J Clin Pathol 1988;41:103-1 10

Technical methodsImmunoblot fingerprinting ofcoagulase negative staphylococci

JP BURNIE, W LEE, RC MATTHEWS, R BAYSTON* Fromthe Department of Medical Microbiology, StBartholomew's Hospital, London, and the *Institute ofChild Health, London

Coagulase negative staphylococci have becomeprevalent nosocomial pathogens following theimplantation of foreign bodies such as prostheticvalves, cerebrospinal fluid shunts, joint prostheses andcentral venous catheters. They are also the mostcommon contaminants of blood cultures so that ameans of differentiating contamination from a truebacteraemia is important.' Assessment of the clinicalimportance of an individual blood culture isolate iscurrently based on the clinical condition of the patientand the presence of similar isolates with identicalantibiograms from subsequent blood cultures.2Unrelated isolates from different patients, however,may have the same antibiogram,3 and retesting thesame isolate does not always give reproducibleresults.4

Baird-Parker5 produced a scheme for differentiatingsix biotypes ofcoagulase negative Staphylococcus (SI-SVI) and eight biotypes of Micrococcus. Kloss et alused these for species specific groups,6 but the problemremained that isolates of biotype SII (S epidermidis)comprised 75% of all clinically important isolates sothat further typing methods were required to sub-divide these isolates.7 Results with phage typing havebeen inadequate both in terms of the number ofisolates typable and in the degree of reproducibility.8Examination of protein profiles obtained by sodiumdodecyl sulphate polylacrylamide gel electrophoresis(SDS-PAGE) showed that these were species specificbut could not subdivide isolates within the species.9SDS-PAGE typing with sulphur-35 methionine hasbeen of some value but this is an expensive techniquerequiring the use of radiolabels and has only beenapplied to a few isolates.'" Isolates of Staphylococcusaureus have been typed by immunoblotting culturesupernatants against human antiserum."

In this paper we report the results of immunoblotfingerprinting of isolates of coagulase negative sta-phylococci. Isolates were examined from 17 unrelatedpatients with systemic infections to assess the ability ofthe technique to discriminate between different strains.Multiple isolates were also available from seven

Accepted for publication 8 July 1987

patients in whom there was a clinical problem thoughtto be related to coagulase negative staphylococcusinfection. In all cases multiple isolates were obtainedfrom blood cultures, and it was important to decidewhether these were several different contaminants or asingle predominant pathogenic strain. All isolateswere probed by a rabbit hyperimmune antiserumraised against the predominant strain of coagulasenegative staphylococcus, Staphylococcus epidermidis,from one of these cases (case 3). Individual isolatesfrom the 17 patients with systemic infections and allisolates from three of the multiply infected systemiccases (cases 5, 6, and 7) were also probed by the humanantiserum from case 5 and a rabbit hyperimmuneantisera raised against Staphylococcus aureus. Theresults were then compared with those obtained fromphage typing, biochemical studies, and antibiograms.

Material and methods

Seventeen isolates were collected from 17 differentpatients with systemic coagulase negative sta-phylococcal infections. Seven additional patients hadmultiple isolates from blood cultures (table 1).The coagulase negative staphylococcus from case 3

and an isolate of the epidemic methicillin resistant Saureus were grown overnight on blood agar plates.Each was harvested, washed in distilled water, andfragmented by a French press at 12 mPa. Fiftymilligrams of the supernatant obtained by spinning at12000 g for 15 minutes was mixed with I ml ofcomplete Freund's adjuvant. New Zealand whiterabbits were immunised on day 0 and day 14 bysubcutaneous injection. The rabbits were bled at 28days.

Table I Details ofseven patients with multiple blood cultureisolates

No positiveNo of forblood coagulase

Case samples negative ClinicalNo taken staphylococci comment

I 18 18 Aortic and mitralvalve endocarditis

2 6 6 Aortic valveendocarditis

3 12 12 Hickman line infection4 9 3 Candida septicaemia

treated byamphotericin B

5* 14 12 Aortic valve endocarditis6* 2 2 Infected central line7* 3 3 Infected central line

*These three cases were all on the intensive care unit at StBartholomew's Hospital at the same time.

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104Pooled human antiserum was obtained from case 5

and used to fingerprint the 17 individual isolates fromdifferent patients and all the isolates from cases 5, 6,and 7.

IMMUNOBLOT FINGERPRINTINGPreparation ofisolatesEach isolate was grown overnight at 37°C in tryptone-soya broth. After centrifuging pellets were resuspen-ded in 100 pl sterile distilled water and 100 p1lysostaphin (Sigma; 200 pg/ml) and incubated at 37°Cfor 30 minutes. Protein concentration was measuredby the Lowry method and standardised to 10 mg/ml.

Gel electrophoresisThe enzyme extracts were solubilised in 2-5% sodiumdodecyl sulphate (SDS) and 1-3% 2-mercaptoethanolat 1 00°C for four minutes. Electrophoresis was carriedout on a 10% polyacrylamide gel in a discontinuousbuffer system. Extract (60 p1) was applied to each well.The following molecular weight standards were used:myosin, 200 000; betagalactosidase, 11 650; phos-phorylase B, 92 500; bovine serum albumin, 66 200;ovalbumin 45 000; carbonic anhydrase 31 000;soybean trypsin inhibitor, 21 500, and lysozyme,14 400.

ImmunoblottingThe preparations were transferred from gel tonitrocellulose paper in a LKB Trans-Blot cell with acurrent of06A for 45 minutes at 25°C. A 25 mM= Tris,192 mM glycine buffer (pH 8 3) containing 20%methanol was used. Free protein sites were saturatedby incubation in 3% bovine serum albumin in bufferedsaline (0 9% sodium chloride and 10mM Tris (p1- 7 4)at 4°C overnight. The nitrocellulose was thenincubated at 25°C for two hours with the rabbit orhuman antiserum, diluted I in 50 in 3% bovine serumalbumin and 0 05% Tween 20. After washing fivetimes in 0 9% sodium chloride and 0 05% Tween 20the nitrocellulose was incubated for one hour withalkaline phosphatase conjugated goat antirabbit IgG(Sigma), or alkaline phosphatase conjugated goatantihuman IgG and IgM (Sigma), used at a 1 in 1000dilution in 3% bovine serum albumin. After washingagain this was incubated for 15 minutes with equalvolumes of naphthol AS-MX phosphate (Sigma; 0 4mg/ml in distilled water) and fast red TR salt (Sigma; 6mg/ml in 0-2 M, Tris, pH 8.2).2

OTHER TYPING SYSTEMSAntibiogramsThis was performed by a disc susceptibility method onIsosensitest agar (Oxoid CM 471). Ten ug discs ofmethicillin, tetracycline, gentamicin, erythromycin,fusidic acid and chloramphenicol were used as well as

Technical methods2 ug discs of rifampicin and clindamycin. Penicillinwas tested with a 1 ug disc. All tests were performed at35°C except methicillin which was performed at 30°C.

Biochemistry andphage typingIsolates were biotyped by the commercially availableAPI Staph system (API SA System, La Balme, lesGrottes, France) and phage typed by the Division ofHospital Infection, Colindale.

Results

Immunoblot fingerprinting detected multipleantigenic bands with molecular weights varying from27 to 250 kD. The technique was evaluated by threecriteria: typability, reproducibility, and ability todiscriminate between two isolates that were con-sidered to be distinct on clinical and bacteriologicalgrounds. All isolates were typable by this method.Reproducibility has presented problems whenanalogous techniques have been applied to othermicro-organisms.'3 To compensate for this in the caseof C albicans, all the isolates were run on the same geland prepared with the same batch ofenzyme. When allthe isolates of coagulase negative staphylococci wereexamined twice, however, the degree ofreproducibilitywas sufficient for a direct comparison between twoimmunoblots.A difference in the position and intensity of three

antigenic bands has been the criterion used to decidewhether two isolates are definitely different.'4 Theresults of nine of the 17 systemic isolates from 17different patients are shown in figs I to 3. The anti-Sepidermidis rabbit hyperimmune antiserum detected19 antigenic bands and showed at least three antigenicband differences between all isolates with the excep-tion of isolates 5 and 7 which were identical (fig 1). The

08

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FiglI Nine systemic isolates stained by anti-S epidermidisserum.

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105

250) + / ; .!:.:. .4s 250230-220 ~22 0 230185 190

12 8120~ ~ 120120nn...* ., 5n~~~~~~~~698

90 V! ."!,

~' .- . 68-

64

520 ~ i ...5~~ ... jz ... _|> ^ . ~4640 *+" ~ 4

35 ... .. .....

1 2 3 4 5 6 7 8 9

Fig 2 Nine systemic isolates stained by rabbit anti-S aureusserum.

anti-MRSA rabbit antiserum detected 18 antigenicbands and found at least three differences between allisolates with the exception of isolates 5 and 7 whichwere identical (fig 2). The human hyperimmune serumfrom case 5 identified 13 antigenic bands only andproduced less than three antigenic band differencesbetween isolates 5, 7, and 8 (fig 3). Isolates 5, 7, and 8(table 2) were all non-typable by phages and werebiotype SVI(I). Isolates 5 and 7 had the sameantibiogram (table 2). The SII biotype isolates, shownby tracts 2, 4, 6 and 9, are clearly differentiated fromeach other by all three antisera. In the remaining eightisolates (data not shown) there were more than threedifferences between each set of isolates. Only two ofthe 17 isolates were typable by phages and eight werebiotype SI1. Antibiograms produced 11 different typescompared with the 16 seen by fingerprinting with therabbit anti-S epidermidis hyperimmune antiserum.

72 _ _68 _N4"

e:w ... _IM 64

37 _ .. 3735 II-4!t3 5

30_n O _ _ _3029 _o 29

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1 2 3 4 5 6 7 8 9

Fig 3 Nine systemic isolates stained by human antisernm.

The multiple isolates from individual patients werethus fingerprinted by this antiserum and in cases 5-7by the human antiserum from case 5.The results of immunoblot fingerprinting of the

multiple isolates from the patients correlated well withantibiograms, biotyping, phage typing and clinicalhistory (table 3). In cases 1, 2, and 3 a pattern of apredominant isolate, which was untypable by phagetyping, indistinguishable by biotyping, and identicalby antibiogram and immunoblot typing, was observed(table 3). Fifteen of the 18 isolates from case one wereidentical on all the test systems examined and two ofthe remaining three showed distinct immunoblotpatterns, phage types, biotypes and antibiograms. Thefinal isolate had a distinct immunoblot pattern. Fourisolates from case 3 are shown in fig 4. In case 4 allthree isolates were untypable by phage typing andindistinguishable by biotyping but were distinguish-

Table 2 Details ofphage typing, biotype, and antibiogramsfrom 17 systemic isolates

AntibiogramIsolate PhageNo typable Biotype P M F R C E G V

- M3 R R R S S S S S2 - SIl R S S S S S S S3 - SV R R R R S S R S4 - Sl R S R S R R R S5 - SVI(I) R R S S R R R S6 + Sil R S S S S S S S7 - SVI(I) R R S S R R R S8 - SVI(I) R S S S S S S S9 +t SIl R S S S S S R S10 - SVI(3) S S S S S S S S11 - SV R R S R S S S S12 - SIl R R R S R R R S13 - SVI(3) S S S S S S S S14 - SV R R S R S S R S15 - SIl R R S S S S S S16 - Sl R S S S S S S S17 - S1l S S S S S S S S

*phage type 27/1 55/A6C/A9C/7 1/275/48/456/471 /A/459/275A/B 1; tphage type 1 55/A6C/A9C/456/459.P: penicillin; M: methicillin; F: fusidic acid; R: rifampicin; C: chloramphenicol; E: erythromycin; G: gentamicin; and V: vancomycin

Technical methods

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Technical methods25., 9, and 10). These were different from each other,2 202f confirming the existence of a cluster of isolates on the

unit rather than an outbreak. The pattern of results18 1' with the human antiserum was identical, although

only eight antigenic bands were detected.1 2S

38949088

Fig 4 Tracts 1-3, isolatesfrom case 4; tracts 4-7, isolatesfrom case three; tract 8, biotype SII isolatesfrom casefive;tract 9, biotype SII isolatefrom case six; tract 10, biotypeSII isolatefrom case seven; All isolates stained by anti-Sepidermidis serum.

able by antibiograms and immunoblotting (fig 4, tracts1-3). There was greater than three antigenic banddifferences between each of the isolates (fig 4, tracts 1-3). This was compatible with the clinical history, whichsuggested that the blood culture isolates were con-taminants.

Cases 5, 6, and 7 formed a cluster on the intensivecare unit at St Bartholomew's Hospital. All 17 isolatesproduced the same antibiogram and 13 were biotypeSII (table 3). Phage typing split the isolates from case 5into two main groups, but all produced the samepattern on immunoblot fingerprinting. The non-biotype SII isolates from all three cases producedgreater than six differences from each other and the SIIisolates on immunoblotting. An example of an SIIisolate from each ofthe cases is shown in fig 4 (tracts 8,

Discussion

Coagulase negative staphylococci are the organismsmost commonly isolated from blood cultures.'5 Theidentification of a true infection rather than multiplecontaminants is based on the repeated isolation oforganisms with an identical phenotype from severalsets of blood culture bottles. This phenotype has beendefined by markers which have included antibiograms,plasmid profiles, biotypes and phage types.Antibiograms can be difficult to reproduce andisolates from different patients may have the same pat-tern.416 In this study the 17 systemic isolates producedonly 11 different antibiograms (table 2); the isolatesfrom cases 5, 6, and 7 had identical antibiograms (table3).

Plasmid profile analysis has been regarded as a moreprecise method, but is only marginally better insensitivity, and difficult to interpret.'7 Antibiotic resis-tant plasmids transfer between coagulase negative andcoagulase positive staphylococci and between strainsof coagulase negative staphylococci, which is animportant factor to consider if they are the basis of atyping scheme.'8Compared with phage typing, the immunoblot

technique has the advantage that all isolates aretypable. It was clearly more sensitive than biotyping asbiotype SIT has been shown to cause up to 75% ofinfections.7 In the present series 42 isolates had thisbiotype so that it was essential to be able to subdivide

Table 3 Details ofphage typing, biotype, and antibiograms ofmultiple isolatesfrom cases 1-7

AntibiogramCase Isolate PhageNo No typable Biotype P M F R C E G V

I 1-16 SV R R S S S S R S17 SVI(2) S S S S S S S S18 +* SVI(3) S S S S S S S S

2 1-6 - SII S S S S S S S S3 1-12 - SII R R S S S S R S4 1 - SIl S S S S S S S S

2 - SI R S S S S S S S3 - SIl R R S S S S R S

5 1-4 - SIT R R R R R R R S5-10 +1tSI R R R R R R R S11 M6 R R R R R R R S12 - SVI R R R R R R R S

6 1 SIT R R R R R R R S2 +: SI1 R R R R R R R S

7 1 SV R R R R R R R S2 - SVI R R R R R R R S3 - SIl R R R R R R R S

*phage A6c; inhibition reaction 155/B1I; tphage A6c; tphage 1 55/A9c.P: penicillin; M: methicillin; F: fusidic acid; R: rifampicin; C: chloramphenicol; E: erythromycin; G: gentamicin; and V: vancomycin

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Technical methods 107

it. On clinical grounds, excluding multiple identicalisolates from the same patients, this represented 17different isolates. Antibiograms identified nineseparate patterns and four isolates produced distinctphage types. Immunoblot fingerprinting with therabbit anti-S epidermidis serum as probe produced atleast three antigenic band differences between each ofthese isolates. Reproducibility has been a problemwith other fingerprinting systems,'3 but with sta-phylococci the lysostaphin based breakdownproduced sufficiently similar extracts for gel to gelcomparison.'4 We confirmed this, provided that thesame batch of antiserum was the antibody probe. Therabbit hyperimmune antisera were better than thehuman sera at differentiating between isolates. Isolates5 and 7 of the 17 systemic cases were identical with allthree antisera, isolate 8 could only be differentiated bythe rabbit hyperimmune antisera.Immunoblot fingerprinting also showed the

similarity of multiple isolates from the same patientwhen these were thought to be a single pathogenicstrain rather than multiple different contaminants. Incase 1 ofthe 16 isolates which were biotype SV, 15 wereidentical on immunoblot fingerprinting. This confir-med the clinical importance of the strain and allowedone isolate to be selected for further antibiotic sen-sitivity testing. In case 5, another case of endocarditis,immunoblot fingerprinting showed the existence ofone predominant strain despite the presence of twodistinct phage types.The clinical and epidemiological applications of the

technique are well illustrated by the results from cases5, 6, and 7. All three cases occurred on the same unitwithin seven days of each other. The antibiogramswere identical, suggesting a cross infection problem.The isolates were fingerprinted and this rapidly confir-med that: 10 of the 12 isolates from case 5 wereidentical despite the presence of two phage types; thebiotype SII, non-phage typable isolates of case 6 weredifferent from those of case 5 despite identicalantibiograms; the isolates from case 7 were all differentfrom each other and from the isolates from cases 5 and6. This indicated that the cluster was not due to crossinfection from a single source. The problem resolvedspontaneously and there had been no further cases onthe unit at the time of writing (four months).

We thank Dr R Marples and the Division of HospitalInfection, Colindale, for phage and biotyping all theisolates.

References

1 MacGregor RR, Beaty HN. Evaluation of positive blood cultures.Arch Intern Med 1972;130:84-7.

2 Harsteine AJ, Valvano MA, Marthland VH, Fuchs PC, Potter SA,Crosa JH. Antimicrobic susceptibility and plasmid profileanalysis as identity tests for multiple blood isolates ofcoagulasenegative Staphylococci. J Clin Microbiol 1987;25:589-93.

3 Archer GL, Karchmer AW, Vischniavsky R, Johnston JL.Plasmid pattern analysis for the differentiation ofinfecting fromnoninfecting Staphylococcus epidermis. J Infect Dis1984;149:913-20.

4 Christensen GD, Parisi JT, Bisno AL, Simpson WA, Beachey EH.Characterization of clinically significant strains of coagulasenegative Staphylococci. J Clin Microbiol 1983;18:258-9.

5 Baird Parker AC. The classification of Staphylococci andmicrococci from world-wide sources. J Gen Microbiol1965;38:363-87.

6 Kloos WE, Tornabene TG, Schleifer KH. Isolation and character-isation of micrococci from human skin including two newspecies Micrococcus lylae and Micrococcus kristinae. Int JSystem Bacteriol 1974;24:79-101.

7 Marples RR. Laboratory assessment in the epidemiology ofinfections caused by coagulase negative staphylococci. J MedMicrobiol 1986;22:285-6.

8 de Saxe MJ, Crees-Morris JA, Marples R, Richardson JF.Evaluation of current phage-typing systems for coagulasenegative staphylococci. Zentrabl Bacteriol 1981;10(suppl):197-204.

9 Clink J, Pennington TH. Staphylococcal whole cell polypeptideanalysis: evaluation as a taxonomic and typing pool. J MedMicrobiol 1987;23:41-4.

10 Stephenson JR, Tabaqchali S. New method for typing coagulasenegative Staphylococci. J Clin Pathol 1986;39:1271-5.

11 Krikler SJ, Pennington TH, Petrie D. Typing of strains ofStaphylococcus aureus by Western blot analysis of culturesupernatants. J Med Microbiol 1986;21:169-71.

12 O'Connor CG, Ashman LK. Application of the nitrocellulosetransfer technique and alkaline phosphatase conjugated anti-immunoglobulin for determination of the specificity of mono-clonal antibodies to protein mixtures. J Immunol Methods1982;54:267-7 1.

13 Lee W, Burnie J, Matthews R. Fingerprinting Candida albicans.J Immunol Methods 1986;93:177-82.

14 Burnie JP, Matthews RC. Immunoblot analysis: a new method forfingerprinting hospital pathogens. J Immunol Methods1987;100:41-6.

15 Weinstein MP, Reller LB, Murphy JR, Lichtenstein KA. Theclinical significance of positive blood cultures: a comprehensiveanalysis of 500 episodes of bacteremia and fungemia in adults 1.Laboratory and epidemiologic observations. Rev Infect Dis1983;5:35-53.

16 Parisi JT, Hecht DW. Plasmid profiles in epidemiological studiesof infections caused by Staphylococcus epidermidis. Am J Med1980;77:639-44.

17 Archer GL, Johnston JL. Self-transmissible plasmids in Sta-phylococci that encode resistance to aminoglycosides.Antimicrob Agents Chemother 1983;24:70-7.

18 Naidoo J, Noble WC. Transfer of gentamicin resistance betweencoagulase negative and coagulase positive staphylococci onskin. J Hvg 1981;86:183-7.

Requests for reprints to: Dr J P Burnie, Department ofMedical Microbiology, St Bartholomew's Hospital, WestSmithfield, London ECIA 7BE, England.

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