CAMPYLOBACTER I Hin-chung Wong Department of Microbiology Soochow University

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


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


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.


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


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 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


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.


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



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 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


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 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



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


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



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 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


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 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



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.


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

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CAMPYLOBACTER I Hin-chung Wong Department of Microbiology Soochow University