Journal of Bacteriology, January 2003, p . 645-653, Vol . 185, No . 2

Development of Methicillin Resistance in Clinical Isolates of Staphylococcus sciuri by Transcriptional Activation of the mecA Homologue Native to the Species

Isabel Couto,^1,2 Shang Wei Wu,² Alexander Tomasz,² and Hermínia de Lencastre^1,2*

Molecular Genetics Unit, Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal,¹ Laboratory of Microbiology, The Rockefeller University, New York, New York²

Received 19 June 2002/ Accepted 7 October 2002

In a search for the possible original source of mecA, we identified a genetic element closely related to the S . aureus mecA gene in the animal commensal species Staphylococcus sciuri, which is one of the most abundant staphylococcal species on our planet (12) and is only distantly related to S . aureus along taxonomic lines (21, 24) . The broad range of S . sciuri biochemical activities, including the ability to use inorganic nitrogen salts as the sole source of nitrogen, is usually considered the reason for the wide range of host organisms and habitats of this bacterium, which include the skins of several animals as well as environmental reservoirs (6, 12) . The S . sciuri mecA homologue has been found to be native to this species: it is carried by each of the 162 independent S . sciuri isolates tested to date, and these isolates were recovered over a wide range of time periods and from a variety of animal and human hosts (6, 7) .

The mecA homologue identified in S . sciuri showed 79.5% DNA sequence similarity to the mecA gene of a methicillin-resistant S . aureus (MRSA) strain (27), indicating a close evolutionary relationship between these genetic determinants . The overall deduced amino acid sequence identity and similarity between the two elements were 79.98 and 87.69%, respectively . Most differences between the two sequences were found in the transglycosylase domain . When compared separately, the transpeptidase domains showed a similarity of 95.94% and an identity of 91.25%, while for the transglycosylase domains these values were 80.06 and 67.63%, respectively (27) . Despite the similarities between the S . sciuri mecA and the MRSA mecA, the great majority of S . sciuri isolates showed no appreciable resistance to ß-lactam antibiotics (6, 7) . While the native mecA homologue was present in all S . sciuri isolates tested, a careful screen has identified a few S . sciuri isolates which also carried a second copy of mecA (6, 7) . The two copies could be distinguished by cloning and sequencing: one was identified as mecA1, the homologue native to all S . sciuri strains, while the other, mecA2, was similar to the mecA gene found in methicillin-resistant strains of S . aureus (28) .

Recently, Wu et al . (29) described a highly methicillin resistant step mutant obtained in the laboratory by antibiotic selection from the susceptible S . sciuri strain K1, which carries only mecA1, the homologue native to S . sciuri . Sequencing of mecA1 from the resistant mutant K1M200 identified a single point mutation in the -10 consensus of the promoter region which was associated with a striking increase in the level of transcription of the gene and the production of a protein that reacted with a monoclonal antibody against PBP2a, the product of the S . aureus mecA gene . Introduction of the up-regulated mecA1 into an S . aureus strain with an inactivated mecA caused a significant increase in resistance to methicillin and the production of a PBP2a-like protein (29) . These observations implied that mecA1, the gene homologue native to S . sciuri, has the capacity to confer resistance to methicillin and other ß-lactam antibiotics provided that it acquires a more powerful promoter .

During a recent characterization of 28 S . sciuri strains isolated from humans, we found two strains that were particularly interesting in that they showed resistance to methicillin in spite of carrying the single copy of mecA1 only (7) . In the present work, we demonstrate that the mecA gene carried by these strains is indeed the native gene of S . sciuri, which is being constitutively expressed and producing a PBP2a-like protein . This is the first report of S . sciuri strains isolated in vivo, carrying only a copy of the native mecA gene, with the capacity to confer resistance to ß-lactam antibiotics . Characterization of these and additional strains, submitted to in vitro step selection with antibiotic, allowed us to identify several distinct mechanisms by which the S . sciuri mecA1 can be recruited by these bacteria to become an antibiotic resistance determinant .

 
Media and growth conditions. S . sciuri strains were grown in tryptic soy broth (TSB) or agar (TSA) (Difco Laboratories, Detroit, Mich.) at 37°C . Luria-Bertani medium supplemented with 50 µg of ampicillin · ml^-1 was used to grow the E . coli strain carrying the plasmid with the mecA probe .

For analysis of the different types of colonies produced by S . sciuri strain K3, the original culture was plated in TSA and incubated at 37°C for 48 h, followed by incubation at room temperature for 48 to 72 h . The number and color of the colonies were observed every 24 h . For the subsequent study of the different colonies, new cultures were prepared in TSB from distinct and well-isolated colonies .

Antibiotic step selection. For the step selection assay, a colony was picked from a TSA plate containing 3 µg of methicillin/ml and inoculated into TSB with the same concentration of the antibiotic . Upon growth, such cultures were used as inocula of a fresh TSB containing gradually increasing concentrations (in twofold steps) of methicillin . The existence of contaminants among the step-selected cultures was excluded by comparison of their pulsed-field gel electrophoresis (PFGE) patterns with those of the original strains . The ClaI-mecA polymorphisms of the selected cultures were analyzed for possible modifications that might have occurred in the mec region during the step selection procedure .

DNA methods. All routine DNA manipulations were performed by standard methods (2, 20) . Restriction enzymes were purchased from New England Biolabs (Beverly, Mass.) and used as recommended by the manufacturer . Chromosomal DNAs for conventional gel electrophoresis and PFGE were prepared and run in conventional and PFGE gels as previously described (7) .

Preparation of mecA DNA probes. The mecA probe used to hybridize both ClaI and SmaI chromosomal digests was prepared by restricting plasmid pMF13 with XbaI-PstI and isolating the 1.2-kb fragment internal to mecA (17) . The mecA probe used for Northern blot analysis, K1NP, is a 0.5-kb fragment, internal to the mecA of S . sciuri strain K1 .

Preparation of the IS256 DNA probe. The IS256 probe was prepared by PCR amplification of a 1.14-kb fragment by using primers ICP17 (5'-CCCAGGAGGACTTTTACATG) and ICP12 (5'-GCCTCACGCGCTAAGTTAAT) .

Hybridization of chromosomal DNA digests with the mecA or IS256 probe. Following restriction of the chromosomal DNA with ClaI or SmaI, the digestion fragments were resolved in gels (conventional or PFGE, respectively) and transferred to nylon membranes by vacuum transfer . Membranes containing the digested DNAs were hybridized with the mecA or IS256 probe under high-stringency conditions using the ECL Random Prime nonradioactive labeling system (RPN 3030; Amersham, Little Chalfont, Buckinghamshire, United Kingdom) according to the manufacturer's recommendations .

PCRs. PCR amplification of DNA for sequencing or probe preparation was performed with TTh polymerase (HT Biotechnology Ltd., Cambridge, United Kingdom) on a Perkin-Elmer Applied Biosystems (Cheshire, United Kingdom) Gene Amp PCR System 9600 . When necessary, PCR products were purified with the Wizard PCR Preps DNA purification system (Promega, Madison, Wis.) .

Determination of DNA sequence. DNA sequencing was performed with automated DNA sequencing systems at the sequencing facilities located at the Gulbenkian Institute (Oeiras, Portugal) and The Rockefeller University . Nucleotide and amino acid sequences were analyzed with DNAStar software (Lasergene, Madison, Wis.) .

RNA preparation and Northern blot analysis. RNA was prepared with the FastRNA isolation kit (Bio 101, Vista, Calif.) according to the recommendations of the manufacturer . RNA samples (5 µg each) were resolved in a 1.2% agarose-0.66 M formaldehyde gel in MOPS (morpholinepropanesulfonic acid) running buffer (Sigma Chemical Company, St . Louis, Mo.), with RNA size markers (Promega) . RNAs were blotted onto a nitrocellulose membrane with the Turboblotter alkaline transfer system (Schleicher & Schuell, Keene, N.H.) . Probe labeling with [-³²P]dCTP was carried out with the Megaprime System (Amersham Life Science Inc., Arlington Heights, Ill.), and hybridization took place under high-stringency conditions .

Primer extension analysis. The 5' ends of transcripts of S . sciuri mecA were determined by primer extension as previously described (3, 29) . Briefly, the MAK1PE oligonucleotide, 5'-TTCAATGGCATCAATTGTTTCG (29), was end labeled with [-³²P]dATP by using a 5'-end labeling kit (Amersham Life Science Inc.) and was hybridized with 25 µg of each RNA preparation . Reverse transcription was carried out with SuperScript RT (Gibco-BRL, Gaithersburg, Md.) . After heat inactivation of the enzyme, the reaction product was incubated with RNase H (Roche Diagnostics GmbH, Mannheim, Germany), purified by extraction with phenol-chloroform-isoamyl alcohol, and ethanol precipitated . Analysis of the extended products was carried out on 7.5% polyacrylamide-urea gels . Sequencing reaction mixtures containing K1 DNA primed by oligonucleotide MAK1PE were carried out with the Sequenase system (U.S . Biochemical Corp., Cleveland, Ohio) and applied in parallel lanes on the gel .

Membrane purification. Membrane proteins were prepared from bacterial cultures as previously described (23) . Proteins were quantified by using the Dc Protein Assay kit (Bio-Rad Laboratories, Hercules, Calif.) .

Western blotting. For detection of PBP2a in the membrane protein fraction, 30 µg of each membrane protein preparation was resolved on 8% acrylamide gels and transferred to nitrocellulose membranes by Western blotting as previously described (29) . Incubation with a monoclonal antibody against PBP2a of a MRSA strain (Eli Lilly & Co., Indianapolis, Ind.) was carried out with the ECL Western blot analysis system (Amersham Pharmacia Biotech) (29) .

During repeated plating and restreaking of colonies, we noted yet another unusual property of this strain . Plating strain K3 on TSA, we observed that the predominantly yellow colonies were occasionally mixed with white colonies (Fig . 1) . We followed up this observation by plating cultures of the original strain K3 and also cultures grown from two distinct colonies picked from the TSA plate: a yellow colony (K3y) and a white colony (K3w) . The three cultures were plated on TSA at various dilutions, and the numbers of yellow and white colonies were counted after 48 h of incubation at 37°C . Table 2 shows the results of two such experiments . The results indicate that the yellow colonies kept segregating both yellow and white colonies, although the yellow ones predominated . On the other hand, the white colonies produced only white colonies . Following additional incubation at room temperature for 2 to 3 days, white sectors of growth appeared in some of the yellow colonies (Fig . 1) . Determination of the antibiotic resistance of colony variants K3y and K3w by population analysis showed that K3y was resistant to methicillin and had retained the heterogeneous population structure of the original strain K3, while in cultures of K3w the majority of cells had lost resistance (Table 1) .

 
The SmaI PFGE profiles of chromosomal DNAs from pure cultures of the yellow (K3y) and white (K3w) colony variants showed differences in three bands: two fragments of approximately 160 and 30 kb present in the yellow variant were absent from the white segregant, which instead had a new band of 120 kb . The remaining SmaI fragments were identical (Fig . 2A) . Testing with the mecA DNA probe identified two hybridizing bands, of 160 and 145 kb, in strain K3y and a single hybridizing band of 145 kb in strain K3w (Fig . 2B) . The change of one SmaI band from 160 kb in K3y to 120 kb in K3w, corresponding to the loss of approximately 40 kb in the chromosome of the white culture, was accompanied by the loss of the 160-kb mecA-hybridizing band carried by the original strain K3 (Fig . 2B) . Hybridization of chromosomal ClaI digests with the same mecA probe confirmed that during segregation of the white variant one of the mecA copies was lost and also demonstrated that the copy lost was the S . aureus type mecA2 . This conclusion was based on the fact that the S . aureus type mecA2 comprises a ClaI restriction site producing two mecA-hybridizing fragments after ClaI digestion, while the S . sciuri mecA1 has no recognition site for ClaI (27) . While strain K3y produced three ClaI-mecA fragments (one from mecA1 and two from mecA2) of 13.5, 5.4, and 2.2 kb, strain K3w showed a single ClaI-mecA fragment, of 13.5 kb (Fig . 2C), which corresponded to the mecA1 copy . The nature of the 30-kb band, which was also lost during segregation of the K3 white variants, is unknown .

 
Five S . sciuri strains were selected for more detailed characterization: strain K3y (new designation for strain K3 producing predominantly yellow colonies); strain K3w, the nonpigmented (white) segregant of K3; K3wM200, a highly methicillin resistant mutant of K3w obtained in the laboratory by step selection; and two naturally occurring methicillin-resistant S . sciuri strains, SS-37 and SS-41, each of which carried only the gene homologue mecA1 native to S . sciuri . Strains SS-37 and SS-41 expressed heterogeneous resistance to methicillin: methicillin MICs for the majority of cells in cultures of SS-37 and SS-41 were 25 and 3 µg/ml, respectively, and subpopulations of cells capable of growing at 800 (SS-37) or 25 (SS-41) µg of methicillin/ml were present in cultures of these strains (7) (Table 1) .

PFGE analysis of chromosomal SmaI digests was used to test the relatedness of the five S . sciuri strains studied . As already described, strains K3y and K3w and the original strain, K3, showed closely related PFGE patterns (Fig . 2A), shared by the resistant laboratory derivative K3wM200 (data not shown) . Unexpectedly, the same PFGE pattern was also shared by strain SS-37 . The similarity of PFGE patterns was surprising in view of the different origins of these strains: SS-37 was isolated from the nasopharynx of a child in 1997 (7), and strain K3 was recovered from skin at a neonatal ward of a hospital in East Africa in 1992 (6, 12) . Strain SS-41, recovered from the nares of an elderly patient in Cape Verde in 1996 (7), had a completely different PFGE pattern .

Sequence analysis of mecA and upstream region. The parallel losses of methicillin resistance and the second, S . aureus type copy of mecA during segregation of white K3w colonies indicates that the methicillin-resistant phenotype of K3y was associated with the presence of mecA2 in this strain . In order to determine the basis for the methicillin resistance in the other three strains studied, we sequenced the mecA gene and the region upstream in K3wM200 and in strains SS-37 and SS-41 . Each of these strains carried a sequence virtually identical to that of mecA1, identified earlier as the mecA homologue native to all S . sciuri isolates (27, 28) . Minor deviations from this common sequence were a change in 2 bp within the mecA-coding region (551 to 552 bp) in strain SS-37, leading to the replacement of an isoleucine by a valine residue, and the replacement of a thymine by a guanine residue at position 1474 of the coding sequence in both strains SS-37 and SS-41, leading to the replacement of a tyrosine by aspartate . The presence of an aspartate residue in this position was previously described for S . sciuri type strain K1 (27) . For each of the three strains studied, a well-conserved ribosome-binding site (GGGAGG) was detected, 6 bp upstream of the putative mecA initiation codon .

Examination of the DNA sequence further upstream of mecA revealed that in both SS-37 and K3wM200, an additional 1.3-kb fragment was inserted in the promoter region of mecA. This element was identified as a complete copy of IS256 that inserted in slightly different positions in the two genomes: at 109 and 92 bp upstream of the start codons of SS-37 and K3wM200 mecA, respectively . In both cases, the insertion element was oriented in the same transcriptional direction as the mecA gene, and it was flanked by an 8-bp duplication: TGATTTTA for SS-37 and TTTTAATT for K3wM200 .

No IS256 was detected in the third methicillin-resistant strain, SS-41 . Examination of 618 bp of DNA sequence upstream of the SS-41 mecA revealed only three differences in comparison to K3 mecA1: a replacement of cytosine by thymine at 143 bp upstream of the start codon and two additional single-base differences further upstream at 449 and 464 bp .

Transcriptional analysis of mecA. A DNA probe internal to the S . sciuri mecA1 was used to analyze transcription of this gene by Northern blot analysis . Single transcriptional units were detected in the methicillin-resistant strains SS-37, SS-41, and K3wM200, while strain K3w (and strain K1, used as a negative control) showed no hybridization signal under the conditions used . The transcripts identified for strains SS-37, SS-41, and K3wM200 varied in size and intensity of the hybridization signal (Fig . 3) . Strain K3wM200 showed partial RNA degradation in the Northern blot assay, probably resulting from the relative instability of the transcript in this laboratory-selected mutant .

 
In order to determine if these strains shared the same transcriptional start site, we mapped the 5' ends of all transcripts by primer extension . As a control, we used the pair of S . sciuri strains K1 and K1M200; a point mutation in the -10 region of the step-selected mutant K1M200 was associated with increased transcription of mecA (29) . K1 DNA was used as a sequencing ladder to determine the sizes of the extended products .

Strains SS-37, K3wM200, and SS-41 all showed different transcriptional starts, which also differed from the one previously determined for K1M200 (29) . The transcription starts for strains SS-37 and K3wM200 were mapped 80 and 64 bp upstream of the mecA start codon, respectively, close to the 3' end of IS256 (Fig . 4) . Based on the +1 sites determined for SS-37 and K3wM200, the -35 promoter region should correspond to the outward-facing 3' end of IS256 (nucleotides TTGACT) as already described for S . aureus (15) . The -10 region for strains SS-37 and K3wM200 would correspond to nucleotides TTTAAT and TATTAT, respectively (Fig . 4) . The transcriptional initiation site of strain SS-41 was located 113 bp upstream of the mecA start codon . Based on the +1 site identified for SS-41, the consensus -10 and -35 regions would be, respectively, TATAGT and TTCCTT . The -35 region would include the cytosine-thymine substitution previously identified for this strain (see Fig . 4) .

 
Western blot analysis. Expression of the mecA gene homologue carried by strains SS-37, SS-41, K3w, and K3wM200 was tested in membrane protein preparations with a monoclonal antibody against PBP2a . Membrane preparations of strains K1 and K1M200 were used as negative and positive controls, respectively (29) . All three resistant strains, SS-37, SS-41, and K3wM200, produced a protein that reacted with the antibody for PBP2a, with the same molecular mass as the protein produced by K1M200, i.e., 84 kDa (Fig . 5) . (29) . The amounts of protein produced by strains SS-37, SS-41, and K3wM200 were greater than the amount produced by strain K1M200 . The methicillin-susceptible strains K1 and K3w showed no detectable production of protein reacting with the antibody (Fig . 5) .

 
IS256 hybridization patterns. The presence of IS256 in the chromosomes of the S . sciuri strains was analyzed by hybridizing SmaI digests with a DNA probe for this insertion sequence . Since IS256 does not contain any restriction site for SmaI (4), we concluded that the number of hybridizing bands represented the minimum number of copies of IS256 in the chromosome . Strain SS-37 showed three hybridizing SmaI bands of approximately 145, 170, and 265 kb . Strains K3y and K3w hybridized with the IS256 probe in two SmaI fragments of 170 and 265 kb . In strain K3wM200 two additional DNA fragments of approximately 145 and >480 kb—i.e., a total of four SmaI bands—hybridized with this probe (Fig . 6) . Most interestingly, the 145-kb SmaI fragment in which one of these additional IS256 copies was found was the one carrying the mecA gene (see Fig . 2) .

 
The additional observation that the size of the single ClaI-mecA hybridization band was reduced from 13.5 kb in the parental strain K3w to 8 kb in K3wM200 (data not shown) is consistent with the insertion of one IS256 copy upstream of mecA during the step selection procedure, since this insertion sequence contains a ClaI recognition sequence (4) .

The putative number of IS256 copies carried by each strain was confirmed by hybridizing chromosomal EcoRI digests with a probe for IS256, which does not contain EcoRI restriction sites (data not shown) .

We studied three S . sciuri isolates recovered from humans (one from colonization and two from clinical specimens) that showed a significant degree of methicillin resistance . This phenotype represents a deviation from the typical susceptibility profile of the S . sciuri species, most members of which are completely susceptible to ß-lactam antibiotics, including methicillin (6) . The observations described in this paper suggest that each of these three methicillin-resistant natural isolates of S . sciuri has followed distinct strategies to adapt to the pressure of antimicrobial agents in the human-clinical environment .

The mecA region of the first of these drug-resistant strains, strain SS-41, was compared to the already characterized mecA1 region of strain K3 . Differences within a 618-bp region sequenced upstream of the mecA of strain SS-41 were minor: one single-base change each at positions 449 and 464, and a change of cytosine to thymine at 143 bp upstream of the start codon . The transcription of mecA in strain K3w was very poor in contrast to the strong transcription of the gene in strain SS-41 (Fig . 3) . We suggest that the striking increase in the transcription rate (and the appearance of the drug-resistant phenotype) in SS-41 is related to the change from cytosine to thymine upstream of mecA . This interpretation is based on the overall similarity of the mecA upstream regions in strains K3w and SS-41 and on the primer extension data positioning the transcription start site close to the thymine residue at position 143 . Testing this hypothesis would require cloning and expression of SS-41 mecA DNA in a methicillin-susceptible strain . The methicillin resistance mechanism proposed for strain SS-41 is analogous to the mechanism recently identified in a laboratory mutant of S . sciuri, in which increased transcription of the mecA homologue and appearance of drug resistance were caused by a single-base alteration in the -10 region of the promoter (29) . The fact that SS-41 was isolated from a clinical source suggests that this survival strategy may also be used by strains exposed to antibiotic pressure in vivo .

In the second heterogeneously methicillin resistant S . sciuri strain, SS-37, the native mecA1 gene appears to be mobilized to become a potential antibiotic resistance gene by up-regulation of its transcription, resulting in the appearance of large quantities of a PBP2a-like protein in the cell extract . Sequencing of mecA in this strain revealed that the upstream region contained an IS256 element . The formation of highly active hybrid promoters by IS256 has been previously described in S . aureus (15) . The transcriptional data obtained indicate that insertion of IS256 in the upstream region of SS-37 mecA has indeed created such a hybrid promoter, which was hijacked by the mecA homologue as a surrogate promoter . The presence of IS256 in two of the methicillin-resistant natural isolates was surprising, since this insertion sequence has not been detected before in S . sciuri isolates and our own data indicate that it is rarely carried by this species (I . Couto, unpublished data) . Therefore, it seems reasonable to hypothesize that S . sciuri strains carrying IS256, in particular strain SS-37, which carries several copies of IS256, must have acquired this insertion sequence from an exogenous source, possibly from some human species of staphylococci (8, 14) or other bacteria (18, 19) known to carry multiple copies of IS256 .

The third strain analyzed in this study, strain K3, had already been shown to carry two mecA genes: mecA1, typical of all S . sciuri isolates, and a second copy, mecA2, indistinguishable from the S . aureus mecA determinant (28) . The new observations described in this paper demonstrate that strain K3 is unstable: it segregates methicillin-susceptible nonpigmented (white) and methicillin-resistant yellow colonies . The yellow colonies continue to segregate yellow and white colonies, while the white colonies breed white progeny only . Our observations further show that the loss of antibiotic resistance in the white colonies was associated with loss of the S . aureus type mecA2 gene from these segregants, indicating that the source of methicillin resistance in strain K3 was mecA2 .

Additional revealing observations were made by further studying K3w, the nonpigmented, methicillin-susceptible segregant of strain K3 from which the S . aureus type mecA2 was lost . We were interested in determining what type of drug resistance mechanism—if any—such methicillin-susceptible segregants could generate during antibiotic selection in vitro . Exposure of K3w to gradually increasing concentrations of methicillin rapidly yielded the laboratory mutant K3wM200, a strain for which the original methicillin MIC of 3 µg/ml characteristic of strain K3w was increased to 200 µg/ml . Examination of this step mutant for the transcription rate of the S . sciuri mecA homologue showed a striking increase over that of the parental strain K3w . Cell extracts from K3wM200 contained a protein that reacted with a monoclonal antibody prepared against PBP2a, the S . aureus mecA gene product .

Both the SmaI-IS256 and the ClaI-IS256 hybridization results suggest that during the step pressure, K3wM200 has recruited one of the IS256 elements carried by K3w—presumably through an intrachromosomal translocation or duplication—such that this insertion sequence could assume the role of a surrogate promoter for increased expression of the mecA homologue, as described for strain SS-37 . Autonomous movement of IS256 and multiple independent insertions of this element into the staphylococcal chromosome have been observed (8, 14) . Ziebuhr et al . (30) have shown that phase variation in biofilm production by Staphylococcus epidermidis was caused by alternating insertion and excision of IS256 at different sites of the ica gene cluster; other authors have suggested that IS256 transposition into the llm gene is involved in methicillin heteroresistance in S . aureus (15) . Our own results suggest that IS256 may also play a role in the modulation of methicillin resistance in S . sciuri .

Putting all observations described in this paper together allows one to suggest that strains SS-37, K3w, and K3 are related to one another along a putative evolutionary sequence of events, summarized in Fig . 7 . We propose that the ancestral strain in this sequence was a methicillin-susceptible S . sciuri strain with the genotype of K3w or SS-37 (PFGE pattern X1 in Table 1) carrying solely the native mecA homologue . This ancestral strain drifted over into the human-clinical environment, where it acquired the IS256 element . We propose that this strain (the hypothetical SS-37* in Fig . 7), equipped with several copies of IS256, was in fact the direct precursor of strain SS-37, which under exposure to some ß-lactam antibiotic in the environment recruited one of the acquired IS256 elements as a promoter, allowing overexpression of the mecA homologue and consequent expression of methicillin resistance . A second lineage of SS-37* did not recruit the acquired IS256 sequence for resistance but rather evolved another resistance strategy, which involved acquisition of a complete copy of the mecA2 gene from a MRSA or methicillin-resistant coagulase-negative staphylococcus strain, and thus emerged as strain K3 or K3y, equipped with two copies of mecA—one from MRSA and one from the S . sciuri native mecA . Strain K3y is able to cope with antibiotic pressure, as shown by its heterogeneous resistance profile . However, in the absence of antibiotic pressure, this strain is unstable and segregates white colonies which are antibiotic susceptible and from which the S . aureus copy of mecA2 is lost; by this process K3w, which carries only the S . sciuri copy of mecA1 plus the IS256 sequences, is generated . When such a strain is exposed to antibiotic pressure in the laboratory environment, the bacterium again uses the first strategy for resistance: the hijacking of the IS256 element to provide a strong promoter driving the overexpression of the mecA gene, resulting in the methicillin-resistant phenotype of strain K3wM200 . The reversibility of the K3y/K3w system may be considered a model to illustrate an early stage in the evolution of a drug resistance mechanism in which maintenance requires continuous drug selection; otherwise, the mechanism is lost . The implication is that at this reversible stage of evolution, compensatory ("fitness") mutations have not yet stabilized the resistance mechanism, and the biological price of maintaining it in a nonselective environment is not worth its "cost."

 
The segregation of white K3 colonies resembles the spontaneous formation of nonpigmented or sectored colonies in S . aureus cultures reported earlier by Servin-Massieu (22) . The mechanism of cosegregation of pigmentation with methicillin susceptibility observed for this strain is not clear . The loss of the yellow color together with the loss of the second, S . aureus type copy of mecA suggests that genetic determinants of pigmentation may be located in the same 40-kb DNA which is deleted from the chromosome of strain K3w .

The observations described in this report provide further evidence suggesting that the mecA homologue ubiquitous in the animal commensal species S . sciuri may very well have been the evolutionary precursor of the mecA determinant in MRSA . In this view the assembly of the mec element, i.e., the large 30- to 60-kb "foreign" DNA in which the mecA determinant of S . aureus is embedded, may have occurred in a parallel, or possibly a later, evolutionary sequence of events .

We gratefully acknowledge the help of A . M . Ludovice and S . Gardete (ITQB/UNL) in DNA sequencing and Northern blot analysis, as well as the help of M . Rocheta (ITQB/UNL) in the preparation of color photographs and the technical assistance of M . C . Lopes (ITQB/UNL) .

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