bacterial colonization and alters properties of the gut

1

Microbial manipulation of the rat dam changes 1

bacterial colonization and alters properties of the gut 2

in her offspring3

Frida Fåk*, Siv Ahrné⊥, Göran Molin⊥, Bengt Jeppsson^ and Björn Weström*4

5

*Department of Cell and Organism Biology, Animal Physiology, Lund University, Helgonavägen 3B, SE-223 62 Lund6

⊥ Food Hygien, Department of Food Technology, Engineering and Nutrition, Lund University, Box 124, SE-221 00 Lund7

^ Department of Surgery, Malmö University Hospital, SE-205 02 Malmö8

9

10

Corresponding author:11

Frida Fåk12

Department of Cell and Organism Biology, Animal Physiology, Lund University, 13

Helgonavägen 3B, SE-223 62 Lund, Sweden14

E-mail: [email protected]

Tel: +46-46-222 97 3316

Fax: +46-46-222 45 3917

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Running head: Maternal microbiota disturbances alters gut growth19

of 35Articles in PresS. Am J Physiol Gastrointest Liver Physiol (October 25, 2007). doi:10.1152/ajpgi.00023.2007

Copyright © 2007 by the American Physiological Society.

2

ABSTRACT20

21

The impact of an altered bacterial colonization on gut development has not been thoroughly 22

studied, despite the increased risk of certain diseases with a disturbed microbiota after birth. 23

This study was conducted to determine the effect of microbial manipulation, i.e. antibiotic 24

treatment or Escherichia coli (E. coli) exposure, of the dam on bacterial colonization and gut25

development in the offspring. Pregnant rats were administered either broad-spectrum 26

antibiotics three days prior to parturition, or live non-pathogenic E. coli CCUG 29300T one 27

week before parturition and up to 14 days of lactation in the drinking water. Caecal bacterial 28

levels, gut growth, intestinal permeability, digestive enzyme levels and intestinal 29

inflammation were studied in two-week old rats.30

Pups from dams that were antibiotic-treated had higher densities of Enterobacteriaceae 31

which correlated with a decreased stomach growth and function, lower pancreatic protein 32

levels, higher intestinal permeability and increased plasma levels of the acute phase protein, 33

haptoglobin, compared with pups from untreated mothers. Exposure of pregnant/lactating 34

mothers to E. coli CCUG 29300T, also resulting in increased Enterobacteriaceae levels, gave 35

in the offspring similar results on the stomach and an increased small intestinal growth as 36

compared to the control pups. Furthermore, E. coli pups showed increased mucosal 37

disaccharidase activities, increased liver, spleen and adrenal weights, as well as increased 38

plasma concentrations of haptoglobin. These findings indicate that disturbing the normal 39

bacterial colonization after birth, by increasing the densities of caecal Enterobacteriaceae, 40

appear to have lasting effects on the postnatal microflora which affects gut growth and 41

function.42

43

Keywords: Bacteria, Gut permeability, Enterobacteriaceae, Neonatal, Intestine44

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INTRODUCTION45

46

The digestive tract of newborn mammals is exposed to various luminal factors, including 47

nutrients and the colonizing microbiota after birth. A rapid colonization of the gut by 48

primarily aerobic bacteria occurs as soon as the neonate is exposed to the world outside the 49

protective uterus, and the maternal fecal, vaginal and cutaneous microflora, as well as the 50

environmental bacteria, contributes. In fact, it appears as the mother’s bacterial flora is shared 51

to a large degree by the offspring, at least in laboratory mice (18). The introduction of solid 52

food at time of weaning leads to changes in the gut microbial flora. In rats, bacteria favored53

by the mother’s milk, i.e. Gram-positive lactobacilli and bifidobacteria, are followed by 54

colonizing Gram-negative and anaerobic bacteria (33). 55

Rodents are born with an immature gastrointestinal (GI) system, which is relatively 56

permeable to macromolecules due to a high endocytotic activity of the small intestinal 57

enterocytes (25). With age, the high permeability declines until a more mature gut barrier is 58

established at weaning (gut closure). During the early suckling period, the gut undergoes 59

morphological as well as functional development, with rapid growth of the intestine and an 60

increased expression of digestive enzymes, such as lactase. At weaning, crypt hyperplasia 61

along with intestinal closure and an increased expression of maltase, sucrase and pancreatic 62

trypsin denotes vast changes of the gastrointestinal tract (25).63

Studies with germ-free animals have revealed that bacteria are essential to the growth and 64

development of the GI system. In animals without a microflora the intestinal weight and 65

surface area are decreased (3). Additionally, the intestinal villi are thinner and the enterocytes 66

show an abnormal shape. However, the caecum can be almost eight times larger in germ-free 67

animals compared to conventionally raised animals, due to mucus and fiber accumulation and 68

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subsequent water retention (23). Additionally, pancreas protein content is increased in germ-69

free animals, reflecting higher amounts of some digestive enzymes (19).70

An aberrant gut microflora may be just as detrimental for the individual as having no 71

microflora at all. Diarrhea, inflammatory bowel diseases, pancreatitis and even allergies have 72

been shown to be related to the bacterial flora (22, 24, 34). The effect of antibiotics during 73

pregnancy on the newborn has not been extensively studied, but one report showed that by 74

destabilizing the maternal digestive microflora, the newborn rat pups had significantly lower 75

numbers of intestinal staphylococci and lactobacilli up to five days after birth (5). However, 76

the impact of the microfloral alteration on the GI system was not studied, neither whether the 77

changes in bacterial numbers persisted (5). 78

The present study was designed to elucidate the effects of the maternal microflora on 79

bacterial colonization and GI development of the neonatal rat. We hypothesized that 80

disturbing the normal colonization sequence by administering antibiotics in the drinking water 81

to the dam during late pregnancy would have an effect on GI growth and maturation of her 82

offspring. Thus, just before the normal weaning process starts, at two weeks of age, rat pups 83

from antibiotic-treated mothers were compared with pups from untreated mothers. Numbers 84

of two important members of the gut microflora, Enterobacteriaceae and lactobacilli, in the 85

gut of both pups and mothers were analyzed, as well as gut organ growth, small intestinal 86

permeability and disaccharidase activities, as well as pancreas enzyme activities and intestinal 87

inflammation. After establishing that the antibiotic treatment of the dams elevated 88

Enterobacteriaceae levels in the pups and influenced the GI system, we exposed additional 89

pregnant and lactating dams to Escherichia coli, strain CCUG 29300T, a major member of the 90

Enterobacteriaceae family, in order to further investigate this aspect.91

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MATERIALS AND METHODS92

93

Animals and experimental procedure94

Sprague-Dawley rats (Taconic, Ry, Denmark) were used and the dams were chosen to be of 95

similar age and weight to minimize differences in their gut microflora. The dams were housed 96

separately from one week before parturition in polycarbonate cages on a good laboratory 97

practice chopped aspen wood bedding (Beeky bedding, Scanbur BK AB, Sollentuna, Sweden) 98

with free access to a breeding diet (SDS, RM1, Essex, England, containing 22.5 % protein, 5 99

% fat, vitamins and minerals and the rest carbohydrates) and tap water in our animal facility 100

under a controlled environment (21±1°C, 50±10 RH%, 12-12 hour light-dark cycle). After 101

birth (the day of birth was assigned as day 0) each litter was restricted to a number of 8 to 13 102

pups and was held with its respective dam for two weeks. The local Ethical Review 103

Committee for Animal Experiments had approved the study.104

In the antibiotic study, from 3-4 days prior to the expected day of delivery and up to day 0 105

(birth) or 1, a mixture of the broad-spectrum antibiotics (metronidazole 0.5 mg/ml, neomycin 106

2.5 mg/ml and polymyxin B 0.09 mg/ml) was administered in the drinking water (31). The 107

mean water consumption of the dams (n=3) was approximately 20 ml/day, while the control108

dams (n=3) given water without antibiotics, had a consumption of about 40 ml/day.109

In the bacterial exposure study, E. coli CCUG 29300T (CCUG = Culture Collection of 110

University of Göteborg; T = type strain) was administered in the drinking water at 1.8x108111

CFU per ml, from one week prior to expected parturition and continuing during the suckling 112

period until two weeks of age. Both treated (n=2) and control dams given water without 113

bacteria (n=2) consumed an average of approximately 40 ml water/day.114

115

116

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Bacterial preparation117

E. coli CCUG 29300T was grown in Luria Broth Medium (tryptone 10 g/l, yeast extract 5 g/l 118

and NaCl 5 g/l) in a shaking water bath at 37°C for 20 hours. The cells were harvested by 119

centrifugation at 3000 x g for 5 minutes, resuspended in freezing buffer (3.6 mM K2HPO4, 1.3 120

mM KH2PO4, 2.0 mM Na-citrate, 1.0 mM MgSO4, 12% glycerol) and kept at –70°C until 121

feeding. At each day of administration, new bacterial suspensions were thawed, washed with 122

saline, and centrifuged. The cell pellet was dissolved in tap water and added to the dams’ 123

water bottles. Viable count was performed on the water bottles containing bacteria, which 124

gave a final concentration of 1.8x108 CFU/ml.125

126

Procedure at sacrifice127

At the end of the experiment, when the pups were two weeks of age, they were separated from 128

their mothers and gavaged by use of a teflon stomach tube with a marker solution containing 129

bovine serum albumin (1.25 mg BSA/g b. wt) and bovine immunoglobulin (0.25 mg BIgG/g 130

b. wt.) (both Sigma-Aldrich Co, St Louis, USA). After 3 hours, the pups were anaesthetized131

with a mixture of Ketamin (Ketalar, Parke-Davis, Solna, Sweden, 0.5 mg/g b. wt.) and 132

Azaperon (Stresnil, Janssen-Cilag Pharma, Wien, Austria, 0.4 mg/g b. wt) in 0.15 M NaCl. 133

The bowel and chest was cut open and blood samples were taken by heart puncture into tubes 134

containing 1.5 mg EDTA and 20 000 IU aprotinin (Trasylol; Bayer, Leverkusen, Germany) 135

and ice-chilled. The pancreas was then carefully dissected, rinsed in ice-cold saline, weighed136

and immediately frozen. After this, the small intestine (divided into two halves of equal 137

length, proximal and distal SI) and stomach were dissected, flushed with ice-cold saline, 138

weighed and frozen. The contents of the stomach was collected and saved on ice. After 139

dissection of the spleen, thymus, liver and adrenals, their weights were recorded. Finally, the 140

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caecum with contents was removed, weighed and frozen in freezing buffer (3.6 mM K2HPO4, 141

1.3 mM KH2PO4, 2.0 mM Na-citrate, 1.0 mM MgSO4, 12% glycerol) at -70 °C.142

After completion of the necropsy, blood samples were centrifuged (3,000 g for 15 min at 143

4°C) and plasma was removed and stored at -70 °C until further analysis. The stomach 144

content was mixed in 0.5 ml 0.9 % NaCl and centrifuged (3,000 g for 15 min at 4°C) after 145

which the pH was measured.146

Fecal samples collected freshly from the mothers at three time points, the day before start 147

of antibiotic or bacterial treatments, at parturition and after two weeks at the end of 148

experiment (n=2-6 at each time point and treatment group), were frozen in freezing buffer and 149

stored at -70 °C until analysis.150

151

Analyses152

The ceacal and fecal samples153

The caecum with its content or fecal samples was thawed and homogenized in the freezing 154

medium using a sterile pipette. After vortexing the samples, serial dilutions were made in 155

dilution medium (9 mg/ml NaCl, 1 mg/ml peptone, 0.2 mg/ml cysteine, 1 ml Tween/1000 ml 156

distilled water) and spread on violet red bile glucose (VRBG) and Rogosa agar plates,157

respectively (Oxoid Ltd, Basingstoke, Hampshire, England). After incubating VRBG plates 158

for 24 hours aerobically and Rogosa plates for 48 hours anaerobically (AnaeroGen, Oxoid 159

Ltd, Basingstoke, Hampshire, England), the number of colonies was estimated and calculated 160

as colony forming units (CFU) per gram caecum with content. Colonies found growing on 161

VRBG agar plates were considered to be enterobacteria belonging to the family 162

Enterobacteriaceae, while colonies found on Rogosa agar plates were considered to be 163

lactobacilli (Lactobacillus-like bacteria). 164

165

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166

Randomly amplified polymorphic DNA (RAPD)167

Random colonies from VRBG plates were randomly picked from E. coli pups and control 168

pups and washed twice in sterile water. By shaking with glass beads (2 mm) for 45 min at 4 169

°C, the cells were disintegrated and DNA recovered. The samples were centrifuged at 20817 x 170

g for 5 min after which the supernatant (1 µl) was used for PCR. The reaction mixture 171

contained PCR buffer (Boehringer-Mannheim Scandinavia, Bromma, Sweden), 0.2 mmol/L 172

of each nucleotide (Perkin-Elmer, Branchburg, NJ, USA), Taq DNA polymerase (Boehringer-173

Mannheim), and 1 mg/ml primer (a 9-mere with the sequence 5`ACG CGC CCT-3`, 174

synthesized by Scandinavian Gene Synthesis, Köping, Sweden). The PCR reaction was 175

performed in a Perkin-Elmer thermal cycler, with the following temperature profile: four 176

cycles consisting of 94°C for 45 s, 30°C for 120 s, 72°C for 60 s, followed by 26 cycles 177

consisting of 94°C for 5 s, 36°C for 30 s, 72°C for 30 s (1 s extension per cycle). The PCR 178

session was terminated at 72°C for 10 min, followed by cooling to 4°C.179

Gel electrophoresis was run by applying 20 µl of the PCR product on a horizontal, 1.5 % 180

agarose gel for 2.5 hours at 80 V in TB buffer (89 mM boric acid, 2.5 mmol/L EDTA, pH 8.3) 181

with a DNA molecular weight standard VI (0.5 µg, Boehringer Mannheim). The gel was 182

stained in ethididum bromide for 15 min, followed by washing for 2x10 min. Bands were 183

visualized at 302 nm with a UV transilluminator/UVP Inc (San Gabriel, CA, USA) and184

pattern from E. coli pups and control pups were compared with the band pattern from the E. 185

coli CCUG 29300T given to the dams.186

187

The pancreas188

The pancreas protein content was determined according to the Lowry method (20) modified 189

for 96-well microplates. Briefly, the pancreata were homogenized in ice-cold 0.2 mol/L TRIS-190

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HCl buffer + 0.05 mol/L CaCl2 (pH 7.8) using a glass/glass homogenizer, followed by 191

centrifugation at 15,000 x g for 20 min at 4 °C. The protein concentration was determined in 192

the supernatant by reading the absorbance of the samples at 690 nm using a plate reader and 193

bovine serum albumin as the standard. To estimate the trypsin amount, the pancreatic194

supernatants were activated with enterokinase and thereafter incubated with the substrate Bz-195

Arg-pNA (Sigma-Aldrich Co, St Louis, USA), and the absorbance change was then measured 196

at 405 nm (10). The amount of enzyme causing transformation of 1.0 µmol of substrate/min at 197

25 °C was defined as one unit. 198

199

The small intestine200

After homogenizing the proximal small intestine in 9 volumes of ice-cold NaCl using a knife-201

homogenizer, the protein amount was determined as described above. In addition, the 202

Dahlqvist method (7) was used to measure the intestinal disaccharidase activities. The 203

substrates lactose, sucrose and maltose were incubated with the intestinal homogenates for 204

one hour, after which the reaction was stopped with a glucose oxidase reagent (Sigma-205

Aldrich) and the amount of generated glucose was determined. Glucose (0.05-1.0 mg/ml) was 206

used as standard. The disaccharidase activities were estimated by reading the absorbance at 207

450 nm. 208

209

The blood210

The intestinal macromolecular permeability was determined by measuring the concentrations 211

of the marker molecules BSA and BIgG in blood samples taken three hours after gavage, by 212

electroimmunoassay (rocket electrophoresis) (2, 17) using specific antisera for BSA (rabbit 213

anti-cow albumin, Dako A/S, Denmark) and BIgG (rabbit anti-BIgG, Dako A/S, Denmark). 214

Purified BSA and BIgG (Sigma-Aldrich Co, St Louis, USA) were used as standards.215

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The concentration of the plasma acute phase protein, haptoglobin, was analyzed using a 216

commercially available kit (PhaseTM Range Haptoglobin Assay, Tridelta Development Ltd, 217

Ireland) according to the manufacturer’s instructions. In short, plasma was incubated with 218

haemoglobin which bound to any haptoglobin present in the samples leading to preservation 219

of peroxidase activity of the haemoglobin. A colorimetric reaction showing the peroxidase 220

activity in the samples was then compared with a haptoglobin standard (0-2 mg/ml). 221

Absorbance was measured at 630 nm. The assay sensitivity was reported to be 0.05 mg/ml 222

haptoglobin.223

224

Calculations225

Student’s t-test was performed (unpaired, two-tailed) on all of the results, where p-values < 226

0.05 were considered significant. Exact p-values are reported, unless below 0.001. The 227

antibiotic group (n=11) was compared to the controls (n=11) run in parallel, whereas the E.228

coli pups (n=16) were compared to their respective control pups (n=12). The effect of 229

antibiotic or E. coli CCUG 29300T treatments on the dams’ fecal flora was estimated by 230

comparing bacterial numbers of treated dams with controls dams at one week before 231

treatment, at the day of parturition and at the final day of the experiment.232

To compensate for body weight differences all organ parameters are given per g body 233

weight, giving a relative organ weight that could be compared between groups.234

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RESULTS235

236

Effects of microbial manipulation on the microflora of dams237

No diarrhea was noticed among the antibiotic-treated mothers, but the stool consistence 238

became softer during the treatment, an effect that did not persist after 2 weeks. At the end of 239

the 3-4-day treatment period, antibiotics significantly reduced the numbers of lactobacilli in 240

the fecal samples, while two weeks later, the numbers of lactobacilli were restored (Figure 241

1a). The fecal numbers of Enterobacteriaceae were not altered by the antibiotic treatment, 242

although an insignificant increase (p = 0.46) was found at the end of the treatment at 243

parturition (Figure 1b).244

Exposure to live Escherichia coli CCUG 29300T via the drinking water during three weeks 245

did not appear to affect the stool consistency of the mothers. At day of parturition as well as 246

two weeks later, fecal samples from E. coli dams showed a higher amount of 247

Enterobacteriaceae, though not significantly due to a low number of female samples. The 248

numbers of lactobacilli did not change due to the E. coli CCUG 29300T exposure (Figure 1a). 249

250

Effects of microbial manipulation in the offspring251

Neither the antibiotic treatment nor the E. coli CCUG 29300T exposure of rat dams had any 252

impact on the body weight of their pups as compared to pups of the untreated control mothers 253

(Table 1). Two pups out of 13 in the antibiotic group died at the age of nine days for unknown 254

reasons, while there was a 100 % survival in the E. coli and the control groups.255

256

Effects on the caecum and microflora of the pups257

At sacrifice, when the pups were two weeks old, no difference in the weight of the caecum258

including its contents was found between any of the groups (Table 1). The number of 259

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Enterobacteriaceae, however, was significantly higher in ceacal samples from pups from the 260

antibiotic-treated dams as compared to pups from untreated dams, while in the numbers of 261

lactobacilli no differences were found (Figure 2). In the E. coli CCUG 29300T exposure 262

study, higher numbers of Enterobacteriaceae was found in the E. coli pups as compared to the 263

control pups, while no difference was found with regard to the lactobacilli numbers (Figure 264

2). The RAPD analysis showed that the E. coli CCUG 29300T given to the mothers was not 265

recovered in the pups’ caecal flora.266

267

Effects on the stomach of the pups268

The weight of the stomach tissue was significantly lower in the pups born from the antibiotic-269

treated mothers as compared to the control pups. In addition, the pH of the stomach contents 270

was significantly higher in the antibiotic group (Table 1). Similarly, in the E. coli exposure271

CCUG 29300T study, the stomach pH was found to be significantly higher in the E. coli pups 272

than in the control pups, but the stomach weight did not differ between groups (Table 1).273

274

Effects on the small intestine of the pups275

Antibiotic treatment of the dams did not significantly affect the weight of the small intestine 276

of the pups (Table 1). The small intestinal protein content and the intestinal lactase and 277

maltase activities did not differ between groups, although the sucrase activity was 278

significantly higher in the antibiotic group in comparison with the control group (Table 3).279

The E. coli CCUG 29300T exposure of the mothers did, however, affect their pups’ 280

intestinal growth. Both the small intestinal weight (both proximal and distal part) and the 281

protein content of the proximal part were significantly increased in the E. coli pups as 282

compared to the control pups (Table 1 and Table 3). Also the activities of the disaccharidases 283

lactase, maltase and sucrase were significantly higher in E. coli pups (Table 3).284

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The plasma level of the macromolecular markers at 3 hours after gavage was significantly 285

higher in the antibiotic group of pups with regard to BIgG (Figure 3), while no significant 286

difference was found for BSA (antibiotic group: 14.7 (2.1), control group: 14.5 (3.7)). In the 287

E. coli group, no significant differences were observed for either BIgG (Figure 3) or BSA (E. 288

coli group:12.6 (2.4), control group: 13.6 (4.0)) between the groups.289

290

Effects on the pancreas and the liver of the pups291

No difference was found in the pancreas weight between the antibiotic and the control groups. 292

The pancreatic protein content, however, was significantly lower in the antibiotic group 293

compared to the control group, while no differences were found for the trypsin content (Table 294

2). The E. coli CCUG 29300T exposure study showed a significant increase in the pancreas 295

weight in the E. coli group in comparison with the control group. No significant differences 296

were found in the protein or trypsin content of the pancreas between groups (Table 2). The 297

liver weighed significantly more in the antibiotic group in comparison with the control group 298

(Table 1), and similarly, the E. coli pups had an increased liver weight as compared to the 299

control pups.300

301

Effects on plasma haptoglobin of the pups302

The acute-phase protein haptoglobin was found to be significantly elevated in plasma from 303

both the antibiotic pups and the E. coli pups in comparison with their respective controls 304

(Figure 4). 305

306

Effects on the thymus and the spleen of the pups307

The weights of the two lymphoid organs, the thymus and the spleen, were not altered in the 308

pups by the antibiotic treatment of the dams (Table 1). Treatment with E. coli CCUG 29300T309

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had no effect on the pups’ thymus weight, but significantly heavier spleens were found in the 310

E. coli pups as compared to control pups (Table 1).311

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DISCUSSION312

313

Effect of microbial manipulation on the bacterial flora of the dam and her offspring314

Microbial manipulations by antibiotic treatment or E. coli CCUG 29300T exposure of 315

pregnant and lactating rats lead to transient changes in their gut microflora that also affected 316

their offspring with increased numbers of ceacal Enterobacteriaceae at two weeks of age.317

Treating the dams with broad spectrum antibiotics resulted in an increase in the 318

Enterobacteriaceae levels and significantly reduced the numbers of fecal lactobacilli at 319

parturition, but after two weeks, these numbers had normalized. Similarly, exposure to E. coli 320

CCUG 29300T via the drinking water lead to increases in the levels of fecal321

Enterobacteriaceae, while no significant effect was found in the lactobacilli numbers.322

The resulting microflora in the offspring from the antibiotic-treated mothers showed an323

increase in the numbers of Enterobacteriaceae, but no effect on the lactobacilli as compared 324

to the offspring from control dams. It is plausible that the overgrowth of antibiotic-resistant 325

bacteria, i.e. Enterobacteriaceae, in the dams’ microflora favored the colonization of 326

Enterobacteriaceae in the offspring. In a study with penicillin treated lactating mice,327

enterococci, coliforms and clostridia were found in the offspring’s caeca, while lactobacilli 328

did not colonize pups of treated mothers (27). It is interesting to note that although the rat 329

dams restored their numbers of lactobacilli and Enterobacteriaceae after two weeks, their330

pups had remaining increased Enterobacteriaceae levels at two weeks of age, indicating that 331

the microflora disturbances induced after birth were maintained during the suckling period. In 332

contrast, a recent study in rabbits showed that the bacteria colonizing during the suckling 333

period had a greater impact on the caecal microbial communities than those colonizing at 334

birth (1).335

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In the E. coli CCUG 29300T exposure study, also increased levels of Enterobacteriaceae 336

were found in the two week-old pups. However, the situation was slightly different since 337

these pups were exposed to E. coli CCUG 29300T also during the suckling period, either 338

directly via consumption of drinking water or indirectly via the mother. The DNA profile of 339

the Enterobacteriaceae found in the pups was different from the E. coli strain consumed by 340

the dams, indicating that the E. coli 29300 T given to the mothers did not directly transfer to 341

the pups, but increased the numbers of other Enterobacteriaceae species transferred.342

Increase in Enterobacteriaceae levels early in life appears occasionally and independently343

of any antibiotic regimen. According to Wang et al, 2004, babies may become solely 344

colonized with E. coli a week after birth, which might not only influence their gastrointestinal 345

development, but also their health status in adulthood (32). Our findings that elevated 346

Enterobacteriaceae levels induced after birth persist during the suckling period, warrants 347

further investigations concerning the significance of an early colonization of 348

Enterobacteriaceae and their role in the pathogenesis of later disease.349

350

Effects on gut organ growth and development in the offspring351

Disrupting the microbial colonization sequence by use of broad-spectrum antibiotics in 352

pediatric care has been shown to modulate the expression of important gastrointestinal 353

developmental related genes (28). In addition, there appears to be a correlation between 354

antibiotic treatment after birth and development of allergy (24), but the influence of antibiotic 355

therapy during the neonatal period on development of the digestive and immune function of 356

the gut has not been investigated thoroughly.357

The findings of the present study, using the neonatal rat model, show that an increased level358

of Enterobacteriaceae during the suckling period has an impact on the gastrointestinal growth 359

and development. The stomach weight was decreased in the antibiotic group of pups and both 360

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the E. coli and the antibiotic groups had a decreased HCl secretion, reflecting a decreased 361

growth and functional maturation of the stomach. This may weaken the animals’ defense362

against ingested pathogenic bacteria. Germ-free rats have been found to have decreased 363

plasma levels of gastrin, and altered levels of gastrin might have been responsible for the 364

stomach effects seen in the rat pups.365

The small intestine was significantly heavier in the E. coli pups, while no difference was 366

found in the antibiotic pups as compared to the control group. This was correlated with an 367

increase in the mucosal disaccharidase activities as well as an increased protein content of the 368

small intestine. Similarly, a stimulation of mucosal sucrase and lactase activities has been 369

shown in suckling pigs by treatment with either an antibiotic or a probiotic (6), but it has also 370

been observed that some probiotic bacteria possess endogenous lactase activity (26). Anyhow, 371

it has been shown previously that the gut microflora can influence intestinal proliferation and 372

enzyme expression and our results confirm that Enterobacteriaceae can affect intestinal 373

growth (15).374

In addition, an increased intestinal marker permeability was found in the antibiotic pups 375

whereas this was not seen in the E. coli pups. Whatever the mechanism behind this is, it 376

remains clear that a decreased barrier function during early life can predestine to diseases later 377

in life, such as allergies (4, 24). In contrast, feeding probiotic lactobacilli have been shown to 378

decrease intestinal permeability in the same model with suckling rat pups (11). Furthermore, a 379

mix of probiotic strains have been found to be able to improve the barrier integrity in mice 380

with colitis (21) and Lactobacillus paracasei exposure was able to counteract stress-induced 381

elevated gut permeability in rats (9). Infants later developing allergies show an increased 382

colonization of aerobic bacteria and a delayed colonization of anaerobic bacteria with a 383

concurrent increased intestinal permeability, further supporting our findings above (4, 16).384

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The gut-associated organ, the exocrine pancreas, also appeared to have been influenced by 385

the antibiotic treatment of the dams, where the pups had lower total pancreatic protein content386

than the control group. The slightly heavier pancreata found in the E. coli pups was not 387

reflected in any changes in the pancreatic content of protein or trypsin, suggesting that there 388

was an increased growth but no functional maturation of the pancreas. The altered gut 389

microflora might have influenced the degradation of pancreatic enzymes in the intestine, 390

thereby possibly influencing the pancreatic enzyme production similar to what has been 391

observed in germ-free rats (13, 19).392

Also the liver was affected, where both the antibiotic and the E. coli pups had heavier livers 393

than the control pups. It appears likely that this was due to an enhanced fat deposition in the 394

liver because of an inflammatory response to endotoxin exposure due to the increased levels 395

of Enterobacteriaceae in the gut (8). This is most likely also true for the E. coli group, since 396

exposure to lipopolysaccharide could possibly have initiated an inflammatory reaction leading 397

to an increased liver weight. The increased weight of the spleen in the E. coli group of pups 398

further implies that this might be the case. In addition, both the antibiotic and the E. coli399

groups showed increased levels of the acute phase protein, haptoglobin, at 14 days of age, 400

which strongly supports that an inflammatory response triggered by the bacterial flora had 401

indeed taken place (14). An aberrant colonization with an overgrowth of bacteria after birth 402

could lead to an inflammatory cascade triggering damage of the small intestine and 403

necrotizing enterocolitis (NEC) in infants (12). Moreover, the adrenals showed an increased 404

weight in the E. coli group and it is possible that the physiological stress induced by the 405

inflammation lead to an increased production of adrenal stress hormones. Possibly, the local 406

immune system in the gut was activated, in a manner similar to what happens during weaning 407

when activation of T cells occurs and pro-inflammatory cytokines increase, leading to an 408

increase in growth of the small intestine (29, 31).409

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One could argue that the effects seen on GI organs in the antibiotic experiments were due 410

to direct toxic effects of the antibiotics pre- or postnatally, since Brunel and Gouet (1993) 411

reported in a similar experiment, that some antibiotics might cross the placental barrier and 412

thus be found in the newborn. However, they claimed that the antibiotics used, ampicillin, did 413

not transfer from the tissues to the intestinal lumen affecting the microflora. Furthermore, 414

since similar effects on gut growth and maturation were found in the E. coli CCUG 29300T415

exposure study it is likely that also the effects seen in the antibiotic study were, in fact, caused 416

by effects on the bacterial colonization.417

In summary, disturbing the maternal microflora had vast effects on her offspring, with 418

elevated caecal numbers of opportunistic Enterobacteriaceae, leading to intestinal 419

inflammation, altered gastrointestinal properties and decreased barrier properties. The 420

mechanism behind the effects remains to be further elucidated, but we can conclude that the 421

inflammation per se was probably not responsible for the stimulated growth of the GI tract in 422

the E. coli pups, since the antibiotic pups showed no such growth increase of the intestine 423

despite their elevated haptoglobin levels. 424

It was intriguing that only a three-fold increase in the levels of Enterobacteriaceae could 425

have such vast effects on gut growth and development in the laboratory (specific pathogen-426

free) rats. This is, to our knowledge, the first study reporting effects on postnatal 427

gastrointestinal development by increasing the levels of a single family of bacteria. More 428

studies are needed that investigate the influence of the mother’s intestinal microflora on the 429

newborn, as well as the effects of antibiotic treatment during the neonatal period (28) and how 430

this affects the health in adulthood.431

, 432

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ACKNOWLEDGEMENTS433

The authors wish to thank Mrs Inger Mattson and Mrs Camilla Björklöv for expert technical 434

assistance. The Royal Physiographic Society in Lund, Sweden is greatly acknowledged for 435

financial support.436

437

438

439

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441

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53-69, 2004.527

33. Wenzl H H, Schimpl G, Feierl G and Steinwender G. Time course of spontaneous 528

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245, 1998.533

534

535

536

537

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Figure 1. Numbers (mean +SD) of lactobacilli (a) and Enterobacteriaceae (b) in fecal 538

samples taken from the rat dams (exposed to either antibiotics or E. coli CCUG 29300T)539

one day before treatment, at the day of parturition and two weeks later (n=2-6 per 540

treatment group and sample time) as compared to control dams. Significant differences 541

were calculated between treated dams and control dams at each time period using 542

Student’s t test.543

544Figure 2. Numbers (mean +SD) of Enterobacteriaceae and lactobacilli in caecum of pups 545

born from either antibiotic-treated dams, E. coli-exposed dams or respective control 546

dams. Significant differences were calculated between the antibiotic group or the E. coli547

group as compared to the respective controls using Student’s t test.548

549

Figure 3. Plasma levels (mean +SD) of the marker molecule bovine immune globulin 550

(BIgG) at 3 hours after marker gavage of pups born from either antibiotic-treated dams, 551

E. coli-exposed dams or respective control dams. The only significant difference 552

calculated using Student’s t-test was between the antibiotic group and their controls for 553

BIgG.554

555

Figure 4. Plasma haptoglobin levels (mean +SD) in pups born from either antibiotic-556

treated dams, E. coli-exposed dams or control dams. Significant differences were 557

calculated between the antibiotic group or the E. coli group and their respective controls558

using Student’s t test.559

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560

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Table 1. Body weight (g), weight of stomach, small intestine (SI, proximal and distal halves), caecum including its contents, liver,

adrenal, thymus and spleen (mg/g b. wt) and pH of stomach contents from 14-day old rats born from either antibiotic-treated or E. coli-

exposed dams or from untreated dams (controls).

Treatment Body wt. Stomach Stomach pH Proximal SI Distal SI Ceacum Liver Adrenals Thymus Spleen

Antibiotic group (n=11) 28.8 (2.9)

N.S.

6.4 (0.5)

p = 0.005

4.3 (0.8)

p = 0.02

14.7 (1.3)

N.S.

13.1 (2.0)

N.S.

3.0 (0.5)

N.S.

32.5 (3.0)

p = 0.007

0.2 (0.05)

N.S.

4.9 (0.6) 3.9 (0.4)

N.S.

Controls (n=11) 27.6 (1.6) 7.0 (0.3) 3.6 (0.4) 14.7 (1.5) 13.9 (1.4) 3.2 (0.5) 29.5 (1.4) 0.2 (0.1) 4.8 (0.5) 4.1 (0.5)

E. coli group (n=16) 30.0 (1.4)

N.S.

7.3 (0.5)

N.S.

5.0 (0.3)

p = 0.01

18.3 (1.0)

p < 0.001

15.8 (1.0)

p = 0.001

3.6 (0.5)

N.S.

31.0 (1.5)

p = 0.0001

0.21 (0.1)

p = 0.04

4.5 (0.5)

N.S

4.6 (0.4)

p = 0.03

Controls (n=12) 30.4 (1.3) 6.9 (0.6) 4.4 (0.8) 15.2 (1.4) 14.2 (1.4) 3.6 (0.5) 28.3 (1.6) 0.15 (0.1) 4.4 (0.4) 4.4 (0.3)

Values are expressed as mean (SD). Significant differences were found between the antibiotic group or the E. coli group and their respective control groups. N.S. = not significant.

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Table 2. Effect of antibiotic treatment or E. coli exposure of rat dams as compared to control dams on the pups’ pancreas weight (mg/g

b. wt), protein (mg/g b. wt) and trypsin (U/g b. wt) content.

Treatment Weight Protein Trypsin

Antibiotic group (n=11) 3.7 (0.2)

N.S.

146 (24)

p < 0.001

10.4 (2.2)

N.S.

Controls (n=11) 3.5 (0.3) 214 (17) 13.1 (3.4)

E. coli group (n=16) 3.1 (0.4)

p = 0.003

181 (40)

N.S.

13.3 (3.4)

N.S.

Controls (n=12) 2.6 (0.5) 222 (133) 13.1 (4.1)

Values are expressed as mean (SD). Significant differences were found between the antibiotic group or the E. coli group and their respective control groups. N.S. = not significant.

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Table 3. Effect of antibiotic treatment or E. coli exposure of rat dams as compared to control dams on the pups’ small intestinal

(proximal part) protein content (mg/g b. wt) and disaccharidase activities (U/g b. wt).

Treatment Protein Lactase Maltase Sucrase

Antibiotic group (n=11) 145 (48)

N.S.

126 (40)

N.S.

66 (34)

N.S.

2.7 (3.2)

p = 0.04

Controls (n=11) 131 (31) 104 (13) 58 (14) 0.8 (0.8)

E. coli group (n=16) 301 (83)

p < 0.001

105 (15)

p = 0.01

106 (14)

p < 0.001

6.4 (3.3)

p < 0.001

Controls (n=12) 182 (49) 92 (13) 76 (12) 2.3 (2.3)Values are expressed as mean (SD). Significant differences were found between the antibiotic or the E. coli group and their respective control groups. N.S. = not significant.

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254x190mm (96 x 96 DPI)

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bacterial colonization and alters properties of the gut

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