Evaluation of Simultaneous Growth of O157:H7, spp., and in Ground Beef Samples in Different Growth Media.

Several multiplex approaches for the simultaneous detection of pathogens in food have been developed in recent years, but the use of a single enrichment medium remains a problem. In this study, six enrichment broths (five non-selective media, tryptic soy broth (TSB), brain heart infusion broth (BHI), buffered peptone water (BPW), universal pre-enrichment broth (UPB), no. 17 broth, and a selective, broth (SEL)), were studied for the simultaneous detection of O157:H7, spp.

Oligonucleotide probes for imaging and diagnosis of bacterial infections.

The rise of infectious diseases as a public health concern has necessitated the development of rapid and precise diagnostic methods. Imaging techniques like nuclear and optical imaging provide the ability to diagnose infectious diseases within the body, eliminating delays caused by sampling and pre-enrichments of clinical samples and offering spatial information that can aid in a more informed diagnosis. Traditional molecular probes are typically created to image infected tissue without accurately identifying the pathogen.

In vitro selection of DNA aptamers against staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA.

Antifungal Susceptibility and sp. Biofilm Production in Clinical Isolates of HIV-Positive Brazilian Patients under HAART Therapy.

The aim of the present study was to characterize biofilms formed by spp. clinical isolates (n = 19), isolated from the oral mucosa of HIV-positive patients. For characterizing the biofilms formed by several sp.

Exploring the Antibiotic Resistance Profile of Clinical Isolates in Portugal.

While antibiotic resistance is rising to dangerously high levels, resistance mechanisms are spreading globally among diverse bacterial species. The emergence of antibiotic-resistant Klebsiella pneumoniae, mainly due to the production of antibiotic-inactivating enzymes, is currently responsible for most treatment failures, threatening the effectiveness of classes of antibiotics used for decades. This study assessed the presence of genetic determinants of β-lactam resistance in 102 multi-drug resistant (MDR) K.

Development of a Novel Peptide Nucleic Acid Probe for the Detection of spp. in Water Samples.

are opportunistic intracellular pathogens that are found throughout the environment. The contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires' disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection.

The role of Nucleic Acid Mimics (NAMs) on FISH-based techniques and applications for microbial detection.

Fluorescent in situ hybridization (FISH) is a powerful tool that for more than 30 years has allowed to detect and quantify microorganisms as well as to study their spatial distribution in three-dimensional structured environments such as biofilms. Throughout these years, FISH has been improved in order to face some of its earlier limitations and to adapt to new research objectives. One of these improvements is related to the emergence of Nucleic Acid Mimics (NAMs), which are now employed as alternatives to the DNA and RNA probes that have been classically used in FISH.

Modelling aptamers with nucleic acid mimics (NAM): From sequence to three-dimensional docking.

Aptamers are single-stranded oligonucleotides, formerly evolved by Systematic Evolution of Ligands by EXponential enrichment (SELEX), that fold into functional three-dimensional structures. Such conformation is crucial for aptamers' ability to bind to a target with high affinity and specificity. Unnatural nucleotides have been used to develop nucleic acid mimic (NAM) aptamers with increased performance, such as biological stability.

Improving aptamer performance with nucleic acid mimics: de novo and post-SELEX approaches.

Aptamers are structural single-stranded oligonucleotides generated in vitro to bind to a specific target molecule. Aptamers' versatility can be enhanced with nucleic acid mimics (NAMs) during or after a selection process, also known as systematic evolution of ligands by exponential enrichment (SELEX). We address advantages and limitations of the technologies used to generate NAM aptamers, especially the applicability of existing engineered polymerases to replicate NAMs and methodologies to improve aptamers after SELEX.

Friends with Benefits: An Inside Look of Periodontal Microbes' Interactions Using Fluorescence In Situ Hybridization-Scoping Review.

Fluorescence in situ hybridization (FISH) has proven to be particularly useful to describe the microbial composition and spatial organization of mixed microbial infections, as it happens in periodontitis. This scoping review aims to identify and map all the documented interactions between microbes in periodontal pockets by the FISH technique. Three electronic sources of evidence were consulted in search of suitable articles up to 7 November 2020: MEDLINE (via PubMed), Scopus (Elsevier: Amsterdam, The Netherlands), and Web of Science (Clarivate Analytics: Philadelphia, PA, USA) online databases.

Lipoplexes to Deliver Oligonucleotides in Gram-Positive and Gram-Negative Bacteria: Towards Treatment of Blood Infections.

Bacterial resistance to antibiotics threatens the ability to treat life-threatening bloodstream infections. Oligonucleotides (ONs) composed of nucleic acid mimics (NAMs) able to inhibit essential genes can become an alternative to traditional antibiotics, as long as they are safely transported in human serum upon intravenous administration and they are carried across the multilayered bacterial envelopes, impermeable to ONs. In this study, fusogenic liposomes were considered to transport the ONs and promote their internalization in clinically relevant bacteria.

Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?

The emergence of bacterial resistance to traditional small-molecule antibiotics is fueling the search for innovative strategies to treat infections. Inhibiting the expression of essential bacterial genes using antisense oligonucleotides (ASOs), particularly composed of nucleic acid mimics (NAMs), has emerged as a promising strategy. However, their efficiency depends on their association with vectors that can translocate the bacterial envelope.

Establishment of a New PNA-FISH Method for Identification: First Insights for Future Use in Pulmonary Samples.

is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence hybridization (PNA-FISH) is described.

FISH and chips: a review of microfluidic platforms for FISH analysis.

Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function.

Minimum information guideline for spectrophotometric and fluorometric methods to assess biofilm formation in microplates.

The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the necessary information that a manuscript should include for the experiments in a study to be clearly interpreted and independently reproduced.

Quantitative assessment of individual populations within polymicrobial biofilms.

Selecting appropriate tools providing reliable quantitative measures of individual populations in biofilms is critical as we now recognize their true polymicrobial and heterogeneous nature. Here, plate count, quantitative real-time polymerase chain reaction (q-PCR) and peptide nucleic acid probe-fluorescence in situ hybridization (PNA-FISH) were employed to quantitate cystic fibrosis multispecies biofilms. Growth of Pseudomonas aeruginosa, Inquilinus limosus and Dolosigranulum pigrum was assessed in dual- and triple-species consortia under oxygen and antibiotic stress.

Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides.

Background: Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specific miRNA expression profile, with increasing evidence of the role of miRNA-9 in this disease. miRNA-9 upregulation has been shown to influence the expression of E-cadherin-encoding gene, triggering cell motility and invasiveness.

Anti-miRNA oligonucleotides: A comprehensive guide for design.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally. As a consequence of their function towards mRNA, miRNAs are widely associated with the pathogenesis of several human diseases, making miRNAs a target for new therapeutic strategies based on the control of their expression. Indeed, numerous works were published in the past decades showing the potential use of antisense oligonucleotides to target aberrant miRNAs (AMOs) involved in several human pathologies.

Developing a model for cystic fibrosis sociomicrobiology based on antibiotic and environmental stress.

Cystic fibrosis (CF) infections are invariably biofilm-mediated and polymicrobial, being safe to assume that a myriad of factors affects the sociomicrobiology within the CF infection site and modulate the CF community dynamics, by shaping their social activities, overall functions, virulence, ultimately affecting disease outcome. This work aimed to assess changes in the dynamics (particularly on the microbial composition) of dual-/three-species biofilms involving CF-classical (Pseudomonas aeruginosa) and unusual species (Inquilinus limosus and Dolosigranulum pigrum), according to variable oxygen conditions and antibiotic exposure. Low fluctuations in biofilm compositions were observed across distinct oxygen environments, with dual-species biofilms exhibiting similar relative proportions and P.

Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique.

Purpose: We aim to develop peptic nucleic acid (PNA) probes for the identification and localization of Aggregatibacter actinomycetemcomintans and Porphyromonas gingivalis in sub-gingival plaque and gingival biopsies by Fluorescence in situ Hybridization (FISH).

Methods: A PNA probe was designed for each microorganism. The PNA-FISH method was optimized to allow simultaneous hybridization of both microorganisms with their probe (PNA-FISH multiplex).

Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared.

Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model.

Introduction: In this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting the C57BL/6 mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary to a sequence of the H.

Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes.

In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach.