Vol. 30, No. 7
OF CLINICAL MICROBIOLOGY, JUIY 1992, P. 1728-1733 0095-1137/92/071728-06$02.00/0 Copyright © 1992, American Society for Microbiology
JOURNAL
Rapid Detection of the mecA Gene in Methicillin-Resistant Staphylococci by Enzymatic Detection of Polymerase Chain Reaction Products KIMIKO UBUKATA,l* SATORU NAKAGAMI,2 AKIHIRO NITTA,2 AKIO YAMANE 2 SAYOKO KAWAKAMI,1 MUTUMI SUGIURA,' AND MASATOSHI KONNO' Department of Clinical Pathology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo, 173,' and Institute for Biotechnology Research, Wakunaga Phannaceutical Co., Ltd., Koda-cho, Takata-gun, Hiroshima, 729-64, Japan Received 30 January 1992/Accepted 10 April 1992
In order to identify methicillin-resistant staphylococci from clinical sources with ease and reliability, enzymatic detection of polymerase chain reaction (ED-PCR) was applied. ED-PCR is based on the capture of amplified products via biotin-streptavidin affinity and the detection of an incorporated hapten in amplified products with an enzyme-linked antibody. In order to identify methicillin-resistant staphylococci of all species, a 150-bp fragment of the mecA gene was targeted for ED-PCR. After PCR was performed with a pair of biotin and dinitrophenol 5'-labeled primers, the reaction mixture was applied to a microtiter well precoated with streptavidin. Thereafter, bound PCR products were detected colorimetrically with alkaline phosphataseconjugated anti-dinitrophenol antibody. The extraction of DNA from staphylococcal cells for PCR was simplified so that it could be performed within one tube. The total assay, including PCR, took less than 3 h. The sensitivity of mecA gene detection ranged from >5 x 102 CFU per tube for Staphylococcus aureus to >5 x 103 CFU per tube for Staphylococcus epidermidis. Genotyping results obtained by ED-PCR of 161 tested strains from the colonies (97 strains of S. aureus and 64 strains of coagulase-negative staphylococci) were compared with the phenotypic susceptibilities of the strains to oxacillin. The results of ED-PCR showed excellent agreement with the MICs of oxacillin with very few exceptions; only one strain of S. aureus and two strains of coagulase-negative staphylococci were found to possess the mecA gene, which was discrepant with their phenotypes. Fifty-five blood culture samples were also tested by ED-PCR. For staphylococcal isolates in 33 of the cultures, oxacillin MICs were >4 ,ug/ml; 31 of the 33 staphylococcal isolates were determined by ED-PCR to be mecA gene positive. These results suggest that ED-PCR can be used with reasonable confidence in the clinical microbiological laboratory.
Inasmuch as methicillin-resistant Staphylococcus aureus (MRSA) strains also tend to show resistance toward additional antibiotics such as beta-lactams, aminoglycosides, and macrolides (10), only a small number of antibiotics are clinically available for the treatment of MRSA infections. Therefore, whenever a staphylococcal strain is isolated from patients with severe infections and who are immunodeficient, it is necessary to identify rapidly whether the isolate is MRSA. Unfortunately, when susceptibilities to various antibiotics are measured by standard methods (14), MICs of oxacillin or methicillin tend to be ambiguous for certain of the staphylococcal strains, including coagulase-negative staphylococci (2, 4).
derived from MRSA was used as a probe has been reported to be a means of identifying methicillin resistance (1). More recently, several attempts to detect the presence of the mecA gene by the polymerase chain reaction (PCR) have also been reported (9, 12, 21). In order to more rapidly and simply detect the mecA gene, we established a new method that relies on enzymatic detection of the PCR product (ED-PCR) (Fig. 1) by the use of a pair of primers labeled with biotin and dinitrophenol (28). In this report, we describe the results of ED-PCR tested on colonies as well as blood cultures.
MATERIALS AND METHODS
The main cause of methicillin resistance is the production of an alternative enzyme, named penicillin-binding protein 2' (PBP 2') (13, 26, 27) or PBP 2a (5, 16), in addition to the usual PBPs. The gene encoding PBP 2', designated mecA, has been cloned (7, 11, 20, 23) and sequenced by Song et al. (19) and Ryffel et al. (17). The mecA gene is not present in methicillin-susceptible strains but is almost invariably present in resistant strains (15, 24, 25), including low-levelresistant ones (2), although there are a few exceptions (8, 22). In view of the information presented above, a hybridization method in which a 32P-labeled mecA DNA fragment
*
Strains. MRSA TK2566, Staphylococcus epidermidis TK1645, and Staphylococcus haemolyticus TK2384 were used as standard strains for detection of the mecA gene by ED-PCR. Staphylococcal colonies grown on tryptic soy II blood agar plates (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) were directly used as clinical isolates from the patients. The staphylococcal species listed in Table 1 were identified by using the Posi Combo panel for the auto SCAN-4 system (Baxter Healthcare Corp., West Sacramento, Calif.). Preparation of blood culture samples for ED-PCR. The possibility of directly detecting the presence of the mecA gene in blood cultures was examined by using culture medium in 16A and 17A culture bottles (Becton Dickinson
Corresponding author. 1728
VOL. 30, 1992
DETECTION OF mecA GENE BY ED-PCR
1) DNA AMPLIFICATION (PCR)
2) AMPLIFIED DNA CAPTURING AND ENZYMATIC DETECTION ALP-CONJUGATED ANTI-DNP ANTIBODY
B.LL
L
B
D
1
PNPP
D D D D ~) YELLOW D25) and in which organisms were observed by Gram staining. A total of 500 ,u1 of each culture medium was sterilely transferred to a 1.5-ml Eppendorf tube, and the tube was centrifuged at 1,500 rpm (158 x g) for 3 min to remove blood cell components. Then, 200 ,ul of the supernatant was transferred to another tube, and the tube was centrifuged at 10,000 rpm (7,000 x g) for 5 min to harvest the organisms. After the supernatant was discarded, the pellet was suspended in 200 p.l of phosphate-buffered saline (pH 7.2) and 2 pI of the suspension was used for ED-PCR. Oligonucleotides. On the basis of the DNA sequence of the mecA gene derived from S. aureus TK784 (19), a few pairs of primers were used to try to amplify the mecA gene by PCR. As a result, the primers that corresponded to nucleotides 181 to 200 as the sense strand and 311 to 330 as the antisense strand were selected for their specificities and efficiencies. The primers were named MRS1 (5'-GAAATGACTGAACG TCCGAT) and MRS2 (5'-GCGATCAATGTTACCGTAGT), respectively. This set of primers amplifies a 150-bp-long segment of the mecA gene. The labeling of primers was performed by using Aminolink II (Applied Biosystems, Foster City, Calif.) for synthesis; this was followed by purification and the reactions with biotin N-hydroxysuccinimide ester or dinitrofluorobenzene (3). Cell lysis and DNA amplification. The first lysis solution, which contained 200 p.g of lysostaphin (Sigma Chemical Co., St. Louis, Mo.) per ml and 50 mM Tris-HCI (pH 7.5), was dispensed in 30-,ul aliquots into a microcentrifuge tube (0.5 ml). The solution was overlaid with 2 drops of mineral oil and was stored at -20°C. Each colony on the agar plate was TABLE 1. Clinical staphylococcal isolates used for ED-PCR No. of strains Species S. aureus............................................................ S. epidermidis...................................................... S. haemolyticus................................................... S. capitis............................................................ 4 S. hominis........................................................... 2 S. warneri........................................................... 1 S. simulans......................................................... 1 S. saprophyticus.................................................. Total ..........................................
161
1729
picked with a sterilized pick and suspended in the first lysis solution. Similarly, 2 p.l of bacterial suspension from a blood culture instead of a colony was added to the first lysis solution. The suspension was incubated at 50°C for 5 min by using a DNA thermal cycler (Perkin-Elmer Cetus, Emeryville, Calif.). Then, 10 p.1 of the second lysis solution, which consisted of 110 mM Tris-HCI (pH 8.9), 1.5 mM MgCl2, 80 mM KCl, 500 p.g of bovine serum albumin per ml, 0.1% sodium cholate, 0.1% Triton X-100, 200 p.g of proteinase K per ml, 0.45% Tween 20, and 0.45% Nonidet P-40, was added to each tube. The mixture was incubated at 60°C for 10 min and subsequently at 95°C for S min. Finally, 10 p.l of the ED-PCR mixture, which was composed of 110 mM Tris-HCl (pH 8.9); 1.5 mM MgCl2; 80 mM KCI; 500 p.g of bovine serum albumin per ml; 0.1% sodium cholate; 0.1% Triton X-100; 5 p.g of each of the primers per ml; 1 mM (each) dATP, dGTP, dCTP, and TTP; and 100 U of Tth DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) per ml, was added to each tube and subjected to 30 cycles of PCR by using a DNA thermal cycler (Perkin-Elmer Cetus). The conditions of the PCR were as follows: denaturation at 94°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 5 s.
Detection of amplified DNA. A total of 10 p.1 of the amplified mixture was transferred to a streptavidin-coated microtiter well with 100 p.1 of alkaline phosphatase-conjugated anti-dinitrophenol antibody diluted in a washing solution that was composed of 50 mM Tris-HCI (pH 7.5), 150 mM NaCl, and 0.05% Tween 20. The mixture was left at room temperature for 30 min. The mixture was aspirated and the well was washed three times with 200 p.1 of the washing solution. Finally, 100 p.1 ofp-nitrophenyl phosphate solution (4 mg/ml in diethanoleamine [pH 9.8]-0.5 mM MgCl2) was added to each well. After 30 min at room temperature, the A405 was read. The ED-PCR procedures are shown in Fig. 2. Antimicrobial susceptibility tests. The susceptibilities of staphylococci to antimicrobial agents were determined by the broth dilution method by using the Posi Combo panel in parallel with mecA gene detection. The final inoculum size of the bacteria placed into the panel was 105 CFU/ml. The susceptibilities of these strains to oxacillin and methicillin were also measured by an agar dilution method by using Mueller-Hinton agar (Eiken Chemical Co., Ltd., Tokyo, Japan) containing 2% NaCl. The bacterial cultures in Mueller-Hinton broth were diluted to give a final inoculum of 104 CFU per spot. All of the agar plates were incubated at 32°C for 24 h, and the MIC was defined as the lowest concentration of antibiotic that inhibited visible microbial growth on the agar plate. Induction of PBP 2' production. Ten milliliters of cells that were cultured overnight was inoculated into 200 ml of tryptic soy broth with or without 1 p.g of ceftizoxime per ml as the inducer. The cells were grown at 32°C to the logarithmic growth phase with shaking. Subsequently, the bacterial cells were harvested and the membrane fractions were collected by a previously described method (26). Membrane proteins were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Production of PBP 2' was detected by using a densitometer set at 530 nm. RESULTS Sensitivity of ED-PCR for detection of the mecA gene. The results of tests of the sensitivity of ED-PCR for mecA gene detection are shown in Table 2 and Fig. 3. The strains used
1730
J. Cl-IN. MICROBIOL.
UBUKATA ET AL. 2) Lysis of cells ® 50°C, 5min Addition of 2nd lysis sol. (1Oji) 2 60°C, 10min (2 94°C, 5min
1) Transfer of colony into 1 st lysis sol.(30lxl)
4) Amplification of DNA ( 94C, 15secE 2 55C, 15sec 30 cycles () 72°C, 5sec J
3) Addition of primer mix sol.(1 Oji)
(90min)
(20min)
6) Enzyme reaction 5) Capturing of PCR products and Immuno reaction PCR products(1 01±1) \\ ALP-labeled anti DNP antibody ,0C PNPP (100i) (100 1±) Washing with buffer
7) Read at 405nm mecA Positive:Yellow color
Leave at room temp (30 min)
Leave at room temp (30 min)
Streptavidin coated plate
FIG. 2. Procedure for detection of the mecA gene by ED-PCR. Samples from colonies were lysed in two steps (lysostaphin and proteinase K treatments). Amplification started with the addition of the ED-PCR mixture, which contained a pair of primers labeled with biotin and dinitrophenol (DNP). Capture of the amplified DNA and immunoreaction were performed simultaneously. The bound alkaline phosphatase (ALP) activity was measured by usingp-nitrophenyl phosphate (PNPP) as a chromogenic substrate. All heating reactions were performed on a thermal cycler, and the total assay (from picking the colonies to judging the results) was done within 3 h.
standards were MRSA TK2566, S. epidennidis TK1645, and S. haemnolyticus TK2384. Blood culture samples were prepared by the procedures described above. Detection of the mecA gene was possible when bacterial cells were present at >5 x 102 CFU per tube in the cases of S. aureus and S. haemolyticus, and the mecA gene was even detected in blood culture. After color development, microtiter wells with A405 values of more than 0.200 were a distinct yellow color. In the case of S. epidennidis, however, EDPCR was positive when the bacterial counts were greater than 5 x 10i CFU per tube; this was an order of magnitude difference in the sensitivity of ED-PCR between S. aureus and S. epidennidis. The color developed after the addition of p-nitrophenyl phosphate solution and was readily seen with the naked eye. No detectable signal by ED-PCR was given when Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were used as samples. as
From these results, the clinical isolates or samples that showed an optical density at 405 nm of >0.200 were considered to be positive for the mecA gene. Correlation between the mecA gene and oxacillin susceptibility. Figure 4 shows the relationship between the presence of the mecA gene and the oxacillin susceptibilities of 97 strains of S. aureus derived from clinical sources. The MICs of oxacillin measured routinely by the broth dilution method were also compared with those measured by the agar dilution method. On the basis of the breakpoints for oxacillin, the strains for which the MICs of the antibiotics were >4 ,ug/ml were regarded as methicillin resistant. Of the 97 strains, 51 strains were phenotypically methicillin resistant, and all 51 of the strains were mecA gene positive. Of the 46 strains that were susceptible to oxacillin, only one was mecA gene positive. The production of PBP 2' by this strain could
TABLE 2. Sensitivity of ED-PCR for detection of the mecA gene from staphylococci
A405 (mean + SD)" CFU per tube
10,(00 5,000 1,000 500 100
S. aureus TK2556
1.897 1.397 0.753 0.347 0.007
± + ± + ±
0.059 0.121 0.075 0.005 0.005
The A405 was read after color development for 30 min.
S. epidermidis TK1645 1.027 ± 0.009 0.337 ± 0.033 0.047 ± 0.026 0.020 ± 0.007 0.010 + 0.005
S.
haernolyticuis TK2384
1.263 1.263 0.747 0.276 0.040
± ± ± + +
0.054 0.047 0.075 0.011 0.014
Blood culture (S. alureius) 1.005 ± 0.062 1.091 ± 0.136 0.518 ± 0.084 0.213 ± 0.067 0.061 ± 0.021
VOL. 30, 1992
DETECTION OF mecA GENE BY ED-PCR
pg/mi
ABC DE FG HIJKLM NO P
. >8 -*-*4,870 2,016 1360N
8 p.g/ml by the broth dilution method. Therefore, these 10 strains were oxacillin resistant by the broth dilution method. Yet, as measured by the agar dilution method, the MICs for the same 10 strains were 4 4 10i CFU per tube. It appears that it is possible to detect the presence of the mecA gene directly in clinical materials such as throat swab, sputum, urine, and pus specimens by ED-PCR with slight modifications of the procedure (data not shown). In conclusion, we believe that ED-PCR is particularly useful in differentiating staphylococcal strains with low-level resistance to oxacillin from susceptible strains in a timely manner.
DISCUSSION
REFERENCES
Phenotypic expression of methicillin resistance is sometimes heterogeneous (6). Therefore, particular growth conditions, such as an incubation temperature lower than 35°C, addition of NaCl to the medium, and large inoculum size, are required for the susceptibility tests routinely performed by clinical microbiology laboratories. Even then it is still difficult to determine the methicillin resistance of the borderlineresistant strains (18), despite the recommendations of the National Committee for Clinical Laboratory Standards (14). Certain attempts at using PCR for the detection of the mecA gene, and therefore the identification of methicillinresistant staphylococcal strains, have been reported (9, 12, 21). However, detection of the products by these PCR methods requires a tedious electrophoresis procedure following amplification of the mecA gene. In addition, preparation of template DNA from staphylococcal cells is not always simple and effective. The first virtue of the ED-PCR described in this report is the establishment of procedures for efficient bacteriolysis so that the lysates can be used directly for amplification. Consequently, preparation for PCR was simplified and consisted of only three steps. The first step was the transfer of colonies into the first lysis solution (lysostaphin), the second step was the addition of the second lysis solution (proteinase K), and the third step was the addition of the ED-PCR mixture (all reagents for PCR). Because of these procedures, it was possible to perform all heating reactions on a thermal cycler within a single tube. The second virtue of the ED-PCR was in the elimination of agarose gel electrophoresis for the detection of PCR products. Instead, detection was performed by a standard enzyme-linked immunosorbent assay. Evaluation for the presence of the mecA gene became possible by determination of theA405. Consequently, the total assay, including PCR, took about 3 h. The sensitivity of detection of the mecA gene by ED-PCR ranged from 5 x 102 CFU per tube for S. aureus to 5 x 103 CFU per tube for S. epidermidis. This range of sensitivity should pose no problem when amplification of the mecA gene is carried out with bacterial colonies. In tests with colonies of clinical isolates, the mecA gene was detected by ED-PCR in all samples for which MICs
1. Archer, G. L., and E. Pennel. 1990. Detection of methicillin resistance in staphylococci by using a DNA probe. Antimicrob. Agents Chemother. 34:1720-1724. 2. Chambers, H. F., G. L. Archer, and M. Matsuhashi. 1989.
Low-level methicillin resistance in strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 33:424-428. 3. Coull, J. M., H. L. Weith, and R. Bischoff. 1986. A novel method
for the introduction of an aliphatic primary amino group at the 5' terminus of synthetic oligonucleotides. Tetrahedron Lett. 27: 3991-3994. 4. Hackbarth, C. J., and H. F. Chambers. 1989. Methicillinresistant staphylococci: detection methods and treatment of
infections. Antimicrob. Agents Chemother. 33:995-999. 5. Hartman, B. J., and A. Tomasz. 1984. Low-affinity penicillin-
binding protein associated with P-lactam resistance in Staphylococcus aureus. J. Bacteriol. 158:513-516.
6. Hartman, B. J., and A. Tomasz. 1986. Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 29:85-92. 7. Inglis, B., P. R. Matthews, and P. R. Stuart. 1988. The expression in Staphylococcus aureus of cloned DNA encoding methicillin resistance. J. Gen. Microbiol. 134:1465-1469. 8. Lencastre, H., A. M. Figueiredo, C. Urban, J. Rahal, and A. Tomasz. 1991. Multiple mechanisms of methicillin resistance and improved methods for detection in clinical isolates of Staphylococcus aureus. Antimicrob. Agents Chemother. 35: 632-639. 9. Ligozzi, M., G. M. Rossolini, E. A. Tonin, and R. Fontana. 1991. Nonradioactive DNA probe for detection of gene for methicillin
resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 35:575-578. 10. Maple, P. A. C., J. M. T. Hamilton-Miller, and W. Brumfitt. 1989. Worldwide antibiotic resistance in methicillin-resistant
Staphylococcus aureus. Lancet i:537-540. 11. Matsuhashi, M., M. D. Song, F. Ishino, M. Wachi, M. Doi, M. Inoue, K. Ubukata, N. Yamashita, and M. Konno. 1986. Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to f-lactam antibiotics in Staphylococcus aureus. J. Bacteriol. 167:975-980. 12. Murakami, K., W. Minamide, K. Wada, E. Nakamura, H. Teraoka, and S. Watanabe. 1991. Identification of methicillinresistant strains of staphylococci by polymerase chain reaction. J. Clin. Microbiol. 29:2240-2244. 13. Murakami, K., K. Nomura, M. Doi, and T. Yoshida. 1987. Production of low-affinity penicillin-binding protein by low- and high-resistance groups of methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 31:1307-1311.
DETECTION OF mecA GENE BY ED-PCR
VOL. 30, 1992 14. National Committee for Clinical Laboratory Standards. 1990. Performance standards for antimicrobial disk susceptibility testing. M2-A4, 4th ed., vol. 10, no. 7. National Committee for Clinical Laboratory Standards, Villanova, Pa. 15. Pierre, J., R. Williamson, M. Bornet, and L. Gutmann. 1990. Presence of an additional penicillin-binding protein in methicillin-resistant Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus simulans with a low affinity for methicillin, cephalothin, and cefamandole. Antimicrob. Agents Chemother. 34:1691-1694. 16. Rossi, L., E. Tonin, Y. R. Cheng, and R. Fontana. 1985. Regulation of penicillin-binding protein activity: description of a methicillin-inducible penicillin-binding protein in Staphylococcus aureus. Antimicrob. Agents Chemother. 27:828-831. 17. Ryffel, C., W. Tesch, I. Birch-Machin, P. E. Reynolds, L. Barberis-Maino, F. H. Kayser, and B. Berger-Bachi. 1990. Sequence comparison of mecA genes from methicillin-resistant Staphylococcus aureus and Staphylococcus epidennidis. Gene 94:137-138. 18. Sierra-Madero, J. G., C. Knapp, C. Karaffa, and J. A. Washington. 1988. Role of ,-lactamase and different testing conditions in oxacillin-borderline-susceptible staphylococci. Antimicrob. Agents Chemother. 32:1754-1757. 19. Song, M. D., M. Wachi, M. Doi, F. Ishino, and M. Matsuhashi. 1987. Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion. FEBS Lett. 221:167-171. 20. Tesch, W., A. Strassle, B. Berger-Bachi, D. O'Hara, P. Reynolds, and F. H. Kayser. 1988. Cloning and expression of methicillin resistance from Staphylococcus epidermidis in Staphylococcus carnosus. Antimicrob. Agents Chemother. 32:14941499. 21. Tokue, Y., S. Shoji, S. Satoh, A. Watanabe, and M. Motomiya. 1991. Detection of methicillin-resistant Staphylococcus aureus
22.
23.
24.
25.
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(MRSA) using polymerase chain reaction amplification. Tohoku J. Exp. Med. 163:31-37. Tomasz, A., H. B. Drugeon, H. M. de Lencastre, D. Jabes, L. McDougal, and J. Bille. 1989. New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity. Antimicrob. Agents Chemother. 33:1869-1874. Ubukata, K., R. Nonoguchi, M. Matsuhashi, and M. Konno. 1989. Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureusspecific penicillin-binding protein. J. Bacteriol. 171:2882-2885. Ubukata, K., R. Nonoguchi, M. Matsuhashi, M. D. Song, and M. Konno. 1989. Restriction maps of the regions coding for methicillin and tobramycin resistances on chromosomal DNA in methicillin-resistant staphylococci. Antimicrob. Agents Chemother. 33:1624-1626. Ubukata, K., R. Nonoguchi, M. D. Song, M. Matsuhashi, and M. Konno. 1990. Homology of mecA gene in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans to that of Staphylococcus aureus. Antimicrob. Agents Chemother.
34:170-172. 26. Ubukata, K., N. Yamashita, and M. Konno. 1985. Occurrence of P-lactam-inducible penicillin-binding protein in methicillin-resistant staphylococci. Antimicrob. Agents Chemother. 27:851857. 27. Utsui, Y., and T. Yoklita. 1985. Role of an altered penicillinbinding protein in methicillin- and cephem-resistant Staphylococcus aureus. Antimierob. Agents Chemother. 28:397-403. 28. Yamane, A., T. Ka4d§ti, and S. Nakagami. 1989. Rapid detection of specific gene sequences by PCR with two functional primers. The Fourth San Diego Conference on Nucleic Acid Research, paper no. 13.
OF CLINICAL MICROBIOLOGY, JUIY 1992, P. 1728-1733 0095-1137/92/071728-06$02.00/0 Copyright © 1992, American Society for Microbiology
JOURNAL
Rapid Detection of the mecA Gene in Methicillin-Resistant Staphylococci by Enzymatic Detection of Polymerase Chain Reaction Products KIMIKO UBUKATA,l* SATORU NAKAGAMI,2 AKIHIRO NITTA,2 AKIO YAMANE 2 SAYOKO KAWAKAMI,1 MUTUMI SUGIURA,' AND MASATOSHI KONNO' Department of Clinical Pathology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo, 173,' and Institute for Biotechnology Research, Wakunaga Phannaceutical Co., Ltd., Koda-cho, Takata-gun, Hiroshima, 729-64, Japan Received 30 January 1992/Accepted 10 April 1992
In order to identify methicillin-resistant staphylococci from clinical sources with ease and reliability, enzymatic detection of polymerase chain reaction (ED-PCR) was applied. ED-PCR is based on the capture of amplified products via biotin-streptavidin affinity and the detection of an incorporated hapten in amplified products with an enzyme-linked antibody. In order to identify methicillin-resistant staphylococci of all species, a 150-bp fragment of the mecA gene was targeted for ED-PCR. After PCR was performed with a pair of biotin and dinitrophenol 5'-labeled primers, the reaction mixture was applied to a microtiter well precoated with streptavidin. Thereafter, bound PCR products were detected colorimetrically with alkaline phosphataseconjugated anti-dinitrophenol antibody. The extraction of DNA from staphylococcal cells for PCR was simplified so that it could be performed within one tube. The total assay, including PCR, took less than 3 h. The sensitivity of mecA gene detection ranged from >5 x 102 CFU per tube for Staphylococcus aureus to >5 x 103 CFU per tube for Staphylococcus epidermidis. Genotyping results obtained by ED-PCR of 161 tested strains from the colonies (97 strains of S. aureus and 64 strains of coagulase-negative staphylococci) were compared with the phenotypic susceptibilities of the strains to oxacillin. The results of ED-PCR showed excellent agreement with the MICs of oxacillin with very few exceptions; only one strain of S. aureus and two strains of coagulase-negative staphylococci were found to possess the mecA gene, which was discrepant with their phenotypes. Fifty-five blood culture samples were also tested by ED-PCR. For staphylococcal isolates in 33 of the cultures, oxacillin MICs were >4 ,ug/ml; 31 of the 33 staphylococcal isolates were determined by ED-PCR to be mecA gene positive. These results suggest that ED-PCR can be used with reasonable confidence in the clinical microbiological laboratory.
Inasmuch as methicillin-resistant Staphylococcus aureus (MRSA) strains also tend to show resistance toward additional antibiotics such as beta-lactams, aminoglycosides, and macrolides (10), only a small number of antibiotics are clinically available for the treatment of MRSA infections. Therefore, whenever a staphylococcal strain is isolated from patients with severe infections and who are immunodeficient, it is necessary to identify rapidly whether the isolate is MRSA. Unfortunately, when susceptibilities to various antibiotics are measured by standard methods (14), MICs of oxacillin or methicillin tend to be ambiguous for certain of the staphylococcal strains, including coagulase-negative staphylococci (2, 4).
derived from MRSA was used as a probe has been reported to be a means of identifying methicillin resistance (1). More recently, several attempts to detect the presence of the mecA gene by the polymerase chain reaction (PCR) have also been reported (9, 12, 21). In order to more rapidly and simply detect the mecA gene, we established a new method that relies on enzymatic detection of the PCR product (ED-PCR) (Fig. 1) by the use of a pair of primers labeled with biotin and dinitrophenol (28). In this report, we describe the results of ED-PCR tested on colonies as well as blood cultures.
MATERIALS AND METHODS
The main cause of methicillin resistance is the production of an alternative enzyme, named penicillin-binding protein 2' (PBP 2') (13, 26, 27) or PBP 2a (5, 16), in addition to the usual PBPs. The gene encoding PBP 2', designated mecA, has been cloned (7, 11, 20, 23) and sequenced by Song et al. (19) and Ryffel et al. (17). The mecA gene is not present in methicillin-susceptible strains but is almost invariably present in resistant strains (15, 24, 25), including low-levelresistant ones (2), although there are a few exceptions (8, 22). In view of the information presented above, a hybridization method in which a 32P-labeled mecA DNA fragment
*
Strains. MRSA TK2566, Staphylococcus epidermidis TK1645, and Staphylococcus haemolyticus TK2384 were used as standard strains for detection of the mecA gene by ED-PCR. Staphylococcal colonies grown on tryptic soy II blood agar plates (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) were directly used as clinical isolates from the patients. The staphylococcal species listed in Table 1 were identified by using the Posi Combo panel for the auto SCAN-4 system (Baxter Healthcare Corp., West Sacramento, Calif.). Preparation of blood culture samples for ED-PCR. The possibility of directly detecting the presence of the mecA gene in blood cultures was examined by using culture medium in 16A and 17A culture bottles (Becton Dickinson
Corresponding author. 1728
VOL. 30, 1992
DETECTION OF mecA GENE BY ED-PCR
1) DNA AMPLIFICATION (PCR)
2) AMPLIFIED DNA CAPTURING AND ENZYMATIC DETECTION ALP-CONJUGATED ANTI-DNP ANTIBODY
B.LL
L
B
D
1
PNPP
D D D D ~) YELLOW D25) and in which organisms were observed by Gram staining. A total of 500 ,u1 of each culture medium was sterilely transferred to a 1.5-ml Eppendorf tube, and the tube was centrifuged at 1,500 rpm (158 x g) for 3 min to remove blood cell components. Then, 200 ,ul of the supernatant was transferred to another tube, and the tube was centrifuged at 10,000 rpm (7,000 x g) for 5 min to harvest the organisms. After the supernatant was discarded, the pellet was suspended in 200 p.l of phosphate-buffered saline (pH 7.2) and 2 pI of the suspension was used for ED-PCR. Oligonucleotides. On the basis of the DNA sequence of the mecA gene derived from S. aureus TK784 (19), a few pairs of primers were used to try to amplify the mecA gene by PCR. As a result, the primers that corresponded to nucleotides 181 to 200 as the sense strand and 311 to 330 as the antisense strand were selected for their specificities and efficiencies. The primers were named MRS1 (5'-GAAATGACTGAACG TCCGAT) and MRS2 (5'-GCGATCAATGTTACCGTAGT), respectively. This set of primers amplifies a 150-bp-long segment of the mecA gene. The labeling of primers was performed by using Aminolink II (Applied Biosystems, Foster City, Calif.) for synthesis; this was followed by purification and the reactions with biotin N-hydroxysuccinimide ester or dinitrofluorobenzene (3). Cell lysis and DNA amplification. The first lysis solution, which contained 200 p.g of lysostaphin (Sigma Chemical Co., St. Louis, Mo.) per ml and 50 mM Tris-HCI (pH 7.5), was dispensed in 30-,ul aliquots into a microcentrifuge tube (0.5 ml). The solution was overlaid with 2 drops of mineral oil and was stored at -20°C. Each colony on the agar plate was TABLE 1. Clinical staphylococcal isolates used for ED-PCR No. of strains Species S. aureus............................................................ S. epidermidis...................................................... S. haemolyticus................................................... S. capitis............................................................ 4 S. hominis........................................................... 2 S. warneri........................................................... 1 S. simulans......................................................... 1 S. saprophyticus.................................................. Total ..........................................
161
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picked with a sterilized pick and suspended in the first lysis solution. Similarly, 2 p.l of bacterial suspension from a blood culture instead of a colony was added to the first lysis solution. The suspension was incubated at 50°C for 5 min by using a DNA thermal cycler (Perkin-Elmer Cetus, Emeryville, Calif.). Then, 10 p.1 of the second lysis solution, which consisted of 110 mM Tris-HCI (pH 8.9), 1.5 mM MgCl2, 80 mM KCl, 500 p.g of bovine serum albumin per ml, 0.1% sodium cholate, 0.1% Triton X-100, 200 p.g of proteinase K per ml, 0.45% Tween 20, and 0.45% Nonidet P-40, was added to each tube. The mixture was incubated at 60°C for 10 min and subsequently at 95°C for S min. Finally, 10 p.l of the ED-PCR mixture, which was composed of 110 mM Tris-HCl (pH 8.9); 1.5 mM MgCl2; 80 mM KCI; 500 p.g of bovine serum albumin per ml; 0.1% sodium cholate; 0.1% Triton X-100; 5 p.g of each of the primers per ml; 1 mM (each) dATP, dGTP, dCTP, and TTP; and 100 U of Tth DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) per ml, was added to each tube and subjected to 30 cycles of PCR by using a DNA thermal cycler (Perkin-Elmer Cetus). The conditions of the PCR were as follows: denaturation at 94°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 5 s.
Detection of amplified DNA. A total of 10 p.1 of the amplified mixture was transferred to a streptavidin-coated microtiter well with 100 p.1 of alkaline phosphatase-conjugated anti-dinitrophenol antibody diluted in a washing solution that was composed of 50 mM Tris-HCI (pH 7.5), 150 mM NaCl, and 0.05% Tween 20. The mixture was left at room temperature for 30 min. The mixture was aspirated and the well was washed three times with 200 p.1 of the washing solution. Finally, 100 p.1 ofp-nitrophenyl phosphate solution (4 mg/ml in diethanoleamine [pH 9.8]-0.5 mM MgCl2) was added to each well. After 30 min at room temperature, the A405 was read. The ED-PCR procedures are shown in Fig. 2. Antimicrobial susceptibility tests. The susceptibilities of staphylococci to antimicrobial agents were determined by the broth dilution method by using the Posi Combo panel in parallel with mecA gene detection. The final inoculum size of the bacteria placed into the panel was 105 CFU/ml. The susceptibilities of these strains to oxacillin and methicillin were also measured by an agar dilution method by using Mueller-Hinton agar (Eiken Chemical Co., Ltd., Tokyo, Japan) containing 2% NaCl. The bacterial cultures in Mueller-Hinton broth were diluted to give a final inoculum of 104 CFU per spot. All of the agar plates were incubated at 32°C for 24 h, and the MIC was defined as the lowest concentration of antibiotic that inhibited visible microbial growth on the agar plate. Induction of PBP 2' production. Ten milliliters of cells that were cultured overnight was inoculated into 200 ml of tryptic soy broth with or without 1 p.g of ceftizoxime per ml as the inducer. The cells were grown at 32°C to the logarithmic growth phase with shaking. Subsequently, the bacterial cells were harvested and the membrane fractions were collected by a previously described method (26). Membrane proteins were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Production of PBP 2' was detected by using a densitometer set at 530 nm. RESULTS Sensitivity of ED-PCR for detection of the mecA gene. The results of tests of the sensitivity of ED-PCR for mecA gene detection are shown in Table 2 and Fig. 3. The strains used
1730
J. Cl-IN. MICROBIOL.
UBUKATA ET AL. 2) Lysis of cells ® 50°C, 5min Addition of 2nd lysis sol. (1Oji) 2 60°C, 10min (2 94°C, 5min
1) Transfer of colony into 1 st lysis sol.(30lxl)
4) Amplification of DNA ( 94C, 15secE 2 55C, 15sec 30 cycles () 72°C, 5sec J
3) Addition of primer mix sol.(1 Oji)
(90min)
(20min)
6) Enzyme reaction 5) Capturing of PCR products and Immuno reaction PCR products(1 01±1) \\ ALP-labeled anti DNP antibody ,0C PNPP (100i) (100 1±) Washing with buffer
7) Read at 405nm mecA Positive:Yellow color
Leave at room temp (30 min)
Leave at room temp (30 min)
Streptavidin coated plate
FIG. 2. Procedure for detection of the mecA gene by ED-PCR. Samples from colonies were lysed in two steps (lysostaphin and proteinase K treatments). Amplification started with the addition of the ED-PCR mixture, which contained a pair of primers labeled with biotin and dinitrophenol (DNP). Capture of the amplified DNA and immunoreaction were performed simultaneously. The bound alkaline phosphatase (ALP) activity was measured by usingp-nitrophenyl phosphate (PNPP) as a chromogenic substrate. All heating reactions were performed on a thermal cycler, and the total assay (from picking the colonies to judging the results) was done within 3 h.
standards were MRSA TK2566, S. epidennidis TK1645, and S. haemnolyticus TK2384. Blood culture samples were prepared by the procedures described above. Detection of the mecA gene was possible when bacterial cells were present at >5 x 102 CFU per tube in the cases of S. aureus and S. haemolyticus, and the mecA gene was even detected in blood culture. After color development, microtiter wells with A405 values of more than 0.200 were a distinct yellow color. In the case of S. epidennidis, however, EDPCR was positive when the bacterial counts were greater than 5 x 10i CFU per tube; this was an order of magnitude difference in the sensitivity of ED-PCR between S. aureus and S. epidennidis. The color developed after the addition of p-nitrophenyl phosphate solution and was readily seen with the naked eye. No detectable signal by ED-PCR was given when Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were used as samples. as
From these results, the clinical isolates or samples that showed an optical density at 405 nm of >0.200 were considered to be positive for the mecA gene. Correlation between the mecA gene and oxacillin susceptibility. Figure 4 shows the relationship between the presence of the mecA gene and the oxacillin susceptibilities of 97 strains of S. aureus derived from clinical sources. The MICs of oxacillin measured routinely by the broth dilution method were also compared with those measured by the agar dilution method. On the basis of the breakpoints for oxacillin, the strains for which the MICs of the antibiotics were >4 ,ug/ml were regarded as methicillin resistant. Of the 97 strains, 51 strains were phenotypically methicillin resistant, and all 51 of the strains were mecA gene positive. Of the 46 strains that were susceptible to oxacillin, only one was mecA gene positive. The production of PBP 2' by this strain could
TABLE 2. Sensitivity of ED-PCR for detection of the mecA gene from staphylococci
A405 (mean + SD)" CFU per tube
10,(00 5,000 1,000 500 100
S. aureus TK2556
1.897 1.397 0.753 0.347 0.007
± + ± + ±
0.059 0.121 0.075 0.005 0.005
The A405 was read after color development for 30 min.
S. epidermidis TK1645 1.027 ± 0.009 0.337 ± 0.033 0.047 ± 0.026 0.020 ± 0.007 0.010 + 0.005
S.
haernolyticuis TK2384
1.263 1.263 0.747 0.276 0.040
± ± ± + +
0.054 0.047 0.075 0.011 0.014
Blood culture (S. alureius) 1.005 ± 0.062 1.091 ± 0.136 0.518 ± 0.084 0.213 ± 0.067 0.061 ± 0.021
VOL. 30, 1992
DETECTION OF mecA GENE BY ED-PCR
pg/mi
ABC DE FG HIJKLM NO P
. >8 -*-*4,870 2,016 1360N
8 p.g/ml by the broth dilution method. Therefore, these 10 strains were oxacillin resistant by the broth dilution method. Yet, as measured by the agar dilution method, the MICs for the same 10 strains were 4 4 10i CFU per tube. It appears that it is possible to detect the presence of the mecA gene directly in clinical materials such as throat swab, sputum, urine, and pus specimens by ED-PCR with slight modifications of the procedure (data not shown). In conclusion, we believe that ED-PCR is particularly useful in differentiating staphylococcal strains with low-level resistance to oxacillin from susceptible strains in a timely manner.
DISCUSSION
REFERENCES
Phenotypic expression of methicillin resistance is sometimes heterogeneous (6). Therefore, particular growth conditions, such as an incubation temperature lower than 35°C, addition of NaCl to the medium, and large inoculum size, are required for the susceptibility tests routinely performed by clinical microbiology laboratories. Even then it is still difficult to determine the methicillin resistance of the borderlineresistant strains (18), despite the recommendations of the National Committee for Clinical Laboratory Standards (14). Certain attempts at using PCR for the detection of the mecA gene, and therefore the identification of methicillinresistant staphylococcal strains, have been reported (9, 12, 21). However, detection of the products by these PCR methods requires a tedious electrophoresis procedure following amplification of the mecA gene. In addition, preparation of template DNA from staphylococcal cells is not always simple and effective. The first virtue of the ED-PCR described in this report is the establishment of procedures for efficient bacteriolysis so that the lysates can be used directly for amplification. Consequently, preparation for PCR was simplified and consisted of only three steps. The first step was the transfer of colonies into the first lysis solution (lysostaphin), the second step was the addition of the second lysis solution (proteinase K), and the third step was the addition of the ED-PCR mixture (all reagents for PCR). Because of these procedures, it was possible to perform all heating reactions on a thermal cycler within a single tube. The second virtue of the ED-PCR was in the elimination of agarose gel electrophoresis for the detection of PCR products. Instead, detection was performed by a standard enzyme-linked immunosorbent assay. Evaluation for the presence of the mecA gene became possible by determination of theA405. Consequently, the total assay, including PCR, took about 3 h. The sensitivity of detection of the mecA gene by ED-PCR ranged from 5 x 102 CFU per tube for S. aureus to 5 x 103 CFU per tube for S. epidermidis. This range of sensitivity should pose no problem when amplification of the mecA gene is carried out with bacterial colonies. In tests with colonies of clinical isolates, the mecA gene was detected by ED-PCR in all samples for which MICs
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