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Antimicrobial Agents and Chemotherapy, March 2003, p. 883-888, Vol. 47, No. 3
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.3.883-888.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Diversity of Tetracycline Resistance Genes in Bacteria from Chilean Salmon Farms

Claudio D. Miranda,1 Corinna Kehrenberg,2 Catherine Ulep,3 Stefan Schwarz,2 and Marilyn C. Roberts3*

Department of Aquaculture, Universidad Católica del Norte, Coquimbo, Chile,1 Institute for Animal Science, Federal Agricultural Research Center (FAL), Neustadt-Mariensee, Germany,2 Department of Pathobiology, University of Washington, Seattle, Washington 981953

Received 18 July 2002/ Returned for modification 28 October 2002/ Accepted 20 December 2002


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ABSTRACT
 
Twenty-five distinct tetracycline-resistant gram-negative bacteria recovered from four Chilean fish farms with no history of recent antibiotic use were examined for the presence of tetracycline resistance (tet) genes. Sixty percent of the isolates carried 1 of the 22 known tet genes examined. The distribution was as follows. The tet(A) gene was found in six isolates. The tet(B) gene was found in two isolates, including the first description in the genus Brevundimonas. Two isolates carried the tet(34) and tet(B) genes, including the first description of the tet(34) gene in Pseudomonas and Serratia and the first description of the tet(B) gene in Pseudomonas. The tet(H) gene was found in two isolates, which includes the first description in the genera Moraxella and Acinetobacter. One isolate carried tet(E), and one isolate carried tet(35), the first description of the gene in the genus Stenotrophomonas. Finally, one isolate carried tet(L), found for the first time in the genus Morganella. By DNA sequence analysis, the two tet(H) genes were indistinguishable from the previously sequenced tet(H) gene from Tn5706 found in Pasteurella multocida. The Acinetobacter radioresistens isolate also harbored the Tn5706-associated 1,063-bp IS element IS1597, while the Moraxella isolate carried a 1,026-bp IS-like element whose 293-amino-acid transposase protein exhibited 69% identity and 84% similarity to the transposase protein of IS1597, suggesting the presence of a novel IS element. The distribution of tet genes from the Chilean freshwater ponds was different than those that have previously been described from other geographical locations, with 40% of the isolates carrying unidentified tetracycline resistance genes.


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INTRODUCTION
 
Intensive fish farming is done in Chile, which is the second-largest producer of farm-raised salmon in the world (25). Oxytetracycline is the most frequently used antimicrobial agent in the Chilean salmon industry, which has resulted in increased tetracycline resistance (Tcr) in gram-negative bacteria associated with all aspects of fish farming, from the water entering and leaving the ponds to the fish food pellets themselves. Previous studies of Tcr Acinetobacter spp., Aeromonas hydrophila, Edwardsiella tarda, Pasteurella piscicida (which has been reclassified as Photobacterium damselae subsp. piscicida) (7), Vibrio anguillarum, and Vibrio salmonicida from fish farms have been characterized in other geographical areas (2, 8, 23). A few studies have characterized nonpathogenic bacteria isolated from catfish ponds (5) or from polluted and unpolluted marine sediments (1). In these previous studies, 66 to 94% of the total isolates carried one of five known Tcr genes: tet(A), tet(B), tet(C), tet(D), and tet(E).

A recent report describes the isolation of Tcr bacteria from fish farm influents, salmon culture tanks, farm effluents, surface water, salmon, and unmedicated fish food pellets (15). From that study, 25 Tcr isolates were selected for characterization of the tetracycline resistance genes by using oligonucleotide probes representing 23 of the known tetracycline resistance genes (3). In selected cases, mating and transformation experiments were done, and tet genes were sequenced.


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MATERIALS AND METHODS
 
Bacteria. Originally, 103 oxytetracycline-resistant gram-negative isolates were recovered from four freshwater Chilean salmon farms located in the southern part of the country (14). From this collection, 25 isolates were obtained that represented all four fish farms and the various locations of sample collection (fish farm influents, salmon culture tanks, farm effluents, surface water, salmon fingerlings, and unmedicated fish food pellets). In particular, isolates representing genera not previously examined for tet genes were also included. These isolates had previously been identified and included Acinetobacter spp. (4), Aeromonas hydrophila (1), Brevundimonas vesicularis (2), Escherichia coli (1), Enterobacter sakazakii (1), Moraxella sp. (1), Morganella morganii (1), Pseudomonas fluorescens (4), Pseudomonas sp. (3), Pantoea sp. (1), Providencia rettgeri (1), Ralstonia pickettii (1), Serratia liquefaciens (1), Sphingomonas paucimobilis (1), and Stenotrophomonas maltophilia (1) (Table 2). The tetracycline MICs ranged from 128 to 2,048 µg/ml (14). The isolates were maintained on L agar supplemented with 25 µg of tetracycline per ml at either room temperature (25°C) or at 37°C, depending on the species.


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  		TABLE 2. Characterization of Chilean gram-negative bacteria

DNA-DNA hybridization. Whole bacteria and whole DNA dot blots were prepared as previously described (13). Twenty-three tet gene probes were used for hybridization of whole bacteria dots and whole DNA dots. The specific oligonucleotide probes for tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(M/O/S), tet(P), and tet(Q) have been previously characterized (18-22, 26) (Table 1). We also included some tet genes commonly found in gram-positive bacteria as well as some of the newer tet genes (Table 1).


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  		TABLE 1. Primers used in this study

MICs. The oxytetracycline MICs were previously done (15). The doxycycline and minocycline MICs were determined by agar dilution following National Committee for Clinical Laboratory Standards (NCCLS) protocols (16), with final concentrations ranging from 2 to 256 µg/ml. Plates were incubated at room temperature for 48 h. E. coli reference strain ATCC 25922 was used as a control, incubated at 37°C, and read after 24 h. NCCLS breakpoints for all tetracyclines are defined as follows: susceptible (S), <=4 µg/ml; intermediate (I), 8 µg/ml; and resistant (R), >=16 µg/ml (16).

PCR assay. Those isolates positive by DNA-DNA hybridization were verified by PCR assay with hybridization of the PCR products with an internal probe by using previously described PCR assays for the tet(A), tet(B), tet(E), tet(L), and tet(H) genes (6, 18) and/or by PCR sequencing (Table 1).

Transfer experiments. The isolates selected for use as donors included A. hydrophila carrying tet(E); Brevundimonas vesicularis carrying tet(B); Enterobacter sakazakii carrying tet(B); Pseudomonas fluorescens carrying tet(A); P. pseudoalcaligenes carrying tet(B); Serratia liquefaciens carrying tet(B) and tet(34); Moraxella spp. and Acinetobacter radioresistens, both carrying tet(H); Morganella morganii carrying tet(L); Stenotrophomonas maltophilia carrying tet(35); and Acinetobacter sp., Brevundimonas vesicularis, Providencia rettgeri, and Ralstonia pickettii carrying unknown genes (Table 2). Transfer of the tetracycline resistance phenotype was done with the recipient E. coli strains HB101 and DH5{alpha}, which had been selected for resistance to streptomycin (1,000 µg/ml), nalidixic acid, fusidic acid, and rifampin at 25 µg/ml each and with the Chilean strains as donors as previously described. All matings were done at 37°C a minimum of two times as previously described (5). Selected transconjugants' tetracycline genes were verified by DNA-DNA hybridization and/or PCR. Experiments involving transformation into E. coli JM107 as well as electrotransformation into tetracycline-susceptible Mannheimia haemolytica M3000 and Pasteurella multocida P4000 strains were performed as described previously (11). Selection of the transformants was done on Luria-Bertani agar or sheep blood agar supplemented with 20 µg of tetracycline per ml.

Partial sequence of the tet(L) PCR product. The tet(L) PCR product was sequenced and compared by using the Biological Information's Resource Software at the University of Washington as previously described (13).

Sequencing and location of tet(H) genes and Tn5706-associated IS elements. The tet(H) genes and the Tn5706-associated IS elements were amplified by PCR. For tet(H) amplification, the previously described primers (Table 1) were used, which resulted in a 1,076-bp internal segment of the tet(H) gene. For amplification of the IS elements, a single 18-bp oligonucleotide that corresponded exactly to the perfect 18-bp inverted repeats at the termini of IS1596/IS1597 was used. The PCR products were cloned into pCR-Blunt II-TOPO (Invitrogen, Groningen, The Netherlands). The cloned PCR amplicon of the novel IS1599 element was used as a specific gene probe in subsequent hybridization experiments. Confirmation of the plasmid location of the tet(H) genes and the IS1597 and IS1599 elements was achieved by Southern blot hybridization experiments. For this, plasmid profiles of the Acinetobacter and Moraxella isolates were prepared with the Qiagen midi kit (Qiagen, Hilden, Germany).

Nucleotide sequence accession number. The sequences of the tet(H) amplicons and the IS elements of the Acinetobacter and Moraxella isolates have been deposited in the EMBL database under the following accession numbers: Acinetobacter tet(H), AJ487672; Moraxella tet(H), AJ487674; Acinetobacter IS1597, AJ487673; and Moraxella IS1599, AJ487675.


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RESULTS
 
MICs. Twenty-two (88%) of the 25 isolates were doxycycline resistant (>=16 µg/ml), while 2 of the 3 remaining isolates showed intermediate resistance (8 µg/ml), and 1 isolate was susceptible to doxycycline (Table 2). Of the four isolates carrying the tet(B) gene, three were minocycline resistant (16 µg/ml), with E. sakazakii being intermediate to minocycline (8 µg/ml). Only one other isolate, the A. hydrophila strain carrying a tet(E) gene, was resistant to minocycline, while two P. fluorescens isolates carrying a tet(A) gene and one R. pickettii isolate carrying an unknown gene were also intermediate to minocycline (Table 2).

Distribution of the tet genes. Initially the 25 isolates were examined for the presence of tet(A) through tet(G), since these genes have previously been found in bacteria isolated from freshwater ponds (4, 5, 8, 23). Only three of the six tet genes were found in the isolates, and 11 (44%) of the 25 isolates were positive for the tet(A) gene (4 Pseudomonas fluorescens isolates, 1 Pseudomonas sp. isolate, and 1 E. coli isolate); the tet(B) gene (1 Brevundimonas vesicularis isolate, 1 Enterobacter sakazakii isolate, 1 Pseudomonas pseudoalcaligenes isolate, and 1 Serratia liquefaciens isolate), and the tet(E) gene (1 Aeromonas hydrophila isolate). The presence of these tet genes in the respective isolates was verified by PCR assays. The isolates carrying the tet(A) gene were from three of the four fish farms, while those with the tet(B) gene were from two of the fish farms (Table 2).

The isolates were then screened for 17 additional tet genes with probes for tet(H) to tet(Z), except for tet(V) and tet(U), tet(30), tet(31), tet(34), and tet(35) (Table 1). These tet genes were chosen because (i) we had cloned controls available in the laboratory that served as positive controls, (ii) they represented the majority of tet genes currently characterized, or (iii) they have recently been described in other water bacteria (3, 17, 24). Six isolates hybridized with these additional probes. One isolate each of Moraxella sp. and A. radioresistens carried the tet(H) gene, and these isolates were from two different farms. An S. liquefaciens isolate and a Pseudomonas pseudoalcaligenes isolate, each carrying tet(B) and tet(34), were from the same farm. We also identified an M. morganii isolate carrying the tet(L) gene and an S. maltophilia isolate carrying the tet(35) gene. All six of these isolates carried multiple plasmids. Additional oligonucleotide probes were used to verify the presence of the tet(34) and tet(35) genes. The presence of the tet(L) and tet(H) genes was verified by PCR, Southern blotting, and sequence analysis of the PCR products. All three genes appeared to be associated with plasmids, as confirmed by hybridization. The tet(L) PCR product showed 100% amino acid homology with the tet(L) gene from the plasmid pTHT15 from Bacillus stearothermophilus (data not shown). The two isolates with the tet(H) genes are described below. The isolates that did not carry one of the known genes were found in similar numbers from all four farms.

Mobility of the tet genes. Selected isolates were used as donors in mating experiments. We were unable to transfer the tet(H) from either the A. radioresistens or Moraxella sp. donors using E. coli or Pasteurella as recipients, although the genes were associated with plasmids. Similarly, transfers of the tet(L) gene from M. morganii, the tet(A) genes from four different P. fluorescens isolates, the tet(E) gene from A. hydrophila, and the unknown genes from Acinetobacter sp., A. radioresistens, R. pickettii, and B. vesicularis were not detected, although the rate of transfer could be <1 x 10-10 per recipient (data not shown). The P. pseudoalcaligenes and S. liquefaciens isolates carrying both tet(B) and tet(34) genes transferred both genes to the E. coli recipient at frequencies of 5.0 x 10-5 to 1.3 x 10-6 per recipient, respectively. The 10 individual transconjugants examined received both genes from both matings. The B. vesicularis isolate carrying the tet(B) gene and the P. rettgeri isolate with the unknown tet gene transferred at frequencies 1.0 x 10-6 to 9.6 x 10-6 per recipient. The S. maltophilia isolate with the tet(35) gene transferred tetracycline resistance at similar frequencies, and multiple plasmids were transferred. However, the resulting transconjugants did not carry the tet(35) gene. Similarly, the E. sakazakii isolate carrying the tet(B) gene transferred tetracycline resistance, but the transconjugants did not carry the tet(B) gene (Table 2).

Hybridization studies revealed the location of the tet(H) genes in the Moraxella and A. radioresistens isolates on plasmids of less than 12 kb. Since these plasmids are too small for conjugation, transformation into CaCl2-competent E. coli strain JM107 and electrotransformation into the recipient strains Mannheimia haemolytica M3000 and Pasteurella multocida P4000 were repeatedly performed. None of these experiments yielded Tcr transformants.

Characterization of the Tn5706-associated tet(H) genes and insertion elements. The tet(H) gene has previously been found exclusively in isolates of the two genera Pasteurella and Mannheimia (9-11). This gene has been well characterized and has previously been shown to be part of a nonconjugative transposon, Tn5706, in which the area from tetR(H) to tet(H) is bracketed by the almost identical insertion sequences IS1596 and IS1597 (12). The tet(H)-specific PCR amplicons of the Acinetobacter sp. and Moraxella sp. isolates were both 1,076 bp in size and proved to be indistinguishable by DNA sequence from the corresponding structural tet(H) gene from Tn5706. In addition, the Acinetobacter isolate also carried the Tn5706-associated 1,063-bp insertion sequence IS1597. This was confirmed by DNA hybridization and complete sequence analysis of the element. The tet(H) gene and IS1597 element were confirmed by Southern blot hybridization to be located on the same plasmid (data not shown). The Moraxella sp. isolate had a slightly smaller amplicon, and sequence analysis identified a 1,026-bp element that closely resembled an insertion sequence. This element, tentatively designated IS1599, had the same perfect 18-bp inverted repeats as IS1596 and IS1597 at its ends. While IS1596 and IS1597 exhibited two open reading frames for proteins of 70 and 228 amino acids (aa), IS1599 only had a single open reading frame of a 293-aa protein. This protein had 69% identity and 84% similarity to the 228-aa transposase proteins of the aforementioned IS elements. The difference in size between IS1599 and IS1596/IS1597 is due to the absence of a 34-bp direct repeat located at positions 282 to 315 in IS1596/IS1597, as well as the loss of a single triplet located at positions 685 to 687 in IS1596/IS1597 (Fig. 1). Hybridization experiments confirmed that the IS1599 element and the tet(H) genes were on the same plasmid in the Moraxella sp. isolate.



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  		FIG. 1. DNA and amino acid sequence comparison between IS1597 and IS1599, the IS sequence described in this study. The 18-bp inverted repeats at both ends of the IS sequences and translational start and stop codons are underlined. The 34-bp direct repeat in IS1597 is displayed in lowercase letters. The dashes in the IS1599 sequence indicate missing bases compared to the IS1597 sequence.


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DISCUSSION
 
Tetracyclines are the most frequently used antimicrobial agents in veterinary medicine in many parts of the world. Oxytetracycline is the most commonly used antimicrobial in freshwater salmon farming in Chile (14, 15, 24). The spread of tet genes is often facilitated by their location on mobile genetic elements, such as plasmids and transposons (5). Of the various tet genes currently known, tet(A), tet(B), tet(D), tet(E), and tet(G) have previously been found in bacteria from fish farms (2, 4, 5, 8, 23). Three of these genes, tet(A), tet(B), and tet(E), were found in 11 (44%) of the isolates from this study. A number of tet genes were found in new genera in this study, including the tet(L) gene in Morganella morganii. The tet(L) gene was originally found in various Bacillus spp., but more recently it has been described in five other gram-positive genera, Mycobacterium spp., and Streptomyces spp. and in the gram-negative anaerobes Fusobacterium spp. and Veillonella spp. (3). However this is the first description of the tet(L) gene in a facultative anaerobic species. The detection of tet(B) in the genus Brevundimonas is also a novel observation (3). This strain was minocycline resistant, as were two of the three other isolates carrying the tet(B) gene, which has previously been associated with minocycline resistance (3). The detection of the tet(E) gene in Aeromonas hydrophila has previously been reported from fish (5). One other isolate, A. hydrophila, was resistant to minocycline. Unfortunately, whether this tet(E) gene confers minocycline resistance cannot easily be tested, since we could not transfer the gene (Table 2). This was not unexpected, since previously, no one had been able to transfer tet(E) genes from other resistant strains and species examined (5, 23). The tet(34) and tet(35) genes have recently been described in Vibrio spp. (16, 23). In the present study, the tet(34) gene was found in S. liquefaciens and P. pseudoalcaligenes along with the tet(B) gene, and both isolates came from the same farm. Whether these two genes are on the same plasmid in each host and whether the plasmids from each species are related to each other is under investigation. Initial Southern blots show both the tet(34) and tet(35) genes hybridizing with the chromosomal DNA in the original isolates and their transconjugants. Clearly more work needs to be done with these isolates. The S. maltophilia isolate carrying the tet(35) gene was also isolated in this fish farm. These data extend the host range, since both isolates came from the same farm, and suggest that both tet(34) and tet(35) genes may be common in bacteria of cold water animals and their environment.

The tet(H) gene has previously been identified as part of the small composite transposon Tn5706 (12), which was found in a complete or truncated form either on plasmids or in the chromosomal DNA of Pasteurella and Mannheimia spp. The finding that the tet(H) gene was present in isolates of Acinetobacter radioresistens and Moraxella sp. from salmon farms was the first detection of this gene in bacteria other than Pasteurella and Mannheimia. Sequence analysis of a 1,076-bp fragment, which comprised almost the entire tet(H) gene, revealed no differences in the nucleotide sequence of the tet(H) gene (10-12). Copies of the Tn5706-associated insertion sequence IS1597 were present on the tet(H)-carrying plasmid in A. radioresistens. A novel IS element, IS1599, lacked a 34-bp direct repeat present found in IS1596/1597 and exhibited only a single open reading frame for a putative transposase protein of 298 aa. The IS1599-borne transposase protein showed highest similarity to the 228-aa transposase proteins of IS1596/1597 and was considered to be a member of the same family of IS elements.

Tcr bacteria from fish bacteria in previous studies have had a variable ability to transfer the Tcr phenotype, with the exception of the tet(E) gene, which has been associated with nonconjugative plasmids (5, 23). The strains carrying the tet(A), tet(H), and tet(L) genes, as well as those carrying one of the unknown genes, did not transfer, while the strain carrying both tet(B)and tet(34) transferred both genes, and the strain carrying tet(35) transferred the Tcr phenotype, but not the tet(35) gene, suggesting that a novel tet gene may also be present in this isolate. Unlike the two isolates carrying tet(H) in this study, previously described tet(H)-carrying strains carry plasmids that replicate and express the Tcr phenotype in Pasteurella, Mannheimia, and E. coli recipients (10-12). The tet(H) gene is part of a transposon (12) that might have integrated into a limited-host-range plasmid of A. radioresistens and Moraxella, which are replication deficient in Pasteurella, Mannheimia, and E. coli recipients, which could account for the lack of Tcr transformants in the transformation and electrotransformation experiments conducted in this study.

In summary, the data from this study showed that gram-negative bacteria from the salmon farm environment harbor a variety of tet genes. A number of new genera were found to carry known tet genes, while 10 isolates may carry novel tetracycline resistance genes. The finding of the tet(H), tet(L), tet(34), and tet(35) genes in gram-negative bacteria from these farms extends our knowledge on the distribution of tet genes and suggests that a wide spectrum of tet genes, rather than the genes tet(A) to tet(G), should be used when future studies are done. Clearly surveillance studies of fish farms and other food-producing farms outside of Japan, Europe, and North America are needed to monitor the continuing evolution in the distribution of tet genes in this environment.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathobiology, University of Washington, Seattle, WA 98195. Fax: (206) 543-3873. E-mail: marilynr{at}u.washington.edu. Back


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Antimicrobial Agents and Chemotherapy, March 2003, p. 883-888, Vol. 47, No. 3
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.3.883-888.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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