Segaran Pillai

Common Ancestry and Novel Genetic Traits of Francisella novicida-Like Isolates from North America and Australia as Revealed by Comparative Genomic Analyses

ABSTRACT Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains.

Whole-genome relationships among Francisella bacteria of diverse origins define new species and provide specific regions for detection

ABSTRACT Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. IMPORTANCE DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.

Genetic diversity within the genus Francisella as revealed by comparative analyses of the genomes of two North American isolates from environmental sources

BackgroundFrancisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4.ResultsThe size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1,700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus Francisella (e.g., F. novicida strain U112 and F. tularensis strain Schu S4), several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism that was absent in the genomes of strain ATCC 25017, F. novicida strain U112, and F. tularensis strain Schu S4. The polymorphic nature of polysaccharide biosynthesis/modification gene clusters among different Francisella strains was also evident from genome analyses.ConclusionsFrom genome comparisons, it appeared that genes encoding novel functions have contributed to the metabolic enrichment of the environmental lineages within the genus Francisella. The inability to acquire new genes coupled with the loss of ancestral traits and the consequent reductive evolution may be a cause for, as well as an effect of, niche selection of F. tularensis. Sequencing and comparison of the genomes of more isolates are required to obtain further insights into the ecology and evolution of different species within the genus Francisella.

A Rapid Antimicrobial Susceptibility Test for Bacillus anthracis

ABSTRACT An effective public health response to a deliberate release of Bacillus anthracis will require a rapid distribution of antimicrobial agents for postexposure prophylaxis and treatment. However, conventional antimicrobial susceptibility testing for B. anthracis requires a 16- to 20-h incubation period. To reduce this time, we have combined a modified broth microdilution (BMD) susceptibility testing method with real-time quantitative PCR (qPCR). The growth or inhibition of growth of B. anthracis cells incubated in 2-fold dilutions of ciprofloxacin (CIP) (0.015 to 16 μg/ml) or doxycycline (DOX) (0.06 to 64 μg/ml) was determined by comparing the fluorescence threshold cycle (CT) generated by target amplification from cells incubated with each drug concentration with the CT of the no-drug (positive growth) control. This ΔCT readily differentiated susceptible and nonsusceptible strains. Among susceptible strains, the median ΔCT values were ≥7.51 cycles for CIP and ≥7.08 cycles for DOX when drug concentrations were at or above the CLSI breakpoint for susceptibility. For CIP- and DOX-nonsusceptible strains, the ΔCT was <1.0 cycle at the breakpoint for susceptibility. When evaluated with 14 genetically and geographically diverse strains of B. anthracis, the rapid method provided the same susceptibility results as conventional methods but required less than 6 h, significantly decreasing the time required for the selection and distribution of appropriate medical countermeasures.

Identification and Analysis of Informative Single Nucleotide Polymorphisms in 16S rRNA Gene Sequences of the Bacillus cereus Group

ABSTRACT Analysis of 16S rRNA genes is important for phylogenetic classification of known and novel bacterial genera and species and for detection of uncultivable bacteria. PCR amplification of 16S rRNA genes with universal primers produces a mixture of amplicons from all rRNA operons in the genome, and the sequence data generally yield a consensus sequence. Here we describe valuable data that are missing from consensus sequences, variable effects on sequence data generated from nonidentical 16S rRNA amplicons, and the appearance of data displayed by different software programs. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of the Bacillus cereus group, i.e., Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis. These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP) previously reported to be specific to B. anthracis was detected in some B. cereus strains. However, a different SNP, at position 1139, was identified as being specific to B. anthracis, which is a biothreat agent with high mortality rates. Compared with visual analysis of the electropherograms, basecaller software frequently missed gene sequence variations or could not identify variant bases due to overlapping basecalls. Accurate detection of 16S rRNA gene sequences that include intragenomic variations can improve discrimination among closely related species, improve the utility of 16S rRNA databases, and facilitate rapid bacterial identification by targeted DNA sequence analysis or by whole-genome sequencing performed by clinical or reference laboratories.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Culture-Independent Diagnostics for Health Security.

The past decade has seen considerable development in the diagnostic application of nonculture methods, including nucleic acid amplification-based methods and mass spectrometry, for the diagnosis of infectious diseases. The implications of these new culture-independent diagnostic tests (CIDTs) include bypassing the need to culture organisms, thus potentially affecting public health surveillance systems, which continue to use isolates as the basis of their surveillance programs and to assess phenotypic resistance to antimicrobial agents. CIDTs may also affect the way public health practitioners detect and respond to a bioterrorism event. In response to a request from the Department of Homeland Security, Los Alamos National Laboratory and the Centers for Disease Control and Prevention cosponsored a workshop to review the impact of CIDTs on the rapid detection and identification of biothreat agents. Four panel discussions were held that covered nucleic acid amplification-based diagnostics, mass spectrometry, antibody-based diagnostics, and next-generation sequencing. Exploiting the extensive expertise available at this workshop, we identified the key features, benefits, and limitations of the various CIDT methods for providing rapid pathogen identification that are critical to the response and mitigation of a bioterrorism event. After the workshop we conducted a thorough review of the literature, investigating the current state of these 4 culture-independent diagnostic methods. This article combines information from the literature review and the insights obtained at the workshop.

Comparative analyses of a putative Francisella conjugative element

A large circular plasmid detected in Francisella novicida-like strain PA10-7858, designated pFNPA10, was sequenced completely and analyzed. This 41,013-bp plasmid showed no homology to any of the previously sequenced Francisella plasmids and was 8-10 times larger in size than them. A total of 57 ORFs were identified within pFNPA10 and at least 9 of them encoded putative proteins with homology to different conjugal transfer proteins. The presence of iteron-like direct repeats and an ORF encoding a putative replication protein within pFNPA10 suggested that it replicated by the theta mode. Phylogenetic analyses indicated that pFNPA10 had no near neighbors in the databases and that it may have originated within an environmental Francisella lineage. Based on its features, pFNPA10 appears to be a novel extra-chromosomal genetic element within the genus Francisella. The suitability of pFNPA10 as a vector for transformation of species of Francisella by conjugation remains to be explored.

Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples.

We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert(®) test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 10(6) spores/mL (ca. 1.5 × 10(5) spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores.

Genome Sequences of Multidrug-Resistant, Colistin-Susceptible and -Resistant Klebsiella pneumoniae Clinical Isolates from Pakistan

ABSTRACT The emergence and spread of colistin resistance among multidrug-resistant (MDR) Klebsiella pneumoniae represent a critical threat to global health. Here, we report the complete genome sequences of 10 MDR, colistin-susceptible and -resistant K. pneumoniae clinical isolates obtained in Pakistan between 2010 and 2013.