This longitudinal study was carried out between August and December 2013. The neonates were recruited from the labor and postnatal wards of the University College Hospital (UCH), Ibadan within 6–12 h of birth (day 1). The neonates were subsequently seen on days 3, 9, and 14 after birth. The UCH provides primary and specialist care to Ibadan and other parts of the South-West region of Nigeria. The labor and postnatal wards have a total of 75 beds, where healthy neonates and those who do not require intensive care are nursed besides their mothers after delivery. In the absence of maternal and neonatal complications, neonates are usually discharged within 48–72 h after delivery.

All healthy neonates were considered eligible to participate in the study. A neonate was regarded as “healthy or needing no intensive care” if there were no clinical features indicating the need for admission into the special care baby unit, but well enough to be nursed by the mother’s bedside. Seventy-two neonates were enrolled at birth but two mothers declined further participation of their babies after the first day, giving a dropout rate of 2.8%.

The required sample size was determined using the colonization rate from the previous study by Rotimi et al. (14) in 1985 which identified Escherichia coli as the commonest organism in 18 out of 23 babies by the first day of life, giving 78.2% colonization rate. It was hypothesized that the percentage of neonates with Escherichia coli colonization rate would be ±10% than the previously reported. At 95% level of confidence, 65 babies were required. Considering the fact that each neonate was sampled four times, 264 rectal swabs samples were expected.

Systematic random sampling method was used to recruit every fifth live-born neonate in the UCH who met the inclusion criteria during the study period. Neonates who had anorectal anomalies were not eligible to participate in the study.

Two trained research assistants collected data using a structured interviewer-administered questionnaire. All rectal swab samples were collected by two of the investigators (AK and AEO). Information recorded on questionnaire included mothers’ demographic characteristics, pregnancy and delivery history; infant’s characteristics including APGAR scores, mode of resuscitation, feeding methods, and antibiotic/antimycotic use. The 5-min APGAR scores of ≤3, 4–6, and 7–10 were regarded as indicative of low (severe asphyxia), mild–moderate abnormal, and reassuring (no asphyxia), respectively (15). We determined the gestational ages of the neonates using the date of the last menstrual period (LMP) reported by mothers corroborated with ultrasound report. In the event the mother’s LMP was unknown, the gestational age of the baby was estimated by the pediatrician using the New Ballard score system (16). Preterm neonates were those whose gestational ages were less than 37 weeks.

Rectal swabs were taken from the neonates on the first day within 6–12 h after birth while on the ward. Subsequent samples were collected on days 3, 9, and 14 when mothers brought their babies for follow-up. Samples were collected in an aseptic manner using sterile gel coated soft cotton tipped swab sticks (Transwab^® Amies; Medical Wire & Equipment, Corsham, Wiltshire, England). The Transwab^® is a sterile pack containing a gel-coated soft cotton tipped swab and a container containing Amies transport medium and recommended for recovery of aerobes, anaerobes, and fastidious organisms. All specimens were collected following the procedures described in Centre for Diseases Control manual (17). Each tube containing the swab was placed in a transport pack and in a cold box and transported to the laboratory within 3 h after collection by a laboratory motorcycle dispatcher. Five mothers who failed to present their babies for follow-up at different times were located through their addresses and phone numbers with the rectal swabs samples collected through home visits.

The rectal swabs on arrival in the laboratory were plated onto MacConkey agar and MacConkey Crystal Violet agar alongside Salmonella Shigella agar plates. Each sample was also inoculated onto a Selenite F broth and further processed in accordance with the standard practice for bacteria isolation. Other media used were Columbia blood agar and Amikacin blood agar, which were both incubated anaerobically at 37°C for 2 days. Inoculation of the plates was carried out using standard methods such that a semiquantitative assessment of the density of growth could be made on the solid media. All primary plates were reincubated after 18 h, regardless of the culture results.

Upon isolation of organisms, gram staining was done, which divided isolates into gram-negative and -positive bacteria. The complete identification and speciation of all the gram-negative isolates were done using the Analytical Profile Index (API) 20E^® and API 20NE^® (API^® bioMérieux Clinical Diagnostics Kits, USA) identification system in accordance with the standard procedures for bacteria classification. The API^® is a miniaturized identification system that contains dehydrated sugars that gives a characteristic color change upon addition and incubation of substrates. Miniaturized reagents are also incorporated in the system. API 20E^® identified members of the Enterobacteriaceae while API 20NE^® identified the nonfermenters and biochemically inert gram-negative rods. Gram-positive cocci were identified biochemically using the catalase test and coagulase test; Pastorex for Staphylococci (18). Anaerobes were conclusively identified employing the use of AnaeroGen a miniaturized GasPak™ gas regenerating kit system (19). All plates were kept for 40 h before being read. Indicator strips were added into the anaerobic jars to ascertain the presence of anaerobiosis.

The main outcome was the frequency of the types of isolates cultured on days 1, 3, 9, and 14 while independent variables included characteristics of neonates and mothers. Chi-square test was used to test associations between categorical variables, while comparisons between continuous variables were performed using Student’s t-test. The patterns of organisms identified on the different days were displayed in charts. All p < 0.05 were considered statistically significant. The data were analyzed using Statistical Package for Social Sciences (SPSS) for Windows 21 (IBM SPSS statistics 2012, Chicago, IL, USA).

Approval for the study was obtained from the University of Ibadan/UCH Ethics Review Committee (approval number: UI/EC/13/0161), and written informed consent was obtained from each mother.

Of the 68 mothers whose neonates participated in the study, two had twin deliveries. The mothers’ age ranged from 17 to 40 years (mean = 28.5 ± 5.1 years). Twenty-six (38.2%) of the mothers were primipara, 65 (95.6%) booked and received antenatal care at the study center. Pregnancy-related medical conditions recorded in the hospital notes and corroborated by mothers’ reports were hypertension (n = 3; 4.4%), antepartum hemorrhage (n = 1; 1.5%), postpartum hemorrhage (n = 1; 1.5%), and polyhydramnios (n = 1; 1.5%). Only two (2.9%) mothers had PROMs both lasting >24 h and they were given intravenous Augmentin (amoxicillin and clavulanate) prophylactically for 48 h, then orally to complete a 7–10-day course. Three mothers (4.4%) had prolonged labor lasting 19 to 24 h. Sixty-six (97.1%) of the mothers reported no illness or complications during pregnancy, and only two (2.9%) were “HIV positive.”

Table 1 shows the characteristics of the neonates. There were 32 (45.7%) males and 38 (54.3%) female neonates. There were 66 singletons and two sets of twins. Sixty-one neonates (87.1%) were exclusively breastfed, and 9 (12.9%) were given water and/or formula in addition to breast milk. Two neonates required hospital admission, which occurred during the second week of life, with aspiration pneumonitis and acyanotic congenital cardiac defect, respectively. One mother reported administering oral antibiotics to her neonate (1.4%). Five (7.1%) neonates were delivered by elective cesarean section. One female term neonate had pustular skin rash on the third day of life was given a course of oral cephalexin, which resolved by the ninth day of life.

Bacteria were isolated from the rectal swab samples of 65 (92.9%) on day 1 mainly facultative anaerobes (15 of 16 different species). Figure 1 shows the species of isolates. The most frequently isolated bacteria was coagulase-negative Staphylococcus (n = 27; 38.6%); followed by Staphylococcus aureus (n = 22; 31.4%) and Escherichia coli (n = 21; 30.0%). Serratia fanticola, Serratia liquefaciens, Citrobacter koseri, and Proteus mirabilis were found in samples from one neonate each. With the exception of Escherichia coli in preterm neonates (n = 9; 50.0%) compared with term neonates 12 (23.1) (p = 0.031), there was no significant differences in the bacterial isolates in preterm and term neonates (Table 1).

Table 2 also shows comparisons of frequency of bacterial isolates from the neonates between neonates who were preterm versus term, had mild to moderate asphyxia versus no asphyxia, and delivered per vaginal versus cesarean section. On the first day of life, the odds of isolating Escherichia coli (OR = 3.3; 95% CI: 1.1, 10.2) from the rectal swabs of preterm neonates was significantly higher than that of term neonates.

Different species of isolates were obtained from all 70 (100.0%) rectal samples on day 3 of life (Figure 2). Coagulase-negative Staphylococcus (58.6%) was the most frequently isolated bacteria on day 3. Table 3 shows the bacteria isolates from neonates by maturity, presence or absence of asphyxia at birth, mode of delivery, and whether or not neonate was exclusively breastfed for the first 3 days of life. The coagulase-negative Staphylococcus was more frequently (p = 0.042) isolated in nonasphyxiated neonates (64.6%) than asphyxiated neonates (27.3%). Asphyxiated neonates had significantly higher colonization by Enterobacter cloacae (36.4%) than nonasphyxiated neonates (8.5%) (p = 0.029). On day 3, the odds of isolating coagulase-negative Staphylococcus from the rectal swabs of nonasphyxiated neonates were significantly higher than that of asphyxiated neonates (OR = 4.83; 95% CI: 1.15, 20.16). Asphyxiated neonates had significantly higher odds of colonization by Enterobacter cloacae (OR = 6.1; 95% CI: 1.33, 28.57) than nonasphyxiated neonates.

The isolates obtained from all 70 (100.0%) neonates on the ninth day of life were as displayed in Figure 3. The most frequently isolated bacteria were coagulase-negative Staphylococcus (44.3%). Table 4 shows comparisons of bacterial isolates from the neonates by maturity, presence or absence of asphyxia at birth, mode of delivery, and whether or not exclusively breastfed for the first 9 days of life. Remarkably, the odds of isolating coagulase-negative Staphylococcus (OR = 4.17; 95% CI: 1.21, 7.38) was significantly higher in nonasphyxiated than asphyxiated neonates as shown in Table 4.

Figure 4 and Table 5 show the bacteria isolates obtained from the samples of all 70 (100.0%) neonates on day 14. Coagulase-negative Staphylococcus (47.1%) remains the most frequently isolated organism, it was isolated more from those who were not exclusively breastfed than those who had exclusively breastfeeding indicating significantly higher odds of Staphylococcus aureus (OR = 7.38; 95% CI: 1.62, 33.61) from nonexclusively breastfed than that of exclusively breastfed neonates.

Table 6 shows the trend and comparisons of the difference in colonization patterns between days 1, 3, 9, and 14. It appears that bacteria colonization in the neonates became established at slightly different times. On day 1, bacteria isolates were predominantly facultative anaerobes comprising coagulase-negative Staphylococcus, Staphylococcus aureus, Escherichia coli, and Enterobacteria other than Escherichia coli forming a large contingent (Enterobacter cloacae, Enterobacter aerogenes, Raoultella ornithinolytica, and Klebsiella oxytoca). On days 3 and 9, the bacteria isolates were heterogeneous but still dominated by coagulase-negative Staphylococcus and the Enterobacteria, Escherichia coli, and Raoultella ornithinolytica. On day 14, there was more diversity in the isolates with 23 different species but the dominance of coagulase-negative Staphylococcus and Staphylococcus aureus was maintained. Most of the remaining constituent bacteria were other members of the Enterobacteriaceae family. Statistically significant increasing trends were observed for Staphylococcus aureus and Klebsiella pneumoniae, respectively, as the neonates got older.