What does SPP mean in medical terms

DETECTION AND DISTINCTION OF LIVE AND DEAD BACTERIA WITH PCR USING THE EXAMPLE OF CAMPYLOBACTER SPP *

An official procedure according to §64 LFGB is available for the detection of germs and the determination of the germ count from food. However, the cultivation is very time-consuming and takes about 6 to 7 days. PCR-based methods, on the other hand, can significantly shorten the examination time, but also detect DNA of dead cells. Therefore, Campylobacter spp. be culturally affirmed. The aim of the research project is to develop a standardized quantitative detection method for Campylobacter spp. to develop on poultry meat and in water samples. The method is a combination of real time PCR and bacterial treatment with the differentiation agent ethidium monoazide (EMA) or propidium monoazide (PMA). The aim of this treatment is to detect only living cells as far as possible. Shifts in the detection curves in the PCR, however, require an adjustment using standard curves. The results show that the detection and quantification of live Campylobacter spp. are possible using real-time PCR.

Summary

Campylobacter has emerged as the most common cause of bacterial food borne disease in Germany. Transmission of Campylobacter normally occurs via contaminated food of animal origin or surface water. According to the “Official collection of investigation procedures according to § 64 LFGB” a direct cultivation method has been approved for the detection and quantification of the pathogen. Nevertheless, the cultivation is time-consuming and takes up to 6 to 7 days. PCR-based methods give fast results, but are not able to differentiate between viable and nonviable cells. Therefore, it was aim of the present study to develop a rapid method for detection of Campylobacter. It is designed as a real time PCR combined with the use of the intercalating dyes ethidium monoazid (EMA) and propidium monoazid (PMA), each other. This allows only the detection and quantification of viable cells. Using intercalating dyes, a shift of the sample detection curves during real time-PCR is observed, but can be compensated by type curves. This allows a calculation of viable Campylobacter cell counts by real time PCR

1. Introduction

Campylobacter spp. are among the most important bacterial food infectious agents worldwide. While they are among the natural inhabitants of the intestines in some domestic animals, especially poultry, and only cause clinical manifestations there in exceptional cases, they can pose a serious health hazard to humans. As a rule, the pathogen is absorbed through food and then leads to gastrointestinal diseases, some of which are severe, with watery to bloody diarrhea. In some cases, serious complications such as joint inflammation or the occur Guillain BarréSyndrome on. This term refers to a degenerative-inflammatory disease of the peripheral nervous system, which can cause severe paralysis of the patient and even death.

While salmonella was long regarded as the most important bacterial food infectious agent, this picture has changed over the past few years. In the meantime, salmonellosis has been ousted from first place by campylobacteriosis. According to statistics from the Robert Koch Institute (RKI), the latter caused 71,307 cases of illness in 2011 [1]. In 2011 there were only 24 512 reported cases of salmonella. Campylobacteriosis is thus reported to the RKI as the second most common diarrheal disease after norovirus infections and is at the same time the most important food-borne disease.

Campylobacter is usually transmitted to humans through contaminated food of animal origin, especially through inadequately cooked poultry meat and products, which can also lead to cross-contamination of other products if the kitchen is poorly hygienic. Raw milk and surface water are also important in the transmission. The majority of diseases occur in children under 5 years of age and young adults between the ages of 20 and 24, with men in all age groups being affected somewhat more frequently than women [2]. A large number of soldiers belong to this age group and are therefore potentially at risk. Accumulations of illnesses were also recorded, with deliveries from large kitchens and participation in communal catering in some cases playing a causal role.

1.1. Campylobacter - demanding but still a survivor

Compared to other food infectious agents, Campylobacter can be viewed as a rather unusual germ. The survival of this pathogen in the environment and outside of its natural habitat, the intestinal tract of the colonized animal, initially seems rather unlikely. A microaerobic atmosphere with only 5% oxygen and an increased carbon dioxide content of 10% is necessary for the germ to grow. Due to their sensitivity to oxygen, demanding nutrient media that contain protective substances against oxygen radicals must be used for culturing the pathogens. Furthermore, Campylobacter spp. sensitive to dehydration, acids, heat and high salt concentrations. However, their metabolic activity is retained at temperatures around 4 ° C, so that the bacteria can tolerate storage at refrigerator temperatures better than at room temperature, for example. Campylobacter can also partially survive deep freezing. They are also able to take on a special stage when exposed to external stress factors, which is referred to as “viable-but-non-culturable” (VBNC). In this form, these bacteria can no longer be cultured and have a reduced metabolic activity, but when the external survival conditions change, they can return to a reproductive stage. The bacteria remain potentially infectious for humans. Campylobacter has thus found ways and niches to survive in the environment, although the demands on the growth conditions are actually quite high.

Fig. 1: Morphology of thermophilic Campylobacter species: colonies on carbonaceous medium (left); microscopic preparation (phase contrast): typical spiral shape (circled) (center); Gram stain: Gram-negative slender rods (right).

 

 

 

 

 

1.2. Species pathogenic to humans and their occurrence

The most important human pathogenic Campylobacter species are Campylobacter (C.) jejuni, C. coli and C. lari. These are gram-negative, screw-twisted rod bacteria of the genus Campylobacter (Fig. 1), which have a width of 0.2-0.9 µm and a length between 0.5 and 5.0 µm. These germs multiply at an optimum temperature of 42 ° C, so that they are counted among the thermophilic species. These thermophilic species were found in 99% of Campylobacter diseases in humans in 2010. Mostly C. jejuni was identified as the causative agent [1].

Birds and especially farm poultry such as chickens are ideal hosts for Campylobacter, as their intestinal tract provides optimal conditions for survival. Large amounts of the pathogen are often detected in the caecum contents of broilers, up to 106 bacteria / g intestinal contents can be determined. Typically, the animals themselves do not become ill, but are merely asymptomatic carriers of the germ. The pathogen can enter the fattening herd via rodents, insects or staff. A colonization of the entire stock can then take place in a very short time. As a result, the entire population is often Campylobacter-positive within a week. Slaughtering is a critical point for the contamination of poultry meat as a foodstuff. Especially during the process steps of scalding, de-feathering and evisceration, the surface of the animal body can be contaminated by the contents of the appendix colonized with Campylobacter. Cross-contamination from rinsing water and equipment is also possible (Fig. 2).

In order to be able to enjoy poultry meat as a “safe” food, it is therefore urgently advisable to safely kill the pathogens through complete heating or boiling. It is also important to store and prepare the meat separately from other foods so that the pathogen is not carried from one food to another. Cutting boards and knives that have been used to process raw meat, for example, should no longer be used to prepare other dishes.

 

 

 

 

 

 

2. Methods and results

For the detection of Campylobacter spp. In food of animal origin, a microbiological method for the detection and counting of germs is available in the official collection of test methods according to Section 64 of the Food and Feed Code (LFGB) and the ISO standard. The disadvantage of this method is the long time it takes to obtain a sample result and the final confirmation (duration: 6-7 days). Due to this time delay, it is hardly possible to intervene quickly and directly in the production and marketing process of the food.

The detection of the pathogen at the DNA level, for example with the polymerase chain reaction (PCR), enables faster sample results within a few hours. However, this method has the disadvantage that Campylobacter DNA is detected, regardless of whether it is a living and therefore infectious germ, or whether the bacterium has already died and can no longer lead to disease [9 ]. So only the entire DNA of all germs from living and dead Campylobacter is determined. A statement about the vitality status of the cells cannot be made with the PCR [3, 6].

In order to solve this problem, the aim of the contract research project * "Quantification and differentiation of living and dead food infectious agents using PCR in action" was to develop a rapid method for the detection of Campylobacter and to establish it in the Federal Armed Forces' main institute for veterinary medicine in Kiel. The approach for this was the combination of the real-time PCR method with the use of so-called DNA-intercalating substances. If they are added to the bacteria, such substances can bind irreversibly to the DNA after exposure to light. Different DNA-intercalating substances such as ethidium monoazide (EMA) and propidium monoazide (PMA) were used to determine which were suitable for the above question [4]. Binding to the DNA is only possible with membrane-permeable cells with a defective or missing cell wall or with exposed DNA, i.e. cells that are no longer viable. In the case of living and membrane-intact cells, on the other hand, they cannot pass through the cell membrane and thus their DNA remains unbound and free of these inhibiting substances (Fig. 3 A). Incubation with the substances takes place for 15 min in the dark. Before the DNA is purified, the intercalating substance is firmly bound to the bacterial DNA by irradiation with a high-energy halogen lamp and the excess is inactivated. If the DNA of these bacteria is isolated in a subsequent step, one obtains, on the one hand, bound DNA, which originates from dead, membrane-damaged cells, and unbound DNA, which was isolated from living cells (Fig. 3 B). The next step is real-time PCR. Here it is not possible to amplify the DNA bound to intercalating substances, so that only DNA from living cells, i.e. unbound DNA, can be detected (Fig. 3 C). With this 2005 by Rudi, Moen, Droemtorp et al. [8] published method can be used to distinguish living, infectious bacteria from dead cells by pretreating the cells with the intercalating substances and make their germ count visible in "real time" with the help of real-time PCR (Fig 4) [5].

 

 

 

 

 

 

 

3. Discussion

The flawless quality of the food is an important component of consumer health protection and is therefore also of crucial importance in the Bundeswehr for the deployment and performance of soldiers. The microbiological status of the food should be emphasized in this regard. In addition to spoilage pathogens, which often lead to strong, sensory recognizable changes, pathogenic germs play a decisive role. The medically significant pathogens include Campylobacter spp., Which has displaced salmonella as the most common food infectious agent in Germany. Many people are often affected in communal catering. Good food hygiene therefore plays a major role in the prevention of such incidents, especially in the countries of use with high temperatures that are favorable for microorganisms and in some cases high air humidity.

The aim of the research project was to establish the method of distinguishing between live and dead bacteria for Campylobacter and to test it on various foods such as poultry meat and water samples. With the help of these studies it was possible to develop a method for determining the live bacteria count of Campylobacter on poultry meat. This pathogen was selected for the investigations because of its medical importance and complex and time-consuming cultivation. The principle could also be applied to other bacteria and represent a universal method for differentiating living and dead bacteria. By establishing this method, in addition to material and personnel savings, a very fast sample result can be achieved compared to the conventional cultural method. This enables prompt and direct intervention in food safety and thus also guarantees greater consumer protection for the consumer.

 

 

 

 

 

 

 

 

4. Conclusions

With the introduction of extensive cultural and, more recently, molecular biological methods, food safety in the Bundeswehr can be checked to a high standard in the veterinary medicine laboratory (VetMedLab). With the introduction of PCR evidence in the VetMedLab in use, the range of diagnostic options could be expanded considerably here as well. In the research project, fundamental work was carried out to establish a PCR detection to differentiate between dead (non-infectious) and living (infectious) bacteria. The procedure for differentiating between living and dead bacteria is a considerable advance in diagnostics and can also be used for the detection of other bacteria, including for special questions in the field of clinical microbiology. This is a universal method of diagnostics and, in this case, especially of food hygiene. The safety of the products can be checked promptly from manufacture to consumption.

Literature:

  1. Anonymous: Infection epidemiological yearbook of reportable diseases for 2011. Robert Koch Institute: 44; 165.
  2.  Anonymous: Infection epidemiological yearbook of reportable diseases for 2010. Robert Koch Institute. Epid Bull 2011; 15: 130-133.
  3. Cenciarini shelves C, Courtois S, La Scola B: Nucleic acids as viability markers for bacteria detection using molecular tools. Future Microbiol 2009; 4: 45-64.
  4. Hein I, Schneeweiss W, Stanek C, Wagner M: Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR. J Microbiol Methods 2007; 71: 336-339.
  5. Hey Y, Chen CY: Quantitative analysis of viable, stressed and dead cells of Campylobacter jejuni strain 81-176. Food Microbiol 2010; 27: 439-446.
  6. Nocker A, Camper A: Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. FEMS Microbiol Lett 2009; 291: 137-142.
  7. Reich F, Atanassova V, Haunhorst E, Klein G: The effects of Campylobacter numbers in caeca on the contamination of broiler carcasses with Campylobacter. International Journal of Food Microbiology 2008; 127: 116-120.
  8. Rudi K, Moen B, Drømtorp SM, Holck AL: Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. Appl Environm Microbiol 2005; 71: 1018-1024.
  9. Wolffs P, Norling B, Rådström P: Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells. J. Microbiol Methods 2005; 60: 315-323.

 

* Developed as part of a research project (project number M-SABX-9A006, University of Veterinary Medicine, Hanover, Institute for Food Quality and Safety) for the Bundeswehr medical service.

 

Date: 21.09.2012

Source: Military Medical Monthly 2012 / 8-9