Modelling the bacterial communities associated with cystic fibrosis lung infections.

In many human diseases that cystic fibrosis (CF) patients suffer from, for example, lung infections, bacteria have been considered to grow as biofilms. The ability of key CF pathogens such as Pseudomonas aeruginosa to resist antibiotic therapies may be due to the poor drug penetration of these biofilms. The overall aim of this study was to develop biofilm models in vitro that resembled the bacterial species composition of CF sputa. Here, this was a step towards a longer term goal of forming multiple bacterial biofilm models in vitro that would serve, in turn, as better assays of antibiotic susceptibilities than conventionally grown cells. Biofilm models were constructed from 31 CF sputum samples, using a modified microtitre plate assay. Three forms of assessment of these biofilms were made, namely, the mass, microscopic analysis and species composition. Species composition in sputa and biofilms, characterised by terminal restriction fragment length polymorphism (T-RFLP) analysis of ribosomal gene polymerase chain reaction (PCR) products amplified from directly extracted nucleic acids, indicated that the bacterial community in sputa was well reproduced in the biofilm models. Typically, fresh sputa contained 4.6 +/- 2.3 bacterial species, with the species number decreasing to 4.0 +/- 1.6 over 5 days-this was not statistically significant (p = 0.29). This study outlines a novel methodology by which to generate and study bacterial biofilms communities. It is also hoped that the versatility of this in vitro approach, combined with its simplicity and high reproducibility, will make it an effective system to study CF sputum biofilm development and, in the longer term, serve as a means of assessing antibiotic susceptibilities.

Complications of obstetric regional analgesia: how much information is enough?

Two hundred parturients who had received epidural analgesia during labour (100 in Melbourne, Australia and 100 in London, UK) were asked on the first postnatal day about their sources of antenatal information on pain relief in labour, their awareness of potential complications of epidural analgesia and the level of risk at which they would wish to be informed before consenting to a procedure. Sources of antenatal information were similar in the two countries although more women in Australia received information from an anaesthetist or obstetrician than in the UK, whilst more women in the UK received information from the media than in Australia. Knowledge of risks was also similar although the Australian subjects were more aware of infective complications while those in the UK were more aware of intravascular injection of local anaesthetic; these differences may reflect recent high-profile cases in the two countries. The preferred level of risk at which women wanted to be informed about a complication varied from 1:1 to 1:1,000,000,000 in all three centres. The majority of women considered that the benefits of epidural analgesia outweighed each of the potential complications. Women differ in their requirements for antenatal information about regional analgesia and its complications, with some wanting to know every complication, however rare. Anaesthetists should be flexible in their disclosure of information when obtaining consent for regional analgesia and consider the particular wishes of each patient rather than follow rigid centralised guidelines.

Population dynamics and gene transfer in genetically modified bacteria in a model microcosm.

The horizontal transfer and effects on host fitness of a neutral gene cassette inserted into three different genomic loci of a plant-colonizing pseudomonad was assessed in a model ecosystem. The KX reporter cassette (kanamycin resistance, aph, and catechol 2, 3, dioxygenase, xylE) was introduced on the disarmed transposon mini-Tn5 into: (I) the chromosome of a spontaneous rifampicin resistant mutant Pseudomonas fluorescens SBW25R; (II) the chromosome of SBW25R in the presence of a naturally occurring lysogenic-phage (phage Phi101); and (III) a naturally occurring plasmid pQBR11 (330 kbp, tra+, Hgr) introduced into SBW25R. These bacteria were applied to Stellaria media (chickweed) plants as seed dressings [c. 5 x 104 colony-forming units (cfu)/seed] and the seedlings planted in 16 microcosm chambers containing model plant and animal communities. Gene transfer to pseudomonads in the phyllosphere and rhizosphere was found only in the plasmid treatment (III). Bacteria in the phage treatment (II) initially declined in density and free phage was detected, but populations partly recovered as the plants matured. Surprisingly, bacteria in the chromosome insertion treatment (I) consistently achieved higher population densities than the unmanipulated control and other treatments. Plasmids were acquired from indigenous bacterial populations in the control and chromosome insertion treatments. Plasmid acquisition, plasmid transfer from inocula and selection for plasmid carrying inocula coincided with plant maturation.

Ecological and molecular maintenance strategies of mobile genetic elements.

This review considers the influence of selection pressure, fitness and population structures on the evolution of mobile genetic elements (including plasmids, phage, pathogenicity islands, transposons and insertion sequences) that constitute the horizontal gene pool of bacteria. These are considered at different scales using examples from in vitro evolutionary studies of Escherichia coli and associated bacteriophage, detailed molecular analyses of the broad host-range IncP-1 plasmids, population surveys of pseudomonad plasmids and genomic comparisons of members of the Rhizobiaceae. All biological systems show genetic redundancy (the existence of allelic variation) at some population level, i.e. within a cell, a clone, population or community. We consider the level(s) at which redundancy is expressed and how this will affect and has influenced the evolution of mobile genetic elements.

The transfer dynamics of Pseudomonas sp. plasmid pQBR11 in biofilms.

Plasmid pQBR11 (294 kb) is one of a group of genetically similar conjugative plasmids that are common to and persist in pseudomonad populations colonising leaves and roots of crops and wild plants. This plasmid was marked by insertion of a green fluorescent protein reporter cassette to facilitate epifluorescence microscopy tracking and enumeration of transconjugant formation in biofilms. The intrinsic transfer activity of the plasmid, the conditions which affect this activity and the patterns of infectious spread of the plasmid in simple biofilms were evaluated. Transfer was observed over a range of nutritional and other conditions but no transitory de-repression of plasmid transfer was observed from new transconjugants and re-transfer of the plasmid by transconjugant cells played only a secondary role in the establishment of the plasmid in biofilm populations. As the plasmid was poorly invasive the parasitic persistence of pQBR11 by transfer alone is highly unlikely. The key factors for establishment of this plasmid in these biofilm populations were the size and activity of the donor population and the effect of plasmid carriage on host fitness.

Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot).

The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m). Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis. The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February. The structure of the sampled community was described using: species richness, Simpson's index and the Shannon-Wiener index. All three indices detected a number of significant differences with depth demonstrating stratification. The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity. Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure. Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera. Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates. Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

Ecological and physiological analyses of Pseudomonad species within a phenol remediation system.

A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial treatment plant was made to select potential strains as microbial biosensors. Pseudomonads were the most abundant group, of which 48 selected from the liquor or suspended solids were assessed for their physiological response to phenolic pollutant loading and niche specialisation. By FAME-MIS identification the Pseudomonads were clustered into six major species groups. Those isolates able to utilise phenol as a sole carbon source predominantly belonged to a non-clonal Pseudomonas pseudoalcaligenes cluster determined by REP-PCR genotyping. Rapid microtitre based respiration assays were developed to contrast activity in response to increasing concentrations of phenol. A considerable range in response for both phenol degrader and non-degrader strains was observed. This natural phenotypic and physiological heterogeneity could facilitate the selection of isolates for the development of a suite of ecologically relevant, custom designed sensors with predictable toxicity susceptibilities to monitor process efficacy.

Activation of neurokinin NK(2) receptors by tachykinin peptides causes contraction of uterus in pregnant women near term.

The aim of this study was firstly to elucidate whether the mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)-regulated contractility of myometrium obtained from near-term pregnant women, and secondly to investigate the receptor subtype(s) responsible. In the presence of peptidase inhibitors, i.e. thiorphan (3 micromol/l; endopeptidase 24.11 inhibitor), captopril (10 micromol/l; angiotensin converting enzyme inhibitor) and bestatin (10 micromol/l; aminopeptidase inhibitor); all three mammalian tachykinins elicited concentration-related contractions of isolated myometrial preparations. The rank order of agonist potency of the mammalian tachykinins in the presence of the peptidase inhibitors was NKA > SP = NKB, indicating that the contractile effects were mediated by activation of an NK(2) receptor. The NK(2) receptor-selective agonist, [Lys(5), MeLeu(9), Nle(10)]NKA(4-10), produced concentration-related contractile responses, while the respective NK(1) and NK(3) receptor-selective agonists, [Sar(9), Met(O(2))(11)]SP and [N-MePhe(7)]NKB, had no effect either in the absence or presence of the peptidase inhibitors. The NK(2) receptor-selective antagonist, SR48968, produced concentration-related rightward shift in the log concentration curve to [Lys(5), MeLeu(9), Nle(10)]NKA(4-10). This study shows that tachykinins elicit contractile effects on human myometrium obtained from pregnant women near term, and that these effects are mediated by an NK(2) receptor. An excitatory effect of the tachykinins on these preparations could indicate a physiological role for these peptides in enhancing contractility of the uterus in women at term.

Description of a novel plasmid replicative origin from a genetically distinct family of conjugative plasmids associated with phytosphere microflora.

A novel replicative origin (oriV) from a conjugative, mercury resistance plasmid (pQBR11, 304 kbp) has been cloned and sequenced. Homology to the pQBR11 oriV-containing 3.55 kbp BamHI fragment (pCV1200) was restricted to one of five genetically distinct classes (group I) of narrow host range, mega-plasmids that persist as a genetic component of the pseudomonad community indigenous to the microflora of sugar beet. The oriV of pQBR11 was located within a unique sequence of 300 bp which initiated the replication of pUC derived suicide vectors in Pseudomonas putida UWC1. The limited size of the DNA sequence required to initiate replication, and the presence of two 15/16 bp directly repeated motifs, indicate that this group of mega-plasmids contain a single origin of replication, which initiates replication via a host-polymerase dependent rolling circle mechanism.

The acquisition of indigenous plasmids by a genetically marked pseudomonad population colonizing the sugar beet phytosphere is related to local environmental conditions.

The transfer of naturally occurring conjugative plasmids from the indigenous microflora to a genetically modified population of bacteria colonizing the phytospheres of plants has been observed. The marked strain (Pseudomonas fluorescens SBW25EeZY6KX) was introduced as a seed dressing to sugar beets (Beta vulgaris var. Amethyst) as part of a field experiment to assess the ecology and genetic stability of deliberately released bacterial inocula. The sustained populations of the introduced strain, which colonized the phytosphere, were assessed throughout the growing season for the acquisition of plasmids conferring mercury resistance (Hg(supr)). Transconjugants were isolated only from root and leaf samples collected within a narrow temporal window coincident with the midseason maturation of the crop. Conjugal-transfer events were recorded during this defined period in two separate field release experiments conducted over consecutive years. On one occasion seven of nine individual plants sampled supported transconjugant P. fluorescens SBW25EeZY6KX, demonstrating that conjugative gene transfer between bacterial populations in the phytosphere may be a common event under specific environmental conditions. The plasmids acquired in situ by the colonizing inocula were identified as natural variants of restriction digest pattern group I, III, or IV plasmids from five genetically distinct groups of large, conjugative mercury resistance plasmids known to persist in the phytospheres of sugar beets at the field site. These data demonstrate not only that gene transfer may be a common event but also that the genetic and phenotypic stability of inocula released into the natural environment cannot be predicted.

Impact of Plasmid pQBR103 Acquisition and Carriage on the Phytosphere Fitness of Pseudomonas fluorescens SBW25: Burden and Benefit.

The phytosphere population densities of Pseudomonas fluorescens SBW25EeZY6KX (lacZX aph xylE) carrying pQBR103 (Hg(supr) Tra(sup+), 330 kbp) declined significantly relative to plasmid-free populations after seed inoculation. As the sugar beet plants matured, ca. 100 days after planting, simultaneous selections for plasmid-carrying hosts were observed in the phyllospheres and rhizospheres of field-grown plants. The recovery of these populations to densities indistinguishable from the densities of plasmid-free inocula (4 x 10(sup5) CFU/g in the rhizosphere) demonstrates that phytosphere-associated plasmids confer a specific fitness advantage to host bacteria.

Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer.

A plasmid-free, non-pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes. By site directed homologous recombination a KX cassette [kanamycin resistance (kanr) and catechol 2,3 dioxygenase (xylE)] and a ZY cassette [lactose utilization (lacZY, beta-galactosidase, lactose permease)] were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome. Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro-organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation.