Search

Cronobacter spp. is an important foodborne pathogen that can cause life-threatening diseases in infants and immunocompromised adults. The present study was carried out to understand the prevalence and characterization of Cronobacter spp. in dried edible mushrooms in Jiangsu province, China. Cronobacter isolates were identified and genotyped by multilocus sequence typing (MLST); the antimicrobial susceptibility of Cronobacter strains was determined by the disk diffusion method; the biofilm formation ability of Cronobacter spp. was assessed using the microtiter plate method. The overall prevalence of Cronobacter spp. in dried edible mushrooms was 14.8%, with the highest contamination rate of after 37.2% found in Auricularia auricular. The Cronobacter isolates were identified as C. sakazakii (n = 26), C. malonaticus (n = 2), C. dublinensis (n = 2) and C. turicensis (n = 1). The MLST scheme produced 20 sequence types (STs), two of which were newly identified. ST148 was the most prevalent ST (n = 5), followed by ST4 (n = 3), ST17 (n = 3), ST64 (n = 3), and ST540 (n = 2). One (3.2%) and 15 (48.4%) Cronobacter isolates were resistant to tetracycline and meropenem, respectively. In contrast, all of the tested isolates were susceptible to the remaining 14 antibiotics. Moreover, 20 (64.5%) Cronobacter isolates showed weak ability to produce biofilm, but no isolates showed strong or moderate biofilm-forming ability. PRACTICAL APPLICATION: Our findings revealed a high genetic diversity of Cronobacter spp. in dried edible mushrooms and provided new epidemiological evidence for the widespread existence of Cronobacter spp. in such products. The presence of Cronobacter spp. in dried edible mushrooms may pose potential risks to human health and enhancing the hygiene of such products are necessary to ensure food safety., (© 2022 Institute of Food Technologists®.)

Cronobacter spp. have been linked to clinical cases of infection in both adults and infants. Enrichment of Cronobacter spp. before detection has been necessary but is quite time consuming. Hence, we sought to develop an immunomagnetic separation (IMS) PCR method that could shorten the time of enrichment before the detection of Cronobacter spp. The polyclonal antibody used in this immunomagnetic separation was prepared based on the outer membrane protein A of Cronobacter sakazakii China Center of Industrial Culture Collection 21560 and had high specificity to the target. The primers used in the IMS-PCR method also showed high specificity. The detection limit of IMS-PCR for pure C. sakazakii culture was 5.2 × 10 2 cfu/mL. Cronobacter sakazakii in artificially contaminated powdered infant formula (PIF) was also detected at a detection limit of 5.2 × 10 2 cfu/mL. After 8 h of enrichment, the detection limit in PIF was lower than 5.2 × 10 1 cfu/mL. An interference test using Escherichia coli in artificially contaminated PIF showed that the IMS-PCR method developed in this study had a good ability to resist interference. Finally, the IMS-PCR method was applied to the detection of Cronobacter in food samples and was shown to be reliable. Thus, this newly developed IMS-PCR detection method was quite sensitive, rapid, and reliable and could be applied to the detection of Cronobacter in foods., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Monoclonal antibody and polyclonal antibody specific to Cronobacter spp. had been reported in previous studies. However, the preparation of single-chain variable fragment (scFv) was faster and convenient. Hence, the aim of this study was to construct a scFv using outer membrane protein A (OmpA) of C. sakazakii as antigen. The protein sequences of OmpA of Cronobacter spp. were analyzed first. The results showed protein OmpA with length of 347 amino acids was conserved in Cronobacter genus (94.83%-100% of protein identity) and was greater than that observed for the other genera tested (8.28-91.64% of protein identity). Then, purified protein OmpA expressed in E. coli was used to prepare hybridoma and to construct scFv further. The scFv was named scFvH81 and analyzed by bioinformatics. The model of scFvH81 built by homologous modeling had a good quality (residues in disallowed regions: 3%) and showed that scFvH81 had a standard pocket-like site. Purified scFvH81 was prepared by denaturation and renaturation of inclusion body and it showed a good specificity and its affinity of Ka=2.39×10(6)M(-1). Therefore, it could be used in the detection and the pathogenesis study of Cronobacter spp., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A novel real-time PCR (qPCR) assay with internal amplification control based on the rpoB gene was developed for the detection and quantification of Cronobacter spp. Inclusivity and exclusivity of the qPCR assay were tested on a strain collection containing 19 Cronobacter and 26 non-Cronobacter strains. All Cronobacter strains were successfully identified, whereas no cross-reactivity was observed with non-Cronobacter strains. The sensitivity of the qPCR assay for pure culture and powdered infant formula (PIF) without enrichment was 3.44 log CFU/ml(g) (2.74 × 10(3) CFU/ml(g)). When the qPCR assay was applied to artificially contaminated PIF after a 12-h enrichment step, as few as 0.03 log CFU/ml (1.06 × 10(0) CFU/ml) of C. sakazakii could be detected. The limit of detection of the qPCR assay was not reduced by the presence of 8 log CFU/ml of Salmonella Enteritidis in PIF. A total of 70 food samples were analyzed for the presence of Cronobacter spp., out of which 3 dry cereal products, 5 maternal milk, and 1 infant food formula were found as positive by qPCR. The results obtained by qPCR were consistent with those obtained by culture-based method. Results from this study demonstrate that the qPCR assay is a rapid, specific, and accurate method suitable for Cronobacter detection in foods.

Here, we report the RNA polymerase beta-subunit gene (rpoB) as a new molecular marker for the identification of the Cronobacter species. The results indicated that members of the Cronobacter genus are more easily discriminated by rpoB sequencing than 16S rRNA sequencing, and reliable identification could be achieved by rpoB gene sequence comparison., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Abstract: Cronobacter spp. are important foodborne pathogens that can cause severe diseases such as meningitis, sepsis, and necrotizing enterocolitis in neonates. In this study, 195 food samples, including cereals, cereal products, powdered infant formula (PIF), infant food formula, herbs, spices, vegetables, and fruits, were analyzed for the presence of Cronobacter spp. by culture-based method. The presumptive isolates were further confirmed by targeting the 16S rDNA gene using PCR. Out of 195 samples, 13 samples (6.7%) were positive for Cronobacter species. 12 of 85 cereal and cereal products (14.1%), and 1 of 22 herbs and spices (4.5%) were contaminated. In contrast, no Cronobacter was detected in commercial powdered infant formula, infant food formula, vegetables, or fruits. Alignment of 16S rRNA gene sequences showed that 13 isolates was most closely related to the genus Cronobacter. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that Cronobacter sakazakii was the only Cronobacter species isolated from various food samples. The antimicrobial susceptibility of 13 Cronobacter isolates was determined by the standard disk diffusion method. All isolated strains, except one resistant to ampicillin, were sensitive or displayed intermediate susceptibility to the 10 antimicrobial agents investigated. No multiple drug resistance was observed. [Copyright &y& Elsevier]

Abstract: In this study, a PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed for the detection and quantification of Cronobacter spp. in powdered infant formula (PIF). The method uses digoxigenin- and biotin-labelled primers targeting the rpoB gene, and PCR products were detected using ELISA with antidigoxigenin-detecting antibodies. The specificity of the PCR-ELISA assay was evaluated with 39 strains, including 19 strains of Cronobacter spp. After 25 cycles of amplification, the PCR-ELISA assay showed a good linear relationship between cell numbers and PCR-ELISA values. The sensitivity of PCR-ELISA assay was 1.06 × 102 and 1.06 × 103 cfu mL−1 for pure cultures and PIF without enrichment, respectively. When the PCR-ELISA system was applied to artificially contaminated PIF, as few as 100 cfu mL−1 of Cronobacter sakazakii could be detected after 10 h enrichment. The PCR-ELISA assay is thus a sensitive and accurate method for the rapid detection and quantification of Cronobacter spp. in PIF. [Copyright &y& Elsevier]