Evaluation of a novel porous antimicrobial media for industrial and HVAC water biocontrol.

A novel treatment method, consisting of pea-gravel with a marine coating supplemented with alkyldimethylbenzylammonium chloride (ADBAC or benzalkonium chloride), has been examined for its antimicrobial performance and coating stability in aqueous environments. Initial column studies examining the porous media's ability to reduce bacterial loads in heating, ventilation, and air conditioning (HVAC) water found average reductions of 94% from pre-flush levels (106 colony forming unit (CFU)/mL) when assessed with R2A spread plates and 83% reductions with SimPlates. There was no observed statistical difference between the average of pre- and post-flush waters from four tests of the media without ADBAC. Taxonomic identification, by 16S rRNA gene sequencing, of colonies drawn from pre- and post-ABDAC R2A plates showed similarities with taxa observed in high frequency from prior cultivation-independent surveys of other cooling tower systems. With this proof of concept, two versions of the media were evaluated for potential coating components released during aqueous exposure. Neither released measurable volatile organic compounds (VOC) components, but one did release bisphenol A and ABDAC compounds. Subsequent column tests of the more durable coating were conducted using cultures of interest in industrial water and demonstrated significant reductions in neutralized post-column Enterococcus faecalis samples and near complete loss of Legionella pneumophila in non-neutralized fluids, but lower reductions in Pseudomonas aeruginosa.

Antibiotic-resistant bacteria in the Hudson River Estuary linked to wet weather sewage contamination.

Heterotrophic bacteria resistant to tetracycline and ampicillin were assessed in waterways of the New York City metropolitan area using culture-dependent approaches and 16S rRNA gene sequence analysis of resultant isolates. Resistant microbes were detected at all 10 sampling sites in monthly research cruises on the lower Hudson River Estuary (HRE), with highest concentrations detected at nearshore sites. Higher frequency sampling was conducted in Flushing Bay, to enumerate resistant microbes under both dry and wet weather conditions. Concentrations of ampicillin- and tetracycline-resistant bacteria, in paired samples, were positively correlated with one another and increased following precipitation. Counts of the fecal indicator, Enterococcus, were positively correlated with levels of resistant bacteria, suggesting a shared sewage-associated source. Analysis of 16S rRNA from isolates identified a phylogenetically diverse group of resistant bacteria, including genera containing opportunistic pathogens. The occurrence of Enterobacteriaceae, a family of enteric bacteria, was found to be significantly higher in resistant isolates compared to total heterotrophic bacteria and increased following precipitation. This study is the first to document the widespread distribution of antibiotic-resistant bacteria in the HRE and to demonstrate clearly a link between the abundance of antibiotic-resistant bacteria and levels of sewage-associated bacteria in an estuary.

Local environmental pollution strongly influences culturable bacterial aerosols at an urban aquatic superfund site.

In polluted environments, when microbial aerosols originate locally, species composition of the aerosols should reflect the polluted source. To test the connection between local environmental pollution and microbial aerosols near an urban waterfront, we characterized bacterial aerosols at Newtown Creek (NTC), a public waterway and Superfund site in a densely populated area of New York, NY, USA. Culturable bacterial aerosol fallout rate and surface water bacterial concentrations were at least an order of magnitude greater at NTC than at a neighboring, less polluted waterfront and a nonurban coastal site in Maine. The NTC culturable bacterial aerosol community was significantly different in taxonomic structure from previous urban and coastal aerosol studies, particularly in relative abundances of Actinobacteria and Proteobacteria. Twenty-four percent of the operational taxonomic units in the NTC overall (air + water) bacterial isolate library were most similar to bacterial 16S rRNA gene sequences previously described in terrestrial or aquatic environments contaminated with sewage, hydrocarbons, heavy metals, and other industrial waste. This study is the first to examine the community composition and local deposition of bacterial aerosols from an aquatic Superfund site. The findings have important implications for the use of aeration remediation in polluted aquatic environments and suggest a novel pathway of microbial exposure in densely populated urban communities containing contaminated soil and water.

Environmental controls on coastal coarse aerosols: implications for microbial content and deposition in the near-shore environment.

Coarse aerosols (particle diameter (D(p)) > 2 µm) produced in coastal surf zones carry chemical and microbial content to shore, forming a connection between oceanic, atmospheric, and terrestrial systems that is potentially relevant to coastal ecology and human health. In this context, the effects of tidal height, wind speed, and fog on coastal coarse aerosols and microbial content were quantified on the southern coast of Maine, USA. Aerosols at this site displayed clear marine influence and had high concentrations of ecologically relevant nutrients. Coarse aerosol concentrations significantly increased with tidal height (i.e., decreasing distance from waterline), onshore wind speed, and fog presence. As onshore wind speeds rose above 3 m s(-1), the mean half-deposition distance of coarse aerosols increased to an observed maximum of 47.6 ± 10.9 m from the water's edge at wind speeds from 5.5-8 m s(-1). Tidal height and fog presence did not significantly influence total microbial aerosol concentrations but did have a significant effect on culturable microbial aerosol fallout. At low wind speeds, culturable microbial aerosols falling out near-shore decreased by half at a distance of only 1.7 ± 0.4 m from the water's edge, indicating that these microbes may be associated with large coarse aerosols with rapid settling rates.

Bacterial quality of groundwater downgradient of onsite wastewater disposal systems and the influence on eastern Long Island's embayments.

Onsite wastewater disposal systems (OWDS) can introduce bacterial and chemical contaminants, via groundwater, into aquifers and adjacent waterways. We evaluated the concentration of fecal indicator bacteria (FIB) and antibiotic resistant bacteria (ARB) in the shallow groundwater of Eastern Long Island, New York, downgradient of OWDS using cultivation approaches and analysis of 16 S rRNA genes. While FIB and ARB were detected in 80% and 67% of groundwater samples, respectively, concentrations were low, suggesting that, at least at the time of sampling, groundwater was not a large-scale source of fecal bacterial contamination to adjacent embayments. ARB isolates did not include common fecal associated genera and the concentration of FIB and ARB did not correlate well with the concentration of pharmaceutical contaminants, suggesting that bacterial contaminants were poorly linked to OWDS discharge. Concentrations of FIB in the studied embayments were significantly greater in nearshore compared to mid-channel environments, suggesting that land-based sources are likely to be the major contributors of bacterial contamination.

Patterns of sediment-associated fecal indicator bacteria in an urban estuary: Benthic-pelagic coupling and implications for shoreline water quality.

Estuarine and coastal waterways are commonly monitored for fecal and sewage contamination to protect recreator health and ecosystem functions. Such monitoring programs commonly rely on cultivation-based counts of fecal indicator bacteria (FIB) in water column samples. Recent studies demonstrate that sediments and beach sands can be heavily colonized by FIB, and that settling and resuspension of colonized particles may significantly influence the distribution of FIB in the water column. However, measurements of sediment FIB are rarely incorporated into monitoring programs, and geographic surveys of sediment FIB are uncommon. In this study, the distribution of FIB and the extent of benthic-pelagic FIB coupling were examined in the urbanized, lower Hudson River Estuary. Using cultivation-based enumeration, two commonly-measured FIB, enterococci and Escherichia coli, were widely distributed in both sediment and water, and were positively correlated with each other. The taxonomic identity of FIB isolates from water and sediment was confirmed by DNA sequencing. The geometric mean of FIB concentration in sediment was correlated with both the geometric mean of FIB in water samples from the same locations and with sediment organic carbon. These two positive associations likely reflect water as the FIB source for underlying sediments, and longer FIB persistence in the sediments compared to the water, respectively. The relative representation of other fecal associated bacterial genera in sediment, determined by 16S rRNA gene sequencing, increased with the sequence representation of the two FIB, supporting the value of these FIB for assessing sediment contamination. Experimental resuspension of sediment increased shoreline water column FIB concentrations, which may explain why shoreline water samples had higher average FIB concentrations than samples collected nearby but further from shore. In combination, these results demonstrate extensive benthic-pelagic coupling of FIB in an urbanized estuary and highlight the importance of sediment FIB distribution and ecology when interpreting water quality monitoring data.

Comparison of Bacterial Diversity in Air and Water of a Major Urban Center.

The interaction of wind with aquatic and terrestrial surfaces is known to control the creation of microbial aerosols allowing for their entrainment into air masses that can be transported regionally and globally. Near surface interactions between urban waterways and urban air are understudied but some level of interaction among these bacterial communities would be expected and may be relevant to understanding both urban air and water quality. To address this gap related to patterns of local air-water microbial exchange, we utilized next-generation sequencing of 16S rRNA genes from paired air and water samples collected from 3 urban waterfront sites and evaluated their relative bacterial diversity. Aerosol samples at all sites were significantly more diverse than water samples. Only 17-22% of each site's bacterial aerosol OTUs were present at every site. These shared aerosol OTUs included taxa associated with terrestrial systems (e.g., Bacillus), aquatic systems (e.g., Planktomarina) and sewage (e.g., Enterococcus). In fact, sewage-associated genera were detected in both aerosol and water samples, (e.g., Bifidobacterium, Blautia, and Faecalibacterium), demonstrating the widespread influence of similar pollution sources across these urban environments. However, the majority (50-61%) of the aerosol OTUs at each site were unique to that site, suggesting that local sources are an important influence on bioaerosols. According to indicator species analysis, each site's aerosols harbored the highest percentage of bacterial OTUs statistically determined to uniquely represent that site's aquatic bacterial community, further demonstrating a local connection between water quality and air quality in the urban environment.

Spatial patterns of pharmaceuticals and wastewater tracers in the Hudson River Estuary.

The widespread use of pharmaceuticals by human populations results in their sustained discharge to surface waters via wastewater treatment plants (WWTPs). In this study, 16 highly prescribed pharmaceuticals were quantified along a 250 km transect of the Hudson River Estuary and New York Harbor to describe their sources and spatial patterns. Sampling was conducted over two dry weather periods in May and July 2016, at 72 sites which included mid-channel and nearshore sites, as well as locations influenced by tributaries and WWTP outfalls. The detection frequency of the study pharmaceuticals was almost identical between the May and July sampling periods at 55% and 52%, respectively. Six pharmaceuticals were measurable at 92% or more of the sites during both sampling periods, illustrating their ubiquitous presence throughout the study area. Individual pharmaceutical concentrations were highly variable spatially, ranging from non-detect to 3810 ng/L during the study. Major factors controlling concentrations were proximity and magnitude of WWTP discharges, inputs from tributaries and tidal mixing. Two compounds, sucralose and caffeine, were evaluated as tracers to identify wastewater sources and assess pharmaceutical behavior. Sucralose was useful in identifying wastewater inputs to the river and concentrations showed excellent correlations with numerous pharmaceuticals in the study. Caffeine-sucralose ratios showed potential in identifying discharges of untreated wastewater occurring during a combined sewage overflow event. Many of the study pharmaceuticals were present throughout the Hudson River Estuary as a consequence of sustained wastewater discharge. Whereas some concentrations were above published effects levels, a more complete risk assessment is needed to understand the potential for ecological impacts due to pharmaceuticals in the Hudson River Estuary.

Culturable bioaerosols along an urban waterfront are primarily associated with coarse particles.

The source, characteristics and transport of viable microbial aerosols in urban centers are topics of significant environmental and public health concern. Recent studies have identified adjacent waterways, and especially polluted waterways, as an important source of microbial aerosols to urban air. The size of these aerosols influences how far they travel, their resistance to environmental stress, and their inhalation potential. In this study, we utilize a cascade impactor and aerosol particle monitor to characterize the size distribution of particles and culturable bacterial and fungal aerosols along the waterfront of a New York City embayment. We seek to address the potential contribution of bacterial aerosols from local sources and to determine how their number, size distribution, and taxonomic identity are affected by wind speed and wind direction (onshore vs. offshore). Total culturable microbial counts were higher under offshore winds (average of 778 CFU/m3 ± 67), with bacteria comprising the majority of colonies (58.5%), as compared to onshore winds (580 CFU/m3 ± 110) where fungi were dominant (87.7%). The majority of cultured bacteria and fungi sampled during both offshore winds (88%) and onshore winds (72%) were associated with coarse aerosols (>2.1 µm), indicative of production from local sources. There was a significant correlation (p < 0.05) of wind speed with both total and coarse culturable microbial aerosol concentrations. Taxonomic analysis, based on DNA sequencing, showed that Actinobacteria was the dominant phylum among aerosol isolates. In particular, Streptomyces and Bacillus, both spore forming genera that are often soil-associated, were abundant under both offshore and onshore wind conditions. Comparisons of bacterial communities present in the bioaerosol sequence libraries revealed that particle size played an important role in microbial aerosol taxonomy. Onshore and offshore coarse libraries were found to be most similar. This study demonstrates that the majority of culturable bacterial aerosols along a New York City waterfront were associated with coarse aerosol particles, highlighting the importance of local sources, and that the taxonomy of culturable aerosol bacteria differed by size fraction and wind direction.

Community level analysis: genetic and biogeochemical approaches to investigate community composition and function in aerobic ammonia oxidation.

Aerobic ammonia oxidation is the process that converts ammonium to nitrate and thus links the regeneration of organic nitrogen to fixed nitrogen loss by denitrification. It is performed by a phylogenetically restricted group of Proteobacteria (ammonia-oxidizing bacteria, AOB) that are autotrophic and obligately aerobic. This chapter describes methods for the measurement of ammonia oxidation in the environment, with a focus on seawater systems and stable isotopic tracer methods. It also summarizes the current state of molecular ecological approaches for detection of AOB in the environment and characterization of the composition of AOB assemblages.

Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics.

The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station's history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

Aeration remediation of a polluted waterway increases near-surface coarse and culturable microbial aerosols.

Aeration remediation is currently used in polluted urban waterways to increase oxygen levels in the water column. Recent studies have provided increasing evidence that the bursting of bubbles at water surfaces introduced by aeration, or other surface disturbances, can transfer viable bacteria to the air. In heavily sewage-polluted waterways these water-originated bacterial aerosols may pose as a health risk to recreators in small boats or residents inhabiting the shoreline. Nonetheless, few studies have explored aerosols above active aeration remediation projects in waterways or investigated how bacterial aerosols change with vertical distance from aeration activities. This study, conducted at the Newtown Creek superfund site in Brooklyn, NY, USA, measured coarse aerosol particles and culturable bacteria in near-surface air above waters undergoing aeration remediation. Regardless of aeration operation culturable bacterial fallout was greater near-surface (0.6m above water) than previously-reported measurements made at 2.5m. Molecular analysis of the 16S rRNA gene sequences from isolated bacteria demonstrates that water and air shared a large number of bacterial genera and that the genera present in the near-surface aerosols (0.6m) contained water-associated Vibrio and Caulobacter, which were not present at 2.5m, despite the smaller sequence library size from the near-surface. Also, the near-surface microbial assemblage had significantly greater association with sequences detected previously in aquatic environments compared to the 2.5m library. We found compelling evidence that aeration activity contributed to this vertical gradient in bacterial aerosol concentrations and identity. Similar to results from 2.5m, concentrations of near-surface respirable coarse aerosols (<10 um) increased significantly when aeration was occurring. Culturable bacterial aerosol fallout was also greater near-surface when the aerator was on compared to simultaneous measurements made at 2.5m. Furthermore, when the aerator was operating, the near-surface bacterial aerosol assemblage was statistically more similar to water assemblages than when the aerator was off. These findings highlight the potential for aeration remediation to increase exposure to viable bacterial aerosols in recreators (e.g. kayakers), a problem of greater concern where surface water is heavily polluted with sewage, as in Newtown Creek.

Transitions in nirS-type denitrifier diversity, community composition, and biogeochemical activity along the Chesapeake Bay estuary.

Chesapeake Bay, the largest estuary in North America, can be characterized as having steep and opposing gradients in salinity and dissolved inorganic nitrogen along the main axis of the Bay. In this study, the diversity of nirS gene fragments (encoding cytochrome cd 1-type nitrite reductase), physical/chemical parameters, and benthic N2-fluxes were analyzed in order to determine how denitrifier communities and biogeochemical activity vary along the estuary salinity gradient. The nirS gene fragments were PCR-amplified, cloned, and sequenced from sediment cores collected at five stations. Sequence analysis of 96-123 nirS clones from each station revealed extensive overall diversity in this estuary, as well as distinct spatial structure in the nirS sequence distributions. Both nirS-based richness and community composition varied among stations, with the most dramatic shifts occurring between low-salinity (oligohaline) and moderate-salinity (mesohaline) sites. For four samples collected in April, the nirS-based richness, nitrate concentrations, and N2-fluxes all decreased in parallel along the salinity gradient from the oligohaline northernmost station to the highest salinity (polyhaline) station near the mouth of the Bay. The vast majority of the 550 nirS sequences were distinct from cultivated denitrifiers, although many were closely related to environmental clones from other coastal and estuarine systems. Interestingly, 8 of the 172 OTUs identified accounted for 42% of the total nirS clones, implying the presence of a few dominant and many rare genotypes, which were distributed in a non-random manner along the salinity gradient of Chesapeake Bay. These data, comprising the largest dataset to investigate nirS clone sequence diversity from an estuarine environment, also provided information that was required for the development of nirS microarrays to investigate the interaction of microbial diversity, environmental gradients, and biogeochemical activity.

Worldwide distribution of Nitrosococcus oceani, a marine ammonia-oxidizing gamma-proteobacterium, detected by PCR and sequencing of 16S rRNA and amoA genes.

Diversity of cultured ammonia-oxidizing bacteria in the gamma-subdivision of the Proteobacteria was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first Nitrosococcus oceani-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of NITROSOCOCCUS: These findings imply that organisms similar to the cultivated N. oceani strains have a worldwide distribution.

Environmental acquisition of thiotrophic endosymbionts by deep-sea mussels of the genus bathymodiolus.

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally "leaky" system of vertical transmission could not be excluded.