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Journal of Clinical Microbiology, August 2006, p. 2728-2732, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.00376-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

New Staphylococcus aureus Genotyping Method Based on Exotoxin (set) Genes

Fábio Aguiar-Alves,^1,2 Fred Medeiros,² Octavio Fernandes,^2,3 Rossiane Maria Gudziki Pereira,³ Françoise Perdreau-Remington,⁴ and Lee W. Riley^1*

Division of Infectious Diseases and Immunity, School of Public Health, University of California, Berkeley, California,¹ Department of Tropical Medicine, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil,² Diagnosticos da America S.A., Rio de Janeiro, Brazil,³ Department of Molecular Epidemiology, University of California at San Francisco, San Francisco, California⁴

Received 20 February 2006/ Returned for modification 15 March 2006/ Accepted 4 June 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
A variety of methods for genotyping Staphylococcus aureus isolates exists: the two most widely used methods are pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Here, we describe a sequence-based genotyping method based on genes encoding S. aureus superantigen-like proteins, which belong to a family of exotoxins called staphylococcal exotoxins. The sequences of PCR-amplified internal fragments of three different set genes (set2, set5, and set7) of 61 well-characterized clinical methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates and reference strains were compared. Phylogenetic analysis was performed based on single-nucleotide polymorphisms (SNP). The SNP dendrograms of the set gene sequences differentiated the 61 isolates into 22 distinct subgroups, designated exotoxin sequence types (ETST), while the standard seven-gene MLST profiles differentiated the same 61 isolates into 19 subgroups. Of the 19 different MLST subgroups, 16 corresponded to 16 distinct ETST groups. However, three MLST subgroups, ST1, ST30, and ST36, were each further separated into more than one ETST subgroup. The exotoxin-based genotyping method was able to discriminate MRSA and MSSA isolates according to their specific epidemiological characteristics. This SNP analysis of the three set genes is thus equally or more discriminatory than the seven-gene MLST method, providing a good alternative typing tool for a laboratory that has sequencing capability.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Staphylococcus aureus isolates, especially methicillin-resistant S. aureus (MRSA) isolates, are a leading cause of hospital-acquired infections (19) and are increasingly recognized as a cause of variety of community-acquired infections associated with poor hygiene, antimicrobial drug use, wounds, and crowding, as well as those with no obvious underlying medical conditions (2, 4-6, 9, 14-16, 20, 22). Community-acquired MRSA differs from hospital-associated MRSA in several ways, including epidemiologic associations, drug resistance determinants, and putative virulence factors (3, 18, 25, 28). Much of what we know about these differences came from studies based on genotypic characterizations of clinical isolates of S. aureus. Hence, simple and affordable methods to subtype S. aureus could further contribute to better understanding of the epidemiology of infections caused by this organism.

MRSA is typically genotyped by pulsed-field gel electrophoresis (PFGE), combined with staphylococcal cassette chromosome mec (SCCmec) typing, or multilocus sequence typing (MLST). While highly discriminating, PFGE is labor-intensive and has poor interlaboratory reproducibility and interpretability (8, 27). Despite its high reproducibility and standardization, MLST is relatively expensive due to the large number of targets that need to be sequenced (26).

Comparative genomic analysis of S. aureus strains revealed a high degree of interstrain variation (1, 11-13). One such variable region is called RD13, which, in strains N315 and Mu50, contains exotoxin gene-containing genomic islands SaPIn2 and SaPIm2, respectively (12). Although the RD13 locus is found in all S. aureus strains, in the eight reference strains, the RD13 region contains 5 (in strain NCTC6571) to 10 (in strain NCTC 8325) Staphylococcus exotoxin-like protein genes (12). These Staphylococcus exotoxin-like protein genes are allelic among different strains (1, 24), indicating that they are subject to selective pressures encountered during infection. This led Baba et al. to propose that S. aureus isolates may be typed by comparing allelic differences in these set genes (allotyping) (1).

Here, we describe one allotyping method based on set genes and compared it with the standard MLST method using an epidemiologically well-characterized collection of MRSA and MSSA isolates.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial isolates. Sixty-one S. aureus isolates were studied: 8 MRSA isolates and 13 MSSA clinical isolates from different neighborhoods in Rio de Janeiro, Brazil (Diagnosticos da America S.A.); 22 MRSA isolates and 11 MSSA isolates from the San Francisco Bay area, California; and 7 reference strains from the Network of Antimicrobial Resistance in Staphylococcus aureus, with known genome sequences (NCTC8325, MW2, MSSA476, MRSA252, Mu50, USA300, and N315). The clinical isolates were selected from different geographical regions and clinical manifestations to ensure strain diversity and minimize epidemiologic linkages (Table 1). Isolates from patients with invasive disease were defined as MRSA or MSSA isolates recovered from blood, urine, skin abscess, or soft tissue ulcers.

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TABLE 1. S. aureus isolates from Rio de Janeiro, Brazil, and San Francisco, Calif., and reference strains from the Network of Antimicrobial Resistance in Staphylococcus aureus used in this study

set gene allotyping. (i) Design of three exotoxin gene primers. The sequences of three genes, set2, set5, and set7, from the strains Mu50 and N315, were matched through BLAST analysis with eight publicly available S. aureus genome sequences to obtain their consensus gene sequences (COL, NCTC8325, MW2, MSSA476, MRSA252, Mu50, USA300, and N315). The COL strain lacks set5 and set7, so this strain was not included in the analysis in this study (12). The nucleotide sequence analysis (blastn) was performed with a program provided on the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/). Sequences of the set genes were aligned, and we found that set5 and set7 genes exhibit the most diversity among the different sequenced reference strains. set2 is found in all reference strains. Thus, primers were targeted against set2, set5, and set7. The primers were designed to target the most divergent region in these genes. Table 2 summarizes the primer sequences used in this study and shows expected amplicon sizes.

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TABLE 2. Synthetic oligonucleotide primers used for set allotyping by PCR

(ii) DNA extraction and PCR. S. aureus DNA was prepared for PCR by boiling. Briefly, cells were scraped off an overnight blood agar plate with a sterile loop, washed twice in 1.5 ml of 1 x Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA [pH 7.5]), resuspended in 0.5 ml of water, and immersed in boiling water for 15 min. The cell debris was pelleted by centrifugation at 8,000 x g for 5 min, and the supernatant containing the released DNA was transferred to a fresh microcentrifuge tube. PCR amplification for the three different exotoxins genes (set2, set5, and set7) was performed with the oligonucleotide primers (Sigma-Genosys, Woodlands, TX) listed in Table 2. The PCR mixture (50 µl) contained 0.3 µl primer 1 (100 pmol), 0.3 µl primer 2 (100 pmol), 0.4 µl deoxynucleoside triphosphates (10 mM), 5.0 µl of 10 x thermophilic buffer (Promega Corporation, Madison, Wis.), 0.2 µl Taq DNA polymerase (5 U/µl) (Promega Corporation, Madison, Wis.), and 46 µl of distilled water. Finally, 1.0 µl of the template DNA suspension was added to each 0.2-ml reaction tube. The DNA amplification conditions were 1 min of initial denaturation at 94°C; 32 cycles consisting of 30 s at 94°C, 30 s at 52°C, and 1 min at 72°C; and a final extension for 5 min at 72°C. The presence of a PCR product was determined by electrophoresis of 6 µl of the reaction product in a 1% agarose gel, in Tris-acetate-EDTA buffer (Tris-acetate-2 mM EDTA [pH 8.3]) at 110 V, visualized under UV light by the Gel Doc 1000 system (Bio-Rad). The size of the PCR products was determined by comparison to a 100-bp DNA molecular weight marker (Promega Corporation, Madison, Wis.). The primers and PCR cycling conditions used for MLST analysis were the same as those described by Enright et al. (10) and are updated on the S. aureus MLST website (http://www.mlst.net).

(iii) Sequence analysis. The PCR products were purified by the QIAquick PCR purification kit (QIAGEN Sciences, Maryland) and sequenced in both directions. Sequencing was performed at the University of California—Berkeley sequencing facility. The facility runs a 25-cycle sequencing reaction with the following program: 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 min. The sizes of the amplicons for set2, set5, and set7 were 499 bp, 511 bp, and 573 bp, respectively (Table 2). The sequences used to generate a phylogenetic tree consisted of a region shorter than the total amplicon sizes (484 bp, 414 bp and 491 bp for set2, set5, and set7, respectively) (Fig. 1). All sequences reported by the sequencing facility were reviewed visually, and analysis was performed by Seqman and EditSeq (DNA Star/Lasergene, Madison, WI).

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FIG. 1. Phylogenetic analysis based on concatenated sequences of the three set genes of S. aureus. Each strain is abbreviated with the isolate designation and place of isolation, followed by the MLST (ST) designation. *, MLST subgroup with more than one ETST genotype.

(iv) Analysis of gene sequences and genetic relationship among isolates. As is done with MLST nomenclature, for each set gene locus, the sequences obtained from all 61 isolates were compared and any new set gene sequence was assigned a numerical allotype designation (10). Then, strains with an identical set of allotype numbers assigned to set2, set5, and set7 were classified to belong to the same exotoxin sequence type (ETST) and given a numerical designation (Table 1).

PFGE. All of the MRSA isolates (n = 30) were also typed by PFGE, according to the method described by Charlesbois et al. (7). The extracted DNA was digested by SmaI endonuclease and the DNA fragments were resolved in 1% agarose in 0.5x Tris-borate-EDTA buffer for 22 h at 14°C and 6 V/cm in a CHEF DR-II apparatus (Bio-Rad, Hercules, CA).

SCCmec typing. SCCmec typing was performed on all the MRSA isolates by the PCR method described by Oliveira and de Lencastre. (21). Briefly, SCCmec type was determined by multiplex PCR amplifications with the following parameters: predenaturation for 4 min at 94°C; 30 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min; postextension for 4 min at 72°C; and soaking at 4°C. PCR products (10 µl) were resolved in a 2% MetaPhor (Cambrex Bioscience, Rockland, ME) agarose gel in 0.5 x Tris-borate-EDTA buffer (Bio-Rad, Hercules, CA) at 100 V and visualized with ethidium bromide.

The phylogenetic trees based on the concatenated sequences of the three gene sequences were reconstructed by neighbor-joining algorithm based on the Kimura 2 parameter, calculated by MEGA, version 3.0 (17). One concatenated sequence from each ETST group was included in the tree. Estimates of the sampling variance were made by bootstrap analysis with 1,000 repetitions.

The work in this study was performed at the Division of Infectious Diseases, School of Public Health, University of California, Berkeley, Calif.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Genotype analysis. By the seven-gene standard MLST genotyping method, the 61 isolates were differentiated into 19 different genotypes (Table 1). Sixteen of them corresponded to 16 distinct ETST genotypes. However, three MLST subgroups, ST1, ST30, and ST36, were each further separated into more than one genotype. The ST1 subgroup containing nine isolates was separated into three ETST subgroups, ETST6, ETST7, and ETST15 (Table 1). The ST30 subgroup containing four isolates was composed of ETST14 and ETST22. The ST36 subgroup containing four isolates was separated into ETST10 and ETST14. The ST36 isolates showed the most heterologous set7 gene sequence (10%) among all isolates tested. Even with only one set gene (set5 or set7), the 61 isolates were separated into 16 subgroups (Table 1).

The neighbor-joining tree showed a good correlation with results obtained by the exotoxin allotyping and MLST. However, the exotoxin allotyping method showed a higher number of allotypes than did MLST for the 61 isolates tested. The MLST method generated 19 genotypes, while the exotoxin allotyping generated 22 genotypes (Fig. 1). The nucleotide sequences of all of the isolates are shown in the supplemental data.

PFGE. The PFGE analysis of 30 MRSA isolates differentiated them into 14 genotypes. ETST differentiated the same set of MRSA isolates into 10 genotypes, while the MLST generated 8 distinct subtypes.

SCCmec. The SCCmec typing of 30 MRSA isolates generated three distinct types. The combination of SCCmec with MLST differentiated these MRSA isolates into eight subtypes.

Epidemiological analysis. The 13 MSSA isolates from Rio de Janeiro were separated into seven MLST groups and seven corresponding ETST subgroups. All eight MRSA isolates from Rio de Janeiro belonged to one distinct MLST group and one ETST group. Thus, there was greater heterogeneity among MSSA isolates than MRSA isolates from Rio de Janeiro. The 22 MRSA isolates from San Francisco were separated into 7 MLST subgroups and 10 ETST subgroups.

Of 21 Brazilian clinical S. aureus isolates, 10 came from nasal swabs, 10 came from invasive disease sources, and 1 had an unknown source (Table 3). MLST and ETST each separated the nasal colonization isolates into six corresponding subgroups. Two of the six nasal MSSA isolates belonged to the same ST and ETST genotypes (ETST2 [ST5] and ETST8 [ST74]) found among 10 invasive disease isolates. The most common genotype among the Brazilian MSSA isolates was ETST6 (ST1), accounting for 3 of 13 Brazilian MSSA isolates, and they were isolated only from invasive disease sources. On the other hand, there was only one genotype among the Brazilian MRSA isolates (ETST1 [ST239]), and they were found in both colonization and invasive disease sources.

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TABLE 3. Comparison of exotoxin (ETST) and MLST sequence types of the S. aureus isolates by resistance profile (colonization and invasive disease source)

Among the San Francisco isolates, 12 were from colonization sources and 21 were from invasive disease sources. All of the genotypes among the MRSA colonization isolates were shared among MRSA invasive disease isolates (Table 3). ETST2 (ST5) and ETST6 (ST1) genotypes were found among both United States and Brazilian isolates.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The multiple set genes in S. aureus and their sequence heterogeneity provided an opportunity to target them for genotyping clinical isolates of S. aureus. The discrimination of 61 isolates based on three exotoxin genes agreed well with that obtained by the standard MLST genotyping method. Sequences of the isolates in the exotoxin gene-based phylogenetic tree clustered into either identical or more discriminatory groupings than those based on MLST. However, when a subset of the isolates (MRSA) was tested by PFGE, the three-exotoxin-gene-based allotyping method was not as discriminating as the PFGE-typing method. The 30 MRSA isolates were comprised of 14 distinct PFGE types, compared to 10 ETST genotypes.

Nevertheless, since the set gene allotyping is based on only three gene targets, it may serve as an affordable and quick typing method for epidemiological assessment of S. aureus infections, such as during outbreak investigations or hospital- or community-based surveillance, especially if the outbreak or hospital or community infections were caused by one of the strains (ST1, ST30, or ST36) found to be further separable by ETST. This allotyping method was indeed able to identify and distinguish recognized international clones of MRSA (ST1 and ST36) and identify common genotypes in two widely separate geographic regions. Thus, the method shows both epidemiologic and typing system concordance with the established MLST method (23).

 
We thank Jose Carlos Amorin and Tanya Glaser for technical support and Amy White for logistical support. Binh an Diep is thanked for careful help in the sequence analysis and advice.

This work was supported by the Brazilian Ministry of Health CAPES Program and the NIH's Fogarty International Center (grant number NIH/D43 TW006563).

 
* Corresponding author. Mailing address: Division of Infectious Diseases, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720. Phone: (510) 642-9200. Fax: (510) 642-6350. E-mail: lwriley{at}berkeley.edu.

Supplemental material for this article may be found at http://jcm.asm.org/.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, August 2006, p. 2728-2732, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.00376-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Aguiar-Alves, F. Articles by Riley, L. W. Search for Related Content PubMed PubMed Citation Articles by Aguiar-Alves, F. Articles by Riley, L. W.