Transcript Jordan University of Science and Technology. Department of
JORDAN UNIVERSITY OF SCIENCE AND TECHNOLOGY PROF AKRAM AL ABBOODI
E. COLI
O157:H7 CHARACTERISTICS, PATHOGENICITY, SPREAD AND CONTROL
E. COLI
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.
MANY STRAINS
Serotypes Antibody – antigen rxn 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 4
E. COLI
THAT CAUSE HUMAN GASTROINTESTINAL ILLNESS 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 toxin factors( aea,hly,stxs ) Intimin,Hemolysin,,Shiga 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 serotype 1.
cytotoxin produced by Shigella dysenteriae 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).
SOURCES of E coli Primary habitat: large intestine, recto-anal junction?
• • •
warm, constant nutrient rich vigorous growth Secondary habitat: water, soil, sediment
• •
cool, fluctuating nutrient limiting
•
survival Re-colonization Death Excretion Environment
Excretion Re-colonization Death Environment
Has HACCP led to a reduction in human incidence?
INDIRECT CONTACT (ENVIRONMENTAL) TRANSMISSION
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.
EXAMPLE OF A CASE STUDY
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 2013.
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
SEROGROUPS OF 23 NON-O157 STEC OUTBREAKS, U.S., 1990-2007
Green shows most common serogroups of sporadic cases
Serogroups O111
(one outbreak also had O157)
O121 O26 O45 O27, O103 , O104, O153 O26 and O121 No. outbreaks 10 3 3 2 1 each 1
PERSONS WITH HUS RARELY HAD NON-O157 STEC STRAINS THAT PRODUCED ONLY SHIGA TOXIN 1 Isolates with clinical information submitted to CDC, 1983-2002
STEC toxin profile Only Shiga toxin 1 HUS (n= 21 ) 5% No HUS (n=271) 68% Shiga toxin 2 (+/- Shiga toxin 1) 95% 32% Total 100% 100% Overall, 61% of human non-O157 STEC produced only Shiga toxin 1
Brooks, JID 2005
FOOD VEHICLES IN NON-O157 STEC OUTBREAKS, U.S., 1990 2007
N = 11
Food Vehicle No. outbreaks Salad bar 1 Salad and ice Berries Milk Cider Punch Unknown 1 1 1 1 1 5
Pyramid of Surveillance
Reported to health department & CDC STEC isolated Clinical lab tests for STEC Specimen obtained Person seeks care Person becomes ill Exposed to STEC
CONTROL
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 40-140 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 doneness, only trust the thermometer.
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
PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY OF ESCHERICHIA COLI O157:H7 ON BEEF CATTLE SLAUGHTERED IN AMMAN ABATTOIR AKRAM R. ALABOUDI ,TAREQ M. OSAILI, MAJDI RAHAHLAH B DEPARTMENT OF PATHOLOGY AND ANIMAL HEALTH, FACULTY OF VETERINARY MEDICINE, JORDAN UNIVERSITY OF SCIENCE AND TECHNOLOGY, IRBID, 22110, JORDAN 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.
CLINICAL LAB TESTING FOR STEC
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
CT-SMAC medium Incubation at 37 o C for 18h-24h Incubation at 37 o C for 24 hrs Confirmation of pure colonies by indole formation , biochemical and serological with antiserum (O157,H7) ( MUG) Identification by PCR; Stx1, Stx2, Hly, eae ,O157,H7
GROWTH ON SELECTIVE MEDIA
Table 1: Number of samples with presumptive colonies of
E.coli
O157:H7 on CT-SMAC and CHROM agar.
Sample type Carcasses Hides Feces Total isolates No. animals tested No. positive samples on CT-SMAC 180 17 ( 9.4 %) 180 24 ( 13.3 % ) 180 22 (12.2 %) No. positive samples on CHROM agar 17 ( 24 ( 22 9.4% ) 13. 3%) (12. 2%) 540 63 63
CONFIRMATION OF
E.COLI
O157:H7
Confirmatory test Latex O157 Latex H7 Spot indole No. (%) of positive isolates
63 ( 100 ) 55 ( 87 ) 63 ( 100 )
CONFIRMATION OF
E.COLI
CONVENTIONAL PCR O157:H7 USING
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 )
63
Flich7 ( H7 )
55
rfbE + Flich7( O157+H7 )
55 51 (83) 50 (91) 50 (91)
TOXIGENICITY OF 50 PCR CONFIRMED
E. COLI
O157: H7
Source of isolates number of AE(aea) isolates HLY STX1 Carcasses
14 (7.8%) 14 (100%) 14 (100%) 9 (64%)
STX2
1 (7%)
STX1+STX NO STX 2
4 (28.5%) 0 (0%)
Hides Feces T otal
19 17 (10%) (8.3%) 50 19 17 (100%) (100%) 19 (100%) 17 (100%) 13 (68%) 8 (47%) 3 2 (15.8%) (11.8%) 50 (
100%)
50 (
100
% 30 (
60%
) 6 (
12%)
2 5 (10.5%) (29.4%) 1 2 (5.3%) (11.8%) 11 (
22
%) 3 (
6%
)
Figure 3: Multiplex PCR for detection of virulent and toxigenic gene for analysis (2% agarose gel) of PCR patterns for
E.coli
E.coli
O157:H7. Electrophoresis 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 reporting an
E. coli
that commonly varied from
E.coli
O157: H7in hides comes in agreement with other studies O157 occurrence in hide 4.5-56% (Borhom et al., 2002, Tutenel et al. 2003b).
FECAL PREVALENCE
E. coli
O157:H7 ranges from fecal prevalence 5 to 17% 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 ( 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).
DISTRIBUTION OF
E.COLI
CARCASSES O157: H7 IN HIDES, FECES AND 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 as the main source of were observed between contamination level of carcasses, hides and feces. This seem to be quite logical
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 hides or fecal content contamination source in abattoir are both of from not to be neglected.
. However possible utensils and cross employers has 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 dressing.
depends mainly on level of hygienic precautions taken during slaughtering; hide removal evisceration and carcass This could be shown when six animals revealed presence of carcasses.
E. coli
O157 from intestinal contents but not from either hide or
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 complications ( ), and being more frequently associated with human disease Boerling et al., 1999 ).
6% NON TOXIGENIC ISOLATES
Non- pathogenic researchers
E.coli
O 157:H7 strains (without toxins factors) have also been identified from feces or beef carcass by other (Cerqueira et al., 1999; Guyon et al., 2001; Rogerie et al., 2001; Genevieve et al., 2005).
SUSCEPTIBILITY TO ANTIMICROBIAL AGENTS
The minimum inhibitory concentration ( MIC ) values for 40 O157:H7 isolates : chosen E.coli 1 All isolates were susceptible to ( Gentamicin, Ampicillin, Tetracycline, Ciprofloxacin ).
2 50% of the isolates were resistant Vancomycin and Neomycin ).
to three antibiotics ( Erythromycin, 3 25% were multi resistant to four antibiotics ( Erythromycin, Vancomycin, Doxycycline ,Neomycin ).
4 5% r esistant 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 goat Jordanian isolates from (Tarawneh et al., 2008 sheep and ) 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 Greece. ewes’ milk, fresh sausages and swine intestines in
CONCLUSION
The uses of IMS showed increased sensitivity of the isolation method.
E.coli
O157and
E.coli
O157:H7 latex agglutination confirmation of presumptively identification of
E.coli
test are O157:H7.
highly sensitive for
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 .
RECOMMENDATION
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
E.coli
to overcome the spread O157:H7 among non carrier animals.
of 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 antimicrobials of a strict policy for use is in need to control the of antimicrobial resistant pathogens.
of emergence .
6- Establish national record and documentation system for food borne outbreaks concerning
E. coli
O157:H7.