Comparative Outer Membrane Protein Analysis of High and Low-Invasive Strains of Cronobacter malonaticus

Aldubyan, Maha A; Almami, Ibtesam S; Benslimane, Fatiha; Alsonosi, Abdlrhman; Forsythe, Stephen

doi:10.3389/fmicb.2017.02268

Cited by 19 publications

(16 citation statements)

References 44 publications

(51 reference statements)

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“…Hoeflinger and Miller [ 62 ] evaluated flagella-mediated autoaggregation and determined that the flagella play an important role in the pathogenesis of C. sakazakii . Aldubyan et al [ 63 ] established the significant role of flagella in the adherence and invasion of pathogens that contain fliC . Decreased motility has been related to the loss of the fliC gene.…”

Section: Resultsmentioning

confidence: 99%

Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk

Holý

Parra‐Flores

Lepuschitz

et al. 2020

Foods

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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.

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Section: Resultsmentioning

confidence: 99%

Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk

Holý

Parra‐Flores

Lepuschitz

et al. 2020

Foods

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“…Flagella are implicated in many mechanisms of adhesion to host cells and pathogenicity; the FliR gene is therefore instrumental in both adherence and invasion (Du et al, 2016). The fliC gene has also been strongly associated with adherence and virulence traits of the pathogens (Dingle et al, 2011; Aldubyan et al, 2017; Holý et al, 2019). The FlhA gene is implicated as required for export of flagellin and therefore assembly of flagella; it therefore has been described to play a role in invasion into and adhesion to epithelial cells by various bacteria, such as Pseudomonas aeruginosa, Bacillus thuringiensis and B. cereus and C. sakazakii (Fleiszig et al, 2001; Ramarao and Lereclus, 2006; Du et al, 2016).…”

Section: Resultsmentioning

confidence: 99%

Whole Genome Sequencing-Based Comparison of Food Isolates of Cronobacter sakazakii

et al. 2019

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Cronobacter sakazakii is an emerging foodborne pathogen, which is linked to life-threatening infections causing septicemia, meningitis, and necrotizing enterocolitis. These infections have been epidemiologically connected to ingestion of contaminated reconstituted powder infant formula. Even at low water activity C. sakazakii can survive for a long time; it is capable of protective biofilm formation and occasionally shows high virulence and pathogenicity even following stressful environmental conditions. Hence it is a challenging task for the food industry to control contamination of food ingredients and products through the entire production chain, since an increasing number of severe food-related outbreaks of C. sakazakii infections has been observed. The seemingly great capability of C. sakazakii to survive even strict countermeasures combined with its prevalence in many food ingredients requires a greater in depth understanding of its virulence factors to master the food safety issues related to this organism. In this context, we present the whole genome sequence (WGS) of two different C. sakazakii isolated from skimmed milk powder (C7) and ready-to-eat salad mix (C8), respectively. These are compared to other, already sequenced, C. sakazakii genomes. Sequencing of the fusA allele revealed that both isolates were C. sakazakii . We investigated the molecular characteristics of both isolates relevant for genes associated with pathogenesis and virulence factors, resistance to stressful environmental conditions (e.g., osmotic and heat), survival in desiccation as well as conducted a comparative genomic analysis. By using multi-locus sequence typing (MLST), the genetic type of both isolates is assessed and the number of unique genes is determined. DNA of C. sakazakii C8 is shown to hold a novel and unique sequence type; the number of unique genes identified in the genomic sequence of C. sakazakii C7 and C8 were 109 and 188, respectively. Some of the determined unique genes such as the rhs and VgrG genes are linked to the Type VI Secretion System cluster, which is associated with pathogenicity and virulence factors. Moreover, seven genes encoding for multi-drug resistance were found in both isolates. The finding of a number of genes linked to producing capsules and biofilm are likely related to the observed resistance to desiccation.

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“…In summary, the invasion frequency is dependent on the bacterial strain and the cell line [44,51,52]. Flagellar proteins expressed only in the highly invasive C. malonaticus strains maintain a strong correlation with their motility ability and significantly promote bacterial invasion [53]. Interestingly, a significant increase (p=0.0004) was observed in CS WT strain adherence to N1E-115 cells.…”

Section: Discussionmentioning

confidence: 92%

Proteomics Profile of Cronobacter sakazakii and fliF Mutant: Adherence and Invasion to Mouse Neuroblastoma Cells

Esteban-‬Kenel

Curiel-Quesada²,

Quezada

et al. 2019

Preprint

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Background: Cronobacter sakazakii is an opportunistic foodborne pathogen associated with necrotizing enterocolitis, bacteraemia, and meningitis in infants. A comparative proteomic study of C . sakazakii ATCC BAA-894 (CS WT) and isogenic mutant of flagella were performed; including the ability of both strains to adhere and invade to N1E-115 cells. Results: To achieve this goal, a non-motile C. sakazakii ATCC BAA-894 fliF ::Tn5 (CS fliF ::Tn5) strain was generated using an EZ-Tn5Tnp Transposome kit. Analysis of differential protein expression showed that 81.49% (361/443) of the proteins were identified in both strains, 8.35% (37/443) were exclusively expressed in the CS WT strain and 10.16% (45/443) in the CS fliF ::Tn5 strain. The main exclusive proteins from the CS WT strain were classified into the following subcategories: “cell motility” and “signal transduction mechanisms”. In contrast, the exclusive proteins from the CS fliF ::Tn5 strain were classified into the following subcategories: “intracellular trafficking, secretion, and vesicular transport”, “replication, recombination, and repair”, “nucleotide transport and metabolism”, “carbohydrate transport and metabolism”, “coenzyme transport and metabolism”, and “lipid transport and metabolism”. Expression of the Cpa protein was shared by both strains but was more abundant in the CS WT strain than in the CS fliF ::Tn5 strain. Significant increases (p=0.0001) in adherence to N1E-115 cells were observed for the non-motile CS fliF ::Tn5 strain, with 31.3×10 6 CFU/mL, relative to the CS WT strain, with 14.5×10 6 CFU/mL. Additionally, for infection of N1E-115 cells, the CS WT strain showed a 0.17% invasion frequency, which was significantly increased (p=0.01) compared to that of the non-motile CS fliF ::Tn5 strain. Conclusion : The proteins involved in motility were mainly identified by proteomic analysis in CS WT strain when compare to CS fliF ::Tn5 strain. Our data showed that flagella are required to promote invasion to N1E-115 cells and absence of flagella significantly increases the adherence to N1E-115 cells when compare to CS WT strain.

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Comparative Outer Membrane Protein Analysis of High and Low-Invasive Strains of Cronobacter malonaticus

Cited by 19 publications

References 44 publications

Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk

Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk

Whole Genome Sequencing-Based Comparison of Food Isolates of Cronobacter sakazakii

Proteomics Profile of Cronobacter sakazakii and fliF Mutant: Adherence and Invasion to Mouse Neuroblastoma Cells

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