Occurrence of Tetracycline-Resistant and Tetracycline- Degrading Bacteria in Wastewater Treatment Plant Effluent and Environmental Water Systems

There is a need to conduct more research on the degradability of pharmaceuticals and personal care products in environmental waters for controlling water pollution and sustaining water system. In this study, we added tetracycline to 6 water samples, incubated the samples in the laboratory, and determined the degradation rates and bacterial growth in each sample for analysis of the growth of tetracycline-resistant or tetracycline-degrading bacteria and the mechanism of tetracycline degradation. The main conclusions obtained in this study can be summarized as follows: (1) The maximum degradation of tetracycline was in the Unoke River water samples with 60% degradation in the sample with the initial tetracycline concentration of 1 mg/L. (2) Not all bacteria growing in the water environment containing tetracycline were capable of degrading the antibiotic. (3) The ability of bacteria to degrade tetracycline in environmental water systems may be useful in microbial source tracking.

Journal of Water and Environment Technology, Vol. 8, No.4, 2010 Address correspondence to Koji Tosa, Department of Applied Chemistry, College of Bioscience and Chemistry, Kanazawa Institute of Technology, Email: tosa@neptune.kanazawa-it.ac.jp Received May 8, 2010, Accepted July 7, 2010. - 321 - Occurrence of Tetracycline-Resistant and Tetracycline- Degrading Bacteria in Wastewater Treatment Plant Effluent and Environmental Water Systems Takahiro OOISHI*, Koji TOSA** *Program in Bioscience and Applied Chemistry, Graduate School of Engineering, Kanazawa Institute of Technology, Ishikawa 921-8501 Japan **Department of Applied Chemistry, College of Bioscience and Chemistry, Kanazawa Institute of Technology, Ishikawa 921-8501 Japan ABSTRACT There is a need to conduct more research on the degradability of pharmaceuticals and personal care products in environmental waters for controlling water pollution and sustaining water system. In this study, we added tetracycline to 6 water samples, incubated the samples in the laboratory, and determined the degradation rates and bacterial growth in each sample for analysis of the growth of tetracycline-resistant or tetracycline-degrading bacteria and the mechanism of tetracycline degradation. The main conclusions obtained in this study can be summarized as follows: (1) The maximum degradation of tetracycline was in the Unoke River water samples with 60% degradation in the sample with the initial tetracycline concentration of 1 mg/L. (2) Not all bacteria growing in the water environment containing tetracycline were capable of degrading the antibiotic. (3) The ability of bacteria to degrade tetracycline in environmental water systems may be useful in microbial source tracking. Keywords: antibiotic resistance, biodegradation, microbial source tracking, tetracycline. INTRODUCTION Varieties of pharmaceuticals and personal care products (PPCPs) are widely used, and are released into the water environment. These PPCPs are detected at concentrations of 10 pg/L to 2 mg/L in the rivers of Japan (Nakata et al., 2008; Sugishita et al., 2008). When PPCPs is released into environmental water systems, they trigger the spread of antibiotic-resistant bacteria. Tetracyclines are widely used as antibiotics for animals in the livestock industry and the fish farming industry (Nonaka and Suzuki, 2007). In 2001 tetracyclines accounted for 43% of the total antibiotics used for animals in Japan (Tamura, 2003). There has been a rise in the amount of antibiotic-resistant bacteria in the environment because of the widespread use of antibiotics in large amounts (Kummerer, 2004). These widespread antibiotic-resistant bacteria frequently cause serious nosocomial infections, and the control of antibiotic-resistant bacteria is a significant challenge to the public. An important method for controlling the growth of antibiotic-resistant bacteria is microbial source tracking, because the method can identify the sources of bacterial contamination of environmental waters. Recently, a number of microbial source tracking methods have been developed (Simpson et al., 2002; Stapleton et al., 2007). These methods depend on phenotypic and genotypic bacterial characteristics including antibiotic resistance and DNA types. However, antibiotic-degrading ability is not determined in these microbial source tracking methods. Bacteria from a source with a high concentration of antibiotics may have the ability to degrade antibiotics which may be useful in microbial source tracking. Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 322 - Many studies have been conducted on the physicochemical or biochemical removal of PPCPs in environmental water systems and wastewater treatment systems. These include coagulation, sedimentation, chlorination, ozonation, activated carbon adsorption, activated sludge processes and sludge treatment (Kubota et al., 2008; Shimazaki et al., 2008; Urase, 2008; Jia et al., 2009). Many studies have also been conducted on acute and chronic toxicity tests using Daphnia spp., algae and small fish (Yamamoto et al., 2008). However, only a few studies have been conducted on the microbial degradation of PPCPs in river or lake waters while the mechanism of PPCPs degradation in environmental waters is not yet well-known. There is a need to conduct more research on the degradability of PPCPs in environmental waters for controlling water pollution and sustaining water systems. In this study, we added tetracycline to 6 water samples, incubated the samples in the laboratory, and determined the degradation rates and bacterial growth in each sample for the analysis of the growth of tetracycline-resistant or tetracycline-degrading bacteria and the mechanism of tetracycline degradation. MATERIALS AND METHODS Water samples were collected from the effluent of a small wastewater treatment plant with an activated sludge process (Jokaso), from rain water, and from Tedori River (Tedori Bridge), Unoke River (Konan Bridge) and Lake Tedori; deionized water in the laboratory was also sampled. The wastewater treatment plant is located at Kanazawa Institute of Technology and the sample collected was neither chlorinated nor disinfected. Unoke River is located near some stockbreeding farms in Kahokugata reclaimed land, Ishikawa. The samples were stored in an ice box or in a refrigerator maintained at 4°C. We started the experiment within 24 h of sampling. Tetracycline hydrochloride (Tokyo Chemical Industry, Japan) was dissolved in predetermined concentration in 100 mL of each water sample contained in an Erlenmeyer flask. The flasks were plugged with silicon carbide porous plugs and incubated in the dark with shaking at 20°C for 4 d. The tetracycline concentration and density of bacteria were determined before, after, and during the incubation. Tetracycline concentration was analyzed by high-performance liquid chromatography (HP1100; Agilent) with an Eclipse XDB C8 column (4.6 × 150 mm, 5 µm) at 40°C and a flow rate of 1 mL/min, and the mobile phase used was acetic acid at 0.01M concentration in a 75 : 25 (v/v) water/methanol mixture. Spectrophotometric detection of tetracycline in the samples was conducted at 356 nm. Bacterial density in the samples was determined by the agar- plate culture method using nutrient agar medium (Eiken Chemical, Japan) with incubation at 36°C for 24 h. For the determination of the density of tetracycline-degrading bacteria, triplicate 0.0072, 0.072, 0.72 and 7.2 mL water samples with an initial tetracycline concentration of 10 mg/L were incubated with shaking at 20°C for 7 d. The sample whose absorbance at 356 nm decreased after incubation was considered to show tetracycline degradation. The most probable number of bacterial density of tetracycline-degrading heterotrophic bacteria was calculated from the results using a computer program (Kohno and Suzuki, 1999). The density of tetracycline resistant bacteria was also determined. The density of Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 323 - tetracycline resistant heterotrophic bacteria was determined by the agar-plate culture method using the R2A agar medium (Eiken Chemical, Japan) containing 100 mg/L tetracycline. The inoculated plates were incubated at 20°C for 7 d. After incubation, the colonies obtained on the plates were counted. The colonies were inoculated in sterilized water samples containing 10 mg/L tetracycline and incubated with shaking at 20°C for 7 d. The samples whose absorbance at 356 nm decreased after incubation were considered to show tetracycline degradation. The colonies formed on the plate were observed under a microscope, and the size and shape of microbial cells were studied to determine whether the colonies were bacterial or fungal. RESULTS AND DISCUSSION The changes in the tetracycline concentration in water samples are shown in Fig. 1. The concentration values written in parentheses for the succeeding results represent the initial concentration of tetracycline in the samples. No degradation was observed in the deionized water (1 mg/L) and the wastewater treatment plant effluent samples (1 mg/L). Less than 10% degradation was observed in the following water samples: deionized water (5 and 10 mg/L), Tedori River water (1 mg/L) and wastewater treatment plant effluent (5 mg/L). A considerable 20% degradation was observed in the following samples: Tedori River water (5 and 10 mg/L), wastewater treatment plant effluent (10 mg/L), and rain water (5 and 10 mg/L). More than 20% degradation was observed in all Lake Tedori water and Unoke River water samples and 1 rain water sample (1 mg/L). 40 50 60 70 80 90 100 01234 Residual Tetracycline (%) Incubation Time (day) Deionized Water 40 50 60 70 80 90 100 01234 Residual Tetracycline (%) Incubation Time (day) Rain Water 40 50 60 70 80 90 100 01234 Residual Tetracycline (%) Incubation Time (day) Lake Tedori 40 50 60 70 80 90 100 01234 Residual tetracycline (%) Incubation Time (day) Tedori River 40 50 60 70 80 90 100 01234 Residual Tetracycline (%) Incubation Time (day) Unoke River 40 50 60 70 80 90 100 01234 Residual Tetracycline (%) Incubation Time (day) WTP Effluent ○ : 1mg/L, □ : 5 mg/L, ▲ : 10 mg/L of Initial Concentration of Tetracycline Fig. 1 - Tetracycline degradation in water samples Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 324 - Maximum tetracycline degradation, 60%, was observed in the Unoke River water sample (1 mg/L). Tedori River and wastewater treatment plant effluent reduced tetracycline better at higher tetracycline concentration, in contrast with Unoke River and rain water. Some conditions such as nutrients might be sufficient for tetracycline-degrading bacteria in Tedori River and wastewater treatment plant effluent but not sufficient in Unoke River and rain water. The bacterial density in the water samples before and after incubation within 4 d is shown in Fig. 2. The density increased in all wastewater treatment effluent samples, Lake Tedori water samples (1, 5, and 10 mg/L), all deionized water samples, and Unoke River water samples (5 and 10 mg/L), after 4 d of incubation. However, the density decreased in all rain water samples and the Unoke River water sample (1 mg/L). The bacterial density in most samples before incubation decreased with tetracycline concentration except for wastewater treatment plant effluent. The tetracycline added to the tested water was carried into the agar medium and affected the number of bacterial colony on the plate. The relationship between bacterial density and tetracycline degradation is shown in Fig. 3. There was no correlation between both initial and final bacterial densities and tetracycline degradation. Not only tetracycline but also organic matter in the test water is utilized for the bacterial growth. These results suggest substrate selection by bacteria in water systems and that not all bacteria growing in water systems with tetracycline can degrade this antibiotic. 1 10 100 1000 10000 100000 01510 Bacterial Density (CFU/mL) Tetracycline (mg/L) Deionized Water 1 10 100 1000 10000 100000 01510 Bacterial Density (CFU/mL) Tetracycline (mg/L) Rain Water 1 10 100 1000 10000 100000 01510 Bacterial Density (CUF/mL) Tetracycline (mg/L) Lake Tedori 1 10 100 1000 10000 100000 01510 Bacterial Density (CFU/mL) Tetracycline (mg/L) Tedori River 1 10 100 1000 10000 100000 01510 Bacterial Density (CFU/mL) Tetracycline (mg/L) Unoke River 1 10 100 1000 10000 100000 01510 Bacterial Density (CFU/mL) Tetracycline (mg/L) WTP Effluent ■ : before incubation, □ : after incubation for 4 d Fig. 2 - Bacterial density in water samples before and after incubation Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 325 - The results of the tetracycline degradation test for the Unoke River water and wastewater treatment plant effluent samples are listed in Table 1. No tetracycline degradation was observed in the wastewater treatment plant effluent samples. Significant degradation of tetracycline was observed in all three Unoke River water samples with 7.2 and 0.72 mL sample volumes and 1 of 3 samples with the 0.072 mL sample volume. The density of tetracycline-degrading bacteria in the Unoke River samples was calculated to be 6.9 MPN/mL. The density of tetracycline-resistant heterotrophic bacteria in the Unoke River and the wastewater treatment plant effluent water samples was 375 CFU/mL and 885 CFU/mL, respectively. The density of the wastewater treatment plant effluent sample that showed no tetracycline degradation was higher than that of the Unoke River water sample that showed degradation. Unoke River is located near some stockbreeding farms in Kahokugata reclaimed land and the water discharged from the farms may have affected the results of this study. Tetracycline degrading ability was exhibited by 2 of 15 colonies of resistant bacteria isolated from the Unoke River water samples, and the density of these tetracycline-degrading bacteria was calculated to be 50 CFU/mL. This value is not consistent with the value of the most probable number of bacteria mentioned previously (6.9 MPN/mL) because the 2 different detection methods probably had a different accuracy of detection. Many types of bacterial genes for tetracycline resistance are already known, most of which code for proteins mediating tetracycline efflux or ribosomal protection, but some code for tetracycline-degrading enzymes (Yang et al., 2004; Nonaka and Suzuki, 2007; Park and Choung, 2007). Degradation of tetracycline by some fungal enzymes was also recently reported (Wen et al., 2009). Microscopic observation of the tetracycline- degrading colonies isolated in our study revealed that the colonies were bacterial and not fungal. These results suggest that the ability of bacteria to degrade antibiotics in environmental water may be useful in microbial source tracking. 0 20 40 60 80 100 1 10 100 1000 10000 Degraded Tetracycline (%) Initial Bacterial Density (CFU/mL) 0 20 40 60 80 100 1 10 100 1000 10000 Degraded Tetracycline (%) Final Bacterial Density (CFU/mL) Fig. 3 - Relationship between bacterial density and tetracycline degradation Table 1 - Results of tetracycline degradation test for Unoke River water and wastewater treatment plant effluent Sample Volume (mL) Unoke River Water Wastewater Treatment Plant Effluent 7.2 3/3 0/3 0.72 3/3 0/3 0.072 1/3 0/3 0.0072 0/3 0/3 Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 326 - CONCLUSIONS The main conclusions obtained in this study can be summarized as follows: (1) The maximum degradation of tetracycline was in the Unoke River water samples with 60% degradation in the sample with the initial tetracycline concentration of 1 mg/L. 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