Half plate amplicon sequencing
Microbiome changes in Response to Antibiotic Therapies in a Single CF patient
Quantitative PCR (qPCR) and 16S rRNA gene pyrosequencing were used to characterize the microbial community in one adult cystic fibrosis patient in multiple sputum samples collected before, during and after antibiotic treatments over a 276-day period.  Although the dominant pathogens in all samples were Pseudomonas and Burkholderia, sequencing revealed a rich community with 105 taxa from six different phyla.  Comparison of the microbial community between conditions and over time identified a strong microbiome response to repeated antibiotic treatment. Characterization of the microbial community requires both pyrosequencing to measure relative abundance of each taxa and qPCR targeting the 16S rRNA gene to measure total abundance.  Combining 16S rDNA pyrosequencing with qPCR quantification produced data consistent with a model in which: (i) the inhaled antibiotic Tobramycin had limited effectiveness in reducing bacterial load; (ii) orally taken Ciprofloxacin and Trimethoprim-Sulfamethoxazole (TMP-SMZ) substantially reduced bacterial load, (iii) Burkholderia was more susceptible to these antibiotics than was Pseudomonas; (iv) total bacterial load increased rapidly upon cessation of antibiotic treatment with the increase being driven primarily by an increase in Burkholderia; and (v) species richness of taxa other than Pseudomonas and Burkholderia responded to antibiotics with a depression in overall species richness that recovered more slowly than did Burkholderia abundance.  While the findings from one patient should not be generalized to all CF patients, our results illustrate that metagenomic techniques and frequent longitudinal sampling can yield fresh insights into well-studied problems.