Partial 16S rRNA Gene Sequences Research Articles

Characterization of Ornithobacterium rhinotracheale field isolates from Hungary

ABSTRACTOrnithobacterium rhinotracheale is a widely distributed rod-shaped Gram-negative bacterium that infects several avian species including chickens and turkeys. It is associated with respiratory signs, growth retardation, mortality, and reduced egg production, thus causing severe economic losses to the poultry industries. In this study, 37 field isolates of O. rhinotracheale, collected from various locations in Hungary between 1997 and 2015, were identified and characterized by the analysis of partial 16S rRNA gene sequences, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and random amplified polymorphic DNA (RAPD) PCR assays with the OPG11, OPH19, and M13 primers. Most of the field isolates were serotype A, one was serotype B, and four were serotype D. One isolate could not be typed with antisera against serotypes A–E. In a phylogenetic analysis of the 16S rRNA sequences, the isolates formed two clusters. Thirteen distinct patterns were identified with ERIC-PCR, and the RAPD assay with the M13 primer assigned the isolates to 10 different patterns. The other two RAPD assays were unsuitable for distinguishing and grouping the isolates. Neither ERIC type nor RAPD pattern correlated with the place or year of isolation. However, the strains isolated from chickens were more heterogeneous on ERIC-PCR than the isolates recovered from turkeys. In this study, ERIC-PCR was the most discriminatory method for investigating the genetic diversity of O. rhinotracheale isolates.

Improvement of Growth, Yield, and Pigmentation of Mung Bean Plants UsingOchrobactrum intermediumCP‐2 as Bioinoculant

The selection of strains for production of biofertilizers is of primary importance. A strain with diverse growth promoting activities, especially the nitrogen fixation and phosphate solubilization, has highest preference over those with just one activity. In the present study, a symbiotic nitrogen fixing bacterium was isolated from the root nodules of chickpea and characterized asOchrobactrum intermediumCP‐2 on the basis of biochemical tests and partial 16S rRNA gene sequencing and blast analysis. Besides fixing nitrogen, the CP‐2 strain also showed the ability of phosphate solubilization and indole acetic acid production. It was also found positive for ammonia and siderophore production. A significant increase in the lengths of radical and plumule and percent germination was observed when the rhizobacterial strain selected for the study was inoculated in vitro for seed assay.O. intermediumCP‐2 significantly (p < 0.05) increased shoot and root lengths, fresh and dry weights, and yield of mung bean plants both in pot trials and under field conditions. The nutrient uptake and microbial count also increased significantly with inoculation of the strain during the experiment. This study revealed the positive effect ofO. intermediumCP‐2 on mung bean crop.

In vitro and In vivo biofilm formation by Azotobacter isolates and its relevance to rhizosphere colonization

Twelve Azotobacter isolates exhibited varying levels of biofilm formation in a microtiter plate assay. The most efficient biofilm-forming isolate, as identified by partial 16S rRNA gene sequencing analysis, was Azotobacter vinelandii (AZCH6). Two isolates (AZCH5 and AZCH6), showing similar plant growth-promoting traits but differing in in vitro biofilm forming capacity, were further investigated for biofilm characterization on glass coverslips and on roots of wheat seedlings using confocal laser scanning microscopy and scanning electron microscopy. Rhizosphere colonization by AZCH6 in terms of CFUg−1 of soil, was significantly higher (P≤0.05) as compared to AZCH5. The data revealed that AZCH6 and AZCH5 exhibits similar pattern between biofilm formation and rhizosphere colonization. Therefore, strong biofilm-forming ability of Azotobacter on plant roots should be considered an important criterion for selection of effective isolates for enhanced root colonization.

The presence of Rhipicephalus muhsamae north of the Sahara

The occurrence of male and female Rhipicephalus muhsamae feeding on dogs and a horse in Upper Egypt is described. The morphological identification was verified using partial 16S rRNA gene sequences. By applying respective real-time PCRs, no evidence neither for Coxiella burnetii nor for spotted-fever rickettsia infections were found in the ticks.

Use of municipal solid wastes for chemical and microbiological recovery of soils contaminated with metal(loid)s

Iron-rich water treatment residues (Fe-WTRs) and municipal solid waste compost (MSWC) were added together at two different total rates (i.e. 0.5% Fe-WTRs+0.5% MSWC and 1% Fe-WTRs+1% MSWC) to a degraded sub-alkaline soil (pH 8.0) contaminated with Sb (∼110 mg kg−1 soil), Pb (∼1200 mg kg−1), Cd (∼23 mg kg−1), and Zn (∼5400 mg kg−1). A large number of chemical and biological endpoints were evaluated to assess the efficacy of the treatments after five months of incubation. Both treatments significantly reduced the labile fractions of the metal(loid)s in soil, especially Sb, while increasing the abundance of culturable heterotrophic bacteria, actinomycetes and fungi (i.e. up to 6.3-, 1.6- and 4.1-fold higher than control respectively). Soil enzyme activities, i.e. dehydrogenase, β-glucosidase and urease, were also significantly enhanced in the treated soils (i.e. up to ∼12-, 3- and 2-fold higher than control respectively). The amendment addition affected the structure of the soil microbial community as highlighted by the higher metabolic potential and catabolic versatility of treated soils (Biolog CLPP) and by the significantly higher α-diversity values based on high throughput partial 16S rRNA gene sequencing. Moreover, analysis of the dominant operational taxonomic units (OTUs) showed differences in the microbial communities of untreated and treated soils. Plant growth (Helichrysum italicum) in the treated soils was greatly stimulated while metal(loid)s uptake was significantly reduced. Overall, the results indicated that the applied treatment could be ideal for the chemical and (micro)biological recovery of sub-alkaline soils contaminated with Sb and co-occurring metals, and H. italicum appears to be a promising plant species for aided phytostabilisation of such soils.

Genetic characterization of Rhipicephalus sanguineus (sensu lato) ticks from dogs in Portugal

BackgroundThe taxonomic status of the brown dog tick Rhipicephalus sanguineus (sensu stricto) is a subject of on-going debate; there is a consensus that populations of this tick species should be referred to as R. sanguineus (sensu lato) until its taxonomic status is resolved. Recent genetic studies revealed the existence of more than one lineage of R. sanguineus (s.l.) in temperate countries. In this study, we assessed the genetic identity of ticks collected from rural dogs living in several areas located in all major geographical regions of Portugal.MethodsA total of 347 ticks were collected from rural dogs living in different regions of Portugal. These ticks were morphologically identified and partial mitochondrial 16S rRNA gene sequences (~300 bp) were obtained from representative specimens.ResultsThe ticks were morphologically identified as Ixodes ricinus (seven males and 27 females), Rhipicephalus bursa (one male), Rhipicephalus pusillus (one female) and R. sanguineus (s.l.) (two larvae, 101 nymphs, 108 males and 100 females). Partial mitochondrial 16S rRNA gene sequences were obtained from 58 R. sanguineus (s.l.) specimens, and all of them were genetically identified as belonging to the so-called temperate lineage of R. sanguineus (s.l.)ConclusionsThese results strongly suggest that the temperate species of R. sanguineus (s.l.) is the only representative of this tick group found on dogs in Portugal. It also adds weight to the hypothesis that Rhipicephalus turanicus is not present in this country, although further investigations are necessary to confirm this.

Soil formation and initial microbiological activity on a foreland of an Arctic glacier (SW Svalbard)

The initial microbiological activity on the forefield of the continuously retreating Werenskiold glacier (West Spitsbergen) along the chronosequence was investigated using both culture-dependent and culture-independent approaches. The prokaryotic cell parameters: total cell number (TCNg−1 d.w.), biomass (μgCg−1 d.w.), average cell volume (ACV, μm3), morphological structure (% cocci, rods, curved cells in TCN) and the contribution of viable cells with intact membrane (% in TCN) were established. The number of opportunistic bacteria (CFUg−1 d.w.) was counted using plate culture methodology. The PCR-DGGE of amplified 16S rRNA gene fragments and sequencing were also used to analyse the total (native soil samples) and culturable (plate wash samples) bacterial community structure. A total of 33 partial 16S rRNA gene sequences were obtained from the excised DGGE bands, with the majority of the sequences closely related to Actinobacteria, Bacteroidetes, and Proteobacteria (β and γ) groups. A high proportion of cultured and uncultured Arthrobacter in the studied glacier foreland soil confirms their role in the initiation of soil formation processes. Changes in the structure of both the native soil samples and wash plate samples communities along the chronosequence indicated their participation in young soil formation. Although initial microbial activity is closely associated with a higher proportion of non-cultivated bacteria, less numerous cultivated bacterial strains also feature significantly in the biodiversity. The main factors positively affecting TCN, BB, CFU, and the contribution of viable cells were water, nitrogen, carbon and organic matter content in the soil. In our study we have shown that a large share of the finest-particle-size fractions in the soil (dust, clay) negatively affects the BB and CFU. The native soil samples number of operational taxonomic units (OTUs) was mostly dependent on the pore water composition, and the C:N ratio in the soil, the wash plate samples number of OTUs was mostly connected with organic matter and soil water content.

Molecular characterization of Hepatozoon felis in Rhipicephalus sanguineus ticks infested on captive lions (Panthera leo).

Hepatozoon spp. are protozoan parasites that infect a wide range of domestic and wild animals. The infection occurs by ingestion of an infected tick. This study was carried out to detect and characterize Hepatozoon spp. in ticks collected from captive lions (Panthera leo) in Thailand based on the partial 18S rRNA gene sequence. A total of 30 ticks were collected and identified as Rhipicephalus sanguineus. The collected ticks were separated into 10 tick pools by sex and life stages. Of the 10 tick pools examined, only one (10%) was found to be infected with the Hepatozoon species. Sequencing and phylogenetic analysis showed a clustering of the partial 18S rRNA gene sequence like that of H. felis from the GenBank database. This is the first report of H. felis in R. sanguineus ticks collected from captive lions in Thailand. Our results indicated that R. sanguineus may be a possible vector of feline Hepatozoon in Thailand.

Isolation of a non-traditional sulfate reducing-bacteria Citrobacter freundii sp. and bioremoval of thallium and sulfate

A novel non-traditional sulfate reducing bacterium (Sr 10) was isolated from an up-flow anaerobic sludge bed for acid mine drainage treatment which contains thallium (Tl) and sulfate. Phylogenetic analysis of partial 16S rRNA gene sequence of isolate Sr 10 revealed that it was identified to Citrobacter freundii species. Sr 10 was visually rod-shaped and very motile with peri-flagellum according to transmission microscopy. The Fourier transform infrared spectroscopy analysis indicated that the stains had various functional groups for Tl and sulfate removal, including hydroxyl, carboxyl, amide and phosphate. Under anaerobic conditions, the optimized growth conditions for the stain were obtained at temperature of 35°C and initial pH value of 7.0. Sr 10 was able to remove both Tl(I) and SO42− simultaneously with the removal efficiency up to 99.60% and 89.80%, respectively. This strain might be used for Tl and sulfate removal in the process of bioremediation restoration.

MALDI‐TOF MS identification of microbiota associated with pest insect Diabrotica speciosa

Abstract Resistance development in pest insects has guided the advance of cleaner and more effective strategies for pest control. An interesting and promising strategy is the manipulation of insects via their gut microbiota. To evaluate the feasibility of this strategy, Diabrotica speciosa, a highly polyphagous pest insect from South America that causes substantial damage to several important crops, was reared under controlled conditions. Aerobic culturable bacteria were isolated from the gut of D. speciosa and identified using proteomic fingerprints obtained by matrix‐assisted laser desorption ionization coupled to time‐of‐flight mass spectrometry (MALDI‐TOF MS), as well as by genomic methods via partial sequencing of the 16S rRNA gene. Seventy‐three strains belonging to 17 genera and up to 29 different species were isolated. γ‐Proteobacteria of the orders Pseudomonadales and Enterobacteriales were the predominant. A core gut microbiome for the genus Diabrotica could be inferred when microbiotas from different species from the genus were compared. Molecular and spectrometric techniques indicated complete agreement of genera classification, although cluster analysis revealed distinct taxonomic grouping patterns. MALDI‐TOF MS provided reliable identification of culturable gut bacteria, demonstrating similar efficacy, with cheaper and faster results relative to partial 16S rRNA gene sequencing, and also showed an interesting and unexpected phyloproteomic correlation.

Comparison of growth on mannitol salt agar, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, VITEK® 2 with partial sequencing of 16S rRNA gene for identification of coagulase-negative staphylococci.

Mannitol salt agar (MSA) is often used in resources' limited laboratories for identification of S.aureus however, coagulase-negative staphylococci (CoNS) grows and ferments mannitol on MSA. 171 strains of CoNS which have been previously misidentified as S.aureus due to growth on MSA were collected from different locations in Nigeria and two methods for identification of CoNS were compared i.e. ViTEK 2 and MALDI-TOF MS with partial 16S rRNA gene sequencing as gold standard. Partial tuf gene sequencing was used for contradicting identification. All 171 strains (13 species) grew on MSA and ferments mannitol. All tested strains of S.epidermidis, S. haemolyticus, S. nepalensis, S. pasteuri, S.sciuri,, S. warneri, S. xylosus, S. capitis were correctly identified by MALDI-TOF while variable identification were observed in S.saprophyticus and S.cohnii (90%, 81%). There was low identification of S.arlettae (14%) while all strains of S.kloosii and S.gallinarum were misidentified. There is absence of S. gallinarum in the MALDI-TOF database at the period of this study. All tested strains of S.epidermidis, S. gallinarum, S. haemolyticus, S. sciuri,, S. warneri, S. xylosus and S.capitis were correctly identified by ViTEK while variable identification were observed in S.saprophyticus, S. arlettae, S. cohnii, S. kloosii, (84%, 86%, 75%, 60%) and misidentification of S.nepalensis, S. pasteuri. Partial sequencing of 16S rRNA gene was used as gold standard for most strains except S.capitis and S.xylosus where the two species were misidentified by partial sequencing of 16S rRNA contrary to MALDI-TOF and ViTEK identification. Tuf gene sequencing was used for correct identification. Characteristic growth on MSA for CoNS is also identical to S.aureus growth on the media and therefore, MSA could not differentiate between S.aureus and CoNS. The percentage accuracy of ViTEK was better than MALDI-TOF in identification of CoNS. Although partial sequencing of 16S rRNA gene was used as gold standard in this study, it could not correctly identify S.capitis and S.xylosus.

A new species of Hepatozoon Miller, 1908 (Apicomplexa: Adelerina) from the snake Philodryas nattereri Steindachner (Squamata: Dipsadidae) in northeastern Brazil

Based on both unique morphological characteristics of the gamont, distinct changes caused to the host erythrocyte and analysis of partial 18S rRNA gene sequences, a new parasite of the genus Hepatozoon Miller, 1908 is described from the snake Philodryas nattereri Steindachner (Squamata: Dipsadidae) in northeastern Brazil. The new species, Hepatozoon musa n. sp., is characterized by large and curved mature gamonts (18.9 ± 0.9 μm in length and 3.8 ± 0.3 μm in width) that considerably engorge infected host erythrocytes and displace the nucleus laterally, which become longer and thinner. Phylogenetic estimates indicate the new species is more closely related to the recently described Hepatozoon cuestensis O'Dwyer, Moço, Paduan, Spenassatto, Silva & Ribolla, 2013, from Brazilian rattlesnakes. These recent findings highlight the need for further studies of Hepatozoon to better determine the biodiversity of this common but poorly-studied parasite group.

Characterization and phylogenetic affiliation of Actinobacteria from tropical soils with potential uses for agro-industrial processes.

Secondary metabolites produced by Actinobacteria of tropical soils represent a largely understudied source of novel molecules with relevant application in medicine, pharmaceutical and food industries, agriculture, and environmental bioremediation. The present study aimed to characterize sixty-nine Actinobacteria isolated from compost and tropical soils using morphological, biochemical, and molecular methods. All the isolates showed high variation for morphological traits considering the color of pigments of the aerial and vegetative mycelium and spore chain morphology. The enzymatic activity of amylase, cellulase, and lipase was highly variable. The amylase activity was detected in 53 (76.81%) isolates. Eighteen isolates showed enzymatic index (EI) > 4.0, and the isolates ACJ 45 (Streptomyces curacoi) and ACSL 6 (S. hygroscopicus) showed the highest EI values (6.44 and 6.42, respectively). The cellulase activity varied significantly (P ≤ 0.05) among the isolates. Twenty-nine isolates (42.02%) showed high cellulase activity, and the isolates ACJ 48 (S. chiangmaiensis) and ACJ 53 (S. cyslabdanicus) showed the highest EI values (6.56 for both isolates). The lipase activity varied statistically (P ≤ 0.05) with fourteen isolates (20.29%) considered good lipase producers (EI > 2.0). The isolate ACSL 6 (S. hygroscopicus) showed the highest EI value of 2.60. Molecular analysis of partial 16S rRNA gene sequencing revealed the existence of 49 species, being 38 species with only one representative member and 11 species represented by one or more strains. All species belonged to three genera, namely Streptomyces (82.61%), Amycolatopsis (7.25%), and Kitasatospora (10.14%). The present results showed the high biotechnological potential of different Actinobacteria from tropical soils.

Microbial Community Composition of Two Environmentally Conserved Estuaries in the Midorikawa River and Shirakawa River

To provide a general overview of the microbial communities in environmentally conserved estuaries, the top 5 cm of sediment was sampled from the sandy estuary of the Shirakawa River and from the muddy estuary of the Midorikawa River. Higher amounts of organic matter were detected in the Midorikawa estuary sample than in the Shirakawa estuary sample. Measurement of redox potential revealed that the Shirakawa estuary was aerobic and the Midorikawa estuary was much less aerobic. Clone analysis was performed by targeting partial 16S rRNA gene sequences and using extracted DNA from the samples as a template. Various bacteria were detected, among which Gammaproteobacteria was dominant at both estuaries. Unclassified clones were detected in the Gammaproteobacteria group, mainly among samples from the Midorikawa estuary. Other detected bacterial groups were Alphaproteobacteria , Deltaproteobacteria , Chloroflexi , Actinobacteria , and Bacteroidetes . All the Deltaproteobacteria clones were anaerobic sulfate-reducing bacteria. Those aerobic and anaerobic bacteria coexisted in the top 5 cm of the estuary sediments indicating the surface layer have active sulfur and carbon cycle. Abundance of aerobic Gammaproteobacteria may be an indicator for conserved estuaries. Keywords: conserved environment, clone analysis, estuary, microbial community, 16S rRNA gene.

Cnemaspis flaviventralis, a new species of gecko (Squamata: Gekkonidae) from the Western Ghats of Maharashtra, India

Cnemaspis flaviventralis, a new species of day gecko, is described from the forests of Amboli, Sindhudurg District, Maharashtra State, northern Western Ghats, India. The new species was previously confused with the sympatric species Cnemaspis girii, C. indraneildasii, C. kolhapurensis and C. goaensis. It is distinguished from C. giri by having spine-like tubercles on flanks, granular dorsal scales intermixed with large, depressed, slightly keeled scales (vs. lack of spine-like tubercles on flanks, granular dorsal scales, intermixed with large smooth scales); from C. indraneildasii by having dorsal scales heterogeneous (vs. homogenous), lacking a series of enlarged median sub-caudal scales, and 28–29 (vs. 20) ventral scales across mid-body; from C. kolhapurensis by having heterogeneous (vs. homogenous) dorsal scalation, lacking spine-like tubercles on flanks and lacking pre-cloacal pores (vs. 24–28 pre-cloacal-femoral pores); and from C. goaensis by lacking pre-cloacal pores and lacking a series of enlarged median sub-caudal scales. We further provide partial mitochondrial 16S rRNA gene sequences for the new species and for the sympatric species C. girii, C. kolhapurensis and C. goaensis, and show that the new species is genetically distinct.

Variations in the bacterial community compositions at different sites in the tomb of Emperor Yang of the Sui Dynasty

To fully understand the bacterial processes in tomb environments, it is necessary to investigate the details of the bacterial communities present under such oligotrophic conditions. Here, high-throughput sequencing based on partial 16S rRNA gene sequences was used to fully evaluate the bacterial communities at different sites in the tomb of Emperor Yang of the Sui Dynasty. We also aimed to identify the soil factors that were significant related to bacterial diversity and community composition. The results showed the presence of a broad taxonomic diversity that included nine major phyla. Actinobacteria, Firmicutes and Proteobacteria dominated the bacterial profiles in all tomb soil samples. However, significant differences between deposited soils (DS) and covering soils (CSA, CSB and CSC) were revealed by chemistry-based principal component analysis (PCA), the number of OTUs, and the Chao 1 and Shannon indexes. At the family level, hierarchically clustered heatmap and LefSe analyses showed differences in the bacterial community compositions at different sampling sites. Notably, CSA contained significant populations of Nocardioidaceae, Pseudonocardiaceae and Streptomycetaceae, which are often reported to be associated with biodeterioration in cave environments. Further, the most abundant group (>10%) in all soil samples was Streptococcaceae, whose abundance decreased from 34.66% to 13.43% with increasing soil depth. The results of redundancy analysis (RDA) and the Monte Carlo permutation test indicated that soil pH and Cu and Mn levels were significantly related to the bacterial communities in this tomb. This research offers new insight into bacterial communities in cave environments and also provides important information for the protection of this historically important tomb.

Correlating methane production to microbiota in anaerobic digesters fed synthetic wastewater

A quantitative structure activity relationship (QSAR) between relative abundance values and digester methane production rate was developed. For this, 50 triplicate anaerobic digester sets (150 total digesters) were each seeded with different methanogenic biomass samples obtained from full-scale, engineered methanogenic systems. Although all digesters were operated identically for at least 5 solids retention times (SRTs), their quasi steady-state function varied significantly, with average daily methane production rates ranging from 0.09 ± 0.004 to 1 ± 0.05 L-CH4/LR-day (LR = Liter of reactor volume) (average ± standard deviation). Digester microbial community structure was analyzed using more than 4.1 million partial 16S rRNA gene sequences of Archaea and Bacteria. At the genus level, 1300 operational taxonomic units (OTUs) were observed across all digesters, whereas each digester contained 158 ± 27 OTUs. Digester function did not correlate with typical biomass descriptors such as volatile suspended solids (VSS) concentration, microbial richness, diversity or evenness indices. However, methane production rate did correlate notably with relative abundances of one Archaeal and nine Bacterial OTUs. These relative abundances were used as descriptors to develop a multiple linear regression (MLR) QSAR equation to predict methane production rates solely based on microbial community data. The model explained over 66% of the variance in the experimental data set based on 149 anaerobic digesters with a standard error of 0.12 L-CH4/LR-day. This study provides a framework to relate engineered process function and microbial community composition which can be further expanded to include different feed stocks and digester operating conditions in order to develop a more robust QSAR model.

Influence of the Po River runoff on the bacterioplankton community along trophic and salinity gradients in the Northern Adriatic Sea

AbstractThe Po River runoff strongly affects the oceanographic and ecological characteristics of the Northern Adriatic Sea. Catalysed reported deposition ‐ fluorescence in situ hybridization (CARD‐FISH) analysis was employed to assess how the composition of the coastal bacterioplankton community is influenced by the river runoff in two different seasons (spring and autumn). Samples were collected from the water column along a coastal–offshore transect in the Northern Adriatic Sea at different depths. Four clone libraries were then constructed from coastal (0 m) and offshore waters (−65 m). Higher abundances of bacteria were recorded in coastal waters as compared with the offshore samples. This result was mainly due to the trophic state of the water column, and it was related to salinity. Particularly, Actinobacteria and Gammaproteobacteria were affected by the riverine inputs, whereas Bacteroidetes and Alphaproteobacteria showed only minor responses. This was particularly clear in the autumn sample, in which a clear difference between the coastal and the offshore samples was found due to a strong influence of the less saline river water with high nutrient concentrations. Analysis of 205 partial length 16S rRNA gene sequences indicated a high diversity with the dominance of Alphaproteobacteria, Bacteroidetes and Gammaproteobacteria mainly affiliated to coastal and marine bacterioplankton clades. Actinobacteria were also detected and, together with Gammaproteobacteria, strongly follow the freshwater intrusion. Overall, our results indicate that the seasonal difference in the Po River discharge greatly affects the bacterioplankton community. In spring we observed a smooth transition from coastal to open‐sea conditions owing to the more superficial freshwater plume. In autumn the deeper freshwater intrusion in the coastal site and thermal stratification offshore resulted in a marked difference between the coastal and offshore microbial communities.

Diversity shifts of rumen bacteria induced by dietary forages in dairy cows and quantification of the changed bacteria using a new primer design strategy

The partial 16S rRNA gene sequences (100 to 500 bp) were widely used to reveal rumen bacterial composition influenced by diets, while quantification of the changed uncultured bacteria was inconvenient due to difficult designing of specific primers based on short sequences. This study evaluated the effect of forage resources on rumen bacterial diversity and developed new strategy for primer design based on short sequences to quantify the changed uncultured bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis and subsequent band sequencing were used to reveal the distinct rumen bacteria composition in cows fed with two forage sources (single corn stover vs. mixed forages including alfalfa hay and corn silage). The bacterial diversity in the rumen of dairy cows fed with corn stover was lower than that with mixed forages (P<0.05). The bacterium named R-UB affiliating to uncultured Succinivibrionaceae was identified, and it was abundant in the rumen of cows fed with mixed forages compared to corn stover. The full length 16S rRNA gene sequences with identity of >97% to the R-UB 16S rRNA gene sequence were obtained from GenBank and used to design specific primers to quantify uncultured bacterium R-UB. All sequences of amplicon from the new primers were of 100% identity to R-UB sequences indicating the high specificity of new primers. Quantitative PCR confirmed that abundance of R-UB in the rumen of cows fed with corn stover was lower than those fed with mixed forages (P<0.01). New strategy for designing primers based on partial 16S rRNA genes to quantify targeted uncultured bacteria was successfully developed. The rumen bacteria descending significantly in the cows fed corn stover compared to those fed mixed forages was identified as uncultured R-UB from Succinivibrionaceae.

Bacterial diversity and antibiotic resistances of abundant aerobic culturable bacteria in input and output samples of 15 German biogas plants.

Determination of the diversities and antibiotic resistances of the most abundant aerobic culturable bacteria and their survival in 15 German biogas plants (BGPs). Microbiological standard media (complex and selective media) were applied to enumerate mesophilic and oligotrophic bacteria (Enterobacteriaceae, Salmonella, staphylococci and enterococci) in input and output samples of BGPs. Concentrations of aerobic culturable bacteria in outputs were mostly one to two orders of magnitude lower than in input samples. In total, 852 isolates from input and 902 from output samples were identified either by partial 16S rRNA gene sequencing or Matrix-assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) analysis. Antibiotic resistance screening of target families against veterinary relevant antibiotics resulted in similar resistance pattern in input and output samples. Potentially pathogenic bacteria were detected in output samples of BGPs, but in decreased abundance. The selectivity of the most applied clinical standard media was insufficient. Only little is known about the release of antibiotic resistant and potentially pathogenic bacteria from BGPs. However, this study indicates the detection of several potentially pathogenic bacteria in output samples, which showed several antibiotic resistances.