Jayedul Hassan

称号及び氏名博士（獣医学）

学位授与の日付

平成３１年３月３１日

論Development of a multiplex PCR targeting eae, stx and cdt

文

名genes, and characterization of cdt gene-positive Providencia
rustigianii isolated from a diarrheic child in Japan（eae, stx および cdt 遺伝子を検出できるマルチプレックス
PCR の構築と日本の小児下痢症患者から分離された cdt 遺伝子陽性プロビデンシア・ラスティゲニーの性状解析）

論文審査委員

主査

山崎伸二

副査

向本雅郁

副査

三宅眞実

論文要旨

Introduction
Food borne zoonoses is a major concern worldwide. Zoonotic transmission is considered to
be a key driver in the current emergence and re-emergence of novel diseases. It has been estimatedthat 60% of all communicable and 75% of emerging infectious diseases of people were originatedfrom animals.

Recently, zoonoses due to Salmonella spp. Campylobacter spp. and pathogenic E.

coli seems to be increased significantly. E. coli in general are normal flora of human and animal
intestine, however, some of them are pathogenic to human and animals. Pathogenic E. coli aregrouped into two groups, diarrheagenic E. coli (DEC) and extra-intestinal pathogenic E. coli(ExPEC), which are associated with intestinal and extra-intestinal infections, respectively. Someof the DEC such as enterohemorrhagic E. coli (EHEC) or Shiga toxin (Stx)-producing E. coli(STEC) carrying eae (attaching and effacing) and/or stx genes and enteropathogenic E. coli(EPEC) carrying bfp (bundle forming pili) and/or eae genes are zoonotic pathogens. EPEC can befurther divided into two groups such as typical EPEC (tEPEC) having both bfp and eae genes andatypical EPEC (aEPEC) having eae gene only. aEPEC has been considered to be originated fromanimals while tEPEC is originated from human. Stx is divided into two types such as Stx1 andStx2. Stx2-producing E. coli are associated with more severe clinical conditions such as
hemolytic-uremic syndrome (HUS) and neurological disorder than Stx1-producing E. coli.
Furthermore, EHEC (eae gene-positive STEC) is generally more virulent than STEC (eaegene-negative). It should be noted that aEPEC would be automatically converted to EHEC if
Stx2-phage lysogenize aEPEC. Therefore, it is considered that aEPEC is more virulent than tEPEC.
Along with these E. coli pathotypes, cytolethal distending toxin (CDT)-producing E. coli (CTEC)
has been also isolated from children with diarrhea. CDT consists of 3 subunits such as CdtA,CDtB and CdtC. Among 5 types of CDT (I to V)-producing E. coli so far known,
CDT-II-producing E. coli has been demonstrated to be Escherichia albertii, which is also known
to be an emerging zoonotic pathogen. Because several E. albertii strains have been isolated fromwild finch, pigeons and raccoons, and have been associated with various outbreaks offood-poisoning.
Recently, aEPEC, CTEC, STEC and E. albertii are increasing reported from clinical cases
but the association of those bacterium with diarrhea is not epidemiologically established. To
understand the epidemiological significance of these pathogens in diarrheal diseases, detection
and isolation of causative bacteria from stool samples is necessary. Several simplex and multiplex
PCR have been described for the detection of those bacteria based on their important virulence
determinants (eae, stx and cdt). However, none of the existing PCR systems are suitable to detect
eae, stx, particularly stx2, and cdt genes in a single PCR. Thus, to facilitate rapid detection ofthese bacteria in diarrheic stool samples, this study was designed to develop a multiplex PCR(m-PCR) which can amplify eae, stx and cdt in a single reaction mixture. By using the m-PCR,
untypable cdtB gene was detected from stool of diarrheic child during routine surveillance, anduntypable cdtB gene-positive bacterium was isolated and identified as Providencia rustigianii. cdt
gene-positive P. rustigianii was further analyzed genetically and phenotypically.
Chapter 1. Development of a multiplex PCR for the detection of eae, stx and cdt genes, and its
application to clinical samples
There are different variants of eae, stx and cdt genes with distinct biological features.
Specific PCR primers for each gene were designed and a multiplex PCR (m-PCR) for the detectionof these 3 genes was developed. For this purpose, primers for eae and stx were designed aligningsequences of all the reported variants of the target genes. In case of cdt gene amplification,primers were taken from Hinenoya et al. (2009), which can detect all the 5 subtypes of cdtB genes
in E. coli. This m-PCR could amplify eae genes and all the reported subtypes of stx and cdtB
genes except stx2f with 100% sensitivity (58/ 58 isolates) and specificity (51/ 51 isolates).
Detection limit of the m-PCR for eae, stx and cdtB were 100, 10-100, and 1-10 CFU/assay,respectively. This m-PCR could also detect eae, stx and cdtB genes in spiked stool samples withconsiderable detection limits. Thus, to determine the prevalence of eae, stx and cdt genes indiarrheic children in Japan, this m-PCR was employed. A total of 555 rectal swab samples wereexamined upon enrichment in TSB where 4.9 and 5.8% of the samples were positive for eae andcdt genes, respectively. Further, restriction fragment length polymorphism (RFLP) assay(Hinenoya et al., 2009) was used to subtype the cdt genes in positive samples. From 3 samples
cdt-II positive bacteria which were also positive for eae genes were isolated and identified as E.
albertii. In addition, one untypable cdtB (UT-cdtB) gene having similar RFLP pattern to that of P.
alcalifaciens was detected. The results indicated that this m-PCR can be used for surveillance of
diarrheic stool samples not only for eae, stx and cdt gene-positive aEPEC, STEC, CTEC, E.
albertii but also Providencia spp. The UT-cdtB was detected in a stool

sample collected from a

15 months old infant where no other pathogenic virus (Adenovirus, Norovirus, Rotavirus) andbacterial pathogens (Aeromonas spp., Campylobacter spp. Klebsiella spp., Salmonella spp., andYersinia spp.) has been detected indicating that the UT-cdtB carrying bacteria might be associatedwith diarrhea in that infant. The UT-cdtB gene sequence had 95% homology with cdtB gene of P.
alcalifaciens. CDT production has been reported in very limited number of P. alcalifaciens strains
and the association of CDT producing P. alcalifaciens with diarrhea has not been elucidated. Thus,
this study was further extended to isolate and characterize the UT-cdtB bearing bacterium anddetermine its association with diarrhea by in-depth genotypic and phenotypic analysis.
Chapter 2. Identification the untypable cdtB positive bacterium as P. rustigianii and its
genotypic analysis
P. alcalifaciens like UT-cdtB positive bacteria was isolated using Providencia specific
PMXMP media. Interestingly, this isolate when further characterized by biochemical tests, 16SrRNA and rpoB gene sequencing was identified as P. rustigianii (strain JH-1) but not P.
alcalifaciens. This is the first report regarding the detection of cdtB gene in P. rustigianii. P.
rustigianii is rarely isolated from human diseases and strain JH-1 is the only known strain in P.
rustigianii having cdtB gene. Thus, the whole cdt gene cluster of strain JH-1 was sequenced byprimer walking which revealed a 2,223 bp sequences including 2,143 bp long cdtABC gene cluster.
The deduced amino acid sequences of CdtA, CdtB and CdtC were 94, 97 and 94% homologous toCDT of previously reported P. alcalifaciens respectively. As cdt is not frequently associated withProvidencia, we have speculated that Providencia spp. might have acquired cdt genes throughhorizontal transfer events. Interestingly, S1-PFGE and Southern hybridization revealed thepresence of cdtB gene on a large plasmids of P. rustigianii strain JH-1 and other 4 cdt
gene-positive P. alcalifaciens strains tested. Furthermore, cdt bearing plasmid in strain JH-1(pJH-1) could be transferred to other enterobacteria including cdt negative P. rustigianii strain
GTC1504. This in vitro transferability suggests that this plasmid is horizontally transferable. To
further characterize the plasmid, P. rustigianii strain JH-1 was sequenced by whole genome
sequencing (WGS) using MiSeq and PacBio RSII platform. WGS revealed a 3,992,833 bp
chromosome and 168,819 bp plasmid with 41.2 and 38.7% GC contents, respectively. Annotation

of the plasmid sequences confirmed the presence of cdt genes on the plasmid along with type threesecretion system (T3SS) and conjugation related genes. Further analysis revealed another T3SS
related genes on the chromosome. The T3SS on the chromosome (cT3SS) belonged to Esc family
whereas plasmid T3SS (pT3SS) belonged to the Inv/Mxi-Spa family. In-silico analysis revealed
that cT3SS of strain JH-1 was ≥64% homologous to the T3SS’s of other Providencia spp. butpT3SS was found to be unique. Furthermore, cT3SS-spaL (cspaL) like gene sequences could be
detected on other strains of P. rustigianii and P. alcalifaciens by S1-PFGE, but pT3SS was present
only on the plasmids of cdt positive Providencia tested. This result indicated that cT3SS might beconserved among Providencia strains but pT3SS might be associated with the cdt bearing plasmid.
Presence of cdt and pT3SS genes on the same plasmid indicated that the cdt genes positive
Providencia spp. might be sharing similar type of plasmids. From the findings of genetic analysis
it could be said that P. rustigianii strain JH-1 carried all the machineries to be a potential virulent
bacteria and its virulence might be regulated by the transferable cdt bearing plasmid (pJH-1).
However, in-vitro and in-vivo experiments are necessary to establish the virulence and role of cdt
and T3SS genes on the virulence of P. rustigianii strain JH-1.
Chapter 3. Phenotypic characterization of P. rustigianii strain JH-1
To understand the virulence of strain JH-1 biological activity of cdt and T3SS gene-products
were examined. Western blotting and cytotoxicity assay with CHO cells revealed production ofbiologically active CDT by strain JH-1. T3SS is reported to be associated with invasion inSalmonella Typhimurim. As strain JH-1 possesses two T3SSs similar to S. Typhimurium, we haveattempted to determine the association of the T3SSs with the invasiveness of this strain. HeLa cell
invasion assay under gentamicin protection revealed strain JH-1 as invasive. In addition to T3SS,CDT production is also associated with the invasion in Campylobacter spp. To determine theassociation of CDT and T3SS’s with the invasiveness, cdtB and spaL (ATPase) gene mutants ofcT3SS (cspaL) and pT3SS (pspaL) were used. Invasion assay with the mutants revealed that, cdtBand cspaL mutation did not but pspaL gene mutants (∆pspaL) lost their invasiveness. Interestingly,
invasiveness was increased in ∆cspaL. To understand the underlying mechanisms of invasion loss
or increase, relative expression of pT3SS genes were determined by qRT-PCR. Relative
expression of 10 tested pT3SS genes were significantly downregulated in ∆pspaL, whereas in∆cspaL relative expression of genes downstream of pspaL were significantly up-regulated,indicating that pspaL might be essential for the expression of pT3SS gene but cT3SS possibly
suppress pT3SS through interfering the regulation of pT3SS genes, however, the actual
mechanism of interaction between the T3SSs could not be uncovered. In P. alcalifaciens invasion
was considered as the major mechanism of diarrhea induction. As strain JH-1 could invade HeLacells, further rabbit ileal loop test was performed to determine its diarrheagenicity. Strain JH-1
could cause fluid accumulation in the ileal loop. In addition, ∆cspaL could but ∆pspaL could not
induce fluid accumulation, indicating that pT3SS was associated with diarrheagenicity of strain

JH-1. Furthermore, CDT and T3SS activity could be determined in transconjugants carrying
pJH-1.
Conclusions
Taken together, it can be concluded that, m-PCR developed in this study would be useful indetecting the target genes in diarrheic stool samples. P. rustigianii strain JH-1 could producefunctional CDT and diarrheagenic. The diarrheagenicity of strain JH-1 was regulated by a T3SS
located on a plasmid which was horizontally transferable to other enterobacteria with its
properties. As cdt producing P. alcalifaciens strains also carries similar plasmid and pT3SS likesequences we assume that P. alcalifaciens also use similar T3SS to invade and induce diarrhea,
however, further descriptive studies are necessary to establish this assumption.

審査結果の要旨

動物および動物性由来食品による食中毒が多発し、特に先進国において大きな問題となっている。腸管出血性大腸菌（EHEC）はその代表格であるが、近年、腸管病原性大腸菌（EPEC）や Escherichia albertii（ Ea）も問題となっている。腸管病原性大腸菌（EPEC）は、
eae と bfp の両病原遺伝子を有する定型腸管病原性大腸菌（tEPEC）と eae 遺伝子のみを保有し、動物が感染源となる非定型腸管病原性大腸菌（aEPEC）が存在する。また、斃死した野鳥や健康な野生動物から分離され，新興人獣共通感染症菌として注目を集めている Eaによる集団食中毒事例が過去 7 年間、我が国で多発している。しかしながら、Ea は細菌学的性状が EPEC や EHEC と酷似していることから、過去の事例において EPEC や EHEC と誤同定されていた。EHEC、EPEC 及び Ea の共通な病原因子として eae 遺伝子を有すること、また EHEC の主要な病原因子である stx 遺伝子を保有する Ea も一部存在することもその一因である。一方、ある種の大腸菌は細胞膨化致死毒素を産生し、病気の家畜、健康な家畜及び下痢症患者から分離されている。近年、II 型の細胞膨化致死毒素（CDT）を産生する大腸菌（CTEC）は、大腸菌でなく Ea であることが明らかとなった。Ea が共通に保有する病原因子として eae と cdt 遺伝子がある。このように、誤同定が起こる背景として Ea 、EHEC と EPEC を鑑別し特異的に検出できる系が無いことが原因である。
第一章では、EHEC、EPEC、Ea と CTEC の疫学的調査を目的にこれら 4 種類の細菌の病原因子である eae、stx 及び cdt 遺伝子を検出できるマルチプレックス PCR（M-PCR）の構築を試みた。構築した M-PCR の感度、特異性を当該遺伝子陽性及び陰性の 58 及び 55株を用いて調べた結果、それぞれ 100%であった。検出下限は、純培養菌では eae、stx 及び cdt 遺伝子、PCR チューブあたりそれぞれ 100、10-100、1-10 CFU であった。糞便を用いたスパイクサンプルでは、それぞれ 10 5 、10 5 と 10 4 CFU/mL、増菌培養後では、それぞれ 10 3 、10 2 と 10 2 CFU/mL であった。さらに、小児下痢症患者便 555 検体を用いて当該遺伝子を検出したところ、4.9%、5.5%の検体で eae と cdt 遺伝子が検出されたが、stx 遺伝子は全く検出されなかった。興味深いことに型別できない cdt (UT-cdt)遺伝子が検出され、その塩基配列は Providencia alcalifaciens（Pa）の cdt 遺伝子と高い相同性を有していた。
第 2 章では、UT-cdt 遺伝子が Pa の cdt 遺伝子と高い相同性を示したことから、UT-cdt遺伝子陽性菌を Providencia 属菌の選択培地を用いて分離し、菌種同定を試みた。その結果、
UT-cdt 遺伝子陽性菌は P. rustigianii（Prus）であることが明らかとなった。さらに、cdt 遺伝子の全塩基配列を明らかとし、cdt 遺伝子は染色体上でなく、接合伝達性プラスミド上に見つかった。Cdt 遺伝子以外の病原遺伝子が存在しているかどうかを調べるため、本菌の全ゲノム配列を解析した。その結果、染色体は 3,992,833 bp から、プラスミドは 168,819 bpからなり、プラスミド上には cdt 遺伝子に加え３型分泌装置（pT3SS）に関わる遺伝子と接合伝達に関わる遺伝子も存在することを明らかとした。さらに染色体上にもプラスミド性とは異なる T3SS（cT3SS）をコードした遺伝子を見出した。これらは、それぞれサルモネ
ラで見出された T3SS と類似したものであった。以上の結果は、Prus の病原因子として cdt遺伝子及び T3SS 関連遺伝子の存在を初めて示す結果であった。
第 3 章では、第 2 章で見出した Prus の病原遺伝子が実際に病原性の発現に関与しているかについて調べた。CDT 活性は CHO 細胞を用いて証明され、T3SS の細胞侵入性は HeLa細胞によって確認された。しかし、pT3SS の変異体は細胞侵入性を失ったが、cT3SS の変異体は細胞侵入性を失わせるどころかむしろ細胞侵入性を促進した。この変異体は、pT3SSの遺伝子発現を促進していた。さらに、Prus の下痢原性をウサギの腸管ループ試験で調べた結果、液体貯留活性が認められ、下痢原性が確認された。しかも、pT3SS の変異体はウサギの腸管ループ試験による液体貯留活性が消失したのに対し、cT3SS の変異体は野生型の株を投与した時と同様、液体貯留活性を示した。ウサギ腸管組織の病理組織学的解析を行なった結果、腸管組織の傷害が観察された。また、Prus の病原性プラスミドは、非病原性の P. ruttgeri（ Pret）にも接合伝達され CDT の産生及び細胞侵入性を示すことを明らかとした。以上の結果より、Prus の病原遺伝子は in vitro および in vivo において実際に Prus の病原性に関わっていることが示された。
以上の結果は、誤同定が問題となっていた EHEC、EPEC 及び Ea のみならず、人獣共通感染症菌の可能性のある病原性 Providencia 属菌の分離にも有用な検出系を構築したことに加え、Providencia 属菌の病原性に CDT と T3SS が関わっていることを示唆するものである。特に T3SS については Providencia 属菌に 2 コピー存在し、pT3SS が病原性発現に直接関与する一方、cT3SS は pT3SS の発現調節を介して病原性に関与していることを明らかとした。これらの研究成果は獣医学の分野のみならず医学の分野においても多大な貢献をすると考えられる。従って、本論文の審査ならびに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。