称号及び氏名

博士（獣医学）

学位授与の日付

平成２３年３月３１日

論Comparison of molecular methods for the species identificationof clinical Campylobacter strains and their antimicrobial
resistance（カンピロバクター属菌の遺伝学的菌種同定法の比較と臨床分離菌の薬剤耐性）

文

名

論文審査委員

主査
副査
副査

S. M. Lutful Kabir

山崎 伸二
小崎 俊司
笹井 和美

論文要旨

Introduction
Campylobacters are microaerophilic spiral Gram-negative bacteria. They are motile andpossessing a single flagellum at one or both poles and they are non-sporeformer. Among more than17 species currently recognized in the genus Campylobacter, C. jejuni and C. coli can infect poultry,
livestock and pets and these animals are source of human infection. Most infections are believed toresult from the ingestion of contaminated food such as undercooked meats, contaminated water, or
raw milk. Furthermore, C. jejuni and C. coli infections are considered to be the leading causes ofhuman gastroenteritis in the developed countries particularly in Japan.
The accurate identification of C. jejuni and C. coli is needed for the identification of theirdiseases spectrum, tracing their source of infection as well as their route of transmission. However,conventional identification methods for Campylobacter species are tedious and time-consuming,
because these organisms are slow growing, biochemically less reactive and require microaerobic
conditions. In particular, it is often difficult to differentiate C. jejuni from C. coli by biochemical
methods and 16S rRNA gene sequence because of the extensive similarity between these twospecies. Hippuricase activity is the only key marker for the differentiation of C. jejuni; however,this activity is sometimes very weak in C. jejuni and hippuricase-negative strains of this species
are also well recognized. Therefore, detection of hippuricase (hipO) gene by PCR is more reliable
than biochemical test for the differentiation of C. jejuni from C. coli. However, deletion andmutation of the primer binding sites in hipO gene resulting in no amplification by PCR has been
also noticed. In addition, several PCR-based assays targeting 16S or 23S rRNA gene or
species-specific genes have been developed to facilitate the differentiation of C. jejuni from C. coli,
however, the ability of these assays for exact identification of Campylobacter species has not yet
been established.
Cytolethal distending toxin (CDT), which is one of the well-characterized virulence factors
in Campylobacter spp., is encoded by three linked genes termed cdtA, cdtB and cdtC. The cdt geneclusters in C. jejuni, C. coli and C. fetus were demonstrated to be ubiquitously distributed in eachspecies in a species-specific manner. Recently, a cytolethal distending toxin (cdt) gene-based
species specific multiplex PCR assay has been developed for the detection and identification of C.

jejuni, C. coli and C. fetus. However, among the cdtA/B/C gene-based multiplex PCR, which one isthe best for the monitoring of clinical samples is also a matter of great concern.

Campylobacter infections pose a serious public health problem for which many countries
are monitoring their infection and antimicrobial resistance patterns. There are some reports about
increasing trend in antimicrobials resistance particularly to quinolones in Campylobacter in many
parts of the world. The emergence of quinolone resistance is mediated by several mechanisms.
Resistance to fluoroquinolones typically arises as a result of alterations in the target enzymes(DNA gyrase and topoisomerase IV) and its decreased accumulation inside the bacteria due to an
over expression of efflux pump systems. Both of these mechanisms are chromosomally mediated.
Considering the potential threat of Campylobacter infections associated with their risks of
antimicrobial resistance in Japan, this study was designed for the Campylobacter strains isolated
from diarrheal patients in Japan to achieve the following objectives: (1) evaluation of a cdt
gene-based species-specific multiplex PCR assay, (2) comparison of other molecular methods and(3) genotypic characterization of C. jejuni and C. coli strains resistant to fluoroquinolone.

Chapter 1: Evaluation of a cdt gene-based species-specific multiplex PCR assay for theidentification of Campylobacter strains isolated from diarrheal patients in Japan
A total of 325 Campylobacter strains isolated from stool specimens of patients withdiarrhea who visited the Tokyo Women’s Medical University Hospital, Japan during the period
between 1997 and 2005, were used in this study. These strains were identified as Campylobacter
spp., C. jejuni or C. coli by culture methods, microscopy and biochemical tests (catalase, oxidaseand hippuricase activity) at the Tokyo Women’s Medical University Hospital. In this study,
bacteria were grown on blood base agar at 37oC for 48 h under microaerobic conditions and DNA
templates were prepared by boiling method. DNA templates were used for hipO gene PCR, 16SrRNA gene sequence and cdtA/B/C gene-based multiplex PCR. A total of 314 strains wereidentified as C. jejuni by hipO gene PCR and 11 strains were negative by the same PCR. These 11

strains were subjected to 16S rRNA gene sequencing (1,335 bp) followed by homology search using
BLAST program and all were identified as C. jejuni/C. coli. Later, all the (n=325) strains were
subjected to cdtA/B/C gene- based multiplex PCR for further identification and all the strains wereidentified as it were by combined methods of hipO and 16S rRNA gene-based identification except
5, 4, and 1 strains that were negative by cdtA, cdtB and cdtC gene-based multiplex PCR,
respectively. In order to elucidate the reasons for getting these negative results, amplification
using common primers, sequencing and sequence data analyses of the cdt gene clusters of thesestrains were carried out. Clustal V alignment of the sequences from test strains with the published

cdt genes sequence of C. jejuni 81-176 (Acc. No. U51121) revealed 667-bp deletion between cdtAand cdtB plus additional 51-bp deletion within the cdtB genes in 4 C. jejuni strains, consistent
with the shorter PCR amplicon size than expected size. Furthermore, two strains comprising C.

jejuni and C. coli that did not yield specific PCR products contained mutations in the primer
binding sites of cdtA and cdtC genes, respectively. These data indicate that cdtC gene-based
multiplex PCR is more accurate and reliable than cdtA or cdtB gene-based multiplex PCR to
identify the species of Campylobacter strains such as C. jejuni and C. coli.
In order to investigate the genetic diversity of randomly selected 50 C. jejuni and 11 C. coli
strains, pulsed-field gel electrophoresis (PFGE) was employed. C. jejuni (n=50) and C. coli (n=11)
strains were classified into 42 pulsotypes and 4 subtypes, and 8 pulsotypes, respectively. These
data indicate that the C. jejuni and C. coli strains used in this study were genetically diverse.
Interestingly, the 4 C. jejuni strains that carried deletion between cdtA and cdtB as well as in cdtB
gene, 2 of them were clonal even isolated at different time point. These data demonstrate that such
type of C. jejuni strains having deletions in cdt gene clusters might be spread from the common ordifferent sources.

Chapter 2: Comparison of other molecular methods for the species identification ofCampylobacter strains
To compare the sensitivity and specificity of the cdt gene-based multiplex PCR with thoseof other PCR-based species identification methods, the applicability of different published PCR
assays targeting different genes such as 23S rRNA, ceuE, hipO, ask, lpxA and mapA were further
tested with 58 C. jejuni of clinical origin, 31 C. coli of different origins (clinical and environment)
and 14 reference strains (3 C. jejuni, 2 C. coli, 2 C. fetus and 5 other Campylobacters and 2

Arcobacter spp.). When C. jejuni specific 23S rRNA gene-based PCR was used out of 58 C. jejuni,
31 C. coli and 14 reference strains, 56 C. jejuni, 1 C. coli and 3 C. jejuni reference strains gave
specific amplification, respectively, although size of the amplified fragments was variable (590 bp,
640 bp and 740 bp) and different from the previously described. On the other hand, when C. coli
specific 23S rRNA gene-based PCR was used out of 58 C. jejuni, 31 C. coli and 14 reference strains,
2 C. jejuni, 31 C. coli and 3 reference strains (2 C. coli and 1 C. lari) gave specific amplification,
respectively. When C. jejuni specific ceuE gene-based PCR was applied to the same set of thestrains, 51 C. jejuni and 1 C. jejuni reference strains gave specific amplification, respectively. On
the other hand, when C. coli specific ceuE gene-based PCR was applied to the same set of thestrains, 30 C. coli and 2 C. coli reference strains gave specific amplification, respectively. When C.

jejuni specific lpxA gene-based PCR was applied, 58 C. jejuni, 3 C. coli and 3 C. jejuni reference
strains gave specific amplification, respectively. On the other hand, when C. coli specific lpxA
gene-based PCR was applied, 4 C. jejuni, 31 C. coli and 2 C. coli reference strains gave specificamplification, respectively. When mapA gene-based PCR was used, 55 C. jejuni, 3 C. coli and 4
reference strains (3 C. jejuni and 1 C. coli) gave specific amplification, respectively. However, all C.

jejuni and C. coli strains used in this study were accurately identified by combination of hipO andask gene-based PCR.
In order to verify the failure of the species identification by 23S rRNA, ceuE, lpxA andmapA gene-based PCR, new primers covering the flanking regions of these genes were designed forchecking the nucleotide sequences of primer binding sites of PCR negative strains.

A few C.

jejuni strains negative by C. jejuni specific ceuE gene-based PCR and 1 C. coli strains negative by
C. coli specific ceuE gene-based PCR were amplified and sequenced by using newly designed
primers. These strains were found to have mutations in both primers binding sites. On the other
hand, 3 C. coli strains positive (faint band) by C. jejuni specific lpxA gene-based PCR and 4 C.

jejuni strains positive (faint band) by C. coli specific lpxA gene-based PCR were amplified andsequenced by using newly designed primers. These strains were found to have a few mutations in
the forward primer-binding site but there were no mutations in the reverse primer-binding site.
However, a few C. jejuni strains negative by 23S rRNA gene-based PCR didn’t give any specificamplification by newly designed primers as well. In addition, a few C. jejuni strains negative bymapA gene-based PCR, 3 C. coli and 1 C. coli reference strains positive by mapA gene-based PCR
also didn’t give any specific amplification by newly designed primers. As several investigators
already reported the less sensitivity of hipO gene-based PCR and there is no evidence yet for thesimultaneous occurrence of deletions or mutations in the cdtA, cdtB and cdtC genes of the same

Campylobacter strain, combination of cdtC and cdtB or cdtA gene-based multiplex PCR might be
one of the best option for the accurate identification of C. jejuni, C. coli and C. fetus.

Chapter 3: Antimicrobial resistance profiles and genotypic characterization offluoroquinolone resistance in C. jejuni and C. coli strains isolated from diarrheal
patients in Japan
Since the antimicrobial resistant Campylobacter strains have been increasing worldwide,
analysis of antimicrobial resistance profile was carried out with the 325 Campylobacter strains
including 314 C. jejuni and 11 C. coli by disk diffusion method. Although fluoroquinolones andmacrolides are the antimicrobial agents of choice when therapeutic intervention is warranted andtetracyclines have been suggested as an alternative choice in the treatment of clinicalcampylobacteriosis, a series of antimicrobial agents such as ampicillin, tetracycline, azithromycin,
erythromycin, chloramphenicol, gentamicin, nalidixic acid, ciprofloxacin, levofloxacin, ofloxacin,
fosfomycin and sulphamethoxazole-trimethoprim were used in this study. Tetracycline andfluoroquinolone resistances were observed in both C. jejuni (29 and 24%, respectively) and C. coli
(36 and 36%, respectively) strains analyzed in this study. On the other hand, most of the C. jejuni

and C. coli strains analyzed in this study were susceptible to macrolides, gentamicin andchloramphenicol. These data suggest that macrolides could be used as first choice of the drug forthe treatment of campylobacteriosis in Japan. However, multidrug resistance (when considered
resistance to 3 or more agents) was found in 28 and 46% of C. jejuni and C. coli strains,respectively. These data suggest that the prevalence of multidrug resistance may be higher in C.

coli in comparison to C. jejuni strains in Japan.
In order to analyze genetic background of fluoroquinolone resistant C. jejuni and C. coli
strains, PCR amplifications of gyrA and parC genes were performed. However, there was no strain
positive for parC gene in this study. On the other hand, the gyrA genes in 75 C. jejuni and 4 C. coli
strains were sequenced to investigate whether there was any mutation. A total of 73 out of 75 C.

jejuni strains which were resistant to CIP (MIC, 8-64 µg/ml) had the T to I substitution at codon 86
in GyrA that is considered to be the main contributing factor for fluoroquinolone resistance inCampylobacter spp. The additional mutations were observed at codons 203 (N203S), 203 (N203S)
and 206 (A206V), 149 (V149I) and 203 (N203S), and 90 (D90N) and 203 (N203S) in 23, 11, 5, and 1
strains, respectively. On the other hand, no point and silent mutations were found in 1 strain(K278) although this strain was phenotypically resistant and only silent mutations were noticed in
1 strain (K170). Furthermore, in case of C. coli, a total of 4 strains, which were resistant to CIP(MIC, 16-64 µg/ml), had the T to I substitution at codon 86 in GyrA. These data suggest that theamino acid substitution at codon 86 (T86I) in the quinolone resistance determining region (QRDR)
of GyrA may play a major role in the acquisition of resistance to fluoroquinolone in the C. jejuni
and C. coli strains tested.
To elucidate the role of efflux pump in the acquisition of resistance to fluoroquinolone in
the C. jejuni and C. coli strains, PCR amplifications of cmeR genes in 75 C. jejuni and 4 C. coli
strains were performed. One strain (K170; MIC:16 µg/ml) having silent mutations in the gyrA
sequence displayed mutations in the HTH DNA binding motif of CmeR as well as in the promoter
region of CmeABC efflux pump. Another strain (K278; MIC: 8 µg/ml) having no mutations in the

gyrA sequence displayed mutations in the CmeR as well as in the inverted repeat of the promoter
region of CmeABC efflux pump. Overall, there were no mutations in the CmeR and promoter
region of CmeABC efflux pump in the 17 fluoroquinolone resistant C. jejuni strains and in theremaining 60 fluoroquinolone resistant Campylobacter strains, there were mutations either in
CmeR or in promoter region of CmeABC efflux pump or in both regions. These observations
suggest that generally low level of fluoroquinolone resistance occurred due to point mutations of
the gyrA gene but high level of fluoroquinolone resistance might be occurred due to point
mutations in the gyrA gene as well as mutations in the CmeR and promoter region of CmeABC
efflux pump. Furthermore, PFGE analysis of 45 fluoroquinolone resistant C. jejuni strains
revealed 32 pulsotypes and 3 subtypes, indicating that these fluoroquinolone resistant C. jejuni
strains were genetically diverse. However, a few fluoroquinolone resistant C. jejuni strains wereclonal and this was the indication of spreading of a few fluoroquinolone resistant C. jejuni strainsfrom the common sources.

Conclusions
1. cdtC gene-based multiplex PCR appears to be the most useful for the simple and rapid
identification of C. jejuni and C. coli.
2. Among several PCR-based species identification methods of C. jejuni and C. coli, hipO and ask
gene-based method appeared to be the best.
3. But combination of cdtC and cdtB or cdtA gene-based multiplex PCR may be more accurate
and might be best for the accurate identification of C. jejuni and C. coli.
4. Emergence of resistance of C. jejuni and C. coli to a variety of antimicrobials especiallytetracycline and fluoroquinolones among the diarrheal patients in Japan.
5. Generally low level of fluoroquinolone resistance occurs due to point and silent mutations of
the gyrA gene only but high level of fluoroquinolone resistance may occur due to point
mutations in the gyrA gene as well as mutations in the CmeR and promoter region of
CmeABC.

審査結果の要旨
Campylobacter 属細菌は、我が国においても最も問題となっている食中毒原因菌の一つである。現在Campylobacter 属細菌は、少なくとも 17 菌種知られているが、食中毒原因菌に指定され、特に重要なのが C. jejuni と C. coli の２菌種である。特に家禽の腸管内に常在する C. jejuni や C. coli で汚染した鶏肉を加熱不十分な状態で食することにより、食中毒を引き起こすと考えられている。C. jejuni は末梢神経障害であるギランバレー症候群の原因となることから C. jejuni と C. coli を鑑別して検査することは重要である。C. jejuni や C. coli は（1）微好気性細菌であり、培養には特殊な装置が必要で、（2）増殖が遅く、（3）
生化学的性状が酷似しており菌種同定が容易でないなどの理由から、本菌の分離・同定には時間を要するだけでなく、誤同定や同定できないなどの重要な問題がある。これらを克服するため、C. jejuni や C.

coli の特異的な遺伝子を PCR で検出することにより、この２菌種を鑑別する方法が多数開発されている。
しかしながら、どの方法が最適かについては充分検討されていない。一方、家畜や医療現場における抗菌薬の不適切な使用の結果、ニューキノロンやマクロライドを含む多剤耐性菌の出現が医療現場で大きな問題となっている。それゆえ、患者から分離される C. jejuni や C. coli の薬剤感受性を把握することは重要である。
本研究では、我々の研究グループが既に開発し報告した細胞膨化致死毒素（ cdt）遺伝子を検出する
PCR 法について 325 株の臨床分離株を用いて、その有用性の評価を行い、さらにその他の菌種特異的遺伝子を検出する PCR 法と比較した。さらに、わが国の下痢症患者から分離された C. jejuni や C. coli
について薬剤感受性について解析した。以下はそれらの結果の概要である。
第１章では、我が国の小児下痢症患者から分離され、Campylobacter 属様細菌として同定された株について、16S rRNA 遺伝子の解析と馬尿酸水解酵素（hipO）遺伝子の有無を調べ、得られた結果を総合的に判定し 314 株を C. jejuni、11 株を C. coli と同定した。また得られた結果は、cdtA、cdtB 及び cdtC

gene-based Multiplex PCR 法で得られた結果と一致した。しなしながら、cdt gene-based Multiplex PCR 法
で PCR 産物を得られなかった株が少数認められ、その株の cdt 遺伝子の解析から、プライマー結合部位の変異と cdt 遺伝子の一部が欠失していることが原因であることがわかった。以上の結果より、cdtC 遺伝
子と cdtA 或いは cdtB 遺伝子を標的とした PCR 法を組み合わせることが最良であることを示した。
第２章では、既に報告されている C. jejuni と C. coli の菌種を鑑別できる PCR 法即ち、C. jejuni を特異的に検出できる 23S rRNA、ceuE、lpxA、hipO、mapA PCR あるいは、C. coli を特異的に検出できる 23S
rRNA、ceuE、lpxA、ask PCR と cdt gene-based multiplex PCR 法について 61 株の C. jejuni、33 株の C.

coli、それ以外の Campylobacter 属菌 7 株と Arocbacter 属菌 2 株を含む 103 株を用いて評価した。その結果、hipO と ask は、C. jejuni と C. coli を 100%の特異性及び感度で検出できたが、その他の PCR 法は特異性と感度に問題があった。hipO と ask を標的とした PCR 法は個別に行う必要があり、cdt 遺伝子を標的とした PCR 法は一度の PCR で C. jejuni と C. coli 鑑別することができ、cdt 遺伝子を標的とした PCR
法の方がより実用的であることを示した。
第３章では、わが国の下痢症患者から分離された 314 株の C. jejuni と 14 株の C. coli の合計 325 株について薬剤感受性を調べた。その結果、C. jejuni 及び C. coli はニューキノロン及びテトラサイクリンに対して約 30%前後の耐性率と一部の菌で高い MIC 値を示した。一方、マクロライドに対しては C. jejuni、C. coli
共にほとんどが感受性であり、マクロライドが治療に用いる第一選択薬となることを明らかにした。また、ニューキノロンに対する耐性について遺伝子レベルで解析した結果、gyrA 遺伝子の変異或いは薬剤排出ポンプをコードした遺伝子のプロモーター領域或いはその転写を調節している遺伝子の変異が関与している可能性を示した。
以上の結果は、cdt gene-based Multiplex PCR 法、特に cdtC 遺伝子を標的とした PCR 法が他の菌種特異的な PCR 法と比べ、C. jejuni や C. coli の菌種を簡便、迅速、正確に同定・鑑別できること、及びわが国で分離された C. jejuni や C. coli がニューキノロンとテトラサイクリンにある程度の割合で耐性化しているが、マクロライドに関してはほとんどの株が感受性であることを示した。これらの成果は、C. jejuni や C.

coli による食中毒の制御に寄与するとともに、細菌学および感染制御学に多大な貢献をすると考えられる。
従って、最終試験の結果と併せて、博士（獣医学）の学位を授与することを適当と認める。