Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

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e Although the source of drinking water (DW) used in hospitals is commonly disinfected, biofilms forming on water pipelines are a refuge for bacteria, including possible pathogens that survive different disinfection strategies. These biofilm communities are only beginning to be explored by culture-independent techniques that circumvent the limitations of conventional monitoring efforts. Hence, theories regarding the frequency of opportunistic pathogens in DW biofilms and how biofilm members withstand high doses of disinfectants and/or chlorine residuals in the water supply remain speculative. The aim of this study was to characterize the composition of microbial communities growing on five hospital shower hoses using both 16S rRNA gene sequencing of bacterial isolates and whole-genome shotgun metagenome sequencing. The resulting data revealed a Mycobacterium-like population, closely related to Mycobacterium rhodesiae and Mycobacterium tusciae, to be the predominant taxon in all five samples, and its nearly complete draft genome sequence was recovered. In contrast, the fraction recovered by culture was mostly affiliated with Proteobacteria, including members of the genera Sphingomonas, Blastomonas, and Porphyrobacter. The biofilm community harbored genes related to disinfectant tolerance (2.34% of the total annotated proteins) and a lower abundance of virulence determinants related to colonization and evasion of the host immune system. Additionally, genes potentially conferring resistance to ␤-lactam, aminoglycoside, amphenicol, and quinolone antibiotics were detected. Collectively, our results underscore the need to understand the microbiome of DW biofilms using metagenomic approaches. This information might lead to more robust management practices that minimize the risks associated with exposure to opportunistic pathogens in hospitals.

Section: Methodsmentioning

confidence: 99%

Biofilms on Hospital Shower Hoses: Characterization and Implications for Nosocomial Infections

Soto-Girón

Rodriguez-R

Luo

et al. 2016

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“…faecalis (Faecalis (37), and human-specific Bacteroidales (HF183 and BacHum assays) (38,39). The qPCR assays were performed as previously described by Ryu et al (37). Standard curves were generated by using plasmids containing the sequences for each of the targeted genes.…”

Section: Methodsmentioning

confidence: 99%

“…(Entero1 assay) (35), Ent. faecalis (Faecalis (37), and human-specific Bacteroidales (HF183 and BacHum assays) (38,39). The qPCR assays were performed as previously described by Ryu et al (37).…”

Section: Methodsmentioning

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

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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...

“…Bootstrap values were based on 1,000 replications. (33), and E. casseliflavus (33) were performed against water and fecal DNA extracts in 25-l reaction mixtures containing 1ϫ TaqMan universal PCR master mix with AmpErase uracil-N-glycosylase (Applied Biosystems, Foster City, CA) and 0.2 g/l bovine serum albumin (Table 1). Tenfold dilutions of each DNA extract were used to test for PCR inhibition (21,34).…”

Section: Methodsmentioning

confidence: 99%

Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay

Ryu

Grond

Verheijen

et al. 2014

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dUsing 16S rRNA gene sequencing analysis, we examined the bacterial diversity and the presence of opportunistic bacterial pathogens (i.e., Campylobacter and Helicobacter) in red knot (Calidris canutus; n ‫؍‬ 40), ruddy turnstone (Arenaria interpres; n ‫؍‬ 35), and semipalmated sandpiper (Calidris pusilla; n ‫؍‬ 22) fecal samples collected during a migratory stopover in Delaware Bay. Additionally, we studied the occurrence of Campylobacter spp., enterococci, and waterfowl fecal source markers using quantitative PCR (qPCR) assays. Of 3,889 16S rRNA clone sequences analyzed, the bacterial community was mostly composed of Bacilli (63.5%), Fusobacteria (12.7%), Epsilonproteobacteria (6.5%), and Clostridia (5.8%). When epsilonproteobacterium-specific 23S rRNA gene clone libraries (i.e., 1,414 sequences) were analyzed, the sequences were identified as Campylobacter (82.3%) or Helicobacter (17.7%) spp. Specifically, 38.4%, 10.1%, and 26.0% of clone sequences were identified as C. lari (>99% sequence identity) in ruddy turnstone, red knot, and semipalmated sandpiper clone libraries, respectively. Other pathogenic species of Campylobacter, such as C. jejuni and C. coli, were not detected in excreta of any of the three bird species. Most Helicobacter-like sequences identified were closely related to H. pametensis (>99% sequence identity) and H. anseris (92% sequence identity). qPCR results showed that the occurrence and abundance of Campylobacter spp. was relatively high compared to those of fecal indicator bacteria, such as Enterococcus spp., E. faecalis, and Catellicoccus marimammalium. Overall, the results provide insights into the complexity of the shorebird gut microbial community and suggest that these migratory birds are important reservoirs of pathogenic Campylobacter species.