16 Micrococcus

Friederike Hilbert and Hans-Jiirgen Busse University of Veterinary Medicine Vienna

CONTENTS

16.1 III trod uc tiou

".,,221

!6.1. I Classification of the Genus. MlcHJCOCCllS .. _._ ..... ._._. ,_,_ .... 2.21

!6.1.1 Biology, Parhogcucsis, and Medical Importance. ,,_,_. 22.2

!6.1.3 Identification end Diagnosi s of thc Genus MlcrocOCCl1S _ _ •. _ •. _. ,_.,_., 223

16,2 Method, ",."., .. ,"''', •. ,."."."."."., .. , .. , ... , .. ,."."."."., .. , .. , .. , .. ,."."."."."., •. ,", .. , .. ,."."."."."., .. , .. , .. , .. ,."."."."., .. , .. , .. , .. ,.".".," 22.3

!6.2.1 Sample Collection and Preparation _ _ .. _. ._ __ _.,_.,_., ,,_, .. 223

16.2.1.1 Sample Collection "., .. 223

16.2.1.2 Preparation of the Food Matrix _._.,_,

16.2.1

Dc: teetion Procedu TC 5 _". _". _" __ . _ .. _ __ . __ . _ •. _ •. _

16.3 Conclusions and Future: Perspectives _ .

References .. ,._ _ .. _ .. _ .. _. _ .. _ .. _ _ .. _ .. _. __ . __ ._ .. _ __ ._ .. _ .. _ .. _ ..

16.1 INTRODUCTION

16.1,1 CLA55IF[CATlON OF THE GENUS MICROCOCCUS

The _genO:5 MiclOC'occus was originally described by Cojm' ::i~ mo st ·140 ye nrs ag.Q. A ftc r n umerou s reorgan i zation s of the: _gcllUS eud rcclcssifcruions of certain SPCCie:5 into new gellem!3 the: gcllUS now is encompassing a clade of four spe:cics including MjcmcOC'clIs 'ulell.r the: type species of rhe genus, Micrococcus l'ylae,~ Mjcmcoccl1s afllarcticl1s,5 and Micrococclf:S jJaI>'IIS.6

The: species are sep nmrcd f rom e i:9C h orhe r at 16S rR N A gene: similarities between 96.9 and '98.3% but rcprcscntauvcs of rei etcd species of the: geno:s ex h ibir peptidoglycan 11j pe A 2 {Iy.""i neb ased pe: P ticloglycun and all in tc r:pe p Lid e bridge consisting of the: stem peptide) or A4u (IY5i nc based pcptidcglycnn nnd aspartic acid as the inrnrpcpridc bridge) according to Schleifer and Kandler.' Quinone: sysLenlscon.'ii.""L predominantly of mcnaquincnc with seven end/or eight i soprc noic "LIn it.~ in th c side ch ai u and one of th esc u n ~ ts may 1>0 dihydrogcnatcd such as M K-7 (HJ and M K-8 (H J. M K-8 and MK-8 (H,) 0' MK-8 (H,). Chemcmxonorutc ch a r ectc ri st ic 5 such ;.'i, pe: p L id ogtyc un t 1j pc nnd qui none system arc con side: red to be h igh ly conserved nmOll_g .""pe:.cecs of a single _gelH.JS ..... ith only fe ..... know n exceptions. Al reedy based on the characteristics so far collected for four specie: ... beth quinone .""y.""Lenl and pcprtdoglycun type are unusu ally variable nn-long species of the: genus. The quinone .... ysiem may be useful for ideutificarion or strains of M. jft.1i11llS W h ich ha .... been reponed to contain a quinone system consisting of rhe m::ijor.compoullcL~ MK-7 (H~ and M K - 8: (H 2). On L he other h. a nd ~1. iytaa nnd M. GIll a m u: us are characterized by quinone systems atsc Found in cerrei n M. I.IIU!IIS struius (Table ·16.1) and hence rhcy cnnncr

".,.223 ".,.224 ".,.226

. "".,,226

be identified based on this trait alone. On the other hand, peptidoglycan type A2. nlily be indicative for straiu s of M. trtU!IIS biovcr 1_ Fatty acid profiles predominantly shew co-C 15:O(]O cud ameiso-C~:s:(] acids.~--6 Other fatty acid .... were reported only in minor amounts, Differences useful fordiffcrcuti ation between MfCmCOCClls species arc not reported. Hence, also fatly acid analysis broadly applied in clinical diagnostics for rapid idcutificatiou of pnrhogcns nppnrcrnly is not useful for refenble idenrtficruicn of Mjcn-){"occrts species and especially for identification of M. !werts and M. lylae. variability in physiologicn] traits among strains of" M. lul(.'us4ma!kes.iL almost impossible to reliably disriaguish L h is s.pec ic 5 from the oth er :s.pec ic 5 of L h c gell u s, Howcvc r, optima] growth temperatures suggest that M, lmt:lls and M. tytae 1"1.1 ay pO.""5C.S.5 the poreatinl to cause human infcclion, (Table 16.1).

A ruon_g csmbliehcd bacterial specie: ... M. 'Ulew~ is quite unique. Strains of th i."" species exhibit stgnificanr variability in ph y siological rra i ts as men ticned ilOO\'C bu t il L"iO in both peptidoglycan type nnd quinone system. Strains of this species either contain n peptidoglycan type A2 or A4o:. and the quinone: system either consists or two rnejcr compounds M K-8 and MK-8 (1-1,) or only MK-8 (HJ.' Mainly based on this, variabitity the species. was recently subdivided into three bjovars. Interestingly, strains of one or these biovnre, bicvar 11 exhibit a unique: baud after REP-PCR which t,,,,, lacking in represcamtives of biovar I and biovnr lll.~

M. uaeus i~"" nn inhabitant of the huruau . skin whereas M. lylG(.' is only rarely isol ercd from this hilbiLilLIi: lmercsriugly, at EM BL gene ball k more than 50 sequence entries can be found which arc sharing 99.5% or even higher t6S rRNA gene 5[]"Uilarity w~th: the type strum of this species. [J] contrast, only one: entry can be found in the _gene bank. which shares 9'9_5%

221

222

TABLE 16.1

Phenotypic uffferentjarlon Between Micrococcus Species

Molecul" Detection of Foodborne ~'Ihogem

Characte rlsf!c

M.lalerrs:

o ptirnal ~ rowth 'le1DJl"!irutu re (·C) Nitmte reduction \'Og.:=:rPra;b.er reaction M:3joIqlJ.jJ1on~~l

Peptidog ~_I'mn L~' pe

11

MK-8. MIK-S (Hl) or M K-8 (HJ :\~ crAac

D-M!ll1nofli!ie D-M!llLc&:= L-M:.dat(:

D-T«:haI05ol:

+

M.lrIOJ~

M . .ilp"plI'a,rdicus

37

1.6.8 +

11

MK-8(H21 :\4U

MK-S (H]). MK- 7 (H2) .'''''

M K-S. MK-$ (H2) NOlL ~~I11:3I):.xd

Hydrolysis-OIl:

Aselrnllaflon ol:

+

Sm~(I"':-':-.s.; Wi,C:~T. M,l:=h.l[ . 11M. i. :i).f/. lr~o~. M«:m'~~., 52.629.2002; Lin. tL C:L:1i.,frn'. J. S.I·n. t".'m .. 11.iuobil'l'f .• 50. 715.2COllund Liu. X. Y. ~I al .. Jrjl. i. S.,".\·I. EI'I'2f . .w.n:l"obi.-~ .• j7. 66. 2007.

+

16S rRNA gene: scC]uc:ncc: similarity with the: type stmiu of M. (y'cre w here as several c: n tries can fouu d de: sign arcd M. Iylae but sharing les, than 99_5% similarity. In bactertnl system atics 16S rR NA gene .seC]:uc noes a rc not ncce pled as a tool for spec ie 5 ide n ti fie ction bee ause c uses arc: know n w he: re gcnoruically and phenotypically clearly scpnratcd species share 99.9% 16S rRNA gene similarity," However, value.", of 99. j% or higher lllay be suitable for prel imiaary idcntification of" L 1,1 ese strains as members of M. 'mer~s or at least a very closely but so far undescribed species.

16.1'.2 B:IOLOG'r', PATHO GENESIS.!' AND MEDICAL IMPORTANCE

The family Micror.:occaceoe includes Gram-positive bacteria found ill LI. variety of differ-ent habitats c.g.~ water, various soils, iJlSCCt5T skin of humans and most other mammals. and as a cOIlscqtlCnCe are found on a diversity of difIerent food. Nonspecific tsol atiou of Mjcrococcus strains from grain, fruits, seeds, plants, milk, cheese, fresh meat an d fe mien ted meat, all d A sh P roo uc t sis. com tuon in food microbiology.KH3011 food some members of this gcnLl.s, (Ire commensnle in othercases different strains fuuction as ferme n te rs and ri pe rs DU t also as. spo i 1 ers as. \Ii ell as food borne pathogens mninjy caused by thci r production of biogeu ic eruines (8A).14.15 M. 1~lrerts as. well as M. tytoe arc kuowu to be involved ill ripening of ccrteiu naturally ripened produ-cts. c_g.., cl1CCSCT fermented fish products (e.g., .s..I.1 ickara, cassava), and salami type sausages, M, {wells [5 used as a starter cultu re strain for fermented fish products uud ferme n ted dry- sau sage 5. Neve rt he] ess, Mil" I'DC'OCC' us spp, will never re present the m ai n flora in ne ith er 'Of these prod uct s, w h ic 1.1 ar-c 1 no tobac i II i, pcd iococc i, stephylococc i , and for

certain products ycasts. and molds. However, in some fermented products MicnxocclIs spp. are considered to be e sscn t i nl fo r the deve 1 oprucnt of aroma and FI avor, 1.(]-1.3. ~-I5i Microbial enzymcs produced by MiclvcocL'rts s.pp. which are i nvol vcd in th e dcvelcpmeu t of f a\I"Q r and a rom a a rc a sail resistant glutamiu use, an amylase, cnzynlc5 to degrade L - meth iou ill C to vel cti lc su I f u: r com pou nd s. Ell :z.ymes. for protein hydrolysis and espccinlty enzyrae s for lipolytic activity to modify generated carbonyl compounds ar-c e ssenl i ill fo r the dcvelopmc n t of FI avor, The sc ac l i \i' it ies 1.1 ave bee n shewn to be c ssentinl for the SPCC[n.c odor of tong air dried CLi red 5ausa_gcs,l'(]

The action of cn zyrucs rcquircd for ripcuing of fermented fcods cnn cause spoilage of food as. well. Hence, MrCIfX:OCCIIS spp, as halotolerant microorganisms (j-IO% NaCll,' have been fcuud [[lJ different spoiled food stuff Fre.01 meat, sea food and insufficient thermal processing of products rich in fat are at risk to contnin higb numbersof M~cJlJC'oCCUS s.pp. especially in the absence of 'Other microbial spoilers as. arc pscodcmonads and lnctcbecilli. Nevertheles: s, spoilage by Micrococcus spp. is rare. Typical. CI.1 ill.l,gc:S include protein hydrolysis and lipolysi,."·"

Probably the greatest health threat for humans. is related to the capability of micrococci to produce HA. As. MrclDCoccWii spp. arc r ou nd on d i ffcrcut raw, ferrnen ted, and somcti mes CVCJ} in heat treated foods. many studies include MrCI'DCOCCllS as reference stmius to rest the effect of antiruicrobi ill activi ty of d i ffc ren l prcduc t s or l rear men t 5 'Or to study the cffcc t of transfer from one environment to another which might be important fcr cxtraiutcstinnl !llfection5.!];·u

BA ar-c formed by decarboxylation of free amino acids, Many different bacterial species found! in food are able to prod uce MI ffic ic n t amo Ij n rs of (fcc a rbox yl use to be rele ased

Micrococcus

in L 1,1 e feed mat ri x and produce SA su-ch a s hi st am i ne, putrescine, cadnveriae, tyramine, phcnyjethyl atu i ne, and funhe:r.15-E Certain strains of M_ llileus have also bccu .,,"-1,1 ow n to prod u cc cad ave ri ne ill food s bu t t 1,1 ere i 5 J10 ev ideuce from t[ te rat 0: rc 0 f cap ubi 1 it y fo r prod uc rion of orhe r BA.17 Cadaverine itself has only little toxic effects but it potentiates the toxicity of histaru i ne in food! by inh ibiiing hist aminc-rnct abol izing e:nzymes. MICI,l as.diaruiuc oxidase end histami ne Ncrncthyl-transfcrnsc. 2E

The toxicity of histamine and other BA in hum nus arc: based Oll the: iutcrucrion of hismruiue with cellular merubrane receptors, effecting the cardiovascul ar system, smooth m uscle 5, and even to: any motor nee ron sti mu I arion. Sym ptoru s in humans of biogenic amine poisoning are urticarta, Hushing, headache, abdominal cramp.s.! diarrhea, cud vomiting, The effect varies individually (hismmiue intolerance) and it is. dose and m arrfix dependent. Th us, the: sarue amount of B A may cause 110 to severe symptoms if administered with difJere n 1 food m atrice s.1:i ~ The: whole: f am i l"j of Mic rococc rts strai n s may ill so IPt ily an i mportan L role as norm at gut m ic roflora and mL1y also be involved in symptoms of digestive disorders in humans." Thus, the role of Mtcnxoccus spp_ in food is highly diverse as the gc:llUS irsel f.

Additionally, stmins of the genus. arc: kJIDWn as.opport:u.ni sric path ogen s for cxtrai n tcstiu ill disc ase m niul y ill i m munocornpronuscd persc:ms,:H.l2 meuingius," septic arthrius," cndocard i tis!] 5-37 ill f ccrion s of leu kern r a patients with ind ,welling lines and patients undergoing continuous umulatory peritoneal dialysis or with ventrieulo-pcritonenl shuut," in L racrnu ial su p pu ration ~J~ b acteracm r a,.w·41 ch roll ic c utaneou s infections in H IV positive piltieJJts,-Il nud catheter infection."

16.1.3 IDENTIFICATION AND D'AGN'051S OF THE GENUS MIC~OCOCCU5

For the sampling of food cud food stuff-c-if no specific advice is gDVCll-the: spcci fie lnrcru etional Standard has to be followed dealing with the product concerned c.g., DIN 10162. A portion of the p reduct ~ s hcmogeu i zed with 1: 10 of adequate dilution medium e.g., peptone solution ([SO 6887) using LI. stomacher 400 or by Uln-a LUHax. A dilutiou series (depending of expected colony counts) arc: plated. fur meat end me at prod IlC ts, chee se as. well as for f sh and f sh prodocrs itu which MrclDcoccllS spp_ may be found the detection

• md isolation of MjcrocOC'cil~ spp, i:s difficult as. most strains. are not able to compete with S,crpJry'oeocclIs s.pp.trJ . .!O In most cases Mieftxoccus spp, arc n01 able to grow on Baird-Parker il,gar~ icrlco:batcd ac:robically ilt 3CI or 3~C for 43 hand Lhus, canIlot be isotilted ill ~amll.d with S'crJJ/ry'ococcus spp. e:vell though! some ar~iclc:!'. aJ1d books. refi'::r Lo it:u [ll 5Ome: cas.cs. M. 'weus ill.ld M. lylac:: Cil11 be dcLecLed on colony cOLmt il,gar especially if the product harbors a low DLLlllbe:r of bacrerria, Sugges.tions.for the isolilti0J1 of M. lWe-IIs wi1h the addiLion of 20 ~glDnl fUril101[done to corony counL ngar arc: confirmed ill oo:r srudie:s_4:i4.0 Howevc:r, sl.JICe: tlhe existellcc of furazotid0l1e sen si Live m ie-rococci was. rc ported 47 no L all me:l.1.l be r,s of r"he

223

micrococci community will be detected using this. selective medium. In productscoutaining a ratherhigh mrmbcr of other bactcri ill spe:c ies a combiu ction of 5% eodi um c 1,110 ride and 20 Ilg/ml furazolidone has. been fouud 0: se fu 1.-1.6 Furthermore, verificntion of colonies by aerobic/anaerobic growth tOo distinguish between facultative au aerobes and Grmu smrniug has to be done. Discriruinarion From e.g., Kocuria Vll,rjans and diffcrcutiatiou between the: Mtcnxoeeus species by biochemical reactions such il.r,;, tests for k atalasc reaction, lysestnphin resistance rcsriug, assimilation of lactose, glucose, gclutiu, glycerol, and nitrate reduction (see Table 16.1) is complicated by highly similar reaction profile"

Thus there is. au essential need for molecular tools allowing identification of members of the: genu,r,;, MicmcoccllS as well as to discriminate between species. To the bcsr of our knowledge no molecular detection method for Microcoeeus from food h as been dcvclo pcd.

16.2 M ETHO[)S

16.:2.1 SA"PL' COlLKTION AND PREPA"ATION

16.2 .1.1 Sa m ple Collect! (m

As M. 'weus cud M. IJlcre are found in a variety of different food with uupredicmble aruonnrs runghig from one to [0' cfu per gramlml, respectively, the sampling procedure and pre: p nration are dcpc ndca L on the: product il.r,;, well 011 the purpo5C of detection. Hence, not only general procedures 1,1a,"'C to be followed, such as cooling and! fast transport to L he I abcratory as 'W cl [ as rapid sample au ill Y si s bu L ill so to control these specific food products nt different processing steps, at different times of fermentation, or during shelf life. Further chemical analysis on the food matrix might be neee.r,;,sary e,g.t analysis of BA to define safety, accurate shelf life, or consistent sensori ill quality. In some: cases iL might be ne:ces..~ilry to analyze: MjCJDeOC'CllS isolates for their ilb[lity to c au se bio gIL: n ic am r ne produ ction ill food .. to de fine L 1,1 ci r spectrum of lipolyric activity, protein hydrolysis and/or their carbohydrate dcgradnticu ilbrlity to categorize the isolate as spoiler, ferruenter or pathogen. However, it appears to be feasible to ecpl ace some of these grOow th de: pcndc n 1 ex am in aLions. by development of PCR!; directed to genes coding for ce rt a in en zy rues such as oru ith r ne - and ly si II e -dcc arbor yl use or argin i ne-d i 11 yd rol ase .

16.2 ,1'.2 Pre pa ration of the Food M.atr;ix

The complexity of food matric:e~ the::rr preparatioll and the choice of LI,le beM pos..r,;jbJc: way to de:tect dirffc:rc:nt bac.k:ri il or elute bacterial DNA are Mill oCOll.r,;,jderoo as. one of the upper 1110 5l r mpo r L ill1 t problem sin food m [cro biolo,gy and th U,r,;,! ha,"'C been describcd! experimelltally proce5scd alld exLensi vely di se:u ssc:d for most food bornc p iltho gcru s,-I&.l.£) no: L SCI far 1l0t for MjcmeOC'cl1s s.pp. Nevcrthelc:ss..,. one: can assumc that protocol s fo r prep :rration of food m atr[ce 5 U sed for the:: j sol ilLion aJ1d idelltificatron of other foodbome pil1hogc:ns might be u sc:f 0: I al:so for III ic rocooc.i.

224

Meat and meat products. MJ-croco['crts :spp_ OCCLI rs [[1: fermcutcd meat products. as well as. prcducre ccnserved "by high salt concentrations cud low tClllpcmu.uc thermal processing. Examples of these products arc dry-fermented :sau:!'.agc:~ trad i l ion al made and ternperat LI rc: sen sitive p rodu cts 1 i ke pate contain ing liver or tard_II.Jl.llt I'"

These prod uc ts are dime u It to process f u rt her fo r m [embi a 1 DNA an ajysi S j],1j. they COIl t ai n: so bst euces c.g., protei n ases th at arc: able to di stu rb m [ern bi al DNA eatrac rion a 5 well il.lj. DNA polymerase activity for arnpt[fiCaLiou.:W.!"(] Additionally the: removal of fru, proteins as wcl l as. DNA from meat species [5 challenging. Nonclhde,s,5, there arc: methods that iHC COIl sidcrcd to a llow d i rec l DNA [sol arion from meat and meat products and further amplification e.g.t real-time peR end rhus, qu.antjficution of microorgilllisrns.:5I}-:5]; III the: case of dry fermented salJ:!;age as well es products contain ing lard, (he separation of Fat i s most important. Various filtration and centrifugnticu steps prior to DNA prcpamticu are eSf;C:lltiat.:50[,:5];

Dalry products, Mjcrococcus spp, arc reponed to be found on smear ripened cheese c.g..t Gubeen cheese on trnditionally made goat and sheep cheese, and OIl Cnmcrabcrt-like cud bluc-vci ned cheeses at 00_ 111. L7 ] n some c ases i l is not essen ,ei al to homogenize cheese: if only the surface flor-a needs to be illla]yz:c:d.5.I But to aenjyze total cheese one hns to homogcni z.e a spc:c D fie '""01 u me of the d ai ry prod uc l in edcq u: nte su spcusicu c.g., 0.1% peptone solution or physiological sodium chloride solu lion. Li kc ce rt a in me at P roduc ts d ai ry food m atriccs tu ay harbor su bstanee s to red uce the amou n l of ex l rae ted DNA and/or inhibit further DNA amplification.

Fish and seafood. Mi[,JlJC'OC'cus spp_ iHC reported to be m ai n ly found in t red i t ion ally fe rruen led fish (c .g., c assav a Heh for further production of lauhouin) or fish. sausa_gc:.(;I·JIi:

As. fi sh au d especi ally fish produc t s a re at ri.1i& to con l ni n significant high values. of BA; the detection of MjcI"Vcoccrts .~pp. which are able to produce BA in fish and sea food is. rc lev au t to !h u: m au he LLl th.15 TI,l:U s, nex t to the: i sol ntiou andl or detection of Micrococcus .r,;.pp. it is important to identify their ability to produce BA by molecular OD'" tradition al based methods.

A suitable and easy isolation method for Micmco['C'rts DNA ex l rae lion from all above mel] tioned! food m ctricc 5 is. given 'by Stevens and layku:.s.55 nud by Rcssmanith et al.:5G Both methods arc: described below end arc: slightly adapted :to fil the: pu:rpose:

(1) Eleven gram,r,;. of the: sample is diluted ill 2j ml sterile 0_9% sa] ill e sol u rion and 8 ru 1 of '2 j% w Iv sod! i u m ci t rate sol ut ion su pplc men ted w ith pclyc t hyl eae gl ycol to a final conceu trat ion of 4% and l reared by a stomacher for at least "2 min af room temperatu:re_ For me: at and f sh produ cts coutai n i ng large r p nrtic te the homogcll i led product is filtc",d tEtro"g)l choo,",,· cloth. Silmples. i:'Ire ce:ntriflllgc:di LLl9700xg for I j m[ll .1 4"C, Tho pellel is I,"o,ferred to DNA cXIroclioll using either DNAzol or a commerciill kiL for tolill m ic robi al DNA eUrac t ion.

Molecul;n Detection of Foodborne HJ;3thogem,

(2) The: scruple is diluted 1:2 ill lysD,r,;. buffer ccnteiuiug Ix PBS, 8 M urea and 1% SOS. The sample is t reared by a stem ac her for 3 m iu. Lysi 5 DU ffcr is added to a volume of 40 rul and [yS[S isc arricd out at 4TC for 30 mill ,I constant sh aking at 200 rpm following a ceutritugation step for 30 min LLl room ternperature aud 3220xg. The supernntnnt is discharged nod the pellet dissolved ill 'i00-1500 p·1 of lx PBS. Hard cheese and sausage: samples are scdimcntatcd for 5 mill and the: supc:mil.tillll is further ccntrifugcd nt 800Dxg for 5 mill. DNA isolation from the remniuiug pellet [S performed using a commercjal kit for total microbial DNA isol etion.

16 • .2 .2 D HKI 10 N P ao cmUK~S

(i) peR detection of M" IUlCUS: So far there arc: no stnnd ard protocols. used for dctcctiou and identification of MiclfX'OCCIIS spp, OIl a molecular level but the: following proccdu rc cau be used for obtaining template DNA for proposed PCR nrnpliflcation (see T.IDIe 16.n

Suspccuvecolonics on e_g.~ LB ngar ccutaining 5% sodium chloride cud 20 pgiml furazolidone can be directly used for template DN_A extraction il,r,;. described by three cycles of" frc:e:zc:-thawing..51

([) One loop of biomess is suspended in :50-'100 ~Il.~ter-

ile ddH,D

(2) rapidly frozen in liquid nitrogen.

(3) followed by melting at 600(; for Smin. (4) Repeat s.teps 2 au d 3 twice.

(5) CeH debris is. pcllctcd in a micruccntrifugc at npproximately IOOOOxg. The supernatant is ready for usc in PCR (and can be stored al-20CC for severa] weeks).

If colony counts are expected at 10::1. per _gfml of food a direct molecular detection might be possible and totnl microbial DNA isol arion can be: performed il,r,;. described in Section is.z.z.

PCR uud sequencing of the: 16S rRNA gc:ne might be performed as Jol low s.;

([) Qu ami fie at ion of tempt nte DNA_ (2) Use ) ng (e'trocled from c"II""'). (3) lOx dNTP, (2 tu M each),

(4)100 mNl forward pri me L OIl d 100 11] M reverse pri mer

(stock) (see Tobie ]6.3).

(5) PCR reaction buffer including MgCl, (W4 (6) Taq polyruemsc (e.g., 5 UI~l,

(7) A typical reaction of 2j pl includes: [-5 ~Il template DNA, 2,) ~l lOx dNTP", OJ ~I forward primer, 0.1 ~I reverse primer, 2.5 pi lOx PCR buffer, 0.5 U Thq po~ymera,,,, (0.1111 of 'i Uilll) adjust with ddHP to 2) I'l.

(8) peR condiitions ilrc: gl,"'Cn in Tilb[e '16_3.

Micrococcus

225

TABLE 16.2

Prlmees and peR Conditions

frirner

Sequence (5.'-]'1 c~'lgc:lgDrg~I:":·lImwc

ccg [('~wtlC'~ul;!1::lg LLL g>[cctg~Drgl:": lC't:.'lCgl[ccl:": cgccgmcnc tcgcgorD.gc ~g

Ta~l!~ GC=M" 16s R N:'\' gil: nc variabk region V+:5

=A

Com t

Com2 MlL-rN.-\-for MlL-rN.-\-re\'

Amp" [0 n 51:z~ {hpj variable

Annl!allng Ternpemture

Schwi~er ct nl." T11i~:;'LUd}"

)10

TABLE 16.3

Characteristics Differen1:latJng Micrororrus tuteu« and Kocuria vari'ans (formerly Micrococcus 'Varlall7s)

Chemctertsttcs

.~.c robjc L'IC id from I:": lueose L\-itrnlell:dllc~tolllilritc

SiilD mOIlll~ ~iilrn1e agar Il1org:3l11i~ nilrogelll agar

S~u:r r, \·: Koce r. M. _ 1 n B ergt=~· "a M!ln uat of S~·;!i.1f m:3lic B 8.('1eriolt::tg~·.

Sneath. P.H.:\. e t al. ,cd!>.). William!> &. wilkins s. Baltimore. [986-.

+

(9) Amplicons can be purified directly or after detection in gc 1. elec trcphorc 5C::S cud Cl.C ised from t he matrix using different commercial kits.

(10) Subject to automated sequencing using the above mentioned forward and/or reverse primers, mruch, uud an al y zc :sequence to ex i sri n g d at nb use.

COn!;L r u ction of speci fic pri me: rs for the: detection of micrococci is hampered by the: 1 imitcd genomic :sequences. deposited "gene banks. The 15S rRNA gene is most likely no L su it able to re ach th i s goal bee au se of the h ighly COIlserved chnraceer cf th is gt:nc_ Morc promising might be primcrs directed to so-called house-keeping gc:ne.s. which .HC less conserved then 16S rRNA gcne:5 but sufficiently conserved to construct MiC'.fDC'OCcUS-s.pecific primers. However, among commonly studied house-keeping _gc:J1CS. we: could only And nine meA gene soql.LCJ1CC:S of micrococci in gc:JIC banks of which only two were of we~l characterized strains, M, luler,s ATCC 331 (A FlI4783) cud Micrococcus Iytae ATCC 27>66 (AFlI4778}. After alignment with corresponciag gene of two Arthrobacter s.pp. (clo:sc:ly related to MiCltx'OCCIIS) we were: able to construct a primer pair (see Tobie 16.3) which .• hould allow specific amplification of partial meA 'With M. /ulellSand M. tytae resultingin LUI: amplicouof Slu bp. However the suitabil it)', selectivity, and specificity of th[s approach have not been tes s ted so f ar bee ause of ill n avai I albi 1 i ty or :su: ffic iCD L n u mher of wei I c h arne teri zed strai n 5 or bot ft spec ies

(ii) Molecular detection or biogenic amine producjeg bacteria: Some Grum-positive species (e:.g_, Clost ridjllJJP, Mi[,JDCOCCUS, Lactobocittus, Le ucoeowoc, cud Te.rmgt:l1- o.cO[,[,IIS) generate BA including histamine, tyramine,

phenylalanine, putrescine, and cadaverine from free am ino acid s in ferme n ted food ,So a nd beve rages a s a Ie::SU I t of their amino acid dccarbcxylnsc activities. Ah!hou:gh BA sue h as h i st am ine and tyram i J1C do not nffec t the: 5C: n :sory q:u nl i ty of the food prcduc ts, the: se com pou n d s 11 ave bee: n implicated in a number of food poiscnmg caSC5. These bacteria harbor gc:ne::s hdc and t yrdc encoding for bistidine and tyrosine dccarbcxyluscs, respectively, which arc: involved in the production of BA from free amino acids, By employing primers targeting lsdc and ryrdc gc:ne:s il,r,;. well as. [6S rRNA _gc:ne of bacteria ns au intern ill peR control, Coren, and COLonoo developed a ruulriplex PC"R for the: early detection of potential tyratu.inc and histamine producbng bacteria directly on bacterial colonies. Specifically, the following three primer scrs are utilized:

HDC3 (5'-GATGGTATTGTITCKTATGA-3') .J1d f-UDC4 (5'-CAAACACCAGCATCITC-3') for a 44O-bp tutc gono fragment; TD2 (5'-·ACATAGTCAACCATRITGAA-3') and TD5 (5' -CAAATGGA AGAAGAAGTAGG-3') fora IIOO-bp t yrdc genc frugmcnts; and ·16S :rRNA universal primers BSF3 (5'-AGAGTITGATCCTGGCTCAG-3') .J1d BSRI5 41 (5'-AAGGAGGTGATCCAGCCGCA-3') for a 1'i30-bp fragment as. the peR internal control. Primer concentralion, are 20 pmol for HDC3, HDC4, TD2, and TD5 and 5 pmol for BSFB cud BSR1541; and tho PCR amplification progJilm consists of OJ1C cycle of 9.soc for 5 ruin; 32 cycles of 9.soc for 45 sec, 520C for 45 sec, and! 720C ·1 mi n I j sec; and one cycle of 720(; for 5 min. After completion, 18 pi of each peR sample arc separated on 0.8% (w t/vol) agarose gels in I X TEE buffer at 100 V fOT 50 min aud then visu ,1- i zcd with cthi.diunu bromide sminiag using a GclDoc 2000. The: au: L hors succ cssf ull y de tee ted HDC+iTy:rDC+ ,~1 r a ins of C tost ridi 11 III, E nte rococc us, Lacr obac i II.IIS, 01.:: tsococc ns, and Ter J"agt' tsococc rts, Un fortunately, while pyruvoyl-clcpcndcnt HDC activity h as been observed in the: genus Mic;roco{;cUS, L h e tutc , and r yrde -e:q u: ivalen t gc: ne scq:u enc es a [C u 01 f ou nd in micrococci, which prevent the PCR application of the HDC3, HDC4~ TDlt and ill5 primers for the detection of tyramine and histamiuc producing micrococci, Perhaps, the gene responsible for pyruvoyl-dcpcudcru HDC activity in micrococci nny have evolved over time: so that it differs significantly from thnt in Clostridium, Enterococcus, Lactobocittus, Oenococcus, and Tevmge nococcns. Further ex am i n ation of m ic rococo al genome ~:s c 1 e il r1 Y needed to identify distinct gc:ne scq:ucncc ncecunring for its. pyruvcyldependent HOC ncttvity for subsequent PCR appl ic aticn.

22b

16.3 CONC LUSIONS AND FUTURE PE Ii. SPECTIVES

In food microbiology species of the: _gc:llUS Mi['rococ(,GceCl{! h ave bee n dcsc ribed as fermcu te rs, spoi lers, cud pathogen s. BU:L because: they do not attach major importance in one of these multifarious functions not much research has beeu undertaken to clarify their role: ill food. ln most cases iso laticn of micrococci [S mainly coincidently Ll.5 no standard protocols ex i Sl [11 food III ic rob iology for isol ericn or eveu further characterization. The TC(:cn1 reclassifications of cerrain species. especially the classification of c.&l' M. varians into K. Vf1ricws 5C:L.1j. lJCW requirements in di ffcrcutiarion of m ic rococc i. The d i ffe rcn ri arion be l wee n M. 'rtl e us and K_ ~'a1"ial!:5" is csecutial to studying fermentation and spoilage: in food microbiology, as. they have different f unctions in food Icrmcutation, production of BA audjor spoilage. For instance K. varilms is known to be a beneficial microcrgnntsrus of m ai n i mpo[t ance in food f e rmen ration au dill te ruction wit h other fcrmcutcrs c.g.~ staphylococci_ill. Nevertheless, the differentiation toO micrococci win cause problems because both specie shave si mil ar groOw th rc:q u i re men t s and hence, are difficult to discriminate (sec Table ·16.3). Therefore, new me l hod s 1.1 ave to be dcsc ribed, adopted, aud cv nl u ated for detecting this ge:nus iu food.

ln order 10 develop a peR .r,;.ystc:m for rapid idcnrificmiou two strategies appear to be most promising:

(i) Developmen t of a PCR w h ic h gene rates genom ic fiugcrprin ts characteristic for Micrococcus species, Recently, it has been demonstmied thilt M. lraens biovnr 11 strains exhibit a ehuractcristic bnud after REP-PCR which was not detected in rc:pre. sentarive s of biova r I and III srraiu s, Ttl i s observation i ndic mes a rather high genomic variability among strains of this species which is also derucnstrnted by the high. degree of vnrinbility in phenotypic c 1.1 arncteri sties and it may !be d i ffie ult to construe l primers which in fl'C:R produce M. luleJ"ls-spc:cific fingerprints, (ii) De ve lcprue n t of a spec ies spcci fie -PC R. ~ n orde r to deve lop . such all approach .sequence infcematicn from house-keeping gt:lle.r,;. are: urgently needed. These: ge:nes should be present ill il~l Mjc..'ftJ['OC'cu'! species and strains and sufficiently vnrinble to COIl struc t prnucrs fo r spee i fie klcn t ific arion at the spec ics leve 1_ Un fort uu ate: 1 y, q u ite a few seq ueu ces other th an 16S rR NA _gc:lle seq uc nces arc: av ni 1 abte from gene ban k 5 W h ic h hamper the design of such a PCR-approilch. Since the b[gge.~l thre:al for ntlmans is the capability oOf M, lulerts lo decllrbony] ilte lysille and to produce the bio_gc:ll i.e am[lle cadaverine llle moM "sdul rool would be a peR .pociAcally dCleding Il,C gclle coding for lys[ne-dccarDo::ri.ylase bUl also for this ge:ne no sequence informat:[on is avaDtable c'01.1cerning M_ lulens_ So far .. i.l is UnkJ10Wn whether lysiJle decarbm.ytL9tion ilctivity is. a commoOn trail of M. IlltertS Of OJ]l)," eertain sLrains.of lbis .~pccie s and hell ce10 Mle h il PCR wou Id! have: l he adivan l age to discr[mill illc betweell hazardous and nonhaz.ilrdous stra[n!i. of the species... Howevert:ill.so f'Or thisgcnc no scquel1ce information from M. 'lIleusis LLCcessiblc:. Hopeflilly, in the neiJ.rfulure the com ple fe genome of :Ji re: pre: sen l ilti ,"'C 'OF a Mj~_: mcoC'C' rls

Molecul;n Detection of Foodborne HJ;3thogem,

species wilt be sequenced. This infcrmnriou would be a huge step forward to identify house-keeping _gc:JleS or other gene largel cud their &c:q]llences and! to construct species- or ge:nusspecific primers. However, for il1.11' of these developments a 1 argc number of reliable identified strains of the species [1.1 que st ion will h ave to be su bjcctcd to the sy stern to test its suitability,

The two species M. Imeus and K. varilms share: in the the 16S rRNA ge:ne.s. less than 95% simil erity and hence a PCR may be developed to distinguish the two species. Since the 16S rRNA similarities are qjU ire low it appeiHs to be achicvable IQ develop 0 d u plcx peR wi th t ru roc or lou r prnuers which amplifies two 16S rRNA gene fragments of different sizes specific for M, hlterts and K. IVtlrimrs, respectively.

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