Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%–60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.

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Evaluation of MicroScan Bacterial Identification Panels for Low-Resource Settings

Diagnostics ◽

10.3390/diagnostics11020349 ◽

2021 ◽

Vol 11 (2) ◽

pp. 349

Author(s):

Sien Ombelet ◽

Alessandra Natale ◽

Jean-Baptiste Ronat ◽

Olivier Vandenberg ◽

Liselotte Hardy ◽

...

Keyword(s):

Bacterial Species ◽

Ease Of Use ◽

Bacterial Identification ◽

Gram Positive ◽

Gram Negative ◽

Low Resource Settings ◽

Low Resource ◽

Bacillus Spp ◽

Gram Negative Organisms ◽

Instructions For Use

Bacterial identification is challenging in low-resource settings (LRS). We evaluated the MicroScan identification panels (Beckman Coulter, Brea, CA, USA) as part of Médecins Sans Frontières’ Mini-lab Project. The MicroScan Dried Overnight Positive ID Type 3 (PID3) panels for Gram-positive organisms and Dried Overnight Negative ID Type 2 (NID2) panels for Gram-negative organisms were assessed with 367 clinical isolates from LRS. Robustness was studied by inoculating Gram-negative species on the Gram-positive panel and vice versa. The ease of use of the panels and readability of the instructions for use (IFU) were evaluated. Of species represented in the MicroScan database, 94.6% (185/195) of Gram-negative and 85.9% (110/128) of Gram-positive isolates were correctly identified up to species level. Of species not represented in the database (e.g., Streptococcus suis and Bacillus spp.), 53.1% out of 49 isolates were incorrectly identified as non-related bacterial species. Testing of Gram-positive isolates on Gram-negative panels and vice versa (n = 144) resulted in incorrect identifications for 38.2% of tested isolates. The readability level of the IFU was considered too high for LRS. Inoculation of the panels was favorably evaluated, whereas the visual reading of the panels was considered error-prone. In conclusion, the accuracy of the MicroScan identification panels was excellent for Gram-negative species and good for Gram-positive species. Improvements in stability, robustness, and ease of use have been identified to assure adaptation to LRS constraints.

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The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms

Micromachines ◽

10.3390/mi9080367 ◽

2018 ◽

Vol 9 (8) ◽

pp. 367 ◽

Cited By ~ 6

Author(s):

Yuguang Liu ◽

Dirk Schulze-Makuch ◽

Jean-Pierre de Vera ◽

Charles Cockell ◽

Thomas Leya ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Bacterial Species ◽

Cell Manipulation ◽

Microfluidic Platforms ◽

Gram Positive ◽

Gram Negative ◽

Genome Amplification ◽

Single Cell Sequencing ◽

Wide Range

Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

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Evaluating Flinders Technology Associates card for transporting bacterial isolates and retrieval of bacterial DNA after various storage conditions

Veterinary World ◽

10.14202/vetworld.2020.2243-2251 ◽

2020 ◽

Vol 13 (10) ◽

pp. 2243-2251

Author(s):

Azhar G. Shalaby ◽

Neveen R. Bakry ◽

Abeer A. E. Mohamed ◽

Ashraf A. Khalil

Keyword(s):

Nucleic Acid ◽

Bacterial Species ◽

Storage Conditions ◽

Animal Origin ◽

Cell Detection ◽

Bacterial Cells ◽

Gram Positive ◽

Bacterial Dna ◽

Gram Negative ◽

Fta Cards

Background and Aim: Flinders Technology Associates (FTA) cards simplify sample storage, transport, and extraction by reducing cost and time for diagnosis. This study evaluated the FTA suitability for safe transport and storage of Gram-positive and Gram-negative bacterial cells of animal origin on its liquid culture form and from organ impression smears (tissues) under the same routine condition of microbiological laboratory along with detecting their nucleic acid over different storage conditions. Materials and Methods: Increase in bacterial count from 104 to 107 (colony-forming units/mL) of 78 isolates representing seven bacterial species was applied onto cards. FTA cards were grouped and inoculated by these bacteria and then stored at different conditions of 24-27°C, 4°C, and –20°C for 24 h, for 2 weeks, for 1 and 3 month storage, respectively. Bacteriological examination was done, after which bacterial DNA was identified using specific primers for each bacterial type and detected by polymerase chain reaction (PCR). Results: The total percentage of recovered bacteria from FTA cards was 66.7% at 24-27–C for 24 h, the detection limit was 100% in Gram-positive species, while it was 57.4% in Gram-negative ones. Regarding viable cell detection from organ impression smears, it was successful under the previous conditions. No live bacterial cells were observed by bacteriological isolation rather than only at 24-27°C for 24 h storage. All bacterial DNA were sufficiently confirmed by the PCR technique at different conditions. Conclusion: Overall, the FTA card method was observed to be a valid tool for nucleic acid purification for bacteria of animal origin in the form of culture or organ smears regardless of its Gram type and is used for a short time only 24 h for storage and transport of live bacteria specifically Gram-positive type. Moreover, the bacterial nucleic acid was intact after storage in –20°C for 3 months and was PCR amplifiable.

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Effect of Seasonality on Chemical Composition and Antibacterial and Anticandida Activities of Argentine Propolis. Design of a Topical Formulation

Natural Product Communications ◽

10.1177/1934578x1200701015 ◽

2012 ◽

Vol 7 (10) ◽

pp. 1934578X1200701 ◽

Cited By ~ 4

Author(s):

María Inés Isla ◽

Yanina Dantur ◽

Ana Salas ◽

Carolina Danert ◽

Catiana Zampini ◽

...

Keyword(s):

Antibacterial Activity ◽

Candida Species ◽

Pharmaceutical Preparation ◽

Pharmaceutical Formulations ◽

Resistant Bacteria ◽

Topical Formulation ◽

Gram Positive ◽

Gram Negative ◽

Propolis Extract ◽

Phenolic And Flavonoid

The effect of seasonality on Argentine propolis collected during one year on its phenolic and flavonoid content and on the growth of Gram-positive and Gram-negative antibiotic resistant bacteria and Candida species was evaluated. Extracts of propolis samples collected in the summer and spring showed higher phenolic and flavonoid contents than the samples collected in other seasons (5.86 to 6.06 mg GAE/mL and 3.77 to 4.23 mg QE/mL, respectively). The propolis collected in summer and autumn showed higher antibacterial activity (30 μg/mL) than the other samples (MIC values between 30 and 120 μg/mL). No antibacterial activity was detected against Gram-negative bacteria. Also, these extracts were able to inhibit the development of five Candida species, with MFC values of 15-120 μg/mL. Pharmaceutical formulations containing the more active propolis extract were prepared. The hydrogel of acrylic acid polymer containing summer propolis extract as an antimicrobial agent showed microbiological, physical and functional stability during storage for 180 days. The pharmaceutical preparation, as well as the propolis extracts, was active against Candida sp. and antibiotic-multi-resistant Gram-positive bacteria. These results reveal that propolis samples collected by scraping in four seasons, especially in summer in Calingasta, San Juan, Argentina, can be used to obtain tinctures and hydrogels with antibacterial and antimycotic potential for topical use.

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Diversity profiling of xenic cultures of Dientamoeba fragilis following systematic antibiotic treatment and prospects for genome sequencing

Parasitology ◽

10.1017/s0031182019001173 ◽

2019 ◽

Vol 147 (1) ◽

pp. 29-38

Author(s):

Rory Gough ◽

Joel Barratt ◽

Damien Stark ◽

John Ellis

Keyword(s):

Antibiotic Treatment ◽

Genome Sequencing ◽

Ribosomal Dna ◽

Bacterial Species ◽

Nutritional Requirement ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Dientamoeba Fragilis ◽

The Impact

AbstractThe presence of bacterial DNA in Dientamoeba fragilis DNA extracts from culture poses a substantial challenge to sequencing the D. fragilis genome. However, elimination of bacteria from D. fragilis cultures has proven difficult in the past, presumably due to its dependence on some unknown prokaryote/s. This study explored options for removal of bacteria from D. fragilis cultures and for the generation of genome sequence data from D. fragilis. DNA was extracted from human faecal samples and xenic D. fragilis cultures. Extracts were subjected to 16S ribosomal DNA bacterial diversity profiling. Xenic D. fragilis cultures were then subject to antibiotic treatment regimens that systematically removed bacterial species depending on their membrane structure (Gram-positive or Gram-negative) and aerobic requirements. The impact of these treatments on cultures was assessed by 16S amplicon sequencing. Prior to antibiotic treatment, the cultures were dominated by Gram-negative bacteria. Addition of meropenem to cultures eliminated anaerobic Gram-negative bacteria, but it also led to protozoan death after 5 days incubation. The seeding of meropenem resistant Klebsiella pneumoniae strain KPC-2 into cultures before treatment by meropenem prevented death of D. fragilis cells beyond this 5 day period, suggesting that one or more species of Gram-negative bacteria may be an essential nutritional requirement for D. fragilis. Gram-positive cells were completely eliminated using vancomycin without affecting trophozoite growth. Finally, this study shows that genome sequencing of D. fragilis is feasible following bacterial elimination from cultures as the result of the major advances occurring in bioinformatics. We provide evidence on this fact by successfully sequencing the D. fragilis 28S large ribosomal DNA subunit gene using culture-derived DNA.

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Transition of Bacterial Diversity and Composition in Tongue Microbiota during the First Two Years of Life

mSphere ◽

10.1128/msphere.00187-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 1

Author(s):

Shinya Kageyama ◽

Mikari Asakawa ◽

Toru Takeshita ◽

Yukari Ihara ◽

Shunsuke Kanno ◽

...

Keyword(s):

Oral Cavity ◽

Bacterial Diversity ◽

Bacterial Species ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Oral Microbiota ◽

Bacterial Composition ◽

Content Type ◽

Operational Taxonomic Units ◽

Rrna Gene Sequencing

ABSTRACTNewborns are constantly exposed to various microbes from birth; hence, diverse commensal bacteria colonize the oral cavity. However, how or when these bacteria construct a complex and stable ecosystem remains unclear. This prospective cohort study examined the temporal changes in bacterial diversity and composition in tongue microbiota during infancy. We longitudinally collected a total of 464 tongue swab samples from 8 infants (age of <6 months at baseline) for approximately 2 years. We also collected samples from 32 children (aged 0 to 2 years) and 73 adults (aged 20 to 29 years) cross-sectionally as control groups. Bacterial diversities and compositions were determined by 16S rRNA gene sequencing. The tongue bacterial diversity in infancy, measured as the number of observed operational taxonomic units (OTUs), rapidly increased and nearly reached the same level as that in adults by around 80 weeks. The overall tongue bacterial composition in the transitional phase, 80 to 120 weeks, was more similar to that of adults than to that of the early exponential phase (EEP), 10 to 29 weeks, according to analysis of similarities. Dominant OTUs in the EEP corresponding toStreptococcus perorisandStreptococcus lactariusexponentially decreased immediately after EEP, around 30 to 49 weeks, whereas several OTUs corresponding toGranulicatella adiacens,Actinomyces odontolyticus, andFusobacterium periodonticumreciprocally increased during the same period. These results suggest that a drastic compositional shift of tongue microbiota occurs before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years.IMPORTANCEEvaluating the development of oral microbiota during infancy is important for understanding the subsequent colonization of bacterial species and the process of formation of mature microbiota in the oral cavity. We examined tongue microbiota longitudinally collected from 8 infants and found that drastic compositional shifts in tongue microbiota occur before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years. These results may be helpful for preventing the development of various diseases associated with oral microbiota throughout life.

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Effect of Organochlorine Pesticides on Growth and Biological Activities of Bacterial Species Important to the Dairy Food Industry

Journal of Milk and Food Technology ◽

10.4315/0022-2747-39.2.90 ◽

1976 ◽

Vol 39 (2) ◽

pp. 90-94 ◽

Cited By ~ 1

Author(s):

MARY L. SANDFORD ◽

B. E. LANGLOIS

Keyword(s):

Food Industry ◽

Biological Activities ◽

Bacterial Species ◽

Inhibitory Effect ◽

Growth Patterns ◽

Gram Positive ◽

Dairy Food ◽

Gram Negative ◽

Generation Times ◽

Slight Inhibition

Three growth patterns (no effect, slight inhibition, or complete inhibition) were observed when bacterial species common to the dairy-food industry were grown in media containing 50 or 100 ppm DDT, dieldrin, or endrin. The pattern obtained appeared to depend on species and type and concentration of pesticide. All pesticides studied had a greater inhibitory effect on gram positive species than they had on gram negative species when grown in broth. Acid production by lactic acid bacteria was inhibited in broth plus 5 ppm chlordane or heptachlor but unaffected in skimmilk plus up to 100 ppm of these pesticides. Generation times for gram negative species grown in broth plus 10 ppm chlordane or heptachlor were similar to those obtained in controls. Growth of gram positive species was inhibited in broth plus 10 ppm of these pesticides but unaffected in skimmilk containing similar pesticide concentrations. Generation times for several gram negative species were increased by 10 ppm heptachlor in skimmilk.

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In VitroActivity of AZD0914, a Novel Bacterial DNA Gyrase/Topoisomerase IV Inhibitor, against Clinically Relevant Gram-Positive and Fastidious Gram-Negative Pathogens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01016-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6053-6063 ◽

Cited By ~ 12

Author(s):

Douglas J. Biedenbach ◽

Michael D. Huband ◽

Meredith Hackel ◽

Boudewijn L. M. de Jonge ◽

Daniel F. Sahm ◽

...

Keyword(s):

Bacterial Species ◽

Dna Gyrase ◽

Species Group ◽

Coagulase Negative Staphylococci ◽

Topoisomerase Iv ◽

Gram Positive ◽

Bacterial Dna ◽

Gram Negative ◽

Content Type

ABSTRACTAZD0914, a new spiropyrimidinetrione bacterial DNA gyrase inhibitor with a novel mode of inhibition, has activity against bacterial species commonly cultured from patient infection specimens, including fluoroquinolone-resistant isolates. This study assessed thein vitroactivity of AZD0914 against key Gram-positive and fastidious Gram-negative clinical isolates collected globally in 2013. AZD0914 demonstrated potent activity, with MIC90s for AZD0914 of 0.25 mg/liter againstStaphylococcus aureus(n= 11,680), coagulase-negative staphylococci (n= 1,923), streptococci (n= 4,380), andMoraxella catarrhalis(n= 145), 0.5 mg/liter againstStaphylococcus lugdunensis(n= 120) andHaemophilus influenzae(n= 352), 1 mg/liter againstEnterococcus faecalis(n= 1,241), and 2 mg/liter againstHaemophilus parainfluenzae(n= 70). The activity againstEnterococcus faeciumwas more limited (MIC90, 8 mg/liter). The spectrum and potency of AZD0914 included fluoroquinolone-resistant isolates in each species group, including methicillin-resistant staphylococci, penicillin-resistant streptococci, vancomycin-resistant enterococci, β-lactamase-producingHaemophilusspp., andM. catarrhalis. Based on thesein vitrofindings, AZD0914 warrants further investigation for its utility against a variety of Gram-positive and fastidious Gram-negative bacterial species.

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In VitroAntibacterial Activity of AZD0914, a New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-Positive, Fastidious Gram-Negative, and Atypical Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04124-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 467-474 ◽

Cited By ~ 44

Author(s):

Michael D. Huband ◽

Patricia A. Bradford ◽

Linda G. Otterson ◽

Gregory S. Basarab ◽

Amy C. Kutschke ◽

...

Keyword(s):

Antibacterial Activity ◽

Legionella Pneumophila ◽

Bacterial Species ◽

Dna Gyrase ◽

Cross Resistance ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Topoisomerase Inhibitor

ABSTRACTAZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potentin vitroantibacterial activity against key Gram-positive (Staphylococcus aureus,Staphylococcus epidermidis,Streptococcus pneumoniae,Streptococcus pyogenes, andStreptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzaeandNeisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance inS. aureus, and if mutants were obtained, the mutations mapped togyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration andin vitrotime-kill studies. Inin vitrocheckerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potentin vitroantibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development.

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Identification and Characterization of the First Cholesterol-Dependent Cytolysins from Gram-Negative Bacteria

Infection and Immunity ◽

10.1128/iai.00927-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 216-225 ◽

Cited By ~ 42

Author(s):

Eileen M. Hotze ◽

Huynh M. Le ◽

Jessica R. Sieber ◽

Christina Bruxvoort ◽

Michael J. McInerney ◽

...

Keyword(s):

Cell Structure ◽

Bacterial Species ◽

Cytolytic Activity ◽

Opportunistic Pathogens ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Positive Type ◽

Secretion Signal ◽

Anaerobic Soils

The cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that have been exclusively associated with a wide variety of bacterial pathogens and opportunistic pathogens from theFirmicutesandActinobacteria, which exhibit a Gram-positive type of cell structure. We have characterized the first CDCs from Gram-negative bacterial species, which includeDesulfobulbus propionicustype species Widdel 1981 (DSM 2032) (desulfolysin [DLY]) andEnterobacter lignolyticus(formerlyEnterobacter cloacae) SCF1 (enterolysin [ELY]). The DLY and ELY primary structures show that they maintain the signature motifs of the CDCs but lack an obvious secretion signal. Recombinant, purified DLY (rDLY) and ELY (rELY) exhibited cholesterol-dependent binding and cytolytic activity and formed the typical large CDC membrane oligomeric pore complex. Unlike the CDCs from Gram-positive species, which are human- and animal-opportunistic pathogens, neitherD. propionicusnorE. lignolyticusis known to be a pathogen or commensal of humans or animals: the habitats of both organisms appear to be restricted to anaerobic soils and/or sediments. These studies reveal for the first time that the genes for functional CDCs are present in bacterial species that exhibit a Gram-negative cell structure. These are also the first bacterial species containing a CDC gene that are not known to inhabit or cause disease in humans or animals, which suggests a role of these CDCs in the defense against eukaryote bacterial predators.

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