Pain reflects the informational value of nociceptive inputs.

ABSTRACT: Pain perception and its modulation are fundamental to human learning and adaptive behavior. This study investigated the hypothesis that pain perception is tied to pain's learning function. Thirty-one participants performed a threat conditioning task where certain cues were associated with a possibility of receiving a painful electric shock. The cues that signaled potential pain or safety were regularly changed, requiring participants to continually establish new associations. Using computational models, we quantified participants' pain expectations and prediction errors throughout the task and assessed their relationship with pain perception and electrophysiological responses. Our findings suggest that subjective pain perception increases with prediction error, that is, when pain was unexpected. Prediction errors were also related to physiological nociceptive responses, including the amplitude of nociceptive flexion reflex and electroencephalography markers of cortical nociceptive processing (N1-P2-evoked potential and gamma-band power). In addition, higher pain expectations were related to increased late event-related potential responses and alpha/beta decreases in amplitude during cue presentation. These results further strengthen the idea of a crucial link between pain and learning and suggest that understanding the influence of learning mechanisms in pain modulation could help us understand when and why pain perception is modulated in health and disease.

Anti-phage defence through inhibition of virion assembly.

Bacteria have evolved diverse antiviral defence mechanisms to protect themselves against phage infection. Phages integrated into bacterial chromosomes, known as prophages, also encode defences that protect the bacterial hosts in which they reside. Here, we identify a type of anti-phage defence that interferes with the virion assembly pathway of invading phages. The protein that mediates this defence, which we call Tab (for 'Tail assembly blocker'), is constitutively expressed from a Pseudomonas aeruginosa prophage. Tab allows the invading phage replication cycle to proceed, but blocks assembly of the phage tail, thus preventing formation of infectious virions. While the infected cell dies through the activity of the replicating phage lysis proteins, there is no release of infectious phage progeny, and the bacterial community is thereby protected from a phage epidemic. Prophages expressing Tab are not inhibited during their own lytic cycle because they express a counter-defence protein that interferes with Tab function. Thus, our work reveals an anti-phage defence that operates by blocking virion assembly, thereby both preventing formation of phage progeny and allowing destruction of the infected cell due to expression of phage lysis genes.

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa.

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa.

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

Cluster-based psychological phenotyping and differences in anxiety treatment outcomes.

The identification of markers of mental health illness treatment response and susceptibility using personalized medicine has been elusive. In the context of psychological treatment for anxiety, we conducted two studies to identify psychological phenotypes with distinct characteristics related to: psychological intervention modalities (mindfulness training/awareness), mechanism of action (worry), and clinical outcome (generalized anxiety disorder scale scores). We also examined whether phenotype membership interacted with treatment response (Study 1) and mental health illness diagnosis (Studies 1-2). Interoceptive awareness, emotional reactivity, worry, and anxiety were assessed at baseline in treatment-seeking individuals (Study 1, n = 63) and from the general population (Study 2, n = 14,010). In Study 1, participants were randomly assigned to an app-delivered mindfulness program for anxiety for two months or treatment as usual. Changes in anxiety were assessed 1 and 2 months post-treatment initiation. In studies 1-2, three phenotypes were identified: 'severely anxious with body/emotional awareness' (cluster 1), 'body/emotionally unaware' (cluster 2), and 'non-reactive and aware' (cluster 3). Study 1's results revealed a significant treatment response relative to controls (ps < 0.001) for clusters 1 and 3, but not for cluster 2. Chi-square analyses revealed that phenotypes exhibited significantly different proportions of participants with mental health diagnoses (studies 1-2). These results suggest that psychological phenotyping can bring the application of personalized medicine into clinical settings.Registry name and URL: Developing a novel digital therapeutic for the treatment of generalized anxiety disorder https://clinicaltrials.gov/ct2/show/NCT03683472?term=judson+brewer&draw=1&rank=1 .Trial registration: Registered at clinicaltrials.gov (NCT03683472) on 25/09/2018.

App-Based Mindfulness Training Predicts Reductions in Smoking Behavior by Engaging Reinforcement Learning Mechanisms: A Preliminary Naturalistic Single-Arm Study.

Mindfulness training (MT) has been shown to influence smoking behavior, yet the involvement of reinforcement learning processes as underlying mechanisms remains unclear. This naturalistic, single-arm study aimed to examine slope trajectories of smoking behavior across uses of our app-based MT craving tool for smoking cessation, and whether this relationship would be mediated by the attenuating impact of MT on expected reward values of smoking. Our craving tool embedded in our MT app-based smoking cessation program was used by 108 participants upon the experience of cigarette cravings in real-world contexts. Each use of the tool involved mindful awareness to the experience of cigarette craving, a decision as to whether the participant wanted to smoke or ride out their craving with a mindfulness exercise, and paying mindful attention to the choice behavior and its outcome (contentment levels felt from engaging in the behavior). Expected reward values were computed using contentment levels experienced from the choice behavior as the reward signal in a Rescorla−Wagner reinforcement learning model. Multi-level mediation analysis revealed a significant decreasing trajectory of smoking frequency across MT craving tool uses and that this relationship was mediated by the negative relationship between MT and expected reward values (all ps < 0.001). After controlling for the mediator, the predictive relationship between MT and smoking was no longer significant (p < 0.001 before and p = 0.357 after controlling for the mediator). Results indicate that the use of our app-based MT craving tool is associated with negative slope trajectories of smoking behavior across uses, mediated by reward learning mechanisms. This single-arm naturalistic study provides preliminary support for further RCT studies examining the involvement of reward learning mechanisms underlying app-based mindfulness training for smoking cessation.

Awareness drives changes in reward value which predict eating behavior change: Probing reinforcement learning using experience sampling from mobile mindfulness training for maladaptive eating.

BACKGROUND AND AIMS: Maladaptive eating habits are a major cause of obesity and weight-related illness. The development of empirically-based approaches, such as mindfulness training (MT) that target accurate mechanisms of action to address these behaviors is therefore critical. Two studies were conducted to examine the impact of MT on maladaptive eating and determine the involvement of reinforcement learning mechanisms underlying these effects. METHODS: In Study1, maladaptive eating behaviors were assessed using self-report questionnaires at baseline and 8 weeks after an app-based MT intervention (n = 46). A novel mindful eating craving tool was embedded in our intervention to assess: eating behaviors (intake frequency/magnitude), and reward (contentment ratings) experienced after eating. Using a well-established reinforcement learning (Rescorla-Wagner) model, expected reward values (EV) were estimated as a function of contentment levels reported after eating. In Study2 (n = 1,119), craving tool assessments were examined in an independent sample using the app in a real-world naturalistic context. RESULTS: Study 1's results revealed a significant decrease in EV and eating behaviors across craving tool uses. In addition, changes in reward values predicted decreases in eating behaviors. Finally, Study 1's results revealed significant pre-post intervention reductions in self-reported eating behaviors. In Study2, we observed a significant decrease in EV, but not in eating behaviors, across craving tool uses. Study 2 also revealed a predictive relationship between EV and eating behaviors. DISCUSSION AND CONCLUSIONS: These results support the implementation of MT to prevent and treat maladaptive eating behaviors, which target reinforcement learning processes as mechanisms of action.

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.

Effects of Brief Mindfulness Interventions on the Interference Induced by Experimental Heat Pain on Cognition in Healthy Individuals.

Background: Pain captures attention and interferes with competing tasks demanding cognitive effort. Brief mindfulness interventions involving both conceptual learning and meditation exercises have been shown to improve attention and reduce pain sensitivity, and could potentially reduce pain interference. This study assesses the effect of a 5-day mindfulness intervention (20 min/day) on the interference produced by thermal pain on working memory performance using a 2-back task. Methods: Healthy participants were randomized into three groups exposed to mindfulness meditation training (n = 15), an active educational control intervention comprising only conceptual information on mindfulness (n = 15), or no intervention (n = 15). The two active interventions were administered in a dual-blind fashion and outcomes were assessed by research personnel blind to this allocation. Evaluation sessions were conducted before and after the interventions to assess the effect of pain on 2-back performance (pain interference). Importantly, both pain stimuli and the 2-back task were calibrated individually and in each session before assessing pain interference, thereby controlling for possible changes in baseline pain sensitivity and cognitive performance. Secondary outcomes included heat pain sensitivity, cold pain tolerance, cognitive inhibition, cognitive flexibility, and divided attention. Results: Manipulation checks confirmed that heat pain interferes with the performance of the working-memory task. Compared to the no-intervention control group, pain interference was significantly reduced following the conceptual intervention but not the meditation intervention, although a corollary analysis suggests the effect might be due to regression toward the mean caused by baseline imbalance in pain interference. Secondary outcomes also suggested an increase in pain tolerance in the conceptual learning group only. Discussion: A short mindfulness meditation intervention was insufficient to reduce pain interference but conceptual learning about mindfulness produced some unexpected benefits. Although the generalization of experimental findings to clinical pain conditions may be premature, these results highlight the importance of distinguishing the contribution of mindfulness education and meditation training in future studies. Understanding the effects of mindfulness training on pain regulation and management must take into consideration the multiple factors underlying this complex intervention.

Expected value and sensitivity to punishment modulate insular cortex activity during risky decision making.

The exact contribution of the insula to risky decision making remains unclear, as are the specific outcome parameters and inter-individual characteristics that modulate insular activity prior to a risky choice. This fMRI study examines the contributions of outcome valence, magnitude, probability, and expected value (EV) to insular activity during risky decision making, and explores the influence of sensitivity to reward and to punishment, and anxiety, to insular activity. Participants (N = 31) performed a gambling task requiring choice between two roulettes with different outcome magnitude, probability and EV, under gain and loss conditions separately, and filled questionnaires assessing sensitivity to punishment/reward, and state/trait anxiety. Parametric analyses were conducted to examine the modulation of brain activity during decision making in relation to each task parameter. Correlations were examined between insular activity and psychometric questionnaires. EV of the selected roulette was associated with right posterior insula activation during decision making. Higher sensitivity to punishment was associated with lower bilateral insular activation. These findings suggest that the right posterior insula is involved in tracking the EV of a risky option during decision making. The involvement of the insula when making risky decisions also appears to be influenced by inter-individual differences in sensitivity to punishment.

The Diverse Impacts of Phage Morons on Bacterial Fitness and Virulence.

The viruses that infect bacteria, known as phages, are the most abundant biological entity on earth. They play critical roles in controlling bacterial populations through phage-mediated killing, as well as through formation of bacterial lysogens. In this form, the survival of the phage depends on the survival of the bacterial host in which it resides. Thus, it is advantageous for phages to encode genes that contribute to bacterial fitness and expand the environmental niche. In many cases, these fitness factors also make the bacteria better able to survive in human infections and are thereby considered pathogenesis or virulence factors. The genes that encode these fitness factors, known as "morons," have been shown to increase bacterial fitness through a wide range of mechanisms and play important roles in bacterial diseases. This review outlines the benefits provided by phage morons in various aspects of bacterial life, including phage and antibiotic resistance, motility, adhesion and quorum sensing.

Reduced Fear-Conditioned Pain Modulation in Experienced Meditators: A Preliminary Study.

OBJECTIVE: Mindfulness-based practice is a form of cognitive/affective training that may help reduce suffering by attenuating maladaptive anticipatory processes. This study's objective was to examine the pain modulating impact of classical fear learning in meditation practitioners. METHODS: The hyperalgesic effects of pain expectation and uncertainty were assessed outside formal meditation in 11 experienced meditators (>1000 hours) compared with meditation-naive controls during a Pavlovian classical fear-conditioning paradigm involving two visual stimuli (CS+/CS-), one of which (CS+) co-terminated with a noxious electrical stimulus (unconditioned stimulus) on 50% of trials. A Rescorla-Wagner/Pearce-Hall hybrid model was fitted onto the conditioned skin conductance responses using computational modeling to estimate two learning parameters: expected shock probability and associability (i.e., uncertainty). RESULTS: Using a scale ranging between 0 (no pain) and 100 (extremely painful), meditators reported less pain (M = 19.9, SE = 5.1 for meditators, M = 32.4, SE = 2.4 for controls) but had comparable spinal motor responses (nociceptive flexion reflex) to the unconditioned stimulus. Multilevel mediation analyses revealed that meditators also exhibited reduced hyperalgesic effects of fear learning on higher-order pain responses but comparable effects on the nociceptive flexion reflex. These results suggest that mindfulness affects higher-order perceptual processes to a greater extent than from descending inhibitory controls. Furthermore, meditators showed reduced hyperalgesic effects of fear conditioning with no significant group difference in conditioned learning as evidenced by discriminative anticipatory skin conductance responses and learning parameters derived from computational modeling. CONCLUSIONS: These results highlight potential mechanisms underlying mindfulness-related hypoalgesia, relevant to clinical conditions in which repeated pain exposure might reinforce hyperalgesic processes through fear conditioning.

Phage Morons Play an Important Role in Pseudomonas aeruginosa Phenotypes.

The viruses that infect bacteria, known as phages, play a critical role in controlling bacterial populations in many diverse environments, including the human body. This control stems not only from phages killing bacteria but also from the formation of lysogens. In this state, the phage replication cycle is suppressed, and the phage genome is maintained in the bacterial cell in a form known as a prophage. Prophages often carry genes that benefit the host bacterial cell, since increasing the survival of the host cell by extension also increases the fitness of the prophage. These highly diverse and beneficial phage genes, which are not required for the life cycle of the phage itself, have been referred to as "morons," as their presence adds "more on" the phage genome in which they are found. While individual phage morons have been shown to contribute to bacterial virulence by a number of different mechanisms, there have been no systematic investigations of their activities. Using a library of phages that infect two different clinical isolates of P. aeruginosa, PAO1 and PA14, we compared the phenotypes imparted by the expression of individual phage morons. We identified morons that inhibit twitching and swimming motilities and observed an inhibition of the production of virulence factors such as rhamnolipids and elastase. This study demonstrates the scope of phage-mediated phenotypic changes and provides a framework for future studies of phage morons.IMPORTANCE Environmental and clinical isolates of the bacterium Pseudomonas aeruginosa frequently contain viruses known as prophages. These prophages can alter the virulence of their bacterial hosts through the expression of nonessential genes known as "morons." In this study, we identified morons in a group of Pseudomonas aeruginosa phages and characterized the effects of their expression on bacterial behaviors. We found that many morons confer selective advantages for the bacterial host, some of which correlate with increased bacterial virulence. This work highlights the symbiotic relationship between bacteria and prophages and illustrates how phage morons can help bacteria adapt to different selective pressures and contribute to human diseases.

Learned expectations and uncertainty facilitate pain during classical conditioning.

Pain spontaneously activates adaptive and dynamic learning processes affecting the anticipation of, and the responses to, future pain. Computational models of associative learning effectively capture the production and ongoing changes in conditioned anticipatory responses (eg, skin conductance response), but the impact of this dynamic process on unconditional pain responses remains poorly understood. Here, we investigated the dynamic modulation of pain and the nociceptive flexion reflex by fear learning in healthy human adult participants undergoing a classical conditioning procedure involving an acquisition, reversal and extinction phase. Conditioned visual stimuli (CS+) coterminated with a noxious transcutaneous stimulation applied to the sural nerve on 50% of trials (unconditioned stimuli). Expected pain probabilities and cue associability were estimated using computational modeling by fitting a hybrid learning model to skin conductance response elicited by the CS+. Multilevel linear regression analyses confirmed that trial-by-trial changes in expected pain and associability positively predict ongoing fluctuations in pain outcomes. Mediation analysis further demonstrated that both expected probability and associability affect pain perception through a direct effect and an indirect effect mediated by descending modulatory mechanisms affecting spinal nociceptive activity. Moderation analyses further showed that hyperalgesic effects of associability were larger in individuals reporting more harm vigilance and less emotional detachment. Higher harm vigilance was also associated with a stronger mediation of hyperalgesic effects by spinal processes. These results demonstrate how dynamic changes in pain can be explained by associative learning theory and that resilient attitudes towards fear/pain can attenuate the adverse impact of adaptive aversive learning processes on pain.

Membrane Translocation and Assembly of Sugar Polymer Precursors.

Bacterial polysaccharides play an essential role in cell viability, virulence, and evasion of host defenses. Although the polysaccharides themselves are highly diverse, the pathways by which bacteria synthesize these essential polymers are conserved in both Gram-negative and Gram-positive organisms. By utilizing a lipid linker, a series of glycosyltransferases and integral membrane proteins act in concert to synthesize capsular polysaccharide, teichoic acid, and teichuronic acid. The pathways used to produce these molecules are the Wzx/Wzy-dependent, the ABC-transporter-dependent, and the synthase-dependent pathways. This chapter will cover the initiation, synthesis of the various polysaccharides on the cytoplasmic face of the membrane using nucleotide sugar precursors, and export of the nascent chain from the cytoplasm to the extracellular milieu. As microbial glycobiology is an emerging field in Gram-positive bacteria research, parallels will be drawn to the more widely studied polysaccharide biosynthesis systems in Gram-negative species in order to provide greater understanding of these biologically significant molecules.

A Bacteriophage-Acquired O-Antigen Polymerase (Wzyß) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα.

Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and ß, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a ß-polymerase (Wzyß). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyß was determined using a dual phoA-lacZα reporter system wherein random 3' gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to ß-galactosidase activity. The topology of Wzyß developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg(254), Arg(270), Arg(272), and His(300) were found to be essential for Wzyß function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzy ß knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce ß-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzy ß under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and ß-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyß utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.

Application of Whole-Genome Sequencing Data for O-Specific Antigen Analysis and In Silico Serotyping of Pseudomonas aeruginosa Isolates.

Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed the Pseudomonas aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability often associated with P. aeruginosa isolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst, P. aeruginosa serotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data.

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium.

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

The Widespread Multidrug-Resistant Serotype O12 Pseudomonas aeruginosa Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters.

UNLABELLED: The O-specific antigen (OSA) in Pseudomonas aeruginosa lipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classified P. aeruginosa into 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how the P. aeruginosa OSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83 P. aeruginosa strains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinct P. aeruginosa strains. Specifically, we identified a "serotype island" ranging from 62 kb to 185 kb containing the P. aeruginosa O12 OSA gene cluster, an antibiotic resistance determinant (gyrA(C248T)), and other genes that have been transferred between P. aeruginosa strains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related to P. aeruginosa PA7. Acquisition and recombination of the "serotype island" resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings. IMPORTANCE: Infection rates in hospital settings by multidrug-resistant (MDR) Pseudomonas aeruginosa clones have increased during the past decades, and serotype O12 is predominant among these epidemic strains. It is not known why the MDR phenotype is associated with serotype O12 and how this clone type has emerged. This study shows that evolution of MDR O12 strains involved a switch from an ancestral O4 serotype to O12. Serotype switching was the result of horizontal transfer and genetic recombination of lipopolysaccharide (LPS) biosynthesis genes originating from an MDR taxonomic outlier P. aeruginosa strain. Moreover, the recombination event also resulted in acquisition of antibiotic resistance genes. These results impact on our understanding of MDR outbreak strain and serotype evolution and can potentially assist in better monitoring and prevention.

The D3 bacteriophage α-polymerase inhibitor (Iap) peptide disrupts O-antigen biosynthesis through mimicry of the chain length regulator Wzz in Pseudomonas aeruginosa.

Lysogenic bacteriophage D3 causes seroconversion of Pseudomonas aeruginosa PAO1 from serotype O5 to O16 by inverting the linkage between O-specific antigen (OSA) repeat units from α to ß. The OSA units are polymerized by Wzy to modal lengths regulated by Wzz1 and Wzz2. A key component of the D3 seroconversion machinery is the inhibitor of α-polymerase (Iap) peptide, which is able to solely suppress α-linked long-chain OSA production in P. aeruginosa PAO1. To establish the target specificity of Iap for Wzyα, changes in OSA phenotypes were examined via Western immunoblotting for wzz1 and wzz2 single-knockout strains, as well as a wzz1 wzz2 double knockout, following the expression of iap from a tuneable vector. Increased induction of Iap expression completely abrogated OSA production in the wzz1 wzz2 double mutant, while background levels of OSA production were still observed in either of the single mutants. Therefore, Iap inhibition of OSA biosynthesis was most effective in the absence of both Wzz proteins. Sequence alignment analyses revealed a high degree of similarity between Iap and the first transmembrane segment (TMS) of either Wzz1 or Wzz2. Various topology prediction analyses of the Iap sequence consistently predicted the presence of a single TMS, suggesting a propensity for Iap to insert itself into the inner membrane (IM). The compromised ability of Iap to abrogate Wzyα function in the presence of Wzz1 or Wzz2 provides compelling evidence that inhibition occurs after Wzyα inserts itself into the IM and is achieved through mimicry of the first TMS from the Wzz proteins of P. aeruginosa PAO1.