ABSTRACTThe ability ofEscherichia coliandBacillus subtilisto 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. TheE. colicytoplasmic 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 anE. colibiofilm, 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. subtiliscells were acid shifted from pH 7.5 to pH 6.0. InB. subtilis, unlike the case withE. coli, cytoplasmic pH showed no “overshoot” but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individualB. subtiliscells (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.

BackgroundNewborns 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.ResultsTo 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).ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0126-1) contains supplementary material, which is available to authorized users.

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.

Mice born by C-section developed increased body weight and lacked microbiota developmental changes observed in control 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.