Detection of Human Intestinal Catalase-Negative, Gram-Positive Cocci by rRNA-Targeted Reverse Transcription-PCR

Abstract

An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) for enumeration of catalase-negative, Gram-positive cocci was established. Subgroup-or species-specific primer sets targeting 16S or 23S rRNA from Enterococcus, Streptococcus, and Lactococcus were newly developed. The RT-qPCR method using these primers together with the previously reported primer sets specific for the Enterococcus genus, the Streptococcus genus, and several Streptococcus species was found to be able to quantify the target populations with detection limits of 10 3 to 10 4 cells per gram feces, which was more than 100 times as sensitive as the qPCR method (10 6 to 10 8 cells per gram feces). The RT-qPCR analysis of fecal samples from 24 healthy adult volunteers using the genus-specific primer sets revealed that Enterococcus and Streptococcus were present as intestinal commensals at population levels of log 10 6.2 ؎ 1.4 and 7.5 ؎ 0.9 per gram feces (mean ؎ standard deviation [SD]), respectively. Detailed investigation using species-or subgroup-specific primer sets revealed that the volunteers harbored unique Enterococcus species, including the E. avium subgroup, the E. faecium subgroup, E. faecalis, the E. casseliflavus subgroup, and E. caccae, while the dominant human intestinal Streptococcus species was found to be S. salivarius. Various Lactococcus species, such as L. lactis subsp. lactis or L. lactis subsp. cremoris, L. garvieae, L. piscium, and L. plantarum, were also detected but at a lower population level (log 10 4.6 ؎ 1.2 per gram feces) and prevalence (33%). These results suggest that the RT-qPCR method enables the accurate and sensitive enumeration of human intestinal subdominant but still important populations, such as Gram-positive cocci.

Yakult Intestinal Flora-SCAN: A Novel Culture-Independent Analytical Method for Detection of Bacteria in the Bloodstream

“…Furthermore, while DNA-based PCR detects both viable and dead microorganisms, YIF-SCAN quantifies viable microorganisms, because rRNA molecules are better indicators of the status of viable microorganisms than rRNA genes [11] . Also, the data obtained by YIF-SCAN are almost equivalent to those obtained by fluorescence in situ hybridization targeting rRNA molecules [9,10,[12][13][14][15] .…”

Salivaricin D, a Novel Intrinsically Trypsin-Resistant Lantibiotic from Streptococcus salivarius 5M6c Isolated from a Healthy Infant

“…Although S. salivarius is predominantly found in the human oral cavity (23,40), it has also been occasionally isolated from the gut (25,33,35,51), and salivaricin D is produced by a strain which was isolated from the feces of a healthy human infant, indicating that it forms part of the normal flora in the gut as well. In addition, the producer strain appears to occur in high numbers in fecal microflora, suggesting that it might have traits that make it competitive and dominant in the gut flora.…”

“…It has also been observed in the nasopharynx and intestinal tract and isolated from the human feces (25,35,44) and breast milk of healthy women (1,46). S. salivarius strains produce a number of bacteriocins, most of which are lantibiotics (22,39,(52)(53)(54).…”

Sensitive Quantification of Clostridium difficile Cells by Reverse Transcription-Quantitative PCR Targeting rRNA Molecules

“…This RT-qPCR method covers a wide variety of intestinal bacterial populations, including subdominant bacteria, and has several advantages, such as sensitivity, rapidity, and accuracy (23,28). We have developed a specific primer set for C. difficile targeting the 23S rRNA gene, and here we examined the population levels of this bacterium in adult intestines by RT-qPCR.…”