Influence of Intrapartum Antibiotic Prophylaxis for Group B Streptococcus on Gut Microbiota in the First Month of Life : Journal of Pediatric Gastroenterology and Nutrition

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Original Articles: Gastroenterology

Influence of Intrapartum Antibiotic Prophylaxis for Group B Streptococcus on Gut Microbiota in the First Month of Life

Corvaglia, Luigi*; Tonti, Giacomo*; Martini, Silvia*; Aceti, Arianna*; Mazzola, Giuseppe; Aloisio, Irene; Di Gioia, Diana; Faldella, Giacomo*

Author Information
Journal of Pediatric Gastroenterology and Nutrition 62(2):p 304-308, February 2016. | DOI: 10.1097/MPG.0000000000000928
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What Is Known

  • Antibiotics in early life can impair the balance between health and disease by altering commensal gut microbiota.
  • The effect of intrapartum antibiotic prophylaxis for group B Streptococcus on bacterial colonization of the infant's gut has not been investigated extensively.

What Is New

  • At 7 days of life, infants who had not received intrapartum antibiotic prophylaxis and were exclusively human milk–fed had higher counts of Bifidobacteria.
  • Furthermore, regardless of intrapartum antibiotic prophylaxis treatment, infants fed exclusive human milk had higher Lactobacillus spp counts both at 7 and 30 days of life.

The colonization of the gastrointestinal tract is thought to begin during the birth process, when the infant's gut is exposed to maternal and environmental bacteria (1). Studies performed in preterm fetuses and infants, however, have shown that amniotic fluid and meconium are not sterile, thus suggesting an intrauterine origin of gut microbiota (2,3). At birth, the neonatal gastrointestinal tract is rapidly colonized by bacteria from the mother and the environment; the first colonizers are generally aerobes and facultative anaerobes (4,5), followed by strict anaerobes such as Bifidobacterium spp, Bacteroides spp, and Clostridium spp (6). The composition of gut microbiota is influenced by several factors, including mode of delivery, gestational age (GA), maternal microbiota of the intestine, vagina and epidermis, hospitalization after birth, type of infant feeding, and use of antibiotics and probiotics (6–9). Gut microbiota of term infants, born by vaginal delivery (VD) and exclusively breast-fed, is considered to be ideally healthy, with its low count of Clostridium difficile and Escherichia coli, and high number of beneficial bacteria such as Bifidobacteria and Lactobacilli(10).

Group B Streptococcus (GBS), mainly represented by Streptococcus agalactiae strains, is one of the most important causes of infection and sepsis in the neonatal period. Infants born by VD may acquire GBS during the birth process from maternal vagina, cervix, or rectum, where it resides in ∼10% to 20% of pregnant women (11,12). The incidence of early-onset GBS sepsis declined significantly in the last decade, because of the introduction of GBS universal screening during late pregnancy (13) and consequent intrapartum antibiotic prophylaxis (IAP) in GBS-positive women (14,15).

Recent data suggest that the use of antibiotics in early life can impair the balance between health and disease later in life by altering commensal gut microbiota (16). The effect of IAP on bacterial colonization of the infant's gut has not been investigated extensively (4,17,18). Studies performed up to now have shown that IAP does not increase the amount of antibiotic resistant enterobacteria (4) but could reduce vertical transmission of lactic acid bacteria from IAP-treated mothers (17); however, both these studies were performed using culture-based techniques, which are known to have several limitations, in particular in counting and isolation of anaerobic bacteria (19).

A recent study evaluated, by means of 16S ribosomal RNA gene sequence–based analysis and quantitative polymerase chain reaction, the development of gut microbiota in preterm, extremely-low-birth-weight infants, using a small group of term infants as the control group. Data from these latter infants suggested a profound impact of IAP on gut microbiota composition (18).

In a preliminary study, we evaluated at 7 days of life the effect of IAP on gut microbiota in a relatively small sample of exclusively breast-fed term infants born by VD; selected microbial groups were quantified by real-time PCR, and further analysis was performed within the Bifidobacterium genus. A significant reduction of Bifidobacteria counts was documented in newborns born to IAP-treated mothers; furthermore, Bifidobacteria were found to be affected by IAP also qualitatively because IAP led to a reduction in the frequency of B breve, B bifidum, and B dentium(6).

The aim of the present paper was to evaluate these differences in further detail, expanding the initial number of subjects and following up infants until 1 month of age. The influence of type of feeding on microbiota composition was also explored.


The study was performed in the Nursery of S. Orsola-Malpighi Hospital in Bologna, Italy, and was approved by the institutional ethics committee (study ID 12/2013/U/Oss).


Between October 2012 and June 2013, 84 healthy term infants, born by VD, with birth weight adequate for GA, and whose mothers had been screened for GBS at 35 to 37 weeks gestation, were enrolled in the study. The exclusion of preterm or small/large for GA infants, infants born by caesarean section, and infants admitted to the neonatal intensive care unit was made to minimize potential confounding factors (20).

Infants were excluded also in the following cases:

  1. The mother had received any antibiotic other than IAP in the 4 weeks before delivery.
  2. Maternal IAP was performed for reasons other than GBS positivity (ie, prolonged rupture of membranes in GBS-negative women).
  3. Maternal IAP was performed with antibiotics other than ampicillin, such as erythromycin.
  4. The infant had major congenital malformations.
  5. The infant developed signs of infection and/or received any antibiotic treatment after birth.
  6. The infant had, or developed at birth, any serious clinical conditions that contraindicated participation in the study.

Infants were divided into 2 groups according to maternal GBS status and IAP:

  1. IAP group: infants born to GBS-positive mothers who had received IAP. According to the institutional treatment protocol for GBS prophylaxis (derived from the Centers for Disease Control and Prevention guidelines (13)), intravenous ampicillin was given every 4 hours until delivery (first dose 2 g, following doses 1 g each).
  2. Control group: infants born to GBS-negative mothers, who thus did not receive any antibiotic treatment before/at delivery.

Written informed consent was obtained from each infant's parent/legal guardian when the infant was about to be discharged from the nursery (48–72 hours of life). Patients’ characteristics, including GA, birth weight, sex, and Apgar score at 1’ and 5’ after birth, were summarized in a specific case report form.

Fecal Samples’ Collection and Analysis

Follow-up visits were performed at 7 and 30 days of life. At each visit, information on infants’ weight gain, clinical conditions (feeding tolerance, infections, etc), and ongoing treatments (ie, use of prebiotics, probiotics, antibiotics) was collected. Furthermore, the characteristics of the infants’ feeding (exclusive breast-feeding, exclusive formula feeding, or mixed feeding) were recorded.

Fecal samples were collected at each follow-up visit. After collection, they were put into numbered screw-capped sterile plastic containers, which were immediately frozen at −80°C, until they were processed for DNA extraction.

Microbiological analyses were performed at the Laboratory of Microbiology, Department of Agricultural Sciences, University of Bologna (Italy), according to previously published methods (6). Investigators who performed the analyses were blind to the group identity of the infants.

A total of 200 mg of feces were used for DNA extraction using the QIAamp DNA Stool Mini Kit (QIAGEN, West Sussex, UK). Extracted DNA was stored at −80°C. The purity and concentration of extracted DNA were determined by measuring the ratio of the absorbance at 260 and 280 nm (Infinite 200 PRO NanoQuant; Tecan, Mannedorf, Switzerland).

Quantification of selected microbial groups (Bifidobacterium spp, Lactobacillus spp, and Bacteroides fragilis) was carried out with real-time PCR. The assays were performed as previously described (6). Data obtained from amplification were transformed to obtain the number of bacterial cells per gram of feces, expressed as Log colony-forming unit per gram.

Statistical Analysis

Data were analyzed using SPSS version 20.0.0 (IBM SPSS Statistics, Armonk, NY). Data distribution was evaluated by the Kolmogorov-Smirnov test. Nonparametric tests were used because data did not follow a normal distribution. Baseline characteristics in the IAP and control groups were compared using the independent-samples Mann-Whitney U test for continuous variables and the χ2 test for categorical variables. The influence of IAP on fecal bacterial count at 7 and 30 days of life was evaluated using the independent-samples Mann-Whitney U test.

Furthermore, multiple regression analysis was performed to estimate the effect of IAP on fecal bacterial count after controlling for the type of infant feeding. Specifically, a hierarchical regression analysis was performed; IAP was entered first, followed by type of feeding. For the analysis, feeding type was coded as a binary categorical variable; exclusive breast-feeding versus any formula feeding (the latter includes infants receiving exclusive formula or a variable proportion of breast milk and formula). A P < 0.05 was considered as statistically significant.



During the study period, 84 newborns were recruited (35 in the IAP and 49 in the control group). Neonatal characteristics did not differ between infants in the IAP and control groups (Table 1). There was no difference between groups in terms of duration of rupture of membranes.

Baseline characteristics of the enrolled infants

All the recruited infants were evaluated at 7 and 30 days after birth. The characteristics of the infants at the 2 follow-up visits are shown in Table 2. No difference between groups in terms of weight gain and rate of exclusive breast-feeding was documented. None of the infants was receiving, or had received since birth, any treatment with prebiotics, probiotics, and antibiotics.

Characteristics of the enrolled infants evaluated at 7 and 30 days of life

Influence of IAP on Fecal Bacterial Counts

The count of Bifidobacterium spp was significantly lower in the IAP group than in the control group at 7 days of life (independent-samples Mann-Whitney U test; median [interquartile range] 6.01 Log colony-forming unit per gram [5.51–6.98] vs 7.80 [6.61–8.26], respectively; P = 0.000), whereas no difference was documented at 30 days (8.41 [7.71–8.80] vs 8.39 [7.96–8.86], respectively; P = 0.842). No difference was documented between the 2 groups at any time point in the count of Lactobacillus spp (5.56 [4.94–6.14] vs 5.45 [4.81–6.14] at 7 days, P = 0.518; 5.29 [4.68–6.01] vs 5.25 [4.60–6.15] at 30 days, P = 0.818) and the B fragilis group (7.71 [5.80–9.33] vs 7.75 [5.87–9.61] at 7 days, P = 0.618; 7.36 [5.80–9.09] vs 8.51 [5.86–9.37] at 30 days, P = 0.479).

Hierarchical multiple regression was performed for each bacterial group, both at day 7 and day 30. The results of these analyses for Bifidobacterium spp and Lactobacillus spp are provided in Table 3. At 7 days of life, IAP and feeding type were significantly and independently associated with Bifidobacterium spp count, with higher counts in infants who had not received IAP and were exclusively human milk (HM)–fed. IAP accounted for ∼22% of the variance of the outcome (R2 = 0.216 in step 1) and feeding type contributed for an additional 8% (R2 = 0.301 in step 2). At 30 days of life, Bifidobacterium spp count was unrelated to IAP or feeding type.

Results of hierarchical multiple regression analysis

Hierarchical regression analysis confirmed that IAP was unrelated to Lactobacillus spp counts either at 7 and 30 days of life. This analysis, however, showed a significant effect of feeding type on Lactobacillus spp counts; regardless of IAP treatment, infants fed exclusive HM had higher Lactobacillus spp counts both at 7 and 30 days of life. Feeding type gave the main contribution to the variability of the outcome (∼6% at 7 days [R2 = 0.003 in step 1 and R2 = 0.065 in step 2] and 11% at 30 days [R2 = 0.000 in step 1 and R2 = .105 in step 2]). No significant influence of IAP or feeding type was documented for B fragilis group, either at 7 or 30 days of life (data not shown).


Three groups of bacteria were monitored in this work: members of the Bifidobacterium genus, which had been previously shown to decrease at 7 days of life in infants born to IAP-treated mothers (6), members of the Lactobacillus genus, which showed in a previous study a decreased vertical transmission from IAP-treated mothers (17), and members of the B fragilis group. This latter group comprises species such as Bacteroides thetaiotaomicron and B fragilis, which have been shown to be pioneer bacteria in the majority of neonates, particularly the breast-fed ones (7).

The present study confirms previous data according to which the fecal count of Bifidobacteria is reduced by maternal IAP in the first week of life (6); however, the effect of prenatal antibiotic treatment proves to be transient because the fecal count of Bifidobacteria gets back to normal at 1 month of life. The present study also examines the effect of type of feeding on gut microbiota, showing that, in infants born to GBS-positive mothers, the use of formula represents an additional and independent negative factor in terms of Bifidobacteria colonization. The counts of Lactobacilli and B fragilis group are not influenced by IAP; however, regardless of IAP, exclusively HM-fed infants have a higher Lactobacilli count both at 7 and 30 days of life. A recent study, primarily aimed at evaluating gut microbiota in extremely-low-birth-weight infants, reached similar conclusions, showing that IAP influences gut microbiota composition also in term infants; however, the number of included term infants exposed to IAP was relatively small (18).

The introduction of universal screening for GBS and consequent IAP which followed the Centers for Disease Control and Prevention updated guidelines (13) has dramatically reduced the incidence of early-onset GBS sepsis both in the US and in Europe, as most countries launched national guidelines for GBS prevention (11). Despite the clinical benefit of IAP, however, little is known on how it affects neonatal bacterial gut colonization and whether alterations of gut microbiota related to IAP could have short- and long-term consequences in terms of health and disease.

Most previous studies on this topic have important limitations, such as the reduced number of samples considered, the nonstandardization of potential confounding factors (such as mode of delivery, GA, and prolonged rupture of membranes) and the use of culture-dependent techniques that may have drawbacks when counting fecal bacteria, in particular anaerobic ones (19). Our study was therefore designed to overcome these problems; a highly selective population of healthy, adequate for GA, and term infants born by VD, was recruited, and microbial populations were counted with the use of molecular techniques. To our knowledge, this is the first study investigating the effect of IAP on gut microbiota in term infants in the first month of life by means of molecular techniques. Only 1 previous study was focused specifically on IAP (4): 25 three-day-old infants born to GBS-positive mothers who had received intravenous IAP with amoxicillin were compared with 25 controls, matched for GA, mode of delivery, and type of feeding. No differences in the count of Bifidobacteria and Bacteroides were documented; however, fecal samples were analyzed by culture-dependent methods, and it was not possible to document any specific effect of feeding. In the study by Keski-Nisula (17), which investigated vertical transmission of Lactobacillus spp, IAP and longer rupture of membranes were associated with a lower transmission rate of Lactobacilli. Nevertheless, the study was not comparable to ours because a relatively unselected population of term infants was recruited, and the Lactobacillus population was studied using a neonatal oral swab and analyzed by culture-based methods.

One further study (21) examined by means of molecular techniques the influence of prenatal and neonatal antibiotic treatments on infants’ gut microbiota during the first 2 months of life; similar to the results of our study, colonization by Bifidobacteria was initially attenuated in infants exposed to prenatal or neonatal antibiotics and got back to normal at 2 months of life. The group treated with antibiotics, however, consisted of only 8 infants, and no control for confounders such as feeding or mode of delivery was made.

Any alteration on the development of gut microbiota in early life is presumably associated with a divergent immunological starting point in the host, with potential implications for the development of disease later in life (16). Several events in early life can lead to a perturbation in the physiological development of a healthy microbiota. In the present study, type of feeding was found to have a great impact on gut colonization in the first days of life; exclusive HM feeding had a positive and persistent effect on the count of Lactobacilli, which was independent from antibiotic exposure; furthermore, the use of formula had a negative effect, which was additional to the effect of IAP, on the count of Bifidobacteria at 7 days of life. Although available data regarding differences in gut microbiota composition in breast-fed and formula-fed infants are often contradictory (22), results obtained with culture-independent methods showed that infants fed HM have higher count of Bifidobacteria compared with formula-fed infants (23,24).

HM is a complex biofluid, which has both probiotic and prebiotic properties; it represents a unique source of bacteria, such as Bifidobacteria and Lactobacilli, which are able to colonize the infant's gut and to promote health benefits for the host (25), and also contains specific oligosaccharides that exert a prebiotic effect on gut microbiota, stimulating beneficial microorganisms such as Bifidobacteria and Lactobacilli(26). The development of gut microbiota is driven by the so-called “pioneer bacteria”; in this perspective, alterations in the composition of gut microbiota in early life have strong implications in terms of later health and disease. The results of our study show that IAP alters the infant's microbiota by reducing the count of Bifidobacteria, and that this is further affected in infants receiving formula feeding. Whether these alterations could have long-term consequences on health and disease is unknown; however, the promotion of exclusive breast-feeding appears to be important for reducing the alterations in the count of Bifidobacteria induced by IAP and also for promoting the infant's colonization with beneficial bacteria such as Lactobacilli. A recent study also documented a long-term effect of exclusive breast-feeding; at 4 months of age, exclusively breast-fed infants had increased levels of taxa that are used as probiotics compared with formula-fed infants; in addition, the cessation of breast-feeding shifted gut microbiota toward an adult-like composition (27).

These findings suggest that, to reproduce the beneficial effects of breast-feeding, further studies should investigate the opportunity of giving a formulation containing potential probiotic bacteria, such as Lactobacilli and Bifidobacteria, in the first weeks of life to infants born to GBS-positive mothers and not receiving exclusive breast-feeding. The results of the present study are partially limited by the evaluation of only 3 bacterial groups; future studies should be performed using more comprehensive techniques aimed at evaluating whether IAP has wider effects on gut microbiota.


Gut microbiota was evaluated in healthy term infants born by VD. A total of 84 infant–mother pairs were studied. At 7 days of life, infants whose mothers had received IAP had a significantly lower Bifidobacteria count compared with controls; furthermore, infants who had not received IAP and were exclusively HM-fed had higher counts of Bifidobacteria. There were no differences in Bifidobacteria count at 30 days or in Lactobacilli and B fragilis counts at any time point. Regardless of IAP treatment, infants fed exclusively HM had higher Lactobacillus spp counts both at 7 and 30 days of life.


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group B Streptococcus; human milk; infant; intrapartum antibiotic prophylaxis; microbiota

© 2016 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,