scholarly journals In VitroCulture of Previously Uncultured Oral Bacterial Phylotypes

2015 ◽  
Vol 81 (24) ◽  
pp. 8307-8314 ◽  
Author(s):  
Hayley Thompson ◽  
Alexandra Rybalka ◽  
Rebecca Moazzez ◽  
Floyd E. Dewhirst ◽  
William G. Wade
Keyword(s):  
Culture Media ◽  
Pcr Primers ◽  
Oral Bacteria ◽  
Subgingival Plaque ◽  
Community Studies ◽  
Shotgun Metagenomics ◽  
Content Type ◽  
Specific Pcr ◽  
Proteose Peptone

ABSTRACTAround a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use anin vitrobiofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA).In vitrobiofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxaBacteroidetesbacteria HOT 365 and HOT 281,Lachnospiraceaebacteria HOT 100 and HOT 500, andClostridialesbacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype,Actinomycessp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected inin vitro-cultivated biofilms.Lachnospiraceaebacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture withParvimonas micraorVeillonella dispar/parvulaafter colony hybridization-directed enrichment. The establishment ofin vitrobiofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.

scholarly journals Specific PCR detection of Peptostreptococcus magnus

2003 ◽  
Vol 52 (4) ◽  
pp. 309-313 ◽  
Author(s):  
M.P. Riggio ◽  
A. Lennon
Keyword(s):  
Pcr Primers ◽  
Oral Bacteria ◽  
Rrna Gene ◽  
Subgingival Plaque ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Specific Pcr ◽  
Wide Range ◽  
Pcr Products ◽  
Peptostreptococcus Magnus

Peptostreptococcus magnus is the most pathogenic and one of the most common Gram-positive anaerobic cocci found in human clinical specimens. The organism has been isolated in pure culture from a range of serious infections, including meningitis and endocarditis. However, isolation of Peptostreptococcus magnus from the oral cavity has rarely been attempted. Identification of Peptostreptococcus magnus in clinical specimens is reliant upon microbiological culture and biochemical methods, which often give ambiguous results. The aim of this study was to develop a PCR assay for the specific detection of Peptostreptococcus magnus in oral clinical specimens. PCR primers specific for Peptostreptococcus magnus DNA were derived by comparison of 16S rRNA gene sequences and selection of primers that demonstrated specificity at their 3′ ends for Peptostreptococcus magnus. PCR positivity for Peptostreptococcus magnus DNA was indicated by the amplification of a 553 bp product. The PCR assay was then used to attempt detection of Peptostreptococcus magnus DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. The PCR assay was demonstrated to be highly specific for Peptostreptococcus magnus DNA, since no PCR products were obtained when genomic DNA from a wide range of other oral bacteria, including closely related Peptostreptococcus species, was used in the PCR assay. Confirmation of specific amplification of Peptostreptococcus magnus DNA was obtained by digestion of PCR products with the restriction endonuclease RsaI, which gives a unique restriction profile for Peptostreptococcus magnus. Of the 33 subgingival plaque samples analysed, 2 (6 %) were positive for Peptostreptococcus magnus DNA. None of the 60 pus aspirates analysed was positive for Peptostreptococcus magnus DNA. It is concluded that Peptostreptococcus magnus is not a major pathogen in adult periodontitis or dento-alveolar abscesses. The PCR assay provides a more rapid, specific and sensitive alternative to conventional methods for identification of Peptostreptococcus magnus in clinical specimens.

scholarly journals Effects of Methanogenic Inhibitors on Methane Production and Abundances of Methanogens and Cellulolytic Bacteria inIn VitroRuminal Cultures

2011 ◽  
Vol 77 (8) ◽  
pp. 2634-2639 ◽  
Author(s):  
Zhenming Zhou ◽  
Qingxiang Meng ◽  
Zhongtang Yu
Keyword(s):  
Methane Production ◽  
Volatile Fatty Acids ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Cellulolytic Bacteria ◽  
Bacterial Populations ◽  
Content Type ◽  
Specific Pcr ◽  
Methanogen Population ◽  
Propynoic Acid

ABSTRACTThe objective of this study was to systematically evaluate and compare the effects of select antimethanogen compounds on methane production, feed digestion and fermentation, and populations of ruminal bacteria and methanogens usingin vitrocultures. Seven compounds, including 2-bromoethanesulphonate (BES), propynoic acid (PA), nitroethane (NE), ethyltrans-2-butenoate (ETB), 2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were tested at a final concentration of 12 mM. Ground alfalfa hay was included as the only substrate to simulate daily forage intake. Compared to no-inhibitor controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to 99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate (79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23% without a significant effect on total VFAs, acetate, or propionate. BES significantly reduced the propionate concentration but not the production of methane, total VFAs, or acetate. ETB or NE had no significant effect on any of the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed that none of the inhibitors significantly affected total bacterial populations but that they did reduce theFibrobacter succinogenespopulation. SN reduced theRuminococcus albuspopulation, while PA and 2NEOH increased the populations of bothR. albusandRuminococcus flavefaciens. Archaeon-specific PCR-denaturing gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the methanogen population structure, while archaeon-specific qPCR revealed a significant decrease in methanogen population in all treatments. These results showed that EB, ETB, NE, and BES can effectively reduce the total population of methanogens but that they reduce methane production to a lesser extent. The results may guide futureinvivostudies to develop effective mitigation of methane emission from ruminants.

Transcriptional control of delta-crystallin gene expression in the chicken embryo lens: demonstration by a new method for measuring mRNA metabolism

1993 ◽  
Vol 13 (6) ◽  
pp. 3282-3290
Author(s):  
X Li ◽  
D C Beebe
Keyword(s):  
Chicken Embryo ◽  
Transcriptional Control ◽  
Cultured Cells ◽  
Pcr Primers ◽  
High Refractive Index ◽  
In Vitro Transcription ◽  
Equilibrium Density ◽  
Specific Pcr ◽  
High Concentrations

Crystallins are proteins that accumulate to very high concentrations in the fiber cells of the lens of the eye. Crystallins are responsible for the transparency and high refractive index that are essential for lens function. In the chicken embryo, delta-crystallin accounts for more than 70% of the newly synthesized lens proteins. We used density labeling and gene-specific polymerase chain reaction (PCR) to determine the mechanism regulating the expression of the two very similar delta-crystallin genes. Newly synthesized RNA was separated from preexisting RNA by incubating the lenses with 15N- and 13C-labeled ribonucleosides and then separating newly synthesized, density-labeled RNA from the bulk of light RNA by equilibrium density centrifugation in NaI-KI gradients. The relative abundances of the two crystallin mRNAs in the separated fractions were then determined by PCR. This method permitted the quantitation of newly synthesized processed and unprocessed delta-crystallin mRNAs. Additional studies used intron- and gene-specific PCR primers to determine the relative expression of the two delta-crystallin genes in processed RNA and unprocessed RNA extracted from different regions of the embryonic lens. Results of these tests indicated that the differential expression of the delta-crystallin genes was regulated primarily at the level of transcription. This outcome was not expected on the basis of the results of previous studies, which used in vitro transcription and transfection methods to evaluate the relative strengths of delta-crystallin promoter and enhancer sequences. Our data suggest that the cultured cells used in these earlier studies may not have provided an accurate view of delta-crystallin regulation in the intact lens.

A review of in vitro attempts to develop the axenic culture of Treponema pallidum and genomics-based suggestions to achieve this elusive goal

2021 ◽  
Vol 70 (7) ◽  
Author(s):  
Souad Belkacemi ◽  
Maryam Tidjani Alou ◽  
Saber Khelaifia ◽  
Didier Raoult
Keyword(s):  
Microscopic Observation ◽  
Type Species ◽  
Culture Media ◽  
Treponema Pallidum ◽  
Axenic Culture ◽  
Dark Field ◽  
Content Type ◽  
Link Type ◽  
Oral Microbiology

To date, the axenic culture of Treponema pallidum remains a challenge in the field of microbiology despite countless attempts. Here, we conducted a comprehensive bibliographic analysis using several databases and search engines, namely Pubmed, Google scholar, Google, Web of Science and Scopus. Numerous unsuccessful empiric studies have been conducted and evaluated using as criteria dark-field microscopic observation of motile spiral shaped cells in the culture and virulence of the culture through rabbit infectivity. All of these studies failed to induce rabbit infectivity, even when deemed positive after microscopic observation leading to the misnomer of avirulent T. pallidum . In fact, this criterion was improperly chosen because not all spiral shaped cells are T. pallidum . However, these studies led to the formulation of culture media particularly favourable to the growth of several species of Treponema, including Oral Microbiology and Immunology, Zürich medium (OMIZ), Oral Treponeme Enrichment Broth (OTEB) and T-Raoult, thus allowing the increase in the number of cultivable strains of Treponema . The predicted metabolic capacities of T. pallidum show limited metabolism, also exhibited by other non-cultured and pathogenic Treponema species, in contrast to cultured Treponema species. The advent of next generation sequencing represents a turning point in this field, as the knowledge inferred from the genome can finally lead to the axenic culture of T. pallidum .

scholarly journals Development of a Rapid, Sensitive, and Field-Deployable Razor Ex BioDetection System and Quantitative PCR Assay for Detection of Phymatotrichopsis omnivora Using Multiple Gene Targets

2013 ◽  
Vol 79 (7) ◽  
pp. 2312-2320 ◽  
Author(s):  
M. Arif ◽  
J. Fletcher ◽  
S. M. Marek ◽  
U. Melcher ◽  
F. M. Ochoa-Corona
Keyword(s):  
Rna Polymerase Ii ◽  
Quantitative Pcr ◽  
Target Genes ◽  
Pcr Primers ◽  
Internal Transcribed Spacers ◽  
Diagnostic Tools ◽  
Purification Method ◽  
Content Type ◽  
Specific Pcr ◽  
Phymatotrichopsis Omnivora

ABSTRACTA validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection ofPhymatotrichopsis omnivora.This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. BecauseP. omnivorais difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based onP. omnivoranucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection ofP. omnivorain infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (∼30-min) on-site assay capability forP. omnivoradetection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.

scholarly journals Identification by PCR of Fusarium culmorum Strains Producing Large and Small Amounts of Deoxynivalenol

2002 ◽  
Vol 68 (11) ◽  
pp. 5472-5479 ◽  
Author(s):  
B. Bakan ◽  
C. Giraud-Delville ◽  
L. Pinson ◽  
D. Richard-Molard ◽  
E. Fournier ◽  
...  
Keyword(s):  
Intergenic Region ◽  
Sequence Data ◽  
Fusarium Culmorum ◽  
Pcr Primers ◽  
Wheat Grains ◽  
Specific Pcr ◽  
Trichodiene Synthase

ABSTRACT Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.

Production of collagenase and inhibitor (TIMP) by intracranial tumors and dura in vitro

1983 ◽  
Vol 59 (3) ◽  
pp. 461-466 ◽  
Author(s):  
Ahmed N. Halaka ◽  
Rowena A.D. Bunning ◽  
Colin C. Bird ◽  
Myles Gibson ◽  
John J. Reynolds
Keyword(s):  
Organ Culture ◽  
Tumor Invasion ◽  
Culture Media ◽  
Intracranial Tumors ◽  
Short Term ◽  
Content Type ◽  
Collagenase Inhibitor ◽  
Tissue Degradation ◽  
Fine Print

✓ The production of collagenase and collagenase inhibitor (TIMP) by various intracranial tumors (25 meningiomas, eight gliomas, seven metastases, four pituitary adenomas, and five others) was studied in short-term organ culture. While meningiomas produced negligible amounts of collagenase, two metastatic carcinomas of bronchial and breast origin produced significant amounts of the enzyme. Cultures of dura from an invasive meningioma and of bone invaded by a meningioma also produced collagenase. In varying amounts, TIMP was detected in culture media from most of the tumors studied; invasive tumors tended to produce less TIMP than noninvasive tumors. The results are discussed in relation to current views on tissue degradation and mechanisms of tumor invasion.

scholarly journals Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

2013 ◽  
Vol 57 (6) ◽  
pp. 2768-2779 ◽  
Author(s):  
Darren J. Creek ◽  
Brunda Nijagal ◽  
Dong-Hyun Kim ◽  
Federico Rojas ◽  
Keith R. Matthews ◽  
...  
Keyword(s):  
Cell Culture ◽  
Trypanosoma Brucei ◽  
Culture Medium ◽  
Culture Media ◽  
Culture Methods ◽  
Content Type ◽  
Drug Activity ◽  
Rational Development ◽  
High Concentrations

ABSTRACTIn vitroculture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream formTrypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supportsin vitrogrowth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.

scholarly journals Comparative Genome Analyses of Lactobacillus crispatus Isolates from Different Ecological Niches Reveal an Adaptation of This Species to the Human Vaginal Environment

2021 ◽  
Vol 87 (8) ◽  
Author(s):  
Leonardo Mancabelli ◽  
Walter Mancino ◽  
Gabriele Andrea Lugli ◽  
Christian Milani ◽  
Alice Viappiani ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Vaginal Microbiota ◽  
Microbial Consortia ◽  
Microbial Composition ◽  
Vaginal Infections ◽  
Shotgun Metagenomics ◽  
Content Type ◽  
Lactobacillus Crispatus ◽  
Vaginal Tract

ABSTRACT The vaginal microbiota is defined as the community of bacteria residing in the human vaginal tract. Recent studies have demonstrated that the vaginal microbiota is dominated by members of the Lactobacillus genus, whose relative abundance and microbial taxon composition are dependent on the healthy status of this human body site. Particularly, among members of this genus, the high prevalence of Lactobacillus crispatus is commonly associated with a healthy vaginal environment. In the current study, we assessed the microbial composition of 94 healthy vaginal microbiome samples through shotgun metagenomics analyses. Based on our results, we observed that L. crispatus was the most representative species and correlated negatively with bacteria involved in vaginal infections. Therefore, we isolated 15 L. crispatus strains from different environments in which this species abounds, ranging from vaginal swabs of healthy women to chicken fecal samples. The genomes of these strains were decoded and their genetic content was analyzed and correlated with their physiological features. An extensive comparative genomic analysis encompassing all publicly available genome sequences of L. crispatus and combined with those decoded in this study revealed a genetic adaptation of strains to their respective ecological niche. In addition, in vitro growth experiments involving all isolated L. crispatus strains, together with a synthetic vaginal microbiota, reveal how this species is able to modulate the composition of the vaginal microbial consortia at the strain level. Overall, our findings suggest that L. crispatus plays an important ecological role in reducing the complexity of the vaginal microbiota by depleting pathogenic bacteria. IMPORTANCE The vaginal microbiota is defined as the community of bacteria residing in the human vaginal tract. Recent studies have demonstrated that the high prevalence of Lactobacillus crispatus strains is commonly associated with a healthy vaginal environment. In the current study, we assessed the microbial composition of 94 public healthy vaginal samples through shotgun metagenomics analyses. Results showed that L. crispatus was the most representative species and correlated negatively with bacteria involved in vaginal infections. Moreover, we isolated and sequenced the genomes of new L. crispatus strains from different environments, and the comparative genomics analysis revealed a genetic adaptation of strains to their ecological niche. In addition, in vitro growth experiments display the capability of this species to modulate the composition of the vaginal microbial consortia. Overall, our findings suggest an ecological role exploited by L. crispatus in reducing the complexity of the vaginal microbiota toward a depletion of pathogenic bacteria.

scholarly journals Influential Parameters for the Analysis of Intracellular Parasite Metabolomics

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. e00097-18 ◽  
Author(s):  
Maureen A. Carey ◽  
Vincent Covelli ◽  
Audrey Brown ◽  
Gregory L. Medlock ◽  
Mareike Haaren ◽  
...  
Keyword(s):  
Drug Treatment ◽  
Antimalarial Drug ◽  
Malaria Parasite ◽  
Culture Media ◽  
Untargeted Metabolomics ◽  
Metabolic Effects ◽  
Intracellular Parasite ◽  
Metabolomics Data ◽  
Content Type

ABSTRACT Metabolomics is increasingly popular for the study of pathogens. For the malaria parasite Plasmodium falciparum, both targeted and untargeted metabolomics have improved our understanding of pathogenesis, host-parasite interactions, and antimalarial drug treatment and resistance. However, purification and analysis procedures for performing metabolomics on intracellular pathogens have not been explored. Here, we purified in vitro-grown ring-stage intraerythrocytic P. falciparum parasites for untargeted metabolomics studies; the small size of this developmental stage amplifies the challenges associated with metabolomics studies as the ratio between host and parasite biomass is maximized. Following metabolite identification and data preprocessing, we explored multiple confounding factors that influence data interpretation, including host contamination and normalization approaches (including double-stranded DNA, total protein, and parasite numbers). We conclude that normalization parameters have large effects on differential abundance analysis and recommend the thoughtful selection of these parameters. However, normalization does not remove the contribution from the parasite’s extracellular environment (culture media and host erythrocyte). In fact, we found that extraparasite material is as influential on the metabolome as treatment with a potent antimalarial drug with known metabolic effects (artemisinin). Because of this influence, we could not detect significant changes associated with drug treatment. Instead, we identified metabolites predictive of host and medium contamination that could be used to assess sample purification. Our analysis provides the first quantitative exploration of the effects of these factors on metabolomics data analysis; these findings provide a basis for development of improved experimental and analytical methods for future metabolomics studies of intracellular organisms. IMPORTANCE Molecular characterization of pathogens such as the malaria parasite can lead to improved biological understanding and novel treatment strategies. However, the distinctive biology of the Plasmodium parasite, including its repetitive genome and the requirement for growth within a host cell, hinders progress toward these goals. Untargeted metabolomics is a promising approach to learn about pathogen biology. By measuring many small molecules in the parasite at once, we gain a better understanding of important pathways that contribute to the parasite’s response to perturbations such as drug treatment. Although increasingly popular, approaches for intracellular parasite metabolomics and subsequent analysis are not well explored. The findings presented in this report emphasize the critical need for improvements in these areas to limit misinterpretation due to host metabolites and to standardize biological interpretation. Such improvements will aid both basic biological investigations and clinical efforts to understand important pathogens.

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