Induction of β-Lactamase Activity and Decreased β-Lactam Susceptibility by CO2 in Clinical Bacterial Isolates

Antimicrobial susceptibility testing of clinical isolates is a crucial step toward appropriate treatment of infectious diseases. The clinical isolate Francisella philomiragia 14IUHPL001, recently isolated from a 63-year-old woman with atypical pneumonia, featured decreased susceptibility to β-lactam antibiotics when cultivated in 5% CO2. Quantitative β-lactamase assays demonstrated a significant (P < 0.0001) increase in enzymatic activity between bacteria cultivated in 5% CO2 over those incubated in ambient air. The presence of β-lactamase genes blaTEM and blaSHV was detected in the clinical isolate F. philomiragia 14IUHPL001 by PCR, and the genes were positively identified by nucleotide sequencing. Expression of blaTEM and blaSHV was detected by reverse transcription-PCR during growth at 5% CO2 but not during growth in ambient air. A statistically significant alkaline shift was observed following cultivation of F. philomiragia 14IUHPL001 in both ambient air and 5% CO2, allowing desegregation of the previously reported effects of acidic pH from the currently reported effect of 5% CO2 on blaTEM and blaSHV β-lactamases. To ensure that the observed phenomenon was not unique to F. philomiragia, we evaluated a clinical isolate of blaTEM-carrying Haemophilus influenzae and found parallel induction of blaTEM gene expression and β-lactamase activity at 5% CO2 relative to ambient air. IMPORTANCE β-Lactamase induction and concurrent β-lactam resistance in respiratory tract pathogens as a consequence of growth in a physiologically relevant level of CO2 are of clinical significance, particularly given the ubiquity of TEM and SHV β-lactamase genes in diverse bacterial pathogens. This is the first report of β-lactamase induction by 5% CO2

Bones

I heal in the absence of words with the quiet scratching of charcoal on paper, and with the familiar texture of my thumb blending and erasing shadows with dust. I push my boundaries with new mediums, like watercolors. I experiment often and usually fail. I put my anger into harsh lines, I choose colors based on an emotion and I fill a canvas with the things I cannot say. I heal in brush strokes and in the contours of a piece that says what I want it to say without having to ever say a word. Medium: Charcoal and pen on paper

A novel allele of Alx4 results in reduced Fgf10 expression and failure of eyelid fusion in mice.

Normal fusion of developing eyelids requires coordination of inductive signals from the eyelid mesenchyme with migration of the periderm cell layer and constriction of the eyelids across the eye. Failure of this process results in an eyelids open at birth (EOB) phenotype in mice. We have identified a novel spontaneous allele of Alx4 that displays EOB, in addition to polydactyly and cranial malformations. Alx4 is expressed in the eyelid mesenchyme prior to and during eyelid fusion in a domain overlapping the expression of genes that also play a role in normal eyelid development. We show that Alx4 mutant mice have reduced expression of Fgf10, a key factor expressed in the mesenchyme that is required for initiation of eyelid fusion by the periderm. This is accompanied by a reduced number of periderm cells expressing phosphorylated c-Jun, consistent with the incomplete ablation of Fgf10 expression. Together, these data demonstrate that eyelid fusion in mice requires the expression of Alx4, accompanied by the loss of normal expression of essential components of the eyelid fusion pathway. Mamm Genome 2015 Apr; 26(3-4):173-8

Kidney adysplasia and variable hydronephrosis, a new mutation affecting the odd-skipped related 1 gene in the mouse, causes variable defects in kidney development and hydronephrosis.

Many genes, including odd-skipped related 1 (Osr1), are involved in regulation of mammalian kidney development. We describe here a new recessive mutation (kidney adysplasia and variable hydronephrosis, kavh) in the mouse that leads to downregulation of Osr1 transcript, causing several kidney defects: agenesis, hypoplasia, and hydronephrosis with variable age of onset. The mutation is closely associated with a reciprocal translocation, T(12;17)4Rk, whose Chromosome 12 breakpoint is upstream from Osr1. The kavh/kavh mutant provides a model to study kidney development and test therapies for hydronephrosis. Am J Physiol Renal Physiol 2015 Jun 15; 308(12):F1335-42

Differential Evolutionary Selection and Natural Evolvability Observed in ALT Proteins of Human Filarial Parasites.

The abundant larval transcript (ALT-2) protein is present in all members of the Filarioidea, and has been reported as a potential candidate antigen for a subunit vaccine against lymphatic filariasis. To assess the potential for vaccine escape or heterologous protection, we examined the evolutionary selection acting on ALT-2. The ratios of nonsynonymous (K(a)) to synonymous (K(s)) mutation frequencies (ω) were calculated for the alt-2 genes of the lymphatic filariasis agents Brugia malayi and Wuchereria bancrofti and the agents of river blindness and African eyeworm disease Onchocerca volvulus and Loa loa. Two distinct Bayesian models of sequence evolution showed that ALT-2 of W. bancrofti and L. loa were under significant (P<0.05; P < 0.001) diversifying selection, while ALT-2 of B. malayi and O. volvulus were under neutral to stabilizing selection. Diversifying selection as measured by ω values was notably strongest on the region of ALT-2 encoding the signal peptide of L. loa and was elevated in the variable acidic domain of L. loa and W. bancrofti. Phylogenetic analysis indicated that the ALT-2 consensus sequences formed three clades: the first consisting of B. malayi, the second consisting of W. bancrofti, and the third containing both O. volvulus and L. loa. ALT-2 selection was therefore not predictable by phylogeny or pathology, as the two species parasitizing the eye were selected differently, as were the two species parasitizing the lymphatic system. The most immunogenic regions of L. loa and W. bancrofti ALT-2 sequence as modeled by antigenicity prediction analysis did not correspond with elevated levels of diversifying selection, and were not selected differently than predicted antigenic epitopes in B. malayi and O. volvulus. Measurements of ALT-2 evolvability made by χ2 analysis between alleles that were stable (O. volvulus and B. malayi) and those that were under diversifying selection (W. bancrofti and L. loa) indicated significant (P<0.01) deviations from a normal distribution for both W. bancrofti and L. loa. The relationship between evolvability and selection in L. loa followed a second order polynomial distribution (R2 = 0.89), indicating that the two factors relate to one another in accordance with an additional unknown factor. Taken together, these findings indicate discrete evolutionary drivers acting on ALT-2 of the four organisms examined, and the described variation has implications for design of novel vaccines and diagnostic reagents. Additionally, this represents the first mathematical description of evolvability in a naturally occurring setting

Selection and Evolvability of ALT-2.

<p>Values for selection (ω) and evolvability (E) were calculated for each amino acid residue in the ALT-2 sequence. The single E value was plotted against the ω value for pairwise comparisons between all species. Residues that were both significantly evolvable and under diversifying selection appear in the blue-shaded boxes. The two conserved ALT-2 sequences (<i>O</i>. <i>volvulus</i>, [filled circles] and <i>B malayi</i> [filled diamonds], <b>l A</b>) and the two diversified ALT-2 sequences (<i>L</i>. <i>loa</i> [open squares] and <i>W</i>. <i>bancrofti</i> [open triangles], <b>B</b>) served as positive and negative controls, respectively. Selection and evolvability of ALT-2 from <i>L</i>. <i>loa</i> followed a second-order polynomial distribution when compared to either <i>B</i>. <i>malayi</i> (<b>C</b>) or <i>O</i>. <i>volvulus</i> (<b>E</b>). Only a small number of residues in <i>W</i>. <i>bancrofti</i> were both diversified and evolvable when compared to <i>B</i>. <i>malayi</i> (<b>D</b>), but none were detected when compared to <i>O</i>. <i>volvulus</i> (<b>F</b>).</p

Phylogenetic Analysis.

<p>The evolutionary history of ALT-2 consensus sequences from <i>B</i>. <i>malayi</i>, <i>W</i>. <i>bancrofti</i>, <i>O</i>. <i>volvulus</i>, and <i>L</i>. <i>loa</i> was inferred using (<b>A</b>) the Neighbor-Joining method and (<b>B</b>) the Maximum Parsimony method (N = 500 bootstrap replicates each). Regardless of method, the branching order was the same and was consistent with ribosomal RNA-derived branching order.</p

Antigenicity and Selection Projection.

<p>Calculated ω values using the M8 model (Y axis, bar graph) are projected onto graphical measurements of antigenicity predictions (Z axis, line graph) across all sites in ALT-2 (X axis, marked by position number). Predicted antigenic epitopes are denoted with arrows. The cutoff ω value of 1 is depicted as a black line; values extended above the line represent residues under diversifying selection, and values below the line represent residues under purifying selection. Areas under diversifying selection in ALT-2 of <i>L</i>. <i>loa</i> and <i>W</i>. <i>bancrofti</i> have minimal overlap with predicted antigenicity.</p

Pairwise Evolvability (E) Analysis Design.

<p>Pairwise Evolvability (E) Analysis Design.</p