Altered Antibiotic Tolerance in Anaerobic Digesters Acclimated to Triclosan Or Triclocarban

Bench-scale anaerobic digesters were amended to elevated steady-state concentrations of triclosan (850 mg/kg) and triclocarban (150 mg/kg) using a synthetic feed. After more than 9 solids retention time (SRT) values of acclimatization, biomass from each digester (and a control digester that received no antimicrobials) was used to assess the toxicity of three antibiotics. Methane production rate was measured as a surrogate for activity in microcosms that received doses of antibiotics ranging from no-antibiotic to inhibitory concentrations. Biomass amended with triclocarban was more sensitive to tetracycline compared to the control indicating synergistic inhibitory effects between this antibiotic and triclocarban. In contrast, biomass amended with triclosan was able to tolerate statistically higher levels of ciprofloxacin indicating that triclosan can induce functional resistance to ciprofloxacin in an anaerobic digester community

The Role of Household Antimicrobials in the Proliferation of Antibiotic Resistance During Anaerobic Digestion

Antimicrobial chemicals in consumer personal care products have been found to increase antibiotic resistance in pure culture studies. Although many studies focus on antibiotic resistance development pertinent to medical scenarios, resistance developed in natural and engineered environments might be significant and has become an emerging concern for human health. This dissertation focuses on the antimicrobial chemicals triclosan and triclocarban. These compounds are distinctly different from antibiotics and are used in products like soaps that are labelled as “antibacterial”. Municipal wastewater treatment plants receive triclocarban and triclosan loads higher than most contaminants of emerging concern because they are frequently used in consumer products and then discharged into the sewerage system. This research specifically focused on the impact of triclosan and triclocarban in lab-scale anaerobic digesters and investigated how they influenced digester function, the relative abundance of resistance genes, microbial community structure, and cross-resistance to antibiotics. Lab-scale anaerobic digesters were operated for 180 days and loaded with concentrations of triclocarban or triclosan ranging zero to inhibitory concentrations. Both triclosan and triclocarban selected for mexB, a gene that confers multidrug resistance in bacteria, at environmentally relevant concentrations. This is the first research to demonstrate that triclocarban can select for a multidrug resistance gene in anaerobic digesters. Relatively higher concentrations of these chemicals inhibited function in anaerobic digesters and further selected for some resistance genes and against others. The functional inhibition was not reversible when chemicals were removed. When these chemicals were removed from functioning digesters the mexB concentrations were no longer different from the control digesters suggesting that a decrease in consumer usage could have impacts on environmental antibiotic resistance. At higher concentrations of triclosan, and all concentrations of triclocarban, digester microbial community structures irreversibly shifted away from the control. In a separate set of experiments, addition of these antimicrobials altered how anaerobic digester microbial communities responded to the presence of three other antibiotics. Triclosan-amended communities had increased resistance to ciprofloxacin; triclocarban-communities were more sensitive to tetracycline and chloramphenicol. This research demonstrates that antimicrobials should be considered along with antibiotics when determining the role of chemical stress on the proliferation of antibiotic resistance

Recovery of Agricultural Nutrients from Biorefineries

This review lays the foundation for why nutrient recovery must be a key consideration in design and operation of biorefineries and comprehensively reviews technologies that can be used to recover an array of nitrogen, phosphorus, and/or potassium-rich products of relevance to agricultural applications. Recovery of these products using combinations of physical, chemical, and biological operations will promote sustainability at biorefineries by converting low-value biomass (particularly waste material) into a portfolio of higher-value products. These products can include a natural partnering of traditional biorefinery outputs such as biofuels and chemicals together with nutrient-rich fertilizers. Nutrient recovery not only adds an additional marketable biorefinery product, but also avoids the negative consequences of eutrophication, and helps to close anthropogenic nutrient cycles, thereby providing an alternative to current unsustainable approaches to fertilizer production, which are energy-intensive and reliant on nonrenewable natural resource extraction

Triclocarban Influences Antibiotic Resistance and Alters Anaerobic Digester Microbial Community Structure

Triclocarban (TCC) is one of the most abundant organic micropollutants detected in biosolids. Lab-scale anaerobic digesters were amended with TCC at concentrations ranging from the background concentration of seed biosolids (30 mg/kg) to toxic concentrations of 850 mg/kg to determine the effect on methane production, relative abundance of antibiotic resistance genes, and microbial community structure. Additionally, the TCC addition rate was varied to determine the impacts of acclimation time. At environmentally relevant TCC concentrations (max detect = 440 mg/kg), digesters maintained function. Digesters receiving 450 mg/kg of TCC maintained function under gradual TCC addition, but volatile fatty acid concentrations increased, pH decreased, and methane production ceased when immediately fed this concentration. The concentrations of the mexB gene (encoding for a multidrug efflux pump) were higher with all concentrations of TCC compared to a control, but higher TCC concentrations did not correlate with increased mexB abundance. The relative abundance of the gene tet(L) was greater in the digesters that no longer produced methane, and no effect on the relative abundance of the class 1 integron integrase encoding gene (intI1) was observed. Illumina sequencing revealed substantial community shifts in digesters that functionally failed from increased levels of TCC. More subtle, yet significant, community shifts were observed in digesters amended with TCC levels that did not inhibit function. This research demonstrates that TCC can select for a multidrug resistance encoding gene in mixed community anaerobic environments, and this selection occurs at concentrations (30 mg/kg) that can be found in full-scale anaerobic digesters (U.S. median concentration = 22 mg/kg, mean = 39 mg/kg)

Chronic Exposure to Triclosan Sustains Microbial Community Shifts and Alters Antibiotic Resistance Gene Levels in Anaerobic Digesters

Triclosan, an antimicrobial chemical found in consumer personal care products, has been shown to stimulate antibiotic resistance in pathogenic bacteria. Although many studies focus on antibiotic resistance pertinent to medical scenarios, resistance developed in natural and engineered environments is less studied and has become an emerging concern for human health. In this study, the impacts of chronic triclosan (TCS) exposure on antibiotic resistance genes (ARGs) and microbial community structure were assessed in lab-scale anaerobic digesters. TCS concentrations from below detection to 2500 mg kg−1 dry solids were amended into anaerobic digesters over 110 days and acclimated for \u3e3 solid retention time values. Four steady state TCS concentrations were chosen (30–2500 mg kg−1). Relative abundance of mexB, a gene coding for a component of a multidrug efflux pump, was significantly higher in all TCS-amended digesters (30 mg kg−1 or higher) relative to the control. TCS selected for bacteria carrying tet(L) and against those carrying erm(F) at concentrations which inhibited digester function; the pH decrease associated with digester failure was suspected to cause this selection. Little to no impact of TCS was observed on intI1 relative abundance. Microbial communities were also surveyed by high-throughput 16S rRNA gene sequencing. Compared to the control digesters, significant shifts in community structure towards clades containing commensal and pathogenic bacteria were observed in digesters containing TCS. Based on these results, TCS should be included in studies and risk assessments that attempt to elucidate relationships between chemical stressors (e.g. antibiotics), antibiotic resistance genes, and public health

Syntroph Diversity and Abundance in Anaerobic Digestion Revealed Through a Comparative Core Microbiome Approach

Anaerobic digestion is an important biotechnology treatment process for conversion of waste to energy. In this study, a comparative core microbiome approach, i.e., determining taxa that are shared in functioning digesters but not shared in non-functioning digesters, was used to determine microbial taxa that could play key roles for effective anaerobic digestion. Anaerobic digester functions were impaired by adding the broad-spectrum antimicrobial triclosan (TCS) or triclocarban (TCC) at different concentrations, and the core microbiomes in both functioning and non-functioning anaerobic digesters were compared. Digesters treated with high (2500 mg/kg) or medium (450 mg/kg) TCS and high (850 mg/kg) TCC concentrations lost their function, i.e., methane production decreased, effluent volatile fatty acid concentrations increased, and pH decreased. Changes in microbial community diversity and compositions were assessed using 16S rRNA gene amplicon sequencing. Microbial richness decreased significantly in non-functioning digesters (p \u3c 0.001). Microbial community compositions in non-functioning digesters significantly differed from those in functioning digesters (p = 0.001, ANOSIM). Microbes identified as potentially key taxa included previously known fatty acid-degrading syntrophs and amino acid-degrading syntrophs. A diverse group of syntrophs detected in this study had low relative abundance in functioning digesters, suggesting the importance of rare microbes in anaerobic digester operation. The comparative microbiome approach used in this study can be applied to other microbial systems where a community-driven biological phenomena can be observed directly

Metagenomics Reveal Triclosan-Induced Changes in the Antibiotic Resistome of Anaerobic Digesters

Triclosan (TCS) is a broad-spectrum antimicrobial used in a variety of consumer products. While it was recently banned from hand soaps in the US, it is still a key ingredient in a top-selling toothpaste. TCS is a hydrophobic micropollutant that is recalcitrant under anaerobic digestion thereby resulting in high TCS concentrations in biosolids. The objective of this study was to determine the impact of TCS on the antibiotic resistome and potential cross-protection in lab-scale anaerobic digesters using shotgun metagenomics. It was hypothesized that metagenomics would reveal selection for antibiotic resistance genes (ARGs) not previously found in pure culture studies or mixed-culture studies using targeted qPCR. In this study, four different levels of TCS were continuously fed to triplicate lab-scale anaerobic digesters to assess the effect of TCS levels on the antibiotic resistance gene profiles (resistome). Blasting metagenomic reads against antibiotic/metal resistance gene database (BacMet) revealed that ARG diversity and abundance changed along the TCS concentration gradient. While loss of bacterial diversity and digester function were observed in the digester treated with the highest TCS concentration, FabV, which is a known TCS resistance gene, increased in this extremely high TCS environment. The abundance of several other known ARG or metal resistance genes (MRGs), including corA and arsB, also increased as the concentrations of TCS increased. Analysis of other functional genes using SEED database revealed the increase of potentially key genes for resistance including different types of transporters and transposons. These results indicate that antimicrobials can alter the abundance of multiple resistance genes in anaerobic digesters even when function (i.e. methane production) is maintained. This study also suggests that enriched ARGs could be released into environments with biosolids land application

Removal of Antibiotic Resistance Genes in an Anaerobic Membrane Bioreactor Treating Primary Clarifier Effluent at 20 °C

Anaerobic membrane bioreactors (AnMBR) play a key role in future plans for sustainable wastewater treatment and resource recovery because they have no energy-intensive oxygen transfer requirements and can produce biomethane for renewable energy. Recent research results show that they can meet relatively stringent discharge limits with respect to BOD5 and TSS when treating municipal wastewater primary effluent. Sustainable used water recovery plans should also consider removal of unregulated pollutants. Antibiotic resistance genes (ARGs) represent an important emerging contaminant due to public health concerns surrounding the spread of infections resistant to common antibiotics. Conventional activated sludge processes have demonstrated mixed results regarding ARG removal. The objective of this research was to determine the impact of an AnMBR on ARG removal when treating municipal primary clarifier effluent at 20 °C. AnMBR treatment resulted in 3.3 to 3.6 log reduction of ARG and the horizontal gene transfer determinate, intI1, copies in filtrate. Membrane treatment significantly decreased the total biomass as indicated by a decrease in 16S rRNA gene concentration. Microbial community analysis via Illumina sequencing revealed that the relative abundance of putative pathogens was higher in membrane filtrate compared to primary effluent although the overall bacterial 16S rRNA gene concentrations was lower in filtrate. Membrane treatment also substantially reduced microbial diversity in filtrate compared to anaerobic reactor contents

Characterizing Habitat Suitability for a Central‐Place Forager in a Dynamic Marine Environment

Characterizing habitat suitability for a marine predator requires an understanding of the environmental heterogeneity and variability over the range in which a population moves during a particular life cycle. Female California sea lions (Zalophus californianus) are central‐place foragers and are particularly constrained while provisioning their young. During this time, habitat selection is a function of prey availability and proximity to the rookery, which has important implications for reproductive and population success. We explore how lactating females may select habitat and respond to environmental variability over broad spatial and temporal scales within the California Current System. We combine near‐real‐time remotely sensed satellite oceanography, animal tracking data (n = 72) from November to February over multiple years (2003–2009) and Generalized Additive Mixed Models (GAMMs) to determine the probability of sea lion occurrence based on environmental covariates. Results indicate that sea lion presence is associated with cool (\u3c14°C), productive waters, shallow depths, increased eddy activity, and positive sea‐level anomalies. Predictive habitat maps generated from these biophysical associations suggest winter foraging areas are spatially consistent in the nearshore and offshore environments, except during the 2004–2005 winter, which coincided with an El Niño event. Here, we show how a species distribution model can provide broadscale information on the distribution of female California sea lions during an important life history stage and its implications for population dynamics and spatial management

Subsequent Surgery After Revision Anterior Cruciate Ligament Reconstruction: Rates and Risk Factors From a Multicenter Cohort

BACKGROUND: While revision anterior cruciate ligament reconstruction (ACLR) can be performed to restore knee stability and improve patient activity levels, outcomes after this surgery are reported to be inferior to those after primary ACLR. Further reoperations after revision ACLR can have an even more profound effect on patient satisfaction and outcomes. However, there is a current lack of information regarding the rate and risk factors for subsequent surgery after revision ACLR. PURPOSE: To report the rate of reoperations, procedures performed, and risk factors for a reoperation 2 years after revision ACLR. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: A total of 1205 patients who underwent revision ACLR were enrolled in the Multicenter ACL Revision Study (MARS) between 2006 and 2011, composing the prospective cohort. Two-year questionnaire follow-up was obtained for 989 patients (82%), while telephone follow-up was obtained for 1112 patients (92%). If a patient reported having undergone subsequent surgery, operative reports detailing the subsequent procedure(s) were obtained and categorized. Multivariate regression analysis was performed to determine independent risk factors for a reoperation. RESULTS: Of the 1112 patients included in the analysis, 122 patients (11%) underwent a total of 172 subsequent procedures on the ipsilateral knee at 2-year follow-up. Of the reoperations, 27% were meniscal procedures (69% meniscectomy, 26% repair), 19% were subsequent revision ACLR, 17% were cartilage procedures (61% chondroplasty, 17% microfracture, 13% mosaicplasty), 11% were hardware removal, and 9% were procedures for arthrofibrosis. Multivariate analysis revealed that patients aged <20 years had twice the odds of patients aged 20 to 29 years to undergo a reoperation. The use of an allograft at the time of revision ACLR (odds ratio [OR], 1.79; P = .007) was a significant predictor for reoperations at 2 years, while staged revision (bone grafting of tunnels before revision ACLR) (OR, 1.93; P = .052) did not reach significance. Patients with grade 4 cartilage damage seen during revision ACLR were 78% less likely to undergo subsequent operations within 2 years. Sex, body mass index, smoking history, Marx activity score, technique for femoral tunnel placement, and meniscal tearing or meniscal treatment at the time of revision ACLR showed no significant effect on the reoperation rate. CONCLUSION: There was a significant reoperation rate after revision ACLR at 2 years (11%), with meniscal procedures most commonly involved. Independent risk factors for subsequent surgery on the ipsilateral knee included age <20 years and the use of allograft tissue at the time of revision ACLR