Journal of Medical Microbiology (2012), 61, 1428–1434
DOI 10.1099/jmm.0.041046-0
Autolytic activity and molecular characteristics of
Staphylococcus haemolyticus strains with induced
vancomycin resistance
Jung Wook Kim, Gyung Tae Chung, Jung Sik Yoo, Yeong Seon Lee
and Jae Il Yoo
Correspondence
Jae Il Yoo
Division of Antimicrobial Resistance, Center for Infectious Diseases, Korea National Institute of
Health, Osong, Republic of South Korea
[email protected]
Received 27 November 2011
Accepted 14 July 2012
The aim of this study was to investigate the molecular characteristics of induced vancomycin
resistance in Staphylococcus haemolyticus. Autolytic properties and phenotypic
characteristics of passage-selected vancomycin-resistant S. haemolyticus strains were
examined. In addition, expression of autolysis-related genes (atl, lrgAB, sarA and lytS) was
investigated using the RNase protection assay (RPA). The RPA results indicated that only the
expression of the atl gene was significantly upregulated (2.5- to 6-fold increase) in
vancomycin-intermediate and vancomycin-resistant strains. The vancomycin-resistant strains
exhibited lower expression of murein hydrolase proteins and reduced autolytic activity
compared with the parent strain. In addition, a reduced growth rate, cell wall thickening and
higher survival rate in the presence of lysostaphin were observed in vancomycin-intermediate
and vancomycin-resistant induced strains compared with the parent strain. In conclusion,
altered autolytic properties, in particular upregulation of the atl gene, may contribute to
vancomycin resistance in S. haemolyticus.
INTRODUCTION
Coagulase-negative staphylococci (CoNS) have been recognized as important causative agents of nosocomial bacteraemia. In particular, the frequency of Staphylococcus
haemolyticus infection is second only to that of Staphylococcus epidermidis among clinical isolates of CoNS (Vignaroli
et al., 2006). Some recent reports have recognized S.
haemolyticus as the cause of severe infections, including
meningitis, skin and skin structure infections, prosthetic
joint infections, bacteraemia and even endocarditis (Yu
et al., 2010). Because of the rising incidence of meticillin
resistance, glycopeptides have been recommended as
therapeutic agents for serious staphylococcal infections
(Nunes et al., 2006). However, the extensive use of
glycopeptides has decreased the susceptibility of staphylococcal species to these agents. In addition to the tendency
of S. haemolyticus to develop resistance to multiple
antibiotics, it was the first Gram-positive pathogen to
acquire glycopeptide resistance; this occurred earlier than
in other staphylococcal species and enterococci (Biavasco
et al., 2000). However, the molecular characteristics of
glycopeptide resistance in S. haemolyticus are not yet
completely understood.
Abbreviation: RPA, RNase protection assay.
1428
Various mechanisms of vancomycin resistance have been
reported in vancomycin-intermediate Staphylococcus aureus (VISA) (Cui et al., 2003; Finan et al., 2001; Pfeltz et al.,
2000; Sieradzki & Tomasz, 1997, 1999). Furthermore, a
few reports have indicated that the mechanism underlying glycopeptide resistance in S. epidermidis, S. haemolyticus, Staphylococcus hominis and Staphylococcus
sciuri is similar to that described in VISA and heteroVISA strains (Sieradzki et al., 1998, 1999). An S.
haemolyticus strain with reduced vancomycin susceptibility has been reported to exhibit a thicker cell wall
than that of the parent strain and variable rates of
autolysis (Nunes et al., 2006). However, the molecular
characteristics of vancomycin resistance in S. haemolyticus are poorly documented. In this study, we induced
vancomycin resistance in susceptible S. haemolyticus and
investigated the phenotypic characteristics and expression of autolytic activity-related genes in the induced
strains.
METHODS
Clinical strains. Staphylococcal isolates were collected from tertiary
hospitals in Korea. The staphylococcal strains were identified as
CoNS by catalase, coagulase and DNase testing. The CoNS were
identified to the species level by using the VITEK system (bioMérieux). In this study, 10 staphylococcal strains were identified as
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S. haemolyticus with induced vancomycin resistance
S. haemolyticus. Among the S. haemolyticus strains, the S.
haemolyticus GS52 strain showed a vancomycin-susceptible MIC
(MIC 4), which was the lowest MIC for vancomycin among the
collected S. haemolyticus strains. To induce vancomycin resistance in
a susceptible strain, the S. haemolyticus GS52 strain was selected for
further study. This strain has been described in a previous study
(Yoo et al., 2010).
Generation of mutants. Mutants were selected from the clinical S.
haemolyticus GS52 strain by streaking 10 ml of an overnight culture
onto brain heart infusion (BHI) plates containing 4 mg vancomycin
ml21 at 35 uC. Cultures were grown in BHI broth (Difco) containing
4 mg vancomycin ml21. This procedure was repeated with increasing
gradient concentrations of vancomycin until stable mutants with
vancomycin MIC values of 16 (GS52V16) and 48 (GS52V48) mg ml21
were obtained.
Growth curves. The S. haemolyticus strains were grown in BHI broth
at 37 uC with aeration. In each experiment, overnight cultures were
diluted 1000-fold in 10 ml fresh BHI broth medium and grown at
37 uC with shaking at 200 r.p.m. Growth was followed by monitoring
the OD600 at hourly intervals.
Antimicrobial susceptibility testing. The MICs of vancomycin,
teicoplanin, oxacillin, penicillin, imipenem, linezolid, daptomycin,
tigecycline, erythromycin and rifampicin for all derivatives and parent
strains were determined by Etest (AB Biodisk) according to the
manufacturer’s instructions.
Autolysis assay. Autolysis assays were performed for mutant and
parental strains as previously described (Fournier & Hooper, 2000).
Cells were grown exponentially to an OD600 of about 0.3. The cultures
were then chilled rapidly and centrifuged; cells were then washed once
with ice-cold distilled water and resuspended in 50 mM Tris/HCl
(pH 7.5) and 0.1 % Triton X-100 to obtain an OD600 of 1.0. The cells
were then incubated at 35 uC with shaking, and the changes in OD600
were measured. Results were normalized to OD600 at time zero
(OD0), i.e. per cent lysis at time t5[(OD0 ODt)/OD0]6100. All the
results shown represent the mean values of at least two independent
determinations.
Zymographic analysis. Zymographic analysis of peptidoglycan
hydrolase profiles was performed as previously described (Zheng
et al., 2007). Briefly, overnight cultures were centrifuged at 10 000 g at
4 uC for 10 min, and the supernatants were collected, filter-sterilized
and concentrated 100-fold with a Centricon concentrator (Millipore).
The concentration of total proteins in each sample was determined
using the Bradford assay (Bio-Rad) according to the manufacturer’s
instructions. A total of 30 mg of proteins from each sample was
resolved on an 8 % SDS-PAGE gel containing 0.2 % autoclaved and
lyophilized wet S. haemolyticus cells or Micrococcus lysodeikticus cells
(Sigma-Aldrich). Following electrophoresis, the gels were washed with
water and incubated overnight in renaturation buffer [25 mM Tris/
HCl (pH 7.0) containing 1 % Triton X-100] at 37 uC. The gel was
stained with 1 % methylene blue in 0.01 % KOH and destained in
deionized water. Zones of hydrolysis appeared as white bands in the
gels; black bands indicating regions of murein hydrolase activity were
also observed.
RNase protection assay (RPA). The strains were cultured in 10 ml
BHI and the cells were incubated at 37 uC with shaking until they
reached an OD600 of 0.4. Cells were harvested by centrifugation, and
total RNA was isolated from the pellet by using an RNeasy mini kit
(Qiagen) according to the manufacturer’s instructions. RNA
antisense probes were prepared using a MAXIscript kit (Ambion).
RNA antisense probes were transcribed with a template containing a
T7 phage promoter. The antisense probe template was prepared by
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PCR with genomic DNA as the template, and primer sets with the T7
phage promoter were incorporated into the downstream primer
(Table 1). PCR products were eluted, dried and resuspended in 20 ml
water. The template was transcribed with 2 U T7 RNA polymerase in
a reaction mixture containing Ambion 16 transcription buffer, 1 mg
template, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.3 mM biotinlabelled UTP and water in a final reaction volume of 20 ml. The
reaction mixture was incubated at 37 uC for 1 h and then run on a
10 % polyacrylamide gel to purify the probe. S. haemolyticus RNA
(10 mg) was hybridized overnight with the labelled RNA probes
(150 pg) at 42 uC in hybridization buffer. The unhybridized probe
was digested with RNase A/T1 (1 : 100 dilutions) at 37 uC for
30 min, the RNases were inactivated, and the remaining RNA was
precipitated. The pellet was resuspended in formamide gel loading
buffer. Then, the sample was run on a 10 % polyacrylamide/7 M
urea gel, and transferred to a positively charged nylon membrane.
The membrane was incubated with anti-digoxigenin antibody
(1 : 10 000; Ambion) conjugated to alkaline phosphatase. Subsequently, the protected fragments were detected using CDP-Star
(Ambion). Chemiluminescent signals were quantified using Labworks software (UVP).
RT-PCR. Expression of target genes was confirmed by RT-PCR. Five
micrograms of total RNA was reverse-transcribed using random
hexamers and Moloney murine leukemia virus reverse transcriptase
for 60 min at 42 uC. The resulting cDNA fragments (1 ml) were used
as templates for PCR amplification of target genes. The PCR cycling
reaction was performed under the following conditions: 94 uC for
5 min; 30 cycles of 94 uC for 30 s, 50 uC for 30 s, 72 uC for 30 s; and
72 uC for 7 min.
Transmission electron microscopy (TEM). All strains were
inoculated on BHI agar at 35 uC for 24 h and were then fixed with
2 % paraformaldehyde and 2 % glutaraldehyde in 0.05 M sodium
cacodylate buffer (pH 7.2) for 18–24 h. Samples were post-fixed for
2 h at room temperature with 1 % osmium tetroxide in 0.05 M
sodium cacodylate buffer (pH 7.2). Cells were then washed,
dehydrated with ethanol and embedded. Ultrathin sections (60–
70 nm) were stained with uranyl acetate and lead citrate and observed
using a JEM 1010 (JEOL) transmission electron microscope operating
at 80 kV. Morphometric evaluation of the cell wall was performed
using images at a final magnification of 6100 000. At least 30 cells
from each strain with nearly equatorial cut surfaces were measured;
the results are expressed as mean±SD values. The statistical
significance of increases in the cell wall thickness was evaluated by
Student’s t-test (P,0.001).
Lysostaphin resistance determination. Strains were cultured
overnight in BHI broth and diluted in BHI medium containing
256 mg lysostaphin ml21 to an OD600 of 1.0. After 1 h, the bacterial
survival rate was determined by measuring the OD600.
RESULTS
Strains and antimicrobial susceptibility testing
Using the S. haemolyticus GS52 strain, derivative strains
were selected (S. haemolyticus GS52V16, GS52V48) in the
presence of vancomycin. After 30–40 serial passages,
stable vancomycin-resistant strains (MIC 16, MIC 48)
were obtained. The origin of derivative strains was confirmed by PFGE patterns of the parent strain (data not
shown). These strains showed decreased susceptibilities
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1429
J. W. Kim and others
Table 1. Primers used in this study
Gene
atl
lrgB
lrgA
lytS
sarA
16S rRNA gene
Primer sequences (5§A3§)
Product size (bp)
GGATGGGTTAGCCAAGCAT
GGTACCCAACCACGTGAAG
CGGAATACTGTTATCCGTTATAC
AAGCACTTCACGCTTTTTATAT
CCCTCCCCAAGTTTAGTTTG
TCCTATTATGCACTGGTGTGG
GGCCACGTGCGAGTTGTTT
ATTTTAACACATGTGGGCGC
GCAATGATCACATACGCAG
GTGTACTATTTACGAG
AACGCGAAGAACCTTACC
CATTGTAGCACGTGTGTAGC
284
to vancomycin, teicoplanin, tigecycline and daptomycin
compared with the parent strain (Table 2). The MIC of
teicoplanin increased in proportion to that of vancomycin.
The daptomycin MIC increased sixfold; however, this change
was within the susceptible MIC range. The tigecycline MIC
changed from susceptible to resistant in the S. haemolyticus
GS52V48 strain. The MICs of other antimicrobial agents were
unchanged.
Characterization of the vancomycin-resistant
strains
Cell wall thickening is a common feature of vancomycinresistant staphylococci. TEM was performed on S. haemolyticus GS52V4, GS52V16 and GS52V48 strains to assess
cell wall thickness. TEM revealed that the cell wall thickness
235
271
266
226
275
of all the derivative strains was significantly higher
(P,0.001) than that of the parent strain. The parental
strain GS52V4 exhibited a cell wall thickness of 21.48 nm,
and the derivatives GS52V16 and GS52V48 exhibited an
increase in thickness of 28 % and 89 %, respectively (Fig.
1a). We examined the survival rate for the derivatives
GS52V16 and GS52V48 in 256 mg lysostaphin ml21. The
survival rate of GS52V4 in lysostaphin was 34.4 %, while the
survival rates for the GS52V16 and GS52V48 strains were
79.8 % and 95.9 %, respectively (Fig. 1c).
The growth characteristics of the GS52V4, GS52V16 and
GS52V48 strains were examined in BHI broth medium
without antibiotics. The S. haemolyticus GS52V16 and GS52V48
strains grew slower than the parent strain in the exponential
phase (Fig. 1b).
Table 2. Antimicrobial susceptibility profiles of clinical and derivative strains of S. haemolyticus
VAN, Vancomycin; TEI, teicoplanin; OXA, oxacillin; PG, penicillin; IMP, imipenem; LIN, linezolid; DAP, daptomycin; TIG, tigecycline; EM,
erythromycin; RIF, rifampicin.
MIC (mg ml”1)
S. haemolyticus strain
9307
SH 39
GY 717
NS 4479
NS 4479
NS 3412
HS 011
SC 392
SA 2
CS 110
GS52V4
GS52V16
GS52V48
1430
VAN
TEI
OXA
PG
IMP
LIN
DAP
TIG
EM
RIF
12
8
8
12
8
8
8
8
8
4
16
48
12
8
8
8
8
8
12
12
8
4
16
32
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢32
¢32
¢32
¢32
¢32
¢32
¢32
¢32
¢32
¢32
¢32
¢32
8
0.5
8
8
¢32
¢32
¢32
¢32
¢32
¢32
¢32
¢32
0.38
0.25
0.25
0.5
0.25
0.5
0.75
0.5
0.25
0.75
0.75
0.75
0.5
0.5
0.5
0.38
0.5
0.75
0.75
0.5
0.38
0.125
0.75
0.75
0.25
0.5
1
1
0.5
0.25
0.5
0.25
0.5
0.5
0.5
1.5
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
¢256
0.012
0.012
0.012
0.016
0.016
0.012
0.016
0.012
0.016
0.016
0.012
0.012
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Journal of Medical Microbiology 61
S. haemolyticus with induced vancomycin resistance
(a)
GS52V4
GS52V16
GS52V48
21.48±2.71
27.54±6.54*
40.6±12.11*
(b)
120
% Survival to lysostaphin
(c)
3
100
OD600
2
GS52V4
GS52V16
GS52V48
1
0
80
60
40
20
0
0
1
2
3 4 5
Time (h)
6
7
8
GS52V4
GS52V16
GS52V48
Fig. 1. TEM images of representative strains. (a) Magnification, ¾100 000. The values under each panel represent mean±SD
values for cell wall thickness (nm). Asterisks denote statistical significance (P,0.001) compared with GS52V4. (b) Growth
curves. (c) Lysostaphin resistance analysis.
Susceptibility to Triton X-100-induced autolysis
Autolysis rates are shown in Fig. 2. The vancomycinresistant derivative strains, S. haemolyticus GS52V16 and
GS52V48, exhibited lower autolysis rates than that of their
parent strain (Fig. 2a). In the presence of Triton X-100,
72 % of the parent strain cells lysed after 4 h incubation. In
contrast, only 12 % of the S. haemolyticus GS52V48 and
30 % of the S. haemolyticus GS52V16 cells lysed after 4 h
incubation. Zymographic analysis showed that the hydrolase protein bands at 105 kDa, 57 kDa and 87 kDa were
reduced in the derivative strains (arrow in Fig. 2b). SDSPAGE also showed that proteins corresponding to the lytic
bands were decreased in these strains (Fig. 2c).
Identification of the expression levels of
autolysis-associated genes
To further determine whether reduced autolysis resulted
from changes in the transcription of autolysis-related
genes, we investigated the expression levels of genes
involved in autolysis by RPA. The expression of genes
(atl, lrgAB, lytSR, sarA) encoding enzymes with previously documented bacteriolytic activities in S. aureus
was confirmed in our S. haemolyticus strain. Among
these genes, the transcription of the atl gene was much
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higher (sixfold increase) than that in the parent strain,
whereas little or no changes in lrgAB, lytSR and sarA gene
transcription were observed in the S. haemolyticus
GS52V16 and GS52V48 strains (Fig. 3). The change in
atl transcript level identified by RPA was confirmed by
RT-PCR (Fig. 3c).
DISCUSSION
Resistance to teicoplanin and vancomycin in S. haemolyticus has been previously reported (Schwalbe et al., 1990;
Veach et al., 1990), but little is known about its autolytic
activity or about the expression of autolytic genes. In this
study, we investigated phenotypic changes, including
expression of several autolytic activity-related genes, in S.
haemolyticus strains with laboratory-induced vancomycin
resistance.
Our results demonstrate a characteristic change in cell wall
thickness similar to that reported in previous studies (Cui
et al., 2003; Nunes et al., 2006) on S. haemolyticus strains
with induced vancomycin resistance. In the present study,
the cell wall thickness of the strain with induced vancomycin resistance was almost 1.9-fold higher than that of its
parent strain. Cell wall thickness changes led to increased
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1431
J. W. Kim and others
100
(b) kDa
199
116
80
79
% Initial OD600
(a)
1
2
(c) kDa
3
199
2 3
116
79
60
52
40
GS52V4
GS52V16
GS52V48
20
0
1
0
1
52
2
Time (h)
3
37
29
4
Fig. 2. Triton X-100-induced autolysis and zymographic analysis of S. haemolyticus strains. (a) Autolytic activity profiles of
S. haemolyticus parent and derivative strains GS52V4, GS52V16 and GS52V48. (b) Zymographic patterns and (c) extracellular
proteins from S. haemolyticus parent and derivative strains. Lanes: 1, GS55V4; 2, GS52V16; 3, GS52V48.
survival rates of vancomycin-intermediate and vancomycin-resistance-induced strains when exposed to lysostaphin. The strain with induced vancomycin resistance also
showed decreased susceptibility to teicoplanin, tigecycline
and daptomycin. The increased MIC of daptomycin, which
is known to target the bacterial cell membrane, may be
influenced by the cell wall changes.
Autolysis is linked to the process of cell division, and
is therefore related to the growth of the cell and the
expression of autolysins, which hydrolyse cell wall
(a)
1
(b)
2
(c)
3
2
atl
lrgA
lrgA
lrgB
lrgB
lytS
lytS
sarA
sarA
16S rRNA
16S rRNA
7
GS52V4
GS52V16
5
GS52V48
4
3
2
1
0
1432
3
atl
6
Fold Induction
1
components. The autolysis rates of S. haemolyticus with
induced vancomycin resistance were less than those of the
parent strain. This suggests that resistance to autolysis
indicates reduction of cell wall turnover (Pfeltz et al.,
2000; Nunes et al., 2006). In addition, the results of the
SDS-PAGE and zymographic experiments showed that
expression of autolysin proteins had decreased in S.
haemolyticus with induced vancomycin resistance. While
several autolysin genes have been reported and studied in
S. aureus, little is known of autolysin genes and their
expression in vancomycin-resistant S. haemolyticus. Here,
atl
lrgA
lrgB
lytS
sarA
Fig. 3. Results of RNA expression for atl,
lrgAB, lytS and sarA genes from S. haemolyticus. (a) RPA and (c) RT-PCR analysis of
parent and derivative strains of S. haemolyticus. Lanes: 1, GS55V4; 2, GS52V16; 3,
GS52V48. (b) Expression of mRNAs by RPA
was normalized to the 16S rRNA gene and
represented as fold induction relative to an S.
haemolyticus GS52V4 level of 1. The values
represent the mean±SD values from three
separate experiments.
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Journal of Medical Microbiology 61
S. haemolyticus with induced vancomycin resistance
we confirmed the presence of the atl, lrgAB, lytS and sarA
genes in vancomycin-resistance-induced S. haemolyticus
and compared the expression of these genes in derivative
strains and the parent strain. Other reported autolysis
genes, such as the cidABC operon, arlRS and mgrA genes
of S. haemolyticus, were not detected. In this study, the
expression of the atl gene increased in the vancomycinresistance-induced S. haemolyticus, but the expression of
the other autolysis-related genes – lrgAB, lytS and sarA –
did not change. In S. aureus, the atl gene plays a fundamental role in cell division and separation. Decreased
atl gene expression in vancomycin-resistant S. aureus may
produce a build-up of peptidoglycan layers contributing
to a thickened cell wall. However, in the present study,
expression of autolysis-related genes such as lrgAB, lytS
and sarA showed little change; only atl gene expression
was upregulated in the vancomycin-resistance-induced
S. haemolyticus strains, while cell wall thickness was
much greater than that in the parent strain. This indicates
that the atl gene is involved in vancomycin resistance of
S. haemolyticus. In this study, decreased autolytic activity and increased expression of atl were observed in
vancomycin-resistance-induced S. haemolyticus. In previous studies, the correlation of expression of atl and
autolytic activity has been investigated in vancomycinresistant S. aureus strains, and some showed increased atl
gene expression or lower autolytic activity than that of
parent strains (Mongodin et al., 2003; Wootton et al., 2005;
Nunes et al., 2006). Though the majority of studies have
focused on S. aureus, the exact mechanism has not been
answered yet.
bloodstream isolates. Antimicrob Agents Chemother 44, 3122–
3126.
In conclusion, vancomycin-resistance-induced S. haemolyticus exhibited a typically thick cell wall, decreased cell growth
phenotype, decreased autolysis activity and upregulation of
the atl gene. Because our study investigated S. haemolyticus
strains specifically induced for vancomycin resistance, our
findings may have somewhat limited application to other
vancomycin-resistant S. haemolyticus strains.
and autolysis by vancomycin in a highly vancomycin-resistant mutant
of Staphylococcus aureus. J Bacteriol 179, 2557–2566.
However, to our knowledge, this is the first investigation of the
expression of autolysis-related genes in vancomycin-resistant S.
haemolyticus. Because S. haemolyticus is prevalent in hospitals
and is regarded as an important nosocomial pathogen with a
tendency to develop multiple resistance, further understanding
of the mechanisms underlying vancomycin resistance should
be acquired through future detailed studies.
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ACKNOWLEDGEMENTS
Vignaroli, C., Biavasco, F. & Varaldo, P. E. (2006). Interactions
between glycopeptides and b-lactams against isogenic pairs of
This study was supported by the Korea Centres for Disease Control
and Prevention (intramural research grant 2007-N44003-00).
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