Br. |. exp. Path. (I988), 69, 813-831

Streptococcal necrotizing fasciitis: development of ananimal model to study its pathogenesis

D.V. Seal and D. KingstonMicrobial Pathogenicity Research Group, Division of Communicable Diseases Clinical Research Centre,

Watford Road, Harrow, Middlesex HAI 3UJ, UK.

Received for publication 20 July I987Accepted for publication 23 July I988

Summary. Necrotizing fasciitis is a serious and increasingly common human disease whichcan be caused by an infection with ,B-haemolytic streptococci (BHS) ofLancefield groups A, C or

G, spreading rapidly in the loose connective tissue over the muscle fascia. To facilitate study ofits pathogenesis, we have developed an animal model for the production of a spreadinginfection with BHS in the loose connective tissue over the muscle layer in the skin of NewZealand White rabbits. Intradermal injection of group A BHS alone into the flank was

unsatisfactory in that a spreading lesion occurred on only 12% ofoccasions. When the group ABHS were co-injected with cultures of Staphylococcus aureus, the results depended on the strainof S. aureus used: an abscess-producing strain isolated from pigs gave rise to a spreading lesionon 50% of occasions. When BHS were injected with the a-lysin of S. aureus at a titre whichproduced inflammation without necrosis, spreading lesions occurred on 75% of occasions.However, both inoculated and uninoculated broth acted synergistically with the a-lysin inpotentiating the spread of the streptococci. This demonstration of synergy between BHS and a-

lysin of S. aureus may reflect the clinical situation in the human, as both organisms have beenfound to occur together at sites where spreading streptococcal infections have originated.

Keywords: necrotizing fasciitis, P-haemolytic streptococci, Staphylococcus aureus ac-lysin

Haemolytic streptococci (BHS) give rise to avariety of different infections of the skin andsubcutaneous tissues, of which the mostserious is necrotizing fasciitis (Seal & LeppardI982). Other bacteria may cause this diseasebut we are here concerned only with necro-tizing fasciitis caused by BHS, either alone orsynergistically with other organisms. Thesalient feature is that the streptococcal infec-tion spreads rapidly through the loose con-nective tissue overlying the fascia of muscle,but without involving the muscle, causing aspreading gangrenous process. To help

understand the pathogenesis, we haveextablished an animal model, of which apreliminary account has been published(Seal et al. I985). Another anirnal model ofspreading infection was described by Mer-genhagen et al. (I958), but differed in somerespects from ours, and used anaerobic nothaemolytic streptococci. Such models areimportant because they may help to identifythe virulence factors of the causative orga-nism and so allow dangerous strains to berecognized and preventive immunization tobe developed. There are many accounts of

Correspondence: Mr D. Kingston, Microbial Pathogenicity Research Group, Division ofCommunicableDiseases, Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ, UK.

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D.V. Seal & D. Kingstonstreptococcal virulence factors (e.g. Ginsburg1972, Alouf I980, Kimura et al. I985), buttheir role in the pathogenesis of disease hasreceived only limited direct study, and wehope that the model described here willfacilitate such work.Our clinical observations suggested that

necrotizing fasciitis may be initiated, at leaston some occasions, by a mixed streptococcaland staphylococcal infection (Seal et al.I985; Barker et al. I987). We cultured S.aureus as a mixed infection from six ofour 36patients, Hammar and Wanger (I977) fromfour of their eight patients. Meleney (I924)who made the first major study of thedisease, isolated BHS from all 20 of hispatients with necrotizing fasciitis, from nineof these in mixed culture, nearly alwaysincluding S. aureus. We show later (Results)that we could only occasionally reproducethe disease in rabbits by intradermal injec-tion ofBHS alone. We therefore attempted todevelop an animal model by injecting rabbitsintradermally with cultures of haemolyticstreptococci isolated from these infections, towhich cultures ofvarious strains of Staphylo-coccus aureus (S. aureus) and, later, staphylo-coccal a-lysin, were added. Our aim has beento produce conditions under which a spread-ing infection would occur that resembled thehuman disease. The importance of bacterialsynergy in the causation of disease hasreceived only erratic attention, partlybecause of the natural assumption that theadditional organisms are present only ascontaminants.

Materials and Methods

Bacterial strains

P-Haemolytic streptococci (BHS) of Lance-field groups A, C (Streptococcus equisimilis)and G (large colony) were isolated fromclinical cases of necrotizing fasciitis (Gaunt etal. I984, Barker et al. I987). Streptococcalcolonies were identified by haemolysis andgrowth on blood and bile-aesculin agar andwere grouped by a co-agglutination method

(Phadebact or Streptex); strains were typedwith M antisera by Dr G. Colman at theCentral Public Health Laboratory, Colindale,NW9 5HT. Str. sanguis II (API 426144I)was isolated from blood cultures of a child.The human strain of S. aureus was a clinicalisolate from a case of staphylococcal necro-tizing fasciitis (Leppard & Seal I983). Thiswas untypable by the basic phage set (RTDand RTD Xioo) but reacted strongly withtwo experimental phages. A mouse (phagetype 6/47/53/54/75/77/83A/84/85) and apig (phage type 29/52/80/8I) strain of S.aureus, both known to cause experimentalabscesses in their respective species werekindly provided by Professor J. Jones of theRoyal Veterinary College, Potters Bar. TheWood 46 strain (NCTC 7I2I) was used tocompare toxin production. The bacteriawere stored in IO% glycerol broth frozen inliquid nitrogen.

Preparation of cultures for rabbit inoculation

Frozen ampoules were thawed and the orga-nisms subcultured onto blood agar and thegrowth from these plates used to inoculatebroth cultures. Streptococci were grown inTodd-Hewitt broth (Oxoid or Difco) with I or2% added neopeptone (Difco) (THN). Inpreparation for future studies some work wasdone with BHS grown in Difco Todd-Hewittwith 2% Difco neopeptone which had beenfiltered through an Amicon or Milliporemembrane to remove molecules larger thanIO 000 molecular weight ('ioK broth'). Thedissolved powders were first filtered througha large fluted Whatman no. i filter paper(essential), then through a 0.22 gm Milliporemembrane and finally through the ultra-filter. At no stage was this broth heated;sterilization was by passage through a 0.22,um Millipore membrane. Staphylococci weregrown in Oxoid nutrient broth no. 2. Allcultures were incubated at 3 7TC overnight.Streptococci were injected either in brothculture or after centrifugation, washing andresuspension in fresh broth, in phosphate-buffered saline (PBS) or in their own culture

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Spreading streptococcal infection in rabbits

supernatant fluid. Staphylococci wereinjected as broth cultures or diluted inculture supernatant, either alone or, more

commonly, injected with the streptococci.Bacterial cultures were counted by standardmethods as colonies on the surface of bloodagar plates; the numbers of colony-formingunits injected varied from io6 to 108. In theResults section, a short analysis is presentedsuggesting that this substantial variationhad no major effect.

Preparation and production of a-lysin

Crude a-lysin of S. aureus was obtained infreeze-dried form from SCLAVO sp A, Divi-sione Diagnostici, Via Fiorcutina I, 53100

Siena, Italy. This a-lysin was diluted in PBSfrom 1:5 to i: 8o and injected in 0.2 ml

amounts to establish the dilution whichwould cause an area of inflammation with-out necrosis (IWN) of about i cm diameteraround the site of injection. It was then usedat this dilution (I: 1 5 equivalent to I/70 IU/ml) for the experiments in association withstreptococci; a control injection was includedfor each animal to show that injection oftoxin alone gave IWN. Polyclonal rabbitantisera to purified staphylococcal a and f,-lysin were kindly supplied by Dr Adlam,Wellcome Research Laboratories, Becken-ham, Kent.

Production of staphylococcal a-lysin bythe pig and human strains of S. aureus was

estimated by comparison with the Wood 46strain using a procedure based on Burnet(I930). Organisms were grown in THN in6% CO2 or on the surface ofblood agar in 4%CO2 or in air. The toxin produced on the agarplates was harvested in io ml of PBS and thesupernatant estimated. Serial doubling dilu-tion in PBS was used in a microtitre plateusing rabbit erythrocytes and taking 50%haemolysis as the endpoint.

Establishment of rabbit model

Rabbits were young adult New ZealandWhite (on one occasion Sandy-lop) which

were injected intradermally in the flank. Thevolume injected was either O.I, 0.2 or 0.4

ml. Initially, rabbits were given only one or

two injections but it was found possible(provided that serious lesions were likely todevelop after only a proportion of the injec-tions) to give three or four injections in eachflank I cm ventrally to the spine.

For this study I68 inoculations were madewith group A BHS, using four different Mtypes (I, 3, 22 and 64). Seventy-one of theseinoculations were made without additionaltoxin, and 48 with staphylococcal a-lysin(including eight with anti-a-lysin), as de-scribed in Table i. On 49 occasions group ABHS were co-injected with S. aureus: I 8 withhuman S. aureus (including eight with anti-cxlysin), 28 with pig S. aureus and three withmouse S. aureus, as described in Table 2. On13 occasions group C or G BHS were inocu-lated without additional toxin, on i6 withadditional toxin (Table i) and on i6 occa-sions with human S. aureus (Table 2). Str.sanguis was inoculated alone on four occa-

sions and with a-lysin on six occasions. Thepig S. aureus was inoculated alone on 12

occasions. The rabbits were usuallyinspected daily and the appearance of thelesions recorded. They were killed whenexperience suggested that no further de-velopment of this lesion would take place, or

earlier if seriously ill.Bacteriological examinations of pus and

blister fluid was carried out by standardmethods. Swabs from the lesions were cul-tured on blood agar and colonies identifiedand estimated. Histological material was

collected at post-mortem, fixed in neutralformol saline and stained with haemotoxylinand eosin or by Gram's stain. In the generalstudy I05 slides from 64 lesions were exam-

ined. They were graded for severity into fivecategories (-, ±, +, + +, + + +) for thefollowing pathological features: muscle des-truction, haemorrhage, inflammatory cells,pus, vessels thrombosed, vessels engorged,streptococcal debris and streptococcalchains. The grading was based on generalvisual impression. Reliability of grading was

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Table i. P-haemolytic streptococci injected into the rabbit flank with and without additional toxin:number of lesions in each clinical category

Number of lesions

SpreadingBHS

cellulitisM type Non-spreading or gangrene*

M typeI 3 22 64 Total no. minimal lump or

group (no. of injections) Suspension injections apparent ulcer I 3 22 64 C/G

Without additional toxinA 4 I 2 43 own culture broth 50 21 23 0 0 I 5A 0 0 0 7 washed & suspended

in fresh broth 7 4 2 0 0 0 IA 0 I 4 9 washed & suspended

in PBS I4 5 9 00 0 0C own culture broth 5 4 0OG own culture broth 8 7 I 0

With staphylococcal a-lysin at IWNt titreA 3 0 2 I9 own culture broth

with a-lysin 24 0 6 3 0 0 15A 4 0 0 4 ditto+ anti-a-lysin 8 0 8 0 0 0 0A 0 0 0 7 washed & suspended

in fresh brothwith a-lysin 7 0 2 0 0 0 5

A 0 0 0 9 washed & suspendedinPBS with a-lysin 9 I 6 0 0 0 2

C own culture brothwith a-lysin 4 0 4 0

C ditto+ anti-a-lysin 4 3 I 0G own culture broth

with a-lysin 4 0 3 IG ditto+ anti-a-lysin 4 2 2 0

* Massive synergistic necrosis did not occurt IWN, inflammation without necrosis

checked by re-scoring every tenth slide (byprocessing number) without the previousscoring being available. Reproducibility wasfound to be good except for a category(general tissue destruction) which was there-fore eliminated. Dilatation and thrombosis ofthe vessels was scored by each of us indepen-dently. The lesions were also classified asearly (post-mortem at 4-6 days, 36 lesions)or late (post-mortem at I2-I8 days, 28lesions).To follow the spread offluid from the site of

injection, 0.4 ml of I% trypan blue in PBSwas injected intradermally into two sites(front and back), in the same positions asthose that were used for the BHS. After iomin the skin was reflexed under anaesthesiausing a paramedian abdominal incision andthe inner surface photographed.

Results

It became clear that the same injection intothe rabbit flank could give rise to different

D. V. Seal & D. Kingston8I6

Spreading streptococcal infection in rabbitsTable 2. f,-haemolytic streptococci injected into the rabbit flank with cultures of S. aureus: number oflesions in each clinical category

BHS Number of lesions

M type Non-spreadingSpreading

I 3 22 64 No. of minimal lump or cellulitisgroup (no. of injections) Suspension injections apparent ulcer or gangrene*

A 4 0 0 6 own culture broth withhuman S. aureus IO I 7 2t

A 4 0 0 4 ditto + anti-a-lysin 8 2 6 0A o o 2 26 own culture broth with

pig S. aureus 28 5 9 I4*pig S. aureus alone in own

broth 12 5 2 5A o o 0 3 own culture broth with

mouse S. aureus 3 0 2 IC own culture broth with

human S. aureus 4 2 2 0C ditto + anti-a-lysin 4 2 2 0G own culture broth human

S. aureus 4 3 I 0G ditto + anti-oa-lysin 4 3 I 0

* Massive synergistic necrosis occurred on two occasions (M types 22 and 64). Both M type 22 strainswere among these I4.

t Both due to M type i strains.

clinical responses. The results have thereforebeen analysed by taking all the clinicalresponses following a given injection andrecording the numbers which fell into eachof four different categories: (i) massive syner-gistic necrosis (rapid development in under24 hours of superficial and deep necrosisover an area greater than 4 cm in lengthwith gross purulent discharge), (ii) spreadinglesion of cellulitis or necrotizing fasciitis(development over 2 to 5 days of an acutelyinflamed area extending from the site ofinjection to the belly with necrosis, if present,limited to the dermis), (iii) lump or ulcer and(iv) minimal apparent lesion.

Injection of streptococci alone or with ax-lysin

Table i summarizes these results and showsthat group A BHS injected in their own

culture broth only gave rise to a spreadinginfection on I2% of occasions; group C andgroup G BHS (with a smaller number ofinjections) never gave rise to a spreadinglesion. However, if group A BHS wereinjected with a-lysin at the IWN titre, spread-ing lesions arose much more frequently: 75%of occasions when injection was made inown culture broth, 7I% of occasions whenwashed and resuspended in fresh broth, and22% of occasions when washed and resus-pended in PBS. When group C BHS wereinjected in own culture broth with a-lysin,0/4 injections gave a spreading lesion; forgroup G BHS the number ofspreading lesionswas 1/4. The effects of the crude a-lysin wereneutralized by specific anti-oe-lysin. Thus it isclear that both a-lysin and broth greatlypotentiated the production of spreadinglesions.

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D.V. Seal & D. KingstonStr. sanguis was tested in the rabbit skin in

broth culture and in broth culture with a-lysin (in the same way as group A BHS).When 0.2 ml of an overnight culture in THN(4 x io0 colony-forming units per ml) was

injected in four sites, no clinical response wasseen. When the same injection was madewith a-lysin at IWN into six sites, five smallulcers and one 'bleb' (very small lump) were

seen.

It was possible that in animals which hadto be killed early (6 days after inoculation orless-see Methods) not all the lesions haddeveloped to their fullest extent. To checkthis the results in Table i for group A BHSinjected in broth without additional toxinwere split into groups, animals killed early (6days or less) and those killed late (7 days or

more). In late-killed animals 6/39 injectionswere found to give spreading lesions, inearly-killed animals I/I 8. Thus the fact thatsome animals had to be killed early mighthave reduced slightly the proportion ofspreading lesions developing, but does notaffect the general picture. However, there is a

difference in that there were more lesionsdiagnosed as lump or ulcer (rather thanminimal apparent) in late-killed animals(22/39 compared with 3/I8). In this paperwe are concerned with the production ofspreading lesions and this effect will bedisregarded. The results following injectionof BHS in broth with a-lysin were similarlyanalysed and it was found that there were

I I/I 5 spreading lesions in the animals killedearly and I2/I6 in those killed late. Thuswhen a-lysin was co-injected, there was noeffect on the results because some animalshad to be killed early.The infecting dose was derived from an

overnight broth culture and the bacterialcounts were therefore variable, but this didnot seem to matter. Thus, for example,injecting group A BHS in broth with x-lysingave 19/28 spreading lesions with counts of5 x io6 or I07 and 6/9 spreading lesionswith counts of 5 x I07 or 108. Similarly therewere 19/29 spreading lesions where thevolume injected was 0.2 ml and 6/II wherethe volume injected was 0.4 ml.

Table 3. Bacteriological results for injection of group A BHS and pig S. aureus into the rabbit flank

Suspension (no. perml) Culture results*Volume No. of

Clinical category gp A BHS S. aureus injected (ml) rabbits gpA BHS S. aureus

Massive synergisticnecrosis 2X108 2XIO8 0.4 2 +++/+++ +++/+++

Necrotizing fasciitis 2 X I08 3 x I08 0.4 2 ++ ++/++ + -/_Spreading cellulitis 4 x I07 7x I08 0.2 3 +/+ +/- +/+/+ + +

8 x I07 6 x io8 0.2 2 + ++/+++ ++ + +/+ + +Ulcer with necrosis 4x 107 7X I08 0.2 I + + + + +Medium/small lump 4 x 107 7X I08 0.2 2 -/- + + +/+ + +

8 x I07 6 x Io8 0.2 2 +++/+++ ++/+++2 X 108 5 x I08 0.4 I + +

* Where the spreading infections occurred, cultures were made from the spreading edge.-No growth.+ <Io colonies.+ I0-50 colonies.+ + 50-200 colonies.+ + + > 200 colonies.

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Spreading streptococcal infection in rabbits

Fig. i. Normal rabbit skin with blood vessels overlying the continuous muscle layer (platysma muscle).Loose connective tissue not separated from the muscle by dense connective tissue fascia. H & E x 50.

Injection of BHS with cultures of S. aureus

Table 2 summarizes these results; it needs tobe read in conjuction with Table i. Of thethree strains of S. aureus used, the pig S.aureus gave rise to spreading lesions mostfrequently. When co-injected with group ABHS, spreading lesions occurred on 50% ofoccasions, but they also occurred on 5/12occasions when the pig S. aureus was injectedwithout BHS. Spreading lesions alsooccurred when the human strain of S. aureus

was injected with group A BHS, but notwhen injected with group C or group G BHS.Comparing toxin production showed thatthe pig strain gave yields similar to that oftheWood 46 strain but the human strain gaveamounts I/3 2-I/64 of those strains and thetoxin was detectable only from culturesgrown on blood agar in 4% CO2. Thus co-injection ofBHS with S. aureus increased theincidence of spreading lesions over thatgiven by the injection ofBHS alone, and thisseemed to depend on toxin production.

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D.V. Seal & D. Kingston

Fig. 2.a. Section from necrotizing fasciitis H & E x 50.

Table 3 summarizes the bacteriology oflesions produced by the simultaneous injec-tion of S. aureus and group A BHS where thedetailed sampling was carried out. In thistable a distinction is made between two typesof spreading lesion, necrotizing fasciitis andspreading cellulitis: this will be discussedlater. It can be seen that in the two examplesof necrotizing fasciitis the S. aureus did notspread with the BHS, but in the five examplesof the latter it did.

Necrotizing fasciitis and spreading cellulitis

This distinction between two types of spread-ing lesion has been disregarded in Tables Iand 2. It appeared to be associated with thesite of the injection. Considering only theinjection of group A BHS in broth togetherwith a-lysin at IWN, at the injection sitenearest the head there were IO/13 spreadinglesions, of which 6/io were diagnosed asnecrotizing fasciitis. At other injection sites

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Spreading streptococcal infection in rabbits

Fig. 2.b. Section from necrotizing fasciitis. Gram stain showing cocci clustered round vessels x 50

there were I3/I8 spreading lesions, ofwhichI/I3 was diagnosed as necrotizing fasciitis.It was very noticeable that necrotizing fascii-tis developed rapidly (within 2 days) as

against spreading cellulitis which graduallyincreased over 8 days.

Spread from injection site

Study of the skin into which blue dye hadbeen injected showed strong staining of theveins and lymphatics running more or lessvertically from the spine to the abdomen.

Histology

Normal rabbit skin is illustrated in Figure i,where it can be seen that the loose connec-tive tissue is not separated from the muscleby a dense fascial layer as occurs, for exam-ple, in the human limb. The injection ofgroup A BHS was always followed by loca-lized tissue destruction and pus formation,although this was not always obvious onclinical inspection. For example, there was+ + + localized muscle destruction and pusin sections taken from two sites in which

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D.V. Seal & D. Kingston

Fig. 2.c. As Fig. 2b at high power, x 5oo.

injection of I07 group A BHS had producedno clinical lesion. Pus graded at + + or+ + + was present following the injection ofgroup A BHS in 41/42 sections from 'spread-ing lesions', in I 7/17 sections from 'lumps orulcer' and in 22/26 sections from 'minimalapparent lesion'. In contrast, when bacteria-free toxins were injected, there was no pusformation. Thus, six sections from the cate-gory 'inflammation without necrosis' (allfollowing the injection of a-lysin) and foursections in the category 'minimal apparentlesion' (following the injection of various

bacteria-free toxins) had no detectable pus.In an attempt to identify a factor whichmight correlate with the severity of thelesion, a rough grading was made of strepto-cocci in sections from sites of injection ofBHS. The differences were large, and veryvariable from field to field, so no exactquantitation was attempted. The Gram-stained sections were scored from - to+ + + in two categories: intact cocci withsome chains apparent and Gram positivedebris without streptococcal morphology.When tabulated, the results seemed at first to

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Spreading streptococcal infection in rabbits

Fig. 3.a. Section from spreading cellulitis H& E x 50. This figure corresponds closely to the human disease(Barker et al. I98 7), but is only one of a pattern of distributions of pus and cocci found.

correlate with the severity of the lesion butcloser analysis showed that could beexplained by the different lengths of timeafter injection that the animals had beenkilled and sections prepared. Animals keptalive for longer had on average fewer strepto-cocci in the sections either intact ordegraded, but especially the former. Theeffect of the division between early-killed andlate-killed animals was also noted for theoccurrences of thrombosis in the vessels. The

type of minimal apparent lesion described as'bleb' was analysed for the histology from IOearly-killed and I4 late-killed animals.Thrombosis graded as +, + + or + + + wasfound in four of the former but none of thelatter. In the spreading lesion described asspreading cellulitis similar thrombosis wasfound in 6/9 early-killed animals but i/6late-killed animals (these figures are forsections taken from the top of the lesion, butsimilar proportions apply to sections taken

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D.V. Seal & D. Kingston

Fig. 3.b. As Fig. 3.a, Gram stain showing localization of cocci, x 50.

further down). In necrotizing fasciitis, allkilled early, the figures were 1/3. Typicalhistological appearances are illustrated inFigs 2 and 3.The histology of lesions where Str. sanguis

was injected together with a-lysin at IWN,taken IO days after injection, was essentiallythat ofthe a-lysin alone; pus and streptococciwere not found, in complete contrast to theinjection of BHS.

Toxin studies

Antisera specific for staphylococcal a- and f-

lysin were used to show that the IWNreaction was caused by the a-lysin compo-nent (or predominantly so). Crude a-lysin attwice the concentration for IWN wasinjected into eight sites, four with specificanti-ax-lysin diluted I: 2, and four with speci-fic anti-fl-lysin dilution I: 2. At all the siteswith anti-,B-lysin there was inflammationwith necrosis, whereas at those with anti-a-lysin no clinical lesions were seen.Group A BHS were grown in ioK broth for

injection into the rabbit on I8 occasions.Because of the variable nature of the re-sponse to injected organisms, only gross

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Spreading streptococcal infection in rabbits

tt L~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1.0...

V"'''.'- AaL;~~~~~~~~~~~A A

Fig. 4. Rabbit skin 24 h after injection of crudestaphylococcal a-lysin. Note neutrophil infiltra-tion with marked perivascular cuffing. H & Ex40.

differences in their pathogenicity could beidentified. As such differences were notapparent, the results with organisms grown

in ioK broth have been pooled with thosefrom THN broths. However, we note thatinjection ofgroup A BHS grown in ioK brothwith a-lysin at IWN titre gave a spreadinglesion on three out of six occasions. Someexperiments were carried out on the growthof four different group A BHS in this mediumas compared with THN. Final counts were

equal, but growth was faster in the ioKbroth. We found that a haemolysin requiringthiol activation (streptolysin 0) was pro-

duced, as was a proteinase (and its precur-

sor), but further analysis of toxin productionwas not carried out.

Since crude staphylococcal a-lysin poten-

Fig. 5. As Fig. 4 at high power showing damagedvessel with neutrophils. H & E x 400.

tiated the development of the spreadinglesions sections were prepared from the skinofa rabbit injected with various dilutions andkilled 24 h later. Sections showed changeswhere the severity paralleled the titre of thetoxin. There was widespread extravasationof neutrophils into the tissue with perivascu-lar cuffing very prominent, illustrated atIWN titre in Figs 4 and 5. There was alsoinflammation of the vessel walls accompa-nied by congestion in the vessels and somehaemorrhage into the tissues. There was noevidence of intracapillary clotting. Changeswere also detected in sections of skin col-lected close to the injection site where theinfection would have run; these were of lessseverity, sometimes amounting only to ac-cumulation of neutrophils in capillaries. Wewere not able to tell from the histological

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D. V. Seal & D. KingstonTable 4. Comparison ofstreptococcal necrotizing fasciitis in man with the spreading lesion induced in theflank of the rabbit

Category Human infection Rabbit model

ClinicalBlistering necrosis with pus frequent presentSubacute type associated with occasional presentsevere cellulitisSpread follows venous drainage follows venous and lymphatic

drainageRate and extent of spread rapid and severe or sub-acute rapid and severe or sub-acute

(slow spreading cellulitis)Outcome life-threatening life-threateningBacteriologicalInitiated by group A BHS (occasionally group A BHS + staphylococcal

group C or G) + /-S. aureus a-lysin, or +pig S. aureus,occasionally group A BHS alone

Maintained by group A BHS group A BHS(occasionally group C or G) (occasionally group G)

HistologicalPresence of staphylococcal not known at IWN*a-lysin at initial sitePus frequent presentThrombus formation frequent occasionalHaemorrhage into tissue present occasionalMuscle necrosis nil occasional-localized onlyGram-positive cocci at spreading often found presentedge

* Inflammation without necrosis.

picture whether the neutrophils had beendamaged. No changes were found in asection taken from the site ofinjection ofPBS.

Because THN acted synergistically withthe crude a-lysin, a comparison was madebetween the results of three intradermalinjections of PBS, three of THN, four of a-lysin at IWN in PBS and four of a-lysin at thesame dilution in THN. The injections weredistributed between two rabbits and the skinreactions noted 24 h later. The animals werethen killed and strips of skin excised forhistological examination. The histologicalsections (and the skin reactions) wereassessed blind. Injection ofPBS alone gave nodetectable skin reaction or histological

change. Injection of THN produced slightreddening and the corresponding histologi-cal appearance was of slight general poly-morph infiltration; perivascular cuffing wasnot found. Injection of a-lysin at IWN titre inPBS produced reddening (two injections) ormarked reddening (two injections), one onlyof the latter recorded as running down-wards. In contrast, injection of the samedilution of a-lysin in THN gave a similardegree ofreddening, but on all four occasionswas recorded as running downwards. Theamount of polymorph infiltration was notconsistently higher for injection of a-lysin inTHN as against PBS. Injection of a-lysin ineither caused marked perivascular cuffing.

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Spreading streptococcal infection in rabbits

Discussion

It is probably impossible to produce a perfectanimal model for a human disease. We choseto work with the rabbit because it is readilyavailable, large enough for three or fourinjections to be made into each flank, andalso large enough for a spreading infection tobe followed. The essential feature of thehuman infection that we aimed to mimic wasthe rapid spread of BHS infection over theconnective tissue fascia covering the musclebundles mostly in the limbs. To achieve thiswe have examined the conditions underwhich a spreading infection is developed inthe loose connective tissue of the rabbitdermis, where it is not separated from themuscle layer by a dense fascial sheath. Thecrucial point is that a spreading necroticstreptococcal lesion in the rabbit flank was

only obtained reliably if staphylococccal a-lysin, at a titre that did not cause necrosis,was co-injected with a BHS isolated fromnecrotizing fasciitis (Table I); co-injection ofcultures of S. aureus also potentiated theproduction of the spreading lesion, but was

not so effective. We have not yet analysed thecharacteristics of the streptococcus whichare necessary for this to occur, for exampleby testing strains isolated from a differentsource e.g. throat, or strains which havebeen genetically manipulated, to determine ifthey are more or less able to set up spreadinginfection. The infection in the human andanimal model is compared in Table 4 forclinical, bacteriological and histological fea-tures.Our animal model is directly relevant only

to necrotizing fasciitis caused by BHS.Feingold (I98I) makes the distinction thatthe form of the disease caused by intestinalorganisms (often mixed), usually followsabdominal surgery, as against that caused byBHS which usually arises in the extremitieseither spontaneously or following minortrauma. The European literature reportscases almost exclusively in the second cate-gory (Hammar & Wanger 1977; Cruick-shank et al. I98I; Hawkey et al. I980;

Redding I980; Colebunders et al. I984;Barker et a]. I987), though we reported acase originating in the foot of a diabeticcaused by an anaerobic streptococcus andone in the leg of an elderly patient caused byS. aureus. The American literature (White19 5 3; Crosthwait et a]. I 964; Rea & WyrickI970; Stone & Martin I972; Wilson &Haltalin I973; Defore et a]. 1977; Giuliano etal. 1977; Casali et al. I980; Janevicius et al.i982; Goldberg et al. i984; Lally et al. I984)implicates a much wider variety of orga-nisms including even Haemophilus influenzaetype b (Collette et al. I987). The bacteriologyis not always easy to interpret and strepto-coccal serology, which we considered to bevaluable in establishing the aetiology (Lep-pard & Seal I983) is rarely reported; neitheris distinction between acute and sub-acuteinfection often made. However, it seems clearthat BHS, often isolated with other orga-nisms, are an important cause but thatmixed infections with a wide variety oforganisms including anaerobes are oftenimplicated. Further, Feingold's (I98I) dis-tinction seems to be an over-simplification.

There have been several studies in animalsof other synergistic infections. Brewer &Meleney (I926), Meleney (I93I) and Mer-genhagen et al. (I958) studied synergybetween S. aureus and microaerophilic oranaerobic streptococci in the production oflesions in animals related to necrotizingfasciitis. Roberts (I967a, b, I969) studiedthe synergy between Fusiformis necrophorusand Corynebacterium pyogenes to produce aform of foot-rot (infective bulbar necrosis) insheep, which was, in part at least, due to theimportance for F. necrophorus of a growthfactor produced by C. pyogenes and theimportance for C. pyogenes of the leucocidinproduced by F. necrophorus. Synergybetween fusobacteria and spirochaetes wasinvestigated by Hampp & Mergenhagen(I 963). There have been a number of studieson the potentiation of bacterial disease byviral infection (Larson et al. I977; Mackow-iak I978; Porter et al. I98I). In particular,that by Porter et al. (I98I) considered

827

D.V. Seal & D. Kingstonsynergy between myositis caused by a picor-navirus and BHS septicaemia, resulting inacute streptococcal rhabdomyolysis which isusually fatal. Of particular interest also arethe mixed infections between BHS and S.aureus found in tropical pyoderma (Allen etal. I971) and impetigo. In the latter, orga-nisms are commonly present in the naturallesion, which can be caused by either inde-pendently (Dajani et al. 1972). Similarly theexperimental lesion in the hamster can becaused by either or both organisms and theinteraction between the organisms can beantagonistic (Dajani & Wannamaker 1970,I 9 71 ). Necrotizing fasciitis is not a sequel ofimpetigo, possibly because of host factors orbecause the organisms which cause theselesions are regarded as forming a separategroup of 'skin' strains, and may differ in theirarmamentarium. It would thus be of interestto test impetigo strains in our model.We tried three strains of S. aureus in our

animal model, two fairly extensively. Theisolate from the patient with necrotizingfasciitis was much less effective than the pigstrain which produced much higher levels oftoxin in vitro. The pig strain also spreadwithout the presence of BHS, which couldreflect its high level of toxin production: S.aureus by itselfcan cause necrotizing fasciitis,but our experience is that this is rare (Lep-pard & Seal I983). As we obtained a moresatisfactory and better controlled modelusing the cx-lysin preparation we concen-trated on this approach.The a-lysin we used was not specially

purified. However, the ability of the crude a-lysin to give rise to inflammation in therabbit skin was blocked by specific anti-x-lysin but not by specific anti-,B-lysin. Thespecific anti-ac-lysin also blocked the ability ofthe crude a-lysin to cause a spreading lesionwhen co-injected with group A BHS (TableI) and so a-lysin played the major role, but aminority contribution from other staphylo-coccal products in this preparation cannot beexcluded. It possibly had the same effectwhen human S. aureus was co-injected withgroup A BHS (Table 2), but it is difficult to

interpret results with co-injection of specificantisera using live organisms which con-tinue to elaborate toxin in the animal.

Injection of crude staphylococcal toxininto the rabbit skin in amounts well in excessofIWN produces local infarction and dermo-necrosis, with an actue inflammatory reac-tion with extensive polymorphonuclear infil-tration only at the boundary (Thal & Egner1954). When we injected crude a-lysin atlevels which produced inflammation with-out necrosis, we saw only the marked inflam-matory reaction, potentiated by co-injectionof THN. We have not found a detailedinvestigation of the reactions in the skinfollowing the injection of a-lysin. Moststudies have been concerned with the manydifferent cell types affected (Jeljaszewicz et al.1978), the biochemical lesion produced bythe a-lysin being the formation of holes inlipid membranes (Freer & Arbuthnott I983).It is possible that other agents which producelocal damage, including other bacterial tox-ins, would have the same potentiating effect.A number of studies on infection with S.aureus suggest this. For example, Elek &Conen (1957) found that sterile black silksutures did, though broth filtrates did not,potentiate the development of a staphylococ-cal abscess in humans. Goshi et al. (I96I)found that heat-induced coagulationnecrosis enormously potentiated the abilityof S. aureus to set up infection in the rabbitskin. The additional potentiating effect in S.aureus infection of a cell wall componentwhich impedes the inflammatory response(Easmon et al. I973) in part supports thisidea. However, when we injected a-lysin intothe skin, the histological examinationshowed an inflammatory response withprominent extravasation of neutrophils andthat this spread in a minor form down thetrack which spreading lesions (if they deve-loped) were known to take, in direct contrastto the impedin described by Easmon et al.These observations are similar to those ofBurke and Miles (I958). They described a 3-4 h increase in leucocytosis and permeabilitywhich occurred when a variety of bacterial

828

Spreading streptococcal infection in rabbitsspecies were injected intracutaneously inguinea-pigs, and the area over which thisoccurred corresponded to that of the indur-ated 24 h lesion.A point of interest was the absence of

thrombus formation 24 h after a-lysin injec-tion. Damaged tissue can activate the bloodclotting system by a variety of ways (Muller-Berghaus I977) and conversely thrombusformation can cause ischaemic damage,which might well potentiate infection. In ourpaper on the human infection (Barker et al.I980) we drew attention to thrombi presentin the affected tissue, and similar observa-tions have been made by others (Bahna &Canalis I980). We found thrombi present ina proportion of both small and severe lesionsin the rabbit provided that the histologicalmaterial was taken within 6 days of theinjection. This is similar to the humandisease when sections of material taken latefrom sub-acute infection showed evidence ofrecanalization of thrombosed vessels.Although this study is not definitive on thepoint, the absence of thrombi followinginjection of crude staphylococcal a-lysin atIWN and their presence in small non-spread-ing lesions suggests that thrombus formationis a product of the infection rather than itscause.One of the apparent oddities of this model

is that spread of infection is invariably downthe flank of the belly and never sideways.This appears to correspond to lymph andvenous drainage, since trypan blue injectedin the same way tracks down the veins andlymphatics in this direction. In humanpatients, spreading streptococcal infectionsmay take different routes depending on thesite from which the infection arose (Seal &Leppard I982). We think that, although thedirection of spread may be different in differ-ent anatomical areas, this does not invali-date our model. In our model a giveninjection did not always give the sameclinical response, but we need to rememberthat patients also give variable reactions.Occasional injections of streptococci without

additional factors (except broth) gave rise tospreading lesions (Table i), and about 25%of injections with a-lysin did not. It is possiblethat anatomical factors play a role in this, asthey appeared to do in deciding whether aspreading lesion was spreading cellulitis ornecrotizing fasciitis. Also, in the animalspreading lesions contained a tube of pus ofvarying conformation running down theflank between the epidermis and the musclelayer: we do not often observe this in humans(Barker et al. I987). Other comparisonsbetween the human and rabbit model aresummarized in Table 4.The special ioK broth appeared on a

limited number of results to be a suitablegrowth medium for further investigation ofthis model. By using a medium which con-tains only low molecular weight compo-nents, it is comparatively easy to separatethem from the exotoxins by ultrafiltration.We have not checked toxin production ex-tensively in this medium, although streptoly-sin 0 and proteinase were produced. Thebetter growth that occurred in it is likely to bedue to the removal of the inhibitory factorsproduced when glucose is autoclaved withpeptone (Meynell & Meynell 1970) or per-haps to the avoidance of heat sterilization inthe prepared medium.

Str. sanguis was tested because of its use asa host for the virulence factors of group ABHS. It was found that in its native state,without genetic manipulation, it was unableto persist or cause a pyogenic response in thismodel, with or without x-lysin. Thus, if astrain carrying genetic elements for produc-tion of virulence factors such as streptolysinO were to cause a more serious lesion in thismodel, it would be evidence for the impor-tance ofthat virulence factor. The traditionalmouse challenge test consists of direct inocu-lation into the peritoneal cavity followed byearly septicaemic death (Dochez et al. I 9 I 9).This contrasts with our model which wethink approximates well to spreading softtissue infection caused by ,B-haemolyticstreptococci.

829

830 D. V. Seal & D. Kingston

Acknowledgements

We thank Dr F.G. Barker for advice oncertain aspects of the histopathology and DrC. Coid for helpful discussions. We also thankDr C. Adlam for specific antisera, Professor J.Jones for animal strains of S. aureus, and bothfor helpful discussions. We are most gratefulto Mrs Janet Gilbert for frequent and carefultyping of the text.

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