Cronobacter Sakazakii Strains Research Articles

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

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

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

Cronobacter sakazakii is an opportunistic pathogen that can cause significant morbidity and mortality in neonates. Its transmission dynamics are poorly understood, though powered infant formula (PIF) is thought to be the major transmission vehicle. How the PIF becomes contaminated remains unknown. Our survey shows that roughly 1/4 of US homes are contaminated with Cronobacter sakazakii, particularly in the kitchen setting. Our analyses suggest that the domestic environment may contribute to contamination of PIF and provides insights into mitigating the risk of transmission.

Characterization of Cronobacter sakazakii and Cronobacter malonaticus Strains Isolated from Powdered Dairy Products Intended for Consumption by Adults and Older Adults.

The objective of this study was to characterize Cronobacter spp. and related organisms isolated from powder dairy products intended for consumption by adults and older adults using whole-genome sequencing (WGS), and to identify genes and traits that encode antibiotic resistance and virulence. Virulence (VGs) and antibiotic resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder, and MOB-suite tools. Susceptibility testing was performed using disk diffusion. Five presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal MLST. Three C. sakazakii strains were of the clinical pathovar ST1, one was ST31, and the remaining isolate was C. malonaticus ST60. In addition, Franconibacter helveticus ST345 was identified. The C. sakazakii ST1 strains were further distinguished using core genome MLST based on 2831 loci. Moreover, 100% of the strains were resistant to cefalotin, 75% to ampicillin, and 50% to amikacin. The C. sakazakii ST1 strains were multiresistant (MDR) to four antibiotics. Additionally, all the strains adhered to the N1E-115 cell line, and two invaded it. Eighteen ARGs mainly involved in antibiotic target alteration and antibiotic efflux were detected. Thirty VGs were detected and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, and genes involved in metabolism and stress. The pESA3, pSP291-1, and pCMA1 plasmids were detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52, and IS26. The isolates of C. sakazakii and C. malonaticus exhibited multiresistance to antibiotics, harbored genes encoding various antibiotic resistance proteins, and various virulence factors. Consequently, these contaminated powdered dairy products pose a risk to the health of hypersensitive adults.

Comparative genomics of Cronobacter sakazakii strains from a powdered infant formula plant reveals evolving populations

Cronobacter sakazakii is an opportunistic pathogen causing life-threatening infections in newborns. C. sakazakii strains (N = 26) isolated from a powdered infant formula (PIF) plant over a 15-years period were sequenced. Seventeen strains belonged to sequence type ST83 and nine to ST4. Core-genome MLST revealed clonality of most ST83 and some ST4 strains, showing that these stains derive from persistent populations. A genome comparison between the ST83 and ST4 strains identified population-specific genes, of which some are potentially involved in persistence, such as aggregation and stress-resistance genes in ST83 and fimbrial genes in ST4 strains.A single nucleotide polymorphisms comparison revealed 20 SNPs in a lactose operon. This lactose operon was shown to be transposon associated and was present in all PIF isolates, yet only in 171 of 449 public available C. sakazakii genomes, suggesting a role for lactose as evolutionary force in a PIF environment.Eighteen and 20 antimicrobial resistance genes were found in the ST83 and ST4 strain set, respectively. Four ST4 strains possessed a mcr6.1 gene acquired via HGT. encoding colistin resistance.We show that evolving C. sakazakii populations persist concomitantly in an industrial environment, which has a potential impact on public health.

Screening of Antibiotic and Virulence Genes from Whole Genome Sequenced Cronobacter sakazakii Isolated from Food and Milk-Producing Environments.

The objective of this study was to use whole-genome sequencing (WGS) to screen for genes encoding for antibiotic resistance, fitness and virulence in Cronobacter sakazakii strains that had been isolated from food and powdered-milk-producing environments. Virulence (VGs) and antibiotic-resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder and PlasmidFinder tools. Susceptibility testing was performed using disk diffusion. Fifteen presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal-MLST. Nine C. sakazakii strains were found in the meningitic pathovar ST4: two were ST83 and one was ST1. The C. sakazakii ST4 strains were further distinguished using core genome MLST based on 3678 loci. Almost all (93%) strains were resistant to cephalotin and 33% were resistant to ampicillin. In addition, 20 ARGs, mainly involved in regulatory and efflux antibiotics, were detected. Ninety-nine VGs were detected that encoded for OmpA, siderophores and genes involved in metabolism and stress. The IncFIB (pCTU3) plasmid was detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52 and ISEhe3. The C. sakazakii isolates analyzed in this study harbored ARGs and VGs, which could have contributed to their persistence in powdered-milk-producing environments, and increase the risk of infection in susceptible population groups.

"Multidimensional correlation analysis of temperature and contact time on eradication of biofilms of Cronobacter sakazakii on abiotic surfaces by combination of hypochlorite and malic acid".

In the present study, malic acid in combination with sodium hypochlorite is evaluated for eradication of biofilms formed by Cronobacter sakazakii strains individually and in a cocktail on different abiotic surfaces. The biofilm formation by five strains of C. sakazakii and their cocktail culture on different substrates was studied in Tryptone Soy Broth (TSB) and reconstituted Powdered Infant Formula (PIF). Further, the effect of temperature (4, 27, 37 and 50°C) and contact time (10, 20, 30, 40, 50 and 60min) on antibiofilm potential of test solution (0.0625mol l-1 malic acid and 0.00004mol l-1 sodium hypochlorite) against biofilm formed by C. sakazakii cocktail culture was investigated on these surfaces. The effect was evaluated in terms of viable cell count and biofilm texture using scanning electron microscopy (SEM). Principal Component Analysis (PCA) revealed that the maximum biofilm reduction was observed for stainless steel at 4°C after 60min of contact whereas at 25, 37 and 50°C, maximum biofilm reduction was observed for polycarbonate. For glass and polyurethane, maximum log reductions were observed at 50°C. The SEM images revealed cell surface deformation and disruption in biofilms after treatment with the test solution. The antibiofilm potential was observed to be greatly affected by contact time and temperature. These results indicated that the combination of malic acid NaOCl can effectively kill and remove C. sakazakii biofilms from food contact surfaces and enteral feeding tubes.

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

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

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

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. 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. 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. 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.

Characterization of Cronobacter sakazakii Strains Originating from Plant-Origin Foods Using Comparative Genomic Analyses and Zebrafish Infectivity Studies.

Cronobacter sakazakii continues to be isolated from ready-to-eat fresh and frozen produce, flours, dairy powders, cereals, nuts, and spices, in addition to the conventional sources of powdered infant formulae (PIF) and PIF production environments. To understand the sequence diversity, phylogenetic relationship, and virulence of C. sakazakii originating from plant-origin foods, comparative molecular and genomic analyses, and zebrafish infection (ZI) studies were applied to 88 strains. Whole genome sequences of the strains were generated for detailed bioinformatic analysis. PCR analysis showed that all strains possessed a pESA3-like virulence plasmid similar to reference C. sakazakii clinical strain BAA-894. Core genome analysis confirmed a shared genomic backbone with other C. sakazakii strains from food, clinical and environmental strains. Emerging nucleotide diversity in these plant-origin strains was highlighted using single nucleotide polymorphic alleles in 2000 core genes. DNA hybridization analyses using a pan-genomic microarray showed that these strains clustered according to sequence types (STs) identified by multi-locus sequence typing (MLST). PHASTER analysis identified 185 intact prophage gene clusters encompassing 22 different prophages, including three intact Cronobacter prophages: ENT47670, ENT39118, and phiES15. AMRFinderPlus analysis identified the CSA family class C β-lactamase gene in all strains and a plasmid-borne mcr-9.1 gene was identified in three strains. ZI studies showed that some plant-origin C. sakazakii display virulence comparable to clinical strains. Finding virulent plant-origin C. sakazakii possessing significant genomic features of clinically relevant STs suggests that these foods can serve as potential transmission vehicles and supports widening the scope of continued surveillance for this important foodborne pathogen.

Complete genome sequences and genomic characterization of five plasmids harbored by environmentally persistent Cronobacter sakazakii strains ST83 H322 and ST64 GK1025B obtained from powdered infant formula manufacturing facilities

BackgroundCronobacter sakazakii is a foodborne pathogen that causes septicemia, meningitis, and necrotizing enterocolitis in neonates and infants. The current research details the full genome sequences of two extremely persistent C. sakazakii strains (H322 and GK1025B) isolated from powdered infant formula (PIF) manufacturing settings. In addition, the genetic attributes associated with five plasmids, pH322_1, pH322_2, pGK1025B_1, pGK1025B_2, and pGK1025B_3 are described.Materials and MethodsUsing PacBio single-molecule real-time (SMRT®) sequencing technology, whole genome sequence (WGS) assemblies of C. sakazakii H322 [Sequence type (ST)83, clonal complex [CC] 83) and GK1025B (ST64, CC64) were generated. Plasmids, also sequenced, were aligned with phylogenetically related episomes to determine, and identify conserved and missing genomic regions.ResultsA truncated ~ 13 Kbp type 6 secretion system (T6SS) gene cluster harbored on virulence plasmids pH322_2 and pGK1025B_2, and a second large deletion (~ 6 Kbp) on pH322_2, which included genes for a tyrosine-type recombinase/integrase, a hypothetical protein, and a phospholipase D was identified. Within the T6SS of pH322_2 and pGK1025B_2, an arsenic resistance operon was identified which is in common with that of plasmids pSP291_1 and pESA3. In addition, PHASTER analysis identified an intact 96.9 Kbp Salmonella SSU5 prophage gene cluster in pH322_1 and pGK1025B_1 and showed that these two plasmids were phylogenetically related to C. sakazakii plasmids: pCS1, pCsa767a, pCsaC757b, pCsaC105731a. Plasmid pGK1025B_3 was identified as a novel conjugative Cronobacter plasmid. Furthermore, WGS analysis identified a ~ 16.4 Kbp type 4 secretion system gene cluster harbored on pGK1025B_3, which contained a phospholipase D gene, a key virulence factor in several host–pathogen diseases.ConclusionThese data provide high resolution information on C. sakazakii genomes and emphasizes the need for furthering surveillance studies to link genotype to phenotype of strains from previous investigations. These results provide baseline data necessary for future in-depth investigations of C. sakazakii that colonize PIF manufacturing facility settings and genomic analyses of these two C. sakazakii strains and five associated plasmids will contribute to a better understanding of this pathogen's survival and persistence within various “built environments” like PIF manufacturing facilities.

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.

Antibiotic Resistance and Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Infant Formula Milk.

Background: Cronobacter sakazakii is a new emerging foodborne bacterial pathogen associated with severe lethal diseases such as meningitis, necrotizing enterocolitis, and septicemia in infants and neonates. Powdered infant formula milk (PIFM) has been recognized as one of the main transmission vehicles and contaminated sources of this pathogen. This study aimed to investigate the prevalence rate, genotypic and phenotypic antibiotic resistance profile, and clonal relatedness of C. sakazakii strains isolated from 364 PIFM samples collected from Tehran city, Iran. Methods: Culture-based methods, Kirby–Bauer disk diffusion antibiotic resistance testing, conventional Polymerase Chain Reaction (PCR), and Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) assays were used in this study to detect and characterize the C. sakazakii isolates. Results: We isolated 25 C. sakazakii strains from PIFM samples (6.86%). The isolates were highly resistant to amoxicillin-clavulanic acid, amoxicillin, ampicillin, cefoxitin, cefepime, erythromycin, ceftriaxone, ciprofloxacin, and chloramphenicol and susceptible to gentamicin, tetracycline, norfloxacin, and azithromycin antibiotics. The blaCTX-M-1 gene was detected in 96% of the isolates. The isolates were categorized into eight distinct clonal types using the ERIC-PCR method, showing a high genetic diversity among the isolates. However, there was a significant correlation between the genotypic and phenotypic antibiotic resistance properties of the isolates. Conclusions: Novel microbial surveillance systems for detecting multi-drug-resistant C. sakazakii are required to control the contamination of this foodborne pathogen in infant foods.

Chemotaxis and Shorter O-Antigen Chain Length Contribute to the Strong Desiccation Tolerance of a Food-Isolated Cronobacter sakazakii Strain.

Cronobacter sakazakii is an opportunistic pathogen causing a lethality rate as high as 80% in infants. Desiccation tolerance ensures its survival in powdered infant formula (PIF) and contributes to the increased exposure to neonates, resulting in neonatal meningitis, septicemia, and necrotizing enterocolitis. This study showed that a food-isolated C. sakazakii G4023 strain exhibited a stronger desiccation tolerance than C. sakazakii ATCC 29544 strain. Considering the proven pathogenicity of G4023, it could be a big threat to infants. Transcriptome and proteome were performed to provide new insights into the desiccation adaptation mechanisms of G4023. Integrated analyses of these omics suggested that 331 genes were found regulated at both transcriptional and protein levels (≥2.0- and ≥1.5-fold, respectively). Deletion of chemotaxis system encoded genes cheA and cheW resulted in decreased tolerance in both short- and long-term desiccation. Reduced O-antigen chain length contributed to the biofilm formation and desiccation tolerance in the short term rather than the long term. In addition, biosynthesis of flagella, arginine and its transport system, and Fe/S cluster were also observed regulated in desiccated G4023. A better understanding of desiccation adaptation mechanisms of G4023 could in turn guide the operations during production and preservation of PIF or other food to reduce survival odds of G4023 and lower its exposure to get to infants.

Prevalence, characterization, and antibiotic susceptibility of Cronobacter spp. in a milk powder processing environment: The first reported case in Serbia.

Cronobacter spp. are opportunistic foodborne pathogens that most often infect neonates and infants through contaminated powdered infant formula. No reports have been published in Serbia on the prevalence of Cronobacter spp. in powdered milk production environments. Consequently, this study aimed to determine the prevalence, molecular characterization, antimicrobial susceptibility, and biofilm‐forming ability of Cronobacter spp. isolated from a milk powder plant. Hundred samples were collected from the production facility. Fifteen Cronobacter sakazakii strains were isolated and identified, giving a contamination rate of 15%. Using multi‐locus sequence typing, the isolates were divided into five sequence types (STs). Cronobacter sakazakii ST4 (50%), ST1 (16.67%), and ST83 (16.67%) were the dominant STs isolated. A novel sequence type (ST759) was identified and registered in the Cronobacter MLST database. The results of the antibiotic susceptibility testing indicated that C. sakazakii strains were susceptible to piperacillin/tazobactam, ampicillin/sulbactam, and amoxicillin/clavulanate, especially to meropenem and cefotaxime. Most of the ST4 showed moderate‐to‐strong biofilm‐forming ability. The presence of clinically relevant isolates (ST4, ST1, ST83, and ST8) revealed that the production plant is likely a potential concern for public health. Finally, finding new sequence types like the one detected in this study (ST759) underlines evolving genetic changes in C. sakazakii.

Draft Genome Sequences of Seven Cronobacter sakazakii Strains Carrying the mcr 9.1 Gene Isolated in Chile.

ABSTRACTCronobacter sakazakii is a pathogen that causes severe diseases such as meningitis and necrotizing enterocolitis in infants under 12 months, associated with the consumption of contaminated rehydrated powdered infant formula (PIF). We present seven C. sakazakii genome sequences isolated from PIF and dairy products in Chile in 2017.

Profiling the Virulence and Antibiotic Resistance Genes of Cronobacter sakazakii Strains Isolated From Powdered and Dairy Formulas by Whole-Genome Sequencing.

Cronobacter sakazakii is an enteropathogen that causes neonatal meningitis, septicemia, and necrotizing enterocolitis in preterm infants and newborns with a mortality rate of 15 to 80%. Powdered and dairy formulas (P-DF) have been implicated as major transmission vehicles and subsequently the presence of this pathogen in P-DF led to product recalls in Chile in 2017. The objective of this study was to use whole genome sequencing (WGS) and laboratory studies to characterize Cronobacter strains from the contaminated products. Seven strains were identified as C. sakazakii, and the remaining strain was Franconibacter helveticus. All C. sakazakii strains adhered to a neuroblastoma cell line, and 31 virulence genes were predicted by WGS. The antibiograms varied between strains. and included mcr-9.1 and blaCSA genes, conferring resistance to colistin and cephalothin, respectively. The C. sakazakii strains encoded I-E and I-F CRISPR-Cas systems, and carried IncFII(pECLA), Col440I, and Col(pHHAD28) plasmids. In summary, WGS enabled the identification of C. sakazakii strains and revealed multiple antibiotic resistance and virulence genes. These findings support the decision to recall the contaminated powdered and dairy formulas from the Chilean market in 2017.

Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk.

Cronobacter spp. are opportunistic pathogens of the Enterobacteriaceae family. The organism causes infections in all age groups, but the most serious cases occur in outbreaks related to neonates with meningitis and necrotizing enterocolitis. The objective was to determine the in silico and in vitro putative virulence factors of six Cronobacter sakazakii strains isolated from powdered milk (PM) in the Czech Republic. Strains were identified by MALDI-TOF MS and whole-genome sequencing (WGS). Virulence and resistance genes were detected with the Ridom SeqSphere+ software task template and the Comprehensive Antibiotic Resistance Database (CARD) platform. Adherence and invasion ability were performed using the mouse neuroblastoma (N1E-115 ATCCCRL-2263) cell line. The CRISPR-Cas system was searched with CRISPRCasFinder. Core genome MLST identified four different sequence types (ST1, ST145, ST245, and ST297) in six isolates. Strains 13755-1B and 1847 were able to adhere in 2.2 and 3.2 × 106 CFU/mL, while 0.00073% invasion frequency was detected only in strain 1847. Both strains 13755-1B and 1847 were positive for three (50.0%) and four virulence genes, respectively. The cpa gene was not detected. Twenty-eight genes were detected by WGS and grouped as flagellar or outer membrane proteins, chemotaxis, hemolysins, and invasion, plasminogen activator, colonization, transcriptional regulator, and survival in macrophages. The colistin-resistance-encoding mcr-9.1 and cephalothin-resis-encoding blaCSA genes and IncFII(pECLA) and IncFIB(pCTU3) plasmids were detected. All strains exhibited CRISPR matrices and four of them two type I-E and I-F matrices. Combined molecular methodologies improve Cronobacter spp. decision-making for health authorities to protect the population.

Expression of stress regulator and virulence genes of Cronobacter sakazakii strain Yrt2a as a response to acid stress

This research aimed to evaluate the effect of acid stress on the expression of stress regulator (grxB and rpoS) and virulence (ompA, hfq, and cpa) genes of Cronobacter sakazakii Yrt2a. The results showed that C. sakazakii Yrt2a experienced decrease in number during acid stress and was no longer culturable 90min post exposure to pH 3.0. During acid stress, the expression of grxB, rpoS, ompA, cpa and hfq was upregulated by 2.15; 2.19; 1.55; 1.1 and 1.41 log, respectively. However, all genes expression was downregulated when the bacteria entered the unculturable state. The expression of gene grxB, rpoS, ompA, cpa decreased to 1.04; 0.37; 0.84 and 1.71 log, respectively; while hfq gene expression reached a level lower than that of control. This research implies a supposition that during acid stress, C. sakazakii was capable of maintaining its culturability and pathogenicity until they are no longer culturable.

Hyperspectral imaging of common foodborne pathogens for rapid identification and differentiation.

Hyperspectral imaging (HSI) provides both spatial and spectral information of a sample by combining imaging with spectroscopy. The objective of this study was to generate hyperspectral graphs of common foodborne pathogens and to develop and validate prediction models for the classification of these pathogens. Four strains of Cronobacter sakazakii, five strains of Salmonella spp., eight strains of Escherichia coli, and one strain each of Listeria monocytogenes and Staphylococcus aureus were used in the study. Principal component analysis and kNN (k‐nearest neighbor) classifier model were used for the classification of hyperspectra of various bacterial cells, which were then validated using the cross‐validation technique. Classification accuracy of various strains within genera including C. sakazakii, Salmonella spp., and E. coli, respectively, was 100%; except within C. sakazakii, strain BAA‐894, and E. coli, strains O26, O45, and O121 had 66.67% accuracy. When all strains were studied together (irrespective of their genus) for the classification, only C. sakazakii P1, E. coli O104, O111, and O145, S. Montevideo, and L. monocytogenes had 100% classification accuracy, whereas E. coli O45 and S. Tennessee were not classified (classification accuracy of 0%). Lauric arginate treatment of C. sakazakii BAA‐894, E. coli O157, S. Senftenberg, L. monocytogenes, and S. aureus significantly affected their hyperspectral signatures, and treated cells could be differentiated from the healthy, nontreated cells.