Biochemical but not compositional recovery of skin mucosal microbiome communities after disruption

Abstract

Background: The microbiomes of animals are complex communities that strongly affect the health of the hosts. Microbiomes on mucosal surfaces have the highest densities and most extensive biochemical exchanges with the hosts. Although antibiotics are potent tools to manage infections, they can disrupt the normal microbiota, causing numerous side effects.

Materials and methods: Taking a community ecology approach, mucosal microbiome community responses to five disruptive conditions (two broad-spectrum antibiotics, a biocide, elevated temperature, and rinsing) were analyzed. Skin of the fish Gambusia affinis was the mucosal model. Microbiome recovery was measured by culturable counts, community biochemical profiles, genetic fingerprinting, and community 16S gene sequencing (rinsing condition only).

Results: Following all disruptions, the total counts rose and then returned to the pre-treatment (PT) level. This overgrowth was confirmed via direct staining and community metabolic activity measurements. After rinsing, diversity decreased and one taxon dominated (family Aeromonadaceae) temporarily, the findings similar to numerous other studies with antibiotics. While the community did not return to the PT taxonomic composition, the biochemical profile did.

Conclusion: This suggests that the biochemical pathways in a community are important during recovery, and a return to the original composition is not required to restore original function.

Keywords: Gambusia affinis; chlorhexidine; rifampicin; tetracycline.

Figure 1

Treatment protocols for the five disruptions. Notes: a: 30-minute exposure to chlorhexidine. b: 3-minute rinsing procedure. Abbreviations: d, day; RIF, rifampicin; temp, temperature; TET, tetracycline.

Figure 2

Sensitive and resistant culturable numbers. Notes: (A) Data from the PT fish shown at −1 on the X-axis. Data from the fish after 3 days of RIF treatment shown at 0 on the X-axis. All other points shown at days after recovery. PT resistant count is 0. All five graphs share the same axes for ease of comparison. (B) Data from the PT fish shown at −1 on the X-axis. Data from the fish after 3 days of TET treatment shown at 0 on the X-axis. All other points shown at days after recovery. There were no TET-resistant colonies from PT fish or at the 1 day time point for the control fish. (C) Data from the PT fish shown at −1 on the X-axis. Counts after 30-minute CHX treatment shown at 0 on the X-axis. All other points shown at days after recovery. (D) Data from the PT fish shown at −1 on the X-axis. Data at 10 hours, just after final temperature reached, shown at −0.5 on the X-axis. Data from the fish after 3 days of 34°C treatment shown at 0 on the X-axis. All other points shown at days after recovery. (E) Data from the PT fish shown at 0 on the X-axis. All other points shown at days after recovery. This first time point is after 10 hours of recovery. Abbreviations: CFU, colony-forming unit; CHX, chlorhexidine; PT, pre-treatment; RIF, rifampicin; TET, tetracycline; treat, treatment.

Figure 3

Community composition rinse experiment. Notes: Normalized abundance of community members at most precise level of taxonomic identification (mostly genus). OTUs that are identified at the family level have a single asterisk, ones identified at the order level have two asterisks, and class level with three asterisks. Names without an asterisk are at the genus level. Only shown are members with >1% abundance in at least one sample. The pre-treatment fish skin microbiome is an average abundance from three fish. All other columns represent one fish. Abbreviation: OTU, operational taxonomic unit.

Figure 4

Community fingerprint during and following elevated temperature. Notes: Community fingerprint before, during, and after exposure of the fish skin microbiome to elevated temperatures. A: 100 bp DNA ladder, B: pre-treatment fish skin microbiome sample, C: pre-treatment aquarium water sample, D: 10-hour treatment, E: 10-hour treatment APW water sample, F: 3-day treatment, G: 3-day treatment APW sample, H: 24-hour recovery, I: 3-day recovery, J: 3-day recovery APW sample, K: 7-day recovery, L: 7-day recovery APW sample, M: 12-day recovery, N: empty lane, and O: 1 kb DNA ladder. Lanes with arrows are fish skin microbiome, other lanes are surrounding water samples. Abbreviation: APW, artificial pond water.

Figure 5

Community fingerprint during rinse experiment. Notes: (A) RISA pattern from physical rinse disruption experiment. A: 100 bp DNA ladder, B: pre-treatment fish skin microbiome sample, C: 5-hour recovery, D: 10-hour recovery, E: 24-hour recovery, F: 48-hour recovery, G: 4-day recovery, H: 7-day recovery, and I: 1 kb DNA ladder. (B) ERIC PCR pattern from physical rinse disruption experiment. A: 1 kb DNA ladder, B: pre-treatment fish skin microbiome sample, C: 10-hour recovery, D: 1-day recovery, E: 2-day recovery, F: 4-day recovery, G: 5-hour recovery, H: 7-day recovery, and I: 1 kb DNA ladder. Abbreviations: ERIC, enterobacterial repetitive intergenic consensus; RISA, ribosomal intergenic spacer analysis.

Figure 6

Venn diagrams comparing community compositions during rinse recovery (A–C). Notes: Numbers represent bacterial families. PT is pre-treatment community. Other communities are days of recovery.

Figure 7

NMDS comparing community compositions during rinse recovery. Notes: NMDS is non-metric multidimensional scaling, performed by the PAST program, version 3.21. PT is pre-treatment, and all communities represented by a number with D are days of recovery.