Fate and Behavior of Tetracycline Resistance Genes in Activated Carbon Adsorption

Sri Anggreini¹, Alma Rizky Aurellya², Wenqing Li¹, Fusheng Li^1,3*

¹Graduate School of Engineering, Gifu University, Gifu, Japan.
²Graduate School of Natural Science and Technology, Gifu University, Gifu, Japan.
³River Basin Research Center, Gifu University, Gifu, Japan.

DOI: 10.4236/jwarp.2024.161001                

Abstract

The accessibility of tetracycline resistance gene (tetG) into the pores of activated carbon (AC), as well as the impact of the pore size distribution (PSD) of AC on the uptake capacity of tetG, were investigated using eight types of AC (four coal-based and four wood-based). AC showed the capability to admit tetG and the average reduction of tetG for coal-based and wood-based ACs at the AC dose of 1 g·L^-1 was 3.12 log and 3.65 log, respectively. The uptake kinetic analysis showed that the uptake of the gene followed the pseudo-second-order kinetics reaction, and the uptake rate constant for the coal-based and wood-based ACs was in the range of 5.97 × 10^-12 - 4.64 × 10^-9 and 7.02 × 10^-11 - 1.59 × 10^-8 copies·mg^-1·min^-1, respectively. The uptake capacity analysis by fitting the obtained experiment data with the Freundlich isotherm model indicated that the uptake constant (K_F) values were 1.71 × 10³ - 8.00 × 10⁹ (copies·g^-1)^1-1/n for coal-based ACs and 7.00 × 10⁸ - 3.00 × 10¹⁰ (copies·g^-1)^1-1/n for wood-based ones. In addition, the correlation analysis between K_F values and pore volume as well as pore surface at different pore size regions of ACs showed that relatively higher positive correlation was found for pores of 50 - 100 Å, suggesting ACs with more pores in this size region can uptake more tetG. The findings of this study are valuable as reference for optimizing the adsorption process regarding antibiotic resistance-related concerns in drinking water treatment.

Keywords

Antibiotic Resistance Genes, Adsorption, Activated Carbon, Drinking Water Treatment

1. Introduction

Antibiotics have been widely used since the 1940s to treat bacterial infections in humans, livestock, and aquaculture, as well as feed additives in animal husbandry [1] . The overuse of antibiotics has resulted in the proliferation of antibiotic resistance genes (ARGs) in the aquatic environment. ARGs can survive in water even in the absence of selective pressure and spread their antibiotic resistance traits through vertical gene transfer (i.e. via cell division) and horizontal gene transfer (i.e. via conjugation, transduction, and transformation) [2] [3] . The emergence of ARGs has raised significant concerns due to their potential implications on public health. ARGs can directly impact the effectiveness of antibiotics, which are crucial medications for treating diseases. Bacteria evolve antibiotic resistance to enhance their survival, thus reducing treatment options to treat infections, prolonging illness, increasing healthcare costs, and elevating mortality rates in the human population. Antibiotic resistance has caused more than 25,000, 38,000, 58,000 and 23,000 deaths in the European Union, Thailand, India, and the United States each year, respectively [4] . Nowadays, antibiotic resistance contributes to approximately 700,000 deaths worldwide each year [5] . Antibiotic resistance is expected to produce a significant public health crisis by 2050, resulting in over 10 million deaths and a global cost burden of approximately US$100 trillion [6] .

Tetracycline is a frequently utilized antibiotic in humans and animals due to its broad-spectrum activity and low cost [7] . The widespread application of tetracyclines in aquaculture, animal husbandry, and healthcare has led to the spread of tetracycline resistance genes in the environment. Previous studies reported that tetracycline resistance genes were predominantly detected in the aquatic environment [8] [9] [10] . Tetracycline resistance prevalence in certain European countries was observed to be 66.9% and 44.9% for Escherichia coli and Klebsiella (spp.) species, respectively [11] . In addition, WHO has added tetracycline resistance to its priority list of critical antibiotic-resistant bacteria (ARB) in drinking water [6] . Therefore, mitigating and controlling the proliferation of tetracycline resistance genes is essential for ecological safety and human health.

ARGs have been broadly detected in various environmental contexts, including surface water, groundwater, hospital and livestock sewage, as well as wastewater treatment effluents [12] [13] [14] [15] . ARGs originating from different sources will accumulate in the drinking water source and be carried along with raw water to the drinking water treatment plant (DWTP). Nevertheless, DWTP is not able to eliminate ARGs completely, leading to the presence of ARGs in tap water [16] . Thus, there is a possibility of exposure during bathing, cooking, and other uses. The issue of ARGs in drinking water deserves attention because it is closely related to people’s daily lives and threatens human health. The understanding of the fate and behavior of ARGs in various treatment processes, including coagulation, sedimentation, sand filtration, chlorination, and activated carbon (AC) adsorption, has not been identified. Concerning the adsorption process, the implications of the adsorption of ARGs on AC have not been well defined. Most studies have reported that granular AC filtration increases the diversity and absolute abundance of ARGs since the biofilm on the AC surface can act as a nutrient source for ARBs and be an ideal site for the dissemination of ARGs [17] [18] . Meanwhile, powdered AC, which is commonly added to DWTP receiving wells, resulted in a low reduction of ARGs. Previous research reported that the reduction of 27 ARGs by powdered AC in DWTP only reached 0.23 log toward influent concentrations of 1.83 × 10⁹ - 3.51 × 10⁹ copies·L⁻¹ [16] . Powdered AC can also increase the spread of ARGs during the recycling of water from drinking water treatment sludge as raw water. ARGs may be able to enter the AC pores and possibly be released from the pores during the recycling process. The released ARGs can transfer their resistance trait to bacteria, leading to the spread of ARGs during the recycling process. Thus, the penetration of ARGs into the pores of AC can cause problems in both granular and powdered AC. This emphasizes the need for comprehensive investigations to elucidate the fate and behavior of ARGs during the adsorption process, which is critical for optimizing water treatment strategies and tackling concerns related to antibiotic resistance.

AC adsorption is widely used in drinking water treatment to remove various contaminants in water due to its larger surface area and pore volume. The uptake of ARGs on AC might be affected by various properties of AC such as total surface area, total pore volume, pore size distribution (PSD), charge density, and functional groups of AC [19] . Depending on the physicochemical features of AC, the uptake of ARGs on AC may differ. One of the most critical properties influencing the adsorption process is the PSD of AC [20] . According to previous studies, the pore size of AC below 15 Å was found to be more effective for the adsorption of small organic micropollutants [21] . Large molecular weight humic substances (1 - 10 kDa) are mostly adsorbed in the mesopores of AC (20 - 500 Å) [22] . The different sizes of AC pores provide varying access routes for molecules. Larger pores accommodate larger molecules, while smaller pores are suitable for smaller molecules. So far, several studies have been focused on the occurrence and removal of ARGs in different treatment processes in DWTP [17] [23] [24] . However, little is known about the accessibility of ARGs on the AC pores and the fate of ARGs during the adsorption process.

Hence, the objectives of this study were to investigate the possible access of the tetracycline resistance gene into the pores of AC and the impact of the PSD of AC on the uptake capacity changes of the gene. For this purpose, batch adsorption experiments were conducted for uptake of tetG by eight commercially available ACs (four coal-based and four wood-based) with different PSDs. Experiment data were analyzed using adsorption kinetic and isotherm models. The uptake rate of tetG on different types of ACs was determined using adsorption kinetics. The behavior of tetG on different types of ACs was described by adsorption isotherms. Furthermore, the correlation analysis between the uptake capacity of the gene and the PSD of AC was performed to evaluate the impact of PSD on the admission of the gene. The concentration of the gene before and after adsorption was quantified using a quantitative polymerase chain reaction (qPCR). This study can serve as a reference in the selection and design of ACs to improve the elimination of ARGs and reduce the risk of spreading ARGs in DWTP.

2. Materials and Methods

2.1. Activated Carbon

Four coal-based ACs (AC-1, AC-2, AC-3, AC-4) and four wood-based ACs (AC-5, AC-6, AC-7, AC-8) were purchased from the market. These ACs are steam-activated ones and the main difference regarding their physiochemical properties is PSDs. The PSD of each AC was measured by high-resolution Micromeritics 3Flex instrument (Micromeritics 3Flex, USA) based on nitrogen adsorption. The pore volume and pore surface values of the ACs are shown in Table 1 and Table 2, respectively. The pore size region of AC was categorized based on previous studies that investigated the effect of PSD of AC on the adsorption capacity of dissolved organic matter in drinking water sources [25] . The majority of pores are concentrated in the size range below 20 Å for all eight ACs. For AC-2, the total pore volume and surface area are obviously lower than other types of AC, and this AC has more pores in the size region below 10 Å both its pore volume and pore surface.

Further, the functional groups on the surface of the AC were examined by Fourier transform infrared spectroscopy (FTIR). The FTIR spectra of all eight ACs are displayed in Figure 1. The existence of a strong peak at 3500 cm⁻¹ indicates the presence of hydroxyl groups. In addition, amide groups and carboxylic acid groups were detected at wavenumbers of 1650 and 1400 cm⁻¹, respectively [26] . All eight ACs had similar functional groups, suggesting these ACs had similar chemical structure. The different properties of all types of ACs were clearly seen in the total surface area, total pore volume and PSD of ACs, demonstrating these properties might contribute more to the uptake of tetG.

Table 1. Total pore volume and the pore volume percentages in different pore size regions of eight ACs used in this study.

Table 2. Total pore surface area and the surface area percentages in different pore size regions of eight ACs used in this study.

Figure 1. The FTIR spectra of eight ACs used in this study.

2.2. Antibiotic Resistance Genes (ARGs) Preparation

The tetracycline resistance gene (tetG) was prepared by referring to the TA cloning method (TaKaRa, Japan). The non-quantitative PCR instrument was used to amplify the target gene. Further, the Gel and PCR Clean-up kit (Macherey-Nagel, Germany) was utilized to purified PCR product. The ligation reaction of the target gene was performed with plasmid pMD20-T (TaKaRa, Japan). The resulting product was transformed into E. coli DH5α competent cells and cultured overnight in LB medium with tetracycline. Further, white colonies were picked for PCR amplification to confirm the insert of the target gene. The confirmed colonies containing the desired plasmid were cultured overnight in LB medium with tetracycline. Then, the plasmid was extracted from E. coli DH5 using a plasmid extraction kit (Macherey-Nagel, Germany), and its concentration was measured using a Quantus Fluorometer. Further, the qPCR analysis was performed to amplify tetG at 133 bp with designed primers (forward primer: TTA TCG CCG CCG CCC TTC T, reverse primer: TCA TCC AGC CGT AAC AGA AC) and utilizing SYBR^® Premix Ex TaqTM (TaKaRa, Japan) based on the manufacturer’s protocol [27] . The amplification product was isolated by gel electrophoresis and purified using a Gel and PCR Clean-up kit. The purified amplification product was diluted with sterile deionized water to obtain the amplicon solution and used for adsorption experiments.

2.3. Adsorption Experiments

Adsorption experiments were carried out using the batch equilibrium technique in 5 mL vials at 20˚C. Adsorption kinetics experiments were conducted for 180 minutes until equilibrium was attained using an AC dose of 1 g·L⁻¹ and an initial tetG concentration of 6.86 × 10¹³ copies·L⁻¹. Adsorption isotherm experiments were conducted at AC doses within the range of 0.01 - 1 g·L⁻¹, and the initial concentration of tetG was 6.86 × 10¹³ copies·L⁻¹. After shaking for 180 minutes, centrifugation was performed for 10 minutes at 12,000 g to remove the AC particles. The obtained supernatant was subjected to tetG quantification. The concentration of tetG before and after adsorption was determined by qPCR.

3. Results and Discussion

3.1. Uptake Rate of tetG on ACs

The changes of the uptake rate of tetG with time are shown in Figure 2. The uptake was rapid at the beginning and gradually slowed as the contact time increased until equilibrium was reached in 150 minutes. For the coal-based ACs, as displayed in Figure 2(a), compared to AC-2, the uptake rate for AC-1, AC-3, and AC-4 was higher, being about 4.04, 3.80, and 3.90 log at 180 min, respectively. AC-2 showed a lower uptake rate, which only reached 0.75 log after 180 min. It was found that uptake rate by AC-2 was approximately 5 times lower than other types of coal-based AC. Further, the uptake rate of tetG for wood-based ACs is presented in Figure 2(b). AC-7 had the highest uptake rate of tetG (4.50 log) compared to the other wood-based ACs at 180 min. Meanwhile, the uptake rates for AC-5, AC-6, and AC-8 were 3.20, 3.44, and 3.46 logs, respectively. The differences in the uptake rate of tetG on AC were attributed to the different surface properties of the ACs, such as surface chemistry, total pore volume, total surface area, etc.

Pseudo-first-order and pseudo-second-order kinetic models were used in this study to determine the uptake rate and possible mechanisms involved in the uptake of tetG. The pseudo-first-order kinetic model is given as [28] :

$\frac{d{q}_t}{d_t}=k_1\left(q_e-q_t\right)$ (1)

where q_t (copies·mg⁻¹) and q_e (copies·mg⁻¹) are the uptake capacity of tetG onto AC at time t and equilibrium, respectively. The k₁ (min⁻¹) and t (min) are the pseudo-first-order rate constant and time of reaction, respectively. The k₁ and q_e were obtained from linear plot of ln (q_e – q_t) with time as displayed in Figure 3. Further, the pseudo-second-order kinetic model is exhibited by the following equation [28] :

$\frac{d{q}_t}{d_t}=k_2{\left(q_e-q_t\right)}^2$ (2)

where k₂ (copies·mg⁻¹·min⁻¹) is the pseudo-second-order rate constant. The k₂ and q_e were computed from the intercept and slope of the linear plot of t/q_t against t as demonstrated in Figure 4.

Figure 2. Changes of the uptake rate of tetG with time by (a) 4 coal-based and (b) 4 wood-based ACs (AC dose: 1 g·L⁻¹, initial tetG concentration: 6.86 × 10¹³ copies·L⁻¹).

Figure 3. Analysis of the uptake of tetG based on the pseudo-first-order kinetic model for (a) coal-based and (b) wood-based ACs.

Figure 4. Analysis of the uptake of tetG based on the pseudo-second-order kinetic model for (a) coal-based and (b) wood-based ACs.

As shown in Figure 3 and Figure 4, pseudo-second-order fitted well with the experimental data compared to the pseudo-first-order kinetic model, suggesting the experimental data follows the pseudo-second-order reaction. The R² value of the pseudo-second-order was very high (R² > 0.975), and the calculated q_e value was in good agreement with the experimental ones as shown in Table 3, indicating the applicability of the pseudo-second-order kinetic model to describe the uptake rate of tetG on AC. The highest uptake rate constant was found in AC-1 (4.64 × 10⁻⁹ copies·mg⁻¹·min⁻¹) for coal-based AC and AC-7 (1.59 × 10⁻⁸ copies·mg⁻¹·min⁻¹) for wood-based one. While AC-2 had the smallest uptake rate constant compared to all types of AC (coal-based and wood-based), which was (5.97 × 10⁻¹² copies·mg⁻¹·min⁻¹). Moreover, as displayed in Table 3, the uptake capacity (q_e) of tetG for coal-based and wood-based ACs was in the range 6.40 × 10⁹ - 1.12 × 10¹⁰ copies·mg⁻¹ and 8.91 × 10⁹ - 1.82 × 10¹⁰ copies·mg⁻¹, respectively.

The pseudo-second-order reaction assumes that the adsorption process is controlled by chemisorption, involving valence forces by sharing electrons between the adsorbent and the adsorbate [29] . As mentioned earlier, hydroxyl groups and carboxylic acid groups are present in eight ACs. These functional groups may allow for H-bonding interactions between the H atom of the AC and the N atom of the tetG DNA base. Moreover, π- π interactions may occur between the aromatic structure of AC and the DNA base of tetG. All DNA bases are aromatic and can easily interact with AC through this interaction. AC also consists of aromatic sheets that are flat and broken in places by slit-shaped pores. Heteroatoms (e.g. oxygen, nitrogen) exist in individual particles inserted between the aromatic sheets or incorporated as functional groups [30] . Similar findings were also obtained in other studies [31] .

3.2. Uptake Capacity of tetG on ACs

The capability of AC to adsorb tetG was evaluated with the Freundlich isotherm model. The equation of this model is described as follows [32] .

Table 3. Estimated pseudo-second-order kinetic model parameters for the uptake of tetG on all eight ACs used in this study.

$q_e=K_F{C}_e{}^{1/n}$ (3)

where K_F is the Freundlich constant representing the adsorption strength, and 1/n reflects the affinity of tetG with AC.

The Freundlich isotherm data for the uptake of tetG on coal-based and wood-based ACs are shown in Figure 5. The Freundlich isotherm parameters and R² values are summarized in Table 4. The experimental data for all eight ACs were fitted well with this model, except AC-2. The 1/n value was found below 1, indicating the uptake of tetG on AC is favorable [33] . AC-2 had the highest 1/n value compared to the other types of ACs. This implies that weak affinity between tetG and AC-2 during the adsorption process. Meanwhile, other types of ACs exhibit small 1/n values compared to AC-2. A smaller 1/n value indicates a higher level of affinity or a stronger interaction between tetG and AC [33] . The K_F values for coal-based and wood-based ACs varied in the range of 1.71 × 10³ - 8.00 × 10⁹ (copies·g⁻¹)^1−1/n and 7.00 × 10⁸ - 3.00 × 10¹⁰ (copies·g⁻¹)^1−1/n, respectively. The highest K_F value was found from AC-1 and AC-7 for coal based and wood-based AC, respectively. Compared to all types of ACs, AC-2 possessed the smallest K_F value. The K_F value follows the order AC-7 > AC-1 > AC-5 > AC-6 > AC-4 > AC-8 > AC-3 > AC-2. While the 1/n value follows the order AC-2 > AC-3 > AC-4 > AC-8 > AC-5 > AC-6 > AC-1 > AC-7. Generally, wood-based ACs were found to have a higher K_F value than coal-based ACs, indicating wood-based ACs have a higher ability to accept ARGs compared to coal-based ones.

3.3. Correlation Analysis of the Relations of the Uptake Capacity of tetG with Pore Volume and Pore Surface in Different Size Regions of ACs

Table 5 shows the Pearson correlation analysis between the estimated K_F and the pore volume as well as the pore surface in different pore size regions of eight ACs. The highest positive correlation was noticed between K_F and the pore size region of 50 - 100 Å in both pore volume and pore surface, suggesting pores within this size region might be effective for the uptake of tetG. Pores in the range of 50 - 100 Å probably provide ideal accessibility for tetG, allowing the gene to enter and be fixed inside the pore. Pore sizes of 50 - 240 Å also generated a good positive relation with K_F but the correlation coefficient was slightly smaller compared to pores of 50 - 100 Å. It indicated that tetG is preferentially adsorbed on pores slightly larger than its size due to the greater attractive force and more contact points between tetG and the active site of AC. This result is attributed to the potential force overlap formed when the opposite pore wall is slightly larger than the size of the adsorbed molecule [34] . Pore sizes of 50 - 240 Å may be able to adsorb tetG, but the available active sites for uptake are smaller due to the limited surface area at larger pore sizes.

Figure 5. Analysis of the uptake capacity of tetG based on the Freundlich isotherm model for (a) 4 coal-based and (b) 4 wood-based ACs.

Table 4. Estimated Freundlich isotherm model parameters for the uptake capacity of tetG on all eight ACs used in this study.

Table 5. Pearson correlation coefficient (r) obtained from the correlation analysis of the estimated K_F with the pore volume and pore surface in different pore size regions of eight ACs. The symbol “-” indicates the correlation is negative.

Pore sizes below 20 Å showed a negative correlation with K_F as displayed in Table 5, indicating these pore sizes are not accessible by tetG. Hence, it is obvious that AC-2 has the lowest K_F value compared to other types of ACs due to more pore sizes in the size region below 20 Å, with the percentage of pore volume and pore surface being 98.1% and 99.9%, respectively (Table 1 and Table 2). The size exclusion effect might prevent tetG from entering the pores of AC-2. Thus, the accessibility of tetG to the AC pores is crucial since the appropriate pore size can allow the penetration of tetG into the pore, resulting in more effective gene uptake. This result showed that micropores (<20 Å) were less effective for the uptake of tetG [35] . The micropore may have a larger surface area but it has no entry point and insufficient transport pathways for the gene.

4. Conclusion

The possible access of tetG and the impact of the PSD of AC on the uptake of tetG were evaluated. The results suggested that the uptake of tetG varied greatly with the type of AC. The uptake kinetic analysis indicated that the uptake of tetG followed pseudo-second-order reaction. The uptake capacity analysis demonstrated that the Freundlich isotherm model described the uptake capacity data quite well. In addition, correlation analysis between the estimated K_F value and PSD of ACs suggested that pores with sizes of 50 - 100 Å were more effective in uptake of tetG. Pores below 20 Å were too small for the uptake of tetG. This study can serve as a reference for AC selection or material design for optimizing the uptake of tetG from water.

Acknowledgements

This study was conducted with the support of JSPS for Grant-in-Aid for Scientific Research (A) (20H00261).

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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