Introduction

Staphylococcus aureus is one of the most common causes of nosocomial and community-acquired infections worldwide [2, 6]. Since the emergence of methicillin-resistant strains of S. aureus (MRSA), the glycopeptide vancomycin has been the antibiotic of choice for serious MRSA infections [5]. In 1996, a S. aureus strain with intermediate resistance to vancomycin (VISA) (vancomycin MIC= 8μg /ml) was first isolated from a patient in Japan [10]. Shortly afterward, VISA strains were isolated in USA, Europe and other Asian countries [9, 24], arousing considerable concern regarding the emergence of S. aureus strains for which there will be no effective therapy. Characterization of these VISA strains indicates that the mechanisms of resistance are complex and involve changes in cell wall content and composition [3, 22]. In addition, the appearance of a powerful glycopeptide resistance mechanism among clinical isolates of enterococci, associated with the vanA and vanB gene clusters [4, 13, 15, 21], and the demonstration of experimental transfer of the vanA gene cluster from Enterococcus faecalis to S. aureus [16] have raised fears about the occurrence of such genetic transfer in clinical isolates of methicillin-resistant staphylococci. This type of transfer has already been reported in clinical isolates of Streptococcus bovis (vanB) [20] and Bacillus circulans (vanA) [12].

In June 2002, the world's first reported clinical infection due to S. aureus with high resistance to vancomycin (VRSA) (vancomycin MIC>128 μg/ml) was diagnosed in a patient in the USA [23]. A second isolate was reported in October of the same year [14]. These isolates contain the vanA genes from enterococci and the methicillin-resistance gene mecA. The possible emergence and dissemination of VRSA strains is a serious health threat and makes it absolutely necessary to optimize prevention strategies and fast detection methods.

In the present study, we developed a multiplex PCR protocol that allows the simultaneous detection of the two most widespread, transferable vancomycin resistance genes, vanA and vanB, and the methicillin resistance gene mecA, as well as the identification of S. aureus (femB). In addition, since mupirocin has been suggested as a reasonable adjuvant agent to prevent staphylococcal infections, and high resistance is already widespread [5, 18, 19], detection of the ileS-2 gene was also included.

Material and methods

Bacterial strains and DNA extraction

As there were no vancomycin-resitant S. aureus isolates available for our study, mixtures of enterococcal and staphylococcal colonies that harbored the different resistance markers were used to show that these genes could be detected simultaneously. Bacterial strains and susceptibility testing methods were previously described [17, 18]. The following strains were used: E. faecalis V583 (vancomycin-resistant, VanB), E. faecalis ATCC 29212 (vancomycin-susceptible), E. faecium BM4147 (vancomycin-resistant, VanA), S. aureus ATCC 29213 (methicillin- and mupirocin-susceptible), S. aureus SEIMC (methicillin-resistant and mupirocin-susceptible), S. aureus isolate 242 (methicillin- and mupirocin-resistant) and Staphylococcus epidermidis 8859-65 (methicillin- and mupirocin-resistant). For DNA extraction, bacteria were grown overnight on BHI agar plates. One colony from a Staphylococcus strain and one from a Enterococcus strain were mixed and resuspended in 25 μl of sterile distilled water, and heated at 100 °C for 15 min. A 5-μl aliquot of this suspension was directly used as template for PCR amplification.

Simultaneous detection of vanA, vanB, femB, mecA and ileS-2 by PCR

The primers used in this study are listed in Table 1. Multiplex PCR assays were carried out with the DNA suspension obtained from the Staphylococcus/Enterococcus mixtures. We ensured that our PCR protocol was adequate for the individual amplification of each DNA fragment: vanB (1,145 bp), vanA (732 bp), femB (651 bp), ileS-2 (456 bp) and mecA (310 bp) (not shown). Multiplex PCR was then done using the following mixtures: E. faecalis V583 (vanB)/S. aureus 242 (femB, mecA, ileS-2), E. faecium BM4147 (vanA)/S. aureus 242 (femB, mecA, ileS-2), E. faecalis ATCC 29212/S. aureus ATCC 29213 (femB), E. faecalis V583 (vanB)/S. aureus ATCC 29213 (femB), E. faecium BM4147 (vanA)/S. aureus ATCC 29213 (femB), E. faecalis V583 (vanB)/S. aureus SEIMC (femB, mecA), E. faecium BM4147 (vanA)/S. aureus SEIMC (femB, mecA), E. faecalis V583 (vanB)/S. epidermidis (mecA, ileS-2), E. faecium BM4147 (vanA)/S. epidermidis (mecA, ileS-2). The PCR conditions used were modifications of protocols previously described by us [17, 18]. A 5-µl aliquot of the DNA suspension was added to 28.5 μl of PCR mixture consisting of 1× reaction buffer [16 mM (NH4)2SO4, 67 mM Tris-HCl (pH 8.8)], 0.2 mM concentration of each of the four dNTPs (Promega Corp., Madison, Wis.), 3 mM MgCl2, 1.5 μM of each femB primer, 0.5 μM of each vanA and vanB primer, 0.4 μM of each ileS-2 primer and 0.4 μM of each mecA primer, and 1.25 U of Taq DNA polymerase (Bioline, London, UK). The femB primers were used to identify S. aureus strains, and the mecA, ileS-2, vanA and vanB primers to detect the respective resistance markers. In order to reduce the formation of nonspecific extension products, the protocol included a "hot-start". DNA amplification was carried out in a GeneAmp PCR system 2700 (Applied Biosystems, Foster City, Calif., USA) with the following thermal cycling profile: an initial denaturation step at 94 °C for 5 min was followed by 10 cycles of amplification (denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 45 s), and another 25 cycles of amplification (denaturation at 94 °C for 30 s, annealing at 50 °C for 45 s, and extension at 72 °C for 2 min), ending with a final extension step at 72 °C for 10 min. After PCR amplification, 5 μl were removed and subjected to agarose gel electrophoresis (2% agarose, 1× TBE, 100 V) to estimate the sizes of the amplification products by comparison with a 100-bp DNA ladder (Roche Diagnostics, Mannheim, Germany) .The gel was stained with ethidium bromide and the amplicons were visualized under a UV light.

Table 1. PCR primers used in this study
Full size table

Results and discussion

The reaction conditions for the multiplex PCR assay were optimized to ensure that each target sequence was satisfactorily amplified. The primers used in this study differ in annealing temperatures, which increased the possibility of occurrence of unwanted bands that originated from nonspecific amplification. Therefore, two rounds of amplification with different annealing temperatures were carried out. Multiplex PCR with targets that differ widely in size often favors amplification of the shorter target over the longer ones [18]. Thus, to ensure amplification of each target, different primer concentrations, template DNA preparations, and MgCl2 concentrations were assayed, and those described above were chosen.

Figure 1 shows the results obtained with the optimized multiplex PCR assay as described in Material and methods; amplification of vanA, vanB, femB, ileS-2 and mecA targets produced distinct bands corresponding to their respective molecular size that were easily recognizable. vanA or vanB could be detected in mixtures containing femB (lanes 4 and 5), femB and mecA (lanes 6 and 7), and femB, mecA, and ileS-2 (lanes 1 and 2). vanA, vanB, ileS-2 and mecA were also simultaneously detected in the presence of the S. epidermidis (femB–) DNA. No amplification of the antibiotic resistance markers was observed in the control without DNA (lane 10) or in the mixture containing two susceptible strains (E. faecalis ATCC 29212 and S. aureus ATCC 29213) (lane 3). The entire protocol for the multiplex PCR assay, including the DNA extraction and electrophoresis, can be completed in less than 5 h.

Fig. 1.
figure 1

Agarose gel electrophoresis of multiplex PCR amplification products from different enterococcal/staphylococcal mixes. Lanes: M 100-bp DNA ladder (Roche Diagnostics, Mannheim, Germany), 1 Enterococcus faecalis V583 (vanB1)/Staphylococcus aureus 242 (femB, mecA, ileS-2), 2 Enterococcus faecium BM4147 (vanA)/S. aureus 242 (femB, mecA, ileS-2), 3 E. faecalis ATCC 29212/S. aureus ATCC 29213 (femB), 4 E. faecalis V583 (vanB1)/S. aureus ATCC 29213 (femB), 5 E. faecium BM4147 (vanA)/S. aureus ATCC 29213 (femB), 6 E. faecalis V583 (vanB1)/S. aureus SEIMC (femB, mecA), 7 E. faecium BM4147 (vanA)/S. aureus SEIMC (femB, mecA), 8 E. faecalis V583 (vanB1)/S. epidermidis (mecA, ileS-2), 9 E. faecium BM4147 (vanA)/S. epidermidis (mecA, ileS-2), 10 control without a DNA template

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Since vancomycin is frequently the drug of choice for the treatment of infections caused by now common MRSA, the recent appearance of a fully vancomycin-resistant strain of S. aureus has confronted us with the possible existence of non-tractable infections. The protocol described here could be used to facilitate the detection and identification of S. aureus or MRSA strains carrying vanA or vanB.