A means and method for treating bacterial infection, providing antioxidant activity, and chelatin... more A means and method for treating bacterial infection, providing antioxidant activity, and chelating copper using a copper binding compound produced by methanotrophic bacteria is described. The compound, known as methanobactin, is the first of a new class of antibiotics having gram-positive activity. Methanobactin has been sequenced, and its structural formula determined.
Effective domestic wastewater treatment is among our primary defenses against the dissemination o... more Effective domestic wastewater treatment is among our primary defenses against the dissemination of infectious waterborne disease. However, reducing the amount of energy used in treatment processes has become essential for the future. One low-energy treatment option is anaerobic− aerobic sequence (AAS) bioreactors, which use an anaerobic pretreatment step (e.g., anaerobic hybrid reactors) to reduce carbon levels, followed by some form of aerobic treatment. Although AAS is common in warm climates, it is not known how its compares to other treatment options relative to disease transmission, including its influence on antibiotic resistance (AR) in treated effluents. Here, we used metagenomic approaches to contrast the fate of antibiotic-resistant genes (ARG) in anaerobic, aerobic, and AAS bioreactors treating domestic wastewater. Five reactor configurations were monitored for 6 months, and treatment performance, energy use, and ARG abundance and diversity were compared in influents and effluents. AAS and aerobic reactors were superior to anaerobic units in reducing ARG-like sequence abundances, with effluent ARG levels of 29, 34, and 74 ppm (198 ppm influent), respectively. AAS and aerobic systems especially reduced aminoglycoside, tetracycline, and β-lactam ARG levels relative to anaerobic units, although 63 persistent ARG subtypes were detected in effluents from all systems (of 234 assessed). Sulfonamide and chloramphenicol ARG levels were largely unaffected by treatment, whereas a broad shift from target-specific ARGs to ARGs associated with multi-drug resistance was seen across influents and effluents. AAS reactors show promise for future applications because they can reduce more ARGs for less energy (32% less energy here), but all three treatment options have limitations and need further study.
Debate exists about whether agricultural versus medical antibiotic use drives increasing antibiot... more Debate exists about whether agricultural versus medical antibiotic use drives increasing antibiotic resistance (AR) across nature. Both sectors have been inconsistent at antibiotic stewardship, but it is unclear which sector has most influenced acquired AR on broad scales. Using qPCR and soils archived since 1923 at Askov Experimental Station in Denmark, we quantified four broad-spectrum β-lactam AR genes (ARG; bla TEM , bla SHV , bla OXA and bla CTX-M) and class-1 integron genes (int1) in soils from manured (M) versus inorganic fertilised (IF) fields. " Total " β-lactam ARG levels were significantly higher in M versus IF in soils post-1940 (paired-t test; p < 0.001). However, dominant individual ARGs varied over time; bla TEM and bla SHV between 1963 and 1974, bla OXA slightly later, and bla CTX-M since 1988. These dates roughly parallel first reporting of these genes in clinical isolates, suggesting ARGs in animal manure and humans are historically interconnected. Archive data further show when non-therapeutic antibiotic use was banned in Denmark, bla CTX-M levels declined in M soils, suggesting accumulated soil ARGs can be reduced by prudent antibiotic stewardship. Conversely, int1 levels have continued to increase in M soils since 1990, implying direct manure application to soils should be scrutinized as part of future stewardship programs. Mass use of antibiotics for treating infectious disease over the 20 th century has improved human health and agricultural productivity in the developed world, enhancing the quality of life of millions 1. However, antibiotic use, especially in medicine and agriculture, also has fuelled increasing acquired antibiotic resistance (AR) in exposed organisms to a point where many antibiotics are compromised; multi-resistance pathogens are common; and new antibiotic development has become uneconomical 2–4. Despite growing awareness of the AR crisis (e.g., ref. 5), debate continues over who and which activities are most responsible for increased AR in nature, largely because it is hard to link specific causative actions with explicit AR consequences 6,7. AR is ancient with VanX proto-resistance and multi-resistance genes being detected in ~30,000-year old DNA from permafrost 8. However, increased antibiotic use has clearly mobilised AR genes and accelerated bacterial AR evolution in strains not previously intrinsically resistant 9,10 , including human pathogens 11. Antibiotic overuse and poor water quality in some parts of the world have further altered the environmental resistome (i.e., the pool of all AR genes; ARG) 12,13 , which increases the probability of AR acquisition in any exposed bacteria 14. Despite the above, debate continues about the relative role of each sectoral driver of acquired AR: medicine, agriculture or environmental pollution. This debate often follows parochial bias, but it also stems from substantial difficulties in identifying root causes of detected AR in most scenarios. AR often results from acquisition or mutation of a defence gene, which allows an organism to better protect itself. However, phenotypic detection of AR is only observed when a strain is exposed to an antibiotic. Therefore, organisms might acquire the potential for AR (e.g., an ARG or mutation) in one place, but phenotypic AR only becomes apparent when the " next " antibiotic treatment fails, possibly great distances away 15. This reality has led many to falsely presume AR acquisition
Bacterial populations conditionally display nonlinear dynamic behaviour in bioreactors with stead... more Bacterial populations conditionally display nonlinear dynamic behaviour in bioreactors with steady inputs, which is often attributed to varying habitat conditions or shifting intracellular metabolic activity. However, mathematical modelling has predicted that such dynamics also might simply result from staggered birth, growth, and death events of groups of cells within the population, causing density oscillations and the cycling of live and dead cells within the system. To assess this prediction, laboratory experiments were performed on Escherichia coli strain K12-MG1655 grown in chemostats to first define fine-scale population dynamics over time (minutes) and then determine whether the dynamics correlate with live-dead cell cycles in the system. E. coli populations displayed consistent oscillatory behaviour in all experiments. However, close synchronisation between OD 600 and live-dead cell oscillations (within~33-38 min cycles) only became statistically significant (p<0.01) when pseudo-steady state operations approaching carrying capacity existed in the bioreactor. Specifically, live cells were highest at local OD 600 maxima and lowest at local OD 600 minima, showing that oscillations followed live-dead cell cycles as predicted by the model and also consistent with recent observations that death is non-stochastic in such populations. These data show that oscillatory dynamic behaviour is intrinsic in bioreactor populations, which has implications to process operations in biotechnology.
Molecular techniques have clear value for community characterization; however, almost all previou... more Molecular techniques have clear value for community characterization; however, almost all previous datasets are based upon non-molecular measurements and it is hard to compare "old" data with "new" data because few correlations have been made. Therefore, the purpose of this evaluation was to simultaneously use molecular and non-molecular methods within the same sampling program to determine how data compare. Three methods were used for characterizing microbial populations in Lake 260 (L260) at Experimental Lakes Area (Ontario, Canada) during a whole-lake exposure study. Methods included wholecell microscopic counts (for bacteria, cyanobacteria, algae and zooplankton), chlorophyll a, and small subunit (ssu)-rRNA hybridization using EUB-338, EUB-785, CYAN-785-a/b, EUCA-1379 and UNIV-1390 gene probes. Strong correlations were found between the EUB-785 probe signal and "bacteria minus cyanobacteria" direct counts, and the EUB-338 probe signal and "bacteria plus cyanobacteria" counts. Furthermore, the difference in probe signal between EUB-338 and EUB-785 (a presumptive signature for cyanobacteria and plastids) correlated with cyanobacterial direct counts and also with chlorophyll a. However, EUCA-1379 probe signal did not correlate with algae counts, and UNIV-1390 probe signal only correlated with total bacteria counts. Results suggest that, although ssu-rRNA methods are fast, reproducible, and specifically detect "viable" organisms, their use may be limited to non-eukaryotic populations unless new probes are developed that are more specific.
The incorporation of microbial diversity in design would ideally require predictive theory that w... more The incorporation of microbial diversity in design would ideally require predictive theory that would relate operational parameters to the numbers and distribution of taxa. Resource ratio-theory (RRT) might be one such theory. Based on Monod kinetics, it explains diversity in function of resource-ratio and richness. However, to be usable in biological engineered system, the growth parameters of all the bacteria under consideration and the resource supply and diffusion parameters for all the relevant nutrients should be determined. This is challenging, but plausible, at least for low diversity groups with simple resource requirements like the ammonia oxidizing bacteria (AOB). One of the major successes of RRT was its ability to explain the 'paradox of enrichment' which states that diversity first increases and then decreases with resource richness. Here, we demonstrate that this pattern can be seen in lab-scale-activated sludge reactors and parallel simulations that incorpora...
Many studies have quantified antibiotics and antibiotic resistance gene (ARG) levels in soils, su... more Many studies have quantified antibiotics and antibiotic resistance gene (ARG) levels in soils, surface waters, and waste treatment plants (WTPs). However, similar work on municipal solid waste (MSW) landfill leachates is limited, which is concerning because antibiotics disposal is often in the MSW stream. Here we quantified 20 sulfonamide (SA), quinolone (FQ), tetracycline (TC), macrolide (ML) and chloramphenicol (CP) antibiotics, and six ARGs (sul1, sul2, tetQ, tetM, ermB, and mefA) in MSW leachates from two Shanghai transfer stations (TS; sites Hulin (HL) and Xupu (XP)) and one landfill reservoir (LR) in April and July 2014. Antibiotic levels were higher in TS than LR leachates (985 ± 1965 ng/L vs. 345 ± 932 ng/L, n = 40), which was because of very high levels in the HL leachates (averaging at 1676 ± 5175 ng/L, n = 40). The mean MLs (3561 ± 8377 ng/L, n = 12), FQs (975 ± 1608 ng/L, n = 24), and SAs (402 ± 704 ng/L, n = 42) classes of antibiotics were highest across all samples. AR...
Numerous studies have quantified antibiotic resistance genes (ARG) in rivers and streams around t... more Numerous studies have quantified antibiotic resistance genes (ARG) in rivers and streams around the world, and significant relationships have been shown that relate different pollutant outputs and increased local ARG levels. However, most studies have not considered ambient flow conditions, which can vary dramatically especially in tropical countries. Here, ARG were quantified in water column and sediment samples during the dry-and wetseasons to assess how seasonal and other factors influence ARG transport down the Almendares River (Havana, Cuba). Eight locations were sampled and stream flow estimated during both seasons; qPCR was used to quantify four tetracycline, two erythromycin, and three beta-lactam resistance genes. ARG concentrations were higher in wet-season versus dry-season samples, which combined with higher flows, indicated much greater ARG transport downstream during the wet-season. However, water column ARG levels were more spatially variable in the dry-season than the wet-season, with the proximity of waste outfalls strongly influencing local ARG levels. Results confirm that dry-season sampling provides a useful picture of the impact of individual waste inputs on local stream ARG levels, whereas the majority of ARGs in this tropical river were transported downstream during the wet-season, possibly due to re-entrainment of ARG from sediments.
Alachlor (2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide) is a moderately toxic herbic... more Alachlor (2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide) is a moderately toxic herbicide that is frequently found in agriculturally impacted surface waters. To assess primary mechanism(s) that affect its fate in aquatic systems, two field experiments were performed using large mesocosms (n ¼ 39) and smaller microcosms with and without microbial inhibitors (n ¼ 16). The mesocosm experiment tested the effect of fertility conditions on alachlor fate, assessing alachlor disappearance over time under oligotrophic (total phosphorus (TP) o12 mg/L) through hypereutrophic (TP>80 mg/L) water conditions. Whereas, the microcosm experiment assessed alachlor fate in the presence of microbial inhibitors that selectively blocked eubacterial (chloroamphenicol, streptomycin, and penicillin combined), eukaryotic (cycloheximide), and universal (all inhibitors) microbial activity. First-order alachlor transformation rate coefficients ranged from 0.006 to 0.042 day À1 when microbial inhibitors were not present (halflives from 16 to 122 days) with the highest rates occurring in hypereutrophic waters. Statistics indicated that mean TP, and universal and eubacterial small sub-unit rRNA level most closely correlated with transformation rate. Further, the inhibitor study indicated that alachlor transformation was biotic (>90%), but that high transformation rates only occurred when eubacterial and eukaryotic domains were both metabolically active. Our results confirm that alachlor transformation is primarily biotic; however, efficient biotransformation only occurs when both major microbial domains in aerobic systems are active. r
Proceedings of the National Academy of Sciences, 2012
Methane-oxidizing bacteria are nature's primary biological mechanism for suppressing atmospheric ... more Methane-oxidizing bacteria are nature's primary biological mechanism for suppressing atmospheric levels of the second-most important greenhouse gas via methane monooxygenases (MMOs). The copper-containing particulate enzyme is the most widespread and efficient MMO. Under low-copper conditions methane-oxidizing bacteria secrete the small copper-binding peptide methanobactin (mbtin) to acquire copper, but how variations in the structures of mbtins influence copper metabolism and species selection are unknown. Methanobactins have been isolated from Methylocystis strains M and hirsuta CSC1, organisms that can switch to using an iron-containing soluble MMO when copper is limiting, and the nonswitchover Methylocystis rosea. These mbtins are shorter, and have different amino acid compositions, than the characterized mbtin from Methylosinus trichosporium OB3b. A coordinating pyrazinedione ring in the Methylocystis mbtins has little influence on the Cu(I) site structure. The Methylocystis mbtins have a sulfate group that helps stabilize the Cu(I) forms, resulting in affinities of approximately 10 21 M −1 . The Cu(II) affinities vary over three orders of magnitude with reduction potentials covering approximately 250 mV, which may dictate the mechanism of intracellular copper release. Copper uptake and the switchover from using the ironcontaining soluble MMO to the copper-containing particulate enzyme is faster when mediated by the native mbtin, suggesting that the amino acid sequence is important for the interaction of mbtins with receptors. The differences in structures and properties of mbtins, and their influence on copper utilization by methaneoxidizing bacteria, have important implications for the ecology and global function of these environmentally vital organisms. C opper is an essential protein cofactor involved in many important cellular processes (1, 2), and copper-trafficking systems have been extensively studied (1, 3-8). Although copper uptake by eukaryotes is well defined (1, 4,, acquisition of this metal by prokaryotes remains poorly understood. Methane-oxidizing bacteria secrete the small copper-binding molecule methanobactin (mbtin) when copper is limiting (10-18), presumably for sequestration of this metal. These organisms have conditionally high requirements for copper , primarily for the active site (20) of the particulate methane monooxygenase (pMMO). Almost all known methane-oxidizing bacteria use pMMO for the consumption of methane , an important greenhouse gas. A subclass of "switchover" organisms exists that can also produce a less efficient iron-containing soluble MMO (sMMO) under copperdeficient conditions, with pMMO expression up-regulated in response to an increase in the copper-to-cell ratio .
To provide deeper insights into nitrification process within aerobic bioreactors containing suppl... more To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors). Three bench-scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5-day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (&amp;amp;gt;95%). However, when HRT was reduced to 2.5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia-oxidizing bacteria, AOB; nitrite-oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB-NOB activity were less apparent, resulting in incomplete nitrification. Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification. This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.
Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing... more Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing bacteria (methanotrophs) that have been linked to the substantial copper needs of these environmentally important microorganisms. The only characterized mbs are those from Methylosinus trichosporium OB3b and Methylocystis strain SB2. M. trichosporium OB3b produces a second mb (mb-Met), which is missing the C-terminal Met residue from the full-length form (FL-mb). The as-isolated copper-loaded mbs bind Cu(I). The absence of the Met has little influence on the structure of the Cu(I) site, and both molecules mediate switchover from the soluble iron methane mono-oxygenase to the particulate copper-containing enzyme in M. trichosporium OB3b cells. Cu(II) is reduced in the presence of the mbs under our experimental conditions, and the disulfide plays no role in this process. The Cu(I) affinities of these molecules are extremely high with values of (6-7) × 10(20) M(-1) determined at pH ≥ 8.0. The affinity for Cu(I) is 1 order of magnitude lower at pH 6.0. The reduction potentials of copper-loaded FL-mb and mb-Met are 640 and 590 mV respectively, highlighting the strong preference for Cu(I) and indicating different Cu(II) affinities for the two forms. Cleavage of the disulfide bridge results in a decrease in the Cu(I) affinity to ∼9 × 10(18) M(-1) at pH 7.5. The two thiolates can also bind Cu(I), albeit with much lower affinity (∼ 3 × 10(15) M(-1) at pH 7.5). The high affinity of mbs for Cu(I) is consistent with a physiological role in copper uptake and protection.
There is growing concern worldwide about the role of polluted soil and water environments in the ... more There is growing concern worldwide about the role of polluted soil and water environments in the development and dissemination of antibiotic resistance. Our aim in this study was to identify management options for reducing the spread of antibiotics and antibiotic-resistance determinants via environmental pathways, with the ultimate goal of extending the useful life span of antibiotics. We also examined incentives and disincentives for action. We focused on management options with respect to limiting agricultural sources; treatment of domestic, hospital, and industrial wastewater; and aquaculture. We identified several options, such as nutrient management, runoff control, and infrastructure upgrades. Where appropriate, a cross-section of examples from various regions of the world is provided. The importance of monitoring and validating effectiveness of management strategies is also highlighted. Finally, we describe a case study in Sweden that illustrates the critical role of communication to engage stakeholders and promote action. Environmental releases of antibiotics and antibiotic-resistant bacteria can in many cases be reduced at little or no cost. Some management options are synergistic with existing policies and goals. The anticipated benefit is an extended useful life span for current and future antibiotics. Although risk reductions are often difficult to quantify, the severity of accelerating worldwide morbidity and mortality rates associated with antibiotic resistance strongly indicate the need for action.
Bacterial populations conditionally display nonlinear dynamic behaviour in bioreactors with stead... more Bacterial populations conditionally display nonlinear dynamic behaviour in bioreactors with steady inputs, which is often attributed to varying habitat conditions or shifting intracellular metabolic activity. However, mathematical modelling has predicted that such dynamics also might simply result from staggered birth, growth, and death events of groups of cells within the population, causing density oscillations and the cycling of live and dead cells within the system. To assess this prediction, laboratory experiments were performed on Escherichia coli strain K12-MG1655 grown in chemostats to first define fine-scale population dynamics over time (minutes) and then determine whether the dynamics correlate with live-dead cell cycles in the system. E. coli populations displayed consistent oscillatory behaviour in all experiments. However, close synchronisation between OD 600 and live-dead cell oscillations (within~33-38 min cycles) only became statistically significant (p<0.01) when pseudo-steady state operations approaching carrying capacity existed in the bioreactor. Specifically, live cells were highest at local OD 600 maxima and lowest at local OD 600 minima, showing that oscillations followed live-dead cell cycles as predicted by the model and also consistent with recent observations that death is non-stochastic in such populations. These data show that oscillatory dynamic behaviour is intrinsic in bioreactor populations, which has implications to process operations in biotechnology.
In wastewater treatment plants, nitrifying systems are usually operated with elevated levels of a... more In wastewater treatment plants, nitrifying systems are usually operated with elevated levels of aeration to avoid nitrification failures. This approach contributes significantly to operational costs and the carbon footprint of nitrifying wastewater treatment processes. In this study, we tested the effect of aeration rate on nitrification by correlating ammonia oxidation rates with the structure of the ammonia-oxidizing bacterial (AOB) community and AOB abundance in four parallel continuous-flow reactors operated for 43 days. Two of the reactors were supplied with a constant airflow rate of 0.1 liter/min, while in the other two units the airflow rate was fixed at 4 liters/min. Complete nitrification was achieved in all configurations, though the dissolved oxygen (DO) concentration was only 0.5 ؎ 0.3 mg/liter in the low-aeration units. The data suggest that efficient performance in the low-DO units resulted from elevated AOB levels in the reactors and/or putative development of a mixotrophic AOB community. Denaturing gel electrophoresis and cloning of AOB 16S rRNA gene fragments followed by sequencing revealed that the AOB community in the low-DO systems was a subset of the community in the high-DO systems. However, in both configurations the dominant species belonged to the Nitrosomonas oligotropha lineage. Overall, the results demonstrated that complete nitrification can be achieved at low aeration in lab-scale reactors. If these findings could be extended to full-scale plants, it would be possible to minimize the operational costs and greenhouse gas emissions without risk of nitrification failure.
A means and method for treating bacterial infection, providing antioxidant activity, and chelatin... more A means and method for treating bacterial infection, providing antioxidant activity, and chelating copper using a copper binding compound produced by methanotrophic bacteria is described. The compound, known as methanobactin, is the first of a new class of antibiotics having gram-positive activity. Methanobactin has been sequenced, and its structural formula determined.
Effective domestic wastewater treatment is among our primary defenses against the dissemination o... more Effective domestic wastewater treatment is among our primary defenses against the dissemination of infectious waterborne disease. However, reducing the amount of energy used in treatment processes has become essential for the future. One low-energy treatment option is anaerobic− aerobic sequence (AAS) bioreactors, which use an anaerobic pretreatment step (e.g., anaerobic hybrid reactors) to reduce carbon levels, followed by some form of aerobic treatment. Although AAS is common in warm climates, it is not known how its compares to other treatment options relative to disease transmission, including its influence on antibiotic resistance (AR) in treated effluents. Here, we used metagenomic approaches to contrast the fate of antibiotic-resistant genes (ARG) in anaerobic, aerobic, and AAS bioreactors treating domestic wastewater. Five reactor configurations were monitored for 6 months, and treatment performance, energy use, and ARG abundance and diversity were compared in influents and effluents. AAS and aerobic reactors were superior to anaerobic units in reducing ARG-like sequence abundances, with effluent ARG levels of 29, 34, and 74 ppm (198 ppm influent), respectively. AAS and aerobic systems especially reduced aminoglycoside, tetracycline, and β-lactam ARG levels relative to anaerobic units, although 63 persistent ARG subtypes were detected in effluents from all systems (of 234 assessed). Sulfonamide and chloramphenicol ARG levels were largely unaffected by treatment, whereas a broad shift from target-specific ARGs to ARGs associated with multi-drug resistance was seen across influents and effluents. AAS reactors show promise for future applications because they can reduce more ARGs for less energy (32% less energy here), but all three treatment options have limitations and need further study.
Debate exists about whether agricultural versus medical antibiotic use drives increasing antibiot... more Debate exists about whether agricultural versus medical antibiotic use drives increasing antibiotic resistance (AR) across nature. Both sectors have been inconsistent at antibiotic stewardship, but it is unclear which sector has most influenced acquired AR on broad scales. Using qPCR and soils archived since 1923 at Askov Experimental Station in Denmark, we quantified four broad-spectrum β-lactam AR genes (ARG; bla TEM , bla SHV , bla OXA and bla CTX-M) and class-1 integron genes (int1) in soils from manured (M) versus inorganic fertilised (IF) fields. " Total " β-lactam ARG levels were significantly higher in M versus IF in soils post-1940 (paired-t test; p < 0.001). However, dominant individual ARGs varied over time; bla TEM and bla SHV between 1963 and 1974, bla OXA slightly later, and bla CTX-M since 1988. These dates roughly parallel first reporting of these genes in clinical isolates, suggesting ARGs in animal manure and humans are historically interconnected. Archive data further show when non-therapeutic antibiotic use was banned in Denmark, bla CTX-M levels declined in M soils, suggesting accumulated soil ARGs can be reduced by prudent antibiotic stewardship. Conversely, int1 levels have continued to increase in M soils since 1990, implying direct manure application to soils should be scrutinized as part of future stewardship programs. Mass use of antibiotics for treating infectious disease over the 20 th century has improved human health and agricultural productivity in the developed world, enhancing the quality of life of millions 1. However, antibiotic use, especially in medicine and agriculture, also has fuelled increasing acquired antibiotic resistance (AR) in exposed organisms to a point where many antibiotics are compromised; multi-resistance pathogens are common; and new antibiotic development has become uneconomical 2–4. Despite growing awareness of the AR crisis (e.g., ref. 5), debate continues over who and which activities are most responsible for increased AR in nature, largely because it is hard to link specific causative actions with explicit AR consequences 6,7. AR is ancient with VanX proto-resistance and multi-resistance genes being detected in ~30,000-year old DNA from permafrost 8. However, increased antibiotic use has clearly mobilised AR genes and accelerated bacterial AR evolution in strains not previously intrinsically resistant 9,10 , including human pathogens 11. Antibiotic overuse and poor water quality in some parts of the world have further altered the environmental resistome (i.e., the pool of all AR genes; ARG) 12,13 , which increases the probability of AR acquisition in any exposed bacteria 14. Despite the above, debate continues about the relative role of each sectoral driver of acquired AR: medicine, agriculture or environmental pollution. This debate often follows parochial bias, but it also stems from substantial difficulties in identifying root causes of detected AR in most scenarios. AR often results from acquisition or mutation of a defence gene, which allows an organism to better protect itself. However, phenotypic detection of AR is only observed when a strain is exposed to an antibiotic. Therefore, organisms might acquire the potential for AR (e.g., an ARG or mutation) in one place, but phenotypic AR only becomes apparent when the " next " antibiotic treatment fails, possibly great distances away 15. This reality has led many to falsely presume AR acquisition
Bacterial populations conditionally display nonlinear dynamic behaviour in bioreactors with stead... more Bacterial populations conditionally display nonlinear dynamic behaviour in bioreactors with steady inputs, which is often attributed to varying habitat conditions or shifting intracellular metabolic activity. However, mathematical modelling has predicted that such dynamics also might simply result from staggered birth, growth, and death events of groups of cells within the population, causing density oscillations and the cycling of live and dead cells within the system. To assess this prediction, laboratory experiments were performed on Escherichia coli strain K12-MG1655 grown in chemostats to first define fine-scale population dynamics over time (minutes) and then determine whether the dynamics correlate with live-dead cell cycles in the system. E. coli populations displayed consistent oscillatory behaviour in all experiments. However, close synchronisation between OD 600 and live-dead cell oscillations (within~33-38 min cycles) only became statistically significant (p<0.01) when pseudo-steady state operations approaching carrying capacity existed in the bioreactor. Specifically, live cells were highest at local OD 600 maxima and lowest at local OD 600 minima, showing that oscillations followed live-dead cell cycles as predicted by the model and also consistent with recent observations that death is non-stochastic in such populations. These data show that oscillatory dynamic behaviour is intrinsic in bioreactor populations, which has implications to process operations in biotechnology.
Molecular techniques have clear value for community characterization; however, almost all previou... more Molecular techniques have clear value for community characterization; however, almost all previous datasets are based upon non-molecular measurements and it is hard to compare "old" data with "new" data because few correlations have been made. Therefore, the purpose of this evaluation was to simultaneously use molecular and non-molecular methods within the same sampling program to determine how data compare. Three methods were used for characterizing microbial populations in Lake 260 (L260) at Experimental Lakes Area (Ontario, Canada) during a whole-lake exposure study. Methods included wholecell microscopic counts (for bacteria, cyanobacteria, algae and zooplankton), chlorophyll a, and small subunit (ssu)-rRNA hybridization using EUB-338, EUB-785, CYAN-785-a/b, EUCA-1379 and UNIV-1390 gene probes. Strong correlations were found between the EUB-785 probe signal and "bacteria minus cyanobacteria" direct counts, and the EUB-338 probe signal and "bacteria plus cyanobacteria" counts. Furthermore, the difference in probe signal between EUB-338 and EUB-785 (a presumptive signature for cyanobacteria and plastids) correlated with cyanobacterial direct counts and also with chlorophyll a. However, EUCA-1379 probe signal did not correlate with algae counts, and UNIV-1390 probe signal only correlated with total bacteria counts. Results suggest that, although ssu-rRNA methods are fast, reproducible, and specifically detect "viable" organisms, their use may be limited to non-eukaryotic populations unless new probes are developed that are more specific.
The incorporation of microbial diversity in design would ideally require predictive theory that w... more The incorporation of microbial diversity in design would ideally require predictive theory that would relate operational parameters to the numbers and distribution of taxa. Resource ratio-theory (RRT) might be one such theory. Based on Monod kinetics, it explains diversity in function of resource-ratio and richness. However, to be usable in biological engineered system, the growth parameters of all the bacteria under consideration and the resource supply and diffusion parameters for all the relevant nutrients should be determined. This is challenging, but plausible, at least for low diversity groups with simple resource requirements like the ammonia oxidizing bacteria (AOB). One of the major successes of RRT was its ability to explain the 'paradox of enrichment' which states that diversity first increases and then decreases with resource richness. Here, we demonstrate that this pattern can be seen in lab-scale-activated sludge reactors and parallel simulations that incorpora...
Many studies have quantified antibiotics and antibiotic resistance gene (ARG) levels in soils, su... more Many studies have quantified antibiotics and antibiotic resistance gene (ARG) levels in soils, surface waters, and waste treatment plants (WTPs). However, similar work on municipal solid waste (MSW) landfill leachates is limited, which is concerning because antibiotics disposal is often in the MSW stream. Here we quantified 20 sulfonamide (SA), quinolone (FQ), tetracycline (TC), macrolide (ML) and chloramphenicol (CP) antibiotics, and six ARGs (sul1, sul2, tetQ, tetM, ermB, and mefA) in MSW leachates from two Shanghai transfer stations (TS; sites Hulin (HL) and Xupu (XP)) and one landfill reservoir (LR) in April and July 2014. Antibiotic levels were higher in TS than LR leachates (985 ± 1965 ng/L vs. 345 ± 932 ng/L, n = 40), which was because of very high levels in the HL leachates (averaging at 1676 ± 5175 ng/L, n = 40). The mean MLs (3561 ± 8377 ng/L, n = 12), FQs (975 ± 1608 ng/L, n = 24), and SAs (402 ± 704 ng/L, n = 42) classes of antibiotics were highest across all samples. AR...
Numerous studies have quantified antibiotic resistance genes (ARG) in rivers and streams around t... more Numerous studies have quantified antibiotic resistance genes (ARG) in rivers and streams around the world, and significant relationships have been shown that relate different pollutant outputs and increased local ARG levels. However, most studies have not considered ambient flow conditions, which can vary dramatically especially in tropical countries. Here, ARG were quantified in water column and sediment samples during the dry-and wetseasons to assess how seasonal and other factors influence ARG transport down the Almendares River (Havana, Cuba). Eight locations were sampled and stream flow estimated during both seasons; qPCR was used to quantify four tetracycline, two erythromycin, and three beta-lactam resistance genes. ARG concentrations were higher in wet-season versus dry-season samples, which combined with higher flows, indicated much greater ARG transport downstream during the wet-season. However, water column ARG levels were more spatially variable in the dry-season than the wet-season, with the proximity of waste outfalls strongly influencing local ARG levels. Results confirm that dry-season sampling provides a useful picture of the impact of individual waste inputs on local stream ARG levels, whereas the majority of ARGs in this tropical river were transported downstream during the wet-season, possibly due to re-entrainment of ARG from sediments.
Alachlor (2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide) is a moderately toxic herbic... more Alachlor (2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide) is a moderately toxic herbicide that is frequently found in agriculturally impacted surface waters. To assess primary mechanism(s) that affect its fate in aquatic systems, two field experiments were performed using large mesocosms (n ¼ 39) and smaller microcosms with and without microbial inhibitors (n ¼ 16). The mesocosm experiment tested the effect of fertility conditions on alachlor fate, assessing alachlor disappearance over time under oligotrophic (total phosphorus (TP) o12 mg/L) through hypereutrophic (TP>80 mg/L) water conditions. Whereas, the microcosm experiment assessed alachlor fate in the presence of microbial inhibitors that selectively blocked eubacterial (chloroamphenicol, streptomycin, and penicillin combined), eukaryotic (cycloheximide), and universal (all inhibitors) microbial activity. First-order alachlor transformation rate coefficients ranged from 0.006 to 0.042 day À1 when microbial inhibitors were not present (halflives from 16 to 122 days) with the highest rates occurring in hypereutrophic waters. Statistics indicated that mean TP, and universal and eubacterial small sub-unit rRNA level most closely correlated with transformation rate. Further, the inhibitor study indicated that alachlor transformation was biotic (>90%), but that high transformation rates only occurred when eubacterial and eukaryotic domains were both metabolically active. Our results confirm that alachlor transformation is primarily biotic; however, efficient biotransformation only occurs when both major microbial domains in aerobic systems are active. r
Proceedings of the National Academy of Sciences, 2012
Methane-oxidizing bacteria are nature's primary biological mechanism for suppressing atmospheric ... more Methane-oxidizing bacteria are nature's primary biological mechanism for suppressing atmospheric levels of the second-most important greenhouse gas via methane monooxygenases (MMOs). The copper-containing particulate enzyme is the most widespread and efficient MMO. Under low-copper conditions methane-oxidizing bacteria secrete the small copper-binding peptide methanobactin (mbtin) to acquire copper, but how variations in the structures of mbtins influence copper metabolism and species selection are unknown. Methanobactins have been isolated from Methylocystis strains M and hirsuta CSC1, organisms that can switch to using an iron-containing soluble MMO when copper is limiting, and the nonswitchover Methylocystis rosea. These mbtins are shorter, and have different amino acid compositions, than the characterized mbtin from Methylosinus trichosporium OB3b. A coordinating pyrazinedione ring in the Methylocystis mbtins has little influence on the Cu(I) site structure. The Methylocystis mbtins have a sulfate group that helps stabilize the Cu(I) forms, resulting in affinities of approximately 10 21 M −1 . The Cu(II) affinities vary over three orders of magnitude with reduction potentials covering approximately 250 mV, which may dictate the mechanism of intracellular copper release. Copper uptake and the switchover from using the ironcontaining soluble MMO to the copper-containing particulate enzyme is faster when mediated by the native mbtin, suggesting that the amino acid sequence is important for the interaction of mbtins with receptors. The differences in structures and properties of mbtins, and their influence on copper utilization by methaneoxidizing bacteria, have important implications for the ecology and global function of these environmentally vital organisms. C opper is an essential protein cofactor involved in many important cellular processes (1, 2), and copper-trafficking systems have been extensively studied (1, 3-8). Although copper uptake by eukaryotes is well defined (1, 4,, acquisition of this metal by prokaryotes remains poorly understood. Methane-oxidizing bacteria secrete the small copper-binding molecule methanobactin (mbtin) when copper is limiting (10-18), presumably for sequestration of this metal. These organisms have conditionally high requirements for copper , primarily for the active site (20) of the particulate methane monooxygenase (pMMO). Almost all known methane-oxidizing bacteria use pMMO for the consumption of methane , an important greenhouse gas. A subclass of "switchover" organisms exists that can also produce a less efficient iron-containing soluble MMO (sMMO) under copperdeficient conditions, with pMMO expression up-regulated in response to an increase in the copper-to-cell ratio .
To provide deeper insights into nitrification process within aerobic bioreactors containing suppl... more To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors). Three bench-scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5-day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (&amp;amp;gt;95%). However, when HRT was reduced to 2.5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia-oxidizing bacteria, AOB; nitrite-oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB-NOB activity were less apparent, resulting in incomplete nitrification. Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification. This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.
Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing... more Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing bacteria (methanotrophs) that have been linked to the substantial copper needs of these environmentally important microorganisms. The only characterized mbs are those from Methylosinus trichosporium OB3b and Methylocystis strain SB2. M. trichosporium OB3b produces a second mb (mb-Met), which is missing the C-terminal Met residue from the full-length form (FL-mb). The as-isolated copper-loaded mbs bind Cu(I). The absence of the Met has little influence on the structure of the Cu(I) site, and both molecules mediate switchover from the soluble iron methane mono-oxygenase to the particulate copper-containing enzyme in M. trichosporium OB3b cells. Cu(II) is reduced in the presence of the mbs under our experimental conditions, and the disulfide plays no role in this process. The Cu(I) affinities of these molecules are extremely high with values of (6-7) × 10(20) M(-1) determined at pH ≥ 8.0. The affinity for Cu(I) is 1 order of magnitude lower at pH 6.0. The reduction potentials of copper-loaded FL-mb and mb-Met are 640 and 590 mV respectively, highlighting the strong preference for Cu(I) and indicating different Cu(II) affinities for the two forms. Cleavage of the disulfide bridge results in a decrease in the Cu(I) affinity to ∼9 × 10(18) M(-1) at pH 7.5. The two thiolates can also bind Cu(I), albeit with much lower affinity (∼ 3 × 10(15) M(-1) at pH 7.5). The high affinity of mbs for Cu(I) is consistent with a physiological role in copper uptake and protection.
There is growing concern worldwide about the role of polluted soil and water environments in the ... more There is growing concern worldwide about the role of polluted soil and water environments in the development and dissemination of antibiotic resistance. Our aim in this study was to identify management options for reducing the spread of antibiotics and antibiotic-resistance determinants via environmental pathways, with the ultimate goal of extending the useful life span of antibiotics. We also examined incentives and disincentives for action. We focused on management options with respect to limiting agricultural sources; treatment of domestic, hospital, and industrial wastewater; and aquaculture. We identified several options, such as nutrient management, runoff control, and infrastructure upgrades. Where appropriate, a cross-section of examples from various regions of the world is provided. The importance of monitoring and validating effectiveness of management strategies is also highlighted. Finally, we describe a case study in Sweden that illustrates the critical role of communication to engage stakeholders and promote action. Environmental releases of antibiotics and antibiotic-resistant bacteria can in many cases be reduced at little or no cost. Some management options are synergistic with existing policies and goals. The anticipated benefit is an extended useful life span for current and future antibiotics. Although risk reductions are often difficult to quantify, the severity of accelerating worldwide morbidity and mortality rates associated with antibiotic resistance strongly indicate the need for action.
Bacterial populations conditionally display nonlinear dynamic behaviour in bioreactors with stead... more Bacterial populations conditionally display nonlinear dynamic behaviour in bioreactors with steady inputs, which is often attributed to varying habitat conditions or shifting intracellular metabolic activity. However, mathematical modelling has predicted that such dynamics also might simply result from staggered birth, growth, and death events of groups of cells within the population, causing density oscillations and the cycling of live and dead cells within the system. To assess this prediction, laboratory experiments were performed on Escherichia coli strain K12-MG1655 grown in chemostats to first define fine-scale population dynamics over time (minutes) and then determine whether the dynamics correlate with live-dead cell cycles in the system. E. coli populations displayed consistent oscillatory behaviour in all experiments. However, close synchronisation between OD 600 and live-dead cell oscillations (within~33-38 min cycles) only became statistically significant (p<0.01) when pseudo-steady state operations approaching carrying capacity existed in the bioreactor. Specifically, live cells were highest at local OD 600 maxima and lowest at local OD 600 minima, showing that oscillations followed live-dead cell cycles as predicted by the model and also consistent with recent observations that death is non-stochastic in such populations. These data show that oscillatory dynamic behaviour is intrinsic in bioreactor populations, which has implications to process operations in biotechnology.
In wastewater treatment plants, nitrifying systems are usually operated with elevated levels of a... more In wastewater treatment plants, nitrifying systems are usually operated with elevated levels of aeration to avoid nitrification failures. This approach contributes significantly to operational costs and the carbon footprint of nitrifying wastewater treatment processes. In this study, we tested the effect of aeration rate on nitrification by correlating ammonia oxidation rates with the structure of the ammonia-oxidizing bacterial (AOB) community and AOB abundance in four parallel continuous-flow reactors operated for 43 days. Two of the reactors were supplied with a constant airflow rate of 0.1 liter/min, while in the other two units the airflow rate was fixed at 4 liters/min. Complete nitrification was achieved in all configurations, though the dissolved oxygen (DO) concentration was only 0.5 ؎ 0.3 mg/liter in the low-aeration units. The data suggest that efficient performance in the low-DO units resulted from elevated AOB levels in the reactors and/or putative development of a mixotrophic AOB community. Denaturing gel electrophoresis and cloning of AOB 16S rRNA gene fragments followed by sequencing revealed that the AOB community in the low-DO systems was a subset of the community in the high-DO systems. However, in both configurations the dominant species belonged to the Nitrosomonas oligotropha lineage. Overall, the results demonstrated that complete nitrification can be achieved at low aeration in lab-scale reactors. If these findings could be extended to full-scale plants, it would be possible to minimize the operational costs and greenhouse gas emissions without risk of nitrification failure.
Chapter in Heritage 2010: Heritage and Sustainable Development, Chapter: The resilience and care of ancient stone monuments in changing environments, Editors: R Ameda, S Lira, C Pinheiro, 2010
Ancient stone monuments (ASMs), such as standing stones and rock art panels, are powerful and ico... more Ancient stone monuments (ASMs), such as standing stones and rock art panels, are powerful and iconic expressions of Britain's rich prehistoric past that have major economic and tourism value. However, ASMs are under pressure due to increasing anthropogenic exposure and changing climatic conditions, which accelerate their rates of disrepair. Although scientific data exists on the integrity of stone monuments, most applies to "built" systems; therefore, additional work specific to ASMs in the countryside is needed to develop better-informed safeguarding strategies. Here, we use Neolithic and Bronze Age rock art panels across Northern England as a case study for delineating ASM management actions required to enhance monument preservation. The state of the rock art is described first, including factors that led to current conditions. Rock art management approaches then are described within the context of future environments, which models suggest to be more dynamic and locally variable. Finally, a Condition Assessment and Risk Evaluation (CARE) scheme is proposed to help prioritise interventions; an example of which is provided based on stone deterioration at Petra in Jordon. We conclude that more focused scientific and behavioural data, specific to deterioration mechanisms, are required for an ASM CARE scheme to be successful.
Globally increasing antibiotic resistance (AR) will only be reversed through a suite of multidisc... more Globally increasing antibiotic resistance (AR) will only be reversed through a suite of multidisciplinary actions (One Health), including more prudent antibiotic use and improved sanitation on international scales. Relative to sanitation, advanced technologies exist that reduce AR in waste releases, but such technologies are expensive, and a strategic approach is needed to prioritize more affordable mitigation options, especially for Low- and Middle-Income Countries (LMICs). Such an approach is proposed here, which overlays the incremental cost of different sanitation options and their relative benefit in reducing AR, ultimately suggesting the “next-most-economic” options for different locations. When considering AR gene fate versus intervention costs, reducing open defecation (OD) and increasing decentralized secondary wastewater treatment, with condominial sewers, will probably have the greatest impact on reducing AR, for the least expense. However, the best option for a given country depends on the existing sewerage infrastructure. Using Southeast Asia as a case study and World Bank/WHO/UNICEF data, the approach suggests that Cambodia and East Timor should target reducing OD as a national priority. In contrast, increasing decentralized secondary treatment is well suited to Thailand, Vietnam and rural Malaysia. Our approach provides a science-informed starting point for decision-makers, for prioritising AR mitigation interventions; an approach that will evolve and refine as more data become available.
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Papers by David W Graham