Presentation on theme: "Gram-negative rod, facultative anaerobe Normal flora of the mouth and intestine Protects the intestinal tract from bacterial infection Assists in digestion."— Presentation transcript:
Gram-negative rod, facultative anaerobe Normal flora of the mouth and intestine Protects the intestinal tract from bacterial infection Assists in digestion Produces small amounts of vitamins B 12 and K Colonizes newborns GI tract within hours after birth E. coli 0157:H7 on the other hand produces toxins that cause severe damage to the lining of the intestine, and produce illness in humans.
4 O antigen Somatic (on LPS) 174 antigens H antigen Flagella 57 antigens K antigen Capsule and or fimbria antigen 80 antigens There are more than 700 different serotypes of E. coli Distinguished by different surface proteins and polysaccharides Serotypes Antibody – antigen rxn
Shiga toxin-producing (STEC), also called Enterohemorrhagic (EHEC) E. coli O157 serogroup Non-O157 serogroups Enteropathogenic (EPEC) Enterotoxigenic (ETEC) Enteroinvasive (EIEC) Other types, less well characterized
E coli O157 Pathogenic E. coli are classified according to virulence factors(aea,hly,stxs) Intimin,Hemolysin,,Shiga toxin mechanisms of diseases(adhering) clinical signs(HUS) Hemolytic-uremic syndrome presence of O and H antigens (174 O, 57H)
The Shiga toxin-producing E. coli (STEC) pathotype refers to those strains of E. coli that are capable to produce one class of cytotoxins called Shiga toxin. Shiga toxin-producing E. coli (Sub set) that cause hemorrhagic colitis and hemolytic uremic syndrome are commonly knows as enterohemorrhagic E. coli (EHEC).
The STEC are also named verotoxin producing E. coli (VTEC). The name Shiga toxin (Stx), derived from similarity to a cytotoxin produced by Shigella dysenteriae serotype 1. verotoxin (VT), means they are cytotoxic to Vero cells.
Shiga toxin-producing E. coli (STEC) serotypes commonly isolated from beef cattle or their products. Therefore beef cattle or it is products can cause severe gastrointestinal disease in humans, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS).
Excretion Re-colonization Environment Death Secondary habitat: water, soil, sediment cool, fluctuating nutrient limiting survival SOURCES of E coli Primary habitat: large intestine, recto-anal junction? warm, constant nutrient rich vigorous growth
Excretion Re-colonization Environment Death Has HACCP led to a reduction in human incidence?
An estimated 73,000 cases occur annually in the United States. That number is probably many times multiplied due to poor reporting in less industrialized countries.
In February 2004, the Okinawa Prefectural Chabu Health Center and the Okinawa Prefectural Institute of Health and Environment reported multiple cases of E. coli 0157:H7 in a single family possibly after eating contaminated meat bought at a U.S. military commissary in Okinawa. Positive ID of E. coli 0157:H7 was made on 02/17/2004, after OCHC gathered samples of the frozen meat and analyzed it by means of pulse field gel electrophoresis (PFGE),. The samples were sent to Pulse Net USA for comparison to U.S. isolates, and matched E. coli 0757:H7. To exclude the possibility of contamination after opening of the meat packages, the U.S. Naval Hospital, Okinawa, Jp. Obtained unopened packages, leftovers, and the samples of human isolates, and compared all the samples.
Example of a case study Outbreak of Shiga toxin-producing E. coli O157 associated with consumption of watercress, United Kingdom, August to September 2013 N Launders () 1, L Byrne 1, N Adams 1, K Glen 1, C Jenkins 2, D Tubin-Delic 3, M Locking 4, C Williams 5, D Morgan 1, An increase in the number of cases of Shiga toxin-producing Escherichia coli O157 PT 2 stx2 infection was reported in the United Kingdom on 9 September Of the 19 cases, 13 were interviewed, of which 10 reported consuming watercress purchased from one retailer. The retailer recalled pre-packed bagged salads containing watercress on 12 September. The descriptive epidemiology was supported by a case–case study performed after control measures were implemented. The outbreak strain was intimin (eae)positive and haemolysin (hylA) positiv e
SerogroupsNo. outbreaks O111 (one outbreak also had O157)10 O1213 O263 O452 O27, O103, O104, O1531 each O26 and O1211 Green shows most common serogroups of sporadic cases
STEC toxin profileHUS (n= 21) No HUS (n=271) Only Shiga toxin 15%68% Shiga toxin 2 (+/- Shiga toxin 1) 95%32% Total100% Overall, 61% of human non-O157 STEC produced only Shiga toxin 1 Brooks, JID 2005 Isolates with clinical information submitted to CDC,
Food VehicleNo. outbreaks Salad bar1 Salad and ice1 Berries1 Milk1 Cider1 Punch1 Unknown5 N = 11
Pyramid of Surveillance Exposed to STEC Person becomes ill Person seeks care Specimen obtained Clinical lab tests for STEC STEC isolated Reported to health department & CDC
Being educated about foods that hold a certain risk to consumers is very important in preventing outbreaks of E.coli as well as other food borne pathogens. Get off to a CLEAN start: One of the best ways to prevent the spreading of and illness is hand washing. This needs to be done before and after food handling as well as when switching between different foods. CHILL food and stop bacteria cold: The danger zone for bacterial growth is between degrees F. Chilling foods does not kill the bacteria it only stops growth. Cooking kills. SEPARATE don’t cross contaminate: Raw meat should be placed on the bottom shelf in the fridge so it cannot drip onto other foods. When shopping, storing or preparing food, raw meat should be placed away from ready to eat foods. COOK safely: Cooking meat to the safe temperature, 160 degrees F, kills E.coli. Hamburger can turn brown before it is at this safe temperature. The look, color, or feel of the meat is not a test for doneness, only trust the thermometer.
Meat Science 93 (2013) 463–468
Sources of Carcass Contamination 1- Hide Cattle hides is an important source of microbial contamination of carcasses ( McEvoy et al., 2000 ). E.coli O157:H7 and non-O157:H7 STEC can get it is way from cattle hides to the carcass ( Barkocy-Gallagher et al., 2003, Nostosijevic et al 2008).
2- Feces Many surveyed studies suggested that more than 20% of cattle shed VTEC in their feces and it might that these animals are the principle source of contamination ( Elder et al. 2000; Smith et al. 2001). The carcasses could be contaminated with VTEC when the gut contents or fecal materials get in contact with meat surfaces.
E. coli O157 Unusual feature: does not ferment sorbitol streak stool specimen onto plate containing Sorbitol-MacConkey (SMAC) medium select clear colonies (others are pink) O157 strains agglutinate when O157 antisera is added Non-O157 STEC Lack unusual features, look like good E. coli
25 gm. hide, feces or meat 225 ml M- TSB+ NOVOBIOCIN homogenization incubation 6 hrs at 41,5 o C Concentration of Escolar 0157 capture onto immunomagnetic particles washing with sterile wash buffer +
Incubation at 37 o C for 24 hrs Incubation at 37 o C for 24 hrs CT-SMAC medium Incubation at 37 o C for 18h-24h Incubation at 37 o C for 18h-24h Confirmation of pure colonies by indole formation, biochemical (MUG) and serological with antiserum (O157,H7) Confirmation of pure colonies by indole formation, biochemical (MUG) and serological with antiserum (O157,H7) Identification by PCR; Stx1, Stx2, Hly, eae,O157,H7 Identification by PCR; Stx1, Stx2, Hly, eae,O157,H7
Table 1: Number of samples with presumptive colonies of E.coli O157:H7 on CT-SMAC and CHROM agar. Sample type No. animals tested No. positive samples on CT-SMAC No. positive samples on CHROM agar Carcasses (9.4 %) Hides (13.3 %) Feces (12.2 %) Total isolates
Confirmatory testNo. (%) of positive isolates Latex O (100) Latex H7 55 (87) Spot indole63 (100)
Table 2: PCR confirmation of O157 and H7 latex agglutination positive isolates. Type of gene No. of agglutination positive isolates No (%) of positive isolates rfbE (O157) 6351 (83) Flich7 (H7) 5550 (91) rfbE + Flich7(O157+H7) 5550 (91)
Source of isolates number of isolates AE(aea)HLYSTX1STX2 STX1+STX 2 NO STX Carcasses14 (7.8%)14 (100%) 9 (64%)1 (7%)4 (28.5%)0 (0%) Hides19 (10%)19 (100%) 13 (68%)3 (15.8%)2 (10.5%)1 (5.3%) Feces17 (8.3%)17 (100%) 8 (47%)2 (11.8%)5 (29.4%)2 (11.8%) Total 5050 (100%)50(100%30 (60%)6 (12%)11 (22%)3 (6%)
Figure 3: Multiplex PCR for detection of virulent and toxigenic gene for E.coli O157:H7. Electrophoresis analysis (2% agarose gel) of PCR patterns for E.coli O157:H7 detection. Lane M= DNA ladder marker (Promega, USA). Lane 1 = positive control E.coli O157:H7 (ATCC # 43895). Lane 4, 6, 8, and 10 = positive result for (hly, Stx1, aea, Stx2,) with expected band size 166, 210, 397, 484 respectively. Lane 2, 3 = negative control. Lane 5, 7, and 9 No sample.
PREVALENCE ON HIDE The 10% prevalence rate of E.coli O157: H7in hides comes in agreement with other studies reporting an E. coli O157 occurrence in hide that commonly varied from % (Borhom et al., 2002, Tutenel et al. 2003b).
FECAL PREVALENCE E. coli O157:H7 fecal prevalence was 8.3% and this again comes in agreement with many studies that found a fecal prevalence in cattle at slaughter or at the farm that ranges from 5 to 17% ( Laegreid et al., 1999; Bonardi et al., 2001; Chapman et al., 2001; Conedera et al., 2001; Meyer-Broseta et al., 2001; Paiba et al., 2002).
The Prevalence of E.coli O157:H7 in Dressed Beef Carcass A 7.8% prevalence was higher than 0.2% reported In the U.S ( FSIS, 1994 ). Or 0.47% and 1.4% reported in the U.K ( Richards et al., 1998; Chapman et al., 2001). But closed to 12% in Italy (Bonardi et al., 2001).
The prevalence of E.coli O157:H7 on beef sample under study were 10%, 8.3%, and 7.8% for hides, feces and carcasses respectively. These prevalence rates are closely related and no significant differences were observed between contamination level of carcasses, hides and feces. This seem to be quite logical as the main source of E.coli is the fecal material which found it is way to animal external surface in a way or another.
Contamination of carcasses with VTEC can occur when gut contents or fecal matter come into contact with meat surfaces. These observations comes in close agreement with other findings in that a significant correlation do exist between the prevalence of E. coli O157 in feces and hides and carcass contamination ( Chapman et al 1994; Elder et al 2000; Bonardi et al 2001., Barkocy-Gallagher et al 2003).
Therefore the main sources of possible carcass contamination source in abattoir are both of hides or fecal content. However possible cross contamination from utensils and employers has not to be neglected. This might be apparent from following observation recorded in this study, where in two cases the pathogens were recovered from carcasses only, but not from hides or feces.
The distribution of E.coli O157:H7 among hides and carcasses depends mainly on level of hygienic precautions taken during slaughtering; hide removal evisceration and carcass dressing. This could be shown when six animals revealed presence of E. coli O157 from intestinal contents but not from either hide or carcasses.
The apparently high 7.8% prevalence on carcasses surfaces reported in this study is most likely related to no good hygienic measures applied in studied abattoir.
TOXIGENESITY OF ISOLATES Out of 47 toxigenic isolates, 30 (64%) isolates harboring Stx1; 6 (13%) Stx2; and 11(23%) reveled both toxin. Different studies showed that Shiga toxins 2 producing strains of EHEC are more virulent than Stx1 or Stx1 and Stx2 producing EHEC ( Donnenberg, 2002 ), and being more frequently associated with human disease complications ( Boerling et al., 1999 ).
Non- pathogenic E.coli O 157:H7 strains (without toxins factors) have also been identified from feces or beef carcass by other researchers (Cerqueira et al., 1999; Guyon et al., 2001; Rogerie et al., 2001; Genevieve et al., 2005).
The minimum inhibitory concentration (MIC) values for 40 chosen E.coli O157:H7 isolates : 1- All isolates were susceptible to (Gentamicin, Ampicillin, Tetracycline, Ciprofloxacin) % of the isolates were resistant to three antibiotics (Erythromycin, Vancomycin and Neomycin) % were multi resistant to four antibiotics (Erythromycin, Vancomycin, Doxycycline,Neomycin). 4- 5% resistant to five antibiotics (Erythromycin, Vancomycin, Doxycycline, Neomycin, Streptomycin).
The results of present study indicated that E. coli O157:H7 were susceptible to gentamicin and ciprofloxacin, which come in agreement with local Jordanian isolates from sheep and goat (Tarawneh et al., 2008 ) and other studies in USA among E. coli O157:H7 isolates from humans, cattle, swine and food, where all E. coli O157:H7 isolates, regardless of the source of isolation were susceptible to gentamicin and ciprofloxacin ( Schroeder et.al, 2001; Steve et.al, 2005 ).
all E.coli O157:H7 tested were also susceptible to tetracycline which again comes in parallel with Dontorou et al.,2003, testing three isolates obtained from ewes’ milk, fresh sausages and swine intestines in Greece.
The uses of IMS showed increased sensitivity of the isolation method. E.coli O157and E.coli O157:H7 latex agglutination test are highly sensitive for confirmation of presumptively identification of E.coli O157:H7.
The results of this study may help in establishing a risk based analysis systems for E.coli and developing criteria for routine microbiological food standardization.
1- Further studies could follow to determine the prevalence of E. coli O157:H7 in other animal’s species from different farms and slaughterhouses 2- A good hygienic practices or HACCP system should be applied equally at all levels along the food chain, especially in slaughterhouses to overcome the spread of E.coli O157:H7 among non carrier animals. 3- It becomes necessary to add additional barriers or hurdles to control the presence of E.coli O157:H7 throughout the food chain.
3- Further studies are needed to determine other E.coli serotypes and shiga toxins subtypes. 4- Implementation of a strict policy for use of antimicrobials is in need to control the emergence of antimicrobial resistant pathogens. . 6- Establish national record and documentation system for food borne outbreaks concerning E. coli O157:H7.