Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK
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Abstract |
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Introduction |
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Most of the integrons studied to date have been identified in clinical isolates, where they contribute significantly to the prevalence and dissemination of antibiotic resistance genes. 15 ,16 ,17 ,18 ,19 The frequency of occurrence of integrons in bacteria other than those of clinical importance is unknown. In this study we examined 3000 Gram-negative bacteria from an estuarine environment for the presence of class 1 integrons. The structure of the integrons identified was examined and the nature of the DNA inserted in the variable region of 74 of the integrons was determined.
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Materials and methods |
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The Tay Estuary is the outlet to the North Sea for the Rivers Tay and Earn which provide virtually all the freshwater flow into the estuary. The sample site, Invergowrie Bay, is tidal and located 16 km from the river mouth, to the west of the city of Dundee. Water samples, collected in sterile bottles at high tide from the shore line of the Bay during October and November 1993, were transported back to the laboratory, on ice, and processed within 2 h of collection. Culturable bacteria were recovered by spread plating 0.1 mL volumes of appropriately concentrated or diluted water samples onto agar plates selective for coliforms (MacConkey agar, IDG Ltd., Bury, UK), Pseudomonas spp. (Pseudomonas spp. agar base supplemented with X108 (IDG Ltd.) and Vibrio spp. (thiosulphate-citrate-bile salts-sucrose agar (TCBS), IDG Ltd.). Duplicate sets of plates were incubated at 37 and 26°C for 24 and 48 h, respectively. A total of 3000 isolates (1000 each of coliform, Pseudomonas-like and Vibrio-like isolates) were subcultured and studied further.
Taxonomic tests
Organisms were termed coliform isolates if they formed red or pink colonies on MacConkey agar, were Gram-negative and bacillus-shaped, oxidase-negative and fermentative with glucose by the oxidation/fermentation test. Organisms were termed Pseudomonas-like isolates if they formed colonies on Pseudomonas spp. selective medium, were Gram-negative and bacillus-shaped, oxidase-positive and oxidative with glucose by the oxidation/fermentation test. Organisms were termed Vibrio-like isolates if they formed 2-3 mm yellow or green colonies on TCBS medium, were Gram-negative and curved-bacillus-shaped, oxidase-positive and fermentative with glucose by the oxidation/fermentation test.
Colony and dot blot hybridization
Bacterial colonies were cultured, at appropriate incubation temperatures, on the surface of
Hybond N+ membranes (Amersham Pharmacia Biotech UK
Ltd., Little Chalfont, UK) placed on nutrient agar (IDG Ltd.) plates. Cells were lysed and the
liberated DNA bound to the membranes by the method of Maniatis et al.
20 The filters were baked at 80°C for 2 h to
immobilize the DNA before hybridization. DNA dot blots were prepared by spot inoculation of
heat-denatured genomic DNA, isolated from 1 mL overnight cultures with the Puregene DNA
Isolation Kit (Flowgen Instruments Ltd., Lichfield, UK) according to the manufacturer's
instructions (Amersham Pharmacia Biotech UK Ltd., Amersham, UK). A 1.289 kb fragment of
the recombinant plasmid pLKO26
21 was used as the specific probe for the integrase gene, intI1, of Tn21. The probe was labelled with fluorescein-dUTP by the ECL
random-prime labelling system (Amersham Pharmacia Biotech UK Ltd.) in conditions
recommended by the manufacturer. Pre-hybridization and hybridization were performed at
60°C with hybridization buffers recommended by Amersham Pharmacia Biotech UK Ltd.
After hybridization, the filters were washed in 1 x SSC with SDS 0.1% for 30 min
at
60°C and in 0.5 x SSC with SDS 0.1% for 30 min at 60°C. An
oligonucleotide
probe, CASS2 (Table I), specific for the qacEand
qacE1
genes of class 1 integrons, was 3'-end labelled with fluorescein-dUTP
by the ECL
oligo-labelling system (Amersham Pharmacia Biotech UK Ltd.) in conditions recommended by
the manufacturer. Pre-hybridization and hybridization were performed at 42°C with
hybridization buffers recommended by Amersham. After hybridization, the filters were washed
twice in 2 x SSC with SDS 0.1% for 5 min at room temperature and twice in 0.5
x
SSC with SDS 0.1% for 15 min at 53°C. Detection of a positive hybridization result
was by
means of an ECL detection kit (Amersham Pharmacia Biotech UK Ltd.) with the conditions and
protocols recommended by the manufacturer.
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Escherichia coli K12 strains carrying plasmids R751 (In16) and R388 (In3), which
contain class 1 integrons were used as positive controls for the intI1, qacE(In16) andqacE1(In3) genes
5 and E. coli K12 strains carrying plasmids R483
(Tn7) and pUK163 (Tn4132), which contain class 2 integrons (intI2*)
4
,23 were used as
negative controls.
PCR amplification
Oligonucleotide primers used for PCR amplification are listed in Table I and their annealing sites and estimated product sizes are shown in the Figure. Standard PCRs were carried out in 50 µL volumes containing 5 µL of 10 x PCR buffer (100 mM Tris-HCl (pH 8.8), 500 mM KCl, 15 mM MgCl 2, 1% Triton X-100), 5 µL of 10 x deoxynucleotide triphosphate mix (2 mM each dATP, dCTP, dGTP and dTTP) (Amersham Pharmacia Biotech UK Ltd.), 2 µL of each primer stock solution (25 pmol/µL), 35 µL sterile distilled water and 1 µL BIOTAQ polymerase (0.5 U/µL diluted solution) (Bioline UK Ltd, London, UK). A portion of bacterial colony, cultured on a nutrient agar plate, was added to provide the DNA template. Cell lysis and DNA amplification were performed in an Omn-E thermal cycler (Hybaid Ltd., Teddington, UK) using the following cycle conditions: 96°C for 5 min, 55°C for 1 min, 70 °C for 3 min (one cycle); 96°C for 15s, 55°C for 30s, 70°C for 3 min (24 cycles) and a final extension of 70 °C for 5 min (one cycle). PCR products >2 kb in length were generated following amplification of purified genomic DNA templates with Expand Long Template PCR System according to the manufacturer's instructions (Roche Diagnostics Ltd., Lewes, UK).
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PCR-amplified DNA was visualized and the fragment sizes estimated by electrophoresis of reaction products in 1-2% agarose gels containing 1 µg of ethidium bromide per mL using TAE running buffer (40 mM Tris acetate (pH 7.8), 5 mM sodium acetate, 1 mM disodium EDTA). PCR products were purified with the Wizard PCR Preps DNA purification system according to the manufacturer's instructions (Promega UK Ltd., Southampton, UK) prior to digestion with restriction endonucleases (Life Technologies Ltd, Paisley, UK) according to the manufacturer's instructions. Routinely, reaction mixtures contained 10 µL of the PCR product and 5-10 U of enzyme in a total volume of 20 µL. Incubation was for 1-2 h at 37°C. PCR products were either cloned directly into the vector plasmid pGEM-T according to the manufacturer's instructions (Promega UK Ltd.) or, following digestion, fragments were ligated into suitably digested pUC19 vector DNA.
DNA sequencing of PCR products
Recombinant plasmids for DNA sequencing were prepared with the Qiagen Plasmid Mini Kit according to the manufacturer's instructions (Qiagen Ltd., Crawley, UK) Sequences were determined on one DNA strand only, using an ABI PRISM 377 Automated DNA Sequencer. Primers HS89 in the 5' conserved segment and HS120 in the 3' conserved segment, 24 were used in addition to the standard forward and reverse sequencing primers.
Antimicrobial susceptibility testing
Isolates, revealed by sequence analysis to contain antibiotic resistance genes within an integron structure, were tested for phenotypic expression of resistance by the breakpoint method of sensitivity testing. 25 Briefly, approximately 104 cfu were inoculated on to the surface of Iso-Sensitest agar (Oxoid, Basingstoke, UK) containing ampicillin, amikacin, chloramphenicol, gentamicin, sulphamethoxazole, tobramycin and trimethoprim, at concentrations recommended for breakpoint sensitivity testing of Enterobacteriaceae and Pseudomonas spp., 25 erythromycin and streptomycin at 8 mg/L, and spectinomycin at 100 mg/L. All drugs were obtained from Sigma Aldrich Co. Ltd, Poole, UK.
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Results |
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The presence of a class 1 integron was detected by a combination of colony hybridization with the intI1 specific gene probe followed by PCR amplification of the intI1 gene in those strains which gave a positive hybridization result. PCR products were confirmed to be intI1 by restriction with SphI which results in two fragments of 393 bp and 499 bp. Only those isolates which gave positive results in both hybridization and PCR analysis were recorded as harbouring a class 1 integron. The intI1 gene was present in 109 of the 3000 isolates screened. Forty of the integron-positive isolates were coliforms, 35 were Pseudomonas-like and 34 were Vibrio-like.
The occurrence and nature of the 3'-conserved segment of 85 of the integrons was
investigated. The sulI resistance gene was detected by production of an intragenic 408
bp fragment following PCR with the sul1/sul1.rev primer pair. The presence of an adjacent qacE1 gene was inferred if a 900 bp DNA fragment was produced
following PCR with the orf4/sul1.rev primer pair. All isolates shown by PCR to contain the sulI gene expressed phenotypic resistance to sulphamethoxazole. Isolates shown to lack the sulI gene were subsequently tested for the presence of a qacE gene by dot-blot
hybridization with a CASS2 oligonucleotide probe. The results of these experiments are
summarized in Table II. Less than half (36/85) of the class 1 integrons
were found to contain
both the qacE
1 and sulI genes. Forty-nine integrons lacked
the sulI resistance gene but of these, 38 probed positive for the qacE gene.
Although such integrons were prevalent in all three types of isolate, a greater proportion of the
integrons identified in Vibrio-like isolates (68%) showed this genotype compared
with
those identified in Pseudomonas-like isolates (39%) and coliform isolates
(32%).
Eleven integrons did not appear to possess either a sulI or qacE gene.
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The 74 class 1 integrons, comprising an intI1 gene and part or all of the 3' conserved region, were analysed for the presence of inserted DNA in the variable region of the integron. Since several reports have indicated that the aadA1 gene (encoding resistance to streptomycin/spectinomycin) is the most frequently integrated gene cassette in class 1 integrons, 17 ,18 three separate PCR amplifications were performed with three different primer pairs (Figure): (i) primer pair CASS1/CASS2 enabled amplification of the entire variable region; (ii) primer pair CASS1/aadA.rev enabled amplification of DNA between the 5'-conserved segment and an aadA cassette if present; and (iii) primer pair aadA/CASS2 enabled amplification of DNA between the 3'-conserved segment and an aadA cassette if present. The size of each product was estimated by agarose gel electrophoresis and products of similar size were compared by restriction endonuclease mapping of the PCR fragments.
The results are summarized in Table II. The integrons were nominally assigned to five major groups according to the composition of the 3'-conserved segment and the presence or absence of integrated DNA in the variable region. Nineteen integrons (Groups 2a and 2b) lacked any integrated gene cassettes in their variable region. A further 21 integrons (Group 2c), were shown to contain a 1100 bp DNA insert. Preliminary DNA sequence analysis of this insert suggests it to be a novel IS30-related insertion sequence, indicating that these integrons do not contain integrated gene cassettes in their variable region. Twenty-eight integrons (Groups 3 and 4) were shown to contain an aadA gene cassette which conferred phenotypic resistance to streptomycin and spectinomycin in the host. In 15 of the integrons (Group 3) aadA was the only cassette present in the variable region. The nature of the aadA gene was determined by digestion of the CASS2/aadA PCR product with either PvuI (which releases a 240 bp end fragment from products containing aadA1a DNA), or PstI (which releases a 180 bp end fragment from products containing aadA1b DNA) or EcoRI (which releases a 315 bp end fragment from products containing aadA2 DNA). The majority of the integrons (25/28) carried the aadA1a gene. The remaining three integrons (Group 4d) carried an aadA2 cassette. None of the integrons were found to carry an aadA1b gene. Six integrons (Group 5) did not carry an integrated aadA gene cassette but were found to contain DNA inserts ranging in size from 600 to 1700 bp.
Characterization of Group 4 and 5 integrons
The variable regions of the 20 integrons in Groups 4 and 5 were restriction mapped and partially
sequenced to reveal the presence of previously characterized gene cassettes and identify any
novel inserted DNA sequences. Routinely, 400-700 nucleotides of integron specific DNA
sequence were determined from each sequencing run. The results are summarized in Table III. A
number of complete gene cassettes, defined by the presence of a core sequence (GTTRRRY) at
the 5'-end of the gene and a 59 base-element at the 3'-end of the gene
4 were identified, most of which were found to encode
phenotypic resistance to antimicrobial drugs (Table III). In addition to
cassettes catB3
and catB5 (encoding resistance to chloramphenicol),
dfr1a, dfrIIc, dfrV,dfrVII and dfrXII
(encoding resistance to trimethoprim), aacA4andaacC1 (encoding resistance to aminoglycosides), and oxa2 (encoding resistance to
ß-lactams), the cassettes orfDand orfF, of unknown function, were also
identified. The integron identified in isolate SJ118 was found to contain an incomplete sat1 resistance gene cassette (designated sat11) in addition to a
complete aadA1a gene cassette. The sat1 gene was truncated by approximately
600 bp, consisting only of the first 464 bp of the published 1379 bp structural gene.
26 Four integrons, identified in isolates SJ111, SJ098,
SJ150 and SJ003, were found to contain identical DNA inserts comprising aacC1 and aadA1a gene cassettes flanking a 650 bp DNA segment of which 450 bases were
determined. Comparison with sequences held in the GENBANK database revealed homology
with bases 2039-2345 of the putative gene cassette orfX (GENBANK accession no.
AJ009820). No function has been asigned to this open reading frame.
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Discussion |
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Almost all class 1 integrons characterized to date contain a 3'-conserved segment
comprising a sulI resistance gene in addition to the qacE1
gene. The exceptions are the integron found in plasmid R751 which contains a complete qacE gene cassette and no sulI resistance gene,
11 and the recently described class 1 integron identified in
the Pseudomonas aeruginosa Mus clinical isolate which contains a 5'-conserved
segment, two resistance gene cassettes (aacA4 and oxa20) but no
3'-conserved segment.
28 In contrast to the integrons found in clinical isolates, the
majority of integrons identified in this study lack a sulI resistance gene, but contain a
complete qacE gene in the 3'-conserved segment. This difference in the basic
structure of the integrons found in clinical and environmental bacteria may reflect differences in
the selective pressures the bacteria are exposed to. Since sulphonamides are synthetic
antimicrobials, natural bacterial isolates are less likely to have been exposed to these drugs and
hence the bacteria may not yet have found a need to integrate a sulI resistance gene
cassette into their integron. The distribution of the qacE and qacE
1
genes within Gram-negative bacteria has recently been surveyed by Kazama .
29 Interestingly, these authors identified the qacE gene only in clinical isolates of P. aeruginosa. However, 14 of the 15 strains
with the qacE gene also possessed the qacE
1 gene and were
resistant to sulphamethoxazole, supporting the proposal by Bissonnette & Roy
5 that class 1 integrons may have evolved from an ancestral
integron, InA, through integration of a sulI-orf5 unit at a crossover point within the
3'-end of the qacE gene. The significant number of integrons which lack a sulI resistance gene identified in this survey lend further support to the proposal for an InA
ancestral integron structure.
The role of integrons in the capture and expression of antibiotic resistance genes in Gram-negative clinical bacteria is now well established. Characterization of the integrons identified in this survey revealed that the majority lack integrated gene cassettes in the variable region. Although the bacteria in this study were cultured from estuarine samples, and were thus deemed to be environmental isolates, it is possible that some of the strains, particularly those identified as coliforms, may have originated from human bacterial flora. The River Tay, which is tidal, receives untreated domestic effluent, including that from a large general hospital in Dundee, and hence some of the isolates recovered may represent those from human domestic waste. The lack of integrated gene cassettes in a significant proportion of the integrons may therefore be a consequence of previously integrated cassettes being excised from the integron 30 ,31 when antibiotic selective pressures are diluted out in the natural environment, or alternatively may represent a reservoir of ancestral integrons which have not yet acquired integrated gene cassettes. A number of the integrons contained a 1.1 kb insert in the variable region which was tentatively identified as a novel IS30-like insertion sequence. 32 DNA sequencing showed the inserted DNA to comprise an open reading frame which translated to a protein with >40% identity with the transposase of IS30-related elements. The boundaries of this putative IS-element were identified in one of the integrons and showed that the inserted DNA had not been recombined into the attI crossover point into which gene cassettes are normally integrated. Detailed analysis of this element is now underway.
As with other surveys, the aadA1 gene cassette was the most frequently found resistance gene in the variable region of integrons, and was often the only cassette present. It has been reported that cassettes preferentially recombine into the attI site of an integron, rather that the attC site at the 3'-end of cassettes already present in the integron. 3 ,33 The predominance of the aadA1 gene cassette and the observation that additional gene cassettes, when present in integrons carrying the aadA1 gene, were generally found upstream of the aadA1 resistance gene suggests that the aadA1 gene may be either the first cassette to be acquired by an integron and/or may be more stably integrated into the integron than other gene cassettes. After aminoglycoside resistance gene cassettes, the most prevalent resistance gene cassettes found were those encoding resistance to trimethoprim. This is interesting since, like sulphonamides, trimethoprim is a synthetic antimicrobial and hence direct selective pressure to retain these resistance genes in bacteria in a natural habitat would be expected to be lower than that for genes encoding resistance to antibiotics such as aminoglycosides, chloramphenicol, ß-lactams and erythromycin produced by soil microbes.
The integron identified in isolate SJ290 contained the erythromycin resistance gene, ereA, 34 in addition to the trimethoprim resistance gene cassette, dfrV. A cassette structure for the ereA gene has not previously been reported. The size of the ereA-containing insert identified here was estimated, from sequencing of one DNA strand only, to be 1379 bp. A potential 59 base element was identified downstream of the ereA structural gene and a possible core sequence (GTTATGC) was found at the junction with the dfrV gene cassette. More comprehensive sequence data are required to confirm the structure of this potentially novel cassette.
The results of this study reveal that integron structures are prevalent in bacteria in natural habitats. The frequent identification of empty integrons may support the view that antibiotic selective pressures play a significant role in promoting the incorporation and maintenance of gene cassettes in the variable region of integrons and in the absence of sustained antibiotic pressures resistance gene cassettes are excised from the integron. The predominance of selected aminoglycoside and trimethoprim resistance gene cassettes may, however, indicate that some gene cassettes are more stably incorporated within integron structures than others.
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Acknowledgments |
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Notes |
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* Corresponding author. Tel: +44-(0)1382-344270; Fax:
+44-(0)1382-344275; E-mail: h.k.young{at}dundee.ac.uk
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Received 11 October 1998; returned 27 January 1999; revised 15 February 1999; accepted 8 March 1999