Bovine lactoferrin decreases cholera-toxin-induced intestinal fluid accumulation in mice by ganglioside interaction.

Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT's B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea

Effect of bovine lactoferrin (bLF) on CT-induced fluid accumulation in ileal loops.

<p>CT (1.5 µg) was added to the mouse ileal loop with bLF (10 mg/mL). Weigh/length ratios were calculated. A) bLF added before CT. Each loop was injected with 0.1 mL of sample solution (phosphate-buffered saline [PBS], 2 mg/loop bLF, PBS control) and 10 min later CT (1.5 µg) was added, B) bLF added together or at the same time with CT, and C) bLF added 10 min after CT. *, <i>p</i><.05; **, <i>p</i><.01 for the comparison of bLF group vs. CT groups, by Mann–Whitney U test.</p

Effect of lactoferrin concentrations on ETEC H10407 growth with and without an excess of ferric chloride.

<p>Bacteria were incubated on Luria Broth (control) (▪) or bovine lactoferrin at 1.0 mg/mL (Δ) or 10.0 mg/mL (▴) or bovine lactoferrin at 10 mg/mL with an excess of ferric chloride (□) (4∶1 molar ratio of iron to lactoferrin) for 12 h. Three dilutions of the bacterial culture were plated on agar to determine the number of colonization forming units (CFU) (mean ± SD of 3 experiments).</p

Inhibitory ability of bovine lactoferrin (bLF) on the binding of CT (gradient of concentration) to GM1 by GM1-ELISA.

<p>CT was added at a two fold increase in doses (3.91–500 ng) after (black bars), together (grey bars) or before (striped boxes) the bovine lactoferrin administration, and incubated in total for 3 h. The white bars represent the control group (CT alone). The absorbance was read at 450 nm in an ELISA plate reader. Values are mean ± standard desviation of three independent assays. *, <i>p</i><.05; **, <i>p</i><.01; ***, <i>p</i><.001 for the comparison of bLF groups vs. CT group (CT alone), by unpaired t test.</p

Effect of bovine lactoferrin concentrations on ETEC H10407 growth.

<p>A) Effect of two lactoferrin concentrations on ETEC H10407 growth. Bacteria were incubated on Luria Broth (control) (▪) or bovine lactoferrin at 1.0 mg/mL (Δ) or 10.0 mg/mL (▴) for 8 h. Bacterial growth was monitored spectrophotometrically at OD600 for 8 h. B) Effect of iron and lactoferrin on ETEC growth. Bacteria were incubated in Luria Broth (control) with (▪) or without iron (□), or bovine lactoferrin at 10 mg/mL with (▴) and without iron (Δ) (4∶1 molar ratio of iron to lactoferrin). Bacterial growth was monitored spectrophotometrically at OD600 for 8 h (mean ± SD of 3 experiments). The SD bars are not seen in the majority of the samples because they fall inside the symbol.</p

Inhibitory effect of bovine lactoferrin (bLF) on CT-induced fluid accumulation in the mouse ileal loop.

<p>Control (phosphate-buffered saline, pH7.4) or CT (1.5 µg) without or with bLF (10 mg/mL) were simultaneously injected into the ileal loops, and mice were sacrificed 24 h later.</p

Comparative analysis of antimicrobial resistance in enterotoxigenic Escherichia coli isolates from two paediatric cohort studies in Lima, Peru

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado.Background Antibiotic resistance is increasing worldwide, being of special concern in low- and middle-income countries. The aim of this study was to determine the antimicrobial susceptibility and mechanisms of resistance in 205 enterotoxigenic Escherichia coli (ETEC) isolates from two cohort studies in children <24 months in Lima, Peru. Methods ETEC were identified by an in-house multiplex real-time PCR. Susceptibility to 13 antimicrobial agents was tested by disk diffusion; mechanisms of resistance were evaluated by PCR. Results ETEC isolates were resistant to ampicillin (64%), cotrimoxazole (52%), tetracycline (37%); 39% of the isolates were multidrug-resistant. Heat-stable toxin producing (ETEC-st) (48%) and heat-labile toxin producing ETEC (ETEC-lt) (40%) had higher rates of multidrug resistance than isolates producing both toxins (ETEC-lt-st) (21%), p<0.05. Only 10% of isolates were resistant to nalidixic acid and none to ciprofloxacin or cefotaxime. Ampicillin and sulfamethoxazole resistance were most often associated with blaTEM (69%) and sul2 genes (68%), respectively. Tetracycline resistance was associated with tet(A) (49%) and tet(B) (39%) genes. Azithromycin inhibitory diameters were ≤15 mm in 36% of isolates, with 5% of those presenting the mph(A) gene. Conclusions ETEC from Peruvian children are often resistant to older, inexpensive antibiotics, while remaining susceptible to ciprofloxacin, cephalosporins and furazolidone. Fluoroquinolones and azithromycin remain the drugs of choice for ETEC infections in Peru. However, further development of resistance should be closely monitored.Revisión por pare

Diarrheagenic Escherichia coli in Human Immunodeficiency Virus (HIV) Pediatric Patients in Lima, Perú

We conducted a prospective study in three hospitals in Lima in human immunodeficiency virus (HIV) children to determine the frequency of diarrheagenic Escherichia coli. Five E. coli colonies/patients were studied by a multiplex real-time polymerase chain reaction to identify the six currently recognized groups of diarrhea-associated E. coli. We have analyzed 70 HIV-associated diarrheal and 70 control samples from HIV-infected children without diarrhea. Among the diarrheal episodes 19% were persistent, 3% dysenteric, and 33% were associated with moderate or severe dehydration. The diarrheagenic E. coli were the most commonly isolated pathogens in diarrhea (19%) and control samples (26%) (P = 0.42), including enteroaggregative (6% versus 10%), enteropathogenic (6% versus 10%), and enterotoxigenic E. coli (4% versus 3%), respectively. The HIV-infected children with diarrhea had the worse age-related immunosuppression, higher viral loads, and were on highly active antiretroviral treatment (HAART) less often than HIV-infected children without diarrhea. Diarrheagenic E. coli were highly resistant to ampicillin (74%) and cotrimoxazole (70%)