Growth and Toxin Production by Clostridium botulinum in

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1976, p Copyright 1976 Americn Society for Microbiology Vol. 32, No. 5 Printed in U.S.A. Growth nd Toxin Production by Clostridium botulinum in Moldy Tomto Juice C. N. HUHTANEN, J. NAGHSKI,* C. S. CUSTER, AND R. W. RUSSELL Estern Regionl Reserch Center, Agriculturl Reserch Service, Phildelphi, Pennsylvni 19118,* nd Met nd Poultry Inspection Progrm, Animl nd Plnt Helth Inspection Service, Beltsville, Mrylnd Received for publiction 14 June 1976 Tomto juice inoculted with Cldosporium sp. or Penicillium sp. developed ph grdients with the upper portions ner the mold mts hving ph vlues ner neutrlity nd the lower portions remining more cid. Clostridium botulinum spores in these moldy tomto juices germinted, grew out, nd produced toxin. Botulism is usully ssocited with the ture medi, on the other hnd, the inhibiting ingestion of improperly seled, home-cnned, ph ws bout 0.2 units higher. Studies on the neutrl or slightly cidic foods. There hve effect of ph on C. botulinum 62A (7) indicted been, however, number of reported outbreks tht spores would consistently grow out nd from foods generlly considered too cid to support germintion, growth, or toxin production growth would occur t ph 4.9 fter long incu- produce toxin t ph 5.0 or higher wheres by spores of Clostridium botulinum. Stone (11) btion. Roberts nd Ingrm (9) indicted tht described n outbrek of botulism presumbly ph 5.4 ws inhibitory to C. botulinum type A or cused by eting ripe persimmon; Meyer nd B in pork mcerte medium. Inhibition occurred t higher ph vlues when the slt or Gunnison (6) confirmed pers s the source of ftl botulism in two people; nd Slocum et l. nitrite concentrtions were incresed. Bird- (10) indicted tht tomtoes were the source of Prker nd Freme (1) indicted tht 5.3 ws toxin ftl to two persons. The ph of the tomto the minimum ph for germintion nd outgrowth of types A, B, or E in culture medi. juice ws They isolted erobic bcilli nd diplococci from the incriminted food, but could Mushrooms, cidified with cetic cid, becme not isolte C. botulinum. Inocultion of fresh toxic t ph 4.77 (5). cnned tomtoes with the incriminted juice The possibility of certin, recently developed did not produce toxin. The sme workers summrized reports of nine outbreks of botulism ner the minimum for growth of C. botulinum vrieties of tomtoes hving ph vlues t or from cid foods tht included three from pricots, three from tomto products, nd three thn low cidity might be responsible for out- prompted us to determine whether fctors other from the report on pers by Meyer nd Gunnison (6). One of the pricot reports noted the breks of botulism from tomtoes. presence of mold on the surfce of the cnned MATERIALS AND METHODS fruit. Another report referred to by these uthors indicted tht incriminted tomtoes (our designtion) were obtined from the CDC, At- C. botulinum cultures. Type A cultures C, nd C2 were "obviously fermented." lnt, G.; C3 nd C4 were obtined from the Animl A recent review on botulism in the United nd Plnt Helth Inspection Services (APHIS), Beltsville, Md., nd Sttes from 1899 to 1973 (2) indicted tht C9 ws from the Northern Regionl Reserch Center, Agriculturl Reserch totl of 27 outbreks were trced to fruits nd Service (this ws type 62A, NRRL B1218); C,0 ws 20 more to condiments including tomto relish, Americn Type Culture Collection (ATCC) chili peppers, nd chili suce. Other recent Type B cultures were C5 nd C6 from APHIS, C7 ws cses ttributed to home-preserved tomtoes or from U.S. Army Lbs, Ntick, Mss., nd C,, ws tomto juice were reported by Vrtnynts from the ATCC (7949). The cultures were mintined in cooked-met medium lyered with Vspr. (14), Popugilo et l. (8), nd the Center for Disese Control (CDC) (3, 4). Spore suspensions were prepred by the method of Townsend et l. (12) investigted the effect of Tsuji nd Perkins (13); the concentrtions were djusted to 104/ml. ph on the germintion nd growth of C. botulinum spores in vriety of food substrtes. For by blending rw, fresh tomtoes nd plting in to- Mold cultures. The mold cultures were obtined ll food substrtes tested, ph of 4.70 ppered mto juice gr (Difco). Four dissimilr-ppering to inhibit growth nd toxin production; in cul- colonies were picked nd trnsferred to tomto juice 711

2 712 HUHTANEN ET AL. gr slnts. These were identified s two species of Cldosporium nd two of Penicillium. The slnts were incubted t room temperture for 10 dys, 3.5 ml of wter ws dded, nd the spores were triturted with loop. The suspensions were mde up to contin pproximtely 104 spores/ml. Confi'rmtion of growth nd toxin production. Brewer nerobic gr, 5 ml in 15- by 125-mm tubes, ws used to detect nerobic bcteri by inoculting loopful of tomto juice into melted gr. These were incubted for 3 dys t 35 C nd observed for the presence of lrge numbers of nerobic colonies nd gs production. Further confirmtion of growth in tomto juice ws by Grm stin. Toxin in the tomto juice ws determined by dding n equl volume of geltin-phosphte buffer (0.2% geltin, 1.6% N2HPO4 7H20), centrifuging t 1,000 reltive centrifugl force for 20 min, nd injecting 0.5 ml intrperitonelly into two mice. Tomto juice. Tomto juice ws obtined from locl supermrket. For experiments with mold spore suspensions, the tomto juice ws djusted to ph 4.2, 4.6, nd 5.2 with 40% KOH, dispensed in 20-ml mounts into 20- by 150-mm tubes, nd inoculted with 2 ml of the spore suspension of C. botulinum (1,000 spores/ml finl concentrtion). The tubes were then heted to 68 C for 30 min. After heting nd cooling the tubes, mold spores were dded to finl concentrtion of bout 1,000 spores/ml. Incubtion ws t room temperture (22 to 28 C). Experiments for determining minimum ph vlues for growth were mde in 15- by 125-mm test tubes contining 4 ml of tomto juice with 1,000 spores/ml (0.4 ml of 104/ml suspension). The tubes were heted in flowing stem (100 C) for 4 min. The tomto juice ws overlid with Vspr, nd the tubes were incubted t 35 C. The ph vlues in these experiments were determined on noninoculted tubes of tomto juice fter 2 dys of incubtion. These were invribly bout 0.2 to 0.4 units higher thn when originlly djusted. becme evident for the different strins of C. botulinum (Tble 1). Three of the type A cultures (C2, C3, nd C,) filed to produce gs, typicl Grm stins, or nerobic colonies in Brewer nerobic gr t the highest ph tested (5.87). C, nd Clo hd minimum ph of 5.84, nd C4 grew t 5.68, with skip t Of the type B strins, C1, filed to grow t ph 5.87, nd C5 grew t 5.84, C6 t 5.49, nd C7 t In second experiment the minimum ph vlues were 5.66 for C1, greter thn 5.72 for C2 nd C3, 5.44 for C4, 5.72 for C5, 5.34 for C6, 5.24 for C7, nd 5.57 for Cg nd Clo. Growth of C. botulinum with molds. Three of the cultures (C6, C7, nd C9) were selected for further study in mold-contminted commercil tomto juice (Tble 2). Without mold, there ws no evidence for growth (i.e., no gs in the tomto juice nd no nerobic colonies in gr) of C. botulinum in 60 dys, the durtion of the test. In tomto juice lso inoculted with Cldosporium sp. (no. 1), ll three cultures produced visible gs, typicl Grm smers, nd nerobic gs-producing colonies 4 dys fter the mold hd formed hevy mts of growth on the surfce. Similr, lthough slightly delyed, results were obtined with Cldosporium sp. (no. 2). On the other hnd, the two Penicillium species, which did not form compct mt of growth on the surfce, llowed growth of C. botulinum only with C7 nd one replicte of C". Toxin production. The Cldosporium sp. (no. 1) culture ws studied further with C. botulinum cultures C6, C7, nd C9 (Tble 3). When the mold ws dded t the beginning of the test, gs production from C6 nd C7 ws evident within two weeks from the time of hevy mt formtion (3 dys) t the three ph RESULTS levels (4.2, 4.6, nd 5.2). Cg grew more slowly. Minimum ph for growth. There ws lrge When mold ws dded fter 35 dys t room difference in the minimum ph t which growth temperture, the C. botulinum spores showed TABLE 1. Time (dys) required for gs production by C. botulinum in tomto juice t vrious ph vlues DySb required for gs production t ph: Culture Type C, A C _- - - C C C5 B C C Cg C Cll - - Four milliliters of commercil tomto juice in 15- by 125-mm tubes with 1,000 spores/tube, seled with APPL. ENVIRON. MICROBIOL. Vspr, nd heted t 212 F (1000C) for 4 min. bdys from time of inocultion. c -, No gs production in 120 dys; no gs ws produced in ny of the cultures t ph vlues of 4.76, 4.92, nd 5.04.

3 VOL. 32, 1976 similr germintion nd outgrowth ptterns. Toxin production ppered to be correlted with erly gs production; the highest titers were with C9 t ph 4.6 nd 5.2. C7 grew fster thn C6, but the toxin titers were lower. Development of ph grdient under mold mt. A grdul chnge in the ph of tomto juice without C. botulinum, under the mt of Cldosporium sp., is shown in Tble 4. Three dys fter the mold mt formed on juice with n initil ph of 4.2, the ph of the underside of the mt ws When the strting ph ws 4.60, tht of the underside of the mt ws Grdients of ph decresing with depth were observed. In tomto juice with n initil ph of 4.2, TABLE 2. Time required for gs production by C. botulinum in moldy tomto juice" Moldb Dys requiredc by culture: C6 C7 C9 None _d _ No. 1 Cldosporium sp No. 2 Cldosporium sp No. 3 Penicillium sp No. 4 Penicillium sp Commercil tomto juice (ph 4.2) incubted t room temperture. Results re from duplicte 20- by 150-mm tubes contining 20 ml of tomto juice nd 1,000 spores of C. botulinum per ml. b Cldosporium sp. (no. 1) showed hevy mt of growth in 3 dys; Cldosporium sp. (no. 2) nd the penicilli formed less dense mts. c Dys from strt of experiment; in this cse 4 dys fter formtion of the mold mt. Growth ws verified by Grm stins nd the formtion of mny nerobic colonies in Brewer nerobic gr with gs production. d _, No gs production in 60 dys. TABLE 3. BOTULISM IN TOMATO JUICE 713 in only 6 dys the top 0.5 ml hd reched ph (5.33) tht ws higher thn the minimum ph llowing growth of C7 (5.24 from Tble 1). When the tomto juice strted t ph 4.6, the minimum ph ws t depth of 1.5 ml in 6 dys, nd t 5.2 initil ph the minimum ph ws t depth of 3.5 ml. The ph grdully incresed with length of incubtion. Inhibition of mold growth with sorbic cid. Sorbic cid t 100,ug/ml dded to commercil tomto juice inoculted with Cldosporium sp. showed complete inhibition of the mold for the durtion of the experiment (6 months). Prtil inhibition ws obtined with 50,ug/ml. Spores of C. botulinum culture C, did not grow out or produce gs when mold growth ws prtilly or totlly inhibited. DISCUSSION The work reported here indictes tht the minimum ph for outgrowth of spores in tomto juice vries gretly for different strins of C. botulinum type A or B; the lowest we observed ws 5.24 with one type B strin (C7, Tble 1). Other strins filed to outgrow even t ph Our results were chieved with spore concentrtions of 103/ml s contrsted to 106/ml used in the Ntionl Cnners Assocition study (7). They found the inhibitory ph to rnge from 4.8 for spores obtined from vegettive cells grown t ph 4.75 in tomtoes nd sporulted t ph 7.0 to ph 5.5 for cells sporulted t ph 5.5 in tomtoes. Without tomto pssge, the inhibitory ph ws 4.9. Huschild et l. (5) indicted tht the inhibitory ph for toxinogenesis ws 4.77 for mushrooms in oil mrinted with cetic cid. Town- Effect ofph on growth nd toxin production of C. botulinum in moldy tomto juice Culture ph Mold dded t: C6 C, C, Gsb Toxinc Gs Toxin Gs Toxin 4.2 Strt 17 _ d Strt dys _e dys Strt Strt dys dys Strt Strt dys dys Commercil tomto juice incubted t room temperture. b Dys from strt of experiment for gs bubbles to form under mold mt (which formed in 3 dys). creciprocl of titer. d-, Toxin determintion negtive. eno gs produced in 180 dys.

4 714 HUHTANEN ET AL. TABLE 4. Effect of mold growth on ph of tomto juice ph fter 6, 9, nd 19 dys of incubtion Portion tested Strting ph 4.2 Strting ph 4.6 Strting ph 5.2 6c Under side mold mtd of Topmost 0.5 mle Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Next 0.5 ml Commercil tomto juice incubted t room temperture; strting volume ws pproximtely 20 ml. b Hevy mold mt evident in 3 dys. c A different tube ws used for the mesurement on different dys. d Mold mt ws removed nd inverted, nd the ph ws determined by contct with combintion probe. e Successive portions removed in order from top. send et l. (12) studied the minimum ph for growth in vriety of substrtes. They indicted tht type A strins were s tolernt s type B in most substrtes, lthough in one food (strined fruit pudding with frin) type B ws more cid tolernt. They found toxin production in some cses without noticeble gs formtion. The lowest ph llowing growth ws 4.80 in pinepple rice pudding. Higher inhibitory ph vlues for growth were obtined in culture medi thn in cidic foods. These reports indicte tht the minimum ph for outgrowth of spores depends on fctors such s previous history of the vegettive form, especilly regrding cid exposure, size of spore inoculum, prticulr strin used, nd incubtion temperture. Our work indictes tht the originl ph of the food my be only one fctor in preventing growth of C. botulinum in tomtoes or other cid foods. If mold develops on the surfce, the APPL. ENVIRON. MICROBIOL. ph of the underlying food is rised, even with very low initil ph vlues. In fct, the onset nd rte of toxin production ws s rpid t ph 4.2 s t ph 5.2, indicting tht even high-cid foods my be unsfe when molds re present. A frugl, uninformed person might remove the mold mt nd consume portion of the product tht contins the preformed toxin. Routine nlysis of the reminder of the product fter routine mixing could esily show it to be in the high-cid rnge. This ppered to be the cse in n outbrek of nine cses ofbotulism (two ftlities) in the USSR reported by Popugilo et l. (8). They trced the toxin to improperly processed tomto juice which showed overt mold growth nd ph vlues rnging from 3.8 to 4.5 in severl cns exmined. No ttempt ws pprently mde to determine the ph t vrious levels of the juice. The report of Slocum et l. (10) lso indicted tht mold ws present on the surfce of pricots implicted in n outbrek.

5 VOL. 32, 1976 They reported ph of 4.21 in cn of tomtoes implicted in nother outbrek. Meyer nd Gunnison (6) reported ph of 3.86 in pers cusing n outbrek of botulism in Cliforni. Interestingly, they found the pers to contin yest cells but no molds. Dowell et l. (Intergency Botulism Reserch Coordinting Committee, Rockville, Md., 1974) described outbreks from home-cnned blueberries with ph of 3.5, tomtoes with ph of 4.5, nd tomto juice with ph of 4.2. Our results nd those of the bove workers indicte tht one hzrd from botulism in cid foods my not be the initil level of cidity per se but the sequentil growth of cid-destroying orgnisms (molds from this study, but other orgnisms could lso consume cid) nd C. botulinum. The work reported here indictes tht 100,ug of sorbic cid per ml might effectively control the mold in tomto juice. This is under further investigtion. ACKNOWLEDGMENTS We pprecite the id of J. Eye of APHIS nd D. Fennel of the Northern Regionl Reserch Center in the identifiction of the molds. LITERATURE CITED 1. Bird-Prker, A. C., nd B. Freme Combined effect of wter ctivity, ph nd temperture on the growth ofclostridium botulinum from spore nd vegettive cell inocul. J. Appl. Bcteriol. 30: Center for Disese Control Botulism in the BOTULISM IN TOMATO JUICE 715 United Sttes, Hndbook for epidemiologists, clinicins, nd lbortory workers. Center for Disese Control, Atlnt. 3. Center for Disese Control Foodborne nd wterborne disese outbreks. Annul summry (issued Jnury 1976). Center for Disese Control, Atlnt. 4. Center for Disese Control Morbid. Mortl. Week. Rep. 23: Huschild, A. H. W., B. J. Aris, nd R. Hilsheimer Clostridium botulinum in mrinted products. Cn. Inst. Food Sci. Technol. J. 8: Meyer, K. F., nd J. B. Gunnison Botulism due to home cnned Brtlett pers. J. Infect. Dis. 45: Ntionl Cnners Assocition Conditions for growth of Clostridium botulinum in tomtoes. Finl Report. Ntionl Cnners Assocition, Wshington, D.C. 8. Popugilo, V. M., S. P. Zhurvsky, E. E. Yroshevsky, V. I. Sukhkov, L. N. Zhukov, nd T. A. Kon'shin Cses of botulism cused by preserved tomto juice. Gig. Snit. 37(Feb.): Roberts, T. A., nd M. Ingrm Inhibition of growth of C. botulinum t different ph vlues by sodium chloride nd sodium nitrite. J. Food Technol. 8: Slocum, G. G., H. Welch, nd A. C. Hunter An outbrek of botulism cused by home-cnned tomtoes. Food Res. 6: Stone, W. J Botulism from ingestion of ripe fruit. J. Am. Med. Assoc. 92: Townsend, C. T., L. Yee, nd W. A. Mercer Inhibition of the growth of Clostridium botulinum by cidifiction. Food Res Tsuji, K., nd W. E. Perkins Sporultion of Clostridium botulinum. I. Selection of sporultion prticulte medium. J. Bcteriol. 84: Vrtnynts, I. S Botulism cused by home preserved tomtoes. Zdrvookhr. Turkm. 16:25-26.