International Journal of Systematic and Evolutionary Microbiology (2016), 66, 536–541
DOI 10.1099/ijsem.0.000750
Description of ‘Candidatus Berkiella aquae’
and ‘Candidatus Berkiella cookevillensis’,
two intranuclear bacteria of freshwater amoebae
Yohannes T. Mehari,1 B. Jason Hayes,13 Katherine S. Redding,24
Parvadha V. G. Mariappan,3§ John H. Gunderson,3 Anthony L. Farone1
and Mary B. Farone1
Correspondence
1
Mary B. Farone
2
[email protected]
Biology Department, Middle Tennessee State University, Murfreesboro, TN, USA
Center for the Management, Utilization, and Protection of Water Resources,
Tennessee Technological University, Cookeville, TN, USA
3
Department of Biology, Tennessee Technological University, Cookeville, TN, USA
Two novel bacteria of the phylum Proteobacteria were isolated during searches for amoebaresistant micro-organisms in natural and constructed water systems. Strain HT99 was isolated
from amoebae found in the biofilm of an outdoor hot tub in Cookeville, Tennessee, USA, and
strain CC99 was isolated from amoebae in the biofilm of a cooling tower in the same city. Both
bacteria were Gram-stain-negative cocci to coccobacilli, unculturable on conventional laboratory
media, and were found to be intranuclear when maintained in Acanthamoeba polyphaga. The
genomes of both isolates were completely sequenced. The genome of CC99 was found to be
3.0 Mbp with a 37.9 mol% DNA G+C content, while the genome of HT99 was 3.6 Mbp with a
39.5 mol% DNA G+C content. The 16S rRNA gene sequences of the two isolates were 94 %
similar to each other. Phylogenetic comparisons of the 16S rRNA, mip and rpoB genes, the DNA
G+C content and the fatty acid composition demonstrated that both bacteria are members of
the order Legionellales, and are most closely related to Coxiella burnetii. The phenotypic and
genetic evidence supports the proposal of novel taxa to accommodate these strains; however,
because strains HT99 and CC99 cannot be cultured outside of the amoeba host, the respective
names ‘Candidatus Berkiella aquae’ and ‘Candidatus Berkiella cookevillensis’ are proposed.
Free-living amoebae are natural environmental predators of
bacteria and other micro-organisms found in soil and
water, including anthropogenic systems such as drinking
water systems, air conditioners and cooling towers, spas
3Current address: Biology Program, Sinclair Community College,
Mason, OH 45040, USA.
4Current address: Department of Dermatology, University
Tennessee Health Science Center, Memphis, TN 38163, USA.
of
§Current address: Biology Department, East Central College, Union,
MO 63084, USA.
Abbreviations: SSW, filter-sterilized spring water; TEM, transmission
electron microscopy.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA
gene sequences of CC99 and HT99 are EF492067 and AY741401,
respectively. Those for the mip and rpoB sequences of CC99 and HT99
are KP177956, KP177957, KP177958 and KP177959, respectively.
Two supplementary figures and one supplementary table are available
with the online Supplementary Material.
536
and swimming pools (Rodrı́guez-Zaragoza, 1994; Benkel
et al., 2000; Greub & Raoult, 2004; Berk et al., 2006).
Some bacteria only survive engulfment by amoebae whereas
other bacteria not only survive but multiply within the
amoebal host (Barker & Brown, 1994; Horn & Wagner,
2004; Molmeret et al., 2005). Recent reviews list over 40
genera of bacterial symbionts of amoebae that are human
pathogens, making amoebae important environmental
reservoirs of virulent bacteria (Lamoth & Greub, 2010;
Moliner et al., 2010). Evidence also suggests that amoebae
can aid in the selection of virulence traits and the adaptation of bacteria for intracellular survival in human macrophages and other cell types (Cirillo et al., 1994; Greub &
Raoult, 2004; Evstigneeva et al., 2009). This report describes
two bacterial isolates, HT99 and CC99, that were recovered
from amoebae in biofilm samples from constructed aquatic
environments (Berk et al., 2006). Both bacteria are amoebaresistant, intranuclear bacteria. We have determined, on the
basis of both genotypic and phenotypic characteristics, that
these two bacteria do not belong to known genera, justifying the proposal of novel taxa. However, due to the
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Intranuclear bacteria of amoebae
intracellular lifestyle of the bacteria and the lack of pure cultures, we propose the classification of these two bacteria as
representatives of ‘Candidatus Berkiella aquae’ and ‘Candidatus Berkiella cookevillensis’ and in this report describe
the characteristics of both novel bacteria.
Bacterial strain HT99 was recovered from an amoeba isolated from an outdoor hot tub spa, and strain CC99 was
recovered from an amoeba isolated from a hospital cooling
tower. The amoebae were found in biofilm material that
was swabbed from the sides of a cooling tower and an
uncovered hot tub spa that were both filled with chlorinated water from a municipal water system in Cookeville,
Tennessee, USA. The biofilm material was made into a
slurry and plated onto the centre of non-nutrient agar
plates seeded with UV-killed Escherichia coli and processed
as previously described (Berk et al., 2006). After 48 h,
plates were examined microscopically for the presence of
amoebae. Plates positive for amoebae were washed with
approximately 2 ml of sterile Tris-buffered saline solution
and 0.1 ml aliquots of the wash were added to individual
wells of a 96-well flat-bottom microplate. Microplates
were incubated at the temperature of the original agar
plate and wells were checked daily for the presence of
infected amoebae. Both CC99 and HT99 were recovered
from samples incubated at 30 8C that contained native
amoebae infected with highly motile bacteria. Aliquots
from microplate wells positive for infected amoebae were
added to Acanthamoeba polyphaga monolayers. Monolayers of A. polyphaga (ATCC 30461) were grown in
25 cm2 flat-bottomed cell culture flasks in 5 ml tryptic
soy broth (TSB, Becton Dickinson). Once amoebae were
confluent, the growth medium was removed, and the
monolayer washed and medium replaced with 5 ml of
filter-sterilized spring water (SSW; Carolina Biological
Supply), which is osmotically balanced but nutrient poor
to encourage amoebae to feed. After addition of 0.1 ml aliquots from microplate wells to the monolayers, incubation
was continued at 30 8C, which increased the number of
infected amoebae although contaminating bacteria were
still present. Contaminating bacteria were eliminated by
passage of 3- to 5-day-old amoeba lysates through
0.45 mm filters, antibiotic treatment with 50 mg gentamicin
ml21 and 100/50 mg piperacillin/tazobactam ml21 (all
from Sigma-Aldrich), and dilution of contaminating bacteria to extinction. Following each attempt to eliminate
contaminants, aliquots of the treated sample were added
to monolayers of A. polyphaga in SSW as above to confirm
that the infecting bacteria were still present. Additional aliquots were plated onto surfaces of BCYE differential agar,
tryptic soy agar (TSA) and TSA II sheep blood agar (all
from Becton Dickinson) to assess the numbers of contaminants and to check individual colony types for their infectivity for amoebae. For both of the described isolates,
contaminating bacteria were eliminated using these
described techniques. However, the infecting bacteria
were unable to be cultured on conventional laboratory
medium as determined by plating amoeba lysates of the
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co-cultures onto BCYE differential agar, TSA and TSA II
sheep blood agar with incubation at temperatures from
25 to 37 8C in ambient air, 5 % CO2, anaerobic (90 %
H2, 10 % CO2; GasPak, Becton Dickinson) and microaerophilic (5 % O2, 10 % CO2, 85 % N2; CampyPak, Becton
Dickinson) atmospheres. Cultivation of the micro-organisms was also attempted with the more recently described
ACCM-2 medium (kindly provided by Dr Robert Heinzen)
for axenic growth of Coxiella burnetii (Omsland et al.,
2011). Gram, endospore, capsular and RYU flagellar
(Remel) staining were performed on bacteria in amoeba
lysates.
Because isolates CC99 and HT99 did not grow on the conventional laboratory media described above, their growth
in A. polyphaga was examined by Giemsa staining and
transmission electron microscopy (TEM). A. polyphaga
monolayers were maintained in TSB and transferred to
SSW for infection with the bacteria. Both Giemsa staining
and TEM were performed periodically following infection
of A. polyphaga. For Giemsa staining, a 0.1 ml aliquot of
dislodged cell suspension was centrifuged through a Shandon cytofunnel (Thermo Scientific) onto a microscope
slide and Giemsa stained (Sigma-Aldrich) as described by
Newsome et al. (1998). For TEM, bacteria and
A. polyphaga co-cultures were fixed in 2.5 % (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h, followed by post-fixation with 2 % (w/v) aqueous osmium
tetroxide for 2 h at room temperature. Fixed specimens
were dehydrated in an ascending ethanol series from 30
to 100 % (v/v) with final dehydration in propylene oxide.
Samples were embedded in Epon-Araldite (Electron
Microscopy Sciences) and 70 nm sections stained with
5 % (w/v) uranyl acetate and lead citrate. Microscopy
was performed with a Hitachi H-7650-II transmission electron microscope.
The 16S rRNA genes from isolates CC99 and HT99 were
amplified from amoeba co-cultures by the PCR using
universal eubacterial primers 8F (59-AGTTACTCTTCACCATTGATCCTGGCTCAG-39) and 1541R (59-GCTAAGGATCCAAGCTTAAGGAGGTGWTCCA-39). PCR products
were cloned into plasmid vectors and sequenced using
internal primers 515F (59-GTGCCAGCMGCCGCGGTAA-39), 911F (59-TCAAAKGAATTGACGGGGGC-39),
806R (59-GGACTACCAGGGTATCTAAT-39) and 1392R
(59-ACGGGCGGTGWGTRC-39) as previously described
(Berk et al., 2006). The plasmid inserts were sequenced in
both directions, combined into a single consensus
sequence, and compared with sequences available in the
GenBank database using the BLASTN program on the
NCBI website (http://www.ncbi.nlm.nih.gov). For whole
genome sequencing, genomic DNA was purified from
both isolates following their growth in A. polyphaga co-culture. After complete lysis of the amoebae, CC99 and HT99
cells were separated from host cell debris using Renografin
density-gradient centrifugation as described by Shannon &
Heinzen (2008). Genomic DNA was extracted from the
purified isolates using a DNeasy Blood and Tissue kit
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Y. T. Mehari and others
(Qiagen). The genomic DNA was sequenced at the University of Maryland School of Medicine, Institute for Genome
Sciences (IGS), Genome Resource Center. The genome
sequences were generated using genomic paired-end
sequencing on an Illumina MiSeq and sequence reads
were assembled using Newbler software v2.5.3 (Roche).
The contig data were annotated using the annotation pipeline at the IGS Informatics Resource Center (http://www.
igs.umaryland.edu/). Protein coding sequences were identified using Glimmer3 algorithm as part of the IGS annotation engine. Non-coding RNA genes were predicted using
RNAmmer, and tRNA-scanSE was utilized to predict
tRNA genes. Searches for all predicted genes were performed using NCBI BLAST (http://www.ncbi.nlm.nih.gov/
blast/). Phylogenetic trees were reconstructed from conserved gene sequences, 16S rRNA (1356 bp), mip
(587 bp) and rpoB (384 bp). The 16S rRNA, mip and
rpoB gene sequences were aligned with similar sequences
from the NCBI nucleotide database using CLUSTAL _X 2.0
and MEGA6 software (Tamura et al., 2011). Bayesian analyses were run using MrBayes (v3.2.2) under the general
time reversible (GTR) model along with gamma-distributed rates of evolution and a proportion of invariant sites
(Ronquist & Huelsenbeck, 2003). Analyses were performed
for 2 000 000 generations using four Monte Carlo Markov
chains, and sample trees were taken every 1000 generations.
Posterior probabilities calculated over 5000 best trees were
used as support values for nodes in the tree. Maximumlikelihood analyses were performed using PhyML 3.0
(a)
with input tree generated by BIONJ with the GTR model
of nucleotide substitution incorporating invariable sites
and a discrete gamma distribution. The node reliabilities
of the phylogenetic tree generated were estimated using
bootstrap values calculated over 1000 replicates.
Cellular fatty acid analysis was performed using bacterial
isolates purified from amoebal co-cultures using Renografin density-gradient centrifugation as described above.
Fatty acid methyl esters (FAME) were obtained by saponification, methylation and extraction as described in the
MIDI standard protocol. FAME analyses were carried out
by MIDI using a Hewlett Packard 5890 gas chromatograph.
The MIDI software package was used to automatically integrate peaks as well as to name the predominant fatty acids
and determine their composition as a percentage of total
peak area.
Both strains CC99 and HT99 were transferred to cultures
of A. polyphaga and initially resembled Legionella-like
amoebal pathogens (Adeleke et al., 1996) due to their failure to be cultivated on traditional culture media and their
intracellular growth and high motility within A. polyphaga.
No growth of either bacterium occurred on any of the culture media tested under any incubation conditions
employed for 30 or more days, including the ACCM-2
growth medium, as determined by both optical density
measurements and PCR amplification of 16S rRNA gene.
TEM (Fig. 1) and Giemsa staining (Fig. S1, available in
the online Supplementary Material) showed that both
(b)
Fig. 1. Transmission electron micrographs of cells of CC99 (a) and HT99 (b). Bacteria are seen within the double nuclear
membrane of A. polyphaga (arrows; bars, 2 mm). Although bacteria often appear in the cytoplasm as they travel to the nucleus
or extracellular following cell lysis, the structures present in the cytoplasm seen in these micrographs are mitochondria, which
are similar in size to the bacteria. Insets of the bacteria show the condensed chromatin and that their sizes are between 300
and 500 nm (inset bars, 0.5 mm).
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Intranuclear bacteria of amoebae
isolates were contained within the nuclei of amoebal cells.
Bacteria were visible in nuclei as early as 12 h post-infection (p.i.) and filled the nuclei over the following
24–48 h, resulting in cytopathogenicity of the amoeba
population within 72 h. Bacteria remained motile for
12–24 h, after which time, the bacterial cells became nonmotile and adhered tightly to the bottom of culture
flasks. Gram staining of lysed amoebal cultures showed
that both CC99 and HT99 were Gram-stain-negative. Bacterium CC99 was coccoid with diameters ranging from
0.30 to 0.60 mm, occurring singly or as diplococci. HT99
was a coccobacillus, 0.45–0.65 mm in length and 0.30–
0.55 mm in width, occurring singly or in pairs. Both
CC99 and HT99 exhibited electron-dense, condensed chromatin. Neither endospores nor capsules were detected by
staining. Flagella staining revealed polar flagella in both
isolates, although no flagella were observed for either bacterium by TEM. Because TEM only showed intracellular
bacteria, it is likely that, as with Legionella pneumophila,
motility is associated with their growth phase such that
bacteria multiplying within the host are non-motile,
while bacteria from later stages of infection or in cell lysates
are flagellated and highly motile (Pruckler et al., 1995;
Byrne & Swanson, 1998). CC99 and HT99 were infectious
for at least 50–60 days when in suspensions of lysed amoebae in SSW. When tested for intracellular growth at various
temperatures, both CC99 and HT99 grew in A. polyphaga
and were detected in the nuclei at 28, 30 and 37 8C.
The genomes of both isolates were sequenced for total reads
of 11 839 536 and 11 009 736 for CC99 and HT99, respectively. The reads were assembled into 44 contigs containing
2 990 361 bp for CC99 and 56 contigs containing
3 626 027 bp for HT99. The DNA G+C content of the
CC99 and HT99 genomes was 37.9 mol% and 39.5 mol%,
respectively, and is similar to those reported for other species
of the genus Legionell (Pagnier et al., 2014). The 16S rRNA
gene sequences were 95–100 % (CC99) and 90–96 %
(HT99) similar to sequences from uncultured organisms
in the NCBI GenBank database. CC99 had 100 % identity
to an uncultured isolate from a Korean oyster shell waste
pile. Of cultured organisms, CC99 and HT99 were most
similar to C. burnetii (90 % of 1354 and 91 % of 1355
bases, respectively). Both CC99 and HT99 showed only
88 % identity to L. pneumophila sequences and had 94 %
identity to each other, thus demarcating them as representatives of separate species according to the 3 % delineation for
16S rRNA divergence between species (Stackebrandt &
Goebel, 1994). The Bayesian and maximum-likelihood phylogenetic analyses based on 16S rRNA (Fig. 2), mip and rpoB
genes (Fig. S2) showed that isolates CC99 and HT99 appear
within the order Legionellales but occupy a distinct phylogenetic position clustered within the family Coxiellaceae.
Strains CC99 and HT99 had similar cellular fatty acids with
different proportions (Table S1). The predominant fatty
acids on both CC99 and HT99 were monounsaturated,
18 : 1 v9c, 18 : 1 v7c and 16 : 1 v7c, with a combined
content of 51 % and 55 %, respectively. The major fatty
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acid was 18 : 1 v9c for both CC99 (19 %) and HT99
(30 %). The other moderate fatty acid components
included 16 : 0, 18 : 0, 18 : 2 v6c, 20 : 4 v6c and 20 : 2
v6c. Unlike species of the genera Coxiella, Aquicella and
Legionella, CC99 and HT99 only contained trace amounts
of branched-chain fatty acids (Tzianabos et al., 1981;
Diogo et al., 1999; Santos et al., 2003).
In summary, based on phylogenetic, physiological and
morphological evidence, we conclude that strains CC99
and HT99 belong to unique species within a novel candidate genus in the family Coxiellaceae. The two bacteria
form a distinct phyletic lineage within the family Coxiellaceae with less than 94 % sequence similarity to one
another, as well as distinct cellular fatty acid profiles, distinguishing them as representatives of separate species.
However, because both CC99 and HT99 exhibit similar
morphologies, intranuclear growth and fatty acid compositions, they have been placed within the same genus. The
invasion of the amoeba host nucleus by both strains
distinguishes them from other members of the family
Coxiellaceae, and they contain minimal amounts of
branched-chain fatty acids, which is unlike C. burnetii and
species of the genus Aquicella of the family Coxiellaceae and
species of the genus Legionella of the family Legionellaceae.
Description of Candidatus Berkiella aquae
‘Candidatus Berkiella aquae’ [Berk.i.el9la. L. dim. suff. -ella;
N.L. fem. n. Berkiella after the US microbiologist, Sharon
G. Berk, for her contributions to the study of interactions
between protozoa and bacteria; a9quae. L. fem. gen. n. aquae
of water, referring to the source of isolation and for the
Roman term, aquae, referring to settlements with mineral
springs such as Aquae Sulis, now Bath, England, and Aquae
Spadanae, near Liège, Belgium (Sarton, 1954; Bunson, 1991)].
Represented by strain HT99, which was isolated from an
amoeba in the biofilm of an outdoor hot tub spa in Cookeville, TN, USA. The strain is in axenic culture with the
amoeba host A. polyphaga. Cells stain Gram-negative and
are short coccobacillus-shaped cells, 0.45–0.65 mm in
length and 0.30–0.55 mm in width, occurring singly or in
pairs. Non-spore-forming, motile and exhibits intranuclear
growth in the amoeba host. Phylogenetic analyses of the
16S rRNA, mip and rpoB genes indicate that this strain is
different from all other recognized genera and belongs to
the class Legionellales, family Coxiellaceae. The mole
G+C ratio of the DNA is 39.5 mol%. Predominant fatty
acids are monounsaturated, 18 : 1 v9c, 18 : 1 v7c and
16 : 1 v7c. The bacterium does not grow on conventional
laboratory culture medium, but grows in A. polyphaga at
temperatures ranging from 25 to 37 uC.
Description of Candidatus Berkiella
cookevillensis
‘Candidatus Berkiella cookevillensis’ (cooke9vill.ensis. L.
adj. cookevillensis referring to Cookeville, TN, USA, where
the first strain was isolated).
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539
Y. T. Mehari and others
78/1.00
98/0.83
0.71
Legionella wadsworthiiT (NR_044970)
Fluoribacter dumoffiiT (NR_119025)
Legionella bozemanae (M36031)
70/0.90
0.2
Fluoribacter gormaniiT (NR_026521)
91/1.00
Legionella longbeachaeT (AY444740)
86/1.00
Legionella lyticaT (NR_026334)
77/0.98
1.00
0.94
Legionellaceae
Legionella pneumophila Philadelphia 1T (NR_074231)
'Legionella donaldsonii' (Z49724)
Legionella tunisiensisT (NR_109416)
0.93
Legionella geestiana (Z49723)
Legionella massiliensisT (NR_109415)
100/1.00
0.69 100/1.00
Legionella nagasakiensisT (NR_116420)
Legionella oakridgensisT (X73397)
0.88
Legionella maceacherniiT (NR_041790)
100/1.00
Legionella micdadeiT (NR 041791)
Legionella jamestowniensisT (NR_044959)
'Candidatus Berkiella cookevillensis' CC99 (EF492067)
'Candidatus Berkiella aquae HT99' (AY741401)
80/1.00
Coxiella burnetii RSA 493 (NR_074154)
98/1.00
84/1.00
54/0.88
100/1.00
93/1.00
'Candidatus Berkiella'
Coxiella
'Rickettsiella tipulae' (EU180598)
'Rickettsiella melolonthae' (EF408231)
Rickettsiella
Rickettsiella grylli (U97547)
Coxiellaceae
100/1.00
Diplorickettsia massiliensisT (GQ857549) Diplorickettsia
100/1.00
Aquicella siphonisT (NR_025764)
Aquicella lusitanaT (NR_025763)
Aquicella
Francisella tularensis subsp. mediasiatica (NR_074666)
Fig. 2. 16S rRNA gene phylogenetic tree inferred using maximum-likelihood and Bayesian analyses. The tree depicts phylogenetic relationships between bacterial strains CC99 and HT99 and sequences in the NCBI database, representing recognized bacterial species in the order Legionellales. The tree was rooted by using Francisella tularensis as an outgroup. Values
at nodes represent maximum-likelihood bootstrap values in percentage (first value) and Bayesian posterior probabilities
(second value); only bootstrap values above 50 % are shown. Bar, nucleotide substitutions per site.
Represented by bacterial strain CC99, which was isolated
from an amoeba in the biofilm of a cooling tower in Cookeville, TN, USA. The strain is in axenic culture with
A. polyphaga. Cells stain Gram-negative and are coccoid
shaped with diameters ranging from 0.30 to 0.60 mm,
occurring singly or as diplococci. Non-spore-forming,
motile and exhibits intranuclear growth in the amoeba
host. Phylogenetic analyses of the 16S rRNA, mip and
rpoB genes indicate that this strain is different from all
other recognized genera and from ‘Ca. Berkiella aquae’,
and belongs to the order Legionellales, family Coxiellaceae.
The mole G+C ratio of the DNA is 39.5 mol%. Predominant fatty acids are monounsaturated, 18 : 1 v9c, 18 : 1
v7c and 16 : 1 v7c. The bacterium does not grow on
540
conventional laboratory culture medium, but grows in
A. polyphaga at temperatures ranging from 25 to 37 uC.
Acknowledgements
This work has been supported by grants from the US Environmental
Protection Agency Science to Achieve Results (STAR) grant program
(nos R82711101 and R833102) and FRCAC grants from Middle Tennessee State University, as well as continued support from the Biology
Department at Middle Tennessee State University and the Department of Biology and Center for the Management, Utilization, and
Protection of Water Resources at Tennessee Technological University.
The authors would like to thank Witold Skolasinski of Middle Tennessee State University for assistance with characterizing bacterium
HT99, and Joyce Miller and Marion Wells of the MIMIC Center of
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Intranuclear bacteria of amoebae
Middle Tennessee State University and Amy Knox of Tennessee Technological University for their technical assistance with TEM and
figure preparation for this project.
Molmeret, M., Horn, M., Wagner, M., Santic, M. & Abu Kwaik, Y.
(2005). Amoebae as training grounds for intracellular bacterial
pathogens. Appl Environ Microbiol 71, 20–28.
Newsome, A. L., Scott, T. M., Benson, R. F. & Fields, B. S. (1998).
References
Isolation of an amoeba naturally harboring a distinctive Legionella
species. Appl Environ Microbiol 64, 1688–1693.
Adeleke, A., Pruckler, J., Benson, R., Rowbotham, T., Halablab, M. &
Fields, B. (1996). Legionella-like amebal pathogens—phylogenetic
Omsland, A., Beare, P. A., Hill, J., Cockrell, D. C., Howe, D., Hansen,
B., Samuel, J. E. & Heinzen, R. A. (2011). Isolation from animal tissue
Barker, J. & Brown, M. R. W. (1994). Trojan horses of the microbial
and genetic transformation of Coxiella burnetii are facilitated by
an improved axenic growth medium. Appl Environ Microbiol 77,
3720–3725.
status and possible role in respiratory disease. Emerg Infect Dis 2, 225–230.
world: protozoa and the survival of bacterial pathogens in the
environment. Microbiology 140, 1253–1259.
Pagnier, I., Croce, O., Robert, C., Raoult, D. & La Scola, B. (2014).
Benkel, D. H., McClure, E. M., Woolard, D., Rullan, J. V., Miller, G. B. Jr.,
Jenkins, S. R., Hershey, J. H., Benson, R. F., Pruckler, J. M. & other
authors (2000). Outbreak of Legionnaires’ disease associated with a
Genome sequence of Legionella anisa, isolated from a respiratory
sample, using an amoebal coculture procedure. Genome Announc 2,
e00031–e00014.
display whirlpool spa. Int J Epidemiol 29, 1092–1098.
Pruckler, J. M., Benson, R. F., Moyenuddin, M., Martin, W. T. &
Fields, B. S. (1995). Association of flagellum expression and
Berk, S. G., Gunderson, J. H., Newsome, A. L., Farone, A. L., Hayes,
B. J., Redding, K. S., Uddin, N., Williams, E. L., Johnson, R. A. & other
authors (2006). Occurrence of infected amoebae in cooling towers
intracellular growth of Legionella pneumophila. Infect Immun 63,
4928–4932.
compared with natural aquatic environments: implications for
emerging pathogens. Environ Sci Technol 40, 7440–7444.
Rodrı́guez-Zaragoza, S. (1994). Ecology of free-living amoebae. Crit
Bunson, M. (1991). A Dictionary of the Roman Empire. Oxford, UK:
Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian
Rev Microbiol 20, 225–241.
Byrne, B. & Swanson, M. S. (1998). Expression of Legionella
phylogenetic inference under mixed models. Bioinformatics 19,
1572–1574.
pneumophila virulence traits in response to growth conditions.
Infect Immun 66, 3029–3034.
Santos, P., Pinhal, I., Rainey, F. A., Empadinhas, N., Costa, J., Fields,
B., Benson, R., Verı́ssimo, A. & Da Costa, M. S. (2003). Gamma-
Cirillo, J. D., Falkow, S. & Tompkins, L. S. (1994). Growth of
proteobacteria Aquicella lusitana gen. nov., sp. nov., and Aquicella
siphonis sp. nov. infect protozoa and require activated charcoal
for growth in laboratory media. Appl Environ Microbiol 69,
6533–6540.
Oxford University Press.
Legionella pneumophila in Acanthamoeba castellanii enhances
invasion. Infect Immun 62, 3254–3261.
Diogo, A., Verı́ssimo, A., Nobre, M. F. & da Costa, M. S. (1999).
Usefulness of fatty acid composition for differentiation of Legionella
species. J Clin Microbiol 37, 2248–2254.
Sarton, G. (1954). Book review of Médecine et Médicins au Pays de
Evstigneeva, A., Raoult, D., Karpachevskiy, L. & La Scola, B. (2009).
Shannon, J. G. & Heinzen, R. A. (2008). Infection of human
Amoeba co-culture of soil specimens recovered 33 different bacteria,
including four new species and Streptococcus pneumoniae.
Microbiology 155, 657–664.
monocyte-derived macrophages with Coxiella burnetii. Methods Mol
Biol 431, 189–200.
Greub, G. & Raoult, D. (2004). Microorganisms resistant to free-living
Liège by Marcel Florkin. J Hist Med Allied Sci IX, 471–475.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for
Horn, M. & Wagner, M. (2004). Bacterial endosymbionts of free-living
DNA-DNA reassociation and 16S rRNA sequence analysis in the
present species definition in bacteriology. Int J Syst Bacteriol 44,
846–849.
Lamoth, F. & Greub, G. (2010). Amoebal pathogens as emerging
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. &
Kumar, S. (2011). MEGA5: molecular evolution genetics analysis
Moliner, C., Fournier, P.-E. & Raoult, D. (2010). Genome analysis of
using maxiumum likelihood, evolutionary distance, and maximum
parsiomony methods. Mol Biol Evol 28, 2731–2739.
amoebae. Clin Microbiol Rev 17, 413–433.
amoebae. J Eukaryot Microbiol 51, 509–514.
causal agents of pneumonia. FEMS Microbiol Rev 34, 260–280.
microorganisms living in amoebae reveals a melting pot of
evolution. FEMS Microbiol Rev 34, 281–294.
http://ijs.microbiologyresearch.org
Tzianabos, T., Moss, C. W. & McDade, J. E. (1981). Fatty acid
composition of rickettsiae. J Clin Microbiol 13, 603–605.
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