16S Ribosomal RNA Gene-Based Phylogenetic Analysis of Abundant Bacteria in River, Canal and Potable Water in Bangkok, Thailand

Abstract

In Southeast Asian countries, industrialization and urbanization is occurring rapidly, and water pollution in rivers and canals poses serious problems in some areas, especially in cities. Excess inflow of domestic, agricultural, and industrial wastewater to freshwater environments disturbs the aquatic microbial ecosystem, which can further pollute water by inhibiting biodegradation of pollutants. Therefore, monitoring of microbes in freshwater environment is important to identify changes in indigenous microbial populations and to estimate the influence of wastewater inflows on them. Polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE) analysis is suitable for monitoring changes in microbial communities caused by human activities, but this method can be difficult in eutrophic freshwater samples that contain PCR inhibitors. In this study, we optimized DNA extraction procedures and PCR conditions for DGGE analysis of bacterial populations in freshwater samples (canal, river, and tap water) collected in Bangkok, Thailand. A simple freeze–thaw procedure was effective for extracting DNA from bacterial cells in the samples, and LA Taq with added bovine serum albumin provided the best PCR amplification. The PCR–DGGE approach revealed that the most common bacteria in freshwater samples belonged to Gammaproteobacteria, while a Gram-positive bacterium was present at Bangkok Noi Canal. Temporally and spatially continuous analyses of bacterial populations in Bangkok canals and rivers by PCR–DGGE approach should be useful to recognize disturbances of microbial ecosystems caused by excess inflows of wastewater.

In Southeast Asian countries, industrialization and urbanization is occurring rapidly, and water pollution in rivers and canals has become a serious problem in some areas, especially in cities.^1–4) Sewage plants have been constructed in these areas, but the total number is not yet sufficient, and drainpipes are sometimes incomplete. Canals and rivers are therefore polluted by untreated or incompletely treated sewage in these areas. Excess inflow of domestic, agricultural, and industrial wastewater to freshwater environments disturbs aquatic microbial ecosystems, which can further collapse the ecosystem, because bacterial populations are essential in degrading pollutants derived from human activities. Therefore, monitoring of freshwater microbes is important to recognize changes in indigenous microbial populations and to estimate the influence of wastewater inflows on them. Microbial ecology in Southeast Asian countries is therefore being explored; however, current knowledge is limited.

Culture-dependent methods such as plate counting and most probable number (MPN) estimates are often used to evaluate the microbiological quality of freshwater and drinking water.⁴⁾ Determination of bacterial numbers and identification of major bacteria based on solely on culturing yields valuable information. However, bacterial colony formation is dependent on growth conditions, such as nutrient media, incubation time, and temperature. Additionally, most bacteria in natural aquatic environments cannot be cultured by conventional techniques.^5,6) Thus, culture-independent methods should be used for microbiological monitoring in addition to culture-dependent methods.^7,8)

Several molecular microbiological techniques have been developed to identify bacterial cells and analyze their diversity and community structure in natural environments without the need for isolation. In particular, denaturing gradient gel electrophoresis (DGGE)⁹⁾ has been used to determine the genetic diversity of natural microbial communities and to identify the phylogenetic affiliation of community members.^10–12) Polymerase chain reaction (PCR)-amplified DNA fragments of the eubacterial 16S ribosomal RNA (rRNA) gene that have the same length but different sequences can be separated by DGGE. However, PCR–DGGE analysis is often difficult to implement with freshwater samples from eutrophic environments that contain PCR-inhibitors, such as humic acid.

In this study, we optimized PCR conditions for PCR–DGGE analysis of bacteria in freshwater collected from canals and rivers as well as tap water samples from metropolitan Bangkok, Thailand. The abundant bacteria in these samples were then phylogenetically analyzed using the rRNA-targeted PCR–DGGE approach.

Materials and Methods

Sampling Sites

Water samples were collected in Bangkok, Thailand from Bangkok Noi Canal (samples 1–5 and 12 in Table 1), Chao Phraya River (samples 8–11 in Table 1), and Bangkok taps (samples 6 and 7 in Table 1) on February 24, 2002 (Supplementary Fig. 1). The canal is 10–20 m wide, while the river spans more than 100 m. The water quality of the Chao Phraya River has been monitored by the Pollution Control Department of Thailand, and biochemical oxygen demand (BOD) was 1.1–8 mg/L (average 3.5 mg/L) in metropolitan Bangkok.¹³⁾

Fig. 1. Amplification of Bacterial 16S Ribosomal RNA Genes in Freshwater Samples Taken in Bangkok, Thailand

Bacterial DNA was extracted by a modified Teske method (lanes 1–5) or a simple freeze–thaw method (lanes 6–10). M: pHY marker. Lanes 1 and 6: Bangkok Noi Canal; lanes 2–4 and 7–9: Chao Phraya River; lanes 5 and 10: tap water. N: Distilled water (negative control).

Table 1. Physicochemical Characteristics of Freshwater Samples

Sample	Location	A.T.a) (°C)	W.T.b) (°C)	pH	D.O.c) (mg L−1)
1	Canal; Taling Chan Floating market (13°46′16.5″N, 100°27′22.1″E)	31.3	29.3	7.5	0.9
2	Canal; Bangkok Noi District Office (13°45′31.7″N, 100°28′37.4″E)	31.8	29.3	7.3	1.4
3	Canal; Bangkok Noi (13°48′8.2″N, 100°28′34.4″E)	33.6	29.7	7.4	1.3
4	Canal; Khlong Ban Yai (Nonthaburi) (13°50′47.4″N, 100°25′25.7″E)	37.1	29.8	7.4	2.7
5	Canal; near temple (13°51′3.5″N, 100°25′28.9″E)	36.6	29.7	7.6	3.8
6	Tap water; riverside house (13°51′3.5″N, 100°25′28.9″E)	35.0	29.6	7.5	6.6
7	Tap water; house (reservoir) (13°49′57.5″N, 100°28′22.4″E)	34.8	33.1	7.2	4.5
8	River; Wat Chaloem Phra Kiat (13°50′40.0″N, 100°29′5.4″E)	34.9	30.7	7.4	1.2
9	River; Krung Thon Bridge (13°46′36.4″N, 100°30′3.2″E)	35.6	29.7	7.4	6.7
10	River; Pra Pin Klao pier (13°45′28.4″N, 100°29′25.1″E)	35.6	29.5	7.4	—d)
11	River; Thonburi station (13°45′15.2″N, 100°29′13.7″E)	35.6	29.2	7.4	—
12	Canal; Thonburi station (13°45′21.1″N, 100°29′9.7″E)	34.6	29.8	7.5	1.3

a) A.T., ambient temperature. b) W.T., water temperature. c) D.O., dissolved oxygen. d) —, not determined.

One tap water sample stored in a plastic bucket at a riverside house and one from a water reservoir at a house in the city were also collected as oligotrophic aquatic environmental samples.

The collected samples were stored immediately in ice and used for the following experiments within two hours after sampling.

DNA Extraction for 16S rRNA Gene Analysis

Bacterial DNA was extracted using a simple freeze–thaw¹⁴⁾ or modified Teske method.¹⁵⁾ Bacterial cells in water samples were vacuum-filtered onto polycarbonate white filters (pore size: 0.2 µm). In the simple freeze–thaw method, these filters were placed into sterilized tubes with 400 µL of sterile DNA-free water. The samples were mixed thoroughly, frozen in liquid nitrogen, and then thawed at room temperature. This freeze–thaw cycle was repeated twice, and the extracted DNAs were directly used for PCR.

In the modified Teske method, each filter was added to 2 mL of AE buffer (20 mm sodium acetate (pH 5.5), 1 mm ethylenediaminetetraacetic acid (EDTA)), frozen in liquid nitrogen, and then thawed at room temperature. This freeze–thaw cycle was repeated once. After this lysis treatment, each filter and buffer were added to 6 mL of Tris–EDTA (TE)-buffered phenol–chloroform–isoamyl alcohol (25 : 24 : 1, pH 8.0) and 60 µL of 25% (w/v) sodium dodecyl sulfate. After 5 min of incubation at 60°C, each solution was cooled on ice for 3 min and then centrifuged for 5 min at 3500×g. The supernatant was transferred to a new tube and 250 µL of 2 m sodium acetate (pH 5.2) was added. Nucleic acids were precipitated with 2.5 volumes of 100% ethanol for 3 h at −20°C and then recovered by centrifugation. Each pellet was washed with 75% ethanol, dried, and redissolved in 50 µL of sterile distilled water. The suspensions were used for PCR.

Primers and PCR Amplification

Fragments of the 16S rRNA gene, including the V6–V8 region, were amplified with primers GC-clamp-EUB f933 (5′-GC-clamp-GCA CAA GCG GTG GAG CAT GTG G-3′) and EUB r1387 (5′-GCC CGG GAA CGT ATT CAC CG-3′), which are specific for universally conserved bacterial 16S rRNA gene sequences.¹⁵⁾ For DGGE analysis of the PCR products, a GC-clamp (5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G-3′) was attached to the 5′ end of primer EUB f933. PCR was performed with one of the following enzymes according to the manufacturer’s guidelines: Ampli Taq Gold (Applied Biosystems, Foster City, CA, U.S.A.), LA Taq (TaKaRa Bio, Shiga, Japan), rTaq (Toyobo, Osaka, Japan), KOD Plus (Toyobo), or Platinum Pfx (Invitrogen, Carlsbad, CA, U.S.A.). One or more of the following additives were used to optimize amplification: bovine serum albumin (BSA) solution¹⁶⁾ (0.2 mg/mL; TaKaRa Bio), betaine solution (0.5 m; Sigma-Aldrich, St. Louis, MO, U.S.A.), and dimethyl sulfoxide (DMSO; 1% (v/v); Nacalai Tesque, Kyoto, Japan).

Denaturing Gradient Gel Electrophoresis

PCR products were loaded onto a 6.5% (w/v) polyacrylamide gel in 1×TAE (40 mm Tris, 20 mm acetic acid, and 1 mm EDTA (pH 8.0)). The polyacrylamide gels (37.5 : 1 acrylamide–bisacrylamide) were made with denaturing gradients ranging from 50–70% (100% denaturant contained 7 m urea and 40% formamide). The gels were run at 55°C for 10 min at 20 V and subsequently for 14 h at 100 V.

Sequencing of DGGE Fragments

Bands of 16S rRNA gene fragments in the DGGE gel were excised with a sterile razor blade. The DNA was reamplified with EUB f933 and EUB r1387 primers, and the PCR products were ligated for cloning. Plasmid inserts were reamplified by PCR with primers GC-clamp-EUB f933 and EUB r1387 and analyzed by DGGE. Plasmids inserts that produced a DGGE band at the same position as the excised band were selected and their sequences were analyzed by a sequencer (CEQ-8000, Beckman Coulter, Indianapolis, IN, U.S.A.).

Nucleotide Sequence Accession Numbers

The sequences obtained in this study have been deposited in the DDBJ database under accession Nos. AB769961 to AB769969.

Results and Discussion

To monitor bacteria in river and canal water in Bangkok with a PCR-based approach, we first optimized the DNA extraction method and PCR conditions for these samples, because eutrophic freshwater samples often inhibit PCR. For DNA extraction, simple freeze–thaw and modified Teske methods were compared. The modified Teske method involves DNA purification steps after a freeze–thaw treatment while the simple freeze–thaw method does not. Different DNA polymerases and additives were tested for effective PCR amplification.

Figure 1 shows electrophoretic gels comparing the DNA extraction procedures and PCR conditions. Rather clear bands were obtained when bacterial DNAs were extracted by the modified Teske method, while the simple freeze–thaw method was effective in almost all samples. Thus, this simple method is suitable for analyzing many samples simultaneously. TaqGold is often used for PCR amplification of eubacterial 16S rRNA genes from freshwater samples^10,11,14,15); however, this DNA polymerase was not very effective in our study. Among the five DNA polymerases tested, LA Taq yielded the best amplification, while rTaq, KOD Plus, and Platinum Pfx amplified non–specific products (data not shown). The PCR additives BSA, betaine, and DMSO were also examined. Neither betaine nor DMSO was effective (data not shown), while BSA enhanced amplification with both Taq Gold and LA Taq. Thus, we concluded that LA Taq with BSA provided the best amplification for our samples.

Bacterial 16S rRNA genes in the freshwater samples were then amplified with the optimized conditions and analyzed by DGGE (Fig. 2). More than 10 bands appeared in each sample, and eight were selected for further analysis: two that were common in all freshwater samples, one that was specific to all Bangkok Noi Canal water samples, three that were specific to household tap water stored in a plastic bucket, and two specific to household tap water in a reservoir.

Fig. 2. Denaturing Gradient Gel Electrophoresis Band Patterns of Freshwater Bacteria from Bangkok Noi Canal (Lanes 1–5 and 12), Chao Phraya River (Lanes 8–11), and Tap Water (Lanes 6 and 7) Taken in Bangkok, Thailand, Using the Primer Combination EUBf933-GC/EUBr1387

M: pHY marker. Arrows indicate bands excised for sequencing. Bands 1AL0202 and 2AL0202: common in all freshwater samples; bands 3RH0202, 4RH0202, and 5RH0202: bands specific to purified tap water stored in a plastic tank at a riverside house; bands 6TW0202 and 7TW0202: specific to tap water in a reservoir; band 8BN0202: specific to Bangkok Noi Canal water.

Figure 3 shows the phylogenetic positions of the eight clones. The two bacteria common to all freshwater samples belonged to Gammaproteobacteria; 1AL0202 had 99% similarity with a partial sequence isolated from Lake Biwa, Japan’s largest freshwater lake, and 2AL0202 belonged to Acinetobacter sp. Kenzaka used fluorescence in situ hybridization to analyze bacterial communities in freshwater samples from Chao Phraya River and reported that 30–40% of bacteria belonged to Gammaproteobacteria,¹³⁾ similar to our results. The dominant bacterium in Bangkok Noi Canal (8BN0202) belonged to Gram-positive bacteria and was closely related to Staphylococcus sciuri. This bacterium is sometimes found in urine and related to urinary tract infections.¹⁷⁾ This sampling site was probably affected by the wastewater inflow. The dominant bacteria in tap water (3RH0202, 4RH0202, 5RH0202, and 6TW0202) belonged to Alphaproteobacteria, which are often found in oligotrophic environments.¹⁴⁾ The bacterium found in tap water stored in the reservoir (7TW0202) had 99% similarity with a partial sequence of Rhodococcus aetherovorans, which is often found in soil. Interestingly, 6TW0202 was very close to bacteria found in pharmaceutical purified water used in Osaka, Japan, which was prepared from tap water.¹⁴⁾ This bacterium may be widely distributed in tap water.

Fig. 3. Phylogenetic Affiliations within the Freshwater Bacteria in Bangkok Noi Canal, Chao Phraya River, and Tap Water Taken in Bangkok, Thailand

Sequences determined in this study are shown in boldface (compare with Fig. 2 for location of bands on the gel). The scale bar corresponds to 0.1 substitutions per nucleotide position.

In this study, we optimized a DNA extraction procedure and PCR conditions for DGGE analysis of bacterial populations in freshwater samples collected in Bangkok, Thailand. Bacterial populations in both eutrophic (e.g., canal and river) and oligotrophic (e.g., tap water) aquatic environments could be analyzed by the optimized PCR–DGGE procedure. DGGE is suitable to detect changes in microbial communities caused by human activities,¹⁵⁾ and temporally and spatially continuous analyses of bacterial populations in Bangkok canals and rivers by PCR–DGGE should be useful to recognize disturbances in microbial ecosystems caused by excess inflow of wastewater.

Acknowledgment

This study was partly supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research [A]) Grant Numbers 13376003 and 21256002.

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