ARS | Publication request: Specificity and sensitivity of common cultivation conditions selective for Campylobacter spp. in the poultry microbiome as determined by 16S rRNA gene pyrosequencing

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 28, 2011
Publication Date: May 21, 2011
Citation: Oakley, B., Morales, C., Line, J.E., Hiett, K.L., Seal, B.S. 2011. Specificity and sensitivity of common cultivation conditions selective for Campylobacter spp. in the poultry microbiome as determined by 16S rRNA gene pyrosequencing . Meeting Abstract. Volume: III Page 135.

Technical Abstract: Background: The poultry gastrointestinal microbiome remains poorly sampled relative to the capabilities of current DNA sequencing methods. Traditionally, a wide variety of selective methods for cultivation have been used to screen poultry samples for specific bacterial taxa of interest, generally human pathogens such as Campylobacter, but several important questions need to be addressed with the comprehensive community censuses that are now possible using high-throughput sequencing: 1) How sensitive and specific are cultivation methods relative to the initial community being screened, and 2) How do commonly used recovery methods compare to one another? Methods: We used 16S rRNA tagged-pyrosequencing to compare bacterial recovery in a factorial design with five commonly used Campylobacter-selective plating media (Cefex, Capetown, MCCDA, CLA, and CVA), two incubation atmospheres (10% CO2/5% O2/85% N2 and 10% CO2/10% H2/80% N2), and two incubation temperatures (37° and 42° C), and compared these results to direct sequencing of the poultry gut microbiome. Results: Analysis of 487,096 raw sequence reads revealed that the fecal bacterial community was dominated by Bacteroides, Brevibacterium, and Lactobacillus, and was quite species-rich with >500 taxa at a 3% sequence similarity cutoff. Campylobacter sequences encountered in the feces represented only a small proportion (<0.04%) of the whole community. In contrast, sequences recovered from selective media were dominated by a single Campylobacter sequence type which accounted for 88-97% of the sequences. Recovery of Campylobacter was very similar across the five media types compared and sequences with significant matches to 14 Campylobacter strains were recovered on all five media. Incubation atmosphere had little effect on recovery, but significant differences in media sensitivity (fewer Camplyobacter phylotypes; p=0.035) and specificity (more non-Campylobacter phylotypes; p=0.011) was found for media incubations at 42° versus 37° C. Conclusions: These results provide insight into the bacterial community structure of the poultry gut microbiome and suggest that commonly used media selective for Campylobacter perform equivalently but may have reduced sensitivity and specificity at higher incubation temperatures.