194
Group B streptococcal infection
The Journal of Pediatrics August 1976
Antibiotic-killing kinetics of group B streptococci Group B streptococci are uniformly susceptible to penicillin or ampicillin. Nevertheless, morbidity and mortality in newborn infants infected with group B streptococci !s a major clinical problem. Bacteriologic determinants in the outcome of this infection were studied. Streptococcal kilOng kinetics were studied for ampicillin alone and in combination with gentamicin. In all cases killing was accelerated by the combination. The practice of discontinuing administration of the aminoglycoside antibiotic when group B streptococcal infection is confirmed should be re-evaluated.
V. Schauf, M.D.,* A. Deveikis, M.D., L. Rift, M.D., and A. Scrota, Chicago, Ill.
GROUP B STREPTOCOCCI are uniformly killed by overnight incubation with concentrations of penicillin or ampicillin that are easily achievable in blood and cerebrospinal fluid, 1-~ With widespread use of these apparently effective antibiotics in sick neonates, the reported case fatality rates of group B streptococcal infection, acquired from the maternal genital tract or from nosocomial sources, vary from 14 to 70%. 4. ~ Delays in diagnosis and treatment, low birth weight, 6 and immunologic factors 7 may be responsible for the poor outcome in some patients. Other factors, however, which may bear on the interaction between the treated host and microbe are largely unexplored. Therefore, detailed antibiotic susceptibility testing and kinetic studies were performed to deterfnine conditions required for eradication of group B streptococci in vitro. The combination of ampicillin and gentamicin was shown to accelerate killing of these organisms when compared to ampicillin alone. The present recommendation for the use of penicillin alone in treatment of group B streptococcal infection is discussed in relation to the data presented. From the Departments of Pediatrics and Medicine, Abraham Lincoln School of Medicine, University of Illinois, College o f Medicine. Support was provided also by the Schering Corporation. Dr. Deveikis is supported by a National institutes of Health training grant No. NO AI 00208, Department of Health, Education and Welfare, National Institute of Allergy and Infectious Diseases. *Reprint address': 840 South Wood St., Chicago, Ill. 60612.
MATERIALS AND METHODS Streptococci. Nine strains of group B streptococci isolated from the blood or CSF of infants were identified definitely as group B by capillary precipitation reactivity with Lancefield group B grouping serum. Reference strains of group B streptococci types Ia, Ib, Ic, II, and IlI were supplied by Dr. Facklam of the Center for Disease Control, Atlanta, Georgia. Two isolates of group A beta hemolytic streptococci, identified by Lancefield grouping, were used for comparison to group B streptococci in timed antibiotic-killing studies. Abbreviations used CSF: cerebrospinal fluid TP: tryptose phosphate CFU: colony-forming units MIC: minimal inhibitory concentration MBC: minimal bactericidal concentration Fresh log phase cultures from tryptose phosphate broth were diluted in fresh TP broth to give an inoculum size of approximately 10~ colony-forming units per milliliter for all studies. Previous studies demonstrated that group B streptococcal growth rates were similar in TP broth and in Todd-Hewitt broth supplemented with 5% of red blood cells from dogs. Antibiotic laboratory standards of penicillin G, ampicillin, methicillin, kanamycin (Bristol), clindamycin (Upjohn), and gentamicin (Schering) (provided by the manufacturers) were reconstituted to 2 mg/ml, dispensed in aliquots, and stored at - 2 0 ~ Tube dilution sensitivity. Determinations of minimal
Volume 89 Number 2
Group B streptococcal infection
19 5
Table I. Antibiotic susceptibility of group B streptococci
Minimal inhibitory concentrations (tLg/ml) I Cflnicalisolates
Reference strains Antibiotic
la I Ib
Ic i
H
Minimal bactericidal concentration (l~g/ml)
111
Range
Reference strains
Median
!a [ lb
Cfinical isolates
lc I 11
1II
Range I Median
i
Penicillin G Ampicillin Methicillin Clindamycin Kanamycin Gentamicin ND
= not
0.03 0.15 0.5 0.015 64 8
0 . 0 3 250 125->500 125 125 250 125 64-250 125 16 32 125 125 125 32-125 16 32 16 16 4-16 16
0.25 0.5 4.0 0.25 500 64
done.
inhibitory concentration and minimal bactericidal concentration of penicillin G, ampicillin, methicillin, clindamycin, kanamycin, and gentamicin were performed for all strains using standard twofold tube dilution. The lowest concentration of antibiotic preventing the development of turbidity is designated the MIC. The lowest concentration of antibiotic resulting in less than 10 C F U / ml is designated the MBC. Studies for synergism. Antibiotic susceptibility titrations were performed for group B streptococci using twofold dilutions of ampicillin and gentamicin. Some strains were also tested with penicillin-gentamicin combinations. A n additive effect of two antibiotics was considered to be present when inhibition or killing was produced by a concentration of each drug in the combination that was twofold less than either antibiotic alone. A synergistic effect produced inhibition or killing when each drug in the combination was fourfold less than either antibiotic alone. Kinetic studies. Timed killing assays were performed with two isolates o f group A streptococci and with all strains of group B streptococc i. Bactericidal rates of group A and group B strains were compared using fixed amounts of ampicillin (1 and 10 /~g/ml) and using 50 times the previously determined MBC for each strain. Bactericidal rates of group B strains were compared using ampicillin, 1 and l0/zg/ml alone, a n d i n combination with gentamicin, 2, 5 and 10 t~g/ml, respecti,vely. After i n c u b a tion, aliquots were withdrawn at time zero and at one- to four-hour intervals for 24 hours, and quantitatively plated. RESULTS Tube dilution sensitivity. The range and median of MICs and MBCs of nine isolates and the M1Cs and MBCs for the five reference strains for the antibiotics studied are shown in Table I. Penicillin G, ampicillin, and clin-
9-
~
:
:
8' ~.....O 7 .
g 8 6
.
.
.
.
.
.
.
.
.
..O .
.
9 ............
.
.
9
.~ ........... 9 .....
O 5 O_
_~4
~-------O.
\
. 4
8
12 HOURS
16
\
"-,: 20
24
Fig. 1. Growth of group A ('"') and group B ( ) streptococci without antibiotic. Killing of group A streptococcus by 1 /xg/ml ampicillin (--). Killing of group B streptococcus strains Ja and Tr with ampicillin: Ja 1 ~g/ml (.... ), Ja 10/xg/ml ~ - - - - ) , Tr 1/,g/ ml (. . . . . . ). damycin are effective bactericidal agents in vitro at very low concentrations which can easily be achieved in patients' serum. Bactericidal concentrations of methicillin varied considerably among the strains. All were resistant to kanamycin and gentamicin. Studies for synergism. Antibiotic susceptibility titrations showed synergism at achievable concentrations between ampicillin and gentamicin for two of four strains. Ampicillin and gentamicin in combination were no more effective than ampicillin alone for one strain and only additive in effect for one additional strain. Penicillin and gentamicin were additive in effect for two strains. Kinetic studies. Timed killing assays with ampicillin were performed for three clinical isolates and reference strain III of group B streptococci and for two isolates of group A streptococci. Represe~tative data are shown in Fig 1. In the absence of antibiotic, the growth rate of
196
Group B streptococcal infection
The Journal of Pediatrics August 1976
i1
j
8 84
r.
.
.
.
.
.
.
.
6
J 0 0
iii .... ,w""
L
5~
t9
0,4
2
4
"2 S
",% g4
a
HOURS
Fig. 2A. Growth of group B streptococci without antibiotics (--) and with 10/Lg/ml gentamicin (,...). Reference strain III with 10/~g/ml ampicillin (. . . . ) and with 10/~g/ml ampicillin and 2/~g/ml gentamicin (---).
..e
~176 ~176 ~176176 ~176
I-
g 0 r / 0 0 0 (.9
0 .J
~ ~176176 6
.,t,
**"~
51
4'
N `%O
3
N9
,Ik`%
9 ~"0
9 .....
9
"~`%
i
~
'
xxxx 6D ~ HouRs
"`%
;
24
Fig. 2B. Growth of group B streptococci without antibiotics (--) and with 10/~g/ml gentamicin (....). Strain Da with 1 ~g/ml ampicillin ( . . . . ) and with 1 ~g/ml ampicillin and 10 t~g/ml gentamicin ( ..... ).
group B streptococci was greater than that of group A. In the presence of t /~g/ml of ampicillin, sterilization of group A strains was complete at four hours. Sterilization of group B strains, however, did not occur until 20 to 24 hours, even in the presence of 1 or 10 ~g/m! of ampicillin. In additional studies using 50 times the MBC of ampicillin for each strain, killing of group A strains occurred within four hours. In contrast, virtually no killing of group B strains was observed during this time. Killing of grouP B streptococci by ampicillin alone was
compared to the killing of the same strains using the same amount of ampicilli n in combination with gentamicin. Representative data for two studies are shown in Fig 2. Growth of bacteria occurred in the absence of antibiotics and in the presence of 10 ~g/ml gentamicin. Killing was complete with 1 and 10/~g/ml ampicillin in 10 to 24 hours. Killing occurred at a much more rapid rate when 2 or 10 /~g/ml gentamicin was added to the ampicillin. Table II shows the log10 reduction in colony count after four hours incubation and the time required to achieve
Volume 89 Number 2 99.99% killing with 1 to 2 t~g/ml ampicillin compared to 1 to 2 ~g/ml ampicillin plus 10 t~g/ml gentamicin for each strain. In all strains, a 1 to 4 lOglo greater reduction in colony count at four hours was produced by the combination than by ampicillin alone. In each of the strains tested, killing was achieved more rapidly with the antibiotic combination than with ampicillin alone. Five strains of group B streptococci were tested with 10 /~g/ml of ampicillin alone and in combination with 5/~g/ ml of gentamicin, in each case killing was accelerated by the combination. The effect, however, was not as marked as when the combination contained 10 /~g/ml gentamicin. DISCUSSION The observed susceptibility of group B streptococci to penicillin and to ampicillin and their resistance to aminoglycosides confirm previous observations. 1-:'The killing of group B streptococci by ampicillin is much slower than killing of group A streptococci, even at concentrations of ampicillin many times in excess of the MBC. Addition to ampicillin after growth was established did not result in more rapid killing of group B streptococci (unpublished data). Since group B streptococci growth rates were faster than those of group A, the slow bactericidal activity does not relate to inadequate nutrition of group B streptococci, but rather seems to be intrinsic to the o[ganism. This property of group B streptococci may partly explain the pathogenicity of these susceptible organisms in nurseries where ampicillin and penicillin are used frequently. The difficulty observed in eradication of pulmonary infection in newborn infants 4 and of the carrier state in pregnant women 8 with penicillin or ampicillin may also result from the relatively slow action of ampicillin on these organisms. Bacterial killing was complete with low concentrations of ampicillin after 18 to 24 hours incubation, and it is not contradictory that synergy between penicillin and aminoglycosides was not always demonstrated. Tests for synergistic activity are determined after 18 hours incubation and refer to the amount of antibiotics required for bactericidal activity, and not to the rate.of kiiling. When killing kinetics were examined, it was possible to show in every case more rapid killing of group B streptococci with ampicillin and gentamicin in combination compared to ampicillin alone even when ampicillin concentration was as great as 50 times the MBC. Acceleration of bacterial killing by the addition of gentamicin was observed with as little as 2 /~g/ml and occurred reproducibly at 5 /~g/ml. The effect was most pronounced at 10 ~g/mi of gentamicin.
Group B streptococcal infection
19 7
Table II. Timed killing of streptococcal strains by ampicillin or ampicillin and gentamicin
Loglo reduction in 4 hr
Time to 99.99% killing (hr)
' Ampicillin*I Ampicillin*Strain I Ampicillin* I~gentamicint Ampicillin* gentamicin~ Ia Ib Ic II !II Pe Ja Jo Do Da Ga Tr Ru Jc
1 2 1 3 2 1 1 1 1 1 2 1 1 1
2 3 4 4 5 2 5 3 4 3 4 3 3 3
16 16 8 8 16 20 18 18 23 18 8 8 18 18
10 7 3 4 3 18 3 7 2 5 4 5 7 5
*1-2/~g/ml ampicillin. tl0/~g/ml gentamicin. Antibiotic concentrations were chosen in the range of clinically achievable levels. The peak serum concentration of gentamicin recommended for patients is 10/xg/ml. The levels of gentamicin tested are not achievable in CSF. Ampicillin at 1 /~g/ml represents a minimum level that can be maintained even in CSF, and at 10 ~g/ml represents a level commonly achieved clinicallyY In all cases, enhance d killing by 5 or 10/~g/ml of gentamicin occurred. Further experimentation in vivo is required to determine the relevance of these observations. The slow bactericidal response to ampicillin, with enhancement of killing by the addition of an aminoglycoside, seen with group B streptococci is similar to observations with enterococci and Listeria. 1~ The acceleration of bacterial killing is independent of the aminoglycoside MBC. 1~ The mechanism of enhanced killing of group B streptococci by penicillin-aminoglycoside combinations is unknown; however, we postulate that gentamicin may enter and may act on cells minimally damaged by penicillin. Further in vitro studies, development of animal models, and, possibly, studies of eradication of the group B streptococcal carrier state in healthy adult volunteers would help determine if penicillin-aminoglycoside combinations offer a clinical advantage. In view of the accelerated killing observed With ampicillin and gentamicin, the current recommendation for the ~se of a penicillin alone in treatment of group B streptoccccal infection should be re-evaluated.
19 8
Group B streptococcal infection
We thank Mr. A. Simonaitis for technical assistance. Valuable discussion and supplies were provided by Dr. LeBeau, Dr. G. Jackson, V. Zimelis, and D. Matulionis.
The Journal of Pediatrics August 1976
6.
7. REFERENCES
1. Anthony BF, and Concepcion NF: Group B Streptococcus in a general hospital, J Infect Dis 132:561, 1975. 2. Bergqvist G, Hurvell B, Malmborg A, Rylander M, and Turnell R: Neonatal infections caused by group B streptococci, Scand J Infect Dis 3:157, 1971. 3. Matsen JM, and Coghlan CR: Antibiotic testing and susceptibility patterns of streptococci, in Wannamaker LW, and Matsen JM, editors: Streptococci and streptococca! diseases; recognition, understanding, and management, New York, 1972, Academic Press, Inc, pp 189-204. 4. Franciosi RA, Knostman JD, and Zimmerman RA: Group B streptococcal neonatal and infant infections, J PEDIATR 82:707, 1973. 5. Baker CJ, Barrett FF, Gorden RC, and Yow MD: Suppurative meningitis due to streptococci of Lancefield group B: A study of 33 infants, J PEDIATR 82:724, 1973.
8.
9.
10.
11. 12.
Quirante J, Ceballos R, and Cassady G: Group B /?-hemolytic streptococcal infection in the newborn, Am J Dis Child 128:659, 1974. Klesius PH, Zimmerman RA, Matthews JH, and Krushak, DH: Cellular and humoral immune response to group B streptococci, J PEDrATR 83:926, 1973. Hall R, Barnes W, Sumathy V, Harris D, Rhodes P, and Fayez J: Antibiotic treatment of parturient women colonized with group B streptococci, J PEDIATR 86:975, 1975 (abstr). Kaplan JM, McCracken GH, Horton L J, Thomas ML, and Davis N: Pharmacologic studies in neonates given large doses of ampicillin, J PEDIATR 84:571, 1974. Watanakunakorn C: Penicillin combined with gentamicin or streptomycin: Synergism against enterococci, J Infect Dis 124:581, 1971. Hunter, TH: Treatment of some bacterial infections of the heart and pericardium, Bull NY Acad Med 28:213, 1952. Gorden RC, Barrett FF, and Clark DJ: Influence of several antibiotics, singly and in combination, on the growth of Listeria monocytogenes, J PEDIATR 80:667, 1972.
Group B streptococcal infection
The Journal of Pediatrics August 1976
Antibiotic-killing kinetics of group B streptococci Group B streptococci are uniformly susceptible to penicillin or ampicillin. Nevertheless, morbidity and mortality in newborn infants infected with group B streptococci !s a major clinical problem. Bacteriologic determinants in the outcome of this infection were studied. Streptococcal kilOng kinetics were studied for ampicillin alone and in combination with gentamicin. In all cases killing was accelerated by the combination. The practice of discontinuing administration of the aminoglycoside antibiotic when group B streptococcal infection is confirmed should be re-evaluated.
V. Schauf, M.D.,* A. Deveikis, M.D., L. Rift, M.D., and A. Scrota, Chicago, Ill.
GROUP B STREPTOCOCCI are uniformly killed by overnight incubation with concentrations of penicillin or ampicillin that are easily achievable in blood and cerebrospinal fluid, 1-~ With widespread use of these apparently effective antibiotics in sick neonates, the reported case fatality rates of group B streptococcal infection, acquired from the maternal genital tract or from nosocomial sources, vary from 14 to 70%. 4. ~ Delays in diagnosis and treatment, low birth weight, 6 and immunologic factors 7 may be responsible for the poor outcome in some patients. Other factors, however, which may bear on the interaction between the treated host and microbe are largely unexplored. Therefore, detailed antibiotic susceptibility testing and kinetic studies were performed to deterfnine conditions required for eradication of group B streptococci in vitro. The combination of ampicillin and gentamicin was shown to accelerate killing of these organisms when compared to ampicillin alone. The present recommendation for the use of penicillin alone in treatment of group B streptococcal infection is discussed in relation to the data presented. From the Departments of Pediatrics and Medicine, Abraham Lincoln School of Medicine, University of Illinois, College o f Medicine. Support was provided also by the Schering Corporation. Dr. Deveikis is supported by a National institutes of Health training grant No. NO AI 00208, Department of Health, Education and Welfare, National Institute of Allergy and Infectious Diseases. *Reprint address': 840 South Wood St., Chicago, Ill. 60612.
MATERIALS AND METHODS Streptococci. Nine strains of group B streptococci isolated from the blood or CSF of infants were identified definitely as group B by capillary precipitation reactivity with Lancefield group B grouping serum. Reference strains of group B streptococci types Ia, Ib, Ic, II, and IlI were supplied by Dr. Facklam of the Center for Disease Control, Atlanta, Georgia. Two isolates of group A beta hemolytic streptococci, identified by Lancefield grouping, were used for comparison to group B streptococci in timed antibiotic-killing studies. Abbreviations used CSF: cerebrospinal fluid TP: tryptose phosphate CFU: colony-forming units MIC: minimal inhibitory concentration MBC: minimal bactericidal concentration Fresh log phase cultures from tryptose phosphate broth were diluted in fresh TP broth to give an inoculum size of approximately 10~ colony-forming units per milliliter for all studies. Previous studies demonstrated that group B streptococcal growth rates were similar in TP broth and in Todd-Hewitt broth supplemented with 5% of red blood cells from dogs. Antibiotic laboratory standards of penicillin G, ampicillin, methicillin, kanamycin (Bristol), clindamycin (Upjohn), and gentamicin (Schering) (provided by the manufacturers) were reconstituted to 2 mg/ml, dispensed in aliquots, and stored at - 2 0 ~ Tube dilution sensitivity. Determinations of minimal
Volume 89 Number 2
Group B streptococcal infection
19 5
Table I. Antibiotic susceptibility of group B streptococci
Minimal inhibitory concentrations (tLg/ml) I Cflnicalisolates
Reference strains Antibiotic
la I Ib
Ic i
H
Minimal bactericidal concentration (l~g/ml)
111
Range
Reference strains
Median
!a [ lb
Cfinical isolates
lc I 11
1II
Range I Median
i
Penicillin G Ampicillin Methicillin Clindamycin Kanamycin Gentamicin ND
= not
0.03 0.15 0.5 0.015 64 8
0 . 0 3 250 125->500 125 125 250 125 64-250 125 16 32 125 125 125 32-125 16 32 16 16 4-16 16
0.25 0.5 4.0 0.25 500 64
done.
inhibitory concentration and minimal bactericidal concentration of penicillin G, ampicillin, methicillin, clindamycin, kanamycin, and gentamicin were performed for all strains using standard twofold tube dilution. The lowest concentration of antibiotic preventing the development of turbidity is designated the MIC. The lowest concentration of antibiotic resulting in less than 10 C F U / ml is designated the MBC. Studies for synergism. Antibiotic susceptibility titrations were performed for group B streptococci using twofold dilutions of ampicillin and gentamicin. Some strains were also tested with penicillin-gentamicin combinations. A n additive effect of two antibiotics was considered to be present when inhibition or killing was produced by a concentration of each drug in the combination that was twofold less than either antibiotic alone. A synergistic effect produced inhibition or killing when each drug in the combination was fourfold less than either antibiotic alone. Kinetic studies. Timed killing assays were performed with two isolates o f group A streptococci and with all strains of group B streptococc i. Bactericidal rates of group A and group B strains were compared using fixed amounts of ampicillin (1 and 10 /~g/ml) and using 50 times the previously determined MBC for each strain. Bactericidal rates of group B strains were compared using ampicillin, 1 and l0/zg/ml alone, a n d i n combination with gentamicin, 2, 5 and 10 t~g/ml, respecti,vely. After i n c u b a tion, aliquots were withdrawn at time zero and at one- to four-hour intervals for 24 hours, and quantitatively plated. RESULTS Tube dilution sensitivity. The range and median of MICs and MBCs of nine isolates and the M1Cs and MBCs for the five reference strains for the antibiotics studied are shown in Table I. Penicillin G, ampicillin, and clin-
9-
~
:
:
8' ~.....O 7 .
g 8 6
.
.
.
.
.
.
.
.
.
..O .
.
9 ............
.
.
9
.~ ........... 9 .....
O 5 O_
_~4
~-------O.
\
. 4
8
12 HOURS
16
\
"-,: 20
24
Fig. 1. Growth of group A ('"') and group B ( ) streptococci without antibiotic. Killing of group A streptococcus by 1 /xg/ml ampicillin (--). Killing of group B streptococcus strains Ja and Tr with ampicillin: Ja 1 ~g/ml (.... ), Ja 10/xg/ml ~ - - - - ) , Tr 1/,g/ ml (. . . . . . ). damycin are effective bactericidal agents in vitro at very low concentrations which can easily be achieved in patients' serum. Bactericidal concentrations of methicillin varied considerably among the strains. All were resistant to kanamycin and gentamicin. Studies for synergism. Antibiotic susceptibility titrations showed synergism at achievable concentrations between ampicillin and gentamicin for two of four strains. Ampicillin and gentamicin in combination were no more effective than ampicillin alone for one strain and only additive in effect for one additional strain. Penicillin and gentamicin were additive in effect for two strains. Kinetic studies. Timed killing assays with ampicillin were performed for three clinical isolates and reference strain III of group B streptococci and for two isolates of group A streptococci. Represe~tative data are shown in Fig 1. In the absence of antibiotic, the growth rate of
196
Group B streptococcal infection
The Journal of Pediatrics August 1976
i1
j
8 84
r.
.
.
.
.
.
.
.
6
J 0 0
iii .... ,w""
L
5~
t9
0,4
2
4
"2 S
",% g4
a
HOURS
Fig. 2A. Growth of group B streptococci without antibiotics (--) and with 10/Lg/ml gentamicin (,...). Reference strain III with 10/~g/ml ampicillin (. . . . ) and with 10/~g/ml ampicillin and 2/~g/ml gentamicin (---).
..e
~176 ~176 ~176176 ~176
I-
g 0 r / 0 0 0 (.9
0 .J
~ ~176176 6
.,t,
**"~
51
4'
N `%O
3
N9
,Ik`%
9 ~"0
9 .....
9
"~`%
i
~
'
xxxx 6D ~ HouRs
"`%
;
24
Fig. 2B. Growth of group B streptococci without antibiotics (--) and with 10/~g/ml gentamicin (....). Strain Da with 1 ~g/ml ampicillin ( . . . . ) and with 1 ~g/ml ampicillin and 10 t~g/ml gentamicin ( ..... ).
group B streptococci was greater than that of group A. In the presence of t /~g/ml of ampicillin, sterilization of group A strains was complete at four hours. Sterilization of group B strains, however, did not occur until 20 to 24 hours, even in the presence of 1 or 10 ~g/m! of ampicillin. In additional studies using 50 times the MBC of ampicillin for each strain, killing of group A strains occurred within four hours. In contrast, virtually no killing of group B strains was observed during this time. Killing of grouP B streptococci by ampicillin alone was
compared to the killing of the same strains using the same amount of ampicilli n in combination with gentamicin. Representative data for two studies are shown in Fig 2. Growth of bacteria occurred in the absence of antibiotics and in the presence of 10 ~g/ml gentamicin. Killing was complete with 1 and 10/~g/ml ampicillin in 10 to 24 hours. Killing occurred at a much more rapid rate when 2 or 10 /~g/ml gentamicin was added to the ampicillin. Table II shows the log10 reduction in colony count after four hours incubation and the time required to achieve
Volume 89 Number 2 99.99% killing with 1 to 2 t~g/ml ampicillin compared to 1 to 2 ~g/ml ampicillin plus 10 t~g/ml gentamicin for each strain. In all strains, a 1 to 4 lOglo greater reduction in colony count at four hours was produced by the combination than by ampicillin alone. In each of the strains tested, killing was achieved more rapidly with the antibiotic combination than with ampicillin alone. Five strains of group B streptococci were tested with 10 /~g/ml of ampicillin alone and in combination with 5/~g/ ml of gentamicin, in each case killing was accelerated by the combination. The effect, however, was not as marked as when the combination contained 10 /~g/ml gentamicin. DISCUSSION The observed susceptibility of group B streptococci to penicillin and to ampicillin and their resistance to aminoglycosides confirm previous observations. 1-:'The killing of group B streptococci by ampicillin is much slower than killing of group A streptococci, even at concentrations of ampicillin many times in excess of the MBC. Addition to ampicillin after growth was established did not result in more rapid killing of group B streptococci (unpublished data). Since group B streptococci growth rates were faster than those of group A, the slow bactericidal activity does not relate to inadequate nutrition of group B streptococci, but rather seems to be intrinsic to the o[ganism. This property of group B streptococci may partly explain the pathogenicity of these susceptible organisms in nurseries where ampicillin and penicillin are used frequently. The difficulty observed in eradication of pulmonary infection in newborn infants 4 and of the carrier state in pregnant women 8 with penicillin or ampicillin may also result from the relatively slow action of ampicillin on these organisms. Bacterial killing was complete with low concentrations of ampicillin after 18 to 24 hours incubation, and it is not contradictory that synergy between penicillin and aminoglycosides was not always demonstrated. Tests for synergistic activity are determined after 18 hours incubation and refer to the amount of antibiotics required for bactericidal activity, and not to the rate.of kiiling. When killing kinetics were examined, it was possible to show in every case more rapid killing of group B streptococci with ampicillin and gentamicin in combination compared to ampicillin alone even when ampicillin concentration was as great as 50 times the MBC. Acceleration of bacterial killing by the addition of gentamicin was observed with as little as 2 /~g/ml and occurred reproducibly at 5 /~g/ml. The effect was most pronounced at 10 ~g/mi of gentamicin.
Group B streptococcal infection
19 7
Table II. Timed killing of streptococcal strains by ampicillin or ampicillin and gentamicin
Loglo reduction in 4 hr
Time to 99.99% killing (hr)
' Ampicillin*I Ampicillin*Strain I Ampicillin* I~gentamicint Ampicillin* gentamicin~ Ia Ib Ic II !II Pe Ja Jo Do Da Ga Tr Ru Jc
1 2 1 3 2 1 1 1 1 1 2 1 1 1
2 3 4 4 5 2 5 3 4 3 4 3 3 3
16 16 8 8 16 20 18 18 23 18 8 8 18 18
10 7 3 4 3 18 3 7 2 5 4 5 7 5
*1-2/~g/ml ampicillin. tl0/~g/ml gentamicin. Antibiotic concentrations were chosen in the range of clinically achievable levels. The peak serum concentration of gentamicin recommended for patients is 10/xg/ml. The levels of gentamicin tested are not achievable in CSF. Ampicillin at 1 /~g/ml represents a minimum level that can be maintained even in CSF, and at 10 ~g/ml represents a level commonly achieved clinicallyY In all cases, enhance d killing by 5 or 10/~g/ml of gentamicin occurred. Further experimentation in vivo is required to determine the relevance of these observations. The slow bactericidal response to ampicillin, with enhancement of killing by the addition of an aminoglycoside, seen with group B streptococci is similar to observations with enterococci and Listeria. 1~ The acceleration of bacterial killing is independent of the aminoglycoside MBC. 1~ The mechanism of enhanced killing of group B streptococci by penicillin-aminoglycoside combinations is unknown; however, we postulate that gentamicin may enter and may act on cells minimally damaged by penicillin. Further in vitro studies, development of animal models, and, possibly, studies of eradication of the group B streptococcal carrier state in healthy adult volunteers would help determine if penicillin-aminoglycoside combinations offer a clinical advantage. In view of the accelerated killing observed With ampicillin and gentamicin, the current recommendation for the ~se of a penicillin alone in treatment of group B streptoccccal infection should be re-evaluated.
19 8
Group B streptococcal infection
We thank Mr. A. Simonaitis for technical assistance. Valuable discussion and supplies were provided by Dr. LeBeau, Dr. G. Jackson, V. Zimelis, and D. Matulionis.
The Journal of Pediatrics August 1976
6.
7. REFERENCES
1. Anthony BF, and Concepcion NF: Group B Streptococcus in a general hospital, J Infect Dis 132:561, 1975. 2. Bergqvist G, Hurvell B, Malmborg A, Rylander M, and Turnell R: Neonatal infections caused by group B streptococci, Scand J Infect Dis 3:157, 1971. 3. Matsen JM, and Coghlan CR: Antibiotic testing and susceptibility patterns of streptococci, in Wannamaker LW, and Matsen JM, editors: Streptococci and streptococca! diseases; recognition, understanding, and management, New York, 1972, Academic Press, Inc, pp 189-204. 4. Franciosi RA, Knostman JD, and Zimmerman RA: Group B streptococcal neonatal and infant infections, J PEDIATR 82:707, 1973. 5. Baker CJ, Barrett FF, Gorden RC, and Yow MD: Suppurative meningitis due to streptococci of Lancefield group B: A study of 33 infants, J PEDIATR 82:724, 1973.
8.
9.
10.
11. 12.
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