Bioenergetics of simultaneous oxygen and nitrate respiration and nitric oxide production in a Pseudomonas aeruginosa agar colony biofilm.

Pseudomonas aeruginosa is a biofilm forming pathogen commonly associated with infection of the cystic fibrosis (CF) lung, chronic wounds and indwelling medical devices. P. aeruginosa is a facultative aerobe that can use nitrate (NO3-) found in healthy and infected tissues and body fluids to generate energy through denitrification. Further, P. aeruginosa the expression of denitrification genes has been found in specimens from people with CF. The main aim of this study was to determine the relative energy contribution of oxygen (O2) respiration and denitrification in single Pseudomonas aeruginosa PAO1 biofilm colonies under different O2 concentrations to estimate the possible relative importance of these metabolic processes in the context of biofilm infections. We showed that the used strain PAO1 in biofilms denitrified with nitrous oxide (N2O), and not nitrogen (N2), as the end product in our incubations. From simultaneous O2 and N2O microprofiles measured with high spatial resolution by microsensors in agar colony biofilms under air, N2 and pure O2, the rates of aerobic respiration and denitrification were calculated and converted to ATP production rates. Denitrification occurred both in the oxic and anoxic zones, and became increasingly dominant with decreasing O2 concentrations. At O2 concentrations characteristic for tissues and wounds (20-60 µM), denitrification was responsible for 50% of the total energy conservation in the biofilm. In addition the formation of nitric oxide (NO), a precursor of N2O and an important regulator of many cellular processes, was strongly influenced by the local O2 concentrations. NO production was inhibited under pure O2, present under anoxia (∼1 µM) and remarkably high (up to 6 µM) under intermediate O2 levels, which can be found in infected tissues. Possible impacts of such NO levels on both the host and the biofilm bacteria are discussed.

Nitrate respiration occurs throughout the depth of mucoid and non-mucoid Pseudomonas aeruginosa submerged agar colony biofilms including the oxic zone.

Pseudomonas aeruginosa is an opportunistic pathogen and well characterized biofilm former. P. aeruginosa forms strong oxygen gradients inside biofilms due to rapid oxygen respiration in the top layers and the poor solubility of oxygen coupled with diffusion limited transport. Transcriptomic evidence from in vitro and ex vivo sampling suggests that denitrification is occurring in biofilms in ostensibly oxic environments. It is hypothesized that in the presence of nitrate there is stratification with aerobic respiration occurring in the outer oxic layer and denitrification in the lower anoxic zone. We used submerged agar colony biofilms grown from mucoid (FRD1) and non-mucoid (PAO1) strains to simultaneously measure depth microprofiles of oxygen and nitrous oxide in the same colony with microelectrodes. Oxygen respiration occurred at the top of the colony as expected but denitrification occurred throughout the entire depth, even in the oxic region. Local denitrification rates were highly variable suggesting heterogenous metabolic activity within the colony. We also assessed the short-term influence of tobramycin on aerobic respiration within a PAO1 colony. Although there was an immediate reduction in respiration it was never completely arrested over a 2 h period. On tobramycin removal the oxygen gradient steadily reestablished, demonstrating immediate recovery of metabolic activity.

Calculating expected effects of treatment effectivity and river flow rates on the contribution of WWTP effluent to the ARG load of a receiving river.

Concentrations of genetic markers for antibiotic resistance genes (ARGs) were measured in the effluents of three Norwegian wastewater treatment plants (WWTPs) and in a receiving river upstream and downstream of the discharge point of one WWTP. Calculations based on mass balances were carried out to evaluate the impact of river flow rates and treatment effectivity on the WWTP's contribution to the load of genetic markers in the river. At average river flow rates, the WWTP effluent contributes 5-15% to the genetic marker load of the respective river. However, at minimum river flow rates, the WWTP effluent contributes 22-55% to the loads of different genetic markers. Scenarios of an improved or worsened removal of genetic markers in the WWTP showed that a further 1-log removal using additional treatment would be sufficient to improve considerably the river water quality with respect to genetic markers. Then, at an average flow rate, the contribution of the WWTP effluent to the load of the river would be less than 2%. However, in the case of low treatment effectivity or malfunction of the WWTP, the marker load of the river would increase dramatically. Even at average flow rate, 75-92% of the marker load would then originate from the WWTP. The results demonstrate the importance of considering the flow rates and hydrologic characteristics of the recipient water body when deciding on priorities regarding the upgrade of WWTPs for further removal of ARGs.

A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum ß-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Antibiotic resistance genes in treated wastewater and in the receiving water bodies: A pan-European survey of urban settings.

There is increasing public concern regarding the fate of antibiotic resistance genes (ARGs) during wastewater treatment, their persistence during the treatment process and their potential impacts on the receiving water bodies. In this study, we used quantitative PCR (qPCR) to determine the abundance of nine ARGs and a class 1 integron associated integrase gene in 16 wastewater treatment plant (WWTP) effluents from ten different European countries. In order to assess the impact on the receiving water bodies, gene abundances in the latter were also analysed. Six out of the nine ARGs analysed were detected in all effluent and river water samples. Among the quantified genes, intI1 and sul1 were the most abundant. Our results demonstrate that European WWTP contribute to the enrichment of the resistome in the receiving water bodies with the particular impact being dependent on the effluent load and local hydrological conditions. The ARGs concentrations in WWTP effluents were found to be inversely correlated to the number of implemented biological treatment steps, indicating a possible option for WWTP management. Furthermore, this study has identified blaOXA-58 as a possible resistance gene for future studies investigating the impact of WWTPs on their receiving water.

Removal of antibiotic resistant E. coli in two Norwegian wastewater treatment plants and by nano- and ultra-filtration processes.

The effectivity of different treatment stages at two large wastewater treatment plants (WWTPs) located in Oslo, Norway, to remove antibiotic resistant Escherichia coli from municipal wastewater was investigated. The WWTPs were effective in reducing the total cultivable E. coli. The E. coli in WWTP samples were mainly resistant to ampicillin (6-27%) and trimethoprim-sulfamethoxazole (5-24%), and, to a lesser extent, tetracycline (3-14%) and ciprofloxacin (0-7%). In the first WWTP, a clear decrease in the percentage of E. coli resistant to these antibiotics was found, with the main removal occurring during physical/chemical treatment. In the second WWTP, the percentage of cultivable resistant E. coli did not display a considerable change. During laboratory-scale membrane filtration of WWTP effluents using ultrafiltration (UF) and nanofiltration (NF) membranes, all E. coli, including those resistant to antibiotics, were removed completely. The results imply that UF and NF processes are potent measures to remove antibiotic resistant bacteria (ARB) during post-treatment of WWTP effluents, thus reducing the potential spread of antibiotic resistance in the receiving aquatic environment.

High Throughput Analysis of Integron Gene Cassettes in Wastewater Environments.

Integrons are extensively targeted as a proxy for anthropogenic impact in the environment. We developed a novel high-throughput amplicon sequencing pipeline that enables characterization of thousands of integron gene cassette-associated reads, and applied it to acquire a comprehensive overview of gene cassette composition in effluents from wastewater treatment facilities across Europe. Between 38 100 and 172 995 reads per-sample were generated and functionally characterized by screening against nr, SEED, ARDB and ß-lactamase databases. Over 75% of the reads were characterized as hypothetical, but thousands were associated with toxin-antitoxin systems, DNA repair, cell membrane function, detoxification and aminoglycoside and ß-lactam resistance. Among the reads characterized as ß-lactamases, the carbapenemase blaOXA was dominant in most of the effluents, except for Cyprus and Israel where blaGES was also abundant. Quantitative PCR assessment of blaOXA and blaGES genes in the European effluents revealed similar trends to those displayed in the integron amplicon sequencing pipeline described above, corroborating the robustness of this method and suggesting that these integron-associated genes may be excellent targets for source tracking of effluents in downstream environments. Further application of the above analyses revealed several order-of-magnitude reductions in effluent-associated ß-lactamase genes in effluent-saturated soils, suggesting marginal persistence in the soil microbiome.

Effect of nitrate on sulfur transformations in sulfidogenic sludge of a marine aquaculture biofilter.

The effect of NO(3)(-) addition on dissimilatory SO(4)(2-) reduction and sulfide conversion in organic-rich sludge from the digestion basin of a recirculating marine aquaculture system was studied. SO(4)(2-) reduction could only explain a minor fraction (up to 4-9%) of the observed total sulfide production (up to 35 mmol L(-1) day(-1)), indicating that the main source of sulfide in the sludge was not SO(4)(2-) reduction, but desulfuration during the decomposition of organic matter. Although NO(3)(-) inhibited SO(4)(2-) reduction, but not desulfuration, the primary NO(3)(-) mitigation effect was the onset of NO(3)(-)-mediated sulfide oxidation (up to 75 mmol L(-1) day(-1)), partially to elemental sulfur (S(0)). Above NO(3)(-) concentrations of 0.6 mM in the bulk water, the net sulfide production and oxidation zones were moved deeper into flocs and sludge cores, which effectively prevented sulfide from entering the water column. However, the sulfide efflux from the sludge instantly recovered after NO(3)(-) depletion. Thus, the NO(3)(-) level in the water column controls the zonation and magnitude of sulfur transformations in the sludge. The effect of NO(3)(-) relies therefore on its sustained presence in the water column, which in turn depends on a well-functioning nitrification in the mariculture system.

Sulfide-induced nitrate reduction in the sludge of an anaerobic digester of a zero-discharge recirculating mariculture system.

The anaerobic digester is a vital component in a zero-discharge mariculture system as therein most of the organic matter is mineralized and nitrogen-containing compounds are converted to gaseous N(2). Although denitrification is a major respiratory process in this nitrate-rich treatment stage, also sulfate respiration takes place and may cause undesirable high sulfide concentrations in the effluent water. To examine the effect of sulfide on nitrate reduction, in situ depth profiles of inorganic nitrogen and sulfur compounds were determined. Additionally, nitrate reduction was examined as a function of ambient sulfide concentrations in sludge collected from different locations in the anaerobic reactor. Depth profiles showed high concentrations of nitrate and low concentrations of sulfide and ammonia in the aqueous layer of the reactor. A sharp decrease of nitrate and an increase in sulfide and ammonia concentrations was measured at the water-sludge interface. Nitrate reduction was highest in this interface zone with rates of up to 8.05+/-0.57 micromol NO(3)(-)h(-1)g((sludge))(-1). Addition of sulfide increased the nitrate reduction rate at all sludge depths, pointing to the important role of autotrophic denitrification in the anaerobic reactor. Dissimilatory nitrate reduction to ammonia (DNRA) was found to be low in all sludge layers but was enhanced when sludge was incubated at high sulfide concentrations. Although nitrate reduction rates increased as a result of sulfide addition to sludge samples, no differences in nitrate reduction rates were observed between the samples incubated with different initial sulfide concentrations. This as opposed to sulfide oxidation rates, which followed Michaelis-Menten enzymatic kinetics. Partial oxidation of sulfide to elemental sulfur instead of a complete oxidation to sulfate, could explain the observed patterns of nitrate reduction and sulfide oxidation in sludge incubated with different initial sulfide concentrations.

Impact of nitrate on the structure and function of bacterial biofilm communities in pipelines used for injection of seawater into oil fields.

We studied the impact of NO(3)(-) on the bacterial community composition, diversity, and function in in situ industrial, anaerobic biofilms by combining microsensor profiling, (15)N and (35)S labeling, and 16S rRNA gene-based fingerprinting. Biofilms were grown on carbon steel coupons within a system designed to treat seawater for injection into an oil field for pressurized oil recovery. NO(3)(-) was added to the seawater in an attempt to prevent bacterial H(2)S generation and microbially influenced corrosion in the field. Microprofiling of nitrogen compounds and redox potential inside the biofilms showed that the zone of highest metabolic activity was located close to the metal surface, correlating with a high bacterial abundance in this zone. Upon addition, NO(3)(-) was mainly reduced to NO(2)(-). In biofilms grown in the absence of NO(3)(-), redox potentials of <-450 mV at the metal surface suggested the release of Fe(2+). NO(3)(-) addition to previously untreated biofilms induced a decline (65%) in bacterial species richness, with Methylophaga- and Colwellia-related sequences having the highest number of obtained clones in the clone library. In contrast, no changes in community composition and potential NO(3)(-) reduction occurred upon subsequent withdrawal of NO(3)(-). Active sulfate reduction was below detection levels in all biofilms, but S isotope fractionation analysis of sulfide deposits suggested that it must have occurred either at low rates or episodically. Scanning electron microscopy revealed that pitting corrosion occurred on all coupons, independent of the treatment. However, uniform corrosion was clearly mitigated by NO(3)(-) addition.

Nitrosomonas Nm143-like ammonia oxidizers and Nitrospira marina-like nitrite oxidizers dominate the nitrifier community in a marine aquaculture biofilm.

Zero-discharge marine aquaculture systems are an environmentally friendly alternative to conventional aquaculture. In these systems, water is purified and recycled via microbial biofilters. Here, quantitative data on nitrifier community structure of a trickling filter biofilm associated with a recirculating marine aquaculture system are presented. Repeated rounds of the full-cycle rRNA approach were necessary to optimize DNA extraction and the probe set for FISH to obtain a reliable and comprehensive picture of the ammonia-oxidizing community. Analysis of the ammonia monooxygenase gene (amoA) confirmed the results. The most abundant ammonia-oxidizing bacteria (AOB) were members of the Nitrosomonas sp. Nm143-lineage (6.7% of the bacterial biovolume), followed by Nitrosomonas marina-like AOB (2.2% of the bacterial biovolume). Both were outnumbered by nitrite-oxidizing bacteria of the Nitrospira marina-lineage (15.7% of the bacterial biovolume). Although more than eight other nitrifying populations were detected, including Crenarchaeota closely related to the ammonia-oxidizer 'Nitrosopumilus maritimus', their collective abundance was below 1% of the total biofilm volume; their contribution to nitrification in the biofilter is therefore likely to be negligible.