ABSTRACT
With the emergence of RNA sequencing technologies, metatranscriptomic studies are rapidly gaining attention as they simultaneously provide insight into gene expression profiles and therefore disease association pathways of microbial pathogens and their hosts. This approach, therefore, holds promise for applicability in infectious disease diagnostics. A challenge of this approach in the clinical setting is the low amount and quality of RNA, especially microbial RNA in most clinically-infected specimens. Here, we compared two commercially available stranded cDNA library preparation kits, the NuGEN Ovation SoLo RNA-Seq System and the Illumina TruSeq Stranded Total RNA, using RNA extracted from synovial and sonicate fluids from a subject with periprosthetic joint infection. The Ovation SoLo RNA-Seq System provided more useful transcriptomic data for the infecting bacterium, whereas the TruSeq Stranded Total RNA kit provided more useful human transcriptomic data.
Subject(s)
Gene Library , Infections/diagnosis , RNA, Bacterial/analysis , Sequence Analysis, RNA/methods , Arthroplasty , Gene Expression , Genes, Bacterial/genetics , Humans , Infections/genetics , Infections/microbiology , Periprosthetic Fractures/microbiology , RNA, Bacterial/isolation & purification , Reagent Kits, Diagnostic , Sequence Analysis/methods , Streptococcus sanguis/genetics , Streptococcus sanguis/pathogenicity , Synovial Fluid/microbiology , TranscriptomeABSTRACT
A relationship between hyperammonemia and Ureaplasma infection has been shown in lung transplant recipients. We have demonstrated that Ureaplasma urealyticum causes hyperammonemia in a novel immunocompromised murine model. Herein, we determined whether Ureaplasma parvum can do the same. Male C3H mice were given mycophenolate mofetil, tacrolimus, and prednisone for 7 days, and then challenged with U. parvum intratracheally (IT) and/or intraperitoneally (IP), while continuing immunosuppression over 6 days. Plasma ammonia concentrations were determined and compared using Wilcoxon rank-sum tests. Plasma ammonia concentrations of immunosuppressed mice challenged IT/IP with spent broth (median, 188 µmol/L; range, 102-340 µmol/L) were similar to those of normal (median, 226 µmol/L; range, 154-284 µmol/L, p > 0.05), uninfected immunosuppressed (median, 231 µmol/L; range, 122-340 µmol/L, p > 0.05), and U. parvum IT/IP challenged immunocompetent (median, 226 µmol/L; range, 130-330 µmol/L, p > 0.05) mice. Immunosuppressed mice challenged with U. parvum IT/IP (median 343 µmol/L; range 136-1,000 µmol/L) or IP (median 307 µmol/L; range 132-692 µmol/L) had higher plasma ammonia concentrations than those challenged IT/IP with spent broth (p < 0.001). U. parvum can cause hyperammonemia in pharmacologically immunocompromised mice.
Subject(s)
Hyperammonemia/pathology , Immunocompromised Host , Ureaplasma Infections/complications , Ureaplasma/growth & development , Aged , Animals , Disease Models, Animal , Humans , Male , Mice, Inbred C3H , Plasma/chemistryABSTRACT
In vitro studies demonstrate that oxacillin minimal inhibitory concentrations (MICs) of methicillin-resistant S. aureus (MRSA) strains USA300 and 400 decrease in the presence of cefoxitin. The aim of this study was to characterize the activity of cefoxitin plus ß-lactams against a collection of MRSA isolates. We assessed the in vitro antimicrobial activity of a selection of ß-lactams alone and together with subinhibitory concentrations of cefoxitin against a collection of MRSA, methicillin-susceptible S. aureus (MSSA), and vancomycin-intermediate S. aureus (VISA) isolates using MICs and time kill assays. For community-associated (CA) MRSA strains USA300 and USA400, MICs of nafcillin, cefazolin, cephalexin, cefuroxime, ceftriaxone and cefotaxime decreased by 8- to 64-times in the presence of 10 µg/ml cefoxitin. In contrast, for hospital-associated (HA) strains COLn, N315, and Mu50, there was no change in any ß-lactam MIC in the presence of cefoxitin. When combined with cefoxitin, the cephalexin MIC decreased for eight CA-MRSA and five MSSA sequence types but did not change for seven HA-MRSA sequence types. ß-lactam/cefoxitin combinations were synergistic against CA- but not HA-MRSA strains in time kill assays. Cefoxitin combined with a variety of ß-lactams enhances their activity against CA-MRSA strains in vitro. Further studies of combination ß-lactam therapy may provide insight into ß-lactam biology, penicillin binding protein cooperativity, and novel therapeutic strategies against MRSA.
