Virulence of Environmental Stenotrophomonas maltophilia Serologically Cross-reacting with Shigella-specific Antisera
This research involved an environmental strain of Stenotrophomonas maltophilia which has been reported to produce serological cross-reactivity with Shigella dysenteriae type 8 specific antisera. Since clinical diagnosis of shigellosis is largely based on culture and serology, the investigation was aimed at in vivo and in vitro virulence comparison between the culturally similar environmental S. maltophilia isolate and the reference S. dysenteriae strains. The findings of this study revealed the absence of virulent genes of Shigella sp. like ipaH, virA and stx1 and characteristic invasive large plasmid in the test isolate. The Western blot analysis revealed that serological cross-reactivity of Stenotrophomonas maltophilia was due to certain protein component(s) in its outer membrane. The isolate was capable of producing extracellular protease, exhibited alpha hemolysis and was negative for hemagglutinating assay. The isolate gave negative reaction with rabbit ileal loop and Sereny tests. The S. maltophilia isolate did not possess any enterotoxic or invasive property as that of virulent S. dysenteriae strains. Further characterizations and adequate genetic manipulations of this environmental isolate may contribute to the development of a potential vaccine candidate for shigellosis.
Shigellosis or bacillary dysentery caused by Shigella sp. is endemic
in many developing countries including Bangladesh. Among at least 80 million
cases, 700,000 deaths occur each year due to shigellosis in developing countries.
Seventy percent of these cases occur in children less than 5 years of age (WHO,
2005). By the age of 2, about 37% of the children in Bangladesh have acquired
the infection (Seidlein et al., 2006). The poor
sanitary conditions in developing countries like Bangladesh contribute to the
spread of the disease and increasing antibiotic resistance further complicates
the treatment. A safe and effective Shigella vaccine offers great potential
as a means of preventing shigellosis.
Laboratory diagnosis of Shigella is often based on the isolation of
the organism from feces of the patients by means of cultural and biochemical
characteristics (Kelly et al., 1985). The slide
agglutination test using a number of commercially available antisera specific
to different groups and types, is a common and indispensable test for serological
typing of Shigella sp. (Ewing and Lindberg, 1984;
Evins et al., 1988).
Bacterial strains carrying identical or similar antigenic components on their
surface structure might also react with the antibodies produced against other
strains (Grubor et al., 2006; Houpikian
and Raoult, 2003; Khan et al., 2003). It
was found that various types of microorganisms such as Hafnia alvei,
Plesiomonas shigelloides, Providencia alcalifaciens and Yersinia
enterocolitica serotype 03 and E. coli (O114:H32, O157:H7, O157:H19,
etc.) gave serological cross-reactivity with polyclonal group-specific Shigella
antisera (Lefebvre et al., 1995). A number of
environmental isolates were also found to cross-react with different types of
Shigella-specific monovalent antisera. But the major cellular components
giving rise to such cross-reactivity remains to be identified. Rahman
et al. (2007) demonstrated that various environmental isolates cross-reacted
with six different serotypes of Shigella and were, in each case, highly
type-specific. Amongst those isolates, two environmental S. maltophilia exhibited
significant serological cross-reactivity with S. dysenteriae type 8 specific
monovalent antisera. These environmental isolates producing a high cross-reactivity
with various types of Shigella antisera represent a potential for development
of a shigellosis vaccine based on live cells, because they probably are not
virulent like clinical organisms (Rahman et al.,
The present investigation involved the comparison of virulent properties of
the previously reported environmental Stenotrophomonas maltophilia isolate
(Rahman et al., 2007) with Shigella dysenteriae
strains and to identify the putative cellular components giving rise to this
type of serological cross-reactivity.
MATERIALS AND METHODS
Bacterial strains: The test organism Stenotrophomonas maltophilia (RBSD4), was an environmental isolate and three clinical Shigella dysenteriae strains (S. dysenteriae type 2 (602), type 3 (C253) and type 4 (613)) were used as reference. All the organisms were obtained from the Environmental Microbiology Laboratory, Department of Microbiology, University of Dhaka, Bangladesh in 2009.
Biotyping of the test isolate Stenotrophomonas maltophilia: Biotyping of the test isolate S. maltophilia along with the reference Shigella dysenteriae strains were performed to reveal similarities or dissimilarities in their biochemical behavior.
Detection of Shigella-specific virulence genes (ipaH, virA
and stx1) by PCR
Extraction of chromosomal DNA: Chromosomal DNA of the test isolate and
the reference strains were extracted and purified according to the method described
by Sambrook et al. (1989). Following this method,
pellets from overnight fresh culture were collected by centrifugation at 10,000
rpm for 5 min and were resuspended in 100 μL of solution I containing 50
mM Tris-HCl buffer (pH: 7.5), 50 mM EDTA and 20% (w/v) sucrose. This was followed
by addition of 300 μL of solution II containing 1% (w/v) SDS and 50 mM
NaCl and 5 μL of proteinase K (20 mg mL-1) to each eppendorf.
After gentle shaking, the pellet suspension was incubated at 55°C for 60
min in a water bath. DNA was extracted by using 400 μL phenol: chloroform:
isoamylalcohol mixture (25:24:1). The and the top phase was recovered carefully
into fresh tubes were mixed well, centrifuged for 10 min at 10,000 rpm eppendorf
tubes. Double volume of ice-cold 100% ethanol followed by 1/10th volume of the
3 M sodium acetate (pH: 5.2) were added to the DNA solution, mixed well and
then kept at -20°C overnight. DNA pellet collected by centrifugation at
10,000 rpm for 10 min was washed again with 70% ethanol and was finally dried
in a desiccator under vacuum and resuspended in 50 μL TE buffer.
PCR: The PCR was performed in a 25 μL volume consisting of 0.5 μg of genomic DNA, 0.5 mM of each of the oligonucleotide primers (Table 1) for ipaH, virA and stx1, 2.5 μL of a 10xPCR reaction buffer (500 mM Tris-Cl, pH 8.9, 500 mM KCl and 25 mM MgCl2), 0.5 μL 10 mM dNTPs, 1.25 units of AmpliTaq DNA polymerase (Invitrogen, Life Technologies, USA) and an appropriate volume of sterile MilliQ water. Thermal cycling conditions were set for denaturation at 94°C for 1 min, annealing of primers at 55°C for 1 min 30 sec and primer extension at 72°C for 1 min 30 sec. Amplification was performed for 35 cycles, The expected sizes of the amplicons were ascertained by electrophoresis in 1.0% agarose gels (10 μL PCR product loaded) with an appropriate molecular size marker (1-kb DNA ladder; Sigma, USA).
Plasmid profile analysis: Plasmid extraction from the test isolates
was carried out according to the procedure described by Kado
and Liu (1981). Overnight fresh culture was centrifuged at 12,000 rpm for
7 min and pellets were resuspended in 100 μL autoclaved solution I (50
mM glucose, 25 mM Tris-HCl, 10 mM EDTA at 4°C and pH: 8.0) and kept at room
temperature for 5 min. Freshly prepared 200 μL of solution II (0.2 N NaOH,
1% SDS in deionized water) was then added, mixed gently and kept in ice for
10 min. This was followed by addition of 150 μL of solution III (3 M potassium
acetate, 5 M glacial acetic acid, pH: 4.8), mixed gently and kept in ice for
5 min. The suspension was centrifuged at 12,000 rpm for 5 min and 300 μL
of supernatant was collected in fresh eppendorf tubes. Double volume of ice
cold ethanol (95%) was added to the supernatant, mixed well and kept at room
temperature for 10 min. Following centrifugation at 12,000 rpm for 5 min, the
pellet was taken out and mixed with 1.0 mL ethanol (70%). This was centrifuged
again at 12,000 rpm for 5 min. Finally, the collected pellet was dried and resuspended
in 50 μL TE buffer and kept at 4°C.
Plasmid DNA was separated by horizontal electrophoresis in 1.0% agarose gel
slab in Tris-acetate EDTA (TAE) buffer at room temperature at 50 volts for 3
h. The gel was stained with ethidium bromide and DNA bands were visualized using
UV transilluminator (Gel Doc, Bio-Rad, USA).
Extraction of Outer Membrane Protein and Lipopolysaccharide (LPS): The
outer membrane proteins were extracted from the isolates by the water extraction
method described by Hale et al. (1985). Briefly,
an isolated colony from a fresh culture in Nutrient Agar (NA) was inoculated
into brain heart infusion broth and incubated at 37°C for 24 h in an orbital
shaker. Following incubation, the whole culture media was harvested by centrifugation
at 8,000 rpm for 15 min. The pellet was washed in normal saline and resuspended
in 5.0 mL of sterilized deionized water and was shaken (100 oscillations per
min) for 6 h at room temperature. The suspension was then centrifuged at 10,000
rpm for 20 min and the supernatant was collected and filtered through a Millipore
filter having pore size of 0.45 μm. LPS was extracted using extraction
kit (iNTRON Biotechnology, http://eng.intronbio.com/product/LPS.htm).
Finally, 70 μL of 10 mM Tris-HCl buffer (pH: 8.0) was added to the LPS
pellet and was dissolved by boiling for 1 min.
Western blot analysis: Outer Membrane Proteins (OMP) and LPS were fractionated
separately by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
using 15% polycrylamide gels following the procedure described by Laemmli
(1970). Twelve microlitre of OMP and LPS were loaded. These were followed
by Western blot analyses (Towbin and Gordon, 1984).
LPS and OMP were translocated from the gel to the nitrocellulose membrane by
overnight transfer in transfer buffer (1.8% glycine, 0.36% Tris, 25% methanol
in deionized water). The membrane was then cut into strips, washed with PBS
(Phosphate Buffer Saline), PBS-Tween20 (0.1%) and subjected to blocking solution
(3% skim milk in PBS). Commercially available antisera specific to S. dysenteriae
type 2, 3, 4 and 8 were used at a concentrations of 1:50 dilutions as primary
antisera (Denka Seiken, Tokyo, Japan). Horse radish peroxidase (HRP)-conjugated
antibody (anti-rabbit IgG; Sigma) at 1:2000 dilutions was used as secondary
antibody. Diaminobenzoic acid (DAB) (0.1%) dissolved in 100 mM citrate buffer
(2.94% Na-citrate in deionized water, pH 5.2) with 0.15% H2O2
was used as the substrate.
Proteolytic, hemolytic and hemagglutinating activity: Proteolytic activity
was determined based on the ability to hydrolyze milk protein casein. Fresh
cultures were inoculated in skim milk agar plate and incubated overnight at
37°C (Murray et al., 1999).
The hemolytic activities of the test S. maltophilia isolate and reference
S. dysenteriae strains were determined by streaking aseptically on the
blood agar plates. After incubation at 37°C for 18-20 h in aerobic and anaerobic
jar incubator, the hemolysis zone and type under both the conditions were determined
respectively (Atlas, 2006).
The hemagglutinating study was performed in microtiter plate. The chicken blood
cells were washed thrice in 0.01 M cold PBS and finally, suspended to a concentration
of 10% (v/v). Fifty microlitre of chicken blood cell suspension was added to
each well. Bacterial suspension containing 1010 colony forming unit
(cfu) mL-1 was subjected to two-fold serial dilution in 0.01 M PBS.
Fifty microlitres of serially diluted test isolate and Shigella strains
were then added to the wells and the microtiter plate was incubated at 22°C
for 1 h. The highest dilution giving complete hemagglutination was recorded
as the end point (Franzon and Manning, 1986).
Test of invasiveness: Sereny (keratoconjunctivitis) test was used as
confirmatory test of invasiveness. Bacterial suspension adjusted to a concentration
of 3x108 cfu mL-1 was used as inocula for Sereny test.
Thirty microliter of this suspension from each organism was dispensed into the
left eye of each guinea pig. Cell suspensions of Shigella flexneri 2a
was used as positive control (Sereny, 1955).
Rabbit Ileal Loop (RIL) assay for enterotoxicity: Preparation of inocula
was done following the method of De and Chatterje (1953).
Both live cells and culture filtrates were used as inocula. Prepared inoculum
cell suspension and culture filtrate (1.0 mL each) of the test isolate and reference
strain together with positive (Vibrio cholerae 569B) and negative (PBS)
controls were injected separately in the ligated loops of an Albino rabbit.
The same method was repeated twice to check for reproducibility of results.
The animals were sacrificed after 18 h by intravenous administration of 10%
magnesium sulfate. Enterotoxicity was determined according to the fluid accumulation
ratio suggested by De and Chatterje (1953).
Antibiotic susceptibility pattern: Susceptibility of the test isolate
and the reference strains were determined by using the standardized agar-disc-diffusion
method known as Kirby-Bauer (Barry et al., 1985).
A total of seventeen different commercially available (Oxoid Limited, England)
antibiotic discs were used in this study. The zone diameter for individual antimicrobial
agent was then translated into sensitive, intermediate or resistant categories
according to the interpretation table (Oxoid Limited, England).
Biotyping of the test isolate: The test isolate Stenotrophomonas maltophilia and the reference Shigella dysenteriae strains had many similar biochemical properties. But in contrast to the reference strains, the test isolate was unable to utilize any of the carbohydrates and possessed lysine decarboxylase activity (Table 2).
Detection of Shigella-specific virulence genes and plasmid profile analysis: The test isolate S. maltophilia did not harbor ipaH, virA or stx1 genes (Fig. 1, 2), whereas all the reference strains demonstrated ipaH and virA positive results and 421 and 215 bp amplified bands were observed in the agarose gel respectively. In addition, the reference strain S. dysenteriae type 3 (C253) was also stx1 positive and gave 384 bp amplified band.
Analysis of plasmid DNA by agarose gel electrophoresis revealed that the test isolate S. maltophilia had no plasmid. Only the reference strain S. dysenteriae type 4 (613) had large plasmid and other reference strains contained plasmid bands ranging between approximately 2.9 and 14 kb (Fig. 3).
LPS and OMP profile analysis: Western blot analysis of the LPS of the
test isolate S. maltophilia did not reveal any cross-reactivity with
S. dysenteriae type 8 specific antisera. This suggested that, the cross
reactivity of this isolate was not due to its LPS. Any other outer membrane
protein or surface protein might contribute to such type of serological cross-reactivity.
Therefore, this assumption was confirmed by the OMP profile analysis (Fig.
4) which showed antigenic bands on the nitrocellulose membrane.
Proteolytic, hemolytic and hemagglutinating activity: The test isolate S. maltophilia gave clear zone of proteolysis on skim milk agar whereas all the reference Shigella dysenteriae strains demonstrated negative result.
In case of the test Stenotrophomonas maltophilia isolate, zones of α-hemolysis
(greenish hue) were detected in blood agar plates incubated under aerobic as
well as anaerobic conditions.
||PCR analysis of the test S. maltophilia isolate for
Shigella-specific virulence gene, ipaH. Products of a PCR
assay for the ipaH gene are shown. Lane 1: 1 kb+ DNA ladder marker;
Lane 3: S. dysenteriae type 2 (602); Lane 4: S. dysenteriae
type 5 (613); Lane 5: S. dysenteriae type 3 (C253); Lane 6: S.
maltophilia; Lane 7: Negative control
||Biochemical characteristics and antibiotic susceptibility
pattern of the environmental test isolate S. maltophilia along with
the reference S. dysenteriae strains
|*K: Alkaline reaction, A: Acid reaction, S: Sensitive to antibiotic
||PCR analysis of the test S. maltophilia isolate for
Shigella-specific virulence gene, virA. Products of
a PCR assay for the virA gene are shown. Lane 1: 1 kb+ DNA ladder
marker; Lane 2: S. dysenteriae type 2 (602); Lane 3: S. dysenteriae
type 4 (613); Lane 4: S. dysenteriae type 3 (C253); Lane 5: S.
maltophilia, Lane 6: Negative control
||Plasmid profile analysis. Lane 1: S. dysenteriae type
3 (C253); Lane 2: S. dysenteriae type 2 (602); Lane 3: S. dysenteriae
type 4 (613); Lane 5: S. maltophilia; Lane 6: Supercoiled DNA ladder
But the reference strains (S. dysenteriae type 2, 3 and 4) did not show
any hemolysis (gamma hemolysis) on blood agar plate incubated either in aerobic
or in anaerobic condition.
||OMP profile of the test isolate S. maltophilia (RBSD4)
along with three clinical S. dysenteriae strains. Lane 1: protein
marker; Lane 2 and 3: test isolate S. maltophilia; Lane 4 and 5:
S. dysenteriae type 2 (602); Lane 6 and 7: S. dysenteriae
type 4 (613); Lane 8 and 9: S. dysenteriae type 3 (C253)
||Bar chart showing the ratio of fluid accumulation (mL cm-1)
by the test isolate, reference strains and positive control in RIL assay.
Key: 1: V. cholerae 569B live cells (positive control); 2: V.
cholerae 569B culture filtrate (positive control); 3: Test S. maltophilia
live cells; 4: Test S. maltophilia culture filtrate; 5: S. dysenteriae
type 2 (602); 6: S. dysenteriae type 3 (C253); 7: S. dysenteriae
type 4 (613) live cells
Neither the test isolate S. maltophilia (RBSD4) nor the reference strains (S. dysenteriae type 2, 3 and 4) exhibited any hemagglutinating activity in vitro.
Sereny (keratoconjunctivitis) assay: Sereny test revealed that with the test S. maltophilia did not cause any sign of inflammation in guinea pig eyes. Only those inoculated with the reference strain S. dysenteriae type 4 (613) and positive control S. flexneri 2a exhibited typical symptoms of keratoconjunctivitis.
Rabbit Ileal Loop (RIL) assay: Level of enterotoxicity was determined according to the ratio of fluid accumulation (mL) per unit (cm) length of ileal loop. Response is considered positive when the fluid accumulation reflects≥0.4 mL cm-1. Live cell and culture filtrate of the test S. maltophilia (RBSD4) did not cause any fluid accumulation. Enterotoxicity was evident in RIL only with the reference strain S. dysenteriae type 4 (613) and the positive control Vibrio cholerae 569B (Fig. 5).
Antibiotic susceptibility pattern of the test and reference strains: The test isolate S. maltophilia showed resistance to methicillin, erythromycin, amoxycillin, cefotaxime, ceftriaxone and penicillin G. It also showed intermediate resistance to antibiotics like tetracycline, nalidixic acid, rifampicin, polymyxin B and kanamycin. The test isolate was sensitive to gentamicin, ciprofloxacin, chloramphenicol and trimethoprim sulfamethoxazole (Table 2).
Shigellosis is one of the dreadful diarrheal diseases due to its invasive character,
systemic manifestations, severe nutritional impact and tendency to recur over
prolonged periods (Mata et al., 1970). In the
present study, the virulent properties of serologically cross-reactive environmental
Stenotrophomonas maltophilia were compared with reference Shigella
dysenteriae strains. The serologically cross-reactive S. maltophilia
isolate revealed many similarities and dissimilarities in their biochemical
behavior. In contrast to the reference strains, the S. maltophilia isolate
was unable to utilize carbohydrates, e.g., glucose, lactose, sucrose, maltose
and arabinose. This finding is not in conformity to another report which showed
that some clinical S. maltophilia strains were capable of utilizing glucose,
lactose, sucrose, maltose, xylose and citrate (Travassos
et al., 2004). Another distinguishing characteristic of the test
isolate was that it possessed lysine decarboxylase activity while all the reference
Shigella strains were lysine decarboxylase negative. Lysine decarboxylase
(LDC) activity is present in ≈90% of E. coli strains (Edwards
and Ewing, 1972) but all the strains of EIEC and Shigella sp. are
LDC negative (Silva et al., 1980). When the gene
for LDC, cadA was introduced into Shigella flexneri 2a, virulence
became attenuated and enterotoxin activity was inhibited significantly. The
enterotoxin inhibitor was identified as cadaverine, a product of the reaction
catalyzed by LDC. Comparison between the S. flexneri 2a and E. coli
K-12 genomes in the region of cadA revealed a large deletion in Shigella.
Representative strains of Shigella sp. and enteroinvasive E. coli
displayed similar deletions of cadA (Maurelli et
al., 1998). This study is consistent with the present findings with
the test S. maltophilia isolate which was lysine decarboxylase positive
but lacked enterotoxic property.
Presence of Shigella-specific virulence genes have been reported in
closely related Escherichia coli virotypes (Pass
et al., 2000). In this study, serologically cross-reactive environmental
S. maltophilia isolate demonstrated PCR negative results for Shigella-specific
virulence genes like ipaH, virA and stx1 indicating that
this organism did not possess any invasive or enterotoxigenic property. Absence
of Shigella-specific virulence genes is a prerequisite for a vaccine
candidate against shigellosis (DuPont et al., 1972).
This serologically cross-reactive environmental S. maltophilia isolate
fulfils the criterion and may open up the scope for development of shigellosis
vaccine based on live cells.
Invasive organisms like E. coli (EIEC) and Shigella sp., cause a rapid keratoconjunctivitis when placed on the conjunctiva of the guinea pig eye (known as Sereny test). Manifestations of keratoconjunctivitis include white or yellowish discharge from the eye, crustiness around the eye, redness or swelling in and around the eye and behavior such as pawing at the eyes or sneezing. Sereny test-positive isolates carry large (usually 140-MDa) plasmid responsible for this property. In the present study, the test isolate S. maltophilia gave negative response in the Sereny test indicating that it was noninvasive. Only the reference strain of S. dysenteriae type 4 (613) produced typical symptoms of keratoconjunctivitis in inoculated guinea pig eyes. This result is consistent with the plasmid profile analysis which showed the presence of a large virulence plasmid only in S. dysenteriae type 4 (613) and its absence in all the other reference strains including the test isolate.
The serological cross-reactivity is mostly due to the heat stable LPS antigens
or common outer membrane protein antigens (Peterfi et
al., 2007). In the present study, Western blot analysis revealed that
the cross-reactivity with S. maltophilia isolate was not due to its LPS
component. As many protein components in the outer membrane are also shared
among different Gram negative bacteria, the cross-reaction observed in the test
isolate might be due to any outer membrane protein(s). This assumption was eventually
confirmed by Western blot analysis involving outer membrane protein (OMP) of
the bacterial isolate. The environmental S. maltophilia isolate yielding
a high cross-reactivity with Shigella antisera represents potential for
development of a shigellosis vaccine.
The ability of organisms to produce enterotoxins and hence their diarrhoeagenic potential was tested using the rabbit ileal loop assay. The test isolate S. maltophilia live cells and its culture filtrate did not cause any fluid accumulation indicating that it was incapable of producing enterotoxin. Absence of enterotoxic property is consistent with the finding that it did not possess any toxin encoding gene.
Active extracellular protease was secreted by the test isolate. Some clinical
S. maltophilia isolate also produce alkaline serine protease detected
as a major secretion product (Windhorst et al., 2002).
Stenotrophomonas maltophilia G2 isolated from a soil sample producing
extracellular serine protease have been documented in a separate study (Huang
et al., 2009). The roles played by protease in this environmental
S. maltophilia isolate remains to be explored. The test S. maltophilia
isolate exhibited alpha hemolysis on blood agar plate indicating that it was
capable of partially degrading hemoglobin, giving rise to the typical greenish
hue around bacterial growth. An earlier study which reported clinical S.
maltophilia presenting a cell-free hemolytic activity similar to the 'hot-cold'
hemolysins (Figueiredo et al., 2006) is in conformity
to the present findings. Hemolysins may be channel-forming proteins or phospholipases
or lecithinases that destroy red blood cells and other cells by lysis (Figueiredo
et al., 2006). No hemagglutinating activity in vitro was exhibited
by the test isolate and the reference strains. This indicates the absence of
specific hemagglutinin on the bacterial cell surface which interacts with the
chicken red blood cell membrane component.
Resistance to β-lactam antibiotics was confirmed previously in a study
reporting approximately 200 kb plasmid purified from clinical isolates of S.
maltophilia (Avison et al., 2001). Transformation
of a 5.6 kb plasmid, designated pTHB, derived from a S. maltophilia isolate
into Escherichia coli K-12 HB101 resulted in the expression of resistance
to the penicillin and to cefazolin (Kelly et al.,
1995). However, the occurrence of multiple antibiotic resistance devoid
of any plasmid in the test isolate indicates that these resistance genes might
be chromosome mediated. This type of antibiotic resistance might be due to the
occurrence of certain genes like Smqnr, a new chromosome-borne quinolone resistance
gene, in S. maltophilia (Shimizu et al., 2008).
The overall findings of this study suggest the S. maltophilia isolate to be noninvasive and nonenterotoxic. Multiple antibiotic resistance and production of hemolysin and extracellular protease are some prominent features of the test isolate. Routine procedures to identify S. dysenteriae must incorporate virulence tests in addition to culture and serology in order to rule out any possibility of misdiagnosis. Finally, further investigations are necessary to fully characterize the virulent properties of this environmental isolate and its potential as a vaccine candidate against shigellosis.
This study was supported by The Norwegian Programme for Development, Research and Higher Education (NUFU), grant No NUFUPRO-2007/10063.
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