Yong Qiu

Gradient Microfluidics Enables Rapid Bacterial Growth Inhibition Testing

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask).

Aptameric detection of quinine in reclaimed wastewater using a personal glucose meter

Quantification of small-molecule analytes in an aqueous environment remains expensive due to the high costs of instruments and human resources. An aptamer–invertase biosensor coupled with a personal glucose meter (PGM) is a precise and promising method to substitute the traditional instruments. Herein, using quinine as an example, we demonstrated the feasibility of using an aptamer–invertase biosensor to quantify quinine in reclaimed wastewater. The analytical procedure consisted of building the structure of aptamer–DNA–invertase on magnetic beads, competition between quinine and invertase, catalysis of sucrose by the released invertase, and quantification of glucose by a PGM. The dissociation coefficient of aptamer and quinine was estimated to be 0.36 μM by isothermal titration calorimetry, indicating very strong affinity between them. The linear calibration curve of quinine was in the range from 0 to 2 μM. The limit of detection (LOD) was estimated to be 0.13 μM for quinine in pure water and 0.32 μM for quinine in reclaimed wastewater. The selectivity for quinine detection in the presence of other pharmaceutics was satisfactory. Thus, the aptamer–invertase–PGM method is an inexpensive, affordable and feasible choice for quinine analysis in wastewater management.

A novel microfluidic system enables visualization and analysis of antibiotic resistance gene transfer to activated sludge bacteria in biofilm

Antibiotic resistance genes (ARGs) in environment have become a growing public concern, due to their potential to be obtained by pathogens and their duplication along cell division. Horizontal gene transfer (HGT) was reported to be responsible for ARGs dissemination in microbes, but the HGT feature in environmental biofilm was still unclear due to insufficient assay tools. To address this challenge, we applied a novel microfluidic system to cultivate thin biofilm by continuous supply of nutrients and close contact between cells. Resembling the living state of biofilm in open environment, this chip visualized the transfer of ARG-encoded plasmids RP4 and pKJK5 to the receptors, e.g., activated sludge bacteria. The average plasmid transfer frequency per receptor (T/R) from RP4-hosted Pseudomonas putida KT2440 to activated sludge bacteria was quantified to be 2.5 × 10-3 via flow cytometry, and T/R for pKJK5-hosted Escherichia coli MG1655 was 8.9 × 10-3, while the corresponding average frequencies per donor (T/D) were diverse for the two host strains as 4.3 × 10-3 and 1.4 × 10-1 respectively. The difference between T/R and T/D was explained by the plasmid transfer kinetics, implying specific purposes of the two calculations. Finally, we collected the transconjugants by fluorescent activated cell sorting and further sequenced their 16S rDNA. Bacteria from phyla Proteobacteria and Firmicutes were found more susceptible to be transconjugants than those from Bacteroidetes. Our work demonstrated that microfluidic system was advantageous in biofilm HGT study, which can provide more insights into environmental ARG control.

Single bacteria studies using microfluidics

The importance of individual heterogeneity within a genetically identical population has become well recognized. However, single bacteria studies have been beset by a number of challenges ranging from single-cell handling to detection. Most of these stem from bacteria’s microscale dimensions and the complexity of their natural environment. In recent years, microfluidics has emerged as a powerful tool to manipulate single cells and their immediate microenvironments and is well suited to address these challenges. The protocols below will describe the creation of microfluidic devices for monolayer cell culture and long-term tracking of morphological dynamics from individual bacteria under precisely delivered perturbations. Step-by-step procedures for on-chip assays and morphological-based image analysis are described in detail, and these approaches enable fast quantification of bacteria growth and morphological changes under a broad range of conditions within a single experiment. Importantly, these methods do not require labeling of cells, thereby offering unique advantages in the investigation of naturally occurring microbes.

Selective effect and elimination of antibiotics in membrane bioreactor of urban wastewater treatment plant

Analyzing the temporal dynamics of antibiotics, antibiotic resistance genes (ARGs) and the functional community could contribute to the regulation and optimization of wastewater treatment plant (WWTP) operation to achieve better antibiotics and ARGs removal performances during different seasons. However, there is little research in this area. Therefore, in this study, samples were collected from the influent, activated sludge (AS), and permeate of the membrane bioreactor (MBR) in a WWTP located in Beijing, China, biweekly over 13 months, and then analyzed systematically. The removal efficiency for all detected antibiotics through biodegradation and adsorption was 59.25 ± 2.79%, with the highest rate of 64.79 ± 4.68% observed in summer, indicating that the higher temperature in summer may promote biodegradation in MBR. In contrast, the elimination of antibiotics through microfiltration was negligible and unfavorable, with negative overall removal rates. However, a positive rejection rate of 9.48 ± 8.92% was only observed in winter, indicating that a colder temperature might lead to better, but still limited, antibiotics rejection. Sulfonamides (SAs) were more likely to impose a selective pressure on their corresponding ARGs. However, due to the degradability of tetracyclines (TCs) and potential selection of ARGs in wastewater before entering WWTP, there were still TC resistances with non-detectable TCs. Significantly negative relationships were observed between the relative abundance of nitrifying bacteria (Nitrosomonas and Nitrospira) and the concentrations of certain antibiotic classes, indicating that nitrifying bacteria could be involved in the co-metabolic biodegradation of certain antibiotics through enzyme catalyzation during nitrification.

Removal of antibiotic resistance genes in four full-scale membrane bioreactors

Antibiotic resistance genes (ARGs) discharged through wastewater treatment plants (WWTPs) has aroused growing public concern for its risk to human health and ecological safety. Membrane bioreactor (MBR) has been recognized as an effective approach to remove ARGs in full-scale WWTPs, but its advantage over traditional processes was not clearly quantified. To address this, we investigated four full-scale WWTPs containing parallel MBR and traditional processes (oxidation ditch or sequencing batch reactor) to compare the reduction of eight types of ARGs (blaTEM, ermB, tetW, tetO, sul1, sul2, addD, and qnrS) and int1. In general, MBRs reduced the ARGs (1.1-7.3 log removal) better than parallel processes (0.4-4.2 log removal). Notably, the dominant ARGs in the influent, such as ermB, sul1 and int1 (106.39-107.79 copies/mL), were more effectively reduced by MBRs (1.5-7.3 log removal) than traditional processes (0.8-3.4 log removal). Meanwhile, the distribution of those ARGs in activated sludge was not significantly different between aforementioned processes (p > 0.05). The separation coefficient (Ksw) was proposed to represent the contribution of solid separation on ARG removal, subsequent analysis revealed surprisingly strong correlation between Ksw values of dominant ARGs (ermB, sul1 and int1) and their log removal by MBR (R = 0.79-0.96, p < 0.05), while such correlation was much weaker in traditional process (R = 0.33-0.37), indicating solid separation was the major pathway for removal of dominant ARGs and int1. According to the canonical correlation analysis between process operation and ARG removal in MBR, sludge retention time (SRT) seemed to be the major factor affecting removal of dominant ARGs and int1. This comparative study can be helpful for further understanding and operating MBR process to reduce the ARGs in effluent.

Simple Urea Immersion Enhanced Removal of Tetracycline from Water by Polystyrene Microspheres

Antibiotics pose potential ecological risks in the water environment, necessitating their effective removal by reliable technologies. Adsorption is a conventional process to remove such chemicals from water without byproducts. However, finding cheap adsorbents with satisfactory performance is still a challenge. In this study, polystyrene microspheres (PSM) were enhanced to adsorb tetracycline by surface modification. Simple urea immersion was used to prepare urea-immersed PSM (UPSM), of which surface groups were characterized by instruments to confirm the effect of immersion. Tetracycline hydrochloride (TC) and doxycycline (DC) were used as typical adsorbates. The adsorptive isotherms were interpreted by Langmuir, Freundlich, and Tempkin models. After urea immersion, the maximum adsorption capacity of UPSM at 293 K and pH 6.8 increased about 30% and 60%, achieving 460 mg/g for TC and 430 mg/g for DC. The kinetic data were fitted by first-order and second-order kinetics and Weber–Morris models. The first-order rate constant for TC adsorption on UPSM was 0.41 /h, and for DC was 0.33 /h. The cyclic urea immersion enabled multilayer adsorption, which increased the adsorption capacities of TC on UPSM by two to three times. The adsorption mechanism was possibly determined by the molecular interaction including π–π forces, cation-π bonding, and hydrogen bonding. The simple surface modification was helpful in enhancing the removal of antibiotics from wastewater with similar structures.

Real-time study of rapid spread of antibiotic resistance plasmid in biofilm using microfluidics

Gene transfer in biofilms is known to play an important role in antibiotic resistance dissemination. However, the process remains poorly understood. In this study, microfluidics with time-lapse imaging was used for real-time monitoring of plasmid-mediated horizontal gene transfer (HGT) in biofilms. Pseudomonas putida KT2440 harboring an antibiotic resistance plasmid RP4 was chosen as the donor while Escherichia coli and activated sludge bacteria were used as the recipient cells. Dynamic features of the transfer process, including the transfer rate, cell growth rate and kinetic changes of the transfer frequency, were determined. It was found that the routes for gene transfer strongly depend on the structure and composition of a biofilm. While intraspecies HGT is essential to initiate a transfer event, the secondary retransfer from transconjugants to the same species is more efficient and can cause cascading gene spread in single-strain biofilms. For the activated sludge biofilm, only small and scattered colonies formed and vertical gene transfer appears to be the dominant route after initial intraspecies transfer. Furthermore, more than 46% of genera in the activated sludge were permissive to plasmid RP4, many of which are associated with human pathogens. These phenomena imply early prevention and interruptions to biofilm structure could provide an effect way to inhibit rapid antibiotic resistance gene spread and reduce the likelihood of catastrophic events associated with antibiotic resistance.

Aptamer-based detection of melamine in milk using an evanescent wave fiber sensor

Rapid detection of melamine has been achieved in past studies to support the quality control of milk and formula products. Aptamer-based biosensors are a promising choice for melamine analysis due to their advantages over instrumental analysis and immunoassays. A long-chained aptamer with 88 nucleotides has been validated for its recognition of melamine. However, signal conversions of such aptamers have not been sufficiently explored for the purpose of their application in practice. Here, in order to extend the feasibility of detecting melamine using aptamers, we attempted to use an aptamer-based evanescent wave fiber (EWF) sensor to quantify melamine in milk. The duration of the analytical procedure was about 17 min. The calibration curve was interpreted by the dual-log function in the range from 0.1 to 20 μM. The limit of detection (LOD) of melamine was 0.15 μM in the buffer and 3.0 μM in milk. The selectivity of the method to melamine was verified using analogues such as cyanuric acid, ammelide, and ammeline. The recovery rates of melamine in liquid milk ranged from 92% to 108%, and the relative standard deviations were less than 7.5%. Thus, aptamer-based EWF sensing is a feasible method to detect melamine in milk.

The Estimate of Influence of Drainage Pipe Network Operation on Influent of WWTP

The drainage pipe network plays a very important role that takes on the collection and transportation of wastewater in sewer system. The well status of drainage pipe network is necessary to guarantee better operation efficiency of a wastewater treatment plant. The study aims to estimate the influence of the drainage pipe network operation status on the influent of wastewater treatment plant (WWTP). The long-term and short-term and shock effect were investigated in the study. The result indicated that the influent wastewater component variation is obviously by the year and season rule in the long-term effect. The daily variation rule of wastewater quality and quantity is significant in the short-term effect. The shork effect is significant and the flow variation caused by rainfall became weak normally after 2-3 days of rain.