Surveillance and characterisation of Cronobacter spp. in Czech retail food and environmental samples

Cronobacter spp. (formerly Enterobacter sakazakii) are emerging, opportunistic pathogens that are linked with food-borne infections in neonates and infants. In the present study, 291 samples of food, 36 samples from a dairy farm and 140 samples of dust from vacuum cleaners were examined for the presence of Cronobacter spp. using chromogenic media and biochemical tests. Altogether, 72 Cronobacter spp. strains were isolated in accordance with the reference standard ?SN P ISO/TS 22964 (2006). No Cronobacter spp. strains were detected in 10 samples of infant milk formula or in samples from a dairy farm. Twelve out of 20 positive food samples were dry products. The incidence of Cronobacter spp. in instant and powdered products and spices (12 positive isolates out of 82 samples) was significantly higher than that in other foods (P?=?0.002), but lower than that in samples of dust (52 isolates; P?<?0.001). The incidence of Cronobacter spp. in dust from restaurants, bars and hotels (13 positive isolates in 20 samples) was significantly higher than that in dust from households (P?=?0.010). The polymerase chain reaction assay for the species-specific detection of the rpoB gene was performed in 49 isolates. Thirty-four Cronobacter spp. isolates were identified as Cronobacter sakazakii, nine isolates as Cronobacter malonaticus and one isolate as Cronobacter turicensis.     

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product.     

Biochemical and molecular characterization of <Emphasis Type="Italic">Cronobacter</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> (formerly <Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>) isolated from foods

The aim of this study was to identify and characterize Cronobacter spp. isolated from a range of foods. A total of 71 Cronobacter strains were isolated from 602 foods in our laboratory. The highest contamination was observed in foods of plant origin, e.g. spices, teas, chocolate, nuts, pastries and vegetables. On the basis of genus and species identification performed using genus-specific PCR, 16S rRNA sequencing and AFLP genotyping, most of the strains belonged to Cronobacter sakazakii. Biochemical profiling by the tests included in API 20E, complemented with relevant additional tests, classified the strains into 13 biogroups. AFLP genotyping facilitated discrimination of six main groups at the 70% similarity level and strain grouping correlated clearly with species identification. Our results indicate that molecular typing by AFLP may be applied as a useful tool not only for direct comparison of Cronobacter isolates, providing traceability, but also for the reliable species classification. Moreover, tracing of these bacteria in a wider variety of foods should be important to enhance the knowledge of their transmission.     

The Genotypic Characterization of Cronobacter spp. Isolated in China

Cronobacter spp. (Enterobacter sakazakii) is an important pathogen contaminating powdered infant formula (PIF). To describe the genotypic diversity of Cronobacter isolated in China, we identified the isolates using fusA allele sequencing, and subtyped all of the isolates using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multiple-locus variable-number tandem-repeat analysis (MLVA). A total of 105 isolates were identified, which included C. sakazakii (58 isolates), C. malonaticus (30 isolates), C. dublinensis (11 isolates), C. turicensis (5 isolates), and C. muytjensii (1 isolate). These isolates were showed to have 85 PFGE-patterns, 71 sequence types (STs), and 55 MLVA-patterns. Comparisons among the three molecular subtyping methods revealed that the PFGE method was the most distinguishable tool in identifying clusters of Cronobacter spp. through DNA fingerprinting, and MLST method came second. However, ESTR-1, ESTR-2, ESTR-3, and ESTR-4 were not effective loci for subtyping Cronobacter spp. such that the MLVA method requires further improvement.     

Genome Sequence of Cronobacter sakazakii BAA-894 and Comparative Genomic Hybridization Analysis with Other Cronobacter Species

Background

The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified.

Methodology/Principal Findings

We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10–17% absence of genes.

Conclusions/Significance

CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.     

Microbial contamination of food products consumed by infants and babies in Korea

Aims: The objectives of this study were to investigate the microbiological safety of various foods intended for consumption by infants and babies. Methods and Results: The incidence of Cronobacter spp. and Enterobacteriaceae from powdered infant formula (PIF, n = 75) and baby soy milk (n = 10) was examined. Additionally, aerobic plate count, coliforms and the prevalence of foodborne pathogens were investigated in 230 samples from a variety of infant and baby foods, including cereal‐based follow‐up formulas (FUF), liquid FUF and other infant foods. High APCs were observed in nutrient supplements and cereal‐based FUF. Coliforms were found in 6 (2·6%) products, and Cronobacter spp. was isolated in 10 (4·4%) samples, including four PIF and six cereal‐based FUF. Bacillus cereus was detected in 48 (20·9%) samples: cereal‐based FUF items (23·0%), rice soups (20·6%), honey samples (40·0%), biscuits (40·0%) and liquid FUF (7·4%). Conclusions: New safety criteria, along with hygienic control measures and consumer education strategies, are essential to improve the microbiological safety of infant or baby foods. Significance and Impact of the Study: This study provides comprehensive information about the prevalence and level of contamination of infant and baby food products by Cronobacter spp. and other major foodborne pathogens.     

Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes

Background  

The Cronobacter genus (Enterobacter sakazakii) has come to prominence due to its association with infant infections, and the ingestion of contaminated reconstituted infant formula. C. sakazakii and C. malonaticus are closely related, and are defined according their biotype. Due to the ubiquitous nature of the organism, and the high severity of infection for the immunocompromised, a multilocus sequence typing (MLST) scheme has been developed for the fast and reliable identification and discrimination of C. sakazakii and C. malonaticus strains. It was applied to 60 strains of C. sakazakii and 16 strains of C. malonaticus, including the index strains used to define the biotypes. The strains were from clinical and non-clinical sources between 1951 and 2008 in USA, Canada, Europe, New Zealand and the Far East.     

Genes Involved in Cronobacter sakazakii Biofilm Formation

Cronobacter spp. are opportunistic food-borne pathogens that can cause severe and sometimes lethal infections in neonates. In some outbreaks, the sources of infection were traced to contaminated powdered infant formula (PIF) or contaminated utensils used for PIF reconstitution. In this study, we investigated biofilm formation in Cronobacter sakazakii strain ES5. To investigate the genetic basis of biofilm formation in Cronobacter on abiotic surfaces, we screened a library of random transposon mutants of strain ES5 for reduced biofilm formation using a polystyrene microtiter assay. Genetic characterization of the mutants led to identification of genes that are associated with cellulose biosynthesis and flagellar structure and biosynthesis and genes involved in basic cellular processes and virulence, as well as several genes whose functions are currently unknown. In two of the mutants, hypothetical proteins ESA_00281 and ESA_00282 had a strong impact on flow cell biofilm architecture, and their contribution to biofilm formation was confirmed by genetic complementation. In addition, adhesion of selected biofilm formation mutants to Caco-2 intestinal epithelial cells was investigated. Our findings suggest that flagella and hypothetical proteins ESA_00281 and ESA_00282, but not cellulose, contribute to adhesion of Cronobacter to this biotic surface.Biofilms are interface-associated consortia of microorganisms that are typically embedded in an endogenous slimy matrix referred to as extracellular polymeric substance (EPS). It is generally accepted that growth as a biofilm is the predominant microbial lifestyle in nature. Biofilms have several phenotypic characteristics that clearly set them apart from planktonic cultures, most notably increased resistance to a variety of environmental influences (16), which makes their eradication more difficult. Microbial biofilms are of special concern to the food industry, as biofilms on raw materials or food contact surfaces represent possible sources of product contamination with spoilage or pathogenic microorganisms (for a recent review, see reference 4).Cronobacter spp. are opportunistic food-borne pathogens that can cause severe disease in neonates which may present as septicemia, meningitis, or necrotizing enterocolitis (NEC). In several outbreaks, the source of infection was traced to contaminated powdered infant formula (PIF) or to spoons and blenders used in preparation of PIF (8, 10). The genus Cronobacter currently comprises six species: Cronobacter sakazakii, Cronobacter dublinensis, Cronobacter turicensis, Cronobacter malonaticus, Cronobacter muytjensii, and Cronobacter genomospecies 1 (20). Cronobacter spp. display remarkable resistance to desiccation compared to other Enterobacteriaceae (7), which may contribute to their long-term survival in PIF and on surfaces. Few studies of biofilm formation by Cronobacter spp. have been conducted so far. It has been observed that some strains are able to form biofilms on glass, stainless steel, polyvinyl chloride (PVC), polycarbonate, silicone, and enteral feeding tubes in different media (19, 24, 28). Like biofilm formation in other bacteria, biofilm formation is different for different strains and is highly dependent on the medium and surface used. Furthermore, the survival of C. sakazakii in biofilms under different environmental conditions has been investigated (23), and increased resistance of Cronobacter biofilms to disinfectants has been demonstrated (25). Cellulose has been described as a component of the Cronobacter extracellular matrix (15, 28, 51).In this study, we performed a genetic analysis of biofilm formation by Cronobacter sakazakii strain ES5, a clinical isolate, by using random transposon mutagenesis and subsequent screening of a mutant library for altered biofilm phenotype using a microtiter assay system. In addition, the biofilm structure of the wild type and selected mutants in a continuous-culture flow cell system was investigated by using confocal laser scanning microscopy (CLSM). Finally, we tested whether for selected mutants the defects in biofilm formation observed on the abiotic surface had an influence on the capacity of C. sakazakii to adhere to Caco-2 intestinal epithelial cells.     

Development of resistance in Cronobacter sakazakii ATCC 29544 to thermal and nonthermal processes after exposure to stressing environmental conditions

Aims: The objective was to study the response of Cronobacter sakazakii ATCC 29544 cells to heat, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) and ultraviolet light (UV‐C) treatments after exposure to different sublethal stresses that may be encountered in food‐processing environments. Methods and Results: Cronobacter sakazakii stationary growth‐phase cells (30°C, 24 h) were exposed to acid (pH 4·5, 1 h), alkaline (pH 9·0, 1 h), osmotic (5% NaCl, 1 h), oxidative (0·5 mmol l^?1 H₂O₂, 1 h), heat (47·5°C, 1 h) and cold (4°C, 4 h) stress conditions and subjected to the subsequent challenges: heat (60°C), PEF (25 kV cm^?1, 35°C), MS (117 μm, 200 kPa, 35°C) and UV‐C light (88·55 mW cm^?2, 25°C) treatments. The inactivation kinetics of C. sakazakii by the different technologies did not change after exposure to any of the stresses. The combinations of sublethal stress and lethal treatment that were protective were: heat shock–heat, heat shock–PEF and acid pH–PEF. Conversely, the alkaline shock sensitized the cells to heat and UV‐C treatments, the osmotic shock to heat treatments and the oxidative shock to UV‐C treatments. The maximum adaptive response was observed when heat‐shocked cells were subjected to a heat treatment, increasing the time to inactivate 99·9% of the population by 1·6 times. Conclusions: Cronobacter sakazakii resistance to thermal and nonthermal preservation technologies can increase or decrease as a consequence of previous exposure to stressing conditions. Significance and Impact of the Study: The results help in understanding the physiology of the resistance of this emerging pathogen to traditional and novel preservation technologies.     

Analysis of the DNA region mediating increased thermotolerance at 58°C in <Emphasis Type="Italic">Cronobacter</Emphasis> sp. and other enterobacterial strains

Cronobacter spp. are opportunistic pathogens associated with serious infections in neonates. The increased stress tolerance, including thermoresistance, of some Cronobacter strains can promote their survival in production facilities and thus raise the possibility of contamination of dried infant milk formula, which has been identified as a potential source of infection. In this study, we characterized a DNA region which is present in some Cronobacter strains and which contributes to their prolonged survival at 58°C. The 18 kbp long region containing 22 open reading frames was sequenced in Cronobacter sakazakii ATCC 29544. The major feature of the region contained a cluster of conserved genes, most of them having significant homologies with bacterial proteins involved in some type of stress response, including heat, oxidation and acid stress. The same thermoresistance DNA region was detected in strains belonging to the genera Cronobacter, Enterobacter, Citrobacter and Escherichia and its presence positively correlated with increased thermotolerance.     

Stress tolerant virulent strains of Cronobacter sakazakii from food

Background

Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh.

Result

Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress.

Conclusion

Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-63) contains supplementary material, which is available to authorized users.     

Prevalence and Characterization of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. from Various Foods,Medicinal Plants,and Environmental Samples

Dairy or non-dairy based products were explored to determine the prevalence, molecular characterization, and antibiotic susceptibility of Cronobacter spp. The isolation was done as per ISO 22964:2006 on chromogenic media followed by further confirmation by biochemical- and 16S rRNA-based identification. From 219 samples, the chromogenic agar assay and biochemical tests yielded presumptive 45 isolates. Among them, only 36 isolates showed 282 bp band amplified from ITS-G gene confirming as Cronobacter sakazakii. The Cronobacter spp. prevalence was highest in herbs and spices (34 %) while environmental samples had contamination rates of 23 % indicating plants as a possible reservoir of this pathogen. All the isolates were resistant to β-lactam derivatives (68 %), macrolides (88.6 %), and aminoglycosides (79.9 %) but susceptible to phenicoles (31.6 %) and tetracyclines (15 %) derivatives. The results emphasize the screening of plant materials before their incorporation in food matrices.     

A new cost-effective, selective and differential medium for the isolation of Cronobacter spp

The objective of the present study was to develop a new selective, differential and cost-effective medium (Kim and Rhee — KR-medium) for the isolation of Cronobacter spp. In this new medium, which contained salicin as a differential agent, Cronobacter spp. generated typical colonies with characteristic violet-colored centers surrounded by a transparent to opalescent border, and the growth of other microorganisms (40 strains) was inhibited or produced visually distinguishable colonies. Using healthy and heat- and desiccation-injured cells, the quantity of nutrients was adjusted to determine the optimal recovery rate, selectivity, differentiation and cost-effectiveness. Peptone and salicin concentrations were established as 10 and 8 g/L, respectively. The KR medium was then validated using salicin fermenting organisms, including Cronobacter spp. (52 strains), Enterobacter cloacae (50 strains) and Klebsiella pneumonia (10 strains) isolated from clinical and food specimens. All strains of Cronobacter spp. produced typical colonies and other salicin fermenting organisms were easily distinguishable from Cronobacter spp. with the exception of 2 E. cloacae strains. The verification of KR medium was carried out in powdered infant formula artificially inoculated with healthy, heat-injured, and desiccation-injured Cronobacter spp. and the expected typical colonies were appeared. The KR medium had a high specificity (98%) and sensitivity (100%), with no false-negative results. Moreover, we show that the cost of the KR medium is much lower than that of other selective and differential media. The use of the KR medium for the selective isolation of Cronobacter spp. in laboratories and food industry settings may therefore lessen the financial burden of Cronobacter spp. detection.     

Development of a loop-mediated isothermal amplification assay for detection of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>)

Contamination of Cronobacter spp. (Enterobacter sakazakii) in infant formulas and other food products is a severe problem. Here a loop-mediated isothermal amplification (LAMP) assay was developed for rapidly detecting Cronobacter spp. in powdered infant formula. Sequences of 16S/23S rDNA internal intergenic spacer of Cronobacter spp. were used as the target template to design LAMP primers. The detection outcome can be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay was further analyzed in comparison with that of regular PCR and real time quantitative PCR. The results showed that all of Cronobacter spp. strains display positive reaction to the detections while all of the non-Cronobacter spp. strains were negative, and that the LAMP assay exhibits a high sensitivity of 9.1 fg/μL (The sensitivity of regular PCR and real time quantitative PCR is 91 and 9.1 pg/μL, respectively.). The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for the detection of Cronobacter spp. in infant formula.     

Isolation of Cronobacter spp. (formerly Enterobacter sakazakii) from infant food, herbs and environmental samples and the subsequent identification and confirmation of the isolates using biochemical, chromogenic assays, PCR and 16S rRNA sequencing

Background  

Cronobacter spp. (formerly Enterobacter sakazakii), are a group of Gram-negative pathogens that have been implicated as causative agents of meningitis and necrotizing enterocolitis in infants. The pathogens are linked to infant formula; however, they have also been isolated from a wide range of foods and environmental samples.     

Structures and genetics of Kdo-containing O-antigens of Cronobacter sakazakii G2706 and G2704, the reference strains of serotypes O5 and O6

Cronobacter sakazakii G2706 and G2704 are the reference strains of serotypes O5 and O6 in the serological classification of this species proposed recently. Mild acid degradation of the lipopolysaccharides of both strains resulted in cleavage of the O-polysaccharide chains at the acid-labile linkage of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to yield oligosaccharides representing repeating units of the O-polysaccharides. The oligosaccharides and alkali-degraded lipopolysaccharides were studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy, and the following O-polysaccharide structures were established:The structure of strain G2706 is unique among the known bacterial polysaccharide structures, whereas that of strain G2704 is identical to the structure of Cronobacter malonaticus 3267 [MacLean, L. L.; Vinogradov, E.; Pagotto, F.; Farber, J. M.; Perry, M. B. Biochem. Cell Biol.2009, 87, 927–932], except for that the latter lacks O-acetylation. Putative functions of the genes in the O-antigen gene clusters of C. sakazakii strains studied are in agreement with the O-polysaccharide structures.     

Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order

Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.     

Detection and characterization of Shigella species isolated from food and human stool samples in Nabeul, Tunisia, by molecular methods and culture techniques

Aims: This study was designed to isolate Shigella spp. strains from food and stool samples by a combination of PCR and culture methods and characterize their serotypes, antibiotic resistance profiles, virulence genes and pulsed‐field gel electrophoresis (PFGE) patterns to investigate possible clonal relationships amongst strains circulating. Methods and Results: Six Shigella spp. strains were isolated from 280 food samples against 16 Shigella isolates from 236 stool samples of symptomatic patients and asymptomatic food handlers during the period from January 2007 to December 2009 in Public Health Regional Laboratory of Nabeul. The detection of ipaH, ipaBCD, ial, ShET‐1 and ShET‐2 was performed by a PCR technique with specific primers. Conclusions: The use of PCR technique improved the rate of detecting Shigella in stool samples from 6·7 to 14% and in food samples from 2·1 to 8·6%. Percentage of Shigella isolates and ipaH‐specific PCR demonstrated a marked pattern of seasonality, increasing in summer and fall seasons for human and food isolates. Amongst the environmental strains, 50% of isolates were invasive. However, for the 16 clinical strains isolated, nine were found to be positive for both ial and ipaBCD gene and 11 were found to produce ShET‐1 and/or ShET‐2. XbaI PFGE analysis revealed the presence of a predominant clone amongst Shigella sonnei strains recovered from different sources circulating in Nabeul, Tunisia, throughout the years 2007–2009. Significance and Impact of the Study: This study demonstrated the existence of Shigella in food samples and dispersion of different virulence genes amongst these isolates, which appear to constitute an environmental source of epidemic spread. The clonal relationships amongst strains isolated from food elements and human stools indicate the incrimination of different kinds of foods as vehicle of transmission of Shigella, which are usually escaped from detection by traditional culture methods.     

Immunoproteomic identification of immunogenic proteins in Cronobacter sakazakii strain BAA-894

Cronobacter spp. are emerging opportunistic pathogens. Cronobacter sakazakii is considered as the predominant species in all infections. So far, our understanding of the species’ immunogens and potential virulence factors of Cronobacter spp. remains limited. In this study, an immunoproteomic approach was used to investigate soluble and insoluble proteins from the genome-sequenced strain C. sakazakii ATCC BAA-894. Proteins were separated using two-dimensional electrophoresis, detected by Western blotting with polyclonal antibodies of C. sakazakii BAA-894, and identified using tandem mass spectrometry (MALDI-MS and MALDI-MS/MS, MS/MSMS). A total of 11 immunoreactive proteins were initially identified in C. sakazakii BAA-894, including two outer membrane proteins, four periplasmic proteins, and five cytoplasmic proteins. In silico functional analysis of the 11 identified proteins indicated three proteins that were initially described as immunogens of pathogenic bacteria. For the remaining eight proteins, one protein was categorized as a potential virulence factor involved in protection against reactive oxygen species, and seven proteins were considered to play potential roles in adhesion, invasion, and biofilm formation. To our knowledge, this is the first time that immunogenic proteins of C. sakazakii BAA-894 have been identified as immunogens and potential virulence factors by an immunoproteomics approach. Future studies should investigate the roles of these proteins in bacterial pathogenesis and modulation of host immune responses during infection to identify their potential as molecular therapeutic targets.