Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species.

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.

Susceptibility to Cronobacter sakazakii decreases with increasing age in neonatal CD-1 mice.

Neonatal, premature, or very low birth weight infants fed reconstituted powdered infant formula contaminated with Cronobacter (Enterobacter sakazakii) may develop infections resulting in severe outcomes such as septicemia, necrotizing enterocolitis, meningitis, or death. Infants who recover from infection may have morbidities such as hydrocephalus, mental retardation, or developmental delays. Although increasing age appears to reduce susceptibility to Cronobacter infection, it is not known at what age or why these infants become less susceptible. Our study objectives were to compare the susceptibilities of neonatal mice of different ages to Cronobacter sakazakii infection. Timed-pregnant CD-1 mice were allowed to give birth naturally. Neonatal mice were orally gavaged at postnatal days (PNDs) 1.5, 5.5, and 9.5 with a single dose of vehicle or 10(3), 10(7), or 10(10) CFU/ml C. sakazakii strain MNW2 in reconstituted powdered infant formula. Pups were euthanized 7 days after challenge. Brains, livers, and ceca were excised and analyzed for C. sakazakii invasion, and blood was collected for serum amyloid A analysis as a biomarker of infection. C. sakazakii invasion was age dependent; the pathogen was isolated from brains, livers, and ceca of neonatal mice treated at PNDs 1.5 and 5.5 but not from those of pups treated at PND 9.5. C. sakazakii was more invasive at PND 1.5 in brains than in livers and ceca and was isolated from 22, 14, and 18% of these tissue samples, respectively. Serum amyloid A was detected in only one treated neonate. Mortality was observed only in neonates treated at PND 1.5. In conclusion, neonatal mice had a time-dependent susceptibility to C. sakazakii infection, with resistance increasing with increasing age.

Time course of fetal tissue invasion by Listeria monocytogenes following an oral inoculation in pregnant guinea pigs.

Pregnant women are 20 times more likely to develop listeriosis than are members of the general population, and infection can result in abortion, stillbirth, or neonatal illness. The objective of this study was to orally challenge pregnant guinea pigs with Listeria monocytogenes to assess maternal and fetal tissue invasion at postinoculation days 2, 6, 9, and 21. The time course of invasion was followed by fluorescence microscopy and a traditional culture method. Guinea pigs were treated on gestation day 35 with L. monocytogenes doses ranging from 10(4) to 10(8) CFU. L. monocytogenes was isolated and viewed in maternal and fetal tissues as early as 2 days postinoculation. L. monocytogenes was isolated from placentas, fetal livers and brains, and maternal spleens at similar rates, suggesting that invasion of the spleen could be indicative of fetal invasion. When comparing fecal shedding, all animals treated with 10(4) CFU were shedding L. monocytogenes by postinoculation day 7, and all animals treated with the higher doses (10(6) or 10(8) CFU) were shedding L. monocytogenes by postinoculation day 5. These data suggest that L. monocytogenes crosses the fetoplacental barrier and invades the fetus by day 2 after maternal ingestion. When comparing the sensitivities of microscopy and culture, neither method consistently detected L. monocytogenes at a higher rate. However, detection in individual tissues differed. Microscopy was significantly more sensitive with fetal liver (P<0.001) and brain (P<0.001) at the highest dose of 10(8) CFU, but at the lowest dose of 10(4) CFU culture was significantly more sensitive with maternal spleen (P=0.04).

Comparison of virulence of three strains of Cronobacter sakazakii in neonatal CD-1 mice.

Cronobacter sakazakii (Enterobacter sakazakii) is an emerging pathogen that has been isolated from powdered infant formula and associated with outbreaks of infection in infants in neonatal intensive care units. In a previous study, we observed that neonatal CD-1 mice are susceptible to C. sakazakii infection and that the pathogen invades brain, liver, and cecum tissues. The study objective was to compare the virulence of three strains of C. sakazakii in neonatal CD-1 mice. The strains tested were MNW2 (a food isolate), SK81 (a clinical isolate), and 3290 (a clinical isolate). Timed-pregnant CD-1 mice were allowed to give birth on gestation day 19 or 20. Neonatal mice were sexed and culled to 10 per litter, each having five males and five females. Neonates were orally gavaged with C. sakazakii strains MNW2, SK81, or 3290 at doses ranging from 10(2.8) to 10(10.5) CFU on postnatal day 3.5. Pups surviving to postnatal day 10.5 were euthanized, and brain, liver, and cecum tissues were excised. C. sakazakii was isolated from all three tissues in mice treated with C. sakazakii, regardless of strain. C. sakazakii strain 3290 was significantly more invasive in brains (42.1% of mice) than were strains MNW2 (6.7%) and SK81 (15.9%). Mortality was observed for all strains of C. sakazakii tested, with SK81 being significantly more lethal (5.6%) than MNW2 (1.2%) or 3290 (0.6%). Our findings suggest that invasiveness does not necessarily correlate with mortality among different strains of C. sakazakii, and the clinical isolates are more virulent than the food isolate.

Neonatal mice as models for Cronobacter sakazakii infection in infants.

Cronobacter sakazakii is an opportunistic pathogen that has been isolated from powdered infant formulas. C. sakazakii infection can result in serious illnesses such as bacteremia, septicemia, meningitis, and death in at-risk infants who are orally fed contaminated reconstituted powdered infant formulas. The objective of this study was to compare the susceptibilities of BALB/c, C57BL/6, and CD-1 mice to C. sakazakii strain MNW2. We acquired timed-pregnant CD-1 mice and allowed them to give birth naturally. On postnatal day 3.5, each pup was administered a total dose of approximately 10(2) to 10(11) CFU C. sakazakii strain MNW2 in reconstituted powdered infant formula. Mice were observed twice daily for morbidity and mortality. At postnatal day 10.5, the remaining pups were euthanized, and brain, liver, and cecum were excised and analyzed for the presence of C. sakazakii. C. sakazakii was isolated from brains, livers, and ceca in all three mouse strains. The CD-1 mouse strain was the most susceptible of the three, with the lowest infectious dose (10(2) CFU) and the lowest lethal dose (also 10(2) CFU).