-- tuberculosis

-- anthrax

-- staph

-- e. coli

-- typhoid

-- salmonella

-- urinary tract infection

-- peritonitis

-- strep throat

-- stomach ulcers

-- tularemia

-- lyme disease

Juan C. Robles1,

Larry Koreen1,2,

Steven Park1 and

David S. Perlin1,2,*

+Author Affiliations

1Public Health Research Institute, International Center for Public Health

2Department of Microbiology and Molecular Genetics, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103

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ABSTRACT

Multilocus sequence typing (MLST) has emerged as a powerful new DNA-typing tool for the evaluation of intraspecies genetic relatedness. This method relies on DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. However, the results of the MLST scheme for Candida albicans have heretofore never been formally compared to those of other established typing techniques. To assess the value of MLST relative to those of other DNA fingerprinting tools for discriminating among strains of C. albicans, we applied it to a previously well-characterized set of 29 C. albicans isolates evaluated by the random amplified polymorphic DNA (RAPD), multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization probe techniques. MLST identified three clusters of genetically related isolates, with 82.3% direct concordance with MLEE, 82.7% with RAPD analysis, and 86.2% with the Ca3 Southern hybridization technique. When MLST was applied to a subset of 22 isolates of unrelated origins, it identified 21 independent diploid sequence types (DSTs), resulting in a discriminatory power of 99.6%. These DSTs were 96.9, 99.6, and 99.6% concordant with the genotypes identified by RAPD analysis, MLEE, and Ca3 Southern hybridization, respectively. These results demonstrate that MLST is a highly effective technique that performs at least comparably to other established DNA fingerprinting techniques.

The introduction of novel antifungal agents has helped stem the steady rise of systemic fungal infections observed over the years (4, 16, 17, 28). Nevertheless, nosocomial Candida albicans infections remain a major cause of morbidity and mortality among immunosuppressed patients (7, 22). In fact, a recent study showed that the mortality rate for patients with nosocomial candidemia is 61%, a 49% increase over that for other matched hospitalized patients (9). Successful treatment and prevention of these infections within the hospital setting depend not only on improved therapy but also on limitation of their spread through rapid and accurate detection of these pathogens. For this purpose, several image-based genotyping techniques have been developed and are widely used to characterize C. albicans strains. Unfortunately, these techniques are not well suited for rapid and high-throughput sample processing. They are also technically demanding and often require assumptions about hybridization and/or gel migration efficiency.

As opposed to image-based techniques, DNA sequence-based genotyping techniques are rapid and often rely on the nucleotide sequences of genes that are under stabilizing selective pressure (e.g., housekeeping genes). Typing schemes that use DNA sequence size and nucleotide polymorphisms have been shown to be effective for the identification of Candida species. For example, the nucleotide polymorphisms of a 396-bp fragment of the mitochondrial cytochrome b gene accurately distinguish between isolates of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. lusitaniae, and C. stellatoidea (1, 30). Similarly, amplicon size variations of the CaACT1 gene intron discriminate among isolates of C. dubliniensis and C. albicans (5). In addition, some degree of intraspecies discrimination was achieved by sequence analysis of these genes, underlining the utility of DNA sequencing for the accurate characterization of yeast pathogens.

In much the same way that it has been used for bacterial pathogens (27, 29), multilocus sequence typing (MLST) has emerged as an alternative typing tool that has a high degree of resolution and that has the capacity to rapidly characterize large numbers of clinical C. albicans isolates. MLST is based on the DNA sequence analysis of nucleotide polymorphisms within housekeeping genes, and it has shown a high degree of intraspecies discriminatory power for bacterial pathogens (13, 14, 23) and, most recently, fungal species, such as C. albicans (2, 26). MLST studies of C. albicans isolates (2, 3, 26) demonstrated that this technique is applicable to a diploid species and can effectively characterize sets of unrelated and related isolates.

However, MLST has not been formally validated by comparison to other conventional fingerprinting methods. Random amplified polymorphic DNA (RAPD) analysis, multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization, among others, have been shown to be effective for the study of both local and global epidemiological populations of Candida spp. (18, 24). MLEE can effectively identify genetic macrovariations that accumulate very slowly and that can thus be used to assess the phylogeny of C. albicans (24). Ca3 Southern hybridization can detect both rapidly and slowly accumulating genetic microvariations within strains, making it a suitable technique for characterization of both local and global populations of C. albicans (19, 25). It is important to determine if MLST is as reliable as other established DNA fingerprinting techniques, and in this study MLST was applied to a panel of C. albicans isolates (n = 29) that had previously been analyzed by RAPD analysis, MLEE, and Ca3 Southern hybridization (18).

Genomic Pathogen Typing

Larry H. Bernstein, MD, FCAP, Curator

LPBI

  Genomic Pathogen Typing Using Solid-State Nanopores

Allison H. Squires, Evrim Atas, Amit Meller

PLOS  Nov 12, 2015   DOI: http://dx.doi.org:/1371/journal.pone.0142944

Citation: Squires AH, Atas E, Meller A (2015) Genomic Pathogen Typing Using Solid-State Nanopores. PLoS ONE 10(11): e0142944.   http://dx.doi.org:/10.1371/journa…

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