JAC Advance Access originally published online on April 14, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):65-71; doi:10.1093/jac/dkn166
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Original research |
Complete sequence of the floR-carrying multiresistance plasmid pAB5S9 from freshwater Aeromonas bestiarum
1 INRA, ENVN, UMR1035 Chimiothérapie Aquacole et Environnement, F-44307 Nantes, France 2 INRA, UR1282 Infectiologie Animale et Santé Publique, F-37380 Nouzilly, France 3 Institut für Nutztiergenetik, Friedrich-Loeffler-Institut (FLI), D-31535 Neustadt-Mariensee, Germany 4 Unité de Biodiversité des Bactéries Pathogènes Emergentes, Institut Pasteur, F-75724 Paris Cedex 15, France
* Corresponding author. Tel: +33-2-40-68-78-62; Fax: +33-2-40-68-78-28; E-mail: gordon{at}vet-nantes.fr
Received 7 January 2008; returned 14 February 2008; revised 11 March 2008; accepted 21 March 2008
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Abstract |
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Objectives: A multiresistant Aeromonas bestiarum strain, shown to be persistent and spreading in a freshwater stream, was investigated for the presence, location and organization of antimicrobial resistance genes.
Methods: The plasmid pAB5S9 was transferred by electroporation into Escherichia coli TG1. The resistance phenotype mediated by pAB5S9 was determined. Moreover, the plasmid was sequenced completely and analysed for its structure and organization of reading frames.
Results: Plasmid pAB5S9 mediated resistances to phenicols, sulphonamides, streptomycin and tetracycline. The analysis of the 24.7 kb sequence revealed the presence of 20 predicted coding sequences (CDSs), which included the floR, sul2 and strA-strB resistance genes and a tetR-tet(Y) determinant. Approximately 7.5 kb of pAB5S9 showed 100% nucleotide sequence identity to three non-contiguous segments of the SXT element of Vibrio cholerae. Regions identical to SXT comprised the floR gene, flanked upstream by a complete and downstream by a truncated ISCR2 element, and the region of the sul2 and strA-strB genes. Other CDSs of pAB5S9 related to plasmid replication and partitioning, metabolic and gene regulation functions as well as conjugative transfer showed homology to sequences from diverse bacterial species, indicating a mosaic structure.
Conclusions: This study provides the first report of a floR-carrying plasmid in the genus Aeromonas and the first description of a tetR-tet(Y) determinant. The analysis of the multiresistant A. bestiarum strain indicates that strains of this species, some of which are opportunistic pathogens for fish, might also act as a resistance gene reservoir in the freshwater environment.
Keywords: antibiotics , florfenicol resistance , tetracycline resistance , fish-pathogenic bacteria , aquatic environment
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Introduction |
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Aeromonas spp. are ubiquitous aquatic bacteria that commonly host resistance plasmids.1–3 In humans, these bacteria have been associated with diarrhoeal disease and opportunistic wound infections.4 Some members of the phenotypic ‘Aeromonas hydrophila’ complex, which includes the species A. hydrophila, Aeromonas bestiarum and Aeromonas salmonicida (the latter being considered as non-motile, unlike the other two), are also major aetiological agents of fish diseases.5 In aquaculture farms, bacterial disease outbreaks, including Aeromonas-associated ones, are often prevented or treated with antimicrobials agents. The natural susceptibility of Aeromonas spp. to antimicrobials (except to ampicillin for a majority of strains) renders them interesting indicators for the survey of antimicrobial resistance in freshwater environments.3,6 A study by Bruun et al.7 points at the possible role of environmental aeromonads as providers of resistance genes for human pathogens. Indeed, freshwater streams are receptors of many domestic, industrial and agricultural wastes, which are known to contain antimicrobial agents and antimicrobial-resistant bacteria.6,8 Because they host microbial ecosystems in which bacteria of very diverse phylogenetic and ecological origins can meet, freshwater environments likely provide favourable conditions for the spread of antimicrobial resistance. Autochthonous aquatic bacteria such as Aeromonas spp. may acquire resistance plasmids from allochthonous bacteria introduced by wastes and may, in turn, behave as reservoirs of resistance genes for other susceptible autochthonous and possibly pathogenic bacteria. In the aquatic context, multiresistance plasmids offer an important selective advantage to their bacterial hosts, as they increase their chance to be selected for in a wide range of contaminated environments.9
In a previously described survey conducted along a French river receiving effluents of fish farms, we had isolated Aeromonas spp. strains on the basis of their resistance to florfenicol.6 In France and other European Union (EU) countries, the use of florfenicol was approved only for cattle and swine. Nevertheless, in several non-EU member states, it is also approved for use in fish, mainly for the treatment of furunculosis caused by A. salmonicida and rainbow trout fry syndrome caused by Flavobacterium psychrophilum.10,11 In the present study, we characterized a multiresistance plasmid responsible for the florfenicol resistance of an environmental strain of A. bestiarum.
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Bacterial strains isolation, identification and typing
The isolation of nine florfenicol-resistant Aeromonas in freshwater sediments sampled in a river in Brittany, France, which receives domestic and agricultural effluents, was described previously.6 Species identification was performed by sequencing 940 nucleotides of the rpoB housekeeping gene and comparing the results with a database held at the Pasteur Institute, Paris, France. The rpoB genes of the isolates were sequenced between positions 1490 and 2430 (numbering of the Escherichia coli rpoB sequence; GenBank accession no. U76222 [GenBank] ). For their typing by PFGE, genomic DNA was prepared as described previously,12 restricted with XbaI (Promega, Charbonnières, France) and separated using a CHEF DR III system (Bio-Rad, Marnes-la-Coquette, France) at 14°C for 23 h at 220 V with a pulse time of 0.5–18 s.
The E. coli TG1 and the rifampicin-resistant E. coli J5 strains were used as recipients in electrotransformation and conjugation experiments, respectively. E. coli ATCC 25922 and A. hydrophila ATCC 35654 were used as quality control strains in all susceptibility testing procedures. The E. coli BN10660-1 strain, which carries the floR gene on the BN10660 conjugative plasmid,13 was used as control in PCR experiments.
Antimicrobial susceptibility testing
Antimicrobial susceptibility patterns of the original Aeromonas isolates and the E. coli TG1 transformed with pAB5S9 were determined by disc diffusion following the recommendations given in the documents M42-A and M31-A2 of the Clinical and Laboratory Standards Institute (CLSI).14,15 The following antimicrobial agents were tested (disc contents in parentheses): florfenicol (30 µg), chloramphenicol (30 µg), ampicillin (10 µg), ceftriaxone (30 µg), ceftazidime (30 µg), aztreonam (30 µg), streptomycin (10 µg), spectinomycin (100 µg), gentamicin (15 µg), kanamycin (30 µg), neomycin (30 IU), sulfamethoxazole (300 µg), trimethoprim (5 µg), tetracycline (30 µg), ciprofloxacin (5 µg), enrofloxacin (5 µg), marbofloxacin (5 µg) and nalidixic acid (30 µg). All antimicrobial discs except for florfenicol were purchased from Bio-Rad. Florfenicol discs were obtained from Schering-Plough Animal Health (Segré, France).
MICs of florfenicol, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline were determined for the original A. bestiarum 5S9 strain and the E. coli TG1 strain transformed with pAB5S9, according to the recommendations given in the CLSI documents M31-A2 and M49-A, respectively.15,16 To investigate the possible inducibility of tetracycline resistance, tested strains were also cultivated in Mueller–Hinton broth supplemented with 1 mg/L tetracycline, prior to the determination of tetracycline MICs.17
Standard PCRs were performed to detect the floR gene with the primers floR1 (5'-CCCGCTATGATCCAACTCAC-3') and floR2 (5'-ACCCACATCGGTA GGATGAA-3') and an annealing temperature of 55°C. The expected size of the amplicon was 803 bp. The presence of circular genetic elements carrying the floR gene was also investigated with the florCirc1 and florCirc2 primers, as described previously.13
Plasmid DNA was purified from the A. bestiarum strains by an alkaline lysis procedure.18 Electrocompetent E. coli TG1 cells were prepared from 200 mL of a fresh culture (OD600 of 0.5) in LB broth. Cells were submitted to several washes, first with cold sterile water and then with cold 10% glycerol. Electrocompetent cells were finally resuspended with 400 µL of cold 10% glycerol and frozen at –70°C until use. Electroporation of 40 µL of E. coli TG1 cells in the presence of the plasmid extracts was carried out in sterile 0.1 cm gap electroporation cuvettes (Eurogentec, Seraing, Belgium) and using a BioRad E. coli Pulser with the following parameters: 25 µF, 1.25 kV and 200 
. Transformants were selected by plating electroporated cells on LB agar plates supplemented with 32 mg/L florfenicol. Conjugation experiments were performed at 22 and 37°C in liquid medium, as previously described.13
Plasmid DNA was extracted and purified from cultures of E. coli transformants with a QIAGEN Plasmid Midi Kit (Courtaboeuf, France) used according to the manufacturer's instructions. Plasmid DNA was digested with the restriction enzyme EcoRI or BglI and submitted to electrophoresis in a 0.8% agarose gel. Southern blot hybridization was carried out with the ECL direct nucleic acid labelling and detection system (Amersham Biosciences, Orsay, France). The digested DNA was then electrotransferred onto a Hybond-N+ membrane (Amersham Biosciences). After fixation by heating, the membrane was hybridized using as probe the 6522 bp EcoRI–BamHI floR-carrying fragment from the E. coli BN10660 plasmid (GenBank accession no. AF231986 [GenBank] ).19 The signals were detected by exposure of the membrane to Hyperfilm ECL film (Amersham Biosciences).
Plasmid pAB5S9 was sequenced by Genome Express (Grenoble, France) by primer walking, starting with primers designed from the known floR gene sequences. Homology searches were carried out using the BLAST suite of programs.20 Coding sequences (CDSs) were detected with the GeneMark gene prediction software, version 2.5 (http://exon.gatech.edu/genemark/) and the ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The complete nucleotide sequence of pAB5S9 has been deposited in the GenBank database under accession number EF495198.
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Isolation of an A. bestiarum strain carrying a multiple antimicrobial resistance plasmid
Originally, nine florfenicol-resistant Aeromonas spp. strains were isolated from sediments sampled at diverse locations along a river in Brittany, France, during two sampling campaigns conducted in October 2004 and March 2005.6 All these isolates were shown by PCR to carry the floR gene conferring resistance to chloramphenicol and florfenicol (data not shown).21 PFGE typing experiments and species identification by rpoB sequencing revealed that five florfenicol-resistant Aeromonas strains represented the same A. bestiarum clone (data not shown), designated 5S9. These findings indicated that the A. bestiarum 5S9 clone has persisted for at least 5 months and has spread over a distance of at least 14 km along the stream.6 The four other florfenicol-resistant isolates were identified as Aeromonas media and represented the same clone, as shown by PFGE typing (data not shown). Attempts to transfer, by conjugation or electrotransformation, the florfenicol resistance of those isolates to E. coli were unsuccessful.
The florfenicol resistance of A. bestiarum 5S9 was plasmid-mediated, as we could transfer this resistance to an E. coli TG1 recipient strain by electroporation. With the exception of ampicillin resistance, which is a characteristic of most aeromonads, and nalidixic acid resistance, which is presumably due to chromosomal mutations, all the other antimicrobial resistances of the A. bestiarum strain were transferred with the pAB5S9 plasmid (Table 1). In addition to the expected florfenicol/chloramphenicol resistance, pAB5S9-transformed E. coli were also resistant to streptomycin, sulfamethoxazole and tetracycline.
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Plasmid pAB5S9 sequence analysis
The complete sequence of plasmid pAB5S9 consisted of 24 716 bp and revealed an average G+C content of 54.0%. Standard nucleotide BLAST revealed that more than 7.5 kb out of 24.7 kb was identical to sequences of the SXT integrative conjugative element present in the Vibrio cholerae genome sequences.22 The annotation of the pAB5S9 plasmid nucleotide sequence showed that it contained 20 predicted CDSs (Table 2 and Figure 1). All of them appeared to be complete, except one truncated putative transposase. Putative functions could be assigned to 17 of the 19 apparently complete CDSs on the basis of protein homology.
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Five CDSs were associated with antimicrobial resistance (CDSs 9 and 13–16), two with plasmid replication and partitioning functions (CDSs 3 and 4), four with metabolic and gene regulation functions (CDSs 1, 7, 10 and 12), four with conjugative transfer (CDSs 17–20) and two with transposition (CDSs 5 and 8). The gene map of plasmid pAB5S9 is presented in Figure 1(a), and the essential features of the predicted CDSs are given in Table 2.
Genetic environment of the floR gene
Plasmid digestions with BglI and EcoRI and Southern blot hybridization experiments first revealed that the close genetic environment of floR on the pAB5S9 plasmid was similar to that previously described for the E. coli BN10660 plasmid (data not shown).13,19 On the BN10660 plasmid, floR was originally reported to be flanked upstream by a truncated transposase gene (
tnpA) and downstream by a complete transposase gene (tnpA).13 These transposase sequences are now recognizable as parts of ISCR2 elements.23 Sequencing showed that the situation on pAB5S9 is opposite to that observed on the BN10660 plasmid, as the floR gene is flanked upstream by a complete ISCR2 element and downstream by a truncated one (Figure 1). For the BN10660 plasmid, the assumption was made that floR is part of a novel transposon.13 The authors detected a circular form by PCR, which was interpreted as an intermediate of transposition of this putative transposon named TnfloR.13 In view of the in silico analysis of ISCR elements, Toleman et al.24 suggested that homologous recombination between the repeated ISCR2 sequences could as well generate such circular forms of the expected size. Interestingly, we were also able to amplify by PCR the same circular form obtained for the putative circular form of TnfloR (data not shown), using the pAB5S9 plasmid as template and the same divergent primers as used by Doublet et al.13 Indeed, the presence of these complete and truncated ISCR2 results in the presence of a 524 bp duplicated region on both sides of floR, which can act as substrates for homologous recombination events.
Moreover, the region spanning CDSs 8–11, which contains the floR gene, showed 100% nucleotide identity with two segments of the SXT element of V. cholerae (Figure 1a). In pAB5S9, the complete ISCR2 element located 0.9 kb upstream of floR was found to be identical to that present, although more than 5 kb upstream of floR, on the SXT element.22 Downstream of floR, the situation was strictly identical in pAB5S9 and in SXT, with the presence of a putative LysR transcriptional repressor gene and the truncated ISCR2 element.
Both the complete and the truncated ISCR2 exhibited a 16 bp terIS sequence, a putative replication terminator, located 100 bp upstream of the start codon of the transposase gene (Figure 1b). In contrast, only the complete ISCR2 was found to possess a 19 bp oriIS sequence, a putative origin of replication, located 214 bp downstream of the stop codon of the transposase gene (Figure 1b). However, the functionality of the ISCR2 element for mobilizing resistance genes has to be elucidated. Although experimental evidence is still lacking, it is thought that ISCR elements could transpose through a rolling circle (RC)-replication mechanism as IS91-like transposable elements do and could mobilize DNA located upstream of the transposase gene.23,24 This may occur when the RC-replication mechanism misidentifies the cognate termination sequence terIS and proceeds to replicate the DNA adjacent to terIS. However, in the case of plasmid pAB5S9, all the resistance genes are located downstream of the complete ISCR2 and are, therefore, not adjacent to the terIS.
Tetracycline, streptomycin and sulphonamide resistances genes
Interestingly, streptomycin and tetracycline MICs were very different between the A. bestiarum 5S9 strain and the E. coli TG1 transformed with pAB5S9 (Table 1). The streptomycin MIC was 16-fold higher for A. bestiarum 5S9 than for the E. coli transformant (256 versus 16 mg/L, respectively). Streptomycin susceptibility in the presence of the strAB resistance determinant, or other streptomycin resistance genes, has been observed previously in E. coli.25 In contrast, the tetracycline MIC was 8-fold higher for E. coli TG1 carrying pAB5S9 than for A. bestiarum 5S9. However, we observed that tetracycline resistance was highly inducible in A. bestiarum, with an increase in the MIC from 16 to 128 mg/L after pre-incubation with 1 mg/L tetracycline. In contrast, the pre-incubation of E. coli TG1 carrying pAB5S9 in the presence of tetracycline only increased the MIC from 128 to 256 mg/L. This latter observation suggested that tetracycline resistance was expressed at a high level in the E. coli background, whereas it was inducibly expressed in the A. bestiarum background.
Downstream of the ISCR2- and floR-carrying region, a tet resistance determinant was identified, consisting of the divergently oriented tetR repressor gene (CDS 12) and the tet(Y) efflux protein gene (CDS 13). To the best of our knowledge, this is the first report of a tet(Y)-associated tetR gene. BLAST analysis indicated that the corresponding TetR(Y) protein shared only 64% identity (and 75% similarity) with the next closely related TetR protein, namely TetR(G), encoded by a Listonella anguillarum plasmid (Table 2). Considering the inducibility of the tetracycline resistance phenotype that we demonstrated in A. bestiarum, it can be assumed that this tetR gene is functional.
The region spanning the two streptomycin phosphotransferase genes strB and strA (CDS 14 and 15, respectively) and the sul2 gene coding for a type II dihydropteroate synthase (CDS 16) showed 100% identity to a 2.9 kb segment of the V. cholerae SXT element.22 In the SXT element, this region is located only a few nucleotides downstream of the 3'-end of the floR-carrying segment (Figure 1a). Actually, the presence of the tet determinant in pAB5S9 appeared to be the result of an insertion in the SXT sequences between the truncated ISCR2 element and the streptomycin resistance genes. This hypothesis was supported by the significantly lower GC content of the tetR and tet(Y) genes, when compared with the flanking sequences (Table 2).
Other genes present on pAB5S9 also indicated a mosaic structure, which is likely the result of interplasmid recombination events. Two non-contiguous CDSs, namely repA (CDS 3), which codes for a putative replication initiation protein, and resA (CDS 5), which codes for a putative resolvase, showed highest amino acid homology to a Bordetella bronchiseptica plasmid,26 with amino acid identities of 
80% (Table 2). The region encompassing CDSs 6 and 7 showed total nucleotide identity to sequences of a Corynebacterium glutamicum plasmid, coding for a hypothetical protein and a putative trypsin-like serine protease, respectively.
The four tra genes related to conjugative transfer, which are organized in a single complex (CDSs 17–20), showed highest amino acid identities between 64% and 75% to their counterparts in Pseudomonas aeruginosa (Table 2). Those four tra genes are likely not sufficient for pAB5S9 to be a self-transmissible plasmid. This may explain why no transfer could be observed by conjugation in repeated attempts, and the transfer of plasmid pAB5S9 was obtained only by the artificial means of electroporation. However, effective conjugal transfer of resistance plasmids has already been reported in Aeromonas.27,28 Rhodes et al.29 have also shown that resistance-encoding plasmids have disseminated between different Aeromonas species and E. coli and between the human and aquaculture environments. The transfer of resistance plasmids from motile Aeromonas spp. to E. coli under conditions mimicking the natural aquatic environment was also experimentally demonstrated.7
In conclusion, we provide the first description of a multiresistance plasmid in A. bestiarum, an opportunistic pathogenic bacterium for fish which might also act as a resistance gene reservoir for other freshwater bacteria. The presence of mobile and exchangeable resistance genes in the aquatic bacterial communities is a key factor for the acquisition of resistance and the development of novel multiresistance plasmids. In this regard, the resistance genes found on plasmid pAB5S9 have been detected on mobile elements such as the SXT element of V. cholerae, but also on plasmids in a wide variety of bacteria including those from aquatic environments, such as the fish pathogen Photobacterium damselae subsp. piscicida where the plasmid-borne floR gene was first encountered.30 It is plausible that the pAB5S9 multiresistance plasmid has contributed to the observed persistence and spread of the A. bestiarum 5S9 strain along the studied river. As a matter of fact, this strain would likely be positively selected if placed in the context of a fish farm applying an oxolinic acid, or a florfenicol, or a tetracycline treatment. Bacterial diversity and richness in aquatic environments likely provide favourable conditions for bacteria to develop multiresistance plasmids. Therefore, activities leading to imposition of antimicrobial selective pressure on aquatic bacteria should be considered with caution.
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This work was financially supported by the French National Institute for Agricultural Research (INRA) and the National Veterinary School of Nantes (ENVN).
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None to declare.
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Acknowledgements |
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We thank the National Veterinary School of Nantes (ENVN) for hosting this work and the French National Institute for Agricultural Research (INRA) and the Pays de la Loire Region for grant support.
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