Bacterial 16S ribosomal DNA in house dust mite cultures.
BACKGROUND
Allergen extracts prepared from Dermatophagoides farinae contain significantly more endotoxin than Dermatophagoides pteronyssinus extracts, and extracts from both mite extracts contain more endotoxin than pollen extracts. Attempts to culture bacteria from mite cultures have failed to establish the sources of the endotoxin.


OBJECTIVE
To determine the bacterial sources of endotoxin in mite extracts.


METHODS
Live mites of both species were obtained from 2 sources, DNA was extracted from the mites, and DNA encoding bacterial 16S ribosomal RNA was amplified by using specific primers. The amount of bacterial DNA in each mite DNA sample was determined by quantitative PCR using an internal standard, and sequence homologies were determined from amplifications performed by using a high-fidelity DNA polymerase.


RESULTS
DNA from D farinae appeared to contain between 11-fold and 24-fold more 16S ribosomal gene copies than the genomic DNA from D pteronyssinus (P < or = .003). Sequence analysis indicated the dominant presence of at least 3 phylogenetic clusters of Bartonella species (henselae, quintana, vinsonii, and grahamii), as well as uncharacterized alpha-proteobacteria, from both D farinae and D pteronyssinus. In a few clones, sequences from Escherichia coli, Pseudomonas species, and Acinetobacter species were also identified.


CONCLUSIONS
House dust mite DNA contains evidence of Bartonella and other Gram-negative species. These Gram-negative species are likely to be the sources of the endotoxin found in mite allergenic extracts.
Valerio
Cherry R
CR
Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD, USA.
Murray
Patrick
P
Arlian
Larry G
LG
Slater
Jay E
JE
D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis*; D004391:Dust; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification; D008070:Lipopolysaccharides / Q000032:analysis; D008925:Mites / Q000235:genetics* / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004391:Dust; D008070:Lipopolysaccharides; D012336:RNA, Ribosomal, 16S