The highly infectious and zoonotic pathogen Francisella tularensis is the etiologic agent of tularemia, a potentially fatal disease if untreated. Despite the high average nucleotide identity, which is >99.2% for the virulent subspecies and >98% for all four subspecies, including the opportunistic microbe Francisella tularensis subsp. novicida, there are considerable differences in genetic organization. These chromosomal disparities contribute to the substantial differences in virulence observed between the various F. tularensis subspecies and subtypes. The methods currently available to genotype F. tularensis cannot conclusively identify the associated subpopulation without using time-consuming testing or complex scoring matrices. To address this need, we developed both single and multiplex quantitative real-time PCR (qPCR) assays that can accurately detect and identify the hypervirulent F. tularensis subsp. tularensis subtype A.I, the virulent F. tularensis subsp. tularensis subtype A.II, F. tularensis subsp. holarctica (also referred to as type B), and F. tularensis subsp. mediasiatica, as well as opportunistic F. tularensis subsp. novicida from each other and near neighbors, such as Francisella philomiragia, Francisella persica, and Francisella-like endosymbionts found in ticks. These fluorescence-based singleplex and non-matrix scoring multiplex qPCR assays utilize a hydrolysis probe, providing sensitive and specific F. tularensis subspecies and subtype identification in a rapid manner. Furthermore, sequencing of the amplified F. tularensis targets provides clade confirmation and informative strain-specific details. Application of these qPCR- and sequencing-based detection assays will provide an improved capability for molecular typing and clinical diagnostics, as well as facilitate the accurate identification and differentiation of F. tularensis subpopulations during epidemiological investigations of tularemia source outbreaks.

Introduction: Menaquinone is used for transporting electrons and is essential for the aerobic and anaerobic respiratory systems of all pathogens and prokaryotes. Many Gram-positive bacteria use only menaquinone in the electron transport system. Thus, menaquinone biosynthesis is a potential target for the development of inhibitors against bacteria including drug-resistant pathogens. Results: After modeling, synthesis and in vitro testing, we determined that 7-methoxy-2-naphthol-based inhibitors targeted the MenA enzyme of the menaquinone biosynthesis pathway. The developmental compounds 1 and 2 were active against Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus with a minimal inhibitory concentration of 3–5 μg/ml. Conclusion: Nontraditional bicyclic inhibitors, compounds 1 and 2 could serve as lead compounds for the development of an antimicrobial agent, with activities against M. tuberculosis and methicillin-resistant S. aureus.

Tularemia is a potentially fatal disease that is caused by the highly infectious and zoonotic pathogen Francisella tularensis. Despite the monomorphic nature of sequenced F. tularensis genomes, there is a significant degree of plasticity in the organization of genetic elements. The observed variability in these genomes is due primarily to the transposition of direct repeats and insertion sequence (IS) elements. Since current methods used to genotype F. tularensis are time-consuming and require extensive laboratory resources, IS elements were investigated as a means to subtype this organism. The unique spatial location of specific IS elements provided the basis for the development of a differential IS amplification (DISA) assay to detect and distinguish the more virulent F. tularensis subsp. tularensis (subtypes A.I and A.II) and subsp. holarctica (type B) strains from F. tularensis subsp. novicida and other near neighbors, including Francisella philomiragia and Francisella-like endosymbionts found in ticks. Amplicon sizes and sequences derived from DISA showed heterogeneity within members of the subtype A.I and A.II isolates but not the type B strains. These differences were due to a 312-bp fragment derived from the IS element ISFtu1. Analysis of wild-type F. tularensis isolates by DISA correlated with pulsed-field gel electrophoresis genotyping utilizing two different restriction endonucleases and provided rapid results with minimal sample processing. The applicability of this molecular typing assay for environmental studies was demonstrated by the accurate identification and differentiation of tick-borne F. tularensis. The described approach to IS targeting and amplification provides new capability for epidemiological investigations and characterizations of tularemia source outbreaks.

Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed F. tularensis type A genomes. In contrast with the F. tularensis A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced F. tularensis A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96-3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that F. tularensis A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving F. tularensis genetic diversity and plasticity is needed.

These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near neighbors.