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Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Christina Casas , Elizabeth C. Anderson , Kayode K. Ojo , Ian Keith , Daryl Whelan , Don Rainnie , Marilyn C. Roberts
DOI: http://dx.doi.org/10.1016/j.femsle.2004.10.039 59-63 First published online: 1 January 2005


Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tcr) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tcr plasmids to an Escherichia coli recipient at 15 °C. Five of the isolates, with genetically related Tcr plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7 × 10−4 to 2.8 × 10−6 per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 °C is the lowest temperature that has been documented in bacteria.

  • Aeromonas salmonicida
  • Tetracycline resistance
  • tet(A)
  • tet(B)
  • pRAS1

1 Introduction

Oxytetracycline resistant Aeromonas sp. have been isolated from aquaculture environment [15,8,9], as well as from polluted and unpolluted marine sediment [10]. In 1989 an atypical Aeromonas salmonicida, which causes the salmonid disease furunculosis, was isolated. This strain carried a 45-kb plasmid, pRAS1, which conferred resistance to tetracycline, sulfonamides and trimethoprim. This plasmid belonged to the IncU incompatibility group, and was able to be conjugally transfer to an Escherichia coli recipient in laboratory matings, at 22 °C [11]. IncU plasmids have been isolated from Aeromonas sp. from aquatic environments around the world [11], however, these plasmids are not limited to Aeromonas sp. since a pRAS1-like plasmid had previously been identified in a German E. coli[9]. Currently, plasmids carrying tet(A), tet(B), tet(C), tet(D), tet(E) and tet (31), have been found in different Aeromonas isolates [12,13]. Some of these plasmids could be conjugally transferred to an E. coli or other Gram-negative recipients at temperatures ranging from 20 to 37 °C [3,4]. However, less is known about gene transfer from psychrophilic slow-growing marine bacteria, such as the atypical A. salmonicida, which are able to grow between 4 and 15 °C and a mesophilic bacterium such as E. coli, which have a much broader environmental range.

In this report seven Tcr pRAS1-like and two Tcr non pRAS1-like plasmids from atypical A. salmonicida were isolated between 1992 and 2001 from diseased salmon and trout isolated from hatcheries or seacage sites in Northeastern North America. These plasmids were characterized based on the specific tetracycline resistance (tet) gene carried, presence of the IncU group and a complete Tn1721, sul1 coding for sulfonamide resistance, and dfrA16 coding for trimethoprim resistance, as well as their ability to transfer their Tcr plasmids to an E. coli recipient at 15 °C. Using all these markers, the nine plasmids could be divided into four groups. Seven plasmids from two groups could transfer their plasmids at 15 °C to E. coli, while the other three plasmids did not. This describes the lowest temperature (15 °C) documented for conjugal transfer in bacteria and suggests that direct gene exchange between psychrophilic and mesophilic bacteria may occur in nature under rare conditions.

2 Materials and methods

2.1 Bacterial isolates

Nine atypical Tcr (MIC of 64–128 μg ml−1 oxytetracycline) A. salmonicida were obtained from diseased salmon and trout grown in hatcheries or seacage sites in Northeastern North America. The isolates were from 1992–2001 and were selected to be representative of 28 isolates taken during this time period (Table 1). These strains grew at temperatures ranging between 4 and 15 °C and were considered psychrophilic. They were slow growing requiring 2–4 weeks at 4 °C for visible growth to appear on Brucella Agar plates supplemented with 5% sheep blood (BA) (Difco Laboratories, Division Becton Dickinson & Co. Sparks, MD, USA).

View this table:

Characteristics of the A. salmonicida plasmids and frequency of transfer to E. coli recipient

Strain isolatedYeartet geneIncUIS6100Tn1721 (complete)sul1dfrA16pRAS1-likeaTransfer frequencyGroupd
U10216-921992A++No1.9 × 10−31
U15194-951995A++++Yes5.7 × 10−43
U18470-951995A++++Yes8.7 × 10−53
U12652-971997A++++Yes2.8 × 10−63
U15828-981998A++++Yes7.9 × 10−53
U13675-991999A++++Yes8.3 × 10−53
  • apRAS1-like was defined as positive for IS6100, IncU, sul1, dfrA16 plus the 13 kb region containing these genes had the same restriction pattern as pRAS1.

  • bpRAS1-like but also has complete Tn1721.

  • cNo transformants were detected in multiple experiments.

  • dbased on results of tet gene, presence of IncU replicon IS6100, complete Tn1721, presence of sul1 and dfrA16 genes.

2.2 PCR, DNA–DNA hybridization and Southern blots

The TcrA. salmonicida were screened for the presence of the tet(A), tet(B), tet(C), tet(D) and tet(E) genes by DNA–DNA hybridization and specific PCR assays as previously described [4]. The IncU, IncM and IncP incompatibility was determined by DNA–DNA hybridization of plasmid with specific probes, as was the presence of IS6100, the sul1 and dfrA16 genes and the complete Tn1721. PCR assays and cloning of PCR products were done to verify the presence of the IncU incompatibility, the IS6100, the presence of sulI and dfrA16 genes and the complete Tn1721. Whether the plasmid was pRAS1-like was based on the presence of the IncU incompatibility group, the presence of IS6100, and the sul1 and the dfrA16 genes. If the plasmid carried all four traits they were considered to be pRAS1-like (Table 1). Primers used for the various assays are listed in Table 2. Primers were combined for PCR assays and appropriate controls were used. Plasmids from the A. salmonicida and E. coli HB101 transconjugants were prepared as previously described [12].

View this table:

Primers used for detection of various genes, transposon and IS sequences

GenePrimerSequence 5′–3′Reference
sul1sulFCTT CGA TGA GAG CCG GCG GCThis study
Tn17211721orf1FTTA TCC TCG TTT TTC CCA AAA CThis study

Southern blots of the plasmid preparations were made and blots hybridized with 32P-lableled primers as previously described to verify location of the tet genes [7,14].

2.3 Mating experiments

Mating experiments were done using a modified E. coli HB101 as the recipient [3,4]. This strain had previously been selected for chromosomal resistance to fusidic acid, nalidixic acid, rifampicin, (25 μg ml−1 for each) and streptomycin (1000 μg ml−1) [3,4]. The recipient was grown at 36.5 ° C over night, while growth of the atypical A. salmonicida was at 4 °C on BA (Difco Laboratories) until growth was visible (2–4 weeks). The A. salmonicida cells were removed from the plates with a sterile swab and a suspension that was equal to a 3 McFarland standard was prepared for the A. salmonicida donors and E. coli recipient. The mating mixture included 0.5 ml of A. salmonicida and 0.1 ml of the E. coli (5:1 ratio) which was mixed and placed on a BA agar slant in a 5 ml sterile glass tube. The tubes were placed in a chiller set at 15 °C and incubated for 7 days, while those that did not yield transconjugants were incubated for 14 days, to allow for low frequency events. At lower temperatures the E. coli HB101, used as the recipient, did not adequately replicate. Each mating was done a minimum of two times. The bacteria were removed from the slant with a sterile swab, serially diluted and plated onto selective media to determine the frequency of transfer as previously described [4,6]. Transconjugants were selected on L agar supplemented with 20 μg ml−1 tetracycline plus 500 μg ml−1 streptomycin (Sigma Co., St. Louis, MO, USA) and the E. coli recipient was selected on L agar supplemented with streptomycin (500 μg ml−1), both were incubated at 36.5 °C. The resulting transconjugants were re-streaked onto L agar with rifampicin (25 μg ml−1) plates and incubated at 36.5 °C to confirm the transfer of Tcr into E. coli recipients. Susceptiblity to tetracycline and the sulfamethoxazole/trimethoprim combination was determined in the transconjugants. Plasmids were isolated from selected transconjugants for further study.

2.4 Disk susceptiblity

Susceptiblity testing was performed on E. coli transconjugants using Mueller–Hinton agar (Difco Laboratories) with the antimicrobial disks for tetracycline and sulfamethoxazole/trimethoprim using NCCLS protocols and standards [15].

2.5 Restriction endonuclease analysis

The plasmid DNA was digested with EcoRI and separated on a 0.7% agarose gel. The restriction profiles of the A. salmonicida plasmids were done and the region carrying the genes and DNA sequences of interest were identified. The restriction profiles for this region was compared to that of pRAS-1 plasmid (Table 1) [9].

3 Results and discussion

The eight A. salmonicida isolated between 1992 and 1999 carried a tet(A) gene, while the 2001 A. salmonicida carried a tet(B) gene (Table 1). Plasmid preparations from all nine isolates identified a single plasmid of approximately 58 kb in each isolate; no other plasmids were found (Fig. 1). Hybridization with 32P-labeled probes for the tet(A) or the tet(B) genes demonstrated that the large plasmids carried the tet genes (data not shown). Based on the type of tet gene carried, the plasmids could be divided into two groups (Table 1).


Agarose gel of plasmids from A. salmonicida. Lane 1, E. coli V517 with multiple plasmid of known size; lane 2, A. salmonicida 18470-95; lane 3, A. salmonicida 13675-99; lane 3, A. salmonicida 15194-95.

Recently a 12 kb region of pRAS1 was shown to carry the IncU replicon, IS6100 sequences, the sul1 and dfrA16 genes and a fragment rather than the complete Tn1721[11]. Using these same characteristics we could divide the nine plasmids into four groups (Table 1). The U10216-92 plasmid carried a complete Tn1721 and the sul1 gene but was negative for the other genes and the IS element, while the U19158-01 plasmid was negative for all probes tested and carried a tet(B) gene (Table 1). Plasmids from U12141-95 and U13042-95 represented a third plasmid group, which carried a complete Tn1721 and all the characteristics of a pRAS1-like plasmid (13). Plasmids from isolates U15194-95, U18470-95, U12652-97, U15828-98 and U13675-99 represented a fourth group. Restriction analysis with EcoRI, of the plasmid from U10216-92 was distinct from those of U15194-95, U18470-95, U12652-97, U15828-98 and U13675-99. These five plasmids had identical EcoRI restriction patterns to each other and to pRAS1 and carried the tet(A) gene on a 5.5 kb EcoRI fragment similar to pRAS1 (data not shown) [11]. In contrast, the initial pulsed-field gel electrophoresis (PFGE) analysis of the chromosomal DNA using XbaI restriction enzyme [16], indicted that the eight isolates from 1992–1999 carrying the tet(A) gene were related while the isolate from 2001 was distinct.

The ability to conjugally transfer their plasmids at 15 °C to an E. coli recipient also varied. The plasmids from group 3 transferred at frequencies ranging from 5.7 × 10−4 to 2.8 × 10−6/recipient, while the plasmid from group 1 transferred at a higher frequency (1.9 × 10−4) (Table 1). These transfer frequencies are similar to what we found for TcrBrevundimonas vesicularis, Pseudomonas pseudoalcaligens, Providencia rettgeri and Serratia liquefaciens isolated from Chilean fish farms, though short incubation times were used for the Chilean isolates because they grew much more quickly than did the Aeromonas isolates [4].

A single plasmid of approximately 58 kb was found in both donors and selected Tcr transconjugants, from the matings with the six A. salmonicida donors (Fig. 2(a)). The 32P-labeled tet(A) probe hybridized with the large plasmid for each of these transconjugants verifying that the plasmids do carry the tet(A) gene (Fig. 2(b)). Except for transconjugants from the U10216-92 mating, the Tcr transconjugants were resistant to trimethoprim/sulfamethoxazole. All plasmids from the Tcr transconjugants had the same characteristics as the donor strains. The strains carrying plasmids from group 2 and group 4 had no detectable transconjugants after multiple mating experiments, although the rate of transfer could be <1 × 10−10/recipient which is below the detection limit of the assay. The group 1 and 2 plasmids carried a complete Tn1721 element, however the plasmid from isolate U10216-92 was not of the IncU incompatibility group and did not carry the IS6100 sequences or the dfrA16 gene. Jones et al. [17] found a limited correlation between tet(A) carrying plasmids and IncM and IncP incompatibility groups, however the plasmid from U10216-92 did not hybridize with either, nor did the plasmid from U19158-01. In contrast, the two group 2 plasmids were IncU incompatibility and had all the same characteristics as the group 3 plasmids but carried a complete Tn1721, while the group 3 plasmids did not.


(a) Agarose gel of plasmids from transconjugants from various matings. Lane 1, E. coli V517; lane 2, transconjugant from mating with A. salmonicida 18470-95; lane 3, E. coli transconjugants from mating with A. salmonicida 15194-95; lane 4, E. coli transconjugants from mating with A. salmonicida 13675-99; lane 5 E. coli transconjugants from mating with A. salmonicida 12652-97; lane 6, E. coli transconjugants from mating with A. salmonicida 10216-92; lane 7, E. coli transconjugants from mating with A. salmonicida 15828-98. (b) X-ray film of the Southern blot from (a) hybridized with 32P-labeled tet(A) probe.

The nine plasmids could be divided into four groups with groups 1 and 4 carrying non-pRAS1-like plasmids and groups 2 and 3 carrying pRAS1-like plasmids. The group 3 pRAS1-like plasmids, and the group 1 non-pRAS1-like plasmid, were able to transfer their plasmids. The plasmids from groups 1, 2 and 3 shared characteristics in common and the isolates carrying these plasmids had identical PFGE patterns. In contrast, the group 4 plasmid had no characteristic in common with the other eight plasmids and the isolate U19158-01 had a distinct by PFGE (Table 1).

This is the first description of psychrophilic bacteria exchanging antibiotic resistant plasmids directly with a mesophilic laboratory E. coli at 15 °C, though pRAS1 plasmids have been shown to transfer at higher temperature [22 °C] into E. coli[9]. This suggests that these atypical isolates have the potential to directly participate in antibiotic resistance gene exchange with the broader bacterial population and this should be considered when examining the potential for gene exchange in water and related environments.


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