CAMPYLOBACTER II

1. CAMPYLOBACTERII Hin-chung Wong Department of Microbiology Soochow University

2. 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

3. CONTROL OF CAMPYLOBACTER IN FOODS • The effects of various disinfectants were tested. The killing time depended on the size of the inoculum

4. 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

5. 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

6. 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

7. ISOLATION AND ENUMERATION

8. 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)

9. ISOLATION AND ENUMERATION

10. 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

11. 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.

12. Selective Media Moran and Upton, 1987

13. Identification • A rapid latex agglutination (LA) identification kid known as Campyslide (BBL Microbiology Systems) was developed and evaluated

14. Identification • Analysis of the electrophoretic profiles of the outer membrane proteins (OMP) could be used to differentiate C. jejuni from C. coli.

15. 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).

16. 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

17. Identification

18. Identification • 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.

19. 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

20. Immunofluorescence Microscopy • Fecal material was emulsified in 1% Formalin-PBS 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

21. 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.

22. Bioluminescence Assay

23. Others • Most Probable Number Method and Direct Plate Count • Filtration Method • Enzyme-linked Immunosorbent Assay • Confirmation by latex agglutination

24. 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

25. Detection of toxin • CDT

26. Detection of toxin • CDT

27. 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

28. Detection of toxin genes

29. Detection of toxin genes • Cytolethal distending toxin (cdt) gene-based 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

30. 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.

31. 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

32. 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

33. Chemotaxis • Positive chemotactic responses of C. jejuni were directed toward only L-fucose (of 20 carbohydrates tested) and L-aspartate, L-cysteine, 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

34. 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, N-acetylgalactosamine, and the non-sugar carbohydrate sorbitol

35. Adhesion and Invasion

36. Adhesion and Invasion • The adherence was also inhibited partially by treating the bacterial cells with proteases or glutaraldehyde

37. 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

38. 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

39. Adhesion and Invasion

40. 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

41. 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).

42. 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.

43. 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

44. Cytotoxins • In another study, the filtrates of 12 polymyxin-treated (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

45. 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

46. 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

47. Cytotoxins

48. Cytotoxins

49. 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

50. Cytotoxins