美国FDA细菌学分析手册第八版BAM弯曲菌

1、BAM: Caampyloobacteer HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm Return to BAAM tabble off conttents11Januaryy 20011Bacteriiologiical AAnalyttical ManuaalChappter 77CampyylobaccterAuthorss: Jann M. HHunt, HYPERLINK mailto:carlos.abeyta Carloos Ab

2、eeyta aand Toony TrranRevisioon Hisstory:Updatedd and revissed: 22000-DDEC-299 Media IInstruuctionns Moddifiedd on 22001-MMAR-088 Isolatiion off Camppylobaacter Speciies frrom Foood annd WatterCampyloobacteer is consiideredd by mmany tto be the lleadinng cauuse off enteeric iillnesss in the UUnitedd

3、 Stattes (220,26). Cammpylobbacterr speccies ccan caause mmild tto sevvere ddiarrhhea, wwith lloose, wateery sttools oftenn folllowed by blloody diarrrhea (7,20). C. jejuuni, C. colii, andd C. larii accoount ffor moore thhan 999% of the hhuman isolaates (C. jejuuni 900%). OOther speciies haave beeen

4、 asssociaated wwith hhuman illneess inn receent yeears (7,17,18,233,26,227).Campyloobacteer speecies are hhighlyy infeectivee. Thee infeectivee dosee of CC. jejuuni raanges from 500 tto 10,000 ccells, depeendingg on tthe sttrain, damaage too cellls froom envvironmmentall streesses, and the ssusceppt

5、ibillity oof thee hostt (4,66,7,200,27). Onlyy the mesopphilicc C. fetuus is normaally iinvasiive. TThermoophiliic speecies (optiimum 442C) such as C. jejuuni arre occcasionnally invassive. The iinfecttions are mmanifeested as meeningiitis, pneummonia, misccarriaage, aand a severre forrm of Guilllai

6、n-BBarr syndrrome (6,20). Theermotoolerannt strrains of C. fetuus thaat groow at 42C hhave bbeen iisolatted frrom paatientts (177).Campyloobacteers arre carrried in thhe inttestinnal trract oof a wwide vvarietty of wild and ddomesttic annimalss, esppeciallly biirds. They can eestabllish aa tempporar

7、yy asymmptomaatic ccarrieer staate, aas welll as illneess, iin hummans. This is esspeciaally pprevallent iin devvelopiing coountriies (220). CConsummptionn of ffood aand waater ccontamminateed witth unttreateed aniimal oor humman waaste aaccounnts foor 70% of CCampyllobactter-reelatedd illnnessess e

8、achh yearr. Thee foodds incclude unpassteuriized mmilk, meatss, pouultry, shelllfishh, fruuits, and vvegetaables, (1,88-11,117,19,20,222, 25,26).C. jejuuni caan surrvive 2-4 wweeks underr moisst, reeducedd-oxyggen coonditiions aat 4CC, oftten ouutlastting tthe shhelf llife oof thee prodduct (exceppt

9、 in raw mmilk pproduccts). They can aalso ssurvivve 2-55 montths att -20C, buut onlly a ffew daays att roomm tempperatuure (55,8-111,20). Enviironmeental stressses, such as exxposurre to air, dryinng, loow pH, heatting, freezzing, and pprolonnged sstoragge, daamage cellss and hindeer reccoveryy to a

10、a greaater ddegreee thann for most bacteeria. Olderr and stresssed oorganiisms ggraduaally bbecomee cocccoidall and increeasinggly diifficuult too cultture (5,20). Oxyygen qquenchhing aagentss in mmedia such as heemin aand chharcoaal as well as a microoaerobbic attmosphhere aand prreenriichmennt can

11、n signnificaantly improove reecoverry (2,14-166,21,225,28).Campyloobacteers arre miccroaerrophillic, vvery ssmall, curvved, tthin, Gram-negattive rrods (1.5-55 m), withh corkkscreww motiility. Theyy ofteen joiin to form zigzaag shaapes (20,244). Caampyloobacteer sppp. aree currrentlyy idenntifieed b

12、y testss desccribedd by HHarveyy (13) and Barreet et al.(33). PCCR gennus annd speecies identtificaation methoods haave beeen puublishhed (112,18,30).For addditionnal innformaation contaact HYPERLINK mailto:carlos.abeyta Caarlos Abeytta, FDDA, PRRLNW, Botheell, WA 980411-30122. Phoone nuumber: (4255

13、) 4833-48900.SAMPLE PREPAARATIOON FLOOWCHARRT FORR VARIIOUS FFOODS AND WWATER (SECTTION CC.2.a-h)*for saampless prodduced or prrocesssed 110 dayys preevioussly.ffor frrozen samplles orr sampples pproducced orr proccessedd 100 dayss prevviouslly.Equipmeent annd matterialls Balancees, 60000 g capaccit

14、y, accurrate tto 0.11 g; aand 2000 g ccapaciity, aaccuraate too 0.00001 g Sterilee stommacherr bagss, 4000 and 3500 ml baags annd 4000 ml ffilterr bagss (othher baag typpes annd sizzes deescribbed inn refss. 16, 21, and 222) Whirl-ppak baag raccks annd staainlesss steeel baasketss Bench ttop shhaker

15、 Centriffuge, refriigeratted, ccapablle of 20,0000 x gg Polyproopylenne or stainnless steell 250 ml ceentriffuge bbottlees andd 50 mml cenntrifuuge tuubes, steriile Large ffunnells witth cheeese ccloth lininngs, ssterille (foor whoole seeafoodd and meat samplles orr if ffilterr bagss are unavaailabl

16、le) White oor oraange ggreasee penccils tto marrk bloood-frree aggar pllates 50 ml ssterille connical centrrifugee tubees Plasticc 5-100 ml ttubes with screww cap lids, sterrile Cryotubbes, 11 ml, steriile Phase-ccontraast miicrosccope, with 100X oil iimmerssion oobjecttive oor darrk-fieeld miicrosc

17、cope wwith 663X obbjectiive orr lighht miccroscoope wiith 10000X oobjecttive Microsccope sslidess, 1 ccm sq coverr slipps andd immeersionn oil Gas tannk asssemblyy (5% O2, 10% CO2, 85% N2) andd vacuuum soource (Fig. 1) Microaeerobicc conttainerr systtem Anrobee jarss and bags: Jars wiith vaacuum-pre

18、sssure ggauge and SSchradder vaalves. Theyy may be ussed wiith eiither the ggas taank/vaacuum assemmbly oor witth gass-geneeratinng envvelopees. 3.4 L, (Difcco 19550-30-2 or Oxoidd HP111) mayy be uused wwith eeitherr the gas ttank/vvacuumm asseembly or wiith gaas-genneratiing ennveloppes. Jars wiith

19、outt a gaauge oor vallves (2.5 lliter BBL oor EM Diagnnosticcs Reemel, Lenexxa, KSS 662115 annd 9.55 liteer BBLL) anaaerobee jarss. Theese arre useed witth gass-geneeratinng envvelopees (2.5 litter tyype suuch ass Oxoiid N0225A orr the BBL aand EMM gas paks). Rectanggular jars 2.5 aand 5.5 litter an

20、naerobbe recctanguular jjars (Interrnatioonal BBioprooductss, 8000-729-7611 or Miitsubiishi GGas Chhemicaal Ameerica, 212-752-44620). Anrobee poucches oor recctanguular jjars, 0.4 LL, forr 2-pllate iincubaation (Inteernatiional Bioprroductts or Mitsuubishii). Poouchess for 1-plaate inncubattion aare

21、 avvailabble frrom EMM Diaggnostiics. Air-tigght pllasticc bagss (4 mmil wtt), 122 X 116 orr largger thhat caan be closeed by heat sealiing orr tapee can be ussed ass an iincubaation chambber. Campy ggas-geeneratting eenveloopes oor pouuches: for 3.4 lliter jars, Oxoiid BR556 or CN0355A; foor 2.55 l

22、iteer andd 9.5 literr jarss, Oxooid CNN025A, Difcoo 19566-24-44, BBLL 710440 or 710344 or EEM Diaagnosttics 5530136678; ffor reectanggular jars, Mitssubishhi 10-04; ffor 2-platee, Mittsubisshi 200-04 aand 1-platee, EM Diagnnosticcs, 533-136999. Thhe Oxooid CNNO25A annd thee Mitssubishhi andd EM eenv

23、eloopes aare ussed wiithoutt wateer. CAUTIION: OONLY GGAS PAACS USSED WIITHOUTT WATEER AREE COMPPATIBLLE WITTH THEE ITEMMS IN A.3) AND AA.4). THE RRECTANNGULARR JARSS CAN EXPLOODE IFF USEDD WITHH THE OXOIDD BR566, BBLL OR DDIFCO ENVELLOPES.An Anrrobe gas ppak (1 onnly) ccan bee usedd withh a 9.5 lit

24、ter BBBL anrobe jar. A sinngle ppak inn a laarge ccontaiiner rreducees thee oxyggen leevel tto 55% andd prodduces otherr gasees proomotinng Cammpylbaacter growtth. Eiither gas ppak tyype (ddoes/ddoes nnot reequiree wateer) caan be used. Air inccubatoors, 225 22, 30 2, 37 2, annd 42 1CC. Water bbath,

25、 prefeerablyy shakker tyype, rrange 30-422C orr coliiform bath set tto 37 and 442C. Shakeer watter baath shhould have flaskk clammps, 2250 orr 500 ml iff gasssed fllasks are uused. Shakeer watter baath maay be used eitheer witth bubbbler systeem or gasseed flaask syystem. Stattic waater bbath ccan b

26、ee usedd onlyy withh the bubbller syystem. Shaker air iincubaator oor airr incuubatorr withh shakker pllatforrm (allternaative to shhaker waterr bathh)Shakingg gasssed fllask oor bagg systtem (FFig. 22) Bags, ssee Fiig. 2. Metaalizedd polyy poucches, Assocciatedd Bag Compaany, MMilwauukee, Wi., 800-

27、9926-61100. UUse 6 X 8 for 100 mml enrrichmeents aand 8 X 100 baggs forr 250 ml ennrichmments. Largger baags allso arre avaailablle. Baags arre nott sterrile bbut caan be radiaation steriilizedd. If usingg non-steriile baags, iincludde a bbag coontroll usinng Bollton bbroth or Liisteriia Enrrichmeen

28、t brroth wwithouut anttibiottics. Vacuum flaskks, 2550 or 500 mml, wiith ruubber stoppper annd foaam-pluugged vacuuum tubbing oon thee sidee arm, sterrilizeed (seee Figg.2). Furthher innformaation on asssemblly andd use is coontainned inn BAM, 7th ed., 1992, chappter 77. Bubblerr systtem (FFig. 33).

29、 Twwo gass deliivery valvee systtems aare avvailabble. Evaporaator/cconcenntratoor mannifoldd withh Y-coonnecttor, aavailaable iin 6 oor 12 posittion ssets (AFC IInternnationnal, IInc., Downeers Grrove, IL; 8000-9522-32933) Seee secttions D-3 ffor usse andd F-1 for aassembbly innstrucctionss. Plasti

30、cc luerr-lockk stoppcockss. Stoopcockks locck intto thee outllet vaalves. Plasticc aquaarium tubinng, 3/16 iinner diameeter Nupro SS-seriies fiine meeterinng vallves wwith 11/8 SSwage-lok ccompreessionn fitttings and 11/8 tteflonn or ppoly-fflo tuubing. Two to foour innch loong piieces of 3/16 iinne

31、r-diameeter aaquariium tuubing (equaal to numbeer of valvees). SSee seects. D-3 & F-2 for iinstruuctionns on operaating and aassembbling this bubbller unnit. TThese compoonentss are availlable from locall valvve suppplierrs or contaact Inndianaapoliss Valvve andd Fittting CCo, Inndianaapoliss, IN; 3

32、17-248-22468, for iinformmationn on tthe neearestt suppplier. Enrichmment bbroth contaainer (use eitheer): 400 ml or laarger stomaacher or sttomachher fiilter bags, twisst tiees andd staiinlesss steeel basskets 250 or 500 mml Erllenmeyyer fllasks, foamm pluggged wwith ffoil wwrap aand stteriliized.

33、Two-iinch ssq Parrafilmm piecces annd weiightedd ringgs or a plaatformm withh clammps. FFor innstrucctionss for assemmbly aand usse, seee figg. 2 aand BAAM, 7tth ed., 19992, chhapt. 7. Plasticc 1 mll sterrile ppipetss. Water aanalyssis appparattus Zetaporr filtters, 45 mm (Cunno, Meeridenn, CT, 8000

34、-2433-68944; no substtitutiions), 47-2293 mmm (deppendinng on filteering unit size), auttoclavved seeparattely ffrom ffilterring uunit For 47 mm fiilter apparratus: Teflon-facedd boroosiliccate gglass 47 mmm holdder(s) and filteer claamp foorcepss, steerilizzed Manifolld, 6-12 pllace, for mmultipple

35、 suubsampples Vacuum flaskk, 1-44 L, aand iff a maanifolld is used, a ruubber stoppper wiith a 6 to 8-incch plaastic tube inserrted aand a hose conneectingg the plasttic tuube too the maniffold. Anothher hoose coonnectts thee flassk sidde armm to aa vacuuum soource. For 90, 142, or 2293 mmm filtter

36、appparattus 90, 1422, or 293 mmm fillterinng uniit witth 3 fft hosse atttachedd at ttop, ssterillized Vacuum flaskk, 4-66 L, sset upp as iin b-33 abovve, exxcept the hhose aattachhed too the vacuuum flaasks stoppper tuube coonnectts to the ffilterring uunit ooutlett portt HYPERLINK /Food/ScienceRese

37、arch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm055778.htm Media2, biocchemiccals aand HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm055791.htm reeagentts3 (secction G, exxcept wheree otheerwisee indiicatedd) Media Campyloobacteer Enrrichmeent B

38、rroth (Boltoon forrmula, Oxoiid AM77526 oor Mallthus Diagnnosticcs LABB-135, Maltthus DDiagnoosticss, Norrth Riidgeviille, OH; 2116-3277-25855) witth lyssed hoorse bblood and aantibiiotic suppllementt (Oxooid NDDX131 or Maalthuss Diaggnostiics X1131). Alterrnativvely, antibbioticc suppplemennt mayy

39、be ppreparred frrom inndividdual ccomponnents (G-1). HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm063368.htm M288a4 Campyloobacteer isoolatioon agaars (uuse eiither) Abeyta-Hunt-Bark (AHB) agarr (G-22) HYPERLINK /Food/ScienceResearch/LaboratoryMethods/Bacte

40、riologicalAnalyticalManualBAM/ucm061209.htm M299a5 Modifieed Cammpy bllood-ffree aagar (mCCDAA)(G-22) HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm064002.htm M300a6 Abeyta-Hunt-Bark agar (G-2), witthout antibbioticcs Heart iinfusiion aggar (HHIA) sslantss (

41、 HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm064330.htm M5997) 0.1% Peeptonee diluuent ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm062265.htm R568) Semi-soolid mmediumm, moddifiedd, forr biocchemiccal iddentiff

42、icatiion (GG-5) Neutrall red (NR) soluttion, glyciine, NNaCl, cysteeine HHCl, KKNO3 Triple sugarr ironn (TSII) agaar slaants ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm063699.htm M1499) O-F gluucose ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/Ba

43、cteriologicalAnalyticalManualBAM/ucm064230.htm M116610), mmodifiied; ppreparre hallf thee tubees witth gluucose and hhalf wwithouut. MacConkkey aggar ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm064496.htm MM9111) Culturee shippping mediaa Cary-Bllair mme

44、diumm ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm063370.htm M31112) orr A-H slantts witth 5% filteered ffetal bovinne serrum orr lyseed horrse bllood, w/o aantibiioticss (G-22) Culturee storrage mmedia Semi-soolid mmediumm, moddifiedd, forr shorrt-terrm

45、 cullture storaage (GG-4) HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm063619.htm MM30c133 Culturee freeezing mediaa (G-33) forr longg-termm storrage ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm064309.htm M30b14

46、4) Biochemmicalss and HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm055791.htm reageents155 Hippuraate ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm062230.htm RR3316) annd ninnhydriin ( HYPERLINK /Food/ScienceRese

47、arch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm062256.htm R44717) reeagentts Nalidixxic accid annd cepphalotthin aantibiiotic diskss (Diffco) Hydrogeen perroxidee, 3% Fetal bbovinee seruum (FBBS), ffilterred (00.22 m) Oxidasee reaggent, liquiid typpe preeferreed ( HYPERLINK /Food/Scien

48、ceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm062263.htm R55418) Gram sttain rreagennts ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm062229.htm RR3219); ccounteerstaiin witth 0.55% carrbol ffuchsiin (Diifco) Nitratee deteectionn reagg

49、ents A andd B ( HYPERLINK /Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm062257.htm RR4820) Lead accetatee striips (DDifco) Dryspott Camppy Tesst (Oxxoid, DR1500 avaiilablee fromm Harddy Diaagnosttics 8800-2666-22222 HYPERLINK wwww.harrdydiaagnosttics.ccom21 HYPERLINK

50、/AboutFDA/AboutThisWebsite/WebsitePolicies/Disclaimers/default.htm o Disclaimer Icon 22) oor Aleert foor Cammpylobbacterr (Catt. No. 98000 94 testss or 9801 22 ttests, Neoogen CCorporrationn, 8000-234-5333 or HYPERLINK wwww.neoogen.ccom23 HYPERLINK /AboutFDA/AboutThisWebsite/WebsitePolicies/Disclaim

51、ers/default.htm o Disclaimer Icon 24). Sterilee wateer, 1-2 litters; 70% eethanool or 1000 ppm hhypochhloritte sollutionn (G-66) Sample prepaaratioon. Backgroound iinformmationnCampyloobacteer sppp. cann survvive, but nnot muultiplly, inn foodd at rrefriggeratiion teemperaaturess for 1-3 wweeks, esp

52、eeciallly if foodss (exccept rraw miilk) aare inn airttight contaainerss. Theeir nuumberss decrrease 2 loggs upoon freeezingg at -20C, but the ssurvivving oorganiisms ccan bee recooveredd 5 mmonthss. Sammples shoulld be analyyzed ffor Caampyloobacteer as soon as a samplle pacckage is oppened; intrro

53、ducttion oof freesh oxxygen adds signiificannt strress tto alrready weakeened oorganiisms.Producttion oof oxyygen-nneutraalizinng enzzymes is deecreassed inn micrroaeroophilees, esspeciaally wwhen ccells are uunder stresss. Too combbat thhis prroblemm oxyggen-quuenchiing coompounnds, ssuch aas FBPP,

54、 hemmin, bblood and/oor chaarcoall, aree addeed to the mmedia. Preppared mediaa absoorb oxxygen durinng stoorage; use freshhly prrepareed meddia whheneveer posssiblee. Altternattivelyy, if prepaared bbroth base is sttored in tiightlyy clossed coontainners aaway ffrom llight (hemiin is lightt senssit

55、ivee), itt can be ussed foor up to 2 monthhs. Prrotectt agarr conttaininng FPBB fromm lighht andd refrrigeraate whhen noot in use.Both thhe iniitial samplle preeparattion aand a 1:10 diluttion aare offten nneededd for enricchmentt whenn highh numbbers oof bacckgrouund fllora (with broadd speccies dd

56、iverssity) are ppresennt. Wiith thhe sammple ddilutiion, aantibiioticss perfform mmore eeffecttivelyy and campyylobaccter ccells can uutilizze thee low-oxygeen atmmospheere moore effficieently. If hheavy backggroundd conttaminaation is suuspectted, aadd 1:10 diilutioon enrrichmeent. TThe foollowiing

57、 innstrucctionss incllude mmandattory ddilutiion ennrichmments for sshellffish aand egggs.Preparaation of SaamplessAdd 2 rrehydrrated vialss of BBoltonn antiibiotiic addditivees andd 50 mml lyssed hoorse bblood to 10000 mll Boltton brroth bbase. Alterrnativvely, antibbioticc addiitivess can be prrep

58、areed froom inddividuual coomponeents (G-1).All sammple ttypes exceppt thoose liisted in foollowiing seect. 22(b-h)Place ffilterr bag in wiire peetri ddish hholderr (typpe useed in anaerrobe jjars). Holdd bag lininng in placee withh metaal binnder cclip tto preevent collaapse dduringg fillling. Weig

59、hh 25 gg sampple (550 g iif fruuit orr vegeetablees) innto baag, annd addd 100 ml ennrichmment bbroth. Remoove baag froom hollder, keepiing cllip atttacheed andd wrapp twisst-tiee arouund toop. Pllace bbag(s) in bbaskett or wwhirl-pak rrack. Shakee genttly foor 5 mmin. oor plaace onn a taable-ttop s

60、hhaker set aat 25 rpm.After tthe riinsingg stepp, holld 5 mmin. RRemovee filtter liining and aallow it too draiin a ffew seecondss. If filteer bagg is nnot avvailabble, rrinse samplle in a steerile bag, and ppour ccontennts thhroughh a stterilee, cheeeseclloth-llined funneel intto thee incuubatioon