Medical Hypotheses 103 (2017) 29–31

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Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy

In Brucella: Selective pressure may turn some genes on instead of default

off position
Gamal Wareth a,b,⇑, Falk Melzer a, Heinrich Neubauer a
a
OIE Reference Laboratory for Brucellosis, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str.
96a, 07743 Jena, Germany
b
Faculty of Veterinary Medicine, Benha University, Moshtohor, Toukh 13736, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Brucellosis is a major zoonosis with worldwide prevalence; more than half a million new human cases
Received 11 January 2017 are expected every year. The genus Brucella (B) encompasses 12 accepted nomo-species and brucellae
Accepted 3 April 2017 were described as Gram negative, aerobic, non-motile and non-haemolytic facultative intracellular bac-
teria. The Brucella genome contains flagella-specific genes and various hemolysins, but no flagella are
formed and nor hemolysis is seen. Selective pressure can cause accumulation of mutations that turn
those genes on instead of default off position and provoked the motile and hemolytic phenotypes. The
ability of brucellae to change from a non-haemolytic to a haemolytic phenotype might influence their
pathogenicity and could provide a substantial insight to explain the correlation of acute brucellosis
and hemolytic anemia in humans.
Ó 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background Brucellosis is one of the most significant zoonotic diseases

Turning genes on and off is a major activity of all living cells. In
the human genome, almost 10 percent of the genes produce pro-
teins that regulate the expression of other genes. Cells express
‘turns on’ only a fraction of their genes at a certain time point,
while repress ‘turned off’ the rest of the genes. Microbes regulate
the expression of many of their genes in response to selective pres-
sure, environmental stress and nutritional sources available.
Microbes face various challenges in their natural habitat e.g. host
immune system, nutrient limitations, or interspecies competition,
but adjust their metabolism to nutrient rich monoculture condi-
tions of the laboratory and may acquire genetic modifications to
cope with selective pressure [1,2]. The Nobel laurates Francois
Jacob and Jacque Monod explored for the first time the mecha-
nisms for turning genes on and off in bacteria. They reported, that
bacterial genes responsible for digestion can be turned on and off
according to nutritional content in different media. However, gene
regulation is critical for life and the complicated process is not yet
fully understood.
worldwide. Disease in farm animals has been known from ancient
time and is caused by B. melitensis (small ruminants as hosts), B.
abortus (bovidae as hosts) and B. suis (pigs as hosts). Only recently,
wild type adapted species have been described including B. pinni-
pedialis from cetaceans causing sever disease but with an unknown
zoonotic impact. Twelve accepted homogenous species were rec-
ognized in the genus Brucella. Dissociation, colony morphology
and cultural characterizations are the most basic features used to
distinguish between the different brucellae in the laboratory. Com-
plete genome sequencing of B. melitensis strain 16 M revealed the
presences of flagella-specific genes and several genes that encode
for various types of hemolysins [3], but those genes are not active
and motile or hemolytic phenotypes of B. melitensis have not been
reported yet. Multiple sub-cultivations of brucellae under adverse
conditions is resulting in a changed appearance colonies leading to
a changed appearance of colonies due to gene functions.
Hypothesis

Multiple passages of a Brucella strain in vitro induce changes in

⇑ Corresponding author at: OIE Reference Laboratory for Brucellosis, Friedrich-
Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial
Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany.
E-mail addresses: gamalwareth@hotmail.com, gamal.wareth@fli.de (G. Wareth).
its cultural characteristics. We hypothesize that selective pressure
may cause accumulation of some mutations that turn some genes
on instead off position. Such events could play a significant role in
pathogenicity/virulence of Brucella and might result in changing of
colony characteristics also.

http://dx.doi.org/10.1016/j.mehy.2017.04.006
0306-9877/Ó 2017 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
30 G. Wareth et al. / Medical Hypotheses 103 (2017) 29–31
Discussion
Brucellosis is a debilitating zoonotic disease affecting humans
and animals. Although the genomes of Brucella spp. contain several
hemolysins and flagellar operons, no evidence for motility and
hemolysis was observed by coincidence until now. Thus brucellae
are considered as a non-motile and non-hemolytic intracellular
bacterium. Accumulations of mutations during growth may lead
to the repair of non-functional genes present in the Brucella gen-
ome and may result in changes pathogenesis. We would like to
support our postulate by the following observations:
(1) Motility characteristics: Earlier it has been reported that
under specific growth conditions, non-motile B. melitensis
was able to form a sheathed flagellum-like structure by an
unknown mechanism. Recently, Brucella strains isolated
from a Pac-Man frog (Ceratophyrus ornate) in Texas showed
for the first time high motility [4]. Though brucellae were
described as non-motile bacteria, B. melitensis expresses dis-
tal and basal parts of the flagellum [5]. The whole genomic
sequencing of non-motile brucellae revealed the presence
of flagella pseudogenes or flagella-specific type III, type IV
genes [3] but without function. On the other hand, the anal-
ysis of flagella genes in the recently described motile strains
revealed that all genes were fully functional [4]. Phenomena
occurring during passage of strains may have led to reactiva-
tion and expression of the turned off genes.
(2) Hemolysis characteristics: Although brucellae are described
as non-hemolytic bacteria, genes involved in the production
of hemolysin, b-hemolysin and hemolysin III were found in
B. melitensis, B. abortus, B. suis [3], and B. pinnipedialis. Here,
we report for the first time in vitro generated B. pinnipedialis
cause hemolysis on blood agar after 30 serial passages under
non-optimal laboratory conditions. After twenty serial pas-
sages on blood agar, B. pinnipedialis colonies produced weak
b-hemolysis on blood agar media containing 7.5% blood. The
hemolysis was clear in the dense part of the plate. The inten-
sity of the hemolysis increased from passage to passage.
After 30 passages, the colonies caused strong b-hemolysis
even around single colonies (Fig. 1). To the best of our
knowledge, this is the first report on Brucella isolates which
cause hemolysis on blood agar media. The complete geno-
mic sequencing of all isolates of the current study revealed
the presence of different hemolysin genes in all B. pinnipedi-
alis strains before and after serial passages. However, the
activity of hemolysins became obvious only after subculture.
Hemolysin is considered to be a potential virulence factor.
The regulation of hemolysin gene expression and conse-
quently the hemolytic activity of microbes is dependent on
the availability of iron ions [6]. Thus, hemolysins are pro-
duced only when their production is needed. Hemolysin
genes are also present in B. abortus, B. melitensis and B. suis,
but this genes are turned off. It is worth to mention, that the
Fig. 1. Brucella pinnipedialis reference strain cultivated on blood agar showing no
hemolysis (A); showing weak b-hemolysis on blood agar containing 7.5% blood (B),
and showing strong b-hemolysis around single colonies on Columbia blood agar
containing 10% blood (C).
expression of hemolysin due to in vitro mutations was
reported for Staphylococcus aureus [7]. Thus, the hemolysin
genes of B. pinnipedialis may have accumulated mutations
during growth on blood agar that resulted in the repair of
default genes. The results presented here show the ability
of these bacteria to express hemolysin genes which will have
an effect reflect on pathogenicity. Haemolytic anemia during
Brucella infection in humans is reported but a conclusive
explanation for this complication is missing [8].
(3) Rough colonial morphology: After ten serial passages on
blood agar, the smooth B. pinnipedialis reference strain con-
verted to a rough phenotype. This result was confirmed by
dissociation studies with tryptoflavin and crystal violet test
according to Alton et al. [9]. Brucellae devoid of the O-PS
are called rough or ‘‘R” brucellae. They are mutants derived
from smooth ‘‘S” progenitors. Conversion of smooth to rough
occurs spontaneously depending on the strain and growth
conditions. Thus, serial passages of the brucellae in vivo or
in vitro have been used to obtain less virulent ‘‘R” vaccines
[10].
Clinical implication of phenotypic changes
The ability of a pathogen to respond to environmental changes
is crucial to its virulence. Motility could increase the virulence of
brucellae by enables it to reach to a favorable niche, to invade
and colonize host cells, and to infect new hosts [4]. Moreover,
motility enhances bacterial uptake and help the bacteria to reach
the site of entry which can elevate the rate of infection [11]. On
the other hand, expression of hemolysins by Brucella either alone
or in combination with other virulence factors poses a serious risk
to the host or may even threaten its life. The main consequence of
hemolysin expression is the destruction of erythrocytes (hemoly-
sis) and their removal from the blood stream resulting in hemolytic
anemia. Recently, several cases of hemolytic anemia in humans
were reported accompanied by purpura, spontaneous hemor-
rhages, bleeding and hematuria. This untypical clinical presenta-
tion was seen in B. abortus and B. melitensis infection [8,12–22].
The relapse of hemolysis in some patients was accompanied by
brucellosis relapse also [12]. In deed brucellae were the assumed
cause of auto-immune hemolytic anemia in humans [14,15] after
a Coombs positive autoimmune hemolytic anemia was diagnosed
and recovery following corticosteroid therapy and recommended
specific antimicrobial regimen for brucellosis. Our hypothesis is
corroborated by the case of a patient who suffered from severe
microangiopathic hemolytic anemia with hemorrhagic syndrome
e.g. purpura, epistaxis and hematuria, and who completely recov-
ered after receiving anti-brucellae therapy [16]. Our hypothesis
can be helpful to investigate the correlation of acute brucellosis
and occurrence of hemolytic anemia. Understanding the role of
hemolysin expression and motility in the virulence of Brucella
strains will require further genome wide studies and complete pro-
teomic analysis as well as involving animal models.
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