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Critical Reviews in Food Science and Nutrition


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Shigella as a Foodborne Pathogen and Current Methods


for Detection in Food
a b a
B. R. Warren , M. E. Parish & K. R. Schneider
a
University of Florida, Department of Food Science and Human Nutrition , 359 FSHN Bldg,
Newell Drive, Gainesville, FL, 32611, USA
b
University of Maryland, Department of Nutrition and Food Science , 0112 Skinner Bldg.,
College Park, MD, 20742, USA
Published online: 18 Jan 2007.

To cite this article: B. R. Warren , M. E. Parish & K. R. Schneider (2006) Shigella as a Foodborne Pathogen and
Current Methods for Detection in Food, Critical Reviews in Food Science and Nutrition, 46:7, 551-567, DOI:
10.1080/10408390500295458

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Critical Reviews in Food Science and Nutrition, 46:551567 (2006)
Copyright C Taylor and Francis Group, LLC
ISSN: 1040-8398
DOI: 10.1080/10408390500295458

Shigella as a Foodborne Pathogen


and Current Methods for Detection
in Food

B.R. WARREN
Downloaded by [Montana State University Bozeman] at 03:41 18 August 2014

University of Florida, Department of Food Science and Human Nutrition, 359 FSHN Bldg, Newell Drive, Gainesville, FL
32611, USA

M.E. PARISH
University of Maryland, Department of Nutrition and Food Science, 0112 Skinner Bldg., College Park, MD 20742, USA

K.R. SCHNEIDER
University of Florida, Department of Food Science and Human Nutrition, 359 FSHN Bldg, Newell Drive, Gainesville, FL
32611, USA

Shigella, the causative agent of shigellosis or bacillary dysentery, has been increasingly involved in foodborne outbreaks.
According to the Centers for Disease Control and Preventions Emerging Infections Program, Foodborne Diseases Active
Surveillance Network (FoodNet), Shigella was the third most reported foodborne bacterial pathogen in 2002. Foods are
most commonly contaminated with Shigella by an infected food handler who practices poor personal hygiene. Shigella is
acid resistant, salt tolerant, and can survive at infective levels in many types of foods such as fruits and vegetables, low pH
foods, prepared foods, and foods held in modified atmosphere or vacuum packaging. Survival is often increased when food
is held at refrigerated temperatures. Detection methods for Shigella include conventional culture methods, immunological
methods, and molecular microbiological methods. Conventional culture of Shigella in foods is often problematic due to
the lack of appropriate selective media. Immunological methods for Shigella have been researched, yet there is only one
commercially available test kit. Molecular microbiological methods such as PCR, oligonucleotide microarrays, and rep-PCR
have also been developed for the detection and identification of Shigella. This manuscript reviews the general characteristics,
prevalence, growth and survival, and methods for detection of Shigella in food.

Keywords conventional, polymerase chain reaction, PCR, media

SHIGELLA AS A FOODBORNE PATHOGEN non-motile rods that typically do not ferment lactose. In addition,
they are lysine-decarboxylase, acetate, and mucate negative and
Background Information do not produce gas from glucose (some S. flexneri 6 serotypes
may produce gas) (Echeverria et al., 1991; ICMSF, 1996).
Shigella, the causative agent of shigellosis or bacillary There are four serogroups of Shigella: S. dysenteriae
dysentery, was first discovered over 100 years ago by Kiyoshi (serogroup A) serotypes 115, S. flexneri (serogroup B)
Shiga, a Japanese scientist (Anonymous, 2002). Shigellae, mem- serotypes 18 (9 subtypes), S. boydii (serogroup C) serotypes
bers of the family Enterobacteriaceae, are nearly identical ge- 119, and S. sonnei (serogroup D) serotype 1. The four
netically to Escherichia coli (E. coli) and are closely related serogroups of Shigella differ in epidemiology (Ingersoll et al.,
to Salmonella and Citrobacter spp. (Lampel, 2001). Shigellae 2002). S. dysenteriae is primarily associated with epidemics
are Gram-negative, facultatively anaerobic, non-sporeforming, (Ingersoll et al., 2002) with serotype 1 associated with the
highest fatality rate (515%) (CDC, 2003c). S. flexneri pre-
dominates in areas of endemic infection, while S. sonnei has
Address correspondence to Keith Schneider, FSHN Department, 359 FSHN been implicated in source outbreaks in developed countries
Bldg, Newell Drive, Gainsville, FL 32611. Email: krschneider@ifas.ufl.edu (Hale, 1991). S. boydii is associated with source outbreaks in
551
552 B. R. WARREN ET AL.

Central and South America and is rarely isolated in North are fully invasive and all virulence plasmid polypeptides are
America. encoded (Sansonetti, 1991).
Shigella has been classically characterized as a waterborne Once ingested, Shigellae move through the gastrointestinal
pathogen (Smith, 1987) and outbreaks have occurred from con- tract to the colon, where they translocate the epithelial barrier via
taminated community water sources that are un- or under- M cells that overlay the solitary lymphoid nodules (Suzuki and
chlorinated (Blostein, 1991; Fleming et al., 2000; CDC, 2001). Sasakawa, 2001). Upon reaching the under side of the M cells,
Foodborne outbreaks of Shigella are also common, especially Shigella infect macrophages and induce cell apoptosis (Suzuki
with foods that are subjected to processing or preparation and Sasakawa, 2001). Infected macrophages release interleukin-
by hand, are exposed to a limited heat treatment, or are 1, which elicits a strong inflammatory response (Zychlinsky
served/delivered raw to the consumer (Wu et al., 2000). Sev- et al., 1994). Once released from the macrophage, Shigella en-
eral examples of food products from which Shigella has been ters neighboring epithelial cells (enterocytes), through a process
isolated are potato salad, ground beef, bean dip, raw oysters, known as macropinocytosis. This involves the host cell extend-
fish, and raw vegetables. ing its membrane into formations known as pseudopodia, which
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The infective dose for Shigella is very low: 10 cells of S. engulf large volumes and close around them forming a vac-
dysenteriae and 500 cells of S. sonnei (Kothary and Babu, 2001). uole. Macropinocytosis requires actin polymerization and the
Populations such as the very young, the very old, or persons presence of myosin, an actin-binding protein (Stendahl et al.,
with decreased immune function may be at more risk for infec- 1980). Common stimuli that induce macropinocytosis include
tion. Due to the low infective dose of Shigella, person-to-person cytokines and bacterial antigens. In response to invasion, epithe-
transmission is common. Typical symptoms of infection include lial cells produce pro-inflammatory cytokines which contribute
bloody diarrhea, abdominal pain, fever, and malaise. Although to inflammation of the colon (Suzuki and Sasakawa, 2001).
the mechanism is unknown, seizures have been reported in 5.4% Once engulfed by epithelial cells, Shigellae immediately
of shigellosis cases involving children (Galanakis et al., 2002). disrupt the vacuole, releasing them to the host cell cytoplasm
Chronic sequelae from S. dysenteriae serotype 1 infections can where they multiply rapidly. Sansonetti et al. (1986) observed
include hemolytic uremic syndrome (HUS), while S. flexneri the generation time of S. flexneri in HeLa cells to be approx-
infections are associated with later development of Reiters syn- imately 40 minutes. In order to sustain efficient intracellular
drome, especially in persons with the genetic marker HLA-B27 growth requires the acquisition of host cell nutrients. Since little
(CDC, 2003c). Reiters syndrome is characterized by joint pain, free iron exists within mammalian host cells, Shigella spp. must
eye irritation, and painful urination (CDC, 2003c). also express high-affinity iron acquisition systems. In order to
obtain iron, Shigella synthesize and transport the siderophores
aerobactin and enterobactin (Vokes et al., 1999) and utilize
Pathogenesis of Shigella a receptor/transport system in which iron is obtained from
heme. Siderophores are low molecular weight, iron-binding
Shigellae invade the local epithelium of the colon (large in- compounds that remove iron from host proteins. Enterobactin
testine) which results in the symptoms of shigellosis. The inva- is produced by some but not all Shigella spp. (Perry and San
sion of epithelial cells involves four steps: entry into epithelial Clemente, 1979; Payne, 1983), while aerobactin is synthesized
cells, intracellular multiplication, intra- and intercellular spread- by S. flexneri, S. boydii (Lawlor and Payne, 1984), and some S.
ing, and killing of the host cell (Sansonetti, 1991). The invasion sonnei (Payne, 1988). Headley et al. (1997) demonstrated that
process is controlled by a 180 to 220-kDa virulence plasmid. aerobactin systems, although active in extracellular environ-
The virulence plasmid contains invasion plasmid antigen (ipa) ments, are not expressed intracellularly. This suggests that
genes which encode four highly immunogenic polypeptides; siderophore-independent iron acquisition systems can provide
IpaA, IpaB, IpaC, and IpaD. Studies in which Tn5 insertions essential iron during intracellular multiplication (Headley et al.,
affecting the expression of the ipa genes reveal that expression 1997).
of ipaB, ipaC, and ipaD is strongly associated with entry, while The capacity for Shigella to spread intracellularly and in-
ipaA is not (Sansonetti, 1991). fect adjacent epithelial cells is critical in the infection process
Invasion is also mediated by the virF gene, which is located (Sansonetti, 1991). Intra- and intercellular spreading is con-
on the virulence plasmid, and the virR gene, which is located trolled by the icsA (virG) gene located on the virulence plasmid.
on the chromosome. The virF gene encodes a 30-kDa protein The icsA gene encodes the protein IcsA, which enables actin-
that positively regulates the expression of the ipa genes and based motility (Bernardini et al., 1989) and intercellular spread
icsA (also known as virG), another virulence plasmid gene that (Makino et al., 1986). IcsA is a surface-exposed outer membrane
encodes intra- and intercellular spread. Environmental factors protein consisting of three distinctive domains: a 52 amino acid
which affect the expression of virF are not known. The virR gene N-terminal signal sequence, a 706 amino acid -domain, and a
is a repressor of the plasmid invasion genes in a temperature- 344 amino acid C-terminal -core (Goldberg et al., 1993; Lett
dependant manner (Sansonetti, 1991). When Shigellae are et al., 1989; Suzuki et al., 1995). The -domain is the exposed
grown at 30 C they do not express the Ipa polypeptides and portion while the -core is embedded in the outer membrane
are therefore non-invasive, however, Shigellae grown at 37 C (Suzuki et al., 1995).
REVIEW OF SHIGELLA DETECTION METHODS 553

IcsA is distributed at one pole of the outer membrane surface. rapidly. Early killing of host cells involves metabolic events
This asymmetrical distribution allows the polar formation of that rapidly drop the intracellular concentration of ATP, increase
actin tails, and thus directional movement of Shigella within pyruvate, and arrest lactate production (Sansonetti and Mounier,
host cell cytoplasm. The polar localization of IcsA is primarily 1987). Shiga toxin (STX), a potent toxin produced by S. dysen-
affected by two events: teriae serotype 1, does not play a role in the early killing of host
cells. Fontaine et al. (1988) constructed a Tox-mutant strain of
(i) the rate of diffusion of outer membrane IcsA (Sandlin et al., S. dysenteriae serotype 1 and found that the mutant killed as
1995; 1996; Sandlin and Maurelli, 1999; Robbins et al., efficiently as the wild-type strain.
2001) and
(ii) the specific cleavage of IcsA by the protease IcsP (SopA) Toxins Produced by Shigella spp
(dHauteville et al., 1996; Egile et al., 1997; Steinhauer et al.,
1999). S. dysenteriae serotype 1 strains produce the potent toxin
The rate of diffusion of outer membrane IcsA is directly af- STX. Although STX is not necessary to sustain an infection,
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fected by the O side chains of the membrane lipopolysaccharide its expression increases the severity of disease. Three biologic
(LPS). Sandlin et al. (1996) demonstrated this relationship with activities associated with STX are cytotoxicity, enterotoxicity,
a S. flexneri LPS mutant, which did not produce the O-antigen. and neurotoxicity, while the one known biochemical effect is
Without O side chains, the LPS mutant polymerized actin in the inhibition of protein synthesis (Donohue-Rolfe et al., 1991).
a non-polar fashion. In addition to O side chains, the correct STX is considered the prototype to a family of toxins known
composition of the IcsA C-terminus domain is also required as Shiga-like toxins (SLT), which are similar in structure and
for polar localization and polar movement of S. flexneri (Suzuki function, and share the same receptor sites. Perhaps the most
et al., 1996). A S. flexneri mutant, in which a segment of the IcsA widely known human pathogen that produces SLTs is E. coli
C-terminus domain had been deleted, was unable to polymer- O157:H7, which has two toxin variants (SLT I and SLT II).
ize actin in a polar fashion or move unidirectionally (Suzuki STX is composed of two polypeptides: an A subunit (32,225
et al., 1996). MW) and five B subunits (7,691 MW, each) (Donohue-Rolfe
The icsP gene encodes the outer membrane protease IcsP et al., 1991). The B subunits mediate binding to cell surface
(also called SopA) which cleaves laterally diffused IcsA, thus receptors, which have been identified as glycolipids containing
promoting polar localization (Egile et al., 1997). An E. coli terminal galactose-(1-4)galactose disaccharides, such as gal-
K-12 strain, engineered to express the icsA gene, was shown abiosylceramide and globotriasylceramide (Gb3) (Brown et al.,
to diffuse IcsA along its outer membrane (Monack and Theriot, 1991; Keusch et al., 1991). The A subunit, once inside the host
2001). When the same E. coli K-12 strain was engineered to cell cytoplasm, acts enzymatically to cleave the N-glycosidic
express the icsP gene with the icsA gene, the number of bacte- bond of adenine at nucleotide position 4324 in the 28S rRNA of
ria which polymerized actin at one pole increased (Monack and the 60S ribosomal unit (Donohue-Rolfe et al., 1991).
Theriot, 2001). The reader is directed to the following references Recently, two enterotoxins, shigella enterotoxin 1 (ShET 1)
for information on molecular mechanisms of actin assembly in and shigella enterotoxin 2 (ShET 2), have been characterized
Shigella: Goldberg, 1993; Miki et al., 1996; Loisel et al., 1999; and are believed to play a role in the clinical manifestation of
Suzuki and Sasakawa, 2001; Shibata et al., 2002. shigellosis (Yavzori et al., 2002). ShET 1, which is chromoso-
Intercellular spreading is dependent upon an actin-based mally encoded, was only prevalent in isolates of S. flexneri 2a.
motility mechanism (Monack and Theriot, 2001). Shigella cells ShET 2, however, is encoded on the virulence plasmid, and was
first form a membrane bound protrusion into an adjacent cell. detected in all Shigella isolates tested, except several isolates
This protrusion must distend two membranes: one from the that had been cured of the virulence plasmid.
donor cell, and another from the recipient cell (Parsot and
Sansonetti, 1996). As the protrusion pushes further into the re- Foodborne Outbreaks Involving Shigella
cipient cell, it is taken up by the recipient cell resulting in the
bacteria enclosed in a double-membrane vacuole (Monack and A summary of selected recent foodborne shigellosis out-
Theriot, 2001). Intercellular spread is completed when Shigella breaks is given in Table 1. Characteristic of foodborne shigel-
rapidly escapes from the double-membrane vacuole, releasing it losis, several recent outbreaks have been associated with foods
into the cytosol of the secondary cell. Monack and Theriot (2001) consumed raw (Martin et al., 1986; Fredlund et al., 1987; Davis
observed intercellular spread of an E. coli K-12 strain expressing et al., 1988; Cook et al., 1995; Dunn et al., 1995; Frost et al.,
the icsA and icsP genes in HeLa cells. After invading adjacent 1995; Kapperud et al., 1995; Public Health Laboratory Service
cells, the E. coli K-12 strain was observed both enclosed in a (PHLS), 1997; CDC, 1999) and processed or prepared by hand
double-membrane vacuole and free in the cytosol of host cells. (Martin et al., 1986; Lee et al., 1991; Lew et al., 1991; Yagupsky
The early killing of host cells by Shigella requires invasion et al., 1991; Dunn et al., 1995; Chicago Department of Pub-
and is mediated by the virulence plasmid. Sansonetti (1991) lic Health (CDPH), 1999; Trevejo et al., 1999; Toronto Public
demonstrated the inability of non-invasive S. flexneri to kill Health (TPH), 2002). In 2000, a multi-state outbreak of shigel-
host cells, whereas the invasive phenotype killed efficiently and losis was traced to a commercially prepared five-layer bean dip
554 B. R. WARREN ET AL.

Table 1 Selected foodborne outbreaks involving Shigella

Year Serogroup Food product(s) implicated Reference(s)

1983 S. sonnei Tossed salad Martin et al., 1986


1986 S. sonnei Shredded lettuce Davis et al., 1988
1986 S. sonnei Raw oysters Reeve et al., 1989
1987 S. sonnei Watermelon Fredlund et al., 1987
1988 S. sonnei Uncooked tofu salad Yagupsky et al., 1991; Lee et al., 1991
1989 S. flexneri 4a German potato salad Lew et al., 1991
1992 S. flexneri 2 Tossed salad Dunn et al., 1991
1994 S. sonnei Iceberg lettuce Long et al. 2002; Kapperud et al., 1995; Frost et al., 1995
19956 S. sonnei Fresh pasteurized milk cheese Garca-Fulgueiras et al., 2001
1996 S. flexneri Salad vegetables PHLS, 1997
1998 S. sonnei Uncooked, chopped curly parsley CDC, 1999
1998 S. flexneri Restaurant-associated, source unknown Trevejo et al., 1999
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1999 S. boydii 18 Bean salad (parsley or cilantro) CDPH, 1999


2000 S. sonnei Five layer bean dip CDC, 2000; Kimura et al., 2004
2001 S. sonnei Raw oysters Terajima et al., 2004
2002 Shigella spp. Greek-style pasta salad TPH, 2002

(Kimura et al., 2004). This outbreak involved 406 ill persons et al. (1983) examined foods from Mexican homes, commer-
(14 hospitalizations, 0 deaths) across 10 states. After extensive cial sources in Guadalajara, Mexico, and from restaurants in
epidemiological investigation, numerous problems were identi- Houston, TX, for contamination with bacterial enteropathogens.
fied in the manufacturing process and investigators determined While no Shigella was isolated from foods sampled from 12
that the source of the outbreak was most likely an infected food- Houston restaurants or from food commercially prepared in
handler (Kimura et al., 2004). This outbreak demonstrates the Guadalajara, Mexico, four isolates were obtained from meals
vulnerability of our food supply to point-source contamination prepared in Mexican homes. These studies demonstrate the im-
with Shigella followed by wide distribution and subsequent in- portance of proper food handling and the role of food handlers
fection of many consumers (Kimura et al., 2004). in the transmission of Shigella.
In response to President Clintons National Food Safety Ini-
tiative (January 1997) and Produce & Imported Foods Safety Ini-
Prevalence of Shigella: Food and Food Handlers tiative (October 1997), the U.S. Food and Drug Administration
(FDA) has investigated the presence of foodborne pathogens,
According to the CDC Emerging Infections Program, including Shigella, on imported and domestic produce (FDA,
Foodborne Diseases Active Surveillance Network (FoodNet), 2001a; FDA, 2001b; FDA, 2003). An FDA survey of imported
Shigella was the third most reported foodborne bacterial broccoli, cantaloupe, celery, parsley, scallions, loose-leaf let-
pathogen in 2002 (CDC, 2003b). Of 16,580 laboratory- tuce, and tomatoes found Shigella contamination in nine of 671
diagnosed cases, Shigella accounted for 3,875 cases (23.4%) be- total samples: three of 151 cantaloupe samples, two of 84 celery
hind only Salmonella (6,028 cases) and Campylobacter (5,006 samples, one of 116 lettuce samples, one of 84 parsley samples,
cases) (CDC, 2003b). From 2001 to 2002, the national inci- and two of 180 scallion samples (FDA, 2001a). Another FDA
dence of Shigella in the U.S. increased from 3.8 cases to 4.5 survey of domestically grown fresh cantaloupe, celery, scallions,
cases per 100,000 (CDC, 2002; CDC, 2003a). The incidence of parsley, and tomatoes found Shigella contamination in five of
each serogroup in the U.S. was similar to previous years with 665 total samples: one of 164 cantaloupe samples, three of 93
serogroup D (S. sonnei) accounting for the majority of all iso- scallion samples, and one of 90 parsley samples (FDA, 2003).
lates (83.5%), followed by serogroup C (S. flexneri, 12.2%), An additional survey of imported produce was conducted; how-
serogroup B (S. boydii, 0.8%), and serogroup A (S. dysenteriae, ever at the time of this publication results were not publicly
0.3%) (CDC, 2003a). available (FDA, 2001b).
Despite the high incidence of shigellosis, there is limited data
on the prevalence of Shigella among food handlers or on food
products. A study investigating the presence of enteropathogens Environmental Factors on Survival of Shigella
among food handlers in Irbid, Jordan, isolated Shigella from the
stools of four out of 283 examined food handlers (al-Lahham Shigella spp. are heat sensitive, acid resistant, salt tolerant
et al., 1990). Mensah et al. (2002) evaluated 511 food items bacteria that can withstand low levels of organic acids (Zaika,
from the streets of Accra, Ghana, from which S. sonnei was 2001; Zaika, 2002a; Zaika 2002b). Zaika (2001) studied the
isolated from one sample of macaroni. It was noted that the survival of S. flexneri strain 5348 in brain heart infusion (BHI)
macaroni was served using bare hands instead of clean uten- broth as a function of pH (2 to 5) and temperature (4 to 37 C).
sils, which may have led to the S. sonnei contamination. Wood When inoculated into BHI broth adjusted to pH 5, S. flexneri
REVIEW OF SHIGELLA DETECTION METHODS 555

demonstrated growth when held at 19, 28, and 37 C, while ICMSF, 1996). Sublethal heat exposure can sensitize Shigella
counts declined over time at temperature of 12 C or lower. When to selective components of microbiological media. Tollison and
inoculated into BHI broth adjusted to pH 2, 3, or 4, inoculated Johnson (1985) demonstrated that S. flexneri sublethally heat-
S. flexneri counts declined over time at all temperatures tested. stressed by exposure to 50.0 C for 30 minutes in phosphate
S. flexneri in BHI broth adjusted to pH 2 reached undetectable buffer became sensitive to 0.85% bile salts and 0.50% sodium
levels in 1 to 3 days when held at temperature of 19 C or lower. desoxycholate. Since these compounds are ingredients in several
In general, S. flexneri survival was greater in BHI broth incu- enrichment and isolation media used for detection of Shigella,
bated at lower temperatures and adjusted to higher pH. This the thermal history of the food sample to be analyzed should be
study suggests that S. flexneri is acid resistant and that acidic known.
foods may support the survival of Shigella over a long period of
time (Zaika, 2001).
Zaika (2002a) studied the survival of S. flexneri strain 5348 Survival of Shigella on Fomites
in BHI broth (pH 4 to 6) containing 0.5 to 8% NaCl. In BHI
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adjusted to pH 6, S. flexneri grew in the presence of 6% NaCl Inanimate objects, or fomites, can serve as vectors for trans-
when held at 19 and 37 C, and in the presence of 7% NaCl mission of Shigella and there have been several reports on
when held at 28 C. Growth of S. flexneri was also observed the survival of Shigella on various surfaces (Spicer, 1959;
in BHI broth adjusted to pH 5 containing 2, 4, 4, 0.5% Nakamura, 1962; Islam et al., 2001). Spicer (1959) studied the
NaCl when held at 37, 28, 19, and 12 C, respectively (Zaika, survival of S. sonnei dried on cotton threads at room tempera-
2002a). S. flexneri populations gradually declined in BHI ad- ture and under refrigeration at various levels of relative humidity.
justed to pH 4 at all incubation temperatures and all levels of In general, survival was better at refrigerated temperatures and
NaCl tested (Zaika, 2002a). Results from this study suggest at high (84%) and low (0%) relative humidity (RH). S. sonnei
that S. flexneri is salt tolerant and may survive in salty foods remained detectable on the cotton threads after 12 days at 5
such as pickled vegetables, caviar, pickled herring, dry cured 10 C (84% RH) (Spicer, 1959). Similar results were observed
ham, and certain cheeses for extended periods of time (Zaika, using 10 different strains of S. sonnei inoculated on cotton, glass,
2002a). wood, paper, and metal at various temperatures (20 C to 45 C)
S. flexneri survival was also studied in BHI broth supple- (Nakamura, 1962). When held at 20 C, most of the strains sur-
mented with organic acids commonly found in fruits and veg- vived for more than 14 days on each surface; however, surfaces
etables (citric, malic, and tartaric acid) or fermentation acids held at 45 C did not support the survival of most strains (Naka-
commonly used as preservatives (acetic and lactic acid) at 0.04M mura, 1962). Investigations on the survival of S. dysenteriae
and adjusted to pH 4 with HCl or NaOH (Zaika, 2002b). Fer- serotype 1 on cloth, wood, plastic, aluminum, and glass ob-
mentation acids (acetic and lactic acid) had a greater effect on jects suggest that 1.5 to 4 hours post-inoculation, S. dysenteriae
survival than citric, malic, and tartaric acids (Zaika, 2002b). serotype 1 enters a viable but non-culturable (VBNC) state
When incubated at 37 C, S. flexneri survived for 1 to 2 days (Islam et al., 2001). While no S. dysenteriae serotype 1 could be
in the presence of each organic acid tested. At 4 C, S. flexneri recovered after 5 days by conventional culture methods, viable
survived in the presence of all the organic acids tested for longer cells could be observed using fluorescent antibody techniques
than 55 days (Zaika, 2002b). This study suggests that organic (Islam et al., 2001). Whether or not Shigella is able to achieve
acids may aid in the inactivation of Shigella, however foods with a true VBNC state or if these results demonstrate the inade-
low levels of acids stored at low temperatures may support the quacy of plating media in recovering viable Shigella has not
survival of the bacterium for extended periods of time (Zaika, been fully investigated. Nevertheless, these studies demonstrate
2002b). that fomites can sustain viable Shigella for an extended time and
Temperature is an important factor in survival of Shigellae. serve as vehicles in the transmission of the pathogen.
Freezing (20 C) and refrigeration (4 C) temperatures sup-
port survival, but not growth, of Shigella (International Com-
mission on Microbiological Specifications for Foods (ICMSF), Survival of Shigella in Water and Food
1996). When studied in nutrient broth, the observed temperature
ranges which permitted growth for S. sonnei and S. flexneri were Shigella can survive in water with little decline in popula-
6.1 to 47.1 C and 7.9 to 45.2 C, respectively (cited in ICMSF, tion. Rafii and Lunsford (1997) inoculated distilled water with
1996), however Shigella can survive for extended periods of time S. flexneri at 2.8 108 colony forming units (cfu)/ml and held the
when held at 20 C or at 4 C. Elevated temperatures are less samples at 4 C. After 26 days, 9.2 107 cfu/ml of S. flexneri sur-
permissive for Shigella survival, and traditional pasteurization vived. This high survival rate of S. flexneri in water supports the
and cooking temperatures are sufficient for inactivation. Evans historical association of shigellosis outbreaks and water sources.
et al. (1970) calculated the decimal reduction time (D-value) Fruits and vegetables can support the growth or survival of
of S. dysenteriae in pasteurized whole milk to be 0.0008 sec at Shigella. Escartin et al. (1989) artificially contaminated fresh cut
82.2 C. When studied in nutrient broth, most strains of S. sonnei papaya, jicama, and watermelon with S. sonnei, S. flexneri, or S.
and S. flexneri were inactivated within 5 min at 63 C (cited in dysenteriae and within 6 hours at room temperature, growth was
556 B. R. WARREN ET AL.

observed. Rafii and Lunsford (1997) inoculated raw cabbage, inoculated S. sonnei remained at level for up to three days, at
onion, and green pepper with S. flexneri and although counts de- which time the populations began to drop. When test samples
creased slightly at 4 C, survival was observed after 12 days on were stored under refrigeration, S. sonnei counts did not drop
onion and green pepper, at which time sampling was terminated after seven days. In the latter study, refrigerated samples main-
due to spoilage (Rafii and Lunsford, 1997). S. flexneri survival tained a constant pH throughout the study, while samples stored
was observed on the cabbage for 26 days. Wu et al. (2000) at room temperature had a drop in pH that may have contributed
studied survival of S. sonnei on whole and chopped parsley to the decline in S. sonnei cell numbers.
leaves. At 21 C, growth on chopped parsley was observed at
a similar rate to that in nutrient broth (Wu et al., 2000). At
4 C, populations declined on both chopped and whole parsley CONVENTIONAL CULTURE METHODS
throughout the 14-day storage period, however S. sonnei sur- FOR SHIGELLA
vived regardless of initial population (Wu et al., 2000). These
studies demonstrate Shigella survival on refrigerated produce Traditional Microbiological Media for Enrichment and
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for periods of time that exceed expected shelf life. Isolation of Shigella
Low pH foods can support survival of Shigella when held
at refrigerated temperatures. Bagamboula et al.(2002) demon- Traditional microbiological techniques make use of selec-
strated S. sonnei and S. flexneri survival in apple juice (pH 3.3 to tive and differential media for the enrichment and isolation of
3.4) and tomato juice (pH 3.9 to 4.1) held at 7 C for 14 days. No Shigella. Many variants of enrichment and plating media have
reduction was observed in the tomato juice, while a 1.2 to 3.1 been investigated for optimal recovery, often with conflicting
log10 cfu reduction was observed in apple juice over the 14 day results. Although Shigella is readily isolated from clinical sam-
study. Rafii and Lunsford (1997) observed S. flexneri survival in ples, food samples are more problematic. Isolation of Shigella
carrot salad (pH 2.7 to 2.9), potato salad (pH 3.3 to 4.4), coleslaw from food samples can be inhibited by indigenous microflora, es-
(pH 4.1 to 4.2), and crab salad (pH 4.4 to 4.5) held at 4 C. Sam- pecially the coliform bacteria and Proteus spp. (ICMSF, 1996).
pling was terminated at day 11 for the carrot and the potato sal- The addition of the antibiotic novobiocin to liquid and solid
ads, at which time S. flexneri counts decreased from 4.3 106 media has been shown to improve the isolation of S. flexneri
to 4.2 102 cfu/g and from 1.32 106 to 8.5 102 cfu/g, and S. sonnei from investigated foods (cited in ICMSF, 1996).
respectively. Sampling of the coleslaw and the crab salads ceased Contamination of food products with Shigella results primarily
due to product spoilage on days 13 and 20, respectively, how- through a food handler with poor personal hygiene; therefore
ever S. flexneri survived at levels of 2.16 104 and 2.4 105 cfu/ the concentration of Shigella may be very low compared to that
g, respectively. These studies indicate that Shigella survived at of the indigenous microflora (Lampel and Maurelli, 2001). Cur-
refrigerated temperatures despite the presence of background rently, selective media are not available that will adequately sup-
microflora and low pH. press the growth of background microflora, therefore Shigella
Prepared foods can also support the survival of Shigella. Islam is often overgrown by competing microorganisms (Lampel and
et al. (1993) investigated the growth and survival of S. flexneri in Maurelli, 2001). More research is needed to determine more
boiled rice, lentil soup, milk, cooked beef, cooked fish, mashed appropriate selective media and enrichment conditions for the
potato, mashed brinjal, and raw cucumber. All food samples, isolation of Shigella from food samples.
except raw cucumber, were autoclaved prior to inoculation. Ten Two enrichment broths initially used for the isolation of
gram or ten ml samples of each food were inoculated with 105 Shigella were Selenite-F (SF) and Tetrathionate (TT) broth.
cells of S. flexneri, incubated at 5, 25, or 37 C and sampled These broths were originally designed for the isolation of
over 72 hrs. All of the foods tested supported growth up to 108 salmonellae, but due to the lack of specific enrichment media
to 1010 cells per g or ml within 6 to 18 hrs after inoculation for Shigellae they were used as all-purpose enteric enrichment
at 25 C and 37 C (Islam et al., 1993). Initial inoculum levels broths (Taylor and Schelhart, 1969). Sodium selenite, although
were maintained throughout the 72 hr holding period for all selective for salmonellae, is toxic to Shigella (and most enter-
foods, except rice and milk. S. flexneri counts in rice decreased ics), therefore it is no longer used in enrichment procedures for
by approximately 1 log after 72 hours at 5 C, whereas counts in Shigella. TT is a peptone base broth with bile salts and sodium
milk dropped after 48 hrs but then returned to the initial inoculum thiosulfate that inhibits growth of most Gram-positives and En-
level by 72 hrs. These results demonstrate the ability of Shigella terobacteriaceae. While TT is routinely used for the enrichment
to grow and survive in a variety of prepared foods that may be of Salmonella, it is rarely used for Shigella. Gram-negative (GN)
contaminated by an infected food handler. broth is a peptone-based broth with glucose and mannitol. The
Shigella is able to survive on produce packaged under vac- concentration of mannitol in GN broth is higher than glucose to
uum or modified atmosphere. Satchell et al. (1990) investigated promote mannitol fermentors, like Shigella. Both TT broth and
the survival of S. sonnei in shredded cabbage packaged under GN broth contain bile salts, which can be inhibitory to stressed
vacuum or in a modified atmosphere of nitrogen and carbon cultures (Tollison and Johnson, 1985). Furthermore, GN broth
dioxide when stored at room temperature or under refrigeration. contains sodium desoxycholate, which has been shown to in-
When test samples were stored at room temperature, counts of hibit heat-stressed Shigellae (Uyttendaele et al., 2001). Still, GN
REVIEW OF SHIGELLA DETECTION METHODS 557

broth is listed as an alternate enrichment medium for the detec- on lactose fermentation. Instead, differentiation on CSPM is
tion of Shigella from food by some standard methods (Lampel, based on proprietary components consisting of select carbo-
2001). hydrates, pH indicators, and chromogens (Dr. Larry Restaino,
Current enrichment procedures (FDA, 1998; Lampel, 2001, personal communication). Shigella spp. produce white to clear
Health Canada, 2004) use a low carbohydrate medium, Shigella colonies on CSPM while competitors produce colored colonies.
broth (SB) with the addition of novobiocin, for the detec- CSPM has been compared to MAC and SSA for the recovery
tion/isolation of Shigella. One study reported that acids produced of S. boydii and S. sonnei from tomato surfaces with no signif-
by other Enterobacteriaceae during the fermentation of carbohy- icant differences in recovery observed (Warren, 2003; Warren
drates were toxic to Shigellae (Mehlman et al., 1985); however, et al., 2005). Further evaluation of CSPM against other strains of
other studies have shown that Shigella can grow at pH 4.5 to 4.75 S. boydii and S. sonnei as well as the other serogroups of Shigella
(Bagamboula et al., 2002) and survive at pH 4.0 (Zaika, 2002). is needed.
Since SB contains very little carbohydrate, the effect of a low
pH environment during enrichment is limited. SB is also less
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stringent than the TT broth and the GN broth for the enrichment The BAM Culture Method for Detection of Shigella in Foods
of Shigella since it contains neither bile salts nor sodium des-
oxycholate. A recent study investigated SB, GN broth, tryptic The FDA Bacteriological Analytical Manual (BAM) culture
soy broth, and Enterobacteriaceae Enrichment (EE) broth with method for the isolation and detection of Shigella spp. from
the addition of novobiocin for enrichment/detection of Shigel- food utilizes a combination of low carbohydrate enrichment,
lae (Uyttendaele et al., 2001). When incubated in GN broth, anaerobic conditions, and elevated temperature (FDA, 1998).
S. sonnei was unable to grow to comparable levels as observed Briefly, a 25 g sample of the food product is transferred to 225
in SB and EE broths. EE broth, however, has been reported in- ml of Shigella broth (SB) to which novobiocin (0.5 g/ml for
hibitory to S. boydii (Warren, 2003; Warren et al., in press). S. sonnei; 3.0 g/ml for other Shigella spp.) has been added.
Multiple plating media with differing selectivity can be used Samples are held at room temperature for 10 minutes and pe-
to increase the chances of Shigella isolation. The most common riodically shaken. Sample supernatants are transferred to an
low selectivity medium used for plating Shigella is MacConkey Erlenmeyer flask and the pH adjusted to 7.0 0.2 with ster-
Agar (MAC). Eosin methylene blue (EMB) or Tergitol-7 (T7) ile 1 N NaOH or 1 N HCl. Flasks are incubated anaerobi-
agars can also be used. Since differentiation on MAC is solely cally for 20 hours (44 C for S. sonnei; 42 C for all other
based on lactose fermentation, Shigella colonies look similar to Shigella spp.) and the enrichments are streaked on MAC.
those of many lactose negative competitors (Uyttendaele et al., Confirmation of suspicious colonies involves tests for motil-
2001). On MAC, Shigella colonies are translucent or slightly ity, H2 S, gas formation, lysine decarboxylase, and fermenta-
pink, with or without rough edges. Shigella produce colorless tion of sucrose or lactose. Isolates negative for all confirma-
colonies on EMB and bluish colonies on the yellowish-green T7 tory tests are further tested using biochemical reactions in-
agar (Lampel, 2001). cluding adonitol, inositol, lactose, potassium cyanide, mal-
Intermediate selectivity media useful in isolating Shigella onate, citrate, salicin, and methyl red. Shigellae are negative
are desoxycholate citrate agar (DCA) and xylose lysine desoxy- for all except methyl red. Antisera agglutination is then used
cholate agar (XLD). Shigella produce colorless colonies on both to identify any culture displaying typical Shigella charact-
DCA and XLD. Bhat and Rajan (1975) reported XLD superior eristics.
to DCA for the isolation of Shigella since DCA required a 48- June et al. (1993) evaluated the effectiveness of the BAM
hour incubation to show clear colony morphology as opposed to culture method for Shigella. Two strains of S. sonnei, strains
overnight incubation for XLD. Unfortunately, D-xylose, which 9290 and 25931, were inoculated on potato salad, chicken salad,
serves as a differentiating agent on XLD agar, is fermented by cooked shrimp salad, lettuce, raw ground beef, and raw oys-
some strains of S. boydii while most Shigella cannot ferment ters. Using either unstressed or chilled stressed cells, acceptable
xylose (Bhat and Rajan, 1975). Thus some strains of Shigella recovery was achieved for both strains from the potato salad,
will be missed if XLD is used as the sole plating medium. chicken salad, cooked shrimp salad, and lettuce samples, but
Highly selective media for Shigella spp. include Salmonella- not from the ground beef and the raw oyster samples. Approxi-
Shigella agar (SSA) and Hektoen Enteric agar (HEA). A problem mately 4 log differences in recovery from ground beef samples
associated with SSA and HEA is that they may be too stringent were observed between the two strains suggesting high strain
for some strains of Shigella, especially if the culture is stressed variability. Given the low infective dose of Shigella (as low as
(Lampel, 2001; Uyttendaele et al., 2001). Shigella produce 10 cells), the BAM was considered ineffective for the evaluation
colorless, translucent colonies on SSA and green colonies on of raw ground beef and raw oysters.
HEA. In 2002, the BAM culture method for Shigella was evaluated
A newly developed plating medium, Chromogenic Shigella using two strains of unstressed, chill-stressed, or freeze-stressed
spp. Plating Medium (CSPM) (R&F Laboratories, West S. sonnei (strains 357 and 20143) on selected types of pro-
Chicago, IL) offers medium selectivity (bile salts, antibiotic duce (Jacobson et al., 2002). Acceptable recovery of unstressed
supplementation) with an alternative to differentiation based cells (<1.0 101 cfu/25g) and chill-stressed or freeze-stressed
558 B. R. WARREN ET AL.

Figure 1 Lipopolysaccharide (LPS) structure of Gram-negative bacteria. The O-antigen polysaccharide unit is repeated n times.

cells (<5.2 101 cfu/25g) were observed for all produce types IMMUNOLOGICAL METHODS FOR
tested (Jacobson et al., 2002). In our laboratory, similar tests of a SHIGELLA DETECTION
modified protocol showed unacceptable recovery of unstressed
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S. sonnei(patient isolate from an outbreak involving an unknown LPS of Shigella


source) and S. boydii (patient isolate from an outbreak involving
bean salad) on tomatoes at 1.9 102 cfu/tomato and >5.3 105 Gram-negative bacterial LPS consists of three distinct re-
cfu/tomato, respectively (Warren, 2003). These results support gions: lipid A, core oligosaccharide, and a serotype-specific
the observation that significant variation exists among strains O-polysaccharide chain (O-antigen) (Fig. 1) (Neidhardt, 2004).
of S. sonnei and demonstrate the importance of including the The lipid A portion anchors the LPS molecule to the outer mem-
other serogroups of Shigella when evaluating culture methods brane. The core oligosaccharide is composed of two regions: the
for detection in food. inner core and the outer core. The inner core is common to many
enterobacterial species and is composed of heptose and 2 keto-3
deoxyoctulosonate (KDO), while the outer core is rich in hex-
Other Culture Methods for the Detection of Shigella in Foods ose and tends to be more species-specific (Tsang et al., 1987).
Studies of the core structure of S. flexneri have indicated that
Alternate culture methods for the detection of Shigella in serotypes 1 to 5 and variants X and Y share the E. coli R3 core
foods can be found in Health Canadas Compendium of Ana- (Carlin and Lindberg, 1986), while S. flexneri serotype 6 and
lytical Methods (Health Canada, 2004) and the American Public S. sonnei share the E. coli R1 core (Gamian and Romanowska,
Health Associations Compendium of Methods for the Microbi- 1982; Carlin and Lindberg, 1986; Viret et al., 1992) (Fig. 2). An-
ological Examination of Foods (CMMEF) (Lampel, 2001). The tibodies have been developed with specificity to the inner core
Health Canada method is based on the BAM culture method with of Shigella spp. (Rahman and Stimson, 2001). The O-antigen
a few modifications; most notably enrichments for all Shigella is a polymer of repeating saccharides which is highly variable
spp. (including S. sonnei) are supplemented with 0.5 g/ml among species. Strains sharing identical O-antigen repeating
novobiocin and incubated anaerobically at 42 C. The CMMEF units are of the same serotype. The O-antigens of all S. flexneri
culture method is also similar to the BAM culture method, how- serotypes are a repeating tetrasaccharide (Carlin and Lindberg,
ever, the level of novobiocin in S. sonnei enrichment media
is lower (0.3 g/ml) and enrichment conditions are aerobic at
37 C. The CMMEF further suggests that two to three plates of
various selective media be used to streak the enriched cultures:
MAC for low selectivity, XLD for intermediate selectivity, and
HEA for high selectivity. Confirmation of suspicious colonies
is similar to methods described above for the BAM culture
method.
Recently, the CMMEF culture method and an enrichment
procedure involving EE broth have been compared to the BAM
culture method for the detection of S. boydii and S. sonnei on
tomato surfaces (Warren, 2003). Natural tomato microflora was
found to have a great impact on recovery of S. sonnei and
completely inhibited recovery of S. boydii in all three culture
methods. No significant differences (P > 0.05) were observed
among the culture methods for detection of S. sonnei, or between Figure 2 Structural differences between hexose regions of Shigella sonnei
the BAM and CMMEF culture methods for the detection of S. and Shigella flexneri lipopolysaccharides. A) S. sonnei and S. flexneri serotype 6
share the hexose region of the E. coli R1 core. B) S. flexneri serotypes 15 and X
boydii. EE broth was found to be inhibitory to S. boydii. These and Y variants have a hexose region identical to the E. coli R3 core. (Structures
results suggest the need for more selective enrichment protocols compiled from Jansson et al., (1981)). Abbr.: Gal = galactose, Glc = glucose,
for the evaluation of Shigella spp. in food. GlcNAc = 2-acetamido-N-deoxyglucose.
REVIEW OF SHIGELLA DETECTION METHODS 559

other Shigella species, the LPS genes (rfc and rfb) of S. sonnei are
located on a large virulence plasmid, which can be spontaneously
lost at high frequency. Sansonetti et al. (1981) investigated the
stability of form I plasmids and observed 1 to 45% plasmid loss
from re-streaking form I colonies onto MAC and incubating
Figure 3 O-antigen repeating subunits of S. sonnei and S. flexneri 24 hrs at 37 C. When the large virulence plasmid is lost, rough
lipopolysaccharide. (A) The disaccharide repeating subunit of the S. sonnei (avirulent) colonies, termed form II, are produced that express
O-antigen, (B) the tetrasaccharide repeating subunit of S. flexneri serotype the Enterobacteriaceae R1 lipopolysaccharide core (Sansonetti
6, and (C) the tetrasaccharide repeating subunit of S. flexneri serotypes et al., 1981; Gamian and Romanowska 1982). A defective mu-
1-5 and X and Y variants. (Structures compiled from Carlin and Lindberg,
(1986); Dmitriev et al., (1979); and Gamian and Romanowska, (1982)).
tant of form II S. sonnei LPS, termed R-form, is characterized
Abbr: LAltNAcA = N -acetyl-L-altrosaminuronic acid, 4-N -DFucNac = 2- by an incomplete core region (Gamian and Romanowska, 1982)
acetamido-4-amino-2,4,6-trideoxy-D-galactose, GalA = galacturonic acid, (Fig. 4). As antibodies specific for S. sonnei O-antigen will bind
GalNAc = N -acetylgalactosamine, Ac3 Rha = 3-O-acetylrhamnose, Rha = form I, but not form II or R-form LPS, immunological detec-
rhamnose, GlcNAc = 2-acetamido-N-deoxyglucose.
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tion methods with specificity for the O-antigen of Shigella are


compromised when the virulence plasmid is lost.
1986), while the O-antigen of S. sonnei is a repeating disaccha-
ride (Kenne et al., 1977) (Fig. 3).
Immunological Detection Methods for Bacteria

LPS Differences in S. Sonnei Form I and Form II Immunological methods, such as latex agglutination (LA),
enzyme immunoassay (EIA), or immunomagnetic separation
Virulent S. sonnei produce smooth colonies, termed form I, (IMS), have been utilized for the detection of many foodborne
which result from the expression of the O-antigen. Unlike the bacterial pathogens. LA assays involve latex particles coated

Figure 4 Lipopolysaccharides of S. sonnei. A) Form I, B) form II, and C) R-form. (Structures compiled from Gamien and Romanowska, (1982)).
560 B. R. WARREN ET AL.

with antibodies specific for target bacteria. Binding causes a LA Methods for Shigella
visible clumping of the latex particle-bacteria complexes that
can be seen with the naked eye. EIA is a term used to describe LA requires the prior isolation of a suspect colony on solid
many assay formats in which enzyme-labeled antibodies are media. For this reason LA methods cannot be used for detection
used to bind antigens with detection via a colorimetric reac- of Shigella directly from a food sample, but rather can confirm
tion using the enzyme label. Microplate readers are generally or aid in the characterization of suspected Shigella colonies.
used for the detection of the colorimetric signal and these as- Several latex agglutination serotyping kits for Shigella are com-
says can be performed in a high-throughput format. IMS in- mercially available, however, only one representative kit will be
volves small paramagnetic beads coated with antibodies against discussed in this review.
the surface antigens of bacterial cells. Paramagnetic beads ex- The Wellcolex Color Shigella Test (WCT-Shigella, Remel
hibit magnetic properties only when placed within a magnetic Inc., Lenexa, KS) allows the identification of isolates to the
field and show no residual magnetism when removed from this species level using only two reagents, each consisting of a mix-
field. After target bacteria are bound by the antibody-coated ture of red and blue latex particles coated with antibodies specific
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beads, a magnetic field is used to separate the bead-bacteria com- for one of the four different Shigella serogroups. Each reagent is
plexes. Once the magnetic field is removed, the bead-bacteria added to one of two identical sample spots of the suspect isolate.
complexes return to suspension (Olsvik et al., 1994). Detection The presence of homologous antigen results in the agglutination
of bead-bacteria complexes can be performed via direct plating of one color coupled with a change in background color. Color
on microbiological media, direct microscopy, or nucleic acid change combinations due to any of the four Shigella serogroups
amplification. LA, EIA, and IMS methods require the use of are easily distinguishable as is the negative reaction in which
monoclonal or polyclonal antibodies to bind specific antigens the particles remain in smooth purple suspension. Non-specific
present on the surface of bacterial cells. For this reason, devel- agglutination results in a purple agglutination with a clear back-
opment of antibodies with sufficient specificity is critical for the ground.
performance of immunological methods. A summary of these Bouvet and Jeanjean (1992) tested the WCT-Shigella against
and other rapid methods for Shigella detection is presented in 100 Shigella isolates from human stools and observed specificity
Table 2. and sensitivity at 100% and 98%, respectively. The two Shigella

Table 2 Selected rapid methods for detection of Shigella

Serogroup Target Matrix Sample Preparation Detection Detection Limit Reference

S. sonnei ipaH Tomatoes FTA filtration Nested PCR <101 cfu/tomato Warren, 2003
S. boydii
S. flexneri ipaH Water SE, boiled extract Semi-nested PCR 1.1101 cfu/ml Theron et al., 2001
S. dysenteriae 1 virA Mayonnaise CEP PCR 102 103 cfu/ml Villalobo and Torres,
1998
S. dysenteriae virA Mussels PE, CEP PCR 101 102 cfu/ml Vantarakis et al.,
2000
S. flexneri plasmid DNA Lettuce Alkaline denaturation PCR 1.0 104 cfu/g Lampel et al., 1990
S. flexneri ial Various foods SE, BDC Nested PCR 1.0 101 cfu/g Lindqvist, 1999
S. sonnei form I LPS, ial Feces IMS PCR 1.01.5101 cfu/ml Achi-Berglund and
Lindberg, 1996
S. dysenteriae LPS Rectal swabs PE, DPSM EIA ND Sonjai et al., 2001
S. flexneri
S. boydii
S. sonnei
S. dysenteriae LPS Various foods None Biosensor 4.9 104 cfu/ml Sapsford et al., 2004
S. dysenteriae1 LPS, ial Feces IMS PCR ND Achi et al., 1996
S. flexneri
S. sonnei
S. dysenteriae1 LPS, ial Feces IMS PCR 1.0 101 cfu/g Islam and Lindberg,
1992
S. flexneri
S. dysenteriae LPS, 16S rRNA Sewage Immunocapture UPPCR 5.0 101 cfu/ml Peng et al., 2002
S. flexneri
S. boydii
S. sonnei
S. dysenteriae1 LPS Feces IMS EIA 1.0 103 cfu Islam et al., 1993
S. flexneri

Abbreviations: SEselective enrichment, PEpre-enrichment, NDnot determined, LPSlipopolysaccharide, EIAenzyme immunoassay, BDCbuoyant density
centrifugation, UPPCRuniversal primer PCR, DPSMdirect plate on selective media, CEPchemical extraction/ ethanol precipitation.
REVIEW OF SHIGELLA DETECTION METHODS 561

strains that did not give visible aggregates were S. dysenteriae et al., 1996) from feces prior to PCR. The detection limit for
type 2 and S. flexneri type 4. These results were supported by the IMS-EIA and IMS-PCR methods were 1.0 103 cfu/ml
Kocka et al. (1992). Using the WCT-Shigella, 42 of 42 clinical (Islam et al., 1993) and 1.01.5 101 cfu/ml (Islam and
Shigella isolates and 7 of 8 stock Shigella cultures were correctly Lindberg, 1992; Achi-Berglund and Lindberg, 1996), respec-
grouped (Kocka et al., 1992). The stock Shigella culture that was tively. In addition, the IMS-PCR method was more than two
not correctly grouped had been repeatedly passed in culture that times as effective then the conventional culture method for the
may have resulted in the loss of some antigenicity. Lefebvre diagnosis of shigellosis in children with severe diarrhea (Achi
et al. (1995) evaluated WCT-Shigella and six commercial slide et al., 1996). These studies have significance with respect to
agglutination Shigella serogrouping kits for accuracy. The WCT- Shigella detection in foods, however further investigations are
Shigella was easily performed and the interpretation of results needed to evaluate the suitability of the IMS techniques for use
was less subjective than the other tests. WCT-Shigella met a with food samples.
performance standard of 90% accuracy in these evaluations.
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Additional Technologies for Immunological


EIA Methods for Shigella Detection of Shigella

Although the EIA methods for the detection of foodborne In a recent report, Sapsford et al. (2004) describe detection
pathogens are common, the only commercially available kits of S. dysenteriae in buffer and on chicken carcasses using an ar-
for Shigella spp are the Shigel-Dot A (for S. dysenteriae), B (for ray biosensor developed at the Naval Research Laboratory. The
S. flexneri), C (for S. boydii), and D (for S. sonnei) test kits (Sci- array biosensor measures total internal reflection fluorescence
ence Development and Management. Ltd., Bangkok, Thailand). using a 25-minute sandwich immunoassay for antigen detection.
The Shigel-Dot kits are membrane dot-blot enzyme-linked im- An advantage of the array biosensor is that little or no sample
munosorbent assays (ELISAs). These test kits have been vali- preparation is required prior to analysis. The detection limit of
dated using 500 rectal swabs and have been compared to conven- the array biosensor for S. dysenteriae in ground turkey, chicken
tional culture isolation and Western blot analysis. The diagnostic carcass wash, buffered milk, and a lettuce leaf wash was ob-
accuracy of the Shigel-Dot A, B, C, and D was 99.2%, 95.0%, served at 7.8 105 cfu/g, 4.9 104 cfu/ml, 7.8 105 cfu/ml,
94.0%, and 96.4%, respectively, when compared to conventional and 2.0 105 cfu/ml, respectively. When tested in buffer, the
culture isolation, and all were 100% when compared to the con- array biosensor did not respond as efficiently to the other
ventional culture isolation and Western blot combined (Sonjai serogroups of Shigella and cross-reacted with E. coli, suggesting
et al., 2001). Monoclonal antibodies included in the Shigel-Dot the specificity of the polyclonal antibodies was a limiting factor.
D kit are reported to detect both S. sonnei form I and form II The use of a monoclonal antibody with higher specificity may
LPS. improve the diagnostic ability of this testing format.

IMS Methods for Shigella Detection MOLECULAR MICROBIOLOGICAL METHODS FOR


SHIGELLA DETECTION IN FOOD
IMS has been investigated for the concentration and purifi-
cation of bacterial pathogens from food samples (Cudjoe and PCR Detection of Shigella in Foods
Krona, 1997; Hsih and Tsen, 2001; Drysdale et al., 2004; Lynch,
et al., 2004) and are reportedly more sensitive than comparable Polymerase chain reaction (PCR) methods for detection of
conventional culture methods (Cudjoe and Krona, 1997; Hsih Shigella in food have previously demonstrated higher sensi-
and Tsen, 2001). Anti-Salmonella, anti-E. coli O157:H7, anti- tivity than comparable culture methods (Warren, 2003). PCR
Campylobacter, and anti-Listeria IMS beads are commercially assays for Shigella spp. have targeted the invasion associated lo-
available (Matrix MicroScience, Cambridgeshire, UK; Dynal cus (ial) (Islam and Lindberg, 1992; Lindqvist, 1999), the virA
Biotech, Oslo, Norway) however, anti-Shigella IMS beads are gene (Villalobo and Torres, 1998; Vantarakis et al., 2000), or
not yet commercially available. the ipaH gene (Sethabutr et al., 1993; Sethabutr et al., 2000;
IMS has been used as a technique to concentrate Shigella Theron et al., 2001; Lampel and Orlandi, 2002; Warren, 2003)
from clinical samples for downstream detection processes such for amplification. The same PCR primers are used to detect
as EIA (Islam et al., 1993) or PCR (Islam and Lindberg, each of the serogroups of Shigella and enteroinvasive E. coli.
1992; Achi et al., 1996; Achi-Berglund and Lindberg, 1996). The ial and virA genes are located on the large virulence plas-
By using IMS, PCR inhibitors inherent to fecal samples were mid (sometimes referred to as the invasion plasmid), however,
successfully eliminated. Briefly, IMS was used to concentrate sequencing of the S. flexneri genome (Jin et al., 2002) revealed
S. sonnei (Achi et al., 1996; Achi-Berglund and Lindberg, 1996), the ipaH gene to be encoded multiple times on both the chro-
S. flexneri(Islam and Lindberg, 1992; Achi et al., 1996), and mosome and the large virulence plasmid. Thus, detection of the
S. dysenteriae serotype 1 (Islam and Lindberg, 1992; Achi ipaH gene is possible in the event the large virulence plasmid
562 B. R. WARREN ET AL.

has been lost, which has been shown to occur when food sam- water, 1.4 101 cfu/ml for lake water, 5.8 102 cfu/ml for
ples are stored for a prolonged periods of time prior to analysis river water, 6.1 102 cfu/ml for treated sewage water, and 1.1
(Lampel and Orlandi, 2002). The discussion below will be lim- 101 cfu/ml for tap water. Variability in results among the water
ited to PCR detection of Shigella in food samples, however sev- samples was attributed to the presence of humic substances that
eral reports of detection in fecal samples appear in the literature. inhibited PCR. Humic substances are transformed products from
These methods are summarized along with other rapid methods soil organic matter that do not belong to the known classes of bio-
in Table 2. chemistry. These include humic acids, fulvic acids, and humins.
Vantarakis et al. (2000) developed a multiplex PCR method Pre-enrichment in GN broth served to dilute humic substances
to detect both Shigella spp. and Salmonella spp. in mussels. while allowing the S. flexneri to multiply, thereby increasing the
Multiplex PCRs involve the use of two or more sets of primers concentration of target DNA.
in the same PCR such that multiple targets can be amplified
in the same reaction. Artificially inoculated S. dysenteriae and

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S.Typhimurium were recovered by homogenizing mussel meat Improved PCR Detection of Shigella by FTA Filtration
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with peptone water. DNA from an aliquot of the homogenate



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was purified using a guanidine isothionate method and concen- FTA cards (Whatman, Clifton, NJ) have been used in tem-
trated via ethanol precipitation. The virA gene of Shigella spp. plate preparation for PCR detection of pathogenic microorgan-
and the invA genes of Salmonella spp. were amplified. Sam- isms (Lampel et al., 2000; Orlandi and Lampel, 2000; Lampel
ple homogenates were inoculated at various concentrations to and Orlandi, 2002; Warren, 2003; Warren et al., 2005). Mois-

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establish the lowest detection limit for the method. When sam- ture in the sample activates chemicals in the FTA card that
ples were not pre-enriched prior to analysis, the multiplex PCR lyse cells, denature enzymes, inactivate pathogens, and immo-
method was able to detect S. dysenteriae at 1.0 103 cfu/ml bilize genomic DNA (Whatman website, 2004). Chemicals in

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in the homogenate. Following a 22 hour incubation in buffered the FTA cards also protect DNA and RNA from light, free
peptone water the multiplex PCR methods was able to S. dysen- radicals, enzymes, and pathogens during dry, room-temperature

teriae detect levels as low as 1.0 101 to 1.0 102 cfu/ml
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storage. FTA filtration as sample preparation for PCR has been
in the homogenate. Similar results were observed for developed for detection of Cyclospora and Cryptosporidium in
S. Typhimurium. water (FDA, 1998). Recently, this sample preparation technique
Villalobo and Torres (1998) investigated PCR for the detec- has been investigated for detection of S. boydii and S. sonnei
tion of S. dysenteriae serotype 1 in mayonnaise. Samples were from tomato rinses by nested PCR (Warren, 2003; Warren et al.,
homogenized in buffered peptone water and artificially contam- 2005). Briefly, tomato rinses were passed through a two-stage
inated with various concentrations of S. dysenteriae serotype 1. filter where the first stage contained filter paper for size exclusion
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Bacterial cells were lysed with detergent, the DNA extracted and the second stage contained FTA filter paper. After purifi-

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with phenol-chloroform and precipitated with ethanol. A multi- cation and washing, 6mm punches are taken from the FTA
plex PCR was then used to amplify regions from the virA gene filters and used directly as a template in the first step of the

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and the 16S rRNA gene. The lowest level of inoculation de- nested PCR. The FTA filtration/nested PCR (FTA-PCR) as-
tected by this method was 1.0 102 to 1.0 103 cfu/ml in the say detected S. boydii and S. sonnei at 6.2 100 cfu/tomato and
homogenate. 7.4 100 cfu/tomato, respectively (Warren, 2003; Warren et al.,
In a study by Lindqvist (1999), nested PCR was investigated 2005).
for the detection of the ial locus of Shigella spp. from spiked Although FTA-PCR had excellent sensitivity when used to
lettuce, shrimp, milk, and blue cheese samples. Nested PCR analyze tomato rinses, which are relatively clean, similar results
involves the use of two sequential PCRs in which the target se- were not observed when the recovery of inoculated S. boydii
quence in the second PCR lies within the amplified sequence and S. sonnei was attempted from cantaloupes, strawberries, or
in the first PCR. Nested PCRs can be used to achieve extreme retail Valencia oranges (Dr. K.R. Schneider, unpublished data).
low sensitivities. Food samples inoculated were homogenized FTA-PCR was unable to detect inoculated Shigella from straw-
in physiological saline and bacteria were isolated by buoyant berries, suggesting that the purification and washing protocol
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density centrifugation (separation of components based on den- used on the FTA filters may not be adequate to remove PCR in-
sity). Single PCR, using only the external primer sets, was able hibitors (Warren, 2003). Despite several size exclusion filtering
 
to detect S. flexneri in aqueous solution at 0.51.0 105 cfu/ml.
R R
techniques applied prior to the FTA filtration, the FTA filters
Nested PCR was more sensitive and was able to detect 1.0 were routinely clogged by cantaloupe fibers or the wax coating
103 cfu/ml. The nested PCR assay in combination with buoyant from retail oranges, which entered the buffer rinse during recov-
density centrifugation was able to detect S. flexneri inoculated ery procedures (Warren, 2003). The advantage of the FTA-PCR
onto all four foods at 1.0 101 cfu/g (Lindqvist, 1999). technique is that no enrichment was necessary to obtain low de-
Theron et al. (2001) investigated a semi-nested PCR for the tection limits due to the large volume of the sample analyzed by
detection of the ipaH gene of S. flexneri in spiked environ- the filters. The technique warrants further investigation for the
mental water samples. The detection limits in the various en- analysis of foods for Shigella contamination as improvements
vironmental water samples were 2.0 103 cfu/ml for well in pre-filtration techniques could improve the sensitivity.
REVIEW OF SHIGELLA DETECTION METHODS 563

Additional Molecular Microbiological Techniques for DiversiLabTM System (Spectral Genomics, Inc., Houston, TX)
Detection of Shigella generates such fragments by rep-PCR that are then analyzed
using microfuidics lab-on-a-chip and the Agilent 2100 Bioana-
An interesting new method, immunocapture universal primer lyzer (Agilent Technologies, Inc. Palo Alto, CA). The results can
PCR (iUPPCR), has been recently reported (Peng et al., 2002). A be visualized in several formats including dendograms, electro-
universal primer PCR employs primers designed against highly pherograms, gel-like image, or scatter plots (Lising et al., 2004).
conserved regions, such as 16S rRNA genes, thus the result- Rep-PCR technology has been shown to be reproducible and
ing PCR can be used for amplification of almost any bacte- can allow the differentiation of species, subspecies, and strains
ria. Typically, the sequence of the resulting amplicon would be in as little as 4 hours. Some Shigella serogroups show > 95%
further analyzed for identification of the bacteria. In iUPPCR similarity with the DiversiLabTM Enteric Kit Beta version, how-
however, the immunocapture is employed for specificity and the ever the DiversiLabTM Shigella kit offers greater differentia-
universal primer PCR is used for detection of captured bacte- tion among serogroups and strains (Lising et al., 2004). Strains
ria. Monoclonal antibodies specific for individual serotypes of of E. coli (non-pathogenic, enterohemorragic, and enterotoxi-
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S. dysenteriae, S. flexneri, S. boydii, or S. sonnei were coated genic) were effectively differentiated from Shigella spp. using
into the wells of a 96-well polystyrene plate. Cross-reactivity rep-PCR (Raza et al., 2003; Lising et al., 2004), however, no
tests demonstrated the specificity of the monoclonal antibodies enteroinvasive E. coli strains were included in the studies.
to the strain level. Wells were challenged with bacteria and cap-
tured bacteria were detected using the universal primer PCR, i.e.
PCR with primers specific for 16S rRNA sequences conserved CONCLUDING REMARKS
among closely related bacteria. The detection limit for Shigellae
in broth was 5.0 102 cfu/ml (Peng et al., 2002). Presumably, Shigella spp. continues to be a significant agent of foodborne
detection of up to 96 pathogens sharing the conserved 16S rRNA illness in the U.S. While Shigella can survive in many types
sequence could be accomplished in the same plate using specific of food, there is not much information about the prevalence
monoclonal antibodies for bacterial capture (differentiation of of Shigella on food or among food handlers. Despite much
cell types) and the universal primer PCR for detection. Further research, an appropriate conventional culture protocol for the
investigation of this method with respect to food samples as detection of Shigella in foods is still needed. As of this pub-
opposed to bacteria in broth are required. lication, few immunological-based detection kits for Shigella
Advances in oligonucleotide microarrays have further en- are commercially available. Several molecular microbiological
abled the simultaneous detection and identification of bacte- methods, such as PCR, microarrays, and rep-PCR, have been
rial foodborne pathogens. Oligonucleotide microarrays involve developed; however, more research is needed. While the devel-
the ordered immobilization of specific probes to a solid sur- opment of rapid test methods will facilitate more timely and cost
face followed by hybridization with labeled sample DNA. After effective testing, a more profound reduction of foodborne shigel-
hybridization, the development of the label can allow identifi- losis may depend on the education of employees and consumers
cation/enumeration of complementary sample DNA sequences. as to proper personal hygiene.
In a recent report, Kakinuma et al. (2003) describes an oligonu-
cleotide microarray using probes specific for the gyrB gene to ACKNOWLEDGEMENTS
detect and differentiate E. coli, Shigella, and Salmonella. PCR
amplified regions of the gyrB gene were fluorescently labeled This work was funded by USDA-CSREES IFAFS Grant num-
and hybridized to detection probes immobilized on glass slides. ber 00-52102-9637. This is Florida Agricultural Experiment Sta-
Based on reaction patterns, 3 species of Shigella, 7 serovars of tion Journal Series number R-10735.
Salmonella, and 1 strain of E. coli were correctly identified at the
species level. Identification at the subspecies level of Salmonella
was problematic when multiple serovars were present in the NON-ENDORSEMENT OF COMMERCIAL PRODUCTS
same sample due to the overlap of microarray patterns. Assay
formats such as this could be expanded to potentially detect and References to all products are provided for information only
identify a wide range of enteric pathogens. and do not constitute endorsement or warranty, express or im-
Finally, repetitive sequence-based PCR (rep-PCR) has been plied, by the authors and/or their employers or the publishers of
demonstrated for the identification and molecular typing of this work, as to their suitability, content, usefulness, functioning,
members of the family Enterobacteriaceae, including Shigella completeness, or accuracy.
(Raza et al., 2003; Lising et al., 2004). In rep-PCR, primers
are designed complementary to bacterial interspersed repetitive
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