Development of a Multilocus Sequence Typing System for Medically Relevant Bipolaris Species

Abstract

cMultilocus sequence typing (MLST) is the gold standard genotyping technique for many microorganisms. This classification approach satisfies the requirements for a high-resolution, standardized, and archivable taxonomic system. Here, we describe the development of a novel MLST system to assist with the investigation of an unusual cluster of surgical site infections caused by Bipolaris spp. in postoperative cardiothoracic surgery (POCS) patients during January 2008 to December 2013 in the southeastern United States. We also used the same MLST system to perform a retrospective analysis on isolates from a 2012 Bipolaris endophthalmitis outbreak caused by a contaminated product. This MLST system showed high intraspecies discriminatory power for Bipolaris spicifera, B. hawaiiensis, and B. australiensis. Based on the relatedness of the isolates, the MLST data supported the hypothesis that infections in the POCS cluster were from different environmental sources while confirming that the endophthalmitis outbreak resulted from a point source, which was a contaminated medication. Dematiaceous fungi belonging to the genus Bipolaris are abundant in the environment. Many species in this genus are known to cause devastating disease in plants and staple crops around the world (1). In humans, they are common etiological agents of fungal sinusitis. Surgical site infections (SSIs) and deep tissue and invasive infections by members of this genus do occur, but they are extremely rare and typically associated with immunocompromised patients (2-7). The common disease-causing Bipolaris species in humans are Bipolaris spicifera, B. hawaiiensis, and, occasionally, B. australiensis (3). A recent proposal transfers these species to the genus Curvularia (1), but this new name has not yet been widely accepted.In the clinical setting, Bipolaris infections are usually diagnosed using microscopy to distinguish the morphological characteristics of the fungus following culture. In recent years, the sequence comprising the internal transcribed spacer (ITS) regions and the 5.8S nuclear rRNA genes of the ribosomal cistron has been exploited for species assignment of dematiaceous fungi (8, 9). In most cases, ITS-based sequence identification is sufficient for determining species. However, ITS-based sequencing is limited by its dependence on the completeness and accuracy of available DNA sequence databases. A DNA typing system employing two or more loci, such as multilocus sequence typing (MLST), can be used when it is necessary to distinguish between isolates within a species (10, 11). MLST has been successfully implemented for species identification within a species complex as well as strain differentiation within a species for various fungal pathogens of humans such as Aspergillus fumigatus, Candida albicans, Candida glabrata, Candida tropicalis, Cryptococcus gattii, Cryptococcus neoformans, and Fusarium sp. (12-18). The recent investigations of infections caused by dematiaceous fungi (5, 19) have highlighted the importance of high-resolution, sta...

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