Download Possible virulence factors of Staphylococcus sciuri

FEMS Microbiology Letters 199 (2001) 47^53
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Possible virulence factors of Staphylococcus sciuri
Srdjan Stepanovic̈
a
a;
*, Dragana Vukovic̈ a , Vladimir Trajkovic̈ a , Tanja Samardz­ic̈ b ,
è upic̈ a , Milena Sívabic̈-Vlahovic̈ a
Maja C
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr Subotic̈a 1, 11000 Belgrade, Yugoslavia
b
Institute for Biological Research `Sinis­a Stankovic̈', 29 Novembra 142, 11000 Belgrade, Yugoslavia
Received 13 December 2000 ; received in revised form 7 March 2001; accepted 19 March 2001
Abstract
Staphylococcus sciuri is an opportunistic pathogen of controversial clinical significance. The factors that contribute to colonization and/or
infection caused by this bacterium have not been studied intensively so far. The present research was carried out in order to study the
presence of potential virulence factors in 121 human and animal isolates of this bacterium. Isolates were examined for biofilm formation,
hemagglutination, presence of clumping factor, production of spreading factors and exotoxins, cytotoxicity and capacity to stimulate nitric
oxide production. The results showed that S. sciuri is highly capable of biofilm production, that it displays strong proteolytic and DNase
activities, produces hemolysins and stimulates nitric oxide production by rat macrophages. Although the present study showed existence of
a wide spectrum of possible virulence determinants of S. sciuri, their exact contribution to virulence of this bacterium in vivo remains to be
determined. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Virulence; Pathogenicity ; Staphylococcus sciuri
1. Introduction
Staphylococcus sciuri, the coagulase-negative species,
was ¢rst described by Kloos et al. in 1976 [1]. This bacterium is widespread in nature and is associated with a variety of wild and domestic animals [1^4]. S. sciuri has been
shown to be an invasive pathogen for animals causing
wound infections and mastitis [3,5]. Although principally
animal species, S. sciuri may colonize humans and its isolation from clinical samples such as skin, vagina, blood,
urine, central venous catheters has been reported [4,6^8].
Moreover, diseases in humans caused by this bacterium,
such as wound infections, soft tissue infections, abscesses,
boils, peritonitis and endocarditis, have been described
[5,6,9^11]. However, apart from the studies of Hedin
and Widerstrom [10] and Wallet et al. [11] that clearly
identi¢ed S. sciuri as a causative agent of endocarditis
and peritonitis, respectively, no other reports did prove
that this bacterium actually caused the infections reported.
Thus, clinical relevance of S. sciuri for humans is still
* Corresponding author. Tel. : +381 (11) 685961;
Fax: +381 (11) 656950; E-mail: [email protected]
controversial. It should be noted that the rare recognition
of S. sciuri as a human pathogen is partly due to di¤culties in its identi¢cation in routine laboratory practice since
many clinical laboratories do not identify coagulase-negative staphylococci (CoNS) to the species level [12]. Moreover, it has been shown that commercial systems for identi¢cation could misidentify S. sciuri [13,14].
The possible virulence factors of S. sciuri have not been
studied intensively so far. In the present study S. sciuri
animal and human isolates were examined for bio¢lm formation, hemagglutination, presence of clumping factor,
production of spreading factors, toxins, and hemolysins,
toxigenicity and capacity to stimulate nitric oxide (NO)
production.
2. Materials and methods
2.1. Strains
In total, 121 strains of S. sciuri were used in this study:
the reference strain S. sciuri ATCC 29062, four clinical
isolates [8], and 116 isolates sampled from the skin and
mucous membranes of dogs. The clinical isolates were
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 5 0 - 1
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identi¢ed by the Becton Dickinson commercial set Sceptor
Staphylococcus MIC/ID panel. Previously described
screening procedure [8] was used for identi¢cation of
S. sciuri strains of animal origin. Brie£y, each tube coagulase-negative and esculin-positive staphylococcal isolate
was tested for novobiocin susceptibility, oxidase activity,
and acid production from ra¤nose. All novobiocin-resistant, oxidase-positive strains that did not produce acid
from ra¤nose were preliminary identi¢ed as S. sciuri. In
order to con¢rm the identi¢cation, the biochemical properties of these strains were further analyzed on a set of
22 carbohydrates. Prior to inoculation, all strains were
transferred from the stock cultures to P agar [15] and
incubated aerobically overnight at 35³C.
2.2. Bio¢lm formation
2.3. Hemagglutination
Hemagglutination test was performed in a manner similar to the method described by Rupp and Archer [17]. The
hemagglutination was performed in U-shaped 96-well microtiter plates (Spektar, Cacak, Yugoslavia). The bacterial
suspensions in phosphate-bu¡ered saline were adjusted to
McFarland standard 1. Two serial dilutions of the bacterial suspension were made in the microtiter plates to give a
total volume of 50 Wl per well. Then, 50 Wl of the 1%
human O erythrocyte suspension in phosphate-bu¡ered
saline were added to each well. After shaking, the plates
were incubated at room temperature for 2 h, and the hemagglutination was recorded as positive or negative.
2.4. Clumping factor
The clumping factor was determined with human plasma on glass slides [15]. Formation of visible clusters within
60 s was regarded as a positive result.
2.5. Proteinase activity
Proteinase activity was detected using the casein and
gelatin media according to the procedures described by
Koneman et al. [18] and Oberhofer [19], respectively. Caseinase activity was observed as a transparent halo surrounding the growth, while the zone of clouding around
the growth indicated the gelatinase activity.
Splitting of Tween 80, Tween 40 and Tween 20 was
examined in tryptose blood agar base (TBAB) (Difco laboratories, Detroit, MI, USA) enriched with 0.01%
CaCl2 W2H2 O and 1% Tween 80 (Difco), Tween 40 and
Tween 20 (Serva), respectively [15]. Splitting of Tweens
was characterized by an opacity around the culture. Spirit
blue agar (Difco) supplemented with Bacto lipase reagent
(Difco), and TBAB enriched with 1% glycerol tributyrate
(tributyrin) (BDH, Pool, UK), were also used in the study.
The presence of zones of clearing was taken as an indication of positive enzyme activity. The inoculated media
used for detection of lipolytic activity were incubated for
48 h at 35³C and, thereafter for 72 h at room temperature.
2.7. Lecithinase activity
Lecithinase activity was examined on Baird^Parker medium containing 5% egg-yolk emulsion [15]. Inoculated
plates were incubated for 48 h at 35³C and thereafter for
24 h at room temperature. The lecithinase activity was
characterized by an opaque zone of precipitate in the medium around the growth.
2.8. Starch hydrolysis
The ability of S. sciuri strains to degrade starch was
determined by culturing bacteria on the plates containing
TBAB plus 1% starch soluble (Mallinckrodt Chemical
Works, Saint Louis, MO, USA). After incubation at
35³C for 2 days, the plates were £ooded with Grams's
iodine solution ; a clear zone around the growth indicated
hydrolysis of starch [19].
2.9. DNase activity
For detection of DNase activity, strains were inoculated
on TBAB, and incubated for 24 h at 35³C. Thereafter, the
plates were £ooded with melted DNase agar containing
toluidine blue (Torlak, Belgrade, Yugoslavia). After a further 24 h of incubation at 35³C, the plates were examined
for a pink zone surrounding the growth on a blue background [20].
2.10. Fibrinolysin activity
For demonstration of ¢brinolysin activity, sterile human
plasma was added to molten TBAB to give the ¢nal concentration of 12% [21]. After heating in water bath at 56³C
for 15 min, the mixture was poured into Petri dishes.
Tested strains were spot-inoculated onto dried poured
plates, and incubated for 24 h at 35³C and a further 24 h
at room temperature. Fibrinolysin-positive isolates showed
clearing around the spot inocula.
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Quanti¢cation of bio¢lm formation by S. sciuri strains
cultivated in brain heart infusion broth (BBL, Becton
Dickinson Microbiology Systems, Cockeysville, MD,
USA) was determined by the modi¢ed microtiter-plate
test, as previously described [16]. Upon the optical density
(OD) of bacterial ¢lms, all strains were classi¢ed into the
following categories: no bio¢lm producers, weak, moderate, or strong bio¢lm producers [16]. The test was carried
out two times and the results were averaged.
2.6. Lipase activity
S. Stepanovic̈ et al. / FEMS Microbiology Letters 199 (2001) 47^53
2.11. Urease activity
Urease activity was tested on Christensen agar according to the recommendations given by Freney et al. [15].
Spot-inoculated plates were incubated for 72 h at 35³C.
The purple color of the inoculated agar was interpreted as
a positive result.
2.12. Hemolytic activity
2.13. Toxins
The production of K, L and N toxins was determined on
TBAB with 5% de¢brinated sheep or fresh human blood
(fresh human O blood, from one of the authors, S.S.).
Tested strains were inoculated perpendicularly to the
streak of a L toxin-producing Staphylococcus aureus [15].
Inoculated plates were incubated at 35³C for up to 3 days.
The results were evaluated as antagonistic (L^K hemolysins) or synergistic (L^N hemolysins) e¡ects as determined
by the shapes of hemolytic zones produced.
2.14. Test for cytotoxic activity
Cytotoxicity of S. sciuri strains was examined using
Vero cells in 96-well microtiter trays. Suspensions of
S. sciuri strains in distilled water were adjusted to
0.5 McFarland standard. Then, 20 Wl of bacterial suspensions were added to 3.5 ml of brain heart infusion broth
(BBL, Becton Dickinson Microbiology Systems). The
tubes were incubated 2 days at 35³C, and thereafter for
2 days at room temperature. After centrifugation of bacterial broth cultures, 20 Wl of supernatants were added in
triplicate to 180 Wl of cell culture medium. After incubation for 24 h at 37³C, in humidity atmosphere and 5%
CO2 , wells were examined for cytopathic e¡ect (CPE)
under microscope, and cell viability was determined by
the dye developing method using MTT (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany) [22]. After formazan
extraction by DMSO (Sigma-Aldrich Chemie GmbH,
Steinheim, Germany), the absorbance was measured at
570 nm by using an automated ICN Flow Titertek Multiscan Plus reader.
ical isolates and one dog isolate) and S. aureus ATCC
25923, as the positive control, were incubated overnight,
at 35³C on tryptose agar (Difco). Thereafter, cells were
suspended in pyrogen-free 0.9% NaCl, at concentration
of 1U109 colony forming units (CFU) per ml. Suspended
bacteria were heat-killed (120 min at 65³C), stored at 4³C,
and used within 7 days. Resident rat macrophages were
obtained and cultured (20 000 per well), as previously described [23], and stimulated by staphylococcal cells. The
appropriate dilutions of bacterial suspensions were made
on the day of the experiment in order to obtain ratios of
10, 100 or 1000 bacteria per macrophage. All strains were
tested in triplicate, and incubated for 24 h and 48 h at
37³C in humidi¢ed atmosphere with 5% CO2 . Nitrite accumulation, an indicator of NO production, was measured
using the Griess reagent [23]. The absorbance at 570 nm
was measured by using an automated ICN Flow Titertek
Multiscan Plus reader. The statistical signi¢cance of di¡erences was analyzed using the Student's t-test and P values
less than 0.05 were considered signi¢cant.
3. Results and discussion
Summarized results of examination for S. sciuri factors
that possibly contribute to the virulence of this organism
are presented in Table 1.
The virulence factors of staphylococci have been studied
most extensively in the species of S. aureus. Although
CoNS have emerged as important pathogens, particularly
as the cause of medical devices infections [24], little is
known about the virulence factors of these bacteria [25].
The most important virulence factor of Staphylococcus
epidermidis and other CoNS is assumed to be bio¢lm formation on indwelling medical devices [24,26]. Thus, it has
been proposed that testing for bio¢lm production could be
a useful marker for the pathogenicity of CoNS [27,28].
Medical device infections caused by S. sciuri have not
been reported until today. Only isolation of S. sciuri
from central venous catheters has been described, but it
2.15. Capacity of S. sciuri to generate NO in
rat macrophages
Six strains of S. sciuri (S. sciuri ATCC 29062, four clin-
Fig. 1. Levels of NO produced by rat peritoneal macrophages cultured
for 24 and 48 h with 10, 100 and 1000 S. sciuri cells per macrophage.
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Hemolytic activity was determined on TBAB supplemented with 5% de¢brinated rabbit blood, 5% de¢brinated
sheep blood (obtained from Torlak, Belgrade, Yugoslavia), or 2.5% washed human O erythrocytes (obtained
from Institute for Transfusiology, Belgrade, Yugoslavia),
according to the recommendations given by Freney et al.
[15]. Plates were incubated at 35³C.
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Table 1
Possible virulence factors of S. sciuri
Factor
Number of strains showing
Negative reaction
107 (88.43%)
0 (0%)
57 (47.11%)a
14 (11.57%)
121 (100%)
64 (52.89%)
Proteinase activity:
Gelatinase hydrolysis
Casein hydrolysis
121 (100%)
116 (95.87%)
0 (0%)
5 (4.13%)
Lipase activity:
Splitting of Tween 80
Splitting of Tween 40
Splitting of Tween 20
Lipase reaction on Spirit Blue Agar
Splitting of Tributyrin
Lecithinase activity
Starch hydrolysis
DNase activity
Fibrinolysin activity
Urease activity
0 (0%)
121 (100%)
121 (100%)
0 (0%)
121 (100%)
0 (0%)
0 (0%)
117 (96.69%)
0 (0%)
0 (0%)
121 (100%)
0 (0%)
0 (0%)
121 (100%)
0 (0%)
121 (100%)
121 (100%)
4 (3.31%)
121 (100%)
121 (100%)
Hemolytic activity:
On rabbit blood
On sheep blood
On human blood
0 (0%)
0 (0%)
119 (98.35%)b
121 (100%)
121 (100%)
2 (1.65%)
Toxins:
K Toxin
L Toxin
N Toxin
Toxigenicity toward Vero cells
0 (0%)
0 (0%)
119 (98.35%)
0 (0%)
121 (100%)
121 (100%)
2 (1.65%)
121 (100%)
a
Among clumping factor-positive strains, eight (14.04%) gave strong reaction, 35 (61.4%) moderate reaction and 14 (24.56%) weak reaction.
Among strains showing hemolytic activity on human blood, 112 (94.12%) showed strong hemolysis (zone of hemolysis s 2.5 mm from the culture
streak), seven (5.88%) showed weak hemolysis (zone of hemolysis 6 2.5 mm).
b
was not established whether these strains were pathogens
or contaminants [8]. In the present study 107 (88.43%) S.
sciuri strains were shown to be capable of bio¢lm production. Among these, 26 (24.30%) were strong, 31 (28.97%)
moderate, and 50 (46.73%) were weak bio¢lm producers.
As far as S. aureus is concerned, bio¢lm production has
not been considered as a virulence factor. However, it was
shown that 88.9% of S. aureus strains isolated from orthopedic implants (infected knee and hip prostheses) were
capable of bio¢lm formation [29]. The studies on bio¢lm
formation by S. epidermidis, the most signi¢cant pathogen
among CoNS, showed that up to 76.7% of strains of this
bacterium produced bio¢lm [29,30]. When other CoNS of
minor clinical signi¢cance, e.g. Staphylococcus haemolyticus, Staphylococcus xylosus, and Staphylococcus simulans,
were tested, 47.8% of strains produced bio¢lm [30]. In the
present study we observed the rate of bio¢lm producers
among the tested S. sciuri strains comparable to those of
pathogenic staphylococci. This indicates that capacity for
bio¢lm formation may be considered as a possible virulence factor of this bacterium.
The ability of S. epidermidis to produce bio¢lm on plas-
tic surfaces was shown to be associated with its capacity to
mediate hemagglutination of erythrocytes of di¡erent species [17,31]. The similar association for S. sciuri has not
been shown in our study. Hemagglutination with human
O erythrocytes was not observed in any of the tested S.
sciuri strains, while nearly 90% of them were able to form
bio¢lm. This probably excludes hemagglutinin as an additional possible virulence factor important for bio¢lm formation by S. sciuri. In general, the rate of agglutination
for non-S. epidermidis CoNS is low (33%) [17]. Still, it is
possible that S. sciuri could agglutinate erythrocytes of
sheep or some other animal species, as it was described
for Staphylococcus saprophyticus isolates [26].
The ability of S. aureus to initiate colonization by attachment to ¢brinogen-containing substrates, and to
clump in the presence of ¢brinogen and thus increase resistance to phagocytosis, is primarily due to the presence
of clumping factor [32,33]. S. epidermidis, and most other
CoNS do not produce clumping factor [15]. Clumping
factor has recently been described in S. sciuri [4]. In the
present study clumping factor was detected in 57 (47.11%)
tested strains of S. sciuri, although the majority of the
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Positive reaction
Bio¢lm formation
Hemagglutination
Clumping factor
S. Stepanovic̈ et al. / FEMS Microbiology Letters 199 (2001) 47^53
activity, starch hydrolysis, ¢brinolysin activity and urease
activity were not found in the tested S. sciuri strains.
We did not establish S. sciuri hemolytic activity on
plates with 5% de¢brinated rabbit blood or 5% de¢brinated sheep blood, which is in agreement with previous
studies [1,2,6]. However, when grown on plates with 2.5%
washed human O erythrocytes, 119 strains showed clearly
detectable complete hemolysis. When full human blood
was used, no hemolysis was observed. In the strains showing hemolytic activity, only N toxin was detected. The fact
that tested S. sciuri strains displayed N hemolysin only,
whose hemolytic properties are inhibited by serum components [39], partly explains the absence of hemolysis on full
human blood agar. On the contrary, cooperative hemolytic action between S. sciuri N and S. aureus L toxins
was visible only on full human blood agar plates after
prolonged incubation for 3 days. When 2.5% washed human O erythrocytes were used, clearly detectable hemolysis could be seen around the culture streak of tested
S. sciuri strains, but synergistic L^N hemolysis was not
observed. There is no apparent explanation for this ¢nding.
It has been shown that N toxin is produced by 97% of
S. aureus strains and 50^70% of CoNS [40]. Although this
toxin causes a wide spectrum of cytotoxic e¡ects, its importance for course of infections caused by staphylococci
remains unclear [40]. Since we established that almost all
tested strains produced N toxin, it seemed reasonable to
assume that this product may be of certain importance for
S. sciuri virulence. However, when the cytotoxic activity of
S. sciuri was tested in vitro using Vero cells monolayer, no
CPE was observed. As far as cytotoxic e¡ects of other
staphylococci are concerned, it has been shown that they
considerably vary depending on the cell type used in the
study [25,41,42]. When S. aureus cytotoxicity to Vero cells
was examined, only six out of 20 tested strains exhibited
cytotoxic e¡ect [42]. Thus, S. sciuri cytotoxicity should be
further evaluated using cell lines other than Vero.
The capacity of a bacterium to stimulate NO production may also be considered as a factor of importance for
the course of an infection because of its numerous biological e¡ects. NO or its derivatives exhibit antiproliferative
e¡ects [43], suppress protein synthesis [44], and in£uence
the processing of nascent collagen [45], and, therefore,
may a¡ect the process of wound repair [46]. It has also
been shown that an enhanced formation of NO contributes to the circulatory failure, multiple organ failure and
shock [47]. We evaluated S. sciuri capacity to generate NO
in rat macrophages. As there were no statistically signi¢cant di¡erences among tested staphylococcal strains in NO
production, only the results of one clinical strain are presented in Fig. 1. The results of the present study showed
S. sciuri ability to induce NO synthesis by rat peritoneal
macrophages in a dose-related manner. Similar results
were obtained in studies with S. aureus [48], and S. epidermidis [49].
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clumping factor-positive strains (85.96%) gave only weak
to moderate reaction. The presence of the clumping factor
was not detected in any of 30 clinical isolates of S. sciuri
[6] as well as in the strain which caused endocarditis [10].
The overall data suggest that clumping factor occurs in S.
sciuri, but its exact role in pathogenesis of infections
caused by this organism is still unclear.
As far as S. epidermidis and other CoNS are concerned,
it has been shown [34^36] that extracellular products like
protease, DNase, lipase, hemolysins, and other exoenzymes may be responsible for tissue degradation and
spreading of an infection caused by these bacteria. The
results of the present study show that S. sciuri is a bacterium with strong proteolytic activity. All tested strains
were gelatinase-producing and nearly 96% showed caseinolytic activity, which is in agreement with results of previous studies [1,4]. While S. sciuri proteolytic activity is
comparable to that of S. aureus [37], it is interesting to
note that only 43.3% of S. epidermidis strains produced
gelatinase and 3.3% displayed caseinolytic activity [30].
Although the status of DNase as a virulence factor is
debatable, Daghistani et al. [37] showed that signi¢cantly
more wound isolates of S. aureus produced DNase (97.5%)
than those isolated from healthy nasal £ora (77%). In
contrast to other mainly DNase-negative CoNS [20], we
established that absolute majority (96.69%) of the tested
S. sciuri strains produced DNase. Thus, DNase activity of
S. sciuri is comparable to that of S. aureus wound isolates.
Among CoNS only S. chromogenes, species which can
cause more severe infections than the majority of other
CoNS species [25], showed similar DNase activity [20].
The contribution of lipolytic activity to virulence of
staphylococci is not fully understood. However, it has
been shown that 79% of S. aureus strains isolated from
wounds produced lipase [37]. The proportion of strains
producing lipase in the majority of CoNS ranged from
10 to 60% [38]. The exceptions were Staphylococcus schleiferi and S. saprophyticus which resembled S. aureus in that
80% of the strains produced lipase, and Staphylococcus
lugdunensis and S. haemolyticus strains with no detectable
lipase activity [38]. The evaluation of S. sciuri lipolytic
activity showed that all strains were capable of splitting
of Tween 20, Tween 40 and tributyrin. No lipolytic activity toward Tween 80 and Difco lipase reagent was observed. Previous reports on S. sciuri lipolytic activity are
somewhat contradictory. In the study of Devriese et al.,
splitting of tributyrin was detected in 10 out of 48 tested
strains of animal origin, while no lipolytic activity toward
Tween 80 was observed [2]. According to Kloos et al.,
S. sciuri does not exhibit lipolytic activity at all [4]. It is
well known that staphylococcal lipases show substrate speci¢city, which may explain the discrepant results obtained
in di¡erent studies. However, the exact contribution of
lipolytic enzymes to S. sciuri virulence as well as to virulence of other staphylococci needs to be further clari¢ed.
In contrast to the factors discussed above, lecithinase
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S. Stepanovic̈ et al. / FEMS Microbiology Letters 199 (2001) 47^53
S. sciuri has attracted special attention after it was suggested that mecA gene of methicillin-resistant strains of
staphylococci originated from an evolutionary relative of
the mecA homolog that has been identi¢ed in S. sciuri
[4,50,51]. Moreover, the ability of S. sciuri to transfer
genes of resistance to other bacteria, pathogenic for
men, has been established [51]. On the other hand, the
virulence factors of this bacterium are not yet well understood. The results of the present study showed that S.
sciuri has a wide spectrum of possible virulence factors.
Moreover, some of the factors displayed activities similar
to those of pathogenic staphylococci. However, it remains
unclear whether they are, indeed, virulence factors or simply common features of this bacterium. Thus, the exact
contribution of the examined factors to S. sciuri virulence
in vivo remains to be determined.
[12]
[13]
[14]
[15]
[16]
We thank Dr. Vera Pravica (School of Biological Sciences, University of Manchester, UK) whose support and
friendship made this study possible.
[17]
[18]
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