Phage SEP1 hijacks Staphylococcus epidermidis stationary cells' metabolism to replicate.

In nature, bacteria often survive in a stationary state with low metabolic activity. Phages use the metabolic machinery of the host cell to replicate, and, therefore, their efficacy against non-dividing cells is usually limited. Nevertheless, it was previously shown that the Staphylococcus epidermidis phage SEP1 has the remarkable capacity to actively replicate in stationary-phase cells, reducing their numbers. Here, we studied for the first time the transcriptomic profiles of both exponential and stationary cells infected with SEP1 phage using RNA-seq to gain a better understanding of this rare phenomenon. We showed that SEP1 successfully takes over the transcriptional apparatus of both exponential and stationary cells. Infection was, however, delayed in stationary cells, with genes within the gp142-gp154 module putatively implicated in host takeover. S. epidermidis responded to SEP1 infection by upregulating three genes involved in a DNA modification system, with this being observed already 5 min after infection in exponential cells and later in stationary cells. In stationary cells, a significant number of genes involved in translation and RNA metabolic and biosynthetic processes were upregulated after 15 and 30 min of SEP1 infection in comparison with the uninfected control, showing that SEP1 activates metabolic and biosynthetic pathways necessary to its successful replication.IMPORTANCEMost phage-host interaction studies are performed with exponentially growing cells. However, this cell state is not representative of what happens in natural environments. Additionally, most phages fail to replicate in stationary cells. The Staphylococcus epidermidis phage SEP1 is one of the few phages reported to date to be able to infect stationary cells. Here, we unveiled the interaction of SEP1 with its host in both exponential and stationary states of growth at the transcriptomic level. The findings of this study provide valuable insights for a better implementation of phage therapy since phages able to infect stationary cells could be more efficient in the treatment of recalcitrant infections.

Phage-Host Interaction Analysis Using Flow Cytometry.

Phage-host interactions are commonly evaluated by culture-based methods. However, these techniques are very laborious and time-consuming. Therefore, other time-efficient, not labor-intensive, and cost-effective methods have been developed.This chapter describes the methodology used to assess the susceptibility of planktonic cultures of bacteria to phage infection and to study their interactions over time by flow cytometry.

Genomic and proteomic characterization of vB_SauM-UFV_DC4, a novel Staphylococcus jumbo phage.

Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses. KEY POINTS: • vB_SauM-UFV_DC4 is a new viral species belonging to a new genus within the class Caudoviricetes. • vB_SauM-UFV_DC4 carries a set of RNA polymerase subunits and an RNA-directed DNA polymerase. • vB_SauM-UFV_DC4 and closely related jumbo phages showed a similar set of lytic proteins.

Isolation and Characterization of the Acadevirus Members BigMira and MidiMira Infecting a Highly Pathogenic Proteus mirabilis Strain.

Proteus mirabilis is an opportunistic pathogen and is responsible for more than 40% of all cases of catheter-associated urinary tract infections (CAUTIs). Healthcare-associated infections have been aggravated by the constant emergence of antibiotic-resistant bacterial strains. Because of this, the use of phages to combat bacterial infections gained renewed interest. In this study, we describe the biological and genomic features of two P. mirabilis phages, named BigMira and MidiMira. These phages belong to the Acadevirus genus (family Autographiviridae). BigMira and MidiMira are highly similar, differing only in four missense mutations in their phage tail fiber. These mutations are sufficient to impact the phages' depolymerase activity. Subsequently, the comparative genomic analysis of ten clinical P. mirabilis strains revealed differences in their antibiotic resistance profiles and lipopolysaccharide locus, with the latter potentially explaining the host range data of the phages. The massive presence of antimicrobial resistance genes, especially in the phages' isolation strain P. mirabilis MCS, highlights the challenges in treating infections caused by multidrug-resistant bacteria. The findings reinforce BigMira and MidiMira phages as candidates for phage therapy purposes.

Combining phages and antibiotic to enhance antibiofilm efficacy against an in vitro dual species wound biofilm.

Chronic wound management is extremely challenging because of the persistence of biofilm-forming pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which are the prevailing bacterial species that co-infect chronic wounds. Phage therapy has gained an increased interest to treat biofilm-associated infections, namely when combined with antibiotics. Here, we tested the effect of gentamicin as a co-adjuvant of phages in a dual species-biofilm wound model formed on artificial dermis. The biofilm-killing capacity of the tested treatments was significantly increased when phages were combined with gentamicin and applied multiple times as multiple dose (three doses, every 8 h). Our results suggest that gentamycin is an effective adjuvant of phage therapy particularly when applied simultaneously with phages and in three consecutive doses. The multiple and simultaneous dose treatment seems to be essential to avoid bacterial resistance development to each of the antimicrobial agents.

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.

Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2.

Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had Kd values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 104-7.0 × 107 CFU mL-1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 104 CFU mL-1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.

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.

Analysis of intact prophages in genomes of Paenibacillus larvae: An important pathogen for bees.

Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a highly contagious and worldwide spread bacterial disease that affects honeybee brood. In this study, all complete P. larvae genomes available on the NCBI database were analyzed in order to detect presence of prophages using the PHASTER software. A total of 55 intact prophages were identified in 11 P. larvae genomes (5.0 ± 2.3 per genome) and were further investigated for the presence of genes encoding relevant traits related to P. larvae. A closer look at the prophage genomes revealed the presence of several putative genes such as metabolic and antimicrobial resistance genes, toxins or bacteriocins, potentially influencing host performance. Some of the coding DNA sequences (CDS) were present in all ERIC-genotypes, while others were only found in a specific genotype. While CDS encoding toxins and antitoxins such as HicB and MazE were found in prophages of all bacterial genotypes, others, from the same category, were provided by prophages particularly to ERIC I (enhancin-like toxin), ERIC II (antitoxin SocA) and ERIC V strains (subunit of Panton-Valentine leukocidin system (PVL) LukF-PV). This is the first in-depth analysis of P. larvae prophages. It provides better knowledge on their impact in the evolution of virulence and fitness of P. larvae, by discovering new features assigned by the viruses.

Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica.

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori.

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.

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 [...].

Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages.

Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are highly needed. Flow cytometry (FC) allows a quick differentiation and enumeration of bacterial cell populations and has been used to assess in vitro the activity of antimicrobial compounds. In this work, we propose FC as a rapid and reliable method to assess the susceptibility of a bacterial population to phage infection. For that, the interaction of phages vB_PaeM_CEB_DP1 and vB_PaeP_PE3 with Pseudomonas aeruginosa PAO1 was characterized by FC. Synchronous infection assays were performed, and samples were collected at different time points and stained with SYTO BC and PI before analysis. Part of the collected samples was used to characterize the expression of early, middle, and late genes by qPCR. Both FC and qPCR results were correlated with phage propagation assays. Results showed that SYTO BC median fluorescence intensity (MFI) values increased in the first 25 min of PE3 and DP1 infection. The increase of fluorescence is due to the expression of phage genes observed by qPCR. Since SYTO BC MFI values increase with gene expression, it allows the determination of host susceptibility to a phage in a short period of time, avoiding false positives caused by lysis from without. In conclusion, this method may allow for a quick and high-throughput real-time screening of different phages to a specific host, which can be crucial for a quick phage selection in clinical practice.

Targeted Antimicrobial Photodynamic Therapy of Biofilm-Embedded and Intracellular Staphylococci with a Phage Endolysin's Cell Binding Domain.

Bacterial pathogens are progressively adapting to current antimicrobial therapies with severe consequences for patients and global health care systems. This is critically underscored by the rise of methicillin resistant Staphylococcus aureus (MRSA) and other biofilm-forming staphylococci. Accordingly, alternative strategies have been explored to fight such highly multidrug resistant microorganisms, including antimicrobial photodynamic therapy (aPDT) and phage therapy. aPDT has the great advantage that it does not elicit resistance, while phage therapy allows targeting of specific pathogens. In the present study, we aimed to merge these benefits by conjugating the cell-binding domain (CBD3) of a Staphylococcus aureus phage endolysin to a photoactivatable silicon phthalocyanine (IRDye 700DX) for the development of a Staphylococcus-targeted aPDT approach. We show that, upon red-light activation, the resulting CBD3-700DX conjugate generates reactive oxygen species that effectively kill high loads of planktonic and biofilm-resident staphylococci, including MRSA. Furthermore, CBD3-700DX is readily internalized by mammalian cells, where it allows the targeted killing of intracellular MRSA upon photoactivation. Intriguingly, aPDT with CBD3-700DX also affects mammalian cells with internalized MRSA, but it has no detectable side effects on uninfected cells. Altogether, we conclude that CBD3 represents an attractive targeting agent for Staphylococcus-specific aPDT, irrespective of planktonic, biofilm-embedded, or intracellular states of the bacterium. IMPORTANCE Antimicrobial resistance is among the biggest threats to mankind today. There are two alternative antimicrobial therapies that may help to control multidrug-resistant bacteria. In phage therapy, natural antagonists of bacteria, lytic phages, are harnessed to fight pathogens. In antimicrobial photodynamic therapy (aPDT), a photosensitizer, molecular oxygen, and light are used to produce reactive oxygen species (ROS) that inflict lethal damage on pathogens. Since aPDT destroys multiple essential components in targeted pathogens, aPDT resistance is unlikely. However, the challenge in aPDT is to maximize target specificity and minimize collateral oxidative damage to host cells. We now present an antimicrobial approach that combines the best features of both alternative therapies, namely, the high target specificity of phages and the efficacy of aPDT. This is achieved by conjugating the specific cell-binding domain from a phage protein to a near-infrared photosensitizer. aPDT with the resulting conjugate shows high target specificity toward MRSA with minimal side effects.

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).

Understanding the Complex Phage-Host Interactions in Biofilm Communities.

Bacteriophages and bacterial biofilms are widely present in natural environments, a fact that has accelerated the evolution of phages and their bacterial hosts in these particular niches. Phage-host interactions in biofilm communities are rather complex, where phages are not always merely predators but also can establish symbiotic relationships that induce and strengthen biofilms. In this review we provide an overview of the main features affecting phage-biofilm interactions as well as the currently available methods of studying these interactions. In addition, we address the applications of phages for biofilm control in different contexts.

New Insights on Biofilm Antimicrobial Strategies.

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

Selection of aptamers against triple negative breast cancer cells using high throughput sequencing.

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with [Formula: see text] values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.