Download Biofilm formation by Staphylococcus capitis strains isolated from

Journal of Medical Microbiology (2013), 62, 1051–1059
DOI 10.1099/jmm.0.050500-0
Biofilm formation by Staphylococcus capitis strains
isolated from contaminated platelet concentrates
Valerie S. Greco-Stewart,1 Hamza Ali,1 Dilini Kumaran,1 M. Kalab,2
Ineke G. H. Rood,3 Dirk de Korte3 and Sandra Ramı́rez-Arcos1
Correspondence
1
Sandra Ramı́rez-Arcos
2
[email protected]
Canadian Blood Services, Ottawa, Ontario, Canada
Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada
3
Sanquin Blood Supply Foundation, Amsterdam, The Netherlands
Received 31 July 2012
Accepted 3 April 2013
Bacterial contamination of platelet concentrates (PCs) poses the greatest infectious risk in
modern transfusion medicine despite the implementation of measures such as improved skin
disinfection and first aliquot diversion. The majority of PC contaminants are commensal skin flora
introduced by venipuncture at the time of blood collection. The predominant organisms are Grampositive coagulase-negative staphylococci such as Staphylococcus capitis. This bacterium has
been implicated in numerous instances of infection and sepsis, likely for its ability to form surfaceassociated communities of micro-organisms encased in extracellular materials, known as biofilms.
In the present study, five strains of S. capitis isolated from contaminated PCs were assessed for
their ability to produce extracellular polysaccharide (slime), a canonical indicator of biofilmformation ability, on Congo red agar plates. Biofilm formation was evaluated in both glucoseenriched trypticase soy broth (TSBg) and in PCs by using a crystal violet staining assay. The
chemical nature of the biofilms was evaluated by disruption assays using sodium metaperiodate
and proteinase K. In addition, biofilm architecture was observed by scanning electron microscopy.
The presence of the biofilm-associated icaR and icaADBC genes was also examined by PCR.
While only two out of the five S. capitis strains formed biofilms in TSBg, all strains formed biofilms
in PCs. The ability of strains to produce extracellular polysaccharide and their possession of wildtype ica genes were not exclusive predictors of biofilm formation in TSBg or PCs; different profiles
of biofilm markers were observed among isolates. This is likely due to the proteinaceous
composition of the S. capitis biofilm matrix. Interestingly, an ica-negative, non-slime-producing
isolate was capable of biofilm formation in PCs. Together, these data indicate that the platelet
storage environment stimulates biofilm formation in S. capitis in the absence of extracellular
polysaccharide production and that multiple bacterial factors and regulatory elements are likely
involved in biofilm formation in this milieu.
INTRODUCTION
Transfusion-associated bacterial infections have been
significantly reduced in recent years by the introduction
of several interventions, including the implementation of
improved skin disinfection methods, first aliquot diversion
and bacterial testing (Corash, 2011). However, despite
these strategies, bacterial contamination of blood products
currently poses the most significant infectious risk in
transfusion medicine. Platelet concentrates (PCs) are
exquisitely susceptible to contamination since their storage
conditions of 20–24 uC with agitation are particularly
Abbreviations: CoNS, coagulase-negative staphylococci; CRA, Congo
red assay/agar; TSBg, glucose-enriched trypticase soy broth; PC,
platelet concentrate; PIA, polysaccharide intercellular adhesin.
A supplementary table is available with the online version of this paper.
050500 G 2013 SGM
amenable to bacterial growth (Braine et al., 1986). While
Gram-negative organisms are typically implicated in the
most severe adverse transfusion reactions, Gram-positive
bacteria constitute the majority of organisms responsible for
PC contamination (Brecher & Hay, 2005; Corash, 2011).
Specifically, coagulase-negative staphylococci (CoNS), the
most abundant group of species composing the commensal
microflora of human skin, are most frequently implicated in
bacterial contamination of PCs. These bacteria are likely
introduced at the time of venipuncture during blood
collection despite the implementation of the aforementioned prophylactic measures to prevent contamination
(Corash, 2011). S. capitis is a common CoNS that is often
recovered from contaminated PCs (Eder et al., 2009; Pearce
et al., 2011; Rood et al., 2011). Since 2009, Canadian Blood
Services has detected four strains of S. capitis by routine PC
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
Printed in Great Britain
1051
V. S. Greco-Stewart and others
screening, preventing transfusion of the contaminated
platelet units.
A ubiquitous constituent of the human skin microbiome
(Kloos & Schleifer, 1975), S. capitis accounts for ~5 % of all
clinical CoNS isolates (Oren & Merzbach, 1990). While S.
capitis is typically apathogenic, it has occasionally been
shown to cause illness such as skin infection, pneumonia
and urinary tract infection (Oren & Merzbach, 1990; Pal &
Ayyagari, 1989). Serious instances of S. capitis infection,
including prosthetic valve-related endocarditis (Nalmas
et al., 2008) and shunt-related and community-acquired
meningitis (Oud, 2011; Oren & Merzbach, 1990), have also
been reported. Furthermore, it has been estimated that S.
capitis accounts for up to 20 % of cases of neonatal sepsis
(Van Der Zwet et al., 2002), a life-threatening illness in this
highly vulnerable population. S. capitis has been shown to
survive and proliferate in blood components (WaltherWenke et al., 2010) and has been known to escape
automated bacterial detection methods (Murphy et al.,
2008), posing a potential threat to blood product recipients.
While bacteria associated with the skin microflora are
traditionally considered non-pathogenic, the ability of
certain strains to form biofilms, surface-associated populations of cells encased in an extracellular polymeric matrix,
increases their virulence by enhancing their capacity to
adhere to and colonize biotic and abiotic surfaces, evade
the immune response, and resist antimicrobial chemotherapy [reviewed by Flemming & Wingender (2010)]. We have
previously demonstrated that several strains of Grampositive (Staphylococcus epidermidis) and Gram-negative
(Serratia marcescens) bacteria recovered from contaminated platelet units form biofilms in the platelet storage
environment; remarkably, some strains that were unable to
form biofilms in traditional laboratory medium adopt a
biofilm-forming phenotype when grown under platelet
storage conditions (Greco et al., 2007; Greco-Stewart et al.,
2012). Additionally, strains of S. marcescens which form
biofilms in PCs demonstrate an increased propensity to
evade detection by automated culture screening (GrecoStewart et al., 2012).
Many genes have been identified in staphylococcal species,
including CoNS such as S. epidermidis, which contribute to
biofilm formation [reviewed by Götz (2002) and by Fey &
Olson (2010)]. Proteins, carbohydrates, teichoic acids and
DNA have all been shown to compose staphylococcal
biofilms; thus a variety of biofilm-associated genes and
operons involved in the four stages of biofilm growth,
namely adherence (primary attachment), accumulation,
maturation and dispersal, have been identified. While little
is known about biofilm formation in S. capitis, homologues
of some biofilm-associated genes, such as the icaADCB
operon, have been identified (de Silva et al., 2002). The ica
genes compose a genetic locus whose products are
responsible for the synthesis and export of polysaccharide
intercellular adhesin (PIA) in S. epidermidis (Heilmann
et al., 1996). PIA is a b-1,6-linked N-acetylglucosamine
1052
homoglycan (Mack et al., 1996) involved in the adhesion
and accumulation phases of biofilm growth and accounts
for the ‘slime’ produced in biofilm-positive isolates of S.
epidermidis (Heilmann et al., 1996; McKenney et al.,
1998).
The ambition of the present study was to characterize
strains of S. capitis (Rood et al., 2011) recovered from
contaminated platelet units with respect to their capacity to
form biofilms under platelet storage conditions and to
determine which bacterial factors contribute to biofilm
formation in this environment.
METHODS
Bacterial strains and growth conditions. Five strains of S. capitis
captured during routine platelet screening by automated culture using
the BacT/ALERT System (bioMérieux) were recovered from contaminated PCs at Sanquin Blood Supply Foundation (strains 512, 517,
521 and 525; Rood et al., 2011) and Canadian Blood Services (strain
07/2010). Prior to experimentation, strains were examined using API
Staph identification strips (bioMérieux) according to the manufacturer’s protocols to verify culture purity and to assess urease
production. Two reference strains of S. epidermidis, ATCC 35984
and ATCC 12228, obtained from the American Type Culture
Collection, were included as biofilm-positive and biofilm-negative
controls, respectively. All strains were cultivated in trypticase soy
broth (TSB; Difco) and grown on trypticase soy agar plates incubated
overnight at 37 uC unless otherwise stated. Strains were preserved at
280 uC in brain–heart infusion (BHI) broth (BD Biosciences)
supplemented with 15 % glycerol (w/v).
Platelet concentrates (PCs). Blood was obtained from healthy,
deferred volunteer donors after receipt of signed, informed consent.
Whole blood units were collected, screened for transmissible
diseases, and processed at the Canadian Blood Services Network
Centre for Applied Development (netCAD). PCs were prepared by
either the buffy-coat or apheresis methods in accordance with
Canadian Blood Services standard operating procedures. The use of
PCs and experimental approaches were approved by the Canadian
Blood Services Research Ethics Board.
Biofilm assays. A semiquantitative crystal violet (CV) staining assay
was used to assess the amounts of biofilm-related materials and cells
deposited on the surface of six-well tissue culture-treated polystyrene
plates (Fisher Scientific), similar to methods originally described by
Christensen et al. (1985) and modified in our laboratory (Greco et al.,
2007; Greco-Stewart et al., 2012). Biofilm assays were performed on
cultures grown in either TSB supplemented with 0.5 % glucose (TSBg;
Difco) or in in-date (,5 day old) PCs. Each well was inoculated with
~108 c.f.u. ml21 of each bacterial strain in duplicate. Strains grown in
TSBg were incubated at 37 uC without agitation overnight (~16 h)
while bacteria grown in PCs were incubated under platelet storage
conditions [22±2 uC for 5 days with agitation of ~64 rocks min21 on
a thermal rocker (Fisher)]. Following incubation, planktonic cells and
culture medium were removed and wells were washed three times
with sterile PBS (pH 7.4) and stained with 3 ml of a 3 % Gram CV
solution (BD Biosciences) for 30 min with agitation (~60 r.p.m. on a
horizontal shaker). Upon removal of the dye, wells were rinsed three
times with PBS and materials were eluted with 3 ml of 80 : 20
ethanol : acetone (v/v) solution. Measurements of samples from each
well were taken in triplicate at 492 nm using a microplate reader
(Expert Plus). Independent experiments were performed a minimum
of nine times on different days.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
Journal of Medical Microbiology 62
S. capitis biofilm formation in platelets
Scanning electron microscopy (SEM). Biofilm formation of S.
capitis strains on polystyrene pegs was assessed by SEM. Biofilms
were formed on the 96-peg lid of a 96-well plate known as the
Minimum Biofilm Eradication Concentration (MBEC) device
(Physiology & Genetics, Innovotech). MBEC devices were inoculated
with ~108 c.f.u. ml21 of each strain in either TSBg or PCs and
incubated as described above. Following incubation, pegs were
removed with sterile forceps, rinsed five times in PBS, preserved in
0.1 M cacodylate [Na(CH3)2AsO2?3H2O; J. B. EM Services] containing 2.5 % glutaraldehyde, and stored at 4 uC prior to examination.
Pegs were critical-point dried, sputtered with a 20 nm layer of gold
particles, and visualized by using the XL30 ESEM microscope
(Phillips) with an accelerating voltage of 7.5 kV, beam spot of 2, and
working distance 7.5 mm as previously described (Greco-Stewart
et al., 2012).
Biofilm disruption experiments. The chemical nature of the S.
capitis biofilm matrix was investigated by treatment with sodium
metaperiodate (disrupts polysaccharides) or proteinase K (disrupts
proteins) following previously published protocols (Stevens et al.,
2009; Fredheim et al., 2009). Pre-formed S. capitis biofilms grown in
TSBg or in PCs in six-well plates (as described above) were washed
with sterile 0.9 % saline solution (SS), followed by the addition of
2.9 ml of either 10 mM sodium metaperiodate in 50 mM sodium
acetate buffer (pH 4.5) or 100 mg ml21 proteinase K in 20 mM Tris
(pH 7.5) and 100 mM NaCl. The plates were then incubated at
37 uC for 2 h or overnight with proteinase K or sodium
metaperiodate, respectively. The treated biofilm cells were washed
with SS, air-dried and stained with CV as described above. The
assays were repeated in duplicate (non-disrupted control and
disrupted sample) four and two independent times in PCs and
TSBg, respectively.
Assessment of slime production. Congo red assay (CRA) for the
assessment of slime (extracellular polysaccharide) production was
performed based on the protocol of Freeman et al. (1989) and
modified as previously described (Greco et al., 2008). Congo red dye
(Sigma-Aldrich) and sucrose (Sigma-Aldrich) were dissolved in sterile
water, filter-sterilized, and added to autoclaved BHI agar to achieve
final concentrations of 0.8 g l21 and 36 g l21, respectively. Strains
were plated in duplicate and incubated for 24 h at 37 uC. Positive
slime production was scored when the majority of colonies had a
rough, black morphology whereas slime-negative strains appeared as
smooth, red-to-pink colonies. Assessment was performed in duplicate
with the assistance of a blinded volunteer who was not involved in the
research and was unaware of strain identity. The assay was performed
four times in duplicate.
PCR and DNA sequencing. PCR was used to amplify the icaAD
region and its cognate promoter (~1.6 kb). Primers were designed
based on published sequence data (AY146582; Møretrø et al., 2003)
and comprised a forward and reverse primers (59-gcgc ctt caa ttc taa
aat ctc ccc-39 and 59-gcgc acg acc ttt ctt aat ttt ttg g-39, respectively).
Similarly, primers to amplify icaR (icaR-FW: 59-gcgc ggg gga gat ttt
aga att gaa gaa-39 and icaR-REV: 59-gcgc ctc caa gta att gta taa aat
tcg-39) and icaBC (icaCB-FW: 59-gcgc tta gtg tga ttt cca act agg-39
and icaCB-REV: 59-gcgc aag aaa gaa agg tgg cta tgc tac-39) were
designed based on the sequence of the S. capitis ica locus available at
the accession number JF930147.1 (Cui et al., 2011). Reactions were
performed in a final reaction volume of 50 ml using OneTaq
Polymerase (New England Biolabs) according to the manufacturer’s
recommended protocol. Template DNA (5 ml per reaction) was
obtained from cell suspensions of S. capitis strains lysed in sterile,
nuclease-free water adjusted to a McFarland turbidity standard of
0.5. The reaction was as follows: 94 uC for 30 s, 30 cycles of 94 uC
for 30 s/53 uC for 45 s/68 uC for 1.45 min, 68 uC for 5 min, and
hold at 4 uC. Products were resolved by electrophoresis in 1 %
http://jmm.sgmjournals.org
agarose and purified using the QIAquick PCR Purification kit
(Qiagen). Sequencing was performed by Stemcore Laboratories
(Ottawa, ON) using the forward primer from the PCR as well as
internal primer 59-gcgc gtt ggt tac tgg gat act gat atg-39 (annealing at
position 830 of icaAD) to ensure complete gene coverage.
Sequencing of the icaR and icaBC genes was accomplished by using
the amplification primers and internal primers icaCB1358-FW (59gcgc aat gcg tcg tta ctt act-39) and icaCB1935-FW (59-gcgc att caa att
ctt tat ctg tca cac-39). Multiple alignment of the Ica proteins was
performed using Multalin (Corpet, 1988) and CLUSTAL W (Thompson
et al., 1994) web-based software. Sequences of the S. capitis Ica
proteins available at JF930147.1 (Cui et al., 2011) were included as
references.
Statistical analysis. For comparison of biofilm formation in TSBg
and PCs, means and standard deviations were calculated using
Statistical Analysis System SAS 9.1.3 software (SAS Institute) and
plotted using Microsoft Excel. Error bars represent the mean plus one
standard deviation. Statistical significance (P-value) was calculated by
pairwise comparison of data by non-parametric t-test using SAS and a
value of P,0.05 was interpreted as statistically significant. For the
biofilm disruption experiments, means and SDs were calculated by
treatments and strain types for each environment. Differences and
their 95 % confidence intervals were calculated to show the
magnitude of the difference. For each type of strain, the paired ttest was applied to find if the difference between treatment and
control was statistically significant. Overall treatment effects were
evaluated through the mixed model analysis. In the model, potential
correlation between paired control and treatment units was
controlled by fitting random effects. A P-value of ,0.05 was
considered statistically significant.
RESULTS
Clinical isolates of S. capitis form biofilms in PCs
Five strains of CoNS recovered from contaminated PCs
that were previously identified as S. capitis (Rood et al.,
2011) were confirmed to be S. capitis subspecies capitis
by their negative urease reaction (Bannerman & Kloos,
1991). Biofilm formation assays showed that S. capitis
strains 512 and 525 do not form biofilms in TSBg while
strains 517, 521 and 07/2010 were biofilm-positive in
this medium (Fig. 1; P,0.0005). In the PC environment,
all clinical strains of S. capitis formed biofilms based on
the biofilm-negative threshold established using the
negative control strain S. epidermidis ATCC 12228
grown in TSBg (Fig. 1, baseline indicated with dotted
line). Notably, S. capitis strains 512 and 525 became
biofilm-positive when grown in PCs as previously
reported with S. epidermidis ATTC 12228 (Greco et al.,
2007); biofilm formation was significantly different
between growth in TSBg and in PCs for these strains
(P,0.05). Biofilm-positive control strain S. epidermidis
ATCC 35984 remained biofilm-positive in PCs although
it exhibited diminished biofilm formation capacity
compared with when grown in TSBg (P,0.001). While
lack of biofilm formation in TSBg correlated with lack of
slime production on CRA for strains 512 and 525,
biofilm formation by strain 521 was not associated with
slime production on CRA (Table 1).
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
1053
V. S. Greco-Stewart and others
2.0
TSBg
PC
1.8
1.6
A492 nm
1.4
1.2
*
*
*
*
1.0
0.8
0.6
0.4
0.2
0
35984
12228
512
S. epidermidis
517
521
525
07/2010
S. capitis clinical isolates
Fig. 1. Biofilm formation of clinical S. capitis strains grown in laboratory medium (TSBg) and in platelet concentrates (PCs).
Asterisks represent strains for which biofilm formation in TSBg differed significantly from those formed in PCs (P,0.05). The
dotted line indicates the threshold for classification of a biofilm-negative phenotype in TSBg as compared with control strain S.
epidermidis 12228.
S. capitis biofim matrix is mainly composed of
proteins
525) an increase in absorbance at 492 nm was observed
after treatment (data not shown).
All S. capitis pre-formed biofilms were disrupted when
treated with proteinase K (Fig. 2, Table 2). A mixed model
analysis showed that treatment with proteinase K significantly reduced pre-formed biofilms by all strains in
comparison with the non-treated controls in both TSBg
(P50.0038) and PCs (P,0.0001). In addition, no differences in susceptibility to treatment were observed between
biofilms formed in TSBg (P50.0745) or in PCs
(P50.1615). Only biofilms formed by strain 07/2010 in
PCs were significantly disrupted after treatment with
sodium metaperiodate (P,0.0001), indicating that a mix
of polysaccharide and proteins is present in the biofilm
matrix of this strain. Interference of the platelet milieu with
the sodium metaperiodate treatment is suggested by the
observation that in some cases (e.g. strains 512, 521 and
Biofilm architecture varies between cells grown in
TSBg and in PCs
To further characterize biofilm formation by S. capitis
strains in different environments, SEM was used to observe
differences in biofilm architecture and composition.
Macroscopically, S. capitis biofilms grown in PCs acquire
a pale tan to orange hue when grown under platelet storage
conditions whereas S. epidermidis biofilms remain white;
biofilms are visible by dissection microscopy for biofilmpositive strains, whereas biofilm-negative pegs appear
smooth. SEM of representative fields of biofilm-positive
and -negative strains are shown in Fig. 3. It was observed
that biofilms grown on pegs in TSBg existed primarily as
confluent (ATCC 35984) or patchy (517) monolayers in
Table 1. Biofilm-related characteristics of S. capitis strains isolated from contaminated PCs
NA, Not applicable; BF, biofilm formation; CRA, Congo red agar; TSBg, trypticase soy broth supplemented with 0.5 % glucose; PC, platelet
concentrate.
Strain BF in TSBg BF in PCs Slime production
on CRA
Presence of ica genes
icaR
Amino acid substitutions in Ica proteins
icaADBC
IcaR
IcaA
512
517
521
No
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
Yes
Yes
No
Yes
Yes
NA
NA
None
None
525
07/2010
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
None
None
None
N107D, G162D,
A197D L288I
None
None
1054
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
IcaD
IcaB
IcaC
NA
NA
NA
None
None
None
None K136E, I150V L67F,
M326V
None
None
None
None
None
None
Journal of Medical Microbiology 62
S. capitis biofilm formation in platelets
(a)
2.00
OD492
1.50
Control
Proteinase K-treated
1.00
0.50
0.00
517
521
07/2010
(b)
OD492
0.50
0.00
512
517
521
525
biofilm-forming strains whereas scattered cells or small cell
clusters were observed for biofilm-negative strains (ATCC
12228 and 512). When strains were grown in PCs, biofilms
appeared as confluent masses composed of cells and
extracellular materials which were several layers thick.
Discrete platelets were not observed although much of the
extracellular matrix was consistent with previous observations of platelet-derived materials (Greco-Stewart et al.,
2012).
Fig. 2. S. capitis biofilm disruption by treatment with proteinase K. (a) TSBg and (b) PCs.
07/2010
2011). Of the clinical isolates, only strain 521 deviated
from the IcaA (N107D, G162D, A197D and L288I), IcaB
(K136E and I150V) and IcaC (L67F and M326V)
consensus sequences (Table 1). Potential regulatory
elements in the region upstream of the icaA start codon
were also examined and the presence of a GAT
transversion at the 210 position and a GAA transition
at position 229 was noted in strain 521. The IcaR and
IcaD sequences were completely identical among clinical
and reported strains.
Profile of biofilm-associated genes present in
clinical strains of S. capitis
Many genes have been identified in S. aureus and S.
epidermidis that contribute to various stages of biofilm
formation (reviewed by Fey & Olson, 2010). Since little is
known about biofilm-related genes in S. capitis, we chose to
selectively amplify genes bearing homology to biofilmassociated genes of S. epidermidis, a closely related CoNS.
Genes of the icaADBC locus and its transcriptional
regulator icaR were successfully amplified for all strains
with the exception of S. capitis 512 (Table 1). The upstream
regulatory sequence of icaADBC was also amplified since
regulatory elements are contained therein; specifically, the
IcaR binding region is located in a 28 nt region positioned
17 nt upstream from the start codon of icaA (Jeng et al.,
2008).
S. capitis icaR, icaADBC and cognate upstream element
were sequenced and genes translated for strains 517, 521,
525 and 07/2010 and compared with each other and with
a previously reported sequence (JF930147.1; Cui et al.,
http://jmm.sgmjournals.org
Table 2. Disruption of the S. capitis biofilm matrix
NA, Not applicable; TSBg, trypticase soy broth supplemented with
0.5 % glucose; PC, platelet concentrates.
Strain
Biofilm formation
Biofilm disruption
Na metaperiodate Proteinase K
512
517
521
525
07/2010
TSBg
PCs
TSBg
PCs
TSBg
PCs
TSBg
PCs
TSBg
PCs
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
NA
NA
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
NA
NA
No
No
Yes
Yes
Yes
Yes
1055
V. S. Greco-Stewart and others
TSBg
the matrix of S. capitis biofilms must be of a heterogeneous
nature including other components such as DNA. Similar
observations have been reported for the coagulase-negative
Staphylococcus haemolyitcus, another recognized neonate
pathogen (Fredheim et al., 2009).
PC
35984
5 µm
5 µm
5 µm
5 µm
5 µm
5 µm
5 µm
5 µm
12228
512
517
Fig. 3. SEM of CoNS strains grown in TSBg and in PCs. Control
strains S. epidermidis ATCC 35984 (biofilm-positive) and ATCC
12228 (biofilm-negative) are shown as well as clinical isolates
512 (biofilm-negative) and 517 (biofilm-positive) of S. capitis.
Representative fields are shown at 5000¾ magnification.
DISCUSSION
In the present study, we have characterized the biofilm
formation properties of strains of S. capitis, an important
neonate pathogen (Van Der Zwet et al., 2002), recovered
from contaminated platelet units, using several criteria and
have observed various unique profiles with respect to
biofilm-associated phenotypes among these strains. We
have also noticed that strains which display a biofilmnegative phenotype in conventional laboratory media
convert to a biofilm-positive phenotype in PCs, as observed
for other Gram-negative (Greco-Stewart et al., 2012) and
Gram-positive species (Greco et al., 2007). Differences were
unrelated to subspecies, since all strains were determined to
be S. capitis subspecies capitis, and unrelated to plasma
sensitivity since all strains were able to survive and grow in
PCs.
Biofilm disruption studies indicate that, with the exception
of S. capitis 07/2010, all strains have a biofilm matrix
composed mainly of proteins. Since incomplete biofilm
disruption was accomplished by proteinase K treatment
and disruption with sodium metaperiodate had little effect,
1056
In the present study, the S. capitis biofilm phenotype does
not always bear the hallmark properties of classic PIAmediated biofilm formation. The presence of wild-type or
mutant Ica proteins, responsible for PIA biosynthesis, does
not have a relevant impact on biofilm formation by this
species. The phenotypes of strains 521 and 525, for example,
are suggestive of PIA-independent biofilm formation in PCs.
While S. capitis 521 has multiple mutations in the IcaA, IcaB
and IcaC proteins, its ability to form biofilms in TSBg and in
PCs is not impaired. Strain 525 possesses wild-type Ica
proteins and yet is still unable to form biofilms in regular
media. Notably, ica-negative S. capitis strain 512 displayed a
typical biofilm-negative phenotype in TSBg but adopted a
biofilm-positive phenotype when grown in PCs, similar to
what has been observed with the biofilm-negative strain S.
epidermidis ATCC 12228 (Greco et al., 2007). It thus appears
that ica-negative strains of S. capitis are indeed capable of
biofilm formation under certain circumstances and that
environmental cues might contribute to ica-independent
biofilm formation in these strains.
Biofilms confer protective benefits to bacteria, including
reduced susceptibility to antimicrobials, protection from
the immune system and resistance to sheer stresses.
However, it has been demonstrated that S. epidermidis
ica-positive strains are at a disadvantage with respect to
skin colonization when compared with ica-negative
commensal strains (Rogers et al., 2008). It can be
speculated that strains lacking the ica operon, but that
are able to form biofilms in vivo, would have a heightened
potential for opportunistic pathogenicity. This could
explain why ica-negative biofilm-forming strains are often
isolated from clinical samples (Petrelli et al., 2006; Rohde
et al., 2007; Qin et al., 2007; Fredheim et al. 2009). It has
also been shown that biofilm-associated proteins such as
Embp are sufficient to stimulate staphylococcal biofilm
formation in vitro in the absence of the icaADBC operon
and other biofilm-related genes such as aap (Christner
et al., 2010; Los et al., 2010). These observations suggest
that a delicate balance exists with respect to the possession
and expression of biofilm-associated genes in order to
optimize bacterial survival under various physiological
conditions.
It has been shown that the ica genes are required for
attachment of S. epidermidis to polystyrene (and potentially
other hydrophilic surfaces; Heilmann et al., 1996);
however, pre-conditioning these surfaces with biomaterials
(such as by the deposition of serum proteins resulting from
incubation with PCs) could stimulate biofilm initiation in
ica-negative strains. Serum conditioning has been shown to
promote binding of bacteria to surfaces via microbial
adhesins recognising extracellular matrix macromolecules
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
Journal of Medical Microbiology 62
S. capitis biofilm formation in platelets
(MSCRAMMs; Patti & Höök, 1994). S. epidermidis has
been shown to bind, aggregate and activate platelets via
SdrG, a protein involved in the initial attachment phase of
biofilm formation (Brennan et al., 2009). S. capitis
possesses an SdrG homologue, SdrX, that has been shown
to bind collagen VI (Liu et al. 2004), a component of the
extracellular matrix that is also secreted by macrophages
(Schnoor et al., 2008). It is thus expected that some
proteinaceous factor(s) facilitate biofilm attachment and
accumulation in S. capitis ica-negative isolates, and that
platelet storage conditions are conducive to biofilm
formation in numerous strains that would typically appear
biofilm-negative in vitro in conventional media.
We have also shown that slime production on Congo red
agar is not an accurate tool for determining the presence
of the ica genes or the production of polysaccharide
intercellular adhesin. Similar observations were previously
reported by de Silva et al. (2002). In an examination of 180
CoNS strains isolated from a neonatal intensive care unit,
the authors demonstrated that the presence of ica genes is
not a definitive predictor of biofilm production in these
organisms. In this study, it was observed that only 59 % of
ica-positive strains formed biofilms, and that presence of
the ica genes did not always correlate with slime
production on Congo red agar. These results suggest that
genetic regulation of the ica operon and not the mere
presence of these genes is the determinate factor in biofilm
production among clinical isolates. Though S. capitis strain
525 does not possess any mutations in the IcaR binding
region upstream from icaA (Jeng et al., 2008), it can be
speculated that its inability to form biofilms could result
from aberrant regulation of ica operon expression and that
growth in PCs stimulates biofilm formation in an icaindependent manner.
S. capitis 521 is remarkable as it forms biofilms but fails to
react with Congo red agar despite harbouring multiple
mutations in the Ica proteins (Table 1). This could be due
either to polysaccharide-independent biofilm formation as
described above or to differences in polysaccharide
composition. The use of Congo red in revealing polysaccharide production associated with biofilm production in
S. aureus is not advisable since the majority of strains fail to
react with the dye due to molecular differences in their
polysaccharide but form robust biofilms when submitted
to analysis by crystal violet assay (Knobloch et al., 2002; Taj
et al., 2012).
Our data show that all isolates of S. capitis recovered from
contaminated PCs form biofilms in the platelet storage
environment. These observations are congruous with other
examinations in our laboratory that show that the clinical
isolates of both Gram-positive and Gram-negative organisms demonstrate enhanced biofilm-forming potential in
this milieu (Greco et al., 2007; Greco-Stewart et al., 2012).
This poses an increased danger in transfusion medicine
since biofilm-forming strains are more likely to subvert
detection by automated culture (Greco-Stewart et al., 2012)
http://jmm.sgmjournals.org
and have increased virulence if tainted PC units are
transfused. During transfusion, dislodged biofilms can be
transferred to the recipient and then become foci of
infection, particularly if the patient has implanted biomedical devices. S. capitis is particularly dangerous to
premature neonates, a vulnerable demographic who often
receive multiple transfusions to treat complications
associated with preterm birth and who are particularly
susceptible to development of sepsis and death as a result
of staphylococcal infection (de Silva et al., 2002; Van Der
Zwet et al., 2002). Characterization of biofilm formation of
common blood contaminants is thus recommended in
order to develop improved methods of detection and
eradication of these organisms, contributing to the
ultimate goal of improving the safety of our blood supply.
ACKNOWLEDGEMENTS
We thank the staff of Canadian Blood Services netCAD (Vancouver,
BC) for the collection, manufacture and shipment of PCs, and the
volunteer donors from whom samples were collected. We are grateful
to Dr Judy Hannon and the staff at the Canadian Blood Services
Edmonton site for providing S. capitis strain 07/2010 and for their
input during the initial characterization of this isolate. We also thank
Dr Q.-L. Yi for performing statistical analyses of biofilm data. V. G. S. was the recipient of a Post Doctoral Fellowship Award from
Canadian Blood Services and Health Canada. H. A. was funded by a
scholarship from the Faculty of Applied Medical Sciences, Taibah
University, Madina, Saudi Arabia. Funding of this research was
provided by Canadian Blood Services and Health Canada.
REFERENCES
Bannerman, T. L. & Kloos, W. E. (1991). Staphylococcus capitis subsp.
ureolyticus subsp. nov. from human skin. Int J Syst Bacteriol 41, 144–
147.
Braine, H. G., Kickler, T. S., Charache, P., Ness, P. M., Davis, J.,
Reichart, C. & Fuller, A. K. (1986). Bacterial sepsis secondary to
platelet transfusion: an adverse effect of extended storage at room
temperature. Transfusion 26, 391–393.
Brecher, M. E. & Hay, S. N. (2005). Bacterial contamination of blood
components. Clin Microbiol Rev 18, 195–204.
Brennan, M. P., Loughman, A., Devocelle, M., Arasu, S., Chubb, A. J.,
Foster, T. J. & Cox, D. (2009). Elucidating the role of Staphylococcus
epidermidis serine-aspartate repeat protein G in platelet activation.
J Thromb Haemost 7, 1364–1372.
Christensen, G. D., Simpson, W. A., Younger, J. J., Baddour, L. M.,
Barrett, F. F., Melton, D. M. & Beachey, E. H. (1985). Adherence of
coagulase-negative staphylococci to plastic tissue culture plates: a
quantitative model for the adherence of staphylococci to medical
devices. J Clin Microbiol 22, 996–1006.
Christner, M., Franke, G. C., Schommer, N. N., Wendt, U., Wegert, K.,
Pehle, P., Kroll, G., Schulze, C., Buck, F. & other authors (2010). The
giant extracellular matrix-binding protein of Staphylococcus epidermidis mediates biofilm accumulation and attachment to fibronectin.
Mol Microbiol 75, 187–207.
Corash, L. (2011). Bacterial contamination of platelet components:
potential solutions to prevent transfusion-related sepsis. Expert Rev
Hematol 4, 509–525.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
1057
V. S. Greco-Stewart and others
Corpet, F. (1988). Multiple sequence alignment with hierarchical
clustering. Nucleic Acids Res 16, 10881–10890.
Cui, B., Smooker, P. M., Rouch, D. A., Daley, A. J. & Deighton, M. A.
(2013). Differences between two clinical Staphylococcus capitis
subspecies as revealed by biofilm, antibiotic resistance, and pulsedfield gel electrophoresis profiling. J Clin Microbiol 51, 9–14.
de Silva, G. D. I., Kantzanou, M., Justice, A., Massey, R. C., Wilkinson,
A. R., Day, N. P. J. & Peacock, S. J. (2002). The ica operon and biofilm
production in coagulase-negative staphylococci associated with
carriage and disease in a neonatal intensive care unit. J Clin
Microbiol 40, 382–388.
Los, R., Sawicki, R., Juda, M., Stankevic, M., Rybojad, P., Sawicki, M.,
Malm, A. & Ginalska, G. (2010). A comparative analysis of phenotypic
and genotypic methods for the determination of the biofilm-forming
abilities of Staphylococcus epidermidis. FEMS Microbiol Lett 310, 97–
103.
Mack, D., Fischer, W., Krokotsch, A., Leopold, K., Hartmann, R.,
Egge, H. & Laufs, R. (1996). The intercellular adhesin involved in
biofilm accumulation of Staphylococcus epidermidis is a linear beta1,6-linked glucosaminoglycan: purification and structural analysis.
J Bacteriol 178, 175–183.
McKenney, D., Hübner, J., Muller, E., Wang, Y., Goldmann, D. A. &
Pier, G. B. (1998). The ica locus of Staphylococcus epidermidis encodes
Eder, A. F., Kennedy, J. M., Dy, B. A., Notari, E. P., Skeate, R.,
Bachowski, G., Mair, D. C., Webb, J. S., Wagner, S. J. & other authors
(2009). Limiting and detecting bacterial contamination of apheresis
production of the capsular polysaccharide/adhesin. Infect Immun 66,
4711–4720.
platelets: inlet-line diversion and increased culture volume improve
component safety. Transfusion 49, 1554–1563.
Møretrø, T., Hermansen, L., Holck, A. L., Sidhu, M. S., Rudi, K. &
Langsrud, S. (2003). Biofilm formation and the presence of the
Fey, P. D. & Olson, M. E. (2010). Current concepts in biofilm
intercellular adhesion locus ica among staphylococci from food and
food processing environments. Appl Environ Microbiol 69, 5648–5655.
formation of Staphylococcus epidermidis. Future Microbiol 5, 917–933.
Flemming, H. C. & Wingender, J. (2010). The biofilm matrix. Nat Rev
Microbiol 8, 623–633.
Fredheim, E. G. A., Klingenberg, C., Rohde, H., Frankenberger, S.,
Gaustad, P., Flaegstad, T. & Sollid, J. E. (2009). Biofilm formation by
Staphylococcus haemolyticus. J Clin Microbiol 47, 1172–1180.
Freeman, D. J., Falkiner, F. R. & Keane, C. T. (1989). New method for
detecting slime production by coagulase negative staphylococci. J Clin
Pathol 42, 872–874.
Götz, F. (2002). Staphylococcus and biofilms. Mol Microbiol 43, 1367–
1378.
Greco, C., Martincic, I., Gusinjac, A., Kalab, M., Yang, A. F. &
Ramı́rez-Arcos, S. (2007). Staphylococcus epidermidis forms biofilms
under simulated platelet storage conditions. Transfusion 47, 1143–
1153.
Greco, C., Mastronardi, C., Pagotto, F., Mack, D. & Ramı́rez-Arcos, S.
A. (2008). Assessment of biofilm-forming ability of coagulase-negative
staphylococci isolated from contaminated platelet preparations in
Canada. Transfusion 48, 969–977.
Greco-Stewart, V. S., Brown, E. E., Parr, C., Kalab, M., Jacobs, M. R.,
Yomtovian, R. A. & Ramı́rez-Arcos, S. M. (2012). Serratia marcescens
strains implicated in adverse transfusion reactions form biofilms in
platelet concentrates and demonstrate reduced detection by automated culture. Vox Sang 102, 212–220.
Heilmann, C., Schweitzer, O., Gerke, C., Vanittanakom, N., Mack, D.
& Götz, F. (1996). Molecular basis of intercellular adhesion in the
biofilm-forming Staphylococcus epidermidis. Mol Microbiol 20, 1083–
1091.
Jeng, W. Y., Ko, T. P., Liu, C. I., Guo, R. T., Liu, C. L., Shr, H. L. & Wang,
A. H. (2008). Crystal structure of IcaR, a repressor of the TetR family
implicated in biofilm formation in Staphylococcus epidermidis. Nucleic
Acids Res 36, 1567–1577.
Kloos, W. E. & Schleifer, K. H. (1975). Isolation and characterization
of staphylococci from human skin II. Descriptions of four new
species: Staphylococcus warneri, Staphylococcus capitis, Staphylococcus
hominis, and Staphylococcus simulans. Int J Syst Bacteriol 25, 62–
79.
Murphy, W. G., Foley, M., Doherty, C., Tierney, G., Kinsella, A.,
Salami, A., Cadden, E. & Coakley, P. (2008). Screening platelet
concentrates for bacterial contamination: low numbers of bacteria
and slow growth in contaminated units mandate an alternative
approach to product safety. Vox Sang 95, 13–19.
Nalmas, S., Bishburg, E., Meurillio, J., Khoobiar, S. & Cohen, M. N.
(2008). Staphylococcus capitis prosthetic valve endocarditis: report of
two rare cases and review of literature. Heart Lung 37, 380–384.
Oren, I. & Merzbach, D. (1990). Clinical and epidemiological
significance of species identification of coagulase-negative staphylococci in a microbiological laboratory. Isr J Med Sci 26, 125–128.
Oud, L. (2011). Community-acquired meningitis due to
Staphylococcus capitis in the absence of neurologic trauma, surgery,
or implants. Heart Lung 40, 467–471.
Pal, N. & Ayyagari, A. (1989). Species identification & methicillin
resistance of coagulase negative staphylococci from clinical specimens. Indian J Med Res 89, 300–305.
Patti, J. M. & Höök, M. (1994). Microbial adhesins recognizing
extracellular matrix macromolecules. Curr Opin Cell Biol 6, 752–758.
Pearce, S., Rowe, G. P. & Field, S. P. (2011). Screening of platelets for
bacterial contamination at the Welsh Blood Service. Transfus Med 21,
25–32.
Petrelli, D., Zampaloni, C., D’Ercole, S., Prenna, M., Ballarini, P., Ripa,
S. & Vitali, L. A. (2006). Analysis of different genetic traits and their
association with biofilm formation in Staphylococcus epidermidis
isolates from central venous catheter infections. Eur J Clin Microbiol
Infect Dis 25, 773–781.
Qin, Z., Yang, X., Yang, L., Jiang, J., Ou, Y., Molin, S. & Qu, D. (2007).
Formation and properties of in vitro biofilms of ica-negative
Staphylococcus epidermidis clinical isolates. J Med Microbiol 56, 83–93.
Rogers, K. L., Rupp, M. E. & Fey, P. D. (2008). The presence of
icaADBC is detrimental to the colonization of human skin by
Staphylococcus epidermidis. Appl Environ Microbiol 74, 6155–6157.
Rohde, H., Burandt, E. C., Siemssen, N., Frommelt, L., Burdelski, C.,
Wurster, S., Scherpe, S., Davies, A. P., Harris, L. G. & other authors
(2007). Polysaccharide intercellular adhesin or protein factors in
Evaluation of different detection methods of biofilm formation in
Staphylococcus aureus. Med Microbiol Immunol (Berl) 191, 101–106.
biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections.
Biomaterials 28, 1711–1720.
Liu, Y., Ames, B., Gorovits, E., Prater, B. D., Syribeys, P., Vernachio,
J. H. & Patti, J. M. (2004). SdrX, a serine-aspartate repeat protein
Rood, I. G. H., de Korte, D., Ramı́rez-Arcos, S., Savelkoul, P. H. M. &
Pettersson, A. (2011). Distribution, origin and contamination risk of
expressed by Staphylococcus capitis with collagen VI binding activity.
Infect Immun 72, 6237–6244.
coagulase-negative staphylococci from platelet concentrates. J Med
Microbiol 60, 592–599.
Knobloch, J. K., Horstkotte, M. A., Rohde, H. & Mack, D. (2002).
1058
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
Journal of Medical Microbiology 62
S. capitis biofilm formation in platelets
Schnoor, M., Cullen, P., Lorkowski, J., Stolle, K., Robenek, H., Troyer,
D., Rauterberg, J. & Lorkowski, S. (2008). Production of type VI
collagen by human macrophages: a new dimension in macrophage
functional heterogeneity. J Immunol 180, 5707–5719.
through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Stevens, N. T., Greene, C. M., O’Gara, J. P. & Humphreys, H. (2009).
Van Der Zwet, W. C., Debets-Ossenkopp, Y. J., Reinders, E., Kapi,
M., Savelkoul, P. H. M., Van Elburg, R. M., Hiramatsu, K. &
Vandenbroucke-Grauls, C. M. J. E. (2002). Nosocomial spread of a
Biofilm characteristics of Staphylococcus epidermidis isolates associated with device-related meningitis. J Med Microbiol 58, 855–862.
Staphylococcus capitis strain with heteroresistance to vancomycin in a
neonatal intensive care unit. J Clin Microbiol 40, 2520–2525.
Taj, Y., Essa, F., Aziz, F. & Kazmi, S. U. (2012). Study on biofilm-
Walther-Wenke, G., Schrezenmeier, H., Deitenbeck, R., Geis, G.,
Burkhart, J., Höchsmann, B., Sireis, W., Schmidt, M., Seifried, E. &
other authors (2010). Screening of platelet concentrates for bacterial
forming properties of clinical isolates of Staphylococcus aureus. J Infect
Dev Countries 6, 403–409.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
http://jmm.sgmjournals.org
contamination: spectrum of bacteria detected, proportion of
transfused units, and clinical follow-up. Ann Hematol 89, 83–91.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Fri, 28 Oct 2016 22:52:07
1059