Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Gastroenterology
Efficacy of Different Probiotic Combinations on Death and Necrotizing Enterocolitis in a Premature Rat Model
Wu, Shu-Fen†; Chiu, Hsiao-Yu*; Chen, An-Chyi†; Lin, Hsiang-Yu*; Lin, Hung-Chih‡; Caplan, Michael§
*Department of Pediatrics, China Medical University Hospital
†School of Medicine, China Medical University
‡School of Chinese Medicine, China Medical University, Taichung, Taiwan
§Pritzker School of Medicine, University of Chicago, Chicago, IL.
Address correspondence and reprint requests to Hung-Chih Lin, MD, Department of Pediatrics, China Medical University Hospital, No. 2 Yuh-Der Road, Taichung 404, Taiwan (e-mail: email@example.com).
Received 10 May, 2012
Accepted 16 January, 2013
Drs Wu and Chiu participated equally in this study.
This study was supported by the National Science Council of Taiwan (Grant NSC 95–2314-B-039–034) and China Medical University Hospital (grant DMR96–100).
The authors report no conflicts of interest.
Objective: The aim of the present study was to investigate the most effective probiotic combinations to prevent death and necrotizing enterocolitis (NEC) in a premature rat model.
Methods: One hundred fifty-eight premature Sprague-Dawley premature rats were enrolled. Probiotic strains Bifidobacterium bifidum, B longum, Lactobacillus acidophilus, L plantarum, and B breve were fed as a single strain or mixture with 2 or 3 strains for a total of 9 study groups; control groups received no exogenous probiotic supplement. Fecal samples were collected for 72 hours to detect probiotic strains and pathologic strains by real-time polymerase chain reaction. Colony counts of probiotic strains Escherichia coli and Klebsiella were compared between groups before and after 36 hours of the study period. The incidence of death and NEC were compared via Fisher exact test between groups.
Results: The results demonstrated that L plantarum alone (P = 0.0026) and B bifidum with B longum together (P = 0.0017) were more effective in reducing NEC as compared with the control group. All of the study groups except B breve and B bifidum with B breve definitely prevented death compared with controls. B bifidum and B longum together had significantly lower mortality than the control group (P < 0.0001). Colony counts of E coli and Klebsiella in stool samples were significantly decreased in the B bifidum, B longum, and L plantarum group compared with the other study and control groups after 36 hours.
Conclusions: Administration of a mixture of probiotic strains with B bifidum and B longum was most effective in preventing death and NEC in this animal model, and these observations provide an evidence-based strategy for designing further neonatal clinical trials.
See “Probiotics for the Prevention of Necrotizing Enterocolitis: Not Just Which Ones But Also Why?” by Martin on page 3.
Necrotizing enterocolitis (NEC) is the most commonly acquired catastrophic gastrointestinal disease primarily affecting preterm very-low-birth-weight (VLBW; birth weight <1500 g) infants. The incidence of NEC varies from country to country, reportedly affecting 2.6% to 28.0% of preterm VLBW infants (1), with mortality of 16% to 42% (2). Given that NEC frequently progresses from early signs of intestinal inflammation to extensive necrosis within a matter of hours, it is desirable to determine strategies to prevent NEC rather than relying on treatment strategies alone. Eighteen clinical trials and 5 meta-analyses (3–7) found oral probiotics effective in preventing NEC and death; however, these clinical trials used a variety of different strains and mixtures, and therefore the optimal strain or combination for premature infants remains unknown. Studies with adequate sample size are vital to interpret this expanding literature, and we earlier performed a meta-analysis comparing single or mixed probiotic strains for efficacy in reducing death and NEC in preterm VLBW infants (7a). Results showed that a mixture of strains was more effective in reducing death and NEC; this was compatible with another meta-analysis by Guthmann et al (8). Yet these retrospective analyses could not directly identify any mixture as most effective and cited the need for further clinical trials. Nonetheless, clinical trials take time and resources, whereas an animal model is a fast and effective way to investigate this issue.
Probiotic strains such as Bifidobacterium bifidus, B breve, B longum, Lactobacillus acidophilus, and L plantarum have been used to prevent NEC in animal models (9–11) and in clinical trials as described by a meta-analysis (3). The aforementioned strains are often found in intestinal tracts of breast-fed preterm infants (12–14). A recent original study showed NEC linked with a lack of microbiota diversity (15); a review article suggested mixtures of probiotics as more beneficial than single strains on gut and immune function (16).
We thus hypothesized that a mixture of these probiotic strains would increase microbiota diversity and thus be most effective in reducing death and NEC. Our animal model investigated single and/or mixed probiotic strains to find the most effective probiotic combination in preventing death and NEC.
Time-dated pregnant Sprague-Dawley rats were anesthetized with CO2 (30–45 seconds) on the 21st day of gestation (2–3 days before expected day), and pups were delivered via cesarean section. Premature rats were collected and placed in a neonatal incubator for humidity and temperature control, then fed orogastrically 0.1 mL every 3 hours (Esbilac formula; 200 kcal · kg−1 · day−1; Pet-Ag, New Hampshire, IL) using a siliconized catheter (24 gauge; Gesco International, San Antonio, TX), with volume advanced as tolerated up to 0.4 mL by 72 hours of life. At 30 minutes of life (30 minutes before asphyxia exposure), all of the rats were challenged with 108 colony-forming units (CFUs) per animal of Escherichia coli and Klebsiella via an orogastric tube. Additional inocula of 108 CFU organisms per rat daily were given to all groups at 8 AM before asphyxia exposure daily during the study period. Rats were stressed with asphyxia twice daily by breathing 100% nitrogen gas for 50 seconds, then exposed to 4°C for 10 minutes.
Using this protocol, 70% to 80% of rats manifested clinical and pathological signs similar to neonatal NEC by the third day of life, according to a previous animal model (17). We used the same protocol several times with 10 research assistants to ascertain whether the success rate of NEC was as previously reported before conducting our study with the same team members. Nine study groups were treated with probiotics (108 CFU organisms per animal daily); 3 control groups (n = 12, 13, and 13) received no exogenous probiotic supplement. Duplicate controls validated similar conditions across experiments. Study groups were fed with diverse probiotics: group 1, B bifidum (PM-A0218, ProMD; n = 13); group 2, B longum (PM-A0101, ProMD; n = 16); group 3, L acidophilus (BCCM8151; n = 14); group 4, L plantarum (PM-A0087, ProMD; n = 16); group 5, B breve (ATCC15700; n = 11); group 6, B bifidum and B longum (n = 12); group 7, B bifidum and B breve (n = 12); group 8, B bifidum, B longum, and L acidophilus (n = 14); group 9, B bifidum, B longum, and L plantarum (n = 12). Because of the large sample size, we performed the same protocol 4 times with 3 pregnant rats delivered each time for a total of 12 pregnant rats. Active pups distributed among 12 groups were randomly cared for by 5 experienced research assistants. To examine the composition of the microbial community in the stool sample, fecal samples were collected for 72 hours to detect probiotic strains and pathologic strains such as E coli and Klebsiella by real-time polymerase chain reaction (PCR) in the study and control groups. Colony counts of probiotic strains E coli and Klebsiella were compared between groups before and after 36 hours of study period.
Species-Specific Quantitative Real-Time PCR
The primers and probes for the assays were based on sequences of the 16S-23S intergenic spacer regions of the different Bifidobacterium species and Lactobacillus species according to previous studies (18,19). In addition, the primers and probes for E coli were based on 16S rRNA gene sequences, whereas those for K pneumoniae were based on the phoE gene sequence. Species-specific sequences were used to design primers and probes for B bifidum, B longum, L plantarum, E coli(20), and K pneumoniae(21). All bifidobacteria and Lactobacillus strains were also detected using previously designed primers and probes (18,19).
A research assistant observed and recorded every 3 hours whether rats developed cyanosis, abdominal distention, respiratory distress, lethargy, or death. All of the animals surviving the entire 72-hour study protocol were euthanized via decapitation; the protocol was reviewed and approved by the China Medical University Animal Experimental Center and Use Committee (protocol no. 95–42-N).
Tissue Harvest and NEC Evaluation
After natural death or artificial termination, the gastrointestinal tract was removed and small intestine visually evaluated for typical signs of NEC (intestinal discoloration, intestinal hemorrhage, ileal distention, and/or ileal stenosis). A 2-cm section of distal ileum proximal to the ileocecal valve from each animal was fixed overnight in 70% ethanol, paraffin-embedded, sectioned at 4 to 6 μm, and stained with hematoxylin and eosin for histologic evaluation of NEC. Histologic changes in ileal architecture were scored by a blinded evaluator and graded: 0, normal, no damage; 1, slight submucosal and/or lamina propria separation; 2, moderate separation of submucosa and/or lamina propria and/or edema in submucosal and muscular layers; 3, severe separation of submucosa and/or lamina propria and/or severe edema in submucosa and muscular layers, region villous sloughing; and 4, loss of villi with necrosis (Fig. 1). To assess incidence of NEC, animals with histological scores of ≥2 were scored as having NEC.
Intergroup NEC incidence and mortality rates were compared via Fisher exact test. We applied the Kaplan-Meier method to estimate survival probability and log-rank tests to compare the survival curves between groups. Three control groups were pooled to make comparisons with other groups. To evaluate differences in incidence of NEC, mortality, and survival probability among groups, P = 0.05 was considered significant for all evaluations. The Mann-Whitney U test was used to compare E coli and Klebsiella fecal colony counts between the study and control groups. All of the analyses were performed using SPSS statistical software (SPSS Inc, Chicago, IL). A P value of <0.05 was considered significant.
In total, 158 preterm Sprague-Dawley rats were enrolled. Table 1 shows the incidence of NEC, mortality rate, and survival time in each study group versus controls. Results show all of the study groups except groups 3 (L acidophilus) and 5 (B breve) statistically reduced the incidence of NEC compared with controls. Groups 4 (L plantarum) and 6 (B bifidum and B longum) exhibited the greatest effect in reducing NEC compared with other groups. The mortality rate was lower statistically than that of the control group in all of the study groups except 5 (B breve) and 7 (B bifidum and B breve); however, a mixture of probiotics with 2 (B bifidum and B breve) or 3 strains did not statistically reduce the incidence of death significantly compared with a single strain. As for survival time in all of the probiotic groups except groups 5 (B breve), 7 (B bifidum and B breve), and 8 (B bifidum, B longum, and L acidophilus), animals lived significantly longer than in the control group. Table 2 shows the colony counts of probiotic strains E coli and Klebsiella in stool samples from animals at 72 hours. L acidophilus, L plantarum and B bifidum, and B longum with L plantarum significantly inhibited proliferation of E coli statistically compared with the control group. In addition, B bifidum, B longum, and L plantarum significantly inhibited the proliferation of Klebsiella in stool samples compared with other study and control groups after 36 hours.
This complex study demonstrates that the mixed probiotic strain containing B bifidum and B longum was the most effective in preventing death and NEC in our premature rat model, and that B bifidum, B longum, and L plantarum together significantly decreased the colony counts of E coli and Klebsiella in fecal samples after 36 hours of study.
Bifidobacterium species are important in maintaining general health; they contribute to beneficial microflora in the intestinal tract and provide stability of the human intestinal microflora. Khailova et al (9) reported that B bifidum had a protective effect against NEC injury in a neonatal rat model, such that alterations in the ileum epithelial tight junction and adherent junction structure may be a crucial factor in improvement of intestinal integrity by reducing inflammatory reactions in the ileum. They also found B bifidum downregulated apoptosis in both in vivo and in vitro NEC models by a COX-2–dependent mechanism and reduced mucosal injury (9). B longum can block the growth of harmful bacteria by lowering the pH in the intestinal tract through production of acetic and lactic acids and boosting the immune system (22). B longum purportedly prevents NEC in gnotobiotic quails (23) and in the rat model (11) via inhibition of the growth of clostridial species and the reduced inflammatory response of the intestine. Clinical studies show that probiotic strains including B longum can significantly reduce morbidity because of NEC in VLBW newborns, with suggested possible mechanisms being the alteration of microbial flora following enteral feeding of probiotics, as well as establishment of early full enteral feeding (24). Our study echoed these results: B bifidum alone and B longum alone, or multiple strains including B longum could reduce the NEC incidence in an animal model. Oral administration of B breve reduces butyric acid production, which may protect low-birth-weight infants from NEC (25). Nonetheless, no clinical trial proved this speculation. Our animal study demonstrated B breve was ineffective in the prevention of NEC, and further study would be necessary to delineate the discrepancy between possible human effects and the lack of a clear effect in our neonatal rat model.
Intestinal Lactobacillus strains can inhibit the growth of pathogens by lowering the pH because of production of lactic and acetic acid (26) or by competing for nutrients and epithelial adhesion sites (27). L acidophilus is the predominant Lactobacillus species of the gastrointestinal tract; it inhibits other pathologic bacteria such as K pneumoniae and Candida albicans by adhering to the intestinal wall (28,29). One clinical study showed early gut colonization with dead or living L acidophilus lowered the incidence of NEC, but challenges in study design and inadequate sample size (30) make conclusions highly speculative. Our study did not show that L. acidophilus reduced the incidence of NEC, which seemingly contradicts human studies that show mixtures containing L acidophilus as effective for disease prevention (24,31,32). Differences in these outcomes may be explained by synergistic effects between mixed probiotics in the prevention of NEC. L plantarum can be isolated from the intestinal tract in preterm infants and mediate adherence to human colonic cells (33). In a recent study using neonatal piglets, early administration of a probiotic mixture containing 4 Lactobacillus strains including L plantarum (and 1 Bifidobacterium species) promoted colonization of beneficial commensal microbiota capable of limiting formula-induced mucosal atrophy, dysfunction, and pathogen load, and reduced NEC incidence and severity (10). Our study is the first to highlight a clear protective role for L plantarum alone against NEC and death.
In a recent publication, probiotic supplementation with L paracasei, B animalis, and Streptococcus thermophilus (2.4 × 1010 CFU/day) appeared to increase the incidence of NEC in preterm pigs (34). Intriguingly, this conflicted with their previous study in which probiotics decreased the incidence of NEC in preterm pigs using B animalis and L acidophilus, L casei, L pentosus, and L plantarum (5 × 109 CFU) (10). It may be that dosage >1010 CFU could be a detrimental factor for NEC and death, and this was consistent with our initial studies in the animal model (unpublished). This finding further illustrates specific strains and doses of probiotics as critical determinants in the setting of NEC prevention. Careful planning and interpretation of clinical studies are vital before incorporating specific dosages and specific strains into clinical practice. Our animal model could thus provide key support for considering doses of 108 CFU for further clinical trials.
We observed lower mortality in all of the study groups compared with controls, except for groups 5 (B breve) and 7 (B bifidum and B breve). No other study has examined B breve or B bifidum and B breve in reducing mortality in an animal model or clinical trial. The mechanism whereby probiotics reduce the incidence of mortality is unknown. Research evidence indicates that the intestine of preterm infants appears far more sensitive to stimuli that induce interleukin (IL)-8 production compared with adult intestines (35). Overproduction of IL-8 is a critical component of systemic inflammatory responses in neonates resulting from NEC or sepsis (36,37), and in vitro studies showed that probiotics could inhibit IL-8 production, thereby providing a potential reason why probiotics could reduce death (38).
This study demonstrated that the combination of B bifidum and B longum was more effective than any single or other combined strain in preventing death and NEC, similar to results from clinical trials with multiple strains (24,31,39,40). Nonetheless, our data were surprising that demonstrated that B bifidum, B longum, and L acidophilus or B bifidum, B longum, and L plantarum together were less effective than the 2-strain combination. We observed that using 3 strains of probiotics resulted in a high concentration of milk-induced gastric outlet block with severe gastric distension and resulting aspiration pneumonia, and these premature rats died before development of NEC. We speculate that a mixture of 3 strains of probiotics with 3 × 108 CFU may be too concentrated for the neonatal rat stomach to properly digest and propel downward toward the distal intestinal tract.
There are some limitations to this study that warrant additional consideration. First, the number of animals in each group seems relatively small; however, the results of 4 different experimentation times were consistent without significant differences in the incidence of NEC. The incidence of death and NEC in the control group was 0.8 and that of the treatment group 0.3, using the Fisher exact test with a significance level of 0.05; testing the intergroup proportions with sample size 12 would attain a power with 0.83.
Second, this study was not designed to investigate the mechanism by which probiotics reduce the incidence of NEC and death. Nonetheless, because microbial flora has been shown to contribute to NEC pathogenesis, we analyzed probiotic and pathologic microorganisms in fecal samples in all of the groups, and found that the colony counts of E coli and K pneumonia in the B bifidum–, B longum–, and L plantarum–treated group were significantly lower than other study and control groups by real-time PCR. Although the combination of B bifidum, B longum, and L plantarum seemed to be more effective in reducing the quantity of pathologic flora, this combination was not more effective in reducing death and NEC in this animal model. Although probiotics may influence NEC and death by affecting the proliferation of pathologic flora, other mechanisms such as inhibition of the proinflammatory response, reduction in mucosal permeability, and activation of anti-inflammatory responses by binding to Toll-like receptors may contribute to this discrepancy. Further study on probiotic-specific mechanisms in this animal model may provide additional insight and is clearly warranted.
Third, animal models usually aim to test hypotheses or explore pathomechanisms, and there is no perfect animal model of NEC clearly described. Nonetheless, this neonatal rat model is a reasonable and reproducible approach designed to simulate clinical situations to identify the most effective probiotic strain in the prevention of death and NEC. This study made many comparisons, and it is well known that repeated testing heightens type 1 error rates. False discovery rate control procedures (41) served to correct for comparison between probiotic and control groups. One asterisk indicates comparisons rejecting the null hypothesis after false discovery rate adjustment. The before- and after-adjustment results gave consistent conclusions: the combination of B bifidum and B longum and L. plantarum alone differed greatly from controls in NEC incidence, whereas mortality rates of single strains such as B bifidum, B longum, L acidophilus, L plantarum, and the combination of B bifidum and B longum were statistically different from the control group.
In summary, this study showed that a mixture of B bifidum and B longum and L. plantarum alone prevented NEC compared with controls. All single probiotics except B breve and all combinations except those containing B bifidum and B breve prevented death compared with controls. A mixture of B bifidum and B longum was the most effective in reducing the incidence of death and NEC in the premature rat model. These findings may suggest the most effective probiotic strains to prevent death and NEC for further clinical trials.
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death; necrotizing enterocolitis; premature rat; probiotics
© 2013 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
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