Journal of Hospital Infection (1992) 22 ( S u p p l e m e n t A), 89-97

In-vitro b a c t e r i c i d a l activity of four oral antibiotics against p a t h o g e n s responsible for a c u t e otitis m e d i a in c h i l d r e n N. L a m b e r t - Z e c h o v s k y , P. M a r i a n i - K u r k d j i a n , C. Doit, F. B o u r g e o i s a n d E. B i n g e n

Hdpital Robert Debrg, Service de Bact~riologie, Paris, France Summary: T h i s s t u d y was designed to test the i n - v i t r o activity of four oral antibiotics against t h e four m i c r o o r g a n i s m s most f r e q u e n t l y isolated in acute otitis media: ~ - l a c t a m a s e - p o s i t i v e Haemophilus influenzae ( N = 10), [3-1actamase-positive Moraxella catarrhalis ( N = 10), penicillin-sensitive Streptococcus pneumoniae ( N = l l ) and m e t h i c i l l i n - s e n s i t i v e Staphylococcus aureus ( N = 10), by the bactericidal curve m e t h o d . Bactericidal kinetics were determ i n e d for c o n c e n t r a t i o n s of antibiotic equivalent to those f o u n d in the m i d d l e ear after t r e a t m e n t : a m o x y c i l l i n - c l a v u l a n i c acid (2'5 m g 1-1/0"6 m g I 1 and 2 " S m g 1 - 1 / 1 . 2 m g l ~), cefaclor ( l m g l - ~ ) , e r y t h r o m y c i n (0.Smg1-1) and e r y t h r o m y c i n / s u l f i s o x a z o l e (0"2/3 m g 1-1). T h e i n o c u l u m was of 10 ~ colonyf o r m i n g units (cfu) ml-~. T h e bacterial c o u n t s were p e r f o r m e d after 5 h a n d 24 h using a spiral i n o c u l a t o r system. T h e results s h o w e d that amoxycillinclavulanic acid h a d rapid bactericidal activity ( < 24 h) on the tested organisms at each of the doses used (reductions

Oral dose

Mean middle-ear concentration at 2 h (rag l ~)

250 + 62"5 mg 10 mg kg -1 15 mg kg ~ 12"5+ 37"5 mg kg-~

2.5 + 0.6-1 2 1 0.5 0"2 + 3

In-vitro activity of antibiotics against otitis media pathogens

91

O r g a n i s m s w e r e g r o w n in c u l t u r e m e d i a as follows: S. aureus: u n s u p p l e m e n t e d M u e l l e r - H i n t o n b r o t h ; S . pneumoniae: B r a i n - h e a r t infusion b r o t h + 1 0 % ascites; H. influenzae a n d M . catarrhalis: Mueller-Hinton broth + G supplement. T h e c u l t u r e s w e r e p l a c e d in a stirring w a t e r - b a t h at 37~ Bactericidal activity was d e t e r m i n e d b y c o u n t i n g s u r v i v o r s w i t h a s e m i - a u t o m a t i c S p i r a l p l a t e r at 5 and 24 h; S. pneumoniae was n o t t e s t e d at 24 h b e c a u s e it is r a p i d l y lysed. T h e d e t e c t i o n t h r e s h o l d of the s y s t e m is 1 0 2 c f u m l -I, a l l o w i n g a p o t e n t i a l c a r r y o v e r effect to be d e t e c t e d . Killing c u r v e s w e r e e s t a b l i s h e d f r o m the m e a n log10 c o u n t s for e a c h a n t i b i o t i c and each strain; b a c t e r i c i d a l activity was d e f i n e d as a 3 log10 cfu m l - 1 r e d u c t i o n in the initial i n o c u l u m at 24 h. Results

M i n i m u m inhibitory concentrations T h e M I C results for each a n t i b i o t i c against the t e s t e d strains are s h o w n in T a b l e II. H . influenzae R e g a r d l e s s of the c o n c e n t r a t i o n of C A (0"6 or 1 - 2 m g m l - l ) , A M X + C A gave a 1 lOgl0cfum1-1 r e d u c t i o n at 5 h, a n d a r e d u c t i o n of at least 3 log10 cfu ml 1 at 24 h. T h e t h r e e r e m a i n i n g a n t i b i o t i c s w e r e b a c t e r i o s t a t i c at 5 h and ineffective at 24 h ( F i g u r e 1). M . catarrhalis R e g a r d l e s s of the c o n c e n t r a t i o n o f CA, the c o m b i n a t i o n A M X + C A gave a r e d u c t i o n of 2 log10 cfu m l 1 at 5 h and a b o u t 3 log10 cfu m l - 1 at 24 h; killing was slightly g r e a t e r w h e n C A was used at l ' 2 m g 1 - 1 C e f a c l o r h a d no b a c t e r i c i d a l activity, w i t h a r e d u c t i o n of o n l y 0"5 log10 cfu m l - 1 at 5 h a n d no effect at 24 h. E r y t h r o m y c i n , alone and in c o m b i n a t i o n w i t h sulfisoxazole, gave a slight r e d u c t i o n at 5 h; at 24 h, e r y t h r o m y c i n alone was bacteriostatic,

Table II. MIC~,, (mgl ~) of the studied strains Organisms (no.)

Amoxycillin clavulanic acid

tt. influenzae(10) S. pneumoniae (11) iVI. catarrhalis (10) ,9. a u r e u s (10)

0"5 (0"25-1)* 0.03 (0.01 0'06)

* Range.

Cefaclor

Erythromycin sulfisoxazole

8 (2 16) 4 (1 16) 1 (0"2-8) 0.5 (0125 0-5) 0"05 (0.012 0-4) 0.12 (0.008 0-5)

0"25 (0"012 0"5) 16 0"5 (0"25 2)

Erythromycin

(8-64)

0"5 (0"125-2)

0"5 (0-25 1)

0"5 (0"25 1)

0"5 (0"0t 0'5)

0"5 (0"01-0"5)

N. Lambert-Zeehovsky

92

et al.

H. influenzao ~-lactomase (+) /V:rO strains I0

u E

g ...I

21

I

I

I

0

5 Hours

24

F i g u r e 1. C o l o n y c o u n t s o f Haemophilus influenzae at d i f f e r e n t t i m e s a f t e r a n t i b i o t i c a d d i t i o n ( m g l t). I = A m o x y c i l l i n (AMX) 2"5mg+clavulanic acid ( C A ) I'2mg,+=AMX 2"5 m g + C A 0 ' 6 rag, * = C e f a c l o r ( C E F ) 1 m g , [] = E r y t h r o m y c i n ( E R Y ) 0"5 rag, • = E R Y 0"2 r a g + S u l f i s o x a z o l e ( S F S ) 3 rag, 9 = C o n t r o l .

while the bacterial count continued to fall moderately with the combination (Figure 2).

S. pneumoniae Regardless of the concentration of CA, the combination A M X + CA was strongly bactericidal against S. pneumoniae at 5 h, with a 4 logl0 cfu m l - l reduction. A reduction of about 2 logu~ cfu m l - 1 was obtained with cefaclor, e r y t h r o m y c i n and e r y t h r o m y c i n + sulfisoxazole (Figure 3). S . allreLlS

A M X + CA gave a reduction of 2 logu~ cfu ml I at 5 h, regardless of the amoxycillin concentration; the combination was bactericidal at 24 h, with a

In-vitro activity of antibiotics against otitis media pathogens

93

l~l co'torrholis /~-Iactamase ( + ) N = I0 strains I0

~A

7

-g o

g, -J

0

I 0

1 5

I 24

Hours

Figure 2. Colony counts of Moraxella catarrhalis at different times after antibiotic addition. F o r key see legend to Figure 1.

reduction > 3 logl0 c f u m l -I. Cefaclor and e r y t h r o m y c i n showed no bactericidal activity against S . aureus; similarly, ERY + S F S only gave a 1 log m cfu m1-1 reduction at 24 h (Figure 4). In s u m m a r y , cefaclor, erythromycin, and erythromycin-sulfisoxazole when tested at the concentrations close to those found in the middle ear, caused an initial reduction in bacterial counts of 1-2 l o g l 0 c f u m l - 1 ; for A M X + CA a greater bactericidal effect was seen with a reduction of /> 3 logu) cfu m1-1 being achieved at 24 h. It should be noted, however, that in the case of S . aureus the pre-selected test concentration of A M X + CA were higher than the expected middle ear concentrations. Discussion

T h i s study showed that only amoxycillin-clavulanic acid is bactericidal 24 h

94

N. Lambert-Zechovsky

al.

et

Streptococcus pneumon/oe N : II strains rO

J & 6

T

--I

1 0

1 5 Hours

Figure 3. Colony counts of Streptococcus pneumoniae at different times after antibiotic addition. F o r key see legend to Figure 1.

post-dose against the four species of bacteria most frequently isolated in acute otitis media, including certain [3-1actamase-producing strains. Bonte and Carlier ~ have shown that after only 3 h, amoxycillin has greater bactericidal activity (as measured by bacterial A T P ) than cefaclor when tested against these same organisms. T h e bactericidal activity of amoxycillin-clavulanic acid on H. influenzae has been confirmed by other authors 9'1~ but it is rather surprising that no effect was observed with cefaclor, even at 24 h, since Bartmann and T a r b u c found bactericidal activity 11. T h e r e are several possible reasons for this: (i) increased resistance of H. influenzae to cefaclor; or (ii) instability of cefaclor in solution 12 and especially in H. influenzae culture medium. F u r t h e r m o r e Bartmann and T a r b u c used a concentration of 4 mg 1 1, higher than in this study, and greater than would be expected in middle-ear fluid.

In-vitro activity of antibiotics against otitis m e d i a pathogens

95

Gtaphy/ococcus oureus methiciilin- $ N = I O strains I0

/

T ~ 51

[

I

0

5

__

I

24

Hours

F i g u r e 4. C o l o n y c o u n t s o f S t a p h y l o c o c c u s aureus at d i f f e r e n t t i m e s a f t e r a n t i b i o t i c a d d i t i o n . 9 = A M X 4 nag + C A 1 '5 r a g , + = A M X 8 m g + C A 1.5 nag, * = C E F 1 nag, [~ = E R Y 0' 5 nag, x = E R Y 0.2 n a g + S F S 3 nag, 9 = C o n t r o l . F o r a b b r e v i a t i o n s see l e g e n d to F i g u r e 1.

It is particularly difficult to test for bactericidal activity against S.

pneumoniae as it lyses spontaneously in vitro, thus interfering with the interpretation of the results. 13 Therefore, in the present study the test was restricted to 5 h. T h e strains tested were penicillin sensitive ( M I C