Georg H. Reischer

Quantitative PCR Method for Sensitive Detection of Ruminant Fecal Pollution in Freshwater and Evaluation of This Method in Alpine Karstic Regions

ABSTRACT A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 × 109 marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 105 marker equivalents per liter).

A quantitative real‐time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area

Aims:  The aim of the study was the development of a sensitive human‐specific quantitative real‐time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human‐specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan® minor groove binder probes.

Quantitative microbial faecal source tracking with sampling guided by hydrological catchment dynamics

The impairment of water quality by faecal pollution is a global public health concern. Microbial source tracking methods help to identify faecal sources but the few recent quantitative microbial source tracking applications disregarded catchment hydrology and pollution dynamics. This quantitative microbial source tracking study, conducted in a large karstic spring catchment potentially influenced by humans and ruminant animals, was based on a tiered sampling approach: a 31-month water quality monitoring (Monitoring) covering seasonal hydrological dynamics and an investigation of flood events (Events) as periods of the strongest pollution. The detection of a ruminant-specific and a human-specific faecal Bacteroidetes marker by quantitative real-time PCR was complemented by standard microbiological and on-line hydrological parameters. Both quantitative microbial source tracking markers were detected in spring water during Monitoring and Events, with preponderance of the ruminant-specific marker. Applying multiparametric analysis of all data allowed linking the ruminant-specific marker to general faecal pollution indicators, especially during Events. Up to 80% of the variation of faecal indicator levels during Events could be explained by ruminant-specific marker levels proving the dominance of ruminant faecal sources in the catchment. Furthermore, soil was ruled out as a source of quantitative microbial source tracking markers. This study demonstrates the applicability of quantitative microbial source tracking methods and highlights the prerequisite of considering hydrological catchment dynamics in source tracking study design.

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study.

The bacterial load of Ureaplasma parvum in amniotic fluid is correlated with an increased intrauterine inflammatory response

Ureaplasma spp. are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), and preterm labor (PL). We analyzed 118 samples from amniotic fluid of preterm infants before 34 weeks of gestation by quantitative polymerase chain reaction (qPCR). Bacterial load, Ureaplasma biovar discrimination (Ureaplasma urealyticum and Ureaplasma parvum), and the level of inflammation were correlated with short-term clinical outcome. U. parvum was the predominant biovar, and increased bacterial load was significantly linked to histologic chorioamnionitis, PROM + PL, early-onset sepsis, and bronchopulmonary dysplasia. Furthermore, there was a positive correlation between the amount of U. parvum and the magnitude of inflammatory response inside the amniotic cavity observed by elevated interleukin 8 levels. We postulate that the bacterial load of Ureaplasma spp. measured by qPCR should be determined in studies investigating the potential clinical impact of intrauterine Ureaplasma spp. on the outcome of preterm infants.

Bacterial diversity along a 2600 km river continuum

Summary The bacterioplankton diversity in large rivers has thus far been under‐sampled despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along the midstream of the Danube River and its tributaries. Using 16S rRNA‐gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free‐living and particle‐associated bacterial communities. These shifts were also supported by beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwaters. Dominated by the acI lineage, the freshwater SAR11 (LD12) and the P olynucleobacter group, typical freshwater taxa increased in proportion downriver and were accompanied by a decrease in soil and groundwater‐affiliated bacteria. Based on views of the meta‐community and River Continuum Concept, we interpret the observed taxonomic patterns and accompanying changes in alpha and beta diversity with the intention of laying the foundation for a unified concept for river bacterioplankton diversity.

Escherichia coli and enterococci are sensitive and reliable indicators for human, livestock and wildlife faecal pollution in alpine mountainous water resources

Aims:  This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring.

Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid

Infection with Toxoplasma gondii during pregnancy is often asymptomatic and may cause severe fetal damage. A quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) assay was developed for the specific and sensitive detection of the previously described 529-bp repeat element occurring up to 200 to 300 times in T. gondii genome. The qualitative and quantitative detection limits determined were 6 and 20 marker copies (1/30 to 1/50 of 1 parasite) per PCR, respectively. In addition to standard PCR cycling conditions, 3 different fast PCR protocols were evaluated to minimize run time. A higher variability but no loss of specificity was observed. For the evaluation of clinical applicability, a total of 135 amniotic fluid samples were analyzed targeting both 529-bp and B1 gene. The sensitivity and specificity were 88.0% and 100.0% for B1, and 100.0% and 98.2% for 529-bp PCR assay (positive predictive value and negative predictive value: 100.0% and 97.4%, and 92.6% and 100.0%, respectively). Our results demonstrated an increased sensitivity of the 529-bp PCR assay even in a faster protocol.

Performance characteristics of qPCR assays targeting human- and ruminant-associated Bacteroidetes for microbial source tracking across sixteen countries on six continents

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

Hypothesis-Driven Approach for the Identification of Fecal Pollution Sources in Water Resources

Water resource management must strive to link catchment information with water quality monitoring. The present study attempted this for the field of microbial fecal source tracking (MST). A fecal pollution source profile based on catchment data (e.g., prevalence of fecal sources) was used to formulate a hypothesis about the dominant sources of pollution in an Austrian mountainous karst spring catchment. This allowed a statistical definition of methodical requirements necessary for an informed choice of MST methods. The hypothesis was tested in a 17-month investigation of spring water quality. The study followed a nested sampling design in order to cover the hydrological and pollution dynamics of the spring and to assess effects such as differential persistence between parameters. Genetic markers for the potential fecal sources as well as microbiological, hydrological, and chemo−physical parameters were measured. The hypothesis that ruminant animals were the dominant sources of fecal pollution in the catchment was clearly confirmed. It was also shown that the concentration of ruminant markers in feces was equally distributed in different ruminant source groups. The developed approach provides a tool for careful decision-making in MST study design and might be applied on various types of catchments and pollution situations.