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.

Computational resources and strategies to assess single-molecule dynamics of the translation process in S. cerevisiae.

This work provides a systematic and comprehensive overview of available resources for the molecular-scale modelling of the translation process through agent-based modelling. The case study is the translation in Saccharomyces cerevisiae, one of the most studied yeasts. The data curation workflow encompassed structural information about the yeast (i.e. the simulation environment), and the proteins, ribonucleic acids and other types of molecules involved in the process (i.e. the agents). Moreover, it covers the main process events, such as diffusion (i.e. motion of molecules in the environment) and collision efficiency (i.e. interaction between molecules). Data previously determined by wet-lab techniques were preferred, resorting to computational predictions/extrapolations only when strictly necessary. The computational modelling of the translation processes is of added industrial interest, since it may bring forward knowledge on how to control such phenomena and enhance the production of proteins of interest in a faster and more efficient manner.

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.

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.

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).

A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes (MPs) move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different MPs to diffuse across multiple cellular barriers, as well as the reaction time associated with the formation of the duplex molecular probe-RNA, were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 7.94 × 102 - 4.39 × 103 s), whereas for animal cells, the limiting step should be the diffusion of the probe through the bulk (1.8-5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis for a more rational development of FISH protocols in the future.

Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes.

Listeria monocytogenes is one of the most important foodborne pathogens due to the high hospitalization and mortality rates associated to an outbreak. Several new molecular methods that accelerate the identification of L. monocytogenes have been developed, however conventional culture-based methods still remain the gold standard. In this work we developed a novel Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH) method for the specific detection of L. monocytogenes. The method was based on an already existing PNA probe, LmPNA1253, coupled with a novel blocker probe in a 1:2 ratio. The method was optimized for the detection of L. monocytogenes in food samples through an evaluation of several rich and selective enrichment broths. The best outcome was achieved using One Broth Listeria in a two-step enrichment of 24 h plus 18 h. For validation in food samples, ground beef, ground pork, milk, lettuce and cooked shrimp were artificially contaminated with two ranges of inoculum: a low level (0.2-2 CFU/25 g or mL) and a high level (2-10 CFU/25 g or mL). The PNA-FISH method performed well in all types of food matrices, presenting an overall accuracy of ≈99% and a detection limit of 0.5 CFU/25 g or mL of food sample.

Computational resources and strategies to construct single-molecule metabolic models of microbial cells.

Recent computational methodologies, such as individual-based modelling, pave the way to the search for explanatory insight into the collective behaviour of molecules. Many reviews offer an up-to-date perspective about such methodologies, but little is discussed about the practical information requirements involved. The biological information used as input should be easily and routinely determined in the laboratory, publicly available and, preferably, organized in programmatically accessible databases. This review is the first to provide a systematic and comprehensive overview of available resources for the modelling of metabolic events at the molecular scale. The glycolysis pathway of Escherichia coli, which is one of the most studied pathways in Microbiology, serves as case study. This curation addressed structural information about E. coli (i.e. defining the simulation environment), the reactions forming the glycolysis pathway including the enzymes and the metabolites (i.e. the molecules to be represented), the kinetics of each reaction (i.e. behavioural logic of the molecules) and diffusion parameters for all enzymes and metabolites (i.e. molecule movement in the environment). Furthermore, the interpretation of relevant biological features, such as molecular diffusion and enzyme kinetics, and the connection of experimental determination and simulation validation are detailed. Notably, the information from classical theories, such as enzymatic rates and diffusion coefficients, is translated to simulation parameters, such as collision efficiency and particle velocity.

Agent-based model of diffusion of N-acyl homoserine lactones in a multicellular environment of Pseudomonas aeruginosa and Candida albicans.

Experimental incapacity to track microbe-microbe interactions in structures like biofilms, and the complexity inherent to the mathematical modelling of those interactions, raises the need for feasible, alternative modelling approaches. This work proposes an agent-based representation of the diffusion of N-acyl homoserine lactones (AHL) in a multicellular environment formed by Pseudomonas aeruginosa and Candida albicans. Depending on the spatial location, C. albicans cells were variably exposed to AHLs, an observation that might help explain why phenotypic switching of individual cells in biofilms occurred at different time points. The simulation and algebraic results were similar for simpler scenarios, although some statistical differences could be observed (p < 0.05). The model was also successfully applied to a more complex scenario representing a small multicellular environment containing C. albicans and P. aeruginosa cells encased in a 3-D matrix. Further development of this model may help create a predictive tool to depict biofilm heterogeneity at the single-cell level.

Impact of polymicrobial biofilms in catheter-associated urinary tract infections.

Recent reports have demonstrated that most biofilms involved in catheter-associated urinary tract infections are polymicrobial communities, with pathogenic microorganisms (e.g. Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) and uncommon microorganisms (e.g. Delftia tsuruhatensis, Achromobacter xylosoxidans) frequently co-inhabiting the same urinary catheter. However, little is known about the interactions that occur between different microorganisms and how they impact biofilm formation and infection outcome. This lack of knowledge affects CAUTIs management as uncommon bacteria action can, for instance, influence the rate at which pathogens adhere and grow, as well as affect the overall biofilm resistance to antibiotics. Another relevant aspect is the understanding of factors that drive a single pathogenic bacterium to become prevalent in a polymicrobial community and subsequently cause infection. In this review, a general overview about the IMDs-associated biofilm infections is provided, with an emphasis on the pathophysiology and the microbiome composition of CAUTIs. Based on the available literature, it is clear that more research about the microbiome interaction, mechanisms of biofilm formation and of antimicrobial tolerance of the polymicrobial consortium are required to better understand and treat these infections.

Critical review on biofilm methods.

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH).

Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) is a highly specific molecular method widely used for microbial identification. Nonetheless, and due to the detection limit of this technique, a time-consuming pre-enrichment step is typically required before identification. In here we have developed a lab-on-a-chip device to concentrate cell suspensions and speed up the identification process in yeasts. The PNA-FISH protocol was optimized to target Saccharomyces cerevisiae, a common yeast that is very relevant for several types of food industries. Then, several coin-sized microfluidic devices with different geometries were developed. Using Computational fluid dynamics (CFD), we modeled the hydrodynamics inside the microchannels and selected the most promising options. SU-8 structures were fabricated based on the selected designs and used to produce polydimethylsiloxane-based microchips by soft lithography. As a result, an integrated approach combining microfluidics and PNA-FISH for the rapid identification of S. cerevisiae was achieved. To improve fluid flow inside microchannels and the PNA-FISH labeling, oxygen plasma treatment was applied to the microfluidic devices and a new methodology to introduce the cell suspension and solutions into the microchannels was devised. A strong PNA-FISH signal was observed in cells trapped inside the microchannels, proving that the proposed methodology works as intended. The microfluidic designs and PNA-FISH procedure described in here should be easily adaptable for detection of other microorganisms of similar size.

Discriminating typical and atypical cystic fibrosis-related bacteria by multiplex PNA-FISH.

This study aims to report the development of peptide nucleic acid (PNA) probes to specifically detect the cystic fibrosis (CF)-associated traditional and atypical species Pseudomonas aeruginosa and Inquilinus limosus, respectively. PNA probes were designed in silico, developed and tested in smears prepared in phosphate-buffer saline (PBS), and in artificial sputum medium (ASM). A multiplex fluorescent in situ hybridization (FISH) approach using the designed probes was further validated in artificially contaminated clinical sputum samples and also applied in polymicrobial 24 h-old biofilms involving P. aeruginosa, I. limosus, and other CF-related bacteria. Both probes showed high predictive and experimental specificities and sensitivities. The multiplex PNA-FISH assay, associated with non-specific staining, was successfully adapted in the clinical samples and in biofilms of CF-related bacteria, allowing differentiating the community members and inferring about microbial-microbial interactions within the consortia. This study revealed the great potential of PNA-FISH as a diagnostic tool to discriminate between classical and less common CF-associated bacteria, being suitable to further describe species-dependent prevention strategies and deliver more effective target control therapeutics. Biotechnol. Bioeng. 2017;114: 355-367. © 2016 Wiley Periodicals, Inc.

The cystic fibrosis microbiome in an ecological perspective and its impact in antibiotic therapy.

The recent focus on the cystic fibrosis (CF) complex microbiome has led to the recognition that the microbes can interact between them and with the host immune system, affecting the disease progression and treatment routes. Although the main focus remains on the interactions between traditional pathogens, growing evidence supports the contribution and the role of emergent species. Understanding the mechanisms and the biological effects involved in polymicrobial interactions may be the key to improve effective therapies and also to define new strategies for disease control. This review focuses on the interactions between microbe-microbe and host-microbe, from an ecological point of view, discussing their impact on CF disease progression. There are increasing indications that these interactions impact the success of antimicrobial therapy. Consequently, a new approach where therapy is personalized to patients by taking into account their individual CF microbiome is suggested.

Impact of Delftia tsuruhatensis and Achromobacter xylosoxidans on Escherichia coli dual-species biofilms treated with antibiotic agents.

Recently it was demonstrated that for urinary tract infections species with a lower or unproven pathogenic potential, such as Delftia tsuruhatensis and Achromobacter xylosoxidans, might interact with conventional pathogenic agents such as Escherichia coli. Here, single- and dual-species biofilms of these microorganisms were characterized in terms of microbial composition over time, the average fitness of E. coli, the spatial organization and the biofilm antimicrobial profile. The results revealed a positive impact of these species on the fitness of E. coli and a greater tolerance to the antibiotic agents. In dual-species biofilms exposed to antibiotics, E. coli was able to dominate the microbial consortia in spite of being the most sensitive strain. This is the first study demonstrating the protective effect of less common species over E. coli under adverse conditions imposed by the use of antibiotic agents.

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH).

Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.

Microbiome in cystic fibrosis: Shaping polymicrobial interactions for advances in antibiotic therapy.

Recent molecular methodologies have demonstrated a complex microbial ecosystem in cystic fibrosis (CF) airways, with a wide array of uncommon microorganisms co-existing with the traditional pathogens. Although there are lines of evidence supporting the contribution of some of those emergent species for lung disease chronicity, clinical significance remains uncertain for most cases. A possible contribution for disease is likely to be related with the dynamic interactions established between microorganisms within the microbial community and with the host. If this is the case, management of CF will only be successful upon suitable and exhaustive modulation of such mixed ecological processes, which will also be useful to predict the effects of new therapeutic interventions.

Interaction between atypical microorganisms and E. coli in catheter-associated urinary tract biofilms.

Most biofilms involved in catheter-associated urinary tract infections (CAUTIs) are polymicrobial, with disease causing (eg Escherichia coli) and atypical microorganisms (eg Delftia tsuruhatensis) frequently inhabiting the same catheter. Nevertheless, there is a lack of knowledge about the role of atypical microorganisms. Here, single and dual-species biofilms consisting of E. coli and atypical bacteria (D. tsuruhatensis and Achromobacter xylosoxidans), were evaluated. All species were good biofilm producers (Log 5.84-7.25 CFU cm(-2) at 192 h) in artificial urine. The ability of atypical species to form a biofilm appeared to be hampered by the presence of E. coli. Additionally, when E. coli was added to a pre-formed biofilm of the atypical species, it seemed to take advantage of the first colonizers to accelerate adhesion, even when added at lower concentrations. The results suggest a greater ability of E. coli to form biofilms in conditions mimicking the CAUTIs, whatever the pre-existing microbiota and the inoculum concentration.

Water-induced modulation of Helicobacter pylori virulence properties.

While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium's ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.