Campylobacter spp. are the most frequently identified bacterial cause of acute diarrhea in the developed world and can cause post infectious complications such as reactive arthritis and Guillain-Barré syndrome. Reported cases in England and Wales recorded at the Health Protection Agency (now Public Health England, or PHE) have risen from approximately 58,000 in 2000 to about 65,000 in 2012. This has fueled the recent media storm regarding the prevalence of the organism in fresh chickens from supermarkets and butchers. Results as high as 73 percent of chickens testing positive for the presence of Campylobacter—according to Food Standards Agency—may come as a shock to many consumers; but for those who work in foodborne zoonoses, like Paul Wigley, PhD, professor at University of Liverpool in U.K., these levels are not a surprise.
“The reality is around one in 100 people get Campylobacter infection each year and most of these cases are associated with chicken,” comments Prof. Wigley. “The reason why Campylobacter has become the main issue in poultry microbiology is widely due to the host; it can colonize the chicken very well—the raised body temperature of birds over mammals and the low oxygen in the gut suit the bacterium well, meaning it can grow to levels of 1010 colony-forming unit per gram of intestinal content.”
The most frequently isolated Campylobacter spp. associated with human disease is Campylobacter jejuni, accounting for around 90 percent of cases, followed by Campylobacter coli, accounting for many of the remaining cases. Other species are reported (such as C. lari and C. upsaliensis), but these are rarely associated with human campylobacteriosis cases.
During the 1980s and 1990s, Salmonella enterica subspecies enterica serotype Enteritidis phage type 4 caused an epidemic that was frequently associated with the consumption of poultry meat and eggs. According to research published in the Journal of Applied Microbiology, the decline of incidence was widely attributed to the extensive vaccination of egg-laying hens against the serovar. Current efforts to reduce incidence of human campylobacteriosis cases largely focus around control strategies, such as hygiene and biosecurity measures of broiler flocks. However, this approach can only be effective if the control strategy efforts are focused throughout the food production chain, commonly referred to as farm to fork strategies. Efforts are underway into the feasibility of a vaccine for Campylobacter, however strategies are limited due to an incomplete understanding of the organism’s pathogenesis and extensive rate of horizontal gene transfer.
Approaches in Detection
If Campylobacter is detected at unacceptable levels within a flock, there is currently not a great deal poultry producers can do pre-slaughter to effectively reduce the levels, especially since there is huge concern regarding the over use of antibiotics that can lead to development of resistance.
A number of post-slaughter controls are being developed, such as freezing, irradiation, steam, or hot water treatment. However, these measures can only be effective if carried out at the right time and no recontamination event occurs.
For food producers the main weapon against Campylobacter remains surveillance through testing to properly implement control measures. In Europe the main standard observed for detection and enumeration of Campylobacter spp. from poultry is ISO 10272. Originally published in 2006, it is now currently under revision to incorporate several important changes. The basic format of testing includes selective enrichment in broth followed by selective isolation on solid media with further confirmation of characteristic colonies. One of the most important changes is the description of the detection procedure based on the sample type and purpose of the test.
The standard for detection is split into three groups: A, B, and C. This separation of testing protocols recognizes the challenge of radically different test samples and helps improve the ability to detect Campylobacter. All procedures use modified charcoal cefoperazone deoxycholate (mCCD) agar as the isolation medium but differ in the enrichment step.
Detection procedure A is designed for samples with low number and/or stressed Campylobacter with a low non-target background microflora, such as cooked or frozen products. Procedure A uses a 1:10 dilution of the sample in Bolton broth, which utilizes a cocktail of cefoperazone, vancomycin, trimethoprim and amphotericin B to select for Campylobacter spp. The sample is incubated at 37 degrees Celsius for four to six hours, followed by a 44-hour incubation at 41.5 degrees Celsius. All incubation is in a microaerobic atmosphere. The lower temperature pre-incubation is to allow for resuscitation of stressed cells prior to the more selective higher temperature. The prolonged 44-hour enrichment is to allow for sufficient multiplication of a low number of target cells.
Procedure B is designed for samples with a low number of Campylobacter in the presence of a high level of non-target background microflora, such as raw meat or milk. Procedure B uses a 1:10 dilution of the sample in Preston broth, which utilizes a cocktail of polymyxin B, rifampicin, trimethoprim and amphotericin B to select for Campylobacter spp. The sample is incubated at 41.5 degrees Celsius for 24 hours in a microaerobic atmosphere. The main difference between Bolton and Preston broths is that Bolton is formulated to better cope with the resuscitation of stressed microorganisms, whereas Preston broth is more selective to deal with a high background challenge.
Procedure C does not utilize an enrichment step and is a direct plating method for products with high numbers of Campylobacter, such as poultry caecal content. Procedure C can be used with the second part of ISO 10272 concerned with enumeration of Campylobacter in the test material.
As previously mentioned, the plating medium of choice in all protocols is mCCD agar, which, unlike both Preston and Bolton broths, is a blood-free medium. In the formulation, blood is replaced by charcoal, ferrous sulfate, and sodium pyruvate to aid in the recovery and growth of Campylobacter. The agar is made selective with the addition of the bile salt sodium deoxycholate, the third generation cephalosporin antibiotic cefoperazone and the antifungal amphotericin B.
Typical colonies of Campylobacter on mCCD agar are grey/white, often with a metallic sheen, and are flat and moist. Further confirmation is done by examination of morphology and motility, presence of oxidase, and absence of aerobic growth.
A very similar organism that is often mistaken for Campylobacter is Arcobacter. Like Campylobacter, Arcobacter is also a member of the family Campylobacteraceae and exhibits a very similar morphology on mCCD agar. A way to differentiate the two, however, is by testing duplicate plates both aerobically and microaerobically. Arcobacter can tolerate the aerobic environment and will grow, but Campylobacter cannot. The clinical significance of Arcobacter is debatable; however, in this case, it is a cause of false positive results and/or incorrect counts.
Preston and Bolton broths have been favored by ISO as the enrichment media of choice, although there are other media that have been shown to be highly effective at selectively enriching Campylobacter from other sample types. Exeter broth has been used successfully to enrich Campylobacter from samples such as poultry house boot socks used for monitoring levels of the organism in and around the poultry house. Similar to Preston, Exeter uses a nutrient broth base, supplemented with lysed horse blood, but instead uses trimethoprim, rifampicin, polymyxin, cefoperazone, and amphotericin b as selective antibiotics. Again plating is done using mCCD agar, but reduction of background microflora can be achieved with the use of a 0.45-micrometer disk filter. By placing a filter on the agar surface and placing 100 microliters of enriched sample on top, the Campylobacter can pass through whilst most other enteric microorganisms are retained. The filter can then be discarded and the plates incubated as normal.
Whilst traditional microbiology methods remain at the forefront of Campylobacter testing, rapid methods are being further more utilized as a detection tool. ISO guidelines are available regarding the detections of pathogens using polymerase chain reaction (PCR)—ISO 22174:2005 and ISO 20838:2006—and many commercial products are available for Campylobacter that can give a result in around 90 minutes after 24 hour pre-enrichment. Unique DNA targets are amplified to create millions of copies that can be analyzed to give a result. Real-time PCR can be used to amplify and simultaneously detect amplified target by using florescent-labeled probes that generate reporter molecules. These molecules are excited by light and detected by the machine. This process allows for much quicker results without further analysis.
The technology does, however, have its drawbacks. Besides the cost implications, the major issue is the effect of the sample matrices. Many sample matrices, such as water or raw meat, contain interfering inhibitory substances that can reduce or completely prevent the amplification process. It is possible to reduce the effect of such compounds by undergoing a secondary enrichment to dilute out the problem; however this makes the methods somewhat less rapid.
Other technologies exist, such as enzyme-linked immunosorbent assay, or ELISA, and other immunological methods. These methods rely on specific antibodies that capture the target organism, which, in combination with further enzyme chemistry, yields a detectable signal. For this technology to perform, the antibodies utilized must be able to capture all species of interest, which can have highly variable immunological statuses, meaning the antibodies will have variable performance. The detection limit is also lower than that of PCR, but the technology is often cheaper.
Another technology that is gaining greater approval in the food and water testing industry is matrix-assisted laser desorption ionization time-of-flight, or MALDI-TOF. Here laser irradiation is used to vaporize a sample (in the form of biomass from an agar plate), releasing charged ions, which are attracted to a detector. The speed at which the ions reach the detector yields a pattern specific to a given organism from a database, thereby giving an identification. Though this technology is proving popular, its reproducibility and reliability is only as good as the library database it is linked to, and the results can be affected by the state of the cell prior to analysis.
Finally, the most exciting technologies to emerge in pathogen testing as a whole are single nucleotide polymorphisms (SNP) analysis and whole genome sequencing (WGS). These technologies, whilst not quite ready for widespread microbiological food testing, represent a giant leap forward in modern microbiology and further progress our understanding of Campylobacter and its pathogenesis.
An example of implementation and successful use of WGS is the PHE food pathogen reference laboratory. After several years of development, infrastructure building and protocol validation, PHE successfully implemented WGS of Salmonella spp. sent to the reference laboratory. This replaced the lengthy conformation protocol including serotyping and phage typing. A combination of multilocus sequence typing and SNP analysis provide a powerful tool to correctly identify and characterize food pathogens faster and more in depth than able to do before. As well as allowing faster identification and greater ability to source and deal with outbreaks, this technology gives a better understanding of the organism and its source by assessing similarity between genomes. The greatest disadvantage with the technology is, however, the cost and time required to develop a working system for any given organism.
A drawback of all the rapid methods is that, without effective control measures in place to deal with Campylobacter once detected, they might not be able to be utilized as well as they could be.
Clearly there is a great deal of work and development ahead for dealing with Campylobacter. It is evident from current efforts that there is no single step that will solve the problem. It will mostly like be a collection of efforts from all aspects of the food industry—from production all the way to final product testing—that will prevent the prevalence of Campylobacter in poultry.
Elmerhebi is the technical product specialist at Lab M. Reach him at email@example.com.