JOURNAL OF CLINICAL MICROBIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Apr. 2001, p. 1540–1548Vol. 39, No. 4
Identification and Characterization of Phage Variants
of a Strain of Epidemic Methicillin-Resistant
Staphylococcus aureus (EMRSA-15)
G. L. O’NEILL,1* S. MURCHAN,1A. GIL-SETAS,2AND H. M. AUCKEN1
Laboratory of Hospital Infection, Central Public Health Laboratory, London, NW9 5HT, United Kingdom,1
and Microbiologia, Virgin del Campino Hospital, Pamplona, Spain2
Received 18 September 2000/Returned for modification 11 November 2000/Accepted 11 January 2001
EMRSA-15 is one of the most important strains of epidemic methicillin-resistant Staphylococcus aureus
(EMRSA) found in the United Kingdom. It was originally characterized by weak lysis with phage 75 and
production of enterotoxin C but not urease. Two variant strains of EMRSA-15 which show a broader phage
pattern than the progenitor strain have emerged. A total of 153 recent clinical isolates representing classical
EMRSA-15 (55 isolates) or these phage variants (98 isolates) were compared by SmaI macrorestriction profiles
in pulsed-field gel electrophoresis (PFGE) as well as by urease and enterotoxin C production. Eight of the 98
isolates were shown to be other unrelated strains by both PFGE and their production of urease, a misidenti-
fication rate of 8% by phage typing. Seventy-one EMRSA-15 isolates were enterotoxin C negative, and the
majority of these were sensitive to phage 81. Examination of PFGE profiles and Southern blotting studies
suggest that the enterotoxin C gene locus is encoded on a potentially mobile DNA segment of ca. 15 kb. After
elimination of the eight non-EMRSA-15 isolates, the remaining 145 were characterized by PFGE, yielding 22
profiles. All profiles were within five band differences of at least one other profile. Classical EMRSA-15 isolates
showed nine PFGE profiles, with the majority of isolates (68%) in profile B1. Six of these nine PFGE profiles
were unique to the classical EMRSA-15 isolates. Among the phage variants of EMRSA-15, 16 profiles were
seen, but the majority of isolates (83%) fell into 1 of 4 profiles (B2, B3, B4, and B7) which correlated well with
phage patterns. The most divergent PFGE profiles among the EMRSA-15 isolates had as many as 12 band
differences from one another, suggesting that in examining isolates belonging to such a temporally and
geographically disseminated epidemic strain, the range of PFGE profiles must be regarded as a continuum and
analyzed by relating the profiles back to the most common or progenitor profile.
Staphylococcus aureus is the leading cause of surgical wound
infections and the second most frequent cause of bacteremia in
the hospital setting (7). Methicillin-resistant S. aureus (MRSA)
first appeared in the early 1960s but virtually disappeared dur-
ing the 1970s in the United Kingdom. It reappeared in the
early 1980s (18), and over the last two decades, strains with
resistance to an extended range of antibiotics have emerged to
pose a major threat to public health. MRSA isolates may com-
prise as much as 45% of the total number of S. aureus isolated
from patients with bacteremia (2). Thousands of isolates are
sent each year to the S. aureus Reference Service (SaRS) at
the Central Public Health Laboratory (CPHL) for typing;
there they are subdivided primarily on the basis of their sus-
ceptibilities to 27 phages (19, 21). Where additional discrimi-
nation between strains is required, SmaI digests of chromo-
somal DNA are subjected to pulsed-field gel electrophoresis
Epidemic strains of MRSA are defined as those which have
been identified in two or more patients in two or more hospi-
tals (13). The first epidemic MRSA (EMRSA) strain, desig-
nated EMRSA-1, was recognized in 1981 (17) and continued
to cause outbreaks in hospitals until the late 1980s. A second
EMRSA strain, EMRSA-2, emerged in the late 1980s (22) and
was followed closely by 12 other EMRSA strains described-
during a survey carried out in 1987 and 1988 (13). EMRSA-15
emerged during 1991 and rapidly displaced most of the other
EMRSA strains (1). It has now spread to, and is endemic in,
hundreds of hospitals across the United Kingdom; it has also
recently been identified as causing outbreaks in Australia, New
Zealand, Germany, Sweden, and Finland. The strain classically
is characterized by weak lysis with phage (?) 75 of the inter-
national set, production of enterotoxin C, and nonproduction
of urease (24).
In recent years two “variant” strains of EMRSA-15 have
been recognized by SaRS. They were identified as EMRSA-15
variants despite producing additional phage reactions, notably
with phages 42E, 81, 83C, and 90, because they were pheno-
typically similar to classical EMRSA-15 isolates (i.e., in colo-
nial morphology, toxin production, and lack of urease produc-
tion) and arose in hospitals with large circulating EMRSA-15
populations. Subsequent investigation of some of these isolates
by PFGE revealed that they gave SmaI digestion patterns iden-
tical or closely related to those of the classical EMRSA-15
isolates, and they were coded as phage variants “42E” and
“83C.” Subsequently, some of these variants have lost the re-
action with phage 75 which was initially a defining character-
istic of EMRSA-15.
This study was undertaken to determine, firstly, whether all
isolates classified as EMRSA-15 phage variants are genotypi-
* Corresponding author. Present address: Division of Environmen-
tal Health and Risk Management, Public Health Building, University
of Birmingham, Birmingham B15 2TT, United Kingdom. Phone: 44
121 414 7750. Fax: 44 121 414 3078. E-mail: firstname.lastname@example.org.
cally EMRSA-15; secondly, whether variation in phage pattern
is related to variation in PFGE profile; and finally, whether any
other characteristics of the strain vary with particular changes
in phage pattern and/or PFGE profile.
MATERIALS AND METHODS
Bacterial strains. In total, 153 clinical isolates of EMRSA-15 and the
EMRSA-15 phage variants “42E” and “83C” were examined. Isolates were
selected for inclusion in the following manner. Phage typing records of isolates
from 1998 were used to identify all 55 hospitals from disparate geographical
locations in England, Wales, and the Republic of Ireland which had sent phage
variants of EMRSA-15 to SaRS. In some hospitals, multiple different phage
variants (i.e., patterns showing reaction differences from one another) were
found, so a single representative of each phage variant pattern was included from
each hospital. In total, 98 isolates representing the phage variants of EMRSA-15
were selected for further study. In addition, classical EMRSA-15 isolates (based
on a weak reaction with phage 75 only) were selected as controls from 49 of the
55 hospitals (no classical EMRSA-15 isolates were present in 6 of the hospitals).
Upon subsequent enterotoxin testing, the “classical” isolates from six hospitals
were found to be enterotoxin C negative. Due to this finding, an additional
classical isolate from each of these hospitals, which was enterotoxin C positive,
was included. The EMRSA-15 type strain (NCTC 13142) was included as a
control strain for enterotoxin C production, PFGE, and phage typing. Additional
controls were NCTC 8325, ATCC 29213 (susceptibility testing), and the enter-
otoxin-producing strains NCTC 10652 (enterotoxin A; gene sea), NCTC 10654
(enterotoxin B; seb), NCTC 10655 (enterotoxin C; sec), NCTC 10656 (entero-
toxin D; sed), and NCTC 11963 (toxic shock syndrome toxin [TSST-1], tst).
Isolates were stored on nutrient agar (NA) slopes at room temperature and
recovered by subculture on NA plates followed by overnight incubation at 37°C.
All isolates were maintained on Microbank beads (Prolab, UK) at ?70°C.
Phenotypic characterization of strains. Strains were phage typed in triplicate
using the international phage set (21) and local experimental phages 88A, 90,
83C, and 932 (19). Phage typing of the isolates was performed at 100 times the
routine test dilution (100? RTD) because most United Kingdom MRSA isolates
are nontypeable at RTD (23). Isolates were tested for coagulase, urease produc-
tion, and antimicrobial susceptibility by standard methods as previously de-
The following agents were tested by the agar dilution method on Isosensitest
agar (Oxoid, Ltd., Basingstoke, United Kingdom) with 2% horse blood and
interpreted using the following resistance breakpoints: ciprofloxacin, ?1 mg/
liter; erythromycin, ?0.5 mg/liter; fusidic acid, ?1 mg/liter; gentamicin, ?1
mg/liter; kanamycin, ?4 mg/liter; neomycin, ?4 mg/liter; methicillin, ?4 mg/
liter; mupirocin, ?8 mg/liter; streptomycin, ?4 mg/liter; rifampin, ?0.12 mg/
liter; teicoplanin, ?4 mg/liter; tetracycline, ?1 mg/liter; and vancomycin, ?4
mecA PCR. Isolates which were sensitive to methicillin were examined for
carriage of the mecA gene by PCR according to the method of Bignardi et al. (6).
Enterotoxin detection. All isolates were screened for the presence of entero-
toxin genes A through D (sea through sed) and the TSST-1 gene (tst) by the
method of Johnson et al. (10), but DNA was extracted by boiling in 5% Chelex
100 (Bio-Rad Laboratories, Hercules, Calif.) and lysates were centrifuged to
remove cell debris. The supernatant was transferred to a fresh tube, and 5 ?l was
used as a template in the PCRs (20). Selected isolates were also tested for the
production of enterotoxins A through D using a reverse passive latex agglutina-
tion kit (SET-RPLA; Oxoid Ltd.) according to the manufacturer’s instructions.
PFGE. PFGE was performed by the method of Kaufmann (11). Briefly, DNA
was extracted from overnight cultures grown at 37°C on NA and restriction
digested with SmaI (Boehringer GmbH, Mannheim, Germany) overnight at 30°C
according to the manufacturer’s instructions. Digested DNA was electropho-
resed in 1.2% agarose gels for 30 h with a ramped pulse time of 1 to 80 s using
a CHEF DRII or CHEF Mapper (Bio-Rad Laboratories). DNA fragments were
visualized by staining with 0.5 ?g of ethidium bromide/ml. Gels were photo-
graphed under UV illumination, and the data were saved to a floppy disk prior
to analysis. Gel data were analyzed with GelCompar software (Applied Maths,
Probe generation, Southern blotting, and hybridization. A subset of isolates
were examined for enterotoxin C gene (sec) carriage by Southern blotting. A
699-bp biotin-labeled sec probe was generated by PCR using the following
primers: TGT ATC AGC AAC TAA AGT TAA GTC and AAA GGCAAG
CAC CGA AG. PCR was performed in a total volume of 100 ?l containing 2.5
U of Taq polymerase, 200 ?M deoxynucleoside triphosphates, 68 ?M biotin-16-
dUTP, 2 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 5 ?l of
template DNA (prepared as described above). Cycling conditions consisted of 1
cycle of denaturation at 96°C for 2 min followed by 30 cycles of denaturation at
94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 1 min, with
a final extension at 72°C for 5 min. The PCR product was purified using a
QIAquick PCR purification kit (Qiagen Ltd., Crawley, United Kingdom), and
the probe concentration was estimated spectrophotometrically at 260 nm. DNA
fragments from PFGE gels were transferred to nylon membranes (Hybond N;
Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by vacuum
blotting according to the method of Kaufmann et al. (12) with the following
modifications: the fragmentation solution (0.25 M HCl) was applied for 25 min,
the denaturation solution (0.5 M NaOH–1.5 M NaCl) was applied for 1 h, the
neutralization solution (1.5 M NaCl–0.5 M Tris) was applied for 30 min, and then
blotting was undertaken using 20? SSC (1? SSC is 0.15 M NaCl plus 0.015 M
sodium citrate) for 2 h at 5 ? 103Pa. Hybridization was performed by the
method of Kaufmann et al. (12) using the sec probe at a concentration of 1 ?g/ml.
Commercially available biotinylated ? DNA digested with HindIII at a concen-
tration of 1 ?g/ml was used as a probe to detect the ? concatamer size standards
on the PFGE gels. Hybridization was detected with the BlueGene Nonradioac-
tive Detection System (Gibco BRL, Life Technologies, Paisley, United King-
Coagulase production and methicillin resistance. All iso-
lates were coagulase positive, and 149 of 153 isolates were
resistant to methicillin. Of the four methicillin-sensitive iso-
lates, one was positive for the mecA gene by PCR. The other
three isolates were included in the study despite being methi-
cillin sensitive because their phage patterns were as expected
and loss of the mecA gene upon storage is known to occur (14).
Susceptibility to other antimicrobial agents. The sensitivity
patterns for the classical and variant EMRSA-15 isolates were
similar, with all isolates susceptible to gentamicin, neomycin,
teicoplanin, and vancomycin and the majority susceptible to
fusidic acid, kanamycin, mupirocin, rifampin, streptomycin,
and tetracycline (Table 1). However, almost all classical
EMRSA-15 isolates (94%) were resistant to ciprofloxacin, and
the majority were resistant to erythromycin (80%). Resistance
to these two antibiotics was less frequent among the variant
isolates (76 and 62%, respectively).
Phage typing. All 55 clinical isolates included as classical
EMRSA-15 isolates gave the expected phage reaction of 75wk
(coded as phage pattern 1). Isolates of the variant strains
reacted with a combination of the expected phages: 42E, 75,
81, 83C, and 90 (Table 2). Certain weak reactions were vari-
TABLE 1. Antimicrobial susceptibilities of EMRSA-15 isolates
No. (%) of isolates resistant
(n ? 55)
(n ? 90)
(n ? 145)
Fusidic acid (?1)
VOL. 39, 2001CHARACTERIZATION OF PHAGE VARIANTS OF EPIDEMIC MRSA-151541
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1548 O’NEILL ET AL. J. CLIN. MICROBIOL.