Transcript CAMPYLOBACTER

CAMPYLOBACTER
II
Hin-chung Wong
Department of Microbiology
Soochow University
Content
 CONTROL OF CAMPYLOBACTER IN FOODS
 ISOLATION AND ENUMERATION
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Enrichment Procedures
Selective Media
Identification
Most Probable Number Method and Direct Plate count
Filtration Method
Immunofluorescence Microscopy
Bioluminescence Assay
Enzyme-linked Immunosorbent Assay
Confirmation by latex agglutination
Detection of toxin
Detection of toxin genes
 PATHOGENICITY AND VIRULENCE FACTORS
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Campylobacter Enteritis
Animal Model
Chemotaxis
Adhesion and Invasion
Enterotoxins
Cytotoxins
 MOLECULAR STUDIES OF ANTIBIOTIC RESISTANCE
 CONCLUSIONS
 REFERENCES
CONTROL OF CAMPYLOBACTER IN
FOODS
The effects of various disinfectants were
tested. The killing time depended on the
size of the inoculum
CONTROL OF CAMPYLOBACTER IN
FOODS
 Results of experiments in which an
antibiotic-containing medium was used
suggest that a high proportion of the
remaining cells were injured
ISOLATION AND ENUMERATION
Injury may occur in response to a number
of stresses associated with food processing,
such as heating, freezing, desiccation,
acidulation and others.
Cells of C. jejuni exposed to heating or
freezing were progressively less able to
grow at 43C, particularly on selective media
ISOLATION AND ENUMERATION
 Detection of injured cells (by heating,
freeze/thawing) frequently requires special
recovery procedures:
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Culture the injured cells at 37C in brucella broth
supplemented with succinate, cysteine and antibiotics,
excluding polymyxin B. Polymyxin B was added after 6
h and the incubation temperature was shifted to 42C.
Culture the injured cells on brucella broth
supplemented with pyruvate, ferrous sulfate, and
sodium bisulphite (FBP) at 37C or 42C for 4 h.
Pre-enrichment in non-selective culture broth (nutrient
broth plus blood and aerotolerant supplement only) at
37C for 2 h before the addition of antibiotics
ISOLATION AND ENUMERATION
ISOLATION AND ENUMERATION
Thus enrichment, or selective enrichment
methods are essential for accurate
detection of the organism in foods.
 Most enrichment incubation procedures
recommend 42C for 48h under
microaerobic condition (5% O2, 10% CO2,
85% N2)
ISOLATION AND ENUMERATION
ISOLATION AND ENUMERATION
A biphasic culture system containing 4 ml of
brucella agar and 6 ml of brucella broth in
25 cm2 tissue culture flasks was developed
for rapid Campylobacter cultivation
B, brucella broth; A, brucella agar; F, supplements; a, atmosphere air; g, gas mixture
Selective Media
A variety of selective media have been
developed for primary isolation of the
thermophilic campylobacters.
The most widely used media contain
peptone supplemented with yeast extract,
sodium metabisulphite, and blood.
The campylobacters are non-hemolytic, but
in general, the addition of blood enhances
the survival and growth of these organisms.
Selective Media
Moran and Upton, 1987
Identification
 A rapid latex agglutination (LA)
identification kid known as Campyslide
(BBL Microbiology Systems) was
developed and evaluated
Identification
Analysis of the electrophoretic profiles of
the outer membrane proteins (OMP) could
be used to differentiate C. jejuni from C. coli.
Identification
A PCR method for the rapid identification
and discrimination of thermophilic C. jejuni
and C. coli was developed by using a gene
encoding a protein involved in siderophore
transport (ceuE).
Identification
The omp50 gene and the Omp50 protein
are prevalent in Campylobacter strains
(Table 12).
Immunodetection assays and DNA-DNA
hybridizations showed that most C. coli
strains tested were negative and most C.
jejuni and C. lari strains tested were
positive.
A PCR assay was developed, using the
omp50 gene as a species-specific target
Identification
Identification
A cytolethal distending toxin (cdt) genebased species-specific multiplex PCR
assay for the detection of cdtA, cdtB or
cdtC gene of C. jejuni, C. coli or C. fetus,
respectively, was developed and evaluated
with 76 Campylobacter strains belonging to
seven different species and 131 other
bacterial strains of eight different genera.
Identification
A multiplex polymerase chain reaction
(PCR) to detect and differentiate food-borne
pathogens of the three genera
Campylobacter, Arcobacter and
Helicobacter in a single step procedure was
developed base on one common reverse
primer and three genus-specific forward
primers were designed by hybridizing to the
16S rRNA of selected reference strains
Immunofluorescence Microscopy
 Fecal material was emulsified in 1% FormalinPBS to prepare about a 10% suspension.
Samples were spotted onto five wells of a
multiwell slide and air dired, heat fixed, and then
flooded with 10% formalin-PBS for 10 min and
stained with the conjugate for 30 min
 Murine monoclonal antibodies to C. jejuni which
recognized a flagellin epitope common to most
Campylobacter spp. and an epitope restricted to
C. jejuni and C. coli were developed
Bioluminescence Assay
ATP has been used to estimate microbial
load based on the facts that bacterial cells
contain a fairly constant amount of ATP.
The ATP is first released in its free soluble
state and reacts with luciferin in the
presence of luciferase, magnesium, and
oxygen to produce light.
Bioluminescence Assay
Others
 Most Probable Number Method and Direct
Plate Count
 Filtration Method
 Enzyme-linked Immunosorbent Assay
Confirmation by latex agglutination
Detection of toxin
Among the isolates of 117 C. jejuni isolates
from Danish turkeys:
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97.4% produced cytolethal distending toxin
(CDT) in Vero cell assays,
89.7% in Colon 205 assays, and
93.2% in chicken embryo cell assays
Detection of toxin
CDT
Detection of toxin
CDT
Detection of toxin genes
A total of 117 C. jejuni isolates from Danish
turkeys were tested for the presence of
seven virulence and toxin genes by PCR
Detection of toxin genes
Detection of toxin genes
Cytolethal distending toxin (cdt) genebased species-specific multiplex PCR
assay for identifying C. jejuni, C. coli and C.
fetus has been developed and evaluated
with 34 Campylobacter-like organisms
isolated from poultry in Thailand for species
identification and was compared with other
assays including API Campy, 16S rRNA
gene sequence, and hippuricase (hipO)
gene detection
Detection of toxin genes
In another study, a cytolethal distending
toxin (cdt) gene-based species-specific
multiplex PCR assay for the detection of
cdtA, cdtB or cdtC gene of C. jejuni, C. coli
or C. fetus, respectively, was developed
and evaluated with 76 Campylobacter
strains belonging to seven different species
and 131 other bacterial strains of eight
different genera.
PATHOGENICITY AND VIRULENCE FACTORS
 The consequences of C. jejuni infection vary considerably
from asymptomatic excretion to severe bloody diarrhea,
high fever, and prostration.
 Most often, the illlness appears to last from 2 to 7 days,
with diarrhea, abdominal cramping, and fever as the most
significant symptoms.
 Bloody stools are common for hospitalized patients. The
diarrhea may be so severe as to mimic acute ulcerative
colitis, and the abdominal pain may mimic acute
appendicitis
 However, extra intestinal infections including meningitis,
cholecystitis, and urinary tract infection have been
reported
Animal Model
Infant Chicken Model
Removable Intestinal Tie Adult Rabbit
Diarrhea Model (RITARD)
Chicken Embryo Model
 ICR adult mice, Adult athymic (去胸線) and
euthymic germfree BALB/c mice
Chemotaxis
Positive chemotactic responses of C. jejuni
were directed toward only L-fucose (of 20
carbohydrates tested) and L-aspartate, Lcysteine, L-glutamate, and L-serine (of 15
amino acids tested).
The organism was also attracted to
pyruvate, succinate, fumarate, citrate,
malate, and α-ketoglutarate. Most
constituents of bile tested were
chemorepellents
Adhesion and Invasion
 The HeLa adhesive strains of C. jejuni and C. coli were
more frequently isolated from patients with diarrhea and
fever
 Although C. jejuni lacks fimbriae, it may possess other
adhesions.
 Adhesion has been demonstrated by using cell lines such
as HeLa, INT 407 and HEp-2 cells
 The adhesion was interfered by L-fucose, asparagus pea
lectin (which recognizes L-fucose determinants on cells),
or partial inhibited by other carbohydrates such as glucose,
galactose, mannose N-acetylglucosamine, Nacetylgalactosamine, and the non-sugar carbohydrate
sorbitol
Adhesion and Invasion
Adhesion and Invasion
The adherence was also inhibited partially
by treating the bacterial cells with proteases
or glutaraldehyde
Adhesion and Invasion
 The flagellum may contain adhesions for
epithelial cells, since an aflagellated variant
of C. jejuni adhered poorly to cells.
Aflagellated organisms still attach to cells to
some extent, suggesting the possibility of
multiple adhesions
Shearing of the bacterial cells to remove
the flagella reduced bacterial adhesion,
whereas immobilization of the flagellum
with KCN increased adhesion
Adhesion and Invasion
 Assaying by HEp-2 cells, clinical isolates of C.
jejuni were more invasive than the nonclinical
strains studied
 When HEp-2 cells were treated with cytochalasin
B, the invasiveness of C. jejuni was reduced,
indicating active participation of the host cell in
the uptake of these organisms (phagocytosis)
 The number of intracellular C. jejuni isolates
decreased when the Campylobacter whole-cell
lysate were adsorbed onto HEp-2 cell monolayers.
It suggested special invasive ligand appears to be
dependent upon an intact carbohydrate moiety
Adhesion and Invasion
Enterotoxins
Cytotonic effects of Campylobacter cultures
could be determined in Vero and CHO cells,
GM1 ELISA, and cyclic AMP accumulation
in cells exposed to these culture filtrates
 was demonstrated by using antitoxins to
cholera toxin and E. coli heat-labile
enterotoxin
Enterotoxins
 C. jejuni produces enterotoxins which is
related to the heat-labile enterotoxins of E.
coli. The B subunits of C. jejuni enterotoxin
(by dissoication techniques involving gel
filtration in the presence of guanidine) was
functional and immunological properties
resemble those of the B subunits of cholera
toxin and E. coli heat-labile toxin (LT).
Enterotoxins
 Enterotoxin of C. jejuni was produced by
culturing in a Casamino acids-yeast extract
broth (Difco) containing 1.0 μg/ml of ferric
chloride and incubated at 37C in the
presence of 10% carbon dioxide
 Maximum enterotoxin production was
achieved by growth at 42C for 24 h under
agitation
 Addition of polymyxin enhanced the
recovery of toxin.
Enterotoxins
 This enterotoxin was partial purified by gel
filtration, anti-cholera toxin immunoglobulin
and ganglioside affinity column
chromatographies.
A 68-kDa polypeptide was shown to have
immunological relationship with cholera
toxin, and the 68- and 54-kDa polypeptides
might be responsible for the recognition of
ganglioside
Cytotoxins
 In another study, the filtrates of 12 polymyxintreated (to release toxin) isolates of C. jejuni were
placed on HeLa cells and CHO cells and showed
significant cytotoxicity similar to Shiga-like toxin
but not neutralized by antisera against either
Shiga-like toxin I or II.
 This cytotoxin was unstable at temperature above
50C and its activity decreased by the treatment of
trypsin.
 This cytotoxin may contribute to the colonic
mucosal invasive process that characterizes C.
jejuni enteritis
Cytotoxins
 The toxin, called cytolethal distending toxin
(CDT), which causes direct DNA damage
leading to invocation of DNA damage
checkpoint pathways.
CDT consists of three protein subunits,
CdtA, CdtB, and CdtC, with CdtB recently
identified as a nuclease. Both CdtA and
CdtC bound with specificity to the surface of
HeLa cells, whereas CdtB did not
Cytotoxins
 C. jejuni induces oncotic（膠質的swelling）,
rather than apoptotic death of T84 enterocytes.
 C. jejuni-treated enterocytes exhibited extensive
cytoplasmic vacuolation, rapid (3-6 h) loss of
plasma membrane integrity ('cytotoxicity'), loss of
mitochondrial transmembrane potential, and ATP
depletion.
 Enterocytes also exhibited increased
oligonucleosomal DNA fragmentation, a feature
characteristic of apoptosis
Cytotoxins
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Cytotoxins
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Cytotoxins
Quorum sensing is known to be related to
regulation of virulence factors in some
pathogens. Function of luxS is related to
the regulation of cdt in C. jejuni
The reverse transcriptase-polymerase
chain reaction (RT-PCR) showed that cdtA,
cdtB, and cdtC genes constitute a
polycistronic operon in C. jejuni
A decrease in cdt transcription was
observed in the luxS null mutant
Cytotoxins
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Cytotoxins
C. jejuni is capable of extensive replication
within human monocytic cell vacuoles and
induces apoptotic death via cytolethal
distending toxin
Regulation of virulence
 Maximal host cell invasion requires the
secretion of proteins termed Campylobacter
invasion antigens (Cia). The virulence
potential of Campylobacter may be
triggered by the bile acid deoxycholate
(DOC).
Regulation of virulence
MHD contain DOC
Regulation of virulence
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MOLECULAR STUDIES OF ANTIBIOTIC
RESISTANCE
Partial sequence analysis of a tet(O)
plasmid from a multiple-drug-resistant
clinical isolate of C. jejuni revealed 10
genes or pseudogenes encoding different
aminoglycoside inactivating enzymes,
transposase-like genes, and multiple
unknown genes from a variety of
pathogenic and commensal bacteria
MOLECULAR STUDIES OF ANTIBIOTIC
RESISTANCE