The Incidence of Necrotizing Enterocolitis Is ...

Supplemental Material can be found at: http://jn.nutrition.org/content/suppl/2011/01/20/jn.110.12856 1.DC1.html

The Journal of Nutrition Nutrition and Disease

The Incidence of Necrotizing Enterocolitis Is Increased Following Probiotic Administration to Preterm Pigs1–3 Malene S. Cilieborg,4,5 Thomas Thymann,4 Richard Siggers,4 Mette Boye,5 Stine B. Bering,4 Bent B. Jensen,6 and Per T. Sangild4* 4 Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen DK-1870 Frederiksberg C, Denmark; 5National Veterinary Institute, Technical University of Denmark, DK-1790 Copenhagen, Denmark; and 6Department of Animal Health and Bioscience, Faculty of Agricultural Sciences, University of Aarhus, 8830 Tjele, Denmark

Abstract Preterm birth and necrotizing enterocolitis (NEC) is associated with inappropriate gut colonization and immunity, which intestinal structure, function, microbiology, and immunology in the immediate postnatal period. Just after birth, caesareandelivered preterm pigs were inoculated with Lactobacillus paracasei, Bifidobacteria animalis, and Streptococcus thermophilus (total 2.4 3 1010/d) either as live (ProLive, n = 14) or gamma-irradiated dead bacteria (ProDead, n = 12) and compared with controls (n = 14). All pigs received parenteral nutrition for 2 d followed by enteral formula feeding until tissue collection on d 5. Compared with control pigs, intestinal weight was lower and NEC incidence was higher in both groups given probiotics (64–67 vs. 14%; P,0.01). Hexose absorption, brush border enzyme activities, and gut barrier function were lower in the ProDead group compared with the other groups (P , 0.05), whereas live probiotics induced higher expression of the proinflammatory cytokines IL-1a and IL-6 (P , 0.05). Probiotics minimally affected gut colonization, except that live probiotics induced a higher density of B. animalis and lower bacterial diversity in the distal intestinal mucosa and lower SCFA concentrations in the colon (P , 0.05). The detrimental effects of probiotic bacteria in this study may relate to the specific strain and dose combination and may have involved the very immature gut immune system and low NEC incidence in the control group. It remains to be determined whether similar adverse responses to probiotics occur in preterm infants. J. Nutr. 141: 223–230, 2011.

Introduction Necrotizing enterocolitis (NEC)7 is a multi-factorial gastrointestinal disease in preterm infants associated with high morbidity and mortality. The etiology is not fully understood, but intestinal colonization plays a crucial role, because NEC occurs only when bacteria are present (1). Although bacterial overgrowth is a common observation in NEC patients, it remains unclear how the gut microbiota, or specific bacterial species, predispose to NEC. Probiotic bacteria are thought to suppress colonization of potential pathogens by competition for adhesion sites or substrates, by production of antimicrobial metabolites, or by modulating the host immune defense (2–4). The immu1 Supported by the Danish Strategic Research Council under the program Health, Food, and Welfare and by Chr Hansen A/S. 2 Author disclosures: M. S. Cilieborg, T. Thymann, R. Siggers, M. Boye, S. B. Bering, B. B. Jensen, and P. T. Sangild, no conflicts of interest. 3 Supplemental Table 1 is available with the online posting of this paper at jn. nutrition.org. 7 Abbreviations used: FISH, fluorescence in situ hybridization; NEC, necrotizing enterocolitis; PN, parenteral nutrition; ProDead, treatment group given dead probiotics; ProLive, treatment group given live probiotics; TLR, toll-like receptor; T-RF, terminal restriction fragment. * To whom correspondence should be addressed. E-mail: [email protected].

nomodulatory actions of probiotics may be mediated by host cell receptors for bacterial patterns and antigens, and dead bacteria may thus have similar effects to live bacteria. One Cochrane analysis (5) and several meta-analyses (6–9) concluded that administration of probiotics decreases NEC incidence in preterm infants and studies in rodent and pig NEC models have supported the NEC preventive effects of probiotics (2–4,10,11). In an earlier study with preterm pigs, also published in this journal, a mix of 5 probiotic strains [Lactobacillus acidophilus, L. paracasei, L. pentosus, L. plantarum, and Bifidobacterium animalis (Bb12)] at a total daily dose of 1.3– 2.6 3 1010 CFU did not reduce NEC incidence (69 vs. 89%) but significantly reduced NEC severity and improved digestive function (10). Despite these supportive results, the optimal probiotic treatment for this sensitive population remains unknown, and it is critical to investigate the optimal strain(s), dose, and time of administration before probiotics can be routinely given in clinical practice to preterm infants. The mechanisms of probiotic action in the preterm gut remain uncertain, and each treatment strategy may work differently and thus require independent health and safety investigations.

ã 2011 American Society for Nutrition. Manuscript received July 02, 2010. Initial review completed August 26, 2010. Revision accepted November 26, 2010. First published online December 22, 2010; doi:10.3945/jn.110.128561.

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may be improved by probiotic bacteria. Using a preterm pig model of NEC, we investigated the effects of probiotics on

With the aim to further investigate the NEC-preventive effects of probiotics, we used NEC-sensitive preterm pigs to investigate the effects of a relatively high total daily dose (2.4 3 1010 CFU) of 3 probiotic strains that have previously been used to prevent NEC (12,13) or infant diarrhea (14): L. paracasei, Streptococcus thermophilus, and B. animalis, Bb12. The probiotic mix was given as live or dead bacteria immediately after birth. We hypothesized that administration of these probiotics would improve gut structure (villus heights, mucosal mass, integrity) and function (digestive enzymes, sugar absorption) and reduce pathogen colonization and expression of proinflammatory cytokines and decrease the incidence of NEC.

Materials and Methods

Clinical evaluation and tissue collection. The pigs were checked every 3 h for NEC symptoms (bloody diarrhea, lethargy, respiratory distress, and abdominal distension) until tissue collection. Irrespective of time, pigs that developed clear NEC symptoms were immediately euthanized and tissues were collected. For all pigs, the stomach, 3 regions of the small intestine (proximal, middle, and distal), and the colon were scored for NEC lesions according to our standard macroscopic evaluation system: 1 = normal; 2 = local hyperaemia and edema; 3 = hyperaemia, extensive edema, and local hemorrhage; 4 = extensive hemorrhage; 5 = local necrosis and pneumatosis intestinalis; and 6 = extensive necrosis and pneumatosis intestinalis. A minimum score of 3 in a minimum of 1 gastrointestinal region was defined as NEC (15). Organs were weighed and tissue from the 3 small intestinal regions together with stomach, cecum, and colon contents were collected and snap-frozen for later biochemical and microbiological analyses. Tissue from the distal small intestine (the region usually affected most by NEC lesions) was formalin fixed and paraffin embedded for later histology and fluorescence in situ hybridization (FISH) analyses. Nutrient digestive function and mucosal integrity. Before and 24 h after transition to full enteral feeding, in vivo sugar absorption tests were performed as previously described (16). Blood samples were collected 224

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Microbiology. The dead probiotic mix was tested by plating on unselective calf blood agar (SSI Diagnostika), lactic acid bacteria selective MRS agar (deMan, Rogosa, Sharp; National Veterinary Institute), and on Streptococci selective M-17 agar (National Veterinary Institute). The same media were used for enumeration of total anaerobes, total lactic acid bacteria, and Streptococci spp. in cecum contents. Clostridium perfringens were counted as hemolytic colonies on calf blood agar. Paraffin embedded cross sections (3 mm) of distal small intestine were hybridized with CY3 labeled oligonucleotide probes (Eurofins MWG Operon) for general bacteria targeting of 16S rRNA [5’-GCTGCCTCCCGTAGGAGT-39 (17)]. Bacterial abundance was visualized by scanning as above. Tissues with positive staining (n = 17) were further hybridized with specific probes for the probiotic bacteria [L. paracasei: 5’-GTTCCATGTTGAATCTCGG-39 (10) B. animalis: 5’-ATTTCCCACCCCACCATG-39 (10) S. thermophilus: (5’-CACCCGTTCTTGACTTAC-39, designed using the software ARB (18) and the database Greengenes (19) and a probe for Streptococcaceae (5’-CACTCTCCCCTTCTGCAC-39 (20)]. Specificity was tested by BLAST (21) and affinity was confirmed on porcine lung tissue injected with S. thermophilus. Based on bacterial appearance, the tissue sections were given a FISH score where 1 = no signal, 2 = few micro colonies, 3 = abundant bacteria in the mucosal periphery or intestinal lumen and 4 = numerous bacteria located between the villi and/or translocating the epithelia. The tissue-associated microbiota of the distal small intestine was characterized by terminal restriction fragment (T-RF) length polymorphism with restriction enzyme CfoI as previously described (10,17). After sequencing (Applied Biosystems Genetic Analyzer 3130/3130xl) and analyses in BioNumerics 4.5 (Applied Maths), the relative intensity of T-RF was calculated as the individual T-RF intensity divided by the total sample intensity. The identity of dominating T-RF (mean relative intensities . 0.01) was proposed based on in silico digest performed in the MiCA software (22) and the RDPII database (Release 9, Update 37, Bacterial SSU 16S rRNA). As a marker of bacterial metabolic activity, concentrations of 16 SCFA in stomach and colon contents were measured by HPLC as previously described (23). Inflammatory responses. Expression of proinflammatory mediators, IL-1a, IL-6, IL-8, IFNg, TNFa, and of the LPS receptor, toll-like receptor (TLR)-4 in the distal small intestine was investigated by qPCR. RNA was extracted (RNeasy Mini Kit, QIAGEN) followed by cDNA synthesis (QuantiTect Reverse Transcription kit, QIAGEN) and qPCR from 30 ng cDNA (Fast Real-Time PCR System and TaqMan Universal PCR Master Mix, Applied Biosystems) using custom primers and probes (Supplemental Table 1). The target genes were normalized to endogenous reference genes [phosphoglycerate kinase 1 (PGK1) and TATA box binding protein (TBP)] based on geNorm analysis (24). Primary cells from the proximal small intestine were isolated and stimulated ex vivo with LPS to measure enterocyte resistance to secondary endotoxin stimulation (25). Primary cells were isolated and cultured by modification of (25), using HyQtase (Thermo Scientific) for separation of cells, and subsequent incubation on Transwell membranes (106 cells/cm2, 0.4 m, Corning). Cells were stimulated with 10 g/L LPS


Animal protocol and experimental design. Forty pigs from 3 sows (Large White 3 Danish Landrace) were delivered by caesarean section at 90% gestation and reared individually in temperature- and oxygenregulated incubators (Air-Shields). Still sedated from the caesarean surgery, the pigs were fitted with umbilical catheters (4F; Portex) and orogastric feeding tubes (6F; Pharmaplast). Mother’s serum (12 mL/kg intraarterial during the first 24 h) was provided for passive immunological protection. All pigs were nourished for 2 d with parenteral nutrition (PN) through the umbilical catheter [4–6 mL/(kg.h); Nutriflex lipid plus, Braun] with added amino acids (Vamin, Fresenius Kabi) to meet the requirement of pigs. PN was followed by 2 d of full enteral feeding [15 mL/(kg×3 h)] by orogastric tubes with a formula made from 3 commercial products (Pepdite, Maxipro, and Liquigen-MCT, kindly donated by Nutricia, Allerød, Denmark) until killing (pentobarbitone, 60 mg/kg, i.v.) and tissue collection on d 5. The above procedures follow our standard protocol as previously described in detail (1,10,15) and were approved by the National Committee on Animal Experimentation, Denmark. According to birth weight and sex, the pigs were stratified in a split litter design into a control group (n = 14) and 2 groups inoculated daily with a mix of live (ProLive, n = 14) or dead (ProDead, n = 12) gammairradiated (1 3 10 kGg, Sterigenics) probiotic strains: L. paracasei (ATCC55544), B. animalis (Bb12, DSM15954), and S. thermophilus (DSM15957). The mixed, freeze-dried bacteria-maltodextrin powdered product (kindly donated by Chr. Hansen, Hørsholm, Denmark), was dissolved in formula prior to each inoculation and 2 mL with 3 3 109 CFU was given every 3 h via the orogastric catheter (to a total of 2.4 3 1010 CFU/d). Inoculation started from 6 h after birth and continued until tissue collection on d 5. Pigs in the control group received identical amounts of formula and maltodextrin powder.

from the umbilical catheter before and after administration of an oral bolus (15 mL/kg) of 5% galactose and 2% mannitol. Four hours prior to tissue collection and urine sampling from the bladder, the pigs were fed an oral bolus (15 mL/kg) of lactulose (0.5 g/kg) and mannitol (0.3 g/kg) to measure the intestinal permeability as the urinary ratio of the 2 sugars, as previously described (15). The activities of mucosal disaccharidases (maltase, sucrase, and lactase) and peptidases (aminopeptidase N and A and dipeptidylpeptidase IV) were investigated on homogenates of small intestinal tissue (proximal, middle, and distal sections) using specific substrates as previously described (1). Mucosa was scraped from 10-cm sections of the distal small intestine to determine dry weight proportions. Villus height and crypt depth were measured on scanning pictures obtained from paraffin embedded sections of distal small intestine (ArrayWoRxe microarray scanner, Applied Precision) using the software of SoftWoRx Explorer version 1.1 (Applied Precision).

(E. coli K-235, Sigma-Aldrich) for 3 h until harvest, snap freezing, and storage (2808C) until qPCR analysis of TNFa and IFNg expressions as above, with 18S subunit ribosomal RNA (RSP18S) used as reference gene. Calculations and statistical analyses. All results are presented as means and SEM. Group differences in NEC incidences were tested using Fisher’s exact test (SAS/STAT version 9.1; SAS Institute). Group differences in bacterial enumeration were tested pair wise using Student’s t test. The mean relative T-RF intensities were tested using WILCOXON Monte Carlo Estimates Exact Test in SAS. Based on overall T-RF similarity, principal component analysis was generated in BioNumerics version 4.5 (Applied Maths) to evaluate overall colonization differences between groups of pigs. All remaining variables were tested with a 2-way ANOVA using the SAS PROC MIXED procedure with treatment and sex as fixed variables and sow and pig (nested within litter) as random variables. When relevant, the pooled values for pigs given both live and dead probiotics were tested against the values from control pigs. For qPCR data, statistics were performed on raw data and fold expression relative to control data are presented. For all analyses, P , 0.05 was used as the critical level of significance.

Clinical outcome. Five ProLive pigs, 3 ProDead pigs, and 1 control pig developed NEC before the scheduled tissue collection, which resulted in shorter mean lifespans for the 2 groups given probiotics (77 6 4 h) compared with controls (93 6 3 h) (P,0.05). A total of 19 pigs were diagnosed with NEC at the end of the experiment, with higher incidences among probiotics-treated pigs compared with controls [ProLive, 64% (9/14) and ProDead, 67% (8/12) vs. 14% (2/14); P,0.05] and the intestinal lesion score was higher than in controls (Fig. 1A). Treatment groups did not differ in birth weight (1131 6 43 g), daily weight gain (213 6 4 g/d), relative organ weights [(g/kg body): stomach, 6.1 6 0.2; colon, 9.1 6 0.3; lungs, 21.3 6 0.8;

FIGURE 1 NEC lesion scores for 5 gastrointestinal regions (A), intestinal relative weight (B), intestinal permeability (C ), mucosal proportion (dry weight) of the distal small intestine (D), villus height in the distal small intestine (E ), and villus height and crypt depth ratio (F ) in pigs inoculated with live or dead probiotics and in controls. Values are means 6 SEM, n = 14 or 12 (ProDead). Labeled means without a common letter differ, P , 0.05.

Mucosal structure and digestive function. The dry mucosa proportion was significantly lower in ProDead pigs compared with ProLive pigs and controls, and intestinal villi in control pigs were significantly higher than the pooled means of ProLive and ProDead pigs (Fig. 1E). Pigs with NEC had mucosal atrophy with a mean villus height of 451 6 61 mm compared with 803 6 37 mm for healthy pigs (P , 0.05). Intestinal permeability was significantly greater for ProDead pigs compared with controls (Fig. 1C). In vivo hexose absorption and passive sugar diffusion did not differ among treatment groups prior to enteral feeding. After 24 h of enteral formula feeding, hexose absorption decreased 30–80% in all 3 groups. The plasma galactose concentration at 20 min in the ProDead group was 0.09 6 0.04 mmol/L and in the ProLive and control groups, 0.24 6 0.95 and 0.30 6 0.92, respectively (P = 0.2). Lactase and aminopeptidase N and A activities were significantly lower in the proximal and middle small intestine in the ProDead group compared with controls, whereas there were no difference in the distal section (Fig. 2). Microbiology. Cultivation demonstrated that gamma irradiation efficiently killed the probiotic bacteria. Bacterial numbers in

FIGURE 2 Brush border enzyme activities in the proximal (A), middle (B), and distal (C ) region of the small intestine of pigs inoculated with live or dead probiotics and in controls. Values are means 6 SEM, n = 14 or 12 (ProDead). Labeled means without a common letter differ, P , 0.05. Probiotics and necrotizing enterocolitis

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Results

liver, 30.9 6 0.6; spleen, 1.9 6 0.1; heart, 8.9 6 0.3; kidneys, 9.4 6 0.3; adrenals, 0.20 6 0.01] and relative length of the small intestine (298 6 11 cm/kg body). The relative weight of the small intestine was significantly lower in the probiotic groups compared with controls (Fig. 1B). For all clinical, functional, structural, microbiological, and immunological variables, there were no litter or sex effects.

This difference was mostly due to higher lactate concentrations (Table 2). Pigs with NEC had significantly higher concentrations of acetate in the stomach and significantly more acetate and butyrate, but less lactate in the colon, than healthy pigs (Table 2). Inflammatory gene responses. qPCR on intestinal tissues showed 2-5 fold higher mean expression of all measured immune mediators (TNFa, IFNg IL-1a, IL-6, IL-8, and TLR-4) in the 2 probiotic groups compared with control pigs (Table 3). For the ProLive group, expressions of only IL-1a and IL-6 were significantly greater than in controls (P , 0.05). The ex vivo inflammatory response of isolated cells showed a higher IFNg expression in LPS-stimulated cells from ProLive pigs compared with controls (P , 0.05), whereas TNFa expression was greater than in controls in unstimulated cells from ProDead pigs (P , 0.01) (Table 4).

Discussion NEC reflects an exaggerated tissue response to enteral feeding and colonizing gut bacteria. The possibility to improve colonization in preterm infants by probiotic bacteria has therefore been investigated in several randomized controlled trials. Systematic reviews of these studies (6–9) conclude that administration of probiotics decreases the risk of NEC across the widely differing inclusion criteria, probiotics strain(s), doses, and times of administration. Animal studies (2–4,10,26) and cell models (27–29) indicate that probiotics prevent NEC through mechanisms such as antiinflammatory properties (2,4), improved barrier function, mucus production and cytoprotection, and decreased apoptosis (3). Considering this background, the present results were surprising and we rejected our hypothesis that the selected combination of probiotic strains would reduce NEC

FIGURE 3 Bacterial abundance in cecum from pigs inoculated with live or dead probiotics and in controls, n = 14 or 12 (ProDead) and from pigs with (n = 19) and without (n = 21) NEC. Lactic acid bacteria (MRS), Streptococci (M17), total anaerobes (blood) ,and Clostridium perfringens (CP) (A). FISH scores of general bacteria (Eu bact), B. animalis (B. anim), and Streptococcaceae (Strept) in the distal small intestine (B). FISH of general bacterial (C1, C2) and B. animalis (C3) in the distal small intestine of ProLive pigs with NEC. Values are means 6 SEM. Means labeled with letters without a common letter differ, P , 0.05. *Different from healthy pigs, P , 0.05. 226

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cecum contents were not different among the groups, whereas pigs with NEC had significantly higher densities of Streptococci spp., total anaerobes, and C. perfringens than healthy pigs (Fig. 3A). FISH showed no effect of live or dead probiotics on overall bacterial abundance, but abundance was significantly higher in pigs with NEC compared with healthy pigs (Fig. 3B). B. animalis was detected with FISH (Fig. 3C3) in several pigs and the abundance was significantly higher in ProLive pigs than in the 2 other groups (Fig. 3B) with positive staining in 5/6 analyzed tissues from ProLive, 2/6 tissues from ProDead, and 0/5 tissues from control pigs. L. paracasei was only detected at very low density, but S. thermophilus was not detected. In tissues from some of the NEC pigs given live or dead probiotics, a highly invasive bacterium was visualized with the general probe, but it could not be identified with the probiotic or Streptococcaceae probes (Fig. 3C1,2). The total number of T-RF, indicating microbial diversity, was lower in ProLive pigs (37 6 4 T-RF) than in control pigs (60 6 6 T-RF) (P , 0.01), whereas the number in ProDead pigs (50 6 7) did not differ from the other groups. Principal component analysis did not reveal treatment effects on overall bacterial composition (data not shown), and intensities of only a few dominating T-RF differed significantly among treatment groups (Table 1). On the other hand, NEC affected the abundance of several bacteria with either higher or lower relative intensities of several T-RF in pigs with NEC compared with healthy pigs (Table 1). The administered probiotics strains L. paracasei and B. animalis could, according to the MiCA digest, be represented by T-RF 600 and T-RF 372, respectively, both observed in the 2 probiotic groups. With a theoretical length of 581 bp, S. thermophilus was not observed in any of the groups. SCFA concentrations in stomach contents (Table 2) did not differ among treatment groups, whereas control pigs had higher total concentrations of colonic SCFA than ProLive pigs (P , 0.05) and the pooled means from both probiotic groups (P = 0.02).

TABLE 1

Relative intensities of dominating T-RF from the T-RF length polymorphism analysis of bacteria associated with the distal intestine in control, ProLive, and ProDead pigs and in those with and without NEC1 T-RF2 (intensity 3 10-2)

Pigs Control ProLive ProDead NEC Healthy

n 14 14 12 19 21

206 11.8 6.8 7.5 7.1 10.3

6 3.4 6 3.7 6 3.8 6 3.1 6 2.8

217 3.6 2.4 13.4 4.7 7.2

231

6 1.5 6 0.6 6 5.3 6 2.3 6 2.7

0.3 10.0 2.9 5.0 4.1

6 0.2 6 5.7 6 1.2 6 1.7 6 3.6

3.5 7.4 8.2 9.4 3.6

233

372

406

570

584

6 2.0 6 2.5 6 2.2 6 2.2 6 1.4*

6 2.9 6 4.8 6 3.9 6 3.7 6 2.0*

6 1.9 6 1.7 6 1.6 6 1.0 6 1.5*

6 1.2 6 2.4 6 1.6 6 1.9 6 0.3*

6 4.6 6 6.1b 6 2.9b 6 2.9 6 4.6*

10.4 15.2 14.2 20.5 7.1

6.6 3.1 3.3 1.9 6.5

2.5 4.8 4.7 7.3 1.1

28.7 13.5 14.1 12.0 2.1

a

5.6 1.8 3.0 2.0 4.8

598

600

6 2.2 6 0.7 6 2.0 6 1.3 6 1.5*

6 1.9 6 3.8a 6 1.5ab 6 1.0 6 2.8

1.9 9.2 2.7 1.6 7.4

607 b

2.9 0.7 1.5 0.6 2.7

6 0.8a 6 0.4b 6 0.6ab 6 0.3 6 0.6

Values are means 6 SEM. Means in a column in the first 3 rows with superscripts without a common letter differ, P , 0.05. In the last row, an asterisk indicates different from pigs with NEC, P , 0.05. 2 Proposed identities for individual T-RF: 206: Acinetobacter calcoaceticus or Burkholderia spp. 217: Enterococcus faecium; 231: Clostridium spp.; 233: C. perfringens; 372: Klebsiella pneumoniae, E. coli or Bifidobacterium spp; 406: Lactobacillus spp.; 570: Burkholderia spp. 584: Streptococci spp. or Lactococcus spp.; 598: Lactobacillus spp.; 600: Lactobacillus spp. or Bifidobacterium spp.; 607: Lactobacillus spp. 1

TABLE 2

previous studies (10,15,31,32). This suggested that a low, rather than high, NEC sensitivity could relate to the unexpected adverse effects of probiotic administration, which is consistent with the suggestion that probiotics exhibit the greatest beneficial effects under conditions where NEC incidence is high (33). In preterm PN-fed pigs, NEC lesions develop 1–2 d after the start of full enteral formula feeding with an incidence of 50–80%, although the incidence during some periods can be much lower (10–25%) (12,13). This variability in the NEC prevalence in preterm pigs is consistent with the epidemic nature of infant NEC (34) and consistent with the fact that NEC in preterm pigs develops spontaneously without artificial disease induction, such as hypoxia, hypothermia, or bacterial toxin administration, often used to induce NEC in other animal models (11,35,36). Different environmental bacterial exposure may explain the epidemic nature of NEC. In this study, we implemented a very high hygiene level to avoid cross-contamination of probiotic bacteria. This could provide a tentative explanation for the low NEC incidence among control pigs, and thus explain why the probiotics did not have the expected effect. Preterm neonates are initially colonized by bacteria of low diversity and total number and their gut immune system is immature. This may render the intestine hypersensitive to administration of probiotic bacteria, consistent with increased bacterial translocation and neonatal mortality in immunodeficient mice after probiotic administration (37). However, comparison with our previous studies reveals that bacterial density and diversity in this study were similar to those in previous studies for both general and specific bacteria with densities of 109–1010 CFU/g distal intestinal contents (10,31). Hence low bacterial density and diversity cannot explain the low NEC sensitivity in the control group.

SCFA concentrations in stomach and colon contents in control, ProLive, and ProDead pigs and in those with and without NEC1 Stomach

Pigs

n

Control ProLive ProDead NEC Healthy

14 14 12 19 21

Total 57.9 48.0 49.8 55.4 47.9

6 4.5 6 10.3 6 20.7 6 13.0 6 5.3

Lactate 23.5 6 21.3 6 20.8 6 23.2 6 20.4 6

5.2 4.0 6.0 4.1 3.9

Acetate 1.3 3.6 2.3 3.2 1.6

6 0.4b 6 0.8a 6 0.5ab 6 0.6 6 0.4*

Colon Butyrate 1.5 6 4.3 6 0.9 6 3.1 6 1.5 6

0.6 2.5 0.4 1.8 0.5

Others2 0.3 0.4 1.1 0.5 0.7

6 0.2 6 0.3 6 0.7 6 0.4 6 0.2

Total 75.7 6 47.2 6 60.0 6 50.7 6 68.5 6

6.6a 8.9b 9.2ab 8.6 5.7

Lactate 66.4 33.2 45.7 32.6 60.1

6 6 6 6 6

8.0a 9.5b 9.0ab 8.5 6.3*

Acetate 3.7 6.7 5.5 9.1 2.6

6 1.6 6 1.3 6 1.1 6 1.2 6 0.5*

Octanoate

Formate

2.2 6 2.2 6 4.2 6 2.7 6 2.8 6

2.6 6 2.7 6 3.2 6 3.1 6 2.6 6

0.8 1.6 0.8 1.2 0.7

0.5 0.7 0.5 0.6 0.4

Others3 0.8 1.5 2.3 3.2 0.3

6 0.5 6 1.0 6 0.6 6 0.8 6 0.2*

Values are means 6 SEM, Means in a column in the first 3 rows with superscripts without a common letter differ, P , 0.05. In the last row, an asterisk indicates different from pigs with NEC, P , 0.05. 2 Pooled values for formate, succinate, octanoate. 3 Pooled values for propionate, butyrate, iso-butyrate, valeric acid, iso-capronic acid, and succinate. 1

Probiotics and necrotizing enterocolitis

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in preterm pigs. The probiotics used in this study increased the incidence and severity of NEC, even in the group given dead bacteria, where colonization and metabolism of probiotic bacteria was prevented. The reduced mucosal integrity and digestive function, together with increased expression of proinflammatory mediators, support our conclusions and highlight a possibility of adverse consequence of administering probiotics to preterm newborns. There is great need to screen different mixtures of probiotic bacteria in clinically relevant animal models and to define the mechanisms of action before routine use of probiotics for preterm infants can be recommended. In a previous study with preterm pigs, administration of 5 probiotic strains (B. animalis, L. acidophilus, L. paracasei, L. pentosus, and L. plantarum) increased digestive function and reduced NEC severity without significant reduction in NEC incidence (69 vs. 89% in the control group) (10). Experimental procedures for these 2 pig studies were almost identical. Inoculation started a few hours after birth with doses of 1.3– 2.6 3 1010 CFU in the previous study compared with 2.4 3 1010 CFU in the present study. Both doses were in the upper range of most studies with preterm infants, but comparable doses of B. infantitis, B. bifidum, B. longum, and L. acidophilus (total 2 3 1010 CFU/d) resulted in decreased sepsis, death, and feeding intolerance in preterm infants (13). In the present study, we excluded L. acidophilus due to its possible excessive production of D-lactate (30), whereas S. thermophilus was included based on the observed beneficial effects in infant studies (12). Hence, neither the chosen strains nor the total dose can explain the observed adverse effects. In the present study, the NEC incidence in the control group was relatively low across all 3 litters (2/14, 14%) relative to

TABLE 3

Treatment n

Control ProLive ProDead

Gene expression of 5 inflammatory cytokines and TLR-4 in the distal small intestine in control, ProLive and ProDead pigs1 IFNg

TNFa

IL-1a

IL-6

IL-8

TLR-4

mRNA abundance, fold of control 14 1.0 6 0.5 1.0 6 0.3 1.0 6 0.5b 1.0 6 0.3b 1.0 6 1.3 1.0 6 0.3 14 2.2 6 3.0 2.6 6 0.9 4.1 6 3.2a 5.3 6 6.7a 2.4 6 2.2 2.5 6 1.7 12 1.9 6 2.0 1.5 6 0.3 1.9 6 1.1b 2.3 6 2.3ab 3.1 6 8.5 1.4 6 0.5

1 Values are means 6 SEM. Means in a column with superscripts without a common letter differ, P , 0.05.

Acknowledgements We thank Mette Schmidt, Louise Fangel Juhl, and Elin Skytte for assistance with the animal procedures. We also thank Kristina Møller for assistance with mRNA analyses and Annie Ravn Pedersen, Lars Mølbak, Sophia Rasmussen, Joanna Amenuvor, and Thomas Boye Pihl at the National Veterinary Institute, DTU for assistance with the microbiological analyses. Dr. Thomas Leser and Benedicte Flambard from Chr Hansen A/S are thanked for helpful discussions and support with the probiotics formulation. M.S.C., T.T., R.S., M.B., and P.T.S. designed research; M.S.C., T.T., R.S., S.B.B, and B.B.J. conducted research; M.S.C., R.S., and S.B.B. analyzed data; M.S.C. and P.T.S. wrote the paper; and M.S.C. had primary responsibility for final content. All authors read and approved the final manuscript.

Literature Cited TABLE 4

Gene expression (mRNA abundance) of 2 proinflammatory cytokines from epithelial cells isolated from the proximal small intestine, with or without LPS stimulation1 IFNg

Treatment

Control, n = 14 ProLive, n = 14 ProDead, n = 12

–LPS

1.0 6 0.2 1.3 6 0.3 0.7 6 0.3

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2.

TNFa +LPS

mRNA abundance, 1.0 6 0.2b 1.9 6 0.3a 1.0 6 0.3b

–LPS fold of control 1.0 6 0.3b 0.8 6 0.4b 2.4 6 0.5a

+LPS 3. 1.0 6 0.4 1.3 6 0.5 0.7 6 0.5

1 Values are means 6 SEM. Means in a column with superscripts without a common letter differ, P , 0.05.

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Sangild PT, Siggers RH, Schmidt M, Elnif J, Bjornvad CR, Thymann T, Grondahl ML, Hansen AK, Jensen SK, et al. Diet- and colonizationdependent intestinal dysfunction predisposes to necrotizing enterocolitis in preterm pigs. Gastroenterology. 2006;130:1776–92. Khailova L, Dvorak K, Arganbright KM, Halpern MD, Kinouchi T, Yajima M, Dvorak B. Bifidobacterium bifidum improves intestinal integrity in a rat model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol. 2009;297:G940–9. Lin PW, Nasr TR, Berardinelli AJ, Kumar A, Neish AS. The probiotic Lactobacillus GG may augment intestinal host defense by regulating apoptosis and promoting cytoprotective responses in the developing murine gut. Pediatr Res. 2008;64:511–6. Caplan MS, Miller-Catchpole R, Kaup S, Russell T, Lickerman M, Amer M, Xiao Y, Thomson R Jr. Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model. Gastroenterology. 1999;117:577–83.


SCFA concentrations in stomach and colon were also similar in this and previous studies (10,15,31,32). Administration of the live probiotic mix affected gut colonization as indicated by decreased bacterial diversity and colonic SCFA concentrations and it increased in situ abundance of B. animalis. However, species-specific differences were only minor, and it was not possible to associate the different treatments or NEC with a particular microbiota, although NEC is associated with elevated overall bacterial density in the mucosa. Only B. animalis colonized the intestine, but the competitive properties of probiotics potentially lowered the microbial diversity and thereby increased the NEC sensitivity in preterm pigs, consistent with the lowered microbial diversity in infants with NEC (38). Administration of live probiotics further increased the tissue expression of IL-1a, IL-6, and LPS dependent ex vivo expression of IFNg, indicating that live probiotics stimulated the intestinal epithelium to a proinflammatory response. Administration of the dead probiotic mix led to mucosal atrophy, greater intestinal permeability, and reduced digestive enzyme activities and sugar absorptive capacity. These effects of dead bacteria may result from epithelial interaction with bacterial antigens. Ex vivo cell studies indicated that the dead probiotics induced an LPS-independent proinflammatory response involving TNFa. This is further supported by a preliminary study showing significantly increased TNFa expression in IPEC-J2 cells (differentiated crypt cells derived from newborn pig jejunum) after stimulation with a pool of all the 3 probiotic strains compared with cells stimulated with LPS alone (3.3-fold; P , 0.05; S. Bering, M. Cilieborg, and P. Sangild, unpublished results). Together this indicates that probiotics may be provocative to an intestinal epithelium that has not yet adapted to colonizing bacteria, which is also the condition for cesareandelivered preterm pigs reared in a high-sanitary environment. Infant studies differ widely in their inclusion criteria, choice of probiotic strain(s), doses, timing and duration of administra-

tion, and the measured outcomes. The available systematic reviews (5–9) are based on 14 randomized studies where 9 found no effect on NEC (39–42) or did not comment on NEC (43–47). Five studies observed a significant reduction in relative NEC risk (26,28,48–50), consistent with a study not included in any review due to use of retrospective controls (51). Three additional studies, not included in reviews, found no effect (52) or did not comment on NEC (53,54). Probiotics were well tolerated in these studies and often colonized and/or altered gut colonization (43,53). Nevertheless, probiotics have been reported to induce sepsis in preterm infants (55,56), compromised children (57,58) and adults (59), and increased mortality in adult pancreatitis patients (60). Also, in a rat model of indomethacin-induced enteropathy, provision of Lactobacillus GG increased intestinal ulceration (61). Thus, in some circumstances, certain probiotic strain(s) may be detrimental for immunocompromised patients with disturbed gut function. More studies are needed to investigate beneficial or adverse effects of different probiotic strategies (timing, dose, strains) to prevent NEC in preterm infants. Also, more animal and cell studies are needed to elucidate the interactions with the immature intestinal epithelium of different mammalian species. This research could include studies using gnotobiotic models to differentiate between probiotic effects on modulation of the commensal gut microbiota from the effect on modulation of the host immune response. Because natural gut colonization is influenced by birth mode, infant age, genetics, diet, hospital environment and antibiotics treatment, it is a large but necessary task to ensure that only beneficial probiotic treatments are used for this highly sensitive patient group.

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