Mortality in adult intensive care patients with severe systemic inflammatory response syndromes is strongly associated with the hypo-immune TNF -238A polymorphism.

The systemic inflammatory response syndrome (SIRS) is associated with activation of innate immunity. We studied the association between mortality and measures of disease severity in the intensive care unit (ICU) and functional polymorphisms in genes coding for Toll-like receptor 4 (TLR4), macrophage migratory inhibitory factor (MIF), tumour necrosis factor (TNF) and lymphotoxin-alpha (LTA). Two hundred thirty-three patients with severe SIRS were recruited from one general adult ICU in a tertiary centre in the UK. DNA from patients underwent genotyping by 5' nuclease assay. Genotype was compared to phenotype. Primary outcome was mortality in ICU. Minor allele frequencies were TLR4 +896G 7%, MIF 173C 16%, TNF -238A 10% and LTA +252G 34%. The frequency of the hypoimmune minor allele TNF -238A was significantly higher in patients who died in ICU compared to those who survived (p = 0.0063) as was the frequency of the two haplotypes LTA +252G, TNF -1031T, TNF -308G, TNF -238A and LTA +252G, TNF-1031T, TNF-308A and TNF-238A (p = 0.0120 and 0.0098, respectively). These findings re-enforce the view that a balanced inflammatory/anti-inflammatory response is the most important determinant of outcome in sepsis. Genotypes that either favour inflammation or its counter-regulatory anti-inflammatory response are likely to influence mortality and morbidity.

Bacteria associated with cysts of the soybean cyst nematode (Heterodera glycines).

The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world. The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops. To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means. Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst. Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 +/- 0.5) x 10(5) bacteria (mean +/- standard deviation) with various cellular morphologies. Filamentous fungi were also observed. Live-dead staining indicated that the majority of cyst bacteria were viable. The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature. Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition. Bands shared by all eight samples included the actinobacterium genera Actinomadura and STREPTOMYCES: A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium. These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence. Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp. This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.

The triazine hydrolase gene trzN from Nocardioides sp. strain C190: cloning and construction of gene-specific primers.

Using oligonucleotides derived from the N-terminal sequence of a triazine hydrolase from Nocardioides sp. strain C190, two DNA fragments containing trzN were cloned into Escherichia coli and their nucleotide sequences were determined. The 456-amino acid polypeptide predicted from the 1356-bp trzN ORF displayed significant similarity to triazine hydrolases from Pseudomonas and Rhodococcus isolates and belonged to the same amidohydrolase family. The trzN gene was flanked by two DNA sequences possessing 57 and 69% identity, respectively, at the protein level to Rhodococcus erythropolis sequences for a transposase and a transposase helper protein. Amplification primers specific to trzN were tested in soils inoculated with strain C190. The results demonstrated that the primers were specific to trzN, and could detect populations at 10(8) cfu g(-1) soil using 250-mg soil samples.

Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts.

The nodC and nifH genes were characterized in a collection of 83 rhizobial strains which represented 23 recognized species distributed in the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium, as well as unclassified rhizobia from various host legumes. Conserved primers were designed from available nucleotide sequences and were able to amplify nodC and nifH fragments of about 930 bp and 780 bp, respectively, from most of the strains investigated. RFLP analysis of the PCR products resulted in a classification of these rhizobia which was in general well-correlated with their known host range and independent of their taxonomic status. The nodC and nifH fragments were sequenced for representative strains belonging to different genera and species, most of which originated from Phaselous vulgaris nodules. Phylogenetic trees were constructed and revealed close relationships among symbiotic genes of the Phaseolus symbionts, irrespective of their 16S-rDNA-based classification. The nodC and nifH phylogenies were generally similar, but cases of incongruence were detected, suggesting that genetic rearrangements have occurred in the course of evolution. The results support the view that lateral genetic transfer across rhizobial species and, in some instances, across Rhizobium and Sinorhizobium genera plays a role in diversification and in structuring the natural populations of rhizobia.