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Associations between leguminous plants and symbiotic nitrogen-fixing rhizobia are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Although this symbiosis is in part species specific, different rhizobial strains may colonize the same nodule. Some rhizobial strains are commonly known as better competitors than others, but detailed analyses that aim to predict rhizobial competitive abilities based on genomes are still scarce. Here, we performed a bacterial genome-wide association (GWAS) analysis to define the genomic determinants related to the competitive capabilities in the model rhizobial species Sinorhizobium meliloti. For this, 13 tester strains were green fluorescent protein (GFP) tagged and assayed versus 3 red fluorescent protein (RFP)-tagged reference competitor strains (Rm1021, AK83, and BL225C) in a Medicago sativa nodule occupancy test. Competition data and strain genomic sequences were employed to build a model for GWAS based on k-mers. Among the k-mers with the highest scores, 51 k-mers mapped on the genomes of four strains showing the highest competition phenotypes (>60% single strain nodule occupancy; GR4, KH35c, KH46, and SM11) versus BL225C. These k-mers were mainly located on the symbiosis-related megaplasmid pSymA, specifically on genes coding for transporters, proteins involved in the biosynthesis of cofactors, and proteins related to metabolism (e.g., fatty acids). The same analysis was performed considering the sum of single and mixed nodules obtained in the competition assays versus BL225C, retrieving k-mers mapped on the genes previously found and on vir genes. Therefore, the competition abilities seem to be linked to multiple genetic determinants and comprise several cellular components. IMPORTANCE Decoding the competitive pattern that occurs in the rhizosphere is challenging in the study of bacterial social interaction strategies. To date, the single-gene approach has mainly been used to uncover the bases of nodulation, but there is still a knowledge gap regarding the main features that a priori characterize rhizobial strains able to outcompete indigenous rhizobia. Therefore, tracking down which traits make different rhizobial strains able to win the competition for plant infection over other indigenous rhizobia will improve the strain selection process and, consequently, plant yield in sustainable agricultural production systems. We proved that a k-mer-based GWAS approach can efficiently identify the competition determinants of a panel of strains previously analyzed for their plant tissue occupancy using double fluorescent labeling. The reported strategy will be useful for detailed studies on the genomic aspects of the evolution of bacterial symbiosis and for an extensive evaluation of rhizobial inoculants.

In this study, we aimed to characterize the population structure, drug resistance mechanisms, and virulence genes of Enterococcus isolates in Estonia. Sixty-one Enterococcus faecalis and 34 Enterococcus faecium isolates were collected between 2012 and 2014 across the country from various sites and sources, including farm animals and poultry (n = 53), humans (n = 12), environment (n = 24), and wild birds (n = 44). Clonal relationships of the strains were determined by whole-genome sequencing and analyzed by multi-locus sequence typing. We determined the presence of acquired antimicrobial resistance genes and 23S rRNA mutations, virulence genes, and also the plasmid or chromosomal origin of the genes using dedicated DNA sequence analysis tools available and/or homology search against an ad hoc compiled database of relevant sequences. Two E. faecalis isolates from human with vanB genes were highly resistant to vancomycin. Closely related E. faecalis strains were isolated from different host species. This indicates interspecies spread of strains and potential transfer of antibiotic resistance. Genomic context analysis of the resistance genes indicated frequent association with plasmids and mobile genetic elements. Resistance genes are often present in the identical genetic context in strains with diverse origins, suggesting the occurrence of transfer events.

Associations between leguminous plants and symbiotic nitrogen fixing bacteria (rhizobia) are a classical example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Though being in part species-specific, different strains may colonize the same plant symbiotic structure (nodule). It is known that some rhizobial strains are better competitor than others, but detailed analyses aimed to predict from the rhizobial genome its competitive abilities are still scarce. Here we performed a bacterial genome wide association (GWAS) analysis to define the genomic determinants related to the competitive capabilities in the model rhizobial species Sinorhizobium meliloti. Thirteen tester strains were GFP-tagged and assayed against three reference competitor strains RFP-tagged (Rm1021, AK83 and BL225C) in a Medicago sativa nodule occupancy test. Competition data in combination with strains genomic sequences were used to build-up a model for GWAS based on k-mers. The model was then trained and applied for competition capabilities prediction. The model was able to well predict the competition abilities against two partners, BL225C, Rm1021 with coefficient of determination of 0.96 and 0.84, respectively. Four strains showing the highest competition phenotypes (> 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) versus BL225C were used to identify k-mers associated with competition. The k-mers with highest scores mapped on the symbiosis-related megaplasmid pSymA and on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (i.e. glycerol, fatty acids) suggesting that competition abilities reside in multiple genetic determinants comprising several cellular components.IMPORTANCEDecoding the competitive pattern that occurs in the rhizosphere is challenging in the study of bacterial social interaction strategies. To date, single-gene approach has been mainly used to uncover the bases of nodulation, but there is still a gap about the main features that a priori turn out rhizobial strains able to outcompete indigenous rhizobia. Therefore, tracking down which traits make different rhizobial strains able to win the competition for plant infection over other indigenous rhizobia will allow ameliorating strain selection and consequently plant yield in sustainable agricultural production systems. We have proven that a k-mer based GWAS approach can effectively predict the competition abilities of a panel of strains, which were analyzed for their plant tissue occupancy by using double fluorescent labeling. The reported strategy could be used for detailed studies on the genomic aspects of the evolution of bacterial symbiosis and for an extensive evaluation of rhizobial inoculants.

We have developed an easy-to-use and memory-efficient method called PhenotypeSeeker that (a) identifies phenotype-specific k-mers, (b) generates a k-mer-based statistical model for predicting a given phenotype and (c) predicts the phenotype from the sequencing data of a given bacterial isolate. The method was validated on 167 Klebsiella pneumoniae isolates (virulence), 200 Pseudomonas aeruginosa isolates (ciprofloxacin resistance) and 459 Clostridium difficile isolates (azithromycin resistance). The phenotype prediction models trained from these datasets obtained the F1-measure of 0.88 on the K. pneumoniae test set, 0.88 on the P. aeruginosa test set and 0.97 on the C. difficile test set. The F1-measures were the same for assembled sequences and raw sequencing data; however, building the model from assembled genomes is significantly faster. On these datasets, the model building on a mid-range Linux server takes approximately 3 to 5 hours per phenotype if assembled genomes are used and 10 hours per phenotype if raw sequencing data are used. The phenotype prediction from assembled genomes takes less than one second per isolate. Thus, PhenotypeSeeker should be well-suited for predicting phenotypes from large sequencing datasets. PhenotypeSeeker is implemented in Python programming language, is open-source software and is available at GitHub (https://github.com/bioinfo-ut/PhenotypeSeeker/)., Author summary Predicting phenotypic properties of bacterial isolates from their genomic sequences has numerous potential applications. A good example would be prediction of antimicrobial resistance and virulence phenotypes for use in medical diagnostics. We have developed a method that is able to predict phenotypes of interest from the genomic sequence of the isolate within seconds. The method uses a statistical model that can be trained automatically on isolates with known phenotype. The method is implemented in Python programming language and can be run on low-end Linux server and/or on laptop computers.