축산물유래 Listeria monocytogenes의 virulence marker 및 gene 조사

이철현,송현호,김미령,강호조,손원근 한국식품위생안전성학회 2008 한국식품위생안전성학회지 Vol.23 No.3

To investigate the epidemiological characteristics of 68 Listeria monocytogenes isolates, including 11 reference strains and 57 isolates from imported US beef, domestic meats (beef, pork, chicken meat), raw milk, and milk plants. L. monocytogenes was to evaluate the production of virulence proteins, such as hemolysin (LLO) and lecithinase (LCP), the adsorption of Congo red (CRA), and to detect virulence genes using the polymerase chain reaction (PCR). In the study of virulence protein production, 68(100%), 62(91.2%), and 54(79.4%) of the 68 L. monocytogenes strains were positive for LLO production, the LCP test, and the CRA test, respectively, while strains of other species, such as L. innocua, L. gray, L. murrayi, and L. welshimeri, were not. There were no significant differences between L. monocytogenes serotypes and the ability to produce LLO or LCP. L. monocytogenesstrains had very high hemolytic titers (2 to 16 fold), while the other Listeria species, other than L. ivanovii and L. seeligeri, did not. The hemolysin activities of L. monocytogenes, L. ivanovii, and L. seeligeri usually exceeded 1.0 HU/mg, while those of other Listeria spp. were less than 0.04 HU/mg. In the PCR assay, all of the L. monocytogenes strains contained the hlyA, plcA, plcB, inlA, and inlB virulence genes and produced a product of the expected size. In the PCR of the actA gene, the expected 385-bp product was seen in 39(57.4%) L. monocytogenesstrains, while an unexpected 268-bp product was seen in 29(42.6%) strains. Most L. monocytogenes strains isolated from Hanwoo beef produced the 385- bp actA gene product, while strains of imported US beef usually produced the 268-bp actA gene product. By contrast, no virulence gene products were amplified in the other Listeria spp. To investigate the epidemiological characteristics of 68 Listeria monocytogenes isolates, including 11 reference strains and 57 isolates from imported US beef, domestic meats (beef, pork, chicken meat), raw milk, and milk plants. L. monocytogenes was to evaluate the production of virulence proteins, such as hemolysin (LLO) and lecithinase (LCP), the adsorption of Congo red (CRA), and to detect virulence genes using the polymerase chain reaction (PCR). In the study of virulence protein production, 68(100%), 62(91.2%), and 54(79.4%) of the 68 L. monocytogenes strains were positive for LLO production, the LCP test, and the CRA test, respectively, while strains of other species, such as L. innocua, L. gray, L. murrayi, and L. welshimeri, were not. There were no significant differences between L. monocytogenes serotypes and the ability to produce LLO or LCP. L. monocytogenesstrains had very high hemolytic titers (2 to 16 fold), while the other Listeria species, other than L. ivanovii and L. seeligeri, did not. The hemolysin activities of L. monocytogenes, L. ivanovii, and L. seeligeri usually exceeded 1.0 HU/mg, while those of other Listeria spp. were less than 0.04 HU/mg. In the PCR assay, all of the L. monocytogenes strains contained the hlyA, plcA, plcB, inlA, and inlB virulence genes and produced a product of the expected size. In the PCR of the actA gene, the expected 385-bp product was seen in 39(57.4%) L. monocytogenesstrains, while an unexpected 268-bp product was seen in 29(42.6%) strains. Most L. monocytogenes strains isolated from Hanwoo beef produced the 385- bp actA gene product, while strains of imported US beef usually produced the 268-bp actA gene product. By contrast, no virulence gene products were amplified in the other Listeria spp.

증균배지에서의 Listeria Interspecies의 경쟁생육 비교

이다연,조용선 한국식품위생안전성학회 2018 한국식품위생안전성학회지 Vol.33 No.1

Monitoring of Listeria monocytogenes, the causative agent of listeriosis, in food is inportant for public health. The Korean Food Standards Codex has adopted a ‘zero-tolerance’ policy for L. monocytogenes. The standard detection method of L. monocytogenes is based on enrichment. Thus, proper enrichment methods need to be instituted to ensure quality control of the detection procedures. In this study, the growth of L. monocytogenes and Listeria innocua as a mixed culture in Listeria enrichment broth (LEB) was monitored during artificial contamination of enrichment culture. We confirmed competitive growth or interspecies inhibitory activity of L. monocytogenes and L. innocua. Interspecies growth differences and the inhibitory activity of different inoculation and mixtures L. innocua against L. monocytogenes were examined. The concentration of L. monocytogenes must be 2.0 log CFU/mL or more than L. innocua to grow better than L. innocua. It is known that Listeria spp. and L. monocytogenes show growth difference during LEB, resulting in the risk of false-negative results. The inhibition of L. monocytogenes by L. innocua was always observed when present at lower concentrations. However, it was confirmed that L. innocua suppressed when L. monocytogenes was present at a higher concentration. Therefore if a mixture of Listeria spp. is present, detecting L. monocytogenes is difficult. Thus, a new enrichment broth to improve the detection rate of L. monocytogenes is needed.

Listeria monocytogenes의 신속검출을 위한 선택배지 및 multiplex PCR 기법 개발

정병열,임현숙,정석찬,Jung, Byeong-yeal,Lim, Hyun-sook,Jung, Suk-chan 대한수의학회 2003 大韓獸醫學會誌 Vol.43 No.2

Listeria (L.) monocytogenes in samples could not be detected occasioally by faster growth of other Listeria spp. especially L. innocua. The aim of this study was to develop the differential media and multiplex polymerase chain reaction (PCR) assays for the rapid detection of L. monocytogenes. L. monocytogenes colonies were characterized by their ${\beta}$-hemolysis with fluorescence under 366 nm UV light on the Listeria hemolysis agar (LHA). L. innocua, a species commonly present in foods, did not produce ${\beta}$-hemolysis on LHA. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua. The multiplex PCR assays were developed to distinguish from L. monocytogenes and other Listeria spp. with two pairs of primers. The primers were designed in 16S rRNA and listeriolysin O gene for specific amplification of all members of the genus Listeria and L. monocytogenes, respectively. The multiplex PCR assays produced 560 and 938 bp products in L. monocytogenes; only 938 bp products in the genus Listeria. The multiplex PCR assays could detect as little as 50 pg of L monocytogenes DNA. These results indicated that the differential media and multiplex PCR assays might be useful diagnostic tools for the rapid detection of L. monocytogenes.

위해평가를 통한 치즈에서의 Listeria monocytogenes 식중독 발생 가능성 분석

하지명,이지연 한국식품위생안전성학회 2020 한국식품위생안전성학회지 Vol.35 No.6

L. monocytogenes는 고병원성 식중독 세균으로 치즈, 식육 및 식육가공품, 훈제연어 등을 통해 식중독을 일으킨다. 현재 국내 식품공전 상 치즈에서는 L. monocytogenes에 대하여 불검출 기준을 적용하고 있으나, 이에 대한 과 학적 근거가 확실하지 않고 L. monocytogenes 식중독에 대한 국민의 경각심을 일깨우기 위하여 기존에 수행된 연구들을 토대로 위해평가를 실시하였다. 그 결과, 치즈에 L. monocytogenes의 초기오염수준은 -4.0 Log CFU/g으로 확인되었다. 2018년 국민건강영양조사 결과 치즈의 섭취자 비율은 11.8%이고, 치즈 섭취량에 대한 최적확률분포는 Lognormal distribution이며 평균 32.5 g의 치즈를 섭취 하는 것으로 확인되었다. 치즈 섭취로 인한 L. monocytogenes 식중독 발생 가능성은 일반군에서 평균 5.09×10-7, 민감군에서 평균 4.32×10-6로 분석되어, 민감군에서의 식중독 발생 가능성이 다소 높은 것으로 확인되었다. 식중독 발생 가능성에는 섭취자 비율이 가장 큰 영향을 미치지만 보관 및 운송 시간 또한 영향을 미치는 것으로 확인되어 유통환경에 대한 철저한 관리가 중요한 것으로 사료된다. 본 위해평가를 통하여 치즈에서의 L. monocytogenes의 기준규격을 뒷받침 할 수 있는 과학적인 데이터를 확보할 수 있었다. 향후 다양한 식품에서의 L. monocytogenes 위해평가 연구가 수행되어야 할 것으로 사료된다. Listeria monocytogenes is a highly pathogenic gram-positive bacterium that is easily isolated from cheese, meat, processed meat products, and smoked salmon. A zero-tolerance (n=5, c=0, m=0/25 g) criteria has been applied for L. monocytogenes in cheese meaning that L. monocytogenes must not be detected in any 25 g of samples. However, there was a lack of scientific information behind this criteria. Therefore, in this study, we conducted a risk assessment based on literature reviews to provide scientific information supporting the baseline and to raise public awareness of L. monocytogenes foodborne illness. Quantitative risk assessment of L. monocytogenes for cheese was conducted using the following steps: exposure assessment, hazard characterization, and risk characterization. As a result, the initial contamination level of L. monocytogenes was -4.0 Log CFU/g in cheese. The consumption frequency of cheese was 11.8%, and the appropriate probability distribution for amount of cheese consumed was a Lognormal distribution with an average of 32.5 g. In conclusion, the mean of probabilities of foodborne illness caused by the consumption of cheese was 5.09×10-7 in the healthy population and 4.32×10-6 in the susceptible population. Consumption frequency has the biggest effect on the probability of foodborne illness, but storage and transportation times have also been found to affect the probability of foodborne illness; thus, management of the distribution environment should be considered important. Through this risk assessment, scientific data to support the criteria for L. monocytogenes in cheese could be obtained. In addition, we recommend that further risk assessment studies of L. monocytogenes in various foods be conducted in the future.

팽이버섯에서 Listeria monocytogenes의 온도별 생존과 유기산에 의한 저감화

김세리,김원일,윤재현,정도영,최송이,황인준,나겐드란 라잘링감 한국식품위생안전성학회 2020 한국식품위생안전성학회지 Vol.35 No.6

본 연구는 수출 팽이버섯에서 L. monocytogenes가 검출되어 리콜 됨에 따라 팽이버섯의 안전성을 확보하기 위하여 유통 온도에 따른 팽이버섯 중 L. monocytogenes의 생존을 조사하고 유기산을 이용한 팽이버섯 중 L. monocytogenes 의 저감화 기술을 개발하였다. 저장 온도에 따른 팽이 버섯 중 L. monocytogenes의 생존을 조사하기 위하여 L. monocytogenes가 오염된 팽이버섯(초기 농도 4.5 Log CFU/g)을 1-35 ̊C에 각각 저장하고 균수변화를 조사하였다. 그 결과 팽이버섯을 1 ̊C에 저장하였을 때 L. monocytogenes 는 1개월 내에 증식하지 않았고 30 ̊C, 35 ̊C에서는 각각 36시간, 24시간이내에 3.0 log CFU/g 증가하였다. 팽이버섯 중 L. monocytogenes의 저감화를 위하여 1-3% 유기산 3종 (초산, 젖산, 말산)에 10-30분 동안 처리하였을 때 팽이버섯 중 L. monocytogenes는 초산보다는 말산, 젖산의 저감효과가 높았고 젖산과 말산의 처리 농도가 증가할수록 저감효과는 증가하는 것으로 나타났다. 3% 젖산과 말산을 10분 이상 처리하면 약 3.0 log CFU/g이상 감소하였다. 따라서 팽이버섯의 안전성을 확보하기 위해서는 수출할 때는 1 ̊C 내외의 저온을 유지하고 소비단계에서는 3% 젖산과 말산에서 10분 정도 처리 후 섭취하는 것이 필요하다. Listeria monocytogenes (L. monocytogenes) was responsible for several recall cases owing to its incidence in mushrooms exported from the Republic of Korea. In this study, we investigated the survival of L. monocytogenes in enoki mushroom (Flammulina velutipes) at different temperatures and the antilisterial effect of its organic acids. Enoki mushrooms were innoculated with L. monocytogenes (initial concentration 4.5 log CFU/g) and stored at 1-35oC, No growth of L. monocytogenes in enoki mushrooms was observed at 1oC for 30 days. 3.0 log CFU/ g growth of L. monocytogenes was also achieved after 36 h and 24 h at 30oC and 35oC, respectively. To evaluate the antilisterial effect of the organic acids (acetic acid, lactic acid, malic acid), enoki mushrooms were treated with 1-3% of each acid for 10-30 min. The efficacy of malic acid and lactic acid was significantly higher than that of acetic acid. Over 3.0 log reductions were observed when L. monocytogenes in enoki mushrooms was immersed in 3% lactic acid and malic acid over 10 minutes or more. Therefore, it is necessary to keep enoki mushrooms at 1oC during the export process and treat them with 3% lactic acid and malic acid for 10 min prior to consumption.

Effect of Fat Contents on Thermal Resistance, Antibiotic Sensitivity, and Caco-2 Cell Invasion of Listeria monocytogenes

Lee, Jinhee,Yoon, Hyunjoo,Lee, Sunah,Lee, Heeyoung,Yoon, Yohan Korean Society for Food Science of Animal Resource 2013 한국축산식품학회지 Vol.33 No.4

This study evaluates the effects of fat contents on the thermal resistance, antibiotic sensitivity, and Caco-2 cell invasion of Listeria monocytogenes. Ten strain mixture of L. monocytogenes in milk (0, 1, and 4% fat) and pork sausage patties (10, 20, and 30% fat) were exposed to $63^{\circ}C$. To evaluate effects of fat on the antibiotic sensitivity of L. monocytogenes, the L. monocytogenes strains NCCP10811 (most antibiotic resistant to streptomycin) and NCCP10943 (most antibiotic sensitive to streptomycin) were exposed to different fat contents in milk and pork sausage patties, and L. monocytogenes from the foods were used for antibiotic sensitivity assays. The most invasive L. monocytogenes strains (NCCP10943) was exposed to different fat contents in milk or pork sausage patties, and L. monocytogenes from the foods were used for the Caco-2 cell invasion assays. The reductions of L. monocytogenes populations were not generally influenced by fat contents. The L. monocytogenes subjected to milk fat had increased sensitivities (p<0.05) due to some antibiotics. In addition, Caco-2 cell invasion efficiency of L. monocytogenes NCCP10943 increased (p<0.05) as fat contents increased. These results indicated that higher fat contents may be related to L. monocytogenes invasions and heat resistances in pork sausage patties, but the relationship between fat and antibiotic sensitivity varied according to antibiotics, strains, and fat contents.

Listeria monocytogenes에 대한 길경의 물 및 에탄올 추출물의 항균효과

김복자 한국식품조리과학회 2000 한국식품조리과학회지 Vol.16 No.5

길경중 물과 에탄올을 이용하여 얻은 추출물을 5종의 식중독세균(Listeria monocytogenes ATCC 15313, Staphylococcus aureus 196E ATCC 13565, Escherichia coli O157:H7 ATCC 43895, Salmonella typhimurium ATCC 13311, Yersinia enterocolitica)에 대하여 항균효과를 검색한 결과 Gram양성균인 L. monocytogenes에 대하여 물 및 에탄을 추출물 모두 MIC가 60$\mu\textrm{g}$/ml으로 극히 우수한 항균효과를 보여주었으며 항균효과가 높은 추출물의 열 안정성을 살펴보기 위하여 Listeria monocytogenes균 ATCC 15313, 19111 및 19113등 3종에 대하여 실험한 결과 길경 내 항균성물질은 121$^{\circ}C$ 에서 15분간 처리 시에도 항균효과가 나타남을 보아 열 안정성이 있는 것으로 나타났다. 또한 길경 추출물의 농도에 따른 항균효과를 실험한 결과 추출물의 농도가 500$\mu\textrm{g}$/m1일 때 Listeria monocytogenes균 ATCC 15313, 19111 및 19113 균 모두에 거의 안정된 항균효과를 가지고 있었으며 특히 Listeria monocytogenes ATCC 15313에 대하여 더욱 강한 항균효과를 보여주었다. Antimicrobial activity to the extracts of Platycodon glandiforum A. De. candolle was investigated against various food poisioning microorganisms(Listeria monocytogenes ATCC 15313, Staphylococcus aureus 196E ATCC 13565, Escherichia coli O157:H7 ATCC 43895, Salmonella typhimurium ATCC 13311 and Yersinia enterocolitica). The water and 70% ethanol extracts of Platycodon glandiforum A. De. candolle showed the strongest antimicrobial activities against Listeria monocytogenes in concentration of 500∼1000 $\mu\textrm{g}$/ml into culture broth (TSB) and was very stable over heat at 121$^{\circ}C$ for 15 min.

Antimicrobial effect of phytochemicals to Listeria monocytogenes isolated from slaughterhouses

Hyeji Kim,Hyemin Oh,Heeyoung Lee,Sejeong Kim,Jimyeong Ha,Jeeyeon Lee,Yukyung Choi,Yewon Lee,Yujin Kim,Yohan Yoon,Soomin Lee 한국식품위생안전성학회 2018 한국식품위생안전성학회지 Vol.33 No.4

본 연구에서는 도축장에서 분리된 Listeria monocytogenes를 저해할 수 있는 식물성 항균 물질을 탐색하고, 각 항균 물질의 최소살균농도(MBC)를 확인하였다. 잠재적인 항균 물질로 자몽종자 추출물, 감귤류 추출물, 생강 추출물, 배 추출물, 매실 농축액, 도라지 추출물, 대추 추출물, 오미자 추출물을 선정하였고, 대상 균주로는 국내 도축장에서 분리한 15 개의 L. monocytogenes 균주를 혼합하여 사용하였다. MBC 확인 결과, 최대 4.0 μg/mL 농도의 생강 추출물, 배 추출물, 매실 농축액, 도라지 추출물, 대추 추 출물, 오미자 추출물은 고농도의 L. monocytogenes (7 Log CFU/mL)에 대해 균 저해 효과가 전혀 나타나지 않았다. 반면, 7 Log CFU/mL의 L. monocytogenes에 대한 자몽종자 추출물과 감귤류 추출물의 MBC는 0.002 μg/mL인 것으로 확인되었다. 3 Log CFU/mL의 L. monocytogenes에 대한 MBC를 시험해 본 결과, 자몽종자 추출물과 감귤류 추출물의 MBC는 0.001 μg/mL로 나타났다. 이상의 결과로 볼 때 자몽종자 추출물과 감귤류 추출물은 L. monocytogenes를 제어하기 위해 적용될 수 있을 것으로 판단된다. This objective of this study was to investigate inhibitory effect of natural antimicrobials on Listeria monocytogenes isolated from Korean slaughterhouses. A mixture of 15 strains of L. monocytogenes at low (3 Log CFU/mL)and high (7 Log CFU/mL) concentration was exposed to various extracts (grapefruit seed extract, citrus fruits extract, ginger extract, pear extract, Japanese apricot concentrate, balloon flower extract, jujube extract, and omija extract) at 0.001-4.0μg/mL . Ginger extract, pear extract, Japanese apricot concentrate, balloon flower extract, jujube extract, and omija extract showed no antimicrobial effects to high-concentration of L. monocytogenes (7 Log CFU/mL). However, grapefruit seed extract and citrus fruit extract showed antibacterial effects against L. monocytogenes at 3 and 7 Log CFU/mL with MBCs of 0.001 and 0.002 μg/mL, respectively. These results indicate that grapefruit seed extract and citrus fruits extract can be used to control L. monocytogenes as natural antimicrobials.

Listeria monocytogenes YM-7의 생리적 특성

김영목 ( Young Mog Kim ),박욱연 ( Uk Yeon Park ),목종수 ( Jong Soo Mok ),장동석 ( Dong Suck Chang ) 한국수산학회 1995 한국수산과학회지 Vol.28 No.4

리스테리아 증세로 의심 되는 환자의 혈액으로부터 분리한 균은 저온에서의 운동성 시험, 용혈능 검정시험, 당 이용성, CAMP 시험의 결과 전형적인 L. monocytogenes의 특성을 보여 L. monocytogenes YM-7로 동정하고 명명하였다. L. monocytogenes YM-7의 세균학적 특성과 이 균이 생산하는 용혈독소의 이화학적 특성은 다음과 같다. L. monocytogenes YM-7 균주는 Gram양성 간균으로, 운동성 측정 결과 반고체 배지에서 우산 모양의 층이 생겼고, 최적증식조건은 37℃, 식염 농도 0%, pH 8.0이었고, 이 균의 생육 가능범위는 8~40℃, 식염 농도 0~7%, pH 5.0~10.0였다. L. monocytogenes YM-7 균주가 생산하는 용혈독소는 균 생육곡선의 정지기 부근에서 최대 활성을 나타내었다. 이 용혈독소는 비교적 열에 불안정 하였으나, 37℃에서 180분 후에도 용혈활성이 남아 있었다. 그리고 용혈독소는 pH 6.0~8.0에서 안정하였으나 그 외의 pH 영역에서는 매우 불안정 하였다. Listeria monocytogenes has been increasingly recognized as an important food poisoning and pathogenic bacterium which is Gram positive, non spore forming and facultative anaerobic rod shape. Bacteriological and physiological characterization of L. monocytogenes YM-7 isolated from a patient was performed. Optimum growth condition of L. monocytogenes YM-7 was at 37℃, pH 8.0 and 0% sodium chloride in tryptic soy broth, and then it grew pretty well in the range of 8~40℃, pH 5.0~10.0 and up to 7% of sodium chloride in the medium. The highest hemolysin activity of hemolysin produced by L. monocytogenes YM-7 was shown in the stationary phase of its growth. Hemolysin produced by the isolated strain was stable at 4℃ and pH 6.0~8.0, while it was gradually unstable by increasing the storage temperature.

Antimicrobial Effect of Phytochemicals to Listeria monocytogenes Isolated from Slaughterhouses

Kim, Hyeji,Oh, Hyemin,Lee, Heeyoung,Kim, Sejeong,Ha, Jimyeong,Lee, Jeeyeon,Choi, Yukyung,Lee, Yewon,Kim, Yujin,Yoon, Yohan,Lee, Soomin The Korean Society of Food Hygiene and Safety 2018 한국식품위생안전성학회지 Vol.33 No.4

본 연구에서는 도축장에서 분리된 Listeria monocytogenes를 저해할 수 있는 식물성 항균 물질을 탐색하고, 각 항균 물질의 최소살균농도(MBC)를 확인하였다. 잠재적인 항균 물질로 자몽종자 추출물, 감귤류 추출물, 생강 추출물, 배 추출물, 매실 농축액, 도라지 추출물, 대추 추출물, 오미자 추출물을 선정하였고, 대상 균주로는 국내 도축장에서 분리한 15 개의 L. monocytogenes 균주를 혼합하여 사용하였다. MBC 확인 결과, 최대 $4.0{\mu}g/mL$ 농도의 생강 추출물, 배 추출물, 매실 농축액, 도라지 추출물, 대추 추출물, 오미자 추출물은 고농도의 L. monocytogenes (7 Log CFU/mL)에 대해 균 저해 효과가 전혀 나타나지 않았다. 반면, 7 Log CFU/mL의 L. monocytogenes에 대한 자몽종자 추출물과 감귤류 추출물의 MBC는 $0.002{\mu}g/mL$인 것으로 확인되었다. 3 Log CFU/mL의 L. monocytogenes에 대한 MBC를 시험해 본 결과, 자몽종자 추출물과 감귤류 추출물의 MBC는 $0.001{\mu}g/mL$로 나타났다. 이상의 결과로 볼 때 자몽종자 추출물과 감귤류 추출물은 L. monocytogenes를 제어하기 위해 적용될 수 있을 것으로 판단된다. The objective of this study was to investigate the inhibitory effect of natural antimicrobials on Listeria monocytogenes isolated from Korean slaughterhouses. A mixture of 15 strains of L. monocytogenes at low (3 Log CFU/mL) or high (7 Log CFU/mL) concentration was exposed to various extracts (grapefruit seed extract, citrus fruit extract, ginger extract, pear extract, Japanese apricot concentrate, balloon flower extract, jujube extract, and omija extract) at $0.001-4.0{\mu}g/mL$. Ginger extract, pear extract, Japanese apricot concentrate, balloon flower extract, jujube extract, or omija extract showed no antimicrobial effects on high-concentration of L. monocytogenes (7 Log CFU/mL). However, grapefruit seed extract and citrus fruit extract showed antibacterial effects against L. monocytogenes at 3 and 7 Log CFU/mL with MBCs of 0.001 and $0.002{\mu}g/mL$, respectively. These results indicate that grapefruit seed extract and citrus fruits extract can be used to control L. monocytogenes as natural antimicrobials.