Advances in our understanding and distribution of the Cronobacter genus in China.

This review considers how research in China has progressed our understanding and subsequent improved control of Cronobacter. This emergent bacterial pathogen is associated with neonatal infections through the ingestion of contaminated prepared feed. The review includes large-scale surveys of various sources of the organism, including infant formula production facilities. The analysis of over 20,000 samples is presented. Over 10,000 being from powdered infant formula and other infant foods as well as environmental sampling of production facilities, the remaining being from food, food ingredients, and human carriage. A major advance in China was adopting DNA-sequence-based methods (that is, multilocus sequence typing, clustered regularly interspaced short palindromic repeats-cas array profiling, and single-nucleotide polymorphism analysis) for the identification and genotyping of the organism. These methods have considerably advanced our understanding of the taxonomy, ecology, and virulence of this organism. In turn, this has improved source tracking of the organism both in infant formula production facilities and epidemiological investigations. Furthermore, whole-genome sequencing has revealed a range of virulence and persistence mechanisms as well as plasmid-borne multidrug resistance traits. China now has reliable and robust methods for accurate microbial source tracking of Cronobacter for use both in the food production environment and epidemiological analysis.

Cytotoxicity profile of Cronobacter species isolated from food and clinical specimens in Brazil.

AIMS: The objective of this study was to evaluate the cytotoxic activity of Cronobacter strains isolated from foods (n = 50) and clinical samples (n = 6) in Brazil and genotype selected strains (n = 18) using multi-locus sequence typing (MLST) METHODS AND RESULTS: The cytotoxic activity of C. sakazakii (n = 29), C. dublinensis (n = 13), C. malonaticus (n = 6), C. turicensis (n = 6) and C. muytjensii (n = 2) was screened using Vero, RK13, Hep2c, NCTC clone 929 and BHK-21 cell lines. Selected Cronobacter strains were assigned to C. sakazakii ST 21, C. turicensis ST 252, C. sakazakii ST 647, and three newly assigned STs: C. turicensis STs 738-740. The maximum death caused by non-heat-treated filtrates was 20·4, 86·2, 47·0 and 84·0%, in Vero, RK13, Hep2c and NCTC clone 929 cells, respectively. These were caused by C. sakazakii strains C291 and C292 (ST 494) which had been isolated during neonatal Cronobacter meningitis infection, and C110 (ST 395) isolated from flaxseed flour. Thermal treatment (100°C/20 min) significantly reduced the cytotoxicity activity in NCTC clone 929 and Vero cells (P ≤ 2 × 10-6 ), but not in RK13 (P = 0·12) and Hep2c (P = 0·85), indicating the cytotoxin(s) were probably proteinaceous. Electron microscopy revealed that cytotoxic compounds from C. sakazakii induced several cell death characteristics, including loss of cell-cell contact, microvilli reduction and cellular lysis. Autophagic vacuoles and mitochondrial damage were the most common ultrastructural features observed. CONCLUSIONS: It was concluded that Cronobacter strains, especially C. sakazakii, could produce heat-labile cytotoxic compounds in cell filtrates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study providing insights into the pathogenesis of the Cronobacter genus. Cytotoxins were identified in excreted filtrates of C. sakazakii strains isolated from food and clinical specimens. The presence of Cronobacter strains that can produce cytotoxins in foods can be a potential threat to human health and highlight the need for high levels of hygiene.

Antibiotic Resistance, Virulence Factors, Phenotyping, and Genotyping of Non-Escherichia coli Enterobacterales from the Gut Microbiota of Healthy Subjects.

Non-Escherichia coli Enterobacterales (NECE) can colonize the human gut and may present virulence determinants and phenotypes that represent severe heath concerns. Most information is available for virulent NECE strains, isolated from patients with an ongoing infection, while the commensal NECE population of healthy subjects is understudied. In this study, 32 NECE strains were isolated from the feces of 20 healthy adults. 16S rRNA gene sequencing and mass spectrometry attributed the isolates to Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Enterobacter kobei, Citrobacter freundii, Citrobacter amalonaticus, Cronobacter sp., and Hafnia alvei, Morganella morganii, and Serratia liquefaciens. Multiplex PCR revealed that K. pneumoniae harbored virulence genes for adhesins (mrkD, ycfM, and kpn) and enterobactin (entB) and, in one case, also for yersiniabactin (ybtS, irp1, irp2, and fyuA). Virulence genes were less numerous in the other NECE species. Biofilm formation was spread across all the species, while curli and cellulose were mainly produced by Citrobacter and Enterobacter. Among the most common antibiotics, amoxicillin-clavulanic acid was the sole against which resistance was observed, only Klebsiella strains being susceptible. The NECE inhabiting the intestine of healthy subjects have traits that may pose a health threat, taking into account the possibility of horizontal gene transfer.

The occurrence and distribution characteristics of Cronobacter in diverse cereal kernels, flour, and flour-based products.

Cronobacter was positive in cereals at a relatively high rate. In the present study, we investigated the occurrence and characteristics of this pathogen systematically in diverse cereals. All sampled food (N = 467) contained Cronobacter with a high positive rate of 54.0%. The enumeration experiment showed the concentration ranged from 0.3 to more than 110 MPN/100 g, and 87.9% of 127 samples were less than 10 MPN/100 g. There were significant differences (p < 0.05) in positive rates for Cronobacter among cereal kernels (40.2%), cereal flour (66.7%), cereal products made from raw cereal flour (87.6%), and cereal products made from flour (ready-to-eat) (17.4%). The dominant Cronobacter species was C. sakazakii and C. dublinensis, followed by C. malonaticus and C. turicensis. Two interesting clusters with more than 90% similarities were identified by pulsed-field gel electrophoresis (PFGE). The C1 cluster (four isolates) indicated these strains were derived from a common source and persisted in the food production environment for an extended time. The C2 cluster (six isolates) indicated the pathogen could be transmitted via cereal processing. Our research provided baseline data for Cronobacter in diverse cereals and was helpful for understanding Cronobacter transmission. The results also indicate that additional control measures should be developed to reduce the risk of infection by these opportunistic pathogenic bacteria.

Characterization of the pathogenicity of clinical Cronobacter malonaticus strains based on the tissue culture investigations.

Cronobacter malonaticus is a member of the genus Cronobacter which is considered an opportunistic pathogen. The significance of C. malonaticus has recently increased since it was documented to be involved in several serious neonatal infections. However, the virulence factors of C. malonaticus including their ability to adhere, invade and overcome host barriers have not been studied before. Unlike previous Cronobacter research, this study is mainly focused on C. malonaticus and is aimed to investigate its virulence characteristics that enable this species to cause adult and neonatal infections. Altogether, 20 strains were included in this study (19 clinical and one environmental strain). Our data showed that the clinical C. malonaticus has an ability to adhere and invade Caco-2, HBMEC, A549 and T24 cell lines. Moreover, the result showed that certain strains of C. malonaticus (including 1827 and 2018) were able to persist well in macrophages. However, ST7 strains 1827 and 2018 proved to be the most invasive strains among all used strains. The CDC strain 1569 (ST307) which was isolated from the blood of a fatal neonatal case showed also significant results in this study as it was able to invade all used human cells and survive and replicate within microphages. Finally, the findings of this study confirm the potential ability of C. malonaticus to cause serious infections in neonates or adults such as necrotising enterocolitis, meningitis, bacteraemia, pneumonia and urinary tract infection.

Occurrence of virulence factors in Cronobacter sakazakii and Cronobacter malonaticus originated from clinical samples.

BACKGROUND: Cronobacter spp. are Gram-negative, facultative-anaerobic, non-spore forming, enteric coliform bacteria, which belongs to the Enterobacteriaceae family. Cronobacter spp. are opportunistic pathogens that have brought rare but life-threatening infections such as meningitis, necrotizing enterocolitis and bloodstream infections in neonates and infants. Information on the diversity, pathogenicity and virulence of Cronobacter species obtained from various sources is still relatively scarce and fragmentary. The aim of this study was to examine and analyse different pathogenicity and virulence factors among C. sakazakii and C. malonaticus strains isolated from clinical samples. METHODS: The thirty-six clinical Cronobacter strains have been used in this study. This bacterial collection consists of 25 strains of C. sakazakii and 11 strains of C. malonaticus, isolated from different clinical materials. Seven genes (ompA, inv, sip, aut, hly, fliC, cpa) were amplified by PCR. Moreover, the motility and the ability of these strains to adhere and invade human colorectal adenocarcinoma (HT-29) and mouse neuroblastoma (N1E-115) cell lines were investigated. RESULTS: Our results showed that all tested strains were able to adhere to both used cell lines, HT-29 and N1E-115 cells. The invasion assay showed that 66.7% (24/36) of isolates were able to invade N1-E115 cells while 83% (30/36) of isolates were able to invade HT-29 cells. On the average, 68% of the C. sakazakii strains exhibited seven virulence factors and only 18% in C. malonaticus. All strains amplified ompA and fliC genes. The other genes were detected as follow: sip 97% (35/36), hlyA 92% (33/36), aut 94% (34/36), cpa 67% (24/36), and inv 69% (25/36). CONCLUSIONS: C. sakazakii and C malonaticus strains demonstrate the diversity of the virulence factors present among these pathogens. It is necessary to permanently monitor the hospital environment to appropriately treat and resolve cases associated with disease. Furthermore, in-depth knowledge is needed about the source and transmission vehicles of pathogens in hospitals to adopt pertinent prevention measures.

Interaction of matrix metalloproteinase-9 and Zpx in Cronobacter turicensis LMG 23827T mediated infections in the zebrafish model.

Bacteria belonging to the genus Cronobacter have been recognized as causative agents of life-threatening systemic infections primarily in premature and low-birthweight neonates. Validation of putative bacterial virulence components as well as host factors potentially involved in the response to infection has been hampered in the past by the availability of suitable neonatal animal models. In the current study, the zebrafish embryo model was employed to study the interaction of the zinc metalloproteinase Zpx present in Cronobacter turicensis LMG 23827T , with the eukaryotic MMP-9, a proteinase that functions to cleave extracellular matrix gelatin and collagen. Cleavage and activation of the human recombinant pro-MMP-9 by zpx-expressing C. turicensis cells were demonstrated in vitro, and the presence and increase of the processed, active form of zebrafish pro-MMP-9 were shown in vivo. We provided evidence that Zpx induces the expression of the mmp-9 but also increases the levels of processed MMP-9 during infection. The involvement of the MMP-9 in induction of the expression of the bacterial Zpx was shown in zebrafish mmp-9 morphant experiments. This study identified MMP-9 as a substrate of Zpx and demonstrated yet-undescribed mutual cross-talk between these two proteases in infections mediated by C. turicensis LMG 23827T .

Phenotypic characterization of pathogenic Cronobacter spp. strains.

BACKGROUND: Cronobacter species are Gram-negative opportunistic foodborne pathogens that may cause enterocolitis, bacteremia and meningitis in neonates and premature neonates. Lipopolysaccharide (LPS) serves as the major component of the outer membrane of cell, is a potential virulence factor for Cronobacter. METHODS: Given the potential importance of this molecule in infection and virulence, SDS-PAGE of LPS, MS and TLC characterization of phospholipids and phenotypic characterization of Cronobacter spp. strains were carried out. RESULT: The phospholipids from Cronobacter yielded four major peaks at m/z 719.9, 733.9, 747.9 and 773.9 in the spectrum. All Cronobacter showed O-antigen bands except C. muytjensii ATCC 51329. When Cronobacter defect O-antigen, the outer membrane permeability and cell surface hydrophobicities are increased. All Cronobacter are able to grow under pH 5.0 condition and able to grow under 6% NaCl concentration. C. dublinensis DSM 18705 has a higher infection rate to Caco-2 cells than other Cronobacter. CONCLUSION: Invasion of pathogens into a host cell is critical component to an infectious case. And C. dublinensis DSM 18705 has a higher infection rate to Caco-2 cells than other Cronobacter.

Reconstituting the History of Cronobacter Evolution Driven by Differentiated CRISPR Activity.

Cronobacter strains harboring the CRISPR-Cas system are important foodborne pathogens causing serious neonatal infections. However, the specific role of the CRISPR-Cas system in bacterial evolution remains relatively unexplored. In this study, we investigated the impact of the CRISPR-Cas system on Cronobacter evolution and obtained 137 new whole-genome Cronobacter sequences by next-generation sequencing technology. Among the strains examined (n = 240), 90.6% (193/213) of prevalent species Cronobacter sakazakii, Cronobactermalonaticus, and Cronobacterdublinensis strains had intact CRISPR-Cas systems. Two rare species, Cronobactercondimenti (n = 2) and Cronobacteruniversalis (n = 6), lacked and preserved the CRISPR-Cas system at a low frequency (1/6), respectively. These results suggest that the presence of one CRISPR-Cas system is important for a Cronobacter species to maintain genome homeostasis for survival. The Cronobacter ancestral strain is likely to have harbored both subtype I-E and I-F CRISPR-Cas systems; during the long evolutionary process, subtype I-E was retained while subtype I-F selectively degenerated in Cronobacter species and was even lost by the major Cronobacter pathovars. Moreover, significantly higher CRISPR activity was observed in the plant-associated species Cdublinensis than in the virulence-related species C. sakazakii and Cmalonaticus Similar spacers of CRISPR arrays were rarely found among species, suggesting intensive change through adaptive acquisition and loss. Differentiated CRISPR activity appears to be the product of environmental selective pressure and might contribute to the bidirectional divergence and speciation of CronobacterIMPORTANCE This study reports the evolutionary history of Cronobacter under the selective pressure of the CRISPR-Cas system. One CRISPR-Cas system in Cronobacter is important for maintaining genome homeostasis, whereas two types of systems may be redundant and not conducive to acquiring beneficial DNA for environmental adaptation and pathogenicity. Differentiated CRISPR activity has contributed to the bidirectional divergence and genetic diversity of Cronobacter This perspective makes a significant contribution to the literature by providing new insights into CRISPR-Cas systems in general, while further expanding the roles of CRISPR beyond conferring adaptive immunity and demonstrating a link to adaptation and species divergence in a genus. Moreover, our study provides new insights into the balance between genome homeostasis and the uptake of beneficial DNA related to CRISPR-based activity in the evolution of Cronobacter.

Updates on the Cronobacter Genus.

There has been considerable concern related to Cronobacter spp. in foods, especially due to their highlighted association with neonatal infections through the ingestion of reconstituted powdered infant formula (PIF). This concern resulted in improved microbiological criteria recommendations by the Codex Alimentarius Commission and revised WHO advice on the preparation of infant feeds. In recent years, the diversity of the genus has been well described, and various detection and typing methods have been developed. This review considers our current knowledge of the genus and how DNA-sequence-based methods have contributed considerably to research into improved detection methods and more reliable identification procedures, genotyping schemes, and genomic analysis. The broader occurrence of Cronobacter in food ingredients, finished products, and food manufacturing environments is covered. This review also highlights the significance of clonal lineages in microbial source tracking and the use of CRISPR-cas array profiling.

Prevalence of curli genes among Cronobacter species and their roles in biofilm formation and cell-cell aggregation.

Cronobacter species are food-borne opportunistic pathogens that cause sepsis, meningitis, and necrotizing enterocolitis in neonates. Bacterial pathogens such as pathogenic Escherichia coli and Salmonella species express extracellular curli fimbriae that are involved in rugosity, biofilm formation, and host cell adherence. csgBAC operon encodes the major curli structural subunit CsgA and the nucleator protein CsgB. csgDEFG operon encodes the regulatory protein CsgD and putative assembly factors. In this study, the curli operons were analyzed in the sequences of 13 Cronobacter strains and other enteric bacterial pathogens. Isogenic mutants of csgA and csgB were generated in C. turicensis LMG23827 (z3032). csgA and csgB mutants did not express curli fimbriae as indicated by a lack of Congo red binding and absence of curli by electron microscopic evaluation. Compared to the wild type strain, biofilm formation and cell-cell aggregation of csgA and csgB mutants were remarkably decreased. The prevalence of curli operons were investigated in 231 Cronobacter strains isolated from different sources using polymerase chain reaction (PCR) assay. The results of the PCR analysis showed that csgA and csgG were present in 30% clinical isolates, 8% food, and 11% environmental isolates. These genes were present in C. dublinensis, C. malonaticus, C. turicensis, and C. universalis, but not in C. muytjensii and C. sakazakii. Our data indicate that csgBAC and csgDEFG operons were present about three fold higher in clinical isolates than in isolates from other sources. The csgA and csgB genes were shown to be involved in the early stages of biofilm development and cell-cell aggregation in Cronobacter.

Phenotypic characterization of Cronobacter spp. strains isolated from foods and clinical specimens in Brazil.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. This study evaluated the phenotypic characteristics of 57 Cronobacter strains (C. sakazakii n=41, C. malonaticus n=10, C. dublinensis n=4, and C. muytjensii n=2) isolated from food (n=54) and clinical specimens (n=3) in Brazil. These strains included sequence types (ST): ST395-ST398, ST402, ST413 and ST433-ST439, isolated from food samples, and three C. malonaticus clinical strains previous isolated from an outbreak which were ST394 (n=1) and ST440 (n=2). Strains were tested for capsule production, biofilm formation, protease activity, hemolytic activity, cell-cell aggregation, and desiccation resistance. Capsule formation was observed with all Cronobacter strains. Forty-four (77.2%) strains showed proteolytic activity on milk agar. All strains showed ß-hemolysis against erythrocytes from guinea pig, horse and rabbit. Using erythrocytes from sheep, the majority of strains (53/57; 92.9%) showed α-hemolysis and the remaining, ß-hemolysis. All Cronobacter strains produced weak biofilms in microtiters polystyrene plates, which were independent of temperature (4, 25 and 37°C) and/or growth conditions. In glass tubes, formation of either a moderate or strong biofilm was observed in 15/57 (26.3%), 19/57 (33.3%) and 27/57 (47.4%), at 4, 25 and 37°C, respectively. Desiccation treatment decreased Cronobacter viability by 1.55 to >3.87Log10CFU/mL. Cell-cell aggregation was observed in 17 (29.8%) strains. This study showed that the Cronobacter species evaluated showed differing phenotypes, independent of their origin (clinical or not) and ST. Further studies are necessary to elucidate the factors affecting phenotype expression. This may identify novel bacterial targets that could be useful in the development of strategies to control Cronobacter in food chain and to prevent cases of infections.

The DSF Family of Quorum Sensing Signals: Diversity, Biosynthesis, and Turnover.

The diffusible signaling factor (DSF)-based quorum sensing (QS) system has emerged as a widely conserved cell-cell communication mechanism in Gram-negative bacteria. Typically, signals from the DSF family are cis-2-unsaturated fatty acids which regulate diverse biological functions. Recently, substantial progress has been made on the characterization of new members of this family of signals. There have also been new developments in the understanding of the biosynthesis of these molecules where dual enzymatic activities of the DSF synthase and the use of various substrates have been described. The recent discovery of a naturally occurring DSF turnover mechanism and its regulation provides a new dimension in our understanding of how DSF-dependent microorganisms modulate virulence gene expression in response to changes in the surrounding environment.

Cronobacter spp.

The Cronobacter group of pathogens, associated with severe and potentially life-threatening diseases, until recently were classified as a single species, Enterobacter sakazakii. The group was reclassified in 2007 into the genus Cronobacter as a member of the Enterobacteriaceae. This chapter outlines the history behind the epidemiology, analyzes how our understanding of these bacteria has evolved, and highlights the clinical significance the Cronobacter spp. have for neonatal and elderly patient populations and treatment of the associated infections.

The DSF type quorum sensing signalling system RpfF/R regulates diverse phenotypes in the opportunistic pathogen Cronobacter.

Several bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals to control biofilm formation and virulence. Previous work showed that in Burkholderia cenocepacia the RpfFBc/RpfR system is involved in sensing and responding to DSF signals and that this signal/sensor gene pair is highly conserved in several bacterial species including Cronobacter spp. Here we show that C. turicensis LMG 23827(T) possesses a functional RpfF/R system that is involved in the regulation of various phenotypes, including colony morphology, biofilm formation and swarming motility. In vivo experiments using the zebrafish embryo model revealed a role of this regulatory system in virulence of this opportunistic pathogen. We provide evidence that the RpfF/R system modulates the intracellular c-di-GMP level of the organism, an effect that may underpin the alteration in phenotype and thus the regulated phenotypes may be a consequence thereof. This first report on an RpfF/R-type QS system of an organism outside the genus Burkholderia revealed that both the underlying molecular mechanisms as well as the regulated functions show a high degree of conservation.

[Lactobacillus rhamnosus GG inhibits Cronobacter-induced meningitis in neonatal rats].

OBJECTIVE: To investigate the inhibitory effect of Lactobacillus rhamnosus GG ( LGG) against Cronobacter-induced meningitis in neonatal rats. METHODS: The cell adhesion and invasion capacities of Cronobacter were assayed in Caco-2 cells, and the optimal time length and concentration of the bacterium for infection were determined. The suppressive effects of LGG on the adhesion and invasion of Cronobacter in caco-2 cells were tested by competitive and exclusion experiments, and its inhibitory effect against Cronobacter-induced meningitis was evaluated in neonatal rats. RESULTS: Cronobacter showed aggressive adhesion to caco-2 cells with an optimal infection time of 3 h. LGG produced a concentration-dependent inhibition of Cronobacter adhesion and invasion by competing with and excluding the latter for cell adhesion. In neonatal rats, LGG showed an obvious preventive effect and also a moderate therapeutic effect against Cronobacter-induced meningitis. CONCLUSION: LGG can inhibit Cronobacter entry across the intestinal barrier to achieve preventive and therapeutic effects against Cronobacter-induced meningitis.

Evaluation of zebrafish as a model to study the pathogenesis of the opportunistic pathogen Cronobacter turicensis.

Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827(T), a clinical isolate responsible for two fatal sepsis cases in neonates. Here, the microinjection of approximately 50 colony forming units (CFUs) into the yolk sac resulted in the rapid multiplication of bacteria and dissemination into the blood stream at 24 h post infection (hpi), followed by the development of a severe bacteremia and larval death within 3 days. In contrast, the innate immune response of the embryos was sufficiently developed to control infection after the intravenous injection of up to 10(4) CFUs of bacteria. Infection studies using an isogenic mutant devoid of surviving and replicating in human macrophages (ΔfkpA) showed that this strain was highly attenuated in its ability to kill the larvae. In addition, the suitability of the zebrafish model system to study the effectiveness of antibiotics to treat Cronobacter infections in zebrafish embryos was examined. Our data indicate that the zebrafish model represents an excellent vertebrate model to study virulence-related aspects of this opportunistic pathogen in vivo.

A Cronobacter turicensis O1 antigen-specific monoclonal antibody inhibits bacterial motility and entry into epithelial cells.

Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited.

Cronobacter: an emergent pathogen causing meningitis to neonates through their feeds.

The recognition of Cronobacter as a public health concern was raised when powdered infant formula (PIF) was linked to several neonatal meningitis outbreaks. It is an opportunistic pathogen that causes necrotising enterocolitis, infantile septicaemia, and meningitis which carries a high mortality rate among neonates. It has been also linked with cases of infection in adults and elderly. Over the past decade, much focus has been made on developing sensitive and specific characterisation, detection, and isolation methods to ascertain the quality of foods, notably contamination of PIF with Cronobacter and to understand its ability to cause disease. Whole genome sequencing has unveiled several putative virulence factors, yet the full capacity of the pathogenesis of Cronobacter has not yet been elucidated.

Cronobacter spp.--opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits.

The genus Cronobacter consists of a diverse group of Gram-negative bacilli and comprises seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti. Cronobacter are regarded as opportunistic pathogens, and have been implicated in newborn and infant infections, causing meningitis, necrotizing enterocolitis and bacteraemia or sepsis. Cronobacter virulence is believed to be due to multiple factors. Some strains were found to produce diarrhoea or cause significant fluid accumulation in suckling mice. Two iron acquisition systems (eitCBAD and iucABCD/iutA), Cronobacter plasminogen activator gene (cpa), a 17 kb type VI secretion system (T6SS), and a 27 kb filamentous haemagglutinin gene (fhaBC) and associated putative adhesins locus are harboured on a family of RepFIB-related plasmids (pESA3 and pCTU1), suggesting that these are common virulence plasmids; 98% of 229 tested Cronobacter strains possessed these plasmids. Even though pESA3 and pCTU1 share a common backbone composed of the repA gene and eitCBAD and iucABCD/iutA gene clusters, the presence of cpa, T6SS and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type and species. Other factors were observed, in that Cronobacter form biofilms, and show unusual resistance to heat, dry and acid stress growth conditions. The outer-membrane protein A is probably one of the best-characterized virulence markers of Cronobacter. Furthermore, it was reported that Cronobacter employ phosphatidylinositide 3-kinase/Akt signalling, which activates protein kinase C-α and impairs the host cell's mitogen-activated protein kinase pathway, in order to invade cells. Cronobacter can also use immature dendritic cells and macrophages to escape the immune response. This review addresses the various virulence and environmental-adaptive characteristics possessed by members of the genus Cronobacter.