Moraxella
VSHFLHVDVSRWHQWLDOVRXUFHVRI0&5OLNH
SRO\P\[LQUHVLVWDQFHGHWHUPLQDQWV
1LFRODV.LHIIHU3DWULFH1RUGPDQQDQG/DXUHQW3RLUHO
Emerging Antibiotic Resistance, Medical and Molecular Microbiology Unit, Department of
Medicine, University of Fribourg, Fribourg1; INSERM European Unit (IAME, France),
University of Fribourg, Fribourg, Switzerland2; Swiss National Reference Center for Emerging
Antibiotic Resistance (NARA), Fribourg, Switzerland3; Insitute for Microbiology, University of
Lausanne and University Hospital Centre, Lausanne, Switzerland4
&RUUHVSRQGLQJ DXWKRU 0DLOLQJ DGGUHVV 0HGLFDO DQG 0ROHFXODU 0LFURELRORJ\
8QLWီ'HSDUWPHQW RI 0HGLFLQHီ)DFXOW\ RI 6FLHQFHီ8QLYHUVLW\ RI )ULERXUJီUXH $OEHUW
*RFNHO ီ&+ )ULERXUJ 6ZLW]HUODQG 3KRQH (PDLO
ODXUHQWSRLUHO#XQLIUFK 5XQQLQJWLWOH3RWHQWLDORULJLQRIWKH0&5OLNHGHWHUPLQDQWV .H\ZRUGVSRO\P\[LQSODVPLGUHVLVWDQFHUHVHUYRLUDQLPDO0&5
http://doc.rero.ch
Published in "Antimicrobial Agents and Chemotherapy doi: 10.1128/AAC.00129-17,
2017" which should be cited to refer to this work.
3ODVPLGPHGLDWHG UHVLVWDQFH WR SRO\P\[LQV PHGLDWHG E\ WKH 0&5
GHWHUPLQDQWVKDVEHHQUHFHQWO\UHSRUWHGLQEnterobacteriaceae ZRUOGZLGH8VLQJ
3&5EDVHG DQG FORQLQJ VWUDWHJLHV D VHULHV RI Moraxella VSHFLHV ZHUH VFUHHQHG
IRU mcrOLNH JHQHV Moraxella VSS WKDW DUH PDLQO\ DQLPDO SDWKRJHQV EXW PD\
DOVR EH KXPDQ SDWKRJHQV ZHUH LGHQWLILHG DV SRWHQWLDO UHVHUYRLUV RI mcrOLNH
JHQHV
5HVLVWDQFH WR SRO\P\[LQV LQ Enterobacteriaceae UHVXOWV PRVWO\ IURP
FKURPRVRPDOPXWDWLRQVLQJHQHVLQYROYHGLQPRGLILFDWLRQRIWKHOLSRSRO\VDFFKDULGH
/36,QSDUWLFXODULWKDVEHHQVKRZQWKDWPXWDWLRQVWUXQFDWLRQVRULQVHUWLRQV
LQWR JHQHV HQFRGLQJ /36PRGLI\LQJ HQ]\PHV PD\ EH UHVSRQVLEOH IRU DFTXLUHG
UHVLVWDQFH WR SRO\P\[LQV LQ Enterobacteriaceae Acinetobacter baumannii
DQG Pseudomonas aeruginosa +RZHYHU WKH SODVPLGPHGLDWHG SRO\P\[LQ
UHVLVWDQFHGHWHUPLQDQW0&5KDVEHHQUHSRUWHGUHFHQWO\IURPEscherichia coliDQG
Klebsiella pneumoniae LVRODWHV UHFRYHUHG IURP KXPDQV DQG DQLPDOV LQ &KLQD
7KHQ WKH mcr-1 JHQH KDV EHHQ UHSRUWHG ZRUOGZLGH LQ RWKHU HQWHUREDFWHULDO JHQXV
LQFOXGLQJ Enterobacter Salmonella DQG Shigella ,W KDV EHHQ LGHQWLILHG LQ
(QWHUREDFWHULDFHDH LVRODWHG IURP YDULRXV DQLPDO VSHFLHV LQFOXGLQJ FDWWOH FKLFNHQ
DQGSLJVDQGIURPULYHUVDPSOHVDQGYHJHWDEOHV
0&5LVDDPLQRDFLGORQJSKRVSKRHWKDQRODPLQHWUDQVIHUDVHWKDWDGGV
SKRVSKRHWKDQRODPLQH WR WKH OLSLG $ PRLHW\ RI WKH /36 OHDGLQJ WR D PRUH FDWLRQLF
/36VWUXFWXUHDQGFRQVHTXHQWO\WRUHVLVWDQFHWRSRO\P\[LQV5HFHQWO\WKH0&5
SURWHLQ DPLQRDFLG ORQJ DPLQRDFLG LGHQWLW\ ZLWK 0&5 KDV EHHQ
LGHQWLILHGLQ%HOJLXPIURPFROLVWLQUHVLVWDQWE. coli$GGLWLRQDOO\WKH0&5
DQG 0&5 YDULDQWV ERWK H[KLELWLQJ VLQJOH DPLQR DFLG VXEVWLWXWLRQV FRPSDUHG WR
0&5 KDYH EHHQ LGHQWLILHG LQ ,WDO\ DQG &KLQD UHVSHFWLYHO\ 6HYHUDO
SODVPLG W\SHV FDUU\LQJWKH mcr-1 JHQH KDYHEHHQ LGHQWLILHGEHORQJLQJ HLWKHU WR WKH
,QF,,QF+,RU,QF;LQFRPSDWLELOLW\JURXSV,QDGGLWLRQERWKWKHmcr-1.2DQGmcr-2 JHQHVKDYHDOVREHHQLGHQWLILHGRQWR,QF;SODVPLGVFDIIROGV
,WLVKLJKO\VXJJHVWHGWKDWDQLPDOVWUHDWHGZLWKSRO\P\[LQVPLJKWUHSUHVHQWD
VLJQLILFDQWUHVHUYRLURISRO\P\[LQUHVLVWDQW*UDPQHJDWLYHLVRODWHVDQGLQSDUWLFXODU
RI 0&5SURGXFLQJ LVRODWHV ,Q IDFW SRO\P\[LQV DUH KHDYLO\ XVHG LQ YHWHULQDU\
PHGLFLQH ZLWK 6SDLQ DQG ,WDO\ EHLQJ WKH FRXQWU\ ZLWK WKH KLJKHVW FRQVRPSWLRQ IRU
IRRGSURGXFLQJ DQLPDOV LQ (XURSH 7KH IUHTXHQW LGHQWLILFDWLRQ RI 0&5
SURGXFLQJ LVRODWHV IURP DQLPDOV IXUWKHU HQKDQFHV WKH K\SRWKHVLV WKDW WKH DQLPDO
ZRUOGPLJKWEHWKHKRWVSRWRIWKLVUHVLVWDQFHGHWHUPLQDQW
,QWKHLQLWLDOSXEOLVKHGZRUNVUHSRUWLQJ0&5DQG0&5UHVSHFWLYHO\
D FHUWDLQ GHJUHH RI VHTXHQFH LGHQWLW\ EHWZHHQ WKH mcr-1 DQG mcr-2 JHQHV DQG
VRPH LQWULQVLF FKURPRVRPDO JHQHV IURP Moraxella VSHFLHV ZDV KLJKOLJKWHG 2XU
K\SRWKHVLV ZDV WKDW WKRVH ODWWHU PLJKW EH FDQGLGDWHV WR EH FRQVLGHUHG DV SRWHQWLDO
SURJHQLWRUVRIGLIIHUHQWmcrOLNHJHQHV
1RW RQO\ VHTXHQFH DQDO\VHV RI mcrOLNH JHQHV ZDV FRQVLGHUHG EXW DOVR WKH
IROORZLQJ IHDWXUHV ILUVW WKH mcr-1 JHQH LV YHU\ RIWHQ DVVRFLDWHG WR WKH LQVHUWLRQ
VHTXHQFH,6,6Apl1WKDW PLJKWEHUHVSRQVLEOHIRULWV PRELOL]DWLRQ7KDW,6KDV
EHHQ RULJLQDOO\ LGHQWLILHG LQ Actinobacillus pleuropneumoniae EHORQJLQJ WR WKH
Pasteurellaceae IDPLO\7KDWVSHFLHVLVUHVSRQVLEOHIRUSRUFLQHSOHXURSQHXPRQLD
DQGLVZLGHO\IRXQGLQSLJV6HFRQGPDQ\mcr-1-SRVLWLYHLVRODWHVLGHQWLILHGFR
KDUERUUHVLVWDQFHGHWHUPLQDQWVFRUUHVSRQGLQJWRVSHFLILFDQWLELRWLFVXVHGLQYHWHULQDU\
PHGLFLQH VXFK DV WKH floR JHQH HQFRGLQJ UHVLVWDQFH WR IORUIHQLFRO 0DQ\
SXEOLVKHG VWXGLHV VKRZHG WKH ZLGH GLVVHPLQDWLRQ RI mcr-1SRVLWLYH E. coli LVRODWHV
PRVWO\LQDQLPDOV)LQDOO\WKH,QF;W\SHSODVPLGVWKDWDUHRIWHQLGHQWLILHG
DVVXSSRUWVRIWKHmcr-1JHQHKDYHEHHQLGHQWLILHGLQHQWHUREDFWHULDOVWUDLQVUHFRYHUHG
IURPSLJVUHJDUGOHVVRIWKHSUHVHQFHRImcr-1
2XUIRFXVZDVWKHUHIRUH PDGHRQVSHFLHVEHORQJLQJWRWKHMoraxellaJHQXV
Moraxella VSHFLHV DUH *UDPQHJDWLYH EDFWHULD EHLQJ FRPPHQVDOV RU SDWKRJHQV RI
DQLPDOVFDWWOHVKHHSFDWVGRJVUDEELWVDQGSLJVZLWKMoraxella catarrhalis EHLQJ
DPXFRVDOFRPPHQVDORUSDWKRJHQIRUKXPDQV,QVLOLFRDQDO\VLVRYHU*HQEDQN
GDWDEDVHV UHYHDOHG WKDW D JHQH HQFRGLQJ D SXWDWLYH 0&5OLNH SURWHLQ ZDV ORFDWHG
RQWRWKHFKURPRVRPHRIWKHM. catarrhalisJHQRPHVKDULQJDQGDPLQRDFLG
LGHQWLW\ZLWK0&5DQG0&5UHVSHFWLYHO\
*HQRPLF'1$VZHUHH[WUDFWHGIURPDVHULHVRIVWUDLQVEHORQJLQJWRGLIIHUHQW
Moraxella VSHFLHV FRUUHVSRQGLQJ WR Moraxella lincolnii &,3, Moraxella
bovoculi &,37 Moraxella porci &,37 Moraxella lacunata
&,3 Moraxella saccharolytica &,3 DQG &,3 Moraxella bovis
&,37 DQG &,3 Moraxella equi &,37 Moraxella canis
&,37 Moraxella caprae &,37 Moraxella boevrei &,37
Moraxella nonliquefaciens &,3 Moraxella laennata &,3 DQG
Moraxella osloensis &,3 3&5EDVHG H[SHULPHQWV ZHUH SHUIRUPHG E\ XVLQJ
VSHFLILFDQGLQWHUQDOSULPHUVIRUERWKWKHmcr-1DQGmcr-2 JHQHVQDPHO\0&5
)RU ¶7$7&*&7$7*7*&7$$$*&&¶ DQG 0&55HY ¶
7&77**7$777**&**7$7&¶ 7KH DQQHDOLQJ WHPSHUDWXUH ZDV GHFUHDVHG WR
& LQ RUGHU WR DOORZ WKH DPSOLILFDWLRQ RI '1$ WDUJHWV WKDW PLJKW EH QRW IXOO\
KRPRORJRXV WR WKH mcr JHQHV )RU WKRVH 3&5 JLYLQJ SRVLWLYH UHVXOWV RQO\ LQWHUQDO
DPSOLILFDWLRQVRIWKHmcr-OLNHJHQHVZHUHREWDLQHG,QRUGHUWRJDLQNQRZOHGJHDERXW
WKHHQWLUHmcrOLNHJHQHVDQLQYHUVH3&5VWUDWHJ\ZDVXVHGDVUHSRUWHG%ULHIO\
RXWZDUG SULPHUV ZHUH GHVLJQHG IURP WKH LQWHUQDO VHTXHQFHG IUDJPHQW LQ RUGHU WR
DPSOLI\DGMDFHQW'1$VHTXHQFHV7HPSODWHVFRUUHVSRQGHGWRJHQRPLF'1$VRIWKH
VWUDLQVWKDWKDGEHHQSUHYLRXVO\GLJHVWHGZLWKGLIIHUHQWUHVWULFWLRQHQ]\PHV%DP+,
+LQG,,,(FR5,DQGVHOIFLUFXODUL]HGXVLQJ'1$7OLJDVH
3&5DVVD\VZLWKLQWHUQDOmcr-1-VSHFLILFJHQHVJDYHSRVLWLYHUHVXOWVIRUWKUHH
VWUDLQV FRUUHVSRQGLQJ WR M. lincolnii, M. porci DQG M. osloensis 6XEVHTXHQWO\ WKH
HQWLUH mcrOLNH JHQHV ZHUH LGHQWLILHG XVLQJ WKH LQYHUVH3&5 VWUDWHJ\ 7KH
FRUUHVSRQGLQJSURWHLQVUHVSHFWLYHO\QDPHG0&5/,1 IRUM. lincolnii0&5325 IRU
M. porci DQG 0&526/ IRU M. osloensis VKDUHG VLJQLILFDQW DPLQR DFLG LGHQWLWLHV
ZLWK 0&5 DQG 0&5 UDQJLQJ IURP WR UHVSHFWLYHO\ 7DEOH
1RWHZRUWK\ WKH PRVW FORVHO\UHODWHG YDULDQW FRPSDUHG WR 0&5 ZDV LGHQWLILHG LQ
M. porci ZKLOH WKH PRVW FORVHO\UHODWHG YDULDQW RI 0&5 ZDV LGHQWLILHG LQ M.
osloensis. 7KRVH LGHQWLWLHV ZHUH DOVR FDOFXODWHG IRU WKH FRUUHVSRQGLQJ JHQHV DW WKH
QXFOHRWLGHOHYHOVDQGIXUWKHUFRQILUPHGWKHVLJQLILFDQWGHJUHHRIVLPLODULW\ZLWKmcr-OLNHJHQHVVKDULQJFDLGHQWLW\ZLWKWKHmcr-1DQGmcr-2JHQHV7DEOH.
,QWHUHVWLQJO\ ZKHQ FRQVLGHULQJ WKH UHFHQWO\ GHWHUPLQHG VWUXFWXUH RI WKH
FDWDO\WLF GRPDLQ RI 0&5 DOO 0&5OLNH SURWHLQV LGHQWLILHG LQ WKLV VWXG\
SRVVHVVWKH VL[F\VWHLQHUHVLGXHVIRUPLQJ WKHWKUHHGLVXOILGHEULGJHVRI WKH FDWDO\WLF
GRPDLQ)LJXUH7KHWKUHRQLQHUHVLGXHVKRZQWREHWKHFDWDO\WLFQXFOHRSKLOH
ZDVDOVRFRQVHUYHGLQDOOWKRVHSURWHLQV$SK\ORJHQHWLFWUHHZDVHODERUDWHGZLWK
QXFOHRWLGH VHTXHQFHV RI WKH JHQHV HQFRGLQJ WKH GLIIHUHQW 0&5OLNH HQ]\PHV DORQJ
ZLWK RWKHU SXWDWLYH SKRVSKRHWKDQRODPLQH WUDQVIHUDVHSURWHLQV ,QWHUHVWLQJO\ 0&5
DQG 0&5 VLJQLILFDQWO\ FOXVWHUHG ZLWK WKRVH QHZO\LGHQWLILHG 0&5OLNH
GHWHUPLQDQWV IURP Moraxella VSS )LJXUH SDQHO $ 1RWHZRUWK\ WKH
SKRVSKRHWKDQRODPLQH WUDQVIHUDVH IURP Enhydrobacter aerosaccus DOVR FOXVWHUHG LQ
WKDWJURXS7KLVUHVXOWLVLQDFFRUGDQFHZLWKWKHUHFHQWWD[RQRPLFUHFODVVLILFDWLRQRI
WKDW VSHFLHV LQWR WKH IDPLO\ 0RUD[HOODFHDH ,Q SDUDOOHO WKH SK\ORJHQHWLF WUHH
HODERUDWHGZLWKVHTXHQFHVRIWKH651$JHQHVHTXHQFHVRIWKRVHGLIIHUHQWEDFWHULDO
VSHFLHVFRQILUPHGWKDWIHDWXUH)LJXUHSDQHO%
:KHQ DQDO\]LQJ WKH QXFOHRWLGH VHTXHQFHV ORFDWHG GRZVWUHDP RI mcrOLNH
JHQHV WKH VLJQLILFDQW LGHQWLW\ EHWZHHQ WKH PRELOL]HG IUDJPHQW HQFRPSDVVLQJ HLWKHU
mcr-1 RU mcr-2 DQG WKRVH LGHQWLILHG LQ WKH FKURPRVRPH RI WKH Moraxella VSHFLHV
ZHUHHYLGHQFHG,QGHHGKRPRORJXHVRIWKHDPLQRDFLGORQJRSHQUHDGLQJIUDPH
SUHYLRXVO\ LGHQWLILHG GRZQVWUHDP RI mcr-1 ZHUH DOVR LGHQWLILHG GRZQVWUHDP RI WKH
mcrOLNH JHQHV VKDULQJ FD DPLQR DFLG LGHQWLW\ 7DEOH 7KLV SURWHLQ
HQFRGHVDSXWDWLYH3$3PHPEUDQHDVVRFLDWHGOLSLGSKRVSKDWDVH $3&5EDVHGVFUHHQLQJRIWKHGLIIHUHQWMoraxella VWUDLQVIRUWKHSUHVHQFHRI WKHUHSOLFDVHJHQHRI,QF;SODVPLGVZDVSHUIRUPHGZLWKSULPHUV;)ZDQG;5Y DVSXEOLVKHG,QWHUHVWLQJO\DSRVLWLYHVLJQDOZDVREWDLQHGXVLQJWKHJHQRPLF'1$ RIM. lacunataWKDWZDVIXUWKHUFRQILUPHGWREHLGHQWLFDOWRWKH,QF;UHSOLFDVH JHQH LGHQWLILHG LQ DVVRFLDWLRQ ZLWK WKH mcr-1 JHQH WKHUHIRUH VKRZLQJ WKDW M. lacunata PD\EHDOVRDUHVHUYRLURI,QF;W\SHSODVPLGV
http://doc.rero.ch
6LQFHWKHmcr-1JHQHLVPRVWRIWHQDVVRFLDWHGWRWKH,6Apl1HOHPHQWD3&5
VSHFLILF IRU WKDW ,6 HOHPHQW ZDV SHUIRUPHG ZLWK DOO Moraxella VWUDLQV $ SRVLWLYH
VLJQDO ZDV IRXQG ZLWK WKH M. porcii VWUDLQ DQG VHTXHQFLQJ FRQILUPHG D SHUIHFW
LGHQWLW\ ZLWK ,6Apl1 1RWLFHDEO\ ,6Apl1 ZDV QRW ORFDWHG XSVWUHDP RI WKH mcr-por
JHQH E\ FRQWUDVW WR ZKDW LV REVHUYHG IRU ERWK mcr-1 DQG mcr-2 JHQHV. 7KLV UHVXOW
KLJKOLJKWVWKDWVRPHMoraxellaVSHFLHVPD\SRVVHVVLQWKHLUJHQRPHERWKWKHSXWDWLYH
UHVLVWDQFH JHQH DORQJ ZLWK WKH JHQHWLF WRRO OLNHO\ LQYROYHG LQ WKHLU PRELOL]DWLRQ
WKHUHIRUH UHLQIRUFLQJ WKH SRVVLELOLW\ RI RFFXUUHQFH RI VXFK PRELOL]DWLRQ SURFHVV
1RWHZRUWK\ZHUHFHQWO\GHPRQVWUDWHGWKDW,6Apl1HOHPHQWVFRXOGPRELOL]HWKHmcr-1
JHQHE\WUDQVSRVLWLRQPDQXVFULSWLQUHYLVLRQ
7KH GLIIHUHQW mcrOLNH JHQHV ZHUH FORQHG LQ WKH VDPH /DUDELQRVH LQGXFLEOH
YHFWRU S%$'E 5HFRPELQDQW SODVPLGV ZHUH H[SUHVVHG LQWR E. coli 723 E\
DGGLQJ/DUDELQRVHDQG0,&YDOXHVRIFROLVWLQZHUHGHWHUPLQHGE\EURWKGLOXWLRQ
PHWKRGV DV UHFRPPHQGHG E\ &/6, 3URGXFWLRQ RI 0&5/,1 DQG 0&5325
FRQIHUUHGRUIROGLQFUHDVHG0,&YDOXHVRIFROLVWLQLQE. coli IURPWRRU
JPO DQG KLJKHU IROG ZDV WKH 0,& LQFUHDVH FRQIHUUHG E\ 0&526/
JPO 1RQHWKHOHVV XVLQJ WKH H[DFW VDPH EDFNJURXQG WKH KLJKHVW 0,& YDOXH RI
FROLVWLQ ZDV DFKLHYHG ZLWK 0&5 WKXV KLJKOLJKWLQJ WKDW 0&5 ZDV WKH PRVW
HIILFLHQWHQ]\PHWRFRQIHUDFTXLUHGUHVLVWDQFHWRFROLVWLQLQE. coli. 1RWHZRUWK\ERWK
M. lincolniiDQGM. porci VKRZHGKLJK0,&YDOXHVRIFROLVWLQJPO
7KLV UHSRUW LQGLFDWHV WKDW Moraxella FRQVWLWXWH D UHVHUYRLU RI mcrOLNH JHQHV
WKDW PLJKW EH PRELOL]HG IURP WKHLU RULJLQDO KRVW WR EHFRPH DFTXLUHG UHVLVWDQW
GHWHUPLQDQWVLQFOLQLFDOO\VLJQLILFDQWVSHFLHVDVSUHYLRXVO\VKRZQIRURWKHUUHVLVWDQFH
JHQHV ZLWK Kluyvera VSS EHLQJ WKH VRXUFHV RI &7;0W\SH H[WHQGHGVSHFWUXP
ODFWDPDVH JHQHV Citrobacter freundii Morganella morganii DQG Hafnia
alveiRI$PS&W\SHODFWDPDVHJHQHVShewanellaVSSRI2;$DQG
2;$ FDUEDSHQHPDVH JHQHV DQG Shewanella algae RI WKH SODVPLG
PHGLDWHGTXLQRORQHUHVLVWDQFHGHWHUPLQDQWV4QU$
,WLVWHPSWLQJWRVSHFXODWHWKDWWKHKHDY\XVDJHRISRO\P\[LQVLQDQLPDOVPD\EHD
VHOHFWLYH IDFWRU IRU PRELOL]LQJ WKRVH QDWXUDOO\RFFXUULQJ 0&5 UHVLVWDQFH
GHWHUPLQDQWV IURP MoraxellaOLNH VSHFLHV WR (QWHUREDFWHULDFHDH ,Q DGGLWLRQ ZH
VKRZHGKHUHWKDWDOOJHQHWLFIHDWXUHVUHTXHVWHGIRUPRELOL]DWLRQRIWKHmcr-like JHQHV
EDFWHULDO SURJHQLWRU JHQHWLF WRROV DQG VHOHFWLYH SUHVXUH DUH SUHVHQW LQ Moraxella WKDWPD\EHSURYLGHUVRIIXUWKHUFOLQLFDOO\UHOHYDQWDQGSODVPLGPHGLDWHG0&5OLNH GHWHUPLQDQWVLQWKHIXWXUH7KHH[DFWVSHFLHVDFWLQJDVSURJHQLWRUVRIWKH0&5DQG 0&5HQFRGLQJJHQHVUHPDLQVKRZHYHUWREHGHWHUPLQHG )XQGLQJ 7KLVZRUNKDVEHHQIXQGHGE\WKH8QLYHUVLW\RI)ULERXUJE\JUDQWVIURPWKH
$1,:+$ (5$1(7 SURMHFW 6ZLW]HUODQG E\ WKH 2)63 %HUQ 6ZLW]HUODQG JUDQW
QDQGE\WKH1RYDUWLV)RXQGDWLRQIRUPHGLFDOELRORJLFDO5HVHDUFK
$FNQRZOHGJPHQWV
:HDUHJUDWHIXOWR&)\IHDQG7+*URVVPDQIRUWKHJLIWRISODVPLGS%$'E
5()(5(1&(6
3RLUHO / -D\RO $ 1RUGPDQQ 3 3RO\P\[LQV DQWLEDFWHULDO DFWLYLW\ VXVFHSWLELOLW\
WHVWLQJ SODVPLG DQG FKURPRVRPDOO\HQFRGHG UHVLVWDQFH PHFKDQLVPV &OLQ 0LFURELRO
5HY,QSUHVV
<X=4LQ:/LQ-)DQJ64LX-$QWLEDFWHULDOPHFKDQLVPVRISRO\P\[LQDQG
EDFWHULDOUHVLVWDQFH%LRPHG5HV,QW
-D\RO$3RLUHO/%ULQN$9LOOHJDV09<LOPD]01RUGPDQQ35HVLVWDQFH
WRFROLVWLQDVVRFLDWHGZLWKDVLQJOHDPLQRDFLGFKDQJHLQSURWHLQ3PU%DPRQJKlebsiella
pneumoniaeLVRODWHVRIZRUOGZLGHRULJLQ$QWLPLFURE$JHQWV&KHPRWKHU
3RLUHO/-D\RO$%RQWURQ69LOOHJDV092]GDPDU07XUNRJOX61RUGPDQQ3
7KH mgrB JHQH DV D NH\ WDUJHW IRU DFTXLUHG UHVLVWDQFH WR FROLVWLQ LQ Klebsiella
pneumoniae-$QWLPLFURE&KHPRWKHU
3RWURQ $ 3RLUHO / 1RUGPDQQ 3 (PHUJLQJ EURDGVSHFWUXP UHVLVWDQFH LQ
Pseudomonas aeruginosaDQGAcinetobacter baumanniiPHFKDQLVPVDQGHSLGHPLRORJ\
,QW-$QWLPLFURE$JHQWV
/LX<<:DQJ<:DOVK75<L/;=KDQJ56SHQFHU-'RL<7LDQ*'RQJ%
+XDQJ ; <X /) *X ' 5HQ + &KHQ ; /Y / +H ' =KRX + /LDQJ = /LX -+
6KHQ-(PHUJHQFHRISODVPLGPHGLDWHGFROLVWLQUHVLVWDQFHPHFKDQLVP0&5LQ
DQLPDOV DQG KXPDQ EHLQJV LQ &KLQD D PLFURELRORJLFDO DQG PROHFXODU ELRORJLFDO VWXG\
/DQFHW,QIHFW'LV
3RLUHO / .LHIIHU 1 /LDVVLQH 1 7KDQK ' 1RUGPDQQ 3 3ODVPLGPHGLDWHG
FDUEDSHQHPDQGFROLVWLQUHVLVWDQFHLQDFOLQLFDOLVRODWHRIEscherichia coli/DQFHW,QIHFW
'LV
3RLUHO/.LHIIHU1%ULQN$&RHW]H--D\RO$1RUGPDQQ3*HQHWLFIHDWXUHV
RI 0&5SURGXFLQJ FROLVWLQUHVLVWDQW Escherichia coli LVRODWHV 6RXWK $IULFD
$QWLPLFURE$JHQWV&KHPRWKHU
'RXPLWK0*RGEROH*$VKWRQ3/DUNLQ/'DOOPDQ7'D\0'D\00XOOHU
3HERG\%(OOLQJWRQ0-GH3LQQD(-RKQVRQ$3+RSNLQV./:RRGIRUG1
'HWHFWLRQ RI WKH SODVPLGPHGLDWHG mcr-1 JHQH FRQIHUULQJ FROLVWLQ UHVLVWDQFH LQ KXPDQ
DQG IRRG LVRODWHV RI Salmonella enterica DQG Escherichia coli LQ (QJODQG DQG :DOHV -
$QWLPLFURE&KHPRWKHU
0F*DQQ 3 6QHVUXG ( 0D\EDQN 5 &RUH\ % 2QJ $& &OLIIRUG 5 +LQNOH 0
:KLWPDQ 7 /HVKR ( 6FKDHFKHU .( Escherichia coli KDUERULQJ mcr-1 DQG
bla&7;0RQDQRYHO,QF)SODVPLG)LUVWUHSRUWRImcr-1LQWKH8QLWHG6WDWHV$QWLPLFURE
$JHQWV&KHPRWKHU
=HQJ .- 'RL < 3DWLO 6 +XDQJ ; 7LDQ *% (PHUJHQFH RI SODVPLGPHGLDWHG
mcr-1 JHQH LQ FROLVWLQUHVLVWDQW Enterobacter aerogenes DQG Enterobacter cloacae
$QWLPLFURE$JHQWV&KHPRWKHU
+DHQQL 0 3RLUHO / .LHIIHU 1 &KkWUH 3 6DUDV ( 0pWD\HU 9 'XPRXOLQ 5
1RUGPDQQ 3 0DGHF -< &RRFFXUUHQFH RI H[WHQGHG VSHFWUXP ȕ ODFWDPDVH DQG
0&5HQFRGLQJJHQHVRQSODVPLGV/DQFHW,QIHFW'LV
3HUUHWHQ96WUDXVV&&ROODXG$*HUEHU'&ROLVWLQUHVLVWDQFHJHQHmcr-1LQ
DYLDQSDWKRJHQLF Escherichia coli LQ 6RXWK $IULFD $QWLPLFURE $JHQWV &KHPRWKHU
2ODLWDQ $2 7KRQJPDOD\YRQJ % $NNKDYRQJ . 6RPSKDYRQJ 6 3DERULERXQH 3
.KRXQV\ 6 0RUDQG 6 5RODLQ -0 &ORQDO WUDQVPLVVLRQ RI D FROLVWLQUHVLVWDQW
Escherichia coliIURPDGRPHVWLFDWHGSLJWRDKXPDQLQ/DRV-$QWLPLFURE&KHPRWKHU
=XUIXK . 3RLUHO / 1RUGPDQQ 3 1XHVFK,QGHUELQHQ 0 +DFKOHU + 6WHSKDQ 5
2FFXUUHQFHRIWKHSODVPLGERUQHmcr-1FROLVWLQUHVLVWDQFHJHQHLQ(6%/SURGXFLQJ
Enterobacteriacae LQ ULYHU ZDWHU DQG LPSRUWHG YHJHWDEOH VDPSOHV LQ 6ZLW]HUODQG $QWLPLFURE$JHQWV&KHPRWKHU
;DYLHU %% /DPPHQV & 5XKDO 5 .XPDU6LQJK 6 %XWD\H 3 *RRVVHQV +
0DOKRWUD.XPDU6,GHQWLILFDWLRQRIDQRYHOSODVPLGPHGLDWHGFROLVWLQUHVLVWDQFH
JHQHmcr-2LQEscherichia coli%HOJLXP-XQH(XUR6XUYHLOO
'L3LODWR9$UHQD)7DVFLQL&&DQQDWHOOL$+HQULFL'H$QJHOLV/)RUWXQDWR6
*LDQL 7 0HQLFKHWWL ) 5RVVROLQL *0 mcr-1.2 D QHZ mcr YDULDQW FDUULHG RQ D
WUDQVIHUDEOH SODVPLG IURP D FROLVWLQUHVLVWDQW .3& FDUEDSHQHPDVHSURGXFLQJ Klebsiella
pneumoniaeVWUDLQRI6HTXHQFH7\SH$QWLPLFURE$JHQWV&KHPRWKHU
/X ; +X < /XR 0 =KRX + :DQJ ; 'X < /L = ;X - =KX % ;X ; .DQ %
0&5 D QHZ 0&5 YDULDQW FDUULHG E\ DQ ,QF3 SODVPLG LQ D FROLVWLQUHVLVWDQW
Salmonella entericaVHURYDU7\SKLPXULXPLVRODWHGIURPDKHDOWK\LQGLYLGXDO$QWLPLFURE
$JHQWV&KHPRWKHU,QSUHVV
3RLUHO/1RUGPDQQ3(PHUJLQJSODVPLGHQFRGHGFROLVWLQUHVLVWDQFHWKHDQLPDO
ZRUOGDVWKHFXOSULW"-$QWLPLFURE&KHPRWKHU
&DWU\%&DYDOHUL0%DSWLVWH.*UDYH.*UHLQ.+ROP$-XNHV+/LHEDQD(
/RSH]1DYDV$0DFND\'0DJLRUDNRV$30RUHQR5RPR0$0RXOLQ*0XxR]
0DGHUR & 0DWLDV )HUUHLUD 3RPED 0& 3RZHOO 0 3\|UlOl 6 5DQWDOD 0
5XåDXVNDV 0 6DQGHUV 3 7HDOH & 7KUHOIDOO (- 7|UQHNH . YDQ 'XLMNHUHQ (
7RUUHQ(GR-8VHRIFROLVWLQFRQWDLQLQJSURGXFWVZLWKLQWKH(XURSHDQ8QLRQDQG
(XURSHDQ(FRQRPLF$UHD(8(($GHYHORSPHQWRIUHVLVWDQFHLQDQLPDOVDQGSRVVLEOH
LPSDFWRQKXPDQDQGDQLPDOKHDOWK,QW-$QWLPLFURE$JHQWV
7HJHWPH\HU +( -RQHV 6& /DQJIRUG 35 %DOWHV 1 ,6Apl1 D QRYHO LQVHUWLRQ
HOHPHQW RI Actinobacillus pleuropneumoniae SUHYHQWV $S[,9EDVHG VHURORJLFDO
GHWHFWLRQRIVHURW\SHVWUDLQ$39HW0LFURELRO
5\FURIW$1*DUVLGH/+ActinobacillusVSHFLHVDQGWKHLUUROHLQDQLPDOGLVHDVH
9HW-
+X < /LX ) /LQ ,< *DR *) =KX % 'LVVHPLQDWLRQ RI WKH mcr-1 FROLVWLQ
UHVLVWDQFHJHQH/DQFHW,QIHFW'LV
/R :8 &KRZ .+ /DZ 3< 1J .< &KHXQJ << /DL (/ +R 3/ +LJKO\
FRQMXJDWLYH,QF;SODVPLGVFDUU\LQJbla&7;0LQEscherichia coliIURPKXPDQVDQGIRRG
DQLPDOV-0HG0LFURELRO
6XQ - 'HQJ + /L / &KHQ 0< )DQJ /; <DQJ 4( /LX <+ /LDR ;3
&RPSOHWH QXFOHRWLGH VHTXHQFH RI cfrFDUU\LQJ ,QF; SODVPLG S6' IURP Escherichia
coli$QWLPLFURE$JHQWV&KHPRWKHU
$HEL & Moraxella catarrhalis SDWKRJHQ RU FRPPHQVDO" $GY ([S 0HG %LRO
%HOODLV 6 3RLUHO / /pRWDUG 6 1DDV 7 1RUGPDQQ 3 *HQHWLF GLYHUVLW\ RI
FDUEDSHQHPK\GURO\]LQJPHWDOORODFWDPDVHVIURPChryseobacteriumFlavobacterium
indologenes$QWLPLFURE$JHQWV&KHPRWKHU
6WRMDQRVNL 9 6DQNDUDQ % 3UDVDG %9 3RLUHO / 1RUGPDQQ 3 3DO]NLOO 7
6WUXFWXUH RI WKH FDWDO\WLF GRPDLQ RI WKH FROLVWLQ UHVLVWDQFH HQ]\PH 0&5 %0& %LRO
.DZDPXUD < )XMLZDUD 1 1DND 7 0LWDQL $ .XERWD + 7RPLGD - 0RULWD <
+LWRPL - *HQXV Enhydrobacter 6WDOH\ HW DO VKRXOG EH UHFRJQL]HG DV D
PHPEHURIWKHIDPLO\5KRGRVSLULOODFHDHZLWKLQWKHFODVV$OSKDSURWHREDFWHULD0LFURELRO
,PPXQRO
)\IH & /H%ODQF * &ORVH % 1RUGPDQQ 3 'XPDV - *URVVPDQ 7+
(UDYDF\FOLQHLVDFWLYHDJDLQVWEDFWHULDOLVRODWHVH[SUHVVLQJWKHSRO\P\[LQUHVLVWDQFHJHQH
mcr-1$QWLPLFURE$JHQWV&KHPRWKHU
&OLQLFDO DQG /DERUDWRU\ 6WDQGDUGV ,QVWLWXWH 3HUIRUPDQFH VWDQGDUGV IRU
DQWLPLFURELDO VXVFHSWLELOLW\ WHVWLQJ WZHQW\IRXUWK LQIRUPDWLRQDOVXSSOHPHQW 06
&/6,:D\QH3$
+XPHQLXN & $UOHW * *DXWLHU 9 *ULPRQW 3 /DELD 5 3KLOLSSRQ $ %HWD
ODFWDPDVHVRIKluyvera ascorbataSUREDEOHSURJHQLWRUVRIVRPHSODVPLGHQFRGHG&7;
0W\SHV$QWLPLFURE$JHQWV&KHPRWKHU
3RLUHO / .lPSIHU 3 1RUGPDQQ 3 &KURPRVRPHHQFRGHG $PEOHU FODVV $
ODFWDPDVH RI Kluyvera georgiana D SUREDEOH SURJHQLWRU RI D VXEJURXS RI &7;0
H[WHQGHGVSHFWUXPODFWDPDVHV$QWLPLFURE$JHQWV&KHPRWKHU
3RLUHO / *XLEHUW 0 *LUOLFK ' 1DDV 7 1RUGPDQQ 3 &ORQLQJ VHTXHQFH
DQDO\VHVH[SUHVVLRQDQGGLVWULEXWLRQRIampCampRIURPMorganella morganiiFOLQLFDO
LVRODWHV$QWLPLFURE$JHQWV&KHPRWKHU
*LUOLFK ' 1DDV 7 %HOODLV 6 3RLUHO / .DULP $ 1RUGPDQQ 3 %LRFKHPLFDO
JHQHWLF FKDUDFWHUL]DWLRQ DQG UHJXODWLRQ RI H[SUHVVLRQ RI DQ $&&OLNH FKURPRVRPH
ERUQHFHSKDORVSRULQDVHIURPHafnia alvei$QWLPLFURE$JHQWV&KHPRWKHU
:X 6: 'RUQEXVFK . .URQYDOO * 1RUJUHQ 0 &KDUDFWHUL]DWLRQ DQG
QXFOHRWLGH VHTXHQFH RI D Klebsiella oxytoca FU\SWLF SODVPLG HQFRGLQJ D &0<W\SH
ODFWDPDVH FRQILUPDWLRQ WKDW WKH SODVPLGPHGLDWHG FHSKDP\FLQDVH RULJLQDWHG IURP WKH
Citrobacter freundii$PS&ODFWDPDVH$QWLPLFURE$JHQWV&KHPRWKHU
3RLUHO / +pULWLHU & 1RUGPDQQ 3 &KURPRVRPHHQFRGHG DPEOHU FODVV '
ODFWDPDVH RI Shewanella oneidensis DV D SURJHQLWRU RI FDUEDSHQHPK\GURO\]LQJ
R[DFLOOLQDVH$QWLPLFURE$JHQWV&KHPRWKHU
3RWURQ$3RLUHO/1RUGPDQQ32ULJLQRI2;$DQHPHUJLQJFDUEDSHQHP
K\GURO\]LQJR[DFLOOLQDVHDVDFKURPRVRPDOJHQHLQShewanella xiamenensis$QWLPLFURE
$JHQWV&KHPRWKHU
3RLUHO/5RGULJXH]0DUWLQH]-00DPPHUL+/LDUG$1RUGPDQQ32ULJLQ
RI SODVPLGPHGLDWHG TXLQRORQH UHVLVWDQFH GHWHUPLQDQW 4QU$ $QWLPLFURE $JHQWV
&KHPRWKHU
*RX\0*XLQGRQ6*DVFXHO26HD9LHZYHUVLRQ$PXOWLSODWIRUPJUDSKLFDO
XVHU LQWHUIDFH IRU VHTXHQFH DOLJQPHQW DQG SK\ORJHQHWLF WUHH EXLOGLQJ 0RO %LRO (YRO
/HJHQGRI)LJXUH
)LJXUH6HTXHQFHFRPSDULVRQRISODVPLGPHGLDWHG0&5OLNHGHWHUPLQDQWV7KHSODVPLG PHGLDWHG0&5DQG0&5GHWHUPLQDQWVDUHIURP0&5/,1LVIURPM. lincolnii 0&5&$7 IURP M. catarrhalis, 0&526/ IURP M. osloensis DQG 0&5325 IURP M.
porci. 'RWVDUHIRULGHQWLFDODPLQRDFLGUHVLGXHV7KHFRQVHUYHGF\VWHLQHUHVLGXHVIRUPLQJ
WKH WKUHH GLVXOILGH EULGJHV RI WKH 0&5 FDWDO\WLF GRPDLQ DUH VKDGHG 7KH WKUHRQLQH WKDWZDVVKRZQWREHWKHFDWDO\WLFQXFOHRSKLOHLQ0&5LVDOVRFRQVHUYHGDPRQJ DOOWKRVH0&5SURWHLQVDQGEUDFNHWHG
)LJXUH 3K\ORJHQHWLF WUHH REWDLQHG IRU D VHULHV RI SKRVSKRHWKDQRODPLQH WUDQVIHUDVH HQFRGLQJ JHQHV SDQHO $ DQG 6 51$ JHQHV SDQHO % E\ WKH GLVWDQFH PHWKRG XVLQJ 1HLJKERU-RLQLQJDOJRULWKP6HD9LHZYHUVLRQVRIWZDUH%UDQFKOHQJWKVDUHGUDZQWR VFDOH DQG DUH SURSRUWLRQDO WR WKH QXPEHU RI DPLQR DFLG FKDQJHV ZLWK ERRWVWUDS UHSOLFDWLRQV7KHGLVWDQFHDORQJWKHYHUWLFDOD[LVKDVQRVLJQLILFDQFH
7DEOH3HUFHQWDJHVRILGHQWLW\DWWKHDPLQRDFLGDQGQXFOHRWLGHOHYHOVEHWZHHQSODVPLG PHGLDWHG0&50&5SRO\P\[LQUHVLVWDQFHGHWHUPLQDQWVDQGWKRVHLGHQWLILHGLQWKH
GLIIHUHQWMoraxellaVSHFLHVQDPHO\M. porciiM. osloensisM. lincolniiDQGM. catarrhalis
0&5 0&5 0&5325 0&526/ 0&5/,1
0&5 0&5325 0&526/ 0&5/,1 0&5&$7
http://doc.rero.ch
7DEOH$QDO\VLVRIWKHSXWDWLYHRSHQUHDGLQJIUDPH25)UHJLRQVORFDWHGGRZQVWUHDP RIWKHmcrOLNH JHQHV 'LVWDQFHIURP *HQH 25)OHQJWK mcrVWRSFRGRQ ,GHQWLW\DPLQRDFLGV
DPLQRDFLGV QXFOHRWLGHV RI25)0&5FRYHUDJHD
mcr-1 mcr-2 D F mcr26/ mcr&$7 mcr/,1 mcr325
D7KH FRYHUDJH UHSUHVHQWV WKH SHUFHQWDJH RI WKH TXHU\ VHTXHQFH WKDW RYHUODSV WKH
VXEMHFWVHTXHQFHLH25)0&5E3XWDWLYH25)RI0&5LVWUXQFDWHGE\DFRS\RI
,61595F&RYHUDJHZDVFDOFXODWHGIURPWKHDOLJQPHQWRIWKHWUXQFDWHG25) 0&5DQG
WKHILUVWDPLQRDFLGVRI25)0&5 7KHmcr26/ mcr&$7 mcr/,1DQG mcr325JHQHV
DUHUHVSHFWLYHO\IURPM. osloensisM. catarrhalisM. lincolniiDQGM. porcii
0&5 004+769:<556963)9/9$69$9)/7$7$1/7))'.,647<3,$'1/*)9/7,$99/)* 0&5 076++6<,10*/$($0$996,,60$90 0&5/,1 06/.+147+177)7.)/.616):6.*):615+57.'/.*/'$</)0,,9449069/1+$/,$6/7 0&5&$7 06/.+147+177)7.)/.616):6.*):615+57.'/.*/'$</)0,,9449069/1<$/,$6/7 0&526/ 0691165:/$544*,1$<00*,//,$7(9)$4+,,*6/3/ 0&5325 0/1)/+3.+,1</0/,,69.499/9+/$6/7& 0&5 $0//,77//66<5<9/.39/,///,0*$976<)7'7<*79<'770/41$/47'4$(7.'//1$$),05,,*/*9/36//9$)9.9'<3 0&5 99/60/)999$1 0&5/,1 ,///9/*+77$&),/,$)$*+760.//,99/$*,&:,,*43/6)* 0&5&$7 9///9/*+77$&),/,$)$*+760.//,99/$*,&:,,*43/6)* 0&526/ 99,9/7$/)/7$7$.6$)91///9::47)3 0&5325 9/$9,*)7)/)0,09$,76667/*/9//,/./3+)$ 0&5 7:*.*/055/*/,9$6/$/,//399$)66+<$6))59+.3/56<913,03,<69*./$6,(<..$6$3.'7,<+$.'$94$7.3'05. 0&5 ,4$07:*99/9,*/)49),77776( 0&5/,1 /.96.97</9$9*,//4())79790$10617.7(01,.7$67 0&5&$7 /.$6.97</9$9*,/4())79790$10617.7(01,.7$67 0&526/ 3,.56,/47</9*99,/0.1(47$79$/4/44740796177 0&5325 1).71$)4<///*9,/6($)(7$$7/7$77674 0&5 35/99)99*(7$5$'+96)1*<(5'7)34/$.,'*971)61976&*767$<693&0)6</*$'(<'9'7$.<4(19/'7/'5/*9 0&5 4*(9/$44' 0&5/,1 $4$+0'/,/*4+*$,(.'+, 0&5&$7 $4$+0'/,/*4+*(.'+, 0&526/ *4/170$7$14,$90.'1+. 0&5325 /6.9660+/6.,'11+6+
http://doc.rero.ch
0&5 6,/:5'116'6.*90'./3.$4)$'<.6$711$,&1713<1(&5'9*0/9*/'')9$$11*.'0/,0/+40*1+*3$<) 0&5 *$7<)7<6 0&5/,1 $15$.<4163/4**7+'+.+$14,9< 0&5&$7 $15$.<4163/4**7+'+.+$14,9< 0&526/ 16715$'97570<.4$1417/1479 0&5325 19$'/<4176'/+(7$+4(,,<7$.161499< 0&5 .5<'(.)$.)739&(*1(/$.&(+46/,1$<'1$//$7''),$46,4:/47+61$<'960/<96'+*(6/*(1*9</+*031$)$3.( 0&5 4.'.($1$ 0&5/,1 '(4/7664579/.7'$$47+$7$/.. 0&5&$7 '(4/766(579/.7'$$47+$7$/.. 0&526/ .4(746'39)/.791'.<'67+4$9,<.,$ 0&5325 1$(75'''76+9/.7'.4$6+7/7$ 0&5 45693$)):7'.47*,730$7'79/7+'$,737//./)'97$'.9.'57$/+5 0&5 $6117).769*$),4 0&5/,1 /,///*$'3)$9$1637$*)61674$7$.)913/' 0&5&$7 /,///*$'3)$9$1637$*)61674$7$.)913/' 0&526/ .+$60$*+64$9361(5474*.3/),. 0&5325 .+$/$13$+$9614$35.7.$7(14$0),(
http://doc.rero.ch
DZͲϭ DZͲϮ ĂŵƉLJůŽďĂĐƚĞƌũĞũƵŶŝƉƚ ĐŝŶĞƚŽďĂĐƚĞƌďĂƵŵĂŶŶŝŝƉƚ WƐĞƵĚŽŵŽŶĂƐĂĞƌƵŐŝŶŽƐĂƉƚ sŝďƌŝŽĐŚŽůĞƌĂĞƉƚ ŶƚĞƌŽďĂĐƚĞƌĐůŽĂĐĂĞƉƚ <ůĞďƐŝĞůůĂƉŶĞƵŵŽŶŝĂĞƉƚ <ůƵLJǀĞƌĂĂƐĐŽƌďĂƚĂƉƚ ƐĐŚĞƌŝĐŚŝĂĐŽůŝƉƚ WĂƐƚĞƵƌĞůůĂŵƵůƚŽĐŝĚĂƉƚ EĞŝƐƐĞƌŝĂŐŽŶŽƌƌŚŽĞĂĞ>Ɖƚ EĞŝƐƐĞƌŝĂŵĞŶŝŶŐŝƚŝĚŝƐ>Ɖƚ DŽƌĂdžĞůůĂŽƐůŽĞŶƐŝƐDZͲK^> ŶŚLJĚƌŽďĂĐƚĞƌĂĞƌŽƐĂĐĐƵƐƉƚ DŽƌĂdžĞůůĂƉŽƌĐŝDZͲWKZ DŽƌĂdžĞůůĂůŝŶĐŽůŶŝŝDZͲ>/E DŽƌĂdžĞůůĂĐĂƚĂƌƌŚĂůŝƐDZͲd DŽƌŐĂŶĞůůĂŵŽƌŐĂŶŝŝƉƚ ŶƚĞƌŽďĂĐƚĞƌĐůŽĂĐĂĞƉƚ