Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.     

Futility without a dichotomy: towards an ideal physician-patient relationship

The futility debate may be considered as an effort to provide a clear and justified borderline between physician and patient decision–making authority. In this paper we argue that the search for a definition of futility that provides physicians with a final argument in discussions about life–prolonging treatment, is misplaced. An acceptable and meaningful criterion of futility that satisfies this effort seems impossible. As a consequence, we reject a dichotomous domain of decision–making power as the starting point for definitions of futility. A good decision about withholding life–sustaining treatment should be justified from the perspectives of both physician and patient. In this light, a range of definitions of futility is still useful as it can clarify intuitions that a treatment is inappropriate.     

Nucleic acid testing (NAT) in high prevalence–low resource settings

Blood screening by NAT for major transfusion transmitted viral infections (TTIs) was originally intended to complement serology for detection of infected donations. Reports from developed countries showed limited marginal value to NAT blood screening in improving blood safety. Reports on NAT results from Europe indicated yield of 1:0.6 million donations for HBV, <1:M for HCV and HIV-1-related to low prevalence of TTI. In contrast, prevalence of TTI in resource-limited countries is almost always high. As a result, more incident cases can be expected among first-time blood donors. Most reports of NAT blood donation screening in these countries showed NAT confirmed yield as high as 1/2800 for HBV and 1/3100 blood donations for HCV as reported from Thailand and Egypt, respectively. The issues for low resource countries are mostly the high cost of NAT but also the requirements of staff qualification, adequate facilities, reagent procurement and maintenance of delicate equipment. Alternatives to commercial NAT are the use of combos antigen-antibody for HIV and HCV, anti-HBc for HBV and in-house NAT. Most of these alternatives have been reported but very few comparisons are available. Once yield data is available, models for estimation of feasibility and cost-effectiveness are proposed to help decision-making.     

Endophytic bacterial diversity in poplar trees growing on a BTEX-contaminated site: the characterisation of isolates with potential to enhance phytoremediation

The diversity of endophytic bacteria found in association with poplar was investigated as part of a larger study to assess the possibility and practicality of using endophytic bacteria to enhance in situ phytoremediation. Endophytic bacteria were isolated from the root, stem and leaf of two cultivars of poplar tree growing on a site contaminated with BTEX compounds. They were further characterised genotypically by comparative sequence analysis of partial 16S rRNA genes and BOX-PCR genomic DNA fingerprinting, and phenotypically by their tolerance to a range of target pollutants, heavy metals and antibiotics. One hundred and 21 stable, morphologically distinct isolates were obtained, belonging to 21 genera, although six isolates could not be identified with confidence to a genus. The endophytic bacteria exhibited marked spatial compartmentalisation within the plant, suggesting there are likely to be species-specific and non-specific associations between bacteria and plants. A number of isolates demonstrated the ability to degrade BTEX compounds or to grow in the presence of TCE. This study demonstrates that within the diverse bacterial communities found in poplar several endophytic strains are present that have the potential to enhance phytoremediation strategies.     

DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria

A denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of dsrB (dissimilatory sulfite reductase beta-subunit)-genes in sulfate-reducing communities. For this purpose a PCR primer pair was optimized for the amplification of a approximately 350 bp dsrB gene fragment that after DGGE gel electrophoresis enabled us to discriminate between dsrB genes of different SRB-subgroups,-genera and -species. The dsrB-DGGE method revealed considerable genetic diversity when applied to DNA extracts obtained from aquifer samples that were derived from monitoring wells of an in situ metal precipitation (ISMP) pilot project conducted at the site of a non-ferrous industry or from environmental heavy metal contaminated samples. The sequences of the excised and sequenced DGGE bands represented dsrB genes of different SRB-subgroups,-genera and -species, thus confirming the broad applicability of the PCR primer pair. Linking the results of the physico-chemical follow-up of the field and lab experiments to the dsrB-DGGE data will provide a better understanding of the contribution of the SRB populations to the ongoing ISMP processes.     

Use of single-point genome signature tags as a universal tagging method for microbial genome surveys

We developed single-point genome signature tags (SP-GSTs), a generally applicable, high-throughput sequencing-based method that targets specific genes to generate identifier tags from well-defined points in a genome. The technique yields identifier tags that can distinguish between closely related bacterial strains and allow for the identification of microbial community members. SP-GSTs are determined by three parameters: (i) the primer designed to recognize a conserved gene sequence, (ii) the anchoring enzyme recognition sequence, and (iii) the type IIS restriction enzyme which defines the tag length. We evaluated the SP-GST method in silico for bacterial identification using the genes rpoC, uvrB, and recA and the 16S rRNA gene. The best distinguishing tags were obtained with the restriction enzyme Csp6I upstream of the 16S rRNA gene, which discriminated all organisms in our data set to at least the genus level and most organisms to the species level. The method was successfully used to generate Csp6I-based tags upstream of the 16S rRNA gene and allowed us to discriminate between closely related strains of Bacillus cereus and Bacillus anthracis. This concept was further used successfully to identify the individual members of a defined microbial community.