Transcript CAMPYLOBACTER

Campylobacter jejuni
黃顯宗
Campylobacter jejuni
• 早期名為Vibrio fetus，屬於動物病菌，
1970年中期才有乳品與水污染中毒發生
• 主要是兩種C. jejuni，C. coli及大多數中
毒為前者（95%）
• G-，vibriod，microaerophilic，在21%O2
下不長，最適生長溫度42度，生長範圍在
25~50度，菌體小，生長慢
• 禽類普遍可分離到
INTRODUCTION
• Campylobacter could be isolated from
the normal flora of barnyard animals and
fowl and caused periodically diarrheal
diseases.
• Therefore, much of the early
investigation of Campylobacter was done
by the veterinary community
• Originally classified in the genus Vibrio
and the new genus, Campylobacter, was
created by Sebald and Veron in 1963
Campylobacter jejuni
• 培養時空氣成份是5%O2、10%CO2、
85%N2
• 潛伏期2~5天，症狀：發熱，下痢，
腹痛，血便
• 在美國地區發生率高，幾乎與
Salmonella、Shigella相當
世界食品安全趨勢
全球食品安全發展趨勢
INTRODUCTION
• Campylobacter also caused human
abortion and tissue and blood infections
including septic arthritis, bacterial
endocarditis, phlebitis (血栓靜脈炎),
meningoencephalitis, prolonged febrile
illness (發燒), stillbirth, etc.
• Most of the early illnesses attributed to
Campylobacter was caused by C. fetus
INTRODUCTION
• Epidemics involving C. jejuni have been
reported from both developed and
underdeveloped countries and it is also
one of the causative agents of traveller's
diarrhea
• Children in the underdeveloped countries
could be asymptomatic carriers and also
the leading cause of diarrhea
TAXONOMY AND GENERAL
CHARACTERISTICS
• The genus Campylobacter is composed
of Gram-negative, vibroid shaped
bacteria which, when examined
microscopically in wet mounts, exhibit a
corkscrew-like darting motility
Classification and Biotyping
Classification and Biotyping
• The genus is divided initially into two groups
based on the presence or absence of catalase.
The catalase-negative species are generally
considered to be saphrophytes
• All "C. upsaliensis" strains were hippurate
negative, and six of seven were susceptible to
cephalothin. This new species could be a
potential human pathogen associated with
gastroenteritis and bacteremia in normal
individuals as well as with opportunistic
infections in immunocompromised patients
Classification and Biotyping
• The catalase-positive species include C. jejuni,
C. coli, and C. fetus subspecies fetus and
venerealis, C. laridis and C. hyointestinalis.
• Aerotolerant Campylobacter strains have also
been identified from aborted porcine, ovine,
and equine fetus, and DNA homology studies
suggest them to be a new species.
Classification and Biotyping
• C. jejuni and C. coli are the most common,
human intestinal pathogens, although other
catalase-positive species are occasionally
responsible for cases of human gastroenteritis
• Generally, the human intestinal pathogens (C.
jejuni, C. coli) can be distinguished by their
thermophilic growth properties (i.e. growth at
42C).
• The non-intestinal pathogens grow at 25C, but
not at 42C
Biotyping
DNase activity
• The bacteria were preincubated in
polymyxin B (which has been used to
release the cell-associated secretory
proteins, such as enterotoxins, protease,
alkaline phosphatase, DNase, etc.) and
suspended into the well in the DNase
test agar plate (DNase agar of Difco with
0.01% toluidine blue O). A strong pink
zone indicating DNA hydrolysis was
seen around the well after 20 to 24 h of
aerobic incubation at 37C.
Microaerophilism
• These bacteria fail to grow aerobically in
air atmospheres (i.e. 21% O2, v/v), but
can be cultured successfully under
microaerobic conditions in atmospheres
containing 6-10% oxygen supplemented
with 5% carbon dioxide, either nitrogen
or hydrogen may be used to make up the
difference in partial pressure
Microaerophilism
• The microaerophilic nature of the
campylobacters may be related to their
sensitivity to toxic reduced forms of
oxygen, such as superoxide radicals and
hydrogen peroxide. Such toxic
substances may be formed by
photoreaction and in the presence of air.
Microaerophilism
Light and Air
Microaerophilism
• Addition of ferric iron, sodium metabisulphite,
sodium pyruvate to brucella-based medium
permitted most strains of C. jejuni to grow
aerobically (i.e. at 21% oxygen) in a CO2
incubator
• The addition of bovine erythrocyte superoxide
dismutase (Cu/Zn enzyme) to brucella agar is
even more effective in enhancing
aerotolerance
• Catalase (catalase and superoxide dismutase
are present in the cytoplasm of catalasepositive campylobacters) also enhance
aerotolerance, but to a lesser extent.
Microaerophilism
• The chemicals added to enhance
aerotolerance scavenge superoxide radicals
(ferric iron binding dihydroxyphenyl
compounds, ferric iron bisulphate coordination
complexes)
• or decompose hydrogen peroxide (e.g.
pyruvate)
• Addition of active superoxide dismutase, a
superoxide anion scavenging enzyme,
minimizes production of cocccoid forms in
suspensions stored in air
Microaerophilism
• Addition of charcoal trap for free radicals
generated by photochemical oxidative
mechanisms, therefore, it is
recommended that media used for the
primary isolation of campylobacters be
stored in the dark to prevent peroxide
build-up in the medium
Microaerophilism
• Proteins involved in the oxidative stress
of campylobacters have been studied.
• Of the three groups of haemoglobins
identified in microorganisms (singledomain globins, flavohaemoglobins and
truncated globins), the last group is
involved in moderating O2 flux within C.
jejuni.
Microaerophilism
• The function of the truncated
haemoglobin (Ctb) encoded by C. jejuni
was investigated by constructing a ctb
mutant and characterizing its phenotype
• Cells mutated in ctb were disadvantaged
when grown under conditions of high
aeration and defective in survival in air
Microaerophilism
Microaerophilism
• Pathogenic bacteria experience nitrosative
stress from NO generated in the host and from
nitrosating species such as Snitrosoglutathione.
• The food-borne pathogen C. jejuni responds
by activating gene expression from a small
regulon under the control of the NO-sensitive
regulator, NssR.
• The NssR regulon up-regulated two
hemoglobins (Cgb and Ctb) and more than 90
other genes, notably those encoding heat
shock proteins and proteins involved in
oxidative stress tolerance and iron
metabolism/transport
Microaerophilism
• The putative global posttranscriptional
regulator csrA was mutated in C. jejuni.
• In E. coli, CsrA is involved in regulating
stationary-phase metabolism, represses
glycogen biosynthesis, gluconeogenesis,
peptide transport, and biofilm formation.
• The csrA mutant was attenuated in
surviving oxidative stress.
Microaerophilism
• In another study, the oxidative stress response after
exposure to paraquat, a strong oxidising agent, was
analyzed by two-dimensional protein electrophoresis
and Maldi-ToF mass spectrometry
• Oxidative stress and redox-related proteins were
overexpressed: FldA flavodoxin and a pyruvateflavodoxin oxidoreductase encoded by cj1476c.
• No increase in SodB expression was observed.
• An additional quantitative RT-PCR analysis showed an
increase in katA but not in sodB expression
• However, the sodB mutant was very sensitive to
paraquat,
Morphology
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Several morphology forms of C. jejuni are found in
culture. Rod forms, which include spirals, S-shaped
and characteristically curved cells predominate in fresh
young cultures
whereas non-culturable coccoid forms occur mainly in
old cultures
In addition, rods transform to coccoid forms when
conditions are unfavorable for growth
Compared with stored media, production of coccoid
forms was less on freshly prepared media
Addition of supplements used as detoxifying agents
(ferrous sulphate, sodium metabisulphite and sodium
pyruvate) minimized production of these forms on
media.
Morphology
• Viable but
nonculturable
• VBNC
Morphology
Growth Temperature and Heat Resistance
• Optimal temperature for the growth of
Campylobacter is at 42C.
• C. jejuni is not a heat-stable bacterium,
with low D value.
• 受到基質影響
• The D value in chicken medium was
higher than in peptone medium
Growth Temperature and Heat Resistance
• D value測定方法
• Z value
Effects of Iron and Other Minerals
• Cells grown in low-iron medium
exhibited slower growth rates and altered
cellular morphology
• Increased numbers of longer, more
filamentous forms were seen in Gramstain smears
Effects of Iron and Other Minerals
Effects of Iron and Other Minerals
• Three proteins, with apparent Mol.Wt. of
82,000, 76,000, and 74,000, were
consistently present in the outer
membrane of cells grown in low-iron
medium
• 鐵的攝取
– Siderophore
– transporter
Plasmid Profile and Antibiotic Resistance
• Serotyping, phage typing, plasmid profile and antibiotic
resistance could be useful epidemiological markers
• Usually high percentage of Campylobacter spp.
contain plasmid, e.g. 53% of isolates from animals
contained plasmid DNA ranged in size from less than
1 to 86 MDa
• Resistance to tetracycline and gentamicin were
probably plasmid mediated
• Most strains were susceptible to kanamycin,
clindamycin, chloramphenicol, kanamycin, tobramycin,
streptomycin, erythromycin, gentamicin, tetracycline,
and compound sulfonamide
Plasmid Profile and Antibiotic Resistance
• In another study, tetracycline resistance was
detected in six out of nine C. coli isolates (67
%) and 13 out of 74 C. jejuni isolates (18 %).
• Both low- and high-level tetracycline
resistance was associated with the presence
of the tet(O) gene.
• In C. jejuni, tet(O) was plasmid-encoded in 54
% of tetracycline-resistant isolates
• whereas in C. coli, tet(O) appeared to be
located on the chromosome
Plasmid Profile and Antibiotic Resistance
Plasmid Profile and 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
Plasmid Profile and Antibiotic Resistance
• The plasmid in Lactococcus and
Lactobacillus species contains two
putative antibiotic resistance homologs,
an ermB gene encoding erythromycin
and clindamycin resistance, and a
streptomycin resistance gene, aadE.
• Of particular note is the aadE gene
which holds 100% identity to an aadE
gene found in C. jejuni plasmid but which
probably originated from a Gram-positive
source
Serotyping
• Serological typing of the campylobacters
is complex and several schemes have
been devised, however, no uniform
serotyping scheme has been adopted,
and there are over 50 recognized
serotypes of C. jejuni
• Antigens used for serological typing
include heat stable and heat labile
antigenic factors, outer membrane
proteins and flagellar antigens
Phage typing
• A bacteriophage typing system was
developed with phages isolated from
poultry feces
• Selection of 14 phages from the 47
phages available was assisted by
determination of the Sneath-Jaccard
similarity coefficients and subsequent
unweighted pair-group arithmetic
averaging cluster analysis
• This typing set was reproducible and
stable in the isolates from Illinois
Typing with lectins
• Bacterial suspension, 50 ul, reacted with
50 ul of lectin solutions (lectins from
different plants) in U-bottomed microtiter
wells
• Different reaction patterns were
observed (Table 5). Heating of the
cultures to 100C and holding for 30-60
min greatly enhanced their reactivity with
lectins
Typing with lectins
Restriction fragments
• On the basis of restrictive digest, six
types were identified with AfaI, seven
types with MboI and five types with
HaeIII.
• With a combination of these three
enzymes, 22 types were found
Restriction fragments
PCR methods
• PCR methods are also developed.
Based on a 10-mer primer (5'CCTGTTAGCC-3'), a random amplified
polymorphic DNA (RAPD) method for
typing C. coli isolated from pigs was
developed
PCR methods
• Amplified fragment length polymorphism
(AFLP) patterns was applied to type C.
jejuni isolates from human and chicken
• AFLP原理
PCR methods
• The published genome sequence of C.
jejuni strain NCTC 11168 was used to
model an accurate and highly
reproducible fluorescent amplified
fragment length polymorphism (FAFLP)
analysis.
• Predicted and experimentally observed
amplified fragments (AFs) generated
with the primer pair HindIII+A and
HhaI+A were compared
PCR methods
PCR methods
• A molecular typing approach for C. jejuni
and C. coli was developed with
restriction fragment length polymorphism
analysis of a 9.6-kb PCR-amplified
portion of the lipopolysaccharide gene
cluster.
PCR methods
Ribotying
• Strains from diverse sources of C. jejuni were
examined for polymorphism around the 16S
rRNA genes.
• Complete typeability was obtained; 30 distinct
PstI and 42 HaeIII polymorphisms were found
• Three bands were detected in almost all
strains with these enzymes, confirming that
three copies of the 16S rRNA gene are typical
for C. jejuni
• By combination of the two enzyme
polymorphisms, 77 16S ribotypes were defined
among the 261 strains analyzed
Pulsed-field gel
electrophoresis
• The DNA of C. jejuni, with its low G+C content,
was found to have no restriction sites for
enzymes NotI and SfiI, which cut a high-G+C
regions.
• With SalI, six restriction fragments with
average values of 48.5, 80, 110, 220, 280, and
980 kilobases (kb) were obtained.
• With SmaI, nine restriction fragments with
average values ranging from 39 to 371 kb,
which yielded an average genome size of
1.726 Mb were obtained.
• With KpnI, 11 restriction fragments with sizes
ranging from 35 to 387.5 kb, which yielded an
average genome size of 1.717 Mb were
obtained
Multilocus sequencing
• A multilocus sequence typing (MLST)
system for this organism is described to
exploit the genetic variation present in
seven housekeeping loci to determine
the genetic relationships among isolates
of C. jejuni
Multilocus sequencing
• non-synonymous （dN）synonymous
（dS）突變的機率（dN/dS）
OCCURRENCE IN ENVIRONMENT,
ANIMALS AND FOODS
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Water and soil may be contaminated with
Campylobacter, possibly reflecting contamination of
the environment with animal excreta
• Organisms in feces, or those inoculated into water, soil,
or milk, may survive for several weeks when ambient
temperatures are low
• Water is potentially an important reservoir of the
thermophilic campylobacters and is an established
vehicle for the transmission of these organisms to man
and domestic animals
• Water-borne outbreaks of Campylobacter enteritis
have been reported from various countries
In animal
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C. jejuni and C. coli are frequently present as part of
the normal interstinal flora of various animals and fowl
Both species have been isolated from intestinal
contents of turkeys, chickens, ducks and also wild
birds.
Poultry is contaminated to a substantial extent with
Campylobacter.
Even eggs may be contaminated with Campylobacter
due to material from infected chickens.
However, C. jejuni may not be a significant pathogen
for chickens under normal conditions. C. jejuni was
isolated from 106 of 200 samples of pigeon feces
In Food
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Poultry
Meat
Raw milk
Antibiotic resistance, cytotoxicity,
virulence genes, typing
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
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 Shigalike 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
• 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
Detection of toxin
• CDT
Detection of toxin
• CDT
Detection of toxin
• Among the isolates of 117 C. jejuni
isolates from Danish turkeys:
– 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
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
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:
– 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
• 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
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
• Analysis of the electrophoretic profiles of
the outer membrane proteins (OMP)
could be used to differentiate C. jejuni
from C. coli.
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
• 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
• 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 foodborne 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
Others
• Most Probable Number Method and
Direct Plate Count
• Filtration Method
• Enzyme-linked Immunosorbent Assay
• Confirmation by latex agglutination
Identification
• A rapid latex agglutination (LA)
identification kid known as Campyslide
(BBL Microbiology Systems) was
developed and evaluated
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