Polyphasic Taxonomic Study of Clinically Significant Actinomadurae Including the Description of Actinomadura latina sp. nov.

Zbl. Bakt. 285, 212-233 (1997) © Gustav Fischer Verlag, Jena

Polyphasic Taxonomic Study of Clinically Significant Actinomadurae Including the Description of Actinomadura latina sp. nov. M. E. TRUJILLO and M. GOODFELLOW Department of Microbiology, The Medical School, Newcastle-upon-Tyne, UK

Summary Thirty-one strains received either as Actinomadura madurae or Actinomadura pelletieri were assigned to four phena, clusters 1 to 4, in a numerical phenetic survey of the genus Actinomadura. Clusters 2 and 4 corresponded to the validly described species A. madurae and A. pelletieri whereas clusters 1 and 3 encompassed strains received as A. madurae and A. pelletieri, respectively. The two clusters that contained A. madurae strains formed a single taxon when a dataset lacking the antibiotic sensitivity entries was examined. Results from pyrolysis mass spectrometric and DNA amplification fingerprinting analyses underpinned the taxonomic status of clusters 2, 3 and 4 and suggested that the A. madurae strains accounted for a relatively wide range of variation. It is proposed that the cluster 3 strains be given species status within the genus Actinomadura given the congruence found between the chemical, molecular and numerical phenetic data. The name Actinomadura latina is proposed for the new taxon. The type strain is DSM 43382.

Introduction The taxonomy of the genus Actinomadura (26) has undergone marked revision given the application of chemical (9, 25, 30), numerical phenetic (1, 11, 12, 14,30) and molecular systematic methods (9, 25, 31). The genus is now well defined and encompasses twenty-six validly described species (25), the members of which characteristically form non-fragmenting, extensively branched substrate mycelia and aerial hyphae that carry up to fifteen arthrospores. Actinomadurae have meso-diaminopimelic acid as the major diamino acid of the wall peptidoglycan, which is of the A1 y type, contain major proportions of hexahydrogenated menaquinones with nine isoprene units saturated at sites II, III and VIII, diphosphatidylglycerol and phosphatidylinositol as major phospholipids, and complex mixtures of fatty acids with hexadecanoic, 14-methylpentadecanoic and 10-methyloctadecanoic acids predominating (9, 25). Most Actinomadura species have been defined using a small number of biochemical and morphological properties with little attempt made to confirm their taxonomic status. However, there is evidence from DNA relatedness and numerical taxonomic studies that Actinomadura madurae and Actinomadura pelletieri, causal agents of actinomycete mycetoma, are heterogeneous (1, 9, 11, 12, 17). Actinomadura madurae has been reported to be responsible for non-mycetomic infections (44), including one

Taxonomy of clinically significant actinomadurae

213

involving an immunocompromised patient (29). It is becoming increasingly more important to clarify the taxonomy of clinically significant actinomadurae. The application of numerical taxonomic methods has led to significant improvements in bacterial classification (13). The main contribution has been the circumscription of homogeneous clusters that can be equated with taxospecies. It is essential to evaluate the status of numerically defined taxa using independent taxonomic criteria as similarities between strains can be distorted by factors such as the choice and number of strains and tests, test reproducibility and the selection of data handling techniques (32). Several rapid chemical and molecular fingerprinting methods are available for this purpose, including Curie-point pyrolysis mass spectrometric (15) and amplification fragment length polymorphism (AFLP) techniques (3). The speed and reproducibility of pyrolysis mass spectrometry (PyMS) and its applicability to a wide range of bacteria make it an attractive method for evaluating the integrity of taxa circumscribed using conventional taxonomic criteria. In general, good agreement has been found between classifications derived using PyMS and more standard procedures, for example, in comparative studies on Bacteroides (6), Corynebacterium (20), Fusobacterium (27) and Streptococcus (23, 42). There is evidence that the technique provides a quick and effective way of checking the taxonomic status of numerically circumscribed species of Streptomyces (15, 33). It is also encouraging that PyMS and DNA pairing experiments give similar patterns of relatedness (10, 33). Amplification fragment length polymorphism analyses involve the enzymatic amplification of template DNA directed by one or more arbitrary oligonucleotide primers to produce a characteristic spectrum of products, some of which may be polymorphic. The procedure is rapid, independent of prior genetic and biochemical knowledge of the test strains and also allows tailoring of the number of resultant products and polymorphisms. Several variations of the method have been developed (4, 39, 40). The AFLP procedure introduced by Caetano-AnolUs et al. (4), termed DNA amplification fingerprinting (DAF), offers the most advantages, and provides the highest resolution of fingerprint products. Depending on the amplification parameters, the DAF technique can be used to generate a complex but characteristic spectrum of products, many of which are polymorphic (5). The more complex patterns are well suited for DNA fingerprinting. The present investigation was designed to clarify the taxonomy of representative A. madurae and A. pelletieri strains. Members of these taxa were examined using standard numerical taxonomic procedures with representatives of the resultant clusters being the subject of AFLP and PyMS analyses.

Methods Numerical taxonomy. Thirty-one strains (Table 1), received either as A. madurae or A. pelletieri, were examined for 293 unit characters (Table 2) using procedures described in earlier studies (2, 14, 19). Inoculated plates were incubated at 30 DC for 21 days, apart from

tests designed to determine temperature requirements. The organisms were maintained on modified Bennett's agar (24) at room temperature and as glycerol suspensions (20%, v/v) at -20 DC. Nearly all of the characters existed in one of two mutually exclusive states and were scored plus (1) or minus (0). Quantitative multivariate characters, such as resistance to antibiotics and chemical inhibitors, were coded using the additive method of Sneath and Sokal (37). The coded data were examined using the CLUSTAN IC program (43) using the

214

M. E. Trujillo and M. Goodfellow

simple matching coefficient (SSM; 38), which includes positive and negative similarities, the Jaccard coefficient (SJ; 34), which takes into account positive matches only, and the pattern coefficient (Dp; 43), which allows for differences in growth rates, periods of incubation and similar factors that can distort similarity values (35). Clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) algorithm (37). Three strains (Table 1) were examined in duplicate and an estimate of test variance calculated (formula 15: 36), this was used to determine the average probability (p) of an erroneous test result (formula 4:36). Tests shown to have a high intra-operator test error were deleted from the final data matrix, as were the data on the duplicated strains and tests found to give all positive or all negative results. Pyrolysis mass spectrometry. The glycerol stock cultures were used to inoculate sterile nitrocellulose membrane filters placed over modified Bennett's agar (24) plates. The inoculated plates were incubated at 30 DC for 6 days, apart from the slow-growing A. pelletieri strains (A7, A8, A9, A13, A19\ A24, A35, A36, A169, A183, A185, A187, A246) which were incubated for 9 days. Single colonies were chosen to smear ferro-nickel alloy foils (Horizon Instruments Ltd., Heathfield, East Sussex, UK) using a sterilised plastic loop. The inoculated foils were inserted into pyrolysis tubes (Horizon Instruments) and the samples heated at 80 DC for 5 minutes. Each culture was analysed in triplicate. The samples were processed in a single batch on a Horizon Instruments RAPyD 400X pyrolysis mass spectrometer. Curie-point pyrolysis was at 530 DC for 2.4 seconds with a temperature rise time of 0.6 of a second. The inlet heater was set at 160 DC. The pyrolysate was ionised by a low energy (20eV) electron beam and separated in the quadrupole mass spectrometer at scanning intervals of 0.35 seconds. Integrated ion counts for each sample at unit mass intervals from 51 to 400 were recorded and stored on hard disk, together with total ion counts and the sample pyrolysis sequence, without background removal. Data analysis. Variation between spectra due to inoculum size was normalised by iterative re-normalisation (21) and individual masses ranked according to their characteristicity values (7) prior to principal component analysis (PCA). Principal components (PC's) accounting for less than 0.1 % of the total variance were discarded. Canonical variate analysis (CVA) was then performed to generate sample groups on the basis of the retained PC's taking into account the sets of triplicates (41). The data from the PC-CVA analyses were presented as Mahalanobis distances which were transformed to a percentage similarity matrix using the SG coefficient (18) and used to construct a dendrogram with the UPGMA algorithm (37). Comparative analyses were initially performed on all of the strains and subsequently on strains labelled A. madurae. Random primer DNA amplification. Glycerol stock cultures were used to inoculate sterile nitrocellulose membrane filters placed on modified Bennett's agar (24) plates. The inoculated plates were incubated at 30 DC for 10 days. Two colonies from each culture were suspended in 36 ilL ofTE buffer (pH 8.0) supplemented with proteinase K (50 Ilg mL-I, Sigma) and incubated at 55 DC for 15 minutes then transferred to a boiling bath for 5 minutes to complete the lysis. Debris was removed by centrifugation and the supernatant kept in ice until required for PCR amplification. The M13 forward 17-mer primer (5'-3') GTAAAACGACGGCCAGT (Cancer Research Unit, University of Newcastle-upon-Tyne, UK) was used for the amplification. Each PCR reaction (25 ilL comprised 1 X Taq reaction buffer (Hoefer, UK), dNTPs (200 mM each), primer (311M), Biotaq DNA polymerase (0.5 units, Hoefer, UK), and DNA template (5 ilL, about 20-25 ng). The reaction mix was overlaid with a drop of mineral oil and PCR amplification carried out in a HybAid Omnigene automated thermocycler (HybAid Limited, Teddington, Middlesex, UK) according to the following temperature profiles: 1 cycle at 94 DC (5 min) for initial denaturation; 4 cycles at 94 DC (1 min), 30 DC (2 min), 72 DC (2 min) followed by 30 cycles at 94 DC (1 min), 30 DC (2 min), 72 DC (2 min) and a final extension period at 72 DC for 5 minutes. Reproducibility was checked by analysing each sample three times on different days and by using control assays, in which the addition of template DNA or primer were omitted.

Taxonomy of clinically significant actinomadurae

215

Table 1. Description, source and history of strains assigned to clusters defined at the 85% SSM, UPGMA level Laboratory Number

Name on receipt

Source and strain history

Cluster 1 Actinomadura sp. A.madurae CBS 331.54 A38 A. madurae M.A. Gordon, State of New York Department of Health, AlA133 * bany, N. Y., USA, 291A (DSM 44028) A137 A. madurae DSM 44029 A. madurae D. Frey, Institute of Medical Research, Royal North Shore HosA165 pital, Crows Nest, NSW, Australia, RNSH 201; Dr. Plunkett (1965); actinomycetomic mycetoma (DSM 44030) A176 A. madurae R. Vanbreuseghem, Institute of Tropical Medicine, Antwerp, Belgium, RV 12875; IP 380 (DSM 44033) Cluster 2 Actinomadura madurae A16T NCTC 5654; J. T. Duncan (1938), mycetoma pedis (ATCC 19425, CCM 136, DSM 43067, IMRU 1190) A11* C Philpot, London School of Hygiene and Tropical Medicine, London, U. K., LSHTM 393 A17 NCTC 1070; J. T. Duncan (1934), madura foot (DSM 43236) A22, A25 E Mariat, Institut Pasteur, Paris, France, IP 725 (DSM 44021); IP 393 (DSM 44022) A30, A31, A32 EMariat, IP 703, mycetoma, Mexico (DSM 44023); IP 363; madura foot, Tunis (DSM 44024); IP 364; madura foot (DSM 43380)

Cluster 3 Actionmadura sp. A. pelletieri C. Philpot, 1065; E. C. Smith (1929), Nigeria, mycetoma of the

A10

arm (DSM 43382)

Al16

A. pelletieri D. Frey, RNHS 203; K. Murray, LSHTM 1067 (1964); myceto-

A122

A. pelletieri

A130 A167

A. pelletieri A.pelletieri

ma (DSM 46198) H. Prauser, Zentralinstitut fur Mikrobiologie und Experimentelle Therapie, lena, Germany, IMET 9592 (DSM 46199) M. A. Gordon, 295; N. E Conant D. Frey, RNSH 204; K. Murray, LSHTM 1068 (1964); mycetoma (DSM 46201)

Cluster 4 Actinomadura pelletieri A19 T NCTC 4162 (Nocardia pelletieri); J. T. Duncan (1933); E. C. Smith (1928), mycetoma of the arm (DSM 43383) C.Philpot, CP 377 (DSM 44038); 388S; 368; 388H (DSM A7, A8, A9, A13 44039) E Mariat, IP 385 (DSM 44040); IP 308 (DSM 43384); IP 381 A24, A35, A36* R. Vanbreuseghem, RV 7053; A. Gonzalez-Ochoa, Mexico 1185 A169 E Mariat, IP 389; M. Andre, mycetoma of the foot; IP 394, myA183, A185, A187 cetoma of the foot (DSM 46196); IP 729; Dr. Resilliot, mycetoma of the foot, Cameroon D. MacKenzie, London School of Hygiene and Tropical MediA246 cine, London, U. K., NCPF 1066, Aden (1946) TType strain; * Duplicated culture; Sources: ATCC; American Type Culture Collection, Rockville, Maryland, USA, CBS Centralbureau voor Schimmelcultures, Oosterstraat 1, Baarn, The Netherlands; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; NCTC, National Collection of Type Cultures, Central Public Health Laboratories, London, England, U. K., NCPF, Mycological Reference Laboratory, Bristol, England, U. K.

216

M. E. Trujillo and M. Goodfellow

Table 2. Distribution of positive characters to clusters defined at the 85% similarity level in the SSM, UPGMA analysis Character

Cluster Number of strains Degradation Tests Aesculin Arbutin Casein Chitin DNA Elastin Gelatin Hypoxanthine Pectin RNA Starch Testosterone Tributyrin Tyrosine Tween 20 Tween 40 Tween 60 Tween 80

1 5

8

3 5

4 13

100 100 100 o 0 25 40 40 50 80 100 40 50 o 12 38 60 80 0 20 25 62 100 80 88 100 88 100 100 100 88 60· 75

100 100 100 20 60 40 100 40 80 80 80 60 100 100 100 100 100 80

8 16 100 0 38 84 100 23 0 16 23 0 77 84 69 84 100 84

o

8 100 84

o

100 8

2

100 100 40

Enzyme Tests Substrates based on 7-amino-4-methylcoumarin (7AMC) • Endopeptidase substrates L-Alanine-L-alanine-L-phenylalanine-7-AM C 40 Acetyl-L-alanine-L-alanine-L-tyrosine-7-AM C l O D Methoxysuccinyl-L-alanine-L-phenylalanine-L-lysine-7-AMC 100 L-Arginine-7-AMC 40 Glutaryl-L-phenylalanine-7-AMC 100 Benzyloxycarbonyl-glycine-L-proline-7-AMC 20 100 t-Butyloxycarbonyl-L-valine-glycine-L-arginine-7-AMC • Exopeptidases ~-Alanine-7-AMC

D-Alanine-7-AMC L-Alanine-7-AMC L-Asparagine-7-AMC Glycine-7-AMC (HBr)

80 100 60 100 100

12 88 100 38 100 12 100 50 100 25 100 75

100 100 20 20 100

100 100 60 100 80

8

92

100 100 23 92

30

Taxonomy of clinically significant actinomadurae

217

Table 2. Continued Character

'" ~

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~

~

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:!

<:s ~

ci. Vl

~

~

~

~

~

ci. Vl

:!

:!

<:s ~

<:s ~ 0

~

~<:s

~<:s

0

0 l:!

~

0

~

,:; ....

,:; ....

,:; ....

'';::

~

~

~

~

'"

Cluster Number of strains

'"

~

'"

'"

'"

1 5

2 8

3 5

4 13

80 60 60 40

100 88

92

12

100 100 20 20

100

88

20

38

100 100

100 88

20 60

100 18

12

4-Mu-~-D-Xyloside

80 100 100 20 40 100 80 100 100 100 100 100 100 100 100

50 25 100 100 0 0 100 100 100 100 100 75 100 100 100 100

0 0 100 100 100 0 0 0 100 100 100 0 80 20 0 100 100

8 0 8 0 0 0 54 0 0 0 0 8 0 82 0

• Inorganic ester 4-MU-Sulphate

100

100

0

0

• Organic esters 4-MU-Eicosanoate 4-MU-Heptanoate 4-MU-Lignocerate 4-MU-Myristate 4-MU-Nonanoate

60 100 0 60 100

25 100 12 38 88

0 80 0 20 100

0 38 0 0 77

L-Isoleucine-7 -AM C L-Pyroglutamate-7-AMC L-Serine-7-AMC L-Valine-7-AMC • Miscellaneous Haloxon

12

8 16 16

Substrates based on 4-methylumbelliferone (4MU) • Glycosides 4-Mu -2-Acetamido- 2-deoxy-~- D-galactopyranoside 4-MU-2-Acetamido-2-deoxy-~-D-glucopyranoside 4-MU-2-Acetamido-4,6,O-benzylidine-7-deoxy-~-D-

glucopyranoside 4-MU-N -Acetyl-~- D-galactosaminide 4-MU-a-L-Arabinofuranoside 4-MU-a-L-Arabinopyranoside 4-MU -~- D-Cellobiopyranoside 4-MU -~- L-Fucopyranoside 4-MU-a-D-Galactoside 4-MU -~- D-Galactoside 4-MU-a-D-Glucoside 4-MU+D-Glucoside 4-MU -~- D-Glucuronide 4- MU-~- D-Lactoside 4- MU-~- D-Maltoside 4-MU-a-D-Mannopyranoside 4- MU-~- D-Mannopyranoside 4-MU-~-D-Ribofuranoside

0 0

8 92

218

M. E. Trujillo and M. Goodfellow

Table 2. Continued

0;:: 0'';"::

l::;:!'"

Character

~

0.. 00

0.. 00

l::;:!

l::;:!

!i:

l:: ~':!

~

~

!i:

!i:

!i:

0:; .....

0:; .....

0:; .....

':!

0

\.l

~

'"

~

l:: ~':!

':!

0

!i:

0

\.l

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Cluster Number of strains

':!

0

0';::;::

\.l

-«:

\.l

-«:

-«:

1 5

2 8

3 5

4 13

100 20 80 100 40 100

100 50 50 100 75

80 0 0 100 0 60

8 8 8 84 0 23

0

12

0

0

100 0 0 0

25 0 62

12

40 20 0 40

8 54 8 38

• Spores Present

0

0

20

8

• Spore arrangement Hooks Straight

0 0

0 0

20 0

0 8

Monohydric Methanol Propanol

40 20

0 0

0 0

0 0

Polyhydric Glycerol

100

100

100

16

4-MU-Oleate 4-MU-Palmitate 4-MU-Pentadecanoate 4-MU-Methyl-ethyl-acetate 4-MU-Methyl-ethyl-octanoate 4-MU-Methyl-ethyl-stearate • Miscellaneous 7-Ethoxycoumarin-4-MU

12

Morphological Tests • Substrate mycelium colour Grey/olive-grey Pink/orange Red/purple White/cream/yellow Micromorphology

Nutritional Tests Sole carbon and energy sources (1 %, w/v) • Alcohols

• Carbohydrates

Taxonomy of clinically significant actinomadurae

219

Table 2. Continued .>::

:::;::'"

Character

ci.

;:

'"I:l

'"I:l ~

;:

.:; ..,

.:; ..,

0

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;: 0 .:; .., «:"

0

«:"

~

'"I:l

~

;:

'"

:::;::'"

:::;::

:::;::

~

ci.

~

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Cluster Number of strains

..,

.~

'"I:l

«:"

0

.:; ..,

«:"

1 5

2 8

3 5

4 13

100 60 100

100 0 100

100 60 100

8 0 18

100 100 100

100 100 100

100 100 60

0 8 0

100

100

100

16

100 60 100 100

100 0 88 100

100 40 100 100

82 23 31 59

D(+)-Melezitose D( +)-Raffinose

60 0

12 12

100 0

0 0

Polysaccharide Glycogen

100

100

80

82

0 20

12 0

40 20

0 8

80

88

0

0

100

100

100

8

20

25

40

0

Monosaccharides Pentoses L( +)-Arabinose D(-)-Ribose D(+)-Xylose Hexoses D(-)-Fructose D( +)-Galactose D(+)-Mannose Deoxy-hexose L(+)-Rhamnose Disaccharides D( +)-Cellobiose Lactose Maltose D(+)-Trehalose Trisaccharides

Glycosides D( +)-Glucosamine Salicin Suger alcohols Pentitol Adonitol Hexitol Mannitol Sole carbon and energy sources (0.1 %, w/v) • Amino acids L-Arginine

220

M. E. Trujillo and M. Goodfellow

Table 2. Continued .;:

'"

Character

...

~

ci..
~

:i:

I:S

~

~

~

.-...

I:S

~

.-...

...

.5

--(

--(

--(

0

~

\l

~

~

.5

0

L-Glutamine L-Glycine L-Histidine L-Lysine L-Methionine L-Phenylalanine L-Valine

~

~I:S

"I:l

~ .-:... '"

ci..
~

~

Cluster Number of strains

.~

~I:S

I:S

~

0

0

...

\l

~

\l

\l

--(

1 5

2 8

3 5

4 13

40 20 20 20 0 40 80

88 38 75 0 0 75 75

20 40 0 0 20 100 0

0 16 59 0 0 59 8

20

0

0

0

40 80

50 62

40 0

0 16

20

12

0

0

20

0

20

0

40 0 0 100 0 20 0 0 20 0 100 60 20 80 20 0

25 25 0 100 25 88 0 0

60 0 0 100 0 100 0 20 60 0 100 60 20 100 60 0

32 0 8 69 0 31 8 31 23 23 31 0 8 69 69 31

• Carboxylic acids

Aromatic acid

Sodium hippurate

Aliphatic acids

Lactic acid Sodium propionate

Hydroxy acid

Sodium citrate

Keto acid

Sodium pyruvate Physiological Tests • Growth in the presence of chemical inhibitors (%, w/v) 4 Adenine Crystal violet 0.00002 0.0001 0.01 Phenol 0.1 Phenyl ethanol (%, v/v) 0.1 0.2 0.005 Potassium tellurite 0.01 0.02 Sodium azide 0.01 0.02 Sodium chloride 4 0.01 Teepol (%, v/v) 0.001 Tetrazolium 0.01

12

0

12 0 12 100 50 0

Taxonomy of clinically significant actinomadurae

221

Table 2. Continued Character

.;::

~

...

l::

~

ell

;:

ci,

l::

l::

l::~

ci,

;:

;:

0

~

;:

;: 0

...

0

;:

0:;

..:::"

l::~

~

~

0

.:; ...

<:>..

~

~

~

~~

ell

-B

-B

Cluster Number of strains

.~

-B

;:

0';::

..:::"

0';::

..:::"

..:::"

1 5

2 8

3 5

4 13

60 0 0

62 12 12

100 20 0

77 8 8

pH 5.5 9.0 10.0

0 80 0

0 25 12

40 60 0

0 8 0

Temperature lOoC 20°C 25°C 3rC 45°C

60 80 80 80 0

62 100 100 100 0

100 100 100 100 0

16 38 85 92 69

2 4

0 0

12 0

0 0

16 8

20 30 35

0 0 0

0 38 0

0 0 0

16 8 8

5 10 15 25 90 100 120 20 30

40 20 0 0 0 0 0 40 0

100 88 75 88 88 38 75 75 12

40 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

Thallous acetate

0.0001 0.001 0.005

• Growth at:

• Resistance to antibiotics (f.lg mL-1 ) Aminoglycosides Streptomycin sulphate

~-Lactams

Cephalosporins Cephalotin

Penicillins Ampicillin Benzylpenicillin Carbenicillin Cefamandole

222

M. E. Trujillo and M. Goodfellow

Table 2. Continued Character

':!

ci.

~

'"

~

~

~

....;:s

;:s

<;)

Oleandomycin phosphate Rifampicin Ticarcillin

40 10 25 20 2 6 10 70 75 80

':!

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-:;.... <;)

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~

~

~

;:

0

<;)

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;:s

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0

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':!

Cluster Number of strains

....

-~

~

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Lincomycin hydrochloride

-;::

';:s~"

0

<;)

1 5

2 8

5

3

4 13

0 0 0 0 80 80 60 20 20 20

12 12 0 12 88 75 62 100 100 100

0 60 20 0 100 100 60 0 0 0

0 0 0 0 16 0 0 16 16 16

All of the test strains cleaved succinyl-L-alanine-L-alanine-L-valine-7-AMC, benzyloxycarbonyl-L-arginine-7 -AM C, L-arginine-L-arginine-7 -AM C(H2 0), L-arginine-L-arginine-Lphenylalanine-7-AMC, glutaryl-glycine-glycine-L-phenylalanine-7-AMC, glycine-L-alanine-7-AMC, glycine-L-arginine-7-AMC (2.5 AcOH), glycine-L-proline-7-AMC (HCI), succinyl-L-leucine-L-tyrosine-7 -AMC, L-lysine-L-alanine-7 -AMC, N-a-acetyl-L-lysine-7AM C, N -acetyl-L-phenylalanine-L-arginine-7 -AM C, benzyloxycarbonyl-L-proline-L-arginine-7-AMC (HCI), trans-4-hydroxy-L-proline-7-AMC (endopeptidase substrates); Lglutamine-7-AMC (HCI), L-leucine-7-AMC, L-methionine-7-AMC, L-ornithine-7-AMC, L-phenylalanine-7-AMC, L-proline-7-AMC (HBr), L-tyrosine-7-AMC (exopeptidase substrates) and 4-MU-N-acetyl-~-D-glucopyranoside (glycoside substrate) 4-MU-phosphate, 4-MU-bis-phosphate (inorganic esters) and grew at pH 8.0 and in the presence of sodium azide (0.001 % w/v). Nearly all of the strains (2: 99%) cleaved L-citrulline-7-AMC, L-cysteine-benzylocarbonyl-7-AMC and L-glutamic acid-7-AMC (exopeptidase substrates). None of the strains cleaved 4-MU-butyrate, 4-MU-propionate (organic esters), 4-MU-~-D­ fucopyranoside, 4-MU-a-L-fucoside (glycoside substrates), 4-MU-acetamidohexanoate, 4MU-guanidinoaminobenzoate (miscellaneous); degraded xanthine or xylan, produced aerial hyphae, diffusible pigments, or spore chains; grew on melibiose, sorbitol, L(-)-sorbose, and xylitol (1 %, w/v) or L-asparagine, benzamide, butane-1,4,-diol, p-cresol, L-cystine, ethanolamine, L-leucine, sodium benzoate and sodium malonate, toluene, and trimethylamine oxide (0.1 %, w/v) as sole carbon sources or at pH 4.5,5.0,11.0 or 12.0, at 4°C, 50°C, 55°C or 60°C or in the presence of (%, w/v) crystal violet (0.0001), phenyl ethanol (0.3 and 0.4%, v/v), sodium chloride (7,13), testosterone (0.1), tetrazolium (0.1), thallous acetate (0.01), cephaloridine (2 and 10 f.lg mL-1 ), neomycin sulphate (3 and 7, f.lg mL-1 ), streptomycin sulphate (16 f.lg mL-1 ), tetracycline (20, 30 and 40 f.lg mL-1 ) or tobramycin sulphate (0.5, 1 and 5 f.lg mL- 1 ). Nearly all of the organisms (2:99%) were unable to cleave 4MU-7-ethoxycoumarin or use cholesterol, phenol, resorcinol and L-threonine as sole carbon sources for energy and growth.

Taxonomy of clinically significant actinomadurae

223

DNA amplification fragments were separated by polyacrylamide gel electrophoresis in NuSieve 3: 1 agarose (4%). Gels and running buffer were prepared in TBE buffer (pH 8.0). The PCR products (5 f.LL) were loaded with 1 f.LL of a gel loading solution (Sigma Chemical Co., Poole, Dorset, UK) and electrophoresis performed on a GNA-200 apparatus (Pharmacia LKB, Cambridge, UK) at 4 Vlcm; usually until the dye front reached about 1 em from the end of the gel. The band sizes were estimated by comparing with either
.

Results

Numerical classification Quality of final data matrix. Experimental test error was estimated from data collected on the duplicated cultures (see Table 1). The average probability (p) of an erroneous test result, calculated from the pooled variance (S? = 0.051) of the unit characters, was 5.4%. The duplicated strains showed a mean observed similarity of 89.2% SSM. Most tests were reliable and gave S? values below 0.150. The results for the rapid enzyme tests N-acetyl-7AMC, 4MU-a-L-iduronide, 4MU-laurate, and 4MU-pyrophosphate, resistance to benzylpenicillin (10, 20 Ilg mL- i ), demeclocycline (2,7 f.Lg mL-i), gentamycin (2,4,10 f.Lg mL-i), neomycin sulphate (1 f.Lg mL-i), oleandomycin phosphate (10 f.Lg mL- i ), and potassium tellurite (0.001 % w/v), and growth on a-alanine, L-citrulline, gluconic acid, L-glutamic acid, L-proline, L-serine, sodium acetate, sodium butyrate, sodium citrate, sodium succinate, sucrose, and tyrosine as sole carbon sources, were excluded from the numerical analyses as they were not reproducible. A further 105 unit characters were deleted from the raw data matrix as they showed little or no separation value (Table 2). The final data matrix, excluding the duplicated strains, contained information on 31 organisms and 160 unit characters. The cophenetic correlation values were 0.9107 (SSM, UPGMA), 0.9294 (SJ, UPGMA) and 0.7657 (Sp, UPGMA). A second numerical analysis was performed on the final data set minus the 36 entries for the antibiotic sensitivity tests. Clustering of strains using the Sf' Sp, and SSM coefficients and the UPGMA algorithm. The 31 strains received either as A. madurae or A. pelletieri were assigned to four clusters defined at or above the 85% similarity (S) level (Fig. 1). Clusters 2 and 4, which encompassed 8 and 13 strains, respectively were equated with the species A. madurae and A. pelletieri as they included the type strains of these taxa. Cluster 1 contained 5 strains received as A. madurae and cluster 3 the remaining 5 strains received as A. pelletieri. The four clusters were recovered in their entirety in the corresponding SJ and Sp, UPGMA analyses. Three clusters were recognised at the 83 % S-level in the corresponding analyses based on the reduced data set (Fig.2). High cophenetic correlation values were again

224

M. E. Trujillo and M. Goodfellow Strain number

Percentage similarity 65

75

85

95

Cluster Identity number

100

~~i~}

A133 A38 A25 A30 A32 A31 A22 A17 A16 T A11

~~~~

A167 A116 A10 A246 A187 A169 A24 A183 A35 A185 A19T A36 A9 A13

}

1

Actinomadura sp.

2

Actinomadura madurae

3

Actinomadura sp.

4

Actinomadura pelletier;

AS

A7 Fig. 1. Abridged dendrogram showing relationships between clusters defined at the 85% similarity level using the SSM coefficient and the UPGMA algorithm. T denotes clusters containing type strains. recorded, namely 0.9152 (SSM, UPGMA), 0.9334 (S1' UPGMA) and 0.7429 (Sp, UPGMA). Two of the original four numerically defined taxa, namely clusters 3 (Actinomadura sp.) and 4 (A. pelletieri) were recovered in their entirety, but clusters 1 (Actinomadura sp.) and 2 (A. madurae) formed a new taxon which encompassed all of the strains received as A. madurae. The same three clusters were defined in the corresponding SJ and Sp, UPGMA analyses. Actinomadura madurae A176 was found on the periphery of cluster 1 in the second analysis. Pyrolysis mass spectrometry Excellent agreement was found between the results of the triplicate analyses of each strain (results not shown). The 31 test strains were assigned to three homogeneous groups when the pyrolysis data were presented as a dendrogram derived from the Ma-

Taxonomy of clinically significant actinomadurae Strain number

Percentage similarity

75

85

95

~~~~ A167

A116 ' - - - - A10 A187 A246 L..-_ _ A24 A169 A36 A183 A185 A35 A19T A9 A13 A8 A7 85

95

Identity

100

A176 A38 A165 A137 A133 A30 A32 A31 A25 A22 A17 A16T A11

75

Cluster number

225

}

1&2

Actinomadura madurae

3

Actinomadura sp.

4

Actinomadura pelletieri

100

Fig.2. Abridged dendrogram showing relationships between clusters defined at the 83 % similarity level using the SSM coefficient, the UPGMA algorithm and the reduced data set.

halanobis distances (Fig. 3). Group 1 encompassed the 5 strains originally assigned to cluster 3 (Actinomadura sp.) together with A. madurae A176 from cluster 1 (Actinomadura sp.). The composition of the second group corresponded exactly with that of cluster 4 (A. pelletier;). The final group, contained all of the strains received as A. madurae, apart from strain A176. All but one of the group 3 strains were found in two subgroups, labelled 3A and 3B, when the data for these organisms were examined separately (Fig. 4). The exception, A. madurae A2S, was recovered as an outlier in an initial analysis of the group 3 strains.

Random primer DNA amplification The number of DNA bands obtained for the 27 test strains varied from 4 to 14 with the length of the products lying between 100 and 700 bp. In general, good congruence

226

M. E. Trujillo and M. Goodfellow Percentage similarity

60

70

80

90

Strain number

70

80

90

Identity

100

A122 A10 A167 A130 A116 A176 A169 A246 A30 A9 A36 A13 A8 A185 A24 A183 A19T A187 A7 A32 A31 A25 A30 A22 A165 A38 A17 A16 T A137 A133 A11 60

Group number

1

Actinomadura sp.

2

Actinomadura pelletieri

3

Actinomadura madurae

100

Fig. 3. Dendrogram showing relationships between the representative Actinomadura strains based on pyrolysis mass spectral data. The dendrogram is derived from similarity values based on Mahalanobis distances with clustering achieved using the UPGMA algorithm. was found between the results of the triplicate analyses though some minor variations were found particularly in the density of some of the bands. In all cases, several strongly amplified fragments were detected. Amplified products were not observed in the control assays where either the cells or primers were omitted. Most of the test strains were assigned to four groups both on visual inspection (Fig. 5) and by statistical analysis of spectra derived from scanning the gels (Fig. 6). The number of DNA bands generated by the 3 representative cluster 1 strains (Actinomadura sp.), that is the group 1 strains, varied from seven for A. madurae A133 to nine for A. madurae A176. All three strains showed 3 common bands of about 100 to 200 bp. Most of the group 2 strains (cluster 2; A. madurae) had up to

Taxonomy of clinically significant actinomadurae Percentage similarity

65

85

Strain number

Group number

227

Identity

100

A165 A137 3a

Actinomadura sp.

3b

Actinomadura madurae

A133 A38 A32 A31 A30 A22 A17 A16 T A11 65

85

100

Fig.4. Dendrogram representing relationships between representative Actinomadura madurae strains based on pyrolysis mass spectral data. See legend to Figure 3 for details. 2 bands of approximately 150 and 200 bp in common, one of which was also found in the group 1 strains. The number of bands for this group varied from four for A. madurae A17 to nine bands for A. madurae A31. The bands were within the range 100 to 500 bp. Actinomadura madurae A30 was recovered as a single membered group although this strain shared two common bands with the remaining A. madurae strains (cluster 2). Most of the A. pelletieri strains of group 4 (= cluster 4) showed a characteristic banding pattern. The members of this group generated the highest number of bands ranging from seven for strain A. pelletieri A169 up to fifteen for A. pelletieri strains A36 and A246 with sizes ranging from 70 to 500 bp. Five bright bands seen in the region of 100 to 200 bp were found on all eleven strains. The group 3 strains (= cluster 3; Actinomadura sp.) also showed a characteristic pattern. At least seven bands were 15

Zbl. Bakt. 285/2

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M. E. Trujillo and M. Goodfellow

Taxonomy of clinically significant actinomadurae

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found in each DAF profile though variation in the intensity of the bands was observed. Three of the strains, namely Actinomadura sp. A116, A122 and A130, gave an extra band of approximately 500 bp. Actinomadura pelletieri A167 from cluster 3 (A. pelletieri) was recovered with the group 4 strains; this organism did not have the extra band of around 500 bp that was found in Actinomadura strains Al16, A122 and A130.

Percentage similarity

Strain number

~g~} A165 A133 A32 A25 A31 A11 A17 A22 A16T

~~~2} A130 A116 A246 A8 A13 A183 A36 A19 T A185 A167 A7 A169 A9 A30

Cluster number

Identity

Group

1

Actinomadura sp.

1

2

Actinomadura madurae

2

3

Actinomadura sp.

3

4

Actinomadura pelletieri

4

Actinomadura madurae

Fig.6. Dendrogram showing relationships between representative Actinomadura strains based on DNA amplification fingerprint data. The similarity between the strains was calculated by comparing the spectral traces, derived from the electrophoretic DNA profiles, using the correlation coefficient (r) with clustering achieved using the UPGMA algorithm.

... Fig. 5. DNA amplification fingerprints of Actinomadura strains analysed using the primer M-13; Sa, representatives of clusters 1 and 2; Sb, representatives of clusters 3 and 4.

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M. E. Trujillo and M. Goodfellow

Discussion Our findings confirm and extend those from previous studies which suggested that clinically significant actinomycetes might be underspeciated (1, 9, 11, 12). In the present investigation, representative A. madurae and A. pelletieri strains were assigned to four numerically defined clusters that were well separated from additional clusters equated with validly described species of Actinomadura. Two of the four clusters corresponded to A. madurae and A. pelletieri, the remaining two, clusters 1 and 3, contained isolates received as A. madurae and A. pelletieri, respectively. The separation of clusters 1 and 2, each of which encompassed the strains received as A. madurae, may be more apparent than real as the constituent organisms were recovered in a single cluster when the numerical analyses were repeated on the reduced data set. The separation of the A. madurae strains into two closely related taxa in the earlier analyses could be due to the discontinuous distribution of plasmids; it has been shown that plasmid mediated characters can distort relationships between otherwise related strains (16). In contrast, the separation of the strains received as A. pelletieri into two distinct clusters was strongly supported by the results of the numerical phenetic analyses on the reduced data set. The taxonomic status of the phena corresponding to A. madurae (cluster 2), A. pelletieri (cluster 4) and Actinomadura sp. (cluster 3) is strongly supported by the PyMS and DNA amplification fingerprinting data. The congruence found between the numerical phenetic and PyMS data is in good agreement with the findings from earlier comparative studies (15, 33, 42) and thereby provides further evidence that pyrolysis mass spectrometry provides a rapid and effective way of evaluating the integrity of numerically defined taxospecies. Similarly, the good agreement found between the numerical taxonomic and DAF data provides further evidence that random primer DNA amplification can be used to evaluate the status of taxospecies (8,23,28). It is also interesting that both the PyMS and DAF data indicated that the A. madurae strains encompass a wide range of variation. Further comparative taxonomic studies, including DNA relatedness experiments, are needed to resolve the taxonomic status of A. madurae. It is evident from the numerical taxonomic, DAF and PyMS data that cluster 3 (Actinomadura sp.) forms a taxon equivalent in rank to the phena equated with A. madurae and A. pelletieri. It is also clear that the cluster 3 strains can readily be distinguished from representatives of A. madurae and A. pelletieri by several phenotypic properties based on degradative, enzymatic, nutritional and physiological tests. The separation of strains received as A. pelletieri into two distinct clusters is also supported by chemotaxonomic and DNA relatedness data (9). Although few strains are common to the present study and that of Fischer et al. (9) it is clear that A. pelletieri A1D (cluster 3) shows little DNA relatedness with authentic representatives of A. madurae and A. pelletieri. It is also clear from both of these studies that strain A1D was wrongly assigned to the cluster equated with A. pelletieri by Athalye et al. (2). It is evident from the genotypic and phenotypic data that the cluster 3 strains merit species status within the genus Actinomadura (26) as emended by Kroppenstedt et al. (25). Description of Actinomadura latina Trujillo and Goodfellow sp. nov. lao ti' na. L. fem. adj. latina Latin, ex America Latina, since many clinically significant strains of actinomadurae have been isolated in Latin America. The description is taken from the present and earlier studies (2,9). Colonies cream to pink, convex and wrinkled. Well developed non-fragmenting substrate mycelium. Aerial mycelium absent or rare, no diffusible pigments produced. De-

Taxonomy of clinically significant actinomadurae

231

grades aesculin, arbutin, casein, gelatin, tributyrin, tyrosine, Tweens 20, 40 and 60; cleaves the conjugated fluorogenic substrates acetyl-L-alanine-L-alanine-L-tyrosine7AMC, methoxysuccinyl-L-alanine-L-phenylalanine-L-Iysine7AMC, tertbutyloxycarbonyl-L-valine-glycine-L-arginine-7AMC (endopeptidase substrates); ~-alanine7AMC, D-alanine-7AMC, L-asparagine-7AMC, L-isoleucine-7AMC, L-pyroglutamate-7AMC (exopeptidase substrates); 4MU-a-L-arabinofuranoside, 4MU-a-L-arabinopyranoside, 4 MU-~- D-cello biopyranoside, 4 MU-a-D-glucoside, 4 MU-~- D-glucoside, 4MU-~-D-ribofuranoside, 4MU-~-D-xyloside (glycoside substrates); 4MU-methylacetyl-acetate, 4MU-nonanoate (organic esters); utilises L-arabinose, D-cellobiose, Dfructose, D-galactose, glycerol, maltose, mannitol, D-melezitose, L-phenylalanine, Lrhamnose, D-trehalose, and D-xylose as sole carbon sources. Tolerant to phenol (0.01 %, w/v), phenyl ethanol (0.1 %, v/v), sodium azide, (0.01 %, w/v), teepol (0.01 %, v/v), thallous acetate (0.0001 %, w/v); resistant to rifampicin (2, 6 flg mL-I ). Temperature range for growth: 10 to 3rc. The cell wall contains meso-2-6-diaminopimelic acid as the diamino acid. The principal wall sugars are arabinose, glucose, madurose and ribose. The organism is rich in straight-chain saturated fatty acids, with hexadecanoic, heptadecanoic and pentadecanoic acids predominating. Menaquinones are predominantly of the MK-9(H6) type with lesser proportions of MK-9(Hs) and MK-9(~) also present. The mol%, G + C composition of the DNA of the type strain is 67%. Causes actinomycete mycetoma. Type strain: Actinomadura latina AI0 (DSM 43382) Acknowledgements. The authors are indebted to Mr. C. S. Hetherington and Dr. G. P. Manfio for help with the PyMS analyses, to Dr. T. O. MacAdoo for checking the etymology of the epithet latina and to those who provided the test strains (Table 1). M. E. T. was supported by a grant from Consejo Nacional de Ciencia y Tecnologia (CONACYT, MEXICO).

References

1. Alderson, G. and M. Goodfellow: Classification and identification of actinomycetes causing mycetoma. Post. Hig. I Med. D6sw. 44 (1979) 109-124 2. Athalye, M., M. Goodfellow, J. Lacey, and R. P. White: Numerical classification of Actinomadura and Nocardiopsis. Int. J. Syst. Bacteriol. 35 (1985) 86-98 3. Bassam, B.]., G. Caetano-Anolles, and P. M. Gresshoff: DNA amplification fingerprinting of bacteria. Appl. Microbiol. Biotechnol. 38 (1992) 70-76 4. Caetano-A no lies, G., R. J. Bassam, and P. M. Gresshoff: DNA amplification using very short arbitrary oligonucleotide primers. BiolTechnol. 9 (1991) 553-557 5. Caetano-Anolles, G., R.J. Bassam, and P. M. Gresshoff: DNA amplification fingerprinting: A strategy for genome analysis. Plant Mol. BioI. Rep. 9 (1991) 292-305 6. Duerden, B. I., A. Eley, L. Goodwin, J. T. Magee, J. M. Hindmarch, and K. W. Bennett: A comparison of Bacteroides ureolyticus isolates from different clinical sources. J. Med. Microbiol. 29 (1989) 63-73 7. Eshuis, w., P. G. Kistemaker, and H. L. C. Meuzelaar: Some numerical aspects of reproducibility and specificity. In: Analytical pyrolysis, pp.151-166, eds. C. E. R. Jones and C. A. Cramers. Elsevier, Amsterdam (1977) 8. Fekete, A., J. A. Bantle, S. M. Halling, and R. W. Stich: Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers. J. Bacteriol. 174 (1992) 7778-7783

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