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ABSTRACT: A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.
FEMS Microbiology Letters 05/2004; 233(1):131-8. · 2.04 Impact Factor
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ABSTRACT: Microbial 16S rDNA from babies' fecal samples were amplified by PCR, and analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. PCR-DGGE profiles were used to follow in time the colonization of the intestine by bacteria. Four healthy babies, one baby who received antibiotics and their parents participated to the present study to determine the extent to which administration of antibiotics can modify the bacterial colonization of neonatal human gut and verify the influence of parental factors on the formation of the fecal bacterial community. In the healthy babies, Escherichia coli or bacteria belonging to Clostridium spp. were the initial colonizers rapidly followed by Bifidobacterium, Bacteroides, Clostridium, Streptococcus, Enterococcus and Actinomyces. Bifidobacterium species appeared already after five days in the breast-fed babies while there was a delay in the baby who received a formula based diet during only one day after birth. In each baby two or three bifidobacterial species including B. infantis were found. The observed variations in species were not associated with the feeding changes. The comparison of DGGE profiles of the babies and their parents patterns showed bands with equal migration suggesting a vertical transmission determined by genetic and environmental factors. The brief appearance of pioneer bacteria determined as being E. coli and Enterococcus spp. in the profile from the baby under antibiotic therapy, was succeeded by a small stable community consisting of Ruminococcus species. No Bifidobacterium sequences were detectable in this antibiotic-treated baby in spite of a partly breast-milk diet.
Anaerobe 11/2003; 9(5):219-29. · 2.41 Impact Factor
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ABSTRACT: The establishment of bacterial communities in two healthy babies was examined for more than the first 10 months of life by monitoring 16S ribosomal DNA (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE) and by analyzing the sequences of the major ribotypes. DGGE profiles of the dominant populations in the intestines of the infants were obtained by analyzing daily or weekly fecal samples. After delivery, the germfree infant gastrointestinal tracts were rapidly colonized, and the succession of bacteria in each ecosystem was monitored. During the first few days of life the profiles were simple, but they became more complex as the bacterial diversity increased with time in both babies. Clone libraries of amplified 16S rDNA fragments from baby feces were constructed, and these libraries allowed identification of the bacterial types by comparative DNA sequence analysis; the bacteria identified included members of the genera Bifidobacterium, Ruminococcus, Enterococcus, Clostridium, and Enterobacter: Species most closely related to the genera Bifidobacterium and Ruminococcus in particular dominated the intestinal microbiota based on the stability over time and the numbers, as estimated by the intensities of the bands. However, 19 of the 34 cloned rDNA sequences exhibited less than 97% identity with sequences of known bacteria or cloned sequences in databases. This study showed that using PCR-DGGE and 16S rDNA sequence analysis together resulted in a dynamic description of bacterial colonization in the infant intestinal ecosystem and allowed visualization of bacteria that are difficult to cultivate or to detect by other methods.
Applied and Environmental Microbiology 02/2002; 68(1):219-26. · 3.83 Impact Factor
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ABSTRACT: At the time of weaning, major quantitative and qualitative changes occur in the composition of the intestinal microbiota of piglets, influenced by diet, environmental factors, and the host. Within a short period of time, the intestinal microbiota must ultimately develop from a simple, unstable community into a complex and stable one. Here we present data on the development of the intestinal microbiota based on 16S rRNA gene sequence diversity. In addition to a PCR-based analysis of the 16S rRNA gene by cloning and denaturing gradient gel electrophoresis (DGGE), data on fluorescent in situ hybridisation (FISH) are presented to quantify the total bacterial communities, major Lactobacillus populations and specific Lactobacillus species. The results reported here indicate that the addition of non-digestible, fermentable carbohydrates (= prebiotics) leads to an enrichment of lactobacilli in the small intestine, and increased stability and diversity of the bacterial community in the colon. The data support the hypothesis that changes of the diet can modulate the composition of the microbiota in the intestine. These findings may have potentially major implications for the development of dietary strategies aiming to improve animal health during the weaning process. Études de la diversité microbienne de l’écosystème gastro-intestinal porcin au moment du sevrage. Au moment du sevrage, des changements majeurs, influencés par des facteurs alimentaires et environnementaux et par l’hôte, interviennent dans la composition de la flore intestinale des porcelets. A partir de la naissance, celle-ci doit se développer d’une communauté simple et instable à une autre, complexe et stable. Dans cette revue, nous présentons des résultats portant sur le développement de la flore intestinale et basés sur l’analyse de la diversité des séquences d’ADNr 16S. En plus des analyses d’ADNr 16S basées sur la PCR par clonage et électrophorèse en gel de gradient de dénaturation (DGGE), des résultats d’hybridation in situ (FISH) sont présentés pour quantifier les populations majeures de Lactobacillus. Les résultats indiquent que l’addition de glucides non digestibles mais fermentescibles (= prébiotiques) ont conduit à un développement de lactobacilli dans l’intestin grêle, et à une diversité et une stabilité accrues de la flore dans le colon. Ces résultats supportent l’hypothèse selon laquelle des changements alimentaires peuvent modifier la composition de la flore de l’intestin. Ces observations ont des implications majeures pour la santé pendant le processus de sevrage.
http://dx.doi.org/10.1051/animres:2004019.
