Whole genome sequencing and genotyping Klebsiella pneumoniae multi-drug resistant hospital isolates from Western Kenya.

Objectives: Klebsiella pneumoniae are a frequent cause of nosocomial infections worldwide. Sequence type 147 (ST147) has been reported as a major circulating high-risk lineage in many countries, and appears to be a formidable platform for the dissemination of antimicrobial resistance (AMR) determinants. However, the distribution of this pathogen in Western African hospitals has been scarcely studied. The main objective of this work was to perform whole genome sequencing of K. pneumoniae isolates from a referral hospital in Kakamega (Kenya) for genotyping and identification of AMR and virulence determinants. Methods: In total, 15 K. pneumoniae isolates showing a broad spectrum antimicrobial resistance were selected for whole genome sequencing by Illumina HiSeq 2500 platform. Results: ST147 was the dominant lineage among the highly-resistant K. pneumoniae isolates that we sequenced. ST147 was associated with both community- and the hospital-acquired infections, and with different infection sites, whereas other STs were predominantly uropathogens. Multiple antibiotic resistance and virulence determinants were detected in the genomes including extended-spectrum ß-lactamases (ESBL) and carbapenemases. Many of these genes were plasmid-borne. Conclusions: Our data suggest that the evolutionary success of ST147 may be linked with the acquisition of broad host-range plasmids, and their propensity to accrue AMR and virulence determinants. Although ST147 is a dominant lineage in many countries worldwide, it has not been previously reported as prevalent in Africa. Our data suggest an influx of new nosocomial pathogens with new virulence genes into African hospitals from other continents.

More than just a gel: the extracellular matrixome of Pseudomonas aeruginosa.

Armed with an arsenal of protein secretion systems, antibiotic efflux pumps, and the occasional proclivity for explosive self-destruction, Pseudomonas aeruginosa has become a model for the study of bacterial pathogenesis and biofilm formation. There is accruing evidence to suggest that the biofilm matrix-the bioglue that holds the structure together-acts not only in a structural capacity, but is also a molecular "net" whose function is to capture and retain certain secreted products (including proteins and small molecules). In this perspective, we argue that the biofilm matrixome is a distinct extracellular compartment, and one that is differentiated from the bulk secretome. Some of the points we raise are deliberately speculative, but are becoming increasingly accessible to experimental investigation.

Impact of transient acquired hypermutability on the inter- and intra-species competitiveness of Pseudomonas aeruginosa.

Once acquired, hypermutation is unrelenting, and in the long-term, leads to impaired fitness due to its cumulative impact on the genome. This raises the question of why hypermutators arise so frequently in microbial ecosystems. In this work, we explore this problem by examining how the transient acquisition of hypermutability affects inter- and intra-species competitiveness, and the response to environmental insults such as antibiotic challenge. We do this by engineering Pseudomonas aeruginosa to allow the expression of an important mismatch repair gene, mutS, to be experimentally controlled over a wide dynamic range. We show that high levels of mutS expression induce genomic stasis (hypomutation), whereas lower levels of induction lead to progressively higher rates of mutation. Whole-genome sequence analyses confirmed that the mutational spectrum of the inducible hypermutator is similar to the distinctive profile associated with mutS mutants obtained from the airways of people with cystic fibrosis (CF). The acquisition of hypermutability conferred a distinct temporal fitness advantage over the wild-type P. aeruginosa progenitor strain, in both the presence and the absence of an antibiotic selection pressure. However, over a similar time-scale, acquisition of hypermutability had little impact on the population dynamics of P. aeruginosa when grown in the presence of a competing species (Staphylococcus aureus). These data indicate that in the short term, acquired hypermutability primarily confers a competitive intra-species fitness advantage.

Therapeutic interventions alter ecological interactions among cystic fibrosis airway microbiota.

The airways of people with cystic fibrosis (CF) often harbor a diverse microbiota and in recent years, much effort has been invested in cataloguing these. In spite of providing a wealth of insight, this cataloguing tells us little about how the organisms interact with one another in the CF airways. However, such relationships can be inferred using the theoretical framework of the Lotka-Volterra (LV) model. In the current work, we use a generalized Lotka-Volterra model to interrogate the nationwide data collected and curated by the UK CF Registry. This longitudinal dataset (covering the period 2008-2020) contains annual depositions that record the presence/absence of microbial taxa in each patient, their medication, and their CF genotype. Specifically, we wanted to identify trends in ecological relationships between the CF microbiota at a nationwide level, and whether these are potentially affected by medication. Our results show that some medications have a distinct influence on the microbial interactome, especially those that potentially influence the "gut-lung axis" or mucus viscosity. In particular, we found that patients treated with a combination of antimicrobial agents (targeting the airway microbiota), digestive enzymes (assisting in the assimilation of dietary fats and carbohydrates), and DNase (to reduce mucus viscosity) displayed a distinctly different airway interactome compared with patients treated separately with these medications.

Distribution and molecular evolution of the anti-CRISPR family AcrIF7.

Anti-clustered regularly interspaced short palindromic repeats (CRISPRs) are proteins capable of blocking CRISPR-Cas systems and typically their genes are located on mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPR's function and evolution. Here, we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered 5 subclusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological, and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families.

Systems-Wide Dissection of Organic Acid Assimilation in Pseudomonas aeruginosa Reveals a Novel Path To Underground Metabolism.

The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive under diverse environmental conditions and ecological niches; yet, little is known about the central metabolic pathways that sustain its growth during infection or precisely how these pathways operate. In this work, we used a combination of 'omics approaches (transcriptomics, proteomics, metabolomics, and 13C-fluxomics) and reverse genetics to provide systems-level insight into how the infection-relevant organic acids succinate and propionate are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (2-MCS; PrpC) and its paralogue citrate (CIT) synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then derepresses prpC expression. Our findings provide a clear example of how "underground metabolism," facilitated by enzyme substrate promiscuity, "rewires" Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that, due to its unrivalled resistance to antibiotics, ubiquity in the built environment, and aggressiveness in infection scenarios, has acquired the somewhat dubious accolade of being designated a "critical priority pathogen" by the WHO. In this work, we uncover the pathways and mechanisms used by P. aeruginosa to grow on a substrate that is abundant at many infection sites: propionate. We found that if the organism is prevented from metabolizing propionate, the substrate turns from being a convenient nutrient source into a potent poison, preventing bacterial growth. We further show that one of the enzymes involved in these reactions, 2-methylcitrate synthase (PrpC), is promiscuous and can moonlight for another essential enzyme in the cell (citrate synthase). Indeed, mutations that abolish citrate synthase activity (which would normally prevent the cell from growing) can be readily overcome if the cell acquires additional mutations that increase the expression of PrpC. This is a nice example of the evolutionary utility of so-called "underground metabolism."

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate ß-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Decreased efficacy of antimicrobial agents in a polymicrobial environment.

The airways of people with cystic fibrosis (CF) often harbour diverse polymicrobial communities. These airway infections can be impossible to resolve through antibiotic intervention, even though isolates of the individual species present are susceptible to the treatment when tested in vitro. In this work, we investigate how polymicrobial cultures comprised of key CF-associated pathogens respond to challenge with species-specific antimicrobial agents; colistin (targets Pseudomonas aeruginosa), fusidic acid (targets Staphylococcus aureus), and fluconazole (targets Candida albicans). We found that growth in a polymicrobial environment protects the target microorganism (sometimes by several orders of magnitude) from the effect(s) of the antimicrobial agent. This decreased antimicrobial efficacy was found to have both non-heritable (physiological) and heritable (genetic) components. Whole-genome sequencing of the colistin-resistant P. aeruginosa isolates revealed single nucleotide polymorphisms and indels in genes encoding lipopolysaccharide (LPS) biosynthesis and/or pilus biogenesis, indicating that a previously undescribed colistin resistance mechanism was in operation. This was subsequently confirmed through further genetic analyses. Our findings indicate that the polymicrobial nature of the CF airways is likely to have a significant impact on the clinical response to antimicrobial therapy.

The methylation-independent mismatch repair machinery in Pseudomonas aeruginosa.

Over the last 70 years, we've all gotten used to an Escherichia coli-centric view of the microbial world. However, genomics, as well as the development of improved tools for genetic manipulation in other species, is showing us that other bugs do things differently, and that we cannot simply extrapolate from E. coli to everything else. A particularly good example of this is encountered when considering the mechanism(s) involved in DNA mismatch repair by the opportunistic human pathogen, Pseudomonas aeruginosa (PA). This is a particularly relevant phenotype to examine in PA, since defects in the mismatch repair (MMR) machinery often give rise to the property of hypermutability. This, in turn, is linked with the vertical acquisition of important pathoadaptive traits in the organism, such as antimicrobial resistance. But it turns out that PA lacks some key genes associated with MMR in E. coli, and a closer inspection of what is known (or can be inferred) about the MMR enzymology reveals profound differences compared with other, well-characterized organisms. Here, we review these differences and comment on their biological implications.

Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykF PA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the "ring loop" covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent "locked on" and "locked off" allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.

The Pseudomonas aeruginosa whole genome sequence: A 20th anniversary celebration.

Toward the end of August 2000, the 6.3 Mbp whole genome sequence of Pseudomonas aeruginosa strain PAO1 was published. With 5570 open reading frames (ORFs), PAO1 had the largest microbial genome sequenced up to that point in time-including a large proportion of metabolic, transport and antimicrobial resistance genes supporting its ability to colonize diverse environments. A remarkable 9% of its ORFs were predicted to encode proteins with regulatory functions, providing new insight into bacterial network complexity as a function of network size. In this celebratory article, we fast forward 20 years, and examine how access to this resource has transformed our understanding of P. aeruginosa. What follows is more than a simple review or commentary; we have specifically asked some of the leaders in the field to provide personal reflections on how the PAO1 genome sequence, along with the Pseudomonas Community Annotation Project (PseudoCAP) and Pseudomonas Genome Database (pseudomonas.com), have contributed to the many exciting discoveries in this field. In addition to bringing us all up to date with the latest developments, we also ask our contributors to speculate on how the next 20 years of Pseudomonas research might pan out.

An in vitro model for the cultivation of polymicrobial biofilms under continuous-flow conditions.

The airways of people with cystic fibrosis (CF) are often chronically colonised with a diverse array of bacterial and fungal species. However, little is known about the relative partitioning of species between the planktonic and biofilm modes of growth in the airways. Existing in vivo and in vitro models of CF airway infection are ill-suited for the long-term recapitulation of mixed microbial communities. Here we describe a simple, in vitro continuous-flow model for the cultivation of polymicrobial biofilms and planktonic cultures on different substrata. Our data provide evidence for inter-species antagonism and synergism in biofilm ecology. We further show that the type of substratum on which the biofilms grow has a profound influence on their species composition. This happens without any major alteration in the composition of the surrounding steady-state planktonic community. Our experimentally-tractable model enables the systematic study of planktonic and biofilm communities under conditions that are nutritionally reminiscent of the CF airway microenvironment, something not possible using any existing in vivo models of CF airway infection.

Short-chain reactive probes as tools to unravel the Pseudomonas aeruginosa quorum sensing regulon.

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed "quorum sensing" (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C4-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a 'click' tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.

Virulence and antimicrobial resistance genes are enriched in the plasmidome of clinical Escherichia coli isolates compared with wastewater isolates from western Kenya.

Many low-middle income countries in Africa have poorly-developed infectious disease monitoring systems. Here, we employed whole genome sequencing (WGS) to investigate the presence/absence of antimicrobial resistance (AMR) and virulence-associated (VA) genes in a collection of clinical and municipal wastewater Escherichia coli isolates from Kakamega, west Kenya. We were particularly interested to see whether, given the association between infection and water quality, the isolates from these geographically-linked environments might display similar genomic signatures. Phylogenetic analysis based on the core genes common to all of the isolates revealed two broad divisions, corresponding to the commensal/enterotoxigenic E. coli on the one hand, and uropathogenic E. coli on the other. Although the clinical and wastewater isolates each contained a very similar mean number of antibiotic resistance-encoding genes, the clinical isolates were enriched in genes required for in-host survival. Furthermore, and although the chromosomally encoded repertoire of these genes was similar in all sequenced isolates, the genetic composition of the plasmids from clinical and wastewater E. coli was more habitat-specific, with the clinical isolate plasmidome enriched in AMR and VA genes. Intriguingly, the plasmid-borne VA genes were often duplicates of genes already present on the chromosome, whereas the plasmid-borne AMR determinants were more specific. This reinforces the notion that plasmids are a primary means by which infection-related AMR and VA-associated genes are acquired and disseminated among these strains.

Arming the troops: Post-translational modification of extracellular bacterial proteins.

Protein secretion is almost universally employed by bacteria. Some proteins are retained on the cell surface, whereas others are released into the extracellular milieu, often playing a key role in virulence. In this review, we discuss the diverse types and potential functions of post-translational modifications (PTMs) occurring to extracellular bacterial proteins.

Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in elongation factor, fusA1.

Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression.

Divergent Synthesis of Novel Cylindrocyclophanes that Inhibit Methicillin-Resistant Staphylococcus aureus (MRSA).

The cylindrocyclophanes are a family of macrocyclic natural products reported to exhibit antibacterial activity. Little is known about the structural basis of this activity due to the challenges associated with their synthesis or isolation. We hypothesised that structural modification of the cylindrocyclophane scaffold could streamline their synthesis without significant loss of activity. Herein, we report a divergent synthesis of the cylindrocyclophane core enabling access to symmetrical macrocycles by means of a catalytic, domino cross-metathesis-ring-closing metathesis cascade, followed by late-stage diversification. Phenotypic screening identified several novel inhibitors of methicillin-resistant Staphylococcus aureus. The most potent inhibitor has a unique tetrabrominated [7,7]paracyclophane core with no known counterpart in nature. Together these illustrate the potential of divergent synthesis using catalysis and unbiased screening methods in modern antibacterial discovery.