Charles W. Knapp

Evidence of Increasing Antibiotic Resistance Gene Abundances in Archived Soils since 1940

Mass production and use of antibiotics and antimicrobials in medicine and agriculture have existed for over 60 years, and has substantially benefited public health and agricultural productivity throughout the world. However, there is growing evidence that resistance to antibiotics (AR) is increasing both in benign and pathogenic bacteria, posing an emerging threat to public and environmental health in the future. Although evidence has existed for years from clinical data of increasing AR, almost no quantitative environmental data exist that span increased industrial antibiotic production in the 1950s to the present; i.e., data that might delineate trends in AR potentially valuable for epidemiological studies. To address this critical knowledge gap, we speculated that AR levels might be apparent in historic soil archives as evidenced by antibiotic resistance gene (ARG) abundances over time. Accordingly, DNA was extracted from five long-term soil-series from different locations in The Netherlands that spanned 1940 to 2008, and 16S rRNA gene and 18 ARG abundances from different major antibiotic classes were quantified. Results show that ARG from all classes of antibiotics tested have significantly increased since 1940, but especially within the tetracyclines, with some individual ARG being >15 times more abundant now than in the 1970s. This is noteworthy because waste management procedures have broadly improved and stricter rules on nontherapeutic antibiotic use in agriculture are being promulgated. Although these data are local to The Netherlands, they suggest basal environmental levels of ARG still might be increasing, which has implications to similar locations around the world.

Experimental demonstration of chaotic instability in biological nitrification

Biological nitrification (that is, NH3 → NO2− → NO3−) is a key reaction in the global nitrogen cycle (N-cycle); however, it is also known anecdotally to be unpredictable and sometimes fails inexplicably. Understanding the basis of unpredictability in nitrification is critical because the loss or impairment of this function might influence the balance of nitrogen in the environment and also has biotechnological implications. One explanation for unpredictability is the presence of chaotic behavior; however, proving such behavior from experimental data is not trivial, especially in a complex microbial community. Here, we show that chaotic behavior is central to stability in nitrification because of a fragile mutualistic relationship between ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), the two major guilds in nitrification. Three parallel chemostats containing mixed microbial communities were fed complex media for 207 days, and nitrification performance, and abundances of AOB, NOB, total bacteria and protozoa were quantified over time. Lyapunov exponent calculations, supported by surrogate data and other tests, showed that all guilds were sensitive to initial conditions, suggesting broad chaotic behavior. However, NOB were most unstable among guilds and displayed a different general pattern of instability. Further, NOB variability was maximized when AOB were most unstable, which resulted in erratic nitrification including significant NO2− accumulation. We conclude that nitrification is prone to chaotic behavior because of a fragile AOB–NOB mutualism, which must be considered in all systems that depend on this critical reaction.

Antibiotic resistance gene abundances associated with waste discharges to the Almendares River near Havana, Cuba

Considerable debate exists over the primary cause of increased antibiotic resistance (AR) worldwide. Evidence suggests increasing AR results from overuse of antibiotics in medicine and therapeutic and nontherapeutic applications in agriculture. However, pollution also can influence environmental AR, particularly associated with heavy metal, pharmaceutical, and other waste releases, although the relative scale of the “pollution” contribution is poorly defined, which restricts targeted mitigation efforts. The question is “where to study and quantify AR from pollution versus other causes to best understand the pollution effect”. One useful site is Cuba because industrial pollution broadly exists; antibiotics are used sparingly in medicine and agriculture; and multiresistant bacterial infections are increasing in clinical settings without explanation. Within this context, we quantified 13 antibiotic resistance genes (ARG; indicators of AR potential), 6 heavy metals, 3 antibiotics, and 17 other organic pollutants at 8 locations along the Almendares River in western Havana at sites bracketing known waste discharge points, including a large solid waste landfill and various pharmaceutical factories. Significant correlations (p < 0.05) were found between sediment ARG levels, especially for tetracyclines and β-lactams (e.g., tet(M), tet(O), tet(Q), tet(W), blaOXA), and sediment Cu and water column ampicillin levels in the river. Further, sediment ARG levels increased by up to 3 orders of magnitude downstream of the pharmaceutical factories and were highest where human population densities also were high. Although explicit links are not shown, results suggest that pollution has increased background AR levels in a setting where other causes of AR are less prevalent.

Antibiotic Resistance Gene Abundances Correlate with Metal and Geochemical Conditions in Archived Scottish Soils

The vast majority of antibiotic resistant genes (ARG) acquired by human pathogens have originated from the natural environment. Therefore, understanding factors that influence intrinsic levels of ARG in the environment could be epidemiologically significant. The selection for metal resistance often promotes AR in exposed organisms; however, the relationship between metal levels in nature and the intrinsic presence of ARG has not been fully assessed. Here, we quantified, using qPCR, the abundance of eleven ARG and compared their levels with geochemical conditions in randomly selected soils from a Scottish archive. Many ARG positively correlated with soil copper levels, with approximately half being highly significant (p<0.05); whereas chromium, nickel, lead, and iron also significantly correlated with specific ARG. Results show that geochemical metal conditions innately influence the potential for AR in soil. We suggest soil geochemical data might be used to estimate baseline gene presence on local, regional and global scales within epidemiological risk studies related to AR transmission from the environment.

Quantification of Tetracycline Resistance Genes in Feedlot Lagoons by Real-Time PCR

ABSTRACT A new real-time PCR method is presented that detects and quantifies three tetracycline resistance (Tcr) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tcr gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tcr genes present in seven different cattle feedlot lagoons, to validate the method, and to assess whether resistance gene concentrations correlate with free-tetracycline levels in lagoon waters. The method proved to be sensitive across a wide range of gene concentrations and provided consistent and reproducible results from complex lagoon water samples. The log10 of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r2 = 0.50, P < 0.001; n = 18), with the geometric means of individual resistance concentrations ranging from 4- to 8.3-fold greater in lagoon samples with above-median tetracycline levels (>1.95 μg/liter by enzyme-linked immunosorbent assay techniques) than in below-median lagoon samples. Of the three Tcr genes tested, tet(W) and tet(Q) were more commonly found in lagoon water samples. Successful development of this real-time PCR assay will permit other studies quantifying Tcr gene numbers in environmental and other samples.

Methane monooxygenase gene expression mediated by methanobactin in the presence of mineral copper sources

Methane is a major greenhouse gas linked to global warming; however, patterns of in situ methane oxidation by methane-oxidizing bacteria (methanotrophs), natures main biological mechanism for methane suppression, are often inconsistent with laboratory predictions. For example, one would expect a strong relationship between methanotroph ecology and Cu level because methanotrophs require Cu to sustain particulate methane monooxygenase (pMMO), the most efficient enzyme for methane oxidation. However, no correlation has been observed in nature, which is surprising because methane monooxygenase (MMO) gene expression has been unequivocally linked to Cu availability. Here we provide a fundamental explanation for this lack of correlation. We propose that MMO expression in nature is largely controlled by solid-phase Cu geochemistry and the relative ability of Cu acquisition systems in methanotrophs, such as methanobactins (mb), to obtain Cu from mineral sources. To test this hypothesis, RT-PCR expression assays were developed for Methylosinus trichosporium OB3b (which produces mb) to quantify pMMO, soluble MMO (the alternate MMO expressed when Cu is “unavailable”), and 16S-rRNA gene expression under progressively more stringent Cu supply conditions. When Cu was provided as CuCl2, pMMO transcript levels increased significantly consistent with laboratory work. However, when Cu was provided as Cu-doped iron oxide, pMMO transcript levels increased only when mb was also present. Finally, when Cu was provided as Cu-doped borosilicate glass, pMMO transcription patterns varied depending on the ambient mb:Cu supply ratio. Cu geochemistry clearly influences MMO expression in terrestrial systems, and, as such, local Cu mineralogy might provide an explanation for methane oxidation patterns in the natural environment.

Effects of biochar and activated carbon amendment on maize growth and the uptake and measured availability of polycyclic aromatic hydrocarbons (PAHs) and potentially toxic elements (PTEs).

With the aim of investigating the effects of carbonaceous sorbent amendment on plant health and end point contaminant bioavailability, plant experiments were set up to grow maize (Zea mays) in soil contaminated with polycyclic aromatic hydrocarbons (PAHs) and metals. Maize and pine derived biochars, as well as a commercial grade activated carbon, were used as amendments. Plant growth characteristics, such as chlorophyll content and shoot to root biomass, improved with sorbent amendment to varying extents and contaminant uptake to shoots was consistently reduced in amended soils. By further defining the conditions in which sorbent amended soils successfully reduce contaminant bioavailability and improve plant growth, this work will inform field scale remediation efforts.

Correlations between in situ denitrification activity and nir-gene abundances in pristine and impacted prairie streams.

Denitrification is a process that reduces nitrogen levels in headwaters and other streams. We compared nirS and nirK abundances with the absolute rate of denitrification, the longitudinal coefficient of denitrification (i.e., Kden, which represents optimal denitrification rates at given environmental conditions), and water quality in seven prairie streams to determine if nir-gene abundances explain denitrification activity. Previous work showed that absolute rates of denitrification correlate with nitrate levels; however, no correlation has been found for denitrification efficiency, which we hypothesise might be related to gene abundances. Water-column nitrate and soluble-reactive phosphorus levels significantly correlated with absolute rates of denitrification, but nir-gene abundances did not. However, nirS and nirK abundances significantly correlated with Kden, as well as phosphorus, although no correlation was found between Kden and nitrate. These data confirm that absolute denitrification rates are controlled by nitrate load, but intrinsic denitrification efficiency is linked to nirS and nirK gene abundances.

Increased Waterborne blaNDM-1 Resistance Gene Abundances Associated with Seasonal Human Pilgrimages to the Upper Ganges River

Antibiotic resistance (AR) is often rooted in inappropriate antibiotic use, but poor water quality and inadequate sanitation exacerbate the problem, especially in emerging countries. An example is increasing multi-AR due to mobile carbapenemases, such as NDM-1 protein (coded by blaNDM-1 genes), which can produce extreme drug-resistant phenotypes. In 2010, NDM-1 positive isolates and blaNDM-1 genes were detected in surface waters across Delhi and have since been detected across the urban world. However, little is known about blaNDM-1 levels in more pristine locations, such as the headwaters of the Upper Ganges River. This area is of particular interest because it receives massive numbers of visitors during seasonal pilgrimages in May/June, including visitors from urban India. Here we quantified blaNDM-1 abundances, other AR genes (ARG), and coliform bacteria in sediments and water column samples from seven sites in the Rishikesh-Haridwar region of the Upper Ganges and five sites on the Yamuna River in Delhi to contrast blaNDM-1 levels and water quality conditions between season and region. Water quality in the Yamuna was very poor (e.g., anoxia at all sites), and blaNDM-1 abundances were high across sites in water (5.4 ± 0.4 log(blaNDM-1·mL–1); 95% confidence interval) and sediment (6.3 ± 0.7 log(blaNDM-1·mg–1)) samples from both seasons. In contrast, water column blaNDM-1 abundances were very low across all sites in the Upper Ganges in February (2.1 ± 0.6 log(blaNDM-1·mL–1)), and water quality was good (e.g., near saturation oxygen). However, per capita blaNDM-1 levels were 20 times greater in June in the Ganges water column relative to February, and blaNDM-1 levels significantly correlated with fecal coliform levels (r = 0.61; p = 0.007). Given that waste management infrastructure is limited in Rishikesh-Haridwar, data imply blaNDM-1 levels are higher in visitor’s wastes than local residents, which results in seasonally higher blaNDM-1 levels in the river. Pilgrimage areas without adequate waste treatment are possible “hot spots” for AR transmission, and waste treatment must be improved to reduce broader AR dissemination via exposed returning visitors.

Copper-Binding Properties and Structures of Methanobactins from Methylosinus trichosporium OB3b

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.