Animals devoid of pulmonary system as infection models in the study of lung bacterial pathogens

Abstract

Biological disease models can be difficult and costly to develop and use on a routine basis. Particularly, in vivo lung infection models performed to study lung pathologies use to be laborious, demand a great time and commonly are associated with ethical issues. When infections in experimental animals are used, they need to be refined, defined, and validated for their intended purpose. Therefore, alternative and easy to handle models of experimental infections are still needed to test the virulence of bacterial lung pathogens. Because non-mammalian models have less ethical and cost constraints as a subjects for experimentation, in some cases would be appropriated to include these models as valuable tools to explore host-pathogen interactions. Numerous scientific data have been argued to the more extensive use of several kinds of alternative models, such as, the vertebrate zebrafish (Danio rerio), and non-vertebrate insects and nematodes (e.g., Caenorhabditis elegans) in the study of diverse infectious agents that affect humans. Here, we review the use of these vertebrate and non-vertebrate models in the study of bacterial agents, which are considered the principal causes of lung injury. Curiously none of these animals have a respiratory system as in air-breathing vertebrates, where respiration takes place in lungs. Despite this fact, with the present review we sought to provide elements in favor of the use of these alternative animal models of infection to reveal the molecular signatures of host-pathogen interactions.

Full text

REVIEW ARTICLE
published: 04 February 2015
doi: 10.3389/fmicb.2015.00038
Animals devoid of pulmonary system as infection models
in the study of lung bacterial pathogens
Yamilé López Hernández1*†, Daniel Yero 2,3 *†, Juan M. Pinos-Rodríguez1and Isidre Gibert2,3
1Centro de Biociencias, Universidad Autónoma de San Luis Potosí, San Luis de Potosí, Mexico
2Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain
3Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain
Edited by:
Zsolt Molnár, University of Szeged,
Hungary
Reviewed by:
Paras Jain, Albert Einstein College of
Medicine, New York, USA
Roman Zahorec, Comenius
University, Slovakia
*Correspondence:
Yamilé López Hernández, Centro de
Biociencias, Universidad Autónoma
de San Luis Potosí, Km 14.5,
Carretera San Luis Potosí-Matehuala,
Soledad de Graciano Sánchez,
San Luis de Potosí, Mexico
e-mail: yamile.lopez@uaslp.mx;
Daniel Yero, Institut de Biotecnologia
i de Biomedicina, Universitat
Autònoma de Barcelona, Campus
Universitari, Bellaterra, Barcelona
08193, Spain
e-mail: daniel.yero@uab.cat
†These authors have contributed
equally to this work.
Biological disease models can be difficult and costly to develop and use on a routine basis.
Particularly, in vivo lung infection models performed to study lung pathologies use to be
laborious, demand a great time and commonly are associated with ethical issues. When
infections in experimental animals are used, they need to be refined, defined, and validated
for their intended purpose.Therefore, alternative and easy to handle models of experimental
infections are still needed to test the virulence of bacterial lung pathogens. Because non-
mammalian models have less ethical and cost constraints as a subjects for experimentation,
in some cases would be appropriated to include these models as valuable tools to explore
host–pathogen interactions. Numerous scientific data have been argued to the more
extensive use of several kinds of alternative models, such as, the vertebrate zebrafish
(Danio rerio), and non-vertebrate insects and nematodes (e.g., Caenorhabditis elegans)
in the study of diverse infectious agents that affect humans. Here, we review the use of
these vertebrate and non-vertebrate models in the study of bacterial agents, which are
considered the principal causes of lung injury. Curiously none of these animals have a
respiratory system as in air-breathing vertebrates, where respiration takes place in lungs.
Despite this fact, with the present review we sought to provide elements in favor of the
use of these alternative animal models of infection to reveal the molecular signatures of
host–pathogen interactions.
Keywords: alternative model, pneumonia, zebrafish, C. elegans,Drosophila melanogaster,Galleria mellonella,
tuberculosis, cystic fibrosis
INTRODUCTION: A GOOD ANIMAL MODEL
To study the pathology, host immune response and the com-
plex interactions between host and pathogen, the use of animal
models have been invaluable, but for many years, it have pre-
sented strong public, scientific concerns, as well as philosophical
contradictions (Lipscomb et al., 2010). From the great contri-
butions of Luis Pasteur and Robert Koch in the use of animal
models to decipher the causal agents of several diseases, includ-
ing Bacillus anthracis,Mycobacterium tuberculosis or the rabies
virus, concepts related to the animal use and handle and bioethics
have arise (Baumans, 2004). Russell and Burch (1959) developed
the “Three Rs” principle (Replacement, Reduction, and Refine-
ment), although their work remained largely ignored well into
the 1970s (Balls and Halder, 2002). At this time, rodents were
considered despicable animals and consequently they were treated
without consideration, wide spreading its use as research model
(Damy et al., 2010). In 1999, the Declaration of Bologna reaf-
firmed that “humane science is a prerequisite for good science,
and is best achieved in relation to laboratory animal procedures by
the vigorous promotion and application of the Three Rs” (Balls,
2009). In 2002, the genome sequence of mouse was completed
(first mammal after humans; Watkins-Chow and Pavan, 2008).
This fact largely contributed to the great use of mouse as ani-
mal model. However, and taken into account the principle of
Replacement, several alternative models far different from the
mammals classic ones, have been developed in the last years
(Hendriksen, 2002).
A perfect animal model would be a model that satisfied not
only scientific, but also ethical criteria (Zak and O’Reilly, 1993).
Among the most controversial experimental animal models from
the point of view of ethics, lung infections induced by bac-
teria are considered by far the most aggressive. For instance,
several pathogens are able to kill non-human mammals due to
lung infections. For this reason, these models need to be opti-
mized to better reproduce human infections and acute lung
damages. In addition, when an animal model is used in preclinical
research, we have to consider that not all results will be success-
fully extrapolated from animal studies to humans (Fuchs et al.,
2009;Evans et al., 2010). Some important anatomical, physiologi-
cal, genetic, and molecular differences are clearly present between
species.
The pathogenesis of infection is a result of the balance of the
host–pathogen interaction (Mason et al., 1995). The pathogen and
host genetic backgrounds are a relevant determinant of this out-
come, as continuously several genes are activated or repressed
depending on environmental changes during experiments. For
many years it was thought that mammalian models of infection
were the unique choice to study host–pathogen interactions as
well as for pre-clinical evaluation of vaccines and drugs before
their use in humans (Means and Aballay, 2011). However, there
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López Hernández et al. Lung pathogens alternative infection models
are presently numerous scientific data that have been argued to
the more extensive use of several kinds of alternative infection
models, such as, small vertebrates, insects, and nematodes (Kurz
and Ewbank, 2007). The present review summarizes, compare and
discuss the published experience in classical animal models, such
as mice, and alternative animal models, particularly the zebrafish
(Danio rerio), Caenorhabditis elegans and the insects Drosophila
melanogaster and Galleria mellonella (the greater wax moth) in
the study of bacterial agents which are considered the principal
causes of lung injury.
ANIMAL MODELS TO STUDY BACTERIAL LUNG PATHOGENS,
SAFETY AND ETHICAL ISSUES AND THE NEED OF
ALTERNATIVE METHODS
OVERVIEW OF MAMMALIAN MODELS FOR RESEARCH ON PULMONARY
INFECTION
The respiratory system of most non-human mammals mimics
in general a lung environment in humans, in terms of chemi-
cal and physical conditions and spatial structures. In addition,
the pulmonary defenses to respiratory infections in non-human
mammals are somehow similar to humans. Hence, most of the
pneumonia animal studies are carry out in mammals to facil-
itate some types of investigation (Mizgerd and Skerrett, 2008).
However, extrapolation of results to humans is not straightfor-
ward owing to significant anatomical and physiological differences
between species. The different mammals do not appear to
present similar mucociliary clearance and alveolar macrophage
morphometry (Fernandes and Vanbever, 2009).
To produce experimental pulmonary infection, mammalians
offer a wide diversity of inoculation routes [reviewed and com-
pared in Bakker-Woudenberg (2003)], being intranasal (i.n.) and
intratracheal (i.t.) inoculations the infection routes seem to be the
most “naturally acquired.” The intratracheal model of infection
requires a complex and invasive technique for disease induc-
tion but offers the advantage of allowing almost total delivery
of the bacterial inoculum to the lungs. The model of infection
through intranasal aspiration is the most commonly used, as
it is fast and easier to perform without invasive surgical proce-
dures and also because it mimics the natural route of infection in
humans. When the inoculum is administered intranasally it could
be applied as an aerosol (passive inhalation) or as nasal droplets.
The intranasal aerosol model instead requires an exposure cham-
ber with a nebulizer, and permits the simultaneous infection of
several mice (Mizgerd and Skerrett, 2008). However, aerosol stud-
ies carry the greatest potential risk of infection with airborne
pathogens (Chen et al., 2011), the inhaled dose varies considerably
and the equipment to perform the infection is not always available
in research laboratories. Most of these routes may require anes-
thetize animals and sometimes post-administration of pain relief
drugs.
Rodents, more than other animals, are commonly used to
study pneumonia. Mice are the most used in infection experi-
ments and offer many advantages, including that they are relatively
inexpensive, easy to maintain, easy to handle and their genome
can be manipulate (Denny, 2005;Leung et al., 2013). Despite of
this, surgical and invasive techniques are required to reproduce
some acute or chronic lung diseases in mice. However, the use
of general anesthesia with their side effects in host is considered
as a great disadvantage of these techniques (Mizgerd and Sker-
rett, 2008). Guinea pigs, rats and hamsters apart of mice, are
some of the other animals employed as models of lung infections
(Bakker-Woudenberg, 2003).
There are well-documented relevant differences relative to lung
infection between mice and humans. Differences in the structural
anatomy, cellular composition of the tracheobronchial epithelium,
local phagocytic and chemical defenses, inflammatory or immune
response, need to be analyzed at the moment of interpreting the
experimental results. Mice are different in terms of their lower
complexity of the airway branches and a relatively large caliber
of the airway lumen (Irvin and Bates, 2003). Furthermore, they
have different cellular expression and ligand binding for selected
Toll like receptors (TLRs; Mizgerd and Skerrett, 2008;Apt and
Kramnik, 2009). In contrast with humans, this species has a well-
developed broncus associated lymphoid tissues (BALTs) system,
may be due to their life habits. In healthy humans, this system is
practically absent (Hyde et al., 2009). However, the availability of
immunological reactives as well as several mice transgenic lines has
made their use almost indispensable in the majority of infection
studies.
Mice are resistant (not susceptible) too many human pathogens
(Lyons et al., 2004;Di Pietrantonio and Schurr, 2005;Aziz et al.,
2007). Consequently, in addition to rodents, several other mam-
malian animal models have been explored in respiratory diseases
studies: cattles, goats, sheeps, pigs, dogs, and non-human pri-
mates. The fact that non-human mammalian species are more
phylogenetically related to humans justifies at least in part the
use of these species by researchers. However, none of them com-
pletely reproduces all the aspects of lung diseases in humans.
The most clinically relevant model has been the primate infec-
tion models, due to the high genetic and structural similarity
with humans (Bem et al., 2011;Kaushal et al., 2012). Nonethe-
less, the cost, time, logistic and ethical considerations and the risk
of zoonoses of this model mean that it can only be used in a limited
fashion.
Despite the simplicity of their immune system, and their evolu-
tionary distance to human, some non-mammalian models using
small animals (fishes, nematodes, and insects) are characterized
by their short generation time which redundance in the low cost
of experiments. Curiously none of these animals have a respi-
ratory system as in air-breathing vertebrates, where respiration
takes place in lungs. However, such models have allowed success-
ful screening for virulence genes in the most common bacterial
lung pathogens (Tab l e 1 ).
ZEBRAFISH (Danio rerio) AS AN ALTERNATIVE VERTEBRATE MODEL
FOR STUDYING LUNG INFECTION AGENTS
In adult fish, respiration takes place mainly through the gills.
In embryo zebrafish gill development begins by 3 days post-
fertilization, in the meantime cutaneous respiration accounts for
nearly all gas exchange (Rombough, 2007). Naturally, zebrafish
get infected by pathogens through the digestive route, the dam-
aged fish surface or through the gills (Cantas et al., 2012). Over
the past decade, several bacteria and viruses have been studied
in their ability to infect zebrafish (Martin and Renshaw, 2009).
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López Hernández et al. Lung pathogens alternative infection models
Table 1 |Most significant contributions of alternative animal models for the study of some relevant lung pathogens.
Lung
pathogen
Alternative model Relevant contribution to pulmonary infection in mammals Reference
Streptococcus
pneumoniae
Zebrafish embryos Pneumococci evade immune clearance by interfering with phagocytic functions. Rounioja et al. (2012)
Adult zebrafish Virulence attenuated mutants are defective in polysaccharide capsule, autolysin, or
pneumolysin.
Saralahti et al. (2014)
Galleria mellonella larvae Non-capsulated and pneumolysin defective strains were less virulent than their respective
wild types. The role of antimicrobial peptide activity and resistance has been addressed.
Evans and Rozen (2012)
Staphylococcus
aureus
Zebrafish embryos Role of Macrophages in the ingestion of S. aureus during in vivo infections. Prajsnar etal. (2008)
Caenorhabditis elegans Several genes encoding virulence factors, including biofilm-related, identified in S. aureus
were relevant for mammalian pathogenesis.
Sifri et al. (2003,2006),
Begun et al. (2005,2007)
G. mellonella larvae Both bacterial glycolysis and gluconeogenesis have important roles in virulence. Purves et al. (2010)
Mycobacterium
tuberculosis
Zebrafish infected with Mycobacterium marinum Infection with M. marinum induces host proteases in epithelial cells and enhances recruitment
of macrophages in order to promote granuloma formation to facilitate bacterial dissemination.
Volkman etal. (2010)
Zebrafish infected with M. marinum The importance of Th2-type response in controlling mycobacterial infection. Hammarén et al. (2014)
Drosophila melanogaster infected with M.
marinum
Model to reveal the relationship between phagocytes and bacteria. Dionne etal. (2003)
Pseudomonas
aeruginosa
C. elegans (Slow killing) Positive correlation between P. aeruginosa genes required to kill C. elegans and those for
pathogenesis in mammals.
Tan etal. (1999a)
(Continued)
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López Hernández et al. Lung pathogens alternative infection models
Table 1 |Continued
Lung pathogen Alternative model Relevant contribution to pulmonary infection in mammals Reference
C. elegans (Fast killing) The diversity of toxic molecules produced and released by P. aeruginosa facilitates its
pathogenicity and contributes to impair lung function in CF.
Cezairliyan et al. (2013)
Zebrafish embryos The T3SS, biofilm formation and quorum-sensing systems are involved in virulence, and these
systems correlate with increased P. aeruginosa virulence in murine models and in humans.
Clatworthy et al. (2009)
Zebrafish embryos Helped to support a connection between the cystic fibrosis transmembrane conductance
regulator (CFTR) and the innate immune response.
Phennicie et al. (2010)
D. melanogaster The model allowed in vivo study of P. aeruginosa biofilm infections. Mulcahy et al. (2011)
G. mellonella larvae Identification of mammalian virulence factors of P. aeruginosa, particularly biofilm-related genes. Jander et al. (2000)
Burkholderia
pseudomallei
C. elegans Disruption of calcium signal transduction, a second messenger in the epithelial response to
bacteria, as mechanism for nematode neuromuscular intoxication caused by Burkholderia spp.
O’Quinn et al. (2001)
C. elegans Identification of virulence factors further validated in an intranasal infection model in BALB/c
mice.
Gan et al. (2002)
D. melanogaster infected with Burkholderia
thailandensis
Potential model host to study the role of innate immunity in melioidosis. Pilátová and Dionne
(2012)
Stenotrophomonas
maltophilia
Zebrafish adults Abundance of protein Ax21, a quorum-sensing factor, proved correlation to mortality in the
zebrafish infection model.This protein triggers innate immunity in both plants and animals.
Ferrer-Navarro et al.
(2013)
C. elegans (Slow killing) Diffusible signal factor (DSF) that controls cell–cell communication, is involved in virulence,
biofilm formation, and motilities.
Huedo et al. (2014)
G. mellonella larvae The model confirms protease StmPr1 as relevant virulence factor of S. maltophilia. Nicoletti etal. (2011)
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López Hernández et al. Lung pathogens alternative infection models
A major advantage for its use has been that during the first days
after fertilization (≈48 h until hatching) the embryos look trans-
parent and until 3 weeks the larvae are quite translucent (Ali et al.,
2011). Therefore, it is possible to follow in real time the pro-
gression of infected living embryos, using fluorescent techniques
(Redd et al., 2006). However, adult fishes are gaining recognition
as a model for bacterial infections because they possess a fully
developed adaptive immune system (Meijer and Spaink, 2011).
The second main advantage of this model is the great
possibilities that it offers for genomic and large-scale mutant
analysis. Zebrafish genome is already available (Ramachan-
dran et al., 2010) and quite well-annotated (ZF version 9;
http://www.ensembl.org/Danio_rerio/Info/Index). More than
26,000 genes encoding proteins have been sequenced and anno-
tated, showing high conservation between innate and adaptative
related genes with the respective orthologs in humans (Meeker
and Trede, 2008;Cui et al., 2011;Rauta et al., 2012).
Most of the mammalian immune system components and
molecules have been identified in zebrafish or in other teleost
species (Mitra et al., 2010;Alejo and Tafalla, 2011;Xu et al., 2012;
Page et al., 2013), including a population of antigen-presenting
cells very similar to the mammalian dendritic cells (Lugo-Villarino
et al., 2010). Innate immunity is functional, with macrophages
and neutrophils that are active at 48 h post-fertilization. These
species have an active complement system which can be started
/initiated by the same ways presents in mammals (Holland and
Lambris, 2004;Sunyer et al., 2005). The adaptive immune sys-
tem also consists of T cells and B cells although the main site
for antigen presentation and T cell maturation is the spleen. Fur-
thermore, multiple waves of hematopoiesis in zebrafish occur at
distinct anatomical sites analogous to mammalian hematopoiesis
(Kanther and Rawls, 2010).
Renshaw et al. (2007) established a model of inflammation,
injuring to the zebrafish tailfin and inducing a characteristic
neutrophilic inflammatory response, which resolves with a similar
FIGURE 1 |Time course of implementation of mice and alternative models for bacterial infections. Persistent infection in adult zebrafish refers to a latent
disease Mycobacterium marinum model (Parikka etal., 2012).
kinetics as in mammals. These authors defined a new model for in
vivostudy of inflammation resolution and their link with apoptosis
(Renshaw et al., 2007). By other way, Benyumov et al. (2012) devel-
oped an in vivo zebrafish model to test phenotypic differences
between human fibroblasts that participate in physiological and
pathological process.
Additionally, zebrafish has been considered as replacement
method for animal experiments because they present some charac-
teristics such as high rate of fecundity, small size, easy maintenance,
fast development and less stringent regulatory and ethical consid-
erations since it has been considered that fish embryos in early
developmental stages do not experiment pain, suffering, or dis-
tress. Although the ethical constraints become apparent, one study
suggests that experiments with zebrafish should be subject to reg-
ulation from 5 days post-fertilization onward (Strähle et al., 2012)
since is between days 5 and 6 when larvae start to feed. Thus, an
animal protocol should be required to infect zebrafish older than
the time the animals become free feeding. On the other hand,
some recent studies say that common anesthetics are not the most
“human” or humanize option for zebrafish euthanasia and could
cause animal suffering (Cressey, 2014), adding evidences that it is
necessary to minimize distress or death.
As in preclinical researches with mammalian models, adult
animals should be allocated for several days under special con-
ditions before beginning the experimental procedures, in order
to reach their adaptation to new laboratory conditions and to
recoverfromstress(Figure 1). When zebrafish embryos are used,
this time is relatively short because they develop very rapidly. To
reach a pathogenic dose, as for traditional infection in mice mod-
els, zebrafish infection involves a single dose of bacteria requiring
an initial population of pathogens able to proliferate avoiding,
long enough, the detection by immune cells. However, when
adult fish are less susceptible hosts for bacterial infection, higher
amount of live bacteria are required when compared to infection in
mice. For instance, intraperitoneal infection of zebrafish with the
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López Hernández et al. Lung pathogens alternative infection models
Pseudomonas aeruginosa PAO1 strain or with Stenotrophomonas
maltophilia clinical isolates reported median lethal dose (LD50)
values of approximately 5 ×107cfu/dose or 5 ×108cfu/dose
respectively (Ferrer-Navarro et al., 2013;Huedo et al., 2014;Ruyra
et al., 2014). Infection models in zebrafish usually are conducted
within the time frame of days rather than weeks to months
(Figure 1), but in the adult persistent mycobacterial infection
model a timeline of progression of infection could reach to weeks
post-infection (Cronan and Tobin, 2014;Hammarén et al., 2014).
Also in zebrafish embryos and larvae, P. aeruginosa requires higher
dose of pathogens to establish a virulent infection because many
of these pathogens are killed by macrophages and neutrophils
(Brannon et al., 2009).
Caenorhabditis elegans AS A NON-VERTEBRATE MODEL FOR
STUDYING OF LUNG BACTERIAL AGENTS
In 1963 Sydney Brenner proposed the nematode C. elegans as
an experimental organism for pursuing research in developmen-
tal biology and neurology (Ankeny, 2001). C. elegans has fewer
than 1000 body cells when completely grown. C. elegans possesses
remarkable advantages that make it an ideal model, for example,
low cost, simple growth conditions, and a short generation time
with an invariant cell lineage. By other way, at present, there are
many molecular and genetic methods for its manipulation (Kurz
and Ewbank, 2000). The genome of C. elegans was completely
sequenced at the end of 1998 (Schulenburg and Ewbank, 2007). In
comparison with mice, is obvious that culture and maintenance of
C. elegans is far simpler and cheaper. Besides, ethical regulations
are practically absent for experimentation with worms. Regarding
to cell cultures, C. elegans contaminated stocks are easily identified
and cleaned better than mammalian contaminated cells (Hulme
and Whitesides, 2011).
Fascinatingly, this worm shares similarity to mammalian
immune system, particularly signaling cascades in innate immu-
nity in response to pathogen invasion (Alper et al., 2008). Because
nematodes consume microorganisms as their food source, there
is presumably selection pressure to evolve and maintain immune
defense mechanisms (Kim et al., 2002;Alper et al., 2010;Shivers
et al., 2010). While the human lung and the nematode intestine
clearly differ in several anatomical, cellular and biochemical char-
acteristics, in the nematode intestine it is possible to identify
pathogen-specific virulence factors that interact with epithelial
surfaces. To reinforce the wide field of applications of such non-
vertebrate models, the study carried out by Green et al. (2009)
describe, despite of the absence of lungs in the nematode, a practi-
cal model to evaluate the impact of the cigarette smoke exposure on
innate immunity. In this study, C. elegans responded to nicotine,
which is one of the main components of cigarette smoke, con-
verting nicotine to cotinine, in a similar manner to mammals and
opening the path to demonstrate that the animals are absorbing the
smoke. In this model, C. elegans was subjected to whole cigarette
smoke exposure, overcoming several aspects impossible to eluci-
date using human epithelial cell lines, showing down-regulation in
many genes in response to the smoke, mainly several host defense
genes (Green et al., 2009).
Additional advantage of using nematodes as animal model
of bacterial infection lies in reduced experimental time without
the need of animal acclimation before infection, and time-kill
curves taking only few hours (fast killing) or days (slow killing)
post-infection depending on the killing mechanism (Figure 1).
However, nematodes need to be synchronized about 1 week prior
to infection to reduce the variation in results associated to dif-
ferences of age. There are two methods to synchronize worms:
egg preparation via bleaching and egg lay (Sulston and Hodgkin,
1988). The first method produces more progeny than the latter;
however, egg lay can generate a better synchronized population.
Scientists take also advantage of temperature-sensitive sterile C.
elegans mutants (e.g., the CF512 strain) to avoid production of a
brood that would complicate the scoring of death of the infected
worms. These animals do not produce progeny at working tem-
perature (25◦C), thus simplifying the procedure. However, these
strains appear to be slightly more resistant to bacterial infections
(Feinbaum et al., 2012).
More than 40 human pathogens, or their close relatives, are
known to cause disease in C. elegans (Sifri et al., 2005). P. aerug-
inosa was the first broad host-range pathogen, able to kill C.
elegans (Tan et al., 1999a,b). C. elegans infection process has
the advantage to closely resemble chronic infection, because the
host is usually exposed to maximal dose of pathogen. On the
other hand, among killing mechanisms, slow killing involves
a process similar to those related to an infection process. At
present, there are five distinctive mechanisms of worm killing
identified: infection with intestinal colonization, persistent infec-
tion, invasion, biofilm formation, and toxin-mediated killing
(Smith et al., 2002;Beale et al., 2006;Dunbar et al., 2012). Obvi-
ously, in the C. elegans model will be possible to study only
those human diseases caused by pathogens able to infect the
nematodes. In addition to the bacterial species, the killing mech-
anism is in most cases dependant to the way the bacterium is
prepared prior to infection. For instance, depending on the com-
position of the agar medium where the bacterium is grown,
the rate of P. aeruginosa-mediated killing of C. elegans will be
different (Tan et al., 1999a). If the P. aeruginosa tested strain
is grown on minimal medium, the killing will occur in the
course of several days; by the contrary, if a high-osmolarity
medium is used, the killing will occur in the course of several
hours.
INSECT AS MODELS OF INFECTION
In opposite to mammals, the respiration process in insects occurs
by a network of tubes called tracheae and tracheoles. Despite this,
insects have recently been shown to be a valuable alternative to
animal models for bacterial pathogenesis studies. This is mainly
due to the fact that insects have a relatively advanced system of
antimicrobial defenses. Like mammals, insects possess a complex
innate immune system and display evolutionary conservation of
signaling cascades (Lemaitre and Hoffmann, 2007). In addition,
as with other non-vertebrate models, the advantages are the low
cost of maintenance and no ethical concerns. There are multiple
genetic and molecular tools available. By other way, these models
have a precise endpoint. The low cost of maintenance and the rapid
development, allow their use in high animal number for proper
statistical analysis of results. Among these models, the common
fruit fly D. melanogaster and the larvae of G. mellonella,havebeen
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López Hernández et al. Lung pathogens alternative infection models
shown to be relevant for several fungal and bacterial mammalian
pathogens (Limmer et al., 2011;Sprynski et al., 2014).
Most insects have a very rapid life cycle, which consists of
four clearly defined stages: the embryo, the larva, the pupa,
and the adult. In addition, insect rearing is easy and relatively
cheap (Ramarao et al., 2012). For larva, drugs can be administered
directly injecting the organism or mixing with media (solid or
liquid with 2% yeast paste). For adults, drugs may be delivered
as aerosol, mixed with food, injected or applied directly to the
nerve cord. In the injection method, a needle or a nanoinjector
preloaded with pathogen culture is used to prick the body cav-
ity (insect hemocoel). Injection requires anesthetization, which
is usually done with carbon dioxide, and requires the transfer of
insects into vials containing food, where the worms incubated at
25–30◦C and their survival is evaluated (Igboin et al., 2012). For
ingestion, it is common to introduce the insects into small labora-
tory tubes containing filter disks embedded with media containing
pathogens of interest.
Galleria mellonella larvae are cost effective, widely available and
the results can be obtained within 2 or 3 days (Figure 1). There
are three main ways in which Galleria fight bacterial infections:
circulating phagocytic hemocytes that patrol the hemolymph;
proteolytic cascades that can be quickly triggered, activating the
melanization response and inducing antimicrobial immune effec-
tors such as lysozymes, as well as antimicrobial peptides which can
be rapidly synthesized by the fat body (Yuen and Ausubel, 2014).
An added benefit of using Galleria for pathogenesis studies is that
infections can be carried out at 37◦C or higher, as Galleria tolerates
relatively high temperatures, unlike the Zebrafish,D. melanogaster
and C. elegans (maximum 25–28◦C; Glavis-Bloom et al., 2012).
The larger size of the Galleria larva, compared to other inverte-
brate models, also allows it to be infected with larger and more
controlled doses of the pathogen without significantly traumatiz-
ing the insect. Some disadvantages of the G. mellonella model rely
in the fact that genetic methods for generating recombinant organ-
isms and to sequence them are not completely available. However,
this model could be improved in the next years and hopefully,
could be used for more pathogens, for which no alternative models
of infection exist.
FROM CLASSIC TO ALTERNATIVE MODELS IN STUDYING
RELEVANT BACTERIAL LUNG PATHOGENS
The most common causes of bacterial lung infections in nor-
mal human hosts include Streptococcus pneumoniae,Haemophilus
influenzae and Staphylococcus aureus, and the recent increase of
M. tuberculosis. Pneumonia is classified according the source of
infection into community-acquired pneumonia (CAP), hospital-
acquired or nosocomial, aspiration of foreign material and
immunocompromised host (Woodhead, 2013). In basis of their
presentation, pathogens have been classified into “typical” and
“atypical.” Typical organisms in CAP include S. pneumoniae,
H. influenzae,S. aureus,Moraxella catarrhalis, and P. aerugi-
nosa (Musher and Thorner, 2014). Atypical organisms include
Legionella species, Mycoplasma pneumoniae, Burkholderia spp.,
Chlamydia spp., Chlamydophila spp.,Coxiella burnetii and viruses
(Jones, 2010). In almost all epidemiological studies of hospital-
acquired pneumonia (Jones, 2010;Barbier et al., 2013;Polverino
et al., 2013;Erdem et al., 2014), a consistent six organism groups
(S. aureus,P. aeruginosa,Klebsiella species, Escherichia coli,
Acinetobacter species, and Enterobacter species) caused ∼80% of
episodes, with lower prevalences of Serratia species, S. maltophilia,
and community-acquired pathogens, such as pneumococci and
H. influenzae. In compromised hosts, the bacterial causes of
pneumonia are much broader, including species not usually
considered of high virulence in humans. For instance, Mycobac-
terium sp.,Burkholderia spp.,P. aeruginosa, and S. aureus are the
most important infectious agents in cystic fibrosis (CF) patients
(Coutinho et al., 2008).
For the most frequent bacteria causing pneumonia, scien-
tists have developed animal models of infection, mainly using
mice (Figure 2 and Tab l e 1 ). However, the introduction of
alternative non-mammalian models is still at its beginning and
obviously for host-permissive pathogens the contribution would
be higher. For instance, despite several mouse M. tuberculosis
lung infection models are utilized, and Mycobacterium marinum
infection of fishes results in chronic granulomatous diseases sim-
ilar to mycobacterioses in mammals (Cosma et al., 2006b), C.
elegans, a well-established model host, is resistant to mycobac-
terial infection (Couillault and Ewbank, 2002). The most extreme
example is that of pathogens for which there are no or very
few alternative models of infection, such as H. influenzae,M.
catarrhalis, and M. pneumoniae (Figure 2). On the other side
as will be discussed later, P. aeruginosa, a versatile and ubiqui-
tous bacterium, is capable to survival and colonize various living
host organisms facilitating the development of infection mod-
els spanning from nematodes to small vertebrates (Figure 2).
Here, we discuss the models that have been developed for study-
ing most common human lung pathogens by comparing the
mouse model with alternative ones in zebrafish, nematodes, and
insects.
ANIMAL MODELS OF PNEUMOCOCCAL INFECTION
Streptococcus pneumoniae is considered the most common bac-
terial agent in CAP and a great number of animal models of
pneumococcal diseases are available. Some review articles from the
past decade have specifically mentioned the value of animal mod-
els to test pneumococcal vaccines (Briles et al., 2000;Adamou et al.,
2001;Bogaert et al., 2004;Kadioglu and Andrew, 2004;Morsczeck
et al., 2008) and the use of murine models of pneumonia to eval-
uate protein-mediated antimicrobial responses (Srivastava et al.,
2007), the mouse genetic susceptibility to pneumococcal disease
(Proft and Fraser, 2003) and for investigating the pathophysiology
of bacterial meningitis (Teles et al., 1997;Gerber et al., 2001;Blair
et al., 2005;Endo et al., 2012).
On the other hand, as there are several evidences about the
positive correlation between infection with Streptococcus iniae
and Streptococcus pyogenes in zebrafish and mammalian mod-
els, there is simple to assume the use of zebrafish to evaluate
host–pathogen interactions during pneumococcal infection (Borst
et al., 2013). In a recent work, Rounioja et al. (2012) reported
the use of zebrafish embryo to evaluate the response of the host
immune system against challenge with pneumococci. They also
reported that this response is dependent on whether the pneumo-
cocci could evade clearance by interfering with host phagocytic
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López Hernández et al. Lung pathogens alternative infection models
FIGURE 2 |Comparative number of publications for different lung
pathogens employing mice, Caenorhabditis elegans, zebrafish, and
insects as animal models of infection.The number of publications is shown
between parentheses. A systematic search was conducted using PubMed
(http://www.ncbi.nlm.nih.gov/pubmed). Literature searches were conducted
to identify studies published until 1 November, 2014.
function. Moreover, pneumococcal mutants defective in impor-
tant virulence factors were attenuated in this in vivo model
system (Rounioja et al., 2012). The adult zebrafish model can be
also used to investigate pneumococcal diseases (Saralahti et al.,
2014). The authors showed that S. pneumoniae mutants defective
in polysaccharide capsule are also attenuated and that elimina-
tion of pneumococci depends mainly on host innate immune
responses.
To our knowledge, little is the information available up to
day related to the use of C. elegans to study pathogenesis of
Streptococcus.Garsin et al. (2001,2003), in two separate works,
demonstrated the suitability of using C. elegans as a model host
for Gram-positive infection, including Enterococcus faecalis,S.
aureus, and S. pneumoniae.Jansen et al. (2002) demonstrated that
S. pyogenes kills C. elegans, both on solid and in liquid medium,
mediated by the hydrogen peroxide production (Jansen et al., 2002;
Bolm et al., 2004).
The potential application of the larvae of G. mellonella as an
informative infection model for S. pneumoniae has been also stud-
ied (Evans and Rozen, 2012) since strains differing in known
virulence factors could be distinguished in this host. Strains lack-
ing capsule or pneumolysin showed less virulence than their
respective wild types. Particularly, pneumolysin plays a role in
damaging human lung epithelium, allowing the establishment of
infection (Rayner et al., 1995).
ANIMAL MODELS OF STAPHYLOCOCCAL INFECTION
As nasal colonization is a main requisite for the establishment of
S. aureus infection in humans (Baur et al., 2014), mice infected
by using this route is a useful model for characterizing early
host responses. However, this model has failed in mimic the
whole natural route of infection, resulting in self-limited dis-
ease (Alami et al., 1968;Bartell et al., 1968;Anatoli˘
Iet al., 1971;
Ansfield et al., 1977;Bragonzi et al., 2004;Bloemendaal et al.,
2011). The Bolus infection models, where mice are challenged
by i.t. and i.n. inoculation have been more successful in produc-
ing intrapulmonary infection and host mortality (Jakab, 1976;Hu
et al., 2006;Rajagopalan et al., 2006;Huzella et al., 2009;Fernandez
et al., 2011;Park et al., 2011). Sawai et al. (1997) described a murine
model of acute staphylococcal pneumonia inoculating mice by
intravenous (i.v.) injection of a suspension containing bacteria
enmeshed in agar-beads. This model allows the bacteria to remain
in the lung for several weeks, and it is reproducible and simple
(Sawai et al., 1997).
Li and Hu (2012) developed a zebrafish embryo infection
model with S. aureus at 36 h post-fertilization. These researches
inoculated the bacteria directly into the pericardial cavity, eye,
and yolk body. By using GFP-expressing S. aureus and trans-
genic zebrafish lines along with multicolored confocal fluorescence
methods, they could analyze different phases of bacterial infec-
tion. As important conclusion from this work, the dynamic of
infection clearly depends on the bacterial entry routes (Li and
Hu, 2012). Prajsnar et al. (2008), using a similar model, identi-
fied staphylococcal virulence genes, whose respective mutants are
attenuated in zebrafish. The virulence factors include a peroxide
regulon repressor, a protein involved in starvation survival and a
response regulator involved in controlling exoproteinsproduction.
They also demonstrated that in zebrafish embryos, macrophages
phagocytize S. aureus during in vivo infections. Accordingly to
Kubica etal. (2008), these immune cells act as a reservoir dur-
ing infection. This model in both zebrafish embryos and adults
also allowed rapid screening of mutants for those strains with
attenuated pathogenicity, identifying relevant factors of pathogen
virulence and host immunity (Lin et al., 2007;Li and Hu, 2012;
Lü et al., 2012).
On the other hand, several studies used the C. elegans model
to assess virulence levels between some different methicillin-
resistant S. aureus strains, demonstrating the suitability of this
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López Hernández et al. Lung pathogens alternative infection models
model for studying the virulence and pathogenicity of S. aureus
strains (Wu et al., 2010,2012,2013). In addition, several S. aureus
virulence determinants recognized as important in mammalian
pathogenesis are also identified as relevant for full pathogenicity
in nematodes, including agr (a quorum-sensing global virulence
regulatory system), sarA (global virulence regulator), the alter-
native sigma factor B, alpha-hemolysin, and V8 serine protease
(Sifri et al., 2003,2006). However, Polakowska et al. (2012) showed
that there is no substantial variation in virulence among different
staphylococcal strains using this experimental model, questioning
the usefulness of it.
In another work, JebaMercy et al. (2011) performed solid and
liquid assays for the infection of C. elegans with S. aureus, demon-
strating that S. aureus took ∼90 h for the complete killing of C.
elegans and thereby postulating that colonization with live bacte-
ria was necessary for worm killing. Using an interactive genetic
approach, Begun et al. (2007) established a novel in vivo exper-
imental model to explain the interaction between the bacteria
biofilm matrix and components of the innate immune system.
This study demonstrates the ancient conserved function against
predation linked to the protective activity of biofilms. In another
work based on C. elegans infections, the same authors identi-
fied staphylococcal genes relevant for mammalian pathogenesis,
including the product of the nagD gene, which was not previously
described as a virulence factor (Begun et al., 2005).
Larvae of the greater wax moth also have provide insights into
the pathogenesis of S. aureus, principally as a suitable host for test-
ing the in vivo efficacy of antimicrobial agents (Gao et al., 2010;
Desbois and Coote, 2011;Gibreel and Upton, 2013;Apolónio
et al., 2014). In addition, using this models authors have demon-
strated for the first time that both glycolysis and gluconeogenesis
have important roles in virulence (Purves et al., 2010). Their
results showed that two glyceraldehyde-3-phosphate dehydroge-
nase (GAPDH) homologs (GapA and GapB) are required for the
virulent phenotype of S. aureus in this model. In S. pyogenes
surface-associated GAPDH was associated with antiphagocytic
properties and host cell adherence (Boël et al., 2005).
ANIMAL MODELS FOR MYCOBACTERIAL PATHOGENESIS
Due to the complex interaction between the pathogen and the
host, it has been very difficult to find out an ideal model to
study mycobacterial pathogenesis. Some mice strains can be easily
infected via aerosol with a low dose of M. tuberculosis, multiplying
in the lungs and subsequently spreading to liver and spleen. The
infection is controlled but not eliminated, by cell-mediated immu-
nity, mainly T cell responses, and the infection is well-tolerated for
more than 1 year (Beamer and Turner, 2005;Aguilar et al., 2007,
2010;Andreevskaia et al., 2007). Hence, the mouse model has been
largely a suitable infection model (Orme, 2005b;Cooper, 2014).
Surprisingly, there is still no ideal and validated model of exper-
imental tuberculosis disease (Vilaplana and Cardona, 2014), and
the mechanisms leading to latency and reactivation of are still
unclear (Parikka et al., 2012).
BALB/C mice infection models by i.t. injection using a high
dose of bacilli (Hernández-Pando et al., 1996) is one of the most
employed models. This model has greatly contributed to eluci-
date the role of antibodies in the protection against mycobacterial
infections (López et al., 2009,2010;Alvarez et al., 2013), and to
the screening and validation of new vaccine candidates (Castillo-
Rodal et al., 2006;López et al., 2006). Alternatively, C57BL mice
have been infected i.n. via aerosol with a low dose of M. tuber-
culosis, which produces a well-tolerated infection dominated by
Th1 response (Kelly et al., 1996;Cardona et al., 2003;Aldwell
et al., 2005;Saini et al., 2012). For this reason, this is actu-
ally a model of slow progressive disease, and the animal death
is produced by excessive inflammation or immunopathology
(Mustafa et al., 1999a,b,2000). The model of latent tuberculo-
sis has been also partially reproduced experimentally in mice
(Phyu et al., 1998;Shi et al., 2011;Zhang et al., 2011)whichare
injected intratracheally with relatively low numbers of the viru-
lent strain H37Rv (Hernandez-Pando et al., 1998). After that, low
and stable bacillary counts with few granulomas appear and the
mice continue gaining weight and appear healthy for more than
2 years.
Although mice are the most employed animal model for study-
ing human TB, it has important drawbacks. Due to the fact that
M. tuberculosis is not a natural pathogen of mice, the pathological
development of TB will be clearly different from that in human. As
relevant characteristic, we can mention the absence of granulomas
formation in lungs from mice (Grumbach et al., 1967;Karakousis
et al., 2004;Mollenkopf et al., 2004;Orme, 2005a,b;Hunter et al.,
2007;Dharmadhikari and Nardell, 2008;Young, 2009;Apt, 2011).
The immune response elicited in mice after mycobacterial infec-
tion is able to control bacillary load even without causing marked
lesions (Vilaplana and Cardona,2014). Therefore, mice are gener-
ally resistant to TB infection when compared with other rodents,
and even humans, as evidenced by their ability to tolerate relatively
large bacterial numbers within their lungs without signs of disease
(Be et al., 2008;Dharmadhikari and Nardell, 2008;De Steenwinkel
et al., 2009). Also, ethically, these models in conjunct appear to be
more aggressive to the mice.
Zebrafish and fruit fly are emerging as alternative models and
have provided new insights into the pathogenesis of the tuber-
culosis disease. By the contrary, C. elegans, to our knowledge,
seems to be not a feasible model for infection with M. tubercu-
losis. The zebrafish has been a key model in our understanding
of mycobacterial infection. Studies on this model employ a fish
pathogen, M. marinum, a close relative to M. tuberculosis (Tob i n
and Ramakrishnan, 2008). This bacterium is a natural pathogen
of fish and amphibian (van der Sar et al., 2004a,b). The M. mar-
inum infection produced in these hosts is quite similar to those
produced in humans, mainly in the granuloma formation (Prouty
et al., 2003;Berg and Ramakrishnan, 2012). Low doses (<100 bac-
teria) of M. marinum lead to a chronic infection in adult zebrafish
(Parikka et al., 2012), while higher doses cause a fatal acute infec-
tion (Cosma et al., 2006b). Besides, M. marinum grows faster
than M. tuberculosis, it can be easily manipulated and requires
only common laboratory precautions (biosafety level 2). The
zebrafish/M. marinum infection model changed the old concep-
tion that granuloma formation requires lymphocytes and by the
contrary, postulated that the granuloma actually functions as a
bacterial tool for disseminating the disease.
Experiments conducted by Ramakrishnan and colleagues using
the zebrafish/M. marinum infection model (Davis et al., 2002;
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López Hernández et al. Lung pathogens alternative infection models
Davis and Ramakrishnan, 2009;Ramakrishnan, 2013), demon-
strated for the first time new mechanisms of bacterial dissemi-
nation: the bacterial transfer between two macrophages through
membrane tethers and re-phagocytosis of bacteria associated with
dead macrophages in tissues. They also observed that, as early
as 72 h post-injection, the extravasated infected macrophages
began to form granuloma-like aggregates in the tissues, establish-
ing that there is not needed the participation of adaptive immunity
to initiate granuloma formation (Davis et al., 2002). Using this
model, and taking advantages of the powerful live imaging of
the zebrafish model, it was determined that an efficient bacte-
rial expansion depends on the mycobacterial region of deletion
1 (RD1) locus. The researchers also demonstrated that the bac-
terial protein ESAT6 elicits the expression of metalloproteinase 9
(MMP9) in the host, and both proteins act together for granuloma
formation (Pozos et al., 2004;Volkman et al., 2004;Cosma et al.,
2006a;Berg and Ramakrishnan, 2012;Takaki et al., 2013;Cam-
bier et al., 2014). These findings showed that one protein from the
host and one from the bacteria, constitute a virulence axis which
evade the host’s early immune responses and lead to mycobac-
terial dissemination. This study is, from our point of view, the
most important evidence of how the zebrafish model can be used
to validate and to re-postulate host–pathogen interactions during
mycobacterial infection.
On the other hand, Swaim et al. (2006) reported that lym-
phocytes play the same critical role in controlling mycobacterial
infection in fishes and mammals by the use of a defective zebrafish
mutant in the rag1 gene. They also demonstrated that bacte-
ria defective in RD1 region are also attenuated in zebrafish. In
addition, the zebrafish/M. marinum model has proven to be
useful for studying the latency, dormancy, and reactivation of
latent or subclinical tuberculosis (Parikka et al., 2012). This group
has recently studied using this model the T cell responses in
mycobacterial infection and they have found associations between
the disease severity (bacterial load) and the type and magni-
tude of T cell responses, particularly an adequate Th2-type
response (Hammarén et al., 2014). This infection model also
helped to demonstrate that mycobacterial antigensAg85B, CFP-10,
and ESAT-6 protect adult zebrafish from mycobacterial infection
(Oksanen et al., 2013), paving a new way in tuberculosis vaccine
research.
Mycobacterium marinum also causes a lethal infection in the
fly D. melanogaster characterized by a widespread tissue dam-
age, even at significant low bacterial doses (Dionne et al., 2003).
These initial stages of the infection were very similar to the early
stages in frogs and fishes infected with M. marinum (Pozos et al.,
2004). This model may be valuable in testing the activity of new
antimycobacterial agents (Oh et al., 2013).
ANIMAL MODELS TO STUDY THE VIRULENCE FACTORS OF
Pseudomonas aeruginosa
Pseudomonas aeruginosa is an opportunistic human pathogen
that can also infect several diverse organisms, such as plants,
nematodes, insects, and mammals. Thus, we counted on good
mammalian and non-mammalian models for studying virulence
factors of P. aeruginosa. In humans, P. aeruginosa is widely associ-
ated with nosocomial infections in CF patients (Moreau-Marquis
et al., 2008) and other immunocompromised individuals, and the
resolution of the infections is hampered due to the formation
of drug resistance biofilms. Until the moment, a difficult exists
on studying biofilms formation in the context of animal and
human lungs. However, several non-mammalian models have pro-
vided compelling data regarding P. aeruginosa biofilm formation
(reviewed in Lebeaux et al., 2013).
Acute and chronic models of lung P. aeruginosa infection have
been developed using several mammalian species (rats, guinea
pigs, hamsters, mice, sheep, rabbits, and baboons; Seidenfeld
et al., 1986;Starke et al., 1987;Pier et al., 1990;Collins et al., 1991;
Iwata and Sato, 1991;Hart et al., 1993;Terashima et al., 1995;
Bakker-Woudenberg et al., 2002;Luna et al., 2007,2009;Ku ra-
hashi et al., 2009;Rodríguez-Rojas et al., 2009;Collie et al., 2013).
The chronic infection model has been extensively used and charac-
terized, showing certain similarities with human pathology due to
the persistence of the inoculum and the resultant lung pathology
(van Heeckeren and Schluchter, 2002). Depending on the route,
dose administered, and the frequency of dosing, acute lung infec-
tion with either rapid clearance of the bacteria or acute sepsis and
death could take place (George et al., 1991). Using this model, it has
been shown that P. aeruginosa must express several key virulence
factors (Balloy et al., 2007).
A literature survey about acute vs. chronic P. aeruginosa lung
infections clearly shows that to induce an infection for more than
1 month, it is necessary to use an immobilizing agent such as
agar, agarose, or seaweed alginate together with the bacterial sus-
pension (Iwata and Sato, 1991;Hart et al., 1993;McMorran et al.,
2001;Moser et al., 2002). The initial agar-beads model of chronic
pulmonary infection with P. aeruginosa was modified for its use in
mice by Starke et al. (1987) and has been widely used to study CF
lung disease, bacterial pathogenesis (Gosselin et al., 1995,1998;
Morissette et al., 1995;Stevenson et al., 1995;Moser et al., 1997;
Sapru et al., 1999;Tam et al., 1999;McMorran et al., 2001), and
for the evaluation of new therapies (Pier et al., 1990;Staczek
et al., 1998;Chmiel et al., 1999;Wilmott et al., 2000) and viru-
lence factors (Rodríguez-Rojas et al., 2009). Researchers have also
developed a mouse model with a lung pathology similar to human
CF. One group generated mice that absorb excess sodium in the
airways (Mall et al., 2004;Mall, 2008) and these animals devel-
oped airway obstruction with dehydrated mucus. This CF model
promises to answer important questions about the cause of the
inflammation that leads to lung damage and failure in CF (Mall
et al., 2004;Mall, 2008). Both acute and chronic models require
extensive use of animals and labor-exhausting techniques to pre-
pare and immobilizing bacteria in agar, as well as good surgery
skills.
Brannon et al. (2009) developed a zebrafish embryo infection
model for the study of systemic P. aeruginosa infection, and for
evaluating the virulence of a type 3 secretion system (T3SS)
mutant. By fluorescence microscopy it was possible to follow
in real time P. aeruginosa infection in transgenic zebrafish with
fluorescently labeled neutrophils and macrophages (Hall et al.,
2007). Clatworthy et al. (2009) demonstrated that lethal infec-
tion requires quorum-sensing and the T3SS for full virulence in
late-stage zebrafish embryos infected with P. aeruginosa. Curi-
ously, T3SS expression has been associated also to increased risk
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López Hernández et al. Lung pathogens alternative infection models
of P. aeruginosa infection in hospitalized patients (Ledizet et al.,
2012) and it is also associated with initial infections in patients
with CF (Jain et al., 2008). They demonstrated that the infection
outcome could be influenced on the pathogen side, by both the
inoculum size and the presence of known virulence determinants
(lasR, mvfR, and psc D) and on the host side by developmental
stage and the modulation of the immune system (Clatworthy et al.,
2009). They also showed that the infection process can be mod-
ified through the use of morpholinos or antibiotics, which were
used to shift immune cell numbers or rescue embryos from lethal
challenge respectively.
In one study performed by Phennicie et al. (2010) in zebrafish,
the role of the cystic fibrosis transmembrane conductance regu-
lator (CFTR) in the innate immune response to acute infection
with P. aeruginosa was evaluated. The authors found that the P.
aeruginosa bacterial load was significantly higher in cftr morphants
(knockdown of the zebrafish ortholog to human cftr ) than in con-
trol embryos, according with similar studies performed with mice
and human bronchial epithelial cells.
The P. aeruginosa-zebrafish infection model has allowed con-
ducting chemical screens for small molecules or antimicrobial
compounds. For instance, the treatment of infected embryos
with front line antipseudomonal agents could save zebrafish
embryos from a lethal P. aeruginosa challenge (Clatworthy et al.,
2009). More recently, Ruyra et al. (2014) reported the use of
immunostimulant-loaded nanoliposomes to protect adult fishes
against bacterial or viral infections. In a model of adult zebrafish
infection developed by these researchers, nanoliposomes protected
zebrafish against otherwise lethal bacterial (P. aeruginosa PAO1)
and viral (SprinViraemia of Carp Virus) infections.
Tan et al. (1999a,1999b) conducted several studies (Tan and
Ausubel, 2000;Tan, 2002) showing for the first time the use of C.
elegans in the study of P. aeruginosa pathogenesis. They demon-
strated that accumulation of P. aeruginosa cells in intestines is
crucial to explain the killing mechanism. In addition, as other
authors have demonstrated, they showed that bacterial genes
required for this killing were also described in mammalian or plant
hosts pathogenesis. Feinbaum et al. (2012) screened mutants with
reduced ability to kill C. elegans using a mutant library represent-
ing ∼80% of the non-essential P. aeruginosa PA14 genes. They
described a set of 180 P. aeruginosa genes necessary for normal
levels of virulence. The principal contributors to P. aeruginosa
virulence in the C. elegans infection model were genes that play
key roles in survival of P. aeruginosa within the host intestine,
particularly regulatory genes that are involved in quorum-sensing
(Feinbaum et al., 2012).
Insects have been also surrogate model systems for identifying
mammalian virulence factors of P. aeruginosa. Previous studies
showed that this bacterium is a virulent pathogen of fruit flies
(Boman et al., 1972). Using the D. melanogaster as model host,
D’Argenio et al. (2001) have identified mutants of P. aeruginosa
with reduced virulence. Among these mutants, the pil-chp signal
transduction system is particularly relevant also in mammals and
is involved in type IV pilus synthesis and biofilm formation (Kato
et al., 2008). The D. melanogaster model allowed in vivo study of
P. aeruginosa biofilm infections by oral administration (Mulcahy
et al., 2011). By the other hand, several studies summarized by
Jander et al. (2000) point out similarities between virulence of
P. aeruginosa mutants in mice and G. mellonella. This infection
model helped to demonstrate that human anti-microbial peptides
that inhibited the initial steps in biofilm formation could be used
in the development of new therapies for P. aeruginosa infection
(Dean et al., 2011).
ALTERNATIVE MODELS FOR TWO LESS COMMON BACTERIAL LUNG
PATHOGENS
Previous works suggest a limited invasiveness of Stenotrophomonas
maltophilia in mice, as indicated by a transient and minimal
presence of the bacteria in animal organs after infection. S. mal-
tophilia CF strains were shown to cause no mortality in a neonatal
mouse model of respiratory tract infection (Waters et al., 2007).
Despite this lack of strong invasiveness, mouse models of S. mal-
tophilia infection have been useful answering questions about
immune response against this pathogen (Brooke, 2012). Addi-
tionally, a model of acute respiratory infection in DBA/2 mice
inoculated with aerosolized S. maltophilia has allowed the study
of lung pathology and the mechanisms of infection resolution
(Di Bonaventura et al., 2010). However, in this model, most of
the animals were able to control the infection in a short time
period, even at high doses of virulent inoculums, being the ani-
mal weight the best criterion to evaluate the virulence of tested
strains (Di Bonaventura et al., 2010;Pompilio et al., 2011). One
study also showed bacterial colonization in rat lungs after 7 days
post-infection (McKay et al., 2003).
S. maltophilia has also been isolated from channel catfish
(Ictalurus punctatus) with infectious intussusception syndrome
(Geng et al., 2010), suggesting that the use of fish as a model to
evaluate the pathogenicity and susceptibility of S. maltophilia to
available antimicrobial agents is adequate. Recently, a model of
intraperitoneal infection in zebrafish confirms the attenuation of
aS. maltophilia collection strains when compared with recent clin-
ical isolates (Ferrer-Navarro et al., 2013), paving the way for new
approaches to gain relevant information on pathogenesis of this
bacterium. An infection model using C. elegans has been proposed
for routine screening of S. maltophila isolates for pathogenesis
(Thomas et al., 2013). In this work the in vivo killing efficiency
was evaluated by four different methods: classical fast killing assay,
filter-based fast killing assay, slow killing assay and virulence assay
using heat inactivated bacteria. Moreover, virulence regulation in
S. maltophilia mediated by a quorum-sensing system has been
recently studied in the C. elegans and zebrafish infection models
(Huedo et al., 2014). In that work, it has been demonstrated that
S. maltophilia inoculated by intraperitoneal route in zebrafish is
characterized by rapid body dissemination. By the other hand, one
study using the insect G. mellonella suggests the proteolysis as a
possible pathogenic mechanism in S. maltophilia isolates from CF
infections (Nicoletti et al., 2011).
On the other hand, respiratory pathogens like Burkholderia
pseudomallei has the same type of tropism in mice than that
observed in humans, regardless of its acute or chronic output
(Stundick et al., 2013). Modeling of experimental melioidosis has
been conducted in numerous biologically relevant models includ-
ing mammalian and invertebrate hosts (reviewed in Warawa,
2010). Non-mammalian models have been explored since the
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López Hernández et al. Lung pathogens alternative infection models
mechanisms of Burkholderia virulence may be conserved during
evolution from worms to mammals. Drosophila and G. mellonella
have also shown to be useful alternative infection models for
Burkholderia spp. (Castonguay-Vanier et al., 2010;Wand et al.,
2011;Pilátová and Dionne, 2012). Particularly, Burkholderia thai-
landensis is highly virulent in the fruit fly (Pilátová and Dionne,
2012), a closely related organism to B. pseudomallei known to be
avirulent in humans, thus being a useful model for mammalian
melioidosis.
Data shown by O’Quinn et al. (2001) suggest that the disease
phenotype observed in nematode after exposure to B. pseudo-
mallei may be also valuable for investigating the pathogenesis of
these bacteria. Burkholderia species are able to cause ‘disease-like’
symptoms and kill the nematode C. elegans either by infection or
intoxication (O’Quinn et al., 2001;Darby, 2005) or suppressing
worm immunity by specific degradation of a GATA transcrip-
tion factor (Lee et al., 2013). The study of O’Quinn et al. (2001)
suggests that the neuromuscular intoxication caused by B. pseu-
domallei is related to a signal transduction mechanism involving
calcium. It is well-known that bacterial toxins can increase the
content of free calcium (Ca2+) in the cytosol of the host (TranVan
Nhieu et al., 2004). In this sense, calcium acts as a second messen-
ger in several physiological processes and immune mechanisms.
The common respiratory bacterial pathogens, P. aeruginosa and
S. aureus, activate Ca2+fluxes after contact with y epithelial cells
from the respiratory tract, activating proinflammatory signaling
events (Ratner et al., 2001). The Ca2+fluxes mediate the expres-
sion of proinflammatory cytokines and chemokines necessary to
recruit leukocytes to the lung and also to initiate modifications
in the epithelial junctions to facilitate leukocyte transmigration
into the airway lumen (Chun and Prince, 2009). The C. elegans
system has been used to screen for new virulence factors in B.
pseudomallei, and selected attenuated bacterial mutants were fur-
ther evaluated in an intranasal infection model in BALB/c mice
(Gan et al., 2002). The results in mice validate positively the use
and clinical relevance of C. elegans as an alternative model in the
screening of virulence factors in B. pseudomallei.
CONCLUDING REMARKS
The nematode was the first invertebrate alternative model
described, followed by the larvae and adult fruit fly models
(Drosophila sp.) and more recently, the wax moth larvae (G. mel-
lonella) model. Non-mammalian vertebrates, as fish and amphib-
ians, which are able to mount an adaptive immune response,
are now available as excellent tools. These kinds of models are
usually criticized as being too distant from human. Several limita-
tions such as their reduced complexity and the simplicity of their
immune system, differences in temperature, target organs, or par-
ticular receptors have impaired the use of these models. However,
these have been mentioned also for the murine models of other
diseases. No model is perfect, each one has its specific strengths
and weaknesses, but the most important thing is to combine the
information gained from one to other, taking advantages of the
incredible genomics and bioinformatics tools on-hand, before to
extrapolate to humans beings. The belief that vertebrates are nec-
essary the best available models in biomedical research was called
the high-fidelity fallacy (Stevens, 1992;Russell, 1995). To avoid
some of the limitations of these models, researchers begin to study
infection and immunity in non-mammalian models. Since our
knowledge of the immune system and their evolutionary conser-
vation has increased, the usefulness of alternative models different
for mammals has been accepted more. Although could seems irra-
tional to study lung diseases in animals who do not have lungs,
several evidences support the benefits that these studies, if carried
properly,may to bring in the elucidation of human lung pathology
diseases (Tab l e 1 ). Thus, these non-mammalian organisms have
been successfully employed to elucidate conserved and universal
immune mechanisms. In addition, the small size of the most used
non-mammalian organisms enables to perform high throughput
and automated studies (Letamendia et al., 2012). Most of these
model organisms have their genome completely sequenced, offer-
ing the possibility to do genetic studies both on the bacteria and
the host. And what we consider one of the most practical advan-
tages, the alternative models described here provide a way to easily
bypass the ethical limitations of some types of studies in higher
animal models.
What we do recommend to the scientific community facing the
design of experimental infection with bacterial pathogens? Firstly,
we have to consider several aspects related to the immunopathol-
ogy of the diseases that we want to reproduce in an animal model.
The relationship between the host and the guest in terms of molec-
ular interactions is crucial to determine which type of response we
will observe and consequently, to plan strategies for measuring
it. But, a question arises. Which, among the methods that we
will employ, are better in terms of cause less injury or damage to
the animal? Is this response only measurable in vertebrate animal
models? There are now sufficient evidences about the similarities
of non-vertebrates immune systems and mammals. By other way,
it has been demonstrated that the handling and maintenance of
non-mammals organisms is easier and shaper. It is common to
think in mice immediately when we are planning an in vivo exper-
imental infection. Certainly, it is the most employed animal model
in biomedical research. However, we could be aware about ethical
restrictions that the use of large amount of animals and certain
experimental procedures could implicate for the investigation. So,
we modestly recommend being in mind the possibility of consid-
ering the use of no vertebrate animal models, as worms or insect
as a first screening when it is reasonable. For the latter stages of
the research we may to use other mammalian models. However,
for certain lung diseases, conclusive points will arise more prop-
erly from the conjunction of one or more experimental studies
carrying on in different species.
AUTHOR CONTRIBUTIONS
Yamilé L. Hernández and Daniel Yero authored first draft of
manuscript with academic input and expertise provided by Isidre
Gibert and Juan M. Pinos-Rodríguez. All authors were involved in
reviewing manuscript and have approved the final version.
ACKNOWLEDGMENTS
The authors would like to acknowledge the significant review and
the many valuable suggestions made by Nerea Roher, head of the
group of Evolutive Immunology at IBB (Institut de Biotecnologia
i de Biomedicina) and professor at the Departament de Biologia
Frontiers in Microbiology |Infectious Diseases February 2015 |Volume 6 |Article 38 |12

López Hernández et al. Lung pathogens alternative infection models
Cellular, Immunologia i Fisiologia Animal, Universitat Autònoma
de Barcelona, Spain. Isidre Gibert and Daniel Yero acknowledge
support from the Generalitat de Catalunya AGAUR (2014-SGR-
1280) and Ministerio de Economía y Competitividad, Spain (grant
BFU2010-17199).
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 24 November 2014; accepted: 12 January 2015; published online: 04 February
2015.
Citation: López Hernández Y, Ye roD, Pinos-Rodríguez JM and Gibert I (2015) Animals
devoid of pulmonary system as infection models in the study of lung bacterial pathogens.
Front. Microbiol. 6:38. doi: 10.3389/fmicb.2015.00038
This article was submitted to Infectious Diseases, a section of the journal Frontiers in
Microbiology.
Copyright © 2015 López Hernández, Yero, Pinos-Rodríguez and Gibert. This is an
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ted, provided the original author(s) or licensor are credited and that the original
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No use, distribution or reproduction is permitted which does not comply with these
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