• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.5
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• pp.764-768
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• 2010

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.

• Journal of Dairy Science and Biotechnology
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• v.39 no.3
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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).

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.229-239
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• 2012

Cronobacter spp. (formerly Enterobacter sakazakii), a Gram-negative bacillus, is a rare cause of meningitis and central nervous system infections. In England, the first case infected by this organism occurred in 1958. By July 2008, approximately 120 documented cases of Cronobacter spp. infection and at least 27 deaths have been identified from all around the world in the published literature and in reports submitted by public health sectors. In 2007, it was proposed by European organizations that the original taxonomy of E. sakazakii would be revised, to consist of five new species moved to a new genus, and identified as "Cronobacter". E. sakazakii has thus now been reclassified as 6 separate species in the new genus, Cronobacter, gen. nov., within the Enterobacteriaceae family. The new species are presently Cronobacter sakazakii, C. turicensis, C. malonaticus, C. muytjensii, and C. dublinensis; the sixth species is identified simply as genomospecies I, as currently including only two representative strains. The objectives of this review are to provide insight on (1) the classification and taxonomy of Cronobacter spp., (2) its clinical etiology and pathogenicity, (3) prequency of Cronobacter spp. in different categories of ready-to-eat food other than infant formula, (4) methods for detecting, isolating and typing Cronobacter spp., and (5) recent research trends for detecting Cronobacter spp.

• Journal of Dairy Science and Biotechnology
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• v.39 no.3
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• pp.104-112
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• 2021

This study was conducted to evaluate the efficacy of commercial disinfectants at inactivating Enterobacter sakazakii (Cronobacter spp.) in water. Disinfectant I contained 6.15% sodium hypochlorite, and disinfectant II contained both 2.25% n-alkyl dimethylbenzyl ammonium chloride and 2.25% n-alkyl ethylbenzyl ammonium chloride. Disinfectant I was added to distilled water to obtain a range of residual chloride concentrations at 50 ppm intervals with a maximum of 1-1,000 ppm. Disinfectant II was prepared at concentrations ranging from 1-200 ppm with 5 ppm intervals. Exposure time for all solutions was 10 min. In total, 58 E. sakazakii (Cronobacter spp.) strains were tested in this study. Nine isolates were obtained from clinical samples, and 49 isolates were obtained from environmental samples. Seven strains (6 clinical and 1 environmental) were able to survive in 100 ppm disinfectant I, and a maximum of 5 ppm of disinfectant II. Fifty one strains (3 clinical and 48 environmental) were not killed in 10 ppm of disinfectant I and 1 ppm of disinfectant II in water. In conclusion, this study demonstrated that clinical E. sakazakii (Cronobacter spp.) strains displayed 5- to 10-fold higher resistance to disinfectants than environmental E. sakazakii (Cronobacter spp.) strains. Disinfectant II, containing quaternary ammonium compounds, was shown to be more potent in inactivating E. sakazakii (Cronobacter spp.) in water used to clean infant formula manufacturing equipment than disinfectant I.

• Journal of Dairy Science and Biotechnology
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• v.41 no.3
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• pp.103-112
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• 2023

This study aimed to assess the effectiveness of 10 different pre-enrichment methods using Real-Time polymerase chain reaction (PCR) in support of the FDA method. When the initial Cronobacter spp. (Enterobacter sakazakii) inoculation was 7.2 CFU/g, the Ct values were observed in the following order: 21.37 (Enterobacteriaceae enrichment [EE] broth), 21.95 (brain heart infusion [BHI]), 22.72 (tryptic soy broth [TSB]), 23.02 (violet red bile lactose [VRBL]), 22.31 (TSB-0.1% sodium pyruvate [SP]), 23.43 (distilled water [DW]), 24.34 (phosphate buffered saline [PBS]), 24.95 (nutrient broth [NB]), 25.82 (TSB-0.6% yeast extract [YE]), and 28.27 (violet red bile glucose [VRBG]). For an inoculation of 1.82% CFU/g of Cronobacter spp. (E. sakazakii), the Ct values were recorded in this sequence: 20.34 (EE broth), 22.16 (TSB-0.6% YE), 22.37 (BHI), 22.71 (VRBL), 22.88 (TSB), 23.01 (DW), 23.19 (NB), 23.79 (TSB-0.1% SP), 24.66 (VRBG), and 24.70 (PBS). Finally, when the inoculum of Cronobacter spp. (E. sakazakii) was 0.182 CFU/g, the Ct values followed this order: 21.93 (VRBL), 23.07 (TSB-0.6% YE), 23.31 (DW), 23.47 (PBS), 23.70 (BHI), 24.14 (TSB-0.1% SP), 25.14 (TSB), 29.00 (VRBG), 31.55 (EE broth), and were undetected in the case of NB. Consequently, these results indicate that there were no significant differences among the 10 different pre-enrichment broths. Future studies should focus on exploring pre-enrichment broths that can improve the limit of detection at very low Cronobacter spp. (E. sakazakii) concentrations and enhance the selective recovery of Cronobacter spp. (E. sakazakii) under acid, antibiotic, cold, and heat damage conditions.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1855-1862
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• 2016

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 $10^7CFU/ml$ 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.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.757-762
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• 2010

Cronobacter is a major foodborne pathogen in powdered infant formula and can lead to serious developmental after-effect and death to infants. The contamination of Cronobacter may be a high risk for the powdered foods. To isolate and identify Cronobacter from the powdered foods such as powdered infant formula and Saengsik in Korea, a conventional culture method, rapid identification system, PCR, and 16S rDNA sequencing were performed. As the results of isolation, seven Cronobacter spp. were isolated from seven out of 102 powdered infant formulas and 41 Cronobacter were isolated from 41 out of 86 Saengsiks. Forty-eight Cronobacter isolates were identified to be C. sakazakii and C. dublenisis by 16S rDNA sequence analysis. Most of the isolates were C. sakazakii and 13% of the isolates were C. dublinesis. One fourth of the C. sakazakii isolates showed different biochemical characteristics of negative nitrate reduction and nonmotility activities compared with the other strains reported previously.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1629-1635
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• 2016

A novel bacteriophage, PBES 02, infecting Cronobacter sakazakii was isolated and characterized. It has a spherical head of 90 nm in diameter and a tail of 130 nm in length, and belongs to Myoviridae as observed under a transmission electron microscope. The major virion protein appears to be 38 kilodaltons (kDa) in size. The latent period of PBES 02 is 30 min and the burst size is 250. Infectivity of the phage remained intact after exposure to temperatures ranging from 4℃ to 55℃ for 1 h. It was also stable after exposure to pHs ranging from 6 to 10 for 1 h. The phage effectively removed contaminating Cronobacter sakazakii from broth infant formula. PBES 02 has a double-stranded DNA genome of 149,732 bases. Its GC ratio is 50.7%. Sequence analysis revealed that PBES 02 has 299 open reading frames (ORFs) and 14 tRNA genes. Thirty-nine ORFs were annotated, including 24 related to replication and regulation functions, 10 related to structural proteins, and 5 related to DNA packaging. The genome of PBES 02 is closely related to that of two other C. sakazakii phages, CR3 and CR8. Comparison of DNA sequences of genes encoding the major capsid protein revealed a wide geographical distribution of related phages over Asia, Europe, and America.

• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.509-514
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• 2011

This study was conducted to investigate the phenotypic and genotypic characteristics of Korean isolates of Cronobacter spp. (Enterobacter sakazakii). A total of 43 Cronobacter spp., including 5 clinical isolates, 34 food isolates, 2 environmental isolates, and 2 reference strains (C. sakazakii ATCC 29004 and C. muytjensii ATCC51329) were used in this study. Korean isolates of Cronobacter spp. were divided into 11 biogroups according to their biochemical profiles and 3 genomic groups based on the analysis of their 16S rRNA gene sequences. Biogroups 1 and 2 contained the majority of isolates (n=26), most of which were contained in 16S rRNA cluster 1 (n=34). Korean isolates of Cronobacter spp. showed diverse biochemical profiles. Biogroup 1 contained C. sakazakii GIHE (Gyeonggido Research Institute of Health and Environment) 1 and 2, which were isolated from babies that exhibited symptoms of Cronobacter spp. infection such as gastroenteritis, sepsis, and meningitis. Our finding revealed that Biogroup 1, C. sakazakii, is more prevalent and may be a more pathogenic biogroup than other biogroups, but the pathogenic biogroup was not represented clearly among the 11 biogroups tested in this study. Thus, all biogroups of Cronobacter spp. were recognized as pathogenic bacteria, and the absence of Cronobacter spp. in infant foods should be constantly regulated to prevent food poisoning and infection caused by Cronobacter spp.

• Korean Journal of Food Science and Technology
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• v.43 no.2
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• pp.176-181
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• 2011

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 $52{\pm}5PFU/cell$ for ES2 phage and $21{\pm}3PFU/cell$ for ST2 phage after latent period of 30-40 minutes. ST2 phage showed higher heat stability at $60^{\circ}C$ 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.