Abstract

Our study showed that enteral administration of prophylactic probiotics in neonatal intensive care setup could significantly reduce morbidity due to necrotising enterocolitis in very low birth weight newborn. It also helps in establishing early full enteral feeding and reduces hospital stay.

Introduction

Necrotizing enterocolitis (NEC) is a significant cause of mortality and morbidity in pre-term, very low birth weight (VLBW) newborn. It is the most common gastrointestinal emergency in newborns [1]. Regarding treatment, current recommendation is prompt and early diagnosis and supportive management, but no effective preventive strategy is as yet available. Despite the achievements observed in perinatal care over the last decade, the prognosis of newborns with NEC has not improved. In fact, over recent years, it has been observed that the number of deaths from this condition is on the increase and mortality rates range from between 18% and 45%, depending on the degree of prematurity and the severity of infection [2].

NEC is a multifactorial disease that results from an interaction between the loss of mucosal integrity (due to ischemia, inflammation and infection) and the host response to that injury (in the form of circulatory, inflammatory and immune-mediated response). The most common risk factors cited are prematurity, enteral feeding and colonization by bacteria [3] such as Escherichia coli, Klebsiella, Clostridium perfringes, Staphylococcal epidermis and Rotavirus.

The infant's intestine is initially colonized by complex flora that reflects maternal vaginal and large intestinal flora [4, 5] Breast milk is the first source of bacteria such as Lactobacillus and Bifidobacteria which compete with pathogens. The mode of birth, surrounding environment and dietary factors influence initial gut colonization in a neonate. Although a range of aerobic and anaerobic flora colonize the gut of a normal infant by 10 days of life, an infant in the NICU undergoes delayed colonization following the use of broad spectrum of antibiotics on the limited number of bacterial species present species, which tend to be virulent [6–8] and will enhance an inflammatory cascade, facilitating the development of NEC.

A probiotic is a live anaerobic bacterial food supplement that benefits the recipient by improving the intestinal flora balance. Studies have shown that orally administered non-enteropathogenic bacteria can colonize the gut of the of the newborn and reduce colonization with pathogenic strains [9].

Animal studies have suggested that the administration of bifidobacterium decreases the incidence of NEC through the modulation of the inflammatory cascade [10].

Hence, we tried to evaluate whether incidence, severity and death due to NEC can be reduced, which is thought to lead to a reduction of the load of prophylactic administration of probiotics by reducing pathogenic organisms in the enteric mucosa of pre-term VLBW.

Objective

The objective of this study is the evaluation of the efficacy of probiotics in promoting food tolerance and reducing the incidence and severity of NEC and death related to NEC in pre-term VLBW infants.

Subjects and Methods

This prospective randomized double-blind control trial was carried out in the Neonatal Care Unit of Medical College and Hospital, Kolkata. Pre-term infants (gestational age <32 weeks) and VLBW infants (<1500 g) born between October 2007 and March 2008, who fulfilled the eligibility criteria (started feed enterally and survived beyond 48 h of life) were included in the study. Babies with major congenital and gastrointestinal anomalies and babies who expired due to other neonatal illnesses were excluded. Gestation was assessed from history of last menstrual period and after birth by new Ballard scores [11].

Feeding was started when vitals were stable, active bowel sounds were heard and no abdominal distension or blood/bile in NG tube suction was noticed. After entering the study, the infants were randomly assigned to two groups by random number table sequence. Parental written informed consents were taken. The probiotic-fortified group received a probiotic mixture (Bifidobacteria infantis, Bifidobacteria bifidum, Bifidobacteria longum and Lactobacillus acidophilus, each 2.5 billion CFU) with expressed breast milk twice daily, the dosage being 125 g kg−1 [12] till discharge. The control group was fed with breast milk only.

The demographic and clinical variables that are potential risk factors for NEC were compared in both the study and the control group. A mother receiving two doses of betamethasone or dexamethasone given 24 h before delivery was considered to have been on prenatal steroids. Infants with birth weight >2 SDs below the mean for gestational age were considered small for gestational age. Prolonged rupture of amniotic membrane was defined as rupture of the amniotic membrane >18 h before delivery. Chorioamnionitis was defined as maternal fever, foul-smelling amniotic fluid and left shift of the white blood cell differential count and was confirmed by the obstetrician. Asphyxia was defined by the following criteria: (i) an umbilical or scalp blood pH 7.0, (ii) an APGAR score of 3 at 5 min, (iii) neurologic manifestations including hypotonic seizures or hypoxic-ischaemia encephalopathy and (iv) multiple organ failure. Surfactant was used for respiratory distress syndrome within 2 h after birth in cases of ventilated infants needing oxygen supplementation with fractional inspired oxygen of 0.40 and showing radiologic changes typical of respiratory distress syndrome. Indomethacin was indicated in infants with patent ductus arteriosus showing left-to-right shunt by echocardiography.

Primary outcome measures were (i) feed tolerance in terms of days required to reach full enteral feeding, (ii) length of hospital stay and (iii) morbidities such as NEC, sepsis and death due to NEC or sepsis.

Babies were weighed daily and monitored for daily increment in feed volume, abdominal girth, appearance of erythema of abdominal wall, loose stools with blood, vomiting and orogastric tube suction >50% of previous feed. NEC was graded according to Bell's staging [13]—stage I (suspicion), stage II (definite) and stage III (advanced). Bell's staging ≥II was considered to be severe. Sepsis was confirmed by positive blood culture or positive CSF culture taken beyond 5 days of age.

Two groups were compared using unpaired student t-test for continuous variables and chi-square test for categorical variables. Values were expressed for mean and standard deviation. P-value <0.05 was considered significant.

Results

In all, 274 preterm VLBW newborns were admitted to the NICU in the study period, among them 186 infants were randomized, of which 91 were exposed to probiotics and 95 were not. Birth weight (1172 ± 143 g vs. 1210 ± 143 g, P-value 0.07) and gestational age (30.12 ± 1.63 weeks vs. 30.14 ± 1.59 weeks, P-value 0.90) were not significantly different. Other demographic and clinical variables of both mother and infant did not differ between the two groups (Tables 1 and 2).

Table 1

Maternal clinical and infant's demographic and clinical characteristics

Characteristics Study group (N = 91) Control group (N = 95) P-value 
Birth weight (grams) 1172 ± 143 1210 ± 143 0.07 
Gestational age (week) 30.12 ± 1.63 30.14 ± 1.59 0.90 
Preeclampsia, n (%) 21 (22.1) 15 (15.78)  
Prenatal steroid, n (%) 58 (63.73) 61 (64.12)  
Cesarean section, n (%) 42 (46.15) 47 (49.47)  
Multiplepregnancy, n (%) 14 (15.13) 19 (20)  
Chorioamnionitis, n (%) 9 (9.89) 12 (12.63)  
Small for gestational age, n (%) 34 (37.36) 30 (31.57)  
Apgar (5 min)    
    <3  
    4–6 21 29  
    >7 66 61  
Asphyxia, n (%) 4 (4.39) 5 (5.26)  
pH 7.29 ± 1 7.31 ± 1.1  
Characteristics Study group (N = 91) Control group (N = 95) P-value 
Birth weight (grams) 1172 ± 143 1210 ± 143 0.07 
Gestational age (week) 30.12 ± 1.63 30.14 ± 1.59 0.90 
Preeclampsia, n (%) 21 (22.1) 15 (15.78)  
Prenatal steroid, n (%) 58 (63.73) 61 (64.12)  
Cesarean section, n (%) 42 (46.15) 47 (49.47)  
Multiplepregnancy, n (%) 14 (15.13) 19 (20)  
Chorioamnionitis, n (%) 9 (9.89) 12 (12.63)  
Small for gestational age, n (%) 34 (37.36) 30 (31.57)  
Apgar (5 min)    
    <3  
    4–6 21 29  
    >7 66 61  
Asphyxia, n (%) 4 (4.39) 5 (5.26)  
pH 7.29 ± 1 7.31 ± 1.1  

None of the differences are statistically significant (P > 0.05).

Table 2

Clinical variables in study infants

Variables Study group (N = 91) Control group (N = 95) 
Age at enrolment (days) 5.97 ± 1.41 5.45 ± 1.26 
Use of surfactant, n (%) 12 (13.18) 9 (9.47) 
Umbilical artery catheter (n18 (19.78) 21 (22.1) 
Umbilical venous catheter (n18 (19.78) 21 (22.1) 
Intermittent mandatory ventilation (days) 5 ± 3.7 5.0 ± 4.5 
Use of dopamine, n (%) 53 (58.24) 49 (53.5) 
Dopamine (days) 3.5 ± 2.5 3 ± 3.1 
Indomethacin, n (%) 26 (28.57) 30 (31.57) 
Intraventricular hemorrhage, grades 3–4, n (%) 4 (4.39) 7 (7.36) 
Variables Study group (N = 91) Control group (N = 95) 
Age at enrolment (days) 5.97 ± 1.41 5.45 ± 1.26 
Use of surfactant, n (%) 12 (13.18) 9 (9.47) 
Umbilical artery catheter (n18 (19.78) 21 (22.1) 
Umbilical venous catheter (n18 (19.78) 21 (22.1) 
Intermittent mandatory ventilation (days) 5 ± 3.7 5.0 ± 4.5 
Use of dopamine, n (%) 53 (58.24) 49 (53.5) 
Dopamine (days) 3.5 ± 2.5 3 ± 3.1 
Indomethacin, n (%) 26 (28.57) 30 (31.57) 
Intraventricular hemorrhage, grades 3–4, n (%) 4 (4.39) 7 (7.36) 

The number of days required to reach full enteral feeding (13.76 ± 2.28 vs. 19.2 ± 2.02; P < 0.001) was significantly low in babies who received probiotics. Therefore, feeding tolerance was better in the probiotic-exposed group. Duration of hospital stay was (17.17 ± 3.23 vs. 24.07 ± 4, P < 0.001) also significantly low in the probiotic-exposed group compared with the control. The incidences of NEC was significantly low in the probiotic-exposed group when compared with non-exposed group (5 of 92 [5.5%] vs. 15 of 95 [15.8%], respectively; P = 0.042). Compared among the two groups (Bell stage ≥II) of NEC was compared in the two groups, it showed no difference (2.46 ± 0.51 vs. 2.6 ± 0.54, P-value 0.62).

The study group suffered significantly low incidences of culture-proven sepsis (13 of 91 [14.3%] vs. 28 of 95 [29.5%]; p = 0.020). Death rate was reduced in exposed group when compared with control group (4 of 91 [4.4%] vs. 14 of 95 [14.7%]; P = 0.032) (Table 3). None of the blood cultures taken grew B. infantis, B. bifidum, B. longum or L. acidophilus.

Table 3

Outcome variables after oral probiotics

Variables Study group (N = 91) Control group (N = 95) P-value 
Full enteral feeding (days) 13.76 ± 2.28 19.2 ± 2.02 <0.001 
Hospital stay (days) 17.17 ± 3.23 24.07 ± 4 <0.001 
Severity of NEC (Bell stage ≥2) 2.46 ± 0.51 2.6 ± 0.54 0.62 
Death 4 (4.4) 14 (14.7) 0.032 
NEC grade 2 or 3 5 (1.1) 15 (15.8) 0.042 
Sepsis (culture proven) 13 (14.3) 28 (29.5) 0.020 
Variables Study group (N = 91) Control group (N = 95) P-value 
Full enteral feeding (days) 13.76 ± 2.28 19.2 ± 2.02 <0.001 
Hospital stay (days) 17.17 ± 3.23 24.07 ± 4 <0.001 
Severity of NEC (Bell stage ≥2) 2.46 ± 0.51 2.6 ± 0.54 0.62 
Death 4 (4.4) 14 (14.7) 0.032 
NEC grade 2 or 3 5 (1.1) 15 (15.8) 0.042 
Sepsis (culture proven) 13 (14.3) 28 (29.5) 0.020 

Discussion

Our study shows that probiotic mixture containing B. infantis, B. bifidum, B. longum and L. acidophilus reduces incidence and death due to NEC in pre-term VLBW newborns and improves feed tolerance.

There have been few clinical trials that have reported the outcomes for pre-term infants given probiotics. In the study by Hung-Chin Lin et al. [12], the study group was fed with infloran (Lactobacillus and B. infantis) at 125 mg kg−1 dose−1 twice daily with breast milk till discharge and outcomes were measured in terms of NEC, sepsis and death due to NEC or sepsis. This study showed that infloran as a probiotic is protective of NEC in VLBW infants. There is no standard dose schedule for probiotics in NEC. We have used a probiotic dose similar to that used in the Hui-Ching et al. study.

In the study by Bin-Nun et al. [14] in Israel, neonates received daily feeding supplementation with probiotic mixture (B. infantis, B. bifidus, Streptococcus thermophillus) of 109 CFU day−1. It was found that incidence, severity and death due to NEC was less in the study group than in the control group. Our results were also comparable. In Italy, 585 infants of <33 weeks gestational age or birth weight <1500 g were randomized to receive Lactobacillus rhamnosus GG (6 × 109 CFU) once a day from the start of feeds to the time of discharge, or a placebo. Outcome measures included the incidence of urinary tract infection, bacterial sepsis and NEC.

The numbers of babies with any of the three outcomes were surprisingly low and there were no significant differences between the probiotic and placebo groups [15]. One major difference from ours is that we used B. infantis, B. bifidum, B. longum and L. acidophilus, which are normal commensals of breast milk [6, 7].

Although Wagner et al. [16] suggested that safety issues of probiotics treatment needed to be addressed in immunodeficient hosts such as neonates, in a study by Hoyos et al. [17] in South America, neonates received daily dose of 250 million live L. acidophillus and 250 million B. infantis. No complications were attributed to daily administration of probiotics. A similar observation was found here too, as none of the blood cultures grew Lactobacillus and Bifidobacteria.

Probiotics shorten the minimum duration for the establishment of full enteral feeding, however, the severity of NEC was similar in both groups. Here, we hypothesized that once NEC developed, its progression could not be stopped by probiotics and there could be other factors responsible for progression of NEC. Our result differs from that of Bin-Nun et al., who observed that probiotics reduce both incidence and severity of NEC.

To conclude, the evidence from experimental data appears to support that microbial invasion is an important factor in pathogenesis of NEC. Henceforth, alteration of microbial flora following enteral feeding of probiotics may be beneficial especially in pre-term VLBW infants. In a study by Li et al. [18], it was proved that probiotics are an important independent protective factor for NEC.

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