Authors: Balasubramanian Chellammal Muthubharathi, Velayutham Ravichandiran, Krishnaswamy Balamurugan...

Cronobacter sakazakii, an opportunistic foodborne pathogen prevalently detected in contaminated powdered infant formula, is associated with different diseases, including meningitis. It can cross the blood-brain barrier and affects the CNS. The impact of C. sakazakii on host neuronal cells and behavior is largely unknown. Hence, detailed molecular data are required to understand its severity. Caenorhabditis elegans is a unique model for studying chemical communication, as it relies on chemosensation for searching nutritional supplements. Although, C. sakazakii is pathogenic to C. elegans, our analysis indicated that C. elegans was highly attracted toward C. sakazakii compared to its food source, E. coli OP50. To study the cue for the attraction, bioactive components (RNA/Protein/Lipopolysaccharides/Metabolites) of C. sakazakii were isolated and used for observing the chemotaxis behavior of C. elegans. The results signified that C. elegans was more attracted toward acid extracted metabolites than those of the other extraction methods. The combined action of acid extracted metabolites of C. sakazakii and a candidate pathogen drastically reduced the survival of C. elegans. In addition, analysis suggested that the exposure of isolated metabolites through acid extraction to C. elegans for 24 h modified the candidate immune regulatory genes involved in pathogen recognition and kinase activity such as , , , , , and .

Topics: Humans; Infant; Animals; Cronobacter sakazakii; Caenorhabditis elegans; Escherichia coli; Cues; Infant Formula; Cronobacter

PubMed: 36377894
DOI: 10.1128/iai.00281-22

Authors: Aboubaker M Garbaj, Samira A Farag, Jihan A Sherif...

BACKGROUND

sspecies are the most significant foodborne pathogen in infant milk formula (IMF). These pathogens have been incriminated in severe forms of neonatal meningitis, sepsis, and necrotizing enterocolitis with a high mortality rate.

AIM

This study was performed to elucidate the effect of heat stress on spp. ( and ) in reconstituted IMF (RIMF).

METHODS

The reconstituted formula was inoculated with five isolates and four isolates separately. The nine isolates of spp. were heated in RIMF at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C. The - and -values were determined by using linear regression analysis.

RESULTS

The -values of all isolates of (CS1, CS3, CS4, CS5, and CS6) at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C were in the ranges 7.29-23.47, 2.77-15.50, 0.62-1.04, 0.62-1.02, 0.62-1.00, 0.62-1.00 minutes, respectively; while, the -values extended from 2.50°C to 4.28°C. The - values of isolates (CP1, CP2, CP3, CP4) were in the ranges 7.60-22.32, 1.42-8.45, 0.62-1.08, 0.62-0.78, 0.62-0.78, 0.62-0.79 minutes at 48°C, 52°C, 56°C, 60°C, 64°C, 66°C, respectively and the calculated -values ranged from 3.33°C to 4.89°C.

CONCLUSION

This study may contribute to improving the understanding of the behavior of and isolates in RIMF at various heat stress temperatures and may participate in the effective control of these pathogens in infant food production.

Topics: Infant Formula; Food Microbiology; Enterobacteriaceae; Animals; Cronobacter sakazakii; Milk

PubMed: 36777432
DOI: 10.5455/OVJ.2023.v13.i1.11

Review

Authors: Esteban-Kenel Veronica, Ochoa Sara A, Curiel-Quesada Everardo...

Cronobacter sakazakii is an opportunistic foodborne pathogen associated with necrotizing enterocolitis, bacteremia, and meningitis in infants. A comparative proteomic study of C. sakazakii ATCC BAA-894 (CS WT) and a fliF::Tn5 mutant was performed, including the ability of both strains to adhere to and invade N1E-115 cells. To achieve this goal, a nonmotile C. sakazakii‬ ATCC BAA-894 fliF::Tn5 (CS fliF::Tn5) strain was generated using an EZ-Tn5 Tnp Transposome kit. Analysis of differential protein expression showed that 81.49% (361/443) of the proteins were expressed in both strains, 8.35% (37/443) were exclusively expressed in the CS WT strain, and 10.16% (45/443) were exclusively expressed in the CS fliF::Tn5 strain. The main exclusively expressed proteins in the CS WT strain were classified into the "cell motility" and "signal transduction mechanisms" subcategories. The proteins exclusively expressed in 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 detected in both strains, but Cpa was more abundant in the CS WT strain than in the CS fliF::Tn5 strain. A significant increase (p = 0.0001) in adherence to N1E-115 cells was observed in the nonmotile CS fliF::Tn5 strain (31.3 × 10 CFU/mL) compared to the CS WT strain (14.5 × 10 CFU/mL). Additionally, the CS WT strain showed a 0.17% invasion frequency in N1E-115 cells, which was significantly higher (p = 0.01) than that of the nonmotile CS fliF::Tn5 strain. In conclusion, the proteins involved in the motility were mainly identified by proteomic analysis in the CS WT strain compared to the CS fliF::Tn5 strain. Our data indicate that flagella are required to promote the invasion of N1E-115 cells and that the absence of flagella significantly increases the adherence to N1E-115 cells.

Topics: Animals; Cronobacter sakazakii; Mice; Neuroblastoma; Proteomics

PubMed: 33157215
DOI: 10.1016/j.micpath.2020.104595

Authors: Haoran Wang, Yulu Li, Zhuo Li...

essential oil (LC-EO) has anti-insecticidal, antioxidant, and anticancer proper-ties; however, its antimicrobial activity toward has not yet been researched extensively. The objective of this study was to investigate the antimicrobial and antibiofilm effects of LC-EO toward , along with the underlying mechanisms. The minimum inhibitory concentrations of LC-EO toward eight different strains ranged from 1.5 to 4.0 μL/mL, and LC-EO exposure showed a longer lag phase and lower specific growth compared to untreated bacteria. LC-EO increased reactive oxygen species production, decreased the integrity of the cell membrane, caused cell membrane depolarization, and decreased the ATP concentration in the cell, showing that LC-EO caused cellular damage associated with membrane permeability. LC-EO induced morphological changes in the cells. LC-EO inhibited in reconstituted infant milk formula at 50 °C, and showed effective inactivation of biofilms on stainless steel surfaces. Confocal laser scanning and attenuated total reflection-Fourier-transform infrared spectrometry indicated that the biofilms were disrupted by LC-EO. These findings suggest a potential for applying LC-EO in the prevention and control of in the dairy industry as a natural antimicrobial and antibiofilm agent.

PubMed: 36496708
DOI: 10.3390/foods11233900

Authors: Babak Pakbin, Wolfram Manuel Brück, Samaneh Allahyari...

BACKGROUND

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 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 isolates.

RESULTS

We isolated 25 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 are required to control the contamination of this foodborne pathogen in infant foods.

PubMed: 35454680
DOI: 10.3390/foods11081093

Authors: Xi Chen, Juan Xue, Xiaoli Dong...

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 , a pathogenic bacterium known for causing severe infections, particularly in infants and immunocompromised individuals. By employing a combination of genetic screening, comparative proteomics, and validation using zebrafish and rat models, we rapidly screened highly virulent strains and identified two genes, and , as potential regulators of toxicity toward zebrafish and rats. Proteomic profiling revealed upregulated proteins upon knockout of and , 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 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 validation to rapidly identify specific genes and proteins associated with the virulence of , a highly pathogenic bacterium responsible for severe infections in vulnerable populations. The identification of two genes, and , as potential regulators of toxicity toward zebrafish and rats and the proteomic profiling upon knockout of and provides novel insights into the mechanisms underlying bacterial virulence. The findings contribute to our understanding of '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.

Topics: Humans; Infant; Rats; Animals; Virulence Factors; Cronobacter sakazakii; Zebrafish; Proteomics; Enterobacteriaceae Infections; Anti-Bacterial Agents; Genetic Testing; Cronobacter

PubMed: 37750707
DOI: 10.1128/aem.01028-23

Authors: Yuan-Song Zhang, Lei Yuan, Fedrick C Mgomi...

Cronobacter sakazakii, a foodborne pathogen, can contaminate powdered infant formula (PIF) and cause life-threatening meningitis, necrotizing colitis and meningoencephalitis in infants. Bacteriophages are increasingly considered an efficient approach to target pathogenic microorganisms. In the current study, four virulent phages that can infect C. sakazakii were isolated from sewage samples, and their biological and complete genomic characteristics were analyzed. A comparative genomic analysis was performed to investigate the functional genes and phylogenetic evolution of the four phages. The results revealed that all four phages belonged to the Ackermannviridae family. Notably, the viral burst size of the phages ranged from 10 to 250 PFU/cell, following a latent period of 5 min to 20 min. Moreover, phages were stable over a pH range of 4 to 10 and a temperature range of 50 ℃ to 60 ℃. The full length of the complete genomes of the four phages ranged from 41,929 bp to 146,806 bp, containing lysis genes but no virulence genes. Phylogenetic tree analysis showed that the four phages were members of two distinct genetic groups with a significant genetic evolutionary distance between each C. sakazakii phage. Furthermore, the antibacterial assay revealed that all phages could inhibit the growth of C. sakazakii for up to 24 h. Taken together, the four phages have huge prospects as additives in dairy products to counter C. sakazakii.

Topics: Infant; Humans; Bacteriophages; Cronobacter sakazakii; Phylogeny; Genomics; Genome, Viral

PubMed: 36963724
DOI: 10.1016/j.virusres.2023.199102

Review

Authors: Audrey Feeney, Kai A Kropp, Roxana O'Connor...

A characteristic feature of the opportunistic foodborne pathogen Cronobacter sakazakii is its ability to survive in extremely arid environments, such as powdered infant formula, making it a dangerous opportunistic pathogen of individuals of all age groups, especially infants and neonates. Herein, we provide a brief overview of the pathogen; clinical manifestations, environmental reservoirs and our current understanding of stress response mechanisms and virulence factors which allow it to cause disease.

Topics: Animals; Cronobacter sakazakii; Enterobacteriaceae Infections; Foodborne Diseases; Humans; Microbial Viability; Virulence

PubMed: 25562731
DOI: 10.4161/19490976.2014.983774

Authors: Haiyan Zeng, Chengsi Li, Wenjing He...

strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing , however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of , , and were used to verify the feasibility of the method. Results showed that 161 strains could be divided into 129 CRISPR types (CTs), among which CT15 ( = 7) was the most prevalent CT followed by CT6 ( = 4). Further, 65 strains were divided into 42 CTs and CT23 ( = 8) was the most prevalent followed by CT2, CT3, and CT13 ( = 4). Finally, 31 strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of food-borne outbreaks, from clinical cases to food sources and the production sites.

PubMed: 31555228
DOI: 10.3389/fmicb.2019.01989

Authors: Jiří Novotný, Barbora Svobodová, Jiří Šantrůček...

Members of the genus are responsible for severe infections in infants and immunosuppressed individuals. Although several virulence factors have been described, many proteins involved in the pathogenesis of such infections have not yet been mapped. This study is the first to fractionate Cronobacter sakazakii cells into outer membrane, inner membrane, periplasmic, and cytosolic fractions as the basis for improved proteome mapping. A novel method was designed to prepare the fractionated samples for protein identification. The identification was performed via one-dimensional electrophoresis-liquid chromatography electrospray ionization tandem mass spectrometry. To determine the subcellular localization of the identified proteins, we developed a novel Python-based script (Subcelloc) that combines three web-based tools, PSORTb 3.0.2, CELLO 2.5, and UniProtKB. Applying this approach enabled us to identify 1,243 C. sakazakii proteins, which constitutes 28% of all predicted proteins and 49% of all theoretically expressed outer membrane proteins. These results represent a significant improvement on previous attempts to map the C. sakazakii proteome and could provide a major step forward in the identification of virulence factors. spp. are opportunistic pathogens that can cause rare and, in many cases, life-threatening infections, such as meningitis, necrotizing enterocolitis, and sepsis. Such infections are mainly linked to the consumption of contaminated powdered infant formula, with Cronobacter sakazakii clonal complex 4 considered the most frequent agent of serious neonatal infection. However, the pathogenesis of diseases caused by these bacteria remains unclear; in particular, the proteins involved throughout the process have not yet been mapped. To help address this, we present an improved method for proteome mapping that emphasizes the isolation and identification of membrane proteins. Specific focus was placed on the identification of the outer membrane proteins, which, being exposed to the surface of the bacterium, directly participate in host-pathogen interaction.

Topics: Bacterial Outer Membrane Proteins; Cronobacter; Cronobacter sakazakii; Food Microbiology; Humans; Infant; Infant Formula; Infant, Newborn; Proteome; Proteomics; Virulence Factors

PubMed: 35435719
DOI: 10.1128/aem.02508-21