Using PCR-Based Detection and Genotyping to Trace Streptococcus salivarius Meningitis Outbreak Strain to Oral Flora of Radiology Physician Assistant

Velusamy Srinivasan,Patricia L Shewmaker,Priti Patel,Robert E Gertz Jr,Alice Y Guh,Judith Noble-Wang,Sarah Patrick,Amit S Chitnis,Bernard Beall,Isaac Benowitz,George Turabelidze,Heather O'Connell,Ramy K Aziz

doi:10.1371/journal.pone.0032169

Abstract

We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA). We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF) specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis.

Highlights

Streptococcus salivarius is a predominant bacterial species of human oral flora [1], [2], and has been the most commonly identified species causing bacterial meningitis cases that occur after spinal injection procedures because of contamination of the procedure site with saliva [3]
Alpha-hemolytic streptococcal meningitis has rarely been reported in connection with the performance of lumbar puncture for various purposes including myelograms and spinal anesthesia
For both the prior New York outbreak associated with an anesthesiologist [6] and our further investigation of the outbreak involving the radiology physician assistant (RPA) [9] presented here, compelling evidence on the route of transmission was supplied based upon unique 16 locus profiles shared solely between oral S. salivarius isolates of the administering anesthesiologist or RPA and the patient receiving the spinal injection procedure

Summary

Introduction

Streptococcus salivarius is a predominant bacterial species of human oral flora [1], [2], and has been the most commonly identified species causing bacterial meningitis cases that occur after spinal injection procedures because of contamination of the procedure site with saliva [3]. Analysis based upon sequences of multiple chromosomal loci indicates that S. salivarius is a distinct, genetically diverse species that undergoes frequent intraspecies horizontal exchange events [4], [5], [6]. There is cumulative sequence-based data that reveal the likelihood of frequent genetic exchange between the two species and supports their separation into distinct species [5]. Independent cases of S. salivarius meningitis epidemiologically linked to the same spinal injection procedure provider have previously been reported [6], [7], [8]. In two situations genetically indistinguishable strains were recovered from the case CSF specimen and the oral flora of the individual carrying out the lumbar puncture [6], [8]

Methods

Results

Conclusion