In this article we will discuss about:- 1. Introduction to Campylobacter 2. The Organism of Campylobacter and its Characteristics 3. Pathogenesis and Clinical Features 4. Isolation and Identification 5. Association with Foods.
Contents:
- Introduction to Campylobacter
- The Organism of Campylobacter and its Characteristics
- Pathogenesis and Clinical Features of Campylobacter
- Isolation and Identification of Campylobacter
- Campylobacter’s Association with Foods
1. Introduction to Campylobacter:
Campylobacter has been known as a veterinary problem since the early years of the Century when the original isolate, known then as Vibrio fetus, was associated with infectious abortion in sheep and cattle. In 1931 the species Vibrio jejuni was described as the cause of winter dysentery in calves and in 1946 a similar organism was isolated from blood cultures of patients in a milk-borne outbreak of acute diarrhoea.
Later King, working with human blood isolates, distinguished two groups on the basis of their optimum growth temperature. One group corresponded to Vibrio fetus and the second, ‘thermophilic’, group, which grew best at 42 °C, came from patients with preceding diarrhoea.
Both groups differ from the cholera and halophilic vibrios biochemically, serologically and in their mol% G + C ratio, and were reclassified into the new genus Campylobacter in 1963. In the 1970s, with the development of suitable selective media, it was established that Campylobacter jejuni, and to a lesser extent Campylobacter coli, are a major cause of diarrhoeal illness, rivaling and even surpassing Salmonella in importance in many countries.
Campylobacter laridis, C. concisus and C. hyointestinalis have also been isolated occasionally from patients with diarrhoea and C. pylori, now reclassified as Helicobacter pylori, has been associated with gastritis and stomach and duodenal ulcers.
2. The Organism of Campylobacter and its Characteristics:
Campylobacters are non-sporeforming, oxidase-positive, Gram-negative rods. Cells are pleomorphic and may be 0.5-8 µm in length and 0.2-0.5 µm in width.
Log- phase cells have a characteristic slender, curved or spiral shape and one or more polar or amphitrichous flagella which confer a rapid, darting motility and may be an important feature in pathogenesis (Figure 7.1). As cultures age, spiral or curved bacilli are replaced by round forms.
Campylobacters cannot ferment or oxidize sugars and are oxygen-sensitive microaerophiles, growing best in an atmosphere containing 5-10% oxygen and 3-5% carbon dioxide.
All Campylobacter species grow at 37 °C; C. jejuni and C. coli have optima at 42-45 °C but cannot survive cooking or pasteurization temperatures (D55 a 1 min). They do not grow below 28 °C and survive poorly at room temperature.
Although their viability declines during chill or frozen storage, they may nevertheless persist under these conditions for prolonged periods; survival has been recorded in milk and water at 4 °C after several weeks storage and in frozen poultry after several months. They are also particularly sensitive to other adverse conditions such as drying and reduced pH.
The principal environmental reservoir of pathogenic campylobacters is the alimentary tract of wild and domesticated animals and birds and it is a commonly found commensal of rodents, dogs, cats, dairy cattle, sheep, pigs, poultry and wild birds.
The high optimum growth temperature of C. jejuni and C. coli could be an adaptation to the higher body temperature of birds and reflect their importance as a primary reservoir of the organism. Asymptomatic human carriage also occurs.
Though they would not appear to survive particularly well outside an animal host, campylobacters can be commonly isolated from surface water.
Survival is enhanced by low temperatures and studies conducted in Norway have shown that strains of C. jejuni, C. coli and C. laridis remained viable in un-chlorinated tap water at 4 °C for 15 days (10 days at 12 °C) and 10-15 days in polluted river water at the same temperature (6-12 days at 12 °C).
Under adverse environmental conditions campylobacters have been reported to adopt a ‘viable non-culturable’ state where the organism cannot be isolated by cultural methods but nevertheless remains infective. Evidence for this is conflicting but one study has shown that viable non-culturable C. jejuni can revert to a culturable state by passage through an animal host.
3. Pathogenesis and Clinical Features of Campylobacter:
Enteropathogenic campylobacters cause an acute enterocolitis which, in the absence of microbiological evidence, is not easily distinguished from illness caused by other pathogens. The incubation period is from 1 to 11 days, most commonly 3-5 days, with malaise, fever, severe abdominal pain and diarrhoea as the main symptoms.
The diarrhoea produces stools containing 106-109 cells g-1, which are often foul- smelling and can vary from being profuse and watery to bloody and dysenteric.
Gastrointestinal symptoms are sometimes preceded by a prodromal stage of fever, headache and malaise which lasts about a day. The diarrhoea is self-limiting and persists for up to a week, although mild relapses often occur. Excretion of the organism continues for up to 2-3 weeks. Vomiting is a less common feature.
Complications are rare although reactive arthritis can develop and Campylobacter has been shown to cause the serious neurological disease, Guillain-Barre syndrome.
As with other pathogens the infective dose will depend upon a number of factors including the virulence of the strain, the vehicle with which it is ingested and the susceptibility of the individual. Young adults (15-24 years old) and young children (1 – 4 years) appear particularly susceptible.
In an outbreak at a boys’ school in England caused by contamination of a water-holding tank with bird droppings, the infective dose was estimated as 500 organisms and in a separate study, a similar dose in milk caused illness in a volunteer.
Motility, chemo-taxis and the corkscrew morphology of the cells are all important factors in the virulence of Campylobacter, enabling it to penetrate the viscous mucus which covers the epithelial surface of the gut.
Studies with C. jejuni have demonstrated a chemotactic response toward the sugar L-fucose, a number of amino acids, and intestinal mucus from mice and pigs. Although Campylobacter does not possess fimbriae it probably possesses other adhesins that enable it to adhere to epithelial cells once the mucosal barrier has been penetrated.
The different symptoms that characterize Campylobacter infection probably reflect the operation of different pathogenic mechanisms. It seems likely that the profuse watery diarrhoea is due to elaboration of a secretory enterotoxin similar to cholera toxin.
Some strains of C. jejuni and C. coli have been shown to produce a heat-labile, acid-sensitive enterotoxin (Mr 60-70 kDa) which stimulates adenylate cyclase activity and disrupts normal ion transport in the enterocytes.
Campylobacter enterotoxin also resembles the cholera and Escherichia coli LT toxins serologically, since it is inactivated by antibodies to these toxins and some nucleotide sequence homology has been demonstrated between the gene coding the enterotoxin and that coding E. coli LT.
Toxin production in vitro is stimulated by levels of iron in excess of those required for optimal growth and this may be an important factor in virulence expression in vivo.
In the dysenteric syndrome, the cells of the terminal ileum and colon are the important sites of infection with invasion and necrosis producing blood- and pus- containing stools. Production of a heat-labile, trypsin-sensitive cytotoxin has been demonstrated in more than 70% of C. jejuni and C. coli strains.
The toxin is not neutralized by antibodies to Shiga or Clostridium difficile toxins. Evidence for a correlation between isolation of cytotoxin-producing strains and clinical symptoms is contradictory at present so the precise significance of cytotoxin in pathogenesis remains to be determined.
4. Isolation and Identification of Campylobacter:
Although most of the isolation procedures and media used were designed for C. jejuni, they are also suitable for C. coli and C. laridis. Pathogenic campylobacters have a reputation for being difficult to grow but in fact their nutritional requirements are not particularly complex and they can be grown on a number of peptone-based media including nutrient broth.
Where problems can sometimes arise is in their sensitivity to oxygen and its reactive derivatives.
Although pathogenic Campylobacters possess catalase and superoxide dismutase, the accumulation of peroxides and superoxide in media during storage or incubation can inhibit growth. For this reason an incubation atmosphere of 5- 6% oxygen with about 10% carbon dioxide and media containing oxygen scavenging compounds such as blood, pyruvate, ferrous salts, charcoal and meta-bisulfite are commonly used.
A number of selective enrichment media are used which include cocktails of antibiotics such as polymyxin B, trimethoprim and others as selective agents. In many cases cells isolated from food or other environmental sources have been sub- lethally injured as a result of stresses such as freezing, drying or heating and, as a result, are more sensitive to antibiotics and toxic oxygen derivatives.
This can mean that they will not grow on the usual selective media unless allowed a period for recovery and repair in which case a resuscitation stage of 4 h at 37 °C in a nonselective environment is recommended.
After selective enrichment for 24 and 48 h under micro-aerobic conditions at 42- 43 °C, samples are streaked on to selective plating media.
These normally contain a nutrient-rich basal medium supplemented with oxygen scavengers such as blood and/or FBP (a mixture of ferrous sulfate, sodium meta-bisulfite, and sodium pyruvate), and a cocktail of antibiotics similar to those used for selective enrichment. It is important to store pre-prepared media under nitrogen, at 4 °C and away from light to reduce the build-up of toxic oxides.
Colonies are non-haemolytic and have a rather unimpressive flat, watery appearance with an irregular edge and a grey or light-brown coloration. Suspect colonies are examined microscopically for motility and morphology and subjected to a range of tests after purification.
C. jejuni, C. coli, and C. laridis are catalase and oxidase positive, reduce nitrate to nitrite, grow at 42 °C but not at 25 °C micro-aerobically and cannot grow aerobically at 37° C. C. laridis is resistant to nalidixic acid while C. jejuni and C. coli are not. C. jejuni and C. coli can be distinguished by the ability of the former to hydrolyse hippurate.
Various bio-typing, phage typing and serotyping schemes have been proposed but are not used routinely as an epidemiological tool.
5. Campylobacter’s Association with Foods:
Campylobacter infection can be acquired by a number of routes. Direct transmission person-to-person or from contact with infected animals, particularly young pets such as kittens or puppies, has been reported, as have occasional waterborne outbreaks. However food is thought to be the principal vehicle.
As a common inhabitant of the gastrointestinal tract of warm-blooded animals, Campylobacter inevitably finds its way on to meat when carcasses are contaminated with intestinal contents during slaughter and evisceration.
Numbers are reduced significantly as a result of chilling in the abbatoir; the incidence of Campylobacter- positive beef carcasses in Australia was found to decrease from 12.3% to 2.9% on chilling and a similar survey of pig carcasses in the UK found a decrease from 59% down to 2%.
This is primarily a result of the sensitivity of Campylobacter to the dehydration that takes place on chilling. Subsequent butchering of red-meat carcasses will spread the surviving organisms to freshly cut, moist surfaces where viability will decline more slowly.
Poultry carcasses which cool more rapidly due to their size suffer less surface drying when air-chilled and this, probably coupled with the surface texture of poultry skin, enhances survival. Surveys in Australia, the UK and the USA have found 45%, 72% and 80% respectively of chilled poultry carcasses at the abattoir to contain Campylobacter.
The incidence of campylobacters on retail meats in several countries has been found to vary from 0-8.1% for red meats and from 23.1-84% for chicken. Adequate cooking will assure safety of meats but serious under-cooking or cross- contamination from raw to cooked product in the kitchen are thought to be major routes of infection.
Despite its frequent occurrence in poultry, eggs do not appear to be an important source of Campylobacter. Studies of eggs from flocks colonized with C. jejuni have found the organism on around 1 % of egg shells or the inner shell and membranes. Prolonged survival on the dry egg surface is unlikely and egg albumin has been shown to be strongly bactericidal.
Milk can contain Campylobacter as a result of faecal contamination on the farm or possibly Campylobacter mastitis. The bacterium cannot survive correct pasteurization procedures and the majority of outbreaks, many quite large, have involved unpasteurized milk.
More than 2500 children aged 2-7 years in England were infected by consumption of free school milk which is thought to have by-passed pasteurization. In Switzerland, a raw-milk drink was associated with an outbreak at a fun-run which affected more than 500 participants. It is not on record whether any personal best times were achieved that day.
Post-pasteurization contamination may always re-introduce the organism to milk. For example, pecking of pasteurized milk delivered to the doorstep by birds of the crow family has been strongly implicated in a number of cases of Campylobacter enteritis in the UK. Dairy products other than fresh milk do not pose a threat due to the low resistance of Campylobacter to conditions of reduced pH or aw.
Other foods recognized as potential sources of Campylobacter infection include shellfish and mushrooms. C. jejuni and C. coli were detected in 14% of oyster flesh tested, although 2 days depuration was sufficient to cleanse oysters artificially contaminated to contain 800 cfu of campylobacters g-1. An outbreak in the USA was ascribed to raw clams.