Antibiotic Resistance Genes Contamination Research Articles

Occurrence and distribution of antibiotic resistance genes in lakes and reservoirs from water-receiving area of Eastern Route of the South-to-North Water Diversion Project, Northern China

Abstract To characterize antibiotic resistance genes (ARGs) in surface waterbodies in Shandong Province, and to understand the occurrence, diversity and resistance mechanism of ARGs, nine aquatic ecosystems were selected and evaluated regarding occurrence of ARGs in the period from 2018 to 2019. Thirty-three types and 242 subtypes of ARGs were detected in the nine waterbodies with the most subtypes in Wohushan (242) and Jihongtan (235), and the fewest ARGs in Datun (213) and Mishan (221). Among the top nine ARG types, MacB, BcrA, CarA and TetW were the dominant ARGs in these samples. Four groups of the study sites were distinguished by cluster analysis, indicating different distribution characteristics of ARGs in these waterbodies, and regional differences in ARG contamination. Efflux pumps were the predominant resistance mechanism in all the waterbodies in terms of both subtype number (49.2%) and abundance (70.4%), followed by operon, antibiotic inactivation and others. Our findings provide comprehensive information to better understand the contamination level of ARGs on a local level as well as for global considerations, and may be practically helpful for the prevention and control of ARG pollution.

Family livestock waste: An ignored pollutant resource of antibiotic resistance genes.

The random discharge of livestock waste from family farms without utilization and treatment has caused great pressure on the rural ecological environment and gravely increased the environmental pollution. In this study, we targeted 26 family livestock farms to assess the occurrence characteristics of antibiotic resistance genes (ARGs) in livestock waste and its receiving farmland environment in Erhai Lake basin of China by real-time fluorescence quantitative PCR. The results showed that various common ARGs and some high-risk ARGs (i.e., blaampC, blaOXA-1 and blaTEM-1) were prevalent in family livestock waste, and the pollution of tetracycline resistance genes was the most serious in these family livestock farms. Meanwhile, we also found that the ARG levels were higher in family chicken farms than that in pig and cattle farms, and ARGs pollution in layer waste and sow waste was more severe than that in broiler waste and piglet/fattening pig waste, respectively. Troublesomely, significant ARGs levels could be discharged via manure application, further causing the increase of ARGs abundance in soil environment (approximately 11-36 times). This study demonstrated the high prevalence and severity of ARGs contamination in family livestock farms, also emphasizing that family livestock waste was a non-ignored important pollutant resource of ARGs in the environment.

Manure fertilization increase antibiotic resistance in soils from typical greenhouse vegetable production bases, China

A large quantity of manure is applied in greenhouse vegetable production (GVP) soils, while manure fertilization often leads to the proliferation of antibiotic resistance genes (ARGs) in soils. However, comprehensive study on the effects of different types of manure on ARGs in GVP soils remains unknown, and the baseline level of ARGs in GVP soil is poorly quantified. This study conducted a comprehensive survey of ARGs in GVP soils using high-throughput quantitative PCR. We found elevated ARG diversity and absolute abundance in fertilized soil, whereas no significant difference in soil ARGs amended with different types of manure. Redundancy analysis indicated that the change of bacterial community compositions and environmental factors contributed partially to the shift in ARG profiles. Bipartite network analysis indicated that one ARG was detected in non-manured soils, while 50 ARGs and 4 mobile gene elements were exclusively detected in fertilized soils, suggesting introduction of ARGs from manure into soils largely explained the increased ARG diversity in fertilized soil. By comparison of ARG absolute abundance between manured and non-manured soil, we estimated the typical level of ARG absolute abundance in non-manured soil, which provided the first rough baseline level of ARGs to assess ARG contamination in GVP soils.

Contamination of antibiotic resistance genes (ARGs) in a typical marine aquaculture farm: source tracking of ARGs in reared aquatic organisms

Although the prevalence and concentrations of antibiotic resistance genes (ARGs) in aquaculture is receiving increasing scientific interest, there is little understanding of the direct sources and dissemination pathways of ARGs in marine aquaculture-reared organisms. This study investigated the dynamics of ARGs and the bacterial community throughout the rearing period in a typical marine aquaculture farm in South China. The results demonstrated that sul1 and qnrD were predominant in the sediment, and qnrD and qnrA were predominant in the intestinal tracts of shrimps. Network analysis showed that the chemical oxygen demand, total organic carbon, dissolved organic carbon, suspended solids, and total phosphorus were positively correlated with the predominant ARGs. The results of the network and source tracking analyses indicate that environmental factors and the bacterial community may drive the dissemination of ARGs dissemination in the environment and in shrimp reared by marine aquaculture, and sediment is the most direct and important medium in this dissemination. These results aid in improving our understanding of the sources, level, and dissemination of ARGs in marine aquaculture.

Occurrence and distribution of antibiotic resistance genes in the water and sediments of Qingcaosha Reservoir, Shanghai, China

BackgroundQingcaosha Reservoir is China’s largest river impoundment and an important drinking water resource for Shanghai city. Although antibiotics contamination in the reservoir has been reported, little is known about the presence of antibiotic resistance genes (ARGs). In this study, the occurrence and distribution of 12 ARGs and the class 1 integron gene (intI-1) in water and sediments collected from the reservoir were investigated for 1 year.ResultsThe 12 ARGs were detected in both water and sediment samples, and no significant temporal or spatial variations were observed. Sulfonamide resistance genes were predominant in the reservoir with a detection frequency of 100%. Statistical analysis indicated a positive correlation in relative abundance between some ARGs such as sul1, sul2, and intI-1. Most ARGs were negatively correlated with total nitrogen and positively correlated with chemical oxygen demand and chlorophyll-a.ConclusionIn general, ARGs were found to be prevalent in Qingcaosha Reservoir, among which sulfonamide resistance genes were the most dominant and tetB was the least. intI-1 may facilitate the proliferation and propagation of some ARGs, especially sul1 and sul2. The abundance of ARGs was well correlated with aquatic environmental factors in water, providing potential clues for the control of ARG contamination.

Dynamics of Antibiotic Resistance Genes During the Municipal Solid Waste Leachate Treatment

Antibiotic resistance genes (ARGs) are considered to be emerging environmental contaminants. ARGs are frequently detected in municipal solid waste landfill and its leachate. In this study, high-throughput quantitative PCR techniques were used to investigate the dynamics of ARGs during the treatment process of landfill leachate. The results showed that ARGs in leachate were diverse and abundant, and leachate treatment could effectively reduce their abundance; the absolute abundance of ARGs was reduced to 9.2×1010 copies·L-1, which was five orders of magnitude less than the abundance of the influent. However, the range and abundance of antibiotic resistance genes in the leachate effluents were still high compared to the natural environment. Therefore, the secondary contamination of ARGs might be caused by leachate being directly discharged into municipal sewage system. In this regard, ecological and environmental risk is an inconvenient truth. Furthermore, strong correlations indicate that heavy metals and mobile genetic elements (MGEs) in landfill leachate might influence the transfer of ARGs.

The occurrence of sulfonamide and quinolone resistance genes at the Fildes Peninsula in Antarctica.

Antibiotic resistance pollution is globalizing. However, little quantitative data exists regarding the occurrence of antibiotic resistance genes (ARGs) in Antarctica, which is central to assessing the level of global contamination of ARGs. Soil, sediment, and animal feces were sampled from 12 sites in the Fildes Peninsula in January and February 2017. The occurrence and distribution of qnrS, sul1 and sul2 were investigated. The results showed that sul1 was the predominant ARG and that all the ARGs were detected in animal feces. The total absolute abundance of the ARGs in animal feces (5.86 × 107 copies) was the highest. Strong and positive correlations between sul1 and int1 were found, indicating that int1 might play an important role in the creation and spread of ARGs. This data and the analysis are critical for filling the data gap regarding ARGs in Antarctica and for improving understanding of the globalization of antibiotic resistance pollution.

Cleavage and transformation inhibition of extracellular antibiotic resistance genes by graphene oxides with different lateral sizes

Due to excessive consumption of antibiotics, antibiotic resistance genes (ARGs) become a ubiquitous pollutant in aquatic environments. Graphene oxide (GO), an emerging 2D nanomaterial, was used for potential control of ARG contamination in the present work. We systematically investigated the interaction of GOs with Kanamycin resistance gene (aphA)-containing plasmid DNA, and related the inhibition of ARG transformation by GOs. Four GOs with different lateral sizes (1.0, 0.60, 0.17, and 0.08 μm2) were prepared, and defined as GO1.0, GO0.60, GO0.17 and GO0.08. It is found that all the four GOs could effectively intercalate into plasmid DNA, and the intercalation abilities followed the order: GO0.08 > GO0.17 > GO0.60 > GO1.0. Based on circular dichroism (CD) spectrum analysis, all GOs disturbed the base stacking mode and double helix structure of DNA, which is positively related to the intercalation activities of GOs. For GO0.08 at 25 μg/mL, the supercoiled plasmid DNA was partially cleaved, and the nicked and linear structures were observed based on agarose gel electrophoresis analysis. Moreover, the amplification and transformation of aphA gene were both inhibited due to GO-plasmid DNA interactions, and the inhibition was stronger with increasing GO concentrations and smaller lateral sizes. The inhibition of aphA transformation after GO0.08 (25 μg/mL) exposure achieved 50%. The size-dependent interaction of GOs with ARGs-containing plasmid DNA will be useful for guiding the environmental applications of GOs in reducing extracellular ARG transformation.

Distribution, diffuse, and removal of tetracyclines and sulfonamide antibiotic resistance genes in wastewater treatment plant: A review.

The contamination of antibiotic resistance genes (ARGs) caused by abuse of antibiotics has attracted more and more attention. Due to their low price, tetracyclines and sulfonamides are widely used. The plenty of residual tetracyclines and sulfonamides is discharged into wastewater treatment plant (WWTPs), with consequent ARGs pollution. To understand the current status of ARGs contamination and removal efficiency, we summarized the distribution and spread mechanism of tetracyclines and sulfonamides ARGs, and further emphasized the ARGs removal efficiency across different treatment technologies. Based on the current knowledge and lack of ARGs, future work were proposed, such as investigating ARGs contamination in WWTPs, improving ARGs removal technologies, exploring spread mechanisms of ARGs.

Synergistic effects of key parameters on the fate of antibiotic resistance genes during swine manure composting

Livestock manure is a reservoir of antibiotic resistance genes (ARGs), posing a potential risk to environment and human health. However, there has been no optimization study about the comprehensive composting treatment for livestock manure ARGs based on multiple operation factors. In this study, anaerobic composting of swine manure in light was conducted under different combined conditions of composting time, temperature, water content, pH, heavy metal passivators and wheat straw. The diversity and relative abundance of ARGs in the compost were detected using high throughput quantitative real-time PCR, and the concentrations of antibiotics and heavy metals were determined. The results showed that under the optimized conditions (composting time, 30 d; temperature, 50 °C; water content, 50%; pH 9.0; heavy metal passivators and wheat straw), compared with the control, the detected number of ARGs and mobile genetic elements in the compost was reduced by 45% and 27.3%, and their relative abundance decreased by 33.9% and 36.9%, respectively. Moreover, the exchangeable heavy metal content of the compost declined by 34.7–57.1%, and the antibiotic level decreased by 28.8–77.8%. This study proposes that synergistic effects of key parameters can effectively mitigate the combined contamination of ARGs, antibiotics, and heavy metals in swine manure. Main findingOptimized parameters (anaerobic composting time 30 d, temperature 50 °C, water content 50%, pH 9.0) effectively mitigated the combined pollution of ARGs, antibiotics, and heavy metals in swine manure.

Dissemination of antibiotic resistance genes (ARGs) by rainfall on a cyclic economic breeding livestock farm

Livestock farms are a propagating source of antibiotic resistance genes (ARGs) via the discharge of animal waste carrying resistant bacteria. Groundwater is widely used in traditional livestock farming. However, there is little available information on ARGs in pumped well water. To investigate the dissemination of ARGs in well water and to assess the impact of rainfall on ARGs, the abundance and diversity of ARGs on a long-term co-livestock feedlot farm where both pig and fish farming is conducted were analysed during the dry and wet seasons in this study, and twenty ARGs were detected. The total concentrations of the target ARGs (∑ARGs) in piggery wastewater and fishponds were approximately 109 copies/mL and 106 copies/mL, respectively. The wastewater treatment plant at this farm failed to avoid ARG contamination, and the biogas digester and lagoon treatment showed low abilities to eliminate ∑ARGs. The open operation mode of the lagoon and fishpond facilitated the dissemination of ARGs, which was assisted by the nearby stream, especially in the wet season. Additionally, the tetracycline resistance gene tetB showed the highest levels among the target ARGs in well water, which were higher in the wet season than the dry season. Affected by rainfall, the concentrations of ARGs in the samples rose, and the correlation between surface water and groundwater increased. These results suggested that both the surface water and the well water in the livestock farming area harboured serious ARGs contamination, which could pose ecological risks and cause food safety problems.

Review on the removal of antibiotic resistance genes from livestock manure by composting

In recent years, due to the abuse of antibiotics in livestock industry, the antibiotic resistance genes (ARGs) are induced to produce in animals. ARGs are excreted into the environment with the feces, and contaminate the soil and groundwater through horizontal gene transfer (HGT). This in turn causes ARGs contamination. Composting, as an excellent traditional method of recycling manure, has a positive effect on the removal of ARGs from the environment. This article focuses on the removal effects of livestock manure composting (including aerobic composting and anaerobic composting) on ARGs, and then evaluates and prospects the removal of ARGs during manure composting.

High abundances of class 1 integrase and sulfonamide resistance genes, and characterisation of class 1 integron gene cassettes in four urban wetlands in Nigeria.

There is little information about environmental contamination with antibiotic resistance genes (ARG) in Sub-Saharan Africa, home to about 1 billion people. In this study we measured the abundance of three genes (sul1, sul2, and intI1) used as indicators of environmental contamination with ARGs in the sediments of four urban wetlands in southwestern Nigeria by qPCR. In addition, we characterised the variable regions of class 1 integrons in sulfamethoxazole/trimethoprim (SMX/TRI)-resistant bacteria isolated from the wetlands by PCR and DNA sequencing. The indicator ARGs were present in all wetlands with mean absolute copy numbers/gram of sediment ranging between 4.7x106 and 1.2x108 for sul1, 1.1x107 and 1x108 for sul2, and 5.3x105 and 1.9x107 for intI1. The relative abundances (ARG/16S rRNA copy number) ranged from about 10−3 to 10−1. These levels of ARG contamination were similar to those previously reported for polluted environments in other parts of the world. The integrase genes intI1 and intI2 were detected in 72% and 11.4% SMX/TRI-resistant isolates, respectively. Five different cassette array types (dfrA7; aadA2; aadA1|dfrA1; acc(6’)lb-cr|arr3|dfrA27; arr3|acc(6’)lb-cr|dfrA27) were detected among 34 (59.6%) intI1-positive isolates. No gene cassettes were found in the nine intI2-positive isolates. These results show that African urban ecosystems impacted by anthropogenic activities are reservoirs of bacteria harbouring transferable ARG.

Inflow water is a major source of trout farming contamination with Salmonella and multidrug resistant bacteria

The impact of European aquaculture, namely trout farms, in the spread of antibiotic resistance and/or zoonotic pathogens has been scarcely addressed. Moreover, aquaculture contamination sources and bacterial dissemination routes have been barely explored. In this study, we assessed the contribution of Portuguese land-based intensive rainbow trout farms and retailed market trout to the spread of Salmonella and bacteria carrying clinically-relevant antibiotic resistance genes (ARGs) as well as inflow water and feed as possible sources of those contaminants. Cultural and molecular methods were used to analyse 53 fish farm samples (upstream/downstream water and sediments, tanks and trout) and 25 marketed trout. Plasmid-mediated quinolone resistance (PMQR) genes were found in 21% (n = 11/53) of samples (water/sediment/feed/trout), from all collection points (upstream/within/downstream tanks) and seasons, as well as in 12% (n = 3/25) of marketed trout (3 supermarkets). PMQR genes (qnrS1-S2-S3, qnrB7-B19, qnrD1, oqxAB) were detected in Enterobacteriaceae or Aeromonas hydrophila. An E. coli strain producing extended-spectrum-beta-lactamase SHV-12 was detected in all sampled points of a fish farm. Salmonella (4 serotypes, including S. Newport-ST118) was detected in 26% (n = 14/53) of the samples from both farms (water/sediment upstream/within tanks). The clinically-relevant plasmid-mediated colistin resistance mcr genes were not detected. However, colistin resistant S. Abony with new mutations in the chromosomal pmrA and pmrB genes was observed. Identical Salmonella and SHV-12-producing E. coli strains (by PFGE/MLST) in water upstream and within trout tanks points to inflow-water of trout farms as an important source of pathogenic bacteria and ARG contamination. These results highlight the need to define microbiological standards for water supplying fish farms in the EU and to establish surveillance and control strategies to limit bacterial transmission associated with this fastest growing food sector worldwide.

Antibiotic resistome promotion in drinking water during biological activated carbon treatment: Is it influenced by quorum sensing?

The contamination of antibiotic resistance genes (ARGs) in drinking water may pose a direct threat to human health. This study applied high-throughput qPCR and sequencing to investigate the dynamics of ARGs and bacterial communities during the advanced treatment of drinking water using biological activated carbon. The promotion of ARGs was observed, and the normalized copy number of ARGs increased significantly after BAC treatment, raising the number of detected ARGs from 84 to 159. Twenty-nine ARGs were identified as biofilm-influencing sources in the BAC, and they persisted after chlorination. The shift of bacterial communities primarily had effects on the changes in resistome. Firmicutes, Cyanobacteria were related to persistent ARGs mostly in the BAC biofilm. Meanwhile, the Acyl-Homoserine Lactones (AHLs), quorum sensing molecules, and bacteria that produced AHLs were identified to understand the promotion of ARGs. The isolated AHL-producing bacteria belonged to the Proteobacteria, Firmicutes and Bacteroidetes phyla. Six detectable AHLs had an influence on plasmid-based horizontal gene transfer in the intragenus mating systems, indicating that the dynamics of ARGs were strongly affected by quorum sensing between specific bacteria in the biofilm. These results provide new insight into the mechanism of antibiotic resistome promotion in BAC biofilms.

Occurrence, abundance, and distribution of sulfonamide and tetracycline resistance genes in agricultural soils across China

The prevalence and proliferation of antibiotic resistance genes (ARGs) have been identified as an emerging contaminant of concern and a crucial threat to public health worldwide. In this study, we carried out a nation-wide sampling campaign across China to investigate the distribution and abundances of 8 major ARGs in agricultural soils. The levels of sulfonamide (sul) and tetracycline (tet) resistance genes in China's agricultural soils ranged from 10−6–10−2 to 10−8–10−2 gene copies/16S rRNA gene copies, respectively. Northeast China is the hot-spot of ARGs, likely due to long-term wastewater irrigation in the area. Redundancy analysis was further performed to assess the influences of environmental variables on ARG abundances. Sulfonamide resistance genes displayed strong correlations with meteorological parameters (mean annual precipitation and temperature), and decreased from north to south. In comparison, tetracycline resistance genes were more closely related to soil organic matter and pH. Co-selection between heavy metals and ARGs was significant among Cu, Hg and sulfonamide resistance genes. This study highlighted the current status of ARG contamination and their influencing factors in China's agricultural soils. Findings are valuable to identify effective management options for reducing the release of antibiotics and control ARG spread in the agriculture sector across the world.

Graphene Oxide Inhibits Antibiotic Uptake and Antibiotic Resistance Gene Propagation.

Antibiotics and antibiotic resistance genes (ARGs) in the natural environment have become substantial threats to the ecosystem and public health. Effective strategies to control antibiotics and ARG contaminations are emergent. A novel carbon nanomaterial, graphene oxide (GO), has attracted a substantial amount of attention in environmental fields. This study discovered the inhibition effects of GO on sulfamethoxazole (SMZ) uptake for bacteria and ARG transfer among microorganisms. GO promoted the penetration of SMZ from intracellular to extracellular environments by increasing the cell membrane permeability. In addition, the formation of a GO-SMZ complex reduced the uptake of SMZ in bacteria. Moreover, GO decreased the abundance of the sulI and intI genes by approximately 2-3 orders of magnitude, but the global bacterial activity was not obviously inhibited. A class I integron transfer experiment showed that the transfer frequency was up to 55-fold higher in the control than that of the GO-treated groups. Genetic methylation levels were not significant while sulI gene replication was inhibited. The biological properties of ARGs were altered due to the GO-ARG noncovalent combination, which was confirmed using multiple spectral analyses. This work suggests that GO can potentially be applied for controlling ARG contamination via inhibiting antibiotic uptake and ARG propagation.

Does human activity impact the natural antibiotic resistance background? Abundance of antibiotic resistance genes in 21 Swiss lakes

Antibiotic resistance genes (ARGs) are emerging environmental contaminants, known to be continuously discharged into the aquatic environment via human and animal waste. Freshwater aquatic environments represent potential reservoirs for ARG and potentially allow sewage-derived ARG to persist and spread in the environment. This may create increased opportunities for an eventual contact with, and gene transfer to, human and animal pathogens via the food chain or drinking water. However, assessment of this risk requires a better understanding of the level and variability of the natural resistance background and the extent of the human impact. We have analyzed water samples from 21 Swiss lakes, taken at sampling points that were not under the direct influence of local contamination sources and analyzed the relative abundance of ARG using quantitative real-time PCR. Copy numbers of genes mediating resistance to three different broad-spectrum antibiotic classes (sulfonamides: sul1, sul2, tetracyclines: tet(B), tet(M), tet(W) and fluoroquinolones: qnrA) were normalized to copy numbers of bacterial 16S rRNA genes. We used multiple linear regression to assess if ARG abundance is related to human activities in the catchment, microbial community composition and the eutrophication status of the lakes. Sul genes were detected in all sampled lakes, whereas only four lakes contained quantifiable numbers of tet genes, and qnrA remained below detection in all lakes. Our data indicate higher abundance of sul1 in lakes with increasing number and capacity of wastewater treatment plants (WWTPs) in the catchment. sul2 abundance was rather related to long water residence times and eutrophication status. Our study demonstrates the potential of freshwater lakes to preserve antibiotic resistance genes, and provides a reference for ARG abundance from lake systems with low human impact as a baseline for assessing ARG contamination in lake water.

Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake

Antibiotic-resistance genes (ARGs) are currently discussed as emerging environmental contaminants. Hospital and municipal sewage are important sources of ARGs for the receiving freshwater bodies. We investigated the spatial distribution of different ARGs (sul1, sul2, tet(B), tet(M), tet(W) and qnrA) in freshwater lake sediments in the vicinity of a point source of treated wastewater. ARG contamination of Vidy Bay, Lake Geneva, Switzerland was quantified using real-time PCR and compared with total mercury (THg), a frequently particle-bound inorganic contaminant with known natural background levels. Two-dimensional mapping of the investigated contaminants in lake sediments with geostatistical tools revealed total and relative abundance of ARGs in close proximity of the sewage discharge point were up to 200-fold above levels measured at a remote reference site (center of the lake) and decreased exponentially with distance. Similar trends were observed in the spatial distribution of different ARGs, whereas distributions of ARGs and THg were only moderately correlated, indicating differences in the transport and fate of these pollutants or additional sources of ARG contamination. The spatial pattern of ARG contamination and supporting data suggest that deposition of particle-associated wastewater bacteria rather than co-selection by, for example, heavy metals was the main cause of sediment ARG contamination.

Prevalence of antibiotic resistance genes and their relationship with antibiotics in the Huangpu River and the drinking water sources, Shanghai, China

The prevalence of antibiotic resistance genes (ARGs) and their relationship with antibiotics in the surface waters of the Huangpu River and the drinking water sources of Shanghai, China, were investigated. 39 ARGs, including four sulfonamide ARGs, 23 tetracycline ARGs, four chloramphenicol ARGs, five β–lactam ARGs and three penicillin ARGs were targeted in this study. Two sulfonamide ARGs (sul I and sul II), eight tetracycline ARGs (tet(A), tet(B), tet(C), tet(G), tet(M), tet(O), tet(W) and tet(X)), and one β–lactam ARG (TEM) were detected to be present in water samples by polymerase chain reaction (PCR), with the detection frequencies ranging from 42.86% to 100%. The average concentrations of the 11 ARGs ranged from 3.66×101copy/mL (tet(B)) to 1.62×105copy/mL (sul II), quantified by quantitative real-time PCR (QPCR). The number of detected ARGs and the ARG concentrations were generally higher in suburban sampling sites than in urban sites. Being in or near suburban rural areas, some raw drinking water sources were observed to have comparatively higher ARG contamination, drawing an urgent attention for the concern of public health. Generally consistent relations were observed between the concentrations of tet genes and the tetracycline levels and between the concentrations of sul genes and the sulfonamide levels.