- Results: Analysis of the genomic content of 58 S. - Phylogenetic network analysis of the protein families encoded by the pan-genome indicated that genetic exchange between Salmonella and Citrobacter may have led to the acquisition of similar traits and also diversification within the genera.. - Comparative analysis of the core and pan-genomes was able to define the genetic features that distinguish Salmonella from Citrobacter and highlight niche specific adaptations.. - Salmonella enterica subspecies enterica is one of the leading causes of foodborne illnesses in the world. - 1 Bureau of Microbial Hazards, Health Canada, Ottawa, Ontario, Canada Full list of author information is available at the end of the article. - However, these differences can also exist among strains of the same serovar [7–9]. - The inclusion of additional inter- mediate strains bearing similarities to other bacterial species significantly increased the complexity of the genus [19]. - Subsequent analysis of the 16S rRNA sequence and multilocus sequence typing (MLST), indicated three phylogenetic groupings for Citrobacter: I (C. - Different regions of the ge- nomes, having different rates of recombination, may undergo genetic isolation due to sequence divergence and ecological adaptation. - The objectives of the analysis were to define the core and pan genome of this population of isolates and to identify regions contributing to their di- versity that may have impacted the evolution of Salmon- ella and Citrobacter.. - Characterization of Citrobacter strains falsely identified as Salmonella. - API20E provided identification profiles for four of the five strains as C. - A maximum likelihood phylogenetic tree based on the 16S rRNA sequence of the five false positive Citrobacter strains, seven unspeciated Citrobacter produce isolates and members of the family Enterobacteriaceae (Table S2 and Figure S1) demonstrated that members of the Citro- bacter genus did not form a distinct clade. - A comparative analysis of the 58 Salmonella and 37 Citrobacter genomes was performed to gain insight into the genetic differences between these two groups of bac- teria. - enterica strains chosen for this study encompass the diversity of the subspecies and include strains that may be associated with fresh pro- duce in Canada (Table 1). - Of the 38 serovars selected, 19 included representatives of serovars frequently associ- ated with clinical salmonellosis in Canada. - The genome size of the individual S. - Analysis of the orthologous relationships be- tween the pan-genome revealed the presence of 195 Citrobacter gene sequences that did not have ortholo- gues in Salmonella (i.e. - A maximum likelihood phylogenetic tree based on the genomic variations in the amino aicd sequences of the Salmonella-Citrobacter core genome effectively divided the 95 strains into one of two clades (Fig. - The top- ology of the Salmonella taxon differed from that of Citrobacter, showing low bootstrap values in some of the internal nodes and shorter genetic distances. - highest support included the clade consisting of the five Enteritidis and one Gallinarum strain, a result consistent with previous studies, as well as clades containing Typhi- murium and Heidelberg [24, 25]. - The core genes of serovar Pasing were the most divergent of the 58 Salmonella strains and formed the outgroup for this population.. - Overall, the topology of the Citrobacter group bore re- semblance to previous 16S rRNA and MLST-based trees that grouped 11 Citrobacter genomospecies into three previously described species complexes [15, 16]. - This portion of the tree had well-supported nodes with the population falling into one of three major clades. - farmeri strains that were closely related to four of the five strains falsely identified as Salmonella (S646, S647, S648, and S1285) (Fig. - Strain S1284 (red dot) was located in the third clade. - 1 Estimation of the Salmonella-Citrobacter pan- and core genome size. - The heterogeneity and polyphyletic origin of Citro- bacter species precluded the accurate taxonomic classification of the unspeciated strains in Fig. - Therefore, the core genomes of the Citrobacter strains in this study were compared to those of 160. - A maximum likelihood phylogenetic tree based on the variations in the amino acid sequences of the core genome resolved the population into twelve clades (Fig. - This grouping pat- tern enabled the identification of many of the. - unspeciated Citrobacter strains (Fig. - Eleven of the twenty-one unspeciated strains clus- tered with C. - Although this strain was found in the same cluster, where the majority of the strains were C. - Four of the five. - A maximum likelihood phylogenetic tree constructed from the concatenated core amino acid sequences of 197 Citrobacter strains was used to determine the taxonomic designation of the unspeciated Citrobacter strains in this study (yellow dots).. - For each triangle, the letters represent the group of related strains found in the branches of the node. - Functional analysis of Salmonella - Citrobacter core genes The functional classification of the following three sets of gene sequences were analyzed (Table S7). - A significant number of these sequences, ap- proximating 11% of the predicted core gene functions were designated as either poorly characterized or could not be classified into a KEGG pathway or BRITE. - Poorly characterized genes made up a respective 28 and 16% of the Salmonella specific and Citrobacter specific unique gene sequences.. - The majority of the core gene sequences were pre- dicted to encode proteins with roles in the major KEGG categories of metabolism (60. - Of the metabolic functions, the three major categories represented were for the metabolism of carbohydrates (17. - In comparison, the major KEGG categories repre- sented in the Salmonella-specific unique gene set were metabolism (54. - Of the metabolic processes, the three major categories were carbohydrate metabolism (13. - In contrast, the majority of the functions unique to Citrobacter were. - Network analysis of the Salmonella-Citrobacter pan- genome provided a complementary view of the evolu- tionary relationships between the population of strains.. - Figure 5 illustrates the genetic network of the Salmon- ella and Citrobacter strains based on the proportion of protein families shared between the two groups of bacteria. - At 26% shared gene families, all of the strains, regardless of their taxonomic designation, formed one community of interconnected members (Fig. - This recapitulates the results of the Salmonella-Citrobacter comparative genome analysis, wherein the core gene se- quences made up ~ 30% of individual genomes. - However, the network also depicted the separation of four of the Citrobacter strains that were falsely identified as Salmonella (red) from the rest of the Citrobacter network. - In contrast, all of the Salmon- ella strains maintained one network due to their genetic similarity.. - Analysis of the core and pan-genome of a population of Salmonella and Citrobacter strains offered a reliable de- lineation of the fuzzy taxonomic boundaries between these closely related bacteria. - As the most stable portion of the pan-genome, the core genome encodes character- istics common to all members of a species [28, 29].. - A recent study on the genomic structure of the Rickettsiales defined criteria to define genus and species assignments based on core genome alignments, with alignments ≥10% of the average input genome length and ≥ 96.8% identity respectively [30]. - A consensus tree based on se- quence variations of the amino acid sequences encoded by the Salmonella-Citrobacter core genes was able to re- solve the two bacterial species into two distinct clades and provided sufficient delineation to infer the taxo- nomic affiliation of the Citrobacter strains. - Analysis of 37 Citrobacter strains proved insufficient in determining the taxonomic assignment of many of the unspeciated strains in this study. - Adding more Citrobacter genomes to the analysis greatly increased the resolving power of the analysis. - Of the Citrobacter genomes investigated, the ones belonging to C. - freundii appeared to be the most divergent, appearing in seven of the 12 clades. - According to the results of the rarefaction analysis, the core genome defined in this study is believed to reflect the diversity of Salmonella and Citrobacter strains that may be associated with food commodities, specifically fresh produce in Canada. - A limitation of the current study is that the KEGG database is primarily intended to catalogue eukaryotic (human) cellular func- tions. - Just over half of the 1112 sequences identified as unique to our collection of Salmonella was mapped to cellular pathways. - The stability of the core genome makes it an indis- pensable tool for defining the genetic integrity of bacter- ial populations. - There was greater support for the evolutionary relationships observed between many of the Citrobacter strains in our study, presumably due to the greater diversity of that population. - Accessory and singleton gene sequences represent the most dynamic regions of the genomes. - Moreover, closely related species that are found in the same envir- onment, such as Salmonella and Citrobacter, are more interconnected and genetically similar [38]. - Genetic ex- change between members of the community network, through recombination or HGT, can contribute to the acquisition of traits enabling survival in fluctuating envi- ronments and potentially giving rise to strains with atyp- ical biochemical, phenotypic, and antigenic properties [20, 39]. - Phenotypic characterization of the five Citrobacter strains that were misidentified as Sal- monella provided variable results and were unable to un- ambiguously determine the species identity. - This method is also limited by the compre- hensiveness of the database used to compare spectra, which to date are heavily biased towards medically rele- vant pathogens [42]. - The limited variability in the conserved region of the. - Our results showed that four of the five Citrobacter strains falsely identified as Salmonella were phylogenetically similar and the prevalence of these strains in fresh produce and other food products deserves further investigation to help improve the detection of Salmonella. - Bacterial strains, genome sequencing, and assembly Table 1 provides a list of the 37 Citrobacter spp. - enterica strains included in the study.. - enterica enterica strains in this study were chosen to encompass the diversity of the subspecies and represent serovars frequently associated with outbreaks as well as serovars isolated from food and rarely associ- ated with human disease (grey box) [2, 3]. - Of the 38 serovars selected, 19 included representatives of serovars frequently associated with clinical salmonellosis in Canada. - All strains were sequenced at the Plateforme d’Ana- lyses Génomiques of the Institute for Integrative and Systems Biology (IBIS), Université Laval, Quebec, Canada using the procedures described by the Salmon- ella Syst-OMICS consortium [44]. - The complete set of gene sequences identified in all of the genomes was defined as the pangenome. - The core genome represented se- quences that were present in all of the analyzed genomes and the accessory genome was the set of sequences that were present in a sub-set of at least two genomes. - A rarefaction analysis was performed to estimate the size of the core genome of Salmonella and Citrobacter.. - The analysis was achieved by randomly selecting subsets (ranging 2–95 genomes) of the input samples and calcu- lating the core- and pan- counts. - The point of the curve at which additional number of genes incorporated into the core genome relative to the num- ber of analyzed genomes begins to plateau indicates the value needed for complete core genome representation for all included genomes.. - Phylogenetic trees based on variations in the amino acid sequences encoded by the core genome were con- structed using the tree module of centreseq [27]. - The numbers on the internal nodes of the tree represent the propor- tion of individual trees that are congruent with the con- sensus tree and indicate support for the separation of the taxa at that particular node.. - Of the remaining RefSeq genomes, 160 sequences were chosen for further analysis to represent the 13 Citrobac- ter species depicted on the tree (Table S6).. - Functional activities were assigned to the predicted amino acid sequences of the Salmonella-Citrobacter core. - The percentage of shared genes among genomes of the 96 strains were visualized using the pairwise report and network chart features of centreseq [27]. - Number of open reading frames (ORFS) and coding sequences (CDS) in the genomes of SalFoS strains.. - Genes present in the pan-genome of 95 Salmonella and Citrobacter strains. - We thank members of the Goodridge lab for identifying the false positive strains using the VITEK 2 system.. - design, the collection, analysis and interpretation of data, nor in the writing of the manuscript.. - World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, Protozoal, and viral diseases, 2010: a data synthesis. - Population dynamics and antimicrobial resistance of the most prevalent poultry-associated Salmonella serotypes. - Phylogenetic diversity of the enteric pathogen Salmonella enterica subsp.. - Outbreak of nosocomial infections due to extended-spectrum beta- lactamase-producing strains of enteric group 137, a new member of the family Enterobacteriaceae closely related to Citrobacter farmeri and Citrobacter amalonaticus. - Utility of the Citrobacter rodentium infection model in laboratory mice. - Genomic comparison of the closely-related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum. - Antigenic formulae of the Salmonella serovars. - Pan-genome analyses of the species Salmonella enterica, and identification of genomic markers predictive for species, subspecies, and Serovar. - Ecological and temporal constraints in the evolution of bacterial genomes. - Multicenter evaluation of the Bruker MALDI Biotyper CA system for the identification of clinically important Bacteria and yeasts
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