Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections

Abstract

The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis, Comamonas terrigena, and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis, dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection.

FIG. 1.

Mixed electropherogram which indicates polymicrobial infection and should be followed by a systematic cloning step.

FIG. 2.

Schematic strategy for the reconciliation of conventional culture and PCR assays.

FIG. 3.

Comparison of the data obtained for the analysis of 525 bone or joint samples using conventional culture and 16S rRNA gene PCR followed by sequencing.

FIG. 4.

(A) Phylogenetic tree of anaerobic gram-positive bacteria inferred from comparison of 16S rRNA gene sequences. Sequences (900 nucleotides) were aligned by using the multisequence alignment program Clustal X (version 1.8). Phylogenetic relationships were determined by using MEGA, version 2.1. Distance matrices were determined by using Kimura's parameters. These matrices were used to elaborate dendrograms by using the neighbor-joining method. Numbers at nodes are the proportion of 100 resamplings that support the topology shown. Listeria monocytogenes was used as the out-group. Bar, 0.02 nucleotide changes per nucleotide position. (B) Phylogenetic tree of Prevotella spp. inferred from comparison of 16S rRNA gene sequences. Sequences (900 nucleotides) were aligned by using the multisequence alignment program Clustal X (version 1.8). Phylogenetic relationships were determined by using MEGA, version 2.1. Distance matrices were determined by using Kimura's parameters. These matrices were used to elaborate dendrograms by using the neighbor-joining method. Number at nodes are the proportion of 100 resamplings that support the topology shown. Bacteroides thetaiotaomicron was used as the out-group. Bar, 0.02 nucleotide changes per nucleotide position.

FIG. 5.

Strategy for the use of 16S rRNA gene PCR for the diagnosis of bone or joint infections.