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Article

Effects of Nano-Titanium Dioxide on the Horizontal Transfer of Antibiotic Resistance Genes in Microplastic Biofilms

by
Yangyuan Zhou
1,2,
Guosheng Zhang
1,2,
Dawei Zhang
1,2,
Weiying Li
1,2,3,*,
Ningzheng Zhu
2,
Jinpei Bo
2,
Xiangzhou Meng
1,2,
Yao Chen
3,
Yu Qin
3 and
Huajie Liu
4
1
State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
2
Jiaxing Key Laboratory of Environmental Risk Source Identification and Control, Jiaxing-Tongji Environmental Research Institute, 1994 Linggongtang Road, Jiaxing 314051, China
3
School of River and Ocean Engineering, Chongqing Jiaotong University, Chongqing 400074, China
4
School of Chemical Science and Engineering, Tongji University, Shanghai 200092, China
*
Author to whom correspondence should be addressed.
Water 2024, 16(15), 2155; https://doi.org/10.3390/w16152155
Submission received: 3 July 2024 / Revised: 27 July 2024 / Accepted: 29 July 2024 / Published: 30 July 2024
Browse Figures
Review Reports Versions Notes

Abstract

:
Emerging pollutants such as microplastics in water environments readily accumulate microorganisms on their surfaces, forming biofilms and concentrating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Consequently, microplastic biofilms have attracted the attention of researchers. Horizontal gene transfer (HGT) of ARGs is one of the primary ways that bacteria acquire antibiotic resistance. Most studies focus on the effects of nanomaterials on suspended bacteria, but microplastic biofilms as hotspots for horizontal gene transfer also warrant significant investigation. This study primarily explored and compared the effects of nano-titanium dioxide on the conjugation transfer frequency of ARGs in suspended bacteria and microplastic biofilms. Nano-titanium dioxide could promote ARG conjugation in both suspended bacteria and microplastic biofilms, with a greater effect on the former. The mechanism involved nano-titanium dioxide promoting the production of reactive oxygen species (ROS) in suspended and biofilm bacteria, increasing the synthesis of outer membrane proteins, enhancing the cell membrane permeability, and elevating the expression levels of conjugation-related genes, thereby facilitating the conjugation transfer of ARGs. Biofilm bacteria, being heavily encased and protected by extracellular polymeric substances (EPS), exhibit greater resistance to external environmental pressure, resulting in the weaker impact of nano-titanium dioxide on biofilm bacteria compared to suspended bacteria. This study reveals the risk of ARG conjugation transfer within microplastic biofilms induced by nanomaterials, providing valuable insights into the risks of microplastic and antibiotic resistance dissemination in water environments.

1. Introduction

The issue of antibiotic resistance has garnered widespread attention from scholars all over the world [1,2], as antibiotics simultaneously eradicate pathogenic bacteria and contribute to the emergence of resistance [3,4]. The presence of antibiotic-resistant bacteria (ARB) diminishes or nullifies the efficacy of antibiotics in treating bacterial infections [5]. Additionally, antibiotic resistance genes (ARGs) spread among bacteria through vertical gene transfer (VGT) and horizontal gene transfer (HGT), leading to the emergence of multidrug-resistant superbugs [6,7,8]. It is predicted that, by 2050, ARB infections will result in the deaths of 10 million people globally [9]. The persistent presence and spread of ARGs in water environments pose a significant threat to human health, with the potential risks surpassing those of the antibiotics themselves [10,11].
Microplastics (<5 mm) are emerging pollutants in water environments [12]. Researchers have detected microplastics in surface water worldwide (Tables S1 and S2). For example, large amounts of microplastics have been detected in the surface seawater of the Northwest Pacific, with concentrations ranging from 640 to 42,000 particles/km2 [13]. Common types of microplastics found in the oceans include polyvinyl chloride (PVC), polypropylene (PP), polyethylene (PE), and polystyrene (PS) [14,15]. In the Maowei Sea, the concentration of microplastics in sediments is approximately 500–900 particles/kg dry weight (dw), which is significantly lower than the microplastic concentration in the inflow area (1800–2300 particles/kg dw) [16], indicating that the lower water flow rate at the estuary accelerates the deposition of microplastics from water to sediments. Therefore, water environments are one of the major reservoirs of microplastics [17,18].
Microorganisms of various sizes and types in water easily attach to the surfaces of microplastics [19], forming microplastic biofilms. The community structure of microplastic biofilms significantly differs from that of natural biofilms on surfaces like stones and wood in water, with the α-diversity (richness and evenness) of these communities being lower than in nature and with larger amounts of microorganisms from the Pyrus genus and algae [20]. Microplastics can serve as carriers, accumulating ARB and ARGs on their surfaces, leading to a significant increase in ARG abundance [21]. The unique three-dimensional structure of microplastic biofilms reduces the effectiveness of sterilization techniques in removing ARB and ARGs [22]. ARB and ARGs can adhere to the surfaces of microplastics and persist in various water environments [23], such as estuaries and oceans, for extended periods, and they can also spread through the aquatic food chain with microplastics, posing a threat to aquatic ecosystems. Therefore, compared to natural biofilms in water, microplastic biofilms are unique in their widespread distribution, large quantity, and ease of spreading. ARGs in microplastic biofilms are more difficult to remove than suspended ARGs in water, posing a greater threat to human health.
Horizontal gene transfer is one of the most crucial pathways through which bacteria acquire antibiotic resistance, encompassing conjugation, transformation, and transduction [1,24]. Water environments are significant sites for the horizontal transfer of ARGs [25,26]. Studies have shown that most ARGs in wastewater treatment plants are located on mobile elements such as plasmids, and the risk of antibiotic resistance’s spread through HGT in water environments has garnered considerable attention from researchers [27,28].
Nano-titanium dioxide is a white, loose powder known for its UV resistance, antibacterial properties, self-cleaning ability, and anti-aging performance. It is utilized in various fields, including cosmetics, functional fibers, plastics, inks, coatings, paints, fine ceramics, and water treatment [29,30,31]. When nano-titanium dioxide is released into water environments, its concentration is relatively low, but it may be higher when used in water treatment processes. Consequently, the potential hazards of nano-titanium dioxide in water environments have raised concern. In 2012, Qiu et al. first reported that nano-oxides in water environments could induce bacterial oxidative stress, increase the cell membrane permeability, alter the expression of genes related to conjugation transfer, and promote the horizontal transfer of ARGs mediated by the RP4 plasmid [32]. Subsequent studies have reported the impact of environmental factors like nanomaterials on the conjugation transfer of ARGs among suspended bacteria [33,34,35]. However, microplastic biofilms, as hotspots for ARG conjugation transfer, may pose a greater risk for ARG dissemination compared to suspended bacteria. The effects of nano-titanium dioxide on the conjugation transfer of ARGs between suspended bacteria and microplastic biofilm bacteria in water environments are less reported and require urgent investigation to elucidate the mechanisms involved.
In summary, this study primarily compared the effects of nano-titanium dioxide on the conjugation transfer of ARGs between suspended bacteria and microplastic biofilm bacteria. First, the effect of nano-titanium dioxide on ARG conjugation was examined. Next, the effects of nano-titanium dioxide on intracellular oxidative stress, bacterial outer membrane proteins, and cell membrane permeability were explored. Finally, the influence of nano-titanium dioxide on the expression levels of conjugation-related genes was investigated, with a particular focus on elucidating the mechanisms by which nano-titanium dioxide promotes ARG conjugation.

2. Materials and Methods

2.1. Bacterial Strains and Microplastics

The donor bacteria used in this study were Escherichia coli (E. coli) HB101 (RP4) carrying the RP4 plasmid, which harbors ARGs for kanamycin, tetracycline, and ampicillin resistance. This strain was kindly provided by researcher Li Junwen from the Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, China. HB101 (tra-mutant RP4), which carries a tra-mutant RP4 plasmid [32], differs from the former in that the genes regulating conjugative transfer have been removed by restriction enzyme digestion. Consequently, it cannot express or synthesize conjugative pili and is incapable of conjugation, but it can still undergo transformation. This strain was used to exclude the possibility of transformation transfer occurring through the natural release of the RP4 plasmid by bacteria. The recipient bacteria used were E. coli NK5449, resistant to rifampicin and purchased from the China General Microbiological Culture Collection Center (catalog number 1.1437). The microplastic type used in this study was PVC, purchased from the International Plastics Market in Dongguan, China, with dimensions of 4.0 × 4.0 × 0.2 mm. Nano-titanium dioxide was purchased from Shanghai Aladdin Biochemical Technology Co. Ltd., Shanghai, China. Nano-titanium dioxide is anatase and hydrophilic, with a particle size of 5–10 nm. An electron microscopy image of nano-titanium dioxide is shown in Figure S5.

2.2. Bacterial Culture

The glycerol stock of E. coli was removed from the low-temperature freezer and thawed at room temperature. In a sterile hood, a small amount of the bacterial suspension was added to LB liquid medium containing 50 mg/L kanamycin, 32 mg/L tetracycline, and 100 mg/L ampicillin. The medium was incubated overnight in a constant-temperature shaker. Then, 20 mL of the bacterial suspension was transferred into a sterilized centrifuge tube and centrifuged at high speed (8000 rpm, 10 min). The supernatant was discarded, and the bacterial pellet at the bottom of the centrifuge tube was washed with sterilized simulated estuarine water to remove residual antibiotics and medium. The bacterial pellet was resuspended in an appropriate amount of simulated estuarine water (specific details are described in the Supporting Information) to achieve a suitable concentration of the bacterial suspension. The glycerol stock of E. coli NK5449 was thawed at room temperature, and a small amount of the bacterial suspension was added to LB liquid medium containing 160 mg/L rifampicin in a sterile hood. After overnight incubation, a suitable concentration of bacterial suspension was prepared following the same steps as described above.

2.3. Effects of Nano-Titanium Dioxide on Conjugation Transfer of ARGs

HB101 (RP4) was selected as the donor bacterium, and NK5449 was chosen as the recipient bacterium. The experimental conditions included an initial bacterial concentration of approximately 108 CFU/mL, pH = 7.4, and a PVC microplastic concentration of 1.0 particles/mL, with a donor/recipient ratio of 1:1. Nano-titanium dioxide was added to the reaction system to achieve final concentrations of 0, 0.005, 0.05, 0.5, 5.0, and 50 mmol/L. The mixture was placed in a constant-temperature shaker at 25 °C and 160 rpm for 72 h. Microplastics were randomly selected from the reaction system; each microplastic was placed in 5 mL PBS and washed slowly for 3–5 min. This was repeated 3–5 times, ensuring that no bacteria were detected in the final PBS wash. Bacteria were eluted from the microplastics using previously established methods [36,37], with treated biofilms dispersed in PBS and ultrasonically treated at 4 °C in a water bath sonicator at 50 kHz for 5 min and then resuspended in PBS solution. The mixture was diluted using a gradient dilution method with PBS buffer solution. Then, 100 microliters of the diluted mixture were placed onto LB agar plates (containing 160 mg/L rifampicin) to culture the recipients and onto LB solid plates containing 160 mg/L rifampicin, 50 mg/L kanamycin, 32 mg/L tetracycline, and 100 mg/L ampicillin to culture the transconjugants. The mixture was spread evenly using a spreader, and the LB plates were inverted and incubated in a constant-temperature incubator at 37 °C. Colony counts were conducted after 24 h, with each experiment repeated three times. The detection of transconjugants in suspended bacteria followed the same method as for biofilm bacteria, with each experiment repeated five times. Additionally, HB101 (tra-mutant RP4), which carries an RP4 plasmid with the conjugation transfer region removed, was used as a control to exclude the transformation transfer of the RP4 plasmid. Experimental verification showed that the impact of RP4 transformation transfer in the conjugation experiment was negligible. The frequency of conjugative transfer was calculated using the following formula:
Frequency   of   conjugative   transfer = N u m b e r   o f   t r a n s c o n j u g a n t s   ( C F U / m L ) N u m b e r   o f   r e c i p i e n t s   ( C F U / m L )
where the number of recipients refers to the number of E. coli NK5449, and the number of transconjugants refers to the number of E. coli that obtain ARGs through conjugation.

2.4. mRNA Expression Analysis

The relevant gene expression information that was detected is shown in Table S3. After the reaction was completed, mRNA was extracted from the reaction using a bacterial RNA extraction kit from TianGen Biotech Co. Ltd., Beijing, China, following the kit’s instruction manual. The purified RNA had an A260/A280 ratio of approximately 2.0 and an A260/A230 ratio of over 2.0 (measured with a NanoDrop 2000C, NanoDrop Technologies, Wilmington, DE, USA), indicating that the purified RNA was free from contaminants such as DNA and proteins. The RNA was then reverse-transcribed into DNA using a rapid reverse transcription kit from TianGen Biotech (Beijing) Co., Ltd., with operations conducted strictly according to the instructions. The purified DNA had an A260/A280 ratio between 1.6 and 1.8 and an A260/A230 ratio greater than 2.0. Using 16S rRNA as the internal reference gene, a real-time fluorescence quantitative PCR was performed with an ABI 7500 real-time fluorescence quantitative PCR instrument (Applied Biosystems) to detect the relative expression levels of the target genes. The changes in the expression of the target genes under different conditions were expressed as fold changes in gene expression, calculated using the following formula:
Δ C T = Δ C T ( T a r g e t   g e n e ) − Δ C T ( 16 S   r R N A )
Δ Δ C T = Δ C T ( e x p e r i m e n t a l   g r o u p ) − Δ C T ( c o n t r o l   g r o u p )
F C = 2 − Δ Δ C T
where CT represents the cycle threshold, which is the number of cycles required for the fluorescence signal in each PCR reaction well to reach the set threshold; the target gene refers to the specific genes listed in Table S3; FC (fold change) represents the fold change in the gene expression level before and after different treatments.

2.5. Other Analytical Methods and Data Analysis

Superoxide dismutase (SOD), lactate dehydrogenase (LDH), glutathione peroxidase (GSH-Px), reactive oxygen species (ROS), catalase (CAT), and adenosine triphosphate (ATP) were detected using corresponding kits from the Nanjing Jiancheng Bioengineering Institute. The specific experimental procedures were carried out according to the instructions provided with the kits. Detailed information is described in the Supporting Information.
Each experiment was conducted five times to ensure reliability. Statistical analysis to determine significant differences between the experimental and control groups was performed using Student’s t-test, with the significance threshold set at p < 0.05. For multiple comparisons among the experimental groups, the Student–Newman–Keuls (S-N-K) test was employed. All statistical analyses were performed using SPSS version 19.0. The null hypothesis, which posited no difference in frequency between different samples, was rejected at a value less than or equal to 0.05.

3. Results and Discussion

3.1. Effects of Nano-Titanium Dioxide on Conjugation of ARGs in Microplastic Biofilms

As shown in Figure 1A, the conjugation transfer frequency of the RP4 plasmid among suspended bacteria significantly increased, up to approximately 31-fold, with the addition of nano-titanium dioxide (>0.5 mmol/L). When the concentration of nano-titanium dioxide reached 50 mmol/L, the conjugation frequency of ARGs decreased, due to the inactivation effect of high concentrations of nano-titanium dioxide on suspended bacteria (Figure S2; 96% suspended bacteria were inactivated), leading to bacterial death and a subsequent decrease in the conjugation frequency. In the absence of nano-titanium dioxide, the conjugation transfer frequency of the RP4 plasmid in biofilms was about 9.8 times higher than in suspended bacteria, indicating a higher frequency of antibiotic plasmid conjugation transfer in biofilms compared to suspended bacteria. This was due to the higher density of the bacteria in the biofilms, facilitating greater donor and recipient bacteria interactions and material exchange, or due to the unique gene expression profiles in biofilm bacteria that favor conjugation transfer. In the microplastic biofilms, there was no significant difference in the conjugation frequency between the experimental group and the control group when the concentration of nano-titanium dioxide was below 5.0 mmol/L. Only when the concentration of nano-titanium dioxide exceeded 50 mmol/L did the conjugation frequency in the biofilm experimental group significantly increase, up to approximately 9.7-fold. This was much lower than the 31-fold increase observed in suspended bacteria. This suggests that biofilm bacteria are much less affected by nano-titanium dioxide compared to suspended bacteria. This resistance is attributed to the aggregation of microorganisms in biofilms, which are encased in extracellular polymeric substances (EPS) that provide resistance to external stresses such as nano-titanium dioxide. When the concentration of nano-titanium dioxide was less than or equal to 0.5 mmol/L, the conjugation transfer frequency in the biofilms was significantly higher than in the suspended bacteria, primarily due to biofilm formation rather than the effect of nano-titanium dioxide. At a concentration of 5 mmol/L, the conjugation transfer frequency in the biofilms was significantly lower than in the suspended bacteria, indicating that both biofilm formation and nano-titanium dioxide influence conjugation, reaffirming that biofilm bacteria are less affected by nano-titanium dioxide than suspended bacteria. This also indicates that biofilm formation does not always promote conjugation transfer; for example, at a nano-titanium dioxide concentration of 5 mmol/L, the formation of microplastic biofilms inhibited ARG conjugation compared to suspended bacteria. At higher concentrations of nano-titanium dioxide (>50 mmol/L), the conjugation frequency of ARGs in microplastic biofilms was higher than in suspended bacteria, because high concentrations of nano-titanium dioxide inactivate suspended bacteria but have limited inactivation effects on microplastic biofilm bacteria.
As shown in Figure 1B, the conjugation transfer frequency of ARGs in the microplastic biofilms initially increased rapidly with the conjugation reaction time and then maintained slow growth or even remained unchanged. During the reaction time of 0–12 h, since the microplastic biofilms had not yet begun to form and there were no stable bacterial attachments on the microplastic surface, the conjugation frequency could not be detected. When the reaction time exceeded 24 h, the microplastic biofilms had preliminarily formed and gradually matured, allowing the detection of the conjugation frequency. During the conjugation transfer process of ARGs, donor bacteria HB101 and recipient bacteria NK5449 establish contact through their cell membranes and form a pilus channel, through which gene transfer occurs. The RP4 resistance plasmid, carrying ARGs, acts as a vector and can move from the donor bacteria to the recipient bacteria through this channel. This conjugation transfer process depends on the expression of a series of specific genes; the proteins encoded by these genes play crucial roles in establishing the gene transfer channel and facilitating gene transfer during conjugation. This process is similar to active transport, requiring protein involvement and a certain amount of time. Additionally, the conjugation transfer process is often regulated by cellular oxidative stress. Therefore, subsequent investigations will focus on the levels of oxidative stress, the outer membrane proteins, and the expression of genes related to the conjugation transfer process to elucidate the mechanisms by which microplastic biofilms promote ARG conjugation.

3.2. Effects of Nano-Titanium Dioxide on ROS

Next, the effects of nano-titanium dioxide on intracellular catalase, superoxide dismutase, glutathione peroxidase, and reactive oxygen species in bacteria were examined (Figure 2). The experimental results indicated that nano-titanium dioxide could enhance the activity of oxidative stress enzymes within the cells. For instance, nano-titanium dioxide increased the SOD activity in suspended bacteria by 6.2 times and in microplastic biofilm bacteria by 3.7 times. At a nano-titanium dioxide concentration of 50 mmol/L, a large number of suspended bacteria died, resulting in a decrease in SOD activity, whereas nano-titanium dioxide barely inactivated the microplastic biofilm bacteria (Figure S2) but could enhance the SOD activity within them. These results are consistent with the trends observed in Figure 1. Additionally, nano-titanium dioxide promoted an increase in the intracellular ROS content. These findings suggest that nano-titanium dioxide can induce oxidative stress in cells, increase the intracellular ROS concentrations, potentially enhance the cell membrane permeability, and alter the expression levels of genes related to conjugation, thereby affecting ARG conjugation transfer. Moreover, at concentrations below 5 mmol/L, the oxidative stress effect of nano-titanium dioxide on suspended bacteria was greater than on microplastic biofilm bacteria, indicating that the formation of microplastic biofilms can mitigate the oxidative stress impact of nano-titanium dioxide on bacteria. Consequently, the expression levels of genes associated with bacterial oxidative stress were subsequently investigated to further explore the oxidative stress conditions in bacteria.

3.3. Effects of Nano-Titanium Dioxide on Oxidative Stress

The expression levels of oxidative stress genes in the suspended bacteria and microplastic biofilm bacteria are shown in Figure 3. The expression levels of rpoS, oxyR, soxR, and marA [38], which are related to oxidative stress, were studied. Overall, the expression of oxidative stress-related genes in the suspended and biofilm bacteria was upregulated to varying degrees after the application of nano-titanium dioxide, with trends consistent with those in Figure 1. The oxyR regulatory protein, encoded by the oxyR gene, is involved in various physiological and metabolic activities, including the antioxidant response, the inhibition of spontaneous mutations, pathogenicity, iron metabolism, and outer membrane protein phase transition. The upregulation of oxyR gene expression indicates that nano-titanium dioxide induces the production of a large amount of oxidative substances within the bacteria. In response, the bacteria upregulate oxyR gene expression to produce the OxyR regulatory protein, attempting to regulate their physiological balance and maintain normal life activities.
These experimental results suggest that nano-titanium dioxide induces oxidative stress in both suspended and microplastic biofilm bacteria, potentially increasing the biofilm permeability to promote ARG conjugation transfer. Additionally, nano-titanium dioxide can promote intracellular ATP production (Figure S3), enhancing the energy supply during the conjugation transfer process and thereby facilitating ARG conjugation transfer. The formation of microplastic biofilms can mitigate the effects of nano-titanium dioxide on the expression levels of oxidative stress-related genes and the intracellular ATP concentration in bacteria, potentially reducing the impact of nano-titanium dioxide on ARG conjugation transfer.

3.4. Effects of Nano-Titanium Dioxide on Outer Membrane Proteins and Cell Membrane Permeability

Next, the expression levels of genes related to outer membrane protein synthesis (ompA, ompC, and ompF) were examined (Figure 4). The results showed that nano-titanium dioxide could promote the expression of the ompA, ompC, and ompF genes, with biofilm bacteria exhibiting unique gene expression levels distinct from those of suspended bacteria. For example, 5.0 mmol/L nano-titanium dioxide increased the expression levels of ompA among suspended bacteria by 19.6 times but had no significant effect on biofilm bacteria. At 50 mmol/L nano-titanium dioxide, the expression levels of ompA among suspended bacteria decreased, while the expression levels in microplastic biofilms increased by 11.6 times. These experimental results are consistent with those in Figure 1. Outer membrane proteins are a class of proteins located in the outer membranes of bacteria, primarily functioning to form channels in the bacterial outer membrane, through which nutrients, drugs, and toxins can be transported. OmpA, OmpC, and OmpF are three common outer membrane proteins [39]. The protein encoded by the ompA gene, known as outer membrane protein A (OmpA), is a monomeric protein of approximately 36 kDa, mainly found in the outer membranes of Gram-negative bacteria such as E. coli. OmpA has various biological functions, including being a major structural component of the cell exterior, promoting cell adhesion and the invasion of host cells, and stimulating immune responses. The ompA gene is widely present in many bacteria and is considered an important marker of bacterial pathogenicity. OmpC and OmpF are two other common outer membrane proteins, with sizes of 40 kDa and 42 kDa, respectively. They are also mainly found in the outer membranes of Gram-negative bacteria and belong to the porin family. These proteins can form water channels that help bacteria to regulate the osmotic pressure between the inside and outside of the cell under adverse conditions. The expression of the ompC gene in some bacteria is also associated with antibiotic resistance. The functions of these proteins include regulating the permeability of the bacterial outer membrane and maintaining the stability of the intracellular environment. An increase in outer membrane proteins implies enhanced cell membrane permeability, facilitating the conjugation transfer of ARGs. Therefore, nano-titanium dioxide can promote the conjugation transfer of ARGs by accelerating the synthesis of outer membrane proteins.
When the bacterial cell membrane is damaged, its permeability increases, leading to the release of lactate dehydrogenase (LDH) into the external environment. Therefore, changes in LDH release can be used to indicate changes in bacterial cell membrane permeability [40]. As shown in Figure 4D, nano-titanium dioxide can promote the release of LDH from both suspended bacteria and microplastic biofilm bacteria, thereby increasing the cell membrane permeability. The effect of nano-titanium dioxide on microplastic biofilms is less pronounced than on suspended bacteria. These experimental results indicate that nano-titanium dioxide can cause damage to bacterial cell membranes (Figure S4), promote the synthesis of outer membrane proteins, increase the porin channels for material exchange between bacteria and the external environment, and enhance the cell membrane permeability, thereby facilitating the conjugation transfer of ARGs.

3.5. Mechanism of Microplastic Biofilms Promoting Conjugation of ARGs

Finally, the expression levels of genes related to the conjugative transfer process were analyzed (Figure 5). Nano-titanium dioxide significantly promoted the expression levels of these genes. For instance, nano-titanium dioxide at 50 mmol/L increased the expression level of the traG gene in microplastic biofilm bacteria by 9.2 times, while 5.0 mmol/L of nano-titanium dioxide increased the expression level of the traG gene in suspended bacteria by 7.6 times. The same concentration of titanium dioxide had a greater impact on suspended bacteria than on microplastic biofilms. The traA gene encodes 121 amino acids with a molecular weight of 13 kDa, and its main peptide sequence comprises the first 51 amino acids. The mature pilin protein has a molecular weight of 7 kDa and contains 70 amino acid residues. The conjugation transfer of ARGs requires the formation of a gene transfer channel between the donor and recipient bacteria via conjugative pili, which are primarily composed of pilin subunits (the expression product of the traA gene). Therefore, an increase in conjugative pili synthesis facilitates the conjugation transfer process.
When donor bacteria synthesize the pilin proteins encoded by resistance plasmids, these pili can rapidly establish contact with surrounding cells, inducing a connection between the two cells [41]. During this process, the partial fusion of the cell membranes occurs, forming a structure known as the conjugation bridge. The plasmid DNA in the donor bacterium is cleaved at the oriT region, forming single-stranded DNA, which then traverses the conjugation bridge to enter the recipient bacterium. Subsequently, the single-stranded DNA in both the donor and recipient bacteria is replicated to form complete double-stranded plasmids. The traG and trbBp genes regulate the conjugation pairing process, and the formation of microplastic biofilms can promote their expression, thereby facilitating the conjugation transfer of ARGs.
After the conjugation pairing system is formed, the plasmid DNA in the donor bacterium is cleaved at the oriT region to form a single strand, which crosses the conjugation bridge to enter the recipient bacterium. Subsequently, the single-stranded DNA in both the donor and recipient bacteria is replicated to form complete double-stranded plasmids. Therefore, the DNA transfer and replication system represents the final stage of conjugation transfer. The trfAp gene, related to plasmid transfer and replication, encodes a transcriptional regulatory factor that belongs to a subtype of the Rep family. This regulatory factor binds to specific sequences on plasmid DNA and regulates plasmid replication and transfer by influencing gene expression, playing a crucial role during cell growth and division.
In summary, the mechanism by which nano-titanium dioxide promotes ARG conjugation can be elucidated (Figure 6). Nano-titanium dioxide promotes the production of ROS within suspended and biofilm bacteria, increases the cell membrane permeability, enhances the bacteria’s ability to transfer genes across the cell membrane, raises the intracellular ATP levels, increases the synthesis of porin proteins, accelerates the assembly of conjugative pili, speeds up the formation of the conjugation pairing system, and accelerates the DNA replication and transfer processes, thereby facilitating the conjugation transfer of ARGs. Biofilm bacteria are extensively encased and protected by EPS, which reduces the bacteria’s contact with external environmental factors and enhances their resistance to environmental stress, resulting in a weaker impact of nano-titanium dioxide on biofilm bacteria compared to suspended bacteria.

4. Conclusions

Nano-titanium dioxide (at concentrations lower than 5 mmol/L) can increase the conjugation frequency of ARGs in suspended bacteria by 31 times, with no significant effect on microplastic biofilm bacteria. At a concentration of 50 mmol/L, a large number of suspended bacteria die, resulting in a decrease in the conjugation frequency between suspended bacteria, but the microplastic biofilm bacteria still survive, with the ARG conjugation frequency increasing by 9.7 times. Nano-titanium dioxide promotes the production of ROS in both suspended and biofilm bacteria, increases the cell membrane permeability, enhances the bacteria’s ability to transfer genes across the cell membrane, raises the intracellular ATP levels, increases the synthesis of outer membrane proteins, accelerates the assembly of conjugative pili, speeds up the formation of the conjugation pairing system, and accelerates the DNA replication and transfer processes, thereby facilitating the conjugation transfer of ARGs. Biofilm bacteria are extensively encased and protected by EPS, which reduces their contact with external environmental factors and enhances their resistance to environmental stress, resulting in a weaker impact of nano-titanium dioxide on biofilm bacteria compared to suspended bacteria. Nano-titanium dioxide poses a risk of increasing antibiotic resistance in water environments, and its potential to promote ARG conjugation transfer should be considered when it is used in water treatment.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w16152155/s1, Figure S1: The amount of bacteria in microplastic biofilms at different nano-titanium dioxide concentrations; Figure S2: The amount of suspended bacteria at different nano-titanium dioxide concentrations; Figure S3: Effects of nano-titanium dioxide concentration on intracellular adenosine triphosphate (ATP); Figure S4: SEM of microplastic biofilms at 24 h with 50 mmol/L nano-titanium dioxide; Figure S5: Electron microscopy image of nano-titanium dioxide particles; Table S1: Distribution of microplastics in oceans around the world; Table S2: Distribution of microplastics in freshwater around the world; Table S3: Primer information for qPCR reaction. References [42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64] are cited in Supplementary Materials.

Author Contributions

Conceptualization, G.Z. and W.L.; methodology, Y.Z.; software, G.Z.; validation, D.Z., N.Z. and J.B.; formal analysis, Y.Z.; investigation, Y.Z.; resources, G.Z. and W.L.; data curation, Y.Z.; writing—original draft preparation, Y.Z. and G.Z.; writing—review and editing, W.L., X.M., Y.C., Y.Q. and H.L.; visualization, D.Z., N.Z. and J.B.; supervision, G.Z.; project administration, G.Z.; funding acquisition, W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This project was funded by the National Key Research and Development Program of China (Grant No.: 2021YFC32013-04), the National Natural Science Foundation of China (51979194), the Scientific Research Project of Fuzhou Water Supply Co., Ltd. (Grant No.: 20203000), and the Scientific Research Project of Wuxi Water Supply Co., Ltd. (Grant No.: kh0040020240152).

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors extend their gratitude to Shiyanjia Lab (www.shiyanjia.com, accessed on 15 June 2023) for providing invaluable assistance with the SEM analysis.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Water 16 02155 g001
Figure 1. Effects of nano-titanium dioxide on ARG conjugation in suspended bacteria and microplastic biofilms. (A). Effects of nano-titanium dioxide concentration on conjugation of ARGs. (B). Effects of time on conjugation of ARGs. “*” indicates that there are significant differences between the experimental groups and all the other groups.
Figure 1. Effects of nano-titanium dioxide on ARG conjugation in suspended bacteria and microplastic biofilms. (A). Effects of nano-titanium dioxide concentration on conjugation of ARGs. (B). Effects of time on conjugation of ARGs. “*” indicates that there are significant differences between the experimental groups and all the other groups.
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Figure 2. Effects of nano-titanium dioxide on oxidative stress. (A) Effects on superoxide dismutase (SOD), (B) catalase (CAT), and (C) glutathione peroxidase (GSH-Px). (D) Effects of nano-titanium dioxide on ROS. “*” indicates that there are significant differences between the experimental groups and all the other groups.
Figure 2. Effects of nano-titanium dioxide on oxidative stress. (A) Effects on superoxide dismutase (SOD), (B) catalase (CAT), and (C) glutathione peroxidase (GSH-Px). (D) Effects of nano-titanium dioxide on ROS. “*” indicates that there are significant differences between the experimental groups and all the other groups.
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Figure 3. Effects of nano-titanium dioxide on gene expression levels associated with oxidative stress. Effects on (A) rpoS, (B) oxyR, (C) soxR, and (D) marA expression levels. “*” indicates that there are significant differences between the experimental groups and all the other groups.
Figure 3. Effects of nano-titanium dioxide on gene expression levels associated with oxidative stress. Effects on (A) rpoS, (B) oxyR, (C) soxR, and (D) marA expression levels. “*” indicates that there are significant differences between the experimental groups and all the other groups.
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Figure 4. Effects of nano-titanium dioxide on outer membrane proteins and cell membrane permeability. Effects on the expression levels of (A) ompA, (B) ompC, and (C) ompF. (D) Effects on lactic dehydrogenase (LDH) release. “*” indicates that there are significant differences between the experimental groups and all the other groups.
Figure 4. Effects of nano-titanium dioxide on outer membrane proteins and cell membrane permeability. Effects on the expression levels of (A) ompA, (B) ompC, and (C) ompF. (D) Effects on lactic dehydrogenase (LDH) release. “*” indicates that there are significant differences between the experimental groups and all the other groups.
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Figure 5. Effects of nano-titanium dioxide on gene expression levels associated with conjugation. Effects on the expression levels of (A) traA, (B) traG, (C) trbBp, and (D) trfAp. “*” indicates that there are significant differences between the experimental groups and all the other groups.
Figure 5. Effects of nano-titanium dioxide on gene expression levels associated with conjugation. Effects on the expression levels of (A) traA, (B) traG, (C) trbBp, and (D) trfAp. “*” indicates that there are significant differences between the experimental groups and all the other groups.
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Figure 6. The mechanisms by which nano-titanium dioxide promotes ARG conjugation.
Figure 6. The mechanisms by which nano-titanium dioxide promotes ARG conjugation.
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Zhou, Y.; Zhang, G.; Zhang, D.; Li, W.; Zhu, N.; Bo, J.; Meng, X.; Chen, Y.; Qin, Y.; Liu, H. Effects of Nano-Titanium Dioxide on the Horizontal Transfer of Antibiotic Resistance Genes in Microplastic Biofilms. Water 2024, 16, 2155. https://doi.org/10.3390/w16152155

AMA Style

Zhou Y, Zhang G, Zhang D, Li W, Zhu N, Bo J, Meng X, Chen Y, Qin Y, Liu H. Effects of Nano-Titanium Dioxide on the Horizontal Transfer of Antibiotic Resistance Genes in Microplastic Biofilms. Water. 2024; 16(15):2155. https://doi.org/10.3390/w16152155

Chicago/Turabian Style

Zhou, Yangyuan, Guosheng Zhang, Dawei Zhang, Weiying Li, Ningzheng Zhu, Jinpei Bo, Xiangzhou Meng, Yao Chen, Yu Qin, and Huajie Liu. 2024. "Effects of Nano-Titanium Dioxide on the Horizontal Transfer of Antibiotic Resistance Genes in Microplastic Biofilms" Water 16, no. 15: 2155. https://doi.org/10.3390/w16152155

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