Identification of staphylococcal phage with reduced transcription in human blood through transcriptome sequencing.

Tasha M Santiago-Rodriguez,Marcus B Jones,Mayuri Naidu,David T Pride,Melissa Ly

doi:10.3389/fmicb.2015.00216

Abstract

Many pathogenic bacteria have bacteriophage and other mobile genetic elements whose activity during human infections has not been evaluated. We investigated the gene expression patterns in human subjects with invasive Methicillin Resistant Staphylococcus aureus (MRSA) infections to determine the gene expression of bacteriophage and other mobile genetic elements. We developed an ex vivo technique that involved direct inoculation of blood from subjects with invasive bloodstream infections into culture media to reduce any potential laboratory adaptation. We compared ex vivo to in vitro profiles from 10 human subjects to determine MRSA gene expression in blood. Using RNA sequencing, we found that there were distinct and significant differences between ex vivo and in vitro MRSA gene expression profiles. Among the major differences between ex vivo and in vitro gene expression were virulence/disease/defense and mobile elements. While transposons were expressed at higher levels ex vivo, lysogenic bacteriophage had significantly higher in vitro expression. Five subjects had MRSA with bacteriophage that were inhibited by the presence of blood in the media, supporting that the lysogeny state was preferred in human blood. Some of the phage produced also had reduced infectivity, further supporting that phage were inhibited by blood. By comparing the gene expression cultured in media with and without the blood of patients, we gain insights into the specific adaptations made by MRSA and its bacteriophage to life in the human bloodstream.

Highlights

Staphylococcus aureus is a pathogen that is considered normal human flora, and often takes advantage of breaks in protective skin barriers to cause disease (Chaffin et al, 2012)
All ex vivo and in vitro Methicillin Resistant Staphylococcus aureus (MRSA) isolates were grown to log phase (Supplemental Figure 1), and total RNA was isolated from each subject/MRSA isolate under both growth conditions
Because the ex vivo samples were cultured in the presence of blood from each human subject, we enriched to remove any RNA that may have been derived from the human host

Summary

Introduction

Staphylococcus aureus is a pathogen that is considered normal human flora, and often takes advantage of breaks in protective skin barriers to cause disease (Chaffin et al, 2012). While S. aureus strains were primarily treatable with beta-lactam antibiotics in the past, their widespread use has resulted in the emergence of Methicillin-Resistant S. aureus (MRSA) strains. CA-MRSA generally has been responsible for many invasive soft tissue infections, and has several virulence. Staphylococcal phage inhibited in blood factors including Panton-Valentine leukocidin (PVL), which are thought to contribute greatly to its pathogenesis. PVL, which is not regularly detected in Hospital Acquired (HA)-MRSA strains, have been associated with epidemics of several CA-MRSA strains in the United States (Pan et al, 2003; Vandenesch et al, 2003). Virulence factors are of particular interest in invasive bloodstream infections, and many are derived from mobile genetic elements, including plasmids, bacteriophage, transposons, and pathogenicity islands (Bae et al, 2006; Baba et al, 2008; Diep et al, 2008b)

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