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Antimicrobial Agents and Chemotherapy, June 2000, p. 1568-1574, Vol. 44, No. 6
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Antibiotic Resistance in the ECOR Collection: Integrons and Identification of a Novel aad Gene

Didier Mazel,1 Broderick Dychinco,2,dagger Vera A. Webb,2,Dagger and Julian Davies2,*

Unité de Programmation Moléculaire et Toxicologie Génétique, Institut Pasteur, 75724 Paris cedex 15, France,1 and Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada2

Received 23 September 1999/Returned for modification 17 December 1999/Accepted 13 March 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The 72 Escherichia coli strains of the ECOR collection were examined for resistance to 10 different antimicrobial agents including ampicillin, tetracycline, mercury, trimethoprim, and sulfonamides. Eighteen strains were resistant to at least one of the antibiotics tested, and nearly 20% (14 of 72) were resistant to two or more. Several of the resistance determinants were shown to be carried on conjugative elements. The collection was screened for the presence of the three classes of integrons and for the sul1 gene, which is generally associated with class 1 integrons. The four strains found to carry a class 1 integron also had Tn21-encoded mercury resistance. One of the integrons encoded a novel streptomycin resistance gene, aadA7, with an attC site (or 59-base element) nearly identical to the attC site associated with the qacF gene cassette found in In40 (M.-C. Ploy, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 42:2557-2563, 1998). The conservation of associated attC sites among unrelated resistance cassettes is similar to arrangements found in the Vibrio cholerae superintegrons (D. Mazel, B. Dychinco, V. A. Webb, and J. Davies, Science 280:605-608, 1998) and supports the hypothesis that resistance cassettes are picked up from superintegron pools and independently assembled from unrelated genes and related attC sites.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The ability of bacteria to acquire and disseminate exogenous genes via mobile genetic elements such as plasmids and transposons has been the major factor in the development of multiple drug resistance over the last 50 years. Integrons have been implicated in this spread among the gram-negative bacteria, especially in enteric bacteria and pseudomonads. Integrons contain gene expression elements that incorporate open reading frames (gene cassettes) and convert them to functional genes (45, 46). Variations of integrons that have been described include the multiresistant integrons (MRI), usually carried on mobile elements (for reviews see references 20, 21, and 45), and the chromosomal superintegrons, which have been identified in the chromosomes of several Vibrio species (36; for a review, see reference 35).

All known integrons have three key components necessary for the procurement of exogenous genes: (i) a gene coding for an integrase (intI), (ii) a primary recombination site (attI), and (iii) a promoter (19, 22, 46, 56). Integron integrases recombine gene cassettes downstream of the proximal attI site of the resident promoter, permitting expression of their encoded proteins. Most of the nearly 60 resistance cassettes known to date (20, 35) contain a single gene associated with a specific recombination sequence, the attC site (or 59-base element). The most common cassettes are those for aminoglycoside or trimethoprim resistance, of which 14 and 12, respectively, have been identified. The attC sites (59-base elements) comprise a family of diverse sequences which vary in size from 57 to 141 bp; only the boundaries are conserved sequences. Three classes of MRI have been defined based on the homology of the integrase genes, and each appears to be able to acquire the same gene cassettes. Some MRI carrying as many as five different resistance determinants have been characterized. Plasmid-borne integrons owe their mobility to association with transposable elements, and many class 1 integrons are found on Tn21-like transposons which also encode mercury and tetracycline resistances (29, 55). The class 1 integron found associated with Tn21 contains the aadA1 gene and has been designated In2 (for a review, see reference 29). The mechanism by which these multiple-drug-resistance cassette arrays are built up has been elucidated previously (10).

Several studies of integron distribution in clinically significant gram-negative isolates have been described (28, 33, 34); in all but one case, selection was for their aminoglycoside resistance phenotype. However, such an analysis has not, to our knowledge, been described for clinically unselected enterobacteria. The ECOR collection is a widely used set of 72 reference Escherichia coli strains isolated between 1973 and 1983 from a variety of animal hosts and a variety of geographic locations (41). The strains have been well characterized biochemically and represent the range of genotypic variation in the species as a whole (see, for example, references 1, 3, 5, 6, 11-18, 23-25, 27, 32, 37-40, 42, 44, 50, 52, 53, 57, 58, and 60). Pulsed-field gel electrophoresis studies have shown that the genome sizes of these natural isolates of E. coli vary considerably (4), and many carry plasmids (7, 47, 48); some of these carry colicins and colicin-related genes (47, 48). However, despite extensive examination of this collection, there are no reports of its overall antibiotic resistance patterns, although Summers and colleagues have investigated mercury resistance (30, 59).

We screened the ECOR strains for resistance to 10 different antimicrobial agents including ampicillin, tetracycline, mercury, trimethoprim, and sulfonamides. In addition, PCR was used to assay the collection for the presence of the three classes of mobile integrons and the sul1 gene, which is generally associated with class 1 integrons. Four strains were found to carry a class 1 integron.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains and plasmids. The 72 strains of the ECOR collection were kindly provided by H. Ochman. In conjugation experiments the recipient strain was the nalidixic acid-resistant E. coli K802NR (2). Products from PCR studies were cloned into the vector pCR2 (Invitrogen, Carlsbad, Calif.).

Antibiotic sensitivity testing. The antibiotic sensitivity profile of each ECOR strain was tested by spreading two Mueller-Hinton agar plates with 100 µl each of an overnight culture grown in Luria-Bertani medium (LB). Difco sensitivity disks for the following antibiotics were placed on the plates (six disks per plate): ampicillin (10 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), neomycin (30 µg), oxacillin (1 µg), penicillin G (2 U), streptomycin (10 µg), sulfisoxazole (300 µg), tetracycline (30 µg), tobramycin (10 µg), and trimethoprim (5 µg). Disks containing mercuric chloride (0.125, 0.25, 0.5, or 1 µmol), ethidium bromide (50 µg), paromomycin (20 or 50 µg), astromycin (20 or 50 µg), lividomycin (20 or 50 µg), or butirosin integron (20 or 50 µg) were made from sterile blank disks. The plates were incubated at 37°C overnight. Antibiotic resistance was scored by comparing the zone of inhibition to those of sensitive strains of E. coli.

PCR procedures. PCR was performed in 50-µl volumes in 96-well plates. Reaction conditions were as follows: 9 µl of Ultratherm buffer containing 50 mM MgCl2, each deoxynucleoside triphosphate (dNTP) to 2 mM, 25 pmol of each primer, 10 µl of template DNA, and 0.4 µl of Ultratherm enzyme (Bio/Can Scientific, Mississauga, Ontario, Canada). Template DNA was prepared as follows: a cell pellet from 1 ml of overnight culture was resuspended in 0.5 ml of water and boiled for 10 min. The primers for PCR are listed in Table 1. Conditions for amplification using the intI1, intI2, intI3, and sul1 primers were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 60 s. Conditions for amplification using the qac primers were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s. Conditions for amplification using the primer combinations IRI and int1.F, IRT and merA1, and 38/- and merT1 were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 3 min. In all experiments an additional 8-min extension at 72°C was included after the 30th cycle. Amplification using the mer primers was carried out as described previously (30).

                              
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  		TABLE 1.   Primer sequences for PCR analysis

Conjugation studies. All ECOR collection strains that exhibited resistance to any of the antibiotics tested were further examined for the ability to transfer resistance by conjugation. The recipient E. coli strain K802NR and each donor ECOR strain tested were grown to mid-log phase. Equal volumes (25 µl) of donor and recipient were spread on a Mueller-Hinton plate and incubated at 37°C overnight. The resulting biomass was harvested, plated on Mueller-Hinton agar plates containing nalidixic acid (30 µg/ml) and the selective antibiotic (indicated in Table 3), and incubated overnight at 37°C.

Nucleotide sequencing. Nucleotide sequencing reactions were performed using the ABI PRISM Dye Terminator cycle sequencing Ready Reaction Kit with AmpliTaq DNA polymerase FS (Perkin-Elmer [PE] Applied Biosystems, Foster City, Calif.) and electrophoresed on a 373 Stretch (PE Applied Biosystems).

Computer analysis. Initial nucleotide sequence analysis was performed by BLAST (National Center for Biotechnology Information [NCBI]). Alignments and phylogenetic analysis were performed using the PAUP and PAUPTREES programs of the Wisconsin package (Genetics Computer Group, Madison, Wis.) or CLUSTAL W1.7.

Nucleotide sequence accession numbers. The aadA7 cassette nucleotide sequence has been deposited in GenBank under accession number AF224733. Nucleotide and amino acid sequences presented in Fig. 1 were retrieved from GenBank.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Antibiotic resistance profiles. The antibiotic resistance profiles of the ECOR collection strains are summarized in Table 2. Of the 14 antimicrobial agents tested, resistance to 8 was observed. One-quarter of the strains in the collection (18 of 72) were resistant to at least one antibiotic, and nearly 20% (14 of 72) were resistant to two or more antimicrobial agents. Strains 3, 31, 37, and 48 and their transconjugants were tested for resistance to both streptomycin and spectinomycin. All were resistant to both antibiotics (Tables 2 and 3). The most common resistances were to sulfisoxazole (14 strains), tetracycline (12 strains), and streptomycin (11 strains).

                              
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  		TABLE 2.   Antibiotic resistance patternsa

Conjugation studies. The 18 antibiotic-resistant strains were tested for their abilities to transfer their resistance phenotypes by conjugation as described in Materials and Methods. The results are summarized in Table 3.

                              
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  		TABLE 3.   Summary of conjugation experiments

Distribution of integrons. PCR-based examination for the presence of integrons was carried out on the entire collection. The results of this analysis, which used oligonucleotide primers specific for the three different intI genes, are summarized in Table 4. None of the strains showed an amplification product when the int2 or int3 primers were used. A class 1 integron, however, was found in four strains, strains 3, 31, 37, and 48, based on the presence of PCR products with the three primer sets specific for a class 1 integron, namely, int1, sul, and qac primers. All four of these strains were resistant to streptomycin and to the sulfonamide drug sulfisoxazole. When assayed with the int1.R and qac.R primers, all four produced similar 2.3-kb PCR products, indicating that they each harbored a single resistance cassette. When amplified with intI1.R and aadA.R (a primer specific for the aminoglycoside adenylyltransferase cassette aadA1a, formerly named aadA [20]), ECOR strains 3, 37, and 48 were positive in the amplification reaction, suggesting that they carried In2. Interestingly, strain 31 was negative in this assay.

                              
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  		TABLE 4.   Summary of PCR experimentsa

For additional characterization of these integrons, the four strains carrying a class 1 integron were screened for the presence of a mer locus typical of Tn21-like transposons using mer-specific primers and primers specific for either the 5' or 3' ends of the inverted repeats of In2 (primers IRI and IRT) or the inverted repeat of Tn21 IRmer (primer 38/-). All four strains were positive with the mer primers (Table 4). In agreement with these results, ECOR strains 3, 31, and 48 were phenotypically mercury resistant (Hgr); however, ECOR strain 37 was mercury sensitive (Hgs). When primers specific for the inverted repeat regions of In2 and Tn21 were used in combination with the int1.F and mer primers, only strains 3, 37, and 48 produced the expected products (Table 4). Thus, ECOR strains 3, 37, and 48 contain the typical Tn21 carrying the In2 integron (59). The structure harboring the integron in ECOR strain 31 is similar to Tn21 but differs from it in two ways: first, the cassette carried by the integron is not aadA1 (see below), and the IRT region is not identical to Tn21 (as shown by the absence of PCR amplification with IRT plus merA1 primers) (29). The KH802NR streptomycin-resistant transconjugants obtained from mating with ECOR strain 3 and ECOR strain 31 were also assayed and gave results identical to those for donor strains (data not shown).

Characterization of the cassette carried by the ECOR strain 31 integron. As ECOR strain 31 carried an integron different from In2, the PCR product from the assay containing the int1.R and qac.R primers was cloned and further characterized. According to its location in the so-called 5' and 3' conserved segments (22), the full cassette array of a class 1 integron is theoretically contained in such an amplification product. The nucleotide sequence of the entire fragment was determined, and analysis revealed a single cassette inserted at the attI1 site. This cassette encoded an aminoglycoside adenylyltransferase not described previously, which conferred streptomycin and spectinomycin resistance on a sensitive E. coli DH5alpha strain. This novel AAD(3")-like enzyme is related to the known AAD(3") enzymes, its closest relatives being those encoded by aadA1a, aadA1b, and aadA2 (about 70% identity), which are also integron cassettes (see Fig. 1A). This gene has been named aadA7. The attC site carried by this cassette is located just downstream of the aadA7 stop codon. This attC site is homologous to those found in six other antibiotic resistance cassettes (see Fig. 1), its closest relative being the one found in the qacF cassette (43).


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We have screened the ECOR collection of Escherichia coli strains for antibiotic resistance phenotypes and their genetic organization (integrons). The 72 strains were collected between 1973 to 1983, from primarily healthy human and animal hosts, although 11 were isolated from infected human urinary tracts (41). Of the 18 strains found to be resistant to at least one antibiotic, all but 1 (ECOR strain 48) were from healthy individuals and most (10 strains) belonged to group A (Table 2). These results provide another striking example of the spread of resistance genes in bacterial populations. Nearly all of the animal hosts were bred under human control, either as domestic animals or in zoos. Animals may acquire resistant strains via many routes, e.g., through selection by antimicrobial therapy, drug supplements in the food, or contamination from a worker. Forty percent of the group A strains (10 of 25) were resistant to at least one antimicrobial agent.

The most frequent phenotypes were sulfonamide resistance, tetracycline resistance, and streptomycin resistance. Because sulfonamide has been used since the 1930s, resistance to this compound is now widespread. Tetracyclines were the first major group of "broad-spectrum" antibiotics (49), and they have been used worldwide in both human and animal medicine. Streptomycin has also been used for a considerably long period of time, and even if it has been largely abandoned for the treatment of infections caused by gram-negative bacteria, resistance is still frequent in enteric bacteria, particularly in E. coli. Two independent studies of Enterobacteriaceae isolated in the 1990s showed an average of 23% streptomycin resistance for E. coli isolates (9, 31).

Since many resistance genes are carried on conjugative plasmids, we assayed the transferability of the different resistance markers found in the ECOR strains in a simple conjugation assay. Natural isolates of E. coli harbor an average of four plasmids (54), and many of the ECOR strains have been shown to carry colicinogenic and IncF-related plasmids (7, 47, 48). In tests of conjugative transfer at 37°C, we observed at least one transferable resistance determined in half of the resistant strains. Notably, all of these conjugative elements were carried by strains that had been shown to contain an IncF plasmid (see Table 3), some belonging to the R1 subclass (7). Further analysis of the transconjugants is required to show if they carry the F-like plasmids of the donor strains.

Integron acquisition is considered the major cause of multiple resistance in gram-negative species, mainly in enteric bacteria and pseudomonads. Several studies have shown that 43 to 50% of recent European clinical isolates carried detectable integrons and that these strains were statistically more likely to be resistant to antibiotics than integron-negative strains (26, 33). Previously, Roy and colleagues found that about 75% of aminoglycoside-resistant clinical isolates carried an integron (28). Integrons are less frequently represented in the ECOR strains; only 4 out of the 11 aminoglycoside-resistant strains carry a class 1 integron (Table 4), and all 4 contain an aadA cassette encoding streptomycin and spectinomycin resistance. These strains were found in groups A, D, and E (Tables 2 and 4).

Three of these strains (ECOR strains 3, 37, and 48) carried an In2 integron, while the fourth (ECOR strain 31) carried an integron containing a single aadA cassette not previously described (see below). All four gave positive results in PCR screening for the mer-1 locus carried by the family of Tn21-like transposons. These transposons also contain a class 1 integron (8, 29). We have confirmed the presence of Tn21 in strains 3, 37, and 48; however, only three strains (3, 31, and 48), were found to be resistant to mercuric chloride (Table 2). Our results differ from those obtained by Summers and colleagues, who found seven Hgr strains in the ECOR collection (ECOR strains 3, 31, 34, 37, 41, 48, and 65) (30, 59). This discrepancy is likely due to differences in the protocols followed in the two studies.

The integron of ECOR strain 31 has been found to contain a novel resistance cassette, aadA7, encoding an aminoglycoside adenylyltransferase. Its product is closely related to other AAD(3") enzymes found in gram-negative bacteria; it has about 70% identity with the enzymes encoded by aadA1a, aadA1b, and aadA2, 55% identity with that encoded by aadA3, and 44% identity with that encoded by aadA(Sch) (Fig. 1A). Notably, with the exception of aadA(Sch) from Salmonella enterica serovar Choleraesuis, all the gram-negative AAD(3") enzymes are carried on integron cassettes. The attC site associated with aadA7 is closely related to those found in six other resistance cassettes (Fig. 1B). Three correspond to the aadA1a, aadA1b, and aadA2 cassettes, whose genes are also highly similar to aadA7. However, the qacF-associated attC site (43) is most similar, with only one difference out of 51 nucleotides (from the inverse core site to the G of the core site; see Fig. 1B). Other related attC sites are those found in the aacA cassette, which encodes an aminoglycoside 6'-acetyltransferase, and the aadA1a cassette (Fig. 1B). This family of related attC sites seems to be the type most represented among resistance cassettes. Conservation of associated attC sites among unrelated cassettes is typical of the situation found in the Vibrio cholerae and related superintegrons (36), supporting the hypothesis that resistance cassettes are picked up from superintegron cassette pools. Furthermore, this suggests that such cassettes are independently assembled from unrelated genes and related attC sites, probably from the wide variety of bacterial species which contain superintegrons (51).


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  		FIG. 1.   (A) Sequence alignment of the AAD(3")-like enzyme encoded by the aadA7 gene cassette with its homologues; (B) sequence alignment of the different attC sites related to the aadA7-associated attC site. Only the bases of the inverse core site (ICS) and core site (CS) which are conserved in all of the attC sites are shown, i.e., the TAAC of the ICS and the GTTA of the CS. The lowercase letters of the CS (G tta) correspond to the functional CS of the cassette, i.e., the one that is located upstream of the aadA gene and is linked to the G in the circularized cassette. Invariable positions in sequences are indicated by asterisks. Dashes, gaps; asterisks, invariable positions; periods and colons, different levels of similarity. The sequences used came from the following sources (given as GenBank accession numbers): AadA1a, X12870; AadA1b, M95287; AadA2, X68227; AadA3, Z50802; AadA.Sch, X68089; aacA, M29695; aadB, X04555; qacF, AF034958.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of British Columbia, Room 300, Wesbrook Building, 6174 University Blvd., Vancouver, British Columbia, Canada, V6T 1Z3. Phone: (604) 882-9308. Fax: (604) 822-6041. E-mail: jed{at}unixg.ubc.ca.

dagger Present address: School of Public Health, University of Michigan, Ann Arbor, MI 48109-2029.

Dagger Present address: Lookfar Solutions Inc., Tofino, British Columbia V0R2Z0, Canada.


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Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Antimicrobial Agents and Chemotherapy, June 2000, p. 1568-1574, Vol. 44, No. 6
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