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Prevalence and molecular characterization of
CTX-M extended-spectrum β-lactamase-producing
Escherichia coli from 2000 to 2010 in Japan
TAKAFUMI HARA, TAKAFUMI SATO, TSUKASA HORIYAMA,SACHI KANAZAWA, TAKAHIRO YAMAGUCHI and HIDEKI MAKI
Infectious Diseases, Discovery Research Laboratory for Core Therapeutic Areas,
Shionogi & Co., Ltd., Toyonaka, Osaka 561–0825, Japan(Received for publication February 16, 2015)

The prevalence of extended-spectrum β-lactamase (ESBL) in Enterobacteriaceae
has been increasing worldwide. The aims of this study were to determine theprevalence of ESBLs among clinical isolates of Escherichia coli obtained from 2000
to 2010 in Japan, and to characterize the sequence type (ST) and antimicrobial
susceptibility of the blaCTX-M-carrying strains. The genes for β-lactamases were
determined by conventional PCR and sequencing, and the antimicrobial
susceptibility test was performed by the broth microdilution method. Among the 948
strains, 35 were judged as ESBL-positive strains. The positive rates ranged from
0.6% to 3.9% until 2008, but surged to 10.3% in 2010. Thirty-three of them carried
blaCTX-M, but all were negative for ESBL-type blaTEM and blaSHV. blaCTX-M-14 was themost prevalent (18/33) among blaCTX-M-carrying strains, followed by blaCTX-M-15(7/33) of which ﬁve were isolated in 2008 and 2010. Additionally, blaCTX-M-27
appeared in 2010 for the ﬁrst time in this study and accounted for more than a thirdof the blaCTX-M-carrying strains. From the MLST analysis, ST131 known as a world
pandemic clone, has been predominantly isolated since 2006. The major types of
ESBLs carried by ST131 strains clearly shifted from blaCTX-M-14 to blaCTX-M-15 and/or
blaCTX-M-27 between 2006 and 2010. Most of these isolates were still susceptible todoripenem, latamoxef (moxalactam), ﬂomoxef and cefmetazole. Our results suggestthat a change of the dominant type of ESBL among Enterobacteriaceae is currently
in progress in Japan, and therefore further periodic surveillance is needed.

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Introduction
Recently, there has been an explosive increase in the prevalence of Enterobacteriaceae withhigh-level resistance to various β-lactams due to the presence of extended-spectrum β-lactamase(ESBL) enzymes worldwide1). ESBLs can hydrolyze penicillins as well as oxyimino-cephalosporins, such as ceftazidime and cefotaxime, which have played a valuable role in the treatment ofGram-negative infections. Some researchers have reported that the ESBL-producing Enterobacteriaceae are associated with infections that result in poor clinical outcomes, delayed initiation of
appropriate antibacterial therapy, longer hospital stays and greater hospital expenses2, 3). Indeed,
the infections caused by these bacteria are an emerging public health concern worldwide.
Most ESBLs belonging to Ambler Class A can be classiﬁed into three types: TEM, SHV, andCTX-M4). During the 1990s, ESBLs corresponded approximately to the derivatives of TEM- andSHV-type β-lactamases in Escherichia coli worldwide5). However, since the late 1990s, CTX-Mtype ESBLs have dramatically increased and become predominant. These have been found in both
hospital-acquired and community-acquired infections. The CTX-M-type of ESBL can be further
classiﬁed into ﬁve groups according to their amino acid sequences: CTX-M-1, CTX-M-2, CTXM-8, CTX-M-9 and CTX-M-25 groups. Some of the CTX-M enzymes have speciﬁc geographicaldistributions: CTX-M-9 and CTX-M-14 are found in Spain, and CTX-M-14 in South East Asia
and North America. Both of these enzymes belong to the CTX-M-9 group. As for the CTX-M-1
group, CTX-M-1 is found in Italy and CTX-M-3 in Poland, while CTX-M-15 is currently themost common variant distributed worldwide6). Additionally, molecular epidemiology studies reveal that one speciﬁc E. coli pandemic clone, ST131, has been closely associated with the spread of
CTX-M enzymes including CTX-M-157). In Japan, the dominant CTX-M-group carried by E. coli
underwent a shift from CTX-M-2 to CTX-M-14 in the early 2000s8). Several studies have recently reported the isolation of global pandemic CTX-M-15-carrying strains in Japan, suggestingthat an epidemiological change might be starting9, 10). However, there have been few reports oflong-term nationwide surveillance in Japan about the changes in dominance of CTX-M enzymes.
In this study, we investigated the prevalence and molecular characteristics of ESBLs among
E. coli isolates obtained from 20 Japanese hospitals between 2000 and 2010, including their antimicrobial susceptibility to commonly used antimicrobials.

Materials and Methods
Bacterial strains
A total of 948 non-duplicate clinical isolates of E. coli collected from 20 hospitals in various
regions in Japan every two years between 2000 and 2010 were examined.

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Screening for ESBL
ESBL production was conﬁrmed by the broth microdilution method according to the Clinicaland Laboratory Standards Institute (CLSI) guidelines11). All isolates with MIC of either cefotaxime or ceftazidime of ≥2 μg/mL and with a ≥8-fold MIC reduction of either cefotaxime orceftazidime occurring in the presence of clavulanic acid (4 μg/mL) were identiﬁed as potential
ESBL producers. These isolates were subjected to polymerase chain reaction (PCR) analyses forthe detection of ESBL genes.
Detection of ESBL genes and DNA sequencing
PCR was performed using a single conventional PCR assay with GoTaq® Master Mix(Promega) to identify ESBL genes, including blaTEM, blaSHV, blaOXA-1 group, blaCTX-M-1 group,
blaCTX-M-2 group, blaCTX-M-9 group, blaCTX-M-8 group, blaCTX-M-25 group and blaCMY. Crude genomic DNA
was extracted from the isolates by heat lysis. Brieﬂy, colonies were suspended in 100 μL of distilled water, and the cells were lysed by heating at 95°C for 10 min. Cellular debris was removedby centrifugation, and then supernatant containing DNA was subjected to PCR. The PCR ampliﬁcation consisted of a pre-PCR stage at 94°C for 5 min, and then 25 cycles at 94°C for 30 s, at
55°C for 30 s and at 74°C for 1 min, and a ﬁnal extension stage at 74°C for 7 min using C1000TM
Thermal Cycler (Bio-Rad). The nine sets of primers used for ampliﬁcation and sequencing areshown in Table 1. Direct sequencing of PCR products was performed by Euroﬁns Genomics(Tokyo, Japan), and the obtained sequences that were truncated approximately 30 base of bothﬂanking region was used to classify consistent sub-types.
Antimicrobial susceptibility testing
The susceptibilities to 12 antimicrobials such as ampicillin-sulbactam, tazobactam-piperacillin, cefepime, cefmetazole, cefotaxime, ceftazidime, ceftriaxone, ﬂomoxef, latamoxef (moxalactam), doripenem, levoﬂoxacin, and sulfamethoxazole-trimethoprim were determined by the brothmicrodilution method with cation-adjusted Mueller-Hinton broth (Difco, USA), according to the
CLSI guidelines11). MIC interpretation of susceptibility was determined based on the CLSI breakpoint (as the MIC breakpoints of ﬂomoxef and cefozopran were unavailable, those of latamoxef
and cefepime, respectively, were applied instead)12).
Multilocus sequence typing (MLST)
MLST was carried out on all blaCTX-M-carrying strains according to the protocol and primer
sets speciﬁed on the E. coli MLST web site (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). After performing the gene ampliﬁcation and sequencing of 7 standard housekeeping genes (adk, fumC,
gyrB, icd, mdh, purA, and recA), the allelic proﬁle and ST determinations were identiﬁed.

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Table 1. Primers used in this study

Results
Detection rates of ESBL-positive strains and distribution of ESBL genes
Among the 948 E. coli strains isolated between 2000 and 2010, 35 were identiﬁed as ESBLpositive strains. The detection rates each year ranged from 0.6% (1/164) to 3.9% (6/155) until
2008, however, the rate in 2010 surged to 10.3% (16/156) (Fig. 1). Determining the ESBL genes
by PCR and sequencing showed that all the strains except two were positive for at least one
blaCTX-M gene. One of the blaCTX-M-negative strains carried blaTEM-1 (non-ESBL gene), while theother did not carry any of the β-lactamase genes tested in this study. Figure 1 also shows the distribution of blaCTX-M each year, indicating that the most frequently detected ESBL gene was
blaCTX-M-14, followed by blaCTX-M-15 and blaCTX-M-2 throughout the observation period, with the exception of blaCTX-M-27. In 2010, strains carrying blaCTX-M-27 were detected for the ﬁrst time in thisstudy and accounted for 43.8% (7/16), of which one strain concurrently carried blaCTX-M-15. The

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Fig. 1. Number of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli anddistribution of blaCTX-M genes among ESBL-positive isolates between 2000 and 2010
Numbers in parentheses represent the total number of isolates examined.

Table 2. Number of ST131 strains carrying blaCTX-M genes

other types of ESBL genes (blaTEM, blaSHV, blaCTX-M-8 group and blaCTX-M-25 group) were not detectedin any strains tested in this study.
MLST
Among the 33 blaCTX-M-carrying E. coli, 17 were identiﬁed as the global pandemic clone
ST131. The rest strains were belonged to several ST types including ST38, ST354 and ST648. In
this study, the ST131 strains were ﬁrst isolated in 2006, and became predominant (10/16) in 2010
among blaCTX-M-carrying strains (Table 2). All ﬁve ST131 types isolated in 2006 were equivalent
to all blaCTX-M-14-carrying strains isolated in 2006. However, the number of ST131 among
blaCTX-M-14-carrying strains decreased to one in three in 2008 and one in seven in 2010. In 2008

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and 2010, ST131 was dominated by blaCTX-M-15- and/or blaCTX-M-27-carrying strains; all the
blaCTX-M-15- and/or blaCTX-M-27-carrying strains isolated in 2010 belonged to ST131.
Susceptibility for antimicrobials
As for the injectable antimicrobials, almost all the blaCTX-M-carrying E. coli were susceptible
to doripenem (carbapenem), ﬂomoxef, latamoxef (oxacephem), and cefmetazole (cephamycin).
They were also comparatively susceptible to tazobactam-piperacillin, but not to some cephalosporins such as ceftriaxone, cefotaxime, ceftazidime and cefepime (Fig. 2). The numbers of susceptible strains were 8 out of 33 strains for levoﬂoxacin (Table 3). Focusing on each blaCTX-M
type, the numbers of strains carrying blaCTX-M-15 which were susceptible to cephalosporins as wellas β-lactam-β-lactamase inhibitor combinations were relatively lower than those of blaCTX-M-14and blaCTX-M-27-carrying strains. None of the blaCTX-M-27-carrying strains were susceptible toceftazidime, and the susceptibility rate was much lower than that of blaCTX-M-14-carrying strains(13 of the 18 strains). One blaCTX-M-14-carrying strain showing low susceptibility to some antimicrobials, including cefmetazole and ﬂomoxef, carried the blaCMY-2 Class C β-lactamase gene,
known to be responsible for resistance to them.

Fig. 2. Cumulative percentage of isolates inhibited against blaCTX-M-carrying Escherichia coli
(n=33) for 10 injectable antimicrobialsaﬁxed tazobactam concentration of 4 μg/mL; b ﬁxed ratio of 2 : 1.

Table 3. Antimicrobial susceptibility of blaCTX-M-carrying Escherichia coli

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Discussion
Since the late 1990s, CTX-M-type β-lactamase have become the predominant ESBL among
clinical isolates of E. coli worldwide, and infections caused by these pathogens have become an
emerging public health concern5). Many epidemiological studies of CTX-M have been performed
in different countries. In this study, we investigated the prevalence of Japanese clinical isolates
and compared the results with those of past reports.
Our date revealed the detection rates of ESBL-carrying E. coli gradually increased andsurged to 10.3% in 2010. A similar observations were reported by CHONG, et al. and NAKAMURA,
et al.13, 14). These results, together with our ﬁndings, indicate that there is no longer any doubt
about the increase of ESBL-carrying E. coli in Japan in recent years. Both previous reports also
showed that the most frequently identiﬁed ESBL genes were blaCTX-M-type, with the blaCTX-M-9
group predominating, followed by the blaCTX-M-1 group, thus showing the same tendency as our result.
Although these two studies did not determine the detailed sub-types of blaCTX-M in ESBL-carriers,
we classiﬁed them by sequence analysis.
We found that blaCTX-M-27 appeared in 2010 for the ﬁrst time in this study and the numbers of
isolation reached almost the same levels as blaCTX-M-14. Both blaCTX-M genes belong to the
blaCTX-M-9 group. On the other hand, as for the blaCTX-M-1 group, only blaCTX-M-15 was detected in 2008
and 2010. CHONG, et al. and NAKAMURA, et al. also deduced the existence of blaCTX-M-15 in E. coli
isolates carrying the blaCTX-M-1 group, but they did not refer to the emergence of blaCTX-M-27-carrying
strains among the identiﬁed blaCTX-M-9 group-carrying strains because it is well-known that
blaCTX-M-14 was dominant in Japan in 2000s13–15). It has been reported that the clonal outbreaks of
blaCTX-M-27-carrying E. coli ST131 isolates were occurred in Japan even though the area was limited9, 10). YANO, et al. reported that as many as 21 out of 71 E. coli isolates collected from tertiary
hospitals in the Tohoku district between 2008 and 2011 were positive for blaCTX-M-279). KURODA, etal. also identiﬁed 22 blaCTX-M-27-carrying strains collected between 2008 and 2009, though these
isolates were restricted to the Tohoku and Kanto districts in Japan10). Thus, clonal outbreaks of
blaCTX-M-27-carrying E. coli ST131 have deﬁnitely occurred centering around eastern Japan, andour ﬁndings demonstrate the emergence of blaCTX-M-27 found in various regions in Japan.
The emerging CTX-M-27-producers are less susceptible to ceftazidime compared to CTXM-14-producers according to the previous report16). Our result also demonstrated that 3rd generation cephalosporins including ceftazidime as well as levoﬂoxacin were completely inactive
against blaCTX-M-27-carrying strains. Also, the β-lactam-β-lactamase inhibitor combinations in addition to ampicillin-sulbactam was less active against blaCTX-M-15-carrying strains. As these antimicrobials are frequently used for treatment in many countries including Japan, there is a concernthat these drugs will apply selection pressure to these isolates. Under such circumstances, it isnoteworthy that most of these blaCTX-M-carrying strains were still susceptible to doripenem, oxa-

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cephems and cefmetazole.
In conclusion, our study reveals a sharp rise of blaCTX-M-carrying E. coli in recent years inJapan and the spread of not only blaCTX-M-15 (the same as in other countries) but also blaCTX-M-27(differing from other countries) among clinical isolates of E. coli in Japan. The spread of
blaCTX-M-27 is closely associated with ST131. In fact, a change of the dominant type of ESBL in
Japan may be currently in progress. Further periodic surveillance is needed.
Acknowledgments
The authors thank all medical facilities that submitted bacterial strains for the performanceof this study, and Y. JINUSHI, K. HIROOKA, K. UOTANI, I. HIGASHIYAMA, A. MAKINO, T. HORI, M.
TSUJI, and A. NAITO for experimental technical support and/or helpful discussion.
Conﬂict of interest
The authors declare no conﬂict of interest.

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