PCR-based ribotyping of Staphylococcus aureus

Genotyping techniques are valuable tools for the epidemiologic study of Staphylococcus aureus infections in the hospital setting. Pulsedfield gel electrophoresis (PFGE) is the current method of choice for S. aureus strain typing. However, the method is laborious and requires expensive equipment. In the present study, we evaluated the natural polymorphism of the genomic 16S-23S rRNA region for genotyping purpose, by PCR-based ribotyping. Three primer pairs were tested to determine the size of amplicons produced and to obtain better discrimination with agar gel electrophoresis and ethidium bromide staining. The resolution of the typing system was determined using sets of bacteria obtained from clinical specimens from a large tertiary care hospital. These included DNA from three samples obtained from a bacteremic patient, six strains with known and diverse PGFE patterns, and 88 strains collected over a 3-month period in the same hospital. Amplification patterns obtained from samples from the same patient were identical, and PFGE from samples known to be different produced three genotypes. Amplification of DNA from 61 methicillinresistant isolates produced only one pattern. Methicillin-sensitive strains yielded a diversity of patterns, pointing to a true polyclonal distribution throughout the hospital (22 unique patterns from 27 strains). Computer-based software can be used to differentiate among identifiable strains, given the low number of bands and good characterization of PCR products. PCR-based ribotyping can be a useful technique for genotyping methicillin-sensitive S. aureus strains, but is of limited value for methicillin-resistant strains. Correspondence


Introduction
Staphylococcus aureus is a major pathogen in both community and hospital settings (1).Phenotyping and genotyping techniques are important for tracing the epidemiology of S. aureus infections.Phenotyping techniques, such as antibiotic susceptibility and phage typing, are based on unstable characteristics and can show poor reproducibility.Genotyping techniques are based on chromosomal or plasmid material and can provide better results in terms of reproducibility and strain differentiation.
Pulsed-field gel electrophoresis (PFGE) is considered the genotyping technique of choice by reference laboratories such as the CDC (2), but this methodology is labor-intensive, time-consuming, and requires expensive equipment.In contrast, PCR-based genotyping technologies are simple and fast.The primers used in the reactions can be arbitrary, i.e., not complementary to any specific region (RAPD), or can be complementary to known sequences of the organism's genome (REP-PCR).
The amplification of the genomic 16S-23S rRNA spacer region was first described as a reliable technique for typing Pseudomonas cepacia, Staphylococcus aureus, Enterococcus faecium, Escherichia coli, and Enterobacter sp (3).In the present study we explored the natural polymorphism of the genomic 16S-23S rRNA region from S. aureus as a genotyping tool.Three primer pairs were tested to check for the size of amplicons produced, and best visualization upon agar gel electrophoresis and ethidium bromide staining, typeability, and discriminatory power were determined using a collection of bacteria obtained from clinical specimens from Hospital Municipal Dr. Mário Gatti, a 200-bed tertiary care hospital in Campinas, São Paulo State, Brazil, during a 3-month period.

Susceptibility tests
Methicillin resistance was determined by the NCCLS protocol (7,8) in terms of the width of the inhibition zone produced by a 5 µg oxacillin disk on a 5% NaCl-supplemented Mueller-Hinton agar plate.

Electrophoresis
PCR products were run on 2% agarose gels (85 V at room temperature).Molecular sizes were determined based on a 100-bp ladder molecular mass marker.Kodak Digital Science 1D Image Analysis software was used to estimate sizes and hierarchical analyses were performed using an appropriate statistical software (SPSS 7.5).

Results
Amplification profiles of three isolates taken randomly using the three sets of primer pairs (A,B,C) are shown in Figure 1 (lanes 1 to 9).The amplicons obtained with primer pair C (6) corresponded to the smallest fragments which were best resolved under the electrophoretic conditions used.This primer pair was subsequently used in all reactions.
Isolates from the same patient with acute endocarditis (A1, A2, A3) produced a single pattern that can be seen in Figure 1 (lanes 10 to 12).Initial and subcultured ATCC iso-lates, designated B1, B2, and B3 (1st, 10th and 30th passage), yielded the same amplification profile, as can be seen in Figure 2.This figure also shows the amplicons ob-

PCR ribotyping of Staphylococcus aureus
tained from six isolates with different known PFGE profiles (C1 to C6).Three distinct amplification patterns found.Twenty-seven of 88 isolates (37%) collected in the hospital were methicillin-sensitive.DNA amplification of these isolates yielded three to nine bands per isolate with molecular weights ranging from 360 to 733 bp.All isolates were typeable.Methicillinsensitive isolates showed 22 different patterns, some of which are illustrated in Figure 3. Hierarchical analysis showed a similarity ranging from 45 to 100% (simple matching).
The amplification of the methicillin-resistant S. aureus (MRSA) isolates produced the same band pattern as shown in Figure 4.The molecular weights ranged from 431 to 644 bp.All isolates were also typeable.

Discussion
Staphylococcus aureus is a major nosocomial pathogen that causes a range of diseases, including endocarditis, osteomyelitis, pneumonia, toxic-shock syndrome, food poisoning, carbuncles, and boils.In the early 1950's, acquisition and spread of beta-lactamase-producing plasmids thwarted the effectiveness of penicillin for treating S. aureus infections.In 1959, methicillin, a synthetic penicillin, was introduced.However, by 1960, MRSA strains were identified as a direct result of S. aureus acquiring the mecA gene, which encodes for an altered penicillin-binding protein gene (PBP2a).
Bacterial strain typing, or subspeciation, has become an important clinical tool to investigate suspected outbreaks and to evaluate nosocomial transmission.
Previous studies have shown that MRSA strains are the result of a recent evolutionary change linked to the acquisition of the mecA gene (9).Several typing methods have been used to confirm this hypothesis, such as mecA RFLP and Tn554 blot-hybridization.
PCR ribotyping is a fast and reliable typing method with good typeability and repro-ducibility for several bacterial species, such as P. cepacia, E. coli and Enterobacter sp.This approach targets the 16S-23S rRNA region which is polymorphic and repetitive in the genome of such bacteria.Earlier reports have shown that S. aureus can be typed with the use of this technique.In the present study, we evaluated the basic characteristics of this methodology, i.e., typeability, reproducibility and discriminatory power using isolates collected from a general tertiary care hospital.
When typing methicillin-sensitive S. aureus this technique yielded amplification patterns consistent with good attributes such as typeability, reproducibility, and discriminatory power.However, amplification of DNA from MRSA produced a pattern with low discriminatory power.In fact, strains from different PFGE groups could not be distinguished with the use of PCR ribotyping.The short interval (3 months) for the collection of isolates in this study could explain these findings.Another possibility is the existence of just one clone of SAMR largely disseminated in the hospital.
Otherwise, these findings corroborate others that point to a recent evolutionary change of this microorganism dating back to the early 1960's, when they probably acquired genetic material that encodes for resistance to methicillin (mecA gene).This acquisition had a major role in the complexity of the S. aureus genome, since many integration and deletion events occur in this resistance island.The recent description of the whole genome sequences of MRSA and vancomycin-resistant S. aureus by Japanese researchers certainly contributed to this hypothesis (10).
MRSA genotyping still poses a challenge to several research teams.The use of a single method such as PFGE, Southern blot ribotyping, Tn554 and mecA hybridization, and others recently described, like spa-and coatyping, which are based on sequencing, has yielded disappointing results (11).The com-bination of two or more of these methods seems to be the best approach to establish the clonal nature of organisms from hospital settings (12).

Figure 1 .
Figure 1.Electrophoretic patterns of PCR ribotypes of three S. aureus strains obtained with the three different primer pairs: lanes 1 to 3 with primer pair A, described by Gürtler and Stanisich (5), lanes 4 to 6 with primers B, described by Kostman et al. (3), and lanes 7 to 9 with primers C, described by Jensen et al. (6).Lanes 10 to 12 show PCR ribotypes from three S. aureus isolates collected from the same patient on different days.Lane M: 100-bp ladder.