Identification of Brevibacterium flavum genes related to receptors involved in bacteriophage BFK20 adsorption.

Phage infection of bacterial cells is a process requiring the interaction between phage receptor binding proteins and receptors on the bacterial cell surface. We prepared a Brevibacterium flavum CCM 251 EZ-Tn5 transposon insertional library and isolated phage-resistant mutants. Analysis of the DNA fragments produced by single-primer PCR was used to determine the EZ-Tn5 transposon insertion sites in the genomes of phage-resistant B. flavum mutants. Seven disrupted genes were identified in forty B. flavum mutants. The phage resistance of these mutants was demonstrated by cultivation analysis in the presence of BFK20, and the adsorption rate of BFK20 to these mutants was tested. B. flavum mutants displayed significantly reduced adsorption rates; the lowest rate was observed for mutants containing interrupted major facilitator superfamily (MFS) protein and glycosyltransferase genes. Uninterrupted forms of these genes were cloned into corynebacterial vector pJUP06 and used for in trans complementation of the corresponding B. flavum mutants. The growth of these complemented mutants when infected with BFK20 closely resembled that of wild-type B. flavum. These complemented mutants also exhibited similar BFK20 adsorption as the wild-type control. We infer that the disrupted MFS protein and glycosyltransferase genes are responsible for the phage-resistant phenotype of these B. flavum transposition mutants.

Contribution of the thermotolerance genomic island to increased thermal tolerance in Cronobacter strains.

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 thrB-Q genes and a shorter version (6 kbp) harbouring only the thrBCD and thrOP 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 rpoS, 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.

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.