Infectious pathogens could spread to the environment through various transmission routes (e.g. direct contact, aerosol, and oral) . It is important to note that sewage systems from facilities and institutions receiving pathogen carriers are at risks of contamination due to the presence of these organisms at high concentrations . Hence, wastewater treatment methods implemented to eliminate hazardous pathogens require proper attention. However, it was reported that only 54% of the hospitals in Vietnam possessed such systems, thus increasing the risk of spreading dangerous persistent pathogens, including Mycobacterium tuberculosis (MTB) , . More than 130 years since Robert Koch’s discovery and 22 years after being declared by the World Health Organization as a global threat, the disease caused by MTB (tuberculosis or TB) now ranks as the first leading cause of death from an infectious disease worldwide, surpassing AIDS . In 2014, there were 9.6 million new TB cases and 1.5 million TB deaths . In Vietnam, the rapid increase of antibiotic resistance together with the occurrence of multidrug resistance (MDR) strains have further complicated the situation . More importantly, if protected from light, MTB could survive up to 74 days and thus might contribute to its widespread and possible disease transmission .
The most common wastewater treatment methods are divided into physical (heating, ultraviolet radiation, etc.), chemical (e.g. hypochlorite/chlorination and ozonation), and biological categories. The major disadvantages of the first two are complex implementation and high maintenance cost (e.g. due to corrosion of the pipe systems) , . Biological methods have become valuable alternatives, with the additional advantage of being environmental friendly. Most of these methods use live microorganisms in either fermentation processes to remove toxic chemicals or filtration applications through the formation of biofilm  and thus are without specific targets. This study is a further development from our previous results obtained with a complex composed of magnetic amino nanoparticles and anti-TB antibody molecules. The complex referred to as NP-NH2-anti-TB was shown to be capable of specifically capturing MTB , which, with the addition of lysing enzymes, would be killed. This paper also reported the initial assessment of the developed method in a wastewater model by spiking the wastewater samples collected from a hospital and a facility receiving TB patients with Mycobacterium bovis from Bacillus Calmette-Guérin (BCG) vaccine. The results of this research indicated the potential applications of the NP-NH2-anti-TB complex combined with enzymes to efficiently treat MTB-contaminated wastewater.
2 Materials and methods
2.1 Wastewater collection
The wastewater samples were collected from a local hospital specialized in pulmonary diseases and a facility admitting TB patients in Hanoi, Vietnam. After the absence of MTB was confirmed by the standard culture method, the spike samples were prepared using 1 ml of wastewater to dissolve lyophilized BCG vaccine (Institute of Vaccines and Medical Biologicals, Nha Trang, Vietnam) containing 0.5 mg M. bovis. Later, the samples were used for the enrichment and lysis of TB bacteria.
2.2 Nanoparticle fabrication
The nanoparticles were the superparamagnetic iron oxide composed of ferric iron (Fe3+) and ferrous iron (Fe2+). The iron oxide particles were coated with a protective layer of silica. Then, the magnetic nanoparticles were functionalized with amino groups by adding to a water solution of aminopropyl triethoxysilane (APTS) . The concentration of the NH2 group was estimated to be ~24 nmol/mg nanoparticles and the final concentration of magnetic amino nanoparticles was 5 mg/ml.
2.3 EDC coupling
Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC or EDAC) was prepared at a concentration of 250 mg/ml (fresh stock; Sigma) to create a zero-length crosslink between the nanoparticles and the proteins (anti-TB antibody and enzymes) . Two different types of complexes were generated: single complexes and dual complexes. Single complexes included individual complexes of NP-NH2-anti-TB, NP-NH2-lipase, and NP-NH2-lysozyme. Immediately before use, 2 μl particles were withdrawn and washed three times with coupling buffer [phosphate-buffered saline (PBS); pH 6.5] and the supernatant was discarded. After the final wash, 2 μl of either lysozyme or lipase (20 mg/ml; Sigma) and 2 μl EDC (250 mg/ml) were added into 71 μl buffer. Then, 2 μl antibody (7 mg/ml; supplied by Abcam) was added to the NP-NH2-enzyme complex. The dual complexes were produced by conjugating the enzymes and antibody onto the nanoparticles either simultaneously (one step) or consecutively (two step). 2 μl antibody, 2 μl enzyme (lysozyme or lipase), and 4 μl EDC were added into 67 μl buffer. The coupling reactions were performed at 22°C for 30 min; the same amounts of EDC (2 or 4 μl) were added after each 15 min during the course of the reaction. The supernatant was then discarded and the complexes were used in the enrichment and cell lysis of Mycobacterium as described below.
In the NP-NH2-anti-TB complex stability experiment, a 10-reaction volume was combined in the coupling step and the resulting complexes were kept in storage buffer [PBS-TBN (pH 7.4), PBS with the addition of 0.01% Tween 20, 0.1% bovine serum albumin (BSA), 0.05 M NaN3] at 4°C. A one-reaction volume was withdrawn and used to enrich the bacteria at indicated time points.
2.4 Mycobacterium enrichment, cell lysis, and IS6110 amplification
The NP-NH2-anti-TB complex was incubated with 50 μl BCG vaccine for 45 min at room temperature (RT). After the enrichment step, BCG supernatant was discarded and 25 μl buffer [Tris-EDTA: 50 mM Tris, 1 mM EDTA (pH 7.5)] was added. The mixture was then incubated for 30 min at 37°C for cell lysis by lysing enzymes. After being separated from the nanoparticles, genomic DNA was used as template for polymerase chain reaction (PCR) amplification using IS6110 primers (purchased from Integrated DNA Technologies) with the following thermocycle: 1×95°C, 5 min; 40×(95°C, 40 s; 65°C, 30 s; 72°C, 30 s); 1×72°C, 7 min; 4°C, ∞. PCR products were run on 2% agarose gel.
2.5 Immunoblotting analysis
Hybond membrane (Amersham) was used in this experiment. BCG vaccine samples were spotted on the membrane (soaked in methanol then equilibrated with water before use), which was later blocked in 5% BSA PBS, washed three times in PBS-Tween (10 min each), and then incubated with primary antibody [anti-TB antibody (Abcam); 1:10,000 dilution, 1 h, RT]. After three 10-min washes, the membrane was incubated with secondary antibody [Alkaline phosphatase-conjugated anti-mouse (Promega), 1:4000 dilution, 1 h, RT]. Finally, the signal was developed using BCIP/NBT substrates (used 6.6 μl NBT/ml and 3.3 μl BCIP/ml) in alkaline phosphatase buffer (pH 9.5; 100 mM NaCl, 5 mM MgCl2, and 100 mM Tris).
3 Results and discussion
3.1 EDC catalysis optimization
In our previous optimization, the coupling reaction was incubated for 30 min at 22°C in the presence of EDC . Longer reaction times, up to 120 min, were performed; however, the result showed decreases in coupling catalytic productivity. In this study, to generate the NP-NH2-anti-TB, NP-NH2-lipase, and NP-NH2-lysozyme complexes, especially the dual complexes with two proteins immobilized onto the nanoparticles, the reaction was extended to 45 min and fresh EDC was added after each 15 min during the course of the reaction.
As shown in Figure 1, the coupling catalytic productivity, as indicated by the intensity of the IS6110 specific PCR product, was optimal in the 30-min-long reaction. In the 45-min-long reaction, the catalytic productivity was decreased, probably due to the effect of larger EDC amounts, causing unwanted formation of intermolecular and intramolecular amide bonds, thus reducing the number of functional complexes. The effect of adding excessive amounts of EDC in disrupting the three-dimensional structure of the conjugated protein was also reported elsewhere , .
3.2 Single- and dual-complex formation and specific mycobacterial cell lysis efficiency
Before evaluating the capturing capability of the generated complexes by PCR amplification, an immunoblotting experiment was conducted. As shown in Figure 2A, the bind of anti-TB antibody on NP was stable, indicated by almost no signal detected in BCG supernatant (lower row, lane BCG) compared to strong signal from the beads (lane B). This was consistent with the result reported in Chu et al, showing efficient biding of MTB onto the NP-NH2-anti-TB complexes through the antibodies, demonstrated by the absence of PCR products in the unbound fraction (sputum supernatant) .
Single complexes, including the capturing complex (conjugated with anti-TB antibody) and lysing complexes (coupled to either lysozyme or lipase), were used with same volumes of the BCG sample. As shown in Figure 2B, the capturing complex combined with heat lysis and lysozyme complex produced bands of similar intensities (lanes 1 and 3). The lipase complex was less efficient in lysing M. bovis cell as indicated by a faint band (lane 2). Lysozyme has been used in many mycobacterial cell lysis protocols; however, lipase is rarely included , , . The result of this experiment, indicating significantly weaker activity of lipase, was consistent with this fact.
Different strategies were used to generate the dual complexes with anti-TB antibody and either lipase or lysozyme onto the nanoparticles: (1) both antibody and enzyme were coupled at the same time in one step to the nanoparticles; and (2) coupling in two consecutive steps: antibody then enzyme or reverse. As shown in Figure 2C, the one-step reaction combining antibody with lysozyme was most efficient (lane 1; 48.8% compared to positive control regarding band intensity quantified by ImageJ). This experiment also confirmed the higher lysing activity of lysozyme compared to lipase (Figure 2C and D, odd lanes vs. even lanes). The dual complex was expected to possess both capturing and lysing capabilities at different levels. In the one-step reactions, the amounts of antibody and enzyme molecules conjugated to nanoparticles would be more likely close to 1:1 ratio; thus, the complexes formed might have equivalent capturing and lysing activities. On the contrary, in the two-step reactions, the dual complexes formed could incline toward either stronger capturing or higher lysing due to the difference in the available surface areas for conjugation with either more antibody or enzyme molecules, respectively. The first protein coupled onto the surface of nanoparticles could hinder assessing area for the second one.
3.3 Stability and performance of the complex in an established wastewater model
The stability of the complexes was an important characteristic for wastewater treatment. As shown in Figure 3, after 9 weeks, the NP-NH2-anti-TB complex was comparably stable. Compared to the previous study, the stability of the NP-NH2-anti-TB complex was doubled. However, this stability might be decreased in wastewater due to the presence of chemicals, such as detergents, unfavorable pH values, or ionic strength. Hence, a wastewater model was established to test the performance of the complexes generated in this study.
Because PCR was used to confirm the presence of MTB in wastewater samples, first we determined the optimal sample volume in PCR. Unless the wastewater volume was higher than 8% of the total reaction volume, the PCR efficiency would not be affected. However, the result was negative for the wastewater samples collected. This was consistent with the result using the standard culturing method. As an alternative, the wastewater samples were spiked with BCG as described in Materials and methods.
The preliminary results with the capturing complexes (Figure 4) showed that the capturing activity retained in the wastewater model (88% compared to positive control; ImageJ). Furthermore, the testing result of single complexes (NP-NH2-anti-TB complex integrated with either NP-NH2-lipase or NP-NH2-lysozyme complexes) and dual complex (NP-NH2-anti-TB-enzyme) indicated the competence in eliminating MTB in the wastewater (data not shown).
In this study, single and dual complexes possessing specific capturing and lysing capabilities for Mycobacterium were successfully generated. These complexes were stable for more than 2 months in the laboratory settings and the complexes were preliminarily tested in treating wastewater spiked with M. bovis. With further validation, the results of this research indicate potential for applications in wastewater treatment.
The Center for Food Security and Public Health. Disease Pathogen Routes of Transmission. Iowa State University: USA, 2008. Google Scholar
Francy DS, Stelzer EA, Bushon RN, Brady AMG, Mailot BE, Spencer SK, Borchardt MA, Elber AG, Riddell KR, Gellner TM. U.S. Geological Survey Scientific Investigations Report. 2011–5150, p 44. Google Scholar
Velayati AA, Farnia P, Mirsaeidi M. Int. J. Mycobacteriol. 2014, 4, 2212–5531. Google Scholar
Fine AE, Bolin CA, Gardiner JC, Kaneene JB. Vet. Med. Int. 2011, article ID 765430. Google Scholar
Global Tuberculosis Report 2015, 20th ed., WHO. Google Scholar
Viet Nam: Optimism for Multidrug-Resistant TB Patients. WHO, 2013. Available from: http://www.who.int/features/2013/viet_nam_tuberculosis/en/. Accessed January 2016.
Duffield BJ, Young DA. Vet. Microbiol. 1985, 10, 193. Google Scholar
U.S. EPA. Wastewater Technology Fact Sheet. Ultraviolet Disinfection, Washington, DC, 1999. Google Scholar
U.S. EPA. Wastewater Technology Fact Sheet. Chlorine Disinfection, Washington, DC, 1999. Google Scholar
Sewage Water Treatment Vat. Available from: https://microbewiki.kenyon.edu/index.php/Sewage_Water_Treatment_Vat#Harmful_Bacteria. Accessed January 2016.
Yen P, Nguyen ATV, Tuan-Nghia P, Chu LL, Nguyen DQ, Nguyen HM, Nguyen HN, Nguyen HL. Int. J. Nanotechnol. 2015, 12, 355. Google Scholar
Nguyen HL, Nguyen HN, Nguyen HH, Luu MQ, Nguyen MH. Adv. Nat. Sci. Nanosci. Nanotechnol. 2015, 6, 015008. Google Scholar
Hermanson GT. Bioconjugate Techniques. 3rd ed., Elsevier, Inc./Academic Press, 2013, pp 261–263. Google Scholar
Xing Y, Chaudry Q, Shen C, Kong KY, Zhau HE, Chung LW, Petros JA, O’Regan RM, Yezhelyev MV, Simons JW, Wang MD, Nie S. Nat. Prot. 2007, 2, 1152. Google Scholar
Vocadlo DJ, Davies GJ, Laine R, Withers SG. Natural 2001, 412, 835–838. Google Scholar
Phillips DC. Proc. Natl. Acad. Sci. U. S. A. 1967, 57, 483. Google Scholar
Razin S, Argaman M. J. Gen. Microbiol. 1963, 30, 155. Google Scholar
About the article
Dao Q. Nguyen
Dao Q. Nguyen has been an undergraduate research assistant at the Enzymology and Bioassay Unit at the KLEPT since 2011. He received his bachelor’s degree in biotechnology-biochemistry from the VNU University of Science (Hanoi, Vietnam) in 2013. He is currently an MSc student at the same university.
Phuong T. Duong
Phuong T. Duong is a technician at the Enzymology and Bioassay Unit at the KLEPT. She graduated from the Vietnam National University of Science (Hanoi, Vietnam) with a BSc in biotechnology-biochemistry in 2015. Her fields of interest include biochemistry, molecular biology, and environmental biology. She has been working on other projects on enzymatic colorimetric assay and pneumonic agent detection.
Hieu M. Nguyen
Hieu M. Nguyen has been working as a researcher at the Center of Materials Science, Faculty of Physics, VNU University of Science. He received his Master’s degree in solid-state physics from the VNU University of Science in 2012. Shortly after, he became a member of the Nano and Energy Center (NEC). His research interests include the preparation of nanomaterials such as magnetic nanoparticles (Fe3O4, Fe3O4-SiO2, Fe2O3), metallic nanoparticles (Ag, Au), semiconductor nanoparticles (ZnS), and functionalization of nanomaterials for biological, pharmaceutical, and environmental applications.
Nguyen Hoang Nam
Nguyen Hoang Nam completed his Master’s thesis at the Physics Department of Eotvos Lorand University (Hungary) in 2004 and received his PhD in physics from Osaka University (Osaka, Japan) in 2008. He worked as a postdoc at the School of Science, Osaka University, from 2008 to 2009. He is now a lecturer at the Faculty of Physics, VNU University of Science. His subjects include fabricating, characterization, and application of nanomaterials in the field of life science.
Nguyen Hoang Luong
Nguyen Hoang Luong graduated from the Physics Department of Kishinev State University in 1977. He received his PhD in solid-state physics from the University of Hanoi (now known as VNU University of Science) in 1984. He received the degree of Doctor of Habilitation from the University of Mining and Metallurgy (Krakow, Poland) in 1999. He was promoted to the rank of Professor in 2002. His research interests include synthesis, characterization and application of nanomaterials.
Yen Pham received her PhD in biochemistry and molecular biology/enzymology from the Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill in 2009 and finished her postdoctoral training in 2011 here. She is a lecturer/researcher in the Key Laboratory of Enzyme and Protein Technology (KLEPT, Hanoi, Vietnam), working on nanomaterials and their application in medicine. Her most recent grants focus on gene mutation detection and recombinant protein production for anti-Helicobacter pylori drug screening.
Published Online: 2016-09-01
Published in Print: 2016-10-01