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    1994, 32(1):143. J. Clin. Microbiol. J Vuopio-VarkilaP Kuusela, P Hildén, K Savolainen, M Vuento, O Lyytikäinen and  by slide agglutination tests.Staphylococcus aureus strains not identified Rapid detection of methicillin-resistant http://jcm.asm.org/content/32/1/143Updated information and services can be found at: These include:  CONTENT ALERTS  more»cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:  http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:  onA  pr i  l  1  9  ,2  0 1 4  b  y P E N N  S T A T E  U N I  V h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om  onA  pr i  l  1  9  ,2  0 1 4  b  y P E N N  S T A T E  U N I  V h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om   Vol. 32, No. 1 OURNAL OF CLNICAL MICROBIOLOGY, Jan. 1994, p. 143-147 0095-1137/94/ 04.00+0 Copyright © 1994, American Society for Microbiology Rapid Detection of Methicillin-Resistant Staphylococcus aureus Strains Not Identified by Slide Agglutination Tests P. KUUSELA,l* P. HILDtN,l K. SAVOLAINEN,1 M. VUENTO,2 0. LYYTIKAINEN,3 AND J. VUOPIO-VARKILA4 Department of Bacteriology andImmunology, University of Helsinki,4 and Departments of Infection Epidemiology3 and Special Bacterial Pathogens,' National Public Health Institute, Helsinki, and Department of Biology, University of Jyvaskyla, Jyvaskyli,2 Finland Received 22 June1993/Returned for modification 10 August1993/Accepted 15 October 1993 Seventy-nine methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated during 1980 to 1990, were classified as MRSA Aggl- (14 strains) and MRSA Aggl+ (65strains) strains on the basis of test resultsin slide agglutination assays designed to detect fibrinogen-bindingprotein (clumping factor) and protein A on the staphylococcal surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that lysostaphin digests of MRSA Aggl- strains contained a high-molecular-weight protein which was not detected in digests of MRSA Aggl+ strains. Immunization of rabbits withan MRSAAggl strain produced anantiserum which agglutinated all MRSA Aggl strains and also 64 of 65 MRSA Aggl+ strains. Only 1 of 68 coagulase-negative staphylococci showed agglutination inthis assay. The anti-MRSA Aggl antiserum reacted mainly witha 230-kDa staphylococcal surface protein but also with a 175-kDa protein, probably formed by proteolysis of the former and a few slightly smaller proteins. These could not be immunologically detected in lysostaphin digests of MRSA Aggl+ strains. Purifiedantibodies reacting with the 230-kDa proteinagglutinated all MRSA Aggl- strains, indicating that the protein is located on the surfaces of staphylococci. The results suggest a tentativerole for the 230-kDa protein or its fragments as a novel target to develop more efficient rapid identification methods for S. aureus, including MRSA. Identification of Staphylococcus aureus,an important human pathogen, is based on typical morphology, positivecoagulation reaction, production of thermostable nuclease, and utilization ofvarious sugars asa carbohydrate source (14). Thesemethods are laborious andtime-consuming, requiring incubation for several hours before the reaction result can be recorded. To overcome these drawbacks, slide agglutination tests employing particles coated either with fibrinogen or with fibrinogen andimmunoglobulin G havebeen developed for rapid detection of protein A and/or the fibrinogen-bindingprotein (clumping factor)associated with the surface of S. aureus, respectively. In numerous compar- ative studies, these tests have shown high sensitivities and specificities for S. aureus  1, 2, 4,6,8, 27). A few reports, however, indicate that 1 to 25 of methicillin-resistant S. aureus (MRSA) strains are not detected by these assays (MRSA Aggl- strains) (4, 17, 21,22, 26). In this article we describe the identification of a high- molecular-weight protein present in lysostaphin digests of MRSA Aggl- strains. A similar type of protein was also found in MRSA strainsidentified by slide agglutination tests (MRSA Aggl+), albeitin much lower concentrations. We further demonstrate that a direct bacterial agglutination assay employing antiserum against an MRSA Aggl- strain detects both types of MRSA strains with high sensitivity and specificity. * Corresponding author. Mailing address: Department of Bacte- riology and Immunology, University of Helsinki, P.O. Box 21, 00014 Helsinki, Finland. Phone: 358-0-434 61. Fax: 358-0-434 6382. Electronicmail address: pkuusela@cc.helsinki.fi. MATERIALS AND METHODS Bacterial strains. A total of79 methicillin-resistant S. aureus strains were collected during the period from 1980 to 1990 at the Department ofBacteriology and Immunology, University of Helsinki,Helsinki, Finland. The strains were isolated from clinical samples obtained in 12 different hospi- tals or outpatient health centers in the southern part of Finland. Seventy-eight of the isolates wererecoveredfrom different patients; from one patient, both an MRSA Aggl- strain and an MRSA Aggl+ strain were isolated at a 1-week interval. In order to minimize the possibility of dealing with the same bacterial strain in different patients, a period of at least 3 months was required between the isolation dates for samples srcinating from the same hospital. Also, 20methi- cillin-susceptible S. aureus (MSSA) strains per year were collected as controls for slide agglutination tests. The strains were stored in milk-glycerol at -70°C and cultivated for experiments onsheep blood agar plates for 20 to 24 h at 37°C. All the strains were coagulase, DNase, and urease producersandformed acid from maltose and trehalose. MRSA Aggl- strains were additionally confirmed by API- Staph(BioMerieux S.A.) as S. aureus strains. ATCC strains (9144,12600, 25923, and 29213 [S. aureus]; 27840 [S. capi- tis]; 35538 [S. caprae]; 29974 [S. cohnii]; 12228 and 14990 [S. epidermidis]; 35539 [S. gallinarum]; 29970 [S. haemolyti-cus]; 29885 [S. hominis]; 11249 [S. hyicus]; 29663 [S. inter- medius]; 43809 [S. lugdunensis]; 15305 [S. saprophyticus];43808 [S. schleiferiJ; 29060 [S. sciuri]; 27851 [S. simulans]; 27836 [S. warneri]; and 29971 [S. xylosus]) and neonatal septicemia S. epidermidis strains collected during a nation- wide surveillance of bacteremic diseases in children since 1985 (7) were obtained from the collection of the National Public Health Institute, Helsinki, Finland. The strains were stored at -70°C in 10 skim milk until use. Antimicrobial susceptibility. Antimicrobial susceptibility 143   onA  pr i  l  1  9  ,2  0 1 4  b  y P E N N  S T A T E  U N I  V h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om   144 KUUSELA ET AL. was determined with Neo-Sensitabs disks (A/S Rosco) and Mueller-Hinton II medium (BBL, Becton Dickinson Micro- biologySystems). Methicillinresistance was identified with 1-,ug oxacillin disks on Mueller-Hinton II agar platesincu- bated at 30°C. Oxacillin MICs were determined by the plate dilution method on Mueller-Hinton II agar plates with 4 NaCl and incubation at 37°C. Strains for which the MIC of oxacillin was >4 p,g/ml were regarded as methicillin resis- tant Phage typing. Phage typing was performed with the inter- national phage set (5)in the StaphylococcusReferenceLaboratory at the National Public Health Institute. Agglutination tests. For all agglutination experiments, strains were cultivated on sheepblood agar plates overnight at 37°C. The slide agglutination tests were performed accord- ing to the instructions of the manufacturers. Staphyslide- Test (BioMerieux) is a hemagglutination test employing fibrinogen-coated (test reagent) and uncoated (control re- agent) sheep erythrocytes to detect the clumping factor on the S. aureus surface. Staphaurex (Wellcome Diagnostics) and ANI S. aureus TEST (Ani Biotech OY, Helsinki, Finland) are latex agglutination tests in which particles are coatedwith fibrinogen and immunoglobulin G to detectthesurface-associated clumping factor and protein A, respec- tively. Latex particles are either suspended (Staphaurex) or driedreagentdots on a card (ANI S. aureus TEST). All three tests are sensitive and specific for S. aureus (20). Direct bacterial agglutination tests were performed by mixing two to three coloniesof staphylococci with absorbed and diluted (1:7) anti-MRSA Aggl- antiserum or withconcentrated purified anti-230-kDa-protein antibodies on a coverslip. Ag- glutination was recorded after 10 to 30 s. Serum from nonimmunized rabbits was used as a control. Antiserum against MRSA Aggl- strains. Antiserum against a representative MRSA Aggl- strain was produced by immunizing rabbits twice subcutaneously at 2-week intervals with 109 heat-killed bacteria mixed in Freund s complete adjuvant. Ten days after the last booster, blood was col- lected and serum was isolated. The antiserum was absorbed twice with intact S. epidermidis ATCC 12228 (2 x 109 bacteria per ml of antiserum for 2 h at 4°C) grown in Todd-Hewitt broth. For a few experiments, theantibodies against the 230-kDa protein were isolated from antiserum by adsorbing theantibodies to nitrocellulose membranes con- taining the protein band. After washings with phosphate- buffered saline (PBS), theantibodies were eluted by incu- bating the membranes for 10 min in 1 M acetate, pH 2.0, and then theeluate was neutralized and concentrated. Analysis of lysostaphin digests. For lysostaphin digestion, staphylococci were grown in Todd-Hewitt broth overnight at 37°C, collected by centrifugation, and washed twice with PBS. Finally, the bacterial density was adjusted to approx- imately 2 x 1010 bacteria per ml. Digestion was accom- plished by incubating 0.5 ml of bacterial suspension for 2 h at 37°C with 10 p,g of recombinantlysostaphin (Applied Micro- biology, Inc., New York,N.Y.) and 4 pg each of RNase and DNase (Sigma) in the presence of 0.5 mM phenylmethylsul- fonyl fluoride (Sigma)andethylmaleimide (Sigma). Unbro-ken bacterial cells were removed by centrifugation, and the supernatants were incubated for 15 min at 80°C to inhibit the enzymes. Finally, protein concentrations in the digests were determined as described previously (19). Lysostaphin digests were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (16) with slabs containing 8 acrylamide. The gels were stained with Coomassie blue for protein or, when needed, subse- TABLE 1. Characteristics of MRSA Agg- strains Susceptibility category' Strain No. of type isolates Erythro- Clinda- Tobra- Phage type mycinmycinmycin a 4 R R R 81/42E/47/54/75/84/85 b 1 SS R 81/42E/47/54/75/84/85 c 2 SSS 81/42E/47/54/75/84/85 d 2 RRR 85 e 5 R S R 85 aTotal of 14 isolates were used. I All strains were susceptible to vancomycin, rifampin, fusidicacid,netilm- icin, tetracycline, and co-trimoxazole. S,susceptible; R, resistant. C Read at 100times theroutine testdilution. The following phages were used: 3A, 3C, 6, 29, 42E, 47, 52, 52A, 53, 54, 55, 71,75,77,79,80,81, 83A, 84, 85,94, 95, and 96. quently transferredelectrophoretically tonitrocellulose membranes (24). Membranes were pretreated for 1 h at room temperature with PBS containing 5 (wt/vol) defatted milk powder and 1 (vol/vol)Triton X-100and then washed twicewith TEN-Tween buffer(0.05 M Tris-HCl [pH 7.5], 0.025 M EDTA, 0.15 M NaCl, 0.5 [vol/vol] Tween 20). Themembranes were first probed with a predetermined dilution of anti-MRSA Aggl- antiserum or control serumand thenwith horseradish peroxidase-conjugated F(ab')2 fragments of sheep antibodies to rabbit immunoglobulin G (Jackson Im- munoResearch); all probes were diluted in TEN-Tween buffer. Finally, the membranes were washed four timeswith TEN-Tween buffer and oncewith PBS. The bands were visualized by incubating the membranes in 50 ml of50 mM acetate buffer, pH 5.0, containing 3-amino-9-ethylcarbazole (10 mg), N,N -dimethylformamide (2.5 ml), and 30 hydro-genperoxide (30 ,ul . Statistics. Statistical comparisonbetween MICs for MRSA Aggl+ and MRSA Aggl- strains was done by Student's t test. RESULTS Characterization of MRSA Aggl- strains. A total of 79 MRSA strains, isolated during 1980 to 1990, were included in the study. Thenumber of isolates varied between 2 and 12 each year. Eleven of these strains showed no agglutinationreaction with three commercial slide agglutination assays designed for detection of S. aureus. Three MRSA strains displayed variable agglutination results in repeated testings with different assays. These strains were, however, recorded as MRSA Aggl- strains in theanalysis. The proportion of MRSA Aggl- strains among all MRSA strains was 17.7 . There was no statistical difference between the MICs of oxacillinfor the MRSA Aggl+ group (median, 128 tLg/ml; range, 4 to 512 p,g/ml) and that for the MRSA Aggl- group (median, 128 ,uglml; range, 64 to 256 ,ug/ml) (data not shown). All 220 MSSA strainscollected during the same period were correctly identified with these assays. Susceptibility to antibiotics and phage typing. The suscep- tibilities of MRSA Aggl- strains to various antibiotics are shown in Table 1. To studywhether the isolates represented individualstrains, the antibiotic susceptibility patterns andphage types of the strains weredetermined (Table 1 . The MRSA Aggl- strains were shown to represent five different strain types (a through e) consisting of two phage types. Neither of the phage types was common among MRSA Aggl+ strains collected during the same interval. J. CLIN. MICROBIOL.   onA  pr i  l  1  9  ,2  0 1 4  b  y P E N N  S T A T E  U N I  V h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om   RAPID DETECTION OF MRSA 145 A 1 2 3 4 5 6 7 8 910 11 12 1314 200 116- 97- A wi s v O 4w . .i 42   | SZ ~~t~W t .W S.,, -k 0- B 200 116- 97-66- :j ., 42   ... : 30   ft aw :>v.. If; b Z 4 x . . . s.NNo -e aX- ;=   _ at j: \,,.;|. +:. FIG. 1. SDS-PAGE analysis of lysostaphin digests (48 p.g of protein per slot) of 14 MRSA Agg- (A) and 14 representative MRSA Aggl+ (B) strains. The arrow indicates the 230-kDa protein notdetected in the digests of MRSA Aggl+ strains. Migration of molecular weight markers is shown on the left (weights are in thousands). SDS-PAGE analysis of lysostaphin digests. When cell wall lysostaphin digests of various MRSA strains wereanalyzed by SDS-PAGE, a clear difference was seen between digests of MRSA Aggl- strains and thoseof MRSA Aggl+ strains. Digests of 11 MRSA Aggl- strains contained a protein with a molecular weight of 230,000 which could not be visualized by protein staining in digests of the MRSA Aggl+ strains (Fig. 1A, lanes 1 through 11, and 1B, lanes 1 through 14). In thedigest of one MRSA Aggl- strain, the respectiveprotein band migrated slightlyfaster, corresponding to an approxi- mate molecular weight of 190,000 (Fig. 1A, lane 12). In digests of two MRSA Aggl- strains, no respectiveprotein band could be seen inthis region of thegel(Fig. 1A, lanes 13 and 14). Immunoblotting. In order to study the expressionof the 230-kDa protein,rabbits wereimmunized with an MRSA Aggl- strain harboring the protein. In immunoblotting oflysostaphin digests of MRSA Aggl- strains, anti-MRSA Aggl- antibodies absorbedwith S. epidennidis visualized principally the 230-kDa protein and additionally a 175-kDa protein (Fig. 2A). Thesewere notdetected in immunoblots of digests from MRSA Aggl+ strains. In MRSA Aggl- digests, the antiserum also detected two smaller proteins with approximate molecularweights of110,000 and 80,000 (Fig. 2A, lanes 1 through 11). Inthe digest of one MRSA Aggl- strain which gave alternating results inslide aggluti- nation assays, the antiserum stained two polypeptides withapproximate molecularweights of190,000 and 97,000 (Fig. 2A, lane 12). Similarly, in digests of two other alternating MRSA Aggl- strains, the antiserum detected mainly a 175-kDa polypeptide (Fig. 2A, lanes13 and 14). These polypeptides were not detected in digests of MRSA Aggl+ strains. With high concentrationsof MRSA Aggl+ digests, the antiserum detected two proteins with approximate mo- lecular weights of 120,000 to 125,000 and 100,000 to 105,000 which,however, stained much less intensively (data not shown). Nonimmunized rabbit serum did not stain any of these proteins (Fig. 2A, lower panel, lanes 1 through 14). One polypeptide was visualized with control serum in di- gests of MRSA Aggl-and MRSA Aggl+ strains(Fig. 2A and B, respectively). In MRSA Aggl- digests, the molecular weight seemed to be constant in contrast to the one found in MRSA Aggl+ digests, which varied slightly.Purified anti- bodies to the 230-kDa protein stained not only the corre- sponding230-kDaband but also the 175-kDa protein, indi- cating that the onewith a lowermolecular weight is most probablygeneratedfrom the larger one by proteolytic deg- radation(data not shown). Directbacterialagglutination assay. In directbacterial agglutination assays,rabbit antiserumobtained by immuni- zation with an MRSA Aggl- strain andabsorbed with S. epidermidis detected all 14 MRSA Aggl- strains (Table 2 . The antiserum also detected 64 of 65 MRSA Aggl+ strains and 20 of 32 MSSA strains. Interestingly, none of the 52 S. epidennidis strains, which included both methicillin-resis- tant and methicillin-susceptible strains, and only 1 strain (an S. hominis isolate) of16other coagulase-negativestaphylo- cocci gave a positive result in direct agglutination assay A 1 2 3 4567 8 9 10 1112 13 14 200 -   97 - 66 -gggggggp--- up 44 - 30   200 - 97 - 66 - 44 - 30 - B 1 2 3 4 5 6 7 8 9 10 11 12 13 14   I*e is X,4  l   MlS  m man FIG. 2. Immunoblotting analysis of lysostaphin digests(5.2 to6.0 pg of protein per slot) of14 MRSA Aggl- (A) and 14 representative MRSA Aggl+ (B) strains with absorbed anti-MRSA Aggl- antiserum(upper panels) andnormal rabbit serum asa control (lower panels). Migration of molecular weight markers is shown on the left (weights are in thousands). For details, see Materials and Methods. VOL. 32, 1994   onA  pr i  l  1  9  ,2  0 1 4  b  y P E N N  S T A T E  U N I  V h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om 
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