Reduced subgenomic RNA expression is a molecular indicator of asymptomatic SARS-CoV-2 infection.

Background: It is estimated that up to 80% of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are asymptomatic and asymptomatic patients can still effectively transmit the virus and cause disease. While much of the effort has been placed on decoding single nucleotide variation in SARS-CoV-2 genomes, considerably less is known about their transcript variation and any correlation with clinical severity in human hosts, as defined here by the presence or absence of symptoms. Methods: To assess viral genomic signatures of disease severity, we conducted a systematic characterization of SARS-CoV-2 transcripts and genetic variants in 81 clinical specimens collected from symptomatic and asymptomatic individuals using multi-scale transcriptomic analyses including amplicon-seq, short-read metatranscriptome and long-read Iso-seq. Results: Here we show a highly coordinated and consistent pattern of sgRNA expression from individuals with robust SARS-CoV-2 symptomatic infection and their expression is significantly repressed in the asymptomatic infections. We also observe widespread inter- and intra-patient variants in viral RNAs, known as quasispecies frequently found in many RNA viruses. We identify unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Moreover, these frequently occurring structural variants in SARS-CoV-2 genomes serve as a mechanism to further induce SARS-CoV-2 proteome complexity. Conclusions: Our results indicate that differential sgRNA expression and structural mutational burden are highly correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.

Issues for microbial regulation: Aeromonas as a model.

The decision by public health agencies to regulate specific microorganisms that may be found in drinking water can only be made if specific criteria find that a microorganism poses a health risk. These criteria should include: (1) there is a clinical history of an organism causing disease from the ingestion of drinking water; (2) there is epidemiological evidence that drinking water rather than food or other vectors is a major source of disease; (3) there is sufficient evidence that the target organism, if found in water, possesses virulence factors capable of causing disease in humans; (4) there is sufficient evidence that the target organism is not readily removed or inactivated by multi-barrier conventional water treatment process (e.g., coagulation-filtration-disinfection); (5) there is sufficient evidence that the target organism, if surviving conventional treatment, will be viable, virulent, and present in sufficient numbers to cause disease; (6) there are robust analytical methods for the target organism which have acceptable sensitivity, specificity, and reproducibility to measure accurately the presence of the target organism in treated water; and (7) the performance criteria of analytical method(s) for the target organism have been certified by the appropriate public health agency, and there is intra-laboratory field-test performance data to base this certification.

Life-threatening sepsis caused by Burkholderia cepacia from contaminated intravenous flush solutions prepared by a compounding pharmacy in another state.

We report 2 life-threatening cases of Burkholderia cepacia sepsis caused by infusate contamination during compounding. Bacterial isolates from the patients' blood cultures and the infusate were indistinguishable by pulsed-field gel electrophoresis. Proper quality controls at a local and national level are important for ensuring safe delivery of compounded medications to patients in all settings, including those outside health care facilities.

Antipneumococcal activity of DK-507k, a new quinolone, compared with the activities of 10 other agents.

Agar dilution MIC determination was used to compare the activity of DK-507k with those of ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, sitafloxacin, amoxicillin, cefuroxime, erythromycin, azithromycin, and clarithromycin against 113 penicillin-susceptible, 81 penicillin-intermediate, and 67 penicillin-resistant pneumococci (all quinolone susceptible). DK-507k and sitafloxacin had the lowest MICs of all quinolones against quinolone-susceptible strains (MIC at which 50% of isolates were inhibited [MIC50] and MIC90 of both, 0.06 and 0.125 microg/ml, respectively), followed by moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin. MICs of beta-lactams and macrolides rose with those of penicillin G. Against 26 quinolone-resistant pneumococci with known resistance mechanisms, DK-507k and sitafloxacin were also the most active quinolones (MICs, 0.125 to 1.0 microg/ml), followed by moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin. Mutations in quinolone resistance-determining regions of quinolone-resistant strains were in the usual regions of the parC and gyrA genes. Time-kill testing showed that both DK-507k and sitafloxacin were bactericidal against all 12 quinolone-susceptible and -resistant strains tested at twice the MIC at 24 h. Serial broth passages in subinhibitory concentrations of 10 strains for a minimum of 14 days showed that development of resistant mutants (fourfold or greater increase in the original MIC) occurred most rapidly for ciprofloxacin, followed by moxifloxacin, DK-507k, gatifloxacin, sitafloxacin, and levofloxacin. All parent strains demonstrated a fourfold or greater increase in initial MIC in <50 days. MICs of DK-507k against resistant mutants were lowest, followed by those of sitafloxacin, moxifloxacin, gatifloxacin, ciprofloxacin, and levofloxacin. Four strains were subcultured in subinhibitory concentrations of each drug for 50 days: MICs of DK-507k against resistant mutants were lowest, followed by those of sitafloxacin, moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin. Exposure to DK-507k and sitafloxacin resulted in mutations, mostly in gyrA.

Antibacterial susceptibility of a vancomycin-resistant Staphylococcus aureus strain isolated at the Hershey Medical Center.

Staphylococcus aureus strain HMC3 isolated at the Hershey Medical Center, was resistant to vancomycin (VRSA) through the presence of the vanA resistance gene; it also contained mecA, erm(A), erm(B), tet(K) and aac(6')-aph(2"), conferring resistance to licensed beta-lactams, macrolides, tetracycline and aminoglycosides. HMC3 also had alterations in GyrA and GrlB and was resistant to available quinolones. Experimental drugs with low MICs (<2 mg/L) for VRSA HMC3 included cephalosporins BAL9141 and RWJ-54428; glycopeptides oritavancin and dalbavancin; the lipopeptide daptomycin; the glycolipodepsipeptide ramoplanin; new fluoroquinolones WCK 771 A, WCK 1153, DK-507k and sitafloxacin; and the DNA nanobinder GS02-02. These agents were all bactericidal as were trimethoprim/sulfamethoxazole and teicoplanin (MIC 4 mg/L). Oxazolidinones linezolid and ranbezolid; the injectable streptogramin quinupristin/dalfopristin; DNA nanobinders GS2-10547 and GS02-104; peptide deformylase inhibitors NVP-PDF713 and GS02-12; tetracycline derivative tigecycline; the antifolate iclaprim; mupirocin and fusidic acid were all active in vitro but bacteriostatic.

Activity of nine oral agents against gram-positive and gram-negative bacteria encountered in community-acquired infections: use of pharmacokinetic/pharmacodynamic breakpoints in the comparative assessment of beta-lactam and macrolide antimicrobial agents.

BACKGROUND: The application of pharmacokinetic (PK) and pharmacodynamic (PD) data in conjunction with minimum inhibitory concentrations (MICs) of antibacterial agents has been shown to allow for improved selection and appropriate dosing of antimicrobial agents for specific infections, increasing the likelihood of bacteriologic cure and, through this, reducing the risk for the development of resistant organisms. OBJECTIVES: This study was undertaken to provide data on current levels of resistance among common community-acquired bacterial species to 7 betalactam antimicrobial agents (including the combination product amoxicillin/clavulanate), azithromycin, and clarithromycin, determined through application of the PK/PD breakpoints based on time-above-MIC for the beta-lactams and the nonazalide macrolide clarithromycin, and on 24-hour serum area under the curve divided by MIC for the azalide macrolide azithromycin. METHODS: The antimicrobial products tested were amoxicillin/clavylanate, cefpodoxime, cefdinir, cefditoren, cefprozil, cefuroxime, cefixime, azithromycin, and clarithromycin. The bacterial species comprised 70 penicillin-susceptible, 68 penicillin-intermediate, and 69 penicillin-resistant strains of Streptococcus pneumoniae; 46 beta-lactamase-positive and 54 beta-lactamase-negative strains of Haemophilus influenzae; 49 strains of Moraxella catarrhalis; and 100 methicillin-sensitive strains of Staphylococcus aureus (MSSA). Strains were isolated from clinical specimens obtained from outpatient-acquired infections in 1 clinical center in the Northeast and 1 in the north-central area of the United States within the past 2 years. National Committee for Clinical Laboratory Standards microdilution MIC methodology was used. PK/PD breakpoints were obtained from previously published studies and were based on blood values. RESULTS: Amoxicillin/clavulanate was the product to which the greatest percentage of susceptible, intermediate, and resistant strains of pneumococci were sensitive at the PK/PD breakpoint, followed by cefditoren, cefpodoxime, cefuroxime, cefdinir, and cefprozil. None of the cephalosporins were active against penicillin-resistant pneumococci. Cefditoren and cefpodozime were the agents to which the greatest percentage of beta-lactamase-positive and beta-lactamase-negative strains of H influenzae were sensitive, followed by amoxicillin/clavulanate, cefdinir, and cefuroxime. Cefprozil was inactive against H influenzae. All of the beta-lactam products were active against M catarrhalis. All but cefpodoxime, cefditoren, and cefixime were active against MSSA. CONCLUSIONS: In this study, based on PK/PD breakpoints, amoxicillin/clavulanate had the best overall activity of the 9 antimicrobial products tested. Cefpodoxime and cefditoren were active against >or=90% of strains of penicillin-susceptible and penicillin-intermediate pneumococci, H influenzae, and M catarrhalis. The macrolides azithromycin and clarithromycin were active against penicillin-susceptible and penicillin-intermediate pneumococci and M catarrhalis; they were inactive against H influenzae and penicillin-resistant pneumococci.

Single and multi-step resistance selection study in Streptococcus pneumoniae comparing ceftriaxone with levofloxacin, gatifloxacin and moxifloxacin.

Attempts were made to select resistant pneumococcal mutants by sequential subculturing of 12 clinically isolated pneumococci, [four were penicillin sensitive (MIC) 0.03-0.06 mg/l, four penicillin intermediate (MIC 0.25-0.5 mg/l) and four penicillin resistant (MIC 2-4 mg/l)] in sub-inhibitory concentrations of ceftriaxone, levofloxacin, gatifloxacin and moxifloxacin. Subculturing in gatifloxacin, levofloxacin, moxifloxacin and ceftriaxone selected 12 mutants (12/12), 10 mutants (10/12), 10 mutants (10/12) and three mutants (3/12), respectively. DNA sequencing of the quinolone-resistant mutants showed that most strains had mutations in GyrA at E85 or S81. This in vitro mutation study demonstrates a clear distinction between the low frequency of development of resistance with ceftriaxone exposure as opposed to the high frequency with quinolone exposure.

Scedosporium apiospermum (Pseudallescheria boydii) endocarditis.

Scedosporium apiospermum, the asexual state of Pseudallescheria boydii, is increasingly recognized as an opportunistic pathogen. We report a case of native valve endocarditis due to this organism that developed in an elderly patient following a prolonged hospitalization. Literature on endocarditis caused by S. apiospermum and P. boydii is reviewed.