An In Silico Exploration of the Factors That Affect the Precision of the Bethesda Assay

Objectives Despite more than 40 years of experience performing the Bethesda assay (BA), poor intra- and interlaboratory precision remains the biggest laboratory challenge to date. Methods The BA procedure was modeled using stochastic simulation techniques to determine the precision of the BA up to dilutions of 1:4,096, to estimate the minimum significant relative change at various inhibitor titers, and to understand the laboratory procedural variables that could significantly affect the performance of the BA at high dilutions. Results Selecting the lowest dilution tube with a residual activity closest to 25% for calculating the reported Bethesda titer (BT), using a factor activity assay with a coefficient of variation less than or equal to 7.5% in the range of 15% to 50% factor activity level, performing the factor activity measurement in replicates, and minimizing pipette volumetric error resulted in the lowest imprecision in the reported BT. The factor neutralization kinetics of the inhibitor appear to have little impact on the precision of the assay if the incubation time is greater than 90 minutes. Conclusions This in silico model will assist future laboratory efforts in standardizing the quantification of specific coagulation factor inhibitors and improving the precision of the reported results.

Evaluation of the Sensitivity of Staphylococcus aurous Isolated from Nasal Swabs to Natural Honey

Introduction: Bacterial resistance to antibacterial agents is a very serious threat to public health. Where some antibacterial agents prove ineffective, the antibacterial properties of honey have been shown to be highly efficacious against several human bacterial pathogens. The purpose of this study is to investigate the sensitivity of Staphylococcus aureus isolated from the nursing staff of a hospital to natural honey. Methods: In this study, 35 strains of methicillin-resistant S. aurous samples were selected from hospital staff's nasal swabs. Two strains were vancomycin-resistant. The serial dilution tube test methodwas used to determine minimum inhibitory concentration (MIC) .The susceptibility of each strain of staph bacteria to natural honey without wax was determined and compared with that of a glucose solution with the same density. Results: In all strains, except for the two strains resistant to vancomycin, MIC level was < 8.3% (v/v). The MIC of glucose as dense as honey was four times higher. The two vancomycin-resistant strains were completely resistant to natural honey. Conclusions: This study has therefore demonstrated that inhibiting bacterial growth is not merely done by purely natural honey not because of osmolality, but vancomycin-resistant bacteria are not sensitive to natural honey. Keywords: sensitivity, Staphylococcus aurous, natural honey, minimum inhibitory concentration

Molecular Study of Quinolone Resistance Determining Regions of gyrA Gene and parC Genes in Clinical Isolates of Acintobacter baumannii Resistant to Fluoroquinolone

Introduction: Acinetobacterb aumannii (A. baumannii) is an important pathogen in health care associated infections. Quinolone resistance has emerged in this pathogen. Aims & Objectives: The aim of the present study was to determine the presence of mutations of gyrA gene and parC genes by Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP-PCR) among clinical isolates of A. baumanii. Materials and Methods: The study was carried out on 140 clinical isolates of A. baumannii. The isolates were subjected to molecular study of mutations of gyrA gene and parC genes by RFLP–PCR beside determination of Minimal Inhibitory Concentration (MIC) by macro dilution tube method. Results: The isolates of A. baumannii were resistant to ciprofloxacine and levofloxacin at MIC >4 µg/ml. The most isolates had MIC >128 µg/ml (42.3%). All resistant strains to ciprofloxacin of A. baumannii had mutations in gyrA and parC. The most frequent mutations were combined mutations in both genes (85.5%) and 5% had single mutation either in gyrA or parC. The most frequently combined mutations were associated with MIC >128 µg/ml (42.3%). Conclusion: From this study we can conclude that resistance to ciprofloxacin was common in clinical isolates of A. baumannii. The most frequent mutations were present in gyrA and parC. However, mutations in parC alone were not uncommon. Further large scale studies are required to elucidate the resistance pattern of A. baumannii and its molecular mechanisms.

Checking the Validity of the Harvesting and Disaggregating Steps in Laboratory Tests of Surface Disinfectants

A chemical disinfectant against surface-associated bacteria typically uses carriers (e.g., glass disks) that are purposely contaminated with bacteria prior to disinfection. After disinfection, the bacteria are harvested by mechanically separating them from the carrier surface to form a suspension of cells in a dilution tube. Bacterial clumps in the tube are disaggregated using mechanical or chemical techniques, thereby creating a well-mixed suspension of single cells suitable for enumeration. Efficacy is quantified by comparing the viable cell count for a disinfected carrier to the viable cell count for sham-disinfected (control) carrier. A test is said to be biased (invalid) if the observed efficacy measure is systematically higher or lower than the true efficacy. It is shown here for the first time that the bias attributable to the harvesting and disaggregating steps of a disinfectant test can be measured. For some conventional biofilm harvesting and disaggregating techniques, laboratory checks showed either negligible bias or important bias, depending on the disinfectant. Quantitative bias checks on the harvesting and disaggregating steps are prudent for each combination of carrier material, microorganism, and disinfectant. The quantitative results should be augmented by microscopic examination of harvested disinfected and control carriers and of the disaggregated suspensions.

Neutrophilic Fe-Oxidizing Bacteria Are Abundant at the Loihi Seamount Hydrothermal Vents and Play a Major Role in Fe Oxide Deposition

ABSTRACT A number of hydrothermal vent sites exist on the summit of the Loihi Seamount, a shield volcano that is part of the Hawaiian archipelago. The vents are 1,100 to 1,325 m below the surface and range in temperature from slightly above ambient (10°C) to high temperature (167°C). The vent fluid is characterized by high concentrations of CO2 (up to 17 mM) and Fe(II) (up to 268 μM), but there is a general paucity of H2S. Most of the vents are surrounded by microbial mats that have a gelatinous texture and are heavily encrusted with rust-colored Fe oxides. Visually, the Fe oxides appeared homogeneous. However, light microscopy revealed that the oxides had different morphologies, which fell into three classes: (i) sheaths, (ii) twisted or irregular filaments, and (iii) amorphous oxides. A morphological analysis of eight different samples indicated that the amorphous oxides were overall the most abundant; however, five sites had >50% sheaths and filamentous oxides. These latter morphologies are most likely the direct result of microbial deposition. Direct cell counts revealed that all of the oxides had abundant microbial populations associated with them, from 6.9 × 107 to 5.3 × 108 cells per ml of mat material. At most sites, end point dilution series for lithotrophic Fe oxidizers were successful out to dilutions of 10−6 and 10−7. A pure culture was obtained from a 10−7 dilution tube; this strain, JV-1, was an obligate, microaerophilic Fe oxidizer that grew at 25 to 30°C. A non-cultivation-based molecular approach with terminal-restriction fragment length polymorphism also indicated the common presence of Fe-oxidizing bacteria at Loihi. Together, these results indicate that Fe-oxidizing bacteria are common at the Loihi Seamount and probably play a major role in Fe oxidation. A review of the literature suggests that microbially mediated Fe oxidation at hydrothermal vents may be important globally.