Relevance of genetic causes and environmental adaptation of Cronobacter spp. isolated from infant and follow-up formula production factories and retailed products in China: A 7-year period of continuous surveillance based on genome-wide analysis.

The possible contamination routes, environmental adaptation, and genetic basis of Cronobacter spp. in infant and follow-up formula production factories and retailed products in mainland China have been determined by laboratory studies and whole-genome comparative analysis in a 7-year nationwide continuous surveillance spanning from 2012 to 2018. The 2-year continuous multicenter surveillance of the production process (conducted in 2013 and 2014) revealed that the source of Cronobacter spp. in the dry-blending process was the raw dry ingredients and manufacturing environment (particularly in the vibro sieve and vacuum cleaner), while in the combined process, the main contamination source was identified as the packing room. It is important to note that, according to the contamination control knowledge obtained from the production process surveillance, the contamination rate of retail powdered infant formula (PIF) and follow-up formula (FUF) products in China decreased significantly from 2016 onward, after improving the hygiene management practices in factories. The prevalence of Cronobacter spp. in retailed PIF and FUF in China in 2018 was dramatically reduced from 1.55 % (61/3925, in 2012) to an average as low as 0.17 % (13/7655 in 2018). Phenotype determination and genomic analysis were performed on a total of 90 Cronobacter spp. isolates obtained from the surveillance. Of the 90 isolates, only two showed resistance to either cefazolin or cefoxitin. The multilocus sequence typing results revealed that C. sakazakii sequence type 1 (ST1), ST37, and C. malonaticus ST7 were the dominant sequence types (STs) collected from the production factories, while C. sakazakii ST1, ST4, ST64, and ST8 were the main STs detected in the retailed PIF and FUF nationwide. One C. sakazakii ST4 isolate (1.1 %, 1/90) had strong biofilm-forming ability and 13 isolates (14.4 %, 13/90) had weak biofilm-forming ability. Genomic analysis revealed that Cronobacter spp. have a relatively stable core-genome and an increasing pan-genome size. Plasmid IncFIB (pCTU3) was prevalent in this genus and some contained 14 antibacterial biocide- and metal-resistance genes (BMRGs) including copper, silver, and arsenic resistant genes. Plasmid IncN_1 was predicted to contain 6 ARGs. This is the first time that a multi-drug resistance IncN_1 type plasmid has been reported in Cronobacter spp. Genomic variations with respect to BMRGs, virulence genes, antimicrobial resistance genes (ARGs), and genes involved in biofilm formation were observed among strains of this genus. There were apparent differences in copies of bcsG and flgJ between the biofilm-forming group and non-biofilm-forming group, indicating that these two genes play key roles in biofilm formation. The findings of this study have improved our understanding of the contamination characteristics and genetic basis of Cronobacter spp. in PIF and FUF and their production environment in China and provide important guidance to reduce contamination with this pathogen during the production of PIF and FUF.

Assessment of the Sensitivity of Foodborne Cronobacter spp. Strains and Other Enterobacteria to Temperature Stresses and Chlorine-Containing Biocides.

We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.

Diverse Genotypes of Cronobacter spp. Associated with Dairy Farm Systems in Jiangsu and Shandong Provinces in China.

Cronobacter spp. are the most concerning foodborne pathogen in infant formula milk powder. Currently, there are many reports on the prevalence of Cronobacter spp. in infant formula milk and its processing environment, but there are few studies on the prevalence of Cronobacter spp. on dairy farms. We have, therefore, undertaken this study to investigate and track genomic epidemiology of Cronobacter spp. isolates from Chinese dairy farms in the provinces of Jiangsu and Shandong. In this study, forty Cronobacter spp. strains, consisting of thirty Cronobacter sakazakii, eight Cronobacter malonaticus, and two Cronobacter dublinensis, were obtained from 1115 dairy farm samples (raw milk, silage, bedding, and feces), with a prevalence rate of 3.57%. These isolates were classified into 10 Cronobacter serotypes and 31 sequence types (STs), including three novel STs which were isolated for the first time. Notably, pathogenic Cronobacter STs 7, 8, 17, 60, and 64, which are associated with clinical infections, were observed. Antimicrobial susceptibility testing showed that all the Cronobacter spp. were highly resistant to cephalothin and fosfomycin, which was consistent with the antimicrobial genotype. All isolates carried core virulence genes related to adherence, invasion, endotoxin, immune evasion, secretion system, and regulation. Approximately half the isolates were also able to produce a strong biofilm. Twenty-one prophages and eight plasmids were detected, with the most common prophage being Cronobacter_ENT47670 and the most common plasmid being IncFIB (pCTU1). In addition, two isolates harbored the transmissible locus of stress tolerance (tLST) which confers high environmental persistence. Phylogenetic analysis showed strong clustering by species level and sequence types. Isolates from different sources or regions with a similar genomic background suggests the cross-contamination of Cronobacter spp. The presence of diverse genotypes of Cronobacter spp. in dairy farms in Jiangsu and Shandong provinces indicates that surveillance of Cronobacter spp. on dairy farms should be strengthened, to prevent and control transmission and ensure the quality and safety of raw dairy products.

High-affinity truncated aptamers for detection of Cronobacter spp with magnetic separation-assisted DNAzyme-driven 3D DNA walker.

After optimizing the original aptamer sequence by truncation strategy, a magnetic separation-assisted DNAzyme-driven 3D DNA walker fluorescent aptasensor was developed for detecting the food-borne pathogen Cronobacter species. Iron oxide magnetic nanoparticles (MNPs) modified with a hybrid of truncated aptamer probe and DNAzyme strand (AP-E1) denoted as MNPs@AP-E1, were employed as capture probes. Simultaneously, a DNAzyme-driven 3D-DNA walker was utilized as the signal amplification element. The substrate strand (Sub) was conjugated with the gold nanoparticles (AuNPs), resulting in the formation of AuNPs@Sub, which served as a 3D walking track. In the presence of the target bacteria and Mg2+, E1-DNAzyme was activated and moved along AuNPs@Sub, continuously releasing the signal probe. Under optimized conditions, a strong linear correlation was observed for Cronobacter sakazakii (C. sakazakii) in the concentration range 101 to 106 CFU mL-1, with a low detection limit of 2 CFU mL-1. The fluorescence signal responses for different Cronobacter species exhibited insignificant differences, with a relative standard deviation of 3.6%. Moreover, the aptasensor was successfully applied to determine  C. sakazakii in real samples with recoveries of 92.86%-108.33%. Therefore, the novel method could be a good candidate for ultra-sensitive and selective detection of Cronobacter species without complex manipulation.

Group selective aptamers: Broad-spectrum recognition of target groups in Cronobacter species and implementation of electrochemical biosensors as receptors.

Aptamers are a versatile class of receptors with a high affinity and selectivity for specific targets. Although their ability to recognize individual targets has been extensively studied, some scenarios require the development of receptors capable of identifying all target groups. This study investigated the use of aptamers to achieve the broad-spectrum recognition of groups instead of individual targets. Aptamers were screened for selectively distinct groups of Cronobacter species associated with foodborne diseases. Seven Cronobacter spp. were divided into Group A (C. sakazakii, C. malonaticus, C. turicensis, and C. muytjensii) and Group B (C. dublinensis, C. condimenti, and C. universalis). Aptamers with exclusive selectivity for each group were identified, allowing binding to the species within their designated group while excluding those from the other group. The screened aptamers demonstrated reliable affinity and specificity with dissociation constants ranging from 1.3 to 399.7 nM for Group A and 4.0-24.5 nM for Group B. These aptamers have also been successfully employed as receptors in an electrochemical biosensor platform, enabling the selective detection of each group based on the corresponding aptamer (limit of detection was 7.8 and 3.2 CFU for Group A and Group B, respectively). The electrochemical sensor effectively detected the extent of infection in each group in powdered infant formula samples. This study highlights the successful screening and application of group-selective aptamers as sensing receptors, emphasizing their potential for diverse applications in different fields such as food safety, environmental monitoring, and clinical diagnostics, where the selective biosensing of target groups is crucial.

The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland.

The present study aimed to determine the genotyping diversity and hemolytic properties of 24 strains of Cronobacter spp. (15 Cronobacter sakazakii, 6 Cronobacter malonaticus, 2 Cronobacter turicensis, and 1 Cronobacter condimenti) isolated from commercial ready-to-eat leaf vegetables, sprouts, nuts, and dried fruits. The multilocus sequence typing (MLST) method was used to determine the sequence types (ST) and clonal complexes (CC) of these strains. The study demonstrated the high genotypic diversity of the Cronobacter genus bacteria isolated from plant-based foods. Five novel sequence types (804, 805, 806, 807, and 808) and the presence of novel alleles in the ppsA, gltB, gyrB, and infB loci were detected. In total, 16 of the 24 strains were assigned to the sequence types ST99, ST258, ST17, ST648, ST21, ST494, and ST98. One C. sakazakii strain (s12) isolated from alfalfa sprouts was assigned to the clonal complex CC4, which encompasses strains often associated with severe infections leading to meningitis in infants. In addition, 87.5% and 16.7% of the Cronobacter spp. strains showed ß-hemolysis of equine and sheep red blood cells, respectively. The presence of the pathogenic species C. sakazakii, C. malonaticus, and C. turicensis in ready-to-eat plant-derived food products shows they are potential sources of infection, especially to those with compromised immunity, which substantiates their further multi-faceted characterization. The significance of this study may prove useful not only in epidemiological investigations, but also in assessing the risk of infections caused by the presence of Cronobacter.

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.

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.

Modeling the UV-C Inactivation Kinetics and Determination of Fluence Required for Incremental Inactivation of Cronobacter spp.

ABSTRACT: A study was undertaken to model the UV-C inactivation kinetics and determine the fluences required for the incremental inactivation of several strains of Cronobacter spp. suspended in clear phosphate-buffered saline (PBS). In total, 13 strains of Cronobacter spp. were individually suspended in PBS and treated with UV-C doses of 0, 2, 4, 6, 8, and 10 mJ cm-2 with a collimated beam device emitting UV-C at 253.7 nm. The log reduction from each treatment was identified using the plate count method and plotted against the UV-C dose and then curve fitted using several mathematical models. The UV-C dose required for incremental inactivation of each isolate was determined using both linear and nonlinear regression. For the 13 strains tested, a UV-C dose of 10 mJ cm-2 inactivated between 3.66 ± 0.101 and 5.04 ± 0.465 log CFU mL-1. The survival behavior of all strains was best fitted to the Weibull+tail model, with correlation coefficients between 97.17 and 99.71%, and was used to determine the fluences required for incremental inactivation. The UV-C fluences needed to inactivate 1 log (D10-value) of Cronobacter spp. in buffer were between 3.53 and 5.50 mJ cm-2, whereas a fluence greater than 6.57 mJ cm-2 was required to achieve a 4-log inactivation. A clear understanding of the UV-C dose-response of several strains of Cronobacter spp. lays the foundation to design effective UV-based disinfection systems.

Identification and antibiotic resistance of Cronobacter spp. isolated from dried edible mushrooms.

Cronobacter spp. is an important foodborne pathogen that can cause life-threatening diseases in infants and immunocompromised adults. The present study was carried out to understand the prevalence and characterization of Cronobacter spp. in dried edible mushrooms in Jiangsu province, China. Cronobacter isolates were identified and genotyped by multilocus sequence typing (MLST); the antimicrobial susceptibility of Cronobacter strains was determined by the disk diffusion method; the biofilm formation ability of Cronobacter spp. was assessed using the microtiter plate method. The overall prevalence of Cronobacter spp. in dried edible mushrooms was 14.8%, with the highest contamination rate of after 37.2% found in Auricularia auricular. The Cronobacter isolates were identified as C. sakazakii (n = 26), C. malonaticus (n = 2), C. dublinensis (n = 2) and C. turicensis (n = 1). The MLST scheme produced 20 sequence types (STs), two of which were newly identified. ST148 was the most prevalent ST (n = 5), followed by ST4 (n = 3), ST17 (n = 3), ST64 (n = 3), and ST540 (n = 2). One (3.2%) and 15 (48.4%) Cronobacter isolates were resistant to tetracycline and meropenem, respectively. In contrast, all of the tested isolates were susceptible to the remaining 14 antibiotics. Moreover, 20 (64.5%) Cronobacter isolates showed weak ability to produce biofilm, but no isolates showed strong or moderate biofilm-forming ability. PRACTICAL APPLICATION: Our findings revealed a high genetic diversity of Cronobacter spp. in dried edible mushrooms and provided new epidemiological evidence for the widespread existence of Cronobacter spp. in such products. The presence of Cronobacter spp. in dried edible mushrooms may pose potential risks to human health and enhancing the hygiene of such products are necessary to ensure food safety.

Virulence and DNA sequence analysis of Cronobacter spp. isolated from infant cereals.

Cronobacter spp. is an opportunistic pathogen that causes severe infections, affecting newborns and infants, and is also an emerging cause of hospital-acquired infection in elderly populations. These infections are mainly associated with the consumption of infant formulas, even though these bacteria have been isolated from other foods as well. Cronobacter spp. invades epithelial cells and escapes the immune response mechanisms, multiplying inside macrophages. However, the pathogenesis and virulence factors of these bacteria have not been fully elucidated and need to be further studied. Therefore, this study aimed to evaluate the ability of Cronobacter spp. strains isolated from infant cereals to invade and survive within macrophages, investigate the virulence phenotype using the Galleria mellonella model, and identify possible genes involved in bacterial pathogenesis through pan-genome analysis. All the isolates were able to invade macrophages and the survival of bacteria decreased over a 72 h period, with bacterial cell counts reaching up to 106 CFU/ml. Cronobacter sakazakii isolate 112 exhibited a similar mortality rate (40-70%) to the ATCC BAA 894 strain (Cronobacter sakazakii) in G. mellonella assay. In addition, some unique virulence genes (isolate 7, ada_2, tcmA_1, acrB_3; isolate 78, ampC_2, rihC_1 and isolate 112, fimH, ylpA, gtrA) were identified within isolates with the invasive profile in the in vivo and in vitro assays. Furthermore, isolates from different species were grouped into seven distinct clusters in the pan-genome analysis. The most virulent isolates (7, 78, and 112) were grouped in distinct subclusters in the cladogram. This work revealed potential Cronobacter spp. pathogenic strains recovered from infant cereals.

Detection of Genus and Three Important Species of Cronobacter Using Novel Genus- and Species-Specific Genes Identified by Large-Scale Comparative Genomic Analysis.

The genus Cronobacter includes seven species; however, the strains of Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter turicensis were highly correlated with clinical infections. Rapid and reliable identification of these three species of Cronobacter is important in monitoring and controlling diseases caused by these bacteria. Here, we identified four pairs of novel marker genes for the Cronobacter genus, C. sakazakii, C. malonaticus, and C. turicensis based on large-scale comparative genomic analysis from 799 Cronobacter and 136,146 non-Cronobacter genomes, including 10 Franconibacter and eight Siccibacter, which are close relatives of Cronobacter. Duplex and multiplex PCR methods were established based on these newly identified marker genes. The reliability of duplex and multiplex PCR methods was validated with 74 Cronobacter and 90 non-Cronobacter strains. Strains of C. sakazakii, C. malonaticus, and C. turicensis could be detected accurately at both the genus and species level. Moreover, the newly developed methods enable us to detect 2.5 × 103 CFU/ml in pure culture. These data indicate that the accurate and sensitive established methods for Cronobacter can serve as valuable tools for the identification of these strains recovered from food, environmental, and clinical samples.

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.

Processing environment monitoring in low moisture food production facilities: Are we looking for the right microorganisms?

Processing environment monitoring is gaining increasing importance in the context of food safety management plans/HACCP programs, since past outbreaks have shown the relevance of the environment as contamination pathway, therefore requiring to ensure the safety of products. However, there are still many open questions and a lack of clarity on how to set up a meaningful program, which would provide early warnings of potential product contamination. Therefore, the current paper aims to summarize and evaluate existing scientific information on outbreaks, relevant pathogens in low moisture foods, and knowledge on indicators, including their contribution to a "clean" environment capable of limiting the spread of pathogens in dry production environments. This paper also outlines the essential elements of a processing environment monitoring program thereby supporting the design and implementation of better programs focusing on the relevant microorganisms. This guidance document is intended to help industry and regulators focus and set up targeted processing environment monitoring programs depending on their purpose, and therefore provide the essential elements needed to improve food safety.

Invited review: Stress resistance of Cronobacter spp. affecting control of its growth during food production.

Members of the Cronobacter genus include food-borne pathogens that can cause infections in infants, with a mortality rate as high as 40 to 80%. The high fatality rate of Cronobacter and its isolation from numerous types of food, especially from powdered infant formula, demonstrate the serious nature of this organism. The source tracking of Cronobacter spp. and the analysis of high-frequency species from different sources are helpful for a more targeted control. Furthermore, the persistence during food processing and storage may be attributed to strong resistance of Cronobacter spp. to environment stresses such as heat, pH, and desiccation. There are many factors that support the survival of Cronobacter spp. in harsh environments, such as some genes, regulatory systems, and biofilms. Advanced detection technology is helpful for the strict monitoring of Cronobacter spp. In addition to the traditional heat treatment, many new control techniques have been developed, and the ability to control Cronobacter spp. has been demonstrated. The control of this bacteria is required not only during manufacture, but also through the selection of packaging methods to reduce postprocessing contamination. At the same time, the effect of inactivation methods on product quality and safety must be considered. This review considers the advances in our understanding of environmental stress response in Cronobacter spp. with special emphasis on its implications in food processing.

Microbiological Quality of Nuts, Dried and Candied Fruits, Including the Prevalence of Cronobacter spp.

Cronobacter genus bacteria are food-borne pathogens. Foods contaminated with Cronobacter spp. may pose a risk to infants or immunocompromised adults. The aim of this study was to determine the microbiological quality of nuts, seeds and dried fruits with special emphasis on the occurrence of Cronobacter spp. Analyses were carried out on 64 samples of commercial nuts (20 samples), dried fruits (24), candied fruits (8), seeds (4), and mixes of seeds, dried fruits and nuts (8). The samples were tested for the total plate count of bacteria (TPC), counts of yeasts and molds, and the occurrence of Cronobacter spp. Cronobacter isolates were identified and differentiated by PCR-RFLP (Polymerase Chain Reaction - Restriction Fragments Length Polymorphism) and RAPD-PCR (Random Amplified Polymorphic DNA by PCR) analysis. TPC, and yeasts and molds were not detected in 0.1 g of 23.4%, 89.1%, and 32.8% of the analyzed samples. In the remaining samples, TPC were in the range of 1.2-5.3 log CFU g-1. The presence/absence of Cronobacter species was detected in 12 (18.8%) samples of: nuts (10 samples), and mixes (2 samples). The 12 strains of Cronobacter spp. included: C. sakazakii (3 strains), C. malonaticus (5), and C. turicensis (4). The results of this study contribute to the determination of the presence and species identification of Cronobacter spp. in products of plant origin intended for direct consumption.

Isolation, comparison of identification methods and antibiotic resistance of Cronobacter spp. in infant foods.

Cronobacter spp. are opportunistic pathogens that cause serious infections, especially in infants, elderly, and immunocompromised people. Dehydrated infant foods are the main vehicle associated with infections caused by these bacteria. Thus, this study aims to investigate the occurrence of Cronobacter spp. in 152 commercial samples of dehydrated infant formulas (77 samples) and dehydrated infant cereals (75 samples), as well as characterize the isolates. Polymerase Chain Reaction (PCR) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) methods for isolate identification were used, and their results compared. Furthermore, the susceptibility to 11 antibiotics was tested, and DNA sequencing of one isolate with multi-drug resistance was analyzed. No contamination in the infant formula samples was found, whereas 17.33% (13/75) of the infant cereal samples presented contamination with Cronobacter sakazakii. The identification results by PCR and MALDI-TOF/MS were divergent for some isolates. The antimicrobial resistance results showed a high incidence of resistance to cefazolin (94.4%) besides resistance to amoxicillin (9.45%), cefpodoxime (5.55%), streptomycin (1.35%), and trimethoprim/sulfamethoxazole (1.35%). Whole genome sequencing of one multi-drug resistant isolate showed six genes associated with antimicrobial resistance and an 82% possibility of being a human pathogen based on the presence of virulence factors. The presence of Cronobacter spp. in infant foods represents a risk for the infant's health. Moreover, the presence of a pathogenic multi-drug resistant isolate in infant's food reinforces the necessity of improving food safety policies to protect young children.

Multi-locus sequence typing and antimicrobial susceptibility profile of Cronobacter sakazakii and Cronobacter malonaticus isolated from corn-based farinaceous foods commercialized in Brazil.

The aim of this study was to evaluate the microbiological quality of 45 samples of corn-based farinaceous foods commercialized in Brazil. The bacteriological analysis performed were: detection of Salmonella and Cronobacter, and enumeration of faecal coliforms and Bacillus cereus. The Cronobacter isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene and MLST. No sample presented contamination by Salmonella or B. cereus (<102 UFC/g). Faecal coliforms were detected in two (4.4%) samples but in low concentration (≤23.0 MPN/g), and 20 samples (44.4%) contained Cronobacter. Twenty-nine unique Cronobacter isolates were identified as C. sakazakii (n = 18), C. malonaticus (n = 2); that presented 11 different fusA alleles, including new fusA 183. MLST analysis revealed 17 sequence types (STs), six of which were newly identified (ST687-690, 693, and 694). Resistance or intermediary resistance were found to ceftazidime (15.0%), aztreonam (15.0%), nalidixic acid (15.0%), nitrofurantoin (15.0%), cefepime (10.0%), gentamicin (5.0%), and tetracycline (5.0%). The presence of Cronobacter in corn-based farinaceous foods could be a significant risk to infants as these products are used as alternatives to commercially available infant formula. Strategies to manage the risk of Cronobacter infections due to the consumption of these alternative feeds need to be developed by the regulatory agencies.

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk.

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.

Prevalence and Genetic Diversity of Cronobacter Species Isolated From Four Infant Formula Production Factories in China.

The aim of this study was to investigate the prevalence and genotypic characteristics of Cronobacter isolated from powdered infant formula (PIF) manufacturing facilities and to identify a potential source of contamination. A total of 42 Cronobacter isolates (5%) were detected in 835 environmental samples collected during the surveillance study. These isolates included C. sakazakii (n = 37), C. malonaticus (n = 3), and C. turicensis (n = 2). The isolates were divided into 14 sequence types (STs) by multi-locus sequence typing (MLST) and 21 pulsotypes (PTs) using pulsed-field gel electrophoresis (PFGE). The dominant C. sakazakii sequence types were ST3 (n = 12) and ST21 (n = 10), followed by ST136 (n = 6). The major PTs were PT22 (n = 12) and PT17 (n = 4) based on 100% similarity. Strains isolated from samples collected at the same production facility showed closer phylogenetic relation than those collected from distinct facilities. The result of extensive traceback sampling showed that PIF residues (PIF dust in production areas), fluid beds, drying areas, floors, and soil samples collected adjacent to the production facilities were the primary positive areas for Cronobacter. The present study outlines an effective approach to determine prevalence and genetic diversity of Cronobacter isolates associated with contamination of PIF.