J . clin. Path., 1972, 2 5 , 799-803 T h e erythrasma microorganism i n situ: studies using t h e skin surface biopsy technique R . N. D. RAMNARAIN, B . BHOGAL, AND N . T. MOORE From t h e Institute o f Dermatology, S t . John's Hospitalfor Diseases o f t h e Skin, London, a n d t h e Depart- ment of Crystallography, Birkbeck College, London SYNOPSIS T h e skin surface biopsy technique has been used t o investigate t h e erythrasma organism in situ i n t h e stratum corneum i n 11 patients. Staining b y P A S a n d Gram stain showed t he presence of a large number o f organisms arranged haphazardly i n some areas a n d i n microcolonies i n others. With t h e scanning electron microscope i t was possible t o se e that smooth filamentous chains of microorganisms h a d penetrated horn cells a n d caused disturbance o f t h e surface structure o f these cells. Enzyme histochemical tests showed that the erythrasma microorganism possessed a strong react- ivity f o r NAD diaphorase a n d other mitochondrial enzymes. T he reactivity wa s focal confirming a complex subcellular organization o f organelles. I t i s suggested that t h e erythrasma microorganism secretes a mucopolysaccharide sheath i n some circumstances. Erythrasma i s a chronic scaly dermatosis affecting t h e body flexures a n d intertriginous areas o f adults. Previously it w a s believed t o b e d u e either t o a type o f dermatophyte (Microsporum minutissimum) o r a n actinomycete (Nocardia minutissimum). I n 1969, a diphtheroid termed Corynebacterium minutissimum w a s isolated from lesions o f erythrasma an d thought t o b e the causative agent (Sarkany, Taplin, a nd Blank, 1961). Still more recently Somerville (1970) suggested that several strains o f fluorescent diphther- oid organisms were responsible f o r t h e clinical disorder that i s recognized a s erythrasma. In this study w e describe results o f o u r investigations into t h e causative organism o f erythrasma using t h e skin surface biopsy technique, which removes an intact layer o f stratum corneum with a cyano- acrylate adhesive (Marks a n d Dawber, 1971). A general account o f t h e us e of this method f o r demonstrating pathogenic microorganisms in t h e stratum corneum i s documented elsewhere (Marks a n d Dawber, 1972). Methods PATIENTS T h e clinical details o f t h e 1 1 patients with erythrasma wh o were investigated i n this study are as follows: Received f o r publication 8 June 1972. a l l 1 1 patients were men, t h e average age wa s 4 8 a nd t h e a g e range 2 4 t o 6 3 . I n nine t h e groins were involved, in four t h e t oe webs involved, a n d i n four t h e axillae were involved. I t c a n b e seen that t h e usual male predominance o f t h e disease i s evident i n o ur group o f patients. A coral pink fluorescence w a s detected i n t he involved sites when they were viewed i n ultraviolet light a n d small fluorescing colonies o f C . minutissimum were isolated o n 1 99 plates from skin scales in a l l patients. SKIN SURFACE BIOPSIES These specimens were obtained using ethyl cyano- acrylate (Permabond, Staines, Middx). Drops o f t h e adhesive were placed o n glass microscope slides or coverslips. Samples taken onto coverslips were stored i n t h e deep freeze a t -40'C before histo- chemical examination. Samples o n microscope slides were examined b y light microscopy after alcohol fixation (two minutes) a n d staining with periodic acid Schiff reagent (periodic acid 1 0 minutes, Schiff reagent 2 0 minutes) o r Gram stain (methyl violet t w o minutes, Gram's iodine t w o minutes, with differentiation i n equal parts o f alcohol a n d acetone f o r four seconds a n d counterstaining i n neutral r e d t o minutes). These preparations were made per- manent b y mounting them with a coverslip a nd neutral mounting medium. 7 9 9 group.bmj.com on February 28, 2011 - Published by jcp.bmj.com Downloaded from
J . c l i n . P a t h . , 1 9 7 2 , 2 5 , 7 9 9 - 8 0 3
T h e e r y t h r a s m a m i c r o o r g a n i s m i n s i t u : s t u d i e su s i n g t h e s k i n s u r f a c e b i o p s y t e c h n i q u e
R . MARKS, N. D. RAMNARAIN, B . BHOGAL, AND N. T . MOORE
From t h e I n s t i t u t e o f D e r m a t o l o g y , S t . J o h n ' s H o s p i t a l f o r D i s e a s e s o f t h e S k i n , L o n d o n , a n d t h e D e p a r t -
ment of C r y s t a l l o g r a p h y , B i r k b e c k C o l l e g e , L o n d o n
SYNOPSIS T h e s k i n s u r f a c e b i o p s y t e c h n i q u e h a s b e e n u s e d t o i n v e s t i g a t e t h e e r y t h r a s m a o r g a n i s mi n s i t u i n t h e s t r a t u m corneum i n 11 p a t i e n t s . S t a i n i n g b y PAS a n d Gram s t a i n s h o w e d t h e presence
o f a l a r g e n u m b e r o f o r g a n i s m s a r r a n g e d h a p h a z a r d l y i n some areas a n d i n m i c r o c o l o n i e s i n o t h e r s .W i t h t h e s c a n n i n g e l e c t r o n m i c r o s c o p e i t was p o s s i b l e t o se e t h a t s m o o t h f i l a m e n t o u s c h a i n s o f
m i c r o o r g a n i s m s h a d p e n e t r a t e d h o r n c e l l s a n d c a u s e d d i s t u r b a n c e o f t h e s u r f a c e s t r u c t u r e o f t h e s e
c e l l s .E n z y m e h i s t o c h e m i c a l t e s t s s h o w e d t h a t t h e e r y t h r a s m a m i c r o o r g a n i s m p o s s e s s e d a s t r o n g r e a c t -
i v i t y f o r NAD d i a p h o r a s e a n d o t h e r m i t o c h o n d r i a l enzymes. T h e r e a c t i v i t y was f o c a l c o n f i r m i n ga c o m p l e x s u b c e l l u l a r o r g a n i z a t i o n o f o r g a n e l l e s .
I t i s s u g g e s t e d t h a t t h e e r y t h r a s m a m i c r o o r g a n i s m s e c r e t e s a m u c o p o l y s a c c h a r i d e s h e a t h i n some
c i r c u m s t a n c e s .
E r y t h r a s m a i s a c h r o n i c s c a l y d e r m a t o s i s a f f e c t i n gt h e b o d y f l e x u r e s a n d i n t e r t r i g i n o u s a r e a s o f a d u l t s .P r e v i o u s l y i t w a s b e l i e v e d t o b e d u e e i t h e r t o a t y p e
o f d e r m a t o p h y t e ( M i c r o s p o r u m m i n u t i s s i m u m ) o r a na c t i n o m y c e t e ( N o c a r d i a m i n u t i s s i m u m ) . I n 1 9 6 9 , a
d i p h t h e r o i d t e r m e d C o r y n e b a c t e r i u m m i n u t i s s i m u m
w a s i s o l a t e d f r o m l e s i o n s o f e r y t h r a s m a a n d t h o u g h t
t o b e t h e c a u s a t i v e a g e n t ( S a r k a n y , T a p l i n , a n dB l a n k , 1 9 6 1 ) . S t i l l more r e c e n t l y S o m e r v i l l e ( 1 9 7 0 )s u g g e s t e d t h a t s e v e r a l s t r a i n s o f f l u o r e s c e n t d i p h t h e r -o i d o r g a n i s m s w e r e r e s p o n s i b l e f o r t h e c l i n i c a ld i s o r d e r t h a t i s r e c o g n i z e d a s e r y t h r a s m a . I n t h i ss t u d y we d e s c r i b e t h e r e s u l t s o f o u r i n v e s t i g a t i o n si n t o t h e c a u s a t i v e o r g a n i s m o f e r y t h r a s m a u s i n g t h es k i n s u r f a c e b i o p s y t e c h n i q u e , w h i c h r e m o v e s a n
i n t a c t l a y e r o f s t r a t u m c o r n e u m w i t h a c y a n o -
a c r y l a t e a d h e s i v e ( M a r k s a n d D a w b e r , 1 9 7 1 ) . A
g e n e r a l a c c o u n t o f t h e u s e o f t h i s m e t h o d f o rd e m o n s t r a t i n g p a t h o g e n i c m i c r o o r g a n i s m s i n t h es t r a t u m c o r n e u m i s d o c u m e n t e d e l s e w h e r e ( M a r k sa n d D a w b e r , 1 9 7 2 ) .
M e t h o d s
PATIENTS
T h e c l i n i c a l d e t a i l s o f t h e 1 1 p a t i e n t s w i t h e r y t h r a s m awho w e r e i n v e s t i g a t e d i n t h i s s t u d y a r e a s f o l l o w s :R e c e i v e d f o r p u b l i c a t i o n 8 J u n e 1 9 7 2 .
a l l 1 1 p a t i e n t s w e r e m e n , t h e a v e r a g e a g e w a s 4 8 a n dt h e a g e r a n g e 2 4 t o 6 3 . I n n i n e t h e g r o i n s w e r e
i n v o l v e d , i n f o u r t h e t o e w e b s w e r e i n v o l v e d , a n d i n
f o u r t h e a x i l l a e w e r e i n v o l v e d . I t c a n b e s e e n t h a t t h eu s u a l m a l e p r e d o m i n a n c e o f t h e d i s e a s e i s e v i d e n ti n o u r g r o u p o f p a t i e n t s . A c o r a l p i n k f l u o r e s c e n c ew a s d e t e c t e d i n t h e i n v o l v e d s i t e s w h e n t h e y w e r e
v i e w e d i n u l t r a v i o l e t l i g h t a n d s m a l l f l u o r e s c i n gc o l o n i e s o f C . m i n u t i s s i m u m w e r e i s o l a t e d on 1 9 9
p l a t e s f r o m s k i n s c a l e s i n a l l p a t i e n t s .
SKIN SURFACE BIOPSIES
T h e s e s p e c i m e n s w e r e o b t a i n e d u s i n g e t h y l c y a n o -
a c r y l a t e ( P e r m a b o n d , S t a i n e s , M i d d x ) . D r o p s o f t h ea d h es i v e w e r e p l a c e d o n g l a s s m i c r o s c o p e s l i d e s o r
c o v e r s l i p s . S a m p l e s t a k e n o n t o c o v e r s l i p s w e r e
s t o r e d i n t h e d e e p f r e e z e a t -40'C b e f o r e h i s t o -c h e m i c a l e x a m i n a t i o n . S a m p l e s o n m i c r o s c o p e s l i d e sw e r e e x a m i n e d b y l i g h t m i c r o s c o p y a f t e r a l c o h o l
f i x a t i o n ( t w o m i n u t e s ) a n d s t a i n i n g w i t h p e r i o d i ca c i d S c h i f f r e a g e n t ( p e r i o d i c a c i d 1 0 m i n u t e s ,S c h i f f r e a g e n t 2 0 m i n u t e s ) o r Gram s t a i n ( m e t h y lv i o l e t t w o m i n u t e s , G r a m ' s i o d i n e t w o m i n u t e s , w i t h
d i f f e r e n t i a t i o n i n e q u a l p a r t s o f a l c o h o l a n d a c e t o n e
f o r f o u r s e c o n d s a n d c o u n t e r s t a i n i n g i n n e u t r a l r e d
t w o m i n u t e s ) . T h e s e p r e p a r a t i o n s w e r e made p e r -m a n e n t b y m o u n t i n g t h e m w i t h a c o v e r s l i p a n d
n e u t r a l m o u n t i n g m e d i u m .
7 9 9
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R . M a r k s , N . D . R a m n a i r a i n i , B . B h o g a l , a n d N . T . Moore
ENZYME HISTOCHEMICAL INVESTIGATIONS
S k i n s u r f a c e b i o p s i e s f r o m s i x p a t i e n t s were t a k e n
onto c o v e r s l i p s i n s t e a d o f m i c r o s c o p e s l i d e s a n d w e r e
t e s t e d w i t h t h e f o l l o w i n g enzyme h i s t o c h e m i c a l
r e a c t i o n s : ( a ) n o n - s p e c i f i c e s t e r a s e ( i n d o x y l a c e t a t e
e s t e r a s e m e t h o d o f H o l t , 1 9 5 4 ) ; ( b ) l e u c i n e a m i n o -
p e p t i d a s e ( s i n g L - l e u c y l 4 m e t h o x y-
n a p h t h y l a -m i d e as s u b s t r a t e a n d t h e m e t h o d o f N a c h l a s ,
C r a w f o r d , a n d S e l i g m a n , 1 9 5 7 ) ; ( c ) NADd i a p h o r a s e ,
l a c t i c d e h y d r o g e n a s e , s u c c i n i c d e h y d r o g e n a s e , a n d
b e t a - h y d r o x y b u t y r i c d e h y d r o g e n a s e ( u s i n g NBT
m e t h o d o f P e a r s e , 1 9 6 0 ) ; a n d ( d ) a d e n o s i n e t r i -
p h o s p h a t a s e ( u s i n g m e t h o d o f W a c h s t e i n , M e i s e l , a n d
N i e d z w i e d z , 1 9 6 0 ) .
SCANNING ELECTRON MICROSCOPE STUDIES
S k i n s u r f a c e b i o p s i e s f r o m t h r e e o f t h e s u b j e c t s we re
t a k e n onto g l a s s m i c r o s c o p e s l i d e s . A r e a s f r o m t h es p e c i m e n - b e a r i n g s i t e s o f t h e s e s l i d e s we re c u t t o
s i z e , m o u n t e d on ' s t u b s ' , a n d t h e n c o a t e d w i t h
c a r b o n i n an E d w a r d s c o a t i n g u n i t . T h e c o a t e d
s p e c i m e n s w e r e t h e n v i e w e d i n a C a m b r i d g e
s t e r e o s c a n s c a n n i n g e l e c t r o n m i c r o s c o p e (SEM) a t a n
a c c e l e r a t i n g v o l t a g e o f 2 0 k v a t a 4 5 ° t i l t .
R e s u l t s
S k i n s u r f a c e b i o p s i e s f r o m t h e a f f e c t e d s i t e s s t a i n e d
w i t h PAS r e a g e n t o r Gram s t a i n d e m o n s t r a t e d t h epresence o f numerous f u s i f o r m m i c r o o r g a n i s m s
w h i c h w e r e a r r a n g e d i n c l u s t e r s , c h a i n s , o r s i n g l y ,
a n d s c a t t e r e d over v a r i o u s l y s i z e d areas ( F i g s . 1 a a n db ) . T h e c h a i n s t h a t we re seen u s u a l l y c o m p r i s e d
t h r e e , f o u r , o r o c c a s i o n a l l y m o r e , i n d i v i d u a lb a c t e r i a l c e l l s . T h e m i c r o o r g a n i s m s a p p e a r e d
a p p r o x i m a t e l y t h r e e t o f o u r t i m e s as l o n g as t h e y
we re b r o a d . W i t h t h e PAS s t a i n t h e r e was n o t i c e a b l e
d i f f u s e s t a i n i n g o f t h e s i t e s c o n t a i n i n g t h e m i c r o -
o r g a n i s m s w h i c h was v i s i b l e m a c r o s c o p i c a l l y . T h e r e
d i d n o t appear t o b e a p a r t i c u l a r a c c e n t u a t i o n a r o u n d
h a i r f o l l i c l e s or s w e a t g l a n d o p e n i n g s .
T h e r e s u l t s o f t h e enzyme h i s t o c h e m i c a l i n -v e s t i g a t i o n s are g i v e n i n T a b l e 1 . I t can b e seen t h a tt h e s e b a c t e r i a possess a w i d e range o f enzyme
r e a c t i v i t i e s . M i t o c h o n d r i a l enzyme a c t i v i t i e s s e e m e d
p a r t i c u l a r l y s t r o n g , e s p e c i a l l y NAD d i a p h o r a s e a n d
l a c t i c d e h y d r o g e n a s e a c t i v i t i e s . T h e r e a c t i o n pro-
d u c t s f r o m t h e t e s t s p e r f o r m e d we re n o t d i s t r i b u t e dd i f f u s e l y w i t h i n t h e i n d i v i d u a l b a c t e r i a l c e l l s , b u t
a p p e a r e d a g g r e g a t e d i n w e l l d e f i n e d f o c i w i t h i n e a c h
c e l l ( F i g . 2 ) .W i t h t h e SEM s i n g l e r o d - s h a p e d a n d o v a l s t r u c -
t u r e s a n d c h a i n s we re seen l y i n g on, a n d e m b e d d e d
i n , t h e s u r f a c e o f t he i n d i v i d u a l c o r n i f i e d c e l l s .
I n d i v i d u a l b a c t e r i a l c e l l s m e a s u r e d a p p r o x i m a t e l y
2 , x 1 I s a l t h o u g h t h e r e was q u i t e a w i d e range o f
Enzyme N o . N o . N o F u t n g u s T o t a l
A c t i v i t y P o s i t i v e N e g a t i v e
T e s t e d
N o n - s p e c i f i c 4 0 1 5e s t e r a s e
L e u c i n e 1 1 0 2
a m i n o
p e p t i d a s eNAD 5 0 0 5d i a p h o r a s e
L a c t i c 4 0 1 5
d e h y d r o -
g e n a s e
ATPase I 0 0 1S u c c i n i c 2 0 2
d e h y d r o -g e n a s e
3 - H y d r o x y - 2 0 0 2b u t y r i c
d e h y d r o -
g e n a s e
T a b l e I S u m m a r y o f t h e r e s u l t s o f t h e e n z y m e h i s t o -c h e m i c a l t e s t s
s i z e s p r e s e n t ( F i g . 3 ) . T h e c h a i n s w e r e f o r m e d b y t h ea p p a r e n t l i n k i n g o f s e v e r a l o r g a n i s m s . I t i s i n t e r e s t i n gt o n o t e t h a t a l t h o u g h i n d i v i d u a l b a c t e r i a w e r e e a s i l ys e e n when t h e y w e r e i s o l a t e d o r s i n g l e c e l l s , i t w a s
o n l y j u s t p o s s i b l e t o make o u t t h e s h a p e o f t h ei n d i v i d u a l m i c r o o r g a n i s m s w h e n t h e y w e r e a r r a n g e d
i n a c h a i n . T h e s u r f a c e o f t h e b a c t e r i a l c e l l s a p p e a r e d
r e g u l a r l y s m o o t h w i t h o u t a n y o b v i o u s s p e c i a l i z e da n a t o m i c a l f e a t u r e s a t m a g n i f i c a t i o n s u p t o 2 5 0 0 0 .T h e n u m b e r o f o r g a n i s m s s e e n b y SEM w a s s m a l l
c o m p a r e d t o t h e l a r g e n u m b e r s e e n i n s i m i l a r p r e -p a r a t i o n s v i e w e d w i t h t h e l i g h t m i c r o s c o p e a f t e rs t a i n i n g . T h e c h a i n s o f b a c t e r i a l c e l l s w e r e f r e q u e n t l ys e e n t o b e p e n e t r a t i n g t h e c o r n i f i e d c e l l s . A t t h e s i t e o f
p e n e t r a t i o n t h e r e w e r e , i n s e v e r a l s c a l e s , o p e n i n g s
t h a t w e r e t w o o r t h r e e t i m e s l a r g e r t h a n t h e d i a m e t e r
o f t h e m i c r o o r g a n i s m s .
T h e i n v o l v e d c e l l s o f t h e s t r a t u m c o r n e u m h a d a n
i r r e g u l a r r i d g e p a t t e r n , m a i n l y c o m p o s e d o f
l o w , b r o k e n , u n d u l a t i n g r i d g e s w h i c h s o m e t i m e s
b r a n c h e d . T h e s u r r o u n d i n g u n i n v o l v e d c e l l s o f t e n
p o s s e s s e d p r o m i n e n t v i l l i r e s e m b l i n g t h e s u r f a c es t r u c t u r e o f t h e s c a l e i n p s o r i a s i s a n d s o m e o t h e r
p a r a k e r a t o t i c d i s o r d e r s ( D a w b e r , M a r k s , a n d S w i f t ,1 9 7 2 ) .
Comment
T h e s k i n s u r f a c e b i o p s y m e t h o d i s w e l l s u i t e d t o t h es t u d y o f c e r t a i n a s p e c t s o f t h e m i c r o b i o l o g y o f t h es k i n . I n p a r t i c u l a r , a g o o d v i e w i s o b t a i n e d o f v a r i o u s
i n v a d i n g m i c r o o r g a n i s m s i n t h e i r ' n a t u r a l h a b i t a t 's o t h a t t h e i n v a s i v e p r o c e s s c a n b e s t u d i e d . I n
a d d i t i o n , t h e n u m b e r o f p a t h o g e n s p r e s e n t c a n b e
d i r e c t l y e s t i m a t e d a n d i t i s a l s o p o s s i b l e t o o b s e r v e
8 0 0
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R . M a r k s , N . D . R a m n a r a i n , B . B h o g a l , a n d N . T . M o o r e
F i g . 2 P h o t o m i c r o g r a p h f r o m s k i n s u r f a c e b i o p s y t e s t e dw i t h NAD d i a p h o r a s e r e a c t i o n . N o t e f o c a l a g g r e g a t i o n
o f f o r m a z a n r e a c t i o n p r o d u c t ( x 2 5 0 ) .
a n y m o r p h o l o g i c a l o r h i s t o c he m i c a l c h a n g e s c a u s e d
b y t h e r a p e u t i c m e a s u r e s .
I n t h i s s t u d y o f e r y t h r a s m a , s e v e r a l f e a t u r e s h a v e
come t o l i g h t t h a t a r e w o r t h y o f c o m m e n t . T h e r e
w a s a r e m a r k a b l e d i s p a r i t y b e t w e e n t h e n u m b e r s o f
b a c t e r i a s e e n i n s t a i n e d p r ep a r a t i o n s w i t h t h e l i g h t
m i c r o s c o p e a n d t h e n u m b e r s s e e n b y SEM. T h i si s
p r o b a b l y a r e f l e c t i o n o f t h e f a c t t h a t t h e ' s u r f a c e 's c a n n e d b y SEM u s i n g t h e s k i n s u r f a c e b i o p s y
m e t h o d i s f i v e o r s i x c e l l l a y e r s down i n t o t h es t r a t u m c o r n e u m , w h i l e w i t h t h e l i g h t m i c r o s c o p e t h ew h o l e t h i c k n e s s o f t h e s p e c i m e n r e m o v e d i s e x a -
m i n e d . M o n t e s , B l a c k , a n d M c B r i d e ( 1 9 6 7 ) d e m o n -
s t r a t e d b y c o n v e n t i o n a l EM t h a t t h e g r e a t m a j o r i t y
o f t h e m i c r o o r g a n i s m s o f e r y t h r a s m a d i d n o t i n v a d e
f u r t h e r t h a n t h e s u p e r f i c i a l p a r t o f t h e s t r a t u m
c o r n e u m . T h i s m u s t a l s o b e a m a j o r c o n t r i b u t i n g
r e a s o n f o r t h e r e l a t i v e l y s m a l l n u m b e r o f m i c r o -
o r g a n i s m s s e e n b y S EM i n t h i s s t u d y .
T h e s u r f a c e s o f t h e h o r n c e l l s i n t o w h i c h t h em i c r o o r g a n i s m s h a d p e n e t r a t e d p o s s e s s e d a d i s -o r g a n i z e d r i d g e p a t t e r n , s u g g e s t i n g t h e p o s s i b i l i t y o f
F i g . 3 S c a n n i n g e l e c t r o nm i c r o g r a p h f r o m s k i n s u r f a c eb i o p s y o f e r y t h r a s m a . N o t e
f i l a m e n t o u s c h a i n o f m i c r o -o r g a n i s m s ( A ) e m e r g i n g f r o ma s c a l e w i t h a d i s o r g a n i z e ds c a l e p a t t e r n ( B ) ( x 4 0 0 0 ) .
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T h e e r y t h r a s m a m i c r o o r g a n i s m i n s i t u : s t u d i e s u s i n g t h e s k i n s u r f a c e b i o p s y t e c h n i q u e
d i s r u p t i o n o f t h e s y s t e m o f t o n o f f l a m e n t - d e s m o s o m ec o m p l e x e s . I n a d d i t i o n , t h e i m p r e s s i o n w a s g a i n e d
t h a t a t t h e s i t e o f p e n e t r a t i o n t h e o p e n i n g w a s , i ns o m e c a s e s , much w i d e r t h a n t h e m i c r o o r g a n i s m s .
T h i s m i g h t s u g g e s t t h a t p e n e t r a t i o n w a s a c c o m p l i s h e d
b y a c h e m i c a l d i s s o l u t i o n r a t h e r t h a n b y p u r e l y
p h y s i c a l p r e s s u r e . M o n t e s e t a l ( 1 9 6 7 ) n o t e d d i s -o r g a n i z a t i o n o f ' k e r a t i n f i b r i l s ' i n h o r n c e l l s i n v a d e d
b y C . m i n u t i s s i m u m a n d t h i s w o u l d b e i n a c c o r d w i t h
o u r o b s e r v a t i o n s .S t r o n g e n z y m e r e a c t i v i t y o f i n d i v i d u a l b a c t e r i a l
c e l l s w a s d e t e c t e d a n d t o o u r k n o w l e d g e t h i sm e t a b o l i c a c t i v i t y h a s n o t b e e n r e p o r t e d i n t h i s wayb e f o r e w i t h t h e e r y t h r a s m a o r g a n i s m . Some o f t h er e a c t i o n p r o d u c t s w e r e o b v i o u s l y a g g r e g a t e d i n w e l ld e f i n e d a r e a s , c o n f i r m i n g a c o m p l e x s u b c e l l u l a ra r r a n g e m e n t a k i n t o mammalian c e l l s . A s i m i l a ra r r a n g e m e n t h a s b e e n n o t e d w i t h e n z y m e r e a c t i o n s i nt h e d e r m a t o p h y t e f u n g i ( M e i n h o f , 1 9 6 8 ) .
We b e l i e v e t h a t t h e s m o o t h e x t e r i o r o f t h eo r g a n i s m s o b s e r v e d b y SEM, w i t h o n l y a s l i g h tc o n s t r i c t i o n a t t h e s i t e o f t h e j u n c t i o n s b e t w e e n
m i c r o o r g a n i s m s i n a c h a i n , p l u s t h e PAS r e a c t i v i t yo f t h e m i c r o o r g a n i s m ( a n d i t s i m m e d i a t e e n v i r o n -m e n t ) r e f l e c t s t h e s e c r e t i o n o f a m u c o p o l y s a c c h a r i d es h e a t h b y t h e o r g a n i s m i n s o m e s i t u a t i o n s . S o m e r -
v i l l e ( 1 9 7 2 ) d i s c u s s e d t h e m i c r o b i o l o g y o f t h ec u t a n e o u s d i p h t h e r o i d s a n d s t a t e d t h a t t h e d i p h -t h e r o i d s r e s p o n s i b l e f o r t r i c h o m y c o s i s a x i l l a r i sp r o d u c e d s e c r e t i o n s t h a t s t i c k t h e m t o g e t h e r a n d
t o t h e h a i r , a n d c a u s e , i n a d d i t i o n , d e s t r u c t i o n o f t h e
h a i r k e r a t i n . A s i m i l a r m a t e r i a l may w e l l b e p r o d u c e d
b y C . m i n u t i s s i m u m .
We a r e g r a t e f u l t o Dr Y . M . C l a y t o n a n d t h e s t a f f o f
t h e M y c o l o g y D e p a r t m e n t f o r h e l p i n t h i s i n v e s t i -g a t i o n , a n d t o D r s W. C . N o b l e a n d D . A . S o m e r -v i l l e f o r t h e i r h e l p f u l s u g g e s t i o n s .
R . M . i s i n r e c e i p t o f a g r a n t f r o m t h e MRC.
R e f e r e n c e s
D a w b e r , R . P . R . , M a r k s , R . , a n d S w i f t , J . A . ( 1 9 7 2 ) . S c a n n i n ge l e c t r o n m i c r o s c o p y o f t h e S t r a t u m c o r n e u m . B r i t . J . D e r m . , 8 6 ,
2 7 2 - 2 8 1 .
H o l t , S . J . ( 1 9 5 4 ) . A ne w a p p r o a c h t o t h e c y t o c h e m i c a l l o c a l i z a t i o n o fe n z y m e s . P r o c . r o y . S o c . B . , 1 42 , 1 6 0 - 1 6 9 .
M a r k s , R . , a n d D a w b e r , R . P . R . ( 1 9 7 1 ) . S k i n s u r f a c e b i o p s y . B r i t . J .D e r m . , 8 4 , 1 1 7 - 1 2 3 .
M a r k s , R . , an d D a w b e r , R . P . R . ( 1 9 7 2 ). I n s i t u m i c r o b i o l o g y o f t h e
s t r a t u m c o r n e u m . A r c h . D e r m . , 1 0 5 , 2 1 6 - 2 2 1 .
M e i n h o f , W. ( 1 9 6 8 ) . Zu m h i s t o c h e m i s c h e n N a c h w e i s v o n Enzymen d e s
e n e r g i e l i e f e r n d e n S t o f f w e c h s e l s i n D e r m a t o p h y t e n d e r
G a t t u n g e n M i c r o s p o r u m E p i d e r m o p h y t o n u n d K e r a t i n o m y c e s .A r c h . k l i n . e x p . D e r m . , 2 3 2 , 2 7 9 - 2 9 4 .
M o n t e s , L . F . , B l a c k , S . H . , and M c B r i d e , M. E . ( 1 9 6 7 ) . B a c t e r i a l
i n v a s i o n o f t h e S t r a t u m corneum i n e r y t h r a s m a . J . i n v e s t .D e r m . , 4 9 , 4 7 4 - 4 8 5 .
N a c h l a s , M. M . , C r a w f o r d , D . T . , a n d S e l i g m a n , A . M. ( 1 9 5 7 ) . Th eh i s t o c h e m i c a l d e m o n s t r a t i o n o f l e u c i n e a m i n o p e p t i d a s e . J .H i s t o c h e m . C y t o c h e m . , 5 , 2 6 4 - 2 7 8 .
P e a r s e , A . G . E . ( 1 9 6 0 ) . H i s t o c h e m i s t r y ; T h e o r e t i c a l a n d A p p l i e d ,
2nd e d . C h u r c h i l l , L o n d o n .S a r k a n y , I . , T a p l i n , D ., and B l a n k , H . ( 1 9 6 1 ) . Th e e t i o l o g y a n d
t r e a t m e n t o f e r y t h r a s m a . J . i n v e s t . D e r m . , 3 7 , 2 8 3 - 2 9 0 .S o m : r v i l l e , D . A . ( 1 9 7 2 ) . Th e m i c r o b i o l o g y o f t h e c u t a n e o u s
d i p h t h e r o i d s . B r i t . J . D e r m . , 8 6 , S u p p l . 8 , 1 6 - 2 0 .
S o m e r v i l l e , D . A . ( 1 9 7 0 ) . E r y t h r a s m a i n n o r m a l y o u n g a d u l t s . J . m e d .M i c r o b i o l . , 3 , 5 7 - 6 4 .
W a c h s t e i n , M . , M e i s e l , E . , a n d N i e d z w i e d z , A . ( 1 9 6 0 ) . H i s t o c h e m i c a l
d e m o n s t r a t i o n o f m i t o c h o n d r i a l a d e n o s i n e t r i p h o s p h a t a s ew i t h t h e l e a d - a d e n o s i n e t r i p h o s p h a t e t e c h n i q u e . J . H i s t o c h e m .
C y t o c h e m . , 8 , 3 8 7 - 3 8 8 .
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