Isolation of Pathogenic Cronobacter Species as Bacteriological Risks Indicator in Powdered Infant Formula Available to Deprived Infants in Lagos Metropolis, Nigeria.

Powdered infant formula (PIF) is rich in nutrients that support the survival and growth of bacteria that trigger food safety disorders in deprived infants through life-threatening illnesses. The study aims to examine and identify the incidence of pathogenic bacteria of concern in PIF upon reconstitution in lukewarm water. A total of 172 samples consisting of 38 brands of PIF available in the Lagos metropolis were sampled, suspended in water (10 g in 100 mL), and bacteria strains were isolated using combinational enrichment and selective culture techniques. Pure bacterial strains were characterized and identified based on their physiology and 16S rRNA gene sequence homology. While 85 bacterial strains were isolated from the enriched culture system, 20 strains were selectively isolated based on tolerance to sodium deoxycholate. Approximately 13% of the selected bacteria were identified as Cronobacter spp., exhibiting virulence traits including extracellular protease production, coagulation and proteolysis of casein, haem-agglutination, and ß-haemolysis of human blood. Approximately 82% of the Cronobacter strains tolerated NaCl (10%) and bile salt; and exhibited resistance to cefotaxime, ceftriaxone, gentamicin, and Amoxicillin-clavulanic acid antibiotics. The presence of Cronobacter spp. in 13% of the PIF brands available to infants calls for concern about the safety of deprived infants that might be fed with such PIF. Consequently, PIF safety alerts need to be activate while further studies on critical points at which the pathogens get introduced to the PIFs need to be identified.

Microbiological survey and genomic analysis of Cronobacter sakazakii strains isolated from US households and retail foods.

Cronobacter species are opportunistic pathogens that are capable of causing morbidity and mortality, particularly in infants. Although the transmission dynamics involved in Cronobacter infections remain largely unknown, contaminated powdered infant formula (PIF) has been linked to 30% of Cronobacter sakazakii cases involving invasive illness in infants. As several lines of evidence have implicated the domestic environment in PIF contamination, we undertook a microbiological survey of homes (N = 263) across the US. Cronobacter spp. and C. sakazakii were isolated from 36.1% and 24.7% of US homes, respectively, with higher recovery rates observed for floor and kitchen surfaces. Multi-locus sequence typing indicated that the dominant strain was C. sakazakii ST4, the sequence type most commonly associated with neonatal meningitis. For comparison purposes, retail foods (N = 4,009) were also surveyed, with the highest contamination frequencies (10.1%-26.3%) seen for nut products, seeds, and grains/baked goods/flours. The sequence type profile of isolates recovered from homes mirrored that of isolates recovered from retail foods, with increased representation of ST1, ST4, ST13, ST17, and ST40. Analysis of 386 whole genomic sequences revealed significant diversity. Redundancies were only observed for isolates recovered from within the same domicile, and there were no identical matches with sequences archived at the NCBI pathogen database. Genes coding for putative virulence and antibiotic resistance factors did not segregate with clinically significant sequence types. Collectively, these findings support the possibility that contamination events occurring within the home should not be overlooked as a contributor to community-onset Cronobacter infections. IMPORTANCE: Cronobacter sakazakii is an opportunistic pathogen that can cause significant morbidity and mortality in neonates. Its transmission dynamics are poorly understood, though powered infant formula (PIF) is thought to be the major transmission vehicle. How the PIF becomes contaminated remains unknown. Our survey shows that roughly 1/4 of US homes are contaminated with Cronobacter sakazakii, particularly in the kitchen setting. Our analyses suggest that the domestic environment may contribute to contamination of PIF and provides insights into mitigating the risk of transmission.

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.

Genome Mining of Novel Targets and Construction of Ladder-shaped melting temperature isothermal amplification Assays for the Identification of Cronobacter sakazakii and Cronobacter malonaticus.

Cronobacter species are potential pathogens that can contaminate powdered infant formula. C. sakazakii and C. malonaticus are the most common species of Cronobacter associated with infections. This study mined new molecular targets for the detection of C. sakazakii and C. malonaticus by using comparative genome approaches. Specific target genes mngB and ompR were obtained and used to detect C. sakazakii and C. malonaticus, respectively. A novel detection method, termed ladder-shape melting temperature isothermal amplification (LMTIA), was developed and evaluated. The detection limit for pure C. sakazakii DNA was 1 pg per reaction and 1 fg per reaction for C. malonaticus. The C. sakazakii, C. malonaticus, and the reference stains were all correctly identified. The amplicons can be successfully visualized and identified by naked eyes when hydroxy naphthol blue dye (HNB dye) was used in the reaction. Therefore, the LMTIA assays developed in this study showed potential application for microorganism identification and detection.

Diurnal rhythmicity of infant fecal microbiota and metabolites: A randomized controlled interventional trial with infant formula.

Microbiota assembly in the infant gut is influenced by diet. Breastfeeding and human breastmilk oligosaccharides promote the colonization of beneficial bifidobacteria. Infant formulas are supplemented with bifidobacteria or complex oligosaccharides, notably galacto-oligosaccharides (GOS), to mimic breast milk. To compare microbiota development across feeding modes, this randomized controlled intervention study (German Clinical Trial DRKS00012313) longitudinally sampled infant stool during the first year of life, revealing similar fecal bacterial communities between formula- and breast-fed infants (N = 210) but differences across age. Infant formula containing GOS sustained high levels of bifidobacteria compared with formula containing B. longum and B. breve or placebo. Metabolite and bacterial profiling revealed 24-h oscillations and circadian networks. Rhythmicity in bacterial diversity, specific taxa, and functional pathways increased with age and was strongest following breastfeeding and GOS supplementation. Circadian rhythms in dominant taxa were further maintained ex vivo in a chemostat model. Hence, microbiota rhythmicity develops early in life and is impacted by diet.

Evaluation of the biocontrol potential of a collagen peptide/trehalose-based Cronobacter sakazakii phage powder in rehydrated powdered infant formula.

Cronobacter sakazakii is a major foodborne pathogen that is mainly transmitted through powdered infant formula (PIF) and has a high mortality rate of up to 80%, particularly in fetuses and neonates. Bacteriophages have emerged as an effective biocontrol agent for antibiotic-resistant bacteria. In this study, lytic phage SG01 was newly characterized and loaded into collagen peptide/trehalose-based powders to develop an antibacterial agent against C. sakazakii contamination in PIF. The phage belongs to the Siphoviridae family, has an icosahedral head and a flexible tail, and showed rapid and persistent antibacterial activity up to 17 h. It was specifically active against C. sakazakii and also exhibited effective anti-biofilm properties. The phage was freeze-dried to a collagen peptide/trehalose-based powder and the phage was tested for viability, storage stability, and antibacterial activity. The optimal composition was 5% (w/v) collagen peptides and 1% (w/v) trehalose, which demonstrated the highest phage viability after freeze-drying. The phage remained stable in the collagen peptide/trehalose-based powder for up to four weeks at 4 °C and 25 °C, indicating that this is a desirable formulation for phage protection. Furthermore, the phage powder showed significant antibacterial efficacy in PIF, with a 4-log CFU/mL reduction within 6 h. Overall, the tested phage powder has the potential to be used as an antimicrobial agent in the food industry, particularly in powdered foods such as PIF.

Co-culture of Cronobacter sakazakii and Staphylococcus aureus: Explore the influence of mixed biofilm formation and regulation of Cronobacter sakazakii biofilm formation genes.

Bacterial biofilm is a protective matrix composed of metabolites secreted by bacteria that envelop bacteria. By forming a biofilm, bacteria can considerably improve their environmental tolerance. In food-related processing environment, different types of microorganisms are often present in biofilms. The main contaminating strain in the powdered infant formula (PIF) processing environment, Cronobacter sakazakii and Staphylococcus aureus continues to pollute the PIF processing environment after biofilm production. This study selected Cronobacter sakazakii with a weak biofilm-forming ability as one of the test organisms. The coexistence of Cronobacter sakazakii and Staphylococcus aureus on the surface of production equipment was simulated to analyze the interaction. Biofilm formation in the co-culture group was significantly higher than the others. In-depth study of the effect of Staphylococcus aureus on the biofilm formation genes of Cronobacter sakazakii. Results show two bacteria can coexist on the surface of a metal device, forming a more compact hybrid biofilm structure. Under co-culture conditions, S. aureus increased bcsA and fliD expression in Cronobacter sakazakii, whereas decreased bcsC expression. Signaling molecules produced by Staphylococcus aureus (Autoinducer 2) significantly promoted the biofilm formation of Cronobacter sakazakii at the concentration of 0-500 ng/mL (0.099-0.177) and up-regulated the expression of bcsA, filD and flhD genes.

Depletion of reactive oxygen species induced by beetroot (Beta vulgaris) extract leads to apoptosis-like death in Cronobacter sakazakii.

This research aimed to disclose the antibacterial activity of beetroot extract (Beta vulgaris) against Cronobacter sakazakii and its possible mechanisms. We evaluated its antibacterial activity by measuring the minimum inhibitory concentration (MIC) and time-kill kinetics. We also evaluated the intracellular ATP levels, bacterial apoptosis-like death (ALD), and reactive oxygen species (ROS) levels to reveal the possible antibacterial mechanisms. Our results showed that the MIC of beetroot extract against C. sakazakii was 25 mg/mL and C. sakazakii (approximately 8 log cfu/mL) was completely inhibited after treatment with 2 MIC of beetroot extract for 3 h. Beetroot extract reduced intracellular ATP levels and facilitated characteristics of ALD in C. sakazakii, such as membrane depolarization, increased intracellular Ca2+ levels, phosphatidylserine externalization, caspase-like protein activation, and DNA fragmentation. Additionally, and different from most bacterial ALD caused by the accumulation of ROS, beetroot extract reduced the intracellular ROS levels in C. sakazakii. Our experimental data provide a rationale for further research of bacterial ALD and demonstrate that beetroot extract can inhibit C. sakazakii in food processing environments.

Effects of a Novel Infant Formula on the Fecal Microbiota in the First Six Months of Life: The INNOVA 2020 Study.

Exclusive breastfeeding is highly recommended for infants for at least the first six months of life. However, for some mothers, it may be difficult or even impossible to do so. This can lead to disturbances in the gut microbiota, which in turn may be related to a higher incidence of acute infectious diseases. Here, we aimed to evaluate whether a novel starting formula versus a standard formula provides a gut microbiota composition more similar to that of breastfed infants in the first 6 months of life. Two hundred and ten infants (70/group) were enrolled in the study and completed the intervention until 12 months of age. For the intervention period, infants were divided into three groups: Group 1 received formula 1 (INN) with a lower amount of protein, a proportion of casein to whey protein ratio of about 70/30 by increasing the content of α-lactalbumin, and with double the amount of docosahexaenoic acid/arachidonic acid than the standard formula; INN also contained a thermally inactivated postbiotic (Bifidobacterium animalis subsp. lactis). Group 2 received the standard formula (STD) and the third group was exclusively breastfed (BF) for exploratory analysis. During the study, visits were made at 21 days, 2, 4, and 6 months of age, with ±3 days for the visit at 21 days of age, ±1 week for the visit at 2 months, and ±2 weeks for the others. Here, we reveal how consuming the INN formula promotes a similar gut microbiota composition to those infants that were breastfed in terms of richness and diversity, genera, such as Bacteroides, Bifidobacterium, Clostridium, and Lactobacillus, and calprotectin and short-chain fatty acid levels at 21 days, 2 and 6 months. Furthermore, we observed that the major bacteria metabolic pathways were more alike between the INN formula and BF groups compared to the STD formula group. Therefore, we assume that consumption of the novel INN formula might improve gut microbiota composition, promoting a healthier intestinal microbiota more similar to that of an infant who receives exclusively human milk.

Limosilactobacillus reuteri DSM 17938-Containing Infant Formulas and the Associations with Gastrointestinal Tolerance: A Cross-Sectional Observational Study.

Limosilactobacillus (L.; previously Lactobacillus) reuteri has been shown to influence gastrointestinal (GI) tolerance. This study was a secondary analysis of GI tolerance data from a multi-country, cross-sectional, observational study in healthy infants using the validated Infant Gastrointestinal Symptom Questionnaire (IGSQ) and a gut comfort questionnaire. Breastfed infants (BFI; n = 760) were compared to formula-fed infants receiving either L. reuteri-containing formula (FFI + LR; n = 470) or standard formula without any probiotic or prebiotic (FFI-Std; n = 501). The IGSQ composite scores (adjusted mean ± SE) in FFI + LR (22.17 ± 0.39) was significantly lower than in FFI-Std (23.41 ± 0.37) and similar to BFI (22.34 ± 0.30;), indicating better GI tolerance in FFI + LR than in FFI-Std. Compared with FFI-Std, FFI + LR had lower reports of difficulty in passing stools (11% vs. 22%; adjusted-odds ratio (OR) (95%CI) = 0.46 (0.31-0.68)), fewer hard stools (mean difference = -0.12 (-0.21, -0.02)) and less physician-confirmed colic (OR = 0.61 (0.45-0.82)), and similar to BFI. Parent-reported crying time (mean difference = -0.15 (-0.28, -0.01)), frequency of spitting-up/vomiting (mean difference = -0.18 (-0.34, -0.03)), volume of spit-up (mean difference = -0.20 (-0.32, -0.08)) and fussiness due to spitting-up/vomiting (mean difference = -0.17 (-0.29, -0.05)) were lower in FFI + LR versus FFI-Std and similar to BFI. In this study, L. reuteri-containing formula was associated with improved digestive tolerance and behavioral patterns.

Lack of correlation between growth rate and sequence type among Cronobacter sakazakii.

Species identification and growth rates for a collection of Cronobacter strains from clinical and non-clinical sources have been previously reported. However, advancements in DNA sequencing-based identification methods now allow for more accurate identification. Here we report the sequence types (STs) for 24 strains of Cronobacter sakazakii and examine any possible correlation between sequence type and growth rate, which could influence risk through greater pathogen multiplication and reach of infectious doses during time between formula preparation and feeding. The most common clonal complexes (CCs) identified were C. sakazakii CC1 and CC4. CC1 strains belonged to ST1 (n = 8) and ST391 (n = 1), while CC4 included ST4 (n = 4), ST255 (n = 1) and ST295 (n = 1). Three strains were found to belong to CC100 and two were found to belong to ST64. The remaining STs identified were represented by single strains. CC4 strains have a slightly not significant tendency for faster growth rates at 25 °C; however, the small sample size suggests that more strains need to be analysed to determine if this is a true result. In conclusion, the growth rates of C. sakazakii strains do not appear to be strongly correlated to ST.

Visual detection of Cronobacter sakazakii on a microfluidic chip fabricated by a 3D molding method.

Cronobacter sakazakii (C. sakazakii) is a pathogenic bacterium associated with life-threatening neonatal infections that have been linked to contaminated powdered infant formula (PIF). Most C. sakazakii testing is still limited in microbiology laboratories due to the need for sophisticated equipment and professional technicians. Microfluidic chips combined with isothermal amplification analysis are shown to be one of the most attractive microbiological on-site detection platforms. In this study, PDMS microfluidic chips were fabricated by a simple 3D molding method and sealed with "PDMS glue". The chip consisted of an inlet, a microchannel, six reaction wells, and six vent holes. And based on the 16S rRNA and ITS genes of C. sakazakii, we have successfully proposed a multiplex competitive annealing mediated isothermal amplification (mCAMP) assay on the microfluidic chip for the visual detection of C. sakazakii in PIF samples. The primers were fixed in the reaction wells of the chip before detection, which can be preserved for 60 days at 4 °C. The results showed that the established mCAMP assay had high specificity, and the limit of detection was 2.2 × 103 CFU g-1. With enrichment culture, even if the initial inoculation level is 1 CFU g-1, the mCAMP assay can still detect the presence of C. sakazakii in spiked PIF samples. The test results are visible to the naked eye, which is suitable for rapid analysis in resource-limited settings.

Preparation of silver nanoparticles/polymethylmethacrylate/cellulose acetate film and its inhibitory effect on Cronobacter sakazakii in infant formula milk.

Cronobacter sakazakii is a harmful foodborne pathogen, and its contaminated food will pose a huge threat to human health. Prevention of C. sakazakii contamination of food is valuable for food safety as well as for human health. In this study, silver nanoparticles (AgNP) were successfully immobilized on the surface of cellulose acetate (CA) and polymethylmethacrylate (PMMA) composite to obtain AgNP/PMMA/CA film. Through the inhibition zone and growth curve experiments, we found that AgNP/PMMA/CA films has excellent antibacterial activity on C. sakazakii. The AgNP/PMMA/CA film can prolong the lag phase of the growth curve of C. sakazakii from 2 to 8 h. The antibacterial films were found to reduce the survival of C. sakazakii in Luria-Bertani and infant formula by combining it with a mild heat treatment (45°C, 50°C, and 55°C). The AgNP/PMMA/CA film combined with 55°C water bath can completely inactivate C. sakazakii in infant formula within 120 min. Finally, the potential mechanism by which AgNP/PMMA/CA films reduce the heat tolerance of C. sakazakii was investigated by quantitative real-time PCR. The results showed that AgNP/PMMA/CA films could reduce the expression of environmental tolerance-related genes in C. sakazakii. The current research shows that AgNP/PMMA/CA film has strong antibacterial activity, and the antibacterial film combined with mild heat treatment can accelerate the inactivation of C. sakazakii and effectively reduce the harm of foodborne pathogens. The AgNP/PMMA/CA film can be used as a potential packaging material or antibacterial surface coating.

Validation of the Assurance® GDS for Cronobacter Tq II in Infant Formulas, Infant Cereals, Ingredients, and Environmental Samples Collaborative Study: First Action 2021.08.

BACKGROUND: The Assurance® GDS for Cronobacter Tq II assay is a nucleic acid amplification system for the qualitative detection of Cronobacter. The method uses an upfront concentration of the target organism from the enrichment by immunomagnetic separation (IMS) using the PickPen® device. OBJECTIVE: The Assurance GDS for Cronobacter Tq II method was evaluated for Official Methods of AnalysisSM certification. METHOD: The matrix was compared to the ISO 22964:2017: Microbiology of the Food Chain-Horizontal Method for the Detection of Cronobacter spp. standard and using an alternative confirmation procedure. The alternative method was evaluated using 10 g test portions in an unpaired study design for powdered infant formula (milk based with iron and DHA) containing probiotics. Eleven technicians from eight laboratories located within the United States and Europe participated in the collaborative study. Statistical analysis was conducted according to the probability of detection (POD) statistical model as presented in the AOAC validation guidelines. The difference in laboratory POD (dLPODC) values with 95% confidence intervals across collaborators was calculated for each level between the candidate and reference method results and between the candidate presumptive and confirmed results. RESULTS: Results obtained for the low inoculum level test portions produced a dLPOD value with a 95% confidence interval of 0.03 (-0.18, 0.15). The dLPOD results indicate equivalence between the candidate method and reference method for the matrix evaluated. The method also demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. There were no false negative results; the false positive rate was determined and produced a value of <2%. CONCLUSIONS: Based on the data generated, the method demonstrated Assurance GDS for Cronobacter Tq II assay produced acceptable interlaboratory reproducibility data and statistical analysis. HIGHLIGHTS: The Assurance GDS for Cronobacter Tq II method is suitable for the rapid qualitative detection of Cronobacter in infant formulas, infant cereals, ingredients, and environmental samples.

Emerging of Multidrug-Resistant Cronobacter sakazakii Isolated from Infant Supplementary Food in China.

Cronobacter is a foodborne pathogen associated with severe infections in restricted populations and particularly with high mortality in neonates and infants. The prevalence and antimicrobial resistance (AMR) phenotype of Cronobacter cultured from powdered infant formula and supplementary food were studied. The virulence factors, AMR genes, and genomic environments of the multidrug-resistant isolates were further studied. A total of 1,055 Cronobacter isolates were recovered from 12,105 samples of powdered infant formula and supplementary food collected from 29 provinces between 2018 and 2019 in China. Among these, 1,048 isolates were from infant supplementary food and 7 were from powdered infant formula. Regarding antimicrobial resistance susceptibility, 11 (1.0%) isolates were resistant and two showed resistance to four antimicrobials (ampicillin [AMP], tetracycline [TET], sulfamethoxazole-trimethoprim [SXT], and chloramphenicol [CHL]), defined as MDR. These two MDR isolates were subsequently identified as Cronobacter sakazakii sequence type 4 (ST4) (C. sakazakii Crono-589) and ST40 (C. sakazakii Crono-684). Both MDR isolates contain 11 types of virulence genes and 7 AMR genes on their genomes. Meanwhile, the IncFIB plasmids of both MDR C. sakazakii isolates also harbored 2 types of virulence genes. Results of the genomic comparative analysis indicated that food-associated C. sakazakii could acquire antimicrobial resistance determinants through horizontal gene transfer (HGT). IMPORTANCE As a foodborne pathogen, Cronobacter can cause serious infections in restricted populations and lead to death or chronic sequelae. Although a number of investigations showed that Cronobacter isolates are susceptible to most antimicrobial agents, MDR Cronobacter isolates, isolated mainly from clinical cases but occasionally from foods, have been reported in recent years. In this study, we successfully identified two MDR Cronobacter sakazakii isolates from infant foods based on nationwide surveillance and genome sequencing in China. Genomic analysis revealed that these two MDR C. sakazakii strains acquired resistance genes from other species via different evolution and transmission routes. It is important to monitor MDR C. sakazakii isolates in infant foods, and appropriate control measures should be taken to reduce the contamination with and transmission of this MDR bacterium.

A 16S rRNA Sequencing Study Describing the Environmental Microbiota of Two Powdered Infant Formula Built Facilities.

Microbial safety is critically important for powdered infant formula (PIF) fed to neonates, with under-developed immune systems. The quality and safety of food products are dictated by those microorganisms found in both raw materials and the built production environment. In this study, a 2-year monitoring program of a production environment was carried out in two PIF factories located in the Republic of Ireland, and the environmental microbiome in different care areas of these sites was studied by using a 16S ribosomal RNA (rRNA)-based sequencing technique. Results highlighted a core microbiome associated with the PIF factory environment containing 24 bacterial genera representing five phyla, with Acinetobacter and Pseudomonas as the predominant genera. In different care areas of the PIF factory, as hygiene standards increased, deciphered changes in microbial community compositions became smaller over time and approached stability, and bacteria dominating the care area became less influenced by the external environment and more by human interactions and raw materials. These observations indicated that the microbial composition can be altered in response to environmental interventions. Genera Cronobacter and Salmonella were observed in trace amounts in the PIF factory environment, and bacterial genera known to be persistent in a stressed environment, such as Acinetobacter, Bacillus, Streptococcus, and Clostridium, were likely to have higher abundances in dry environment-based care areas. To our knowledge, this is the first study to characterize the PIF production environment microbiome using 16S rRNA-based sequencing. This study described the composition and changing trends of the environmental microbial communities in different care areas of the PIF manufacturing facility, and it provided valuable information to support the safer production of PIF in the future.

Evaluation of Potential for Butyl and Heptyl Para-Hydroxybenzoate Enhancement of Thermal Inactivation of Cronobacter sakazakii during Rehydration of Powdered Infant Formula and Nonfat Dry Milk.

ABSTRACT: In previous studies, parabens in model systems enhanced the thermal inactivation of foodborne pathogens, including Cronobacter sakazakii, Salmonella enterica serotype Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes. However, few studies have been conducted to evaluate this phenomenon in actual food systems. In the present study, the potential enhancement of thermal inactivation of C. sakazakii by butyl para-hydroxybenzoate (BPB) was evaluated in powdered infant formula (PIF) and nonfat dry milk (NFDM) in dry and rehydrated forms. When PIF was rehydrated with water at designated temperatures (65 to 80°C) in baby bottles, BPB did not enhance thermal inactivation. When rehydrated NFDM and lactose solutions with BPB were inoculated and heated at 58°C, BPB enhancement of thermal inactivation of C. sakazakii was negatively associated with the concentration of NFDM solutions in a dose-dependent manner, whereas thermal inactivation was enhanced in the presence of lactose regardless of its concentration, suggesting an interaction between proteins and BPB. Fluorescence testing further indicated an interaction between BPB and the proteins in PIF and NFDM. In inoculated dry NFDM with and without BPB stored at 24 and 55°C for 14 days, BPB did not substantially enhance bacterial inactivation. This study suggests that BPB is not likely to enhance mild thermal bacterial inactivation treatments in foods that have appreciable amounts of protein.

A novel photoreactive DNA-binding dye for detecting viable Klebsiella pneumoniae in powdered infant formula.

In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA-RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA-RAA assay can detect as low as 2.6 × 103 cfu/mL of K. pneumoniae in pure culture and 2.3 × 104 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 100 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA-RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.

Membrane Proteins and Proteomics of Cronobacter sakazakii Cells: Reliable Method for Identification and Subcellular Localization.

Members of the genus Cronobacter are responsible for severe infections in infants and immunosuppressed individuals. Although several virulence factors have been described, many proteins involved in the pathogenesis of such infections have not yet been mapped. This study is the first to fractionate Cronobacter sakazakii cells into outer membrane, inner membrane, periplasmic, and cytosolic fractions as the basis for improved proteome mapping. A novel method was designed to prepare the fractionated samples for protein identification. The identification was performed via one-dimensional electrophoresis-liquid chromatography electrospray ionization tandem mass spectrometry. To determine the subcellular localization of the identified proteins, we developed a novel Python-based script (Subcelloc) that combines three web-based tools, PSORTb 3.0.2, CELLO 2.5, and UniProtKB. Applying this approach enabled us to identify 1,243 C. sakazakii proteins, which constitutes 28% of all predicted proteins and 49% of all theoretically expressed outer membrane proteins. These results represent a significant improvement on previous attempts to map the C. sakazakii proteome and could provide a major step forward in the identification of Cronobacter virulence factors. IMPORTANCECronobacter spp. are opportunistic pathogens that can cause rare and, in many cases, life-threatening infections, such as meningitis, necrotizing enterocolitis, and sepsis. Such infections are mainly linked to the consumption of contaminated powdered infant formula, with Cronobacter sakazakii clonal complex 4 considered the most frequent agent of serious neonatal infection. However, the pathogenesis of diseases caused by these bacteria remains unclear; in particular, the proteins involved throughout the process have not yet been mapped. To help address this, we present an improved method for proteome mapping that emphasizes the isolation and identification of membrane proteins. Specific focus was placed on the identification of the outer membrane proteins, which, being exposed to the surface of the bacterium, directly participate in host-pathogen interaction.

Selection of a Potential Synbiotic against Cronobacter sakazakii.

ABSTRACT: Cronobacter sakazakii is an opportunistic foodborne pathogen that can be fatal to infants; it is commonly associated with powdered infant formula due to contamination during manufacturing processes or during preparation in hospitals or homes. This project aimed to select a potential synbiotic, a combination of probiotic strains with a prebiotic product, to inhibit the growth of C. sakazakii in an in vitro dynamic infant gut model (Simulator of the Human Intestinal Microbial Ecosystem). A total of 16 lactic acid bacteria (LAB) were tested for their inhibitory properties against four different C. sakazakii strains by a zone of inhibition test. Lactobacillus and Pediococcus species were able to inhibit the growth (>15-mm inhibition zones) of all C. sakazakii strains tested, and only one strain from the two genera exhibited atypical resistance to tetracycline. All C. sakazakii strains and the selected LAB strains, which inhibited C. sakazakii and did not exhibit atypical antibiotic resistance, were grown in Luria-Bertani or de Man Rogosa Sharpe broth, respectively, containing 1% dextrose or 1% commercial prebiotic (w/v) to compare their ability to metabolize the prebiotic product. Overall, based on the growth inhibition of C. sakazakii, antibiotic susceptibility, and prebiotic metabolism, 6 of the 16 LAB were chosen to be part of a potential synbiotic. This study has provided valuable information that will help with the development of a synbiotic that can be used in powdered infant formula to reduce the potential for C. sakazakii-related illnesses in infants.