Of 203 human clinical isolates of Campylobacter jejuni from Alberta, Canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 μg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 μg/ml). In total, the MICs for 37% of tetracycline-resistant isolates (256 to 512 μg/ml) were higher than those previously reported in C. jejuni (64 to 128 μg/ml). In the tetracycline-resistant clinical isolates, 67% contained plasmids and all contained the tet(O) gene. Four isolates resistant to high levels of tetracycline (MIC = 512 μg/ml) contained plasmids carrying the tet(O) gene, which could be transferred to other isolates of C. jejuni. The tetracycline MICs for transconjugants were comparable to those of the donors. Cloning of tet(O) from the four high-level tetracycline-resistant isolates conferred an MIC of 32 μg/ml for Escherichia coli DH5α. In contrast, transfer to a strain of C. jejuni by using mobilization conferred an MIC of 128 μg/ml. DNA sequence analysis determined that the tet(O) genes encoding lower MICs (64 to 128 μg/ml) were identical to one other, although the tet(O) genes encoding a 512-μg/ml MIC demonstrated several nucleotide substitutions. The quinolone resistance determining region of four ciprofloxacin-resistant isolates (2%) was analyzed, and resistance was associated with a chromosomal mutation in the gyrA gene resulting in a Thr-86-Ile substitution. In addition, six kanamycin-resistant isolates contained large plasmids that carry the aphA-3 marker coding for 3′-aminoglycoside phosphotransferase. Resistance to erythromycin was not detected in 203 isolates. In general, resistance to most antibiotics in C. jejuni remains low, except for resistance to tetracycline, which has increased from about 8 to 50% over the past 20 years

Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.

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The emergence of drug-resistant bacteria is a severe problem in aquaculture. The ability of drug resistance genes to transfer from a bacterial cell to another is thought to be responsible for the wide dissemination of these genes in the aquaculture environment; however, little is known about the gene transfer mechanisms in marine bacteria. In this study, we show that a tetracycline-resistant strain of Photobacterium damselae subsp. damselae , isolated from seawater at a coastal aquaculture site in Japan, harbors a novel multiple drug resistance plasmid. This plasmid named pAQU1 can be transferred to Escherichia coli by conjugation. Nucleotide sequencing showed that the plasmid was 204,052 base pairs and contained 235 predicted coding sequences. Annotation showed that pAQU1 did not have known repA , suggesting a new replicon, and contained seven drug resistance genes: bla CARB-9 -like, floR , mph (A)-like, mef (A)-like, sul2 , tet (M) and tet (B). The plasmid has a complete set of genes encoding the apparatus for the type IV secretion system with a unique duplication of traA . Phylogenetic analysis of the deduced amino acid sequence of relaxase encoded by traI in pAQU1 demonstrated that the conjugative transfer system of the plasmid belongs to MOB H12 , a sub-group of the MOB H plasmid family, closely related to the IncA/C type of plasmids and SXT/R391 widely distributed among species of Enterobacteriaceae and Vibrionaceae. Our data suggest that conjugative transfer is involved in horizontal gene transfer among marine bacteria and provide useful insights into the molecular basis for the dissemination of drug resistance genes among bacteria in the aquaculture environment.

We found increased numbers of oxytetracycline (OTC)-resistant bacteria in sediment and seawater around a marine aquaculture site after OTC therapy. Samples were collected at an aquaculture site along the coast of the Seto Inland Sea, Japan in 2004. In April, the percentage of bacteria resistant to 60 µg mL −1 OTC in the surface sediment was 6.8%-20.0%. The percentages increased during OTC therapy in the summer reaching 53.3%-60.7% in September. Ninety-two days after drug cessation, the percentages decreased to below 22.9%. Tet(M)-positive bacteria were detected in the sediment and seawater samples. Tet(M) was evident in both Gram-positive and Gram-negative bacteria from various genera, and was newly identified in Paenibacillus, Sporosarcina, Shewanella, and Pseudoalteromonas. The dominant tet(M)-positive isolates were strains of Vibrio suggesting that this genus is an important reservoir for tet(M) in the marine environment. Two different alleles were found, tet(M)-A and tet(M)-B, each in isolates from five genera. The data suggests drug therapy used in the aquaculture acted as a selective pressure promoting increased numbers of resistant bacteria.

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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 microg 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.

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10 -7 to 10 -3 ; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10 -6 to 10 -5 ; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.