Microbes and Infection, 3, 2001, 1345−1352

© 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

S1286457901014964/REV

Use of mixed infections with Salmonella strains to

study virulence genes and their interactions in vivo
Carmen R. Beuzón, David W. Holden*
Department of Infectious Diseases, Imperial College School of Medicine, Flowers Building, Armstrong Road, London SW7 2AZ, UK

ABSTRACT – In the Salmonella–mouse model of systemic infection, high dose inoculation results in
the multiplication of many of the cells present in the inoculum, rather than the clonal amplification of a
small number. This characteristic has allowed the development of methods to screen multiple strains for
either virulence attenuation or gene expression within the same animal. Mixed infections with mutant
and wild-type strains are used to provide a sensitive measure of virulence attenuation referred to as the
competitive index. We have recently used a variation of this method, involving mixed infections of
single and double mutant strains, to study virulence gene interaction in vivo. © 2001 Éditions
scientifiques et médicales Elsevier SAS
competitive index / gene interactions / COI / STM / IVET

1. Introduction responds to changes in pH, osmolarity and temperature

Salmonella typhimurium encounters a diversity of envi-
ronments in the mouse throughout the course of systemic
infection. After oral inoculation and survival in the stom-
ach and intestine, it penetrates the gut epithelium by
invading the M cells in the Peyer’s patches of the ileum [1],
and reaches the spleen and liver where it multiplies intra-
cellularly [2, 3], eventually resulting in fatal bacteraemia.
S. typhimurium pathogenesis is a complex, multifactorial
process that results from the activity of many bacterial
gene products: it has been estimated that at least 4% of its
genome is involved in virulence [4]. Many of these genes
are clustered together on ’pathogenicity islands’ (PAIs)
which appear to have been acquired by horizontal transfer
from unknown sources. SPI-1 and SPI-2 are two PAIs that
encode structurally similar but functionally distinct type III
secretion systems (TTSS) which translocate virulence pro-
teins from bacterial to host cells during the infectious cycle
[5]. SPI-1 plays an important role in invasion of epithelial
cells [6, 7], whereas SPI-2 is required for bacterial replica-
tion within macrophages [8] and systemic growth in the
mouse [9, 10]. In addition to these, numerous other genes
have been shown to be necessary for S. typhimurium
virulence [11]. Many of them are involved in intracellular
survival and replication; these include the spv locus,
present on a large plasmid [12], the ompR/envZ genes,
which encode a two-component regulatory system that
*Correspondence and reprints.
E-mail address: d.holden@ic.ac.uk (D.W. Holden).
[13, 14], and the phoP/phoQ genes which encode another
two-component regulatory system that controls the expres-
sion of more than 40 virulence genes [15]. Although the
biochemical functions of some of these genes have been
elucidated, little is known about their regulation in vivo
and how they interact during the infectious process.
The study of interactions between bacterial strains and
cultured host cells has led to the identification of genes
involved in specific aspects of virulence (e.g., invasion of
epithelial cells [16]). However, the complexity of Salmo-
nella virulence obviously limits the use of this type of
analysis. In recent years there has been a shift towards
more sophisticated in vivo virulence studies, and some of
these rely on the use of mixed infections. The application
of mixed infections in the analysis of the Salmonella–
mouse interaction will be the subject of this review, with
particular emphasis on our recent application of the com-
petitive index to the analysis of gene interactions in vivo.
2. Use of mixed infections
in the analysis of Salmonella virulence
Intraperitoneal (i.p.) inoculations of mice with a mix-
ture of different Salmonella strains were first used more
than 40 years ago to study fundamental aspects of the
infection process. Meynell and Stocker [17] inoculated
mice with a mixture of S. paratyphi-B strains carrying one
of three different flagellar antigens, or a mixture of Xyl+
Gal+ and Xyl– Gal– S. typhimurium strains. All these strains
have a similar growth rate in vivo. They demonstrated that

Microbes and Infection 1345

2001, 1345-0
Forum in Immunology
at high dose inocula, the ratio between these strains in the
blood after infection was the same as their ratio in the
inoculum. They concluded from these results that if the
inoculum dose is sufficiently high, systemic S. typhimu-
rium infection of mice results from the multiplication of
many of the cells present in the inoculum, rather than the
clonal expansion of a small number. Therefore, mice can
support the growth of multiple Salmonella strains simulta-
neously, and at high dose inoculum the probability of any
given bacterium to multiply in a mixed infection will be
equivalent to its ability to multiply when infecting by itself.
These findings have allowed the recent development of
several techniques for the identification and characterisa-
tion of virulence functions of Salmonella and other patho-
gens in vivo.
2.1. High-throughput screening methods
to identify Salmonella virulence factors
in vivo based on the use of mixed infections
Characterisation of virulence genes frequently involves
analysis of levels of attenuation by infection studies with
individual mutant strains [18]. Individual analysis of bac-
terial strains carrying reporter gene fusions has also been
used to study gene expression in vivo [19]. The fact that
the Salmonella–mouse model can support mixed systemic
infections where any given strain has the same probability
to multiply as in single infection has made possible the
simultaneous high-throughput analysis of either virulence
attenuation or gene expression of multiple strains within
the same animal. These new methods use the mouse as a
selective environment: in vivo expression technology
(IVET) is based on positive selection of in vivo-induced
genes [20], whereas signature-tagged mutagenesis (STM)
is based on in vivo negative selection of attenuated strains
[9].
2.1.1. IVET
When a pathogen enters the host, altered patterns of
gene expression enable it to respond to the challenges of
the new environment. Therefore, a method to select pro-
moters activated in vivo provides a means to identify
genetic loci that could be important for bacterial viru-
lence. The first IVET method consisted of the inoculation
of mice with a pool of strains carrying chromosomal
fusions to a promoterless reporter gene essential for growth
within the host, whose expression could therefore be
positively selected for in vivo [20]. Several versions of the
IVET method have been used in different pathogen–host
systems, and the results and implications from these have
been discussed in detail in other reviews [21–24].
2.1.2. STM
STM is a technique that labels mutants with different
identifying DNA ’signature-tags’ [9]. Strains carrying
tagged mutations can be identified on the basis of their
tags, allowing them to be individually detected in mixed
infection. PCR amplification of the tags from a pool of STM
mutants is carried out using primers common to all of
them, and the resulting products are used as hybridisation
probes. Comparative hybridisation with probes obtained
prior to inoculation and after bacterial recovery from the
host enables attenuated mutants to be identified [9]. STM
1346
Beuzón and Holden
therefore takes advantage of the capability of the animal–
pathogen interaction model to support the simultaneous
growth of multiple bacterial strains. To date, over 50
attenuated mutants have been identified by STM in S. ty-
phimurium and hundreds of them in several other patho-
gens [25]. The technical aspects, results and limitations of
STM in Salmonella and other pathogens have been dis-
cussed in detail previously [22, 25–29].
3. Functional characterisation
of virulence determinants
The use of techniques such as IVET and STM has
identified a large number of genes that are important for
Salmonella virulence. In some cases, DNA sequence
analysis has provided information on their specific func-
tions. However, in other cases the information obtained by
sequence analysis is not sufficient to suggest a function for
the genes. In these cases, phenotypic characterisation of
mutant strains is a useful way to shed light on gene
function. In the case of Salmonella, a series of assays can
be done to relate these genes to one or more aspects of
Salmonella virulence, for example: resistance to serum
complement and antimicrobial peptides, invasion of epi-
thelial cells, and survival and replication within macro-
phages. In addition to these, more sophisticated in vivo
studies can be done to establish the time and the body site
where the virulence attenuation first becomes apparent.
3.1. Competitive index
Historically, in vivo determination of the contribution
of a given gene to virulence has relied on the use of the
LD50 test. The LD50 is defined as the number of bacteria
necessary to kill 50% of the infected animals. The LD50 is
a useful measure of virulence, but it has limitations: it is
relatively crude and reflects only the cumulative effect of
all the steps involved in causing lethality. A failure of a
mutation to raise the LD50 does not necessarily mean that
the mutation did not affect an important virulence deter-
minant, but could reflect a situation in which the effect
was not sufficient to be detected by the assay.
The competitive index (CI) is an alternative measure
that uses mixed infections to determine the degree of
virulence attenuation caused by a given mutation. Equiva-
lent numbers of two strains (the wild-type and a mutant
strain) are combined (input) and used to inoculate an
animal host. Bacteria are recovered after an appropriate
period of time from a target organ (output) and the num-
bers of each strain are enumerated by colony-forming
units (CFU); strains are usually distinguished by different
antibiotic resistance markers. The CI is defined as the ratio
between the mutant strain and the wild type in the output
divided by the ratio of the two strains in the input [30, 31].
The CI is a more sensitive measure of virulence attenu-
ation than the LD50 because it reflects bacterial numbers
directly rather than survival of the host animals, and it can
therefore distinguish between mutant strains whose attenu-
ation is too subtle to be detected by LD50. By testing the
virulence levels of mutant versus wild-type strains within
the same animal, animal to animal variation is decreased.
Microbes and Infection
2001, 1345-0
Salmonella virulence gene interactions in vivo
Furthermore, the LD50 is calculated from the number of
animals surviving the infection whereas the CI is obtained
from the numbers of bacteria before and after the infec-
tion, providing a figure that is based on a much larger
sample size. Therefore, the CI constitutes a more accurate
indication of virulence attenuation using fewer animals.
An obvious theoretical disadvantage of CI is that the defect
of some attenuated strains might be complemented in
trans by the wild-type strain present in the same inoculum.
The CI is also a versatile method where route of infection,
dose, strain combination and ratio, time and site of recov-
ery can be modified to provide a more detailed description
of the contribution to virulence of a given gene. All these
factors have made CI analysis an increasingly popular
method to determine virulence attenuation [32–35].
4. In vivo analysis of gene interactions
in Salmonella by mixed infections
An in vivo assay to establish links between a new
virulence determinant and known virulence genes or regu-
latory circuits operating in the Salmonella infection pro-
cess could be a useful source of information about its
potential function. Two approaches have been described
to study gene interactions in vivo, both relying on the
additivity of phenotypic effects of mutations. Because
Salmonella virulence is multifactorial, combinations of
mutations in genes with different virulence functions
should result in strains with increased attenuation.
4.1. Early uses of competitive index
to study gene interactions
Bäumler and collaborators [36] were the first to apply
mixed infections to study Salmonella gene interactions in
vivo. They wished to determine if the functions of S. typh-
imurium lpfC (encoding long polar fimbriae) and invA
(encoding a structural component of the SPI-1 TTSS) in
colonisation and penetration of the mouse gut epithelium
were independent. They constructed an invA– lpfC– double
mutant strain and compared it after oral mixed inoculation
to the wild-type strain. If these gene functions are indepen-
dent, the attenuation of an invA– lpfC– mutant strain is
predicted to be similar to the combined attenuation of an
invA– mutant strain plus a lpfC– mutant. They infected
mice by the oral route, with mixed inocula of the wild-type
strain and either the double or the single mutant strains.
Bacteria were recovered from the spleen 5 days later and
the bacterial CFU of each strain determined, distinguished
by antibiotic resistance markers. The attenuation of the
double mutant strain was greater than the predicted com-
bined effect of the single mutations, suggesting that muta-
tions resulting in the loss of function of these systems have
a synergistic effect on the attenuation of S. typhimurium
virulence. The simplest explanation for these results is
therefore that S. typhimurium possesses two independent
mechanisms to colonise and penetrate the gut epithelium
and that the absence of one of them can be partially
compensated by the action of the other.
Microbes and Infection
2001, 1345-0
Forum in Immunology
4.2. In vivo ’cancelling out’ to analyse Salmonella
gene interactions by mixed infections
Bäumler and collaborators [36] showed that mixed
infection with double mutant and wild-type strains could
be a useful tool to study Salmonella gene interactions in
vivo. However, determination of phenotypic additivity by
comparison to the wild-type strain is difficult in cases
where the level of attenuation caused by a single mutation
is very high (C.R. Beuzón and D.W. Holden, unpublished
results).
To detect differences in attenuation between double
and single mutant strains affected in systemic infection, we
have developed a modification of the competitive index
which involves co-infecting the same animal with single
and double mutant strains [3, 37]. In a mixed infection
with a strain carrying mutations in two genes and a strain
carrying only one of these mutations, the attenuation
caused by the shared mutation ought to be equivalent and
therefore ’cancel out’. The CI obtained after cancelling out
the effect of one mutation represents the contribution that
the other mutation has to the attenuation of the double
mutant strain. For the sake of clarity we will refer to this
modified competitive index as COI (’cancelled out’ com-
petitive index). COI is defined as the ratio of the double
mutant to single mutant strain in the output, divided by the
ratio of the two strains in the input.
Direct comparison of virulence of single and double
mutant strains within the same animal gives a more reli-
able account of the functional relationship of the genes
than comparisons carried out using results from different
animals, because it eliminates the effects of animal to
animal variation. Furthermore, the use of COI to analyse
gene interactions in vivo provides a more relevant envi-
ronment in which to assess the significance of gene func-
tion and interaction than in vitro models.
4.2.1. COI predictions
If we consider that two virulence genes, a and b,
contribute independently to Salmonella virulence, the
presence or absence of a functional copy of gene a would
not affect the way in which gene b would contribute to
virulence. Therefore the COI(a– versus a– b–) would be
similar to the corresponding CI(wt versus b–) (this situation
is represented in figure 1).
On the other hand, null mutations in two genes that
contribute equally to the same specific function (e.g., by
encoding different steps of the same biosynthetic pathway
or components of a macromolecular structure) would
cause a similar attenuation of virulence singly or com-
bined. Therefore, the double mutant strain would be no
more attenuated than the single mutants. In this case, the
CIs of the single mutant strains would be lower than 1.0
and similar to each other, whereas the corresponding COIs
would be similar to 1.0 (figure 2). The analysis of two gene
products not involved in the same specific function, but
linked because the action of one is completely dependent
on the action of the other would potentially give similar
results. Hence, any analysis resulting in COIs of approxi-
mately 1.0 requires further study before it can be con-
cluded that the genes are involved in the same function.
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Forum in Immunology
Figure 1. COI determination and theoretical predictions in the
case of two genes contributing independently to virulence. This
figure has been adapted from Beuzon et al., Infect. Immun. 69
(2001) 7254–7261 with permission from the American Society
of Microbiology.
There are clearly other possibilities apart from those
discussed above. For example, gene products that are
components of regulatory circuits can partially overlap in
some of the functions they regulate, or contribute to dif-
ferent degrees to the same function. In these cases, the
predicted COIs would not correspond to those shown in
figures 1 and 2. In the case of genes involved in alternative
but independent processes, where the loss of one of the
processes is compensated by the action of the other (such
as lpfC and invA), COIs would be greater than the corre-
sponding CI.
4.3. Analysis of gene interaction
in systemic infection by COI
To determine if COI could be used to indicate pheno-
typic additivity of mutations affecting independent func-
tions involved in systemic infection, i.p. mixed infections
of mice were carried out using single and double mutant
strains affected in purD (a gene encoding a component of
the purine biosynthetic pathway [38]) and ssaV (a gene
encoding a structural component of the SPI-2 secretion
[39], necessary for secretion [40]). The COI(ssaV– versus
purD– ssaV–) in the spleen, 48 h after inoculation was not
1348
Beuzón and Holden
Figure 2. COI determination and theoretical predictions in the
case of two genes involved in the same specific function.This
figure has been adapted from Beuzon et al., Infect. Immun. 69
(2001) 7254–7261 with permission from the American Society
of Microbiology.
statistically different from the CI(purD– versus wild-type)
(table I). Similarly, the reciprocal COI(purD– versus purD–
ssaV) was not statistically different from CI(ssaV– versus
wild-type). These results indicate that a mutation in purD
causes the same attenuation to a strain independently of
whether that strain has a functional SPI-2 TTSS or not, and
vice versa; in other words, the functions carried out by
their gene products contribute independently to Salmo-
nella virulence [3, 37]. Strains carrying mutations in either
ssaV or sseB (a gene predicted to encode a component of
the SPI-2 translocon) [32, 40], were similarly inoculated
i.p. in mixed infections with a strain carrying mutations in
both genes. The COIs obtained were similar to 1.0 (table I),
as predicted from the knowledge that their gene products
contribute to the same Salmonella virulence function [32,
39, 41]. Similar results were obtained from the analysis of
ssaV and sseC, another gene from SPI-2 (table I) [3].
4.3.1. Independent functions
In the original work by Shea et al. [3], the COI was used
to analyse the possibility of a functional relationship
between the spv operon and SPI-2 TTSS. The spv and SPI-2
genes have been shown to be important for systemic
infection, and mutations in both systems cause a similar
Microbes and Infection
2001, 1345-0
Salmonella virulence gene interactions in vivo
Table I. COI analysis of S. typhimurium mutants.
Mixed infection
Controls
wt v ssaV–
wt v purD–
ssaV– v ssaV–, purD–
purD– v ssaV–, purD–
wt v sseB–
ssaV– v ssaV–, sseB–
sseB– v ssaV–, sseB–
wt v sseC–
sseC– v ssaV–, sseC–
spv
wt v spvA–
ssaV– v ssaV–, spvA–
ompR/envZ
wt v ompR–
ssaV– v ssaV–, ompR–
ompR– v ssaV–, ompR–
sifA
wt v sifA–
ssaV– v ssaV–, sifA–
sifA– v ssaV–, sifA–
a
Not significantly different to 1.0 (P < 0.05). b Not significantly differ-
ent to the CI of the corresponding single mutant strain versus wild type
strain.
This table has been adapted from Beuzón et al. [37] by permission of
Oxford University Press.
level of attenuation. The spv genes are located on the
virulence plasmid [12] and are necessary for intracellular
replication in mice [42]. Although it has been recently
shown that SpvB has ADP-ribosyltransferase activity [43],
its target, and the overall virulence function of the spv
locus remains to be determined. The CI of an ssaV– mutant
strain versus the wild-type strain was not significantly
different from its corresponding COI(spvA– versus spvA–
ssaV–) indicating that these genes contribute indepen-
dently to Salmonella virulence.
4.3.2. Related functions
COI analysis has also been carried out to determine a
functional link between the SPI-2 TTSS, OmpR/EnvZ and
SifA [37]. Strains carrying mutations in ompR/envZ are
highly attenuated in systemic infection of mice, but muta-
tions in genes originally known to be regulated by OmpR
could not account entirely for its virulence defect [44, 45].
It has been shown recently that transcription of ssrA, a
gene encoding a component of SsrA/SsrB, the SPI-2 two-
component regulatory system [46–48], is modulated by
OmpR in infected tissue cultured cells [49]. It was there-
fore possible than the attenuation of ompR mutants was
attributable to an in vivo role in regulation of SPI-2 expres-
sion. To analyse this possibility, strains carrying mutations
in either ssaV or ompR were combined in mixed infections
with a strain carrying both mutations. The COIs for the
double mutant strain were not significantly different from
1.0, suggesting that these genes are involved in the same
virulence function, which provides an explanation for the
attenuation of an ompR– mutant strain.
SifA is encoded on a PAI located within the potABCD
operon on the Salmonella chromosome [41]. Strains car-
Microbes and Infection
2001, 1345-0
Forum in Immunology
rying mutations in sifA, ompR or envZ are all defective for
the formation of tubular membranous structures known as
CI or COI Sifs, which are normally induced in epithelial cells by
wild-type bacteria [41, 50, 51]. sifA– mutant strains are
0.006 attenuated in virulence following oral or i.p. inoculation
0.0005 of mice [37, 41]. The COIs for a strain carrying mutations
0.0038b in sifA and ssaV were similar to 1.0 (table I). These results
0.019b suggested that these genes are involved in the same func-
0.016 tion in S. typhimurium virulence [37].
1.2a Further analysis was carried out to determine the nature
0.82a of this potential interaction. The expression of sifA was
0.013 found to be strictly regulated by SsrA/SsrB. A sifA– mutant
1.28a strain has an intracellular replication defect in macro-
phages similar to that of some SPI-2 mutants [37]. Analysis
0.068
of the N-terminal region of the predicted sequence of SifA
0.052b
showed similarities to proteins that are secreted by Salmo-
0.008 nella TTSSs. This sequence has been proposed to be a
0.823a secretion and translocation signal [52]. All these results
0.21a suggest that SifA could be secreted by the SPI-2 TTSS. The
failure of strains carrying mutations in SPI-2 TTSS genes to
0.012 induce Sif formation in epithelial cells supports this possi-
1.34a bility [37]. Furthermore, the failure of a sifA– mutant strain
1.17a to induce Sif formation can be complemented in epithelial
cells transfected with a plasmid expressing SifA, suggest-
ing that the site of action of SifA is in the host cell cytosol
or on the cytosolic face of the vacuolar membrane [37].
SifA was also found to be necessary to maintain the
membrane of the Salmonella-containing vacuole, since
sifA– mutant bacteria lose their vacuolar membrane and
are released into the cytosol of the host cell by 10 h after
infection [37]. These results confirmed that, as indicated
by COI analysis, sifA and SPI-2 are involved in the same
virulence function.
4.3.3. Compensatory functions
COI has been recently used in our laboratory to study
the functional relationship between two different iron
uptake systems (ABC transporters) of Streptococcus pneu-
moniae in vivo (Brown et al., Mol. Microbiol. 40 (2001)
572–585). Mixed infections of mice with strains carrying
null mutations in genes encoding either one or both trans-
porters were carried out by two different routes of inocu-
lation and recovery: intranasal inoculation recovering bac-
teria from lungs and spleen, and i.p. inoculation recovering
bacteria from spleen. The results are consistent in showing
that these two transport systems act independently to
provide the iron necessary for bacterial growth in vivo.
The COIs for these systems were significantly lower than
the corresponding CIs. Therefore, the loss of both systems
reduces dramatically the ability of the organism to survive
and replicate in vivo, whereas the loss of function of one
system can be partially compensated for by the other.
4.4. Conclusions
From its original use to study the aetiology of Salmo-
nella systemic infection [17], to its more recent applica-
tions in high-throughput screening methods [9, 20] or in
vivo analysis of gene interactions [3, 36, 37], mixed infec-
tions have proved a valuable tool in advancing our under-
standing of Salmonella virulence.
The length, dose and route of infection are important
factors to consider when using mixed infections to obtain
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Forum in Immunology
representative recovery of inoculated strains. For example,
with respect to the route of inoculation, M cells of the
Peyer’s patches are considered a bottleneck that restricts
the number of bacteria that can translocate across the gut
epithelium (discussed in [22]). Nevertheless, it has been
shown that up to 30 different S. typhimurium strains are
capable of reaching the spleen after oral inoculation [53].
To obtain representative numbers of virulent strains in
the output from a mixed infection, the mice have to be
inoculated with a relatively high dose of bacteria [17].
High dose inoculation raises the potential problems that
some attenuated strains might be complemented in trans
by the wild-type strain in the same animal, or might benefit
from immune breakdown caused by such a high inocu-
lum. In either case, this could lead to attenuated strains
being mistaken as virulent. However, it is interesting to
note that the frequency with which avirulent mutants of
S. typhimurium were detected by STM [9] is very similar to
the frequency obtained by Bowe et al. [4] when screening
individual mutant strains at a lower dose.
When standard CI analysis is used to address the attenu-
ation of mutant strains, the CI value must be determined
with accuracy to be used as a reference for the virulence
attenuation caused by that mutation. The number of ani-
mals used needs to be sufficiently high to provide a low
standard deviation for the CI value. On the other hand,
COI values only need to be accurate enough to be assigned
to one of three categories: similar to 1.0, similar to the
corresponding CI, or lower than the corresponding CI.
Once the COI has been deemed to fall into one of these
categories, the actual numerical value of the index is
irrelevant. This approach therefore reduces the number of
animals needed in each analysis. For the COI studies
described here, in most cases three mice were sufficient to
obtain a meaningful interpretation.
The recent application of COI analysis to study viru-
lence gene interaction in S. pneumoniae (Brown et al.,
Mol. Microbiol. 40 (2001) 572–585) shows that this
method can be applied to other host–pathogen interac-
tions. In the case of Salmonella, the diversity of serovars
and animal models of infection provides a fertile ground
for the application of this method, and could potentially
allow a rapid assessment of gene interactions which might
differ between the specific host–serovar combinations,
and thereby help in studies of host specificity.
Acknowledgments
We would like to express our gratitude to Herb Arst for
help in making COI predictions, and for critical review.
We thank members of our laboratory for helpful discus-
sion. Work from our laboratory described in this review
was supported by the Medical Research Council and
Wellcome Trust (UK).
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Beuzón and Holden
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