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

Matsuda, Kazunori; Tsuji, Hirokazu; Asahara, Takashi; Takahashi, Takuya; Kubota, Hiroyuki; Nagata, Satoru; Yamashiro, Yuichiro; Nomoto, Koji

doi:10.1128/aem.07990-11

Cited by 42 publications

(46 citation statements)

References 56 publications

(67 reference statements)

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“…After incubation at room temperature for 10 min, the fecal samples were cooled to -20 ° C and then mailed to the Yakult Central Institute. The bacterial counts were determined using the Yakult Intestinal Flora-SCAN (YIF-SCAN) according to the RT quantitative PCR technique [13,14]. The specificity of the RTquantitative PCR assay group-, species-, the Staphylococcus spa gene, and the Staphylococcus mecA gene-specific primers was determined as described previously [15] .…”

Section: The Pre-specified Secondary Outcome: the Intestinal Microbiomentioning

confidence: 99%

The Effectiveness of Lactobacillus Beverages in Controlling Infections among the Residents of an Aged Care Facility: A Randomized Placebo-Controlled Double-Blind Trial

Nagata

Asahara²,

Wang³

et al. 2015

Ann Nutr Metab

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Backgrounds/Aims: To clarify the usefulness of Lactobacillus casei strain Shirota (LcS)-fermented milk in the normalization of bowel movements and improvement of infection control for the elderly residents and staff of facilities for the elderly. Methods: A randomized placebo-controlled double-blind test was performed among the elderly residents (average age, 85) and staff members (average age, 37) of facilities for the elderly. The participants randomly received either LcS-fermented milk or a placebo beverage once daily for 6 months. Clinical data and enteric conditions were compared between the 2 groups. Results: A significantly lower incidence of fever and improved bowel movements were seen in the LcS-fermented milk group (n = 36) in comparison to the placebo group (n = 36). The numbers of Bifidobacterium and Lactobacillus were significantly higher (p < 0.01), the numbers of destructive bacteria such as Clostridium difficile were significantly lower (p < 0.05), and the fecal acetic acid concentration and total acidity were significantly higher in the LcS group. A significant difference in the intestinal microbiota, fecal acetic acid, and pH was also observed between the LcS and placebo groups among the facility's staff members. Conclusions: The long-term consumption of LcS-fermented milk may be useful for decreasing the daily risk of infection and improving the quality of life among the residents and staff of facilities for the elderly.

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Section: The Pre-specified Secondary Outcome: the Intestinal Microbiomentioning

confidence: 99%

The Effectiveness of Lactobacillus Beverages in Controlling Infections among the Residents of an Aged Care Facility: A Randomized Placebo-Controlled Double-Blind Trial

Nagata

Asahara²,

Wang³

et al. 2015

Ann Nutr Metab

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“…We theorize that in order to better assess the impact of fecal pollution it is important to target active cells via rRNA-based detection of gene markers in fecal source tracking studies. Since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells (26), it is not a surprise that recent studies have shown that rRNA-targeted RT-qPCR assays can be more sensitive than rRNA gene-based qPCR assays (27,28). In addition, the rRNA/rRNA gene ratio from each target bacterium is an important indicator of its metabolic status within bacterial communities and has been used to provide an estimate of in situ bacterial growth rates (29,30).…”

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confidence: 99%

Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

Kapoor

Pitkänen

Ryu

et al. 2015

Appl Environ Microbiol

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The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as "naked DNA" in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources. Sewage overflows and stormwater runoff introduce high levels of fecal bacteria into surface waters and are considered the primary cause of water quality impairments in urban watersheds, particularly those affected by combined sewer overflows (CSOs) (1, 2). Sewage contamination of surface waters poses a serious risk to human and environmental health via waterborne disease outbreaks (3-5), deterioration of recreational and drinking water quality (6, 7), and degradation of aquatic ecology (8, 9). Hence, identifying the primary source(s) of fecal contamination is imperative to enable best management practices for mitigating pollution and public health risks.Microbial source tracking (MST) methods targeting fecal bacteria have recently been used to identify the sources of fecal contamination impacting water systems (10, 11). Many of these MST methods are based on quantitative PCR (qPCR) assays targeting the bacterial rRNA genes present within water DNA extracts (12, 13). However, the value of DNA-based monitoring in microbial ecology studies is limited by the possibility of DNA being associated with dead cells or the extent to which "naked DNA" may survive in the environment once bacteria are lysed (14-16). These facts pose a significant challenge to the environmental fate and transport of fecal bacteria which are often assess...

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“…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] .…”

Section: Principle Of Yif-scanmentioning

confidence: 90%

“…Focusing on the possibility of using YIF-SCAN to improve the accuracy of the diagnosis of pathogenic microorganisms, we developed primer sets not only for indigenous intestinal bacteria [11,12,15,17,[39][40][41][42] , but also for various pathogenic microorganisms that can be detected in clinical settings ( Table 1 ). Along with the intestinal bacterial analysis, we have also developed a highly sensitive system for quantifying Streptococcus such as Streptococcus pyogenes , S. agalactiae , and S. pneumoniae/ mitis (frequently detected in sepsis) [43] ; Clostridium difficile (pathogenic bacterium of antibiotic-induced diarrhea) [13] ; Candida albicans , C. glabrata , C. tropicalis , C. parapsilosis , and C. krusei (pathogenic fungi of mycoses) [44] ; Vibrio cholerae/mimicus , V. parahaemolyticus/alginolyticus , and Campylobacter jejuni/coli (pathogenic bacteria of food poisoning) [16] ; Lactobacillus crispatus , L. gasseri , L. jensenii , and L. iners (indigenous vaginal bacteria); Atopobium vaginae , Gardnerella vaginalis , and Mobiluncus curtisii (pathogens of bacterial vaginosis), Mobiluncus curtisii , and Chlamydia trachomatis/muridarum (pathogenic bacteria of sexually transmitted disease) [45] .…”

Section: Detection Of Bacteria In Blood By Yif-scanmentioning

confidence: 99%

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

Tsuji

Nomoto²

2017

Ann Nutr Metab

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We have developed a highly sensitive microbial analytical system, the Yakult Intestinal Flora-SCAN (YIF-SCAN), based on reverse transcription-quantitative PCR, to quantify the intestinal microbiota. Targeting ribosomal RNA “molecules,” which are present in abundant copies in the microorganisms, YIF-SCAN is at least hundreds of times more sensitive (detection limits: 10^2-3 cells/g feces, 1 cell/mL blood) than conventional DNA-based methods, and hence enables highly sensitive and precise detection of viable microorganisms in various types of samples, such as stools, blood etc. Specifically, this high analytical sensitivity enables more accurate and reliable detection (compared with conventional culture methods) of microorganisms migrating in the blood, bacteremia, in patients with different diseases such as pediatric patients with febrile neutropenia, neonates with clinically diagnosed sepsis, and patients with type 2 diabetes mellitus or ischemic stroke. Such detection of bacteremia during gastrointestinal surgery can also be particularly effective for preventing postoperative infectious complications. Herein, we review some of the studies corroborating the potential application and clinical significance of YIF-SCAN in diagnosing bacteremia (even at marginable levels) in patients with different diseases.

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

Cited by 42 publications

References 56 publications

The Effectiveness of <b><i>Lactobacillus</i></b> Beverages in Controlling Infections among the Residents of an Aged Care Facility: A Randomized Placebo-Controlled Double-Blind Trial

The Effectiveness of <b><i>Lactobacillus</i></b> Beverages in Controlling Infections among the Residents of an Aged Care Facility: A Randomized Placebo-Controlled Double-Blind Trial

Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

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

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