Browsing by Subject "microbiology"

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Ammonia-Oxidizing Bacteria In Biofilters Removing Trihalomethanes Are Related To Nitrosomonas Oligotropha
(2011-04) Wahman, David G.; Kirisits, Mary Jo; Katz, Lynn E.; Speitel, Gerald E.; Kirisits, Mary Jo; Katz, Lynn E.; Speitel, Gerald E.
Ammonia-oxidizing bacteria (AOB) in nitrifying biofilters degrading four regulated trihalomethanes-trichloromethane, bromodichloromethane, dibromochloromethane, and tribromomethane-were related to Nitrosomonas oligotropha. N. oligotropha is associated with chloraminated drinking water systems, and its presence in the biofilters might indicate that trihalomethane tolerance is another reason that this bacterium is dominant in chloraminated systems.
Amplification Of Dna-Polymerase Gene Fragments From Viruses Infecting Microalgae
(1995-04) Chen, Feng; Suttle, Curtis A.; Chen, Feng; Suttle, Curtis A.
Nested PCR with three highly degenerate primers was used for amplification and identification of DNA polymerase (pol) genes from viruses which infect three genera of microalgae. Group specific primers (AVS1 and AVS2) were designed on the basis of inferred amino acid sequences unique to the DNA pol genes of viruses (PBCV-1 and NY-2A) that infect an endosymbiotic Chlorella-like alga (Chlorophyceae) and a virus (MpV-SP1) which infects the photosynthetic flagellate Micromonas pusilla (Prasinophyceae). In addition, a nested primer (POL) was designed on the basis of the highly conserved amino acid sequence YGDTDS found in most B-family (alpha-like) DNA pol genes. These primers were used to amplify DNA from the three viruses, PBCV-1, NY-2A, and MpV-SP1, for which the primers were designed, as well as eight clonal isolates of genetically distinct viruses which infect M. pusilla and others which infect Chrysochromulina spp. (Prymnesiophyceae), suggesting that these are a group of related viruses. In contrast, no product resulted from using DNA from viruses which infect the marine brown algae Ectocarpus siliculosis and Feldmannia sp. (Phaeophyceae), suggesting that these viruses may not be closely related to those that infect microalgae. These primers were also used to amplify DNA from natural virus communities. Our results indicate that nested PCR, even under low-stringency conditions, can be used as a rapid method to verify the presence in seawater of a group of related viruses which infect microalgae. Sequence analysis of these fragments should provide information on the genetic diversity and potentially the phyletic relationships among these viruses. This is the first example of a PCR-based technique designed to detect viruses which infect eukaryotic algae.
Antibodies Recognizing Protective Pertussis Toxin Epitopes are Preferentially Elicited by Natural Infection Versus Acellular Immunization
(2011-06) Sutherland, Jamie N.; Chang, Christine; Yoder, Sandra M.; Rock, Michael T.; Maynard, Jennifer A.; Sutherland, Jamie N.; Chang, Christine; Maynard, Jennifer A.
Despite more than 50 years of vaccination, disease caused by the bacterium Bordetella pertussis persists, with rates increasing in industrialized countries over the past decade. This rise may be attributed to several factors, including increased surveillance, emergence of vaccine escape variants, waning immunity in adults, and the introduction of acellular subunit vaccines, which include chemically detoxified pertussis toxin (PTd). Two potently protective epitopes on pertussis toxin (PTx) are recognized by the monoclonal antibodies 1B7 and 11E6, which inhibit catalytic and cell-binding activities, respectively. In order to determine whether the PTx exposure route affects antibody responses to these epitopes, we analyzed sera from 30 adults with confirmed pertussis exposure and from 30 recently vaccinated adults for specific anti-PTx antibody responses and in vitro CHO cell neutralization titers. While overall titers against PTx and the genetically detoxified variant, PTg, containing the R9K and E129G substitutions, were similar in the two groups, titers against specific epitopes depended on the exposure route. Natural infection resulted in significantly higher titers of anti-PTx-subunit 1, 1B7-like, and 11E6-like antibodies, while acellular vaccination resulted in significantly higher titers of antibodies recognizing PTd. We also observed a correlation between in vitro protection and the presence of 1B7-like and 11E6-like antibodies. Notably, chemical detoxification, as opposed to genetic inactivation, alters the PTx tertiary and quaternary structure, thereby affecting conformational epitopes and recognition of PTx by 1B7 and 11E6. The lower levels of serum antibodies recognizing clinically relevant epitopes after vaccination with PTd support inclusion of PTg in future vaccines.
Antifungal Susceptibilities Among Different Serotypes Of Cryptococcus Gattii And Cryptococcus Neoformans
(2009-01) Thompson, George R.; Wiederhold, Nathan P.; Fothergill, Annette W.; Vallor, Ana C.; Wickes, Brian L.; Patterson, Thomas F.; Wiederhold, Nathan P.
We measured antifungal activity against 128 cryptococcal isolates (86 of C. neoformans and 42 of C. gattii) to determine if differences in serotype susceptibility exist. Contrary to previous results, we found no serotype susceptibility differences. Isavuconazole, posaconazole, and voriconazole demonstrated excellent potency against each isolate and serotype, including isolates with reduced fluconazole susceptibilities.
Assessment Of Serum (1 -> 3)-Beta-D-Glucan Concentration As A Measure Of Disease Burden In A Murine Model Of Invasive Pulmonary Aspergillosis
(2008-03) Wiederhold, Nathan P.; Najvar, Laura K.; Vallor, Ana C.; Kirkpatrick, William R.; Bocanegra, Rosie; Molina, Destiny; Olivo, Marcos; Graybill, John R.; Patterson, Thomas F.; Wiederhold, Nathan P.
Serum (1 -> 3)-beta-D-glucan concentrations were serially measured in the presence and absence of antifungal therapy in a murine model of invasive pulmonary aspergillosis. Serum (1 -> 3)-beta-D-glucan was detected early during the course of infection, and reductions in this biomarker were associated with improved survival in animals treated with antifungal agents.
Caspofungin Dose Escalation For Invasive Candidiasis Due To Resistant Candida Albicans
(2011-07) Wiederhold, Nathan P.; Najvar, Laura K.; Bocanegra, Rosie A.; Kirkpatrick, William R.; Patterson, Thomas F.; Wiederhold, Nathan P.
Previous in vivo studies have reported caspofungin dose escalation to be effective against Candida glabrata with reduced susceptibility. We hypothesized that higher doses of caspofungin would be effective against invasive candidiasis caused by the more virulent species Candida albicans, including isolates resistant to this echinocandin. Immunocompetent mice were inoculated with one of three C. albicans isolates, including one susceptible and two resistant isolates with different FKS1 hot spot 1 point mutations. Mice received daily caspofungin treatment for 7 days and were then followed off therapy for 2 weeks to assess survival. Kidney tissue and blood were collected, and fungal burden and serum (1 -> 3)-beta-D-glucan were measured. Significant differences in virulence were observed among the three C. albicans isolates, which translated into differences in responses to caspofungin. The most virulent of the resistant isolates studied (isolate 43001; Fks1p F641S) did not respond to caspofungin doses of up to 10 mg/kg of body weight, as there were no differences in survival (survival range, 0 to 12% with treatment), tissue burden, or (1 -> 3)-beta-D-glucan concentration compared to those for untreated controls. Higher doses of caspofungin did improve survival against the second resistant isolate (53264; Fks1p S645P) that demonstrated reduced virulence (5 and 10 mg/kg; 80% survival). In contrast, caspofungin doses as low as 1 mg/kg improved survival (85 to 95%) and reduced tissue burden and (1 -> 3)-beta-D-glucan concentration against the susceptible isolate (ATCC 90028). These data suggest that caspofungin dose escalation for invasive candidiasis may not be consistently effective against resistant C. albicans isolates, and this may be associated with the virulence of the strain.
Comparison Of Cefazolin Versus Oxacillin For Treatment Of Complicated Bacteremia Caused By Methicillin-Susceptible Staphylococcus Aureus
(2014-09) Li, Julius; Echevarria, Kelly L.; Hughes, Darrel W.; Cadena, Jose A.; Bowling, Jason E.; Lewis, James S.; Li, Julius; Echevarria, Kelly L.; Hughes, Darrel W.; Lewis, James S.
Contrary to prior case reports that described occasional clinical failures with cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infections, recent studies have demonstrated no difference in outcomes between cefazolin and antistaphylococcal penicillins for the treatment of MSSA bacteremia. While promising, these studies described low frequencies of high-inoculum infections, such as endocarditis. This retrospective study compares clinical outcomes of cefazolin versus oxacillin for complicated MSSA bacteremia at two tertiary care hospitals between January 2008 and June 2012. Fifty-nine patients treated with cefazolin and 34 patients treated with oxacillin were included. Osteoarticular (41%) and endovascular (20%) sources were the predominant sites of infection. The rates of clinical cure at the end of therapy were similar between cefazolin and oxacillin (95% versus 88%; P = 0.25), but overall failure at 90 days was higher in the oxacillin arm (47% versus 24%; P = 0.04). Failures were more likely to have received surgical interventions (63% versus 40%; P = 0.05) and to have an osteoarticular source (57% versus 33%; P = 0.04). Failures also had a longer duration of bacteremia (7 versus 3 days; P = 0.0002), which was the only predictor of failure. Antibiotic selection was not predictive of failure. Rates of adverse drug events were higher in the oxacillin arm (30% versus 3%; P = 0.0006), and oxacillin was more frequently discontinued due to adverse drug events (21% versus 3%; P = 0.01). Cefazolin appears similar to oxacillin for the treatment of complicated MSSA bacteremia but with significantly improved safety. The higher rates of failure with oxacillin may have been confounded by other patient factors and warrant further investigation.
Comparison of Lateral Flow Technology and Galactomannan and (1 -> 3)-Beta-D-Glucan Assays for Detection of Invasive Pulmonary Aspergillosis
(2009-12) Wiederhold, Nathan P.; Thornton, Christopher R.; Najvar, Laura K.; Kirkpatrick, William R.; Bocanegra, Rosie; Patterson, Thomas F.; Wiederhold, Nathan P.
We compared a lateral flow device to galactomannan and (1 -> 3)-beta-D-glucan assays to detect invasive aspergillosis in an established guinea pig model of pulmonary disease. The lateral flow device became positive earlier ( =day 3) than the (1 -> 3)-beta-D-glucan and galactomannan assays ( day 5), with all samples positive by each assay on day 7.
Continuous Versus Intermittent Infusion Of Oxacillin For Treatment Of Infective Endocarditis Caused By Methicillin-Susceptible Staphylococcus Aureus
(2009-05) Hughes, Darrel W.; Frei, Christopher R.; Maxwell, Pamela R.; Green, Kay; Patterson, Jan E.; Crawford, George E.; Lewis, James S.; Hughes, Darrel W.; Frei, Christopher R.; Maxwell, Pamela R.; Lewis, James S.
Infective endocarditis (IE) is the fourth leading cause of life-threatening infection in the United States and imposes significant morbidity and mortality. The American Heart Association guidelines for the diagnosis and treatment of IE do not address continuous-infusion (CI) oxacillin. This retrospective study compares outcomes between CI oxacillin and intermittent-infusion (II) oxacillin in the treatment of IE caused by methicillin-susceptible Staphylococcus aureus (MSSA). A total of 709 medical records were reviewed for inpatients with definitive IE treated between 1 January 2000 and 31 December 2007. Continuous data were analyzed by Student's t test or the Wilcoxon rank sum test. The chi-square test or Fisher's exact test was used to compare nominal data. A multivariate logistic model was constructed. One hundred seven patients met eligibility criteria for inclusion into the study. Seventy-eight patients received CI oxacillin, whereas 28 received II oxacillin. CI and II groups were similar with respect to 30-day mortality (8% versus 10%, P = 0.7) and length of stay (20 versus 25 days, P = 0.4) but differed in 30-day microbiological cure (94% versus 79%, P = 0.03). Sixty-three patients received synergistic gentamicin, whereas 44 did not. The gentamicin and no-gentamicin groups were similar with respect to 30-day mortality (11% versus 4%, P = 0.2) and 30-day microbiological cure (90% versus 89%, P = 0.8); however, times to defervescence (4 versus 2 days, P = 0.02) were significantly different. CI oxacillin is an effective alternative to II oxacillin for the treatment of IE caused by MSSA and may improve microbiological cure. This convenient and pharmacodynamically optimized dosing regimen for oxacillin deserves consideration for patients with IE caused by MSSA.
Degradation Of Trichloroethylene By Methanol-Grown Cultures Of Methylosinus Trichosporium Ob3B Pp358
(1996-03) Fitch, Mark W.; Speitel, Gerald E.; Georgiou, George; Fitch, Mark W.; Speitel, Gerald E.; Georgiou, George
A soluble methane monooxygenase-constitutive mutant strain of Methylosinus trichosporium OB3b, strain PP358, was grown with methanol as the carbon source, and the kinetics of trichloroethylene (TCE) degradation were determined. PP358 exhibited high TCE degradation rates under both oxygen- and carbon-limiting conditions. The optimal pseudo first-order rate constant for TCE was comparable to the values measured for cells grown,vith methane. We found that growth under oxygen-limiting conditions results in increased accumulation of polyhydroxybutyrate, which in turn correlates,vith higher transformation capacities for TCE. It was also shown that methanol inhibits TCE degradation only at high concentrations. Thus, methanol-grown cultures of PP358 represent an efficient system for the biodegradation of chlorinated hydrocarbons.
Development Of An Engineered Bioluminescent Reporter Phage For Detection Of Bacterial Blight Of Crucifers
(2012-05) Schofield, David A.; Bull, Carolee T.; Rubio, Isael; Wechter, W. Patrick; Westwater, Caroline; Molineux, Ian J.; Molineux, Ian J.
Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant >light>-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wildtype phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.
Development Of Caspofungin Resistance Following Prolonged Therapy For Invasive Candidiasis Secondary To Candida Glabrata Infection
(2008-10) Thompson, George R.; Wiederhold, Nathan P.; Vallor, Ana C.; Villareal, Nyria C.; Lewis, James S.; Patterson, Thomas F.; Wiederhold, Nathan P.
We report a case of Candida glabrata invasive candidiasis that developed reduced susceptibility to caspofungin during prolonged therapy. Pre- and posttreatment isolates were confirmed to be isogenic, and sequencing of hot spots known to confer echinocandin resistance revealed an F659V substitution within the FKS2 region of the glucan synthase complex.
Directed Evolution Of Xylose Isomerase For Improved Xylose Catabolism And Fermentation In The Yeast Saccharomyces Cerevisiae
(2012-08) Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S.; Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S.
The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tall and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.
Discovery Of Ethanol-Responsive Small Rnas In Zymomonas Mobilis
(2014-07) Cho, Seung Hee; Lei, Roy; Henninger, Trey D.; Contreras, Lydia M.; Cho, Seung Hee; Lei, Roy; Henninger, Trey D.; Contreras, Lydia M.
Zymomonas mobilis is a bacterium that can produce ethanol by fermentation. Due to its unique metabolism and efficient ethanol production, Z. mobilis has attracted special interest for biofuel energy applications; an important area of study is the regulation of those specific metabolic pathways. Small RNAs (sRNAs) have been studied as molecules that function as transcriptional regulators in response to cellular stresses. While sRNAs have been discovered in various organisms by computational prediction and experimental approaches, their discovery in Z. mobilis has not yet been reported. In this study, we have applied transcriptome analysis and computational predictions to facilitate identification and validation of 15 novel sRNAs in Z. mobilis. We furthermore characterized their expression in the context of high and low levels of intracellular ethanol. Here, we report that 3 of the sRNAs (Zms2, Zms4, and Zms6) are differentially expressed under aerobic and anaerobic conditions, when low and high ethanol productions are observed, respectively. Importantly, when we tested the effect of ethanol stress on the expression of sRNAs in Z. mobilis, Zms2, Zms6, and Zms18 showed differential expression under 5% ethanol stress conditions. These data suggest that in this organism regulatory RNAs can be associated with metabolic functions involved in ethanol stress responses.
Dynamics And Distribution Of Cyanophages And Their Effect On Marine Synechococcus Spp
(1994-09) Suttle, Curtis A.; Chan, Amy M.; Suttle, Curtis A.; Chan, Amy M.
Cyanophages infecting marine Synechococcus cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting Synechococcus strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 x 10(5) ml(-1) near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and Synechococcus concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 x 10(3) ml(-1)). When Synechococcus concentrations exceeded ca. 10(3) ml(-1), cyanophage concentrations increased markedly (ca. 10(2) to > 10(5) ml(-1)), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity d (per day), as a function of solar irradiance I (millimoles of quanta per square meter per second), were used to develop a relationship (d = 0.2610I-0.00718; r(2) = 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of Synechococcus cells would be infected daily by viruses. Calculations based on contact rates between Synechococcus cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most Synechococcus cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of Synechococcus cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the Synechococcus population on a daily basis.
Effect of CSLM imaging rate on biofilms of P. aeruginosa and S. aureus
(2015-05) Du, Reginald; Gordon, Vernita
Biofilms are sessile communities of bacteria that can be found in an wide range of environments. Their inhabitants are phenotypically distinct from plank- tonic bacteria and are capable of forming complex, three-dimensional structures. Biofilms are studied using confocal scanning laser microscopy, or CSLM. This technique uses lasers and Novel Fluorescent Proteins (NFPs) to measure growth and structure formation of single- and multi-species biofilms in situ in three dimensions. We investigate the effects of slow and fast rates of image acquisition on mono- and co-cultures of biofilm forming bacteria: Pseudomonas aeruginosa and Staphylococcus aureus. After calculating growth rates and lag times, we find that fast scanning rates reduce the growth rate of P. aeruginosa in co-culture. Additionally, co-culture speeds up P. aeruginosa growth relative to monoculture when imaged at a slow rate, and fast scanning reverts co-culture growth to monoculture-like behavior. Additionally, a significant lag time is observed for P. aeruginosa grown in co-culture. The observed influence of confocal imaging rate on population dynamics should be considered in future studies to ensure accurate measurement of bacterial phenomena.
Effects Of Experimental Exclusion Of Scavengers From Carcasses Of Anthrax-Infected Herbivores On Bacillus Anthracis Sporulation, Survival, And Distribution
(2013-06) Bellan, Steve E.; Turnbull, Peter C. B.; Beyer, Wolfgang; Getz, Wayne M.; Bellan, Steve E.
Scavenging of anthrax carcasses has long been hypothesized to play a critical role in the production of the infectious spore stage of Bacillus anthracis after host death, though empirical studies assessing this are lacking. We compared B. anthracis spore production, distribution, and survival at naturally occurring anthrax herbivore carcasses that were either experimentally caged to exclude vertebrate scavengers or left unmanipulated. We found no significant effect of scavengers on soil spore density (P > 0.05). Soil stained with terminally hemorrhaged blood and with nonhemorrhagic fluids exhibited high levels of B. anthracis spore contamination (ranging from 103 to 108 spores/g), even in the absence of vertebrate scavengers. At most of the carcass sites, we also found that spore density in samples taken from hemorrhagic-fluid-stained soil continued to increase for >4 days after host death. We conclude that scavenging by vertebrates is not a critical factor in the life cycle of B. anthracis and that anthrax control measures relying on deterrence or exclusion of vertebrate scavengers to prevent sporulation are unlikely to be effective.
Efficacy Of Posaconazole As Treatment And Prophylaxis Against Fusarium Solani
(2010-03) Wiederhold, Nathan P.; Najvar, Laura K.; Bocanegra, Rosie; Graybill, John R.; Patterson, Thomas F.; Wiederhold, Nathan P.
Invasive fusariosis is a highly aggressive fungal infection associated with high mortality in heavily immunocompromised patients. Although posaconazole is efficacious as salvage therapy against infections caused by Fusarium species, concerns remain regarding this agent in the setting of reduced potency. To evaluate the efficacy of posaconazole as treatment or prophylaxis against invasive fusariosis caused by Fusarium solani, we utilized a neutropenic murine model of disseminated disease. ICR mice were administered escalating doses of posaconazole (6.25, 12.5, 25, or 50 mg/kg of body weight twice daily [BID]) by oral gavage beginning 2 days prior to inoculation in the prophylaxis studies or beginning 12 h after inoculation as treatment. Therapy was continued until day 9 postinoculation, and animals were monitored off therapy until day 15 for survival. Fungal burden was assessed as CFU in the kidneys. A clear dose-response relationship was observed, as the highest dose of posaconazole (50 mg/kg) was the most effective in prolonging survival and reducing tissue fungal burden both as prophylaxis and as treatment. This dose response was associated with high posaconazole serum concentrations as measured by bioassay. However, the extent of efficacy was also dependent on the infecting inoculum, as greater increases in survival and reductions in fungal burden were observed with the lower inocula tested. In this model high dosages of posaconazole were effective as treatment and prophylaxis against disseminated fusariosis caused by F. solani.
Evaluation Of Etest Method For Determining Isavuconazole MICs Against Cryptococcus Gattii And Cryptococcus Neoformans
(2008-08) Thompson, George R.; Fothergill, Annette W.; Wiederhold, Nathan P.; Vallor, Ana C.; Wickes, Brian L.; Patterson, Thomas F.; Wiederhold, Nathan P.
We compared Etest with broth microdilution testing for isavuconazole activity against 92 Cryptococcus isolates. A 97.8% agreement was found between these methods, without major discrepancies (> 2-well dilution difference). Our findings support the use of the Etest methodology as a reliable method for the determination of MICs against Cryptococcus spp.
Exonuclease Removal Of Dideoxycytidine (Zalcitabine) By The Human Mitochondrial DNA Polymerase
(2008-01) Hanes, Jeremiah W.; Johnson, Kenneth A.; Hanes, Jeremiah W.; Johnson, Kenneth A.
The toxicity of nucleoside analogs used for the treatment of human immunodeficiency virus infection is due primarily to the inhibition of replication of the mitochondrial genome by the human mitochondrial DNA polymerase (Pol gamma). The severity of clinically observed toxicity correlates with the kinetics of incorporation versus excision of each analog as quantified by a toxicity index, spanning over six orders of magnitude. Here we show that the rate of excision of dideoxycytidine (zalcitabine; ddC) was reduced fourfold (giving a half-life of similar to 2.4 h) by the addition of a physiological concentration of deoxynucleoside triphosphates (dNTPs) due to the formation of a tight ternary enzyme-DNA-dNTP complex at the polymerase site. In addition, we provide a more accurate measurement of the rate of excision and show that the low rate of removal of ddCMP results from both the unfavorable transfer of the primer strand from the polymerase to the exonuclease site and the inefficient binding and/or hydrolysis at the exonuclease site. The analogs ddC, stavudine, and ddATP (a metabolite of didanosine) each bind more tightly at the polymerase site during incorporation than normal nucleotides, and this tight binding contributes to slower excision by the proofreading exonuclease, leading to increased toxicity toward mitochondrial DNA.