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Promises and failures of gallium as an antibacterial agent

Article  in  Future Microbiology · March 2014

DOI: 10.2217/fmb.14.3 · Source: PubMed

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 Review

014
Promises and failures of gallium as an
antibacterial agent

Fabrizia Minandri1, Carlo Bonchi1, Emanuela Frangipani1, Francesco Imperi2

& Paolo Visca*,1

of
Abstract: Gallium has a long history as a diagnostic and chemotherapeutic agent. The
pharmacological properties of Ga(III) rely on chemical mimicry; when Ga(III) is exogenously
supplied to living cells it can replace Fe(III) within target molecules, thereby perturbing
bacterial metabolism. Ga(III)-induced metabolic distresses are dramatic in fast-growing

ro
& Visca

cells, like bacterial cells. Interest in the antibacterial properties of Ga(III) has been raised by
the compelling need for novel drugs to combat multidrug-resistant bacteria and by the
shortage of new antibiotic candidates in the pharmaceutical pipeline. Ga(III) activity has
been demonstrated, both in vitro and in animal models of infections, on several bacterial
rP
pathogens, also including intracellular and biofilm-forming bacteria. Ga(III) activity is
affected by iron availability and the metabolic state of the cell, being maximal in iron-poor
media and in respiring cells. Synergism between Ga(III) and antibiotics holds promise as last
B.14.3
resort therapy for infections sustained by pandrug-resistant bacteria.
ho

uture Background Keywords 

Metals such as copper, mercury and silver have been known for their ability to suppress microbial • Acinetobacter • antibiotic
growth since antiquity. The antimicrobial properties of metals have been theorized by the Swiss chemist resistance • anti-infectives
lium as Nägeli [1] with the ‘oligodynamic effect’ (from the Greek: oligos = few, dynamis = force), which is a • Burkholderia • Ganite®
toxic effect observed at very low concentrations. Metal compounds have been exploited for a number of • iron metabolism
clinical applications, and have become an essential component of the anti-infective armamentarium [2] . • Mycobacterium
ut

The semimetal gallium (Ga) is not novel to clinical applications, as it has been extensively used as a • Pseudomonas
diagnostic for the localization of malignant cells and inflammatory lesions, or as a therapeutic for the • Staphylococcus
treatment of cancer, autoimmune diseases and bone resorption disorders [3–7] . The pharmacological
properties of Ga(III) are attributable to its similarity with Fe(III) at the level of nuclear radius, coordi-
A

nation chemistry and ionization potential. These features allow Ga(III) to substitute for Fe(III) in the
prosthetic group of several enzymes (reviewed in [8]). Iron transport proteins, such as transferrin (Tf)
and lactoferrin (Lf), are able to form complexes with Ga(III) and deliver it to the cell [9,10] . Inside the
cell, Ga(III) perturbs iron metabolism since it is incorporated into essential proteins and enzymes in
place of iron. Unlike iron, Ga(III) cannot be reduced under physiological conditions and, therefore,
it cannot take part in redox reactions, ultimately inhibiting a number of essential functions. Several
metabolic pathways, involving both Fe(III)-dependent and -independent enzymes, are targeted by
Ga(III), and Ga(III)-induced metabolic distresses are dramatic in fast-growing cells, like cancer and
bacterial cells [3,11–17] .
y Ga(III), in the form of citrate-buffered gallium nitrate [Ga(NO3)3], is approved by the US FDA
(Ganite®, Genta, NJ, USA) for the treatment of cancer-associated hypercalcemia. Other gallium
ure
UK 1
Department of Sciences, University “Roma Tre”, Viale Guglielmo Marconi 446, 00146 Rome, Italy,
2
Department of Biology & Biotechnology “C. Darwin”, “Sapienza” University of Rome, Rome, Italy
*Author for correspondence: Tel.: +39 06 57336347; Fax: +39 06 57336321; paolo.visca@uniroma3.it part of

10.2217/FMB.14.3 © 2014 Future Medicine Ltd Future Microbiol. (2014) 9(3), 379–397 ISSN 1746-0913 379
Review  Minandri, Bonchi, Frangipani, Imperi & Visca
compounds, such as gallium chloride and gal-
lium maltolate, have been developed to improve
antimetabolite activity while mitigating toxic
effects, and some of these compounds are under
preclinical investigation [18,19] .
The importance of iron in bacterial metabo-
lism and virulence has fostered the exploitation
of Ga(III) as an anti-infective [20–30] . Hereafter,
we will provide a comprehensive review of recent
findings on the in vitro and in vivo antibacterial
activity of Ga(III), and discuss the pros and cons
of Ga(III) as an anti-infective.
Effect of gallium on bacterial metabolism
Ga(III) acts as an iron mimetic and is supposed
of
to exploit Fe(III) transport systems to enter bac-
terial cells. Basic information on Fe(III) uptake
and metabolism in bacteria are therefore instru-
mental to understanding the mechanism(s) of
ro
action and uptake routes of Ga(III).
●●Bacterial iron uptake & metabolism
Iron is scarcely bioavailable under aerobic con-
ditions since it is almost completely present as
rP
an oxidized and insoluble oxyhydroxide Fe(III)
polymer [31] . Bacteria have evolved a multiplicity
of strategies to cope with low iron bioavailability.
Almost all mechanisms rely on proteins at the cell
surface that bind and transport Fe(III) or heme-
ho
containing molecules, or Fe(II) under anaerobic
conditions. The many iron uptake strategies used
by Gram-positive and Gram-negative bacteria
have been exensively reviewed [32,33] and are sum-
marized in Figure 1. The Fe(III) and heme acqui-
sition systems of bacteria can either involve direct
ut
contact between cells and Fe(III)/heme sources,
or the synthesis and secretion of siderophores and
hemophores, which scavenge Fe(III) and heme,
respectively, from natural sources and/or host
A
proteins [33] . Hemophore-based heme uptake is
exploited by several Gram-negative bacteria [34]
and by Bacillus anthracis [35] . Heme can either
be incorporated into heme-requiring proteins
or cleaved by cytoplasmic heme oxygenases to
release iron [36] . Conversely, Fe(III) must always
be dissociated from siderophores either by hydrol-
ysis, or modification of the siderophore scaffold,
or reduction of Fe(III) to Fe(II). The majority of
Fe(III)-siderophore complexes are internalized
into the cytoplasm prior to iron release, although
some undergo periplasmic reduction of Fe(III) to
Fe(II), followed by Fe(II) transport into the cyto-
sol by dedicated inner membrane transporters
(reviewed in (Figure 1) [37]).
380 Future Microbiol. (2014) 9(3)
Under anaerobic or reducing conditions,
Fe(II) is the prevalent iron form and, different
from Fe(III), it freely diffuses through outer
membrane porins of Gram-negative bacteria,
and it is transported through the cytoplasmic
membrane by an ABC Fe(II) transporter, called
Feo (Figure 1) [38] .
Iron acquisition must be finely controlled,
since both iron deficiency and iron overload
can be detrimental to cells. In fact, while too
low intracellular iron cannot support bacterial
growth, too high intracellular Fe(II) concentra-
tions cause the production of hydroxyl radicals
(via Fenton-type reactions) and oxidative dam-
age [31] . Therefore, bacteria must sense intracel-
lular iron levels, and regulate iron uptake accord-
ingly. In all bacteria iron homeostasis is ensured
by transcriptional repressors of the Fur or DtxR
family, which require Fe(II) as a cofactor to
efficiently bind the promoter of target genes [39]
(Figure 1) . In addition, small regulatory RNAs
(sRNAs) controlling the expression of iron-con-
taining proteins in response to intracellular iron
level variations, have been identified in different
bacterial species (Figure 1) (reviewed in [40,41]).
●●Mechanism of action & uptake routes of
gallium in bacteria
Given the similarities between Ga(III) and
Fe(III) ions and the inability of the former to
undergo redox cycles, it is plausible that Ga(III)
can substitute for Fe(III) in iron-containing
enzymes, thus repressing their activity [8] . This
is consistent with the observation that, in any
bacterial species investigated to date, the anti-
microbial activity of Ga(III) is counteracted by
an excess of Fe(III). Since many iron-containing
enzymes are involved in critical functions in bac-
teria, such as DNA synthesis and repair, metabo-
lism, respiration and oxidative stress response
[42] , Ga(III) is likely to cause multiple deleteri-
ous effects to bacterial cells. Whether the anti-
bacterial activity of Ga(III) relies on a general
perturbation of bacterial iron metabolism or on
the inhibition of a specific enzyme and/or cellu-
lar pathway remains an open question. A recent
study on the antibacterial activity of Ga(III)
against Pseudomonas aeruginosa reported a strong
influence of the carbon source, indicating that
some metabolic pathways may be more iron-
demanding, and thus more Ga(III)-sensitive,
than others [29] . Moreover, pioneering studies
on the neighbour species Pseudomonas fluorescens
revealed that bacterial cells adapted to grow in
future science group
 Promises & failures of gallium as an antibacterial agent  Review

Diderm bacteria
(Gram-negative)

TonB

TonB
ExbD
ExbB

ExbD
B

ExbB
n
To

bB
bD

Ex Iron-uptake
Ex

OM genes
PS
CM ryhB gene

CM
RyhB-like sRNAs

of
mRNAs for iron-using proteins

Monoderm bacteria
(Gram-postive)

Fe(III) Fe(II) Siderophore ro

Ferri-siderophore Heme Host iron-binding proteins
rP
Figure 1. Iron-uptake strategies and iron-dependent gene regulation in bacteria. Fe(III) is delivered to bacterial cells by host iron-
binding proteins (transferrin or lactoferrin), heme or heme-containing proteins (e.g., hemoglobin or hemopexin) and endogenous or
exogenous siderophores. These iron sources are recognized by specific surface receptors on the OM or CM of Gram-negative or Gram-
positive bacteria, respectively. In Gram-positive bacteria, Fe(III) or Fe(III)-chelator complexes are transported into the cytosol by ABC
ho

transporters coupled to specific CM-anchored lipoprotein receptors. In Gram-negative bacteria, OM receptors transport Fe(III) or Fe(III)-
chelator complexes into the PS thanks to the interaction with the Ton complex (TonB, ExbB, ExbD), which transduces the energy of the
proton gradient on the CM into conformational changes of the receptors. Once in the PS, Fe(III), heme and Fe(III)-loaded siderophores
(ferrisiderophores) are sequestered by specific periplasmic binding proteins that deliver them to cognate ABC transporters (functionally
homologous to those of Gram-positive bacteria). The soluble form Fe(II) can plausibly cross the OM of Gram-negative bacteria through
ut

porins, and is then translocated to the cytosol by the Fe(II) transporter FeoB, which is also present in some Gram-positive bacteria. In
the presence of sufficient amounts of intracellular iron, Fe(II) binds Fur-like repressor proteins (or DtxR-like proteins in some Gram-
positive bacteria). Fe(II)–Fur directly or indirectly represses transcription of genes involved in iron uptake that, therefore, are expressed
only under iron-depleted conditions, in other words, when Fur is mainly present in its apo form (⊥ indicates repression). Fe(II)–Fur also
A

represses the expression of specific small RNAs (e.g., RyhB in Escherichia coli) that inhibit translation of mRNAs encoding a number of
iron-using proteins. As a result, these proteins are mainly expressed under iron-replete conditions.
CM: Cytoplasmic; OM: Outer membrane; PS: Periplasmic space.

the presence of very high Ga(III) concentrations
(up to 1 mM) display enhanced reactive oxygen
species (ROS) production and increased expres-
sion of ROS-detoxifying enzymes, suggesting
that Ga(III) toxicity is not only due to perturba-
tion of iron metabolism, but also to the genera-
tion of oxidative stress [11,14] . Recently, a spon-
taneous mutant of P. aeruginosa with 12-fold
increased resistance to Ga(III) was obtained
through sequential subculturing in the presence
of an inhibitory concentration of Ga(NO3)3 [16] .
Genome sequencing revealed 64 nonsilent point
mutations in this mutant, mostly occurring in
genes involved in metabolism, membrane trans-
port, cell division, DNA recombination/repair,
signal transduction, energy production and
synthesis of siderophores and phenazines [16] .
Since several cellular processes were found to
be involved in adaptation/resistance to Ga(III),
this study suggests that Ga(III) has multiple
targets and resistance mechanisms in bacterial
cells. The effect of Ga(III) on the expression

future science group www.futuremedicine.com 381

Review  Minandri, Bonchi, Frangipani, Imperi & Visca
of P. aeruginosa iron-repressible virulence fac-
tors (i.e., factors induced by iron limitation) is
still controversial, and molecular mechanisms
of interference with gene regulation remain
obscure [26,43] .
Irrespective of the mechanism of action,
Ga(III) needs to enter bacterial cells to reach its
molecular targets. However, the way by which
Ga(III) is internalized by bacteria is unclear.
Ga(III) has no known role in microbial physi-
ology and biological systems do not discriminate
between Fe(III) and Ga(III), hence the most
plausible hypothesis is that Ga(III) crosses the
cytoplasmic membrane of bacteria by exploit-
ing Fe(III)-uptake routes, rather than through
of
dedicated transport system(s). However, Ga(III)
is efficiently acquired by both wild-type and
siderophore-defective P. aeruginosa mutants,
even under iron-replete conditions that repress
ro
the expression of Fe(III)-uptake genes [26] , sug-
gesting that siderophore-mediated transport
is not the main route for Ga(III) entrance in
P. aeruginosa. Unfortunately, this work did not
assess the actual distribution of cell-associated
rP
Ga(III) between the periplasmic and cytoplas-
mic compartments [26] , thus the possibility exists
that the amount of Ga(III) measured in P. aer-
uginosa cells does not represent the fraction that
actually reaches the cytosol.
ho
It can be supposed that Ga(III) can cross
the outer membrane of Gram-negative bacte-
ria by passive diffusion through porins, as also
suggested in the case of soluble Fe(II) ion [38] .
However, the impermeability of the cytoplasmic
membrane of both prokaryotic and eukaryotic
ut
cells to ions suggests that Ga(III) needs specific
transport mechanism(s) to reach the cytoplasm.
It has indeed been shown, by small-angle x-ray
scattering and transmission electron micros-
A
copy, that Ga(III) mainly accumulates in the
cell-envelope compartment (outer membrane,
periplasm and inner membrane) of P. fluores-
cens cells exposed to millimolar concentrations
of Ga(III) [44] , confirming that Ga(III) trans-
port across the cytoplasmic membrane is the
rate-limiting step for internalization by Gram-
negative bacteria. In Escherichia coli expressing
the Haemophilus influenzae heterologous pro-
teins FbpABC, corresponding to a periplasmic
Fe(III)-binding protein (FbpA) and the cognate
inner membrane ABC transporter (FbpBC) for
delivery of Fe(III) from the periplasm to the
cytoplasm, both internalization of Ga(III) and
susceptibility were significantly increased [45] .
382 Future Microbiol. (2014) 9(3)
Thus, at least in E. coli, Ga(III) activity depends
on its active transport into the cytoplasm, and
Fe(III) uptake mechanisms can contribute to
this process. Accordingly, it has been shown
that P. aeruginosa transposon mutants defec-
tive in the periplasmic iron-binding protein
HitA or in the cognate inner membrane per-
mease HitB display a three- to fourfold increase
in resistance to Ga(III) and >tenfold decrease
of the Ga(III) intracellular content [16] , further
confirming that Ga(III) can exploit endogenous
iron-uptake mechanisms to reach the cytoplasm
of bacterial cells.
●●Iron chelators as Trojan horses for gallium
delivery to bacterial cells
Recently, there has been a renewed interest in
the use of natural or synthetic Fe(III) chelators
as antimicrobial agents [46] . By competing with
siderophores for Fe(III) binding, exogenous che-
lators can deprive bacterial cells of essential iron.
This is not a general rule, however, since many
bacterial species have evolved the ability to use
exogenous chelators as vehicles to deliver Fe(III)
to the cell. A paradigmatic example is provided
by the iron chelator deferoxamine B (DFO,
Desferal®, Novartis Pharma, Switzerland),
which can be used as an exogenous iron carrier
by several bacteria, including P. aeruginosa [47,48] .
Although the route(s) by which Ga(III) enters
the bacterial cell is uncertain, several studies have
explored the possibility of using chelators from
both microbial and chemical sources as Trojan
horses to enhance Ga(III) delivery to bacteria.
This strategy has recently been revisited, and
several Ga(III)–siderophore and Ga(III)–heme
complexes have been evaluated in vitro as new
anti-infectives [49] .
Almost all studies on siderophore-mediated
Ga(III) delivery have been performed on P. aer-
uginosa as model organism. P. aeruginosa is a par-
adigmatic example of iron-uptake capability, and
iron metabolism has recently been exploited as
an ideal target for new anti-P. aeruginosa drugs
[22,26,29,50,51] . Banin et al. demonstrated that a
complex between Ga(III) and the heterologous
siderophore DFO was slightly more effective
than Ga(III) alone in killing P. aeruginosa cells,
both in established biofilms and in vivo, even
though the in vitro minimum inhibitory concen-
trations (MICs) of DFO–Ga(III) and Ga(III)
alone were similar [22] . Although it has been
demonstrated that DFO receptors present on
the P. aeruginosa outer membrane were required
future science group

for antibacterial activity, the uptake process
and the fate of the DFO–Ga(III) complex in
P. aeruginosa cells were not investigated [22] . The
anti-P. aeruginosa activity of Ga(III) complexed
with either DFO or the iron chelator citrate has
also been compared [29] . The Ga(III)–citrate
complex showed higher bacteriostatic and bac-
tericidal activities than DFO–Ga(III), and was
also more effective in protecting macrophages
from infection, plausibly due to a more efficient
uptake of Ga(III)–citrate by P. aeruginosa cells
[29] . However, the activities of Ga(III)–citrate
and Ga(III) alone were not compared in this
study, hampering the direct evaluation of the
effect of chelator-mediated transport of Ga(III)
on bacterial growth. Notably, Ga(III)–citrate
was found to exert a negligible inhibitory activ-
ity on a number of other species, including E.
coli [29] . This is of particular interest considering
that a functional Fe(III)-citrate transport system
is present in E. coli K-12 [52] , and indicates that
citrate-mediated active uptake of Ga(III) may
not be sufficient for Ga(III)–citrate toxicity.
Different from heterologous siderophores or
rP
chemical chelators, endogenous siderophores
have no apparent effect on Ga(III) activity in
P. aeruginosa. Although both Ga(III)–pyo-
verdine and Ga(III)–pyochelin complexes are
recognized and actively internalized by cognate
ho
outer membrane receptors [53,54] , the inhibitory
activity of Ga(III) on P. aeruginosa was not
influenced by the presence of these siderophores
and their uptake systems [26,53,54] . However, the
anti-P. aeruginosa effect of preformed Ga(III)–
pyoverdine and Ga(III)–pyochelin complexes
ut
has neither been tested nor compared with that
of Ga(III) alone. Thus, the few data available
indicate that Ga(III) complexation with sidero-
phores and/or iron chelators does not signifi-
A
cantly increase its antibacterial activity, suggest-
ing that the internalization process is not the
limiting step for the activity of Ga(III).
Promising results were obtained with the
Ga(III)–protoporphyrin IX (PPIX or apo-
heme) complex. It has been demonstrated that
Ga(III)–PPIX has potent antibacterial activ-
ity against a number of Gram-negative and
Gram-positive bacteria [30,49,55–57] . Although
Ga(III) –PPIX can exploit heme receptors
for transport across the outer membrane of
Gram-negative bacteria [57,58] , heme uptake
systems on the cytoplasmic membrane are not
required for Ga(III)–PPIX-mediated inhibi-
tion of both Gram-negative and Gram-positive
future science group
Promises & failures of gallium as an antibacterial agent  Review
bacteria, suggesting that the main target(s) of
Ga(III)–PPIX could be located on the peri-
plasmic (extracellular) side of the cytoplasmic
membrane [58] . Different from what is observed
with Ga(III) alone, the inhibitory activity of the
Ga(III)–PPIX complex is not significantly coun-
teracted by an excess of Fe(III) [30] . Moreover,
obligate and facultative anaerobic bacteria using
fermentation for energy production were found
to be resistant to Ga(III)–PPIX, while E. coli
mutants impaired in ROS detoxification mecha-
nisms were hypersensitive to this compound [30] .
Altogether, these observations led to the hypoth-
esis that the inhibitory activity of Ga(III)–PPIX
does not rely on intracytoplasmic release of free
of
Ga(III), but on insertion of Ga(III)–PPIX into
membrane-bound cytochromes, with conse-
quent disruption of the electron flow, ROS
overproduction and oxidative stress [58] .
ro
In vitro & in vivo antibacterial activity of
gallium compounds
The interest in Ga(III) as an antibacterial agent
has been resuscitated by the paucity of effec-
tive antibiotics to combat emerging multid-
rug-resistant (MDR) bacterial pathogens (i.e.,
isolates nonsusceptible to at least one agent in
three or more antimicrobial categories accord-
ing to [59]). Several investigations indicate that
Ga(III) is able to inhibit bacterial growth both
in vitro and in vivo [20–30,56], prompting clinical
investigations of the efficacy of Ga(III)-based
antimicrobial chemotherapy.
●●Activity on planktonic bacterial pathogens
Most pathogenic bacteria are able to multiply
in biological fluids and/or in the environment
as free planktonic cells, a mode of growth that
is mimicked by cultures in liquid media, as
for antimicrobial susceptibility tests. Several
Ga(III) compounds have been tested against
a variety of bacterial species in liquid cultures,
under an array of conditions that often hamper
the direct comparison of results from different
sources. Hereafter, the most relevant data will
be discussed.
Two independent studies have recently dem-
onstrated strong activity of Ga(NO3)3 on the
nosocomial pathogen Acinetobacter bauman-
nii [20,60] . Low concentrations were effective
in inhibiting the growth of both laboratory
and MDR A. baumannii isolates in iron-poor
media (inhibitory concentrations by 90%
[IC 90 s] ranged from 2 to 80 μM) (Table 1) ,
www.futuremedicine.com 383
Review  Minandri, Bonchi, Frangipani, Imperi & Visca

Table 1. In vitro antibacterial activity of gallium.

Effect on Microorganism Ga(III) Growth condition (medium)§ Compound inhibitory Ga(III) concentration Ref.
compound concentration (μM)†
Planktonic Acinetobacter Ga(NO3)3 Chemically-undefined medium IC90 >100 μM >100 [60]
growth baumannii (10% TSB)
Chemically-defined medium IC90 = 100 μM 100 [60]
(RPMI 1640)
Chemically-defined medium IC90 = 3.2 μM 3.2 [60]
(RPMI + 10% serum)
Chemically-defined medium (M9- IC90 = 2–80 μM 2–80 [20]
DIP)
Chemically-undefined medium IC90 = 4–64 μM 4–64 [20]
(human serum)
Bacillus subtilis Ga(III)–PPIX Chemically-undefined medium MIC = 0.2 μg/ml 0.3 [30]
(NB)

of
Burkolderia cepacia Ga(NO3)3 Chemically-defined medium MIC = 250 μM 250 [28]
complex (CDM no iron)
Enterococcus Ga(III)–Cit Chemically-undefined medium IC90 = 1280–2560 μM 1280–2560 [29]
faecalis (Iso-Sensitest; Fisher Scientific,
UK)

Escherichia coli
DFO–Ga(III)

Ga(III)–Cit,
DFO–Ga(III) ro
Chemically-undefined medium
(Iso-Sensitest)
Chemically-undefined medium
(Iso-Sensitest)
IC90 = 1280–5120 μM

IC90 = 2560–5120 μM
1280–5120

2560–5120
[29]

[29]
rP
Ga(III)–Cit, Chemically-defined medium IC90 = 1280–5120 μM 1280–5120 [29]
DFO–Ga(III) (HHW)
Ga(III)–PPIX Chemically-undefined medium MIC <0.5 μg/ml <0.8 [30]
(NB + DIP)
Helicobacter pylori Ga(III)–PPIX Chemically-undefined medium MIC = 0.19 μg/ml 0.3 [30]
(Brucella broth + 2% serum)
ho

Klebsiella Ga(III)–Cit Chemically-defined medium IC90 = 1280–5120 μM 1280–5120 [29]

pneumonia (HHW)
Chemically-undefined medium IC90 = 1280–5120 μM 1280–5120 [29]
(Iso-Sensitest)
DFO–Ga(III) Chemically-defined medium IC90 = 1280–2560 μM 1280–2560 [29]
(HHW)
ut

Chemically-undefined medium IC90 = 2560–5120 μM 2560–5120 [29]

(Iso-Sensitest)
Ga(III)–PPIX Chemically-undefined medium MIC = 2 μg/ml 3.2 [30]
(NB)
A

Lysteria Ga(III)–PPIX Chemically-undefined medium MIC = 0.2 μg/ml 0.3 [30]

monocytogenes (NB)
Mycobacterium Ga(NO3)3 Chemically-undefined medium IC50 ≈ 250 μM‡ 250 [27]
avium complex (BACTEC™; BD, NJ, USA)
M. avium subsp. Ga(NO3)3 Chemically-undefined medium IC90 = 200–743 μM 200–743 [62]
paratuberculosis (BACTEC)
Mycobacterium Ga(III)–PPIX Chemically-undefined medium MIC = 0.4 μg/ml 0.6 [30]
bovis (NB)
†
Actual concentration of the Ga3+ ion after complex dissociation.
‡
IC50s were not clearly stated, but extrapolated from the references.
§
Refer to citations for composition of culture media.
7H9: Middlebrook 7H9 broth; BACTEC: BACTEC 12b broth; BBC: Biofilm bactericidal concentration; BIC: Biofilm inhibitory concentration; BM2 succinate: BM2 minimal medium
supplemented with 34 mM succinate; CDM: Chemically-defined medium; DFO–Ga(III): Desferoxamine–gallium; DIP: 2’2-dipyridyl; Ga(III)–PPIX: Gallium–protoporphyrin
IX; Ga(III)–Cit: Gallium–citrate; GaM: Gallium maltolate; HHW: Hussain, Hastings & White medium; IC50: Compound concentrations that inhibit growth by 50%; IC90: Compound
concentrations that inhibit growth by 90%; LB: Luria-Bertani broth; M9-DIP: Mimimal medium 9 supplemented with 0.1 mM 2,2’-dipyridyl; MBC: Minimum bactericidal
concentration; MBBC: Minimum biofilm bactericidal concentration; MBIC: Minimum biofilm inhibitory concentration; MIC: Minimum inhibitory concentration (lowest
concentration causing no visible growth); MM: Minimal medium; NB: Nutrient broth; RPMI: Roswell Park Memorial Institute medium; Tf–Ga(III): Transferrin–gallium; TSB: Tryptic soy
broth.

384 Future Microbiol. (2014) 9(3) future science group

 Promises & failures of gallium as an antibacterial agent  Review

Table 1. In vitro antibacterial activity of gallium (cont.).

Effect on Microorganism Ga(III) Growth condition (medium)§ Compound inhibitory Ga(III) concentration Ref.
compound concentration (μM)†
Planktonic Mycobacterium Ga(III)–PPIX Chemically-undefined medium MIC = 0.4 μg/ml 0.6 [30]
growth smegmatis (LB)
(cont.)
Mycobacterium Ga(NO3)3 Chemically-undefined medium IC50 ≈ 60–250 μM‡ 60–250 [27]
tuberculosis (BACTEC)
Chemically-defined medium IC50 = 1.25–2.50 μM 1.25–2.50 [27]
(7H9)
Neisseria spp. Ga(III)–PPIX Chemically-undefined medium MIC = 0.2 μg/ml 0.3 [30]
(NB)
Proteus mirabilis Ga(III)–Cit, Chemically-undefined medium IC90 = 1280 μM 1280 [29]
DFO–Ga(III) (Iso-Sensitest)
Ga(III)–Cit, Chemically-defined medium IC90 = 1280 μM 1280 [29]

of
DFO–Ga(III) (HHW)
Pseudomonas Ga(NO3)3 Chemically-defined medium IC90 = 1–40 μM 1–40 [26]
aeruginosa (BM2 succinate)
Chemically-defined medium MIC = 62.5 μM 62.5 [28]

ro
(CDM no iron)
GaCl3, DFO– Chemically-undefined medium MIC = 32 μM 32 [22]
Ga(III) (1% TSB)
Ga(III)–Cit Chemically-defined medium IC90 = 20–40 μM 20–40 [29]
(HHW)
rP
Chemically-undefined medium IC90 = 80–640 μM 80–640 [29]
(Iso-Sensitest)
DFO–Ga(III) Chemically-defined medium IC90 = 40–640 μM 40–640 [29]
(HHW)
Chemically-undefined medium IC90 = 160–1280 μM 160–1280 [29]
(Iso-Sensitest)
ho

Rhodococcus equi Ga(NO3)3 Chemically-defined medium MBC = 50 μM 50 [24]

(MM)
GaM Chemically-defined medium MIC90 = 8 μM 8 [69]
(minimal medium without iron)
Staphylococcus Ga(III)–Cit Chemically-defined medium IC90 = 320–5120 μM 320–5120 [29]
ut

aureus (HHW)
Chemically-undefined medium IC90 = 2560–5120 μM 2560–5120 [29]
(Iso-Sensitest)
DFO–Ga(III) Chemically-defined medium IC90 = 320–5120 μM 320–5120 [29]
(HHW)
A

Chemically-undefined medium IC90 = 2560–5120 μM 2560–5120 [29]

(Iso-Sensitest)
GaM Chemically-defined medium MIC = 375–2000 μg/ml 847–4494 [21]
(RPMI)
Chemically-defined medium MIC = 0.06–4.00 mg/ml 140–8988 [64]
(RPMI 1640)
Ga(III)–PPIX Chemically-undefined medium MIC = 1.0–2.5 μg/ml 1.6–4.0 [30]
(NB)
†
Actual concentration of the Ga3+ ion after complex dissociation.
‡
IC50s were not clearly stated, but extrapolated from the references.
§
Refer to citations for composition of culture media.
7H9: Middlebrook 7H9 broth; BACTEC: BACTEC 12b broth; BBC: Biofilm bactericidal concentration; BIC: Biofilm inhibitory concentration; BM2 succinate: BM2 minimal medium
supplemented with 34 mM succinate; CDM: Chemically-defined medium; DFO–Ga(III): Desferoxamine–gallium; DIP: 2’2-dipyridyl; Ga(III)–PPIX: Gallium–protoporphyrin
IX; Ga(III)–Cit: Gallium–citrate; GaM: Gallium maltolate; HHW: Hussain, Hastings & White medium; IC50: Compound concentrations that inhibit growth by 50%; IC90: Compound
concentrations that inhibit growth by 90%; LB: Luria-Bertani broth; M9-DIP: Mimimal medium 9 supplemented with 0.1 mM 2,2’-dipyridyl; MBC: Minimum bactericidal
concentration; MBBC: Minimum biofilm bactericidal concentration; MBIC: Minimum biofilm inhibitory concentration; MIC: Minimum inhibitory concentration (lowest
concentration causing no visible growth); MM: Minimal medium; NB: Nutrient broth; RPMI: Roswell Park Memorial Institute medium; Tf–Ga(III): Transferrin–gallium; TSB: Tryptic soy
broth.

future science group www.futuremedicine.com 385

Review  Minandri, Bonchi, Frangipani, Imperi & Visca

Table 1. In vitro antibacterial activity of gallium (cont.).

Effect on Microorganism Ga(III) Growth condition (medium)§ Compound inhibitory Ga(III) concentration Ref.
compound concentration (μM)†
Planktonic Staphylococcus Ga(III)–Cit Chemically-defined medium IC90 = 1280–2560 μM 1280–2560 [29]
growth epidermidis (HHW)
(cont.)
Chemically-undefined medium IC90 = 2560–5120 μM 2560–5120 [29]
(Iso-Sensitest)
DFO–Ga(III) Chemically-defined medium IC90 = 640–2560 μM 640–2560 [29]
(HHW)
Chemically-undefined medium IC90 = 1280–5120 μM 1280–5120 [29]
(Iso-Sensitest)
GaM Chemically-defined medium MIC = 100–200 μg/ml 225–450 [21]
(RPMI)
Staphylococcus GaM Chemically-defined medium MIC = 0.25–4.00 mg/ml 562–8988 [64]

of
pseudintermedius (RPMI 1640)
Vibrio cholerae Ga(III)–Cit Chemically-defined medium MIC = 1280–2560 μM 1280–2560 [29]
(HHW)
DFO–Ga(III) Chemically-defined medium MIC = 1280 μM 1280 [29]

ro
(HHW)
Ga(III)–Cit, Chemically-undefined medium MIC = 1280 μM 1280 [29]
DFO–Ga(III) (Iso-Sensitest)
Yersinia Ga(III)–PPIX Chemically-undefined medium MIC = 0.4 μg/ml 0.6 [30]
enterocolitica (NB + DIP)
rP
Yersinia Ga(III)–PPIX Chemically-undefined medium MIC = 0.4 μg/ml 0.6 [30]
pseudotuberculosis (NB + DIP)
Biofilm P. aeruginosa Ga(NO3)3 Flow cells (1% TSB) BIC = 0.5 μM 0.5 [26]
BBC = 100 μM 100
GaCl3, DFO– Flow cells (1% TSB) BIC = 1 μM 1 [22]
Ga(III)
ho

DFO–Ga(III) Flow cells (1% TSB) BBC = 1 mM 1000 [22]

Ga(III)–Cit Microtitre plates MBIC = 10 μM 10 [29]
(20% Iso-Sensitest)
B. cepacia complex Ga(NO3)3 Microtitre plates (CDM no iron) MBBC = 32 mg/l 125 [28]
S. aureus GaM Microtitre plates (RPMI) MBIC = 3000–6000 6741–13482 [21]
ut

μg/ml
S. epidermidis GaM Microtitre plates (RPMI) MBIC = 94–3000 μg/ml 211–6741 [21]
Intracellular M. tuberculosis Ga(NO3)3 Human macrophages IC50 = 30–100 μM 30–100 [27]
growth
A

MBC = 500 μM 500
Tf–Ga(III) Human macrophages IC50 = 62.5 μM 125.0 [27]
†
Actual concentration of the Ga3+ ion after complex dissociation.
‡
IC50s were not clearly stated, but extrapolated from the references.
§
Refer to citations for composition of culture media.
7H9: Middlebrook 7H9 broth; BACTEC: BACTEC 12b broth; BBC: Biofilm bactericidal concentration; BIC: Biofilm inhibitory concentration; BM2 succinate: BM2 minimal medium
supplemented with 34 mM succinate; CDM: Chemically-defined medium; DFO–Ga(III): Desferoxamine–gallium; DIP: 2’2-dipyridyl; Ga(III)–PPIX: Gallium–protoporphyrin
IX; Ga(III)–Cit: Gallium–citrate; GaM: Gallium maltolate; HHW: Hussain, Hastings & White medium; IC50: Compound concentrations that inhibit growth by 50%; IC90: Compound
concentrations that inhibit growth by 90%; LB: Luria-Bertani broth; M9-DIP: Mimimal medium 9 supplemented with 0.1 mM 2,2’-dipyridyl; MBC: Minimum bactericidal
concentration; MBBC: Minimum biofilm bactericidal concentration; MBIC: Minimum biofilm inhibitory concentration; MIC: Minimum inhibitory concentration (lowest
concentration causing no visible growth); MM: Minimal medium; NB: Nutrient broth; RPMI: Roswell Park Memorial Institute medium; Tf–Ga(III): Transferrin–gallium; TSB: Tryptic soy
broth.

and the Ga(III) effect was gradually reversed
by increasing FeCl 3 concentration [20,60] .
Interestingly, Ga(NO3)3 was even more effec-
tive when A. baumannii was grown in human
serum, showing IC90 values within the range of
Ga(III) plasma levels achievable with standard
Ganite regimens [20] . These findings represent
an important advance in antibacterial chemo-
therapy, given the paucity of antibiotics retain-
ing activity against A. baumannii, and the risk
posed by the emergence and dissemination
of pandrug-resistant (PDR) clones [61] (i.e.,

386 Future Microbiol. (2014) 9(3) future science group


nonsusceptibile to all agents in all antimicrobial
categories according to [59]).
Ga(III) activity was investigated in-depth
on planktonic cells of the opportunistic patho-
gen P. aeruginosa [22,26] . Ga(NO3)3 was highly
effective in inhibiting the growth of clini-
cal P. aeruginosa isolates, showing IC90 values
between 1 and 40 μM, and in this case Fe(III)
also counteracted Ga(III) activity [26] . Similar
inhibitory activity was reported using gallium
chloride (GaCl3) on the reference P. aeruginosa
strain PAO1, with MIC = 32 μM (Table 1) [22] .
Moreover, Ga(NO3)3 exerted a dose-dependent
bactericidal effect on P. aeruginosa starting from
concentrations as low as 10 μM [26] .
These data suggest that existing resistance to
conventional antibiotics does not compromise
the activity of Ga(III) against MDR pathogens
like A. baumannii and P. aeruginosa [20,26] .
Ga(NO3)3 activity was also investigated in the
Burkholderia cepacia complex species, but the
MICs were 250 μM for all species tested (Table 1)
[28] . Intriguingly, P. aeruginosa PAO1 grown in
the same medium as an internal control, showed
rP
a MIC much higher (62.5 μM) than previously
reported inhibitory concentrations [22,26] , pre-
sumably owing to different composition and
iron content of the media.
The ability of Ga(NO3 ) 3 to inhibit the
ho
planktonic growth of mycobacteria has been
investigated on the Mycobacterium avium com-
plex, M. avium subsp. paratuberculosis (MAP)
and Mycobacterium tuberculosis strains. High
Ga(NO3 ) 3 concentrations (>200 μM) were
needed to reduce planktonic growth of M.
ut
avium complex and MAP strains, likely as a
consequence of the high iron content of the
media used (Table 1) [27,62] . In fact, the Ga(NO3)3
concentration required to inhibit M. tuberculo-
A
sis planktonic growth in a chemically-defined
iron-poor medium was much lower (concentra-
tion that inhibited the growth by 50% [IC50]
was ∼2.0 μM) (Table 1) [27,62] .
In addition to inorganic Ga(III) salts, a num-
ber of Ga(III) complexes have been tested on
bacterial pathogens with the aim of enhancing
the antimicrobial efficacy of Ga(III).
As mentioned above, although P. aeruginosa
uses DFO as an iron carrier, DFO–Ga(III) (1:1
molar ratio) was bacteriostatic and bactericidal at
almost the same concentrations as GaCl3 alone
[22] . The antibacterial activity of Ga(III)–citrate
(1:2 molar ratio) and DFO–Ga(III) complexes
was also compared in another study; however,
future science group
Promises & failures of gallium as an antibacterial agent  Review
very high MICs (in the mM range) were observed
with both complexes for all but one species tested
(Table 1) [29] . P. aeruginosa was the most sensitive
to Ga(III)–citrate (IC90 range 20–40 μM) and
DFO–Ga(III) (IC90 range 40–640 μM) in mini-
mal medium [29] . However, these IC90 values are
higher than those observed with Ga(NO3)3 or
GaCl3 alone [22,26] .
Gallium maltolate (GaM, 1:3 gallium:maltol
molar ratio) is an orally active Ga(III) compound
endowed with high bioavailablity and low toxic-
ity [63] , whose activity has been assessed against
different Staphylococcus species [21,64] . In general,
Staphylococcus aureus showed poor susceptibil-
ity to GaM, as also reported for Ga(III)–cit-
of
rate (Table 1) [21,29] . GaM activity was also dis-
couraging for Staphylococcus pseudintermedius
veterinary isolates (Table 1) [64] .
GaM was tested on Salmonella spp. with the
ro
purpose of reducing Salmonella fecal shedding
in cattle [25] . The growth of Salmonella enterica
serovar Newport was inhibited by GaM in a
time- and dose-dependent manner, albeit a sig-
nificant effect was evident only at high GaM
concentrations (250–500 μM) in minimal
growth medium [25] .
As anticipated , Ga(III)–PPIX is endowed
with strong antibacterial activity against a wide
range of Gram-positive and Gram-negative
bacterial pathogens, even in iron-sufficient
growth media [30,49,55,56] . Among the bacte-
ria tested, Bacillus subtilis, E. coli, Helicobacter
pylori, Listeria monocytogenes, Mycobacterium
bovis, Neisseria gonorrhoeae, Neisseria menin-
gitidis, Yersinia enterocolitica and Yersinia pseu-
dotuberculosis, were the most sensitive [MICs ≤
0.5 μg/ml, corresponding to 0.8 μM Ga(III)]
(Table 1) [30] . Good Ga(III)–PPIX inhibitory
activity was also recently reported for A. bau-
mannii [49] . Conversely, some bacterial species
were found to be resistant to Ga(III)–PPIX,
and these were Gram-negative species lack-
ing heme uptake systems (S. enterica serovar
Typhimurium) and/or microorganisms that
do not contain cytochromes such as facultative
anaerobes (Streptococcus pyogenes and Enterococcus
faecalis) [30] .
●●Activity on bacterial biofilms
Bacterial cells living in biofilms are more resist-
ant to antibiotics than their planktonic counter-
part, mostly owing to the composition and low
permeability of the extracellular matrix, which
makes biofilms often responsible for persistent
www.futuremedicine.com 387
Review  Minandri, Bonchi, Frangipani, Imperi & Visca
or chronic infections. Given the favorable diffu-
sion properties of Ga(III) complexes, a number
of studies have exploited the possibility of using
Ga(III) as a biofilm-perturbing agent (Table 1) .
While P. aeruginosa biofilms are particularly
difficult to eradicate by means of antibiotic
therapy, they were found to be highly sensi-
tive to Ga(III). It was shown that a sub-MIC
concentration of Ga(NO3)3 strongly inhibited
biofilm formation by P. aeruginosa (Table 1) , and
that inhibition was reversed by biofilm perfusion
with FeCl3 [26] . Impressively, 100 μM Ga(NO3)3
killed P. aeruginosa cells deeply embedded in the
biofilm matrix, where most antibiotics lose their
efficacy due to poor permeation (Figure 2) [26] .
of
Other Ga(III) compounds, like DFO–Ga(III),
GaCl 3, and Ga(III)–citrate, showed potent
suppressive activity on P. aeruginosa biofilm
formation in the micromolar range [22,29] .
ro
Similar effects were also observed using 64 mg/l
Ga(NO3)3 (corresponding to 250 μM Ga(III))
on preformed biofilms of bacteria belonging to
the B. cepacia complex [28] .
Consistent with the poor activity of Ga(III)
rP
on planktonic staphylococcal cells, the minimal
biofilm inhibitory concentration of GaM was
very high (mM range) for both laboratory and
clinical Staphylococcus strains (Table 1) [21] .
ho
●●Activity on intracellular bacterial
pathogens
Several pathogenic bacteria such as M. tubercu-
losis, Francisella spp. and Rhodococcus equi have
evolved the ability to infect and replicate in a
variety of human cells, especially macrophages,
ut
where intracellular iron is essential for their life
[27,65,66] . Bacterial iron-uptake systems also play
a role intracellularly, allowing iron retrieval from
Tf and Lf within macrophages [27] . At the site of
A
infection, iron uptake by activated macrophages
prevalently occurs via specific Lf and Tf recep-
tors, and therefore Tf–Fe(III) and Lf–Fe(III)
complexes represent an important component
of the macrophage intracellular iron pool [67,68] .
Ga(NO3)3 treatment of human macrophages
infected with both antibiotic-susceptible and
MDR M. tuberculosis resulted in dose-depend-
ent growth inhibition, with IC50s ranging from
ca. 30 to ca. 100 μM, depending on the strain
(extrapolated from [27]). Moreover, Ga(NO3)3
exerted a bactericidal effect on M. tuberculosis
within macrophages at a concentration of 500
μM, [27] . Challenge of infected macrophages
with Tf–Ga(III) (molar ratio 1:2) showed >50%
388 Future Microbiol. (2014) 9(3)
reduction of M. tuberculosis growth at 62.5
μM (corresponding to 125 μM Ga(III)) [27] ,
comparable with the effect of Ga(NO3)3 alone.
Growth inhibition >70% was achieved at
Tf–Ga(III) or Lf–Ga(III) concentrations as low
as 5 μM (corresponding to 10 μM of Ga(III))
for Francisella tularensis and Francisella novicida
strains [extrapolated from [66] , concomitant with
decreased activity of key iron-containing antiox-
idant enzymes, such as catalase and superoxide
dismutase [66] . Within macrophages, F. tula-
rensis was inhibited by 100 μM Tf–Ga(III)
or Lf–Ga(III) (corresponding to 200 μM
Ga(III)), although the activity of Tf–Ga(III)
and Lf–Ga(III) were not compared with that of
Ga(III) alone [66] .
Potent inhibitory activity on the extracellular
planktonic growth of R. equi was reported using
both GaM (IC90 = 8 μM) [69] and Ga(NO3)3
(bactericidal effect at 50 μM) [24] . Accordingly,
treatment of murine macrophage-like cells with
10 μM GaM caused >threefold decrease in
intracellular R. equi concentrations [70] .
Finally, Ga(III)–PPIX showed potent inhi-
bition of cell invasion and intracellular multi-
plication of the oral pathogen Porphyromonas
gingivalis [56] .
As a whole, these results (Table 1) demonstrate
that Ga(III) affects the growth of relevant
bacterial pathogens in different cellular models.
●●Protective activity of gallium in animal
models of bacterial infection & veterinary
applications
The majority of animal studies on the in vivo
efficacy of Ga(III) compounds were conducted
in mice infected by different routes with a given
bacterial species, and then treated with Ga(III)
compounds. In addition, for in vivo studies,
direct comparison of outcomes is complicated
by differences in the type of infection, thera-
peutic dosage, and routes of Ga(III) administra-
tion. Most remarkably, the relevance for human
applications, that is the translation from human
therapeutic dose to animal dose, has rarely been
addressed.
The earliest evidence of the in vivo antimicro-
bial efficacy of Ga(III) dates back to 1931, when
Levaditi et al. demonstrated a protective effect
of Ga(III)–tartrate from Treponema pallidum
or Trypanosoma evansi infections in different
animal models (Table 2) [71] .
More recently, the in vivo activity of Ga(NO3)3
against A. baumannii was investigated in
future science group
 Promises & failures of gallium as an antibacterial agent  Review

0 µM Ga(III) 0.5 µM Ga(III)

of
24 h 48 h 72 h

Cross section
ro
rP
Side view
ho

Figure 2. Effect of Ga(III) on Pseudomonas aeruginosa biofilms. (A) Ga(III) prevents Pseudomonas
aeruginosa biofilm formation. Confocal microscopic images of green-fluorescent protein-labeled
P. aeruginosa in flow cells perfused with growth medium without (left) or with (right) Ga(NO3)3 at the
ut

indicated concentration, 5 days after inoculation. (B) Ga(III) kills established biofilm. Biofilm of GFP-
labeled P. aeruginosa were grown for 3 days in the absence of Ga(NO3)3 and then treated with 100 μM
Ga(NO3)3 for the indicated time period. Cells were stained with propidium iodide (30 μM) to label
dead cells. Images in (A) and (B, top) are top-down views (x–y plane); (B, bottom) images are side
A

views (x–z plane); scale bars: 50 μm.

Adapted with permission from [26].
For color images please see www.futuremedicine.com/doi/full/10.2217/fmb.14.3

two different models, the insect Galleria mel-
lonella and the neutropenic mouse [20,60] .
Administration of Ga(NO3 ) 3, at concentra-
tions mimicking the human therapeutic dose,
protected G. mellonella from A. baumannii-
mediated killing [20] . Coherently, intraperito-
neal administration of Ga(NO3)3 was effective
in reducing A. baumannii load in the lungs of
neutropenic mice [60] . Putting together in vitro
and in vivo results (Tables 1 & 2) , it can be con-
cluded that Ga(NO3)3 holds promise as a new
anti-A. baumannii agent, raising new hope
for treatment of patients infected with PDR
A. baumannii.
Kaneko et al. tested the antibacterial activ-
ity of Ga(NO3)3 against P. aeruginosa in both
acute and chronic mouse lung infection. In the
acute model, fatal sepsis due to intratracheal
infection with a lethal dose of P. aeruginosa
was prevented by a single intranasal adminis-
tration of Ga(NO3)3 3 h postinfection (Table 2)
[26] . As expected, intranasal administration of
Fe(III) prior to infection abrogated Ga(III)
activity [26] . Similarly, Ga(NO3)3 was effective

future science group www.futuremedicine.com 389

Review  Minandri, Bonchi, Frangipani, Imperi & Visca

Table 2. In vivo antibacterial activity of gallium.

Ga(III) Microorganism Therapeutic regimen (route of Concentration in μmol/kg Type of infection (animal Ref.
compound administration) of single dose model)
Ga(III) tartrate Treponema 30–45 mg/kg (im.) 136–205 Acute infection (rabbit) [71]
pallidum 15 mg/kg (iv.) 68 Acute infection (rabbit) [71]
Trypanosoma 225 mg/kg (iv.) 1024 Acute infection (mice) [71]
evansi
Ga(NO3)3 Acinetobacter Single dose of 1200 μmol/kg 1200 Hemolymph infection [20]
baumannii (hemolymph injection) (Galleria mellonella)
Two doses, prior and after 98 Pulmonary infection [60]
infection, of 25 mg/kg (ip.) (neutropenic mouse)
Pseudomonas Single dose of 50 μl of a 250 mM 600† Acute pulmonary infection [26]
aeruginosa solution (in.) (mouse)
Three daily doses of 50 μl of 600† Chronic airway infection [26]
a 250 mM solution for 3 days model (mouse)

of
(intranasal)
Mycobacterium Two doses, prior and after 78 Intestinal infection (calves) [72]
avium subsp. infection, of 20 mg/kg (oral)
paratuberculosis

ro
Francisella novicida Single dose of 50 μl of 250 mg/ 2330† (single dose) + 40† Pulmonary infection [66]
ml solution (in.) + daily doses of (daily dose) (mouse)
10 mg/kg for 15 days (ip.)
GaM P. aeruginosa Single dose of 25 mg/kg (sc.) 56 Wound infection (burned [23]
mouse)
rP
A. baumannii Single dose of 100 mg/kg (sc.) 225 Wound infection (burned [23]
mouse)
Staphylococcus Single dose of 100 mg/kg (sc.) 225 Wound infection (burned [23]
aureus mouse)
Rhodococcus equi Daily dose, prior and after 22.5 Peritoneum infection [24]
infection, of 10 mg/kg for (mouse)
ho

16 days (oral)
Ga(III)–PPIX Neisseria Single dose prior infection with ND Vaginal infection (mouse) [55]
gonorrhoeae 2 μg (topic)
Values extrapolated considering a mouse average weight of 21 g.
†

Ga(III)–PPIX: Gallium–protoporphyrin IX; GaM: Gallium maltolate; im.: Intramuscular; in.: Intranasal; ip.: Intraperitoneal; iv., Intravenous; ND: Not determinable; sc.: Subcutaneous.
ut

in a murine model of chronic airway infection,
where administration of Ga(NO3)3 starting 5 h
postinfection, three times/day by inhalation
for 9 days, reduced the bacterial load in the
A
lymph node tissues was significantly decreased
(Table 2) [72] .
GaM has been evaluated against P. aerugi-
nosa, using a thermally injured mouse model of

lung (Table 2) [26] . These in vivo results are fully
consistent with in vitro studies , reporting high
susceptibility of P. aeruginosa to Ga(III)-based
compounds.
In vivo activity of Ga(NO3)3 has also been
assayed in a mouse model of F. novicida lung
infection. Multiple Ga(NO3 ) 3 administra-
tions protected mice from lethal infection [66] ,
although at relative high dosages (Table 2) .
Ga(NO3 ) 3 activity has also been tested in
calves, in order to determine protection from
infection with MAP and assess putative collat-
eral effects of treatment [72] . No adverse effects
were observed in calves orally treated with
Ga(NO3)3, whilst MAP burden in intestinal and
acute infection. Subcutaneously administered
GaM prevented fatal infection, indicating that
P. aeruginosa proliferation and systemic spread
were blocked [23] . The protective activity of GaM
in vivo was also observed for A. baumannii and
S. aureus burn eschar infection, albeit at higher
locally injected doses of GaM than those used
to suppress P. aeruginosa (Table 2) [23] . Despite
the poor susceptibility reported in vitro (Table 1),
this study holds promises for the use of GaM, a
Ga(III)-compound endowed with low toxicity
[63] , against S. aureus infection.
Oral GaM reduced R. equi burden in spleen,
lung and liver in a mouse model of infection
(Table 2) [24] . The use of GaM in veterinary

390 Future Microbiol. (2014) 9(3) future science group


medicine was also extended to cattle experi-
mentally infected with S. enterica; however, no
reduction in Salmonella fecal level was observed
[25] . These results, together with in vitro data
(Table 1) , discourage the use of Ga(III) against
Salmonella spp.
Ga–PPIX is endowed with strong in vitro
activity against N. gonorrhoeae [30] . Ga–PPIX
activity was also confirmed in vivo, resulting in
substantial reduction of gonococcal coloniza-
tion in an experimental murine model of vaginal
infection (Table 2) [55] .
In conclusion, different animal models
of infection provided evidence of an over-
all good efficacy and tolerability of Ga(III)-
based compounds. The most promising results
were obtained in laboratory animals infected
with MDR bacteria, such as P. aeruginosa and
A. baumannii. MDR P. aeruginosa infections
represent the main cause of death and compli-
cation in an array of chronic pulmonary dis-
eases, thus the possibility of developing new
Ga(III)-based anti-pseudomonas therapies is
fascinating. The growing interest in this area is
rP
testified by the recent set up of a Phase 1 clini-
cal trial aimed at monitoring the effect of the
FDA-approved Ga(NO3)3 formulation (Ganite)
in cystic fibrosis patients chronically infected by
P. aeruginosa [73] .
ho
Synergistic activity of Ga(III) with
antimicrobials
Several studies have explored the activity of
Ga(III) in combination with antibiotics, in
search for useful synergistic effects (i.e., the
ut
reduction in the active concentration of both
antibiotic and gallium compared with individual
components used alone, according to [74]).
The antimicrobial efficacy of DFO–Ga(III)
A
in combination with gentamicin (GEN) has
been tested on both stationary phase and bio-
film-growing P. aeruginosa cells [22] . The DFO–
Ga(III)–GEN combination caused a higher
reduction of bacterial viability than GEN or
DFO–Ga(III) supplied alone [22] . These prom-
ising results were confirmed in vivo using a rabbit
scratched cornea infection model: reduction in
P. aeruginosa corneal injury was much more evi-
dent upon treatment with DFO–Ga(III)–GEN,
than with individual components [22] . Similarly,
the β-lactam antibiotic loracarbef, conjugated
with DFO–Ga(III), displayed enhanced growth
inhibitory effect against Micrococcus luteus,
compared with individual components [75] .
future science group
Promises & failures of gallium as an antibacterial agent  Review
To further enhance the delivery of the
Ga(III)–GEN combination, the activity of these
compounds co-encapsulated in liposomes has
been evaluated on clinical P. aeruginosa isolates
[76] . The formulation, called Lipo–Ga–GEN,
displayed lower MIC and MBC than Lipo–GEN
alone on planktonic growing P. aeruginosa [76] .
Moreover, Lipo–Ga–GEN successfully killed
P. aeruginosa biofilms, while the correspond-
ing concentrations of GEN (8 mg/l), Ga(III)
(0.6 μM), Lipo–Ga (0.6 μM), or Lipo–GEN
(8 mg/l) supplied alone, had much weaker
effects [76] .
Another example of Ga(III) –antibiotic
synergism was offered by the high activity of
of
Ga(NO3)3 in combination with colistin (poly-
myxin E) [20] , which represents the last therapeu-
tic option for PDR A. baumannii. Since colistin
acts by perturbating the outer membrane integ-
ro
rity, it has been hypothesized that it could facili-
tate Ga(III) diffusion into A. baumannii cells,
thereby promoting growth inhibition [20] .
Conclusion & future perspective
The necessity of iron for bacterial growth and
the requirement of efficient iron assimilation sys-
tems for infection have made iron transport and
metabolism very attractive targets for the devel-
opment of new antibacterial therapies. Many
efforts have been put forth to assess the drug-
gability of bacterial Fe(III) transport systems;
however, their diversity, functional redundancy
and species-specificity have so far precluded
the development of broad-spectrum antibacte-
rial agents targeting iron uptake [46] . On the
other hand, the Fe(III)-mimetic properties of
Ga(III) provided a shortcut to efficient target-
ing of bacterial iron metabolism, circumventing
the problem of Fe(III) uptake specificity. The
repositioning of Ga(III) as an antibacterial agent
offered the opportunity to take advantage of the
wealth of knowledge on the already established
pharmacological properties of Ga(III)-based
formulations, thus accelerating clinical testing
of their antibacterial properties.
A remarkable feature making Ga(III) a prom-
ising candidate in antibacterial chemotherapy is
its ability to accumulate within inflammatory
sites [4,5,77] . Multiple factors contribute to the
accumulation and retention of Ga(III) in inflam-
matory lesions, many of which are attributable to
the nonspecific response of injured tissues. Acute
inflammation is characterized by different vas-
cular events, including vasodilation, increased
www.futuremedicine.com 391
Review  Minandri, Bonchi, Frangipani, Imperi & Visca
permeability and augmented blood flow. Owing
to these vascular changes and release of lysoso-
mal content by pathogen-stimulated neutrophils,
high concentration of Tf and Lf are present at
the site of inflammation [78] . Local production
of bacterial and neutrophil proteases and the low
pH could facilitate Ga(III) release from Tf and
Lf, resulting in remarkably high free Ga(III) con-
centration in the infected tissue. These effects,
which are the basis of radiogallium scanning
of septic foci [8,9] , could also explain the good
protective activity of Ga(III) in animal infected
with bacterial species that show poor sensitivity
to Ga(III) in vitro (Tables 1 & 2) .
The wealth of in vitro and in vivo data available
of
suggest that some Ga(III) formulations could in
the future represent a promising complement
and/or alternative to the use of conventional
antimicrobials for treatment of certain bacterial
ro
infections. As an example, infections sustained
by P. aeruginosa and A. baumannii are extremely
resistant to currently available antibiotic thera-
pies, whilst both species are very sensitive to
the inhibitory activity of Ga(III) both in vitro
rP
and in vivo. It is tempting to correlate the high
susceptibility of these species with their obli-
gate respiratory metabolism, where iron plays
a pivotal role in electron transfer to and from
cytochrome complexes. Accordingly, facultative
ho
anaerobes that undertake fermentative processes
(e.g., enterobacteria, enterococci, staphylococci
and streptococci) are less susceptible. This poses
the problem of how the metabolic state of the
cell and the culture condition (e.g., iron levels
and pO2) affect Ga(III) susceptibility in vitro.
ut
The diverse growth conditions used in vitro to
assess the inhibitory activity of Ga(III) on dif-
ferent bacterial pathogens, especially the vari-
able iron content of culture media, hamper a
A
straightforward comparison between studies. As
a general rule, the poorer the iron content of the
medium, the higher the Ga(III) activity, irrespec-
tive of the tested bacterial species. Ga(III) specia-
tion in liquid media should also be taken into
account, since extensive Ga(III) precipitation can
occur at neutral pH, that is the pH optimum
for growth of bacterial pathogens. Ingredients of
culture media can also influence speciation and
biological effects of Ga(III) by ligand exchange,
for example between iron complexes and bacte-
rial siderophores [29] . Therefore, it is mandatory
that in vitro antimicrobial susceptibility testing of
Ga(III) compounds are performed in chemically
defined media under standard assay conditions,
392 Future Microbiol. (2014) 9(3)
in order to improve interlaboratory reproducibil-
ity of results and avoid the bias introduced by
ill-defined iron content and Ga(III) speciation
in complex bacteriological media.
There are a number of open issues that deserve
further consideration for the appropriate usage
of Ga(III) as an antibacterial agent. First of all,
Ga(III) bioavailability appears to be a major issue,
and future research should focus on the devel-
opment of optimized Ga(III) carriers endowed
with increased capacity to reach infective foci
and permeate bacterial membrane(s). It should
be taken into account that bacteria generate bio-
films in vivo as an adaptive response to the hostile
environment encountered during the infection.
Bacterial cells embedded in biofilms exist in dif-
ferent metabolic states owing to decreasing oxy-
gen and nutrient gradients from the surface to the
bottom of the biofilm. Cells growing on the bio-
film surface are more metabolically active than
those living in deeper layers, and markedly differ
in their response to toxic compounds, including
antimicrobials [79] . Although the biofilm texture
contains large water-filled channels that allow
free diffusion of ions, the biofilm matrix shows
a net negative charge due to the presence of
DNA and anionic polysaccharides that can trap
positively-charged ions such as Ga(III). Thus,
both the resting metabolic state of the bacte-
rial cell and the scavenging effect of the matrix
could contribute to reducing the antimicrobial
activity of Ga(III) in biofilm-growing bacteria.
If translated into clinical practice, higher Ga(III)
dosages than those predictable from in vitro anti-
microbial susceptibility testing on planktonic
cells would be required for successful therapy of
biofilm-related infections. Related to the above
issues, Ga(III)-coated biomaterials and deliv-
ery formulations for slow Ga(III) release have
recently been developed, providing new tools to
suppress or prevent bacterial growth and biofilm
formation on medical implants [80,81] .
Neither the problem of bacterial resistance to
Ga(III) nor the duration of bacteriostasis have
yet been fully addressed. In vitro activity data for
Ga(III) compounds demonstrate bacteriostasis
for short exposure periods, but it cannot be ruled
out that bacteria would outgrow after prolonged
exposure times, either by mutant selection or
adaptation. Unlike most antibiotics, which have
a single target within the bacterial cell, Ga(III)
is a typical multitarget drug that is predicted
to impair several Fe(III)-dependent functions.
Therefore, resistance mechanisms such as target
future science group

mutation, drug modification or development of
alternative metabolic pathways are unlikely to
develop in bacteria to overcome Ga(III) inhibi-
tion. Conversely, reduced permeability by muta-
tion of membrane transporter(s), active efflux,
extracytoplasmic scavenging (e.g., by sidero-
phores) and intracellular trapping into storage
(e.g., bacterioferritin-like) molecules would,
in theory, be effective mechanisms for Ga(III)
resistance; however, for all of them the similarity
between Ga(III) and Fe(III) could imply a severe
fitness cost to the resistant cell. This represents
another area for future investigation.
At the molecular level, further studies are
required to shed more light on the route(s) by
which Ga(III) enters bacterial cells, as well as
on the mechanism(s) responsible for growth
inhibition and, eventually, bacterial death. A
promising strategy to obtain information on
Ga(III) mechanism(s) of action is the selection of
Ga(III)-insensitive variants upon Ga(III) adap-
tation, and their genetic and functional char-
acterization through omics technologies. This
approach has recently been exploited to address
rP
the mechanisms of Ga(III) resistance in P. aer-
uginosa [16] . As to the mechanism of Ga(III)-
induced bacterial death, it has been shown that
Ga(III) enhances ROS production and generates
oxidative stress [11,14] ; however, further research
ho
is needed to determine to what extent these cel-
lular responses play a prominent role in bacte-
rial death, as proposed for many conventional
bactericidal antibiotics [82] .
Regarding the clinical applications of Ga(III)
as an antibacterial agent, it should be noted
ut
that Ga(III) is endowed with immunosuppres-
sive activity on macrophage and T-cell func-
tions [83,84] , and could weaken the host immune
response upon long-term administration. The
A
problem is exacerbated in immunocompromised
patients, where the immune deficit impairs effi-
cient clearance of infecting bacteria, and who
require bactericidal treatments for successful
eradication of infection. Current therapeutic
regimens allow attainment of Ga(III) plasma
levels of 10–20 μM, depending on the formula-
tion, with predictably higher levels in infective
foci due to Ga(III) release from Tf and Lf. Such
concentrations are bactericidal for few bacterial
species and strains (Table 1) , posing the need for
careful determination of the bactericidal concen-
tration of Ga(III) compounds against infecting
bacteria, which should be reached and constantly
maintained at the infected site to attain bacterial
future science group
Promises & failures of gallium as an antibacterial agent  Review
eradication. The iron status of the patient should
also be considered, since pathological iron over-
load, as in hemochromatosis and thalassemia,
would predictably impair the antibacterial effi-
cacy of Ga(III), while chelation therapy has been
shown to lower Ga(III) levels due to increased
urinary secretion [85] . Lastly, nearly all data on
the protective activity of Ga(III) in vivo have
been generated by infecting laboratory animals
that had been treated before or concomitant with
bacterial challenge [20,23,24,55,60,66,72] and, to our
knowledge, only one paper has addressed the effi-
cacy of Ga(III) administered a few hours postin-
fection [26] . These experimental setups are pro-
foundly different from the clinical setting, where
of
therapy is usually initiated after the infection is
diagnosed, and call for further in vivo investiga-
tions on the efficacy of Ga(III) in the treatment
of pre-established bacterial infections.
ro
The end of the ‘golden age’ of antibiotic
discovery left us with a dramatic shortage of
new drugs to combat MDR pathogens [86,87] .
Meanwhile, infections caused by MDR bacteria
continue to increase in frequency and cause wor-
ryingly high morbidity and mortality worldwide
[88] . In this scenario, the compelling question is
whether and when Ga(III) could be used as an
antibacterial agent. Clinical trials still do not pro-
vide the conclusive answer to this question; how-
ever, in vitro data and animal infection studies
indicate a potential use of Ga(III) as a last resort
drug, in combination therapy with other anti-
microbials, to treat local or systemic infections
sustained by PDR, otherwise untreatable bacte-
ria. The activity of Ga(III) can be potentiated
in vivo (i.e., the therapeutic dose can be lowered)
by combination with antibiotics showing syner-
gism in vitro (e.g., aminoglycosides and colistin),
thus expanding the therapeutic potential to those
species or strains that are resistant to either of
the compounds alone. Lastly, the possibility of
using Ga(III) for topical treatments and coat-
ing of biomaterials and implants is an attractive
perspective.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involve-
ment with any organization or entity with a financial interest
in or financial conflict with the subject matter or materials
discussed in the manuscript. This includes employment, con-
sultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of
this manuscript.
www.futuremedicine.com 393
Review  Minandri, Bonchi, Frangipani, Imperi & Visca

Executive summary
Properties of gallium as an antimetabolite
●● Gallium (Ga(III)), is a group IIIA semimetal possessing extensive chemical similarity with iron (Fe(III)).
●● Ga(III) can substitute for Fe(III) in several iron-containing enzymes, host iron-binding proteins, and bacterial
siderophores.
●● Unlike Fe(III), Ga(III) cannot undergo redox cycles under physiological conditions.
●● Replacement of Fe(III) with Ga(III) in the prosthetic group of iron-containing enzymes disrupts essential cellular
functions.
Medical applications of Ga(llI)
●● Ga(III) compounds are used as diagnostic and therapeutic tools in clinical medicine.
●● Citrate-buffered gallium nitrate (Ganite®, Genta, NJ, USA) is approved by US FDA for treatment of cancer-associated
hypercalcemia.

of
Fate of Ga(III) into bacterial cells
●● Ga(III) is likely to exploit Fe(III)-uptake routes to enter bacterial cells.
●● Ga(III) perturbs bacterial iron metabolism and generates oxidative stress.
In vitro & in vivo antibacterial activity of Ga(llI)
●●
ro
Ga(III), in the forms of nitrate, chloride and maltolate salts, is endowed with a potent growth-inhibitory activity, both
in vitro (on planktonic- and biofilm-living bacteria) and in vivo. A number of Gram-negative and Gram-positive bacterial
species, including Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia cepacia complex, Mycobacterium
rP
tuberculosis, Francisella tularensis and Rhodococcus equi are severely inhibited by Ga(III).
●● Staphylococcus spp., Salmonella enterica serovars and other facultative anaerobic species are poorly inhibited by Ga(III).
●● Ga(III) activity is influenced by the metabolic state of the cell, and growth inhibition is invariably reversed by Fe(III).
●● A number of Ga(III) complexes have been tested in order to enhance Ga(III) acquisition by bacteria (“Trojan horse”
ho

strategy).
●● Among carriers, Ga(III)–protoporphirin IX showed potent antibacterial activity both in vitro and in vivo.
Considerations for the use of Ga(III) as a new anti-infective
●● Ga(lIl) is more effective against bacterial species that preferentially adopt aerobic metabolism, while facultative
ut

anaerobes undertaking fermentative processes are less susceptible.

●● Pre-existing resistance to conventional antibiotics does not compromise Ga(III) activity.
●● Concerns remain regarding side effects of Ga(III) on host immune response.
A

●● The mechanism(s) of antibacterial action of Ga(III) and its molecular targets deserve more in-depth investigation.
●● Development of a standard method for Ga(III) susceptibility testing will facilitate harmonization of susceptibility data.
●● Definition of minimal bacteriostatic and bactericidal concentrations for individual species and strains is mandatory.

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