove any growth-inhibiting components, did not alter the abilityof medium madewitheither supplement tosupportM. hyopneumoniae.However, when eitherPurifiedagar oragarose wereusedasthesolidifyingagent,Friismedium,butnottheME supplement,grewcoloniesofM.hyopneumoniae(Table1).
MEsolidmediumsupportedthegrowthofM.hyorhinis.When charcoal-treated agar, agarose or Purified agar were used, ME supplementdidnotgrowM.hyorhiniscolonieswhileFriismedium supportedM.hyorhiniswhensolidifiedwitheachofthedifferent agars.
BasedonthefindingsofTauraso(1967)andasnotedbyFriis (1975)&KobischandFriis(1996),DEAE-dextranwasaddedtothe agar while molten and the growth of two strains of
M.hyopneumoniaeandM.hyorhinis werecompared. Resultsare showninTable1.TheinclusionofDEAE-dextranintoPurifiedagar further enhanced the growth of colonies for strains of
M.hyopneumoniae(strains277/94and 325/95)andappearedto besuperiortothemediumcommerciallyavailable(Fig.3).
3.4.SelectivecultureofM.hyopneumoniae
ThesusceptibilityofM.hyopneumoniae277/94and325/95,and
M.hyorhinisMhr1/09wasmeasuredonsolidmediumcontaining fourantimicrobialsoverarangeofconcentrations.Theresultsare showninTable 2.The MICofkanamycinfor M.hyopneumoniae
isolates 325/95 and 277/94 was shown to be 8.0
m
g/ml whileM.hyorhinisappearedtobesusceptibletoalowerconcentration, withanMICof1.0
m
g/ml.BothstrainsofM.hyopneumoniaegrew on medium containing 6.5m
g/ml. A mixed culture (equal numbers) of M. hyopneumoniae strain 277/94 and M. hyorhinisMhr1/09wasusedtoassesstheabilityofkanamycintoselectively suppressthegrowthofM.hyorhinisonsolidmedium.After8days incubation in the absence of kanamycin, M. hyorhinis colonies outgrewM.hyopneumoniaeasrecognisedbycolonymorphology (Fig.4A&B).However,withadditionof2
m
g/mlkanamycinonlyM.hyopneumoniaecoloniesappearedtogrow(Fig.4C&D). TherelativesensitivityofMhr1/09tokanamycinpromptedthe testingofmorestrains.Afurther19M.hyopneumoniaeand12M. hyorhinisfieldisolatesfromtheUKandDenmark,whoseidentity wasconfirmedbybothPCRandgrowthcharacteristics,weretested for their susceptibility tokanamycin on MH selectivemedium (Table3).AllM.hyopneumoniaeisolatesgrewinthepresenceof 2
m
g/mlkanamycin.Theisolatesappearedtobeunaffectedbythe incorporationofkanamycinintothemediumwiththeexceptionof isolateMp258/94inwhichasmallamountofgrowthsuppression wasnotedintermsofcolonysize.Thegrowthofall12M.hyorhinisisolateswascompletelyinhibitedbytheadditionofkanamycinin solidmedium.Samplesofpneumoniclunglesionswereobtained fromtheabattoirovera periodof12months.Culturesofthese grownonMHselectivemediumwerefreefromM.hyorhiniswhile approximately60%ofsamplesgrownonnon-selectiveFriisliquid and solid medium contained M. hyorhinis and became rapidly overgrown(Table3).
4.Discussion
M.hyopneumoniae hasbeenlong recognisedas difficultand unreliabletoculture(Friis,1971).Growthonsolid mediumhas been considered particularly difficult with colonies growing to only 0.3–0.5mm diameter in 7–10days. This study was a systematic investigation to improvethe medium for cultureof thispathogen.Theresultingimprovedmediumwascrucialinthe successfuldevelopmentoftransformationandtransposon muta-genesis of M.hyopneumoniae as described byMaglennon et al. (2013a,b).
Establishmentof thegrowth curve forM. hyopneumoniaein Friis medium was a pre-requisite for studies toallow efficient transformation. Using viable counts on the solid medium described here,rather than colourchange unitsor theindirect methods of colour shift (Friis, 1975) or measuring ATP by luminometry (Stemke and Robertson,1990; Calus et al., 2010), thegrowthcurvecouldbeaccuratelydetermined.
Ithasbeenconsideredthatglucoseistheprimarycarbonand energy source for M. hyopneumoniae. Supplementation of the mediumwithadditionalglucosedidnotimprovetheviabilityor growthrate.ThiswasconsistentwiththeresultsofStemkeand Fig.2.EffectofglucoseandalkalionthegrowthcurveofM.hyopneumoniae.Viable
Robertson(1990) whofoundnoincreaseingrowthrateorpeak viability. Theyused27mMglucose. Since physiologicalglucose concentrationsarenormallybetween3and5mM,weconsidered an increase to4 and 8mM tobe appropriate for themedium.
QuenchingoftheacidformedinthemediumusingNaOHhadonly a minoreffectonthe viability,which by8days incubation had reachedthelevelofthecontrolwithoutaddedNaOH.Thusit is likelythatdepletionofkeynutrientsratherthanacidinhibitionisa factorlimitinggrowthinculture.
Initialexperimentsindicatedthatthepurityoftheagarusedto solidify modified Friis medium greatly affected the growth of
M.hyopneumoniae.Bacteriologicalagarwasfoundtopreventthe growth of M. hyopneumoniae colonies. Gould et al. (1944) had reported a similar inhibitory effect on the growth of Neisseria gonorrhoeae following the solidification of liquid medium with agar.LeyandMueller(1946)identifiedacomponent,similartoa fatty acid, present in agar following methanol extraction. This extractedcomponentpresentwithinagarwasfoundtoinhibitthe growth of N. gonorrhoeae in the absence of starch. Activated charcoalisknowntoadsorbseveralimpuritiessuchasfattyacids.
Table1
GrowthofM.hyopneumoniaeandM.hyorhinisondifferentsolidmediafollowing10and5daysincubationat37C.
Sample Liquidmedium Testagar M.hyopneumoniae M.hyorhinis
1 MEsupplement MEagar +++ +++
2 1.3%AgarNo.1. NG NG
3 Charcoal-treatedNo.1 NG NG
4 1.3%agarose NG NG
5 1.3%Purifiedagar NG NG
6 ModifiedFriis(x2.8) MEagar +++ +++
7 1.3%AgarNo.1. NG +++
8 Charcoal-treatedNo.1 NG +++
9 1.3%agarose +++ +++
10 1.3%Purifiedagar ++ +++
Fig.3. ColoniesofM.hyopneumoniae325ondifferentsolidmediapreparations.Colonieswereincubatedfor7daysat37Cinahumidifiedboxandphotographedusinga dissectingmicroscope.A,MEsupplementsolidifiedwithMEagar;B,modifiedFriismediumsolidifiedwith0.85%Purifiedagar;C,modifiedFriismediumsolidifiedwith0.8% Purifiedagarand0.1mg/mlDEAE-dextran;D,modifiedFriismediumsolidifiedwith0.9%agarose.ArrowsindicatesmallM.hyopneumoniaecolonies..
Table2
MICsofantimicrobialagentsforM.hyopneumoniaestrain277/94and325/95,and
M.hyorhinisMhr1/09forkanamycin,tetracycline,chloramphenicolandpuromycin
determinedonsolidmedium.
Antimicrobialagent MICmg/ml
277/94 325/95 Mhr1/09
Kanamycin 8.0 8.0 1.0
Tetracycline 0.125 0.125 0.06
Chloramphenicol 2.5 2.5 2.5
Therefore, attempts were made to remove potential inhibitory factorsfrom the agar suchas free fatty acid-like inhibitorsby treatmentwithactivatedcharcoal.Sinceactivatedcharcoalhadno effectontheagar,we concludethat this wasfailuretoremove inhibitoryfactor(s).
WhenthecommercialMEsupplementwassolidifiedwithagar otherthanthesuppliedagarcomponentitwasunabletosupport the growth of either M. hyorhinis or M. hyopneumoniae. This suggestedthatanessentialconstituentforgrowthwasabsentfrom theliquidsupplementandonlypresentwithintheagar compo-nent. Solidifying Friis medium with Purified agar treated with DEAE-dextranconfersasubstantialimprovementonthe commer-ciallyavailable medium forgrowth of M. hyopneumoniae. Since DEAEisapositivelychargedgroupabletobindnegativelycharged ions,itsactionmaybeexplainedbychelationoftheagaropectin present in agar which may be inhibitory to growth of M. hyopneumoniae.Agaropectinisthemixtureofsmallermolecules, comprising alternating residues of L- and D-galactose modified
withacidic side-groups(particularly sulfate). Thisis consistent withthebeneficialuseofpurifiedagarose,whichisdepletedof agaropectin, over agar no. 1 in this study. No equivalent improvement was seen from DEAE in culture of M. hyorhinis, suggestingthat it is less sensitive totheacidic components of agaropectin.
Friis (1971) described a selective medium to suppress M. hyorhinis.Thisrequiresrabbitantiserumwhichisexpensiveand
not always available for regular culture. In testing the relative sensitivity of M. hyopneumoniae and M. hyorhinis to four antimicrobials,ourresultsshowedthatthelatterwasconsistently more sensitive to kanamycin. Williams (1978) had previously shown that both M. hyorhinis and M. hyopneumoniae were susceptible to kanamycin (0.1 and 0.5
m
g/ml respectively). However,differentisolateswereused,anddeterminationofthe MIC byWilliams was in liquid medium.Nevertheless, incorpo-rationof2m
g/mlofkanamycinintotheagarmediumappearedto prevent thegrowthof M.hyorhinisfollowing 8daysincubation. WhenmixedinequalvolumeswithM.hyopneumoniaestrain325, theM.hyorhinisdidnotformcolonieswhilecontrols,withoutthe additionofkanamycin,yieldedonlyM.hyorhiniscoloniesfromthe mixedculture.Screeningof M.hyopneumoniaeandM.hyorhinisstrainsshowedthatthisdifferenceinkanamycinsusceptibilitywas present betweenallM.hyopneumoniaeand M. hyorhinisstrains tested.
The addition of kanamycin can be used in place of the hyperimmune rabbit serum and gives a clean selection for the cultureofM.hyopneumoniae,effectivelypreventingthegrowthof
M.hyorhiniswhileallowingcoloniesofM.hyopneumoniaetoform. TherelativekanamycinresistanceofM.hyopneumoniaehasgreatly improvedtheprimaryisolationofM.hyopneumoniaefromabattoir tissuesamplesoriginatingfrom15differentfarms.Falsenegatives, resultingfromM.hyorhinisovergrowthhavebeenovercomeusing thisselectivemedium.
Fig.4.SuppressionofM.hyorhinisgrowthfollowingkanamycinselectiononsolidmedium.Growthofamixedmycoplasmaculturecontaining1:1equalvolumesofM.
hyopneumoniaeandM.hyorhinismid-logphaseculture,following9daysincubationonFriissolidmediumwithandwithout2mg/mlkanamycinat37Cinahumidifiedbox.
A,M.hyopneumoniae277andM.hyorhinis;B,M.hyopneumoniae325andM.hyorhinis;C,M.hyopneumoniae277andM.hyorhiniswith2mg/mlkanamycin;D,M.
Conflictsofinterest
None.
Acknowledgements
TheauthorsacknowledgetheBBSRCandPfizerAnimalHealth (nowZoetis)forfinancialsupportofCASEstudentshipstoBSCand LM-S. ANR, GAM and JGB weresupported byBBSRC grant BB/ G020744/1ofwhich5%ofthefundswerecontributedbyZoetis.
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Table3
GrowthofM.hyopneumoniaeandM.hyorhinisisolatesonsolidmediumwithandwithout2mg/mlkanamycin.
M.hyopneumoniaeisolate Source Growthonsolidmedium
Nokanamycin 2mg/mlkanamycin
MH001 UKfieldisolatesfromlunglesions2009–2010 +++ ++
MH002 +++ +++
MH003 +++ +++
MH005 +++ +++
MH008 +++ +++
MH010 +++ +++
MH011 +++ +++
MH015 +++ ++
MH016 +++ +++
MH031 +++ +++
MH033 +++ +++
MS2 Danishfieldisolatesfromlunglesions1969 +++ +++
MS3 +++ +++
MS8 +++ +++
MS13 +++ +++
MS15 +++ +++
MS25 +++ +++
MS29 +++ +++
Mp258/94 Danishfieldisolatesfromlunglesions1994–2007. +++ +
Mp277/94 ++ ++
Mp473/95 + +
Mp738/95 +++ +++
Mp325/95 ++ ++
Mp79/06 +++ ++
Mp87/06 ++ +
Mp92/06 +++ +++
Mp96//06 +++ +++
Mp85/07 + +
Mp261/07 +++ +++
M.hyorhinisisolate Source Growthonsolidmedium
Nokanamycin 2mg/mlkanamycin
MHR01 UKfieldisolatesfromlunglesions2009–2010. +++ NG
MHR02 +++ NG
MHR04 +++ NG
MHR05 +++ NG
MHR06 +++ NG
MHR07 +++ NG
MHR08 +++ NG
MHR09 +++ NG
MHR10 +++ NG
MHR11 +++ NG
MHR12 +++ NG
P450 +++ NG
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