Theresa J. Ochoa

New insights into the epidemiology of enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of persistent diarrhea. EPEC were originally serogroup-defined E. coli associated with infantile diarrhea. As various mechanisms of pathogenesis have been discovered, EPEC classification has come to be based on the presence of specific genes. The eae (intimin) and bfpA (bundle-forming pilus) genes have both been used for identification of EPEC and for subdivision of this group of bacteria into typical and atypical strains. For many years typical EPEC have been considered to be the leading cause of infantile diarrhea in developing countries and were considered rare in industrialized countries. However, current data suggests that atypical EPEC are more prevalent than typical EPEC in both developing and developed countries. Moreover, the duration of diarrhea in patients infected with atypical EPEC is significantly longer than that caused by other pathogens. When comparing the isolation rates of EPEC among children with diarrhea and healthy controls without diarrhea, in general, there is a higher isolation rate in diarrhea, although not significantly higher in all studies. These inconsistencies probably are related to the study patient populations, reflecting a possible age-related susceptibility to infection.

Enteropathogenic Escherichia coli infection in children.

Purpose of review Enteropathogenic Escherichia coli (EPEC) is an important diarrheal pathogen of young children. As the diagnosis of EPEC is now based mainly on molecular criteria, there has been an important change in its prevalence. The purpose of this study is to review the current epidemiology of EPEC infection and the new insights into its physiopathology. Recent findings Recent epidemiological studies indicate that atypical EPEC (aEPEC) is more prevalent than typical EPEC (tEPEC) in both developed and developing countries, and that aEPEC is important in both pediatric endemic diarrhea and diarrhea outbreaks. Therefore, it is important to further characterize the pathogenicity of these emerging strains. The virulence mechanisms and physiopathology of the attaching and effacing lesion (A/E) and the type three secretion-system (T3SS) are complex but well studied. A/E strains use their pool of locus of enterocyte effacement (LEE)-encoded and non-LEE-encoded effector proteins to subvert and modulate cellular and barrier properties of the host. However, the exact mechanisms of diarrhea in EPEC infection are not completely understood. Summary Remarkable progress has been made to identify virulence determinants required to mediate the pathogenesis of EPEC. However, fast, easy, and inexpensive diagnostic methods are needed in order to define optimal treatment and prevention for children in endemic areas.

Detection of Diarrheagenic Escherichia coli by Use of Melting-Curve Analysis and Real-Time Multiplex PCR

ABSTRACT Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx1 and stx2 for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.

Effect of lactoferrin on enteric pathogens

Much has been learned in recent years about the mechanisms by which breastfeeding improves child health and survival. However, there has been little progress in using these insights to improve pediatric care. Factors that are important for protecting the breast fed infant might be expected to decrease the adverse effects of weaning on diarrhea, growth, and development. Lactoferrin, an iron-binding protein with multiple physiological functions (anti-microbial, anti-inflammatory, and immunomodulatory), is one of the most important proteins present in mammalian milk. Protection against gastroenteritis is the most likely biologically relevant activity of lactoferrin. Multiple in vitro and animal studies have shown a protective effect of lactoferrin on infections with enteric microorganisms, including rotavirus, Giardia, Shigella, Salmonella and the diarrheagenic Escherichia coli. Lactoferrin has two major effects on enteric pathogens: it inhibits growth and it impairs function of surface expressed virulence factors thereby decreasing their ability to adhere or to invade mammalian cells. Thus, lactoferrin may protect infants from gastrointestinal infection by preventing the attachment by enteropathogens in the gut. Recently several clinical trials in children have started to address this issue. Whether lactoferrin can prevent a significant portion of diarrheal disease remains to be determined.

Community-associated methicillin-resistant Staphylococcus aureus in pediatric patients.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections increased from 2000 to 2003 in hospitalized pediatric patients in Houston. CA-MRSA was associated with greater illness than was infection with methicillin-susceptible strains. Children with CA-MRSA were younger and mostly African American. Of MRSA isolates, 4.5% had the inducible macrolide-lincosamide-streptogramin B phenotype.

Age-Related Susceptibility to Infection with Diarrheagenic Escherichia coli among Infants from Periurban Areas in Lima, Peru

BACKGROUND Diarrheagenic Escherichia coli strains are being recognized as important pediatric enteropathogens worldwide. However, it is unclear whether there are differences in age-related susceptibility to specific strains, especially among infants. METHODS We conducted a passive surveillance cohort study of diarrhea that involved 1034 children aged 2-12 months in Lima, Peru. Control stool samples were collected from randomly selected children without diarrhea. All samples were analyzed for common enteric pathogens and for diarrheagenic E. coli with use of multiplex real-time polymerase chain reaction. RESULTS The most frequently isolated pathogens in 1065 diarrheal episodes were diarrheagenic E. coli strains (31%), including enteroaggregative (15.1%) and enteropathogenic E. coli (7.6%). Diarrheagenic E. coli, Campylobacter species, and rotavirus were more frequently isolated from infants aged >or=6 months. Among older infants, diffusely adherent E. coli and enterotoxigenic E. coli were more frequently isolated from diarrheal samples than from control samples (P <.05). Children aged >or=6 months who were infected with enterotoxigenic E. coli had a 4.56-fold increased risk of diarrhea (95% confidence interval, 1.20-17.28), compared with younger children. Persistent diarrhea was more common in infants aged <6 months (13.5% vs 3.6%; P <.001). Among children with diarrheagenic E. coli-positive samples, coinfections with other pathogens were more common in children with diarrhea than in control children (40.1% vs 15.6%; P <.001). CONCLUSIONS Diarrheagenic E. coli strains were more frequently isolated in samples from older infants. In this setting with high frequency of pathogen exposure and high frequency of breastfeeding, we hypothesize that the major age-related differences result from decreased exposure to milk-related protective factors and from increased exposure to contaminated food and water.

Drug-resistant Diarrheogenic Escherichia coli, Mexico

Diarrheogenic Escherichia coli isolates from 45 (73%) of 62 hospitalized patients were resistant to common antimicrobial drugs. Sixty-two percent were multidrug resistant, and >70% were resistant to trimethoprim-sulfamethoxazole and ampicillin. Ciprofloxacin and cefotaxime were uniformly active. Effective and safe oral agents are needed to treat children with bacterial diarrhea.

Human Lactoferrin Impairs Virulence of Shigella flexneri

Lactoferrin is a glycoprotein present in most human mucosal secretions, including human milk. Lactoferrin is bacteriostatic in low iron media and, in some settings, bactericidal. Lactoferrin impairs ability of Shigella flexneri serotype 5 strain M90T to invade HeLa cells. To determine the mechanism by which lactoferrin decreases invasiveness of Shigella organisms, its effect on the major virulence proteins responsible for bacterial uptake by host cells was evaluated. Lactoferrin induced degradation of invasion plasmid antigens IpaB and, to a lesser extent, IpaC, the key proteins responsible for bacteria-directed phagocytosis by mammalian cells. The lipid A-binding N-terminal portion of lactoferrin (residues 1-33) induces release of invasion antigens but does not induce degradation of IpaBC. Lactoferrin does not directly degrade previously released invasion plasmid antigens but works by making IpaBC susceptible to breakdown by surface-expressed protease(s).

Lactoferrin impairs type III secretory system function in enteropathogenic Escherichia coli

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries. EPEC uses a type III secretory system to deliver effector proteins into the host cell. These proteins cause the characteristic attaching and effacing lesion on enterocytes. Lactoferrin, a glycoprotein present in human milk, inhibits EPEC adherence to mammalian cells. To determine the effect of lactoferrin on the initial host cell attachment step that is mediated by the type III secretory system, we focused on EPEC-induced actin polymerization in HEp2 cells, on the hemolytic activity, and on measurement of E. coli secreted proteins A, B, and D (EspABD). Lactoferrin blocked EPEC-mediated actin polymerization in HEp2 cells and blocked EPEC-induced hemolysis. The mechanism of this inhibition was lactoferrin-mediated degradation of secreted proteins necessary for bacterial contact and pore formation, particularly EspB. The proteolytic effect of lactoferrin was prevented by serine protease inhibitors. This disruption of the type III secretory system implies that lactoferrin could provide broad cross protection against the enteropathogens that share this mechanism.

Impact of lactoferrin supplementation on growth and prevalence of Giardia colonization in children.

We conducted a randomized, double-blind, placebo-controlled trial comparing supplementation with bovine lactoferrin versus placebo for the prevention of diarrhea in children. Comparison of overall diarrhea incidence and prevalence rates found no significant difference between the 2 groups. However, there was a lower prevalence of colonization with Giardia species and better growth among children in the lactoferrin group.