Molecular detection of transcriptionally active bacteria from failed prosthetic hip joints removed during revision arthroplasty.
The purpose of this study was to use microbiological culture and bacterial 16S rRNA gene sequencing methods to detect transcriptionally active bacteria present on the surface of failed prosthetic hip joints removed during revision arthroplasty. Five failed prosthetic hip joints were sonicated to dislodge adherent bacteria and subjected to microbiological culture. Bacterial RNA was extracted from each sonicate, cDNA prepared by reverse transcription and the 16S rRNA gene amplified using universal primers. Polymerase chain reaction (PCR) products were cloned, assigned to distinct groups by restriction fragment length polymorphism (RFLP) analysis and one representative clone from each group was sequenced. Bacteria were identified by comparison of the obtained 16S rRNA gene sequences with those deposited in public access sequence databases. All five specimens were positive for the presence of bacteria by both culture and PCR. Culture methods identified species from eight genera. Molecular detection of transcriptionally active bacteria identified a wider range of species. A total of 42 phylotypes were identified, of which Lysobacter gummosus was the most abundant (31.6%). Thirty-four clones (14.5%) represented uncultivable phylotypes. No potentially novel species were identified. It is concluded that a diverse range of transcriptionally active bacterial species are present within biofilms on the surface of failed prosthetic hip joints.
Riggio
M P
MP
Infection and Immunity Research Group, Level 9, Glasgow Dental Hospital & School, 378 Sauchiehall Street, Glasgow G2 3JZ, UK. m.riggio@dental.gla.ac.uk
Dempsey
Kate E
KE
Lennon
Allan
A
Allan
David
D
Ramage
Gordon
G
Bagg
Jeremy
J
D000328:Adult; D000368:Aged; D001178:Arthroplasty / Q000009:adverse effects*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D003001:Cloning, Molecular; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D006621:Hip Joint / Q000601:surgery*; D006801:Humans; D008297:Male; D050296:Microbial Viability; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D016459:Prosthesis-Related Infections / Q000382:microbiology*; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D018076:DNA, Complementary; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S
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