Sakazakii In Powdered Infant Formula Research Articles

Immunoinformatics assisted design of a multi-epitope kit for detecting Cronobacter sakazakii in powdered infant formula

Abstract Objectives Cronobacter sakazakii, formerly Enterobacter sakazakii, is an emerging ubiquitous and opportunistic foodborne pathogen with a high mortality rate. It has been implicated in cases of meningitis, septicaemia, and necrotizing enterocolitis among infants worldwide in association with powdered infant formula (PIF). This study was an insilico designed peptide base kit framework, using immunoinformatic techniques for quick detection of C. sakazakii in PIF. Materials and Methods In the present study, a peptide-based kit was designed with a bioinformatic technique to rapidly identify C. sakazakii in PIF using flhE, secY, and bcsC, which are genes responsible for its biofilm formation, as target genes. The antigenicity, membrane topology, and the presence of signal peptides of the target genes were analysed using VaxiJen, DeepTMHMM, and SignalP servers. To provide stability and flexibility to the multiple-epitope construct, the linear B cells and helper T cells (IL-4 (interleukin 4) and IL-10 (interleukin 10) inducing epitopes) were linked with a GSGSG linker followed by the addition of protein disulphide bonds. To ascertain specificity, the multi-epitope construct was molecularly docked against genes from sources other than PIF, like alfalfa, and the environment, with PIF being the highest: –328.48. Finally, the codons were modified using the pET28a(+) vector, and the resultant multi-epitope construct was successfully cloned in silico. Results The final construct had a length of 486 bp, an instability index of 23.26, a theoretical pI of 9.34, a molecular weight of 16.5 kDa, and a Z-score of –3.41. Conclusions The multi-epitope peptide construct could be a conceptual framework for creating a C. sakazakii peptide-based detection kit, which has the potential to provide fast and efficient detection. However, there is a need for additional validation through the in vitro and in vivo techniques.

Directional immobilization of phage on the palladium-based nanozyme for colorimetric detection of Cronobacter sakazakii in powdered infant formula

Cronobacter sakazakii in powdered infant formula (PIF) can cause infections in newborns that are life-threatening. Establishing a High-performance evaluation method for C. sakazakii in the PIF is urgently needed. In this study, a chromogenic system based on phage-directed immobilized nanozyme for colorimetric measurement of C. sakazakii in PIF was constructed. Initially, our acquired phage EspYZU15 was electrostatically immobilized on the surface of a palladium-based nanozyme to produce EspYZU15 directionally immobilized Pd (EspYZU15@Pd). EspYZU15@Pd possesses outstanding peroxidase-like properties, catalyzing the chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The ability of EspYZU15@Pd to recognize and trap host bacteria allows C. sakazakii to specifically inhibit the TMB chromogenic process catalyzed by EspYZU15@Pd. Based on this detection strategy, a visual quantification method for C. sakazakii ranging from 3.2 × 102 to 3.2 × 107 CFU mL−1 was developed, yielding a low limit of detection (LOD) of 3.6 × 101 CFU mL−1. This method was able to successfully detect C. sakazakii in PIF, with outstanding recoveries ranging from 95.8% to 104.9%. These characteristics indicate that the EspYZU15@Pd-based chromogenic system has considerable promise for the colorimetric determination of C. sakazakii in PIF.

Rapid and Visual Determination of Cronobacter sakazakii in Powdered Infant Formula Using Competitive Annealing Mediated Isothermal Amplification (CAMP)

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen that causes serious infections, particularly in infants. Neonatal infections with this pathogen have an 80% mortality rate. One mechanism that C. sakazakii infects infants is through contaminated powdered infant formula (PIF). Here, based on the 16S rRNA gene, competitive annealing-mediated isothermal amplification (CAMP) is reported for the determination of C. sakazakii to reduce the risk of infection. The established CAMP method was able to accurately identify all target bacterial strains and did not yield false positive results with non-target bacterial strains. Furthermore, accelerating primers were designed and included in the CAMP reaction to enhance the amplification efficiency. The sensitivity of the assay for C. sakazakii in spiked PIF was 1.9 × 103 CFU/g. The CAMP assay with enrichment accurately detected the presence of C. sakazakii in PIF even at an initial inoculation level of 1 CFU/g. Additionally, to make the procedure simpler, hydroxynaphthol blue was employed as a visual indicator. For positive results, the reaction solution turns blue instead of violet, while the negative samples remain violet, which is suitable for rapid analysis in resource-limited settings.

A sensitive visual DNAzyme-based strategy for Cronobacter sakazakii in PIF by aRPA

Cronobacter sakazakii, as a most important foodborne pathogen in powdered infant formula (PIF), can cause diseases with high mortality in infants and young children, and has become a key monitoring target in dairy industry. In this study, a visual detection strategy based on DNAzyme and asymmetry recombinase polymerase amplification (aRPA) was developed for monitoring C. sakazakii in PIF. A large amount of single-stranded DNA (ssDNA) rich of guanine (G) was produced in this process and ssDNA could bend and fold into intramolecular parallel G-quadruplex in the presence of potassium chloride. The complex of G-quadruplex and hemin, described as DNAzyme, has strong peroxidase-like activity and catalyze the reaction of H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB), making the color of solution blue. To our knowledge, this is the first attempt in the detection of C. sakazakii based on aRPA and DNAzyme. The limits of detection (LOD) of this method for C. sakazakii were as low as 2.2 CFU mL−1 in pure culture and 5.4 × 101 CFU g−1 in artificially contaminated PIF, respectively. Besides, good stability of the method was shown in the on-site simulation evaluation, which was realized by only warm water, indicating that such strategy may have bright application prospects for field test without heating equipment.

Isolation and characteristics of new phage JK004 and application to control Cronobacter sakazakii on material surfaces and powdered infant formula

Cronobacter sakazakii (C. sakazakii) is a food-borne pathogen that can be spread by powdered infant formula (PIF) to cause illness in infants. Bacteriophages (phages) are an effective potential strategy to control food-borne pathogens. Phage JK004 which isolated from the native environment of C. sakazakii was a novel virulent phage with short latent period and large burst size. JK004 had tolerance to heat, pH and osmotic-stress. The phage JK004 did not contain known virulence, transduction or antibiotic resistance genes, indicating that JK004 is safe in the practical application. In addition, because of the biofilms formation, C. sakazakii could adhere to material surfaces and was difficult to remove, resulting in persistent contamination of the PIF. JK004 could effectively remove the biofilms that have formed on sterile 96-well plates with 87% inhibition rate. While the inhibition rate of bacteria adhered on the material surfaces such as polyethylene, rubber and stainless steel was 99.95%, 99.83%, 99.84%, respectively, with significant difference between the JK004 treated group and the control group (p < 0.05). The antibacterial activity of JK004 against C. sakazakii in PIF was also excellent. This study provides a basis for the potential application of JK004 for controlling of C. sakazakii in food production.

Effect of 405-nm light-emitting diode on environmental tolerance of Cronobacter sakazakii in powdered infant formula

Cronobacter sakazakii is an opportunistic pathogen that can survive extreme desiccation, heat, acid, and osmotic stress. This can increase the risk of infection, resulting in severe diseases, mainly in neonates. The inactivation effect of 405 ± 5-nm light-emitting diode (LED) illumination on C. sakazakii with different initial concentrations and C. sakazakii strains isolated from powdered infant formula (PIF) and baby rice cereal (BRC) were firstly evaluated. Then, the effect of 405 ± 5-nm LED on the tolerance of diverse environmental conditions of C. sakazakii in PIF was investigated. Conditions involving desiccation [PIF, Water activity (aw): 0.2–0.5], heat (45, 50, and 55 °C), acid (simulated gastric fluid: SGF, pH 4.75 ± 0.25), and bile salt (0.2%, bile salt solution) were used to study the effects of 405-nm LED on C. sakazakii resistance. The transcription levels of ten tolerance-associated genes and changes in bacterial cell membrane were examined to understand the response of C. sakazakii to LED illumination. The results showed that 405-nm LED effectively inactivated C. sakazakii ATCC 29544 with initial concentration from 8 to 1 log CFU/g in PIF and strains isolated from PIF and BRC. Moreover, 405-nm LED could decrease the tolerance of C. sakazakii in PIF to desiccation, heat treatment at 50 and 55 °C, SGF, and bile salt to different degrees, but the resistance to the heat treatment at 45 °C was not influenced by LED illumination. In addition, the transcription levels of the ten tolerance-associated genes measured in the LED-illuminated C. sakazakii cells were significantly downregulated compared with those in unilluminated controls. The damage on cell membrane was confirmed for LED-treated cells by LIVE/DEAD® assay. These results indicate that 405-nm LED illumination may be effective at reducing the environmental resistance of C. sakazakii in PIF. Furthermore, this study suggests the potential for applying 405-nm LED technology in the prevention and control of pathogens in food processing, production, and storage environments.

Specific detection of Cronobacter sakazakii in powdered infant formula using ssDNA aptamer.

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen associated with bacterial meningitis, sepsis, and necrotizing enterocolitis in premature and immuno-compromised infants. C. sakazakii is typically acquired by ingesting contaminated powdered infant formula (PIF). The growing demand for a safe food supply requires rapid detection of foodborne pathogens for delivering safe-to-consume food to consumers. In the present study, we isolated C. sakazakii-specific aptamers using a centrifugation-based partitioning method (CBPM) instead of systematic evolution of ligands by exponential enrichment (SELEX) process. Unlike SELEX, the CBPM reduces the evolution-loop time to obtain enriched probes, allowing the isolation of target-specific aptamers in a shorter time. The two aptamers (SC25 and SC45) isolated using the CBPM showed high affinity and specificity for C. sakazakii (Kd: 34 and 66 nM). Among the two aptamers, SC25 aptamer detected efficiently C. sakazakii in PIF with less cross-reactivity. Our results indicate that the isolated aptamers could be used for detecting C. sakazakii in PIF and reducing the overall testing time compared with the conventional C. sakazakii detection method.

Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula

Cronobacter sakazakii is an opportunistic foodborne pathogen that can infect newborns through powdered infant formula (PIF). In this study, we developed a novel enhanced lateral flow immunoassay (LFA) with enhanced sensitivity for detection of C. sakazakii in PIF by the naked eye. The proposed strategy for signal enhancement of the traditional LFA used concentrated gold nanoparticles (AuNP) as the enhancer to conjugate with capture antibodies, which could increase the immobilized capture antibodies concentration at the detection zone to improve capture efficiency. Besides, the detection signal was further amplified by accumulated AuNP as the C. sakazakii labeled with AuNP probes was captured by antibodies conjugated with enhancer at the test line. We also studied the effect of different concentrations of capture antibodies and concentrated AuNP on detection performance, and found that 2.2 mg/mL of capture antibodies and 0.06 nM concentrated AuNP were the optimal combination that could avoid a false-positive signal and maximally amplify the detection signal of the enhanced LFA. Using this strategy, the detection sensitivity of the enhanced LFA was 103 cfu/mL and improved 100-fold compared with traditional LFA. The strip was highly specific to C. sakazakii, and the time for detection of C. sakazakii in PIF was shortened by 3 h. In summary, the enhanced LFA developed by the addition of concentrated AuNP as the enhancer can be used as a sensitive, rapid, visual qualitative and point-of-care test method for detecting target analytes.

A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula.

A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5µg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMA-qPCR assay was 3.3×102 cfu/mL and 4.4×102 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6×102 cfu/g was obtained in the presence of the Staphylococcus aureus (107 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF.

Tea polyphenols inactivate Cronobacter sakazakii isolated from powdered infant formula

This study evaluated the antimicrobial activity of tea polyphenols (TP) against 4 Cronobacter sakazakii strains with different sequence types (ST) isolated from powdered infant formula (PIF). The results showed that in normal saline, 5mg/mL of TP (pH 3.44) could eliminate approximately 7.0 log cfu/mL of C. sakazakii within 1 h; in rehydrated PIF, after acidification with HCl (pH 3.55), TP showed a stronger antibacterial activity compared with the controls (malic acid, ascorbic acid, and citric acid). Further, some differences were obvious in tolerance to TP between C. sakazakii strains with different ST. The tolerance of C. sakazakii CE1 (ST4) to TP was found to be greater than that of the other 3 C. sakazakii strains (ST1, ST8, and ST64). The results of recovered test and transmission electron microscope analysis revealed that the action of TP against C. sakazakii was an irreversible bactericidal process caused by leakage of cytoplasm. Taken together, these results indicated that TP had an effective bactericidal effect against C. sakazakii, and provided a new idea for preventing and inactivating C. sakazakii in PIF.

Mining for sensitive and reliable species-specific primers for PCR for detection of Cronobacter sakazakii by a bioinformatics approach

Although several studies have reported PCR assays for distinguishing Cronobacter sakazakii from other species in the genus, reports regarding assay sensitivity and specificity, as well as applications for food testing, are lacking. Hence, the objective of this study was to develop a sensitive and reliable PCR-based method for detection of C. sakazakii by screening for specific target genes. The genome sequence of C. sakazakii in the GenBank database was compared with that of other organisms using BLAST. Thirty-eight DNA fragments unique to C. sakazakii were identified, and primers targeting these sequences were designed. Finally, 3 primer sets (CS14, CS21, and CS38) were found to be specific for C. sakazakii by PCR verification. The detection limit of PCR assays using the 3 pairs of primers was 1.35 pg/μL, 135 fg/μL, and 135 fg/μL, respectively, for genomic DNA, and 5.5×105, 5.5×103, 5.5×103 cfu/mL, respectively, using pure cultures of the bacteria, compared with 13.5 pg/μLand 5.5×105 cfu/mLfor primer set SpeCronsaka, which has been previously described. Cronobacter sakazakii were detected in artificially contaminated powdered infant formula (PIF) by PCR using primer sets CS21 and CS38 after 8h of enrichment. The detection limit was 5.5×10−1 cfu/10g of PIF. Thus, the PCR assay can be used for rapid and sensitive detection of C. sakazakii in PIF.

Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection ofCronobacter sakazakii

Cronobacter sakazakii is an emerging pathogen associated with the ingestion of contaminated reconstituted formula, which causes necrotizing enterocolitis, sepsis, and meningitis in low-birth-weight preterm neonatal infants. Sensitive and specific detection methods are needed to better control C. sakazakii infections. This study aims to develop a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting C. sakazakii in powdered infant formula (PIF). A set of four LAMP primers were designed based on the published C. sakazakii ompA gene sequence. Specificity of the assay was evaluated using a panel of 22 C. sakazakii, 27 Enterobacteriaceae family except C. sakazakii, and 25 other strains. Assay sensitivity was determined using serial dilutions of C. sakazakii American Type Culture Collection 51329 culture ranging from 10(6) colony-forming units (CFU)/mL to extinction. The assay was also tested in experimentally inoculated PIF samples. The ompA-based LAMP assay was able to detect specifically all of the 22 C. sakazakii strains without amplification from 52 non-C. sakazakii strains. The detection limit was 10(1) CFU/mL in pure culture, up to 10-fold more sensitive than that of the ompA-polymerase chain reaction (PCR). When applied to PIF, sensitivity was 10(2) CFU/mL, up to 10-fold that of the ompA-PCR. The ompA-based LAMP assay developed in this study was sensitive, specific, and low cost with great potential for future field detection of C. sakazakii in PIF.