Insights into U-to-C RNA editing from the lycophyte Phylloglossum drummondii.

The lycophyte Phylloglossum drummondii is the sole inhabitant of its genus in the Huperzioideae group and one of a small minority of plants which perform uridine to cytidine RNA editing. We assembled the P. drummondii chloroplast and mitochondrial genomes and used RNA sequence data to build a comprehensive profile of organellar RNA editing events. In addition to many C-to-U editing events in both organelles, we found just four U-to-C editing events in the mitochondrial transcripts cob, nad1, nad5 and rpl2. These events are conserved in related lycophytes in the genera Huperzia and Phlegmariurus. De novo transcriptomes for three of these lycophytes were assembled to search for putative U-to-C RNA editing enzymes. Four putative U-to-C editing factors could be matched to the four mitochondrial U-to-C editing sites. Due to the unusually few numbers of U-to-C RNA editing sites, P. drummondii and related lycophytes are useful models for studying this poorly understood mechanism.

Genomic description of acquired fluconazole- and echinocandin-resistance in patients with serial Candida glabrata isolates.

Candida glabrata is one of the most common causes of systemic candidiasis, often resistant to antifungal medications. To describe the genomic context of emerging resistance, we conducted a retrospective analysis of 82 serially collected isolates from 33 patients from population-based candidemia surveillance in the United States. We used whole-genome sequencing to determine the genetic relationships between isolates obtained from the same patient. Phylogenetic analysis demonstrated that isolates from 29 patients were clustered by patient. The median SNPs between isolates from the same patient was 30 (range: 7-96 SNPs), while unrelated strains infected four patients. Twenty-one isolates were resistant to echinocandins, and 24 were resistant to fluconazole. All echinocandin-resistant isolates carried a mutation either in the FKS1 or FKS2 HS1 region. Of the 24 fluconazole-resistant isolates, 17 (71%) had non-synonymous polymorphisms in the PDR1 gene, which were absent in susceptible isolates. In 11 patients, a genetically related resistant isolate was collected after recovering susceptible isolates, indicating in vivo acquisition of resistance. These findings allowed us to estimate the intra-host diversity of C. glabrata and propose an upper boundary of 96 SNPs for defining genetically related isolates, which can be used to assess donor-to-host transmission, nosocomial transmission, or acquired resistance. IMPORTANCE In our study, mutations associated to azole resistance and echinocandin resistance were detected in Candida glabrata isolates using a whole-genome sequence. C. glabrata is the second most common cause of candidemia in the United States, which rapidly acquires resistance to antifungals, in vitro and in vivo.

Risk factors for community-associated Clostridioides difficile infection in young children.

The majority of paediatric Clostridioides difficile infections (CDI) are community-associated (CA), but few data exist regarding associated risk factors. We conducted a case-control study to evaluate CA-CDI risk factors in young children. Participants were enrolled from eight US sites during October 2014-February 2016. Case-patients were defined as children aged 1-5 years with a positive C. difficile specimen collected as an outpatient or ⩽3 days of hospital admission, who had no healthcare facility admission in the prior 12 weeks and no history of CDI. Each case-patient was matched to one control. Caregivers were interviewed regarding relevant exposures. Multivariable conditional logistic regression was performed. Of 68 pairs, 44.1% were female. More case-patients than controls had a comorbidity (33.3% vs. 12.1%; P = 0.01); recent higher-risk outpatient exposures (34.9% vs. 17.7%; P = 0.03); recent antibiotic use (54.4% vs. 19.4%; P < 0.0001); or recent exposure to a household member with diarrhoea (41.3% vs. 21.5%; P = 0.04). In multivariable analysis, antibiotic exposure in the preceding 12 weeks was significantly associated with CA-CDI (adjusted matched odds ratio, 6.25; 95% CI 2.18-17.96). Improved antibiotic prescribing might reduce CA-CDI in this population. Further evaluation of the potential role of outpatient healthcare and household exposures in C. difficile transmission is needed.

Invasive Methicillin-Resistant Staphylococcus aureus USA500 Strains from the U.S. Emerging Infections Program Constitute Three Geographically Distinct Lineages.

USA500 isolates are clonal complex 8 (CC8) Staphylococcus aureus strains closely related to the prominent community- and hospital-associated USA300 group. Despite being relatively understudied, USA500 strains cause a significant burden of disease and are the third most common methicillin-resistant S. aureus (MRSA) strains identified in the U.S. Emerging Infections Program (EIP) invasive S. aureus surveillance. To better understand the genetic relationships of the strains, we sequenced the genomes of 539 USA500 MRSA isolates from sterile site infections collected through the EIP between 2005 and 2013 in the United States. USA500 isolates fell into three major clades principally separated by their distribution across different U.S. regions. Clade C1 strains, found principally in the Northeast, were associated with multiple IS256 insertion elements in their genomes and higher levels of antibiotic resistance. C2 was associated with Southern states, and E1 was associated with Western states. C1 and C2 strains all shared a frameshift in the gene encoding AdsA surface-attached surface protein. We propose that the term "USA500" should be used for CC8 strains sharing a recent common ancestor with the C1, C2, and E1 strains but not in the USA300 group.IMPORTANCE In this work, we have removed some of the confusion surrounding the use of the name "USA500," placed USA500 strains in the context of the CC8 group, and developed a strategy for assignment to subclades based on genome sequence. Our new phylogeny of USA300/USA500 will be a reference point for understanding the genetic adaptations that have allowed multiple highly virulent clonal strains to emerge from within CC8 over the past 50 years.

Epidemiology and factors associated with candidaemia following Clostridium difficile infection in adults within metropolitan Atlanta, 2009-2013.

We assessed prevalence of and risk factors for candidaemia following Clostridium difficile infection (CDI) using longitudinal population-based surveillance. Of 13 615 adults with CDI, 113 (0·8%) developed candidaemia in the 120 days following CDI. In a matched case-control analysis, severe CDI and CDI treatment with vancomycin + metronidazole were associated with development of candidaemia following CDI.

Electron tomographic structure and protein composition of isolated rat cerebellar, hippocampal and cortical postsynaptic densities.

Electron tomography and immunogold labeling were used to analyze similarities and differences in the morphology and protein composition of postsynaptic densities (PSDs) isolated from adult rat cerebella, hippocampi, and cortices. There were similarities in physical dimensions and gross morphology between cortical, hippocampal and most cerebellar PSDs, although the morphology among cerebellar PSDs could be categorized into three distinct groups. The majority of cerebellar PSDs were composed of dense regions of protein, similar to cortical and hippocampal PSDs, while others were either composed of granular or lattice-like protein regions. Significant differences were found in protein composition and organization across PSDs from the different brain regions. The signaling protein, ßCaMKII, was found to be a major component of each PSD type and was more abundant than αCaMKII in both hippocampal and cerebellar PSDs. The scaffold molecule PSD-95, a major component of cortical PSDs, was found absent in a fraction of cerebellar PSDs and when present was clustered in its distribution. In contrast, immunogold labeling for the proteasome was significantly more abundant in cerebellar and hippocampal PSDs than cortical PSDs. Together, these results indicate that PSDs exhibit remarkable diversity in their composition and morphology, presumably as a reflection of the unique functional demands placed on different synapses.

Prevalence of blaZ gene types and the inoculum effect with cefazolin among bloodstream isolates of methicillin-susceptible Staphylococcus aureus.

We sought to define the prevalence of blaZ gene types and the inoculum effect to cefazolin among methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections. The blaZ gene was present in 142/185 (77%) isolates. A total of 50 (27%) isolates had a ≥4-fold increase in the cefazolin MIC from a standard to a high inoculum, and 8 (4%) demonstrated a nonsusceptible cefazolin MIC, all type A blaZ strains. The efficacy of cefazolin in the presence of the inoculum effect requires further study.

Electron cryotomography of postsynaptic densities during development reveals a mechanism of assembly.

Postsynaptic densities (PSDs) are responsible for organizing receptors and signaling proteins that regulate excitatory transmission in the mammalian brain. To better understand the assembly and 3D organization of this synaptic structure, we employed electron cryotomography to visualize general and fine structural details of PSDs isolated from P2, P14, P21 and adult forebrain in the absence of fixatives and stains. PSDs at P2 are a loose mesh of filamentous and globular proteins and during development additional protein complexes are recruited onto the mesh. Quantitative analysis reveals that while the surface area of PSDs is relatively constant, the thickness and protein occupancy of the PSD volume increase dramatically between P14 and adult. One striking morphological feature is the appearance of lipid raft-like structures, first evident in PSDs from 14 day old animals. These detergent-resistant membranes stain for GM1 ganglioside and their terminations can be clearly seen embedded in protein "bowls" within the PSD complex. In total, these results lead to the conclusion that the PSD is assembled by the gradual recruitment and stabilization of proteins within an initial mesh that systematically adds complexity to the structure.

Vascular access thrombosis and interventions in patients missing hemodialysis sessions.

BACKGROUND: Vascular access (VA) failure is a major complication in patients with end-stage renal disease (ESRD) receiving hemodialysis (HD). Thrombosis is the most common cause of VA dysfunction, but the risk factors for VA thrombosis are not well established. While the practice of missing HD sessions (HDs) is associated with increased morbidity and mortality, its impact on VA outcomes is unknown. We evaluated the impact of missing HDs on thrombosis and intervention rates in arteriovenous (AV) accesses. METHODS: Retrospective review of prevalent HD patients using AV access was done in 2 outpatient HD centers at The Ohio State University over a one-year period. RESULTS: A total of 142 patients underwent a total of 15,692 HDs, missing 1,602 HDs. Of the 78 patients who met the inclusion criteria, 50 patients missed at least 1 HD. Those with AVF demonstrated no significant association between missing HDs and VA thrombosis. Also, the incidence rate (IR) of intervention was not significantly different for those missing and not missing HDs. However, in the AVG group, those missing HDs were more likely to experience VA thrombosis (OR 9.48, p ≈ 0.041) and had a higher IR of intervention. CONCLUSION: The practice of missing HDs was prevalent. Those missing dialysis sessions with AVG were more likely to experience VA thrombosis and needed more interventions to maintain VA patency. Our study reveals a differential impact of missing HDs on thrombosis in AVG and AVF, depicting a need to explore mechanistic explanations that may eventually help develop specific preventive strategies.

A high-density SNP genome-wide linkage scan in a large autism extended pedigree.

We performed a high-density, single nucleotide polymorphism (SNP), genome-wide scan on a six-generation pedigree from Utah with seven affected males, diagnosed with autism spectrum disorder. Using a two-stage linkage design, we first performed a nonparametric analysis on the entire genome using a 10K SNP chip to identify potential regions of interest. To confirm potentially interesting regions, we eliminated SNPs in high linkage disequilibrium (LD) using a principal components analysis (PCA) method and repeated the linkage results. Three regions met genome-wide significance criteria after controlling for LD: 3q13.2-q13.31 (nonparametric linkage (NPL), 5.58), 3q26.31-q27.3 (NPL, 4.85) and 20q11.21-q13.12 (NPL, 5.56). Two regions met suggestive criteria for significance 7p14.1-p11.22 (NPL, 3.18) and 9p24.3 (NPL, 3.44). All five chromosomal regions are consistent with other published findings. Haplotype sharing results showed that five of the affected subjects shared more than a single chromosomal region of interest with other affected subjects. Although no common autism susceptibility genes were found for all seven autism cases, these results suggest that multiple genetic loci within these regions may contribute to the autism phenotype in this family, and further follow-up of these chromosomal regions is warranted.

Dual proline labeling protocol for individual "baseline" and "response" biosynthesis measurements in human articular cartilage.

OBJECTIVE: The heterogeneity of biosynthesis in human-derived cartilage explants poses a challenge to its use in experiments. The aim of this study was to determine the consistency with which two consecutive measures of biosynthesis could be made in individual human articular cartilage explants using a dual proline radiolabeling protocol. METHODS: Full-thickness cartilage explants were harvested from young bovine or human (total knee replacement) tibial plateaus. Two consecutive measurements of biosynthesis were obtained by measuring (3)H-proline and (14)C-proline incorporation. Each sample's ratio of (14)C-/(3)H-proline incorporation was computed. For comparison to traditional experimental designs, the (14)C-proline incorporation ratio was computed for adjacent cartilage samples. The number of samples needed to observe a change in the proline incorporation ratio of 10, 20, and 50% was determined for both methods. RESULTS: The dual-label ratio was consistent across samples from the same plateau [95% confidence interval (CI): +/-20% (human) and +/-30% (bovine) of median]. Adjacent human sample pairs had much greater variability in their (14)C-proline incorporation (95% CI: +/-50% of median). Adjacent bovine sample pairs had CIs that were similar in magnitude to those for the dual-label approach. In the human plateaus, ratio changes of 10, 20 and 50% could be detected using dramatically fewer samples than the adjacent pair method. For bovine samples, the two methods required a similar number of samples per group. CONCLUSION: The consistency of the dual-label approach may overcome the difficulties in studying the effects of interventions on biosynthesis in human cartilage in vitro.

Changing characteristics of invasive pneumococcal disease in Metropolitan Atlanta, Georgia, after introduction of a 7-valent pneumococcal conjugate vaccine.

BACKGROUND: The rate of invasive pneumococcal disease (IPD) has decreased among both immunized children and nonimmunized adults since the licensure of a heptavalent pneumococcal conjugate vaccine (PCV7) for use in infants in the United States in 2000. METHODS: Temporal trends in IPD incidence, clinical syndromes, and underlying conditions were analyzed using active laboratory- and population-based surveillance data from the Centers for Disease Control and Prevention-sponsored Georgia Emerging Infections Program for the 20-county Metropolitan Atlanta, Georgia, for the period of July 1997 through June 2004. P values were determined by test for trend. RESULTS: Since 2000, there have been significant decreases in the rates of invasive pneumococcal pneumonia (relative risk [RR], 0.80; P=.002) and meningitis (RR, 0.41; P=.003) in adults and for primary bacteremia, invasive pneumonia, and meningitis in children (RR, 0.16 [P<.001], 0.60 [P=.003], and 0.70 [P=.009], respectively). Among human immunodeficiency virus-infected persons, there were significant decreases in the overall rates of IPD (decrease of 43%; P<.001) and invasive pneumonia (decrease of 44%; P<.001) since 2000-2001, although the rate of IPD increased significantly (increase of 53%; P=.022) among patients with acquired immunodeficiency syndrome. There was a concurrent increase in the proportion of adults aged > or = 40 years with underlying comorbidities. Rates of non-PCV7 serotypes increased 1.61-fold and 1.28-fold from 2000-2001 to 2003-2004 in children and adults (P=.005 for both). CONCLUSIONS: The decreasing incidence of IPD in Atlanta since 2000-2001 was associated with decreases in cases of pneumonia and meningitis in adult and pediatric subjects and in cases of primary bacteremia in children. The burden of serotype-replacement disease remained small. Adults with comorbidities represent a growing proportion of patients with IPD.

Clinical outcomes of bacteremic pneumococcal pneumonia in the era of antibiotic resistance.

Limited data are available about the impact of antimicrobial resistance on clinical outcomes in cases of pneumococcal pneumonia. This was studied in 146 persons hospitalized with invasive pneumonia due to Streptococcus pneumoniae (minimum inhibitory concentration of cefotaxime, > or = .25 microg/mL) who were identified through population-based active surveillance for the period of November 1994 through April 1996. Compared with matched control subjects who had infection with more-susceptible S. pneumoniae, the proportion of subjects who died or who were admitted to an intensive care unit did not differ significantly. Multivariable analysis showed no significant contribution of antimicrobial resistance to mortality or the requirement for care in an intensive care unit. The ability to detect an effect of antimicrobial resistance on these important outcome measures may have been influenced by aggressive multidrug empirical therapy in this group of hospitalized patients. Factors other than resistance, such as severity of illness at presentation and advance directive status ("do not resuscitate" orders), appear to have a stronger influence on pneumococcal pneumonia outcomes.

Group B streptococcal disease in nonpregnant adults.

Group B streptococcal (GBS) disease in nonpregnant adults is increasing, particularly in elderly persons and those with significant underlying diseases. Diabetes, neurological impairment, and cirrhosis increase risk for invasive GBS disease. Skin, soft-tissue, and osteoarticular infections, pneumonia, and urosepsis are common presentations. Meningitis and endocarditis are less common but associated with serious morbidity and mortality. Disease is frequently nosocomial and may be related to the placement of an iv catheter. Recurrent infection occurs in 4.3% of survivors. Capsular serotypes Ia, III, and V account for the majority of disease in nonpregnant adults. Although group B streptococci are susceptible to penicillin, minimum inhibitory concentrations are 4-fold to 8-fold higher than for group A streptococci. Resistance to erythromycin and clindamycin is increasing. The role of antibodies in protection against GBS disease in nonpregnant adults is unresolved. However, the immunogenicity of GBS vaccines being developed for prevention of neonatal disease should be assessed for adults who are at risk.

Active bacterial core surveillance of the emerging infections program network.

Active Bacterial Core surveillance (ABCs) is a collaboration between the Centers for Disease Control and Prevention and several state health departments and universities participating in the Emerging Infections Program Network. ABCs conducts population-based active surveillance, collects isolates, and performs studies of invasive disease caused by Streptococcus pneumoniae, group A and group B Streptococcus, Neisseria meningitidis, and Haemophilus influenzae for a population of 17 to 30 million. These pathogens caused an estimated 97,000 invasive cases, resulting in 10,000 deaths in the United States in 1998. Incidence rates of these pathogens are described. During 1998, 25% of invasive pneumococcal infections in ABCs areas were not susceptible to penicillin, and 13.3% were not susceptible to three classes of antibiotics. In 1998, early-onset group B streptococcal disease had declined by 65% over the previous 6 years. More information on ABCs is available at www.cdc.gov/ncidod/dbmd/abcs. ABCs specimens will soon be available to researchers through an archive.

Physical symptoms, posttraumatic stress disorder, and healthcare utilization of women with and without childhood physical and sexual abuse.

For four groups of women: no abuse, physical abuse alone, combined sexual and physical abuse, and unclear about memories of abuse, we examined the associations between childhood sexual and physical abuse, chronic physical symptoms in adulthood, PTSD, and health care utilization. Of a randomly selected sample of 600 adult femalc members of a health maintenance organization, 86 (14%) chose to participate. Women with a history of physical and sexual abuse in childhood reported significantly more cardiovascular, immune, musculoskeletal, neurologic, and reproductive symptoms than those without this history. While the Sexual/Physical Abuse group had the most chronic physical symptoms, medical visits, emergency room visits, prescriptions, and severe PTSD, the Unclear Memory group consistently ranked second on these same measures--higher than either Controls or the Physical Abuse group. Findings underscore the importance of screening for trauma history among patients seen in medical clinics, and the importance for psychotherapists of attending to patients' physical as well as psychological symptoms of childhood trauma.

Nucleotide sequence analysis of hypervariable junctions of Haemophilus influenzae pilus gene clusters.

Haemophilus influenzae pili are surface structures that promote attachment to human epithelial cells. The five genes that encode pili, hifABCDE, are found inserted in genomes either between pmbA and hpt (hif-1) or between purE and pepN (hif-2). We determined the sequence between the ends of the pilus clusters and bordering genes in a number of H. influenzae strains. The junctions of the hif-1 cluster (limited to biogroup aegyptius isolates) are structurally simple. In contrast, hif-2 junctions are highly diverse, complex assemblies of conserved intergenic sequences (including genes hicA and hicB) with evidence of frequent recombination. Variation at hif-2 junctions seems to be tied to multiple copies of a 23-bp Haemophilus intergenic dyad sequence. The hif-1 cluster appears to have originated in biogroup aegyptius strains from invasion of the hpt-pmbA region by a DNA template containing the hif-2 genes with termini in the hairpin loop of flanking intergenic dyad sequences. The pilus gene clusters are an interesting model of a mobile "pathogenicity island" not associated with a phage, transposon, or insertion element.

The emergence of Streptococcus pneumoniae resistant to macrolide antimicrobial agents: a 6-year population-based assessment.

From 1994 through 1999, the available isolates (4148 isolates) from active population-based surveillance of invasive pneumococcal disease in metropolitan Atlanta were serotyped and were tested for antimicrobial susceptibility. Macrolide-resistant isolates were studied for the presence of ermAM (a ribosomal methylase gene), mefE (a macrolide efflux gene), and tetM (the class M tetracycline resistance gene). Macrolide resistance increased from 16% of all invasive isolates in 1994 to 32% in 1999. Of the macrolide-resistant pneumococcal isolates studied, 99% contained genomic copies of mefE or ermAM. Isolates with ermAM were mainly serotypes 6B, 23F, 14, or 19F and contained tetM; mefE-associated isolates were predominantly serotypes 14, 6A, or 19F, and most did not contain tetM. The frequency of the ermAM-mediated phenotype in invasive Streptococcus pneumoniae remained stable over the 6-year surveillance. However, the mefE-mediated phenotype increased from 9% in 1994 to 26% of all isolates in 1999 and was noted in new serotypes. By 1999, 93% of the mefE-containing strains had minimum inhibitory concentrations >/=8 microgram/mL. Dissemination of the mefE determinant accounted for the rapid increase in the rate of macrolide resistance in our S. pneumoniae population.