Department of Medical Microbiology, Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB25 2ZN, UK
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Abstract |
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Introduction |
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Methicillin-resistant S. aureus (MRSA) strains do not possess mecI, or have mutations within mecI which prevent it functioning, 8 ,9 ,10 or have mutations within the mec promoter region at the presumptive MecI peptide bind ing site. 9 A small number of mecA-positive S. aureus strains, known as pre-MRSA, have been discovered that are phenotypically susceptible to methicillin. 8 ,11 ,12 In these latter strains the expression of methicillin resistance is fully repressed by mecI and cannot be induced by the presence of a ß-lactam. However, resistant cells arise at
a high frequency resulting from point mutations in the mecI gene.8 Clinical isolates of mecA-positive S. aureus expressing resistance to methicillin have, therefore, circum vented the mecI-mediated repression.
Phenotypically susceptible, mecA-positive, pre-methicillin-resistant CNS (pre-MRCNS) also occur, and are more common than pre-MRSA.9 However, investi gation of the mec region in phenotypically resistant Staphylococcus epidermidis has not revealed the expected mutations in mecI or the mec promoter region.10 Regu lation of methicillin resistance in MRCNS may, therefore, differ from that in MRSA.
The aim of this investigation was to study the distri bution of mecA, mecR1 and mecI in a genetically diverse collection of staphylococcal species. The presence of these genes was correlated with the methicillin MIC for each isolate. Furthermore, the integrity of mecI and the mec promoter region in isolates carrying mecIwas examined by nucleotide sequence analysis. This was performed to locate mutations present in these regions that might explain expression of resistance to methicillin in the presence of the repressor gene.
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Materials and methods |
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The bacteria examined were a collection of 144 isolates of staphylococci. They comprised
42 S. aureus and 102 CNS, including 67 S. epidermidis, 12 Staphylococcus haemolyticus, 12 Staphylococcus simulans, five Staphylococcus warneri, two Staphylococcus xylosus, two Staphylococcus
capitis and two Staphylococcus hominis. The MIC of methicillin for each isolate
by agar incorporation and Etest was available from previous investigations of
methicillin-susceptibility testing techniques.
13
,14 Isolates were classi fied as resistant if the MIC was
8 mg/L.
Preparation of DNA
Staphylococcal cells from a single colony of overnight growth were washed and suspended in 400 µL of lysis solution (50 mM Tris, pH 8, 5 mM EDTA, pH 8, 50 mM NaCl). Lysostaphin (Ambi UK, Trowbridge, UK) was added to a final concentration of 20 mg/L. The suspension was incubated at 37°C, with gentle shaking, for 60 min. The mixture was incubated for a further 2 h at 50°C after the addition of 80 µL of proteinase solution (50 mM Tris, 0.4 M EDTA, pH 8, 0.5% sodium dodecyl sulphate containing 0.5 mg proteinase K (Boehringer Mannheim, Lewes, UK)). DNA was extracted with 200 µL each of phenol and chloroform and the mixture was centrifuged at 13,000g for 5 min. The top layer was removed, mixed with an equal volume of ice-cold ethanol and then centrifuged as before. The DNA pellet was resuspended in 25 µL TE (10 mM Tris, 1 mM EDTA, pH 8) buffer and stored at 20°C until needed.
Primers
The mecA gene was amplified with primers MR1 and MR2 ( Table I), previously described by Tokue et al.15 MR3, located within the region amplified by MR1 and MR2, was designed as a probe by Tokue et al.15 In this study it was used as a third primer with primer MR2 for confirmatory semi-nested PCR using methods described by Falla et al.16
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PCR for mecA, mecR1 and mecI
To amplify the mecA gene, DNA template was diluted 1 in 100. A 20 µL reaction mixture contained 2 µL of DNA template, 0.5 units of Taq polymerase (Advanced Bio technologies, Epsom, UK) and a final concentration of the following: 200 µM of each dNTP (Pharmacia Biotech, St Albans, UK), 1.5 mM MgCl2, 75 mM TrisHCl pH 9.0, 20 mM (NH4)2SO4, 0.01% Tween 20, 250 nM primers MR1 and MR2. Thirty cycles of amplification were performed, each consisting of 60 s at 95°C, 60 s at 55°C and 2 min at 72°C. Each PCR batch was controlled with a known positive and negative isolate and an organism-free sample. Ten microlitres of PCR product was separated by electrophoresis on a 1% agarose gel and visualized under ultraviolet light after staining with ethidium bromide. Positive reactions contained a 1339 bp product.
The primary product underwent a further cycle of semi-nested PCR amplification to confirm its identity. A 2 µL sample of the original reaction mixture was used as DNA template in a further reaction mixture constituted as previously, but with primers MR2 and MR3. Amplifi cation and electrophoresis were performed as before to detect a 785 bp product.
Primers TMW15 and TMW16 were used to detect a 414 bp mecR1 product by the same method as above. To amplify mecI with primers TMW17 and TMW18, the annealing temperature was reduced to 50°C and the 265 bp product was separated on a 2% agarose gel, but other wise conditions were the same. The specificity of the primers was validated by sequencing the products obtained from isolate TME14 by the method described below .
Sequencing of the mecI gene and the mec promoter region
The nucleotide sequence of the mecI gene was determined for 10 mecI-positive S. aureus and 15 CNS isolates chosen as a representative sample. DNA template was diluted 1 in 100 and 4 µL was used in a 40 µL reaction mixture con taining 1 unit of Taq polymerase, 200 µM of each dNTP, 1.5 mM MgCl2, 75 mM TrisHCl pH 9.0, 20 mM (NH4)2SO4, 0.01% Tween 20 and 250 nM each of primers TMW19 and TMW20. Thirty cycles of amplification were performed, each consisting of 60 s at 95°C, 60 s at 50°C and 2 min at 72°C. The 480 bp product included the entire mecI gene.
The primary product was purified using a C-100 Centricon column (Amicon, Gloucester, UK) and then re-amplified with each primer individually in separate reaction mixtures. A total volume of 20 µL was used including 4 µL of Amplitaq DNA polymerase terminator mix (Applied Biosystems, Warrington, UK), 2 µL of primer, and 3090 ng of DNA template. Twenty-four cycles consisted of 30 s at 96°C, 15 s at 50°C and 4 min at 60°C. The product was mixed with 50 µL of 95% ethanol and 2 µL of 3 M sodium acetate and placed on ice for 10 min. It was then centrifuged at 13,000g for 30 min and the ethanol removed before allowing the DNA pellet to dry. The nucleotide sequence was determined on an Applied Biosystems model 373A automated DNA sequencer.
For each isolate, the sequences obtained using each primer were compared using ABI Prism DNA Sequencing Analysis Software (Applied Biosystems) and a consensus sequence was computed. The consensus sequence of mecI for each test isolate was then compared with the published sequence from pre-MRSA strain N315.6
Primers TMW30 and TMW31 were used as above to amplify and sequence the mec promoter region and the consensus sequence from each test isolate was compared with the published sequence from pre-MRSA strain N315.6
Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis (PFGE) was performed on all mecA-positive staphylococci to determine their genetic relatedness. Five colonies of an overnight culture were harvested into 1 mL TEN buffer (0.1 M TrisHCl, 0.15 M NaCl, 0.1 M EDTA pH 8). Cells were washed twice and resuspended in 0.3 mL EC buffer (6 mM TrisHCl, 1 M NaCl, 0.1 M EDTA, pH 8, 0.5% 20 cetyl ether, 0.2% deoxycholate, 0.5% n-lauroylsarcosine). Fifty microlitres of this cell suspension was mixed with 25 µL of lysostaphin (200 mg/L) and then 200 µL of 1% InCert agarose (FMC BioProducts, Rockland, ME, USA) was added. The mixture was allowed to solidify at 4°C for 10 min in a gel plug mould. The solid plug was incubated overnight in 2 mL of EC buffer at 37°C. This was replaced by 2 mL of PB buffer (0.5 M EDTA, pH 8, 1% sarkosyl, proteinase K 1 mg/mL) for a further overnight incubation and then the gel plug was washed six times in 5 mL of TE buffer for 30 min each time to remove proteinase K. Gel plugs were stored in 1 mL of fresh TE buffer at 4°C until needed.
DNA was digested with the restriction endonuclease SmaI (Boehringer Mannheim). A slice of the gel plug, approximately 5 mm x 5 mm, was placed in 200 µL of restriction enzyme mixture (20 U SmaI, 20 µL 10x buffer (as provided with SmaI), 1 µL of bovine serum albumin 1 mg/mL, 5 µL of 20 mM dithiothreitol) for 4 h at 25°C.
Electrophoresis was performed with the CHEF-DR III electrophoresis cell (Bio-Rad, Hemel Hempstead, UK)on a 1% agarose gel with the following electrophoresis parameters: 6 V/cm for 24 h at 14°C; initial pulse time, 10 s; final pulse time, 25 s. A control strain, S. aureus NCTC 8325, with fragments of known sizes,19 was run on each gel to allow comparison. Gels were stained with ethidium bromide 0.5 mg/L and visualized under ultraviolet light.
The resulting band patterns were compared visually. Isolates with indistinguishable patterns were grouped together and those with patterns distinguishable from all other isolates were designated as "unique".
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Results |
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The primary mecA gene product of 1339 bp was found in 21 of the 42 isolates of S. aureus tested and in 44 of the 102 CNS examined. All these positive isolates produced the predicted 785 bp product on confirmatory semi-nested PCR.
Methicillin MIC
All mecA-negative staphylococci had MICs <8 mg/L as determined by
methicillin Etest.14 All mecA-positive
staphylococci had MICs 8 mg/L by at least one of the susceptibility tests performed; none
were classified as pre-MRSA or pre-MRCNS.
13
,14 The MICs determined by methicillin Etest on Columbia
5% NaCl agar are shown in Tables II and III.
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The 414 bp PCR product of mecR1 was found in all 21 mecA- positive S. aureus, but did not occur in the absence of mecA. The 265 bp product of mecI was also detected in ten (48%) of the mecA-positive S. aureus. There was no correlation between methicillin MIC and the presence of mecI (Table II).
mecR1 was detected in 42 of 44 mecA-positive CNS; it was not found in two mecA-positive S. haemolyticus. mecI was detected in 22 (50%) of the mecA-positive CNS. There was no correlation between the methicillin MIC and the regulatory genes detected in each isolate (Table III).
Pulsed-field gel electrophoresis
Analysis of the mecA-positive S. aureus by PFGE revealed 15 different patterns among the 21 isolates examined (Table II). Four isolates gave pattern A, while patterns B, C and D were represented by two isolates each. All other S. aureus isolates had unique patterns.
Four groups of indistinguishable mecA-positive CNS were found after analysis by PFGE (Table III). Patterns E and F were each represented by three isolates of S. epidermidis and pattern G by two isolates. Five S. simulans were included in pattern H. The other 14 S. epidermidis, six S. simulans, eight S. haemolyticus, two S. warneri and one S. hominis examined all produced distinct PFGE patterns.
Sequence of mecI and the mec promoter region
The sequence of the mecI gene was identical to the reference sequence in the four MRSA with PFGE pattern A (Table II). The other six isolates of S. aureus had a cytosine to thymine substitution at nucleotide 202 that produced a new termination codon. In all ten mecI-positive MRSA, the sequence of the mec promoter region was identical to the reference sequence.
The sequence of mecI was the same as that of the reference strain in all the CNS isolates tested (Table III). Seven of the 15 isolates, all of them S. simulans, had a nucleotide substitution, adenine to guanine, in the mec promoter region (Figure).
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Discussion |
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Eleven (52%) of the mecA-positive S. aureus examined in this study did not possess the mecI gene. These isolates were, therefore, able to express methicillin-resistance in the absence of its repressor effect. Six of the S. aureus carrying the gene were found to have a substitution in mecI at nucleotide 202, resulting in an in-frame stop codon in the middle of the open reading frame. This has been noted previously amongst British and European MRSA. 8 ,9 ,10 In these too, the repressor protein would not be produced, thus allowing full expression of methicillin resistance.
The four remaining mecA-positive S. aureus were indis tinguishable by PFGE, all being of pattern A, and were therefore presumed to be the same strain. All were, unexpectedly, found to contain an intact mecI gene and had no mutation within the mec promoter region. This feature should result in a susceptible phenotype. Although the methicillin MICs for these organisms were indeed lower than those for the other MRSA sequenced, none of them were below the breakpoint (Table II). It has pre viously been assumed that a mecI gene with the same sequence as the functioning gene in pre-MRSA N315 and with the same upstream regulatory sequence is capable of producing the MecI peptide, 8 ,9 ,10 although no assay has yet been developed to test this. These results suggest the presence of genetic changes in this strain, not detected in this study or in previous work, which prevent the trans cription of mecI or the function of the MecI peptide.
Previous studies have detected mecI in six species of mecA-positive CNS.8 ,20 The gene sequence has, however, only been determined in 11 S. epidermidis. 10 Despite full expression of methicillin resistance, none of these isolates had a mutation within mecI. Examination of the mecI sequence in phenotypically resistant S. epidermidis, S. simulans and S. hominis in this study confirmed this finding as all had a nucleotide sequence corresponding to the functional gene found in the pre-MRSA N315.6 There is, therefore, no evidence discovered so far to suggest that mutations in mecI play a role in the derepression of mecA in any CNS.
Changes in the presumptive operator for mecA, within the mec promoter region, have been found in MRSA,9 but no mutations were located in the S. epidermidis strains examined.10 In eight of the mecI-positive MRCNS studied here the mec promoter region sequence was the same as that in strain N315.6 It is likely that, as above, there is another mechanism to account for the expression of methicillin resistance in the presence of a functional repression gene in many CNS species.
In this study, a mutation in the mec promoter region was found in every S. simulans sequenced and was not present in any other species examined. The origin of this change is unknown, although several possibilities exist. All these organisms were isolated from the same geographical area and may, therefore, have common ancestry. If this is the case, a mutation at some time in the past has been passed on to subsequent generations of S. simulans. Analysis by PFGE showed that there was one group of five indis tinguishable isolates (pattern H). Other organisms in the same species produced patterns that were clearly distinct, which implies that there was not a recent common ancestor. It is also conceivable that this is the normal sequence for the mec promoter region in S. simulans. If so, function of the region may be normal and the nucleotide substitution may have no effect on the expression of methicillin resistance. The possibility is, therefore, raised that the mec region introduced into this species is different from that in other staphylococci, although this is not com patible with current theories of its molecular evolution.9,21 Until sequence data from S. simulans isolated from other areas are available these questions cannot be answered.
The mutation in S. simulans consisted of a nucleotide substitution in the mecR1 35 promoter sequence (Figure). As mecR1 and mecI form an operon, transcribed from the same start point, the function of both genes may be affected. This mutation is also within the presumptive operator for mecA, the binding site for the MecI peptide. Thus, even if mecI is transcribed, there may be no binding site for the gene product, which will, therefore, be unable to repress mecA.
When mecI is absent, there are also commonly deletions in the penicillin-binding domain at the 3' end of mecR1.8 The primers used in this study amplified a single section of mecR1, within the predicted membrane-spanning domain,22 500900 bp from the 5' end. The presence of the whole gene can, therefore, only be predicted in those isolates found also to carry mecI. When mecR1 was detected in the absence of mecI, it is not known whether the penicillin-binding domain at the 3' end was present. In view of previous findings8 it is likely that most, if not all, of the mecI-negative staphylococci examined have a significant part of mecR1 deleted.
No mecR1 product was detected in two mecA-positive S. haemolyticus in this study. As no separate investigation was made of the 5' terminus, outside the area covered by the primers, it is not known how much, if any, of this gene was present. Suzuki et al.8 have previously described three mecA-positive S. haemolyticus without either the penicillin-binding or the membrane-spanning domains of mecR1. They performed further work that detected the terminal 53 bp at the 5' end, adjacent to mecA, indicating that mecR1 may once have been present. It is possible, therefore, that the isolates studied here have the same genetic organization as the formerly examined S. haemo lyticus.8 This particular configuration had not been found in S. aureus or any other species of CNS, leading to specu lation that the mec region 5' to mecA is always different in S. haemolyticus.21 However, six isolates of S. haemolyticus in this study were found to carry mecR1. Clearly mecR1 is not universally deleted in S. haemolyticus.Neither is the configuration in which the vast majority of the gene is missing confined to this species, as Kobayashi et al.20 have found it in MRSA and methicillin-resistant S. epidermidis.
In summary, examination of this collection has revealed four S. aureus isolates from the same strain, seven S. epidermidis all with different PFGE patterns, and one S. hominis with full expression of methicillin resistance despite the presence of an intact mecI gene and mec promoter region. Phenotypic resistance in these isolates is, therefore, unexplained. The failure of mecI repression must, therefore, result from changes outside the areas examined. As mecI is part of an operon, its expression could be affected by alterations in genes or control regions upstream. Evidence was also found that mecR1 may be deleted more often in S. haemolyticus than in other staphylococcal species. The sequence of the mec promoter region of S. simulans was also consistently different. Further studies on mecA regulation in all staphylococcal species are needed. This may well shed light on some the complexities and ambiguities of expression of methicillin resistance observed here. Certainly it is not simply a product of interaction between the mecA, mecR1 and mecI genes. Some strains, and possibly whole species, seem to have acquired other mechanisms to evade repression of mecA.
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Acknowledgments |
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Notes |
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References |
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2 . Hartman, B. J. & Tomasz, A. (1984). Low-affinity penicillin-binding proteinassociated with beta-lactam resistance in Staphylococcus aureus. Journal of Bacteriology 158, 5136.[ISI][Medline]
3 . Georgopapadakou, N. H., Smith, S. A. & Bonner, D. P. (1982).Penicillin-binding proteins in a Staphylococcus aureus strain resis tant to specific beta-lactam antibiotics. Antimicrobial Agents and Chemotherapy 22, 1725.[ISI][Medline]
4 . Brown, D. F. & Reynolds, P. E. (1980). Intrinsic resistance to beta-lactam antibiotics in Staphylococcus aureus. FEBS Letters 122, 2758.[ISI][Medline]
5 . Suzuki, E., Hiramatsu, K. & Yokota, T. (1992). Survey of methicillin-resistant clinical strains of coagulase-negative staphylo cocci for mecA gene distribution. Antimicrobial Agents and Chemotherapy 36, 42934.[Abstract]
6 . Hiramatsu, K., Asada, K., Suzuki, E., Okonogi, K. & Yokota, T.(1992). Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). FEBS Letters 298, 1336.[ISI][Medline]
7 . Tesch, W., Ryffel, C., Strassle, A., Kayser, F. H. & Berger-Bachi, B. (1990).Evidence of a novel staphylococcal mec-encoded element (mecR)controlling expression of penicillin-binding protein 2'. Antimicrobial Agents and Chemotherapy 34, 17036.[ISI][Medline]
8 . Suzuki, E., Kuwahara-Arai, K., Richardson, J. F. & Hiramatsu, K.(1993). Distribution of mec regulator genes in methicillin-resistant Staphylococcus clinical strains. Antimicrobial Agents and Chemotherapy 37, 121926.[Abstract]
9 . Hiramatsu, K. (1995). Molecular evolution of MRSA. Microbiology and Immunology 39, 53143.[ISI][Medline]
10
.
Kobayashi, N., Taniguchi, K. & Urasawa, S. (1998). Analysis of diversity of
mutations in the mecI gene and mecA promoter/operator region of
methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Antimicrobial Agents and Chemotherapy 42, 71720.
11 . Boyce, J. M., Medeiros, A. A., Papa, E. F. & O'Gara, C. J. (1990). Induction of beta-lactamase and methicillin resistance in unusual strains of methicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 25,73 81.[Abstract]
12 . Archer, G. L., Niemeyer, D. M., Thanassi, J. A. & Pucci, M. J. (1994). Dissemination among staphylococci of DNA sequences associated with methicillin resistance. Antimicrobial Agents and Chemotherapy 38, 44754.[Abstract]
13 . Milne, L. M., Curtis, G. D., Crow, M., Kraak, W. A. & Selkon, J. B. (1987). Comparison of culture media for detecting methicillin resistance in Staphylococcus aureus and coagulase negative staphylococci. Journal of Clinical Pathology 40,1178 81.
14 . Weller, T. M., Crook, D. W., Crow, M. R., Ibrahim, W., Pennington, T. H. & Selkon, J. B. (1997). Methicillin susceptibility testing of staphylococci by Etest and comparison with agar dilution and mecA detection. Journal of Antimicrobial Chemotherapy 39, 2513.[Abstract]
15 . Tokue, Y., Shoji, S., Satoh, K., Watanabe, A. & Motomiya, M.(1992). Comparison of a polymerase chain reaction assay and a conventional microbiologic method for detection of methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 36, 69.[Abstract]
16 . Falla, T. J., Crook, D. W., Brophy, L. N., Maskell, D., Kroll, J. S. & Moxon, E. R.(1994). PCR for capsular typing of Haemophilus influenzae. Journal of Clinical Microbiology 32, 23826.[Abstract]
17 . Berger-Bachi, B., Barberis-Maino, L., Strassle, A. & Kayser, F. H. (1989). FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization. Molecular and General Genetics 219,263 9.[Medline]
18 . Song, M. D., Wachi, M., Doi, M., Ishino, F. & Matsuhashi, M. (1987). Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion. FEBS Letters 221, 16771.[ISI][Medline]
19 . Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. et al. (<1995). Interpreting chromo somal DNA restriction patterns produced by pulsed-field gel electro phoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 22339.
20 . Kobayashi, N., Taniguchi, K., Kojima, K., Urasawa, S., Uehara, N., Omizu, Y. et al. (1996). Genomic diversity of mec regulator genes in methicillin-resistant Staphylococcus aureus and Staphylo coccus epidermidis. Epidemiology and Infection 117, 28995.[ISI][Medline]
21 . Archer, G. L. & Niemeyer, D. M. (1994). Origin and evolution of DNA associated with resistance to methicillin in staphylococci. Trends in Microbiology 2, 3437.[Medline]
22 . Kobayashi, T., Zhu, Y. F., Nicholls, N. J. & Lampen, J. O. (1987). A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis. Journal of Bacteriology 169, 38738.[ISI][Medline]
Received 29 May 1998; returned 20 July 1998; revised 5 August 1998; accepted 12 August 1998