Lipid Composition in the Classification of Rhodococcus equi

Zbl. Bakt. 272, 154-170 (1989)

Lipid Composition in the Classification of Rhodococcus equi M. D. BARTON!, M. GOODFELLOW 2, and D. E. MINNIKIN 3 1 Veterinary

Research Institute, Park Drive, Parkville, Victoria 3052, Australia* Department of Microbiology, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K. 3 Department of Chemistry, The University, Newcastle upon Tyne NEl 7RU, U.K.

2

With 1 Figures· Received July 12, 1989 . Accepted August 21, 1989

Abstract The fatty acid, menaquinone and polar lipid composition of representatives of Rhodoeoeeus equi and related taxa were determined. All of the R. equi strains had major proportions

of straight chain saturated, monounsaturated and 10-methyl branched fatty acids, dihydrogenated menaquinones with eight isoprene units as the predominant isoprenologue, and characteristic polar lipid patterns that contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and glycolipids including a "cord factor"-like compound that was most pronounced in fresh isolates. The mycolic acids of these strains fell within the range C24 to C48 , had 0 to 4 double bonds and released major amounts of C14 : 0 esters on pyrolysis. These lipid data provide further evidence that R. equi strains form a distinct taxospecies within the genus Rhodoeoeeus. The remaining strains also gave lipid profiles consistent with their assignment to the genus Rhodocoeeus. These organisms included strains identified as R. sputi. Zusammenfassung Es wurde die Zusammensetzung der Fettsauren des Menachinons und der polaren Lipide reprasentativer Rhodocoeeus equi- und verwandter Stamme untersucht. Samtliche R. equiStamme hatten hohe Anteile von geradkettigen, einfach ungesattigten und 10-methyl-verzweigten Fettsauren, dihydrogenierten Menachinonen mit 8 Isopren-Einheiten als vorherrschenden Isoprenoid und typische polare Lipidmuster aus Diphosphatidylglycerin, Phosphatidylathanolamin, Phosphatidylinosit, Phosphatidylinositmannosiden und Glycolipiden einschlieBlich einer "cord-factor"-ahnlichen Substanz, die am starksten in frischen Isolaten auftrat. Die Mykolsauren dieser Stamme hatten Kettenlangen zwischen C24 und C48 , wiesen Obis 4 Doppelbindungen auf und setzten bei Pyrolyse grogere Anteile an C14 : o-Estern frei. Aus diesen Lipidanalysen ergeben sich weitere Hinweise, dag R. equi-Stamme eine eigene Spezies im Genus Rhodococcus bilden. Auch die iibrigen Stamme ergaben Lipidmuster, die ihre Zugehorigkeit zum Genus Rhodoeoeeus belegen. Hierzu gehoren die als R. sputi identifizierten Stamme.

* Present address: Central Veterinary Laboratories, Department of Agriculture, G.P.O. Box 1671, Adelaide, South Australia, 5000, Australia

Lipids of Rhodococcus equi

155

Introduction

Corynebacterium equi Magnusson (31) is an important veterinary pathogen that causes bronchopneumonia, ulcerative enteritis and lymphadenitis in foals (4,59). The organism infects other domesticated animals and is being isolated with increasing frequency from human patients, especially from those with generally decreased resistance to infections (14, 23, 45, 55). The species has had a confused taxonomic history (4) and is currently cited under both Corynebacterium and Rhodococcus in the Approved Lists of Bacterial Names (46). Strains labelled C. equi or R. equi have been shown to form a distinct taxospecies within an aggregate group corresponding to the genus Rhodococcus (5, 20, 21). The integrity of this taxon is supported by data from peptidoglycan (54). DNA homology (40, 41, 49) and comparative immunodiffusion studies (43) but even so the name C. equi is still used (24,59). Lipid markers are extensively used in the classification of nocardioform actinomycetes (34), namely mycolic acid-containing bacteria belonging to the genera Corynebacterium, Mycobacterium, Nocardia, Rhodococcus and Tsukamurella (12, 18). Rhodococci, including R. equi, are difficult to distinguish from corynebacteria on either mycolic acid (9) or menaquinone composition (8, 11, 56) but there is some evidence that these organisms can be separated on the basis of fatty acid and polar lipid content (6, 10, 29, 35,48). The primary aim of the present investigation was to determine the value of lipid markers in the classification of R. equi strains.

Methods

Strains and growth conditions. Most strains (Table 1) were grown in magnetically stirred flasks at 25°C for 4 to 5 days in modified Sauton's medium (39) supplemented with thiamine (50 ~g ml- I ). However, cultures prefixed by C, Nand R, and strains MB20, MB37, MB38, MB84, MB139 and MB141, were shaken at 100 revolutions per minute (Orbital incubator, Gallenkamp) for 7 days at 30°C. Cultures were checked for purity, killed with formalin (1 %, v/v), harvested by centrifugation, washed three times with distilled water and freeze-dried. Extraction and analysis of fatty acid methyl esters (FAMES). Dried biomass (about 50 mg) was degraded by acid methanolysis (37) and analytical and preparative TLC of the methanolysates was done as described previously (42). Gas chromatography of petroleum ether solutions of purified FAMES was performed using a non-polar column (3 m, 2.5 OV-1 silicone on 80-100 mesh Chromosorb G. AW-DMCS, 180°C; Phase Separations) fitted to a Pye-UNicam 104 gas chromatograph, with nitrogen as the carrier gas. Some samples (Table 3) were also examined on a Perkin-Elmer Fll flame ionisation gas chromatograph fitted with a stainless steel polar column (6 m, 10% Silar 10C on 100-120 mesh Gas-Chrome Q, 210°C; Applied Science Laboratories) to clarify the identity of components. Retention times and relative proportions of FAMES, expressed as a percentage of the total area, were measured using a Shimadzu CE1B computing integrator. Individual components were identified by comparison of retention times with those of standard mixtures of straight chain and mono-unsaturated fatty acids (C 12-C I8 ); tuberculostearic acid (10-methyloctadecanoic acid) was identified by comparison with a profile of an authentic strain of Mycobacterium avium. The FAMES were also identified by calculating the product of the retention time and peak height for each component in a sample and taking the percentages of the sum of the products for all the components (26).

156

M. D. Barton, M. Goodfellow, and D. E. Minnikin

Table 1. Test strains Description, cluster and laboratory number

Source

Rhodococcus equi Cluster 1" MB2,MB12, MB15, MB28,MB44 MB3 MB10 MB14 MB21,MB24, MB26 MB27, MB80, MB89, MB1l7, MB190 MB60 MB62 MB82 MB90 MB115 MB165 MB171 MB186 Cluster 1F* *

cr

N817 R1+ R120 R128 R134

M. D. Barton, K3s. E2s, D2s, PL, W5s: soil

K. L. Hughes, Veterinary Clinical Centre, Werribee, Victoria, Australia, KLH77; foal lung abscess K. G.Johnston, Ul).iversity of Sydney Rural Veterinary Centre, Camden, New South Wales, Australia, RVC 681/78; foal mesenteric lymph node abscess D. Pemberton, Regional Veterinary Laboratory, Bairnsdale, Victoria, Australia, G78/10727; foal lung abscess J. C. Keast, Veterinary Research Station, Glenfield, New South Wales, Australia, VRS 77/1281, foal lung abscess; VRS 56/ 3712, pig; VRS 77/1881 (RB 66.215m); cat leg abscess E. G. Russell, Veterinary Research Institute, Parkville, Victoria, Australia, VRI 79/565, foal lung abscess; VBI 79/4685A, VRI 79/4685A, VRI 79/1453, VRI 79/5027, VRI 79/11355; pig lymph nodes M. D. Barton, foal joint abscess, Do J. R. Bates, Regional Veterinary Laboratory, Benalla, Victoria, Australia, 79/2859; foal fistulous withers E. G. Russell, VRI 79/5723; adult horse leg wound E. G. Russell, EGR1; soil M. D. Barton, Gg3; goat gut contents B. A. Donald, Animal Research Institute, Yeerongpilly, Queensland, Australia, S7240; cow B. A. Donald, Y250; pig E. G. Russell, rat faeces 1 D.Jones, Department of Microbiology, University of Leicester, U.K., NCTC 1621; foal lung abscess R. E. Gordon, Institute of Microbiology, Rutgers University, New Brunswick, New Jersey, U.S.A., 1257, A.1. Laskin, 2915; soil ATCC 25729; R. E. Gordon, W1621; NCTC 1621 M. Mutimer, Department of Veterinary Pathology and Public Health, University of Queensland, Australia, UQV 161; human faeces M. Mutimer, UQV 1302; bovine rumen M. Mutimer, UQV 1412; porcine faeces

Rhodococcus species Cluster 3" MB37, MB38 MB139, MB141

M. D. Barton, M1s, E3c; soil M.D.Barton, Ed9A, Ed17; horse dung

Cluster 4* MB145

M. D. Barton, Cd19; cattle dung

Lipids of Rhodococcus equi Description, cluster and laboratory number

157

Source

Cluster 1L* N864-N871 Single member clusters

R.E.Gordon, 1066-1070, 1070VV, 1071, 1071VV

Affinity with cluster 1 * MB33

J. B. Woolcock, Department of Veterinary Pathology and Public

MB39, MB155 MBll1

Health, University of Queensland, Australia; cow lymph node M. D. Barton, VRls, Han4; soil E. G. Russell, E6R22; rat brain abscess

Affinity with clusters 3 and 4* MB20, MB43 J. N. Keast, VRS 75/2451110; mare nasal swab; VRS 74.3121; human M.D.Barton, S+; foal mouth ulcer MB84 Single member clusters * * R. E. Gordon (Rhodococcus equi), 390; R. S. Breed; J. S. SimN810 mons; water R. E. Gordon, 358 (Corynebacterium equi); R. S. Robinson N811 (Nocardia sp.) R. E. Gordon, VV3585; S. A. Waksman (Nocardia sp.) N812 R. E. Gordon, 885; D. M. Powelson Uensenia sp.) N813 R. E. Gordon, 1241; J. D. Stout, RS42 (Nocardia sp.); soil N814 R. E. Gordon, 808; L. S. McClung, RH200; R. S. Breed, 6-30 N815 R. E. Gordon, 549; E. N. Ayar (Nocardia aquosus) N818 R. E. Gordon, 588 N829 R. E. Gordon (Corynebacterium equi) , 1256; ATCC 25728; R70 soil E. Wolinsky, Cuyahoga County Hospital, Cleveland, Ohio, R182 U.S.A., M50b; sputum

Extraction and analysis of mycolic acid methyl esters. Mass spectra of the purified mycolic acid methyl esters, extracted from the whole-organism acid methanolysates (37), were recorded on an AEI MS9 instrument using a direct insertion probe, an ionizing voltage of 70eV and a temperature range of 180° to 220°C. Extraction and analysis of polar and isoprenoid quinones. The small scale integrated procedure of Minnikin et al. (38) was used to extract these lipids from biomass samples (about 50 mg). Mass spectra of the purified quinones were recorded on an AEI MS9 instrument using a direct insertion probe, an ionizing voltage of 70eV and a temperature range of 200 to 230°C. Polar lipid patterns were also obtained using published procedures (38). Trehalose dimycolate glycolipids ('cord factor') were resolved using a modification of the solvent described by Silva et al. (45); plates were developed in chloroform-acetonemethanol-water (50: 60: 2.5: 3, v/v) and sprayed with periodate-Schiff reagent (38). Results

Mycolic acids Thin-layer chromatographical analysis of the whole-organism methanolysates revealed the presence of single spots corresponding to mycolic acid methyl esters (MAMES,

158

M. D. Barton, M. Goodfellow, and D. E. Minnikin

Table 2. Overall size and structural features of mycolic acids from Rhodococcus equi and related organisms as determined by mass spectral analyses Designation and laboratory number

Cluster+ Total number of carbons

Main component

Number of double bonds C12

Long chain ester released on pyrolysis ++ Cl4

Cl6

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++

+ + ++ + + + + + + + ++ + ++ + + + + + + + + + ++ + + ++ + ++ +++ +++ +++ +++ ++ + +++ +++ +++ +++ +++ +++ +++

CIS

Rhodococcus equi

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ++ + + ++ ++ + + ++

MB2 MB3, MB89 MBI0 MB12 MB14 MB15, MB90 MB21 MB24 MB26 MB27 MB28 1* MB44 MB60 MB62 MB80 MB82 MBI15 MB117 MB165 MBI71 1* MB186 MB190

38-48 34-48 36-48 36-48 36-48 36-48 36-44 36-46 36-46 34-47 34-48 34-46 34-48 38-48 34-48 34-46 34-48 38-48 ND 38-48 ND 38-48

44 44 44 38 38 44 38 36 40 40 44 42 44 44 36 34 36,42 42 ND 44 ND 44

0-4 0-4 0-4 0-3 0-4 0-3 0-3 0-3 0-3 0-4 0-4 0-3 0-3 0-3 0-3 0-3 0-3 0-3 ND 0-4 ND 0-3

C7 R71 R120 R128 R134 N817

34-44 34-48 34-48 38-48 36-48 34-44

34 42 40 42 42,44 42

0-3 0-4 0-3 0-3 0-3 0-3

32-40 32-40 36-40 36-44

36 36 40 40

0-4 0-4 2-4 2-4

+ +

42-46 42-46 40-46 40-46 40-46 40-46 40-46 40-46 34-40

44 44 44 44 44 42 42 44

0-3 0-3 0-3 0-3 0-3 0-3 0-3 0-3

+++ +++

36

0-2

IF**

Rhodococcus species MB37 MB38 MB139 MB141 N864 N865 N866 N867 N868 N869 N870 N871 MB145

3*

lL*

4*

+ + + +

+ + ++ ++ + + + + + ++ ++ + +

Lipids of Rhodococcus equi Designation and laboratory number

Cluster+ Total number of carbons

Main component

Number of double bonds C12

MB33 MB39 MBll1 MB155 MB20 MB43 MB84

SMC's

34-48 34-47 34-48 34-48 36-44 32-40 32-39

44 40 42 36 40 35,36 35

0-3 0-2 0-3 0-3 2-4

34-40 36-40 32-40 32-38 36-48 34-40 38-46 34-40 38-46 36-42

36 42 36 36 38 38 40 36 42 38

N810 N811 N812 N813 N813 N815 N818 N829 R70 R182

SMC's

SMC's

0-4

0-3 0-4

0-3 0-4 0-4 0-4

0-3

0-4

2-4 2-4

Long chain ester released on pyrolysis ++ C14

C16

++ ++ + ++

+++ +++ +++ ++

+

+++ + +++ ++ ++ + + + + + + +

+ + + +++ ++ + +++ + +++ +++ ++ +++ ++ +++ ++ +++ +++

+ + +++ + + + +

159

C18

+++ ++ + + + + + +++ ++ ++ + +++

+ Clusters defined by Barton and Hughes (5)* and Goodfellow et al. (20)**. SMC's, single member cluster; ++, The main component of each series is denoted by +++, any component greater than 50% of the main peak by + +, and all other significant components by +.

RF 0.2-0.5) and non-hydroxylated fatty acid methyl esters (RF 0.8; 37). The rhodococcal MAMES had a similar mobility to the methyl mycolates prepared from extracts of Corynebacterium diphtheriae PW8. Strains MBll1, N869, N871 and R71 also produced a spot that was shown to be a nocardol (37). Methyl esters of mycolic acids fragment on mass spectrometry in several competing pathways depending on their overall size and structural features (9, 37). The strains of R. equi gave a complex series of fragments corresponding to anhydromycolates in the range mJe 514 to 714, that is, they contained a homologous series of mycolic acids with a carbon range between C34 and C48 • The mycolic acid esters from these strains had nought to four double bonds, in most cases the main component of the parent mycolic acid centred on C40 to C44 • The mycolic acids of the remaining strains were variable in length. Some, including all but one of the strains assigned to clusters 3 and 4, had smaller mycolic acids (C32 to C40 ) but others, notably strains MB33, MB39, MB111, MB155 and N810, were comparable in size to those from R. equi. Seven of the eight representatives of cluster 1L contained mycolic acids with a carbon range between C40 and C46 • The overall size and structural features of the mycolic acids of the test strains is shown in Table 2. The long chain esters released on pyrolysis were predominantly C14 for R. equi and strains MB33, MB39, MBll1 and N810. In contrast, most of the remaining organisms including all but two of those assigned to clusters 3, 4 and 1L contained C 16 as the major component.

MB2 MB3 MBI0 MB12 MB14 MB15 MB21 MB24 MB26 MB27 MB28 MB44 MB60 MB62 MB80 MB82 MB89 MB90 MB115 MB117 MB165 MB171 MB186 MB190

Rhodococcus equi

Description and laboratory number

1

1

Cluster++

Assignment* ECL on OVl column*

5.4 2.8 3.7 2.9 5.8 4.5 4.4 4.4 5.7 4.8 12.6 4.4 7.6 5.0 4.6 6.2

0.2

0.9 0.3

0.4 0.3 0.3

0.2 0.4 0.2

4.7 6.0 5.8 4.5 3.8 4.4 4.4

C0.2 0.2 0.3

0.5 0.9 0.9 1.2 0.6 1.0 1.9 0.6 0.6 2.3 0.8 1.0 1.3

-

0.3

-

0.5 0.7

1.3 tr

tr 0.9 0.3 tr tr tr tr tr tr tr tr tr tr tr tr

1.1

tr tr tr 1.9

1.1

tr 1.6 1.2

47.5 48.5 43.7 45.8 46.5 44.5 40.7 49.8 48.5 47.3 48.1 46.5 49.0 45.3 48.5 45.0 45.6 46.5 42.7 44.7 47.4 47.3 44.7 43.2

7.6 7.1 5.6 4.9 4.5 4.6 4.5 4.9 5.4 4.2 4.8 3.5 4.3 7.2 4.0 5.1 7.1 6.0 5.5 4.7 5.7 5.4 8.4 6.5 1.0 0.8 0.4 0.6 1.3 1.2 0.7 1.6 1.1 0.9 1.2

1.1

0.5 1.4 0.7 0.4 0.4 0.9 0.2 1.2 0.3

1.3

1.1 1.1

1.1 1.2 1.3 04 1.4 0.2 1.2 3.0 1.6 1.8 1.6 1.7 0.9 1.9 1.2 1.5 2.1

1.5 1.5

1.1

1.3

1.7 1.3

1.5 0.9 1.5 1.6

1.1

1.0 0.6 1.4 1.0 0.6 1.4 0.9 1.0 1.2 1.5 0.8

1.2 1.2 0.9 0.8 1.1

1.1

1.5

9.4 8.1 7.5 9.2 11.2 12.1 10.1 11.6 9.1 16.7 9.7 5.8 11.9 6.7 9.3 6.9 7.1 7.2 9.0 10.4 7.8 10.9 5.3 6.6 3.1 2.6 2.0 1.4 1.3

1.1

1.9 1.5 1.9 3.2 1.7 3.2 2.5 1.4 1.9 2.6 1.2 2.9 3.9 1.7 4.5 2.3 1.7 2.3

20.5 20.3 28.7 28.7 28.8 25.9 27.3 28.5 24.2 24.9 24.2 30.2 18.6 25.5 22.9 31.4 27.5 23.8 18.2 27.6 19.4 23.6 29.5 24.5

0.4 2.4 1.3 1.2 1.0 1.0 0.8 0.9 1.3 0.7 0.5 1.4 0.4 2.1

-

1.9 3.5 0.8 0.9

1.1

2.1 1.6 1.6

C17:0 t18:0 C18:1 C18:0 t19:0 17.0 17.4 17.6 18.0 18.4 19.0

1.5

C12:0 C14:0 C15:0 C16:1 C16:0 t17:0 12.0 14.0 15.0 15.6 16.0 16.4 17.0

Table 3. Percentage fatty acid composition of Rhodococcus equi and related taxa

......

~

0::l

2.

~

~

~

::l 0-

~

-~

0-

~

0,...,

0 0

C)

~

F

0

~

...,.,

t:!:'

~

0

0\

N

;:J

N

"'"~

?=

N

-

SMC's**

MB33 MB39 MBll1 MB155 MB20 MB43 MB84 4.9

0.6

1.1 1.8 2.5 0.7 4.1 8.3

8.9

3.6 4.1 1.2 1.9

23.4

1.3 0.6 tr tr tr tr tr

tr

tr tr tr tr

27.0

58.4

45.9 37.4 49.5 48.1 38.4 32.5 30.7

29.1

35.7 35.2 39.1 38.3

0.5

5.3 7.7 5.7 4.1 0.9 1.7 3.1

3.5

1.6 2.1 0.7 0.6

0.2

0.8 3.6 1.4 1.3 1.3 9.8 9.3

10.5

10.5 9.8 2.2 2.3

0.2

0.9 4.3 1.9 4.5 1.7 3.7 6.5

4.9

7.1 4.0 2.2 1.4

0.2

1.2 2.1 1.2 0.9 0.2 3.1 12.1

12.6

0.1

4.3 3.4

14.7

6.2

9.7 7.4 8.4 8.8 25.3 20.2 6.5

6.0

18.6 18.7 26.7 22.4

6.5

6.8

0.5

1.6 2.0 2.3 4.5 4.5

0.9

3.3 5.0

0.3

-

22.8

23.9 23.9 17.7 19.7 20.8 20.5 20.3

20.6

15.1 18.3 17.9 18.7

1.9 0.8 3.0 1.9 1.3 3.1 0.5

1.1

1.5 2.8 1.9 1.8

C17:0 t18:0 C18:1 C18:0 t19:0 17.0 17.4 17.6 18.0 18.4 19.0

+ Abbreviations for fatty acid methyl esters are explained by the following examples: 16:0, straight chain hexadecanoic acid; 18:1, octadecanoic acid; t19:1, 10-methyloctadecanoic acid. ++ Clusters defined by Barton and Hughes (5). * Equivalent chain lengths (ECL) according to Kates (26). * * SMC's, single member clusters.

4.8

Mycobacterium avium D4

7.0 7.4 6.2 3.5 2.6 0.9 1.4

1.3

0.7 0.2 3.7 3.1

Corynebacterium diphtheriae PW8

0.9 0.3 0.2 0.1 0.1

0.2

4

0.1

C12:0 C14:0 C15:0 C16:1 C16:0 t17:0 12.0 14.0 15.0 15.6 16.0 16.4 17.0

MB145

Assignment* ECL on OV1 column *

0.3 0.1

3

Cluster++

MB37 MB38 MB139 MB141

Description and laboratory number

t-'

""......

......

E.

.n

'"rD

n C

()

8

o 0o

~

""

0.: '"o

-6'

Cluster+ mle

N810 N811 N812 N813

MB20 MB43 MB84

MB33 MB39 MBlll

C7 N817 R71

SMC's*

IF**

R. equi MB2 MB3, MBI0, MBI2, MB27, MB80,MB89, MB171,MB190 MB4, MB90 Mb15,MB21, MB60,MB165 1* MB24,MB26,MBI17,MBI86 MB28 MB44 MB62, MB82 MB115

Designation and laboratory number

+ +

+ ++

+

+

+ + + + +

+

+ +

652

+ + + +

+ ++ + + + ++ + ++ ++ + +

+

650

H4

+ + ++ ++

+ + + +

+ + +

+

648

H2

MK-7

+

+

654

H6

+

656

Hs

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ + +++ +++ +++ +++ +++ +++

+ + +

+ + + + + +

+ + + ++ + ++ ++

+

+++ +++

718

+ +

716

H2

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ++ ++ ++ ++ ++ ++

++ ++

720

H4

Mk-8

+

+ + +

+ + + + + + + + + +

+

722

H6

+

784

+++

+

786

H2

++

+

788

H4

MK-9 790

H6

Table 4. Peaks corresponding to molecular ions in the mass spectra of menaquinones of Rhodococcus equi and related taxa. The main component in each series is denoted by + + +, any other component greater than 50% of the main peak by + +" and all other significant components by +.

......

0\

~

::l

C.

5' 2.

~

1'I1

~

::l 0-

I»

~~

0"

5::

0 0 0-

CJ

~

?

I»

... .... 0

I:;I:j

~

N

lL**

SMC**

R182

+ + + + + ++ + + + +

+ + + + + + +

648

+ ++ ++ ++ + ++ ++ + ++ +

+ +

+ + + + ++ ++ + +

Hz 650

+ + + + + ++ + + + +

+

+ + + + + + +

652

H4

MK-7

+

+

H6

654

+

Hs

656

++ +

+ + + + + ++ ++ ++ ++

+

+ ++ + ++ ++ +

716

+++ +++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ ++

+++ +++ +++ +++ +++ +++ +++ +++

Hz 718

++ ++ + +++ ++ ++ ++ ++ ++ ++ ++ ++ +

++ ++ ++ ++ ++ ++ ++ ++

720

H4

Mk-8

++ + + +

+

+ + + +

+

+

722

H6

+ Clusters defined by Barton and Hughes (5), * and Goodfellow et al. (20), * * SMC's, single member clusters.

4*

N864 NB865 NB866, N868 N867 N869 N870 N871

3*

MB145

Rhodococcus species MB37 MB38 MB139 MB141

IF**

SMC's**

N814 N815 N818 N829

R70 R120 R128 R134

Cluster+ mle

Designation and laboratory number

+ + + + + + + +

+ +

784

++ ++ ++ ++ ++ ++ ++ +++

+++ ++

+ +

Hz 786

+ + + + + + + +

++ ++

+ +

H4

788

MK-9

+ ++

790

H6

--~

..... (.,.)

0\

S.

..c

n>

s:: en

(') (')

0 00 (') 0

en 0

f5.:

164

M. D. Barton, M. Goodfellow, and D. E. Minnikin

Fatty acids The R. equi strains contained major amounts of hexadecanoic (C 16 : 0), octadecanoic (C 18 : 1) and tuberculostearic (t 19: 0; 10 methyloctadecanoic) acids with smaller proportions of tetradecanoic acid (C 14 : 0) plus lower homologues of tuberculostearic acid (Table 3). This profile serves to distinguish R. equi from C. diphtheriae PW8 and the remaining rhodococci apart from strains MB33, MB39, MBlll and MBISS. Rhodococcus strains MB20 and MB43, and those assigned to cluster 3, were characterised by the presence of high proportions of octadecanoic acid (C 18 : 1)' Similarly, strains MB84 and MB14S contained relatively large proportions of pentadecanoic acid (C 15 : 0 ).

Isoprenoid quinones Compounds that co-chromatographed with vitamin Kl were the only isoprenoid quinones detected by TLC in extracts of the test strains. The most intense peaks in the mass spectra of the purified menaquinones occurred at mle 187 and 22S and were derived from the naphthoquinone nucleus (3). All of the mass spectra in the high mass region contained strong peaks corresponding to molecular ions (M+) with smaller peaks at M+ -15 denoting the loss of a methyl group from the molecular ion. A summary of the mass spectral data is shown in Table 4. The R. equi strains, and all but three of the remaining rhodococci, contained dihydrogenated menaquinones with eight isoprene units, abbreviated as MK-8(H2)' as the major isoprenologue. In contrast, Rhodococcus strains MB20, MB139 and R182 were characterised by major proportions of MK-9(H2)' Some variation was found in the distribution of minor components.

Polar lipids Examples of the two dimensional polar lipid patterns obtained with R. equi and related strains are shown in Fig. 1 a, b. Most of the lipids were identified by their characteristic chromatographic migration and staining properties with specific spray reagents but some were not positively identified. The R. equi strains and a number of other cultures (MB39, MBll1 and MBISS) contained major amounts of diphosphatidylglycerol and phosphatidylethanolamine, less intense spots corresponding to phosphatidylglycerol, phosphatidylinositol and mono- and diacylated phosphatidylinositol dimannosides, and three characteristic polar glycolipids (G h G2 and G3 ; Fig. 1 a). In addition, a less polar "cord factor" like glycolipid (trehalose mycolate; Fig. 1 b), which characteristically stained turquoise with periodate-Schiff reagent was detected in most strains. The distribution of trehalose mycolates was seen more clearly by single dimensional TLC (Fig. 1 c); higher proportions were noted in extracts of recent isolates, for example MB10 and MBllS (Fig. 1 c). The remaining rhodococci, including those assigned to clusters 3 and 4, had similar polar lipids to those found in the R. equi and associated strains. Some strains, namely MB37 (Fig. 1 b), MB38, MB43, MB84, MB14S, N813, N81S and N829, were especially rich in glycolipids but did not contain phosphatidylethanolamine. Rhodococcus strain R71 and the cluster 1L strains (Fig. 1 d) contained large amounts of a glycolipid (G4 ) that gave a characteristic staining reaction with both a-naphthol and periodateSchiff reagent.

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I" "~'c++

.. (i)

PIM

Fig. 1. Thin-layer chromatography of polar lipids. a) C 7, b) MB 37, c) (i)-(iii); MB 117, MB 115, MB 10, d) N 871. Developing solvents for a), b) and d) were chloroform-methanolwater (65: 25: 4), by vol.) in the first direction and chloroform-acetic acid-methanol-water (80: 15 : 12: 4, by vol.) in the second direction; chloroform-acetone-methanol-water (100: 120: 5: 6, by vol.) was used for c). Periodate-Schiff spray reagent was used for c) and 10% ethanolic molybdophosphoric acid for a), b) and d). Abbreviations: DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; CF, "cord.factor" (trehalose dimycolates); G 1-G4, characteristic glycolipids; G and P, unknown glycolipids and phospholipids.

Discussion The lipid data confirm and extend previous findings which showed that R. (C.) equi formed a well circumscribed taxospecies (5, 17,41,43,49). Similarly, the fatty acid and polar lipid results lend further support to the view (15) that Corynebacterium equi Magnusson (31) be considered as a subjective synonym of Rhodococcus equi (Magnusson) Goodfellow and Alderson 1980 (46).

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The mycolic acids extracted from the R. equi strains exhibited carbon ranges from CZ4 to C48 , 0 to 4 double bonds and released major amounts of C 14 : 0 esters on pyrolysis. These results give weight to those from previous investigations that were based on one or a few strains (9, 57). It is, however, not possible to make a clear distinction between rhodococci and corynebacteria solely on mycolic acid data. Mycolic acids from corynebacteria have carbon ranges from C22 to C38 , 0 to 2 double bonds, and release C8 : 0 to C 18 : 0 esters on pyrolysis (9, 36) whereas those from rhodococci fall within the range C34 to C64 , have 0 to 4 double bonds and yield C 12 : 0 to C 18 : 0 esters (1, 2,9,32). The R. equi strains gave qualitatively similar fatty acid profiles composed of straight-chain saturated, monounsaturated ,and 10-methylbranched fatty acids. The detection of major proportions of hexadecanoic and tuberculostearic acids, together with lesser amounts of the two lower homologues of the latter, is in line with previous results (6, 10,28). It was particularly interesting that all of the R. equi strains contained tuberculostearic acid as this compound is found in all rhodococci (6, 19,33) but not in corynebacteria (10,36), apart from C. ammoniagenes, C. bovis and C. variabilis (7, 10, 13,29). It is also interesting that R. equi strains clustered with rhodococci and not with corynebacteria in a quantitative analysis of fatty acid data (6). It is evident from this and earlier work (8, 11, 56) that R. equi strains contain dihydrogenated menaquinones with eight isoprene units as the predominant isoprenologue. Twelve of the remaining fifteen recognised species of Rhodococcus (16) are also characterised by the presence of major proportions of MK-8(Hz), the exceptions, R. bronchialis, R. rubropertinctus and R. terrae have MK-9(Hz) as the predominant isoprenologue (8, 11). It is not possible to distinguish rho do cocci from corynebacteria solely on the basis of menaquinone composition. Thus, most corynebacteria from animal sources contain major proportions of MK-8(Hz) but C. bovis, C. glutamicum and C. variabilis have MK-9(Hz) as the predominant isoprenologue (11, 19,33). Polar lipid patterns of nocardioform actinomycetes typically contain diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides and, with the exception of corynebacteria, phosphatidylethanolamine (27, 29, 35). It was, therefore, particularly interesting that all of the R. equi strains contained phosphatidylethanolamine in addition to the components common to all nocardioform actinomycetes. It should be noted, however, that phosphatidylethanolamine may not always be a reliable taxonomic marker as proportions of this compound have been found to vary dramatically in extracts of mycolic acid-containing actinomycetes (35). The R. equi strains also contained characteristic glycolipids including a trehalose dimycolate "cord-factor" like compound that was especially pronounced in freshly isolated strains. The polar lipid pattern of R. equi strains is distinctive and serves to distinguish these organisms from both Corynebacterium and Rhodococcus species. examined to date (33,35,36). The present study also showed that the remaining test strains had lipid profiles consistent with their assignment to the genus Rhodococcus. However, further comparative studies are required to determine the taxonomic status of most of these organisms. It is, however, clear from DNA homology experiments (58) that the strains forming cluster 1L (20) should be classified as R. sputi. This species, which was proposed by Tsukamura (51) and redescribed by Tsukamura and Yano (52) is recognised in the current edition of Bergey's Manual of Systematic Bacteriology (16). It has been reported to cause mesenteric lymphadenitis of swine (53). Rhodococci have been assigned to two aggregate taxa using chemical and physiological data (16, 22). Members of the species originally classified in the genus Cardona

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(50), namely R. bronchia lis, R. rubropertinctus and R. terrae, have mycolic acids with between 48 and 66 carbon atoms, contain major proportions of dihydrogenated menaquinones with nine isoprene units and produce mycobactins whereas the remaining species, including R. equi, have shorter chain mycolic acids (34-52 carbons), form dihydrogenated menaquinones with eight isoprene units as the predominant isoprenologue but fail to synthesise mycobactins even under conditions of strictly iron-limited growth. The two groups have also been recognised on the basis of delayed skin test reactions on sensitised guinea pigs and by polyacrylamide gel electrophoresis of bacterial extracts (25). The two aggregate rhodococcal groups have been given generic status by Stackebrandt et al. (47). The genus Rhodococcus has been retained for R. rhodochrous, R. equi and related species and the genus Gordona reintroduced for species that originally bore that name (50). The species assigned to the redefined genus are well circumscribed (16,58) but simple and reliable tests are still needed for the identification of unknown rhodococci. The well characterised strains of R. equi examined in this and earlier studies (5, 20, 21, 40, 43) should be of value in the search and discovery of tests that can be confidently weighted for the identification of Rhodococcus species.

Acknowledgements. Mass spectrometry was carried out by P. Kelly and S. H. Addison. References

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classification of coryneform and nocardioform bacteria and related organisms. J. gen. app!. Microbio!. 22 (1976) 203-214 57. Yano, /., /. Tomiyasu, K. Kaneda, and S. Imaizumi: GC/MS analysis of mycolic acid molecular species and contribution to the chemotaxonomy of new Rhodococcus species. In: Biological, Biochemical and Biomedical Aspects of Actinomycetes, pp. 567-570, Eds. G. Szabo, S. Biro, and M. Goodfellow. Akademiai Kiado, Budapest (1986) 58. Zakrzewska-Czerwinska, J., M. Mordarski, and M. Goodfellow: DNA base composition and homology values in the classification of some Rhodococcus species. J. gen. Microbio!. 134 (1988) 2807-2813 59. Zink, M. c., J. A. Yager, and W. L. Smart: Corynebacterium equi infection in horses, 1958-1984: a review of 131 cases. Can. Vet. J. 27 (1986) 213-217 Dr. M. Goodfellow, Department of Microbiology, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.