Ethical bioprospecting and microbial assessments for sustainable solutions to the AMR crisis.

Antimicrobial resistance (AMR) has been declared one of the top 10 global public health challenges of our age by the World Health Organization, and the World Bank describes AMR as a crisis affecting the finance, health, and agriculture sectors and a major threat to the attainment of Sustainable Development Goals. But what is AMR? It is a phenotype that evolves in microbes exposed to antimicrobial molecules and causes dangerous infections. This suggests that scientists and healthcare workers should be on the frontline in the search for sustainable solutions to AMR.

A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo.

Bacteriophages (phages), viruses capable of infecting and lysing bacteria, are a promising alternative for treating infections from hypervirulent, antibiotic-resistant pathogens like Klebsiella pneumoniae, though narrow host range and phage resistance remain challenges. In this study, the hypervirulent K. pneumoniae NTUH-K2044 was used to purify phage ΦK2044, while two ΦK2044-resistant strains were used to purify two further phages: ΦKR1, and ΦKR8 from hospital sewage.

A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia.

Ventilator-associated pneumonia is defined as pneumonia that develops in a patient who has been on mechanical ventilation for more than 48 hours through an endotracheal tube. It is caused by biofilm formation on the indwelling tube, which introduces pathogenic microbes such as , and into the patient's lower airways. Currently, there is a lack of accurate models of ventilator-associated pneumonia development.

Arginine catabolism is essential to polymyxin dependence in Acinetobacter baumannii.

Polymyxins are often the only effective antibiotics against the "Critical" pathogen Acinetobacter baumannii. Worryingly, highly polymyxin-resistant A. baumannii displaying dependence on polymyxins has emerged in the clinic, leading to diagnosis and treatment failures.

RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat.

The TAM, a Translocation and Assembly Module for protein assembly and potential conduit for phospholipid transfer.

The assembly of β-barrel proteins into the bacterial outer membrane is an essential process enabling the colonization of new environmental niches. The TAM was discovered as a module of the β-barrel protein assembly machinery; it is a heterodimeric complex composed of an outer membrane protein (TamA) bound to an inner membrane protein (TamB). The TAM spans the periplasm, providing a scaffold through the peptidoglycan layer and catalyzing the translocation and assembly of β-barrel proteins into the outer membrane.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'β-signal' imprinted in the final β-strand of the OMP engages the β-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the β-signal are repeated in other, internal β-strands within bacterial OMPs, by peptidomimetic and mutational analysis.

Defining Membrane Protein Localization by Isopycnic Density Gradients.

Bacterial membrane proteins account for around one-third of the proteome in many species and can represent much more than half of the mass of the membranes. Classic techniques in cell biology can be applied to characterize bacterial membranes and their membrane protein constituents, and here we describe a protocol for the purification of outer membranes and inner membranes from Escherichia coli. This allows for compositional analysis of the membranes as well as functional analyses.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of . A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes.

Epitopes in the capsular polysaccharide and the porin OmpK36 receptors are required for bacteriophage infection of Klebsiella pneumoniae.

To kill bacteria, bacteriophages (phages) must first bind to a receptor, triggering the release of the phage DNA into the bacterial cell. Many bacteria secrete polysaccharides that had been thought to shield bacterial cells from phage attack. We use a comprehensive genetic screen to distinguish that the capsule is not a shield but is instead a primary receptor enabling phage predation.

-derived outer membrane vesicles package β-lactamases to promote antibiotic resistance.

causes the sexually transmitted disease gonorrhoea. The treatment of gonorrhoea is becoming increasingly challenging, as has developed resistance to antimicrobial agents routinely used in the clinic. Resistance to penicillin is wide-spread partly due to the acquisition of β-lactamase genes.

Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection.

Surveying membrane landscapes: a new look at the bacterial cell surface.

Recent studies applying advanced imaging techniques are changing the way we understand bacterial cell surfaces, bringing new knowledge on everything from single-cell heterogeneity in bacterial populations to their drug sensitivity and mechanisms of antimicrobial resistance. In both Gram-positive and Gram-negative bacteria, the outermost surface of the bacterial cell is being imaged at nanoscale; as a result, topographical maps of bacterial cell surfaces can be constructed, revealing distinct zones and specific features that might uniquely identify each cell in a population. Functionally defined assembly precincts for protein insertion into the membrane have been mapped at nanoscale, and equivalent lipid-assembly precincts are suggested from discrete lipopolysaccharide patches.

LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention.

Despite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation.

Bacteriophages evolve enhanced persistence to a mucosal surface.

The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut.

A Structurally Characterized Evolutionary Escape Route from Treatment with the Antibiotic Linezolid.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment.

Tall tails: cryo-electron microscopy of phage tail DNA ejection conduits.

The majority of phages, viruses that infect prokaryotes, inject their genomic material into their host through a tubular assembly known as a tail. Despite the genomic diversity of tailed phages, only three morphological archetypes have been described: contractile tails of Myoviridae-like phages; short non-contractile tails of Podoviridae-like phages; and long and flexible non-contractile tails of Siphoviridae-like phages. While early cryo-electron microscopy (cryo-EM) work elucidated the organisation of the syringe-like injection mechanism of contractile tails, the intrinsic flexibility of the long non-contractile tails prevented high-resolution structural determination.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined.

A noncanonical chaperone interacts with drug efflux pumps during their assembly into bacterial outer membranes.

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised.

Targeting bacterial outer-membrane remodelling to impact antimicrobial drug resistance.

The cell envelope is essential for survival and adaptation of bacteria. Bacterial membrane proteins include the major porins that mediate the influx of nutrients and several classes of antimicrobial drugs. Consequently, membrane remodelling is closely linked to antimicrobial resistance (AMR).

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.

The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K.