Antibiotic Resistance Gene Was Increased via Horizontal Transfer in Single and Two-Chamber Microbial Electrolysis Cells

doi:10.21203/rs.3.rs-630388/v1

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Antibiotic Resistance Gene Was Increased via Horizontal Transfer in Single and Two-Chamber Microbial Electrolysis Cells

https://doi.org/10.21203/rs.3.rs-630388/v1

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Microbial electrolysis cells (MECs), have been applied for antibiotic degradation, but simultaneously induced antibiotic resistance genes (ARGs), thus representing a risk to disseminate antibiotic resistance. However, there were few studies on the potential and risk of ARGs transmission in the MECs. In this work, conjugative transfer of ARGs was assessed under three tested conditions (voltages, cell concentration, and donor/recipient ratio) in both single and two-chamber MECs. The results indicated that voltages (> 0.9 V) facilitate the frequency in single-chamber the MECs and two-chamber the MECs (in anode chamber). The donor cell number (donor/recipient ratio was 2:1) showed more favor on the transfer frequency. Furthermore, voltages ranged from 0.9 V to 2.5 V increased ROS production and cell membrane permeability in MECs. These findings offer new insights into the roles of ARGs transfer under different applied voltages in the MECs, which should not be ignored for horizontal transfer of antibiotic resistance.

Environmental Engineering

Environmental Policy

Microbial electrolysis cells

Antibiotic resistant genes

Conjugative transfer

Horizontal gene transfer

Nowadays, the misuse and overuse of antibiotics has entered many water environment matrixes and posed a serious threat to public health (Sanganyado & Gwenzi, 2019). The antibiotics have been found in the municipal environment (Rodriguez-Mozaz et al., 2015; Yi et al., 2019). Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) raised by antibiotics has also called the human attention. ARGs have also been detected in environment matrixes (Sivalingam et al., 2019), such as wastewater treatment plants, farmland and animal wastewater, and even exist in urban runoff (Joy et al., 2013; Sanganyado & Gwenzi, 2019). Horizontal gene transfer (HGT) between bacterial cells considered as the major pathway could favor the transmission of ARGs (Kim et al., 2014). Three mechanisms of HGT were conjugation, transformation, and transduction (Wang et al., 2019). The conjugation made the ARGs transferred from donor to recipient cells by mobile plasmids (Li et al., 2019). The transformation was generally that recipient cells captured the extracellular ARGs (Hasegawa et al., 2018). The transduction was the phage virions obtaining the ARGs and entering the recipient cells (Zhang et al., 2017). Because of the antibiotics vestigital and the risk of ARGs transmission, it is essential to investigate novel approaches that are capable of efficiently degrading antibiotics and reducing abundance of ARB and ARGs.

Microbial electrolysis cells (MECs), including single-chamber MECs and two-chamber MECs, coupled with microbial metabolisms and electrochemical redox reactions that could achieve high-efficiency pollution removal (Xue et al., 2019). Furthermore, the potential of MECs applied on antibiotic removal also performed an excellent potential application (Yan et al., 2019). The capability of antibiotic contaminant removal due to H₂ production in MECs with an applied voltage (Zhang & Angelidaki, 2014). According to the potential of antibiotic contaminants removal and energy production, MECs possessed a better application prospect.

It was worth noting that the ARB and ARGs can be induced under the presence of antibiotics even at a low concentration in MECs (Wang et al., 2016). ARB abundance was increased based on the enhancement of ATP concentration with the current (Li et al., 2018). The abunce of ARGs, compared with ARB, is quite complicated in MECs (Zhang et al., 2020). With high negative cathode potential, the expression of ARGs was reduced in MECs (Guo et al., 2017). In contrast, previous studies showed that the relative abundances of ARGs under cathode potential were significantly increased when the MEC treating antibiotic wastewater (Guo et al., 2018). In addition, DNA carried ARGs whatever the intracellular and extracellular might enter the bacteria without resistance and made it become new ARB by HGT (Liu et al., 2018). Meanwhile, the HGT of ARGs could lead to the production of ARB in MECs (Li et al., 2018; Zhang et al., 2020). ARGs were induced by antibiotic residue and the gene could be transferred to produce new ARB in the system. However, it is not clear that ARGs transmission in MECs.

Thus, this study was focused on the conjugative transfer of ARGs in MECs. The main objective of this paper was to understand the potential risk of ARGs transmission under the electrical stimulation in MECs. Conjugative transfer systems in single-chamber and two-chamber MECs were constructed to illustrate the conjugative transfer frequency, and analysis on the parameters such as applied voltage, bacterial concentration, and donor/recipient ratio affect the frequency. Reactive oxygen species (ROS) and cell membrane permeability of donor and recipient cell were tested by NovoCyte in MECs.

2.1 Construction and operation of MECs

A columnar glass in which 4 cm in diameter and 10 cm in height was set as our single-chamber MECs. The total volume of the container was 100 mL and the utilizing volume was 60 mL. Meanwhile, the two-chamber MECs was 4 cm in diameter and 15 cm in height and assembled in glass columns. The total volume of the two-chamber MECs was 180 mL and the effective working volume (liquid volume) was 120 mL. Besides the chambers were separated with a pretreated cation exchange membrane. Carbon rods (1 cm in thickness, 3 cm in length, Nanjing Zhongdong Chemical Glass Instrument Co., Ltd, Nanjing, China) were wound with stainless steel (1 mm in thickness, 6 cm in length) was installed along the axial center of the reactor as the electrode. Power supply (LW-305KDS, Hong Kong Longwei Instrument Co., Ltd.) provides stable applied voltages.

2.2 Bacterial strains and culture media

E. coli K-12-harboring conjugative RP4 plasmids (60,099 bp) was used as the donor cells. The recipient cells were the E. coli HB101, which contains the streptomycin resistance (Str). The RP4 plasmid does not confer resistance to streptomycin but carries three ARGs against kanamycin (Kan), ampicillin (Amp), and tetracycline (Tet). The strains were incubated in LB-medium (pH of 7.4) and shaken overnight (140 rpm) at 30℃. The strains were collected after centrifugation, the pellets were washed by phosphate-buffered saline (1 × PBS, pH of 7.2) and resuspended in PBS. All antibiotics (Kan, Amp, Tet, and Str) were purchased from Shanghai yuan ye Bio-Technology Co., LTD.

2.3 Conjugative transfer under different conditions

To evaluate the applied voltages on the conjugative transfer frequency of ARGs in the systems, two single-chamber and two-chamber MECs were established according to the experimental design and methods (Fig. 1). Different voltages were carried out in the systems as well. Especially, the effect of electrodes including anode and cathode chamber were also involved in the two-chamber MECs. Besides, the donor/recipient ratio was changed in the MECs. Finally, the frequency of the variation of bacterial concentration was also evaluated in the MECs.

The voltage which the most important factor in MECs, was chosen to evaluate the conjugative transfer frequency of ARGs. Different voltages (including 0.9, 1.5, and 2.5 V) were selected in this study. Donor (10⁸ CFU/mL) and recipient cells (10⁸ CFU/mL) were mixed with the same volume (1:1 ratio).

The donor or recipient cells that show more importance to the conjugative transfer frequency of ARGs in the MECs. Different ratios of donor and recipient cells (1:1, 1:2, and 2:1) at 10⁸ CFU/mL was chosen to explore transfer frequency with 1.5 V voltage and bacterial concentration in the two types MECs. Furthermore, donor and recipient cells at different concentration (10⁶, 10⁷, and 10⁸ CFU/mL) mixed at 1:1 ratio and exposed to 1.5 V in the two types MECs was also evaluated. Besides, the voltage of 0 V was included as the control group. The mixture was incubated for 1, 3, and 6 h at 25 ℃ in MECs.

To furthe revaluate the conjugative transfer frequency of ARGs, the actual water was used in the MCE. The water was token from the river (118°42'39.3''E, 32°12'12.4''N) in NUIST. The water was stored at 4 ℃ and used after filtering with 0.22 μm membrane filters. The quality of river water was shown in Table 1.

Table 1 The quality of the river water

pH	COD (mg/L)	Total nitrogen (mg/L)	Total phosphorus(mg/L)
7.3	45	7.5	0.01

The amount of conjugative frequency was calculated in the following methods. Transconjugants numbers were determined by LB agar selection plates containing Str (30 mg/L), along with the three antibiotics to which RP4 plasmid harbors resistance (100 mg/L Amp, 30 mg/L Kan, and 10 mg/L Tet) (Wang et al., 2019). Accordingly, the LB agar plates containing Str (30 mg/L) were used to account the recipient number. The conjugative frequency was calculated by normalizing the number of transconjugants (CFU/ml) to the total number of recipients (CFU/ml) (Wang et al., 2018).

2.4 ROS and cell membrane permeability test

The dye 2’,7’ - dichlorofluorescein diacetate (DCF - DA, Aladdin, China) was used to investigate the ROS generation change in MECs. Donor and recipient cell individually suspensions in PBS at 0.3 OD₆₀₀ and then mixed. Bacterial cells were treated at different voltages (0, 0.9, 1.5, and 2.5 V) for 2 h in MECs without light. DCF - DA (final concentration of 20 μM) then added to all test samples. The samples were then scanned by a NovoCyte 2040R (ACEA Biosciences Inc., USA) to measure the fluorescence (excitation/emission at 488 nm/525 nm) (Wang et al, 2019).

Applied voltages on cell membrane permeability change were also determined by propidium iodide (PI, YuanYe, China). Bacterial cells containing donor and recipient cells were treated at different voltages (0, 0.9, 1.5, and 2.5 V) for 2 h in MECs. All samples strained with PI (final concentration of 25 mg/L) for 30 min. NovoCyte 2040R at 488 nm excitation and emission at 630 nm determine their fluorescence. All tests were conducted with three samples (Wang et al, 2019, Lu et al, 2020).

3.1 Voltage increases the conjugative transfer frequency

The applied voltage was the main factor contributing to the degradation efficiency of contaminant in the MECs (Yang et al., 2018). Firstly, conjugative transfer frequencies of ARGs were evaluated under different voltages in the single-chamber MECs. As can be seen from Fig. 2, with the increase of applied voltage from 0 to 2.5 V, the conjugative transfer frequency was enhanced in the single-chamber MECs. For instance, the frequency under 0.9 V (1.12 × 10^− 4 per recipient cell) was up to 1.76-fold higher than that of the control group (6.36 × 10^− 4 per recipient cell) after mating 1 h (Fig. 2A). Similarly, the transfer frequencies of ARGs in Fig. 2B were elevated when voltages increased from 0.9 to 2.5 V (p < 0.05). Applied voltage of 2.5 V (1.84 × 10^− 3 per recipient cell) also facilitated the conjugative transfer frequency of ARGs, in which was 7.12-fold higher than that of the control group (2.59 × 10^− 4 per recipient cell) after 6 h mating time (Fig. 2C).

On the other hand, the conjugative transfer frequency change under different applied voltages in the two-chamber MECs was shown in Fig. 2. The frequency trend in the anode chamber was similar to the single-chamber MECs and increased with the applied voltage changed from 0 V to 2.5 V. For example, the frequency under 2.5 V in anode chamber was up to 8.33 × 10^− 4 per recipient cell, and was 8.3-fold higher than that of the control group (1.00 × 10^− 4 per recipient cell) after mating 6 h (Fig. 2F). In contrast to the frequency in the anode chamber, the frequencies in cathode chamber were lower than those in the control groups and significantly decreased in 2.5 V (Fig. 2F). For instance, the average frequency in the cathode chamber (4.18 × 10^− 6 per recipient cell) was one-order lower than that in the control group (6.33 × 10^− 5 per recipient cell) after mating 1 h (Fig. 2D). Furthermore, after mating 3h, the conjugative transfer frequency in the cathode chamber had the peak value under 1.5 V (7.50 × 10^− 6 per recipient cell), which was 1.43-fold and 1.33-fold higher than that under 0.9 V (5.22 × 10^− 6 per recipient cell) and 2.5 V (5.62 × 10^− 6 per recipient cell) respectively (Fig. 2E).

Consequently, these results verified the increased voltages from 0.9 to 2.5 V facilitated the conjugative transfer frequency of ARGs in the single-chamber MECs. Similarly, the frequency of ARGs transmission in the anode chamber was promoted by the increasing applied voltage. This phenomenon may contribute to the fact that the continuous electrical stimulation providing the elections to the chamber and stimulate bacterial activity by direct or indirect election transformation (Dominguez-Benetton et al., 2018; Feng et al., 2015; Liu et al., 2015). The increasing applied voltage might cause the over-production of ROS and the damage of cell membrane permeability, and that could favor the ARGs transmission (Lu et al., 2020). By contrary, the conjugative transfer frequency in the cathode chamber was reduced when the applied voltage surpassed 0.9 V. This result may be attributed to the fact that the applied voltage changes the cell membrane permeability in the cathode chamber and leads to the death of cells. It should be noted that the adenosine triphosphate (ATP) as the indicator of metabolism decreased 46% and 66% under 1 V and 2 V in the cathode chamber respectively, compared to the control group (Ding et al., 2016). The feasibility of hydroxyl radical production has been demonstrated in the cathode of MECs (Wu et al., 2017) and coupled with Fenton reaction for organic degradation (Li et al., 2017a; Li et al., 2017b). However, the production of hydroxyl radicals could cause the reduction of cells number (Kubacka et al., 2014).

3.2 Ratio affects the conjugative transfer frequency

The ratio of donor and recipient cells was changed to evaluate the influence on conjugative transfer frequency in the MECs. Previous studies showed that the theoretical applied voltage in MECs was 0.14 V, the actual voltage could reach as high as 1.2 V (An & Lee, 2013; Ki et al., 2016), thus the 1.5 V as the applied voltage in our following experiment. As shown in Fig. 3, the frequency was increased on the ratio of 1:2 compared with the control group after treating. For example, the frequency on the ratio of 1:2 (3.87 × 10^− 4 per recipient cell) was 1.96-fold higher than that of 1:1 (1.98 × 10^− 4 per recipient cell) after mating 3 h (Fig. 3B). Furthermore, the increasing multiples of the frequency on the ratio of 2:1 was greater than that of 1:2, compared with the control group. For instance, the frequency on the ratio of 2:1 was 4.65-fold, 1.97-fold, 3.10-fold higher that of 1:2 ratio, when the mating time was 1 h, 3 h, 6 h respectively (Fig. 3A B, and C).

Considerately, the frequency of the cathode was lower than that of the anode and the control group in the two-chamber MECs (Fig. 1). Thus, the anode and control group were contrasted when the ratio of donor and recipient cells changed. It was seen that compared with the ratio of 1:1, the frequency on the ratio of 1:2 was higher. For example, at the ratio of 1:2, the frequency of the anode group (6.43 × 10^− 4 per recipient cell) was 2.92-fold higher than that of anode group (2.20 × 10^− 5 per recipient cell) at the ratio of 1:1 respectively after mating 1 h (Fig. 2D). Meanwhile, when the ratio was 2:1, the frequencies of the anode (7.24 × 10^− 4 per recipient cell) and the control group (4.65 × 10^− 4 per recipient cell) were higher than that at the ratio of 1:1 after mating 3 h (Fig. 2E).

The 1:1 ratio of donor/recipient cells often used to access the conjugative transfer frequency in the experiment, but it was not probably confronted in the environment. The above results clarified that when the applied voltage was 1.5 V, the order of frequency under different donor/recipient ratios generally was 2:1 > 1:2 > 1:1. It suggested that frequency was mainly affected by the number of the donor cells in MECs. It should be noted that frequency was calculated by dividing the transconjugant numbers by the recipient numbers. Under the continuous electrical stimulation, more transconjugants were formed as the more donor cells could show more opportunities to contract with recipient cells (Ding et al., 2016). A previous study also found that the donor/recipient ratio show an influence on the frequency, and the highest frequency occurred at the ratio was 1.5:1 comparing to the 0.5:1 and 1:1 ratios (Zhang et al., 2017). Additionally, the increasing number of recipient cells could also slightly promote the frequency and was in accordance with the previous studies (Lécuyer et al., 2018).

3.3 Concentration affects the conjugative transfer frequency

To investigate the effects of bacterial concentrations on the conjugative transfer frequency of ARGs in the MECs, there different bacterial concentrations (10⁶, 10⁷, and 10⁸ CFU/mL) were involved in our study. Likely, the applied voltage was 1.5 V and the ratio of donor/ recipient cells was 1:1. As showed in Fig. 4, bacterial concentration affected the frequencies. It was seen that the frequency of 10⁷ of bacterial concentration was higher than that on 10⁶ and 10⁸ in the signal-chamber MEC (Fig. 4A, B, and C). For example, when the mating time was 1 h (Fig. 4A), the frequency on 10⁷ of bacterial concentration (2.28 × 10^− 3 per recipient cell) was 2.17-fold and 6.88-fold higher than that on 10⁶ (1.05 × 10^− 3 per recipient cell) and 10⁸ (3.31 × 10^− 4 per recipient cell).

Similar to the results obtained from the signal-chamber MECs, frequencies in the two-chamber MECs showed an obvious difference among bacterial concentration, the frequency of 10⁷ bacterial concentrations in the control group showed the highest frequency. For example, the frequency of 10⁷ bacterial concentration (2.73 × 10^− 4 per recipient cell) was higher than 10⁶ (9.78 × 10^− 5 per recipient cell) and 10⁸ (1.61 × 10^− 4 per recipient cell) in the control group after mating 1 h (Fig. 4D). When the mating time was 6 h, the frequencies both on 10⁷ in the anode chamber (5.07 × 10^− 4 per recipient cell) and control group (4.47 × 10^− 4 per recipient cell) were higher than that of 10⁶ and 10⁸ under 1.5 V (Fig. 4F).

The bacterial concentration was found in activated sludge could reach from 10⁵ to 10⁸ CFU/mL (Kim et al., 2014), and bacterial concentrations adapted on conjugative transfer in experiment mostly ranged from 10⁶ to 10⁹ CFU/mL (Guo et al., 2015; Qiu et al., 2012). Generally, the bacterial concentration has a more obvious effect on conjugative transfer frequency in the MECs. The frequency of 10⁷ bacterial concentration was the highest in our study. This phenomenon was in accord with that in the previous study (Zhang et al., 2017). This result may be due to the higher bacterial concentration allowing for the more chances of bacteria contact and lead to the conjugative transfer increase with the bacterial concentration (Zhang et al., 2017). It should be noted that the large standard deviation on 10⁶ CFU/mL bacteria, and its frequency fluctuated at different mating time in the two-chamber MECs. It may be contributed to the nonuniform of transconjugant distribution during sample collection.

3.4 Conjugative transfer frequency in the real wastewater

We confirmed the conjugative transfer frequency in MECs using actual wastewater under the expose of different voltages. The results revealed that voltages could enhance the conjugative transfer frequency in MECs (Fig. 5). For instance, in single-chamber MEC, voltages significantly (p < 0.01) increased the conjugative transfer frequency. The frequency at 2.5 V (5..93 × 10^− 3 per recipient cell) was 421-fold higher than the control group (1.41 × 10^− 4 per recipient cell) after mating 6 h (Fig. 5A). In two-chamber MEC, the conjugative transfer frequency increased when voltage increased from 0.9 V to 1.5V (Fig. 5B). The frequency in the anode chamber significantly (p < 0.05) increase under all voltages compared with the control group, and higher than that in the cathode chamber. For instance, the frequency in anode chamber at 1.5 V (2.28 × 10^− 4 per recipient cell) was 5.61-fold and 11.3-fold higher than that in the cathode chamber (4.04 × 10^− 4 per recipient cell) and control group (2.05 × 10^− 5 per recipient cell) respectively.

The increase in conjugative transfer frequency of ARGs in wastewater might cause by the applied voltages, and this had been found in this study. Alternatively, it is possible that the contaminants in wastewater are likely to accelerate the conjugative transfer in MECs. For example, heavy metals (Zhang et al., 2018), ionic liquid (Lu et al., 2020), and disinfectants (Zhang et al., 2017) could directly or indirectly facilitate the conjugative transfer. Thus, it is reasonable to hypothesize that when MECs was treating various wastewater, voltages and contaminants could interact to accelerate the spread of ARGs.

3.5 Voltages increase ROS production and cell membrane permeability

MECs as an electrochemical reactor were able to induce oxidative stress, which might raise ROS levels, and cause the enhancement of conjugative transfer (Lu et al., 2020). Thus, this study investigated the ROS production and cell membrane permeability changes under different voltages in MECs. As shown in Fig. 6A, ROS production increased with the increase of voltages (from 0.9 V to 2.5 V) in MECs (p < 0.05). ROS production under 2.5V in two-chamber MECs anode was 1.55-flod higher than the control group (0 V) and also higher than that in signal-chamber MECs and two-chamber MECs cathode. Voltages also increased the cell membrane permeability in MECs (Fig. 6B). For example, cell membrane permeability showed a significant increase when the voltage was 1.5 V and 2.5 V (p < 0.01). Cell membrane permeability in signal-chamber MECs under 1.5 V was 1.18-fold higher than that under 0 V.

Previous studies found that the increased conjugative transfer frequency could cause by the enhancement of ROS production and cell membrane permeability. The over-production of ROS could cause the membrane damage and induced the SOS response (Volpe et al., 2018; Zhang et al., 2017), which finally promotes the conjugative transfer frequency. Meanwhile, the extensive cell membrane could accelerate DNA uptake during bacterial transformation (Chen et al., 2004), and the increased cell membrane permeability caused by sub-concentrations disinfectant also promote the horizontal transfer of ARGs (Zhang et al., 2017). In this study, the applied voltages from 0.9 V to 2.5 V were confirmed that enhance the ROS production and cell membrane permeability (Fig. 6), and this may accelerate conjugative transfer in MECs.

4.Conclusion

This study demonstrated that the two types of MECs, such as the single-chamber and the two-chamber, exhibited potential risk of ARGs conjugative transfer. The increased applied voltages could accelerate the ARGs transfer in the two types of systems. Bacterial concentration reached a higher frequency at 10⁷ CFU/mL in the MECs. The donor cells (donor/recipient ratio was 2:1) showed favor on the transfer frequency. Voltages could increase ROS production and cell membrane permeability in MECs. This study clarifies the conjugation of ARGs and helps us understand the fate of ARGs in the MECs. More studies should be conducted to explore the mechanism of ARGs conjugative transfer in the systems.

Acknowledgments

The authors would like to thank Shun Zhu, and KeZhe Gao for excellent experimental support during the TEM test.

Authors’ contributions

Zhao-Bing Guo, Shuai Zhang, and Hai-Liang Song were responsible for the experiment design. Wen-Long Sun, Xiao-Jun Zuo, and Hao Lingperformed the experiments and drafted the manuscript. Zhao-Bing Guo and Shuang Zhang carried out the data processing. All the authors revised the version of the manuscript. All the authors read and approved the version of the manuscript.

Funding

This work was supported by Natural Science Foundation of Jiangsu Province (BK20200816), National Natural Science Foundation of China (41571476), Key University Science Research Project of Jiangsu Province (20KJB610009), and Science and technology innovation project for overseas students in Nanjing. Xiao-Jun Zuo would like to acknowledge the project of Jiangsu Specially-Appointed Professor (1421071801005).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Compliance with ethical standards

Competing interests The authors declare that they have no competing

Ethical approval and consent to participate Not applicable

Consent for publication Not applicable

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Reviews received at journal
27 Aug, 2021
Editor assigned by journal
17 Jun, 2021
First submitted to journal
16 Jun, 2021

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Antibiotic Resistance Gene Was Increased via Horizontal Transfer in Single and Two-Chamber Microbial Electrolysis Cells

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

3. Results

3.1 Voltage increases the conjugative transfer frequency

3.2 Ratio affects the conjugative transfer frequency

3.3 Concentration affects the conjugative transfer frequency

3.4 Conjugative transfer frequency in the real wastewater

3.5 Voltages increase ROS production and cell membrane permeability

4. Conclusion

Declarations

References

Status:

Version 1