Nityananda Chowdhury

称号及び氏名博士(獣医学)

学位授与の日付

平成２２年３月３１日

論Genetic diversity of superintegron and its implication in the文名development of molecular typing tools for epidemiologicalstudies of Vibrio cholerae（スーパーインテグロンの多様性とそれを利用したコレラ菌の分子疫学的解析法の開発）
論文審査委員

主査
副査
副査

山崎
笹井
三宅伸二
和美
眞実

論文要旨

Introduction
Vibrio cholerae is a Gram-negative bacterium. It is an autochthonous
inhabitant of aquatic environments and can cause cholera in human. Cholera is adreadful diarrheal disease killing millions of people since ancient time and ischaracterized by rice-water stool, vomiting etc. Even having more than 200
serogroups of V. cholerae, only two of them O1 and O139 are so far known to be
responsible for epidemics of cholera. The O1 serogroup has two biotypes, El Tor andclassical. Previous six pandemics (1817-1923) were presumably caused by classicalbiotype which is now extinct, whereas El Tor biotype is responsible for the ongoing
7th pandemic starting from 1961. V. cholerae O139 synonym Bengal emerged in
1992 and was thought as an initiator of the 8th pandemic. The non-O1/non-O139
strains are noticed to cause sporadic cases of gastroenteritis.
To trace the epidemic strains rapidly and appropriately, suitable moleculartyping tools for V. cholerae are essential. There are several molecular typing
methods like ribotyping, pulsed-field gel electrophoresis (PFGE), multilocus
sequence typing (MLST) etc. These methods have good discriminative capacity but

are time consuming, complicated and expensive. On the contrary, PCR-based typing
methods like amplified fragment length polymorphism (AFLP), restriction fragment
length polymorphism (RFLP), etc. are rapid, simple and also cost-effective.
Several genetic islands which are present in the genome of V. cholerae playvital role in its evolutionary fitness. One of them is a distinctive class of integron
which is also known as superintegron (SI). The integron is a genetic system which
can capture exogenous DNA and aid in their expression in bacteria. It possesses
three key elements such as (i) a gene encoding for an integrase (intI), (ii) a primary
recombination site (attI) and (iii) a strong promoter. In a V. cholerae O1 El Torstrain (N16961) whose whole genome sequence is now available; the SI is ca 126 kbin size. It is composed of a class 4 integrase gene (intI4) and 179 gene cassettes
which constitutes 3% of the whole genome. Each of the gene cassettes is followed byshort (123 to126 bp) repeat sequence, called V. cholerae repeat (VCR). SI isconsidered as a flexible platform where new gene cassettes can be inserted ordeleted. This flexible nature in gene cassette capturing may result in SI diversity.
However, little is known about the genetic diversity of SI and its implication in thepathophysiology of V. cholerae.
In this study, SI in various strains of V. cholerae from both clinical andenvironmental origins, having differences in serogroup, biotype, geographic
background and isolation time, were analyzed to understand the genetic diversity ofSI, and to develop simple and rapid PCR-fingerprinting methods for V. cholerae on
the basis of SI diversity. The PCR-based methods were also evaluated by comparingwith PFGE. Moreover, the effect of repeated in vitro subculturing of V. cholerae
strains on the stability of SI and PFGE profiles was monitored to evaluate thereliability or usefulness of the PCR-fingerprinting methods in comparison to PFGE.

Chapter 1. Genetic diversity of superintegron among various V. cholerae
strains
To check the genetic diversity of SI, a total of 16 V. cholerae strains including
El Tor (n=7), classical (n=5), O139 (n=2) and non-O1/non-O139 (n=2) isolated fromdifferent geographic origin, source and time were selected. Before proceeding fordiversity analysis, Southern hybridization of BglI-digested genomic DNA of theseselected V. cholerae strains was carried out using 32P-labelled intI4 specific geneprobe. Hybridization results confirmed the existence of intI4 gene among all thestrains tested. This indicates that the test strains may possess SI island in theirgenome. From the genome sequence of strain N16961 (O1, El Tor biotype), total SIisland (ca 126 kb) was extracted, divided into 12 segments and overlapping LA (longand accurate)-PCR primers were designed to scan the whole SI island. All the El Torand O139 strains produced amplicons in respective to the primers of most of thesegments but amplicons were either identical or different in size in comparison to
the control strain N16961. However, primers of most of the SI segments did notwork for V. cholerae classical and non-O1/non-O139 strains. This might be due tothe presence of extensive diverse SI island in these strains. PCR amplicons ofidentical sizes were checked for RFLP using suitable restriction enzymes (BglI,HindIII, MluI, or Sau3AI). PCR amplicons which were different in size weresequenced and analyzed. After careful analysis, it was revealed that the possible
diversity of SI segments were due to the deletion, insertion or rearrangement ofgene cassettes. Possible mechanisms behind gene cassette deletion werehomologous recombination between (i) attI & VCR, (ii) VCR & VCR or (iii)homologous ORF & ORF. Genomic diversity of these strains revealed by PFGE,
were well correlated with SI diversity among El Tor and O139 strains. It might bealso true for classical and non-O1/non-O139 strains, but amplification of more SI
segments are required for better comparison.
PCR scanning, RFLP or sequencing of SI segments revealed that strains
having differences in serogroup, biotype, geographic origin and isolation time had
positive correlation with the diversity of their SI. These findings raised thepossibility to implement the diversity of SI in the development of molecular typing
methods for V. cholerae. On the other hand, the gene rearrangement and theabsence or presence of same gene cassettes in SI island among different V. cholerae
strains raised the question about the stability of SI island as well.

Chapter 2. Development of simple and rapid PCR-fingerprinting methods(PCR-RFLP & VCR-AFLP) for Vibrio cholerae
A total of 177 V. cholerae including El Tor (n=94), O139 (n=54) and classical(n=29) strains isolated between 1948 and 2006 were used in this study. For
PCR-RFLP, comparative ORFs from SI sequences of N16961 (El Tor) and O395
(classical strain whose genome sequence is also available) were analyzed and basedon this analysis, forward and reverse primers were designed from intI4 gene and acommon ORF, respectively. Then PCR products were digested with BglI and RFLP
patterns were analyzed. Out of 94 El Tor strains, 86 strains could generate expectedamplicons. In case of O139 and classical strains, 39 out of 54 and 25 out of 29,
respectively, produced PCR amplicons. PCR-RFLP with BglI was able to generate 8
different fingerprints for El Tor strains, designated arbitrarily as ER1 (El Tor RFLP
type 1) to ER8. For O139 strains, total 5 different fingerprints BR1 (Bengal RFLP
type 1) to BR5, were observed and 2 different RFLP types CR1 (Classical RFLP type1) and CR2 were produced by classical strains. In all cases, the strains that did not
produce any amplicon in LA-PCR were considered to produce at least a single but
different RFLP pattern. As a result, a total of 9 different RFLP patterns for El Tor, 6for O139 and 3 for classical strains were obtained.
For the development of VCR-AFLP, a total of 309 VCR sequences including
178 from the SI sequence of N16961 (O1, El Tor) and 131 from that of O395 (O1,classical) were aligned and two outward oriented primers were designed. All thestrains used for PCR-RFLP analysis, were also tested by VCR-AFLP assay. Fivedifferent fingerprints were obtained from 94 El Tor strains. These patterns weredesignated arbitrarily as EA1 (El Tor AFLP type 1) to EA5. Fifty four O139 strains
produced 2 different patterns, BA1 (Bengal AFLP type 1) and BA2, whereas 29
classical strains were differentiated into 9 patterns, CA1 (Classical AFLP type 1) toCA9.
PCR-RFLP and VCR-AFLP data obtained from each strain were overlappedand merged into a single fingerprint identity to gain better resolution of molecularepidemiological analysis for V. cholerae strains. For example, an El Tor strain that
produced RFLP pattern ER1 and AFLP pattern EA1 were considered as a single
fingerprint and it was denoted as e-I. In this system, all the El Tor strains (n=94)
were found to generate a total of 11 fingerprints which were denoted as e-I to e-XI.
Similarly, the O139 strains (n=54) produced 7 fingerprints, b-I to b-VII, whereas theclassical strains (n=29) produced 10 different fingerprints, c-I to c-X.
To check the feasibility of PCR-RFLP and VCR-AFLP as molecular typing
tools, the strains (n=177) analyzed by PCR-based methods were also analyzed by
PFGE and comparative studies were done. Out of 94 El Tor strains, PFGE could
generate 11 different types, EP1 (El Tor PFGE type 1) to EP11 and 14 subtypes. In
case of the O139 strains (n=54), only 2 different PFGE types, BP1 (Bengal PFGE
type 1) and BP2 could be differentiated, and BP1 type had 19 subtypes. PFGE could
distinguish 29 classical strains into 5 different fingerprints, CP1 (Classical PFGE
type 1) to CP5 and 13 subtypes. Considering Tenover’s criteria, the PFGE subtypes
in El Tor, O139 and classical strains might be merged with their respective parental
type. Thus, only 11, 2 and 5 PFGE types could be considered for El Tor, O139 andclassical strains, respectively.
In a comparative analysis between PCR-based methods and PFGE, thecombined PCR-based assays produced 11 types which were equal to the number ofPFGE types (11 types) for El Tor strains. In case of O139 and classical strains, thePCR-based methods produced comparatively more fingerprints (7 and 10 types,respectively) than those of PFGE (2 and 5 types, respectively). Moreover, PCR-based
methods were effective to distinguish some PFGE types or subtypes into 4 to 6
groups. Conversely, some of the PCR-fingerprints in El Tor, O139 and classicalstrains were discriminated more by PFGE. Comparative analysis indicated thatPCR-based fingerprints data were well correlated with those of PFGE.

Chapter 3. Effect of repeated subculturing of V. cholerae strains on thestability of superintegron (SI) island: evaluation of the SI-based
PCR-fingerprinting methods
The SI island is sedentary and not associated with mobile genetic elements.
However, rearrangement, absence or presence of same gene cassettes in SI island
among different V. cholerae strains have raised the possibility of possessing mobile

gene cassettes that may influence the fingerprinting profiles. In case of Shiga
toxin-producing Escherichia coli (STEC) the variations in PFGE patterns of thesame clone have been reported due to in vivo or in vitro passage. There was no suchreport about V. cholerae previously. Therefore, the effect of repeated in vitro
subculturing of V. cholerae strains on the stability of the SI and change in PFGE
profile (if any) was examined to evaluate the usefulness or reliability of the newly
developed PCR-fingerprinting methods.
Every 3- to 4-day interval repeated subculturing of 3 V. cholerae strains
N16961 (representative of O1 El Tor), O395 (representative of O1 classical), andAP32549 (representative of O139) up to 25-week (total 50 times subculturing) wascarried out. Every 5th week interval, 14 colonies from a single strain were randomly
analyzed by PFGE as well as by newly developed PCR-RFLP and VCR-AFLP assays.
In El Tor strain N16961, in addition to the original ep-Ia (El Tor PFGE type-Ia)
profile, another 3 different profiles (ep-Ib to ep-Id) were observed. The PFGE
profiles differed from each other by 1 to 3 DNA fragments. The original PFGE
profile in classical strain O395 was denoted as cp-Ia (Classical PFGE type-Ia). Like
El Tor strain, the classical strain also produced 3 more PFGE profiles (cp-Ib tocp-Id). The PFGE profiles of classical strain (cp-Ia to cp-Id) differed from each otherby 1 to 6 DNA fragments. In case of O139 strain AP32549, the change in PFGE
profile was also observed but it produced 2 more PFGE profiles (bp-Ib and bp-Ic) inaddition to the original bp-Ia (Bengal PFGE type-Ia). The PFGE profiles in O139
strain were shown to differ from each other by 1 or 2 DNA fragments.
In contrast to PFGE analysis, there was no change in PCR-RFLP
fingerprints and thus the clones of V. cholerae always produced the original
fingerprints er-I (El Tor RFLP type-I), cr-I (Classical RFLP type-I), and br-I (BengalRFLP type-I) by El Tor, classical and O139 strains, respectively. Similarly,
VCR-AFLP assay always produced the original fingerprints ea-I (El Tor AFLPtype-I), ca-I (Classical AFLP type-I), and ba-I (Bengal AFLP type-I) by El Tor,
classical and O139 strains, respectively. There was no visible change in fingerprints
due to the repeated subculturing of V. cholerae strains regardless of theirserogroups or biotypes and PFGE profiles. The combination of both the PCR-assays
is considered to count the diversity of total SI island. Therefore, no change in
PCR-fingerprints by PCR-RFLP and VCR-AFLP indicates the stability of SI in asingle strain. It also indicates that the change in PFGE profiles may be due to theinstability of the part of V. cholerae genome other than SI island.

Conclusions
1. In Vibrio cholerae, there is significant genetic plasticity in the structure ofsuperintegron (SI) owing to the loss, acquisition and rearrangements of gene
cassettes. Therefore, SI island can be targeted for the development of moleculartyping tools for V. cholerae.

2. PCR-fingerprinting methods (PCR-RFLP & VCR-AFLP) developed on the basis of
SI diversity: (i) have good discriminating power and well correlations with PFGE,(ii) are simple, rapid, and cost-effective in comparison to other complicated typing
methods such as PFGE, MLST etc. Therefore, newly developed PCR-fingerprinting
methods may be applicable for molecular epidemiological studies of V. cholerae.
3. The change in PFGE profiles but not in PCR-RFLP or VCR-AFLP fingerprints
due to the repeated subculturing of V. cholerae strains indicates that the SI islandin a single strain is stable under laboratory conditions. Therefore, PCR-RFLP andVCR-AFLP assays developed on the basis of genetic diversity of the SI can be morepractical and reliable than PFGE, when analyzing the genetic lineage of V. cholerae
strains in an etiological manner.

審査結果の要旨

コレラ菌は、汽水域に生息するグラム陰性桿菌であり、ヒトにコレラを引き起こす。コレラは古代から多くの人々の命を奪ってきた非常に恐れられている病気である。現在までに 200 種類以上の O 群血清型が報告されているが、コレラの原因となるのはコレラ毒素を産生する O1 と O139 の O 群血清型のみであると考えられていた。O1 血清型にはさらにエルトール型と古典型の 2 つの生物型に分けられる。第１次から第６次の世界流行では古典型
O1 コレラ菌（古典型 O1）が、1961 年から始まり今日尚続いている第７次ではエルトール型
O1 コレラ菌（エルトール型 O1）が関わっている。1992 年に突如として新型コレラ菌 O139（O139）が出現し、コレラの大流行が引き起こされ、第 8 次のコレラの世界流行かと思われた。一方、O1 と O139 以外のコレラ菌は non-O1、non-O139 コレラ菌（non-O1/O19）と呼ばれ、散発性下痢症の原因菌として問題となっている。
流行型の菌を迅速に検出するためには分子型別が重要である。リボタイピング、PFGE やMLST 等の分子型別法が開発され、優れた解像度を有している。しかしながら、手間、時間やコストがかかること等の問題点がある。一方、PCR 法に基づく分子型別法は簡便、迅速で比較的低コスト等の利点がある。コレラ菌は、スーパインテグロンと呼ばれる全ゲノムの3%に相当する約 126 kb の遺伝子領域を普遍的に保持している。インテグロンは、インテグ
ラーゼ、attI サイトと呼ばれる特定の遺伝子カセットを取り込む領域と、遺伝子カセットの転写に関わるプロモータの３つからなっており、インテグラーゼ近傍の attI サイトに次々に遺伝子を取り込むことから、遺伝子カセットの年輪の様な役割を果たしている。エルトール型 O1 の代表菌株である N16961 株の染色体中 126 kb の領域に 179 個の遺伝子カセットと遺伝子カセットの間に Vibrio cholera repeat（VCR）と呼ばれる多様性のある繰り返し配列を有している。本研究では、スーパーインテグロンの多様性を解析し、コレラ菌スーパーインテグロンを標的とした PCR 法に基づく簡便で迅速な分子型別法を開発した。
さらに開発した PCR 法の解像度と再現性について PFGE と比較した。
第１章では、様々な年代と様々な国から分離されたエルトール型O1、古典型O1、non-O1/O139 を用いて、スーパーインテグロン領域を 12 等分したPCRスキャニング法でその多様性の解析を行った。その結果、増幅産物の分子量に差が認められ、また増幅産物が得られない株や領域があり、コレラ菌のスーパーインテグロンにはかなりな多様性があることが明らかになった。分子量の異なるPCR産物の塩基配列を解析した結果、VCRや同じORFの相同組み換えによる遺伝子の挿入や脱落がこの領域の多様性に関与していることを明らかとした。
第２章では、第 1 章で得られた塩基配列の解析結果やGenBankに登録されているコレラ菌
スーパーインテグロンの塩基配列情報を基に、コレラ菌スーパーインテグロンを標的としたPCR-RFLP法及びVCRを標的としたPCR-AFLP法を実施するために複数のPCRプライマーを設計し、エルトール型O1、94 株、O139、54 株、古典型O1、29 株、合計 177 株のコレラ菌を用いて評価した。最も解像度の高かったPCRプライマーを用いた結果と 177 株のPFGEの結果を比較したところ、PFGEではエルトール型O1 を 11 タイプに、O139 を 2 タイプに、古典型O1を 5 タイプにしか分類できなかった。一方、PCR-RFLP法とPCR-AFLP法の組み合わせでは、エルトール型O1 で 11 タイプ、O139 で 7 タイプ、古典型O1 で 10 タイプと、PFGEよりも高い解像度で分子型別可能となることを明らかとした。
第３章では、同一のクローンであっても実験室内で継代する際にPFGEのPFLPパターンが変化する現象がコレラ菌でも観察されるかどうか、また、本PCR法がその現象により影響を受けるかどうかについて解析した。エルトール型O1、O139 及び古典型O1 の代表株をそれぞれ用いて解析した結果、PFGEでは継代に伴うPFGEパターンの変化が見られたが、PCR-RFLP法やPCR-AFLP法では菌の継代によってパターンが変化することなく、本法はコレラ菌の分子疫学的解析法として有用であることを明らかとした。
以上の結果は、コレラ菌スーパーインテグロンに多様性のあること、またコレラ菌スーパインテグロンがPCR法による分子型別法の標的となりうることを示した。さらに、スーパーインテグロンを標的としたPCR-RFLP法及びPCR-AFLP法を開発し、PFGEに勝るとも劣らない解像度と再現性を有することを明らかとした。これらの成果は、コレラ菌のスーパーインテグロンに多様性があるという新たな知見のみでなく、コレラ菌の新たな分子疫学的解析法の開発としても期待できるものであり、獣医学の分野のみならず医学領域においても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。