Pyrosequencing was used to identify 133 isolates of clinically relevant non-dematiaceous yeasts. These included 97 ATCC strains (42 type strains), seven UAMH strains, and 29 clinical isolates. Isolates belonged to the following genera: Candida (18 species), Trichosporon (10), Cryptococcus (7), Malassezia (3), Rhodotorula (2), Geotrichum (1), Blastoschizomyces (1), and Kodamaea (1). Amplicons of a hyper-variable ITS region were obtained and analyzed using Pyrosequencing technology. The data were evaluated by a BLAST search against the GenBank database and correlated with data obtained by conventional cycle sequencing of the ITS1–5.8S–ITS2 region. Cycle sequencing identified 78.9% of the isolates to the species level. Pyrosequencing technology identified 69.1%. In 90.1% of all of the strains tested, the identification results of both sequencing methods were identical. Most Candida isolates can be identified to the species level by Pyrosequencing. Trichosporon species and some Cryptococcus species cannot be differentiated at the species level. Pyrosequencing can be used for the reliable identification of most commonly isolated non-dematiaceous yeasts, with a reduction of cost per identification compared to conventional sequencing.

Background: Allergen extracts prepared from Dermatophagoides farinae contain significantly more endotoxin than Dermatophagoides pteronyssinus extracts, and extracts from both mite extracts contain more endotoxin than pollen extracts. Attempts to culture bacteria from mite cultures have failed to establish the sources of the endotoxin.Objective: To determine the bacterial sources of endotoxin in mite extracts.Methods: Live mites of both species were obtained from 2 sources, DNA was extracted from the mites, and DNA encoding bacterial 16S ribosomal RNA was amplified by using specific primers. The amount of bacterial DNA in each mite DNA sample was determined by quantitative PCR using an internal standard, and sequence homologies were determined from amplifications performed by using a high-fidelity DNA polymerase.Results: DNA from D farinae appeared to contain between 11-fold and 24-fold more 16S ribosomal gene copies than the genomic DNA from D pteronyssinus (P <= .003). Sequence analysis indicated the dominant presence of at least 3 phylogenetic clusters of Bartonella species (henselae, quintana, vinsonii, and grahamii), as well as uncharacterized alpha-proteobacteria, from both D farinae and D pteronyssinus. In a few clones, sequences from Escherichia coli, Pseudomonas species, and Acinetobacter species were also identified.Conclusion: House dust mite DNA contains evidence of Bartonella and other Gram-negative species. These Gram-negative species are likely to be the sources of the endotoxin found in mite allergenic extracts.

Critical Care Medicine (Masur) Clinical Pathology, National Institutes of Health, Bethesda, MD (Murray)

Rationale We have shown that the endotoxin content of standardized dust mite allergenic extracts appears to be significantly higher in Dermatophagoides farinae (Df) than in D. pteronysinnus (Dp) extracts by limulus amebocyte gel clot (LAL) assay ( J Allergy Clin Immunol 2003;111:777-783). We now examine whether this can be explained by differences in the endosymbiont bacteria residing in the mite species, or by the presence of LAL-inhibitory substances in the Dp extracts. Methods We tested multiple samples of cultured washed mites obtained from three different sources. The mites were washed free of media in either ethanol or water (depending on the source) and were cultured for gram-negative bacteria. We also performed the LAL on mixtures of the extracts (Df alone, Dp alone, Df+Dp, and Df+endotoxin-free water) to detect inhibitory substances in Dp. Results We detected no gram-negative organisms in the 3 Df samples and 2 Dp samples that were washed in ethanol. Among 7 Df samples washed in water, 2 were positive for Ralstonia picketii ; among 7 Dp samples washed in water, 2 were positive for Ralstonia picketii ; and another 2 were positive for Acinetobacter species. The LAL results for the extracts (in EU/mL): Df, 6.8×10 5 ; Dp, 1.7; Df +water, 6.8×10 5 ; Df+Dp, 3.4×10 5 . Conclusions Both Df and Dp cultures seem to carry gram-negative organisms. There is minimal if any inhibition of LAL by the Dp extracts. The observed differences in endotoxin concentration may be due to differences in bioburden, or to fastidious species that we were unable to culture.

RPR 106972 is a novel oral streptogramin combination with reported therapeutic potency against Gram-positive and certain respiratory tract pathogens. MICs for RPR 106972, quinupristin/dalfopristin, and seven comparison drugs were determined by the reference methods against 337 strains selected to define spectrum and potency. RPR 106972 demonstrated antimicrobial activity against oxacillin-susceptible and -resistant Staphylococcus aureus (MIC ranges of 0.12 to 2 micrograms/ml and 0.5 to 2 micrograms/ml, respectively), and coagulase-negative staphylococci were also inhibited by RPR 106972 (MIC90, < or = 0.5 microgram/ml) and quinupristin/dalfopristin (MIC90, < or = 0.25 microgram/ml). Against all streptococcal strains tested (including penicillin-resistant pneumococcus), RPR 106972 was highly active with MIC results at < or = 1 microgram/ml. RPR 106972 inhibited Corynebacterium jeikeium (MIC90, 0.5 microgram/ml). Moraxella catarrhalis (MIC90, 0.25 microgram/ml), and some Haemophilus influenzae (MIC50, 2 micrograms/ml). RPR 106972 and quinupristin/dalfopristin demonstrated little activity against Enterococcus faecalis (MIC90s, 4 to 32 micrograms/ml) as compared to Enterococcus faecium (MIC90s, 0.5 to 1 microgram/ml) and other Enterococcus ssp. (MIC90s, 1 microgram/ml). Studies to establish MIC quality-control guidelines indicated the following ranges: for E. faecalis ATCC 29212, 0.5 to 4 micrograms/ml; for S. aureus ATCC 29213, 0.25 to 1 microgram/ml; and for Streptococcus pneumoniae ATCC 49619, 0.06 to 0.5 microgram/ml. The results of this study indicate that the in vitro activity of RPR 106972 against Gram-positive bacteria and selected Gram-negative respiratory organisms is promising and warrants additional studies of pharmacokinetics, and in vivo infection model dynamics.

Over 2,000 clinical isolates from ten American medical centers were tested for susceptibility to cefotaxine, ceftriaxone, ceftizoxime, and ceftazidime by both broth microdilution and disk diffusion methods. Typically resistant (e.g. enterococci) and highly susceptible (e.g. streptococci) isolates showed no change in susceptibility patterns compared to previous surveys. Pseudomonas aeruginosa and Stenotrophomonas maltophilia exhibited significant decreases in susceptibility to these cephalosporins. Among Escherichia coli and Klebsiella spp isolates, two to three percent were resistant to one of the four drugs whereas they were very susceptible to the other three. Of the four antibiotics, the cefotaxime disk diffusion test results correlated best with the microdilution results. With the other three drugs the disk diffusion test yielded 1 to 9% more susceptible test results and 1 to 20% fewer resistant test results than broth microdilution when testing gram negative bacilli. The clinical significance of such discrepancies is not known, but the impact on antibiotic susceptibility surveys and antibiogram comparisons could be significant.

Quality control guidelines for standardized antimicrobial susceptibility test methods are critical to the continuing accuracy of the tests. In this report, quality control limits were proposed for 22 organism-antimicrobial combinations with minimum inhibitory concentration (MIC) ranges of three or four log2 dilution steps. Disk diffusion zone diameter ranges were proposed for azithromycin compared with Neisseria gonorrhoeae ATCC 49226 and ticarcillin with and without clavulanic acid tested against Staphylococcus aureus ATCC 25923. The data from five or six participating laboratories produced > or = 94.7% of results within proposed MIC limits, and 94.3%-99.0% of zones were found within suggested zone guidelines. These proposed quality control ranges should be validated by in-use results from clinical laboratories.

Forty-three medical centers participated in a national (United States) surveillance study of parenteral antimicrobial agents as empiric therapy of pathogens isolated from blood, skin wounds, respiratory tract, and urine (> 8500 strains, 200 per laboratory). All laboratories tested each organism by the same reagent disks and/or Etest (AB Biodisk, Solna, Sweden) strips. Quality control results validated all laboratories for analyses. The most common isolates were Escherichia coli (1648), Staphylococcus aureus (1408), Pseudomonas aeruginosa (1003), Klebsiella species (792), and the enterococci (684). Among the tested drugs the percent susceptible rates observed were ofloxacin (83.4%), ciprofloxacin (82.0%), and cefuroxime (62.9%) tested against all organisms; cefazolin (54.7%) and ceftazidime (76.7%) tested against all nonfastidious aerobes; gentamicin (91.2%), imipenem (95.3%), ticarcillin-clavulanate (78.2%), and ceftriaxone (66.2%) tested against Gram-negative organisms only; and vancomycin (97.9%) and erythromycin (49.2%) tested against Gram-positive aerobes. Several drug-resistant species appear to be emerging or increasing in the United States: (a) vancomycin-resistant enterococci (7.9%, mostly Enterococcus faecium); (b) oxacillin-resistant S. aureus (21.0%); (c) third-generation cephalosporin-resistant Enterobacteriaceae, including E. coli and Klebsiella species with extended-spectrum β-lactamases (approximately 1.3%–8.6%); (d) penicillin-resistant Strepto-coccus pneumoniae (17.8%); and (e) crprofloxacin-resistant P. aeruginosa (14.9%). Fluoroquinolone resistance among the enteric bacilli was confirmed in 60 of 66 referred strains (0.8% of total strains), and cross-resistance was high among ciprofloxacin, ofloxacin, lomefloxacin, fleroxacin, and norfloxacin (98.3%–100%). Seventeen strains of fluoroquinolone-resistant enteric bacilli (0.2% of total) also harbored an ESBL and resistance to aminoglycosides. Clonal spread within medical centers was observed with the ESBL-producing Klebsiella pneumoniae. This national clinical isolate data base continues to demonstrate broad fluoroquinolone efficacy (ofloxacin > ciprofloxacin) against hospital-based pathogens and many strains of emerging resistant bacteria. Continued US surveillance studies are urged to monitor emerging antimicrobial resistance and to guide interventions to minimize its occurrence.

We read with interest the report by Friedland and colleagues concerning the use of a cefuroxime disk diffusion test to screen for cephalosporin-resistant pneumococci (1). We agree that rapid recognition of such organisms is of paramount importance, but the breakpoints which they propose do not appear to be optimal. Results of a recent several different lots of media. As can be seen in Fig. 1, the breakpoint of 20 mm suggested by Friedland et al. would not detect 4 of 11 strains for which ceftriaxone MICs were .2 ,ug/ml; 8 isolates would be falsely classified as resistant. Perhaps the greatest problem, however, is that 72 of the 235 susceptible organisms (30.6%) fall within the intermediate

Experimental results are interpreted in the light of numerical analaysis, imperfection sensitivity and Design Codes. Two cases are discussed: spherical shells under uniform external pressure and partly filled spherical shells, supported on a continuous equatorial ring. The imperfection sensitivity associated with the first loading case leads to the selection of safety factors that depend on the actual shell stiffness. The second case, in which the load results in a biaxial tension-compression state of stress, is treated approximately in terms of a plate under biaxial load.