David W Graham | Newcastle University (original) (raw)

Papers by David W Graham

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

Applied Microbiology and Biotechnology, 2011

Environmental Monitoring and Assessment, 2008

Microbial biotechnology, Jan 15, 2015

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...

Environmental science & technology, Jan 11, 2015

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...

Frontiers in Microbiology, 2012

TrAC Trends in Analytical Chemistry, 2003

Proceedings of the National Academy of Sciences, 2012

Journal of Applied Microbiology, 2013

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.

Inorganic Chemistry, 2011

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.

Environmental Pollution, 2006

Environmental Health Perspectives, 2013

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.

Clinical Infectious Diseases, 2013

Applied Microbiology and Biotechnology, 2011

Applied and Environmental Microbiology, 2011

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

Applied Microbiology and Biotechnology, 2011

Environmental Monitoring and Assessment, 2008

Microbial biotechnology, Jan 15, 2015

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...

Environmental science & technology, Jan 11, 2015

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...

Frontiers in Microbiology, 2012

TrAC Trends in Analytical Chemistry, 2003

Proceedings of the National Academy of Sciences, 2012

Journal of Applied Microbiology, 2013

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.

Inorganic Chemistry, 2011

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.

Environmental Pollution, 2006

Environmental Health Perspectives, 2013

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.

Clinical Infectious Diseases, 2013

Applied Microbiology and Biotechnology, 2011

Applied and Environmental Microbiology, 2011

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

Water, 2019

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.