Objections to the transfer of Francisella novicida to the subspecies rank of Francisella tularensis.
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ABSTRACT: The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis 'Type B' Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.PLoS ONE 08/2014; 9(8):e104881. · 3.53 Impact Factor
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ABSTRACT: Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.PLoS ONE 09/2014; 9(9):e107964. · 3.53 Impact Factor
- Clinical Infectious Diseases 06/2014; · 9.42 Impact Factor
Objections to the transfer of Francisella novicida to the
subspecies rank of Francisella tularensis
We disagree with a recent proposal by
Huber et al. to transfer Francisella novicida
to the subspecies rank of Francisella
tularensis (Huber et al., 2010). We believe
that the proposal is not appropriate in
light of all currently available knowledge.
In 1989, Hollis et al. (1989) argued that
F. novicida and F. tularensis could be
considered to be one species as judged
from DNA–DNA hybridization
experiments (Hollis et al., 1989). Their
publication was not valid according to the
requirements outlined in the Bacteri-
ological Code (Lapage et al., 1992; Tindall
et al., 2006). As a result, the proposed
elimination of the species F. novicida and
its demotion to a biogroup of F. tularensis
was not included among prokaryotic
names with standing in nomenclature.
Notably, earlier publications considered F.
novicida and F. tularensis to be separate
species based on differences in phenotype
including chemotaxonomic markers,
distinct ecological roles, different clinical
and epidemiological characteristics, and
differing abilities and modes of invasion
and mechanisms of tissue damage in
mammals (Larson et al., 1955; Olsufiev
et al., 1959; Skerman et al., 1980).
From a practical standpoint, separate
species names are useful in a
microbiological laboratory or a clinical
setting and also as a basis for regulations
governing the handling of medically
important organisms. For example,
laboratory handling of F. tularensis, but
not F. novicida, is associated with a high
risk of airborne laboratory-acquired
infection. Importantly, it is fairly easy to
distinguish F. novicida and F. tularensis on
the basis of their different growth and
metabolic requirements on artificial media.
Indeed, in Table 2 of Huber et al. (2010)
data are provided that contradict their own
proposal by presenting 11 metabolic
reactions that are distinct between
F. novicida and F. tularensis (Huber et al.,
Perhaps most importantly, recent findings
from the analysis of multiple genome
sequences of F. tularensis versus F. novicida
have indicated that the increased host-
association of F. tularensis is tied to
evolution as a population lineage
disconnected from F. novicida, even
though genome-wide average nucleotide
identities exceeded 97% (Larsson et al.,
2009). We propose that different
population structures and otherwise
disparate evolutionary patterns in
F. tularensis and F. novicida should be
considered as arguments for retaining
separate species names. A comparison of
17 genomes of members of the genus
Francisella has shown that the emergence
of F. tularensis, in an evolutionary and
population genetic framework, was a
speciation event with no signs of reversals.
For example, there were no traces of
genetic exchange between F. tularensis and
F. novicida. The analysis provided genetic
information that was more precise than
crude DNA–DNA hybridization values for
defining the genetic relationships between
F. tularensis and F. novicida. Recent intense
efforts, including evolutionary and
population criteria, have provided a useful
theoretical framework for defining
prokaryotic species (Achtman & Wagner,
2008; Gevers et al., 2005; Koeppel et al.,
2008). We believe that such a framework
should be taken into consideration in the
taxonomy of the genus Francisella.
Anders Johansson,1Jean Celli,2
Wayne Conlan,3Karen L. Elkins,4
Mats Forsman,5Paul S. Keim,6
Pa ¨r Larsson,5Colin Manoil,7Francis
E. Nano,8Jeannine M. Petersen9
and Anders Sjo ¨stedt1
1Department of Clinical Microbiology,
Umea ˚ University, SE-901 85 Umea ˚,
2Tularemia Pathogenesis Section,
Laboratory of Intracellular Parasites,
Rocky Mountain Laboratories, National
Institute of Allergy and Infectious
Diseases, National Institutes of Health,
Hamilton, MT 59840, USA
3National Research Council Canada,
Institute for Biological Sciences, Ottawa,
ON K1C 2M7, Canada
4Laboratory of Mycobacterial Diseases
and Cellular Immunology, Center for
Biologics Evaluation and Research, US
Food and Drug Administration, Rockville,
MD 20852, USA
5Division of CBRN Defense and Security,
Swedish Defense Research Agency, SE-
901 82 Umea ˚, Sweden
6Department of Biological Sciences,
Northern Arizona University, Flagstaff,
AZ 86011-5640, USA
7Genome Sciences, University of
Washington, Seattle, WA 98195-5065,
8Department of Biochemistry and
Microbiology, University of Victoria,
Victoria, BC V8W 3P6, Canada
9Centers for Disease Control and
Prevention, Division of Vector-Borne
Infectious Diseases, Bacterial Diseases
Branch, 1300 Rampart Road, CSU
Foothills Campus, Fort Collins, CO
Correspondence: Anders Johansson
Achtman, M. & Wagner, M. (2008). Microbial
diversity and the genetic nature of microbial
species. Nat Rev Microbiol 6, 431–440.
Gevers, D., Cohan, F. M., Lawrence, J. G.,
Spratt, B. G., Coenye, T., Feil, E. J.,
Stackebrandt, E., Van de Peer, Y.,
Vandamme, P. & other authors (2005).
Opinion: re-evaluating prokaryotic species. Nat
Rev Microbiol 3, 733–739.
Hollis, D. G., Weaver, R. E., Steigerwalt, A. G.,
Wenger, J. D., Moss, C. W. & Brenner, D. J.
(1989). Francisella philomiragia comb. nov.
(formerly Yersinia philomiragia) and Francisella
tularensis biogroup novicida (formerly
Francisella novicida) associated with human
disease. J Clin Microbiol 27, 1601–1608.
DOI 10.1099/ijs.0.022830-0Printed in Great Britain1717
Huber, B. E., Escudero, R., Busse, H. J.,
Seibold, E., Scholz, H. C., Anda, P., Ka ¨mpfer, P.
& Splettstoesser, W. D. (2010). Description of
Francisella hispaniensis sp. nov., isolated from
human blood, reclassification of Francisella
novicida (Larson et al. 1955) Olsufiev et al. 1959
as Francisella tularensis subsp. novicida comb.
nov., and emended description of the genus
Francisella. Int J Syst Evol Microbiol 60, 1887–
Koeppel, A., Perry, E. B., Sikorski, J.,
Krizanc, D., Warner, A., Ward, D. M., Rooney,
A. P., Brambilla, E., Connor, N. & other authors
(2008). Identifying the fundamental units of
bacterial diversity: a paradigm shift to
incorporate ecology into bacterial
systematics. Proc Natl Acad Sci U S A 105,
Lapage, S. P., Sneath, P. H. A., Lessel, E. F.,
Skerman, V. B. D., Seeliger, H. P. R. & Clark,
W. A. (editors) (1992). International Code of
Nomenclature of Bacteria (1990 Revision).
Bacteriological Code. Washington, DC: American
Society for Microbiology.
Larson, C. L., Wicht, W. & Jellison, W. L. (1955).
A new organism resembling P. tularensis isolated
from water. Public Health Rep 70, 253–258.
Larsson, P., Elfsmark, D., Svensson, K.,
Wikstro ¨m, P., Forsman, M., Brettin, T., Keim, P.
& Johansson, A. (2009). Molecular evolutionary
consequences of niche restriction in Francisella
tularensis, a facultative intracellular pathogen.
PLoS Pathog 5, e1000472.
Olsufiev, N. G., Emelyanova, O. S. & Dunayeva,
T. N. (1959). Comparative study of strains of B.
tularense in the old and new world and their
taxonomy. J Hyg Epidemiol Microbiol Immunol
Skerman, V. B. D., McGowan, V. & Sneath,
P. H. A. (editors) (1980). Approved lists of
bacterial names. Int J Syst Bacteriol 30, 225–420.
Tindall, B. J., Ka ¨mpfer, P., Euze ´by, J. P. &
Oren, A. (2006). Valid publication of names of
prokaryotes according to the rules of
nomenclature: past history and current practice.
Int J Syst Evol Microbiol 56, 2715–2720.
Objections to the transfer of Francisella novicida to the
subspecies rank of Francisella tularensis – response to Johansson
The description of novel species requires
the careful selection and use of a wide
variety of methodologies. As pointed out
by Tindall et al. (2010), experience gained
over the past six decades has continued to
demonstrate the value of comparing
different datasets and also of basing the
description and delineation of taxa on as
wide a dataset as possible. A combination
of data acquired from DNA-based
methods (DNA–DNA hybridization, gene
sequences, genomic fingerprints) and
physiological and morphological traits)
provides a sound basis for the taxonomy of
the prokaryotes (Tindall et al., 2010). The
decision as to whether two bacteria are
members of a single species is still based on
the results from DNA–DNA hybridizations
(Wayne et al., 1987; Stackebrandt et al.,
2002). In general, two bacterial strains are
assigned to the same species if their DNAs
reassociate at levels greater than 70% and
5% or less DTm(Wayne et al., 1987), but
the latter criterion is only rarely applied. In
addition, Wayne et al. (1987) pointed out
‘Subspecies designations can be used for
genetically close organisms that diverge in
Our proposal to transfer Francisella
novicida as a novel subspecies to F.
tularensis subsp. novicida is in agreement
with the above-mentioned
recommendations. As demonstrated by the
results from DNA–DNA reassociation
experiments, F. novicida is genetically close
to F. tularensis (Hollis et al., 1989) and the
phenotypic differences observed (Huber et
al., 2010) are in agreement with the
subspecies concept. Another important
point supporting this taxonomic
rearrangement is the acceptance of the new
combination within the scientific
community. The use of this not yet validly
published new combination may be related
to the fact that in Bergey’s Manual of
Systematic Bacteriology (often erroneously
considered as the ‘bible’ of bacterial
systematics by those interested in bacterial
taxonomy), the transfer of F. novicida to
Francisella tularensis subsp. novicida was
recommended in the chapter dealing with
the genus Francisella (Sjo ¨stedt, 2005).
Although this proposal was never formally
recognized, numerous microbiologists are
already using the name. An online search
survey in ‘Pubmed’ (http://www.ncbi.nlm.
cates that in recent years there is no
significant difference in the frequencies of
the use of the names F. novicida and F.
tularensis subsp. novicida.
From our point of view, it is not consistent
to have a species F. tularensis with three
subspecies supported by DNA–DNA
relatedness data but distinguishable by
phenotypic traits and a separate species F.
novicida that also shares high DNA–DNA
relatedness values (.85%) but which is
phenotypically distinguishable. Based on
the results from the literature and the
results from our investigations, but also for
sake of consistency, it is obvious that our
proposal to assign F. novicida to F.
tularensis as a novel subspecies is well
Below are some additional replies to
certain arguments proposed by Johansson
et al. (2010) to support their stance against
the reclassification of F. novicida.
It is argued, that:
‘From a practical standpoint, sep-
arate species names are useful in a
microbiological laboratory or a
clinical setting and also as a basis
for regulations governing the hand-
ling of medically important organ-
isms. [...] Importantly, it is fairly
easy to distinguish F. novicida and
F. tularensis on the basis of their
different growth and metabolic
requirements on artificial media’.
In contrast to tularaemia caused by F.
tularensis subsp. tularensis or F. tular-
ensis subsp. holarctica, human or
animal infections with strains of F.
tularensis subsp. novicida are extre-
mely rare and there are very few
publications reporting the isolation
of this facultative pathogen. Most of
these reports have shown that it was
very difficult to distinguish those
isolates from strains of F. tularensis,
not only for routine clinical laborat-
Letters to the Editor
1718International Journal of Systematic and Evolutionary Microbiology 60