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Background In silico promoter prediction represents an important challenge in bioinformatics as it provides a first-line approach to identifying regulatory elements to support wet-lab experiments. Historically, available promoter prediction software have focused on sigma factor-associated promoters in the model organism E. coli. As a consequence, traditional promoter predictors yield suboptimal predictions when applied to other prokaryotic genera, such as Pseudomonas, a Gram-negative bacterium of crucial medical and biotechnological importance. Results We developed SAPPHIRE, a promoter predictor for σ70 promoters in Pseudomonas. This promoter prediction relies on an artificial neural network that evaluates sequences on their similarity to the − 35 and − 10 boxes of σ70 promoters found experimentally in P. aeruginosa and P. putida . SAPPHIRE currently outperforms established predictive software when classifying Pseudomonas σ70 promoters and was built to allow further expansion in the future. Conclusions SAPPHIRE is the first predictive tool for bacterial σ70 promoters in Pseudomonas . SAPPHIRE is free, publicly available and can be accessed online at www.biosapphire.com . Alternatively, users can download the tool as a Python 3 script for local application from this site.

According to the latest reports from WHO, the incidence of antibiotic-resistant bacterial infections is increasing worldwide, resulting in increased morbidity and mortality and a rising pressure on health-care systems. However, the development of new antibiotics is an expensive and time-consuming process, urging scientists to seek alternative antimicrobial strategies. Over the past few decades, the concept of therapeutic administration of bacteriophages (also known as phages) has gained popularity worldwide. Although conceptually promising, the widespread implementation of phage therapy in routine clinical practice is restricted by the scarcity of safety and efficacy data obtained according to the strict standards of the applicable clinical trial regulations. In this systematic review, we list clinical data published between Jan 1, 2000 and Aug 14, 2021 on the safety and efficacy of phage therapy for difficult-to-treat bacterial infections, and provide an overview of trials and case studies on the use of phage therapy in several medical disciplines.

Microbiomes from every environment contain a myriad of uncultivated archaeal and bacterial viruses, but studying these viruses is hampered by the lack of a universal, scalable taxonomic framework. We present vConTACT v.2.0, a network-based application utilizing whole genome gene-sharing profiles for virus taxonomy that integrates distance-based hierarchical clustering and confidence scores for all taxonomic predictions. We report near-identical (96%) replication of existing genus-level viral taxonomy assignments from the International Committee on Taxonomy of Viruses for National Center for Biotechnology Information virus RefSeq. Application of vConTACT v.2.0 to 1,364 previously unclassified viruses deposited in virus RefSeq as reference genomes produced automatic, high-confidence genus assignments for 820 of the 1,364. We applied vConTACT v.2.0 to analyze 15,280 Global Ocean Virome genome fragments and were able to provide taxonomic assignments for 31% of these data, which shows that our algorithm is scalable to very large metagenomic datasets. Our taxonomy tool can be automated and applied to metagenomes from any environment for virus classification.Classification of archaeal and bacterial viruses can be automated with an algorithm that identifies relationships on the basis of shared gene content.

Non-model bacteria like Pseudomonas putida , Lactococcus lactis and other species have unique and versatile metabolisms, offering unique opportunities for Synthetic Biology (SynBio). However, key genome editing and recombineering tools require optimization and large-scale multiplexing to unlock the full SynBio potential of these bacteria. In addition, the limited availability of a set of characterized, species-specific biological parts hampers the construction of reliable genetic circuitry. Mining of currently available, diverse bacteriophages could complete the SynBio toolbox, as they constitute an unexplored treasure trove for fully adapted metabolic modulators and orthogonally-functioning parts, driven by the longstanding co-evolution between phage and host. Non-model bacteria offer unique and versatile metabolisms for synthetic biology. In this Perspective, the authors explore the limited availability of well-characterised biological parts in these species and argue that bacteriophages represent a diverse trove of orthogonal parts.

The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile of peripheral blood monocytes from six donors for twelve immunity-related genes ( i . e . CD14 , CXCL1 , CXCL5 , IL1A , IL1B , IL1RN , IL6 , IL10 , LYZ , SOCS3 , TGFBI and TNFA ) induced by Staphylococcus aureus phage ISP and four Pseudomonas aeruginosa phages ( i . e . PNM, LUZ19, 14-1 and GE-vB_Pae-Kakheti25). The phages were able to induce clear and reproducible immune responses. Moreover, the overall immune response was very comparable for all five phages: down-regulation of LYZ and TGFBI , and up-regulation of CXCL1 , CXCL5 , IL1A , IL1B , IL1RN , IL6 , SOCS3 and TNFA . The observed immune response was shown to be endotoxin-independent and predominantly anti-inflammatory. Addition of endotoxins to the highly purified phages did not cause an immune response comparable to the one induced by the (endotoxin containing) phage lysate. In addition, the use of an intermediate level of endotoxins tipped the immune response to a more anti-inflammatory response, i . e . up-regulation of IL1RN and a strongly reduced expression of CXCL1 and CXCL5.

Phages differ substantially in the bacterial hosts that they infect. Their host range is determined by the specific structures that they use to target bacterial cells. Tailed phages use a broad range of receptor-binding proteins, such as tail fibres, tail spikes and the central tail spike, to target their cognate bacterial cell surface receptors. Recent technical advances and new structure–function insights have begun to unravel the molecular mechanisms and temporal dynamics that govern these interactions. Here, we review the current understanding of the targeting machinery and mechanisms of tailed phages. These new insights and approaches pave the way for the application of phages in medicine and biotechnology and enable deeper understanding of their ecology and evolution.

A 30-year-old bombing victim with a fracture-related pandrug-resistant Klebsiella pneumoniae infection after long-term (>700 days) antibiotic therapy is treated with a pre-adapted bacteriophage along with meropenem and colistin, followed by ceftazidime/avibactam. This salvage therapy results in objective clinical, microbiological and radiological improvement of the patient’s wounds and overall condition. In support, the bacteriophage and antibiotic combination is highly effective against the patient’s K. pneumoniae strain in vitro, in 7-day mature biofilms and in suspensions. In this case study of a patient with fracture-related pandrug-resistant Klebsiella pneumoniae infection after long-term antibiotic therapy, the authors use a combination therapy of pre-adapted bacteriophage and antibiotics resulting in clinical, microbiological and radiological improvement.

Post-operative bacterial infections are a leading cause of mortality and morbidity after ongoing liver transplantation. Bacteria causing these infections in the hospital setting can exhibit high degrees of resistance to multiple types of antibiotics, which leads to major therapeutic hurdles. Alternate ways of treating these antibiotic-resistant infections are thus urgently needed. Phage therapy is one of them and consists in using selected bacteriophage viruses – viruses who specifically prey on bacteria, naturally found in various environmental samples – as bactericidal agents in replacement or in combination with antibiotics. The use of phage therapy raises various research questions to further characterize what determines therapeutic success or failure. In this work, we report the story of a toddler who suffered from extensively drug-resistant Pseudomonas aeruginosa sepsis after liver transplantation. He was treated by a bacteriophage-antibiotic intravenous combination therapy for 86 days. This salvage therapy was well tolerated, without antibody-mediated phage neutralization. It was associated with objective clinical and microbiological improvement, eventually allowing for liver retransplantation and complete resolution of all infections. Clear in vitro phage-antibiotic synergies were observed. The occurrence of bacterial phage resistance did not result in therapeutic failure, possibly due to phage-induced virulence tradeoffs, which we investigated in different experimental models. In this study, authors use combinatory bacteriophage-antibiotic therapy, as treatment for extensively drug-resistant Pseudomonas aeruginosa infection in a toddler post liver transplantation. They report on the clinical and microbiological improvement, and present their investigation on how bacterial phage resistance did not result in therapeutic failure.

Spread of multidrug-resistant Pseudomonas aeruginosa strains threatens to render currently available antibiotics obsolete, with limited prospects for the development of new antibiotics. Lytic bacteriophages, the viruses of bacteria, represent a path to combat this threat. In vitro-directed evolution is traditionally applied to expand the bacteriophage host range or increase bacterial suppression in planktonic cultures. However, while up to 80% of human microbial infections are biofilm-associated, research towards targeted improvement of bacteriophages’ ability to combat biofilms remains scarce. This study aims at an in vitro biofilm evolution assay to improve multiple bacteriophage parameters in parallel and the optimisation of bacteriophage cocktail design by exploiting a bacterial bacteriophage resistance trade-off. The evolved bacteriophages show an expanded host spectrum, improved antimicrobial efficacy and enhanced antibiofilm performance, as assessed by isothermal microcalorimetry and quantitative polymerase chain reaction, respectively. Our two-phage cocktail reveals further improved antimicrobial efficacy without incurring dual-bacteriophage-resistance in treated bacteria. We anticipate this assay will allow a better understanding of phenotypic-genomic relationships in bacteriophages and enable the training of bacteriophages against other desired pathogens. This, in turn, will strengthen bacteriophage therapy as a treatment adjunct to improve clinical outcomes of multidrug-resistant bacterial infections. Using a biofilm evolution assay, this study improves the antimicrobial and antibiofilm efficacy of Pseudomonas aeruginosa bacteriophages, while a two-phage cocktail shows further improved antimicrobial efficacy without incurring dual-bacteriophage-resistance.

The bacterial biofilm constitutes a complex environment that endows the bacterial community within with an ability to cope with biotic and abiotic stresses. Considering the interaction with bacterial viruses, these biofilms contain intrinsic defense mechanisms that protect against phage predation; these mechanisms are driven by physical, structural, and metabolic properties or governed by environment-induced mutations and bacterial diversity. In this regard, horizontal gene transfer can also be a driver of biofilm diversity and some (pro)phages can function as temporary allies in biofilm development. Conversely, as bacterial predators, phages have developed counter mechanisms to overcome the biofilm barrier. We highlight how these natural systems have previously inspired new antibiofilm design strategies, e.g., by utilizing exopolysaccharide degrading enzymes and peptidoglycan hydrolases. Next, we propose new potential approaches including phage-encoded DNases to target extracellular DNA, as well as phage-mediated inhibitors of cellular communication; these examples illustrate the relevance and importance of research aiming to elucidate novel antibiofilm mechanisms contained within the vast set of unknown ORFs from phages.