Transcript CAMPYLOBACTER

CAMPYLOBACTER
I
Hin-chung Wong
Department of Microbiology
Soochow University
Content
 INTRODUCTION.
 TAXONOMY AND GENERAL CHARACTERISTICS






Classification and Biotyping
Microaerophilism
Morphology
Growth Temperature and Heat Resistance
Effects of Iron and Other Minerals
Plasmid Profile and Antibiotic Resistance
 SEROTYPING, PHAGE TYPING, INTERACTION WITH LECTINS AND MOLECULAR
TYPING




Serotyping
Phage typing
Typing with lectins
Molecular typing
•
•
•
•
•
Restriction fragments
PCR methods
Ribotying
Pulsed-field gel electrophoresis
Multilocus sequencing
 OCCURRENCE IN ENVIRONMENT, ANIMALS AND FOODS



Environment
In Animals
In Foods
Content
 CONTROL OF CAMPYLOBACTER IN FOODS
 ISOLATION AND ENUMERATION











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






Campylobacter Enteritis
Animal Model
Chemotaxis
Adhesion and Invasion
Enterotoxins
Cytotoxins
 MOLECULAR STUDIES OF ANTIBIOTIC RESISTANCE
 CONCLUSIONS
 REFERENCES
INTRODUCTION
Campylobacters (formerly Vibrio fetus)
were first associated with diseases of cattle
and sheep at the beginning of 20th century.
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
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
Campylobacter enteritis, caused by C.
jejuni and C. coli, is a relatively recent
disease
first recognized following several water and
milk borne epidemics in the mid-1970s
Today, enteritis caused by C. jejuni/coli is
as common as that caused by Salmonella
and Shigella combined.
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
Originally classified in the genus Vibrio and
the new genus, Campylobacter, was
created by Sebald and Veron in 1963
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.
 C. hyointestinalis has been isolated from pigs with
proliferative ileitis, but its role in human disease
has not been established.
 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
Classification and Biotyping
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 610% 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
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 catalase-positive
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 (single-domain
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
Furthermore, the rate at which ctb mutant
cells consumed O2 in an O2 electrode (10200 microM O2) was approximately half the
rate displayed by wild-type cells, reflecting
a role for Ctb in respiration at
physiologically relevant external O2
concentrations
Microaerophilism
Microaerophilism
 Pathogenic bacteria experience nitrosative stress
from NO generated in the host and from
nitrosating species such as S-nitrosoglutathione.
 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
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 pyruvate-flavodoxin
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
 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
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. The D value in chicken
medium was higher than in peptone
medium
Growth Temperature and Heat Resistance
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 Gram-stain
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
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
PCR methods
PCR single-stranded conformational
polymorphism (SSCP) method examines
the hypervariable region of the flaA gene
and as this technique can detect point
mutations, and the SSCP banding patterns
can be used in typing
http://www.nature.com/nprot/journal/v1/n6/fig_tab/nprot.2006.485_F1.html
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
 All but one of the 61 predicted AFs were
reproducibly detected, and no unpredicted
fragments were amplified.
 This FAFLP analysis was used to genotype 74 C.
jejuni strains belonging to the nine heat-stable
(HS) serotypes most prevalent in human disease
in England and Wales.
 The 74 C. jejuni strains exhibited 60 FAFLP
profiles, and cluster analysis of them yielded a
radial tree showing genetic relationships between
and within 13 major clusters
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
OCCURRENCE IN ENVIRONMENT,
ANIMALS AND FOODS
 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
OCCURRENCE IN ENVIRONMENT,
ANIMALS AND FOODS
 In microcosms, aerated with shaking,
exhibited logarithmic decline in recoverable
C. jejuni, while stationary systems
underwent a more moderate rate of
decrease to the noncluturable state
OCCURRENCE IN ENVIRONMENT,
ANIMALS AND FOODS
In animal
 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
Poultry
Meat
Raw milk
Antibiotic resistance, cytotoxicity, virulence
genes, typing