Polymerase chain reaction detection of bacterial 16S rRNA gene in human blood

Abstract

Bacterial 16S ribosomal RNA genes (rDNA) were detected in blood samples from two healthy individuals by PCR under conditions involving 30 cycles that did not produce any visible products from negative control saline. Even from control samples, PCR involving 35-40 cycles yielded visible bands. Major clones detected in the blood samples, but not in control, were the Aquabacterium subgroup, Stenotrophomonas subgroup, Budvicia subgroup, Serratia subgroup, Bacillus subgroup and Flavobacteria subgroup. No clone was located within the bacteroides-clostridium-lactobacillus cluster, which is indigenous to gastrointestinal flora.Key words bacteremia, blood, polymerase chain reaction, 16S ribosomal RNA.Diagnosis of bacterial infection and identification of the agent responsible is essential in clinical medicine, and has been traditionally carried out by inoculating blood or infected tissues into a liquid or solid nutrient medium. This approach has a limitation in that it can detect only bacteria and fungi that are culturable in a laboratory. Recently, PCR using species-specific primers (1-5) and nucleic acid sequence-based amplification (NASBA) (6) have also been used as an alternative approach for detecting agents that are responsible for infection.These techniques can also be used for detailed analysis of microbial biota in the oral cavity and gastrointestinal tract, using universal primers that anneal to conserved regions in the 16S ribosomal RNA gene (rDNA) or gyr B gene (7-9). It is reported that half of the bacteria comprising the oral and intestinal flora have not been previously identified by in vitro culture procedures (10). Moreover, recent studies using PCR have raised the possibility that bacterial DNA may be present in the human bloodstream (11-13). Our ultimate objective is to elucidate the role of such subclinically infecting unculturable or latent bacteria in the pathogenesis of chronic vascular diseases (14-16). As a first step, we investigated whether bacteria can translocate in some way from the oral and intestinal flora to the blood stream in 'healthy' humans. In this preliminary study, blood specimen-specific bacterial sequences were detected by PCR of rDNA. However, they were not representative of sequences found in human intestine.PCR was done with a REDExtract-N-Amp Blood PCR kit (Sigma-Aldrich Japan, Tokyo, Japan) using broadrange 16S ribosomal RNA gene (rDNA)-specific oligonucleotide primers. The PCR kit does not require any type of purification, organic extraction, centrifugation or alcohol precipitation, and can be used with whole blood.For blood sampling, the skin was first sterilized with a cotton swab moistened with popidone iodide, the anterior brachial median vein was punctured using a sterile 21-gauge needle (Terumo Corporation, Tokyo, Japan),

Plasma Levels of Bacterial DNA in HIV Infection: The Limits of Quantitative Polymerase Chain Reaction

“…Moreover, we showed that HIV seronegative subjects have detectable 16S ribosomal DNA in the blood, at a level 1 log greater than that of the control samples, for which the signal is likely due to contamination. These results are in agreement with other previous studies [6,7]. In contrast, Jiang et al [1] found no 16S ribosomal DNA in blood from healthy subjects.…”

Elevated Levels of Circulating DNA in Cardiovascular Disease Patients: Metagenomic Profiling of Microbiome in the Circulation

“…But, in recent decades, presence of bacterial 16S rRNA genes has been reported in the circulation of healthy individuals [24], [25]. Adding to this knowledge are the recent reports on identification of viruses in the circulation of asymptomatic humans [26], [27].…”

Cultural Isolation and Characteristics of the Blood Microbiome of Healthy Individuals

“…Kalfin reported 100% positivity of the blood cultures. Subsequently several other authors questioned the existence of the blood microbiome or DNAemia in healthy individuals, Domingue (1977) [5], (1997) [7], Nikkari et al (2001) [8], Mc Laughlin et al (2002) [9], Moriyama et al (2008) [10], Markova (2015) [11], Damgaard (2015) [12] Dimova et al (2017) [13], Gosiewski et al (2017) [2] and Kowarsky et al (2017) [3]. The authors reported supporting electron microscopy, cultural and molecular data in favour of the existence of the blood microbiota in healthy individuals.…”