2012 Volume 54 Issue 4 Pages 255-262
Objectives: The aim of this study was to identify bacteria in sludge brought by the 2011 tsunami in Japan to determine the necessary precautions for workers who handle the sludge. Methods: Two sludge samples and one water sample were collected from each of two sites in Miyagi Prefecture in June 2011. We also obtained control samples from a paddy field and a dry beach in Fukuoka, Japan. The samples were subjected to physicochemical analyses, conventional cultivation methods, and molecular methods for bacterial flora analysis. The bacterial floras were analyzed using a clone library method employing fragments of the 16S ribosomal RNA gene (rDNA) amplified with universal primers. Results: We detected 51–61 genera in sludge samples and 14 and 17 genera in water samples collected in the tsunami-affected areas. In sludge samples collected in the tsunami-affected areas, more genera belonged to Proteobacteria than to Bacteroidetes, but in water samples collected in these areas, more genera belonged to Bacteroidetes than to Proteobacteria. Non-O1, non-O139 V. cholerae (non-agglutinable vibrio) was found at approximately 104 cells/m/ near the coast of the tsunami affected area. Sulfate-reducing bacteria were detected in sludge collected from the paddy field, and a relatively high concentration of sulfate ions was found in the water sample (258 mg/l). Conclusions: Sludge brought by the tsunami contained some pathogens; therefore, frequent hand washing is recommended for workers who have direct contact with the sludge to minimize their risk of infection. Under the anaerobic conditions of paddy fields, hydrogen sulfide could be produced by sulfate-reducing bacteria metabolizing sulfate ions.
At 14:46 on March 11, 2011, an earthquake occurred off the coast of eastern Japan. This earthquake, which has been named the Great East Japan Earthquake, caused a massive tsunami that resulted in approximately 20,000 deaths1) and left 23 million tons of debris. The tsunami impacted buildings at elevations of up to 40.5 m in Miyako, Iwate Prefecture2). Many workers, volunteers, and residents of tsunami-affected areas have become involved in cleanup efforts.
Sludge brought by the tsunami could contain pathogens with the potential to harm workers3). Fukuda et al. analyzed sediment samples collected from Doukai Bay in Kitakyushu using a culture-independent clone library method and reported that the genera Vibrio, Staphylococcus, and Mycobacterium were present4). Using the same molecular method, Taniguchi et al. identified sulfate-reducing bacteria that could produce hydrogen sulfide, which is very harmful5). To control bacterial hazards associated with sludge brought by the tsunami, it is necessary to identify pathogens and other health risks associated with the sludge. However, to our knowledge, no studies of bacteria in sludge brought by the tsunami have been conducted to date. Therefore, this study was conducted to identify bacteria in sludge brought by the tsunami to determine what precautions workers removing the sludge should take.
We collected three samples at each of two sites in Miyagi Prefecture on June 19, 2011: 1) a paddy field impacted by the tsunami located 300 m from the coast in Shichigahama (A-1 (wet sludge), A-2 (dry sludge), and A-3W (water)) and 2) a waste dump in Tagajo, where debris and sludge were stored for subsequent transport to a final disposal site (B-1 (dry sludge), B-2 (wet sludge), and B-3W (water)). Figure 1 shows a map of the locations from which samples were taken.
Map of the locations where samples were taken.
A and B on the inset map signify the locations where samples A and B were taken.
We also obtained control samples in the city of Fukutsu, Fukuoka Prefecture, Japan, which was not affected by the tsunami, on June 24, 2011. Two samples (C-1 and C-2W) were taken at a paddy field and two samples (C-3 and C-4W) were obtained at a dry beach along the coast of the Genkai Sea.
All samples were collected from the surface at each site and immediately stored in sterile tubes at 4°C until analysis.
Physicochemical testsThe pH, electrical conductivity, total organic carbon, total nitrogen, anions (Cl−, SO42−), and leachability of the samples were determined. Briefly, leachates were obtained from 5 g aliquots of dried soils that had been added to 45 ml of water and then vertically shaken at 170 rpm for 6 h. The leachate was then passed through a filter with a mean pore diameter of 1 μm. All samples were subsequently analyzed using an Iwaki pH meter (model UB-10), a Digital Instruments conductivity meter (model CD-4307), a Shimadzu total organic carbon and total nitrogen analyzer (model TNPC-4110), a Dionex ion chromatograph (model DX-AQ), and a Seiko inductively coupled plasma analyzer (model SPS 1500R). Analytical grade reagents such as metal and anion standards were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Epifluorescence staining and cell lysis efficiencyCell concentrations (cells/ml (water) or g (soil)) were determined by epifluorescence staining using ethidium bromide5–7). Briefly, 100 μl samples were added to 900 μl of ethidium bromide solution (100 μg/ml) and left to stand for 10 min at room temperature, after which the mixture (1.0 ml) was passed through a 0.2 μm pore filter (Millipore, Bedford, MA, USA). Objects shaped like bacteria on the filter were counted with the aid of an Olympus BX50 microscope (Olympus Optical, Tokyo, Japan), and the number of bacteria per milliliter of solution or gram of soil was calculated.
After DNA extraction (described in the “DNA extraction” section), the remaining bacteria were counted in the same manner. The cell lysis efficiency was calculated as the ratio of the number of bacteria remaining after the DNA extraction treatment to the total number before treatment (100–[postextraction number / pre-extraction number] × 100). DNA extracted from samples that showed more than 80% cell lysis was used for analysis of bacteria.
Cultivation methodsAerobic bacteria were counted after incubation on standard agar medium (yeast extract, 0.5 g/l; peptone, 1.0 g/l; glucose, 0.2 g/l; agar, 15 g/l) at 30°C for 6 days4, 5). Samples were also cultivated on bromothymol blue (BTB) lactose agar medium, thiosulfate–citrate–bile salts–sucrose (TCBS) agar medium, SSB agar medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and Wadowsky–Yee–Okuda agar medium supplemented with α-ketoglutarate (WYOα) (Eiken Chemical Co., Ltd., Tokyo, Japan) for at least 48 h at 37°C under aerobic conditions. Colonies on TCBS agar medium were Gram stained, after which the Gram-negative samples were identified using an API 20E system according to the manufacturer's instructions (bioMérieux, Marcy l'Etoile, France). Colonies on WYOα agar medium were subcultured on sheep blood agar to identify Legionella spp.
Serotyping of V. cholerae and PCR of cholera toxin geneOne yellow colony on TCBS agar medium was subjected to serotyping using a V. cholerae AD Seiken kit (Denka Seiken Co., Ltd.) and V. cholerae O139 antiserum (Denka Seiken Co., Ltd.). The colony was also analyzed for the presence of the V. cholerae toxin gene by PCR using the VCT-1&2 primers (Takara Bio Inc.).
DNA extractionTo detect pathogenic bacteria that are difficult to culture, such as mycobacteria, Legionella spp., anaerobes, viable but nonculturable bacteria, sulfur-reducing bacteria, and sulfur-oxidizing bacteria, the bacterial flora of each sample was analyzed by the clone library method with PCR using universal primers specific for the 16S ribosomal RNA gene. The soil sample (0.3 g) was vigorously agitated in 3.0 ml of distilled water. A 900 μl aliquot of sample solution was mixed with 100 μl of 30% SDS solution and approximately 0.3 g of glass beads. The mixtures were then shaken for 5 min at 4,500 rpm using a Micro Smash MS-100 apparatus (Tomy Seiko Co., Ltd., Tokyo, Japan). Next, the samples were centrifuged at 20,000 g for 5 min at room temperature, after the supernatants were collected. This DNA extraction procedure was conducted three times, and the mixture of three supernatants was subsequently extracted with an equal volume of phenol–chloroform–isoamyl alcohol (25: 24: 1 vol/vol). The DNA in the aqueous phases was then concentrated to 30 μl of TE buffer using a Montage PCR centrifugal filter device (Millipore, Bedford, MA, USA).
Clone library construction and nucleotide sequencing analysisUsing the DNA extracts obtained as described above, a fragment of the 16S rDNA (550 bp) was amplified with the primers E341F (5′-CCTACGGGAGGCAGCAG-3′) and E907R (5′-CCGTCAATTCMTTTRAGTTT-3′). PCR amplification was then conducted using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). The cycling conditions were 96°C for 5 min, followed by 30 cycles of 96°C for 30 s, 53°C for 30 s, and 72°C for 1 min, with a final elongation step at 72°C for 7 min. The PCR products were then cloned into Escherichia coli TOP10 cells using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA). A total of 96 white colonies from each sample were randomly selected, and the inserted PCR product was amplified with M13 forward and reverse primers. Sequencing reactions were subsequently conducted using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) with the M13 forward primer. Nucleotide sequences were determined with a 3130 xl Genetic Analyzer (Applied Biosystems).
Highly accurate sequences were trimmed from the primer and vector regions. Only sequences showing good quality and whose primer sequences were successfully trimmed were used for homology analysis. The remaining sequences were compared with an in-house database containing only 16S rRNA gene sequences of type strains (5,878 species) obtained from the Ribosomal Database Project II (http://rdp.cme.msu.edu/) and the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/) using the BLAST algorithm.
EthicsEthics committee approval was not required for this study.
Some environmental physicochemical factors influence bacterial multiplication; therefore, we examined five such factors (Table 1). We found that samples A-2 and A-3W were slightly alkaline. Additionally, the electrical conductivity of A-3W was high (1,346 mS/m). Total organic carbon was also high in A-3W (47.3 mg/l). The chloride (Cl−) concentrations were very high in all samples, especially A-3W (8,503 mg/l) and B-3W (3,396 mg/l), which suggested condensation of seawater. The sulfate (SO42−) concentrations were high in A-3W (258 mg/l and B-1 (329 mg/l).
| A-1 (wet sludge) | A-2 (dry sludge) | A-3W (water) | B-1 (dry sludge) | B-2 (wet sludge) | B-3W (water) | C-1 (wet sludge) | C-2W (water) | C-3 (wet sludge) | C-4W (water) | ||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Potential hydrogen (PH) | — | 7.5 | 8.6 | 8.3 | 7.5 | 7.2 | 7.8 | 6.8 | 7.2 | 6.9 | 7.5 |
| Electric conductivity (EC) | mS/m | 221 | 37 | 1,346 | 198 | 225 | 196 | 6 | ND | 100 | ND |
| Total organic carbon (TOC) | mg/l | 13.5 | 24.6 | 47.3 | 9.7 | 5.5 | 24.9 | 5.4 | 20.9 | 8.2 | 3.7 |
| Total nitrogen (TN) | mg/l | 1.6 | 2.2 | 3.7 | 1.5 | 0.9 | 10.4 | 1.6 | 2.9 | 1.8 | 0.6 |
| Chloride ion (Cl−) | mg/l | 555 | 28 | 8,503 | 337 | 246 | 3,396 | 7 | 278 | 771 | 1,091 |
| Sulfate ion (SO42−) | mg/l | 71 | 24 | 258 | 329 | 117 | 0 | 7 | 133 | 148 | ND |
ND: not detected.
The cell lysis efficiencies varied from 76.9 to 99.8%. Bacteria were enumerated by three methods (Table 2). Bacterial cell numbers were estimated by epifluorescence microscopy, which revealed concentrations of 108–109 cells/g in soil and 106–107 cells/ml in water (including control samples). Aerobic culture on BTB lactose agar medium at 37°C was used to identify lactose-utilizing enteric bacteria. Samples A-1, A-2, B-1, B-2, and C-1 showed approximately 105 colony forming units (CFU) per milliliter (water) or gram (soil), but lactose-utilizing bacteria were found only in B-2, where they accounted for 10% of the colonies. Bacteria were also enumerated by aerobic culture on environmental standard agar medium at 30°C. The results revealed concentrations of 106–108 CFU/g in soil and about 106 CFU/ml in water, even for the control samples.
| A-1 (wet sludge) | A-2 (dry sludge) | A-3W (water) | B-1 (dry sludge) | B-2 (wet sludge) | B-3W (water) | C-1 (wet sludge) | C-2W (water) | C-3 (wet sludge) | C-4W (water) | |
|---|---|---|---|---|---|---|---|---|---|---|
| Cell lysis efficiency (%) | 79.0 (± 2.3) | 96.3 (± 1.4) | 98.6 (± 2.3) | 92.7 (± 0.9) | 99.1 (± 0.5) | 99.8 (± 0.1) | 76.9 (± 0.8) | 85.0 (± 3.0) | 85.9 (± 1.1) | 92.7 (± 2.5) |
| Epifluorescence staining method (cells/m/l (water) or g (soil)) | 6.2 (± 0.4) × 108 | 1.0 (± 0.1) × 109 | 9.9 (± 0.7) × 106 | 7.8 (± 0.1) × 108 | 2.9 (± 0.6) × 108 | 5.3 (± 0.6) × 106 | 1.3 (± 0.2) × 109 | 7.9 (± 0.3) × 106 | 6.8 (± 0.8) × 108 | 7.6 (± 2.5) × 106 |
| Aerobic culture on BTB medium at 37°C (colony forming units/ml (water) or g (soil)) | 8 × 104 | 1 × 105 | 3 × 104 | 1 × 105 | 6 × 105 | 3 × 104 | 7 × 105 | 2 × 104 | 1 × 104 | 1 × 102 |
| Aerobic culture on standard medium for environmental bacteria at 30°C (colony forming units/ml (water) or g (soil)) | 1.0 (± 0.2) × 107 | 6.3 (± 1.0) × 107 | 2.0 (± 0.1) × 106 | 5.9 (± 4.0) × 107 | 9.2 (± 0.3) × 107 | 1.0 (± 0.2) × 106 | 1.0 (± 0.03) × 108 | 1.4 (± 0.4) × 106 | 1.8 (± 0.03) × 106 | 3.1 (± 0.2) × 106 |
Mean ± standard deviation.
Cultivation and Gram staining were conducted to detect Escherichia, Salmonella, Shigella, Vibrio, and Legionella spp. Samples were cultured on SSB agar for detection of Escherichia, Salmonella, and Shigella spp. Aliquots from samples A-3W, B-2, and B-3W produced 104 CFU/ml on SSB agar; however, none of these colonies were suspected pathogens. Aliquots from A-3W produced approximately 104 CFU/ml of suspected V. cholerae on TCBS agar. To discriminate between V. cholerae and other Vibrio species, API 20E tests for Vibrio spp. were conducted. One colony had a 99.5% probability of being V. cholerae and one colony had a 76.8% probability of being V. fluvialis. The suspected V. cholerae colony was then analyzed to determine whether it was serotype O1 and O139. In addition, the colonies were analyzed by PCR to determine whether the cholera toxin gene was present. The analyzed colonies were non-O1, non-O139, and cholera toxin gene negative V. cholerae (non-agglutinable vibrios). Analysis of the samples for the presence of Legionella spp. revealed no positive samples.
Bacterial flora analysesNucleotide sequences of 86 to 95 clones from each sample (total 918 clones) were determined (Table 3). Of these clones, 5–57% and 77–100% showed more than 97 and 80% homology to strains present in an in-house database, respectively. To cover the widest possible range of clones, 80% homology was selected; therefore, taxa above the rank of genus were used for further analyses. The numbers of taxa indicated that the bacterial communities of soil samples were more diverse than those of water samples (Table 3). The numbers of genera detected were as follows: 61 (A-1), 53 (A-2), 57 (B-1), 51 (B-2), 48 (C-1), and 44 (C-3) in soils, and 17 (A-3W), 14 (B-3W), 49 (C-2W), and 45 (C-4W) in water samples. At the phylum level, 10 (A-1), 9 (A-2), 6 (B-1), 4 (B-2), 9 (C-1), and 8 (C-3) taxa were detected in soils, and 4 (A-3W), 3 (B-3W), 7 (C-2W), and 5 (C-4W) taxa were detected in water samples. Most of the genera belonged to Proteobacteria and Bacteroidetes (Table 4). In sludge samples taken in the tsunami-affected areas, more genera belonged to Proteobacteria (37–51%) than to Bacteroidetes (14–27%), but in water samples taken in these areas, more genera belonged to Bacteroidetes (38, 45%) than to Proteobacteria (22, 32%). In control water samples, more genera belonged to Proteobacteria (37, 43%) than to Bacteroidetes (16, 21%).
| A-1 (wet sludge) | A-2 (dry sludge) | A-3W (water) | B-1 (dry sludge) | B-2 (wet sludge) | B-3W (water) | C-1 (wet sludge) | C-2W (water) | C-3 (wet sludge) | C-4W (water) | |
|---|---|---|---|---|---|---|---|---|---|---|
| Number of tested clones | 95 | 90 | 86 | 92 | 88 | 94 | 94 | 94 | 93 | 92 |
| Number of clones with > 97% homology∗1 (% of the total tested clones) | 17 (18%) | 27 (30%) | 4 (5%) | 30 (32%) | 50 (57%) | 36 (38%) | 23 (25%) | 21 (22%) | 6 (6%) | 14 (15%) |
| Number of clones with > 80% homology∗2 (% of the total tested clones) | 73 (77%) | 73 (81%) | 70 (81%) | 77 (84%) | 82 (93%) | 94 (100%) | 82 (87%) | 75 (80%) | 72 (77%) | 72 (78%) |
| Number of phyla | 10 | 9 | 4 | 6 | 4 | 3 | 9 | 7 | 8 | 5 |
| Number of classes | 16 | 15 | 6 | 11 | 10 | 7 | 15 | 11 | 14 | 9 |
| Number of orders | 30 | 27 | 10 | 25 | 24 | 10 | 23 | 23 | 24 | 17 |
| Number of families | 43 | 37 | 13 | 40 | 33 | 12 | 40 | 33 | 32 | 25 |
| Number of genera | 61 | 53 | 17 | 57 | 51 | 14 | 48 | 49 | 44 | 45 |
∗1Number of clones with nucleotide sequences that showed > 97% homology to those present in the database.
∗2Number of clones with nucleotide sequences that showed > 80% homology to those present in the database.
| A-1 (wet sludge) | A-2 (dry sludge) | A-3W (water) | B-1 (dry sludge) | B-2 (wet sludge) | B-3W (water) | C-1 (wet sludge) | C-2W (water) | C-3 (wet sludge) | C-4W (water) | |
|---|---|---|---|---|---|---|---|---|---|---|
| Acidobacteria | 2 | 1 | 9 | 1 | 4 | 17 | 1 | 1 | 4 | 4 |
| Actinobacteria | 5 | 9 | 8 | 9 | 6 | |||||
| Bacteroidetes | 18 | 20 | 38 | 14 | 27 | 45 | 26 | 16 | 12 | 21 |
| Chloroflexi | 1 | 2 | 2 | |||||||
| Cyanobacteria | 2 | 1 | 11 | 8 | 2 | |||||
| Deferribacteres | 1 | 1 | ||||||||
| Firmicutes | 13 | 2 | 3 | 3 | 8 | 6 | 2 | 2 | 2 | |
| Gemmatimonadetes | 2 | |||||||||
| Lentisphaerae | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| Nitrospira | 2 | 2 | 1 | 1 | ||||||
| Proteobacteria | 38 | 37 | 22 | 51 | 44 | 32 | 35 | 37 | 43 | 43 |
| Spirochaetes | 2 | 1 | ||||||||
| Thermomicrobia | 1 | 1 | 1 | |||||||
| Unclassified∗ | 11 | 15 | 14 | 14 | 5 | 12 | 19 | 21 | 20 |
At the family level, some sulfate-reducing bacteria and sulfur-oxidizing bacteria were detected (Table 5). In sample A-1 (wet sludge), 10 clones (11%) of sulfate-reducing bacteria and one clone (2%) of sulfur-oxidizing bacteria were detected among 95 tested clones. One clone of Mycobacterium elephantis (nontuberculous mycobacteria) (97% homology with AJ010747) was also detected in A-1. In sample B-1 (dry sludge), seven clones (8%) of sulfate-reducing bacteria, 11 clones (12%) of sulfur-oxidizing bacteria, and one clone of Legionella impletisoli (94% homology) were detected among 92 tested clones. In sample B-2, two clones (2%) of Clostridiaceae that were possible sulfate-reducing bacteria, two clones (2%) of Vibrionaceae, and one clone of Massilia timonae (97% homology with U54470) were identified among 88 tested clones. In sample B-3W, only sulfur-oxidizing bacteria were detected. Two pathogens were identified in control samples: C-1 contained one clone of Mycobacterium moriokaense (98% homology with AJ429044) and C-4W contained one clone of Vibrio natriegens (100% homology with X74714). Samples C-3 and C-4W, taken from the beach, contained balanced numbers of sulfur-oxidizing and sulfur-reducing bacteria (13 and 14 clones for C-3 and 6 and 9 clones for C-4W).
| Samples | Identified microbes | No of clones |
|---|---|---|
| A-1 (wet sludge) | Mycobacteriaceae (M. elephantis) (P) | 1 |
| Peptococcaceae (Desulfotomaculum) (SRB) | 1 | |
| Desulfobacteraceae (SRB) | 5 | |
| Desulfobulbaceae (SRB) | 2 | |
| Syntrophobacteraceae(Desulfovirga) (SRB) | 1 | |
| Clostridiaceae (Clostridium) (SRB) | 1 | |
| Ectothiorhodospiraceae (SOB) | 1 | |
| A-2 (dry sludge) | Hydrogenophilaceae (SOB) | 1 |
| Chromatiaceae (SOB) | 1 | |
| A-3W (water) | Chromatiaceae (SOB) | 1 |
| B-1 (dry sludge) | Desulfobacteraceae (SRB) | 5 |
| Desulfobulbaceae (SRB) | 2 | |
| Hydrogenophilaceae (SOB) | 3 | |
| Chromatiaceae (SOB) | 5 | |
| Ectothiorhodospiraceae (SOB) | 2 | |
| Piscirickettsiaceae (SOB) | 1 | |
| B-2 (wet sludge) | Clostridiaceae (Clostridium) (SRB) | 2 |
| Oxalobacteraceae (Massilia-timonae) (P) | 1 | |
| Vibrionaceae (V. ichthyoenteri, V. natriegens) (P) | 2 | |
| B-3W (water) | Chromatiaceae (SOB) | 6 |
| Ectothiorhodospiraceae (SOB) | 6 | |
| Thiotrichaceae (SOB) | 4 | |
| C-1 (wet sludge) | Mycobacteriaceae (M. moriokaense) (P) | 1 |
| Clostridium-aldrichii (SRB) | 1 | |
| Clostridium-alkalicellum (SRB) | 1 | |
| Clostridium-cellobioparum (SRB) | 1 | |
| Clostridium-chartatabidum (SRB) | 2 | |
| Desulfovibrio-inopinatus (SRB) | 1 | |
| Ectothiorhodospiraceae (SOB) | 1 | |
| C-2W (water) | Hydrogenophilaceae (SOB) | 1 |
| Chromatiaceae (SOB) | 1 | |
| Ectothiorhodospiraceae (SOB) | 1 | |
| C-3 (wet sludge) | Desulfobacterium-catecholicum (SRB) | 2 |
| Desulfobacterium-indolicum (SRB) | 1 | |
| Desulfosarcina-cetonica (SRB) | 3 | |
| Desulfobulbus-propionicus (SRB) | 3 | |
| Desulfobulbus-rhabdoformis (SRB) | 1 | |
| Desulforhopalus-singaporensis (SRB) | 1 | |
| Desulfotalea-psychrophila (SRB) | 2 | |
| Chromatiaceae (SOB) | 6 | |
| Ectothiorhodospiraceae (SOB) | 6 | |
| Thiotrichaceae (SOB) | 4 | |
| C-4W (water) | Desulfobacter-postgatei (SRB) | 1 |
| Desulfobacterium-catecholicum (SRB) | 1 | |
| Desulfococcus-biacutus (SRB) | 1 | |
| Desulfosarcina-cetonica (SRB) | 1 | |
| Desulfobulbus-propionicus (SRB) | 2 | |
| Chromatiaceae (SOB) | 3 | |
| Ectothiorhodospiraceae (SOB) | 6 | |
| Vibrionaceae (V. natriegens) (P) | 1 |
SRB: Sulfur Reducing Bacteria, SOB: Sulfur Oxidizing Bacteria, P: Pathogen.
(P): M. elephantis and Massilia timonae showed 97% homology to the database type strain.
We analyzed sludge brought by the tsunami that occurred after the Great East Japan Earthquake in 2011 to identify bacterial hazards. To detect bacteria causing enteric or respiratory infection, we used both conventional culture and molecular methods. No significant bacterial proliferation was observed in the samples; however, non-O1, non-O139, and cholera toxin gene negative V. cholerae (non-agglutinable vibrio) was identified by the culture method, and Massilia timonae was found using the molecular method. Additionally, some samples were found to contain sulfate-reducing bacteria and sulfate at concentrations sufficient to generate hydrogen sulfide, which is very toxic to humans. No pathogens that require government notification were identified.
We collected samples from a coastal area in which sludge was stagnant and from a waste dump that contained sludge and debris. Water samples collected from both places contained high levels of chloride ions, which could have been brought from the sea (Table 1). Total organic carbon, which is a nonspecific indicator of water quality, was high in water samples, possibly supporting bacterial growth or metabolic activity8).
Non-agglutinable vibrios, which cause sporadic cases or outbreaks of diarrhea9), have often been found in seawater and were still active in stagnant seawater in the paddy field 3 months after the tsunami (Table 5). We also identified Vibrionaceae in samples B-2 and C-4W. In areas in which the samples for the present study were collected, frequent hand washing and avoiding close contact with the sludge are recommended to prevent infection. There is also potential for the bacteria to proliferate over the summer, when the weather is hot and humid.
Mycobacterium elephantis and Massilia timonae, which have been reported as human pathogens10–13), were found in sludge samples collected from tsunamiaffected areas (Table 5). The most probable reservoir of these bacteria is the outer environment, as we also identified Mycobacterium moriokaense in sample C-114). The site from which sample A was collected was completely immersed after the tsunami and we also identified various bacteria usually found in the sea, such as Marinobacterium (A-1), Neptunomonas (A-1), Marinicola seohaensis (B-2), and Marinobacter (B-2). However, we could not determine whether Mycobacterium elephantis (A-1) and Massilia timonae (B-2) were transferred from the sea or originated from the sample site. These bacteria cause infection, especially among immunocompromised hosts. Therefore, avoiding exposure to the site and frequent hand washing are recommended, especially for immunocompromised individuals. In addition, we identified Legionella impletisoli (B-1) even though the homology was 94%, which has been found in industrial wastes15).
Sulfate-reducing bacteria live in oxygen-deficient environments such as seawater, deep wells, and plumbing systems. They reduce sulfate to form hydrogen sulfide, a colorless gas with an offensive odor. Sulfate-oxidizing bacteria pose no identified health risk and convert hydrogen sulfide gas to sulfuric acid16). In control samples taken from the beach and the sea (C-3 and C-4W), similar numbers of clones were identified for sulfate-reducing and sulfuroxidizing bacteria. However, in a control sample taken from the sludge of a paddy field (C-1), there were more sulfate-reducing bacteria. This was also observed for the sample taken from the sludge of a paddy field in the tsunami-affected area (A-1). Since the sulfate ion levels in sample A-3W were high (Table 1), and this sample was collected near sample A-1, hydrogen sulfide could be produced if these samples became mixed under anaerobic conditions. Even though the sulfate levels in sample B-1 (taken from a waste dump) were higher, hydrogen sulfide would not be produced because the amount of sulfate-reducing bacteria was equal to the amount of sulfate-oxidizing bacteria.
It should be noted that our study had a few limitations. Specifically, a small number of samples were selected for analysis. Further sample analysis is required to clarify the health risks associated with sludge. In addition, the storage method that we used did not allow us to identify cold sensitive bacteria such as Neisseria gonorrhoeae and Neisseria menin-gitides. At the end, we used a clone library that was limited to bacteria; therefore, no conclusions regarding the presence of viruses, fungi, and protozoa can be drawn.
In conclusion, sludge brought by the tsunami following the 2011 earthquake in Japan contained some pathogens. Accordingly, frequent hand washing is recommended for workers that have direct contact with the sludge to minimize the risk of infection. Additionally, hydrogen sulfide could be produced by sulfate-reducing bacteria under anaerobic conditions such as those present in paddy fields. Overall, the results of this study indicate that the sludge should be cleaned up as soon as possible.
Conflict of interest: The authors have declared that no competing interests exist.
Funding: This study was funded by a research fund of the Fit-test research committee. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
