Defining Critical Genes During Spherule Remodeling and Endospore Development in the Fungal Pathogen, Coccidioides posadasii.

Coccidioides immitis and C. posadasii are soil dwelling dimorphic fungi found in North and South America. Inhalation of aerosolized asexual conidia can result in asymptomatic, acute, or chronic respiratory infection. In the United States there are approximately 350,000 new infections per year. The Coccidioides genus is the only known fungal pathogen to make specialized parasitic spherules, which contain endospores that are released into the host upon spherule rupture. The molecular determinants involved in this key step of infection remain largely elusive as 49% of genes are hypothetical with unknown function. An attenuated mutant strain C. posadasii Δcts2/Δard1/Δcts3 in which chitinase genes 2 and 3 were deleted was previously created for vaccine development. This strain does not complete endospore development, which prevents completion of the parasitic lifecycle. We sought to identify pathways active in the wild-type strain during spherule remodeling and endospore formation that have been affected by gene deletion in the mutant. We compared the transcriptome and volatile metabolome of the mutant Δcts2/Δard1/Δcts3 to the wild-type C735. First, the global transcriptome was compared for both isolates using RNA sequencing. The raw reads were aligned to the reference genome using TOPHAT2 and analyzed using the Cufflinks package. Genes of interest were screened in an in vivo model using NanoString technology. Using solid phase microextraction (SPME) and comprehensive two-dimensional gas chromatography - time-of-flight mass spectrometry (GC × GC-TOFMS) volatile organic compounds (VOCs) were collected and analyzed. Our RNA-Seq analyses reveal approximately 280 significantly differentially regulated transcripts that are either absent or show opposite expression patterns in the mutant compared to the parent strain. This suggests that these genes are tied to networks impacted by deletion and may be critical for endospore development and/or spherule rupture in the wild-type strain. Of these genes, 14 were specific to the Coccidioides genus. We also found that the wild-type and mutant strains differed significantly in their production versus consumption of metabolites, with the mutant displaying increased nutrient scavenging. Overall, our results provide the first targeted list of key genes that are active during endospore formation and demonstrate that this approach can define targets for functional assays in future studies.

Transmission of methicillin-resistant Staphylococcus aureus infection through solid organ transplantation: confirmation via whole genome sequencing.

We describe two cases of donor-derived methicillin-resistant Staphylococcus aureus (MRSA) bacteremia that developed after transplantation of organs from a common donor who died from acute MRSA endocarditis. Both recipients developed recurrent MRSA infection despite appropriate antibiotic therapy, and required prolonged hospitalization and hospital readmission. Comparison of S. aureus whole genome sequence of DNA extracted from fixed donor tissue and recipients' isolates confirmed donor-derived transmission. Current guidelines emphasize the risk posed by donors with bacteremia from multidrug-resistant organisms. This investigation suggests that, particularly in the setting of donor endocarditis, even a standard course of prophylactic antibiotics may not be sufficient to prevent donor-derived infection.

Effect of extension of infarction on serial CK activity.

The effects of extension of myocardial infarction by reduction of regional myocardial blood flow (RMBF) to an ischemic region on serum CK activity was examined in 14 awake dogs. Initial infarction was effected by occlusion of the distal left circumflex coronary artery (LCCA) and subsequent extension was produced by occlusion of the proximal LCCA 6, 12 or 18 hours after distal occlusion. Extension was verified by serial measurements of RMBF using radioisotope-labeled microspheres before and after proximal occlusion. Serum CK activity increased initially 2-4 hours after distal coronary occlusion and then increased rapidly and reached peak values 12 hours after occlusion. When the infarction was extended at 6, 12 or 18 hours after initial occlusion, CK appearance was immediately reduced in the 6- and 12-hour experiments, but not in the 18-hour experiments. Extension of infarction at each interval caused delayed increases in CK activity beginning 2-5 hours after proximal occlusion, with peak values occurring 12 hours later. The immediate effects of extension of infarction by reducing blood flow on CK activity are a function of whether the infarcted myocardium continued to release CK, e.g., at 6 and 12 hours after occlusion, or CK release was completed, e.g., 18 hours. The immediate effects of extension of infarction were the result of perfusion on myocardium that is infarcted and continues to release CK, and do not necessarily indicate alterations in the extent of myocardial injury. The delayed effects of proximal and distal occlusion on CK activity were comparable, suggesting that delayed and not immediate alterations in CK activity represent extension of infarction.