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Exposure of Clinical MRSA Heterogeneous Strains to
b-Lactams Redirects Metabolism to Optimize Energy
Production through the TCA Cycle
Mignon A. Keaton2, Roberto R. Rosato1, Konrad B. Plata1, Christopher R. Singh1, Adriana E. Rosato1*
1 Department of Pathology and Genomic Medicine, Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research
Institute, Houston, Texas, United States of America, 2 Metabolon, Inc., Durham, North Carolina, United States of America

Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care
and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a b-lactam-insensitive
penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their
heterogeneous expression of resistance to b-lactam (HeR) in which only a small portion (#0.1%) of the population expresses
resistance to oxacillin (OXA) $10 mg/ml, while in other isolates, most of the population expresses resistance to a high level
(homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression
of mecA and a mutational event that involved the triggering of a b-lactam-mediated SOS response and related lexA and recA
genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of b-lactammediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and
global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass
spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with b-lactammediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillinselected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included
significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative
pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished b-lactam-mediated
HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results
provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to blactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in
turn contributes to the survival response in the presence of b-lactam antibiotics.
Citation: Keaton MA, Rosato RR, Plata KB, Singh CR, Rosato AE (2013) Exposure of Clinical MRSA Heterogeneous Strains to b-Lactams Redirects Metabolism to
Optimize Energy Production through the TCA Cycle. PLoS ONE 8(8): e71025. doi:10.1371/journal.pone.0071025
Editor: Michael Otto, National Institutes of Health, United States of America
Received November 1, 2012; Accepted June 30, 2013; Published August 5, 2013
Copyright: ß 2013 Keaton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by National Institutes of Health grant 5R01AI080688-03 (AER, PI). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: MAK is employed by Metabolon, Inc. The metabolomic analysis for this study was performed by Metabolon, Inc. (Durham, North
Carolina). There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies
on sharing data and materials, as detailed online in the guide for authors.
* E-mail: aerosato@tmhs.org

strains is achieved by growing heterogeneous strains in the
presence of sub-inhibitory concentrations of b-lactams [3–5]. As
we demonstrated in previous studies, HeR MRSA strains clinically
misinterpreted as MSSA (MICs to OXA: 2 mg/ml) were able to
express a homogeneous high level of resistance (MICs: 256 mg/ml;
HoR) when exposed to sub-inhibitory concentrations of OXA
(0.5 mg/ml) [5]. Moreover, we have shown that b-lactammediated HeR/HoR selection was also associated with a
mutational event that involved the triggering of a b-lactammediated SOS response and related lexA and recA genes [5].
Although the mechanism has been explored in detail, less is known
about the cellular physiology of HeR-MRSA strains during the
process of HeR/HoR selection by sub-inhibitory concentrations of
b-lactams. Recent studies suggest that the basic physiology of S.
aureus determines not only growth and survival but also
pathogenicity and adaptation to stress conditions, including
antibiotic pressure [7]. In this sense, it has been shown that S.

Introduction
S. aureus is a main pathogen responsible for a number of diseases
ranging from skin and soft tissue infections to life-threatening
endocarditis in hospitals and community settings [1]. The
prerequisite for methicillin resistance located on SCCmec is mecA,
which encodes a b-lactam-insensitive penicillin binding protein
(PBP), PBP2a, that can continue to cross-link the cell wall once the
native PBPs (i.e., PBP124) have been inactivated [2]. A
characteristic of MRSA strains from hospital and community
associated infections is their heterogeneous expression of resistance
to b-lactam (heterotypic resistance [HeR]) [3–5] in which only a
small portion (#0.1%) of the population expresses resistance to
oxacillin (OXA) $10 mg/ml, in contrast to other isolates in which
most of the population expresses resistance to a high level
(homotypic resistance [HoR]) [3–6]. In addition to mecA, the
process of b-lactam-mediated HeR to HoR selection in MRSA

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Metabolic Adaptations in Clinical MRSA Strains

Table 1. Differential gene expression analysis of metabolism-related pathways between strains SA13011-HoR vs. SA13011-HeR.

ORF

Gene

Fold change

Name

Description

Carbohydrate transport and metabolism
SA0258

rbsK

3.7

ribokinase

Catalyses the phosphorylation of ribose to ribose5-P

SA0259

rbsD

5.3

D-ribose pyranase

Catalyzes the conversion between beta-pyran and
beta-furan forms of D-ribose.

SA0260

rbsU

4.2

hypothetical protein

Putative ribose uptake protein rbsU.

SA0510

araB

3.5

Ribulokinase

Catalyzes the phosphorylation of ribulose to
ribulose-5-P

SA1140

glpF

4.8

glycerol uptake facilitator

Facilitates diffusion of glycerol into the cells.

SA0433

223.1

alpha-glucosidase

Converts trehalose-6-P into D-glucose 6-P.

SAS0431

211.2

sugar-specific PTS transport system, IIBC
component

Phosphotransferase transport system (PTS),

SAS0432
SAR2247

mtlD

SA1336

219.8

putative glycosyl hydrolase

Converts trehalose-6-P into glucose-6-P.

5.2

mannitol-1-phosphate 5-dehydrogenase

Catalyzes: D-mannitol-1-P+NAD+ = D-fructose-6P+NADH

2

glucose-6-phosphate 1-dehydrogenase

Catalyzes: D-glucose-6-P+NADP+ = D-glucono-1,5lactone-6-P+NADPH

SA1065

cfxE

2.3

hypothetical protein

pentose phosphate pathway

SACOL1124

lctP

22.4

hypothetical protein

Transports L-lactate across the membrane.

SA0106

220.3

hypothetical protein

homolog of L-lactate permease lctP

SA2156

24.6

hypothetical protein

Maltose/maltodextrin transport permease

SAS0164

2.9

glucose-specific PTS transporter protein,
IIABC component

Phosphotransferase transport system (PTS),
glucose-specific.

SA0099

25.5

putative PTS transport system, IIABC
component

Putative phosphotransferase transport system
(PTS), mannose specific.

SAS2527
SA2320

glcA

SA2434

8.9

hypothetical protein

Phosphotransferase transport system (PTS)

24.6

PTS system glucose-specific EIICBA
component

Phosphotransferase transport system (PTS,
glucose-specific).

4.2

PTS system EIIBC component

Putative phosphotransferase transport system
(PTS), EIIBC component.

SA0186

ptsG

24

PTS system glucoside-specific IICBA
component

Phosphotransferase transport system (PTS),
glucoside specific.

SA0325

glpT

22.7

glycerol-3-phosphate transporter

Transport of glycerol-3-P.

SA1533

ackA

23.1

acetate/propionate kinase

Involved in pyruvate, propanoate, taurine and
hypotaurine metabolism (conversion of acetate to
acetyl-P and propanoate into propanoyl-P)

SA1236

acyP

3.1

acylphospha-tase

Involved in pyruvate metabolism, glycolysis/
gluconeogenesis.

SA1554

acsA

4

acetyl-coenzyme A synthetase

Conversion of acetate and CoA to acetyl-CoA.

SA1609

pckA

5.6

Phosphoenol-pyruvate carboxykinase

Involved in the TCA cycle and pyruvate
metabolism (catalyzesATP+
oxaloacetate = ADP+phosphoenolpyruvate+CO2)

SA1184

citB

6.3

aconitate hydratase

Involved in the TCA cycle (conversion of citrate to
isocitrate).

SA1244

odhB

2.2

Dihydrolipo-amide acetyltrans-ferase

Involved in the TCA cycle and lysine degradation.

SA1518

citZ

8.9

citrate synthase

Catalyzes the first step in the TCA cycle.

SAS1622

citC

11

isocitrate dehydro-genase

Involved in the TCA cycle (converts isocitrate to
alpha ketoglutarate).

SAR1942

citG

2.5

fumarate hydratase, class-II

Involved in the TCA cycle (converts (S)-malate to
fumarate and water).

SA1089

sucD

3.3

succinyl-CoA synthetase alpha subunit

Catalyzes the only substrate-level
phosphorylation in the TCA cycle.

SA0963

pycA

23

pyruvate carboxylase

Involved in the TCA cycle, alanine and aspartate
metabolism, pyruvate metabolism.

SA1510

gapB

4.8

glyceraldehyde 3-phosphate dehydrogenase

Involved in glycolysis and glyconeogenesis.

22.2

hypothetical protein similar to fructokinase

Catalyzes conversion of fructose to fructose-6-P

SA1845
Energy metabolism

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Metabolic Adaptations in Clinical MRSA Strains

Table 1. Cont.

ORF

Gene

Fold change

Name

Description

SA1927

fbaA

22.3

fructose-bisphosphate aldolase

Involved in gluconeogenesis

SA2204

gpmA

24

2,3-bisphosphoglycerate-dependent
phosphoglycerate mutase

Involved in glycolysis/gluconeogenesis

SAS2401

2.9

hypothetical protein

Putative fructose-1,6-bisphosphatase III involved
in glycolysis/gluconeogenesis, pentose phosphate
pathway and fructose and mannose metabolism

SA0212

23.1

hypothetical protein

Similar to sugar phosphate isomerases/
epimerases

SA2102

24

hypothetical protein

Putative formate dehydrogenase

SA0367

nrfA

2.4

NADPH-dependent oxidoreductase

Involved in maintenance of the cellular redox
state and the disulfide stress response

SA2312

ddh

210.8

D-lactate dehydroge-nase

Catalyzes the formation of pyruvate from lactate

SA0218

pflB

210.5

formate acetyltrans-ferase

Catalyzes a key step in anaerobic glycolysis
(conversion of pyruvate and CoA to
formateacetyl-CoA)

SA0232

lctE

25.7

L-lactate dehydro-genase

Catalyzes conversion of pyruvate (the final
product of glycolysis) to lactate in the absence of
oxygen

Oxidative phosphorylation
SA1241

qoxD

24.8

probable quinol oxidase subunit 4

Involved in oxidative phosphorylation pathway.

SA0910

ppaC

22.7

putative manganese-dependent inorganic
pyrophosphatase

Involved in oxidative phosphorylation pathway,
catalyzes the hydrolysis of pyrophosphate to
phosphate.

SA1735

ctaA

23.5

cytochrome oxidase assembly protein

Cytochrome oxidase assembly protein.

24.8

hypothetical protein

Similar to transmembrane efflux pump protein.

3.3

alanine dehydrogenase

Role in cell wall synthesis, as L-alanine is an
important constituent of the peptidoglycan layer.

SA0684
Aminoacid transport and metabolism
SA1531

Ald

SA1365

gcvPB

2.9

glycine dehydrogenase subunit 2

Catalyzes the degradation of glycine.

SA1366

gcvPA

4.8

glycine dehydrogenase subunit 1

Glycine cleavage system P-protein subunit 1.

SA1367

gcvT

5.3

aminomethyltransferase

Glycine cleavage system aminomethyltransferase
T.

SA2226

7.2

hypothetical protein

Similar to D-serine/D-alanine/glycine transporter.

SA2327

25.9

pyruvate oxidase

Similar to pyruvate oxidase (catalyzes formation of
acetyl phosphate from pyruvate).

SA2318

8.4

hypothetical protein

Similar to L-serine dehydratase (catalyses
deamination of serine to form pyruvate).

SA0818

rocD

4.2

ornithine-oxo-acid transaminase

Involved in urea cycle and metabolism of amino
groups.

SA2341

rocA

4.8

1-pyrroline-5-carboxylate dehydrogenase

Involved in L-proline degradation into Lglutamate

SA1585

3

hypothetical protein

Similar to proline dehydrogenase.

SA1436

8.2

hypothetical protein

Similar to allophanate hydrolase subunit 2

SA1707

3

hypothetical protein

Predicted glutamine amidotransferase

SA0717

2

hypothetical protein

acetyltransferase (isoleucine patch superfamily)

SA2229

22.2

hypothetical protein

Similar to amino acid transporters

SA0180

22.3

hypothetical protein

Similar to branched-chain amino acid transport
system carrier protein.

22.9

oligopeptide transporter putative membrane
permease domain

dipeptide/oligopeptide/nickel transport systems.

SA2200

22.4

hypothetical protein

Similar to ABC transporter

SA2227

7.6

hypothetical protein

Similar to gamma-aminobutyrate permease and
related permeases.

SA2254

opp-1B

SA1169
SA1718

putP

SA0859

22.3

gamma-aminobutyrate permease

Amino acid transporter.

22.6

high affinity proline permease

Proline permease

3

hypothetical protein

Similar to oligoendopeptidase F.

Cell wall associated genes

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Metabolic Adaptations in Clinical MRSA Strains

Table 1. Cont.

ORF

Gene

SA1343

Fold change

Name

Description

2.3

hypothetical protein

Similar to tripeptidase

SA1283

pbp2

2.6

penicillin binding protein 2

Membrane carboxypeptidase

SA0038

mecA

2.8

penicillin binding protein 2A

Membrane transpeptidase

SA1206

femA

3.0

factor essential for expression of
methicillin resistance

Factor essential for expression of methicillin
resistance; involved in the formation of the
staphylococcal pentaglycine interpeptide bridge

SA0244

tagF

5.0

putative glycosyl/glycerophos-phate
transferase

Similar to teichoic acid biosynthesis protein F
(TagF)

SAR2242

glmS

5.3

D-fructose-6-phosphate amidotransferase

Catalyzes the first step in hexosamine metabolism
(converts fructose-6P into glucosamine-6P)

3.0

putative cell shape determinant mreC

Rod shape-determining protein

uppP

3.0

undecaprenyl pyrophosphate phosphatase

Participates in peptidoglycan biosynthesis;
involved in bacitracin resistance

2.8

N-acetylmuramoyl-L-alanine amidase
precursor

Autolysin precursor
Involved in N-acetylmuramic acid degradation

SA1475
SAS0648
SA2437
SA0185

murQ

7.9

N-acetylmuramic acid 6-phosphate
etherase

SA1183

opuD

22.7

glycine betaine transporter

Transporter

SA1987

26.4

hypothetical protein

Probable glycine betaine transporter opuD
homolog.

SA0659

23.6

hypothetical protein

Similar to CsbB stress response protein.

SA0511

22.2

hypothetical protein

Similar to nucleoside-diphosphate-sugar
epimerases.

SA1141

glpK

7.2

glycerol kinase

Involved in the regulation of glycerol uptake and
metabolism, glycerolipid metabolism (catalyzes
glycerol to glycerol 3-P).

SA0432

treP

213.4

PTS enzyme II, phosphoenol-pyruvatedependent, trehalose-specific

Phosphotransferase transport system (PTS),
trehalose-specific.

doi:10.1371/journal.pone.0071025.t001

aureus acquires resistance to vancomycin by adapting both its
physiology and metabolism allowing its growth in the presence of
the antibiotic [8]. In S. aureus, the tricarboxyl citric acid (TCA)
cycle is essential for a majority of metabolic pathways [9–11]. It
serves as a central hub connecting catabolic energy gaining
pathways with anabolic pathways like amino acid, fatty acid and
nucleoside biosynthesis [11]. The central degradation product of
glycolysis, pyruvate, is shunted into TCA cycle via acetyl-CoA
[11]. Pyruvate dehydrogenase complex (PDC) catalyzes then the
conversion of pyruvate to acetyl-CoA with a concomitant
reduction of NAD+ to NADH and release of CO2 [12]. In the
present study we used a combinatorial approach of microarray
analyses with global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in
metabolic pathways and the metabolome associated with blactam-mediated HeR/HoR selection in clinically relevant MRSA
cells. By using these approaches, we found unique features present
in SA13011 after b-lactam-mediated selection (HoR) that included
significant increases in the TCA cycle intermediates citrate, cisaconitate and fumarate and a significant decrease in lactate.
Moreover, mechanistic studies based on the inactivation of acnAcitB (the first gene of the cycle), further demonstrated the
functional significance of altered TCA cycle activity during the
HeR/HoR selection. The present results reveal both the metabolic
cost and the adaptation that HeR-MRSA clinical strains undergo
when exposed to b-lactam pressure, indicating that the energy
production is redirected to supply the cell wall synthesis/
PLOS ONE | www.plosone.org

metabolism, contributing to the cell survival in the presence of
b-lactam antibiotics. These studies involving the analyses of
metabolic pathways in heterogeneous MRSA provide novel
information which may represent an important contribution for
future design of new targets against MRSA infections.

Results
Differential Gene Expression Analysis during OXAmediated SA13011 HeR/HoR Selection Revealed Changes
in Diverse Metabolic Pathways
In an attempt to determine differentially expressed genes
associated with b-lactam-mediated HeR/HoR selection, we
performed gene expression analysis using spotted DNA microarrays as previously described [13]. Pair-wise comparisons were
made in biological triplicates between SA13011- HeR and
SA13011-HoR isogenic strains. SA13011-HeR (OXA MIC:
2 mg/ml) was grown in absence and presence of sub-inhibitory
concentrations of OXA (0.5 mg/ml) leading to SA13011-HoR
(Oxacillin MIC: $256 mg/ml) and collected at similar exponential
growth phase as described both in Methods and previously [5,13].
The extension of microarrays analyses reported here focused
specifically in genes associated with metabolic pathways. These
results are based on a series of statistical analysis (filtering) where
ratios of Cy3 and Cy5 signals were converted to log2 values and
cutoff was set at above 1 (present) or below -1 (absent), as
previously described [13,14]. The ORFs considered here as
differentially expressed are those which log2 ratios of Cy3/Cy5
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Metabolic Adaptations in Clinical MRSA Strains

signals equals 1 (two-fold change) in at least three out of four
independent experiments [13,14].
A total of 230 genes were found differentially expressed, evenly
distributed between up- and down-regulated genes (Table 1); these
genes were classified in six distinct groups. The most represented
group of differentially expressed genes belongs to the functional
category carbohydrate transport and metabolism (22%) followed
by genes involved in amino acid transport and metabolism (16%),
energy production and conversion (10%) and cell wall, membrane,
envelope biogenesis (5%; Table 1).

histidine and nucleotide synthesis [15]. Elevated expression of rbsK,
rbsD, and rbsU may suggest intensified use of D-ribose as energy
and carbon source by SA13011-HeR during the selection process.
In the same group we observed the phosphoenolpyruvatedependent sugar phosphotransferase system (PTS), a major form
of carbohydrate transport involved in the translocation across cell
membrane and phosphorylation of incoming carbohydrates [16].
A pronounced reduction in the expression of trehalose specific,
phosphoenolpyruvate-dependent phosphotransferase system (13fold; treP, SA0432) and a-glucosidase (23-fold; SA0433) that
converts trehalose into D-glucose-6-phosphate was observed.
Moreover, expression of several additional sugar phosphotranspherase transport systems were down-regulated during HeR/
HoR selection including putative mannose specific PTS (SA2527),
glucose specific PTS (glcA, SA0183), putative fructose specific PTS
(SA2434), glucoside specific PTS (ptsG, SA2326) and glucose
specific PTS (SA1566). A group of hypothetical proteins predicted
to be related to carbohydrate metabolism were downregulated
during HeR/HoR selection, including lactate permease (lctP,
SA0106), probable homolog of lactate permease (SA2156),
hypothetical maltose/maltodextrin permease homolog (SA0209)
and glycerol-3-phosphate transporter (glpT, SA0325).

Carbohydrate Transport and Metabolism
Differentially regulated genes in the group of carbohydrate
transport and metabolism included genes involved in the
utilization of ribose as a carbon source, namely rbsK, rbsD, and
rbsU, (SA0258, SA0259 and SA0260, respectively) all of which
were up-regulated during HoR selection. RbsD functions as an
ABC-type ribose transporter which also catalyzes conversion
between b-pyran and b-furan forms of D-ribose [15]; rbsU encodes
for a hypothetical ribose uptake protein and rbsK encodes for
ribokinase that catalyzes the phosphorylation of ribose to ribose-5phosphate, the initial step in ribose metabolism. Ribose-5phosphate serves as the substrate in pentose phosphate pathway
for energy production as well as the carbon source in tryptophan,

Figure 1. Quantitation of mRNA levels of TCA cycle-, amino-acid catabolism-, carbohydrate catabolism- and cell wall-associated
genes by real-time RT-PCR. RNA was prepared from SA13011-HeR and its highly resistant derivative SA13011-HoR (SA13011+ OXA 0.5 mg/ml)
cells, collected at exponential phase of growth, as described in Materials and Methods. Relative fold change values of specific mRNAs in SA13011-HoR
vs. SA13011-HeR (reference value = 1) are shown on the vertical axis. Relative fold change values representing the means of at least three biological
replicates of specific mRNAs 6 standard error of the mean (SEM), sampled in triplicate to minimize error by inter- and intra-samples, are shown on the
vertical axis; 16S rRNA was used as an internal control. Differences between the mean values were analyzed using a one-way analysis of variance
(ANOVA). A P value of ,0.01 was considered statistically significant. Oligonucleotide primers are shown in Table S2.
doi:10.1371/journal.pone.0071025.g001

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Metabolic Adaptations in Clinical MRSA Strains

Figure 2. Heat map of metabolite levels profiled in the heterogeneous SA13011 strain (HeR) and its highly homogeneous
methicillin resistant derivative SA13011-HoR (SA13011+ OXA 0.5 mg/ml). Red indicates high levels and green indicates low levels of each
biochemical arranged on they-axis.
doi:10.1371/journal.pone.0071025.g002

Glycine dehydrogenase subunit 2 (gcvPB, SA1365), subunit 1
(gcvPA, SA1366) and aminotransferase (gcvT, SA1367) are coexpressed and form an operon as judged by their close location,
direction of expression, and existence of the gcv operon in E. coli.
GcvPB, GcvPA and GcvT constitute the glycine cleavage system
involved in glycine degradation, which cleavages glycine into CO2
and NH3, generating NADH and one carbon unit [19]. Elevated
expression of ald and genes that encode for enzymes of glycine
cleavage system may suggest an intensified catabolism of two
major components of peptidoglycan, glycine and alanine, and
implies intense cell wall turn-over during SA13011-HoR selection.
Another gene up-regulated and involved in amino acid catabolism

Amino Acid Transport and Metabolism and Cell Wall
Precursors
The second group of genes displaying differential expression
between SA13011-HeR/2HoR strains included up-regulation of
genes encoding for enzymes involved in degradation of peptidoglycan constituents. Alanine is an important component of S. aureus
cell wall, where it represents three out of five amino acids in the
stem peptides of peptidogylcan [17]. Alanine dehydrogenase (ald,
SA1531), which hydrolyses L-alanine to ammonia, pyruvate and
NADH, were up-regulated 3.3 fold during selection. Three genes
involved in glycine degradation, the constituent of pentaglycine
bridges of peptidoglycan [18], were also found to be up-regulated.

Table 2. Summary showing the number of biochemicals statistically significantly different (p,0.05) between SA13011-HoR vs.
SA13011-HeR.

Welch’s Two Sample t-Tests

Number of biochemicals with
p#0.05

Number of biochemicals
increased p#0.05

Number of biochemicals
decreased p#0.05

SA13011-HoR vs. SA13011-HeR

98

15

83

doi:10.1371/journal.pone.0071025.t002

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Metabolic Adaptations in Clinical MRSA Strains

Figure 3. Analysis of biochemicals associated with the TCA cycle determined by global biochemical profiling across SA13011-HeR
and SA13011-HoR (SA13011+ OXA 0.5 mg/ml) derivative.
doi:10.1371/journal.pone.0071025.g003

encodes for L-serine dehydratase (SA2318). This enzyme catalyzes
the conversion of L-serine to pyruvate and NH3. Up-regulation of
SA2318 suggests intensified serine degradation and generation of
pyruvate which will then serve as the substrate in multiple
metabolic pathways. Its elevated expression also coincides with upregulation of the hypothetical transporter of serine/alanine/
glycine (SA2226).
Catabolism of proline also seems to be intensified during HeR/
HoR selection since the gene encoding for a protein similar to
proline dehydrogenase (SA1585) was up-regulated, as was the gene
encoding the enzyme 1-pyrroline-5-carboxylate dehydrogenase
(rocA, SSA2341) that catalyzes the second step in proline
degradation. Several other genes encoding for proteins involved
in the urea cycle were found to be up-regulated, including
allophanate hydrolase subunit 2 (SA1436) and glutamine amidotransferase (SA1707).

PLOS ONE | www.plosone.org

Energy Production
A number of genes involved in glycolysis, acetate metabolism,
and the TCA cycle were found to be differentially expressed
during HeR/HoR selection. Multiple genes involved in glycolysis/
gluconeogenesis were found to be down-regulated, including
fructose-biphosphate aldolase (fba, SA1927) and 2,3-biphosphoglycerate-dependent phosphoglycerate mutase (gpmA, SA2204).
Fba catalyzes the conversion of fructose-1,6-bisphosphate into
dihydroxyacetone phosphate while glyceraldehyde-3-phosphate
(GpmA) is responsible for converting glycerate-3-phosphate into
glycerate-2-phosphate. We also observed a marked down-regulation of L-lactate dehydrogenase (lctE, SA0232) and D-lactate
dehydrogenase (ddh, SA2312), which interconvert pyruvate and
lactate. Genes encoding enzymes involved in gluconeogenesis were
similarly down-regulated including pyruvate carboxylase (pycA,
SA0963), which catalyzes the conversion of pyruvate into

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Metabolic Adaptations in Clinical MRSA Strains

Figure 4. Analysis of biochemicals associated with sources of acetyl-CoA generation from b-oxidation of fatty acids in SA13011-HeR
and SA13011-HoR (SA13011+ OXA 0.5 mg/ml) during b-lactam mediated HeR/HoR selection.
doi:10.1371/journal.pone.0071025.g004

oxaloacetate, and fructose-1,6-bisphosphatase III (SA2401). The
only glycolytic gene observed to be up-regulated during the HeR/
HoR selection was glyceraldehyde-3-phosphate dehydrogenase
(gapB, SA1510).
In contrast to glycolytic genes, the expression of many TCA
cycle genes was found to be up-regulated. These include citrate
synthase (citZ, SA1518), aconitate hydratase (citB, SA11884),
isocitrate dehydrogenase (citC, SAS1622), and a subunit of aketoglutarate dehydrogenase (odhB, SA1244). Another gene related
to the TCA cycle also found to be up-regulated was acsA (SA1554);
this gene codes for the enzyme responsible of converting acetate
into acetyl-CoA, which can be then be used as carbon or energy
source [20]. Elevated expression of acsA coincided with elevated
expression of aldehyde dehydrogenase homologue (aldA, SA0162)
and acylphosphatase (acyP, SA1236), both involved in acetate
generation. AldA catalyzes the conversion of acetaldehyde into
acetate with simultaneous generation of NADPH while AcyP
generates acetate and ATP from acetyl-phosphate. Elevated
expression of these genes, which generate acetate and energy via
ATP and NADPH, and the concomitant reduction in expression
of acetate/propionate kinase (ackA, SA1533), which stores energy
in the form of acetyl phosphate, suggest that acetate generation
may constitute one of the energy sources that feeds into the TCA
cycle during outgrowth of SA13011-HoR. Interestingly, we
identified several down-regulated genes encoding for enzymes
involved in oxidative phosphorylation including quinolone oxidase
subunit 4 (qoxD, SA0910), manganese-dependent inorganic
pyrophosphatase (ppaC, SA1735), and cytochrome assembly
protein (ctaA, SACOL1124). These changes suggest limited
aerobic metabolism and limited energy production by oxidative
phosphorylation during outgrowth of SA13011-HoR.

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Cell Wall Synthesis
Other genes whose expression was also found elevated during
HeR/HoR selection were glpF and glpK (SA1140 and SA1141,
respectively). These genes encode for glycerol uptake facilitator
and for glycerol kinase, respectively [21]. Glycerol-3-phoshate is
substrate for lipid biosynthesis including lipoteichoic and teichoic
acids, which are important components of both cell membrane
and cell wall. Increased expression of glpF (approximately 5-fold)
and glpK (above 7-fold) during HeR/HoR selection may suggest an
intensified transport of glycerol and synthesis of glycerol-3phosphate during outgrowth of SA13011-HoR. Along with PBP2a
(mecA) up-regulation, expression of pbp2 (PBP2; SA1283) was also
found to be elevated, consistent with the requirement of their
cooperative effect (transglycosylase domain of PBP2 and the
transpeptidase activity of PBP2a) for methicillin resistance in S.
aureus [22]. Another important gene found to be up-regulated in
HoR was femA (SA1206), a factor essential for methicillin
resistance. FemA is responsible for incorporation of glycines 2
and 3 into pentaglycine cross-bridges that allows high crosslinking
of peptidoglycan, a hallmark of the S. aureus cell wall [23]. An
additional interesting gene found to be up-regulated was (SA0244),
a putative glycosyl/glycerphosphate transferase that participates in
teichoic and lipoteichic acids biosynthesis; SA0244 exhibits
homology with TagF which adds glycerol-phosphate units to the
growing chains of poly-glycerols bound to N-acetylglucosamine-b(1–4)-N-acetylmannosamine and linked to lipid carrier, undecaprenyl-pyrophospate [24]. It has been suggested that the large
multienzyme complex of which TagF is a part localizes at sites of
cytoplasmatic membrane determined by the localization of MreC
[24]. These observations may suggest that MreC participates not
only in teichioc/lipteichoic acids biosynthesis, but also in
peptidoglycan biosynthesis by directing the localization of
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Metabolic Adaptations in Clinical MRSA Strains

Figure 5. Analysis of biochemicals corresponding to energy metabolism components NADH, NAD+, FAD, phosphate and
pyrophosphate (PPi) levels during b-lactam mediated SA13011-HeR/HoR selection.
doi:10.1371/journal.pone.0071025.g005

that were observed in the microarray analysis (Table 1). Additionally, we determined increased expression of dltA [D-alaninepoly (phosphoribitol) ligase subunit 1] and dltC [D-alanine-poly
(phosphoribitol) ligase subunit 2] (Figure 1C), both genes encoding
for enzymes involved in ribitol metabolism [25]. Expression
analysis of cell wall associated genes included genes related to the
expression of methicillin resistance including PBP2a (mecA, 6-fold
increase) and PBP2 (pbp2, 4-fold increase), as well as genes
associated with peptidoglycan cross-linking (femA; 5.8-fold increase)
(Figure 1D). Consistent with their role, expression of glucosamine6-phosphate synthase (glmS), important for production of a major
building block of peptidoglycan, was also found up-regulated in
SA13011-HoR strain (Figure 1D). Similar results were obtained
during HeR/HoR selection of the heterogeneous MRSA strain
SA43002 (phenotypically similar to SA13011) (Fig. S3).

enzymatic biosynthetic machineries to proper sites of synthesis.
Proof of the intensity and correlation of these processes may come
from up-regulation of undecaprenyl pyrophosphate phosphatase
(uppP, SA0648). Undecaprenyl phosphate (bactoprenol) is the
carrier lipid on which intermediates of peptidoglycan, teichoic/
lipoteichoic acids and other components of cell wall are assembled
and transported across cytoplasmic membrane. Elevated expression of uppP suggests intensified recycling of the lipid carrier which
is necessary during intense cell wall synthesis. Concurrent
elevation of gene expression involved in peptidoglycan synthesis
(pbp2, pbp2a, femA), teichoic/lipoteichic acids polymerase (tagF) and
mreC, suggests organized, intensified and perhaps coordinated
synthesis of murein, teichoic and lipoteichoic acids during
outgrowth of the HoR derivative.
Validation of the metabolic changes in gene expression
regulated during HeR/HoR selection identified by microarray
analysis was performed by Real-Time RT-PCR by using RNAs
collected from SA13011-HeR and -HoR cells. Consistent with the
microarray analysis, we observed a 10-fold increase in citZ and a 6fold increase in citB expression (Figure 1A). We also measured
expression of mqo2 which encodes malate dehydrogenase and
found its expression was also elevated in SA13011-HoR. We
confirmed changes reporting on acetate metabolism (acyP, acsA,
and ackA; Figure 1A), proline and ornithine metabolism [rocA, (1pyroline-5-carboxylate dehydrogenase); rocD (ornithine-oxo-acid
transaminase; Figure 1B], and ribose metabolism (rbsD; Figure 1C)

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Global biochemical profiling during b-lactam-mediated
HeR/HoR selection. In order to identify and characterize

metabolic changes associated with b-lactam-mediated HeR/HoR
selection, untargeted, global biochemical profiling was performed
for SA13011 during b-lactam-mediated HeR/HoR selection, as
described in Methods. Cells were all collected at similar phase of
growth (OD600 0.7). A total of 194 biochemicals were identified
and categorized into amino acid, carbohydrate, fatty acid,
nucleotide, and cofactor classes. From these 194 metabolites, 98
biochemicals were significantly altered in their levels when
SA13011-HoR was compared to SA13011-HeR (Table 2). Hier-

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Metabolic Adaptations in Clinical MRSA Strains

Figure 6. Schematic representation of genes and/or metabolites related to the TCA cycle summarizing changes in both mRNA
expression and biochemical levels. Red coloration represents up-regulation while green coloration represents down-regulation during b-lactam
mediated HeR/HoR selection.
doi:10.1371/journal.pone.0071025.g006

were unchanged (Table S1). Instead, the majority of medium
chain and long chain free fatty acids were found to be significantly
decreased in SA13011-HoR, indicating increased utilization of this
energy source (Figure 4). Consistent with an increase in fatty acid
oxidation and TCA activity, acetyl-CoA (Figure 3) and NADH
(Figure 5) levels rose after OXA-mediated HeR/HoR selection
(SA13011-HoR). This was accompanied by a decrease in NAD
levels (Figure 5) suggesting that the increased NADH was not
being utilized. Together with diminished phosphate levels
(Figure 5) and the decreased expression of a number of genes
involved in oxidative phosphorylation in SA13011-HoR cells,
including a subunit of FoF1-ATP synthase (atpC) and a protein
required for cytochrome oxidase assembly (ctaA; Table 1), these
changes indicate a decrease in oxidative phosphorylation during
the selection process.

archical cluster analysis of the resulting biochemical profiles
revealed the existence of profound differences associated with blactam heterogeneous resistance (Figure 2). In fact, the SA13011HoR profile was vastly different from SA13011-HeR, clearly
emphasizing the metabolic adaptation that the strain undergoes
when under b-lactam pressure. Importantly, many of the changes
observed in the global biochemical profiles are consistent with the
differential gene expression observed between the SA13011-HeR
and SA13011-HoR strains described above.

Energy Metabolism and Resistance to b-lactam
Consistent with the upregulation of several TCA cycle genes
during HoR selection, many of the TCA intermediates were
elevated in the SA13011-HoR strain as compared to SA13011HeR, with significant increases in the levels of cis-aconitate and
malate (Figure 3; Table S1). These changes suggest that the
SA13011-HoR strain is potentially capable of higher energy
production and increased biosynthetic capabilities than the
parental SA13011-HeR strain that may allow the HoR-derivative
strain to respond to inhibition of wall synthesis by b-lactams.
SA13011-HoR also displayed significantly higher levels of acetylCoA, which can be generated from glycolysis, b-oxidation of fatty
acids, or acetate. Glycolysis did not appear to increase during the
selection process, as 3-phosphoglycerate and phophoenolpyruvate

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Amino Acid Catabolism
Additional biochemical changes associated with SA13011-HoR
included reduction in several amino acids and their metabolites
(Fig. S1; Table S1). These changes may reflect altered utilization,
synthesis, and catabolism. The latter possibility is supported by
increased expression of genes involved in amino acid degradation.
For example, a dramatic reduction in glycine levels was observed
in SA13011-HoR cells (Fig. S1) coincidently with upregulation of

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Metabolic Adaptations in Clinical MRSA Strains

Figure 7. Quantitation of acnA mRNA (A) and TCA cycle-associated genes (B) by Real-Time RT-PCR. RNAs were prepared from SA13011HeR/HoR, acnA-null mutant LMR15 and LMR15 complemented with either the empty-vector (LMR15-EV) or wild-type acnA (LMR17), grown in the
absence or presence of OXA (0.5 mg/ml). Cells were collected at exponential phase of growth as described in Materials and Methods. Relative fold
change values versus SA13011-HeR ( = 1) of specific mRNAs are shown in the vertical axis; 16rRNA was used as an internal control. *, significantly
different than SA13011-HeR (P,0.001).h.
doi:10.1371/journal.pone.0071025.g007

genes that comprise the glycine cleavage system (Table 1).
Similarly, levels of alanine, serine, proline, and ornithine were
reduced in HoR cells, consistent with the increased expression of
SA2318 and ald, SA1585 and rocA, and rocD, respectively.
Catabolism of alanine and serine produce pyruvate that can then
be converted to acetyl-CoA, while catabolism of proline and
ornithine produce glutamate which can be converted to aketoglutarate, and thus contribute to the TCA cycle. Although
most amino acids and their metabolites were decreased in during
HoR selection, glutamine, which is synthesized from glutamate
and is a component of peptidoglycan, was dramatically increased.
Finally, the branched chain amino acid metabolites a-hydroxyisocaproate, a-hydroxyisovalerate, and 2-hydroxy-3-methylvaleratere were increased in HoR cells (Table S1), indicating
catabolism of leucine, isoleucine, and valine which ultimately
produces acetyl-CoA and succinyl-CoA that enter the TCA cycle.

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Markers of Membrane and Cell Wall Remodeling
While most fatty acids decreased compared to the HoR
population (Table S1), several branched-chain fatty acids and
the saturated 20 carbon fatty acid arachidate were significantly
increased in OXA-selected samples (SA13011-HoR) suggesting
they may play a specific role in antibiotic resistance. Since
increases in a free fatty acid can arise from increased synthesis,
remodeling of the cell membrane, or deconjugation from cell wall
components, increases in 15-metylpalmitate/2-methylpalmitate
and arachidate may reflect membrane and cell wall remodeling
mediated by OXA treatment.

Oxidative DNA Damage
Another difference between SA13011-HeR/HoR strains corresponded to a marked decreased in the levels of glutathione (Fig.
S2), which may be associated in these cells with both increased
oxidative stress and activation of a DNA damage response, as we
previously reported during SA13011-HeR/HoR selection [5]. It is
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Metabolic Adaptations in Clinical MRSA Strains

Figure 8. Susceptibility testing of SA13011 aconitase mutant LMR15 and derivatives (2/+ OXA 05 mg/ml; Table 4). Overnight inoculum
was diluted to 0.5 Mc Farland standard and swabbed onto MH agar. glu: cells were grown in media supplemented with glucose 10mM. E-test strips
were placed on the plates and incubated at 37uC for 24 h. A representative picture of repeated (3) experiments is shown.
doi:10.1371/journal.pone.0071025.g008

which resulted in LMR18 (LMR17+ OXA) MIC of 256 mg/ml
(Fig. 8B). Similar results were obtained when pyruvate (20mM) or
ribose (12mM) were used instead of glucose (data not shown).
Supplementation with carbon sources had no effect per se in terms
of resistance acquisition either in SA13011-HeR or LMR17
(Table 3). Expression analysis by Real-Time RT-PCR demonstrated altered regulation of TCA cycle genes in the absence of
acnA during b-lactam-mediated HeR/HoR selection (Fig. 7B).
After acnA inactivation, primary TCA cycle-related genes including citZ, citB, acyP and acsA were down-regulated in LMR16
(LMR15+ OXA), while complementation of the acnA mutant with
the cloned full-length gene determined a regain in their
corresponding expression in LMR18 (LMR17+ OXA). Together,
these results demonstrate the requirement of an active TCA cycle
and its key functional role during during b-lactam-mediated HeR/
HoR selection process.

plausible that the loss of this key antioxidant compound may
contribute to increased mutation rates and the selection of the
homotypic resistant phenotype [5]. Thus, decreased glutathione
may predispose cells to enhancing b-lactam resistance mechanisms
by increasing oxidative DNA damage and consequently the SOS
response that is required for OXA-mediated HeR/HoR selection
[5].
TCA cycle is functionally associated with-b-lactammediated SA13011 HeR/HoR selection. The results from

gene expression and metabolomics analyses described above
demonstrated both a marked increase in the expression of genes
corresponding to the TCA cycle and a redirection of metabolic
activity toward the cycle (see summary Fig. 6). To further
investigate its functional role and the contribution to the b-lactam
mediated HeR/HoR selection, the aconitase gene acnA-citB, which
encodes the second enzyme of TCA cycle, was inactivated in
SA13011-HeR strain (SA13011 DacnA::tetM, LMR15; Table 4).
Expression of acnA in mutant strains was monitored by Real-Time
RT-PCR (Fig. 7A). Phenotypic analysis of acnA-null mutant
LMR15 showed no changes in the susceptibility to OXA after
exposure to sub-inhibitory concentrations of the antibiotic, i.e.
MIC: 1 mg/ml before selection vs. 0.75 mg/ml after OXA
exposure (Table 3, Fig. 8A). These results indicated that
inactivation of the acnA gene impaired b-lactam-mediated HeR/
HoR selection. Complementation of acnA-null mutant LMR15
with a cloned full-length acnA (LMR17) resulted in transcription
levels similar to those corresponding to SA13011-HoR (Fig. 7A).
As a control, LMR15 was complemented with the corresponding
empty-vector (LMR15-EV) and, as expected, this did not rescue
acnA expression (Fig. 7A). Importantly, complementation with fulllength acnA restored the selection of the OXA resistant derivative,
although not to the same degree observed in SA13011-HoR strain
[MICs: 32 mg/ml for LMR18 (LMR17+ OXA) vs. 256 mg/ml, for
SA13011-HoR, Table 3, Fig. 8B). However, higher MICs values
were achieved when full-length acnA-complemented LMR17 strain
undergoing selection with OXA was simultaneously supplemented
with glucose (10mM) to maximize optimal glycolysis coverage,

Discussion
S. aureus is a facultative anaerobe that can survive in aerobic
environment during transmission on the skin and with reduced
levels of oxygen (anaerobic) during abscess [25]. These observations exemplify the capacity of S. aureus to modulate its metabolism
accordingly to the encountered conditions. Under these circumstances, S. aureus preferentially degrades glucose to pyruvate by the
way of the pentose phosphate and glycolic pathways [9]. The
catabolic fate of pyruvate is determined by growth conditions;
under anaerobic growth, pyruvate is reduced to lactic acid [10]
while it is oxidized to acetate and CO2 under aerobic conditions
[26]. Acetate in the form of acetyl-CoA can be oxidized by the
TCA cycle when S. aureus is grown in the presence of certain
intermediates [27]. In the present study, we were interested in
investigating the adaptation of metabolic pathways occurring in
the presence of b-lactam during transition from HeR to HoR
resistant phenotype by using both global microarrays and
metabolomic analyses.
In staphylococci, entry into the post-exponential growth phase
usually coincides with the catabolism of non-preferred carbon

Table 3. MICs to oxacillin (OXA) corresponding to S. aureus aconitase mutants.

STRAIN

MIC OXA (mg/ml)

SA13011-HeR

2

SA13011-HoR (SA13011-HeR+OXA 0.5 mg/ml); SA13011 homogeneous derivative

256

LMR15 (SA13011 DacnA::tetM)

1

LMR-16 (LMR-15+ OXA 0.5 mg/ml); LMR-15 homogeneous derivative

0.75

LMR17 (LMR15+ wild type acnA)

1

LMR18 (LMR17+ OXA 0.5 mg/ml); LMR-17 homogeneous derivative

32

LMR15+ EV

0.5

LMR15+ EV+OXA (0.5 mg/ml)

1

SA13011-HeR+glu

1

SA13011-HeR+OXA (0.5 mg/ml)+glu

256

LMR17+ OXA (0.5 mg/ml)+glu

256

LMR15+ EV+OXA (0.5 mg/ml)+glu

1

doi:10.1371/journal.pone.0071025.t003

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Metabolic Adaptations in Clinical MRSA Strains

Table 4. Strains and plasmids used in this study.

Strain

Relevant Genotype and phenotype

Reference or Source

SA13011-HeR

Heterogeneous [mecA (+), OXA susceptible; ST5, SCCmec type II, spaType 2, TJMBMDMGMK]

[4,5]

SA13011-HoR

SA13011-HeR+OXA (0.5 mg/ml); SA13011 homogeneous derivative

[5]

LMR15

SA13011-HeR DacnA::tetM

This study

LMR16

LMR-15+ OXA (0.5 mg/ml); LMR-15 homogeneous derivative

This study

LMR17

LMR15+ wild type acnA cloned into pSK265

This study

LMR18

LMR-17+ OXA (0.5 mg/ml); LMR-17 homogeneous derivative

This study

RN4220

Restriction deficient Mutagenized RN450

[5]

SA564 DacnA::tetM

acnA mutant

[31]

E.coli (PCR2.1-TOPO)

Ampr Kanr

Invitrogen

S. aureus pSK265

High-copy staphylococcal replicon

[40]

Plasmids

+OXA: indicates the corresponding resistant derivative strain was obtained by growing in the presence of the indicated sub-inhibitory concentrations of OXA.
doi:10.1371/journal.pone.0071025.t004

critical role of the TCA cycle in S. aureus have been previously
reported, showing the significant role played for example, in
evasion of immune response [28]. Inactivation of the TCA cycle
was shown to delay the resolution of cutaneous ulcers in a mouse
soft tissue infection model [28]. Using an in-vitro model of aconitase
mutant these studies revealed changes in the production of nitric
oxide (NO), suggesting that S. aureus may enhance its ability to
survive in the host by altering its metabolism [28]. Similarly, it has
been shown that S. aureus, which requires iron to successfully
colonize the host [29], is able to redirect its central metabolism to
increase iron availability. In a model of iron-starved S. aureus, Fur
protein-mediated increase in the production of lactate as a
fermentative end-product resulted from the concomitant inactivation of TCA cycle enzymes including aconitase [29]. The resulting
process, i.e. increased lactate levels, contributed to decrease pH
which in turn facilitates the release of iron from host transferrin
[29].
Capsule polysaccharide biosynthesis requires TCA cycle intermediates [25]. Inactivation of genes such as citZ (citrate synthase),
citC (isocitrate dehydrogenase) and citB (aconitate hydratase)
prevents capsule formation, without impairing glucose catabolism
but completely inhibited the catabolism of acetate, highlighting the
importance of the energy production in the production of
virulence factors as well [25]. In line with these observations, we
have also found decreased expression of capsule genes in an
aconitase null-mutant generated in our laboratory (Singh, C. and
Rosato, AE; unpublished observations), which further emphasizes
the key role that both the TCA cycle and the re-direction of other
metabolic pathways may have in providing cells the capacity to
develop the high resistant phenotype.
In summary, the present study highlights the importance of
metabolic adaptations of heterogeneous MRSA clinical strains
when undergoing selection to highly resistant HoR derivatives in
the presence of b-lactam antibiotics. These results postulate that blactam-mediated HeR/HoR selection is associated with severe
metabolic stress, as demonstrated by increased production of
acetyl-CoA, increased catabolism of fatty acids (b-oxidation) and
amino acids, and decreased oxidative phosphorylation, altogether
contributing to increased TCA activity that supports and promotes
survival in the presence of b-lactam antibiotics. Importantly, these
observations may identify a promising avenue for combating
multidrug-resistant bacteria, as recently observed with compounds

sources and induction of the TCA cycle [8], which led us to
hypothesize that increased resistance to b-lactams (HoR phenotype) alters intermediary metabolism. From this study, we
demonstrate that b-lactam-mediated HeR/HoR selection is
associated with increased expression of genes that generate
acetate, suggesting that acetate generation is one of the main
sources supplying the TCA cycle activity. In agreement with this
observation, genes encoding for lactate dehydrogenase and alcohol
dehydrogenase, the two enzymes that are central to anaerobic
fermentation, were found down-regulated. Our results provide
strong evidence of the key role played by the TCA cycle during the
HeR/HoR selection. In fact, viable inactivation of the cycle
through knock-down of the aconitase gene (acnA), the second
enzyme of the TCA cycle, abolished the capacity of SA13011HeR to become highly resistant in the presence of b-lactam
antibiotics. Importantly, when complemented cells undergoing
selection with OXA were supplemented with carbon source (e.g.,
glucose or pyruvate), resistance phenotypic levels were comparable
to the parental HoR resistant strain, demonstrating that active
TCA cycle and fueling of it with metabolites entering upper
glycolysis steps favored the HeR to HoR selection in MRSA
strains. This demonstrates the importance, in addition to OXAmediated mecA increased expression [5], of carbon sourcès ability
to be actively metabolized and to allow survival of S. aureus HoR
cells in the presence of b-lactam antibiotics. Moreover, it is
plausible that increased changes in the expression of TCA cycle
genes may represent part of a stress response triggered in response
to b-lactams. In fact, as we showed in this study, impairment of the
TCA cycle and the potential capability of the cell to adapt and
redirect its metabolism (aconitase mutant), dramatically altered
OXA-mediated HeR/HoR selection, providing strong functional
evidence of its involvement and role during the b-lactam-mediated
HeR/HoR selection.
In VISA (Vancomycin Intermediate S. aureus) strains it has been
shown that acetyl-CoA is required for the synthesis of N-acetyl
glucosamine and N-acetyl muramic acids, important constituents
of murein monomer precursor of cell wall synthesis [8]. Consistent
with these observations, we found increased expression of cell wall
genes and cell wall precursors during HeR/HoR selection, notably
elevated expression of ald (alanine dehydrogenase) as well as the
three genes involved in glycine degradation (pentaglycine bridges
of peptidoglycan) were observed, indicating an increased demand
for cell wall biosynthetic components. Numerous examples of the
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Metabolic Adaptations in Clinical MRSA Strains

that were directed against the S aureus pyruvate dehydrogenase
complex [30].

Samples Extraction and Metabolic Profiling
The metabolomic analysis was performed by Metabolon, Inc.
(Durham, NC). The untargeted metabolic profiling platform
employed for this analysis combined three independent platforms:
ultrahigh performance liquid chromatography/tandem mass
spectrometry (UHLC/MS/MS2) optimized for basic species,
UHLC/MS/MS2 optimized for acidic species, and gas chromatography/mass spectrometry (GC/MS). Samples were processed
essentially as described previously [33–38]. Five biological samples
served as replicates throughout the data set; extracted water
samples served as process blanks, and a cocktail of standards
spiked into every analyzed sample allowed instrument performance monitoring. In addition, three types of controls were
analyzed simultaneously with the experimental samples.

Materials and Methods
Bacterial Strains
Clinical MRSA strain SA13011 and derivatives are shown in
Table 4. SA13011 is representative of a heterogeneous MRSA
collection previously described [4,5], which were determined as
OXA susceptible and mecA positive [4,5]. For this study, isogenic
heterogeneous S. aureus 13011 strain (HeR; OXA MIC: 2 mg/ml)
and its highly homogeneous methicillin resistant derivative,
(SA13011-HoR; OXA MIC: 256 mg/ml) were used. SA13011
was part of a group of 25 isolates first described in a previous study
which was identified as ST5, SCCmec type II, spaType 2,
TJMBMDMGMK (13 of the 25 isolates including SA13011
presented these characteristics) [4].

Metabolite Identification and Data Analysis
Metabolites were identified by automated comparison of the ion
features in the experimental samples to a reference library of
chemical standard entries that included retention time, molecular
weight (m/z), preferred adducts, and in-source fragments as well as
associated MS spectra and curated by visual inspection for quality
control using software developed at Metabolon [37].
Experimental samples and controls were randomized across a
one-day platform run. Any missing values were assumed to be
below the limits of detection and for statistical analyses and data
display purposes, these values were imputed with the compound
minimum (minimum value imputation) after normalization to total
protein measurement (Bradford) for each sample. Following log
transformation of protein normalized imputed values, Welch’s
two-sample t-tests were used to identify biochemicals that differed
significantly (p#0.05) between experimental groups using Array
Studio software (OmicSoft) (Table S1). Multiple comparisons were
accounted for by estimating the false discovery rate (FDR) using qvalues [37] (Table S1). Hierarchical clustering was performed with
Array Studio software (OmicSoft) using complete linkage method
and correlation distance metric.

Antibiotics and Chemicals
All the antibiotics and chemicals used in this study including
oxacillin OXA (used at concentrations of 0.5 mg/ml), chloramphenicol (10 mg/ml), tetracycline (5 mg/ml); and carbon sources
pyruvate (10 mM), glucose (20 mM) and ribose (12 mM) were
purchased from Sigma-Aldrich (St. Louis, MO) and ThermoFisher Scientific (Waltham, MA).

Growth Conditions
Selection of SA13011 from the heterotypic (HeR) to the
homotypic (HoR) resistance phenotype was performed as we
previously described [5]. Briefly, bacteria were grown overnight in
5 ml LB broth without antibiotic, diluted to an optical density at
600 nm (OD600) of ,0.025 in 300 ml LB broth, either with or
without 0.5 mg/ml OXA, and grown at 37uC with shaking
(180 rpm). The ODs were monitored every hour for up to 35 h. blactam-mediated HeR to HoR selection was verified by streaking
the cells onto an OXA gradient plate with a concentration ranging
from 0 to 128 mg/ml, as previously shown [5]. SA13011-HoR
resistant cells were proven stable after several passages in freeantibiotic media as previously shown [3,5].

Analysis of Gene Expression by Microarray Transcriptional
Profiling and Real-Time RT-PCR
RNA extractions for real-time RT-PCR were performed as
previously described [5,13]. Total RNA was extracted using a
RNeasy isolation Kit (Qiagen); all RNA samples were analyzed by
A260/A280 spectrophotometry and gel electrophoresis to assess
concentration and integrity, and cleaned of potential DNA
contamination by treating them with DNAse as per manufacturer
recommendations (Ambion, Life Technologies, Austin, TX). Pairwise comparisons were made in biological triplicates between
representative strains and collected at similar exponential growth
phase [14]. Microarray transcriptional profiles were carried out as
previously described [5,14] by using a spotted DNA microarray
(TIGR version 6 S.aureus slides), containing 4546 oligos (70mer)
covering the genomes of S.aureus COL (2654 ORFs), N315 (2623
ORFs), Mu50 (2748 ORFs), MRSA 252 (2744 ORFs), MSSA 476
(2619 ORFs) and pLW043 (62 ORFs), as previously described
[5,13]. TIFF images of the hybridized arrays were analyzed using
TIGR-Spotfinder software (http://www.tigr.org/software/). The
data set was normalized by applying the LOWESS algorithm
(block mode; smooth parameter: 0.33) and using TIGR-MIDAS
(http://www.tigr.org/software/) software, and significant changes
were identified with SAM (significance analysis of microarrays;
http://www-stat.stanford.edu/,tibs/SAM/index.html) software.
Differential expression was defined as a change of more than twofold in transcript versus the comparator strain.

Mutational Insertion Inactivation of Aconitase (acnA-citB)
and Complementation
The acnA-null mutant was constructed by moving acnA::tetM
from strain UAMS-1[31] into SA13011-HeR by general transduction using 80a phage [32]. Trans-complementation of acnA was
performed by using a construct encompassing the complete acnA
gene as well as the upstream region (0.425 kb) including the
putative ribosomal binding site and promoter using acnA primers F
and R shown in Table S2. The 3.7 kb PCR fragment product was
purified using the QIAquick gel extraction kit (Qiagem, Valencia,
CA), ligated into the ligase-independent cloning site of the
PCR2.1-TOPO vector (Invitrogen, Life Technologies, Carlsbad,
CA), and transformed into chemically competent TOP10 E. coli
(Invitrogen). A staphylococcal origin of replication was introduced
by cloning plasmid pSK265, S. aureus replicon [14] into the unique
BamHI site on PCR 2.1-TOPO (Table 4); the construct was moved
into S. aureus RN4220 by electroporation [3]. Trans-complementation of acnA mutant was obtained by transduction of plasmid
psk265 containing wild-type acnA from RN4220 by phage 80a into
SA13011 acnA null mutant (SA13011-DacnA::tet).

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Metabolic Adaptations in Clinical MRSA Strains

Real-time reverse transcription-PCR analysis was done using
the SensiMix SYBR One-Step kit (Quantace/Bioline, Taunton,
MA) according to the manufacturer’s protocol. Gene expression
was compared according to the CT values converted to fold change
with respect of a sample considered as reference (value = 1) using
log2–(DDCt). The change (n-fold) in the transcript level was
calculated using the following equations: DCT = CT(test DNA) 2
CT(reference cDNA), DDCT = DCT(target gene) 2 DCT(16S rRNA), and
ratio = 22DDCT [39]. The quantity of cDNA for each experimental
gene was normalized to the quantity of 16S cDNA in each sample
as determined in a separate reaction. Each RNA sample was run
in triplicate. Values represent the means of at least three biological
replicates 6 standard error of the mean (SEM), sampled in
triplicate to minimize error by inter- and intra-samples. Differences between the mean values were analyzed using a one-way
analysis of variance (ANOVA). A P value of ,0.01 was considered
statistically significant (*). Oligonucleotide primers are shown in
Table S1.

tion. RNA was prepared from SA43002-HeR and its highly
resistant derivative SA43002-HoR (SA43002-HeR+OXA 0.5 mg/
ml) cells, collected at exponential phase of growth, as described in
Materials and Methods. Relative fold change values of specific
mRNAs in SA43002-HoR vs. SA43002-HeR (reference value = 1)
are shown on the vertical axis. Relative fold change values
representing the means of at least three biological replicates of
specific mRNAs 6 standard error of the mean (SEM), sampled in
triplicate to minimize error by inter- and intra-samples, are shown
on the vertical axis; 16S rRNA was used as an internal control.
Oligonucleotide primers are shown in Table S1.
(TIF)
Table S1 Heat map of statistically significant biochemicals profiled in this study. Shaded cells indicate p#0.05 (red
indicates that the mean values are significantly higher for that
comparison; green values significantly lower). Blue-bolded text
indicates 0.05,p,0.10. All data are normalized to Bradford
protein assay values.
(XLSX)

Supporting Information

Table S2 Primers used in this study.

Figure S1 Analysis of biochemicals corresponding to
amino-acid metabolism during b-lactam mediated
HeR/HoR selection.
(TIF)

(DOCX)

Acknowledgments
We acknowledge Dr. G. Somerville for kindly providing the strain SA564
DacnA::tetM. The results from this study have not been presented at any
scientific meetings.

Figure S2 Analysis of biochemicals corresponding to
glutathione metabolism during b-lactam mediated
HeR/HoR selection.
(TIF)

Author Contributions

Figure S3 Quantitation of mRNA levels of TCA cycle-,

Conceived and designed the experiments: AER RRR. Performed the
experiments: MAK RRR AER KBP CRS. Analyzed the data: MAK KBP
RRR AER. Wrote the paper: MAK RRR AER.

carbohydrate catabolism- and cell wall-associated genes
by Real-Time RT-PCR during SA43002 (phenotypically
similar to SA13011) b-lactam induced HeR/HoR selec-

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