• Journal of Microbiology and Biotechnology
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• 제17권3호
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• pp.516-519
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• 2007

Enterobacter sakazakii is an emerging food pathogen, which induces severe meningitis and sepsis in neonates and infants, with a high fatality rate. The disease is generally associated with the ingestion of contaminated infant formula. In this study, we describe the development of a real-time PCR protocol to identify E. sakazakii using a TaqMan probe, predicated on the nucleotide sequence data of the 168 rRNA gene obtained from a variety of pathogens. To detect E. sakazakii, four primer sets and one probe were designed. Five strains of E. sakazakii and 28 non-E. sakazakii bacterial strains were used in order to ensure the accuracy of detection. The PCR protocol successfully identified all of the E. sakazakii strains, whereas the 28 non-E. sakazakii strains were not detected by this method. The detection limits of this method for E. sakazakii cells and purified genomic DNA were 2.3 CFU/ assay and 100 fg/assay, respectively. These findings suggest that our newly developed TaqMan real-time PCR method should prove to be a rapid, sensitive, and quantitative method for the detection of E. sakazakii.

• Journal of Dairy Science and Biotechnology
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• 제39권1호
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• pp.9-19
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• 2021

Enterobacter sakazakii (Cronobacter spp.) causes meningitis, necrotizing enterocolitis, sepsis, and bacteremia in neonates and children and has a high mortality rate. For rapid E. sakazakii detection, various differential and selective media containing α-glucosidase substrates, such as 5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside (BCIG) or 4-methylumbelliferyl-α-D-glucoside (α-MUG), have been developed as only E. sakazakii exhibits α-glucosidase activity in the genus Enterobacter. However, Escherichia vulneris (family: Enterobacteriaceae) can also utilize α-glucosidase substrates, thereby resulting in false positives. Various iron sources are known to promote the growth of gram-negative bacteria. This study aimed to develop a selective medium containing α-glucosidase substrates for E. sakazakii detection that would eliminate false positives, such as those of E. vulneris, and to determine the role of iron source in the medium. Three previously developed (TPD) media, i.e., Oxoid, OK, and VRBG, and the medium developed in this study, i.e., NGTE, were evaluated using 58 E. sakazakii and 5 non-E. sakazakii strains. Fifty-four E. sakazakii strains appeared as fluorescent or chromogenic colonies on all four media that were assessed. Two strains showed colonies on NGTE medium and not on TPD media. In contrast, the remaining two strains showed colonies on TPD media and not on NGTE medium. None of the non-E. sakazakii strains showed fluorescent or chromogenic colonies on any of the evaluated media except E. vulneris, which showed colonies on TPD media and not on NGTE medium. This study demonstrated that the newly developed NGTE medium was not only equally efficient in promoting the growth of bacterial colonies when compared with the currently available media but also eliminated false positives, such as E. vulneris.

• 식품기술
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• 제18권3호
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• pp.45-61
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• 2005

The first cases of neonatal meningitis believed to have been caused by Enterobacter sakazakii were reported in 1961. Prompted by several subsequent outbreaks of E. sakazakii infections in neonates and an increasing number of neonates in intensive care units being fed rehydrated powdered infant formula, considered to be a source of the pathogen, public health authorities and researchers are exploring ways to eliminate the bacterium or control its growth in dry infant formula, processing environments and formula preparation areas in hospitals. Reviewed here are advances in taxonomy and classification of E. sakazakii, methods of detecting, isolating and typing the bacterium, antibiotic resistance, clinical etilogy and pathogenicity. Outbreaks of E. sakazakii infections in neonates and adults are summarized. Reports on the presence of E. sakazakii in clinical settings, the environment and foods and food processing facilities are reviewed.

• 한국식품과학회지
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• 제40권1호
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• pp.101-105
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• 2008

시판되고 있는 선식 원료를 수거하여 최근 새로운 식중독균으로 보고되고 있는 Enterobacter sakazakii 분리 실험을 실시하였다. 그 결과 총 23종의 선식 원료 중 8개의 선식 원료에서 E.sakazakii로 추정되는 콜로니를 분리할 수 있었으며 API 20E kit를 이용하여 1차적으로 동정한 결과, 다시마 분말, 멸치 분말, 현미 분말, 청국장 분말 및 멥쌀 분말에서 E. sakazakii를 분리할 수 있었다. 이후 3 종의 primer를 이용한 PCR을 실시하여 2차적으로 동정하였다. 또한, 분리된 균주에 대한 RAPD-PCR을 실시하여 최종적으로 8종의 분리균으로 molecular typing을 할 수 있었다.

• 한국식품위생안전성학회지
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• 제23권2호
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• pp.157-162
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• 2008

열수(Hot water)를 이용 조제분유 및 선식 준비 시 살균효과를 실험한 결과 $60^{\circ}C$ 이상에서 살균 효과를 나타내었으나 조제분유와 선식에 열수를 가하였을 때 발생하는 온도 저하와 조제분유 및 선식 등의 분말 식품에서 biofilm을 생산하여 내열성이 증가되는 E. sakazakii의 특성을 고려하여 $70^{\circ}C$ 이상의 열수로 조제분유 및 선식 등을 용해하여야 할 것으로 판단된다. 마이크로웨이브를 이용 E. sakazakii의 살균 효과를 실험한 결과 E. sakazakii를 제어하기 위하여 100 mL의 경우 90초 이상 가열하거나 온도측정이 곤란한 경우에는 최초 끓는 시점까지 가열한 후 냉각하여 섭취하여야할 것으로 조사되었다. 용해된 조제분유 및 선식의 저장 안정성을 평가하기 위해 $5{\sim}35^{\circ}C$에 48시간 동안 보관하며 E. sakazakii의 성장곡선을 실험한 결과 $5^{\circ}C$, $10^{\circ}C$ 냉장온도에서는 48시간까지 증식이 미약하였다. 그러나 냉장 온도에서 사멸하지 않고 미약하게 증식하는 E. sakazakii의 특성을 고려하여 조제분유 및 선식의 냉장 보관은 24시간 이내로 제한하여 섭취하여야 할 것으로 판단된다.

• Journal of Dairy Science and Biotechnology
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• 제39권3호
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• pp.113-120
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• 2021

Although the number of incidences of illness caused by ingestion of the bacterial pathogen Enterobacter sakazakii (Cronobacter spp.) has dramatically declined, there remains a need for a robust isolation method to recover this microbe from powdered infant formula (PIF). The current method described in the FDA's Bacteriological Analytical Manual requires multiple steps, and 3-4+ days for complete analysis of PIF isolated E. sakazakii (Cronobacter spp.). We describe a bacteriological method including a one-step enrichment followed by plating on chromogenic agar for presumptive identification of E. sakazakii (Cronobacter spp.). Suspected colonies are confirmed by either biochemical analyses, or a Real-Time PCR-based assay. Using this method, E. sakazakii (Cronobacter spp.) in PIF can be isolated and identified within one day (24 hours).

• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.364-368
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• 2007

Enterobacter sakazakii may be related to outbreaks of meningitis, septicemia, and necrotizing enterocolitis, mainly in neonates. To reduce the risk of E. sakazakii in baby foods, thermal characteristics for Korean E. sakazakii isolates were determined at 52, 56, and $60^{\circ}C$ in saline solution, rehydrated powdered infant formula, and dried baby food. In saline solution, their D-values were 12-16, 3-5, and 0.9-1 min for each temperature. D-values increased to 16-20, 4-5, and 2-4 min in rehydrated infant formula and 14-17, 5-6, and 2-3 min in dried baby food. The overall calculated z-value was 6-8 for saline, 8-10 for powdered infant formula, and 9-11 for dried baby food. Thermal inactivation of E. sakazakii during rehydration of powdered infant formula was investigated by viable counts. Inactivation of cultured E. sakazakii in infant formula milk did not occur for 20 min at room temperature after rehydration with the water at $50^{\circ}C$ and their counts were reduced by about 1-2 log CFU/g at $60^{\circ}C$ and 4-6 log CFU/ml with the water at 65 and $70^{\circ}C$. However, the thermo stability of adapted E. sakazakii to the powdered infant formula increased more than two times. Considering that the levels of E. sakzakii observed in powdered infant formula have generally been 1 CFU/100 g of dry formula or less, contamination with E. sakazakii can be reduced or eliminated by rehydrating water with at least $10^{\circ}C$ higher temperature than the manufacturer-recommended $50^{\circ}C$.

• 한국식품위생안전성학회지
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• 제23권4호
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• pp.309-313
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• 2008

청동합금인 우리 전통 식기 놋그릇 유기 소재의 항미생물 특성을 연구하기 위해 주요한 식중독 세균인 Salmonella spp., E. coli O157, E. sakazakii, B. cereus의 배양액을 유기, 구리, 주석, 스테인레스 스틸 쿠폰에 노출 건조 후 생균정도를 분석하였다. 이들 금속표면에서의 살균력 측정결과 Gram(-) 세균인 S. Typhimurium, E. coli와 E. sakazakii는 구리가 가장 높았고 유기, 주석표면, 스테인레스 스틸 금속표면 순으로 항균성이 높은 것으로 나타났다. 그러나 Gram(+)인 B. cereus의 살균효과는 어느 금속표면에서도 살균효과가 거의 나타나지 않았다. S. Typhimurium, E. sakazakii의 구리이온($Cu^{+2}$ 최소생육저해 농도는 25 ppm이었지만 B. cereus의 경우는 50 ppm으로 높은 것으로 나타났다. 따라서 구리와는 다르게 강한 경도를 갖고 있는 유기 소재를 식품제조 환경에 적용시킬 경우 Salmonella spp.를 포함한 주요한 Gram(-) 세균의 교차오염을 효과적으로 저감화 할 수 있을 것으로 사료된다.

• Food Science and Biotechnology
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• 제15권1호
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• pp.152-157
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• 2006

Enterobacter sakazakii, designated as an unique microbial species in 1980, may cause bacteremia, necrotizing enterocolitis and infant meningitis. The distribution and the thermostability of E. sakazakii in unprocessed ready-to-eat (RTE) agricultural products of 252 and in 25 powdered infant formulas (PIF) were analyzed. Eighty one, 50, 43, and 47% of brown rice, pumpkin, potato, and carrot samples, respectively, had aerobic plate counts (ARC) in the range of 5 log CFU/g or more. Almost all the other products sampled had APC of approximately 2 log CFU/g. Fifty three, 75, 67, and 68% of banana, pumpkin, soybean, and carrot had Enterobacteriaceae counts approximating 3 log CFU/g. Sixty six percent of the brown rice tested had Enterobacteriaceae counts approximating 5-6 log CFU/g. E. sakazakii was isolated from 3/25(12%), 4/23(17%), 1/24(4%), and 1/27(4%) of PIF, brown rice, laver, and tomato samples, respectively. D-values were 3.52-4.79 min at 60 and $D_{60}-values$ were similar as the isolates reported. Thermal inactivation of four thermovariant E. sakazakii strains during the rehydration of PIF with hot water were investigated. At $50^{\circ}C$, the levels of E. sakazakii decreased one log CFU/g for 4-6 min and thereafter the levels remained stable for 20 min. At $60^{\circ}C$, inactivation by about 2 log CFU/g occurred for 20 min. Therefore, the unprocessed agricultural products might be a source of contamination for PIF when used as an ingredient after drying and pulverization. Rehydration of PIF for infant feeding with a water temperature of $60^{\circ}C$ rather than $50^{\circ}C$, as recommended by the manufacturers, may be helpful in the reduction of potential E. sakazakii risk.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.579-584
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• 2008

A commercial chromogenic agar medium (DFI) was supplemented with glucose (mDFI) to enhance the specificity of Enterobacter sakazakii (E. sakazakit) detection. Escherichia vulneris (E. vulneris), a putative false-positive strain on the DFI medium, produces ${\alpha}$-glucosidase. The enzyme ${\alpha}$-glucosidase hydrolyzes a substrate, 5-bromo-4-chloro-3-indolyl-${\alpha}$, D-glucopyranoside $(X{\alpha}Glc)$, producing green colonies. E. sakazakii strains produced green colonies on both DFI and mDFI agar, whereas E. vulneris produced green colonies on DFI agar but small white colonies on mDFI agar. E. sakazakii and E. vulneris were also readily differentiated by colony color when the mixed culture of the two strains was plated on mDFI agar and incubated for 24 h at $37^{\circ}C$. The results indicate that the selectivity of the commercial chromogenic agar medium could be improved by a simple supplementation with glucose.