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Staphylococcal toxins: screening of burn wound isolates and evidence for alphahaemolysin production in the burn wound
462
Burns (1967) 13. (6). 462466
Printedin Great Britain
Staphylococcal toxins: screening of burn wound isolates and evidence for alphahaemolysin production in the burn wound R. C. Fader,*t
Pamela J. Halst and F. C. W. Koo*t
*Department of Microbiology, The University and t The Shriners Burns Institute, Galveston,
Summary Culture lilIr4lcs of 9~~plr~k~~occfts~II(T(‘I~Sstrains isoL&d from burn pilticnts wrrc cxamincd for cytoloxic aclivitics. A I;lrpc molecular weight cytoIoxin (MW=2S.fo(Kl ddtons) III~I cxhihired cytoroxicity (or human foreskin cells ;mtl hacmolytic aclivity against human ;Intl r;M~it crythrocyrcs w;ls idcntilictl. The cytotoxic activily could tic complcIoly ncutr:ilizstl hy antixcrum formal against Ihc cytotoxin. Further ch;,ractcrization of Ihc molcculc hy isoclsctric focusing rcvc;tlctl th;II the cytotoxin wJs composcd of ;II Ic;lxI Iwo toxic faclors Of smdlcr ll~lhXulilr wcighl. I4olh factors cxhihitcd cyIotoxici!y to tissue-culture cells; howcvcr, one factor lyscd r&hit huI noI human crythrocytcs whsrcas the other faclor had the oppsi~r hacmolytic puttcrn. I‘hc CytotoxiciIy of each factor was ncutrdircd hy Ihc antiserum formal apinst the CYIOIoxin. A cytotoxic f;lcIor that sxhibitcd hacmolytic ;tctivity for r;lhhiI erythrocytcs. ;lnd that was ncutralizsd by the cytofoxin antissrum, W;IS idcnlilicd in hurn wound extracts of mice infcctcd with SrupA. uurcu.s. On the hai!, of mdcdlr wcighr and irodsctric focusing data, conclude that the large molecular weight cytotoxin WC was composcd of an aggregation of alpha-hacmolysin and another prescdy unidrntificd toxic molcculc. possibly d&a-toxin. Alpha-hacmolysin appears IO hc produced in vivo during cxperimcntal staphylococcnl hurn wound infection.
INTRODUCTION
hc-w.tm~. infection of the burn wound remains a major cause of morbidity and mortality in thermally injured patients. The burn wound contains a variety of enriched nutrients and provides an ideal environment for the growth of microorganisms. Bacterial infection of the wound im-
of Texas Medical USA
Branch,
Galveston
pedes the haling process and if not controlled can give rise to hacteraemia. The staphylococci ilre normally the first organisms to colonize the burn wound. and although S,t@. (wwts is one of the major pilthogcns infecting burn patients, few studies hilvc been undertaken to investigate the pathogcnesis of burn wound infections caused by the organism. Stlrldt. uurells is known to elaborate ninny rlifferent toxic filctors (Freer d Arbuthnott. IYX3). with a wide spectrum of nctivitics. These factors have been reported to be cytotoxic to tissucculture cdls (McCartney itnd Arbuthnott 117X), erythrocytcs (Arbuthnott. IY75; McCartney ;Ind Arbuthnott, 197X). m;lcrophagcs (McGee et al.. lY83) and human polymorphonuclear Ieukocytos (Gemmel et al.. 1982). Thus, it is conceivitble that toxins elaborated by SIC@. NIIWIIS could exert a wide range of detrimcntd effects on burn patients which. in turn. could fucilit:W the invasion by other Gram-negative organisms and yeasts. In an effort to understand bcttcr the pathogenesis of StiIphylococcal infections after thermal injury, we screened culture filtrates of ii number of burn wound isolntes of SINI~I. uwcws for the prcscnce of toxins. MATERIALS
AND METHODS
Bacterial strains All strains of .Sl@. UIIWIIS wcrc idi!ted from thermally injured patients idmitted to the Shrincrs Burns Institute, Galveston, Texas. The
Fader et al.: Staph. dureus toxms and burn wound mfectlon
isolates were maintained and routinely subcultured on Casamino acids-yeast extract (CYE) agar slants. Preparation of Staph. dureus culture filtrates for toxin testing A seed culture of each Staph. uttreus strain was prepared by inoculating a tube of CYE broth with a loopful of the organisms. Following incubation at 3PC for 18-24 h, 0.1 ml of each seed culture was inoculated into a 125-ml screwcapped Erlenmeyer flask containing 2Sml of CYE medium. The inoculated cultures were incubated at 37°C for 24 h in a New Brunswick Environmental Shaker with an agitation rate of ItN)r/min. Culture filtrates were clarified and separated from bacterial cells by centrifugation at 27ot)og for 15min. The culture tiltrates were then dialysed extensively in phosphate-buffered saline (PBS) and filter-sterilized through Milliport filter units (0.22 urn) before cytotoxicity tcstinp. Cell detachment assay The modified cell detachment assay, described by Koo and Pctcrson (19X.3). was used to examins cytotoxicity. tluman foreskin cells were rcgulilrly maintained in tfanks’ minimum essential medium (MEM) supplemented with IO per cent fetal bovine serum. ItM)units/ml penicillin. and IO)cJml streptomycin. The cells (2x I(r’cclls/ml) were seeded into a Y6-well microtitrc dish and allowed to incubate overnight at 37°C in ;I 5 per cent CO2 incubator to establish monolayers. The monolayered cells were then treated with each Sttiph. uiueu culture filtrate sample (SOpI) and incubated for 24 h. Cells treated with PBS served as a control. After incubation, the monolayers were fixed with 2 per cent formalin solution (Zmin) and stained with crystal violet dye (Xmin). The dye from the stained monolayers was eluted by treatment with I per cent SDS-50 per cent ethanol solution and measured spcctrophotometrically (5YOnm). The following equation was used to quantitate the amount of cytotoxicity: Per cent cell detachment = OD+“, sample l-
OD,+,,, control
x IIH)
Haemolytic assay The haemolytic activity of toxin preparations wx determined In round-bottomed microtitrc plates
463
using the haemolytic assay described by Cumberbatch et al. (1979). Toxin preparations were diluted in PBS and O-I-ml aliquots were added to an equal volume of I per cent rabbit or human erythrocytes. The samples were incubated for 1h at 37°C. followed by incubation at 4°C for another hour. The haemolytic titre was determined by visually estimating 50 per cent haemolysis and using the reciprocal of the dilution to calculate haemolytic units/ml of sample. Preparation of the crude cytotoxin Partial purification of the staphylococcal cytotoxin was accomplished using a Pharmacia Fast Protein Liquid Chromatography (FPLC) system attached to a Bio-Rad TSK Bio-Sil 250 column. Before chromatography. Staph. arcrem culture filtrates were dialysed overnight in O-01 M phosphate buffer, ptf 7.0. Aliquots (O*Sml) of each tiltrate sample were injected into the TSK Bio-Sil 250 column via an injection port of a Pharmacia V-7 valve. Fractions of O.Sml volume were collected and then assayed for cytotoxic activity by ;I cdl detachment or haemolytic assay. Cytotoxic material elutcd from the column was referred to as ‘crude cytotoxin’. Neutralization studies Antiserum against crude cytotoxin Was obtained by injecting rabbits by intrapcritoncal. intramuscular and subcutaneous routes with a total of 2ot)ug of the toxin (Markel et al., lY7Y). Blood samples wcrc taken from the rabbits every 3 weeks. and a booster dose of 2ot)ug of the crude cytotoxin was administcrcd whenever antibody titrcs began to dccre;lse. Serum was stored at -20°C. Neutralization studies were performed by incubating the test samples with equal volumes of preimmune serum or antiserum to the crude cytotoxin. The samples were allowed to incubate with serum at 37°C for I h and then at 4°C overnight. The samples were then tested for cytotoxic and haemolytic activity. Isoelectric focusing Flat-bed isoelectric focusing was accomplished in 3 5 per cent (w/v) gel slurry of Superfine Sephadcx G-75 (Pharmacia Fine Chemicals) with an amopholyte range of ptf 8-10 (Ephortec Servalyt X-IO, flaake Buchler Instruments, Inc., Saddle Brook, NJ, USA). The sample was subjected to isoelectric focusing for 1Xh at 4°C with 8 W constant power. The gel was divided into fractions. placed into syringe columns and, after the addition of 4 ml of sterile distilled water, the
Burns (1967) Vol. 1YNo. 6
eluants were collected. the pH measured. and the fractions dialysed in PBS prior to assaying for cytotoxic and haemolytic activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDWAGE) SDS-PAGE was performed by the method of Laemlli (1970) using a 12-S per cent separating gel. Burned mouse model A 10 per cent total body surface area. full thickness. scald burn in anaesthetized BALB/c mice was produced as described previously (Fader et al.. 1985). At ISmin post-burn. the mice were given Iml of sterile saline intraperitoneally as fluid replacement therapy. Animal inoculation consisted of 0.1 ml of the Stu~h. a~reus suspension (lox organisms/ml) spread onto the surface of the wound. Burn wound extracts The burn wounds of four mice. either infected or uninfected, were excised, minced and homogenized in a Virtis tissue grinder. The homogenate was centrifuged at IS 3Wg for 20 min. the supcrnatant was decanted through sterile gauze. iid passed suhscqucntly through a 1.2brn filter. After 4X h of dialysis in PBS, the remaining filtrate was concentrated. filter stcrilizcd through a 0.22~pm filter and tested for cytotoxic and hacmolytic activities. RESULTS The effect of Stuph. uwws culture filtrates on human foreskin cell monolayers is shown in TubhI. Of the 20 burn isolates tcstcd. culture filtrates of 19 strains were cytotoxic to the cells. The filtrates exhibited cell detachment in the range from 84.54kO.5 per cent to I%+ 1.06 per cent. The onset of cytotoxicity appeared as early as 3-4h (data not shown) and after 24 h of exposure to the filtrates. the cells had degencrated and detached from the monolayer (Fig. 1~). In contrast. control cells treated with PBS exhibited no cytopathic effects (Fig. lb). To characterize the toxic factor further. the filtrates of three isolates exhibiting high levels of activity were subjected to gel filtration and each column fraction (O-5 ml) was tested for cytotoxic activity on human foreskin cells. A similar region of intense cytotoxic activity, corresponding to a molecular weight of 253000 daltons. was observed for all three isolates (Fib. 2). The column fractions were also tested for haemolytic activity and fractions that exhibited cytotoxic
Table 1. Effect of Staph. aufeus culture filtrates on detachment of human foreskin cells
isolate : 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Celldetachment (%1 a434 -+ 0.50 83.32-cO.06 82.81 kO.34 82.27kO.67 82.26+0.11 81.9320.78 81.32kO.17 80.93kO.22 79.83k2.11 79.1Ok1.61 78.66t5.38 78.11 k1.17 77.8320.89 77.78k2.72 76.89k1.28 72.01 +5,49 71.7350.33 10.15kO.06 1.06+ 1.06 0
aclivity for human foreskin cells also haemolyscd rabbit and human erythrocytcs. On the basis of the high molecular weight and the hacmolytic activities of the cytotoxin. further characterization studies were initiated lo dctcrmine if the crude cytotoxin was an aggrcgatc of one or more of the hacmolytic toxins produced The crude cytotoxin was suhby Stq~h. umw. jcctcd to tlat-bed isoelectric focusing and ths resulting fractions were tested for hacmolytic and cytotoxic activities. Fractions 6-K corresponding to pl values of 8.69-9.3 I, exhihitcd cytotoxicity when tested in the cell dctachmcnt assay (Fig. 3). The activity in all three fractions was neutralized by antiserum to the crude cytotoxin (data not shown). Fractions 6 and X were thsn tested in hacmolytic assays using rabbit and human erythrocytss (Tub/e II). Fraction 6. with pl of X-69, haemolyscd rabbit but not human crythrocytes whcrcas Fraction 8, with a pl of 9.31. haemolyscd human but only slightly hnemolysed rabbit erythrocytrs. The haemolytic activity against rabbit orythrocytes could be neutralized by antiserum to the crude cytotoxin but not by preimmune serum. whereas the activity in the fraction with ~19.31 was ncutralizcd by prcimmune and immune strum. Intcrcstingly, the preimmune strum appeared to have no effect on the haemolyric activity of the crude cytotoxin to human erythrocytes.
Fader et al.: Staph. sureus toxins and burn wound tnfect~on
h
to00
o-
‘0 c o-
0 40:
I-
4-
i,
c ,*0
I
J.t0
I,
t
3
.
0
.
,
0
,
10
Fix, 3. Cyloloxic wtivity in frstions rc3ultcd isoclccrric focusing of the crude cytotoxin.
The isoelectric focusing data seemed to suggest that the large molecular weight cytocoxin was composed of alpha-hacmolysin and delta-toxin in an ;rgprcg;titcd form. To attempt to confirm this. tbs crude cytotoxin and fhc fractions with pi X-69
from
and Y.31 were subjected to SDS-PAGE (Fig. 4). Both fhe crude cytotoxin and the material with pIX6Y contained a protein band at M W 35 UN1 dahons that was ahsent in the fraction at pl Y.31. This indicated that the toxin did contain alpha-haemolysin. No protein bands were visible in the molecular weight range of deltatoxin (
Burns (1667) Vol. U/No. 6
466 Table II. Haemolytic activities and neutralization from Staph. aureus culture filtrates Haemolytic
Crude cflotoxin
pi 8.69 pl 9.31
studies of toxic factors
activity IHlJ/ml) Preimmune Immune rabbit/human rabbiffhumant
Rabbit
Human*
1280
640
1280640
o/o
640
0
640/-
Oi-
10
80
-10
-10
Human type 0 erythrocytes. Antiserum to the crude cytotoxin. -, Not tested. ??
t
filtrates. Furthermore. when the extract was subjected to isoelectric focusing and fractions were tested for haemolytic activity (Fig. 5). haemolytic activity for rabbit erythrocytes was identified in the pl range M-8.7. 66.2 K
45.0 K
31.0 K
21.5 K
14,4 K
Fig. 4. SDS-PAGE of Stuph. ut~reus toxin prcparalions. Lane A. crude cytotoxin: Innc B. marari4 at pl841Y; lane C. material at plY.31. The arrow in the left margin points to a prorcin hand (MW=35(Ilodaltons) prcscnt in lnncs A and B, hul ahscnl in lane C.
extracts from uninfected mice were not cytotoxic or haemolytic (Table//f). The cytotoxic and haemolytic activities were neutralized by antiserum to the large molecular weight cytotoxin but not by preimmune serum. suggesting that the toxic activities present in the wound extracts were the same as those isolated from culture
DISCUSSION Although antibiotic therapy has markedly reduced the mortality rate of hurn patients. infections caused by Stqdr. atrretrs arc still a major problem encountered in burn units. This is due in part to the organism acquiring resistance to the commonly used antibiotics. Stuph. uttr~s is a normal inh;lhitant of the skin nnd is thus one of the first microorganisms to colonize the wounds of burn patients. If this colonization is not controlled, the resulting Iocalizcd infection can sometimes Icad to septicaemic complications. In spite of its significance as a pathogen. very little is known ahout the pathogenesis of staphylococcal infections in burns. The purpose of this study was to evaluate burn wound isolates of Stctph. u~~r~ws to determine what toxins might he produced by the organisms and whether the toxins are produced in vivo. The study has demonstrated that toxin production is a common finding among. alpha-haemolysin. Stuph. mretu isolates and that at least one toxin is produced during wound infection. The large molecular weight cytotoxin idcntified in culture filtrates of 1Y of 20 Stctph. uttrerts isolates was determined to be an aggregated molecule of alpha-haemolysin and another unidentified toxin factor, possibly delta-toxin. Both these haemolytic toxins are known to aggregate under certain conditions (Bernheimcr and Schwartz. lY63: Fitton. 19x1). Alpha-haemolysin has been reported to have a molecular weight in the region of 330W daltons. an isoelectric point in the region of N-5 (Freer and Arbuthnott.
467
Fader a al.: Staph. aureus toxins and burn wound infection
Table l/1. Detection and neutralization mice infected with Staph. aweus*
of toxic activity in burn wound extracts of
cytotoxicityon human foreskin cells /% cell detachment)
Haemolytic actiwityt (HU/mll
6584 le,3.66 0 84.90 2 0.90
20 0 20
Infected/burn wound extract Uninfected/burn wound extract Infected/burn wound extract +preimmune serum Infected/burn wound extract +immune serum
0
0
Extracts of burn wounds excised from four mice at 4 days post-burn. t Rabbit erythrocytes.
??
Fig. 5. t hcrnoly~ic activity fur rabbit crythrcrytrs in inkctstl turn wound c’xmcts suhjcckd to isohctric focusing.
IW), and haemolytic activity showing highest sensitivity toward rnhhit rrythrocytcs (Cooper et al.. 1066). These characteristics are all in direct agreement with the toxin identiticd in this study. Since most S/q/r. UIITCIIS strains produce alphahacmolysin (Freer and Arbuthnott, lYX3). a high percentage of burn wound isolates with toxin activity would be expected. The second component of the crude cytotoxin may he delta-toxin, although definitive proof could not be provided. Delta-toxin has been reported to have a molecular weight of 2977 daltons (Fitton et al.. 1980). although molecules of the toxin as large as 2500(W) daltons have been described (Fitton. 1981). After isoelectric focusing, components in the region of ~15.5 and 9.5 have been reported (Krcger et al., lY7l; Turner, 197X). The toxin is thought to act on the surface membranes of cells. thereby accounting for the wide range of activity and rapid action of the toxin (Kreger et al., 1071; Wadstrom and Mollby, 1972). We were unable to demonstrate a low molecular weight protein band on SDS-PAGE of
the isoelectric focusing fraction with ~19.3 and haemolytic activity for human and rabbit erythrocytes. However, further evidence that the material may be delta-toxin is provided by the inhibitory effect of preimmune serum on the haemolytic activity. Delta-toxin is known to be inhibited by phospholipids present in serum (Whitelow and Birkheck. lY7X). This could explain not only the inhihition of the haemolytic activity by preimmune serum but also why the activity was not identified in burn wound extracts, which undoubtably contained .some serum components. In order to establish the involvement of a toxin in an infection, it is imperative to know if the toxin is produced by the infecting organism in situ. This study has provided evidence that alphahacmolysin is produced in the burn wounds of mice infected with S/up/r. uwws. The toxic factor isolated from burn skin extracts contained the same toxic activity as that recovered from culture filtrates, it was neutralized by antiserum made to the cytotoxin, and the activity was localized to the same isoelectric point as alpha-haemolysin. Unfortunately, the immense protein content of the hurned skin extract precluded identifying an alpha-haemolysin band on SDS-PAGE. Furthermore, no toxin activity was found in uninfected burn skin extracts. indicating that the toxin activity did not result from the release of ‘burn toxins’ from the damaged tissue. The precise role of alpha-haemolysin in burn wound infections remains to be determined; however, knowledge that the toxin is present in the burn wound suggests a possible role for the toxin in the infection process. Acknowledgement This work was supported in part by National Institutes of flealth research grant no. I RI!3 A I22670.
Burns (1987) Vol. 1YNo. 6
REFERENCES Arbuthnott J. P. (IY75) Sraphylococcal toxms. In: Schlcssinger D. (ed.). ~~~~crohido,qv. Washington: American Socicly for Microbiology. p. 37. Bcrnhcimer A. W. and Schwartz L. L. (1963) Isolation and composition of staphylococcal alpha-toxin. 1. Gaff. i\/icrohio/. 3Q. 455. Cooper L. Z.. bladoff M. A. and Weinstein L. (1966) tieut stahilitv and species range of purilied staphylo1. Btrcfuriol. 5. IhXh. coccd cd&. Cumhcrhatch N.. Gurwith M. J.. Lunpsfow C. et al. ( lY7Y) Cytoloxic enterotoxin produced by Auromoms Ir~tlrr~pltiltr: relationship of toxigznic isolates to diarrhcal disease. Itrfiw. /mmln. 23. X2Y. Fader R. C.. Nuncz D., Unhehagen J. et al. (10X5) Expcrimcntal cnndidiusis afrcr thermd injury. //rfi,cr. /r?irrIcor. .lY. 7x0. Fitton J. E. (IYXI) Physiochemical studio on &It+ hemolysin. :I stnphylococcal cytolytic polypcptidc. FE&S LPKT. 1.u). 257. Fitton J. E.. Dell A. and Shaw W. V. (IYXO) The amino acid sequence of the delta hacmolysin of Skcplr.h co~cI,s
Freer
J.
u~~rc'us.
tl.
FEBS Lctr. I IS. 2(K).
nnd Arhuthnott
P. (19x2) Toxins of 77wr. IV. ss. Gcmmcll C. G.. Pctcrson P. K.. Schmclling D. J. CI al. ( 1082) Effect of Stnphylococcd u-toxin on ph+cytosis of st;rphylocc~ci by human polynrorphonuclc~lr Icukocytcs. /,t/ia //flnllor. J. 07s. Koo F. C. W. and Pclcnon J. W. (10X.1) Cdl-free cxtrilcts of Scrlrnorrdkr inhibit protsin synthesis :IIKI CilllW cylotoxicily in cukaryolic cull\. ‘litricwtt 21, Sltl~'lI!'k,~oc~cI~~ 1111fw.s.
WI.
J.
I%clrIrrctcol.
Krcgcr A. S.. Kim K. 5. Zuhorersky F. et al. (lY71) Purilicntion and properties of sraphylococcd delta hcmoly\in. /n&r. /rttmrrr. 3. 4-l’). Lncmlli U. K. ( lY70) Clcavagc of structural proteins during the asscmhly of the head of hactcriophagc T-l. Nllrure 222. 6X0. Murkel D. E.. Hcjtmancik K. F.. Pctsr%;on 1. W. et al. (1979) Characterization ol’ the untigcnic dctcrminants of cholera toxin suhunits. Ir~fi*cr. Itr~nrctrr. 25. 615. McCartney A. A. and Arhuthnott J. P. (lY7X) hlodcl of action of membrane-diimagins toxins produced hy Staphylococci. In: Jeljaszewicz J. and Wadstrom T. (e&i). R~creriul Toxins d Cd dMcn~hrut~ev. London. Academic. p. XI). McGee M. P.. Kreger A.. Lenkc E. L ct al. (IYS3) Toxicity ol staphylococcal ;dpha toxin Ior rahhit Irwrrw~. 39. J?Y. alveol;lr macrophages. /@v. Turner W. ti. (lY7X) Purification and characterization of ;Ln immunologicolly distinct delta-hcmolysin from a canine strain of Slu~~h_~~lo~oc~crts uwws. IrrJi’c’l. hinl11ll. 20. 4x5. Wadstrom T. and Mollhy R. ( lY72) Some hi~~logicd propertics of purilicd st;lphyloccKc;ll hamolysins. Tmicotf IO. 5 I I. Whidow D. D. ;Ind Birkhcck T. Il. (107X) Inhibition of st;lphylococcal tlcll;l-h;lcnioly4in by strum lipoprotcinh. IXfIS I.crr. 3. 3.35.
I’q)cr
ucccptctl 2X Mny 10X7.
BRITISH BURN ASSOCIATION 21st Annual Meeting will be held at Stranmillis Training Collcgc, Belfast on Thursday, April 7th and Friday, April 8th. Anticipated cost: Registration f60 Bed and breakfast (on site): X12 per night. Details and applications forms from: Dr J. C. Lawrence, or MRC. Burns Rcscarch Group, Birmingham Accident Hospital. Bath ROW, Birmingham BIS INA.
Mr R. Millar The Ulster Hospital, Dundonald, Belfast BT16 ORH, Northern Irdond.
Burns (1967) 13. (6). 462466
Printedin Great Britain
Staphylococcal toxins: screening of burn wound isolates and evidence for alphahaemolysin production in the burn wound R. C. Fader,*t
Pamela J. Halst and F. C. W. Koo*t
*Department of Microbiology, The University and t The Shriners Burns Institute, Galveston,
Summary Culture lilIr4lcs of 9~~plr~k~~occfts~II(T(‘I~Sstrains isoL&d from burn pilticnts wrrc cxamincd for cytoloxic aclivitics. A I;lrpc molecular weight cytoIoxin (MW=2S.fo(Kl ddtons) III~I cxhihired cytoroxicity (or human foreskin cells ;mtl hacmolytic aclivity against human ;Intl r;M~it crythrocyrcs w;ls idcntilictl. The cytotoxic activily could tic complcIoly ncutr:ilizstl hy antixcrum formal against Ihc cytotoxin. Further ch;,ractcrization of Ihc molcculc hy isoclsctric focusing rcvc;tlctl th;II the cytotoxin wJs composcd of ;II Ic;lxI Iwo toxic faclors Of smdlcr ll~lhXulilr wcighl. I4olh factors cxhihitcd cyIotoxici!y to tissue-culture cells; howcvcr, one factor lyscd r&hit huI noI human crythrocytcs whsrcas the other faclor had the oppsi~r hacmolytic puttcrn. I‘hc CytotoxiciIy of each factor was ncutrdircd hy Ihc antiserum formal apinst the CYIOIoxin. A cytotoxic f;lcIor that sxhibitcd hacmolytic ;tctivity for r;lhhiI erythrocytcs. ;lnd that was ncutralizsd by the cytofoxin antissrum, W;IS idcnlilicd in hurn wound extracts of mice infcctcd with SrupA. uurcu.s. On the hai!, of mdcdlr wcighr and irodsctric focusing data, conclude that the large molecular weight cytotoxin WC was composcd of an aggregation of alpha-hacmolysin and another prescdy unidrntificd toxic molcculc. possibly d&a-toxin. Alpha-hacmolysin appears IO hc produced in vivo during cxperimcntal staphylococcnl hurn wound infection.
INTRODUCTION
hc-w.tm~. infection of the burn wound remains a major cause of morbidity and mortality in thermally injured patients. The burn wound contains a variety of enriched nutrients and provides an ideal environment for the growth of microorganisms. Bacterial infection of the wound im-
of Texas Medical USA
Branch,
Galveston
pedes the haling process and if not controlled can give rise to hacteraemia. The staphylococci ilre normally the first organisms to colonize the burn wound. and although S,t@. (wwts is one of the major pilthogcns infecting burn patients, few studies hilvc been undertaken to investigate the pathogcnesis of burn wound infections caused by the organism. Stlrldt. uurells is known to elaborate ninny rlifferent toxic filctors (Freer d Arbuthnott. IYX3). with a wide spectrum of nctivitics. These factors have been reported to be cytotoxic to tissucculture cdls (McCartney itnd Arbuthnott 117X), erythrocytcs (Arbuthnott. IY75; McCartney ;Ind Arbuthnott, 197X). m;lcrophagcs (McGee et al.. lY83) and human polymorphonuclear Ieukocytos (Gemmel et al.. 1982). Thus, it is conceivitble that toxins elaborated by SIC@. NIIWIIS could exert a wide range of detrimcntd effects on burn patients which. in turn. could fucilit:W the invasion by other Gram-negative organisms and yeasts. In an effort to understand bcttcr the pathogenesis of StiIphylococcal infections after thermal injury, we screened culture filtrates of ii number of burn wound isolntes of SINI~I. uwcws for the prcscnce of toxins. MATERIALS
AND METHODS
Bacterial strains All strains of .Sl@. UIIWIIS wcrc idi!ted from thermally injured patients idmitted to the Shrincrs Burns Institute, Galveston, Texas. The
Fader et al.: Staph. dureus toxms and burn wound mfectlon
isolates were maintained and routinely subcultured on Casamino acids-yeast extract (CYE) agar slants. Preparation of Staph. dureus culture filtrates for toxin testing A seed culture of each Staph. uttreus strain was prepared by inoculating a tube of CYE broth with a loopful of the organisms. Following incubation at 3PC for 18-24 h, 0.1 ml of each seed culture was inoculated into a 125-ml screwcapped Erlenmeyer flask containing 2Sml of CYE medium. The inoculated cultures were incubated at 37°C for 24 h in a New Brunswick Environmental Shaker with an agitation rate of ItN)r/min. Culture filtrates were clarified and separated from bacterial cells by centrifugation at 27ot)og for 15min. The culture tiltrates were then dialysed extensively in phosphate-buffered saline (PBS) and filter-sterilized through Milliport filter units (0.22 urn) before cytotoxicity tcstinp. Cell detachment assay The modified cell detachment assay, described by Koo and Pctcrson (19X.3). was used to examins cytotoxicity. tluman foreskin cells were rcgulilrly maintained in tfanks’ minimum essential medium (MEM) supplemented with IO per cent fetal bovine serum. ItM)units/ml penicillin. and IO)cJml streptomycin. The cells (2x I(r’cclls/ml) were seeded into a Y6-well microtitrc dish and allowed to incubate overnight at 37°C in ;I 5 per cent CO2 incubator to establish monolayers. The monolayered cells were then treated with each Sttiph. uiueu culture filtrate sample (SOpI) and incubated for 24 h. Cells treated with PBS served as a control. After incubation, the monolayers were fixed with 2 per cent formalin solution (Zmin) and stained with crystal violet dye (Xmin). The dye from the stained monolayers was eluted by treatment with I per cent SDS-50 per cent ethanol solution and measured spcctrophotometrically (5YOnm). The following equation was used to quantitate the amount of cytotoxicity: Per cent cell detachment = OD+“, sample l-
OD,+,,, control
x IIH)
Haemolytic assay The haemolytic activity of toxin preparations wx determined In round-bottomed microtitrc plates
463
using the haemolytic assay described by Cumberbatch et al. (1979). Toxin preparations were diluted in PBS and O-I-ml aliquots were added to an equal volume of I per cent rabbit or human erythrocytes. The samples were incubated for 1h at 37°C. followed by incubation at 4°C for another hour. The haemolytic titre was determined by visually estimating 50 per cent haemolysis and using the reciprocal of the dilution to calculate haemolytic units/ml of sample. Preparation of the crude cytotoxin Partial purification of the staphylococcal cytotoxin was accomplished using a Pharmacia Fast Protein Liquid Chromatography (FPLC) system attached to a Bio-Rad TSK Bio-Sil 250 column. Before chromatography. Staph. arcrem culture filtrates were dialysed overnight in O-01 M phosphate buffer, ptf 7.0. Aliquots (O*Sml) of each tiltrate sample were injected into the TSK Bio-Sil 250 column via an injection port of a Pharmacia V-7 valve. Fractions of O.Sml volume were collected and then assayed for cytotoxic activity by ;I cdl detachment or haemolytic assay. Cytotoxic material elutcd from the column was referred to as ‘crude cytotoxin’. Neutralization studies Antiserum against crude cytotoxin Was obtained by injecting rabbits by intrapcritoncal. intramuscular and subcutaneous routes with a total of 2ot)ug of the toxin (Markel et al., lY7Y). Blood samples wcrc taken from the rabbits every 3 weeks. and a booster dose of 2ot)ug of the crude cytotoxin was administcrcd whenever antibody titrcs began to dccre;lse. Serum was stored at -20°C. Neutralization studies were performed by incubating the test samples with equal volumes of preimmune serum or antiserum to the crude cytotoxin. The samples were allowed to incubate with serum at 37°C for I h and then at 4°C overnight. The samples were then tested for cytotoxic and haemolytic activity. Isoelectric focusing Flat-bed isoelectric focusing was accomplished in 3 5 per cent (w/v) gel slurry of Superfine Sephadcx G-75 (Pharmacia Fine Chemicals) with an amopholyte range of ptf 8-10 (Ephortec Servalyt X-IO, flaake Buchler Instruments, Inc., Saddle Brook, NJ, USA). The sample was subjected to isoelectric focusing for 1Xh at 4°C with 8 W constant power. The gel was divided into fractions. placed into syringe columns and, after the addition of 4 ml of sterile distilled water, the
Burns (1967) Vol. 1YNo. 6
eluants were collected. the pH measured. and the fractions dialysed in PBS prior to assaying for cytotoxic and haemolytic activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDWAGE) SDS-PAGE was performed by the method of Laemlli (1970) using a 12-S per cent separating gel. Burned mouse model A 10 per cent total body surface area. full thickness. scald burn in anaesthetized BALB/c mice was produced as described previously (Fader et al.. 1985). At ISmin post-burn. the mice were given Iml of sterile saline intraperitoneally as fluid replacement therapy. Animal inoculation consisted of 0.1 ml of the Stu~h. a~reus suspension (lox organisms/ml) spread onto the surface of the wound. Burn wound extracts The burn wounds of four mice. either infected or uninfected, were excised, minced and homogenized in a Virtis tissue grinder. The homogenate was centrifuged at IS 3Wg for 20 min. the supcrnatant was decanted through sterile gauze. iid passed suhscqucntly through a 1.2brn filter. After 4X h of dialysis in PBS, the remaining filtrate was concentrated. filter stcrilizcd through a 0.22~pm filter and tested for cytotoxic and hacmolytic activities. RESULTS The effect of Stuph. uwws culture filtrates on human foreskin cell monolayers is shown in TubhI. Of the 20 burn isolates tcstcd. culture filtrates of 19 strains were cytotoxic to the cells. The filtrates exhibited cell detachment in the range from 84.54kO.5 per cent to I%+ 1.06 per cent. The onset of cytotoxicity appeared as early as 3-4h (data not shown) and after 24 h of exposure to the filtrates. the cells had degencrated and detached from the monolayer (Fig. 1~). In contrast. control cells treated with PBS exhibited no cytopathic effects (Fig. lb). To characterize the toxic factor further. the filtrates of three isolates exhibiting high levels of activity were subjected to gel filtration and each column fraction (O-5 ml) was tested for cytotoxic activity on human foreskin cells. A similar region of intense cytotoxic activity, corresponding to a molecular weight of 253000 daltons. was observed for all three isolates (Fib. 2). The column fractions were also tested for haemolytic activity and fractions that exhibited cytotoxic
Table 1. Effect of Staph. aufeus culture filtrates on detachment of human foreskin cells
isolate : 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Celldetachment (%1 a434 -+ 0.50 83.32-cO.06 82.81 kO.34 82.27kO.67 82.26+0.11 81.9320.78 81.32kO.17 80.93kO.22 79.83k2.11 79.1Ok1.61 78.66t5.38 78.11 k1.17 77.8320.89 77.78k2.72 76.89k1.28 72.01 +5,49 71.7350.33 10.15kO.06 1.06+ 1.06 0
aclivity for human foreskin cells also haemolyscd rabbit and human erythrocytcs. On the basis of the high molecular weight and the hacmolytic activities of the cytotoxin. further characterization studies were initiated lo dctcrmine if the crude cytotoxin was an aggrcgatc of one or more of the hacmolytic toxins produced The crude cytotoxin was suhby Stq~h. umw. jcctcd to tlat-bed isoelectric focusing and ths resulting fractions were tested for hacmolytic and cytotoxic activities. Fractions 6-K corresponding to pl values of 8.69-9.3 I, exhihitcd cytotoxicity when tested in the cell dctachmcnt assay (Fig. 3). The activity in all three fractions was neutralized by antiserum to the crude cytotoxin (data not shown). Fractions 6 and X were thsn tested in hacmolytic assays using rabbit and human erythrocytss (Tub/e II). Fraction 6. with pl of X-69, haemolyscd rabbit but not human crythrocytes whcrcas Fraction 8, with a pl of 9.31. haemolyscd human but only slightly hnemolysed rabbit erythrocytrs. The haemolytic activity against rabbit orythrocytes could be neutralized by antiserum to the crude cytotoxin but not by preimmune serum. whereas the activity in the fraction with ~19.31 was ncutralizcd by prcimmune and immune strum. Intcrcstingly, the preimmune strum appeared to have no effect on the haemolyric activity of the crude cytotoxin to human erythrocytes.
Fader et al.: Staph. sureus toxins and burn wound tnfect~on
h
to00
o-
‘0 c o-
0 40:
I-
4-
i,
c ,*0
I
J.t0
I,
t
3
.
0
.
,
0
,
10
Fix, 3. Cyloloxic wtivity in frstions rc3ultcd isoclccrric focusing of the crude cytotoxin.
The isoelectric focusing data seemed to suggest that the large molecular weight cytocoxin was composed of alpha-hacmolysin and delta-toxin in an ;rgprcg;titcd form. To attempt to confirm this. tbs crude cytotoxin and fhc fractions with pi X-69
from
and Y.31 were subjected to SDS-PAGE (Fig. 4). Both fhe crude cytotoxin and the material with pIX6Y contained a protein band at M W 35 UN1 dahons that was ahsent in the fraction at pl Y.31. This indicated that the toxin did contain alpha-haemolysin. No protein bands were visible in the molecular weight range of deltatoxin (
Burns (1667) Vol. U/No. 6
466 Table II. Haemolytic activities and neutralization from Staph. aureus culture filtrates Haemolytic
Crude cflotoxin
pi 8.69 pl 9.31
studies of toxic factors
activity IHlJ/ml) Preimmune Immune rabbit/human rabbiffhumant
Rabbit
Human*
1280
640
1280640
o/o
640
0
640/-
Oi-
10
80
-10
-10
Human type 0 erythrocytes. Antiserum to the crude cytotoxin. -, Not tested. ??
t
filtrates. Furthermore. when the extract was subjected to isoelectric focusing and fractions were tested for haemolytic activity (Fig. 5). haemolytic activity for rabbit erythrocytes was identified in the pl range M-8.7. 66.2 K
45.0 K
31.0 K
21.5 K
14,4 K
Fig. 4. SDS-PAGE of Stuph. ut~reus toxin prcparalions. Lane A. crude cytotoxin: Innc B. marari4 at pl841Y; lane C. material at plY.31. The arrow in the left margin points to a prorcin hand (MW=35(Ilodaltons) prcscnt in lnncs A and B, hul ahscnl in lane C.
extracts from uninfected mice were not cytotoxic or haemolytic (Table//f). The cytotoxic and haemolytic activities were neutralized by antiserum to the large molecular weight cytotoxin but not by preimmune serum. suggesting that the toxic activities present in the wound extracts were the same as those isolated from culture
DISCUSSION Although antibiotic therapy has markedly reduced the mortality rate of hurn patients. infections caused by Stqdr. atrretrs arc still a major problem encountered in burn units. This is due in part to the organism acquiring resistance to the commonly used antibiotics. Stuph. uttr~s is a normal inh;lhitant of the skin nnd is thus one of the first microorganisms to colonize the wounds of burn patients. If this colonization is not controlled, the resulting Iocalizcd infection can sometimes Icad to septicaemic complications. In spite of its significance as a pathogen. very little is known ahout the pathogenesis of staphylococcal infections in burns. The purpose of this study was to evaluate burn wound isolates of Stctph. u~~r~ws to determine what toxins might he produced by the organisms and whether the toxins are produced in vivo. The study has demonstrated that toxin production is a common finding among. alpha-haemolysin. Stuph. mretu isolates and that at least one toxin is produced during wound infection. The large molecular weight cytotoxin idcntified in culture filtrates of 1Y of 20 Stctph. uttrerts isolates was determined to be an aggregated molecule of alpha-haemolysin and another unidentified toxin factor, possibly delta-toxin. Both these haemolytic toxins are known to aggregate under certain conditions (Bernheimcr and Schwartz. lY63: Fitton. 19x1). Alpha-haemolysin has been reported to have a molecular weight in the region of 330W daltons. an isoelectric point in the region of N-5 (Freer and Arbuthnott.
467
Fader a al.: Staph. aureus toxins and burn wound infection
Table l/1. Detection and neutralization mice infected with Staph. aweus*
of toxic activity in burn wound extracts of
cytotoxicityon human foreskin cells /% cell detachment)
Haemolytic actiwityt (HU/mll
6584 le,3.66 0 84.90 2 0.90
20 0 20
Infected/burn wound extract Uninfected/burn wound extract Infected/burn wound extract +preimmune serum Infected/burn wound extract +immune serum
0
0
Extracts of burn wounds excised from four mice at 4 days post-burn. t Rabbit erythrocytes.
??
Fig. 5. t hcrnoly~ic activity fur rabbit crythrcrytrs in inkctstl turn wound c’xmcts suhjcckd to isohctric focusing.
IW), and haemolytic activity showing highest sensitivity toward rnhhit rrythrocytcs (Cooper et al.. 1066). These characteristics are all in direct agreement with the toxin identiticd in this study. Since most S/q/r. UIITCIIS strains produce alphahacmolysin (Freer and Arbuthnott, lYX3). a high percentage of burn wound isolates with toxin activity would be expected. The second component of the crude cytotoxin may he delta-toxin, although definitive proof could not be provided. Delta-toxin has been reported to have a molecular weight of 2977 daltons (Fitton et al.. 1980). although molecules of the toxin as large as 2500(W) daltons have been described (Fitton. 1981). After isoelectric focusing, components in the region of ~15.5 and 9.5 have been reported (Krcger et al., lY7l; Turner, 197X). The toxin is thought to act on the surface membranes of cells. thereby accounting for the wide range of activity and rapid action of the toxin (Kreger et al., 1071; Wadstrom and Mollby, 1972). We were unable to demonstrate a low molecular weight protein band on SDS-PAGE of
the isoelectric focusing fraction with ~19.3 and haemolytic activity for human and rabbit erythrocytes. However, further evidence that the material may be delta-toxin is provided by the inhibitory effect of preimmune serum on the haemolytic activity. Delta-toxin is known to be inhibited by phospholipids present in serum (Whitelow and Birkheck. lY7X). This could explain not only the inhihition of the haemolytic activity by preimmune serum but also why the activity was not identified in burn wound extracts, which undoubtably contained .some serum components. In order to establish the involvement of a toxin in an infection, it is imperative to know if the toxin is produced by the infecting organism in situ. This study has provided evidence that alphahacmolysin is produced in the burn wounds of mice infected with S/up/r. uwws. The toxic factor isolated from burn skin extracts contained the same toxic activity as that recovered from culture filtrates, it was neutralized by antiserum made to the cytotoxin, and the activity was localized to the same isoelectric point as alpha-haemolysin. Unfortunately, the immense protein content of the hurned skin extract precluded identifying an alpha-haemolysin band on SDS-PAGE. Furthermore, no toxin activity was found in uninfected burn skin extracts. indicating that the toxin activity did not result from the release of ‘burn toxins’ from the damaged tissue. The precise role of alpha-haemolysin in burn wound infections remains to be determined; however, knowledge that the toxin is present in the burn wound suggests a possible role for the toxin in the infection process. Acknowledgement This work was supported in part by National Institutes of flealth research grant no. I RI!3 A I22670.
Burns (1987) Vol. 1YNo. 6
REFERENCES Arbuthnott J. P. (IY75) Sraphylococcal toxms. In: Schlcssinger D. (ed.). ~~~~crohido,qv. Washington: American Socicly for Microbiology. p. 37. Bcrnhcimer A. W. and Schwartz L. L. (1963) Isolation and composition of staphylococcal alpha-toxin. 1. Gaff. i\/icrohio/. 3Q. 455. Cooper L. Z.. bladoff M. A. and Weinstein L. (1966) tieut stahilitv and species range of purilied staphylo1. Btrcfuriol. 5. IhXh. coccd cd&. Cumhcrhatch N.. Gurwith M. J.. Lunpsfow C. et al. ( lY7Y) Cytoloxic enterotoxin produced by Auromoms Ir~tlrr~pltiltr: relationship of toxigznic isolates to diarrhcal disease. Itrfiw. /mmln. 23. X2Y. Fader R. C.. Nuncz D., Unhehagen J. et al. (10X5) Expcrimcntal cnndidiusis afrcr thermd injury. //rfi,cr. /r?irrIcor. .lY. 7x0. Fitton J. E. (IYXI) Physiochemical studio on &It+ hemolysin. :I stnphylococcal cytolytic polypcptidc. FE&S LPKT. 1.u). 257. Fitton J. E.. Dell A. and Shaw W. V. (IYXO) The amino acid sequence of the delta hacmolysin of Skcplr.h co~cI,s
Freer
J.
u~~rc'us.
tl.
FEBS Lctr. I IS. 2(K).
nnd Arhuthnott
P. (19x2) Toxins of 77wr. IV. ss. Gcmmcll C. G.. Pctcrson P. K.. Schmclling D. J. CI al. ( 1082) Effect of Stnphylococcd u-toxin on ph+cytosis of st;rphylocc~ci by human polynrorphonuclc~lr Icukocytcs. /,t/ia //flnllor. J. 07s. Koo F. C. W. and Pclcnon J. W. (10X.1) Cdl-free cxtrilcts of Scrlrnorrdkr inhibit protsin synthesis :IIKI CilllW cylotoxicily in cukaryolic cull\. ‘litricwtt 21, Sltl~'lI!'k,~oc~cI~~ 1111fw.s.
WI.
J.
I%clrIrrctcol.
Krcgcr A. S.. Kim K. 5. Zuhorersky F. et al. (lY71) Purilicntion and properties of sraphylococcd delta hcmoly\in. /n&r. /rttmrrr. 3. 4-l’). Lncmlli U. K. ( lY70) Clcavagc of structural proteins during the asscmhly of the head of hactcriophagc T-l. Nllrure 222. 6X0. Murkel D. E.. Hcjtmancik K. F.. Pctsr%;on 1. W. et al. (1979) Characterization ol’ the untigcnic dctcrminants of cholera toxin suhunits. Ir~fi*cr. Itr~nrctrr. 25. 615. McCartney A. A. and Arhuthnott J. P. (lY7X) hlodcl of action of membrane-diimagins toxins produced hy Staphylococci. In: Jeljaszewicz J. and Wadstrom T. (e&i). R~creriul Toxins d Cd dMcn~hrut~ev. London. Academic. p. XI). McGee M. P.. Kreger A.. Lenkc E. L ct al. (IYS3) Toxicity ol staphylococcal ;dpha toxin Ior rahhit Irwrrw~. 39. J?Y. alveol;lr macrophages. /@v. Turner W. ti. (lY7X) Purification and characterization of ;Ln immunologicolly distinct delta-hcmolysin from a canine strain of Slu~~h_~~lo~oc~crts uwws. IrrJi’c’l. hinl11ll. 20. 4x5. Wadstrom T. and Mollhy R. ( lY72) Some hi~~logicd propertics of purilicd st;lphyloccKc;ll hamolysins. Tmicotf IO. 5 I I. Whidow D. D. ;Ind Birkhcck T. Il. (107X) Inhibition of st;lphylococcal tlcll;l-h;lcnioly4in by strum lipoprotcinh. IXfIS I.crr. 3. 3.35.
I’q)cr
ucccptctl 2X Mny 10X7.
BRITISH BURN ASSOCIATION 21st Annual Meeting will be held at Stranmillis Training Collcgc, Belfast on Thursday, April 7th and Friday, April 8th. Anticipated cost: Registration f60 Bed and breakfast (on site): X12 per night. Details and applications forms from: Dr J. C. Lawrence, or MRC. Burns Rcscarch Group, Birmingham Accident Hospital. Bath ROW, Birmingham BIS INA.
Mr R. Millar The Ulster Hospital, Dundonald, Belfast BT16 ORH, Northern Irdond.