0095-1137/92/092385-06$02.00/0
Copyright X 1992, American Society for Microbiology
Phenotypic Variation of Staphylococcus epidermidis Isolated
from
a
Patient with Native Valve Endocarditis
MARGARETDEIGHTON,1* STEPHEN PEARSON,2 JILLCAPSTICK,1 DENIS SPELMAN,3 ANDROBERT
BORLAND'
Department ofApplied
Biology
andBiotechnology, RMIT, GPOBox2476V, Melbourne, Victoria3001,1 Departmentof Microbiology, RepatriationGeneral Hospital,Heidelberg,
Victoria 3084,2 and Department ofMicrobiology and InfectiousDiseases, AlfredHospital, Prahran, Victoria
3181,3
Australia Received 18 February 1992/Accepted 1June 1992Twocolonial variants ofStaphylococcus epidernidiswere isolated fromthe valvular tissue of a patient with nativevalveendocarditis.Inaddition todifferingincolonial morphology, the two variants differed in
hemolysis
onblood-containing media, in adherencecapacity,
and in the expression of certain enzymes. Under suitable conditions, both variants were themselvescapable of phenotypic variation, although they differed in the rate atwhichvariants were generated. The variants yielded identicalprofilesonrestriction endonucleaseanalysis
ofplasmid DNA and pulsed-field gel electrophoresis of whole-cell DNA. This report suggests a possible role for phenotypic variation incoagulase-negative staphylococcal virulence. Congo red agar would be an excellent medium forstudying thecontribution ofvariation to the virulence of these organisms.Variation in colonial morphology of coagulase-negative staphylococci
(CoNS)
causing endocarditis has recently been described by Baddour andcolleagues (1, 2, 4). These workers havesuggestedthatphenotypic variationmay con-tribute to virulence by providing the staphylococci with greaterflexibilitytocolonizearangeof environments.CoNS areresponsiblefor up to30% ofcasesofbacterial endocardi-tis, but the majority of infections occur in patients with prostheticvalves (3). In this report, we describea case of native valve endocarditis caused by CoNS in which two colonialphenotypeswere detectedonprimary isolation.CASEREPORT
A 35-year-old man was admitted to the hospital with a 3-week history ofrecurrentfevers,chills, andsweats anda 1-weekhistory of breathlessness.Asystolic cardiacmurmur had been detected on physical examination many years before. He had not undergone dental work or any other invasive procedure before the onset of this illness. There was nohistory ofintravenousdruguse. Onexamination,he wasdyspnoeicat restand febrile(38°C)andhadaheart rate of 120 beats per min. He hadmultiplesplinter hemorrhages, gross cardiomegaly, an audible ejection systolic murmur, and evidence of heartfailure; galloprhythmandcrepitations atbothlungbasesweredetected.Echocardiographyshowed severeaorticincompetence andalarge aortic valve vegeta-tionwith anaortic root abscess. After collectingblood for culture,treatment wascommencedwith intravenous fluclox-acillin, penicillin G, and gentamicin. Flucloxacillin was discontinued when the isolation of a
penicillin-sensitive
CoNS from bloodwas
reported.
This isolatewasunavailable forstudy.Soon afteradmission, thepatientunderwent aortic valve replacement (St. Jude prosthesis) (19)with excision of the vegetation and removal of as much of the abscesscavity as possible.Cultures ofaortic valve tissueyieldedtwocolonial morphotypes of CoNS(A-204 andA-205)which differed in
*Correspondingauthor.
pigmentation and hemolysisbut had thesame antimicrobial susceptibilityprofile. These characteristicsweremaintained onsubculture.Wehypothesized thatthe twocolonial forms representedaline of bacteriathat hadarisennaturallyfrom acommon parent, i.e., theywerevariantsaccordingtothe definition used by Christensenetal. (6).
MATERIALSANDMETHODS
Speciesidentification andantibioticsusceptibility. The var-iants were speciated by using the schemes described by Schleifer (22) and Kloos and Lambe (16). In addition, they were tested with three commercial kits or automated sys-tems,namely,apiSTAPHandID32 STAPH(both fromAPI Systems SA, LaBalme LesGrottes, France)and the Vitek System(Vitek Systems,St.Louis,Mo.).Thesusceptibilities to penicillin, methicillin, chloramphenicol, erythromycin, tetracycline, co-trimoxazole, gentamicin, tobramycin, ri-fampin, and vancomycinwere determined bythe agar dilu-tionprocedure described byFranklin (12).
Qualitativeandquantitativeassessmentof adherence.Slime production was assessed by the tube adherence assay de-scribed by Christensen et al. (7). Quantitative estimations wereperformedbyusing amodification (10)of the spectro-scopictechniquedescribedby Christensenetal.(8)inwhich the degree ofadherence is measured spectroscopicallyand strains are classified as strongly adherent (optical density [OD], >0.6), weaklyadherent (0.3 < OD < 0.6), or nonad-herent(OD, <0.3).
ColonialmorphologyonMAand CRA.Colonial morphol-ogy was studied on Memphis agar (MA) as described by Christensen et al. (6). Congo redagar (CRA)was prepared by incorporating Congo red (0.003%) into blood agar base no. 2(Oxoid).Tomaximizedifferences in colonial morphol-ogy, plateswereincubated for 48 h at35°C and then foran additional 48 h atroom temperature. Colonieswere exam-ined with the aid of a plate microscope against a dark backgroundwith reflectedlight.
Scanning electron microscoscopy. Scanning electron mi-croscopy wasperformedon 24-h cultures oftestorganisms 2385
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in trypticase soy broth (TSB; Oxoid). After being washed twice in normalsaline,cells were fixed in2% glutaraldehyde for 1 h and washed twice in phosphate-buffered saline. Postfixation was performed in1% osmiumtetroxidefor 1 h. This wasfollowedby onewash in10% ethanol,dehydration through a gradedseries of alcohol solutions, and a final wash in95% ethanol. Specimens were vacuumdessicatedfor 24 h, gold coated, and examinedbyscanningelectronmicroscopy (JEOL 35CF).
Extracellular products and enzymes. To assess lipolytic andproteolytic activities, staphylococciwerestreaked onto Baird-Parker medium (Oxoid) and plates were examined after incubation at35°Cfor 3 days. Onthismedium,apearly sheen was interpreted as lipase activity, a zone of opacity was interpreted as phospholipase activity, and clearing aroundsingle colonieswasinterpreted as evidence of prote-olysis. Gelatinase activitywasdetermined by spot inoculat-ing nutrient agar containing 0.4% (wt/vol) gelatin (Difco ingredients) and examining plates for zones of clearing. The hemolysin produced by A-205 was characterized by using the methods described by Hebert and Hancock (15). We assessed hemolysis onhorse, sheep, andbovineblood agar and studied synergisticreactions with (i) ,B-lysin-producing
Staphylococcus
aureus (NCTC 6581) and(ii) Streptococcus agalactiae.Cell surface
hydrophobicity.
Cell surface hydrophobicity was determined bya modification of themethod described byRosenberg (21). Briefly, TSBcultureswere incubated at 35°C and then washed once in hydrophobicity buffer (20). Thiswasan attempt to removemediacomponents but retain exopolysaccharide and other surface structures. After the cultures were resuspended in buffer, p-xylene (10% [vol! vol]) was added. The mixture was incubated for 10 min at 35°Cand vortexedfor1 min to allow cells with a hydropho-bic surface to move into the xylene phase, and then the phaseswere allowed to separatefor 30min. The OD of the aqueousphasewasmeasuredandexpressed as apercentage of the ODof theoriginalsuspension (hydrophobicity index). Ahydrophobicityindex of>80%wasinterpretedasstrongly hydrophobic, and one between 70and80%wasinterpreted ashydrophobic (18).Plasmid profile and restriction enzyme
analysis.
Plasmid DNA wasextractedby the methoddescribedby Lyon etal. (17),except that the finalconcentration oflysostaphin used was 20 U/ml, cleaved with EcoRI and HaeIII (Promega) according to the manufacturer's instructions, andanalyzed by agarosegel electrophoresis (9).Electrophoresis of whole-cell DNA digests. Whole-cell DNA wasprepared anddigestedwith alow-frequencybase cutter
(SmaI),
and fragments were separated bypulsed-field
gel electrophoresis (Pulsaphor 11, Pharmacia) by usinga modi-fication of the method described by Smith andCantor(24).
Toprepare cell suspensions, 200 ,ul of an overnightTSB culture was added to 10 ml of fresh TSB and themixturewas incubated at35°C until the OD at 600 nm reached 0.45(4to 5h).Cells werepelleted by centrifugation at 1,290xg for 15 min, washed twice in Pett IV(10mMTris-HCl [pH 7.6],1 M
NaCl),
and resuspended to a final volume of 500 ,ul. The suspensionwas mixed with an equal volume of2.4% low-melting-point molten Nusieve GTG agarose FMC (Pharma-cia), distributed into molds provided by the manufacturer, and allowed to set.Celllysiswasaccomplished by anovernight incubation of agarose blocks at 37°C with 1 ml of EC lysis solution (6 mM Tris-HCl[pH7.6],1 MNaCl, 100 mM EDTA [pH7.6],0.2% sodium deoxycholate, and 0.5% lauroyl sarcosine [Sigma]
a
S
"
FIG. 1. (a andb) Colonies of S. epidermidis, variants A-204 (a) and
A-205(b),onCRA after incubationat35°C for 48h and then afurther
48 h ofincubation at roomtemperature; (c) coloniesdemonstrating
variation ofstrain A-205 on CRA afterincubationfor 8 weeks in broth.
with freshly addedlysozymechloride [1mg/ml] [Sigma] and lysostaphin [50
,ug/mlJ
[Sigma]). This was followed by an-otherovernight incubation at 50°C with ESP(0.5 M EDTA and1%N-lauroyl sarcosine with freshly addedproteinaseK [1 mg/ml]). Blocks were washed with TE1 buffer (10 mMon April 12, 2020 by guest
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TABLE 1. Species identification and extracellular products of parents (A-204 and A-205)
Test A-204 A-205
Identification apiSTAPH
Profile no. 6716131 6714133
Identification Unidentified Unidentified
ID32STAPH
Profileno. 367132310 367132310
Identification Staphylococcus hominis (99.9%) Staphylococcus hominis(99.9%) Vitek
Profileno. 57466060010 77566060010
Identification Staphylococcus hominis (77%) Staphylococcus simulans (96%) Morphology
Nutrient agar Gray,transparent White, opaque
MA Dark blue, transparent Paleblue, opaque
CRA Dark red Pink
Hemolysis
Horse bloodagar +
Sheep blood agar +
Bovine blood agar Synergistichemolysis
S. aureus +
S. agalactiae Adherence
Tube assay - +
Microtiterassay(OD) 0.29(-) 0.40(+)
Hydrophobicityindex after:
4.5 hof incubation 94 89
48 h ofincubation 91 94
Enzyme activities
Lipase andphospholipase - +
Proteinase +
Gelatinase - +
MICofvancomycin(,ug/ml) 1 2
Tris, 1 mM EDTA[pH8])for 2 hat roomtemperatureand storedat4°C.
Prior to endonuclease digestion, blockswere washed as follows;four 0.5-h washes in 5 ml ofTEl, one15-minwash and then a 30-min wash in 300 ml of restriction buffer (Boehringer incubationbufferA), andfinallya30-min wash in300 ml of restriction buffer containing200
,g
of bovine serumalbumin(BSA)permland 2 mMspermidine(Sigma). Digestion was performed overnight at 25°C with 40 U of SmaI(Boehringer)
in 18 p,l of restriction buffer containing thesameconcentrationsof BSA andspermidine.Quarter blocks were loaded into wells (5 by 1 mm) ina 1.2%agarosegelandelectrophoresedat12°Cfor 10 hwitha pulsetime of20 sand thenfor 14 hwithapulsetime of 5s. Molecular markers(XDNA;pulsed-field gelelectrophoresis, Pharmacia)were included in eachrun.
Phenotypic variation of the original variants. The rate of variation in broth of the two variants was determined by
performing
differentialviablecounts onCRA. Cultureswere prepared by inoculating TSB with growth from a single colony, taking particular care to avoid segmented forms. Thisprecautionwastaken toreduce the risk ofcontamina-tion of inocula with another line of cells. Inoculatedbroths were incubated at 35°C for 24 h, 48h, and 8 weeksatboth 35°C androomtemperature before
appropriate
dilutions for culture onto CRAwere prepared. Colonies which differed morphologically from the parent from which they were derived were defined as variants, and the percentage of variantsin the culturewas calculated.To generate a large collection of variants, segmented colonies and other variant morphological forms were se-quentially subcultured onto CRA and characterized as de-scribed above for the parents.
RESULTS
Colonial morphology of parents. Variant A-205
produced
large (diameter, 1.5 mm) white hemolytic colonies after incubationat35°C for 48 honhorseblood agar. Coloniesof A-204weresmaller(diameter,1mm),gray,transparent, and nonhemolytic. On CRA, A-205 yielded opaque, pale-pink, amorphous colonies (Fig. 1). Someplates
contained occa-sionalpaler-pinkcoloniesorsegmentedforms which resem-bled the predominant morphotype except for a wedge ofon April 12, 2020 by guest
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paler-pink growth. Colonial morphotypes resembling A-204 were also seen occasionally. In contrast, A-204 cultures consisted of dark-red, transparent forms, which had narrow andpalerperipheries (Fig. 1). Sectored colonies andforms resembling thealternativephenotypewere moreprevalent in A-204culturesthan in A-205 cultures.
The colonial morphotypes and sectored colonial forms observed on CRA had their counterparts on MA. On this medium, A-204 colonies were composed of a dark-blue central area surrounded by a pale, transparent peripheral zone; A-205 produced pale-blue, opaque colonies. In our hands,differentiation between phenotypes and the detection ofsegmented colonies wasmore readily achieved on CRA than on MA.
Variation of parents. The rate of colonial variation after subculture of 24- or 48-h broth cultures of either parent onto CRAwaslow(A-204, 0.15%,7,110 colonies counted; A-205, 0.027%, 3,601 colonies counted). In contrast, 2-month-old culturesof bothparentsgenerated variants athigh frequen-cies, regardless ofincubation conditions. Of83broths ex-amined, 52 (63%)gave rise to >10% variant colonial forms and 12 (14%) gave rise to >90% variant forms. It was not uncommon for such cultures to display up to six colonial morphotypes (Fig. 1).
Bysequential subculture ofsectored colonies and other variantforms, itwas possible to derive a large number of second- and later-generation variants. Most variants re-tained their morphologyon subculture andweredefined as stable variants. These stable variants expressed arange of morphological, structural, and biochemical characteristics; someresembledtheopposite phenotype,butothers differed from both parents in one or more properties. Some forms, however,wereunstableandonsubculture eitherrevertedto their parent phenotype or gave rise to a further array of variantcolonial forms.
Species identification and antibiotic
susceptibility
profiles. Byusingtheidentification schemes of Schleifer (22) and of Kloos and Lambe (16), both variants were identified as trehalose-positive Staphylococcus epidernidis. The three commercial systemsfailedtoidentifyeitherstraincorrectly (Table 1). Biochemical reactions of the parentswere identi-calexceptfor
the speed and intensityof certain reactions. StrainA-204 gavesloworweak reactions intestsfor urease, argininedehydrolase, andbacitracinresistance but showed stronger nitratereductase activitythan A-205.Both parents were susceptible to
penicillin,
methicillin, chloramphenicol, erythromycin, tetracycline, cotrimox-azole, gentamicin, tobramicin, rifampin, and vancomycin. There was, however, a small but reproducible one-dilution difference inthevancomycinMIC of thetwoparents(Table1).
Extracellularproductsandenzymes,cell surface hydropho-bicity, and extracellular slime. The parents differed in their abilitytoproduce hemolysinsandcertain other extracellular products (Table 1). Variant A-205 produced a hemolysin which was identified as delta-hemolysin on the basis of activity against sheepand horseerythrocytes and enhanced hemolysison sheep blood agar in the presence of staphylo-coccal
3-lysin.
In addition, A-205 was adherent and pro-ducedlipase,lecithinase, andproteinase.Incontrast,A-204 was less active enzymatically, nonhemolytic, and weakly adherent. Both parents had highlyhydrophobic cell surfaces in thelogarithmic and stationary phases of growth. Scanning electron microscopy of A-205 revealed single cells and clumps of cells surrounded by a matrix of extracellular-94
--646
-4-4
3 0
5
FIG. 2. Plasmidprofiles andrestrictionendonucleaseprofiles of variants A-204and A-205. Lanes: 2 to7,plasmid DNAsof A-204 (lanes 2, 4, and 6)andA-205 (lanes 3, 5, and7), either undigested (lanes2and3), digestedwithEcoRI (lanes4and5),ordigested with HaeIII(lanes6and7); 1,XhoIdigest ofX DNA; 8,HindlIl digest of
ADNA.Molecular sizemarkers areshown inkilobases.
material. Cellsof A-204 appearedsingly or in small groups, withclearly defined cellwalls andno extracellular material. Plasmid,restriction endonuclease, and whole-cell DNA pro-files. The plasmid and restriction endonuclease profiles of A-204 and A-205arepresentedinFig. 2. Onelargeplasmid, approximately55 kb insize,wasidentifiedin both variants. Restriction profiles of plasmid DNA, generated with the endonucleases EcoRI and HaeIII, were identical, as were
whole-cell
DNA profiles analyzed by pulsed-field gel elec-trophoresis (Fig. 3).DISCUSSION
Variation incolonial morphologyof CoNS iswell known tomicrobiologistsbut hasonly recentlybeensuggestedas a possible
pathogenic
mechanism (1, 2, 4). Colonial variants may be observed on standard media, but Christensen and colleagues found that theywere more readily detected on MA (6). In ourhands, CRAwas found to be an excellent medium fordistinguishingcolonialmorphotypesofCoNS.The twovariants of S.
epidermidis
describedin thisarticle wereisolated fromresectedvalvularmaterialfromapatient with native valve endocarditis. They differed in hemolysis, adherencecapacity, and in theproduction oflipase, lecithi-nase, and protease but had similar antibiotic susceptibility patterns.Restriction endonucleaseanalysisofplasmidDNA andpulsed-field gelelectrophoresisof whole-cell DNA sug-gested that the twovariantswere identical or veryclosely related and may have arisen from the same parent. They were isolated from a normally sterile site, and, moreover, both had thecapacitytogenerateafurther array ofvariants, includingforms which closelyresembled the opposite phe-notype.Theproposal (4) that multiple phenotypes ofCoNS may function synergistically in human infections isan attractive
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1 2 3 4
....
_ _-H--.C;;;;i Ba
__
C.
-.
.
- 350
300
-250
---200
- 150
--100
48 5
FIG. 3. Pulsed-fieldgel electrophoresisofSmaI-digested whole-cell DNA from variants A-205 (lanes 1and 2) and A-204(lane 3).
Molecular size standardsarelambdaoligomers (48.5to350kb) (lane 4).
oneand is consistent with the results of thisstudy. Between them, the variantsexpressedarangeof surface
characteris-tics and extracellular products which contribute to CoNS virulence. Bothexpressedahighly hydrophobic cell surface, a property which is believed to mediate early interactions between staphylococcal surfaces and solid substrates (14). One parent (A-205) produced a film of adherent growth on
glassandplastic,acharacteristic that isreportedtopromote adherent microcolonies in vivo (7). Strain A-205 also
pro-duced several potentially tissue-damaging extracellular
en-zymes as well as delta-hemolysin. Although the role of
extracellular products and enzymes in the pathogenesis of CoNS infection isunclear,there issomeevidencetosuggest thattheycontribute tovirulence (13).
It istemptingtospeculatethat theparentsweregenerated
invivo and that each contributed differentproperties tothe infection of the aortic valve. The marked capacity for phenotypic variation invitro, particularly under conditions of nutrientlimitation,suggeststhat suchaprocesscould also
occurinvivo. Indeed,asimilarstrategy has beensuggested for Pseudomonasaeruginosa,which undergoes phenotypic variationwith respect tocolonial morphology, enzyme
pro-duction, and serumsensitivityin the lungsofpatientswith
cysticfibrosisasthe clinical condition progresses (11).
Although bacterial cultures are rarely composed of
uni-form populations of cells (23), the genetic mechanisms responsible for this diversity are incompletely understood.
Phenotypic variation ofgram-negative pathogens has been
the subject of numerous studies, but little is known of the genetic mechanisms responsible forvariation in CoNS. Our results suggest that plasmid loss or gain was not responsible for the observed differences in the variants. Other possible mechanisms include the insertionordeletion of phage DNA, transposition of DNA sequences to a new expression
site,
or promoter inversion (5). For some organisms, programmed generearrangements maycontrol the expression ofsurface antigens and virulence determinants and thus regulate the interaction of the microorganism with its environment (5). The purpose of these programmed gene rearrangements appears to be to prepare a small fraction of cells for environmental changes (5).Insummary, this study demonstratesphenotypic variation of clinical isolates of CoNS invitro, which could indicate a similar invivo mechanism, and will beinvestigated further.
ACKNOWLEDGMENTS
We thank DianneStrazzeri fordeveloping methodsfor the elec-tronmicroscopy used in this study and for examining the isolates. We also thank Victoria Korolik for valuable advice and assistance withthemolecular biologyinthis study.
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