Highlighting the promise of qPCR-based environmental monitoring: response of the ribosomal RNA:DNA ratio of calanoid copepods to toxic cyanobacteria.

Calanoid copepods are integral to aquatic food webs and may drive the bioaccumulation of toxins and heavy metals, spread of infectious diseases, and occurrence of toxic cyanobacterial harmful algal blooms (HABs) in freshwater aquatic systems. However, interrelationships between copepod and cyanobacterial population dynamics and ecophysiology remain unclear. Insights into these relationships are important to aquatic resource management, as they may help guide mitigation efforts. We developed a calanoid copepod qPCR assay to investigate how copepod abundance and physiological status relate to the abundance of cyanobacteria and the concentration of total microcystin in a HAB-prone freshwater multi-use eutrophic lake. Through in silico and in vitro validation of primers and analyses of time series, we demonstrate that our assay can be used as a reliable tool for environmental monitoring. Importantly, copepod RNA:DNA ratios on and shortly after the day when microcystin concentration was at its highest within the lake were not significantly lower (or higher) than before or after this period, suggesting that copepods may have been tolerant of microcystin levels observed and capable of perpetuating bloom events by consuming competitors of toxic cyanobacteria.

Categorical performance characteristics of method ISO 7899-2 and indicator value of intestinal enterococci for bathing water quality monitoring.

Intestinal enterococci indicate the fecal contamination of bathing waters. This study defines the performance characteristics of the reference method ISO 7899-2:2000 with water samples collected from inland and coastal bathing areas in Finland. From a total of 341 bacterial isolates grown on Slanetz and Bartley medium, 63.6% were confirmed as intestinal enterococci on bile aesculin agar. The partial 16S rRNA gene sequences showed that Enterococcus faecium and Enterococcus faecalis clades accounted for 93.1% of the confirmed isolates. The range of the false positive and false negative rate of the ISO 7899-2 was 0.0-18.5% and 5.6-57.1%, respectively, being affected by the presumptive colony count on the membrane. The analysis of multiple sample volumes is proposed to reach 10-100 colonies per membrane when 47 mm diameter membranes are used to prevent overestimation of low counts and underestimation of the high counts.

Corexit 9500 Enhances Oil Biodegradation and Changes Active Bacterial Community Structure of Oil-Enriched Microcosms.

To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25°C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25°C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia were dominated by Vibrio, Idiomarina, Marinobacter, Alcanivorax, and Thalassospira species, while the 5°C consortia were dominated by several species of the genera Flavobacterium, Alcanivorax, and Oleispira Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus, known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5°C consortia, while at 25°C, a decrease in the relative abundance of Marinobacter was observed. At 25°C, Thalassospira, Marinobacter, and Idiomarina were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly, Oleispira was greatly stimulated by the addition of oil at 5°C.IMPORTANCE While dispersants such as Corexit 9500 can be used to treat oil spills, there is still debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on oil-degrading microbial communities. The results of this study provide some insights on the microbial dynamics of hydrocarbon-degrading bacterial populations in the presence of Corexit 9500. Operational taxonomic unit (OTU) analyses indicated that several OTUs were inhibited by the addition of Corexit. Conversely, a number of OTUs were stimulated by the addition of the dispersant, many of which were identified as known hydrocarbon-degrading bacteria. The results highlight the value of using RNA-based methods to further understand the impact of dispersant on the overall activity of different hydrocarbon-degrading bacterial groups.

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

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 ß-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.

The Roles of Biofilm Conductivity and Donor Substrate Kinetics in a Mixed-Culture Biofilm Anode.

We experimentally assessed the kinetics and thermodynamics of electron transfer (ET) from the donor substrate (acetate) to the anode for a mixed-culture biofilm anode. We interpreted the results with a modified biofilm-conduction model consisting of three ET steps in series: (1) intracellular ET, (2) non-Ohmic extracellular ET (EET) from an outer membrane protein to an extracellular cofactor (EC), and (3) ET from the EC to the anode by Ohmic-conduction in the biofilm matrix. The steady-state current density was 0.82 ± 0.03 A/m2 in a miniature microbial electrochemical cell operated at fixed anode potential of -0.15 V versus the standard hydrogen electrode. Illumina 16S-rDNA and -rRNA sequences showed that the Geobacter genus was less than 30% of the community of the biofilm anode. Biofilm conductivity was high at 2.44 ± 0.42 mS/cm, indicating that the maximum current density could be as high as 270 A/m2 if only Ohmic-conduction EET was limiting. Due to the high biofilm conductivity, the maximum energy loss for Ohmic-conduction EET was negligible, 0.085 mV. The energy loss in the second ET step also was small, only 20 mV, and the potential for the EC involved in the second ET was -0.15 V, a value documenting that >99% of the EC was in the oxidized state. Monod kinetics for utilization of acetate were relatively slow, and at least 87% of the energy loss was in the intracellular step. Thus, intracellular ET was the main kinetic and thermodynamic bottleneck to ET from donor substrate to the anode for a highly conductive biofilm.

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.

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.

Impact of Heavy Metals on Transcriptional and Physiological Activity of Nitrifying Bacteria.

Heavy metals can inhibit nitrification, a key process for nitrogen removal in wastewater treatment. The transcriptional responses of amoA, hao, nirK, and norB were measured in conjunction with specific oxygen uptake rate (sOUR) for nitrifying enrichment cultures exposed to different metals (Ni(II), Zn(II), Cd(II), and Pb(II)). There was significant decrease in sOUR with increasing concentrations for Ni(II) (0.03-3 mg/L), Zn(II) (0.1-10 mg/L), and Cd(II) (0.03-1 mg/L) (p < 0.05). However, no considerable changes in sOUR were observed with Pb(II) (1-100 mg/L), except at a dosage of 1000 mg/L causing 84% inhibition. Based on RT-qPCR data, the transcript levels of amoA and hao decreased when exposed to Ni(II) dosages. Slight up-regulation of amoA, hao, and nirK (0.5-1.5-fold) occurred after exposure to 0.3-3 mg/L Zn(II), although their expression decreased for 10 mg/L Zn(II). With the exception of 1000 mg/L Pb(II), stimulation of all genes occurred on Cd(II) and Pb(II) exposure. While overall the results show that RNA-based function-specific assays can be used as potential surrogates for measuring nitrification activity, the degree of inhibition inferred from sOUR and gene transcription is different. We suggest that variations in transcription of functional genes may supplement sOUR based assays as early warning indicators of upsets in nitrification.

Bacterial composition in a metropolitan drinking water distribution system utilizing different source waters.

We investigated the bacterial composition of water samples from two service areas within a drinking water distribution system (DWDS), each associated with a different primary source of water (groundwater, GW; surface water, SW) and different treatment process. Community analysis based on 16S rRNA gene clone libraries indicated that Actinobacteria (Mycobacterium spp.) and α-Proteobacteria represented nearly 43 and 38% of the total sequences, respectively. Sequences closely related to Legionella, Pseudomonas, and Vibrio spp. were also identified. In spite of the high number of sequences (71%) shared in both areas, multivariable analysis revealed significant differences between the GW and SW areas. While the dominant phylotypes where not significantly contributing in the ordination of samples, the populations associated with the core of phylotypes (1-10% in each sample) significantly contributed to the differences between both service areas. Diversity indices indicate that the microbial community inhabiting the SW area is more diverse and contains more distantly related species coexisting with local assemblages as compared with the GW area. The bacterial community structure of SW and GW service areas were dissimilar, suggesting that their respective source water and/or water quality parameters shaped by the treatment processes may contribute to the differences in community structure observed.

Intestinal microbiota and species diversity of Campylobacter and Helicobacter spp. in migrating shorebirds in Delaware Bay.

Using 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.

Genome sequencing reveals the environmental origin of enterococci and potential biomarkers for water quality monitoring.

Enterococci are common members of the gut microbiome and their ease of culturing has facilitated worldwide use as indicators of fecal pollution of waters. However, enterococci were recently shown to persist in environmental habitats, often in the absence of fecal input, potentially confounding water quality assays. Toward resolving this issue and providing a more complete picture of natural enterococci diversity, 11 isolates of Enterococcus faecalis recovered from freshwater watersheds (environmental) were sequenced and compared to 59 available enteric genomes. Phenotypically and phylogenetically the environmental E. faecalis were indistinguishable from their enteric counterparts. However, distinct environmental- and enteric-associated gene signatures, encoding mostly accessory nutrient utilization pathways, were detected among the variable genes. Specifically, a nickel uptake operon was over-represented in environmental genomes, while genes for utilization of sugars thought to be abundant in the gut such as xylose were over-represented in enteric genomes. The distribution and phylogeny of these identified signatures suggest that ancestors of E. faecalis resided in extra-enteric habitats, challenging the prevailing commensal view of enterococci ecology. Thus, habitat-associated gene content changes faster than core genome phylogeny and may include biomarkers for reliably detecting fecal contaminants for improved microbial water quality monitoring.

Microbial community response to chlorine conversion in a chloraminated drinking water distribution system.

Temporary conversion to chlorine (i.e., "chlorine burn") is a common approach to controlling nitrification in chloraminated drinking water distribution systems, yet its effectiveness and mode(s) of action are not fully understood. This study characterized occurrence of nitrifying populations before, during and after a chlorine burn at 46 sites in a chloraminated distribution system with varying pipe materials and levels of observed nitrification. Quantitative polymerase chain reaction analysis of gene markers present in nitrifying populations indicated higher frequency of detection of ammonia oxidizing bacteria (AOB) (72% of samples) relative to ammonia oxidizing archaea (AOA) (28% of samples). Nitrospira nitrite oxidizing bacteria (NOB) were detected at 45% of samples, while presence of Nitrobacter NOB could not be confirmed at any of the samples. During the chlorine burn, the numbers of AOA, AOB, and Nitrospira greatly reduced (i.e., 0.8-2.4 log). However, rapid and continued regrowth of AOB and Nitrospira were observed along with nitrite production in the bulk water within four months after the chlorine burn, and nitrification outbreaks appeared to worsen 6-12 months later, even after adopting a twice annual burn program. Although high throughput sequencing of 16S rRNA genes revealed a distinct community shift and higher diversity index during the chlorine burn, it steadily returned towards a condition more similar to pre-burn than burn stage. Significant factors associated with nitrifier and microbial community composition included water age and sampling location type, but not pipe material. Overall, these results indicate that there is limited long-term effect of chlorine burns on nitrifying populations and the broader microbial community.

Impact of harmful algal bloom severity on bacterial communities in a full-scale biological filtration system for drinking water treatment.

The occurrence of harmful algal blooms (HABs) in freshwater environments has been expanded worldwide with growing frequency and severity. HABs can pose a threat to public water supplies, raising concerns about safety of treated water. Many studies have provided valuable information about the impacts of HABs and management strategies on the early-stage treatment processes (e.g., pre-oxidation and coagulation/flocculation) in conventional drinking water treatment plants (DWTPs). However, the potential effect of HAB-impacted water in the granular media filtration has not been well studied. Biologically-active filters (BAFs), which are used in drinking water treatment and rely largely on bacterial community interactions, have not been examined during HABs in full-scale DWTPs. In this study, we assessed the bacterial community structure of BAFs, functional profiles, assembly processes, and bio-interactions in the community during both severe and mild HABs. Our findings indicate that bacterial diversity in BAFs significantly decreases during severe HABs due to the predominance of bloom-associated bacteria (e.g., Spingopyxis, Porphyrobacter, and Sphingomonas). The excitation-emission matrix combined with parallel factor analysis (EEM-PARAFAC) confirmed that filter influent affected by the severe HAB contained a higher portion of protein-like substances than filter influent samples during a mild bloom. In addition, BAF community functions showed increases in metabolisms associated with intracellular algal organic matter (AOM), such as lipids and amino acids, during severe HABs. Further ecological process and network analyses revealed that severe HAB, accompanied by the abundance of bloom-associated taxa and increased nutrient availability, led to not only strong stochastic processes in the assembly process, but also a bacterial community with lower complexity in BAFs. Overall, this study provides deeper insights into BAF bacterial community structure, function, and assembly in response to HABs.

Molecular diversity of Bacteroidales in fecal and environmental samples and swine-associated subpopulations.

Several swine-specific microbial source tracking methods are based on PCR assays targeting Bacteroidales 16S rRNA gene sequences. The limited application of these assays can be explained by the poor understanding of their molecular diversity in fecal sources and environmental waters. In order to address this, we studied the diversity of 9,340 partial (>600 bp in length) Bacteroidales 16S rRNA gene sequences from 13 fecal sources and nine feces-contaminated watersheds. The compositions of major Bacteroidales populations were analyzed to determine which host and environmental sequences were contributing to each group. This information allowed us to identify populations which were both exclusive to swine fecal sources and detected in swine-contaminated waters. Phylogenetic and diversity analyses revealed that some markers previously believed to be highly specific to swine populations are shared by multiple hosts, potentially explaining the cross-amplification signals obtained with nontargeted hosts. These data suggest that while many Bacteroidales populations are cosmopolitan, others exhibit a preferential host distribution and may be able to survive different environmental conditions. This study further demonstrates the importance of elucidating the diversity patterns of targeted bacterial groups to develop more inclusive fecal source tracking applications.

Tracking the primary sources of fecal pollution in a tropical watershed in a one-year study.

A study was conducted to determine the primary sources of fecal pollution in a subtropical watershed using host-specific assays developed in temperate regions. Water samples (n = 534) from 10 different sites along the Rio Grande de Arecibo (RGA) watershed were collected mostly on a weekly basis (54 sampling events) during 13 months. DNA extracts from water samples were used in PCR assays to determine the occurrence of fecal bacteria (Bacteroidales, Clostridium coccoides, and enterococci) and human-, cattle-, swine-, and chicken-specific fecal sources. Feces from 12 different animals (n = 340) and wastewater treatment samples (n = 16) were analyzed to determine the specificity and distribution of host-specific assays. The human-specific assay (HF183) was found to be highly specific, as it did not cross-react with nontarget samples. The cattle marker (CF128) cross-reacted to some extent with swine, chicken, and turkeys and was present in 64% of the cattle samples tested. The swine assays showed poor host specificity, while the three chicken assays showed poor host distribution. Differences in the detection of host-specific markers were noted per site. While human and cattle assays showed moderate average detection rates throughout the watershed, areas impacted by wastewater treatment plants and cattle exhibited the highest prevalence of these markers. When conditional probability for positive signals was determined for each of the markers, the results indicated higher confidence levels for the human assay and lower levels for all the other assays. Overall, the results from this study suggest that additional assays are needed, particularly to track cattle, chicken, and swine fecal pollution sources in the RGA watershed. The results also suggest that the geographic stability of genetic markers needs to be determined prior to conducting applied source tracking studies in tropical settings.

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.

The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

Functional microbial diversity explains groundwater chemistry in a pristine aquifer.

BACKGROUND: The diverse microbial populations that inhabit pristine aquifers are known to catalyze critical in situ biogeochemical reactions, yet little is known about how the structure and diversity of this subsurface community correlates with and impacts upon groundwater chemistry. Herein we examine 8,786 bacterial and 8,166 archaeal 16S rRNA gene sequences from an array of monitoring wells in the Mahomet aquifer of east-central Illinois. Using multivariate statistical analyses we provide a comparative analysis of the relationship between groundwater chemistry and the microbial communities attached to aquifer sediment along with those suspended in groundwater. RESULTS: Statistical analyses of 16S rRNA gene sequences showed a clear distinction between attached and suspended communities; with iron-reducing bacteria far more abundant in attached samples than suspended, while archaeal clones related to groups associated with anaerobic methane oxidation and deep subsurface gold mines (ANME-2D and SAGMEG-1, respectively) distinguished the suspended community from the attached. Within the attached bacterial community, cloned sequences most closely related to the sulfate-reducing Desulfobacter and Desulfobulbus genera represented 20% of the bacterial community in wells where the concentration of sulfate in groundwater was high (> 0.2 mM), compared to only 3% in wells with less sulfate. Sequences related to the genus Geobacter, a genus containing ferric-iron reducers, were of nearly equal abundance (15%) to the sulfate reducers under high sulfate conditions, however their relative abundance increased to 34% when sulfate concentrations were < 0.03 mM. Also, in areas where sulfate concentrations were <0.03 mM, archaeal 16S rRNA gene sequences similar to those found in methanogens such as Methanosarcina and Methanosaeta comprised 73-80% of the community, and dissolved CH4 ranged between 220 and 1240 µM in these groundwaters. In contrast, methanogens (and their product, CH4) were nearly absent in samples collected from groundwater samples with > 0.2 mM sulfate. In the suspended fraction of wells where the concentration of sulfate was between 0.03 and 0.2 mM, the archaeal community was dominated by sequences most closely related to the ANME-2D, a group of archaea known for anaerobically oxidizing methane. Based on available energy (∆GA) estimations, results varied little for both sulfate reduction and methanogenesis throughout all wells studied, but could favor anaerobic oxidation of methane (AOM) in wells containing minimal sulfate and dihydrogen, suggesting AOM coupled with H2-oxidizing organisms such as sulfate or iron reducers could be an important pathway occurring in the Mahomet aquifer. CONCLUSIONS: Overall, the results show several distinct factors control the composition of microbial communities in the Mahomet aquifer. Bacteria that respire insoluble substrates such as iron oxides, i.e. Geobacter, comprise a greater abundance of the attached community than the suspended regardless of groundwater chemistry. Differences in community structure driven by the concentration of sulfate point to a clear link between the availability of substrate and the abundance of certain functional groups, particularly iron reducers, sulfate reducers, methanogens, and methanotrophs. Integrating both geochemical and microbiological observations suggest that the relationships between these functional groups could be driven in part by mutualism, especially between ferric-iron and sulfate reducers.

Correlative assessment of fecal indicators using human mitochondrial DNA as a direct marker.

Identifying the source of surface water fecal contamination is paramount to mitigating pollution and risk to human health. Fecal bacteria such as E. coli have been staple indicator organisms for over a century, however there remains uncertainty with E. coli-based metrics since these bacteria are abundant in the environment. The relationships between the presence of direct indicator of human waste (human mitochondrial DNA), human-specific Bacteroidales, and E. coli were studied for water samples taken from an urban creek system (Duck Creek Watershed, Cincinnati, OH) impacted by combined sewer overflows. Logistic regression analysis shows that human-specific Bacteroidales correlates much more closely to human mitochondrial DNA (R = 0.62) relative to E. coli (R = 0.33). We also examine the speciation of Bacteroidales within the Duck Creek Watershed using next-generation sequencing technology (Ion Torrent) and show the most numerous populations to be associated with sewage. Here we demonstrate that human-specific Bacteroidales closely follow the dynamics of human mitochondrial DNA concentration changes, indicating that these obligate anaerobes are more accurate than E. coli for fecal source tracking, lending further support to risk overestimation using coliforms, especially fecal coliforms and E. coli.

Determining hot spots of fecal contamination in a tropical watershed by combining land-use information and meteorological data with source-specific assays.

The objective of this study was to combine knowledge of environmental, topographical, meteorological, and anthropologic factors in the Río Grande de Arecibo (RGA) watershed in Puerto Rico with information provided by microbial source tracking (MST) to map hot spots (i.e., likely sources) of fecal contamination. Water samples were tested for the presence of human and bovine fecal contamination in addition to fecal indicator bacteria and correlated against several land uses and the density of septic tanks, sewers, and latrines. Specifically, human sources were positively correlated with developed (r = 0.68), barren land uses (r = 0.84), density of septic tanks (r = 0.78), slope (r = 0.63), and the proximity to wastewater treatment plants (WWTPs) (r = 0.82). Agricultural land, the number of upstream National Pollution Discharge Elimination System (NPDES) facilities, and density of latrines were positively associated with the bovine marker (r = 0.71; r = 0.74; and r = 0.68, respectively). Using this information, we provided a hot spot map, which shows areas that should be closely monitored for fecal contamination in the RGA watershed. The results indicated that additional bovine assays are needed in tropical regions. We concluded that meteorological, topographical, anthropogenic, and land cover data are needed to evaluate and verify the performance of MST assays and, therefore, to identify important sources of fecal contamination in environmental waters.

Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays.

In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (n = 14), surface water (n = 30), and treated source water (n = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for E. coli and Campylobacter spp. in surface waters, and for human-specific Bacteroidales, E. coli, and Enterococcus spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (p < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of E. coli, intestinal enterococci, and thermotolerant Campylobacter spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments.

Metagenomic analyses of drinking water receiving different disinfection treatments.

A metagenome-based approach was used to assess the taxonomic affiliation and function potential of microbial populations in free-chlorine-treated (CHL) and monochloramine-treated (CHM) drinking water (DW). In all, 362,640 (averaging 544 bp) and 155,593 (averaging 554 bp) pyrosequencing reads were analyzed for the CHL and CHM samples, respectively. Most annotated proteins were found to be of bacterial origin, although eukaryotic, archaeal, and viral proteins were also identified. Differences in community structure and function were noted. Most notably, Legionella-like genes were more abundant in the CHL samples while mycobacterial genes were more abundant in CHM samples. Genes associated with multiple disinfectant mechanisms were identified in both communities. Moreover, sequences linked to virulence factors, such as antibiotic resistance mechanisms, were observed in both microbial communities. This study provides new insights into the genetic network and potential biological processes associated with the molecular microbial ecology of DW microbial communities.