Introduction

Necrotizing enterocolitis (NEC) remains a major cause of mortality and morbidity in very preterm (VP) infants [1, 2]. Although the etiopathogenesis is likely multifactorial, inappropriate initial microbial colonization in preterm infants appears to be the most important risk factor for NEC [3]. The immature gut with altered host defense may stimulate an excessive inflammatory response to this dysbiosis, eventually triggering cell injury [4]. The intestinal flora of patients with NEC has a predominance of proteobacteria and significant reduction in diversity of species [5]. Pneumatosis, the pathognomonic sign of NEC is due to abnormal bacterial fermentation.

Probiotics are live microorganisms, when administered in adequate amounts, confer a health benefit on the host [6]. Probiotics protect the immature gut by promoting more diverse intestinal flora, which competitively exclude certain pathogenic bacteria [7]. Therefore, optimizing the preterm gut microbiome with probiotics is a favorable strategy to prevent NEC [8]. A recent systematic review of randomized controlled trials (RCTs) for preventing NEC in preterm infants showed decreased risk of NEC by probiotics with moderate certainty of evidence [9].

Multiple previous meta-analyses have reported that enteral supplementation with probiotics compared to no supplementation or placebo, reduces the risk of NEC, sepsis and mortality [10, 11]. Although numerous trials published and a large number of patients studied, the heterogeneity in study methods and uncertainty regarding efficacy of different probiotic preparations make it difficult to generalize the results. Moreover, given the declining incidence of NEC, it may be challenging to conduct large RCTs adequately powered to detect clinically important reductions [12]. Currently in the United States, the routine use of probiotics in the neonatal intensive care units (NICUs) is limited [13, 14].

The objective of this pre- and post-implementation cohort study was to evaluate the effects of routine prophylactic supplementation of a multi-strain NICU-specific probiotic product in infants born very preterm (<32 weeks of gestation at birth) or very low birth weight (<1500 grams, VLBW) in our practice located in North Texas. We hypothesized that a guideline-driven supplementation of probiotics would be safe, feasible and associated with a decrease in incidence of NEC from the baseline of 6.3% (one year prior to implementation) to 3.3% (one year after implementation) based on estimates derived using data from meta-analysis by Sawh et al. [10, 15].

Methods

Patients and setting

Our neonatology practice cares for approximately 150 infants born VP or VLBW every year. Prior to implementation, probiotics were not utilized for any reason. A guideline was prepared to prophylactically supplement infants born VP or VLBW with Similac® Probiotic Triblend (Abbott, Abbott Park, Illinois; manufactured by Chr. Hansen, HØrsholm, Denmark) from July 1, 2020 in a level IV NICU and from October 1, 2020 in a level III NICU.

One single-use packet (0.5 g powder) of Similac® Probiotic Triblend has a combination of one billion colony forming units (CFU) of Bifidobacterium lactis (BB-12®), Bifidobacterium infantis (BB-02™), and Streptococcus thermophilis (TH-4®).

Guideline

The probiotic packet was mixed in 3 ml of sterile water in a dedicated place by the nurse. The first dose of probiotic was given within 48 h of birth, after first feed of mother’s colostrum (or donor breast milk if colostrum was not available). It was administered through the gavage tube replacing a feed equivalent to the feeding volume if the infant was receiving <3 ml of feed. If the feed was ≥3 ml, the 3 ml probiotic was given in addition to the feed. The probiotic was followed by a flush of 0.5 ml of sterile water. Subsequently, the probiotic was dosed once a day until the infant reached 35 weeks post menstrual age (PMA) or discharge (whichever was earlier). Contraindications to the administration of probiotics included any breach in the gut integrity from conditions such as spontaneous intestinal perforation (SIP), NEC Bell’s stage ≥2.

Study design

This prospective cohort study with a retrospective cohort comparison was conducted after approval by the local institutional review board. Exclusion criteria included death or discharge prior to the first dose of probiotics, death within 48 h after birth or complex congenital or chromosomal anomalies. The prospective and retrospective data were extracted from maternal and neonatal electronic medical records. The prospective study period was 1-year post-implementation, from July 1, 2020 to June 30, 2021 for the level IV NICU and October 1, 2020 to September 30, 2021 for the level III NICU. The study period for the retrospective cohort was also 1-year pre-implementation, from July 1, 2019 to June 30, 2020 for the level IV NICU and October 1, 2019 to September 30, 2020 for the level III NICU.

Data collection

Collected data included maternal demographics, obstetric complications and other labor and delivery variables. Neonatal data such as gestational age, birth weight, gender, Apgar scores, incidence of small for gestational age (SGA, birth weight <10th percentile) and hypothermia (first temperature in the NICU < 96.8°F) were recorded. Nutrition data included the age of first feed, age when probiotics were started, days on parenteral nutrition, central line days and days to full feeds (140 mL/kg/day). Growth velocity was calculated from daily weights by an exponential model and expressed as g/kg/day [16]. Incidence of cholestasis (direct bilirubin >2 mg/dl), postnatal growth failure (discharge weight <10th percentile) and change in z-score between birth and discharge weights were noted.

Outcomes

The primary outcome of interest was NEC. The other outcomes of interest were death and late-onset sepsis (LOS). NEC was described based on clinical and radiographic criteria [17]. Infants must have at least one of the following clinical signs: bilious gastric aspirate or emesis, abdominal distension or occult/gross blood in stool (no fissure) and at least one of the following radiographic findings: pneumatosis intestinalis, portal venous gas, or pneumoperitoneum. NEC needing surgery (surgical NEC) was specifically differentiated from SIP. SIP was defined as focal gastrointestinal perforation diagnosed by visual inspection at the time of surgery or postmortem exam with remainder of the bowel normal [17]. LOS was diagnosed when pathogen was isolated from blood or cerebrospinal fluid >72 h after birth, or a postmortem culture of organ tissue grew a pathogen with concomitant histology of infection. In addition, the number of LOS evaluations >72 h after birth (at least a blood culture obtained plus initiation of antibiotics) were recorded. Other major neonatal outcomes included bronchopulmonary dysplasia, retinopathy of prematurity, and intraventricular hemorrhage. Diagnosis of bronchopulmonary dysplasia was made at 36 weeks post menstrual age if there was any oxygen requirement. In addition, if the infant was on continuous positive airway pressure or ventilator at 36 weeks post menstrual age, it was labeled as severe bronchopulmonary dysplasia [18]. Retinopathy of prematurity needing any treatment was also documented [19]. Intraventricular hemorrhage was graded 1–4 based on the criteria developed by Papile et al. who defined grades 3 and 4 as severe intraventricular hemorrhage [20].

Statistical analyses

In this cohort study a study time period of one year each was chosen for pre- and post-implementation with a goal of 125 VP or VLBW infants in each group. Demographic and outcome variables were compared between the probiotics and no probiotics groups utilizing a 2-tailed Student’s t test or Mann-Whitney U test for continuous variables and Pearson chi-square test or Fisher’s exact test for categorical variables. Logistic regression was performed on primary outcome and other outcomes of interest after adjusting for variables such as gestational age, birth weight, gender, SGA and delayed cord clamping (DCC). These results were reported as odds ratios (ORs) with 95% confidence intervals (CIs). A subgroup analysis was also performed on infants born extremely low birth weight (<1000 grams, ELBW). IBM SPSS version 28 (Armonk, NY) was used to analyze data. A probability value <0.05 was considered to be the threshold of statistical significance.

Results

During the prospective study period, out of 131 VP and/or VLBW infants admitted to the NICU, 6 were excluded (3 died before probiotics were started or within 48 h after birth and 3 were born with complex congenital anomalies). The remaining 125 VP and/or VLBW infants who received probiotics (Probiotics group) were compared to a retrospective cohort of 126 infants who survived beyond 48 h and qualified to receive probiotics, but did not as they were born prior to implementation of routine supplementation (No probiotics group).

No significant differences emerged for any maternal characteristics (Table 1). The median gestational age at birth was 29 weeks and median birth weight was 1200 grams in both the groups. Although there was no significant difference in the incidence of LOS, the median evaluations for suspected LOS were significantly lower in the Probiotics group compared to No probiotics group. The incidence of NEC decreased from 6.3% in the No probiotics group to 1.6% in the Probiotics group. The other characteristics and outcomes, including the incidence of death were similar between both groups (Table 2). The first feed was given significantly earlier after birth in the Probiotics group compared to No probiotics group (15 vs. 17 h of life). The median age of starting probiotics was 33 h of life and were given until 35 weeks PMA. The median number of probiotic doses given were 35. The growth velocity was significantly higher in the Probiotics group compared to No probiotics group (14 vs. 13 g/kg/day). Similarly, median change in z-score between the birth and discharge weights decreased significantly in the Probiotics group compared to No probiotics group (−0.57 vs. −0.80) (Table 3).

Table 1 Maternal characteristics.
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Table 2 Neonatal characteristics and outcomes.
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Table 3 Nutrition data.
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Multiple logistic regression after adjusting for birth weight, gestational age, gender, SGA and DCC revealed no differences in the primary outcome or other outcomes of interest (NEC, death, NEC or death and LOS) for VP or VLBW infants (Table 4). In the subgroup analysis for only ELBW infants, there were no cases of NEC in the Probiotics group compared to the incidence of 14.3% in the No probiotics group. There were no significant differences in the other outcomes of interest between both the groups (Table 5).

Table 4 Outcomes of interest for infants born very preterm or very low birth weight.
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Table 5 Outcomes of interest for infants born extremely low birth weight.
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Discussion

Recent meta-analyses of randomized and non-randomized studies, including 56 RCTs and 30 high quality observational trials respectively comparing probiotics to placebo or no probiotics showed a reduction in NEC with consistent effect size [11, 21]. However, this evidence remains a low to moderate certainty due to heterogeneity in trial designs, small sample sizes in the individual trials, and usage of different probiotic preparations. The RCTs addressing NEC are also confounded with variations in the definition and creating cohorts confounded by SIP [22]. This is quoted as one of the reasons for uncertainty regarding the benefits of probiotics in reducing NEC. Thus, it is very important to improve criteria to define NEC in a consistent way for quality improvement and research purposes [23, 24]. However, it may be challenging to conduct large adequately powered trials to detect significant reduction given the declining incidence of NEC [12].

According to a phone survey performed in 2015, only 8.8% of the participating NICUs in the US were using probiotics routinely for the VLBW infants. When explored, there was no evidence for the safety or efficacy for most of the probiotic products used at that time [13]. Only one product was reported to have protective effect against NEC [25]. This probiotic combination of B. infantis, S. thermophilus and B. lactis, registered with the German Collection of Microorganisms and Cell Cultures as BB-02 96579, Th-4 15957, BB-12 15954 respectively (ABC Dophilus Probiotic Powder for infants, Solgar, Leonia, New Jersey) has been tested in a well-designed RCT (ProPrems study) where 1099 very preterm infants from Australia and New Zealand were randomized to either probiotics or placebo. Although no significant reduction was noted in LOS or mortality, this probiotic combination reduced NEC Bell’s stage ≥2 with a number needed to treat of 43 [26]. It appeared to be safe and did not adversely affect neurodevelopment or behavior in early childhood [27]. Therefore, this combination is conditionally recommended by the panel of experts representing European Society for Pediatric Gastroenterology Hepatology and Nutrition Committee to reduce NEC Bell’s stage ≥2 if all safety conditions are met [28].

A systematic review and network meta-analysis of preterm infants which included 63 trials has shown with high-certainty evidence that compared to placebo or single-strain or other multi-strain probiotic formulations, combinations of one or more Lactobacillus species and one or more Bifidobacterium species are superior for the reduction of mortality and NEC Bell’s stage ≥2 with no difference in the incidence of harm [29]. Another network meta-analysis of preterm infants reported that only a minority of studied strains or combinations had efficacy in reducing mortality or morbidities [30]. The published systematic reviews do recognize that there is not enough information at the strain level in the included trials. Moreover, the validity and viability of the live microbes is rarely reported. In the current study, routine supplementation with multi-strain probiotic blend consisting of Bifidobacterium lactis (BB-12®), Bifidobacterium infantis (BB-02™) and Streptococcus thermophilis (TH-4®) to the infants born VP or VLBW for one year was associated with the reduction of NEC from the baseline of 6.3% to 1.6%. This was better than the aimed risk reduction of 47% (6.3 to 3.3%) based on the published estimates derived from meta-analysis by Sawh et al. (RR of NEC 0.53, 95% CI 0.42–0.66) [10, 15]. Although this reduction of 75% in the current study was not statistically significant, it is clinically relevant. Nonsignificant reductions were also noted with the incidence of LOS decreasing from 10.3% to 5.3% and mortality from 9.5% to 6.4% after introduction of routine probiotics supplementation. For the Similac® Probiotic Triblend utilized in the current study, the manufacturer Chr. Hansen discloses that all three strains were isolated from either their collection of dairy cultures or in-licensed by trusted partners. They underwent genomic sequencing which provide a comprehensive insight of the characteristics and functionality of the strains [31]. These strains are reported to carry no transferable antibiotic-resistant genes. Deoxyribonucleic acid fingerprinting is performed to confirm their validity [32].

Presently, the practice guidelines from major societies affirm the insufficient evidence regarding safety and efficacy of probiotics in preventing NEC in ELBW infants [33, 34]. In a large meta-analysis, only 7 out of 56 RCTs included ELBW infants that showed no significant reduction in NEC, hodeath or LOS with probiotics [11]. However, the meta-analysis of observational trials showed a reduction in NEC Bell’s stage ≥2 with probiotics in a subset of ELBW infants [21]. A cohort study utilizing a multicenter clinical database showed that even though the use increased over a period of two decades (1997–2016), only 4.6% of infants born between gestational age 23–29 weeks were treated with probiotics. In that study, probiotic use was associated with decreased odds of NEC and death [14]. In the current study, benefit was noted in the subset of ELBW infants with reduction in the incidence of NEC from 14.3% prior to 0% after routine implementation of probiotics. However, these results need to be interpreted with caution due to small sample-size.

The action of probiotics at the level of gut epithelium includes upregulation of cellular immunity and cytoprotective genes, enhanced barrier function, downregulation of pro-inflammatory pathways, prevention of apoptosis and competitive exclusion of pathogenic bacteria [7]. The exact mode of action is difficult to assess as it may be strain-dependent [35]. Preterm infants with suboptimal gut epithelial cell barrier and immune function are predisposed to pathogenic bacteria [36]. This in turn can trigger an inflammatory response including toll-like receptor (TLR) signaling. Expression of TLR4 may be critical in the pathogenesis of NEC [37]. Probiotics, by promoting normal commensal gut flora early in life, confers a protective effect against conditions associated with dysbiosis such as NEC and LOS [38]. These microbiota may also interact with immune system to aid in the establishment of normal digestive capacity, gut motility, ability to harvest nutrients and tolerance to food [39]. A RCT reported no effect on postnatal growth in preterm infants receiving Bifidobacterium supplements [40]. In the current study, there was a significant improvement in the growth velocity and significant decrease in the change of z-score between the birth and discharge weight in the group that received probiotics compared to the group that did not. In general, the effect on growth may vary depending on species and strains of microorganisms utilized [41].

Contamination of dietary supplement-grade probiotics products has been noted previously. Fatal gastrointestinal mucormycosis was reported in a preterm infant who received contaminated ABC Dophilus Powder [42]. Therefore, it is pivotal to choose products with elevated safety and quality standards and preferably single-use packets or vials. A recently published systematic review reported that probiotic sepsis is an uncommon event in preterm infants [43]. However, it is crucial that the departments of microbiology and pathology in the hospital are involved for regular surveillance. In the current study, there were no cases of sepsis or adverse events associated with probiotics. Probiotics administration was paused during the events when there was suspicion of compromise in infant’s gut integrity. In such instances like SIP or NEC Bell’s stage ≥2, there is a possibility of gut bacteria seeping into the blood stream. Probiotics were resumed when feeds were restarted. Routine anaerobic cultures were not performed when LOS was suspected. In general, the conventional biochemical methods utilized in the aerobic blood cultures inoculated for 5 days may help to identify the initial growth. If probiotic bacteremia is detected, genomic analysis can help in confirming the strains. This bacteremia is usually susceptible to the regular late-onset sepsis empiric antibiotics.

For the units choosing to introduce routine probiotics into practice, multidisciplinary approach with involvement of key stakeholders, consistent guideline, and methods to monitor outcomes are recommended. It is strongly suggested that other important NEC reduction strategies are in place such as optimization of human milk, standard feeding protocol, antibiotic stewardship and DCC. These initiatives have proven to help reduce NEC and may act in synergism with probiotics [15]. Administering human milk rich in oligosaccharides may improve colonization of the gut with the administered probiotic [44]. In the current study, 100% of the VP or VLBW infants were started on human milk (mother’s milk or donor breast milk) early (within 24 h of life). Approximately 50% infants were discharged on mother’s milk. A recently published meta-analysis suggests that combination of prebiotic and probiotic may have greater treatment efficacy than probiotic alone [45].

As a prospective cohort study with a historic cohort comparison, this study has several limitations. This study reports observational data one-year pre- and one-year post-implementation of routine probiotics supplementation. The enrollment was not based on sample-size calculation (to achieve statistical significance for a reduction of NEC from 6.3% to 3.3%, a sample-size of 1592 infants is needed). Moreover, a RCT may not have been feasible due to possible cross-colonization in the group not receiving probiotics. As a cohort study, the conclusions from this trial may be limited to associations. Also, there is a risk of confounding the results of the study by other practice changes. However, the other NEC reduction practices have been in place in our practice for many years. We did adjust for gestational age, birth weight, gender, SGA and DCC as these variables may influence the outcomes of interest. The radiologists reading the X-rays were not masked formally, but they were not aware of implementation of routine probiotics supplementation. A risk of industry bias cannot be ignored, even though this study did not receive any sponsorship and financial support. Despite these limitations, we feel it is important to share our observations, which are more likely to reflect the real-world clinical practice. In the current study, NEC was consistently defined based on clinical and radiographic criteria in both retrospective and prospective cohorts. In addition, we carefully differentiated SIP and NEC to avoid contamination of data. No other probiotics were used in the unit before or concurrently other than the specific implemented product. As there is limited data from the United States, our experience with a multi-strain probiotic product specifically available for NICU patients may add valuable information to the body of evidence on probiotics supplementation in premature infants. Although meta-analyses suggest benefit with the routine use of probiotics, they offer limited guidance in terms of which probiotics should be used, their dosage, which age group they provide greatest benefit for, and how long they should be given. We believe this observational study provides guidance and addresses some of the concerns that are impeding the widespread practice of routine probiotic supplementation in preterm infants.

In conclusion, routine prophylactic supplementation of a NICU-specific multi-strain probiotic in infants born VP or VLBW was associated with reduction of NEC that is consistent with the literature-reported effect size with no adverse effects such as probiotic sepsis. This study adds to the evidence supporting the routine supplementation of probiotics in infants born VP or VLBW. Further studies need to focus on the optimal length of therapy, additional NICU populations that may benefit, and other effective probiotic and symbiotic combinations.