A core microbiome associated with the peritoneal tumors of pseudomyxoma peritonei

Abstract

Background: Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease.

Methods: To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome.

Main results: We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients.

Conclusions: Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival.

Figure 1

Distribution of prominent bacterial phyla. The relative abundance of each phylum is shown as the number of OTUs that were classified in each phylum out of the total number of bacterial OTUs. Numbers on the X-axis represent patient designations. “T” indicates the sample was taken from tumor tissue and “M” indicates the sample was taken from mucin.

Figure 2

β-diversity between PMP communities. Heatmaps compare the classic Jaccard (Jclass) dissimilarity indices (A) and thetaYC indices (B) for bacterial communities found in PMP patients. Numbering along the left-hand and top sides of each heatmap represent individual patient designations. Each square within the heatmap represents a single pairwise comparison between the corresponding patient communities (labeled at the top and left side of each heatmap). As indicated by the bar below each heatmap, comparisons in which the communities are more dissimilar are more red, whereas communities that are more similar are less red.

Figure 3

Core set of sequences found in all PMP patients. After combining the tumor and mucin sequence sets for each patient, sequences that were present in all patients were classified at a distance of 97%. The relative abundance of these sequences at the phylum level is shown.

Figure 4

Direct detection of bacterial taxa in PMP tumor tissue by ISH. Hybridizations were carried out as described in the Materials and Methods. Using DNA probes complementary to taxon-specific rRNA sequences, we detected the presence of bacteria in additional patients not included in our sequencing study (patients 196 and 244 shown). Images were selected to show positive hybridization signal with a subset of probes used and are not intended to reflect relative abundance within tissue specimens. FIR, Firmicutes; ACT, Actinobacteria; BET, Betaproteobacteria; VER, Verrucomicrobiales.

Figure 5

Bacteria isolated from PMP tissue associates with MUC2. Isolate PMP191F (an unclassified Chitinophagaceae) was cultured with MUC2 secreting HCT-29 cells. The rod shaped bacteria interact with cell-associated MUC2 (left) as well as secreted MUC2 (right). The positive reaction (seen as brown color) for MUC2 staining on the bacterial cells is consistent with interaction or adherence with MUC2. The image shown in the panel on the right is a magnification of the boxed region in the left panel.

Figure 6

Patient survival after antibiotic treatment. Survival of PMP LN negative PMCA patients with or without antibiotic treatment; the red line indicates survival of patients that were treated with antibiotics (n = 6), whereas the black line indicates patients that were not given this treatment (n = 37). Differences in percent survival were compared using a Log-rank statistical test, P = 0.078.