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1. Franco  AA, Hu  L, Grim  CJ, Gopinath  G, Sathyamoorthy  V, Jarvis  KG, Lee  C, Sadowski  J, Kim  J, Kothary  MH, McCardell  BA, Tall  BD,     ( 2011 )

Characterization of putative virulence genes on the related RepFIB plasmids harbored by Cronobacter spp.

Applied and environmental microbiology 77 (10)
PMID : 21421789  :   DOI  :   10.1128/AEM.03023-10     PMC  :   PMC3126477    
Abstract >>
Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, 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. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.
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2. Stoop  B, Lehner  A, Iversen  C, Fanning  S, Stephan  R,     ( 2009 )

Development and evaluation of rpoB based PCR systems to differentiate the six proposed species within the genus Cronobacter.

International journal of food microbiology 136 (2)
PMID : 19467725  :   DOI  :   10.1016/j.ijfoodmicro.2009.04.023    
Abstract >>
Although there are various PCR based methods described in the literature to detect the genus Cronobacter, none of these methods is able to differentiate the six proposed species within this genus. A species differentiation is important for epidemiological studies. Moreover, the different species show differences in sensitivity to chemical agents and antibiotics. Here we report for the first time different rpoB based PCR systems which enable the identification to species level of strains previously confirmed to belong to the genus Cronobacter. Different primer pairs based on the rpoB sequences of the six Cronobacter species type strains were designed. Thereafter, 57 target and non-target strains, previously described in the Cronobacter taxonomy paper, were included for the specificity evaluation. C. turicensis, C. muytjensii, C. dublinensis and C. genomospecies 1 can be reliably identified by the proposed single primer pairs. Only target strains showed a correctly sized amplification product, whereas no amplification product was obtained for all non-target strains used in this study (100% specificity). However, as the rpoB gene sequences of C. sakazakii and C. malonaticus are closely related, a two step procedure is necessary. We therefore recommend a two-step procedure in which the primer pairs Cmalf/Cmalr are used in a follow up PCR on strains that are found to be positive in the amplification with the C. sakazakii specific primers Csakf/Csakr.
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3. Kuhnert  P, Korczak  BM, Stephan  R, Joosten  H, Iversen  C,     ( 2009 )

Phylogeny and prediction of genetic similarity of Cronobacter and related taxa by multilocus sequence analysis (MLSA).

International journal of food microbiology 136 (2)
PMID : 19321218  :   DOI  :   10.1016/j.ijfoodmicro.2009.02.022    
Abstract >>
Multilocus sequence analysis (MLSA) based on recN, rpoA and thdF genes was done on more than 30 species of the family Enterobacteriaceae with a focus on Cronobacter and the related genus Enterobacter. The sequences provide valuable data for phylogenetic, taxonomic and diagnostic purposes. Phylogenetic analysis showed that the genus Cronobacter forms a homogenous cluster related to recently described species of Enterobacter, but distant to other species of this genus. Combining sequence information on all three genes is highly representative for the species' %GC-content used as taxonomic marker. Sequence similarity of the three genes and even of recN alone can be used to extrapolate genetic similarities between species of Enterobacteriaceae. Finally, the rpoA gene sequence, which is the easiest one to determine, provides a powerful diagnostic tool to identify and differentiate species of this family. The comparative analysis gives important insights into the phylogeny and genetic relatedness of the family Enterobacteriaceae and will serve as a basis for further studies and clarifications on the taxonomy of this large and heterogeneous family.
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4. Chen  W, Ai  L, Yang  J, Ren  J, Li  Y, Guo  B,     ( 2013 )

Development of a PCR assay for rapid detection of Cronobacter spp. from food.

Canadian journal of microbiology 59 (10)
PMID : 24102218  :   DOI  :   10.1139/cjm-2013-0243    
Abstract >>
The occurrence of outbreaks of necrotizing meningitis caused by Cronobacter spp. in neonates highlights the need for rapid detection and accurate identification of this pathogenic species. The gold standard for isolation and identification of Cronobacter spp. from powdered infant formula is time consuming and labor intensive. The gyrB gene that encodes the B subunit of DNA gyrase (topoisomerase type II) was found to be suitable for the identification of Cronobacter spp. A region of the gyrB gene of 38 Cronobacter spp. strains and 5 Enterobacter spp. strains was amplified and sequenced, and a pair of primers was designed and synthesized based on the sequence of the gyrB gene. A polymerase chain reaction (PCR) system was developed and optimized to detect Cronobacter spp. The PCR assay amplified a 438 bp DNA product from all 38 Cronobacter spp. strains tested but not from 34 other bacteria. The detection limit was 1.41 pg/PCR (equivalent 282 genomic copies) when the genomic DNA of Cronobacter sakazakii ATCC 29544 was 10-fold diluted. Infant formula powders from 3 different commercial brands were inoculated with strains ATCC 29544 at a level of 56 colony-forming units, and the target fragment were produced after samples were enriched for 6 h at 37 �XC. Twenty-five food samples were evaluated by the PCR assay and the conventional method. A PCR product of the expected size was obtained from 3 samples; however, Cronobacter spp. strains were isolated from only 2 samples by the conventional method. This method is a useful tool for rapid identification of Cronobacter spp. in food and potentially environmental samples.
KeywordMeSH Terms
Food Microbiology
5.     ( 2013 )

Use of novel species-specific PCR primers targeted to DNA gyrase subunit B (gyrB) gene for species identification of the Cronobacter sakazakii and Cronobacter dublinensis.

Molecular and cellular probes 27 (1)
PMID : 22963906  :   DOI  :   10.1016/j.mcp.2012.08.004    
Abstract >>
Cronobacter sakazakii and its phylogenetically closest species are considered to be an opportunistic pathogens associated with food-borne disease in neonates and infants. Neither phenotypic nor genotypic (16S ribosomal DNA sequence analysis) techniques can provide sufficient resolutions for accurately and rapidly identification of these species. The objective of this study was to develop species-specific PCR based on the gyrB gene sequence for direct species identification of the C. sakazakii and Cronobacter dublinensis within the C. sakazakii group. Two pair of species-specific primers were designed and used to specifically identify C. sakazakii and C. dublinensis, but none of the other C. sakazakii group strains. Our data indicate that the novel species-specific primers could be used to rapidly and accurately identify the species of C. sakazakii and C. dublinensis from C. sakazakii group by the PCR based assays.
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