Scand J Infect Dis 25: 3142, 1993

In Vitro Effect on Group A Streptococci of Loracarbef versus Cefadroxil, Cefaclor and Penicillin V

CARL KAMME and ANN-CATHRINE PETERSSON From the Department of Medical Microbiology, University Hospital of Lund, Lund, Sweden

The in vitro activity of loracarbef, penicillin V, cefaclor and cefadroxil against log and stationary phase cultures of group A streptococci was compared. MICs and MBCs were determined with the broth dilution method and by a modified agar plate dilution technique where the beta-lactams were inactivated Pfter the MICs were determined allowing inhibited but not killed organisms to grow on further incubation. The MICs of loracarbef and the two cephalosporins were 16-32 times higher than those of penicillin V. In plate dilution the MBC/MIC ratios of all agents were 5 2 for log phase cultures. With stationary phase cultures, especially in the broth dilution test, the MBC/MIC ratios of loracarbef and the two cephalo- sporins were 2 32 for a large number of strains. The phenotype response of stationary phase cultures to beta-lactam antibiotics may not only be related to the physiological status of the streptococci, to the culture conditions and to the beta-lactam under test. The present investigation indicated that the phenotypic response was also an intrinsic property of certain strains.

C. Kamme, MD, PhD, Department of Medical Microbwbgy, University Hospital of Lund, S6lvegan 23, S-223 62 Lund, Sweden

INTRODUCTION

In streptococcal pharyngitis clinical and bacterial treatment failure following phenoxy- methylpenicillin (penicillin V) therapy is reported to 5-25% (1,2). The minimum inhibitory concentrations (MICs) of group A streptococci for penicillin V are 0.005-0.03 mgh and no resistant strains have been isolated in vivo. Other factors than reduced sensitivity have therefore been suggested in attempts to explain the unsatisfactory results of penicillin treatment. Thus, so called penicillin tolerance and the presence of beta-lactamase producing pathogens in the respiratory tract inactivating the drug have been suggested as factors of significance in this context (3,4). The ecological disturbance by eradicting the highly penicillin sensitive alpha-streptococci has also been suggested to be of importance as the inhibitory activity of the alpha-streptococci against the group A streptococci is lost (5). A number of investigations have shown that cefadroxil treatment is superior to penicillin V treatment regarding both bacterial and clinical cure in streptococcal tonsillitis (6-8).

Loracarbef is a synthetic orally administered carbacephem. It has a beta-lactamase stabil- ity and an antimicrobial spectrum similar to that of cefaclor but possesses significantly greater chemical stability (9-11). The in vitro activity of loracarbef and cefadroxil against group A, B, C and G streptococci is reported to 0.06-1.0 and 0.06-0.12 mgh, respectively

The broth dilution technique, commonly used for determining MIC is subjected to technical pitfalls when used for determining minimum bactericidal concentration (MBC) (12, 13). In order to avoid these pitfalls some investigators have used the agar plate dilution technique and added beta-lactamase to the plates after MIC determination, thus allowing inhibited but not killed organisms to grow on further incubation (14,15). This method thus permits the determination of both MIC and MBC with one single inoculation step and was used in the present investigation for determining the MIC and MBC of loracarbef, cefaclor,

(11).

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

21/1

4Fo

r pe

rson

al u

se o

nly.

38 C. Kamme and A.-C. Petersson Scand J Infect Dis 25

/ C -4 / 7 /.

/

C ,I//

r' /; @ 80- 0

' 4 0 C-.-4-+/d/

L-* - - - - - - * / A ,--t-+- a-

I I a 1 I I I I

0.002 0.008 0.03 0.12 0.5 2.0 3 8.0 mg/ l

Fig. 1. MICs (-) and MBCs (---) of 50 group A streptococci. Plate dilution method. A. Log phase cultures. B. Stationary phase cultures. Penicillin V (M), cefaclor (O), loracarbef (A), cefadroxil (0).

Table I . Group A streptococcal strains with elevated MBCIMIC ratios* 50 strains. Figures denote %

Plate dilution Broth dilution

Log phase Stationary phase Log phase Stationary phase

Penicillin V 0 18 Cefaclor 0 44 Loracarbef 0 34 Cefadroxil 0 38

0 30 24' 76' 2 62 0 72

* Defined as MBClMIC 2 32 a MBUMIC 2 16 for another strain in log phase (2%) and 9 strains in stationary phase (18%), all with

MICs 1 mgA, as the strains grew on the highest concentration used (8 mg/l)

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

21/1

4Fo

r pe

rson

al u

se o

nly.

Scand J Infect Dis 25

cefadroxil and penicillin V. The results were compared with those obtained by the tradi- tional broth dilution test.

Effects of antibiotics on group A streptococci 39

MATERIAL AND METHODS Bacteria 50 isolates of group A streptococci from throat specimens were included.

Antibiotics Loracarbef and cefaclor were kindly provided by Eli Lilly, Sweden, cefadroxil by Bristol-Myers AB, Sweden and penicillin V by AB Kabi, Sweden. The drug solutions, weight compensated for potency, as well as the agar dilution plates were prepared on the day of use.

Plate dilution Log phase and stationary phase cultures in Todd-Hewitt broth were diluted to approximately 5 x lo7 colony-forming units (CFU)/ml. Two @ of the suspensions, approximately 10s CFU, was inoculated with a multipoint inoculator onto the surface of horse blood agar (as controls) and plates containing 2-fold dilutions of the antibiotics. The plates were incubated at 37°C for 20 h when the MICs were determined. MIC was defined as lowest concentration of the drug yielding no visible colony growth. In order to inactivate the antibiotics for determining MBC, beta-lactamases were deposited with a syringe under- neath the agar layer. The agar was released from the edge and the bottom of the dish by entering a long needle at the edge of the plate and the beta-lactamase was evenly spread under the agar. To plates with penicillin V, 0.5 ml(66OOOO units) of Bacto Penase (Difco) was used. For the inactivation of loracarbef, cefaclor and cefadroxil it was necessary to reduce the agar volume from 20 ml to 15 ml and to add 1.5 ml Bacillus cereus beta-lactamases (type I 170 and type I1 17 IU per plate). The plates were left at room temperature for 4 h to allow diffusion of the beta-lactamases through the agar gel. A Micrococcus luteus strain, MIC for all drugs <0.03 mgA, was inoculated onto each plate. The MBCs were read after incubation at 37°C for 24 and 48 h and defined as the lowest concentration of drug permitting no more than 50 visible colonies to grow, that is a killing rate of 99.95%. The M. luteus strain grew uninhibited on the highest concentration of each drug.

Broth dilution From log phase and stationary phase cultures in Todd-Hewitt broth, 2 ml of broths containing 2-fold dilutions of the antibiotics were inoculated to a final concentration of approximately 10s CFUlml. The tubes were incubated at 37°C. The MICs were read after 20 h of incubation and defined as the lowest concentration of the drug that inhibited visible growth. From tubes with no visible growth 10 111 aliquots were inoculated onto horse blood agar plates with 6OOOOO IU beta-lactamases per plate (Bacto Penase) for penicillin V and beta-lactamases from B. cereus 569-H9 (Genenzyme, Maidstone, UK) type I 0.67 IU and type I1 0.08 IU per plate for the 3 other drugs. After incubation at 37°C for 24 and 48 h the MBCs were determined. MBC was defined as the lowest concentration of the drug yielding no growth or growth of no more than 2-3 colonies, that is a killing rate of 99.9%. The precautions recommended for determining MBCMIC in broth dilution tests were followed (13).

RESULTS

Plate dilution The MICs of loracarbef and the two cephalosporins were virtually identical, 0.06-0.25 mg/l, but 16-32 times higher than the MICs of penicillin V, 0.OO4-O.008 mgA. Log phase and stationary phase cultures did not differ in this respect. The MBCs for log phase cultures did not exceed the MICs more than one dilution step for any of the tested agents. For stationary phase cultures the MBCs showed a larger range with MBUMIC ratios 2 32 for 344.4% of the strains for loracarbef and the cephalosporins and for 20% of the strains for penicillin V (Fig. 1, Table I). With few exceptions the same strains showed such elevated ratios. The MBCs recorded after 24 and 48 h of incubation were identical.

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

21/1

4Fo

r pe

rson

al u

se o

nly.

40 C. Karnrne and A.-C. Petersson Scand J Infect Dis 25

i 100- B

.c 0

E U

8 0 - L 9) n

60 -

40 -

2 0 -

0.004 0.016 0.06 0.25 t o 4.0 a 16.0 mg/l

Fig. 2. MICs (-) and MBCs (---) of 50 group A streptococci. Broth dilution method. A. Log phase cultures. B. Stationary phase cultures. Penicillin V (8), cefaclor (O), loracarbef (A), cefadroxil (0).

Broth dilution

For log phase and stationary phase cultures the MICs were the same for penicillin V, loracarbef and cefadroxil, respectively, while most of the strains yielded one dilution step higher MICs for cefaclor in stationary than in log phase. For cefaclor no less than 24% of the strains in log phase culture exhibited a MBUMIC ratio 2 32 (Fig. 2, Table I).

For stationary phase cultures a large number of strains yielded MBCs 2 16 mgA with MBClMIC ratios 2 32. For loracarbef and the cephalosporins 76 and 62-72%, respectively and for penicillin V 30% of the strains showed such elevated ratios (Fig. 2, Table I). With few exceptions this was recorded for the same strains. The MBCs recorded after 24 and 48 h of incubation were identical.

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

21/1

4Fo

r pe

rson

al u

se o

nly.

Scand J Infect Dis 25 Effects of antibiotics on group A streptococci 41

DISCUSSION

In the present investigation the plate dilution technique was used for determining MICs as well as MBCs of beta-lactum antibiotics by adding beta-lactamases to the plates after the MICs were read. The plate dilution method permits determination of both MICs and MBCs by one single inoculation step and the pitfalls of the broth dilution method can thereby be avoided.

Several investigations have shown that MICs in broth and plate dilution tests rarely are identical and for beta-lactam antibiotics slightly higher in the broth than in the plate dilution test (16). This was also the case in the present investigation.

With log phase cultures the broth dilution method showed good agreement with the plate dilution method both regarding the MICs and the MBCs with exception for the MBCs of cefaclor where 30% of the strains showed elevated MBCs with MBC/MIC ratios 2 32 in the broth dilution test. Thus, when determining MBC/MIC ratios of log phase cultures the results may vary between the broth and agar dilution method depending on the beta-lactam under test. Loracarbef on the other hand, chemically closely related to cefaclor yielded virtually identical results as cefadroxil.

Stationary phase cultures will yield a phenotypic response to beta-lactam antibiotics not affecting the inhibitory activity of the drug, the MICs, but diminishing the killing rate, resulting in elevated MBCMIC ratios. The phenotypic response of stationary phase cultures may not only be related to the physiologic status of the organisms, to the conditions provided in the culture media and to the beta-lactam under test. The present investigation indicated that this response was an intrinsic property of certain strains as, with few exceptions, the same strains showed elevated MBCMIC ratios for loracarbef and the two cephalosporins and approximately 50% of them also showed elevated ratios for penicillin V. The phenotypic response or “phenotypic tolerance” of stationary phase cultures shall be distinguished from the rare “genotypic or true tolerance” of actively growing cells (17).

Genotypic penicillin tolerance has been suggested to be related to therapeutic failure in penicillin V treated streptococcal tonsillitis (3, 18) but was not confirmed in a later in- vestigation (19).

REFERENCES 1. Schwartz RH, Wientzen RL Jr, Pedreira F, Feroli EJ, Mella GW, Guandolo VL. Penicillin V for

group A streptococcal pharyngotonsillitis. A randomized trial of seven vs ten days’ therapy. JAMA 246: 1790-1795, 1981.

2. Roos K, Holm SE, Ekedahl C. Treatment failure in acute streptococcal tonsillitis in children over the age of 10 and in adults. Scand J Infect Dis 17: 357-365, 1985.

3. Kim KS, Kaplan EL. Association of penicillin tolerance with failure to eradicate group A strepto- cocci from patients with pharyngitis. J Pediatr 107: 681484, 1985.

4. Brook I. Role of beta-lactamase producing bacteria in the failure of penicillin to eradicate group A streptococci. Pediatr Infect Dis 4: 491495, 1985.

5. Roos K, Grahn E, Holm SE. Evaluation of beta-lactamase activity and microbial interference in treatment failures of acute streptococcal tonsillitis. Scand J Infect Dis 18: 313-319, 1986.

6. Pichichero ME, Disney FA, Aronovitz GH, Talpey WE%, Green JL, Francis AB. Randomized, single-blind evaluation of cefadroxil and phenoxymethyl penicillin in the treatment of streptococcal pharyngitis. Antimicrob Agents Chemother 31: 903-906, 1987.

7. Milatovic D, Knauer J. Cefadroxil versus penicillin in the treatment of streptococcal tonsillopha- ryngitis. Eur J Clin Microbiol Infect Dis 8: 282-288, 1989.

8. Holm SE, Roos K, Str6mberg A. A randomized study of treatment of streptococcal pharyngoton- sillitis with cefadroxil or phenoxymethylpenicillin (penicillin V). Pediatr Infect Dis J 10: S68-s71, 1991.

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

21/1

4Fo

r pe

rson

al u

se o

nly.

42 C. Kamme and A.-C. Petersson Scand J Infect Dis 25

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Cao C, Chin NX, Neu HC. In-vitro activity and beta-lactamase stability of LY 163892. J Antimicrob Chemother 22: 155-165, 1988. Jorgensen JH, Redding JS, Maher LA. Influence of storage and susceptibility test conditions on stability and activity of LY 163892 and four other cephalosporins. Antimicrob Agents Chemother

Howard AJ, Dunkin KT. Comparative in-vitro activity of a new oral carbacephem, LY 163892. J Antimicrob Chemother 22: 445-456, 1988. Ishida K , Guze PA, Kalmanson GM,, Albrandt K , Guze LB. Variables in demonstrating methicillin tolerance in Staphylococcus aureus strains. Antimicrob Agents Chemother 21: 688-690, 1982. Taylor PC, Schoenknecht ED, Sherris JC, Linner EC. Determination of minimum bactericidal concentrations of oxacillin for Staphylococcus aureus influence and significance of technical factors. Antimicrob Agents Chemother 23: 142-150, 1983. Woolfrey BF, Lally RT, Ederer MN. Evaluation of oxacillin tolerance in Staphylococcus aureus a novel method. Antimicrob Agents Chemother 28: 381-388, 1985. Numan M, Banck G, Thore M. Penicillin tolerance in Arcanobacterium haemolyticum. J Infect Dis

Thrupp LD. Susceptibility testing of antibiotics in liquid media. In: Lorian V, ed. Antibiotics in laboratory medicine. Baltimore, 1980. Handwerger S, Tomasz A. Antibiotic tolerance among clinical isolates of bacteria. Rev Infect Dis 7: 368-386, 1985. Grahn E , Holm SE, Roos K. Penicillin tolerance in beta-streptococci isolated from patients with streptococcal tonsillitis. Scand J Infect Dis 19: 421426, 1987. Smith TD, Huskins WC, Kim SK, Kaplan EL. Efficacy of beta-lactamase-resistant penicillin and influence of penicillin tolerance in eradicating streptococci from the pharynx after failure of penicillin therapy for group A streptococcal pharyngitis. J Pediatr 110: 777-782, 1987.

32: 1477-1480, 1988.

161: 261-265, 1990.

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

New

cast

le U

pon

Tyn

e on

12/

21/1

4Fo

r pe

rson

al u

se o

nly.