Use of FISH-FLOW as a Method for the Identification and Quantification of Bacterial Populations.

The gastrointestinal tract (GIT) harbors the largest group of microbiotas among the microbial communities of the human host. The resident organisms typical of a healthy gut are well adapted to the gastrointestinal environment while alteration of these populations can trigger disorders that may affect the health and well-being of the host. Various investigations have applied different tools to study bacterial communities in the gut and their correlation with gastrointestinal disorders such as inflammatory bowel disease (IBD), obesity, and diabetes. This study proposes fluorescent in situ hybridization, combined with flow cytometry (FISH-FLOW), as an alternative approach for phylum level identification of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria and quantification of target bacteria from the GIT based on analysis of fecal samples, where results are validated by quantitative polymerase chain reaction (qPCR) and 16S ribosomal ribonucleic acid (16s rRNA) sequencing. The results obtained via FISH-FLOW experimental approach show high specificity for the developed probes for hybridization with the target bacteria. The study, therefore, suggests the FISH-FLOW as a reliable method for studying bacterial communities in the gut with results correlating well with those of metagenomic investigations of the same fecal samples.

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.

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.

New Insights on Biofilm Antimicrobial Strategies, 2nd Volume.

In biofilms, microorganisms are able to communicate together and assemble by themselves, creating a consortium with different properties from the original free-floating microorganisms [...].

Antimicrobial and Antibiofilm Coating of Dental Implants-Past and New Perspectives.

Regarded as one of the best solutions to replace missing teeth in the oral cavity, dental implants have been the focus of plenty of studies and research in the past few years. Antimicrobial coatings are a promising solution to control and prevent bacterial infections that compromise the success of dental implants. In the last few years, new materials that prevent biofilm adhesion to the surface of titanium implants have been reported, ranging from improved methods to already established coating surfaces. The purpose of this review is to present the developed antimicrobial and antibiofilm coatings that may have the potential to reduce bacterial infections and improve the success rate of titanium dental implants. All referred coating surfaces showed high antimicrobial properties with effectiveness in biofilm control, while maintaining implant biocompatibility. We expect that by combining the use of oligonucleotide probes as a covering material with novel peri-implant adjuvant therapies, we will be able to avoid the downsides of other covering materials (such as antibiotic resistance), prevent bacterial infections, and raise the success rate of dental implants. The existing knowledge on the optimal coating material for dental implants is limited, and further research is needed before more definitive conclusions can be drawn.

Bioinformatic Tools and Guidelines for the Design of Fluorescence In Situ Hybridization Probes.

Fluorescence in situ hybridization (FISH) is a well-established technique that allows the detection of microorganisms in diverse types of samples (e.g., clinical, food, environmental samples, and biofilm communities). The FISH probe design is an essential step in this technique. For this, two strategies can be used, the manual form based on multiple sequence alignment to identify conserved regions and programs/software specifically developed for the selection of the sequence of the probe. Additionally, databases/software for the theoretical evaluation of the probes in terms of specificity, sensitivity, and thermodynamic parameters (melting temperature and Gibbs free energy change) are used. The purpose of this chapter is to describe the essential steps and guidelines for the design of FISH probes (e.g., DNA and Nucleic Acid Mimic (NAM) probes), and its theoretical evaluation through the application of diverse bioinformatic tools.

Application of Nucleic Acid Mimics in Fluorescence In Situ Hybridization.

Traditionally, RNA and DNA probes are used in fluorescence in situ hybridization (FISH) methods for microbial detection and characterization of communities' structure and diversity. However, the recent introduction of nucleic acid mimics (NAMs) has improved the robustness of the FISH methods in terms of sensitivity and specificity. Several NAMs have been used, of which the most relevant are peptide nucleic acid (PNA), locked nucleic acids (LNA), 2'-O-methyl RNA (2'OMe), and phosphorothioates (PS). In this chapter, we describe a protocol using PNA and LNA/2'OMe probes for microbial detection by FISH, pointing out the differences between them. These protocols are easily adapted to different microorganisms and different probe sequences.

Characterization of Social Interactions and Spatial Arrangement of Individual Bacteria in MultiStrain or Multispecies Biofilm Systems Using Nucleic Acid Mimics-Fluorescence In Situ Hybridization.

Biofilms are often composed of different bacterial and fungal species/strains, which form complex structures based on social interactions with each other. Fluorescence in situ hybridization (FISH) can help us identify the different species/strains present within a biofilm , and when coupled with confocal scanning laser microscopy (CSLM), it enables the visualization of the three-dimensional (3D) structure of the biofilm and the spatial arrangement of each individual species/strain within it. In this chapter, we describe the protocol for characterizing multistrain or multispecies biofilm formation using NAM-FISH and CSLM.

Flow-FISH Using Nucleic Acid Mimic Probes for the Detection of Bacteria.

Flow-Fluorescence in situ hybridization (Flow-FISH) enables multiparametric high-throughput detection of target nucleic acid sequences at the single cell-level, allowing an accurate quantification of different cell populations by using a combination of flow cytometry and fluorescent in situ hybridization (FISH). In this chapter, a flow-FISH protocol is described with labeled nucleic acid mimics (NAMs) (e.g. LNA/2'OMe and PNA) acting as the reporter molecules. This protocol allows for the specific detection of bacterial cells. Hence, this protocol can be carried out with minor adjustments, in order to simultaneously detect different species of bacteria in different types of clinical, food, or environmental samples.

Increased Intraspecies Diversity in Escherichia coli Biofilms Promotes Cellular Growth at the Expense of Matrix Production.

Intraspecies diversity in biofilm communities is associated with enhanced survival and growth of the individual biofilm populations. Studies on the subject are scarce, namely, when more than three strains are present. Hence, in this study, the influence of intraspecies diversity in biofilm populations composed of up to six different Escherichia coli strains isolated from urine was evaluated in conditions mimicking the ones observed in urinary tract infections and catheter-associated urinary tract infections. In general, with the increasing number of strains in a biofilm, an increase in cell cultivability and a decrease in matrix production were observed. For instance, single-strain biofilms produced an average of 73.1 µg·cm-2 of extracellular polymeric substances (EPS), while six strains biofilms produced 19.9 µg·cm-2. Hence, it appears that increased genotypic diversity in a biofilm leads E. coli to direct energy towards the production of its offspring, in detriment of the production of public goods (i.e., matrix components). Apart from ecological implications, these results can be explored as another strategy to reduce the biofilm burden, as a decrease in EPS matrix production may render these intraspecies biofilms more sensitive to antimicrobial agents.

Optimizing locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH) procedure for bacterial detection.

Despite the successful application of LNA/2'OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2'OMe hybridization step-hybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria (Enterococcus faecalis and Staphylococcus epidermidis), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2'OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2'OMe and 64% of GC content, should be use in initial optimization of new LNA/2'OMe-FISH protocols.

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.

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.

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.

Minimum information about a biofilm experiment (MIABiE): standards for reporting experiments and data on sessile microbial communities living at interfaces.

The minimum information about a biofilm experiment (MIABiE) initiative has arisen from the need to find an adequate and scientifically sound way to control the quality of the documentation accompanying the public deposition of biofilm-related data, particularly those obtained using high-throughput devices and techniques. Thereby, the MIABiE consortium has initiated the identification and organization of a set of modules containing the minimum information that needs to be reported to guarantee the interpretability and independent verification of experimental results and their integration with knowledge coming from other fields. MIABiE does not intend to propose specific standards on how biofilms experiments should be performed, because it is acknowledged that specific research questions require specific conditions which may deviate from any standardization. Instead, MIABiE presents guidelines about the data to be recorded and published in order for the procedure and results to be easily and unequivocally interpreted and reproduced. Overall, MIABiE opens up the discussion about a number of particular areas of interest and attempts to achieve a broad consensus about which biofilm data and metadata should be reported in scientific journals in a systematic, rigorous and understandable manner.

Improvement of a predictive model of castration-resistant prostate cancer: functional genetic variants in TGFß1 signaling pathway modulation.

Prostate cancer (PC) is the most frequently diagnosed cancer in men. The acquisition of castration-resistant (CR) phenotype is associated with the activation of signaling pathways mediated by growth factors. The TGFß1 and its receptors have an important role in tumor progression, being the pro-apoptotic function modulated by the expression of TGFBR2. A single nucleotide polymorphism -875 G > A in TGFBR2 gene has been described, which may influence the expression levels of the receptor. Our purpose was to investigate the potential role of TGFBR2-875G>A in PC risk and in the response to androgen deprivation therapy (ADT). TGFBR2-875G>A polymorphism was studied by allelic discrimination using real-time polymerase chain reaction (PCR) in 891 patients with PC and 874 controls. A follow-up study was undertaken to evaluate response to ADT. The TGFBR2 and SMAD7 mRNA expression were analyzed by a quantitative real-time PCR. We found that TGFBR2-875GG homozygous patients present lower expression levels of TGFBR2 mRNA (AA/AG: 2(-ΔΔCT) =1.5, P=0.016). GG genotype was also associated with higher Gleason grade (OR=1.51, P=0.019) and increased risk of an early relapse after ADT (HR=1.47, P=0.024). The concordance (c) index analysis showed that the definition of profiles that contains information regarding tumor characteristics associated with genetic information present an increased capacity to predict the risk for CR development (c-index model 1: 0.683 vs model 2: 0.736 vs model 3: 0.746 vs model 4: 0.759). The TGFBR2-875G>A contribution to an early relapse in ADT patients, due to changes in mRNA expression, supports the involvement of TGFß1 pathway in CRPC. Furthermore, according to our results, we hypothesize the potential benefits of the association of genetic information in predictive models of CR development.

Performance of an adipokine pathway-based multilocus genetic risk score for prostate cancer risk prediction.

Few biomarkers are available to predict prostate cancer risk. Single nucleotide polymorphisms (SNPs) tend to have weak individual effects but, in combination, they have stronger predictive value. Adipokine pathways have been implicated in the pathogenesis. We used a candidate pathway approach to investigate 29 functional SNPs in key genes from relevant adipokine pathways in a sample of 1006 men eligible for prostate biopsy. We used stepwise multivariate logistic regression and bootstrapping to develop a multilocus genetic risk score by weighting each risk SNP empirically based on its association with disease. Seven common functional polymorphisms were associated with overall and high-grade prostate cancer (Gleason≥7), whereas three variants were associated with high metastatic-risk prostate cancer (PSA≥20 ng/mL and/or Gleason≥8). The addition of genetic variants to age and PSA improved the predictive accuracy for overall and high-grade prostate cancer, using either the area under the receiver-operating characteristics curves (P<0.02), the net reclassification improvement (P<0.001) and integrated discrimination improvement (P<0.001) measures. These results suggest that functional polymorphisms in adipokine pathways may act individually and cumulatively to affect risk and severity of prostate cancer, supporting the influence of adipokine pathways in the pathogenesis of prostate cancer. Use of such adipokine multilocus genetic risk score can enhance the predictive value of PSA and age in estimating absolute risk, which supports further evaluation of its clinical significance.

Tumor cell-educated periprostatic adipose tissue acquires an aggressive cancer-promoting secretory profile.

BACKGROUND/AIMS: The microenvironment produces important factors that are crucial to prostate cancer (PCa) progression. However, the extent to which the cancer cells stimulate periprostatic adipose tissue (PPAT) to produce these proteins is largely unknown. Our purpose was to determine whether PCa cell-derived factors influence PPAT metabolic activity. METHODS: Primary cultures of human PPAT samples from PCa patients (adipose tissue organotypic explants and primary stromal vascular fraction, SVF) were stimulated with conditioned medium (CM) collected from prostate carcinoma (PC3) cells. Cultures without CM were used as control. We used multiplex analysis and ELISA for protein quantification, qPCR to determine mitochondrial DNA (mtDNA) copy number and zymography for matrix metalloproteinase activity, in order to evaluate the response of adipose tissue explants and SVFs to PC3 CM. RESULTS: Stimulation of PPAT explants with PCa PC3 CM induced adipokines associated with cancer progression (osteopontin, tumoral necrosis factor alpha and interleukin-6) and reduced the expression of the protective adipokine adiponectin. Notably, osteopontin protein expression was 13-fold upregulated. Matrix metalloproteinase 9 activity and mitochondrial DNA copy number were higher after stimulation with cancer CM. Stromovascular cells from PPAT in culture were not influenced by tumor-derived factors. CONCLUSION: The modulation of adipokine expression by tumor CM indicates the pervasive extent to which tumor cells command PPAT to produce factors favorable to their aggressiveness.