The social structure of microbial community involved in colonization resistance

Abstract

It is well established that host-associated microbial communities can interfere with the colonization and establishment of microbes of foreign origins, a phenomenon often referred to as bacterial interference or colonization resistance. However, due to the complexity of the indigenous microbiota, it has been extremely difficult to elucidate the community colonization resistance mechanisms and identify the bacterial species involved. In a recent study, we have established an in vitro mice oral microbial community (O-mix) and demonstrated its colonization resistance against an Escherichia coli strain of mice gut origin. In this study, we further analyzed the community structure of the O-mix by using a dilution/regrowth approach and identified the bacterial species involved in colonization resistance against E. coli. Our results revealed that, within the O-mix there were three different types of bacterial species forming unique social structure. They act as 'Sensor', 'Mediator' and 'Killer', respectively, and have coordinated roles in initiating the antagonistic action and preventing the integration of E. coli. The functional role of each identified bacterial species was further confirmed by E. coli-specific responsiveness of the synthetic communities composed of different combination of the identified players. The study reveals for the first time the sophisticated structural and functional organization of a colonization resistance pathway within a microbial community. Furthermore, our results emphasize the importance of 'Facilitation' or positive interactions in the development of community-level functions, such as colonization resistance.

Figure 1

H₂O₂ production of oral communities when challenged with E. coli. Oral communities derived from the dilution (10⁰ to 10⁻⁸)/regrowth of original O-mix were challenged with (+) or incubated without (−) E. coli (in a 10:1 oral cell-to-E. coli ratio) and the mixtures were spotted onto BHI agar plates containing leuco crystal violet and horseradish peroxidase; blue color development reflecting the H₂O₂ production was monitored after overnight incubation at 37 °C. Three independent replicates were performed and a representative result is shown.

Figure 2

PCR-DGGE analysis of microbial communities derived from dilution/regrowth of original O-mix. Original O-mix was subjected to serial dilution (10⁻⁰–10⁻⁷)/regrowth to establish new bacterial communities. The microbial profile of each derived community was assessed by PCR-DGGE. The major bacterial species within O-mix were identified as indicated by the arrows. Inlet shows the H₂O₂ production of the specific communities in response to the absence (−) and presence (+) of E. coli. Three independent biological replicates were performed and a representative gel image is shown.

Figure 3

Colonization resistance of synthetic communities against E. coli (Ec). E. coli was co-cultivated with synthetic communities comprised different combination of identified bacterial species in a 1:10 ratio, and its viability was monitored after 48 h incubation at 37 °C under anaerobic condition. Two independent experiments were performed in triplicates and the average value±s.d. is shown. The asterisk indicates that the value of that specific experimental setup is significantly lower than that of other setups (Student's t-test P-value <0.05).

Figure 4

Survival of E. coli within synthetic oral communities in two-chamber assay. E. coli alone, or mixed with different oral isolates was cultured in the upper chamber of the two-chamber system, while the rest of the members of the synthetic communities were inoculated to the lower chamber. Viability of E. coli in the upper chamber in each setup (represented by different bars) was monitored after 48 h. Two independent experiments were performed in duplicates, and average value±s.d. is presented. The asterisk indicates significant differences between two values (Student's t-test P-value <0.05).

Figure 5

Tentative model of colonization resistance pathway of the O-mix against integration of E. coli. Green, orange and black balls represent S. saprophyticus, S. infantis and S. sanguinis, respectively.