Characterization of Dev-CD-23823 and Dev-CT57, new Autographivirinae bacteriophages infecting Cronobacter spp.

Cronobacter spp. are opportunistic pathogenic bacteria responsible for severe infections in neonates. Powdered infant formula has been confirmed to be the source of infection in some cases. Bacteriophages offer a safe means for eliminating this pathogen. In the present study, we characterized two closely related Cronobacter-specific bacteriophages of the proposed genus "GAP227virus". The phages Dev-CD-23823 and Dev-CT57 possessed broad host specificity, as they infected 88% and 80% of the Cronobacter strains tested. Genome sequence comparisons of phages Dev-CD-23823 and Dev-CT57 showed different levels of similarity to the prototype GAP227 phage. The Dev-CT57 phage was highly similar, whereas the Dev-CD-23823 phage showed only 75% sequence identity. A phylogenic tree based on the RNA polymerase (RNAP) gene from selected representatives of the subfamily Autographivirinae confirmed the grouping of Dev-CD-23823, Dev-CT57 and GAP227 in one cluster together with phages PP2, Phi80-18 and PhiR8-01. A common conserved motif was also detected in the RNAP promoters of these phages. The functional activity of these RNAP promoters was confirmed experimentally using a promoter probe vector, and a phage-specific signal was observed; however, some cross-specificity of Dev-CD-23823 and Dev-CT57 promoters was also detected. These results will contribute to our understanding of the biology and evolution of Autographivirinae phages.

Molecular Characterization of Clostridium botulinum Harboring the bont/B7 Gene.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the causative agent of botulism, a rare but serious disease that can result in death if not treated. Infant botulism occurs when C. botulinum colonizes the intestinal tract of infants and produces BoNT. It has been proposed that infants under the age of 1 year are uniquely susceptible to colonization by C. botulinum as their intestinal microbiota is not fully developed and provides little competition, allowing C. botulinum to thrive and produce BoNT in the gut. There are seven well-characterized serotypes (A-G) of BoNT identified by the ability of specific antitoxins to neutralize BoNTs. Molecular technology has allowed researchers to narrow these further into subtypes based on nucleic acid sequences of the botulinum toxin (bont) gene. One of the most recently recognized subtypes for bont/B is subtype bont/B7. We identified through whole genome sequencing five C. botulinum isolates harboring bont/B7 from CDC's strain collection, including patient isolates and an epidemiologically linked isolate from an opened infant formula container. In this study, we report the results of whole genome sequencing analysis of these C. botulinum subtype bont/B7 isolates. Average nucleotide identity and high quality single nucleotide polymorphism (hqSNP) analysis resulted in two major clades. The epidemiologically linked isolates differed from each other by 2-6 hqSNPs, and this clade separated from the other isolates by 95-119 hqSNPs, corroborating available epidemiological evidence.

Bacteriocins: Recent Trends and Potential Applications.

In the modern era, there is great need for food preservation in both developing and developed countries due to increasing demand for extending shelf life and prevention of spoilage of food material. With the emergence of new pathogens and ability of micro-organisms to undergo changes, exploration of new avenues for the food preservation has gained importance. Moreover, awareness among consumers regarding harmful effects of chemical preservatives has been increased. Globally, altogether there is increasing demand by consumers for chemical-free and minimal processed food products. Potential of bacteriocin and its application in reducing the microbiological spoilages and in the preservation of food is long been recognized. Bacteriocins are normally specific to closely related species without disrupting the growth of other microbial populations. A number of applications of bacteriocin have been reported for humans, live stock, aquaculture etc. This review is focused on recent trends and applications of bacteriocins in different areas in addition to their biopreservative potential.

Prevalence and Characterization of Cronobacter sakazakii in Retail Milk-Based Infant and Baby Foods in Shaanxi, China.

Cronobacter sakazakii (formerly Enterobacter sakazakii) is an opportunistic pathogen that causes meningitis, sepsis, and necrotizing enterocolitis in neonates and infants through consumption of contaminated milk-based foods. In this study, the prevalence of C. sakazakii in 705 retail milk-based infant and baby food samples was investigated in 12 cities in Shaanxi, China, in 2010 and 2012. One hundred and nineteen samples (16.9%) were C. sakazakii positive. The isolates were further characterized for antimicrobial susceptibility to 14 antibiotics, pulsed-field gel electrophoresis profiles, and presence of the virulence genes. Samples of brand W, Y, A, and G in 2010 and 2012 were C. sakazakii positive. All isolates recovered in 2010 and 2012 were susceptible to levofloxacin and cefoperazone. In 2012, no isolate was resistant to gentamicin, cefoxitin, chloramphenicol, gatifloxacin, ciprofloxacin, and ceftriaxone. Antibiotic resistance of the isolates was most commonly found to rifampicin, amoxicillin-clavulanic acid, streptomycin, tetracycline, and ampicillin in both 2010 and 2012, except to trimethoprim/sulfamethoxazole in 2012. Pulsed-field gel electrophoresis profiles indicated that C. sakazakii isolates were genotypically diverse, although these isolates were prevalent in infant and baby foods with the same brand. A total of 34 virulence gene profiles of the C. sakazakii isolates in 2010 and 2012 were detected. Isolates that co-carried hly-ompX-eitCBAD-iucABCD/iutA genes in 2012 were significantly (p < 0.05) more prevalent than those in 2010. The results added new epidemiological evidence for the widespread occurrence of C. sakazakii in retail milk-based infant and baby foods and this should be an indicator of potential health risk for consumers.

Antimicrobial Activity of Ferulic Acid Against Cronobacter sakazakii and Possible Mechanism of Action.

Cronobacter sakazakii is an opportunistic pathogen transmitted by food that affects mainly newborns, infants, and immune-compromised adults. In this study, the antibacterial activity of ferulic acid was tested against C. sakazakii strains. Minimum inhibitory concentration of ferulic acid against C. sakazakii strains was determined using the agar dilution method. Changes in intracellular pH, membrane potential and intracellular ATP concentration were measured to elucidate the possible antibacterial mechanism. Moreover, SYTO 9 nucleic acid staining was used to assess the effect of ferulic acid on bacterial membrane integrity. Cell morphology changes were observed under a field emission scanning electron microscope. The minimum inhibitory concentrations of ferulic acid against C. sakazakii strains ranged from 2.5 to 5.0 mg/mL. Addition of ferulic acid exerted an immediate and sustained inhibition of C. sakazakii proliferation. Ferulic acid affected the membrane integrity of C. sakazakii, as evidenced by intracellular ATP concentration decrease. Moreover, reduction of intracellular pH and cell membrane hyperpolarization were detected in C. sakazakii after exposure to ferulic acid. Reduction of green fluorescence indicated the injury of cell membrane. Electronic microscopy confirmed that cell membrane of C. sakazakii was damaged by ferulic acid. Our results demonstrate that ferulic acid has moderate antimicrobial activity against C. sakazakii. It exerts its antimicrobial action partly through causing cell membrane dysfunction and changes in cellular morphology. Considering its antimicrobial properties, together with its well-known nutritional functions, ferulic acid has potential to be developed as a supplement in infant formula or other foods to control C. sakazakii.

The growth of Streptococcus mutans in different milks for infant feeding.

After birth various bacterial species, mainly streptococci, colonize the oral cavity and are frequently isolated from carious lesions in children. Previous studies suggest that an earlier colonization of a child's mouth by cariogenic organisms might be related to a higher risk of caries. The aim of this study is to assess the influence of different milks for infant feeding on the growth of Streptococcus mutans (SM) colonies. Three human milk samples from three different mothers and five infant formulas were tested. To prepare the bacterial inoculum, SM was grown in Brain Heart Infusion broth for 18 hours at 37°C in microaerophilic atmosphere. The growth of SM was determined immediately after the inoculation of milks (T0) and after 24 hours (T24) of incubation. After 24 hours of incubation (ΔT= CFUs/ml at T24 – CFUs/ml at T0) the bacterial growth changes were different among milks. Among the complementary milks tested, ΔT of formulas supplemented with Lactobacillus reuteri and with Bifidobacterium lactis was lower than those of non-supplemented formula. In conclusion, on the basis of the reduced SM growth in milks supplemented with probiotics, we may speculate that these formulas have a preventive effect on the development of caries in children.

Identification and characterization of Cronobacter strains isolated from powdered infant foods.

UNLABELLED: Cronobacter spp. (formerly Enterobacter sakazakii) is responsible for rare but fatal cases of infection in neonates and immunocompromised infants. The aim of our study was to characterize Cronobacter strains isolated from powdered infant foods in Public Health Authority of the Slovak Republic in 2009-2010. Powdered infant food products have been analysed using currently available standard method ISO/TS 22964: 2006 for the detection of Cronobacter spp. complemented with qPCR confirmation of positive strains. Thirteen Cronobacter strains were isolated from more than 900 powdered infant formulae, milk-based and cereal-based powdered weaning food products. The strains were assigned to five biogroups and ten multilocus sequence typing (MLST) sequence types. In total, twelve strains were identified as Cronobacter sakazakii and one strain as Cronobacter dublinensis. Multiple strains originated from parallel isolation were obtained in three samples and the variability between strains from the same food was observed twice. The results are in agreement with the hypothesis that the Cronobacter contamination detected in infant powdered food is low and originating in various accidental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterized Cronobacter strains isolated from powdered infant formulae and weaning foods by biotyping and multilocus sequence typing. The later method was shown to be more discriminative and suitable for both species identification and subtyping. Low level (0·9%) of Cronobacter positivity was observed in 916 samples. Multiple sequence types were observed among strains isolated from the same food product. This highlights that multiple isolates from each single sample should be analysed in epidemiological studies, since more than one genetic subtype may be present.

Detection of Cronobacter species in powdered infant formula by probe-magnetic separation PCR.

Cronobacter species are opportunistic foodborne pathogens associated with serious infections in preterm neonates and infants. Based on the epidemiological research, infant formula products are considered to be the main source of infections from this organism. Therefore, accurate methods are required for detection of Cronobacter species. In this study, the specific probe and primers for detection of this organism were designed and verified. The probe-magnetic beads were prepared for sequence capture, followed by PCR assay to detect the target gene. This probe-magnetic separation PCR assay could detect as few as 10(3) cfu/mL of Cronobacter in artificially contaminated infant formulas in less than 4 h. The combination of magnetic beads and PCR showed the potential for the detection of Cronobacter in infant formulas and may have applications in the dairy industry.

[Occurrence of Bacillus cereus in infants and young children foods in 8 provinces in China].

OBJECTIVE: To investigate the contamination status of infants and young children foods by Bacillus cereus. METHODS: 1716 food samples were collected from retail market in 8 provinces in China and examined for Bacillus cereus by the plate count method. RESULTS: Bacillus cereus was isolated from 9.85% of all the samples (169/1716). 14.08% of the powered formula for infants and young children were contaminated with B. cereus as well as 6.01% of the ready-to-eat cereal-based complementary foods for infants and young children. Counts higher than 100 CFU/g were found in 18 samples of the powered formula (2.20%) and 10 samples of the ready-to-eat cereal-based complementary foods (1.11%). CONCLUSION: Inspection and management of infants and young children foods should be enhanced to prevent Bacillus cereus infection.

Systematic risk ranking of microbiological hazards in infant foods.

Ensuring food safety, particularly for vulnerable groups, like infants and young children, requires identifying and prioritizing potential hazards in food chains. We previously developed a web-based decision support system (DSS) to identify specific microbiological hazards (MHs) in infant and toddler foods through a structured five-step process. This study takes the framework further by introducing systematic risk ranking (RR) steps to rank MH risks with seven criteria: process survival, recontamination, growth opportunity, meal preparation, hazard-food association evidence, food consumption habits of infants and toddlers in the EU, and MH severity. Each criterion is given a semi-quantitative or quantitative score or risk value, contributing to the final MH risk calculation via three aggregation methods: semi-quantitative risk scoring, semi-quantitative risk value, and outranking multi-criteria decision analysis (MCDA). To validate the criteria and ranking approaches, we conducted a case study to rank MH risks in infant formula, compared the results of the three risk ranking methods, and additionally evaluated the ranking results against expert opinions to ensure their accuracy. The results showed strong agreement among the three methods, consistently ranking Salmonella non-Typhi and Cronobacter spp. and Shiga-toxin-producing Escherichia coli as the top MH risks in infant formulae, with minor deviations. When MHs were ranked after an initial hazard identification step, all three methods produced nearly identical MH rankings, reinforcing the reliability of the ranking steps and the selected criteria. Notably, the risk value and MCDA methods provided more informative MH rankings compared to the risk scoring method. The risk value and risk scoring methods were implemented into an online tool, called the MIcrobiological hazards risk RAnking decision support system (Mira-DSS), available at https://foodmicrobiologywur.shinyapps.io/MIcrobial_hazards_RAnking/. In conclusion, our framework enables the ranking of MH risks, facilitating intervention comparisons and resource allocations to mitigate MH risks in infant foods, with potential applicability to broader food categories.

Occurrence, evaluation, and human health risk assessment of ochratoxin a in infant formula and cereal-based baby food: a global literature systematic review.

This study reviews global levels of ochratoxin A (OTA) in infant formula and cereal-based foods, using Monte Carlo simulation to assess risks. The review found 24 studies on global OTA levels in infant food and cereal-based products, using databases including PubMed, Scopus, Web of Science and Embase until March 2024. We estimated OTA exposure in infant food based on concentration, intake and body weight. The exposure and hazard quotient margin were calculated using BMDL10 and TDI values. Monte Carlo simulation evaluated human health risks from OTA in infant formula and cereal-based foods. A global study from 14 countries shows varying levels, surpassing EU limits in Tunisia, Ecuador, the USA, and generally in Africa, notably in infant cereals, which had higher levels than formula. Globally, OTA was present in 29.3% of the 3348 samples analyzed, with Lebanon at 95.2% and Brazil at 0%. Analysis indicates only non-carcinogenic risk for infants. While health risks for infants are mostly low, ongoing research and monitoring are vital to minimize OTA exposure in infant food.

Hygiene intervention reduces contamination of weaning food in Bangladesh.

OBJECTIVE: This study was conducted to measure the impact of a hygiene intervention on the contamination of weaning food in Bangladesh. METHODS: Sixty households were selected: 30 study and 30 control households. Samples of weaning food were collected from all the 60 households at baseline and examined for faecal coliforms (FC), faecal streptococci (FS) and Clostridium perfringens (CP) following standard procedures. After cooking, food samples were collected on three occasions before feeding. Following Hazard Analysis Critical Control Point (HACCP) procedures, critical control points were determined. The mothers in the 30 study households were then trained for 4 weeks in how to attain the control point conditions. Then, again the food samples were collected and analysed. RESULTS: At baseline, weaning foods from study and control households were heavily contaminated with FC and FS. The FC and FS counts were 1.84 log(10) and 1.92 log(10) colony-forming unit (cfu)/g, respectively, in the study households, and 0.86 log(10) and 1.33 log(10)  cfu/g, respectively, in the control households in the first feeding. After the intervention, the FC and FS counts in study households had dropped to 0.10 log(10) and 0.09 log(10)  cfu/g, respectively, a statistically significant reduction (P < 0.001). Monitoring the sustainability of the behaviour change after 3 months showed that the mothers were maintaining food hygiene. CONCLUSIONS: A hygiene intervention following the HACCP approach reduced the weaning food contamination significantly. Awareness building among mothers about weaning food hygiene could be an important intervention for preventing weaning food-related diarrhoea in Bangladesh.

Growth inhibition of Cronobacter spp. strains in reconstituted powdered infant formula acidified with organic acids supported by natural stomach acidity.

Cronobacter is associated with outbreaks of rare, but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in newborns. This study was conducted to determine the effect of organic acids on growth of Cronobacter in laboratory medium and reconstituted powdered infant formula (PIF) as well as the bacteriostatic effect of slightly acidified infant formula when combined with neonatal gastric acidity. Inhibitory effect of seven organic acids on four acid sensitive Cronobacter strains was determined in laboratory medium with broth dilution method at pH 5.0, 5.5 and 6.0. Acetic, butyric and propionic acids were most inhibitive against Cronobacter in the laboratory medium. The killing effect of these three acids was partially buffered in reconstituted PIF. Under neonatal gastric acid condition of pH 5.0, the slightly acidified formula which did not exert inhibition effect solely reduced significantly the Cronobacter populations. A synergistic effect of formula moderately acidified with organic acid combined with the physiological infant gastric acid was visible in preventing the rapid growth of Cronobacter in neonatal stomach. The study contributed to a better understanding of the inhibitory effect of organic acids on Cronobacter growth in different matrixes and provided new ideas in terms of controlling bacteria colonization and translocation by acidified formula.

Microbiological assessment and evaluation of rehydration instructions on powdered infant formulas, follow-up formulas, and infant foods in Malaysia.

A total of 90 samples comprising powdered infant formulas (n=51), follow-up formulas (n=21), and infant foods (n=18) from 15 domestic and imported brands were purchased from various retailers in Klang Valley, Malaysia and evaluated in terms of microbiological quality and the similarity of rehydration instructions on the product label to guidelines set by the World Health Organization. Microbiological analysis included the determination of aerobic plate count (APC) and the presence of Enterobacteriaceae and Cronobacter spp. Isolates of interest were identified using ID 32E (bioMérieux France, Craponne, France). In this study, 87% of powdered infant formulas, follow-up formulas, and infant foods analyzed had an APC below the permitted level of <10(4) cfu/g. These acceptable APC ranged between <10(2) to 7.2×10(3) cfu/g. The most frequently isolated Enterobacteriaceae was Enterobacter cloacae, which was present in 3 infant formulas and 1 infant food tested. Other Enterobacteriaceae detected from powdered infant and follow-up formulas were Citrobacter spp., Klebsiella spp., and other Enterobacter spp. No Cronobacter species were found in any samples. Rehydration instructions from the product labels were collated and it was observed that none directed the use of water with a temperature >70°C for formula preparation, as specified by the 2008 revised World Health Organization guidelines. Six brands instructed the use of water at 40 to 55°C, a temperature range that would support the survival and even growth of Enterobacteriaceae.

Bacillus cereus in infant foods: prevalence study and distribution of enterotoxigenic virulence factors in Isfahan Province, Iran.

This study was carried out in order to investigate the presences of Bacillus cereus and its enterotoxigenic genes in infant foods in Isfahan, Iran. Overall 200 infant foods with various based were collected and immediately transferred to the laboratory. All samples were culture and the genomic DNA was extracted from colonies with typical characters of Bacillus cereus. The presences of enterotoxigenic genes were investigated using the PCR technique. Eighty-four of two hundred samples (42%) were found to be contaminated with B. cereus with a ranges of 3 × 10(1)-9.3 × 10(1) spore per gram sample. Totally, entFM had the highest (61.90%) incidences of enterotoxigenic genes while hblA had the lowest (13.09%) incidences of enterotoxigenic genes. Overall, 6.7% of B. cereus isolates had all studied enetrotoxigenic genes while 25.5% of B. cereus strains had all studied enetrotoxigenic genes expectance bceT gene. This study is the first prevalence report of B. cereus and its enterotoxigenic genes in infant foods in Iran. Results showed that the infant food is one of the main sources of enterotoxigenic genes of B. cereus in Iran. Therefore, the accurate food inspection causes to reducing outbreak of diseases.

A rapid and simple screening method of Cronobacter spp. in cell suspension and tofu.

BACKGROUND: Cronobacter spp. causes illnesses such as neonatal meningitis, sepsis and necrotising enterocolitis that can be fatal in infants and neonates. Thus it is of great concern to the food industry, which requires a simple and miniaturised method for the rapid detection of Cronobacter spp. in food. RESULTS: A simple and rapid method was developed for the quantitative estimation of Cronobacter spp. Cell suspension cultures and tofu inoculated with Cronobacter spp. as well as a mixed culture (Cronobacter spp. and other micro-organisms) were serially diluted twofold on microtitre plates. Kim and Rhee broth, containing salicin, was added to each well. After 16-18 h of incubation the colour of broth in the wells changed from purple to yellow owing to salicin fermentation by Cronobacter spp. Bacterial counts were predicted by analysing the end points (the last well number in a series of positive wells). The coefficients of determination (r(2)) between the conventional plating method and the current method had high degrees of correlation (0.954-0.963), suggesting that the latter method offers a reasonable quantification of Cronobacter spp. CONCLUSION: The simple method developed in this study could be used for the efficient and rapid screening of Cronobacter spp. in the food industry.

Quality and safety aspects of infant nutrition.

Quality and safety aspects of infant nutrition are of key importance for child health, but oftentimes they do not get much attention by health care professionals whose interest tends to focus on functional benefits of early nutrition. Unbalanced diets and harmful food components induce particularly high risks for untoward effects in infants because of their rapid growth, high nutrient needs, and their typical dependence on only one or few foods during the first months of life. The concepts, standards and practices that relate to infant food quality and safety were discussed at a scientific workshop organized by the Child Health Foundation and the Early Nutrition Academy jointly with the European Society for Paediatric Gastroenterology, Hepatology and Nutrition, and a summary is provided here. The participants reviewed past and current issues on quality and safety, the role of different stakeholders, and recommendations to avert future issues. It was concluded that a high level of quality and safety is currently achieved, but this is no reason for complacency. The food industry carries the primary responsibility for the safety and suitability of their products, including the quality of composition, raw materials and production processes. Introduction of new or modified products should be preceded by a thorough science based review of suitability and safety by an independent authority. Food safety events should be managed on an international basis. Global collaboration of food producers, food-safety authorities, paediatricians and scientists is needed to efficiently exchange information and to best protect public health.

Quality control throughout the production process of infant food.

The manufacture of infant food is a highly complex process and needs an effective quality control beyond classical in-process parameters and a final microbiological analysis. To ensure a safe end -product, various tools, such as the Hazard Analysis Critical Control Points (HACCP), have been developed to facilitate the management of food safety. Every single infant formula ingredient must have an excellent quality and safety approach because even if an ingredient is used in very small quantities in a single product, serious consequences may arise if the quality and product safety are not taken seriously by the ingredient manufacturer. The purpose of this article was twofold: firstly, to briefly describe existing Quality Management Systems and, secondly, to highlight the consequences of non-quality.

Safety aspects in preparation and handling of infant food.

Powdered infant formula (PIF) can become contaminated during production with harmful bacteria such as Cronobacter spp. (formerly Enterobacter sakazakii). Inadequate conditions of preparation and handling of PIF can therefore exacerbate the risk of severe infection, especially in preterm infants. The WHO emphasized three main interventions for preparation and handling of PIF: (1) to dilute the powdered milk in water at a temperature of at least 70°C to inactivate Cronobacter spp.; (2) to consume milk right after each preparation, and (3) to store reconstituted milk at <5°C. The European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the French Food Safety Agency (Afssa) disagree with the heating process because of possible adverse effects on nutrients and the risk of severe burns. In home settings, PIF should be prepared fresh for each meal and be kept warm in bottle warmers or thermos bottles. In institutional settings, written guidelines for preparation and handling of PIF should be established. The use of sterile liquid formula is encouraged for healthy newborn infants in maternity wards; PIF should be prepared on a daily basis. Safe infant feeding involves the production of microbiologically cleaner infant formula by industry, and both education and support for the caregivers in preparing and handling the formula.

Non-breast milk feeding in developing countries: challenge from microbial and chemical contaminants.

Complementary foods based on cow's milk or gruels consumed by children in developing countries are often contaminated by bacteria during preparation, and ambient temperature rapidly increases microbial load. Thus infant formula or other weaning foods may cause diarrhea in young infants accounting for 25-33% of all deaths <5 years globally. Environmental chemicals such as metals (As, Pb, Cu) and nitrates can cause vomiting/diarrhea. Polychlorinated biphenyls derived from plastics, present in formula and/or breast milk, are endocrine disruptors (the potential threats are not fully quantifiable). The prevailing view is that benefits from breastfeeding outweigh potential risks.