Paving the way forward: Escherichia coli bacteriophages in a One Health approach.

Escherichia coli is one of the most notorious pathogens for its ability to adapt, colonize, and proliferate in different habitats through a multitude of acquired virulence factors. Its presence affects the food-processing industry and causes food poisoning, being also a major economic burden for the food, agriculture, and health sectors. Bacteriophages are emerging as an appealing strategy to mitigate bacterial pathogens, including specific E. coli pathovars, without exerting a deleterious effect on humans and animals. This review globally analyzes the applied research on E. coli phages for veterinary, food, and human use. It starts by describing the pathogenic E. coli pathotypes and their relevance in human and animal context. The idea that phages can be used as a One Health approach to control and interrupt the transmission routes of pathogenic E. coli is sustained through an exhaustive revision of the recent literature. The emerging phage formulations, genetic engineering and encapsulation technologies are also discussed as a means of improving phage-based control strategies, with a particular focus on E. coli pathogens.

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.

Contribution of nanotechnology to greater efficiency in animal nutrition and production.

Feed costs present a major burden in animal production for human consumption, representing a key opportunity for cost reduction and profit improvement. Nanotechnology offers potential to increase productivity by creating higher-quality and safer products. The feed sector has benefited from the use of nanosystems to improve the stability and bioavailability of feed ingredients. The development of nanotechnology products for feed must consider the challenges raised by biological barriers as well as regulatory requirements. While some nanotechnology-based products are already commercially available for animal production, the exponential growth and application of these products requires further research ensuring their safety and the establishment of comprehensive legislative frameworks and regulatory guidelines. Thus, this article provides an overview of the current state of the art regarding nanotechnology solutions applied in feed, as well as the risks and opportunities aimed to help researchers and livestock producers.

Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.

Insights into the genome architecture and evolution of Shiga toxin encoding bacteriophages of Escherichia coli.

BACKGROUND: A total of 179 Shiga toxin-producing Escherichia coli (STEC) complete genomes were analyzed in terms of serotypes, prophage coding regions, and stx gene variants and their distribution. We further examined the genetic diversity of Stx-converting phage genomes (Stx phages), focusing on the lysis-lysogeny decision and lytic cassettes. RESULTS: We show that most STEC isolates belong to non-O157 serotypes (73 %), regardless the sources and geographical regions. While the majority of STEC genomes contain a single stx gene (61 %), strains containing two (35 %), three (3 %) and four (1 %) stx genes were also found, being stx2 the most prevalent gene variant. Their location is exclusively found in intact prophage regions, indicating that they are phage-borne. We further demonstrate that Stx phages can be grouped into four clusters (A, B, C and D), three subclusters (A1, A2 and A3) and one singleton, based on their shared gene content. This cluster distribution is in good agreement with their predicted virion morphologies. Stx phage genomes are highly diverse with a vast number of 1,838 gene phamilies (phams) of related sequences (of which 677 are orphams i.e. unique genes) and, although having high mosaicism, they are generally organized into three major transcripts. While the mechanisms that guide lysis-lysogeny decision are complex, there is a strong selective pressure to maintain the stx genes location close to the lytic cassette composed of predicted SAR-endolysin and pin-holin lytic proteins. The evolution of STEC Stx phages seems to be strongly related to acquiring genetic material, probably from horizontal gene transfer events. CONCLUSIONS: This work provides novel insights on the genetic structure of Stx phages, showing a high genetic diversity throughout the genomes, where the various lysis-lysogeny regulatory systems are in contrast with an uncommon, but conserved, lytic system always adjacent to stx genes.

Bacteriophages to control Shiga toxin-producing E. coli - safety and regulatory challenges.

Shiga toxin-producing Escherichia coli (STEC) are usually found on food products due to contamination from the fecal origin, as their main environmental reservoir is considered to be the gut of ruminants. While this pathogen is far from the incidence of other well-known foodborne bacteria, the severity of STEC infections in humans has triggered global concerns as far as its incidence and control are concerned. Major control strategies for foodborne pathogens in food-related settings usually involve traditional sterilization/disinfection techniques. However, there is an increasing need for the development of further strategies to enhance the antimicrobial outcome, either on food-contact surfaces or directly in food matrices. Phages are considered to be a good alternative to control foodborne pathogens, with some phage-based products already cleared by the Food and Drug Administration (FDA) to be used in the food industry. In European countries, phage-based food decontaminants have already been used. Nevertheless, its broad use in the European Union is not yet possible due to the lack of specific guidelines for the approval of these products. Furthermore, some safety concerns remain to be addressed so that the regulatory requirements can be met. In this review, we present an overview of the main virulence factors of STEC and introduce phages as promising biocontrol agents for STEC control. We further present the regulatory constraints on the approval of phages for food applications and discuss safety concerns that are still impairing their use.

FISH and chips: a review of microfluidic platforms for FISH analysis.

Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory.

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.

Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides.

BACKGROUND: Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specific miRNA expression profile, with increasing evidence of the role of miRNA-9 in this disease. miRNA-9 upregulation has been shown to influence the expression of E-cadherin-encoding gene, triggering cell motility and invasiveness. RESULTS: In this study, we designed LNA anti-miRNA oligonucleotides with a complementary sequence to miRNA-9 and tested their properties to both detect and silence the target miRNA. We could identify and visualize the in vitro uptake of low-dosing LNA-based anti-miRNA oligonucleotides without any carrier or transfection agent, as early as 2 h after the addition of the oligonucleotide sequence to the culture medium. Furthermore, we were able to assess the silencing potential of miRNA-9, using different LNA anti-miRNA oligonucleotide designs, and to observe its subsequent effect on E-cadherin expression. CONCLUSIONS: The administration of anti-miRNA sequences even at low-doses, rapidly repressed the target miRNA, and influenced the expression of E-cadherin by significantly increasing its levels.

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.

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.

Dual-color control of nucleotide polymerization sensed by a fluorescence actuator.

Spatial and temporal control of molecular mechanisms can be achieved using photolabile bonds that connect biomolecules to protective caging groups, which can be cleaved upon irradiation of a specific wavelength, releasing the biomolecule ready-to-use. Here we apply and improve a previously reported strategy to tightly control in vitro transcription reactions. The strategy involves two caging molecules that block both ATP and GTP nucleotides. Additionally, we designed a molecular beacon complementary to the synthesized mRNA to infer its presence through a light signal. Upon release of both nucleotides through a specific monochromatic light (390 and 325 nm) we attain a light signal indicative of a successful in vitro transcription reaction. Similarly, in the absence of irradiation, no intense fluorescence signal was obtained. We believe this strategy could further be applied to DNA synthesis or the development of logic gates.

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.

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.

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.

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.

Molecular characterization and virulence profile of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill cats and dogs in Portugal.

Klebsiella spp. are important pathogens of humans and companion animals such as cats and dogs, capable of causing severe life-threatening diseases. The aim of this study was to characterize the molecular and phenotypic properties of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill companion animals by whole genome sequencing, followed by in vitro assessment of biofilm formation and in vivo pathogenicity using the Galleria mellonella model. Two LPS O-types were identified for all the K. pneumoniae isolates tested (O3B and O1/O2v2) and only one for K. oxytoca isolates (OL104), and the most common STs found were ST11 and ST266. Furthermore, a high diversity of K-locus types was found for K. pneumoniae (KL102; KL105; KL31, and KL13). Within K. pneumoniae, one specific O/K/ST-types combination (i.e., KL105-ST11-O1/O2v2) showed results that were of concern, as it exhibited a high inflammatory response at 12 h post-infection in G. mellonella with 80% of the larvae dead at 72 h post-infection. This virulence potential, on the other hand, did not appear to be directly related to the biofilm-forming capacity. Also, virulence and resistance scores obtained for this set of strains did surpass score 1. The present study demonstrated that Klebsiella spp. isolated from companion animals belonging to STs that can cause human infections and present virulence on an invertebrate model. Thus, this study underscores the role of dogs and cats as reservoirs of resistant Klebsiella spp. that could potentially be transmitted to humans.

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.

Emerging Approaches for Mitigating Biofilm-Formation-Associated Infections in Farm, Wild, and Companion Animals.

The importance of addressing the problem of biofilms in farm, wild, and companion animals lies in their pervasive impact on animal health and welfare. Biofilms, as resilient communities of microorganisms, pose a persistent challenge in causing infections and complicating treatment strategies. Recognizing and understanding the importance of mitigating biofilm formation is critical to ensuring the welfare of animals in a variety of settings, from farms to the wild and companion animals. Effectively addressing this issue not only improves the overall health of individual animals, but also contributes to the broader goals of sustainable agriculture, wildlife conservation, and responsible pet ownership. This review examines the current understanding of biofilm formation in animal diseases and elucidates the complex processes involved. Recognizing the limitations of traditional antibiotic treatments, mechanisms of resistance associated with biofilms are explored. The focus is on alternative therapeutic strategies to control biofilm, with illuminating case studies providing valuable context and practical insights. In conclusion, the review highlights the importance of exploring emerging approaches to mitigate biofilm formation in animals. It consolidates existing knowledge, highlights gaps in understanding, and encourages further research to address this critical facet of animal health. The comprehensive perspective provided by this review serves as a foundation for future investigations and interventions to improve the management of biofilm-associated infections in diverse animal populations.