Unknown Bacterial Strains Research Articles

Changpingibacter yushuensis gen. nov., sp. nov., isolated from fluvial sediment in Qinghai Tibet Plateau of China.

Two facultatively anaerobic, short rod-shaped, non-motile, Gram-stain-positive, unknown bacterial strains (JY-X040T and JY-X174) were isolated from fluvial sediments of Tongtian River in Yushu Tibetan Autonomous Prefecture, Qinghai province, China. Cells formed translucent, gray, round and convex colonies, with a diameter of less than 0.5 mm after 5 days of incubation at 30°C on brain heart infusion-5% sheep blood agar. The 16S rRNA gene sequence similarity between strain JY-X040T and Fudania jinshanensis 313T is 93.87%. In the four phylogenetic trees constructed based on the 16S rRNA gene and 423 core genes, the two isolates form an independent branch, phylogenetically closest to F. jinshanensis 313T, but could not be classified as a member of the genus Fudania or any other genus of the family Arcanobacteriaceae. The DNA G + C content of strain JY-X040T was 57.8%. Calculation results of average nucleotide identity, digital DNA-DNA hybridization value and amino acid identity between strain JY-X040T and F. jinshanensis 313T are 69.9%, 22.9%, and 64.1%. The major cellular fatty acids were C16:0 (23%) and C18:1ω9c (22%). The cell-wall peptidoglycan type was A5α (L-Lys-L-Ala-L-Lys-D-Glu). The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and four unidentified components. The whole-cell sugars contained rhamnose and ribose. MK-10(H4) was the sole respiratory quinone. The minimum inhibitory concentration of streptomycin was 32 μg/ml. All physiological, biochemical, chemotaxonomic and genomic characteristics support that strains JY-X040T and JY-X174 represent members of a novel species in a new genus, Changpingibacter yushuensis gen. nov., sp. nov. The type strain is JY-X040T (GDMCC 1.1996T = KCTC 49514T).

Identification of lactic acid bacteria and rhizobacteria by ultraviolet-visible-near infrared spectroscopy and multivariate classification

The biological processes of interest to agro-industry involve numerous bacterial species. Lactic acid bacteria produce metabolites capable of fermenting food products and modifying their organoleptic properties, and plant-growth-promoting rhizobacteria can act as biofertilizers, biostimulants, or biocontrol agents in agriculture. The protocol of conventional techniques for bacterial identification, currently based on genotyping and phenotyping, require specific sample preparation and destruction. The work presented herein details a method for rapid identification of lactic acid bacteria and rhizobacteria at the genus and species level. To develop the method, bacteria were inoculated on an agar medium and analyzed by near infrared (NIR) and ultraviolet-visible-NIR (UV-Vis-NIR) spectroscopy. Artificial neural network models applied to the UV-Vis-NIR spectra correctly identified the genus (species) of 70% (63%) of the lactic acid bacteria and 67% of the rhizobacteria on an independent prediction set of unknown bacterial strains. These results demonstrate the potential of UV-Vis-NIR spectroscopy to identify bacteria directly on agar plates.

Complete genome assembly data of paenibacillus sp. RUD330, a hypothetical symbiont of euglena gracilis.

An unknown bacterial strain was detected in the cytostome of Euglena gracilis and on the cell surface of Euglena gracilis using transmission electron microscopy. To identify the unknown bacterium and its function, we performed isolation experiments. Here we present the genome sequence of the isolate that was determined to be Paenibacillus sp. The genome of the bacterium was sequenced four times using Illumina technology with pair-end reads, Illumina technology with mate pair reads (inserts 3–4 and 6–8 Kb), and Nanopore technology with long reads (tens of thousands of nucleotides). Assemblies based on Illumina reads including mate-pair reads could not resolve issues caused by long tandem copies of rRNA, other tandem repeats, and extremely GC-rich regions (90–100%). Only long Nanopore reads resolved those gaps and made it possible to complete the entire genome; moreover, we found one plasmid. The length of the genome is 5.56 Mbp, and the average GC content is 59%. The genome of Paenibacillus sp. RUD330 included 8 copies of all the rRNA genes (23S; 16S; 5S), the length of the plasmid was 8.3 Kb.We hope that our genome assembly and the methods used can help other investigators in the assembly of complex genomes. Our reliable assembly could be a good basis for further physiological and genetic engineering studies of similar strains.

Saezia sanguinis gen. nov., sp. nov., a Betaproteobacteria member of order Burkholderiales, isolated from human blood.

The taxonomic position of an unknown bacterial strain designated CNM695-12, isolated from the blood of an immunocompromised subject, was investigated via phenotypic, chemotaxonomic, genotypic and genomic analyses. Bacterial cells were determined to be Gram-stain-negative bacilli, aerobic, non-motile and non-spore-forming. The strain showed catalase activity but no oxidase activity. Optimal growth occurred at 37 °C, pH 7 and with 0-1 % NaCl. C16 : 0, summed feature 8 (comprising C18 : 1ω7c /C18:1 ω6c), and C18 : 1ω9c were the most abundant fatty acids, and ubiquinone 8 was the major respiratory quinone. The polar lipids present included phosphatidylglycerol, phosphatidylethanolamine and other aminophospholipids. The 16S rRNA gene sequence showed approximately 93.5 % similarity to those of different species with validly published names within the order Burkholderiales (e.g. Leptothrix mobilis Feox-1T, Aquabacterium commune B8T , Aquabacterium citratiphilum B4T and Schlegelella thermodepolymerans K14T). Phylogenetic analyses based on 16S rRNA gene sequences and concatenated alignments including the sequences for 107 essential proteins, revealed the strain to form a novel lineage close to members of the family Comamonadaceae. The highest average nucleotide identity and average amino acid identity values were obtained with Schlegelella thermodepolymerans K14T (69.6 and 55.7 % respectively). The genome, with a size of 3.35 Mb, had a DNA G+C content of 52.4 mol% and encoded 3056 predicted genes, 3 rRNA, 1 transfer-messengerRNA and 51 tRNA. Strain CNM695-12 thus represents a novel species belonging to a novel genus within the order Burkholderiales, for which the name Saezia sanguinis gen. nov., sp. nov. is proposed. The type strain is CNM695-12T (=DSM 104959T=CECT 9208T).

Identification of 3 unknown bacterial strains, characterization of copper oxidase genes

Identification of 3 unknown bacterial strains, characterization of copper oxidase genes

Morphological characterization of several strains of the rice-pathogenic bacterium Burkholderia glumae in North Sumatra

Burkholderia glumae is a quarantine seed-borne bacterial pathogen causing panicle blight disease on rice. This pathogen has been detected in some locations in Java, and recently, farmers in North Sumatra have reported rice yield loss with symptoms similar with those on rice infeced by the rice-pathogenic bacterium B. glumae. This research was aimed to isolate several bacterial strains from several rice varieties in various locations in North Sumatra and characterize the morphology of the strains to detect and identify the unknown bacterial strains presumably B. glumae. Several rice seed varieties were collected from Medan and Deli Serdang Districts. The seed samples were extracted, isolated and purified, then grown in semi-selective media PPGA. The morphological characteristics of the bacterial strains were determined including Gram staining, bacterial colony’s and bacterial cell’s morphology. The results showed that of eleven strains isolated, two strains were Gram negative and nine strains were Gram positive. On the basis of colony morphology, all strains had circular form, flat elevation and cream colour while the colony margin varied, i.e. entire and undulate. Most strains had bacillus/rod shape (8 strains) and only 3 strains were coccus.

Lactobacillus colini sp. nov., isolated from Northern Bobwhite (Colinus virginianus).

Biochemical and molecular studies were performed on five unknown bacterial strains isolated from the intestinal contents of Northern Bobwhites (Colinus virginianus) collected from western Texas, USA. The strains were Gram-stain-positive, catalase-negative, non-spore-forming rods arranged in single cells, pairs or short chains. Colonies on Columbia blood agar are circular, flat, entire, approximately 0.5-1.5 mm in diameter and surrounded with a zone of alpha-haemolysis at after incubation for 48 h at 37 °C. Colonies on MRS agar are umbonate with irregular edge, opaque and approximately 1-1.5 mm in diameter after incubation for 48 h. The 16S rRNA gene sequences of the isolates were identical and the highest sequence similarity (97 %) was found to the type strains of Lactobacillus gasseri, L. johnsonii and L. taiwanensis. The strains were distinguishable from related species of the genus Lactobacilluson the basis of carbohydrate fermentation, enzymatic production and fatty acid profiles. The peptidoglycan type is l-Lys-d-Asp (A4α). The DNA G+C content is 35.6 mol%. Major cellular fatty acids are C14 : 0, C16 : 0 and C18 : 1 ω9c. Based on phenotypic, phylogenetic and chemotaxonomic information, the strains represent a novel species of the genus Lactobacillus for which the name Lactobacillus colini sp. nov. is proposed. The type strain is 111144 L1T (=DSM 101872T=KCTC 21086T).

Distribution and isolation of strains belonging to the order Solirubrobacterales.

The number of prokaryotes on earth is estimated to be in the order of 1030 cells,1 and it is well known that most microorganisms in the environment are as-yet-uncultured.2 Many approaches have been tried to isolate unknown bacterial strains. Davis et al.3 reported that medium choice and incubation time are significant for isolation of rare soil bacteria. Patulibacter minatonensis, belonging to the order Solirubrobacterales, was isolated using medium supplemented with superoxide dismutase and proposed as a novel genus in 2006.4 At that time, the order Solirubrobacterales5 consisted of only three families, three genera and three species; Patulibacter minatonensis, Conexibacter woesei6 and Solirubrobacter pauli,7 and presently there are still 11 species including the one species that is isolated in this study. We believe that as-yet-uncultured bacteria may exist within this taxon, and it is possible that additional strains could be used as an untapped resource. As the genera Patulibacter, Conexibacter and Solirubrobacter grow slowly, we tried to investigate their distribution and isolate them by cultivation over a long period of time in order to isolate strains belonging to the order Solirubrobacterales.

Phylogenetic Investigations on the Endosymbiotic Bacteria of Axinella donnani

The present study focuses on the isolation of endosymbiotic bacteria from eight different types of sponges. The sponges were collected from the southern peninsular coast of India and identified as Sigmadocia carnosa, Ircinia fasciculate, Callyspongia diffusa, Zygomycale angulosa, Clathria vulpine, Clathria gorgonids, Phloeodictyon species and Axinella donnani. For the isolation of endosymbionts, the collected sponge species were cultured in three different media such as Nutrient agar media, Zobell marine agar and Zobell marine agar + sponge extracts. The sponge extracts supplemented media produced high bacterial growth than the other media. The sponge A. donnani, recorded the highest bacterial counts. Of which, 13 endosymbiotic bacterial strains (ESB) of A. donnani were screened against common pathogenic bacteria. The strains ESB-3 and ESB-7 were identified to be potential strains exhibiting significant antibacterial property. The antibacterial activity was evaluated through testing against various shrimp pathogens (Vibrio esturiances, Vibrio alginolyticans, Vibrio harvae, Aeromonas hydrophila and Pseudomonas aerogenosa) as well as human pathogens (Streptococcus hemolyticus, Vibrio fisheri, Escherichia coli, Morgenella morgenii, and Bacillus cereus). The strains exhibited significant activity against all the shrimp and human pathogens. The unknown bacterial strain (ESB3 and ESB7) were identified using 16S rRNA gene technique to be Bacillus subtilis, Further, sequencing methodologies verified that the FASTA sequence of ESB3 contains 994 residues and that of ESB7 contains 1023 residues. The result so these findings are presented and elaborately discussed in the following paper.

Identification and characterization of Raoutella ornithilolytica and determination of its herbicide 2,2-dichloropropionate (2,2-DCP) degradation potential

Aims: The goal of the study is to isolate new bacteria species which are capable to utilizing 2,2-dichlropropionic acid (22-DCP) as a sole carbon source from the wastewater sample that was taken from Tioman Island off the coast of Malaysia. Methodology and results: Genomic DNA from unknown bacterial strain (MR1) was extracted and PCR amplification was carried out using universal primers, Fd1 (5’-AGA GTT TGA TCC TGGCTC AG-3’) and rP1 (5’-ACG GTC ATA CCT TGT TAC GAC TT-3’) prior to sequencing. The BLASTn and phylogenetic analysis of the 16S rRNA of the MR1 gene found it shares a 95% homology to an aerobic bacillus Raoutella ornithilolytica. The bacteria which is usually found in an aquatic and hospital environment was found to possess a distinctive feature of being able to utilize 2,2-DCP as sole carbon source. The bacteria has cell doubling time of 23.11 h and maximum release of chloride ion at 0.257 mmol/L in 20 mM 2,2-DCP. Based on morphological and partial biochemical characteristics, the strain was found to be non-motile, Gram negative bacteria with red colonies that gave a positive catalase reaction. Conclusion, significance and impact of study: A better understanding of newly isolated microorganisms from the environment which can potentially be used as bioremediation tools in environmental management. This is the first reported case of Raoutella sp. that has the unique ability to degrade halogenated compound. Hence, the dehalogenation properties and capabilities of the bacteria deserve to be assessed further.

Automatic identification of novel bacteria using Raman spectroscopy and Gaussian processes

Raman spectroscopy is successfully used for the reliable classification of complex biological samples. Much effort concentrates on the accurate prediction of known categories for highly relevant tasks in a wide area of applications such as cancer detection and bacteria recognition. However, the resulting recognition systems cannot always be directly used in practice since unseen samples might not belong to classes present in the training set. Our work aims to tackle this problem of novelty detection using a recently proposed approach based on Gaussian processes. By learning novelty scores for a large bacteria Raman dataset comprising 50 different strains, we analyze the behavior of this method on an independent dataset which includes known as well as unknown categories. Our experiment reveals that non-parametric methods such as Gaussian processes can be successfully applied to the task of finding unknown bacterial strains, leading to encouraging results motivating their further utilization in this area.

Assessment of the bio-preparedness and of the training of the French hospital laboratories in the event of biological threat

A national laboratory network 'Biotox-Piratox' was created in 2003 in France with the purpose of detecting, confirming and reporting potential biological and chemical threat agents. This network is divided into three levels: Level 1 is dedicated to the evaluation of risks (biological, chemical, radiological), to sampling and packing. Level 2 consists of university and military hospitals, who deal with biological specimens, and of environmental and veterinary laboratories, who deal with environmental and animal samples. Level 3 comprises national reference laboratories and the Jean Mérieux biosafety level (BSL)-4 laboratory in Lyon. This report presents the results of four bio-preparedness exercises to check critical points in the processing of samples. These exercises took place in 2007, 2009, 2010 and 2011. Each of them consisted of two parts. The first part was the identification of an unknown bacterial strain and its susceptibility to antibiotics used as a default in case of a bioterrorist event. The second part was the detection of Class III microorganisms, mainly by molecular techniques. The main lesson learnt in these exercises was that the key to successful detection of biological agents in case of a biological threat was standardisation and validation of the methods implemented by all the laboratories belonging to the network.

What bacteria are living in my food?: An open‐ended practical series involving identification of unknown foodborne bacteria using molecular techniques

This open-ended practical series titled "Molecular Identification of Unknown Food Bacteria" which extended over a 6-week period was designed with the aims of giving students an opportunity to gain an understanding of naturally occurring food bacteria and skills in contemporary molecular methods using real food samples. The students first isolated two unknown bacterial strains from two food sources from which they extracted DNA and performed PCR targeting the 16S rRNA gene. Gel electrophoresis was used to analyze both genomic DNA preparations and PCR products. Following purification of PCR products, DNA sequencing was carried out and sequence trace quality was analyzed. The students successfully identified the two unknown bacteria using the BLAST search engine and a wide variety of different organisms were found. Assessment of their understanding of the procedure and ability to explain their findings using supporting primary research literature was via an individually prepared written report. Feedback from students over 2 years (n = 52) in a questionnaire revealed that the practical series was an engaging learning experience and lead to perceived improvements in knowledge of molecular techniques and bioinformatics and also about commonly occurring bacteria in foods.

Application of fluorescence spectra based on radial basis function neural network in identification of bacteria

The results of research on fluorescence spectra of three kinds of probiotic bacteria (lactobacillus acidophilus, streptococcus mutans and lactobacillus bulgaricus) show that the bacteria emit fluorescence when irradiated by ultraviolet light. The spectra are in the range of 300—650 nm with the excitation wavelength of 290 nm. Wavelet transform is used to compress the 150 groups of spectra data, and the number of data points in each group is reduced from 1341 to 168, which not only keeps the character of original spectra unchanged, but also improves the processing speed of neural network. Radial basis function neural network is applied for processing the compressed data. 40 groups of data of each strain are used to be trained, and the other 30 groups of data of which the kinds of bacteria are not given are used for prediction. The result shows that radial basis function neural network can identify the unknown bacterial strain accurately.

New Multiplatform Computer Program for Numerical Identification of Microorganisms

The classification of bacteria by using genomic methods or expensive biochemical-based commercial kits is sometimes beyond the reach of many laboratories that need to perform numerous classifications of unknown bacterial strains in a fast, cheap, and reliable way. A new computer program, Identax, for the computer-assisted identification of microorganisms by using only results obtained from conventional biochemical tests is presented. Identax improves current microbial identification software and provides a multiplatform and user-friendly program. It can be executed from any operating system and can be downloaded without any cost from the Identax website (www.identax.org).

Isolation of Corynebacterium falsenii and description of Corynebacterium aquilae sp. nov., from eagles.

Biochemical, molecular chemical and molecular genetic studies were performed on seven unidentified gram-positive, rod-shaped organisms recovered from eagles. The strains were provisionally identified as Corynebacterium jeikeium with the commercial API Coryne system, but they were able to grow under anaerobic conditions and were non-lipophilic. Comparative 16S rRNA gene sequencing studies demonstrated that the isolates belonged phylogenetically to the genus Corynebacterium. Three strains were identified genotypically as Corynebacterium falsenii; the remaining four strains corresponded to a hitherto unknown lineage within the genus Corynebacterium, associated with a small subcluster of species that included Corynebacterium diphtheriae and its close relatives. The unknown bacterial strains were readily distinguished from these and other species of the genus by biochemical tests. Based on both phenotypic and phylogenetic evidence, it is proposed that the unknown bacterial strains from eagles should be classified as Corynebacterium aquilae sp. nov. (type strain is S-613T = CECT 5993T = CCUG 46511T).

ANALISA URUTAN GEN 16S rRNA DARI BAKTERI ORAL YANG TIDAK DIKENAL

The purpose of this study was to identify five unknown bacterial strains by using 16S rRNA gene sequencing. These strains isolated from endodontic lesions and periodontal pocket are culture-difficult and inert in most biochemical tests, and could not be classified to any established bacterial species by conventional bacteriological method. In the present study, genomic DNA was extracted from the cultured bacterial cells with InstaGene (BIO-RAD), and the 16S rRNA gene was amplified by PCR with universal primers (27F and 1492R) and Premix Taq (Ex Taq version, Takara), then was sequenced by using a Thermo Sequence Fluorescent Labelled Primer Cycle Sequencing Kit (Amersham) and an ALFexpress DNA sequencer (Pharmacin LKB). The segmented nucleotide sequences of 16S rDNA were integrated by using SEQMAN in LASERGENE computer program (DNASTAR). The 16S rDNA sequences of the unknown bacterial strain were applied to GenBank by using BLAST program to search the suspected bacterial species . The MEGALIGN search program showed that the sequence similarities were 89.5% - 91.3% to a type strain of Dialister pneumosintes among the established bacterial species. Based on the phylogenetic data, it is considered that the five unknown strains have to be presented a new bacterial species as Dialister-like bacterium.

Rhizobacterial growth and yield promotion of cucumber plants inoculated with Pythium aphanidermatum

The potential of rhizobacterial strains to reduce the adverse effects of Pythium aphanidermatum on plant growth and yield was examined under in vitro and greenhouse cropping conditions. Rhizobacterial cultures of 10 strains of Pseudomonas putida, 24 strains of Bacillus subtilis, 4 strains of Enterobacter aerogenes, 1 strain of Bacillus cereus, and 1 unknown bacterial strain were evaluated for antagonism against P. aphanidermatum in laboratory and greenhouse conditions. In a dual culture test on potato dextrose agar, only AG-1 of B. subtilis and strains 2xyta75 and B8 of E. aerogenes were antagonistic to P. aphanidermatum. Under sterile in vitro conditions in pouches, 40 bacterial strains were tested for growth promotion of cucumber (Cucumis sativus) seedlings in the absence of P. aphanidermatum. All bacteria except strain PPG-7-1 of P. putida, strains AGS-3 and AGS-1 of B. subtilis, and strain AG-0 of an unidentified bacterium increased root length of cucumber compared with the P. aphanidermatum inoculated controls. Of the 16 bacterial strains evaluated for antagonistic activity against P. aphanidermatum under greenhouse conditions, only one strain (BACT-0) of B. subtilis increased shoot growth by 9% and weight of cucumber plants by 29% compared with the P. aphanidermatum inoculated controls. The same strain increased the fruit yield by 14% and fruit number by 50% of cucumber plants compared with the P. aphanidermatum inoculated controls. This study has demonstrated that the strain BACT-0 of B. subtilis has the potential to increase growth, fruit yield, and fruit number of cucumber plants inoculated with P. aphanidermatum in soilless culture under near commercial greenhouse conditions.