Pathogenic Escherichia coli found in food

International Journal of Food Microbiology, 12 (1991) 103-114 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50

103

FOOD 00368

Pathogenic Escherichia coil found in food Orjan Olsvik 1, Yngvild Wasteson a, Arve Lund 1 and Erik Homes

1.:

I Department of Microbwlogv and Immunology, Norwegian College of VeterinaO" Medicine, Oslo, Norway and 2 Dynal AS, Oslo, Norway (Received 19 September 1990; accepted 25 September 1990) Key words: Escherichia coil; Pathogenic; Food; Detection

The bacteria constituting the species Escherichia coil are commonly found in the intestinal flora of man and animals, and were until late 1950s recognized as non-pathogenic normal cohabitants. However, certain strains might induce disease, and E. coli should therefore be regarded as a potential pathogenic organism. The pathogenic strains can cause distinct disease syndrome as different diarrheal diseases, wound infections, meningitis, septicemia, artherosclerosis, hemolytic uremic syndrome and immunological diseases such as reactive and rheumatoid arthritis. Several different groups of diarrhea-inducing strains are known. The enterotoxigenic E. coil (ETEC) strains produce one or more of toxins from the heat-labile and the heat-stable enterotoxin families. These strains possess specific adhesion fimbria for intestinal attachment and colonization. Some enteropathogenic E. coil strains (EPEC) produce one or more of the cytotoxins, but adhere also to intestinal cells interfering with the electrolyte transport system. The group of strains possessing invasive properties are designated enteroinvasive E. coli (EIEC). Recently. the enterohemorrhagic E. coil (EHEC) strains have been identified and shown to produce one or more of the cytotoxins (vero-cytotoxins. shiga-like toxins). Food originating from warm-blooded animals may be contaminated with E. coil but contamination from human sources are more common for food involved in outbreak of disease. In general, strains causing disease in animals do possess other colonization factors than those found on human pathogenic strains. EIEC strains are, like Shtgella, only known to induce disease in man. However, both healthy and sick cattle are suspected to be a major reservoir for EHEC strains, and several outbreaks have been associated with consumtion of meat or meat products. Cheeses have been the source of outbreaks of both ETEC and EIEC in Europe and the USA, while water seems to be a major source for the different diarrheic E. coil strains affecting children and tourists in the 3rd world. Strains causing non-enteric disease are less known as being transmitted to humans with food as a vector, but the importance of some of these diseases, should implicate further research on what role food plays in spreading these organisms. The recipient of the potential pathogenic E. coil through food, the humans, are also of different risk of contracting diseases. The factors of most importance seems to be the immunological and nutritional status of the host. and recently E. coil strains previously regarded as non-pathogenic, have been implicated in cases of disease in persons with AIDS. Because the species E. coil consists of both pathogenic and non-pathogenic strains, and due to the fact that the latter constitute a part of normal intestinal flora, differentiation between pathogenic and non-pathogenic strains becomes very important. Traditional cultivation from food samples, using selective enrichment broths, has been shown to give growth preference to strains of environmental origin compared to strains of human origin. Isolated strains have a tendency to loose extrachromosomal genes

Correspondence address: O. Olsvik. Department of Microbiology and Immunology, Norwegian College of Veterinary Medicine, Post Box 8146 DEP, 0033 Oslo 1, Norway.

104 encoding important pathogenicity factors during such selective enrichment. It is of the greatest importance to be able to identify the pathogenicity markers such as toxins and adhesins, and differentiate these strains from non-pathogenic strains originating from animals or the environment. This is a difficult task. but genetic probing and PCR technologies can be useful tools in making this type of diagnosis efficient. and provide information of epidemiological importance, specially with respect to routes for spreading pathogenic E. coli strains with food as a vector.

The bacterium and the reservoirs

The bacteria constituting the species Escherichia coil are commonly found in the intestinal flora of man and animals, and were until late 1950s recognized as non-pathogenic normal cohabitants. However, certain strains may induce disease and E. coli should therefore be regarded as a potential pathogenic organism (Sussman, 1985). Because the bacterium's main reservoir is the intestinal tract of warm-blooded animals, the presence of strains in other environments like food and water is used as an indicator of contamination from the previously indicated reservoirs. Under suitable conditions, E. coli can grow in environments like water and food (Doyle and Padhye, 1989). Pathogroups E. coil is among the most frequently isolated bacteria in human and veterinary diagnostic microbiology, but the causative role of the bacterium in a number of diseases is probably yet not recognized or fully understood. The pathogenic strains can cause disease syndromes as distinct as different diarrheal diseases, urinary tract infections, wound infections, meningitis, septicemia, artherosclerosis, hemolytic uremic syndrome, and immunological diseases like reactive and rheumatoid arthritis (Doyle and Padhye, 1989; Sussman, 1985). For several of these diseases in man, the bacterium is among the most frequent cause of infection. E. coli is therefore a multipotent pathogen with ability to cause disease in several body systems. Several different groups of diarrhea-inducing strains are known (Table I). The enterotoxigenic E. coil (ETEC) strains produce one or more toxins from the heat-labile and the heat-stable enterotoxin families (Wasteson et al., 1990). These strains possess specific adhesion fimbria for intestinal attachment and colonization. Such adhesion fimbria are often species specific and are probably one of the most important factors preventing inter-species transmission of enterotoxigenic E. coli strains (Parr?.' and Rooke, 1985). Some enteropathogenic E. coil strains (EPEC) produce one or more of the cytotoxins, but the most important virulence factor might be their ability to adhere to and interfere with the electrolyte transport system in the intestinal epithelial cells. The EPEC strains form a serological group of certain O and H antigens (Doyle and Padhye, 1989, Senerwa et al., 1989a, b). The E. coil strains possessing invasive properties are designated enteroinvasive E. coil (EIEC). These strains often have atypical biochemical characteristics, being lactose-negative or with slow lactose reaction and are often regarded as non-motile. They are often antigenically related to Shigella species and form a distinct serologi-

105 TABLE I Classification of diarrhegenic E. coll. their virulence factors and type of diarrhea Term

Virulence factors

Type of diarrhea

Enterotoxigenic (ETEC)

Enterotoxins: LTIa, LTIb. LT lla. LTIIb STIa, STIb, STII, Specific adhesion fimbria

Rice water

Enteroinvasive (EIEC)

Invasive to intestinal epithelial cells (SLTs?)

Bloody with mucus

Enteropathogenic (EPEC)

Localized adhesion (invasive?) to intestinal epithelial cells (LA. DA) and Some strains SLT I a n d / o r SLT II

Rice water, mucus

Enterohaemorrhagic (EHEC)

Shiga-like toxins: SLT 1 a n d / o r SLT II Colonization factors

Painful diarrhea Hemolytic uremeic syndrome (HUS)

cal group. Invasive E. coil strains have so far only been found to naturally induce disease in humans, similar to Shigella spp. Most of these strains also possess genes encoding one or more of the cytotoxins from the Shiga-like toxin family (SLT I and SLT II) (Doyle and Padhye, 1989; Wasteson et al., 1990). The Shiga-like toxins have also been identified in the enterohemorrhagic E. coli (EHEC) strains. These strains are dominantly of the serogroup O157:H7, but strains of other serogroups have recently been shown to possess the same toxins and to induce disease with the same severe clinical symptoms. A high mortality rate is associated with the hemorrhagic-uremic syndrome (HUS) in young and elderly patients (Wachsmuth et al., 1990). The edema disease in pigs is caused by E. coli strains producing a variant of cytotoxin from the Shiga-like toxin family, SLT IIv. These strains are of different serotypes than the EPEC and EHEC strains, and the clinical symptoms are also different (Wasteson et al., 1990).

Species specificity and transmission routes Food originating from warm-blooded animals may be contaminated with E. coli, but contamination from human sources is more common for food involved in outbreaks of disease. In general, ETEC strains causing disease in animals possess other colonization factors than those found on human pathogenic strains, and are therefore unlikely to induce disease after having crossed the species barrier (Fig. 1). It has also been shown that the heat-labile enterotoxin produced by strains of porcine and human origin are slightly different and have different affinity to the proposed intestinal cell receptor, GM1 (Olsvik et al., 1983). The Shiga-like toxin

106

D? Humans Animals

same species

Food Water Fig. 1. Suggested contamination routes of enteric, pathogenic Escherichia coil strains. The A routes show how humans can be contaminated from sick humans, or through food contaminated with strains from sick humans. The B routes show the same for animals. The C route is the possible contamination of humans with strains from animals through food, and D shows the possible direct contamination of humans directly from sick animals. family have also been shown to have different receptor affinity, although this has not been shown to be of importance for development of disease in the different species. EIEC strains are, like Shigella, only known to induce disease in man (Sussman, 1985). However, both healthy and sick cattle are suspected to be a major reservoir for EHEC strains, and several outbreaks have been associated with consumption of meat or meat products (Wachsmuth et al., 1990). Food as a vector

Cheese has been the source of outbreaks of both ETEC and EIEC in Europe and the U.S.A., while water seems to be a major source for the different diarrheic E. coli strains affecting children and tourists in the 3rd world (Doyle and Padhye, 1989). Strains causing non-enteric disease are less known to be transmitted to humans with food as a vector, but the importance of some of these diseases should initiate further research on what role food plays in spreading these organisms (Doyle, 1985). One should, however, not totally exclude the fact that enterotoxigenic strains have been shown to produce enterotoxins at 22 and 4 ° C temperatures in broths and in food samples (Olsvik et al., 1982; Olsvik and Kapperud, 1982). Intoxication with preformed enterotoxin, like staphylococcal food poisoning, could therefore be a possible way for these organisms to induce diarrhea through foods. Food involved in outbreaks

A survey of the literature shows that different types of food including water have been associated with outbreaks of diarrhea due to E. coil (Doyle and Padhye, 1989).

107

Water was regarded as the source of a large outbreak of ETEC in Crater Lake National Park, Oregon, U.S.A., in 1975. The water supply was suspected to be contaminated from sewage (Rosenberg et al., 1977). A year later, two outbreaks of ETEC, in Japan, were associated with contaminated well water. The drinking water reservoirs of cruise ships have been reported to be the source for several ETEC outbreaks (Doyle and Padhye, 1989). It is, however, difficult to demonstrate whether the patients have been contaminated with strains from drinking water, or from water used in preparing food. Several outbreaks of diarrhea induced by E. coli have been associated with ingestion of cheese. A multistate outbreak of ETEC was investigated in 1983, and French brie cheese was suspected to be the source (MacDonald et al., 1985). Other foods, like turkey mayonnaise, crab meat and scallops, have been reported to be the source of EPEC outbreaks (Doyle and Padhye, 1989). Even consumption of salads containing raw vegetables have been suspected to be the cause of ETEC-diarrhea in members of a medical conference in Mexico (Merson et al., 1977). Several other incidents of ETEC have been linked to certain restaurants or cafeterias (Doyle and Padhye, 1989). For the classical EPEC summer epidemics in hospital during 1920-1950, bottle-feeding of milk has been regarded as a particular risk factor (Doyle and Padhye, 1989). During the last years meat has been found to be a major source for outbreaks of EHEC, and hamburgers and beef sandwiches have most commonly been involved (Wachsmuth et al., 1990).

ETEC, EIEC, EPEC and EHEC strains isolated from food In addition to the food associated with outbreaks of E. coil-induced diarrhea, several investigations of food and water as a potential vehicle have demonstrated the presence of pathogenic strains. ETEC strains have been isolated from drinking and cooking water in several developing countries (Doyle and Padhye, 1989). An investigation in Brazil showed that 5, 7.5 and 10% of beef, hamburgers and sausage, respectively, purchased in a supermarket possessed ETEC strains (Doyle and Padhye, 1989). Such strains were not found in 78 commercial cheese samples from the U.S.A., but a few ETEC strains were isolated from milk products in the F.R.G. (Doyle and Padhye, 1989). Notermans (personal communication) recently tested 274 strains from pork, 248 strains from beef and 278 strains from poultry meat, and found none of them to produce heat-labile enterotoxin, or to possess genes for such toxin. EIEC and EPEC strains have only sporadicly been isolated from food not involved in food poisoning, as EHEC strains have been isolated from beef (Doyle and Padhye, 1989). The recipient Humans and animals have a different risk of contracting potential pathogenic E. coli through food. The factors of most importance seem to be the immunological and nutritional status of the host. Naturally the infectious dose is of importance, and strains growing in foods stored under inappropriate conditions, will be presented to the recipient in far larger doses than strains from food stored under safe conditions.

108 Detection and characterization

Because the species E. coli consists of both pathogenic and non-pathogenic strains, and due to the fact that the latter constitute a part of the normal intestinal flora, differentiation between pathogenic and non-pathogenic strains becomes very important. Traditional cultivation from food samples using selective enrichment broths has been shown to favor strains of environmental origin compared to strains of human origin (Hill et al., 1985). Isolated strains have a tendency to lose extra chromosomal genes encoding important pathogenicity factors during such selective enrichment (Hill and Carhse, 1981; Tables II and III). A recently developed technology, immunomagnetic separation (IMS), using small uniform polystyrene super-paramagnetic beads coated with specific antibodies against surface antigens of the target bacterium, has appeared to be a promising tool for detection of pathogenic bacteria (Gilhuus Moe et al., 1989; Olsvik and Skjerve, 1989). The beads are mixed with the crude sample and after incubation for 10-30 rain, the beads with the bound live bacteria are extracted by using a magnet. The immunomagneticaUy isolated fraction can be washed several times before it is plated on suitable agar plates. Both polyclonal and monoclonal antibodies have been employed with success. The antibodies can be linked to the beads either directly or indirectly using beads pre-coated with anti-mouse or anti-rabbit antibodies. Linking the specific antibody to biotin, streptavidin-coated beads will bind these antibodies specifically. The bacteria bound to the beads appear to grow well, and the technique gives several advantages. First, the target bacteria is separated from the contaminated environment, and is concentrated from being distributed in a large volume to an immunomagnetically purified volume suitable for cultivation on plates or in broths. Growth-preventive reagents in the sample are also removed from the bacteria, which improves cultivation. However, one must have antibodies directed against the surface of the target organism, and these antigens must not be found free in too high concentrations in the sample solution. The negative effects of selective media, as demonstrated by Hill et al. (1985) are not seen using IMS technology. The time used for making a right diagnosis can also be reduced. IMS takes only minutes compared to selective enrichment that could go over days. Fig. 2 shows E. coli bacteria of serotype O157:H7 attaching to Dynabeads coated with polyclonal rabbit anti H7 sera. Enterotoxigenic E. coli possessing the K88 antigens have been isolated using IMS (Lund et al., 1988). The technique has been employed

TABLE II How heterogeneousare E. coil isolated from food? Unique isolates a

Food type Ground beef

No. of samples 6

Total isolates 61

Pork sausage

7

63

39 31

Other

5

29

14

" Biotypes or strains possessing different plasmids were regarded as unique (From Hill et al.. 1985).

109 TABLE III Percentage of LT-positive colonies using the BAM method Strain no.

Before enrichment

After enrichment

K334C2 TD427C2 E2534 1184/68

80 66 80 24

59 53 9.2 1.0

(From Hill et al., 1985.)

for isolation of both Salmonella and Listeria spp. from food (Olsvik and Skjerve, 1989; Skjerve et al., 1990). It is most important to be able to identify the pathogenicity markers like toxins and adhesins, and differentiate these strains from non-pathogenic strains originating from animals or the environment (Sussman, 1985). Genetic probing using polynucleotide fragments appears to be an interesting diagnostic approach (Wachsmuth et al., 1986; Smith et al., 1985; Table IV). During the last years, these polynucleotides have been sequenced, and oligonucleotides produced in an automatic synthesizer have shown that the probing technology soon might be a routine diagnostic

Fig. 2. Escherichia coli O157:H7 strains attaching to Dynabeads M280 coated with sheep anti-rabbit IgG with polyclonalrabbit anti-H7 IgG as secondary antibody.

110 TABLE IV

Polynucleotide probes for diarrhegenic E. coil Pathogroup

Virulence factor

Polynucleotide fragment

Enterotoxigenic

LT I LT I1 ST la ST Ib ST 11

850-bp HinclI fragment of pEWD 299 800-bp HindlI-Pstl fragment of pCP 2725 154-bp Hinfl fragment of pRIT 10036 215-bp HpalI-EcoRI fragment from pSIM 004 460-bp Hinfl fragment from pCHL 6 1750-bp EcoRl fragment of pW 22 1-kb BamHI-Sall fragment from pMAR 22 450-bp Pstl fragment from pW22 3.5-kb HindlI fragment of PCVD 419 l145-bp B a m H l fragment of pJN 37-19 842-bp Pstl-Smal fragment of pNN 110-8 750-bp Hincll fragment of p746 850-bp Aval-Pstl fragment of p363

Inv

Enteroinvasive En teropathogenic

LA (EAF) DA 'EHEC' SLT I SLT 1I VT 1 VT 2

En terohemorrhagic

(From Echeverria et al., 1990; Olsvik et al., 1990a.)

tool in microbiological laboratories (Echeverria et al., 1990; Olsvik et al., 1990; Table V). The latest technique used for identification of pathogenic E. coli strains is the polymerase chain reaction (PCR). Using PCR one might be able to identify genes encoding virulence factors directly in food or stool samples without cultivation of microorganisms at all. Fig. 3 shows a recent development of the PCR technique using a set of nested primers and magnet isolation of the PCR-generated fragments. The signal can be instrumentally quantitated (Olsvik et al., 1990a, b; Rimstad et al., 1990; Uhl6n et al., 1990). The genetic approach appears to give the routine diagnostic microbiological laboratories new tools and making this type of diagnosis both sensitive and efficient (Smith et al., 1985, Wachsmuth et al., 1986). However, during the last years, plasmid profiling and chromosomal restriction endonuclease digest pattern analysis have already shown that genetic methods can provide the laboratory with important

TABLE V

Oligonucleotides for diarrhegenic E. coil Pathogroup

Virulence factor

Sequence

Enterotoxigenic

LTI ST IA ST IB

Enteroinvasive Enteropathogenic Enterohemorrhagic

Inv

5'C ACC TCT AAG TAG TlrG TI'G TTA ATG T 5'GAA CTT TGT AAT CCT GCC TGT GCT GGA TGT 5'GAA TTG TGT AAT CCT GCT TGT ACC GGG TGC 5'CCA TCT ATT GAG ATA CCT GTG 5'TAT GGG GAC CAT GTA TTA TCA 5'CAG TTA ATG TGG TGG CGA AG 5'CT TCG GTA TCC TAT TCC CGG

EAF SLT I SLT II

(From Echeverria et al., 1990; Olsvik et al., 1990a.)

111

5- Native bacterial DNA 1st. PCR using primer P1 and primer P3

P1 5" P1

5"

Y~P P2

P1

P3

~ B

5"

32p P2 P1

2nd. PCR using biotinylated (B) primer Pl and 32p-labelled primer P2

B5"

Magnetic separation using streptavidin (SA)-coated Dynabeads

Fig. 3. Double-nested triple primer PCR with magnetic isolation of generated labelled fragments (Olsvik et al., 1990b).

I A

B

II C

D

A

B

Ill C

D

A

B

C

D

,k

Fig. 4. Plasmid profiles (I) of four enterotoxin-producing Escherichia coil strains isolated from dogs, hybridization with an enzyme-labelled oligonucleotide for ST (II) on Southern blots of I, and IIl the restriction endonuclease digest patterns of plasmids from I.

112 epidemiological i n f o r m a t i o n c o n c e r n i n g E. coli strains i n d u c i n g diarrhea (Mayer, 1988). Plasmid profiles a n d probes have been used to identify E P E C strains causing an o u t b r e a k in a n u r s e r y ward for pre-term n e o n a t e s in N a i r o b i (Senerwa et al., 1989a). Plasmid profile analysis was also used to differentiate E T E C strains isolated from dogs with diarrhea. D N A - D N A h y b r i d i z a t i o n with enterotoxin-specific probes o n S o u t h e r n - b l o t s of p l a s m i d profiles gave, in a d d i t i o n to restriction e n d o n u c l e a s e digest p a t t e r n analysis of the plasmids, valuable epidemiological i n f o r m a t i o n (Wasteson et al., 1988; Fig. 4). T h e enzyme-labelled oligonucleotides used in these experiments have a great potential for being used in r o u t i n e diagnostic laboratories, as radio-isotope labelling requires certain facilities, have a limited shelf-life, a n d are for certain developing countries, j u s t not available.

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