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Stephen J Forsythe - One of the best experts on this subject based on the ideXlab platform.

  • Characterisation of Cronobacter strains isolated from hospitalised adult patients.
    Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2017
    Co-Authors: Veronika Kadlicekova, Stephen J Forsythe, Michal Kajsik, Katarína Šoltys, Tomáš Szemes, Lívia Slobodníková, Lucia Janosikova, Zuzana Hubenakova, Pauline Ogrodzki, Jan Turna
    Abstract:

    Bacteria belonging to the genus Cronobacter are opportunistic pathogens known for causing rare but serious infections in neonates, including meningitis, necrotising enterocolitis and sepsis. Cronobacter infections occur also in adult populations, however, they generally have milder manifestations and their prevalence is uncertain. In this study, the presence of Cronobacter strains from adult patients in the University Hospital in Bratislava was investigated and overall 18 confirmed isolates from 321 patients (5.3%) were recovered. No Cronobacter positive sample was detected in 215 sputum samples from outpatients. The highest occurrence of Cronobacter strains was observed from stroke patients and this may be associated with an abnormal swallowing ability. The isolated strains belonged to the species Cronobacter sakazakii and Cronobacter malonaticus. In silico genotyping (MLST, CRISPR-cas array profiling) of whole genome sequences assigned the strains to three different MLST clones. The majority (12/18) of the isolated strains were sequence type ST513 or single locus variants ST514 and ST515, thereby being members of C. sakazakii pathovar clonal complex CC4. However, according to core genome MLST analysis the ST513-ST515 strains created a unique cluster substantially different from other CC4 strains. The isolated strains were susceptible to 18 tested antibiotics. All strains possess a genomic island encoding for increased thermal tolerance. As Cronobacter strains are frequently present in dried foods of plant origin, spread of a specific clone within a hospital may be caused by food transmission and may be facilitated by its tolerance to environmental stresses such as desiccation and temperature.

  • comparative study of Cronobacter identification according to phenotyping methods
    BMC Microbiology, 2016
    Co-Authors: Emily E Jackson, Stephen J Forsythe
    Abstract:

    Background Microbiological criteria applied to powdered infant formula (PIF) require the absence of all Cronobacter spp. Consequently, misidentification of isolates from finished products can lead to significant financial losses for manufacturers and could increase the risk of neonatal infection. Biochemical identification of suspect isolates using commercially available test panels is recommended for use by PIF manufacturers by both the US FDA and ISO standard methods for Cronobacter species; however, phenotyping can be unreliable, particularly for a genus such as Cronobacter where the taxonomy has been subject to frequent changes. This study compared the predicted identification by commonly used phenotyping kits (API20E and ID32E) for over 240 strains of Cronobacter from diverse sources, which had been identified using DNA sequence analysis. In 2015, the databases associated with the API20E and ID32E biochemical test panels were updated, including the recognition of the Cronobacter genus. Thus, the identifications from multiple versions the databases were compared to each other and to identifications based on DNA sequencing methods.

  • Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order.
    Applied and Environmental Microbiology, 2015
    Co-Authors: Martina Blažková, Stephen J Forsythe, Barbora Javůrková, Pauline Ogrodzki, Jiří Vlach, Sandra Göselová, Ludmila Karamonová, Ladislav Fukal
    Abstract:

    ABSTRACT 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.

  • Cronobacter the emergent bacterial pathogen enterobacter sakazakii comes of age mlst and whole genome sequence analysis
    BMC Genomics, 2014
    Co-Authors: Stephen J Forsythe, Ben Dickins, Keith A Jolley
    Abstract:

    Following the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. This paper presents the application of Next Generation Sequencing (NGS) to Cronobacter which has led to the establishment of the Cronobacter PubMLST genome and sequence definition database ( http://pubmlst.org/Cronobacter/ ) containing over 1000 isolates with metadata along with the recognition of specific clonal lineages linked to neonatal meningitis and adult infections Whole genome sequencing and multilocus sequence typing (MLST) has supports the formal recognition of the genus Cronobacter composed of seven species to replace the former single species Enterobacter sakazakii. Applying the 7-loci MLST scheme to 1007 strains revealed 298 definable sequence types, yet only C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis. This clonal lineage has been confirmed using ribosomal-MLST (51-loci) and whole genome-MLST (1865 loci) to analyse 107 whole genomes via the Cronobacter PubMLST database. This database has enabled the retrospective analysis of historic cases and outbreaks following re-identification of those strains. The Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for researchers. It has the capacity to create new analysis schemes ‘on the fly’, and to integrate metadata (source, geographic distribution, clinical presentation). It is also expandable and adaptable to changes in taxonomy, and able to support the development of reliable detection methods of use to industry and regulatory authorities. Therefore it meets the WHO (2004) request for the establishment of a typing scheme for this emergent bacterial pathogen. Whole genome sequencing has additionally shown a range of potential virulence and environmental fitness traits which may account for the association of C. sakazakii CC4 pathogenicity, and propensity for neonatal CNS.

  • genotypic and phenotypic characteristics of Cronobacter species with particular attention to the newly reclassified species Cronobacter helveticus Cronobacter pulveris and Cronobacter zurichensis
    Food Microbiology, 2014
    Co-Authors: Emily E Jackson, Naqash Masood, Hana Sonbol, Stephen J Forsythe
    Abstract:

    In 2013, Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis, were reclassified as Cronobacter helveticus, Cronobacter pulveris and Cronobacter zurichensis, respectively. Previously these species had been used as negative controls for some Cronobacter detection assays. This study examined cultural, biochemical and molecular Cronobacter detection and identification assays, with emphasis on the new species. Additionally, 32 Cronobacter genomes were examined for the presence of PCR target genes using the BLAST function of the online Cronobacter PubMLST facility. The results of the cultural methods varied and no single medium was able to correctly detect all Cronobacter spp. Since the supporting databases have not been updated to include the Cronobacter genus, Enterobacter sakazakii was returned for four strains of the newly reclassified species with ID32E and none with API 20E. PCR probes targeting rpoB and ompA could not correctly identify the new Cronobacter spp., due to primer specificity or absent target genes. As neonates have been identified as a high-risk group for infection, international standards require the absence of all Cronobacter species in powdered infant formula. However, many conventional detection methods cannot correctly identify the newly recognized species. Conversely, DNA sequence-based methods can adapt to taxonomic revisions and will likely become more common.

Carol Iversen - One of the best experts on this subject based on the ideXlab platform.

  • Genotyping of Cronobacter (Enterobacter sakazakii) strains isolated from an infant formula processing plant.
    Archiv Fur Lebensmittelhygiene, 2020
    Co-Authors: Carol Iversen, Angelika Lehner, Claudia Fricker-feer, Karl Gschwend, Roger Stephan
    Abstract:

    Cronobacter spp. are occasional contaminants of infant food processing establishments and have been implicated in rare cases of neonatal infections. The control of these organisms during the processing of infant formula and identification of possible contaminating sources is of importance to manufacturers. In this study, infant formula processing sites were monitored for the presence of Cronobacter spp. (E. sakazakii) in 2007. Cronobacter were isolated using ISO/TS 22964 and simultaneously using a newly developed differential method, Cronobacter Screening Broth (CSB). Clonal types of Cronobacter strains in the environment, raw materials, and products were investigated using PFGE and RAPD in order to shed light on the possible dissemination routes within the facility. Over the course of the study, a total of 153 Cronobacter isolates were obtained from 46 end-products, 23 raw materials and 44 environmental samples. The 153 Cronobacter isolates represented 71 PFGE pulso-types, with 24 pulso-types containing multiple isolates. Clonal isolates present in products could be matched to indistinguishable pulso-types in raw materials and in the environment, however no strains were identified that were present in both the environment and raw materials. Although the majority of the isolates were obtained from products, the largest diversity of isolates was observed in raw materials. This may indicate raw materials as a significant source of Cronobacter entry into the production facility with subsequent selection for strains that are able to persist within the factory environment.

  • detection and frequency of Cronobacter spp enterobacter sakazakii in different categories of ready to eat foods other than infant formula
    International Journal of Food Microbiology, 2009
    Co-Authors: Andreas Baumgartner, Marius Grand, Marianne Liniger, Carol Iversen
    Abstract:

    Abstract Two hundred sixty eight samples of ready-to-eat foods from retail shops were screened for the presence of Cronobacter by selective enrichment followed by plating on three chromogenic agars (ESIA, ESPM and DFI). Cronobacter was isolated from 14/23 samples of sprouts and fresh herbs/salads (60.9%), 7/26 samples of spices and dried herbs (26.9%) and 3/42 confectionery samples (7.1%). In cases where repeat samples were available, foods positive for Cronobacter were retested twice. In total, 54 Cronobacter isolates from 24 foods were recovered and genetic fingerprint patterns generated using PFGE. Identical PFGE-profiles were generated for Cronobacter isolates from five samples of two confectionery products obtained from a particular bakery shop over a period of 11 months. This may indicate a persistent contamination of the production site. For all other isolates, no clustering by phylogenetic analysis of PFGE-profiles was observed, indicating the sporadic nature of Cronobacter in ready-to-eat foods. Enterobacterial counts varied from a maximum value of 2.9 × 107 CFU/g (in dill) to a minimum value of

  • microarray based comparative genomic indexing of the Cronobacter genus enterobacter sakazakii
    International Journal of Food Microbiology, 2009
    Co-Authors: B Healy, Stephen J Obrien, Carol Iversen, N R Mullane, Angelika Lehner, Roger Stephan, Steven Huynh, Craig T Parker, Seamus Fanning
    Abstract:

    Abstract Cronobacter (Enterobacter sakazakii) is a recently defined genus consisting of 6 species. To extend our understanding of the genetic relationship between Cronobacter sakazakii BAA-894 and the other species of this genus, microarray-based comparative genomic indexing (CGI) was undertaken to determine the presence/absence of genes identified in the former sequenced genome and to compare 276 selected open reading frames within the different Cronobacter strains. Seventy-eight Cronobacter strains (60 C. sakazakii, 8 C. malonaticus, 5 C. dublinensis, 2 C. muytjensii, 1 C. turicensis, 1 C. genomospecies 1, and 1 Cronobacter sp.) representing clinical and environmental isolates from various geographical locations were investigated. Hierarchical clustering of the CGI data showed that the species grouped as clusters. The 5 C. dublinensis and 2 C. muytjensii strains examined formed distinct species clusters. Moreover, all of the C. sakazakii and 3 of 8 C. malonaticus strains formed a large cluster. The remaining C. malonaticus strains formed a sub-group within a larger cluster that also contained C. turicensis, C. genomospecies 1, and an unknown Cronobacter sp. Cronobacter sakazakii and 3 of 8 C. malonaticus strains could be distinguished from the others within the collection by the presence of 10 fimbrial related genes. Similarly, capsule and/or lipopolysaccharide (LPS) related glycosyltransferases differentiated several of the C. sakazakii strains from each other.

  • prevalence of Cronobacter species enterobacter sakazakii in follow on infant formulae and infant drinks
    Letters in Applied Microbiology, 2009
    Co-Authors: Stephen J Obrien, B Healy, Carmen Negredo, W Anderson, Seamus Fanning, Carol Iversen
    Abstract:

    Aims:  To determine the prevalence of Cronobacter spp. (Enterobacter sakazakii) in follow-on formula powders commercially available in European countries. Methods and Results:  A total of 470 samples comprising 31 different products from 18 brand names belonging to seven companies were tested for the presence of Cronobacter species. No milk- or soy-based infant formula powders were found to contain Cronobacter species. However, two cereal-based infant drinks were positive for Cronobacter sakazakii. A review of the published cases spanning the past 48 years did not reveal any fatalities attributable to Cronobacter spp. in children over 3 months. Conclusions:  The low incidence of Cronobacter in infant powdered drinks, the lack of fatal Cronobacter infections in infants greater than 3 months and the low incidence of Cronobacter-related reported illness in this age group indicated that ingestion of these products presents a low risk for the intended consumers. Significance and Impact of the Study:  The risk posed to neonates from the consumption of infant formula contaminated with Cronobacter is clear. Risks associated with powdered follow-on formulae intended for consumption by older infants is now under consideration by the World Health Organization. Our data contributes to the body of knowledge available for the assessment of the risk to consumers from these food products.

  • Prevalence of Cronobacter species (Enterobacter sakazakii) in follow‐on infant formulae and infant drinks
    Letters in Applied Microbiology, 2009
    Co-Authors: Stephen J. O'brien, B Healy, Carmen Negredo, W Anderson, Seamus Fanning, Carol Iversen
    Abstract:

    Aims:  To determine the prevalence of Cronobacter spp. (Enterobacter sakazakii) in follow-on formula powders commercially available in European countries. Methods and Results:  A total of 470 samples comprising 31 different products from 18 brand names belonging to seven companies were tested for the presence of Cronobacter species. No milk- or soy-based infant formula powders were found to contain Cronobacter species. However, two cereal-based infant drinks were positive for Cronobacter sakazakii. A review of the published cases spanning the past 48 years did not reveal any fatalities attributable to Cronobacter spp. in children over 3 months. Conclusions:  The low incidence of Cronobacter in infant powdered drinks, the lack of fatal Cronobacter infections in infants greater than 3 months and the low incidence of Cronobacter-related reported illness in this age group indicated that ingestion of these products presents a low risk for the intended consumers. Significance and Impact of the Study:  The risk posed to neonates from the consumption of infant formula contaminated with Cronobacter is clear. Risks associated with powdered follow-on formulae intended for consumption by older infants is now under consideration by the World Health Organization. Our data contributes to the body of knowledge available for the assessment of the risk to consumers from these food products.

Hana Drahovska - One of the best experts on this subject based on the ideXlab platform.

  • Contribution of the thermotolerance genomic island to increased thermal tolerance in Cronobacter strains
    Antonie van Leeuwenhoek, 2016
    Co-Authors: Maria Orieskova, Michal Kajsik, Tomáš Szemes, Jan Turna, Ondrej Holy, Stephen Forsythe, Hana Drahovska
    Abstract:

    Cronobacter spp. are opportunistic pathogens associated with serious infections in neonates. Increased stress tolerance, including the thermotolerance of some Cronobacter strains, can promote their survival in production facilities and thus raise the possibility of contamination of dried infant formula which has been identified as a potential source of infection. Some Cronobacter strains contain a genomic island, which might be responsible for increased thermotolerance. By analysis of Cronobacter sequenced genomes this determinant was found to be present in only 49/73 Cronobacter sakazakii strains and in 9/14 Cronobacter malonaticus strains. The island was also found in 16/17 clinical isolates originating from two hospitals. Two configurations of the locus were detected; the first one with the size of 18 kbp containing the thr B-Q genes and a shorter version (6 kbp) harbouring only the thr BCD and thr OP genes. Strains containing the thermotolerance island survived significantly better at 58 °C comparing to a C. sakazakii isogenic mutant lacking the island and strains with the longer version of the island were 2–10 times more tolerant than those with the shortened sequence. The function of the genomic island was further confirmed by its cloning into a low-copy vector and transforming it into the isogenic mutant. Different levels of rpo S, encoding for stress-response sigma factor, expression were also associated with variability in strain thermotolerance.

  • characterization of Cronobacter spp isolated from food of plant origin and environmental samples collected from farms and from supermarkets in the czech republic
    International Journal of Food Microbiology, 2016
    Co-Authors: Hana Vojkovska, Renata Karpiskova, Maria Orieskova, Hana Drahovska
    Abstract:

    Abstract The Cronobacter genus (previously known as Enterobacter sakazakii) comprises seven species (Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti) which cause serious infections in neonates and immunocompromised people. Most of the documented outbreaks of these bacteria have been associated with consumption of contaminated powdered infant formula. The plant environment is considered to be the natural habitat of these bacteria. Therefore, a total number of 563 samples of vegetables, fruit, water and environmental swabs were collected from local farms and supermarkets in the Czech Republic and investigated for the presence of Cronobacter spp. The obtained 45 isolates (8.0%) were further characterized by phenotyping (antimicrobial resistance, capsule and pigment production) and genotyping (fusA sequencing, MLST, PCR-serotyping) methods. Most of the Cronobacter isolates (42.2%) were identified as C. sakazakii, followed by C. turicensis (31.1%), C. dublinensis (22.2%), C. malonaticus (2.2%) and C. universalis (2.2%). The 25 identified sequence types, out of which 17 were unique for only one strain, indicated a high diversity of strains. C. sakazakii sequence type 4 (ST 4), which has been associated with many cases of meningitis, was isolated only in one case. A strong association of C. turicensis and C. dublinensis with the plant environment can be deduced from our results.

  • identification and characterization of Cronobacter strains isolated from powdered infant foods
    Letters in Applied Microbiology, 2014
    Co-Authors: A Gicova, Maria Orieskova, Hana Drahovska, Lucia Oslanecova, Eva Kaclíková
    Abstract:

    UNLABELLED: Cronobacter spp. (formerly Enterobacter sakazakii) is responsible for rare but fatal cases of infection in neonates and immunocompromised infants. The aim of our study was to characterize Cronobacter strains isolated from powdered infant foods in Public Health Authority of the Slovak Republic in 2009-2010. Powdered infant food products have been analysed using currently available standard method ISO/TS 22964: 2006 for the detection of Cronobacter spp. complemented with qPCR confirmation of positive strains. Thirteen Cronobacter strains were isolated from more than 900 powdered infant formulae, milk-based and cereal-based powdered weaning food products. The strains were assigned to five biogroups and ten multilocus sequence typing (MLST) sequence types. In total, twelve strains were identified as Cronobacter sakazakii and one strain as Cronobacter dublinensis. Multiple strains originated from parallel isolation were obtained in three samples and the variability between strains from the same food was observed twice. The results are in agreement with the hypothesis that the Cronobacter contamination detected in infant powdered food is low and originating in various accidental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterized Cronobacter strains isolated from powdered infant formulae and weaning foods by biotyping and multilocus sequence typing. The later method was shown to be more discriminative and suitable for both species identification and subtyping. Low level (0·9%) of Cronobacter positivity was observed in 916 samples. Multiple sequence types were observed among strains isolated from the same food product. This highlights that multiple isolates from each single sample should be analysed in epidemiological studies, since more than one genetic subtype may be present.

  • Cronobacter condimenti sp nov isolated from spiced meat and Cronobacter universalis sp nov a species designation for Cronobacter sp genomospecies 1 recovered from a leg infection water and food ingredients
    International Journal of Systematic and Evolutionary Microbiology, 2012
    Co-Authors: Susan Joseph, Hana Drahovska, Esin Cetinkaya, Arturo Levican, M J Figueras, Stephen J Forsythe
    Abstract:

    A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii (strain 1330T), two previously assigned to Cronobacter genomospecies 1 (strains NCTC 9529T and 731) and two as Cronobacter turicensis (strains 96 and 1435) was carried out. The analysis included a phenotypic characterization, sequencing of the 16S rRNA gene and multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). The 16S rRNA gene sequence analysis and MLSA showed strain 1330T, isolated from spiced meat purchased in Slovakia, to form an independent phylogenetic line. Cronobacter dublinensis was the closest neighbour species on the basis of the MLSA. DNA–DNA reassociation and phenotypic analysis revealed that strain 1330T represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed, type strain 1330T = CECT 7863T, = LMG 26250T). The four bacterial strains NCTC 9529T, 731, 96 and 1435, isolated from water, a leg infectionand two food ingredients; onion powder and rye flour, repectively, showed on the phylogenetic tree to cluster together within an independent phylogenetic line, with Cronobacter turicensis as the closest species. The DNA–DNA hybridization data and the phenotypic characterization confirmed that these strains represented a novel species, for which the name Cronobacter universalis sp. nov. is proposed with type strain NCTC 9529T = CECT 7864T, = LMG 26249T.

  • Analysis of the DNA region mediating increased thermotolerance at 58°C in Cronobacter sp. and other enterobacterial strains
    Antonie van Leeuwenhoek, 2011
    Co-Authors: Jana Gajdosova, Eva Kaclíková, Jan Turna, Kristina Benedikovicova, Natalia Kamodyova, Lubomira Tothova, Stanislav Stuchlik, Hana Drahovska
    Abstract:

    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.

Seamus Fanning - One of the best experts on this subject based on the ideXlab platform.

  • Use of a Pan-Genomic DNA Microarray in Determination of the Phylogenetic Relatedness among Cronobacter spp. and Its Use as a Data Mining Tool to Understand Cronobacter Biology.
    Microarrays, 2017
    Co-Authors: Ben D Tall, Christopher J Grim, Venugopal Sathyamoorthy, Laurenda Carter, M H Kothary, Jayanthi Gangiredla, Isha R. Patel, Scott A. Jackson, Mark K. Mammel, Seamus Fanning
    Abstract:

    Cronobacter (previously known as Enterobacter sakazakii) is a genus of Gram-negative, facultatively anaerobic, oxidase-negative, catalase-positive, rod-shaped bacteria of the family Enterobacteriaceae. These organisms cause a variety of illnesses such as meningitis, necrotizing enterocolitis, and septicemia in neonates and infants, and urinary tract, wound, abscesses or surgical site infections, septicemia, and pneumonia in adults. The total gene content of 379 strains of Cronobacter spp. and taxonomically-related isolates was determined using a recently reported DNA microarray. The Cronobacter microarray as a genotyping tool gives the global food safety community a rapid method to identify and capture the total genomic content of outbreak isolates for food safety, environmental, and clinical surveillance purposes. It was able to differentiate the seven Cronobacter species from one another and from non-Cronobacter species. The microarray was also able to cluster strains within each species into well-defined subgroups. These results also support previous studies on the phylogenic separation of species members of the genus and clearly highlight the evolutionary sequence divergence among each species of the genus compared to phylogenetically-related species. This review extends these studies and illustrates how the microarray can also be used as an investigational tool to mine genomic data sets from strains. Three case studies describing the use of the microarray are shown and include: (1) the determination of allelic differences among Cronobacter sakazakii strains possessing the virulence plasmid pESA3; (2) mining of malonate and myo-inositol alleles among subspecies of Cronobacter dublinensis strains to determine subspecies identity; and (3) lastly using the microarray to demonstrate sequence divergence and phylogenetic relatedness trends for 13 outer-membrane protein alleles among 240 Cronobacter and phylogenetically-related strains. The goal of this review is to describe microarrays as a robust tool for genomics research of this assorted and important genus, a criterion toward the development of future preventative measures to eliminate this foodborne pathogen from the global food supply.

  • Survival characteristics of Cronobacter spp. in model bovine gut and in the environment.
    Foodborne Pathogens and Disease, 2010
    Co-Authors: Catherine Molloy, Seamus Fanning, Claire Cagney, Geraldine Duffy
    Abstract:

    Abstract Cronobacter spp. (formally Enterobacter sakazakii) have been linked to illness in infants from contaminated powdered infant formula. The source of the pathogen remains unclear, and it is believed that farm environments and cattle could play a role in the transmission of Cronobacter spp. The aim of this study was to establish if the organism would survive passage through a model of the bovine rumen and abomasum and in bovine feces in the farm environment. Models of the bovine abomasum and rumen were inoculated with Cronobacter strains (final concentrations of 5.7 and 6.5 log10 CFU/mL, respectively), and survival was examined over time in these environments using an adapted ISO/DTS 22964 culture protocol. Fecal samples were inoculated with Cronobacter (final concentration 6.0 log10 CFU/mL), and survival on soil and in containers stored outdoors was examined over time. The results showed no significant changes in the number of Cronobacter in rumen fluid over a 24-h period. Cronobacter were undetecta...

  • microarray based comparative genomic indexing of the Cronobacter genus enterobacter sakazakii
    International Journal of Food Microbiology, 2009
    Co-Authors: B Healy, Stephen J Obrien, Carol Iversen, N R Mullane, Angelika Lehner, Roger Stephan, Steven Huynh, Craig T Parker, Seamus Fanning
    Abstract:

    Abstract Cronobacter (Enterobacter sakazakii) is a recently defined genus consisting of 6 species. To extend our understanding of the genetic relationship between Cronobacter sakazakii BAA-894 and the other species of this genus, microarray-based comparative genomic indexing (CGI) was undertaken to determine the presence/absence of genes identified in the former sequenced genome and to compare 276 selected open reading frames within the different Cronobacter strains. Seventy-eight Cronobacter strains (60 C. sakazakii, 8 C. malonaticus, 5 C. dublinensis, 2 C. muytjensii, 1 C. turicensis, 1 C. genomospecies 1, and 1 Cronobacter sp.) representing clinical and environmental isolates from various geographical locations were investigated. Hierarchical clustering of the CGI data showed that the species grouped as clusters. The 5 C. dublinensis and 2 C. muytjensii strains examined formed distinct species clusters. Moreover, all of the C. sakazakii and 3 of 8 C. malonaticus strains formed a large cluster. The remaining C. malonaticus strains formed a sub-group within a larger cluster that also contained C. turicensis, C. genomospecies 1, and an unknown Cronobacter sp. Cronobacter sakazakii and 3 of 8 C. malonaticus strains could be distinguished from the others within the collection by the presence of 10 fimbrial related genes. Similarly, capsule and/or lipopolysaccharide (LPS) related glycosyltransferases differentiated several of the C. sakazakii strains from each other.

  • prevalence of Cronobacter species enterobacter sakazakii in follow on infant formulae and infant drinks
    Letters in Applied Microbiology, 2009
    Co-Authors: Stephen J Obrien, B Healy, Carmen Negredo, W Anderson, Seamus Fanning, Carol Iversen
    Abstract:

    Aims:  To determine the prevalence of Cronobacter spp. (Enterobacter sakazakii) in follow-on formula powders commercially available in European countries. Methods and Results:  A total of 470 samples comprising 31 different products from 18 brand names belonging to seven companies were tested for the presence of Cronobacter species. No milk- or soy-based infant formula powders were found to contain Cronobacter species. However, two cereal-based infant drinks were positive for Cronobacter sakazakii. A review of the published cases spanning the past 48 years did not reveal any fatalities attributable to Cronobacter spp. in children over 3 months. Conclusions:  The low incidence of Cronobacter in infant powdered drinks, the lack of fatal Cronobacter infections in infants greater than 3 months and the low incidence of Cronobacter-related reported illness in this age group indicated that ingestion of these products presents a low risk for the intended consumers. Significance and Impact of the Study:  The risk posed to neonates from the consumption of infant formula contaminated with Cronobacter is clear. Risks associated with powdered follow-on formulae intended for consumption by older infants is now under consideration by the World Health Organization. Our data contributes to the body of knowledge available for the assessment of the risk to consumers from these food products.

  • Prevalence of Cronobacter species (Enterobacter sakazakii) in follow‐on infant formulae and infant drinks
    Letters in Applied Microbiology, 2009
    Co-Authors: Stephen J. O'brien, B Healy, Carmen Negredo, W Anderson, Seamus Fanning, Carol Iversen
    Abstract:

    Aims:  To determine the prevalence of Cronobacter spp. (Enterobacter sakazakii) in follow-on formula powders commercially available in European countries. Methods and Results:  A total of 470 samples comprising 31 different products from 18 brand names belonging to seven companies were tested for the presence of Cronobacter species. No milk- or soy-based infant formula powders were found to contain Cronobacter species. However, two cereal-based infant drinks were positive for Cronobacter sakazakii. A review of the published cases spanning the past 48 years did not reveal any fatalities attributable to Cronobacter spp. in children over 3 months. Conclusions:  The low incidence of Cronobacter in infant powdered drinks, the lack of fatal Cronobacter infections in infants greater than 3 months and the low incidence of Cronobacter-related reported illness in this age group indicated that ingestion of these products presents a low risk for the intended consumers. Significance and Impact of the Study:  The risk posed to neonates from the consumption of infant formula contaminated with Cronobacter is clear. Risks associated with powdered follow-on formulae intended for consumption by older infants is now under consideration by the World Health Organization. Our data contributes to the body of knowledge available for the assessment of the risk to consumers from these food products.

Susan Joseph - One of the best experts on this subject based on the ideXlab platform.

  • multilocus sequence typing of Cronobacter spp from powdered infant formula and milk powder production factories
    International Dairy Journal, 2013
    Co-Authors: Hana Sonbol, Susan Joseph, Catherine M Mcauley, H M Craven, Stephen J Forsythe
    Abstract:

    Abstract This study applied the Cronobacter spp. multilocus sequence typing (MLST) scheme to three strain collections, then known as Enterobacter sakazakii, which had been isolated between 1988 and 2009 from 14 countries. The results revealed the predominance (85%) of Cronobacter sakazakii (72 strains) in all three collections. The remaining strains were Cronobacter turicensis (10%), Cronobacter malonaticus (4%), and Cronobacter muytjensii (1%). No strains of Cronobacter dublinensis, Cronobacter universalis or Cronobacter condimenti were identified. Twenty-one out of seventy-two C. sakazakii strains were in the clinically significant ST4 clonal complex, and were found in all three strain collections. These results confirm C. sakazakii ST4 is one of the predominant clonal complexes over the past 20 years in several parts of the world. Further understanding of the ecosystem and sources of the organism may be used for the development of improved intervention strategies in the diary industry.

  • comparison of methods for the microbiological identification and profiling of Cronobacter species from ingredients used in the preparation of infant formula
    Molecular and Cellular Probes, 2013
    Co-Authors: Esin Cetinkaya, Susan Joseph, Kamuran Ayhan, Stephen J Forsythe
    Abstract:

    Cronobacter spp. (formerly Enterobacter sakazakii) can be isolated from a wide range of foods and environments, and its association with neonatal infections has drawn considerable attention from regulatory authorities. The principle route of neonatal infection has been identified as the ingestion of contaminated infant formula. A number of methods have been developed to identify Cronobacter spp., however these were before the most recent (2012) taxonomic revision of the genus into seven species. In this study, phenotyping, protein profiling and molecular methods were used to identify Cronobacter strains which had been recently isolated from ingredients used in the preparation of infant formula. Pulsed field gel electrophoresis revealed that different Cronobacter strains had been recovered from the same food products. All isolates were identified as Cronobacter sakazakii according to four genus specific PCR-probes and protein profiling using MALDI-TOF analysis. However, 16S rDNA sequence analyses and fusA allele sequencing gave more accurate identification: four strains were C. sakazakii, one strain was Cronobacter malonaticus and the remaining strain was Cronobacter universalis. Multilocus sequence typing showed the strains were different sequence types. These results demonstrate the presence of different Cronobacter species in food ingredients used in the preparation of infant formula, and also the need for molecular identification and profiling methods to be revised according to taxonomic revisions.

  • Cronobacter condimenti sp nov isolated from spiced meat and Cronobacter universalis sp nov a species designation for Cronobacter sp genomospecies 1 recovered from a leg infection water and food ingredients
    International Journal of Systematic and Evolutionary Microbiology, 2012
    Co-Authors: Susan Joseph, Hana Drahovska, Esin Cetinkaya, Arturo Levican, M J Figueras, Stephen J Forsythe
    Abstract:

    A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii (strain 1330T), two previously assigned to Cronobacter genomospecies 1 (strains NCTC 9529T and 731) and two as Cronobacter turicensis (strains 96 and 1435) was carried out. The analysis included a phenotypic characterization, sequencing of the 16S rRNA gene and multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). The 16S rRNA gene sequence analysis and MLSA showed strain 1330T, isolated from spiced meat purchased in Slovakia, to form an independent phylogenetic line. Cronobacter dublinensis was the closest neighbour species on the basis of the MLSA. DNA–DNA reassociation and phenotypic analysis revealed that strain 1330T represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed, type strain 1330T = CECT 7863T, = LMG 26250T). The four bacterial strains NCTC 9529T, 731, 96 and 1435, isolated from water, a leg infectionand two food ingredients; onion powder and rye flour, repectively, showed on the phylogenetic tree to cluster together within an independent phylogenetic line, with Cronobacter turicensis as the closest species. The DNA–DNA hybridization data and the phenotypic characterization confirmed that these strains represented a novel species, for which the name Cronobacter universalis sp. nov. is proposed with type strain NCTC 9529T = CECT 7864T, = LMG 26249T.

  • Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients.
    International journal of systematic and evolutionary microbiology, 2011
    Co-Authors: Susan Joseph, Hana Drahovska, Esin Cetinkaya, Arturo Levican, M J Figueras, Stephen J Forsythe
    Abstract:

    A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii (strain 1330(T), isolated from spiced meat purchased in Slovakia), two previously assigned to Cronobacter genomospecies 1 (strains NCTC 9529(T) and 731, isolated from water and a leg infection, respectively) and two previously assigned to Cronobacter turicensis (strains 96 and 1435, isolated from onion powder and rye flour, respectively) was carried out. The analysis included phenotypic characterization, 16S rRNA gene sequencing and multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). 16S rRNA gene sequence analysis and MLSA showed that strain 1330(T) formed an independent phylogenetic lineage in the MLSA, with Cronobacter dublinensis LMG 23823(T) as the closest neighbour. DNA-DNA reassociation and phenotypic analysis revealed that strain 1330(T) represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed (type strain 1330(T) = CECT 7863(T) = LMG 26250(T)). Strains NCTC 9529(T), 731, 96 and 1435 clustered together within an independent phylogenetic lineage, with C. turicensis LMG 23827(T) as the closest neighbour in the MLSA. DNA-DNA reassociation and phenotypic analysis confirmed that these strains represent a novel species, for which the name Cronobacter universalis sp. nov. is proposed (type strain NCTC 9529(T) = CECT 7864(T) = LMG 26249(T)).