Detection of Bacterial 16S rRNA and Identification of Four Clinically Important Bacteria by Real-Time PCR

Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and –negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.     

Changing the specificity of a bacterial chemoreceptor

The methyl-accepting chemotaxis proteins are a family of receptors in bacteria that mediate chemotaxis to diverse signals. To explore the plasticity of these proteins, we have developed a simple method for selecting cells that swim to target attractants. The procedure is based on establishing a diffusive gradient in semi-soft agar plates and does not require that the attractant be metabolized or degraded. We have applied this method to select for variants of the Escherichia coli aspartate receptor, Tar, that have a new or improved response to different amino acids. We found that Tar can be readily mutated to respond to new chemical signals. However, the overall change in specificity depended on the target compound. A Tar variant that could detect cysteic acid still showed a strong sensitivity to aspartate, indicating that the new receptor had a broadened specificity relative to wild-type Tar. Tar variants that responded to phenylalanine or N-methyl aspartate, or that had an increased sensitivity to glutamate showed a strong decrease in their response to aspartate. In at least some of the cases, the maximal level of sensitivity that was obtained could not be attributed solely to substitutions within the binding pocket. The new tar alleles and the techniques described here provide a new approach for exploring the relationship between ligand binding and signal transduction by chemoreceptors and for engineering new receptors for applications in biotechnology.     

A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.     

Mechanisms underlying blockade of allograft acceptance by TLR ligands

Immune activation via TLRs is known to prevent transplantation tolerance in multiple animal models. To investigate the mechanisms underlying this barrier to tolerance induction, we used complementary murine models of skin and cardiac transplantation in which prolonged allograft acceptance is either spontaneous or pharmacologically induced with anti-CD154 mAb and rapamycin. In each model, we found that prolonged allograft survival requires the presence of natural CD4(+)Foxp3(+) T regulatory cells (Tregs), and that the TLR9 ligand CpG prevents graft acceptance both by interfering with natural Treg function and by promoting the differentiation of Th1 effector T cells in vivo. We further demonstrate that although Th17 cells differentiate from naive alloreactive T cells, these cells do not arise from natural Tregs in either CpG-treated or untreated graft recipients. Finally, we show that CpG impairs natural Treg suppressor capability and prevents Treg-dependent allograft acceptance in an IL-6-independent fashion. Our data therefore suggest that TLR signals do not prevent prolonged graft acceptance by directing natural Tregs into the Th17 lineage or by using other IL-6-dependent mechanisms. Instead, graft destruction results from the ability of CpG to drive Th1 differentiation and interfere with immunoregulation established by alloreactive natural CD4(+)Foxp3(+) Tregs.     

Genetic mosaic analysis of the equatorial-less mutation in Drosophila mezunogaster

eql (equatorial-less) is a recessive lethal mutation on the second chromosome of Drosophila melanogasfer. J. Campos-Ortega found that eql clones in somatic mosaic flies have reduced numbers of photoreceptor cells, and he suggested that only the R1, R6, and R7 photoreceptor cells were missing in this mutant. These photoreceptor cells help to define the inverted orientation of ommatidial facets along the equatorial midline of the fly eye, hence the mutation was named “equatorial-less”. We have conducted a detailed analysis of the eql mutation, by serial section reconstruction of eql clones marked with bw or w^? in somatic mosaic flies. We found that all photoreceptor cell types (Rl–R8) could be deleted by the eql mutation, and in rare cases the number of photoreceptor cells was increased. The apparent lack of photoreceptor cell type specificity was confirmed by our analysis of genetically mosaic facets, which indicated that no single photoreceptor cell, or subset of photoreceptor cells, was uniquely required to express eql Rather, eql appears to function in all photoreceptor cells, and possibly in all eye precursor cells. The distribution of photoreceptor cell numbers in w eql facets was consistent with the hypothesis that each photoreceptor cell was deleted independently of the others. The eql gene is located on the right arm of chromosome 2 at map location 2 ? 104.5 ± 0.7 and lies between the polytene chromosome bands 59D8 and 60A7. © 1995 Wiley-Liss, Inc.     

Trigenic neural crest-restricted Smad7 over-expression results in congenital craniofacial and cardiovascular defects

Smad7 is a negative regulator of TGFβ superfamily signaling. Using a three-component triple transgenic system, expression of the inhibitory Smad7 was induced via doxycycline within the NCC lineages at pre- and post-migratory stages. Consistent with its role in negatively regulating both TGFβ and BMP signaling in vitro, induction of Smad7 within the NCC significantly suppressed phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo, resulting in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion cells. At the cellular level, increased cell death was observed in pharyngeal arches. However, cell proliferation and NCC-derived smooth muscle differentiation were unaltered. NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were not affected, but subsequent colonization of the OFT was significantly reduced. Induction of Smad7 in post-migratory NCC resulted in interventricular septal chamber septation defects, suggesting that TGFβ superfamily signaling is also essential for cardiac NCC at post-migratory stages to govern normal cardiac development. Taken together, the data illustrate that tightly regulated TGFβ superfamily signaling plays an essential role during craniofacial and cardiac NCC colonization and cell survival in vivo.     

Case–Case Comparison of Candida auris Versus Other Candida Species Bloodstream Infections: Results of an Outbreak Investigation in Colombia

Background

Candida auris is an emerging multidrug-resistant yeast that causes outbreaks in healthcare settings around the world. In 2016, clinicians and public health officials identified patients with C. auris bloodstream infections (BSI) in Colombian healthcare facilities. To evaluate potential risk factors and outcomes for these infections, we investigated epidemiologic and clinical features of patients with C. auris and other Candida species BSI.

Methods

We performed a retrospective case-case investigation in four Colombian acute care hospitals, defining a case as Candida spp. isolated from blood culture during January 2015–September 2016. C. auris BSI cases were compared to other Candida species BSI cases. Odds ratio (OR), estimated using logistic regression, was used to assess the association between risk factors and outcomes.

Results

We analyzed 90 patients with BSI, including 40 with C. auris and 50 with other Candida species. All had been admitted to the intensive care unit (ICU). No significant demographic differences existed between the two groups. The following variables were independently associated with C. auris BSI:?≥?15 days of pre-infection ICU stay (OR: 5.62, CI: 2.04–15.5), evidence of severe sepsis (OR: 3.70, CI 1.19–11.48), and diabetes mellitus (OR 5.69, CI 1.01–31.9).

Conclusion

Patients with C. auris BSI had longer lengths of ICU stay than those with other candidemias, suggesting that infections are acquired during hospitalization. This is different from other Candida infections, which are usually thought to result from autoinfection with host flora.

     

Microarray analysis of cell-free fetal DNA in amniotic fluid: a prenatal molecular karyotype

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.     

The biochemistry of Rubisco in Flaveria

C(4) plants have been reported to have Rubiscos with higher maximum carboxylation rates (kcat(CO(2))) and Michaelis-Menten constants (K(m)) for CO(2) (K(c)) than the enzyme from C(3) species, but variation in other kinetic parameters between the two photosynthetic pathways has not been extensively examined. The CO(2)/O(2) specificity (S(C/O)), kcat(CO(2)), K(c), and the K(m) for O(2) (K(o)) and RuBP (K(m-RuBP)), were measured at 25 degrees C, in Rubisco purified from 16 species of Flaveria (Asteraceae). Our analysis included two C(3) species of Flaveria, four C(4) species, and ten C(3)-C(4) or C(4)-like species, in addition to other C(4) (Zea mays and Amaranthus edulis) and C(3) (Spinacea oleracea and Chenopodium album) plants. The S(C/O) of the C(4) Flaveria species was about 77 mol mol(-1), which was approximately 5% lower than the corresponding value in the C(3) species. For Rubisco from the C(4) Flaverias kcat(CO(2)) and K(c) were 23% and 45% higher, respectively, than for Rubisco from the C(3) plants. Interestingly, it was found that the K(o) for Rubisco from the C(4) species F. bidentis and F. trinervia were similar to the C(3) Flaveria Rubiscos (approximately 650 microM) while the K(o) for Rubisco in the C(4) species F. kochiana, F. australasica, Z. mays, and A. edulis was reduced more than 2-fold. There were no pathway-related differences in K(m-RuBP). In the C(3)-C(4) species kcat(CO(2)) and K(c) were generally similar to the C(3) Rubiscos, but the K(o) values were more variable. The typical negative relationships were observed between S(C/O) and both kcat(CO(2)) and K(c), and a strongly positive relationship was observed between kcat(CO(2)) and Kc. However, the statistical significance of these relationships was influenced by the phylogenetic relatedness of the species.