ciprofloxacin perturbation human gut microbiota
Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation
Stool samples collected from 3 healthy human subjects (52-56 samples per subject) over a 10 month interval including two 5-day courses of the antibiotic ciprofloxacin at 2 months and 8 months into the study. Sampling interval daily for a 19 day interval centered on each course of Cp (1 week before, during 5 days of Cp, and 1 week after), weekly for 3 weeks prior to and subsequent to each period of daily sampling, and monthly at other times. Samples collected by participants and kept at -20C in home freezers for several days until brought to the lab for storage at -80C. Bacterial genomic DNA extracted using QIAamp DNA Stool kit supplemented with 45 sec beadbeating in qiagen lysis buffer prior to initial 95C incubation. Fusion primers adapted from the Titanium Genomic library kit sequences were used to obtain 16S rRNA amplicons spanning the V1-V2-V3 variable regions, with 22-28 cycles of amplification (minimum cycles necessary to generate a distinct band visualizing 3 ul of reaction in agarose with Gelgreen dye and blue illumination).  Proximal primer was A linker - 10mer barcode - dimer spacer - primer complimenting positions 533-515 in reverse orientation; distal primer was B linker - dimer spacer - primer cocktail complimenting positions 8-27 in forward orientation.  Sample amplicon libraries were gel purified, quantified with Picogreen in 96 well plates read on a fluorescent scanner, pooled to include equivalent fluorescence units from each sample,  and sent for sequencing on a full Titanium picotiter plate. Reads screened for human gene sequences vs the Genome Reference Consortium CRCh37 sequence of the human genome (including primary assemblies of all chromosomes plus unassembled portions plus alternative assemblies for polymmorphic loci) and vs the human mitochondrial RefSeq NC_012920.1 using qiime exclude_seqs_by_blast.py command with nondefault thresholds of 1e-4 e value and 90% query length aligned. No reads produced significant hits against human mitochondrial DNA, 14 reads were excluded for matches to human nuclear DNA.
The indigenous human microbiota is essential to the health of the host.  Although the microbiota can be affected by many features of modern life, we know little about its responses to disturbance, especially repeated disturbances, and how these changes compare to baseline temporal variation. We examined the distal gut microbiota of three individuals over 10 months that spanned two courses of the antibiotic ciprofloxacin, analyzing more than 1.7 million bacterial 16S rRNA hypervariable region sequences from 52-56 samples per subject.  Inter-individual variation was the major source of variability between samples. Day to day temporal variability was evident but constrained around an average community composition that was stable over several months in the absence of deliberate perturbation.  The effect of ciprofloxacin on the gut microbiota was profound and rapid, with a loss of diversity and a shift in community composition occurring within 3-4 days of drug initiation.  By one week after the end of each course, communities began to return toward their initial state, but the return was often incomplete.  While broadly similar, community changes after ciprofloxacin varied among subjects and between the two courses within subjects. In all subjects, the composition of the gut microbiota stabilized by the end of the experiment, but was altered from its initial state.  As with other ecosystems, the human distal gut microbiome at baseline is a dynamic regime with a stable average state. Antibiotic perturbation may cause a shift to an alternative stable state, the full consequences of which remain unknown.