A Novel Culturing Chip (cChip) Can Facilitate Culturing of Unculturable Bacteria From Aquatic Environment

2020 ◽  
Author(s):  
Adil Farooq Lodhi ◽  
Ying Zhang ◽  
Maria Adil ◽  
Yulin Deng
Keyword(s):  
Aquatic Environment ◽  
Microbial Growth ◽  
Dilution Method ◽  
Rrna Gene ◽  
Uncultured Bacteria ◽  
Field Samples ◽  
Unculturable Bacteria ◽  
Petri Plate ◽  
Gene Similarity ◽  
Metagenomics Analysis

Abstract Background: Culturing the unculturable microorganisms is an important aspect of microbiology. Once cultured the unculturable microorganisms can be a source of useful antibiotics, enzymes etc. Several studies have been conducted on culturing the unculturable microorganisms from soil. But little knowledge exists about culturing such bacteria from aquatic environment. Therefore, in this study we designed a novel culturing chip (cChip) to facilitate the growth of unculturable aquatic bacterial community. cChip was optimized for microbial growth using known bacteria in the lab, later, microbes from a freshwater lake were concentrated (instead of using the traditional dilution method) and inoculated in cChip before incubating the chip in simulated lake environment. Then further sub-culturing was done on laboratory media. The field samples were also analyzed using traditional culturing and metagenomics for a comparison. Results: Metagenomics analysis showed that 832 microbial species were present in the samples belonging to five different phyla, that is, Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes and Verrucomicrobia. However, traditional culturing yielded only 36 isolates that belonged to four different phyla, that is, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Culturing through cChip yielded 154 isolates belonging to five different phyla, that is, Proteobacteria, Actinobacteria, Bacteroidetes, Verrucomicrobia and Firmicutes. Out of these 154 cChip isolates, 45 were previously uncultured bacteria having a 16S rRNA gene similarity from 91.35 % to 98.7 % to their closest relatives according to NCBI GenBank. Conclusion: This study shows that culturing microorganisms in the cChip from aquatic environment using concentration process before performing the traditional petri plate culturing can result in the successful growth of unculturable bacteria. To the best of our knowledge this is the first study conducted for successful exploration of unculturable aquatic microbial community. This study can have a significant impact on our understanding of the techniques that can be applied for exploring the unexplored microbiome from diverse environments. We also hypothesize that certain modifications in the manufacturing material, keeping the design and techniques of this study intact can result in the application of this technique from gut microbiome to extremophiles in the environment.

scholarly journals Limnohabitans curvus gen. nov., sp. nov., a planktonic bacterium isolated from a freshwater lake

2010 ◽  
Vol 60 (6) ◽  
pp. 1358-1365 ◽  
Author(s):  
Martin W. Hahn ◽  
Vojtěch Kasalický ◽  
Jan Jezbera ◽  
Ulrike Brandt ◽  
Jitka Jezberová ◽  
...  
Keyword(s):  
Fatty Acids ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Uncultured Bacteria ◽  
The Family ◽  
Gene Similarity ◽  
Type Strains

A chemo-organotrophic, aerobic, facultatively anaerobic, non-motile strain, MWH-C5T, isolated from the water column of the oligomesotrophic Lake Mondsee (Austria), was characterized phenotypically, phylogenetically and chemotaxonomically. The predominant fatty acids of the strain were C16 : 1 ω7c/ω6c, C16 : 0, C12 : 1 and C8 : 0-3OH, the major quinone was ubiquinone Q-8 and the G+C content of the DNA of the strain was 55.5 mol%. 16S rRNA gene similarity to the closest related type strains was 96.6 % (Curvibacter delicatus LMG 4328T) and 95.7 % (Rhodoferax fermentans FR3T). Phylogenetic analysis of 16S rRNA gene sequences revealed the affiliation of the strain with the family Comamonadaceae (Betaproteobacteria); however, the phylogenetic position of the strain did not support an affiliation to any previously described genus within this family. A family-wide comparison of traits revealed that the strain possesses a unique combination of DNA G+C content, major fatty acids and major 3-hydroxy fatty acid. Furthermore, the strain differs in several traits from the closest related genera. Based on the phylogeny of the strain and differences from closely related genera, we propose to establish the new genus and species Limnohabitans curvus gen. nov., sp. nov. to accommodate this strain. The type strain of Limnohabitans curvus is MWH-C5T (=DSM 21645T =CCUG 56720T). The type strain is closely related to a large number of uncultured bacteria detected by cultivation-independent methods in various freshwater systems.

scholarly journals Evaluation of Commercial Disinfectants against Staphylococcus lentus and Micrococcus spp. of Poultry Origin

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Otun Saha ◽  
Nadira Naznin Rakhi ◽  
Arif Istiaq ◽  
Israt Islam ◽  
Munawar Sultana ◽  
...  
Keyword(s):  
Micrococcus Luteus ◽  
Bacterial Species ◽  
16S Rrna Gene Sequencing ◽  
Average Diameter ◽  
Diffusion Method ◽  
Dilution Method ◽  
Rrna Gene ◽  
Poultry Farms ◽  
Rrna Gene Sequencing ◽  
Selection Of

Introduction. Effective sanitation strategies for poultry farms require an appropriate selection of the disinfectant based on the contaminants present and their sensitivity to the disinfectants. Aim. The current study investigated the prevalence of streptococci/micrococci in poultry farms of Bangladesh and the efficacy of commercial disinfectants (Savlon, Lysol, Quatovet, Virkon S, and Virocid) along with alcohol against these pathogens to adopt appropriate strategies. Materials and Methods. Conventional approaches and the 16S rRNA gene sequencing were performed to confirm the isolates at the species level along with microtiter biofilm assay to determine their biofilm-forming ability. Efficacy of the disinfectants was tested against those isolates using agar well diffusion and minimum inhibitory concentration (MIC) test by broth dilution method using different dilutions of the disinfectants. Results. Staphylococcus lentus (n = 32), Micrococcus luteus (n = 7), and Micrococcus aloeverae (n = 4) were confirmed among 102 presumptively screened streptococci/micrococci isolates from 43 samples. No single disinfectant showed equally high efficacy against all three bacterial species in agar well diffusion test, although Virocid showed the lowest MIC against all three of them. Lysol was least effective among the commercial disinfectants by both MIC and diffusion method, although each commercial disinfectant was more effective than alcohol. Considering both the average diameter of the inhibition zones and the MIC values, efficacy can be interpreted as Virocid > Quatovet > Savlon > Virkon S > Lysol. Although the efficacy decreased with decreasing concentration, the disinfectants retained a satisfactory level of efficacy at 50% concentration. Among test pathogens, M. aloeverae was the most sensitive to the disinfectants and the weakest biofilm producers, whereas 4/14 S. lentus and 1/5 M. luteus were strong biofilm producers, which may cause more reduction in the efficacy in environmental conditions. Conclusion. As no ideal disinfectant was found in the study, the efficacy of the disinfectants should be routinely evaluated and validated to ensure the sanitation standards in the poultry sector.

scholarly journals Actinocorallia cavernae sp. nov., isolated from a natural cave in Jeju, Korea

2006 ◽  
Vol 56 (5) ◽  
pp. 1085-1088 ◽  
Author(s):  
Soon Dong Lee
Keyword(s):  
Gene Sequence ◽  
Novel Species ◽  
Dilution Method ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Phenotypic Properties ◽  
Polar Lipid Profile ◽  
Phylogenetic Cluster ◽  
Gene Sequence Analysis ◽  
Actinomycete Strain

A novel actinomycete, strain N3-7T, was isolated from a natural cave in Jeju, Republic of Korea, using a dilution method and was subjected to characterization using polyphasic taxonomy. A 16S rRNA gene sequence analysis revealed that the organism belonged to the phylogenetic cluster of the genus Actinocorallia and was most closely related to Actinocorallia glomerata and Actinocorallia longicatena (97.6 and 97.5 % similarity, respectively). The main chemotaxonomic properties of strain N3-7T, such as the principal amino acid of the peptidoglycan, the predominant menaquinone and the polar lipid profile, supported classification in the genus Actinocorallia. The organism was readily differentiated from Actinocorallia species with validly published names on the basis of a broad range of phenotypic properties. Thus the isolate represents a novel species of the genus Actinocorallia, for which the name Actinocorallia cavernae sp. nov. is proposed. The type strain is strain N3-7T (=JCM 13278T=NRRL B-24429T).

scholarly journals Isolation, Phylogenetic and Gephyromycin Metabolites Characterization of New Exopolysaccharides-Bearing Antarctic Actinobacterium from Feces of Emperor Penguin

Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 458
Author(s):  
Hui-Min Gao ◽  
Peng-Fei Xie ◽  
Xiao-Ling Zhang ◽  
Qiao Yang
Keyword(s):  
Genome Mining ◽  
Pairwise Comparison ◽  
Gene Clusters ◽  
Amino Acid Identity ◽  
Core Gene ◽  
Emperor Penguin ◽  
Rrna Gene ◽  
Active Metabolites ◽  
The Antarctic ◽  
Gene Similarity

A new versatile actinobacterium designated as strain NJES-13 was isolated from the feces of the Antarctic emperor penguin. This new isolate was found to produce two active gephyromycin analogues and bioflocculanting exopolysaccharides (EPS) metabolites. Phylogenetic analysis based on pairwise comparison of 16S rRNA gene sequences showed that strain NJES-13 was closely related to Mobilicoccus pelagius Aji5-31T with a gene similarity of 95.9%, which was lower than the threshold value (98.65%) for novel species delineation. Additional phylogenomic calculations of the average nucleotide identity (ANI, 75.9–79.1%), average amino acid identity (AAI, 52.4–66.9%) and digital DNA–DNA hybridization (dDDH, 18.6–21.9%), along with the constructed phylogenomic tree based on the up-to-date bacterial core gene (UBCG) set from the bacterial genomes, unequivocally separated strain NJES-13 from its close relatives within the family Dermatophilaceae. Hence, it clearly indicated that strain NJES-13 represented a putative new actinobacterial species isolated from the gut microbiota of mammals inhabiting the Antarctic. The obtained complete genome of strain NJES-13 consisted of a circular 3.45 Mb chromosome with a DNA G+C content of 67.0 mol%. Furthering genome mining of strain NJES-13 showed the presence of five biosynthetic gene clusters (BGCs) including one type III PKS responsible for the biosynthesis of the core of gephyromycins, and a series of genes encoding for bacterial EPS biosynthesis. Thus, based on the combined phylogenetic and active metabolites characterization presented in this study, we confidently conclude that strain NJES-13 is a novel, fresh actinobacterial candidate to produce active gephyromycins and microbial bioflocculanting EPS, with potential pharmaceutical, environmental and biotechnological implications.

scholarly journals Nocardia jejuensis sp. nov., a novel actinomycete isolated from a natural cave on Jeju Island, Republic of Korea

2006 ◽  
Vol 56 (3) ◽  
pp. 559-562 ◽  
Author(s):  
Soon Dong Lee
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Jeju Island ◽  
Republic Of Korea ◽  
16S Rrna Gene Sequence ◽  
Phylogenetic Position ◽  
Dilution Method ◽  
Rrna Gene ◽  
Rrna Gene Sequence

A novel actinomycete, strain N3-2T, was isolated from a natural cave on Jeju Island, Republic of Korea, using a dilution method and was subjected to polyphasic taxonomy. The almost complete 16S rRNA gene sequence was determined by direct sequencing of the purified PCR product and was compared with those of representatives of the genus Nocardia. It was revealed from the phylogenetic analysis that the organism forms a distinct clade between the Nocardia salmonicida cluster and the Nocardia alba branch within the evolutionary radius occupied by the genus Nocardia of the family Nocardiaceae. The organism showed 16S rRNA gene sequence similarity of 97·4 % with its nearest phylogenetic neighbours, namely N. salmonicida and N. alba. The chemotaxonomic properties, such as the principal amino acid of peptidoglycan, predominant menaquinone and polar lipids, supported the classification in the genus Nocardia. The organism was readily differentiated from Nocardia species with validly published names by a broad set of phenotypic properties and its unique phylogenetic position; the name Nocardia jejuensis sp. nov. is proposed, with N3-2T (=JCM 13281T=NRRL B-24430T) as the type strain.

scholarly journals PCR-RFLP Detection and Genogroup Identification of Piscirickettsia salmonis in Field Samples

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 358
Author(s):  
Pamela Aravena ◽  
Rodrigo Pulgar ◽  
Javiera Ortiz-Severín ◽  
Felipe Maza ◽  
Alexis Gaete ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
In Silico Analysis ◽  
Cost Effective ◽  
Rrna Gene ◽  
Bacterial Composition ◽  
Bacterial Dna ◽  
Phenotypic Differences ◽  
Field Samples ◽  
Pcr Rflp ◽  
Digestion Pattern

Piscirickettsia salmons, the causative agent of piscirickettsiosis, is genetically divided into two genomic groups, named after the reference strains as LF-89-like or EM-90-like. Phenotypic differences have been detected between the P. salmonis genogroups, including antibiotic susceptibilities, host specificities and pathogenicity. In this study, we aimed to develop a rapid, sensitive and cost-effective assay for the differentiation of the P. salmonis genogroups. Using an in silico analysis of the P. salmonis 16S rDNA digestion patterns, we have designed a genogroup-specific assay based on PCR-restriction fragment length polymorphism (RFLP). An experimental validation was carried out by comparing the restriction patterns of 13 P. salmonis strains and 57 field samples obtained from the tissues of dead or moribund fish. When the bacterial composition of a set of field samples, for which we detected mixtures of bacterial DNA, was analyzed by a high-throughput sequencing of the 16S rRNA gene amplicons, a diversity of taxa could be identified, including pathogenic and commensal bacteria. Despite the presence of mixtures of bacterial DNA, the characteristic digestion pattern of the P. salmonis genogroups could be detected in the field samples without the need of a microbiological culture and bacterial isolation.

scholarly journals Identification of Anaerobic Selenate-Respiring Bacteria from Aquatic Sediments

2007 ◽  
Vol 73 (11) ◽  
pp. 3519-3527 ◽  
Author(s):  
Priya Narasingarao ◽  
Max M. Häggblom
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Anaerobic Respiration ◽  
Pseudomonas Stutzeri ◽  
Elemental Selenium ◽  
Rrna Gene ◽  
Terminal Electron Acceptor ◽  
Isolation And Characterization ◽  
Anaerobic Biotransformation ◽  
Gene Similarity

ABSTRACT The diversity population of microorganisms with the capability to use selenate as a terminal electron acceptor, reducing it to selenite and elemental selenium by the process known as dissimilatory selenate reduction, is largely unknown. The overall objective of this study was to gain an in-depth understanding of anaerobic biotransformation of selenium in the environment, particularly anaerobic respiration, and to characterize the microorganisms catalyzing this process. Here, we demonstrate the isolation and characterization of four novel anaerobic dissimilatory selenate-respiring bacteria enriched from a variety of sources, including sediments from three different water bodies in Chennai, India, and a tidal estuary in New Jersey. Strains S5 and S7 from India, strain KM from the Meadowlands, NJ, and strain pn1, categorized as a laboratory contaminant, were all phylogenetically distinct, belonging to various phyla in the bacterial domain. The 16S rRNA gene sequence shows that strain S5 constitutes a new genus belonging to Chrysiogenetes, while strain S7 belongs to the Deferribacteres, with greater than 98% 16S rRNA gene similarity to Geovibrio ferrireducens. Strain KM is related to Malonomonas rubra, Pelobacter acidigallici, and Desulfuromusa spp., with 96 to 97% 16S rRNA gene similarity. Strain pn1 is 99% similar to Pseudomonas stutzeri. Strains S5, S7, and KM are obligately anaerobic selenate-respiring microorganisms, while strain pn1 is facultatively anaerobic. Besides respiring selenate, all these strains also respire nitrate.

scholarly journals Leisingera nanhaiensis sp. nov., isolated from marine sediment

2010 ◽  
Vol 60 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Fengqin Sun ◽  
Baojiang Wang ◽  
Xiupian Liu ◽  
Qiliang Lai ◽  
Yaping Du ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Optimal Growth ◽  
Rrna Gene ◽  
Sandy Sediment ◽  
Phenotypic Properties ◽  
Gram Staining ◽  
Distinct Cluster ◽  
Bacterium Strain ◽  
Gene Similarity

An aerobic, Gram-staining-negative, motile, rod-shaped bacterium, strain NH52FT, was isolated from a sandy sediment sample taken from the South China Sea. On M2 agar medium (a complex medium), colonies were beige in colour. The isolate showed highest 16S rRNA gene sequence similarities to members of the genera Leisingera (96.7 % similarity), Phaeobacter (95.4–96.0 %) and Marinovum (94.1 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NH52FT formed a distinct cluster with Leisingera methylohalidivorans MB2T and Leisingera aquimarina LMG 24366T. Optimal growth was observed at pH 7.0-8.5 and 25 °C and the new isolate required the presence of 1–4 % (w/v) NaCl. The major fatty acids were C18 : 1 ω7c, C16 : 0 2-OH, C10 : 0 3-OH, C12 : 0 3-OH, C16 : 0 and 11-methyl C18 : 1 ω7c. The DNA G+C content was 60.5 mol%. The phylogenetic and chemotaxonomic characteristics of strain NH52FT were similar to those of the genus Leisingera. However, the differences in phenotypic properties and the 16S rRNA gene similarity values demonstrated that the new isolate differed from recognized species of the genus Leisingera. On the basis of phenotypic, chemotaxonomic and phylogenetic data, this organism should be classified as a representative of a novel species in the genus Leisingera, for which the name Leisingera nanhaiensis sp. nov. is proposed. The type strain is NH52FT (=LMG 24841T=CCTCC AB 208316T=MCCC 1A04178T).

scholarly journals Biodiversity of Denitrifying and Dinitrogen-Fixing Bacteria in an Acid Forest Soil

2002 ◽  
Vol 68 (8) ◽  
pp. 3818-3829 ◽  
Author(s):  
Christopher Rösch ◽  
Alexander Mergel ◽  
Hermann Bothe
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nitrite Reductase ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Cloning And Sequencing ◽  
Genetic Traits ◽  
Uncultured Bacteria ◽  
Pcr Products ◽  
The 16S Rrna Gene

ABSTRACT Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd 1-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.

scholarly journals New Site of Modification of 23S rRNA Associated with Clarithromycin Resistance of Helicobacter pylori Clinical Isolates

2002 ◽  
Vol 46 (12) ◽  
pp. 3765-3769 ◽  
Author(s):  
Carla Fontana ◽  
Marco Favaro ◽  
Silvia Minelli ◽  
Anna Angela Criscuolo ◽  
Antonio Pietroiusti ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
23S Rrna ◽  
Dilution Method ◽  
Rrna Gene ◽  
Functional Sites ◽  
Biopsy Specimens ◽  
Clarithromycin Resistance ◽  
Pcr Product ◽  
23S Rrna Gene ◽  
H Pylori

ABSTRACT Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 μg/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an HhaI restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates.

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