Characterizing Differences in Sources of and Contributions to Fecal Contamination of Sediment and Surface Water with the Microbial FIT Framework.

Surface water monitoring and microbial source tracking (MST) are used to identify host sources of fecal pollution and protect public health. However, knowledge of the locations of spatial sources and their relative impacts on the environment is needed to effectively mitigate health risks. Additionally, sediment samples may offer time-integrated information compared to transient surface water. Thus, we implemented the newly developed microbial find, inform, and test framework to identify spatial sources and their impacts on human (HuBac) and bovine (BoBac) MST markers, quantified from both riverbed sediment and surface water in a bovine-dense region. Dairy feeding operations and low-intensity developed land-cover were associated with 99% (p-value < 0.05) and 108% (p-value < 0.05) increases, respectively, in the relative abundance of BoBac in sediment, and with 79% (p-value < 0.05) and 39% increases in surface water. Septic systems were associated with a 48% increase in the relative abundance of HuBac in sediment and a 56% increase in surface water. Stronger source signals were observed for sediment responses compared to water. By defining source locations, predicting river impacts, and estimating source influence ranges in a Great Lakes region, this work informs pollution mitigation strategies of local and global significance.

Manure derived nutrients alter microbial community composition and increase the presence of potential pathogens in freshwater sediment.

AIM: To determine the impact of an acute, pulse disturbance of nutrients from manure on freshwater sediment microbiomes in an experimental system. METHODS AND RESULTS: A controlled freshwater mesocosm experiment was designed to compare the effect of disturbance from nutrients derived from sterile manure (SM), disturbance from equivalent concentrations of laboratory-derived nutrients, and a nondisturbed control on freshwater sediment microbial community composition and function using 16S rRNA amplicon sequencing. Sediment microbiomes impacted by nutrients from SM showed no sign of compositional recovery after 28 days but those impacted by laboratory-derived chemicals lead to a new steady-state (p < 0.05). Carbon and nitrate sources within disturbed mesocosms were the primary drivers of altered microbial community composition. Additionally, multiple potential pathogens (based on exact sequence matching at the species level) were enriched in mesocosms treated with SM. CONCLUSIONS: Nutrient disturbance from SM, in the absence of the manure microbial community, alters the microbiome of sediments without recovery after 28 days and enriches potential pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest manure land application practices should be re-evaluated to account for impact of nutrient disturbance on environmental microbiomes in addition to the impact of the manure microbial community.

Microbial Find, Inform, and Test Model for Identifying Spatially Distributed Contamination Sources: Framework Foundation and Demonstration of Ruminant Bacteroides Abundance in River Sediments.

Microbial pollution in rivers poses known ecological and health risks, yet causal and mechanistic linkages to sources remain difficult to establish. Host-associated microbial source tracking (MST) markers help to assess the microbial risks by linking hosts to contamination but do not identify the source locations. Land-use regression (LUR) models have been used to screen the source locations using spatial predictors but could be improved by characterizing transport (i.e., hauling, decay overland, and downstream). We introduce the microbial Find, Inform, and Test (FIT) framework, which expands previous LUR approaches and develops novel spatial predictor models to characterize the transported contributions. We applied FIT to characterize the sources of BoBac, a ruminant Bacteroides MST marker, quantified in riverbed sediment samples from Kewaunee County, Wisconsin. A 1 standard deviation increase in contributions from land-applied manure hauled from animal feeding operations (AFOs) was associated with a 77% (p-value <0.05) increase in the relative abundance of ruminant Bacteroides (BoBac-copies-per-16S-rRNA-copies) in the sediment. This is the first work finding an association between the upstream land-applied manure and the offsite bovine-associated fecal markers. These findings have implications for the sediment as a reservoir for microbial pollution associated with AFOs (e.g., pathogens and antibiotic-resistant bacteria). This framework and application advance statistical analysis in MST and water quality modeling more broadly.

Effect of benzene and ethylbenzene on the transcription of methyl-tert-butyl ether degradation genes of Methylibium petroleiphilum PM1.

Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds' impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1.

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).

Successful treatment of an MTBE-impacted aquifer using a bioreactor self-colonized by native aquifer bacteria.

A field-scale fixed bed bioreactor was used to successfully treat an MTBE-contaminated aquifer in North Hollywood, CA without requiring inoculation with introduced bacteria. Native bacteria from the MTBE-impacted aquifer rapidly colonized the bioreactor, entering the bioreactor in the contaminated groundwater pumped from the site, and biodegraded MTBE with greater than 99 % removal efficiency. DNA sequencing of the 16S rRNA gene identified MTBE-degrading bacteria Methylibium petroleiphilum in the bioreactor. Quantitative PCR showed M. petroleiphilum enriched by three orders of magnitude in the bioreactor above densities pre-existing in the groundwater. Because treatment was carried out by indigenous rather than introduced organisms, regulatory approval was obtained for implementation of a full-scale bioreactor to continue treatment of the aquifer. In addition, after confirmation of MTBE removal in the bioreactor to below maximum contaminant limit levels (MCL; MTBE = 5 µg L(-1)), treated water was approved for reinjection back into the aquifer rather than requiring discharge to a water treatment system. This is the first treatment system in California to be approved for reinjection of biologically treated effluent into a drinking water aquifer. This study demonstrated the potential for using native microbial communities already present in the aquifer as an inoculum for ex-situ bioreactors, circumventing the need to establish non-native, non-acclimated and potentially costly inoculants. Understanding and harnessing the metabolic potential of native organisms circumvents some of the issues associated with introducing non-native organisms into drinking water aquifers, and can provide a low-cost and efficient remediation technology that can streamline future bioremediation approval processes.

Bacillus subtilis-derived peptides disrupt quorum sensing and biofilm assembly in multidrug-resistant Staphylococcus aureus.

Multidrug-resistant Staphylococcus aureus is one of the most clinically important pathogens in the world, with infections leading to high rates of morbidity and mortality in both humans and animals. The ability of S. aureus to form biofilms protects cells from antibiotics and promotes the transfer of antibiotic resistance genes; therefore, new strategies aimed at inhibiting biofilm growth are urgently needed. Probiotic species, including Bacillus subtilis, are gaining interest as potential therapies against S. aureus for their ability to reduce S. aureus colonization and virulence. Here, we search for strains and microbially derived compounds with strong antibiofilm activity against multidrug-resistant S. aureus by isolating and screening Bacillus strains from a variety of agricultural environments. From a total of 1,123 environmental isolates, we identify a single strain B. subtilis 6D1, with a potent ability to inhibit biofilm growth, disassemble mature biofilm, and improve antibiotic sensitivity of S. aureus biofilms through an Agr quorum sensing interference mechanism. Biochemical and molecular networking analysis of an active organic fraction revealed multiple surfactin isoforms, and an uncharacterized peptide was driving this antibiofilm activity. Compared with commercial high-performance liquid chromatography grade surfactin obtained from B. subtilis, we show these B. subtilis 6D1 peptides are significantly better at inhibiting biofilm formation in all four S. aureus Agr backgrounds and preventing S. aureus-induced cytotoxicity when applied to HT29 human intestinal cells. Our study illustrates the potential of exploring microbial strain diversity to discover novel antibiofilm agents that may help combat multidrug-resistant S. aureus infections and enhance antibiotic efficacy in clinical and veterinary settings. IMPORTANCE: The formation of biofilms by multidrug-resistant bacterial pathogens, such as Staphylococcus aureus, increases these microorganisms' virulence and decreases the efficacy of common antibiotic regimens. Probiotics possess a variety of strain-specific strategies to reduce biofilm formation in competing organisms; however, the mechanisms and compounds responsible for these phenomena often go uncharacterized. In this study, we identified a mixture of small probiotic-derived peptides capable of Agr quorum sensing interference as one of the mechanisms driving antibiofilm activity against S. aureus. This collection of peptides also improved antibiotic killing and protected human gut epithelial cells from S. aureus-induced toxicity by stimulating an adaptive cytokine response. We conclude that purposeful strain screening and selection efforts can be used to identify unique probiotic strains that possess specially desired mechanisms of action. This information can be used to further improve our understanding of the ways in which probiotic and probiotic-derived compounds can be applied to prevent bacterial infections or improve bacterial sensitivity to antibiotics in clinical and agricultural settings.

Cardiovascular multimodality imaging in women: a scientific statement of the European Association of Cardiovascular Imaging of the European Society of Cardiology.

Cardiovascular diseases (CVD) represent an important cause of mortality and morbidity in women. It is now recognized that there are sex differences regarding the prevalence and the clinical significance of the traditional cardiovascular (CV) risk factors as well as the pathology underlying a range of CVDs. Unfortunately, women have been under-represented in most CVD imaging studies and trials regarding diagnosis, prognosis, and therapeutics. There is therefore a clear need for further investigation of how CVD affects women along their life span. Multimodality CV imaging plays a key role in the diagnosis of CVD in women as well as in prognosis, decision-making, and monitoring of therapeutics and interventions. However, multimodality imaging in women requires specific consideration given the differences in CVD between the sexes. These differences relate to physiological changes that only women experience (e.g. pregnancy and menopause) as well as variation in the underlying pathophysiology of CVD and also differences in the prevalence of certain conditions such as connective tissue disorders, Takotsubo, and spontaneous coronary artery dissection, which are all more common in women. This scientific statement on CV multimodality in women, an initiative of the European Association of Cardiovascular Imaging of the European Society of Cardiology, reviews the role of multimodality CV imaging in the diagnosis, management, and risk stratification of CVD, as well as highlights important gaps in our knowledge that require further investigation.

Selenium biotransformations in an engineered aquatic ecosystem for bioremediation of agricultural wastewater via brine shrimp production.

An engineered aquatic ecosystem was specifically designed to bioremediate selenium (Se), occurring as oxidized inorganic selenate from hypersalinized agricultural drainage water while producing brine shrimp enriched in organic Se and omega-3 and omega-6 fatty acids for use in value added nutraceutical food supplements. Selenate was successfully bioremediated by microalgal metabolism into organic Se (seleno-amino acids) and partially removed via gaseous volatile Se formation. Furthermore, filter-feeding brine shrimp that accumulated this organic Se were removed by net harvest. Thriving in this engineered pond system, brine shrimp ( Artemia franciscana Kellogg) and brine fly (Ephydridae sp.) have major ecological relevance as important food sources for large populations of waterfowl, breeding, and migratory shore birds. This aquatic ecosystem was an ideal model for study because it mimics trophic interactions in a Se polluted wetland. Inorganic selenate in drainage water was metabolized differently in microalgae, bacteria, and diatoms where it was accumulated and reduced into various inorganic forms (selenite, selenide, or elemental Se) or partially incorporated into organic Se mainly as selenomethionine. Brine shrimp and brine fly larva then bioaccumulated Se from ingesting aquatic microorganisms and further metabolized Se predominately into organic Se forms. Importantly, adult brine flies, which hatched from aquatic larva, bioaccumulated the highest Se concentrations of all organisms tested.

Identifying sources of antibiotic resistance genes in the environment using the microbial Find, Inform, and Test framework.

Introduction: Antimicrobial resistance (AMR) is an increasing public health concern for humans, animals, and the environment. However, the contributions of spatially distributed sources of AMR in the environment are not well defined. Methods: To identify the sources of environmental AMR, the novel microbial Find, Inform, and Test (FIT) model was applied to a panel of five antibiotic resistance-associated genes (ARGs), namely, erm(B), tet(W), qnrA, sul1, and intI1, quantified from riverbed sediment and surface water from a mixed-use region. Results: A one standard deviation increase in the modeled contributions of elevated AMR from bovine sources or land-applied waste sources [land application of biosolids, sludge, and industrial wastewater (i.e., food processing) and domestic (i.e., municipal and septage)] was associated with 34-80% and 33-77% increases in the relative abundances of the ARGs in riverbed sediment and surface water, respectively. Sources influenced environmental AMR at overland distances of up to 13 km. Discussion: Our study corroborates previous evidence of offsite migration of microbial pollution from bovine sources and newly suggests offsite migration from land-applied waste. With FIT, we estimated the distance-based influence range overland and downstream around sources to model the impact these sources may have on AMR at unsampled sites. This modeling supports targeted monitoring of AMR from sources for future exposure and risk mitigation efforts.

Cardioprotection Using Strain-Guided Management of Potentially Cardiotoxic Cancer Therapy: 3-Year Results of the SUCCOUR Trial.

BACKGROUND: Global longitudinal strain (GLS) can predict cancer therapeutics-related cardiac dysfunction and guide initiation of cardioprotection (CPT). OBJECTIVES: In this study, the authors sought to determine whether echocardiography GLS-guided CPT provides less cardiac dysfunction in survivors of potentially cardiotoxic chemotherapy, compared with usual care at 3 years. METHODS: In this international multicenter prospective randomized controlled trial, patients were enrolled from 28 international sites. All patients treated with anthracyclines with another risk factor for heart failure were randomly allocated to GLS-guided (>12% relative reduction in GLS) or ejection fraction (EF)-guided (>10% absolute reduction of EF to <55%) CPT. The primary end point was the change in 3-dimensional (3D) EF (ΔEF) from baseline to 3 years. RESULTS: Among 331 patients enrolled, 255 (77%, age 54 ± 12 years, 95% women) completed 3-year follow-up (123 in the EF-guided group and 132 in the GLS-guided group). Most had breast cancer (n = 236; 93%), and anthracycline followed by trastuzumab was the most common chemotherapy regimen (84%). Although 67 (26%) had hypertension and 32 (13%) had diabetes mellitus, left ventricular function was normal at baseline (EF: 59% ± 6%, GLS: 20.7% ± 2.3%). CPT was administered in 18 patients (14.6%) in the EF-guided group and 41 (31%) in the GLS-guided group (P = 0.03). Most patients showed recovery in EF and GLS after chemotherapy; 3-year ΔEF was -0.03% ± 7.9% in the EF-guided group and -0.02% ± 6.5% in the GLS-guided (P = 0.99) group; respective 3-year EFs were 58% ± 6% and 59% ± 5% (P = 0.06). At 3 years, 17 patients (5%) had cancer therapeutics-related cardiac dysfunction (11 in the EF-guided group and 6 in the GLS guided group; P = 0.16); 1 patient in each group was admitted for heart failure. CONCLUSIONS: Among patients taking potentially cardiotoxic chemotherapy for cancer, the 3-year data showed improvement of LV dysfunction compared with 1 year, with no difference in ΔEF between GLS- and EF-guided CPT. (Strain Surveillance of Chemotherapy for Improving Cardiovascular Outcomes [SUCCOUR]; ACTRN12614000341628).

Recycled concrete aggregates are an economic form of urban riparian erosion management with limited impacts on freshwater chemistry and microbial diversity.

Urban streams are at high risk of riparian erosion which impacts adjacent infrastructure stability. Methods to prevent stream erosion have been proposed including using recycled concrete (RC) materials to help stabilize the streambed; however, little is known about the environmental and biological impacts of using RC in urban streams. RC, new concrete (NC), and river rock controls were evaluated for their impact on water chemistry, water quality, and microbial community composition over 6.5 months in controlled laboratory mesocosms. Concentrations of 19 metals, nutrients, and pH of mesocosms containing RC were not significantly different from the river rock mesocosm throughout the experiment; however, NC mesocosms contained significantly higher (p < 0.05) concentrations of Co, As, Al, and V in mesocosm water samples compared to both RC and the river rock control. Microbial community diversity was not significantly impacted by mesocosm treatment. Microbial sequences mapping to taxa including Rhodoferax, Acidovorax, Nitrosomonas, and Novosphingobium were significantly more abundant (p < 0.01) in RC and NC mesocosm samples; however, the overall microbial community structure was similar across treatment types. Results from this study suggest that RC does not significantly alter the stream environment including microbial community diversity and is a viable option for use in stream restoration projects.

Similarities in Virulence and Extended Spectrum Beta-Lactamase Gene Profiles among Cefotaxime-Resistant Escherichia coli Wastewater and Clinical Isolates.

The World Health Organization has identified antibiotic resistance as one of the largest threats to human health and food security. In this study, we compared antibiotic resistance patterns between ESBL-producing Escherichia coli from human clinical diseases and cefotaxime-resistant environmental strains, as well as their potential to be pathogenic. Antibiotic susceptibility was tested amongst clinical isolates (n = 11), hospital wastewater (n = 22), and urban wastewater (n = 36, both influent and treated effluents). Multi-drug resistance predominated (>70%) among hospitalwastewater and urban wastewater influent isolates. Interestingly, isolates from clinical and urban treated effluents showed similar multi-drug resistance rates (~50%). Most hospital wastewater isolates were Phylogroup A, while clinical isolates were predominately B2, with a more diverse phylogroup population in urban wastewater. ESBL characterization of cefotaxime-resistant populations identified blaCTX-M-1 subgroup as the most common, whereby blaKPC was more associated with ceftazidime and ertapenem resistance. Whole-genome sequencing of a carbapenemase-producing hospital wastewater E. coli strain revealed plasmid-mediated blaKPC-2. Among cefotaxime-resistant populations, over 60% of clinical and 30% of treated effluent E. coli encoded three or more virulence genes exhibiting a pathogenic potential. Together, the similarity among treated effluent E. coli populations and clinical strains suggest effluents could serve as a reservoir for future multi-drug resistant E. coli clinical infections.

Incremental Value of Myocardial Work over Global Longitudinal Strain in the Surveillance for Cancer-Treatment-Related Cardiac Dysfunction: A Case-Control Study.

The load dependence of global longitudinal strain (GLS) means that changes in systolic blood pressure (BP) between visits may confound the diagnosis of cancer-treatment-related cardiac dysfunction (CTRCD). We sought to determine whether the estimation of myocardial work, which incorporates SBP, could overcome this limitation. In this case-control study, 44 asymptomatic patients at risk of CTRCD underwent echocardiography at baseline and after oncologic treatment. CTRCD was defined on the basis of the change in the ejection fraction. Those with CTRCD were divided into subsets with and without a follow-up SBP increment >20 mmHg (CTRCD+BP+ and CTRCD+BP-), and matched with patients without CTRCD (CTRCD-BP+ and CTRCD-BP-). The work index (GWI), constructive work (GCW), wasted work (GWW), and work efficiency (GWE) were assessed in addition to the GLS. The largest increases in the GWI and GCW at follow-up were found in CTRCD-BP+ patients. The CTRCD+BP- patients demonstrated significantly larger decreases in GWI and GCW than their CTRCD+BP+ and CTRCD-BP- peers. ROC analysis for the discrimination of LV functional changes in response to increased afterload in the absence of cardiotoxicity revealed higher AUCs for GCW (AUC = 0.97) and GWI (AUC = 0.93) than GLS (AUC = 0.73), GWW (AUC = 0.51), or GWE (AUC = 0.63, all p-values < 0.001). GCW (OR: 1.021; 95% CI: 1.001-1.042; p < 0.04) was the only feature independently associated with CTRCD-BP+. Myocardial work is superior to GLS in the serial assessments in patients receiving cardiotoxic chemotherapy. The impairment of GLS in the presence of an increase in GWI and GCW indicates the impact of elevated afterload on LV performance in the absence of actual myocardial impairment.

Antibiotic resistance patterns of Escherichia coli isolates from the clinic through the wastewater pathway.

Antimicrobial resistance (AMR) remains one of the leading global health threats. This study compared antimicrobial resistance patterns among E. coli isolates from clinical uropathogenic Escherichia coli (UPEC) to hospital wastewater populations and throughout an urban wastewater treatment facility - influent, pre- and post-chlorinated effluents. Antibiotic susceptibility of 201 isolates were analyzed against eleven different antibiotics, and the presence of twelve antibiotic resistant genes and type 1 integrase were identified. AMR exhibited the following pattern: UPEC (46.8%) > hospital wastewater (37.8%) > urban post-chlorinated effluent (27.6%) > pre-chlorinated effluent (21.4%) > urban influent wastewater (13.3%). However, multi-drug resistance against three or more antimicrobial classes was more prevalent among hospital wastewater populations (29.7%) compared to other sources. E. coli from wastewaters disinfected with chlorine were significantly correlated with increased trimethoprim-sulfamethoxazole resistance in E. coli compared to raw and treated wastewater populations. blaCTX-M-1 group was the most common extended spectrum beta-lactamase in E. coli from hospital wastewater (90%), although UPEC strains also encoded blaCTX-M-1 group (50%) and blaTEM (100%) genes. Among tetracycline-resistant populations, tetA and tetB were the only resistance genes identified throughout wastewater populations that were associated with increased phenotypic resistance. Further characterization of the E. coli populations identified phylogroup B2 predominating among clinical UPEC populations and correlated with the highest AMR, whereas the elevated rate of multi-drug resistance among hospital wastewater was mostly phylogroup A. Together, our findings highlight hospital wastewater as a rich source of AMR and multi-drug resistant bacterial populations.

Impacts of different N management regimes on nitrifier and denitrifier communities and N cycling in soil microenvironments.

Real-time quantitative PCR assays, targeting part of the ammonia-monooxygenase (amoA), nitrous oxide reductase (nosZ), and 16S rRNA genes were coupled with (15)N pool dilution techniques to investigate the effects of long-term agricultural management practices on potential gross N mineralization and nitrification rates, as well as ammonia-oxidizing bacteria (AOB), denitrifier, and total bacterial community sizes within different soil microenvironments. Three soil microenvironments [coarse particulate organic matter (cPOM; >250 µm), microaggregate (53-250 µm), and silt-and-clay fraction (<53 µm)] were physically isolated from soil samples collected across the cropping season from conventional, low-input, and organic maize-tomato systems (Zea mays L.- Lycopersicum esculentum L.). We hypothesized that (i) the higher N inputs and soil N content of the organic system foster larger AOB and denitrifier communities than in the conventional and low-input systems, (ii) differences in potential gross N mineralization and nitrification rates across the systems correspond with AOB and denitrifier abundances, and (iii) amoA, nosZ, and 16S rRNA gene abundances are higher in the microaggregates than in the cPOM and silt-and-clay microenvironments. Despite 13 years of different soil management and greater soil C and N content in the organic compared to the conventional and low-input systems, total bacterial communities within the whole soil were similar in size across the three systems (~5.15×10(8) copies g(-1) soil). However, amoA gene densities were ~2 times higher in the organic (1.75×10(8) copies g(-1) soil) than the other systems at the start of the season and nosZ gene abundances were ~2 times greater in the conventional (7.65×10(7) copies g(-1) soil) than in the other systems by the end of the season. Because organic management did not consistently lead to larger AOB and denitrifier communities than the other two systems, our first hypothesis was not corroborated. Our second hypothesis was also not corroborated because canonical correspondence analyses revealed that AOB and denitrifier abundances were decoupled from potential gross N mineralization and nitrification rates and from inorganic N concentrations. Our third hypothesis was supported by the overall larger nitrifier, denitrifier, and total bacterial communities measured in the soil microaggregates compared to the cPOM and silt-and-clay. These results suggest that the microaggregates are microenvironments that preferentially stabilize C, and concomitantly promote the growth of nitrifier and denitrifier communities, thereby serving as potential hotspots for N(2)O losses.

Quantitative gene monitoring of microbial tetracycline resistance using magnetic luminescent nanoparticles.

A magnetic/luminescent nanoparticles (MLNPs) based DNA hybridization method was developed for quantitative monitoring of antibiotic resistance genes and gene-expression in environmental samples. Manipulation of magnetic field enabled the separation of the MLNPs-DNA hybrids from the solution and the fluorescence of MLNPs normalized the quantity of target DNA. In our newly developed MLNPs-DNA assay, linear standard curves (R(2) = 0.99) of target gene was determined with the detection limit of 620 gene copies. The potential risk of increased bacterial antibiotic resistance was assessed by quantitative monitoring of tetracycline resistance (i.e., tetQ gene) in wastewater microcosms. The gene abundance and its expression showed a significant increase of tetQ gene copies with the addition of tetracycline, triclosan (TCS), or triclocarban (TCC). A real-time PCR assay was employed to verify the quantification capability of the MLNPs-DNA assay and accordingly both assays have shown strong correlation (R(2) = 0.93). This non-PCR based MLNPs-DNA assay has demonstrated its potential for gene quantification via a rapid, simple, and high throughput platform and its novel use of internal calibration standards.

Survivor microbial populations in post-chlorinated wastewater are strongly associated with untreated hospital sewage and include ceftazidime and meropenem resistant populations.

Wastewater treatment plant (WWTP) effluent has been implicated in the spread of antibiotic resistant bacteria (ARB), including pathogens, as the WWTP environment contains multiple selective pressures that may increase mutation rates, pathogen survivability, and induce gene transfer between bacteria. In WWTPs receiving hospital sewage, this selective effect may be more pronounced due to increased concentrations of antibiotics, ARB, and clinical pathogens from hospital sewage. To determine the extent to which hospital sewage contributes to the microbial community of disinfected wastewater which is released into the environment, we used 16S rRNA sequencing of hospital sewage, WWTP influent, primary effluent, Post-Chlorinated Effluent, and receiving sediments in a combined sewage system to track changes in microbial community composition. We also sequenced the culturable survivor community resistant to ß-lactam antibiotics within disinfected effluent. Using molecular source tracking, we found that the hospital sewage microbiome contributes an average of 11.49% of the microbial community in Post-Chlorinated Effluents, suggesting microorganisms identified within hospital sewage can survive or are enriched by the chlorination disinfection process. Additionally, we identified 28 potential pathogens to the species level, seven of which remained detectable in Post-Chlorinated Effluent and environmental sediments. When Post-Chlorinated Effluents were cultured on media containing ß-lactam antibiotics ceftazidime and meropenem, a diverse antibiotic resistant survivor community was identified including potential human pathogens Bacillus cereus, Bacillus pumilus, and Chryseobacterium indologenes. Together, these results indicate that although wastewater treatment does significantly reduce pathogenic loads and ARBs, their continual presence in disinfected wastewater and receiving sediments suggests additional treatment and microbial tracking systems are needed to reduce human and animal health risks.

Freshwater Sediment Microbial Communities Are Not Resilient to Disturbance From Agricultural Land Runoff.

Microorganisms are critically important for the function of surface water ecosystems but are frequently subjected to anthropogenic disturbances at either acute (pulse) or long-term (press) scales. Response and recovery of microbial community composition and function following pulse disturbance is well-studied in controlled, laboratory scale experiments but is less well-understood in natural environments undergoing continual press disturbance. The objectives of this study were to determine the drivers of sediment microbial compositional and functional changes in freshwaters receiving continual press disturbance from agricultural land runoff and to evaluate the ability of the native microbial community to resist disturbance related changes as a proxy for freshwater ecosystem health. Freshwater sediments were collected seasonally over 1 year in Kewaunee County, Wisconsin, a region impacted by concentrated dairy cattle farming, manure fertilization, and associated agricultural runoff which together serve as a press disturbance. Using 16S rRNA gene amplicon sequencing, we found that sediments in locations strongly impacted by intensive agriculture contain significantly higher abundances (p < 0.01) of the genera Thiobacillus, Methylotenera, Crenotrhix, Nitrospira, and Rhodoferax compared to reference sediments, and functions including nitrate reduction, nitrite reduction, and nitrogen respiration are significantly higher (p < 0.05) at locations in close proximity to large farms. Nine species-level potential human pathogens were identified in riverine sediments including Acinetobacer lwoffi and Arcobacter skirrowii, two pathogens associated with the cattle microbiome. Microbial community composition at locations in close proximity to intensive agriculture was not resistant nor resilient to agricultural runoff disturbance within 5 months post-disturbance but did reach a new, stable microbial composition. From this data, we conclude that sediment microbial community composition is sensitive and shifts in response to chemical and microbial pollution from intensive agriculture, has a low capacity to resist infiltration by non-native, harmful bacteria and, overall, the natural buffering capacity of freshwater ecosystems is unable to fully resist the impacts from agricultural press disturbance.

Antimicrobial Resistance Traits of Escherichia coli Isolated from Dairy Manure and Freshwater Ecosystems Are Similar to One Another but Differ from Associated Clinical Isolates.

Antimicrobial resistance (AMR) is a prevalent global health problem across human and veterinary medicine. The One Health approach to AMR is necessary to mitigate transmission between sources of resistance and decrease the spread of resistant bacteria among humans, animals, and the environment. Our primary goal was to identify associations in resistance traits between Escherichia coli isolated from clinical (n = 103), dairy manure (n = 65), and freshwater ecosystem (n = 64) environments within the same geographic location and timeframe. Clinical E. coli isolates showed the most phenotypic resistance (47.5%), followed by environmental isolates (15.6%) and manure isolates (7.7%), with the most common resistances to ampicillin, ampicillin-sulbactam, and cefotaxime antibiotics. An isolate subset was screened for extended spectrum beta-lactamase (ESBL) production resulting in the identification of 35 ESBL producers. The most common ESBL gene identified was blaTEM-1. Additionally, we found nine different plasmid replicon types including IncFIA-FIB, which were frequently associated with ESBL producer isolates. Molecular phylotyping revealed a significant portion of clinical E. coli were associated with phylotype B2, whereas manure and environmental isolates were more diverse. Manure and environmental isolates were significantly different from clinical isolates based on analyzed traits, suggesting more transmission occurs between these two sources in the sampled environment.