Using probiotics to inhibit in vivo and in vitro growth of Listeria monocytogenes and Cronobacter sakazakii during fetal and neonatal infection
Agyekum, Augustine Kwaku
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Cronobacter sakazakii and Listeria monocytogenes are two pathogens that are known to cause wide range of diseases in infants, elderly people and immune-compromised people. Some of the major infections that both these pathogens can cause include necrotizing enterocolitis, bacteremia, meningitis, fever, headache and pneumonia. Both these pathogens are prevalent in infant food formulas, contaminated food items and surroundings. Newborn infants, elderly people, (especially those above 60 years of old) and immune-compromised people, are vulnerable for the infections of C. sakazakii and L. monocytogenes. Lactobacillus rhamnosus GG has become one of the widely used microorganisms, to treat infections caused by C. sakazakii and L. monocytogenes because of its probiotic uses. Thus, LGG was tested on reducing the pathogen effects of L. monocytogenes and C. sakazakii during fetal and neonatal development. The primary aim of this study was to investigate the ability LGG to reduce growth and invasion of two pathogenic bacteria, L. monocytogenes and C. sakazakii, in vivo and in vitro. To carry out this research study, CD-1 pups were examined by gavaging with both106 LGG or LGG soluble material on gestation days (gd) 1.5, 3.5 and 4.5 and administered by giving single oral dose of (108, 9 or 12) CFU C. sakazakii or vehicle reconstituted powdered infant formula (RPIF) at (gd) 2.5. The results indicated that both LGG and LGG supernatant have helped in reducing the invasion of C. sakazakii in neonatal mice. This study revealed that the brain is the primary tissue, where higher percentage of C. sakazakii was isolated when compared to other tissues such as liver and spleen. This research study has concluded that both LGG and LGG supernatant play an active role in protecting the neonatal mice from death. LGG was also tested for its ability to affect the growth of the intracellular pathogen L. monocytogenes during fetal development. To elucidate the mechanism of probiotic Lactobacillus rhamnosus GG (LGG) inhibition of L. monocytogenes and C. sakazakii, both in vivo and in vitro models were used. Pregnant guinea pig dams treated with yogurt containing probiotics reduced L. monocytogenes that was isolated from tissues compared to L. monocytogenes treatment alone in liver (56% v 100%, respectively) and spleen (14% v 75%, respectively). Fetal tissues from dams receiving yogurt had fewer positive samples when compared to L. monocytogenes treatment alone in fetal liver (17% v 71%, respectively) and fetal brain (21% v 71% respectively). Importantly, stillbirths were reduced in guinea pigs receiving yogurt and L. monocytogenes as compared to L. monocytogenes alone (14% v. 75%). In vitro, L. monocytogenes ranging from 101-7 colony forming units (CFU)/ml PBS were prepared and added to 106 CFU/ml LGG or LGG supernatant. LGG supernatant’s ability to inhibit L. monocytogenes growth was dependent upon L. monocytogenes concentration up to the highest dose tested, 107 CFU/ml. LGG supernatant treatments inhibited L. monocytogenes growth in a dose dependent manner at concentrations up to 105 CFU/ml L. monocytogenes. Changes in pH environment of L. monocytogenes resulting from the addition of LGG supernatant significantly reduced L. monocytogenes growth. In response to exposure to acidic environments both LGG supernatant or lowering of pH significantly reduced L. monocytogenes growth at all L. monocytogenes test concentrations when compared to controls. LGG supernatant’s ability to inhibit L. monocytogenes growth was dependent upon L. monocytogenes concentration up to the highest dose tested, 107 CFU/ml. LGG supernatant treatments inhibited L. monocytogenes growth in a dose dependent manner at concentrations up to 105 CFU/ml L. monocytogenes. Neither LGG supernatant nor LGG treatments yielded any consistent pattern on L. monocytogenes growth at 107 CFU/ml. The data suggests under the conditions tested, LGG and LGG supernatant significantly reduce C. sakazakii growth and viability and the anti-microbial activity of LGG supernatant is due to (a) heat-stable, non-proteinaceous compound(s). Further, a dose dependent relationship was found with lactic acid treated LGG supernatant and reduced C. sakazakii growth. In conclusion, both an acidic pH environments and LGG supernatant treatments affected L. monocytogenes and C. sakazakii growth and viability in vitro thus, further studies will be needed to investigate the mechanisms involved.