JOURNAL OF BACTERIOLOGY, Apr. 2008, p. 2275–2278
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 7
Role of Staphylococcus aureus Catalase in Niche Competition against
Bonggoo Park,1Victor Nizet,2and George Y. Liu1*
Division of Pediatric Infectious Diseases and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles,
California 90048,1and Department of Pediatrics, Division of Pharmacology & Drug Discovery, School of
Medicine, University of California at San Diego, La Jolla, California 920932
Received 2 January 2008/Accepted 15 January 2008
Nasal colonization by Staphylococcus aureus is a major predisposing factor for subsequent infection. Recent
reports of increased S. aureus colonization among children receiving pneumococcal vaccine implicate Strepto-
coccus pneumoniae as an important competitor for the same niche. Since S. pneumoniae uses H2O2to kill
competing bacteria, we hypothesized that oxidant defense could play a significant role in promoting S. aureus
colonization of the nasal mucosa. Using targeted mutagenesis, we showed that S. aureus expression of catalase
contributes significantly to the survival of this pathogen in the presence of S. pneumoniae both in vitro and in
a murine model of nasal cocolonization.
Staphylococcus aureus causes a wide range of infections
ranging from minor skin infections to life-threatening invasive
diseases. The emergence of methicillin-resistant strains with
high virulence potential in both hospital and community set-
tings is contributing to a current public health crisis (9, 12, 13).
A major risk factor for S. aureus infection is antecedent colo-
nization of the nasal mucosa (19). Successful colonization de-
pends not only on the ability of S. aureus to survive host factors (4,
6) but also on coexistence with other bacteria (16, 21).
The latter concept has been underscored by two recent re-
ports that implicate Streptococcus pneumoniae as a primary
competitor for niche colonization (3, 15).
Specifically, one surveillance study performed in an area
where pneumococcal vaccination was not practiced showed
that the S. pneumoniae carriage rate in children was negatively
associated with S. aureus nasal carriage (15). The other study
showed that children with recurrent otitis media vaccinated
with the 7-valent pneumococcal vaccine had an increased in-
cidence of S. aureus-related acute otitis media and S. aureus
colonization after vaccination (3), suggesting that there is a
natural competition for colonization between S. aureus and S.
S. pneumoniae produces H2O2as an antimicrobial factor to
reduce competition by other upper respiratory pathogens, such
as Haemophilus influenzae, Neisseria meningitides, Moraxella
catarrhalis, and S. aureus (14, 16). Since S. aureus is a natural
colonizer of the human nares, we hypothesized that its success
derives in part from a relative resistance to H2O2killing by
other microflora. Here we tested this hypothesis by generating
a catalase knockout mutant strain of S. aureus and examining
the role of enzymatic H2O2inactivation in niche competition
with S. pneumoniae.
MATERIALS AND METHODS
Bacterial strains, media, and mice. S. aureus strains were cultured at 37°C in
Todd-Hewitt broth (THB) or on Todd-Hewitt agar (THA) (Difco). S. pneu-
moniae TIGR4 was cultured in THB with 0.5% yeast extract (THY) at 37°C in
a 5% CO2incubator. Eight- to 10-week old female CD1 mice were purchased
from Charles River Laboratories, Wilmington, MA. When included, antibiotics
were added at the following concentrations: 100 ?g ampicillin/ml, 50 ?g eryth-
romycin/ml, and 100 ?g spectinomycin/ml.
Generation of catalase-deficient S. aureus ?KatA mutant. In-frame allelic
replacement of the S. aureus katA gene with a spectinomycin adenyltransferase
(spec) cassette was performed using PCR-based methods as described previously
(11), with minor modifications. Primers were designed based on the previously
N315 (10). PCR was used to amplify 500 bp upstream of katA with primers katAupF
(5?-ATGGTCGACTATGACATCAACACTTGTAAC-3?) and katAupR (5?-TCA
AATATATCCTCCTCATCCCTCCACAATTTATAATAAT-3?) along with 500
bp of sequence immediately downstream of katA with primers katAdownF (5?-AA
katAdownR (5?-ATCGGATCCTACCCAGAATTACTTCGTACT-3?).The katAupR
and katAdownF primers were constructed with 25-bp 5? extensions corresponding to
the 5? and 3? ends of the spec gene, respectively. The upstream and downstream
PCR products were then combined with a 650-bp amplicon of the complete spec
gene for use as templates in a second round of PCR using primers katAupF and
katAdownR. The resultant PCR amplicon, containing an in-frame substitution of
katA with spec, was subcloned into temperature-sensitive vector pMAD (1) to
create the knockout plasmid. This vector was transformed initially into permis-
sive S. aureus strain RN4220 and then into S. aureus strain Newman by electro-
poration. Transformants were grown at 30°C and shifted to the nonpermissive
temperature for plasmid replication (40°C), and differential antibiotic selection
and blue-white color selection with 5-bromo-4-chloro-3-indolyl-?-D-galacto-
pyranoside (X-Gal) were used to identify candidate mutants. Allelic replacement
of the katA allele was confirmed unambiguously by PCRs that documented
targeted insertion of spec and the absence of katA in chromosomal DNA isolated
from the final mutant, which was designated the ?KatA mutant.
Complementation studies. Primers katAF_KpnI (5?-ATAGGTACCTCCCAT
GGTAAAGCCAAGAG-3?) and katAR_BamHI (5?-ATAGGATCCTTTACGC
GCACGTTAAACAC-3?) were used to amplify the katA gene from the chro-
mosome of wild-type (WT) S. aureus strain Newman. The fragment was
directionally cloned into the shuttle expression vector pDCerm (8), and the
recombinant plasmid (pKatA) was used to transform the S. aureus ?KatA mu-
tant by electroporation. For the complementation studies, the isogenic WT and
?KatA S. aureus strains were transformed with the control pDCerm plasmid.
Strains containing the pDCerm or pKatA plasmid were maintained in THB or on
THA containing erythromycin.
H2O2susceptibility assay. H2O2susceptibility assays were performed using
overnight S. aureus cultures grown at 37°C with shaking. Bacteria were harvested
by centrifugation, suspended in phosphate-buffered saline (PBS) at a concentra-
* Corresponding author. Mailing address: Department of Pediatrics,
Cedars-Sinai Medical Center, 8700 Beverly Blvd., Room 4221, Los
Angeles, CA 90048. Phone: (310) 423-4471. Fax: (310) 423-8284. E-
?Published ahead of print on 25 January 2008.