Survival and Expression of rpoS and grxB of Cronobacter sakazakii in Powdered Infant Formula Under Simulated Gastric Conditions of Newborns.

Cronobacter sakazakii can cause severe illnesses in infants, predominantly in preterm newborns, with consumption of contaminated powdered infant formula (PIF) being the major vehicle of infection. Using a dynamic human gastrointestinal simulator called the SHIME, this study examined the effects of gastric acidity and gastric digestion time of newborns on the survival and expression of stress genes of C. sakazakii. Individual strains, inoculated at 7 log CFU/mL into reconstituted PIF, were exposed to gastric pH values of 4.00, 5.00 and 6.00 for 4 h with gradual acidification. The survival results showed that C. sakazakii grew in the stomach portion of the SHIME during a 4-h exposure to pH 4.00, 5.00 and 6.00 by 0.96-1.05, 1.02-1.28 and 1.11-1.73 log CFU/mL, respectively. The expression of two stress genes, rpoS and grxB, throughout gastric digestion was evaluated using reverse transcription qPCR. The upregulation of rpoS and grxB during the 4-h exposure to simulated gastric fluid at pH 4.00 showed that C. sakazakii strains may be experiencing the most stress in the pH 4.00 treatment. The gene expression results also suggest that C. sakazakii strains appeared to develop an acid adaptation response during the 4-h exposure that may facilitate their survival. Altogether, this study highlights that a combination of low gastric acidity, long digestion time in the presence of reconstituted PIF, created a favorable environment for the adaptation and survival of C. sakazakii in the simulation of a newborn's stomach. This study gives directions for future research to further advance our understanding of the behavior of C. sakazakii in the GI tract of newborns.

Global and regional prevalence of Cronobacter sakazakii in powdered milk and flour.

Cronobacter sakazakii (Cz) infections linked with powdered milk/flour (PMF) are on the increase in recent times. The current study aimed at assessing worldwide and regional prevalence of Cz in PMF. Cz-PMF-directed data were conscientiously mined in four mega-databases via topic-field driven PRISMA protocol without any restriction. Bivariate analysis of datasets was conducted and then fitted to random-intercept logistic mixed-effects regressions with leave-one-study-out-cross-validation (LOSOCV). Small-study effects were assayed via Egger's regression tests. Contributing factors to Cz contamination/detection in PMF were determined using 1000-permutation-bootstrapped meta-regressions. A total of 3761 records were found out of which 68 studies were included. Sample-size showed considerable correlation with Cz positivity (r = 0.75, p = 2.5e-17), Milkprod2020 (r = 0.33, p = 1.820e-03), and SuDI (r = - 0.30, p = 4.11e-03). The global prevalence of Cz in PMF was 8.39% (95%CI 6.06-11.51, PI: 0.46-64.35) with LOSOCV value of 7.66% (6.39-9.15; PI: 3.10-17.70). Cz prevalence in PMF varies significantly (p < 0.05) with detection methods, DNA extraction method, across continents, WHO regions, and world bank regions. Nation, detection method, world bank region, WHO region, and sample size explained 53.88%, 19.62%, 19.03%, 15.63%, and 9.22% of the true differences in the Cz prevalence in PMF, respectively. In conclusion, the results indicated that national will power in the monitoring and surveillance of Cz in PMF matched with adequate sample size and appropriate detection methods will go a long way in preventing Cz contamination and infections.

Antibacterial action of slightly acidic electrolytic water against Cronobacter sakazakii and its application as a disinfectant on high-risk contact surfaces.

Powdered infant formula (PIF) is prone to Cronobacter sakazakii (C. sakazakii) contamination, which can result in infections that endanger the lives of newborns and infants. Slightly acidic electrolytic water (SAEW) has shown antibacterial effects on a variety of foodborne pathogens and has a wide applicability in the food industry. Here, the antibacterial activity of SAEW against C. sakazakii and its use as a disinfectant on contact surfaces with high infection transmission risk were investigated. The inactivation of SAEW on C. sakazakii was positively correlated to the SAEW concentration and treatment time. The antibacterial effect of SAEW was achieved by decreasing the intracellular adenosine triphosphate (ATP), K+, protein, and DNA contents of C. sakazakii, reducing the intracellular pH (pHin) and destroying the cell morphology, which led to inactivation of C. sakazakii ultimately. To test the applicability of this study, the results showed that approximately 103 CFU/cm2 of C. sakazakii were successfully inactivated on stainless steel and rubber surfaces after a 30 mg/L SAEW treatment for 20 s. These results indicate the antibacterial mechanism and potential application of SAEW against C. sakazakii, as well as a new strategy for the prevention and control of C. sakazakii on stainless steel and rubber surfaces.

Prevalence and phylogenetic traits of nitrite-producing bacteria in raw ingredients and processed baby foods: Potential sources of foodborne infant methemoglobinemia.

Nitrite, which has been mainly regarded as a chemical hazard, can induce infant methemoglobinemia. As for nitrite as a product of microbial metabolism, the contribution of the oral or gut microbiome has mostly received attention, whereas the role of nitrite-producing bacteria (NPBs) in food has been less elucidated. In this study, mesophilic NPBs were isolated from food samples (n = 320) composed of raw ingredients for weaning foods (n = 160; beetroot, broccoli, carrot, lettuce, rice powder, spinach, sweet potato, and honey) and processed baby foods (n = 160; cereal snack, cheese, yogurt, powdered infant formula, sorghum syrup, vegetable fruit juice, and weaning food). The phylogenetic diversity of the NPB strains was analyzed via 16S rRNA sequencing. All 15 food items harbored NPBs, with a prevalence of 71.9 % and 34.4 % for the raw ingredients and processed foods, respectively. The NPBs isolated from the foods were identified as Actinomycetota (Actinomycetes), Bacteroidota (Flavobacteriia, Sphingobacteriia), Bacillota (Bacilli), or Pseudomonadota (Alpha-, Beta-, and Gammaproteobacteria). Among the raw and processed foods, beetroot (85.0 %) and powdered infant formula (70.0 %) showed had the highest NPB prevalence (P > 0.05). Bacillota predominated in both types of food. The contamination source of Pseudomonadota, which was another major phylum present in the raw ingredients, was presumed to be the soil and endophytes in the seeds, whereas that of Bacillota was the manufacturing equipment used with the raw ingredients. Common species for probiotics, such as Lacticaseibacillus, Leuconostoc, Enterococcus, and Bacillus, were isolated and identified as NPBs. To our knowledge, this is the first study to reveal the taxonomical diversity and omnipresence of NPBs in food for babies. The results of this study highlight the importance of food-mediated microbiological risks of infant methemoglobinemia which are yet underrecognized.

Effect of RpoS on the survival, induction, resuscitation, morphology, and gene expression of viable but non-culturable Salmonella Enteritidis in powdered infant formula.

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS-deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (aw) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS, katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low aw foods.

The safety of at home powdered infant formula preparation: A community science project.

Formula fed infants experience gastrointestinal infections at higher rates than breastfed infants, due in part to bacteria in powdered infant formula (PIF) and bacterial contamination of infant feeding equipment. The United Kingdom National Health Service (UK NHS) has adopted the World Health Organization recommendation that water used to reconstitute PIF is ≥70°C to eliminate bacteria. We used community science methods to co-design an at home experiment and online questionnaire ('research diary') to explore the safety of PIF preparation compared to UK NHS guidelines. 200 UK-based parents of infants aged ≤12 months were recruited; 151 provided data on PIF preparation, and 143 were included in the analysis of water temperatures used to reconstitute PIF. Only 14.9% (n = 11) of 74 PIF preparation machines produced a water temperature of ≥70°C compared with 78.3% (n = 54) of 69 kettle users (p < 0.001). The mean temperature of water dispensed by PIF preparation machines was 9°C lower than kettles (Machine M = 65.78°C, Kettle M = 75.29°C). Many parents did not always fully follow NHS safer PIF preparation guidance, and parents did not appear to understand the potential risks of PIF bacterial contamination. Parents should be advised that the water dispensed by PIF preparation machines may be below 70°C, and could result in bacteria remaining in infant formula, potentially leading to gastrointestinal infections. PIF labelling should advise that water used to prepare PIF should be ≥70°C and highight the risks of not using sufficiently hot water, per WHO Europe advice. There is an urgent need for stronger consumer protections regarding PIF preparation devices.

Sensitive detection of viable Cronobacter sakazakii by bioluminescent reporter phage emitting stable signals with truncated holin.

As outbreaks of foodborne illness caused by the opportunistic pathogen Cronobacter sakazakii (Cs) continue to occur, particularly in infants consuming powdered infant formula (PIF), the need for sensitive, rapid, and easy-to-use detection of Cs from food and food processing environments is increasing. Here, we developed bioluminescent reporter bacteriophages for viable Cs-specific, substrate-free, rapid detection by introducing luciferase and its corresponding substrate-providing enzyme complex into the virulent phage ΦC01. Although the reporter phage ΦC01_lux, constructed by replacing non-essential genes for phage infectivity with a luxCDABE reporter operon, produced bioluminescence upon Cs infection, the emitted signal was quickly decayed due to the superior bacteriolytic activity of ΦC01. By truncating the membrane pore-forming protein holin and thus limiting its function, the bacterial lysis was delayed and the resultant engineered reporter phage ΦC01_lux_Δhol could produce a more stable and reliable bioluminescent signal. Accordingly, ΦC01_lux_Δhol was able to detect at least an average of 2 CFU/ml of Cs artificially contaminated PIF and Sunsik and food contact surface models within a total of 7 h of assays, including 5 h of pre-enrichment for Cs amplification. The sensitive, easy-to-use, and specific detection of live Cs with the developed reporter phage could be applied as a novel complementary tool for monitoring Cs in food and food-related environments for food safety and public health.

Application of a novel phage LPCS28 for biological control of Cronobacter sakazakii in milk and reconstituted powdered infant formula.

Contamination of infant formula with Cronobacter sakazakii (C. sakazakii) can cause fatal infections in neonates. Phages have emerged as promising antibacterial agents for food safety, but their effectiveness may be limited by thermal processing. In this study, we isolated 27 C. sakazakii phages from environmental water samples and selected LPCS28 due to its broad lysis spectrum. The titer of LPCS28 will not be significantly affected by heating at a temperature of 60 °C for one hour. In both reconstituted powdered infant formula (RPIF) and liquid milk, the pre-added LPCS28, after the thermal processing at 63 °C for 30 min, significantly inhibited the post-contaminated C. sakazakii (103 CFU/mL) and eventually reduced the number of C. sakazakii to below the limit of detection (<10 CFU/mL) within 9 h at 37 °C and significantly delayed the increase of bacterial concentration in the samples at 23 °C. The phylogenetic analysis revealed that LPCS28 belonged to a new genus, we proposed as Nanhuvirus, under the family Straboviridae. These findings suggest that phage LPCS28 is a promising biological control agent for pathogenic C. sakazakii in the dairy industry.

Comparative proteomics reveals the antibiotic resistance and virulence of Cronobacter isolated from powdered infant formula and its processing environment.

Cronobacter species are opportunistic foodborne pathogens that can cause neonatal meningitis, sepsis, and necrotizing enterocolitis. In this genus, certain level strains have high mortality to infant (Cronobacter sakazakii and Cronobacter malonaticus) and antibiotic tolerance. Cronobacter has strong environmental tolerance (acid resistance, high temperature resistance, UV resistance, antibiotic resistance, etc.) and can survive in a variety of environments. It has been isolated in various production environments and products in several countries. However, the relationships between Cronobacter antibiotic tolerance and virulence remain unclear, especially at the molecular level. In this study, 96 strains of Cronobacter were isolated from powdered infant formula and its processing environment and screened for antibiotic tolerance, and proteomic maps of the representative strains of Cronobacter with antibiotic tolerance were generated by analyzing proteomics data using multiple techniques to identify protein that are implicated in Cronobacter virulence and antibiotic resistance. The increase in antibiotic tolerance of Cronobacter had a certain increase in the production of enterotoxin and hemolysin. Only triple tolerated Cronobacter sakazakii decreased the utilization of sialic acid. A total of 16,131 intracellular proteins were detected in eight representative strains, and different proteomes were present in strains with different antibiotic tolerance, including 56 virulence-related proteins. Multiple virulence proteins regulated by unknown genes were also found in the eight isolated representative strains.

Simulation Evaluation of Power of Sampling Plans to Detect Cronobacter in Powdered Infant Formula Production.

Cronobacteris a hazard in Powdered Infant Formula (PIF) products that is hard to detect due to localized and low-level contamination. We adapted a previously published sampling simulation to PIF sampling and benchmarked industry-relevant sampling plans across different numbers of grabs, total sample mass, and sampling patterns. We evaluated performance to detect published Cronobacter contamination profiles for a recalled PIF batch [42% prevalence, -1.8 ± 0.7 log(CFU/g)] and a reference, nonrecalled, PIF batch [1% prevalence, -2.4 ± 0.8 log(CFU/g)]. Simulating a range of numbers of grabs [n = 1-22,000 (representing testing every finished package)] with 300 g total composite mass showed that taking 30 or more grabs detected contamination reliably (<1% median probability to accept the recalled batch). Benchmarking representative sampling plans ([n = 30, mass grab = 10g], [n = 30, m = 25g], [n = 60, m = 25g], [n = 180, m = 25g]) showed that all plans would reject the recalled batch (<1% median probability to accept) but would rarely reject the reference batch (>50% median probability of acceptance, all plans). Overall, (i) systematic or stratified random sampling patterns are equal to or more powerful than random sampling of the same sample size and total sampled mass, and, (ii) taking more samples, even if smaller, can increase the power to detect contamination.

Cronobacter Species in the Built Food Production Environment: A Review on Persistence, Pathogenicity, Regulation and Detection Methods.

The powdered formula market is large and growing, with sales and manufacturing increasing by 120% between 2012 and 2021. With this growing market, there must come an increasing emphasis on maintaining a high standard of hygiene to ensure a safe product. In particular, Cronobacter species pose a risk to public health through their potential to cause severe illness in susceptible infants who consume contaminated powdered infant formula (PIF). Assessment of this risk is dependent on determining prevalence in PIF-producing factories, which can be challenging to measure with the heterogeneity observed in the design of built process facilities. There is also a potential risk of bacterial growth occurring during rehydration, given the observed persistence of Cronobacter in desiccated conditions. In addition, novel detection methods are emerging to effectively track and monitor Cronobacter species across the food chain. This review will explore the different vehicles that lead to Cronobacter species' environmental persistence in the food production environment, as well as their pathogenicity, detection methods and the regulatory framework surrounding PIF manufacturing that ensures a safe product for the global consumer.

Development of multi-reactive aptamers for Cronobacter spp. using the sequential partitioning method to detect them in powdered infant formula.

Cronobacter spp. are opportunistic foodborne pathogens typically detected in contaminated powdered infant formula (PIF). Thus, the rapid detection and control of Cronobacter spp. are required to prevent outbreaks, necessitating the development of specific aptamers. In this study, we isolated aptamers specific to all seven species of Cronobacter (C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C. condimenti, and C. universalis) using a newly proposed sequential partitioning method. This method avoids the repeated enrichment steps, reducing the total aptamer selection time compared with the conventional systematic evolution of ligands by the exponential enrichment (SELEX) process. We isolated four aptamers showing high affinity and specificity for all seven species of Cronobacter, with dissociation constants of 3.7-86.6 nM. This represents the first successful isolation of aptamers for multiple targets using the sequential partitioning method. Further, the selected aptamers could effectively detect Cronobacter spp. in contaminated PIF.

Identification, Typing and Drug Resistance of Cronobacter spp. in Powdered Infant Formula and Processing Environment.

Cronobacter spp. is a food-borne pathogenic microorganism that can cause serious diseases such as meningitis, sepsis, and necrotizing colitis in infants and young children. Powdered infant formula (PIF) is one of the main contamination routes, in which the processing environment is an important source of pollution. In this investigation, 35 Cronobacter strains isolated from PIF and its processing environment were identified and typed by 16S rRNA sequencing and multilocus sequence typing (MLST) technology. A total of 35 sequence types were obtained, and three new sequence types were isolated for the first time. The antibiotic resistance was analyzed, showing that all isolates were resistant to erythromycin but sensitive to ciprofloxacin. Multi-drug resistant strains accounted for 68.57% of the total, among which Cronobacter strains with the strongest drug resistance reached 13 multiple drug resistance. Combined with transcriptomics, 77 differentially expressed genes related to drug resistance were identified. The metabolic pathways were deeply excavated, and under the stimulation of antibiotic conditions, Cronobacter strains can activate the multidrug efflux system by regulating the expression of chemotaxis-related genes, thus, secreting more drug efflux proteins to enhance drug resistance. The study of drug resistance of Cronobacter and its mechanism has important public health significance for the rational selection of existing antibacterial drugs, the development of new antibacterial drugs to reduce the occurrence of bacterial resistance, and the control and treatment of infections caused by Cronobacter.

Are Enterobacteriaceae and Enterococcus Isolated from Powdered Infant Formula a Hazard for Infants? A Genomic Analysis.

Powdered infant formulas (PIF) are the most used dietary substitutes that are used in order to supplement breastfeeding. However, PIF are not sterile and can be contaminated with different microorganisms. The objective of this study was to genomically characterize Enterobacteriaceae (ENT) and Enterococcus strains that were isolated from PIF. Strains were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS). Genomic typing, detection of virulence, and resistance profiles and genes were performed with the Ridom SeqSphere+ software; the comprehensive antibiotic resistance database (CARD) platform; ResFinder and PlasmidFinder tools; and by the disk diffusion method. Nineteen isolates from PIF were analyzed, including ENT such as Kosakonia cowanii, Enterobacter hormaechei, Franconibacter helveticus, Mixta calida, and lactic acid bacteria such as Enterococcus faecium. The strains exhibited resistance to beta-lactams, cephalosporins, and macrolides. Resistance genes such as AcrAB-TolC, marA, msbA, knpEF, oqxAB, fosA, blaACT-7, blaACT-14,qacJ, oqxAB,aac(6')-Ii, and msr(C); and virulence genes such as astA, cheB, cheR, ompA ompX, terC, ironA, acm, and efaAfm, adem were also detected. All the analyzed strains possessed genes that produced heat-shock proteins, such as IbpA and ClpL. In PIF, the presence of ENT and Enterococcus that are multiresistant to antibiotics-together with resistance and virulence genes-pose a health risk for infants consuming these food products.

Draft genome sequence of Cronobacter sakazakii strain MEZCS99 sequence type 3 isolated from chicken in South Africa.

OBJECTIVES: Cronobacter sakazakii is an emerging opportunistic foodborne pathogen that is frequently associated with life-threatening infections such as infantile septicemia, meningitis, and necrotizing enterocolitis. The emergence of antimicrobial-resistant, livestock-associated C. sakazakii is a great public health concern. Here, we report on the first draft genome sequence of C. sakazakii strain MEZCS99 sequence type 3 (ST3) isolated from feces from a healthy chicken in KwaZulu-Natal Province, South Africa. METHODS: The genomic DNA of C. sakazakii was sequenced using an Illumina MiSeq platform (Illumina Inc., San Diego, CA). Generated reads were trimmed and de novo assembled. The assembled contigs were analyzed for virulence and antimicrobial resistance genes, extra-chromosomal plasmids, and multilocus sequence type (MLST). To compare the sequenced strains to other previously sequenced C. sakazakii strains, available raw read sequences of C. sakazakii were downloaded and all sequence files were treated identically to generate a core genome phylogenetic tree. RESULTS: Intrinsic beta-lactam resistance gene blaCSA-1 was detected in MEZCS99. No colistin or other antibiotic resistance genes were detected. MEZCS99 belonged to ST3 and harbored an extra-chromosomal plasmid (IncFIB (pCTU3)). The genome of MEZCS99 strain showed two CRISPR/Cas cluster arrays of I-E (n = 1) and I-F (n = 1) type. CONCLUSION: The genome sequence of strain MEZCS99 will serve as a reference point for molecular epidemiological studies of livestock-associated C. sakazakii in Africa. In addition, this study allows in-depth analysis of the genomic structure and will provide valuable information that helps understand the pathogenesis and antimicrobial resistance of livestock-associated C. sakazakii.

Environmental risk factors associated with the survival, persistence, and thermal tolerance of Cronobacter sakazakii during the manufacture of powdered infant formula.

Cronobacter sakazakii is an opportunistic foodborne pathogen of concern for foods having low water activity such as powdered infant formula (PIF). Its survival under desiccated stress can be attributed to its ability to adapt effectively to many different environmental stresses. Due to the high risk to neonates and its sporadic outbreaks in PIF, C. sakazakii received great attention among the scientific community, food industry and health care providers. There are many extrinsic and intrinsic factors that affect C. sakazakii survival in low-moisture foods. Moreover, short- or long-term pre-exposure to sub-lethal physiological stresses which are commonly encountered in food processing environments are reported to affect the thermal resistance of C. sakazakii. Additionally, acclimation to these stresses may render C. sakazakii resistance to antibiotics and other antimicrobial agents. This article reviews the factors and the strategies responsible for the survival and persistence of C. sakazakii in PIF. Particularly, studies focused on the influence of various factors on thermal resistance, antibiotic or antimicrobial resistance, virulence potential and stress-associated gene expression are reviewed.

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

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.

Genomic Characterization of Cronobacter spp. and Salmonella spp. Strains Isolated From Powdered Infant Formula in Chile.

This study characterized five Cronobacter spp. and six Salmonella spp. strains that had been isolated from 155 samples of powdered infant formula (PIF) sold in Chile and manufactured in Chile and Mexico in 2018-2020. Two strains of Cronobacter sakazakii sequence type (ST) ST1 and ST31 (serotypes O:1 and O:2) and one strain of Cronobacter malonaticus ST60 (O:1) were identified. All Salmonella strains were identified as Salmonella Typhimurium ST19 (serotype O:4) by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST (cgMLST). The C. sakazakii and C. malonaticus isolates were resistant to cephalothin, whereas the Salmonella isolates were resistant to oxacillin and ampicillin. Nineteen antibiotic resistance genes were detected in the C. sakazakii and C. malonaticus isolates; the most prevalent were mcr-9.1, blaCSA , and blaCMA . In Salmonella, 30 genes encoding for aminoglycoside and cephalosporin resistance were identified, including aac(6')-Iaa, ß-lactamases ampH, ampC1, and marA. In the Cronobacter isolates, 32 virulence-associated genes were detected by WGS and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, invasion, plasminogen activator, colonization, transcriptional regulator, survival in macrophages, use of sialic acid, and toxin-antitoxin genes. In the Salmonella strains, 120 virulence associated genes were detected, adherence, magnesium uptake, resistance to antimicrobial peptides, secretion system, stress protein, toxin, resistance to complement killing, and eight pathogenicity islands. The C. sakazakii and C. malonaticus strains harbored I-E and I-F CRISPR-Cas systems and carried Col(pHHAD28) and IncFIB(pCTU1) plasmids, respectively. The Salmonella strains harbored type I-E CRISPR-Cas systems and carried IncFII(S) plasmids. The presence of C. sakazakii and Salmonella in PIF is a health risk for infants aged less than 6 months. For this reason, sanitary practices should be reinforced for its production and retail surveillance.

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.

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.