Long-term evolution of antibiotic tolerance in Pseudomonas aeruginosa lung infections.

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as tolerance. While a variety of resistance mechanisms and underlying genetics are well characterized in vitro and in vivo, an understanding of the evolution of tolerance, and how it interacts with resistance in situ is lacking. We assayed for tolerance and resistance in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to 40 years of evolution, with 3 clinically relevant antibiotics: meropenem, ciprofloxacin, and tobramycin. We present evidence that tolerance is under positive selection in the lung and that it can act as an evolutionary stepping stone to resistance. However, by examining evolutionary patterns across multiple patients in different clone types, a key result is that the potential for an association between the evolution of resistance and tolerance is not inevitable, and difficult to predict.

Disentangling host-microbiota complexity through hologenomics.

Research on animal-microbiota interactions has become a central topic in biological sciences because of its relevance to basic eco-evolutionary processes and applied questions in agriculture and health. However, animal hosts and their associated microbial communities are still seldom studied in a systemic fashion. Hologenomics, the integrated study of the genetic features of a eukaryotic host alongside that of its associated microbes, is becoming a feasible - yet still underexploited - approach that overcomes this limitation. Acknowledging the biological and genetic properties of both hosts and microbes, along with the advantages and disadvantages of implemented techniques, is essential for designing optimal studies that enable some of the major questions in biology to be addressed.

A cognitive-behavioural intervention to reduce driving anger: Evaluation based on a mixed-method approach.

Road anger is an increasing problem that does not only lead to discomfort but is also associated with aggressive driving and an increased risk of crash involvement. Based on a cognitive-behavioural approach, we developed and tested a short group intervention (90 min) that aimed at decreasing road anger among drivers. We used a before-after control group design, surveying people who did (EG) and who did not (CG) participate in the group intervention. The sample (n = 126) consisted of 80 men and 46 women with a mean age of 54 years (SD = 13). In addition, a sub-sample (n = 37) participated in a driving simulator study before and after the intervention. To enrich the survey- and simulator-based findings, we conducted a focus group discussion about post-intervention experiences in traffic. Comparisons of pre and post survey results showed that constructive expression as measured by a slightly revised sub-scale of the Driving Anger Expression Inventory (DAX-short), increased significantly for EG participants but not within the CG. In addition, the involvement in milder forms of road anger (e.g., yelling) decreased significantly in the EG but not in the CG. In the simulator study, we observed that EG participants decreased the extent of aggressive driving, commenting and gesturing - however, none of these differences were statistically significant. EG participants self-assessed the intervention overall positive and 44 % reported that they had changed their way of thinking or reacting in traffic. Overall, results indicate a positive effect of the cognitive-behavioural group intervention. Study limitations, possibilities to improve the intervention and different areas of application are discussed.

Microbial evolutionary medicine: from theory to clinical practice.

Medicine and clinical microbiology have traditionally attempted to identify and eliminate the agents that cause disease. However, this traditional approach is becoming inadequate for dealing with a changing disease landscape. Major challenges to human health are non-communicable chronic diseases, often driven by altered immunity and inflammation, and communicable infections from agents which harbour antibiotic resistance. This Review focuses on the so-called evolutionary medicine framework, to study how microbial communities influence human health. The evolutionary medicine framework aims to predict and manipulate microbial effects on human health by integrating ecology, evolutionary biology, microbiology, bioinformatics, and clinical expertise. We focus on the potential of evolutionary medicine to address three key challenges: detecting microbial transmission, predicting antimicrobial resistance, and understanding microbe-microbe and human-microbe interactions in health and disease, in the context of the microbiome.

Privatisation rescues function following loss of cooperation.

A single cheating mutant can lead to the invasion and eventual eradication of cooperation from a population. Consequently, cheat invasion is often considered equal to extinction in empirical and theoretical studies of cooperator-cheat dynamics. But does cheat invasion necessarily equate extinction in nature? By following the social dynamics of iron metabolism in Pseudomonas aeruginosa during cystic fibrosis lung infection, we observed that individuals evolved to replace cooperation with a 'private' behaviour. Phenotypic assays showed that cooperative iron acquisition frequently was upregulated early in infection, which, however, increased the risk of cheat invasion. With whole-genome sequencing we showed that if, and only if, cooperative iron acquisition is lost from the population, a private system was upregulated. The benefit of upregulation depended on iron availability. These findings highlight the importance of social dynamics of natural populations and emphasizes the potential impact of past social interaction on the evolution of private traits.

Surveillance of Foodborne Pathogens: Towards Diagnostic Metagenomics of Fecal Samples.

Diagnostic metagenomics is a rapidly evolving laboratory tool for culture-independent tracing of foodborne pathogens. The method has the potential to become a generic platform for detection of most pathogens and many sample types. Today, however, it is still at an early and experimental stage. Studies show that metagenomic methods, from sample storage and DNA extraction to library preparation and shotgun sequencing, have a great influence on data output. To construct protocols that extract the complete metagenome but with minimal bias is an ongoing challenge. Many different software strategies for data analysis are being developed, and several studies applying diagnostic metagenomics to human clinical samples have been published, detecting, and sometimes, typing bacterial infections. It is possible to obtain a draft genome of the pathogen and to develop methods that can theoretically be applied in real-time. Finally, diagnostic metagenomics can theoretically be better geared than conventional methods to detect co-infections. The present review focuses on the current state of test development, as well as practical implementation of diagnostic metagenomics to trace foodborne bacterial infections in fecal samples from animals and humans.

Gene-Based Pathogen Detection: Can We Use qPCR to Predict the Outcome of Diagnostic Metagenomics?

In microbial food safety, molecular methods such as quantitative PCR (qPCR) and next-generation sequencing (NGS) of bacterial isolates can potentially be replaced by diagnostic shotgun metagenomics. However, the methods for pre-analytical sample preparation are often optimized for qPCR, and do not necessarily perform equally well for qPCR and sequencing. The present study investigates, through screening of methods, whether qPCR can be used as an indicator for the optimization of sample preparation for NGS-based shotgun metagenomics with a diagnostic focus. This was used on human fecal samples spiked with 10³ or 106 colony-forming units (CFU)/g Campylobacter jejuni, as well as porcine fecal samples spiked with 10³ or 106 CFU/g Salmonella typhimurium. DNA was extracted from the samples using variations of two widely used kits. The following quality parameters were measured: DNA concentration, qPCR, DNA fragmentation during library preparation, amount of DNA available for sequencing, amount of sequencing data, distribution of data between samples in a batch, and data insert size; none showed any correlation with the target ratio of the spiking organism detected in sequencing data. Surprisingly, diagnostic metagenomics can have better detection sensitivity than qPCR for samples spiked with 10³ CFU/g C. jejuni. The study also showed that qPCR and sequencing results may be different due to inhibition in one of the methods. In conclusion, qPCR cannot uncritically be used as an indicator for the optimization of sample preparation for diagnostic metagenomics.

Diversity, Prevalence, and Longitudinal Occurrence of Type II Toxin-Antitoxin Systems of Pseudomonas aeruginosa Infecting Cystic Fibrosis Lungs.

Type II toxin-antitoxin (TA) systems are most commonly composed of two genes encoding a stable toxin, which harms the cell, and an unstable antitoxin that can inactivate it. TA systems were initially characterized as selfish elements, but have recently gained attention for regulating general stress responses responsible for pathogen virulence, formation of drug-tolerant persister cells and biofilms-all implicated in causing recalcitrant chronic infections. We use a bioinformatics approach to explore the distribution and evolution of type II TA loci of the opportunistic pathogen, Pseudomonas aeruginosa, across longitudinally sampled isolates from cystic fibrosis lungs. We identify their location in the genome, mutations, and gain/loss during infection to elucidate their function(s) in stabilizing selfish elements and pathogenesis. We found (1) 26 distinct TA systems, where all isolates harbor four in their core genome and a variable number of the remaining 22 on genomic islands; (2) limited mutations in core genome TA loci, suggesting they are not under negative selection; (3) no evidence for horizontal transmission of elements with TA systems between clone types within patients, despite their ability to mobilize; (4) no gain and limited loss of TA-bearing genomic islands, and of those elements partially lost, the remnant regions carry the TA systems supporting their role in genomic stabilization; (5) no significant correlation between frequency of TA systems and strain ability to establish as chronic infection, but those with a particular TA, are more successful in establishing a chronic infection.

Towards diagnostic metagenomics of Campylobacter in fecal samples.

BACKGROUND: The development of diagnostic metagenomics is driven by the need for universal, culture-independent methods for detection and characterization of pathogens to substitute the time-consuming, organism-specific, and often culture-based laboratory procedures for epidemiological source-tracing. Some of the challenges in diagnostic metagenomics are, that it requires a great next-generation sequencing depth and unautomated data analysis. RESULTS: DNA from human fecal samples spiked with 7.75 × 101-7.75 × 107 colony forming unit (CFU)/ml Campylobacter jejuni and chicken fecal samples spiked with 1 × 102-1 × 106 CFU/g Campylobacter jejuni was sequenced and data analysis was done by the metagenomic tools Kraken and CLARK. More hits were obtained at higher spiking levels, however with no significant linear correlations (human samples p = 0.12, chicken samples p = 0.10). Therefore, no definite detection limit could be determined, but the lowest spiking levels found positive were 7.75 × 104 CFU/ml in human feces and 103 CFU/g in chicken feces. Eight human clinical fecal samples with estimated Campylobacter infection loads from 9.2 × 104-1.0 × 109 CFU/ml were analyzed using the same methods. It was possible to detect Campylobacter in all the clinical samples. CONCLUSIONS: Sensitivity in diagnostic metagenomics is improving and has reached a clinically relevant level. There are still challenges to overcome before real-time diagnostic metagenomics can replace quantitative polymerase chain reaction (qPCR) or culture-based surveillance and diagnostics, but it is a promising new technology.

Sociomics: Using Omic Approaches to Understand Social Evolution.

All of life is social, from genes cooperating to form organisms, to animals cooperating to form societies. Omic approaches offer exceptional opportunities to solve major outstanding problems in the study of how sociality evolves. First, omics can be used to clarify the extent and form of sociality in natural populations. This is especially useful in species where it is difficult to study social traits in natural populations, such as bacteria and other microbes. Second, omics can be used to examine the consequences of sociality for genome evolution and gene expression. This is especially useful in cases where there is clear variation in the level of sociality, such as the social insects. Major tasks for the future are to apply these approaches to a wider range of non-model organisms, and to move from exploratory analyses to the testing of evolutionary theory.

Fast Selective Detection of Pyocyanin Using Cyclic Voltammetry.

Pyocyanin is a virulence factor uniquely produced by the pathogen Pseudomonas aeruginosa. The fast and selective detection of pyocyanin in clinical samples can reveal important information about the presence of this microorganism in patients. Electrochemical sensing of the redox-active pyocyanin is a route to directly quantify pyocyanin in real time and in situ in hospitals and clinics. The selective quantification of pyocyanin is, however, limited by other redox-active compounds existing in human fluids and by other metabolites produced by pathogenic bacteria. Here we present a direct selective method to detect pyocyanin in a complex electroactive environment using commercially available electrodes. It is shown that cyclic voltammetry measurements between -1.0 V to 1.0 V reveal a potential detection window of pyocyanin of 0.58-0.82 V that is unaffected by other redox-active interferents. The linear quantification of pyocyanin has an R² value of 0.991 across the clinically relevant concentration range of 2-100 µM. The proposed method was tested on human saliva showing a standard deviation of 2.5% ± 1% (n = 5) from the known added pyocyanin concentration to the samples. This inexpensive procedure is suggested for clinical use in monitoring the presence and state of P. aeruginosa infection in patients.

Long-term social dynamics drive loss of function in pathogenic bacteria.

Laboratory experiments show that social interactions between bacterial cells can drive evolutionary change at the population level, but significant challenges limit attempts to assess the relevance of these findings to natural populations, where selection pressures are unknown. We have increasingly sophisticated methods for monitoring phenotypic and genotypic dynamics in bacteria causing infectious disease, but in contrast, we lack evidence-based adaptive explanations for those changes. Evolutionary change during infection is often interpreted as host adaptation, but this assumption neglects to consider social dynamics shown to drive evolutionary change in vitro. We provide evidence to show that long-term behavioral dynamics observed in a pathogen are driven by selection to outcompete neighboring conspecific cells through social interactions. We find that Pseudomonas aeruginosa bacteria, causing lung infections in patients with cystic fibrosis, lose cooperative iron acquisition by siderophore production during infection. This loss could be caused by changes in iron availability in the lung, but surprisingly, we find that cells retain the ability to take up siderophores produced by conspecifics, even after they have lost the ability to synthesize siderophores. Only when cooperative producers are lost from the population is the receptor for uptake lost. This finding highlights the potential pitfalls of interpreting loss of function in pathogenic bacterial populations as evidence for trait redundancy in the host environment. More generally, we provide an example of how sequence analysis can be used to generate testable hypotheses about selection driving long-term phenotypic changes of pathogenic bacteria in situ.

Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics.

The efficiency of ten widely applied DNA extraction protocols was evaluated for suitability for diagnostic metagenomics. The protocols were selected based on a thorough literature study. Chicken fecal samples inoculated with about 1×10(3) and 1×10(6) CFU/g Campylobacter jejuni were used as a model. The evaluation was performed based on total DNA yield measured by fluorometry, and quality and quantity of C. jejuni DNA measured by real-time PCR. There was up to a 25-fold variance between the lowest (NucliSens miniMAG, BIOMÉRIEUX) and highest (PowerLyzer PowerSoil DNA Isolation Kit, MO BIO Laboratories) yielding protocols. The PowerLyzer PowerSoil DNA Isolation Kit performed significantly better than all other protocols tested. Selected protocols were modified, i.e., extended heating and homogenization, resulting in increased yields of total DNA. For QIAamp Fast DNA Stool Mini Kit (Qiagen) a 7-fold increase in total DNA was observed following the protocol for human DNA analysis and including a 5 min heating step at 70°C. For the PowerLyzer PowerSoil and the PowerFecal DNA Isolation Kit (MO BIO Laboratories) the total DNA fold increase was 1.6 to 1.8 when including an extra 10 min of bead-vortexing. There was no correlation between the yield of total DNA and the amount of PCR-amplifiable DNA from C. jejuni. The protocols resulting in the highest yield of total DNA did not show correspondingly increased levels of C. jejuni DNA as determined by PCR. In conclusion, substantial variation in the efficiency of the protocols to extract DNA was observed. The highest DNA yield was obtained with the PowerLyzer PowerSoil DNA Isolation Kit, whereas the FastDNA SPIN Kit for Feces (MP Biomedicals) resulted in the highest amount of PCR-amplifiable C. jejuni DNA.

Interaction specificity between leaf-cutting ants and vertically transmitted Pseudonocardia bacteria.

BACKGROUND: The obligate mutualism between fungus-growing ants and microbial symbionts offers excellent opportunities to study the specificity and stability of multi-species interactions. In addition to cultivating fungus gardens, these ants have domesticated actinomycete bacteria to defend gardens against the fungal parasite Escovopsis and possibly other pathogens. Panamanian Acromyrmex echinatior leaf-cutting ants primarily associate with actinomycetes of the genus Pseudonocardia. Colonies are inoculated with one of two vertically transmitted phylotypes (Ps1 or Ps2), and maintain the same phylotype over their lifetime. We performed a cross-fostering experiment to test whether co-adaptations between ants and bacterial phylotypes have evolved, and how this affects bacterial growth and ant prophylactic behavior after infection with Escovopsis. RESULTS: We show that Pseudonocardia readily colonized ants irrespective of their colony of origin, but that the Ps2 phylotype, which was previously shown to be better able to maintain its monocultural integrity after workers became foragers than Ps1, reached a higher final cover when grown on its native host than on alternative hosts. The frequencies of major grooming and weeding behaviors co-varied with symbiont/host combinations, showing that ant behavior also was affected when cuticular actinomycete phylotypes were swapped. CONCLUSION: These results show that the interactions between leaf-cutting ants and Pseudonocardia bear signatures of mutual co-adaptation within a single ant population.

The novel somatostatin receptor 2/dopamine type 2 receptor chimeric compound BIM-23A758 decreases the viability of human GOT1 midgut carcinoid cells.

The majority of neuroendocrine tumors (NETs) of the gastroenteropancreatic system coexpress somatostatin receptors (SSTRs) and dopamine type 2 receptors (D2R), thus providing a rationale for the use of novel SSTR2/D2R chimeric compounds in NET disease. Here we investigate the antitumor potential of the SSTR2/D2R chimeric compounds BIM-23A760 and BIM-23A758 in comparison to the selective SSTR2 agonist BIM-23023 and the selective D2R agonist BIM-53097 on human NET cell lines of heterogeneous origin. While having only minor effects on human pancreatic and bronchus carcinoid cells (BON1 and NCI-H727), BIM-23A758 induced significant antitumor effects in human midgut carcinoid cells (GOT1). These effects involved apoptosis induction as well as inhibition of mitogen-activated protein kinase and Akt signaling. Consistent with their antitumor response to BIM-23A758, GOT1 cells showed relatively high expression levels of SSTR2 and D2R mRNA. In particular, GOT1 cells highly express the short transcript variant of D2R. In contrast to BIM-23A758, the SSTR2/D2R chimeric compound BIM-23A760 as well as the individual SSTR2 and D2R agonistic compounds BIM-23023 and BIM-53097 induced no or only minor antitumor responses in the examined NET cell lines. Taken together, our findings suggest that the novel SSTR2/D2R chimeric compound BIM-23A758 might be a promising substance for the treatment of NETs highly expressing SSTR2 and D2R. In particular, a sufficient expression of the short transcript variant of DR2 might play a pivotal role for effective treatment.

Veillonella, Firmicutes: Microbes disguised as Gram negatives.

The Firmicutes represent a major component of the intestinal microflora. The intestinal Firmicutes are a large, diverse group of organisms, many of which are poorly characterized due to their anaerobic growth requirements. Although most Firmicutes are Gram positive, members of the class Negativicutes, including the genus Veillonella, stain Gram negative. Veillonella are among the most abundant organisms of the oral and intestinal microflora of animals and humans, in spite of being strict anaerobes. In this work, the genomes of 24 Negativicutes, including eight Veillonella spp., are compared to 20 other Firmicutes genomes; a further 101 prokaryotic genomes were included, covering 26 phyla. Thus a total of 145 prokaryotic genomes were analyzed by various methods to investigate the apparent conflict of the Veillonella Gram stain and their taxonomic position within the Firmicutes. Comparison of the genome sequences confirms that the Negativicutes are distantly related to Clostridium spp., based on 16S rRNA, complete genomic DNA sequences, and a consensus tree based on conserved proteins. The genus Veillonella is relatively homogeneous: inter-genus pair-wise comparison identifies at least 1,350 shared proteins, although less than half of these are found in any given Clostridium genome. Only 27 proteins are found conserved in all analyzed prokaryote genomes. Veillonella has distinct metabolic properties, and significant similarities to genomes of Proteobacteria are not detected, with the exception of a shared LPS biosynthesis pathway. The clade within the class Negativicutes to which the genus Veillonella belongs exhibits unique properties, most of which are in common with Gram-positives and some with Gram negatives. They are only distantly related to Clostridia, but are even less closely related to Gram-negative species. Though the Negativicutes stain Gram-negative and possess two membranes, the genome and proteome analysis presented here confirm their place within the (mainly) Gram positive phylum of the Firmicutes. Further studies are required to unveil the evolutionary history of the Veillonella and other Negativicutes.

Host specificity of parasite manipulation: Zombie ant death location in Thailand vs. Brazil.

Recently we presented how Camponotus ants in Thailand infected with the fungus Ophiocordyceps unilateralis are behaviorally manipulated into dying where the conditions are optimal for fungal development. Death incurred in a very narrow zone of space and here we compare this highly specific manipulation with a related system in Brazil. We show that the behavioral manipulation is less fine-tuned and discuss the potential explanations for this by examining differences in ant host and environmental characteristics.

Disease dynamics in a specialized parasite of ant societies.

Coevolution between ant colonies and their rare specialized parasites are intriguing, because lethal infections of workers may correspond to tolerable chronic diseases of colonies, but the parasite adaptations that allow stable coexistence with ants are virtually unknown. We explore the trade-offs experienced by Ophiocordyceps parasites manipulating ants into dying in nearby graveyards. We used field data from Brazil and Thailand to parameterize and fit a model for the growth rate of graveyards. We show that parasite pressure is much lower than the abundance of ant cadavers suggests and that hyperparasites often castrate Ophiocordyceps. However, once fruiting bodies become sexually mature they appear robust. Such parasite life-history traits are consistent with iteroparity--a reproductive strategy rarely considered in fungi. We discuss how tropical habitats with high biodiversity of hyperparasites and high spore mortality has likely been crucial for the evolution and maintenance of iteroparity in parasites with low dispersal potential.

Behavioral mechanisms and morphological symptoms of zombie ants dying from fungal infection.

BACKGROUND: Parasites that manipulate host behavior can provide prominent examples of extended phenotypes: parasite genomes controlling host behavior. Here we focus on one of the most dramatic examples of behavioral manipulation, the death grip of ants infected by Ophiocordyceps fungi. We studied the interaction between O. unilateralis s.l. and its host ant Camponotus leonardi in a Thai rainforest, where infected ants descend from their canopy nests down to understory vegetation to bite into abaxial leaf veins before dying. Host mortality is concentrated in patches (graveyards) where ants die on sapling leaves ca. 25 cm above the soil surface where conditions for parasite development are optimal. Here we address whether the sequence of ant behaviors leading to the final death grip can also be interpreted as parasite adaptations and describe some of the morphological changes inside the heads of infected workers that mediate the expression of the death grip phenotype. RESULTS: We found that infected ants behave as zombies and display predictable stereotypical behaviors of random rather than directional walking, and of repeated convulsions that make them fall down and thus precludes returning to the canopy. Transitions from erratic wandering to death grips on a leaf vein were abrupt and synchronized around solar noon. We show that the mandibles of ants penetrate deeply into vein tissue and that this is accompanied by extensive atrophy of the mandibular muscles. This lock-jaw means the ant will remain attached to the leaf after death. We further present histological data to show that a high density of single celled stages of the parasite within the head capsule of dying ants are likely to be responsible for this muscular atrophy. CONCLUSIONS: Extended phenotypes in ants induced by fungal infections are a complex example of behavioral manipulation requiring coordinated changes of host behavior and morphology. Future work should address the genetic basis of such extended phenotypes.