809 DOI:10.13350/j.cjpb.180802 ' ()*+,-./(0 F, GH,I+J ( G,HD I JKX32 L D/ W, GMN750021) @ OR G-. 0. ], @M G-. 0 P,FG ].@, QR I ` OR FG]3OR, FG @M,?@ ST U: <= @ V Q R I ],@AWX EF1 EF2, Y NWI.], N I ] ` OR O R EF1 Z Z [ ]( 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犻狌犿 ),EF2 \ Z [ ]( 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犪犾犻狊 ); ST LR EF1(]^_R `a bc S,EF2(]^_R a a `a c bc S PCR KL EF1U:agg gele efaa,ef2u:esp ace gele efaa 9: ]72h, V 0 9 : P( ),$ P @.F] 2 3F] ] \Z[] ZZ[] V@ ] ` S, 2U:c3 \Z[];ZZ[]; S ; R378.1!"#$ A %& 1673 5234(2018)08 0809 05 [.2018Aug;13(8):809-813.] 犐狊狅犾犪狋犻狅狀, 犻犱犲狀狋犻犳犻犮犪狋犻狅狀, 犪狀犱犪狀犪犾狔狊犻狊狅犳狋犺犲犫犻狅犾狅犵犻犮犪犾犮犺犪狉犪犮狋犲狉犻狊狋犻犮狊狅犳 2 狊狋狉犪犻狀狊狅犳犈狀狋犲狉狅犮狅犮犮狌狊犳狉狅犿犮犪狋犾犲 WANG Xiao yu,ma Wei wu,zhouxue zhang ( 犓犲狔犔犪犫狅狉犪狋狅狉狔狅犳狋犺犲犕犻狀犻狊狋狉狔狅犳犈犱狌犮犪狋犻狅狀犳狅狉犘狉狅狋犲犮 狋犻狅狀犪狀犱犝狋犻犾犻狕犪狋犻狅狀狅犳犛狆犲犮犻犪犾犅犻狅犾狅犵犻犮犪犾犚犲狊狅狌狉犮犲狊犻狀犠犲狊狋犲狉狀犆犺犻狀犪, 犢犻狀犮犺狌犪狀 750021, 犆犺犻狀犪 ) 犃犫狊狋狉犪犮狋 犗犫犼犲犮狋犻狏犲狊 ThepathogenscausingthedeathofcatlefromadairyandaranchinNingxiawereisolatedand identified,andtheirbiologicalcharacteristicswereanalyzed. 犕犲狋犺狅犱狊 Theorgansofcatlewerecolectedfromadairy andacatleranchinningxia,andpathogenswereisolatedandcultured.gramstainingandautomatedbiochemicalidenti ficationwereusedtoidentifythestrains.thebiologicalcharacteristicsofthestrainswereanalyzed,includingculturing characteristics,analysisofdrugsusceptibility,virulencegenesaccordingtopcr,andpathogenicitytoanimals. 犚犲狊狌犾狋狊 TwoGram positivebacteria wereisolatedandpurified,andthey wererespectivelydesignated EF1and EF2.They formedwhite,round,smoothcoloniesonthenutrientmediumandformedblack,round,smoothcoloniesonselectiveme dium.aftergramstaining,bothstrainsappearedaspurplespheres.theef1andef2strainswereidentifiedas 犈狀狋犲狉狅 犮狅犮犮狌狊犳犪犲犮犻狌犿 and 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犪犾犻狊 bytheautomatedbiochemicalidentificationsystem withaconfidenceofgreater than99%.drugsusceptibilitytestingindicatedthattheef1strainisresistanttocephradine,gentamycin,andamikacin andthattheef2strainisresistanttocefradine,lincomycin,ciprofloxacin,oxytetracycline,gentamicin,sulfadiazine,and amikacin.drugsusceptibilitytestingindicatedthatbothstrainshavemulti drugresistance.pcrindicatedthatef1car riestheagg,gele,andefaavirulencegenesandthatef2carriestheesp,ace,gele,andefaavirulencegenes.animal pathogenicitytestingindicatedthatmiceinjectedwiththepathogenicbacteriabegantodiewithin72hours.anatomical observationsofthetwogroupsofmiceindicatedthatorgans(liver,spleen,andkidneys)wereenlargedandblackened. Infectionofthespleenwasespecialysevereinmice.Presumably,Enterococcusismorelikelytomigratetothespleen, andthebacteriaisolatedfromthediseasedorganswereconsistentwiththeinoculatedbacteria. 犆狅狀犮犾狌狊犻狅狀 Thepatho genscausingtheoutbreakinningxiawere 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犪犾犻狊 and 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犻狌犿,whichmayberelatedtothe simultaneouspresenceofmultiplevirulencegenesinthetwoisolates. 犓犲狔狑狅狉犱狊 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犪犾犻狊 ; 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犻狌犿 ;drugresistance;virulencegenes Z[]( 犈狀狋犲狉狅犮狅犮犮狌狊 ) QR ], [ ],Z[], D. 9 @ 30, \Z[] ZZ[]%@ 0,% ' U(No.31660728),E mail:zhouxuezhang@nxu.edu.cn () (1993-),,,! :Y 28 E mail:1072154976@qq.com
810 ] [1] S, 0] F] [2],D E ]. 2 ]!a H ].,$ ]T Ra Z ( ] ;. H [3] ZZ[] b 8, \Z[] [4-5] D 9ZZ[] ] B5239 >\Z[] ZZ[]+ '.,B.,Z[]c/ S / [6],Z[]. bc, +., / b S,Z[]. S 2 [7], Z[] N ] [8],% ], & ],- ] [9] ) S (FDA) R S] 23, BZ[ ] H ) % [10] > Z [ ]. / [11] Z[] 9:Z P!, $Z[].c/ S.23: 4% [12] (. ]FG@ OR= @M, S 2. < *+, 1 *+ 1.1 K 2016 5 G-. 0 P, E A] 1.2 2 L M PfizerZ [ ] (PSE Agar)= MRS (MRSBroth), R $c YUV;F ] DNA 7 5 C $( b) YUV; b $ YUV;ST ; ' $ YU V 6331PCR b, EppendorfUV ;` F] OR, [UV 1.3 N O 3 4 5 * \ Z [ ] ATCC29212 R $ YUV 20 / 20g <9W: G 2 2.1 ( 7 2.1.1 ].@ = _ 9. A] 3 Y,37 12h-18h, ] _, M] Y ^ FG YM] ^ PfizerZ[],37 12h- 18h, ] _ 2.1.2 QR IK 9.M] ;!R,QR IYK, F] _=I / a, D.M], 8 2.1.3 OR 9QR I I[ ].M] ] 0.5, B O R, ` F] OR FGOR 2.2 4 KB, 11 3 < ATCC29212 ( F G,! < = X R! * ] S T " # (EU CAST2017) [13] 2.3 89E 2.3.1 27N $%[14] 7N PCR, >? = S 1, J J ( R)&' YUV7N 71 IJ' 犘犆犚 KLMN OL 犜犪犫犾犲 1 犗犾犻犵狅狀狌犮犾犲狅狋犻犱犲狆狉犻犿犲狉狊狌狊犲犱狋狅犻犱犲狀狋犻犳狔狉犲狊犻狊狋犪狀犮犲犵犲狀犲狊 Gene(s) >? PrimerSequence(5 3 ) ; (bp) Productsize cyla TGGATGATAGTGATAGGAAGT 517 TCTACAGTAAATCTTTCGTCA gele AGTTCATGTCTATTTTCTTCAC 402 CTTCATTATTTACACGTTTG esp TTACCAAGATGGTTCTGTAGGCAC CCAAGTATACTTAGCATCTTTTGGA ace AAAGTAGAATTAGATCCACAC 320 TCTATCACATTCGGTTGCG agg CCAGTAATCAGTCCAGAAACAACC 406 TAGCTTTTTTCATTCTTGTGTTTGTT efaa CGTGAGAAAGAAATGGAGGA 499 CTACTAACACGTCACGAATG 1 / a!9 Diferentsizeduetodiferentnumberofcoreaminoacidrepeats 2.3.2 U. PCR b PCR B6 <(20μl): Mix10μl,ddH 2O8μl,DNA 1μl, 0.5μl PCR B6 >:94 5min;94 30s,52 30s,72 % 45s, 30 ;72 (% 10min PCR 1% KL 2.3.3 L>@M 9 PCR b ) R * $c YUVL>,FG>? (@M 2.4 P Q 4 920 9W:@ 10 20 +0 ( 0, 05 109:, 10 8 CFU/ml. EF1 ],0.2 ml/ ; 209: 10 8 CFU/ml. EF2 ], +0. ATCC29212,( 0 0.15 mol/lpbs, - 2 0 09:G 9:.4
811, 0/ ;A] 0 @ OR 1 ; P? @ 2 ],@AWX EF1 EF2 V0] Y NWI.] ; N I ] 2 ; 34 2.1 R S < T E U V @ ] Q R [ ],& 1 0?( 1 2) 1 犈犉 1QR(STUV,1000 ) 犉犻犵.1 犈犉 1 犵狉犪犿狊狋犪犻狀犻狀犵狉犲狊狌犾狋狊 (1000 ) 2 犈犉 2QR(STUV,1000 ) 犉犻犵.2 犈犉 2 犵狉犪犿狊狋犪犻狀犻狀犵狉犲狊狌犾狋狊 (1000 ) 2.2 )V GP L ; S 2 DOR EF1 ZZ[],EF2 \Z[ ] \Z[] ZZ[]cST AMY( ) LAC( ) AGLU(α- 2 ), S T TyrA(3 4c ),57\ 98% 3 ; WLXY? @ F].ST X S3 EF1(, a,678+, c, a, a, 9:H;_ T;( a T;( ]^_R,`a, bc S EF2(678 +, a, a, 9:H;_ T;( ]^_R, a,, a,`a, c bc S ATCC29212(,678+,`a, a, a 9:H ;_ T;(]^_R,bc T, ( a, a, c S 72 34 9 犜犪犫犾犲 2 犅犻狅犮犺犲犿犻犮犪犾犻犱犲狀狋犻犳犻犮犪狋犻狅狀狋犲狊狋狉犲狆狅狉狋 X Testname EF1 EF2 X Testname EF1 EF2 AMY - + ADH1 + + APPA - - BGAR - - LeuA - - AGAL - - AlaA - - URE - - drib + + NAG + + NOVO + + dmne + + draf - - SAC + + OPTO + + BGAL - - PIPLC - - AMAN - - CDEX + + PyrA + + ProA - - POLYB + + TyrA + - dmal + + ILATk - - MBdG + + NC6.5 + + dtre + + O129R + + AGLU - + dxyl - - PHOS - - AspA + + BGUR - - BGURr - - dgal + + dsor + + BACI + + LAC - + PUL - - dman + + ADH2s + + SAL + + :"+"SP ;"-" SP Note:"+"meanspositive;"-"meansnegative 4 IJ' DE < EF1 EF27. DNA, 6 F G PCR b,x 3 2 ATCC29212 b X ( 4) 5,EF1agg gele efaa b,ef2esp ace gele efaa b,ace cyla gele efaa b; 9 480-550bp 5 IJ' EM (@ ] PCR L> 2\Z[ ] U:. (PAI)` >?FG (,7 2 6. >? E 99.9% M DNA (DL2000) 1~6 EF1 esp agg ace cyla gele efaa b ; 7~12 EF2 agg ace cyl gel efaa b; 3 Z[\] IJ' 犘犆犚 ^_ M DL2000 molecular Markerqualitystandards 1-6 Excre mentef1electrophoresisresults 7-12 Theelectrophoresisresults inef21 esp,2 agg,3 Ace,4 cyla,5 can,6 efaa,7 esp,8 agg,9 Ace,10 cyl.ul,11 gel,12 efaa 犉犻犵.3 犃犿狆犾犻犳犻犮犪狋犻狅狀狉犲狊狌犾狋狊狅犳狋狑狅犲狀狋犲狉狅犮狅犮犮犻狏犻狉狌犾犲狀犮犲犵犲狀犲狊
812 Antibiotic 73 Z[\] `11[@ awx 9 犜犪犫犾犲 3 犜狑狅犲狀狋犲狉狅犮狅犮犮狌狊狋犲狊狋狉犲狊狌犾狋狊狅犳犲犾犲狏犲狀犪狀狋犻犫犻狅狋犻犮狊狌狊犮犲狆狋犻犫犻犾犻狋狔狋犲狊狋狊 EF1 ] (mm) EF1inhibitorydiameter X R Resultdetermination EF2 ] (mm) EF2inhibitorydiameter X R Resultdetermination ]^_R 10 R 8 R a 16 I 14 R 22 S 8 R a 20 S 8 R 678+ 26 S 26 S `a 12 R 10 R c 24 S 0 R a 20 S 20 S a 34 S 22 S bc 10 R 8 R 9:H;_ 38 S 36 S :S T(Sensitivity);I T(Intermecliary);R S(Resistance) H M DNA (DL2000) 1~6 esp agg ace cy la gele efaa b 4 "bc 犃犜犆犆 29212IJ' 犘犆犚 ^_ M DL2000molecularMarkerqualitystandards 1-6 Standard strainsatcc29212electrophoresisresults,1 esp,2 agg,3 ace,4 cy la,5 gele,6 efaa 犉犻犵.4 犛狋犪狀犱犪狉犱狊狋狉犪犻狀狊犃犜犆犆 29212 狏犻狉狌犾犲狀犮犲犵犲狀犲犪犿狆犾犻犳犻犮犪狋犻狅狀狉犲狊狌犾狋狊 6 ;? 109: @ ]Y A,24hY < 1,72h 9: 209: Y =>?@A. %@ B 72h Y9:C/ ( 109: A",$ F +09: Y=DE,> @A9 Y. 09:FG, 10 :., W ; 2 0 +0,( 09:! P I _ 10 20 P @ V 3 ],D E 2 3 ].! FGO R,X ( 5) + 0 9 : @ 5. 3] 5 \] QR(STUV,1000 ) 犉犻犵.5 犐狊狅犾犪狋犲犱犫犪犮狋犲狉犻犪犳狉狅犿狋犺犲犲狓犮狉犲犿犲狀狋犲狀狋犲狉狅犮狅犮犮狌狊犱犲犪狋犺犿犻犮犲犾犻狏犲狉犵狉犪犿狊狋犪犻狀犻狀犵狉犲狊狌犾狋狊 (1000 ) Z[] Z & ]IN#,< % ] > 20 90 <,Z[] N /. ], Z[]. bc G-. 0 0 @ 1 ],DEQR I<= OR R@A \ Z [ ] Z Z [ ] 5 = O R! 16SrDNA >? ( @ M! F,D E ` F ] OR FGORG LM,$ H 8 ( X. I Z[]. S 5./,Z[] 23J 2 S ' S<=.U: ST P, @.ZZ[](]^_ R,`a bc (]^] D D) S,\Z[] ( ] ^ _ R, a,, a,`a, c bc (]^] D KcD L MD D D cd) S,$ S c / S,< \ Z [ ]. S N G O, 2O7JDE J 3 F] 3 F ] S [15] c / S. 2 0 5 G S 3 5, & ]I,! ] Z[] M c/ S! b, 2Z[ ] ' 4. [14] Z [ ] 63, X P EF1 U:agg ( ) gele( ) efaa( & )3 3,EF2U: esp(spvw) ace( VW ) gele efaa43 EF1 EF2 U :gele efaa, gele F G Q VW [16] S 2 Z
813 [] 0/ R S [17] 9: ] P N 2 J, <9: 8 /,T LZ[]G+ U,efaA VP 3. VW, \Z[] ZZ[] 0, \Z[] + %, Z Z [ ]. A R [18],U:WV3 V Z[] R. XC (efaa,esp ace Y X P\Z[]U: 33!ZZ[] U:efaA,W2 ] ST""<=ZZ[]. acm! ace [19-20] Soares [21] esp gele =agg2z[] N,!, F]2 ]S., NF](S.T,!7`F]. S [22] EF1 EF2 U : W D,W Y c/ S 5`. P, ZZ[]. & [ cyla(f G \, ) \ ]F G ^ T [14] \ Z [ ] Z Z [ ]@ cyla \ 5`,STcylA.Z[] ` +> STcylA.]! V Z[]cylA b _ 5 `, 2 @ U:espc3 Mundy [23] \ @.Z [] U:c3, 1 2 `a ST]^EN M. ST R, D E R Z[]. H b,m J c R,! c3 J 2 E.07^ - 0. I, -. K. Z [ ] U :. 3 D [24-25] V Z[] c/ S U:c3, c/ S 2U:,> H F! [1].Z[]@D2OR F [J]. K b,2006,24 (3):228-30. [2] LaukováA,SzabóováR,PlevaP,etal.Decarboxylase posi tive 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犻狌犿狊狋狉犪犻狀狊 isolatedfrom rabbit meatand theirsensitivitytoenterocins[j].foodscinutri,2017,5(1):31-7. [3] Kazun'B,Kazun'K.Probioticsinaquaculture[J].DrugInvention Today,2013,5(1):55-9. [4],,. BZZ[]( Z ] I 8 O. I [J]. Y,2013,25 (5):1069-76. [5],,,.\Z[ ] ( \, 8 P. I[J]. Y,2015,27(6):1832-40. [6]!.K Z[]c >?@+= K L[D]. "#,2015. [7] J.K\Z[]c/ S].KL= [D]. J,2017. [8] RosenthalVD,MakiDG,MehtaA,etal.InternationalNosoco mialinfectioncontrolconsortiumreport,datasummaryfor2002-2007,issuedjanuary2008[j].amjinfectcontrol,2008,36 (9):627-37. [9] SchabergDR,CulverDH,GaynesRP.Majortrendsinthemi crobialetiologyofnosocomialinfection[j].amjmed,1991,91 (3):S72-5. [10] DaviesJ.Inactivationofantibioticsandthedisseminationofre sistancegenes[j].science,1994,264(5157):375-82. [11], T,,. K ZZ[].@ OR= S @M[J].,2017,44(2):530-7. [12] AgersoY,SandvangD.Class1integronsandtetracyclinere sistancegenesin alcaligenes,arthrobacter,and 犘狊犲狌犱狅犿狅狀犪狊狊狆狆.isolatedfrom pigstiesand manuredsoil[j].applenviron Microbiol,2005,71(12):7941-7. [13] KahlmeterG,BrownD,MacgowanA.EUCAST TheEuropean Commiteeon AntimicrobialSusceptibility Testing[J].Antimi crobicnewsleter,1989,6(11):87-9. [14] ^T,,,.\Z[] ZZ[] @. J2 S @M[J]. :b,2008,8(5):433-5. [15] ), ],,.\Z[] ZZ[]. = S ( @M[J].,2014,41(1):125-7. [16] MakinenPL,MakinenKK.The 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犪犾犻狊 extracel lularmetaloendopeptidase (EC3.4.24.30;coccolysin)inacti vateshumanendothelinatbondsinvolvinghydrophobicaminoacid residues[j].biochembiophysrescommun,1994,200(2):981-5. [17] HickeyRM,TwomeyDP,RossRP,etal.Productionofen terolysinabyaraw milkenterococcalisolateexhibitingmultiple virulencefactors[j].microbiology,2003,149(3):655-64. [18] Low YL,Jakubovics NS,FlatmanJC,etal.Manganese de pendentregulationoftheendocarditis associatedvirulencefactor EfaAof 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犪犾犻狊 [J].J Med Microbiol,2003,52 (2):113-9. [19] RamadhanAA,HegedusE.Biofilmformationandespgenecar riageinenterococci[j].jclinpathol,2005,58(7):685-6. [20] NalapareddySR,Qin X,Weinstock GM,etal. 犈狀狋犲狉狅犮狅犮犮狌狊犳犪犲犮犪犾犻狊 adhesin,ace,mediatesatachmenttoextracelularma trixproteinscolagentypeiv and,lamininas welascolagen typei[j].infectimmun,2000,68(9):5218-24. [21] SoaresRO,FediAC,ReiterKC,etal.Correlationbetweenbio filmformationand,gele,esp,and,agggenesin, 犈狀狋犲狉狅犮狅犮犮狌狊狊狆狆.clinicalisolates[J].Virulence,2014,5(5):634-7. [22],, R,. @ Z[]. N =. [J]. b,2016,41 (11):869-73. [23] MundyLM,Sahm DF,Gilmore M.Relationshipsbetweenen terococcalvirulenceandantimicrobialresistance[j].clinmicrobi olrev,2000,13(4):513-22. [24],,. Z [ ] G = @M[J]. b,2017,12(10):1000-2,1006. [25],`.\Z[] JF [J]. b,2012,7(3):231-4. 2018 05 18 2018 08 05