Arbitrarily primed-polymerase chain reaction for identification and epidemiologic subtyping of oral isolates of Fusobacterium nucleatum.
BACKGROUND
Fusobacterium nucleatum is the most frequently isolated bacterium in periodontal disease and plays an important role in serious infections in other parts of the body. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to construct primers for specific identification and subtyping of F. nucleatum. Subtypes may differ in virulence and, hence, are important as periodontal pathogens. Subtypes also may differ in antibiotic susceptibility; therefore, knowing the subtypes may influence choice of treatment.


METHODS
We analyzed 70 DNA samples of F. nucleatum isolated from patients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8) and from healthy carriers (N = 30). From 90 AP-PCR primers screened, five amplification products were selected, cloned in pCR II vector, and sequenced. These sequences were used to design new pairs of specific primers. Sequences were compared to GenBank entries with BLAST and showed no significant matches.


RESULTS
Three primer pairs produced bands of approximately 1 Kb (primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F. nucleatum DNAs tested. PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patients and 9 (30%) healthy controls. Using the same primer pair, 2 other bands of approximately 0.5 Kb and 0.4 Kb were observed with DNA from isolates from 2 (6%) PD and all PD-AIDS patients, but were not observed with DNA samples from healthy controls (P<0.0001). All the primer pairs produced no or different amplicon profiles with DNA samples from bacterial species other than F. nucleatum.


CONCLUSIONS
Our results suggest that PCR primer pairs 5059S, FN5047 or M1211 can be used to specifically identify F. nucleatum isolates and distinguish them from other bacteria. The primer pair M8171 could also be used to differentiate F. nucleatum isolated from periodontal patients or healthy individuals. These specific primers can be used in PCR analysis for specific identification of F. nucleatum and to distinguish it from other bacteria associated with human periodontitis. These approaches appear promising in facilitating laboratory identification, molecular subtyping, and taxonomy of putative periodontopathogens.
Avila-Campos
M J
MJ
Department of Microbiology, University of São Paulo, SP, Brazil.
Sacchi
C T
CT
Whitney
A M
AM
Steigerwalt
A G
AG
Mayer
L W
LW
D017088:AIDS-Related Opportunistic Infections / Q000382:microbiology; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D016967:Fusobacterium nucleatum / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009055:Mouth / Q000382:microbiology*; D010510:Periodontal Diseases / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods
D017931:DNA Primers; D004269:DNA, Bacterial