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Effectiveness of combination therapy with ampicillin
and gentamicin for a 2-month-old boy with group B
streptococcal meningitis
Marie Mitani1,2, *, Miyuki Morozumi3, Masahiro Bamba1,
Makoto Anzo1, Kimiko Ubukata3 and Satoshi Iwata3
1

Department of Pediatrics, Kawasaki Municipal Hospital, Kanagawa, Japan2
Department of Pediatrics, Saitama Municipal Hospital, Saitama, Japan3
Department of Infectious Diseases, Keio University School of Medicine,Tokyo, Japan(Received for publication October 22, 2018)

Group B Streptococcus (GBS, or Streptococcus agalactiae) is among the mostcommon causes of severe bacterial infections in early infancy. Combination therapy
with ampicillin (AMP) plus gentamicin (GM) is recommended for such GBS
infections, although this therapeutic approach has been based on relatively old invitro and in vivo studies. Recently, a synergistic effect from this combination was
found to involve ribosomal binding by GM allowed to enter bacterial cells by cellwall damaged from AMP. Here we report a 2-month-old boy with meningitis causedby GBS, against which AMP plus GM proved clinically effective. GBS isolated fromhis cerebrospinal ﬂuid and blood belonged to capsular type III and sequence type 17,
and possessed hypervirulent GBS adhesin (HvgA); GBS isolated from maternalvaginal samples was the same type. In vitro determinations of time-kill kinetics forthe patientʼs isolate showed enhancement of bactericidal activity by combining AMP
with a small amount of GM. This case report provides clinical and laboratory
evidence supporting effectiveness of combination therapy of AMP plus GM forsevere GBS infection in early infancy.

Introduction
Group B Streptococcus (GBS, or Streptococcus agalactiae) is among the most common causesof severe bacterial infections in early infancy1). While early-onset GBS infection has been decreased*Corresponding author: Marie Mitani, M.D. Tel: ＋81–48–873–4111, E-mail: marymitani@yahoo.co.jp

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Mar. 2019by maternal perinatal prophylaxis, late-onset GBS infection continues to occur unabatedly2).
Conventionally, a penicillin (PEN) has been the ﬁrst-line agent for treatment of invasive GBS
infections. In the 1980s, combined treatment of PEN-susceptible GBS in culture with PEN and an
aminoglycoside, typically gentamicin (GM), was found to enhance bactericidal activity3,4). Addition of GM to ampicillin (AMP) to treat invasive GBS infections is prompted by results of studies
showing accelerated killing of GBS when GM is added to AMP, which is termed a synergistic effect. This synergistic effect has been proven by experiments in vitro and in animals5〜7). Deveikiset al.7) reported that administration of AMP plus GM to GBS-infected mice improved the survival
rate and accelerated clearing of bacteremia beyond treatment with AMP alone. Presently, combination therapy with AMP plus GM is recommended for treatment of invasive GBS infections in early
infancy, but the mechanism of the synergistic effect long has been incompletely understood. Recently, Ebara et al.8) elucidated the mechanism by measuring uptake of GM into cells with liquid
chromatography-tandem mass spectrometry (LC/MS/MS) and by morphologic observations withtransmission electron microscopy (TEM). The synergistic effect against GBS was characterized asinvolving 3 sequential steps. First, bacterial cell walls are damaged by AMP. Next, penetration of
GM into the cells is enhanced by this cell walls damage. Finally, the intracellular GM binds to ribosomes and interferes with protein synthesis, killing the cells.
Here, we report an infant with late-onset GBS meningitis against which the combination of
AMP with GM was highly effective.

Case Report
Clinical course
A Japanese boy was born to a gravida 1, para 0 mother. The mother had been treated for premature labor since the 28th week of gestation. Vaginal GBS colonization was identiﬁed at a gestational age of 32 weeks when premature rupture of membranes occurred, and intravenous cefazolin was initiated. Two days later, the patient was born at a gestational age of 32 weeks and 6days with a birth weight of 2082 g (0.76 standard deviation). GBS colonization was not detectedby culture of skin swab or gastric juice specimens. He was discharged from the hospital at a modiﬁed gestational age of 37 weeks; 32-day-old. He was fed with breast milk.
When he was 2 months old, he presented to our hospital with a 4-day history of fever. He
also had conjugate deviation of the eyes to the right. His activity and appetite were worsening.
On physical examination, his temperature was 38.7°C; heart rate 180 beats/min; and respiratory
rate 40 /min. He appeared pale and hardly moved or cried except upon painful stimulation. Theanterior fontanelle was ﬂat. No signs of meningeal irritation such as Brudzinskiʼs sign were apparent. Conjugate deviation of the eyes to the right was occasionally seen. Laboratory results onadmission were as follows: white blood cell count, 5.16×109/L, including 25% bands and 20%

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72―183（ 83 ）segmented cells; hemoglobin, 9.2 g/dL; platelet count, 596×109/L; C-reactive protein (CRP),
182.3 mg/L (reference, 0–3); and serum glucose, 91 mg/dL. Serum immunoglobulin and complement concentrations were normal. Cerebrospinal ﬂuid (CSF) was yellow and showed pleocytosis
with a white cell count of 15520/μL (lymphocytes: neutrophils, 3520: 12000); a protein concentration of 484 mg/dL; and a glucose concentration of 6 mg/dL. Microscopic examination of CSF
after Gram staining showed Gram-positive diplococci with phagocytosis. The rapid latex agglutination test was positive for GBS. Cranial computed tomography showed a left subdural effusion.
The patient was diagnosed with bacterial meningitis. Intravenous AMP (400 mg/kg/day) andcefotaxime (CTX; 200 mg/kg/day) were started immediately. On the third hospital day, GBS wasisolated from CSF and blood cultures of the day of admission. Since the isolates were susceptible
to β-lactam antibiotics (minimum inhibitory concentration or MIC; 0.125 μg/mL for AMP,
0.031 μg/mL for PEN, and 0.063 μg/mL for CTX, as opposed to 64 μg/mL for GM), CTX was discontinued and AMP was continued alone. The patientʼs general condition improved by the secondday, when repeat CSF examination also showed improvement with a white cell count of 2206/μL(lymphocytes: neutrophils, 203: 2003); protein, 268 mg/dL; and glucose, 34 mg/dL. GBS was nolonger detectable in the CSF by Gram staining. CRP had decreased. However, the patientʼs tem-Figure 1. Clinical course of the patient, whose temperature normalized soon after administration ofgentamicin. Abbreviations: BT, body temperature; CRP, C-reactive protein; WBC, white blood cells;
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Table 1. Bacteriologic characteristics of GBS isolates

perature remained elevated at 38.0°C (Figure 1). We added intravenous GM (5 mg/kg/day) on theeighth day. On the ninth day, his temperature declined. We gave AMP for a total of 21 days andGM for 7 days. The left sided subdural effusion observed on day 8 had decreased on day 25. We
discharged the patient on day 26. Conjugate deviation of the eyes to the right had decreased byday 10 and had disappeared completely at 3 months after onset. Hearing and psychomotor development remain intact to at least 3 years old.
Bacteriologic examination
As shown in Table 1, capsular types of all isolates from the patient and his mother wereidentiﬁed as serotype III using a real-time PCR method devised by Morozumi et al.9). Multilocus
sequence typing (MLST) was performed additionally with reference to the MLST website(https://http//pubmlst.org/sagalactiae/), assigning the isolates to sequence type (ST) 17. The hypervirulent gene (hvgA) encoding the surface protein HvgA was detected by PCR ampliﬁcation asdescribed previously10).
We measured time-kill curves for AMP alone and for the combination of AMP with GM in
the causative isolate using methods described previously8). As shown in Figure 2, viable cellcounts of GBS were decreased more rapidly by the combination than by AMP alone. With thecombination, bactericidal activity was enhanced, showing a greater decrease in viable cells on theorder of 103/mL at 2 h and 104/mL at 4 h.

Discussion
We presented an infant with late-onset GBS meningitis in whom a combination of AMP andGM was highly effective. The patientʼs temperature normalized soon after addition of GM. In
vitro measurement of time-kill kinetics for the isolate demonstrated enhancement of bactericidal

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72―185（ 85 ）Figure 2. Time-kill curves of the isolated GBS strain were measured in vitro for ampicillin (AMP)
and for the combination of AMP with gentamicin (GM) at 2 concentrations. Time-kill curves weremeasured at 2-hour intervals from time zero until 6 h. Bactericidal activity of AMP increased when
GM was included.

activity by adding a small amount of GM to AMP. These clinical and laboratory ﬁndings suggestthat combination therapy with AMP plus GM for GBS infection might be superior to AMP monotherapy.
The synergistic effect of AMP plus GM against GBS has been demonstrated by bactericidal
quantiﬁcations in vitro and by animal experiments in the past. However, details of the mechanisms underlying the clinical advantages were not identiﬁed. Our report provides further clinicalevidence of enhanced effectiveness using combination therapy in the context of mechanisms clariﬁed recently by LC/MS/MS and TEM8).
Moreover, GM has been thought to cross the blood-brain barriers (BBB) only with difﬁculty.
In rabbits, CSF concentration ratios of GM relative to those in serum were reported to be as low
as 21% to 25%11,12). In neonates with meningitis, GM concentrations in CSF following systemic
administration were reported to be below 1.0 μg/mL in most patients according to a bacteriologic
assay with limited sensitivity13). However, the BBB may be disrupted in the presence of meningeal inﬂammation, permitting greater penetration by antibiotics14). Regrettably, we were not able
to measure the AMP and GM concentrations in CSF of this case, but we suspect that GM may
more readily penetrate the BBB in an inﬂammatory state, achieving adequate concentrations inCSF to exert the synergistic effect demonstrated in vitro. GM concentrations in CSF should bestudied using a sensitive assay in the future.

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Prolonged fever in our patient may have resulted from subdural effusion, with defervescencereﬂecting resorption of the effusion. Subdural effusion has been detected in 27% to 42% of patients with meningitis and prolonged fever (over 10 days)15,16). In our patient, however, subdural
effusion seems unlikely to be the cause of prolonged fever, since our patient defervesced before
the effusion fully resolved.
One important ﬁnding in this patient is that the causative GBS was assigned to ST17 by
MLST analysis. GBS of ST17 in capsular type III exhibits hypervirulence17). Speciﬁcally, GBS ofST17 is particularly associated with late-onset infection believed to involve an oral route of infection. An ST17-speciﬁc surface-anchored protein termed hypervirulent GBS adhesin (HvgA) wasidentiﬁed by Tazi et al.18). HvgA enhances ability to adhere to intestinal epithelial cells and also
the microvascular endothelial cells that constitute the BBB. Combination therapy was effective inthe treatment of GBS infection despite this hypervirulence protein.
In this case, horizontal transfer of GBS from mother to infant might exist by the identicalcapsular type and ST in GBS isolates from the mother and the infant. Such horizontal transmission is considered the main cause of late-onset GBS infection in early infancy, which cannot be
prevented by maternal prophylaxis. Toyofuku et al.19) demonstrated more GBS positivity in
1-month-olds than in younger infants. Close observation of neonates with GBS-carrying mothers
is warranted even after perinatal prophylaxis.
According to a nationwide survey of children with GBS infection in Japan, the mortality rateis showing improvement in both early-onset and late-onset cases. However, since sequelae ofmeningitis still are frequent, use of GM together with β-lactam antibiotics should be consideredan option from the early stages of infection in these infants20〜23).
Acknowledgements
We thank all colleagues of Kawasaki Municipal Hospital for helpful discussions.
Conﬂicts of interestThe authors declare no conﬂict of interest. This research did not receive any speciﬁc grantfrom funding agencies in the public, commercial, or not-for-proﬁt sectors.

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