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
able when strains were typed in triplicate, but there was always
a core of conserved reactions for each isolate. Ten major phage
patterns could be delineated. Isolates belonging to phage vari-
ant “83C” inevitably had a strong reaction with this phage and
reacted variably with phages 42E, 81, and 90 (coded as phage
pattern 2). The phage patterns defined among this group were
83C/90 (with or without 42E—phage pattern 2a) and 83C
(phage pattern 2b). Isolates belonging to phage variant “42E”
always reacted strongly with phage 42E and reacted variably
with phages 75, 81, 83C, and 90 (coded as phage pattern 3).
The phage patterns defined among this group were 42E/75/
81/90 (3a), 42E/81/83C/90 (3b), 42E/75/81/83C/90 (3c), 42E/81/
83C (3d), 42E/75/90 (3e), and 42E/75/81/83C (3f). In addition,
one isolate gave the phage pattern 42E/79/81(phage pattern 4).
Patterns were further subdivided on the basis of reaction
strength (Table 2).
PFGE and urease results. Initially PFGE data were ana-
lyzed by comparing band differences between PFGE profiles.
The data were used to generate a dendrogram of percent
relatedness calculated by the Dice coefficient and represented
by UPGMA (Fig. 1), and five clearly different PFGE “clusters”
were identified. The clusters with multiple isolates were coded
B (145 isolates) and C (five isolates), and the others with only
a single representative were considered unique. All isolates
within cluster B showed more than 75% relatedness, with only
40% relatedness to the other four clusters and to NCTC 8325.
The other clusters in turn showed no more than 65% related-
ness to each other. Each of the clusters differed from all other
clusters by at least 14 bands. Cluster B accounted for 145 of
153 (95%) isolates, including all 55 classical EMRSA-15 iso-
lates. All cluster B isolates were urease negative. In contrast,
the cluster C isolates and the three unique isolates were urease
positive. These results suggest that these isolates had been
misclassified by phage typing, i.e., an error rate of 8% for
phage identification of EMRSA-15 variants. One of the me-
thicillin-sensitive isolates belonged to cluster B (giving the clas-
sical phage pattern of 75wk), while the other two had unique
PFGE profiles. PFGE variants occurred within clusters B and
C, and these are described in more detail below. Representa-
tive profiles are shown in Fig. 2.
Subtyping isolates within the B cluster. Among the 145
isolates within the EMRSA-15 cluster (B), 22 PFGE profiles,
differing by at least one band, were seen (B1 through 19, B21,
B23, and 24). A band difference matrix was generated using
these data (Fig. 3). The EMRSA-15 type strain was designated
B1, as were 36 other classical EMRSA-15 isolates (67% of
classical isolates). The remaining 18 classical EMRSA-15 iso-
lates produced eight PFGE profiles with 1 to 3 band differ-
ences from the progenitor pattern, B1 (Table 2). One classical
EMRSA-15 isolate was methicillin sensitive, and its profile,
B24, differed from B1 by a single band shift (a ca. 225-kb band
was replaced by a ca. 190-kb band). Variant EMRSA-15 iso-
lates showed 16 different PFGE profiles including B1, although
this profile accounted for only two isolates. Four profiles, B2,
B3, B4, and B7, accounted for 75 isolates (83% of phage
variants), with the rest of the profiles represented by 1 or 2
isolates. The four main profiles were associated with phage
patterns 2a, 3a, 3b or 3d, and 3c, respectively (Table 2). How-
ever, the correlation between these four phage patterns and
PFGE profiles was not absolute. Four isolates exhibited one of
the phage patterns listed above but produced different PFGE
profiles (B8, B14, and B16). Further, five classical EMRSA-15
isolates had profile B3 and one had profile B7.
Correlation of phage and PFGE patterns with geographical
location and hospital. There was no obvious link between geo-
graphical location and the EMRSA-15 phage variants or PFGE
subtypes (data not shown). Hospitals represented by more than
one isolate usually showed multiple PFGE and phage variants,
and there was no correlation between the PFGE profiles of the
classical and variant isolates in the 49 hospitals represented by
Enterotoxin gene carriage and production. Enterotoxin
genes sea through sed and tst were not detected in isolates from
PFGE cluster C or among those with unique PFGE profiles
(i.e., the eight non-EMRSA-15 isolates). All isolates from
PFGE cluster B were negative for genes sea, seb, sed, and tst,
although 74 carried the sec gene. These included all isolates
belonging to subtypes B1, B2, B5, B6, B18, and B23, which
were sec positive by PCR and/or Southern blotting. All isolates
belonging to the remaining B subtypes were negative for sec
(by PCR and Southern blotting) and did not produce entero-
toxin C. The sec gene was localized to a ca. 110-kb fragment on
PFGE gels (Fig. 4). Isolates carrying the enterotoxin C gene
TABLE 2. Characteristics of phage patterns and
PFGE profiles of isolates
Lytic phage reactions
at 100? RTDa
(phage pattern code)
No. of band
Classical EMRSA-15 isolates
Variant EMRSA-15 isolates
83C/90wk ? 42E (2a)
42E/81/83C ? 90wk
(3b or 3d)
among the variant
42E/81 ? 83C (3d)
aReactions in boldface are strong reactions in the phage type; underlined re-
actions varied in strength between different phage typing runs. wk, weak reaction.
1542 O’NEILL ET AL.J. CLIN. MICROBIOL.
appeared to have a band doublet at this position in their PFGE
profiles, whereas isolates without the gene appeared to have a
single band at this position and an extra band at ca. 95 kb (Fig.
5). There was a clear association between absence of the en-
terotoxin C gene and sensitivity to ?81 (Table 3).
This study was undertaken to extend our knowledge of the
characteristics of one of the two major EMRSA strains in the
United Kingdom. Previously, EMRSA-15 had been defined by
a weak reaction with ?75 of the international set, enterotoxin
C production, and nonproduction of urease. Over the past
decade, phage variants of this strain have arisen across the
United Kingdom, but given that this strain has been circulating
for at least 9 years, it is not suprising that some genetic change
has taken place. A similar broadening of the phage typing
pattern was observed with the epidemic penicillinase-produc-
ing 80/81 strain of the 1950s (21). This highly transmissable and
virulent strain was shown to have spread across, and persisted
in, several continents. The original phage pattern of 80/81
widened to 52/52A/80/81 over time and in different geographic
locations. A defective prophage was responsible for the resis-
tance of the original 80/81 strain to ?52 and ?52A, and re-
placement of this prophage by other phages resulted in the
typing pattern difference (25). Presumably, a similar mecha-
nism is responsible for the alteration of the EMRSA-15 phage
pattern, although we have not yet attempted to identify the
phages responsible for this.
Identification of EMRSA-15 phage variants. More than 90%
of isolates classified as phage variants of EMRSA-15 by phage
typing were shown to be bona fide EMRSA-15 by PFGE, val-
idating the usefulness of this quick and cost-effective method
for typing. In addition, the fact that all the strains misidentified
as EMRSA-15 were urease producers confirms that this simple
test would eliminate the majority of false identifications. Fur-
thermore, the phage patterns of the variant strains were gen-
erally stable and a core of strong reactions was identified.
Although a well-established rule exists for interpreting phage
patterns at RTD (two or more strong reaction differences
define unrelated strains), this has not yet been validated at
100? RTD, and in any case it was intended for use only with
geographically and temporally related isolates. This study sug-
FIG. 1. Dendrogram of percent relatedness of PFGE profiles from putative EMRSA-15 isolates calculated using the Dice coefficient and
represented by UPGMA. Band tolerances were set at 1.0%.
VOL. 39, 2001 CHARACTERIZATION OF PHAGE VARIANTS OF EPIDEMIC MRSA-151543
FIG. 2. SmaI restriction profiles of putative EMRSA-15 isolates. (Top): PFGE profiles B1 through B16; (bottom) PFGE profiles B17 through
B24 and the non-EMRSA-15 profiles (C1 through C5 and unique profiles). A commercial molecular weight marker consisting of concatemers of
lambda DNA, the EMRSA-15 control strain NCTC 13142, and S. aureus strain NCTC 8325 were included as controls on all gels.
1544O’NEILL ET AL.J. CLIN. MICROBIOL.
gests that these reaction difference rules may need to be mod-
ified for strains that require typing at 100? RTD.
Discrimination between EMRSA-15 isolates by PFGE. The
demonstration of different PFGE profiles among, and unique
to, the classical EMRSA-15 isolates illustrates the role that
genetic events such as point mutations, insertions, and dele-
tions may play in altering the PFGE patterns of closely related
isolates. The profile (B24) of one classical EMRSA-15 isolate,
which was methicillin sensitive, differed from the progenitor
pattern B1 by a single band shift from ca. 225 kb to ca. 190 kb.
The size of the shift in molecular weight equates well with that
published for the mec locus in United Kingdom MRSA isolates
(9) and suggests that it is the loss of this genetic element which
has generated subtype B24 from B1. The concordance between
PFGE profile and phage pattern for variant isolates is indica-
tive of the link between loss or gain of prophages from the
genome (resulting in widening of the phage typing pattern) and
changes in either SmaI restriction sites or fragment sizes re-
sponsible for the PFGE banding patterns. Interestingly, three
PFGE profiles, B1, B3, and B7, were represented by both
variant and classical isolates. Most B1 isolates were classical
EMRSA-15 exhibiting the 75wk pattern, but two isolates re-
acted with phages 83C/90 and 42E/75/90 respectively. Con-
versely, although the majority of B3 and B7 isolates were
EMRSA-15 variants, five B3 isolates and one B7 isolate gave
the classical pattern 75wk. These findings suggest that replace-
ment of prophages by other circulating phages may cause
changes in the PFGE profile which are not reflected by
changes in the phage pattern and vice versa. This is sup-
ported by data from Arbeit (3), who showed that different
phages could be obtained from isolates with identical PFGE
patterns and also that the same phage could insert into dif-
ferent fragments, creating strains which had the same ge-
netic composition but PFGE patterns which differed by as
many as 4 bands. These findings illustrate that no typing
system is absolute and that sometimes even a simple system
such as phage typing can give as much, if not more, infor-
mation than a complex DNA-based typing system such as
Criteria for interpretation of PFGE data. For epidemiolog-
ical typing, knowledge of both strain identity and variability
within the strain allows us to make judgments on whether
FIG. 3. Band difference matrix of PFGE profiles of EMRSA-15 isolates. Numbers represent the band differences between PFGE profiles listed
on the x and y axes.
VOL. 39, 2001 CHARACTERIZATION OF PHAGE VARIANTS OF EPIDEMIC MRSA-151545
direct cross-infection or independent acquisition has taken
place. However, interpretation of PFGE data obtained from a
widespread strain such as EMRSA-15 can be problematic with
regard to both strain identity and variability.
For local epidemiological studies, criteria of strain related-
ness such as those described by Tenover et al. (26) with the
modifications of Goering (8) are often applied. These criteria
suggest that isolates showing 1 to 3 band differences from the
outbreak (or progenitor) strain are probably related and part
of the outbreak and that those showing 4 to 6 band differences
may be related. However, these criteria are validated only for
use within epidemiologically and temporally defined (?6
months) outbreaks. Isolates in this study were chosen from
geographically diverse locations and were temporally spaced.
Despite this, the majority of isolates within the B cluster fell
within 1 to 3 band differences from the progenitor pattern, B1.
These findings suggest that most isolates belonging to the B
cluster differ by one genetic event from B1 and that even the
most divergent member of the B cluster, B11, may differ from
B1 by only two genetic events (giving a 6-band difference in the
PFGE profile). How then, do we distinguish between cross-
infection and independent acquisition of a strain such as
EMRSA-15 when closely related PFGE profiles are obtained
from isolates recovered during a suspected outbreak? A recent
study by MacFarlane et al. (16) retrospectively analyzed two
putative outbreaks of EMRSA-15 within a United Kingdom
FIG. 4. Hybridization of a probe for sec to SmaI restriction profiles
of enterotoxin C-positive and -negative EMRSA-15 isolates. Size stan-
dards were visualized by probing with biotin-labeled ? digested with
HindIII. Isolates belonging to PFGE profiles B1 and B2 were sec
positive by PCR, and those belonging to profiles B3 and B4 were
negative. EMRSA-16 was included as a negative control.
FIG. 5. Overlaid densitometric curves of the PFGE profiles of variants B1 and B3 illustrating the difference in peak heights of the band at 110
kb in the profile. Band sizes quoted are approximate.
1546 O’NEILL ET AL.J. CLIN. MICROBIOL.
hospital. In one outbreak they found several different PFGE
profiles differing by 1 to 4 bands, whereas in the second a single
PFGE profile was identified. They suggested that in a highly
clonal organism such as EMRSA-15, perhaps even single band
differences between isolates may be of epidemiological signif-
icance. Prospective epidemiological studies involving hospitals
with a large circulating EMRSA-15 population and known
phage and/or PFGE subtypes may be helpful in attempting to
answer this question.
Although most PFGE profiles obtained from isolates within
the B cluster were closely related to the progenitor profile B1,
higher numbers of band differences were seen when the more-
divergent PFGE variant profiles were compared with each
other. For example, B11 and B18 differ by 12 bands. Compar-
ing these two profiles in isolation and interpreting the results
using the Tenover criteria would lead to their classification as
different strains. Although two isolates with such widely differ-
ing PFGE profiles are unlikely to represent an incident of
cross-infection, classifying them as belonging to different ge-
netic lineages is erroneous. This suggests that in dealing with a
widely disseminated strain such as EMRSA-15, the range of
PFGE profiles must be regarded as a continuum and cutoff
points such as 4 to 6 band differences (which are appropriate
for isolates from putative temporally and geographically re-
lated outbreaks) are too stringent for determining strain iden-
tity. It also emphasizes the importance of relating profiles back
to the most common (or progenitor) profile for analysis.
Carriage of the sec gene. Of particular interest was the ap-
pearance of enterotoxin C-negative variants of EMRSA-15.
These had previously been reported for some isolates with the
classical EMRSA-15 phage pattern (24) but had not been
correlated with a change in PFGE profile. Examination of
sec-negative isolates showed that loss of this gene was associ-
ated with a specific change in PFGE profile, namely, a marked
decrease in the intensity of the ca. 110-kb band hybridizing to
the sec gene probe in sec-positive isolates, concomitant with
the appearance of an extra band at ca. 95 kb. Furthermore, the
sec gene probe failed to hybridize with the remaining ca.
110-kb band in the profile of sec-negative variants. Examina-
tion of peak heights in the densitometric traces of the sec-
positive and -negative isolates supports the view that sec-pos-
itive isolates are characterized by a doublet band at ca. 110 kb,
whereas in sec-negative strains only a single band is present
(Fig. 5). These findings suggest that the sec gene is carried on
a piece of DNA of ca. 15 kb, which appears to have been
excised from the genome in sec-negative variants.
It is known that enterotoxin genes are often located on
mobile elements. Enterotoxin A is phage encoded (5), entero-
toxin D is plasmid borne (4), and TSST-1 is encoded on a
pathogenicity island (15). The relatively small size of the frag-
ment change suggests that enterotoxin C is not phage encoded,
since the average size of the phage genome is ca. 45 to 50 kb.
Studies on the TSST-1 pathogenicity islands SAPI1 and SAPI2
have shown that the tst gene is carried on a 17-kb segment of
DNA that can be mobilized by certain phages (15). It may be,
therefore, that enterotoxin C is encoded on a similar pathoge-
nicity island, since the size of the element appears to be similar
and there is a phage association in that the majority of the
isolates which were sec negative were sensitive to ?81. Further
studies to test this hypothesis are under way.
Overall, this study has shown the usefulness of both phage
typing and PFGE for monitoring the evolution of a prevalent
strain of MRSA within the United Kingdom and has suggested
further avenues of exploration concerning transmission of vir-
ulence factors within S. aureus.
We thank Tyrone Pitt and Barry Cookson for critical comments on
the manuscript and Maria Mena, Mark Ganner, and Marina Warner
for technical assistance.
A. Gil-Setas was supported by a grant from the Spanish Society of
Infectious Diseases and Clinical Microbiology. S. Murchan was funded
by the EU DG-XII HARMONY project and participated in this proj-
ect to facilitate method development for HARMONY.
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No. ?81 resistant
Phage variant EMRSA-15
Total 7371 64
VOL. 39, 2001CHARACTERIZATION OF PHAGE VARIANTS OF EPIDEMIC MRSA-15 1547
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