Characterization of class 1 integrons associated with R-plasmids in clinical Aeromonas salmonicida isolates from various geographical areas

Anja S. Schmidta*,*, Morten S. Bruuna, Jens L. Larsena and Inger Dalsgaardb

a Department of Veterinary Microbiology, and b Danish Institute of Fisheries Research, The Royal VeterinFary and Agricultural University, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Class 1 integrons were found in 26 of 40 antibiotic-resistant isolates of the fish pathogen Aeromonas salmonicida from Northern Europe and North America. Three different dhfr genes, conferring trimethoprim resistance, and one ant(3")1a aminoglycoside resistance gene were identified as gene inserts. The gene cassettes tended to be conserved among isolates from a particular geographical area. Nineteen isolates transferred R-plasmids carrying different tet determinants to Escherichia coli in filter mating assays, and in 15 cases, the class 1 integrons were co-transferred. Transferable sulphadiazine, trimethoprim and streptomycin resistances were invariably encoded by integrons. It thus appears that integron-encoded antibiotic resistance genes contribute substantially to the horizontal spread of antimicrobial resistance within this species, being associated with conjugative plasmids.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The emergence of antimicrobial resistance among fish pathogenic bacteria has, during the past decades, become a major concern in many countries with aquaculture production. Numerous findings of fish pathogens resistant to one or several antibiotic agents have been reported,13 sometimes associated with serious economic losses, when widespread antimicrobial resistance impairs an effective medical treatment of clinical outbreaks. Thus, it has become difficult to effectively control infections with Aeromonas salmonicida subsp. salmonicida (hereafter referred to as A. salmonicida), the causative agent of furunculosis.2,3 Plasmid-borne as well as chromosomally located resistance determinants are commonly found,410 and the spread of R-plasmids within this species appears to contribute to the extensive occurrence of antimicrobial resistance. Although frequently reported, the genetic background of plasmid-borne resistance other than tetracycline resistance is still largely unknown.11 An integron-like drug resistance region was found on the transferable R-plasmid pJA8102-1 from A. salmonicida,7 and Sørum12 described a plasmid-borne class 1 integron in an atypical A. salmonicida isolate. Class 1 integrons are present in a wide range of Gram-negative bacteria, including isolates originating from aquatic environments.13,14 The 3'-conserved segment of class 1 integrons contains a sulI resistance gene, and their site-specific recombination system enables them to collect multiple antimicrobial resistance gene cassettes.15 Although not mobile themselves, they are often associated with conjugative plasmids or transposons.15,16 Thus, the aim of our study was to investigate the presence of these elements among A. salmonicida isolates from different countries. Furthermore, we characterized some of the antibiotic resistance genes involved and their association with transferable plasmids.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates

Sources and designations of 40 A. salmonicida isolates from clinical outbreaks of furunculosis are listed in Table I (Go. One atypical isolate (718) containing the plasmid pRAS1 was also included.8 Biochemical characterization was carried out as described previously.17


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Table I. Antimicrobial resistance phenotypes and plasmids in A. salmonicida isolates from different geographical areas and E. coli transconjugants detected in filter mating assays
 
MIC testing

Following identification, all isolates and transconjugants were tested for their susceptibility to five antimicrobial agents used in aquaculture: amoxycillin, oxytetracycline, oxolinic acid, florfenicol, sulphadiazine and trimethoprim. Streptomycin resistance was also assessed. MIC values were determined by an agar dilution method as described by The National Committee for Clinical Laboratory Standards (NCCLS).18 Mueller–Hinton agar (Difco Laboratories, Detroit, MI, USA) was the basic medium. Stock solutions of the respective antibiotics (oxytetracycline, sulphadiazine and trimethoprim: European Pharmacopoeia 2nd edition, oxolinic acid, amoxycillin and streptomycin: Sigma Chemical Co., Poole, UK; florfenicol: Schering-Plough Animal Health, Kenilworth, NJ, USA) and agar plates were prepared as described,18 with final concentrations ranging from 0.125 to 1024 mg/L. Overnight broth cultures (veal infusion broth, Difco) incubated at 20°C were adjusted to an optical density of 0.5 McFarland standard corresponding to 108 cfu/mL, diluted 1:10 in physiological saline and applied as 1 µL droplets to the plates with a multipoint inoculator (P&R Laboratory Group, St Helens, UK). The inoculum was in this way standardized to contain c. 104 cfu. Each test was run in duplicate on freshly prepared agar plates. Reference strains were included as internal standards in all tests:18 Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212) and Pseudomonas aeruginosa (ATCC 27853). Likewise, a type strain was included: A. salmonicida NCMB 1102. After 2 days of incubation at 20°C the MIC for each isolate was determined as the lowest concentration of the antimicrobial agent able to inhibit bacterial growth.

Isolates were classified as resistant or susceptible to an antimicrobial agent according to their relative MICs (Table I). Breakpoints were determined, with resistant isolates growing in the presence of >=32 mg/L amoxycillin (susceptible isolates: <=0.5 mg/L), >=16 mg/L oxytetracycline [susceptible isolates: <=0.5 (donors) or <=4 mg/L (E. coli)], >=2 mg/L oxolinic acid (susceptible isolates: <=0.25 mg/L), >=2 mg/L florfenicol (<=0.5 mg/L in susceptible isolates), >=512 mg/L sulphadiazine (susceptible isolates: <=128 mg/L), >=512 mg/L trimethoprim (susceptible isolates: <=2 mg/L) and >=64 mg/L (donors) or >=8 mg/L (transconjugants) streptomycin (susceptible donors: <=8 mg/L, susceptible E. coli strain: <=2 mg/L). A few isolates had MICs between these two distinct clusters, and were consequently termed intermediately susceptible.

Plasmid analysis

The antibiotic-resistant A. salmonicida isolates and the transconjugants were screened for their plasmid content, applying the alkaline lysis method described by Kado & Liu,19 followed by agarose gel electrophoresis (Figure 1Go).



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Figure 1.  R-plasmid transfer from A. salmonicida to E. coli. Lanes a and i: strains 39R and V517, respectively, containing reference plasmids of known molecular sizes (kb). Lane b: isolate F5 (tetA positive); lane c: tetA-positive E. coli transconjugant carrying a 50 kb plasmid; lane d: isolate 718 (tetA positive); lane e: tetA-positive E. coli transconjugant with 32 kb plasmid; lane f: isolate MT906 (tetA and tetC positive); lane g: tetA-positive transconjugant containing a 50 kb plasmid; and lane h: tetC-positive transconjugant harbouring a 13 kb R-plasmid.

 
Conjugational gene transfer

All antibiotic-resistant isolates were then included in a filter mating assay as donors in order to detect a putative transfer of R-plasmids to E. coli CSH26Rf.20 Overnight cultures of donor and recipient were adjusted to OD600 = 0.5 with fresh veal infusion broth. Equal volumes (50 µL) of each culture were mixed on a sterile 0.2 µm nitrocellulose filter (Sartorius AG, Goettingen, Germany) placed on a veal infusion agar plate (Difco) and incubated at 20°C overnight. Cells were washed off the filter by vortexing in 10 mL sterile 0.9% NaCl solution, and appropriate 10-fold dilutions were prepared. From each dilution, three 25 µL droplets were applied to selective agar plates, containing 20 mg/L oxytetracycline, 100 mg/L rifampicin (Bie & Berntsen, Rødovre, Denmark) or both. Donor and recipient were also plated on the double selective plates to detect putative mutants. All assays were run in duplicate. Transfer frequencies were calculated as the mean number of transconjugants per recipient (Table I). Transconjugants were screened for the presence of plasmids as described above.

Repeated retransfer experiments were performed as described above, where a tetC-positive E. coli transconjugant with a 13 kb plasmid (originally transferred from isolate MT906, Figure 2Go) was the donor and an amoxycillin- and oxolinic acid-resistant A. salmonicida isolate (96-8-136) the recipient. Selective agar plates contained 20 mg/L oxytetracycline, 2 mg/L oxolinic acid (Sigma) or both. Another assay was run with an oxolinic acid-resistant E. coli CSH26Rf mutant as recipient.



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Figure 2.  Class 1 integrons associated with transferable oxytetracycline R-plasmids among A. salmonicida isolates. Primers targeting the conserved segment of the integron result in different sized PCR products, depending on the inserted gene cassettes: 150 bp, no insert; 550 bp, dhfrIIc; 750 bp, dhfrXVI; 1000 bp, ant(3")1a; 1500 bp, dhfrI combined with a downstream ant(3")1a cassette. Lanes a and s: 100 bp marker; lane b: positive control strain S. typhimurium DT104;22 lane c: negative control; lane d: F5 (Faroes); lane e: transconjugant. Lane f (Norway): 718; lane g: transconjugant; lane h: MT906 (Scotland); lane i: transconjugant with 213 kb R-plasmid; lane j: transconjugant with 13 kb R-plasmid; lane k: 950704-2/1 (Denmark); lane l: transconjugant; lane m: MT900 (Scotland); lane n: transconjugant; lane o: 90644 (Canada); lane p: transconjugant; lane q: 86442 (Canada); and lane r: transconjugant.

 
Detection of tet determinants

In order to screen for tet determinants A–E, we applied a multiplex PCR assay according to Guardabassi et al.21 E. coli strains containing the respective tetracycline resistance genes were included (class A: NCTC50078; class B: HB101/pRT11; class C: DO7/pBR322; class D: C600/ pSL106; class E: HB101/pSL1504). All isolates and transconjugants with MIC values exceeding 16 mg/L oxytetracycline were tested (Table IIGo). Primers are listed in Table IIIGo.


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Table II.  Characterization of tet determinants and antibiotic resistance genes associated with class 1 integrons transferred to E. coli on R-plasmids from clinical A. salmonicida isolates
 

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Table III.  Oligonucleotide primers for PCR analysis of class 1 integrons
 
Analysis of antimicrobial resistance genes associated with integrons

All isolates and transconjugants were screened for the presence of class 1 integrons with specific primers targeting the conserved 5' and 3' segments of the structure as described previously (Table IIIGo and Figure 3Go).22 Thus, the size of a PCR product depends on the number and size of the inserted gene cassettes. Salmonella typhimurium DT104 (9616368) was the positive control strain. PCR products were purified (S-400 HR MicroSpin Columns; Amersham Pharmacia Biotech, Uppsala, Sweden) and sequenced. The nucleotide sequence was determined of both strands of the DNA, using a cycle sequencer 373A (Applied Biosystems, Perkin Elmer, Foster City, CA, USA) as reported earlier.22 With five different sizes ranging between c. 150 bp and 1500 bp, all short amplicons (<1000 bp) and two representatives of the 1000 bp and 1500 bp products, respectively, were fully sequenced. As the 1000 bp and 1500 bp amplicons appeared to contain dhfrI and ant(3")1a inserts alone or in combination, a specific PCR assay targeting the dhfr1 and ant(3")1a genes was then designed to analyse the remaining 1000 bp and 1500 bp PCR products (Figure 3Go). Primer selection was based on published DNA sequences (see Table IIIGo). Cycling conditions were identical for both primer sets: 5 min of initial denaturation at 94°C was followed by 20 cycles of denaturation at 94°C (30 s), annealing at 57°C (30 s) and extension at 72°C (30 s), final extension: 7 min at 72°C (GeneAmp PCR System 9700, Perkin Elmer). Readyto-go PCR beads (Amersham Pharmacia Biotech) were used according to the manufacturer's instructions. Correct amplification was confirmed by sequencing of purified PCR products. The similar Tm values of the two primer sets also allowed determination of the order of the gene cassettes in those cases where two cassettes were inserted, by using dhfr1 F and ant B primers, and ant F and dhfr1 B primers, respectively (Figure 3Go).



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Figure 3. Structure of a class 1 integron with two antibiotic resistance gene inserts and annealing positions of the primers employed in the study (short arrows). The black boxes represent 59 bp elements. IntI, integrase; dhfr1, dihydrofolate reductase (trimethoprim resistance gene); ant(3")1a, aminoglycoside resistance gene; qacE{Delta}1 and sul1 encode disinfectant and sulphonamide resistance, respectively.

 
A separate primer set was used to investigate the 3' conserved segment of the class 1 integrons, containing the qacE{Delta} and sul1 genes (Table IIIGo and Figure 3Go),22 in order to detect defective copies with incomplete sul1 genes, which were frequent findings in the study of aquatic bacteria by Rosser & Young.14


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The plasmids and antibiotic resistance patterns detected in the isolates and transconjugants are presented in Table I, the most prevalent resistotype being resistant to oxytetracycline, sulphadiazine, trimethoprim and streptomycin (14 isolates). When selecting for transfer of oxytetracycline resistance genes, 20 different transconjugants were obtained from 29 A. salmonicida donors.

Most oxytetracycline-resistant isolates (25/29) were also resistant to sulphadiazine, a trait that was transferred to 15 out of 20 different E. coli transconjugants together with the respective tet determinants. Likewise, trimethoprim resistance was found exclusively in combination with oxytetracycline resistance (15 of 29 isolates). A trimethoprim determinant was co-transferred to E. coli in 10 cases, always together with sulphadiazine resistance.

Streptomycin resistance was common and transferable to E. coli on the oxytetracycline resistance plasmid, as 31 isolates and seven transconjugants (Table I) were not susceptible to the drug.

Ten Danish and one Norwegian isolate were amoxycillin resistant, six of them in conjunction with tetracycline resistance but not detected in any transconjugant. Similarly, resistance to oxolinic acid was found in 14 Danish, one Scottish and two Canadian isolates, in nine cases in combination with oxytetracycline resistance. Resistance to florfenicol was found exclusively in seven of the Danish isolates, some of which were oxytetracycline resistant. Again, no co-transfer to E. coli occurred in the filter mating assays. Curiously, most florfenicol-resistant isolates were collected before the introduction of the drug as therapeutic agent in Danish aquaculture in 1996 (Table I).

Most tetracycline resistance genes were located on transferable R-plasmids. Plasmid sizes and transfer frequencies are listed in Table I. Assays with foreign A. salmonicida isolates as donors rendered transconjugants in 16 of 17 different filter matings, in contrast to three of 12 potentially oxytetracycline-resistant Danish donors (Table I). tetA was identified in 17/29 = 58% of the resistant isolates, while three isolates were tetC positive (Table IIGo). Interestingly, one Scottish isolate, MT906, was both tetA and tetC positive. Five isolates from Denmark and three from Canada had unidentified tet determinants. Fourteen of 17 tetA determinants were transferred to E. coli on plasmids, as were the unidentified tet determinants from the three Canadian isolates (Table IIGo). All tetC-positive isolates harboured an unusual 13 kb plasmid (data not shown). Aoki7 has previously noted a non-transferable R-plasmid of a similar size (11.2 kb), carrying a transposon-associated tetC determinant. However, in our study the 13 kb R-plasmid was actually transferred to E. coli at low frequencies from MT906, resulting in a tetC-positive transconjugant (Figure 1Go). No larger plasmids were detected in the transconjugant, and the putative mobilization mechanism is currently being investigated. Interestingly, in repeated filter matings the same donor transferred a 50 kb plasmid carrying a tetA determinant instead of the 13 kb plasmid (Table I and Figure 1Go). No re-transfer to E. coli or an A. salmonicida recipient occurred in repeated matings. One American isolate also rendered a tetC-positive transconjugant, but in this case, the tet gene was located on a large 140 kb plasmid (Table I).

The transferable R-plasmids from Danish and North American isolates were between 140 and 160 kb in size and had lower transfer frequencies compared with the 50 kb resistance plasmids in isolates from Scotland and the Faroes (Table I and Figure 1Go). The atypical A. salmonicida isolate 718 transferred a 32 kb plasmid to E. coli at high frequencies (Figure 1Go), a molecular weight similar to that of the broad host range plasmid pRAS1.8 Typical A. salmonicida strains are also able to transfer R-plasmids within the species as well as to other recipients,4,6 but they tend to be unstable in other bacterial hosts.23 Adams et al.6 propose a common ancestry of oxytetracycline resistance plasmids based on hybridization studies and restriction enzyme profiling of transferable R-plasmids in mainly Scottish A. salmonicida isolates, with tetA being the main genetic determinant. Our results indicate that other tet determinants besides tetA are involved in the spread of oxytetracycline resistance on R-plasmids within this species. The distinct geographical variations might be related to the different usage of antimicrobial agents in the different countries.

Conjugational transfer of sulphadiazine was always associated with the transfer of an integron. All sulphadiazine-resistant isolates harboured class 1 integrons, where sul1 was part of the conserved 3' region (Tables I and II, and Figure 3Go). Likewise, trimethoprim resistance was invariably encoded by one of three different class 1 integron gene cassettes, dhfrI, dhfrIIc and dhfrXVI (Table IIGo). The origin and epidemiology of integron-associated gene cassettes remains unclear.24 Cassette-encoded dihydrofolate reductases have been identified in a broad range of bacterial species from clinical and environmental sources.1315 While dhfrI and dhfrIIc seem to occur commonly in isolates from Denmark and the Faroes, respectively, an unusual dhfr gene insert, dhfrXVI, was detected in the atypical isolate 718 (Table IIGo). To our knowledge, dhfrXVI (first described in a S. typhimurium strain, GenBank accession no. AF077008) has not been reported from an aquatic habitat before. An integron with a dhfrIIc cassette was transferred on the 50 kb R-plasmid (but not with the 13 kb plasmid) (Figure 2Go) of the Scottish isolate, MT906. No dhfr1 or sul1 genes occurred in integron-negative isolates. The high prevalence of plasmid-borne class 1 integrons in this study suggests that the extensive usage of potentiated sulphonamides in aquaculture may have favoured the spread among A. salmonicida isolates of R-plasmids carrying integrons with dhfr cassettes.

Eight Danish isolates with a 1500 bp PCR product had a dhfrI gene inserted into the variable section of the integron in combination with a downstream aminoglycoside resistance gene, ant(3")1a (Table IIGo). A 1000 bp insert consisting of ant(3")1a alone was found in three isolates from Denmark and in four of five Scottish integron-positive isolates. One isolate (86442) contained an integron without insert as well as another with an inserted ant(3")1a gene (Figure 2Go), and transferred both to E. coli together with the plasmid. The aminoglycoside resistance gene ant(3")1a was found inserted into three Danish integrons on 150 kb R-plasmids and four Scottish 50 kb R-plasmids. In the remaining eight integron-positive isolates from Denmark, it was combined with an upstream dhfrI gene, and conjugational transfer involved 150 kb plasmids. The high prevalence of aminoglycoside resistance within the species (Table I) has been noted before,2,7,11 but the genetic background needs further investigation. Aminoglycosides do not occur naturally in the aquatic environment, and are not commonly used as therapeutic agents in aquaculture. A number of aminoglycoside resistance genes are likely to be involved, as some isolates exhibited streptomycin resistance but were ant(3")1a negative (data not shown). However, the transferable streptomycin resistance was encoded exclusively by ant(3")1a (Table IIGo). It has been speculated,14,15 that the wide distribution of the ant(3")1a gene cassette and its preferred location downstream of additional cassettes is due to its prior acquisition and/or a more stable integration.

In our study, conjugative transfer of R-plasmids included the transfer of a class 1 integron (Table IIGo and Figure 2Go), except in two cases, where two ‘empty’ integrons were not transferred to the respective transconjugants from Q203 and 90644. Interestingly, most North American integrons did not contain any resistance gene cassettes. Recent findings of numerous ‘empty’ integrons in bacteria sampled from estuarine waters14 indicate that the resistance gene cassettes may be excised in the absence of selective antibiotic pressures.

Although unable to move on their own, integrons and their inserts contribute to horizontal gene transfer in association with transposons and conjugative plasmids.25,26 It is obvious from our results that class 1 integrons and their resistance gene inserts are highly prevalent on conjugative R-plasmids within A. salmonicida isolates from different countries. Integron-associated sul1 genes were found in all sulphonamide-resistant isolates, often in combination with trimethoprim resistance gene cassettes. Moreover, the transferred streptomycin resistance appeared to be predominantly encoded by ant(3")1a inserts.

Martinez-Freijo et al.16 concluded that the presence of certain cassettes or cassette combinations among human, clinical isolates of enterobacteria was independent of their geographical origin. This study was dealing with very different clinical settings, and the population structure of the pathogen has often been considered to be homogeneous.10,17 The gene cassettes were usually conserved among isolates originating from a particular region, although they differ in resistotype and/or plasmid profile. In contrast, gene cassettes vary between geographical regions. Our results suggest that multiple mechanisms of gene acquisition are involved in the emergence of plasmid-borne antibiotic resistance genes within A. salmonicida, resulting in varying gene patterns in different geographical areas.


    Acknowledgments
 
The skilled technical assistance of Farah S. Bahrani is highly appreciated. We thank Frank M. Aarestrup, Lars B. Jensen and Dorthe Sandvang at the Danish Veterinary Laboratory, Copenhagen, for their generous help and donation of strains, and Henning Sørum, Norwegian College of Veterinary Medicine, for donating isolate 718. We also thank Søren Sørensen (University of Copenhagen) for provision of E. coli CSH26Rf, as well as Gilles Olivier, Dept of Fisheries and Oceans, Canada, and Trevor Hastings, Marine Laboratory, Scotland, for donation of A. salmonicida isolates. This work was supported by the Danish Ministry of Food, Agriculture and Fisheries.


    Notes
 
* Corresponding author. Tel: +45-352-82710; Fax: +45-352-82711; E-mail: ansc{at}kvl.dk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received 8 November 2000; returned 31 January 2001; revised 27 February 2001; accepted 2 March 2001