ABSTRACT
We describe here the genetic organization of the mecelement downstream of the mecA gene in 34 different methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates carrying 13 of the most frequent polymorphisms ofmecA and representing the major epidemic clones of MRSA. All polymorphisms carried three common genetic elements: the hypervariable region, a copy of IS431, and a unique 2-kb sequence (downstream constant segment, or dcs) for which no homologous sequences are found in data banks. Polymorphisms of the downstream region were shown to be caused by the presence of linearized plasmids flanked by insertion sequences (pUB110, pT181, and pI258) and the autonomous insertion sequence IS256.
The 2.1-kb mecA gene is part of a larger (40 to 60 kb) mec element of extraspecies origin which extends both upstream and downstream of themecA determinant. mecA has a singleClaI digestion site, and therefore chromosomalClaI digests of methicillin-resistant Staphylococcus aureus (MRSA) produce two mecA hybridizing fragments, one of which, including sequences downstream of mecA, exhibits considerable strain-to-strain variation in molecular size, ranging from 3.5 to 20 kb. The size of the second smallermecA-hybridizing fragment, produced from the upstream side of mecA, is virtually invariant. Thus, most of the polymorphisms observed in the mecA region reflect variation in DNA downstream of mecA. Up to now, as many as 21 suchmecA polymorphisms have been identified in various MRSA isolates (3, 12, 16, 18, 21, 26), and these polymorphisms have been used as an epidemiological marker and have also been the basis of speculation concerning the evolutionary origin of methicillin resistance in S. aureus (16).
The main purpose of the study described here was to determine the molecular basis of mecA polymorphisms by cloning and sequencing the downstream region of the mecA element in isolates representing the major current clonal types of MRSA.
MATERIALS AND METHODS
Bacterial strains, phage, and plasmids. Escherichia coli host strains, vectors, and recombinant vectors are described in Table 1. MRSA clones and reference strains are listed in Table 2.
E. coli host strains, vectors, and recombinant vectors
ClaI::mecA vicinity organization features of MRSA epidemic clonesa
Media and growth conditions. S. aureus strains were grown in tryptic soy broth or in tryptic soy agar (Difco Laboratories, Detroit, Mich.) with aeration at 37°C for 18 h. Luria-Bertani medium (Difco Laboratories) was used to propagate E. coli DH5α, and ampicillin (100 μg/ml) was added for selection and maintenance of plasmids. E. coli XL1-Blue MRF′ was the host strain for the Lambda ZAP Express vector. All manipulations were performed according to the manufacturer's recommendations (Stratagene Cloning Systems, La Jolla, Calif.).
DNA methods.Standard DNA methodologies were used (2, 25). Restriction endonucleases (New England Biolabs, Beverely, Mass.), calf intestinal alkaline phosphatase, and T4 DNA ligase (Boehringer Mannheim, Indianapolis, Ind.) were used according to the manufacturer's instructions. Plasmid DNA was isolated using the Wizard Plus Minipreps or Wizard Plus Midipreps DNA purification systems (Promega, Madison, Wis.). Lambda DNA was isolated with the Qiagen Lambda Midi kit (Qiagen GmbH, Dusseldorf, Germany). Chromosomal DNAs for PCR were prepared as previously described (11). Southern hybridization, colony blotting, and plaque lifts were performed with the ECL random prime labeling and detection systems (Amersham Pharmacia Biotech Inc., Piscataway, N.J.) according to the manufacturer's recommendations.
PCR.PCR amplifications were performed in a DNA Thermal Cycler 480 (Perkin-Elmer Cetus [PEC], Branchburg, N.J.). The PCR mixture was prepared with a PCR reagent kit according to the manufacturer's recommendations (PEC). In each reaction, 40 pmol of each primer and 1 ng of plasmid or 5 ng of chromosomal template DNA were included. The amplifications were carried out at the following temperature profiles: 94°C for 4 min; 35 cycles of 94°C for 30 s, 47 to 53°C for 30 s, and 72°C for 1 to 4 min; and 72°C for 4 min and 4°C for the remainder of the reaction. For inverted PCR (IPCR), the chromosomal DNA was digested with ClaI; self-ligation and PCR amplification were performed essentially as previously described (9). Long-range PCR was performed by use of a GeneAmp XL kit (PEC) with a magnesium concentration of 1.1 mM, 40 pmol of each primer, and 5 ng of template DNA. The thermal profile was as follows: 94°C for 2 min, 37 cycles of 94°C for 30 s, 65°C for 7 min, 72°C for 10 min, and soaking at 4°C.
Cloning of the ClaI::mecA downstream vicinity of the Iberian MRSA clone (pattern I).The recombinant plasmids and phage are listed in Table 1, and the digestion sites used are depicted in Fig. 1. The 6.0-kb ClaI-EcoRI fragment was cloned into the low-copy-number plasmid vector pACYC177 (recombinant plasmid pDO-1). The PstI-BamHI fragment of pDO-1 was then subcloned into pGEM-3Z (plasmid pDO-3). The 6.5-kb BamHI-BamHI fragment was cloned into bacteriophage λ to generate recombinant bacteriophage λDO-2. The DNA insert of λDO-2 was amplified by long-range PCR using the vector-based primers (T3-AAT TAA CCC TCA CTA AAG GG and T7-GTA ATA CGA CTC ACT ATA GGG C). The amplicon was digested with BamHI andBglII, and the two digests were ligated into pACYC177, creating the plasmids pDO-4 and pDO-5, respectively. The 3′ sequence of the AclI-BamHI fragment in the λDO-2 insert was used to design inverted primers (ABP3-AAT AAC TTG TGG ATA ACT GG and ABP4-TTG GTA ACT GTT TCT CAT GC), and IPCR was performed by use ofAclI-digested chromosomal DNA as a template to obtain sequence data for the further downstream region. Finally, theBamHI-ClaI fragment was ligated with pACYC177 to produce pDO-6. The DNA inserts in plasmids pDO-1, pDO-3, pDO-4, pDO-5, and pDO-6 were sequenced by the primer walking strategy.
Nucleotide sequencing.DNA sequencing was performed at the Rockefeller University Protein/DNA Technology Center with an automated DNA sequencing system (Model 377; Perkin-Elmer Applied Biosystems, Foster City, Calif.). Sequence data were analyzed with the DNA Star software (Lasergene, Madison, Wis.). Homology searches were performed with the BLAST utility available through the National Center for Biotechnology Information website.
Nucleotide sequence accession number.Nucleotide sequences were submitted to the DDBJ/EMBL/GenBank database under accession numbers AF142100 (mecR1 170-bp deletion; strain LHH1),AF142101 (hypervariable region [HVR]; strain HU106), AF142102 (HVR; strain HU41), AF142103 (HVR; strain HU101), and AF18195 (ClaI::mecA downstream vicinity; strain HUC19).
Previously published sequences.Previously published sequences were obtained through the GenBank database website (http://www.ncbi.nlm.nih.gov/). The accession numbers are as follows: for pUB110, M19465; for pT181, J01764; for pI258, L29436; for IS431, X3818; and for IS256, M18086.
RESULTS AND DISCUSSION
Experimental strategy.We first cloned and sequenced the 16-kbClaI::mecA downstream region of the MRSA strain HUC19 (ClaI::mecA pattern I), a prototype of the widely spread Iberian clone (12, 21). Based on the sequence data obtained with HUC19, several primers were designed spanning the entire downstream region (Table3; Fig. 1), and a representative collection of other MRSA clones was screened by PCR in order to detect regions of difference, revealed either by lack of amplification or by shifts in the amplified fragment sizes. When there was no amplification, long-range PCR was used to test for the presence of hypothetical insertion elements in the region. The flanking regions of such polymorphic fragments were then sequenced, and a homology search was performed.
Primers used for screening the mecAdownstream vicinitya
Molecular organization of theClaI::mecA downstream polymorphisms.ClaI-mecA types are displayed on the left; between parentheses are the approximate sizes of theClaI::mecA downstream vicinities in kilobases. ClaI digestion sites are indicated with vertical arrows. Coding sequences for each plasmid are indicated as described in the original references. ΔHVR, deleted HVR; dcs, downstream constant segment; Ins117, the 117-bp sequence flanked by two 15-bp direct repeats; orfX, insertion site of the mec element into the S. aureus chromosome as defined by Ito et al. (15). In pattern I organization the digestion sites used for cloning are indicated, as are the locations of primers used in the screening of the other vicinities (see Table 3 for primer sequences).
The MRSA collection (Table 2) tested included 34 strains with 13 different ClaI::mecA patterns (polymorphisms) covering most of the observed range of variability in the downstream vicinity. The most frequent polymorphisms (I, II, and III) were studied in several strains from different geographic origins. Less-frequent patterns were studied in two strains, the strain for which the pattern was originally described and a second strain from a different geographic origin. Strains sharing the sameClaI::mecA pattern were compared by sizes of PCR fragments and by nucleotide sequencing of the polymorphic regions.
Genetic organization of the mecA downstream region in MRSA strain HUC19 carrying mecA polymorph I.TheClaI::mecA downstream region of this strain was shown to be 15,897 bp, beginning at the ClaI digestion site internal to the mecA gene (Fig. 1). Directly downstream of mecA we found a copy of the HVR which, in relation to the HVR described by Ryffel et al. (24), had a deletion of 1,043 bp spanning the entire dru element. Adjoining the HVR downstream was a copy of the linearized plasmid pUB110 containing the kanamycin and neomycin resistance determinants flanked by two direct repeats of the insertion sequence IS431. The pUB110 linearization caused no interruption of any genes on the plasmid. Therep gene open reading frame contained a nonsense mutation and the rec gene showed a single amino acid substitution in comparison to the published pUB110 sequence (20), which may be a mechanism to stabilize the plasmid-borne antibiotic resistance markers in the MRSA chromosome. The inserted pUB110 was flanked by two external 16-bp inverted repeats, which belong to the ends of IS431, and by two 8-bp internal direct repeats (target sequence duplication). The remaining downstream sequence had some putative open reading frames. Within this region, a short 117-bp sequence flanked by two 15-bp direct repeats (Ins117) was identified.
Genetic organization of the downstream mecA region in MRSA carrying different mecA polymorphisms is shown in Fig. 1. Figure 2 shows two exceptional upstream patterns, one larger and the other smaller than the normal invariant upstream ClaI fragment.
Molecular organization of theClaI::mecA upstream polymorphisms.ClaI-mecA types are displayed on the left; between parentheses are the approximate sizes of theClaI::mecA upstream vicinities in kilobases.ClaI digestion sites are indicated with vertical arrows. Horizontal arrows represent coding sequences and their directions of transcription. The upstream ClaI::mecAfragments were studied by IPCR and sequencing of the amplified fragments.
dcs.A unique 2-kb sequence, the downstream constant segment (dcs) (Fig. 1), was detected in each polymorph and was joined on the downstream side by a 6-kb sequence (Fig. 1, regions between orfX and ClaI), which showed high homology to a sequence in the methicillin-susceptible strain NCTC8325 (Advanced Center for Genome Technology, University of Oklahoma [http://www.genome.ou.edu]), while no sequence homologous to the 2-kbdcs was found in the NCTC8325 genome. Immediately next to the 3′ end of dcs, the same putative orfX—first identified in strain N315 (15)—was present in each strain, suggesting that this proposed integration site (15) is common in all MRSA strains.
Evolutionary relationships.The variable length (up to 16 kb) of DNA extending from the 3′ end of mecA towards theS. aureus chromosomal sequence is composed of two types of genetic elements: conserved blocks (a copy of HVR either complete or carrying a 1043-bp deletion; an IS431 sequence; anddcs) and a variable number of different acquired genetic elements, the nature and arrangement of which can account for the genetic organization of the 3′ vicinity of mecA that is unique to the different polymorphisms of this region.
The simplest downstream organization (polymorphism II) may represent the structure of the earliest MRSA isolates, in which the conserved blocks of foreign DNA are contiguous and bridge the 3′ end ofmecA with the beginning of the host chromosome. The downstream organization of other polymorphisms may have originated by the integration of plasmids through homologous recombination between the IS431 copy downstream to mecA and a copy of IS431 which has previously been transposed into the plasmid. Such a scenario has already been suggested in earlier studies (14, 19, 28) and agrees with our finding that all linearized plasmids were flanked by short 8-bp direct repeats, presumably formed when integration of the insertion sequence into the plasmids occurred. Moreover, this target sequence is the same for different strains with the same integrated plasmid, as is the insertion site, supporting the previously found site-specific insertion of IS431 into each plasmid (27, 28). The IS431 insertion sequence may have provided an efficient tool for the introduction of resistance determinants into the S. aureus chromosome.
Additional possible evolutionary relationships are also apparent. The genetic organization of polymorphism III* characteristic of the pandemic Brazilian clone of MRSA (strain HU25) may have originated from polymorphism III by deletion of pT181. The downstream regions of strains BK739 and HU195 (polymorphism VI) may have originated from the intramolecular recombination between the two IS431copies flanking pT181 in a polymorphism III-like structure with pT181 and pI258 inserted in the mecA downstream vicinity. The potential role of IS431 in these types of genetic exchanges had already been proposed (14).
Our findings support the thesis that the incorporation of themec element into the S. aureus species was a rare event (1, 16). If the opposite were true, one would expect more extensive and diverse structural variation in the mecAdownstream vicinity, other than the presence or absence of mobile elements.
ACKNOWLEDGMENTS
We thank Ana Madalena Ludovice for guidance and advice given during the initial phase of the study and Alexander Tomasz for stimulating discussions and critical revision of the manuscript.
Partial support for this study was provided by projects PRAXIS XXI/2/2.1/BIO/1154/95 and PRAXIS XXI/2/2.2/SAU/1295/95 (Portugal) awarded to H. de Lencastre and by a grant from the Irene Diamond Foundation to A. Tomasz, head of the Laboratory of Microbiology, The Rockefeller University, New York, N.Y. D.C. Oliveira was supported by grant BD/4162/96 from Fundação para a Ciência e Tecnologia, Lisbon, Portugal.
FOOTNOTES
- Received 12 January 2000.
- Returned for modification 6 March 2000.
- Accepted 11 April 2000.
- Copyright © 2000 American Society for Microbiology
