Pathogenic Escherichia coli in Latin America

Escherichia coli are Gram-negative bacteria found as normal commensal flora in the gastrointestinal tract. As a pathogen, E. coli are the most frequent causes of bacterial infections, including urinary tract infections, diarrheal disease, and other clinical infections such as neonatal meningitis, pneumonia and bacteremia. At least six different categories of pathogenic E. coli causing enteric infections have been identified and further characterized. In Latin America, as well as many other developing countries, diarrheal infections caused by E. coli remain an important cause de infant morbidity - mortality. Due to the appearance of the highly virulent strain of E. coli of serotype O157:H7 in the US and Canada in the 1980’s, and subsequently in other Latin American countries, there is an increase need for accurate testing for this and other pathogenic E. coli strains, substantially enhancing detection of virulent strains and, therefore, facilitating identification of sporadic E. coli infections and outbreaks.

The emergence and evolution of pathogenic Escherichia coli strains associated with diarrheal diseases have become a topic of active investigation in recent years due to the emergence of more virulent strains and the association of new serotypes with disease. Outbreak studies indicate that most patients with an intestinal E. coli infection develop mild, uncomplicated diarrhea. However, a significant risk exists that infections caused by highly virulent E. coli isolates, such as the enterohemorrhagic E. coli O157:H7, develop into serious and potentially lifethreatening complications, such as hemolytic uremic syndrome. The relative contribution of recombination events in the generation of new categories of pathogenic E. coli varies among the E. coli population, and it is represented by the wide variety of mobile elements found in different diarrheal strains (e.g. pathogenicity islands, phages, transposons, pathoadaptive mutations, etc). Understanding the population structure of pathogenic E. coli is important, since it impacts the effectiveness of molecular epidemiological studies. Such studies are needed to understand the increasingly recognized diversity of enterotoxigenic E. coli, a leading cause of pediatric and travelers’ diarrhea. In addition, factors underlying the emergence of enteroaggregative and atypical enteropathogenic E. coli strains associated with persistent diarrhea are unknown. Horizontal transfer of genetic elements that affect virulence of diarrheagenic E. coli strains and changes in global agricultural processes, as well as movement of humans and animals, may contribute to the complex natural history of diarrheagenic E. coli.

Enteropathogenic Escherichia coli (EPEC) comprise two groups of distinct organisms classified as typical EPEC (tEPEC) and atypical EPEC (aEPEC). tEPEC were leading infantile diarrheal agents in developing countries, whereas aEPEC prevailed in developed countries. Nowadays, tEPEC are less frequent while aEPEC are emerging enteropathogens of children and adults (including HIV-infected patients) in developing countries. EPEC infections can lead to severe secretory acute and persistent diarrheal diseases. Both EPEC groups contain the locus of enterocyte effacement (LEE), which encodes a Type Three Secretion System and various effector proteins that alter several signaling mechanisms of intestinal cells, leading to the development of attaching and effacing (A/E) lesions. The distinction between tEPEC and aEPEC strains is based on the expression of the bundle-forming pilus (BFP) adhesive-structure, which is restricted to tEPEC. Both EPEC groups lack the Shiga toxin genes of another A/E lesion-producing pathogen, enterohemorrhagic E. coli. aEPEC are much more heterogeneous than tEPEC in terms of phenotypic characteristics and virulence determinants. Humans are the only reservoir of tEPEC, whereas aEPEC strains may be found in humans and diverse animal species. Diagnosis is currently performed in research laboratories that use molecular methods to detect specific virulence properties that distinguish tEPEC from aEPEC strains. Antibiotics are indicated to treat more severe or persistent diarrheal cases, but resistance has been detected worldwide. Prophylactic measures are common to other diarrheal infections and vaccines based on surface or secreted proteins that were shown to induce antibodies (IgG and SIgA) responses in endemic areas are under development.

Enteroaggregative Escherichia coli (EAEC) have been identified in children and adults living in developed and developing countries as well as in travelers returning from developing countries and in patients infected with HIV. In addition to the acute complications of diarrhea, such as dehydration and death, EAEC can also cause persisting diarrhea that can lead in turn to malnutrition and impaired growth and development in children. EAEC strains are defined by the ability to produce a “stacked brick” appearance when placed on HEp-2 epithelial cells in culture, and contaminated food appears to be the main source of EAEC infection. EAEC are genetically heterogeneous. EAEC strain 042 is the prototypical strain for the study of virulence factors and pathogenicity; however, emergence of atypical EAEC has been described. Three major features of EAEC pathogenesis have been defined; abundant adherence to the intestinal mucosa, elaboration of enterotoxins and cytotoxins, and induction of mucosal inflammation. Infection with strains that possess specific virulence factors correlate with elevated levels of fecal cytokines and inflammatory markers. Unfortunately, the diagnosis of EAEC infection remains limited to research laboratories and therefore, are of little use in guiding antibiotic therapy. EAEC induce short-term immunity in healthy individuals which suggests that immunoprophylaxis is a potential option for control of EAEC.

Shiga toxin-producing Escherichia coli (STEC) can produce a wide spectrum of human diseases, being an important cause of both outbreaks and sporadic cases of bloody and non-bloody diarrhea, hemorrhagic colitis, the diarrhea-associated form of hemolytic uremic syndrome (HUS) worldwide. HUS is a major cause of acute renal failure in children. Albeit O157:H7 is by far the most important serotype in human infections, several different O:H serotypes of E. coli can harbor Shiga toxin (stx) genes, and actually some non-O157 strains cause illnesses that are comparable in severity to O157-induced diseases, posing a substantial concern to public health. Infections due to STEC have a proven zoonotic character as these bacteria are largely distributed among both domestic and wildlife animal species. STEC infections are transmitted to humans through contaminated food, water, and contact with infected animals or people. The cascade leading from gastrointestinal infection to renal impairment is complex, being the production of Stx the major pathogenicity determinant of STEC. However, a mosaic of different virulence traits comprising several adhesins and other toxins that may play a role in pathogenesis has also been described. There is no specific treatment to reduce the progression of HUS. Research is still necessary to improve our knowledge on the mechanisms of Stx infections and the pathophysiology of cell injury in HUS to lead to better therapeutic strategies to prevent the acute mortality and the long-term morbidity of HUS.

Enterotoxigenic Escherichia coli (ETEC) have been identified as a major bacterial pathogen responsible for infantile diarrhea in developing nations. ETEC are also the most common bacterial pathogen responsible of acute infectious diarrhea in adults traveling from industrialized nations to less developed countries. After ingestion, ETEC first attaches to epithelial cells lining the small intestine mucosa through an interaction mediated by adhesins known as colonization factor antigens (CFA), ETEC then produce either one or both of the well identified heat-labile (LT) and heat-stable (ST) enterotoxins. The LT of ETEC is structurally and immunologically related to the pentameric V. cholerae enterotoxin while ST is a small, non immunogenic peptide. Polymorphisms in host blood group antigens and the IL-10 gene promoter have been associated with diarrheal disease due to ETEC. The diagnosis of ETEC depends on the identification of the LT and ST toxin by molecular probes or immune based assays. Clinically, diarrhea due to ETEC cannot be differentiated from diarrhea due to other enteropathogens. The correction and maintenance of hydration is essential to prevent dehydration due to ETEC. . Several antimicrobials have demonstrated efficacy to treat ETEC associated travelers’ diarrhea, including Rifaximin, various fluoroquinolones and Azithromycin. Strategies for ETEC vaccines have focused on targeting CFAs of ETEC (CFA/I, CFA/II, and CFA/IV), the immunogenic LT or the use of whole cell killed organisms expressing CFAs.

At least 6 categories of pathogenic E. coli are undoubtedly recognized as being associated with enteric diseases. The importance of diarrheagenic E. coli is probably underestimated due to limited applications of available diagnostic methods in routine laboratories, especially in developing countries. Several strategies have been described for detection and characterization of diarrheagenic E. coli strains, mainly after the development of more advanced techniques. Thus, this chapter presents culture- and DNA-based methods, as well as immunological and biological assays to detect and characterize different diarrheagenic E. coli categories. Additional methods for confirmation and subtyping are also presented. The method of choice will depend very much on the facility of each laboratory, taking into account the sensitivity, specificity, reproducibility, reliability, cost, infrastructural resources and technical skills required by each method. Apart from diagnostic methods we also included some methods, which are more directed to research and epidemiological purposes, but may eventually be useful for a more specific characterization of the E. coli strains.

Shiga toxin--producing Escherichia coli (STEC) are a leading cause of bacterial enteric infections in the United States and have become a significant problem in several countries in Latin America. Prompt, accurate diagnosis of STEC infection is important because appropriate treatment early in the course of infection might decrease the risk for serious complications such as renal damage and improve overall patient outcome. This chapter provides a short review of the clinical experiences of a physician dealing with STEC and Hemolytic Uremic Syndrome patients in Argentina.

Pathogenic strains of Escherichia coli, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), and enterotoxigenic E. coli (ETEC), are responsible for a broad spectrum of diseases, which include local (intestinal) and systemic syndromes. In particular, infection with EHEC strains is also the leading cause of Hemolytic Uremic Syndrome (HUS), a systemic complication that affects to 5-10 % of EHEC infected children. These extracellular bacteria have the ability to intimately attach to the intestinal epithelium, flatten absorptive microvilli (effacement), and cause intestinal damage characterized by cellular necrosis, disruption of the epithelium, diarrhea and occasionally bleeding. The induction of these lesions depends on a type III secretion system (T3SS) encoded within the loci of enterocyte effacement (LEE). In case of EHEC strains, the expression of Shiga toxins (Stx) is one of the major pathogenic factors, which can modulate the severity of the intestinal damage, but also is responsible for HUS development. In thischapter we present and discuss new data helping to understand the role of Stx and the other bacterial pathogenic factors, as well as the involvement of host responses, in the evolution from gastrointestinal disease to systemic HUS complication.

In Argentina, a total of 1,117,718 diarrheal diseases cases were notified in 2008 with an incidence rate of 28.12/100,000 inhabitants, being Diarrheagenic Escherichia coli (DEC) strains the most important etiological agents associated to them. Several community-based studies have assessed the relative frequency of DEC pathotypes, accounting for 6 to 28.8% (EPEC), 9.7 to 24.4% (ETEC), 0.3 to 17.1% (EIEC), 1.2 to 17.1% (STEC), 20 to 31.4% (EAEC), and 27.1 to 29% (DAEC). In the last 10 years, approximately 500 HUS cases were reported annually, with an incidence that ranged between 7.8 and 17/100,000 children less than 5 years of age. STEC O157:H7 was the major serotype isolated (60%), with prevalent genotype stx2/stx2c(vh-a)/eae/ehxA (81.4%), mainly of the phage type 4 (40%). Two XbaI-PFGE patterns are prevalent, AREXHX01.0011 and AREXHX01.0022, representing respectively 9.9% and 5.6% of the E. coli O157 Argentinean isolates in the database. Among the non-O157 STEC strains, genetic profiles were more diverse, but stx2/eae/ehxA (66.2%) was prevalent. STEC strains, mainly Stx2-producers, have been recovered from animals and food, being cattle an important reservoir, with increased risk of illness linked to beef-related dietary habits, and animal exposure. The implementation of integral preventive measures is necessary to decrease the incidence of diarrheal disease in Argentina and the associated human and economic costs.

Data from the health information system of Brazil showed that more than 17 million cases of acute diarrheal diseases (ADD) were notified from 2000 to 2007 (http://portal.saude.gov.br/portal/saude/profissional/area.cfm?id_area=1549). The number of hospitalizations due to ADD is still high in children less than 5 years of age, and varies depending on the region, with several numbers of deaths, representing a serious prejudice to the population health. Among the several bacterial agents, responsible for gastrointestinal infections in Brazil, diarrheagenic E. coli (DEC) accounts for an expressive number of cases. In this chapter we will present an overview on the E. coli situation in Brazil, regarding the most significant DEC pathotypes associated with intestinal infections.

Shiga toxin-producing Escherichia coli (STEC) are emerging pathogens worldwide. Infections associated with STEC have a broad clinical spectrum from asymptomatic infections, acute diarrhea, dysenteric diarrhea and Hemolytic Uremic Syndrome. Some serotypes of STEC are highly virulent for humans due to the presence of two cytotoxins (Stx1, Stx2) and a pathogenicity island called Locus of Enterocyte Effacement (LEE), which is responsible for the adherence to intestinal epithelium, mainly through the intimin protein encoded by eae gene. Other factors have been implicated in virulence, e.g., the products of the efa1, iha, hylA, lpf and saa genes, which are encoded in the genomes of O157 and non-O157. In Chile, STEC Non-O157 serogroups are the most important cause of STEC infections. It has been found that the presence of these specific genes is more relevant than is that of the serogroup. In Chile, a high rate of STEC isolations in swine and bovines as asymptomatic carriers has been reported. Therefore the Chilean Ministry of Health has incorporated STEC as an agent under surveillance. Recently, samples from swine and bovines at slaughter were analyzed and, interestingly, only the bovine carrying STEC exhibited the virulence genes found in human isolates. Several strategies have been proposed to prevent STEC colonization in the animal host, where findings following vaccination with proteins from the LEE locus are encouraging. Thus,, DNA vaccines have been used as a novel strategy, and the immunized animals have shown a strong lymphocyte proliferation against bacterial antigens.

In Mexico, diarrhea diseases are the second leading cause of death in children ≤ 5 years old. Studies regarding enteropathogens associated with severe acute diarrhea, requiring hospitalization, have revealed diarrheagenic Escherichia coli pathotypes (DEC) as the second most prevalent group, only after rotavirus, in children of this age. DEC epidemiology has changed throughout the years, until the mid ‘90s enterotoxigenic E. coli and typical enteropathogenic E. coli (tEPEC) strains were more often isolated from children aged ≤ 5 years, nowadays is enteroaggregative E. coli (EAEC) followed by atypical EPEC. Other DEC identified these days include, ETEC, non-O157:H7 Shiga-toxin producing E. coli (STEC), tEPEC and enteroinvasive E. coli (EIEC). DEC strains have been isolated from local food samples in enough quantities to cause disease. Non-O157:H7 STEC strains have been identified from stool samples from asymptomatic subjects, diarrhea cases and a variety of food items, but O157:H7 serotype prevalence is extremely low. Mexican children and adult serum have shown, cross reactivity against O157:H7, other E. coli serogroups (O7, O116), enteric bacteria and O157:H7 bactericidal activity. Most DECs strains isolated from diarrhea patients and food samples were resistant to TMP-SMX and ampicillin; almost all strains were ciprofloxacin and cefotaxime sensitive. A new E. coli serogroup (64474) sharing only a common O antigen with S. boydii 16, has been identified. Further, substantial reductions in diarrhea related deaths, complications and morbidity due to acute diarrhea episodes, will require sanitation improvement, identification of enteropathogens and development of both new drugs to treat pediatric diarrhea and vaccines against DEC.

The diarrheagenic E. coli (DEC) are important agents of acute and persistent diarrhea in children from Uruguay, Colombia and Peru. The relative importance of each pathotype (and its variants) is variable, and may be attributed to different factors, such as age of children and socioeconomic level, type of study, and laboratory methodology used in each country. However, within the DEC group the most common agents are enterotoxigenic E. coli, enteropathogenic E. coli and enteroaggregative E. coli. Further studies are needed, mainly in food and animal reservoirs, in order to better define the transmission and the local and regional epidemiology of these important diarrheal agents.

Pathogenic Escherichia coli comprise several pathotypes which are able to carry out different manifestations in animals and humans. Some strains cause zoonotic diseases, affecting both animals and humans, while others use the animal as a carrier only, becoming pathogenic for it only circumstantially. A greater understanding of the role of reservoirs, virulence characteristics of the E. coli strains they carry, and transmission routes is necessary for developing methods of epidemiology, control and prevent infectious diseases caused by E. coli.

Chemical communication between pathogens and host mucosal cells corresponds to a dynamic array of molecular interactions. The signature molecules unique to microbial pathogens allow the mammalian immune system to recognize them as a foreign element. This recognition is usually mediated by receptor proteins, which can be classified as toll-like receptors, and recently described as nod-like receptors. These interactions result in innate immune responses targeted against the invading organism. Pathogens also elaborate a variety of proteins that actively engage host signaling pathways and subvert them to facilitate their growth and dispersal. The host function alterations are mediated by microbial pathogens including inflammatory responses, secretory responses, alteration of host cytoskeleton, disruption of epithelial tight junctions and apoptosis. Important interactions between pathogens and host cell involves chemical signaling, that depends on cell density and signaling molecules identified as autoinducers that function as hormone-like molecules in a phenomenon also known as quorum sensing. Pathogens can use these systems to colonize and cause disease in the host, and we will further discuss these mechanisms in this chapter. Chemical signaling involved in these interactions are potential targets for therapeutic strategies against infectious microbes.

In the 21st century, diarrhea is still a leading cause of illness and death especially in children in Latin American countries. ETEC, EHEC, EPEC, and EAEC remain as the major categories of pathogenic E. coli associated with diarrheal disease; however, it is evident that a shift in the serotypes responsible for human disease is occurring in this region. Recent reports implicated atypical EPEC as an important emerging category of E. coli and the association of EHEC O157:H7 as a cause of hemolytic uremic syndrome in Latin America is becoming evident. Significant improvements are required in the area of early diagnosis to increase the likelihood of an effective treatment. In this region, very few studies have addressed the emergence of antimicrobial resistance and high asymptomatic carriage rates for diarrheagenic E. coli (DEC), as well as non-human reservoirs and vehicles of transmission, are largely unknown. It is evident that broadening the epidemiological surveys to include emerging and re-emerging categories of DECs while increasing the capabilities of detection and novel treatment is a priority for the future of the E. coli research in Latin America.