Multidrug resistant pathogens respond differently to the presence of co-pathogen, commensal, probiotic and host cells

Abstract

In light of the ongoing antimicrobial resistance crisis, there is a need to understand the role of co-pathogens, commensals, and the local microbiome in modulating virulence and antibiotic resistance. To identify possible interactions that influence the expression of virulence or survival mechanisms in both the multidrug-resistant organisms (MDROs) and human host cells, unique cohorts of clinical isolates were selected for whole genome sequencing with enhanced assembly and full annotation, pairwise co-culturing, and transcriptome profiling. The MDROs were co-cultured in pairwise combinations either with: (1) another MDRO, (2) skin commensals (Staphylococcus epidermidis and Corynebacterium jeikeium), (3) the common probiotic Lactobacillus reuteri, and (4) human fibroblasts. RNA-Seq analysis showed distinct regulation of virulence and antimicrobial resistance gene responses across different combinations of MDROs, commensals, and human cells. Co-culture assays demonstrated that microbial interactions can modulate gene responses of both the target and pathogen/commensal species, and that the responses are specific to the identity of the pathogen/commensal species. In summary, bacteria have mechanisms to distinguish between friends, foe and host cells. These results provide foundational data and insight into the possibility of manipulating the local microbiome when treating complicated polymicrobial wound, intra-abdominal, or respiratory infections.

Figure 1

Antimicrobial resistance gene expression. Differentially regulated AMR genes in A. baumannii (AB) (a), K. pneumoniae (KP) (b) during bacterial co-culture experiments, and E. hormaechei (EH) in response to co-cultured HDF cells (c) are shown. (CJ) denotes C. jeikeium. Differentially regulated genes shown with edgeR false discovery rate (FDR): <0.05 (red circle) and <0.1 (yellow circle). Log₂-fold change values of gene expression was shown as red (up-regulated) and blue (down-regulated) bar graphs with reference to monocultures. Bar scale from −4 to 4. Averaged gene expression level across control and treatment experiments (log₂ read counts per million, log₂CPM) was shown as green bar graphs. Bar scale from 0 to 15.

Figure 2

Siderophore gene cluster expression in (a) K. pneumoniae (enterobactin), (b) A. baumannii (acinetobactin), and (c) E. hormaechei (enterobactin-like and aerobactin) when co-cultured with individual pathogen/commensal species. K. pneumoniae (KP), A. baumannii (AB), E. hormaechei (EH), L. reuteri (LR), S. epidermidis (SE) and C. jeikeium (CJ). Differentially regulated genes shown with edgeR false discovery rate (FDR): <0.05 (red circle) and <0.1 (yellow circle). Log₂ fold change values of gene expression was shown as red (up-regulated) and blue (down-regulated) bar graphs with reference to monocultures. Bar scale from −4 to 4. Averaged gene expression level across control and treatment experiments (log₂ read counts per million, log₂CPM) was shown as green bar graphs. Bar scale from 0 to 15.

Figure 3

Regulation of A. baumannii type I and type IV pili gene cluster expression when co-cultured with individual pathogen/commensal species. K. pneumoniae (KP), A. baumannii (AB), E. hormaechei (EH), L. reuteri (LR), S. epidermidis (SE) and C. jeikeium (CJ). Differentially regulated genes shown with edgeR false discovery rate (FDR): <0.05 (red circle) and <0.1 (yellow circle). Log₂ fold change values of gene expression was shown as red (up-regulated) and blue (down-regulated) bar graphs with reference to monocultures. Bar scale from −4 to 4. Averaged gene expression level across control and treatment experiments (log₂ read counts per million, log₂CPM) was shown as green bar graphs. Bar scale from 0 to 15.

Figure 4

Expression of K. pneumoniae (a) attachment-related and (b) capsule-related gene clusters in response to co-cultured HDF cells. Differentially regulated genes shown with edgeR false discovery rate (FDR): <0.05 (red circle) and <0.1 (yellow circle). Log₂ fold change values of gene expression was shown as red (up-regulated) and blue (down-regulated) bar graphs with reference to monocultures. Bar scale from −4 to 4. Averaged gene expression level across control and treatment experiments (log₂ read counts per million, log₂CPM) was shown as green bar graphs. Bar scale from 0 to 15.

Figure 5

Human host genes were upregulated when HDF cells were co-cultured with MDROs. Differentially regulated genes shown with edgeR false discovery rate (FDR): <0.05 (red circle). Log₂ fold change values of human gene expression was shown as red (up-regulated) bar graphs with reference to monocultures of HDF cells. Bar scale from 0 to 9. Averaged gene expression level across control and treatment experiments (log₂ read counts per million, log₂CPM) was shown as green bar graphs. Bar scale from 0 to 15.