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The respiratory mycobiome is an important but understudied component of the human microbiota. Like bacteria, fungi can cause severe lung diseases, but their infection rates are much lower. This study compared the bacterial and fungal communities of sputum samples from a large cohort of 56 adult patients with cystic fibrosis (CF) during nonexacerbation periods and under continuous antibiotic treatment. Molecular fingerprinting based on single-strand conformation polymorphism (SSCP) analysis revealed fundamental differences between bacterial and fungal communities. Both groups of microorganisms were taxonomically classified by identification of gene sequences (16S rRNA and internal transcript spacer), and prevalences of single taxa were determined for the entire cohort. Major bacterial pathogens were frequently observed, whereas fungi of known pathogenicity in CF were detected only in low numbers. Fungal species richness increased without reaching a constant level (saturation), whereas bacterial richness showed saturation after 50 patients were analyzed. In contrast to bacteria, a large number of fungal species were observed together with high fluctuations over time and among patients. These findings demonstrated that the mycobiome was dominated by transient species, which strongly suggested that the main driving force was their presence in inhaled air rather than colonization. Considering the high exposure of human airways to fungal spores, we concluded that fungi have low colonization abilities in CF, and colonization by pathogenic fungal species may be considered a rare event. A comprehensive understanding of the conditions promoting fungal colonization may offer the opportunity to prevent colonization and substantially reduce or even eliminate fungus-related disease progression in CF., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Fungi grow under humid conditions and are, therefore, prone to biofilm infections. A 16S rRNA fingerprint analysis was performed on 49 sporocarps of Basidiomycotina in order to determine whether they are able to control these biofilms. Ninety-five bacterial phylotypes, comprising 4 phyla and 10 families, were identified. While ectomycorrhizal fungi harbored the highest bacterial diversity, saprophytic fungi showed little or no association with bacteria. Seven fungal species were screened for antimicrobial and antibiofilm activities. Biofilm formation and bacterial growth was inhibited by extracts obtained from saprophytic fungi, which confirmed the hypothesis that many fungi modulate biofilm colonization on their sporocarps.

The cattle tick Rhipicephalus (Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Background: Asymptomatic bacterial colonisation of medical implants is increasingly recognised. The role of the often unusual bacteria is usually unknown however., Objective and Methods: During a three-year period, patients presenting with clinically evident CIED lead endocarditis and/or pocket infections were included in a prospective database. Using 16s DNA analysis and sequencing, devices and pockets of consecutive patients with evident CIED infection were examined for the presence of bacterial species in addition to the ones grown in conventional culture in order to characterise their clinical role., Results: During the study 77 of a total of 409 consecutive operations involving the explantation of a CIED were performed for clinically evident infection. 71 were included in the study and conventional extended culture and 16s DNA analysis performed and compared. In 42.3% of the patients bacteria were identified by DNA analysis in addition to the culture of the supposedly causative organisms., Conclusion: DNA analysis is more sensitive than conventional culture of swabs in detecting any, or indeed multiple bacterial strains in CIED infection. This may in future influence treatment strategies as e.g. the cohabitation of different strains seems common and is not reflected by classical culture results. Spread of infection along the leads was clearly demonstrated and this strongly supports the recommendation of complete removal of all CIED components in every form of CIED infection. The more unusual bacteria demonstrated here do not appear to play a significant clinical role as suspected earlier.

The wood protection industry has refined their products from chrome-, copper-, and arsenate-based wood preservatives toward solely copper-based preservatives in combination with organic biocides. One of these is Cu-HDO, containing the chelation product of copper and N-cyclohexyldiazenium dioxide (HDO). In this study, the fate of isotope-labeled ((13)C) and nonlabeled ((12)C) Cu-HDO incorporated in wood sawdust mixed with soil was investigated. HDO concentration was monitored by high-pressure liquid chromatography. The total carbon and the δ(13)C content of respired CO(2), as well as of the soil-wood-sawdust mixture, were determined with an elemental analyzer-isotopic ratio mass spectrometer. The concentration of HDO decreased significantly after 105 days of incubation, and after 24 days the (13)CO(2) concentration respired from soil increased steadily to a maximum after 64 days of incubation. Phospholipid fatty acid-stable isotope probing (PFA-SIP) analysis revealed that the dominant PFAs C(19:0)d8,9, C(18:0), C(18:1)ω7, C(18:2)ω6,9, C(17:1)d7,8, C(16:0), and C(16:1)ω7 were highly enriched in their δ(13)C content. Moreover, RNA-SIP identified members of the phylum Acidobacteria and the genera Phenylobacterium and Comamonas that were assimilating carbon from HDO exclusively. Cu-HDO as part of a wood preservative effectively decreased fungal wood decay and overall microbial respiration from soil. In turn, a defined bacterial community was stimulated that was able to metabolize HDO completely.

Many bacteria grow on surfaces forming biofilms but often high dosages of antibiotics can not clear infectious biofilms. Biofilms are controlled by a process called quorum-sensing triggered by species-specific, small, diffusible autoinducers (N-acyl homoserine lactones (AHL), small peptides or a furanosyl borate diester). Several herbal, animal and microbial extracts possess quorum-quenching activity but only few active compounds and synthetic analogues are known. However, quorum-sensing seems not to be the only way to control biofilms. Bacteria have mechanisms to dissolve their biofilms and return to planktonic lifestyle. Only few compounds responsible for the signalling of these processes are known but may open a completely novel line of biofilm control. Furthermore, controlling the attachment of bacterial by inhibiting the formation of pili is another option and some inhibitors have already been characterized. In this article different approaches to control bacterial biofilms are discussed together with the balance between biofilm formation and virulence.

Biofilms are matrix-enclosed microbial aggregations that adhere to biological or non-biological surfaces. They represent a significant and incompletely understood mode of growth for bacteria and fungi. Biofilm infections cause many deaths and high health costs worldwide. Biofilm infections on indwelling devices or implants are difficult to eradicate because of their much better protection against macrophages and antibiotics, compared to free living cells, leading to severe clinical complications often with lethal outcome. One promising approach to combat biofilm infections independent from the conventional control by antibiotics is the generation of functional surfaces preventing the attachment of bacteria. Another aim is the communication machinery used by bacteria to establish a biofilm, the so called quorum-sensing. Here, small diffusible compounds are produced and sensed by the producing cells to measure their concentration and hence cell density. Natural compounds and synthetic analogues have been used successfully to prevent biofilm formation by quorum-quenching. These compounds are still in the preclinical phase, often struggling with toxicity. A principal problem of quorum-quenchers is their high species specificity, resulting in the control of only some pathogenic strains leaving other pathogens untouched. A field still in its infancy is the control of virulence factors expression not preventing the biofilm but suppressing its virulence. This review will give an overview over the pros and cons of the individual targets and an outlook of future developments.

Methanotrophs in the rhizosphere play an important role in global climate change since they attenuate methane emission from rice field ecosystems into the atmosphere. Most of the CH(4) is emitted via transport through the plant gas vascular system. We used this transport for stable isotope probing (SIP) of the methanotrophs in the rhizosphere under field conditions and pulse-labelled rice plants in a Chinese rice field with CH(4) (99% (13)C) for 7 days. The rate of (13)CH(4) loss rate during (13)C application was comparable to the CH(4) oxidation rate measured by the difluoromethane inhibition technique. The methanotrophic communities on the roots and in the rhizospheric soil were analyzed by terminal-restriction fragment length polymorphism (T-RFLP), cloning and sequencing of the particulate methane monooxygenase (pmoA) gene. Populations of type I methanotrophs were larger than those of type II. Both methane oxidation rates and composition of methanotrophic communities suggested that there was little difference between urea-fertilized and unfertilized fields. SIP of phospholipid fatty acids (PLFA-SIP) and rRNA (RNA-SIP) were used to analyze the metabolically active methanotrophic community in rhizospheric soil. PLFA of type I compared with type II methanotrophs was labelled more strongly with (13)C, reaching a maximum of 6.8 atom-%. T-RFLP analysis and cloning/sequencing of 16S rRNA genes showed that methanotrophs, especially of type I, were slightly enriched in the 'heavy' fractions. Our results indicate that CH(4) oxidation in the rice rhizosphere under in situ conditions is mainly due to type I methanotrophs.

The purpose of this work was to determine the extent of microbial metabolic potential for polychlorinated biphenyls (PCBs) in soils that have had no previous exposure to this class of xenobiotic pollutants. Soil and sediment samples of distinct characteristics from six sites in Germany were used to inoculate PCB oil (Aroclor 1242) microdroplets. All samples yielded multispecies biofilms, as revealed by single-strand conformation polymorphism (SSCP) analyses of polymerase chain reaction (PCR) analysis of 16S rRNA genes, and sequence analysis of the main amplicons. Microbes representing 20 different operational taxonomic units (OTUs) were identified in the biofilms, but only a few were common to all biofilms, namely those closely related to Aquabacterium sp., Caulobacter sp., Imtechium assamiensis, Nevskia ramosa, Parvibaculum lavamentivorans and Burkholderia sp. The PCB biofilm communities were always distinct from control biofilms developing from the same samples in the absence of PCB. All PCB droplet-grown biofilms degraded multiple PCB congeners but differed in the congener spectra they degraded. These findings reveal that microbial potential to degrade PCBs is widespread in soils that have not been subjected to PCB contamination, and that this potential is characteristic of consortia of very diverse phylogenetic composition.

Soil contaminated with polychlorinated biphenyls (PCB) was used as an inoculum to grow a complex biofilm community on PCB oil (Aroclor 1242) on a substratum (Permanox). The biofilm was monitored for 31 days by confocal laser scanning microscopy, community fingerprinting using single-strand conformational polymorphism (SSCP), amplicons of the 16S rRNA genes, and chemical analyses of the PCB congeners. SSCP analysis of the young biofilm revealed a rather diverse microbial community with species of the genera Herbaspirillum and Bradyrhizobium as dominant members. The biofilm developing on the PCB droplets displayed pronounced stages of PCB degradation and biofilm development not described before from pure-culture experiments. The first step was the colonization of the substratum while the PCB oil was hardly populated. When a certain density of bacteria was reached on the Permanox, the PCB was colonized, but soon the degradation of the congeners was markedly reduced and many cells were damaged, as seen by LIVE/DEAD staining. Finally, the biofilm formed aggregates and invaded the PCB oil, showing lower numbers of damaged cells than before and a dramatic increase in PCB degradation. This sequence of biofilm formation is understood as a maturation process prior to PCB oil colonization. This is followed by a thin biofilm on the PCB droplet, an aggregation process forming pockets in the PCB, and finally an invasion of the biofilm into the PCB oil. Only the mature biofilm showed degradation of pentachlorinated PCB congeners, which may be reductively dechlorinated and the resulting trichlorobiphenyls then aerobically metabolized.

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.

The salt composition of 14 acidic lakes was analysed, and their microbial communities were compared by their 16S rDNA-based single-strand conformation polymorphism (SSCP) fingerprints. The lakes were grouped into three chemically distinct types, and operational taxonomic units (OTUs) were identified in the 16S rDNA SSCP fingerprints. Discriminant analysis between these groups using a multivariate approach showed that not a single organism, but rather a set of seven OTUs (Ferromicrobium acidophilum, Bacillus pumilus, Acidophilium organovorum, Paenibacillus chibensis, Acidocella sp. and two different alpha-Proteobacteria), was found to be indicative for a given lake type. We conclude that, for these lakes, not single indicator strains but indicator groups exist that are controlled by the ionic composition of the lakes.

Recent advances in the degradation of polychlorinated biphenyls (PCBs) have focussed on the use of experimental enrichment cultures to obtain PCB-degrading communities, and the use of culture-independent approaches to characterize natural and experimental PCB-degrading communities and to identify the key members in this process. PCB-degrading communities can be surprisingly diverse. Novel types of composite bacteria-mineral biofilm communities have been described. Community metabolism of PCBs may lead to the formation of protoanemonin, a dead-end product in some instances but, in others, a seemingly productive intermediate. Analysis of isotope fractionation and preferred enantiomer degradation has provided new information on degradation of PCBs in anaerobic settings. The first defined community capable of dehalorespiration of PCBs has been described, and important community members identified. Here, we provide an overview of the current knowledge of aerobic and anaerobic degradation of PCBs in microbial consortia and in the environment, including novel approaches to determine in situ PCB degradation.

Abstract: 4-Chlorosalicylate (4-CS) can be degraded completely by a bacterial consortium consisting of Pseudomonas reinekei (MT1), Achromobacter spanius (MT3) and Pseudomonas veronii (MT4). The fourth species Wautersiella falsenii (MT2) is thought to act as a ‘necrotizer’ of the community. Single cell approaches were used to follow every species'' degradation activity within the community by assuming that growth and proliferation are activity markers for the utilization of 4-CS and its degradation pathway intermediates as carbon and energy sources. A primary/secondary antibody staining technique for species differentiation was applied and a species-resolved determination of proliferation activity by flow cytometry undertaken. Degradation was followed by quantifying 4-CS and the resulting intermediates by HPLC. A good correlation of HPLC bulk data with the proliferation activity states of every species within the community was found. It was also assumed that reduced activity of strain MT4 and increased proliferation of strain MT2 might have caused an observed breakdown of the consortium grown in the bioreactor. The double staining technique provided the chance to follow bacterial cell states and their roles in mixed cultures without applying labelled substrates. It is therefore in line with single cell techniques already successfully applied in biotechnology for developing strategies to optimize microbially catalyzed production processes. [Copyright &y& Elsevier]

Abstract: Biphenyl degrading bacteria (40 strains) have been isolated along a gradient of chlorobenzene pollution from an aquifer which did not contain any PCB to answer the question of how metabolic/catabolic abilities exist in ecosystems that have not been stressed with the relevant substrates is important for intrinsic bioremediations. Only few of the isolates were characterized by 16S rRNA gene sequence analyses as Pseudomonas species while the majority were Gram-positive, belonging to the order Actinomycetales and representing the genera Rhodococcus and Arthrobacter. The strains could grow on a variety of chlorobenzoates but no pattern of substrate usage and phylogeny or pollution gradient could be found. Strains which were able to grow on 2,5-dichlorobenzoate were often also able to use 3,4- and 3,5-dichloro- and 2,3,5-trichlorobenzoate or those using 2-chlorobenzoate could usually use 2,6-dichlorobenzoate as well. From that results, it is concluded that a highly diverse, basic metabolic activity for PCB degradation existed at this site despite the absence of PCB. [Copyright &y& Elsevier]

A moorland soil site polluted with PCB showed a high diversity of metabolically active bacteria. Beside frequent types of 16S rRNA sequences similar to those of the species of Sphingomonas and the Acidobacterium phylum an unusual high number of sequences from the genus Burkholderia were found. Burkholderia was also the main genus in isolates enriched on biphenyl or various chlorobenzoates. In microcosm experiments sterilized surfaces exposed to PCB polluted soil always showed the presence of clay aggregates formed by bacteria attached to the substratum. The bacteria use the PCB loaded clay colloids as transport medium for the water insoluble substrate to get access to the carbon source. This is a novel mechanism of how bacteria deal with hydrophobic substrates. [ABSTRACT FROM AUTHOR]