*HQHWLFDQGIXQFWLRQDOFKDUDFWHUL]DWLRQRIDQ0&5OLNHSURGXFLQJ
ϭEscherichia coli
UHFRYHUHGIURPVZLQH%UD]LO
Ϯ1LFRODV.LHIIHU3DWULFH1RUGPDQQ$QGUHD0LFNH0RUHQR/XLVD=DQROOL ϯ
0RUHQR5LFKDUG&KDE\$XGH%UHWRQ3LHUUH7LVVLqUHVDQG/DXUHQW3RLUHO ϰ
1
Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science,
ϱ
University of Fribourg, Fribourg, Switzerland, 2INSERM European Unit (IAME, France),
ϲ
University of Fribourg, Fribourg, Switzerland , 3Institute for Microbiology, University of
ϳ
Lausanne and University Hospital Centre, Lausanne, Switzerland, 4Swiss National Reference
ϴ
Center for Emerging Antibiotic Resistance (NARA), University of Fribourg, Switzerland
ϵ
5
Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdadede Medicina
ϭϬ
Veterinária e Zootecnia,São Paulo, Brazil, and 6
Institute for integrative biology of the cell
ϭϭ
(I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Orsay, France
ϭϮ ϭϯ 5XQQLQJWLWOH&KDUDFWHUL]DWLRQRI0&5 ϭϰ .H\ZRUGVSRO\P\[LQSODVPLGmcr ϭϱ ϭϲ ϭϳ
&RUUHVSRQGLQJ DXWKRU 0DLOLQJ DGGUHVV 0HGLFDO DQG 0ROHFXODU 0LFURELRORJ\
ϭϴ
8QLW͒'HSDUWPHQW RI 0HGLFLQH͒)DFXOW\ RI 6FLHQFH͒8QLYHUVLW\ RI )ULERXUJ &KHPLQ GX
ϭϵ
0XVpH ͒&+ )ULERXUJ 6ZLW]HUODQG 3KRQH (PDLO
ϮϬ
ODXUHQWSRLUHO#XQLIUFK Ϯϭ
ϮϮ
http://doc.rero.ch
Published in "Antimicrobial Agents and Chemotherapy doi: 10.1128/AAC.00278-18, 2018"
which should be cited to refer to this work.
Ϯϯ
$ FROOHFWLRQ RI SLJV ZHUH VFUHHQHG IRU FDUULDJH RI FROLVWLQUHVLVWDQW Ϯϰ
(QWHUREDFWHULDFHDH LQ D IDUP LQ 0LQDV *HUDLV %UD]LO 2XW RI WKLV FROOHFWLRQ HLJWK Ϯϱ
FROLVWLQUHVLVWDQW Escherichia coli LVRODWHV ZHUH UHFRYHUHG LQFOXGLQJ RQH IURP 0LQDV Ϯϲ
*HUDLV 6WDWH SURGXFLQJ D QHZ 0&5 YDULDQW 0&5 $QDO\VLV RI WKH Ϯϳ OLSRSRO\VDFFKDULGHUHYHDOHGWKDW0&5KDGDVLPLODUIXQFWLRQDV0&5DQG0&5 Ϯϴ E\DGGLQJDSKRVSKRHWKDQRODPLQHJURXSWRWKHOLSLG$*HQHWLFDQDO\VLVVKRZHGWKDW Ϯϵ WKHmcr-3.12 JHQHZDVFDUULHGE\DQ,QF$&SODVPLGDQGZDVHPEHGGHGLQDQRULJLQDO ϯϬ JHQHWLFHQYLURQPHQW7KLVVWXG\UHSRUWVWKHRFFXUUHQFHRIWKH0&5OLNHGHWHUPLQDQW ϯϭ LQ6RXWK$PHULFDDQGILUVWO\GHPRQVWUDWHVWKHIXQFWLRQDOLW\RIWKLVJURXSRIHQ]\PHVDV ϯϮ DSKRVSKRHWKDQRODPLQHWUDQVIHUDVH ϯϯ ϯϰ ϯϱ
http://doc.rero.ch
,1752'8&7,21 ϯϲ
7KH LQFUHDVLQJ RFFXUUHQFH RI FROLVWLQUHVLVWDQW (QWHUREDFWHULDFHDH LV RI JUHDW FRQFHUQ VLQFH
ϯϳ
FROLVWLQ UHSUHVHQWV RQH RI WKH ODVWUHVRUW WUHDWPHQWV IRU LQIHFWLRQV FDXVHG E\ FDUEDSHQHP
ϯϴ
UHVLVWDQW (QWHUREDFWHULDFHDH &5( ,Q DGGLWLRQ WR FKURPRVRPDOO\HQFRGHG UHVLVWDQFH
ϯϵ
PHFKDQLVPVFRUUHVSRQGLQJWRPXWDWLRQVRUGHOHWLRQVLQJHQHVLQYROYHGLQWKHELRV\QWKHVLVRI
ϰϬ
WKH OLSRSRO\VDFFKDULGH /36 DFTXLUHG UHVLVWDQFH WKURXJK KRUL]RQWDO JHQH WUDQVIHU KDV EHHQ
ϰϭ
UHFHQWO\ GHVFULEHG )LYH GLIIHUHQW SODVPLGPHGLDWHG FROLVWLQ UHVLVWDQFH JHQHV KDYH EHHQ
ϰϮ LGHQWLILHGVRIDULQ(QWHUREDFWHULDFHDHLQFOXGLQJmcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 ϰϯ 7KH\FRGHIRUHQ]\PHVWKDWPRGLI\WKHOLSLG$PRLHW\RIWKH/36RI*UDPQHJDWLYHEDFWHULD ϰϰ DQGFRQVHTXHQWO\FRQIHUULQJUHVLVWDQFHWRSRO\P\[LQ%DQGFROLVWLQ7RGDWHRQO\0&5 ϰϱ DQG0&5KDYHEHHQVKRZQWRIXQFWLRQDVSKRVSKRHWKDQRODPLQHWUDQVIHUDVHV7KHmcr-1 ϰϲ DQGmcr-2JHQHVOLNHO\RULJLQDWHIURPMoraxellaVSHFLHVZLWKMoraxella pluranimalium ϰϳ
EHLQJ WKH SURJHQLWRU RI mcr-2 Aeromonas VSS WKDW RI mcr-3-OLNH JHQHV DQG
ϰϴ
ShewanellaVSSWKDWRImcr-4-OLNHJHQHV7KHRULJLQRIWKHQHZO\GLVFRYHUHGmcr-5JHQH
ϰϵ
UHPDLQV XQNQRZQ 7KH KLJK SUHYDOHQFH RI 0&5SURGXFLQJ E. coli LVRODWHV LQ IRRG
ϱϬ
SURGXFLQJDQLPDOVDQGWKHUHIRUHWKHKLJKUDWHRIFROLVWLQUHVLVWDQWLVRODWHVPD\EHH[SODLQHG
ϱϭ
E\ WKH FRQVWDQW XVH RI FROLVWLQ LQ YHWHULQDU\ PHGLFLQH LQ SDUWLFXODU LQ OLYHVWRFN IRU WKH
ϱϮ
WUHDWPHQWRISRXOWU\VZLQHDQGFDWWOH7RGDWHVL[mcr-3YDULDQWVKDYHEHHQUHSRUWHGVLQFH
ϱϯ
WKH GLVFRYHU\ RI mcr-3.1 LQ -XQH LGHQWLILHG IURP DQ Echerichia coli LVRODWH IURP D
ϱϰ
KHDOWK\SLJLQ&KLQDDQGLQDSalmonella LVRODWHIURPKXPDQLQIHFWLRQVLQ'HQPDUN
ϱϱ
7KH mcr-3.2 YDULDQW ZDV LGHQWLILHG LQ E. coli IURP FDWWOH LQ 6SDLQ 7KH mcr-3.3 WR -3.9
ϱϲ
YDULDQWVZHUHLGHQWLILHGLQAeromonas VSSDQGWKHmcr-3.10 LQE. coliIURPGXFNLQ
ϱϳ
&KLQD)LQDOO\WKHmcr-3.11 JHQHZDVIURPDQE. coliLVRODWHUHFRYHUHGIURPFKLFNHQLQ
ϱϴ &KLQDXQSXEOLVKHG*HQEDQNDFFHVVLRQQXPEHU0*(YHQLIAeromonas VSSZDV ϱϵ GHVFULEHGDVWKHSURJHQLWRURIWKHmcr-3JHQHVWKLVJHQHPLJKWDOVREHIRXQGDVDQDFTXLUHG ϲϬ GHWHUPLQDQWLQWKDWVSHFLHV ϲϭ
+HUH ZH UHSRUW D QRYHO mcr-3 YDULDQW GHWHFWHG LQ DQ E. coli LVRODWH UHFRYHUHG IURP D SRVW
ϲϮ ZHDQLQJGLDUUKHDRIDSLJWKDWZDVSUHYLRXVO\WUHDWHGE\FROLVWLQLQ%UD]LO ϲϯ 5(68/76 ϲϰ &KDUDFWHUL]DWLRQRIDQHZmcr-3YDULDQWDQGVXVFHSWLELOLW\WHVWLQJ2XWRIWKH ϲϱ
SLJ VDPSOHV HLJKW VDPSOHV ZHUH IRXQG WR FRQWDLQ FROLVWLQUHVLVWDQW E. coli LVRODWHV $OO WKH
ϲϲ
DQLPDOVUHFHLYHGWUHDWPHQWLQFOXGLQJFROLVWLQIRUGD\VDIWHUWKHZHDQLQJSHULRG2XWRIWKH
ϲϳ
FROLVWLQUHVLVWDQW E. coli LVRODWHV RQO\ D VLQJOH LVRODWH , ZDV SRVLWLYH E\ 3&5 IRU WKH
ϲϴ
mcr-3JHQH7KHRWKHUFROLVWLQUHVLVWDQWE.
coliLVRODWHVUHPDLQLQJQHJDWLYHIRUWKHRWKHUmcr-ϲϵ
OLNHJHQHV6HTXHQFLQJRIWKH3&5SURGXFWVUHYHDOHGWKDWWKHmcr-3-OLNH JHQHFRUUHVSRQGHGWR
ϳϬ
D QHZ YDULDQW QDPHG mcr-3.12 *HQEDQN DFFHVVLRQ QXPEHU 0* HQFRGLQJ IRU D
ϳϭ
0&5HQ]\PHVKDULQJRIDPLQRDFLGLGHQWLW\ZLWKWKHRULJLQDO0&5 YDULDQWDQG
ϳϮ
EHWZHHQDQGRIDPLQRDFLGLGHQWLW\ZLWKWKHRWKHU0&5OLNHYDULDQWV)LJXUH
ϳϯ
,VRODWH , VKRZHG UHVLVWDQFH WR EURDGVSHFWUXP FHSKDORVSRULQV WHWUDF\FOLQH
ϳϰ
FKORUDPSKHQLFRO IORUIHQLFRO QDOLGL[LF DFLG VXOIRQDPLGHV VXOIRPHWKR[D]ROHWULPHWKRSULP
ϳϱ
DQGNDQDP\FLQ,WZDVIRXQGSRVLWLYHZLWKWKH5DSLG3RO\P\[LQ13WHVWDQGVKRZHGDQ0,&
ϳϲ
RIFROLVWLQDWJPOXVLQJEURWKPLFURGLOXWLRQPHWKRG0/67DQDO\VLVVKRZHGWKDWLVRODWH
ϳϳ
, EHORQJHG WR WKH 67 DQG WR WKH SK\ORJURXS $ $QDO\VLV ZLWK 6HURW\SHILQGHU
ϳϴ
LQGLFDWHGWKDWLWEHORQJHGWRWKH2+VHURW\SH3K\ORJHQHWLFDQDO\VLVRIWKHNQRZQmcr-ϳϵ
3 VKRZHG D VLJQLILFDWLYH GLYHUVLW\ DPRQJ WKH YDULDQWV 7KUHH PDMRU VXEJURXSV FRXOG EH
ϴϬ
LGHQWLILHGLQFOXGLQJL0&50&5DQG0&5LL0&5DQGWKH0&5
ϴϭ
LLL 0&5 0&5 0&5 0&5 DQG 0&5 UHVSHFWLYHO\ 7KH 0&5
ϴϮ
DQG 0&5 HQ]\PHV ZHUH IRXQG WR EH ERWK FORVH WR 0&5 DQG 0&5 YDULDQWV
ϴϯ
)LJXUH
ϴϰ
0&5 LV D SKRVSKRHWKDQRODPLQH WUDQVIHUDVH FRQIHUULQJ UHVLVWDQFH WR FROLVWLQ ϴϱ
0DVVVSHFWURPHWU\DQDO\VLVRIWKH/36VKRZHGWKDWXQOLNHWKH-QHJDWLYHFRQWUROVKRZLQJD
ϴϲ
VLQJOH m/z SHDN FRUUHVSRQGLQJ WR WKH ELVSKRVSKRU\ODWHG KH[D DF\ODWHG OLSLG $ WKH
ϴϳ
0&5 DQG 0&5 SURGXFHUV VKRZHG DQ LGHQWLFDO DGGLWLRQDO SHDN DW m/z ¨m/z
ϴϴ
FRUUHVSRQGLQJWRDQDGGLWLRQRIDSKRVSKRHWKDQRODPLQH3(W1JURXSPHQWWRWKHOLSLG$DVLW
ϴϵ
ZDV SUHYLRXVO\ GHVFULEHG )LJXUH ,QGXFWLRQ RI WKH S%$'Emcr-3like SODVPLG
ϵϬ DOORZHGWRREWDLQDQ0,&RIFROLVWLQDWJPOZKHUHDVWKHQRQLQGXFHGFORQHSUHVHQWHGDQ ϵϭ 0,&DWJPOVKRZLQJWKDWWKHSURGXFWLRQRI0&5FRQIHUUHGDIROGLQFUHDVHRI ϵϮ FROLVWLQ0,&$OWRJHWKHUWKHVHUHVXOWVVKRZHGWKHSKRVSKRHWKDQRODPLQHWUDQVIHUDVHDFWLYLW\ ϵϯ RIWKH0&5HQ]\PHDQGLWVLPSDFWRQWKHFROLVWLQVXVFHSWLELOLW\ ϵϰ
3ODVPLGDQDO\VLV0DWLQJRXWDVVD\VZHUHVXFFHVVIXOZLWKE. coli -DQGKlebsiella ϵϱ
pneumoniae &,3 DV UHFLSLHQWV EXW DOVR ZLWK Aeromonas punctata &,3
ϵϲ
KLJKOLJKWLQJLWVEURDGKRVWUDQJHSURSHUW\%\FRQWUDVWQRWUDQVFRQMXJDQWZDVREWDLQHGXVLQJ
ϵϳ
P. aeruginosa 3D2 DV UHFLSLHQW &RQMXJDWLRQ IROORZHG E\ 3&5 VKRZHG WKDW mcr-3.12 ZDV
ϵϴ
ORFDWHG RQWR D FRQMXJDWLYH SODVPLG QDPHG S 7KDW ODWWHU SODVPLG HQFRGHG UHVLVWDQFH WR
ϵϵ
WHWUDF\FOLQHV VXOIRQDPLGHV FKORUDPSKHQLFRO DQG IORUIHQLFRO 3%57 DQDO\VLV VKRZHG WKDW
ϭϬϬ
SODVPLGSEHORQJHGWRWKH,QF$&LQFRPSDWLELOLW\JURXS.LHVHUH[WUDFWLRQIROORZHGE\
ϭϬϭ
JHOHOHFWURSKRUHVLVLGHQWLILHGLWVVL]HWREHFDNELQVL]H0,&VRIFROLVWLQRIWKHE. coli
ϭϬϮ
DQG K. pneumoniae WUDQVFRQMXJDQWV ZHUH DW DQG JPO UHVSHFWLYHO\ EHLQJ WKHUHIRUH
ϭϬϯ
FDWHJRUL]HGDVUHVLVWDQWDFFRUGLQJWRWKH(8&$67EUHDNSRLQWRULJLQDO0,&VRIWKHEDFWHULDO
ϭϬϰ
KRVWVEHLQJDWDQGJPOUHVSHFWLYHO\KWWSZZZHXFDVWRUJ,QWHUHVWLQJO\0,&V
ϭϬϱ
RIFROLVWLQRIWKH A. punctata WUDQVFRQMXJDQWZDVDWJPORULJLQDO0,&DWJPO
ϭϬϲ
LQGLFDWLQJDYHU\VLJQLILFDQWLPSDFWRI0&5RQFROLVWLQVXVFHSWLELOLW\LQWKDWVSHFLHV ϭϬϳ
%LRLQIRUPDWLF DQDO\VLV DQG JHQHWLF FRQWH[W RI WKH mcr-3-12 gene. :KROH JHQRPH ϭϬϴ
VHTXHQFLQJRIE. coli,GDWDLGHQWLILHGDVHULHVRIUHVLVWDQFHGHWHUPLQDQWVLQFOXGLQJJHQHV
ϭϬϵ
HQFRGLQJUHVLVWDQFHWRȕODFWDPVbla7(0%DQGbla&7;0
JHQHVDPLQRJO\FRVLGHVaph[3’]-ϭϭϬ
Ia, strA DQGstrBWHWUDF\FOLQHVtetASKHQLFROVcatA1DQGfloRVXOSKRQDPLGHVsul2DQG
ϭϭϭ
WULPHWKRSULPdfr187KHmcr-3OLNHJHQHZDVIRXQGLQDVVRFLDWLRQZLWKDJHQHHQFRGLQJIRU
ϭϭϮ
D GLDF\OJO\FHURO NLQDVH dgkA-OLNH VKDULQJ RI QXFOHRWLGH LGHQWLW\ ZLWK WKH dgkA JHQH
ϭϭϯ
LGHQWLILHGLQDVVRFLDWLRQZLWKWKHILUVWmcr-3GHVFULEHGRQSODVPLGS:-
ϭϭϰ
7KHmcr-3.12 JHQHZDVORFDWHGEHWZHHQWZRLQVHUWLRQVHTXHQFHVEHORQJLQJWRWKH,666
ϭϭϱ
DQG ,630 IDPLOLHV UHVSHFWLYHO\ )LJXUH . ,QWHUHVWLQJO\ ES DIWHU WKH HQG RI WKH LQYHUWHG
ϭϭϲ
UHSHDW ULJKW ,55 RI WKH ,630-OLNH DQ ,5/OLNH RI WKH ,666 ZDV GHWHFWHG VKDULQJ RI
ϭϭϳ
QXFOHRWLGH LGHQWLW\ ZLWK WKH ILUVW QW RI WKH ,5/ RI ,666 )LJXUH 7KH SUHVHQFH RI WKLV
ϭϭϴ
,5/OLNHGRZQVWUHDPWKH,630OLNHFRXOGIRUPDSXWDWLYHWUDQVSRVRQZLWKWKH,666
ϭϭϵ
)XUWKHUDQDO\VLVVKRZHGWKDWWKLVSXWDWLYHWUDQVSRVRQZDVHPEHGGHGLQDORQJHUVWUXFWXUHWKDW
ϭϮϬ
ZDVLQVHUWHGEHWZHHQQXFOHRWLGHVDQGRID'1$PHWK\OWUDQVIHUDVHJHQHORFDWHG
ϭϮϭ
RQWKH,QF$&EDFNERQH7KLVVWUXFWXUH ZDVESORQJDQGLVUHSUHVHQWHGLQ)LJXUH)
ϭϮϮ
,W FRXOG EH GHILQHG LQWR WKUHH GLIIHUHQW UHJLRQV L D ¶ UHJLRQ FKDUDFWHUL]HG E\ D ES
ϭϮϯ UHJLRQZLWKD*&FRQWHQWRIFRQWDLQLQJWKUHHSXWDWLYHRSHQUHDGLQJIUDPHVLQFOXGLQJWZR ϭϮϰ HQFRGLQJIRUSXWDWLYHVLWHVSHFLILFLQWHJUDVHVLLWKHSXWDWLYHWUDQVSRVRQFRQWDLQLQJWKHmcr-3 ϭϮϱ YDULDQWDQGWKUHH25)VĮȕDQGȖSUHVHQWLQJD*&FRQWHQWRIDQGLLLD¶UHJLRQRI ϭϮϲ
ES ZLWK D VLPLODU *& DV WKH ILUVW ES UHJLRQ )LJXUH ) 7KH 25) Į ȕ DQG Ȗ
ϭϮϳ
HQFRGHG IRU D UHYHUVH WUDQVFULSWDVH D WUDQVFULSWLRQDO UHJXODWRU DQG D GLJXDQ\ODWH F\FODVH
ϭϮϴ UHVSHFWLYHO\7KHLUSURGXFWVVKRZHGVWURQJDPLQRDFLGLGHQWLW\ZLWKSXWDWLYHSURWHLQV ϭϮϵ IURPAeromonas dhakensis ϭϯϬ ',6&866,21 ϭϯϭ :HUHSRUWKHUHWKHLGHQWLILFDWLRQRIDQRYHOYDULDQWRIWKHmcr-3JHQHGHWHFWHGLQDQE. ϭϯϮ
coli LVRODWH UHFRYHUHG IURP D SLJ LQ %UD]LO ,QWHUHVWLQJO\ SUHYLRXV VWXGLHV DOVR GHVFULEHG
ϭϯϯ
0&5SURGXFHUVUHFRYHUHGIURPDQLPDOVDPSOHVVXJJHVWLQJWKHVDPHOLQNEHWZHHQ
ϭϯϰ
DQLPDODQGFROLVWLQUHVLVWDQFHDVLWKDVEHHQHVWDEOLVKHGIRUWKHmcr-1JHQH7KHSLJVVFUHHQHG
ϭϯϱ
LQ WKLV VWXG\ ZHUH WUHDWHGSUHYLRXVO\ZLWK FROLVWLQ IRU ILIWHHQ GD\V DIWHU WKH ZHDQLQJSHULRG
ϭϯϲ
7KLVVXJJHVWVWKHSRVVLEOHVHOHFWLRQRIFROLVWLQUHVLVWDQWVWUDLQGXULQJWKLVSHULRGDVZHVKRZHG
ϭϯϳ
LQ RXU SUHYLRXV VWXG\ GHVFULELQJ D KLJK SUHYDOHQFH RI 0&5SURGXFHUV LQ D SLJ IDUP LQ
ϭϯϴ
3RUWXJDO ZKHUH DQLPDOV KDG UHFHLYHG FROLVWLQ 7KHUH KDYH EHHQ PDQ\ UHSRUWV RI 0&5
ϭϯϵ
SURGXFHUV LQ %UD]LO ZLWK 0&5 EHLQJ WKH RQO\ YDULDQW V\VWHPDWLFDOO\ LGHQWLILHG 7KHVH
ϭϰϬ
LVRODWHVZHUHDVLQJOHSalmonella enterica VHURW\SH7\SK\PXULXPZDVUHFRYHUHGIURPUHWDLO
ϭϰϭ
PHDWE. coli LVRODWHVUHFRYHUHGIURPFKLFNHQPHDWIURPPLJUDWRU\SHQJXLQV
ϭϰϮ
UHFRYHUHGRQSXEOLFEHDFKHVRUUHFRYHUHGIURPSDWLHQWVZLWKEORRGVWUHDPLQIHFWLRQV
ϭϰϯ
$OVR .3&SURGXFLQJ E. coli DQG .3&SURGXFLQJ Klebsiella pneumoniae
ϭϰϰ
EHORQJLQJWR67DQG67ZHUHLGHQWLILHG$TXLWHH[WHQVLYHVWXG\LGHQWLILHGD
ϭϰϱ
VHULHV RI 0&5SURGXFLQJ E. coli LVRODWHV UHFRYHUHG IURP KXPDQV FKLFNHQ FKLFNHQ
ϭϰϲ PHDWERYLQHWXUNH\VZLQHDQGSHQJXLQ+RZHYHUZHPLJKWVSHFXODWHWKDWPRVWVWXGLHV ϭϰϳ ZHUHGHVLJQHGWRGHWHFWRQO\WKHmcr-1 JHQHVRIDUDQGIHZLQYHVWLJDWLQJWKHRFFXUUHQFHRI ϭϰϴ WKHPRVWUHFHQWO\LGHQWLILHGRWKHUYDULDQWV ϭϰϵ
,VRODWH , FDUULHG D QRYHO mcr-3 YDULDQW QDPHG mcr-3.12 ,W EHORQJHG WR 67
ϭϱϬ
ZKLFKZDVSUHYLRXVO\IRXQGWRFDUU\WKHmcr-1JHQHFRUUHVSRQGLQJWRLVRODWHVUHFRYHUHGIURP
ϭϱϭ
SLJV LQ *HUPDQ\ LQ ,W EHORQJV WR WKH SK\ORJURXS $ RI E. coli WKHUHIRUH
ϭϱϮ
FRUUHVSRQGLQJ WR D FRPPHQVDO VWUDLQ 6HTXHQFH DOLJQPHQW DQDO\VLV VKRZHG WKDW mcr-3.12
ϭϱϯ
VKDUHVRIQXFOHRWLGHLGHQWLW\ZLWKDVHTXHQFHIURPAeromonas veronii.7KLVVXJJHVWVWKDW
ϭϱϰ
WKLV QHZ YDULDQW PD\ KDYH RULJLQDWHG IURP WKDW SDUWLFXODU VSHFLHV RU PD\ KDYH ZLGHO\
ϭϱϱ
GLVVLPLQDWHGDVDQDFTXLUHGUHVLVWDQFHWUDLWZLWKLQWKDWVSHFLHV1RWHZRUWK\ZHVKRZHGKHUH
ϭϱϲ
WKDWWKH,QF$&W\SHSODVPLGEHDULQJWKHmcr-3.12JHQHFRXOGUHSOLFDWHLQAeromonasVS:H
ϭϱϳ
PD\ WKHUHIRUH VSHFXODWH WKDW VXFK SODVPLG W\SH PLJKW KDYH EHHQ LQYROYHG LQ WKH RULJLQDO
ϭϱϴ
VSUHDG RI mcr-3OLNH JHQHV IURP WKHLU SURJHQLWRUV WR RWKHU EDFWHULDO VSHFLHV LQFOXGLQJ
ϭϱϵ PHPEHUVRIWKHEnterobacteriaceaeIDPLO\ ϭϲϬ ,QGXFWLRQH[SHULPHQWVDQGDQDO\VLVRIWKHOLSLG$RIWKHLVRODWHVWURQJO\LQGLFDWHVWKDW ϭϲϭ WKH0&5HQ]\PHFRQIHUVFROLVWLQUHVLVWDQFHWKHVDPHZD\DV0&5DQG0&5HQ]\PHV ϭϲϮ E\DGGLQJDSKRVSKRHWKDQRODPLQHJURXSWRWKHOLSLG$DOWKRXJKWKLVHQ]\PHRQO\VKDUHG ϭϲϯ DQGRIDPLQRDFLGLGHQWLW\ZLWK0&5DQG0&5UHVSHFWLYHO\7KHIDFWWKDW0&5 ϭϲϰ
DQG VKDUH VLPLODU IXQFWLRQV ZDV SUHYLRXVO\ K\SRWKHVL]HG WKURXJK DQ LQVLOLFR SURWHLQ
ϭϲϱ VWUXFWXUHDQDO\VLV ϭϲϲ 7KHmcr-3 JHQHZDVSUHYLRXVO\GHVFULEHGRQWR,QF+,DQG,QF;SODVPLGVZKLFKDUH ϭϲϳ FRPPRQO\IRXQGLQDVVRFLDWLRQZLWKWKHmcr-1DQGmcr-2 JHQHV+HUHZHGHVFULEHGWKHILUVW ϭϲϴ
,QF$& SODVPLG FDUU\LQJ D SODVPLGPHGLDWHG FROLVWLQ UHVLVWDQFH GHWHUPLQDQW 7KLV SODVPLG
ϭϲϵ
EDFNERQHLVFRPPRQO\ LGHQWLILHGDVDVXSSRUWRIPDQ\GLIIHUHQWDQWLELRWLFUHVLVWDQFHJHQHV
ϭϳϬ
+HUH WKH GHWHUPLQDQWV tetA sul2 DQG floR HQFRGLQJ IRU UHVLVWDQFH WR WHWUDF\FOLQH ϭϳϭ VXOIRQDPLGHVDQGSKHQLFROVUHVSHFWLYHO\ZHUHDOVRGHWHFWHGRQWKLVVDPHSODVPLG7KHEURDG ϭϳϮ KRVWUDQJHRIWKLVSODVPLGZDVGHPRQVWUDWHGE\HYLGHQFLQJLWVDELOLW\WRUHSOLFDWHQRWRQO\LQ ϭϳϯ
E. coli DQGK. pneumoniaeEXWDOVRLQA. punctata
ϭϳϰ
7KHmcr-3.12LVORFDWHGLQWRDSXWDWLYHWUDQVSRVRQLQFOXGLQJWKH,666XSVWUHDPRIWKH
ϭϳϱ
mcr-3 JHQH DQG DQ ,630-like GRZQVWUHDP ,QWHUHVWLQJO\ D QW UHJLRQ IRXQG ES
ϭϳϲ
GRZQVWUHDPWKH,630-likeZDVIRXQGLGHQWLFDOWRWKH,5/RIWKH,666)XUWKHUH[SHULPHQWVZLOO
ϭϳϳ
EH FRQGXFWHG WR FRQILUP ZKHWKHU ,666 FRXOG KDYH SOD\HG D UROH LQ WKH DFTXLVLWLRQ RI WKLV
ϭϳϴ SKRVSKRHWKDQRODPLQHWUDQVIHUDVHJHQHE\DPHFKDQLVPVLPLODUWRDRQHKDQGHGWUDQVSRVLWLRQ ϭϳϵ DVLWKDVEHHQGHVFULEHGIRU,6Ecp1LQWKHPRELOL]DWLRQRIbla&7;0 ϭϴϬ 7KHJHQHWLFFRQWH[WRIWKHSXWDWLYHmcr-3WUDQVSRVRQLVFRPSOH[DQGWKHFKURQRORJ\RI ϭϴϭ DFTXLVLWLRQRIWKLVVWUXFWXUHLQWRWKH,QF$&SODVPLGFDQKDUGO\EHH[SODLQHG2QHK\SRWKHVLV ϭϴϮ
LV VXPPDUL]HG LQ WKH )LJXUH 7KH ,666 PLJKW KDYH EHHQ LQYROYHG LQ WKH RULJLQDO
ϭϴϯ
PRELOL]DWLRQ RI WKH mcr-3.12 JHQH IURP Aeromonas VSS. )LJXUHV $' 7KHQ D VHFRQG
ϭϴϰ
PRELOL]DWLRQHYHQWPD\KDYHRFFXUUHGLQYROYLQJDQXQNQRZQPHFKDQLVPEHWZHHQWKHJHQHWLF
ϭϴϱ
VWUXFWXUH FRQWDLQLQJ WKH SXWDWLYH LQWHJUDVHV )LJXUH ( DQG WKH mcr-3-FDUU\LQJ VWUXFWXUH
ϭϴϲ
IRUPLQJDQESLQWHJURQOLNHJHQHWLFFRPSOH[)LQDOO\WKLVZKROHVWUXFWXUHPD\KDYH
ϭϴϳ
EHHQPRELOL]HGDQGLQVHUWHGEHWZHHQWKHQWDQGQWRID'1$PHWK\OWUDQVIHUDVH ϭϴϴ JHQHORFDWHGRQDQ,QF$&SODVPLGEDFNERQH)LJXUH)7KHUHVXOWLQJUHVLVWDQFHSODVPLGLV ϭϴϵ DWWKHHQGRQHRIWKRVHUHVSRQVLEOHIRUWKHVSUHDGRImcr JHQHVDPRQJ(QWHUREDFWHULDFHDH ϭϵϬ 0$7(5,$/$1'0(7+2'6 ϭϵϭ
%DFWHULDO LVRODWH DQG VXVFHSWLELOLW\ WHVWLQJ 6FUHHQLQJ RI FROLVWLQUHVLVWDQW LVRODWHV ϭϵϮ
ZDV SHUIRUPHG IURP GLIIHUHQW SLJV LQ WHQ VZLQH KHUGV LQ GLIIHUHQW VWDWHV RI WKH VWDWH RI
ϭϵϯ
0LQDV*HUDLVLQ%UD]LODOOSLJVSUHVHQWLQJSRVWZHDQLQJGLDUUKHD7KHLVRODWHVZHUHLQLWLDOO\
ϭϵϰ
WHVWHG IRU FROLVWLQ UHVLVWDQFH XVLQJ DJDU GLOXWLRQ PHWKRGV $OO FRORQLHV JURZLQJ RQ SODWHV
ϭϵϱ
VXSSOHPHQWHG ZLWK !JPO RI FROLVWLQ ZHUH FRQILUPHG E\ WKH FRPPHUFLDOL]HG 5DSLG
ϭϵϲ
3RO\P\[LQ 13 WHVW (/,7HFK 0LFURELRORJ\ )UDQFH DQG PLQLPDO LQKLELWRU\
ϭϵϳ
FRQFHQWUDWLRQV0,&VZHUHGHWHUPLQHGE\EURWKPLFURGLOXWLRQPHWKRGXVLQJFDWLRQDGMXVWHG
ϭϵϴ
0+ EURWK $QWLPLFURELDO VXVFHSWLELOLW\ WHVWLQJ IRU RWKHU DQWLELRWLFV IDPLOLHV ZDV SHUIRUPHG
ϭϵϵ
DFFRUGLQJ WR WKH VWDQGDUG GLVN GLIIXVLRQ PHWKRG RQ 0XHOOHU+LQWRQ 0+ DJDU SODWHV
ϮϬϬ
IROORZLQJWKH&/6,UHFRPPHQGDWLRQV
ϮϬϭ
:*6 DQG PROHFXODU DQDO\VLV 3&5 VFUHHQLQJ IRU mcr JHQHV ZDV SHUIRUPHG XVLQJ ϮϬϮ SULPHUVGHVLJQHGWRGHWHFWDOONQRZQYDULDQWVRI0&53ULPHUV0&5DOO)¶*&$777 ϮϬϯ $7*&7*$$&7**&*¶DQG0&5DOO5¶$*&**&777&7*&7*&$$$&¶ ϮϬϰ
http://doc.rero.ch
ZHUHXVHGDQGFRUUHVSRQGLQJDPSOLFRQVZHUHVXEVHTXHQWO\VHTXHQFHG0LFURV\QWK%DOJDFK
ϮϬϱ
6ZLW]HUODQG :KROH JHQRPLF '1$ RI WKH 0&5SRVLWLYH LVRODWH ZDV H[WUDFWHG ZLWK WKH
ϮϬϲ
6LJPD$OGULFK *HQ(OXWH %DFWHULDO *HQRPLF '1$ .LW *HQRPLF OLEUDULHV ZHUH DVVHVVHG
ϮϬϳ
XVLQJ WKH 1H[WHUD;7 OLEUDU\ SUHSDUDWLRQ NLW ,OOXPLQD ,QF 6DQ 'LHJR &$ DQG VHTXHQFLQJ
ϮϬϴ
ZDV SHUIRUPHG XVLQJ WKH ,OOXPLQD 0LQL6HT V\VWHP ZLWK ES SDLUHGHQG UHDGV DQG D
ϮϬϵ
FRYHUDJHRI;*HQHUDWHG)DVW4GDWDZHUHFRPSLOHGDQGDQDO\]HGXVLQJWKH&/&JHQRPLF
ϮϭϬ
ZRUNEHQFK &/& ELR $DUWKXV 'HQPDUN 5HDGV ZHUH GH QRYR DVVHPEOHG ZLWK
Ϯϭϭ
DXWRPDWLF EXEEOH DQG ZRUG VL]H DQG FRQWLJV ZHUH JHQHUDWHG XVLQJ WKH PDSSLQJ PRGH ³PDS
ϮϭϮ
UHDGVEDFNWRFRQWLJV´ZLWKDPLQLPXPFRQWLJOHQJWKRIQXFOHRWLGHV
Ϯϭϯ
7KH UHVXOWLQJ FRQWLJV ZHUH XSORDGHG LQWR WKH &HQWHU IRU *HQRPLF (SLGHPLRORJ\ VHUYHU
Ϯϭϰ
KWWSZZZJHQRPLFHSLGHPLRORJ\RUJ 3ODVPLG UHSOLFRQ W\SLQJ PXOWLORFXV VHTXHQFH
Ϯϭϱ
W\SLQJ VHURW\SH DQG DQWLPLFURELDO UHVLVWDQFH GHWHUPLQDQWV ZHUH GHWHUPLQHG XVLQJ
Ϯϭϲ
3ODVPLG)LQGHU 0/67 6HURW\SH)LQGHU DQG 5HV)LQGHU UHVSHFWLYHO\
Ϯϭϳ
3K\ORJURXS DQDO\VLV ZDV SHUIRUPHG E\ XVLQJ WKH &OHUPRQW PHWKRG 6HTXHQFH
Ϯϭϴ
DOLJQHPHQWV DQG FRQVWUXFWLRQ RI SK\ORJHQHWLF WUHHV ZHUH SHUIRUPHG ZLWK WKH 6HDYLHZ
Ϯϭϵ
DOLJQPHQWWRROYHUVLRQ3UDEL/D'RXD/\RQ)UDQFH
ϮϮϬ
3ODVPLG DQDO\VLV ZDV SHUIRUPHG XVLQJ .LHVHU H[WUDFWLRQ PHWKRG IROORZHG E\ JHO
ϮϮϭ
HOHFWURSKRUHVLVLQRUGHUWRHVWLPDWHWKHVL]HRIWKHSODVPLGFRQWDLQLQJWKH mcr-3 JHQHXVLQJ
ϮϮϮ
WKH E. coli VWUDLQ KDUERULQJ IRXU SODVPLGV RI DQG NE UHVSHFWLYHO\ DV
ϮϮϯ
SODVPLGVL]HPDUNHU7KHGHWHUPLQDWLRQRIWKHLQFRPSDWLELOLW\JURXSZDVFRQILUPHGE\3&5
ϮϮϰ
EDVHGUHSOLFRQW\SLQJ3%57
ϮϮϱ
&RQMXJDWLRQ H[SHULPHQWV ZHUH SHUIRUPHG XVLQJ WKH D]LGHUHVLVWDQW E. coli - VWUDLQ ,Q
ϮϮϲ
DGGLWLRQ FRQMXJDWLRQ ZHUH DOVR SHUIRUPHG LQ WKH WHPRFLOOLQUHVLVWDQW Pseudomonas
ϮϮϳ
aeruginosa3$2 LQ WKH D]LGHUHVLVWDQW Klebsiella pneumoniae &,3 DQG LQ WKH D]LGH
ϮϮϴ
UHVLVWDQWAeromonas punctate&,3UHFLSLHQWVWUDLQV WRWHVWWKHEURDGKRVWUDQJHRIWKH
ϮϮϵ
SODVPLG FRGLQJ IRU WKH mcr-3.12 YDULDQW %RWK GRQRU DQG UHFLSLHQW VWUDLQV ZHUH FXOWXUHG LQ
ϮϯϬ
H[SRQHQWLDOSKDVHWKHQPL[HGRQVROLG/%DJDUXVLQJILOWHUVDWDGRQRUUHFLSLHQWUDWLR
Ϯϯϭ
$IWHUKRILQFXEDWLRQILOWHUVZHUHUHVXVSHQGHGLQ1D&ODQGEDFWHULDOPL[WXUHZHUH
ϮϯϮ
SODWHG RQWRDJDU SODWHVVXSSOHPHQWHG ZLWK FROLVWLQ JPO DQG VRGLXP D]LGH JPO
Ϯϯϯ
IRU E. coli RU ZLWK WHPRFLOOLQ JPO DQG VRGLXP D]LGH JPO IRU P. aeruginosa
Ϯϯϰ
6LQFHWKHSODVPLGEHDULQJWKHmcr-3.12JHQHFRQIHUUHGUHVLVWDQFHWRWHWUDF\FOLQHFRQMXJDWLRQV
Ϯϯϱ
XVLQJ K. penumoniae DQG A. punctata DV UHFLSLHQWV ZHUH DWWHPSWHG XVLQJ WHWUDF\FOLQH
Ϯϯϲ
JPO DQG VRGLXP D]LGH JPO DV VHOHFWLYH PROHFXOHV 6XVFHSWLELOLW\ RI DOO
Ϯϯϳ
WUDQVFRQMXJDQWVWRDQWLELRWLFVZDVFRQILUPHGE\DQWLELRJUDPIROORZHGE\3&5IRUWKHmcr-3
Ϯϯϴ
OLNHJHQH
Ϯϯϵ
$QDO\VLV RI WKH /36 PRGLILFDWLRQ 7KH /36 RI E. coli - XQPRGLILHG OLSLG $ ϮϰϬ
7&$I-mcr-1WUDQVFRQMXJDQWDQG,0&5OLNHSURGXFHUVZHUHDQDO\]HGE\PDVV
Ϯϰϭ
VSHFWURPHWU\ 06 7KH OLSLG $ ZDV REWDLQHG E\ WKH K\GURO\VLV RI PJ RI O\RSKLOL]HG
ϮϰϮ
EDFWHULDLQORILVREXW\ULFDFLGDQG0DPPRQLXPK\GUR[LGHYYKHDWHGIRUK
Ϯϰϯ
DW & DQG FRROHG DW & EHIRUH FHQWULIXJDWLRQ DV SUHYLRXVO\ GHVFULEHG 7KH
Ϯϰϰ
VXSHUQDWDQW ZDV WKHQ GLOXWHG ZLWK ZDWHU DQG O\RSKLOL]HG EHIRUH ZDVK ZLWK PHWKDQRO 7KH
Ϯϰϱ
LQVROXEOH OLSLG $ REWDLQHG ZDV ILQDOO\ H[WUDFWHG LQ D FKORURIRUPPHWKDQROZDWHU
Ϯϰϲ
YYY PL[WXUH 0$/',06 DQDO\VLV ZDV SHUIRUPHG XVLQJ D 3HU6HSWLYH 9R\DJHU 675 3(
Ϯϰϳ
%LRV\VWHPV )UDQFH WLPHRIIOLJKW PDVV VSHFWURPHWHU LQ OLQHDU QHJDWLYH LRQ PRGH
Ϯϰϴ
'LK\GUR[\EHQ]RLFDFLG'+%DWPJPOLQ0FLWULFDFLGLQFKORURIRUPPHWKDQROZDWHU
Ϯϰϵ
YYYZDVXVHGDVPDWUL[ ϮϱϬ
&ORQLQJ DQG RYHUH[SUHVVLRQ RI WKH mcr-3.12 JHQH 7KH QHZ mcr-3 YDULDQW ZDV Ϯϱϭ
FORQHG LQWR WKH DUDELQRVHLQGXFLEOH S%$'EYHFWRU LQ RUGHU WR GHWHUPLQH WKH LPSDFW RI WKH
ϮϱϮ
H[SUHVVLRQ RI WKH 0&5 SKRVSKRHWKDQRODPLQH WUDQVIHUDVH RQ FROLVWLQ VXVFHSWLELOLW\
Ϯϱϯ
,QGXFWLRQ RI S%$'E YHFWRU ZDV SHUIRUPHG XVLQJ 0+ EURWK VXSSOHPHQWHG ZLWK /DUDELQRVH Ϯϱϰ DVSUHYLRXVO\GHVFULEHG Ϯϱϱ Ϯϱϲ $&.12:/('*0(176 Ϯϱϳ
7KLV ZRUN KDV EHHQ IXQGHG E\ WKH 8QLYHUVLW\ RI )ULERXUJ E\ WKH 6ZLVV 1DWLRQDO 6FLHQFH
Ϯϱϴ
)RXQGDWLRQ SURMHFW )16B DQG E\ JUDQWV IURP WKH $1,:+$ (5$1(7
Ϯϱϵ
SURMHFW 6ZLW]HUODQG WKH 2)63 %HUQ 6ZLW]HUODQG JUDQW Q DQG WKH 1RYDUWLV
ϮϲϬ )RXQGDWLRQIRUPHGLFDOELRORJLFDO5HVHDUFK Ϯϲϭ ϮϲϮ Ϯϲϯ
http://doc.rero.ch
5()(5(1&(6 Ϯϲϰ
3RLUHO / -D\RO $ 1RUGPDQQ 3 3RO\P\[LQV DQWLEDFWHULDO DFWLYLW\ Ϯϲϱ
VXVFHSWLELOLW\ WHVWLQJ DQG UHVLVWDQFH PHFKDQLVPV HQFRGHG E\ SODVPLGV RU
Ϯϲϲ FKURPRVRPHV&OLQ0LFURELRO5HY± Ϯϲϳ /LX<<:DQJ<:DOVK75<L/;=KDQJ56SHQFHU-'RL<7LDQ*'RQJ% Ϯϲϴ +XDQJ;<X/)*X'5HQ+&KHQ;/Y/+H'=KRX+/LDQJ=/LX-+ Ϯϲϵ 6KHQ-(PHUJHQFHRISODVPLGPHGLDWHGFROLVWLQUHVLVWDQFHPHFKDQLVP0&5 ϮϳϬ
LQ DQLPDOV DQG KXPDQ EHLQJV LQ &KLQD D PLFURELRORJLFDO DQG PROHFXODU ELRORJLFDO
Ϯϳϭ
VWXG\/DQFHW,QIHFW'LV
ϮϳϮ
;DYLHU %% /DPPHQV & 5XKDO 5 .XPDU6LQJK 6 %XWD\H 3 *RRVVHQV + Ϯϳϯ
0DOKRWUD.XPDU 6 ,GHQWLILFDWLRQ RI D QRYHO SODVPLGPHGLDWHG FROLVWLQ Ϯϳϰ UHVLVWDQFHJHQHmcr-2LQEscherichia coli%HOJLXP-XQH(XUR6XUYHLOO Ϯϳϱ <LQ:/L+6KHQ</LX=:DQJ66KHQ==KDQJ5:DOVK756KHQ-:DQJ Ϯϳϲ <1RYHOSODVPLGPHGLDWHGFROLVWLQUHVLVWDQFHJHQHmcr-3LQEscherichia coli Ϯϳϳ 0%LR Ϯϳϴ &DUDWWROL$9LOOD/)HXGL&&XUFLR/2UVLQL6/XSSL$3H]]RWWL*0DJLVWUDOL Ϯϳϵ
&) 1RYHO SODVPLGPHGLDWHG FROLVWLQ UHVLVWDQFH mcr-4 JHQH LQ Salmonella DQG ϮϴϬ
Escherichiacoli,WDO\6SDLQDQG%HOJLXPWR(XUR6XUYHLOO
Ϯϴϭ
%RURZLDN0)LVFKHU-+DPPHUO-$+HQGULNVHQ566]DER,0DORUQ\% ϮϴϮ
,GHQWLILFDWLRQRIDQRYHOWUDQVSRVRQDVVRFLDWHGSKRVSKRHWKDQRODPLQHWUDQVIHUDVHJHQH
Ϯϴϯ
mcr-5 FRQIHUULQJ FROLVWLQ UHVLVWDQFH LQ GWDUWUDWH IHUPHQWLQJ Salmonella enterica
Ϯϴϰ subspentericaVHURYDU3DUDW\SKL%-$QWLPLFURE&KHPRWKHU Ϯϴϱ 6XQ-;X<*DR5/LQ-:HL:6ULQLYDV6/L'<DQJ56/L;3/LDR;3 Ϯϴϲ /LX<+)HQJ<'HFLSKHULQJ0&5FROLVWLQUHVLVWDQFH0%LR Ϯϴϳ
.LHIIHU 11RUGPDQQ 3 3RLUHO / Moraxella VSHFLHV DV SRWHQWLDO VRXUFHV RI Ϯϴϴ
0&5OLNH SRO\P\[LQ UHVLVWDQFH GHWHUPLQDQWV $QWLPLFURE $JHQWV &KHPRWKHU
Ϯϴϵ
H ϮϵϬ
3RLUHO/.LHIIHU1)HUQDQGH]*DUD\]DEDO-)9HOD$,/DUSLQ<1RUGPDQQ3 Ϯϵϭ
0&5PHGLDWHG SODVPLGERUQH SRO\P\[LQ UHVLVWDQFH PRVW OLNHO\ RULJLQDWHV
ϮϵϮ
IURPMoraxellapluranimalium-$QWLPLFURE&KHPRWKHU
Ϯϵϯ
/LWUXS ( .LLO . +DPPHUXP $0 5RHU / 1LHOVHQ (0 7RUSGDKO 0 Ϯϵϰ
3ODVPLGERUQH FROLVWLQ UHVLVWDQFH JHQH mcr-3 LQ Salmonella LVRODWHV IURP KXPDQ
Ϯϵϱ
LQIHFWLRQV'HQPDUN(XUR6XUYHLOO
Ϯϵϲ
+HUQiQGH] 0 ,JOHVLDV 05 5RGUtJXH]/i]DUR ' *DOODUGR $ 4XLMDGD 1 Ϯϵϳ
0LJXHOD9LOOROGR3&DPSRV0-3tUL]6/ySH]2UR]FR*GH)UXWRV&6iH]-/ Ϯϵϴ
8JDUWH5XL] 0 'RPtQJXH] / 4XHVDGD $ &RRFFXUUHQFH RI FROLVWLQ Ϯϵϵ UHVLVWDQFHJHQHVmcr1DQGmcr-3DPRQJPXOWLGUXJUHVLVWDQWEscherichiacoliLVRODWHG ϯϬϬ IURPFDWWOH6SDLQ6HSWHPEHU(XUR6XUYHLOO ϯϬϭ
/LX / )HQJ < =KDQJ ; 0F1DOO\ $ =RQJ = 1HZ YDULDQW RI mcr-3 LQ DQ ϯϬϮ H[WHQVLYHO\GUXJUHVLVWDQWEscherichia coliFOLQLFDOLVRODWHFDUU\LQJmcr-1DQGbla1'0 ϯϬϯ $QWLPLFURE$JHQWV&KHPRWKHUH ϯϬϰ /LQJ=<LQ:/L+=KDQJ4:DQJ;:DQJ=.H<:DQJ<6KHQ- ϯϬϱ
&KURPRVRPHPHGLDWHG mcr-3 YDULDQWV LQ Aeromonas veronii IURP FKLFNHQ PHDW
ϯϬϲ
$QWLPLFURE$JHQWV&KHPRWKHUH
ϯϬϳ
(LFKKRUQ,)HXGL&:DQJ<.DVSDU+)HOHU$7/ENH%HFNHU$0LFKDHO ϯϬϴ
*% 6KHQ - 6FKZDU] 6 ,GHQWLILFDWLRQ RI QRYHO YDULDQWV RI WKH FROLVWLQ ϯϬϵ
UHVLVWDQFH JHQHmcr-3LQAeromonasVSS IURP WKH QDWLRQDO UHVLVWDQFH PRQLWRULQJ
ϯϭϬ SURJUDPPH*(509HWDQGIURPGLDJQRVWLFVXEPLVVLRQV-$QWLPLFURE&KHPRWKHULQ ϯϭϭ SUHVV ϯϭϮ :DQJ;=KDL:/L-/LX'=KDQJ46KHQ=:DQJ6:DQJ<3UHVHQFH ϯϭϯ
RIDQmcr-3YDULDQWLQAeromonas caviaeProteus mirabilisDQGEscherichia coliIURP
ϯϭϰ
RQHGRPHVWLFGXFN$QWLPLFURE$JHQWV&KHPRWKHUH
ϯϭϱ
/LX<<&KDQGOHU&(/HXQJ/00F(OKHQ\&/0HWWXV576KDQNV504/LX ϯϭϲ
-+ *RRGOHWW '5 (UQVW 5. 'RL < 6WUXFWXUDO PRGLILFDWLRQ RI ϯϭϳ
OLSRSRO\VDFFKDULGH FRQIHUUHG E\ mcr-1 LQ *UDP1HJDWLYH (6.$3( SDWKRJHQV
ϯϭϴ
$QWLPLFURE$JHQWV&KHPRWKHUH
ϯϭϵ
.LHIIHU1$LUHVGH6RXVD01RUGPDQQ33RLUHO/ +LJKUDWHRI0&5 ϯϮϬ
SURGXFLQJ Escherichia coli DQG Klebsiella pneumoniDH DPRQJ SLJV 3RUWXJDO (PHUJ
ϯϮϭ
,QIHFW'LV
ϯϮϮ
5DX 5% GH /LPD0RUDOHV ' :LQN 3/ 5LEHLUR $5 0DUWLQV $) %DUWK $/ ϯϮϯ (PHUJHQFHRImcr-1SURGXFLQJSalmonella entericaVHURYDU7\SKLPXULXPIURP ϯϮϰ UHWDLOPHDWILUVWGHWHFWLRQLQ%UD]LO)RRGERUQH3DWKRJ'LV ϯϮϱ 0RQWH')0HP$)HUQDQGHV05&HUGHLUD/(VSRVLWR)*DOYmR-$)UDQFR ϯϮϲ
%'*0 /LQFRSDQ 1 /DQGJUDI 0 &KLFNHQ PHDW DV D UHVHUYRLU RI FROLVWLQ ϯϮϳ UHVLVWDQWEscherichia coliVWUDLQVFDUU\LQJmcr-1JHQHVLQ6RXWK$PHULFD$QWLPLFURE ϯϮϴ $JHQWV&KHPRWKHUH ϯϮϵ 6HOOHUD)3)HUQDQGHV056DUWRUL/&DUYDOKR03(VSRVLWR)1DVFLPHQWR&/ ϯϯϬ
'XWUD *+ 0DPL]XND (0 3pUH]&KDSDUUR 3- 0F&XOORFK -$ /LQFRSDQ 1 ϯϯϭ
Escherichia coliFDUU\LQJ,QF;SODVPLGPHGLDWHGmcr-1DQGbla&7;0JHQHVLQ
ϯϯϮ
LQIHFWHG PLJUDWRU\ 0DJHOODQLF SHQJXLQV Spheniscus magellanicus - $QWLPLFURE
ϯϯϯ
&KHPRWKHU
ϯϯϰ
)HUQDQGHV 05 6HOOHUD )3 (VSRVLWR ) 6DELQR &3 &HUGHLUD / /LQFRSDQ 1 ϯϯϱ
&ROLVWLQUHVLVWDQW mcr-1SRVLWLYH Escherichia coli RQ SXEOLF EHDFKHV DQ
ϯϯϲ
LQIHFWLRXV WKUHDW HPHUJLQJ LQ UHFUHDWLRQDO ZDWHUV $QWLPLFURE $JHQWV &KHPRWKHU
ϯϯϳ
H ϯϯϴ
5RVVL)*LUDUGHOOR50RUDLV&&XU\$30DUWLQV/)GD6LOYD$0$EGDOD( ϯϯϵ
6HWXEDO -& GD 6LOYD 'XDUWH $- 3ODVPLGPHGLDWHGmcr-1 LQ FDUEDSHQHP ϯϰϬ
VXVFHSWLEOHEscherichia coli67FDXVLQJDEORRGLQIHFWLRQDQXQQRWLFHDEOHVSUHDG
ϯϰϭ
RIFROLVWLQUHVLVWDQFHLQ%UD]LO"&OLQLFV6DR3DXOR
ϯϰϮ
5RFKD ,9 $QGUDGH &$'1 &DPSRV 7/ 5H]HQGH $0 /HDO 1& 9LGDO &)/ ϯϰϯ
;DYLHU '( &LSURIOR[DFLQUHVLVWDQW DQG H[WHQGHGVSHFWUXP ȕODFWDPDVH ϯϰϰ
SURGXFLQJ Escherichia coli 67 VWUDLQ FDUU\LQJ WKHmcr-1 JHQH DVVRFLDWHG ZLWK
ϯϰϱ EORRGVWUHDPLQIHFWLRQ,QW-$QWLPLFURE$JHQWV ϯϰϲ &RQFHLomR1HWR2&$LUHV&$03HUHLUD1)GD6LOYD/+-3LFmR5&6LTXHLUD ϯϰϳ %1$OEDQR50$VHQVL0'&DUYDOKR$VVHI$3''HWHFWLRQRIWKHSODVPLG ϯϰϴ
http://doc.rero.ch
PHGLDWHGmcr-1JHQHLQFOLQLFDO.3&SURGXFLQJEscherichia coliLVRODWHVLQ%UD]LO ϯϰϵ ,QW-$QWLPLFURE$JHQWV ϯϱϬ $LUHV&$0GD&RQFHLomR1HWR2&7DYDUHV(2OLYHLUD75'LDV&)0RQWH]]L ϯϱϭ /)3LFmR5&$OEDQR50$VHQVL0'&DUYDOKR$VVHI$3'(PHUJHQFHRI ϯϱϮ WKHSODVPLGPHGLDWHGmcr-1JHQHLQFOLQLFDO.3&SURGXFLQJKlebsiella pneumoniae ϯϱϯ 6HTXHQFH7\SHLQ%UD]LO$QWLPLFURE$JHQWV&KHPRWKHU ϯϱϰ
'DOPROLQ 79 0DUWLQV $) =DYDVFNL $3 GH /LPD0RUDOHV ' %DUWK $/ ϯϱϱ
$FTXLVLWLRQ RI WKHmcr-1 JHQH E\ D KLJKULVN FORQH RI .3&SURGXFLQJ Klebsiella
ϯϱϲ
pneumoniae67&&%UD]LO'LDJQ0LFURELRO,QIHFW'LV
ϯϱϳ
(VSRVLWR ) )HUQDQGHV 05 /RSHV 5 0XxR] 0 6DELQR &3 &XQKD 03 6LOYD ϯϱϴ
.& &D\{ 5 0DUWLQV :0%6 0RUHQR $0 .Q|EO 7 *DOHV $& /LQFRSDQ 1 ϯϱϵ 'HWHFWLRQRIFROLVWLQUHVLVWDQW0&5SRVLWLYHEscherichia colLE\XVHRIDVVD\V ϯϲϬ EDVHGRQLQKLELWLRQE\('7$DQG=HWDSRWHQWLDO-&OLQ0LFURELRO ϯϲϭ 3XOVV66HPPOHU73UHQJHU%HUQLQJKRII(%DXHUIHLQG5(ZHUV&)LUVW ϯϲϮ
UHSRUWRIDQEscherichia coli VWUDLQIURPVZLQHFDUU\LQJDQ2;$FDUEDSHQHPDVH
ϯϲϯ
DQGWKHFROLVWLQUHVLVWDQFHGHWHUPLQDQW0&5,QW-$QWLPLFURE$JHQWV
ϯϲϰ
3RLUHO / /DUWLJXH 0) 'HFRXVVHU -: 1RUGPDQQ 3 ,6Ecp1BPHGLDWHG ϯϲϱ
WUDQVSRVLWLRQRIbla&7;0LQEscherichia coli$QWLPLFURE$JHQWV&KHPRWKHU
ϯϲϲ ϯϲϳ 3RLUHO//DUSLQ<'RELDV-6WHSKDQ5'HFRXVVHU-:0DGHF-<1RUGPDQQ ϯϲϴ 35DSLG3RO\P\[LQ13WHVWIRUWKHGHWHFWLRQRISRO\P\[LQUHVLVWDQFHPHGLDWHG ϯϲϵ E\WKHmcr-1/mcr-2 JHQHV'LDJQ0LFURELRO,QIHFW'LV ϯϳϬ
&OLQLFDO DQG /DERUDWRU\ 6WDQGDUGV ,QVWLWXWH 3HUIRUPDQFH VWDQGDUGV IRU ϯϳϭ
DQWLPLFURELDO VXVFHSWLELOLW\ WHVWLQJ WK LQIRUPDWLRQDO VXSSOHPHQW 0±6
ϯϳϮ
:D\QH3$7KH,QVWLWXWH
ϯϳϯ
-RHQVHQ .* 7HW]VFKQHU $0 ,JXFKL $ $DUHVWUXS )0 6FKHXW] ) 5DSLG ϯϳϰ
DQG HDV\ LQ VLOLFR VHURW\SLQJ RI Escherichia coli XVLQJ ZKROH JHQRPH VHTXHQFLQJ
ϯϳϱ
:*6GDWD-&OLQ0LFURELRO
ϯϳϲ
&DUDWWROL$=DQNDUL(*DUFtD)HUQiQGH]$9ROGE\/DUVHQ0/XQG29LOOD/ ϯϳϳ
0¡OOHU $DUHVWUXS ) +DVPDQ + ,Q VLOLFR GHWHFWLRQ DQG W\SLQJ RI SODVPLGV ϯϳϴ
XVLQJ 3ODVPLG)LQGHU DQG SODVPLG PXOWLORFXV VHTXHQFH W\SLQJ $QWLPLFURE $JHQWV
ϯϳϵ
&KHPRWKHU
ϯϴϬ
=DQNDUL ( +DVPDQ + &RVHQWLQR 6 9HVWHUJDDUG 0 5DVPXVVHQ 6 /XQG 2 ϯϴϭ $DUHVWUXS)0/DUVHQ09,GHQWLILFDWLRQRIDFTXLUHGDQWLPLFURELDOUHVLVWDQFH ϯϴϮ JHQHV-$QWLPLFURE&KHPRWKHU ϯϴϯ
&OHUPRQW 2 %RQDFRUVL 6 %LQJHQ ( 5DSLG DQG VLPSOH GHWHUPLQDWLRQ RI WKH ϯϴϰ
Escherichia coliSK\ORJHQHWLFJURXS$SSO(QYLURQ0LFURELRO
ϯϴϱ
*RX\ 0 *XLQGRQ 6 *DVFXHO 2 6HD9LHZ YHUVLRQ $ PXOWLSODWIRUP ϯϴϲ
JUDSKLFDO XVHU LQWHUIDFH IRU VHTXHQFH DOLJQPHQW DQG SK\ORJHQHWLF WUHH EXLOGLQJ 0RO
ϯϴϳ
%LRO(YRO
ϯϴϴ
.LHVHU 7 )DFWRUV DIIHFWLQJ WKH LVRODWLRQ RI &&& '1$ IURP Streptomyces ϯϴϵ
lividansDQGEscherichia coli3ODVPLG
ϯϵϬ
&DUDWWROL $ %HUWLQL $ 9LOOD / )DOER 9 +RSNLQV ./ 7KUHOIDOO (- ϯϵϭ
,GHQWLILFDWLRQ RI SODVPLGV E\ 3&5EDVHG UHSOLFRQ W\SLQJ - 0LFURELRO 0HWKRGV
ϯϵϮ
ϯϵϯ
%UHWRQ $ 1RYLNRY $ 0DUWLQ 5 7LVVLHUHV 3 &DURII 0 6WUXFWXUDO DQG ϯϵϰ
ELRORJLFDO FKDUDFWHULVWLFV RI GLIIHUHQW IRUPV RI V. filiformis OLSLG $ XVH RI 06 WR
ϯϵϱ KLJKOLJKWVWUXFWXUDOGLVFUHSDQFLHV-/LSLG5HV ϯϵϲ ϯϵϳ
http://doc.rero.ch
)LJXUH/HJHQGV ϯϵϴ
)LJXUH 3K\ORJHQHWLF WUHH REWDLQHG IRU DOO WKH LGHQWLILHG 0&5OLNH HQ]\PHV LQFOXGLQJ DOO
ϯϵϵ
0&5 YDULDQWV E\ GLVWDQFH PHWKRG XVLQJ 1HLJKERU-RLQLQJ DOJRU\WKP 6HD9LHZ YHUVLRQ
ϰϬϬ
VRIWZDUH %UDQFK OHQJWKV DUH GUDZQ WR VFDOH DQG DUH SURSRUWLRQDO WR WKH QXPEHU RI DPLQR
ϰϬϭ
DFLGVVXEVWLWXWLRQVZLWKERRWVWUDSUHSOLFDWLRQV7KHGLVWDQFHDORQJWKHYHUWLFDOD[LVKDVQR
ϰϬϮ
VLJQLILFDQFH 3HUFHQWDJH RI DPLQR DFLGV LGHQWLW\ VKDUHG EHWZHHQ WKH 0&5 YDULDQW DQG
ϰϬϯ WKHRWKHU0&5OLNHHQ]\PHVLVLQGLFDWHGLQEUDFNHWV ϰϬϰ ϰϬϱ )LJXUH0DVVVSHFWURPHWU\DQDO\VLVRIOLSLG$IURPVWUDLQE. coli-$LWVWUDQVFRQMXJDQW ϰϬϲ
FDUU\LQJ WKH mcr-1 JHQH % DQG WKHFOLQLFDOLVRODWH , H[SUHVVLQJWKH mcr-3.12 JHQH &
ϰϬϳ 7KHDGGLWLRQRID3(W1JURXSLVLQGLFDWHGE\DEODFNDUURZ ϰϬϴ ϰϬϵ )LJXUH3URSRVHGPRGHORIWKHFKURQRORJ\RIWKHDFTXLVLWLRQRIWKHmcr-3.12JHQHLQWRWKH ϰϭϬ
,QF$& SODVPLG. 7KH JHQHV eamA DQG dgkA HQFRGH IRU D PHWDEROLWH WUDQVSRUWHU DQG D
ϰϭϭ
GLDF\OJO\FHURONLQDVHUHVSHFWLYHO\ intADQGintBUHSUHVHQWSXWDWLYHLQWHJUDVHVĮȕDQGȖDUH
ϰϭϮ
WKH 25) HQFRGLQJ IRU D UHYHUVH WUDQVFULSWDVH D WUDQVFULSWLRQDO UHJXODWRU DQG D GLJXDQ\ODWH
ϰϭϯ
F\FODVH UHVSHFWLYHO\ į FRUUHVSRQGV WR WKH 25) HQFRGLQJ IRU D '1$ PHWK\OWUDQVIHUDVH
ϰϭϰ ORFDWHGRQWRWKH,QF$&SODVPLGEDFNERQH ϰϭϱ ϰϭϲ ϰϭϳ ϰϭϴ
http://doc.rero.ch
MCR-3.6 (97%) MCR-3.5 (97%) MCR-3.8 (97%) MCR-3.12 MCR-3.4 (99%) MCR-3.9 (98%) MCR-3.10 (98%) MCR-3.1 (97%) MCR-3.7 (97%) MCR-3.2 (97%) MCR-3.3 (97%) MCR-4 (48%) MCR-5 (35%) MCR-1 (36%) MCR-2 (35%) MCR-3.11 (97%) 0.05
BioNJ 511 sites Poisson 500 repl.
E. coli J53 E. coli J53 + mcr-1 E. coli I112 A B C PEtN m/z m/z m/z 1797,5 1921,8 1921,1 1797,9 PEtN 1798
http://doc.rero.ch
ES ,QF$&EDFNERQH ,666 mcr-3 ,65 eam$ mcr-3 mcr-3 mcr-3 ,666 ,666 ¨eam$ dgk$ ¨,65 ,64
intA orf intB
*&
,64
,64
intA orf intB
¨,65 dgk$ ,630 ,630 ,630 ,630 į