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. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Antifungal Susceptibility and Candida 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 Candida spp. clinical isolates (n = 19), isolated from the oral mucosa of HIV-positive patients. For characterizing the biofilms formed by several Candida sp. strains, isolated from HIV-positive patients, in terms of formed biomass, matrix composition and antifungal susceptibility profile, clinical isolates (n = 19) were collected from oral mucosa and identified. The biofilm of the samples was cultured with fluconazole (1250 mg/L), voriconazole (800 mg/L), anidulafungin (2 mg/L) or amphotericin B (2 mg/L). Afterwards, the quantification of the total biomass was performed using crystal violet assay, while the proteins and carbohydrates levels were quantified in the matrix. The results showed a predominance of C. albicans, followed by C. krusei. Around 58% of the Candida spp. biofilm had susceptibility to fluconazole and voriconazole (800 mg/L), 53% to anidulafungin and 74% to amphotericin B. C. krusei presented both the lowest and the highest biofilm matrix contents in polysaccharides and proteins. The low resistance to antifungal agents reported here was probably due to the fact that none of the participants had a prolonged exposure to these antifungals. A predominance of less virulent Candida spp. strains with low or no resistance to antifungals was observed. This can be attributed to a low fungal selective pressure. This most probably happened due to a low fungal selective pressure but also due to a good adherence to HAART therapy, which guarantees a stable and stronger immune patient response.

Characterization of Oral Candida spp. Biofilms in Children and Adults Carriers from Eastern Europe and South America.

BACKGROUND: Candida albicans and non-Candida albicans Candida species (NCACs) are known to colonize and invade various tissues, including the oral mucosa. In this work, we aimed to characterize mature biofilms of several Candida spp. clinical isolates (n = 33) obtained from the oral mucosa of children, adults, and elders of Eastern Europe and South America. METHODS: Each strain was evaluated for its capacity to form biofilms in terms of total biomass using the crystal violet assay and for matrix components production (proteins and carbohydrates) using the BCA and phenol-sulfuric tests, respectively. The effect of different antifungals on biofilm formation was studied. RESULTS: in the children's group, a predominance of C. krusei (81%) was observed, while, among adults, the main species was C. albicans (59%). Most strains showed a reduced response to antimicrobial drugs when in biofilm form (p < 0.01). Moreover, it was observed that strains isolated from children produced more matrix, with higher levels of protein and polysaccharides. CONCLUSIONS: children were more likely to be infected by NCACs than adults. More importantly, these NCACs were able to form biofilms richer in matrix components. This finding is of clinical importance, particularly in pediatric care, since stronger biofilms are highly associated with antimicrobial resistance, recurrent infections, and higher therapeutic failure.

Exploring the Antibiotic Resistance Profile of Clinical Klebsiella pneumoniae 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 ß-lactam resistance in 102 multi-drug resistant (MDR) K. pneumoniae isolates from patients admitted to two central hospitals in northern Portugal from 2010 to 2020. Antimicrobial susceptibility testing revealed a high rate (>90%) of resistance to most ß-lactam antibiotics, except for carbapenems and cephamycins, which showed antimicrobial susceptibility rates in the range of 23.5−34.3% and 40.2−68.6%, respectively. A diverse pool of ß-lactam resistance genetic determinants, including carbapenemases- (i.e., blaKPC-like and blaOXA-48-like), extended-spectrum ß-lactamases (ESBL; i.e., blaTEM-like, blaCTX-M-like and blaSHV-like), and AmpC ß-lactamases-coding genes (i.e., blaCMY-2-like and blaDHA-like) were found in most K. pneumoniae isolates. blaKPC-like (72.5%) and ESBL genes (37.3−74.5%) were the most detected, with approximately 80% of K. pneumoniae isolates presenting two or more resistance genes. As the optimal treatment of ß-lactamase-producing K. pneumoniae infections remains problematic, the high co-occurrence of multiple ß-lactam resistance genes must be seen as a serious warning of the problem of antimicrobial resistance.

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

Legionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella 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. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents' autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3-4 h) and accurate detection of Legionella.

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. NAMs such as peptide and locked nucleic acids (PNA and LNA) have provided enhanced sensitivity and specificity to the FISH technique, as well as higher flexibility in terms of applications. In this review, we aim to cover the state-of-the-art of the different NAMs and explore their possible applications in FISH, providing a general overview of the technique advancement in the last decades.

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. Prior knowledge of aptamer-target interactions is critical for applying post-SELEX modifications with unnatural nucleotides since it can affect aptamers' structure and performance. Here, we describe an easy-to-apply in silico workflow using free available software / web servers to predict the tertiary conformation of NAM, DNA and RNA aptamers, as well as the docking with the target molecule. Representative 2'-O-methyl (2'OMe), locked nucleic acid (LNA), DNA and RNA aptamers, with experimental data deposited in Protein Data Bank, were selected to validate the workflow. All aptamers' tertiary structure and docking models were successfully predicted with good structural similarity to the experimental data. Thus, this workflow will boost the development of aptamers, particularly NAM aptamers, by assisting in the rational modification of specific nucleotides and avoiding trial-and-error approaches.

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. We also discuss the limitations of existing methods for sequencing NAM sequences and bioinformatic tools to predict NAM aptamer structures. As a conclusion, we suggest that NAM aptamers might successfully compete with molecular tools based on proteins such as antibodies for future application.

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. Studies that showed ex vivo and in situ interactions between, at least, two microorganisms were found eligible. Ten papers were included. Layered or radially ordered multiple-taxon structures are the most common form of consortium. Strict or facultative anaerobic microorganisms are mostly found in the interior and the deepest portions of the structures, while aerobic microorganisms are mostly found on the periphery. We present a model of the microbial spatial organization in sub- and supragingival biofilms, as well as how the documented interactions can shape the biofilm formation. Despite the already acquired knowledge, available evidence regarding the structural composition and interactions of microorganisms within dental biofilms is incomplete and large-scale studies are needed.

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. Locked nucleic acids and 2'-OMethyl RNA were evaluated as model NAMs and formulated into DOTAP-DOPE liposomes followed by post-PEGylation. Our data showed a complexation stability between the post-PEGylated liposomes and the ONs of over 82%, during 24 h in native human serum, as determined by fluorescence correlation spectroscopy. Quantification by a lipid-mixing assay showed that liposomes, with and without post-PEGylation, fused with all bacteria tested. Such fusion promoted the delivery of a fraction of the ONs into the bacterial cytosol, as observed by fluorescence in situ hybridization and bacterial fractionation. In short, we demonstrated for the first time that liposomes can safely transport ONs in human serum and intracellularly deliver them in both Gram-negative and -positive bacteria, which holds promise towards the treatment of bloodstream infections.

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. Vitamin B12 is among the largest molecules known to be taken up by bacteria and has very recently started to gain interest as a trojan-horse vector. Gapmers and steric blockers were evaluated as ASOs against Escherichia coli (E. coli). Both ASOs were successfully conjugated to B12 by copper-free azide-alkyne click-chemistry. The biological effect of the two conjugates was evaluated together with their intracellular localization in E. coli. Although not only B12 but also both B12-ASO conjugates interacted strongly with E. coli, they were mostly colocalized with the outer membrane. Only 6-9% were detected in the cytosol, which showed to be insufficient for bacterial growth inhibition. These results suggest that the internalization of B12-ASO conjugates is strongly affected by the low uptake rate of the B12 in E. coli and that further studies are needed before considering this strategy against biofilms in vivo.

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

Aspergillus fumigatus 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 in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatussensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL-1-1 × 103 conidia·mL-1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL-1 in sputum; 1 × 103 conidia·mL-1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield "fungal ball" clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatussensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.

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. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory.

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. As a result of several discussions between international groups working in the area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm formation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set of recommendations. The intention of the minimum information guideline is to improve the quality of scientific communication that will augment interlaboratory reproducibility in biofilm microplate assays.

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. RESULTS: In this study, we designed LNA anti-miRNA oligonucleotides with a complementary sequence to miRNA-9 and tested their properties to both detect and silence the target miRNA. We could identify and visualize the in vitro uptake of low-dosing LNA-based anti-miRNA oligonucleotides without any carrier or transfection agent, as early as 2 h after the addition of the oligonucleotide sequence to the culture medium. Furthermore, we were able to assess the silencing potential of miRNA-9, using different LNA anti-miRNA oligonucleotide designs, and to observe its subsequent effect on E-cadherin expression. CONCLUSIONS: The administration of anti-miRNA sequences even at low-doses, rapidly repressed the target miRNA, and influenced the expression of E-cadherin by significantly increasing its levels.

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. Quantification methods, that were previously optimized and validated in planktonic consortia, were not always in agreement when applied in multispecies biofilms. Discrepancies in culture and molecular outcomes were observed, particularly for triple-species consortia and antibiotic-stressed biofilms. Some differences were observed, such as the higher bacterial counts obtained by q-PCR and/or PNA-FISH (≤4 log10 cells/cm2) compared to culture. But the discrepancies between PNA-FISH and q-PCR data (eg D. pigrum limited assessment by q-PCR) demonstrate the effect of biofilm heterogeneity in method's reliability. As the heterogeneity in biofilms is a reflection of a myriad of variables, tailoring an accurate picture of communities´ changes is crucial. This work demonstrates that at least two, but preferentially three, quantification techniques are required to obtain reliable measures and take comprehensive analysis of polymicrobial biofilm-associated infections.

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. New classes of chemical-modified-AMOs, including locked nucleic acid oligonucleotides, have recently proved their worth in silencing miRNAs. A correct design of a specific AMOs can help to improve their performance and potency towards the target miRNA by increasing for instance nuclease resistance and target affinity. This review outlines the technologies involved to suppress aberrant miRNAs. From the design strategies used in AMOs to its application in novel miRNA-based therapeutics and detection methodologies.

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. aeruginosa and/or D. pigrum populations dominating three-species consortia. Once exposed to antibiotics, biofilms displayed high resistance profiles, and microbial compositions, distributions, and microbial interactions significantly challenged. The antibiotic/oxygen environment supported such fluctuations, which enhanced for three-species communities. In conclusion, antibiotic therapy hugely disturbed CF communities' dynamics, inducing significant compositional changes on multispecies consortia. Clearly, multiple perturbations may disturb this dynamic, giving rise to various microbiological scenarios in vivo, and affecting disease phenotype. Therefore, an appreciation of the ecological/evolutionary nature within CF communities will be useful for the optimal use of current therapies and for newer breakthroughs on CF antibiotherapy.

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). After being tested on representative strains of P. gingivalis and A. actinomycetemcomitans, the PNA-FISH method was then adapted to detect microorganisms in the subgingival plaque and gingival samples, collected from patients with severe periodontitis. RESULTS: The best hybridization conditions were found to be 59°C for 150min for both probes (PgPNA1007 and AaPNA235). The in silico sensitivity and specificity was both 100% for PgPNA1007 probe and 100% and 99.9% for AaPNA235 probe, respectively. Results on clinical samples showed that the PNA-FISH method was able to detect and discriminate target bacteria in the mixed microbial population of the subgingival plaque and within periodontal tissues. CONCLUSION: This investigation presents a new highly accurate method for P. gingivalis and A. actinomycetemcomitans detection and co-location in clinical samples, in just few hours. With this technique we were able to observe spatial distribution of these species within polymicrobial communities in the periodontal pockets and, for the first time with the FISH method, in the organized gingival tissue.

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. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.