Original Studies

Community Outbreak of Macrolide-resistant Mycoplasma

pneumoniae in Yamagata, Japan in 2009
Yu Suzuki, MT,* Tsutomu Itagaki, MD,† Junji Seto, DVM, PhD,* Akiko Kaneko, MT,*‡
Chieko Abiko, DVM,* Katsumi Mizuta, MD, PhD,* and Yoko Matsuzaki, MD, PhD§

Background: We detected a community outbreak of macrolide-resistant

worldwide,6–9 especially in China, where 90% of strains were resist-
Mycoplasma pneumoniae infection that occurred predominantly among
ant in 2008 to 2009.10 In Japan, macrolide-resistant M. pneumoniae
students at 2 schools in Yamagata, Japan.
isolates were first reported in 2001.11 Since then, the frequency
Methods: Throat swab specimens were collected from patients who were
of macrolide-resistant M. pneumoniae cases has increased annu-
clinically suspected to have M. pneumoniae infection after testing negative
ally: 5.0% in 2003, 30.6% in 2006, 59.1% in 2009 and 89.5% in
for influenza virus by a nasopharyngeal swab rapid antigen test. We per-
2011.12–14
formed cultures for M. pneumoniae, and all isolates were sequenced for the
M. pneumoniae resistance to macrolides is caused by muta-
presence of a mutation of the 23S rRNA gene.
tions in domain V of the 23S rRNA gene that interfere with the
Results: Of 96 specimens collected between July 2009 and January 2010,
binding of macrolides to rRNA.11,15 The A2063G transition in
83 were from students attending junior high school A and primary schools
domain V of the 23S rRNA is the most frequently detected muta-
B, C and D. A total of 47 M. pneumoniae isolates were obtained; among
tion among resistant strains.16 In some reports from Japan, the
them, 25, 15 and 4 were isolated from students attending schools A, B and
A2063G mutation accounts for 82%–91% of all of resistant strains;
D, respectively, and M. pneumoniae could not be isolated from students
it is closely followed by the A2064G mutation.12,16,17 Both mutations
who attended school C. An A2063T mutation in domain V of the 23S rRNA
induce high-level resistance to macrolide with minimum inhibitory
gene, which is associated with macrolide resistance, was identified in 39
concentration (MIC) values of erythromycin of >64 mg/L. Moreo-
(83.0%) isolates. The rates of macrolide resistance at schools A, B and D
ver, one study reported that clarithromycin and erythromycin were
were 96.0%, 86.7% and 0%, respectively. The minimum inhibitory concen-
not effective for children with macrolide-resistant M. pneumoniae
trations for isolates with an A2063T transversion showed high resistance
infections.18
to clarithromycin (minimum inhibitory concentration, 16–64 mg/L), and
Outbreaks of M. pneumoniae are more commonly reported
clarithromycin prescribed initially was clinically ineffective.
in closed or semi-closed settings, such as schools and hospitals,
Conclusions: This school-based cluster of macrolide-resistant M. pneu-
than in open community settings.19–22 Recently, a nursery school
moniae infections, which was identified in 2 geographically close schools,
outbreak of M. pneumoniae caused by macrolide-resistant strains
indicates that the transmission principally occurred by close contact
was reported in China.23 In 2009, under the high alert due to pan-
between students at school. Monitoring the spread of macrolide-resistant M.
demic influenza, we detected a school-based cluster of M. pneumo-
pneumoniae and clinical guidelines for the appropriate medication against
niae infections in a small town with a population of 15,000, which
such infections would be needed to control outbreaks of M. pneumoniae.
was caused by macrolide-resistant strains containing the A2063T
transversion. The A2063T mutation has rarely been found among
Key Words: Mycoplasma pneumoniae, macrolide resistance, school-based macrolide-resistant strains; it was reported in only 1 case among
outbreak, 23SrRNA macrolide-resistant strains in Shanghai (1/90) and Beijing (1/46) in
2008 to 200910,24 and in Beijing (1/64) in 2009.25 We describe here a
(Pediatr Infect Dis J 2013;32: 237–240)
community outbreak of macrolide-resistant M. pneumoniae caused
by strains that possessed this uncommon mutation.

M ycoplasma pneumoniae is one of the most common causes

of upper and lower respiratory tract infections, particularly
in children and young adults.1,2 Several reports have concluded that
M. pneumoniae is responsible for 15%–20% of all cases of commu-
nity-acquired pneumonia3 and 27%–30% of pediatric community-
acquired pneumonia.4,5
M. pneumoniae infections can be treated with macrolides,
which are generally considered to be the first-choice antibiot-
ics for children. The isolation of macrolide-resistant M. pneumo-
niae possessing a 23S rRNA gene mutation has become common
Accepted for publication October 23, 2012.
From the *Department of Microbiology, Yamagata Prefectural Institute of Public
Health; †Yamanobe Pediatric Clinic; ‡Department of Laboratory Medicine,
Yamagata Prefectural Central Hospital; and §Department of Infectious Dis-
eases, Yamagata University Faculty of Medicine, Yamagata, Japan.
The authors have no funding or conflicts of interest to disclose.
Address for correspondence: Yoko Matsuzaki, MD, PhD, Department of Infec-
tious Diseases, Yamagata University Faculty of Medicine, Iida-Nishi 2-2-2,
Yamagata 990–9585, Japan. E-mail: matuzaki@med.id.yamagata-u.ac.jp.
Copyright © 2013 by Lippincott Williams & Wilkins
ISSN: 0891-3668/13/323-0237
DOI: 10.1097/INF.0b013e31827aa7bd
MATERIALS AND METHODS
Clinical Specimens and M. pneumoniae Strains
In July 2009, an increased number of patients with fever
and cough was noted in Yamanobe Pediatric Clinic, located in
the town of Yamanobe, Yamagata Prefecture. The patients were
negative for pandemic influenza by rapid antigen test and were
clinically suspected of M. pneumoniae infection. Between July 13,
2009, and January 31, 2010, a total of 96 throat swab specimens
were collected from children (aged <16 years) who were clinically
suspected of M. pneumoniae infection. The sample collection for M.
pneumoniae analysis was performed after another nasopharyngeal
swab specimen was confirmed to be negative for influenza virus
by a commercial antigen test. Informed consent was obtained from
the participating patients or their guardians. The specimens were
transported to the Yamagata Prefectural Institute of Public Health
for isolation and analysis.
The cultivation of M. pneumoniae was carried out in modi-
fied Hayflick medium. Pleuropneumonia-like organism broth
(Difco, Detroit, MI) was supplemented with horse serum (20%),
yeast extract (15%), glucose (1%), thallium acetate (0.025%),

The Pediatric Infectious Disease Journal  •  Volume 32, Number 3, March 2013 www.pidj.com | 237
Suzuki et al The Pediatric Infectious Disease Journal  •  Volume 32, Number 3, March 2013

phenol red (0.002%) and potassium penicillin G (2000 units/mL). 6

The isolates of M. pneumoniae were identified by a change in the Junior high school A
broth color from red to yellow and by a polymerase chain reaction 5 Primary school B
(PCR) assay as previously described.26 Briefly, 2 sets of primers Primary school D
that were directed against the P1 adhesion gene and against the 16S 4

No. of cases
rRNA gene were used. PCR was carried out in a 15-μL reaction
mixture containing 0.3 U of Go Taq DNA polymerase (Promega,
3
Madison, WI), 160 μM of dNTP, 1.5 mM of MgCl2, 0.5 μM of each
primers, 2 μL of extracted DNA and 3 μL of Green Go Taq reaction
2
buffer (Promega). The primers were annealed to the target at 65°C
for the P1 gene and at 55°C for the 16S rRNA gene, and each gene
was amplified by PCR with 40 cycles. PCR products were analyzed 1
by agarose gel electrophoresis. The isolates were identified as M.
pneumoniae if PCR was positive for both P1 and the 16S rRNA 0
13 20 27 3 10 17 24 31 7 14 21 28 5 12 19 26 2 9 16 23 30 7 14 21 28 4 11 18 25
gene. Jul Aug Sep Oct Nov Dec Jan

Antibiotic Susceptibility Testing FIGURE 1.  The weekly distribution of M. pneumoniae posi-
The MICs of 6 agents for M. pneumoniae were determined tive cases among students from schools A, B and D in Yama-
by a broth microdilution method described previously.11 Erythro- gata from July 13, 2009, through January 31, 2010.
mycin, clarithromycin, azithromycin and minocycline were pur-
chased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. isolates contained the A2063T transversion. The M. pneumoniae
Tosufloxacin was provided by Toyama Chemical Co., Ltd. (Tokyo, isolates that were detected from students at school D did not con-
Japan), and rokitamycin was provided by Asahi Kasei Pharma Co. tain a macrolide-resistant mutation.
(Tokyo, Japan).
Outbreak Within 3 Schools
PCR Amplification and Sequencing The weekly distribution of M. pneumoniae positive cases at
The amplification and sequencing of domain V of the 23S schools A, B and D is shown in Figure 1. The M. pneumoniae infec-
rRNA gene were performed as described by Matsuoka et al.16 The tions among students at school B were concentrated during a 9-week
sequences of the PCR products were compared with the sequence period from July to September, and only 1 case was identified in
of M. pneumoniae M129 (accession no. X68422). both October and November. After the July and August confirma-
tion of M. pneumoniae in students from school A, patients from
RESULTS the school A visited the Clinic continually during a 10-week period
between September and November. The observation of patient clinic
Isolation of M. pneumoniae and Identification visits in an interval of approximately 4–6 weeks is consistent with
of Macrolide Resistance a previous report of a school-based outbreak of M. pneumoniae.20
From July 2009 to January 2010, a total of 47 M. pneumo-
niae isolates were obtained (Table 1). Of the 47 isolates, 25 (53.2%) Patient Diagnosis and Medication
were isolated from students who attended junior high school A, The patients’ age, clinical diagnosis and initial antibiotics
and 15 (31.9%) were isolated from students who attended pri- are summarized in Table 2. Of the students from school A with
mary school B. Schools A and B are located within 0.9 km of the confirmed cases of M. pneumoniae, 17 (68%) were 7th graders
Yamanobe town office. Primary school C is located 2.1 km south who were 12–13 years of age. Similarly, 11 (73%) of the 15 stu-
of school B; however, M. pneumoniae could not be isolated from dents at school B were 4th graders who were 9–10 years old. The
specimens obtained from students who attended school C. Four 5 students at school B who visited the clinic between July 16 and
students at primary school D tested positive for M. pneumoniae. August 6 were first prescribed clarithromycin (10 mg/kg/day).
School D is near the public office of a neighboring town and is 4.2 However, 4 students returned to the clinic because of a persistent
km north of school B in the town of Yamanobe. fever 48 hours after the initiation of clarithromycin, and 3 of the
A macrolide-resistance mutation in domain V of the 23S students were switched to minocycline administration (2.5–3.0 mg/
rRNA gene was detected in 83.0% (39/47) of the children who kg/day). Because the pediatrician had information about the drug
tested positive for M. pneumoniae. All of the macrolide-resistant susceptibility of the isolates, minocycline was initially adminis-
tered to children aged >9 years who were clinically suspected of
M. pneumoniae infection. As a result, 22 (88%) of the 25 students
TABLE 1.  Mycoplasma pneumoniae Isolated in from school A were initially treated with minocycline for 5–7
days, and their condition rapidly improved. Most patients, includ-
Yamagata, Japan
ing those suffering from pneumonia, recovered without hospital
No. of M. pneumoniae Positive treatment. Only 1 student at school D, a 9-year-old male, required
hospitalization due to pneumonia. He had macrolide-susceptible
No. Macrolide- Macrolide-resistant M. pneumoniae and developed atelectasis after receiving treatment
Tested susceptible (A2063T Mutation) with norfloxacin.
Junior high school A 38 1 24
Table 3 shows the MIC ranges of the 6 agents for the 11
Primary school B 31 2 13 isolates with or without the A2063T transversion. The isolates with
Primary school C 10 0 0 the A2063T mutation showed high resistance to erythromycin and
Primary school D 4 4 0 clarithromycin; only minocycline and tosufloxacin had similar
Others 13 1 2 MICs against isolates containing the A2063T mutation to those for
Total 96 8 39
isolates without mutation.

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The Pediatric Infectious Disease Journal  •  Volume 32, Number 3, March 2013 Macrolide-resistant Mycoplasma

TABLE 2.  The Demographics of Patients With M. pneumoniae Infection

Junior High School A Primary School B Primary School D Others

Characteristic
(n = 25) (n = 15) (n = 4) (n = 3)

Grade (age, yr); no. of patients 7th (12–13) 17 (1) 1st (6–7) 2 (1) 1st (6–7) 1 (1) 2-yr 1 (1)
8th (13–14) 5 4th (9–10) 11 3rd (8–9) 2 (2) 5-yr 2
9th (14–15) 3 5th (10–11) 2 (1) 6th (11–12) 1 (1)
Diagnosis; no. of patients
 URI 22 (1) 9 (1) 2 (2) 0
 Bronchitis 1 2 0 0
 Pneumonia 2 4 (1) 2 (2) 3 (1)
Initial antibiotics; no. of patients
 Clarithromycin 1 (1) 5 0 0
 Azithromycin 0 0 1 (1) 0
 Rokitamycin 1 1 0 3 (1)
 Minocycline 22 8 (1) 1 (1) 0
 Norfloxacin 0 1 (1) 2 (2) 0
  Not used 1 0 0 0
The number in parentheses indicates the number of patients with macrolide-susceptible M. pneumoniae infection.
URI indicates upper respiratory tract infection.

DISCUSSION Yamanobe is a small town with a population of 15,000; there are

A community outbreak of macrolide-resistant M. pneu-
moniae that contained an A2063T mutation was identified. The
A2063T transversion was first reported in China in 2008 to 2009. It
is an uncommon mutation and only a few strains have been detected
within China24,25 and Japan.14 Although we cannot determine when
this mutation was introduced into the community, M. pneumoniae
had not been isolated from children in the town of Yamanobe in
2009 until a macrolide-resistant isolate with the A2063T mutation
was detected on July 16, 2009, from a student at school B. The
school-based cluster of M. pneumoniae presented here suggests that
rare resistant strains are spread by close contact between students
at school. Because macrolide-susceptible strains were also isolated
from 3 children at schools A and B, it is apparent that macrolide-
resistant and macrolide-susceptible strains cocirculated within this
community. The dominant spread of the resistant strains with the
A2063T mutation leads us to speculate that this mutation has some
advantage to propagate compared with the macrolide-susceptible
strains, although further experimental analysis is necessary to con-
firm this.
It is known that the household is an important site of
M. pneumoniae transmission.21,27,28 Interestingly, the 2 children
aged <6 years who were infected with an A2063T transversion-con-
taining isolate of M. pneumoniae each had an older sibling in the
4th grade at school B. However, we could not determine whether
disease transmission occurred within households.
In 2009, the pandemic influenza A(H1N1) virus emerged
and rapidly spread throughout the world. The pandemic influenza
A(H1N1) outbreak occurred in Yamanobe in August 2009.
TABLE 3.  In Vitro Activity of the 6 Antibiotic Agents
Against M. pneumoniae Strains
MIC Range of Strains With Mutations in the
23S rRNA Gene (mg/L)
Antibiotic
None (n = 3) A2063T (n = 8)
Erythromycin 0.0039–0.00781 64–256
Clarithromycin 0.00098–0.0039 16–64
Azithromycin 0.00012–0.00049 1–2
Rokitamycin 0.00098 1–2
Minocycline 0.5 0.125–0.5
Tosufloxacin 0.25–0.5 0.125–1
no hospitals, and there is only 1 pediatric clinic. Among patients
who visited the Yamanobe Pediatric Clinic with a fever during the
period between July 2009 and January 2010, a total of 616 children
tested positive for influenza A virus by rapid antigen test. The
children with influenza A virus were widely distributed among all
grades of schools A, B, C and D, whereas the children infected with
macrolide-resistant M. pneumoniae were only identified in schools
A and B. This phenomenon indicates that closer interpersonal
contact is required for transmission of M. pneumoniae than for
influenza A virus. One limitation of this study is that coinfection
of M. pneumoniae with influenza virus could not be considered.
Because unexpected numbers of patients with influenza-like
illnesses visited this clinic in 2009, only the patients who tested
negative for influenza by rapid antigen test were suspected for
M. pneumoniae infection.
The MICs of macrolides against isolates with the A2063T
transversion showed high resistance to clarithromycin. They were
almost equal to the MICs for strains with the A2063G transi-
tion,17 which is the most commonly detected mutation in Japan.
Morozumi et al12 reported that rokitamycin was effective against
strains with the A2063G transition (MIC of 0.25 mg/L) but was
insufficient against strains with the A2064G mutation (MIC of
16 mg/L). The strains with the A2063T mutation presented here
exhibited a slightly higher MIC with rokitamycin (1–2 mg/L) than
the strains with the A2063G mutation. However, it is likely that
rokitamycin was clinically effective for the children infected with
A2063T mutation-containing isolates of M. pneumoniae. Of the 4
children who were infected with an A2063T transversion-contain-
ing isolate and were treated with rokitamycin, 1 child aged 5 years
showed resolution of the fever within 48 hours, and 3 children did
not revisit the clinic after the initiation of rokitamycin. The patients
who received azithromycin or norfloxacin as the initial antibiotic
were not infected with macrolide-resistant M. pneumoniae. Mino-
cycline and tosufloxacin had effective MICs against macrolide-
resistant M. pneumoniae, which were equivalent to the MICs for
susceptible isolates. Neither minocycline nor tosufloxacin are rec-
ommended for pediatric patients; however, pediatricians have little
choice but to prescribe them. It is likely that the administration
of minocycline reduced the patients’ symptoms. However, it is not
clear whether minocycline attenuated the school outbreaks because
the 9- or 10-week outbreak period at school A or B was similar
in length to previously reported outbreaks caused by macrolide-
susceptible M. pneumoniae.19,20

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Suzuki et al The Pediatric Infectious Disease Journal  •  Volume 32, Number 3, March 2013
To avoid treating disease with ineffective antimicrobial
agents, it is important to perform surveillance for macrolide-
resistant M. pneumoniae on a local level, as well as nationally.
For rapid diagnosis, real-time PCR methods for the detection of
the 23S rRNA mutations are available that can test directly for the
presence of macrolide-resistant M. pneumoniae in clinical sam-
ples.6,29,30 Early detection of M. pneumoniae in a clinical setting is
also crucial, but effective methods of detection are lacking. The
development of rapid antigen tests such as those used for influenza
virus, surveillance of the spread of macrolide resistance and the
establishment of clinical guidelines for the appropriate treatment
of macrolide-resistant strains will be needed to control future out-
breaks of M. pneumoniae infection.
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