Probiotic Lactococcus lactis decreases incidence and severity of necrotizing enterocolitis in a preterm animal model

Abbreviations:

1 Introduction

Necrotizing enterocolitis (NEC) persists as the most common and serious gastrointestinal disorder among premature infants. Pathogenesis of this disease process is still not clearly understood, but is thought to be multifactorial [1]. Colonization of the intestine with bacteria from early enteral feeds combined with the inflammatory response of the immune system in the premature infant have been implicated in its development [2]. High mortality rates continue in very low birth weight infants with this disease process; therefore prevention of NEC, rather than treatment, is the ultimate goal [3, 4].

Cronobacter sakazakii (C. sakazakii) has emerged as an opportunistic pathogen implicated in the development of NEC [5, 6]. Since the immature gut is thought to play a key role in the development of NEC, prophylactic probiotics have recently been proposed as a prevention strategy [7–10]. However, no consensus exists regarding which probiotic would be safest and most efficacious in the neonatal population. Lactococcus lactis (L. lactis), a lactic acid producing bacteria commonly found in buttermilk and cheese products, has several unique properties that make it an ideal probiotic for neonates. We evaluated if the probiotic L. lactis prevents development of NEC in the presence of bacterial contamination with C. sakazakii in a preterm rabbit model.

2 Materials and methods

New Zealand white rabbit pups were used in our model based on similar gastrointestinal system properties to that of human neonates. After Institutional Animal Care and Use Committee approval (protocol #3631), two-day preterm pups were delivered via cesarean section and randomly assigned to three diets: a control diet with no additives, a diet seeded with C. sakazakii, and a diet seeded with C. sakazakii and fortified with a live probiotic. The control diet was a commercially available Milk Replacer (Pet AG, Hampshire, IL). For the probiotic diet, Lactococcus lactis subsp. Lactis was obtained in powder form from ATCC (American Type Culture Collection, Manassas, VA). To prepare cultures, 100μL of a 1×10⁹ colony-forming units (CFU)/mL saline suspension of L. lactis was inserted into 40 mL of Trypticase Soy Broth. The mixture was incubated overnight at a temperature of 37ºC and 4% CO₂, centrifuged and the remaining pellet washed in phosphate buffered saline. The pellet was re-suspended in 40 mL of control formula. This technique provided an L. lactis concentration of 2.5×10⁷ cfu/mL diet, which was confirmed by quantitative culture analysis.

Twice daily, pups were administered orogastric gavage bolus feeds of 60 kcal/kg via a 3.5 French umbilical artery catheter, for a total caloric intake of 120 kcal/kg/day. Gram negative bacterial challenge was accomplished by adding C. sakazakii to diet preparations prior to feeding to imitate the bacterial contamination of enterally administered infant formula through feeding tubes, which has been found to occur in neonatal intensive care units [11]. A concentration of 1×10⁸ CFU/mL for the gram-negative bacterial solution was achieved and confirmed using a spectrophotometer. After serial dilutions, the diet was seeded with a final concentration of 1×10⁵ CFU/mL of C. sakazakii. Purity plates and analytical profile index identification of the bacteria (bioMerieux Vitek, Hazelwood, Mo) were used for quality assurance throughout the study.

Oral Ranitidine 20 mg/kg and oral Indomethacin 0.5 mg/kg were administered during morning feeds to reproduce clinical conditions that occur in the neonatal intensive care unit. As a proxy for intestinal dysmotility, anal blockage was performed using Vetbond 3M tissue adhesive; 2 ul of the compound was placed over the anal opening via pipette, and pressure was applied to achieve a seal. Animals were evaluated four times daily to assure blockage remained intact.

Subjects were sacrificed on day four via isoflurane chamber. Intestines were immediately excised, and then underwent 10% formalin perfusion for 48 hours. Tissue samples of distal ileum and proximal colon were extracted and prepared for histological exam using paraffin. Hematoxylin and Eosin staining (Richard-Allan Scientific, Kalamazoo, MI) of 4.0 micron sections were performed. NEC was graded between 0 and 4 based on the pathologic findings (Table 1), and was determined by a pediatric pathologist blinded to group assignments. Outcomes of C. sakazakii versus C. sakazakii + L. lactis groups were compared using Fisher’s exact test with p < 0.05 considered significant.

All pups in the control group survived to sacrifice and none developed NEC (Table 2). Survival inC. sakazakii + L. lactis group was 26% higher compared to C. sakazakii group, and incidence of NEC in C. sakazakii + L. lactis group was 51% lower compared to C. sakazakii group. Of the pups that developed NEC, all pups in the C. sakazakii + L. lactis group had Grade 1 NEC, while one-third of pups in the C. sakazakii group developed Grades 2–4 NEC.

NEC is primarily a disease of prematurity. With advancement in neonatal care leading to improved survival for premature and very low birth weight infants, it is not surprising that several recent studies have reported increased incidence of NEC over time [12, 13]. Unfortunately, this disease continues to have high mortality rates, especially for infants requiring surgical management [4].

Changes in methods of delivery and feeding can alter the normal intestinal flora of infants. Infants are first exposed to bacteria from maternal vaginal and fecal matter when they come in contact with it in the vaginal canal, but increasing rates of cesarean sections in the United States are impeding this process [14, 15]. Maternal microogranisms and other important factors are transferred to infants in breast milk [14]. However, this process is blocked when sterile formula or pasteurized donor breast milk are used for enteral feeds instead. Additionally, extensive antibiotic use in the neonatal intensive care unit can affect infants’ gut flora, specifically leading to a decrease in diversity of intestinal microbiota [16].

In our model, we attempted to mimic normal conditions in the neonatal intensive care unit as closely as possible. Several of these practices have been shown to play a role in development of NEC. Infants in the neonatal intensive care unit are commonly fed using feeding tubes instead of being allowed to feed by mouth. These feeding tubes are known reservoirs for bacterial contamination, yet open gravity feeding systems continue to be employed [11]. Additionally, the acidic environment of the stomach has been shown to be protective against bacterial colonization of the gastrointestinal tract and bacterial translocation [17, 18]. H2 blockers such as Ranitidine, which are routinely used in hospitalized neonates, eliminate this protective barrier. Indomethacin is a prostaglandin inhibitor used to medically treat infants with patent ductus arteriosus. This drug has been shown to increase rates of NEC, likely due to suppression of production of intestinal mucus, which is a protective barrier against bacterial adhesion, and via vasoconstrictive effects that inhibit intestinal blood supply [19]. While the pathogenesis of NEC is multifactorial, intestinal dysmotility of the premature infant is thought to be an important component of this disease process [1]. Therefore, anal blockage was used in our model as a surrogate for intestinal dysfunction and dysmotility.

Probiotics are live organisms that provide a health benefit through several different mechanisms that target immune function and the intestinal microflora [20]. They are used in adults for a variety of gastrointestinal disorders, including inflammatory bowel disease, irritable bowel syndrome, and Clostridium difficile colitis [21]. While use in neonates is beginning to be discussed as a prevention strategy against NEC, there is no consensus regarding which type of probiotic will be superior in safety and efficacy amongst neonates. Since probiotics are live organisms, introduction into the immature neonatal gut can provide a source of infection leading to bacteremia and sepsis [22, 23]. Therefore, the choice of probiotic should be one that is effective in prevention of NEC, but is also not a source for other pathogenic infections in the neonate. Unlike other lactic acid producing bacteria such as Lactobacillus acidophilus, L. lactis produces the L(+) isomer, but lacks the ability to produce the D(–) isomer, which is incompletely metabolized and can cause lactic acidosis [24]. Additionally, two antimicrobial substances produced by L. lactis (nisin and lactocin) protect againstgram-positive bacteria [25, 26]. Therefore, L. lactis is a safe choice for use as a probiotic in neonates.

As C. sakazakii has been discovered as a contaminant in powdered infant formula and has been shown to cause NEC, prevention strategies are needed to combat this pathogen [6, 27]. Our study demonstrated that C. sakazakii led to the development of NEC in 62% of preterm rabbit pups that survived greater than 48 hours. Utilization of the probiotic L. lactis increased survival and decreased incidence of NEC significantly. Grading of NEC showed that one-third of pups who did not receive the probiotic L. lactis and developed NEC had Grades 2–4 NEC, while all pups treated with L. lactis that developed NEC were limited to Grade 1. Therefore, in the presence ofC. sakazakii, L. lactis appears to be protective against development of NEC.

In regards to the study design, the 2-day premature pups were used because they are similar to 24–26-week premature humans in terms of lung development. The 3-day premature pups could not survive due to immaturity of lung development. Pups were sacrificed on day 4 of life because previous studies in our lab established that the intestinal blockage used in our model is the limiting factor for duration of survival. Pups subjected to C. sakazakii and intestinal blockage demonstrated significant drops in survival after 4 days of life [1]. C. sakazakii, previously named Enterobacter sakazakii, was chosen for this study because it has become one of the pathogens commonly associated with the development of NEC in humans [5, 28]. There was asymmetry in the sizes of the groups, which was a planned imbalance. In previous studies in our lab, the CS groups experienced high mortality due to intestinal blockage and bacterial overgrowth. We adjusted our group sizing to account for this.

Routine use of probiotics in neonates may help reduce the incidence and severity of NEC. As L. lactis is a largely non-pathogenic probiotic commonly found in buttermilk and has been proven to decrease rates of NEC in an animal model, its prophylactic use in neonates would be a logical choice. Future studies conducted in the neonatal population would be beneficial to determine its true effect on prevention of development of NEC.

5 Conclusions

In the presence of C. sakazakii, L. lactis appears to be protective against development of NEC in a preterm rabbit model. Future studies are needed that evaluate utilization of prophylactic probiotics in the neonatal intensive care unit to determine if this intervention can successfully decrease rates of NEC in preterm infants.

Disclosures/funding

The authors have nothing to disclose. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Animal research statement

This study was conducted in accordance with guidelines put forth by our Institutional Animal Care and Use Committee.