Korean J. Soil Sci. Fert. Vol.51, No.4, pp.404-411, 2018 Korean Journal of Soil Science and Fertilizer Article https://doi.org/10.7745/kjssf.2018.51.4.404 pissn : 0367-6315 eissn : 2288-2162 Persistence of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in Soil and Compost Amended Soil Kyu-Seok Jung*, Seung-Mi Seo, Hye-Jin Jeon, Bo-Reum Jeong, Eun-Jung Roh, Jae-Gee Ryu, and Kyoung-Yul Ryu National Institute of Agricultural Science, Jeonju 55365, Korea *Corresponding author: win258@korea.kr A B S T R A C T Received: August 13, 2018 Revised: November 30, 2018 Accepted: December 3, 2018 Addition of animal manure to soil can provide opportunity for bacterial pathogens contamination of soil, water, and food. This study was conducted to investigate the survival of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in soil and compost amended soil under the selected environmental conditions. Soil and compost amended soil were inoculated with S. enterica, E. coli O157:H7, and L. monocytogenes. Soil and compost amended soil were incubated at 25 C and consistent moisture content. Samples had been collected during 200 days depending on the given conditions. S. enterica and E. coli O157:H7 survived over 200 days in soil and compost amended soil. L. monocytogenes persisted for 80 days in soil and for 160 days in compost amended soil, respectively. S. enterica and E. coli O157:H7 survived longer than L. monocytogenes at soil and compost amended soil. It is noted that S. enterica, E. coli O157:H7, and L. monocytogenes survived long in soil and compost amended soil. S. enterica, E. coli O157:H7, and L. monocytogenes survived longer in compost amended soil than in soil. Results from these studies provide useful information in identifying manure handling practices to reduce the risk of S. enterica, E. coli O157:H7, and L. monocytogenes transmission to foods produced in the presence of animal waste. Keywords: Soil, Compost, Foodborne pathogens Decimal reduction time (DRT) for S. enterica, E. coli O157:H7, and L. monocytogenes in soil and compost amended soil at 25 C. Treatment Pathogens Regression equation R 2 DRT (days) Soil Compost amended soil S. enterica Log 10 A = -0.0333t + 8.6594 0.92 30.0 E. coli O157:H7 Log 10 A = -0.0276t + 8.7453 0.96 36.2 L. monocytogenes Log 10 A = -0.0769t + 7.618 0.94 13.0 S. enterica Log 10 A = -0.0176t + 8.5789 0.92 56.8 E. coli O157:H7 Log 10 A = -0.0234t + 8.032 0.94 42.7 L. monocytogenes Log 10 A = -0.0355t + 7.4629 0.93 28.1 C The Korean Society of Soil Science and Fertilizer. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Persistence of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in Soil and Compost Amended Soil 405 Introduction 건강에대한관심이고조되면서채소등별도의가공처리없이섭취하는신선농산물에대한소비가꾸준히증가하고있다 (Ahn et al., 1999). 최근신선채소류, 과일류, 비멸균주스등으로인한식중독사고는증가하고있다 (Beuchat, 2001). 식중독균은식물체표면에존재하고특정한환경조건에서는식물체에부착하거나생존할수있다 (Bernstein et al., 2007). 외국에서멜론, 망고, 새싹, 로메인상추, 토마토, 시금치, 고추등농산물에의한식중독사고는최근빈번히발생하고있다 (CDC, 2018). 영국에서는부추에존재하는 E. coli O157:H7에의한식중독사고로 74 명이입원하였고 1명이사망하였다 (Powell, 2011). 병원성대장균에오염된상추로인한식중독사고도미국에서발생하였다 (Ackers et al., 1998). 오염된미생물은수확후소비되기전의저장기간동안신선농산물에서증식하여사람에게식중독을일으킬수있다. 식중독사고를일으키는병원성미생물은토양, 오염된관개수, 가축분변, 먼지, 미숙퇴비등에존재할수있고, 수확하기전몇가지경로로농산물을오염시킬수있다 (Burnett et al., 2001). 특히부숙이충분히되지않은가축분퇴비는재배단계의농산물오염의가장큰오염원이다 (Solomon et al., 2002). 농산물중근채류와엽채류는미부숙퇴비에의한식중독균오염에가장취약할수있다 (Buck et al., 2003). 가축분변유래장내세균의생존이나증식은식물뿌리에서나오는유기물또는근권주위의미생물에영향을받을수있고병원균, 토양미생물, 토양, 식물체뿌리의상호작용의영향을받을수도있다 (Jiang and Shepherd, 2009). 관개수, 퇴비등의농자재등을통해식중독세균이토양으로유입될수있으며, 토양내에서오랫동안생존하여오염이발생할수있다 (Ibenyassine et al., 2006). Jung et al. (2017) 은배추재배토양에서대장균이검출된다고하였다. Erickson et al. (2010) 은 E. coli O157:H7에오염된관개수를토양에접종한결과 76일동안생존하였다고하였다. 병원성대장균은토양과퇴비에서각각 154일, 217일생존할수있다고하였다 (Islam et al., 2004). E. coli O157:H7은토양에서생존하고증식할수있으며가축분퇴비는 E. coli O157:H7의생존능력을더욱증가시킬수있다고하였다 (Gagliardi and Karns, 2000). Holley et al. (2006) 은가축분퇴비를토양에추가하였더니 Salmonella spp. 가더오래생존하였다고하였다. 퇴비가시용된토양에서상추를재배하였을경우, Salmonella는 231일까지생존하였고, S. enterica는상추재배토양에서 63일까지, 파슬리재배토양에서 231일까지생존하였다 (Islam et al. 2004). Gagliardi and Karns (2002) 은 E. coli O157:H7이휴경토양에서 47일까지생존할수있고, 호밀뿌리에서 96일, 알팔파뿌리에서 92일까지생존가능하다고하였다. 병원성미생물에오염된토양이나퇴비가농산물에접촉되어사람이오염농산물을섭취하면식중독이발생할수있다 (Morgan et al., 1988). 본연구는국내토양과가축분퇴비가섞인토양에서병원성미생물 (S. enterica, E. coli O157:H7, L. monocytogenes) 의생존능및생존기간을비교 분석하고, 또한병원성미생물의생존기간과생존에미치는퇴비의영향을분석함으로써농산물안전관리를위한기초자료를제공하고자수행하였다. Materials and Methods 사용균주및시료채취국내축산의주요축종인돼지의분뇨를주원료로생산, 유통되는가축분퇴비를수집하였고접종균주로 ATCC에서분양받은 S. enterica ATCC 13311, E. coli O157:H7 ATCC 43895, L. monocytogenes ATCC 15313를사용하였다. 사용된균주는 tryptic soy broth (Difco Co., Detroit, MI, USA) 에접종한후진탕배양기 (VS 8480, Vision Science, Korea) 를이용하여 37 C, 250-280 rpm에서 18시간배양하였다. 배양액은 10,000 rpm에
406 Korean Journal of Soil Science and Fertilizer Vol. 51, No. 4, 2018 서 2분간원심분리하여상층액은버리고 0.1% buffered peptone water (Difco Co., Detroit, MI, USA) 20 ml을가한후 washing 과정을 2회실시한다음세균수가약 10 8 CFU ml -1 이되도록농도를맞추어사용하였다. 본실험에서는경기도수원에위치한서울대농대실험포장에서노지토양의 0-15 cm 표토깊이에서채취한토양을사용하였으며토양은 2 mm (#10 mesh) 스테인레스체로걸러최대한균질하도록조성한다음, 121 C에서 30분간 2회고압멸균을하여멸균상태로상온에두었다. 토양과퇴비는멸균처리하였으며멸균된토양과가축분퇴비 (250 g) 를첨가한토양각각 500 g을멸균된 polypropylene box (2.3L, Locknlock co., Korea) 에넣고, 균주현탁액 50 ml을가한후스푼으로골고루섞어균질화하였다. 사전실험을통해 polypropylene box (Locknlock) 의수분손실이거의일어나지않는것을확인하였고, 같은수준의토양수분함량 (15%), 퇴비수분함량 (58%) 으로실험을하였다. 토양과가축분퇴비에유해미생물접종후수분손실을막기위하여밀봉상태를유지하면서 25 C의 incubator (VS 1203 PFC, Vision Science, Korea) 에서 200일동안배양하면서매주에 1회씩주기적으로시료를채취하여시험을실시하였고시료채취시외부오염을막기위하여클린벤치 (CLB-201-04, CHC Lab, Korea) 에서작업하였다. 시험에사용한토양과퇴비의이화학적특성은 Table 1, 2과같다. Table 1. Chemical properties of the soil used in the experiment. ph OM Av.P 2 O 5 Exch.(cmol(+) kg -1 ) (1:5) (g kg -1 ) (mg kg -1 NH 4 (mg kg -1 T-N ) ) K Ca Mg Na (%) 6.4 14 181 0.22 6.6 2.4 0.1 10 0.11 Table 2. Chemical properties of livestock manure compost used in the experiment. ph C/N ratio OM T-N T-P 2 O 5 T-K 2 O T-CaO T-MgO ----------------------------------------------------------------------- (%) ----------------------------------------------------------------------- 5.48 45.00 81.70 0.75 0.67 0.69 0.61 0.33 유해미생물계수 S. enterica, E. coli O157:H7, L. monocytogenes의분리는채취한시료를균질화한뒤 3 g을취해서 buffered peptone water (Difco Co., Detroit, MI, USA) 27 ml에접종한후 stomacher (easymix, AES CHEMUNEX, France) 로 2분동안균질화하였다. 균질화된시료는 buffered peptone water (Difco) 를이용하여 10 배씩연속희석하였다. S. enterica, E. coli O157:H7, L. monocytogenes의정량적분석을위해서앞에서준비한시료 1 ml를 xylose lysine deoxycholate agar (Difco), macconkey sorbitol agar (Difco), oxford agar (Difco) 위에각각도말하여 37 C에서 24-48시간배양하였다. 배양후각배지위에형성된 colony를계수하여 colony forming unit (CFU) g -1 으로나타내었다. 의심집락은세균부유액과반응한산물의색변화로판단하는 API test (biomerieux, Marcy 1 Etoile, France), 항원항체의응집반응결과로판단하는 Latex test (Oxoid Ltd., Hampshire, UK) 을수행하여동정하였다. DRT 측정식미생물이 90% 사멸하는데걸리는시간인 DRT (decimal reduction time) 값은다음과같은식에적용하여계산하였다 (Himathongkham et al., 1999). Regression equation은 Microsoft Excel 프로그램을이용하여구하였다.
Persistence of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in Soil and Compost Amended Soil 407 DRT=-log(N 0 /N)/t (Eq. 1) N 0 : 미생물의초기밀도, N: 미생물의최종밀도, t: 시간 Results and Discussion 토양에서유해미생물의생존변화토양과가축분퇴비에존재하는미생물이병원성미생물의생존에미치는영향을배제하기위하여토양과퇴비는멸균처리하였다. 토양에 S. enterica, E. coli O157:H7, L. monocytogenes를접종하여유해미생물종류에따른생존변화양상을조사한결과 (Fig. 1), 같은토양과온도 (25 C) 에서유해미생물의생존양상은유해미생물별로다른경향을나타내었는데 E. coli O157:H7이가장오래생존하였고, L. monocytogenes 은가장빨리사멸하였다. E. coli O157:H7에서 0-50일까지는거의변화가없었고그이후로 200일까지점점감소하는경향을보였다. Ishii et al. (2006) 은대장균이 25 C 이하토양에서 30일이상생존할수있다고하였고, Maule (2000) 은 E. coli O157:H7는토양에서는 130일동안생존할수있다고하였다. S. enterica에서 0-60일까지는거의일정한수준을유지하다가 60일이후부터 200일까지서서히감소하였다. 병원성미생물인 Salmonella는가축분뇨에발견될수있고토양에서 160-200일까지생존할수있다 (Holley et al., 2006). L. monocytogenes에서는 0-30일까지는급격히감소하다가그이후 60일까지는약간감소하였으나 60일이후부터는급격히감소하여 90일정도에는전부사멸하였다. Welshimer (1960) 은 L. monocytogenes가토양에서 67일동안생존할수있다고하였는데본결과에서도비슷한생존기간을나타내었다. 국내토양에서 3종류의병원성미생물중 E. coli O157:H7, S. enterica, L. monocytogenes 순으로생존능력이높았고그중생존능이높은 E. coli O157:H7와 S. enterica는작물재배토양에오염이되지않도록특별한주의가필요하다고생각한다. 작물재배토양에병원성미생물이존재한다면장시간생존할수있어작물이오염될수있다. 오염된작물을인간이섭취하게된다면식중독사고가발생할수있으므로농산물로인한식중독사고를예방하기위하여재배환경을청결하게관리하는것이중요하다. Burnett and Buechat (2001) 은농산물이병원성미생물에의해오염될수있는데그오염원으로는토양, 가축분뇨, 관개수, 먼지, 미숙퇴비등이있다고하였다. 오염된관개수를사용하지않고퇴비를충분히부숙해서사용하는것이농산물로인한식중독사고를예방하는최선의방법이라고생각한다. 토양내 S. enterica, E. coli O157:H7, L. monocytogenes의 DRT (decimal Fig. 1. Survival of S. enterica, E. coli O157:H7, and L. monocytogenes in soil at 25 C (n = 9, error bars = SD).
408 Korean Journal of Soil Science and Fertilizer Vol. 51, No. 4, 2018 Table 3. Decimal reduction time (DRT) for S. enterica, E. coli O157:H7, and L. monocytogenes in soil and compost amended soil at 25 C. Treatment Pathogens Regression equation R 2 DRT (days) Soil Compost amended soil S. enterica Log 10 A = -0.0333t + 8.6594 0.92 30.0 E. coli O157:H7 Log 10 A = -0.0276t + 8.7453 0.96 36.2 L. monocytogenes Log 10 A = -0.0769t + 7.618 0.94 13.0 S. enterica Log 10 A = -0.0176t + 8.5789 0.92 56.8 E. coli O157:H7 Log 10 A = -0.0234t + 8.032 0.94 42.7 L. monocytogenes Log 10 A = -0.0355t + 7.4629 0.93 28.1 reduction time) 값을나타낸것에서 E. coli O157:H7은 36.2일, S. enterica은 30.0일, L. monocytogenes은 13.0일이었다 (Table 3). Jay (2000) 는그람양성균이그람음성균보다양분요구도가크기때문에같은환경조건이라도그람음성균보다생존기간이짧을수있다고하였는데본결과에서도그람음성균인 S. enterica와 E. coli O157:H7는생존기간이길었고그람양성균인 L. monocytogenes는생존기간이짧은경향을보였다. 가축분퇴비가섞인토양에서유해미생물의생존변화가축분퇴비가섞인토양에 S. enterica, E. coli O157:H7, L. monocytogenes를접종하여유해미생물종류에따른생존변화양상을조사한결과 (Fig. 2), 가축분퇴비와토양이섞여있는상태 (25 C) 에서유해미생물의생존양상은유해미생물별로다른경향을나타내었다. S. enterica에서 0-7 일까지는약간증가하였고그이후부터 200일까지서서히감소하는경향을보였다. E. coli O157:H7에서 0-150일까지는서서히감소하다가 150-160일까지는약간증가후다시감소하였다. L. monocytogenes에서는 0-160일까지는점점감소하였고 180일정도에모두사멸하였다. 토양에서병원성미생물의생존기간보다가축분퇴비가섞인토양에서병원성미생물의생존기간이좀더길어지는결과를볼때가축분퇴비가병원성미생물의생존에유리하게작용을한다는것을알수있다. 퇴비에포함되어있는유기물의추가로인해병원성미생물이더오래생존했다고생각한다. 수중에서호기성균은유기물이많은수질에서급성장한다고하였다 (Jang and Nam, 2017). 또한, S. enterica, E. coli O157:H7보다 L. monocytogenes가가축분퇴비의영향이더크다는것도알수있다. 가축분퇴비가토양에섞일때병원성미생물의수는더욱증가한다고하였고 (Unc and Goss, 2004), E. coli O157:H7은토양에서생존하고증식할수있으며가축분퇴비는 E. coli O157:H7의생존능력을더욱증가시킬수있다고하였는데 (Gagliardi and Karns, 2000), 본결과와같았다. Kudva et al. (1998) 은양 ( 羊 ) 의분뇨로생산한퇴비에서 E. coli O157:H7이 4개월동안생존할수있다고보고하였고 You et al. (2006) 은가축분퇴비가포함된토양에서 Salmonella spp. 는 332일까지생존할수있다고하였다. Jone (1986) 은소의분변이포함된토양에서 Salmonella spp. 가 300일동안생존하였고, 가축분뇨가포함된토양에서 256일동안생존했다고보고하였다. 본실험결과중약 5개월이상생존할수있는결과로볼때병원성미생물이우리나라에서생산한퇴비와토양에서도장기간생존할수있다는것을보여주고있다. 가축분뇨에는 S. enterica, E. coli O157:H7, L. monocytogenes 등의병원성미생물이존재할수있다. 가축분뇨는퇴비화과정을거쳐비료로사용하고있는데충분히부숙되지않은퇴비에는병원성미생물이잔존할수있어농산물안전차원에서위험요소가될수있다. 퇴비가섞인토양에서병원성미생물은오랜기간생존가능하므로토양이병원성미생물에오염되지않도록하는것이중요하다. 부숙퇴비사용, 오염되지않은관개수사용, 농작업자의청결한위생, 야생동물출입통제등의방법으로오염예방관리를해야한다고생각한다. Table 3은가축분퇴비가섞인토양내 S. enterica,
Persistence of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in Soil and Compost Amended Soil 409 Fig. 2. Survival of S. enterica, E. coli O157:H7, and L. monocytogenes in compost amended soil at 25 C (n =9, error bars=sd). E. coli O157:H7, L. monocytogenes의 DRT (decimal reduction time) 값을나타낸것이다. S. enterica에서 56.8일, E. coli O157:H7에서 42.7일, L. monocytogenes에서 28.1일이었다. Conclusions 본연구는국내토양과가축분퇴비가섞인토양에서병원성미생물 (S. enterica, E. coli O157:H7, L. monocytogenes) 의생존능및생존기간을비교, 분석하고농산물의안전성을확보하기위하여안전한가축분퇴비의생산과이용에도움을주고자수행하였다. 토양에 S. enterica, E. coli O157:H7, L. monocytogenes를접종하여유해미생물종류에따른생존변화양상을조사한결과, 같은토양과온도 (25 C) 에서유해미생물의생존양상은유해미생물별로다른경향을나타내었는데 E. coli O157:H7이가장오래생존하였고, L. monocytogenes은가장빨리사멸하였다. 가축분퇴비와토양이섞여있는상태 (25 C) 에서유해미생물의생존양상은유해미생물별로다른경향을나타내었는데 S. enterica이가장오래생존하였고, L. monocytogenes은가장빨리사멸하였다. 토양에서병원성미생물의생존기간보다가축분퇴비가섞인토양에서병원성미생물의생존기간이더길어지는결과를볼때가축분퇴비가병원성미생물의생존능력을더욱증가시켰다. 이는퇴비의유기물이병원성미생물의영양원으로작용했기때문으로생각한다. 병원성미생물은토양이나퇴비에서장시간생존할수있고작물을오염시킬수있으므로농산물로인한식중독사고를예방하기위하여병원성미생물이토양에존재하지않도록관리하는것이중요하다. Acknowledgement This work was supported by a grant from the Agenda Program (PJ012009), Rural Development Administration, Republic of Korea.
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