J Infect Chemother (2002) 8:28–32 © Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2002
ORIGINAL ARTICLE
Hiroki Okamoto · Kazuhiro Tateda · Yoshikazu Ishii
Tetsuya Matsumoto · Takao Kobayashi
Shuichi Miyazaki · Keizo Yamaguchi
High frequency of erythromycin A resistance and distribution of mefE and
ermB genes in clinical isolates of Streptococcus pneumoniae in Japan
Received: June 25, 2001 / Accepted: September 19, 2001
Abstract The ermB and mefE genes are important in terms
of their responsibility for macrolide resistance in Strepto-
coccus pneumoniae. We investigated the distribution of the
ermB and mefE genes in erythromycin A-resistant S.
pneumoniae isolated in our hospital during the period 1995–
1998. All amoxicillin-low-susceptible isolates were consid-
ered to be resistant to erythromycin A. All isolates with
resistance to erythromycin A (minimum inhibitory concen-
trations [MICs], ⱖ0.5 µg/ml) possessed ermB or mefE
genes. The MICs of erythromycin A for most mefE-positive
isolates ranged from 0.5 to 2 µg/ml. On the other hand,
approximately 85% of ermB-positive isolates demonstrated
high-level resistance to erythromycin A (MICs, ⱖ4 µg/ml)
while the others showed low-level resistance (MICs, 0.5 to
2 µg/ml). In ermB-positive isolates with low-level resistance,
high-level resistant mutants were selected with a frequency
of 3.1 ⫻ 10⫺6–1.8 ⫻ 10⫺3 on agar containing 4-32 MIC of
erythromycin A, whereas in mefE-positive isolates, no
high-level resistant mutants were detected. Mutants from
ermB-positive isolates with low-level resistance showed
reversibility and heterogeneity. Our data indicated a wide
distribution of erythromycin A-resistant isolates with mefE
or ermB genes among amoxicillin-low-susceptible S.
pneumoniae in Japan. In addition, it is likely that ermB-
positive isolates with low-level resistance show heteroge-
neous high-level resistance.
Key words Streptococcus pneumoniae · Erythromycin A-
resistant · mef and erm gene family
H. Okamoto1 (*) · K. Tateda · Y. Ishii · T. Matsumoto ·
T. Kobayashi · S. Miyazaki · K. Yamaguchi
Department of Microbiology, Toho University School of Medicine,
Tokyo, Japan
Present address:
1
Pharmacology, Lead Optimization, Aventis Pharma Ltd., 1-3-2
Minamidai, Kawagoe, Saitama 350-1165, Japan
Tel. ⫹81-492-43-2421; Fax ⫹81-492-43-4002
e-mail: hiroki.okamoto@aventis.com
Introduction
In recent years, isolates of Streptococcus pneumoniae with
low susceptibility to penicillin have become significant
worldwide problems.1 A number of penicillin-resistant
pneumococci are also resistant to one or more additional
antibiotics, such as cephalosporins and macrolides.2–4 Al-
though the frequency of resistance varies by area and coun-
try, several investigators have reported a high frequency of
macrolide resistance in penicillin-resistant pneumococci.5–8
A nationwide survey in Japan conducted in 1994–1995 (n ⫽
1283) reported that 40.8% of pneumococci were resistant to
penicillin G and 53.7% of the same bacteria were resistant
to erythromycin A.8
Recent studies have identified two important gene fami-
lies responsible for macrolide resistance; erm (a ribosomal
methylase) and mef (a macrolide-specific efflux mecha-
nism).9–13 While ermB confers resistance to most macro-
lides, lincosamides, and streptogramin B antibiotics, mefE
confers resistance only to the 14- and 15-membered macro-
lides.9–11 The mef genes induce low-level resistance, whereas
the erm genes are associated with high-level resistance to
macrolides in pneumococci.9–11 These data clearly demon-
strate the importance of epidemiological and genetic sur-
veys to identify the prevalence and type of macrolide
resistance in clinical isolates of S. pneumoniae. Such surveys
provide valuable information on the choice of antibiotic
therapy in clinical practice.
In the present study, we examined the distribution of erm
and mef genes in erythromycin A-resistant S. pneumoniae
isolated in our hospital. In addition, we studied the fre-
quency of single-step resistance and the reversibility and the
heterogeneity of erm and mef-positive pneumococci.

Materials and methods
Isolates
Erythryomycin A-susceptible S. pneumoniae isolates (n ⫽
32) and -resistant S. pneumoniae isolates (n ⫽ 97) isolated
Toho University Hospital during the period 1995–1998
were used in this study. The isolates were stored at ⫺80°C
until used.
Antibiotics
Amoxicillin (Fujisawa Pharmaceutical, Osaka, Japan),
erythromycin A (Shionogi Pharmaceutical, Osaka, Japan),
and clarithromycin (Taisho Seiyaku, Tokyo, Japan) were
kindly supplied by the indicated companies.
Antimicrobial susceptibility testing
The minimum inhibitory concentration (MIC) of each anti-
biotic was determined by the broth dilution method, using
Mueller-Hinton broth (Difco, Detroit, MI, USA) supple-
mented with 5% lysed horse blood. An inoculum of 5.0 ⫻
104 CFU/well was placed onto a microtiter plate and incu-
bated in air for 18 h at 35°C. The MIC was defined as the
lowest concentration of antimicrobial agent that resulted in
the inhibition of the visible growth of S. pneumoniae.14 The
susceptibility breakpoints for erythromycin were defined
according to the National Committee for Clinical Labora-
tory Standards (NCCLS) year 2000 guidelines;14 strains with
an erythromycin A MIC of 0.5 or more are considered
resistant. That for amoxicillin was determined according to
the NCCLS 1997 guidelines,15 because the latest guidelines
(i.e., year 2000)14 showed a tentative breakpoint for
amoxicillin. Namely, a strain with an amoxicillin MIC of
1 µg/ml or more was considered low-susceptible. For eryth-
romycin A-resistance, strains with an erythromycin MIC of
0.5 to 2 µg/ml and those with an erythromycin MIC of 4 µg/
ml or more were considered low- and high-level resistant,
respectively.
DNA isolation and detection of ermB and mefE genes
Genetic DNA was obtained according to the instructions
provided with SepaGene (Sanko Junyaku, Tokyo, Japan).
The concentration of DNA was adjusted to 1 µg/ml with
distilled water by Optimal Density at 260 nm. The primer
sets used for the genes were 5⬘-GAAAAGGTACTCAAC
CAAATA-3⬘ and 5⬘-AGTAACGGTACTTAAATTGTT
TAC-3⬘ for ermB and 5⬘-AGTATCATTAATCACTAG
TGC-3⬘ and 5⬘-TTCTTCTGGTACTAAAAGTGG-3⬘ for
mefE, based on previously published reports.9,16 The poly-
merase chain reaction (PCR) reaction mixture (50 µl) con-
tained 100 µM primer sets, 2.5 units Taq (Takara, Kyoto,
Japan), and 0.25 mM dNTP mixture (dATP, dCTP, dTTP,
and dGTP). DNA in the reaction mixture was denatured
at 93°C for 3 min. The amplification cycles consisted of
29
extension at 72°C for 60 s, denaturation at 93°C for 60 s, and
annealing at 52°C for 60 s. After 35 amplification cycles, the
last extension step was performed at 72°C for 5 min. The
PCR product was separated by electrophoresis on 3% aga-
rose gels (NuSieve 3 : 1; FMC Bioproducts, Rockland MA,
USA). Strain 3585 (ermB) and strain 02J1175 (mefE),
kindly provided by Dr. Joyce de Azavedo, University of
Toronto, were used for the positive control.
Development of high-level resistance in isolates with low-
level resistance to erythromycin A
ErmB (⫹)/mefE (⫺) and ermB (⫺)/mefE (⫹) isolates
(three isolates each) with low-level resistance to erythromy-
cin A (MICs, 0.5–2 µg/ml) were plated on Muller-Hinton
agar containing 4, 8, 16 or 32 MIC of erythromycin A. After
incubation in air for 48 h at 35°C, the number of resistant
colonies on each plate was counted. We also examined the
reversibility in high-level resistant mutants. For this pur-
pose, the mutants grown on Muller-Hinton agar containing
32 MICs of erythromycin A were subcultured on antibiotic-
free agar and incubated in air for 18 h at 35°C. This subcul-
ture process was repeated twice, and then MICs for each
colony were re-examined. Population analysis of isolates
with low-level resistance was done by spreading 100 µl of
bacterial suspension, which had been prepared to dilute
overnight cultures to about 108 CFU/ml, and the serial dilu-
tions of this suspension over Brain Heart Infusion (BHI)
agar plates containing various concentration of erythromy-
cin A. The plates were incubated in air for 48 h at 35°C, and
the number of mature colonies was counted.
Results
Correlation between MICs of amoxicillin and
erythromycin A
The correlation between the MIC of amoxicillin and that of
erythromycin A was examined in 129 isolates of pneumo-
cocci. All erythromycin A-susceptible isolates (MICs,
ⱕ0.25 µg/ml) were susceptible to amoxicillin (MIC, ⱕ0.5 µg/
ml; Fig. 1). In contrast, among 97 pneumococci resistant to
erythromycin A (MICs, ⱖ0.5 µg/ml), 44 (45.4%) were
amoxicillin-low-susceptible (MICs, ⱖ1 µg/ml). Of a total of
85 pneumococci with an MIC of amoxicillin of 0.5 µg/ml or
less, 53 (62.3%) were resistant to erythromycin A, whereas
all 44 amoxicillin-low-susceptible isolates were also resis-
tant to erythromycin A. We could not find S. pneumoniae
populations that were erythromycin A-susceptible and at
the same time amoxicillin-resistant.
Distribution of ermB and mefE genes
All isolates resistant to erythromycin A possessed either
ermB or mefE genes. On the other hand, erythromycin-
susceptible isolates had neither ermB or mefE genes (data
30

Fig. 1. Correlation of amoxicillin and erythromycin A minimum in- Fig. 2. MICs of erythromycin A and association with presence of mefE
hibitory concentrations (MICs) in Streptococcus pneumoniae isolates. and/or ermB genes in S. pneumoniae. Dotted bars, ermB(⫺)/mefE(⫹);
Small dots, One strain; large dots, Five strains striped bars, ermB(⫹)/mefE(⫺); black bars, ermB(⫹)/mefE(⫹)

Table 1. Frequency of S. pneumoniae mutants highly resistant to erythromycin A

Parent strains Genotype Erythromycin A Selective concentration of Frequency
MIC (µg/ml) erythromycin A (µg/ml)
ermB mef

TOH-58 ⫺ ⫹ 1 4 ⬉5.6 ⫻ 10⫺6

TOH-69 ⫺ ⫹ 2 8 ⬉3.0 ⫻ 10⫺7
TOH-94 ⫺ ⫹ 1 4 ⬉3.0 ⫻ 10⫺7
TOH-103 ⫹ ⫺ 1 4 6.7 ⫻ 10⫺3
8 6.4 ⫻ 10⫺3
16 3.6 ⫻ 10⫺3
32 1.8 ⫻ 10⫺3
TOH-111 ⫹ ⫺ 0.5 2 2.8 ⫻ 10⫺4
4 8.2 ⫻ 10⫺5
8 3.3 ⫻ 10⫺5
16 1.8 ⫻ 10⫺5
TOH-112 ⫹ ⫺ 0.5 2 1.4 ⫻ 10⫺3
4 7.4 ⫻ 10⫺5
8 2.5 ⫻ 10⫺5
16 3.1 ⫻ 10⫺6
MIC, Minimum inhibitory concentration

not shown). The MICs of erythromycin A for mefE(⫹)
S. pneumoniae ranged from 0.5 to 8 µg/ml, and 93.7% of
them were less than 2 µg/ml (Fig. 2). Although the MICs of
erythromycin A for ermB(⫹) isolates varied widely, from
0.5 to more than 128 µg/ml, approximately 85% of the
isolates showed high-level resistance to erythromycin A
(MICs, ⱖ4 µg/ml). A similar correlation between MICs and
the presence of these genes was observed for clarithromycin
(data not shown). We found two double-positive isolates in
this study, and the erythromycin A MICs of both isolates
were more than 128 µg/ml.
Development of high-level resistance in isolates with low-
level resistance to erythromycin A
The rate of mutation with high-level resistance to erythro-
mycin A was examined in mefE (⫹)/ermB (⫺) and mefE
(⫺)/ermB (⫹) isolates whose MICs of erythromycin A
ranged from 0.5 to 2 µg/ml (three isolates each; Table 1). In
mefE (⫹)/ermB (⫺) isolates, no resistant mutants were ob-
tained in any concentrations of erythromycin A examined
(MICs, 4–32). On the other hand, mutants with a high eryth-
romycin A-resistance were selected in mefE (⫺)/ermB (⫹)
isolates, with a frequency of 3.1 ⫻ 10⫺6 to 1.8 ⫻ 10⫺3,
even on plates containing 32-MIC of erythromycin A.
Reversibility of erythromycin A resistance was observed in
three mutants after two passages on antibiotic-free media
(data not shown). A comparison of the population analysis
of TOH-102 (mefE (⫹)/ermB (⫺) strain) and TOH-112
(mefE (⫺)/ermB (⫹) strain) is shown in Fig. 3. TOH-102
was completely inhibited by 4 µg/ml of erythromycin A. On
the other hand, TOH-112 contained high-resistant popula-
tions that grew in the presence of 128 µg/ml of erythromycin
A.

Fig. 3. Population analysis of S. pneumoniae TOH-102 (ermB(⫺)/
mefE(⫹)) and TOH-112(ermB(⫹)/mefE(⫺)). Dots, S. pneumoniae
TOH-112 (ermB(⫹)/mefE(⫺)); triangles, S. pneumoniae TOH-102
(ermB(⫺)/mefE(⫹))
Discussion
In the present study, we examined the distribution of mefE
and ermB genes, and co-resistance between erythromycin A
and amoxicillin in S. pneumoniae isolated in our hospital
during the period 1995–1998. In general, our results were
consistent with those of previous studies and demonstrated
a wide distribution of mefE or ermB-mediated erythromy-
cin A resistance in Japan.
Since the first description of S. pneumoniae with low
susceptibility to penicillin, several reports have indicated
the high prevalence of pneumococci resistant to penicillin as
well as to other antibiotics, such as cephalosporins, tetracy-
clines, and macrolides.2–4 In particular, epidemiological sur-
veys have demonstrated a trend of co-resistance between
penicillin and macrolides.5–7 Barry et al.5 reported that,
more than 50% of penicillin-resistant isolates were resistant
to erythromycin A. Our data were similar to the findings of
the above studies, as shown in Fig. 1. Although the reason is
not clear at present, we could not find isolates of S.
pneumoniae that were erythromycin A-susceptible and
amoxicillin-low susceptible in this study.
Until recently, the only mechanism that could explain
erythromycin A resistance in pneumococci was the methy-
lation in 23S rRNA of bacteria, which results in reduced
affinity between the antibiotic and the ribosome.10,17 This
methylase is encoded in the gene erm (first described in
Streptococcus sanguis), which confers resistance not only to
macrolides but also to lincosamides and streptogramin B
(MLS-type). In 1997, Tait-Kamradt et al.13 discovered
mefE, the gene responsible for the erythromycin A
-resistant M-phenotype (sensitive to clindamycin and
streptogramin B, but resistant to 14- and 15-membered
macrolides) in S. pneumoniae via a putative efflux pump
system. Subsequent studies, by Johnston et al.9 found that
31
57 (38.8%) of 147 erythromycin A-resistant S. pneumoniae
possessed the ermB gene, whereas 81 (55.1%) possessed the
mefE gene.9 Studies by Nishijima et al.11 have recently
shown that 37 (59.6%) of 62 erythromycin-resistant pneu-
mococci isolated in 1995 in their hospital in Japan possessed
the ermB resistant gene and 35 (56.4%) possessed the mefB
gene. The frequency of S. pneumoniae containing ermB and
mefE genes in the present study was similar to that in these
reports.9,11
In the present study, most mefE (⫹) S. pneumoniae iso-
lates expressed low-level resistance to erythromycin A,
whereas many ermB (⫹) isolates exhibited high-level resis-
tance, as shown in Fig. 2. Other investigators have also
reported a strong association between high-level macrolide
resistance and the presence of the ermB gene.9–11 Therefore,
these results confirmed that the mefE gene plays an impor-
tant role in low-level resistance, whereas the ermB gene is
associated with high-level resistance in pneumococci.
However, some ermB-positive S. pneumoniae isolates
with low-level resistance to erythromycin A were found in
our study. Therefore, we compared the frequency of mu-
tants with high-level resistance derived from isolates with
low-level resistance. As a result, we successfully identified
mutants that were highly resistant to erythromycin A, with
a frequency of 10⫺3–10⫺5, from mefE(⫺)/ermB(⫹) isolates,
whereas no such isolates were identified in mefE(⫹)/
ermB(⫺) isolates. According to the result of the population
analysis, these mutants may occur owing to heterogeneity.
These results suggested that the presence of the ermB gene
in pneumococci is a critical risk factor for therapeutic
failure with erythromycin A; thus, a genetic survey is impor-
tant. In the in-vitro selection of macrolide resistance,
Pankuch et al.18 and Davies et al.19 reported the isolation of
highly macrolide-resistant mutants from mefE (⫹) pneumo-
cocci after sequential passages on azithromycin-containing
agar. Interestingly, they also demonstrated the develop-
ment of high-level resistance to azithromycin in mefE (⫺)/
ermB (⫺) isolates. The clinical significance of these
macrolide-resistant mutants, such as their role in the acqui-
sition of resistance during therapy, remains to be deter-
mined in future studies.
Acknowledgments We thank Professor Peter C. Appelbaum for the
critical reading of the manuscript and helpful suggestions. We also
thank Professor Shogo Kuwahara for the valuable advice.
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