Environ. Sci. Technol. 2006, 40, 7141-7149
Occurrence of Escherichia coli O157:H7 and Other Enterohemorrhagic Escherichia coli in the Environment† MAITE MUNIESA,* JUAN JOFRE, C R I S T I N A G A R C IÄ A - A L J A R O , A N D A N I C E T R . B L A N C H Department of Microbiology, University of Barcelona, Diagonal 645, E-08028 Barcelona, Spain
Enterohemorrhagic Escherichia coli (EHEC) (O157 and other serotypes) are zoonotic pathogens linked with severe human illnesses. The main virulence factors of EHEC are the Shiga toxins, among others. Most of the genes coding for these toxins are bacteriophage-encoded. Although ruminants are recognized as their main natural reservoir, water has also been documented as a way of transmission of EHEC. E. coli O157:H7 and other EHEC may contaminate waters (recreational, drinking or irrigation waters) through feces from humans and other animals. Indeed, the occurrence of EHEC carrying the stx2 gene in raw municipal sewage and animal wastewater from several origins has been widely documented. However, the evaluation of the persistence of naturally occurring EHEC in the environment is still difficult due to methodological problems. Methods proposed for the detection and isolation of stx-encoding bacteria, ranging from the classic culture-based methods to molecular approaches, and their application in the environment, are discussed here. Most virulence factors associated with these strains are linked to either plasmids or phages, and consequently they are likely to be subject to horizontal gene transfer between species or serotypes. Moreover, the presence of infectious stx-phages isolated as free particles in the environment and their high persistence in water systems suggest that they may contribute to the spread of stx genes, as they are directly involved in the emergence of new pathogenic strains, which might have important health consequences.
Introduction Escherichia coli belongs to the Enterobacteriaceae and is an endogenous component of the intestinal microbiota of warmblooded animals (1). Although mainly harmless, there are situations in which E. coli can cause illness: (i) when uropathogenic E. coli leave the intestinal tract and enter the urinary tract; (ii) when the bacteria leave the intestinal tract through a perforation into the abdomen; and (iii) when certain pathogenic strains of E. coli transmitted by food or water are ingested (2). Regarding their virulent factors there are five classes of pathogenic E. coli: Enterotoxigenic E. coli (ETEC), which produces enterotoxins; Enteroinvasive E. coli (EIEC), characterized by the invasion of the colonic epithelium; Enteroaggregative E. coli (EAggEC), which is distinguished by †
This review is part of the Emerging Contaminants Special Issue. * Corresponding author phone:+34934039386; fax:+34934039047; e-mail:
[email protected]. 10.1021/es060927k CCC: $33.50 Published on Web 09/02/2006
2006 American Chemical Society
prominent auto-agglutination of bacterial cells; Enteropathogenic E. coli (EPEC), which causes a pedestal-like structure and produces attaching-and-effacing (A/E) histopathology; and enterohemorrhagic E. coli (EHEC), which causes severe abdominal cramps and bloody diarrhea (2).
Enterohemorrhagic E. coli Enterohemorrhagic E. coli (EHEC) causes hemorrhagic colitis and can produce complications associated with this infection, especially in children. Karmali et al. (3) first reported, in 1983, sporadic cases of hemolytic uremic syndrome (HUS), which follows gastrointestinal infection with Shiga toxin-producing E. coli. HUS is defined by a triad of features: acute renal failure especially in children, thrombocytopenia, and microangiopathic hemolytic anemia, occurring in 2-15% of cases, with a high mortality (4). EHEC strains infect with a very low infectious dose (1-102 CFU) (5) and colonize the gut by adherence to epithelial cells. EHEC has caused outbreaks in Canada, the United States, Europe, and Japan, and is also an important pathogen in some parts of the Southern hemisphere (4). The EHEC group is very diverse, and a broad range of O:H serotypes have been associated with human diseases. Serotypes O18, O26, O103, O111, O128, O138, or O157 (5) are the most commonly involved with pathogenicity. Serotype O157:H7 has been reported as the causative agent of a large number of serious infections (2).
Serotype O157:H7 In 1983 Riley et al. (6), described a previously rare serogroup O157:H7, which is the type most commonly associated with large outbreaks worldwide. The significance of E. coli O157:H7 as a public health concern was recognized in 1982 during an investigation into an outbreak of hemorrhagic colitis in the United States. This outbreak was caused by consumption of poorly cooked ground beef in a fast-food restaurant chain in the western U.S. From 1982 to 2002, a total of 350 E. coli O157 outbreaks were reported in the U.S. from 49 states, among which 325 outbreaks included a number of deaths. Serotype O157:H7 alone is responsible for more than 73,000 cases of disease per year (7) in the United States, and has been implicated in 250 deaths (2). In some areas of the United States, E. coli O157:H7 is one of the most commonly isolated pathogens after Campylobacter, Shigella, and Salmonella spp (8). Some studies suggest that O157:H7 may cause only 5080% of all EHEC infections, and because EHEC strains of serotypes other than O157:H7 are not routinely sought, the overall incidence of EHEC infections is difficult to estimate. VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Non-O157:H7 appears to be a more common cause of HUS in Europe (9).
EHEC Virulence Factors EHEC pathogenesis involves expression of toxin genes, along with several other factors. Shiga Toxins. One of the most important pathogenic factors in EHEC is the expression of one or both of the Shiga toxin genes (stx). The Stx family comprises a group of toxins similar to the one produced by Shigella dysenteriae serotype 1, for which reason they were first called “Shiga-like toxins” and later “Shiga toxins” (Stx) (2). The group contains two major toxins, called Stx1 and Stx2, which share approximately 55% aminoacid homology (10). While Stx1 is highly conserved, Stx2 has 11 distinct variants (10), with stx2c, stx2d, stx2e, and stx2f being the most frequently reported (10, 11). Shiga toxins are proteins formed by one catalytic subunit (A) of 32 kDa and five B subunits. The A subunit cleaves a single adenine residue from the 28S rRNA component of the eukaryotic ribosomal 60S subunit. The B subunits mediate binding to cells through interaction with globotriaosylceramide (Gb3) expressed on epithelial and endothelial cell membranes. The action of the toxin inhibits the activity of ribosomal RNA, causing protein synthesis to cease and producing cell death (11). Stx are produced in the colon and travel by the bloodstream to the kidney, where they damage renal endothelial cells and occlude the microvasculature through a combination of direct toxicity and induction of local cytokine and chemokine production, resulting in renal inflammation (9). Stx genes are encoded by temperate lambdoid bacteriophages, which lysogenize EHEC and remain integrated in the host chromosome. Stx-phages also carry some regulator genes (12-14), and are therefore responsible for toxin production and regulation (14). Stx-phages are activated by DNA-damaging agents such as UV light or quinolones, which in turn activate the bacterial SOS system. Specifically, the RecA protein facilitates autocleavage of the phage repressor, causing prophage induction. The stx genes are located downstream of the antiterminator Q, and upstream of the lytic genes S and R necessary for lysis and release of mature phage particles. The activation of the lytic cycle promotes transcription of the Q gene, and the action of the Q antiterminator protein produces activation of SR genes. During this process the stx genes, which are located directly upstream the SR genes, are also transcribed. The production of Stx is then linked to the replication cycle of stx-phages, and the release of Stx is dependent on the phage lysis genes (14). LEE Pathogenicity Island. A number of enteropathogenic bacteria, including both enteropathogenic E. coli (EPEC) and EHEC, insert their own receptor in the host membrane to adhere to the intestinal epithelium. In this process they cause attaching-effacing (A/E) lesions in the small or large intestine and produce diarrhea. EHEC isolates generate A/E lesions by a mechanism similar to that of EPEC and the required bacterial products are encoded within a pathogenicity island known as the locus of enterocyte effacement (LEE) (15). The A/E lesion is characterized by the loss of host cell microvilli (effacement) and intimate attachment of the bacterium to the host membrane on a pedestal of polymerized cytoskeletal elements (15). The LEE locus, which is not present in E. coli K-12, shows differences in length and location between different E. coli groups. In O157:H7 LEE comprises 47 ORFs organized in five operons (LEE1 to 5). The factor of the LEE cassette that promotes intimate adherence is the product of the gene eaeA, a 94-kDa outer membrane protein (OMP) called intimin (16, 17). The bacteria do not use endogenous receptors of the host cells, but produce a host epithelial receptor for intimin 7142
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named Tir (translocated intimin receptor), which is also encoded by LEE (16). The Tir/intimin association induces intracellular signaling and causes cytoskeletal changes in the intestinal cells. It generates the pedestal and the A/E lesions, finally leading to the destruction of the microvilli. The protein is secreted from the bacteria and it is dependent on the type III secretion system and other LEE-encoded proteins called Esp effector proteins (16, 17) for efficient delivery into the host cell. However, the role of Esp proteins in the formation and pathogenicity of the A/E lesion has not been identified. Not all serotypes producing Stx contain LEE and those LEE-negative commonly are not associated with human disease indicating a clear relationship between LEE and pathogenicity. However, some LEE-negative strains (e.g., O103:H21 strains) have been shown to produce illness, suggesting that other factors can also be involved in pathogenicity (17). Other serotypes (O26, O127, O15:H-, or O103:H2) carry LEE loci similar to the LEE locus described in O157, although with some differences (18). Genes Encoded in the Plasmid pO157. Most EHEC strains isolated from humans, especially O157, also carry large (>90 kb) plasmids encoding proteins involved in bacterial pathogenesis. Schmidt et al., (19) characterized the plasmid pO157 and described the presence of relevant pathogenicity factors: the enterohemorrhagic E. coli enterohemolysin (E-hly), which seems to provide iron by the hemoglobin-released production of hemorrhages; KatP catalase-peroxidase, as a part of bacterial defense mechanisms against oxidative stress; the extracellular serine protease EspP, encoding a protease that cleaves pepsin A; and human coagulation factor V, a gene with great similarity to genes of members of the type-II secretion pathway systems of Gram-negative bacteria used to secrete extracellular proteins; and an insertion with high homology with ToxB of Clostridium difficile (17, 19-20). Similar pO157 plasmids have been found in two different O157:H7 strains (7785 and O157 Sakai). Both plasmids are practically identical regarding their distribution and orientation of pO157 genes, although some differences indicate heterogeneous origins (17). Other Factors. Several other pathogenicity factors have been described. Among others are the presence of fimbrial adhesins that reach out from the bacterial surface and enable bacteria to adhere to host cells (7); the presence of Aerobactin, a siderophore used for Fe3+ chelation (16); non-LEE encoded proteins like NleA effector A protein, encoded in a prophageassociated pathogenicity island within the EHEC genome, or the autoagglutinating gene saa (21), an autoagglutinating adhesin isolated from a LEE-negative STEC. Other characteristics that are shared by most EHEC isolates, such as the high acid tolerance or the O157 LPS have been proposed as possible virulent factors; however their implication in infection is still questionable.
EHEC Reservoirs and Transmission Transmission of EHEC has been widely studied and well documented (2, 5, 16, 17, 22). Food-borne transmission of E. coli O157:H7 is the most important means of infection. Transmission is mainly linked to undercooked meat, and, during the 1982 outbreaks, the organism was cultured from a suspected batch of hamburger patties (20). Dairy products like milk and cheese have also been implicated as a vehicle for hemolytic-uremic syndrome (22) and there is evidence that cattle, among other animals, are the most important reservoir for the pathogen (16, 20). Vegetables have also been associated with some outbreaks, including unpreserved apple cider, a seemingly unlikely vehicle (23). Non-foodborne vehicles have also been involved in the spread of E. coli O157:H7, as described for secondary person-to-person contact or nosocomial infections (16).
TABLE 1. Reported Waterborne Outbreaks Caused by Ehec outbreak
occurrence
E. coli serotype
no. cases (deaths)
transmission route
ref
1989 1990 1990 1991 1992 1992 1993 1993 1994 1995 1996 1997 1998 1999 1999 1999 1999 1999 2000 2001 2002 2004 2004
Missouri Saitama (Japan) Shimane (Japan) Oregon Swaziland and South Africa Scotland (U.K.) Rotterdam (The Netherlands) Surrey (U.K.) New York Illinois Scotland (U.K.) Seina¨ joki (Finland) Wyoming New York Connecticu Scotland (U.K.) South Devon (U.K.) Washington Walkerton (Canada) Minessota Kentucky Cornwall (U.K.) British Columbia (Canada)
O157:H7 O157:H7 O26:H11 O157:H7 O157:NM O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O121:H19 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7
243 (4) 174 12 21 >100a 6 4 6 (1) 12 12 6 14 157 775 (2) 11 6 14 (1) 37 2,300 (7) 20 2 7 10
unchlorinated water supply drinking water natural water lake water untreated water paddling pool swimming water paddling pool lake water lake swimming beach drinking water lake water drinking water drinking water lake water unchlorinated water supply beach water lake water drinking water public beach untreated groundwater freshwater stream water spray/recreational park
25 43 46 39 52 27 30 31 62 35 50 32 48 49 38 47 33 37 44 29 28 34 42
a
Undetermined number of cases.
In this review we analyze water-borne transmission, which was first reported in two outbreaks (24, 25) and is now clearly considered as an important transmission pathway.
EHEC Waterborne Outbreaks; Occurrence in Contaminated Water Since 1985, waterborne transmission of EHEC strains has been identified as a probability in two unconnected human cases (26, 27). Microbiological, epidemiological, and environmental studies have associated different uses of water with human outbreaks: recreational, drinking, irrigation, and wastewater (Table 1). Cohort studies of affected people, together with sampling of environmental samples (feces from animals, soils, sludges, water supplies, etc.) from the surroundings where the outbreaks occurred, identified causative EHEC strains by molecular typing using mainly serological, pulsed-field gel electrophoresis, and DNA amplification of virulence genes. Recreational Waters. A high percentage of outbreaks associated with the use of contaminated recreational waters in the United States during 1971-2000 were related to pathogenic E. coli (6%). The outbreaks caused by E. coli O157: H7 were mainly associated with swimming in fresh water (lakes, ponds, and rivers) (28, 29). The source of contamination of recreational waters was related to the bathers themselves, but also to sewage discharges and wild or domestic animals (25, 26). Water is the third greatest known route of transmission after food-borne and person-to-person transmission (27). Outbreaks of E. coli O157:H7 all over the world are strongly related to swimming in lakes, ponds, rivers, or swimming pools (30-34). Poor microbiological conditions of bathing waters or unchlorinated water in swimming pools and paddling pools are usually associated with these outbreaks (35). Several studies reported molecular subtyping using pulsed-field gel electrophoresis performed on isolates from patients, recreational waters and potential fecal sources of contamination. Feces from humans, farm animals, or wildlife, municipal sewage or wastewater, slurries, or manure from animals have been indicated as potential sources of contamination (36, 37), but it is frequently impossible to identify
the source of the pollution. Non-O157 EHEC strains were also isolated from outbreaks related to bathing in lakes or beaches (38). E. coli O121:H9 was isolated from an outbreak of illness in a Connecticut lake community in 1999 (38). The attack rate was highest among bathers younger than 10 years old who were swimming in shallow water. Though E. coli O121:H9 was not isolated from the water after several days of testing, E. coli indicative of fecal contamination was identified from sediment and water samples taken from a storm drain that emptied into the beach area and from a stream flowing into the lake. It was concluded that transient local beach contamination with E. coli O121:H9 serotype had caused the outbreak. But, as in other situations, the lack of surveillance of stx genes in the HUS cases made it difficult to isolate a non-O157:H7 serotype. Routine surveillance for the presence of toxic genes may facilitate the detection of incipient outbreaks. In other swimming-associated outbreaks, E. coli O157:H7 was isolated with other pathogens, such a Shigella sonnei (39). Other recreational activities such as camping close to agricultural grounds accompanied by exceptional rainfall are also related to E. coli O157 outbreaks. Drainage water and mud have been suggested as transmissors of the pathogen from the environment to humans under these circumstances, via contaminated hands of campers, either directly from hand to mouth or via food (40). In these situations, only education in the correct use of recreational waters can reduce the risk of infections. Some infections have occurred at agricultural fairs where beverages made with fairground water were consumed. Several studies have suggested that attendance at summer agricultural fairs in the United States contributes to the seasonal peak in incidence of outbreaks (41). Structural problems and inadequate filtration or disinfection of recirculating water can increase the risk of infections, as described at children’s spray parks (42). Drinking Water. In the epidemiology of E. coli O157:H7 outbreaks in the United States (1982-2002), 10 of the 39 waterborne-related outbreaks were associated with drinking water (29). Though drinking water outbreaks were only 3% of all outbreaks reported during the period studied, it is important to note that they correspond to 15% of all outbreakVOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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related cases (1265 cases). E. coli O157:H7 contaminating the water supply from the well in a nursery school in Japan in 1990 caused a severe outbreak affecting 174 children (43). The use of an unchlorinated water supply was also responsible for a waterborne outbreak of E. coli O157:H7 that affected 243 people in Missouri at the end of 1989 (25). The number of cases decreased rapidly after residents started to boil water and after appropriate chlorination of the water supply. The largest reported outbreak associated with drinking water supplies took place in 2000 in the town of Walkerton in Canada with more than 2,300 cases and 7 deaths (44). The circumstances surrounding the outbreak led to a judicial inquiry into why the fecal contamination of the water supply occurred, and who was responsible. E. coli O157:H7 and Campylobacter spp. were found in a well. Environmental samples were taken from farms surrounding the wells. Later, the molecular subtyping and phage-typing of the E. coli O157: H7 and the Campylobacter spp. isolates from the farm closest to the well were found to be identical to those found in most of the human cases. Several unfortunate circumstances had combined to facilitate the water contamination and he subsequent outbreak: heavy rains accompanied by flooding, E. coli O157:H7 and Campylobacter spp. present in the environment, a well subject to surface water contamination and a water-treatment system that may have been overwhelmed by increased turbidity. Bacteria that contaminated the well could have come from cattle manure on this farm draining into the well with the rain. E. coli serotypes other than O157:H7 have also been associated with outbreaks caused by contaminated water supplies (45, 46). A similar spread from animals to humans through contamination of water supply was also reported in the Highlands of Scotland in 1999 (47). The distribution of an untreated and unprotected private water source in a rural area where animals grazed freely seemed to be the cause of the outbreak. All cases arose in visitors to the area who had limited exposure to the contaminated water. Permanent residents on the same supply were unaffected. Other studies, showing that unchlorinated water supplies had microbiological evidence of fecal microorganisms that contributed to chronic contamination of surface water, calculated that the attack rate among people consuming this unchlorinated water was significantly lower in town residents than in visitors. The lowest attack rate among the exposed residents was among healthy adults. Limited serological data supported the acquisition of partial immunity following long-term exposure in this subpopulation of healthy adults (48). The largest reported waterborne outbreak of E. coli O157: H7 in the United States was also a co-infection outbreak with Campylobacter jejuni affecting 775 persons in New York state following a county fair in 1999 (49). It seems clear that discharges of sewage into streamwater are the major causes of contamination of drinking water supplies and can lead to co-infections with other pathogens (50). Extreme meteorological and climatological conditions are considered a significant risk factor for water contamination. Heavy rainfall occurred several days before the E. coli O157: H7 and Campylobacter outbreaks in Walkerton (51). A large outbreak of E. coli O157 in 1992 in southern Africa was also related to extreme climatological and meteorological situations (52). Measures to avoid discharges of sewage into watersheds, appropriate potabilization treatments, and strict fulfillment of routine sanitation protocols are necessary to prevent occurrences. Irrigation Water. Soils fertilized with contaminated poultry or bovine manure compost or the use of contaminated irrigation water in agriculture have also been reported as transmission routes for E. coli O157:H7 to the vegetables grown in these fields. The consumption of contaminated raw vegetables such as lettuce or other leaf crops could 7144
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transmit the pathogen to humans. Studies spiking E. coli O157:H7 in compost and in irrigation water showed persistence for 154-217 days in soils receiving the compost or irrigation water, with E. coli O157:H7 spiked strains detected in lettuce and parsley for up to 77 and 177 days, respectively (53). It has also been suggested that pre-harvest crop contamination via contaminated irrigation water can occur through plant roots (54).
EHEC in Contaminated Water Origin of EHEC Occurring in the Water Environment. E. coli O157:H7 are excreted by both symptomatic and asymptomatic infected humans (55). Some individuals, for example those developing HUS, excrete O157 a couple of weeks after the onset of the symptoms (9). However, man is not the only origin of these bacteria. It has been shown that different animals excrete them. E. coli O157:H7 has been isolated in different percentages of fecal samples of cattle (56, 57), pigs (57), sheep (58), wild birds (59), and others. As in humans, EHEC O157 is excreted in feces of both sick and healthy animals. Though the fecal excretion of E. coli O157 by cattle is only transient, typically lasting 3 or 4 weeks (60), E. coli O157 can be repeatedly isolated from environmental sources on farms for periods lasting several years (60). Consequently, it may be expected that EHEC O157 reaches and contaminates receiving waters. Though fewer data are available, similar results can be expected with serotypes other than O157 (61), but most authors have focused on O157, probably because of its high relevance in EHEC infections and also because of the availability of specific methods for the detection of this serotype. Much more effort is then needed to evaluate the real extent of non-O157 serotypes, which show high incidence in other geographical areas. Numbers of EHEC O157 and others in the water environment will depend on the amounts that reach the receiving waters and on the persistence of the bacteria. Occurrence of EHEC in the Water Environment. There are few data about the occurrence of EHEC in waters related with an outbreak. Water samples for the enumeration of total coliforms and fecal coliforms have been taken in some E. coli O157 outbreaks. A wide range of values have been found: 10 to 102 CFU/100 mL for fecal coliforms and from 0 to 104 CFU/100 mL for total coliforms in bathing waters (36, 62), and 10 to 102 CFU/100 mL for total coliforms or 10 CFU/100 mL for E. coli in drinking water systems (47, 46). However, the quantification or even the isolation of E. coli O157 has seldom been achieved by traditional or molecular methods (31, 32, 48). Sanderson et al. (63) indicate that the coliform counts should not be used as a marker for E. coli O157 contamination since the results of such coliform counts in feed and water are not associated with prevalence of E. coli O157 in cattle feces or water. However, an estimation of viable E. coli O157 populations in surface water samples was proposed by assuming that the growth rates and lag times of water-borne E. coli O157 and coliforms are similar (64). This estimation has been assayed in watershed samples from Baltimore showing E. coli O157 concentrations of
