International Journal of Systematic and Evolutionary Microbiology (2003), 53, 995–997 DOI 10.1099/ijs.0.

02372-0

Reclassification of ATCC 9341 from Micrococcus

luteus to Kocuria rhizophila
Jane S. Tang1 and Patrick M. Gillevet2
Correspondence American Type Culture Collection1 and George Mason University2, Manassas, VA 20110, USA
Jane S. Tang
jtang@[Link]
Strain ATCC 9341, currently known as Micrococcus luteus, has been designated as a
quality-control strain in a number of applications. It is also cited as the standard culture in
several official methods and manuals, as well as the Code of Federal Regulations. Over the years, it
has become apparent that ATCC 9341 does not resemble other M. luteus strains; however,
its phenotypic characteristics alone were ambiguous. Recently, a polyphasic study was performed
in which molecular data were combined with cytochemical properties and physiological
characteristics. The results clearly indicate that ATCC 9341 is a member of the genus Kocuria.
Thus, it is proposed to reclassify ATCC 9341 as Kocuria rhizophila and to alert users worldwide of
this name change.

Strain ATCC 9341, currently known as Micrococcus luteus (ATCC 15306T) on the RiboPrinter (Qualicon) to analyse
(FDA strain PCI 1001), has been designated as a quality- their DNA fingerprinting patterns. This automated mol-
control strain in a number of applications. The Code of ecular biology workstation performs a Southern hybridiza-
Federal Regulations and the US Pharmacopeia list it as tion using the bacterial rRNA genes as probes after the
a control strain for sterility testing (US National Archives genomic DNA has been digested with EcoRI. The riboprint
and Records Administration, 1997c; US Pharmacopeial from ATCC 9341 did not match either of the fingerprints
Convention, 1995b). The Association of Official Analytical from M. luteus or K. varians (Fig. 1).
Chemists (AOAC) Official Methods, the US Pharmacopeia
and the European Pharmacopoeia use this culture to assay To assess the relationship between the strains in question, we
a variety of antibiotics and fungicide residues (AACC, compared the gene sequences of their 16S rRNA.
1983; AOAC, 1995; British Pharmacopoeia Commission, Amplification of the 16S rRNA genes was accomplished
1993; EDQM, 1997; Huang & English, 1966; Ragheb & by using primers L27F and L1492R (Lane, 1991) and the
Smallidge, 2000a–h; US National Archives and Records PCR products were purified and sequenced. The 16S rRNA
Administration, 1997a; US Pharmacopeial Convention, gene sequence fragments for each strain were assembled by
1995a, c). Furthermore, the Code of Federal Regulations using Sequencher (Gene Codes Corp.) and the consensus
also includes this strain for doxycycline, tetracycline and sequence for each strain was determined. The consensus
chloramphenicol susceptibility disc testing (US National sequence of each strain had at least twofold coverage in
Archives and Records Administration, 1997b). each direction and was used to perform a BLAST search of
GenBank. The 16S rRNA gene sequences from closely
Strain ATCC 9341 was deposited as Sarcina lutea by the US related genera were downloaded and aligned using CLUSTAL
Food and Drug Administration over 40 years ago. Although X (Thompson et al., 1997).
the name was changed to M. luteus in 1977, based on the 8th
edition of Bergey’s Manual of Determinative Bacteriology
(Baird-Parker, 1974), its characteristics were not identical to
those of typical M. luteus strains. Table 1 shows some Table 1. Phenotypic and chemotaxonomic comparison
between strains ATCC 9341 and M. luteus ATCC 4698T
characteristics of ATCC 9341 when compared with ATCC
4698T, the type strain of M. luteus. Character ATCC 9341 ATCC 4698T
During the past few years, users have alerted us that this Oxidase 2 +
culture was identified as Micrococcus varians (now Kocuria Simmons’ citrate + 2
varians) by using the bioMérieux ID 32 Staph kit as well as Acid production from:
the Biolog GP MicroPlate. We ran this strain along with the Glucose + 2
type strains of M. luteus (ATCC 4698T) and K. varians Fructose + 2
Major fatty acids ai-C17 : 0, ai-C15 : 0, ai-C15 : 0, i-C15 : 0
The GenBank/EMBL/DDBJ accession number for the 16S rDNA i-C15 : 0
sequence of strain ATCC 9341 is AF542072.

02372 G 2003 IUMS Printed in Great Britain 995

J. S. Tang and P. M. Gillevet

Arthrobacter separated the Microccocus clade from the

M. luteus Kocuria clade, indicating that these two clades are indeed
ATCC 4698T
separate genera.

In addition to 16S rRNA sequencing, DNA–DNA hybrid-

ization experiments were conducted between ATCC 9341
M. luteus
and the type strains of M. luteus (ATCC 4698T), K. varians
ATCC 9341
(ATCC 15306T) and K. rhizophila (BAA-50T). The results
showed 42?3, 54?7 and 67?6 % similarity with each res-
pective type strain. Thus, it further confirmed that ATCC
9341 does not belong to the genus Micrococcus. Although the
K. varians similarity to K. rhizophila did not quite reach the threshold
ATCC 15306T
of 70 % recommended by Wayne et al. (1987), it definitely
indicated close affiliation with this species. The DNA
fingerprinting pattern of ATCC 9341 did not match exactly
those generated by ATCC BAA-50T, but there was definitely
K. rhizophila
ATCC BAA-50T more similarity to K. rhizophila than to the type strain of
M. luteus (Fig. 1).

Finally, a comparison of phenotypic traits, fatty acid methyl

ester analysis and riboprint patterns between ATCC 9341
Fig. 1. Riboprint pattern of ATCC 9341 compared with and K. rhizophila ATCC BAA-50T also showed agreement
those of M. luteus ATCC 4698T, K. varians ATCC 15306T and with the sequencing results. Both strains grew on Simmons’
K. rhizophila ATCC BAA-50T. citrate and produced acid from glucose and fructose. The
major fatty acids produced by ATCC 9341 were ai-C17 : 0,
ai-C15 : 0 and i-C15 : 0, which are indicative of the genus
Parsimony analyses were conducted using PAUP 4.0b8a Kocuria (Kovács et al., 1999). Furthermore, ATCC 9341 has
(Swofford, 2001) and the data matrix consisted of 1343 base type A11.6 as the peptidoglycan structure, which is identical
pairs with 120 parsimony-informative characters. Bootstrap to that of the type strain of K. rhizophila (Schleifer &
analysis was performed using 100 replicates and the Kandler, 1970; Kovács et al., 1999). Thus, we can conclude
bootstrap values are indicated in Fig. 2. Results indicate that ATCC 9341 is indeed a strain of K. rhizophila.
that ATCC 9341 and the type strain of Kocuria rhizophila In conclusion, we propose to reclassify ATCC 9341 as
(ATCC BAA-50T) were clearly in the same clade and were K. rhizophila. Since this culture is cited in many applications
quite distinct from the M. luteus clade (strains ATCC 4698T, as an official standard strain, we hope to alert users by
D7 and Ballarat). This observation was supported by high publishing the name change in this journal. Manuals,
bootstrap values, and the SSU rRNA gene sequence from procedures and documents need to be updated to reflect
K. rhizophila was 99 % similar to that of ATCC 9341 for this change.
the region aligned. This high similarity also helped in
excluding it from K. varians. Furthermore, several species of

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