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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Modeling the fate of antibiotic resistance genes and class 1 integrons during thermophilic anaerobic digestion of municipal wastewater solids.

This study investigated the use of thermophilic anaerobic digestion for removing antibiotic resistance genes (ARGs) from residual municipal wastewater solids. Four laboratory-scale anaerobic digesters were operated in 8-day batch cycles at temperatures of 40, 56, 60, and 63 °C. Two tetracycline resistance genes (tet(W) and tet(X)), a fluoroquinolone resistance gene (qnrA), the integrase gene of class 1 integrons (intI1), 16S rRNA genes of all Bacteria, and 16S rRNA genes of methanogens were quantified using real-time quantitative PCR. ARG and intI1 quantities decreased at all temperatures and were described well by a modified form of the Collins-Selleck disinfection kinetic model. The magnitudes of Collins-Selleck kinetic parameters were significantly greater at thermophilic temperatures compared to 40 °C, but few statistically significant differences were observed among these parameters for the thermophilic anaerobic digesters. This model allows for the direct comparison of different operating conditions (e.g., temperature) on anaerobic digestion performance in mitigating the quantity of ARGs in wastewater solids and could be used to design full-scale anaerobic digesters to specifically treat for ARGs as a "pollutant" of concern.

The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms.

The bacterial community composition of the full-scale biologically active, granular activated carbon (BAC) filters operated at the St. Paul Regional Water Services (SPRWS) was investigated using Illumina MiSeq analysis of PCR-amplified 16S rRNA gene fragments. These bacterial communities were consistently diverse (Shannon index, >4.4; richness estimates, >1,500 unique operational taxonomic units [OTUs]) throughout the duration of the 12-month study period. In addition, only modest shifts in the quantities of individual bacterial populations were observed; of the 15 most prominent OTUs, the most highly variable population (a Variovorax sp.) modulated less than 13-fold over time and less than 8-fold from filter to filter. The most prominent population in the profiles was a Nitrospira sp., representing 13 to 21% of the community. Interestingly, very few of the known ammonia-oxidizing bacteria (AOB; <0.07%) and no ammonia-oxidizing Archaea were detected in the profiles. Quantitative PCR of amoA genes, however, suggested that AOB were prominent in the bacterial communities (amoA/16S rRNA gene ratio, 1 to 10%). We conclude, therefore, that the BAC filters at the SPRWS potentially contained significant numbers of unidentified and novel ammonia-oxidizing microorganisms that possess amoA genes similar to those of previously described AOB.

Sulfate Reducing Bacteria and Mycobacteria Dominate the Biofilm Communities in a Chloraminated Drinking Water Distribution System.

The quantity and composition of bacterial biofilms growing on 10 water mains from a full-scale chloraminated water distribution system were analyzed using real-time PCR targeting the 16S rRNA gene and next-generation, high-throughput Illumina sequencing. Water mains with corrosion tubercles supported the greatest amount of bacterial biomass (n = 25; geometric mean = 2.5 × 10(7) copies cm(-2)), which was significantly higher (P = 0.04) than cement-lined cast-iron mains (n = 6; geometric mean = 2.0 × 10(6) copies cm(-2)). Despite spatial variation of community composition and bacterial abundance in water main biofilms, the communities on the interior main surfaces were surprisingly similar, containing a core group of operational taxonomic units (OTUs) assigned to only 17 different genera. Bacteria from the genus Mycobacterium dominated all communities at the main wall-bulk water interface (25-78% of the community), regardless of main age, estimated water age, main material, and the presence of corrosion products. Further sequencing of the mycobacterial heat shock protein gene (hsp65) provided species-level taxonomic resolution of mycobacteria. The two dominant Mycobacteria present, M. frederiksbergense (arithmetic mean = 85.7% of hsp65 sequences) and M. aurum (arithmetic mean = 6.5% of hsp65 sequences), are generally considered to be nonpathogenic. Two opportunistic pathogens, however, were detected at low numbers: M. hemophilum (arithmetic mean = 1.5% of hsp65 sequences) and M. abscessus (arithmetic mean = 0.006% of hsp65 sequences). Sulfate-reducing bacteria from the genus Desulfovibrio, which have been implicated in microbially influenced corrosion, dominated all communities located underneath corrosion tubercules (arithmetic mean = 67.5% of the community). This research provides novel insights into the quantity and composition of biofilms in full-scale drinking water distribution systems, which is critical for assessing the risks to public health and to the water supply infrastructure.

Multiple Discharges of Treated Municipal Wastewater Have a Small Effect on the Quantities of Numerous Antibiotic Resistance Determinants in the Upper Mississippi River.

This study evaluated multiple discharges of treated wastewater on the quantities of antibiotic resistance genes (ARGs) in the Upper Mississippi River. Surface water and treated wastewater samples were collected along the Mississippi River during three different periods of 4 days during the summer of 2012, and quantitative real-time PCR (qPCR) was used to enumerate several ARGs and related targets. Even though the wastewater effluents contained 75- to 831-fold higher levels of ARGs than the river water, the quantities of ARGs in the Mississippi River did not increase with downstream distance. Plasmids from the incompatibility group A/C were detected at low levels in the wastewater effluents but not in the river water; synthetic DNA containing an ampicillin resistance gene (bla) from cloning vectors was not detected in either the wastewater effluent or river samples. A simple 1D model suggested that the primary reason for the small impact of the wastewater discharges on ARG levels was the large flow rate of the Mississippi River compared to that of the wastewater discharges. Furthermore, this model generally overpredicted the ARG levels in the Mississippi River, suggesting that substantial loss mechanisms (e.g., decay or deposition) were occurring in the river.

The impacts of triclosan on anaerobic community structures, function, and antimicrobial resistance.

Triclosan is a widespread antimicrobial agent that accumulates in anaerobic digesters used to treat the residual solids generated at municipal wastewater treatment plants; there is very little information, however, about how triclosan impacts microbial communities in anaerobic digesters. We investigated how triclosan impacts the community structure, function and antimicrobial resistance genes in lab-scale anaerobic digesters. Previously exposed (to triclosan) communities were amended with 5, 50, and 500 mg/kg of triclosan, corresponding to the median, 95th percentile, and 4-fold higher than maximum triclosan concentration that has been detected in U.S. biosolids. Triclosan amendment caused all of the Bacteria and Archaea communities to structurally diverge from that of the control cultures (based on ARISA). At the end of the experiment, all triclosan-amended Archaea communities had diverged from the control communities, regardless of the triclosan concentration added. In contrast, over time the Bacteria communities that were amended with lower concentrations of triclosan (5 mg/kg and 50 mg/kg) initially diverged and then reconverged with the control community structure. Methane production at 500 mg/kg was nearly half the methane production in control cultures. At 50 mg/kg, a large variability in methane production was observed, suggesting that 50 mg/kg may be a tipping point where function begins to fail in some communities. When previously unexposed communities were exposed to 500 mg triclosan/kg, function was maintained, but the abundance of a gene encoding for triclosan resistance (mexB) increased. This research suggests that triclosan could inhibit methane production in anaerobic digesters if concentrations were to increase and may also select for resistant Bacteria. In both cases, microbial community composition and exposure history alter the influence of triclosan.

Fate of antibiotic resistance genes and class 1 integrons in soil microcosms following the application of treated residual municipal wastewater solids.

Substantial quantities of antibiotic resistance genes (ARGs) are discharged with treated residual municipal wastewater solids and subsequently applied to soil. The objective of this work was to determine the decay rates for ARGs and class 1 integrons following simulated land application of treated wastewater solids. Treated residual solids from two full-scale treatment plants were applied to sets of triplicate soil microcosms in two independent experiments. Experiment 1 investigated loading rates of 20, 40, and 100 g kg(-1) of residual solids to a sandy soil, while experiment 2 investigated a loading rate of 40 g kg(-1) to a silty-loamy soil. Five ARGs (erm(B), sul1, tet(A), tet(W), and tet(X)), the integrase of class 1 integrons (intI1), 16S rRNA genes, 16S rRNA genes of all Bacteroides spp., and 16S rRNA genes of human-specific Bacteroides spp. were quantified using real-time polymerase chain reaction. ARGs and intI1 quantities declined in most microcosms, with statistically significant (P < 0.05) half-lives varying between 13 d (erm(B), experiment 1, 100 g kg(-1)) and 81 d (intI1, experiment 1, 40 g kg(-1)). These kinetic rates were much slower than have been previously reported for unit operations used to treat wastewater solids (e.g., anaerobic digestion). This research suggests that the design and operation of municipal wastewater treatment facilities with the explicit goal of mitigating the release of ARGs should focus on using technologies within the treatment facility, rather than depending on attenuation subsequent to land application.

Residual disinfectant effectively suppresses Legionella species in drinking water distribution systems supplied by surface water in Minnesota, USA.

Seven public water systems in Minnesota, USA were analyzed from one to five times over a two-year period to assess temporal changes in the concentrations of total bacteria, Legionella spp., and Legionella pneumophila from source (i.e., raw water) through the water treatment process to the end water user. Bacterial biomass was collected by filtering large volumes of raw water (12 to 425 L, median: 38 L) or finished and tap water (27 to 1205 L, median: 448 L) using ultrafiltration membrane modules. Quantitative PCR (qPCR) was then used to enumerate all bacteria (16S rRNA gene fragments), all Legionella spp. (ssrA), and Legionella pneumophila (mip). Total coliforms, Escherichia coli, and L. pneumophila also were quantified in the water samples via cultivation. Median concentrations of total bacteria and Legionella spp. (ssrA) in raw water (8.5 and 4.3 log copies/L, respectively) decreased by about 2 log units during water treatment. The concentration of Legionella spp. (ssrA) in water collected from distribution systems inversely correlated with the total chlorine concentration for chloraminated systems significantly (p = 0.03). Although only 8 samples were collected from drinking water distribution systems using free chlorine as a residual disinfectant, these samples had significantly lower concentrations of Legionella spp. (ssrA) than samples collected from the chloraminated systems (p = 5 × 10-4). There was considerable incongruity between the results obtained via cultivation-independent (qPCR) and cultivation-dependent assays. Numerous samples were positive for L. pneumophila via cultivation, none of which tested positive for L. pneumophilia (mip) via qPCR. Conversely, a single sample tested positive for L. pneumophilia (mip) via qPCR, but this sample tested negative for L. pneumophilia via cultivation. Overall, the results suggest that conventional treatment is effective at reducing, but not eliminating, Legionella spp. from surface water supplies and that residual disinfection is effective at suppressing these organisms within drinking water distribution systems.

Air-drying beds reduce the quantities of antibiotic resistance genes and class 1 integrons in residual municipal wastewater solids.

This study investigated whether air-drying beds reduce antibiotic resistance gene (ARG) concentrations in residual municipal wastewater solids. Three laboratory-scale drying beds were operated for a period of nearly 100 days. Real-time PCR was used to quantify 16S rRNA genes, 16S rRNA genes specific to fecal bacteria (AllBac) and human fecal bacteria (HF183), the integrase gene of class 1 integrons (intI1), and five ARGs representing a cross-section of antibiotic classes and resistance mechanisms (erm(B), sul1, tet(A), tet(W), and tet(X)). Air-drying beds were capable of reducing all gene target concentrations by 1 to 5 orders of magnitude, and the nature of this reduction was consistent with both a net decrease in the number of bacterial cells and a lack of selection within the microbial community. Half-lives varied between 1.5 d (HF183) and 5.4 d (tet(X)) during the first 20 d of treatment. After the first 20 d of treatment, however, half-lives varied between 8.6 d (tet(X)) and 19.3 d (AllBac), and 16S rRNA gene, intI1, and sul1 concentrations did not change (P > 0.05). These results demonstrate that air-drying beds can reduce ARG and intI1 concentrations in residual municipal wastewater solids within timeframes typical of operating practices.