372 Jurnal f Fd Prtectin, Vl. 50, N. 5, Pages 372378 (May 1987) Cpyright^ Internatinal ssciatin f Milk, Fd and Envirnmental Sanitarians Fate f Listeria mncytgenes During the Manufacture and Ripening f Camembert Cheese ELLIOT T. RYSER and ELMER H. MRTH* Department f Fd Science and The Fd Research Institute, University f WiscnsinMadisn, Madisn, Wiscnsin 53706 (Received fr publicatin February 6, 1987) BSTRCT The ability f Listeria mncytgenes t survive the Camembert cheesemaking prcess and grw during ripening f the cheese was examined. Pasteurized whle milk was inculated t cntain abut 500 L. mncytgenes [strain Sctt, V7, Califrnia, (C) r Ohi (OH)] CFU/ml and made int Camembert cheese accrding t standard prcedures. ll wheels f cheese were ripened at 6 C fllwing 10 d f strage at 1516 C t allw prper grwth f Penicillium camemberti. Duplicate wedge (pieshaped), surface and interir cheese samples were analyzed fr numbers f L. mncytgenes by surfaceplating apprpriate dilutins made in Tryptse Brth (TB) n McBride Listeria gar (ML). Initial TB dilutins were stred at 3 C and surfaceplated n ML after 2, 4, 6 r 8 weeks if the rganism was nt quantitated in the riginal sample. Selected Listeria clnies frm duplicate samples were cnfirmed bichemically. Results shwed that numbers f Listeria in cheese increased 5 t 10fld 24 h after its manufacture. Listeria cunts fr strains Sctt, C and OH decreased t <10 t 100 CFU/g in all cheese samples taken during the first 18 d f ripening. In cntrast, numbers f strain V7 remained unchanged during this perid. ll L. mncytgenes strains initiated grwth in cheese after 18 d f ripening. Maximum Listeria cunts f ca. 1 x 10 6 t 5 x 10 7 CFU/g were attained after 65 d f ripening. Generally, a 10 t 100fld increase in numbers f Listeria ccurred in wedge r surface as cmpared t interir cheese samples taken during the latter half f ripening. During this perid, Listeria grwth paralleled the increase in ph f the cheese during ripening. Listeria mncytgenes is an pprtunistic fdbrne pathgen which can cause abrtin in pregnant wmen as well as meningitis in newbrn infants and immuncmprmised adults (20,30,37). Recently, tw large utbreaks f listerisis, which were previusly described (35), resulted frm cnsumptin f cntaminated pasteurized milk (18) r Mexicanstyle cheese (22). Because f resultant increased surveillance, L. mncytgenes was recently islated frm Liederkranz (6,12), Mrbier Rippz (French semisft cheese) (10), Turre de ubier and Frmages des Burns (French sftripened cheese) (10), Mexicanstyle cheese (5,7) and Brie (2,3,4,8,9,11,14). During February and March f 1986, apprximately 60% (ca. 680 millin lb.) f French Brie cheese n the merican market was recalled because f cntaminatin with L. mncytgenes (15). N cases f listerisis were linked t cnsumptin f this cheese in the United States; hwever, several cases did ccur in bth England (13) and Canada. Presently, nly limited infrmatin exists cncerning the fate f L. mncytgenes in cheese. Studies dne in Yugslavia (39,40) and Bulgaria (21) indicated survival f the bacterium in 28dld fully ripened white brined cheese, survival in unsalted cheese made frm skim milk and survival fr 1560 d in white pickled brine cheese, all made frm ewe's milk. Our previus wrk (34) shwed that L. mncytgenes survived during manufacture and strage f bth creamed and uncreamed cttage cheese held 28 d at 3 C. Mre recently (35), we demnstrated that this rganism can survive as lng as 434 d in stirredcurd Cheddar cheese ripened at 6 C. Recently recalls f Lwrenacntaminated Brie cheese have prmpted cncern abut survival and grwth f this rganism in sftripened cheese. Hence, this study was undertaken t determine if L. mncytgenes culd survive during manufacture f Camembert cheese and grw during subsequent ripening f the cheese, thus creating a ptential health hazard. MTERILS ND METHODS Cultures Fur strains f L. mncytgenes were used in this study strain Sctt (clinical islate, sertype 4b), V7 (milk islate, sertype 1), Califrnia (C) (sertype 4b, islated frm Mexicanstyle cheese implicated in a 1985 utbreak f listerisis in Califrnia) and Ohi (OH) (sertype 4b, islated frm recalled Liederkranz cheese manufactured in Ohi). ll cultures were maintained n Tryptse gar (T) slants stred at 4 C and transferred mnthly. Listeria cultures used t inculate milk fr manufacture f Camembert cheese were prepared as previusly described (35). sufficient vlume f the final culture was dispersed in 50 ml f pasteurized milk which was then added t 50 lb. (22.7 kg) f pasteurized whle milk in a small cheese vat t yield abut 5 X 10 2 L. mncytgenes CFU/ml f milk. cmmercial Streptcccus cremris starter culture, CC6, (Marschall Divisin MUes Labratries, Inc., Madisn, WI)
LISTERI IN CMEMBERT CHEESE 373 was used fr cheesemaking. The starter culture was incubated 1618 h at 21 C in previusly steamed (45 min, 100 C) recnstituted nnfat dry milk (10% ttal slids). sufficient amunt f the resulting starter culture was prepared t prvide an inculum f 2% (w/w). Penicillium camemberti was grwn n bttle slants f Czapek gar incubated at 21 C fr 7 d. Mld grwth frm ne agar slant was manually dispersed in 50 ml f sterile distilled water and the resulting suspensin was added t 50 lb. f milk during cheese manufacture. Manufacture and sampling f Camembert cheese Camembert cheese was manufactured accrding t the prcedure utlined in Table 1. Pasteurized [75.0 C (167 F)/16 s] whle milk [50 lb. (22.7 kg)] was placed in a stainless steel cntainer resting in a piltplantsized, steamjacketed cheese vat filled with water. The milk was inculated as described earlier. fter warming the milk t 33.3 C (92 F), 2% (w/w) lactic starter culture was added alng with spres f P. camemberti and 6 ml f the cltting agent Marzyme Single Strength Micrbial Rennet (Marschall Divisin Miles Labratries, Inc., Madisn, WI) diluted 1:40 in tap water. The cagulum was cut int cubes using 1.90cm (0.75in.) cheese knives 50 min after additin f rennet. fter 20 min, curd was dipped int ten 4.5in. (11.4cm) diameter hps and allwed t drain. Hps were turned twice at 2h intervals. fter draining vernight at 2125 C, cheese wheels were drysalted and ripened 10 d at 1516 C (59.060.8 F) with 8595% relative humidity t allw fr prper mld grwth. fter 10 d, cheese wheels were wrapped in fil and ripened at 6 C. TBLE cheese. Step Pasteurizatin ddl. mncytgenes dd starter culture dd P. camemberti dd rennet Cut curd Dip int hps 1st turn 2nd turn Salt Typical manufacturing schedule fr Camembert Time 9:00 M 10:00 11:00 11:00 11:00 11:50 12:10 PM 2:10 4:10 11:00 M Temp.;C ( F) 75.0(167) 10.0 (50) 33:3 (92) In additin t standard labratry practices, several safety precautins were fllwed during Camembert cheese manufacture n recmmendatin f the University f Wiscnsin Bilgical Safety Cmmittee, and were similar t thse previusly described (34). Duplicate samples fr enumeratin f L. mncytgenes and determinatin f ph were taken during manufacture accrding t the fllwing schedule: (a) pasteurized milk, (b) inculated milk, (c) curd at dipping, (d) curd after 2nd turn and (e) curd after salting (0 d). Three different cheese samples wedge (pieshaped), surface and interir were taken in duplicate after 4, 10, 17, 24, 31, 45, 59 and 66 d f ripening and analyzed fr numbers f Listeria and ph. Wedge (pieshaped) samples cnsisted f 20 g f cheese frm the wheel; whereas surface samples cntained 20 g f cheese frm the uter 5 mm f the wheel, including the mat f mld. Interir samples cntained 20 g f cheese, whenever pssible, frm the unripened cre. s ripening prgressed, the size f the interir cheese sample decreased t a single 5g sample at and beynd 31 d f ripening. Enumeratin fh. mncytgenes Duplicate samples f milk (20 ml), curd (20 g) r cheese (20 g) were placed in sterile Stmacher bags f ca. 500ml capacity (Tekmar C., Inc., Cincinnati, OH) and diluted 1:10 with sterile Tryptse Brth (TB). ll samples were prcessed and analyzed fr L. mncytgenes using a cmbinatin f surfaceplating n McBride Listeria gar (28) and, when necessary, cldenrichment in TB, as previusly described (34). In additin, ne clny frm each pair f duplicate samples was cnfirmed as L. mncytgenes accrding t the prcedure f Ryser and Marth (35), which is based n a psitive catalase reactin, tumbling mtility, appearance f bluegreen clnies n T and results f bichemical tests fund n PI 20S test strips (nalytab Prducts, Plainview, NY). Survival fh. mncytgenes in cheese f varius ph values Thirty wheels f Listeriafree Camembert cheese were manufactured and ripened at 6 C t btain cheese at varius ph values. fter 4, 15, 24 and 50 d f ripening, the rind cntaining the mld was remved frm each cheese wheel and the remaining cheese was kneaded by hand in a large Whirl Pak bag t btain fur 100g cheese samples at unadjusted ph values f 4.6, 5.5, 6.1 and 7.4, respectively. The resulting cheese paste was inculated with L. mncytgenes strain Sctt, V7, C r OH at a level f ca. 100 CFU/g, thrughly kneaded by hand as just described and stred at 6 C. t apprpriate intervals, duplicate 5g samples were diluted 1:10 in TB and analyzed fr L. mncytgenes as described earlier using bth directplating and cld enrichment. Selected clnies were cnfirmed as L. mncytgenes as previusly described. The cheese ph was determined as previusly described at the time f bacterilgical analysis and remained cnstant thrughut the experiment. Survival f L. mncytgenes n the surface f Camembert cheese Thirty wheels f Listeriafree Camembert cheese were manufactured as previusly described. Once prper grwth f P. camemberti had ccurred after 10 d f ripening at 1516 C/85 95% R.H., a single 20cm 2 was marked ff n the tp and bttm surface f each cheese wheel and inculated with 0.1 ml f a suspensin f L. mncytgenes strain Sctt, V7, C r OH t prvide ca. 500 CFU/20 cm 2. This inculum level was nt attained in all samples. fter spreading the inculum with a sterile glass rd, the surface was allwed t dry befre the wheels were wrapped in fil and ripened at 6 c. t apprpriate intervals, duplicate 20cm 2 surface areas frm the same cheese were cut deep enugh t give 10 g f cheese which was diluted 1:10 in TB and analyzed fr L. mncytgenes as previusly described. Selected islates were cnfirmed as L. mncytgenes. The cheese ph was als determined at the time f bacterilgical analysis. Determinatin f ph and cntents f misture, milkfat and salt The ph f Camembert cheese was determined at the time f bacterilgical analysis using a ph meter (Crning Mdel 10) equipped with a flatbttmed standard cmbinatin electrde (31). Duplicate misture determinatins were made n tw 3g cheese samples by calculating the percentage f weight JOURNL OF FOOD PROTECTION. VOL. 50, MY 1987
374 RYSER ND MRTH difference in cheese befre and after drying in an atmspheric ven fr 16 h at 110 C, as described in Standard Methds (31). Milkfat determinatins were made n duplidate 9g cheese samples using the Babcck methd (31). Duplicate NaCl determinatins were made n 10g cheese samples using the Quantab Chlride Titratr test strip methd (Miles Labratry, Inc., Elkhart, IN). RESULTS ND DISCUSSION Cmpsitin f cheese Results f misture, fat in the dry matter (FDM) and salt determinatins are in Table 2. The Federal standard fr Camembert cheese prvides that this cheese cannt cntain less than 50% FDM (19). This criterin was met in all trials except trial 10, in which the FDM was 49.0%. The average FDM fr all 13 trials was 50.9% (SD ± 1.45). TBLE 2. Misture, fatindry matter (FDM) and salt cntents f Camembert cheese. Trial Misture (%) FDM (%) Salt (%) 1 2 3 4 5 6 7 8 9 10 11 12 44.1 55.3 59.0 56.6 53.0 60.0 58.3 52.6 54.1 53.1 49.7 57.0 51.5 50.0 53.0 54.2 51.5 50.6 49.0 50.7 3.1 2.3 2.5 3.7 1.7 2.1 1.9 2.2 2.1 2.3 2.2 2.2 N standards exist fr the amunt f misture r salt allwed in Camembert cheese. Cmmercial Camembert cheese cntains apprximately 52.5% misture and 2.5% salt (25). The misture cntent f cheese frm ur 12 trials ranged between 44.1 and 60.0%, with an average f 54.4% (SD ± 4.42). Similarly, salt cntent ranged between 1.7 and 3.7%, with an average f 2.4% (SD ± 0.54). Survival f L. mncytgenes during manufacture f Camembert cheese s in ur previus wrk (35), these results best describe what might be expected if Camembert cheese is manufactured frm milk cntaminated after pasteurizatin. These results als reflect what might happen if pasteurized r partially pasteurized milk was refrigerated befre cheese manufacture, since sublethally injured L. mncytgenes cells might be able t underg repair and grw during refrigerated strage. Several small Camembert cheese factries in France have been heattreating milk at 167 F/10 s (23), which appears t be insufficient t eliminate L. mncytgenes (17) and might therefre lead t cntaminated prduct. The behavir f L. mncytgenes strains Sctt, V7, C and OH during manufacture f Camembert cheese is shwn in Table 3. ll fur strains were recvered at similar levels frm all curd samples taken at dipping when pasteurized milk was inculated t cntain 2.63 t 2.93 lg 10 L. mncytgenes CFU/ml. ccrding t Rsenw and Marth (32), the lag time fr strains Sctt, V7 and C in sterile whle milk incubated at 35 C is abut 2 h. Therefre, grwth during the early stages f cheese manufacture wuld nt be expected, just as was previusly bserved with cttage (34) and Cheddar cheese (35). Earlier studies using Escherichia cli (29) and Enterbacter aergenes (33) shwed that a 10fld increase in numbers typically ccurs during curd frmatin and is a direct result f entrapment f the rganism within curd particles. Hwever, this 10fld cncentratin effect was nt bserved fr L. mncytgenes during manufacture f Camembert as well as Cheddar (35) and cttage cheese (34). During the interval between when samples f curd were taken at dipping and at salting (Table 3), numbers f strains Sctt, V7, C and OH increased 0.65, 0.71, 0.38 and 0.44 lgi CFU/g, respectively. The cmbinatin f a highmisture curd alng with a ph f 5.50 5.30, which decreased vernight at 2125 C t 4.60, prbably allwed mdest grwth f Listeria. Similar results were als bserved during manufacture f Cheddar cheese (35) in which Listeria cunts fr strains Sctt, V7 and C generally increased 0.10.8 lg 10 CFU/g f cheese while the ph decreased frm 5.50 t 5.10 during pressing at rm temperature. Survival fh. mncytgenes during ripening f Camembert cheese Knwledge f the physical, chemical and micrbilgical aspects f Camembert cheese ripening are helpful in understanding the behavir f L. mncytgenes in this prduct. Fur structural znes can be distinguished in Camembert cheese and include the surface, rind, ripe zne and unripened cre (24). Surface and rind characteristics result frm rapid grwth f yeasts and micrccci after salting (26) fllwed by grwth f P. camemberti. The ripe zne results frm diffusin f prtelytic enzymes int cheese, which are prduced by the surface mld (24). fter hydrlysis f casein int watersluble nitrgenus cmpunds, prtelysis cntinues thrugh actin f starter streptccci and P. camemberti, which results in frmatin f lw mlecular weight nitrgenus cmpunds including amines and ammnia. During ripening, milkfat is degraded, lactic acid is degraded, and small amunts f C0 2 are prduced (27). Upn further ripening, the unripened cre decreases in size as the ammnia cncentratin increases in the rind and ripe zne. This results in the typical increase in ph shwn in Fig. 14. During the first 17 d f ripening, numbers f strain Sctt, C and OH (Fig. 1, 3 and 4), all sertype 4b, decreased 10 t 100fld in wedge (pieshaped), surface and interir samples; whereas the level f strain V7,
LISTERI IN CMEMBERT CHEESE 375 TBLE 3. Numbers fl. mncytgenes at varius stages during manufacture f Camembert cheese. Sample Pasteurized milk Inculated milk Curd at dipping Curd at 2nd turn Curd at salting a L. mncytgenes nt detected after 8 weeks at 3 C. b Lg 10 L. mncytgenes!m\ r g; average f 3 trials. Sctt < 1.00 a 2.90 b 3.04 3.21 3.69 V7 <1.00 2.68 2.78 3.08 3.49 Strain C <1.00 2.93 2.97 2.97 3.35 OH <1.00 2.63 2.66 2.57 3.10 5 _ Wedge Surface Interir _ "Cjj ^ < ' ".?*/* ^ ''/"Ss* / rt / / /»v T ' _/ / ^JV *. /< ' / / / %L '>JL~+/^^ V*>^B J* J7 I^^Zr^,' 1 p Z«uf itxxitxx 5.50 6.00 JL I I I I I I I IXJ I I I I I I I I M I I I I.mi 4.50 65 Figure 1. Survival f L. mncytgenes strain Sctt in Camembert cheese. Each pint represents the mean f duplicate determinatins frm 3 different trials. Slid symbls indicate the lgi 0 Listeria/g f cheese. Open symbls indicate ph f the cheese. Slid symbls at <].0!g l0 Listeria/g indicate results fr cld enrichment. Numbers indicate the week at which L. mncytgenes was fund. Figure 3. Survival f L. mncytgenes strain Sctt in Camembert cheese. Each pint represents the mean f duplicate determinatins frm 3 different trials. Slid symbls indicate the lg] 0 Listeria/g f cheese. Open symbls indicate ph f the cheese. Slid symbls at <1.0 lg 10 Listeria/g indicate results fr cld enrichment. Numbers indicate the week at which L. mncytgenes was fund. n ' 'X'' indicates that the rganism was nt detected after 8 weeks f cld enrichment. > z i I I II n l n I I I I H I h M I 1 I I I I I 10 15 20 25 30 36 40 45 50 55 60 65 Figure 2. Survival f L. mncytgenes strain V7 in Camembert cheese. See Fig. 1 fr explanatin f symbls. sertype 1, remained cnstant during the first 30 d f ripening. Lwer Listeria cunts in wedge and interir samples can be attributed t a cheese ph f <5.50 and a strage temperature f 1516 C during the first 10 d f ripening. During this perid a ttal f 13 samples required cld enrichment, f which 5 (38%) were psitive fr Listeria. The rganism was islated frm 3 f 9, 2 6 10 15 20 25 30 35 40 45 50 55 60 66 Figure 4. Survival f L. mncytgenes strain OH in Camembert cheese. See Fig. 3 fr explanatin f symbls. f 3 and 0 f 1 surface, wedge and interir samples, respectively. Further analysis shws that Listeria cunts fr strain Sctt and C decreased mst sharply in surface samples in spite f a rapid ph increase t abut 6.25 in bth instances. In cntrast, grwth f strain OH paralleled the ph increase in all 10 and 17dld cheese samples. L.
376 RYSER ND MRTH mncytgenes wuld be expected t readily grw at a ph f 6.25 (16) and yet grwth f strain Sctt and C was suppressed in surface samples at this ph value. Hence, ther factrs apparently were limiting grwth f Listeria. Recently, we shwed that the fur Listeria strains used in this study grew better in filtersterilized Camembert cheese whey previusly cultured with P. camemberti than in uncultured whey ver a ph range f 5.60 t 6.80 (36). pparently, P. camemberti is nt invlved in reducing the Listeria ppulatin in surface samples during ripening. high cncentratin f NaCl n the cheese surface prbably plays a minr rle in inhibiting Listeria grwth since this rganism can survive in an aqueus slutin f 25.5% (w/v) NaCl (38). fter 17 d f ripening, numbers f all fur L. mncytgenes strains began t increase in all Camembert cheese samples. Listeria cunts increased mst rapidly in surface and wedge samples; whereas grwth was markedly slwer in interir cheese samples which had a lwer ph value. minimum increase f at least 10fld in numbers f strains Sctt, V7, C and OH was bserved between 17 and 30 d f ripening after the ph f wedge and surface samples increased abve 6.30 and 6.70, 5.80 and 6.30, 5.90 and 6.30, and 5.60 and 6.20, respectively. Terplan et al. (41) als bserved similar results with sft mldripened cheese in that numbers f L. mncytgenes near the cheese surface increased > 10 fld between 21 and 28 d f ripening at 5 C. verage ph values f ur wedge and surface samples needed s all strains f Listeria culd increase 10fld were 5.90 (SD ± 0.29) and 6.40 (SD ± 0.22), respectively. In cntrast, grwth f Listeria in interir samples was less prnunced and generally did nt ccur until the ph increased t at least 5.75 t 6.00. Substantially higher cunts fr strain Sctt (Fig. 1), V7 (Fig. 2) and C (Fig. 3) were detected after 30 d f ripening in wedge/surface as cmpared t interir samples. In cntrast, higher cunts fr strain OH (Fig. 4) were seen in wedge/surface as cmpared t interir samples after nly 10 d f ripening. In 65dld cheese, the difference in the Listeria ppulatin between wedge/surface and interir samples fr strains Sctt, V7, C and OH was abut 2.2, 1.5, 1.0 and 1.8 lg cycles, respectively. Similarly, in 56dld mldripened cheese, Terplan et al. (41) bserved Listeria cunts that were 2.5 t 3.3 lg cycles higher in samples frm the surface/near surface as cmpared t the interir f cheese. Our strain OH had the highest cunt in wedge/surface samples taken after 65 d f ripening with abut 3.2 x 10 7 CFU/g fllwed by strain V7, Sctt and C with cunts f 2.0 x 10 7, 6.3 x 10 6 and 2.0 x 10 6 CFU/g, respectively. Survival f L. mncytgenes n the surface f Camembert cheese The surface f 10dld Camembert cheese was inculated with L. mncytgenes in an attempt t simulate pstmanufacture cntaminatin in the ripening rm. fter inculatin, L. mncytgenes was detected at a level f abut 30 CFU/g fr strain Sctt and <10 CFU/g fr strains V7, C and OH, which were detected after cld enrichment (Fig. 5). Strain V7 failed t grw n the cheese surface during 60 d f ripening at 6 C, but was readily detected using cld enrichment. In cntrast, strains Sctt, V7 and C grew during this perid with increases f at least 2 t 3 lg cycles. Fr surface samples analyzed after 70 d f ripening, strains Sctt, C and OH attained maximum levels f 4.9 x 10 4, 7.6 x 10 3 and 8.5 x 10 2 CFU/g, respectively. Maximum Listeria cunts were generally 2 t 4 lg cycles lwer than cunts frm surface samples shwn in Fig. 14. The harsh envirnment n the cheese surface is prbably respnsible fr the lwer Listeria ppulatin since the rganism likely had t penetrate the rind and enter the cheese befre grwth culd ccur. Figure 5. Grwth and survival fl. mncytgenes n the surface f Camembert cheese. Halfslid r slid symbls at <1 lgi Listeria/g indicate that the rganism was detected in ne f tw r tw f tw samples using cld enrichment. Numbers indicate the week at which L. mncytgenes was fund. Survival f L. mncytgenes in cheese at varius ph values Mldripened cheeses such as Camembert are unique in that a wide ph gradient develps in the cheese during ripening. Since rapid grwth f L. mncytgenes cincided with an increase in cheese ph during ripening, an attempt was made t further define the ph requirement fr grwth by inculating cheese samples at 4 different ph values. In unripened cheese at ph 4.6 (Fig. 6), Listeria cunts fr all strains, except Sctt, decreased t <10 CFU/g after 4 d f strage at 6 C. Using cld enrichment, strains V7, C and OH were shwn t survive <4, 21 and 35 d, respectively. In cntrast, strain Sctt was hardier at ph 4.6 and had an apprximate "D" value f 15 d. L. mncytgenes als failed t grw in partially ripened Camembert cheese at ph 5.5 (Table 4). Cld enrichment results indicated that strains Sctt, JOURNL OF FOOD PRO HON, VOL. 50, MY 1987
LISTERI IN CMEMBERT CHEESE 377 m Sctt»V7 C OH Sct t ' /" V7 O C OH V > 2 Q 4,4 i 1 i i t) 8 e 8 ^ 2,3 4 e 8 + 2,2.1.1.1 35 Figure 6. Survival f L. mncytgenes in Camembert cheese at a ph f 4.6. Open symbls at <1 lg, 0 Listeria/g indicate that the rganism was nt detected after 8 weeks f cld enrichment. See Fig. 5 fr explanatin f remaining symbls. Figure 7. Survival and grwth f L. mncytgenes in Camembert cheese at a ph f 6.1. See Fig. 5 and 6 fr explanatin f symbls. TBLE 4. Survival f L. mncytgenes in duplicate samples f Camembert cheese at ph 5.5. Strain Day Sctt V7 C OH 0 7 10 14 21 35 a 1.30 b 2/6 2/x 2/x 2/x 4/4 Sctt»V7» C OH ; a Numbers = weeks f cld enrichment when L. mncytgenes was detected. b Lg 10 CFU/g. C X = L. mncytgenes nt detected after 8 weeks f cld enrichment. V7, C and OH survived 35, 14, 14 and 21 d, respectively. Thus the absence f Listeria grwth at ph 4.6 and 5.5 in these cheese samples agrees with ur ther bservatins (Fig. 14). In partially ripened Camembert cheese at ph 6.1 (Fig. 7) strains Sctt and OH appeared t be hardier than strains V7 and C in that maximum ppulatins f abut 1 X 10 6 CFU/g were reached after 33 d f strage at 6 C. Strains V7 and C were again nly detectable by cld enrichment and failed t survive beynd 14 d. Finally, in cheese at ph 7.4 (Fig. 8), strains Sctt, V7, C and OH grew and reached maximum cunts f abut 3 x 10 4, 6 x 10 2, 1 x 10 3 and 7 x 10 2 CFU/g, respectively. Cunts fr strains Sctt and OH were markedly lwer at ph 7.4 as cmpared t ph 6.1, which suggests that ph 7.4 is abve the ptimal ph fr grwth f L. mncytgenes in this particular envirnment. s a result f the recalls f Brie cheese in 1986, the Fd and Drug dministratin has begun testing all shipments f French sftripened cheese and 20% f all remaining imprted sft cheeses fr L. mncytgenes (8). The French gvernment als has agreed t test sft "tu 1 11 % ~ 5 10 15 20 25 36'" ' 's5 40 45 50 55 Figure 8. Grwth f L. mncytgenes in Camembert cheese at a ph f 7.4. ripened cheese fr Listeria n a temprary basis. Currently, all shipments f sftripened French cheese must be accmpanied by a certificate stating that the cheese is free f L. mncytgenes befre being allwed entry int the merican market. Presently, the FD is advising testing labratries t analyze thin (1/16in.) slices instead f crss sectins f 25dld Camembert cheese fr L. mncytgenes. Our results clearly demnstrate the imprtance f sampling cheese which is at least 25 d ld. Hwever, testing 30dld cheese might yield better results since the numbers f all fur Listeria strains increased at least 10fld between 25 and 30 d f strage. Currently, the ral dse f L. mncytgenes necessary t cause infectin in susceptible individuals pregnant wmen, newbrn infants and immuncmprmised adults is unknwn. Hwever, accrding t the Centers fr Disease Cntrl (1), the Mexicanstyle cheese implicated in the utbreak f listerisis in 1985 cntained
378 RYSER ND MRTH abut 10 2 t 10 3 L. mncytgenes CFU/g. Therefre, it appears that the ripened Camembert cheese manufactured in this study wuld be likely t cause illness if cnsumed by susceptible individuals. Camembert and Brie, as well as Mexicanstyle cheese, nrmally have a ph value abve 6.0 at the time f cnsumptin and thus may pse a significant health hazard if cntaminated with L. mncytgenes. Hence, it is imperative that these cheeses be manufactured under hygienic cnditins frm pasteurized milk and then analyzed fr L. mncytgenes t assure that cnsumers receive a safe prduct. CKNOWLEDGMENT Research supprted by the Cllege f gricultural and Life Sciences, the Natinal Dairy Prmtin and Research Bard thrugh a grant administered by the Dairy Research Fundatin, Rsemnt, IL, and Kraft, Inc., Glenview, IL. We thank R. M. Twedt, FD, Cincinnati, OH fr prviding cultures f L. mncytgenes strains Sctt and V7, J. H. Silliker, Silliker Labratries, Carsn, C fr prviding strain C, M. P. Dyle, The Fd Research Institute, University f WiscnsinMadisn fr prviding strain OH and the late K. B. Raper, Department f Bacterilgy, University f WiscnsinMadisn fr prviding P. camemberti. We als thank M. Rsenbaum, Office f Bilgical Safety, University f WiscnsinMadisn, fr prviding safety guidelines fr the research. REFERENCES 1. jell, G. W. Persnal cmmunicatin. 2. nnymus. 1986. Brie cheese recalled. FD enfrcement reprt. pril 9, 1986. 3. nnymus. 1986. Brie cheese recalls extended by General Fds, U.S. imprter. Fd Chem. News March 3, 1986. 4. nnymus. 1986. FD finds Listeria in Brie frm Frenchcertified plant. Fd Chem. News Feb. 17, 1986. 5. nnymus. 1986. FD finds Listeria in Mexicanstyle sft cheese. Fd Chem. News March 10, 1986. 6. nnymus. 1985. FD is checking facilities f cheese plant after finding Listeria. Fd Chem. News ug. 19, 1985. 7. nnymus. 1985. FD is investigating deaths linked t Mexicanstyle cheese. Fd Chem. News June 17, 1985. 8. nnymus. 1986. 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