Microbes Against Humanity, a workshop game for horrible students: using a creative card game in higher education microbiology teaching.

Introducing creative workshops in higher education curricula, in addition to formal lectures, is an excellent way of reinforcing knowledge and encouraging creative thinking. In particular, the use of card games as a tool for inducing student engagement and enthusiasm has been reported to be a very effective approach. Here, we report an innovative card game-based workshop for use at the intermediate undergraduate level. The name of the game is Microbes Against Humanity and has been adapted from the widely known party game Cards Against Humanity, which is freely available under a creative commons licence. Overall, 64 students and two academics participated in this 2 h workshop. Our students found the workshop to be very enjoyable, considered it to be helpful for their learning and suggested interesting ideas for further improvement. In conclusion, it was shown that such exciting workshops can trigger students' enthusiasm for microbiology and enhance their learning potential.

The Genetics of Prey Susceptibility to Myxobacterial Predation: A Review, Including an Investigation into Pseudomonas aeruginosa Mutations Affecting Predation by Myxococcus xanthus.

Bacterial predation is a ubiquitous and fundamental biological process, which influences the community composition of microbial ecosystems. Among the best characterised bacterial predators are the myxobacteria, which include the model organism Myxococcus xanthus. Predation by M. xanthus involves the secretion of antibiotic metabolites and hydrolytic enzymes, which results in the lysis of prey organisms and release of prey nutrients into the extracellular milieu. Due to the generalist nature of this predatory mechanism, M. xanthus has a broad prey range, being able to kill and consume Gram-negative/positive bacteria and fungi. Potential prey organisms have evolved a range of behaviours which protect themselves from attack by predators. In recent years, several investigations have studied the molecular responses of a broad variety of prey organisms to M. xanthus predation. It seems that the diverse mechanisms employed by prey belong to a much smaller number of general "predation resistance" strategies. In this mini-review, we present the current state of knowledge regarding M. xanthus predation, and how prey organisms resist predation. As previous molecular studies of prey susceptibility have focussed on individual genes/metabolites, we have also undertaken a genome-wide screen for genes of Pseudomonas aeruginosa which contribute to its ability to resist predation. P. aeruginosa is a World Health Organisation priority 1 antibiotic-resistant pathogen. It is metabolically versatile and has an array of pathogenic mechanisms, leading to its prevalence as an opportunistic pathogen. Using a library of nearly 5,500 defined transposon insertion mutants, we screened for "prey genes", which when mutated allowed increased predation by a fluorescent strain of M. xanthus. A set of candidate "prey proteins" were identified, which shared common functional roles and whose nature suggested that predation resistance by P. aeruginosa requires an effective metal/oxidative stress system, an intact motility system, and mechanisms for de-toxifying antimicrobial peptides.

Transmission, adaptation and geographical spread of the Pseudomonas aeruginosa Liverpool epidemic strain.

The Liverpool epidemic strain (LES) is an important transmissible clonal lineage of Pseudomonas aeruginosa that chronically infects the lungs of people with cystic fibrosis (CF). Previous studies have focused on the genomics of the LES in a limited number of isolates, mostly from one CF centre in the UK, and from studies highlighting identification of the LES in Canada. Here we significantly extend the current LES genome database by genome sequencing 91 isolates from multiple CF centres across the UK, and we describe the comparative genomics of this large collection of LES isolates from the UK and Canada. Phylogenetic analysis revealed that the 145 LES genomes analysed formed a distinct clonal lineage when compared with the wider P. aeruginosa population. Notably, the isolates formed two clades: one associated with isolates from Canada, and the other associated with UK isolates. Further analysis of the UK LES isolates revealed clustering by clinic geography. Where isolates clustered closely together, the association was often supported by clinical data linking isolates or patients. When compared with the earliest known isolate, LESB58 (from 1988), many UK LES isolates shared common loss-of-function mutations, such as in genes gltR and fleR. Other loss-of-function mutations identified in previous studies as common adaptations during CF chronic lung infections were also identified in multiple LES isolates. Analysis of the LES accessory genome (including genomic islands and prophages) revealed variations in the carriage of large genomic regions, with some evidence for shared genomic island/prophage complement according to clinic location. Our study reveals divergence and adaptation during the spread of the LES, within the UK and between continents.

Differential transcription of expanded gene families in central carbon metabolism of Streptomyces coelicolor A3(2).

BACKGROUND: Streptomycete bacteria are prolific producers of specialized metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalization or sub-functionalization. To test whether expanded gene families in Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source. RESULTS: RNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialized metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source. CONCLUSIONS: Expression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters.

Genomic plasticity of pathogenic Escherichia coli mediates d-serine tolerance via multiple adaptive mechanisms.

The molecular environment of the host can have profound effects on the behavior of resident bacterial species. We recently established how the sensing and response of enterohemorrhagic Escherichia coli (EHEC) to d-serine (d-Ser) resulted in down-regulation of type 3 secretion system-dependent colonization, thereby avoiding unfavorable environments abundant in this toxic metabolite. However, this model ignores a key determinant of the success of bacterial pathogens, adaptive evolution. In this study, we have explored the adaptation of EHEC to d-Ser and its consequences for pathogenesis. We rapidly isolated multiple, independent, EHEC mutants whose growth was no longer compromised in the presence of d-Ser. Through a combination of whole-genome sequencing and transcriptomics, we showed that tolerance could be attributed to disruption of one of two d-Ser transporters and/or activation of a previously nonfunctional d-Ser deaminase. While the implication of cytoplasmic transport in d-Ser toxicity was unsurprising, disruption of a single transporter, CycA, was sufficient to completely overcome the repression of type 3 secretion system activity normally associated with exposure to d-Ser. Despite the fact that this reveals a mechanism by which evolution could drive a pathogen to colonize new niches, interrogation of sequenced E. coli O157:H7 genomes showed a high level of CycA conservation, highlighting a strong selective pressure for functionality. Collectively, these data show that CycA is a critically important conduit for d-Ser uptake that is central to the niche restriction of EHEC.

Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland.

Multi-locus sequencing typing (MLST) is widely used to monitor the phylogeny of microbial outbreaks. However, several strains of vancomycin-resistant Enterococcus faecium (VREfm) with a missing MLST locus (pstS) have recently emerged in Australia, with a few cases also reported in England. Here, we identified similarly distinct strains circulating in two neighbouring hospitals in Scotland. Whole genome sequencing of five VREfm strains isolated from these hospitals identified four pstS-null strains in both hospitals, while the fifth was multi-locus sequence type (ST) 262, which is the first documented in the UK. All five Scottish isolates had an insertion in the tetM gene, which is associated with increased susceptibility to tetracyclines, providing no other tetracycline-resistant gene is present. Such an insertion, which encompasses a dfrG gene and two currently uncharacterised genes, was additionally identified in all tested vanA-type pstS-null VREfm strains (5 English and 68 Australian). Phylogenetic comparison with other VREfm genomes indicates that the four pstS-null Scottish isolates sequenced in this study are more closely related to pstS-null strains from Australia rather than the English pstS-null isolates. Given how rapidly such pstS-null strains have expanded in Australia, the emergence of this clone in Scotland raises concerns for a potential outbreak.

Genomics of antibiotic-resistance prediction in Pseudomonas aeruginosa.

Antibiotic resistance is a worldwide health issue spreading quickly among human and animal pathogens, as well as environmental bacteria. Misuse of antibiotics has an impact on the selection of resistant bacteria, thus contributing to an increase in the occurrence of resistant genotypes that emerge via spontaneous mutation or are acquired by horizontal gene transfer. There is a specific and urgent need not only to detect antimicrobial resistance but also to predict antibiotic resistance in silico. We now have the capability to sequence hundreds of bacterial genomes per week, including assembly and annotation. Novel and forthcoming bioinformatics tools can predict the resistome and the mobilome with a level of sophistication not previously possible. Coupled with bacterial strain collections and databases containing strain metadata, prediction of antibiotic resistance and the potential for virulence are moving rapidly toward a novel approach in molecular epidemiology. Here, we present a model system in antibiotic-resistance prediction, along with its promises and limitations. As it is commonly multidrug resistant, Pseudomonas aeruginosa causes infections that are often difficult to eradicate. We review novel approaches for genotype prediction of antibiotic resistance. We discuss the generation of microbial sequence data for real-time patient management and the prediction of antimicrobial resistance.

Genomic characterisation of an international Pseudomonas aeruginosa reference panel indicates that the two major groups draw upon distinct mobile gene pools.

Pseudomonas aeruginosa is an important opportunistic pathogen, especially in the context of infections of cystic fibrosis (CF). In order to facilitate coordinated study of this pathogen, an international reference panel of P. aeruginosa isolates was assembled. Here we report the genome sequencing and analysis of 33 of these isolates and 7 reference genomes to further characterise this panel. Core genome single nucleotide variant phylogeny demonstrated that the panel strains are widely distributed amongst the P. aeruginosa population. Common loss-of-function mutations reported as adaptive during CF (such as in mucA and mexA) were identified amongst isolates from chronic respiratory infections. From the 40 strains analysed, 37 unique resistomes were predicted, based on the Resistance Gene Identifier method using the Comprehensive Antibiotic Resistance Database. Notably, hierarchical clustering and phylogenetic reconstructions based on the presence/absence of genomic islands (GIs), prophages and other regions of genome plasticity (RGPs) supported the subdivision of P. aeruginosa into two main groups. This is the largest, most diverse analysis of GIs and associated RGPs to date, and the results suggest that, at least at the largest clade grouping level (group 1 vs group 2), each group may be drawing upon distinct mobile gene pools.

Community-led comparative genomic and phenotypic analysis of the aquaculture pathogen Pseudomonas baetica a390T sequenced by Ion semiconductor and Nanopore technologies.

Pseudomonas baetica strain a390T is the type strain of this recently described species and here we present its high-contiguity draft genome. To celebrate the 16th International Conference on Pseudomonas, the genome of P. baetica strain a390T was sequenced using a unique combination of Ion Torrent semiconductor and Oxford Nanopore methods as part of a collaborative community-led project. The use of high-quality Ion Torrent sequences with long Nanopore reads gave rapid, high-contiguity and -quality, 16-contig genome sequence. Whole genome phylogenetic analysis places P. baetica within the P. koreensis clade of the P. fluorescens group. Comparison of the main genomic features of P. baetica with a variety of other Pseudomonas spp. suggests that it is a highly adaptable organism, typical of the genus. This strain was originally isolated from the liver of a diseased wedge sole fish, and genotypic and phenotypic analyses show that it is tolerant to osmotic stress and to oxytetracycline.

Discovery, characterization and in vivo activity of pyocin SD2, a protein antibiotic from Pseudomonas aeruginosa.

Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity in vivo in a murine model of P. aeruginosa lung infection. In addition, we propose a mechanism of cell targeting and translocation for pyocin SD2 across the P. aeruginosa outer membrane. Pyocin SD2 is concentrated at the cell surface, via binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide (LPS), from where it can efficiently locate its outer membrane receptor FpvAI. This strategy of utilizing both the CPA and a protein receptor for cell targeting is common among pyocins as we show that pyocins S2, S5 and SD3 also bind to the CPA. Additional data indicate a key role for an unstructured N-terminal region of pyocin SD2 in the subsequent translocation of the pyocin into the cell. These results greatly improve our understanding of how pyocins target and translocate across the outer membrane of P. aeruginosa. This knowledge could be useful for the development of novel anti-pseudomonal therapeutics and will also support the development of pyocin SD2 as a therapeutic in its own right.

Genes Required for Free Phage Production are Essential for Pseudomonas aeruginosa Chronic Lung Infections.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infection in patients with cystic fibrosis. The Liverpool Epidemic Strain LESB58 is highly resistant to antibiotics, transmissible, and associated with increased morbidity and mortality. Its genome contains 6 prophages and 5 genomic islands. We constructed a polymerase chain reaction (PCR)-based signature-tagged mutagenesis library of 9216 LESB58 mutants and screened the mutants in a rat model of chronic lung infection. A total of 162 mutants were identified as defective for in vivo maintenance, with 11 signature-tagged mutagenesis mutants having insertions in prophage and genomic island genes. Many of these mutants showed both diminished virulence and reduced phage production. Transcription profiling by quantitative PCR and RNA-Seq suggested that disruption of these prophages had a widespread trans-acting effect on the transcriptome. This study demonstrates that temperate phages play a pivotal role in the establishment of infection through modulation of bacterial host gene expression.

Phenotypic and Genotypic Characteristics of Small Colony Variants and Their Role in Chronic Infection.

Small colony variant (SCV) bacteria arise spontaneously within apparently homogeneous microbial populations, largely in response to environmental stresses, such as antimicrobial treatment. They display unique phenotypic characteristics conferred in part by heritable genetic changes. Characteristically slow growing, SCVs comprise a minor proportion of the population from which they arise but persist by virtue of their inherent resilience and host adaptability. Consequently, SCVs are problematic in chronic infection, where antimicrobial treatment is administered during the acute phase of infection but fails to eradicate SCVs, which remain within the host causing recurrent or chronic infection. This review discusses some of the phenotypic and genotypic changes that enable SCVs to successfully proliferate within the host environment as potential pathogens and strategies that could ameliorate the resolution of infection where SCVs are present.

Strategy for genome sequencing analysis and assembly for comparative genomics of Pseudomonas genomes.

Bacterial genome sequencing has developed rapidly in the last decade and has become a primary method for analyzing the genomic basis of differences in phenotype between strains as well as being a valuable tool for public health epidemiology. This chapter provides a comprehensive workflow for bacterial genome sequencing from experimental design to data suitable for comparative genomics analysis, while mainly focusing on the challenges associated with genome assembly. This approach was successfully applied to 19 Pseudomonas aeruginosa genomes from phenotypically distinct strains.

Transcriptional analysis of Pseudomonas aeruginosa infected Caenorhabditis elegans.

Here, we describe a protocol for the extraction of total RNA from C. elegans infected with P. aeruginosa. The protocol excludes P. aeruginosa cells that have not been ingested by the nematodes and yields total RNA that can be used for detection of transcripts from both host and pathogen.

Pseudomonas aeruginosa can be detected in a polymicrobial competition model using impedance spectroscopy with a novel biosensor.

Electrochemical Impedance Spectroscopy (EIS) is a powerful technique that can be used to elicit information about an electrode interface. In this article, we highlight six principal processes by which the presence of microorganisms can affect impedance and show how one of these--the production of electroactive metabolites--changes the impedance signature of culture media containing Pseudomonas aeruginosa. EIS, was used in conjunction with a low cost screen printed carbon sensor to detect the presence of P. aeruginosa when grown in isolation or as part of a polymicrobial infection with Staphylococcus aureus. By comparing the electrode to a starting measurement, we were able to identify an impedance signature characteristic of P. aeruginosa. Furthermore, we are able to show that one of the changes in the impedance signature is due to pyocyanin and associated phenazine compounds. The findings of this study indicate that it might be possible to develop a low cost sensor for the detection of P. aeruginosa in important point of care diagnostic applications. In particular, we suggest that a development of the device described here could be used in a polymicrobial clinical sample such as sputum from a CF patient to detect P. aeruginosa.

Lectin-like bacteriocins from Pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor.

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of D-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing D-rhamnose and not D-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.

Comparative genomics of isolates of a Pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients.

Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40-50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain's adaptability to varying conditions in the CF lung.

Draft genomes of 12 host-adapted and environmental isolates of Pseudomonas aeruginosa and their positions in the core genome phylogeny.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen particularly associated with the inherited disease cystic fibrosis (CF). Pseudomonas aeruginosa is well known to have a large and adaptable genome that enables it to colonise a wide range of ecological niches. Here, we have used a comparative genomics approach to identify changes that occur during infection of the CF lung. We used the mucoid phenotype as an obvious marker of host adaptation and compared these genomes to analyse SNPs, indels and islands within near-isogenic pairs. To commence the correction of the natural bias towards clinical isolates in genomics studies and to widen our understanding of the genomic diversity of P. aeruginosa, we included four environmental isolates in our analysis. Our data suggest that genome plasticity plays an important role in chronic infection and that the strains sequenced in this study are representative of the two major phylogenetic groups as determined by core genome SNP analysis.

Development of a diagnostic device to detect different Pseudomonas aeruginosa phenotypes in medically relevant contexts.

Pseudomonas aeruginosa, widely present in the environment, is well known for its ability to cause infection in immune compromised individuals. For example, P. aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). Here, we describe how Electrochemical Impedance Spectroscopy (EIS) can be used to detect the presence of four different strains of P. aeruginosa. Using a low cost, screen printed carbon electrode significant changes can be seen in impedance data in the presence of P. aeruginosa after 24 hours. Furthermore, through the use of a normalization technique whereby the phase angle of the impedance (a commonly used parameter) is divided by a starting measurement, it is possible to identify differences between a non-mucoid and mucoid strain of P. aeruginosa. Sensors based upon the techniques described here could be used in a number of healthcare scenarios, where there is a need for low cost, real time detection of P. aeruginosa, such as CF.