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. 2022 Dec 26;10(1):239.
doi: 10.1186/s40168-022-01419-4.

Staphylococcus aureus populations from the gut and the blood are not distinguished by virulence traits-a critical role of host barrier integrity

Elisa J M Raineri  1 , Sandra Maaß  2 , Min Wang  1 , Siobhan Brushett  1 , Laura M Palma Medina  1   3 , Neus Sampol Escandell  1 , Dania Altulea  1   4 , Erwin Raangs  1 , Anne de Jong  5 , Elias Vera Murguia  1 , Edward J Feil  6 , Alex W Friedrich  1 , Girbe Buist  1 , Dörte Becher  2 , Silvia García-Cobos  1   7 , Natacha Couto #  1   6 , Jan Maarten van Dijl #  8
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Staphylococcus aureus populations from the gut and the blood are not distinguished by virulence traits-a critical role of host barrier integrity

Elisa J M Raineri et al. Microbiome. .

Abstract

Background: The opportunistic pathogen Staphylococcus aureus is an asymptomatically carried member of the microbiome of about one third of the human population at any given point in time. Body sites known to harbor S. aureus are the skin, nasopharynx, and gut. In particular, the mechanisms allowing S. aureus to pass the gut epithelial barrier and to invade the bloodstream were so far poorly understood. Therefore, the objective of our present study was to investigate the extent to which genetic differences between enteric S. aureus isolates and isolates that caused serious bloodstream infections contribute to the likelihood of invasive disease.

Results: Here, we present genome-wide association studies (GWAS) that compare the genome sequences of 69 S. aureus isolates from enteric carriage by healthy volunteers and 95 isolates from bloodstream infections. We complement our GWAS results with a detailed characterization of the cellular and extracellular proteomes of the representative gut and bloodstream isolates, and by assaying the virulence of these isolates with infection models based on human gut epithelial cells, human blood cells, and a small animal infection model. Intriguingly, our results show that enteric and bloodstream isolates with the same sequence type (ST1 or ST5) are very similar to each other at the genomic and proteomic levels. Nonetheless, bloodstream isolates are not necessarily associated with an invasive profile. Furthermore, we show that the main decisive factor preventing infection of gut epithelial cells in vitro is the presence of a tight barrier.

Conclusions: Our data show that virulence is a highly variable trait, even within a single clone. Importantly, however, there is no evidence that blood stream isolates possess a higher virulence potential than those from the enteric carriage. In fact, some gut isolates from healthy carriers were more virulent than bloodstream isolates. Based on our present observations, we propose that the integrity of the gut epithelial layer, rather than the pathogenic potential of the investigated enteric S. aureus isolates, determines whether staphylococci from the gut microbiome will become invasive pathogens. Video Abstract.

Keywords: Bacteremia; Barrier; Enteric carriage; S. aureus, Gut; Virulence.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Phylogeny of the 164 BI and GI study isolates. The approximate maximum likelihood phylogeny of the study isolates and the reference strain is shown on the left. Leaves are colored by sequence type as defined in the central legend. The inner ring indicates the isolate source (blue BI, yellow GI), and the outer ring indicates the identified clonal complexes. The arrows indicate the position in the phylogeny of the six isolates that were selected for proteome analyses. The tree and metadata can be found in https://microreact.org/project/5KbM9pyLS5gwpihFXi5ncV-raineri-et-al-2022. Clusters shown on the right are based on the pangenome analysis and were identified with Panini v1.7.1 [1] and Poppunk v2.4.0. using the DBSCAN model
Fig. 2
Fig. 2
S. aureus isolates display high extracellular proteome heterogeneity independently from sequence type or isolation site. A Numbers of identified extracellular proteins of the six investigated S. aureus isolates and their predicted subcellular locations. For all identified extracellular proteins of the investigated strains, the subcellular locations were predicted bioinformatically with GP4 [23]. B Principal component analysis (PCA) based on the LFQ intensities of identified extracellular proteins. The PCA analysis was performed on the identified extracellular proteins of all six S. aureus study isolates. Of note, each strain is presented with the 3 replicates separately and the PCA with the averaged values is presented as Supplementary Figure S 4. Note that the small numbers above each data point refer to the respective replicate measurement. C Extracellular virulence factors of the six investigated S. aureus isolates as identified by proteomics. A total number of 47 virulence factors was identified. Color-coded bars represent the identified proteins and their relative abundance based on the LFQ intensities. *Statistically significant differences in the LFQ intensity of the proteins as assessed ANOVA p < 0.01. Please note that SSL4, SSL7, and SSL11 are listed several times due to significant differences in the respective amino acid sequences. D Heat map based on the LFQ intensities of identified extracellular proteins. Of note, the data for each strain are presented as the average of three replicates. The respective data are presented in Supplementary Table S 1
Fig. 3
Fig. 3
Functional categories and relative abundance of identified extracellular proteins of the investigated S. aureus strains. Voronoi treemaps representing the “top-level functions” (TIGRfam level 1) (A) and “sub-level functions” (TIGRfam level 2) (B). The different functional categories are marked in different colors, and the size of each functional category is proportional to the number of identified proteins with the respective function (A and B). Each protein is represented by a polygon-shaped tile, and its relative amount was assessed based on ratios of the log2-transformed LFQ intensity values per sequence type (ST5/ST1) (C) or per site of isolation (BI/GI) (D). Significant differences in the log2-transformed LFQ intensities per sequence type (ST5/ST1) or per isolation site (BI/GI) were assessed by multiple t tests and subsequent Holm-Sidak, Bonferroni-Dunn, and Sidak-Bonferroni corrections to adjust the P values. Of note, no statistically significant differences were detectable
Fig. 4
Fig. 4
Cellular proteome heterogeneity in the investigated S. aureus isolates. A Numbers of identified cellular proteins of the six investigated S. aureus isolates. B Principal component analysis (PCA) based on the LFQ intensities of identified cellular proteins. The PCA analysis was performed on the identified cellular proteins of all six S. aureus isolates. The PCA with averaged values is presented in Supplementary Figure S 6. Note that the small numbers above each data point refer to the respective replicate measurement. C Heat map based on the LFQ intensities of identified cellular proteins. The data for each strain are based on three independent replicates. The respective data are presented in Supplementary Table S 2
Fig. 5
Fig. 5
Functional categories and relative abundance of identified cellular proteins of the investigated S. aureus strains. Voronoi treemaps representing the “top-level functions” (TIGRfam level 1) (A) and “sub-level functions” (TIGRfam level 2) (B). The different functional categories are marked in different colors, and the size of each functional category is proportional to the number of identified proteins with the respective functions (A and B). Each protein is represented by a polygon-shaped tile, and its relative amount was assessed based on the log2-transformed LFQ intensity values per sequence type (ST5/ST1) (C) or per isolation site (BI/GI) (D) ratios as exported from MaxQuant. Significant differences in the log2-transformed LFQ intensities per sequence type (ST5/ST1) or per isolation site (BI/GI) were assessed by multiple t tests and subsequent Holm-Sidak, Bonferroni-Dunn, and Sidak-Bonferroni corrections to adjust the P values. Of note, no statistically significant differences were detectable
Fig. 6
Fig. 6
Infection of Caco2 cells by S. aureus in the presence or absence of cell–cell junctions. Caco2 cells were seeded in 24-well plates at a density of 200,000 cells per well and cultured for 84 h prior infection. A Confocal fluorescence microscopy images of a Caco2 monolayer stained with antibodies specific for ZO-1 (red) and DAPI (blue). Scale bar: 50 µm. B Disruption of Caco2 cell–cell junctions by treatment for 45 min with EGTA in calcium-free media. C Start of tight junction restoration after EGTA treatment and 1 h incubation in a calcium-free medium and (D) complete restoration of tight junctions after 3 h in a calcium-free medium. E, F, G Infection of the Caco2 monolayer or the EGTA-disrupted Caco2 monolayer upon 3 h infection with GFP-expressing S. aureus. The percentage of living cells (compared to the uninfected control) that resulted in GFP + or GFP − cells after infection was measured by flow cytometry. GFP + Caco2 indicates the proportion of the cell population with GFP-expressing S. aureus and GFP − indicates the population that remained uninfected. F Bacterial adherence was measured immediately upon infection in the absence of lysostaphin and, accordingly, GFP + cells represent both adherent and intracellular bacteria. E, G Upon removal of extracellular bacteria by a 30-min incubation with lysostaphin only intracellular bacteria remain detectable. H, I Confocal fluorescence microscopy images of Caco2 cells in a closed monolayer (H) and upon EGTA treatment (I). Cells were stained with an antibody specific for ZO-1 (red) and DAPI (blue). GFP-expressing S. aureus are represented in green. Scale bar: 50 µm
Fig. 7
Fig. 7
S. aureus infection of human leukocytes and larvae of Galleria mellonella. A Leukocyte infection with the six S. aureus study isolates. To investigate the bacterial virulence and intracellular survival after infection of leukocytes, the course of infection was followed by flow cytometry. Per experiment, the percentage of living cells post-infection was measured compared to the uninfected control. B Living GFP + and GFP − granulocytes following S. aureus infection. GFP + granulocytes indicate the portion of the granulocyte population containing intracellular GFP-expressing S. aureus, and GFP − indicates the population that remained uninfected. Cells were infected with S. aureus for 30 min and subsequently incubated for 30 min with lysostaphin to remove non-internalized bacteria. C Virulence profile of the six S. aureus study isolates in G. mellonella. To investigate their virulence, three independent G. mellonella infection experiments were performed. Per experiment, each strain was used to inoculate 15 G. mellonella larvae (45 larvae/strain in total). Each individual larva was inoculated with 10 μl aliquots of a diluted bacterial suspension (1 × 10.8 CFU/ml) of the respective S. aureus strain. Larval killing was assessed at 24, 48, 72, and 96 h post-inoculation. All values are the mean ± the standard deviation of the three independent infection experiments. Statistical significance of observed differences in virulence between the six S. aureus study strains as assessed using a Gehan-Breslow-Wilcoxon test. *P < 0.03; **P < 0.002; ***P < 0.0002; ****P < 0.0001
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