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There are limited data on meconium and faecal bacterial profiles from African infants and their mothers. We characterized faecal bacterial communities of infants and mothers participating in a South African birth cohort. Stool and meconium specimens were collected from 90 mothers and 107 infants at birth, and from a subset of 72 and 36 infants at 4-12 and 20-28 weeks of age, respectively. HIV-unexposed infants were primarily exclusively breastfed at 4-12 (49%, 26/53) and 20-28 weeks (62%, 16/26). In contrast, HIV-exposed infants were primarily exclusively formula fed at 4-12 (53%; 10/19) and 20-28 weeks (70%, 7/10). Analysis (of the bacterial 16S rRNA gene sequences of the V4 hypervariable region) of the 90 mother-infant pairs showed that meconium bacterial profiles [dominated by Proteobacteria (89%)] were distinct from those of maternal faeces [dominated by Firmicutes (66%) and Actinobacteria (15%)]. Actinobacteria predominated at 4-12 (65%) and 20-28 (50%) weeks. HIV-exposed infants had significantly higher faecal bacterial diversities at both 4-12 (p = 0.026) and 20-28 weeks (p = 0.002). HIV-exposed infants had lower proportions of Bifidobacterium (p = 0.010) at 4-12 weeks. Maternal faecal bacterial profiles were influenced by HIV status, feeding practices and mode of delivery. Further longitudinal studies are required to better understand how these variables influence infant and maternal faecal bacterial composition.

Cronobacter sakazakii has been implicated in causing serious infections in neonates due to consumption of contaminated infant powdered milk. The zoonotic potential of the organism was not clear due to scarce evidence about the role of food animals in the transmission of infection. C. sakazakii was identified in infant powdered milk (n = 100), infant stool (n = 100), and dairy animal feces (n = 100) with the percentages of 1%, 2%, and 4%, respectively. The outer membrane protein A (ompA) gene was characterized in all isolates of different origin, while gene encoding for zinc-metaloprotease (zpx) was only identified in isolates from animal feces. Genotyping of C. sakazakii isolates using enterobacterial repetitive intergenic consensus polymerase chain reaction revealed heterogenicity. The survival and thermotolerance of one potentially virulent C. sakazakii isolate of animal origin were examined at different temperatures. The isolate could survive with a stationary number at refrigeration temperature and the number increased significantly at room temperature after 24 h. The isolate showed thermoresistance when subjected to temperature range from 54°C to 64°C with D values ranged from 13.79 and 4.64 min and z value of 14.42. To the best of our knowledge, this is the first report of C. sakazakii isolation from buffalo feces in Egypt.

The safety and quality of infant milk, whether it is breast milk (BM) or infant formula (IF), are a major concern for parents and public health authorities. BM is recommended as the gold standard at WHO level. However, nowadays IF appears as an essential alternative in Western countries, challenging producers to optimize nutritional quality and safety of IF. The aim of the present article is to give an overview on the assessment and comparison of risks and benefits associated with BM and IF consumption. To date, this intensively debated subject has been mainly investigated. It has been shown that both diets could be sources of beneficial health effects in terms of nutrition and also risks in terms of chemical safety. Moreover, microbiologists have demonstrated that IF consumption can cause illness due to product contamination or inappropriate milk preparation. The article concludes on the bottlenecks and gaps that should be investigated to further progress the quantification of the impact of early diet on infant health. Performing a multi-disciplinary risk-benefit assessment with DALY as endpoint might be a future option to help prioritize management options.

Presence of Cronobacter malonaticus in powdered infant formula (PIF) poses a high risk to infant and public health. Cronobacter malonaticus has been widely distributed in food and food processing environments, and the true origin of C. malonaticus in PIF is poorly understood. Control and prevention of C. malonaticus is necessary for achieving microbial safety of PIF. However, little information about decontamination of C. malonaticus is available. In this study, effects of hydrogen peroxide on inactivation and morphological changes of C. malonaticus cells were determined. Furthermore, inhibitory effects of H 2 O 2 on biofilm formation in C. malonaticus were also performed. Results indicated that H 2 O 2 could completely inactivate C. malonaticus in sterile water with 0.06% H 2 O 2 for 25 min, 0.08% H 2 O 2 for 15 min, and 0.10% for 10 min, respectively, whereas the survival rates of C. malonaticus in tryptic soy broth medium significantly increased with the same treatment time and concentration of H 2 O 2 . In addition, morphological changes of C. malonaticus cells, including cell shrinkage, disruption of cells, cell intercession, and leakage of intercellular material in sterile water after H 2 O 2 treatment, were more predominant than those in tryptic soy broth. Finally, significant reduction in biofilm formation by H 2 O 2 was found using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy detection compared with control samples. This is the first report to determine the effects of H 2 O 2 on C. malonaticus cells and biofilm formation. The findings provided valuable information for practical application of H 2 O 2 for decontamination of C. malonaticus in dairy processing., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Breastfeeding is recommended until 6 months of age, but a wide range of infant formula is available for nonbreastfed or partially breastfed infants. Our aim was to describe infant formula selection and to examine social- and health-related factors associated with this selection. Analyses were based on 13,291 infants from the French national birth cohort Etude Longitudinale Française depuis l'Enfance. Infant diet was assessed at Month 2 by phone interview and monthly from Months 3 to 10 via internet/paper questionnaires. Infant formulas were categorized in 6 groups: extensively or partially hydrolysed, regular with or without prebiotics/probiotics, and thickened with or without prebiotics/probiotics. Associations between type of infant formula used at 2 months and family or infant characteristics were assessed by multinomial logistic regressions. At Month 2, 58.1% of formula-fed infants were fed with formula enriched in prebiotics/probiotics, 31.5% with thickened formula, and 1.4% with extensively hydrolysed formula. The proportion of formula-fed infants increased regularly, but the type of infant formula used was fairly stable between 2 and 10 months. At Month 2, extensively hydrolysed formulas were more likely to be used in infants with diarrhoea or regurgitation problems. Partially hydrolysed formulas were more often used in families with high income, with a history of allergy, or with infants with regurgitation issues. Thickened formulas were used more with boys, preterm infants, infants with regurgitation issues, or in cases of early maternal return to work. The main factors related to the selection of infant formula were family and infant health-related ones., (© 2017 John Wiley & Sons Ltd.)

Benefits of prebiotics for stimulating a healthy intestinal tract are well known. From suppression of pathogens to proliferation of indigenous bacteria of intestines, prebiotics have it all. Since the research on the scope of prebiotics is expanding, new applications are coming up every day thus upgrading the choices consumer has for a healthy living. Incorporation of prebiotics in a wide range of products that food industry offers on shelf is an innovative way to replace fat and sugars along with enhancing the mouthfeel by providing better tongue lubrication. In some cases, the thermal stability of the product is improved along with other sensory, textural and physiological benefits. This paper gives an overview of the various prebiotics available from different sources and their applications in various segments of food industry, notably dairy, beverage, processed fruit-vegetable, bakery, confectionary, extruded snack, sweetener, infant formula, pet food and livestock industry. The effects observed on addition of various prebiotics are also elaborated.

A probabilistic and interdisciplinary risk-benefit assessment (RBA) model integrating microbiological, nutritional, and chemical components was developed for infant milk, with the objective of predicting the health impact of different scenarios of consumption. Infant feeding is a particular concern of interest in RBA as breast milk and powder infant formula have both been associated with risks and benefits related to chemicals, bacteria, and nutrients, hence the model considers these three facets. Cronobacter sakazakii, dioxin-like polychlorinated biphenyls (dl-PCB), and docosahexaenoic acid (DHA) were three risk/benefit factors selected as key issues in microbiology, chemistry, and nutrition, respectively. The present model was probabilistic with variability and uncertainty separated using a second-order Monte Carlo simulation process. In this study, advantages and limitations of undertaking probabilistic and interdisciplinary RBA are discussed. In particular, the probabilistic technique was found to be powerful in dealing with missing data and to translate assumptions into quantitative inputs while taking uncertainty into account. In addition, separation of variability and uncertainty strengthened the interpretation of the model outputs by enabling better consideration and distinction of natural heterogeneity from lack of knowledge. Interdisciplinary RBA is necessary to give more structured conclusions and avoid contradictory messages to policymakers and also to consumers, leading to more decisive food recommendations. This assessment provides a conceptual development of the RBA methodology and is a robust basis on which to build upon., (© 2017 Society for Risk Analysis.)

Background: Early-life colonization of the intestinal tract is a dynamic process influenced by numerous factors. The impact of probiotic-supplemented infant formula on the composition and function of the infant gut microbiota is not well defined. Objective: We sought to determine the effects of a bifidobacteria-containing formula on the healthy human intestinal microbiome during the first year of life. Design: A double-blind, randomized, placebo-controlled study of newborn infants assigned to a standard whey-based formula containing a total of 10 7 colony-forming units (CFU)/g of Bifidobacterium bifidum , Bifidobacterium breve , Bifidobacterium longum , B. longum subspecies infantis (intervention), or to a control formula without bifidobacteria (placebo). Breastfed controls were included. Diversity and composition of fecal microbiota were determined by 16S ribosomal RNA gene amplicon sequencing, and metabolite profiles were analyzed by ultrahigh-performance liquid chromatography-mass spectrometry over a period of 2 y. Results: Infants ( n = 106) were randomly assigned to either the interventional ( n = 48) or placebo ( n = 49) group; 9 infants were exclusively breastfed throughout the entire intervention period of 12 mo. Infants exposed to bifidobacteria-supplemented formula showed decreased occurrence of Bacteroides and Blautia spp. associated with changes in lipids and unknown metabolites at month 1. Microbiota and metabolite profiles of intervention and placebo groups converged during the study period, and long-term colonization (24 mo) of the supplemented Bifidobacterium strains was not detected. Significant differences in microbiota and metabolites were detected between infants fed breast milk and those fed formula ( P < 0.005) and between infants birthed vaginally and those birthed by cesarean delivery ( P < 0.005). No significant differences were observed between infant feeding groups regarding growth, antibiotic uptake, or other health variables ( P > 0.05). Conclusion: The supplementation of bifidobacteria to infant diet can modulate the occurrence of specific bacteria and metabolites during early life with no detectable long-term effects. This trial was registered at germanctr.de as DRKS00003660., (© 2017 American Society for Nutrition.)

Cronobacter species are important foodborne pathogens causing severe infections in neonates through consumption of contaminated powdered infant formula. However, the virulence-associated factors in Cronobacter are largely unknown. In this study, the transcriptome analysis between highly virulent Cronobacter sakazakii G362 and attenuated L3101 strains was used to reveal the potential factors involved in virulence. The total transcripts were grouped into 20 clusters of orthologous group categories and summarized in 3 gene ontology categories (biological process, cellular component, and molecular function). In addition, the differentially expressed genes (DEG) between these isolates were analyzed using Volcano plots and gene ontology enrichment. The predominant DEG were flagella-associated genes such as flhD, motA, flgM, flgB, and fliC. Furthermore, the expression abundance of outer membrane protein or lipoprotein genes (ompW, slyB, blc, tolC, and lolA), potential virulence-related factors (hlyIII and hha), and regulation factors (sdiA, cheY, Bss, fliZ) was also significantly different between G362 and L3101. Interestingly, 3 hypothetical protein genes (ESA&#95;01022, ESA&#95;01609, and ESA&#95;00609) were found to be expressed only in G362. Our findings provide valuable transcriptomic information about potential virulence factor genes, which will be needed in future molecular biology studies designed to understand the pathogenic mechanism of Cronobacter., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This study evaluates the potential of continuous wave Ultraviolet C light (UV-C) and broad-spectrum intense pulsed light (in this study referred to as High Intensity Light Pulses, HILP) for the inactivation of pathogens of public concern in powdered infant formula (PIF) producers. To achieve this goal a sequential set of experiments were performed, firstly in clear liquid media, secondly on the surface of spherical beads under agitation and, finally in PIF. L. innocua was the most sensitive microorganism to both technologies under all conditions studied with reductions exceeding 4 log 10 cycles in PIF. In the clear liquid medium, the maximum tolerance to light was observed for C. sakazakii against UV-C light and for B. subtilis spores against HILP, with a fluence of approximately 17 mJ/cm 2 required for a 1 log 10 cycle inactivation (D value) of each species. In PIF it was possible to inactivate >99% of the vegetative cell populations by HILP with a fluence of 199 mJ/cm 2 and of B. subtilis spores by doubling the fluence. By contrast, for UV-C treatments a fluence of 2853 mJ/cm 2 was needed for 99.9% reduction of C. sakazakii, which was the most light-resistant microorganism to UV-C. Results here obtained clearly show the potential for light-based interventions to improve PIF microbiological safety., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: To examine levels of bacterial contamination in formula feeding bottles in Sidoarjo, East Java, and to assess the preparation practices that may have been responsible., Methods: A cross-sectional study was conducted among 92 randomly selected households with children under the age of two who were bottle-fed formula. In each household, we carried out video observation of mothers/caregivers preparing bottles, and examined samples of formula for coliform bacteria and Escherichia coli (E. coli). In-depth interviews were conducted with a subsample of 20 mothers., Results: A total of 88% of the formula feeds were contaminated with total coliforms at a level >10 MPN/ml, and 45% contained E. coli. These feeds were defined as 'unfit for human consumption'. In the video observations, none of the mothers complied with all five WHO-recommended measures of hygienic formula feed preparation. Only two mothers washed their hands with soap prior to formula preparation. Most mothers also failed to clean or sterilise the bottle and clean the preparation area. In-depth interviews confirmed that such suboptimal hygiene practices were common., Conclusion: The high levels of contamination found highlight that bottles are an important faecal-oral exposure pathway resulting from poor hygiene practices during bottle preparation., (© 2017 John Wiley & Sons Ltd.)

Bacillus cereus is an important foodborne pathogen, which can cause severe food poisoning. The aim of this study was (i) to evaluate the quantitative prevalence of B. cereus in retail prepackaged infant formula and ready-to-eat rice flour in China and (ii) to gain the basic information on pheno- and genotypic characteristics of B. cereus isolates. We found that 40 out of the 587 samples were positive for B. cereus. B. cereus in 3.5% of infant formula samples and 1.0% of rice flour samples outnumbered 100 Colony-Forming Units (CFU)/g. B. cereus level even attained 10 3 -10 4 CFU/g in four infant formula samples and one rice flour sample. Furthermore, we identified the distribution patterns of toxin genes in B. cereus isolates. The results showed that 97.5% of B. cereus isolates harbored at least one enterotoxin gene. Antibiotic susceptibility tests revealed that all isolated B. cereus strains were resistant to penicillin and 50% of them were multidrug resistant. Thirteen new sequence types (STs) and four new alleles were identified via multilocus sequence typing. Clonal Complex (CC) ST-205 and CC ST-142 were predominant clonal complexes. Interestingly, we revealed the special relationship between STs of B. cereus isolates and the geographical distributions of infant food manufacturers for the first time. The data implied that B. cereus of different STs might have a distinct ecological niche in China. In view of relatively high contamination level of enterotoxin- producing B. cereus in a proportion of infant foods, especially in those suitable for the ≤6-month-old infant group, appropriate safety criteria and hygienic control measures for infant foods should be drafted in China to prevent B. cereus infection.

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in Crohn's disease in humans resulting in public concern over the presence of MAP in powdered infant formula, which could contribute towards early human exposure to MAP or MAP components. Testing of representative powdered infant formula samples using effective tests is required to provide information on contamination of infant formula with MAP, so that consumers can make informed decisions. This study aimed to test representative powdered infant formula samples for the presence of MAP using a quantitative PCR and liquid culture method. For this purpose, an efficient DNA extraction method was developed and an optimum decontamination protocol for culture method was identified. A total of 122 powdered infant formula samples were tested, comprising 72 brands produced by 12 manufacturers from 9 countries. Powdered infant formula samples were reconstituted and centrifuged to separate the casein pellet, cream layer and whey fraction. A sensitive qPCR test was performed on DNA extracted from the casein pellet. In addition, the cream layer and casein pellet were cultured in liquid media, following decontamination with the optimum protocol. Of the 122 samples tested, 6 were positive for MAP DNA but none were positive for growth in culture at 12 and 20 weeks. The limit of detection of the quantitative PCR was less than 5 MAP organisms per 1.5g milk powder. The methods developed in the study could be used for quality assurance testing for infant formula and calf milk replacers. The low contamination level of MAP and absence of viable forms in our study suggests a relatively low risk of exposure of infants to MAP components., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cronobacter has been identified as the causative agent of outbreaks or sporadic cases of meningitis, necrotizing enterocolitis, and septicemia associated with powdered infant formula. Food processing environments may provide a possible contamination route. The purpose of this study was to evaluate whole genome mapping (WGM) as a fast and automated molecular epidemiological method for characterizing Cronobacter spp. in the processing environment. This is the first study indicating the applicability of WGM to Cronobacter. WGM was compared with ribotyping, which is often used as an automated typing tool, and with pulsed-field gel electrophoresis, which is a well-known and highly discriminating tool that is also based on restriction site analysis. The comparison of the three tools was carried out on a subset of Cronobacter isolates collected from 2011 to 2014 through a monitoring program. The performance characteristics of WGM have not yet been described; therefore, in the current study its performance was evaluated based on five criteria: typeability, reproducibility, stability, epidemiological concordance, and the discrimination power. WGM was shown to produce typeable, reproducible, and stable results. With a similar cut-off of 98%, WGM was shown to have a discriminatory power equivalent to pulsed-field gel electrophoresis and higher than ribotyping. Future studies are needed to confirm the indicated cut-off level of 98%.

Objective: To develop a Cronobacter spieces identification PCR method based on RNA polymerase beta subunit( rpo B) gene and identify species of Cronobacter isolated from different sources., Methods: A total of 7 primer pairs based on the rpo B were design, and 9 standard strains were set as reference strains to identify species of261 strains isolated from infant formula foods, cereal based foods and clinical diarrhea cases in 2012-2014 in China., Results: The molecular weight of 9 standard strains were as same as expect, 179 of 216 Cronobacter isolates were C. sakazakii, 56 were C. malonaticus, 13 were C. universalis, 11 were C. dublinensis, 2 were C. turicensis, accounted for 68. 58%, 21. 46%, 4. 98%, 4. 21% and 0. 77% of the total isolates, respectively., Conclusion: The Cronobacter species identification PCR system is specific, convenient, sensitive. This system can provide technical support for Cronobacter risk surveillance and control in China.

Cronobacter spp. cause infant disease, several cases have been associated with powdered infant formulae (PIF). In the early 2000s, contamination of German PIF with these opportunistic pathogens was quite common. Before 2008, all isolates Cronobacter spp. had been classified as Enterobacter sakazakii, therefore little is known about species diversity within such isolates. Genetic, serologic, and biochemical traits of 80 Cronobacter isolates, originally obtained 2003-2006 within infant food surveys in Germany, were reassessed in this study. By sequencing of the fusA gene, all isolates were unambiguously assigned to two species, C. sakazakii (n = 73) and C. malonaticus (n = 7). PCR serotyping identified five C. sakazakii serotypes and two C. malonaticus serotypes, biochemical profiling yielded five biogroups. PFGE analysis also showed high heterogeneity in both species. Multilocus sequence typing of 26 selected isolates yielded 16 different sequence types (ST), including C. sakazakii ST 1 (n = 6) and the highly virulent ST 4 (n = 2). The results suggest that just two, but highly heterogeneous species were responsible for the Cronobacter contamination problem which challenged the German PIF industry in the beginning of this century. This fact may have influenced the success of efforts to identify and eliminate sources of contamination., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cronobacter sakazakii and Escherichia coli O157:H7 are well known food-borne pathogens that can cause severe disease. The identification of new alternatives to heating to control these pathogens in foods, while reducing the impact on organoleptic properties and nutritional value, is highly desirable. In this study, nisin and its bioengineered variants, nisin V and nisin S29A, are used alone, or in combination with plant essential oils (thymol, carvacrol and trans-cinnamaldehyde) or citric acid, with a view to controlling C. sakazakii and E. coli O157:H7 in laboratory-based assays and model food systems. The use of nisin variants (30 μM) with low concentrations of thymol (0.015%), carvacrol (0.03%) and trans-cinnamaldehyde (0.035%) resulted in extended lag phases of growth compared to those for corresponding nisin A-essential oil combinations. Furthermore, nisin variants (60 μM) used in combination with carvacrol (0.03%) significantly reduced viable counts of E. coli O157:H7 (3-log) and C. sakazakii (4-log) compared to nisin A-carvacrol treatment. Importantly, this increased effectiveness translated into food. More specifically, sub-inhibitory concentrations of nisin variants and carvacrol caused complete inactivation of E. coli O157:H7 in apple juice within 3 h at room temperature compared to that of the equivalent nisin A combination. Furthermore, combinations of commercial Nisaplin and the food additive citric acid reduced C. sakazakii numbers markedly in infant formula within the same 3 h period. These results highlight the potential benefits of combining nisin and variants thereof with carvacrol and/or citric acid for the inhibition of Gram negative food-borne pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In recent years, the microbiological safety of powdered infant formula has gained increasing attention due to the identification of contaminating C. sakazakii and its epidemiological link with life-threatening neonatal infections. Current intervention strategies have fallen short of ensuring the production of infant formula that is free from C. sakazakii. In this study, we describe the isolation and characterisation of three bacteriophages (phages) and their application as a phage cocktail to inhibit the growth of C. sakazakii in different brands of infant formula, while also assessing the phages ability to prevent biofilm formation. All three phages, isolated from slurry, possess a relatively broad host range, verified by their ability to infect across genera and species. When all three phages were combined and used as part of a phage cocktail, 73% coverage was obtained across all Cronobacter strains tested. Optimum thermo-tolerance and pH stability were determined between 4°C-37°C, and pH6-8, respectively, well within the normal range of application of infant formula. Genome sequencing and analysis revealed all the phages to be free from lysogenic properties, a trait which renders each favourable for phage therapy applications. As such, the combined-phage preparation (3×10 8 pfu/mL) was found to possess a strong bactericidal effect on C. sakazakii/C. sakazakii LUX cells (≤10 4 cfu/mL), resulting in a significant reduction in cell numbers, to below the limit of detection (<10cfu/mL). This was observed following a 20h challenge in different brands of infant formula, where samples in the absence of the phage cocktail reached concentrations of ~10 9 cfu/mL. The phage cocktail also demonstrated promise in preventing the establishment of biofilm, as biofilm formation could not be detected for up to 48h post treatment. These results highlight the potential application of this phage preparation for biocontrol of C. sakazakii contamination in reconstituted infant formula and also as a preventative agent against biofilm formation., (Copyright © 2017. Published by Elsevier B.V.)

The objective of this study was to evaluate the combined effect of citral plus mild heat on nondesiccated and desiccated Cronobacter sakazakii in reconstituted infant formula. Various concentrations of citral (0, 0.3, 0.6, and 0.9%) combined with various temperatures (25, 45, 50, and 55°C) were applied to nondesiccated and desiccated cocktails of three C. sakazakii strains (approximately 6.0 log CFU mL -1 ) in reconstituted infant formula, and the bacterial populations were assayed periodically. The combined treatments had marked antimicrobial effects on C. sakazakii compared with the control. Desiccated cells were more susceptible to citral than were nondesiccated cells in reconstituted infant formula. These findings suggest there is a potential application of citral in combination with mild heat to control C. sakazakii during preparation of reconstituted infant formula.

Background: Infection with Cronobacter spp. leads to neonatal meningitis, necrotizing enterocolitis and bacteremia. Cronobacter spp. are reported to comprise an important pathogen contaminating powdered infant formula (PIF) and follow-up formula (FUF), although little is known about the contamination level of Cronobacter spp. in PIFs and FUFs in China., Results: In total, 1032 samples were collected between 2011 and 2013. Forty-two samples were positive, including 1.6% in PIFs and 6.5% in FUFs. The strains were susceptible to most antibiotics except for cefoxitin. Pulsed-field gel electrophoresis after XbaI digestion produced a total of 36 banding patterns. The 38 strains were found in 27 sequence types (STs), of which nine types (ST454 to ST462) had not been reported in other countries. The clinically relevant strains obtained from the 38 isolates in the present study comprised three ST3, two ST4, two ST8 and one ST1., Conclusion: The contamination rate in the PIF and FUF has stayed at a relatively high level. The contamination rate of PIF was significantly lower than FUF. The isolates had high susceptibility to the antibiotics tested, except cefoxitin. There were polymorphisms between the Cronobacter spp. as indicated by pulsed-field gel electrophoresis and multilocus sequence typing. Therefore, contamination with Cronobacter spp. remains a current issue for commercial infant formulas in China. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Introduction: Patulin has raised the international attention because of its health risk. In fact, it has mutagenic, neurotoxic, immunotoxic, genotoxic and gastrointestinal effects in animals. In the present work, patulin and patulin-producing Penicillium spp. in apple and apple-based products marketed in Qatar were analysed., Methodology: Sampling was carried out using apple fruits and apple-based products. Fungi were isolated from undamaged apples, apple juice and baby apple food. DNA extraction was carried out with DNeasy Plant Mini Kit (QIAGEN, Valencia, USA). The molecular identification of fungal isolates was carried out using ITS1-ITS4 PCR. PCR products were sequenced and blasted. Patulin was extracted and analyzed by LC/MS/MS, then quantified using Agilent 1290UHPLC coupled to 6460 triple quadruple mass spectrometer., Results: Forty-five samples of undamaged fresh apple fruits, apple juice and apple-based baby food products sold in different markets in Qatar were surveyed for both fungal and patulin contamination using Liquid Chromatography Tandem Mass Spectrometery (LC/MS/MS). Twenty-five Penicillium spp. isolates were selected, including 23 P. expansum and one isolate each of P. brevicompactum and P. commune. All the tested Penicillium spp. isolates produced patulin in vitro (from 40 to 100 μg/g on Malt Yeast Extract agar medium). Patulin was detected in 100% of apple juice samples at levels ranging from 5.27 to 82.21 µg/kg. Only 5 samples contained patulin levels higher than European Union recommended limit (50 µg/kg). The average patulin contamination was 30.67 µg/kg and 10.92 µg/kg in baby apple juice and in baby apple compote, respectively.

Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles were newly found in PubMLST database. All isolates except for one singleton (ST-1049), were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and 13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands were compared. It was found the brands might be potentially tracked by the variety of STs, such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they could not be easily tracked just by clonal complex types of the isolates., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pathogen monitoring programs play a crucial role in the verification of the effectiveness of implemented hygiene control measures. Sampling and testing procedures included in pathogen monitoring involve the analysis of multiple test portions where all samples must be negative for the presence of pathogens for a certain test portion size. Many food safety programs require increased testing due to the risks that a pathogen may be present. Analyzing more than one test portion could prove to be expensive and labor intensive. When more than one test portion for a specified food item is to be tested, the test portions could be combined to form a pooled test portion to reduce laboratory workload, costs of reagents and further confirmatory steps, but only when evidence is available that pooling does not affect on the number of false negative results for different matrices. This study has been performed to demonstrate the equivalence of test portion pooling for Salmonella detection with five different methods using cultural, ELISA and Real Time PCR technologies. Twenty-three (23) different food items including confectionary products, meal components, infant formula, pet food and powdered beverages were validated. Other complementary parameters like impact of minimum and maximum incubation time for pre-enrichment, temperature profile, pH and Salmonella concentration after the pre-enrichment and background flora have also been considered in the study. The results showed that pooling test portions up to 375g for Salmonella detection is valid for the methods that were tested. Relative level of detection (RLOD 50 ) values for 22 of the food items tested were acceptable (i.e. lower than 2.5) when comparing the reference sample size (25g) against the alternative pooled sample size (375g), provided the enrichment broth was pre-warmed and maximum incubation time is respected., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background & Aims: Safety and growth adequacy of infant formulae enriched by functional ingredients need stringent evaluation by means of randomized controlled trials (RCTs), therefore we performed a double-blind RCT to evaluate an infant formula enriched with galacto-oligosaccharides, beta-palmitate, and acidified milk vs. a standard infant formula., Methods: Weight, length, head circumference and fecal bacteria (Bifidobacteria, BIF/Clostridia, CLO) were measured in healthy full term infants, at baseline - as before 21 days of life - at 60 and 135 days thereafter. A group of 51 neonates received the enriched formula (ENR), 59 the standard one (ST). Parents were trained to daily register gastrointestinal diseases., Results: All the infants grew homogeneously increasing the anthropometric parameters and complying with WHO and Italian standards: the mean (SD) difference in daily weight between ENR and ST groups was -0.74 (1.13) g/day, corresponding to a 90% CI of -2.62 to 1.13 g/day, well within the postulated interval of equivalence of -3.9 to +3.9 g/day. A statistical improvement in BIF concentration in the microbiota of infants fed by ENR was recorded. There was no between-group change in log 10 CLO, but log 10 BIF increase was higher at T2 vs. T0 in ENR (treatment × time interaction = 0.71, 95% CI 0.08-1.34, p = 0.028) than in ST neonates. This corresponds to estimated mean (95% CI) values of 8.37 (8.04-8.69) log 10 -units for ENR vs. 8.08 (7.77-8.39) log 10 -units for ST neonates. Gastrointestinal effects were mild and similar, with no statistical difference between two groups., Conclusion: Safety and growth ability of the enriched formula has been confirmed. A positive effect on neonatal gut microbiota, consisting of increased fecal BIF counts at T2 vs. baseline has been shown too. Nonetheless, larger RCTs are needed to estimate with greater precision the effective potential attributable to the enriched formula on neonatal microbiota, with particular reference to the mode of delivery., (Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

We show that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast, reliable, and easy method for detection and identification of food-borne bacteria, namely Salmonella spp., Listeria monocytogenes, and Cronobacter spp., in different types of food matrices (salmon, eggs, powdered infant formula milk, mixed herbs, respectively). The main aim of this work was to introduce the SERS technique into three ISO (6579:2002; 11290-1:1996/A1:2004; 22964:2006) standard procedures required for detection of these bacteria in food. Our study demonstrates that the SERS technique is effective in distinguishing very closely related bacteria within a genus grown on solid and liquid media. The advantages of the proposed ISO-SERS method for bacteria identification include simplicity and reduced time of analysis, from almost 144 h required by standard methods to 48 h for the SERS-based approach. Additionally, PCA allows one to perform statistical classification of studied bacteria and to identify the spectrum of an unknown sample. Calculated first and second principal components (PC-1, PC-2) account for 96, 98, and 90% of total variance in the spectra and enable one to identify the Salmonella spp., L. monocytogenes, and Cronobacter spp., respectively. Moreover, the presented study demonstrates the excellent possibility for simultaneous detection of analyzed food-borne bacteria in one sample test (98% of PC-1 and PC-2) with a goal of splitting the data set into three separated clusters corresponding to the three studied bacteria species. The studies described in this paper suggest that SERS represents an alternative to standard microorganism diagnostic procedures. Graphical Abstract New approach of the SERS strategy for detection and identification of food-borne bacteria, namely S. enterica, L. monocytogenes, and C. sakazakii in selected food matrices., Competing Interests: The authors declare that they have no conflict of interests.

In utero and upon delivery, neonates are exposed to a wide array of microorganisms from various sources, including maternal bacteria. Prior studies have proposed that the mode of feeding shapes the gut microbiota and, subsequently the child's health. However, the effect of the mode of feeding and its influence on the development of the neonatal oral microbiota in early infancy has not yet been reported. The aim of this study was to compare the oral microbiota of healthy infants that were exclusively breast-fed or formula-fed using 16S-rRNA gene sequencing. We demonstrated that the oral bacterial communities were dominated by the phylum Firmicutes, in both groups. There was a higher prevalence of the phylum Bacteroidetes in the mouths of formula-fed infants than in breast-fed infants (p = 0.01), but in contrast Actinobacteria were more prevalent in breast-fed babies; Proteobacteria was more prevalent in saliva of breast-fed babies than in formula-fed neonates (p = 0.04). We also found evidence suggesting that the oral microbiota composition changed over time, particularly Streptococcus species, which had an increasing trend between 4-8 weeks in both groups. This study findings confirmed that the mode of feeding influences the development of oral microbiota, and this may have implications for long-term human health.

Necrotising enterocolitis (NEC) is the most commonly acquired gastrointestinal disease of neonates, particularly the very preterm (gestation <32 weeks) and/or very low birth weight (<1500g). It is associated with high morbidity and mortality. Despite improvement in neonatal care and increased use of expressed breast milk (EBM), the incidence remains high in many neonatal intensive care units (NICU), and even shows increasing trend in some countries. Numerous studies have pointed to the infective nature of NEC. Some investigators have reported an increase in the incidence of NEC in their NICU when the percentage of infants with pathogens isolated from their gut increased, and decreased when gut colonisation rate was low. Both bacteria and viruses have been reported to be associated with outbreaks of NEC. The majority (>90%) of the NEC cases occurred in neonates on enteral feeding. Studies have shown that milk (whether EBM or formula) fed to neonates was not sterile and were further contaminated during collection, transport, storage and/or feeding. Other investigators have reported a reduction in the incidence of NEC when they improved infection control measures and hygienic procedures in handling milk. It is, therefore, hypothesised that the most common cause of NEC is due to the feeding of neonates, particularly the vulnerable very preterm small neonates, with milk heavily contaminated during collection at source, transport, storage and/or feeding. Because of the immaturity of the immune system of the neonates, excessive inflammatory response to the pathogen load in the gut leads to the pathogenesis of NEC.

There are several commercial test kits for Mycobacterium avium subspecies paratuberculosis (MAP) detection, each with different advantages, disadvantages, and applications. In the present study, a real-time PCR kit targeting the unique transposon sequence ISMAP02 was evaluated. The analytical sensitivity was determined using the type strain ATCC 19698, and the specificity was validated by testing fifteen MAP isolates, thirteen non-MAP Mycobacterium isolates, and eight non-Mycobacterium isolates. Six spiking experiments were performed using raw milk and reconstituted infant milk artificially contaminated with dilutions containing 10(0)-10(5) MAP cells mL(-1). Sensitivity and specificity were at 100 %. The detection probabilities in raw milk and reconstituted infant milk for the samples (containing 1.4 × 10(1) and 1.7 × 10(1) MAP cell 50 mL(-1)) were 16.6 and 91.6 %, respectively. Thus, the tested kit yielded satisfying results to detect MAP in milk.

This study aimed to optimize the applicability of an immunoliposome-based immunomagnetic concentration and separation assay to facilitate rapid detection of Cronobacter sakazakii in powdered infant formula (PIF). To determine the detection limit, specificity, and pre-enrichment incubation time (0, 4, 6, and 8 h), assay tests were performed with different cell numbers of C. sakazakii (2 × 10 0 and 2 × 10 1 CFU/ml) inoculated in 10 g of PIF. The assay was able to detect as few as 2 cells of C. sakazakii/10 g of PIF sample after 6 h of pre-enrichment incubation with an assay time of 2 h 30 min. The assay was assessed for cross-reactivity with other bacterial strains and exhibited strong specificity to C. sakazakii. Moreover, the assay method was applied to the detection of C. sakazakii in PIF without pre-enrichment steps, and the results were compared with INC-ELISA and RT-PCR. The developed method was able to detect C. sakazakii in spiked PIF without pre-enrichment, whereas INC-ELISA failed to detect C. sakazakii. In addition, when compared with the results obtained with RT-PCR, our developed assay required lesser detection time. The developed assay was also not susceptible to any effect of the food matrix or background contaminant microflora.

Infant formula (IF) requires a strict microbiological standard because of the high vulnerability of infants to foodborne diseases. The current study used the PacBio single-molecule real-time (SMRT) sequencing platform to generate full-length 16S rRNA-based bacterial microbiota profiles of 30 Chinese domestic and imported IF samples. A total of 600 species were identified, dominated by Streptococcus thermophilus, Lactococcus lactis, and Lactococcus piscium. Distinctive bacterial profiles were observed between the two sample groups, as confirmed with both principal coordinate analysis and multivariate analysis of variance. Moreover, the product whey protein nitrogen index (WPNI), representing the degree of preheating, negatively correlated with the relative abundances of the Bacillus genus. This study has demonstrated the application of the PacBio SMRT sequencing platform in assessing the bacterial contamination of IF products, which is of interest to the dairy industry for effective monitoring of microbial quality and safety during production.

Aflatoxin M₁ (AFM₁) is an important biomarker that can be used to evaluate aflatoxin exposure in both humans and animals. The aim of this study was to evaluate the exposure degree of infants to AFM₁ through consumption of breast milk and infant powdered milk in Brazil. For this purpose, the estimated daily intake (EDI) for infants was calculated based on the AFM₁ levels analyzed in 94 breast milk (BM) samples collected in Southern Brazil, and 16 infant powdered milk (IPM) samples commonly commercialized in Brazil. AFM₁ was detected in 5.3% (n = 5) and 43.8% (n = 7) of BM and IPM samples, with mean levels of 0.003 ng/g and 0.011 ng/g, respectively. All the IPM samples showed AFM₁ levels lower than those established by the Brazilian guidelines (5 ng/g), and in most of the samples (81.25%) levels were below the maximum limit tolerated by the European Commission (0.025 ng/g). The EDI of AFM₁ for infants aged zero to 12 months old showed values from 0.018 to 0.069 ng/kg body weight/day for BM, and 0.078 to 0.306 ng/kg body weight/day for IPM. Hazard index (HI) values for BM and IPM were less than one, except for IPM intended for infants up to one month. In conclusion, the exposure of infants to AFM₁ was low, but continuous monitoring of mycotoxin levels is essential to minimize infant health risk., Competing Interests: The authors declare no conflict of interest.

Cronobacter sakazakii, found in foods such as powdered infant formula and plant origin ready-to-eat food, is an opportunistic pathogen to infants, neonates and vulnerable adults. The objective of this study was to monitor the growth of C. sakazakii in fresh-cut 'Royal gala' apple, 'Rocha' pear, and 'Piel de sapo' melon, and the effect of UV-C illumination, acidic electrolyzed water (AEW) and neutral electrolyzed water (NEW) in the reduction of its population. Fresh-cut fruits were inoculated and incubated at different temperatures during 10days while monitoring C. sakazakii. The inhibitory activity of different doses of UV-C (0-10kJ.m(2)), electrolyzed water and sodium hypochlorite (SH) (100ppm chlorine) was evaluated on the fruits inoculated with C. sakazakii. The bacterium showed a significant growth in the fruits at 12 and 20°C, but did not grow at 4°C, despite having survived for 10days. At 8°C, adaptation phases of 0.6-3.9days were estimated in the fruits before exponential growth. The UV-C 7.5 and 10kJ/m(2) produced greater C. sakazakii population decreases (2-2.4logcfu/g) than AEW (1.3-1.8logcfu/g), NEW (1-1.2logcfu/g) and SH (0.8-1.4logcfu/g). The UV-C decontamination system and refrigeration at 4°C, may contribute to the product's safety and quality. The results help better understand the behavior of C. sakazakii on fresh-cut fruit alerting producers of the necessity to respect the high hygienic practices, adequate refrigerating temperature maintenance and caution with the tendency to prolong the validity of this kind of ready-to-eat food., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: DNA methylation of the Th1 and Th2 cytokine genes is altered during cow's milk allergy (CMA). Forkhead box transcription factor 3 (FoxP3) is essential for the development and function of regulatory T cells (Tregs) and is involved in oral tolerance acquisition. We assessed whether tolerance acquisition in children with IgE-mediated CMA is associated with DNA demethylation of the Treg-specific demethylated region (TSDR) of FoxP3., Results: Forty children (aged 3-18 months) were enrolled: 10 children with active IgE-mediated CMA (group 1), 10 children who outgrew CMA after dietary treatment with an extensively hydrolyzed casein formula containing the probiotic Lactobacillus rhamnosus GG (group 2), 10 children who outgrew CMA after treatment with other formulas (group 3), and 10 healthy controls (group 4). FoxP3 TSDR demethylation and expression were measured in mononuclear cells purified from peripheral blood of the four groups of children. FoxP3 TSDR demethylation was significantly lower in children with active IgE-mediated CMA than in either children who outgrew CMA or in healthy children. Formula selection influenced the FoxP3 TSDR demethylation profile. The FoxP3 TSDR demethylation rate and expression level were correlated., Conclusions: Tolerance acquisition in children with IgE-mediated CMA involves epigenetic regulation of the FoxP3 gene. This feature could be a new target for preventive and therapeutic strategies against CMA.

Up-to-date, nonthermal technologies and combinations of them, in accordance with the "hurdle technology" concept, are being applied by different research groups in response to calls by the International Food and Human Health Organizations (ESPGHAN, 2004; FAO/WHO, 2006, 2008) for alternatives to thermal control of Cronobacter sakazakii in reconstituted powdered infant formula milk. This review highlights (i) current knowledge on the application of nonthermal technologies to control C. sakazakii in infant formula milk and (ii) the importance of the application of nonthermal technologies for the control of C. sakazakii as part of the development of strategies in the context of improving food safety and quality of this product.

Background: Microbiological criteria applied to powdered infant formula (PIF) require the absence of all Cronobacter spp. Consequently, misidentification of isolates from finished products can lead to significant financial losses for manufacturers and could increase the risk of neonatal infection. Biochemical identification of suspect isolates using commercially available test panels is recommended for use by PIF manufacturers by both the US FDA and ISO standard methods for Cronobacter species; however, phenotyping can be unreliable, particularly for a genus such as Cronobacter where the taxonomy has been subject to frequent changes. This study compared the predicted identification by commonly used phenotyping kits (API20E and ID32E) for over 240 strains of Cronobacter from diverse sources, which had been identified using DNA sequence analysis. In 2015, the databases associated with the API20E and ID32E biochemical test panels were updated, including the recognition of the Cronobacter genus. Thus, the identifications from multiple versions the databases were compared to each other and to identifications based on DNA sequencing methods., Results: Using previous versions of the API20E database, 90.0 % of strains (216/240) resulted in a match for the species identification; however, version 5.0 produced matches for only 82.3 % of strains (237/288). Similarly, the update to version 4.0 in the ID32E database caused the percentage of matches to drop from 88.9 % (240/270) to 43.2 % (139/322). A smaller study showed that the Vitek GN system identified all 14 strains, belonging all seven Cronobacter species, as members of the 'C. sakazakii group,' but also attributed three strains of Franconibacter helveticus and F. pulveris to this group. In silco analysis of a PCR-based method targeting ompA predicted that amplification would only occur with Cronobacter species and this method may be a feasible alternative to biochemical phenotyping., Conclusions: These results indicate that commercially available biochemical test panels are not sufficiently reliable for speciation of Cronobacter isolates. Although DNA-sequence based methods would be the more reliable approach; however, this is not currently feasible for many food microbiology laboratories. Instead, a previously published PCR-based method targeting ompA is suggested as an alternative for identification of Cronobacter species based on in silico analysis.

It is important to provide formula-fed infants with a bifidobacteria-enriched gut microbiota similar to those of breastfed infants to ensure intestinal health. Prebiotics, such as certain oligosaccharides, are a useful solution to this problem, but the combinational benefits of these oligosaccharides have not been evaluated. This study investigated the benefits of oligosaccharide combinations and screened for an optimal combination of oligosaccharides to promote healthy gut microbiota of formula-fed infants. In vitro and in vivo experiments were performed to assess the bifidogenic effects of lactulose (LAC) alone and LAC combined with raffinose (RAF) and/or galacto-oligosaccharide (GOS), using a mixed culture model and neonatal mice orally administered with these oligosaccharides and Bifidobacterium breve. In the in vitro culture model, the combination of the three oligosaccharides (LAC-RAF-GOS) significantly increased cell numbers of B. breve and Bifidobacterium longum (P<0·05) compared with either LAC alone or the combination of two oligosaccharides, and resulted in the production of SCFA under anaerobic conditions. In the in vivo experiment, the LAC-RAF-GOS combination significantly increased cell numbers of B. breve and Bacteroidetes in the large intestinal content (P<0·05) and increased acetate concentrations in the caecal content and serum of neonatal mice. Genes related to metabolism and immune responses were differentially expressed in the liver and large intestine of mice administered with LAC-RAF-GOS. These results indicate a synergistic effect of the LAC-RAF-GOS combination on the growth of bifidobacteria and reveal possible benefits of this combination to the gut microbiota and health of infants.

This paper describes fabrication of a DNA-based Au-nanoparticle modified pencil graphite electrode (PGE) biosensor for detection of Bacillus cereus, causative agent of two types of food-borne disease, i.e., emetic and diarrheal syndrome. The sensing element of the biosensor was comprised of gold nanoparticles (GNPs) self-assembled with single-stranded DNA (ssDNA) of nheA gene immobilized with thiol linker on the GNPs modified PGE. The size, shape and dispersion of the GNPs were characterized by field emission scanning electron microscope (FESEM). Detection of B. cereus was carried out based on an increase in the charge transfer resistance (Rct) of the biosensor due to hybridization of the ss-DNA with target DNA. An Atomic force microscope (AFM) was used to confirm the hybridization. The biosensor sensitivity in pure cultures of B. cereus was found to be 10(0) colony forming units per milliliter (CFU/mL) with a detection limit of 9.4 × 10(-12) mol L(-1). The biosensor could distinguish complementary from mismatch DNA sequence. The proposed biosensor exhibited a rapid detection, low cost, high sensitivity to bacterial contamination and could exclusively and specifically detect the target DNA sequence of B. cereus from other bacteria that can be found in dairy products. Moreover, the DNA biosensor exhibited high reproducibility and stability, thus it may be used as a suitable biosensor to detect B. cereus and to become a portable system for food quality control., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Cronobacter is an emerging food pathogen, especially in infants and neonates, often associated with the ingestion of contaminated Powdered Infant Formula (PIF). Therefore, regulations require the control of the absence of Cronobacter and of Salmonella, another important food pathogen, in these food products. So far, reference and alternative methods take up to several days, and no validated method exists for the simultaneous detection of these two pathogens. In this work, we propose to address this issue by an innovative and easy-to-operate assay, named Plasmonic Immuno-Assay (PlasmIA), and by producing dedicated polyclonal antibodies. Our approach is based on Surface Plasmon Resonance imaging of antibody-arrays and bacterial growth during a standardized enrichment. Such a single-step assay enables the multiplex detection of both Cronobacter and Salmonella, with concentrations smaller than 30 CFU cells in 25 g PIF samples, in less than 1 day., Significance and Impact of the Study: Among bacterial pathogens involved in food contamination, Cronobacter and Salmonella are of particular interest. Nevertheless, all detection methods used so far require several days to assess food safety. In the present paper, we describe the first multiplex immuno-assay ever described for fast and specific detection of these two pathogens in food samples. Such advances were made possible by combining the advantages of protein microarrays with on-biochip culture of contaminated food samples and an easy-to-operate optical detection. By doing so, we managed to detect both viable Cronobacter and Salmonella occurring during the enrichment phase., (© 2016 The Society for Applied Microbiology.)

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.

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.

Cronobacter sakazakii is a severe virulent strain that is frequently detected in powdered infant formula (PIF). Therefore, it is necessary to develop a fast and specific detection method. The specificity of our newly developed quantitative real-time PCR (qRT-PCR) was validated with DNA from 46 strains. Among them, 12 C. sakazakii strains were correctly amplified, whereas no positive florescent signal was observed from 34 nontarget controls. The detection limit of C. sakazakii was about 110 CFU/mL in broth and 1100 CFU/g in PIF. After enrichment in buffered peptone water for 6 h, our developed qRT-PCR assay could reliably detect C. sakazakii when the inoculation level was as low as 2 CFU/25 g (0.08 CFU/g) in PIF. The growth of C. sakazakii could be inhibited by the presence of Lactobacillus pentosus and Bacillus cereus, which used a longer enrichment period before the isolation was accomplished. However, at 5 and 50 CFU/25 g inoculation levels of C. sakazakii in the presence of 4 × 10(6) CFU L. pentosus/25 g or of 2 × 10(4) CFU B. cereus/25 g, the qRT-PCR assay could detect the presence of Cronobacter even though these artificially spiked samples were negative in culture. Therefore, our results indicated that the qRT-PCR assay could detect samples containing inhibitors and could avoid false negatives by using an internal amplification control.

Powdered infant formulas (PIF) are usually not sterile and may frequently be contaminated by several bacteria strains. Among them, Cronobacter species, previously known as Enterobacter sakazakii, is one of the most harmful, since it might be the causative agent of sepsis and meningitis in newborns and preterm infants during the first weeks of life. The mortality rate of these infections is up to 80 %. Therefore, some precautions are required in the home handling and dilution of PIF. Whereas there is wide consensus about the need that a PIF should be used immediately after being diluted or, if not, stored at < "5 °C", still recently the optimal temperature of the water used to dilute PIF is controversial among scientific societies and health agencies. The current knowledge is reviewed in this paper and provides sufficient evidence to cautiously advise the use of hot water at a temperature of "70 °C" in the dilution of PIF in order to prevent the Cronobacter sp. contamination and growth.

Bacillus cereus is responsible for several outbreaks of foodborne diseases due to its emetic toxin and enterotoxin. Enterotoxins, cytotoxin K (CytK), nonhemolytic enterotoxin (Nhe), and hemolysin BL (Hbl), have been recorded in several diarrheal cases due to food poisoning from B. cereus. The objective of this study was to develop a rapid and accurate method that combines multiplex PCR with propidium monoazide to selectively detect viable cells of enterotoxin-producing B. cereus in milk powder, noodles, and rice, and investigate the distribution of enterotoxins in 62 strains of B. cereus in Jiangxi province, China. The specificity of primers of 3 enterotoxins (i.e., cytK, nheA, and hblD) of B. cereus was verified by inclusivity and exclusivity tests using single PCR. Upon optimization of multiplex PCR conditions, it was found that the detection limit of viable cells was 10(2) cfu/mL of B. cereus in pure culture. By enrichment for 3 or 4 h and propidium monoazide pretreatment, a protocol for detection of viable cells as low as 2.2×10(1) cfu/g in spiked food (e.g., milk powder, noodles, and rice) was established and proved valid even under the interference of non-Bacillus cereus at as high as 10(5) cfu/g. Moreover, the protocol based on multiplex PCR for detection was applied for the analysis of distribution of toxin gene of B. cereus, and the results showed a regional feature for toxin gene distribution, indicating that potential toxigenicity of B. cereus should be evaluated further., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objective: To determine Cronobacter spp. contamination in infant and follow-up powdered formula in China., Methods: All of 2282 samples were collected from the retail markets in China from January 2012 to December 2012, and analyzed for Cronobacter spp. by the Chinese National Food Safety Standard. Characterization of the isolates was analyzed by pulsed-field gel electrophoresis (PFGE) with XbaI and SpeI restriction enzymes., Results: Cronobacter spp. strains were isolated from 25 samples, and the positive rates in infant powdered formulas and follow-up powdered formulas were 0.90% (10/1011) and 1.18% (15/1271), respectively. Analysis of variable data regarding different purchasing store formats, seasonality, and production locations as well as comparison of infant versus follow-up formulas did not reveal statistically significant factors. During the sampling period, one of six surveillance zones did exhibit a statistically significant trend towards higher positive rate. PFGE characterization of Cronobacter spp. to elucidate genetic diversity revealed only three pairs of Cronobacter spp. out of 25 having the same PFGE patterns., Conclusion: The current investigation indicated a lower positive rate of Cronobacter spp. in the powdered formula in China. This evidence suggested contamination originating from multiple different sources during the manufacturing process., (Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The Cronobacter genus (previously known as Enterobacter sakazakii) comprises seven species (Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti)which cause serious infections in neonates and immunocompromised people.Most of the documented outbreaks of these bacteria have been associated with consumption of contaminated powdered infant formula. The plant environment is considered to be the natural habitat of these bacteria. Therefore, a total number of 563 samples of vegetables, fruit, water and environmental swabs were collected from local farms and supermarkets in the Czech Republic and investigated for the presence of Cronobacter spp. The obtained 45 isolates (8.0%) were further characterized by phenotyping (antimicrobial resistance, capsule and pigment production) and genotyping (fusA sequencing,MLST, PCR-serotyping) methods. Most of the Cronobacter isolates (42.2%) were identified as C. sakazakii, followed by C. turicensis (31.1%), C. dublinensis (22.2%), C. malonaticus (2.2%) and C. universalis (2.2%). The 25 identified sequence types, out of which 17 were unique for only one strain, indicated a high diversity of strains. C. sakazakii sequence type 4 (ST 4), which has been associated with many cases of meningitis, was isolated only in one case. A strong association of C. turicensis and C. dublinensis with the plant environment can be deduced from our results.

Aerobic spore forming bacteria are potential milk powder contaminants and are viewed as indicators of poor quality. A total of 738 bacteria, including both mesophilic and thermophilic, isolated from twenty-five powdered milk samples representative of three types of milk powders in China were analyzed based on the random amplified polymorphic DNA (RAPD) protocol to provide insight into species diversity. Bacillus licheniformis was found to be the most prevalent bacterium with greatest diversity (~43% of the total isolates) followed by Geobacillus stearothermophilus (~21% of the total isolates). Anoxybacillus flavithermus represented only 8.5% of the total profiles. Interestingly, actinomycetes represented a major group of the isolates with the predominance of Laceyella sacchari followed by Thermoactinomyces vulgaris, altogether comprising of 7.3% of the total isolates. Out of the nineteen separate bacterial species (except five unidentified groups) recovered and identified from milk powders, twelve proved to belong to novel or previously unreported species in milk powders. Assessment and characterization of the harmful effects caused by this particular micro-flora on the quality and safety of milk powders will be worth doing in the future., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Surveys from different parts of the world have reported that viable Mycobacterium avium subsp. paratuberculosis (MAP) can be cultured from approximately 2% of samples of retail pasteurised milk samples. Pasteurised milk is used for the production of powdered infant formula (PIF) and therefore there is a concern that MAP may also be present in these products. Several studies have previously reported the detection of MAP in PIF using PCR-based assays. However, culture-based surveys of PIF have not detected viable MAP. Here we describe a phage amplification assay coupled with PCR (page-PCR) that can rapidly detect viable MAP in PIF. The results of a small survey showed that the phage-PCR assay detected viable MAP in 13% (4/32) of PIF samples. Culture detected viable MAP in 9% (3/32) PIF samples, all of which were also phage-PCR positive. Direct IS900 PCR detected MAP DNA in 22% (7/32) of PIF samples. The presence of viable MAP in PIF indicates that MAP either survived PIF manufacturing or that post-production contamination occurred. Irrespective of the route of MAP contamination, the presence of viable MAP in PIF is a potential public health concern., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: This study investigated the effect that infant formula had on biofilm growth of Streptococcus mutans. Specifically, it compared biofilm growth in media containing lactose-based and sucrose-based formulas. It also analyzed biofilm formation with formulas of varying iron content. Biofilm growth was tested with the specific infant formula components sucrose, lactose, and ferric chloride. The study was designed to determine if these types of infant formulas and components affected S. mutans biofilm formation differently., Study Design: A 24-hour culture of S. mutans was treated with various concentrations of infant formula diluted in bacteriological media. To test for biofilm formation, S. mutans was cultured with and without the infant formula and formula components. The biofilms were washed, fixed, and stained with crystal violet. The absorbance was measured to evaluate biofilm growth and total absorbance., Results: Sucrose-based formulas provided significant increases in biofilm growth when compared to lactose-based formulas at two dilutions (1:5, 1:20). Similac Sensitive RS (sucrose-based) at most dilutions provided the most significant increase in biofilm growth when compared to the control. Sucrose tested as an individual component provided more of a significant increase on biofilm growth than lactose or iron when compared to the control. A low iron formula provided a significant increase in biofilm growth at one dilution (1:5) when compared to formula containing a normal iron content. There was no significant difference in biofilm growth when comparing high iron formula to normal iron formula or low iron formula. There was no significant difference when comparing Similac PM 60/40 (low iron formula) to Similac PM 60/40 with additional ferric chloride., Conclusion: The results of this study demonstrated that sucrose-based formula provided more of a significant increase in biofilm growth compared to lactose-based formula. Sucrose alone provided a significant increase of biofilm growth at more dilutions when compared to the control than lactose and iron. The amount of iron in formula had a significant effect on biofilm formation only when comparing low iron formula to normal iron formula at the highest concentration (1:5). There was no significant difference in biofilm growth when iron was added to the low iron formula. The information obtained expands current knowledge regarding the influence of infant formula on the primary dentition and reinforces the importance of oral hygiene habits once the first tooth erupts.