Acid-adapted strains of Escherichia coli K-12 obtained by experimental evolution.

Enteric bacteria encounter a wide range of pHs throughout the human intestinal tract. We conducted experimental evolution of Escherichia coli K-12 to isolate clones with increased fitness during growth under acidic conditions (pH 4.5 to 4.8). Twenty-four independent populations of E. coli K-12 W3110 were evolved in LBK medium (10 g/liter tryptone, 5 g/liter yeast extract, 7.45 g/liter KCl) buffered with homopiperazine-N,N'-bis-2-(ethanosulfonic acid) and malate at pH 4.8. At generation 730, the pH was decreased to 4.6 with HCl. By 2,000 generations, all populations had achieved higher endpoint growth than the ancestor at pH 4.6 but not at pH 7.0. All evolving populations showed a progressive loss of activity of lysine decarboxylase (CadA), a major acid stress enzyme. This finding suggests a surprising association between acid adaptation and moderation of an acid stress response. At generation 2,000, eight clones were isolated from four populations, and their genomes were sequenced. Each clone showed between three and eight missense mutations, including one in a subunit of the RNA polymerase holoenzyme (rpoB, rpoC, or rpoD). Missense mutations were found in adiY, the activator of the acid-inducible arginine decarboxylase (adiA), and in gcvP (glycine decarboxylase), a possible acid stress component. For tests of fitness relative to that of the ancestor, lacZ::kan was transduced into each strain. All acid-evolved clones showed a high fitness advantage at pH 4.6. With the cytoplasmic pH depressed by benzoate (at external pH 6.5), acid-evolved clones showed decreased fitness; thus, there was no adaptation to cytoplasmic pH depression. At pH 9.0, acid-evolved clones showed no fitness advantage. Thus, our acid-evolved clones showed a fitness increase specific to low external pH.

Partial convergence of the human vaginal and rectal maternal microbiota in late gestation and early post-partum.

The human vaginal and fecal microbiota change during pregnancy. Because of the proximity of these perineal sites and the evolutionarily conserved maternal-to-neonatal transmission of the microbiota, we hypothesized that the microbiota of these two sites (rectal and vaginal) converge during the last gestational trimester as part of the preparation for parturition. To test this hypothesis, we analyzed 16S rRNA sequences from vaginal introitus and rectal samples in 41 women at gestational ages 6 and 8 months, and at 2 months post-partum. The results show that the human vaginal and rectal bacterial microbiota converged during the last gestational trimester and into the 2nd month after birth, with a significant decrease in Lactobacillus species in both sites, as alpha diversity progressively increased in the vagina and decreased in the rectum. The microbiota convergence of the maternal vaginal-anal sites perinatally might hold significance for the inter-generational transmission of the maternal microbiota.

Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis observed by fluorescence ratio imaging microscopy.

The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis, unlike the case with E. coli, cytoplasmic pH showed no "overshoot" but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.

Bacterial DNA is present in the fetal intestine and overlaps with that in the placenta in mice.

Bacterial DNA has been reported in the placenta and amniotic fluid by several independent groups of investigators. However, it's taxonomic overlap with fetal and maternal bacterial DNA in different sites has been poorly characterized. Here, we determined the presence of bacterial DNA in the intestines and placentas of fetal mice at gestational day 17 (n = 13). These were compared to newborn intestines (n = 15), maternal sites (mouth, n = 6; vagina, n = 6; colon, n = 7; feces, n = 8), and negative controls to rule out contamination. The V4 region of the bacterial 16S rRNA gene indicated a pattern of bacterial DNA in fetal intestine similar to placenta but with higher phylogenetic diversity than placenta or newborn intestine. Firmicutes were the most frequently assignable phylum. SourceTracker analysis suggested the placenta as the most commonly identifiable origin for fetal bacterial DNA, but also over 75% of fetal gut genera overlapped with maternal oral and vaginal taxa but not with maternal or newborn feces. These data provide evidence for the presence of bacterial DNA in the mouse fetus.

Increased weight gain by C-section: Functional significance of the primordial microbiome.

Epidemiological evidence supports a direct association between early microbiota impact-including C-section-and obesity. We performed antibiotic-free, fostered C-sections and determined the impact on the early microbiota and body weight during development. Mice in the C-section group gained more body mass after weaning, with a stronger phenotype in females. C-section-born mice lacked the dynamic developmental gut microbiota changes observed in control mice. The results demonstrate a causal relationship between C-section and increased body weight, supporting the involvement of maternal vaginal bacteria in normal metabolic development.

The first microbial environment of infants born by C-section: the operating room microbes.

BACKGROUND: Newborns delivered by C-section acquire human skin microbes just after birth, but the sources remain unknown. We hypothesized that the operating room (OR) environment contains human skin bacteria that could be seeding C-section born infants. RESULTS: To test this hypothesis, we sampled 11 sites in four operating rooms from three hospitals in two cities. Following a C-section procedure, we swabbed OR floors, walls, ventilation grids, armrests, and lamps. We sequenced the V4 region of the 16S rRNA gene of 44 samples using Illumina MiSeq platform. Sequences were analyzed using the QIIME pipeline. Only 68 % of the samples (30/44, >1000 sequences per site) yielded sufficient DNA reads to be analyzed. The bacterial content of OR dust corresponded to human skin bacteria, with dominance of Staphylococcus and Corynebacterium. Diversity of bacteria was the highest in the ventilation grids and walls but was also present on top of the surgery lamps. Beta diversity analyses showed OR dust bacterial content clustering first by city and then by hospital (t test using unweighted UniFrac distances, p < 0.05). CONCLUSIONS: We conclude that the dust from ORs, collected right after a C-section procedure, contains deposits of human skin bacteria. The OR microbiota is the first environment for C-section newborns, and OR microbes might be seeding the microbiome in these babies. Further studies are required to identify how this OR microbiome exposure contributes to the seeding of the neonatal microbiome. The results might be relevant to infant health, if the current increase in risk of immune and metabolic diseases in industrialized societies is related to lack of natural exposure to the vaginal microbiome during labor and birth.

Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli.

Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2-7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.

Escherichia coli K-12 survives anaerobic exposure at pH 2 without RpoS, Gad, or hydrogenases, but shows sensitivity to autoclaved broth products.

Escherichia coli and other enteric bacteria survive exposure to extreme acid (pH 2 or lower) in gastric fluid. Aerated cultures survive via regulons expressing glutamate decarboxylase (Gad, activated by RpoS), cyclopropane fatty acid synthase (Cfa) and others. But extreme-acid survival is rarely tested under low oxygen, a condition found in the stomach and the intestinal tract. We observed survival of E. coli K-12 W3110 at pH 1.2-pH 2.0, conducting all manipulations (overnight culture at pH 5.5, extreme-acid exposure, dilution and plating) in a glove box excluding oxygen (10% H2, 5% CO2, balance N2). With dissolved O2 concentrations maintained below 6 µM, survival at pH 2 required Cfa but did not require GadC, RpoS, or hydrogenases. Extreme-acid survival in broth (containing tryptone and yeast extract) was diminished in media that had been autoclaved compared to media that had been filtered. The effect of autoclaved media on extreme-acid survival was most pronounced when oxygen was excluded. Exposure to H2O2 during extreme-acid treatment increased the death rate slightly for W3110 and to a greater extent for the rpoS deletion strain. Survival at pH 2 was increased in strains lacking the anaerobic regulator fnr. During anaerobic growth at pH 5.5, strains deleted for fnr showed enhanced transcription of acid-survival genes gadB, cfa, and hdeA, as well as catalase (katE). We show that E. coli cultured under oxygen exclusion (<6 µM O2) requires mechanisms different from those of aerated cultures. Extreme acid survival is more sensitive to autoclave products under oxygen exclusion.