Evaluation of Simultaneous Growth of Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes in Ground Beef Samples in Different Growth Media.

Several multiplex approaches for the simultaneous detection of pathogens in food have been developed in recent years, but the use of a single enrichment medium remains a problem. In this study, six enrichment broths (five non-selective media, tryptic soy broth (TSB), brain heart infusion broth (BHI), buffered peptone water (BPW), universal pre-enrichment broth (UPB), no. 17 broth, and a selective, Salmonella Escherichia Listeria broth (SEL)), were studied for the simultaneous detection of E. coli O157:H7, Salmonella spp., and L. monocytogenes, to validate the suitable enrichment broth to be used for the detection methods. Different ratios of E. coli O157:H7, Salmonella spp., and L. monocytogenes were used. Almost all non-selective broths evaluated in this study showed similar growth parameters and profiles among each other. The only selective enrichment broth under analysis (SEL) showed distinct growth features compared to the non-selective media, allowing for a slower but balanced growth of the three pathogens, which could be beneficial in preventing the overgrowth of fast-growing bacteria. In addition, when tested in ground beef samples, SEL broth seems to be the most distinctive medium with a balanced growth pattern observed for the three pathogens. Overall, this study is intended to provide the basis for the selection of suitable enrichment broths according to the technology detection to be used, the desired time of enrichment, and the expected balanced concentration of pathogens.

Oligonucleotide probes for imaging and diagnosis of bacterial infections.

The rise of infectious diseases as a public health concern has necessitated the development of rapid and precise diagnostic methods. Imaging techniques like nuclear and optical imaging provide the ability to diagnose infectious diseases within the body, eliminating delays caused by sampling and pre-enrichments of clinical samples and offering spatial information that can aid in a more informed diagnosis. Traditional molecular probes are typically created to image infected tissue without accurately identifying the pathogen. In contrast, oligonucleotides can be tailored to target specific RNA sequences, allowing for the identification of pathogens, and even generating antibiotic susceptibility profiles by focusing on drug resistance genes. Despite the benefits that nucleic acid mimics (NAMs) have provided in terms of stabilizing oligonucleotides, the inadequate delivery of these relatively large molecules into the cytoplasm of bacteria remains a challenge for widespread use of this technology. This review summarizes the key advancements in the field of oligonucleotide probes for in vivo imaging, highlighting the most promising delivery systems described in the literature for developing optical imaging through in vivo hybridization.

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.

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.

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.

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.

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.

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.

The Europa Uomo Patient Reported Outcome Study 2.0-Prostate Cancer Patient-reported Outcomes to Support Treatment Decision-making.

BACKGROUND: To further strengthen the voice of patients, Europa Uomo initiated the Europa Uomo Patient Reported Outcome Study 2.0 (EUPROMS 2.0) in October 2021. OBJECTIVE: To collect the self-reported perspective of prostate cancer (PCa) patients on physical and mental well-being after PCa treatment outside a clinical trial setting to inform future fellow patients about the impact of PCa treatment. DESIGN, SETTING, AND PARTICIPANTS: Europa Uomo invited PCa patients to complete a cross-sectional survey including the validated EQ-5D-5L, EORTC-QLQ-C30, and the EPIC-26 questionnaires. Furthermore, the nine-item Shared Decision Making Questionnaire (SDM-Q-9) and diagnostic clinical scenarios were included. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Descriptive statistics was used to assess the demographic and clinical characteristics and to analyze the patient-reported outcome data. RESULTS AND LIMITATIONS: Between October 25, 2021 and January 17, 2022, 3571 men from 30 countries completed the EUPROMS 2.0 survey. The median age of respondents was 70 yr (interquartile range 65-75 yr). Half of the respondents underwent one treatment, most often radical prostatectomy. Men who are treated actively experience lower health-related quality of life than men on active surveillance, mainly regarding sexual function, fatigue, and insomnia. Lower urinary incontinence levels were seen for men who underwent radical prostatectomy (single treatment or in combination with other treatments). Of the respondents, 42% indicated that the determination of the prostate-specific antigen (PSA) value was part of a routine blood test; 25% wanted to undergo screening/early detection for PCa, and 20% indicated that the determination of the PSA value had a clinical reason. CONCLUSIONS: A large sample of 3571 international patients has contributed patient experience after PCa treatment in the EUPROMS 2.0 study, confirming that treatment for PCa mainly affects urinary incontinence, sexual function, fatigue, and insomnia. Such information can be used to direct toward a better patient-doctor relationship, to offer patients ready access to responsible information and a better understanding of their disease and treatment. PATIENT SUMMARY: Through the EUPROMS 2.0 survey, Europa Uomo has strengthened the voice of the patient. Such information can be used to inform future prostate cancer (PCa) patients about the impact of PCa treatment and to engage them in informed and shared decision-making.

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.

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.

Mapping European Association of Urology Guideline Practice Across Europe: An Audit of Androgen Deprivation Therapy Use Before Prostate Cancer Surgery in 6598 Cases in 187 Hospitals Across 31 European Countries.

BACKGROUND: Evidence-practice gaps exist in urology. We previously surveyed European Association of Urology (EAU) guidelines for strong recommendations underpinned by high-certainty evidence that impact patient experience for which practice variations were suspected. The recommendation "Do not offer neoadjuvant androgen deprivation therapy (ADT) before surgery for patients with prostate cancer" was prioritised for further investigation. ADT before surgery is neither clinically effective nor cost effective and has serious side effects. The first step in improving implementation problems is to understand their extent. A clear picture of practice regarding ADT before surgery across Europe is not available. OBJECTIVE: To assess current ADT use before prostate cancer surgery in Europe. DESIGN, SETTING, AND PARTICIPANTS: This was an observational cross-sectional study. We retrospectively audited recent ADT practices in a multicentre international setting. We used nonprobability purposive sampling, aiming for breadth in terms of low- versus high-volume, academic, versus community and public versus private centres. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Our primary outcome was adherence to the ADT recommendation. Descriptive statistics and a multilevel model were used to investigate differences between countries across different factors (volume, centre type, and funding type). Subgroup analyses were performed for patients with low, intermediate, and high risk, and for those with locally advanced prostate cancer. We also collected reasons for nonadherence. RESULTS AND LIMITATIONS: We included 6598 patients with prostate cancer from 187 hospitals in 31 countries from January 1, 2017 to May 1, 2020. Overall, nonadherence was 2%, (range 0-32%). Most of the variability was found in the high-risk subgroup, for which nonadherence was 4% (range 0-43%). Reasons for nonadherence included attempts to improve oncological outcomes or preoperative tumour parameters; attempts to control the cancer because of long waiting lists; and patient preference (changing one's mind from radiotherapy to surgery after neoadjuvant ADT had commenced or feeling that the side effects were intolerable). Although we purposively sampled for variety within countries (public/private, academic/community, high/low-volume), a selection bias toward centres with awareness of guidelines is possible, so adherence rates may be overestimated. CONCLUSIONS: EAU guidelines recommend against ADT use before prostate cancer surgery, yet some guideline-discordant ADT use remains at the cost of patient experience and an additional payer and provider burden. Strategies towards discontinuation of inappropriate preoperative ADT use should be pursued. PATIENT SUMMARY: Androgen deprivation therapy (ADT) is sometimes used in men with prostate cancer who will not benefit from it. ADT causes side effects such as weight gain and emotional changes and increases the risk of cardiovascular disease, diabetes, and osteoporosis. Guidelines strongly recommend that men opting for surgery should not receive ADT, but it is unclear how well the guidance is followed. We asked urologists across Europe how patients in their institutions were treated over the past few years. Most do not use ADT before surgery, but this still happens in some places. More research is needed to help doctors to stop using ADT in patients who will not benefit from it.

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.

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.

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.

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.