Intraspecies Variation in the Emergence of Hyperinfectious Bacterial Strains in Nature

Abstract

Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection. However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in virulence during the infective process- a prospect that has significant implications for human and animal health. Our findings indicate that the capacity to become hypervirulent (100-fold decreased LD(50)) was much more evident in certain S. enterica strains than others. Hyperinfectious salmonellae were among the most virulent of this species; restricted to certain serotypes; and more capable of killing vaccinated animals. Such strains exhibited rapid (and rapidly reversible) switching to a less-virulent state accompanied by more competitive growth ex vivo that may contribute to maintenance in nature. The hypervirulent phenotype was associated with increased microbial pathogenicity (colonization; cytotoxin production; cytocidal activity), coupled with an altered innate immune cytokine response within infected cells (IFN-β; IL-1β; IL-6; IL-10). Gene expression analysis revealed that hyperinfectious strains display altered transcription of genes within the PhoP/PhoQ, PhoR/PhoB and ArgR regulons, conferring changes in the expression of classical virulence functions (e.g., SPI-1; SPI-2 effectors) and those involved in cellular physiology/metabolism (nutrient/acid stress). As hyperinfectious strains pose a potential risk to human and animal health, efforts toward mitigation of these potential food-borne contaminants may avert negative public health impacts and industry-associated losses.

Full text

Intraspecies Variation in the Emergence of
Hyperinfectious Bacterial Strains in Nature
Douglas M. Heithoff
1.
, William R. Shimp
1.
, John K. House
2
, Yi Xie
3
, Bart C. Weimer
3
,
Robert L. Sinsheimer
1
, Michael J. Mahan
1
*
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California, United States of America, 2University of Sydney,
Faculty of Veterinary Science, Camden, New South Wales, Australia, 3Department of Population Health and Reproduction, School of Veterinary Medicine, University of
California, Davis, California, United States of America
Abstract
Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence
of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into
pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection.
However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in
virulence during the infective process- a prospect that has significant implications for human and animal health. Our
findings indicate that the capacity to become hypervirulent (100-fold decreased LD
50
) was much more evident in certain S.
enterica strains than others. Hyperinfectious salmonellae were among the most virulent of this species; restricted to certain
serotypes; and more capable of killing vaccinated animals. Such strains exhibited rapid (and rapidly reversible) switching to
a less-virulent state accompanied by more competitive growth ex vivo that may contribute to maintenance in nature. The
hypervirulent phenotype was associated with increased microbial pathogenicity (colonization; cytotoxin production;
cytocidal activity), coupled with an altered innate immune cytokine response within infected cells (IFN-b; IL-1b; IL-6; IL-10).
Gene expression analysis revealed that hyperinfectious strains display altered transcription of genes within the PhoP/PhoQ,
PhoR/PhoB and ArgR regulons, conferring changes in the expression of classical virulence functions (e.g., SPI-1; SPI-2
effectors) and those involved in cellular physiology/metabolism (nutrient/acid stress). As hyperinfectious strains pose a
potential risk to human and animal health, efforts toward mitigation of these potential food-borne contaminants may avert
negative public health impacts and industry-associated losses.
Citation: Heithoff DM, Shimp WR, House JK, Xie Y, Weimer BC, et al. (2012) Intraspecies Variation in the Emergence of Hyperinfectious Bacterial Strains in
Nature. PLoS Pathog 8(4): e1002647. doi:10.1371/journal.ppat.1002647
Editor: Brad T. Cookson, University of Washington, United States of America
Received February 18, 2010; Accepted March 1, 2012; Published April 12, 2012
Copyright: ß2012 Mahan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by G. Harold & Leila Y. Mathers Foundation, the U.S. Army (W911NF-09-D-0001), and the Cottage Hospital Research Program
(2010-247) (MJM); USDA Agriculture and Food Research Initiative (2008-01452) (MJM/JKH); and USDA CSREES 2006-34526-17001, NIH R01 GM090262-0109,and
NIH R01 HD065122 (BCW). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of this manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mahan@lifesci.ucsb.edu
.These authors contributed equally to this work.
Introduction
Salmonella enterica is a significant food-borne pathogen of humans
causing up to an estimated 1.3 billion cases of disease worldwide,
annually [1,2]. S. enterica is acquired via the fecal-oral route and is
comprised of six subspecies that are subdivided into more than
2500 serovars (serological variants) based on carbohydrate,
lipopolysaccharide (LPS), and flagellar composition [2]. S. enterica
infection can result in any of four distinct syndromes: enteroco-
litis/diarrhea, bacteremia, enteric (typhoid) fever, and chronic
asymptomatic carriage [2–4]. Many serovars infect both humans
and animals wherein the particular syndrome and disease severity
is a function of the serovar and host susceptibility [5,6].
Such host-susceptibility differences present a formidable chal-
lenge to the design of salmonellae control strategies for a number
of reasons: 1) Most infections of livestock are subclinical as
evidenced by the disparity between the frequency and diversity of
isolates from surveillance and clinical submissions [7–9]; 2) Some
isolates are capable of asymptomatic colonization and/or
persistence in a particular animal species while causing acute
disease in another animal species (e.g., different types or classes of
stock) [2–4]; 3) Although a diversity of serotypes are frequently
isolated from intensive livestock production systems, disease
outbreaks are often intermittent and associated with specific
serotypes [8–10]; 4) The capacity of salmonellae to survive and
proliferate in the environment provides a large dynamic reservoir
for infection of livestock and a vehicle for cross-contamination
from animal to human food products [11–14]. These factors are of
particular relevance to the global trend toward intensive livestock
production that favors fecal-oral pathogen transmission, and the
resultant increased risk of animal disease and contamination of
livestock-derived food products [8–10,15].
The diversity of salmonellae present on farms and feedlots, and
the potential for different serovars to possess an array of virulence
attributes, necessitates the use of broad prophylactic strategies that
are efficacious for many serovars simultaneously. An effective
approach for a number of years has been the therapeutic and
prophylactic administration of antibiotics to livestock, but this
option has become limited due to the emergence of multi-drug
resistant pathogenic strains that also present a bona fide risk to
PLoS Pathogens | www.plospathogens.org 1 April 2012 | Volume 8 | Issue 4 | e1002647

human health [1,9,16]. Vaccination is one of the best forms of
prophylaxis against the development of disease caused by
infectious agents. Although vaccination is generally highly specific
in the protection conferred in immunized hosts (protection is
limited to a specific strain or closely-related set of strains), recent
advancements have resulted in the development of vaccines that
elicit cross-protective immunity to multiple strains of the same
species [17–21]. However, currently available vaccines may elicit
limited protection against new pathogens that may express traits
that confer enhanced virulence or compromised host immunity.
The continuing emergence of new virulent strains associated
with an increased incidence and/or severity of disease has yet to be
explained. Insights have been derived from prior animal-passage
studies wherein virulence traits often are increased (reversibly)
following animal passage (e.g., accelerated colonization; hastened
morbidity/mortality; reviewed in [22–24]). For example, host
passage of Vibrio cholerae [25] and Citrobacter rodentium [26] results in
the transition to a hypervirulent state that is maintained for a
limited time after fecal shedding and may contribute to epidemic
spread of the organism [27]. Further, epidemiological evidence
indicates that animals can be infected by natural transmission (via
direct contact with infected animals) with a significantly lower
infectious dose than with organisms obtained from laboratory
culture (e.g., E. coli O157:H7 and S. Choleraesuis) [28–30].
However, many animal passage studies were performed on a
limited number of strains; often only a modest increase in
virulence was observed; multiple rounds of animal passage were
required; and did not address the prospect that animal passage
may lead to markedly increased virulence in some strains and hosts
but not others [25,26,31–39].
In this study, a collection of Salmonella clinical isolates was
screened for those that, following infection, exhibited a pro-
nounced increase in virulence relative to other passaged isolates.
Some salmonellae strains exhibited the hypervirulent phenotype
after in vivo passage, whereas others did not, indicating
intraspecies variation in the capacity for their development. The
resultant hyperinfectious strains were among the most virulent
salmonellae reported and were subsequently shown to be more
capable of infecting vaccinated animals.
Materials and Methods
Strains and media
Salmonella human clinical isolates were obtained from fecal and
blood samples derived from patients with gastroenteritis or
bacteremia, respectively; animal isolates were derived from
different outbreaks, individual cases, or surveillance submissions
to diagnostic laboratories [20]. Virulent S. Typhimurium reference
strain ATCC 14028 (CDC 6516-60) was used in all studies for
comparison. Unless otherwise specified, bacteria were derived
from stationary phase cultures aerated at 37uC containing either
Luria-Bertani (LB) medium [40] or low phosphate, low magne-
sium, pH 5.5 medium supplemented with 0.3% glycerol and 0.1%
casamino acids (LPM pH 5.5) [41,42].
Ethics statement
All animal experimentation was conducted following the
National Institutes of Health guidelines for housing and care of
laboratory animals and performed in accordance with Institutional
regulations after pertinent review and approval by the Institutional
Animal Care and Use Committee at the University of California,
Santa Barbara.
Virulence assays
Oral and Intraperitoneal Lethal Dose
50
(LD
50
): The dose required to
kill 50% of infected animals was determined via the oral (via
gastrointubation) and intraperitoneal (i.p.) routes by infecting at
least 10 mice [43]. Salmonella test strains and wild-type S.
Typhimurium reference strain 14028 were grown overnight in
LB or LPM pH 5.5 medium. Bacterial cells resuspended in
0.2 ml of 0.2M Na
2
HPO
4
pH 8.1 or 0.1 ml of 0.15M NaCl (for
oral and i.p. administration, respectively) were used to infect
mice, which were examined daily for morbidity and mortality up
to 3 weeks post-infection. The oral and i.p. LD
50
for wild-type S.
Typhimurium reference strain 14028 is 10
5
and ,10 organisms,
respectively [43]. Competitive Index (CI): The CI value is the
relative in vivo recovery ratio of test strain/reference strain
obtained from target tissues after equivalent doses are co-
administered by i.p. infection [44]. Salmonella test strains were
grown overnight in either LB or LPM pH 5.5 medium; S.
Typhimurium reference strain MT2057 (a virulent derivative of
strain 14028) was grown in LB [43,44]. Bacterial cells were
resuspended in 0.15M NaCl and an equivalent dose (500
bacterial cells) of a test strain and S. Typhimurium reference
strain MT2057 was co-administered i.p. to at least 5 mice. Five
days post-infection, the bacterial cells were recovered from the
spleen of acutely infected animals. The CI value is the ratio of test
strain/reference strain recovered from the spleen divided by the
ratio of the input inoculum; bacterial cell number was
enumerated by direct colony count. S. Typhimurium reference
strain MT2057 (used in the CI studies) is a virulent derivative of
strain 14028, containing a Lac
+
MudJ transcriptional fusion
which is used to discern it from other Salmonella that are
inherently Lac
2
. Note that the oral and i.p. LD
50
(10
5
and ,10
organisms, respectively), as well as the i.p. competitive index, of
strain MT2057 are indistinguishable from that of the parental
wild-type strain, 14028 [43,44]. Six- to- eight week old BALB/c
mice were used in all virulence studies.
Author Summary
Salmonellosis continues to compromise human health,
animal welfare, and modern agriculture. Developing a
comprehensive control plan requires an understanding of
how pathogens emerge and express traits that confer
increased incidence and severity of disease. It is well-
established that animal passage often results in increased
virulence; however, our findings indicate that the capacity
to undergo a pronounced increase in virulence after
passage was much more prevalent in certain Salmonella
isolates than in others. The resultant hyperinfectious
strains are among the most virulent salmonellae reported;
were restricted to certain serotypes; and were able to
override the immunity conferred in vaccinated animals.
The induction of hypervirulence was responsive to subtle
changes in environmental conditions and, potentially, may
occur in other salmonellae serotypes after passage
through certain hosts and/or exposure to certain environ-
mental variables; a response that may be common across
the microbial realm. Thus, management practices and
environmental conditions inherent to livestock production
have the potential to inadvertently trigger hypervirulence
(e.g., diet; herd size; exposure to livestock waste and/or
antimicrobials). From a farm management perspective,
careful consideration must be given to risk-management
strategies that reduce emergence/persistence of these
potential food-borne contaminants to safeguard public
health and reduce industry-associated losses.
Emerging Hyperinfectious Strains
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Screen for hyperinfectious strains
A collection of 184 Salmonella human and animal clinical isolates
[20] cultured in rich medium was screened for those that were
initially attenuated for virulence via the i.p. route of infection (10
3
-
fold decreased i.p. CI; 10- fold increased i.p. LD
50
); that harbored
the virulence plasmid necessary for systemic disease [45,46]; and
that were competent for virulence via the oral route of infection
(oral LD
50
of 10
5
cells). The 14 isolates that answered this screen
were subjected to oral animal passage whereby bacteria (10
9
cells)
derived from stationary phase cultures containing LB medium
were used to perorally infect mice. Five to seven days post-
infection, spleens were aseptically removed from acutely infected
mice, homogenized in 1 ml of 0.2M Na
2
HPO
4
pH 8.1 (10
8
to
10
9
CFU/g of spleen), and used, without ex vivo growth, to infect
naı
¨ve animals at doses equivalent to, and 10- to 100- fold lower
than, the oral LD
50
of the same strain grown in LB medium (10
5
cells). Such animal passage resulted in the development of
hyperinfectious strains for all (14/14) isolates tested, as confirmed
by a 10- to 100- fold reduced oral and i.p. LD
50
and a 10
3
-to10
4
-
fold increased i.p. CI relative to the values attained after growth in
LB medium. Mice were examined daily following infection for
morbidity and mortality up to 3 weeks post-infection.
Cell culture
The murine macrophage cell line RAW 264.7 (ATCC TIB-71)
was obtained from the American Type Culture Collection,
Rockville, MD., and maintained in minimum essential medium
(MEM) supplemented with L-glutamine and 10% heat-inactivated
bovine growth-supplemented calf serum (HyClone Laboratories,
Logan, UT). Cells were grown in a humidified atmosphere of 5%
carbon dioxide and 95% air at 37uC in 75-cm
2
plastic flasks
(Corning Glass Works, Corning, NY). Cultured murine macro-
phages (RAW 264.7) were harvested by scraping with a rubber
policeman and plated at a density of 2.5610
5
to 5610
5
cells/ml in
4 ml of culture medium in 35 mm-diameter, six-well dishes
(Corning) and grown for 24 h to approximately 80 to 90%
confluence (1610
6
to 5610
6
cells/well) (adapted from [47]).
Bacterial infection of cultured murine macrophages
Bacterial cells were used to infect cultured murine macrophage
(RAW 264.7) monolayers grown in cell culture plates (Corning) at
a multiplicity of infection (MOI) of 10:1 or 100:1. The bacteria
were centrifuged onto cultured monolayers at 1,0006gfor 10 min
at room temperature, after which they were incubated for 30 min
at 37uC in a 5% CO
2
incubator (t = 0 time point). The coculture
was washed once with cell culture medium and incubated for
45 min in the presence of gentamicin (100 mg/ml) to kill
extracellular bacteria, washed once with pre-warmed cell culture
medium, and incubated with gentamicin (10 mg/ml) to the time
points indicated (adapted from [48]).
Bacterial cytocidal activity assay
Macrophage (RAW264.7) cell viability following Salmonella
infection was quantified via a crystal violet dye retention assay in
96 well-plates adapted from references [49,50]. Bacteria derived
from stationary phase cultures containing either LB or LPM
pH 5.5 medium were used to infect cultured macrophage
monolayers (5610
4
to 1610
5
cells/well) at an MOI of 10:1 or
100:1 as described above. At 20 h post-infection, the monolayer
cultures were rinsed twice with PBS, and the adherent cells were
fixed and stained for 10 min with 0.2% crystal violet in 20%
methanol. Monolayers were washed three times with phosphate
buffered saline (PBS) to remove excess crystal violet. Dye retained
by the cells was released using a 50% ethanol/0.1% acetic acid
mixture, diluted 1:2 in PBS, and quantified by absorbance at
577 nm. High cytocidal activity is associated with low dye
retention and vice versa. Data given are representative absorbance
values derived from each condition performed in triplicate.
Standard error of triplicate means is ,20%.
Quantitation of macrophage cytokines post-infection via
qPCR analysis
Bacteria grown overnight in LB or in LPM pH 5.5 medium
were used to infect cultured macrophage (RAW264.7) monolayers
at an MOI of 10:1 in 6-well culture plates as described above.
Total RNA was prepared using the RNeasy Mini kit (Qiagen) as
specified by the manufacturer’s protocol. RNA concentrations
were determined spectrophotometrically. Reverse transcription
was carried out using 2 mg of total RNA with the Superscript
cDNA Synthesis Kit (Invitrogen) as per the manufacturer’s
protocol. qPCR was performed using iQ SYBR Green Supermix
(BioRAD) and an iQ5 real time PCR thermocycler (BioRAD). For
amplification of mouse genes, the primer pairs were those
described in the following studies: IFN-b[51]; IL-1b, IL-6 and
IL-10 [52]; iNOS and GAPDH [53]. Quantification of the qPCR
product was carried out using the iQ5 optical system software
(BioRAD). All target gene transcripts were normalized to that of
the GAPDH gene. The expression ratio value is the level of
transcripts obtained from infected relative to uninfected cells.
Transcriptome analysis
Bacterial RNA/cDNA preparation. Bacterial strains were
grown overnight with aeration at 37uC in LB broth, pelleted and
washed in 0.15M NaCl, and split without dilution into two
cultures containing either LB or LPM pH 5.5 medium. The
cultures were incubated with aeration at 37uC for 4 h after which
approximately 2.5610
10
cells were pelleted via centrifugation,
snap-frozen in an ethanol-dry ice bath, and stored at 280uC.
Bacterial cell pellets were lysed using Max Bacterial Enhancement
Reagent (Invitrogen) at 95uC for 5 min. Total bacterial RNA
($10 mg) was isolated using TRIzol Max Bacterial Isolation Kit
(Invitrogen), and purified with an RNAeasy MinElute kit with on-
column DNase digestion (Qiagen) (A
260/280
ratio of $2.0 and an
A
260/230
ratio of $1.5). Reverse transcription of total RNA was
carried out using Superscriptase II and random hexamers
(Invitrogen). After NaOH treatment to eliminate the RNA
template, single-stranded cDNA was purified with QIAquick
PCR MinElute purification kit (Qiagen).
Array design and hybridization. cDNA (1 mg) was sheared
for 10 min with 0.6 U of DNase I at 37uC (Promega, WI); and
labeled with a custom GeneChip DNA designed by B. C. Weimer
(UC Davis) in conjunction with Affymetrix Inc. (Santa Clara, CA).
Genomic DNA (50 ng) was labeled according to the Escherichia coli
protocol and hybridized onto custom Affymetrix DNA chips
containing probe sets designed for all the annotated coding
sequences (CDSs) and intergenic spaces of S. Typhimurium LT2
genome, resulting in 4,510 probe sets composed of 11 unique 25-
mer probe sequences per CDS. The chips were hybridized and
scanned at the Center for Integrated BioSystems (Utah State
University, Logan, UT), according to the manufacturer’s protocols
for E. coli. Hybridizations for each strain were performed in two
biological replicates.
Data normalization, visualization, and analysis. Gene
expression analysis was performed to identify bacterial gene
transcripts that were significantly altered in hyperinfectious strains
under LB versus LPM pH 5.5 conditions, and not altered, or
altered to the same extent, in a conventionally virulent strain. Raw
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probe-level intensities (.cel files) from all chips were background
corrected using the robust multichip average (RMA) method,
normalized using loess, and summarized using the Bioconductor
Affy package. The raw log
2
gene-level Affymetrix expression
values were transformed to produce log
2
LPM/LB ratio values for
conventionally virulent S. Typhimurium (ST), and hypervirulent S.
Bovismorbificans (SB) and S. Choleraesuis (SC) strains.
Subsequently, log
2
LPM/LB ratio data were loaded into the
CLC Genomics Workbench and further normalized (CLC bio,
Cambridge, MA); and the log
2
LPM/LB ratio statistical
differences between conventionally and hypervirulent strains
were evaluated using the CLC Expression analysis module with
SB and SC grouped together. Two criteria were used as a cutoff to
identify the genes that were significantly altered in hyperinfectious
strains under LB versus LPM pH.5.5 conditions, and not altered,
or altered to the same extent, in a conventionally virulent strain;
i.e., at least a 2-fold expression change in SB, SC or ST; and a 0.05
false discovery rate (FDR) when comparing log
2
LPM/LB ratios
values for SB and SC versus ST. Heat maps were generated from
the resultant list of genes using The Institute for Genomic
Research MultiExperiment Viewer (MeV), version 4.7 [54].
Unsupervised data analysis was performed in MeV using
hierarchical clustering (HCL) [55] modules. All expression
experiments were done in two biological replications.
Statistical analyses
Mouse disease susceptibility. The disease susceptibility in
vaccinated mice infected with hyperinfectious and conventionally
virulent salmonellae was determined by comparing the proportion
of mice surviving virulent challenge using Chi-square (Epicalc
2000 version 1.02, 1998 Brixton Books).
Bacterial cytocidal activity. Cytocidal activity of
hyperinfectious and conventionally virulent salmonellae upon
infection of cultured macrophages was subjected to analysis of
variance in GenStat (13
th
edition, VSN International Ltd, Hemel
Hempstead, UK) using a model that had serotype, media, and
dose as the main effects. The change in cytocidal activity of
hyperinfectious strains (S. Choleraesuis x3246 and S.
Bovismorbificans 158) was individually contrasted to the change
in cytocidal activity of reference S. Typhimurium strain 14028 at
each dose level according to the following ‘a priori’ contrast:
cytocidal activity of the hyperinfectious serovar grown in LB
medium minus the cytocidal activity grown in LPM medium
versus the cytocidal activity of S. Typhimurium 14028 grown in
LB medium minus the cytocidal activity of S. Typhimurium 14028
grown in LPM.
Innate immune cytokine response. Differences in gene
expression displayed by infected relative to uninfected murine
macrophage values were analyzed using residual (or restricted)
maximum likelihood (REML) analysis (Genstat, 13
th
Edition, VSN
International Ltd, Hemel Hempstead, UK). A single variate,
repeated measures model was fitted for the factors media,
organism and time. The Wald chi-square test was used to
determine significant individual effects and interactions between
factors. Differences between the individual means were
determined by calculating an approximate least significant
difference (LSD), using predicted model-based means. Predicted
means are those obtained from the fitted model rather than the
raw sample means, as predicted means represent means adjusted
to a common set of variables, thus allowing valid comparison
between means. A difference of means that exceeded the
calculated LSD was considered significant. For all statistical
analyses, a significance level (P) of less than 0.05 was considered to
be statistically significant.
Gene expression analysis. A description of the
transcriptome statistical analysis is provided in the previous
Materials and Methods section under data normalization,
visualization, and analysis.
Results
Screen for Salmonella strains that exhibit a pronounced
increase in virulence following infection relative to other
animal-passaged isolates
A collection of 184 Salmonella clinical isolates was obtained from
fecal and blood samples derived from human patients with
gastroenteritis or bacteremia; and from animal isolates derived from
different outbreaks, individual cases, or surveillance submissions to
diagnostic laboratories [20]. These isolates were cultured in rich (LB)
medium and screened for those that i) were attenuated for virulence
via the i.p. route of infection (10
3
-fold decreased i.p. CI; 10-fold
increased i.p. LD
50
); ii) harbored the virulence plasmid necessary for
systemic disease [45,46]; and iii) were competent for virulence via the
oral route of infection (oral LD
50
of 10
5
cells). The fourteen isolates
that answered this screen were grown overnight in LB medium and
used to perorally infect mice. Five to seven days post-infection,
bacteria derived from spleens harvested from the resultant acutely
infected animals were used, without ex vivo growth, to orally infect
naı
¨ve animals at doses equivalent to, and 10- to 100-fold lower than,
the oral LD
50
of the same strain grown in LB medium (10
5
cells). The
prior in vivo passage resulted in the development of hyperinfectious
strains for all (14/14) isolates tested, as evidenced by a 10- to 100- fold
reduced oral and i.p. LD
50
and a 10
3
-to10
4
- fold increased i.p. CI
relative to the values attained after growth in LB medium (Table 1).
These isolates comprise some of the most virulent salmonellae strains
reported (i.e., oral LD
50
of 10
3
organisms). In contrast, although in
vivo passage of other clinical isolates exhibited increased virulence
traits after murine passage (increased colonization; decreased time to
morbidity/mortality)- a phenomenon shown previously [39] and
recapitulated here, none (0/7) exhibited a marked change in LD
50
or
CI value relative to that attained after in vitro growth. This was also
the case for conventionally virulent Salmonella reference strain 14028.
Taken together, these data indicate that the 14 hyperinfectious
Salmonella strains are considerably more virulent than other animal-
passaged clinical isolates (100-fold decreased LD
50
); and the display of
increased virulence traits by bacterial strains after murine passage
does not necessarily equate to hypervirulence.
Intraspecies variation in the development of
hyperinfectious salmonellae strains
Most cases of human and livestock salmonellosis are caused by
one Salmonella subspecies, termed S. enterica subsp. enterica [9,56–59].
Here we examined whether there was variation within subsp. enterica
serovars in the capacity for the development of hyperinfectious
strains following murine passage. Our data show that the
hypervirulent phenotype was much more evident in some subsp.
enterica serovars (S. Bovismorbificans [11/11]; S. Choleraesuis [3/3])
(serogroups C2-C3 and C1, respectively), than others (S. Typhi-
murium [0/52]; S. Dublin [0/8]; S. Enteritidis [0/7]) (serogroups B,
D, and D, respectively) (P,0.01). These data suggest that, following
murine infection, Salmonella serovars exhibit intraspecies variation in
the development of hyperinfectious strains.
Hyperinfectious salmonellae exhibit distinct colonization
kinetics relative to that of other animal passaged isolates
To determine the spatio-temporal nature of the development of
hyperinfectious strains, the kinetics of host tissue colonization was
Emerging Hyperinfectious Strains
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followed throughout the infective process. Upon oral infection,
hyperinfectious S. Choleraesuis x3246 grown in LB medium
exhibited a pronounced lag in colonization of mucosal tissues and
visceral organs and did not attain the high bacterial load exhibited
by the same strain after murine passage (open versus closed boxes;
Figure 1). In contrast, conventionally virulent Salmonella reference
strain 14028 grown in LB medium did not display the pronounced
lag in colonization exhibited by S. Choleraesuis x3246 (open
circles versus open boxes). Further, although murine-passaged S.
Typhimurium 14028 exhibited increased colonization (open
versus closed circles) as has been observed with Salmonella and
other enteric pathogens [25,26,39,60], its passage did not result in
the high bacterial load exhibited by murine-passaged S. Choler-
aesuis x3246 at late stages of infection (closed symbols), nor was it
associated with the pronounced decrease in LD
50
associated with
hyperinfectious strains after passage (Table 1). These data indicate
that hyperinfectious strains undergo a switch from a less-virulent to
hypervirulent state following a pronounced lag during the infective
process, and the resultant hyperinfectious strains are much more
virulent than other animal-passaged clinical isolates.
Hyperinfectious salmonellae can be isolated under
defined conditions in vitro, and adopt distinct virulence
states depending on prior growth conditions
Next, we questioned whether strains that exhibited the
hypervirulent phenotype in vivo also had the capacity to enter
the hypervirulent state under defined conditions in vitro. Efforts
were initially focused on conditions reported to reflect that of the
macrophage phagosome, a principal organelle in which salmonel-
lae reside during infection [61,62]; such conditions are character-
ized by low phosphate, low magnesium and mildly acidic medium
(LPM pH 5.5) [41,42]. Growth of S. Choleraesuis x3246 and S.
Bovismorbificans 158 in LPM pH 5.5 medium resulted in the
recovery of hyperinfectious strains similar to those obtained after
murine passage, as evidenced by a 100- fold reduced oral LD
50
and a 10
4
- fold increased i.p. CI value relative to that obtained
after growth in LB medium (Table 2). Further, the degree of
virulence exhibited by the hyperinfectious strains was exquisitely
sensitive to prior growth conditions resulting in low-, medium-,
and high- virulence states as evidenced by the varied i.p. CI values
exhibited in the four media tested (LB; LPM pH 5.5; LPM
pH 7.0; minimal medium pH 5.5). In contrast, growth of
conventionally virulent S. Typhimurium reference strain 14028
in LPM pH 5.5 conditions did not result in a pronounced increase
in virulence relative to LB medium, nor was the degree of
virulence markedly dependent on prior growth conditions as
evidenced by similar i.p. CI values in the four media tested. These
data indicate that the hypervirulent phenotype can be fully
recapitulated in vitro, and hyperinfectious strains are capable of
adopting widely disparate virulence states depending on growth
conditions. Such variability was not observed with conventionally
virulent S. Typhimurium 14028.
Table 1. Comparison of virulence states between hyperinfectious salmonellae and other clinical isolates following laboratory
culture and animal passage.
In vitro passage
b
In vivo passage
b
Strain
a
Serovar Oral LD
50c
i.p. LD
50c
Competitive
index
d
Oral LD
50
i.p. LD
50
Competitive
index
Hyperinfectious strains
x3246 S. Choleraesuis 10
5
10
2
3.0610
24
10
3
,10
1
6.2
3S. Choleraesuis 10
5
10
2
,3.0610
24
10
4
,10
1
0.6
(03)-6339 S. Choleraesuis 10
5
10
2
,3.0610
24
10
3
,10
1
2.4
58 S. Bovismorbificans 10
5
10
2
,3.0610
24
10
4
,10
1
3.0
158 S. Bovismorbificans 10
5
10
2
,3.0610
24
10
3
,10
1
1.5
208 S. Bovismorbificans 10
5
10
2
,3.0610
24
10
3
,10
1
1.8
Other clinical isolates
Lane S. Dublin 10
5
,10
1
0.6 10
5
,10
1
0.4
4973 S. Enteritidis 10
5
,10
1
1.3 10
5
,10
1
9.0
F98 S. Typhimurium 10
5
,10
1
0.5 10
5
,10
1
0.8
UK-1 S. Typhimurium 10
5
,10
1
2.4 10
5
,10
1
0.7
14028 S. Typhimurium ref. strain 10
5
,10
1
0.8 10
5
,10
1
4.6
a
All (184) Salmonella human and animal isolates tested were recovered from different outbreaks or individual cases submitted to diagnostic laboratories, or from
surveillance submissions of on-farm healthy animals [20]. Eighty-one of these strains harbored the virulence plasmid necessary for systemic disease [46] but exhibited an
i.p. virulence defect in a mouse model of typhoid fever; of these isolates, 14 were virulent by the oral route of infection. Conventionally virulent S. Typhimurium
reference strain 14028 was used in all studies for comparison.
b
In vitro/in vivo passage. In vitro passage. Bacteria derived from overnight stationary phase cultures containing LB medium were used to infect BALB/c mice via the oral
or i.p. route of infection as described in Materials and Methods.In vivo passage. Bacteria (10
9
cells) derived from stationary phase cultures containing LB medium were
used to orally infect mice. Five to seven days post-infection, bacteria derived from spleens harvested from acutely infected animals (10
8
to 10
9
CFU/g of spleen
determined by direct colony count) were used, without ex vivo growth, to infect naı
¨ve mice via the oral or i.p. route of infection as described in Materials and Methods.
c
LD
50
virulence assay. The dose required to kill 50% of infected animals (LD
50
) was determined via the oral (via gastrointubation) and i.p. routes by infecting at least 10
mice as described in Materials and Methods.
d
Competitive Index (CI) virulence assay. An equivalent dose (500 bacterial cells) of a test strain and a Lac
+
derivative of S. Typhimurium reference strain 14028 (MT2057)
was co-administered i.p. to at least 5 mice; the CI value is the ratio of test strain/reference wild-type strain recovered from target tissue (spleen) divided by the input
ratio [28] as described in Materials and Methods.
doi:10.1371/journal.ppat.1002647.t001
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The induction of hypervirulence is rapid and rapidly
reversible, and does not require vigorous bacterial cell
growth
Targeting of the actin cytoskeleton during infection by the
Salmonella SpvB cytotoxin promotes intracellular survival, host cell
cytotoxicity, and bacterial dissemination [63,64]. To understand
the mechanistic nature of switching between less-virulent and
hypervirulent states, the kinetics of hypervirulence and Salmonella
cytotoxin (SpvB) production were assessed upon transfer from
nonpermissive (LB medium) to permissive (LPM pH 5.5 medium)
conditions for the hypervirulent phenotype. Transfer of S.
Choleraesuis x3246 from LB to LPM pH 5.5 medium resulted
in a rapid transformation from the virulence-attenuated to the
hypervirulent phenotype, as evidenced by a 10
4
-fold increase in
i.p. CI value 6- to 8- cell generations (cell doublings) post-transfer
(Figure 2). This was accompanied by a 50-fold increase in SpvB
production within 1- to 2- cell generations post-transfer (Figure 2;
inset A). SpvB production was also stimulated in S. Typhimurium
14028 upon transfer from LB to LPM pH 5.5 medium, as was
shown previously after bacterial entry into macrophages and
epithelial cells [65]. However, the resultant protein levels were 8-
fold less than that of S. Choleraesuis x3246 (Figure 2; inset B).
Further, since SpvB production in S. Choleraesuis x3246
occurred more rapidly than that observed for virulence upon
media shift, the full impact of cytotoxin levels on virulence is
either delayed and/or other virulence factors are necessary for
the hypervirulent phenotype. Upon subsequent transfer from
LPM pH 5.5 medium back to LB medium, the hypervirulent
phenotype and associated cytotoxin production was rapidly
reversible to a less-virulent state, as evidenced by a 500-fold
decrease in i.p. CI value and a 30-fold reduction in SpvB within
four generations, and a further return to levels exhibited by
parental cells after 20- to 40- cell generations. The rapid and
rapidly reversible nature of the hypervirulent phenotype suggests
that a non-mutational mechanism controls the switching between
less-virulent and hypervirulent states.
Table 2. Comparison of virulence states between hyperinfectious and conventionally virulent salmonellae following growth under
defined laboratory conditions.
Oral LD
50
i.p. CI
Strain
a
Serovar LB LPM pH 5.5 LB LPM pH 5.5 LPM pH 7.0 Minimal pH 5.5
x3246 S. Choleraesuis 10
5
10
3
3.0610
24
5.0 9.6610
24
5.2610
22
158 S. Bovismorbificans 10
5
10
3
,3.0610
24
1.3 1.3610
21
8.6610
22
14028 S. Typhimurium ref. strain 10
5
10
5
0.8 2.5 0.8 3.6
a
BALB/c mice were orally or i.p. infected with hyperinfectious S. Choleraesuis x3246, S. Bovismorbificans 158 or conventionally virulent S. Typhimurium reference strain
14028 derived from stationary phase cultures containing either LB; low phosphate low magnesium (LPM pH 5.5) [41,42]; or minimal E medium [40] supplemented with
0.2% glucose and 0.1% casamino acids, at the pH indicated. Oral LD
50
and i.p. competitive index (CI) virulence assays were performed as in Table 1.
doi:10.1371/journal.ppat.1002647.t002
Figure 1. Comparison of host site colonization between hyperinfectious and conventionally virulent salmonellae following
laboratory culture and animal passage. BALB/c mice were infected orally (10
7
CFU) with hyperinfectious S. Choleraesuis x3246 (boxes) or
conventionally virulent S. Typhimurium reference strain 14028 (circles). These bacterial cells were derived from either stationary phase cultures
containing LB medium (open symbols); or after in vivo passage (closed symbols), whereby 5 to 7 days post- oral infection, spleens were aseptically
removed from acutely infected mice, and used, without ex vivo growth, to orally infect naı
¨ve animals. PP, Peyer’s Patches; MLN, mesenteric lymph
nodes; CFU, colony forming units. The symbols below the zero CFU value represent the number of mice in which the bacterial load was below the
limits of detection: PP, MLN, spleen ,40 CFU; Liver ,20 CFU.
doi:10.1371/journal.ppat.1002647.g001
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We then examined whether induction of the hypervirulent
state can occur in the absence of rapid bacterial cell growth by
transferring, without dilution, stationary-phase bacterial cells
grown in LB into LPM pH 5.5 medium. It is anticipated that
such a media shift allows for little or no bacterial cell division
since overnight growth in LB medium results in a final cell
density that is 5-fold greater than that obtained in LPM pH 5.5
medium (5610
9
CFU/ml versus 1610
9
CFU/ml, respectively).
Transfer of hypervirulent strains S. Choleraesuis x3246 and S.
Bovismorbificans 158 from LB to LPM pH 5.5 medium, without
dilution, resulted in a rapid transformation from the less-virulent
to hypervirulent state as evidenced by a 500- to 1000- fold
increase in i.p. CI value within 4 h post-transfer (Table 3). No
measurable increase in CFU (5610
9
/ml) or optical density
(OD
600
) was observed over the 10 h time course in permissive
medium (LPM pH 5.5), suggesting little or no bacterial growth is
required for the induction of hypervirulence. Conventionally
virulent Salmonella reference strain 14028 showed no marked
increase in virulence after media switch. Taken together, these
data indicate that the induction of hypervirulence is rapid and
rapidly reversible, and does not require vigorous bacterial cell
growth.
Figure 2. Comparison of the degree of virulence and cytotoxin production in hyperinfectious salmonellae following media shift to
permissive conditions for hypervirulence. The degree of virulence and Salmonella SpvB cytotoxin [63,64] production were assessed as a
function of growth under conditions that were permissive (LPM pH 5.5 medium) or non-permissive (LB medium) for the hypervirulent phenotype.
Insert A. Hyperinfectious S. Choleraesuis x3246 grown in LB medium was transferred to LPM pH 5.5 medium for 10 cell generations (cell doublings);
subsequently, such cells were transferred back into LB medium for 40 cell generations. Bacterial cells were obtained from, and maintained in,
exponential phase cultures diluted periodically such that the cell number was constant at each sampling point. Cell aliquots at the time points
indicated were assessed for virulence via i.p. competitive index (CI) virulence assays in two independent experiments (open and closed circles) and for
SpvB cytotoxin production (representative sample). SpvB cytotoxin was evaluated via whole cell protein extracts corresponding to ,7610
7
Salmonella cells subjected to SDS-PAGE, and transferred to PVDF membrane. Insert B. Conventionally virulent S. Typhimurium reference strain 14028
and hyperinfectious S. Choleraesuis x3246 grown in LB medium were transferred to LPM pH 5.5 medium for 6 cell generations; SpvB cytotoxin was
evaluated via whole cell protein extracts corresponding to ,2610
7
Salmonella cells subjected to SDS-PAGE, and transferred to PVDF membrane.
Membranes were probed with Salmonella rabbit anti-SpvB (Don Guiney, UCSD), and an infrared (IR) dye-conjugated donkey anti-rabbit
immunoglobulin G (IRDye 800CW, Li-Cor Biosciences) was used as secondary antibody. Signal was detected using an Odyssey IR imaging system (Li-
Cor Biosciences).
doi:10.1371/journal.ppat.1002647.g002
Table 3. Comparison of virulence states between
hyperinfectious and conventionally virulent salmonellae
following transfer from nonpermissive to permissive
conditions for the hypervirulent phenotype.
Virulence (i.p. CI)
Time post-transfer (h)
Strain
a
Serovar 0 124810
x3246 S. Choleraesuis 0.0003 0.002 0.008 0.148 0.568 0.570
158 S. Bovismorbificans ,0.0003 0.015 0.028 0.296 0.592 0.813
14028 S. Typhimurium ref.
strain
1.14 1.49 1.49 1.68 1.63 1.93
a
Hyperinfectious S. Choleraesuis x3246 and S. Bovismorbificans 158 as well as
conventionally virulent S. Typhimurium reference strain 14028 were grown
overnight in LB medium. The stationary-phase cells were transferred without
dilution, into permissive conditions for the hypervirulent phenotype (LPM
pH 5.5 medium). Virulence was assessed as a function of time (h) post-transfer
to LPM pH 5.5 medium via i.p. competitive index (CI) virulence assays as in
Table 1.
doi:10.1371/journal.ppat.1002647.t003
Emerging Hyperinfectious Strains
PLoS Pathogens | www.plospathogens.org 7 April 2012 | Volume 8 | Issue 4 | e1002647

Environmental conditions that confer a growth
advantage to hyperinfectious salmonellae in vivo are
associated with a growth disadvantage in vitro
Expression of virulence functions that confer hypervirulence
during the infective process may be deleterious to growth outside
of the host. Thus, we questioned whether environmental
conditions that conferred a growth advantage to hyperinfectious
strains in vivo are associated with a growth disadvantage in vitro
relative to conventionally virulent Salmonella. Hyperinfectious S.
Choleraesuis x3246 and conventionally virulent S. Typhimurium
reference strain 14028 were grown in competition under
conditions that were either permissive (LPM pH 5.5 medium) or
nonpermissive (LB medium) for hypervirulence. An equivalent
dose of both Salmonella strains (5610
7
CFU/ml) were co-cultured
in either LPM pH 5.5 or LB medium following prior growth
individually in the same medium. S. Choleraesuis x3246 was
outcompeted in the mixed population to a far greater extent in
LPM pH 5.5 medium than in LB medium (Figure 3). These data
indicate that growth under environmental conditions that fully
recapitulate the hypervirulent state obtained after in vivo passage
is detrimental to bacterial fitness in vitro- suggesting the possibility
that virulence functions favorable for in vivo growth are
unfavorable ex vivo.
The induction of hypervirulence is associated with an
increased capacity to provoke macrophage cell death
relative to conventionally virulent strains
Salmonella infection of macrophages provokes a caspase-
mediated proinflammatory cell death program, termed pyroptosis
[49,66,67]. Here we examined whether hyperinfectious salmonel-
lae are associated with an increased capacity to initiate
macrophage cell death versus conventionally virulent strains.
Hyperinfectious strains (S. Choleraesuis x3246 and S. Bovismor-
bificans 158) and conventionally virulent S. Typhimurium
reference strain 14028 were grown under conditions that were
permissive (LPM pH 5.5 medium) or nonpermissive (LB medium)
for hypervirulence, and used to infect RAW264.7 murine
macrophage cell cultures at a multiplicity of infection (MOI) of
10:1 or 100:1. A crystal violet dye retention assay was used to
assess the degree of Salmonella cytocidal activity within cultured
macrophages, measured spectrophotometrically 20 h after infec-
tion [49,50]; high cytocidal activity is associated with low dye
retention and vice versa. Infection with hyperinfectious strains (S.
Choleraesuisx3246and S. Bovismorbificans 158) resulted in a dose-
dependent increase in cytocidal activity after prior growth in LPM
pH 5.5 relative to LB medium (2.8-fold and 1.5-fold, respectively;
MOI of 100:1; Table 4). In contrast, infection with conventionally
Figure 3. Comparison of growth rates between hyperinfectious and conventionally virulent salmonellae grown under in vitro
conditions that are permissive for hypervirulence. An equivalent dose of hyperinfectious S. Choleraesuis x3246 and conventionally virulent S.
Typhimurium reference strain 14028 (5610
7
CFU/ml) were co-cultured in either permissive (LB; open boxes) or nonpermissive (LPM pH 5.5 medium;
closed boxes) conditions for the hypervirulent phenotype, following prior growth individually in the same medium. Cell aliquots were sampled for
CFU at the cell generation (cell doubling) indicated. Bacterial cells were obtained from, and maintained in, exponential phase cultures diluted
periodically such that the cell number was constant at each sampling point. The in vitro competition index is the relative ratio of test strain/reference
wild-type strain recovered from the co-culture divided by the input ratio. The values represent the relative ratio of S. Choleraesuis/S. Typhimurium
obtained from 3 independent cultures with the standard error bars designated.
doi:10.1371/journal.ppat.1002647.g003
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virulent S. Typhimurium reference strain 14028 resulted in a dose-
dependent decrease in cytocidal activity after prior growth in LPM
pH 5.5 medium relative to LB (2.5-fold; MOI of 100:1). The
differences in cytocidal activity between hyperinfectious strains
grown in LB versus LPM pH 5.5 relative to that observed with S.
Typhimurium 14028 were statistically significant (P,0.05). Taken
together, these data establish that hyperinfectious strains are
associated with an increased capacity to provoke macrophage cell
death relative to conventionally virulent strains; the induction of
cytocidal activity occurs in a dose-, media-, and strain- dependent
fashion.
Infection of cultured macrophage cells with
hyperinfectious strains is associated with an altered host
innate immune cytokine response
Recognition of conserved pathogen associated molecular
patterns (PAMPs) by host-cell pattern recognition receptors
(PRRs) activates signaling pathways leading to the stimulation of
the innate immune response, characterized by the production of
cytokines and interferon system gene products and their potent
antimicrobial actions [68–72]. To understand the mechanistic
basis of hypervirulence, we examined whether infection of cultured
RAW264.7 macrophage cells with hyperinfectious strains is
associated with an altered innate immune cytokine response. For
this analysis, we assessed the relative transcript levels of cytokine
and interferon (IFN) system genes known to be induced during
Salmonella infection including; the type I IFN system gene, IFN-b
[50,53]; the inflammatory and acute phase response genes,
interleukin-1 beta (IL-1b) and IL-6 [53,73–76]; inducible nitric
oxide synthase (iNOS), a known target of IFN and cytokine
signaling required for resistance to Salmonella infection [53,77–79];
and IL-10, an inhibitory modulator of the inflammatory response
[74,75,80,81]. Hyperinfectious strains (S. Choleraesuis x3246 and
S. Bovismorbificans 158) and conventionally virulent S. Typhi-
murium reference strain 14028 were grown under conditions that
were permissive (LPM pH 5.5 medium) or nonpermissive (LB
medium) for hypervirulence, and used to infect cultured
RAW264.7 murine macrophage cells. At 2, 5 and 8 h post-
infection, RNA was derived from cultured cells and used to assess
relative cytokine transcript levels in infected versus uninfected
cells. Three salient observations were made (Figure 4): 1) Although
reduced induction of all cytokine transcripts tested was observed
upon infection with both hyperinfectious and conventionally
virulent strains grown in LB versus LPM pH 5.5 medium
(P,0.05), only hyperinfectious strains exhibited significant re-
duced stimulation of IFN-b, IL-1b, and IL-6 transcript levels at the
2 h infection time point (2.5- to 3.5- fold; P,0.05). 2) The reduced
stimulation of IL-1band IL-6 exhibited by hyperinfectious strains
at the 2 h time point was followed by a 14- to 30- fold induction at
the 8 h time point. 3) Hyperinfectious strains exhibited signifi-
cantly reduced stimulation of IL-10 relative to S. Typhimurium
14028 irrespective of LB or LPM pH 5.5 growth conditions
(P,0.05); such reduced stimulation was most pronounced at later
infection time points (i.e., 10-fold at t= 8 h under LPM pH 5.5
conditions). These data indicate that hyperinfectious strains confer
altered kinetics/magnitude of the innate immune cytokine
responses that coordinate bacterial clearance via stimulation of
signaling receptors and resultant cellular activation and the
induction of effector mechanisms; e.g., Toll-like receptor recog-
nition/signaling; inflammasome activation; myeloid cell recruit-
ment; and T cell activation [82–84].
Gene expression analysis of Salmonella hyperinfectious
strains
Gene expression analysis was performed to identify bacterial
gene transcripts that were significantly altered in hyperinfectious
strains under LPM pH 5.5 versus LB conditions, and not altered,
or altered to the same extent, in a conventionally virulent strain.
We established that transfer of hypervirulent strains from LB to
LPM pH 5.5 medium resulted in a transformation from the less-
virulent to hypervirulent state within 4 h post-transfer (Table 3)
before proceeding with additional observations. S. Choleraesuis
x3246 and S. Bovismorbificans 158 were grown overnight in LB
medium and transferred, without dilution, to LPM pH 5.5
medium. At 4 h post-transfer, RNA was derived from bacterial
cells and used to assess relative transcript levels in cells grown in
LPM versus LB via hybridization to a custom Salmonella Affymetrix
Genechip (see Materials and Methods). Microarray analysis revealed
that, 4 h post-transfer from LB to LPM pH 5.5 medium,
hyperinfectious strains displayed distinct transcriptional responses
versus those observed in a conventionally virulent strain (Figure 5;
Table S1). At least 3 distinct classes of differentially-regulated
genes are represented, including those under the control of the
PhoP/PhoQ regulatory system, a global regulator of Salmonella
virulence [62,85–87]; the PhoR/PhoB regulatory system involved
in nutrient (phosphate) stress [88,89]; and the ArgR regulatory
system involved in arginine metabolism including acid stress [90–
94] (Table 5). Although differential regulation of these genes was
observed in both hypervirulent and conventionally virulent strains
following transfer from LB to LPM pH 5.5 medium, the degree to
which gene expression is altered differs significantly between them.
For example, several representative genes show a higher level of
induction in hypervirulent strains relative to conventionally
virulent strains (mgtBC; Mg
2+
transport [PhoP/Q]; phoB; PO
4
22
transport [PhoR/B]; argA; artJ; arginine metabolism [ArgR]).
Table 4. Comparison of hyperinfectious and conventionally
virulent salmonellae cytocidal activity upon infection of
cultured macrophages.
Cytocidal activity (A
577
)
a
LB LPM pH 5.5
MOI
Strain Serovar 10 100 10 100
x3246 S. Choleraesuis 0.561 0.261 0.297* 0.094*
158 S. Bovismorbificans 0.246 0.226 0.286 0.151*
14028 S. Typhimurium ref. strain 0.328 0.102 0.560 0.251
a
Hyperinfectious S. Choleraesuis x3246 and S. Bovismorbificans 158 as well as
conventionally virulent S. Typhimurium reference strain 14028 were derived
from stationary phase cultures under permissive (LPM pH 5.5 medium) or
nonpermissive (LB medium) conditions for the hypervirulent phenotype.
Cultured RAW264.7 murine macrophage cells were infected with bacteria at a
multiplicity of infection (MOI) of 10:1 or 100:1 At 20 h post-infection,
macrophages were stained with crystal violet, and bacterial cytocidal activity
was quantified spectrophotometrically (577 nm) as described in Materials and
Methods; high cytocidal activity is associated with low dye retention. Data given
are representative absorbance values derived from each condition performed in
triplicate. Standard error of triplicate means is ,20%.
*Designates statistical significance for changes in cytocidal activity of
hyperinfectious strains grown in LB versus LPM pH 5.5 medium relative to that
found with reference strain S. Typhimurium 14028. Cytocidal activity was
analyzed using analysis of variance; the change in cytocidal activity of the
hyperinfectious S. Choleraesuis and S. Bovismorbificans were individually
contrasted to the change in cytocidal activity of S. Typhimurium 14028 at each
dose level. A significance level (P) of less than 0.05 was considered to be
statistically significant.
doi:10.1371/journal.ppat.1002647.t004
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PLoS Pathogens | www.plospathogens.org 9 April 2012 | Volume 8 | Issue 4 | e1002647

Conversely, other PhoP/Q activated genes show a lower level of
induction (pagK;sifB; SPI-2 effectors) or repression (rtsA; SPI-I
activator) in hypervirulent strains relative to that found in
conventional virulent strains. Increased induction of virulence
functions involved in cellular physiology and metabolism (mgtBC;
phoB; argA) in combination with repression of SPI-1 virulence
Figure 4. Comparison of cytokine transcript levels in cultured macrophages infected with hyperinfectious and conventionally
virulent salmonellae. Innate immune cytokine transcript levels were examined from cultured RAW264.7 murine macrophages infected with
hyperinfectious S. Choleraesuis x3246, S. Bovismorbificans 158 or conventionally virulent S. Typhimurium reference strain 14028 grown under
permissive (LPM pH 5.5; dotted lines) or nonpermissive (LB; solid lines) conditions for the hypervirulent phenotype. (A) IFN-b; (B) IL-1b; (C) IL-6; (D)
iNOS; (E) IL-10. Bacterial cells derived from stationary phase cultures containing either LB or LPM pH 5.5 medium were used to infect cultured RAW
264.7 murine macrophage cells as described in Materials and Methods. The bacteria were centrifuged onto cultured monolayers at 1,0006gfor
10 min at room temperature, after which they were incubated for 30 min at 37uC in a 5% CO
2
incubator (t= 0 time point). The coculture was washed
once and incubated for 45 min with gentamicin (100 mg/ml) at 37uC in a 5% CO
2
incubator, washed once with pre-warmed cell culture medium, and
incubated with gentamicin (10 mg/ml) to the time points indicated (2, 5 and 8 hr). Total RNA was isolated from infected cultured RAW 264.7 murine
macrophage cells, and from mock-infected controls as described in Materials and Methods. RNA samples were analyzed by reverse transcription and
real-time qPCR for: IFN-b; IL-1b; IL-6; iNOS; and IL-10 expression as described in Materials and Methods. Relative target gene transcripts were
normalized to the level of the GAPDH gene, relative to the average of the normalized values obtained for uninfected RAW 264.7 cells. Values given
were obtained from triplicate wells SE ,22%. Although reduced stimulation of all cytokine transcripts tested was observed upon infection with both
hyperinfectious and conventionally virulent strains grown in LPM pH 5.5 medium relative to that exhibited in LB medium (P,0.05), only
hyperinfectious strains exhibited a significant reduced stimulation of IFN-b, IL-1band IL-6 transcript levels at the 2 h infection time point (2.5- to 3.5-
fold; P,0.05).
*
Designates statistical significance for those measures that are specific to hypervirulent strains after growth in LPM pH 5.5 medium
relative to that exhibited in LB medium (P,0.05).
doi:10.1371/journal.ppat.1002647.g004
Emerging Hyperinfectious Strains
PLoS Pathogens | www.plospathogens.org 10 April 2012 | Volume 8 | Issue 4 | e1002647

functions involved in invasion after bacterial entry into host cells
(repression of the hilA activated SPI-1 regulatory cascade via rtsA
down-regulation; phoB up-regulation [reviewed in [95]]) may
increase the capacity of hypervirulent strains to undergo in vivo
adaptation.
Infection with hyperinfectious salmonellae leads to
increased killing of vaccinated animals
Salmonella live attenuated vaccines that contain mutations in the
DNA adenine methylase (dam) confer cross-protective immunity
against virulent challenge with heterologous Salmonella strains in
murine, avian, and bovine models of salmonellosis [19,96–99].
Here, we assessed whether growth of hyperinfectious strains under
permissive conditions for hypervirulence (LPM pH 5.5 medium)
leads to increased killing of vaccinated animals. Mice immunized
with a dam mutant vaccine were more susceptible to infection with
hyperinfectious strains grown under LPM pH 5.5 versus LB
medium in four of five hyperinfectious S. Choleraesuis and S.
Bovismorbificans strains tested (P,0.05) (Table 6). The lone
exception is S. Choleraesuis x3246 to which the vaccine conferred
poor efficacy under either media tested, although a similar trend
was observed (P= 0.20). No change in protection was observed in
vaccinated animals following challenge with conventionally
virulent S. Typhimurium reference strain 14028 grown under
either media condition. These data indicate that hyperinfectious
salmonellae exhibit increased killing of vaccinated animals,
suggesting that immunized populations are more susceptible to
infection by strains bearing the hypervirulent phenotype.
Discussion
Salmonellosis is a principal health concern because of the
endemic prevalence of salmonellae in food and water supplies.
Recent estimates by the CDC and other sources indicate that
Salmonella infections cause 1.4 to 1.6 million foodborne illnesses in
the U.S. annually at an estimated cost of $2.6 to $14.6 billion
[100–104]. This health and economic burden will most likely
continue to expand due to increased multi-drug resistance and the
emergence of new strains that are associated with an increased
incidence and/or severity of disease [1,9,16]. Insights into the
emergence of pathogenic strains have come from animal-passage
studies wherein virulence traits are often increased (reversibly)
following infection (e.g., hastened colonization, morbidity, and/or
mortality; reviewed in [22–24]). Here we show that some Salmonella
strains are considerably more virulent after murine passage
relative to other isolates (100-fold decreased LD
50
); and the
display of increased virulence traits by bacterial strains after
passage does not necessarily equate to hypervirulence. Hyper-
infectious strains are among the most virulent salmonellae
reported, were restricted to certain serovars, and vaccination
conferred poor protection against infection. These strains pose a
potential risk to food safety as the parental isolates- from which
they were derived- originated from diseased livestock. Molecular
characterization of these strains may yield insights into the
emergence of hyperinfectious pathogens and the development of
intervention strategies for human and animal salmonellosis.
Our findings indicate that salmonellae exhibit intraspecies
variation in the development of hyperinfectious strains, as
evidenced by the increased likelihood of particular serovars
displaying the hypervirulent phenotype than others following
murine infection (S. Bovismorbificans [11/11] versus S. Typhi-
murium [0/52]). The hypervirulent phenotype was recapitulated
in vitro with strains adopting distinct virulence states actuated by
prior growth conditions, suggesting that the degree of virulence
Figure 5. Transcriptome analysis of hyperinfectious strains.
Gene expression analysis was performed to identify bacterial gene
transcripts that were significantly altered in hyperinfectious strains
under LPM pH 5.5 versus LB conditions, and not altered, or altered to
the same extent, in a conventionally virulent strain. Hyperinfectious
strains (S. Bovismorbificans 158 [SB] and S. Choleraesuis x3246 [SC]) and
S. Typhimurium reference strain 14028 [ST] were grown overnight in LB
medium, pelleted and washed in 0.15M NaCl, and split without dilution
into two cultures containing either LB or LPM pH 5.5 medium. The
cultures were incubated with aeration for 4 h, after which approxi-
mately 2.5610
10
cells were pelleted via centrifugation. RNA derived
from these bacterial cells was used to assess relative transcript levels in
bacterial cells via hybridization to a custom Salmonella Affymetrix
Genechip as described in Materials and Methods. Each of the 12
columns of the heat map represents an LPM/LB ratio with four pairwise
comparisons provided for each strain. Two criteria were used as a cutoff
to identify the genes that were significantly altered in hyperinfectious
strains (SB; SC) under LB versus LPM pH.5.5 conditions, and not altered,
or altered to the same extent, in a conventionally virulent strain (ST); i.e.,
at least a 2-fold expression change in SB, SC or ST; and a 0.05 false
discovery rate (FDR) when comparing log
2
LPM/LB ratios values for SB
and SC versus ST. Heat maps were generated from the resultant list of
genes using The Institute for Genomic Research MultiExperiment
Viewer (MeV), version 4.7 [54]. All expression experiments were done in
two biological replications.
doi:10.1371/journal.ppat.1002647.g005
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exhibited by these strains can be modified significantly within
different hosts, during different infection states (sub-clinical versus
fulminate infection), or after exposure to certain environmental
variables. Thus, these strains may lead to disease under some
environs but not others [105] (e.g., varied levels of moisture, heat
stress, cell density, salts/nutrients). Consequently, in an outbreak
scenario, although knowledge of the strain serotype is useful
epidemiologically, it may have limited predictive value as to the
clinical disease outcome or whether protection will be provided by
vaccination.
The mechanistic basis for hypervirulence appears to be the
consequence of increased microbial pathogenicity accompanied by
microbe-mediated alterations in innate immune cytokine respons-
es in infected animals. This is evidenced by increased microbial
cytotoxin (SpvB) production, host tissue site colonization, and
cytocidal activity that may coexist in time with a delayed
proinflammatory IFN/cytokine response coupled with a dimin-
ished proinhibitory (IL-10) cytokine response over the entire
infection time course. This immune antagonism strategy is often
employed by viruses, interfering with multiple stages of the innate
immune response; e.g., disruption of pathogen recognition,
downstream signaling pathways, and subsequent repression/
inhibition of a number of innate immune responses [69,106–
108]. Altered innate immunity during the Salmonella infective
process can profoundly impact disease outcome as the bacterium
must strike a balance between initiating inflammatory responses to
Table 5. Bacterial gene transcripts that were specifically altered in hyperinfectious strains under permissive conditions for the
hypervirulent phenotype.
Gene number Gene symbol Log
2
LPM/LB ratio
a
Description
ST SB SC
PhoP/PhoQ
STM4286 lpxO 0.54 1.74 1.88 Lipid A modification [139].
STM3763 mgtB 1.74 4.34 4.40 Magnesium transporter; required for virulence [140].
STM3764 mgtC 2.18 5.13 5.34 Magnesium transport; required for intramacrophage survival and long term systemic infection [141].
STM2585 pagJ 4.26 20.27 0.24 SPI-2 effector; translocated to macrophage cytoplasm [142].
STM1867 pagK 3.12 1.47 0.61 SPI-2 effector; translocated to macrophage cytoplasm [142].
STM1862 pagO 0.67 1.71 1.89 Homology to pagO in Klebsiella pneumoniae drug/metabolite exporter [143].
STM0397 phoB 0.81 2.50 1.72 Response regulator of PhoR/B regulon; represses hilA activated SPI-1 effectors [140,144].
STM4315 rtsA 0.04 21.62 21.39 Activates hilA and downstream SPI-1 effectors; required for cell invasion [88,145].
STM1471 rtsB 21.16 0.72 20.03 Sensory histidine kinase; acts on PhoQ to control PhoP regulated genes [95].
STM1602 sifB 1.47 0.56 0.68 SPI-2 effector; translocated to macrophage cytoplasm [146].
STM0366 yahO 1.22 20.43 21.13 Modification of cell envelope [147].
STM0614 ybdQ 1.46 0.37 20.09 Universal stress protein [144].
PhoB/PhoR
STM4287 phnO 0.16 1.43 0.96 Regulator of phosphocarbonate breakdown [148].
STM0397 phoB 0.81 2.50 1.72 Response regulator of PhoR/B regulon; represses hilA activated SPI-1 effectors [140,144].
STM0320 phoE 0.03 0.87 1.26 Outer membrane pore protein induced in phosphate limiting conditions [149].
STM0384 psiF 1.34 20.40 20.23 Phosphate inducible starvation protein [148].
STM3854 pstB 0.74 2.12 1.81 High affinity phosphate transporter [148].
STM3857 pstS 0.81 3.08 2.38 Induced in macrophage; regulates hilA through phoB [89].
STM4226 yjbA 0.89 1.59 1.60 Induced during macrophage infection [150]; also known as psiE.
ArgR
STM2992 argA 0.51 2.71 3.50 N-acetylglutamate synthase [91].
STM4122 argB 0.56 1.64 2.74 Acetylglutamate kinase [91].
STM3468 argD 1.26 2.84 4.31 Bifunctional N-succinyldiaminopimelate-aminotransferase/acetylornithine transaminase protein [91].
STM3290 argG 20.17 1.54 1.24 Arginosuccinate synthase [91].
STM4123 argH 20.20 1.53 2.10 Arginosuccinate lyase [91].
STM4469 argI 0.36 2.05 3.19 Ornithine transcarbamylase [91].
STM0887 artJ 0.78 1.76 2.61 Arginine transport system component [91].
Other virulence-associated genes
STM4077 yneA 20.26 21.31 20.96 Involved in quorum sensing; encodes periplasmic receptor for AI-2 [151]; also called lsrB.
STM2084 rfbM 2.03 20.13 0.33 Involved in O-antigen synthesis [152]; also known as manC.
a
The log
2
LPM/LB gene expression ratios values for conventionally virulent S. Typhimurium (ST), and hypervirulent S. Bovismorbificans (SB) and S. Choleraesuis (SC)
strains were determined as described in Materials and Methods.
doi:10.1371/journal.ppat.1002647.t005
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promote colonization while avoiding prolonged inflammatory
responses that damage host niches occupied by the microbe during
infection [109–111]. Further, since it is well-established that innate
immune responses stimulate the development of adaptive
immunity [68,112,113], elicitation of an altered IFN/cytokine
signature may contribute to the observed increased disease
susceptibility in vaccinated animals.
Gene expression analysis revealed that transfer from nonpermis-
sive to permissive conditions for the hypervirulent phenotype (LB
versus LPM pH 5.5 medium) resulted in distinct transcriptional
responses in hypervirulent strains that were not altered, or altered to
the same extent, in a conventionally virulent strain. Three major
classes of differentially-regulated genes were identified: those that
reside in the PhoP/PhoQ [62,85–87]; PhoR/PhoB [88,89]; or
ArgR regulons [92–94] that confer changes in the expression of
classical virulence functions (e.g., SPI-1 and SPI-2 effectors) as well
as marked changes in cellular physiology and metabolism (nutrient
and acid stress response). Such altered regulatory circuitry can
contribute in several ways to increased host cell intoxication,
immune evasion, and virulence exhibited by hyperinfectious strains.
1) SPI-1 and SPI-2 effectors are known to harbor potent
immunomodulatory properties resulting in altered host-cell signal-
ing and resultant innate immune cytokine responses [2,114]; down-
regulation of SPI-1 invasion genes upon bacterial entry (rstA; phoB)
may optimize survival/proliferation in the Salmonella containing
vacuole (SCV). 2) Altered physiologic and metabolic changes
(mgtBC; phoB; argA) are known to impact differences in species-
specific lifestyle/behavior; e.g., differential regulation of metabolic,
transporter, and motility functions in Bordetella spp. is thought to
increase the capacity of ex vivo adaptation of B. bronchiseptica [115].
Taken together, altered timing, magnitude, and localization of
bacterial gene expression can have profound effects on virulence
and host immune responses.
Intraspecies variation in the capacity to become hypervirulent
may be due to genes encoded by one serotype but not another
and/or altered expression of preexisting virulence functions.
Acquisition of the viaB locus in S. Typhi provides genes for Vi
capsular biosynthesis (tviBCDE) and a regulatory gene (tviA) that
alters expression of Vi antigen, flagella and the invasion-associated
type III secretion system in response to changes in osmolarity
[116,117]. Such altered expression results in reduced inflamma-
tory responses relative to non-typhoidal serotypes, and introduc-
tion of the viaB locus into S. Typhimurium reduces the
inflammatory response conferred by this pathogen [118]. Addi-
tionally, intraspecies variation in the capacity to become
hypervirulent may be due to differential expression (transcriptional
re-wiring) of preexisting virulence genes as is the case in cross-
species comparisons between BvgA/S regulatory circuit in B.
pertussis and B. bronchiseptica [115] and the PhoP/PhoQ regulatory
circuits in multiple Enterobacteriaceae [119,120]. Thus, intraspe-
cies variation in the capacity to become hypervirulent may be the
consequence of gene acquisition and/or altered expression of
preexisting virulence functions via alterations in principal
regulatory proteins; downstream regulatory proteins; and/or by
cis-acting alterations in target genes [121–124].
Our findings indicate that the phase-variable phenotypes
associated with Salmonella hyperinfectious strains are consistent
with a phenotypic modulation mechanism as switching between
virulence states was rapid and rapidly reversible (non-mutational);
did not require vigorous bacterial cell growth; and was responsive
to subtle differences in environmental signals resulting in multiple
virulence states. Consistent with this suggestion, environmental
conditions that stimulate/inhibit the BvgA/BvgS regulatory
system in Bordetella results in the expression of at least three
distinct phenotypic phases that are each associated with a unique
gene expression profile thought to play an explicit role in the
infectious cycle [125,126]. This provides a potential means to
rapidly adapt to disparate hosts/environments without undergoing
irreversible changes in the genome, and may contribute to the
maintenance of hyperinfectious strains in nature. Additionally,
other serotypes may potentially exhibit hypervirulence in response
to passage through certain hosts or exposure to certain
environments; and this response may be the case across the
microbial realm.
Molecular examination of hyperinfectious strains may provide
insights into i) differences in disease outcomes between closely-
related strains; ii) distinct outbreak scenarios that point to the same
infectious agent; iii) differences in vaccine efficacy between
laboratory versus clinical field trials due to the environmental
complexity of commercial livestock production systems; and iv) the
design of vaccines and therapeutic strategies to improve clinical
disease outcomes.
General implications
From a farm-management perspective, it is desirable to
understand the management and environmental events that lead
to hypervirulence in the context of the production system so that
risk management strategies can be implemented to prevent
disease. It has been established in livestock that host susceptibility
and shedding are dependent on management and environmental
events (herd size, adverse weather conditions, equipment failure,
labor issues, surface water management) that contribute to
compromised host immunity and increased pathogen exposure
[7,12,13,127–129]. Our studies suggest that livestock production
systems have the potential for management and environmental
events to alter pathogen virulence. That is, environmental
conditions inherent to livestock/feedlots (manure, fecal pack and
urine), the influence of diet (high and low protein, fiber, and fat),
and/or exposure to sub-therapeutic concentrations of antimicro-
Table 6. Comparison of disease susceptibility in vaccinated
mice infected with hyperinfectious and conventionally
virulent salmonellae.
Survivors/Total
Strain
a
Serovar LB LPM pH 5.5
x3246 S. Choleraesuis 11/41 8/56
3S. Choleraesuis 14/20 3/23
*
158 S. Bovismorbificans 16/20 10/24
*
174 S. Bovismorbificans 19/22 10/23
*
225 S. Bovismorbificans 18/18 7/20
*
14028 S. Typhimurium ref. strain 20/20 19/22
a
BALB/c mice orally immunized with a live, attenuated dam mutant S.
Typhimurium 14028 vaccine [98]. Vaccinated mice challenged with a dose of
100 LD
50
of hyperinfectious salmonellae derived from stationary phase cultures
under conditions that were permissive (LPM pH 5.5 medium) or nonpermissive
(LB medium) for the hypervirulent phenotype. Nonvaccinated control mice (25/
group) all died by day 21 post-infection. Conventionally virulent S.
Typhimurium reference strain 14028 was used in all studies for comparison.
*Designates statistical significance for the number of survivors obtained after
dam mutant Salmonella vaccinated animals were challenged with salmonellae
grown in LB medium versus LPM pH 5.5 medium. Statistical significance for
difference in proportions was calculated using Chi-square tests; a significance
level (P) of less than 0.05 was considered to be statistically significant.
doi:10.1371/journal.ppat.1002647.t006
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bials may also inadvertently trigger the induction of salmonellae
hypervirulence in livestock.
Epidemiological studies in livestock indicate that the pathoge-
nicity and persistence of S. Typhimurium variants range from
those that cause infections that are relatively mild and geograph-
ically limited, to those that cause small epidemics that circulate in
livestock and humans [130,131], to those that are multi-drug
resistant and have the capacity for pandemic spread and increased
human and animal disease [132,133] (reviewed in [134,135]).
Further, although it is common to find salmonellae on farms [7–9],
a given strain may not be significant from a disease or food safety
perspective. Thus, the development of a means to identify strains
that are likely to be virulent (or hypervirulent) would provide a
better measure of causality and food safety risk and may lead to
the identification of targets for immunoprophylaxis.
Such detection may be complicated by the fact that other
serotypes may potentially become hypervirulent in response to
passage through certain hosts or exposure to certain environments
(e.g., cow, pig, manure, surface water); and this response may be
prevalent in other pathogens. Thus, molecular characterization of
hypervirulence cannot be solely concluded on the basis of
culturing in rich media, and more efforts should be given to
determining virulence characteristics under more physiological
growth conditions and/or in animal models of infection. Of
potential benefit to therapeutic efforts are live-animal infection
model screens for virulence factors and antibiotics that target
microbial functions that confer a growth advantage in vivo relative
to that observed in vitro [136–138].
Future work will focus on the molecular basis of the emergence
of hyperinfectious salmonellae and the development of vaccines, as
well as dietary and environmental management strategies to
mitigate these potential food-borne contaminants before they
cause negative public health impacts and economic losses.
Supporting Information
Table S1 List of Salmonella differentially regulated
genes in hyperinfectious versus conventionally virulent
strains under permissive and nonpermissive conditions
for the hypervirulent phenotype. Gene expression analysis
was performed to identify bacterial gene transcripts that were
significantly altered in hyperinfectious strains under LPM pH 5.5
versus LB conditions, and not altered, or altered to the same
extent, in a conventionally virulent strain as described in Figure 5
legend and Materials and Methods.
(XLSX)
Acknowledgments
We thank Cyril George for assistance with innate immune cytokine
response studies.
Author Contributions
Conceived and designed the experiments: DMH WRS JKH YX BCW
RLS MJM. Performed the experiments: DMH WRS YX. Analyzed the
data: DMH WRS JKH YX BCW RLS MJM. Contributed reagents/
materials/analysis tools: DMH WRS JKH YX BCW RLS MJM. Wrote
the paper: DMH WRS JKH YX BCW RLS MJM.
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