Pseudomonas aeruginosaPAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion,P. aeruginosaPAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.

The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45 ⁺ CD11b ⁺ CD14 ⁺ CD163 ⁺ cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa -mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.

Summary: The population genomics of P seudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P . aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892–7187 open reading frame ( ORFs ; median 6381 ORFs ) in the 20 6.4–7.4 M bp large genomes. The P . aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, G alleria mellonella and murine airway infection models the full spectrum of possible host responses to P . aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat‐independent gradient of virulence in the P . aeruginosa population.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity. The increase in hospital acquired and multi-drug resistant bacterial infections calls for an urgent development of new antimicrobials. As such, the identification and characterization of novel molecular targets involved in bacterial virulence has become a common goal for researchers. The use of non-mammalian hosts, such as the nematode Caenorhabditis elegans, is useful to accelerate this process. In our study, we developed a high-throughput screening method, which further facilitates the use of C. elegans, and allows the rapid screening of a large collection of bacterial mutants at the genomic scale. We have used Pseudomonas aeruginosa, a potent opportunistic pathogen, to perform this study. The screening of more than 2,000 mutant strains allowed the characterization of a mutant affected in the cheB2 gene. Importantly, this mutant was shown to be impaired in a mouse model of infection, supporting that our new screen is a good model to identify virulence genes relevant for infection in mammals. The cheB2 gene encodes a component of a chemotaxis pathway, which is likely involved in the perception of stimuli during the infection process, and allows an appropriate adaptive response for a successful infection. Our method could be applied to other bacterial pathogens and will help researchers discover candidate genes leading to the design of novel antimicrobials.