Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis.

Molecular diagnostic tools in the field of food and water quality analysis are becoming increasingly widespread. Usually, based on DNA amplification techniques such as polymerase chain reaction (PCR), these methods are highly sensitive and versatile but require well-equipped laboratories and trained personnel. To reduce analysis time and avoid expensive equipment, isothermal DNA amplification methods for detecting various target organisms have been developed. However, to make molecular diagnostics suitable for low-resource settings and in-field applications, it is crucial to continuously adapt the working steps associated with DNA amplification, namely sample preparation, DNA extraction, and visualization of the results. Many novel approaches have been evaluated in recent years to tackle these challenges, e.g., the use of ionic liquids for the rapid isolation of nucleic acids from organisms relevant for food and water analysis or the integration of entire analytical workflows on microfluidic chips. In any event, the future of applications in the field of isothermal amplification will probably lie in ready-to-use cartridges combined with affordable handheld devices for on-site analysis. This trend article aims to make prospective users more familiar with this technology and its potential for moving molecular diagnostics from the laboratory to the field. Graphical abstract ᅟ.

Global Distribution of Human-Associated Fecal Genetic Markers in Reference Samples from Six Continents.

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4 400 000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log10 7.2-8.0 marker equivalents (ME) 100 mL-1) and biologically treated wastewater samples (median log10 4.6-6.0 ME 100 mL-1) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.

A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters.

We report a novel molecular assay, based on helicase-dependent amplification (HDA), for the detection of enterococci as markers for fecal pollution in water. This isothermal assay targets the same Enterococcus 23S rRNA gene region as the existing quantitative polymerase chain reaction (qPCR) assays of U.S. Environmental Protection Agency Methods 1611 and 1609 but can be entirely performed on a simple heating block. The developed Enterococcus HDA assay successfully discriminated 15 enterococcal from 15 non-enterococcal reference strains and reliably detected 48 environmental isolates of enterococci. The limit of detection was 25 target copies per reaction, only 3 times higher than that of qPCR. The applicability of the assay was tested on 30 environmental water sample DNA extracts, simulating a gradient of fecal pollution. Despite the isothermal nature of the reaction, the HDA results were consistent with those of the qPCR reference. Given this performance, we conclude that the developed Enterococcus HDA assay has great potential as a qualitative molecular screening method for resource-limited settings when combined with compatible up- and downstream processes. This amplification strategy can pave the way for developing a new generation of rapid, low-cost, and field-deployable molecular diagnostic tools for water quality monitoring.

Bacterial diversity along a 2600 km river continuum.

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 Polynucleobacter 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.

A novel triplex quantitative PCR strategy for quantification of toxigenic and nontoxigenic Vibrio cholerae in aquatic environments.

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6×10(2) to 2.3×10(4) cell equivalents liter(-1), whereas GR-corrected abundances ranged from 4.7×10(3) to 1.6×10(6) cell equivalents liter(-1). GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.

Incorporating expert judgments in utility evaluation of bacteroidales qPCR assays for microbial source tracking in a drinking water source.

Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays.

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.

Short-term microbial release during rain events from on-site sewers and cattle in a surface water source.

The protection of drinking water from pathogens such as Cryptosporidium and Giardia requires an understanding of the short-term microbial release from faecal contamination sources in the catchment. Flow-weighted samples were collected during two rainfall events in a stream draining an area with on-site sewers and during two rainfall events in surface runoff from a bovine cattle pasture. Samples were analysed for human (BacH) and ruminant (BacR) Bacteroidales genetic markers through quantitative polymerase chain reaction (qPCR) and for sorbitol-fermenting bifidobacteria through culturing as a complement to traditional faecal indicator bacteria, somatic coliphages and the parasitic protozoa Cryptosporidium spp. and Giardia spp. analysed by standard methods. Significant positive correlations were observed between BacH, Escherichia coli, intestinal enterococci, sulphite-reducing Clostridia, turbidity, conductivity and UV254 in the stream contaminated by on-site sewers. For the cattle pasture, no correlation was found between any of the genetic markers and the other parameters. Although parasitic protozoa were not detected, the analysis for genetic markers provided baseline data on the short-term faecal contamination due to these potential sources of parasites. Background levels of BacH and BacR makers in soil emphasise the need to including soil reference samples in qPCR-based analyses for Bacteroidales genetic markers.

Vibrio cholerae-An emerging pathogen in Austrian bathing waters?

Vibrio cholerae, an important human pathogen, is naturally occurring in specific aquatic ecosystems. With very few exceptions, only the cholera-toxigenic strains belonging to the serogroups O1 and O139 are responsible for severe cholera outbreaks with epidemic or pandemic potential. All other nontoxigenic, non-O1/non-O139 V. cholerae (NTVC) strains may cause various other diseases, such as mild to severe infections of the ears, of the gastrointestinal and urinary tracts as well as wound and bloodstream infections. Older, immunocompromised people and patients with specific preconditions have an elevated risk. In recent years, worldwide reports demonstrated that NTVC infections are on the rise, caused amongst others by elevated water temperatures due to global warming.The aim of this review is to summarize the knowledge gained during the past two decades on V. cholerae infections and its occurrence in bathing waters in Austria, with a special focus on the lake Neusiedler See. We investigated whether NTVC infections have increased and which specific environmental conditions favor the occurrence of NTVC. We present an overview of state of the art methods that are currently available for clinical and environmental diagnostics. A preliminary public health risk assessment concerning NTVC infections related to the Neusiedler See was established. In order to raise awareness of healthcare professionals for NTVC infections, typical symptoms, possible treatment options and the antibiotic resistance status of Austrian NTVC isolates are discussed.

Vertebrate host phylogeny influences gut archaeal diversity.

Commonly used 16S rRNA gene primers do not detect the full range of archaeal diversity present in the vertebrate gut. As a result, several questions regarding the archaeal component of the gut microbiota remain, including which Archaea are host-associated, the specificities of such associations and the major factors influencing archaeal diversity. Using 16S rRNA gene amplicon sequencing with primers that specifically target Archaea, we obtained sufficient sequence data from 185 gastrointestinal samples collected from 110 vertebrate species that span five taxonomic classes (Mammalia, Aves, Reptilia, Amphibia and Actinopterygii), of which the majority were wild. We provide evidence for previously undescribed Archaea-host associations, including Bathyarchaeia and Methanothermobacter, the latter of which was prevalent among Aves and relatively abundant in species with higher body temperatures, although this association could not be decoupled from host phylogeny. Host phylogeny explained archaeal diversity more strongly than diet, while specific taxa were associated with both factors, and cophylogeny was significant and strongest for mammalian herbivores. Methanobacteria was the only class predicted to be present in the last common ancestors of mammals and all host species. Further analysis indicated that Archaea-Bacteria interactions have a limited effect on archaeal diversity. These findings expand our current understanding of Archaea-vertebrate associations.

Identifying Inorganic Turbidity in Water Samples as Potential Loss Factor During Nucleic Acid Extraction: Implications for Molecular Fecal Pollution Diagnostics and Source Tracking.

Molecular diagnostic methods are increasingly applied for food and environmental analysis. Since several steps are involved in sample processing which can affect the outcome (e.g., adhesion of DNA to the sample matrix, inefficient precipitation of DNA, pipetting errors and (partial) loss of the DNA pellet during DNA isolation), quality control is essential at all processing levels. In soil microbiology, particular attention has been paid to the inorganic component of the sample matrix affecting DNA extractability. In water quality testing, however, this aspect has mostly been neglected so far, although it is conceivable that these mechanisms have a similar impact. The present study was therefore dedicated to investigate possible matrix effects on results of water quality analysis. Field testing in an aquatic environment with pronounced chemo-physical gradients [total suspended solids (TSS), inorganic turbidity, total organic carbon (TOC), and conductivity] indicated a negative association between DNA extractability (using a standard phenol/chloroform extraction procedure) and turbidity (spearman ρ = -0.72, p < 0.001, n = 21). Further detailed laboratory experiments on sediment suspensions confirmed the hypothesis of inorganic turbidity being the main driver for reduced DNA extractability. The observed effects, as known from soil samples, were also indicated to result from competitive effects for free charges on clay minerals, leading to adsorption of DNA to these inorganic particles. A protocol modification by supplementing the extraction buffer with salmon sperm DNA, to coat charged surfaces prior to cell lysis, was then applied on environmental water samples and compared to the standard protocol. At sites characterized by high inorganic turbidity, DNA extractability was significantly improved or made possible in the first place by applying the adapted protocol. This became apparent from intestinal enterococci and microbial source tracking (MST)-marker levels measured by quantitative polymerase chain reaction (qPCR) (100 to 10,000-fold median increase in target concentrations). The present study emphasizes the need to consider inorganic turbidity as a potential loss factor in DNA extraction from water-matrices. Negligence of these effects can lead to a massive bias, by up to several orders of magnitude, in the results of molecular MST and fecal pollution diagnostics.

Large-Scale Metagenome Assembly Reveals Novel Animal-Associated Microbial Genomes, Biosynthetic Gene Clusters, and Other Genetic Diversity.

Large-scale metagenome assemblies of human microbiomes have produced a vast catalogue of previously unseen microbial genomes; however, comparatively few microbial genomes derive from other vertebrates. Here, we generated 5,596 metagenome-assembled genomes (MAGs) from the gut metagenomes of 180 predominantly wild animal species representing 5 classes, in addition to 14 existing animal gut metagenome data sets. The MAGs comprised 1,522 species-level genome bins (SGBs), most of which were novel at the species, genus, or family level, and the majority were enriched in host versus environment metagenomes. Many traits distinguished SGBs enriched in host or environmental biomes, including the number of antimicrobial resistance genes. We identified 1,986 diverse biosynthetic gene clusters; only 23 clustered with any MIBiG database references. Gene-based assembly revealed tremendous gene diversity, much of it host or environment specific. Our MAG and gene data sets greatly expand the microbial genome repertoire and provide a broad view of microbial adaptations to the vertebrate gut.IMPORTANCE Microbiome studies on a select few mammalian species (e.g., humans, mice, and cattle) have revealed a great deal of novel genomic diversity in the gut microbiome. However, little is known of the microbial diversity in the gut of other vertebrates. We studied the gut microbiomes of a large set of mostly wild animal species consisting of mammals, birds, reptiles, amphibians, and fish. Unfortunately, we found that existing reference databases commonly used for metagenomic analyses failed to capture the microbiome diversity among vertebrates. To increase database representation, we applied advanced metagenome assembly methods to our animal gut data and to many public gut metagenome data sets that had not been used to obtain microbial genomes. Our resulting genome and gene cluster collections comprised a great deal of novel taxonomic and genomic diversity, which we extensively characterized. Our findings substantially expand what is known of microbial genomic diversity in the vertebrate gut.

DNA aptamers against bacterial cells can be efficiently selected by a SELEX process using state-of-the art qPCR and ultra-deep sequencing.

DNA aptamers generated by cell-SELEX against bacterial cells have gained increased interest as novel and cost-effective affinity reagents for cell labelling, imaging and biosensing. Here we describe the selection and identification of DNA aptamers for bacterial cells using a combined approach based on cell-SELEX, state-of-the-art applications of quantitative real-time PCR (qPCR), next-generation sequencing (NGS) and bioinformatic data analysis. This approach is demonstrated on Enterococcus faecalis (E. faecalis), which served as target in eleven rounds of cell-SELEX with multiple subtractive counter-selections against non-target species. During the selection, we applied qPCR-based analyses to evaluate the ssDNA pool size and remelting curve analysis of qPCR amplicons to monitor changes in pool diversity and sequence enrichment. Based on NGS-derived data, we identified 16 aptamer candidates. Among these, aptamer EF508 exhibited high binding affinity to E. faecalis cells (KD-value: 37 nM) and successfully discriminated E. faecalis from 20 different Enterococcus and non-Enterococcus spp. Our results demonstrate that this combined approach enabled the rapid and efficient identification of an aptamer with both high affinity and high specificity. Furthermore, the applied monitoring and assessment techniques provide insight into the selection process and can be highly useful to study and improve experimental cell-SELEX designs to increase selection efficiency.

Elucidating fecal pollution patterns in alluvial water resources by linking standard fecal indicator bacteria to river connectivity and genetic microbial source tracking.

A novel concept for fecal pollution analysis was applied at alluvial water resources to substantially extend the information provided by fecal indicator bacteria (FIB). FIB data were linked to river connectivity and genetic microbial source tracking (MST). The concept was demonstrated at the Danube River and its associated backwater area downstream of the city of Vienna, using a comprehensive 3-year data set (10 selected sites, n = 317 samples). Enumeration of Escherichia coli (ISO 16649-2), intestinal enterococci (ISO 7899-2) and Clostridium perfringens (ISO 14189) revealed a patchy distribution for the investigation area. Based on these parameters alone a clear interpretation of the observed fecal contamination patterns was not possible. Comparison of FIB concentrations to river connectivity allowed defining sites with dominating versus rare fecal pollution influence from the River Danube. A strong connectivity gradient at the selected backwater sites became obvious by 2D hydrodynamic surface water modeling, ranging from 278 days (25%) down to 5 days (<1%) of hydraulic connectivity to the River Danube within the 3-year study period. Human sewage pollution could be identified as the dominating fecal source at the highly connected sites by adding information from MST analysis. In contrast, animal fecal pollution proofed to be dominating in areas with low river connectivity. The selection of genetic MST markers was focusing on potentially important pollution sources in the backwater area, using human (BacHum, HF183II), ruminant (BacR) and pig (Pig2Bac) -associated quantitative PCR assays. The presented approach is assumed to be useful to characterize alluvial water resources for water safety management throughout the globe, by allocating fecal pollution to autochthonous, allochthonous, human or animal contamination components. The established river connectivity metric is not limited to bacterial fecal pollution, but can be applied to any type of chemical and microbiological contamination.

Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test.

Over the last decades, various PCR-based methods have been proposed that can identify sources of faecal pollution in environmental waters. These microbial source tracking (MST) methods are powerful tools to manage water quality and support public health risk assessment. However, their application is limited by the lack of specialized equipment and trained personnel in laboratories performing microbiological water quality assessment. Here, we describe a novel molecular method that combines helicase-dependent amplification (HDA) with a strip test for detecting ruminant faecal pollution sources. Unlike quantitative PCR (qPCR), the developed HDA-strip assay only requires a heating block to amplify the ruminant-associated Bacteroidetes 16S rRNA marker (BacR). Following HDA, the reaction mixture can be directly applied onto the test strip, which detects and displays the amplification products by marker-specific hybridization probes via an on-strip colorimetric reaction. The entire assay takes two hours and demands no extensive practical training. Furthermore, the BacR HDA-strip assay achieved comparable results in head-to-head performance tests with the qPCR reference, in which we investigated source-sensitivity and source-specificity, the analytical limit of detection, and the sample limit of detection. Although this approach only yields qualitative results, it can pave a way for future simple-to-use MST screening tools.

Host diet and evolutionary history explain different aspects of gut microbiome diversity among vertebrate clades.

Multiple factors modulate microbial community assembly in the vertebrate gut, though studies disagree as to their relative contribution. One cause may be a reliance on captive animals, which can have very different gut microbiomes compared to their wild counterparts. To resolve this disagreement, we analyze a new, large, and highly diverse animal distal gut 16 S rRNA microbiome dataset, which comprises 80% wild animals and includes members of Mammalia, Aves, Reptilia, Amphibia, and Actinopterygii. We decouple the effects of host evolutionary history and diet on gut microbiome diversity and show that each factor modulates different aspects of diversity. Moreover, we resolve particular microbial taxa associated with host phylogeny or diet and show that Mammalia have a stronger signal of cophylogeny. Finally, we find that environmental filtering and microbe-microbe interactions differ among host clades. These findings provide a robust assessment of the processes driving microbial community assembly in the vertebrate intestine.

Spring Water of an Alpine Karst Aquifer Is Dominated by a Taxonomically Stable but Discharge-Responsive Bacterial Community.

Alpine karst aquifers are important groundwater resources for the provision of drinking water all around the world. Yet, due to difficult accessibility and long-standing methodological limitations, the microbiology of these systems has long been understudied. The aim of the present study was to investigate the structure and dynamics of bacterial communities in spring water of an alpine limestone karst aquifer (LKAS2) under different hydrological conditions (base vs. event flow). The study was based on high-throughput 16S rRNA gene amplicon sequencing, study design and sample selection were guided by hydrology and pollution microbiology data. Spanning more than 27 months, our analyses revealed a taxonomically highly stable bacterial community, comprising high proportions of yet uncultivated bacteria in the suspended bacterial community fraction. Only the three candidate phyla Parcubacteria (OD1), Gracilibacteria (GN02), Doudnabacteria (SM2F11) together with Proteobacteria and Bacteroidetes contributed between 70.0 and 88.4% of total reads throughout the investigation period. A core-community of 300 OTUs consistently contributed between 37.6 and 56.3% of total reads, further supporting the hypothesis of a high temporal stability in the bacterial community in the spring water. Nonetheless, a detectable response in the bacterial community structure of the spring water was discernible during a high-discharge event. Sequence reads affiliated to the class Flavobacteriia clearly increased from a mean proportion of 2.3% during baseflow to a maximum of 12.7% during the early phase of the studied high-discharge event, suggesting direct impacts from changing hydrological conditions on the bacterial community structure in the spring water. This was further supported by an increase in species richness (Chao1) at higher discharge. The combination of these observations allowed the identification and characterization of three different discharge classes (Q1-Q3). In conclusion, we found a taxonomically stable bacterial community prevailing in spring waters from an alpine karst aquifer over the entire study period of more than 2 years. Clear response to changing discharge conditions could be detected for particular bacterial groups, whereas the most responsive group - bacteria affiliated to the class of Flavobacteriia - might harbor potential as a valuable natural indicator of "system disturbances" in karst aquifers.

Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids.

The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.

Opening the black box of spring water microbiology from alpine karst aquifers to support proactive drinking water resource management.

Over the past 15 years, pioneering interdisciplinary research has been performed on the microbiology of hydrogeologically well-defined alpine karst springs located in the Northern Calcareous Alps (NCA) of Austria. This article gives an overview on these activities and links them to other relevant research. Results from the NCA springs and comparable sites revealed that spring water harbors abundant natural microbial communities even in aquifers with high water residence times and the absence of immediate surface influence. Apparently, hydrogeology has a strong impact on the concentration and size of the observed microbes, and total cell counts (TCC) were suggested as a useful means for spring type classification. Measurement of microbial activities at the NCA springs revealed extremely low microbial growth rates in the base flow component of the studied spring waters and indicated the importance of biofilm-associated microbial activities in sediments and on rock surfaces. Based on genetic analysis, the autochthonous microbial endokarst community (AMEC) versus transient microbial endokarst community (TMEC) concept was proposed for the NCA springs, and further details within this overview article are given to prompt its future evaluation. In this regard, it is well known that during high-discharge situations, surface-associated microbes and nutrients such as from soil habitats or human settlements-potentially containing fecal-associated pathogens as the most critical water-quality hazard-may be rapidly flushed into vulnerable karst aquifers. In this context, a framework for the comprehensive analysis of microbial pollution has been proposed for the NCA springs to support the sustainable management of drinking water safety in accordance with recent World Health Organization guidelines. Near-real-time online water quality monitoring, microbial source tracking (MST) and MST-guided quantitative microbial-risk assessment (QMRA) are examples of the proposed analytical tools. In this context, this overview article also provides a short introduction to recently emerging methodologies in microbiological diagnostics to support reading for the practitioner. Finally, the article highlights future research and development needs. This article is categorized under: 1Engineering Water > Water, Health, and Sanitation2Science of Water > Water Extremes3Water and Life > Nature of Freshwater Ecosystems.

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Enterococcus spp. in water.

Faecal pollution of water and the resulting potential presence of human enteric pathogens is a predominant threat to public health. Microbiological water quality can be assessed by the detection of standard faecal indicator bacteria (SFIB) such as E. coli or certain Enterococcus species. In recent years, isothermal amplification methods have become a useful alternative to polymerase chain reaction (PCR), allowing molecular diagnostics with simple or no instrumentation. In this study, a novel screening method for the molecular detection of Enterococcus spp. by loop-mediated isothermal amplification (LAMP) is described. A set of six specific LAMP primers was designed to amplify a diagnostic fragment of the Enterococcus 23S rRNA gene, which is present in several enterococcal species targeted by quantitative PCR (qPCR), which is the standard technique recommended by the US Environmental Protection Agency. Sensitivity and specificity tests were performed using a set of 30 Enterococcus and non-target bacterial reference strains. It is shown that LAMP is equally sensitive and even more specific than the qPCR assay. A dilution series of Enterococcus faecalis DNA revealed that the LAMP method can reliably detect 130 DNA target copies per reaction within 45 min. Additionally, enterococci isolated from Austrian surface waterbodies, as well as a set of DNA extracts from environmental waters, were tested. Contingency analysis demonstrated a highly significant correlation between the results of the developed LAMP assay and the reference qPCR method. Furthermore, a simple staining procedure with a fluorescence dye demonstrated the identification of amplified products by eye. In conclusion, this method is an important component for the efficient screening and testing of water samples in low-resource settings lacking sophisticated laboratory equipment and highly trained personnel, requiring only a simple heating block.