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Campylobacter are naturally occurring pathogen bacterium found in the intestinal tract and faecal matter of wild and domesticated mammals (sheep, cows and pigs) and birds. It is the most common cause of bacterial enteritis across Ireland and Europe and causes more than a million cases of campylobacteriosis each year in the US alone. Campylobacter is an extremely difficult organism to culture and maintain in a laboratory thus there have been many methods developed and modifications proposed for its detection in foods. There are many different methods ranging from selective and differential plating media to PCR and ELISA. There is also a vast range in the time for each test ranging from a couple of hours to a few days. Each test also varies in sensitivity and accuracy. Some tests may be able to detect the minute particles while others may just detect the bare minimum. By being aware of the different methods of detection and identification of the Campylobacter species it makes it possible to improve the technology for better methods and systems in the future.
There are 14 species in the Campylobacter Genus. Each of the species ranges from origin to pathogenicity. They are usually found on contaminated meats such as poultry and are contracted through under cooking of this contaminated meat or by cross-contamination. Campylobacter affect both humans and animals and causes campylobacteriosis which itself is not life threatening but does cause severe complications prior to infection. It is also a growing concern that Campylobacter are becoming increasingly resistant to antibiotics which in the future could prove to be a major problem. By having the ability to rapidly detect and identify Campylobacter species it would avoid unnecessary human and animal sickness and also avoid the possibility of a multi-resistant Campylobacter species.
Campylobacter is a Gram-negative pathogen (red or pink colour when viewed under the microscope after a gram stain). They are s-shaped, curved or thin spiral rods that may also be coccoid in shape (this usually occurring in older cultures) and range in size from 0.2Âµm to 0.9Âµm wide and 0.5Âµm to 5Âµm in length. These organisms can generally have a single polar unsheathed flagellum (monotrichous) or a flagellum at each end (amphitrichous). The motility of the bacteria is characteristically rapid and darting in corkscrew fashions which, when phase-contrast microscopy is used, are very easily detected amongst other bacteria. Campylobacter are microaerophilic organisms. This means that they require oxygen to survive but also requires an environment containing lower levels of oxygen than are present in the atmosphere (approx. 20%). To grow best they require oxygen levels of 5% and lower. They are sensitive to salinity, pH below 5 and freezing conditions. They are considered to be thermophilic microorganisms as they have to ability to grow in temperatures of between 42oC and 45oC but they grow best at 37oC. There are 19 species altogether including biovar and subspecies of the genus Campylobacter.
Table 3.1 Species of Campylobacter, where they are found and the diseases they cause.
C.jejuni subspp. jejuni
Poultry, cattle, sheep & pigs
IID (infectious intestional disease) in humans.
Poultry, cattle, sheep & pigs
Both upper and ower gastrointestinal disease in humans.
Poultry & pigs
IID in humans.
Seagulls, dogs, horses, fish & shellfish
IID and bacteraemia in humans.
Symptomatic and asymptomatic cats and dogs
IID and bacteraemia in humans.
C.fetus subspp. fetus
Sheep & cattle
May cause bacteraemia in humans and can rarely cause IID. Causes sporadic abortion in both pregnant sheep and cattle.
May cause septicaemia in humans, but rarely. Causes abortions on cattle.
Causes ileitis in pigs and rarely causes IID in humans.
Diarrhoea in humans but no known animal source.
May cause periodontal disease and IID in humans, but rarely.
Porcine intestinal disease.
C.sputorum biovar sputorum
Human oral cavity, diarrhoea, abscesses and other infected skin lesions.
Gingivitis in humans.
Sheep faeces & bovine genital tract.
Sheep faeces & bovine genital tract.
Persistent IID in cattle
No known animal source.
Gingivitis and soft tissue abscesses in humans.
No known animal source
Gingivitis and soft tissue abscesses in humans.
Deep tissues infections in humans.
Gingivitis and soft tissue abscesses in humans.
Diarrhoea from asymptomatic cats and dogs
Human oral lesions
Campylobacter, over recent years has become one of the world's most leading cause of foodborne illness and it has become recognized as an important enteric pathogen causing Campylobacter enteritis or campylobacteriosis.
A study was carried out determining the prevalence of Campylobacter species contaminating chicken carcasses (Hue et al.) the overall result showed that 372 out of the 425 chicken carcasses that were tested were positive for Campylobacter. It was also said that the age of the chicken before slaughter was a contributing factor to the risk of contaminated carcasses.
Fig. 4.1 Graph A indicates the prevalence with varying age. Graph B indicates number of bacterium in log10CFU/g with varying age.
The months of the year were also believed to be a prevailing factor of contamination. With reference to Fig. 4.1 the summer months have shown to have the most amounts of contaminated carcass samples where as there was almost an even number of bacterium present, extracted from the caeca of the chickens, measured in log10CFU/g.
Fig. 4.2 Graph A indicates the prevalence of contamination in the varying months. Graph B indicates the prevalence with respect to the number of bacterium present in each sample measured in log10CFU/g
Campylobacteriosis, also known as Campylobacter enteritis, is an infection caused by the Campylobacter species. It affects the intestinal tract and sometimes, but rarely, the blood stream. Campylobacteriosos causes mild or severe diarrhoea most often with blood in the stool and a fever. The latency period of the infection can be between 1-6 days, taking 1-2 days before the symptoms actually start to develop. Symptoms include diarrhoea, as frequently as 10 times per day, many also be watery with traces of blood, inflammation of the intestine and colon, cramps, abdominal pains and a fever as high as 40oC. The infection may last anywhere between 2-10 days and it can be treated with antibiotics but cannot always be used as antibiotic-resistance is a growing concern with the Campylobacter species. Erythromycin may be used in children and tetracycline may be used in adults to treat the infection in severe cases. Campylobacteriosis is usually self-limited, meaning that the primary infection may only last for a given number of days without any mortality although there may be some complications prior to infection. Complications such as Guillain-Barre Syndrome, which is an acute autoimmune neuropathy with ascending paralysis is a severe disorder that can occur prior to Campylobacter infections.
Assays and Specialty Substrate Media for Detection of Campylobacter
SimPlateïƒ¢ Counting Method, developed by BioControl is a media based test developed to improve the quantitative method for Yeasts and Moulds, Coliforms, Campylobacter, Enterobacteriaceae and Total Plate Count.
Fig. 6.1 Campylobacter SimPlate. Note the difference in colour in the wells. This denotes positive and negative result for the presence of the pathogen.
This is an improved counting method developed in attempt to overcome the limitations of other counting methods. The SimPlate system with Binary Detection Technologyïƒ” is the latest achievement in advanced counting methods. It uses a combination of pre-measured media and patent plating devices that provide accurate and easy to read results days before all of the agar plate and film methods of detection. With the use of the Binary Detection Technologyïƒ”, positive and negative results are distinguishable at a glance. After incubation, simply, all the positive wells are counted and then referred to the SimPlate Conversion Table to achieve the number of organisms present in the sample.
Fig. 6.1 Campylobacter SimPlate. Left side shows plate before incubation. Right side shows plate after incubation. Note the difference in colour after incubation.
The SimPlate is also very effective when detecting Yeasts and Molds, Coliforms and Enterobacteriaceae as well as Campylobacter. SimPlate is an effective and effeicient way of detecting how many yeasts, moulds or coliforms etc. are present in a sample. The SimPlate has a maximum counting range of 738 while agar plates and film counting ranges are limited to 300 cfu and less.
Fig. 6.2 Table shows how efficient SimPlate is compared to other standard methods. Note the orange bars and the time differences.
DNA Based Assays for Detection of Campylobacter
GeneQuenceïƒ¢is a DNA probe based assay developed by Neogen for the screening and detection of Campylobacter, Salmonella, Listeria spp. and Listeria monocytogenes.
Neogen's GeneQuenceïƒ¢ Campylobacter is a test developed with the intent of screening poultry carcass rinses for the presence of Campylobacter. Each test kit uses two specific DNA elements to ensure the highest specificity which therefore ensures the most accurste results. The test has been developed to be able to detect 1 - 5 cfu/25g of sample in less than 2 hours and when this test is coupled with an automated plate handling unit it makes it possible to test more than 450 samples in 8 hours. The DNA probe used consists of an oligonucleotide capture probe and an oligonucleotide detector probe. The capture probe is specific to the rRNA sequences of the target organism and it is labelled at the 3' end with polydeoxyadeylic acid. The detector probe is also specific to the rRNA sequences of the target organism and is labelled at the 5' end with and enzyme horseradish peroxidise. The colour change that occurs at the end of the test indicates the presence of the hybridized detector probe in the complex and thus the presence of the rRNA form each target organism.
AccuProbeïƒ¢ developed Gen-Probeïƒ¢ is a DNA probe based test that uses the technique of nucleic acid hybridization for the identification of Campylobacter species from a sample. The principle of this and most nucleic hybridization tests is the ability of complementary nucleic acid strands to specifically align and associate to form double stranded complexes. The AccuProbeïƒ¢ test system uses a single stranded DNA probe with a chemiluminescent label that is complementary to the rRNA of the target organism. The labelled DNA probe combines with the rRNA of the target organism and forms a stable DNA:RNA hybrid. The selection reagent that is supplied in the kit allows for the differentiation of the hybridized and the non-hybridized probes. The labelled probes are then measuered in a Gen-Probe luminometer where a positive result is a luminometer reading that is equal to or greater that the cut-off and a negative result is a reading that is below the cut-off. This particular test can take anywhere between 16 an 24 hours to complete.
Polymerase Chain Reaction (PCR) is another well known DNA based assay that can be used in conjunction with gel electrophoresis to identify and detect microorganisms of interest such as Campylobacter. The principle of PCR is to amplify a specific target sequence of DNA from a complex mixture of as a means of detecting the presence of a particular microorganism. The PCR reaction synthesizes many copies of the targeted DNA sequence in just a few hours. By amplifying the target DNA sequence it becomes possible to detect the microorganism using gel electrophoresis.
There are many different brand of PCR kits sold by many different companies. Campylobacter PCR by Biotecon Diagnostics and Probeliaïƒ¢ PCR by bioControl are just two examples of commercially available kits that can be purchased.
Antibody Based Assays for the Detection of Campylobacter
Latex Agglutination (LA)
This test involves the use of antibody-coated latex beads or colloidal gold particles that clump or agglutinate in the presence of a specific antigen. These tests are fast and easy to perform although they require pure sample cultures for testing and are not very sensitive. They are very useful for quick identification of isolates based on serology. Meritecïƒ¢ by Meridianïƒ”, Microscreenïƒ¢ by Microgenïƒ” and Dryspotïƒ¢ by Oxoidïƒ” are all examples of detection systems and kits that can all be purchased commercially.
Immunomagnetic Seperation (IMS)
This test uses antibodies coupled to magnetic beads to specifically capture the desired target. The use of antibodies in this test allows for high specificity and minimal damage to cells as no harsh reagents are used. This method is much faster that selective enrichment methods as it has the ability of capturing the target cells within minutes as opposed to the overnight incubation of selective enrichment. The Pathatrixïƒ¢ automated detection system that was developed by Matirx Microscienceïƒ”.
Enzyme-Linked Immuosorbent Assay (ELISA)
This is the most popular and commercially test available for identification of food-borne pathogens. The test uses an antibody-coated solid matrix (micro-titre plate) that is used to capture the antigen bacteria or toxin from the enrichment culture. A second antibody that is conjugated with an enzyme is then added to form an antibody-antigen conjugate. The substrate is then added which is cleaved by the enzyme to produce a coloured product that can be read visually or with a spectrophotometer and recorded. There are many commercially available kits available that have the ability of detecting Campylobacter pathogens. These kits include Transia Plateïƒ¢ by BioControlïƒ”, Tecra Campylobacter VIAïƒ” and Tecra Unique Plusïƒ” and Tecra Campylobacter Uniqueïƒ”
The Electoimmunoassay detection mechanism couples specific antibody-antigen binding to the production of an electrical signal. The technology consists of a circuit with a capture antibody attached to the solid surface in the area of the electrode gap. When the sample is added the target antigen binds to the capture antibody. Then a colloidal gold-labelled detection antibody is bound creating a capture-target-detector bound group. The last step is the deposition of silver ions onto the colloidal gold. This produces a conductive silver bridge which closes the circuit resulting in a measured drop in resistance. The Detex Systemïƒ¢ by Molecular Circuitryïƒ” is another available test to detect food-borne pathogens.
Enzyme-Linked Fluorescence Assay (ELFS)
This method follows the same principle as an enzyme inhibition assay (ELISA) except that the enzyme in the reaction catalyzes a fluorescence reaction and not a colour reaction. VIDASïƒ¢ by bioMerieuxïƒ” is an example of a commercially available test kit to detect the Campylobacter pathogen in food samples.
This test uses the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular target protein. It can also be used to concentrate or isolate a specific protein from a sample that may contain thousand of different proteins. After the target protein has been separated, the complexes are disassociated and the proteins are separated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE). The size and quantity of the proteins can then be analyzed by either autoradiography or a gel scanning procedure. This is an extremely sensitive procedure and can detect very small amounts of radiolabeled proteins. The Singlepathïƒ¢ by Merckïƒ” is an example of a commercially available test kit that can be purchased to detect the Campylobacter pathogen.
Biosensors consists of a biological component such as an antibody, coupled to a physicochemical transducer which has the ability to convert minute signal fluctuations resulting from biological interactions into measurable digital electronic readings. There are many different formats of biosensors and several of them have been tested for pathogen testing in foods. One such test is the DIAPROïƒ¢ which has been developed by UNMEDIKïƒ” and is successful in detecting the Campylobacter pathogen.
Plating and Enrichment Media for Detection of Campylobacter
Plating media refers to media that contains agar and are grown in petri-dishes.
Enrichment media refers to the media that is used prior to plating and detection to allow for optimum growth of the bacterial samples that you wish to grow.
Doyle and Roman Broth
VTP FBP Broth
Parker and Sanders Broth
Hunt and Randle Broth
Catalase is a commonly found enzyme, found in almost all living organisms that are exposed to oxygen. Its function is to catalyse the decomposition of hydrogen peroxide to water and oxygen. Hydrogen peroxide is one of the oxidative end products of aerobic carbohydrate metabolism. The test involves aseptically placing a small sample of the organism that is required to be tested onto a clean glass slide. A few drops of approximately 3% hydrogen peroxide are put onto the sample on the slide. The sample is then watched carefully for any signs of gas bubbles. A positive result is indicated by a rapid appearance of gas bubbles visible on the slide and a negative result is no change in the visible appearance, no gas bubbles produced. (Penner, J. L. 1988)
This is a test used to determine the ability of an organism to reduce nitrate to nitrite using the enzyme nitrate reductase and also to test the ability of the microorganism to perform nitrification on nitrate and nitrite to produce molecular nitrogen.
The test is carried out using nitrate broth which contain nutrients and potassium nitrate which is incubated. After incubation a drop of sulfanilic acid and ï¡-naphthylamine is added. If the organism has the ability of reducing the nitrate into nitrite, the nitrites in the medium will form nitrous acid. The sulfanilic acid will react with the nitrous acid to produce diazotized sulfanilic acid and this in turn reacts with the ï¡-naphthylamine to form a red coloured compound. Thus as the medium turn's red after the addition of nitrate reagents it is considered a positive result for nitrate reduction. (Penner, J. L. 1988)
The Triple Sugar Iron test or TSI test is a microbial test used for its ability to test microorganisms for their ability to ferment sugars to produce hydrogen sulphide. It is very often used for the selective identification of enteric bacteria. The TSI slant is in a test tube that contains agar, 1% lactose, 1% sucrose, 0.1% glucose, phenol red (a pH sensitive dye), sodium thiosulphate and ferrous sulphate or ferrous ammonium sulphate. All of the ingredients are mixed together and then solidified in a test tube at an angle.
That slanted angle of the agar allows for the optimum amount of surface area for exposure to both oxygen rich air (aerobic environment) and not exposed at all to air (anaerobic environment).
Microorganisms that ferment any of the three sugars in the medium produce by-products. These by-products are usually acids which will change the colour of the pH sensitive dye (phenol red) to a yellow colour. The position of the colour change in the tube distinguishes the amount of acid production that is associated with the glucose fermentation from the acidic by-products of sucrose or lactose fermentation. A yellow colour visible at the butt of the test tubes indicates enterobacteria that have the ability of fermenting the sugars but in an anaerobic environment. A blackening of the medium is mostly found at the butt of the tube and indicates the utilization of the thiosulphate anion as a terminal electron acceptor and reducing it to sulfide. The hydrogen sulphide then reacts with the ferrous sulphate in the medium and forms ferrous sulphide which is the black precipitate that is visible in the tube. Non-lactose fermentors can result is a yellow/yellow or pink/yellow colour change (if the sucrose is fermented). (Penner, J. L. 1988)
This test is based on the ability of microorganisms to hydrolyse the compound hippurate using ferric chloride indicator to detect benzoic acid which is the first by-product in the hippurate hydrolysis pathway. This test employs ninhydrin as the indicator, which detects glycine which is the second by-product of the hippurate hydrolysis. This test is used for the identification of microorganisms such as Campylobacter jejuni, Listeria monocytogenes and Gardnerella vaginalis. A positive result for the hippurate test is indicated by the appearance of a deep blue colour after 30 minutes and a negative result is indicated by a faint blue colour or no colour change at all. (Penner, J. L. 1988)
Indoxyl Acetate Test
Indoxyl acetate disks are used to determine the ability of microorganisms to hydrolyze indoxyl acetate and are also extremely helpful in the identification and differentiation of Campylobacter, Helicobacter and Wolinella species. The test works on the ability of bacterial hydrolases to release indoxyl from the main compound indoxyl acetate. In the presence of air the indoxyl changes first to indigo white and then to indigo. This is known to be a reliable and rapid method taking only between 5-30 minutes per test.
The results are also easy to read and interpret. A positive result is a blue-green colour that has developed after 20 minutes. It suggests that indoxyl acetate hydrolysis has occurred. A weak-positive result may also occur by the appearance of a pale-blue colour after 10-30 minutes. A negative result has no colour change after 30 minutes. (Penner, J. L. 1988)
The optimum growth temperature for the Campylobacter species is 37oC (as visible form table 1.2). At this temperature the bacterium grow at optimum efficiency. Growth is also visible at temperatures of 25oC and 42oC. The Campylobacter species are classified as thermophilic species of microorganisms. A thermophilic species can be defined as a species that can survive and grow to temperatures of between 45 and 80oC. as observed in table 1.2, there is a definite growth pattern observed at 37oC while at 25oC and 42oC there is growth from some of the species while no growth from other. It is also observed that there is more growth between the species at the higher temperatures of 37oC and 42oC than at 25oC. This would show that the Campylobacter species is a thermotolerant and heat loving bactria thus growing optimally at higher temperatures.
The addition of compounds such as 1% glycine and 0.1% TMAO (Trimethylamine N-oxide) is an addition of stress onto the growing bacterium. Both additions test to see how well the bacteria can grow under osmotic pressure from both the TMAO and the glycine. As visible from table 1.2, there was more growth visible from the 1% glycine than from the 0.1% TMAO. This suggests that few of the species can survive and growth under extreme osmotic conditions. (Penner, J. L. 1988)
Miniturised and Automated Identification Kits for Campylobacter
API Testïƒ¢, developed by bioMerieux, is a well established method for bacterial identification. They give accurate identification of bacterial samples based on vast databases and easy to use, test systems. Each supplied teat contains a strip with 20 miniature wells that are each small biochemical tests that are all quick, safe and easy to do. The API Campy takes only 24 hours and is reliable as it has the optimum growth conditions for this group.
Fig. 7.1 API testing strips, note the wells, each holding a different identification test.
MIS or Sherlockïƒ¢ Microbial ID System, developed by MIDI, is an automated microbial identification system that has the ability of identifying over 1,500 microbial species by FAME analysis. A new technique developed and released in 2007 allows for a more rapid identification of bacteria and yeast in less than 15 minutes from pure cultures.
WalkAwayïƒ¢, developed by Dade MicroScanïƒ” is an automated bacterial identification and susceptibility testing system. This system is very similar to another identification system called the VITEKïƒ¢. They both entail inoculation of a microbial sample into prepared microwell plates for the WalkAwayïƒ¢ system or test cards for the VITEKïƒ¢ system. Both the plates and the cards contain a variety of biochemical substrates and antibiotics. Growth that occurs on the plate or test card results in biochemical substrate changes which can be interpreted by a specialized plate reader for the WalkAwayïƒ¢ or an automated test card reader for the VITEKïƒ¢ system to produce a biochemical profile. This biochemical profile can be compared to the profiles of known microorganisms to identify to unknown microorganisms
Fig. 7.2 Picture of a WalkAwayïƒ¢ automated identification system.
Replianalyzer Systemïƒ¢, developed by Oxoidïƒ” is similar to the WalkAwayïƒ¢ and VITEKïƒ¢ system in the way that each system allows for a biochemical profile to be generated and compared. The Replianalyzer Systemïƒ¢ uses agar plates as opposed to the microwell plates in the WalkAwayïƒ¢ and VITEKïƒ¢ systems.
Malthus Microbial Growth Analyzerïƒ¢ is a system that measures the conductance between two electrodes immersed in media. Any bacterial growth is indicated by a change in the electrical impedance of the growth media. It has been shown that the growth medium is proportional to changes in the conductance component of the impedance. The automated machine is hooked up to a computer and can be programmed to scan the medium at regular time intervals or data can be gotten on demand from each cell on a visual display or in graph format which indicates the change of conductance over time.
This method can be described as both detection, and an identification method. It is a test which has included both conventional molecular and microbiological techniques and produces accurate and efficient results. It has the ability of detecting four faecal pathogens on one single test strip; E. coli 0157, Salmonella, Shigella and Campylobacter. The principle theory is that the characteristic genes that make up each of the four pathogens plus positive internal controls are amplified in a single reaction (multiplex PCR) and then afterwards detected by sandwich hybridization and colour development. The special format of the assay enables for a reduction of the standard protocol for DNA hybridization and processing at ambient temperatures (20-30oC), this therefore allows for a considerable saving of time and avoids the usual time-consuming incubation of the hybridizations in water baths or hybridization ovens.
EntericBioÂ® is an all-in-one test that can be purchased from the manufactures at Serosep Ltd., Limerick, Ireland.
The method itself is based on a four step process:
This step allows for the growth of the pathogenic samples of bacteria under aerobic and microaerophilic conditions using the specially designed EntericBio broth. The incubation is an overnight process which takes 16 hours.
By using a two phase separation system, the inhibitors and bile salts of the faecal pathogens can migrate to the upper phase and the clean DNA can separate into the lower phase.
The ready-to-use PCR tubes that are supplied in the kit improve the accuracy by negating the need to make-up reagents and by eliminating the time consuming pipetting steps that are normally required by other PCR type assays.
The hybridization probes of the kit are specially designed from conserved regions of specific genes for each of the four enteric pathogens which ensure specificity.
The specific probes for each pathogen are present on the test strip and a visible reaction line is formed depending on the pathogen that is present in the faecal sample.
Fig. 8.1 EntericBio test strip with visible indicator blue lines and corresponding bacteria.
Campylobacter is an emerging pathogen meaning that not everything if fully known about the species and there are still being studies done to obtain more knowledge about the species. It is the leading cause of campylobacteriosis which is not life threatening but has serious complications prior to infection and it may become antibiotic resistant if it is not kept under control. Dectection and identification of the bacterium are the best and most efficient way of avoiding unnecessary human and animal sickness. There are many methods of detection and identification, each one varying in size, methodology, sensitivity, reliability, range and price. While the fastest method might be the most desirable it may not be the most accurate or sensitive thus in the long run it will prove to be more expensive and less effective. By being aware of the different available methods improvements can be made to ensure the best and most accurate methods of detection and identification available.