Pseudomonas syringae pv. actinidiae is a pandemic agent of bacterial canker in kiwifruit. The virulence of bacteria can be mediated by horizontal gene transfer in a co-evolutionary process with other microorganisms, enriching their genetic repertoire and enhancing adaptation strategies and fitness. Temperate bacteriophages can integrate into bacterial genomes, increasing genetic diversity and potentially improving bacterial fitness and virulence. To better understand the role of prophages in bacterial fitness and virulence, a bioinformatics analysis was performed on 88 genomes. Prophages were identified, annotated, and analyzed for their genomic content and integration sites. In total, 432 prophage-like sequences were identified, with an average of 4.7 prophages per genome, of which 88 were intact. The GC content of prophages (58.8 %) was similar to that of host genomes (58.5 %), and genome sizes ranged from 19 to 68.2 kb. Genomic and proteomic analysis revealed substantial diversity, grouping into 10 clusters with clonal profiles and broad geographical distribution. These results suggest ancestral acquisition, probably before clonal distribution. Prophages encode putative virulence factors, including metabolic-related proteins (45.7 %), lipoproteins (34.3 %), transporters (8.6 %), transcriptional regulators (37.1 %), and other factors (36 %) which contribute to bacterial stress tolerance, biofilm formation, motility, quorum sensing, metabolism and competition. Some intact prophages (36.4 %) exhibit specific integration sites with homology to host tRNA genes, indicating alternative attachment sites. The study supports the hypothesis that temperate phages contribute to the genetic diversity and pathogenicity by providing accessory genetic material, contributing to bacterial colonization and survival within the host.

Listeriosis is a foodborne infection caused by Listeria monocytogenes that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogenes, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogenes, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria. Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii.

This review provides a comprehensive overview of bacteriophage biology and classification, phage–bacteria interactions, and the mechanisms underlying phage-therapy efficacy, including its activity against biofilms and multidrug-resistant pathogens. It further examines phage–antibiotic synergy, resistance dynamics, safety considerations, pharmacokinetic and pharmacodynamic aspects, and the role of phage banks. Finally, the article discusses clinical evidence supporting phage therapy, emerging applications, and the Portuguese experience, highlighting regulatory, logistical, and clinical pathways that support the integration of phage therapy into modern infectious disease management.

Bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) continues to be a major threat to kiwifruit production worldwide. Current control methods, focused on chemical treatments and orchard hygiene strategies have low efficacy and pose several limitations, necessitating novel sustainable alternatives. In this study, a new lytic Myovirus bacteriophage, Qdp_Psa3, was isolated and characterized from Psa infected kiwifruit orchard soil. The bacteriophage displayed a narrow host range across Psa biovars (1, 2, and 3), strong pH stability and moderate thermal stability being inhibited at temperatures above 25 °C. Genomic sequencing revealed a 115 kb dsDNA genome lacking antibiotic resistance, tRNA, or virulence genes, and the analysis of the DNA packaging cassette suggested an uncharacterized mechanism. Phylogenetic and comparative genomic analyses support its designation as a novel species within the Caudoviricetes class, with proteomic profiling identified 62 virion-associated proteins, 71% of which remains hypothetical. Qdp_Psa3 demonstrated potential as a biocontrol agent, representing a promising candidate for inclusion as a complementary bacteriophage in a phage cocktail. Its discovery provides valuable insights into the hidden viral diversity of kiwifruit soils and expands our understanding of bacteriophage-Psa interactions.

Pseudomonas aeruginosa is a leading cause of chronic lung infections in cystic fibrosis (CF) patients. While bacteriophages hold potential as a treatment for antibiotic-resistant infections, the complex structure and heterogeneity of P. aeruginosa biofilms pose significant challenges to phage therapy. In this study, we investigate the adaptive evolution of the Pbunavirus phage PE1 to biofilms formed by a CF-derived P. aeruginosa isolate. Our findings reveal that biofilm-adapted PE1 mutants exhibit enhanced efficacy in controlling biofilms in vitro under conditions mimicking the CF lung environment. This improvement is attributed to the mutants' increased ability to recognize the diverse populations within the biofilm. Using a combination of cryo-EM, lipopolysaccharide (LPS) profiling, and adsorption assays, we demonstrate that mutations in tail fiber and baseplate genes of the phage improve adsorption and enable recognition of truncated LPS variants. This study highlights the critical role of biofilm heterogeneity in limiting phage effectiveness, identifies mechanisms to overcome this barrier, and pinpoints specific genomic targets for engineering phages tailored for therapeutic applications in CF patients.

Infections caused by carbapenem-resistant Acinetobacter baumannii (A. baumannii; CRAb) are associated with high patient morbidity and mortality. The serious threat for human health imposed by CRAb was recently underscored by identification of close-to-untouchable carbapenem- and tetracycline-resistant isolates. Since outer membrane vesicles (OMVs) of Gram-negative bacteria may contribute to antimicrobial resistance, our present study was aimed at investigating OMVs produced by the first two carbapenem- and tetracycline-resistant A. baumannii isolates in Europe. These isolates, denoted CRAb1 and CRAb2, contain large, nearly identical plasmids that specify multiple resistances. Both isolates produce OMVs that were analyzed by differential light scattering, transmission electron microscopy and proteomics. By comparison with OMVs from the plasmid-free non-carbapenem-resistant A. baumannii isolate Ab1, which is an isogenic ancestor of the CRAb1 isolate, we show that plasmid carriage by the CRAb1 and CRAb2 isolates leads to an increased OMV size that is accompanied by increased diversity of the OMV proteome. Our analyses show that OMVs from CRAb1 and CRAb2 are major reservoirs of proteins involved in antimicrobial resistance, including the plasmid-encoded carbapenemases New Delhi metallo-β-lactamase-1 (NDM-1), and carbapenem-hydrolyzing oxacillinase OXA-97 (OXA-97). Here we report that these OMV-borne carbapenemases hydrolyze imipenem and protect otherwise carbapenem-sensitive A. baumannii and Escherichia coli (E. coli) isolates against this antibiotic. In conclusion, our findings demonstrate that OMVs from highly drug-resistant CRAb confer protection against last-resort antibiotics to non-resistant bacterial pathogens.

Coagulase-negative staphylococci (CoNS) are commensal bacteria of the human skin and mucosal membranes. The incidence of nosocomial infections caused by these species is on the rise, leading to a potential increase in antibiotic tolerance and resistance. Phages are emerging as a promising alternative to combat CoNS infections. Scientists are isolating phages infecting CoNS with a particular interest in S. epidermidis. This review compiles and analyses CoNS phages for several parameters including source, geographical location, host species, morphological diversity, and genomic diversity. Additionally, recent studies have highlighted the potential of these phages based on host range, in vitro evaluation of performance and stability, and interaction with biofilms. This comprehensive analysis enables a better understanding of the steps involved in using these phages for therapeutic purposes.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found in biofilm-associated polymicrobial respiratory infections that are challenging to control. Studies performed in laboratory standard conditions suggest that bacterio(phages) and antibiotic combinations are more active against bacterial communities and biofilms than each agent alone. The purpose of this work was to study the antibacterial efficacy of phage-antibiotic combinations using an in vivo-like three-dimensional lung epithelial model that mimics aspects of the parental tissue, colonized by a mixed bacterial community of P. aeruginosa and S. aureus. The bacterial population was targeted by phages specific to P. aeruginosa and/or gentamicin and ciprofloxacin. The results showed that P. aeruginosa was eradicated from the dual-species community when phage treatment was followed by gentamicin and was significantly reduced when followed by ciprofloxacin. Moreover, applying phages first followed by antibiotics demonstrated superior antibacterial activity compared to simultaneous treatment or treatments with the reverse order of application. This approach also reduced the S. aureus population but not as significant as the P. aeruginosa population. Using an in vivo-like model we demonstrated that phages and antibiotics are effective against dual-species bacterial communities, particularly targeting the P. aeruginosa population. However, but the sequence in which these antimicrobials are applied significantly in fluences the effectiveness of bacterial killing.

Background: Proper hand hygiene is extremely important to control the transmission of pathogens. Although many studies have been undertaken on the effect of washing and drying on bacterial contamination of hands, studies on viral contamination are scarce. Aim: To assess the viral load of artificially contaminated hands after washing and after drying. Methods: Thirty volunteers completed a questionnaire on hand hygiene, and participated in microbial assays testing five different drying approaches, using whole-hand methodology, to quantify viruses on hands. Bacterial assays were also performed for comparison purposes. Results: For both viruses and bacteria, the washing step promoted a significant reduction in the microbial load, while the drying step only promoted a slight reduction, regardless of the drying method used. Hand dryers and paper towels did not induce recontamination of washed hands. Conclusions: Handwashing promoted a reduction in the microbial load of hands, but none of the drying methods tested led to a significant reduction in the microbial load of hands. (c) 2024 The Healthcare Infection Society. Published by Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Listeriosis is an infection caused by the consumption of food contaminated with Listeria monocytogenes. It leads to febrile gastroenteritis, central nervous system infections, and even death in risk populations. Bacteriophage endolysins selectively kill bacteria hydrolyzing their cell walls and have emerged as a potential tool for listeriosis control. Ply511 is an anti-Listeria endolysin that has activity against all serovars of L. monocytogenes. The yeast Saccharomyces cerevisiae has been used to produce endolysins for biocontrol, but prior efforts relied on plasmids, which can lead to gene loss and include selection markers unsuitable for therapeutic use. Integration of endolysins in its genome has also been previously demonstrated, relying however, on selection markers for selection and maintenance of the modifications. This study explores S. cerevisiae as a generally regarded as safe (GRAS) platform for producing and displaying Ply511 through CRISPR-Cas9 integration, offering a marker-free and stable solution for Listeria biocontrol. Our results demonstrate that the surface display of Ply511 does not lead to bacterial reduction. In contrast, we show that yeast secreting endolysin significantly reduces L. monocytogenes in cells, supernatants, and cell extracts. The strongest effect was observed with concentrated spent supernatant and cell extract, which reduced L. monocytogenes below the lower limit of quantification. Additionally, the spent supernatant exhibited active anti-Listeria activity in milk. This study highlights yeast-secreted endolysins as a promising platform for listeriosis control and demonstrates the yeast secretion of endolysins can be used for the biocontrol of pathogenic bacteria. • S. cerevisiae was edited using CRISPR-Cas9 to display or secrete endolysin Ply511. • Cells, supernatants, and extracts of yeast secreting Ply511 act against L. monocytogenes. • Demonstrates the yeast-based delivery of endolysins to control L. monocytogenes.

Pyoderma caused by methicillin-resistant Staphylococcus pseudintermedius poses significant challenges to canine health. Successful therapy requires an integrated approach to prudent antimicrobial use. This report describes the successful treatment of a dog with generalised superficial pyoderma using a topical formulation of S. pseudintermedius phage applied once daily for a period of 30 days. (sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic), (sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic).(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic).(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic), (sic)(sic) 30 (sic), (sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic). La pyodermite caus & eacute;e par Staphylococcus pseudintermedius r & eacute;sistant & agrave; la m & eacute;thicilline pose des d & eacute;fis importants en sant & eacute; canine. Une th & eacute;rapie r & eacute;ussie n & eacute;cessite une approche int & eacute;gr & eacute;e de l'utilisation prudente des antimicrobiens. Ce rapport d & eacute;crit le traitement r & eacute;ussi d'un chien atteint d'une pyodermite superficielle g & eacute;n & eacute;ralis & eacute;e & agrave; l'aide d'une formulation topique de phages de S. pseudintermedius appliqu & eacute;e une fois par jour pendant une p & eacute;riode de 30 jours. Eine Pyodermie, die durch einenMethicillin-resistenten Staphylococcus pseudintermedius verursacht wird, stellt signifikante Herausforderungen f & uuml;r die Gesundheit des Hundes dar. Eine erfolgreiche Therapie bedingt eine integrierte Herangehensweise mit einem vorsichtigen Einsatz von Antibiotika. Dieser Bericht beschreibt die erfolgreiche Behandlung eines Hundes mit einer generalisierten oberfl & auml;chlichen Pyodermie mittels formuliertem S. pseudintermedius Phagen, welcher einmal t & auml;glich f & uuml;r eine Dauer von 30 Tagen aufgetragen wurde. (sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic),(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic).(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic),(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic).(sic)(sic)(sic)(sic)(sic),S. pseudintermedius(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)1(sic)1(sic),30(sic)(sic)(sic)(sic)(sic)(sic),(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic)(sic). A piodermite causada por Staphylococcus pseudintermedius resistente & agrave; meticilina apresenta desafios significativos & agrave; sa & uacute;de canina. A terapia bem-sucedida requer uma abordagem integrada ao uso prudente de antimicrobianos. Este relato de caso descreve o tratamento bem-sucedido de um c & atilde;o com piodermite superficial generalizada utilizando uma formula & ccedil;& atilde;o t & oacute;pica de bacteri & oacute;fago de S. pseudintermedius aplicada uma vez ao dia por um per & iacute;odo de 30 dias. La pioderma causada por Staphylococcus pseudintermedius resistente a la meticilina plantea desaf & iacute;os importantes para la salud canina. Para que el tratamiento sea exitoso, se requiere un enfoque integrado para el uso prudente de los antimicrobianos. Este informe describe el tratamiento exitoso de un perro con pioderma superficial generalizada utilizando una formulaci & oacute;n t & oacute;pica del fago de S. pseudintermedius aplicada una vez al d & iacute;a durante un per & iacute;odo de 30 d & iacute;as.

Pseudomonas syringae pv. actinidiae (Psa) is the causative agent of bacterial canker in kiwifruit (Actinidia spp.). Psa biovar 3 is the most prevalent and virulent, causing frequent and severe outbreaks worldwide. While current treatments have low efficacy, bacteriophages emerge as possible environmentally safe alternative biocontrol agents. In this study, bacteriophage Brt_Psa3 was isolated from the soil of a kiwifruit orchard in Portugal. Morphologically, Brt_Psa3 forms clear plaques and has a Podoviral morphotype. The bacteriophage exhibited broad lytic activity against several plant-pathogenic Pseudomonas strains, including Psa isolates. The isolated bacteriophage has a latent period of 100 min, a burst size of 143 particles/cell, and demonstrates stability at different temperatures and pH values found in kiwifruit orchards. In addition, Brt_Psa3 exhibited tolerance to UVA irradiation during 120 min of incubation. Brt_Psa3 belongs to the Autographiviridae family and Ghunavirus genus, based on full-genome nucleotide alignment and supported by phylogenetic analysis of structural proteins. The phage contains 51 open reading frames with no antibiotic resistance genes identified, within a genome of 40.509 base pairs. In vitro experiments with kiwifruit leaves demonstrated significant reduction of Psa levels (40%) on leaf surfaces, highlighting the bacteriophage's therapeutic potential in managing bacterial canker in kiwifruits.

Oligomeric clusters of amyloid-β (Aβ) are one of the major biomarkers for Alzheimer’s disease (AD). However, proficient methods to detect Aβ-oligomers in brain tissue are lacking. Here we show that synthetic M13 bacteriophages displaying Aβ-derived peptides on their surface preferentially interact with Aβ-oligomers. When exposed to brain tissue isolated from APP/PS1-transgenic mice, these bacteriophages detect small-sized Aβ-aggregates in hippocampus at an early age, prior to the occurrence of Aβ-plaques. Similarly, the bacteriophages reveal the presence of such small Aβ-aggregates in post-mortem hippocampus tissue of AD-patients. These results advocate bacteriophages displaying Aβ-peptides as a convenient and low-cost tool to identify Aβ-oligomers in post-mortem brain tissue of AD-model mice and AD-patients.

ABSTRACT 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. IMPORTANCE Most 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.

Infective endocarditis (IE) is a severe infection of the inner heart. Even with current standard treatment, the mean in-hospital mortality is as high as 15-20%, and 1-year mortality is up to 40% for left-sided IE. Importantly, IE mortality rates have not changed substantially over the past 30 years, and the incidence of IE is rising. The treatment is challenging due to the bacterial biofilm mode of growth inside the heart valve vegetations, resulting in antibiotic tolerance. Achieving sufficient antibiotic anti-biofilm concentrations in the biofilms of the heart valve vegetations is problematic, even with high-dose and long-term antibiotic therapy. The increasing prevalence of IE caused by antibiotic-resistant bacteria adds to the challenge. Therefore, adjunctive antibiotic-potentiating drug candidates and strategies are increasingly being investigated. Bacteriophage therapy is a reemerging antibacterial treatment strategy for difficult-to-treat infections, mainly biofilm-associated and caused by multidrug-resistant bacteria. However, significant knowledge gaps regarding the safety and efficacy of phage therapy impede more widespread implementation in clinical practice. Hopefully, future preclinical and clinical testing will reveal whether it is a viable treatment. The objective of the present review is to assess whether bacteriophage therapy is a realistic treatment for IE.

Recent advances in the synthetic biology field have enabled the development of new molecular biology techniques used to build specialized bacteriophages with new functionalities. Bacteriophages have been engineered towards a wide range of applications including pathogen control and detection, targeted drug delivery, or even assembly of new materials.In this chapter, two strategies that have been successfully used to genetically engineer bacteriophage genomes are addressed: a yeast-based platform and bacteriophage recombineering of electroporated DNA.

Biofilm formation, a strategy of bacterial survival, is a significant concern in different areas, including health, where infectious biofilms are very difficult to combat with conventional antimicrobial therapies. Bacteriophages, the viruses that infect bacteria, are promising agents to prevent and control biofilm-related infections. This chapter describes a series of standard procedures that can be used to study the potential of bacteriophages for biofilm control, from biofilm formation to bacteriophage treatment and evaluation of its efficacy.

Antimicrobial resistance is on the rise globally, prompting increased research and development (R&D) of phage therapy as a strategy to address difficult-to-treat bacterial infections. We review the current state of phage therapy research, including major operational, epistemic, and biological challenges for phage R&D, and discuss some new approaches to developing the technology motivated by recent breakthroughs such as artificial intelligence and synthetic phage production. In addition, we contextualize these R&D challenges and opportunities in light of the ongoing predicament of commercial antimicrobial innovation and current public-private efforts to reinvigorate the pipeline of antimicrobial drug discovery. We conclude with reflections on the potential for new phage therapies to be readily accessible across all income contexts to better ensure broad patient access, and consider possible alternatives to current public and public-private solutions for phage therapy and production.

PURPOSE:Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important nosocomial pathogen. The capsular type (K-type) is considered a major virulence factor, contributing to the evasion of host defenses. The global spread and dissemination dynamics between K-types, sequence types (ST), antibiotic resistance genes, and virulence factors remain largely unknown in Portugal. METHODS:A collection of 96 CRAB clinical samples collected between 2005 and 2019 in the northern region of Portugal were tested for antimicrobial susceptibility profile and screened by polymerase chain reaction for resistance genetic determinants. A subset of 26 representative isolates was subjected to whole-genome sequencing to assess K types, ST types, and genomic relatedness. The pathogenicity of distinct K-types was also tested using Galleria mellonella model. FINDINGS:For the 96 CRAB isolates analyzed, high antimicrobial resistance (>90%) was observed to the carbapenems, fluoroquinolones, and miscellaneous agents. Greater antimicrobial susceptibility (∼30%-57%) was observed for aminoglycosides, particularly tobramycin, and amikacin. Genotypically, 75 strains (78.5%) carried blaOXA-23-like, 18 strains (18.8%) carried blaIMP-like, and 11 strains (14.9%) carried blaOXA-40-like carbapenem resistance genes, respectively. Associations between OXA and ST/capsular locus (KL) types were observed over the years (eg, OXA-40-like/ST46Past/KL120 and OXA-23-like/ST2Past/KL2). ST2Past of clonal complex II was present in most strains, a dominant drug-resistant lineage in the United States and Europe. KL7 was also the most prevalent KL-type (38.5%), followed by KL2 (34.6%), KL120 (23.1%), and KL9 (3.8%). Virulence assessment for different K-types in a Galleria mellonella model revealed a significantly increased virulence for KL120 when compared with KL7, KL9, and KL2. IMPLICATIONS:There are specific CRAB serotypes circulating in Portugal, accounting by the low diversity of acquired carbapenemase genes (OXA-23-like and OXA-40-like), ST types (ST2 and ST46) and KL types (KL2, KL7, KL9, and KL120) identified. The high prevalent of ST2, especially when associated with KL2 and blaOXA-23-like, suggest that antibiotic resistance has been driven by clonal expansion of clonal complex II. Such findings provide useful information on the diversity of multidrug-resistant bacterium that might be relevant for antibacterial interventions.

In the last decade, powerful high-throughput sequencing approaches have emerged to analyse microbial transcriptomes at a global scale. However, to date, applications of these approaches to microbial viruses such as phages remain scarce. Tailoring these techniques to virus-infected bacteria promises to obtain a detailed picture of the underexplored RNA biology and molecular processes during infection. In addition, transcriptome study of stress and perturbations induced by phages in their infected bacterial hosts is likely to reveal new fundamental mechanisms of bacterial metabolism and gene regulation. Here, we provide references and blueprints to implement emerging transcriptomic approaches towards addressing transcriptome architecture, RNA-RNA and RNA-protein interactions, RNA modifications, structures and heterogeneity of transcription profiles in infected cells that will provide guides for future directions in phage-centric therapeutic applications and microbial synthetic biology.