Search

ABSTRACTTwelve Burkholderia pseudomalleiisolates collected over a 32-month period from a patient with chronic melioidosis demonstrated identical multilocus sequence types (STs). However, whole-genome sequencing suggests a polyclonal infection. This study is the first to report a mixed infection with the same ST.

ABSTRACTA retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella entericaserotype Enteritidis and survey the population structure of commonly encountered S. entericaserotype Enteritidis outbreak isolates in the United States. A total of 52 S. entericaserotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. entericaserotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.

ABSTRACTExserohilum rostratumwas the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohiluminfection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratumretrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ~136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum.

ABSTRACTMelioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei. In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1-orf2assay to be superior in detecting B. pseudomalleidirectly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin.

ABSTRACTBrucellaspecies are highly monomorphic, with minimal genetic variation among species, hindering the development of reliable subtyping tools for epidemiologic and phylogenetic analyses. Our objective was to compare two distinct multiple-locus variable-number tandem-repeat analysis (MLVA) subtyping methods on a collection of 101 Brucella melitensisisolates from sporadic human cases of brucellosis in Egypt (n= 83), Qatar (n= 17), and Libya (n= 1). A gel-based MLVA technique, MLVA-15IGM, was compared to an automated capillary electrophoresis-based method, MLVA-15NAU, with each MLVA scheme examining a unique set of variable-number tandem repeats. Both the MLVAIGMand MLVANAUmethods were highly discriminatory, resolving 99 and 101 distinct genotypes, respectively, and were able to largely separate genotypes from Egypt and Qatar. The MLVA-15NAUscheme presented higher strain-to-strain diversity in our test population than that observed with the MLVA-15IGMassay. Both schemes were able to genetically correlate some strains originating from the same hospital or region within a country. In addition to comparing the genotyping abilities of these two schemes, we also compared the usability, limitations, and advantages of the two MLVA systems and their applications in the epidemiological genotyping of human B. melitensisstrains.

ABSTRACTHighly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracismakes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracisthat contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracisstrains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracisAmes strain from the 88 unique B. anthracisstrains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (<100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.

ABSTRACTWe genotyped 15 Bacillus anthracisisolates from Chad, Africa, using multiple-locus variable-number tandem repeat analysis and three additional direct-repeat markers. We identified two unique genotypes that represent a novel genetic lineage in the A cluster. Chadian isolates were susceptible to 11 antibiotics and free of 94 antibiotic resistance genes.

ABSTRACTA TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomalleiand Burkholderia malleiisolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensisand Burkholderia cepacia.

ABSTRACTMolecular epidemiological tools for genotyping clinical isolates of Mycobacterium tuberculosishave been developed and used to help track and contain transmission of tuberculosis. We identified 87 short sequence repeat loci within the genome of the M. tuberculosisH37Rv strain. Nine tandem repeats were found to be variable (variable-number tandem repeats [VNTRs]) in a set of 91 isolates. Fifty-seven of the isolates had only four IS6110bands. The other 34 isolates were members of the Beijing strain family. The number of alleles of each these nine VNTRs was determined by examining each isolate. Six of the loci (Mtb-v1, -v4, -v10, -v15, -v18, and -v20) were able to differentiate the Beijing spoligotype identical isolates into seven distinct genotypes. Five of the loci (Mtb-v3, -v5, -v6, -v10, and -v15) were informative in discriminating the four-band IS6110restriction fragment length polymorphism isolates from each other. The Nei's diversity values of each marker ranged from 0.02 to 0.59, with the number of alleles ranging from two to eight across the entire strain set. These nine loci provide a useful, discriminatory extension of VNTR typing methods for application to molecular epidemiologic studies of M. tuberculosis.

ABSTRACTHuman Lyme borreliosis (LB) is the most prevalent arthropod-borne infection in temperate climate zones around the world and is caused by Borreliaspirochetes. We have identified 10 variable-number tandem repeat (VNTR) loci present within the genome of Borrelia burgdorferiand subsequently developed a multiple-locus VNTR analysis (MLVA) typing system for this disease agent. We report here the successful application of MLVA for strain discrimination among a group of 41 globally diverse Borreliaisolates including B. burgdorferi, B. afzelii, and B. garinii. PCR assays displayed diversity at these loci, with total allele numbers ranging from two to nine and Nei's diversity (D) values ranging from 0.10 to 0.87. The average Dvalue was 0.53 across all VNTR loci. A clear correlation exists between the repeat copy number and the Dvalue (r= 0.62) or the number of alleles (r= 0.93) observed across diverse strains. Cluster analysis by the unweighted pair-group method with arithmetic means resolved the 30 observed unique Borreliagenotypes into five distinct groups. B. burgdorferi, B. afzelii, and B. gariniiclustered into distinct affiliations, consistent with current 16S rRNA phylogeny studies. Genetic similarity and diversity suggest that B. afzeliiand B. gariniiare close relatives and were perhaps recently derived from B. burgdorferi. MLVA provides both phylogenetic relationships and additional resolution to discriminate among strains of Borreliaspecies. This new level of strain identification and discrimination will allow more detailed epidemiological and phylogenetic analysis in future studies.

ABSTRACTFrancisella tularensis, the etiological agent of tularemia, is found throughout the Northern hemisphere. After analyzing the F. tularensisgenomic sequence for potential variable-number tandem repeats (VNTRs), we developed a multilocus VNTR analysis (MLVA) typing system for this pathogen. Variation was detected at six VNTR loci in a set of 56 isolates from California, Oklahoma, Arizona, and Oregon and the F. tularensislive vaccine strain. PCR assays revealed diversity at these loci with total allele numbers ranging from 2 to 20, and Nei's diversity index values ranging from 0.36 to 0.93. Cluster analysis identified two genetically distinct groups consistent with the current biovar classification system ofF. tularensis. These findings suggest that these VNTR markers are useful for identifying F. tularensisisolates at this taxonomic level. In this study, biovar B isolates were less diverse than those in biovar A, possibly reflecting the history of tularemia in North America. Seven isolates from a recent epizootic in Maricopa County, Ariz., were identical at all VNTR marker loci. Their identity, even at a hypervariable VNTR locus, indicates a common source of infection. This demonstrates the applicability of MLVA for rapid characterization and identification of outbreak isolates. Future construction of reference databases will allow faster outbreak tracking as well as providing a foundation for deciphering global genetic relationships.

ABSTRACTA TaqMan-minor groove binding assay designed around a nonsense mutation in the plcRgene was used to genotype Bacillus anthracis, B. cereus, and B. thuringiensisisolates. The assay differentiated B. anthracisfrom these genetic near-neighbors and determined that the nonsense mutation is ubiquitous across 89 globally and genetically diverse B. anthracisstrains.

ABSTRACTWe used multiple-locus variable-number tandem repeat analysis (MLVA) and pagAsequencing to genotype a Bacillus anthracisisolate from a fatal case of human anthrax in Hong Kong. The isolate has a unique MLVA genotype, is related to the Sterne and Ames strains, and is consistent with genotypes identified in China.