A validated pangenome-scale metabolic model for the Klebsiella pneumoniae species complex.

The Klebsiella pneumoniae species complex (KpSC) is a major source of nosocomial infections globally with high rates of resistance to antimicrobials. Consequently, there is growing interest in understanding virulence factors and their association with cellular metabolic processes for developing novel anti-KpSC therapeutics. Phenotypic assays have revealed metabolic diversity within the KpSC, but metabolism research has been neglected due to experiments being difficult and cost-intensive. Genome-scale metabolic models (GSMMs) represent a rapid and scalable in silico approach for exploring metabolic diversity, which compile genomic and biochemical data to reconstruct the metabolic network of an organism. Here we use a diverse collection of 507 KpSC isolates, including representatives of globally distributed clinically relevant lineages, to construct the most comprehensive KpSC pan-metabolic model to date, KpSC pan v2. Candidate metabolic reactions were identified using gene orthology to known metabolic genes, prior to manual curation via extensive literature and database searches. The final model comprised a total of 3550 reactions, 2403 genes and can simulate growth on 360 unique substrates. We used KpSC pan v2 as a reference to derive strain-specific GSMMs for all 507 KpSC isolates, and compared these to GSMMs generated using a prior KpSC pan-reference (KpSC pan v1) and two single-strain references. We show that KpSC pan v2 includes a greater proportion of accessory reactions (8.8 %) than KpSC pan v1 (2.5 %). GSMMs derived from KpSC pan v2 also generate more accurate growth predictions, with high median accuracies of 95.4 % (aerobic, n=37 isolates) and 78.8 % (anaerobic, n=36 isolates) for 124 matched carbon substrates. KpSC pan v2 is freely available at https://github.com/kelwyres/KpSC-pan-metabolic-model, representing a valuable resource for the scientific community, both as a source of curated metabolic information and as a reference to derive accurate strain-specific GSMMs. The latter can be used to investigate the relationship between KpSC metabolism and traits of interest, such as reservoirs, epidemiology, drug resistance or virulence, and ultimately to inform novel KpSC control strategies.

A large-scale genomic snapshot of Klebsiella spp. isolates in Northern Italy reveals limited transmission between clinical and non-clinical settings.

The Klebsiella group, found in humans, livestock, plants, soil, water and wild animals, is genetically and ecologically diverse. Many species are opportunistic pathogens and can harbour diverse classes of antimicrobial resistance genes. Healthcare-associated Klebsiella pneumoniae clones that are non-susceptible to carbapenems can spread rapidly, representing a high public health burden. Here we report an analysis of 3,482 genome sequences representing 15 Klebsiella species sampled over a 17-month period from a wide range of clinical, community, animal and environmental settings in and around the Italian city of Pavia. Northern Italy is a hotspot for hospital-acquired carbapenem non-susceptible Klebsiella and thus a pertinent setting to examine the overlap between isolates in clinical and non-clinical settings. We found no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside the clinical environment. Although we noted occasional transmission between clinical and non-clinical settings, our data point to a limited role of animal and environmental reservoirs in the human acquisition of Klebsiella spp. We also provide a detailed genus-wide view of genomic diversity and population structure, including the identification of new groups.

A curated collection of Klebsiella metabolic models reveals variable substrate usage and gene essentiality.

The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa that are found in a variety of niches and are an important cause of opportunistic health care-associated infections in humans. Because of increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome-scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulfur, and phosphorus substrates. Models were curated and their accuracy was assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions using growth simulations in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species, and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community.

A Phloem-Feeding Insect Transfers Bacterial Endophytic Communities between Grapevine Plants.

Bacterial endophytes colonize the inner tissues of host plants through the roots or through discontinuities on the plant surface, including wounds and stomata. Little is known regarding a possible role of insects in acquiring and transmitting non-phytopathogenic microorganisms from plant to plant, especially those endophytes that are beneficial symbionts providing plant protection properties and homeostatic stability to the host. To understand the ecological role of insects in the transmission of endophytic bacteria, we used freshly hatched nymphs of the American sap-feeding leafhopper Scaphoideus titanus (vector) to transfer microorganisms across grapevine plants. After contact with the vector, sink plants were colonized by a complex endophytic community dominated by Proteobacteria, highly similar to that present in source plants. A similar bacterial community, but with a higher ratio of Firmicutes, was found on S. titanus. Insects feeding only on sink plants transferred an entirely different bacterial community dominated by Actinobacteria, where Mycobacterium sp., played a major role. Despite the fact that insects dwelled mostly on plant stems, the bacterial communities in plant roots resembled more closely those inside and on insects, when compared to those of above-ground plant organs. We prove here the potential of insect vectors to transfer entire endophytic bacterial communities between plants. We also describe the role of plants and bacterial endophytes in establishing microbial communities in plant-feeding insects.

Whole-genome comparative analysis of virulence genes unveils similarities and differences between endophytes and other symbiotic bacteria.

Plant pathogens and endophytes co-exist and often interact with the host plant and within its microbial community. The outcome of these interactions may lead to healthy plants through beneficial interactions, or to disease through the inducible production of molecules known as virulence factors. Unravelling the role of virulence in endophytes may crucially improve our understanding of host-associated microbial communities and their correlation with host health. Virulence is the outcome of a complex network of interactions, and drawing the line between pathogens and endophytes has proven to be conflictive, as strain-level differences in niche overlapping, ecological interactions, state of the host's immune system and environmental factors are seldom taken into account. Defining genomic differences between endophytes and plant pathogens is decisive for understanding the boundaries between these two groups. Here we describe the major differences at the genomic level between seven grapevine endophytic test bacteria, and 12 reference strains. We describe the virulence factors detected in the genomes of the test group, as compared to endophytic and non-endophytic references, to better understand the distribution of these traits in endophytic genomes. To do this, we adopted a comparative whole-genome approach, encompassing BLAST-based searches through the GUI-based tools Mauve and BRIG as well as calculating the core and accessory genomes of three genera of enterobacteria. We outline divergences in metabolic pathways of these endophytes and reference strains, with the aid of the online platform RAST. We present a summary of the major differences that help in the drawing of the boundaries between harmless and harmful bacteria, in the spirit of contributing to a microbiological definition of endophyte.