Occurance of Staphylococcus nepalensis strains in di¡erent sources
including human clinical material
Dana Nováková1, Roman Pantůček2, Petr Petráš3, Dagmar Koukalová4 & Ivo Sedláček1
1
Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Tvrdého 14, 602 00, Brno, Czech Republic; 2Department of Genetics and
Molecular Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic; 3Reference laboratory for Staphylococci, National
Institute of Public Health, Šrobárova 48, 100 42, Praha 10, Czech Republic; and 4Institute of Microbiology, Faculty of Medicine, Palacký University,
Hněvotı́nská 3, 775 15 Olomouc, Czech Republic
Received 11 April 2006; revised 26 June 2006;
accepted 3 July 2006.
First published online 17 August 2006.
DOI:10.1111/j.1574-6968.2006.00408.x
Editor: Jan-Ingmar Flock
Keywords
Staphylococcus nepalensis ; coagulase-negative
staphylococci; ribotyping; whole-cell protein
analysis; macrorestriction analysis; 16S rRNA
gene sequencing.
Abstract
Five isolates of coagulase-negative staphylococci were obtained from human urine,
the gastrointestinal tract of squirrel monkeys, pig skin and from the environment.
All key biochemical characteristics of the tested strains corresponded with the
description of Staphylococcus xylosus species. However, partial 16S rRNA gene
sequences obtained from analysed strains corresponded with those of Staphylococcus nepalensis reference strains, except for two strains which differed in one
residue. Ribotyping with EcoRI and HindIII restriction enzymes, whole cell protein
profile analysis performed by SDS-PAGE and SmaI macrorestriction analysis were
used for more precise characterization and identification of the analysed strains.
Obtained results showed that EcoRI and HindIII ribotyping and whole cell protein
fingerprinting are suitable and reliable methods for the differentiation of
S. nepalensis strains from the other novobiocin resistant staphylococci, whereas
macrorestriction analysis was found to be a good tool for strain typing. The
isolation of S. nepalensis is sporadic, and according to our best knowledge this
study is the first report of the occurrence of this species in human clinical material
as well as in other sources.
Introduction
Coagulase-negative staphylococci (CoNS) are common inhabitants of mucocutaneous sites of humans, mammals and
birds, but they can be isolated from the environment. They
were generally considered as low or non-pathogenic bacteria
for a long time. Increasing interest in this bacterial group
during the last decade has been caused by an increased
occurrence of CoNS as significant pathogens and also by the
emergence of methicilin-resistant CoNS strains (Grattard
et al., 1993; Marsou et al., 2001; Von Eiff et al., 2001).
Although it is evident that CoNS could play an important
role in human as well as animal infections, most of the
routine clinical microbiological laboratories do not identify
CoNS to the species level even when these organisms are
detected as etiological agents in material with high clinical
significance (Weinstein et al., 1998).
Staphylococcus nepalensis is a novobiocin-resistant CoNS
which was originally isolated from goats with pneumonia
in the Himalayan region (Sperger et al., 2003). The recent
work of Fukami et al. (2004) described two S. nepalensis
strains isolated from fish sauce and suggested that
FEMS Microbiol Lett 263 (2006) 163–168
S. nepalensis is able to improve a fish sauce odour. Although
S. nepalensis has been isolated as the pathogen of goats
(Sperger et al., 2003), it has never been found either in
human clinical material or in material of another animal
besides those mentioned above S. nepalensis is phylogenetically closely related to Staphylococcus cohnii subsp. urealyticum, Staphylococcus cohnii subsp. cohnii, Staphylococcus
saprophyticus subsp. saprophyticus, Staphylococcus succinus
and Staphylococcus xylosus. Chemotaxonomic investigation
showed high similarity to S. cohnii, S. saprophyticus and S.
xylosus (Sperger et al., 2003). Biochemical traits of S.
nepalensis are hardly distinguishable from those of
S. xylosus or S. succinus or from Staphylococcus equorum
(Lambert et al., 1998; Nováková et al., 2006; Place et al.,
2002; Schleifer & Kloos, 1975). Because of the similarity of
the biochemical properties among the mentioned related
staphylococci, identification of these bacteria based only on
the biotyping is not sufficient.
The aim of this study was to clarify taxonomic position of
five CoNS identified presumptively as S. xylosus by using
phenotyping, ribotyping, whole-cell protein fingerprinting
and macrorestriction analysis and to evaluate the application
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Correspondence: Dana Nováková, Czech
Collection of Microorganisms, Faculty of
Science, Masaryk University, Tvrdého 14, 602
00, Brno, Czech Republic. Tel.: 1420 549
49 5425; fax: 1420 543 247 339;
e-mail: danan@sci.muni.cz
164
of these methods for the differentiation of phenotypically
similar novobiocin resistant Staphylococcus spp.
Methods
Bacterial strains
Cultivation and phenotypic identification
Analysed strains were grown for 24 h at 37 1C and at 30 1C
on sheep blood agar (HiMedia). The colony size and colony
morphology were observed on P-agar (Kloos et al., 1974)
after 48 h at 37 1C and subsequently after 72 h at laboratory
temperature (Meugnier et al., 1996). Phenotype characterization was achieved by using the commercial kits API
Staph, ID 32 STAPH (bioMérieux) and STAPHYtest16
system (PLIVA-Lachema) according to the manufacturer’s
instructions. Ambiguous and unclear results revealed by a
few tests (production of b-galactosidase and acetoin, hydrolysis of aesculin, acid production from D-xylose, D-mannose,
D-cellobiose, L-arabinose, N-acetylglucosamin, D-raffinose,
D-ribose, D-melezitose, D-melibiose) were verified by conventional testing (Mannerová et al., 2003). All strains were
tested for the production of catalase, oxidase, pyrrolidonyl
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arylamidase (PLIVA-Lachema), clumping factor (Slidex
Staphy Plus, bioMérieux) and coagulase (Itest-Plus) and
hydrolysis of Tween 80, gelatine and casein (Mannerová
et al., 2003). A susceptibility to bacitracin (0.04 IU, ItestPlus), novobiocin (5 mg, Itest-Plus), furazolidone (100 mg,
Itest-Plus) and polymyxin B (300 IU, Oxoid) was examined
by the agar diffusion test on Mueller-Hinton agar (Oxoid)
(Woods & Washington, 1995). The biochemical tests were
evaluated using the online identification tool Apiweb
(BioMérieux, http://apiweb.biomerieux.com); the phenotype characteristics and identification tables described previously (Schleifer & Kloos, 1975; Sperger et al., 2003) were
also used in the absence from Apiweb of any of the less
common staphylococci.
DNA sequencing
The 16S rRNA gene sequencing and phylogenetic analysis
was performed as described previously (Nováková et al.,
2006). The RDP-II Sequence Match tool (Cole et al., 2005)
was used to search for the nearest neighbors of the 16S rRNA
gene sequences. The partial sequences of 16S rRNA gene
were deposited in the GenBank database under the accession
numbers: DQ394098 (CCM 2433); DQ394099 (NRL 04/
478); DQ394100 (NRL 04/522); DQ394101 (CCM 2628) and
DQ394102 (CCM 7317). Those of the type and reference
S. nepalensis strains were deposited under the accession
numbers DQ394096 (CCM 7045T) and DQ394097
(CCM 7046).
Ribotyping
Ribotyping was carried out with the EcoRI and HindIII
restriction endonucleases (Takara Bio.) and the digoxigeninlabelled probe complementary to 16S and 23S rRNA as
described previously (Švec et al., 2001). In brief, EcoRI or
HindIII restriction fragments were transferred to the nylon
membrane Biodyne B (Pall Corp.) by vacuum alcalic blotting,
and then hybridised with the labelled probe overnight at
56 1C. Digitised ribotype patterns were evaluated by GelCompar II software (Applied Maths). Dendrograms were calculated by UPGMA clustering method using the Dice correlation
coefficient. A position tolerance of 1% and optimisation of
0.2% was allowed for the bands. The bacteriophage l DNA
digested by EcoRI and HindIII restriction endonucleases
(Promega) was used as a molecular weight standard.
SDS-PAGE
The bacteria were grown at the standard conditions (37 1C
for 24 h) on nutrient agar (CM3 agar, Oxoid). Isolation
of the whole-cell proteins, SDS-PAGE procedure, and the
densitometric analysis of the protein profiles were performed as described by Pot et al. (1994). The densitometric
FEMS Microbiol Lett 263 (2006) 163–168
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Five presumptive S. xylosus strains isolated at different
geographical areas were tested. Strains CCM 2433 ( = strain
B-P 8; Baird-Parker, 1963) isolated from the skin of a pig
and CCM 2628 ( = strain P. Oeding 1463; Schleifer & Kocur,
1973) retrieved from the environment were obtained from
the Czech Collection of Microorganisms (CCM), Masaryk
University, Brno, Czech Republic. Strain CCM 7317 was
isolated from the urine of a (human) patient treated for
cystitis and stored at National Reference Laboratory for
Staphylococci in Prague (NRL). Two remaining strains NRL
04/522 and NRL 04/478 were isolated from rectal swabs of
healthy South American squirrel monkeys (Saimiri sciureus)
kept in Olomouc Zoo (Czech Republic) in May 2000 and
June 2004, as described previously (Pantůček et al., 2005).
The reference and the type strains of S. xylosus (CCM
2738T, CCM 2725, CCM 4580, NRL 01/431), S. nepalensis
(CCM 7045T, CCM 7046), S. cohnii subsp. urealyticum
(CCM 4294T, CCM 2110), S. cohnii subsp. cohnii (CCM
2736T, CCM 2605), S. saprophyticus subsp. saprophyticus
(CCM 883T, CCM 3318, CCM 3319, CCM 2728) Staphylococcus equorum subsp. equorum (CCM 3832T, CCM 3833,
CCM 7301, CCM 7302), Staphylococcus equorum subsp.
linens (CCM 7278T), Staphylococcus succinus subsp. succinus
(CCM 7157T), Staphylococcus succinus subsp. casei (CCM
7194T) Staphylococcus succinus (CCM 7312, CCM 7313,
CCM 7314) and Staphylococcus gallinarum (CCM 3572T,
CCM 4506) were obtained from the Czech Collection of
Microorganisms (http://www.sci.muni.cz/ccm/).
D. Nováková et al.
165
S. nepalensis strains isolated from different origins
analysis of the digitised protein profiles was performed by
GelCompar II software. The dendrogram was calculated by a
UPGMA clustering method using the Pearson correlation
coefficient. A whole-cell protein extract of Psychrobacter
immobilis CCM 4923 was used as a reference profile and
the Wide Molecular Weight Range marker (Sigma) ranging
from 6500 to 205 000 Da was used as a molecular mass
marker.
Pulsed field gel electrophoresis (PFGE)
Results and discussion
Biotyping
All strains grew well at both 30 1C and 37 1C. White, grey or
yellowish convex colonies with the characteristic elevated
centre were observed on P-agar. The bacteria grew without a
hemolysis on sheep blood agar. All tested strains reduced
nitrates, produced phosphatase, b-galactosidase and b-glucuronidase, and hydrolysed aesculin and Tween 80; none of
them hydrolysed casein. They formed acid from D-xylose,
D-mannose, saccharose, trehalose and L-arabinose; none of
them produced acid from D-cellobiose, D-raffinose, D-melibiose or D-melezitose. The strains NRL 04/478 and NRL 04/
522 did not produce acid from maltose. All tested strains
were resistant to novobiocin and bacitracin and sensitive to
furazolidon and polymyxin B. Based on all these phenotype
results all strains were identified as S. xylosus species; strains
NRL 04/478 and NRL 04/522 were identified as atypical S.
xylosus due to their negative reaction for maltose acidification. Obtained biochemical traits including the antibiotic
susceptibility corresponded with S. xylosus species description. All strains were positive for hydrolysis of aesculin and
Tween 80 and production of pyrrolidonyl arylamidase test,
which are less common properties in the S. xylosus species.
In contrast, these traits are in accordance with description of
S. nepalensis (Sperger et al., 2003). The hydrolysis of Tween
FEMS Microbiol Lett 263 (2006) 163–168
Sequence analysis of 16S rRNA gene
Full or partial sequencing of the 16S rRNA gene is one of the
most useful phylogenetic tools currently available that
allows reliable identification of poorly described or biochemically atypical strains as well as novel species (Clarridge, 2004). Based on the obtained 16S rDNA sequences, all
five isolates were unambiguously identified as S. nepalensis.
The sequences of a 5 0 527-bp fragment of the 16S rRNA gene
determined in strains CCM 2433, NRL 04/522 and NRL 04/
478 were identical to the sequence of the type strain
S. nepalensis CW1T = CCM 7045T (Sperger et al., 2003).
The strains CCM 2628 and CCM 7317 matched the S.
nepalensis type strain, except for one point mutation in
position 213 (Escherichia coli coordinate) with the A residue
in place of G residue that is present in S. nepalensis type
strain. Although sequencing of the 16S rRNA gene is an
effective method, it can be limited by some deficiencies in
the public databases, such as incorrectly named strains,
redundant sequence entries or outdated nomenclatures (Becker et al., 2004).
Ribotyping
The obtained ribotype patterns grouped all five analysed
strains with S. nepalensis reference strains CCM 7045T and
CCM 7046 into a separate cluster (Fig. 1). The similarity
between the EcoRI ribopatterns of the tested strains and the
reference S. nepalensis strains ranged between 55.3–90.1%
(Fig. 1a) and the similarity between the HindIII ribopatterns
ranged from 73.6 to 92.3% (Fig. 1b). The intraspecies
variability of the S. nepalensis strains based on HindIII
ribopatterns was comparable with other staphylococcal
species included in the cluster analysis. The other type and
reference strains used in this study were well differentiated
from S. nepalensis group by using both restriction enzymes,
however our results imply that ribotyping with HindIII is a
better identification tool as it revealed visually very close and
more species-specific ribopatterns than EcoRI restriction
endonuclease. In contrast to these results, Chesneau et al.,
2000 described that EcoRI allows better discrimination of
the staphylococcal species than HindIII does, but there were
no S. nepalensis strains included in that study.
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The preparation of chromosomal DNAs and digestion with
SmaI restriction endonuclease (Roche Diagnostics) were
performed according to the procedure described by
Snopková et al. (1994). SmaI restriction fragments of DNA
were separated in 1.2% (w/v) agarose gel (Serva Electrophoresis), 21 cm in length, in 1 TAE electrophoresis buffer
(0.04 M Tris/acetate, 0.001 M EDTA, pH 8.2) at 14 1C with
CHEF-Mapper (Bio-Rad Laboratories) and program conditions: initial switch time 5 s, final switch time 50 s, voltage
gradient 5.5 V cm1 and run time 28 h. Concatemers of
bacteriophage l (Sigma), l DNA cleaved with HindIII
(Roche Diagnostics) and Staphylococcus aureus NCTC 8325
DNA cleaved with SmaI were used as molecular weight
markers.
80 is generally a rare property among CoNS and seems to be
a helpful test for S. nepalensis identification.
Similar biochemical patterns were observed by Kloos
et al. (1976), who studied the staphylococcal strains isolated
from the skin of the various animals. They described eight
novobiocin-resistant Staphylococcus sp. strains isolated from
squirrel monkey skin that were biochemically close to the
strains studied in the present study. The strains differed in
their lack of ability to produce acid from D-xylose and in the
positive caseinolytic activity.
166
D. Nováková et al.
In this paper, ribotyping proved to be useful for species
discrimination and the identification of strains of staphylococci that are hardly distinguishable biochemically as was
shown previously in the study of Marsou et al. (2001).
SDS-PAGE
Whole-cell protein fingerprinting grouped all tested strains
and S. nepalensis reference cultures into one cluster that was
clearly separated from all other reference strains at the
similarity level of 83.7% (Fig. 2). The intraspecies similarity
within the S. nepalensis cluster ranged from 83.7 to 95.7%.
The individual species included in the cluster analysis were
separated at the similarity levels ranging from 55.8 to 83.7%.
The obtained data partially correspond with the results
published by Thomson-Carter & Pennington (1989) who
observed 76.8–100% intraspecies similarity between different CoNS. In contrast to the present study, the percentage of
similarity between different CoNS found by ThomsonCarter & Pennington did not exceed 68.9%. Good experience with the identification of various CoNS based on
whole-cell protein analysis was also confirmed by the work
of Pennington et al. (1991).
Macrorestriction analysis
Fig. 2. Dendrogram based on whole-cell protein profiles analysis of
tested strains and related staphylococcal species.
The isolates showed four distinct SmaI digest patterns
containing 19–26 restriction fragments sized from 6 to
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FEMS Microbiol Lett 263 (2006) 163–168
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Fig. 1. The cluster analysis of EcoRI (A) and HindIII (B) ribotype patterns of tested strains and related staphylococcal species.
167
S. nepalensis strains isolated from different origins
Acknowledgements
Fig. 3. PFGE banding patterns of SmaI cleaved genomic DNAs representing S. nepalensis strains (lanes 1 to 7) and reference strains of the
related species (lanes 8 to 14).
This work was supported by the long-term research program
of the Ministry of Education of the Czech Republic
(MSM0021622415, MSM0021622416 and MSM6198959205)
and by project of the Ministry of Education number FRVŠ
92658/2006. We thank L. Chrastinová and J. Vokurková
(Zoo Olomouc, Czech Republic) for the microbiological
sampling of squirrel monkeys and K. Káňová for technical
assistance.
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