COMPARISON OF RIDA®QUICK VEROTOXIN/O157 TEST, VEROCYTOTOXICTY ASSAY AND PCR IN DETECTION OF SHIGA TOXINS IN CULTURES OF HUMAN STOOL, ANIMAL FAECES AND MEAT PRODUCTS

COMPARISON OF RIDA^®QUICK VEROTOXIN/O157 TEST, VEROCYTOTOXICTY ASSAY AND PCR IN DETECTION OF SHIGA TOXINS IN CULTURES OF HUMAN STOOL, ANIMAL FAECES AND MEAT PRODUCTS

 ZIDAN S.A^*; TARABEES, R^** and HASSANIN Z.H.^***

^* Department of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, Sadat City University, Al Menofia, Egypt

^** Departememt of Bacteriology, Mycology, and immunology, Faculty of Veterinary Medicine, Sadat City University, Al Menofia, Egypt.

^*** Department of food Hygiene and control, Faculty of Veterinary Medicine, Sadat City University, Al Menofia, Egypt.

Email: shrifzidan@yahoo.com

INTRODUCTION

The Gram-negative bacterium, Escherichia coli is a prominent member of the bacterial microbiota of the environment and in the feces of many species of birds and mammals. Five categories of E. coli have been well associated with diarrhea in several epidemiological studies (Nataro & Kaper, 1998) enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic    E. coli (ETEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC) which mostly regarded as Shiga toxin-producing E. coli (STEC).

The production of Shiga toxins (Verotoxins) is a characteristic trait of STEC. Shiga toxin-producing Escherichia coli (STEC), also called Verotoxin-producing E. coli (VTEC), were first described by Konowalchuk et al. (1977) by their cytotoxic activity on African green monkey kidney (Vero) cells (Konowalchuk et al., 1977). STEC of serotype O157:H7 were linked to cases of Haemorrhagic Colitis (HC) and the consumption of STEC- contaminated meat of bovine origin for the first time in 1982 (Riley et al., 1983; Karmali et al., 2010).

EHEC are capable of initiating life threatening illnesses, particularly in those with immune deficiency, young children and the elderly. The main sources of infection are contaminated, raw or insufficiently heated foods of animal origin, e.g. meat and dairy products, Food-borne outbreaks of STEC disease appear to be increasing and,when mass-produced and mass-distributed foods are concerned, caninvolve large numbers of people (Mohammed, 2010). The reservoir for EHEC is the feces of cattle, sheep and goats. These microorganisms can enter food during the processing of meat and dairy products if hygienic conditions are inadequate (Kudva et al., 1997; Mohammed, 2012).

The drastic increase in the incidence of food infection caused by E. coli demands reliable and rapid methods of detection. In addition to traditional culture methods, immunological techniques are becoming more useful due to their improved specificity and sensitivity (Melton-Celsa et al., 2002).RIDA^®QUICK Verotoxins is an immunochromatographic screening test based on the immune flow principle.

The RIDA^®QUICK Verotoxin O157 test is an immunochromatographic rapid test intended to be used in food analysing laboratories for the qualitative detection of Verotoxins (Shiga-like toxins) 1 and 2 from Verotoxinogenic E.coli (including E. coli O157:H7). This test has been validated and received AOAC(association of analytical communities) approval for detection of Verotoxins 1 and 2 from isolated Verotoxin-producing E.coli(including E. coli O157:H7). The test is also intended to be used in clinical laboratories for the qualitative identification of Verotoxins 1 and 2 (Shiga-like toxins 1 and 2) produced by E. coliisolated in cultures derived from clinical stool specimens and aids in the diagnosis of diseases caused by enterohemorrhagic E. coli  infections.

MATERIALS and METHODS

Isolation of STEC from collected samples:

This study included 623 samples collected from 4 sheep flocks (214 faecal samples), human (209 stool samples) beside 200 samples from different meat products products (40 luncheon, 40 Beef burger, 40 Minced meat, 40 Fresh sausage and 40 Beef kofta. Samples were obtained from animals, patients or products in clean sterile containers then transported with the minimum of delay to the laboratory of animal hygiene and zoonoses department.

A small mass of faecal sample was injected into 5 ml of Tryptic Soy Broth while 25 g of meat product samples were added to 225 ml of Tryptic Soy Broth and incubated overnight at 37 ^oC. Subculture from Tryptic Soya broth was performed on Sorbitol MacConkey Agar (SMAC) with cefixime and tellurite (CT) to obtain the suspected colonies of the bacteria. The obtained colonies were prepared for VCA to detect STEC. Positive samples were confirmed to be STEC by serotyping and PCR reaction to determine the type of Stx. (Konowalchuk et al., 1977).

Vero cell assay of the suspected E. coli strains

The cytotoxicity of the suspected E. coli isolates for vero cells was determined by using tissue culture supernatant and thereby detecting only high level of producers of these cytotoxins based onKonowalchuk et al. (1977).

This test was carried out in 96 well tissue culture plates. 90µL of sterile physiological saline was added to each of the test wells, while 50µL of the physiological saline was added to the negative control wells. 60 µL of the bacterial lysates was added to each well. 50µL of RPMI medium containing 10% calf serum, 2mM L-glutamin, 100 U penicillin/ml and 100 µg streptomycin /ml were added to each one of the test wells. A suspension of vero cells was prepared and 50 µL of this suspension was seeded in each well of the test wells. 50 µL of 1% SDS solution was added to each of the positive control wells. The plates were incubated at 37^oC in 5% CO2 atmosphere, observed daily by using inverted microscope for detection of cell lysis and vacuolation.

Serotyping E. coli isolates

Serotyping of E. coli samples that were positive in Vero cell assay was performed in the (serology unit, Animal Health Research Institute, Dokki). When this reagent is mixed with E. coli strain which has antigens correspondent to the reagent, the antigen antibody reaction occurs to produce agglutination. This reaction is macroscopically observed to determine each serotype (Edwards and Ewing, 1972). Positive Vero cell assay E. coli strains were streaked on Agar media (nutrient agar medium, heart infusion (HI) agar medium, slant or plate medium), previously autoclaved at 121^°C. Pure cultures of the selected     E. coli strains were prepared for determination of their O-groups using slide agglutination test.

Detection of Stx1, Stx2 and eae genes in STEC isolated from faecal samples using Multiplex PCR:

The multiplex PCR was performed as described by Brian et al. (1992) at the laboratory of infectious diseases and Internal medicine, faculty of Veterinary Medicine, Sadat city University.

Genomic DNA extraction:

Chromosomal DNA was isolated from STEC isolates using protienase K extraction. Pure cultures of bacteria in the trypticase soya broth were streaked on MacConkey s agar plates. The inoculated plates were incubated at 37^oC. A single colony from the plate was subcultured into 300 ml of TSB and incubated for 24 hours at 37^oC. After incubation, bacteria were collected by centrifugation at 13000 rpm for 2 minutes and the sediment was suspended in equal volume of Tris-EDTA buffer. Furthermore, 100µl of lysozyme solution (10 mg/L), 100 µl of protienase K enzyme (0.3 mg/L) and 1% dodecyl sulphate were added. The DNA lysate was extracted once with chloroform/isoamyl alcohol (24:1, ratio by volume), then extracted with phenol/ chloroform/isoamyl alcohol (25:24:1, ratio by volume), the aqueous phase was mixed with isopropanol alcohol and incubated at -20C for 30 minutes. The precipitated DNA was spooled out, rinsed in 70 % ethanol and dissolves in 0.5 ml of Tris EDTA buffer.

PCR Amplifiction:

Polymerase chain reaction (PCR) was performed in a thermal cycler model (Biometra). The PCR reaction mix (50 µL) for each sample was consistent of: 10 µL extracted DNA, 3 µL primers mix (0.5 µL from every primer); 1 µL deoxynucleoside triphosphate (dNTP-mix); 5 µL 10x buffer; 1 µL Taq-DNA polymerase enzyme (5000 U/ml) and 30 µL ultra pure deionized water. The reaction mixture was overlaid with mineral oil and was incubated in the thermal cycler as follows: The first initial cycle: 94^oC for 4 minute (initial denaturation). The consequent 35cycles: 94^oC for one minute (denturation), 60^oC for one minute (annealing) and 72^oC for one minute and 30 seconds (extension). The final extension step at 72^oC for 7 minutes then kept at 4^oC (hold temperature) (Brian    et al., 1992).

Table 1: Showing Primer sequences for stx1, stx2 and eae genes.

Detection of amplified products:

10 µl from each PCR amplification were electrophoresed on agarose gel and stained with ethidium bromide solution (0.5 µg/ml), visualized under an ultraviolet transilluminator and photographed. Visible bands of appropriate size of 366 bp (Stx1), 282 bp (Stx2) and 579 bp (eae) were considered positive. Molecular mass markers (100 bp ladder) were electrophoresed simultaneously.

RIDA®QUICK Verotoxin / O157 Combi test (dipsticks):

For in vitro diagnostic use. The RIDA®QUICK Verotoxin / O157 Combi test (RBiopharm, Darmstat, Germany) is an immunochromatographic rapid test for qualitative determination of Vero toxins (shiga toxins) and of Escherichia coli serotype O157 in enrichement cultures.

Modified Tryptic Soya broth (mTSB) containing Mitomycin C (Klie et al., 1997)was inoculated with the test strain and incubated at 37°C for 18 to 24 hours for the production of shiga toxins (Mitomycin C serve as enhancer to stimulate Stx production and release in the culture fluid. after growth, the samples was centrifuged (1500g for 15 minutes) and 1ml of the supernatant fluid is diluted 1:1 with a sample buffer. The mixture was used directly for the test.

The RIDA-QUICK test stick was placed into the test sample and the reaction occurs by the lateral flow of specific antibodies bound to coloured latex particles. Antibodies bind themselves to specific antigens (Stx and/or O157 LPS) if present in the sample and the antigen-antibody complex flows via the membrane to specific collection bands where they are fixed by immobilized, specific antibodies.

RESULTS

Table 2: Comparison of the results of cultivation on SMAC medium with VCA, multiplex polymerase chain reaction (PCR) and RIDA®QUICKVerotoxin / O157 Combi test for detection of STEC infection in sheep, human beings and meat products.

Table 3: Serogroups of verotoxin- producing E. coli isolated from sheep and man.

Table 4: Serogroups of verotoxin- producing E. coli isolated from meat.

Table 5: Occurrence of some virulence genes in serogroups of Shiga toxin-producing E. coli (STEC) isolated from sheep.

Table 6: Occurrence of some virulence genes in serogroups of Shiga toxin-producing E. coli (STEC) from man.

Table 7: Occurrence of some virulence genes in serogroups of Shiga toxin-producing E. coli (STEC) isolated from meat products.

Table 8: The sensitivity and specificity of the RIDA®QUICK Verotoxin / O157 Combi test for detection of samples containing Stx1 and/or stx2 and the O157 antigen:

Fig. (1): Ethidium bromide stained Agarose gel showing the specific amplification products with specific size (366, 282 and 579 bp) primers specific for Stx1, Stx2 and eae genes respectively for STEC isolates from sheep. Lane (M): MW marker = 100 bp DNA ladder (Promega). Lane (1): Positive Control (E. coli O157H7 provided by Animal Health research Institute, Egypt). Lane (2- 6): E. coli isolates from sheep.Lane (6): Lane (7): Negative Control.

Fig. (2): Ethidium bromide stained Agarose gel showing the specific amplification products with specific size (366, 282 and 579 bp) primers specific for Stx1, Stx2 and eae genes respectively for STEC isolates from man. Lane (M): MW marker = 100 bp DNA ladder (Promega). Lane (1): Positive Control. Lane 2-6     E. coli isolates from man. Lane 7: Negative Control.

Fig. (3): Ethidium bromide stained Agarose gel showing the specific amplification products with specific size (366, 282 and 579 bp) primers specific for Stx1, Stx2 and eae genes respectively for STEC isolates from meat products. Lane (M): MW marker = 100 bp DNA ladder (Promega). Lane (1): Positive Control. Lane (2-5)isolates from meat products. Lane (6): Negative Control.

Fig. (4): Intact vero cell monlayer incubated with control medium.

Fig. (5): Cytotoxic effect of shiga toxin cotaining bacterial lysate of STEC on vero cells.

DISCUSSION

Among the E. colihuman pathogens, Verotoxin (Shiga-like toxin) forming strains (VTEC) have gained special importance in recent years. The group of enterohaemorrhagic E. coli(EHEC) with its highly pathogenic serovars 0157:H7, 026, 0103, 0111, 0145, and other strains are of particular concern. Production of Verotoxins is the most common criteria for the detection of this group of bacteria and can be classified into two main categories Verotoxin 1 (VT1, SLT1, Stx1) and Verotoxin 2. EHEC strains may produce either VT1 or VT2 only or both VT1 and VT2 simultaneously (Akiba et al., 2005).

Healthy cattle and sheep are recognized as a major natural reservoir of EHEC and other STEC strains. There are more than 100 serotypes of STEC which have been also isolated from other animals such as goats, pigs, goats, deer, horses, dogs and birds (Kudva et al., 1997 and Gyles 2007).

The results recorded in Table (2)showed that from 623 samples collected from sheep (214 samples), human (209 samples) and meat products (200 samples), 77(12.06 %) samples yielded positive culture SMAC-CT. Further identifications of the isolated colonies were performed by Vero cells cytotoxicity assay which revealed that 37 of E. coli isolates (48.05 %) were verotoxin producing E. coli. The results obtained in this study agreed withRamotar et al. (1995)who reported that SMAC was positive for only 30 % of verocytotoxin-positive samples. On other hand, another study indicated that SMAC resulted in the isolationof E. coli O157 from 80 % of fecal samples which were positivefor Stx by direct cytotoxicity (Ritchie et al., 1992).

In the present study, the most common serogroups of STEC in sheep and man were recorded in table (4). They were O157 (31.82 %), O103 (13.63 %), O20 (13.63 %) and O111 (9.09 %), O8 (9.09 %); O26 (4.54 %); O78 (4.54 %); O126 (4.54 %); O168 (4.55 %); and O1 (4.55 %). While in meat products (luncheon, Beef burger, Minced meat, Fresh sausage and Beef kofta) O111 (26.6%, O26 (20.0 %); O91 (13.3 %); O103 (20.0 %); O145(13.3%) and O86 (0.66%) were the most prevalent (Table, 3). These results are in agreement with Mohammed, (2012) whodemonsrated that non O157 STEC were the most prevalent in meat products sold at Mansoura city, Egypt.

Fecal contamination of meat and milk are important routes through which these pathogens enter the food chain,together with the contamination of vegetables with  animal manure. There are many vectors that can be used to transfer E. coli on and/or into meat products. The feces of the animal can be transferred through hides, carcass, contaminated equipment, non proper hygienic practices, airborne contamination, rodents, insects, and other carrier animals (Mohammed, 2012).

The use of PCR with specific primers for Stx1, Stx2 and eae genes revealed the presence or absence of these genes in the tested isolates. The obtained results recorded in Tables (5,6&7) revealed that isolates ofO26, O103 and one isolate O157 from human; O103 (2 isolates), O126 from sheep as well as O26, O103 and O86 from meat products had only Stx1 while, E. coli O78, O111 O168, O8 (2 isolates), O20 (3 isolates) and O157 (2 isolates) from sheep; O1, O111, 2 isolates O157 from human as well as O111,O26, O91,O103, O145 from meat products  had only Stx2. On the other hand, isolates that possessed both Stx1 and Stx2 were 2 isolates O157 from sheep and O111, O103&O145 from meat products Previous studies have shown that the virulence of STEC for humans may be related to the type of Shiga toxin which is produced by the bacteria. Of the different Shiga toxins, Stx2 (stx2) was found to be related with high virulence and was significantly associated with STEC strains from BD and HUS patients (Paton and Paton, 1998).

Concerning diagnosis of STEC by detection of Shiga toxins, a number of indicator media for the identification of some STEC strains and types have been developed, as well as immunological, genetic and cell culture toxicity assays for Stx detection. The detection of Shiga toxins is the only way to identify all members of the STEC group which consists of strains with different phenotypes and serotypes (Bettelheim and Beutin, 2003).

The Vero cell toxicity assay (VCA) is used as a gold standard for detection of Stx production as it is the most sensitive. However, its specificity must be confirmed in neutralization assays with antisera directed against Stx. As the VCA is laborious, time consuming and demands a specifically designed laboratory and specially trained personnel there is a need for STEC detection systems which are rapid, reliable, standardized and easy to employ (Paton and Paton 2003).

The data presented in Table (2) illustrated  that vero cells cytotoxicity assay showed that 37 of E. coli isolates out of 77(48.05 %) isolates were verotoxin producing E. coli. In PCR study, 37 (100 %) of the 37strains were confirmed to be STEC whereas 22 strains (63.63%) were positive for the VT2 gene, 9 (27.27%) were positive for the VT1 gene and 6 (9.09%) isolates were positive for both genes. In comparison to Vero cells cytotoxicity, the sensitivity of PCR were 100 %. PCR and RIDA®QUICK Verotoxin / O157 Combi test were compared with Vero cytotoxicity assay for a number of reasons. Firstly, The profound sensitivity of Vero cells to Stx which was first observed by Konowalchuk et al. (1977), Secondly, the cytotoxicity for this cellline remains the "gold standard" for confirmation of putativeSTX-producing isolates (Byomi, 1995).

The sensitivity of the RIDA®QUICK Verotoxin / O157 Combi test for detection of samples containing Stx1 and/or stx2 and the O157 antigen was illustrated in Table (8). The sensitivity is the probability that the assay will be positive when the infection is present. The specificity is the probability that the assay will be negative when the infection is absent. They were calculated according to(Agnamey et al., 2011).

We have evaluated the RIDA Quick test for its sensitivity with all the known variants of Stx1 and/or Stx2. For the evaluation, we employed 37 STEC strains of different STEC isolates from sheep, human and different meat sources. The RIDA Quick was compared for its sensitivity with the PCR and Vero cell cytotoxicity test which was used as a gold-standard for toxin activity. The thirty seven  strains of STEC that were positive for VCA and PCR were tested  by RIDA®QUICK Verotoxin / O157 Combi test and  the results showed that 33(89.18%) of them were positive (table, 2 and table,6). The results were very good for the Stx1 (9/9 positive), Stx2 (19/22 positive) and Stx1& Stx2 (6/6). Nearly similar results were obtained by (Melton-Celsa et al., 2002) who detected 5 of 7 positive Stx2 strains (71.4 %).

Detection of the O157 antigen by RIDA®QUICK Verotoxin / O157 Combi test was demonstrated in Table (8). The sensitivity of the assay for the E. coli O157 antigen was tested with 7 strains belonging to sheep (4 strains) and man (3strains). The RIDA Quick assay detected all E. coli O157 strains tested. Strains belonging to the non-O157 serogroups did not react for the O157 antigen in the RIDA®Quick assay indicating a specificity of 100%.These results were in agreement withPark et al. (2003)whodescribedThat  immunochromatographic test of STEC  demonstrated a sensitivity of 100% for the detection of clinical isolates without any cross-reactivity with other enteric organisms

In Conclusion: Sheep are important reservoir of STEC to man  besides, Faecal contamination of meat products by animal manure  is important route through which STEC pathogens enter the food chain and the newly developed  RIDA^®QUICK Verotoxin/O157 test  is a highly sensitive, specific when compared to PCR and VCA. Moreover, it is more rapid method to determine the presence of Stx and serogroup O157 in enrichment cultures. Further studies are needed to investigate the test performance in comparison to Stx targeting ELISA.

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