JAC Advance Access originally published online on May 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):23-30; doi:10.1093/jac/dkl208
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Assessment of allelic variation in the ccrAB locus in methicillin-resistant Staphylococcus aureus clones
1 Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa Oeiras, Portugal 2 Biomathematics Group, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa Oeiras, Portugal 3 Laboratory of Microbiology, The Rockefeller University New York, NY 10021, USA
*Correspondence address. Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal. Tel: +351-21-446-9862; Fax: +351-21-442-8766; E-mail: dco{at}itqb.unl.pt
Received 10 February 2006; returned 11 April 2006; revised 27 April 2006; accepted 28 April 2006
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Abstract |
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Objectives: To evaluate the allelic variability of ccrA and ccrB among methicillin-resistant Staphylococcus aureus (MRSA) strains belonging to different genetic lineages and carrying different SCCmec types.
Methods: Internal fragments from both genes were obtained by PCR amplification with pairs of degenerate primers and the nucleotide sequence was determined for both strands. Nucleotide sequences were aligned and the phylogenetic trees were reconstructed.
Results: The homology scores obtained confirmed the very close relationships among ccrAB alleles associated with each SCCmec type, which further validate this locus as a good target for SCCmec typing strategies. Using this strategy, particularly for the ccrB gene, SCCmec types with the same ccrAB allotype, as detected by PCR, could be discriminated and some strains with non-typeable ccrAB loci were resolved.
Conclusions: We propose that sequencing an internal fragment of ccrB is a strategy for SCCmec typing that may be easily incorporated into other sequence-based MRSA typing strategies, such as multi-locus sequence typing and spa typing.
Keywords: MRSA , ccrAB typing , SCCmec typing
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Introduction |
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Methicillin-resistant Staphylococcus aureus (MRSA) carry a large heterologous mobile genetic element—staphylococcal chromosomal cassette or SCCmec—which includes the central element of methicillin resistance, the mecA gene.1 The genetic organization of the mecA regulon defines the mec gene complex and, in S. aureus, two major classes have been described: class A containing the complete mecA regulon (mecI-mecR1-mecA) and class B containing the mecA regulatory genes disrupted by a truncated copy of IS1272 (
IS1272-
mecR1-mecA).2 The SCCmec element integrates into the S. aureus chromosome at a site-specific location (attBscc), located near the S. aureus origin of replication.3 The mobility of SCCmec is in part due to the presence of the internal recombinase genes, ccrA and ccrB, which encode recombinases of the invertase/resolvase family and constitute the ccr gene complex.2 If fully functional, ccrAB gene-products are able to catalyse the precise excision of the SCCmec element from the S. aureus chromosome. Four allotypes for ccrAB have been identified.4,5 Recently, a new type of ccr gene complex has been reported, which has only one gene (ccrC) and is not closely related to the ccrA or ccrB genes.6
SCCmec may be classified into four major types by combining the class of the mec gene complex with the ccrAB allotype:3,4 type I, class B and ccrAB1; type II, class A and ccrAB2; type III, class A and ccrAB3; and type IV, class B and ccrAB2. Some sporadic SCCmec types structurally related to type IV are characterized by ccrAB allotype 4,5 which has been also identified in a composite SCC structure in a Staphylococcus hominis strain.7
SCCmec typing, coupled with the characterization of the genetic background of MRSA clones, has been used in both local and global MRSA epidemiology studies.8–10 Their key roles in resistance and mobility make the mecA gene complex and ccrAB genes ideal targets for SCCmec typing strategies. The mecA gene itself is highly conserved among different lineages, even among methicillin-resistant strains of other staphylococcal species.4,11,12 Consequently, current SCCmec typing strategies explore polymorphisms in the mec gene complex that result from the presence or absence of the mecA regulatory genes.4,13,14
The four major SCCmec types are characterized mainly by three ccrAB allotypes (SCCmec types II and IV both have ccrAB allotype 2), and a ccrAB typing scheme has been developed based on the PCR detection with primer pairs specific for each ccrAB allotype.4,14 However, except for the sequences of a few reference strains of each SCCmec type, the allelic variation within the different ccrAB allotypes, SCCmec types or MRSA lineages is not known. Moreover, in typing the MRSA in our collection, 7% have non-typeable ccrAB allotype and, therefore, non-typeable SCCmec types. In the present study these issues were addressed by assembling a baseline for the ccrAB allelic variability at the DNA sequence level. For this purpose, internal sequences of ccrA and ccrB were determined using representative MRSA strains previously characterized in terms of their genetic backgrounds and SCCmec types.
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Materials and methods |
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Strain collection
MRSA strains used in the present study are listed in Table 1. All strains have been previously characterized in terms of genetic backgrounds and SCCmec types and included (i) the reference strain for each SCCmec type and subtype; (ii) several isolates belonging to each of three pandemic MRSA clones characterized by different SCCmec types, chosen to maximize geographical and temporal differences; (iii) three representative isolates of MRSA clones with different genetic backgrounds from these three pandemic MRSA clones but having the same SCCmec type; (iv) several SCCmec type IV strains representing most of the genetic lineages that have been described for this structural type; (v) three strains with non-typeable ccrAB allotypes; and (vi) six strains with non-typeable SCCmec.
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Media and growth conditions
Strains were grown overnight at 37°C on tryptic soy agar or tryptic soy broth under aerobic conditions.
DNA isolation
Chromosomal DNA was prepared using the Wizard genomic DNA preparation kit (Promega, Madison, WI, USA), according to the manufacturer's recommendations, except for the addition of lysostaphin at 0.5 mg/mL and RNase at 0.3 mg/mL for the lysis step.
SCCmec typing
Presumptive assignment of SCCmec types was by the multiplex PCR strategy described previously.13 ccrAB PCR typing was performed as described by Ito et al.4 and Okuma et al.14 Some strains listed in Table 1 had also been previously characterized in detail in terms of SCCmec types by PCR and hybridization screenings and DNA sequencing of polymorphic regions.5
ccrAB allelic variation
The allelic variation in the ccrAB locus was evaluated by sequencing internal fragments of ccrA and ccrB amplified by PCR. Based on available sequences at GenBank (http://www.ncbi.nlm.nih.gov) for ccrA and ccrB of S. aureus, pairs of degenerate primers were designed for each gene using the Megalign program (DNA Star software package, Lasergene, Madison, WI, USA) to identify conserved regions. The primer sequences were as follows: ccrA F1, YCC WAA YTA YTG TGG YCG TGT; ccrA R1, TKY TKG TGC RTT KAT NCC T; ccrB F1, CGW YTR GCW MGW AAY ACH TC; and ccrB R1, CTT TTC GWC KYT TWT CRY TCC. The predicted amplicon sizes were 296 bp for ccrA and 496 bp for ccrB. PCR was performed in a GeneAmp PCR System 9600 (Applied Biosystems, Foster City, CA, USA) with the following conditions: 94°C for 4 min; 35 cycles of 94°C for 30 s, 42°C for 60 s and 72°C for 2 min; and a final extension at 72°C for 4 min. In each reaction (final volume of 50 µL), 5 ng of chromosomal template, 1.25 U of Amplitaq DNA polymerase (Applied Biosystems), 1x PCR buffer with 1.5 mM MgCl2 (Applied Biosystems) and 2 mM dNTP mix (MBI Fermentas, Hanover, MD, USA) were used. For ccrA amplification 20 pmol of each primer was used and for ccrB amplification 100 pmol of each primer was used. The Wizard PCR Preps DNA Purification system (Promega) was used to purify amplified fragments. Sequencing of both strands was performed by Macrogen (www.macrogen.com) and the raw data were analysed with DNA Star software (Lasergene).
Nucleotide sequence analysis and phylogenetic tree reconstruction
Multiple sequence alignments were obtained with Clustal X (version 1.8) using default parameters and displayed with the software SEQUENCE OUTPUT for DOS (version 2.0) made available by B. G. Spratt (Imperial College, London, UK).15 Sequence classification was performed using the neighbour-joining (NJ) method based on the per cent divergence between all pairs of sequences from the multiple alignment.16 The corresponding phylogenetic trees were displayed with the NJPLOT program, distributed with Clustal X. Bootstrap NJ values were calculated using 1000 replicates.
Nucleotide sequence accession numbers
For the design of the degenerate primers we used ccrAB sequences available at GenBank. The strains and accession numbers are as follows: strains NCTC10442 and COL (SCCmec type I), AB033763 and CP000046, respectively; strains N315, Mu50 and MRSA252 (SCCmec type II), D86934, BA000017 and BX571856, respectively; strain 85/2082 (SCCmec type III), AB037671; strain 85/3907 (SCCmec type III-inv), AB047088; strains MW2 and CA05 (SCCmec type IVa), BA000033 and AB063172, respectively; strain 8/6-3P (SCCmec type IVb), AB063173; strains MR108 and Q2314 (SCCmec type IVc), AB096217 and AY271717, respectively; and strain HDE288 (SCCmec type IV-var), AF411935. For reference and control purposes, we have included the ccrA and ccrB sequences of strains NCTC10442, N315, 85/2082 and MW2 in the cluster trees and multiple sequence alignments. For control purposes, strains N315 and MW2 were re-sequenced for the internal fragments of ccrAB genes in this study. All nucleotide sequences determined in this study were deposited in GenBank under accession numbers AY918217–AY918255 (ccrA) and AY918256–AY918294 (ccrB).
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Results and discussion |
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Experimental strategy
The allelic variation in the ccrAB locus was evaluated by sequencing internal fragments of ccrA and ccrB. The selected target regions maintained the phylogenetic relationships deduced from the complete nucleotide sequences of ccrA and ccrB, meaning that the internal fragments chosen were representative of the complete gene sequences (see Supplementary data available at http://jac.oxfordjournals.org/, Figures S1 and S2). In terms of discriminatory power and therefore usefulness for typing proposes, ccrB was more suitable than ccrA since strains with ccrAB allotype 2 are properly discriminated in terms of SCCmec type. For instance, control strains for SCCmec type II (N315, Mu50 and MRSA252) are clustered together based on ccrB sequences, whereas, based on ccrA sequences, MRSA252 is clustered together with SCCmec type IV strains. The poor discrimination of ccrAB allotype 2 strains based on ccrA sequences is also illustrated by lower bootstrap support obtained for this region of the tree (see Supplementary data available at http://jac.oxfordjournals.org/, Figures S1 and S2).
ccrAB allelic variability in MRSA
The cluster trees obtained based on the internal sequences of ccrA and ccrB genes for the MRSA strains included in the present study are illustrated in Figures 1 and 2, respectively (also see Supplementary data available at http://jac.oxfordjournals.org/ for the multiple sequence alignments, Figures S3 and S4). In general terms, strains with the same SCCmec and, therefore, the same ccrAB allotype, as defined by PCR detection, fitted into the same cluster. To our knowledge, this is the first set of data for MRSA that truly validates the ccrAB typing scheme based on the PCR detection of the major ccrAB allotypes proposed by Ito et al.4 and Okuma et al.14
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ccrAB allelic variability among strains belonging to the same MRSA lineage
We included multiple isolates of three pandemic MRSA lineages: the Archaic/Iberian clone or ST250/ST247-I/IA; the New York/Japan clone or ST5-II; and the Brazilian clone or ST239-III/IIIA.8,9 The ccrAB sequences from strains belonging to the same MRSA lineage fitted into the same cluster and homology scores of 100% were obtained, suggesting very low mutation rates within the ccrAB locus.
ccrAB allelic variability among strains with different genetic backgrounds but sharing the same SCCmec type
In order to check whether the ccrAB alleles were SCCmec type or MRSA lineage specific we included three strains not related to the three pandemic lineages, but which shared the same SCCmec type: strain HAR21, ST5-I; strain HAR40, ST228-I; and strain HAR24, ST36-II (EMRSA-16).10,17 Based on ccrA sequences, strain HAR21 was discriminated from other SCCmec type I strains by a single point mutation, strain HAR40 was identical to other SCCmec type I strains and strain HAR24 was clustered together with SCCmec type IV strains, which does not validate ccrA (used alone) as a good typing target. However, the ccrB sequences of the three strains were identical to those of other strains sharing the same SCCmec type, suggesting specific ccrB sequences for SCCmec types I and II.
ccrAB allelic variability among SCCmec type IV strains
SCCmec type IV is the smallest structural type of SCCmec and is believed to be the most mobile version. In fact, SCCmec type IV predominates among community-acquired MRSA (CA-MRSA), is disseminated across several genetic lineages of hospital-acquired MRSA (HA-MRSA) and occurs frequently among coagulase-negative staphylococci.3,5,10,18,19 Perhaps as a consequence of its enhanced mobility, SCCmec type IV is also more variable than other SCCmec types and several subtypes have been described.18,20,21 In terms of genetic organization, SCCmec type IV shares with type II the same ccrAB allotype, as defined by PCR detection, and has the same mec gene complex of SCCmec type I. Moreover, some strains have a SCCmec structurally related to SCCmec type IV, sharing the same mecA complex and mecA downstream vicinity but with a different ccrAB allotype—ccrAB 4.5 Due to all these characteristics, SCCmec type IV is a challenge for any multiplex PCR typing strategy, which is a significant issue for MRSA epidemiology since the importance of SCCmec type IV to some extent parallels the increasing problem of CA-MRSA.
The variability among SCCmec type IV strains is well-represented in the MRSA collection studied here. Besides the prototype strains for SCCmec type IV and subtypes IVa, IVb and IVc, we included representative isolates of important CA-MRSA lineages, such as ST1, ST30 and ST80,22 and also some nosocomial lineages associated with SCCmec type IV, such as ST5, ST8 and ST45.10 In spite of the intrinsic variability of SCCmec type IV and the diversity of MRSA lineages, ccrA sequences, with the exception of prototype strain 8/6-3P for SCCmec type IVb, fitted in four closely related clusters, and ccrB sequences, with the exception of three prototype strains, fitted into a single cluster with a homology score of 100%. Based on ccrB sequences SCCmec type IV strains are discriminated from SCCmec type II strains (Figure 2). The discrimination is particularly remarkable if the prototype strains 8/6-3P, CA05 and MW2 and the non-typeable strain JP87 are excluded from the analysis. The discrimination of SCCmec types II and IV based on ccrB sequences is an extremely important refinement of the current ccrAB typing strategies, even though these types are properly discriminated according to the class of the mec gene complex.
Nature of strains with non-typeable ccrAB loci and/or SCCmec types
In our collection, 8% of isolates are non-typeable for SCCmec by the multipex PCR strategy13 and at least 7% for ccrAB according to the Ito et al.4 and Okuma et al.14 strategy. We used low annealing temperatures and degenerate primers targeting different regions, and could amplify and sequence the internal fragments for ccrA and ccrB genes for the three ccrAB non-typeable strains included in this study. Two strains (DEN1451 and DEN2949) were assigned to ccrAB allotype 2, which correlates with the presumptive assignment of these strains to SCCmec type IV by multiplex PCR. The third strain, DEN907, was non-typeable for ccrAB and could not be assigned to a SCCmec type by the multiplex PCR strategy. It clustered with ccrAB allotype 3, which correlates with its genetic background, ST 239, which is predominantly associated with SCCmec type III strains.
We also included some variants of SCCmec type IV which share the same overall structural organization but are not characterized by ccrAB allotype 2: strains HDE288 and IPO92 had been previously described as SCCmec type IV variants with ccrAB allotype 4 and strains POL3 and PL72 as SCCmec type IV variants with ccrAB allotype 1.5 ccrAB allotype 4 has been described as being more phylogenetically distant from the other ccr gene complexes,4 an observation that was confirmed in this study for strains HDE288 and IPO92—see Figures 1 and 2. The ccrAB sequences obtained for strains POL3 and PL72 clustered together with SCCmec type I strains with homologies of 100% or with one base difference, suggesting that these strains would have been more correctly classified as SCCmec type I variants rather than type IV variants.
SCCmec assembly and MRSA evolution
The data obtained in this study also provided some insights into assembly mechanisms of SCCmec and into MRSA evolution. The trees obtained with our data (Figures 1 and 2) indicate that ccrA of SCCmec type I strains is more related to ccrA of SCCmec type IV and II strains than the ccrA of SCCmec type III strains; whereas, ccrB of SCCmec type III strains is more related to ccrB of strains with types IV and II than the ccrB of SCCmec type I strains. These observations suggest the possibility of a separate evolution of the ccrA and ccrB loci, which were later concatenated within the SCCmec, promoting its mobility. Moreover, as previously suggested, SCCmec assembling and evolution seems to be independent of the host phylogenies.9,10,23
As referred to above, the DNA sequences of the internal fragments of ccrA and ccrB are virtually identical among strains that share the same ccrAB allotype. This is particularly relevant for SCCmec type IV (ccrAB allotype 2), which is believed to be the most mobile SCCmec version. In spite of the variability found within SCCmec type IV cassettes, which is mainly located in the mecA upstream region that includes the ccrAB locus, the ccrAB sequences seem to be highly conserved, perhaps due to the selective pressure to keep the recombinase genes, essential for SCCmec mobility, fully functional. It should be stressed that so far, of the ccrAB genes, only allotype 2 has been shown to be functional in promoting the SCCmec excision from the chromosome.6
Interestingly, Hanssen et al.24 also addressed ccrAB sequence variability among clinical coagulase-negative staphylococci (CoNS) from Norway and also 10 MRSA from India, Italy, Finland, the United States and the United Kingdom, and also found high similarity among ccrAB allotypes: 94–100% for ccrAB1, 95–100% for ccrAB2 and 100% for ccrAB3. This is particularly relevant if we take into account that several staphylococcal species were included in the study. Taken together, both studies suggest a strong selective pressure for ccrAB sequence stability among staphylococcal species and provide further evidence for the dissemination of SCCmec sequences among CoNS and MRSA.
ccrB sequencing as a SCCmec typing strategy
In this study we have shown at the DNA sequence level that MRSA strains with the same SCCmec type have very closely related ccrAB gene sequences. To our knowledge this is the first time that the allelic variation on the ccrAB locus in epidemic MRSA clones has been evaluated and the results presented here further validate the ccrAB locus as a good target for SCCmec typing strategies, as previously proposed by Ito et al.4 and Okuma et al.14 Recently a new SCCmec type (type V) has been described with a new ccr gene complex (ccrC), which has only one gene that is not closely related to either ccrA or ccrB.6 Due to the lack of relationship between ccrC and ccrAB, the strategy described in this study is not useful for typing SCCmec type V strains. However, ccrC can be identified easily by PCR, and the epidemiological relevance of SCCmec type V strains remains to be clarified.
The data presented here suggest that sequencing an internal fragment of ccrB may be a useful SCCmec typing strategy since (i) the four major SCCmec types are discriminated, (ii) some strains with non-typeable ccrAB can be resolved and (iii) the nature of some SCCmec variants can be clarified. Depending on the context of MRSA typing studies and on the discriminatory power required, ccrB sequencing can be an addendum to current SCCmec typing strategies or the first-line SCCmec typing strategy. In practical terms this strategy can be easily incorporated into other sequence-based typing protocols, such as MLST and spa typing,25,26 so that MRSA strains are simultaneously typed for genetic background and SCCmec. As for MLST, a central online database may be constructed for deposition of ccrB alleles, which could then be interrogated by researchers in order to assign a particular allele to the appropriate allotype.
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None to declare.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsSupplementary dataReferences |
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Supplementary data (Figures S1–4) are available at JAC Online (http://jac.oxfordjournals.org/).
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Footnotes |
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Present address. Instituto de Engenharia de Sistemas e Computadores: Investigação e Desenvolvimento (INESC-ID), Lisbon, Portugal. 
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Acknowledgements |
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We thank the investigators who kindly provided some of the bacterial strains used in the study: Keiichi Hiramatsu for strain N315, Teruyo Ito for strain Q2314, Robert Daum for strains CA04 and 8/6-3P, Barry Cookson for strains from the Harmony collection (HAR code) and the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for strain MW2. We also thank Alexander Tomasz for stimulating discussions about the data described in the manuscript. Partial support for the study was provided by project POCI/BIA-MIC/58416/2004 from Fundação para a Ciência e Tecnologia (FCT), Lisbon, Portugal, and by project 55068 from Fundação Calouste Gulbenkian, Lisbon, Portugal, both awarded to H. de L. D. C. O. was supported by grant SFRH/BPD/9374/2002 from FCT, Lisbon, Portugal. C. M. was partly supported by a PRODEP fellowship, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal; by project no. 129—IBET, Oeiras Portugal; and by grant SFRH/BD/23010/2005 from FCT, Lisbon, Portugal. S. V. was partly supported by project POCTI/BIO/48333/2002 from FCT, Lisbon, Portugal.
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References |
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 K. Kurt, A. Alderborn, M. Nilsson, B. Strommenger, W. Witte, and U. Nubel Multiplexed Genotyping of Methicillin-Resistant Staphylococcus aureus Isolates by Use of Padlock Probes and Tag Microarrays J. Clin. Microbiol., March 1, 2009; 47(3): 577 - 585. [Abstract] [Full Text] [PDF]
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M. Aires-de-Sousa, B. Correia, H. de Lencastre, and and the Multilaboratory Project Collaborators Changing Patterns in Frequency of Recovery of Five Methicillin-Resistant Staphylococcus aureus Clones in Portuguese Hospitals: Surveillance over a 16-Year Period J. Clin. Microbiol., September 1, 2008; 46(9): 2912 - 2917. [Abstract] [Full Text] [PDF]
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S. Descloux, A. Rossano, and V. Perreten Characterization of New Staphylococcal Cassette Chromosome mec (SCCmec) and Topoisomerase Genes in Fluoroquinolone- and Methicillin-Resistant Staphylococcus pseudintermedius J. Clin. Microbiol., May 1, 2008; 46(5): 1818 - 1823. [Abstract] [Full Text] [PDF]
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 D. C. Oliveira, M. Santos, C. Milheirico, J. A. Carrico, S. Vinga, A. L. Oliveira, and H. de Lencastre ccrB typing tool: an online resource for staphylococci ccrB sequence typing J. Antimicrob. Chemother., April 1, 2008; 61(4): 959 - 960. [Full Text] [PDF]
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N. A. Faria, J. A. Carrico, D. C. Oliveira, M. Ramirez, and H. de Lencastre Analysis of Typing Methods for Epidemiological Surveillance of both Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains J. Clin. Microbiol., January 1, 2008; 46(1): 136 - 144. [Abstract] [Full Text] [PDF]
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Y. Cai, F. Kong, Q. Wang, Z. Tong, V. Sintchenko, X. Zeng, and G. L. Gilbert Comparison of Single- and Multilocus Sequence Typing and Toxin Gene Profiling for Characterization of Methicillin-Resistant Staphylococcus aureus J. Clin. Microbiol., October 1, 2007; 45(10): 3302 - 3308. [Abstract] [Full Text] [PDF]
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C. Milheirico, D. C. Oliveira, and H. de Lencastre Update to the Multiplex PCR Strategy for Assignment of mec Element Types in Staphylococcus aureus Antimicrob. Agents Chemother., September 1, 2007; 51(9): 3374 - 3377. [Abstract] [Full Text] [PDF]
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M. L. Amorim, N. A. Faria, D. C. Oliveira, C. Vasconcelos, J. C. Cabeda, A. C. Mendes, E. Calado, A. P. Castro, M. H. Ramos, J. M. Amorim, et al. Changes in the Clonal Nature and Antibiotic Resistance Profiles of Methicillin-Resistant Staphylococcus aureus Isolates Associated with Spread of the EMRSA-15 Clone in a Tertiary Care Portuguese Hospital J. Clin. Microbiol., September 1, 2007; 45(9): 2881 - 2888. [Abstract] [Full Text] [PDF]
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C. Milheirico, D. C. Oliveira, and H. de Lencastre Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: 'SCCmec IV multiplex' J. Antimicrob. Chemother., July 1, 2007; 60(1): 42 - 48. [Abstract] [Full Text] [PDF]
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A.-M. Hanssen and J. U. E. Sollid Multiple Staphylococcal Cassette Chromosomes and Allelic Variants of Cassette Chromosome Recombinases in Staphylococcus aureus and Coagulase-Negative Staphylococci from Norway Antimicrob. Agents Chemother., May 1, 2007; 51(5): 1671 - 1677. [Abstract] [Full Text] [PDF]
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