Occurrence, genetic characterization, and antibiotic susceptibility of Cronobacter spp. isolated from low water activity functional foods in Brazil.

Cronobacter spp. are bacterial pathogens isolated from a wide variety of foods. This study aims at evaluating the occurrence of Cronobacter spp. in low water activity functional food samples, detect the presence of virulence genes, and determine the antibiotic susceptibility of strains. From 105 samples, 38 (36.2%) were contaminated with Cronobacter spp. The species identified by polymerase chain reaction (PCR) and sequencing analyses (rpoB and fusA genes, respectively) were C. sakazakii (60.3%), C. dublinensis (25.4%), C. turincensis (9.5%), and C. malonaticus (4.8%). Nineteen fusA alleles were identified, including four new alleles. The virulence genes were identified by PCR and all isolates were positive for ompX and sodA genes, 60.3% to cpa gene, and 58.7% to hly gene. Using the disk diffusion method, antibiotic susceptibility to twelve antibiotics was assessed twice, separated by a 19-month period. In the first test, the isolates showed diverse antibiotic susceptibility profiles, with nineteen isolates (30.2%) being multi-drug resistant (resistant to three or more antibiotic classes), in the second, the isolates were susceptible to all antibiotics. Cronobacter spp. in functional foods demonstrates the need for continued investigation of this pathogen in foods, and further research is needed to clarify the loss of resistance of Cronobacter strains.

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.

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.

A universal mechanism on desiccation tolerance of Cronobacter based on intracellular trehalose accumulation regulated by EnvZ/OmpR.

Cronobacter (seven species) can survive in dry powdered infant formula for a long time, but the thorough molecular mechanism of resistance to desiccation remains elusive. Here we examine the regulation mechanism of Cronobacter's tolerance to desiccation by the typical two-component system (TCS) EnvZ/OmpR. When exposed to desiccation conditions, Cronobacter showed higher survival than other pathogens, as well as significantly up-regulated expression of ompR and otsAB genes with markedly decreased survival of their mutants, suggesting their relationship with desiccation tolerance. OmpR directly binds to the promoter of trehalose biosynthesis operon otsBA, significantly enhancing their expression, and boosting the trehalose levels. The ompR-deletion in other six species further confirmed its positive regulation in desiccation tolerance. Our data present a hypothesis that EnvZ/OmpR increases intracellular trehalose levels against damage to the cells, which prompts Cronobacter to survive in desiccation conditions. This study reveals a universal molecular mechanism for desiccation resistance in Cronobacter species.

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

Cronobacter malonaticus (C. malonaticus) is a food-borne pathogen inducing severe infections both in infants and adults, and it could survive in dry powdered infant formula (PIF) for a long time, implying its strong tolerance to desiccation. However, the thorough molecular mechanism of resistance to desiccation remains elusive. When C. malonaticus was exposed to desiccation conditions (7, 15, and 30 d), transcriptomic analysis provided a universal adaptation strategy to withstand desiccation with the increased compatible solutes accumulation, activated stress resistance-related regulators, suppressed protein export and bacterial secretion system, and reduced other unessential survival functions including adhesion, invasion, virulence, and flagellar motility. Importantly, type VI secretion system (T6SS) genes exhibited significantly downregulated expressions, as well as markedly increased survival and viability of their mutants after desiccation treatment, revealing the negative regulation of T6SS in desiccation tolerance. Meanwhile, the decreased expressions of T6SS structure genes in other six species further confirmed the vital role of T6SS in desiccation tolerance of Cronobacter spp. Thus, our studies present a novel hypothesis of desiccation resistance in Cronobacter based on type VI secretion system inhibition, causing the reduction of macromolecule secretion such as effectors and hyperosmolality development within the cytomembrane, which allow Cronobacter to survive in desiccation.

Large-scale genomic survey and characterization of mcr genes carried by foodborne Cronobacter isolates.

IMPORTANCE: Cronobacter is an emerging foodborne opportunistic pathogen, which can cause neonatal meningitis, bacteremia, and NEC by contaminating food. However, the entire picture of foodborne Cronobacter carriage of the mcr genes is not known. Here, we investigated the mcr genes of Cronobacter isolates by whole-genome sequencing and found 133 previously undescribed Cronobacter isolates carrying mcr genes. Further genomic analysis revealed that these mcr genes mainly belonged to the mcr-9 and mcr-10. Genomic analysis of the flanking structures of mcr genes revealed that two core flanking structures were prevalent in foodborne Cronobacter isolates, and the flanking structure carrying IS1R was found for the first time in this study.

Uncovering virulence factors in Cronobacter sakazakii: insights from genetic screening and proteomic profiling.

The increasing problem of antibiotic resistance has driven the search for virulence factors in pathogenic bacteria, which can serve as targets for the development of new antibiotics. Although whole-genome Tn5 transposon mutagenesis combined with phenotypic assays has been a widely used approach, its efficiency remains low due to labor-intensive processes. In this study, we aimed to identify specific genes and proteins associated with the virulence of Cronobacter sakazakii, a pathogenic bacterium known for causing severe infections, particularly in infants and immunocompromised individuals. By employing a combination of genetic screening, comparative proteomics, and in vivo validation using zebrafish and rat models, we rapidly screened highly virulent strains and identified two genes, rcsA and treR, as potential regulators of C. sakazakii toxicity toward zebrafish and rats. Proteomic profiling revealed upregulated proteins upon knockout of rcsA and treR, including FabH, GshA, GppA, GcvH, IhfB, RfaC, MsyB, and three unknown proteins. Knockout of their genes significantly weakened bacterial virulence, confirming their role as potential virulence factors. Our findings contribute to understanding the pathogenicity of C. sakazakii and provide insights into the development of targeted interventions and therapies against this bacterium.IMPORTANCEThe emergence of antibiotic resistance in pathogenic bacteria has become a critical global health concern, necessitating the identification of virulence factors as potential targets for the development of new antibiotics. This study addresses the limitations of conventional approaches by employing a combination of genetic screening, comparative proteomics, and in vivo validation to rapidly identify specific genes and proteins associated with the virulence of Cronobacter sakazakii, a highly pathogenic bacterium responsible for severe infections in vulnerable populations. The identification of two genes, rcsA and treR, as potential regulators of C. sakazakii toxicity toward zebrafish and rats and the proteomic profiling upon knockout of rcsA and treR provides novel insights into the mechanisms underlying bacterial virulence. The findings contribute to our understanding of C. sakazakii's pathogenicity, shed light on the regulatory pathways involved in bacterial virulence, and offer potential targets for the development of novel interventions against this highly virulent bacterium.

[The pathogenicity of Cronobacter in the light of bacterial genomics]. / La patogenicidad de Cronobacter a la luz de la genómica bacteriana.

Introduction: Cronobacter spp. is a genus of Gram-negative bacteria belonging to the family Enterobacteriaceae. Species of the genus Cronobacter, particularly C. sakazakii, are implicated in the development of severe disease in newborns, which occurs with necrotizing enterocolitis, sepsis and meningitis. The disease has been frequently associated with powdered infant formula (PIF) and can therefore occur in the form of outbreaks. The genus Cronobacter has undergone extensive diversification in the course of its evolution, with some species being clearly pathogenic to humans while the impact of other species on human health is uncertain or unknown. Whole genome sequencing is used both in population genetic studies to identify the limited number of genotypes associated with the disease and to detect genes associated with antibiotic resistance or virulence, ultimately allowing more precise epidemiological links to be established between pediatric disease and infant foods.

Introducción: Cronobacter es un género de bacterias gramnegativas perteneciente a la familia Enterobacteriaceae. Algunas especies del género Cronobacter, en particular C. sakazakii, están implicadas en el desarrollo de infecciones neonatales graves, incluyendo meningitis, sepsis y enterocolitis necrotizante. La enfermedad se ha relacionado frecuentemente con los preparados en polvo para lactantes (PPL) y se puede presentar, por tanto, en forma de brotes. El género Cronobacter ha experimentado una amplia diversificación en el curso de su evolución, siendo algunas especies claramente patógenas para los humanos mientras que el impacto de otras especies sobre la salud humana es incierto o desconocido. La secuenciación genómica se utiliza en los estudios de genética de poblaciones tanto para identificar el limitado número de genotipos asociados a la enfermedad como para detectar los genes asociados a la virulencia, la adaptación al estrés o la resistencia a antibióticos, lo que permite, en definitiva, establecer vínculos epidemiológicos más precisos entre la enfermedad pediátrica y los alimentos infantiles.

Genomic insights into Cronobacter spp. recovered from food and human clinical cases in Zhejiang province, China (2008-2021).

AIMS: Cronobacter spp. are emerging food-borne pathogens capable of causing life-threatening illness via several distinct routes. Although endeavors to reduce the incidence of Cronobacter infections are implemented, potential risk of these microorganisms on food safety remains poorly understood. Here, we evaluated the genomic features of clinical Cronobacter and the possible food reservoirs of these infections. METHODS AND RESULTS: Whole-genome sequencing (WGS) data of all human clinical cases (n = 15) during 2008-2021 in Zhejiang were used and compared to sequenced Cronobacter genomes (n = 76) representing various food products. Cronobacter strains exhibited a high degree of genetic diversity by WGS-based subtyping. A variety of serotypes (n = 12) and sequence types (n = 36) were identified, including six novel STs (ST762-ST765, ST798, and ST803) first-time described in this study. Nine clinical clusters representing 12/15 (80%) patients match a potential food source. Genomic insights into virulence genes revealed species/hosts specificity signatures associated with autochthonous populations. Resistance to streptomycin, azithromycin, sulfanilamide isoxazole, cefoxitin, amoxicillin, ampicillin, and chloramphenicol, as well as multidrug resistance, was noted. WGS data can be used to predict resistance phenotypes in amoxicillin, ampicillin, and chloramphenicol, which were extensively used in clinical treatment. CONCLUSIONS: The wide dissemination of pathogenic potential and antibiotic-resistant strains in multiple food sources emphasized the importance of rigorous food safety policies to reduce Cronobacter contamination in China.

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.

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.

The maltose transporter subunit IICB of the phosphotransferase system: An important factor for biofilm formation of Cronobacter.

Cronobacter is a common food-borne opportunistic pathogen, which is easily to form biofilm and difficult to remove. The regulation mechanism on the biofilm formation of Cronobacter has drawn more and more attention. In here, transcriptomic sequencing of free and biofilm states of Cronobacter was performed, and analyzed to identify the differential gene expression through Gene Ontology (GO) function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Based on sequencing analysis of the results, the malX gene encoding maltose transporter subunit IICB in the phosphotransferase system (PTS) might be involved in the formation of Cronobacter biofilm and thus selected for gene knockout. Hereafter, the changes in biofilm formation ability, extracellular polymer and biofilm-related gene expression of malX gene knockout strains were detected to explore the potential mechanism of malX gene on biofilm formation of Cronobacter. From the result, weaken biofilm formation ability of Cronobacter, decreased extracellular polysaccharide content and down-regulated expression of cellulose-related genes were obtained after knockout of malX gene, which verified our deduction. This study is the first to elucidates the regulation mechanism of the PTS on the biofilm formation of Cronobacter, which lays a foundation for the further prevention and control of food contamination caused by Cronobacter.

Genome sequence analysis of Cronobacter phage PF-CE2 and proposal of a new species in the genus Pseudotevenvirus.

The genome of a Cronobacter sakazakii M1 phage named PF-CE2 was characterized in this work, and a new species named "Cronobacter virus PF-CE2", in the genus Pseudotevenvirus of the subfamily Tevenvirinae of the family Myoviridae is proposed. The Gp190 gene of phage PF-CE2 is predicted to encode a bacteriophage-borne glycanase that is capable of degrading fucose-containing exopolysaccharides produced by C. sakazakii M1. Furthermore, we propose changing the taxonomic status of eight additional phages based on nucleotide sequence comparisons. This work provides a theoretical basis for subsequent heterologous expression of the phage PF-CE2 glycanase and provides an important reference for the preservation and sharing of these phages.

In silico species identification and serotyping for Cronobacter isolates by use of whole-genome sequencing data.

Cronobacter spp. are foodborne pathogens that can cause severe infections in neonates through contaminated powdered infant formula. Accurate and rapid pathogen identification and serotyping are crucial to limit the detrimental effects of bacterial infections, and to prevent outbreaks and sporadic infections. Conventional serotyping is tedious, laborious, and time-consuming; however, with whole-genome sequencing (WGS) becoming faster and cheaper, WGS has vast potential in routine typing and surveillance. Hence, in this study, we developed a publicly available tool, CroTrait (CronobacterTraits), for in silico species identification and O serotyping of Cronobacter isolates based on WGS data. CroTrait showed excellent performance in species identification and O serotyping when 810 genomes with known species identities and 276 genomes with known O serotype were tested. Moreover, CroTrait allows rapid prediction of new potential O serotypes. We identified 11 novel potential O serotypes of Cronobacter using CroTrait. Therefore, CroTrait is a convenient and promising tool for species identification and O serotyping of Cronobacter isolates.

A simple and sensitive aptasensor with rolling circle amplification for viable Cronobacter sakazakii detection in powdered infant formula.

Cronobacter sakazakii is a foodborne, emerging opportunistic pathogen that causes severe bacteremia, necrotizing enterocolitis, and sepsis with a mortality rate of up to 80%. In this study, we developed a simple and sensitive fluorescent turn-off aptasensor with rolling circle amplification assay for viable C. sakazakii detection in powdered infant formula. The results showed that the proposed aptasensor has good performance and specificity for detecting viable C. sakazakii in pure culture and powdered infant formula samples within 3 h. Under the optimal reaction conditions, there is a linear relationship between fluorescent intensity at 490 nm and logarithmic concentration of C. sakazakii in the range of 2.7 × 105 to 2.7 × 102 cfu/mL, with a limit of detection of 2.7 × 102 cfu/mL in pure culture. The proposed aptasensor achieved a recovery of 104 to 111% in pure culture, and 96 to 107% in spiked powdered infant formula samples. The proposed aptasensor does not require complicated DNA extraction steps or antibodies, and can be performed at 37°C, making it a convenient and sensitive strategy for C. sakazakii detection.

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.

Comparative genomics reveals environmental adaptation differences between Cronobacter species.

The genus Cronobacter is an opportunistic food-borne pathogen which is able to adapt to diverse environments and shows considerable genetic diversity. Genomic analysis can be used to reveal the variation across the genus with respect to virulence, drug resistance and factors involved in horizontal gene transfer mechanisms, such as integrons, conjugative plasmids, and recombinases. In this study, whole-genome comparative analysis of 27 Cronobacter genomes (12 existing and 15 newly assembled genomes) was performed. A total of 110,010 protein-coding genes were grouped into 11,262 clusters, including 2637 core genes, 4814 strain-specific genes and 3811 dispensable genes. Clusters of Orthologous Groups (COG) analysis indicated that 97.35% of the core genes were for substrate transport and metabolism, and the antibiotic resistance genetic determinants were classified into 136 antibiotic resistance ontologies (AROs). A total of 80 genomic islands (GIs) were identified which contained several type VI secretion system gene clusters, and these were likely to have been acquired by horizontal gene transfer. This study has generated a comprehensive characterization of the genus Cronobacter, leading to a better understanding of the mechanisms and ecological functions among the genome features, speciation, and environmental adaptation strategies.

Occurrence, Virulence and Antimicrobial Susceptibility Profiles of Cronobacter spp. from Ready-to-Eat Foods.

Cronobacter spp. can cause foodborne diseases in infants, but Cronobacter infections in healthy adults and vulnerable people have also been reported. These bacteria have ubiquitous nature and can contaminate various foods. Therefore, we assessed the presence of Cronobacter spp. in popularly consumed ready-to-eat (RTE) food products. In the present study, 51 (15%) of the 340 RTE food samples were contaminated with Cronobacter spp The highest contamination rates were found in spices (46.7%), meat-free cig koftes (44.4%), desserts (23.3%), cereals (23.1%), doners (12.2%), and ice cream (11.1%). Phenotypic and molecular methods, including 16S rRNA, gluA, rpoB, cgcA genes, and fusA allele sequencing were tested to identify Cronobacter species. Of the 51 contaminated samples, 54 isolates were identified as C. sakazakii (n = 43), C. malonaticus (n = 7), C. muytjensii (n = 3) and C. turicensis (n = 1) using fusA analysis. These isolates were assigned to 15 different fusA alleles, two of which (191 and 192) were new alleles. Putative virulence factors such as the ompA and zpx gene, biofilms, and siderophores were detected in most of the Cronobacter isolates (> 85%). Cronobacter isolates were resistant to cephalothin (85.2%), cefoxitin (33.3%), cefotaxime (14.8%), ampicillin (11.1%), cefepime (5.6%), aztreonam (5.6%), and piperacillin (1.9%). The multidrug resistance (against three or more classes of antimicrobial agents) was 7.4%. The results indicated presence of Cronobacter spp. in RTE foods, which may be a risk to human health. It is important to adopt rigorous hygiene and sanitization practices to ensure the microbiological safety of these foods consuming without any processing.