Detection of microbial contaminants of meat

Detection of microbial contaminants of meat

Abstract

Food poisoning is an ever present health issue that is believed to exceed £1.5 billion a year to the UK economy. Microbial contamination of meat is the potential source of infection commonly associated with Campylobacter spp., Salmonella spp., Escherichia coli and Staphylococcus aureus. Epidemiology and pathogenesis of each of these bacterial species are briefly described in this review in order to justify the necessity for improvement of detection methods of bacterial contamination of meat. The final part of review represents the overview of common detection methods such as culture-based methods, immunological methods and DNA-based testing – PCR. 

The plan of laboratory investigation of contamination of meat is present in the design study. The laboratory testing would be focused on PCR procedure for detection of microbial contaminants of meat. The flow chart demonstrates the sequence of work to be carried during research. 
 
 
 
 
 
 

1. Overview

Food borne pathogens are an ubiquitous threat that can cause illness and death and increase the cost of medical and social care. Food poisoning diseases are ever presented thread for human health. For example, in June 2011, in an ongoing large outbreak of food poisoning syndromes caused by an unusual strain of Shiga-toxin–producing Escherichia coli centred on Germany have been reported a total of 3222 cases, including 39 deaths in the period of less than two month(Frank et al, 2011).

Food poisoning can occur in the form of mass outbreaks, involving a large number of people, as well as group and individual cases. Microbial food poisoning is characterised by a sudden onset of disease and typically linked with the consumption of a food containing harmful micro organisms and micro organism derived toxins. Complex and lengthy food supply procedures, changes in eating habits, mass catering and poor hygiene practices are major contributing factors of food poisoning.

Food contamination is caused by different micro organisms and their toxins (Table 1). Products can be contaminated by micro organisms as a result of violations of hygienic and technological production procedures, transportation, storage and retail conditions. Poor personal hygiene, improper cleaning of storage and preparation areas and unclean utensils can cause contamination of raw and cooked foods. These foods are particularly vulnerable to contamination if they are not handled, stored or cooked properly. This accounts for the largest number of food poisoning in the warm season, when the optimal conditions are created for microbial growth (Bentham and Langford, 1994). 

However, the consumption of contaminated food does not always cause food poisoning. As some micro organisms only cause disease in humans following either a massive multiplication in food or a significant accumulation of toxins. Some groups of people, including the very young, and old or immuno-compromised, are at greater risk of severity of effects on their health caused by poisoning.

The symptoms of food poisoning are varied but usually begin a few hours to a few days following consumption of contaminated food (Table 1).

Table 1: FOOD POISONING BACTERIA (P.O.S.T. publications; 1997)

(adapted from “P.O.S.T. Note 101 July 1997” available on-line at http://www.parliament.uk/business/publications/research/post/publications-by-year/pubs1997

Document 1: National food poisoning statistics 1997-2009 (The Chartered Institute of Environmental Health; 2011) available on-line http://www.cieh.org/policy/food_safety.html

Graph 1: National food poisoning statistics 1997-2009 (The Chartered Institute of Environmental Health; 2011)

National food poisoning statistics for 1997-2009 is presented by The Chartered Institute of Environmental Health (Document 1; Graph 1) has shown that Campylobacter is the commonest cause of food poisoning in Great Britain (FSA, 2010; POST, 2003). While the estimated cost to the UK economy is around £500 millions a year, and the total cost of all food poisoning cases is believed to exceed £1.5 billion a year (Lloyds of London, 2010).

The survey of microbiological contamination of fresh red meats completed by the Food Standard Agency on 02 September 2010 established that food poisoning micro organisms found to contaminate red meat include Campylobacter spp., Salmonella spp., E. coli O157, E. coli, Listeria spp., Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfringens, Staphylococcus aureus, and Enterococcus spp (Table 4). (Food and Environment Research Agency, 2010) 

Enterobacteriaceae is detected in 95.88% of the 5,752 samples tested but the prevalence of pathogenic Salmonella and E. coli O157 is low, 0.24% (n=15) and 0.02% (n=1) respectively (Table 4).  

Table 4: Microbiological contamination of red meat at retail sale in the UK (n=5,998)

The numbers of food poisoning cases in the UK recorded by the Health Protection Agency for the period between 1982 and 2009 are displayed in Graph 2. (Health Protection Agency; 2010) 

Graph 2: Food Poisoning Notifications - Annual Totals, England and Wales, 1982 - 2009

There was a significant rise of food poisoning cases between 1982-1997 and moderate decline in number of incidents through the years until 2009 (Graph1).  
 

2. Some common food poisoning causing micro organisms.

Most foodborne illnesses are caused by eating food containing certain types of bacteria. One of the contributing factors of food poisoning is contaminated raw meat. After consumption of contaminated meat, the microorganisms continue to grow, causing an infection. Foods can also cause illness if they contain a toxin or poison produced by bacteria growing in food (Smith et al, 2005). Some of the common bacteria are described below.

Campylobacter spp.,

Species within the genus Campylobacter have emerged as significant clinical pathogens, particularly of human public health concern, where the majority of acute bacterial enteritis is due to these organisms (FSA, 2010; POST, 2003). Campylobacter is a genus of bacteria that are spiral or S-shaped Gram-negative rods, microaerophilic and thermophilic. (Baron, 1996). The organisms are motile, with either uni- or bi-polar flagella (Figure 1). Several species of Campylobacter are associated with human disease but C. jejuni and C.coli is the most common (Moor et al., 2005; Gundogdu et al., 2007; Wellcome Trust, 2011)

Epidemiology. Campylobacter jejuni reside in cattle, sheep, rodens, poultry and birds. It can cause benign or opportunistic infections in animals and birds and while it is rapidly cleared by many strains of laboratory mice, it can cause significant inflammation and enteritis in humans (Skirrow, 1991). Infections are acquired by consumption of contaminated undercooked meat, water or unpasteurised milk (Figure2), but person-to-person spread of infection is rare (Young, et al, 2007).

Figure 2. The sources and outcomes of Campylobacter jejuni infection. (Young, et al, 2007) image is downloaded from http://www.nature.com/nrmicro/journal/v5/n9/fig_tab/nrmicro1718_F1.html   on 28.06.2011  

Pathogenesis. C. jejuni causes an infection of the gastrointestinal tract, Campylobacteriosis. The invasion of cells by bacteria and the production of cytotoxins by C. jejuni cause the gross pathology and histological appearances of ulceration and inflamed bleeding of mucosal surfaces in the jejunum, ileum and colon (Baron, 1996). Symptoms of the infection include malaise, bloody diarrhoea, abdominal pain, fever, nausea and vomiting. The illness usually lasts 2 to 5 days but may be prolonged by relapses (Young et al, 2007).

C. jejuni penetrates the mucus layer and the intestinal epithelial cells in humans causing production of interleukin (IL)-8 by macrophages (Figure 3). IL-8 causes the recruitment of macrophages, neutrophils and dendritic cells (DC).  Immune interactions generate a massive pro-inflammatory response. By contrast, in chickens, C.jejuni can be recognised by immune cells, but the host response does not normally lead to inflammatory diarrhoea in chickens (Young et al, 2007).

Figure 3. Molecular and cellular features of the innate immune response to Campylobacter jejuni in humans and chickens. (Young, et al, 2007) image is downloaded from http://www.nature.com/nrmicro/journal/v5/n9/fig_tab/nrmicro1718_F5.html   on 28.06.2011 

The majority of cases of diseases caused by Campylobacter are sufficiently mild and not to require antibiotic treatment. However, in severe or recurrent cases susceptibility testing is important to ensure appropriate treatment as the emergence of resistance to antibiotic treatments has been reported (Moore et al, 2005).   
 

Salmonella spp.

Salmonella belong to the Enterobacteria, a genus of rod-shaped, Gram-negative, non-spore-forming, predominantly motile bacteria, around 0.7 to 1.5 µm in diameter and are from 2 to 5 µm in lengths (Ryan and Ray, 2004). Salmonella is peritrichous, evenly distributed over the surface of the cell flagella move in all directions. They are facultative anaerobes and obtain their energy from oxidation and reduction reactions using organic sources (Ryan and Ray, 2004).

This genus has been described by several different system of nomenclature. More than 2,500 different serotypes of Salmonella are known. On the basis of serologic and biochemical reactions (Kauffmann-White scheme), there are two recognised species: S.enterica and S.bongori. S. enterica serotype Enteritidis is most common strain that are responsible for the vast majority of salmonellosis infections in humans (Brenner et al., 2000; Sukhnanand et al., 2005)

Epidemiology. Most of Salmonella strains can infect animals, birds and reptiles as well as humans. Salmonella are transmitted via the oral-fecal route. The bacteria are shed in faeces and viable for months in the environment in water, soil, and manure (Ryan and Ray, 2004). Infection generally occurs from eating infected, unclean, or undercooked beef, chicken and eggs. Poor hygiene can benefit for infection to be transmitted from person to person and thus cause secondary spread of disease.

Pathogenesis.   Salmonellosis or Salmonella enterocolitis is one of the most common zoonotic diseases in humans and may be present as one of several syndromes including diarrhoea, fever, vomiting, and abdominal cramps 12 to 72 hours after infection (.A.D.A.M., 2010). Even so, the incubation time for Salmonella infection is typically 12 to 16 hours and the illness can last up to two weeks; it can persist in a carrier for 1 year or more after the infection and be shed with faeces (.A.D.A.M., 2010). Disease is initiated by oral ingestion of foods in which the bacteria are highly concentrated followed by colonization of the intestinal lumen (Figure 4). After the invasion of the intestinal tract, Salmonella multiply within a vesicle inside the epithelial cells in the intestinal wall. The immune system initiates an inflammatory response to this invasion that generally results in diarrhoea. The bacteria can cross the epithelial cell membrane and enter the lymphatic system. Thus it can cause serious or even life-threatening illness (Baron, 1996).

Figure 4: Molecular and cellular features of the innate immune response to Salmonella. (The image is obtained from http://hubpages.com/hub/Salmonella-Food-Poisoning ) 

In most cases, the infection caused by Salmonella spp. do not require any special treatments,  other than plenty of oral fluids.  In case of severe diarrhoea, rehydration with intravenous fluids may be required. If infection spreads from the intestines then antibiotic treatment is offered (Baron, 1996).

Escherichia coli

E. coli is a Gram-negative, facultative anaerobic and non-sporulating bacterium. Cells are rod-shaped, and are about 2– 3.0 μm long and 0.5 μm in diameter (Ryan and Ray, 2004) (Figure 5).

 Most E.coli strains are harmless. Some strains are important part of the normal gut flora in man, but some serotypes can cause serious food poisoning in humans.

A subdivision of E.coli is based on the serotype of its major surface antigens, such as O antigen is a part of lipopolysaccharide layer; H is a flagellin; and K antigenis a capsule (Lawrence and Ochman, 1998). The most studied strain that cause disease in humans is E.coli O157:H7 producing Shiga toxin that is one of the most potent toxins known (Marler, 2011).

Epidemiology. E. coli is a consistent inhabitant in the intestinal tract of different organisms. Some strains of E. coli are often host-specific. However, E. coli develops new strains during the natural biological processes of mutation, gene duplication and horizontal, interspecific gene transfer. Some strains develop virulence characteristics that can be harmful to a host animal (Lawrence and Ochman, 1998). Shiga-like toxin synthesis by E. coli is occurring when bacteria infected with temperate bacteriophage (Baron, 1996) (Figure 6).

E. coli O157:H7 can be transmitted by eating undercooked ground beef, consumption of contaminated vegetables, salami, unpasteurized milk and swimming in or drinking sewage contaminated water. Infection can be transmitted from person to person due to unhygienic practices and thus cause secondary spread of disease.

Pathogenesis.   E. coli can cause infection in the urinary tract and brain stem (meningitis) as well as intestinal diseases.

By the method of pathogenesis E. coli are classified into:

1) Producing toxins (enterotoxigenic) (Figure 7). An infection of gastrointestinal tract by these pathogenic strains is varying in their effects from mild to severe, sometimes fatal, diarrhoea. The E. coli serotypes that are responsible are those that produce Shiga toxin (Stx). Shiga toxin is one of the most potent toxins known (Marler, 2011)

2) Invasive species (enteroinvasive). These organisms do not produce toxins and penetrate the cell wall of the colon causing cell destruction and extreme diarrhoea.

3) Hemorrhagic (enterohemorrhagic). The organisms cause an inflammatory response of the intestinal mucosa.

4) Pathogenic (enteropathogenic) strains are associated with persistent non-bloody diarrhoea and inflammation in young children.

5) Aggregative (clumping or enteroaggregative). 

Staphylococcus aureus

S.aureus is a facultative anaerobic Gram-positive coccal bacterium about 0.5 – 1.0μm in diameter that occur in microscopic clusters resembling grapes ( ). Genus contains at least 15 different species, of which three are of medical importance: S.aureus, S.epidermidis and S.saprophyticus.

Epidemiology. Humans and associated with them animals are natural reservoir for S.aureus, and asymptomatic colonization is far more common than infection. Virulence factor of Staphylococcus is multifactorial. Some strains produce pathogenic cell-associated or/and extracellurar products. Staphylococcal intoxication leads to pathogenesis. The spread of bacteria is by a contact and airborne routes. Organism survives drying and tolerant of salt and nitrites and readily develop resistance to different antibiotic treatments. As a result, the most pathogenic strains resistant to treatments are common in hospitals (MRSA infections).

 Under favourable conditions development of staphylococcal toxin is possible in a variety of foods (dairy, meat, fish, and vegetables).

Pathogenesis. S. aureus can express several different toxins. The leukocidin causes membrane damage to leukocytes. Septic shock is caused by systemic release of α-toxin, while toxic shock is caused by enterotoxins and TSST-1 (Barons,1996).

For the majority of diseases caused by this organism, pathogenesis is multifactorial which depends on the immune status of the host, the strain of bacteria and the number of organisms in the initial exposure. (Figure 8). Hospital strains of S. aureus are often resistant to antibiotics (Barons,1996).

3. Detection methods

Accurate and definitive bacterial identification and pathogen detection is essential for correct disease diagnosis, treatment of infection and establishment of origin of disease outbreaks associated with microbial infections. Detection methods are divided into  three groups:

1. Culture-based methods of identification

Traditional identification methods of bacteria rely on identification of the phenotype of organism using gram staining, culturing on selective media and biochemical methods. However, these methods can be used only for organisms that can be cultivated in vitro. Identification of species by culture-based methods is dependant on the previous knowledge of characteristics of studying organisms such as Gram stain, morphology, culture requirements, and biochemical reactions. If a species does not match known a characteristic of any known genus and species then it may represent a previously unrecognised species (Iwen, 2005). However, these methods are time consuming and not always sufficient if specific treatment requires for adequate response in the treatment of a disease.

2. Immunological techniques

Immunological techniques are used for detection and identification of microorganisms based on their production of specific antigens and for quantitative detection of bacterial toxins. These techniques are widely used as diagnostic tools in medicine and food technology.

Immunological techniques include:

• Western blotting
• Immunoprecipitation
• Immunofluorescence, Immunocytochemistry, Immunohistochemistry
• Enzyme-Linked Immuno Sorbant Assay

Detection of bacteria is based on the ability of antibodies to recognise specific macromolecules, such as proteins or polysaccharides. Monoclonal or polyclonal antibodies are used for observation of a quantitative reaction of an antigen. The use of well selected antibodies is important for sensitivity and specificity of immuno-assays.

3. DNA-based testing: PCR

Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in the process of identification of micro organisms. This is the fastest way to screen bacterial colonies. Most nucleic-acid-based methods cannot distinguish between live and dead cells. However, some infectious diseases caused by a small amount of pathogen and it may be more complicated to establish causative agent by other methods. In case of Salmonella food poisoning cases, the presence of live bacterial cells is significant because only the presence of live cells may be considered significant.

PCR is used to amplify the target DNA about 100-1000 base pairs long. A DNA or RNA target sequence should be unique in order to detect, characterize and identify micro organisms. Artificially synthesized pair of single stranded oligonucleotide primers complementary to the flanking regions of the target sequence (Figure 8) along with a DNA polymerase is used for selective and repeated amplification of target DNA. The primers are complementary to either end of the target sequence but lie on opposite strands. The primers are usually 20-30 nucleotides long and bind to complementary flanking region at 3' end (Rao, 2011)

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Flaking region--------II--------------------Target sequence----------------------II--------Flanking region  5’-------------------------------------------------------------------------------------------------------------------------  A C C C G T T T G G G A T A T T G G G C C T T A T G G T T T A A T -  II  III III III III  II  II  II III III  III II  II  II  II  II  III III III  III III II  II  II  II III  III II  II II  II  II  II  - T G G G C A A A C C C T A T A A C C C G G A A T A C C A A A T T A ------------------------------------------------------------------------------------------------------------------------5’

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Figure 8: Target DNA for amplification by PCR (Rao, 2011) 

Polymerase Chain Reaction is performed in the thermal cycler, which can regulate temperature during amplification and be programmed for the duration of process.

The process involves the repetition of three steps (Vierstraete, 1999) (Figure 9):

• Heat denaturation, which separates the two nucleotide strands of the DNA molecule at 90-95^oC
• primer annealing, in which the primers bind to the single-stranded DNA at a lower temperature (50-65^oC)