Transcriptomic analysis reveals novel desiccation tolerance mechanism of Cronobacter based on type VI secretion system inhibition

Each year, there are 140 million infants born into our world, a planet with finite resources, and they have to be fed. How they are fed has an influence on their health for the rest of their life. The World Health Organization (WHO), United Nations International Children’s Emergency Fund (UNICEF), and the Asia Pacific Academic Consortium for Public Health (APACPH) have all recognized the importance of the first 1000 days of life, beginning at conception for lifetime health.1,2 Prior to birth, adequacy of maternal nutrients, including folate and iodine, an appropriate level of weight gain is very important. All public health and nutrition bodies promote the supremacy of breastmilk in feeding infants. Infants should be given breastmilk exclusively until about 6 months of age, and then complementary foods are needed to provide additional energy and nutrients. Exclusive breastfeeding means no other foods in addition to human milk, and this should be commenced as soon as practicable after birth. The advantages of this feeding regime have been documented in this journal and include lower rates of infection and infant mortality, appropriate growth rates with less obesity, and improved cognitive ability. Many more advantages have been documented throughout the lifespan.2,3 Scaling up breastfeeding could prevent an estimated 800 000 deaths of children under 5 years old and 20 000 breast cancer deaths among mothers each year.4 Despite the advantages of human milk and its availability to almost all infants, a substantial proportion of infants are being fed artificial infant formula. Global sales of infant formula are estimated to have reached $56 billion in 2019. While the rate of exclusive breastfeeding has increased slightly in the last few decades, the use of infant formula has grown more rapidly. The global market is dominated by five large companies who control more than 60% of the sales of infant formula. Much has been written about the marketing tactics used by these multinational companies to promote their products in unethical ways.5

Infant Formula Supply Problems and Public Health


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

Immunoinformatics Assisted Design of a Multi-Epitope Kit for Detecting Cronobacter sakazakii in Powdered Infant Formula (PIF)

Cronobacter sakazakii is an opportunistic pathogen known for causing severe diseases. Mild heat treatment is commonly used in food processing, however, the pathogenic characteristics and underlying mechanisms of Cronobacter sakazakii strains remain poorly understood. In this study, we found that mild heat stress (MHS) at 52 °C can induce several deleterious effects in Cronobacter sakazakii, including damage to the cell wall, genomic DNA breakage, and misfolding of cytoplasmic proteins. These conditions lead to a decreased survival ability under acid, desiccation, and osmotic stress; a reduction in biofilm formation; and diminished motility. Notably, surviving C. sakazakii cells retain their pathogenicity, causing significant intestinal damage in newborn mice. This damage is characterized by epithelial sloughing and disruption of the intestinal structure. Tandem mass tag (TMT)-based proteomics identified 736 proteins with differential abundance across C. sakazakii strains subjected to mild heat stress, highlighting adaptations in biofilm formation, motility, and stress tolerance. Key regulatory changes were observed in phospholipid metabolism and protein synthesis, which underpin this complex stress response. This data illustrates a sophisticated balance between environmental adaptability and pathogenic potential. The metabolic and pathogenic responses of C. sakazakii to mild heat stress are closely linked to its phospholipid metabolism and the production of secretory proteins, both crucial for its virulence and reliant on membrane transport. This complex interplay emphasizes the need to understand these mechanisms to develop effective control strategies. 

Use of proteomics to elucidate characteristics of Cronobacter sakazakii under mild heat stress

Cronobacter spp. Are members of the Enterobacteriaceae family and are important opportunistic pathogens to control during the manufacture of powdered infant formula, as susceptible infants are the most at-risk population for foodborne illness from this bacterium. This review presents the most recent microbiological findings on Cronobacter spp., and gives a view of Cronobacter spp. From the manufacturing and processing perspective. Practical thinking and guidelines for control of this bacterium in the most common parts of the manufacturing process, where contamination may occur, are highlighted, and supported by updated and robust scientific literature. From a practical perspective, all Cronobacter spp. Should be considered as pathogens, and dairy manufacturing plants should respond to its presence, no matter what species is identified. The natural and ubiquitous nature of Cronobacter spp. Suggests that general hygiene and continuous monitoring of the processing environment for this bacterium is important, since complete eradication is unlikely. 

Controlling Cronobacter spp. in dairy manufacturing – Fundamental characteristics and practical guidance

The purpose of this study was to investigate the prevalence of Cronobacter spp. in commercial powdered infant formula (PIF) from nine provinces in China from March 2018 to September 2020, and to reveal the genotype, biofilm-forming ability, and antibiotic susceptibility of these isolates. A total of 27 Cronobacter strains, consisting of 22 Cronobacter sakazakii strains, 3 Cronobacter malonaticus strains, 1 Cronobacter turicensis strain, and 1 Cronobacter dublinensis strain, were isolated from 3,600 commercial PIF samples with a prevalence rate of 0.75%. Compared with the other 8 provinces, PIF from Shaanxi province had a higher prevalence rate (1.25%) of Cronobacter spp. These isolates were divided into 14 sequence types (STs), and 6 Cronobacter serotypes. The main Cronobacter STs were ST4, ST1, and ST64, and the dominant Cronobacter serotype was C. sakazakii serotype O2. Approximately 88.89% of Cronobacter isolates had a strong ability (OD595 > 1) to form biofilms on tinplate, among which the strains with ST4 were more dominant. All isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefotaxime, sulfadiazine, sulfadoxine, trimethoprim-sulfamethoxazole, gentamicin, tetracycline, ciprofloxacin, and colistin, while 55.56 and 96.30% isolates were resistant to cephalothin and vancomycin, respectively. Taken together, our findings highlighted the contamination status and characterization of Cronobacter spp. in commercial PIF from nine provinces of China, and provided guidance for the effective prevention and control of this pathogen in the production of PIF.

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Cronobacter spp. in Commercial Powdered Infant Formula Collected From Nine Provinces in China: Prevalence, Genotype, Biofilm Formation, and Antibiotic Susceptibility

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Cronobacter spp. in Commercial Powdered Infant Formula Collected From Nine Provinces in China: Prevalence, Genotype, Biofilm Formation, and Antibiotic Susceptibility