Transcriptional Regulation of icaADBC by both IcaR and TcaR in Staphylococcus epidermidis.

S. epidermidis is a primary cause of biofilm-mediated infections in humans due to adherence to foreign bodies. A major staphylococcal biofilm accumulation molecule is polysaccharide intracellular adhesin (PIA), which is synthesized by enzymes encoded by the icaADBC operon. Expression of PIA is highly variable among clinical isolates, suggesting that PIA expression levels are selected in certain niches of the host. However, the mechanisms that govern enhanced icaADBC transcription and PIA synthesis in these isolates are not known. We hypothesized that enhanced PIA synthesis in these isolates was due to function of IcaR and/or TcaR. Thus, two S. epidermidis isolates (1457 and CSF41498) with different icaADBC transcription and PIA expression levels were studied. Constitutive expression of both icaR and tcaR demonstrated that both repressors are functional and can completely repress icaADBC transcription in both 1457 and CSF41498. However, it was found that IcaR was the primary repressor for CSF41498 and TcaR was the primary repressor for 1457. Further analysis demonstrated that icaR transcription was repressed in 1457 in comparison to CSF41498, suggesting that TcaR functions as a repressor only in the absence of IcaR. Indeed, DNase I footprinting suggests IcaR and TcaR may bind to the same site within the icaR-icaA intergenic region. Lastly, we found mutants expressing variable amounts of PIA could rapidly be selected from both 1457 and CSF41498. Collectively, we propose that strains producing enhanced PIA synthesis are selected within certain niches of the host through several genetic mechanisms that function to repress icaR transcription, thus increasing PIA synthesis.IMPORTANCEStaphylococcus epidermidis is a commensal bacterium that resides on our skin. As a commensal, it protects humans from bacterial pathogens through a variety of mechanisms. However, it is also a significant cause of biofilm infections due to its ability to bind to plastic. Polysaccharide intercellular adhesin is a significant component of biofilm, and we propose that the expression of this polysaccharide is beneficial in certain host niches, such as providing extra strength when the bacterium is colonizing the lumen of a catheter, and detrimental in others, such as colonization of the skin surface. We show here that fine-tuning of icaADBC transcription, and thus PIA synthesis, is mediated via two transcriptional repressors, IcaR and TcaR.

Evaluation of the BioFire FilmArray® GastrointestinalPanel in a Midwestern Academic Hospital.

The BioFire FilmArray® Gastrointestinal Panel (GIP) was implemented to replace traditional stool culture and enzyme immunoassay (EIA) testing for stool pathogens. The purpose of this study was to evaluate the detection rate, incidence of coinfection, and culture recovery rate of gastrointestinal (GI) pathogens detected by the GIP over a 1-year period. A total of 2257 stools collected from January to December 2015 were tested using the GIP. Clostridium difficile colonization was also evaluated by an antigen/toxin EIA and confirmatory polymerase chain reaction (PCR). The GIP detected one pathogen in 911 (40.4%) specimens. Coinfections were detected in 176 (7.8%) of these specimens. The most frequently detected pathogens were C. difficile (15.2%), norovirus (8.9%), enteropathogenic Escherichia coli (7.1%), enteroaggregative E. coli (3.4%), Campylobacter spp. (2.3%), and sapovirus (2.0%). Each of the remaining GIP targets had a detection rate of ≤1.6%. The recovery of bacteria for public health investigations varied, with rates as high as 77% for Salmonella to as low as 30% for Yersinia enterocolitica. Of stools positive for C. difficile on the GIP that were tested by EIA, only 42.7% (88/206) were found to be producing detectable toxin. Overall, the implementation of the GIP resulted in high detection rates of GI pathogens, including the frequent detection of coinfections. This is a promising test to streamline the testing of agents causing infectious gastroenteritis from multiple tests down to a single order with limited hands-on time. Ongoing studies will need to assess the impact that the GIP has on downstream patient care and public health practices.

Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.

Arginine deiminase in Staphylococcus epidermidis functions to augment biofilm maturation through pH homeostasis.

Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.

Assessment of whole-genome mapping in a well-defined outbreak of Salmonella enterica serotype Saintpaul.

We investigated the use of whole-genome mapping and pulsed-field gel electrophoresis (PFGE) with isolates from an outbreak of Salmonella enterica serotype Saintpaul. PFGE and whole-genome mapping were concordant with 22 of 23 isolates. Whole-genome mapping is a viable alternative tool for the epidemiological analysis of Salmonella food-borne disease investigations.

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Clinical evaluation of the BioFire® Respiratory Panel 2.1 and detection of SARS-CoV-2.

We evaluated the performance of the BioFire® Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison against three other SARS CoV-2 EUA assays. In these studies, the RP2.1 panel had 98 % positive percent agreement (48/49) and 100 % negative percent agreement (49/49). Since 30 % of nasopharyngeal swab specimens have a SARS CoV-2 Ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other EUA approved SARS CoV-2 assays. These data suggested that the BioFire® RP2.1 panel, along with four other SARS CoV-2 assays (Roche cobas, Cepheid Xpert Xpress, BioFire® Defense COVID19, and NECoV19), consistently detected viral RNA at the 10-7 dilution. Overall, these studies suggest that the BioFire® RP2.1 assay can be used to detect acute cases of SARS CoV2 in addition to patients with low viral titer later in disease presentation.

Does trophic level drive organic and metallic contamination in coral reef organisms?

Metallic and organic pollutants constitute a serious threat for coral reef ecosystems, potentially affecting a great number of species interacting within complex trophodynamic processes. Pesticides, PCBs and trace elements were measured on coral reef communities of three Pacific islands (Moorea, Wallis and New Caledonia) in relation with δ15N values, a proxy of trophic level. Several potential sources of organic matter, benthic invertebrates and fish belonging to various trophic strategies were sampled at each island. Wallis and New Caledonia displayed, respectively, the highest concentrations of pesticides and trace elements. In the three islands, most trace element concentrations (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and V) decreased when δ15N was rising (i.e. bioreduction), whereas Hg and Se biomagnified with increasing δ15N values. Only few trace elements in some islands did not show any significant trend in relation with δ15N (i.e., Ag in New Caledonia, Zn in Wallis and As plus Zn in Moorea). PCBs concentrations showed a significant bioreduction in New Caledonia and in Moorea, but a significant biomagnification in Wallis. Aldrin and heptachlor were the only pesticides to show a similar significant bioreduction in the three islands. Other pesticides, such as chlordecone, diazinon, endosulfan I and II, heptachlor-epoxide A and B, lindane and pp'-DDE displayed contrasted patterns (e.g. chlordecone significantly biomagnified in New Caledonia, significantly bioreduced in Wallis and did not displayed any significant trend in Moorea). Finally, for unclear reasons, Moorea displayed only negative significant correlations between δ15N and all pesticides (except pp'-DDT). Our results highlight that trophic level, here assessed through δ15N values, is a good predictor of metallic trace elements biomagnification or bioreduction in coral reef organisms. However, at large spatial scale, trophic level relevance to predict pesticides and PCBs biomagnification or bioreduction should be considered with caution and studied in close relation with local characteristics.

Complete Genome Sequence of Staphylococcus epidermidis CSF41498.

Staphylococcus epidermidis CSF41498 is amenable to genetic manipulation and has been used to study mechanisms of biofilm formation. We report here the whole-genome sequence of this strain, which contains 2,427 protein-coding genes and 82 RNAs within its 2,481,008-bp-long genome, as well as three plasmids.

Staphylococcus epidermidis saeR is an effector of anaerobic growth and a mediator of acute inflammation.

The saeRS two-component regulatory system regulates transcription of multiple virulence factors in Staphylococcus aureus. In the present study, we demonstrated that the saePQRS region in Staphylococcus epidermidis is transcriptionally regulated in a temporal manner and is arranged in a manner similar to that previously described for S. aureus. Studies using a mouse foreign body infection model demonstrated that the virulence of strain 1457 and the virulence of a mutant, strain 1457 saeR, were statistically equivalent. However, histological analyses suggested that the polymorphonuclear neutrophil response at 2 days postinfection was significantly greater in 1457-infected mice than in 1457 saeR-infected mice, demonstrating that SaeR influences the early, acute phases of infection. Microarray analysis demonstrated that a saeR mutation affected the transcription of 65 genes (37 genes were upregulated and 28 genes were downregulated); in particular, 8 genes that facilitate growth under anaerobic conditions were downregulated in 1457 saeR. Analysis of growth under anaerobic conditions demonstrated that 1457 saeR had a decreased growth rate compared to 1457. Further metabolic experiments demonstrated that 1457 saeR had a reduced capacity to utilize nitrate as a terminal electron acceptor and exhibited increased production of lactic acid in comparison to 1457. These data suggest that in S. epidermidis SaeR functions to regulate the transition between aerobic growth and anaerobic growth. In addition, when grown anaerobically, 1457 saeR appeared to compensate for the redox imbalance created by the lack of electron transport-mediated oxidation of NADH to NAD+ by increasing lactate dehydrogenase activity and the subsequent oxidation of NADH.

The presence of icaADBC is detrimental to the colonization of human skin by Staphylococcus epidermidis.

Previous studies have demonstrated that Staphylococcus epidermidis isolates colonizing the skin of healthy humans do not typically encode icaADBC, the genes responsible for the production of polysaccharide intercellular adhesin or biofilms. It was therefore hypothesized that the presence of icaADBC was deleterious to the successful colonization of human skin by S. epidermidis. Using a human skin competition model, it was determined that the strong biofilm-producing S. epidermidis strain 1457 was outcompeted at 1, 3, and 10 days by an isogenic icaADBC mutant (1457 ica::dhfr), suggesting a fitness cost for carriage of icaADBC.

Gene trapping with GFP: the isolation of developmental mutants in the slime mold Polysphondylium.

In order to study how a cell mass undergoes a transition from one symmetry to another in the slime mold Polysphondylium, we developed a genetic screen in which mutant phenotype and gene expression can easily be visualized in the living organism. The screen combines restriction enzyme-mediated integration (REMI) [1,2] and green fluorescent protein (GFP) [3] expression. In REMI, a restriction enzyme is electroporated along with linearized vector into cells, thus determining the site of plasmid insertion and often increasing the integration frequency. A set of transforming plasmids carrying the GFP coding sequence in three reading frames was used for transformation. The plasmids were constructed so that GFP could be expressed only under control of a host promoter. Living transformants expressing GFP spatially and temporally could be rapidly identified in a very large background of non-expressing cells and fruiting bodies. The phenotypes of representative mutants range from cells that cannot aggregate and initiate cell-cell interactions, through mutant fruiting bodies, to apparently wild-type fruiting bodies expressing GFP in all or a subpopulation of cells. The ability to screen mutant living cells and tissues for GFP expression is rapid and effective and likely to have application in many transformable systems where screening by gene and promoter trapping is essential for understanding temporal and spatial gene regulation.

Molecular analysis of Francisella tularensis subspecies tularensis and holarctica.

Rapid methods are needed for public health and military applications to specifically identify Francisella tularensis, the causative agent of tularemia in humans. A comparative analysis of the capabilities of multiple technologies was performed using a well-defined set of organisms to determine which approach would provide the most information in the shortest time. High-resolution molecular techniques, including pulsed-field gel electrophoresis, amplified fragment length polymorphism, and ribotyping, provided subspecies level identification within approximately 24 hours after obtaining an isolate, whereas multilocus variable number tandem repeat analysis with 8 or 25 targets provided strain level discrimination within about 12 hours. In contrast, Raman spectroscopy provided species level identification in 10 minutes but could not differentiate between subspecies tularensis and holarctica.

Molecular motors and membrane traffic in Dictyostelium.

Phagocytosis and membrane traffic in general are largely dependent on the cytoskeleton and their associated molecular motors. The myosin family of motors, especially the unconventional myosins, interact with the actin cortex to facilitate the internalization of external materials during the early steps of phagocytosis. Members of the kinesin and dynein motor families, which mediate transport along microtubules (MTs), facilitate the intracellular processing of the internalized materials and the movement of membrane. Recent studies indicate that some unconventional myosins are also involved in membrane transport, and that the MT- and actin-dependent transport systems might interact with each other. Studies in Dictyostelium have led to the discovery of many motors involved in critical steps of phagocytosis and membrane transport. With the ease of genetic and biochemical approaches, the established functional analysis to test phagocytosis and vesicle transport, and the effort of the Dictyostelium cDNA and Genome Projects, Dictyostelium will continue to be a superb model system to study phagocytosis in particular and cytoskeleton and motors in general.

Plasma choline and betaine and their relation to plasma homocysteine in normal pregnancy.

BACKGROUND: Plasma concentrations of total homocysteine (tHcy) decrease during pregnancy. This reduction has been investigated in relation to folate status, but no study has addressed the possible role of betaine and its precursor choline. OBJECTIVE: We investigated the courses of plasma choline and betaine during normal human pregnancy and their relations to plasma tHcy. DESIGN: Blood samples were obtained monthly; the initial samples were taken at gestational week (GW) 9, and the last samples were taken approximately 3 mo postpartum. The study population comprised 50 women of West African descent. Most of the subjects took folic acid irregularly. RESULTS: Plasma choline (geometric x; 95% reference interval) increased continuously during pregnancy, from 6.6 (4.5, 9.7) micromol/L at GW 9 to 10.8 (7.4, 15.6) micromol/L at GW 36. Plasma betaine decreased in the first half of pregnancy, from 16.3 (8.6, 30.8) micromol/L at GW 9 to 10.3 (6.6, 16.2) micromol/L at GW 20 and remained constant thereafter. We confirmed a reduction in plasma tHcy, and the lowest concentration was found in the second trimester. From GW 16 onward, an inverse relation between plasma tHcy and betaine was observed. Multiple regression analysis showed that plasma betaine was a strong predictor of plasma tHcy from GW 20 onward. CONCLUSIONS: The steady increase in choline throughout gestation may ensure choline availability for placental transfer with subsequent use by the growing fetus. Betaine becomes a strong predictor of tHcy during the course of pregnancy. Both of these findings emphasize the importance of choline and betaine status during normal human pregnancy.

Expression of prestalk and prespore proteins in minute, two-dimensional Dictyostelium slugs.

We show that exceedingly small two-dimensional slugs of Dictyostelium differentiate normally and have an anterior prestalk zone and a posterior prespore zone. Using GFP as a marker attached to the appropriate promoter, prestalk expression is concentrated in the anterior, while prespore expression is produced in the posterior, closely resembling what is found in normal, large slugs.

Green fluorescent protein production in the cellular slime molds Polysphondylium pallidum and Dictyostelium discoideum.

The green fluorescent protein-encoding gene from Aequorea victoria has been cloned into several different transforming vectors and expressed in the cellular slime molds, Polysphondylium pallidum and Dictyostelium discoideum. We find that the protein is stable and non-toxic in both species, can be easily visualized in living and fixed specimens, and can be used to purify rare cells by fluorescence-activated cell sorting (FACS).

Outbreak of vancomycin-resistant Enterococcus faecium in a neonatal intensive care unit.

An outbreak of vancomycin-resistant Enterococcus faecium involving 28 infants in a neonatal intensive care unit was observed. Successful control of the outbreak was achieved following use of patient and staff cohorting, contact isolation precautions, patient and environmental surveillance cultures, environmental decontamination, molecular typing, introduction of an alcohol-based hand disinfectant, and decreased use of vancomycin.

Genome diversity among regional populations of Francisella tularensis subspecies tularensis and Francisella tularensis subspecies holarctica isolated from the US.

Francisella tularensis is a highly infectious facultative intracellular pathogen that is considered a potential agent of bioterrorism. Four different F. tularensis subspecies have been identified and they appear to display different ecological and virulence characteristics as well as differences in geographical distribution. One simple explanation for the variation in ecological and virulence characteristics is that they are conferred by differences in genome content. To characterize genome content among stains isolated from United States, we have used a DNA microarray designed from a shotgun library of a reference strain. Polymorphisms distributed among polyphyletic sets of strains was the most common pattern of genome alteration observed, indicating that strain-specific genome variability is significant. Nonetheless, 13 different contiguous segments of the genome were found to be missing exclusively in each of the subsp. holarctica strains tested. All 13 are associated with repeat sequences or transposases that could promote insertion/deletion events. Comparison of the live vaccine strain to other holarctica strains also identified three regions that are absent exclusively in the live vaccine strain derived from holarctica.