Cronobacter sakazakii 분리배지의 성능 비교

김현정(Hyun-Jung Kim),구민선(Minseon Koo),오세욱(Se-Wook Oh) 한국식품영양과학회 2010 한국식품영양과학회지 Vol.39 No.5

식품공전에 Cronobacter sakazakii 분리배지로 등재되어 있는 3종의 분리배지에 대한 평가실험을 실시하였다. Chromogenic Enterobacter sakazakii agar, Enterobacter sakazakii agar가 VRBG agar에 비하여 뚜렷한 색택과 모양의 집락을 생성하였다. 3종의 분리배지 모두 30종의 Cronobacter sakazakii에 대한 sensitivity가 100%로 측정되었지만 Cronobacter sakazakii 이외의 Enterobacteriaceae 균주를 이용한 specificity 실험에서는 Chromogenic Enterobacter sakazakii agar, Enterobacter sakazakii agar가 100%로 측정되었지만 VRBG agar는 0%로 측정되었다. 인위적으로 접종한 식품에서의 회수율은 실험에 공시된 3종 배지에서 커다란 차이가 없었다. Three different isolation media for Cronobacter sakazakii have been recommended by Korea Food and Drug Administration from 2007. Performance comparison test was carried out between recommended Cronobacter sakazakii isolation medium. Chromogenic Enterobacter sakazakii agar (CESA) and Enterobacter sakazakii agar (ESA) produce more distinctive colonies having characteristic colors and appearance than Violet red bile glucose agar (VRBGA). The sensitivity and specificity of 3 different isolation media was checked. All 3 tested media showed 100% sensitivity when tested with 30 different Cronobacter sakazakii. The CESA and ESA showed 100% specificity when tested with Enterobacteriaceae except Cronobacter sakazakii, However, VRBGA did not show any specificity, showing inadequate selectivity compared to applicable Cronobacter sakazakii isolation medium. Artificially inoculated Cronobacter sakazakii to milk powder was easily recovered with 3 different isolation media and they all showed almost the same recovery activity.

Immunochromatographic Strip Assay for Detection of Cronobacter sakazakii in Pure Culture

( Xinjie Song ),( Shruti Shukla ),( Gibaek Lee ),( Myunghee Kim ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.11

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen, posing a high risk of disease to infants and immunocompromised individuals. In order to develop a quick, easy, and sensitive assay for detecting C. sakazakii, a rabbit anti-C. sakazakii immunoglobulin G (IgG) was developed using sonicated cell protein from C. sakazakii. The developed anti-C. sakazakii (IgG) was of good quality and purity, as well as species-specific. The developed rabbit anti-C. sakazakii IgG was attached to the surface of a sulforhodamine B-encapsulated liposome to form an immunoliposome. A test strip was then prepared by coating goat anti-rabbit IgG onto the control line and rabbit anti-C. sakazakii IgG onto the test line, respectively, of a plastic-backed nitrocellulose membrane. A purple color signal both on the test line and the control line indicated the presence of C. sakazakii in the sample, whereas purple color only on the control line indicated the absence of C. sakazakii in the sample. This immunochromatographic strip assay could produce results in 15 min with a limit of detection of 107 CFU/ml in C. sakazakii culture. The immunochromatographic strip assay also showed very good specificity without cross-reactivity with other tested Cronobacter species. Based on these results, the developed immunochromatographic strip assay is efficient for the detection of C. sakazakii and has high potential for on-site detection.

Cronobacter sakazakii의 산 저항성

장성란,방우석,Jang, Sung-Ran,Bang, Woo-Suk 한국축산식품학회 2011 한국축산식품학회지 Vol.31 No.4

본 연구는 표준균주 C. sakazakii(CAFM2)와 4개의 야생균주(EB 1, EB 5, EB 7, EB 41)를 사용하여 pH를 조정한 산성 환경에서의 저항성을 살펴보았다. C. sakazakii는 pH 4.5 TSB에서 5시간, 10시간 동안 적응한 경우, pH 4.5 TSB에서 적응하지 않은 경우와 비교하여 EB 7을 제외한 모든 균주가 pH 2.5 TSB에서의 D값이 유의적으로 높게 나타났다(p<0.05). 따라서 약산성 환경에서 적응한 균주는 강산성 환경에서의 저항성이 증가하였다. C. sakazakii는 pH 4.5 TSB에서 배양한 경우, pH 7.2 TSB에서 배양한 경우와 비교하여 모든 균주가 pH 2.5 TSB에서의 D값이 유의적으로 높게 나타났다(p<0.05). 따라서 약산성 환경에서 배양한 균주는 강산성 환경에서의 저항성이 증가하였다. 본 연구를 통해 C. sakazakii는 약산성 환경에 적응한 경우 강산성 환경에서의 저항성이 증가하는 것으로 나타났다. 이러한 acid adaptation response는 C. sakazakii와 관련된 식품 가공 산업에서의 hurdle technology를 적용하는데 있어서 안전성 확보와 관련된 시사점을 제공할 수 있을 것으로 판단된다. The objective of this study was to determine the resistance of Cronobacter sakazakii in acidic environments. The D-values of CAFM2 (ATCC 29544), EB 1, EB 5, and EB 41 at pH 2.5 in TSB were significantly (p<0.05) higher when cells were adapted at pH 4.5 in TSB for 5-h then when cells were not adapted at pH 4.5 in TSB. The D-values of CAFM2, EB1, and EB 41 at pH 2.5 in TSB were significantly (p<0.05) higher when cells were adapted at pH 4.5 in TSB for 10-h then when cells were not adapted at pH 4.5 in TSB. The D-values of CAFM2 and EB1 at pH 2.5 in TSB were significantly (p<0.05) higher when cells were adapted at pH 4.5 in TSB for 24-h then when cells were not adapted at pH 4.5 in TSB. The adaptation of C. sakazakii to mild acidic environments may result in increased resistance to severe acidic environments. The D-values of all test strains at pH 2.5 in TSB were significantly (p<0.05) higher when cells were cultured at pH 4.5 then when they were cultured at pH 7.2 in TSB. These data indicate that cells cultured in mildly acidic environments may result in increased resistance to severe acidic environments. The acid adaptation of C. sakazakii showed an increased resistance to acidic environments. The acid adaptation response of C. sakazakii has important implications for food safety, which should be considered when food preservation measures are developed.

사과에 오염된 Cronobacter sakazakii에 대한 유기산의 저해 효과

김민수,박은진 한국식품조리과학회 2018 한국식품조리과학회지 Vol.34 No.1

Purpose: Organic acids are generally recognized as safe (GRAS) and have been used to reduce bacteria in foods. This study aimed to evaluate the inhibitory effects of organic acids on Cronobacter sakazakii on the surfaces of apples using microbiological challenge testing. Methods: To examine the sanitizing effects of four organic acids (acetic acid, citric acid, lactic acid, and propionic acid), we treated four organic acids to C. sakazakii at 1, 3 and 5% and compared the number of C. sakazakii using viable cell counting. We inoculated C. sakazakii to the peel, pulp, stem end, and blossom end of apples, followed by immersion into lactic acid at 1, 2, 3, and 5% for 1 minute. We also immersed the peel and pulp of apples inoculated with C. sakazakii into 1% lactic acid for 30 sec and 1, 3, and 5 minutes. Results: As exposure concentrations and times to the four organic acids increased, the number of viable C. sakazakii decreased significantly (p<0.05). Lactic acid showed the greatest sanitizing effect on C. sakazakii compared to the other organic acids, and the sanitizing effects on the peel of apples were pronounced the most compared to the pulp and two ends of apples. Conclusion: The sanitization of bacteria on the surfaces of fruits may be dependent on the physical structure of fruits and interactions between the fruit matrix and bacteria. Although we suggest 1% lactic acid as the most efficient sanitizer for removing C. sakazakii on the surfaces of apples, fruit type-dependent variability in the sanitizing effects of organic acids needs to be further studied.

Evaluation of commercial probiotic lactic cultures against biofilm formation by Cronobacter sakazakii

( Anubhav Jamwal ),( Kavita Sharma ),( Rajni Chauhan ),( Saurabh Bansal ),( Gunjan Goel ) 대한장연구학회 2019 Intestinal Research Vol.17 No.2

Background/Aims: Cronobacter sakazakii, an emergent pathogen is considered as a major concern to infants and neonates fed on reconstituted powdered infant milk formula. In conjunction with many other factors, biofilm forming capacity adds to its pathogenic potential. In view of the facts that infants are at highest risk to C. sakazakii infections, and emerging antibiotic re-sistance among pathogens, it is imperative to evaluate probiotic cultures for their efficacy against C. sakazakii. Therefore, pure probiotic strains were isolated from commercial probiotic products and tested for their antimicro-bial and anti-biofilm activities against C. sakazakii. Methods: A total of 6 probiotic strains were tested for their antibiotic susceptibility followed by antimicrobial activity using cell-free supernatant (CFS) against C. sakazakii. The inhibitory activity of CFS against biofilm formation by C. sakazakii was determined using standard crystal violet assay and microscopic observations. Results: All the probiotic strains were sensitive to ampicillin, tetracycline, vancomycin and carbenicillin whereas most of the strains were resistant to erythro-mycin and novobiocin. Four of the 6 probiotic derived CFS possessed antimicrobial activity against C. sakazakii at a level of 40 μL. A higher biofilm inhibitory activity (>80%) was observed at initial stages of biofilm formation with weaker activity during longer incubation upto 48 hours (50%.60%). Conclusions: The study indicated the efficacy of isolated commercial probiotics strains as potential inhibitor of biofilm formation by C. sakazakii and could be further explored for novel bioactive molecules to limit the emerging infections of C. sakazakii. (Intest Res 2019;17:192-201.)

분유에 오염된 Cronobacter sakazakii 검출을 위한 중합효소연쇄반응, 실시간중합효소연쇄반응, 등온검출법의 비교

김영주,서승우,왕효우,서동주,이민화,손나리,이복희,최창순,Kim, Young-Joo,Seo, Sheungwoo,Wang, Xiaoyu,Seo, Dong Joo,Lee, Min Hwa,Son, Na Ry,Lee, Bog-Hieu,Choi, Changsun 한국축산식품학회 2013 한국축산식품학회지 Vol.33 No.5

Loop-mediated isothermal amplification (LAMP) is an emerging detection technology for the amplification of DNA under isothermal conditions. The aim of this study was to develop a rapid and reliable LAMP technique for the detection of Cronobacter sakazakii in milk powder. In order to enhance the sensitivity and specificity, LAMP primers targeting outer membrane protein A (ompA) gene of C. sakazakii were designed using Explorer V4 software. Thirty seven C. sakazakii strains and 13 pathogenic microorganisms were used for comparative detection of C. sakazakii using polymerase chain reaction (PCR), real-time PCR, and LAMP. LAMP developed in this study could specifically detect C. sakazakii strains without cross-reactivity with other foodborne pathogens. LAMP products amplified from ompA gene of C. sakazakii were digested with with HhaI and NruI enzyme. The specificity of LAMP was confirmed by restriction fragment length polymorphism (RFLP) analysis. LAMP could detect C. sakazakii within 1 h without bacterial culture and its detection limit was as low as 1 CFU/mL C. sakazakii in milk. In the comparison of the sensitivity, LAMP showed 10,000- and 100-times higher detection limit than PCR or real-time PCR, respectively. Therefore, this study can conclude that LAMP is a rapid and reliable detection technique for C. sakazakii contaminated in powdered milk. 본 연구에서는 영유아에게 치명적인 감염을 일으키는 C. sakazakii에 대하여 LAMP 검출법을 개발하였다. LAMP법에 의한 C. sakazakii의 검출율은 100%였으며 13개의 음성 지표군에 대해서는 모두 음성 반응을 보여 특이도가 매우 높은 것으로 판단되었다. 또한, HhaI과 NruI 두 개의 제한 효소를 LAMP product에 반응시킨 결과, 유전자의 특정 염기서열이 절단되는 것을 확인하였으며, 이를 통해 LAMP 검출법에 의해 증폭된 DNA가 C. sakazakii-specific ompA임을 확인하였다. 조제분유에 오염 된 C. sakazakii를 LAMP법으로 검출 시 검출한계는 $10^0$ CFU/mL이었으며 이는 기존의 PCR법이나 real-time PCR법에 비해 100-10,000배 높은 수준으로 민감도가 매우 높은 것으로 판단되었다. 이와 같이 높은 특이도와 민감도를 가진 LAMP 검출법은 C. sakazakii와 같은 급성 기회 감염균이나 병원성 미생물에 의한 식중독 발생시 현장에서 병원체를 간편하고 신속하게 검출할 수 있는 기술로 기대된다.

조제분유에 오염된 Cronobacter sakazakii에 대한 자외선 살균 효과

김민수,박은진 한국식품조리과학회 2017 한국식품조리과학회지 Vol.33 No.6

Purpose: Powdered infant formula is often exposed to microbial contamination during drying, mixing and packaging, even if it is pasteurized. This study aimed to examine the efficacy of ultraviolet (UV) radiation inactivation of Cronobacter sakazakii causing meningitis in infants through contamination of powdered infant formula. Methods: We applied UV radiation to powdered infant formula contaminated with C. sakazakii at thickness of 1, 3, and 5 mm for 5 min and 10 min and monitored inactivation of C. sakazakii by UV radiation. After stirring the contaminated powdered infant formula every 30 sec, we compared reduction of C. sakazakii between the rotation and non-rotation samples. We applied UV radiation to the contaminated powdered infant formula packaged using three types of polyethylene and low density polyethylene wrapping papers and evaluated the effects of individual packaging on reduction of C. sakazakii by UV radiation. Results: As the thickness of powdered infant formula increased, inactivation of C. sakazakii by UV radiation declined. Reduction of C. sakazakii was significantly higher in the rotation sample than the non-rotation sample. Viable counts of C. sakazakii were reduced with increasing time of exposure to UV radiation, although statistical significance was not obtained. Particularly, sterilization of C. sakazakii by UV radiation was larger in LDPE paper than in PE paper. Conclusion: This study suggests that microbial contamination by C. sakazakii in powdered infant formula can be reduced by UV radiation before and after packaging, thereby contributing to microbiological safety of powdered infant formula.

용균성 박테리오파지에 의한 Cronobacter sakazakii와 Salmonella enterica Typhimurium의 생육저해

이영덕(Young-Duck Lee),박종현(Jong-Hyun Park) 한국식품과학회 2011 한국식품과학회지 Vol.43 No.2

즉석 편이식품에서 위해도가 가장 큰 C. sakazakii와 S. enterica Typhimurium을 박테리오파지로 제어하기 위하여 용균성 박테리오파지를 분리, 동정하였고 조제분유와 채소 주스에 이들 세균에 적용하여 그의 효과를 분석하였다. 박테리오 파지는 돼지 분변에서 C. sakazakii와 S. enterica Typhimurium균을 용해시키는 박테리오파지를 분리하였고 현미경과 그의 특성을 분석, 동정하였다. Cronobacter에 작용하는 ES2 파지와 Salmonella의 ST2 파지는 형태학적 특성이 각각 Myoviridae와 Siphoviridae로 각각 동정되었으며 제한효소지도와 SDS-PAGE 분석에 의하여 서로 다른 파지임을 확인하였다. ES2 파지의 경우 latent period는 약 40분 정도였으며, ST2 파지는 약 30분 정도를 나타냈으며, burst size는 ES2 파지는 약 52±5 PFU/cell, ST2 파지는 약 21±3 PFU/cell로 나타났다. 열안정성은 60℃에서 ST2 파지의 경우 100분 동안 안정한 것으로 나타났으나, ES2 파지는 30분 이후부터는 확인되지 않았다. 따라서 ST2 파지가 ES2 파지에 비해 열안정성이 높은 것을 알 수 있었다. 이 분리 파지를 조제분유와 채소 주스에 직접 적용한 효과는 ES2에 의한 Cronobacter 제어는 접종 후 6시간까지는 균수가 일정하게 유지하였고 균의 증식을 일어나지 않는 것으로 나타났다. ST2 파지에 의한 Salmonella는 생육저해가 잘 일어나 접종시간이 지남에 따라 균수가 감소하는 것을 확인하였다. 그러므로 C. sakazakii와 S. enterica Typhimurium의 생육저해는 이들 용균성 박테리오파지를 활용하여 가능한 것으로 보인다. Cronobacter sakazakii and Salmonella enterica Typhimurium are hazardous pathogens, especially for ready-toeat foods. For control of pathogens, the virulent bacteriophages were isolated, identified, and applied to infant formula milk and vegetable juice. The phages were isolated from swine feces and identified by morphology and molecular characteristics. ES2 phage for C. sakazakii and ST2 phage for S enterica Typhimurium were identified as Myoviridae and Siphoviridae, respectively. Their burst sizes were 52±5 PFU/cell for ES2 phage and 21±3 PFU/cell for ST2 phage after latent period of 30-40 minutes. ST2 phage showed higher heat stability at 60℃ than ES2 phage. ES2 phage held the growth of C. sakazakii untill 6 hr afterwhich the number decreased when applied to the infant formula milk and vegetable juice. ST2 phage also showed growth inhibition so that the number of S. enterica Typhimurium decreased. Therefore, virulent bacteriophages might be an agent for the growth inhibition of C. sakazakii and S. enterica Typhimurium in such the ready-to-eat foods.

저온 환경에서 Cronobacter sakazakii의 저항과 생존

김세훈 ( Se Hun Kim ),장성란 ( Sung Ran Jang ),정현정 ( Hyun Jung Chung ),방우석 ( Woo Suk Bang ) 한국식품저장유통학회(구 한국농산물저장유통학회) 2011 한국식품저장유통학회지 Vol.18 No.4

Cronobacter sakazakii has been isolated from a wide range of environmental sources and from several foods of animal and plant origin. The objective of this study was to determine the resistance of C. sakazakii (ATCC 12868, ATCC 29004, and ATCC 29544) in cold, cold-freeze thaw, cold-acid, and cold starvation-freeze thaw stress. The number of C. sakazakii decreased to 1 log CFU/mL at 5℃ (cold storage) for 10 days. When C. sakazakii was cultivated at a low temperature (13℃), the population of C. sakazakii ATCC 12868 and 29004 increased to 10(9) CFU/mL, and the population of C. sakazakii ATCC 29544 increased to 10(8) CFU/mL. For C. sakazakii ATCC 12868 and 29004, the cold-adapted cells (5℃ 24 hr) decreased by 4 log CFU/mL, and the low-temperature-cultivated cells (13℃) decreased by 0.5 log CFU/mL. In this study, low-temperature cultivation enhanced the freeze-thaw cross-resistance due to the metabolic changes in the cells. Cold stress (5℃ 48 hr, 13℃ cultivation) enhanced the cold-acid cross-resistance. The cold-starved cells in the sterilized 0.1% peptone water enhanced the freeze-thaw cross-resistance with significant differences (p<0.05). Therefore, the increased tolerance of the cold-adapted or low-temperature-cultivated C. sakazakii cells to freeze-thaw, acid, or starvation suggests that such environments should be considered when processing minimally processed foods or foods with extended shelf life.

Two-stage label-free aptasensing platform for rapid detection of <i>Cronobacter sakazakii</i> in powdered infant formula

Kim, Hong-Seok,Kim, Young-Ji,Chon, Jung-Whan,Kim, Dong-Hyeon,Yim, Jin-Hyeok,Kim, Hyunsook,Seo, Kun-Ho Elsevier 2017 Sensors and actuators. B Chemical Vol.239 No.-

<P><B>Abstract</B></P> <P> <I>Cronobacter sakazakii</I> constitutes one of the most life-threatening foodborne pathogens in neonates, and is typically acquired via contaminated powdered infant formula. In this study, we developed a sensitive and convenient two-stage label-free aptasensing platform for colorimetric detection of <I>C. sakazakii</I> in powdered infant formula. In this system, <I>C. sakazakii</I> depletes aptamers from the test solution, and the reduction of aptamers induces aggregation of gold nanoparticles in salt, a process accompanied by a color change from red to purple. Under optimal conditions, <I>C. sakazakii</I> present in PIF at a concentration as low as 7.1×10<SUP>3</SUP> CFU mL<SUP>−1</SUP> could be visually detected within 30min, with a linear range between 7.1×10<SUP>3</SUP> and 7.1×10<SUP>7</SUP> CFU mL<SUP>−1</SUP>. This novel assay provides new opportunities to detect bacteria in real-world samples.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A two-stage label-free aptasensing platform was established to detect <I>C. sakazakii.</I> </LI> <LI> This is the first report of an aptamer against <I>C. sakazakii</I>. </LI> <LI> This assay can detect <I>C. sakazakii</I> in PIF at a 7.1×10<SUP>3</SUP> CFU/mL detection limit. </LI> <LI> This assay requires only 30min or less to complete after sample enrichment. </LI> <LI> This platform may be applicable for detecting other bacteria in real-world samples. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>