Comprehensive genomic analysis reveals virulence factors and antibiotic resistance genes in Pantoea agglomerans KM1, a potential opportunistic pathogen

Abstract

Pantoea agglomerans is a Gram-negative facultative anaerobic bacillus causing a wide range of opportunistic infections in humans including septicemia, pneumonia, septic arthritis, wound infections and meningitis. To date, the determinants of virulence, antibiotic resistance, metabolic features conferring survival and host-associated pathogenic potential of this bacterium remain largely underexplored. In this study, we sequenced and assembled the whole-genome of P. agglomerans KM1 isolated from kimchi in South Korea. The genome contained one circular chromosome of 4,039,945 bp, 3 mega plasmids, and 2 prophages. The phage-derived genes encoded integrase, lysozyme and terminase. Six CRISPR loci were identified within the bacterial chromosome. Further in-depth analysis showed that the genome contained 13 antibiotic resistance genes conferring resistance to clinically important antibiotics such as penicillin G, bacitracin, rifampicin, vancomycin, and fosfomycin. Genes involved in adaptations to environmental stress were also identified which included factors providing resistance to osmotic lysis, oxidative stress, as well as heat and cold shock. The genomic analysis of virulence factors led to identification of a type VI secretion system, hemolysin, filamentous hemagglutinin, and genes involved in iron uptake and sequestration. Finally, the data provided here show that, the KM1 isolate exerted strong immunostimulatory properties on RAW 264.7 macrophages in vitro. Stimulated cells produced Nitric Oxide (NO) and pro-inflammatory cytokines TNF-α, IL-6 and the anti-inflammatory cytokine IL-10. The upstream signaling for production of TNF-α, IL-6, IL-10, and NO depended on TLR4 and TLR1/2. While production of TNF-α, IL-6 and NO involved solely activation of the NF-κB, IL-10 secretion was largely dependent on NF-κB and to a lesser extent on MAPK Kinases. Taken together, the analysis of the whole-genome and immunostimulatory properties provided in-depth characterization of the P. agglomerans KM1 isolate shedding a new light on determinants of virulence that drive its interactions with the environment, other microorganisms and eukaryotic hosts.

Fig 1. The circular genome maps of the P. agglomerans KM1 draft genome.

Genome map showing the features of P. agglomerans KM1 chromosome and plasmids pKM1_1, pKM1_2, and pKM1_3. Circles illustrate the following from outermost to innermost rings: (1) forward CDS, (2) reverse CDS, (3) GC content, and (4) GC skew. All the annotated open reading frames (ORFs) are colored differently according to the COG assignments. Stacked bar chart shows the relative abundance (%) of COG categories calculated based on the total number of predicted ORFs present in the annotated genome.

Fig 2. Phylogenetic analysis of P. agglomerans KM1.

A: Neighbor-joining phylogenetic tree showing the phylogenetic relationship between P. agglomerans KM1 and selected Pantoea strains. The neighbor-joining tree was constructed from an alignment of concatenated fusA, gyrB, leuS, pyrG, rplB, and rpoB gene sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Superscript “T” indicates a type strain. The scale bar represents the number of substitutions per site. Escherichia coli strain K12 substr. MG1655 was used as an outgroup. B: Heatmap showing the OrthoANI values between P. agglomerans KM1 genome and its closely related species. Values greater than 97% indicate that strains belong to the same species.

Fig 3. Genome alignments showing synteny blocks among P. agglomerans strains obtained using progressive Mauve.

P. agglomerans KM1 were compared with other closely related strains namely C410P1, UAEU18, TH81 and L15. Each genome is laid out horizontally with homologous segment outlined as colored rectangles. Each same color block represents a locally collinear block (LCB) or homologous region shared among genomes. Rearrangement of genomic regions was observed between the two genomes in terms of collinearity. Inverted regions relative to KM1 are localized in the negative strand indicated by genomic position below the black horizontal centerline in the Mauve alignment.

Fig 4. Genomic islands (GIs) in P. agglomerans strain KM1 predicted using IslandViewer4.

The predicted genomic islands are colored based on the prediction methods. Red indicates an integrated analysis, blue represents IslandPath-DIMOB prediction, orange represents SIGI-HMM prediction, and green indicates IslandPick analysis. The circular plots show the genomic islands in P. agglomerans KM1 chromosome, and plasmids pKM1_1, pKM1_2 and pKM1_3. GIs are labelled in blue. Prophage regions predicted by PHASTER were indicated in red boxes for prophage region 1 (P1) and region 2 (P2).

Fig 5. Pan-genome analysis of P. agglomerans strains obtained using Gview server.

The innermost circle shows the pan-genome (purple), and outer circlers indicate the genomes of Pantoea agglomerans strains L15 (orange), TH81 (brown), UAEU18 (red), C410P1 (blue), and KM1 (green). Genes with specialized functions were labelled with different colors: virulence-related genes (blue), antibiotic resistance genes (red), and strain-specific regions (black).

Fig 6. Roary matrix-based pan-genome analysis of P. agglomerans strains.

The core-genome tree generated was compared with a matrix where the core and accessory genes were either present (blue) or absent (white).

Fig 7. Cytokine and nitrite production by RAW 264.7 macrophages stimulated with a heat inactivated whole-cell P. agglomerans.

Panels A, D, G and J show TNF-α, IL-6, Nitrite and IL-10 secretion by the simulated RAW 264.7 cells (RAW), TLR4 knock-out RAW 264.7 cells (TLR4KO), RAW 264.7 cells (RAW) in combination with TLR2 inhibitor (RAW+ TLR2 inhib), TLR4 knock-out RAW 264.7 cells in combination with TLR2 inhibitor (TLR4KO + TLR2 inhib.). Additionally stimulations were performed using RAW 264.7 cells in combination with NFκB inhibitor (RAW+NFκB inhib.), TLR4 knock-out RAW 264.7 cells in combination with NFκB inhibitor (TLR4KO + NFκB inhib.) and RAW 264.7 cells in combination with MEK1/2 inhibitor (RAW + MEK inhib.). Panels B, E, H, and K, include control conditions: RAW 264.7 cells stimulated with TLR4 agonist ultrapure LPS (RAW + LPS), TLR4 knock-out RAW 264.7 cells stimulated with TLR4 agonist ultrapure LPS (TLR4KO + LPS), non-stimulated RAW 264.7 cells (RAW control), non-stimulated TLR4 knock-out RAW 264.7 cells (TLR4 control). Panels C, F, I and L include control stimulations: RAW 264.7 cells and TLR4 knock-out RAW 264.7 cells in the presence of inhibitors alone, RAW + TLR2 inhibitor, TLR4KO + TLR2 inhibitor, RAW + NFκB inhibitor, TLR4KO + NFκB inhibitor, and RAW + MEK inhibitor. Values with P < 0.001 were considered as significantly different (****).