Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%-86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size.

Rapid response of the active microbial community to CO2 exposure from a controlled sub-seabed CO2 leak in Ardmucknish Bay (Oban, Scotland)

The response of the benthic microbial community to a controlled sub-seabed CO2 leak was assessed using quantitative PCR measurements of benthic bacterial, archaeal and cyanobacteria/chloroplast 16S rRNA genes. Samples were taken from four zones (epicentre; 25 m distant, 75 m distant and 450 m distant) during 6 time points (7 days before CO2 exposure, after 14 and 36 days of CO2 release, and 6, 20 and 90 days after the CO2 release had ended). Changes to the active community of microphytobenthos and bacteria were also assessed before, during and after CO2 release. Increases in the abundance of microbial 16S rRNA were detected after 14 days of CO2 release and at a distance of 25 m from the epicentre. CO2 related changes to the relative abundance of both major and minor bacterial taxa were detected: most notably an increase in the relative abundance of the Planctomycetacia after 14 days of CO2 release. Also evident was a decrease in the abundance of microbial 16S rRNA genes at the leak epicentre during the initial recovery phase: this coincided with the highest measurements of DIC within the sediment, but may be related to the release of potentially toxic metals at this time point.

The diversity of extradiol dioxygenase (edo) genes in cresol degrading rhodococci from a creosote-contaminated site that express a wide range of degradative abilities.

Analysis of the bacterial population of soil surface samples from a creosote-contaminated site showed that up to 50% of the culturable micro-organisms detected were able to utilise a mixture of cresols. From fifty different microbial isolates fourteen that could utilise more than one cresol isomer were selected and identified by 16S rRNA analysis. Eight isolates were Rhodococcus strains and six were Pseudomonas strains. In general, the Rhodococcus strains exhibited a broader growth substrate range than the Pseudomonas strains. The distribution of various extradiol dioxygenase (edo) genes, previously associated with aromatic compound degradation in rhodococci, was determined for the Rhodococcus strains by PCR detection and Southern-blot hybridization. One strain, Rhodococcus sp. I1 exhibited the broadest growth substrate range and possessed five different edo genes. Gene disruption experiments indicated that two genes (edoC and edoD) were associated with isopropylbenzene and naphthalene catabolism respectively. The other Rhodococcus strains also possessed some of the edo genes and one (edoB) was present in all of the Rhodococcus strains analysed. None of the rhodococcal edo genes analysed were present in the Pseudomonas strains isolated from the site. It was concluded that individual strains of Rhodococcus possess a wide degradative ability and may be very important in the degradation of complex mixtures of substrates found in creosote.

Isolation and characterization of a novel simazine-degrading bacterium from agricultural soil of central Chile, Pseudomonas sp MHP41

s-Triazine herbicides are used extensively in South America in agriculture and forestry. In this study, a bacterium designated as strain MHP41, capable of degrading simazine and atrazine, was isolated from agricultural soil in the Quillota valley, central Chile. Strain MHP41 is able to grow in minimal medium, using simazine as the sole nitrogen source. In this medium, the bacterium exhibited a growth rate of mu = 0.10 h(-1), yielding a high biomass of 4.2 x 10(8) CFU mL(-1). Resting cells of strain MHP41 degrade more than 80% of simazine within 60 min. The atzA, atzB, atzC, atzD, atzE and atzF genes encoding the enzymes of the simazine upper and lower pathways were detected in strain MHP41. The motile Gram-negative bacterium was identified as a Pseudomonas sp., based on the Biolog microplate system and comparative sequence analyses of the 16S rRNA gene. Amplified ribosomal DNA restriction analysis allowed the differentiation of strain MHP41 from Pseudomonas sp. ADP. The comparative 16S rRNA gene sequence analyses suggested that strain MHP41 is closely related to Pseudomonas nitroreducens and Pseudomonas multiresinovorans. This is the first s-triazine-degrading bacterium isolated in South America. Strain MHP41 is a potential biocatalyst for the remediation of s-triazine-contaminated environments.

Stochastic and deterministic processes interact in the assembly of desert microbial communities on a global scale

Extreme arid regions in the worlds' major deserts are typified by quartz pavement terrain. Cryptic hypolithic communities colonize the ventral surface of quartz rocks and this habitat is characterized by a relative lack of environmental and trophic complexity. Combined with readily identifiable major environmental stressors this provides a tractable model system for determining the relative role of stochastic and deterministic drivers in community assembly. Through analyzing an original, worldwide data set of 16S rRNA-gene defined bacterial communities from the most extreme deserts on the Earth, we show that functional assemblages within the communities were subject to different assembly influences. Null models applied to the photosynthetic assemblage revealed that stochastic processes exerted most effect on the assemblage, although the level of community dissimilarity varied between continents in a manner not always consistent with neutral models. The heterotrophic assemblages displayed signatures of niche processes across four continents, whereas in other cases they conformed to neutral predictions. Importantly, for continents where neutrality was either rejected or accepted, assembly drivers differed between the two functional groups. This study demonstrates that multi-trophic microbial systems may not be fully described by a single set of niche or neutral assembly rules and that stochasticity is likely a major determinant of such systems, with significant variation in the influence of these determinants on a global scale.

Low rates of Pseudomonas aeruginosa misidentification in isolates from cystic fibrosis patients

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.

Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes.

The opportunistic human pathogen Propionibacterium acnes is comprised of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II and III, that vary in their production of putative virulence factors, inflammatory potential, as well as biochemical, aggregative and morphological characteristics. Although Multilocus Sequence Typing (MLST) currently represents the gold standard for unambiguous phylogroup classification, and individual strain identification, it is a labour and time-consuming technique. As a consequence, we have developed a multiplex touchdown PCR assay that will, in a single reaction, confirm species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA (all isolates), ATPase (type IA1, IA2, IC), sodA (type IA2, IB), atpD (type II) and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterised by MLST, and representing type IA1 (n=145), IA2 (n=20), IB (n=65), IC (n=7), II (n=45) and III (n=30), the multiplex displayed 100% sensitivity and 100% specificity for the detection of isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. The multiplex assay will provide researchers with a rapid, high-throughput and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, as well as a pre-screening tool to maximise the number of genetically diverse isolates selected for downstream, higher resolution sequence-based analyses.

Microbial Communities Associated with Healthy and White Syndrome-Affected Echinopora lamellosa in Aquaria and Experimental Treatment with the Antibiotic Ampicillin

Prokaryotic and ciliate communities of healthy and aquarium White Syndrome (WS)-affected coral fragments were screened using denaturing gradient gel electrophoresis (DGGE). A significant difference (R = 0.907, p < 0.001) in 16S rRNA prokaryotic diversity was found between healthy (H), sloughed tissue (ST), WS-affected (WSU) and antibiotic treated (WST) samples. Although 3 Vibrio spp were found inWS-affected samples, two of these species were eliminated following ampicillin treatment, yet lesions continued to advance, suggesting they play a minor or secondary role in the pathogenesis. The third Vibrio sp increased slightly in relative abundance in diseased samples and was abundant in non-diseased samples. Interestingly, a Tenacibaculum sp showed the greatest increase in relative abundance between healthy and WS-affected samples, demonstrating consistently high abundance across all WS-affected and treated samples, suggesting Tenacibaculum sp could be a more likely candidate for pathogenesis in this instance. In contrast to previous studies bacterial abundance did not vary significantly (ANOVA, F2, 6 = 1.000, p = 0.422) between H, ST, WSU or WST. Antimicrobial activity (assessed on Vibrio harveyi cultures) was limited in both H and WSU samples (8.1% ±8.2 and 8.0% ±2.5, respectively) and did not differ significantly (Kruskal-Wallis, χ2 (2) = 3.842, p = 0.146). A Philaster sp, a Cohnilembus sp and a Pseudokeronopsis sp. were present in all WS-affected samples, but not in healthy samples. The exact role of ciliates in WS is yet to be determined, but it is proposed that they are at least responsible for the neat lesion boundary observed in the disease.

Short-chain fatty acids cause an IL-8 response in cystic fibrosis airways via increased GPR41

RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune/inflammatory processes.

OBJECTIVES: To investigate the capacity of anaerobes to contribute to CF airway pathogenesis via SCFAs.

METHODS: Samples from 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFAs levels in anaerobe supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of SCFAs receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings, and 16HBE14o- and CFBE41o- cells were evaluated using RT-PCR, western blot, laser scanning cytometry and confocal microscopy. SCFAs-induced IL-8 secretion was monitored by ELISA.

MEASUREMENTS AND MAIN RESULTS: Fifty seven of 109 (52.3%) PWCF were anaerobe-positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF under (n=24) and over 6 years (n=85). All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic and butyric acid. SCFAs levels were higher in BAL samples from adults than children. GPR41 levels were elevated in; CFBE41o- versus 16HBE14o- cells; CF versus non-CF bronchial brushings; 16HBE14o- cells after treatment with CFTR inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells with a higher production of IL-8 in CFBE41o- than 16HBE14o- cells.

CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via upregulated GPR41.

Performance of a Novel Molecular Method in the Diagnosis of Late-Onset Sepsis in Very Low Birth Weight Infants

OBJECTIVE: To compare the use of a generic molecular assay to 'standard' investigations used to assist the diagnosis of late onset bacterial sepsis in very low birth weight infants (VLBW, <1500g).

METHODS: VLBW infants, greater than 48 hours of age, who were clinically suspected to have sepsis were investigated using standard tests (full blood count, C-reactive protein (at presentation) and blood culture), in addition, blood was taken for a universal molecular assay (16S rRNA reverse transcriptase PCR) for comparison. Clinical data were recorded during the suspected infection episode. A validated sepsis score (NEO-KISS) was used to retrospectively determine the presence of sepsis (independent of blood culture). The performance of each of the tests were compared by sensitivity, specificity, positive/negative likihood ratios (+/-LR) and postive/negative predictive values (PPV/NPV).

RESULTS: Sixty-five babies with suspected clinical sepsis were prospectively included. The performance indicators are presented with 95% confidence limits. For the detection of bacteria, blood culture had sensitivity of 0.57 (0.34-0.78), specificity of 0.45 (0.30-0.61); +LR of 1.05 (0.66-1.66) and-LR of 0.94 (0.52-1.7); PPV of 33.3 (18.56-50.97) and NPV of 68.97 (49.17-87.72). Serum CRP had sensitivity of 0.92 (0.64-1) and specificity of 0.36 (0.17-0.59); +LR of 1.45 (1-2.1) and-LR of 0.21 (0.03-1.5); PPV of 44.46 (26.6-66.6) and NPV of 88.9 (51.8-99.7). The universal molecular assay had sensitivity of 0.76 (0.53-0.92), specificity of 0.95 (0.85-0.99); +LR of 16.8 (4.2-66.3) and-LR of 0.25 (0.1-0.5); PPV of 88.9 (65.3-98.6) and NPV of 89.4 (76.9-96.5).

CONCLUSIONS: In VLBW infants this universal molecular assay performed better in the diagnosis of late onset sepsis (LOS) than blood culture and CRP. Further development is required to explore and improve the performance of the assay in real-time diagnosis.

Using microbiological tracers to assess the impact of winter land use restrictions on the quality of stream headwaters in a small catchment.

Diverse land use activities can elevate risk of microbiological contamination entering stream headwaters. Spatially distributed water quality monitoring carried out across a 17km(2) agricultural catchment aimed to characterize microbiological contamination reaching surface water and investigate whether winter agricultural land use restrictions proved effective in addressing water quality degradation. Combined flow and concentration data revealed no significant difference in fecal indicator organism (FIO) fluxes in base flow samples collected during the open and prohibited periods for spreading organic fertilizer, while relative concentrations of Escherichia coli, fecal streptococci and sulfite reducing bacteria indicated consistently fresh fecal pollution reached aquatic receptors during both periods. Microbial source tracking, employing Bacteroides 16S rRNA gene markers, demonstrated a dominance of bovine fecal waste in river water samples upstream of a wastewater treatment plant discharge during open periods. This contrasted with responses during prohibited periods where human-derived signatures dominated. Differences in microbiological signature, when viewed with hydrological data, suggested that increasing groundwater levels restricted vertical infiltration of effluent from on-site wastewater treatment systems and diverted it to drains and surface water. Study results reflect seasonality of contaminant inputs, while suggesting winter land use restrictions can be effective in limiting impacts of agricultural wastes to base flow water quality.

Abundance and diversity of sedimentary bacterial communities in a coastal productive setting in the Western Irish Sea

The bacterial community composition and biomass abundance from a depositional mud belt in the western Irish Sea and regional sands were investigated by phospholipid ester-linked fatty acid profiling, denaturing gradient gel electrophoresis and barcoded pyrosequencing of 16S rRNA genes. The study area varied by water depth (12-111 m), organic carbon content (0.09-1.57% TOC), grain size, hydrographic regime (well-mixed vs. stratified), and water column phytodetrital input (represented by algal polyunsaturated PLFA). The relative abundance of bacterial-derived PLFA (sum of methyl-branched, cyclopropyl and odd-carbon number PLFA) was positively correlated with fine-grained sediment, and was highest in the depositional mud belt. A strong association between bacterial biomass and eukaryote primary production was suggested based on observed positive correlations with total nitrogen and algal polyunsaturated fatty acids. In addition, 16S rRNA genes affiliated to the classes Clostridia and Flavobacteria represented a major proportion of total 16S rRNA gene sequences. This suggests that benthic bacterial communities are also important degraders of phytodetrital organic matter and closely coupled to water column productivity in the western Irish Sea.

A microbiological study of Papillon-Lefévre syndrome in two patients

AIM: To analyse the microflora of subgingival plaque from patients with Papillon-Lefévre syndrome (PLS), which is a very rare disease characterised by palmar-plantar hyperkeratosis with precocious periodontal destruction.

METHODS: Bacterial isolates were identified using a combination of commercial identification kits, traditional laboratory tests, and gas liquid chromatography. Some isolates were also subjected to partial 16S rDNA sequencing. Plaque samples were also assayed for the presence of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in a quantitative enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies.

RESULTS: The culture results showed that most isolates were capnophilic and facultatively anaerobic species-mainly Capnocytophaga spp and Streptococcus spp. The latter included S. constellatus, S. oralis, and S. sanguis. Other facultative bacteria belonged to the genera gemella, kingella, leuconostoc, and stomatococcus. The aerobic bacteria isolated were species of neisseria and bacillus. Anaerobic species included Prevotella intermedia, P. melaninogenica, and P. nigrescens, as well as Peptostreptococcus spp. ELISA detected P gingivalis in one patient in all sites sampled, whereas A. actinomycetemcomitans was detected in only one site from the other patient. Prevotella intermedia was present in low numbers.

CONCLUSIONS: Patients with PLS have a very complex subgingival flora including recognised periodontal pathogens. However, no particular periodontopathogen is invariably associated with PLS.

Biological phosphorus removal during high-rate, low-temperature, anaerobic digestion of wastewater.

We report, for the first time, extensive biologically-mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (~2%) within the sludge bed and fixed-film biofilms. 4’, 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4-1.5 kg COD m-3 d-1 and hydraulic retention times of 8-24 hours, while phosphate removal efficiency ranged from 28-78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12˚C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina Miseq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterised polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter and Acinetobacter was recorded at low numbers. However, it is unknown as yet if these were responsible for the luxury polyP uptake observed in this system. The possibility of efficient phosphate removal and recovery from wastewater during AD would represent a major advance in the scope for widespread application of anaerobic wastewater treatment technologies.

Metagenomic characterisation of the viral community of lough neagh, the largest freshwater lake in Ireland

Lough Neagh is the largest and the most economically important lake in Ireland. It is also one of the most nutrient rich amongst the world's major lakes. In this study, 16S rRNA analysis of total metagenomic DNA from the water column of Lough Neagh has revealed a high proportion of Cyanobacteria and low levels of Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes. The planktonic virome of Lough Neagh has been sequenced and 2,298,791 2×300 bp Illumina reads analysed. Comparison with previously characterised lakes demonstrates that the Lough Neagh viral community has the highest level of sequence diversity. Only about 15% of reads had homologs in the RefSeq database and tailed bacteriophages (Caudovirales) were identified as a major grouping. Within the Caudovirales, the Podoviridae and Siphoviridae were the two most dominant families (34.3% and 32.8% of the reads with sequence homology to the RefSeq database), while ssDNA bacteriophages constituted less than 1% of the virome. Putative cyanophages were found to be abundant. 66,450 viral contigs were assembled with the largest one being 58,805 bp; its existence, and that of another 34,467 bp contig, in the water column was confirmed. Analysis of the contigs confirmed the high abundance of cyanophages in the water column.