Laboratory diagnosis of oxacillin resistance in Staphylococcus aureus by a multiplex-polymerase chain reaction assay

DIAGN MICROBIOLINFECTDIS 1994;19:25-31

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ANTIMICROBIAL SUSCEPTIBILITYSTUDIES

Laboratory Diagnosis of Oxacillin Resistance in Staphylococcus aureus by a Multiplex-Polymerase Chain Reaction Assay Gilles Zambardi, Marie Elisabeth Reverdy, St6phane Bland, Mich61e Bes, Jean Freney, and Jean Fleurette

A polymerase chain reaction (PCR) test was developed in which the mecA gene responsible for the intrinsic resistance to oxacillin in Staphylococcus aureus and the gyrA gene, always present in this species, were amplified in one operation. Among the 468 clinical isolates tested, the results obtained for 454 of the isolates (97%) were consistent with those of MIC determination. Discrepant results were noted for strains with low-level oxacillin resistance (MICs, 4-8 ~g/ml) and mecA gene negative. For these strains, susceptibility to oxacillin was restored in the presence of a B-lactamase inhibitor, which sug-

gested a resistance by penicillinase hyperproduction. In contrast, all of the high-level resistant strains (MICs, >8 ~g/ml) carried the mecA gene. The presence of this gene has frequently been associated with resistance to gentamicin, tetracycline, erythromycin, lincomycin, and pefloxacin. The PCR assay described in this study can be accomplished with ease and total confidence in the clinical microbiologic laboratory for a rapid and effective establishment of antistaphylococcal chemotherapy.

INTRODUCTION

terized (Tomasz et al., 1989; Massida et al., 1992), but their real incidence is not well defined. In the clinical laboratory, detection of strains truly resistant to oxacillin and their differentiation with the penicillinase-hyperproducing strains are slow and sometimes difficult. As the antimicrobial therapy may vary (Massanari et al., 1988; Woods and Yam, 1988; Chambers et al., 1989; Hirano and Bayer, 1991; Pefanis et al., 1993), however, a correct diagnosis of the resistance mechanism is important. In addition, the immediate establishment of an effective and bactericidal therapy is essential to improve the prognosis of patients with S. aureus infections. Consequently, numerous studies have been reported concerning the evaluation of rapid methods for the diagnosis of intrinsic resistance to oxacillin. Among them, DNA probes (Chambers et al., 1989; Archer and Pennel, 1990; De Lencastre et al., 1991) and polymerase chain reaction (PCR) technologies (Murakami et al., 1991; Tokue et al., 1992; Ubukata et al., 1992; Onal et al., 1992) have been successfully used for the detection of the mecA gene. However, these proposed genotyping methods are

To date, two main mechanisms of resistance of Staphylococcus aureus to oxacillin are well documented. One is mediated by an additional lowaffinity penicillin-binding protein (PBP2a) encoded by the mecA gene (Brown and Reynolds, 1980; Hartman and Tomasz, 1984; Ubukata et al., 1985). The other mechanism is associated with a high-level penicillinase production (McDougal and Thornsberry, 1986). More recently, two other (low level) oxacillin resistance mechanisms have been charac-

From the Department of Studies and Research of Medical Bacteriology(EA1655), Alexis Carrel Facultyof Medicine, Lyons, France. Address reprint requests to Dr. G. Zambardi, Centre National de R6f6rencedes Staphylocoques,Facult~ de M6decineAlexis Carrel, Rue GuillaumeParadin, 69372 Lyon Cedex 08, France. Received 22 October 1993; revised and accepted 10 March 1994. © 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/94/$7.00

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not always applicable in a clinical microbiology laboratory as a routine susceptibility test because of the time-consuming processes for staphylococcal DNA extraction or DNA hybridization. In the present study, we report the results obtained with a rapid (<5 h) and reliable multiplex-PCR protocol for the detection of the mecA gene in 468 clinical isolates of S. aureus, by using an internal control for DNA extraction and amplification. The contribution of this procedure is discussed in relation to the conventional phenotyping tests.

MATERIALS A N D METHODS Bacterial Strains and Susceptibility to Antibiotics A total of 474 S. aureus strains isolated in different hospitals in France over a 12-year period were studied. These strains were identified using conventional procedures: clumping-factor, coagulase, heatstable DNase, and, if n e c e s s a r y , biochemical characters (ID 32 Staph; BioM6rieux, Marcy-l'Etoile, France). The minimum inhibitory concentrations (MICs) of oxacillin were determined by a standard procedure using serial dilutions in Mueller-Hinton agar supplemented with 4% sodium chloride. Bacteria were inoculated at a final bacterial density of 104 colony-forming units per surface spot and incubated at 35°C for 24 h before MICs were determined. Concomitantly, the semiautomated API ATB plus (bioM6rieux) method was used for the MIC determination of oxacillin, ampicillin-sulbactam (8 ~g/ml), and oxacillin-sulbactam (8 ~g/ml) in MuellerHinton broth containing 0%, 2%, or 5% NaC1. Susceptibility testing of kanamycin, tobramycin, gentamicin, erythromycin, lincomycin, pristinamycin, tetracycline, minocycline, pefloxacin, rifampin, fosfomycin, trimethoprim-sulfamethoxazol (TMPSMZ), chloramphenicol, fusidic acid, and vancomycin was established by the MIC determination in Mueller-Hinton agar following the instructions of the French Society for Microbiology. Staphylococcus aureus American Type Culture Collection 29213 was included in the study as reference strain.

Phage Typing Strains were phage typed according to the method of Blair and Williams (1961) using the International Basic Set of 23 typing bacteriophages for S. aureus (Marples and Van Leeuwen, 1987): 29, 52, 52A, 79, and 80 (group I); 3A, 3C, 55, and 71 (group II); 6, 42E, 47, 53, 54, 75, 77, 83A, 84, and 85 (group III); 94 and 96 (group V); and 81 and 95.

G. Zambardi et al.

Rapid D N A Extraction Procedure Two bacterial colonies were suspended from agar plates in 100 ~l of Tris-EDTA buffer (Tris-HC1, 10 raM; and EDTA, 1 mM) containing 75 ~g/ml of recombinant lysostaphin (Applied Microbiology, New York City, NY, USA). The bacterial lysis was effected automatically in a DNA thermal cycler (Hybaid TR-1, Teddington, UK) at 37°C for 30 rain and then at 99°C for a further 30 rain. The cells were centrifuged at 13,000 rpm for 5 rain and the supernatant was recovered. Part of this (5 Fxl) was used directly for the PCR assay.

Multiplex-PCR Assays The extracted DNA was amplified by PCR in 50 FL1 reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 2.5 mM MgC12, 0.01% gelatin, 200 ~M of each dNTP, 12.5 pmol and 50 pmol of primers allowing the gyrA and mecA sequences, respectively, 1.25 units of Taq DNA polymerase (PerkinElmer Cetus, Norwalk, CT, USA), and 5 ~1 of sample. The sequences of the primers used have been described by Murakami et al. (1991) and Goswitz et al. (1992). The two target genes were coamplified with the use of a programmable thermal cycler (Hybaid TR-1). The following parameters were used: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and primer extension at 72°C for 1 min, with a total of 30 cycles. A 10-~1 aliquot of amplified product was loaded onto 1.5% agarose gel containing 1 ~g/ml ethidium bromide. The 533-bp (mecA) and 280-bp (gyrA) amplified DNA fragments were separated by electrophoresis and visualized under ultraviolet light (Figure 1).

RESULTS Amplification of mecA and gyrA from S. aureus Figure 1 shows representative data of the PCR assay. The gyrA DNA sequence, which is always detected in the S. aureus genome, acts as an internal control, allowing the quality of the DNA extraction and amplification for each sample to be affirmed unequivocally. With the extraction protocol proposed, only six (1.3%) of the 474 strains consistently failed to release DNA, showing the efficiency of the method.

Concordance Analysis Between the Presence of the mecA Gene and in vitro Resistance to Oxacillin A total of 468 S. aureus clinical isolates covering a wide range of MICs for oxacillin were examined.

Laboratory Diagnosis of Oxacillin Resistance

27

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mecA

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FIGURE 1 Agarose gel analysis of amplified 280-bp (gyrA) and 533-bp (mecA) DNA fragments. Polymerase chain reaction was performed with a lysate of oxacillin-susceptible (S) or -resistant (R) Staphylococcus as a template: (lanes A-C and E-G) intrinsic-resistant [minimum inhibitory concentration (MIC), >16~,g/ml] strains A890466, A890550, A890573, A890639, A890643, and 6477; (lane H) borderline-resistant (MIC, 4 p.g/ ml) strain A890258; (lanes I-N) intrinsic-resistant (MIC, >16 ~g/ml) strains A880503, A880572, A890027, A890071, A890093, and A890134; (lanes O-T) borderline-resistant (MIC, 4 ~g/ml) strains 6740, 6351, A870021, A870111, A870113, and A880359; and (lane D) negative control. The lysis method was not efficient for strain A890027 (lane L) in which the 280-bp fragment is not visible. Results of the PCR technology were compared with the phenotypic susceptibility of the strains to oxacillin (Figure 2). With the phenotypic method, 351 strains were classified as oxacillin susceptible (MIC, <4 ~g/ml) and 117 as resistant, according to the French Society for Microbiology (Acar et al., 1992). The results of the PCR assay were in accordance with the those of the phenotypic method for 97% of

the strains (Table 1). Discrepant results were noted for 14 strains (3%). All of these strains that showed a low-level resistance to oxacillin (MICs, 4 or 8 ~g/ ml) were mecA gene negative. For these strains, the susceptibility to oxacillin or ampicillin was restored in the presence of sulbactam (8 p,g/ml). This suggested a mechanism of resistance to oxacillin by p-lactamase hyperproduction.

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FIGURE 2 Minimum inhibitory concentration (MIC) distribution for oxacillin of Staphylococcus aureus in relation to the presence of the mecA gene.

28

G. Zambardi et al.

TABLE 1

Correlation Between Oxacillin Resistance of Staphylococcus aureus and mecA Gene Polymerase Chain Reaction Result Number of Strains

Characteristic

Oxacillin Susceptible"

Oxacillin Resistant"

mecA positive mecA negative

0 351

103 14

aMinimum inhibitory concentrations were determined by agar dilution assay in the presence of 4% sodium chloride.

I n f l u e n c e of Salt C o n c e n t r a t i o n on the R e s i s t a n c e Level to O x a c i l l i n for the mecA-Gene-Negative Strains

The results of the MICs determined in MuellerHinton broth supplemented with different concencentrations of NaCI (0, 2%, and 5%) were compared with the search for the mecA gene by PCR. Increasing the salt concentration from 0 to 2% led to the detection of all the mecA-gene-positive strains. In contrast, 0, 10, and 47 mecA-negative strains appeared resistant to oxacillin with 0, 2%, and 5% NaC1, respectively (Figure 3). Using the nitrocephin procedure, all of these 47 strains were 13-1actamase producers. The influence of the salt concentration on oxacillin resistance was particularly evident for the strains belonging to phage group V (Figure 3). The phage-typing results for all of the lysotypable strains are presented in Figure 4. When tested with 5% NaC1, 40 (85%) of 47 mecA-gene-negative but oxacillin-resistant (Oxa~; mecA ) l y s o t y p a b l e strains belonged to phage group V. In contrast, the frequency of this phage group was only 19% for the oxacillin-susceptible strains (OxaS; mecA ) and 0 for those presenting an intrinsic resistance to oxacillin (OxaR; mecA +).

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A n t i b i o t i c Resistance A s s o c i a t e d w i t h the Presence of the mecA G e n e (Figure 5)

For all of the antibiotics examined, the mecA-genepositive strains were more resistant than the mecAnegative strains. The presence of the mecA gene was frequently associated with resistance to kanamycin (100%), tobramycin (95%), gentamicin (93%), erythromycin (90%), tetracycline (86%), lincomycin (78%), and pefloxacin (71%). On the other hand, agents such as minocycline, rifampicin, fosfomycin, sulfamethoxazol-trimethoprim (TMP-SMZ), chloramphenicol, fusidic acid, pristinamycin, and vancomycin were active on 76%-100% of these strains (Figure 5).

DISCUSSION The objective of this study was the development of a simple method for a reliable diagnosis of intrinsic resistance of S. aureus to isoxazolyl penicillins. Detection of the mecA gene by the PCR procedure described here is feasible in clinical microbiology laboratories in <5 h. The cost of the reagent for one PCR test, including sample preparation and sample analysis, was estimated at just under $2.5 (US). The results obtained were in agreement for 97% of the strains when compared with MIC results. Discrepant results involved only strains for which a synergism between penicillins and f}-lactamase inhibitor was noted. As these strains remained susceptible to other antistaphylococcal agents (such as gentamicin), in contrast to those harboring the mecA gene, this suggested a resistance mechanism by penicillinase hyperproduction. In our study, no mecA-positive strain appeared falsely susceptible to oxacillin, in contrast to other reports (Murkami et al., 1991; Tokue et al., 1992; Ubukata et al., 1992; l[Jnal et al., 1992). Only the PCR technology offers a rapid and reliable differentiation between heterogeneous intrinsic resistance (mecA-positive strains)

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FIGURE 3 Importance of NaC1 concentration for the detection of penicillinase-hyperproducing strains.

Laboratory Diagnosis of Oxacillin Resistance

29

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FIGURE 4 Phage typing of the Staphylococcus aureus strains. and extrinsic resistance (mecA-negative strains) to oxacillin. Indeed, conventional detection of the extrinsic resistance is based on the MIC determination to oxacillin (MICs, >0.25 p~g/ml and <8 ~g/ml), the synergy between penicillins and ~-lactamase inhibitors, and a rapid hydrolysis of nitrocephin (McDougal and Thornsberry, 1986). These tests could lead to false-negative or false-positive diagnosis of intrin-

sic-oxacillin resistance (Liu and Lewis, 1992). Thus, 19% of the mecA-gene-positive strains of this study appeared susceptible to the ampicillin-sulbactam combination and could be wrongly interpreted as penicillinase hyperproducers. However, detection of the mecA gene does not allow determination of all of the more recently described resistance mechanisms: oxacillinase production (Massida et al., 1992)

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FIGURE 5 Antibiotic susceptibility study in relation to the presence of the mecA gene.

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30

or expression of modified penicillinase-binding proteins (PBPs) different from the PBP2a (Tomasz et al., 1989; De Lencastre et al., 1991). Simple diagnostic m e t h o d s are n e e d e d to d e t e r m i n e the importance and e p i d e m i o l o g y of these two recently described resistance mechanisms. In this study, all of the oxacillin-resistant strains that did not h a r b o r the mecA gene w e r e susceptible to the ampicillin-sulbactam or oxacillin-sulbactam combinations. H o w e v e r , it a p p e a r e d difficult to d e t e r m i n e the real m e c h a n i s m of resistance for each strain, particularly w h e n considering that t h e y m a y be c o m b i n e d in the same staphylococcal strain (De Lencastre et al., 1991; Barg et al., 1991). H e n c e f o r t h , detection of the mecA gene could be e m p l o y e d in conjunction with MIC d e t e r m i n a t i o n for the evaluation of n e w p h e n o t y p i c tests. Indeed, expression of the oxacillin resistance is sometimes h e t e r o g e n e o u s and v e r y d e p e n d e n t o n g r o w t h conditions, such as addition of NaC1 to the m e d i u m (Geberding et al., 1991; Liu et al., 1990; Montanari et al., 1990). In this study, 0-15% of the m e c A - n e g a t i v e strains a p p e a r e d resistant to oxacillin in the presence of 0 and 5% NaC1, respectively. As previously

G. Zambardi et al.

m e n t i o n e d (McMurray et al., 1990; Zierdt et al., 1992), most of t h e m (85%) b e l o n g e d to p h a g e g r o u p V and could express a particular ~-lactamase (McM u r r a y et al., 1990) with an increased excretion (Liu and Lewis, 1992) or activity in relation to salt concentration. A small increase in intrinsic resistance, however, cannot be excluded for these strains (Barg et al., 1991). In clinical practice, detection of the mecA gene facilitates the choice of an antistaphylococcal therapeutic scheme by allowing one to obtain data on the activity of antimicrobial agents o t h e r t h a n f~-lactams, as m e c A - g e n e - p o s i t i v e strains are frequently resistant to kanamycin, gentamicin, e r y t h r o m y c i n , tetracycline, and pefloxacin, as o p p o s e d to those showing an extrinsic resistance. W h e n a treatment must be initiated w i t h o u t delay, the use of inadequate, expensive, toxic, or bacteriostatic antibiotics can n o w be avoided.

The authors acknowledge the technical assistance of Chantal Nervi and Martine Rougier.

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Laboratory Diagnosis of Oxacillin Resistance

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Tomasz A, Drugeon HB, de Lencastre HM, Jabes D, McDougal L, Bille J (1989) New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP2a gene and contain normal penicillinbinding proteins with modified penicillin-binding capacity. Antimicrob Agents Chemother 33:1869-1874. Ubukata K, Yamashita N, Konno M (1985) Occurence of a ~-lactam-inducible penicillin-binding protein in methicillin-resistant staphylococci. Antimicrob Agents Chemother 27:851-857. Ubukata K, Nakagami S, Nitta A, Yamane A, Akwakami S, Sugiura M, Konno M (1992) Rapid detection of the mecA gene in methicillin-resistant staphylococci by enzymatic detection of polymerase chain reaction products. J Clin Microbiol 30:1728-1733. Unal S, Hoskins J, Flokowitsch JE, Wu CYE, Preston DA, Skatrud PL (1992) Detection of methicillin-resistant staphylococci by using the polymerase chain reaction. J Clin Microbiol 30:1685-1691. Woods GL, Yam P (1988) Bactericidal activity of oxacillin against ~3-1actamase hyperproducing Staphylococcus aureus. Antimicrob Agents Chemother 32:1614-1618. Zierdt CH, Hosein IK, Shively R, McLowry JD (1992) Phage pattern-specific oxacillin-resistant and borderline oxacillin-resistant Staphylococcus aureus in US hospitals: epidemiological significance. J Clin Microbiol 30: 252-254.