Thomas G. Cleary

Effect of subinhibitory concentrations of antibiotics on extracellular shiga-like toxin I

SummaryPatients with diarrhea due to strains of enterohemorrhagicEscherichia coli (EHEC) (e. g. O157:H7) might be at a higher risk of developing hemolytic uremic syndrome when treated with antimicrobial agents. It has been suggested that this might be due to an increase of release or production of vero or shiga-like toxin from such organisms, possibly as a stress response to antimicrobial agents. The aim of this study was to detect such increases in extracellular toxinin vitro with a newly developed method that exposed EHEC to high sublethal concentrations followed by a recovery phase at progressively lower concentrations. Five strains of EHEC were exposed to continuously changing concentrations of ciprofloxacin, co-trimoxazole, cefixime and tetracycline. The amount of free shiga-like toxin I (SLT-I) released was compared to the amount released from inocula that were not exposed to antibiotics. There were significant differences between the five EHEC strains in the amount of toxin detected after exposure to antimicrobial agents (p<0.001). Equally important was the type of antibiotic (p<0.001), with ciprofloxacin inducing the largest increase ranging from 169 to 436%, followed by co-trimoxazole, cefixime and tetracycline. In addition, the increases in free toxin correlated with the concentration of the antibiotics (p<0.001). The association between antibiotic-induced increases in SLT-I produced by strains of EHEC and certain classes of antibiotics might influence the analysis of future epidemiological studies on risk factors for HUS.ZusammenfassungDie orale antimikrobielle Behandlung von Patienten mit Durchfällen, die durch enterohämorrhagische Stäme vonEscherichia coli (EHEC), zum Beispiel O157:H7, verursacht werden, ist ein möglicher Risikofaktor für die Entwicklung eines hämalytisch-urämischen Syndroms. Es wird vermutet, daß der Grund hierfür in einer gesteigerten Produktion oder Sekretion von Vero- oder Shiga-like-Toxinen liegt, als Teil einer durch die Antibiotika hervorgerufenen Streßreaktion der Bakterien. Das Ziel dieserIn-vitro-Studie war es, Erhöhungen von extrazellulären Toxinkonzentrationen zu messen nachdem EHEC mit einer Diffusionsmethode wechselnden Konzentrationen von Antibiotika ausgesetzt worden waren. Die Antibiotikakonzentrationen wurden mit der jeweils höchstmöglichen subletalen Konzentration begonnen, die dann stetig abnahm und den Bakterien eine Erholungsphase gestattete. Fünf EHEC- Stämme wurden auf diese Weise Ciprofloxacin, Co-trimoxazol, Cefixim und Tetracyclin ausgesetzt. Zwischen den verschiedenen Stämmen und Antibiotika fanden sich signifikante Unterschiede in den durch die Antibiotika hervorgerufenen Erhöhungen extrazellulärer Toxinkonzentrationen mit p<0,001 für beide Parameter. Ciprofloxacin verursachte die größten Steigerungen von 169 bis 436%, gefolgt von Co-trimoxazol, Cefixim und Tetracyclin. Diese Assoziation von erhöhten Konzentrationen von Vero- und Shiga-like-Toxinen und der Einwirkung von Antibiotika auf verschiedene Keime muß in Zukunft in Studien über die Risikofaktoren, die zur Entwicklung des hämolytischurämischen Syndroms beitragen, berücksichtigt werden.

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.

Recent advances in understanding the pathogenesis of the hemolytic uremic syndromes

One of the requirements for an agent to cause hemolytic uremic syndrome (HUS) is its ability to injure endothelial cells. Shiga-like toxin (SLT) can do this. SLT is produced byEscherichia coli andShigella dysenteriae serotype 1; both have been implicated as causes of typical HUS. Endothelial cells have receptors (GB3) for SLT and the toxin can inhibit eukaryotic protein synthesis, thereby causing cell death. Glomerular endothelial cell injury or death results in a decreased glomerular filtration rate and many of the perturbations seen in HUS. It is no longer certain that hemolysis is the result of a microangiopathy. Cell injury results in release of von Willebrand multimers; if these are ultra-large, thrombosis may ensue. There is also increasing evidence that neutrophils have a role in the pathogenesis of typical HUS.Streptococcus pneumoniae can also cause HUS and care must be taken to avoid giving plasma to patients withS. pneumoniae-associated HUS. There is compelling evidence that types of HUS are inherited by autosomal recessive and autosomal dominant modes. Patients with autosomal recessive HUS may have recurrent episodes. Mortality and morbidity rates are high for the inherited forms.

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.

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.

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.

Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin.

ABSTRACT Hemolytic uremic syndrome (HUS) is associated with intestinal infection by enterohemorrhagic Escherichia coli strains that produce Shiga toxins. Globotriaosylceramide (Gb3) is the functional receptor for Shiga toxin, and tumor necrosis factor alpha (TNF-α) upregulates Gb3 in both human macrovascular umbilical vein endothelial cells and human microvascular brain endothelial cells. TNF-α treatment enhanced Shiga toxin binding and sensitivity to toxin. This upregulation was specific for Gb3 species containing normal fatty acids (NFA). Central nervous system (CNS) pathology in HUS could involve cytokine-stimulated elevation of endothelial NFA-Gb3 levels. Differential expression of Gb3 species may be a critical determinant of Shiga toxin toxicity and of CNS involvement in HUS.

Cytotoxin-producing Escherichia coli and the Hemolytic Uremic Syndrome

Recently, a new class of diarrhea-associated Escherichia coli has been linked to the hemolytic uremic syndrome. The organisms included in this group produce cell-damaging toxins (cytotoxins) related to Shigatoxin made by S. dysenteriae 1. The most common pathogen in this group is E. coli O157:H7.

Concentration of milk secretory immunoglobulin A against Shigella virulence plasmid-associated antigens as a predictor of symptom status in Shigella-infected breast-fed infants

We conducted a prospective, community-based study of healthy breast-fed Mexican infants to determine the protective effects of anti-Shigella secretory IgA antibodies in milk. Milk samples were collected monthly, and stool culture specimens were obtained weekly and at the time of episodes of diarrhea. Nineteen breast-fed infants were found to have Shigella flexneri, Shigella boydii, or Shigella sonnei in stool samples. Ages of the 10 infants with symptomatic infection and the nine with asymptomatic infection did not differ significantly. Milk samples collected up to 12 weeks before infection were evaluated by enzyme-linked immunosorbent assay for secretory IgA antibodies against lipopolysaccharides of S. flexneri, S. boydii serotype 2, S. sonnei, and virulence plasmid-associated antigens. The geometric mean titers of anti-Shigella antibodies to virulence plasmid-associated antigens in milk received before infection were eightfold higher in infants who remained well than in those in whom diarrhea developed. The significance of milk secretory IgA directed against lipopolysaccharide was less clear. We conclude that human milk protects infants against symptomatic shigella infection when it contains high concentrations of secretory IgA against virulence plasmid-associated antigens.