称号及び氏名

博士（獣医学）

学位授与の日付

平成３０年３月３１日

論Prevalence and characterization of extended-spectrum

文

名NAHARAZIMUNbeta-lactamase-producing Escherichia coli and mcr-1
gene-positive E. coli in domestic and imported chickens
in Japan（基質特異性拡張型βラクタマーゼ産生性大腸菌とコリスチン耐性遺伝子陽性大腸菌の国産と輸入鶏肉における汚染状況と分離株の性状解析）

論文審査委員

主査

山﨑伸二

副査

向本雅郁

副査

笹井和美

論文要旨

Introduction
Emergence of antimicrobial resistance (AMR) bacteria, in particular 3 rdgeneration cephalosporins (β-lactam antibiotic), fluoroquinolones, and polymixins(colistin), is a serious global problem. There are several mechanisms by which bacteriaacquire resistance against antimicrobials. Enzymatic cleavage using β-lactamase
enzyme is one of the major mechanisms to acquire resistance against β-lactam
antibiotics. Extended-spectrum β-lactamases (ESBLs) can hydrolyse clinicallyimportant 3rd and 4 th generation cephalosporin and thus, emergence and spread ofESBLs-producing bacteria are serious concern. ESBLs can be divided into three groups
such as TEM, SHV and CTX-M. TEM and SHV were prevalent in 1980s and 1990s andmostly involved in hospital infection caused by ESBL-producing Enterbacteriaceae.
After introduction of 3 rd generation of cephalosporins, CTX-M type has become
dominant and has substituted TEM and SHV. It is interesting to note that CTX-M type
ESBL-producing Enterobacteriaceae is mostly E. coli and associated with communityinfections. Many studies on ESBL-producing E. coli (ESBL-Ec) from both clinicaland food-producing animal have been published worldwide in recent years.
Animal-based foods have been demonstrated as a significant reservoir for ESBL-Ec
whereas meat is considered to be an important source of contamination with ESBL-Ec.
Besides 3 rd generation of cephalosporin, a polymyxin antibiotic (colistin: CST) is also
used widely in poultry production as growth promoter and for therapeutic purpose.
Although CST has serious side effects, it is considered a last-resort antimicrobial andcan be used to treat patients with serious infections caused by multidrug resistant(MDR)
bacteria
suchasNewDelhi
metallo-β-lactamase-1-producing
Enterobacteriaceae. CST resistance conferred by mcr-1 gene is recent emerging issuesince mcr-1 gene located in plasmid and can be transferred which is revealed by recent
study in China. Although E. coli is known to be a commensal in animal intestine
including human, some can cause intestinal and extra-intestinal infection such as
diarrhea, urinary tract infection (UTI), septicemia and neonatal meningitis. Emergenceof AMR in intestinal and extra-intestinal pathogenic E. coli (ExPEC) has become amajor public health concern. Among various meats, the chance of contamination ofchicken with fecal bacteria especially E. coli appears to be higher because of its
processing protocol. Therefore, retail chicken might have more chance to becontaminated with ESBL-Ec and mcr-1 gene-positive E. coli. There are few reports
regarding prevalence of ESBL-Ec and mcr-1 gene-positive E. coli in domestic andimported retail chicken in Japan. In addition, there is no comprehensive study on AMR
pattern and prevalence of virulence genes in ESBL-Ec and mcr-1 gene-positive E. coli
in retail chicken in Japan. Therefore, in this study, prevalence of ESBL-Ec and mcr-1
gene-positive E. coli in domestic and imported retail chicken was examined and theisolates were further characterized for antimicrobial resistant profile, virulence geneprofile and so on.
Chapter 1. Isolation and characterization of ESBL-producing E. coli in domestic
and imported retail chicken
A total of 106 chicken including 56 produced domestically and 50 imported
from abroad (Brazil [n=36], USA [n=8], Thailand [n=6]) were purchased from 9
supermarkets in Osaka prefecture, Japan between August and December 2015.
ESBL producers were identified by double disk diffusion method using cefotaxime(CTX), ceftazidime (CAZ), CTX/clavulanic acid and CAZ/clavulanic acid andconfirmed as E. coli by biochemical test. The result showed that prevalence ofESBL-Ec was 77% (43/56) in domestic chicken (DC) and 52% (26/50) in importedchicken (IC) [72% (26/36), 0% (0/6) and 0% (0/8) in samples from Brazil, Thailandand USA, respectively]. A total of 111 ESBL-Ec was isolated from 43 DC whereas 51
ESBL-Ec were isolated from 26 IC from Brazil. To see whether there was difference inESBL genotypes between isolates of DC and IC, ESBL genotyping (bla C TX-M 1 , 2 , 8/ 25, 9 ,bla TEM and bla S H V ) was done. If more than one isolate from the same chicken carriedsimilar ESBL genes, only representative isolate was selected for further analysis.
Subsequently, 53 out of 111 ESBL-Ec isolates from DC and 30 out of 51 from chickenfrom Brazil were selected. Analysis of ESBL genotype exhibited that ESBL genotypes
were mainly bla CTX-M (91%) including [bla CTX-M-2 (45%), bla CTX-M-1 (34%), blaCTX-M-9(9.5%), bla CTX-M-8 (1.9%)] followed by bla TE M (36%) and bla SHV (15%) in ESBL-Ec
from DC whereas bla CTX-M (100%) including [bla CTX-M-2 (53%), blaCTX-M-8 (43%),
blaCTX-M-1 (3.3%)] followed by bla TE M (20%) were detected in ESBL-Ec from IC.
Antimicrobial susceptibility testing analysis to other antimicrobials showed thatmost of the tested ESBL-Ec from DC were mostly resistant to tetracycline (83%)
followed by streptomycin (70%) and nalidixic acid (62%) whereas most of the tested
ESBL-Ec of IC were resistant to streptomycin (77%) followed by nalidixic acid(63%), tetracycline (57%). Notably, extensive MDR, which is defined to be resistantagainst at least five classes of antimicrobials, was detected in 60% and 70% in
ESBL-Ec isolated from DC and IC, respectively. To see the clonal relationshipamong these isolates, pulsed-field gel electrophoresis (PFGE) was carried out withXbaI digestion. PFGE analysis showed that 53 ESBL-Ec from DC and 30 from IC
generated 44 and 26 pulsotypes, respectively, suggesting high level of genetic
diversity. Taken together, it can be concluded that both DC and IC from Brazil arehighly contaminated with ESBL-Ec with high diversity at genetic level regarding
ESBL groups as well as clonality along with extensive multidrug resistance.
Chapter 2. Isolation and characterization of mcr-1 gene-positive E. coli indomestic and imported retail chicken
Plasmid mediated CST resistance is also recent emerging issue and thus
investigation for the prevalence of mcr-1 gene-positive E. coli in DC and IC is also ofparamount importance. Therefore, retail chickens were investigated for the prevalenceof mcr-1 gene. The results showed that prevalence of mcr-1 gene-positive samples was55% (31/56) in DC while that was 22% (11/50) including 22% (8/36), 33% (2/6) and
13% (1/8) from Brazil, Thailand and the USA, respectively. Subsequently, mcr-1
gene-positive E. coli was isolated from mcr-1 gene-positive retail chicken meats. A
total of 30 and 10 mcr-1 gene-positive E. coli was successfully isolated from DC andIC, respectively and subsequent analysis was done. Furthermore, minimum inhibitory
concentration for CST was determined to be 4-8 µg/ml for mcr-1 gene-positive isolates
from both DC and IC. Antimicrobial susceptibility testing analysis to other
antimicrobials showed that most of the isolates from DC were resistant to tetracycline(63%) followed by kanamycin (50%), sulfamethoxazole/trimethoprim (50%) whereas
most of the isolates from IC were resistant to ampicillin (80%) followed bystreptomycin (70%), kanamycin (70%), ciprofloxacin (70%). Notably, extensive MDR
was detected in 33% and 40% isolates from DC and IC, respectively. PFGE analysis
showed that 30 and 10 mcr-1 gene-positive E. coli isolates from DC and IC generated

25 and 9 pulsotypes, respectively suggesting that high level of genetic diversity.
Taken together, it can be concluded that both DC and IC showed higher prevalence ofmcr-1 gene-positive E. coli with genetic diversity regarding clonality along withextensively MDR.
Chapter 3. Virulence gene profile of ESBL-producing E. coli and mcr-1
gene-positive E. coli isolated from domestic and imported retail chicken
Phylogenetic analyses provide better understanding pathogenicity of the strains.
On the basis of phylogenetic analyses, E. coli strains are characterized into fourphylogenetic lineages (A, B1, B2, and D). Most commensal E. coli belong to lineage Aand B1 and virulent strains belong mainly to lineage B2 and D. So, first to see
phylogenetic groups of isolates of ESBL-Ec and mcr-1 gene-positive E. coli understudy, phylogenetic analysis was done using multiplex PCR (chuA, yjaA andTSPE4.C2). Results suggested that almost half of the ESBL-Ec from both DC and IC
belonged to B2 /D phylogenetic groups but mcr-1 gene-positive E. coli isolates mainly
belonged to A/B1. Next virulence gene profiles were investigated for ESBL-Ec of DC(n=53) and IC (n=30) and mcr-1 gene-positive E. coli of DC (n=30) and IC (n=10).
Virulence genes analyzed includes several genes associated with diarrheagenic E. coli(DEC) such as eaeA (E. coli-attaching and effacing), bfpA (bundle-forming pilus), elt(heat-labile enterotoxin), est (heat-stable enterotoxin), eagg (plasmid ofenteroaggregative E. coli), astA (enteroaggregative E. coli heat-stable enterotoxin 1),stx (Shiga toxin), invE (Invasin of EIEC), daaD (fimbriae adhesion) and cdt (cytolethaldistending toxin) and extra-intestinal pathogenic E. coli (ExPEC) such as genes related
to adhesion (papEF, papC, sfa/focDE and afaBC), toxins (hlyA and cnf), siderophores(fyuA and iroN), protections and invasions (traT and kpsMT) and miscellaneous genes(PAI, usp and ibeA). The results showed that among ESBL-Ec strains prevalence ofastA gene was higher in isolates of IC (47%) compared to that of DC (23%). Similar
trend was also observed in mcr-1 gene-positive E. coli that 60% isolates from IC
possessed astA gene in comparison to 6.7% isolates from DC. Furthermore, cdtB andeaeA genes were detected in one ESBL-Ec and 2 mcr-1 gene-positive E. coli from IC,
respectively. The cdt genes in ESBL-Ec were identified as cdt IV which wasbiologically active by cell culture assay. However, none of the ESBL-Ec or mcr-1
gene-positive E. coli from DC was positive for cdtB and eaeA genes. Thus, it might besaid that IC had higher prevalence of potentially pathogenic E. coli possessing
diarrheic genes. On the other hand, none of the ESBL-Ec or mcr-1 gene-positive E.
coli was positive for bfpA, elt, est, stx1, stx2, invE, eagg and daaD genes irrespectiveof origin of chicken. In prevalence study of virulence genes associated with ExPEC,
traT, which is associated with invasion and protection, showed highest prevalence inall the tested isolates irrespective of the origin. Among ESBL-Ec from DC and IC,
traT prevalence was 60% and 97%, respectively. On the other hand, in mcr-1

gene-positive E. coli from DC and IC, traT prevalence was 80% and 50%, respectively.
The ExPEC genes PapEF and papC are part of pap gene cluster, which is associatedwith adhesion, an important factor of pathogenesis of UTI. The prevalence of pap
genes was 19% and 13% in ESBL-Ec from DC and IC, respectively. Interestingly, noneof the mcr-1 gene-positive E. coli isolated from DC carried pap genes whereas 20%
isolates from IC were positive for pap genes. Prevalence of other ExPEC associatedgenes varied among E. coli isolates analyzed in this study. However, none of theESBL-Ec or mcr-1 gene-positive E. coli was positive for ExPEC associated genes suchas hlyA, cnf, sfa/focDE, afaBC, usp and ibeA examined. Overall, it might be said thatisolates from IC carried more virulence genes in comparison to that from DC.
ESBL-Ec and mcr-1 gene-positive E. coli isolates examined in this study wereextensively MDR at high level and also carried potential virulence genes. Thus, theisolates carrying both virulence and antimicrobial resistance genes might cause
human illness.
Conclusions
ESBL-Ec and mcr-1 gene-positive E. coli isolated from domestic and imported
retail chicken could be a potential reservoir for antimicrobial resistance determinants
and virulence genes. Thus, chicken contaminated with ESBL-Ec and mcr-1
gene-positive E. coli might have potential to cause human illness and requires acontinuous monitoring.

審査結果の要旨

基質特異性拡張型βラクタマーゼ（ESBL）は、様々な種類のβラクタム系薬を分解することから、ESBL 産生菌は医療現場において脅威となっている。ESBLは現在、TEM、SHV と CTX-M の 3 つに分類されている。1980 から 1990 年代は、TEM と SHV が院内でしかも腸内細菌科菌群が主に流行に関わっていた。しかし、2000 年代に入ると第 3 世代セフェムの特許が切れたため価格が低下したことで、家畜の生産現場でも多くのセフェム系抗菌薬が使用されるようになり、流行の中心は CTX-M 型へ、院内から市中へ、腸内細菌科菌群から大腸菌へと変化した。
畜産現場でセフェム系抗菌薬が多用された結果、食肉、特に鶏肉における ESBL産生大腸菌汚染，特に CTX-M 型の ESBL 産生大腸菌の流行が大きな問題となっている。開発途上国を中心に、CTX-M 型の ESBL 産生大腸菌の健康保菌者も多数報告されている。一方、2015 年にプラスミド媒介性のコリスチン耐性菌が中国で見つかり世界を震撼させた。コリスチンはニューデリーメタロプロテアーゼ産生菌など治療が困難な多剤耐性菌の最後の砦として臨床現場で使われている抗菌薬である。また、コリスチン耐性（mcr-I）遺伝子がプラスミド上に見つかったことは、コリスチン耐性が容易に菌株間のみならず菌種間を超えて拡散する可能性がある。以上より、我が国の市販鶏肉における ESBL 産生大腸菌やコリスチン耐性（mcr-I 遺伝子陽性）大腸菌の汚染率や保有する病原遺伝子を調べることは極めて重要である。
第１章では、市販の国産鶏肉 56 検体及び輸入鶏肉 50 検体の合計 106 検体を対象に、ESBL 産生大腸菌の汚染率について調べた。その結果、国産鶏肉の 77%から、輸入鶏肉の 52%から ESBL 産生大腸菌を検出された。輸入鶏肉は、ブラジル産が 36 検体、米国産が 8 検体、タイ産が 6 検体で、検出された全てがブラジル産であり、陽性率は 72%と国産鶏肉に匹敵する割合であった。国産鶏肉 43検体及びブラジル産鶏肉 26 検体から、それぞれ 111 株と 51 株の ESBL 産生大腸菌を分離した。国産鶏肉由来の ESBL 産生大腸菌は 91%が CTX-M 型陽性、36%が TEM 型陽性、15%が SHV 陽性であったのに対し、輸入鶏肉由来の ESBL産生大腸菌は CTX-M 型は 100%、TEM 型が 20%で陽性であった。CTX-M の中でも、CTX-M-2 は国産、輸入に共通して多数検出されたが、国産鶏肉由来株では、CTX-M-１型が、輸入鶏肉では CTX-M-8 型の検出率が高かった。国産鶏肉由来株では、テトラサイクリン耐性が 83%、ストレプトマイシン耐性が 70%、ナリジクス酸耐性が 62%であったのに対し、輸入鶏肉由来株ではストレプトマイシン耐性が 77%、ナリジクス酸耐性が 63%、テトラサイクリン耐性が 57%であった。一方、多剤耐性菌の割合は国産及び輸入鶏肉由来株でそれぞれ 60%と70%であった。以上の結果より、国産、輸入鶏肉ともかなりの割合で多剤耐性の ESBL 産生大腸菌で汚染されていることが明らかとなった。
第 2 章では、コリスチン耐性大腸菌の国産及び輸入鶏肉の汚染状況について解析した。国産鶏肉の 53％から、輸入鶏肉では 22%からコリスチン耐性遺伝子が検出され、それぞれから 30 株と 11 株を分離した。輸入肉の内訳は、ブラジル産 36 検体中 8 検体、タイ産 6 検体中 2 検体、米国産 8 検体中 1 検体で、合計10 株の mcr-I 遺伝子陽性大腸菌を分離した。国産鶏肉由来株の 63%がテトラサイクリン耐性、50%がそれぞれカナマイシン耐性と ST 合剤耐性を示し、輸入鶏肉由来株では 80%がアンピシリン耐性、70%がそれぞれストレプトマイシン耐性、カナマイシン耐性、シプロフロキサシン耐性を示した。多剤耐性菌は国産、輸入鶏肉由来株でそれぞれ 33%と 40%であった。以上の結果より国産、輸入鶏肉とも高率で多剤耐性 mcr-I 遺伝子陽性大腸菌で汚染されていることが明らかとなった。
第 3 章では、第 1 章、2 章で分離した薬剤耐性大腸菌がヒトへの病原性を示す可能性を調べることを目的に、パイロジェニィと病原遺伝子の分布を解析した。その結果、A、B1、B2 と D の 4 種で分類されるパイロジェニィ解析で、由来に関わらず約 50%の ESBL 産生大腸菌がヒトに腸管外感染症を引き起こす可能性のある B2/D であった。一方、mcr-I 陽性大腸菌のほとんどは非病原性の A/B1に属した。次に、病原遺伝子プロファイルを調べた結果、輸入及び国産鶏肉由来 ESBL 産生大腸菌では astA 遺伝子がそれぞれ 47%と 23%で陽性であった。mcr-I陽性大腸菌においても輸入及び国産鶏肉由来で astA 遺伝子が 60%と 6.7%で陽性であった。cdtB 遺伝子や eae 遺伝子も ESBL 産生大腸菌 1 株と mcr-I 陽性大腸菌

2 株で検出されたが、全て輸入鶏肉由来株であった。さらに、腸管外感染症に関わる様々な病原遺伝子が検出され、特に侵入性に関わる traT 遺伝子は国産・輸入に関わらず ESBL 産生大腸菌や mcr-I 陽性大腸菌において 50%から 97%の高い陽性率を示した。
以上の結果は、我が国で市販されている鶏肉は ESBL 産生大腸菌や mcr-I 陽性大腸菌で高率に汚染されており、それらの多くが多剤耐性であり、一部の菌で、特に輸入鶏肉由来株では高率に、腸管内病原性大腸菌や腸管外病原性大腸菌が保持する病原遺伝子を保持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。