Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.     

Normal conjunctival flora in the North American opossum (Didelphis virginiana) and raccoon (Procyon lotor)

We documented the normal conjunctival bacterial flora from 17 opossums (Didelphis virginiana) and 10 raccoons (Procyon lotor) trapped in Manhattan, Kansas (USA) from November 1999 to January 2000. Both raccoons and opossums were free of apparent ocular disease. The inferior conjunctival sacs of each animal were swabbed for aerobic bacterial and Mycoplasma culture and polymerase chain reaction (PCR) for Mycoplasma and Chlamydia detection. All conjunctival samples were positive for one or more species of aerobic bacteria. The most common isolate from opossums was Staphylococcus spp. Other isolates included Streptococcus spp., Bacillus spp., Corynebacterium spp., and Enterococcus faecalis. The most common isolates in raccoons was Bacillus spp. Other isolates included Streptococcus spp., Staphylococcus spp., non-hemolytic Escherichia coli, and Enterococcus faecalis. Mycoplasma culture was negative in samples from opossums and raccoons. Evidence of Mycoplasma and Chlamydia presence was detected by PCR.     

Predominant <Emphasis Type="Italic">Bacillus</Emphasis> spp. in Agricultural Soil under Different Management Regimes Detected via PCR-DGGE

A PCR system for studying the diversity of species of Bacillus and related taxa directly from soil was developed. For this purpose, a specific 24-bp forward primer located around position 110 of the 16S ribosomal RNA gene was designed and combined with a reverse bacterial primer located at the end of the gene. The specificity of this PCR system for bacilli and related taxons was confirmed on the basis of tests with diverse strains as well as with soil DNA. Analysis of a soil DNA derived clone library showed that the amplified fragments affiliated exclusively with sequences of gram-positive bacteria, with up to 95% of the sequences originating from putative Bacillus species. In particular, sequences affiliated to those of B. mycoides, B. pumilus, B. megaterium, B. thuringiensis, and B. firmus, as well as to related taxa such as Paenibacillus, were obtained. A minority, i.e., less than 6%, of the clones affiliated with other gram-positive bacteria, such as Arthrobacter spp., Frankia spp., and uncultured gram-positives. The amplified fragments were used as templates for a second PCR using bacterial 16S rDNA primers, yielding PCR products of about 410 bp, which were separated by denaturing gradient gel electrophoresis (DGGE). Amplicons indicating Bacillus spp. were found in the gel between 45% and roughly 60% denaturant, whereas those representing other, high-G+C% bacteria, were localized in gel regions with denaturant concentrations exceeding about 60%, thus allowing the distinction between these two groups of sequences. We applied this system to compare the group-specific diversity in bacterial communities in an agricultural soil under different regimes, i.e., permanent grassland, grassland recently turned to arable land, and arable land under agricultural rotation. Differences in the Bacillus-related community structures between the treatments were clearly detected. Higher diversities, as judged by Shannon–Weaver indices calculated on the basis of the molecular profiles, were consistently observed in the permanent grassland and the grassland turned into arable land, as compared to the arable land.     

Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.     

Partitioning of bacterial communities between seawater and healthy, black band diseased, and dead coral surfaces

Distinct partitioning has been observed in the composition and diversity of bacterial communities inhabiting the surface and overlying seawater of three coral species infected with black band disease (BBD) on the southern Caribbean island of Cura?ao, Netherlands Antilles. PCR amplification and sequencing of bacterial 16S rRNA genes (rDNA) with universally conserved primers have identified over 524 unique bacterial sequences affiliated with 12 bacterial divisions. The molecular sequences exhibited less than 5% similarity in bacterial community composition between seawater and the healthy, black band diseased, and dead coral surfaces. The BBD bacterial mat rapidly migrates across and kills the coral tissue. Clone libraries constructed from the BBD mat were comprised of eight bacterial divisions and 13% unknowns. Several sequences representing bacteria previously found in other marine and terrestrial organisms (including humans) were isolated from the infected coral surfaces, including Clostridium spp., Arcobacter spp., Campylobacter spp., Cytophaga fermentans, Cytophaga columnaris, and Trichodesmium tenue.     

Bacterial phylogenetic clusters revealed by genome structure.

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.     

Bacterial diversity in water samples from uranium wastes as demonstrated by 16S rDNA and ribosomal intergenic spacer amplification retrievals

Bacterial diversity was assessed in water samples collected from several uranium mining wastes in Ger many and in the United States by using 16S rDNA and ribosomal intergenic spacer amplification retrievals. The results obtained using the 16S rDNA retrieval showed that the samples collected from the uranium mill tailings of Schlema/Alberoda, Germany, were predominated by Nitrospina-like bacteria, whereas those from the mill tailings of Shiprock, New Mexico, USA, were predominated by gamma-Pseudomonas and Frauteria spp. Additional smaller populations of the Cytophaga-Flavobacterium-Bacteroides group and alpha- and delta-Proteobacteria were identified in the Shiprock samples as well. Proteobacteria and Cytophaga-Flavobacterium-Bacteroides were also found in the third uranium mill tailings studied, Gittersee/Coschütz, Germany, but the groups of the predominant clones were rather small. Most of the clones of the Gittersee/Coschütz samples represented individual sequences, which indicates a high level of bacterial diversity. The samples from the fourth uranium waste studied, Steinsee Deponie B1, Germany, were predominantly occupied by Acinetobacter spp. The ribosomal intergenic spacer amplification retrieval provided results complementary to those obtained by the 16S rDNA analyses. For instance, in the Shiprock samples, an additional predominant bacterial group was identified and affiliated with Nitrosomonas sp., whereas in the Gittersee/Coschütz samples, anammox populations were identified that were not retrieved by the applied 16S rDNA approach.     

Partitioning of Bacterial Communities between Seawater and Healthy, Black Band Diseased, and Dead Coral Surfaces

Distinct partitioning has been observed in the composition and diversity of bacterial communities inhabiting the surface and overlying seawater of three coral species infected with black band disease (BBD) on the southern Caribbean island of Curaçao, Netherlands Antilles. PCR amplification and sequencing of bacterial 16S rRNA genes (rDNA) with universally conserved primers have identified over 524 unique bacterial sequences affiliated with 12 bacterial divisions. The molecular sequences exhibited less than 5% similarity in bacterial community composition between seawater and the healthy, black band diseased, and dead coral surfaces. The BBD bacterial mat rapidly migrates across and kills the coral tissue. Clone libraries constructed from the BBD mat were comprised of eight bacterial divisions and 13% unknowns. Several sequences representing bacteria previously found in other marine and terrestrial organisms (including humans) were isolated from the infected coral surfaces, including Clostridium spp., Arcobacter spp., Campylobacter spp., Cytophaga fermentans, Cytophaga columnaris, and Trichodesmium tenue.     

Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting

Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers.     

Impacts of poultry house environment on poultry litter bacterial community composition

Viral and bacterial pathogens are a significant economic concern to the US broiler industry and the ecological epicenter for poultry pathogens is the mixture of bedding material, chicken excrement and feathers that comprises the litter of a poultry house. This study used high-throughput sequencing to assess the richness and diversity of poultry litter bacterial communities, and to look for connections between these communities and the environmental characteristics of a poultry house including its history of gangrenous dermatitis (GD). Cluster analysis of 16S rRNA gene sequences revealed differences in the distribution of bacterial phylotypes between Wet and Dry litter samples and between houses. Wet litter contained greater diversity with 90% of total bacterial abundance occurring within the top 214 OTU clusters. In contrast, only 50 clusters accounted for 90% of Dry litter bacterial abundance. The sixth largest OTU cluster across all samples classified as an Arcobacter sp., an emerging human pathogen, occurring in only the Wet litter samples of a house with a modern evaporative cooling system. Ironically, the primary pathogenic clostridial and staphylococcal species associated with GD were not found in any house; however, there were thirteen 16S rRNA gene phylotypes of mostly gram-positive phyla that were unique to GD-affected houses and primarily occurred in Wet litter samples. Overall, the poultry house environment appeared to substantially impact the composition of litter bacterial communities and may play a key role in the emergence of food-borne pathogens.     

Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets

AIMS: To explore the effect of drug-free poultry production on the intestinal microflora of broiler chickens, the bacterial community of this environment was quantitatively profiled in both conventionally reared birds and birds reared without antibiotic growth promotants (AGPs) on a vegetable-based diet. METHODS AND RESULTS: Quantitative, real-time PCR with group-specific 16S rDNA primer sets was used to enumerate the abundance of the following chicken gastrointestinal (GI) tract phylogenetic groups: the Clostridium leptum-Faecalibacterium prausnitzii subgroup (Clostridium genus cluster IV), the Clostridium coccoides - Eubacterium rectale subgroup (Clostridium cluster XIVa and XIVb), the Bacteroides group (including Prevotella and Porphyromonas), Bifidobacterium spp., the Enterobacteriaceae, the Lactobacillus group (including the genera Leuconostoc, Pediococcus, Aerococcus and Weissella), the Clostridium perfringens subgroup (Clostridium cluster I), Enterococcus spp., Veillonella spp., Atopobium spp., Campylobacter spp. and the domain Bacteria. A species-specific 5'-nuclease (Taqman) assay was also employed to specifically assess Cl. perfringens abundance. Ten birds were sampled from each of two commercial chicken houses, one in which feed was supplemented with AGPs and exogenous animal protein, and the other vegetable-based and drug-free, at 7, 14 and 21 days of age. The ileal community was dominated by two large populations, the lactobacilli and the Enterobacteriaceae, with those taxa much more numerous in drug-free vegetable-based diet fed birds than those conventionally reared at the 7- and 14-day time periods. The progressive changes in microflora in both the conventional and drug-free caeca were similar to each other, with the Enterobacteriaceae sequences dominating at day 7, but being replaced by obligate anaerobe signature sequences by day 14. Of note was the finding that all the day 14 and day 21 replicate caecal samples from the drug-free house were positive for Campylobacter spp. averaging >10(8) 16S rDNA gene copies per gram wet weight. CONCLUSIONS: Quantitative, real-time PCR indicates that the effects of drug-free rearing on the chicken GI tract microbial community are most pronounced in the ileal region, but AGPs may be important in controlling Campylobacter colonization of the caecum. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative taxonomic understanding of the shifting microbial ecology of the broiler chicken gut microbiota is important in the light of AGP withdrawal. AGP withdrawal has occurred in response to concerns over the transfer of antimicrobial-resistant bacteria to humans via the food production chain.     

Molecular microbial diversity of a soil sample and detection of ammonia oxidizers from Cape Evans, Mcmurdo Dry Valley, Antarctica

The aim of our study was to estimate the uncultured eubacterial diversity of a soil sample collected below a dead seal, Cape Evans, McMurdo, Antarctica by an SSU rDNA gene library approach. Our study by sequencing of clones from SSU rDNA gene library approach revealed high diversity in the soil sample from Antarctica. More than 50% of clones showed homology to Cytophaga-Flavobacterium-Bacteroides group; sequences also belonged to alpha, beta, gamma proteobacteria, Thermus-Deinococcus and high GC gram-positive group; Phylogenetic analysis of the SSU rDNA clones showed the presence of species belonging to Cytophaga spp., Vitellibacter vladivostokensis, Aequorivita lipolytica, Aequorivita crocea, Flavobacterium spp., Flexibacter sp., Subsaxibacter broadyi, Bacteroidetes, Roseobacter sp., Sphingomonas baekryungensis, Nitrosospira sp., Nitrosomonas cryotolerans, Psychrobacter spp., Chromohalobacter sp., Psychrobacter okhotskensis, Psychrobacter fozii, Psychrobacter urativorans, Rubrobacter radiotolerans, Marinobacter sp., Rubrobacteridae, Desulfotomaculum aeronauticum and Deinococcus sp. The presence of ammonia oxidizing bacteria in Antarctica soil was confirmed by the presence of the amoA gene. Phylogenetic analysis revealed grouping of clones with their respective groups.     

Identification and phylogenetic analysis of Arsenophonus- and Photorhabdus-type bacteria from adult Hippoboscidae and Streblidae (Hippoboscoidea)

This is the first report of Arsenophonus- and Photorhabdus-type bacteria from Streblidae (bat flies) and Hippoboscidae (louse flies, keds). Strains were detected by means of polymerase chain reaction of 16S rDNA, and phylogenetic analysis determined the relationship of the obtained sequences to previously reported sequences in GenBank. Phylogenetic analysis by means of maximum parsimony revealed that all isolated Arsenophonus spp. 16S rDNA sequences formed a monophyletic sub-clade within other insect Arsenophonus spp., while the Photorhabdus spp. sequences are part of a monophyletic clade including Photorhabdus spp., Xenorhabdus spp. and Proteus spp.     

The Decomposition of Uric Acid in Built Up Poultry Litter

The decomposition of uric acid in built up poultry litter appears to be brought about almost exclusively by the action of aerobic bacteria. Organisms decomposing uric acid usually comprised about one quarter of the bacterial population. They were strains of Corynebacterium and less frequently strains of Nocardia, Streptomyces, Pseudomonas, Alcaligenes and Achromobacter. Uric acid was converted to ammonia by some of the organisms but only to urea by the majority. Hydrolysis of urea to ammonia could be brought about by strains of Corynebacterium, Micrococcus, Alcaligenes, Achromobacter and Cytophaga which had no action on uric acid. It is suggested that the ammoniacal smell and high alkalinity of built up poultry litter result largely from the decomposition of uric acid. The identity of the bacteria concerned is discussed.     

Endophytic Bacterial Diversity in Rice (Oryza sativa L.) Roots Estimated by 16S rDNA Sequence Analysis

The endophytic bacterial diversity in the roots of rice (Oryza sativa L.) growing in the agricultural experimental station in Hebei Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of rice, a pair of bacterial PCR primers (799f–1492r) was selected to specifically amplify bacterial 16S rDNA sequences directly from rice root tissues. Among 192 positive clones in the 16S rDNA library of endophytes, 52 OTUs (Operational Taxonomic Units) were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Cytophaga/Flexibacter/Bacteroides (CFB) phylum, low G+C gram-positive bacteria, Deinococcus-Thermus, Acidobacteria, and archaea. The dominant group was Betaproteobacteria (27.08% of the total clones), and the most dominant genus was Stenotrophomonas. More than 14.58% of the total clones showed high similarity to uncultured bacteria, suggesting that nonculturable bacteria were detected in rice endophytic bacterial community. To our knowledge, this is the first report that archaea has been identified as endophytes associated with rice by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in rice roots.     

APPLICABILITY OF RAPID METHODS FOR DETECTION OF SALMONELLA SPP. IN POULTRY FEEDS: A REVIEW

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. More sensitive and rapid detection of contaminated feedstuffs may lead to more selective and therefore less expensive treatment of feeds, reduced rates of transmission to a poultry host and reduced carcass contamination. In order to interrupt the cycle of Salmonella spp. transmission from feed to poultry to the consumer, more rapid detection methods to monitor these sources are needed that provide conclusive results within the time frame of feed mixing or broiler processing. Within the last decade, new variations of selective media have been investigated to increase selectivity without reducing Salmonella spp. recovery. Immunological assay methods may also decrease assay time from 96 h to within 24–30 h. But all commercially available methods still require 16 to 57 h for preenrichment, enrichment, and in some cases, postenrichment to recover sublethally injured cells before the assay can be performed. Among the molecular methods that are currently available, the polymerase chain reaction (PCR) represents a tremendous potential for the detection of low levels of pathogenic bacteria. Once optimized, rapid methods may be used to quickly, reliably and inexpensively screen a variety of feedstuffs and feed components for the presence of Salmonella spp., with the goal of minimizing both the cost of feed treatment and the horizontal transmission of Salmonella spp. from feed to poultry.     

Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited

The phylogenetic diversity of the intestinal bacterial community in pigs was studied by comparative 16S ribosomal DNA (rDNA) sequence analysis. Samples were collected from a total of 24 pigs representing a variety of diets, ages, and herd health status. A library comprising 4,270 cloned 16S rDNA sequences obtained directly by PCR from 52 samples of either the ileum, the cecum, or the colon was constructed. In total, 375 phylotypes were identified using a 97% similarity criterion. Three hundred nine of the phylotypes (83%) had a <97% sequence similarity to any sequences in the database and may represent yet-uncharacterized bacterial genera or species. The phylotypes were affiliated with 13 major phylogenetic lineages. Three hundred four phylotypes (81%) belonged to the low-G+C gram-positive division, and 42 phylotypes (11.2%) were affiliated with the Bacteroides and Prevotella group. Four clusters of phylotypes branching off deeply within the low-G+C gram-positive bacteria and one in the Mycoplasma without any cultured representatives were found. The coverage of all the samples was 97.2%. The relative abundance of the clones approximated a lognormal distribution; however, the phylotypes detected and their abundance varied between two libraries from the same sample. The results document that the intestinal microbial community is very complex and that the majority of the bacterial species colonizing the gastrointestinal tract in pigs have not been characterized.     

Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus.

The phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy which does not rely on cultivation of the resident microorganisms. Small-subunit rRNA genes (16S rDNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial 16S rDNA sequences showed the existence of well-characterized genera as well as the presence of bacterial species for which no 16S rDNA sequence data are available. Of 55 clones sequenced, 45 were phylogenetically affiliated with four of the major groups of the domain Bacteria: the Proteobacteria, the spirochete group, the Bacteroides group, and the low-G+C-content gram-positive bacteria. Within the Proteobacteria, the 16S rDNA clones showed a close relationship to those of cultivated species of enteric bacteria and sulfate-reducing bacteria, while the 16S rDNA clones in the remaining three groups showed only distant relationships to those of known organisms in these groups. Of the remaining 10 clones, among which 8 clones formed a cluster, there was only very low sequence similarity to known 16S rRNA sequences. None of these clones were affiliated with any of the major groups within the domain Bacteria. The 16S rDNA gene sequence data show that the majority of the intestinal microflora of R. speratus consists of new, uncultured species previously unknown to microbiologists.     

Plant-microbe interactions: identification of epiphytic bacteria and their ability to alter leaf surface permeability

Bacteria were either isolated from leaf surfaces of Hedera helix or obtained from a culture collection in order to analyse their effect on barrier properties of isolated Hedera and Prunus laurocerasus cuticles. On the basis of the 16S rDNA sequences the genera of the six bacterial isolates from Hedera were identified as Pseudomonas sp., Stenotrophomonas sp. and Achromobacter. Water permeability of cuticles isolated from H. helix was measured before and after inoculation with the six bacterial strains. In addition water permeability of cuticles isolated from P. laurocerasus was measured before and after inoculation with the three bacterial strains Pseudomonas aeruginosa, Xanthomonas campestris and Corynebacterium fascians. Rates of water diffusing across isolated cuticles of both species significantly increased by up to 50% after inoculation with all bacterial strains. Obtained results show that epiphytic bacteria have the ability of increasing water permeability of Hedera and Prunus cuticles, which in turn should increase the availability of water and dissolved compounds in the phyllopshere. Consequently, living conditions in the habitat phyllosphere are improved. It can be concluded that the ability to change leaf surface properties will improve epiphytic fitness of leaf surface bacteria.     

Diversity of bacteria associated with grassland soil nematodes of different feeding groups

The bacterial diversity associated with soil nematodes and its relationship with their feeding habits are as yet poorly understood. In the present study the diversity and abundance of bacteria from nematodes and their surrounding soil were analysed and compared. The nematodes were collected from a grassland soil and sorted into bacterial, fungal, plant, predatory and omnivore feeding groups and assigned to taxonomic groups. Total DNA was extracted from the nematodes and partial bacterial 16S rRNA genes were PCR amplified, cloned and sequenced. The abundance and composition of bacterial taxa differed between and within feeding groups. The lowest bacterial diversity was found in the predatory nematodes Prionchulus sp., whereas the highest bacterial diversity was associated with the bacterial-feeding nematode Acrobeles sp. The soil had a more diverse bacterial community than the communities found in the nematode groups. The 16S rRNA gene sequences of bacteria associated with nematodes did not overlap with those detected in soil as determined using the cloning screening approach. However, bacterial sequences identified from nematodes could be detected in the soil with targeted PCR. Our data suggest that the nematodes do not feed on the most abundant bacteria present in soil. Furthermore, several nematodes contained suspected bacterial symbionts and parasites.