Identification of antimicrobial producing Enterococci isolated from Iranian raw milk cheeses using cultural methods

Mohammad Reza Edalatian, Mohammad Bagher Habibi Najafi, Seyed Ali Mortazavi

Department of Food Science and Technology,Agriculture Faculty, Ferdowsi University of Mashhad line

Mashhad, Iran.e-mail: edalatian@um.ac.ir

Abstract— A collection of Enterococci spp. (about 96 isolates) were isolated from two Iranian raw milk cheeses, known as Lighvan and Koozeh cheeses and identified as Ent. faecium,Ent. faecalis, Ent. durans, Ent. casseliflavus and Ent. italicus by 16S rDNA sequencing. These 96 isolates were subjected to Agar- spot and well-diffusion assay in order to detect the bacteriocin- producing ability. According to Agar- spot method, only 48 isolates out of 96, showed bacteriocin-producing ability with clear- zone production on plates against indicator organisms. With well- diffusion Assay, these numbers decreased to 20 isolates which produced clear zone. Then, these 20 isolates (strains) were subjected to rep- PCR for typing and 15 distinct rep- PCR profiles (patterns) were identified.

Keywords-antimicrobial compounds, Lactic flora, raw milk cheese, enterococci, bacteriocin.

I. INTRODUCTION

The enterococci genus (genera) constitutes one of the main part (genera) of Lactic acid bacteria (LAB) which distribute widely in the environment, food and different ecological niches (Kayser, 2003). Among the enterococcus species, Ent. faecium and Ent. faecalis are the two most occurring in food and related habitats. The Enterococci play an important role in the development of the sensory properties of fermented foods (Martin, M., et al., 2006). Also they can be used as starter and adjunct cultures (Giraffa, G.2003; Hugas, M. et. al., 2003).One of the best advantages of enterococi in food is production of a diverse and heterogeneous group of ribosomally synthesized antimicrobial peptides or bacteriocins, (the enterocins) with different spectrum of antagonism activities, structure, and processing and secretion mechanisms (Cintas, L.m, et al., 2001; Franz, et al., 1999; Franz, et al., 2003).Most enterocins belong to class II of the Klaenhammer classification (Klaenhammer, T.R., 1993), although they show a considerable diversity. Many enterococcal bacteriocins are active against the food- borne pathogen Listeria monocytogenes, while other enterocins like enterocin AS-48, show a much broader inhibitory spectrum. Some bacteriocins are also active against Gram- positive food-borne pathogens such as Listeria monocytogenes,

staphylococcus aureus, Bacillus subtilis and spores of Clostridium perfringenes (Klaenhammer 1993; Holzapfel etal. 1995; Stiles 1996).The objective of this work is to screen for enterococci isolated from two Iranian raw milk cheeses with antibacterial properties and enterocin production that may be used as bio-preservative.Most of the enterocins classified as class II bacteriocins including enterocin A (Aymerich, et al., 1996; O’Keeffe, et al., 1999), enterocin B (Casaus, P., et al., 1997;Franz, C.M.A.P. et al., 1999),enterocin P (Cintas,L.M., et al., 1997), enterocin L50 (Cintas, L.M., et al., 1998), enterocin Q (Cintas,L.M., et al., 2000), mundticin (Bennik, M.H.J., et al., 1998) .

II. METHODS AND MATERIALS

A. Strains, Media and culture conditions96 isolates of Enterococci spp. strains isolated during the manufacture and ripening of two Iranian traditional cheeses made from raw milk ( Lighvan and Koozeh) were grouped by typing and identified with (by) ARDRA, sequencing and sequence comparison. These isolates isolated from Lighvan (52) and Koozeh (44) cheeses. 15 isolates as representative were tested for the enterocin production against indicators including gram positive and gram negative bacteria. The indicator strains included Ent. faecalis, Lac. lactis MG1363, Staphylococcus aureus CECT 86, Lactobacillus sakeiCECT 906, Listeria innocua 4202, Lactobacillus plantarum748.All cheese isolates and indicators were recovered on BHI agar, M17 agar (for Lactococci), MRS agar (for lactobacilli), or in Trytone soy broth (TSB) (for Listeria innocua and S.aureus), from -80 °C, then the plates incubated at the corresponding optimum temperature for 24-48 h.

B. Identification and typing of isolatesTotal genomic DNA was extracted from single, isolated ���������������� ������� ������������� ��������water (sigma- Aldrich st. Louis, MO, USA), heated at 98°C for 10 min in a thermo cycler (Bio-Rad Richmond, CA, USA), and some of the isolates, cell extracts were obtainedwith glass beads in a Mini bead Beater apparatus (Bio spec Products, Bartlesville, OK, USA), and centrifuged for 5 min at 16000 rpm. Isolates were identified by ARDRA, followed by sequencing of representative amplicons and comparison of the sequences obtained against those in databases. Enterococci spp. was grouped by repetitive extragenic palindromic (REP) fingerprinting employing the polymerase chain reaction (PCR) and the primer BOXA2R, as reported by koeuth et al. (1995).

180

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il: edalatian@um.ac.c.irirr

Abstract— A collection off EEEEnnttererococcii s ssppppp. . (a(a(a( boboutt 9 9 966 isissololololol tatateses) ttwere isolated from twoooo I I Iraraninianan rrawaww m mililk kkk chchhhheeeeeesesees,s, k kkknnown aas sLighvan and Koozeh hh h chcheeeesses anndd dd ididideenenntititififififieedddde a as s s EEnEnEntttt.t faecium,Ent. faecalis, Ent. dddduuuururananss, EEnt. c casasasssseseliliflflfllaavavavusssus aanand d EnEnEnntt.t ita s by 16S rDNA sequeeeenncncn ing. Theseses 996 isii olo atteeessss w wereereree e suubjjee d to Agar- spot andddd d well-diffuusiionono a asssayy in orrder tot dete ect thebacteriocin- ppppproducucining ababililiti y. AAccccordiiid nggg ttto o Aggar- spoottmethod, on y 8 isol o 96, ed acterioproducing ababababability withh c learar- zone prprododucccttttiononnn ooo n nn pllata es agaagainnstts indicator orororrorganisms. WWitith h wew ll- ddififfufusisionnon A AAsssayayyay, , ththesesse e ee nunumbmbbere sdecreased ttot 220 issolatteses wwhichch proroduduceced d d clclc eeaarr zozozzoooz nnene. Thenen, , ththt ese 20 isolatessss s (strains) wewerere ssububjeectcteded t too reerepp-p-p-p PCCCCRRRR R foffor tyypipiingngngg aaaandndndn 15 distinccccct rep- PCRR prproofiless ( (papattttere ns) wwweweweree iiddedeededentnttntified.d

Keyworddssd -antimimiccroobibialal comompounundsds, , LaLaacctcticicc ffloraa, rararar ww ww mmim lkk cheese, ennnnterocococccci,i, b bactteririococinin.

I. INTRODUCTCTIIIOOONN

The enterrrroococci gennusus ((genenera) consttitiitutuut tes s ononnneee ofofof ttthhehehe mmm aiain npart (geneeeerrarrr ) ) of Lactitic c acidid bacteria (L(LLLLABABA )) ))) whwhicch h didiiststtriribbub te widely in ththt e enviroonmnmenent,t foood d andd d d diddiffffffffferererererereneenennttt t ececece oologgggicici al niches (Kaaayyyyysere , , 200303).) AAmomongng tthe eentnttntn erere ococococoocccuccus s sspspececcieiees, Ent. faecium anaa d Ent. ffaeecacaliliss ara e thhee twtwoo mmomom ststtsts oocccccuuruururriiirinngngn in food and relatededededed hhaba itatts.s. T Thehe E Entntererococococccii ppplalay y anannn i i impmppooro tant role in the deeevvvvevelopmenent of theh ssenensoos rryry ppprrororr pepepertrtrtieies of fermented foods (MMMMMara tin,, M., eett al., 2006)6). AlAlA ssoo ttttthehehehhh y y y cacaaan n be used as starter and adddddjujujujujunct t cucultureses ((GiG raffffaa,a, G G .2.20000 3;33;33;3 HH HHH ugugugasas,, M. et. al., 2003).One of the best advantntntntntagagaaa ess o off enteerocococicicicci ii n n fofoodod ii s sproduction of a diveveeerrrsrsee anand hetterogegeneneouuousss grg ouououp p p off ribosomally syntheheeesizezed d anntit microbial pepttididddidesesesese o o rr rbacteriocins, (the eenttn ereroco inns)s witthh different spectrumm oo oo of antagonism activities,s, sstrt uctuturer , andd prp ocessing andnd secretion mechanisms ((CiCintntasas,, ,, , LLL.L.LLL.m,m,m,m,mm,m e e tt alal.,., 200001;1; F Fraranznz,, etettee al., 1999; Franz, et al., 2003).).).))Most enterocins belong tttto o clc ass s IIIII ooofff ff thththe ee KlKlKlKlKlKlaeaeaa nhnhamammmmmemeeemerr rclassification (Klaenhammmmmemerr, T T.R.RRR.,,. 11 19999993)3)3), , alalala thththhhhough they yyshow a considerable dididiversiity. MMMM anany y eeenenenterococcal bacteriocins are active against the fof od- bborne pathogenListeria monocytogenes, while other enterocins like enterocin AS 48 show a much broader inhibitory spectrum

stst Bacillus subtilis and spores oClClCClososostrtrtrridium perfringenes (Klaenhammer 1993; Holzapfel ealalal. 199995959 ; Stiles 1996).ThThTThe e oboobjejej ctive of this work is to screen for enterococciisisololatatededed from two Iranian raw milk cheeses witantibactc ereriaiaiaia p operties and enterocin production that mbebe used d d asasas bb bioiioio-pp-p-prererer ses rvative.MoMoMoststst of ff ththe ee enenteteroroociciicicinsnsnsnns c lassified as class II bacteriocinininclclcl dududinininggg enenttteteterorroror ciciiccinn n n A AA (AA(A(Aymymymmmmereereericii h, et al., 1996; O’Keeffe, ealal., 1999), enene teteerorocin n B BBBB (C(C(C(( asasa auauuauus,s,s,ss, P ., et al., 1997;FranzC.M.A.P.P eeet t aalal.., 11999999 9)9),e,entnnnn eroccininnin PPPPPP ( ( ( Cintas,L.M., et al1997), entnterocinin L5050 (((CiCiC ntntassasas, , L.L.LLL.L.L MM.M.M.MM , , eeteet aaaaal.l.l.ll , 1998), enterociQ Q (Cinntataas,s,L.M.M., etete a al.l.l., , 2020200200000000 ),) m mmuuununndtdtticicicinininin (( ((BeBeBennik, M.H.J., eal., 199998)8)8)8) .

II. MMMETHETHETHODSODSODSODS ANDDDD MMMMATEATEEERRRIAIAIAIARR LLSLSLS

.. Strains, Media and cultuurere c conno ditioonnss96 isolalatet s of Enteroocoocococccciii spsppp.p.p.p. ss trtraiaainsnsssn ii i issoolalaaateteteteeed d dududurririing thma facture and ripepeenininninnngn fff twtwtwtwoo IrIrananiaiiaian n trtradda iti iooonanananalll l chchhccheesemadee f from raraw w milk (( Ligighvhvanan a andd K KKoooozzezeh)) ww wererrere e grgrgrggrooupeby typyping and idenntit fied wwitittth h h h (b(b(( y)y) A AAARDRDRDRDRARARA, , sssssseqeqee uueuencncccnncinininng g anssequence comparison. These i isosolates isisololateddded ff froromm m LiLiL gghghg va(52) and Koozeh (44) cheeses. 15 isolaatetes as repreresentntntntatattivweweree t tese ted fofor the enterocin productionon against iindicicccatattaata ooroorincludddinini gg gram positive and gramam negattivive e bacttererriaiaiaia. . ThhThTininindidid cator strains included Ent. ffaecalis, LaLacc. lacaccctititisss MGMMGGGM 111131 6633StStapapphyhylococcusus aureus CECT 86, Lactobobbaccca iliillluuluus s s sasaakekCEECEECTCTTC 99 060606, , Listeria innocua 4202, Lacttobobacaca ililluus ss pplplaanantataarruurumm74488.AlA l chheeeese iiiisoss lates and indicatoorsrs wwwererree e rerecocoocoveveredd ononononnononnn B BBBHHHagar, M1M11M177 7 agaggara (fofor Lacttocoococccci),), MMM MRSRSS aagagagagarr r (f(folactobaccilillli), oor in Trytone soyoyyy bbbrrrorothhth ( (TTSSBB)B) (( (fofof r r LLiLiiiiiistssts ereriiinnocua and d S.S.aurereeusuuus),) fromm m -8-8-8800 0 °C°C,, thenen thehee ppplalalateteeincucubabab teeted d at the corrrrereespsppoononondingng o optptp imimimmumum temempeperratuurere ffffoorrrroo 22224444488 h h.

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C. Detection of Antimicrobial activityFor detection of antagonistic activity, an Agar spot test and a well-diffusion assay were applied, successively. The agar spot test was a modification of Fleming et al., method (Fleming et al., 1985). Overnight cultures of the strains to be tested for production of an antimicrobial compound were spotted 5�� ���� ��� � ����� �� ���� ������� ���- agar +0.2%Glucose, M17- agar +0.2%Glucose) and incubated at 30°C for 24 h to allow colonies to develop. Spots were covered with 10 ml of soft-agar (0.75%) inoculated at 0.25% with indicator bacteria. The plates were incubated under the required conditions for indicator bacteria. After incubation for 24 h at 30°C, the plates were checked for inhibition zones. Those strains (isolates) produced clear zone equal to 0.5mm or larger, considered positive in this method (agar spot). Then, in the second method, well-diffusion assay, the positive strains from previous stage, were selected for bacteriocin production evaluation. In this method, firstly, indicator strains were cultured (grown) in broth medium corressponding to each indicator (MRS, M17 and BHI) overnight.������������������� �����!"�����#����������of an overnight culture of the indicator strain and poured into Petri dishes. 1 ml of overnight culture of the producing strain transferred to 1.5 ml microtube and centrifuged. The resulting supernatants were neutralized to pH 6.5-7.0 with NaOH 0.1 M, centrifuged at 14000 rpm for 5 min, and filter-�������$�� ���� �� � �&�� �� ���� ���'���� �*����pore, Bedford, MA, USA). Wells of 3 mm in diameter were cut into ����� ���� ��������� �� �� �� ���� � ��������� ��the potential producer strain was placed into each well. The plates were incubated for 24 h under appropriate conditions and were subsequently examined for zones of inhibition.

III. RESULTS AND DISCUSSION

A. Identification and typing of enterococci spp. isolatesAmong 130 isolates from two Iranian traditional cheeses, Lighvan and Koozeh, 96 isolates were identified as Enterococci by ARDRA and sequencing of some representatives of 16S rDNA PCR-amplicons and comparison of the sequences. From these 96 enterococci spp. 52 and 44 isolates belonged to Lighvan (Ent. faecium(38), Ent. faecalis (11), Ent. durans (1), Ent. italicus (1) and Ent. casseliflavus (1)) and Koozeh cheese (Ent.faecium (36), Ent. faecalis (5), Ent. durans (1) and Ent. casseliflavus (2)), respectively.

B. Antimicrobial activity of enterococci spp.Firstly, the production of enterocin (bacteriocin) by representative isolates of the different strains against some indicators including Listeria. Innocua, Staph. aureus, Ent. faecalis, Lb. plantarum, Lb. sake was analyzed by an agar spot test. Among 96 isolates, 48 isolates showed the inhibitory effect (clear zone) against the different indicator organisms (data not shown). Lb. plantarum CECT 748 was

inhibited by 27 strains. In contrast, S. aureus CECT 86, was inhibited only by 20 strains. Ent. faecalis, Lac. lactis ssp. cremoris MG 1363, Listeria. innocua were inhibited by 38, 3 and 32 strains, respectively. In next stage, the second method, well-diffusion assay, was applied for all strains with antibacterial activity against any of the indicators. Under the conditions of well- diffusion assay, the number of strains which produced clear zone, was decreased as only 20 strains showed clear inhibitory effects (Table 1). These results were in agreement with others’ results; many authors have reported that conformation in liquid media of the inhibition detected by the agar-spot test is not always obtained (Schillinger and Lucke 1989; Larsen et al., 1993; Martinez et al., 1995; Hernandez et al., 2005). Several colony-associated antimicrobial compounds , like fatty acids and H2o2, have been considered to be responsible for the inhibitory for the inhibitory effects observed is solid media (de Vuyst and Leroy, 2007).The number of strains which inhibiting the indicators used in this study were as follows: Ent. faecalis (18/48) strains, Lac. lactis (3/48) strains, Staph. aureus- 0 strain, Listeria innocua- 25 strains (most of them showed strong inhibition), and Lb. plantarum- 2 strains (Table1).Most of these inhibitory strains were shown to belong to Ent. faecium and C34, C35, LR71, KR24 proved to be Ent. faecalis and KR 47 was the only Ent. casseliflavus. Then, all these 20 inhibitory (positive clear zone) strains (isolates)were subjected to rep-PCR for typing with primer BOXA2R (Figure 1).According to rep-PCR profiles, 15 out of 20 strains showed distinct typing profiles.(Figure 2).As we can see in table 1 (well-diffusion assay), some strains can inhibit the Ent. faecalis but with weak inhibition (clear zone), only 2 strains showed the inhibitory effects against Lac. lactis. Most of strains showed the inhibitory influence towards Listeria. innocua. Among all these strains, LR74 (Ent. faecium) showed the widest spectrum of inhibitory since it produced clear zone against Ent. faecalis, Lac. lactis, Listeria innocua and Lb. plantarum. None of the strains showed the antimicrobial activity against Staph. aureus.In conclusion, we detected 20 isolates (producing strains) which produced clear-zone (bacteriocin-like subtances) using Agar- spot and well-diffusion assay. Among these 20 isolates, we detected 15 different distinct isolates according to rep-PCR profiles (patterns). From these 15 bacteriocinogenic strains, 11 of which belonged to Ent. faecium .

181

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inhibition zones. Those strararainininins (isolatees)s) p pror ducececeddd clclcleae r zone equal to 0.5mm orr ll llaarrgeger,r ccononsisisidededeererered d dd poposisiittitiveeve i iiinn thhisis method (agar spot). TTThTThenen,, in ttthehe s ssececececonononnd dddd d mememethththhodod, welll-diffusion assay, thehehehe p pososititivi e ststtrrararaininns s frfrfroomommmo pprrereviviviouus s stage, were selected for r r r babactcteerioociin n ppprprododucctititit ononnnn e vavaalululululuuaation.n. this method, firstly,y,y,y, iindicator sstrrr iaiaiinsns wwere e e cccculttltuurredededdeded ( (grgroown) in broth mediummmm ccorreesssspondddining tto eeach innndidicccc oooorr r (M(M( RSR , M17 and BHI) ov������������������������� �� �����!""!"������������������ #�## ����������� ���� of an overrrrernight cultlturure ofof t he i indndici atatattooror s trtrt aaiaia n n anannd ddd popourured into Petri ddisshes. 1 mml l ofof oovvernnigighth ccuuulllu tutut rere ooooffff f ththt e prrododducuucu iniing gstrain traannnnnsferred to o 1..5 5 mlml mmicicrorotutubebe aanddnd c ccccccenenentrtrifugggeddded. . ThThThThe eeresulting supernrnatatanantsts w werre e neneututralizezeddd d dd tooo p pppppppH HHH 6.5-7...00 00 wiiw hth NaOH 0000.1 MM,, cecentntririfuf geed d at 114040000 rrrrpmpmmmp ff oor 55 5 mm miniinn,, anana d dfilter-�����������$��� �������� � ��� �� �&�&�� ���� ������������ � ����������� ''�'���������� �*�*�*�*�������������pore, Beddddfd ord,d, MMA,A, U SAA).) WWele ls of f 3 3 mmmmmmm iiiin dddiddiddiaamam teteterrer w were ecut into ����������� ������� ����������� �� ��� �������� ���� ��� � � �� � � ��������������������� ����the potenntntttial produccere strtrain n was placceededde i intnto oo o eaeaeaaccchchhch wwweleelll.l. T Theeh plates werrrrre incubateed d for 242 h underr a aaaappppproorooopprppriaiattee cc conononddddiititiioons and were sssssububuu seequenntltly y examamined fforo zzoonnono esese o oooooff ininnhihiihibibibititioon.

IIII. RESUESULTSLTS ANDRR DDISCSCUSUSUSSSSUS IONIONNN

A. Identificaaatititititionn a nd ttypypiningg ofof e entnterrococococccccici spsppp. isiissisollollatteesesAmong 130 isolololllatataaa es froom m twt o Irrana iaiann trtraaadadiitioioonnanaann ll chchcheeee ses, Lighvan and Kooooozozooo ehe , 96 iisosolates weweerrere i idedeededded ntntntnnn ififfieieieieddd d as Enterococci by AAAARRDRRR RARA a ndd s sequeennncccining g ofoofoffo ss omomo e e representatives of 11116S6S666 rrDNA A PCPCR-R-amamamaa plplicicons s anannaa dd comparison of the sequennnnncecc s. F Fror m thhese e 9696969699 eentntererococococcici spp. 52 and 44 isolates s s bbebeb longnged to Lighghvavan n (((EnEnEnEnEnt.t. f ffaeaeaeciciumum(((((38), Ent. faecalis (11111)1 , EnEnt. durranans (1), Ent. iittalicuuss (1(1(1(1(1) ) )) anand d dEnt. casseliflavus (11)))) aandn KKooo zeh chcheee se (Ent.faecium (333(36))6)6)6), ,,Ent. faecalis (5), Ent. dduranans (11)) and Entt. c casa seliflavus (2))))))) , ,respectively.

B. Antimicrobial activityyyy oo o oooffff ff enteerorocococ cccccccccciiiii i ssspspss p.p.Firstly, the production ofofof e enttntteeererococoo ininn (( ( (babab ctctctc eererrrriocin) bbbyyy yrepresentative isolates of ttthhhehhe dddd difififiiffffefereeerentntnnn s ssstrttraiainsn aaaaggggag inst someindicators including Listeria. Innocua, StStStaphhhhh. aureus, Ent. faecalis, Lb. plantarum, Lb. sake was analyzed by an agarspot test Among 96 isolates 48 isolates showed the

3 3 3 3 anaanananandd dd 32 strains, respectively. In next stage, the seconmemmethtththhth ddd, well-diffusion assay, was applied for all strainwiwiththh aantibacterial activity against any of the indicatorsUnUnUUnder r tht e conditions of well- diffusion assay, the number o

ww ich produced clear zone, was decreased as only 2ststs rarainins ss shshshshs owed clear inhibitory effects (Table 1). Thes

sull rrrrr in agreement with others’ results; many authohahhave repepporororteteteteddd dd tht at conformation in liquid media of thinnhihihibibibitit onon d detee eccteteted ddd d bybybybbyby the agar-spot test is not alwayobobbttatainin deded ((((((ScScScScchihihiihillllllll iniinininggeger ananndddd d LuLuLuLLL cke 1989; Larsen et al., 1993MMaMartinez et alal.,. 1 1999999995;5;5;5; HHHHerre nanaandndndndn ez et al., 2005). Severacolony-aassocococociaiaaatetet d aantiit mimiiicrcrobooooo iaiall cocooc mpmpmpmpmpmpounds , like fatty acidand H2H oo22,, hahaveve b eeen n coconsnsidididididdererrere ededdeddee tt t to o bebebeebe r r esponsible for thinhibitory for the inhnhibibbbitititorororory efefeffefeecttctcttcc s ssss obobobbobseses rvrvvedee is solid medi(d(de VuVuysysysst ttt anana d d LLeLeLerrooyy, 22 00000777)7)77 ..The numbmbeer oof f ststrainins s whwhicich h inhibibiii itititingngggg tt t tthehehhhe i indndn icators useinn t his study were aass fofofolllowowowowsss: Enntt.t.t f fffaeaaea caaaacalliliilill ssss (1(1118/8/8//8/48444 ) strains

l i (3/48) s i , SStS h 0000000 Lnnocua- 25 strains (m( osost offof ttheheemm m shshshhsshowwwedededd s tron

inhibitiionon), and Lb. plplpp anannantatarruruumm- 2- 22- 222 ss strtraiaiainsnssn (( ( ((TTaTaT blblbllle1e1e1eee ).))Mostt o of these inhibibitototorrry strtrtraiiaiainns wwerree e shshoown n toto bb b bbeleleleeloonoo g tEnt. ffaeciumm aand C34, C3535, LRLRLRL 7171, KKRR24242 pproor veveed d dd totooo b bbbbee Enfaf ecala is and KR 447 7 was ththhheeee oononlyly EnEnEntt.t. c casassesseseeelililil flff avavavuususss.. ThThenaall these 20 inhibitory (positive clear zozone) ststtrarainins (isososoolalateswere subjected to rep-PCR fof r typing wwitith h primer BBOXXXXA2A2A R(FFigigururee e 1).Accoo drdrdining to rep-PCR profiles, 155 oout of 2200 strainnss s s ss shshshhshowowoweedididistststinct typing profiles.(Figuree 2 ).Ass w wwe e can see e in table 1 (well-diffusion assayyy),), sss ommmmee e ststs raraaaininininicacann n ininhhhibibiit t t ththt e Ent. faecalis but with wweaeaeakk inini hiih bibibbibititititioonon (((cclclcleaezoz nene))), onlnllyy y 22 2 strains showed the inhnhhiibibbitittiti oryyy y y efefe fefecttss agagaggaaiaiinssnLLac. llacaccctittit sss. MoMM st of strains showweded t theheeh ii nhnhibibibititorory inininnflflflflflflflflfluuueueeuenncctowarrdds s LLiLiL steriaia. innocuc a. Ammononng g gg ala l l ththththesese ee sttstrar ininnnsss,s, LL L R7R7(Ent. faeciumm) ) showed the wwiidddddeseesestt tt spsppece trtruumm oo oof if inhhhibibbbibibi iittororsince itit produceced clclclleeeaear r zonee aaagagagagainiinstst EEnnt. ffaeaecaalililiisss,, LLaLaaaccclaactctissi , LLListeria innnnnnococo uaua aand d LbLb. . plplplanantat rurumm. NNoonnene ooo off f fff thththhstrains showed ttheheeehehehehh a a a a nntntimimmiicicrororoobibb al aactctcctivivivitity aggaaiiiaiaiainsnsnnn tt SSStStStStapapapa hhaureus.InInInn cononclusion, we e deetetteeeectctcc ed 22222220 0 isolateses ((((prprrpp ododducucccininingg ststtrraraininnswwhhici h prodducuceded ccclelelel ara -z-zonononoonee ee ((b(bb(b(b(b(( acacacacacaccteteteteeriririririrr ooco ini -llllikikke e sususususus btbttbtana ceesssusing AgAgarar- spot annandd d wewell-d-dififfuufusisiis onono aaa ssayy. AmAmmonono gg ththhhheseseseseseese ee e 222isollateses, wewew d deetetecected dd 15515 ddd ififfefererentnt dd disisstitiit ncncncn ttt isiisisoololatatese accororrrdintotoo rrrepepp-P-P-P-PCCRR p profileess (p(p( atatatteteternnrns)s). . FrFrFromom thesese 111bab ctttererioiocicinonogegeninicc ststrarainins,, 1 111 ofofof w w hichhh b bb lelelononongegegedddd d totototo EnEEEfaeccciiuiumm .

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