Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

Cai, Xiaoming; Yu, Hai-Qiong; ZhouXi, Ruan; Yang, Lei-Liang; Bai, Jian-Shan; Deyi, Qiu; Jian, Zhihua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xingquan

doi:10.1371/journal.pone.0067082

Cited by 32 publications

(43 citation statements)

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“…In order to rapidly and accurately characterize Cronobacter species in PIF and its associated environments, several molecularbased protocols have been developed, which include direct target gene detection and subtyping methods (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Pulsed-field gel electrophoresis (PFGE) is an accepted method for tracking isolates across the food chain, and this approach is generally considered suitable for epidemiological studies (12,(21)(22)(23)(24)(25)(26)(27)(28).…”

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confidence: 99%

Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain

Yan

Wang

Gangiredla

et al. 2015

Appl Environ Microbiol

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cCronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n ‫؍‬ 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments. The epidemiological link between Cronobacter infection in neonates and contaminated powdered infant formula (PIF) has been previously established (4, 5), with C. sakazakii sequence type 4 (ST-4) being linked to cases of meningitis (6). Outbreaks have been associated with contaminated food products and the presence of this bacterium in PIF production environments (5, 7).In order to rapidly and accurately characterize Cronobacter species in PIF and its associated environments, several molecularbased protocols have been developed, which include direct target gene detection and subtyping methods (8-20). Pulsed-field gel electrophoresis (PFGE) is an accepted method for tracking isolates across the food chain, and this approach is generally considered suitable for epidemiological studies (12,(21)(22)(23)(24)(25)(26)(27)(28). A multilocus sequence typing (MLST) scheme for Cronobacter species was developed, which focuses on single nucleotide polymorphisms associated with seven housekeeping genes (including atpD, fusA, glnS, gltB, gyrB, infB, and pps) and identifies their associated alleles (29). This protocol has been used to describe some of the diversity related to the genus (6, 29-31). Both PFGE and MLST have been widely applied to study the genomic diversity of Cronobacter isolated from manufacturing facilities, commercial PIF, and follow-up formula, as we...

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confidence: 99%

Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain

Yan

Wang

Gangiredla

et al. 2015

Appl Environ Microbiol

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“…Therefore, the HRM profiles might be unreliable when the template concentration was too low (0.01 pg), which was consistent with the previous study by Cai et al . ().…”

Section: Resultsmentioning

confidence: 97%

“…Cai et al . () developed an HRM‐PCR method for the rapid detection and identification of Cronobacter spp. based on ompA gene.…”

Section: Discussionmentioning

confidence: 99%

“…Isolation of Cronobacter from PIF was performed as previously described by Cai et al . () and in collaboration with Zhongshan Entry‐Exit Inspection and Quarantine Bureau. All strains listed in Table can be potentially isolated from food samples, and were genetically identified and characterised by 16S RNA sequencing, MLST or conventional methods.…”

Section: Methodsmentioning

confidence: 99%

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Simultaneous detection and identification of pathogenic Cronobacter species by high‐resolution melting analysis in powdered infant formulas

et al. 2017

Int J of Dairy Tech

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A combination method of real-time PCR and high-resolution melting (HRM) analysis for the rapid detection and specific classification of six pathogenic Cronobacter species based on gene cgcA was developed. HRM profiles with distinct T m (melting temperature) peaks were consistently obtained with each species, represented by a single peak ranging from 86.02 to 86.80°C. The detection limit ranged from 0.1 to 1 pg per reaction. Desiccated Cronobacter sakazakii and Cronobacter muytjensii of 2 cfu/25 g were successfully detected after 4-week storage at room temperature. The newly developed method provided a molecular tool for direct detection and simultaneous identification of pathogenic Cronobacter species in powdered infant formulae.

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“…However, diagnostic sequencing methods requires special equipments and are very time‐consuming. As a result, comparing these methods; simple PCR by choosing a suitable part of a gene, could be a simple, quick and cost effective tool for bacterial identification (Cai et al, ).…”

Section: Introductionmentioning

confidence: 99%

A novel primer targeted gyrB gene for the identification of Cronobacter sakazakii in powdered infant formulas (PIF) and baby foods in Iran

Mashoufi

Ghazvini

Hashemi

et al. 2018

Journal of Food Safety

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The aim of this study was to perform a molecular identification and design a polymerase chain reaction (PCR) method based on a specific gene (gyrase subunit B [gyrB]) for rapid detection of Cronobacter sakazakii. In this study, from February 2017 to January 2018, 100 powdered infant formula milks (PIF1‐6) and 100 baby food items (BF1‐8) (total number = 200 samples) were purchased from different commercial brands from different pharmacies of the Mashhad city, Iran from different pharmacies of the Mashhad city, Iran. After isolation of Cronobacter, DNA extraction and PCR assay were performed to detect and confirm genus and species of isolated bacteria with 16S ribosomal RNA (16S rRNA) and designed gyrB primers, respectively. The abundance of C. sakazakii in PIFs and baby foods by culture method were 5/100 (5%) and 8/100 (8%), respectively; also, the final analysis based on gyrB primer pairs in PIF and baby food showed contamination rates of 0/100 (0%) and 3/100 (3%), respectively. These findings indicated that Iranian baby foods were a real threat for children's health. It is also recommended that this designed primer could be used for detection of Cronobacter sakazakii in these products. Practical application Cronobacter sakazakii is an opportunistic bacteria that is known worldwide as a foodborne pathogen especially in Infant Formula and baby foods, therefore the quality of these products has to be evaluated by a rapid, sensitive and specific method. Our findings indicated that Iranian baby foods were a threat for children's health so implementation of adequate sanitary measures to prevent Cronobacter sakazakii illness is necessary. It was also recommended that the designed primer based on gyr B gene could be used for specific detection of Cronobacter sakazakii in these products.

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Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

Cited by 32 publications

References 31 publications

Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain

Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain

Simultaneous detection and identification of pathogenic Cronobacter species by high‐resolution melting analysis in powdered infant formulas

A novel primer targeted gyrB gene for the identification of Cronobacter sakazakii in powdered infant formulas (PIF) and baby foods in Iran

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