称号及び氏名

博士(獣医学) Worada Samosornsuk

学位授与の日付

平成 1９年９月３０日

論「Phenotypic and genotypic characterization of Campylobacter strains

文

名isolated in Thailand（タイで分離されたキャンピロバクター属細菌の表現型および遺伝型による性状解析）」

論文審査委員

主査

山崎 伸二

副査

馬場栄一郎

副査

児玉 洋

論文要旨
Introduction:
Campylobacter is a leading cause of gastroenteritis worldwide. Among 17
species currently known in the genus Campylobacter, C. jejuni, C. coli, C. fetus, C. lari,
C. upsaliensis, and C. hyointestinalis are implicated in human diseases. Many animals,
in particular poultry, pigs and cattle, are reservoirs of these organisms which aretransmitted to human by direct contact or consumption of contaminated food or water.
Campylobacter infections are self-limiting and do not require antimicrobial treatment
except complicated infections which macrolides and fluoroquinolones are often drugs ofchoice. However, resistance of Campylobacter to antimicrobials has substantially
increased in many countries including Thailand and has become a matter of concern.
Campylobacters

are

gram

negative,highlymotile,spiral-shaped

microaerophilic bacteria. Conventional methods for the identification of Campylobacter
species are costly, laborious, time-consuming and considered biochemical inert which isresulted in misidentification or unidentification. A rapid, sensitive, and specific method
for identification of these organisms is required to provide important information for
tracing sources of transmission and selecting an appropriate treatment. Our group
recently developed a multiplex PCR assay for the species-specific detection of C. jejuni,
C. coli, and C. fetus based on cytolethal distending toxin (cdt) genes.
In the present study, firstly Campylobacter strains isolated from human patientsand healthy poultry in Thailand were identified by the phenotypic and genotypic
methods and the results were compared with those obtained by the cdt gene-based
species-specific multiplex PCR. Secondly the cdt genes detected in C. hyointestinalis
were analyzed and biological activity of their gene products were investigated. Finallyantimicrobial resistance profile and mutations in gyrase A (gyrA) gene of C. jejuni and
C. coli strains were analyzed.

Chapter I: Identification of Campylobacter strains isolated in Thailand
A total of 112 Campylobacter strains (78 from human patients and 34 frompoultry) obtained from Thailand between 2001 and 2002 were identified as species levelby API Campy, detection of hippuricase (hipO) gene, and 16S rRNA gene sequence.
The collective results of these three methods were compared with those identified by cdt
genes-based species-specific multiplex PCR.
Among 34 poultry strains, 20, 10, and 1 were identified as C. jejuni, C. coli,
and Arcobacter cryaerophilus, respectively, whereas 3 strains were unidentified by API
Campy. By 16S rRNA gene sequence, all 34 strains were identified as C. jejuni/C. coli.
Thus, the hipO gene, which is known to be specifically present in C. jejuni was
examined. The hipO gene was detected in only 20 strains, which were initially
identified as C. jejuni by API Campy. The collective results of these three methods
could conclude 20 strains as C. jejuni and 14 strains as C. coli, respectively. By contrast,

the cdtA, and cdtC gene-based multiplex PCR could identify as C. jejuni 19 strains, andC. coli 13 strains whereas cdtB gene-based multiplex PCR could identify 20 strains as C.
jejuni and 14 strains as C. coli which is well correlated with the result of the collective
results of the three methods.
On the other hand, among 78 human strains, the cdtA, cdtB, and cdtC
gene-based multiplex PCR could identify 40, 45, and 41 strains as C. jejuni, 29, 31, and30 strains as C. coli, and 0, 1, and 1 strain as C. fetus, respectively. One strain was
unidentified by cdt genes-based multiplex PCR though cdtB common primer yielded a
specific PCR product. In contrast, API Campy identified 45, 23, 2, and 1 strains as C.
jejuni, C. coli, C. fetus, and A. cryaerophilus, respectively and 7 strains were unable tobe identified. By 16S rRNA gene sequence, however, 77 and 1 were identified as C.
jejuni /C. coli, and C. fetus, respectively. One strain which was unable to be identified
by the cdt genes-based multiplex PCR was identified as C. hyointestinalis by 16S rRNA
gene analysis. This is the first report of isolation of C. hyointestinalis from a humanpatient in Thailand. The hipO gene was detected in all 45 strains identified as C. jejuni
by API Campy but not in any other strains. The collective results obtained by threedifferent methods are well correlated with those by the cdtB gene-based multiplex PCR.
Campylobacter strains were further characterized by Pulse-field gelelectrophoresis (PFGE). Although PFGE patterns revealed extensive genetic diversity
which is mostly specific to either human or animal strains, identical and similar PFGE
patterns were also identified in poultry and human strains, suggesting that thetransmission of Campylobacter from poultry to human might occur in Thailand.

Chapter II: Cytolethal distending toxin genes in C. hyointestinalis
There exists the evidence to support an important role for C. hyointestinalis in
human and animal disease. However, the mechanism of the disease has been yet unclear.

To examine the importance of CDT produced by C. hyointestinalis in its pathogenesis,
the cdt genes detected in C. hyointestinalis isolated from a diarrheal patient in Thailand
were sequenced by a genome walking method. The cdt genes of C. hyointestinalis
consist of three closely linked genes termed cdtA, cdtB, and cdtC whose sizes are 798
bp (266 aa), 804 bp (268 aa), and 537 bp (178 aa), respectively. Each Cdt subunit in C.
hyointestinalis has highest homology to those in C. coli (40.9% for CdtA, 61.6% for
CdtB, and 29.8% for CdtC). The cdtB gene of C. hyointestinalis was amplified by PCR
and cloned into an expression vector, pET-28a (+). The recombinant plasmid wastransformed into E. coli BL21 (DE3). The expression of recombinant CdtB protein with
His6-tagged (HisCdtB) was induced in the presence of IPTG. The HisCdtB protein was
purified by an affinity column chromatography followed by gel filtration of FPLC andimmunized against the rabbit for producing antiserum. Anti-HisCdtB serum was
specifically reacted to both purified HisCdtB and ca. 30 kDa protein corresponding to
CdtB in sonic lysate of C. hyointestinalis culture. Subsequently, CDT activities in soniclysate of C. hyointestinalis were investigated by HeLa cell assay and FACS with orwithout antiserum, respectively. Sonic lysate of C. hyointestinalis exhibited progressive
cell distension and death in HeLa cells resulted in G2/M arrest. Both CDT activities
were neutralized by anti-HisCdtB. This is the first report demonstrating the entire cdt
genes sequence and biological activity of cdt genes product in C. hyointestinalis.

Chapter III: Analysis of antimicrobial resistance patterns
Since reports of Campylobacter strains resistant to antimicrobial agents,
particularly macrolides and fluoroquinolones, have been appearing with increasing
frequency worldwide, analysis of antimicrobial resistance profile was conducted with
110 Campylobacter strains isolated from Thailand including 65 C. jejuni (45 humans
and 20 poultry) and 45 C. coli (31 humans and 14 poultry) by E-test. Eightantimicrobial agents such as erythromycin (ERY), azithromycin (AZM), clindamycin(CLI), nalidixic acid (NAL), ciprofloxacin (CIP), tetracycline (TET), chloramphenicol(CHL), and gentamicin (GEN) were included in this study.
High degree of resistances to NAL, CIP, and TET were observed in C. jejuni(93-95%, 89-93%, and 60-85%, respectively) and C. coli (90-93%, 84-93%, and68-86%, respectively) whereas the resistances to ERY, AZM, CLI were observed only in
C. coli strains which resistance rates of poultry strains (57%, 57%, and 57%,
respectively) were higher than those of human strains (13%, 16%, and 13%,respectively). There were no resistant strains of C. jejuni and C. coli to CHL and GEN.
In C. jejuni, most strains isolated from human and poultry showed resistances to NAL
and CIP in the high MIC values of >256 and >32 µg/ml, respectively whereas resistanceto TET in the high MIC values from 64 to >256µg/ml was observed in most strains
isolated from poultry. In C. coli, most strains isolated from human and poultry showed
resistances in the high MIC values of >256µg/ml to ERY, AZM, CLI, NAL, 64 >256µg/ml to TET and >32 µg/ml to CIP.
Furthermore, the gyrA gene, a target of quinolone antimicrobial agents, inquinolone resistant strains was sequenced to investigate its mutation. A Thr86Ile
mutation in the gyrA gene product was identified in all NAL-resistant isolates in
addition to other various different mutations in the gyrA gene in many C. jejuni strains,
indicating that A Thr86Ile mutation in GyrA could be involved in quinolone resistanceas reported previously.

Conclusion
1.

CdtB gene-based multiplex PCR is a simple, rapid and reliable method for
identification of C. jejuni, C. coli, and C. fetus isolated from both healthy animals
and human patients.

2.

PFGE analysis indicated that C. jejuni and C. coli analyzed in this study were
genetically diverse. Some PFGE patterns between humans and poultry strains
showed identical or similar, suggesting that transmission of Campylobacter mightoccur between poultry and human in Thailand.

3.

Analysis of entire cdt genes sequence of C. hyointestinalis revealed the presence of
cdtA, cdtB, and cdtC genes with 798 bp (266 aa), 804 bp (268 aa), and 537 bp (178aa), respectively. CDT was demonstrated to be a potential virulence factor in C.
hyointestinalis.

4.

Most C. jejuni and C. coli strains isolated in Thailand are highly resistant tofluoroquinolone and tetracycline. Some C. coli strains are highly resistant tomacrolides and clindamycin. The Thr86Ile mutation in GyrA could be involved inquinolone resistance in Campylobacter strains isolated in Thailand.

審査結果の要旨
Campylobacter 属細菌による食中毒は我が国の食中毒の発生件数で、近年、常にトップの座を占め、世界的にも増加傾向にある。Campylobacter 属細菌は、家畜、特に家禽の腸管内に常在し、Campylobacter 属細菌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。現在 Campylobacter 属細菌は、17 菌種が知られているがヒトに病気を引き起こすものとして、C. jejuni、C. coli、C. fetus、C. lari、C. upsaliensis と C. hyointestinalis の 6 菌種がある。Campylobacter 属細菌は 1）微好気性細菌であり、培養には特殊な装置が必要である、2）増殖が遅い、3）生化学的性状が菌種間で類似しており菌種同定が容易でない、4) 菌種によって温度感受性が異なるなどの理由から、本菌の分離・同定に時間を要するだけでなく、誤同定や同定できないなどの問題も生じている。一方、家畜や医療現場における抗菌薬の不適切な使用がキノロン薬やマクロライド薬を含め多剤耐性 Campylobacter 属細菌の出現に繋がり医療現場で大きな問題となっている。よって、家禽や患者から分離される Campylobacter 属細菌の薬剤感受性を知ることは重要である。
本研究では、タイの家禽および下痢症患者から分離されたCampylobacter属様細菌について菌
種同定、病原因子および薬剤感受性について解析した。以下はそれらの成績の概要である。

第１章では、タイにおいてCampylobacter属様細菌として分離・同定された 112 菌株（患者由来 78
株、家禽由来 34 株）を従来法、すなわち、生化学的性状試験、16S rRNA遺伝子の塩基配列の解析および馬尿酸水解酵素遺伝子の有無を総合的に判定し、65 株をC. jejuni、45 株をC. coli、そして 1 株をC. fetusと同定した。また得られた結果は、cytolethal distending toxin（cdt）遺伝子に基づく種特異的なMultiplex PCR（ cdtB gene-based Multiplex PCR）法の結果と完全に一致し、 cdtB
gene-based Multiplex PCR法が簡便、迅速でより正確な菌種同定法として有用であることを示した。
また、cdtB gene-based Multiplex PCRで増幅産物が得られず、複数菌種のcdtB遺伝子を増幅出
来 る 共 通 プ ラ イ マ ー で 増 幅 バ ン ド が 得 ら れ た 1 株 は 、 16S rRNA 遺 伝 子 の 解 析 か ら C.

hyointestinalisと同定した。
第２章では、タイの下痢症患者から分離されたC. hyointestinalisが保有するcdt遺伝子の全塩基配列とその遺伝子産物が細胞膨化致死活性を有するかどうかについて解析した。その結果、 C.

hyointestinalis の cdt 遺伝子は他の Campylobacter 属細菌の cdt 遺伝子と同様 cdtA, cdtB およびcdtCの３つの遺伝子から構成され、アミノ酸レベルではC. coliのCdtと最も相同性が高かった。また、組換えC. hyointestinalis CdtB（ChCdtB）を精製し、ChCdtBと特異的に反応する抗体を作成し、C.

hyointestinalisが示す細胞膨化致死活性および細胞周期のG2/M期阻害が抗体により中和されてことから、これらの活性はCdtに基づく特異的なものであることを明らかとした。
第３章では、タイで分離された 65 株のC. jejuni （患者由来 45 株、家禽由来 20 株）及び 45 株のC. coli （患者由来 31 株、家禽由来 14 株）、合計 110 株について薬剤感受性を調べた。その結果、
C. jejuni及びC. coliは家禽及び患者由来株でキノロン薬及びテトラサイクリンに対して高い耐性率と高いMIC値を示した。一方、マクロライド薬に対しては、C. jejuniは家禽及び患者由来株の両方に感受性であったが、C. coliでは患者由来株に比べて家禽由来株で高い耐性率を示した。
以上の結果は、cdtB gene-based Multiplex PCR法がCampylobacter属細菌の菌種を簡便、迅速、
正確に同定できること、C. hyointestinalisで見いだしたCdtが新たな病原因子となる可能性があること及びタイで分離されたC. jejuniやC. coliが高い割合でかつキノロン薬とテトラサイクリンに高度耐性化していることを示した。これらの成果は、Campylobacter属細菌による感染症の制御に寄与するとともに、感染症学および防疫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。