On-site filtration of large sample volumes improves the detection of opportunistic pathogens in drinking water distribution systems.

In this study, we compared conventional vacuum filtration of small volumes through disc membranes (effective sample volumes for potable water: 0.3-1.0 L) with filtration of high volumes using ultrafiltration (UF) modules (effective sample volumes for potable water: 10.6-84.5 L) for collecting bacterial biomass from raw, finished, and tap water at seven drinking water systems. Total bacteria, Legionella spp., Legionella pneumophila, Mycobacterium spp., and Mycobacterium avium complex in these samples were enumerated using both conventional quantitative PCR (qPCR) and viability qPCR (using propidium monoazide). In addition, PCR-amplified gene fragments were sequenced for microbial community analysis. The frequency of detection (FOD) of Legionella spp. in finished and tap water samples was much greater using UF modules (83% and 77%, respectively) than disc filters (24% and 33%, respectively). The FODs for Mycobacterium spp. in raw, finished, and tap water samples were also consistently greater using UF modules than disc filters. Furthermore, the number of observed operational taxonomic units and diversity index values for finished and tap water samples were often substantially greater when using UF modules as compared to disc filters. Conventional and viability qPCR yielded similar results, suggesting that membrane-compromised cells represented a minor fraction of total bacterial biomass. In conclusion, our research demonstrates that large-volume filtration using UF modules improved the detection of opportunistic pathogens at the low concentrations typically found in public drinking water systems and that the majority of bacteria in these systems appear to be viable in spite of disinfection with free chlorine and/or chloramine.IMPORTANCEOpportunistic pathogens, such as Legionella pneumophila, are a growing public health concern. In this study, we compared sample collection and enumeration methods on raw, finished, and tap water at seven water systems throughout the State of Minnesota, USA. The results showed that on-site filtration of large water volumes (i.e., 500-1,000 L) using ultrafiltration membrane modules improved the frequency of detection of relatively rare organisms, including opportunistic pathogens, compared to the common approach of filtering about 1 L using disc membranes. Furthermore, results from viability quantitative PCR (qPCR) with propidium monoazide were similar to conventional qPCR, suggesting that membrane-compromised cells represent an insignificant fraction of microorganisms. Results from these ultrafiltration membrane modules should lead to a better understanding of the microbial ecology of drinking water distribution systems and their potential to inoculate premise plumbing systems with opportunistic pathogens where conditions are more favorable for their growth.

Flushing Temporarily Improves Microbiological Water Quality for Buildings Supplied with Chloraminated Surface Water but Has Little Effect for Groundwater Supplies.

Microbial communities in premise plumbing systems were investigated after more than 2 months of long-term stagnation, during a subsequent flushing event, and during post-flush stagnation. Water samples were collected from showers in buildings supplied with chlorinated groundwater, untreated groundwater, and chloraminated surface water. The building supplied with chlorinated groundwater generally had the lowest bacterial concentrations across all sites (ranging from below quantification limit to 5.2 log copies/L). For buildings supplied with untreated groundwater, bacterial concentrations (5.0 to 7.6 log copies/L) and microbial community diversity index (ACE) values were consistent throughout sampling. Nontuberculous mycobacteria (NTM) and Legionella pneumophila were not detected in any groundwater-supplied buildings. Total bacteria, Legionella spp., and NTM were abundant in the surface water-supplied buildings following long-term stagnation (up to 7.6, 6.2, and 7.6 log copies/L, respectively). Flushing decreased these concentrations by ∼1 to >4 log units and reduced microbial community diversity, but the communities largely recovered within a week of post-flush stagnation. The results suggest that buildings supplied with disinfected surface water are more likely than buildings supplied with treated or untreated groundwater to experience deleterious changes in microbiological water quality during stagnation and that the water quality improvements from flushing with chloraminated water, while substantial, are short-lived.

Multi-scale Investigation of Ammonia-Oxidizing Microorganisms in Biofilters Used for Drinking Water Treatment.

Ammonia-oxidizing microorganisms (AOMs) include ammonia-oxidizing bacteria (AOB), archaea (AOA), and Nitrospira spp. sublineage II capable of complete ammonia oxidation (comammox). These organisms can affect water quality not only by oxidizing ammonia to nitrite (or nitrate) but also by cometabolically degrading trace organic contaminants. In this study, the abundance and composition of AOM communities were investigated in full-scale biofilters at 14 facilities across North America and in pilot-scale biofilters operated for 18 months at a full-scale water treatment plant. In general, the relative abundance of AOM in most full-scale biofilters and in the pilot-scale biofilters was as follows: AOB > comammox Nitrospira > AOA. The abundance of AOB in the pilot-scale biofilters increased with increasing influent ammonia concentration and decreasing temperature, whereas AOA and comammox Nitrospira exhibited no correlations with these parameters. The biofilters affected AOM abundance in the water passing through the filters via collecting and shedding but exhibited a minor influence on the composition of AOB and Nitrospira sublineage II communities in the filtrate. Overall, this study highlights the relative importance of AOB and comammox Nitrospira compared to AOA in biofilters and the influence of filter influent water quality on AOM in biofilters and their release into the filtrate.

Microbial Community Composition in Municipal Wastewater Treatment Bioreactors Follows a Distance Decay Pattern Primarily Controlled by Environmental Heterogeneity.

Understanding spatiotemporal patterns in microbial community composition is a central goal of microbial ecology. The objective of this study was to better understand the biogeography of activated sludge microbial communities, which are important for the protection of surface water quality. Monthly samples were collected from 20 facilities (25 bioreactors) within 442 km of each other for 1 year. Microbial community composition was characterized by sequencing of PCR-amplified 16S rRNA gene fragments. Statistically significant distance decay of community similarity was observed in these bioreactors independent of clustering method (operational taxonomic units [OTUs] at 97% similarity, genus-level phylotypes) and community dissimilarity metric (Sørensen, Bray-Curtis, and weighted Unifrac). Universal colonizers (i.e., detected in all samples) and ubiquitous genus-level phylotypes (i.e., detected in every facility at least once) also exhibited a significant distance decay relationship. Variation partitioning analysis of community composition showed that environmental characteristics (temperature, influent characteristics, etc.) explained more of the variance in community composition than geographic distance did, suggesting that environmental heterogeneity is more important than dispersal limitation as a mechanism for determining microbial community composition. Distance decay relationships also became stronger with increasing distance between facilities. Seasonal variation in community composition was also observed from selected bioreactors, but there was no clear seasonal pattern in the distance decay relationships. IMPORTANCE Understanding the spatiotemporal patterns of biodiversity is a central goal of ecology. The distance decay of community similarity is one of the spatial scaling patterns observed in many forms of life, including plants, animals, and microbial communities. Municipal wastewater treatment relies on microorganisms to prevent the release of excessive quantities of nutrients and other pollutants, but relatively few studies have explored distance decay relationships in wastewater treatment bioreactors. Our results demonstrate a strong distance decay pattern in wastewater treatment bioreactors, regardless of the sequence clustering method or the community dissimilarity metric. Our results suggest that microbial communities in wastewater treatment bioreactors are not randomly assembled but rather exhibit a statistically significant spatial pattern.

Quantifying and predicting antimicrobials and antimicrobial resistance genes in waterbodies through a holistic approach: a study in Minnesota, United States.

The environment plays a key role in the spread and persistence of antimicrobial resistance (AMR). Antimicrobials and antimicrobial resistance genes (ARG) are released into the environment from sources such as wastewater treatment plants, and animal farms. This study describes an approach guided by spatial mapping to quantify and predict antimicrobials and ARG in Minnesota's waterbodies in water and sediment at two spatial scales: macro, throughout the state, and micro, in specific waterbodies. At the macroscale, the highest concentrations across all antimicrobial classes were found near populated areas. Kernel interpolation provided an approximation of antimicrobial concentrations and ARG abundance at unsampled locations. However, there was high uncertainty in these predictions, due in part to low study power and large distances between sites. At the microscale, wastewater treatment plants had an effect on ARG abundance (sul1 and sul2 in water; blaSHV, intl1, mexB, and sul2 in sediment), but not on antimicrobial concentrations. Results from sediment reflected a long-term history, while water reflected a more transient record of antimicrobials and ARG. This study highlights the value of using spatial analyses, different spatial scales, and sampling matrices, to design an environmental monitoring approach to advance our understanding of AMR persistence and dissemination.

Direct Evidence for Deterministic Assembly of Bacterial Communities in Full-Scale Municipal Wastewater Treatment Facilities.

In this study, we investigated whether bacterial community composition in full-scale wastewater treatment bioreactors can be better explained by niche- or neutral-based theory (deterministic or stochastic) and whether bioreactor design (continuous flow versus fill and draw) affected community assembly. Four wastewater treatment facilities (one with quadruplicated continuous-flow bioreactors, two with one continuous-flow bioreactor each, and one with triplicate fill-and-draw bioreactors) were investigated. Bioreactor community composition was characterized by sequencing of PCR-amplified 16S rRNA gene fragments. Replicate bioreactors at the same wastewater treatment facility had largely reproducible (i.e., deterministic) bacterial community composition, although bacterial community composition in continuous-flow bioreactors was significantly more reproducible (P < 0.001) than in fill-and-draw bioreactors (Bray-Curtis dissimilarity, µ = 0.48 ± 0.06 versus 0.58 ± 0.08). Next, we compared our results to previously used indirect methods for distinguishing between deterministic and stochastic community assembly mechanisms. Synchronicity was observed in the bacterial community composition among bioreactors within the same metropolitan region, consistent with deterministic community assembly. Similarly, a null model-based analysis also indicated that all wastewater bioreactor communities were controlled by deterministic factors and that continuous-flow bioreactors were significantly more deterministic (P < 0.001) than fill-and-draw bioreactors (nearest-taxon index, µ = 3.8 ± 0.6 versus 2.7 ± 0.8). Our results indicate that bacterial community composition in wastewater treatment bioreactors is better explained by deterministic community assembly theory; simultaneously, our results validate previously used but indirect methods to quantify whether microbial communities were assembled via deterministic or stochastic mechanisms. IMPORTANCE Understanding the mechanisms of bacterial community assembly is one of the grand challenges of microbial ecology. In environmental systems, this challenge is exacerbated because replicate experiments are typically impossible; that is, microbial ecologists cannot fabricate multiple field-scale experiments of identical, natural ecosystems. Our results directly demonstrate that deterministic mechanisms are more prominent than stochastic mechanisms in the assembly of wastewater treatment bioreactor communities. Our results also suggest that wastewater treatment bioreactor design is pertinent, such that the imposition of feast-famine conditions (i.e., fill-and-draw bioreactors) nudge bacterial community assembly more toward stochastic mechanisms than the imposition of stringent nutrient limitation (i.e., continuous-flow bioreactors). Our research also validates the previously used indirect methods (synchronous community dynamics and an application of a null model) for characterizing the relative importance of deterministic versus stochastic mechanisms of community assembly.

Different Engineering Designs Have Profoundly Different Impacts on the Microbiome and Nitrifying Bacterial Populations in Municipal Wastewater Treatment Bioreactors.

Numerous wastewater treatment processes are designed by engineers to achieve specific treatment goals. However, the impact of these different process designs on bacterial community composition is poorly understood. In this study, 24 different municipal wastewater treatment facilities (37 bioreactors) with various system designs were analyzed by sequencing of PCR-amplified 16S rRNA gene fragments. Although a core microbiome was observed in all of the bioreactors, the overall microbial community composition (analysis of molecular variance; P = 0.001) as well as that of a specific population of Nitrosomonas spp. (P = 0.04) was significantly different between A/O (anaerobic/aerobic) systems and conventional activated sludge (CAS) systems. Community α-diversity (number of observed operational taxonomic units [OTUs] and Shannon diversity index) was also significantly higher in A/O systems than in CAS systems (Wilcoxon; P < 2 × 10-16). In addition, wastewater bioreactors with short mean cell residence time (<2 days) had very low community α-diversity and fewer nitrifying bacteria compared to those of other system designs. Nitrospira spp. (0.71%) and Nitrotoga spp. (0.41%) were the most prominent nitrite-oxidizing bacteria (NOB); because these two genera were rarely prominent at the same time, these populations appeared to be functionally redundant. Weak evidence (AOB:NOB « 2; substantial quantities of Nitrospira sublineage II) was also obtained suggesting that complete ammonia oxidation by a single organism was occurring in system designs known to impose stringent nutrient limitation. This research demonstrates that design decisions made by wastewater treatment engineers significantly affect the microbiome of wastewater treatment bioreactors. IMPORTANCE Municipal wastewater treatment facilities rely on the application of numerous "activated sludge" process designs to achieve site-specific treatment goals. A plethora of microbiome studies on municipal wastewater treatment bioreactors have been performed previously; however, the role of process design on the municipal wastewater treatment microbiome is poorly understood. In fact, wastewater treatment engineers have attempted to control the microbiome of wastewater bioreactors for decades without sufficient empirical evidence to support their design paradigms. Our research demonstrates that engineering decisions with respect to system design have a significant impact on the microbiome of wastewater treatment bioreactors.

Dissolved oxygen concentrations affect the function but not the relative abundance of nitrifying bacterial populations in full-scale municipal wastewater treatment bioreactors during cold weather.

This study aimed to understand the effect of different dissolved oxygen (DO) concentrations on the abundance and performance of nitrifying bacteria in full-scale wastewater treatment bioreactors, particularly during the winter when nitrifying bacterial activity is often negligible. Biomass samples were collected from three parallel full-scale bioreactors with low DO concentrations (<1.3 mg/ L) and from two full-scale bioreactors with higher DO concentrations (~4.0 and ~2.3 mg/ L). The relative abundance of nitrifying bacteria was determined by sequencing of PCR-amplified 16S rRNA gene fragments. In the three bioreactors with low DO concentrations, effluent ammonia concentrations sharply increased with a decline in temperature below approximately 17 °C, while the bioreactors with high DO concentrations showed stable nitrification regardless of temperature. Even with the decline in nitrification during the winter in the three low DO bioreactors, the relative abundance of ammonia oxidizing bacteria (mostly Nitrosomonas spp.) was curiously maintained. The relative abundance of nitrite oxidizing bacteria was similarly maintained, although there were substantial seasonal fluctuations in the relative abundance values of Nitrospira spp. versus Nitrotoga spp. This research suggests that nitrification activity can be controlled during the winter via DO to produce better effluent quality with high DO concentrations or to reduce aeration costs with a concomitant decline in nitrification activity.

Novel class 1 integron harboring antibiotic resistance genes in wastewater-derived bacteria as revealed by functional metagenomics.

Combatting antibiotic resistance is critical to our ability to treat infectious diseases. Here, we identified and characterized diverse antimicrobial resistance genes, including potentially mobile elements, from synthetic wastewater treatment microcosms exposed to the antibacterial agent triclosan. After seven weeks of exposure, the microcosms were subjected to functional metagenomic selection across 13 antimicrobials. This was achieved by cloning the combined genetic material from the microcosms, introducing this genetic library into E. coli, and selecting for clones that grew on media supplemented with one of the 13 antimicrobials. We recovered resistant clones capable of growth on media supplemented with a single antimicrobial, yielding 13 clones conferring resistance to at least one antimicrobial agent. Antibiotic susceptibility analysis revealed resistance ranging from 4 to >50 fold more resistant, while one clone showed resistance to multiple antibiotics. Using both Sanger and SMRT sequencing, we identified the predicted active gene(s) on each clone. One clone that conferred resistance to tetracycline contained a gene encoding a novel tetA-type efflux pump that was named TetA(62). Three clones contained predicted active genes on class 1 integrons. One integron had a previously unreported genetic arrangement and was named In1875. This study demonstrated the diversity and potential for spread of resistance genes present in human-impacted environments.

Flushing of Stagnant Premise Water Systems after the COVID-19 Shutdown Can Reduce Infection Risk by Legionella and Mycobacterium spp.

There is concern about potential exposure to opportunistic pathogens when reopening buildings closed due to the COVID-19 pandemic. In this study, water samples were collected before, during, and after flushing showers in five unoccupied (i.e., for ∼2 months) university buildings with quantification of opportunists via a cultivation-based assay (Legionella pneumophila only) and quantitative PCR. L. pneumophila were not detected by either method; Legionella spp., nontuberculous mycobacteria (NTM), and Mycobacterium avium complex (MAC), however, were widespread. Using quantitative microbial risk assessment (QMRA), the estimated risks of illness from exposure to L. pneumophila and MAC via showering were generally low (i.e., less than a 10-7 daily risk threshold), with the exception of systemic infection risk from MAC exposure in some buildings. Flushing rapidly restored the total chlorine (as chloramine) residual and decreased bacterial gene targets to building inlet concentrations within 30 min. During the postflush stagnation period, the residual chlorine dissipated within a few days and bacteria rebounded, approaching preflush concentrations after 6-7 days. These results suggest that flushing can quickly improve water quality in unoccupied buildings, but the improvement may only last a few days.

A comparison of nitrate removal and denitrifying bacteria populations among three wetland plant communities.

Reed canary grass (Phalaris arundinacea L.) is an invasive, cool-season grass commonly dominating wetlands with high nutrient loads. Its impact on nitrogen removal via denitrification in wetlands is unknown. Most studies of denitrification in treatment wetlands have focused on the effects of physical or chemical variables and not on the effects of plant roots on the soil environment. The purpose of this study was to measure effects of plant type on denitrification rates in typical wetland soils of the midwestern United States by comparing wet prairie mix, switchgrass-dominated, and reed canary grass plant communities. Nitrate (NO3 - ) removal and other parameters were measured in miniature wetlands, or mesocosms, containing each plant community transplanted from a small agricultural treatment wetland in southern Minnesota. Quantitative polymerase chain reaction analysis was used to quantify the total bacteria population (measured with 16S rRNA genes) and denitrifying gene abundance (measured with nosZ genes) from the rhizosphere of each plant community. The wet prairie mix mesocosms on average removed the most NO3 - in each test (p = .01 and .08). Whereas the wet prairie mix removed the most NO3 - from the surface water (p < .01), reed canary grass removed more from the subsurface (p < .01). Ratios of denitrifying to total bacteria were higher in the wet prairie mix than in the other communities' root zones (p < .05). Results suggest that reed canary grass invasion could reduce denitrification in wetlands, especially during the spring and fall when it is growing but other plants are dormant.

Microbiome of Drinking Water Biofilters is Influenced by Environmental Factors and Engineering Decisions but has Little Influence on the Microbiome of the Filtrate.

Bacterial communities in biofilters can improve drinking water quality through the biodegradation of dissolved contaminants but also pose potential risks by harboring and shedding microbes into the drinking water distribution system. In this study, pilot-scale granular activated carbon (GAC)-sand and anthracite-sand pilot-scale biofilters were investigated to determine the effects of filter design and operation on the microbiome of the filter media and its relationship to the microbiome in the filter effluent water. Bacterial abundance in the biofilters was relatively stable over time. Bacterial community composition exhibited spatial variation (i.e., with bed depth) and temporal variation linked to water quality changes. Bacterial community composition was significantly affected by the media type (GAC vs anthracite) and backwashing strategy (chloraminated water vs nonchloraminated water). The biofilters reduced bacterial abundance in the water (∼70%) but had only a minor effect on the bacterial community composition in the filtrate. Overall, our results suggest that the bacterial communities growing on biofilters affect filtered water quality primarily through the biotransformation of pollutants and nutrients rather than by altering the microbial community composition of the water as it passes through the filter.

Effects of geographic location and water quality on bacterial communities in full-scale biofilters across North America.

Spatial patterns of bacterial community composition often follow a distance-decay relationship in which community dissimilarity increases with geographic distance. Such a relationship has been commonly observed in natural environments, but less so in engineered environments. In this study, bacterial abundance and community composition in filter media samples (n = 57) from full-scale rapid biofilters at 14 water treatment facilities across North America were determined using quantitative polymerase chain reaction and Illumina HiSeq high-throughput sequencing targeting the 16S rRNA gene, respectively. Bacteria were abundant on the filter media (108.8±0.3 to 1010.7±0.2 16S rRNA gene copies/cm3 bed volume) and the bacterial communities were highly diverse (Shannon index: 5.3 ± 0.1 to 8.4 ± 0.0). Significant inter-filter variations in bacterial community composition were observed, with weighted UniFrac dissimilarity values following a weak but highly significant distance-decay relationship (z = 0.0057 ± 0.0006; P = 1.8 × 10-22). Approximately 50% of the variance in bacterial community composition was explained by the water quality parameters measured at the time of media sample collection (i.e. pH, temperature and dissolved organic carbon concentration). Overall, this study suggested that the microbiomes of biofilters are primarily shaped by geographic location and local water quality conditions but the influence of these factors on the microbiomes is tempered by filter design and operating conditions.

Nontuberculous Mycobacteria in Two Drinking Water Distribution Systems and the Role of Residual Disinfection.

Nontuberculous mycobacteria (NTM) are frequently found in chloraminated drinking water distribution systems (DWDSs) due to their chloramine tolerance. NTM were investigated in the water-main biofilms and drinking water of a chloraminated DWDS in the United States (initial chloramine residual = 3.8 ± 0.1 mg L-1) and a DWDS in Norway with minimal residual disinfectant (0.08 ± 0.01 mg L-1). Total mycobacteria and Mycobacterium avium complex (MAC) were quantified by qPCR targeting, respectively, atpE genes and the internal transcribed spacer region. Mycobacteria concentrations in drinking water did not differ between the two systems (P = 0.09; up to 6 × 104 copies L-1) but were higher in the biofilms from the chloraminated DWDS (P = 5 × 10-9; up to 5 × 106 copies cm-2). MAC were not detected in either system. Sequencing of mycobacterial hsp65 genes indicated that the chloraminated DWDS lacked diversity and consisted almost exclusively of M. gordonae. In contrast, there were various novel mycobacteria in the no-residual DWDS. Finally, Mycobacterium- and Methylobacterium-like 16S rRNA genes were often detected simultaneously, though without correlation as previously observed. We conclude that, though residual chloramine may increase mycobacterial biomass in a DWDS, it may also decrease mycobacterial diversity.

Comparison of the microbiomes of two drinking water distribution systems-with and without residual chloramine disinfection.

BACKGROUND: Residual disinfection is often used to suppress biological growth in drinking water distribution systems (DWDSs), but not without undesirable side effects. In this study, water-main biofilms, drinking water, and bacteria under corrosion tubercles were analyzed from a chloraminated DWDS (USA) and a no-residual DWDS (Norway). Using quantitative real-time PCR, we quantified bacterial 16S rRNA genes and ammonia monooxygenase genes (amoA) of Nitrosomonas oligotropha and ammonia-oxidizing archaea-organisms that may contribute to chloramine loss. PCR-amplified 16S rRNA genes were sequenced to assess community taxa and diversity. RESULTS: The chloraminated DWDS had lower biofilm biomass (P=1×10-6) but higher N. oligotropha-like amoA genes (P=2×10-7) than the no-residual DWDS (medians =4.7×104 and 1.1×103amoA copies cm-2, chloraminated and no residual, respectively); archaeal amoA genes were only detected in the no-residual DWDS (median =2.8×104 copies cm-2). Unlike the no-residual DWDS, biofilms in the chloraminated DWDS had lower within-sample diversity than the corresponding drinking water (P<1×10-4). Chloramine was also associated with biofilms dominated by the genera, Mycobacterium and Nitrosomonas (≤91.7% and ≤39.6% of sequences, respectively). Under-tubercle communities from both systems contained corrosion-associated taxa, especially Desulfovibrio spp. (≤98.4% of sequences). CONCLUSIONS: Although residual chloramine appeared to decrease biofilm biomass and alpha diversity as intended, it selected for environmental mycobacteria and Nitrosomonas oligotropha-taxa that may pose water quality challenges. Drinking water contained common freshwater plankton and did not resemble corresponding biofilm communities in either DWDS; monitoring of tap water alone may therefore miss significant constituents of the DWDS microbiome. Corrosion-associated Desulfovibrio spp. were observed under tubercles in both systems but were particularly dominant in the chloraminated DWDS, possibly due to the addition of sulfate from the coagulant alum.

Effects of Chloramine and Coupon Material on Biofilm Abundance and Community Composition in Bench-Scale Simulated Water Distribution Systems and Comparison with Full-Scale Water Mains.

The vast majority of bacteria in drinking water distribution systems (DWDSs) reside in biofilms on the interior walls of water mains. Little is known about how water quality conditions affect water-main biofilms because of the inherent limitations in experimenting with drinking water supplies and accessing the water mains for sampling. Bench-scale reactors permit experimentation and ease of biofilm sampling, yet questions remain as to how well biofilms in laboratory reactors represent those on water mains. In this study, the effects of DWDS pipe materials and chloramine residual on biofilms were investigated by cultivating biofilms on cement, polyvinyl chloride, and high density polyethylene coupons in CDC reactors for up to 28 months in the presence of chloraminated or dechlorinated tap water. The bench-scale biofilm microbiomes were then compared with the microbiome on a water main from the full-scale system that supplied the water to the reactors. The presence of a chloramine residual (1.74 ± 0.21 mg/L) suppressed biofilm accumulation and selected for Mycobacterium-like and Sphingopyxis-like operational taxonomic units (OTUs) while the destruction of the chloramine residual resulted in a significant increase in biomass quantity and a shift toward a more diverse community dominated by Nitrospira-like OTUs, which, our results suggest, may be complete ammonia oxidizers (comammox). Coupon material, however, had a relatively minor effect on the abundance and community composition of the biofilm bacteria. Although biofilm communities from the chloraminated water reactor and the water mains shared some dominant populations (namely, Mycobacterium- and Nitrosomonas-like OTUs), the communities were significantly different. This manuscript provides novel insights into the effects of dechlorination and pipe material on biofilm community composition. Furthermore, to our knowledge, it is the first study to compare biofilm in a tap water-fed, bench-scale simulated distribution system to biofilm on water mains from the full-scale system supplying the tap water.

Small and large-scale distribution of four classes of antibiotics in sediment: association with metals and antibiotic resistance genes.

Antibiotic chemicals and antibiotic resistance genes enter the environment via wastewater effluents as well as from runoff from agricultural operations. The relative importance of these two sources, however, is largely unknown. The relationship between the concentrations of chemicals and genes requires exploration, for antibiotics in the environment may lead to development or retention of resistance genes by bacteria. The genes that confer resistance to metal toxicity may also be important in antibiotic resistance. In this work, concentrations of 19 antibiotics (using liquid chromatography tandem mass spectrometry), 14 metals (using inductively coupled plasma-mass spectrometry), and 45 metal, antibiotic, and antibiotic-resistance associated genes (using a multiplex, microfluidic quantitative polymerase chain reaction method) were measured in 13 sediment samples from two large rivers as well as along a spatial transect in a wastewater effluent-impacted lake. Nine of the antibiotics were detected in the rivers and 13 were detected in the lake. Sixteen different resistance genes were detected. The surrounding land use and proximity to wastewater treatment plants are important factors in the number and concentrations of antibiotics detected. Correlations among antibiotic chemical concentrations, metal concentrations, and resistance genes occur over short spatial scales in a lake but not over longer distances in major rivers. The observed correlations likely result from the chemicals and resistance genes arising from the same source, and differences in fate and transport over larger scales lead to loss of this relationship.

Occurrence of Legionella spp. in Water-Main Biofilms from Two Drinking Water Distribution Systems.

The maintenance of a chlorine or chloramine residual to suppress waterborne pathogens in drinking water distribution systems is common practice in the United States but less common in Europe. In this study, we investigated the occurrence of Bacteria and Legionella spp. in water-main biofilms and tap water from a chloraminated distribution system in the United States and a system in Norway with no residual using real-time quantitative polymerase chain reaction (qPCR). Despite generally higher temperatures and assimilable organic carbon levels in the chloraminated system, total Bacteria and Legionella spp. were significantly lower in water-main biofilms and tap water of that system ( p < 0.05). Legionella spp. were not detected in the biofilms of the chloraminated system (0 of 35 samples) but were frequently detected in biofilms from the no-residual system (10 of 23 samples; maximum concentration = 7.8 × 104 gene copies cm-2). This investigation suggests water-main biofilms may serve as a source of Legionella for tap water and premise plumbing systems, and residual chloramine may aid in reducing their abundance.

Sedimentary record of antibiotic accumulation in Minnesota Lakes.

The widespread detection of antibiotics in the environment is concerning because antibiotics are designed to be effective at small doses. The objective of this work was to quantify the accumulation rates of antibiotics used by humans and animals, spanning several major antibiotic classes (sulfonamides, tetracyclines, fluoroquinolones, and macrolides), in Minnesota lake-sediment cores. Our goal was to determine temporal trends, the major anthropogenic source to these lacustrine systems, and the importance of natural production. A historical record of usage trends for ten human and/or animal-use antibiotics (four sulfonamides, three fluoroquinolones, one macrolide, trimethoprim, and lincomycin) was faithfully captured in the sediment cores. Nine other antibiotics were not detected. Ofloxacin, trimethoprim, sulfapyridine, and sulfamethazine were detected in all of the anthropogenically-impacted studied lakes. Maximum sediment fluxes reached 20.5ngcm-2yr-1 (concentration 66.1ng/g) for ofloxacin, 1.2ngcm-2yr-1 (1.2ng/g) for trimethoprim, 3.3ngcm-2yr-1 (11.3ng/g) for sulfapyridine, and 1.0ngcm-2yr-1 (1.6ng/g) for sulfamethazine, respectively. Natural production of lincomycin may have occurred in one lake at fluxes ranging from 0.4 to 1.8ngcm-2yr-1 (0.1 to 5.8ng/g). Wastewater effluent appears to be the primary source of antibiotics in the studied lakes, with lesser inputs from agricultural activities.

Effect of Different Treatment Technologies on the Fate of Antibiotic Resistance Genes and Class 1 Integrons when Residual Municipal Wastewater Solids are Applied to Soil.

Residual wastewater solids are a significant reservoir of antibiotic resistance genes (ARGs). While treatment technologies can reduce ARG levels in residual wastewater solids, the effects of these technologies on ARGs in soil during subsequent land-application are unknown. In this study we investigated the use of numerous treatment technologies (air drying, aerobic digestion, mesophilic anaerobic digestion, thermophilic anaerobic digestion, pasteurization, and alkaline stabilization) on the fate of ARGs and class 1 integrons in wastewater solids-amended soil microcosms. Six ARGs [erm(B), qnrA, sul1, tet(A), tet(W), and tet(X)], the integrase gene of class 1 integrons (intI1), and 16S rRNA genes were quantified using quantitative polymerase chain reaction. The quantities of ARGs and intI1 decreased in all microcosms, but thermophilic anaerobic digestion, alkaline stabilization, and pasteurization led to the most extensive decay of ARGs and intI1, often to levels similar to that of the control microcosms to which no wastewater solids had been applied. In contrast, the rates by which ARGs and intI1 declined using the other treatment technologies were generally similar, typically varying by less than 2 fold. These results demonstrate that wastewater solids treatment technologies can be used to decrease the persistence of ARGs and intI1 during their subsequent application to soil.