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SUMMARYWe investigated the incidence of cases of nosocomial pathogens and risk factors in an intensive treatment unit ward to determine if the number of cases is dependent on location of patients and the colonization/infection history of the ward. A clustering approach method was developed to investigate the patterns of spread of cases through time for five microorganisms [methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter spp., Klebsiella spp., Candida spp., and Pseudomonas aeruginosa] using hospital microbiological monitoring data and ward records of patient-bed use. Cases of colonization/infection by MRSA, Candida and Pseudomonas were clustered in beds and through time while cases of Klebsiella and Acinetobacter were not. We used structural equation modelling to analyse interacting risk factors and the potential pathways of transmission in the ward. Prior nurse contact with colonized/infected patients, mediated by the number of patient-bed movements, were important predictors for all cases, except for those of Pseudomonas. General health and invasive surgery were significant predictors of cases of Candida and Klebsiella. We suggest that isolation and bed movement as a strategy to manage MRSA infections is likely to impact upon the incidence of cases of other opportunist pathogens. [ABSTRACT FROM AUTHOR]

Mining of mineral resources substantially alters both the above and below-ground soil ecosystem, which then requires rehabilitation back to a pre-mining state. For belowground rehabilitation, recovery of the soil microbiome to a state which can support key biogeochemical cycles, and effective plant colonization is usually required. One solution proposed has been to translate microbial inocula from agricultural systems to mine rehabilitation scenarios, as a means of reconditioning the soil microbiome for planting. Here, we experimentally determine both the aboveground plant fitness outcomes and belowground soil microbiome effects of a commercially available soil microbial inocula (SMI). We analyzed treatment effects at four levels of complexity; no SMI addition control, Nitrogen addition alone, SMI addition and SMI plus Nitrogen addition over a 12-week period. Our culture independent analyses indicated that SMIs had a differential response over the 12-week incubation period, where only a small number of the consortium members persisted in the semi-arid ecosystem, and generated variable plant fitness responses, likely due to plant-microbiome physiological mismatching and low survival rates of many of the SMI constituents. We suggest that new developments in custom-made SMIs to increase rehabilitation success in mine site restoration are required, primarily based upon the need for SMIs to be ecologically adapted to both the prevailing edaphic conditions and a wide range of plant species likely to be encountered.

Terrestrial ecosystems in the maritime Antarctic experienced rapid warming during the latter half of the 20 th century. While warming ceased at the turn of the millennium, significant increases in air temperature are expected later this century, with predicted positive effects on soil fungal diversity, plant growth and ecosystem productivity. Here, by sequencing 16S ribosomal RNA genes in 40 soils sampled from along a 1,650 km climatic gradient through the maritime Antarctic, we determine whether rising air temperatures might similarly influence the diversity of soil bacteria. Of 22 environmental factors, mean annual surface air temperature was the strongest and most consistent predictor of soil bacterial diversity. Significant, but weaker, associations between bacterial diversity and soil moisture content, C:N ratio, and Ca, Mg, PO 4 3- and dissolved organic C concentrations were also detected. These findings indicate that further rises in air temperature in the maritime Antarctic may enhance terrestrial ecosystem productivity through positive effects on soil bacterial diversity.

Mining of mineral resources produces substantial volumes of crushed rock based wastes that are characterised by poor physical structure and hydrology, unstable geochemistry and potentially toxic chemical conditions. Recycling of these substrates is desirable and can be achieved by blending waste with native soil to form a 'novel substrate' which may be used in future landscape restoration. However, these post-mining substrate based 'soils' are likely to contain significant abiotic constraints for both plant and microbial growth. Effective use of these novel substrates for ecosystem restoration will depend on the efficacy of stored topsoil as a potential microbial inoculum as well as the subsequent generation of key microbial soil functions originally apparent in local pristine sites. Here, using both marker gene and shotgun metagenome sequencing, we show that topsoil storage and the blending of soil and waste substrates to form planting substrates gives rise to variable bacterial and archaeal phylogenetic composition but a high degree of metabolic conservation at the community metagenome level. Our data indicates that whilst low phylogenetic conservation is apparent across substrate blends we observe high functional redundancy in relation to key soil microbial pathways, allowing the potential for functional recovery of key belowground pathways under targeted management.

Ammonia oxidizing archaea (AOA) and bacteria (AOB) drive nitrification and their population dynamics impact directly on the global nitrogen cycle. AOA predominate in the majority of soils but an increasing number of studies have found that nitrification is largely attributed to AOB. The reasons for this remain poorly understood. Here, amoA gene abundance was used to study the distribution of AOA and AOB in agricultural soils on different parent materials and in contrasting geologic landscapes across Australia (n = 135 sites). AOA and AOB abundances separated according to the geologic age of the parent rock with AOB higher in the more weathered, semi-arid soils of Western Australia. AOA dominated the younger, higher pH soils of Eastern Australia, independent of any effect of land management and fertilization. This differentiation reflects the age of the underlying parent material and has implications for our understanding of global patterns of nitrification and soil microbial diversity. Western Australian soils are derived from weathered archaean laterite and are acidic and copper deficient. Copper is a co-factor in the oxidation of ammonia by AOA but not AOB. Thus, copper deficiency could explain the unexpectedly low populations of AOA in Western Australian soils.

Laccase enzymes produced by both soil bacteria and fungi play important roles in refractory organic matter turnover in terrestrial ecosystems. We investigated the abundance and diversity of fungal laccase genes and bacterial laccase-like genes in soil from subtropical arable lands, and identified which microbial group was associated with laccase activity. Compared with fungal laccase genes, the bacterial laccase-like genes had greater abundance, richness and Shannon-Wiener diversity. More importantly, laccase activity can be explained almost exclusively by the bacterial laccase-like genes, and their abundance had significant linear relationship with laccase activity. Thus, bacterial laccase-like gene has great potential to be used as a sensitive indicator of laccase enzyme for refractory organic matter turnover in subtropical arable lands.

Soils were incubated continuously in an atmosphere of (14)CO2 and the distribution of labeled C into soil organic carbon ((14)C-SOC) was determined at 0-1, 1-5, and 5-17 cm down the profile. Significant amounts of (14)C-SOC were measured in paddy soils with a mean of 1,180.6 ± 105.2 mg kg(-1) at 0-1 cm and 135.3 ± 47.1 mg kg(-1) at 1-5 cm. This accounted for 5.9 ± 0.7% and 0.7 ± 0.2%, respectively, of the total soil organic carbon at these depths. In the upland soils, the mean (14)C-SOC concentrations were 43 times (0-1 cm) and 11 times (1-5 cm) lower, respectively, than those in the paddy soils. The amounts of (14)C incorporated into the microbial biomass (MBC) were also much lower in upland soils (5.0 ± 3.6% and 2.9 ± 1.9% at 0-1 and 1-5 cm, respectively) than in paddy soils (34.1 ± 12.4% and 10.2 ± 2.1% at 0-1 and 1-5 cm, respectively). Similarly, the amount of (14)C incorporated into the dissolved organic carbon (DOC) was considerably higher in paddy soils (26.1 ± 6.9% and 6.9 ± 1.3% at 0-1 and 1-5 cm, respectively) than in upland soils (6.0 ± 2.7% and 4.3 ± 2.2%, respectively). The observation that the majority of the fixed (14)C-SOC, RubisCO activity and cbbL gene abundance were concentrated at 0-1 cm depth and the fact that light is restricted to the top few millimeters of the soil profiles highlighted the importance of phototrophs in CO2 fixation in surface soils. Phylogenetic analysis of the cbbL genes showed that the potential for CO2 fixation was evident throughout the profile and distributed between both photoautotrophic and chemoautotrophic bacteria such as Rhodopseudomonas palustris, Bradyrhizobium japonicum, Rubrivivax gelatinosus and Ralstonia eutropha.

Soils were incubated for 80 days in a continuously labeled (14)CO(2) atmosphere to measure the amount of labeled C incorporated into the microbial biomass. Microbial assimilation of (14)C differed between soils and accounted for 0.12% to 0.59% of soil organic carbon (SOC). Assuming a terrestrial area of 1.4 × 10(8) km(2), this represents a potential global sequestration of 0.6 to 4.9 Pg C year(-1). Estimated global C sequestration rates suggest a "missing sink" for carbon of between 2 and 3 Pg C year(-1). To determine whether (14)CO(2) incorporation was mediated by autotrophic microorganisms, the diversity and abundance of CO(2)-fixing bacteria and algae were investigated using clone library sequencing, terminal restriction fragment length polymorphism (T-RFLP), and quantitative PCR (qPCR) of the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbL). Phylogenetic analysis showed that the dominant cbbL-containing bacteria were Azospirillum lipoferum, Rhodopseudomonas palustris, Bradyrhizobium japonicum, Ralstonia eutropha, and cbbL-containing chromophytic algae of the genera Xanthophyta and Bacillariophyta. Multivariate analyses of T-RFLP profiles revealed significant differences in cbbL-containing microbial communities between soils. Differences in cbbL gene diversity were shown to be correlated with differences in SOC content. Bacterial and algal cbbL gene abundances were between 10(6) and 10(8) and 10(3) to 10(5) copies g(-1) soil, respectively. Bacterial cbbL abundance was shown to be positively correlated with RubisCO activity (r = 0.853; P < 0.05), and both cbbL abundance and RubisCO activity were significantly related to the synthesis rates of [(14)C]SOC (r = 0.967 and 0.946, respectively; P < 0.01). These data offer new insights into the importance of microbial autotrophy in terrestrial C cycling.

This paper identifies key components of the microbial community involved in the mesophilic anaerobic co-digestion (AD) of mixed waste at Rayong Biogas Plant, Thailand. The AD process is separated into three stages: front end treatment (FET); feed holding tank and the main anaerobic digester. The study examines how the microbial community structure was affected by the different stages and found that seeding the waste at the beginning of the process (FET) resulted in community stability. Also, co-digestion of mixed waste supported different bacterial and methanogenic pathways. Typically, acetoclastic methanogenesis was the major pathway catalysed by Methanosaeta but hydrogenotrophs were also supported. Finally, the three-stage AD process means that hydrolysis and acidogenesis is initiated prior to entering the main digester which helps improve the bioconversion efficiency. This paper demonstrates that both resource availability (different waste streams) and environmental factors are key drivers of microbial community dynamics in mesophilic, anaerobic co-digestion., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

The seed oil of Jatropha curcas has been proposed as a source of biodiesel. In plants, seed oil is stored in subcellular organelles called oil bodies (OBs), which are stabilized by proteins. Proteome composition of the J. curcas OBs revealed oleosins as the major component and additional proteins similar to those in other oil seed plants. Three J. curcas oleosins were isolated and characterized at the gene, transcript and protein level. They all contained the characteristic proline knot domain and were each present as a single copy in the genome. The smallest, L-form JcOle3 contained an intron. Isolation of its promoter revealed seed-specific cis-regulatory motifs among others. Spatio-temporal transcript expression of J. curcas oleosins was largely similar to that in other oil seed plants. Immunoassay with antibodies against an Arabidopsis oleosin or against JcOle3, on seed proteins extracted by different approaches, revealed JcOle3 oligomers. Alleles of JcOle3 and single nucleotide polymorphisms (SNPs) in its intron were identified in J. curcas accessions, species and hybrids. Identified alleles and SNPs could serve as markers in phylogenetic or breeding studies., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Palace Leas, a long-term experiment at Cockle Park Farm, Northumberland, UK was established in winter 1896-1897 since when the 13 plots have received regular and virtually unchanged mineral fertiliser and farm yard manure inputs. Fertilisers have had a profound impact on soil pH with the organically fertilised plots showing a significantly higher pH than those receiving mineral fertiliser where ammonium sulphate has led to soil acidification. Here, we investigate the impact of organic and mineral fertilisers on the actinobacterial community structure of these soils using terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene analysis. To differentiate fertiliser effects from seasonal variation, soils were sampled three times over one growing season between May and September 2004 and January 2005. Community profiles obtained using T-RFLP were analysed using multivariate statistics to investigate the relationship between community structure, seasonality and fertiliser management. Soil pH was shown to be the most significant edaphic factor influencing actinobacterial communities. Canonical correspondence analysis, used to investigate the relationship between the 16S rRNA gene community profiles and the environmental parameters, showed that actinobacterial communities also responded to soil water content with major changes evident over the summer months between May and September. Quantitative PCR of the actinobacterial and fungal 16S and 18S rRNA genes, respectively suggested that fungal rRNA gene copy numbers were negatively correlated (P = 0.0131) with increasing actinobacterial signals. A similar relationship (P = 0.000365) was also evident when fatty acid methyl esters indicative of actinobacterial biomass (10-methyloctadecanoic acid) were compared with the amounts of fungal octadecadienoic acid (18:2omega9,12). These results show clearly that soil pH is a major driver of change in actinobacterial communities and that genera such as Arthrobacter and Micrococcus are particularly abundant in soils receiving organic inputs whilst others such as Streptomyces, Acidimicrobium and Actinospica are more prevalent in acid soils. The importance of these findings in terms of fungal abundance and potential disease suppression are discussed.

House mice (Mus domesticus) communicate using scent-marks, and the chemical and microbial composition of these 'extended phenotypes' are both influenced by genetics. This study examined how the genes of the major histocompatibility complex (MHC) and background genes influence the volatile compounds (analysed with Gas Chromatography Mass Spectrometry or GC/MS) and microbial communities (analysed using Denaturating Gradient Gel Electrophoresis or DGGE) in scent-marks produced by congenic strains of mice. The use of Consensus Principal Components Analysis is described and shows relationships between the two types of fingerprints (GC/MS and DGGE profiles). Classification methods including Support Vector Machines and Discriminant Partial Least Squares suggest that mice can be classified according to both background strain and MHC-haplotype. As expected, the differences among the mice were much greater between strains that vary at both MHC and background loci than the congenics, which differ only at the MHC. These results indicate that the volatiles in scent-marks provide information about genetic similarity of the mice, and support the idea that the production of these genetically determined volatiles is influenced by commensal microflora. This paper describes the application of consensus methods to relate two blocks of analytical data.

The human derma emits volatile compounds whose interaction with a receiver's olfactory sensory system may affect individual recognition and mating preferences. Studies suggest that both genes and environmental factors determine characteristic odor of an individual. We used solid-phase microextraction and gas chromatography-mass spectrometry to identify 3-methylbutanal in human axillary odor; we showed that the abundance of this volatile compound varies significantly among individuals and demonstrated that its formation in vitro may be influenced by interaction between human leukocyte antigen peptide and dermal microflora.

The introduction of new approaches for characterizing microbial communities and imaging soil environments has benefited soil microbiology by providing new ways of detecting and locating microorganisms. Consequently, soil microbiology is poised to progress from simply cataloguing microbial complexity to becoming a systems science. A systems approach will enable the structures of microbial communities to be characterized and will inform how microbial communities affect soil function. Systems approaches require accurate analyses of the spatio-temporal properties of the different microenvironments present in soil. In this Review we advocate the need for the convergence of the experimental and theoretical approaches that are used to characterize and model the development of microbial communities in soils.

The incidence of infection by mycobacteria, other than tubercle bacilli (MOTT) is increasing in the United Kingdom, Europe and the United States. These diseases increase morbidity and are an increasing public health concern. However, the epidemiology of disease due to these species is not well characterized. We used space-time clustering approaches and Generalized Linear Modelling to investigate the potential predictors of disease in cases of infection by organisms of the Mycobacterium avium complex (MAC) and M. malmoense recorded in the north of England during 2000-2005. There was significant spatial and temporal clustering in juvenile cases of infection by MAC but not for cases of infection in adults by either species. There were no significant predictors of infection by M. malmoense or juvenile cases of M. avium. Incidence of disease caused by M. avium in adults was significantly related to health deprivation and weakly related to rainfall. We consider possible reasons for the difference in epidemiology in infection by M. avium in adults and juveniles.

Scent marking in mice allows males to communicate information such as territory ownership, male competitive ability and current reproductive, nutritional, social and health status. It has been suggested that female mice eavesdrop on these olfactory cues, using them as a means of selecting mates with dissimilar major histocompatibility complex (MHC) genes, known as H2 in mice. The mechanisms underpinning MHC-dependent olfactory communication remain unresolved. Using congenic mouse strains and molecular methods we explore the involvement of the microbial communities, a known source of odourants, in scent marks to test the hypothesis that the microbial communities and hence the olfactory signals are genetically determined. Here we show that the indigenous microbial community of murine scent marks is genetically determined. Both background genotype and H2 haplotype influence the community structure of the scent mark flora, removing the possibility that community composition is solely orchestrated by the MHC. Qualitative and quantitative components of the bacterial community associated with MHC haplotype and background genotype were identified. The analyses confirm that the four groups of congenic mice tested are distinguishable on basis of the microbiology of their scent marks alone, strengthening the role of microorganisms in the development of MHC-dependent odours.

The spatial location of microorganisms and their activity within the soil matrix have major impacts on biological processes such as nutrient cycling. However, characterizing the biophysical interface in soils is hampered by a lack of techniques at relevant scales. A novel method for studying the distribution of microorganisms that have incorporated isotopically labelled substrate ('active' microorganisms) in relation to the soil microbial habitat is provided by nano-scale secondary ion mass spectrometry (NanoSIMS). Pseudomonas fluorescens are ubiquitous in soil and were therefore used as a model for 'active' microorganisms in soil. Batch cultures (NCTC 10038) were grown in a minimal salt medium containing 15N-ammonium sulphate (15/14N ratio of 1.174), added to quartz-based white sand or soil (coarse textured sand), embedded in Araldite 502 resin and sectioned for NanoSIMS analysis. The 15N-enriched P. fluorescens could be identified within the soil structure, demonstrating that the NanoSIMS technique enables the study of spatial location of microbial activity in relation to the heterogeneous soil matrix. This technique is complementary to the existing techniques of digital imaging analysis of soil thin sections and scanning electron microscopy. Together with advanced computer-aided tomography of soils and mathematical modelling of soil heterogeneity, NanoSIMS may be a powerful tool for studying physical and biological interactions, thereby furthering our understanding of the biophysical interface in soils., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

The impact of inorganic N and P additions on a tropical soil contaminated with petroleum hydrocarbons was investigated using molecular and culture techniques. Microcosms were incubated for 42 days and sampled at 0, 1, 7, 28 and 42 days. Changes in bacterial community structure were determined using denaturing gradient gel electrophoresis (DGGE) of the rRNA following reverse transcription PCR using primers specific to the V3 region of the 16S rRNA gene. To identify which components of the microbial community were changing during incubation, PCR amplicons were resolved using DGGE and the banding patterns analyzed using stepwise discriminant function analysis (SDA). SDA showed that the number of bands needed to recover the differences between samples over time could be reduced from the initial 11 bands for the 16S rRNA transcript to 3 bands. Sequences originating from the rRNA gels (16S rRNA transcripts) were recovered in clades containing known cultured isolates of Bacillus marisflavi, Microbacterium oxydans and Pseudomonas oleovorans. Isolation studies on these soils using lubricant oil as a carbon source yielded 317 bacterial isolates, 3 of which showed high sequence similarity (>96%) with the 16S rRNA transcripts identified using SDA as being important in differentiating between bacterial communities over time. These isolates were then tested singly and in combination for their ability to degrade lubricant oil. These analyses demonstrated that the consortium selected using the combined molecular-SDA approach was more effective at degrading the lubricant in both liquid media and in contaminated sand than the single isolates.

The spatial distribution of an uncultured clade of marine diazotrophic gamma-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of nifH by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline. nifH transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The nifH sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these gamma-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.

Recently developed 13CO2 pulse labelling and stable isotope probing (SIP) methods offer the potential to track 13C-labelled plant photosynthate into phylogenetic groups of microbial taxa in the rhizosphere, permitting an examination of the link between soil microbial diversity and carbon flow in situ. We tested the feasibility of this approach to detect functional differences in microbial communities utilising recently fixed plant photosynthate in moisture perturbed grassland turfs. Specifically, we addressed two questions: (1) How does moisture perturbation (three treatments; continual wetting, drying, and drying followed by rewetting) affect the assimilation of 13C-labelled exudates carbon into the soil microbial community?; (2) Can 13C deposited in soil from pulse-labelled plants be used to identify microbes utilising plant exudates using SIP methodologies? Net CO2 fluxes showed that prior to 13CO2 pulse labelling, all treatments were photosynthetically active, but differences were observed in night time respiration, indicating moisture treatments had impacted on net CO2 efflux. Measurements of pulse-derived 13C incorporated into soil RNA over 2 months showed that there was only evidence of 13C enrichment in the continuously wetted treatments. However, isotopic values represented only a 0.1-0.2 13C at.% increase over natural abundance levels and were found to be insufficient for the application of RNA-SIP. These findings reveal that in this experimental system, the microbial uptake of labelled carbon from plant exudates is low, and further optimisation of methodologies may be required for application of SIP to natural plant-soil systems where 13C tracer dilution is a consideration., (Copyright 2004 Elsevier B.V.)

The inclusion of genetically modified (GM) plants in the human diet has raised concerns about the possible transfer of transgenes from GM plants to intestinal microflora and enterocytes. The persistence in the human gut of DNA from dietary GM plants is unknown. Here we study the survival of the transgene epsps from GM soya in the small intestine of human ileostomists (i.e., individuals in which the terminal ileum is resected and digesta are diverted from the body via a stoma to a colostomy bag). The amount of transgene that survived passage through the small bowel varied among individuals, with a maximum of 3.7% recovered at the stoma of one individual. The transgene did not survive passage through the intact gastrointestinal tract of human subjects fed GM soya. Three of seven ileostomists showed evidence of low-frequency gene transfer from GM soya to the microflora of the small bowel before their involvement in these experiments. As this low level of epsps in the intestinal microflora did not increase after consumption of the meal containing GM soya, we conclude that gene transfer did not occur during the feeding experiment.

The effects of water stress upon the diversity and culturable activity of bacterial communities in the rhizosphere of an established upland grassland soil have been investigated. Intact monoliths were subjected to different watering regimens over a 2-month period to study community adaptation to moisture limitation and subsequent response to stress alleviation following rewetting. Genetic diversity was analyzed with 16S-based denaturing gradient gel electrophoresis (DGGE) of total soil-extracted DNA (rRNA genes) and RNA (rRNA transcripts) in an attempt to discriminate between total and active communities. Physiological response was monitored by plate counts, total counts, and BIOLOG-GN2 substrate utilization analyses. Controlled soil drying decreased the total number of CFU on all the media tested and also decreased the substrate utilization response. Following rewetting of dried soil, culture-based analyses indicated physiological recovery of the microbial population by the end of the experiment. In contrast, DGGE analyses of community 16S rRNA genes, rRNA transcripts and cultured communities did not reveal any changes relating to the moisture regimens, despite the observed physiological effects. We conclude that the imposed moisture regimen modulated the physiological status of the bacterial community and that bacterial communities in this soil are resistant to water stress. Further, we highlight the need for a reexamination of rRNA transcript-based molecular profiling techniques as a means of describing the active component of soil bacterial communities.

The bacterial communities associated with the Caribbean coral Montastrea annularis showing tissue lesions indicative of a White Plague (WP)-like disease were investigated. Two molecular screening techniques using bacterial 16S rDNA genes were used and demonstrated distinct differences between the bacterial communities of diseased and non-diseased coral tissues, and also in relation to the proximity of tissue lesions on diseased corals. Differences between non-diseased corals and the apparently healthy tissues remote from the tissue lesion in affected corals indicates a 'whole coral' response to a relatively small area of infection with a perturbation in the normal microbial flora occurring prior to the onset of visible signs of disease. These whole organism changes in the microbial flora may serve as a bioindicator of environmental stress and disease. There were striking similarities between the 16S rDNA sequence composition associated with the WP-like disease studied here and that previously reported in association with black band disease (BBD) in coral. Similarities included the presence of a potential pathogen, an alpha-proteobacterium identified as the causal agent of juvenile oyster disease (JOD). The WP-like disease studied here is apparently different to WP Type ii because the bacterial species previously identified as the causal agent of WP Type ii was not detected, although the symptoms of the two diseases are similar.

Abstract Temporal and spatial variation of soil bacterial communities was evaluated with both molecular and metabolic profiling techniques. Soil cores (20 cm deep) were taken from an upland grassland in the Scottish Borders (UK) over 3 days in July 1999, and on single days in October 1999, April 2000, and August 2000. Cores were separated into four 5-cm depths to examine vertical spatial distribution. The 0-5-, 5-10- and 10-15-cm samples represented organic horizons whilst the 15-20-cm depths were from a mineral horizon. The potential metabolic activities were analysed using BIOLOG-GN plates, whereas genotypic diversity was evaluated using molecular profiling of amplified 16S rRNA and 16S rDNA gene fragments (denaturing gradient gel electrophoresis (DGGE)). BIOLOG-GN analysis revealed decreased substrate utilisation in the lowest depths, which was coupled with changes in the DNA and RNA DGGE profiles. Seasonal variation was pronounced in the 5-10-cm and 10-15-cm organic horizons for the July samplings whilst the 15-20-cm depths appeared more stable. Potential factors influencing the observed changes in bacterial communities resulting from soil depth and sampling time are discussed.

The bacterial community associated with black band disease (BBD) of the scleractinian corals Diploria strigosa, Montastrea annularis and Colpophyllia natans was examined using culture-independent techniques. Two complementary molecular screening techniques of 16S rDNA genes [amplified 16S ribosomal DNA restriction analysis (ARDRA) of clone libraries and denaturing gradient gel electrophoresis (DGGE)] were used to give a comprehensive characterization of the community. Findings support previous studies indicating low bacterial abundance and diversity associated with healthy corals. A single cyanobacterial ribotype was present in all the diseased samples, but this was not the same as that identified from Phormidium corallyticum culture isolated from BBD. The study confirms the presence of Desulfovibrio spp. and sulphate-reducing bacteria that have previously been associated with the BBD consortium. However, the species varied between diseased coral samples. We found no evidence of bacteria from terrestrial, freshwater or human sources in any of the samples. We report the presence of previously unrecognized potential pathogens [a Cytophaga sp. and an alpha-proteobacterium identified as the aetiological agent of juvenile oyster disease (JOD)] that were consistently present in all the diseased coral samples. The molecular biological approach described here gives an increasingly comprehensive and more precise picture of the bacterial population associated with BBD. To understand the pathogenesis of BBD, our attention should be focused on the pervasive ribotypes identified in this study (the Cyanobacterium sp., the Cytophaga sp. and the JOD pathogen).

Aims: To determine the spatial structure of microbial communities associated with disease lesions of reef corals (Scleractinia)., Methods and Results: Agarose pre-embedding preserved the structure of the disease lesion and surrounding tissues prior to demineralization of the carbonate exoskeleton and embedding in resin. Fluorescence in situ hybridization (FISH) was used to localize bacteria in the lesions of various diseases., Conclusions: The techniques successfully preserved the in situ spatial structure of degenerated coral tissues. In one case (white plague disease), significant bacterial populations were found only in fragmented remnants of degenerated coral tissues at the lesion boundary that would not have been detected using conventional histopathological techniques., Significance and Impact of the Study: Determining the composition, spatial structure and dynamics of microbial communities within the disease lesions is necessary to understand the process of disease progression. The methods described may be applicable to a wide range of diseases involving necrotic lesion formation and requiring extensive tissue processing, such as skeleton demineralization.

A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.

Soil was sampled to a distance of 2.5 mm beneath a root mat of oilseed rape (Brassica napus) in a model rhizosphere system. DNA was extracted and the 16S rDNA amplified, cloned and sequenced. Phylogenetic analysis of these sequences with those held on-line, revealed that 37% of the clones fell within the Holophaga /Acidobacterium phylum, 17% were within the proteobacteria, 14% of the clones were close relatives of Bacillus megaterium and 5% were related to Verrucomicrobium spinosum. An additional eleven clones (21%) could not be assigned to any known phylum and may represent novel bacterial lineages. This study highlights the diverse nature of rhizosphere soils and reinforces the role that molecular approaches play in unravelling such diversity.

We present a method for the selective, physical separation of active and non-active bacterial cells from natural communities. The method exploits the reduction of tetrazolium salts to form insoluble formazan crystals intracellularly in response to the addition of different oxidisable substrates. The intracellular deposition of formazan alters the bouyant density of active cells enabling them to be separated by density gradient centrifugation. The method has been successfully applied to the fractionation and collection of large whole cell sub-populations of active and non-active cells from sea-water samples. Removal of the bands from the density gradient, followed by PCR amplification and DGGE analyses showed distinct differences in the PCR amplicon diversity associated with the active and non-active cell fractions; an indication of changes in bacterial community structure in response to the addition of oxidisable substrate. Thus, based on their in situ respiration potential, the approach enables the cytochemical enrichment and molecular characterisation of mixed bacterial populations in natural environments.

Culturing and molecular techniques were used to monitor changes in the bacterial flora of the avian gastrointestinal (GI) tract following introduction of genetically modified (GM) and unmodified probiotics. Community hybridization of amplified 16S ribosomal DNA demonstrated that the bacterial flora of the GI tract changed significantly in response to the probiotic treatments. The changes were not detected by culturing. Although both GM and non-GM strains of Enterococcus faecium NCIMB 11508 changed the bacterial flora of the chicken GI tract, they did so differently. Probing the community DNA with an Enterococcus faecalis-specific probe showed that the relative amount of E. faecalis in the total eubacterial population increased in the presence of the non-GM strain and decreased in the presence of the GM probiotic compared with the results obtained with an untreated control group.

The maximum in vivo transfer rate of plasmid pAMbeta1 in the gut was 0.03 transconjugant per recipient cell, and this rate could be simulated in vitro only by forced filter mating. Transfer was not detected in liquid culture matings. Our findings demonstrate that in vitro methods, such as forced filter mating and liquid mating, underestimate the in vivo rates of gene transfer.

With the introduction of molecular methods, the past decade has seen renewed interest in soil microbiology. New and exciting molecular technologies and the promise of finally opening the microbial black box in soil drive much of this interest. Although these pioneering studies have added much to our knowledge of microbial diversity in soils, it is debatable whether they have as yet advanced our understanding of the relationship between this diversity and soil processes. Hopefully, over the next few years, the knowledge gained from molecular studies will provide a better understanding of microbial communities in soils and lead ultimately to improvements in land management and to the exploitation of the genetic resources of soil.

We have employed the power of the cyclic NAD-based enzyme amplification system to the determination of 16S rRNA. This generally applicable system employs two oligonucleotide probes, one of which is captured on a microtiter well surface and the other labeled with alkaline phosphatase. The detection of very low levels of hybridization of the capture probe is then achieved by the means of the ultrasensitive enzyme-amplified assay system, resulting in a highly sensitive, convenient, and rapid technology which can be directly employed on unpurified samples. We have been able to demonstrate the detection of 20 amol (10(7) molecules) of pure rRNA, and specific signals from as few as 2000 bacterial cells have also been demonstrated. The total procedural time can be short-5 to 18 h-depending on the dynamic range and sensitivity required. RNA target in the range of 10(12)-10(8) molecules can be assayed within 5 h. Extending the substrate incubation time enables between 10(11) and 10(7) molecules to be determined within 18 h. The system has great potential use with respect to studying the distribution and physiological states of cellular organisms., (Copyright 1998 Academic Press.)

Crystal structure analysis of Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) indicated that the enzyme contained a single calcium binding site that did not exhibit structural features typical of the EF-hand motif. Isothermal titration calorimetry revealed that XYLA binds calcium with a Ka of 4.9 x 10(4) M-1 and a stoichiometry consistent with one calcium binding site per molecule of enzyme. Occupancy of the calcium binding domain with its ligand protected XYLA from proteinase and thermal inactivation and increased the melting temperature of the enzyme from 60.8 to 66.5 degrees C. However, the addition of calcium or EDTA did not influence the catalytic activity of the xylanase. Replacement of the calcium binding domain, which is located within loop 7 of XYLA, with the corresponding short loop from Cex (a Cellulomonas fimi xylanase/exoglucanase), did not significantly alter the biochemical properties of the enzyme. These data suggest that the primary function of the calcium binding domain is to increase the stability of the enzyme against thermal unfolding and proteolytic attack. To understand further the nature of the calcium binding domain of XYLA, four variants of the xylanase, D256A, N261A, D262A, and XYLA"', in which Asp-256, Asn-261, and Asp-262 had all been changed to alanine, were constructed. These mutated enzymes did not show any significant binding to Ca2+, indicating that Asp-256, Asn-261, and Asp-262 play a pivotal role in the affinity of XYLA for the divalent cation. In the presence or absence of calcium, XYLA"' exhibited thermal stability similar to that of the native enzyme bound to Ca2+ ions, although the variant was sensitive to proteinase inactivation. The role of the calcium binding domain in vivo and the possible mechanism by which the domain evolved are discussed.

The widely accepted view that most bacterial species have yet to be cultivated in vitro has gained support from recent ribosomal DNA-based environmental studies. To enable elucidation of the phenotypes of organisms recognized solely by molecular genetic techniques, we developed and evaluated cytochemical methods which colocalize phenotypic properties with in situ rRNA probe hybridization signals. Application of these methods to artificial mixtures of Pseudomonas putida and Escherichia coli or Vibrio vulnificus showed that biochemical properties, such as the cytochrome oxidase reaction and specific substrate-enhanced tetrazolium salt reduction, can be assigned to cells identified by signals from determinative fluorescent rRNA probe binding. By doing the reactions directly on the stage of an inverted microscope and monitoring reaction product formation with a charge-coupled device video camera, it was possible to determine the kinetics of oxidizable substrate utilization in single cells. Analysis of digitized images permitted quantitative study of the relationship between rRNA signal strength and the rate of tetrazolium salt reduction. The approach used in this study opens up new opportunities to investigate the biochemistry, physiology, and behavior of both culturable and nonculturable bacteria in their natural environments.

The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis, Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis, Rhodococcus equi, R. erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis, Lactobacillus plantarum, Escherichia coli and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 30 and 50 min hydrolysis, whereas B. subtilis, E. coli and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.

The quantification of biodiversity among microorganisms has to address both theoretical and practical aspects. Species concepts are often at variance with those applied in macroorganisms, and satisfactory concepts suitable for general use in bacteria and fungi have yet to be formulated. Molecular approaches have not yet provided a universal solution to this key issue. Quantification in habitats such as soil is difficult as isolation procedures yield only a small and skewed selection of the microorganisms present. Indices of taxonomic or phylogenetic diversity have potential in the quantification of microbial diversity at a range of ranks, but the non-equivalence of ranks and representatives of the taxa detected have to be addressed. Chemical and molecular methods have immense potential in the quantification of microbial diversity in environmental samples; 16S rRNA has shown particular promise with bacteria, but as yet the fungi lack a universal probe. A greater awareness of the limitations of existing approaches and methodologies used by microbiologists is needed, but significant progress can be anticipated as new technologies are developed and become more widely adopted.

A genetically-manipulated strain of Lactobacillus plantarum and the unmodified parent strain were introduced into the rumen of sheep at an initial inoculum level of 1 x 10(7) cfu ml-1 of rumen fluid. There were no significant differences between the viable counts of the two inoculants throughout a 24 h sampling period. The rates of loss were 0.36 and 0.29 h-1 (proportion of colony-forming units lost, measured over the first 2 h) for the parent strain and recombinant strain respectively, and within 24 h of inoculation neither of the strains were detectable in rumen fluid. Further experiments in vitro revealed that the inoculants persisted in sterile rumen fluid with a loss rate of 0.044 and 0.057 h-1 for the parent strain and the recombinant strain respectively. Incubations with rumen fluid alone, protozoa-free rumen fluid and protozoa-enriched rumen fluid revealed that protozoal predation was the most significant factor in the loss of the introduced population. The loss rates from protozoa-free rumen fluid were not significantly different (P < 0.05) from those observed in sterile rumen fluid.

Fifty-four carboxydotrophic actinomycetes isolated from soils and composts were compared through 119 unit characters with representative mesophilic and thermophilic streptomycetes. The data were examined using the Jaccard, pattern and simple matching coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. Acceptable cophenetic correlation and test error values allowed confidence to be placed in the resultant numerical taxonomies. The carboxydotrophic actinomycetes, which were distinct from cluster-groups corresponding to the mesophilic and thermophilic streptomycetes, formed two major cluster-groups the members of which were examined for the presence of diagnostic chemical markers. All but two of the carboxydotrophic actinomycetes had a profile of chemical properties consistent with their assignment to the genus Streptomyces. Quantitative fatty acid data were examined using the SIMCA package and the two statistically significant groups obtained corresponded with the cluster-groups circumscribed in the numerical phenetic analysis. Members of the two groups were also distinguished on the basis of their phospholipid composition. The two strains that contained meso-as opposed to LL-diaminopimelic acid in their peptidoglycan also showed a distinct chemotaxonomic profile. It was concluded that the carboxydotrophic actinomycetes form a novel and taxonomically diverse group.

The growth and persistence of two genetically manipulated forms of Lactobacillus plantarum NCDO (National Collection of Dairy Organisms) 1193 have been monitored in grass silage. Both recombinants contained pSA3, a shuttle vector for gram-positive organisms that encodes erythromycin resistance. In one of the recombinants, pSA3 was integrated onto the chromosome, whereas in the other, a pSA3 derivative designated pM25, which contains a Clostridium thermocellum cellulase gene cloned into pSA3, was maintained as an extrachromosomal element. This extrachromosomal element is a plasmid. Rifampin-resistant mutants were selected for the recombinants and the parent strain. When applied to minisilos at a rate of 10 CFU/g of grass, both the recombinants and the parent strain proliferated to dominate the epiphytic microflora and induced an increase in the decline in pH compared with that of the noninoculated silos. The presence of extra genetic material did not appear to disadvantage the bacterium in comparison with the parent strain. The selective recovery of both strains by using rifampin and erythromycin was confirmed by Southern hybridization. Interestingly, the free plasmid (pM25) appeared more stable in silage than was expected from studies in MRS broth. The plasmid was retained by 85% of the rifampin-resistant L. plantarum colonies isolated from a day 30 silo. These data answer an important question by showing that genetically manipulated recombinants of L. plantarum can proliferate and compete with epiphytic lactic acid bacteria in silage.

Seven strains of Rothia dentocariosa were degraded by acid methanolysis and the nonhydroxylated fatty acid methyl esters released were examined by thin-layer and gas chromatography. The fatty acid profiles were composed of iso-, anteiso- and straight chain saturated fatty acids with 12-methyltetradecanoic (anteiso-C15), 14-methylpentadecanoic (iso-C16), 14-methylhexadecanoic (anteiso-C17) and hexadecanoic acid (C16) as major components. A small scale integrated procedure was used for the sequential extraction of isoprenoid quinones and polar lipids. The latter were examined by two-dimensional thin-layer chromatography and all of the test strains contained diphosphatidylglycerol, phosphatidylglycerol and two uncharacterised glycolipids. In all cases the major isoprenoid quinones were unsaturated menaquinones with seven isoprene units. Analyses of the cell wall amino acid composition using gas chromatography showed that the strains contained 2.5 to 5 moles of alanine and 1 mole each of glutamic acid and lysine. The chemical data support the integrity of Rothia dentocariosa and can be used to separate it from all other actinomycetes especially those which contain lysine in the wall peptidoglycan.

Two strains of Arachnia propionica and two Arachnia-like isolates were degraded by alkaline methanolysis and the non-hydroxylated fatty acid esters released were examined by thin-layer and gas chromatography. The fatty acid profiles obtained were both qualitatively and quantitatively similar and were comprised of iso-, anteiso- and straight chain saturated fatty acids with 13-methyltetradecanoic acid (i-15) and 12-methyltetradecanoic acid (ai-15) as major components. One of the Arachnia-like strains (HIK 288) had, in addition to i-15 and ai-15, major amounts of pentadecanoic acid (15:0). All of the strains gave characteristic polar lipid patterns consisting of diphosphatidylglycerol, phosphatidylglycerol and two incompletely characterised glycolipids. Analyses of wall amino acid preparations by gas and thin-layer chromatography showed that Arachnia propionica strains contain major amounts of alanine, glycine, glutamic acid and LL-diaminopimelic acid. The chemical data support the integrity of Arachnia propionica and provide a valuable means of differentiating it from morphologically and physiologically similar strains of Actinomyces israelii.

The existence of subgroups within Bacillus megaterium has been reported previously on the basis of DNA-DNA hybridization and DNA base composition studies. In this study the strains used to define these subgroups have been reanalysed by pyrolysis gas-liquid chromatography. The resultant two-group classification of the test strains was directly comparable with that obtained from the previous nucleic acid analyses at the between-group level. However, comparisons of the test strains at the within-group level proved less successful.

Representative strains of Nocardioides, Arthrobacter simplex and Arthrobacter tumescens were degraded by acid methanolysis and the fatty acid esters released examined by thin-layer and gas chromatography. Branched-chain 14-methylpentadecanoic acid (iso-16) was the predominant component in all but one of the Nocardioides strains. Arthrobacter simplex also contained major amounts of this acid whereas A. tumescens had only minor amounts. All of the test strains possessed 15 and 17 carbon straight chain acids, tuberculostearic acid (10-methyloctadecanoic acid) and its 17 and 18-carbon homologues. The fatty acid profiles of Nocardioides strains lacked 13-methyltetradecanoic and heptadecanoic acids which were both present in Arthrobacter simplex and Arthrobacter tumescens. The profiles of these latter organisms were quantitatively different from each other. The polar lipids of the test strains all contained diphosphatidylglycerol and phosphatidylglycerol but only Arthrobacter tumescens contained phosphatidylinositol and three unidentified polar lipids. Nocardioides and Arthrobacter simplex strains all contained two very characteristic closely related polar lipids. All of the test strains contained tetrahydrogenated menaquinones with eight isoprene units as the major isoprenologue. The results of the present study support the integrity of the genus Nocardioides and provide a reliable way of distinguishing it from other actinomycetes, such as Streptomyces, which also have LL-diaminopimelic acid and glycine in the peptidoglycan. The lipid data, together with results from chemical, genetic and phage host range studies, provide sufficient grounds for the transfer of Arthrobacter simplex to Nocardioides as Nocardioides simplex comb. nov. An emended description of the genus Nocarioides is given.

Representative strains of coagulase-positive and coagulase-negative staphylococci were degraded by acid methanolysis and the resultant fatty acid methyl esters analysed by gas chromatography. The quantitative data obtained were examined by cluster analysis. The coagulase-positive strains formed six major and one single-member cluster at the 90% S-level. The Staphylococcus intermedius aggregate cluster included the single-member cluster and major clusters 1 and 2. The four remaining clusters contained S. aureus strains and were homogeneous and distinct. The coagulase-negative strains were recovered in ten major and three single-member clusters at the 90% S-level. Five of the ten major clusters were reasonably homogeneous with respect to the existing classification. Thus, three S. capitis strains and five of the six S. epidermidis strains, two of the three S. hominis strains and five of the six S. simulans strains were recovered in separate clusters. Cluster 7 was divided into two subclusters; one contained five of the six S. hyicus strains and the other contained the two representatives of S. lentus. The remaining clusters were heterogeneous with regard to the named strains they contained.