The potential of tailed amplicons for SARS-CoV-2 detection in Nucleic Acid Lateral Flow Assays.

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/µL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings.

Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle.

There has been considerable discussion regarding the environmental life cycle of Legionella pneumophila and its virulence potential in natural and man-made water systems. On the other hand, the bacterium's morphogenetic mechanisms within host cells (amoeba and macrophages) have been well documented and are linked to its ability to transition from a non-virulent, replicative state to an infectious, transmissive state. Although the morphogenetic mechanisms associated with the formation and detachment of the L. pneumophila biofilm have also been described, the capacity of the bacteria to multiply extracellularly is not generally accepted. However, several studies have shown genetic pathways within the biofilm that resemble intracellular mechanisms. Understanding the functionality of L. pneumophila cells within a biofilm is fundamental for assessing the ecology and evaluating how the biofilm architecture influences L. pneumophila survival and persistence in water systems. This manuscript provides an overview of the biphasic cycle of L. pneumophila and its implications in associated intracellular mechanisms in amoeba. It also examines the molecular pathways and gene regulation involved in L. pneumophila biofilm formation and dissemination. A holistic analysis of the transcriptional activities in L. pneumophila biofilms is provided, combining the information of intracellular mechanisms in a comprehensive outline. Furthermore, this review discusses the techniques that can be used to study the morphogenetic states of the bacteria within biofilms, at the single cell and population levels.

A New Peptide Nucleic Acid Fluorescence In Situ Hybridization Probe for the Specific Detection of Salmonella Species in Food Matrices.

Salmonella spp. is among the most central etiological agents in foodborne bacterial disorders. To identify Salmonella spp., numerous new molecular techniques have been developed conversely to the traditional culture-based methods. In this work, a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for the specific detection of Salmonella species, allowing a faster analysis compared with the traditional methods (ISO 6579-1: 2017). The method was optimized based on a novel PNA probe (SalPNA1692) combined with a blocker probe to detect Salmonella in food samples through an assessment of diverse-rich and selective enrichment broths. Our findings indicated that the best outcome was obtained using a 24-h pre-enrichment step in buffered peptone water, followed by RambaQuick broth selective enrichment for 16 h. For the enrichment step performance validation, fresh ground beef was artificially contaminated with two ranges of concentration of inoculum: a low level (0.2-2 colony-forming units [CFUs]/25 g) and a high level (2-10 CFUs/25 g). The new PNA-FISH method presented a specificity of 100% and a detection limit of 0.5 CFU/25 g of food sample, which confirms the great potential of applying PNA probes in food analysis.

Promising strategies employing nucleic acids as antimicrobial drugs.

Antimicrobial resistance (AMR) is a growing concern because it causes microorganisms to develop resistance to drugs commonly used to treat infections. This results in increased difficulty in treating infections, leading to higher mortality rates and significant economic effects. Investing in new antimicrobial agents is, therefore, necessary to prevent and control AMR. Antimicrobial nucleic acids have arisen as potential key players in novel therapies for AMR infections. They have been designed to serve as antimicrobials and to act as adjuvants to conventional antibiotics or to inhibit virulent mechanisms. This new category of antimicrobial drugs consists of antisense oligonucleotides and oligomers, DNAzymes, and transcription factor decoys, differing in terms of structure, target molecules, and mechanisms of action. They are synthesized using nucleic acid analogs to enhance their resistance to nucleases. Because bacterial envelopes are generally impermeable to oligonucleotides, delivery into the cytoplasm typically requires the assistance of nanocarriers, which can affect their therapeutic potency. Given that numerous factors contribute to the success of these antimicrobial drugs, this review aims to provide a summary of the key advancements in the use of oligonucleotides for treating bacterial infections. Their mechanisms of action and the impact of factors such as nucleic acid design, target sequence, and nanocarriers on the antimicrobial potency are discussed.

The Role of Flagellum and Flagellum-Based Motility on Salmonella Enteritidis and Escherichia coli Biofilm Formation.

Flagellum-mediated motility has been suggested to contribute to virulence by allowing bacteria to colonize and spread to new surfaces. In Salmonella enterica and Escherichia coli species, mutants affected by their flagellar motility have shown a reduced ability to form biofilms. While it is known that some species might act as co-aggregation factors for bacterial adhesion, studies of food-related biofilms have been limited to single-species biofilms and short biofilm formation periods. To assess the contribution of flagella and flagellum-based motility to adhesion and biofilm formation, two Salmonella and E. coli mutants with different flagellar phenotypes were produced: the fliC mutants, which do not produce flagella, and the motAB mutants, which are non-motile. The ability of wild-type and mutant strains to form biofilms was compared, and their relative fitness was determined in two-species biofilms with other foodborne pathogens. Our results showed a defective and significant behavior of E. coli in initial surface colonization (p < 0.05), which delayed single-species biofilm formation. Salmonella mutants were not affected by the ability to form biofilm (p > 0.05). Regarding the effect of motility/flagellum absence on bacterial fitness, none of the mutant strains seems to have their relative fitness affected in the presence of a competing species. Although the absence of motility may eventually delay initial colonization, this study suggests that motility is not essential for biofilm formation and does not have a strong impact on bacteria's fitness when a competing species is present.

Imaging biofilms using fluorescence in situ hybridization: seeing is believing.

Biofilms are complex structures with an intricate relationship between the resident microorganisms, the extracellular matrix, and the surrounding environment. Interest in biofilms is growing exponentially given its ubiquity in so diverse fields such as healthcare, environmental and industry. Molecular techniques (e.g., next-generation sequencing, RNA-seq) have been used to study biofilm properties. However, these techniques disrupt the spatial structure of biofilms; therefore, they do not allow to observe the location/position of biofilm components (e.g., cells, genes, metabolites), which is particularly relevant to explore and study the interactions and functions of microorganisms. Fluorescence in situ hybridization (FISH) has been arguably the most widely used method for an in situ analysis of spatial distribution of biofilms. In this review, an overview on different FISH variants already applied on biofilm studies (e.g., CLASI-FISH, BONCAT-FISH, HiPR-FISH, seq-FISH) will be explored. In combination with confocal laser scanning microscopy, these variants emerged as a powerful approach to visualize, quantify and locate microorganisms, genes, and metabolites inside biofilms. Finally, we discuss new possible research directions for the development of robust and accurate FISH-based approaches that will allow to dig deeper into the biofilm structure and function.

Fighting Methicillin-Resistant Staphylococcus aureus with Targeted Nanoparticles.

Antimicrobial resistance (AMR) is considered one of the greatest threats to global health. Methicillin-resistant Staphylococcus aureus (MRSA) remains at the core of this threat, accounting for about 90% of S. aureus infections widespread in the community and hospital settings. In recent years, the use of nanoparticles (NPs) has emerged as a promising strategy to treat MRSA infections. NPs can act directly as antibacterial agents via antibiotic-independent activity and/or serve as drug delivery systems (DDSs), releasing loaded antibiotics. Nonetheless, directing NPs to the infection site is fundamental for effective MRSA treatment so that highly concentrated therapeutic agents are delivered to the infection site while directly reducing the toxicity to healthy human cells. This leads to decreased AMR emergence and less disturbance of the individual's healthy microbiota. Hence, this review compiles and discusses the scientific evidence related to targeted NPs developed for MRSA treatment.

Engineered liposomes to deliver nucleic acid mimics in Escherichia coli.

Antisense oligonucleotides (ASOs) composed of nucleic acid mimics (NAMs) monomers are considered as potential novel therapeutic drugs against bacterial infections. However, bacterial envelopes are generally impermeable to naked oligonucleotides. Herein, liposomes loaded with NAMs-modified oligonucleotides (LipoNAMs) were evaluated to deliver ASOs in Escherichia coli. Specifically, we tested several surface modifications that included methoxyPEG conjugated to different lipid anchors or modification of the PEG distal ends with maleimide groups and antibodies. MethoxyPEG coated LipoNAMs showed low delivery efficiency for most bacteria, but maleimide-functionalized PEG LipoNAMs were able to deliver ASOs to nearly half of the bacterial population. Conjugation of antibodies to maleimide-functionalized PEG LipoNAMs increased 1.3-fold the delivery efficiency, enhancing the selectivity towards E. coli and biocompatibility. This work demonstrated for the first time that the coupling of antibodies to PEGylated liposomes can significantly improve the delivery of ASOs in E. coli, which might bring alternative routes for the treatment of bacterial infections in the future.

Advances on diagnosis of Helicobacter pylori infections.

The association of Helicobacter pylori to several gastric diseases, such as chronic gastritis, peptic ulcer disease, and gastric cancer, and its high prevalence worldwide, raised the necessity to use methods for a proper and fast diagnosis and monitoring the pathogen eradication. Available diagnostic methods can be classified as invasive or non-invasive, and the selection of the best relies on the clinical condition of the patient, as well as on the sensitivity, specificity, and accessibility of the diagnostic test. This review summarises all diagnostic methods currently available, including the invasive methods: endoscopy, histology, culture, and molecular methods, and the rapid urease test (RUT), as well as the non-invasive methods urea breath test (UBT), serological assays, biosensors, and microfluidic devices and the stool antigen test (SAT). Moreover, it lists the diagnostic advantages and limitations, as well as the main advances for each methodology. In the end, research on the development of new diagnostic methods, such as bacteriophage-based H. pylori diagnostic tools, is also discussed.

Interactions of microorganisms within a urinary catheter polymicrobial biofilm model.

Biofilms are often polymicrobial in nature, which can impact their behavior and overall structure, often resulting in an increase in biomass and enhanced antimicrobial resistance. Using plate counts and locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH), we studied the interactions of four species commonly associated with catheter-associated urinary tract infections (CAUTI): Enterococcus faecalis, Escherichia coli, Candida albicans, and Proteus mirabilis. Eleven combinations of biofilms were grown on silicone coupons placed in 24-well plates for 24 h, 37°C, in artificial urine medium (AUM). Results showed that P. mirabilis was the dominant species and was able to inhibit both E. coli and C. albicans growth. In the absence of P. mirabilis, an antagonistic relationship between E. coli and C. albicans was observed, with the former being dominant. E. faecalis growth was not affected in any combination, showing a more mutualistic relationship with the other species. Imaging results correlated with the plate count data and provided visual verification of species undetected using the viable plate count. Moreover, the three bacterial species showed overall good repeatability SD (Sr ) values (0.1-0.54) in all combinations tested, whereas C. albicans had higher repeatability Sr values (0.36-1.18). The study showed the complexity of early-stage interactions in polymicrobial biofilms. These interactions could serve as a starting point when considering targets for preventing or treating CAUTI biofilms containing these species.

Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens.

The application of nucleic acid mimics (NAMs), such as locked nucleic acid (LNA) and 2'-O-methyl-RNA (2'OMe), has improved the performance of fluorescence in situ hybridization (FISH) methods for the detection/location of clinical pathogens since they provide design versatility and thermodynamic control. However, an important limitation of FISH techniques is the low number of distinguishable targets. The use of filters in fluorescence image acquisition limits the number of fluorochromes that can be simultaneously differentiated. Recent advances in fluorescence spectral image acquisition have allowed the unambiguous identification of several microorganisms in a single sample. In this work, we aimed to combine NAM-FISH and spectral image analysis to develop and validate a new FISH variant, the spectral imaging-NAM-FISH (SI-NAM-FISH), that allows a multiplexed, robust and rapid detection of clinical pathogens. In the first stage, to implement/validate the method, we have selected seven fluorochromes with distinct spectral properties and seven bacterial species (Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter calcoaceticus). As a strong variation in fluorescence intensities is found between species and between fluorochromes, seven versions of a EUB LNA/2'OMe probe, each conjugated to one of seven fluorochromes, were used to rank species/fluorochromes by FISH and then optimize species/fluorochrome pairing. Then, final validation tests were performed using mixed populations to evaluate the potential of the technique for separating/quantifying the different targets. Overall, validation tests with different proportions of bacteria labeled with the respective fluorochrome have shown the ability of the method to correctly distinguish the species.

Microfluidics combined with fluorescence in situ hybridization (FISH) for Candida spp. detection.

One of the most prevalent healthcare-associated infection is the urinary tract infection (UTI), caused by opportunistic pathogens such as Candida albicans or non-albicans Candida species (NACS). Urine culture methods are routinely used for UTI diagnostics due to their specificity, sensitivity and low-cost. However, these methods are also laborious, time- and reagent-consuming. Therefore, diagnostic methods relying on nucleic acids have been suggested as alternatives. Nucleic acid-based methods can provide results within 24 h and can be adapted to point-of-care (POC) detection. Here, we propose to combine fluorescence in situ hybridization (FISH) with a microfluidic platform for the detection of Candida spp. As a case study we used C. tropicalis, which is reported as the second most common NACS urine isolate obtained from patients suspected with UTI. The microfluidic platform proposed in this study relies on hydrodynamic trapping, and uses physical barriers (e.g., microposts) for the separation of target cells from the suspension. Using a specific peptide nucleic acid (PNA) probe, the FISH procedure was applied onto previously trapped C. tropicalis cells present inside the microfluidic platform. Fluorescence signal intensity of hybridized cells was captured directly under the epifluorescence microscope. Overall, the PNA probe successfully detected C. tropicalis in pure culture and artificial urine (AU) using FISH combined with the microfluidic platform. Our findings reveal that FISH using nucleic acid mimics (PNA) in combination with microfluidics is a reliable method for the detection of microorganisms such as C. tropicalis. As such, this work provides the basis for the development of a POC detection platform in the future.

Liposome Delivery of Nucleic Acids in Bacteria: Toward In Vivo Labeling of Human Microbiota.

Development of specific probes to study the in vivo spatial distribution of microorganisms is essential to understand the ecology of human microbiota. Herein, we assess the possibility of using liposomes loaded with fluorescently labeled nucleic acid mimics (LipoNAMs) to image Gram-negative and Gram-positive bacteria. We proved that liposome fusion efficiencies were similar in both Gram-negative and Gram-positive bacteria but that the efficiency was highly dependent on the lipid concentration. Notably, LipoNAMs were significantly more effective for the internalization of oligonucleotides in bacteria than the fixation/permeabilization methods commonly used in vitro. Furthermore, a structural and morphological assessment of the changes on bacteria allowed us to observe that liposomes increased the permeability of the cell envelope especially in Gram-negative bacteria. Considering the delivery efficiency and permeabilization effect, lipid concentrations of approximately 5 mM should be selected to maximize the detection of bacteria without compromising the bacterial cellular structure.

Prevalence and Diversity of Staphylococcus aureus and Staphylococcal Enterotoxins in Raw Milk From Northern Portugal.

Staphylococcus aureus and staphylococcal enterotoxins are a serious public health concern associated with hospital and community-acquired illnesses. Dairy animals frequently shed S. aureus into the milk supply which can lead to food poisoning in humans. This study aims to investigate the prevalence and genetic diversity of S. aureus and staphylococcal enterotoxins in raw milk from the main dairy region of mainland Portugal. S. aureus was found in 53.0% (95% CI: 40.6-65.4%) of 100 raw cow's milk samples collected from bulk cooling tanks. The highest contamination level was 3.4 log10 CFU.mL-1, and in some samples more than one S. aureus strain was identified. Staphylococcal enterotoxins (SEA-SEE) were detected in one sample. Spa typing revealed 62 distinct S. aureus isolates, being t529 (17.7%, 95% CI: 8.2-27.3%) and t1403 (16.1%, 95% CI: 7.0-25.3%) the predominant types, commonly associated with livestock infection or carriage. The antimicrobial susceptibility test showed that 35.5% of the S. aureus isolates were resistant to at least one antimicrobial agent, with resistance to penicillin being the highest (32.3%, 95% CI: 20.6-43.9%) followed by tetracycline (24.2%, 95% CI: 13.5-34.9%), ciprofloxacin (16.1%, 95% CI: 7.0-25.3%) and chloramphenicol (16.1%, 95% CI: 7.0-25.3%). Moreover, five isolates (8.1%, 95% CI: 1.3-14.8%) were identified as methicillin-resistant S. aureus (MRSA, cefoxitin resistant). Regarding virulence/resistance genes, 46,8% (95% CI: 34.4-59.2%) isolates harbored at least one enterotoxin-encoding gene, and the seg gene was the most frequently detected (41.9%, 95% CI: 29.7-54.2%) followed by the sei (40.3%, 95% CI: 28.1-52.5%), sec (6.5%, 95% CI: 0.3-12.6%), seh (4.8%, 95% CI: 0.0-10.2%), and sea (1.6%, 95% CI: 0.0-4.7%) genes. Five (8.1%, 95% CI: 1.3-14.8%) non-enterotoxigenic isolates carried the mecA gene (corresponding to isolates phenotypically classified as MRSA), and 4.8% (95% CI: 0.0-10.2%) enterotoxigenic strains also had the tsst-1 gene. Our study confirm that raw milk can be a zoonotic source of S. aureus, including enterotoxigenic and MRSA strains. Furthermore, the majority of enterotoxigenic isolates were found to contain genes encoding SEs (SEG, SEH and SEI) not routinely screened. This shows the need for a broader SE screening in food safety control, as well as the relevance of risk mitigation measures to control S. aureus transmission along the food chain in Portugal.

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond.

COVID-19 pandemic ignited the development of countless molecular methods for the diagnosis of SARS-CoV-2 based either on nucleic acid, or protein analysis, with the first establishing as the most used for routine diagnosis. The methods trusted for day to day analysis of nucleic acids rely on amplification, in order to enable specific SARS-CoV-2 RNA detection. This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics, while looking carefully at the concerning virology aspects, steps and instruments a test can involve. Following a theme contextualization in introduction, topics about fundamental knowledge on underlying virology aspects, collection and processing of clinical samples pave the way for a detailed assessment of the amplification and detection technologies. In order to address such themes, nucleic acid amplification methods, the different types of molecular reactions used for DNA detection, as well as the instruments requested for executing such routes of analysis are discussed in the subsequent sections. The benchmark of paradigmatic commercial tests further contributes toward discussion, building on technical aspects addressed in the previous sections and other additional information supplied in that part. The last lines are reserved for looking ahead to the future of NAATs and its importance in tackling this pandemic and other identical upcoming challenges.

Helicobacter pylori infection: from standard to alternative treatment strategies.

Helicobacter pylori is the major component of the gastric microbiome of infected individuals and one of the aetiological factors of chronic gastritis, peptic ulcer disease and gastric cancer. The increasing resistance to antibiotics worldwide has made the treatment of H. pylori infection a challenge. As a way to overhaul the efficacy of currently used H. pylori antibiotic-based eradication therapies, alternative treatment strategies are being devised. These include probiotics and prebiotics as adjuvants in H. pylori treatment, antimicrobial peptides as alternatives to antibiotics, photodynamic therapy ingestible devices, microparticles and nanoparticles applied as drug delivery systems, vaccines, natural products, and phage therapy. This review provides an updated synopsis of these emerging H. pylori control strategies and discusses the advantages, hurdles, and challenges associated with their development and implementation. An effective human vaccine would be a major achievement although, until now, projects regarding vaccine development have failed or were discontinued. Numerous natural products have demonstrated anti-H. pylori activity, mostly in vitro, but further clinical studies are needed to fully disclose their role in H. pylori eradication. Finally, phage therapy has the potential to emerge as a valid alternative, but major challenges remain, namely the isolation of more H. pylori strictly virulent bacterio(phages).

Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods.

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.

Biofilms vs. cities and humans vs. aliens - a tale of reproducibility in biofilms.

Biofilms are complex and dynamic structures that include many more components than just viable cells. Therefore, the apparently simple goal of growing reproducible biofilms is often elusive. One of the challenges in defining reproducibility for biofilm research is that different research fields use a spectrum of parameters to define reproducibility for their particular application. For instance, is the researcher interested in achieving a similar population density, height of biofilm structures, or function of the biofilm in a certain ecosystem/industrial context? Within this article we categorize reproducibility into four different levels: level 1, no reproducibility; level 2, standard reproducibility; level 3, potential standard reproducibility; and level 4, total reproducibility. To better understand the need for these different levels of reproducibility, we expand on the 'cities of microbes' analogy for biofilms by imagining that a new civilization has reached the Earth's outskirts and starts studying the Earth's cities. This will provide a better sense of scale and illustrate how small details can impact profoundly on the growth and behavior of a biofilm and our understanding of reproducibility.

New Insights on Biofilm Antimicrobial Strategies.

Over the last few decades, the study of microbial biofilms has been gaining interest among the scientific community [...].

An Introduction to Fluorescence in situ Hybridization in Microorganisms.

Fluorescence in situ hybridization (FISH) is a molecular biology technique that enables the localization, quantification, and identification of microorganisms in a sample. This technique has found applications in several areas, most notably the environmental, for quantification and diversity assessment of microorganisms and, the clinical, for the rapid diagnostics of infectious agents. The FISH method is based on the hybridization of a fluorescently labeled nucleic acid probe with a complementary sequence that is present inside the microbial cell, typically in the form of ribosomal RNA (rRNA). In fact, an hybridized cell is typically only detectable because a large number of multiple fluorescent particles (as many as the number of target sequences available) are present inside the cell. Here, we will review the major steps involved in a standard FISH protocol, namely, fixation/permeabilization, hybridization, washing, and visualization/detection. For each step, the major variables/parameters are identified and, subsequently, their impact on the overall hybridization performance is assessed in detail.