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Review article
Guidelines and recommendations for antimicrobialminimum inhibitory concentration (MIC)
testing against veterinary mycoplasma species
Peter C.T. HANNAN
Mycoplasma Experience Ltd, 1 Norbury Road, Reigate, Surrey, RH2 9BY, UK
(Received 10 September 1999; accepted 25 February 2000)
Abstract – The absence of standardised procedures for minimum inhibitory concentration (MIC) test-ing of antimicrobial agents against veterinary mycoplasma and ureaplasma species (Mollicutes) hasmade it difficult to compare results originating from different laboratories. This report, prepared onbehalf of the International Research Programme on Comparative Mycoplasmology (IRPCM), offersguidelines and recommendations for veterinary MIC testing of these organisms in an effort to rectifythis problem. The subjects discussed include suitable media for broth and agar MIC assays, storageand preparation of antimicrobial agents, standardisation of mycoplasma inocula for MIC tests, val-idation of equipment, incubation conditions, and determination of MIC end points. A standardmedium for all veterinary mycoplasma MIC tests cannot currently be recommended, owing to the diver-sity of nutritional requirements of different mycoplasma species. Instead mycoplasma broths oragars giving optimal growth of specific mycoplasmas or ureaplasmas are recommended, as sub-optimal growth may lead to falsely low MIC results. The importance of using standardised mycoplasmainocula, for assays using either solid or liquid media is stressed. The growth phase may be lessimportant as lag phase and logarithmic phase cultures of Mycoplasma gallisepticum, M. synoviae, M. bovisand M. hyopneumoniaehave given very similar results in liquid MIC assays. The liquidmethod of Tanner and Wu and the agar method described by Hannan et al. are compared and describedin detail. Methods for calculating MIC50s and MIC90s are described and the interpretation of resultsdiscussed. Methods for assessing mycoplasmacidal (MMC) activity of antimicrobial agents are alsodescribed. Adoption of these guidelines should lead to more consistent MIC results being obtainedbetween laboratories.
MIC / mycoplasmacidal / antibiotic / mycoplasma / ureaplasma
Résumé – Instructions et recommandations pour les tests de concentration minimum inhibitrice(CMI) d’antimicrobiens contre les mycoplasmes en médecine vétérinaire. L’absence de procé-dures standardisées pour le test de concentration minimum inhibitrice (CMI) d’agents antimicro-biens contre les mycoplasmes et uréaplasmes (Mollicutes) présents chez les animaux, rend difficilela comparaison des résultats provenant de différents laboratoires. Ce document, préparé pour le
Vet. Res. 31 (2000) 373–395 373© INRA, EDP Sciences
Tel.: 44 (0) 1737 226662; fax: 44 (0) 1737 224751; e-mail: [email protected]
P.C.T. Hannan374
Programme International de Recherche sur la Mycoplasmologie Comparative (IRPCM), offre des ins-tructions et des recommandations pour les tests d’intérêt vétérinaire de CMI de ces organismes, afinde résoudre ce problème. Les milieux appropriés pour les tests de CMI en milieu liquide et sur geld’agar, le stockage et la préparation des agents antimicrobiens, la standardisation des inoculumspour les tests, la validation des équipements, les conditions d’incubation, et la détermination desvaleurs critiques de CMI, sont les sujets abordés dans ce document. Un milieu standard pour le testde CMI de tous les mycoplasmes d’intérêt vétérinaire ne peut pour le moment pas être recommandé,à cause de la diversité des besoins nutritionnels des différentes espèces de mycoplasmes. Au contraire,des milieux liquides ou sur gel d’agar permettant d’obtenir une croissance optimale spécifique des dif-férentes espèces de mycoplasmes et d’uréaplasmes sont recommandés, une croissance sub-optimalepouvant donner lieu à des résultats de CMI faussement basses. L’importance de l’utilisation d’ino-culums standardisés de mycoplasmes dans des tests en milieu liquide ou solide, est soulignée. La phasede croissance peut être moins importante, les phases de latence et de croissance exponentielle des cul-tures Mycoplasma gallisepticum, M. synoviae, M. boviset M. hyopneumoniaeayant donné des résul-tats très similaires lors de test CMI en milieu liquide. La méthode en milieu liquide de Tanner etWu, et la méthode sur agar décrite par Hannan et al. sont comparées et décrites en détail. Desméthodes pour le calcul des CMI50 et CMI90 sont décrites, et l’interprétation des résultats est discu-tée. Des méthodes pour évaluer l’activité mycoplasmacidale des agents antimicrobiens sont aussidécrites. L’adoption de ces instructions devrait permettre d’obtenir des résultats de CMI plus cohé-rents entre différents laboratoires.
CMI / mycoplasmacidal / antibiotique / mycoplasme / uréaplasme
Table of contents
1. Introduction ............................................................................................................................. 3752. General technical considerations.............................................................................................375
2.1. Media.............................................................................................................................. 3752.2. Identification of mycoplasmas/ureaplasmas................................................................... 3782.3. Standardisation of inocula for MIC tests ........................................................................ 3782.4. Antimicrobial agents ...................................................................................................... 378
2.4.1. Storage ................................................................................................................ 3802.4.2. Preparation of antimicrobial solutions for MIC testing....................................... 3822.4.3. Antimicrobial dilution ranges ............................................................................. 383
2.5. Validation of equipment ................................................................................................. 3832.6. Incubation conditions ..................................................................................................... 385
3. Mycoplasma MIC methods ..................................................................................................... 3853.1. Liquid MIC method........................................................................................................ 386
3.1.1. Preparation of stock mycoplasma cultures.......................................................... 3863.1.2. Preparation of mycoplasma inocula for MIC testing .......................................... 3863.1.3. Viable counting method for liquid MIC assays................................................... 3863.1.4. Preparation of inocula for liquid MIC assays...................................................... 3873.1.5. Modified MIC assay method of Tanner and Wu method [39] ............................ 387
3.2. Solid agar MIC method .................................................................................................. 3883.2.1. Viable counting method for solid MIC assays .................................................... 3883.2.2. Method (modified method of Hannan et al. [15]) ............................................... 388
4. Expression of results ............................................................................................................... 3895. Interpretation of results ........................................................................................................... 3896. Testing for mycoplasmacidal activity ..................................................................................... 3907. Conclusion............................................................................................................................... 392
Veterinary antimycoplasmal testing 375
1. INTRODUCTION
Minimal inhibitory concentration (MIC)data on veterinary mycoplasma species havebeen generated by numerous researcherswho have employed tests using various liq-uid or solid media [1, 4–6, 10, 12, 13, 15–17,19–22, 25–30, 32, 37–39, 42–44, 47, 48,50, 51]. There has been little standardisa-tion of procedures, making it difficult tocompare MIC results from laboratory to lab-oratory [47].
Although guidelines for in vitro suscep-tibility testing criteria and quality controlparameters for veterinary antimicrobialagents have recently been approved for vet-erinary bacteria [46], these do not includemycoplasmas (mollicutes) which are bothslow growing, highly fastidious and do notproduce the turbidity in broth nor sufficientgrowth on agar necessary for conventionalantibacterial testing.
At the 1996 biennial meeting of the Inter-national Organisation for Mycoplasmology(IOM) International Research Programmeon Comparative Mycoplasmology(IRPCM), the Board recommended thatstandard protocols be drawn up for veteri-nary MIC assays.
In mycoplasmology the MIC is definedas the lowest concentration of antimicrobialthat will inhibit visible growth ormetabolism of a mycoplasma species afterits optimal incubation period in vitro.Despite reservations about their clinical rel-evance, MICs are still generally consideredto be the reference point for comparison andevaluation of other sensitivity tests andindeed the efficacy of all antimicrobials isdescribed in terms of MICs. The minimummycoplasmacidal concentration (MMC) isdefined as the lowest concentration ofantimicrobial that prevents growth after sub-culture onto or into antibiotic-free media(in practice, approximately a 99.99% kill).
The wide variation in nutritional require-ments and cultural conditions of veterinarymycoplasmas and the preference of certain
species for agar rather than broth medium,has tended to hamper the development of auniversal veterinary mycoplasmal MICassay. This has been compounded by theneed and preference for different formula-tions of mycoplasma media by workers inthe poultry, swine, cattle, other farm indus-tries and those dealing with companion (pet)animals. These and other problems in rela-tion to the susceptibility testing of molli-cutes in general have been highlightedrecently in “Molecular and Diagnostic Pro-cedures in Mycoplasmology Vol. II” [3, 23].
Recently attempts have been made tostandardise and compare some veterinaryMIC assay methods [4, 10, 42, 47]. The aimof this report is to discuss the factors whichmay influence veterinary MIC testing andto recommend basic procedures for carry-ing out such tests in the hope that consis-tent results can be achieved between labo-ratories in the future.
2. GENERAL TECHNICAL CONSIDERATIONS
2.1. Media
A standard medium for all veterinarymycoplasma MIC tests cannot currently berecommended because of the diversity ofnutritional requirements of differentmycoplasma species. In general,mycoplasma broth or agar giving the optimalgrowth of specific mycoplasmas or ure-aplasmas should be employed in MIC tests.Suboptimal media giving poor growth mayresult in falsely low MIC values.
If penicillin or other β-lactam antibiotics(e.g. ampicillin), which are inactive againstmycoplasmas because of their cell-wall defi-ciency, are incorporated to inhibit bacterialcontamination, it should be borne in mindthat they may interact with the compoundunder evaluation and that antagonism oreven synergism might occur. It is thereforewise to establish if these phenomena occurwith new antimicrobial agents and
P.C.T. Hannan376
β-lactams before carrying out MICs using these bacterial inhibitors. However, penicillin G (1 000 IU⋅mL-1),ampicillin (1 000µg⋅mL-1) or amoxycillin (1 000µg⋅mL-1) with thallium acetate (500 µg⋅mL-1), included in the culturemedium have been found not to influencethe MICs of erythromycin, spiramycin,streptomycin, tetracycline, tiamulin ortylosin against certain avian mycoplasmas[47]. Thallium acetate is inhibitory to ure-aplasmas and should be excluded from testsinvolving these microorganisms.
Many different formulations ofmycoplasma and ureaplasma media havebeen described for the culture of veterinarymycoplasmas. Some of these media, citedin “Methods in Mycoplasmology Vol. 1”[9, 36] are listed in Tables I and II. Othermedia have been used by various workers [1,4, 6, 7, 12, 17, 20, 21, 26, 37, 47, 49, 51].
Most veterinary mycoplasmas can begrown in liquid or on solid media and cantherefore be used in MIC tests employingeither type of medium. An exception to thisis the turkey pathogen, M. meleagridis,par-
ticularly fresh field isolates, which may bereluctant to grow or cause colour changesin liquid medium. MIC tests involving thismycoplasma species should therefore becarried out on solid media, at least until asuitable liquid medium has been developed.
Examples of media which have been usedsuccessfully by some investigators to carryout MIC assays against mycoplasmas fromvarious animal host species, are shown inTable III.
Mycoplasmas develop only faint or noturbidity in broth cultures; consequentlyalternative methods are used to measuremycoplasmal growth. These include incor-poration of substrates such as glucose, argi-nine or urea which are either fermented orhydrolysed by the mycoplasmas causing themedium to become acid or alkaline. ThesepH changes are usually detected by incor-porating the pH indicator phenol red, intothe mycoplasma broth, a colour changedenoting mycoplasmal growth. Somemycoplasmas (e.g. M. iowae) metabolizeboth glucose and arginine and either substrate can be used to detect growth,
Table I. Liquid media used in the isolation and cultivation of veterinary mycoplasmas.
Medium Mycoplasmas cultured
B Modified Hayflick - supports the growth of the majority of mycoplasmas and acholeplasmas.
N Modified Hayflick - superior to medium B forM. anatis, M. bovigenitalium, M. edwardii,M. felis, M. maculosum, M. meleagridis, M. spumans, M. verecundum.
F (Frey) Supports the growth of M. synoviaeand otheravian mycoplasmas.
A26 Supports the growth of M. hyopneumoniaeandM. flocculareand other porcine mycoplasmas.
FF (Friis) Supports the growth of M. hyopneumoniae, M. flocculareand other porcine mycoplasmas.
GS Supports the growth of M. disparand other bovinemycoplasmas.
SP4 For fastidious mycoplasmas including M. dispar,M. synoviae, M. fastidiosum, M. feliminutum, M. alvi and M. sualvi.
These media can be solidified by the addition of agar or agarose (for details refer to Methods in Mycoplasmology,Vol. 1 [9]).
Veterinary antimycoplasmal testing 377
Table II. Media used in the isolation and cultivation of veterinary ureaplasmas.
Liquid MediaUrease colour change Recommended for primary isolation andbroth 10C cultivation of ureaplasmas from animals.(Shepard and Lunceford) [36]Urease colour change broth Developed for bovine ureaplasmas.(Howard et al.) U4 [18]Urea colour change test broth(Taylor-Robinson et al.) [41]
Solid MediaDifferential agar medium A8 Successfully used for the cultivation(Shepard and Combs) [36] of ureaplasmas from primates.Standard agar medium A5K Successfully used for the isolation(Shepard and Lunceford) [36] and cultivation of ureaplasmas from
animals.Standard agar medium Recommended for the isolation and(Taylor-Robinson et al.) [41] culture of animal ureaplasmas.Standard agar medium Developed for the isolation and(Howard et al.) [18] culture of bovine ureaplasmas.
For details refer to Methods in Mycoplasmology,Vol. 1 [36].
Table III. Examples of media which have been used by various researchers to carry out MIC testsagainst mycoplasma species from various animal hosts.
Animal hosts
Medium Poultry Pigs Cattle Sheep/Goats Rodents
MycoplasmasF (Frey) [22]Power and Jordan’s [4, 20, 21]FF (Friis) [5] [5, 10, 15, 42, 48] [5, 10, 43]GS [37]Edward [7] [43] [13]Hayflick [5] [5] [10]B [37] [37]Mycoplasma enrichment [47]broth (Whithear)MEa [16] [16, 17] [16] [16]
UreaplasmasN/HU (Friis) [43]U9B [37] [37] [37] [37]
Bracketed figures under host species: publications in which media were used. a ME (Mycoplasma Experience Ltd) medium is a commercially available medium suitable for growing highlyfastideous mycoplasmas.
P.C.T. Hannan378
however, others, e.g. M. bovisand M.agalactiae, which neither ferment glucosenor hydrolyse arginine, have recently beenfound to metabolize pyruvate, producing apermanent acid colour change [16]. Forthese mycoplasmas it is recommended thatpyruvate be added to glucose-containingmedia for MIC tests. The use of tetrazoliumreduction to facilitate colour changes withM. bovishas been reported but the colourchanges tend to disappear after 1 to 2 days[43].
2.2. Identification of mycoplasmas/ureaplasmas
Ideally the species identification ofmycoplasmas and ureaplasmas should beestablished before, or concurrently with, thetesting of strains for susceptibility to antimi-crobial agents. This involves the isolationand purification of strains to ensure that purecultures are used in MIC tests. The largenumber of mycoplasma species inhabitingthe respiratory and urogenital tracts of farmand domesticated animals and birds, manywith similar colonial morphology, maymake cloning obligatory. If this were notdone, MIC test results would be on mixedcultures which would be misleading. Ifcloning is necessary then it is advisable toselect several different clones and to carryout MIC tests on a mixture of these, to avoidselecting a minority resistant or sensitivesub-population. Cultures obtained frominfection sites which are normally sterile,i.e. the joints, may be pure and in suchinstances broth cultures or agar blocks con-taining several colonies (≥ 5 colonies) can beused to prepare inocula for MIC tests. Thepurity of such cultures should also bechecked in parallel.
2.3. Standardisation of inocula for MIC tests
It is important that mycoplasma inoculafor MIC tests are carefully standardized.
The accepted number of organisms used fortests carried out in liquid and on solid mediais 103 to 105 colour changing units (ccu) permL or colony forming units (cfu) per plate.Use of inocula falling outside this rangemay result in higher or lower MIC values,(higher for >105 ccu/cfu and lower for<103 ccu/cfu) (Tab. IV, Hannan, unpub-lished data). The growth phase of themycoplasmas seems to be less important.Whithear et al. [47] obtained identical MICresults with actively growing and frozencultures of M. synoviae. Mycoplasma cul-tures of M. bovis, M. gallisepticumorM. hyopneumoniaein the lag phase, suchas those obtained from freshly thawed cul-tures from –70 °C diluted and used directly,or after 1–2 h incubation in broth at 36 ± 1 °C, and cultures in the logarithmicgrowth phase have, in our laboratory, givenvery similar results (Tab. V, Hannan unpub-lished data). However such comparisonshave not been made with other mycoplasmaspecies or ureaplasmas. Until it is estab-lished that such a correlation exists withother mollicutes, logarithmic phase culturesshould be used routinely for MIC testing.It has been recommended also thatmycoplasmas in the logarithmic phase ofgrowth be used for initial surveys with newantibiotics [23].
An ATP-dependent luminometry methodhas also been reported recently for deter-mining MICs of species that are highly fas-tidious or have long generation times andfor agents with a short half life [3, 34]. Earlylogarithmic phase cultures are recommendedfor this method.
2.4. Antimicrobial agents
Ideally, antimicrobials should be obtainedin powder form from the manufactureraccompanied by a statement of potency inrelation to base (“as is” or anhydrous, in µgper mg) or as tablets, with details of opti-mum storage conditions, expiry date andsolubility. Some compounds are available
Veterinary antimycoplasmal testing 379
Tab
le IV
. Effe
ct o
f ino
culu
m s
ize
of Myc
opla
sma
galli
sept
icum
stra
in P
G31
and
M
. hyo
pneu
mon
iae st
rain
J o
n th
e M
ICs
(µg⋅m
L-1)a
of e
ight
diff
eren
t ant
imi-
crob
ials
in li
quid
med
iumb .
Myc
opla
sma
Gen
tam
icin
Spi
ram
ycin
Spe
ctin
omyc
inLi
ncom
ycin
Tia
mul
inT
ylos
inE
nrof
loxa
cin
Oxy
tetr
acyc
line
inoc
ulum
(ccu
⋅mL-1
)M
.gM
.hp
M.g
M.h
pM
.gM
.hp
M.g
M.h
pM
.gM
.hp
M.g
M.h
pM
.gM
.hp
M.g
M.h
p
108
NT
10N
T1
NT
5N
T0.
5N
T0.
1N
T0.
25N
T0.
1N
T0.
5
107
>10
010
0.25
2.5
2.5
2.5
101
0.01
0.05
0.05
0.25
0.02
50.
025
0.1
0.25
106
502.
50.
251
2.5
110
0.25
0.01
0.02
50.
050.
10.
025
0.02
50.
10.
25
105
252.
50.
250.
252.
51
100.
250.
005
0.02
50.
025
0.1
0.01
0.02
50.
050.
25
104
252.
50.
250.
52.
52.
510
0.1
0.00
50.
025
0.02
50.
10.
010.
025
0.05
0.25
103
102.
50.
10.
251
15
0.1
0.00
50.
010.
025
0.01
0.00
50.
010.
050.
1
102
101
0.1
0.25
2.5
0.5
50.
10.
010.
025
0.02
50.
050.
005
0.01
0.05
0.1
aM
odifi
ed T
anne
r an
d W
u m
etho
d [3
9].
bM
ycop
lasm
a E
xper
ienc
e gl
ucos
e br
oth
pH 7
.6.
M.g
: M. g
alli
sep
ticu
mst
rain
PG
31.
M.h
p: M
. hyo
pn
eu
mo
nia
estra
in J
.N
T: N
ot te
sted
.
P.C.T. Hannan380
commercially and possible sources of supply in the UK include: Mast Diagnos-tics Ltd. Bootle, Merseyside; Unipath Ltd(oxoid), Basingstoke, Hampshire; SigmaChemical Company, Poole, Dorset. It isessential that antimicrobials are within theirexpiry date and that they are stored accord-ing to the manufacturer’s instructions.
2.4.1. Storage
Most antimicrobial powders can be storedat +4 °C in the dark (quinolones, rifampicinand chloramphenicol are light sensitive),over a drying agent such as silica gel. How-ever, some compounds are best stored at atemperature of –20 °C or below (e.g.netilmicin). Before use they should beallowed to come to room temperature beforethey are opened (to avoid condensation ofwater). Aliquots of stock solutions, at con-centrations in excess of 1 000 mg⋅L-1, canoften be stored at –60 °C for several months[8], but it must be remembered that a stock
solution frozen and then thawed should beused immediately and then discarded(repeated freezing and thawing of solutionsis not acceptable because of loss of potency).Table VI gives a summary of informationon storage, solubility and stability for manycommonly used compounds.
It is preferable to use fresh stock antibi-otic solutions, however some antimicrobialsolutions can be stored at +4 °C, frozen at–20 °C or –70 °C, for limited periods untilrequired for use, without loss of potency.The tetracyclines do not fall into this cate-gory as they tend to precipitate on thawingand should always be tested fresh. It isimportant to check the stability of specificcompounds at –20 °C before testing. Thehalf-lives of antimicrobial compounds undertest conditions may be important when test-ing slow-growing mycoplasmas and can beobtained from the manufacturer or a recentchemical index. For use in MIC tests, freshor freshly thawed, antimicrobial solutions
Table V. Effect of growth phase of Mycoplasma gallisepticum, Mycoplasma bovisand Mycoplasmahyopneumoniae on the MICs (µg⋅mL-1)a of eight different antimicrobials in liquid mediumb.
Antimicrobial Incubation timeagent
Freshly thawed Freshly thawed Freshly thawed(–70 °C) (–70 °C) (–70 °C)
0 h + 2 h at 36 °C + 48 h at 36 °C(lag phase) (lag phase) (log phase)
M.g M. bovis M.hp M.g M. bovis M.hp M.g M.bovis M.hp(PG31) (Donetta) (J) (PG31) (Donetta) (J) (PG31) (Donetta) (J)
Gentamicin 10 5 2.5 25 25 2.5 25 10 2.5 Spiramycin 0.025 0.25 1 0.1 0.5 1 0.1 0.5 0.5 Spectinomycin >1 >1 1 >1 >1 >1 >1 >1 >1 Lincomycin 0.5 0.25 0.25 1 0.25 0.5 1 0.5 0.25 Tiamulin 0.005 0.05 0.05 0.005 0.05 0.05 0.01 0.05 0.025 Tylosin 0.025 0.05 0.25 0.025 0.1 0.1 0.025 0.1 0.25 Enrofloxacin 0.01 0.05 0.025 0.01 0.1 0.025 0.01 0.1 NR Oxytetracycline 0.1 0.1 0.25 0.05 0.1 0.5 0.1 0.1 0.25
a Modified Tanner and Wu method [39].b Mycoplasma Experience (ME) glucose broth pH 7.6 for M. gallisepticumand M. hyopneumoniaestrains and MEglucose broth plus pyruvate pH 7.6 for M. bovis.Inoculum sizes: M. gallisepticum(PG31) 105 ccu⋅mL-1; M. bovis(Donetta) 104–105 ccu⋅mL-1; M. hyopneumoniae(J) 104 ccu⋅mL-1. NR: No result.
Veterinary antimycoplasmal testing 381
Table VI. Preparation and storage of antibiotic solutions (1 000 µg⋅mL-1)a.
Antimicrobial Diluent Storage of Solution Storage of powder
+4 °C –20°C –70°C
Chloramphenicol d alcohol b and 4°C; protect from light (chloromycetin) water
Ciprofloxacin water 2 weeks 3 months 3 months below 25°C for 3 years;(hydrochloride protect from lightmonohydrate)
Clindamycin water 1 day unsuitable room temperature(hydrochloride)
Erythromycin alcohol b and 1 week +5°C stable for 3 years;water protect from moisture
and light
Fusidic acid water or alcohol 25°C
Gentamicin water 6 months NR NR unopened vials 3 years (sulphate) at RT
Metronidazole water 25°C; protect from light
Netilmicin (sulphate) water 6 months 6 months 6 months protect from light
Rifampicin absolute 1 month 1 month +4°C; protect from lightalcohol b
and water
Spectinomycin water(hydrochloride)
Sulphamethoxazole 0.1N NaOH b 1 month 6 months 2 years 5–25°C; protect from light(free acid) and water
Tetracyclinec water NR NR unopened vials 2 years at RT
Tobramycin water 1 week 3 months +5°C; protect from moisture
Trimethoprim lactic acid b 1 month 6 months 2 years 5–25°C; protect from light(lactate) and water
NR: not recommended.RT: room temperature.a Data extracted from “a guide to sensitivity testing”. Report of the working party of Antibiotic Sensitivity Test-ing of the British Society for Antimicrobial Chemotherapy [31].b Enough to dissolve the antimicrobial powder.c Formation of a precipitate on freezing.d Injectable not appropriate.
P.C.T. Hannan382
are diluted in mycoplasma broth or agar togive the final required concentrations inMIC tests.
2.4.2. Preparation of antimicrobial solutions for MIC testing
Compounds can be tested directly as theformulation obtained (e.g. enrofloxacinhydrochloride), or in terms of the active base(e.g. enrofloxacin). Since different salts maydiffer considerably in their molecularweights, their active base contents will vary,weight for weight, and slightly differentMIC values are likely to be obtained for dif-ferent formulations. To avoid this, and toobtain consistent results between laborato-ries, antimicrobials should be tested in termsof their active base component. This isachieved by taking both the manufacturersstated purity and the salt content of the com-pound into account. The compound is thendiluted allowing only for the base content.This is accomplished by applying a correc-tion factor, calculated from the given purityof the compound, its molecular weight andthe molecular weight of the active base. Forexample, if the stated purity of enrofloxacinhydrochloride is 80% and the molecularweights for enrofloxacin HCl andenrofloxacin base are 395.9 and 359.4respectively, the correction factor is calcu-lated as follows:
359.4 (MW enrofloxacin base) ×
80 (purity) = 0.726.
395.9 (MW enrofloxacin HCl) 100
This figure is then applied to the amount ofenrofloxacin HCl weighed to obtain theweight of enrofloxacin base present, e.g.
5 mg (enrofloxacin HCl) × 0.726 = 3.63 mg enrofloxacin base.
In this example, to obtain a convenient finalstock solution containing 1 000 µg⋅mL-1
enrofloxacin base, 5 mg of enrofloxacin HClwould be dissolved in 3.63 mL (consistingof approximately 10% solvent and 90% ster-ile deionized water). Solutions can be ster-
ilized by membrane filtration (0.2 µm poresize) before MIC testing, however the suit-ability of the filters should be checked withthe manufacturers as some antimicrobialscan adsorb to the membranes, Durapore 0.22µm pore size TP cartridge (HydrophilicMembrane) filters (Cat. No. CVGL 75501-Millipore, Bedford, MA, USA) have beenfound to be suitable for filtering difloxacin.This filtration process can be omitted if allsolutions, the compounds themselves andthe apparatus used to carry out the tests aresterile, particularly if an antibacterial β-lac-tam such as ampicillin, is present in the cul-ture medium.
Many commercial preparations of antimi-crobials are water-soluble, but others, par-ticularly those in the research stage, mayrequire organic solvents, acids or alkalis toeffect complete solubilization [31]. In gen-eral the compounds can be dissolved in asmall volume (about one tenth the final vol-ume) of the solvent and then made up tovolume with deionized water. Agitation byvortexing, ultrasonic disintegration using amicroprobe to disperse large particles, ormild warming may be required to obtaincomplete solutions.
Organic solvents which have been usedinclude ethyl alcohol (EtOH), methyl alco-hol (MeOH), dimethyl formamide (DMF)and dimethyl sulphoxide (DMSO), and canbe used for many classes of antimicrobialagent. Acids such as 0.1 N hydrochloric acidor acetic acid can also be used for certainagents. 0.1 N sodium hydroxide is recom-mended for initially dissolving many of thewater-insoluble quinolone antimicrobials.Salts of these latter agents are generallywater-soluble. Following complete solubi-lization under acidic or alkaline conditions,it is often necessary to adjust the pH of the compound solutions to approximately pH 7.0 to avoid non-specific colour changesoccurring in the higher drug concentrationsin MIC tests.
To ensure that solvents are not inhibitoryto the mycoplasmas it is important to include
Veterinary antimycoplasmal testing 383
controls in MIC tests consisting of the sol-vent alone in mycoplasma broth at the sameconcentration as that used in the antimicro-bial dilutions tested.
Where possible, antimicrobials shouldbe tested at the correct pH for optimal in vitro activity, although this is governed bythe ability of the mycoplasmas to grow wellat that particular pH. MIC determinationsbased on colour changes in the medium dueto pH shifts should not be affected if thecorrect end-point controls are included inthe assay. It is debatable whether the in vitropH is pertinent to the in vivo situation sincethe pH at different infection sites and withincells, varies considerably. Macrolides suchas erythromycin and its newer analoguesare more potent under alkaline conditions[2, 11]. A lesser, though significant, effect ofpH on the testing of tetracyclines has alsobeen reported [24]. Robertson et al. foundthat serum had no apparent binding effecton tetracycline, erythromycin or rosaram-icin, but the expected decrease in macrolideactivity against the human species Ure-aplasma urealyticumat pH 6.0 was clearlyevident [35]. This acidity, however, doesreflect the level in the genital tract whereantimicrobial activity is expressed. It may benecessary to adjust media to neutral pH,when dealing with mycoplasma or ure-aplasma species isolated from other sites,although this may result in poorer growthof the microorganism.
2.4.3. Antimicrobial dilution ranges
Antibiotic dilution ranges are usuallyobtained by carrying out doubling dilutions,the highest final drug concentration fre-quently being 64 µg⋅mL-1. In liquid MICtests in microdilution plates, the drug con-centrations are usually prepared at doublethe final concentration (e.g. 128 µg⋅mL-1)to allow for dilution with an equal volume ofdrug-free medium containing themycoplasma inoculum. For MIC tests onsolid medium the drug is incorporated intothe agar plates at the final concentrations
required, allowance being made for thelarger volume of medium, e.g. if the finalvolume of medium per plate is 20 mL then1 mL of drug solution at 20 times its finalrequired concentration is incorporated in 19 mL of molten mycoplasma agar(50± 1 °C) immediately before pouring theplates. In practice, 1 mL of the antimicrobialsolution is pipetted into a sterile plastic uni-versal container; the molten agar is thenadded, mixed by gentle inversion, and the plate poured immediately, to reduce thechance of possible heat inactivation of the compound under test. All plates shouldbe of a uniform thickness. Drug dilutionscan either be prepared in tubes or bottlesusing pipettes or automatic pipetters withsterile filter tips and then transferred tomicrodilution plate wells or molten agar, orthey can be prepared directly in microtitretrays using various types of manual or auto-mated microdilutors.
To minimize the chances of excessivedrug “carry over” and other possible dilutionerrors, compounds may be diluted in tubesusing a combination of doubling dilutionsand dilutions in 10-fold series. This proce-dure involves carrying out a series of 10-fold dilutions of the compound concernedin sterile tubes or bottles; two doubling dilu-tions from each drug concentration producedby the 10-fold dilution procedure are thencarried out. If the highest final required con-centration is 100 µg⋅mL-1, then the rangeof dilutions covered is similar to those pro-duced by the conventional doubling dilu-tion method starting at 64 µg⋅mL-1.
2.5. Validation of equipment
All equipment including chemical bal-ances, automatic pipetters, multi-channelmicro-pipetters and inoculators should beroutinely checked for accuracy of weighingor delivery. Incubators should be checkedregularly for accuracy of temperature.
Table VII. Comparative MICs of five classes of antimicrobial agents against reference strains of avian,porcine and bovine mycoplasma species obtained using liquid and solid media by various investigators.
MIC (µg⋅mL–1)
Organism Liquid Solid Liquid medium Solid medium(type strain) medium (Mod. medium (other workers) (other workers)and antimicrobial Tanner and (Hannan
Wu) 1992 et al.) 1989[39] [15]
M. gallisepticumPG31 Enrofloxacin 0.01 0.025 a,c 0.1 [21]Flumequine 0.5 ≥ 10 a
Tiamulin 0.0025 0.025 a 0.007 [28] 0.01–0.1 [21] Tylosin 0.01 0.5 a 0.016–0.045a [4, 28] 0.1–1 [21] Oxytetracycline 0.1 0.5 a 0.5–1a [4, 39]
M. synoviaeWVU 1853 Enrofloxacin 0.5 0.025 c
Flumequine 10 ≥ 10 0.1–1 [21] Tiamulin 0.1 0.25 Tylosin 0.025 0.025 0.1–1 [11] Oxytetracycline 0.1 0.1 0.1 [11]
M. iowae695 Enrofloxacin 0.005 0.025 b,c
Flumequine 5 > 10 b
Tiamulin 0.005 0.025 b 0.05 [28] Tylosin 0.5 2.5 b 0.05 [28] Oxytetracycline 0.25 0.5 b
M. meleagridis17529Enrofloxacin 0.1 d 0.1 c 1 [21] Flumequine 10 d ≥ 10 Tiamulin 0.1 d 0.25 0.25–1 [21, 28] Tylosin 0.1 d 0.1 0.1–1 [21, 28] Oxytetracycline 1 d 5
M. hyopneumoniaeJEnrofloxacin 0.05 0.01 c ≤ 0.03 [42] Flumequine 0.5 2.5 Tiamulin 0.025 0.05 ≤ 0.03 [19, 42] Tylosin 0.025 0.01 ≤ 0.03 [19, 42] Oxytetracycline 0.25 0.25 0.12 [19, 42, 48]
M. hyosynoviaeS16Enrofloxacin 0.25 0.5 c 0.25 [10] Flumequine 25 ≥ 10 Tiamulin 0.025 0.025 0.0025 [10] Tylosin 0.05 0.025 0.025–0.09 [10, 51] Oxytetracycline 0.5 5
M. hyorhinis BTS-7 Enrofloxacin 0.5 0.25 c 0.8–2 [26, 42] 0.8 [26] Flumequine 5 ≥ 10 Tiamulin 0.05 0.25 ≤ 0.03–0.1 [26, 42] 0.4 [26] Tylosin 0.5 0.25 0.06–0.4 [26, 42] 0.8 [26] Oxytetracycline 0.05 0.25 0.12–0.4 [26, 42, 48] 0.4 [26]
M. bovisDonetta Enrofloxacin 0.25 1 c 1–2 [43] Flumequine 10 ≥ 10 Tiamulin 0.05 0.1 ≤ 0.015 [43] Tylosin 0.05 0.5 0.125 [43] Oxytetracycline 0.1 0.5 4 [43]
a Strain S6.b Strain B11.c Results for ciprofloxacin, chemically very similar to its analogue enrofloxacin.d Unpublished data.
Veterinary antimycoplasmal testing 385
2.6. Incubation conditions
Since most mycoplasmas and ureaplas-mas grow well between 35 °C and 37 °C, atemperature of 36 ± 1 °C is recommendedfor liquid and solid MIC assays, unless it isestablished that optimal growth occurs at atemperature outside this range.
In liquid microtitre MIC assays, it isessential that each well is securely sealedto prevent exchange of gases between wellswhich may result in false colour changesand erroneous MIC end-points. The seal onunused wells should be punctured to avoidlifting of the sealant by air expansion withinthe wells during incubation. If plates areincubated in a humidified cabinet at anappropriate incubation temperature and ina suitable gaseous environment, then allwells should be vented to allow escape ofgaseous products of metabolism of the sub-strates in inoculated wells.
In solid medium assays, plates should beincubated in an atmosphere (e.g. aerobic,95% N2 + 5% CO2, or anaerobic) suitablefor growing the mycoplasma species andstrains under examination. Plates should besuitably dried prior to inoculation to allowabsorption of the mycoplasma inoculum andto prevent cross-contamination of themycoplasma species or strains. This is par-ticularly important when automated multi-point inoculator heads delivering many dif-ferent mycoplasma species or strains areused. It is important that the plates do notdry excessively during incubation as thiscan inhibit mycoplasmal growth and leadto erroneous MIC values. Drying can beavoided by including paper towelling moist-ened with sterile water containing an anti-fungal agent, such as sodium propionate, inthe container containing the MIC plates.
Incubation times of different mycoplasmaspecies and strains vary considerably. In liq-uid MIC tests incubation times are controlledby the time it takes for particular mycoplas-mas or ureaplasmas to cause colour changesequivalent to pre-set pH controls. However,
in agar MIC tests the incubation timesshould be sufficient to allow adequategrowth of the slowest growing mycoplasmaspecies under test.
3. MYCOPLASMA MIC METHODS
Both liquid and solid MIC methods havebeen used in determining MICs against vet-erinary mycoplasma species. Examples ofthese methods are the liquid methoddescribed by Tanner and Wu [39], origi-nally designed to test strains of M. gal-lisepticumand the solid agar methoddescribed by Hannan et al. [15]. Both ofthese methods are also applicable to testingmycoplasmas of human origin and havebeen used to test a variety of differentmycoplasma species from various animalhosts. The results obtained by the two meth-ods are in many instances, in close agree-ment (Tab. VII). This is in accordance withresults obtained for the human species Ure-aplasma urealyticumby Waites et al. [45],where MIC50s and MIC90s obtained in brothwere only four-fold lower than those onagar. It is therefore recommended that theTanner and Wu method be adopted formycoplasma species which grow well inbroth and Hannan et al.’s modified methodfor those which grow either in broth or onagar. The liquid method is simple to carryout and is convenient for testing small num-bers, while the agar system is ideal for eval-uating large numbers of mycoplasma strainsor species on the same agar plate, if a multi-point inoculation system (Denley Ltd,Billingshurst, Sussex, UK) is employed. Thelatter system would be the method of choicefor testing strains of M. meleagridis. Theliquid method could also be applied to tubesif small numbers of strains were to be tested.Further information on the MIC testing ofmollicutes has been published recently byBébéar and Robertson [3].
P.C.T. Hannan386
3.1. Liquid MIC method
3.1.1. Preparation of stock mycoplasmacultures
Following primary isolation and identi-fication, the mycoplasmas are grown at 36 ±1 °C in 10 mL broth medium containing theappropriate substrate (glucose, arginine,pyruvate or urea) and pH indicator (phenolred) until a colour change occurs (pink toorange-yellow for glucose and pyruvate fer-menters, and orange-yellow to pink or redfor arginine and urea catabolizers). Sterileglycerol (cryoprotectant) is then added(optional) to each culture to a final concen-tration of approximately 5% v/v and the cul-tures are divided into 5 × 1 mL aliquots insterile containers and stored at –70 °C.
3.1.2. Preparation of mycoplasma inocula for MIC testing
1. Thaw a 1-mL vial of frozen inoculum(–70 °C) and add 4 mL of the appropriatemedium set at pH 7.6 for glucose or pyru-vate metabolizers, pH 6.8 for arginine orpH 6.0 to 6.5 for urea catabolizers.
2. Incubate the diluted culture at 36± 1 °Cuntil a definite acid or alkaline reaction isobserved.
3. Record the time taken (in hours) for thisreaction to occur.
4. Carry out a viable count immediately. (To compare MICs obtained with lagphase cultures and those of logarithmicphase cultures, the thawed cultures canbe used either directly or after incuba-tion for 2 h at 36°C prior to inoculatingthe antibiotic dilutions. In the firstinstance steps 1 to 3 above for logarith-mic phase cultures are omitted. Onemillilitre of the thawed culture is dilutedin 9 mL of broth, and then vortexed for5 s prior to carrying out a viable count. Inthe second instance, steps 1 to 3 for log-arithmic cultures are also omitted. One
millilitre of the thawed culture is dilutedin 9 mL of growth medium, which aftervortexing for 5 s is incubated for 2 h at 36 °C. Thereafter viable counting proce-dures and MIC testing with the variousinocula are identical).
3.1.3. Viable counting method for liquid MIC assays
1. Add 1 mL of mycoplasma culture to 9 mL of sterile diluent (mycoplasmabroth or broth base) and vortex (Autovor-tex mixer SA1 (or equivalent) Stuart Scientific Company Ltd, Redhill, Sur-rey, UK) the suspension for 5 s to reducepossible clumping of the mycoplasmas.
2. Prepare 10-fold dilutions of the vortexedsuspension from 10-2–10-9 in 0.9 mL vol-umes.
3. Add 0.1 mL of sterile mycoplasma brothat the appropriate pH to 8 consecutivewells in a horizontal row of a round bot-tomed microdilution plate (NUNC A/SRoskilda, Denmark), wells 1–8.
4. Add 0.2 mL of sterile mycoplasma brothto well number 12 (sterility control).
5. Transfer 0.1 mL of each mycoplasmadilution to the 0.1 mL volumes of broth ina microdilution plate beginning at wellnumber 8 and finishing at well number 1.
6. Seal the microdilution plate using anadhesive sealing film (Falcon, BecktonDickenson and Co., Oxnard, CA, USA)and roller, first puncturing the seal on allunused wells to prevent lifting of the filmdue to air expansion during incubation.Complete the sealing by rubbing the sur-face of the film around each well with asmooth object such as the back of a spoon(manufactured from plastic or horn).
7. Incubate the plate at 36 ± 1 °C untilcolour changes are complete.
8. Record the result; the lowest dilution toshow a colour change (approximately 0.2pH unit) denotes the reciprocal of thenumber of colour changing units (ccu)
Veterinary antimycoplasmal testing 387
in 0.1 mL of undiluted mycoplasma cul-ture. For example, if colour changes arerecorded to a dilution of 10-6, then thereare 106 ccu in 0.1 mL of the undilutedculture or 107 ccu⋅mL-1.
3.1.4. Preparation of inocula for liquidMIC assays
1. Repeat the incubation proceduredescribed in 3.1.2 (“Preparation ofmycoplasma inocula for MIC testing”)duplicating exactly the period of incuba-tion.
2. Dilute the culture in mycoplasma broth togive between 103 ccu and 105 ccu permL.
3.1.5. Modified MIC assay method of Tanner and Wu method [39]
1. Transfer 0.1 mL of each compound dilu-tion to separate consecutive wells run-ning horizontally on a microdilution plate(e.g. wells 1 through to 9).
2. To well number 10 add 0.2 mL of sterilemycoplasma broth at the appropriate pH (e.g. pH 7.6, for fermentative species,pH 6.8 for arginine catabolizers or pH 6.0–6.5 for ureaplasmas), as a sterility control.
3. To well number 11, add 0.2 mL of sterilemycoplasma broth adjusted to therequired end-point (pH 6.8 for fermen-tative species, pH 7.6 for arginine catab-olizers or pH 7.0–7.5 for ureaplasmas -end point control).
4. To row number 12 add 0.1 mL of sterilemycoplasma broth at the appropriate pH.
5. In a separate row add 0.1 mL of antibi-otic-free mycoplasma broth at the appro-priate pH containing the antibiotic sol-vent at the same concentration used todissolve the antimicrobial agent (solventcontrol).
6. Add 0.1 mL of mycoplasma inoculum toeach well containing the antibiotic dilu-tions (wells 1 to 9), to the growth con-trol well (well 12) and to the solvent con-trol well.
Each MIC test should include a standardreference mycoplasma (e.g. the type strain ofeach species under investigation) withknown susceptibilities against a range ofantimicrobials, to confirm the validity ofresults and a standard antimicrobial agente.g. (tiamulin) with known potencies againstdifferent species of veterinary mycoplasma.
The viable count of each mycoplasmachallenge inoculum must be tested simul-taneously with the MIC test to confirm theinoculum challenge as being between 103
and 105 ccu per mL.
7. Seal the microdilution plate with adhe-sive film as described above under“Viable counting method for liquid MICassays” and incubate at 36 ± 1 °C untilthe colour in the growth control well firstmatches that of the end-point control. Toachieve this, plates must be examined atfrequent intervals e.g. early morning,noon, late afternoon.
8. Record the initial MICs; the lowestantibiotic concentration to show nochange in colour when the colour of thegrowth control well matches that of thepH end-point control. By definition thisis the MIC value, however if plates are re-incubated the end-point in liquid assayfrequently shifts. For this reason a sec-ond reading is often taken and is referredto as the final MIC. Final readings canbe recorded 7 days later or 14 days laterfor slow growing mycoplasma speciessuch as M. hyopneumoniae, to determinethe permanency of the initial MIC and isthe lowest drug concentration in whichno colour change occurs.
9. Continue incubating the plates untilcolour changes are complete in the wellscontaining the inoculum challenge con-trol viable count.
P.C.T. Hannan388
Where MIC end-points are not achieved,or if the mycoplasma challenge falls out-side the range 103 ccu⋅mL-1 to 105 ccu⋅mL-1,tests must be repeated.
When the initial MIC and final MIC arethe same or very similar, this may be indica-tive of a compound possessing mycoplas-macidal activity. However, this should beconfirmed with MMC tests designed todemonstrate this property, such as dilutingthe contents of wells showing no colourchange into antibiotic free mycoplasmalbroth, followed by incubation, to check theabsence of viable mycoplasmas. The dilutionneeds to be sufficient to prevent carry overof the inhibitory effect of each dilution ofantibiotic and should be in the order of 1:25to 1:50. Alternatively, mycoplasmal killingcurve experiments may be carried out.
3.2. Solid agar MIC method
3.2.1. Viable counting method for solidMIC assays
1. Repeat the incubation proceduredescribed in Section 3.1.2 (“Preparationof mycoplasma inocula for MIC testing”)duplicating exactly the period of incuba-tion.
2. Repeat steps 1 and 2 in Section 3.1.3 forliquid assay viable counts.
3. Transfer 2 µL of each mycoplasma dilu-tion (in triplicate or more for greater accu-racy) to the surface of freshly preparedand dried mycoplasma agar plates usinga micropipetter and filter tip.
4. When the droplets of each suspensionhave been absorbed into the agar, incu-bate the plate at 36 ± 1 °C under theappropriate atmospheric conditions (e.g.aerobic, 95% N2 + 5% CO2 or anaero-bic) in sealed containers containingmoistened paper towelling.
5. Incubate the seeded plates until themycoplasma colonies are well developed
on the plates inoculated with the lowerdilutions of the mycoplasma suspension.
6. Select plates with a moderate growth ofwell separated colonies for counting.Avoid plates with low numbers ofcolonies.
7. Using a plate microscope or invertedmicroscope, count the number of coloniesproduced by each 2 µL droplet. The num-ber of cfu per 2 µL of inoculum is themean count from the three droplets (ormore if additional droplets are counted).
Example:
If the colony counts from an individual 2 µL droplet for a 10-5 dilution of inoculumare 30 cfu, 40 cfu and 35 cfu then the meancount would be 35 cfu per 2 µL. Therefore,to prepare inocula containing between 103
and 105 cfu per 2 µL for MIC testing, theconcentrated culture should be diluted 10-3
to give 3.5 × 103 cfu per 2 µL, which iswithin the recommended number for MICtesting.
3.2.2. Method (modified method of Hannan et al. [15])
1. Prepare agar plates containing a range ofantimicrobial concentrations in 9 cm plastic petri dishes as described in Section 2.4.3. Also, prepare control drug-free plates.
2. Dry the plates.
3. Inoculate each plate with 2 µL containing103–105 cfu, of the mycoplasma cultureto be tested using either micro-pipetterswith filter tips, if only a small number ofstrains are to be tested, or a multi-pointinoculator if larger numbers of strainsare to be tested simultaneously.Whichever method is used, growth con-trol plates (up to four to allow for possi-ble bacterial or fungal contamination)without antibiotics must be included ineach test.
Veterinary antimycoplasmal testing 389
4. Include the following controls:
(a) Reference strain (the type strain ofeach mycoplasma species beingtested).
(b) Plates incorporating a referenceantimicrobial (e.g. tiamulin).
(c) Plates incorporating the solventsused for each antimicrobial at thesame concentration as that used inthe highest concentration of the drug-containing plates.
(d) A challenge inoculum growth con-trol.
5. When the inoculum droplets have beenabsorbed, incubate the plates at 36± 1 °Ceither aerobically or in an atmosphere of95% N2 + 5% CO2 or anaerobically, ifgrowth of the species is dependant onthese latter conditions). Incubation shouldbe maintained in moist conditions, insealed plastic bags or gas jars, for a periodsufficient to allow the slowest growingmycoplasma species to form clearly vis-ible colonies, e.g. 7 days or longer ifspecies such as M. hyopneumoniaeor M. meleagridisare being investigated.
6. Record MICs by comparing the amountof mycoplasmal growth on each platewith that on the antibiotic-free growthcontrol plate using a plate microscope orinverted microscope. The end-point(MIC) is the lowest concentration ofantimicrobial to cause ≥ 50% inhibitionof growth (e.g. reduction in density ofgrowth and size of the colonies) com-pared with that on the control plate. A50% end-point is taken to eliminate platesin which antibiotics show a “tailingeffect” (very small numbers of mycoplas-mas persisting beyond, what appears tobe, the end-point).
Very often readings can be carried outmacroscopically, but end-points shouldalways be checked microscopically.
4. EXPRESSION OF RESULTS
Whether using liquid or solid MIC assays,MICs are expressed either in µg⋅mL-1 ormg⋅L-1. When only small numbers of strains(n ≤ 10) are tested at the same time, it isusual to quote only the range of MICsrecorded (e.g. MIC range = 0.1 µg⋅mL-1 to10µg⋅mL-1) to give an overall impression ofthe variance between strains. However, inlarger studies, it is important to highlightthe percentages of strains susceptible to par-ticular concentrations of antimicrobials. Thisis accomplished by quoting the concentra-tions of compounds to which 50% or 90% ofthe strains are susceptible (MIC50 or MIC90).In comparing in vitro efficacies of marketedor research compounds the geometric meanMIC has been used by some workers. Exam-ples of these calculations are shown in Table VIII.
5. INTERPRETATION OF RESULTS
The designations “sensitive”, “resistant”or “intermediate” (moderately sensitive) arecommon to almost all methods of clinicallaboratory testing, and are distinguished bythe use of in vitro breakpoint antibiotic con-centrations. Sensitive implies that the infec-tion is likely to respond. Intermediateimplies that an intermediate or indetermi-nate response is likely, except perhaps inspecial, defined circumstances, such as whenhigh doses can be used, or the antibiotic isconcentrated at the site of infection. Break-points are determined by attempting to takeimportant pharmacological and microbio-logical factors into account, including serumbinding, and are used routinely in bacterialinfections. These factors have not yet beendetermined for most veterinary mycoplas-mas in the various host species in whichthey may cause disease, although someinvestigators have attempted to establishbreakpoints for some compounds againstcertain mycoplasmal pathogens [16, 22, 42,43] (Tab. IX). This is due to the limited
P.C.T. Hannan390
pharmacological data currently available onmany antimicrobials in different animalspecies.
Until such information becomes avail-able, a standardized assessment of sensi-tive, intermediate and resistant strains is difficult. It is therefore likely that in deter-mining the antimicrobial susceptibilities ofveterinary mycoplasmas, some variance inthese designations will occur between lab-oratories.
6. TESTING FOR MYCOPLASMACIDAL ACTIVITY
Various tests have been used to deter-mine the mycoplasmacidal activity ofantimicrobials. These include:
1. Mycoplasmacidal activity determined byantibiotic dilution.
2. Mycoplasmacidal activity determined byfiltration.
3. Mycoplasmacidal activity determined bysubculture onto agar.
4. Mycoplasma killing curves.
Methods 1 and 2 have been designedspecifically to remove the antibiotic fromthe surviving mycoplasmas in MIC tests andthus give a true indication of the mycoplas-macidal activity of antimicrobial agents.These methods have been described fullyby Taylor-Robinson [40].
Method 3 consists of subculturing all ofthe wells in liquid MIC tests using a 96-pinreplicater/inoculator (Intek Services Ltd,Horley, Surrey, UK) onto mycoplasmal agarat the same time as recording the initialMICs and then incubating the plates to deter-mine the lowest concentration of compoundto inhibit mycoplasmal growth, the MMC.The results of this type of test are frequentlyvery similar to the final MICs in MIC tests.However, the test does not remove theantibiotics from the mycoplasmas and relieson the very small amount of culture trans-ferred on to replicater pins (2 µL) beingdiluted by diffusion into the agar gel. It istherefore possible that, in theory, amycoplasmastatic effect might still be inoperation in high concentrations of antibi-otic. This method has been described byHannan [14].
Method 4, mycoplasmacidal killingcurves, have been reviewed recently byRoberts [33] and are a measure of themycoplasmacidal activity of the antibioticbeing tested, thus providing a dynamic pic-ture of antimicrobial action and interactionover time, based on serial colony counts.However, the repetitive colony counts thatare required are tedious to perform and limitthe number of antimicrobial concentrationsthat can be tested with any isolate. This typeof test has been used to determine themycoplasmacidal activities of certainquinolones and various other antibioticsagainst M. hyopneumoniaeby Hannan et al.[15].
Table VIII. Expression of results in MIC tests:ranges, MIC50s, MIC90s and geometric meanMICs.
Mycoplasma strain No. MIC (µg⋅mL-1)
1 0.01 a, b
2 0.005 a, b
3 0.1 b
4 0.01 a, b
5 0.0025 a, b
6 0.05 a, b
7 0.1 b
8 0.25 b
9 0.5 b
10 1
MIC range 0.0025–1 MIC50 0.05 MIC90 0.5Geometric mean 0.049
a Values used to determine the MIC50 (e.g. 50% of thestrains are susceptible to ≤ 0.05 µg⋅mL-1).b Values used to determine the MIC90 (e.g. 90% of thestrains are susceptible to ≤ 0.5 µg⋅mL-1).To calculate the geometric mean MIC the logarithmof the MIC values for each strain are summed, dividedby the number of strains tested and then antilogged.
Veterinary antimycoplasmal testing 391
Tab
le IX
. MIC
bre
akpo
ints
sug
gest
ed a
gain
st c
erta
in m
ycop
lasm
a sp
ecie
s by
var
ious
inve
stig
ator
s.
Myc
opla
sma
spec
ies
Ant
imic
robi
al a
gent
Bre
akpo
int (
µg⋅m
L-1)
Inve
stig
ator
s
Sen
sitiv
eIn
term
edia
teR
esis
tant
oxyt
etra
cycl
ine
≤4–
8≤
8>
8er
ythr
omyc
in≤
1–4
≤4
> 4
olea
ndom
ycin
≤1–
4≤
4>
4M
. ga
llise
ptic
um
roxi
thro
myc
in≤
1–4
≤4
> 4
M. s
yno
via
esp
iram
ycin
≤2–
8≤
8>
8M
. io
wa
elin
com
ycin
≤2–
8≤
8>
8K
empf
et a
l. 19
89 [2
2]
M. m
ele
ag
rid
isjo
sam
ycin
≤2–
8≤
8>
8ty
losi
n≤
1>
1ki
tasa
myc
in≤
8–17
≤17
> 1
7en
roflo
xaci
n≤
1>
1ox
olin
ic a
cid
≤2–
4≤
4>
4
tetr
acyc
line
≤1
≤4
≥32
chlo
ram
phen
icol
≤
4≤
8≥
16M
. hyo
rhin
iser
ythr
omyc
in≤
1≤
2≥
4te
r La
ak e
t al.
1991
[42]
M
. hyo
pn
eu
mo
nia
ecl
inda
myc
in≤
1≤
2≥
8M
. flo
ccu
lare
oflo
xaci
n≤
1≤
4≥
16ci
prof
loxa
cin
≤1
≤2
≥16
M. b
ovi
ste
trac
yclin
e≤
12–
4≥
8M
. dis
pa
rch
lora
mph
enic
ol≤
48
≥16
ter
Laak
et a
l. 19
93 [4
3]
U. d
ive
rsu
mst
rept
omyc
in≤
48–
16≥
32
M. g
alli
sep
ticu
m≤
0.5
≤1
≥2
M. s
yno
via
e
enro
floxa
cin
≤4
8≥
16M
. io
wa
eox
ytet
racy
clin
e≤
48
≥16
Han
nan
et a
l. 19
97 [1
6]
M. b
ovi
sflu
meq
uine
≤1
≤2
≥4
M. a
ga
lact
iae
tylo
sin
≤8
–≥
16M
. hyo
rhin
istia
mul
in
M. h
yop
ne
um
on
iae
M
. hyo
syn
ovi
ae
The
bre
akpo
int c
ited
for
each
ant
ibio
tic a
pplie
s to
all
of th
e m
ycop
lasm
a sp
ecie
s te
sted
by
each
inve
stig
ator
.
} } }}
P.C.T. Hannan392
7. CONCLUSION
In view of the wide variation in nutri-tional requirements, cultural conditions andgrowth characteristics of veterinarymycoplasmas, it is not surprising that dif-ficulties have been encountered in obtain-ing consistent MIC results from differentlaboratories. There are many factors whichcan influence the results of MIC tests involv-ing veterinary mycoplasma species, includ-ing the composition and pH of the culturemedium, the incubation conditions and thepreference of certain mycoplasma speciesfor agar rather than broth media. Whetherusing a liquid or solid MIC assay system,it is essential that, to avoid obtaining falselylow MIC results, optimal media for growingspecific mycoplasma species are used, thatcultures are pure and that mycoplasma inoc-ula are carefully standardised. The recom-mended viable counts for inocula are 103 to105 ccu⋅mL-1 for liquid assays and 103 to105 cfu per plate for the agar dilutionmethod. The growth phase of the organismsseems to be less important as lag phase cul-tures of M. gallisepticum, M. synoviae, M. bovisand M. hyopneumoniaehave beenshown to give very similar results to cul-tures in the logarithmic growth phase in liq-uid assays.
Antimicrobials should be stored accord-ing to the manufacturers recommendationsand MICs determined in terms of their activebase component, particularly when com-paring results between laboratories. In liquidand solid MIC assays it is important thattests are carefully controlled, with growth,sterility, pH end-point (in liquid assays) andcompound solvent controls and that stan-dard reference mycoplasmas with estab-lished antibiotic sensitivity patterns areincluded in each experiment. In liquid MICassays the microdilution plates must be ade-quately sealed to prevent exchange of gasesbetween wells which might result in falsecolour changes and erroneous MIC end-points and that the reading of liquid or solidMIC tests are carefully standardised. These
factors, coupled with a dearth of standardi-sation of MIC procedures between labora-tories to date, have made it almost inevitablethat significant differences in results wouldoccur. This report should aid in rectifyingthis problem. Inter-laboratory studiesemploying standardised methods with acommon range of antimicrobial agentsagainst standard veterinary mycoplasmaspecies and strains are now needed to deter-mine whether consistent results can beachieved.
ACKNOWLEDGEMENTS
Prepared on behalf of the InternationalResearch Programme on ComparativeMycoplasmology (IRPCM) ChemotherapyTeam of the International Organisation forMycoplasmology (IOM). The author thanksthe following members of the IOM IRPCMChemotherapy working party and consultantsfor their contributions, comments, criticisms,help and suggestions in preparing themanuscript and for reviewing the finaldocument: Dr H.J. Ball (Veterinary SciencesDivision, Stormont, Belfast, Northern Ireland);Dr J.M. Bradbury (Dept. of Avian Pathology,University of Liverpool, Leahurst, Neston,South Wirral, UK); Dr N.F. Friis (DanishVeterinary Laboratory, Copenhagen,Denmark); Dr I. Kempf (Agence Française deSécurité Sanitaire des Aliments (AFSSA),Ploufragan, France); Dr S. Kleven(Department of Avian Medicine, University ofGeorgia, Athens, Ga., USA); Dr S. Levisohn(Division of Poultry Diseases, KimronVeterinary Institute, Bet Dagan, Israel); Dr R.Nicholas (Central Veterinary Laboratory,Addlestone, Surrey, UK); Prof. J.A. Robertson(Department of Medical Microbiology,Immunology, University of Alberta,Edmonton, AB T6G 2H7, Canada); Prof. L.Stipkovits (Veterinary Medical ResearchInstitute, Budapest, Hungary); Dr K.B. Waites(Dept. of Pathology, University of Alabama,Birmingham, AL, USA); Dr J.M. Wilson(Glaxo Laboratories, Greenford, Middlesex,UK); Dr G.D. Windsor (MycoplasmaExperience Ltd, Reigate, Surrey, UK); Prof. K.Whithear (University of Melbourne, School ofVeterinary Science, Werribee, Victoria,Australia). The author also thanks Mrs. S.
Veterinary antimycoplasmal testing 393
Hayes and Mrs. E. Windsor for their parts intyping the manuscript.
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