Vol. 32, No. 1JOURNAL OF CLNICAL MICROBIOLOGY, Jan. 1994, p. 143-1470095-1137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Rapid Detection of Methicillin-Resistant Staphylococcus aureusStrains Not Identified by Slide Agglutination Tests

P. KUUSELA,l* P. HILDtN,l K. SAVOLAINEN,1 M. VUENTO,2 0. LYYTIKAINEN,3AND J. VUOPIO-VARKILA4

Department ofBacteriology and Immunology, University of Helsinki,4 and Departments ofInfectionEpidemiology3 and Special Bacterial Pathogens,' National Public Health Institute, Helsinki,

and Department ofBiology, University ofJyvaskyla, Jyvaskyli,2 Finland

Received 22 June 1993/Returned for modification 10 August 1993/Accepted 15 October 1993

Seventy-nine methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated during 1980 to 1990, wereclassified as MRSA Aggl- (14 strains) and MRSA Aggl+ (65 strains) strains on the basis of test results in slideagglutination assays designed to detect fibrinogen-binding protein (clumping factor) and protein A on thestaphylococcal surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed thatlysostaphin digests of MRSA Aggl- strains contained a high-molecular-weight protein which was not detectedin digests ofMRSA Aggl+ strains. Immunization of rabbits with an MRSAAggl strain produced an antiserumwhich agglutinated all MRSA Aggl strains and also 64 of 65 MRSA Aggl+ strains. Only 1 of 68coagulase-negative staphylococci showed agglutination in this assay. The anti-MRSA Aggl antiserum reactedmainly with a 230-kDa staphylococcal surface protein but also with a 175-kDa protein, probably formed byproteolysis of the former and a few slightly smaller proteins. These could not be immunologically detected inlysostaphin digests ofMRSA Aggl+ strains. Purified antibodies reacting with the 230-kDa protein agglutinatedall MRSA Aggl- strains, indicating that the protein is located on the surfaces of staphylococci. The resultssuggest a tentative role for the 230-kDa protein or its fragments as a novel target to develop more efficient rapididentification methods for S. aureus, including MRSA.

Identification of Staphylococcus aureus, an importanthuman pathogen, is based on typical morphology, positivecoagulation reaction, production of thermostable nuclease,and utilization of various sugars as a carbohydrate source(14). These methods are laborious and time-consuming,requiring incubation for several hours before the reactionresult can be recorded. To overcome these drawbacks, slideagglutination tests employing particles coated either withfibrinogen or with fibrinogen and immunoglobulin G havebeen developed for rapid detection of protein A and/or thefibrinogen-binding protein (clumping factor) associated withthe surface of S. aureus, respectively. In numerous compar-ative studies, these tests have shown high sensitivities andspecificities 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 ofMRSA Aggl- strains. A similar type of protein was alsofound in MRSA strains identified by slide agglutination tests(MRSA Aggl+), albeit in much lower concentrations. Wefurther demonstrate that a direct bacterial agglutinationassay employing antiserum against an MRSA Aggl- straindetects both types of MRSA strains with high sensitivity andspecificity.

* 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.Electronic mail address: pkuusela@cc.helsinki.fi.

MATERIALS AND METHODSBacterial strains. A total of 79 methicillin-resistant S.

aureus strains were collected during the period from 1980 to1990 at the Department of Bacteriology and Immunology,University of Helsinki, Helsinki, Finland. The strains wereisolated from clinical samples obtained in 12 different hospi-tals or outpatient health centers in the southern part ofFinland. Seventy-eight of the isolates were recovered fromdifferent patients; from one patient, both an MRSA Aggl-strain and an MRSA Aggl+ strain were isolated at a 1-weekinterval. In order to minimize the possibility of dealing withthe same bacterial strain in different patients, a period of atleast 3 months was required between the isolation dates forsamples originating from the same hospital. Also, 20 methi-cillin-susceptible S. aureus (MSSA) strains per year werecollected as controls for slide agglutination tests. The strainswere stored in milk-glycerol at -70°C and cultivated forexperiments on sheep blood agar plates for 20 to 24 h at37°C. All the strains were coagulase, DNase, and ureaseproducers and formed 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 neonatalsepticemia S. epidermidis strains collected during a nation-wide surveillance of bacteremic diseases in children since1985 (7) were obtained from the collection of the NationalPublic Health Institute, Helsinki, Finland. The strains werestored at -70°C in 10% skim milk until use.

Antimicrobial susceptibility. Antimicrobial susceptibility

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144 KUUSELA ET AL.

was determined with Neo-Sensitabs disks (A/S Rosco) andMueller-Hinton II medium (BBL, Becton Dickinson Micro-biology Systems). Methicillin resistance was identified with1-,ug oxacillin disks on Mueller-Hinton II agar plates incu-bated at 30°C. Oxacillin MICs were determined by the platedilution method on Mueller-Hinton II agar plates with 4%NaCl and incubation at 37°C. Strains for which the MIC ofoxacillin 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 Staphylococcus ReferenceLaboratory at the National Public Health Institute.

Agglutination tests. For all agglutination experiments,strains were cultivated on sheep blood agar plates overnightat 37°C. The slide agglutination tests were performed accord-ing to the instructions of the manufacturers. Staphyslide-Test (BioMerieux) is a hemagglutination test employingfibrinogen-coated (test reagent) and uncoated (control re-agent) sheep erythrocytes to detect the clumping factor onthe S. aureus surface. Staphaurex (Wellcome Diagnostics)and ANI S. aureus TEST (Ani Biotech OY, Helsinki,Finland) are latex agglutination tests in which particles arecoated with fibrinogen and immunoglobulin G to detect thesurface-associated clumping factor and protein A, respec-tively. Latex particles are either suspended (Staphaurex) ordried reagent dots on a card (ANI S. aureus TEST). All threetests are sensitive and specific for S. aureus (20). Directbacterial agglutination tests were performed by mixing twoto three colonies of staphylococci with absorbed and diluted(1:7) anti-MRSA Aggl- antiserum or with concentratedpurified anti-230-kDa-protein antibodies on a coverslip. Ag-glutination was recorded after 10 to 30 s. Serum fromnonimmunized rabbits was used as a control.Antiserum against MRSA Aggl- strains. Antiserum against

a representative MRSA Aggl- strain was produced byimmunizing rabbits twice subcutaneously at 2-week intervalswith 109 heat-killed bacteria mixed in Freund's completeadjuvant. Ten days after the last booster, blood was col-lected and serum was isolated. The antiserum was absorbedtwice with intact S. epidermidis ATCC 12228 (2 x 109bacteria per ml of antiserum for 2 h at 4°C) grown inTodd-Hewitt broth. For a few experiments, the antibodiesagainst the 230-kDa protein were isolated from antiserum byadsorbing the antibodies to nitrocellulose membranes con-taining the protein band. After washings with phosphate-buffered saline (PBS), the antibodies were eluted by incu-bating the membranes for 10 min in 1.0 M acetate, pH 2.0,and then the eluate was neutralized and concentrated.

Analysis of lysostaphin digests. For lysostaphin digestion,staphylococci were grown in Todd-Hewitt broth overnight at37°C, collected by centrifugation, and washed twice withPBS. 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 at37°C with 10 p,g of recombinant lysostaphin (Applied Micro-biology, Inc., New York, N.Y.) and 4 pg each of RNase andDNase (Sigma) in the presence of 0.5 mM phenylmethylsul-fonyl fluoride (Sigma) and ethylmaleimide (Sigma). Unbro-ken bacterial cells were removed by centrifugation, and thesupernatants were incubated for 15 min at 80°C to inhibit theenzymes. Finally, protein concentrations in the digests weredetermined as described previously (19).

Lysostaphin digests were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (16)with slabs containing 8% acrylamide. The gels were stainedwith Coomassie blue for protein or, when needed, subse-

TABLE 1. Characteristics of MRSA Agg- strains

Susceptibility category'Strain No. oftype isolates" Erythro- Clinda- Tobra- Phage type"

mycin mycin mycin

a 4 R R R 81/42E/47/54/75/84/85b 1 S S R 81/42E/47/54/75/84/85c 2 S S S 81/42E/47/54/75/84/85d 2 R R R 85e 5 R S R 85

aTotal of 14 isolates were used.I All strains were susceptible to vancomycin, rifampin, fusidic acid, netilm-

icin, tetracycline, and co-trimoxazole. S, susceptible; R, resistant.C Read at 100 times the routine test dilution. 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 transferred electrophoretically to nitrocellulosemembranes (24). Membranes were pretreated for 1 h at roomtemperature with PBS containing 5% (wt/vol) defatted milkpowder and 1% (vol/vol) Triton X-100 and then washedtwice with 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 dilutionof anti-MRSA Aggl- antiserum or control serum and thenwith horseradish peroxidase-conjugated F(ab')2 fragments ofsheep antibodies to rabbit immunoglobulin G (Jackson Im-munoResearch); all probes were diluted in TEN-Tweenbuffer. Finally, the membranes were washed four times withTEN-Tween buffer and once with PBS. The bands werevisualized by incubating the membranes in 50 ml of 50 mMacetate buffer, pH 5.0, containing 3-amino-9-ethylcarbazole(10 mg), N,N'-dimethylformamide (2.5 ml), and 30% hydro-gen peroxide (30 ,ul).

Statistics. Statistical comparison between MICs for MRSAAggl+ and MRSA Aggl- strains was done by Student's ttest.

RESULTS

Characterization of MRSA Aggl- strains. A total of 79MRSA strains, isolated during 1980 to 1990, were included inthe study. The number of isolates varied between 2 and 12each year. Eleven of these strains showed no agglutinationreaction with three commercial slide agglutination assaysdesigned for detection of S. aureus. Three MRSA strainsdisplayed variable agglutination results in repeated testingswith different assays. These strains were, however, recordedas MRSA Aggl- strains in the analysis. The proportion ofMRSA Aggl- strains among all MRSA strains was 17.7%.There was no statistical difference between the MICs ofoxacillin for 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 notshown). All 220 MSSA strains collected during the sameperiod were correctly identified with these assays.

Susceptibility to antibiotics and phage typing. The suscep-tibilities of MRSA Aggl- strains to various antibiotics areshown in Table 1. To study whether the isolates representedindividual strains, the antibiotic susceptibility patterns andphage types of the strains were determined (Table 1). TheMRSA Aggl- strains were shown to represent five differentstrain types (a through e) consisting of two phage types.Neither of the phage types was common among MRSAAggl+ strains collected during the same interval.

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RAPID DETECTION OF MRSA 145

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FIG. 1. SDS-PAGE analysis of lysostaphin digests (48 p.g ofprotein per slot) of 14 MRSA Agg- (A) and 14 representativeMRSA Aggl+ (B) strains. The arrow indicates the 230-kDa proteinnot detected in the digests of MRSA Aggl+ strains. Migration ofmolecular weight markers is shown on the left (weights are inthousands).

SDS-PAGE analysis of lysostaphin digests. When cell walllysostaphin digests of various MRSA strains were analyzedby SDS-PAGE, a clear difference was seen between digestsof MRSA Aggl- strains and those of MRSA Aggl+ strains.Digests of 11 MRSA Aggl- strains contained a protein witha molecular weight of 230,000 which could not be visualizedby protein staining in digests of the MRSA Aggl+ strains(Fig. 1A, lanes 1 through 11, and 1B, lanes 1 through 14). Inthe digest of one MRSA Aggl- strain, the respective proteinband migrated slightly faster, corresponding to an approxi-mate molecular weight of 190,000 (Fig. 1A, lane 12). Indigests of two MRSA Aggl- strains, no respective proteinband could be seen in this region of the gel (Fig. 1A, lanes 13and 14).

Immunoblotting. In order to study the expression of the230-kDa protein, rabbits were immunized with an MRSAAggl- strain harboring the protein. In immunoblotting oflysostaphin digests of MRSA Aggl- strains, anti-MRSAAggl- antibodies absorbed with S. epidennidis visualizedprincipally the 230-kDa protein and additionally a 175-kDaprotein (Fig. 2A). These were not detected in immunoblotsof digests from MRSA Aggl+ strains. In MRSA Aggl-digests, the antiserum also detected two smaller proteinswith approximate molecular weights of 110,000 and 80,000(Fig. 2A, lanes 1 through 11). In the digest of one MRSAAggl- strain which gave alternating results in slide aggluti-nation assays, the antiserum stained two polypeptides withapproximate molecular weights of 190,000 and 97,000 (Fig.2A, lane 12). Similarly, in digests of two other alternatingMRSA Aggl- strains, the antiserum detected mainly a175-kDa polypeptide (Fig. 2A, lanes 13 and 14). Thesepolypeptides were not detected in digests of MRSA Aggl+strains. With high concentrations of MRSA Aggl+ digests,the antiserum detected two proteins with approximate mo-lecular weights of 120,000 to 125,000 and 100,000 to 105,000which, however, stained much less intensively (data notshown). Nonimmunized rabbit serum did not stain any ofthese proteins (Fig. 2A, lower panel, lanes 1 through 14).One polypeptide was visualized with control serum in di-gests ofMRSA Aggl- and MRSA Aggl+ strains (Fig. 2A andB, respectively). In MRSA Aggl- digests, the molecularweight seemed to be constant in contrast to the one found inMRSA Aggl+ digests, which varied slightly. Purified anti-bodies to the 230-kDa protein stained not only the corre-sponding 230-kDa band but also the 175-kDa protein, indi-cating that the one with a lower molecular weight is mostprobably generated from the larger one by proteolytic deg-radation (data not shown).

Direct bacterial agglutination assay. In direct bacterialagglutination assays, rabbit antiserum obtained by immuni-zation with an MRSA Aggl- strain and absorbed with S.epidermidis detected all 14 MRSA Aggl- strains (Table 2).The antiserum also detected 64 of 65 MRSA Aggl+ strainsand 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 (anS. hominis isolate) of 16 other coagulase-negative staphylo-cocci gave a positive result in direct agglutination assay

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FIG. 2. Immunoblotting analysis of lysostaphin digests (5.2 to 6.0 pg of protein per slot) of 14 MRSA Aggl- (A) and 14 representativeMRSA Aggl+ (B) strains with absorbed anti-MRSA Aggl- antiserum (upper panels) and normal rabbit serum as a control (lower panels).Migration of molecular weight markers is shown on the left (weights are in thousands). For details, see Materials and Methods.

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146 KUUSELA ET AL.

TABLE 2. Direct bacterial agglutination with antiserum againstMRSA Aggl- strains

No. of strains

Organism(s) Showing agglutination witha:Total Anti-MRSA Anti-230-kDa

Aggl- protein NRS

MSSA 32 20 NT 0MRSA Aggl- 14 14 14 0MRSA Aggl+ 65 64 ob 0S. epidennidisc 52 0 NT 0Coagulase-negative 16 id NT 0

staphylococcia NRS, normal rabbit serum; NT, not tested.b 14 of 65 strains were tested.c 42 methicillin-resistant and 10 methicillin-susceptible strains.d S. hominis.

(Table 2). Purified antibodies to the 230-kDa protein agglu-tinated all 14 MRSA Aggl- strains, indicating that 230- and175-kDa proteins are exposed on the bacterial surface. Ofthe 64 MRSA Aggl+ strains, 14 were tested. None of themagglutinated with the same purified antibodies (Table 2).

DISCUSSION

The present results confirm earlier findings that among

clinical MRSA isolates there are strains which cannot beidentified by slide agglutination assays designed to detectfibrinogen-binding protein (clumping factor) or protein A onthe staphylococcal surface. In our study, their frequencywas 17.7% of MRSA isolates, which is greater than has beenreported earlier (4, 17, 21, 22, 26). Ruane et al. (22) describedfailure rates of 17 and 25% for Staphaurex and Staphyslide-Test, respectively, among 73 MRSA strains obtained fromthree different hospitals in California but also claimed thatthe high percentage of false-negative results might have beendue to a strain endemic to that area. The same possibility isnot completely excluded in the present investigation, al-though five different clones based on phage types andantibiograms could be found among MRSA Aggl- isolates.The MRSA Aggl- strains do not represent a subpopulationwith especially high or, alternatively, only moderate resis-tance towards oxacillin, as the MICs of oxacillin for MRSAAggl- strains and those for MRSA Aggl+ strains do notshow any statistically significant difference.The failure of slide agglutination tests to detect MRSA

Aggl- strains may suggest that protein A and clumpingfactor are poorly available on the bacterial surface. We haveshown that these strains harbor an extra surface protein withan approximate molecular weight of 230,000 which was notdetected in digests of MRSA Aggl+ ones. This raised thepossibility that antibodies against MRSA Aggl- strainswould provide a tool for detecting MRSA strains negative inslide agglutination assays.

In direct bacterial agglutination assays, anti-MRSA Aggl-antiserum detected all the MRSA Aggl- strains and also 64of 65 MRSA Aggl+ strains. The results thus indicate thatMRSA Aggl+ strains also contain the antigenic structures ontheir surfaces. The test also showed a high specificity, sinceonly one S. hominis strain of 68 coagulase-negative staphy-lococci was positive in this assay.

Immunoblotting analysis showed that the immunologicalreactivity of the antiserum was mainly directed toward the230-kDa protein and additionally to a 175-kDa protein. When

larger amounts of protein were loaded onto the gel, smallerproteins could be detected in digests of both MRSA Aggl-and MRSA Aggl+ strains. Together with the finding thatisolated anti-230-kDa-protein antibodies also stained the175-kDa protein in immunoblotting experiments, the presentresults favor the idea that the 230-kDa protein may exist indifferent molecular forms on both MRSA Aggl- and MRSAAggl+ strains, although in much smaller quantities on thelatter ones. This is also in agreement with the finding thatagglutination of MRSA Aggl+ strains by the antiserum wasmuch weaker than that of MRSA Aggl- strains. There is,however, a possibility that the smaller polypeptides detectedin digests of both MRSA groups are not related to either the230- or 175-kDa protein but represent another antigen-antibody system. This has to be studied in more detail byusing antibodies raised against the purified 230-kDa protein.The 48-kDa pentaglycine cross-linking protein and the

74-kDa modified penicillin-binding protein (PBP-2'), prod-ucts of two methicillin resistance genes, femA and mec,respectively, appear to have molecular weights considerablylower than 230,000 (3, 25). Therefore, the 230-kDa proteinidentified in the present study does not seem to be related tothese factors and represents a previously uncharacterizedprotein. It is also expressed on the bacterium, since purifiedantibodies to the 230-kDa protein caused the agglutination ofall MRSA Aggl- strains. Interestingly, two commercial slideagglutination assays have been introduced recently whichtake advantage of specific binding of monoclonal antibodieseither to the surface protein(s) (Slidex; BioMerieux) or totype 5 and 8 capsular polysaccharides (Pastorex Staph-Plus;Sanofi Diagnostics Pasteur) of S. aureus. In a few compar-ative studies, these assays also efficiently detected MRSAstrains which remained negative in slide agglutination tests,strains similar to those used in our study (9, 10, 13). Atpresent, it is not known how the staphylococcal surfaceprotein(s) detected by the Slidex test relates to the 230- and175-kDa proteins described in this article. Guzman et al. (11)also described an enzyme-linked immunosorbent assay em-ploying antigenic differences of excreted staphylococcalglucosaminidase to differentiate various staphylococcal spe-cies. This test was also able to identify MRSA strains whichdid not produce protein A or staphylocoagulase or both.

Studies in progress in our laboratory indicate that theavailability of binding proteins for ligands such as fibronec-tin, laminin, and collagens (12, 15, 18, 23), as well as theavailability of protein A and clumping factor, is reduced onthe surface of the MRSA Aggl- strains included in thisstudy. However, in immunoblots of lysostaphin digests ofMRSA Aggl- strains, the control rabbit serum also detecteda polypeptide with a molecular weight correspondingroughly to the molecular weight of protein A, although itcould not be detected on the staphylococcal surface. Thiscan be explained by the possibility that protein A and/orother binding proteins are prevented by steric hindrancefrom interacting with their counterparts. It remains to beseen whether the 230-kDa protein has this type of inhibitoryrole. It will also be interesting to see whether agglutinationassays which use antibodies to the 230-kDa protein or itssynthetic peptides will provide a more applicable assaysystem to identify S. aureus strains which have surfaceproteins with altered compositions.

ACKNOWLEDGMENTSSirpa Kuisma is acknowledged for excellent technical assistance.

Aino Takala is kindly thanked for providing the neonatal septicemiaS. epidermidis strains.

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The work was supported by the Paulo Foundation.

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