Microbial Diversity within Early-Stage Cultured Panulirus ornatus Phyllosomas

A thorough understanding of the microorganisms and pathogens associated with the larval stage of the tropical ornate rock lobster, Panulirus ornatus, is required to overcome disease outbreaks that currently block aquaculture attempts. This study used microscopy in addition to culture and molecularly based microbiological techniques to characterize the bacterial community associated with cultured, developmental stage PI to PII P. ornatus phyllosomas. Scanning electron microscopy demonstrated colonization of phyllosomas by filamentous, rod-shaped, and coccus-shaped bacteria. A clone library constructed from dead phyllosomas sampled from the larval rearing tank on day 10 was dominated by Thiothrix-affiliated sequences (56% of clones). A comparable library from live phyllosomas also contained Thiothrix-affiliated sequences, though these only represented 19% of clones within the library. Fluorescent in situ hybridization (FISH) confirmed identification of the filamentous bacteria as Thiothrix sp., being present on dead phyllosomas. FISH also identified Leucothrix sp. and Vibrio sp., as well as a range of other rod- and coccus-shaped bacteria, colonizing both live and dead phyllosomas. The development of the microbial community associated with phyllosomas was monitored through a standard larval rearing run using denaturing gradient gel electrophoresis (DGGE). Vibrio sp.-affiliated bands dominated the profiles of live animals through the rearing period and dead phyllosomas sampled on selected days. The population of Vibrio sp. associated with phyllosomas was monitored with culture-based analysis on selective media and demonstrated to increase significantly on day 7, coinciding with the beginning of the larval molt. An isolated Vibrio harveyi strain demonstrated an identical 16S rRNA sequence with retrieved DGGE and clone library sequences. Colonization of phyllosomas with filamentous bacterial species potentially hinders the ability of the animals to molt and, combined with the added stress of the molt process, likely results in reduced immune function, allowing opportunistic pathogenic Vibrio sp. to cause larval mortalities.     

Identification of an antagonistic probiotic combination protecting ornate spiny lobster (Panulirus ornatus) larvae against Vibrio owensii infection

Vibrio owensii DY05 is a serious pathogen causing epizootics in the larviculture of ornate spiny lobster Panulirus ornatus. In the present study a multi-tiered probiotic screening strategy was used to identify a probiotic combination capable of protecting P. ornatus larvae (phyllosomas) from experimental V. owensii DY05 infection. From a pool of more than 500 marine bacterial isolates, 91 showed definitive in vitro antagonistic activity towards the pathogen. Antagonistic candidates were shortlisted based on phylogeny, strength of antagonistic activity, and isolate origin. Miniaturized assays used a green fluorescent protein labelled transconjugant of V. owensii DY05 to assess pathogen growth and biofilm formation in the presence of shortlisted candidates. This approach enabled rapid processing and selection of candidates to be tested in a phyllosoma infection model. When used in combination, strains Vibrio sp. PP05 and Pseudoalteromonas sp. PP107 significantly and reproducibly protected P. ornatus phyllosomas during vectored challenge with V. owensii DY05, with survival not differing significantly from unchallenged controls. The present study has shown the value of multispecies probiotic treatment and demonstrated that natural microbial communities associated with wild phyllosomas and zooplankton prey support antagonistic bacteria capable of in vivo suppression of a pathogen causing epizootics in phyllosoma culture systems.     

Pathogenicity and infection cycle of Vibrio owensii in larviculture of the ornate spiny lobster (Panulirus ornatus)

The type strain of Vibrio owensii (DY05) was isolated during an epizootic of aquaculture-reared larvae (phyllosomas) of the ornate spiny lobster (Panulirus ornatus). V. owensii DY05 was formally demonstrated to be the etiological agent of a disease causing rapid and reproducible larval mortality with pathologies similar to those seen during disease epizootics. Vectored challenge via the aquaculture live feed organism Artemia (brine shrimp) caused consistent cumulative mortality rates of 84 to 89% after 72 h, in contrast to variable mortality rates seen after immersion challenge. Histopathological examination of vector-challenged phyllosomas revealed bacterial proliferation in the midgut gland (hepatopancreas) concomitant with epithelial cell necrosis. A fluorescent-protein-labeled V. owensii DY05 transconjugant showed dispersal of single cells in the foregut and hepatopancreas 6 h postexposure, leading to colonization of the entire hepatopancreas within 18 h and eventually systemic infection. V. owensii DY05 is a marine enteropathogen highly virulent to P. ornatus phyllosoma that uses vector-mediated transmission and release from host association to a planktonic existence to perpetuate transfer. This understanding of the infection process will improve targeted biocontrol strategies and enhance the prospects of commercially viable larviculture for this valuable spiny lobster species.     

Molecular diversity of nitrogen-fixing bacteria associated with Chrysopogon zizanioides (L.) Roberty (vetiver), an essential oil producer plant

The essential oil produced by vetiver can vary in amount and composition depending on the bacterial community associated with its roots. Some of these bacteria could also promote plant growth by fixing nitrogen. This study aimed to analyze the diversity of diazotrophic bacteria tightly associated with roots of different vetiver genotypes. nifH-based PCR-denaturing gradient gel electrophoresis (DGGE) and clone libraries were used. DGGE profiles obtained from bulk and rhizosphere soils and root DNA amplified with nifH primers showed that samples from rhizosphere soil and root were separated at 68% similarity. Twelve bands were excised from the DGGE and sequenced. High similarity with nifH sequences of Bradyrhizobium sp., Pseudacidovorax sp. and Xanthobacter sp. was observed. Moreover, three nifH clone libraries were generated using polF/polR-primers from root DNA samples obtained from vetiver genotypes UFS-VET001, UFS-VET003 and UFS-VET004. In UFS-VET001, 24.2% of 95 clones were affiliated with sequences of Mesorhizobium loti while in UFS-VET003 41.5% of 89 clones were affiliated with Sphingomonas azotifigens, and in UFS-VET004 36.4% of 85 clones were affiliated with Klebsiella pneumoniae. The data obtained can be used to guide the isolation of diazotrophic bacteria, which may contribute to plant growth promotion and improvement of the production of essential oil in vetiver.     

Manipulation of insect gut microbiota towards the improvement of Bactrocera oleae artificial rearing

Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the main pest of olive trees (Olea europaea L.), causing major damages in olive crops. Improvement of mass rearing is a prerequisite for the successful development of large-scale sterile insect technique (SIT) applications. This can be achieved through the enrichment of artificial diets with gut bacteria isolates. We assessed the efficiency of three gut bacteria previously isolated from Ceratitis capitata (Wiedemann), and four isolated from B. oleae, as larval diet additives in both live and inactivated/dead forms. Our results showed that dead Enterobacter sp. AA26 increased pupal weight, whereas both live and dead cells increased pupal and adult production and reduced immature developmental time, indicating that its bacterial cells serve as a direct nutrient source. Live Providencia sp. AA31 improved pupal and adult production, enhanced male survival under stress conditions, and delayed immature development. Dead Providencia sp. AA31, however, did not affect production rates, indicating that live bacteria can colonize the insect gut and biosynthesize nutrients essential for larval development. Live and dead Bacillus sp. 139 increased pupal weight, accelerated immature development, and increased adult survival under stress. Moreover, live Bacillus sp. 139 improved adult production, indicating that Bacillus cells are a direct source of nutrients. Dead Serratia sp. 49 increased pupal and adult production and decreased male survival under stress conditions whereas live cells decreased insect production, indicating that the live strain is entomopathogenic, but its dead cells can be utilized as nutrient source. Klebsiella oxytoca, Enterobacter sp. 23, and Providencia sp. 22 decreased pupal and subsequent adult production and were harmful for B. oleae. Our findings indicate that dead Enterobacter sp. AA26 is the most promising bacterial isolate for the improvement of B. oleae mass rearing in support of future SIT or related population suppression programs.     

Culturability and In situ abundance of pelagic bacteria from the North Sea

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.     

Diversity of bacteria associated with the coral Pocillopora damicornis from the Great Barrier Reef

The microbial community associated with the reef building coral Pocillopora damicornis located on the Great Barrier Reef was investigated using culture-independent molecular microbial techniques. The microbial communities of three separate coral colonies were assessed using clone library construction alongside restriction fragment length polymorphism and phylogenetic analysis. Diversity was also investigated spatially across six replicate samples within each single coral colony using 16S rDNA and rpoB-DGGE analysis. Clone libraries demonstrated that the majority of retrieved sequences from coral tissue slurry libraries affiliated with gamma-Proteobacteria. This contrasted with clone libraries of seawater and coral mucus, which were dominated by alpha-Proteobacteria. A number of retrieved clone sequences were conserved between coral colonies; a result consistent with previous studies suggesting a specific microbe-coral association. rpoB-DGGE patterns of replicate tissue slurry samples underestimated microbial diversity, but demonstrated that fingerprints were identical within the same coral. These fingerprints were also conserved across coral colonies. The 16S rDNA-DGGE patterns of replicate tissue slurry samples were more complex, although non-metric multidimensional scaling (nMDS) analysis showed groupings of these banding patterns indicating that some bacterial diversity was uniform within a coral colony. Sequence data retrieved from DGGE analysis support clone library data in that the majority of affiliations were within the gamma-Proteobacteria. Many sequences retrieved also affiliated closely with sequences derived from previous studies of microbial diversity of healthy corals in the Caribbean. Clones showing high 16S rDNA sequence identity to both Vibrio shiloi and Vibrio coralliilyticus were retrieved, suggesting that these may be opportunist pathogens. Comparisons of retrieved microbial diversity between two different sampling methods, a syringe extracted coral mucus sample and an airbrushed coral tissue slurry sample were also investigated. Non-metric multidimensional scaling of clone library data highlighted that clone diversity retrieved from a coral mucus library more closely reflected the diversity of surrounding seawater than a corresponding coral tissue clone library.     

Identification, detection, and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.     

Bacterial diversity in the bacterioneuston (sea surface microlayer): the bacterioneuston through the looking glass

The bacterioneuston is defined as the community of bacteria present within the neuston or sea surface microlayer. Bacteria within this layer were sampled using a membrane filter technique and bacterial diversity was compared with that in the underlying pelagic coastal seawater using molecular ecological techniques. 16S rRNA gene libraries of approximately 500 clones were constructed from both bacterioneuston and the pelagic water samples and representative clones from each library were sequenced for comparison of bacterial diversity. The bacterioneuston was found to have a significantly lower bacterial diversity than the pelagic seawater, with only nine clone types (ecotaxa) as opposed to 46 ecotaxa in the pelagic seawater library. Surprisingly, the bacterioneuston clone library was dominated by 16S rRNA gene sequences affiliated to two groups of organisms, Vibrio spp. which accounted for over 68% of clones and Pseudoalteromonas spp. accounting for 21% of the library. The dominance of these two 16S rRNA gene sequence types within the bacterioneuston clone library was confirmed in a subsequent gene probing experiment. 16S rRNA gene probes specific for these groups of bacteria were designed and used to probe new libraries of 1000 clones from both the bacterioneuston and pelagic seawater DNA samples. This revealed that 57% of clones from the bacterioneuston library hybridized to a Vibrio sp.-specific 16S rRNA gene probe and 32% hybridized to a Pseudoalteromonas sp.-specific 16S rRNA gene probe. In contrast, the pelagic seawater library resulted in only 13% and 8% of 16S rRNA gene clones hybridizing to the Vibrio sp. and Pseudoalteromonas sp. probes respectively. Results from this study suggest that the bacterioneuston contains a distinct population of bacteria and warrants further detailed study at the molecular level.     

Stimulation or inhibition of growth of the unicellular alga Pavlova lutheri by bacteria isolated from larval turbot culture systems

During growth of larval turbot in aquaculture the first food supplied is usually the rotifer, Brachionus plicatilis and algae are commonly included in the system as food for the rotifers, thereby maintaining their nutrient quality. As bacteria are known to influence markedly the survival of larval turbot, the effect of bacteria, isolated from larval turbot, on growth of Pavlova lutheri was measured over a 3-d period. Of 41 bacteria tested, 23 inhibited growth to various degrees, eight had no effect and 10 were weak growth stimulants. Four bacteria, identified as a Flavobacterium, Vibrio fluvialis, Vibrio natrigens and a Vibrio sp., were strongly inhibitory and the Flavobacterium inhibited growth of Pavlova lutheri from an inoculum of 10³ colony-forming units per ml. Inhibition was due to a heat-labile factor released by the Flavobacterium into the culture medium. The Flavobacterium also produced bacteriocin(s) which inhibited the growth of a range of vibrios. Bacteria antagonistic towards algae would be undesirable in larval rearing and if bacteria are to be selected which are beneficial (probiotics) in larval rearing systems their possible interaction with algae must be considered.     

Microbial community associated with the colonial ascidian Cystodytes dellechiajei

The ascidian Cystodytes dellechiajei (Della Valle, 1877) (phylum Chordata, class Ascidiacea, family Polycitoridae) is a colonial tunicate that inhabits benthic rock environments in the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean Sea. Its life cycle has two phases, the adult sessile colony and the free-living larva. Both adult zooids and larvae are surrounded by a protective tunic that contains several eukaryotic cell lines, is composed mainly of acidic mucopolysacharides associated with collagen and elastin-like proteins, and is covered by a thin cuticle. The microbiota associated with the tunic tissues of adult colonies and larva of C. dellechiajei has been examined by optical, confocal and electron microscopy and by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene clone library analysis. Microscopy analyses indicated the presence inside the tunic, both for the adult and the larva, of a dense community of Bacteria while only the external surface of colony cuticle was colonized by diatoms, rodophyte algae and prokaryotic-like epiphytes. Transmission electron microscopy showed tunic eukaryotic cells that were engulfing and lysing bacteria. 16S rRNA gene analyses (DGGE and clone libraries) and FISH indicated that the community inside the tunic tissues of the adults and larvae was dominated by Alphaproteobacteria. Bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were also detected in the adults. Many of the 16S rRNA gene sequences in the tunic tissues were related to known aerobic anoxygenic phototrophs (AAP), like Roseobacter sp. and Erythrobacter sp. In order to check whether the gene pufM, coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis, was being expressed inside the ascidian tissues, two libraries, one for an adult colony and one for larva, of cDNA from the expressed pufM gene were also constructed. The sequences most frequently (64% for colony and 67% for larva) retrieved from these libraries presented > 90% aa identity with the pufM gene product of the Roseobacter-like group, a cluster of AAP widely detected in marine planktonic environments.     

南美白对虾肠道微生物群落的分子分析

采用分子生物学手段16S rDNA克隆文库方法对实验室养殖条件下的南美白对虾肠道细菌进行了多样性研究。用限制性片段长度多态性(RFLP)方法从文库中筛选出可能不同细菌来源的克隆子12个,测定其16S rDNA片段核甘酸序列,将所获得的序列与GenBank数据库进行BLAST比对,结果表明:南美白对虾肠道的16S rDNA克隆文库中126个克隆子分属2个不同的细菌类群:变形细菌(Proteobacteria)和厚壁细菌(Firmicutes),其中厚壁细菌为优势菌群占到75.4%,且与最相似序列同源性均低于94%;变形细菌占到24.6%,与最相似序列同源性均高于98%,分别为希瓦氏菌属(Shewanella),泛菌属(Pantoea),Aranicola属,假单胞菌属(Pseudomonas)和弧菌属(Vibrio)。     

Identification, Detection, and Spatial Resolution of Clostridium Populations Responsible for Cellulose Degradation in a Methanogenic Landfill Leachate Bioreactor

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.     

A marine bacterium, Micrococcus MCCB 104, antagonistic to vibrios in prawn larval rearing systems

A marine bacterium, Micrococcus MCCB 104, isolated from hatchery water, demonstrated extracellular antagonistic properties against Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, V. fluviallis, V. nereis, V. proteolyticus, V. mediterranei, V cholerae and Aeromonas sp., bacteria associated with Macrobrachium rosenbergii larval rearing systems. The isolate inhibited the growth of V. alginolyticus during co-culture. The antagonistic component of the extracellular product was heat-stable and insensitive to proteases, lipase, catalase and alpha-amylase. Micrococcus MCCB 104 was demonstrated to be non-pathogenic to M. rosenbergii larvae.     

Bacteria Associated with the Gut Tract of Larval Stages of the Aquatic Cranefly Tipula abdominalis (Diptera; Tipulidae)

Scanning electron microscopy, light microscopy, and direct isolations were used to examine the distribution and diversity of bacteria in the gut tracts of larval stages of Tipula abdominalis. The animal had an enlarged hindgut which housed a diverse bacterial community in the lumen and directly attached to the gut wall. Distinct localization was noted, with the most dense and most diverse community anterior to the rectum. A distinct architecture of bacteria occurred in this region, characterized by a layering or a “weblike” array of filamentous bacteria overlying mats of bacteria closely associated with the gut wall. Although morphological diversity was high in the hindgut, filamentous bacteria were the dominant morphology observed. The attached microbiota, sloughed during ecdysis, recolonized to the same density and diversity observed before the molt. The majority of the isolatable bacterial types were facultatively anaerobic. The distinct localization and attached nature of the hindgut bacteria and the recolonization after each molt suggest they are indigenous to this region of the gut tract.     

Phylogenetic Composition of Bacterioplankton Assemblages from the Arctic Ocean

We analyzed the phylogenetic composition of bacterioplankton assemblages in 11 Arctic Ocean samples collected over three seasons (winter-spring 1995, summer 1996, and summer-fall 1997) by sequencing cloned fragments of 16S rRNA genes. The sequencing effort was directed by denaturing gradient gel electrophoresis (DGGE) screening of samples and the clone libraries. Sequences of 88 clones fell into seven major lineages of the domain Bacteria: α (36%)-, γ (32%)-, δ (14%)-, and (1%)-Proteobacteria; Cytophaga-Flexibacter-Bacteroides spp. (9%); Verrucomicrobium spp. (6%); and green nonsulfur bacteria (2%). A total of 34% of the cloned sequences (excluding clones in the SAR11 and Roseobacter groups) had sequence similarities that were <94% compared to previously reported sequences, indicating the presence of novel sequences. DGGE fingerprints of the selected samples showed that most of the bands were common to all samples in all three seasons. However, additional bands representing sequences related to Cytophaga and Polaribacter species were found in samples collected during the summer and fall. Of the clones in a library generated from one sample collected in spring of 1995, 50% were the same and were most closely affiliated (99% similarity) with Alteromonas macleodii, while 50% of the clones in another sample were most closely affiliated (90 to 96% similarity) with Oceanospirillum sp. The majority of the cloned sequences were most closely related to uncultured, environmental sequences. Prominent among these were members of the SAR11 group. Differences between mixed-layer and halocline samples were apparent in DGGE fingerprints and clone libraries. Sequences related to α-Proteobacteria (dominated by SAR11) were abundant (52%) in samples from the mixed layer, while sequences related to γ-proteobacteria were more abundant (44%) in halocline samples. Two bands corresponding to sequences related to SAR307 (common in deep water) and the high-G+C gram-positive bacteria were characteristic of the halocline samples.     

Fungal rDNA signatures in coronary atherosclerotic plaques

Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology.     

Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria

Ticks play an important role in the transmission of arthropod-borne diseases of viral, protozoal and bacterial origin. The present article describes a molecular-biological based method, which facilitated the broad-range analyses of bacterial communities in ixodid ticks (Ixodes ricinus). DNA was extracted both from single ticks and pooled adult ticks. Eubacterial 16S rRNA gene fragments (16S rDNA) were amplified by polymerase chain reaction (PCR) with broad-range ribosomal primers. Sequences spanning the hypervariable V3 region of the 16S rDNA and representing individual bacterial taxons were separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were exised, cloned and sequenced. In addition, we set up a 16S rDNA clone library which was screened by DGGE. Sequences were compared with sequences of known bacteria listed in the GenBank database. A number of bacteria were affiliated with the genera Rickettsia, Bartonella, and Borrelia, which are known to be pathogenic and transmitted by ticks. Two sequences were related to the yet to be cultivated Haemobartonella. To our knowledge, Haemobartonella has never been directly detected in I. ricinus. In addition, members of the genera Staphylococcus, Rhodococcus, Pseudomonas, and Moraxella were detected, which have not been identified in ticks so far. Two bacteria were most closely related to a rickettsial endosymbiont of an Acanthamoeba sp., and to an endosymbiont (Legionellaceae, Coxiella group) of the microarthropod Folsomia candida. The results prove that 16S rDNA genotyping in combination with DGGE analysis is a promising approach for the detection and identification of bacteria infecting ticks, regardless of whether these bacteria are fastidious, obligate intracellular or noncultivable.     

Bacterial populations colonizing and degrading rice straw in anoxic paddy soil

Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the alpha, beta, and gamma Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.     

In situ identification of polyphosphate- and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process

Polyphosphate- and polyhydroxyalkanoate (PHA)-accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods. Results of 16S rDNA clone library and fluorescence in situ hybridization (FISH) with rRNA-targeted, group-specific oligonucleotide probes indicated that the microbial community consisted mostly of the alpha- (9.5% of total cells), beta- (41.3%) and gamma- (6.8%) subclasses of the class Proteobacteria, Flexibacter-Cytophaga (4.5%) and the Gram-positive high G+C (HGC) group (17.9%). With individual phylogenetic groups or subgroups, members of Candidatus Accumulibacter phosphatis in the beta-2 subclass, a novel HGC group closely related to Tetrasphaera spp., and a novel gamma-proteobacterial group were the predominant populations. Furthermore, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups. Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA. In addition, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA. No cellular inclusions were detected in most members of the alpha-Proteobacteria and the Cytophaga-Flavobacterium group. The diversified functional traits observed suggested that different substrate metabolisms were used by predominant phylogenetic groups in EBPR processes.