CAMPYLOBACTERCAMPYLOBACTER I I
Hin-chung Wong Department of Microbiology
Soochow University
ContentContent 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
ContentContent 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 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 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 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 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 TAXONOMY AND GENERAL CHARACTERISTICS 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
Classification and BiotypingClassification 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 BiotypingClassification 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 BiotypingClassification 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
BiotypingBiotyping
Classification and BiotypingClassification and BiotypingThe 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 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 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
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 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 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
MicroaerophilismMicroaerophilism
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.
MicroaerophilismMicroaerophilism
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
MicroaerophilismMicroaerophilism
MicroaerophilismMicroaerophilism
Furthermore, the rate at which ctb mutant cells consumed O2 in an O2 electrode (10-200 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
MicroaerophilismMicroaerophilism
MicroaerophilismMicroaerophilism 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
MicroaerophilismMicroaerophilism
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.
MicroaerophilismMicroaerophilism
MicroaerophilismMicroaerophilism 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 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 Morphology
VBNC
Morphology Morphology
Growth Temperature and Heat Resistance 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 ResistanceGrowth Temperature and Heat Resistance
Effects of Iron and Other Minerals 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
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 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 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
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 ResistancePlasmid 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 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 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 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 Typing with lectins
Restriction fragments 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 Restriction fragments
PCR methods 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 methodsPCR methods
Amplified fragment length polymorphism (AFLP) patterns was applied to type C. jejuni isolates from human and chicken
PCR methodsPCR 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 methodsPCR 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 methodsPCR methods
PCR methodsPCR 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 methodsPCR methods
PCR methodsPCR 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 methodsPCR methods
Ribotying 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 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 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 sequencingMultilocus sequencing
OCCURRENCE IN ENVIRONMENT, OCCURRENCE IN ENVIRONMENT, ANIMALS AND FOODS 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, OCCURRENCE IN ENVIRONMENT, ANIMALS AND FOODSANIMALS 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, OCCURRENCE IN ENVIRONMENT, ANIMALS AND FOODSANIMALS AND FOODS
In animalIn 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 FoodIn Food
PoultryMeatRaw milkAntibiotic resistance, cytotoxicity, virulence
genes, typing