The Foodborne Disease Fbd Biology Essay

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Foodborne disease is one of the most common problems of the world that is the major leading factor to economic loss. The World Health Organization defines foodborne illnesses as diseases, usually infectious and toxic in nature which are caused by agents that enter the body through ingestion of contaminated food. Foodborne infection has become a very important hygienic problem due to rapid expansion of food trade and also increased mobility of today's populations (Loir et al., 2003).

Hundreds of outbreaks of food poisoning cases occur throughout the world. The species of bacteria involved in causing foodborne infection are becoming more diversified. The causative agents usually involve chemicals, heavy metals, parasites, fungi, viruses and bacteria. Plant toxicants and mycotoxins that cause illnesses are also considered as causes of foodborne illnesses. However, many other pathogens that cause these diseases are present in different part of the world depending on the optimum requirement of microorganisms and food habits of people in different geographical locations throughout the world (Loir et al., 2003).

Staphylococcus aureus is one of the most common causes of foodborne infection in most of the countries of the world mainly in India due to warm and humid climate that has a high rate of infection. S. aureus is a facultative anaerobic gram-positive coccus which is non-motile and catalase and coagulase positive. The genus of Staphylococcus is one of the members of the Staphylococcaceae family which is able to grow in a wide range of pH (4.2 to 9.3), temperature (7 to 48.5o C) and sodium concentrations up to 15%. This makes the bacteria grow in a wide variety of foods (Loir et al., 2003).

S. aureus usually inhabit different parts of the body especially present in nasal passages and skin. S. aureus strains are divided into different types of biotype categories, based on their human or animal origin. There are six different biotypes (human, ovine, bovine, avian, non-hemolytic human and nonspecific) according to a biotype schema (Loir et al., 2003).

Staphylococcal enterotoxins are one of the major causes of staphylococcal intoxication due to the ingestion of food contaminated with enterotoxins that are produced by S. aureus. It is a form of gastroenteritis manifesting with vomiting, nausea, diarrhea, abdominal pain, headache and muscle cramps as the major symptoms that occur rapidly (from 30 min to 8h) and usually spontaneous remission is observed (Loir et al., 2003).

Besides, most of the genes responsible for virulence factors are located in strain-specific genetic elements such as plasmids, transposons, bacteriophages and also pathogenic islands that are present in S. aureus. In terms of genetic aspects, mobile genetic elements are one of the most common types of genetic supports in genes encode staphylococcal enterotoxins. SECbovine and see is encoded by a gene located on a pathogenicity island and defective phage, while the sea is encoded on a prophage which is carried by temperate phage (Loir et al., 2003).

Accessory gene regulator (agr) that works with staphylococcal accessory regulator (sar) in the various regulation systems is involved in the determination of S. aureus virulence factors. The seb, sec and sed are agr dependent, while for sea and sej are agr independent. Environmental factors and ability of S. aureus present in high cell density in food product are the main factors in the production of agr-dependent SEs in foodstuffs (Loir et al., 2003).

S. aureus is an extraordinarily versatile pathogen that can cause a large spectrum of infections, from mild to severe and fatal. Staphylococci can produce a group of toxins known as pyrogenic toxins superantigens included toxic-shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxins (SEs), exofoliative toxin A and B ( ETA and ETB) and also Panton-Valentime leukocidin (PVL) are some of 34 different extracellular proteins that can produced by S. aureus strains (Vasconcelos et al., 2010).

This toxin is known as superantigen-mediated disease where S. aureus toxins act as superantigens lead to a massive release of cytokines including interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) that causes capillary leak syndrome that responsible for the development of the symptoms. The wide spectrum of clinical manifestations depends largely on numerous factors that are produced by each strain (Vasconcelos et al., 2010).

Enterotoxins are monomeric, globular water-soluble proteins with low-molecular weight proteins (26900-29600 Da) that are rich in lysine, aspartic and glutamic and with cysteines form the disulfide bridge which consists of 23 different types of SEs been described including staphylococcal enterotoxin A (SEA) to staphylococcal enterotoxin IV (SEIV) is superantigenic and some of them are emetic. Staphylococcal enterotoxins are thermostable where biological activity of toxins remained unchanged even after thermal processing of food and also resistant to gastrointestinal proteases (Vasconcelos et al., 2010).

The detection of staphylococcal enterotoxins has been done by various methods including animal assays, immunological, molecular biological and also by biosensors. Polymerase Chain Reaction (PCR) can also be used to detect a single pathogenic bacterium that could be present in food. It operates by amplifying the target other than signal so it is less prone to produce false-positive (Vasconcelos et al., 2010).

DNA amplification method (PCR) is a useful and reliable tool in detecting genes that code for the production of SEs since the DNA remain unchanged and also it is able to indicate the presence of enterotoxigenic strains of S. aureus. Besides, it is able to detect sources of contamination by detecting the presence of the strains in foods (Vasconcelos et al., 2010).

Reverse transcriptase PCR (RT-PCR) are the sequences of mRNA responsible for enterotoxin production that can be detected. It also yields higher detection sensitivity and ability to detect viable cells of the bacterium. Multiplex PCR (mPCR) method is usually used in typing S. aureus toxins and is able to detect enterotoxin genes in a single procedure (Vasconcelos et al., 2010).

This review focus on the various types of staphylococcal enterotoxins and genetic relatedness of S. aureus strains present in food stuff that are involved in staphylococcal food poisoning.

2.0 Literature review

2.1 Foodborne pathogen and food vehicle

The broad spectrum of foodborne infections had been changed over a period of time, as most of the microbial pathogens were able to contaminated food products which served as vehicle of transmission that caused diseases when toxin are consumed. The spectrum of food pathogen usually including enteric bacteria, aerobes and anaerobes, viral pathogens as well as the parasite that caused an array of illnesses in the human host (Tauxe et al., 2002).

2.1.1 Food and Foodborne pathogens

Food safety is a global health goal and the foodborne diseases take a major crisis on health. Many studies show that food poisoning is an universal problem. Food poisoning occurs in poor countries as well as in developing countries.

Panisello et al. (2000) had investigated the relationship between etiological agents and the vehicle of transmission of the foodborne outbreak between 1992 to 1996 as shown in table 1. The result indicates that S. aureus were most commonly present in poultry 5/98 (5.1%) followed by sandwich 1/21 (4.8%), red meat 4/99 (4.1%), vegetables 1/34 (2.9%) and seafood 1/83 (1.2%). It was not present in other food products in these studies.

While eggs were the highest food vehicle of outbreak accounting for 42/43 (97.7%) in Salmonella followed by desserts 79/83 (95.2%), miscellaneous foods 7/10 (70%), poultry in 66/98 (67.3%), sauces 11/18 (61.1%), sandwiches 11/21 (52.4%) and dairy 9/18 (50%). Red meat was associated with Cl. perfringens about 44.9% total outbreak and food containing cereal usually served as vehicles of transmission related to Bacillus bacteria 14/23 (60.9%) (Panisello et al., 2000).

The higher the percentage the higher was the occurrence of a pathogen as shown in table 1. The occurrences of salmonellosis attributed to eggs should be the highest among food pathogen related to eggs accounting for 97.7% (Panisello et al., 2000).

Other research done by Greig et al. (2009) showed microorganism-specific food source profile (%) for foodborne outbreaks internationally from 1988 to 2007 based on the numbers of outbreaks according to the outbreak investigation. Salmonella, Norovirus and E. coli contributed to 70% among the 4093 foodborne outbreak cases as shown in table 2.

Among the Salmonella species, Salmonella enteritidis was the most frequent involved in foodborne outbreaks that accounted for 991 cases, followed by Salmonella Typhimurium which accounted for only 270 cases of the outbreak. Beside that, multi-ingredient foods, eggs, produce, and beef are also the most common food products that were involved in the foodborne cases as compared to the others (Greig et al., 2009).

Table 2 shows the relationship between different types of food category and also microorganisms. Less than half of the microorganisms in these studies were linked to eggs and other foods as vehicle of transmission, while for multi-ingredient, produce and seafood were heavily linked with almost all types of microorganisms except some of parasitic pathogens. S. aureus were found most commonly present in multi-ingredient food accounting for 22.0%, while produce, seafood, other meats, bakery item, turkey, eggs and other foods contribute only 5% of the total outbreak respectively in the SFP. S. Enteritidis is heavily involved in egg and bakery related outbreak that accounts for 12.1% and 43.3% respectively and Clostridium botulinum contributes to half of the other food outbreaks accounting for 24.1% as shown in table 2 (Greig et al. 2009).

The result shows that only Salmonella and Norovirus were involved in all 13 food categories in which egg category accounts for 43.4% S. enteritidis outbreak and multi-ingredient food was heavily linked to Norovirus outbreak. For the other pathogens, the outbreaks were associated with more than seven food categories in which more than half the number of food categories used in this study with one or two categories being predominant, for example E.coli and 'Beef' (44.2%), L. monocytogenes and 'Dairy product' (41.5%), HAV and Produce' (40.0%) or Clostridium perfringens and 'Beef" (39.1%) in table 2 (Greig et al. 2009).

Panisello et al. (2000) and Greig et al. (2009) have shown various types of foodborne pathogens that can contaminate the food product under favorable conditions. The outbreak of eggs was mostly related to outbreaks of salmonellosis among Salmonella species via vertical and horizontal route. During the formation of the egg, the contents of the eggs easily contaminated with the bacteria before covered with shells is known as vertical transmission (transovarian infection). While in horizontal transmission involved trans shell infection of the contents of the egg during transit through the cloaca and fecal contamination of the external surface of the shell.

These conditions have favored the growth of microorganisms leads to food poisoning. Contamination of food products can occur at any point and anywhere in the food chain from primary production in field to point of consumption by consumers. Cross-contamination between food, recontamination during food processing and preparation including the contamination from food handler can cause food poisoning (Greig et al., 2009).

Table 1: The foodborne outbreak caused by etiology and vehicle of transmission in England and Wales between between 1992 to 1996.

(Panisello et al., 2000)

Table 2: The microorganism-specific food source's profile (%) for foodborne outbreaks reported between 1988 to 2007.

(Greig et al., 2009)

2.2 Methicillin-Resistant S. aureus (MRSA) in food samples

Methicillin-resistant Staphylococcus aureus strains are global health concern that were first discovered in 1961. Staphylococcus aureus is usually present in foods and it is the most common cause of foodborne bacterial intoxication throughout the world. S. aureus becomes methicillin resistant by acquisition of mecA gene which encodes penicillin-binding protein 2a (PBP2a) with a low affinity for all β-lactams antimicrobials. The strains producing PBP2a were resistant to all β-lactams (Normanno et al., 2007).

Consumption of food that contained resistant and multiresistant bacteria will lead to transmission of antibiotic-resistant isolates to human being. Recently, MRSA strains were isolated from several food products or animal-derived food products for instance chickens, cattle and pigs were the most important reservoirs for MRSA and they served as the food vehicle for transmission of S. aureus of animal-derived food products to human being. (Normanno et al., 2007)

Boer et al. (2009) had done a study to estimate the prevalence of MRSA in raw meat samples from retail trade within the period from June 2007 to May 2008. Two-step enrichment in Mueller-Hinton broth+6.5% NaCl for antimicrobial susceptibility testing and phenol red mannitol broth that contained ceftizoxime and aztreonam (PHMB+) followed by isolation on MRSA ID agar (bioMerieux) were the detection methods used for the detection of MRSA in the raw meat samples. Spa typing and multilocus sequence typing (MLST) used to type and differentiate the isolates.

The result showed 264 out of total 2217 (11.9%) MRSA strain isolates in meat samples and 75 samples of meat that showed positive in qualitative test were tested for count of MRSA by plating 1 ml of the 1:10 dilution of the samples of three types of plates of MRSA ID as shown in table 3 (Boer et al., 2009).

Among the MRSA in different types of meat, turkey meat contributed the highest number of positive value accounting for 35.3% (41/116), followed by chicken 16.0% (83/520), veal 15.2% (39/257), pork 10.7% (33/309), beef 10.6% (42/395) and lamb/mutton 6.2% (20/324). Fowl and game showed relatively low percentages of the number of positive results which was less than 4% of the overall meat (Boar et al., 2009).

Table 4 showed the typing results, 85% of the isolated MRSA strains detected in meat had spa-types belonging to multilocus sequence type (ST) 398. In these studies, the majority of the strains originated from spa-types of non-typeable (NT)-MRSA that isolated from the animal food products. This indicated the animals were the main origin of MRSA in raw meats. Among the ST398, biological and imported chicken showed the percentage of spa type ST398 accounted for pork 32/33 (97%), veal 37/39 (95%), turkey 38/41 (93%), (NL+EU) chicken 67/75 (89%), lamb and mutton 14/18 (78%), fowl 3/4 (75%), and beef 25/42 (60%) as compared to game which is the lowest percentage 0/4 (0%) as shown in table 4 (Boar et al., 2009).

This study showed the highest prevalence in turkey, regularly reared chickens and veal as compared to the low prevalence of MRSA in meat from 'biological' reared chickens, fowl and game (no use of growth promoting antibiotics) indicated there were a relationship between the use of antibiotics and dissemination of MRSA in animals. The ST398 was originally methicillin-susceptible in nimals that was spread by the use of antibiotics in stock-farming, while the high prevalence of STnon398 strains isolated from beef due to human had been introduced the STnon398 strains onto raw meat and food handler that contaminate the foods with MRSA strains during food processing and preparation (Boar et al., 2009).

Unhygienic handling of contaminated food with MRSA will cause transmission of MRSA to humans and also colonization of skin and nostrils. In conclusion, the result of the isolated MRSA strains in these studies belonged mostly to spa-types of non-typeable (NT)-MRSA found mainly in food producing animals. This indicates animals are the sources of MRSA. High percentage of STnon398 strains from beef shows that human introduced the strains into raw meat during food preparation and contaminated with other food with MRSA as shown in table 4 (Boar et al., 2009).

Crago et al. (2012) had done a study to determine and investigated the rate of S. aureus and impact of MRSA contamination in food samples in Alberta, Canada within the period between 2007 to 2010. A total of 693 food samples were submitted. Alberta Provincial Laboratory did the microbial testing on the food samples that were suspected to cause food poisoning outbreaks from 1 January 2007 to 31 December 2010. Aerobic plate count (APC) was done to determine the level of background contamination.

Tube coagulase test with rabbit plasma was used to determine the S. aureus colonies and endpoint PCR was used to detect nuc gene to confirm the S. aureus isolates. For antimicrobial susceptibility, disc diffusion was performed on Mueller-Hinton agar plate used to screen for methicillin-resistance present in all S. aureus isolates and endpoint PCR to detect the mecA gene.

A multiplex PCR was carried out by using the S. aureus primer set consists of nucAf and nucA to detect nuc gene and a primer set (mecAf and mecAr) to detect the mecA gene (Crago et al. 2012).

The result showed that only 10.5% (73/693) of the food submitted showed positive for S. aureus which increased within the four year period from 9.2% to 13.6% as shown in table 5. In 2010, 125 amounts of the sample submitted in which 17 of S. aureus showed positive result accounting for 13.6% that is the highest percentage as compared to 2007 that signify the lower percentages accounting for only 9.2% (22/238). 29 meats (poultry n=2, beef n=7, pork n=6 and unknown n=5), 20 prepared foods containing meat (unknown n=4, pork n=1, seafood n=3, poultry n=4 and beef n=8), 11 prepared foods not containing meat, 10 dairy (ice cream n=1, cheese n=1 and milk n=8) and 3 produce are the food samples that showed S. aureus positive as shown in table 5 and 6 (Crago et al., 2012).

Meat showed the highest number of S. aureus contamination as compared to the rest of food accounting for 29 in total within the four year period, while only produce showed the lowest number which was only 3 in total. There were no methicilin-resistance and mecA gene present in any S. aureus isolate from food samples screened by using disc diffusion and PCR in these studies may due to low levels of MRSA in food samples (Crago et al., 2012).

59% (43/73) were found contaminated with more than one type of microorganism on the screening panels among the S. aureus positive food samples. 38 food samples were showed positive with Yersinia enterocolitica (2), Aeromonas spp. (4), C. perfringens (5), B. cereus (1). E. coli O157:H7, Campylobacter spp., Salmonella, or Shigella spp does not present in any S. aureus positive food samples (Crago et al., 2012).

Bacterial co-contamination were shown positive results for S. aureus in food samples by using an aerobic plate count of ≥ 105 CFU/g, 27.4% (20/73) had APCs ≥ 105 CFU/g which shows there were high level of bacterial contamination and 55% contamination caused by more than one enteric pathogen. Among the 73 S. aureus isolates food sample showed that 59% contaminated with more than one type of microorganism on the screening panel (Crago et al., 2012).

Table 6 showed 81% (59/73) of the S. aureus isolates positive samples to be found mainly in dairy produce and meat, protein-rich foods accounting for 81% that associated with staphylococcal food poisoning. It shows that food easily contaminated with S. aureus and MRSA from the production animal and also food handler during food preparation as chicken and pigs were the reservoirs of MRSA that were able to transmit the pathogen to human beings. In this study, S. aureus from prepared food were the main cause of food contamination in which these bacteria present in these foods were due to the contamination of food handlers during food preparations (Crago et al., 2012).

From the studies Boar et al. (2009) and Crago et al. (2012), they stated that animal-derived food product serves as an important vector for the transfer of antibiotic resistances from animals to human being and MRSA plays an important role in contributing to foodborne outbreak. The transfer can occur in three different ways: by the presence of antibiotic residue in food, through the transfer of multiresistant foodborne pathogens, or by consumption of resistant part of original food microflora that transfer to a pathogenic microorganism.

Small amount of MRSA present in foods may constitute a risk for customer and seriously in immunocompromised individuals. Ongoing surveillance of foodborne pathogen and monitoring for the emergence of MRSA and antibiotic resistance in foods is necessary for public health (Crago et al., 2012).

Table 3: The MRSA in meat.

(Boer et al., 2009)

Table 4: MRSA in meat : spa-types.

(Boer et al., 2009)

Table 5: The food submissions and S. aureus positive rates between 2007 to 2010.

(Crago et al., 2012)

Table 6: The food types of S. aureus positive samples from year 2007 to 2010.

(Crago et al., 2012)

2.2.1 Antimicrobial resistance profile of Staphylococcus aureus and MRSA in food sample

Antimicrobial resistance is one of the essential public health concern throughout the world. Due to extended use and misuse of antibiotics in various fields in agriculture and stock-farming cause most of the bacteria to develop resistance to all the antimicrobial agents more rapidly. In the past few years, researchers have found out that the food isolates showed the significant increase in resistance against all the antibiotics. Many studies show that methicilin-resistance S. aureus is one of the bacteria that show resistance not only to methicilin antibiotic but also other antimicrobial agents that are usually present in food-producing animals that cause foodborne disease and also nosocomial infection worldwide.

Pesavento et al. (2007) had done a study to evaluate the rates of food-isolated S. aureus that were resistant to some of antibiotics mainly to methicilin and vancomycin. 176 meat samples of raw meat (42 from poultry, 66 pork meat and 68 beef) that were purchased by retailer area in the Florence area in Italy were subjected to microbiologically analysis. The disk diffusion assay on Muller Hinton agar was used to monitor the antimicrobial sensitivity and the zone of inhibition was recorded in which all the isolates were tested for 12 different types of antimicrobial agents.

176 samples of raw meat that purchased by retailers were analyzed, S. aureus was found presented in 42/176 (23.86%) samples in which 12 (28.57%) were from poultry, followed by 20 (29.41%) from beef and 10 (15.15%) from pork meat. These showed the high prevalence of multiresistant S. aureus in food which had a high risk of infection that can transmitted the resistance to other bacteria as shown in table 7 (Pesavento et al., 2007).

Table 7 showed the antimicrobial sensitivity among the 42 S. aureus extracts showing 13 strains (30.95%) to be sensitive to all the antibiotics while only one strain (2.38%) showed resistance to oxaxillin with intermediate values and 28 (66.67%) of the strains indicated resistance to at least one antibiotic after the inhibition diameter and the relative frequencies was compared. Various pattern of antibiotic-resistance among the strains were seen. All the strain tests showed antimicrobial sensitivity to vancomycin, methicilin and also teicoplani. Only one strain was resistant to cepthalotine (Pesavento et al., 2007).

Only 11 (26.19%) of all the isolates showed one resistant to microbial substances: 1 (2.38%) resistant to both penicillin G and clindamycin, 3 (7.14%) and 6 (14.28%) resistant to ampicilin and oxacillin. While 4 (9.52%) of the strains showed double resistances among the total strains isolated: other 13 strains accounting for (30.95%) showed multiple resistance (more than 3 antibiotic) in which 6 (14.28%) and 3 (7.14%) exhibited triple and quadruple resistance, while 1 and 3 exhibit quintuple and sextuple resistance (Pesavento et al., 2007).

In this study showed a high percentage of resistance level for all the antimicrobial substances in which oxacilin the resistance was seen more frequently in isolates of poultry as compared to isolates of pork and beef (Fisher's exact test=0.018). The occurrences of S. aureus and anti-microbial resistance profile in meat isolates (beef, poultry and pork) showed a relatively large presence of multiresistant S. aureus in food that lead to risk of infection and higher chance of transmission of resistance to the other bacteria (Pesavento et al., 2007).

High resistance to ampicillin present in all isolates accounting for 42.86% due to the natural resistance towards β-lactams of S. aureus induced by exposure to penicillin. Beside that, poultry meat also showed extremely high resistance toward oxacillin due to its usage as a growth promoter in broiler chicken and also used to prevent necrotic enteritis (Pesavento et al., 2007).

Beside that, Narmanno et al. (2007) had done other studies on the occurrence of MRSA strains isolated from animal food products in which 160 S. aureus strains were isolated from 1634 foodstuff samples of animal origin in Italy between 2003 to 2005. The MRSA strain was characterized by detecting the mecA gene using a PCR test and the types of staphylococcal enterotoxins (SEs), resistance properties and ecological origin was determined by biotyping.

Reverse Passive Latex Aggultination (RPLA) and SET-RPLA kit were used for detection of the SEs production among the mecA positive strains. The result showed that 6 out of total 160 S. aureus strains were mecA positive which derived from four isolates that came from different types of food samples (6/1634 or 0.36%). Only one strain was isolated from pecorino cheese and mozzarella cheese, while the remaining four strains came from bovine milk in which all the MRSA strains were able to synthesize staphylococcal enterotoxins (SEs) as shown in table 8. The four isolate (bovine milk) where two of the strains belonged to non-host-specific biovar and ovine biovar and two isolates (mozzaerlla cheese and pecorino cheese) each belonging to non-host-specific biovar and ovine biovar respectively. (Narmanno et al., 2007)

Of all the MRSA strains in these studies able to synthesized SEs in which two strains synthesized SEA and SED (16.6%) and SED (33.3%), while one strain synthesized SEC and SED (16.6%) and SEC (16.6%) as shown in table 8. Of all the biotypes of MRSA strains, half (50%) belonged to non-host-specific (NHS) biovar and half (50%) belonged to ovine bovar. Ovine biovar from bovine milk and pecorino cheese suggested that ruminants act as reservoirs of MRSA strains and that pasteurization of milk will reduce the MRSA infection as cross contamination of the processed dairy environment (Narmanno et al., 2007).

Table 9 showed all the MRSA resistant strains in the study, Narmanno et al. (2007) showed at least resistant to one of the antibiotic, while three strains resistant to three antibiotics and no one resistant to vancomycin and teicoplanin by using the disc agar diffusion method on Mueller-Hinton agar Beside that, among the MRSA strains three strains showed phenotypic resistance to ampicilin and all other strains exhibited resistance (MRSA 5) to nalidixic acids (Narmanno et al., 2007).

Besides, Hammad et al. (2012) had done other studies to determine the prevalence, antibiotic resistance, molecular genetic chacteristic, methicillin-resistant S. aureus (MRSA), methicilin-susceptible S. aureus (MSSA), methicillin-resistant coagulase negative staphylococci (MR-CoNS) that isolated from 200 ready-to-eat food raw fish (sashimi) that collected from 25 retail grocery stores of five supermarket chains in Japanese prefecture of Hiroshima.

The result of Hammad et al. (2012) showed that (93%, 23/25) contained fish were found presence of S. aureus species among the grocery stores surveyed. 170 isolates of MSSA were found in 174 (87%, 174/200) S. aureus isolates samples. These results indicated the high prevalence of S. aureus present in sashimi due to lack of restricted hygienic measure for preparation of sashimi in Japan.

Hammad et al. (2012) also showed the prevalence of MRSA and MR-CoNS in retail ready-to-eat fish as shown in table 10. Different types of Staphylococcus species included 5 came from S. aureus, 2 from S. haemolyticus, 2 from S. warneri and 1 from S. pasteuri which originated from 10 isolate of MRSA and MR-CoNS were obtained from 10/100 samples that collected from 10 shops that belong to four supermarket chains. The analysis of the susceptibility of S. aureus isolates showing that almost 135 isolate susceptible to all the antimicrobial substances and 25 isolates were resistant to one antibiotic, three isolates were resistant to two antibiotic and 12 isolates were resistant to more than three antibiotics.

Majority of multi-drug S. aureus and MR-CoNS isolates showed resistance to macrolides, aminoglycosides and penicillin as shown in the table 10. All the antibiotic resistance gene were resistance to aminoglycosides, tetracyclines, β-lactams, macrolidesm lincosamides and streptogramin B (MLSB) antibiotic was detected (Hammad et al, .2012).

In this study, only one MRSA strain showed resistance to cafoperazon and the remaining of the MRSA and MR-CoNS isolates were susceptible to cefoperazone, cefotaxime and amoxicillin-clavulanic acids. This research has proven that sashimi is one of the vehicle for transmission of multidrug-resistant and toxigenic staphylococci as shown by MRSA and MR-CoNS that isolated from the retail ready-to-eat food in Japan (Hammad et al., 2012).

Other studies conducted by Can et al. (2012) to study the presence of enterotoxigenic, antimicrobial resistant S. aureus present in Turkish cheese and the presence of mecA gene in the S. aureus strains from a total of 200 unpackaged cheeses samples purchased from retail market in Ankara between April 2008 to January 2009.

The result of Can et al. (2012) showed that about 19 (9.5%) out of the 200 cheese samples were contaminated with the coagulase-positive S. aureus in which 12 came from Tulum cheese and 7 come from white cheese samples respectively as shown in table 11. While 7 out of 100 in the Tulum cheese samples examined show the contamination of S. aureus in cheese and of the 100 white cheese samples examined, 5 were contaminated with S. aureus. Beside that, 12 of the CPS isolates were identified as S. aureus over a total of 30 isolates in these studies. By using ELISA test found that 3 out of 12 S. aureus strains were enterotoxigenic and only two of them produced SEC types of staphylococcal enterotoxins and one of them produced SEC and SED together that isolated from only Tulum cheese samples.

In this study, 10 out of 12 strains were shown to be resistant more than one antibiotic tested by disk diffusion method. Only 3 of the strains showed (25%) resistance to single strains which was tetracycline, erythromycin and also ampicilin. While 2 (16.6%) of the strains showed double resistance and the remaining 5 strains showed multiple antibiotic resistance. Ampicillin and erythromycin had the highest resistance rate accounting for 50% as compared to other antimicrobial substances followed by clindamycin (33.3%), teicoplanin and tetracycline (25%), methicillin and oxacillin (16.6%) as shown in the table 12 (Can et al., 2012).

Figure 1 showed the result of mecA positive S. aureus strains on the electrophorese image. M indicated 100bp DNA marker, + indicated positive control (S. aureus ATCC 43300), - indicated Negative control (S. aureus ATCC 25923), 3 and 12 indicated mecA positive S. aureus strains. Polymerase chain reaction detected the mecA positive presence in 2 of the S. aureus strains in which showed phenotypically and genotypically resistance to methicilin. In these studies only Tulum cheese samples indicated the presence of MRSA strains that are found to be enterotoxigenic and have multiple antibiotic resistance properties as well (Can et al., 2012).

From the studies of Pesavento et al. (2007) and Can et al. (2012) showed that all the isolates from food samples were multiresistant and it is necessary an accurate and active control against antibiotic resistances in food mainly on meat in order to detect any expansion of new antibiotic resistance to other bacterias. Therefore, microbiological safety of food has to be guaranteed in order to prevent the transmission of pathogens to customers.

Table 7: The antimicrobial sensitivity of S. aureus isolates (n=42).

(Pesavento et al., 2007)

Table 8: The characteristic of MRSA each isolated from food samples.

(Normanno et al., 2007)

Table 9: The antimicrobial resistance pattern of MRSA isolated from food.

(Normanno et al., 2007)

Table 10: The antibiotic resistance phenotypes and genotypes of multidrug-resistant

Staphylococcus species isolated from sashimi.

(Hammad et al., 2012)

Table 11: The occurrences of S. aureus strains in analyzing cheese samples.

(Can et al., 2012)

Table 12: The antimicrobial sensitivity of S. aureus strains.

(Can et al., 2012)

Figure 1: The electrophorese image of mecA positive of S. aureus.

(Can et al., 2012)

2.3 Staphylococcal food poisoning outbreak

Staphylococcal food poisoning (SFP) is caused by staphylococcal enterotoxins (SEs) produced during a massive growth of Staphylococcus aureus in foods. SFP is a prevalent cause of food-borne disease worldwide. The ability of S. aureus to grow and produce SEs under a wide range of conditions is evident from the variety of foods implicated in the SFP. Raw meat, meat product, including sucuk, raw milk, dairy products and ready-to-eat foods such as bakery product are the among food products reported to be related to S. aureus enterotoxin-induced food poisoning. Egg yolk (EY) reaction-positive strains in Japan are the cause of staphylococcal outbreaks of food poisoning.

Miwa et al. (2001) had done a study on the staphylococcal foodborne outbreak due to an egg yolk (EY) reaction-positive strains that occurred in Japan. Eight of fecal specimens were collected from each patients and streaked on a mannitol salt agar plate containing 5% (v/v) EY Emulsion SR47 to isolated S. aureus. Bright yellow colonies were picked up and subjected to further analysis. Fecal specimen and hand swabs of two food handlers who prepared boxed lunches was subjected to direct and enrichment culture were used to detect the presence of viable S. aureus.

Food samples from boxed lunches were subjected to reverse passive latex agglutination kit (SET-RPLA) used to test for the presence and typing of SE in the food samples. Besides, pulsed-field gel electrophoresis (PFGE) was used for undergo RFLP analysis of the chromosomal DNA preparation from the sample which was digested with the restriction enzyme (SmaI) (Miwa et al., 2001).

The result of the studies showed that no other enteropathogenic organisms were found in which scrambled egg was the food in the boxed lunches. SEA was detected and isolated from four fecal specimens of the eight patients tested. All S. aureus isolates from fecal specimens of patients and the scrambled egg that produced SEA, were shown to be free-coagulase type II and EY reaction-negative by both the tube method and agar plate method. The result of EY reaction of the food isolates and the type strain ATCC 25923 were shown in figure 2 (Miwa et al., 2001).

The S. aureus isolated from scrambled eggs indicated negative EY reaction (no precipitation) by tube method and the colonies with no opaque zone by agar plate method. While for the S. aureus ATCC 25923 which serves as controls indicated positive EY reaction in which there was the appearance of an opaque zone around the colonies by agar plate method and precipitation of EY was observed in tube method (Miwa et al., 2001).

Figure 3 shown the RFLP analysis result in which four isolates from the patients and another one from a scrambled egg specimen. The band 1 indicated the S. aureus isolated from the scrambled eggs; the band 2 to 5 indicated the S. aureus isolated from each of the four patients; the band 6 indicated S. aureus ATCC 25923; while the last band indicated the S. aureus isolated from a patient from another foodborne cases (Miwa et al., 2001).

They show same cleavage pattern along with RE SmaI but show a difference in pattern of the type strain ATCC 25923 and an isolate from another outbreak. They found the scrambled egg to contain SEA (20-40 mg/g) and the number of SEA-producing-foods 3.0Ã-109 CFU/g at a level that was responsible for another outbreak of SFP (Miwa et al., 2001).

In these studies, mishandling of scrambled eggs after cooking is the main cause of contamination with S. aureus in these study. As between 3.8 to 35.3% of food handler were found the presence of S. aureus in their hands by direct and enrichment culture is a significant source of staphylococcal food poisoning (Miwa et al., 2001).

Other studies were done by Sospedra et al. (2012) to detect the production of toxic shock syndrome toxin 1 (TSST-1) in 53 S. aureus isolates collected from food handlers and surfaces of foods in food service establishments in Spain. A total of 908 samples was collected within the period from 2009 to 2011 from different Spanish restaurants that included kitchen knives, clean plates, slicers, cutting boards, dish towels, stainless steel tables and worker's hands were also analyzed.

Table 13 shows 5.8% of the studied samples to be contaminated with S. aureus. Dish towels were the highest in number which was 14 (10.1%) contamination and the slicer was the lowest accounting for 3.4% only. Clean plates and kitchen knife did not show the presence of microorganisms and only (0.1%) one out of 53 isolated S. aureus were found to produce TSST-1 that was isolated from one of the employee's hand who was working in a restaurant among the surfaces studied. These studies reflected the high prevalence of S. aureus in food served in restaurants.

All the studies above showed that S. aureus isolates were present in all kinds of food samples that will lead to staphylococcal food poisoning especially in eggs and poultry. The risk of food poisoning could be reduced if the food handler washed their hands before and after food contact. Food handler hygiene is the most important measure during food preparation because the toxin present in food is very hazardous to consumers and cannot be destroyed even by heat treatment. Hand washing is necessary before start working, before food handling and after finishing the task due to foods cross contamination can occur if contaminated hands with TSST strains touch the food (Sospedra et al., 2012).

Figure 2: The EY reaction of S. aureus isolate and the type strain ATCC 25923 (A) Agar plate method; (B) tube method (a) S. aureus ATCC 25923, (B) S. aureus isolated from causative food.

(Miwa et al., 2001)

Figure 3: The PFGE patterns of S. aureus isolates.

(Miwa et al., 2001)

Table 13: The incidence of Staphylococcus aureus and TSST isolated from them in

food handlers and food service establishment in Spain.

(Sospedra et al., 2012)

2.3.1 Staphylococcal enterotoxins in the food samples

Staphylococcus aureus is one of the most common pathogen related to hospital acquired disease and serious community that considered as a major issues of Public Health. The production of enterotoxins (SEs) by some strains of this microorganism can induced food poisoning during the improper hygiene food preparation (Pereira et al., 2009).

SEs are a group of heat resistant, pepsin resistant enterotoxins that belonging to pyrogenic toxin superantigens (PTSAgs) family that will bind to the outside of the major histocompatibility complex (MHC) class II molecules and Vβ chain of a T-cell receptor (TCR) will formed a complex. Eventually leading to activation of T-cell proliferation in a nonspecific manner (Loir et al., 2003).

In general, SEs have been divided into different serological types included five classical major antigenic types of SEs (SEA, SEB, SEC, SED, and SEE) and other additional SEs (SEG, SEH, SEK, SEJ) have been reported to play an important role in SFP. SEA and SED were commonly involved in food poisoning. While SEK, SEI, SEM, SEN, SEO, SEQ, SER and SET were the newly types of staphylococcal enterotoxins found. The most common causative agents of foodborne were SEs which ranked second or third worldwide in which seafood, meat products and dairy product contain prevalence of S. aureus which have been reported in American, European and also East Asian Countries (Boynukara et al., 2008).

Normanno et al. (2005) had conducted an experiment about the presence of Coagulase-Positive Staphylococci (CPS) and S. aureus in different food samples marketed in Italy. They investigated the enterotoxigenicity of S. aureus strains. 9869 food samples of animal origin marketed in Italy and 1515 swabs from food contact surfaces in the food industry were collected between June 2000 to July 2002. The local health officials collect the food sample from different retail outlet and then subjected for further analyzed. The strains test from food samples grown in tubes with 10 ml of the Brain Heart Infusion Broth and the SET-RPLA kit used to detect staphylococcal enterotoxins. Baird Parker Agar with rabbit plasma fibrinogen (RPF) and API Staph system were used as an isolation medium to identify colonies of coagulase-positive staphylococci in these studies.

The result of Normanno et al. (2005) indicated that the presence of CPS in 1971 samples among the 11384 food samples examined and found that 541 CPS strains and 537 of them (99.3%) were identified as S. aureus. A total of 298 (55.5) out of 537 isolates that will produce enterotoxins as shown in table 14 and 15 in which the characterization of (SEA, SEB, SEC and SED) was performed. The high prevalence of CPS present in the food sample accounting for 17.3% indicated that S. aureus were commonly found in nature and easily contaminated with a food product when exposed to the air as shown in table 14

In these studies, meat products showed the highest prevalence from ripened meat product samples (17.1%) to other meat product samples (48.1%) 1245 out of 5369 (23.1%) meat product samples were contaminated with CPS and 146 were identified as S. aureus and 66 (45.2%) were shown to be enterotoxigenic. The strains assays produced SEA (30.3%), SEB (7.6%), SEC (51.5%), SED (6.1%) , SEA+SEB (1.5%) and SEA+SED (3%) as shown in table 15 (Normanno et al., 2005).

Beside meat product, 3097 of milk and dairy products analyzed, they found 642 (20.7%) were contaminated with CPS. 362 out of 364 isolates were identified as S. aureus and of these, 217 (59.9%) were shown to be enterotoxigenic. In detail, these strains produced SEA (26.7%), SEB (0.9%), SEC (28.1%0, SED (15.7%), SEA+SEB (1.8%), SEA+SED (26%), SEA+SEC (0.5%) and also SEC+SED (0.5%) (Normanno et al., 2005).

Besides that, raw milk also showed high frequent contaminated with CPS (38.4%) that related to cows with mammary infections and SEC (28.1%) in S. aureus strains were the most common staphylococcal enterotoxins produced by strains from milk-producing animals mainly present in milk and dairy product (Normanno et al., 2005).

Beside raw milk samples, out of the 173 pastas samples examined, nine (7.6%) of them were contaminated with coagulase-positive S. aureus and four were identified as S. aureus among the four isolates and one was found to be enterotoxigenic that produced SEC+SED. Among the 119 pasta samples examined, nine of them were found contaminated with CPS. Five isolates examined, four were S. aureus and one of them showed enterotoxigenic that produced SEC (20%), SED (20%), and SEA+SED (60%) (Normanno et al., 2005).

While CPS was found in 22/737 (3%) the fish product samples and 8 of them were identified as S. aureus and only two of them were able to produce two types of SEs that was SEB (50%) and also SEC (50%). Moreover, 1/29 of (3.4%) of the egg product samples were found contaminated with CPS and only one isolate produced SEC enterotoxins. Low amount of CPS contaminated with food contact surfaces (1.6%) indicated good environmental sanitary conditions in the food industry that is able to minimize the S. aureus from being contaminated (Normanno et al., 2005).

Besides that, a total of 1515 swabs from food contact surfaces in the factories was found to have 25 (1.6%) coagulase-positive S. aureus and 3/6 of overall S. aureus were enterotoxigenic that only produced one enterotoxin which is SEC accounting for 50% (Normanno et al., 2005).

Normanno et al. (2005) concluded the total of 298 (55%) of the enterotoxigenic S. aureus strains in these study as shown in table 16 included SEC (33.9%) were the most common SEs isolated in the foodstuff followed by SEA (26.5%), SEA+SED (20.5%), SED (13.4%), SEB (2.7%), SEA+SEB (1.7%), SEC+SED (0.7%), SEA+SEC (0.3%) and SEB+SEC (0.3%). SEA are more common involved in staphylococcal food intoxication followed by SED and SEB. More than half of the S. aureus strains able to produce enterotoxins were hazardous to consumers in which lack of stringent hygienic and sanitary standards in order to prevent S. aureus multiplication in foods.

Normanno et al. (2007) had done a survey to evaluate the occurrence of S. aureus and characterize and biotype of the isolated strains based on the production of SEs and antimicrobial-resistance pattern from each isolated strain in milk, dairy product and meat products marketed in Italy. In this study, they analyzed and collected a total of 1634 food sample including 993 from meat product and 641 from milk and dairy product from retail outlets by local health officials between January 2003 to December 2005 as shown in the table 17 that shows the occurrence of S. aureus in analyzed samples.

Normanno et al. (2007) used buffered peptone water (BPW) to diluted the sample and homogenized in a stomacher, incubated onto Baird-Parker RPF agar and incubated at 35 ℃. The present of coagulase-positive staphylococci were identified and subjected to Gram staining and catalase test. The Gram and catalase positive isolates were identified by using API Staph system.

While the detection of staphylococcal enterotoxin is usually performed by using the reverse passive latex agglutination (RPLA) and subjected to Polymerase chain reaction (PCR) assay. Besides, in the biotyping process they differentiated strains in biovar into human, poultry/poultry-like, bovine, ovine and non-host-specific based on the detection of some properties of the strains tested (Normanno et al., 2007).

The result indicated 209 out of the 1634 (12.8%) samples analyzed were found contaminated with S. aureus in which come from 100/993 (10%) meat product samples and 109/641 (17%) milk and dairy products respectively as shown in table 17 indicated the occurrence of S. aureus in the analyzed sample. Normanno et al. (2007) had found the total prevalence of coagulase positive staphylococci (17.3%) on several foodstuff in Italy to have a lower contamination rate in the range of 17.1% to 48.1% as observed in meat product (Normanno et al., 2007).

In the study, of the 209 strains isolated, 125 (59.8%) of them were found synthesized more than one type of SEs. Among the enterotoxigenic strains detected, SED (42/125-33.6%) was the most frequently detected followed by SEA (23/125-18.4%), SEC (19/125-15.2%) and SEB (8/125-6.4%). 14 of enterotoxigenic strains (14/125-11.2%) synthesized SEA and SED, while 7 of them (7/125-5.6%) synthesized SEB and SEC, 5 of them (5/125-4%) synthesized SEA and SEB, 1 of them (0.8%) synthesized SEB and SED, SEC and SEB (0.8%), SEC and SED (0.8%), SEC and SEE (0.8%). Only one of the strains (0.8%) was identified to synthesize SEB, SEC and SED as shown in table 19 (Normanno et al., 2007).

Among the 125 analyzed strains in which (63/125) belong to the Human ecovar followed by the Ovine (29/125), Non-Host-Specific (22/125), Bovine (9/125) and Poultry-like (2/125) ecovars. The Bovine and Ovine biotypes were presented in dairy products in which the Bovine strains were found in foods come from cow and sheep origin. The SEs most frequently detected from the Bovine biotypes strains were SED (5/9-55.5%), followed by SEA (2/9-22.2%), SEB (1/9-11.1%) and SEC (1/9-11.1%) (Normanno et al., 2007).

The Ovine biotypes strains not only presented in dairy products; however, 14 out of 29 (48%) originated from goat milk and sheep and cheese. While the remaining comes from cow milk (5/29-0.55%), mozzarella (3/29-10.3%) and ricotta cheese (2/29-6.89%). While for Ovine biotypes strains were able to produced SEC alone or association (14/29-48.2%) in which these SEC seems to be the SE specific of this biotypes, followed by SED (7/29-24.1%) and SEA (5/29-0.55%) as shown in table 18 and 19 (Normanno et al., 2007).

22 (17.6%) of the NHS strains were found mainly synthesized SED (15/22-68.1%), SEA (4/22-18.1%), SEB (2/22-9%) and SEA-SED (1/22-45%). These productions of enterotoxigenic strain by NHS strains mainly presented in cow milk (12/22-54.5%), mozzarella cheese (6/22-27.2%), followed by ricotta cheese (2/22-9%), ovine meat (1/22-4.5%) and fresh sausage (1/22-4.5%). For the Poultry-like strains were found in a poultry product and milk accounted for (2/125-1.6%), it most often related to both human and pork meat that usually present in pork meat or noses of industrial worker as well. These strains only synthesized one type of SED, alone or in association (Normanno et al., 2007).

There are positive correlation between biotyping and the ecological origin of the strains in the 100 S. aureus strain that was isolated from a food study by Normanno et al. (2007). These studies provided further evidence to support the relationship between biotype and strains origin as the human strains can detected in handling food (Normanno et al., 2007).

Biotypes isolated from cow, sheep milk and poultry meat are due to the poor hygiene during the production primarily from hand contamination. SEA and also SED are the most common detected SEs produced by Human biotype strains in which indicated that the main factor in causing SFP is contamination of food and also suggested SEA and SED are the common SEs involved in SFP. Table 18 and 19 show 9 Bovine strains and 2 Poultry-like strains that produce SEs. The most frequently SEs produced by the Human Biovar strains were the SEA and SEB, both alone or in association, while Ovine biovar associated with SEC and SED in the NHS biovar (Normanno et al., 2007).

Another research was done by Huong et al. (2010) to investigate the prevalence and genetic diversity of S. aureus and staphylococcal enterotoxins (SEs) in different types of ready-to-eat food in Hanoi, Vietnam. A total of 212 food samples (nem chao, sticky rice, fermented meat, ice cream, grilled pork meat, rice cake and also milk) were collected from different locations of hawkers and food outlets in Hanoi, Vietnam from July to September, 2004. Brain Heart Infusion Broth was used to incubate each isolates of S. aureus of food sample for about 12 hours at 37 o C. And then subjected to centrifugation for 20 min at 3000 rpm and the supernatant was collected. A reversed commercial SET-RPLA was carried out by using a SET-RPLA kit to detect the type A, B,C, and D enterotoxins present in the food samples.

During the ribotyping of S. aureus, standard treatment of lysozyme, sodium dodecyl sulfate 9 (10%), proteinase K and CTAB/NaCl were used to extract and purify the chromosomal DNA of bacteria. The DNA was further purified by using Phenol-chloroform-isoamyl alcohol (24:1) and phenol-chloroform-isoamyl alcohol (25:24:1). HindIII was used as restriction enzyme to digest the chromosomal DNA in order to produced banding pattern in agarose gel electrophoresis and transferred to a nylon membrane and then hybridized with digoxigenin labeled synthetic oligonucleotide probe. The size of each band was determined and the data were collected as 0 (negative) or 1 (positive) (Huong et al., 2010).

The result of Huong et al. (2010) showed that 45 out of 212 (21.2%) of the food samples collected and processed were found to be contaminated with S. aureus. All types of ready-to-eat food product including milk samples showed the highest prevalence (17/48-35.4%) of contamination, followed by ice cream samples (12/48-25%), grilled pork meat (32/48-21.8%). While the S. aureus contamination of the remaining food items fell within the range 12.5% to 16.3%.

This study reflected high occurrence of S. aureus present in the ready-to-eat food samples in Vietnam mainly due to lack of quality control checking for the pathogen. Cross-contamination and also improperly washed container were the main cause of S. aureus contamination. In these studies, Huong et al. (2010) had collected high population of S. aureus bacterium present in milk, rice cake and nem chao. These fall within the range of 1 to 103 CFU/g of all the different types of food samples as shown in the table 20. The detection of SEs in ready-to-eat food samples between 105 and 107 CFU/g of S. aureus is necessary (Huong et al., 2010).

Huong et al. (2010) had reported that 18 out of 45 S. aureus strains isolated produced the targeted classical SEs which was detected by using RPLA enterotoxin typing as shown in the table 20 indicated there was a high prevalence of enterotoxin producing S. aureus strains in different food samples. Among the samples examined, the higher prevalence of S. aureus in milk samples were associated with a high frequency of strains that containing SEs and 9/17 strains were shown positive by the RPLA serotyping (Huong et al., 2010).

Sticky rice of isolated strains did not produce any SEs, while the abundance of SE positive strains fell within the range of 25% to 33.3%. Only one of the strains possessed SEA+SEB among the SE producing S. aureus, while SEB was the highest accounting for 44%, followed by the SEC (33.3%) and SEA (16.7%). SED was not found in any S. aureus strains and SEB was found predominantly distributed in ready-to-eat food samples in this study (Huong et al., 2010).