Persistence of enterohaemorrhagic and nonpathogenic E. coli on spinach leaves and in rhizosphere soil*

Aims: Survival of Escherichia coli O157:H7 and nonpathogenic E. coli on spinach leaves and in organic soil while growing spinach in a growth chamber was investigated. Methods and Results: Spinach plants were maintained in the growth chamber at 20°C (14 h) and 18°C (10 h) settings at 60% relative humidity. Five separate inocula, each containing one strain of E. coli O157:H7 and one nonpathogenic E. coli isolate were applied to individual 4‐week‐old spinach plants (cultivar ‘Whale’) grown in sandy soil. Leaf and soil inocula consisted of 100 μl, in 5 μl droplets, on the upper side of leaves resulting in 6·5 log CFU plant^?1 and 1 ml in soil, resulting in 6·5 log CFU 200 g^?1 soil per plant. Four replicates of each plant shoot and soil sample per inoculum were analysed on day 1 and every 7 days for 28 days for E. coli O157:H7 and nonpathogenic E. coli (by MPN) and for heterotrophic plate counts (HPC). Escherichia coli O157:H7 was not detected on plant shoots after 7 days but did survive in soil for up to 28 days. Nonpathogenic E. coli survived up to 14 days on shoots and was detected at low concentrations for up to 28 days. In contrast, there were no significant differences in HPC from days 0 to 28 on plants, except one treatment on day 7. Conclusions: Escherichia coli O157:H7 persisted in soil for at least 28 days. Escherichia coli O157:H7 on spinach leaves survived for less than 14 days when co‐inoculated with nonpathogenic E. coli. There was no correlation between HPC and E. coli O157:H7 or nonpathogenic E. coli. Significance and Impact of the Study: The persistence of nonpathogenic E. coli isolates makes them possible candidates as surrogates for E. coli O157:H7 on spinach leaves in field trials.     

Plant receptor kinases bind and phosphorylate 14-3-3 proteins

14-3-3 proteins are pSer/pThr-binding proteins that interact with a wide array of cellular ‘client’ proteins. The plant brassinosteroids (BRs) receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is a member of the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that contain cytoplasmic protein kinase domains. At least two LRR-RLKs are involved in BR perception and signal transduction: BRI1 and BRI1-associated receptor kinase 1 (BAK1). We determined that several 14-3-3 proteins bind to BRI1-CD and are phosphorylated by BRI1, BAK1 and At3g21430 receptor kinases in vitro. Moreover, we observed14-3-3 s are phosphorylated on threonine residue(s) with BR-dependent manner. To reveal the function of 14-3-3 proteins interacting with LRR-RLKs, we treated tyrosine phosphatase (PTP1B) to the BRI1-CD recombinant protein, which is autophosphorylated on tyrosine residue(s). Tyrosine autophosphorylation signal was disappeared, suggesting that 14-3-3 proteins cannot protect BRI1 tyrosine phosphorylation from PTP1B phosphatase. Our study suggests that 14-3-3 proteins may be important for plant growth and development through BR signaling.     

Identification of NBS-encoding genes linked to black rot resistance in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis>)

Heading cabbage is a nutritionally rich and economically important cruciferous vegetable. Black rot disease, caused by the bacterium Xanthomonas campestris pv. campestris, reduces both the yield and quality of the cabbage head. Nucleotide binding site (NBS)-encoding resistance (R) genes play a vital role in the plant immune response to various pathogens. In this study, we analyzed the expression and DNA sequence variation of 31 NBS-encoding genes in cabbage (Brassica oleracea var. capitata). These genes encoded TIR, NBS, LRR and RPW8 protein domains, all of which are known to be involved in disease resistance. RNA-seq revealed that these 31 genes were differentially expressed in leaf, root, silique, and stem tissues. Furthermore, qPCR analyses revealed that several of these genes were more highly expressed in resistant compared to susceptible cabbage lines, including Bol003711, Bol010135, Bol010559, Bol022784, Bol029866, Bol042121, Bol031422, Bol040045 and Bol042095. Further analysis of these genes promises to yield both practical benefits, such as molecular markers for marker-assisted breeding, and fundamental insights to the mechanisms of resistance to black rot in cabbage.     

Synthesis of 4-amino-4,5-dideoxy-L-lyxofuranose derivatives and their evaluation as fucosidase inhibitors

The nitrone 4 (4,5-dideoxy-4-hydroxylamino-3,4-O-isopropylidene-L-lyxofuranose) was synthesised from D-ribose and used as key intermediate for the preparation of fucosidase inhibitors. We describe two transformations of 4. Hydrolysis with aqueous sulfur dioxide gave the known potent nanomolar inhibitor 4-amino-4,5-dideoxy-L-lyxofuranose (3). 1,3-Dipolar cycloaddition with enol ethers led to the related 1,2,5,6-tetradeoxy-2,5-imino-L-altroheptonic ester 2a, acid 2b and the corresponding heptitol 2c. The new iminosugars have been evaluated for their inhibitory activity against α-L-fucosidase from bovine kidney. The alcohol 2c turned out to be a potent inhibitor in the same range as the amino-sugar 3 (K(i)=8 vs 10nM).     

Detection of cross-infection associated to a Brazilian PCR-ribotype of <Emphasis Type="Italic">Clostridium difficile</Emphasis> in a university hospital in Rio de Janeiro,Brazil

Clostridium difficile is an important nosocomial enteric pathogen and is the etiological agent of pseudomembranous colites. Recently, the rates of C. difficile infection (CDI) have increased worldwide, but in Brazil few data about this situation and the incidence of clonal types of C. difficile exist. This study aimed to isolate and characterize C. difficile strains from samples obtained of a university hospital (HUCFF) in Rio de Janeiro city, Brazil. CDI was identified by ELISA in 27.1% of HUCFF-in-patients enrolled in the study, and the bacterium was recovered from eight of these fecal samples. All strains, except one, presented tcdA and tcdB genes and presented neither the cdtA and cdtB genes nor any significant deletions in the tcdC gene. All strains were sensitive to metronidazole, vancomycin and moxifloxacin, and resistant to clindamycin, ciprofloxacin and levofloxacin. PCR-ribotyping and PFGE revealed four different clonal types among the isolates. The Brazilian PCR-ribotype 133 accounted for 50% of strains isolated, and PCR-ribotype 233 strains were obtained from 25% of the in-patients. The prevalence and resurgence of the Brazilian PCR-ribotype 133 among the hospitalized patients of HUCFF was established, and cross-infection of different patients associated to the same PCR-ribotypes was detected. Our results emphasize the importance of the diagnosis and control of CDI in order to prevent the emergence of specific clones that can lead to C. difficile-associated outbreaks in Brazilian hospitals.