Donna C. Sullivan

Structure of the genome of equine herpesvirus type 3

Abstract The molecular structure of the genome of equine herpesvirus type 1 (EHV-1) was determined by restriction endonuclease mapping studies. Primary restriction enzyme digestion of purified EHV-1 DNA, either unlabeled, 32P04 labeled, or [3H]TdR labeled, gave the following cleavage patterns: EcoRI yielded 17 fragments of 23.4 to 1.3 megadaltons (Mdalton); BglII, 16 fragments of 24.5 to 1.0 Mdalton; XbaI, 15 major fragments of 18.6 to 1.7 Mdalton; and BamHI,17 fragments of 13.7 to 2.8 Mdalton. Several fragments were present in 0.5 M amounts while all others were 1.0 M no 0.25 M fragments were detected. Secondary restriction enzyme digestion of these isolated fragments with various enzymes, analysis of terminal fragments using both the methods of λ 5′ exonuclease digestion and end labeling with polynucleotide kinase, and blot hybridization experiments with 32P-labeled BamHI fragments indicated that this herpesvirus genome is a 92-Mdalton linear, double-stranded DNA molecule and is comprised of two segments designated as L (Long) and S (Short) which are 71.6 and 20.4 Mdalton, respectively. The 0.5 M fragments are located at the ends of the S region, an arrangement which allows the S region to invert relative to the L region; thus, two structural arrangements (isomers) of the genome exist. In addition, areas of heterogeneity were detected at the L terminus, within the S segment, and at a split variable locus in the L region.

Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay

ABSTRACT Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) contribute to significant hospitalization costs. We report here a droplet digital PCR (ddPCR) assay, which is a next-generation emulsion-based endpoint PCR assay for high-precision MRSA analysis. Reference cultures of MRSA, methicillin-susceptible S. aureus (MSSA), and confounders were included as controls. Copan swabs were used to sample cultures and collect specimens for analysis from patients at a large teaching hospital. Swab extraction and cell lysis were accomplished using magnetic-driven agitation of silica beads. Quantitative PCR (qPCR) (Roche Light Cycler 480) and ddPCR (Bio-Rad QX100 droplet digital PCR system) assays were used to detect genes for the staphylococcal protein SA0140 (SA) and the methicillin resistance (mecA) gene employing standard TaqMan chemistries. Both qPCR and ddPCR assays correctly identified culture controls for MRSA (76), MSSA (12), and confounder organisms (36) with 100% sensitivity and specificity. Analysis of the clinical samples (211 negative and 186 positive) collected during a study of MRSA nasal carriage allowed direct comparison of the qPCR and ddPCR assays to the Cepheid MRSA GeneXpert assay. A total of 397 clinical samples were examined in this study. Cepheid MRSA GeneXpert values were used to define negative and positive samples. Both the qPCR and ddPCR assays were in good agreement with the reference assay. The sensitivities for the qPCR and ddPCR assays were 96.8% (95% confidence interval [CI], 93.1 to 98.5%) and 96.8% (95% CI, 93.1 to 98.5%), respectively. Both the qPCR and ddPCR assays had specificities of 91.9% (95% CI, 87.5 to 94.9%) for qPCR and 91.0% (95% CI, 86.4 to 94.2%) for ddPCR technology.

Vancomycin-Tolerance among Clinical Isolates of Streptococcus pneumoniae in Mississippi during 1999-2001

Background:In recent years, infection with Streptococcus pneumoniae has been a serious worldwide health concern. Antimicrobial-resistant S pneumoniae is increasing in incidence worldwide, posing a potentially serious threat. Resistance to &bgr;-lactams, macrolides, and trimethoprim-sulfamethoxazole represents a major problem in the treatment of pneumococcal infections Methods:Our laboratory conducted a survey of local resistance patterns in S pneumoniae. Clinical isolates from two separate respiratory seasons were collected from representative geographic areas in Mississippi (totaling 28 hospitals) and were tested for antimicrobial resistance to penicillin, amoxicillin, ceftriaxone, cefuroxime, azithromycin, clindamycin, tetracycline, trimethoprim-sulfamethoxazole, levofloxacin, gatifloxacin, moxifloxacin, and vancomycin using reference methods. Vancomycin-tolerant strains of S pneumoniae were initially identified as those in which the vancomycin MIC was 0.5 &mgr;g/mL. Strain tolerance was confirmed by time kill studies Results:For the 1999-2000 respiratory season, 318 isolates were available for testing; for 2001-2002, 166 isolates were available. Of the 484 total isolates tested, two isolates were identified as having increased tolerance to vancomycin. A greater than 2 log10 difference in viability between the tolerant isolates and the nontolerant isolates of S pneumoniae was observed in time kill studies Conclusions:Two vancomycin-tolerant isolates of S pneumoniae were identified and characterized. Antibiotic tolerance is defined as the ability of bacteria to survive but not proliferate in the presence of an antibacterial agent. Tolerance to vancomycin is particularly significant when the incidence of penicillin tolerance or resistance is high. In addition, tolerance to vancomycin is not detected by routine in vitro susceptibility testing.

Plasmodium berghei: Glycolytic enzymes of the infected mouse erythrocyte

Abstract This paper documents the maximal activities of the glycolytic enzymes in the red blood cells of normal mice and mice infected with Plasmodium berghei . There appears to be sufficient parasite-related activity of each glycolytic enzyme to support the increased glycolytic rate, i.e., increased glucose consumption, of the parasite-infected red blood cell. The relative proportions of glycolytic enzyme activities in parasite-infected red cells are different from the proportions in either normal or reticulocyte-rich blood, indicating that the increased enzyme activities associated with infected cells are not due to contaminating host red cells or reticulocytes. A comparison of maximal enzyme activities to the rate of whole cell glucose consumption indicates that different glycolytic control mechanisms are operating in the infected RBC from those in the uninfected cells.

Properties of the genome of equine herpesvirus type 3.

Abstract Methods developed for the isolation and purification of equine herpesvirus type 3 (EHV-3) DNA were shown to yield quantities of intact infectious DNA molecules suitable for characterization by physicochemical, biochemical, and electron microscopic methods. Preparations of EHV-3 DNA were shown by CsCl analytical ultracentrifugation to be comprised of a single species of DNA with a buoyant density of 1.727 g/cm3 which corresponds to a G + C content of 67.9%. Rate velocity centrifugation studies revealed that EHV-3 DNA has a sedimentation coefficient of approximately 55.4 S which corresponds to a molecular weight value of 90 to 100 megadaltons (Md). Intact viral DNA molecules were found to be 96 to 100 Md by electron microscopy, and restriction enzyme analyses with BamHI, EcoRI, and HindIII yielded an average molecular weight of 96.2 Md. Restriction enzyme and blot hybridization methods revealed that: (i) four 0.5 M EcoRI fragments were present, (ii) these four 0.5 M fragments shared significant homology, (iii) three EcoRI terminal fragments were identified and two of these were 0.5 M, and (iv) the two 0.5 M EcoRI end fragments hybridized only to one of the two 1.0 M terminal fragments identified for BamHI or HindIII. These findings indicated that a set of DNA sequences located at one terminus is repeated within the genome and that two populations of molecules exist with regard to the orientation of these repeat sequences. Electron microscopic examination of reannealed EHV-3 DNA molecules revealed structures that contained a single-strand loop equivalent to a duplex molecular weight of 5.3 Md at one end of the molecule contiguous to a double-strand region which terminated in a long single-strand segment. These structures were identical in morphology to those observed for EHV-1 DNA which has an overall genomic structure of an L (long) region covalently linked to an S (short) region; the S region consists of a unique segment (Us) bracketed by inverted repeat sequences (Henry et al., 1981; OCallaghan et al., 1981). The above findings indicate that the 96.2-Md EHV-3 genome is comprised of an L region of approximately 73–76 Md covalently linked to a 20- to 23-Md S region; the S region contains a 5.3-Md Us segment bounded by 8-Md inverted repeat sequences that allow the S region to invert relative to the fixed L region and the genome to exist in two isomeric arrangements.

Antibacterial Activity of Synthetic Fire Ant Venom: The Solenopsins and Isosolenopsins

Background:We determined the in vitro activity of 9 synthetic fire ant venom alkaloids (±)-solenopsin A, (2R, 6R)-solenopsin A, (2S, 6S)-solenopsin B, (±)-isosolenopsin A, (2S, 6R)-isosolenopsin A,(2R, 6S)-isosolenopsin A, (±)-isosolenopsin B, (2S, 6R)-isosolenopsin B, and (2R, 6S)-isosolenopsin B against 6 species of bacteria (Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Stenotrophomonas maltophilia, and Pseudomonas aeruginosa). Methods:The minimum inhibitory concentration and minimum bacteriocidal concentration were determined in accordance with the Clinical Laboratory Standards Institute guidelines. Time kill studies used American Type Culture Collection bacterial isolates tested at 5 times the minimum inhibitory concentration. Results:None of the venom alkaloids inhibited E. coli or P. aeruginosa, whereas all the alkaloids inhibited S. pneumoniae. Only 4 alkaloids inhibited S. pneumoniae, S. aureus, and S. maltophilia. Time-kill kinetics indicates that all 4 active alkaloids had bactericidal activity. Conclusions:Specific isomers of synthetic fire ant venom alkaloids have antibacterial activity against human pathogens.

Can contaminated water be rendered safe for nasal saline irrigations

To compare sterile water to three methods of sterilization (carbon filtration, boiling, and ultraviolet [UV] light) for preparation of nasal saline irrigants free of bacterial and amebic contaminants.

Mechanisms of fosfomycin resistance in carbapenem-resistant Enterobacter sp.

We report on fosfomycin susceptibility and mechanisms of resistance in clinical strains of blaKPC-positive Enterobacter sp. (nu2009=u200919). A total of 14 strains (74%) were susceptible to fosfomycin; 8 strains (42%) were positive for fosA and no strains were positive for FosA3 or FosC2. FosA presence does not appear to correlate with susceptibility.