Occurrence of Two Genotypes of Tetracycline (TC) Resistance Gene tet (M) in the TC-Resistant Bacteria in Marine Sediments of Japan M. HABIBUR RAHMAN, LISA NONAKA, RYOSUKE TAGO, AND SATORU SUZUKI* Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama 790-8577, Japan Received November 30, 2007. Revised manuscript received March 11, 2008. Accepted March 26, 2008. The tetracycline (TC) resistance gene tet(M) was monitored in bacteria isolated from Japanese coastal and off-shore marine sediments. The high rate of occurrence of TC resistant (TC r ) bacteria (120 μg mL -1 TC) was observed at frequency ranges between 0.0-0.08% in Tokyo Bay, 1.67-1.82% in Sagami Bay and 0.0-4.35% in the open Pacific Ocean. The tet(M) gene was PCR amplified from the TC r isolates, showing 127 of 209 isolates (60.8%) as positive. The rate of occurrence of tet(M) was between 32.0-96.0%, 21.1 -28.0% and 0.0-83.3% in the isolates from Tokyo Bay, Sagami Bay and the open Pacific Ocean, respectively. The tet(M) positive isolates belonged to 4 orders of bacteria. Bacillales was the most dominant order (121 strains) among tet(M) possessing bacteria, followed by Actinomycetales (three strains), Flavobacteriales (one strain) and Pseudomonadales (one strain). This indicates that tet(M) is present in various bacterial species and suggests that marine sediments are a natural reservoir of the tet(M) gene. Nucleotide sequence of the tet(M) revealed that two genotypes of tet(M) were found in the bacteria. The two genotypes were placed in genetically distant branches of the phylogenetic tree, suggesting that the two tet(M)s have different origins. Introduction Drug and chemical-resistant bacteria are released from human clinical sources to the natural environment, which can in-turn initiate and spread drug resistance genes to naturally occurring bacteria in the environment (1, 2). Tetracyclines (TCs) are broad-spectrum antibiotics that are used in human therapy, veterinary medicine, agriculture, and aquaculture. The increase of TC resistant bacteria is a serious issue in recent years, not only in human clinical but also in other fields (3, 4). Chronic, low-dose application of TC for the promotion of growth of farm animals, particularly cultured for food, causes an increase in the presence of TC resistant bacteria, not only in pathogenic bacteria but also in commensal environmental bacteria. Bacterial resistance to TC is mediated primarily by two mechanisms; one is the energy dependent efflux pump and the other occurs through the ribosomal protection protein (RPP) (5). Other minor examples of resistance mechanisms are also known such as enzymatic inactivation of TC (6). Among the TC resistance genes, tet(M), one of the RPP genes, has been studied for its distribution in terrestrial environ- ments (7, 8). Recent reports (9–11) showed that the tet(M) gene is distributed in coastal aquaculture areas and sediments in Mekong River, Vietnam. Various genotypes of the tet(M) gene are known to occur as human and animal pathogens (7, 12) in the Mekong River sediments (11), and coastal aquaculture sites in Japan (10). The tet(M) gene is known to associate with mobile genetic elements such as plasmids, transposons, conjugative transposons, and integrons (5, 13, 14). This enables tet(M) to move easily from species to species (15), and thus may result in its wide distribution among numerous bacteria in the environment (7). It is known that drug resistant bacteria occur even in environments without drug contamination (16), suggesting that nonpolluted environment also can contain hidden reservoirs of the TC resistance gene. It is presumed that open Pacific Ocean is not a drug-polluted environment, however, little is known on the occurrence of TC resistance in the marine environment. To examine the potential presence and distribution of TC resistant bacteria and the tet(M) gene in coastal and oceanic environments with little or no known exposure to drugs, we examined the occurrence of TC resistant bacteria and the tet(M) in marine sediments from coastal to off-shore areas of Japan. The tet(M) possessing bacteria were classified by 16S rRNA gene analyses. Sequence of the tet(M) was also analyzed, indicating two types of tet(M) in the isolates. Materials and Methods Sampling Area. Sampling sites are shown in Figure 1. Sampling was performed between 25 June to 1 July, 2004 at two stations in Tokyo Bay (T1 and T2), one station in Sagami Bay (S1) and three stations in the open Pacific Ocean (S4, S3, and S2) in cruice of the R/V Tansei-Maru, Japan Marine Science and Technology Center (JAMSTEC), Japan. Tokyo Bay is located almost in the center of Japan, and has an area of 1000 km 2 with inlet of four rivers. Sagami Bay is located in the southwest of Tokyo Bay. Water exchange rate is high, because Sagami Bay is well connected to the open Pacific Ocean and the Kuroshio current flows strongly offshore. Of the three open Pacific Ocean stations two were located near the northeastern part of Sagami Bay, with one station situated in the center of (S3) and one was out side (S2) of the Kuroshio Current. Depth and water temperature of the sampling area are shown in the Table 2. Sampling Procedure. Sediment core samples were col- lected using a Freger core sampler in Tokyo Bay, and with a Multiple corer in Sagami Bay and the open Pacific Ocean sites. Core samples from 0-3 cm of the surface floor and 15-18 cm below the sea floor (BSF) slices from the same core (Figure 1B), were used for analysis. Sliced samples were transferred into sterile plastic bags and stored at -20 °C on board and at -80 °C in the laboratory until use. Viable Count of Tetracycline Resistant (TC r ) Bacteria. To enumerate the percentage of TC r bacteria, 1 g of wet sediment was thoroughly suspended in 9 mL of sterile phosphate buffered saline (PBS) by vortex and a 10-fold serial dilution was made. Plate counts were performed on marine broth 2216E (Difco, Detroit, MI) plus 1.5% bacto agar (BD and Co., Sparks, MD) containing 0, 60, 120, or 240 μg mL -1 of TC (Sigma, St. Louis, MO). The plates were incubated at 25 °C for 7 days. Duplicate counting was performed, and colony forming unit (CFU) per g sediment was calculated. DNA Extraction from Isolates. For detection of tet(M) in the TC r isolates, a total of 209 TC r isolates were randomly chosen from the plate containing 60 and 120 μg mL -1 of TC. * Corresponding author phone: +81-89-927-8552; fax: +81-89- 927-8552; e-mail: ssuzuki@agr.ehime-u.ac.jp. Environ. Sci. Technol. 2008, 42, 5055–5061 10.1021/es702986y CCC: $40.75  2008 American Chemical Society VOL. 42, NO. 14, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 5055 Published on Web 06/17/2008