Tannins extract from Galla Chinensis can protect mice from infection by Enterotoxigenic Escherichia coli O101.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is classically associated with acute secretory diarrhea, which induces 2 million people death in developing countries over a year, predominantly children in the first years of life. Previously, tannins (47.75%) were extracted from Galla Chinensis and prepared as Galla Chinensis oral solution (GOS) which showed significant antidiarrheal activity in a castor oil-induced diarrhea in mice. Whether the tannins extract were also effective in treatment of ETEC-induced diarrhea was determined in this study. METHODS: Mice were randomly divided into 6 groups (n = 22). The mice in the normal and untreated groups were given normal saline. Three GOS-treated groups were received different concentrations of GOS (5, 10 and 15%, respectively) at a dose of 10 mL/kg. Mice in the positive control group were fed with loperamide (10 mg/kg). The treatment with GOS started 3 days before infection with ETEC and continued for 4 consecutive days after infection. On day 3, mice were all infected with one dose of LD50 of ETEC, except those in the normal group. Survival of mice was observed daily and recorded throughout the study. On days 4 and 7, samples were collected from 6 mice in each group. RESULTS: GOS could increase the survival rate up to 75%, while in the untreated group it is 43.75%. The body weights of mice treated with 15% GOS were significantly increased on day 7 in comparison with the untreated group and the normal group. GOS-treatment recovered the small intestine coefficient enhanced by ETEC-infection. The diarrhea index of mice treated with GOS was significantly decreased. GOS increased the levels of IgG and sIgA in the terminal ileum and decreased the levels of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1ß, IL-6 and IL-8) in serum. GOS could increase the amount of intestinal probiotics, Lactobacilli and Bifidobacteria. GOS could alleviate colon lesions induced by ETEC-infection. GOS showed higher potency than loperamide. CONCLUSIONS: GOS could be a promising drug candidate for treating ETEC infections.

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.).

Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1ß, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1ß, and nitric oxide.

Effects of a microencapsulated formula of organic acids and essential oils on nutrient absorption, immunity, gut barrier function, and abundance of enterotoxigenic Escherichia coli F4 in weaned piglets challenged with E. coli F4.

The objective was to study the effects of microencapsulated organic acids (OA) and essential oils (EO) on growth performance, immune system, gut barrier function, nutrient digestion and absorption, and abundance of enterotoxigenic Escherichia coli F4 (ETEC F4) in the weaned piglets challenged with ETEC F4. Twenty-four ETEC F4 susceptible weaned piglets were randomly distributed to 4 treatments including (1) sham-challenged control (SSC; piglets fed a control diet and challenged with phosphate-buffered saline (PBS)); (2) challenged control (CC; piglets fed a control diet and challenged with ETEC F4); (3) antibiotic growth promoters (AGP; CC + 55 mg·kg-1 of Aureomycin); and (4) microencapsulated OA and EO [P(OA+EO); (CC + 2 g·kg-1 of microencapsulated OA and EO]. The ETEC F4 infection significantly induced diarrhea at 8, 28, 34, and 40 hr postinoculation (hpi) (P < 0.05) in the CC piglets. At 28 d postinoculation (dpi), piglets fed P(OA+EO) had a lower (P < 0.05) diarrhea score compared with those fed CC, but the P(OA+EO) piglets had a lower (P < 0.05) diarrhea score compared with those fed the AGP diets at 40 dpi. The ETEC F4 infection tended to increase in vivo gut permeability measured by the oral gavaging fluorescein isothiocyanate-dextran 70 kDa (FITC-D70) assay in the CC piglets compared with the SCC piglets (P = 0.09). The AGP piglets had higher FITC-D70 flux than P(OA+EO) piglets (P < 0.05). The ETEC F4 infection decreased mid-jejunal VH in the CC piglets compared with the SCC piglets (P < 0.05). The P(OA+EO) piglets had higher (P < 0.05) VH in the mid-jejunum than the CC piglets. The relative mRNA abundance of Na+-glucose cotransporter and B0AT1 was reduced (P < 0.05) by ETEC F4 inoculation when compared with the SCC piglets. The AGP piglets had a greater relative mRNA abundance of B0AT1 than the CC piglets (P < 0.05). The ETEC F4 inoculation increased the protein abundance of OCLN (P < 0.05), and the AGP piglets had the lowest relative protein abundance of OCLN among the challenged groups (P < 0.05). The supplementation of microencapsulated OA and EO enhanced intestinal morphology and showed anti-diarrhea effects in weaned piglets challenged with ETEC F4. Even if more future studies can be required for further validation, this study brings evidence that microencapsulated OA and EO combination can be useful within the tools to be implemented in strategies for alternatives to antibiotics in swine production.

[The first identification of epidemic clone of enterotoxic Escherichia coli O∶6 serogroup highly associated with azithromycin resistance in Shanghai].

Objective: To investigate the molecular characterization of adult diarrhea cases caused by enterotoxic Escherichia coli (ETEC) and explore the practical model of epidemiology for laboratory technique and data needs based on the surveillance network. Methods: Epidemiological design and sampling targeted adult cases ETEC caused diarrhea in epidemic season. The enterotoxin type, serogroup, resistance, colonization factor and molecular type of ETEC were identified. Multiple dynamic phenotypic characteristics of ETEC were indicated by multidimensional and multivariable data. Results: From 2016 to 2018, 84 eligible ETEC strains were detected. The dominant serums/toxins were O∶6 (STh), O∶25 (LT), O∶159 (STh), O∶153 (STh). O∶6 (STh+CS21), which replaced O∶25 and O∶159 as the popular clones in 2018. Six cases of O∶153 (STh+CFA/I+CS8+PT34) in outbreak in 2017 were imported ones. The resistance rates of ETEC strains detected in adults to sulfasoxazole, naproxinic acid, ampicillin and azithromycin were more than 30%, multidrug resistance (MDR) reached 58.3%. Serum/toxin types suggested that attenuated strains were more likely to become MDR. Molecular typing confirmed that the genetic similarity of the dominant clone of O∶6 serogroup (PT20-24) was higher than O∶25 and O∶159. There was a high correlation between the minimal inhibitory concentration (MIC) of azithromycin and the resistant gene mphA (87.5%, 28/32). O∶6 (STh+CS21+mphA) resistant clone was first detected in 2016. Conclusion: A new epidemic clone in adult ETEC diarrhea cases in Shanghai was O∶6 (STh+CS21+mphA). For the first time the association between azithromycin resistance gene mphA and a serum group of ETEC was observed. Multidimensional and multivariate analysis techniques based on epidemiology can help reveal the potential transmission pattern of ETEC for the accurate surveillance and early warning of outbreaks.

Macleaya cordata extract alleviated oxidative stress and altered innate immune response in mice challenged with enterotoxigenic Escherichia coli.

This study examines the effects of dietary Macleaya cordata extract (MCE) on bacterial burden and resistance to enterotoxigenic Escherichia coli (ETEC) in ICR mice. ICR mice were randomly distributed into one of the following groups: (i) basal diet; (ii) basal diet with 200 mg kg-1 MCE; (iii) basal diet challenged with ETEC; and (iv) basal diet with 200 mg kg-1 MCE and challenged with ETEC. Following a 7-day period of pre-treatment, CTRL-ETEC and MCE-ETEC mice were subjected to oral infection using 5×108E. coli SEC 470. The results showed dietary 200 mg kg-1 MCE markedly reduced intestinal ETEC burden (P < 0.05) and the disease-associated mortality was significantly alleviated in the MCE treated group (P < 0.05). In addition, dietary MCE markedly alleviated ETEC-induced oxidative stress, evidenced by the lowered methane dicarboxylic aldehyde (MDA) abundance and enhanced activities of catalase and glutathione peroxidase (P < 0.05). Furthermore, MCE mice exhibited higher immune activity, which might have further mediated ETEC infection. These results indicate MCE plays a preventative role with respect to ETEC infection. Future research should aim to develop MCE as a therapeutic approach to the promotion of intestinal health and a safeguard against ETEC infection.

Antibacterial activity and mode of action of acetone crude leaf extracts of under-investigated Syzygium and Eugenia (Myrtaceae) species on multidrug resistant porcine diarrhoeagenic Escherichia coli.

BACKGROUND: Diarrhoea, a global economically important disease burden affecting swine and, especially piglets, is commonly caused by infection with entero-toxigenic E. coli (ETEC). Adherence of ETEC to porcine intestinal epithelial cells following infection, is necessary for its pathogenesis. While antimicrobials are commonly given as therapy or as feed additives for prophylaxis against microbial infections, the concern over increased levels of antimicrobial resistance necessitate the search for safe and effective alternatives in livestock feed. Attention is shifting to natural products including plants as suitable alternatives to antimicrobials. The activity of acetone crude leaf extracts of nine under-explored South African endemic plants from the Myrtaceae family with good antimicrobial activity were tested against pathogenic E. coli of porcine origin using a microplate serial dilution method. Bioautography, also with p-iodonitrotetrazolium violet as growth indicator was used to view the number of bioactive compounds in each extract. In vitro toxicity of extracts was determined against Caco-2 cells using the 3-(4,5-dimethythiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay. The antimicrobial susceptibility of E. coli isolates was tested on a panel of antimicrobials using the Kirby-Bauer agar diffusion method while the anti-adherence mechanism was evaluated using a Caco-2 cell enterocyte anti-adhesion model. RESULTS: The MIC of the extracts ranged from 0.07-0.14 mg/mL with S. legatii having the best mean MIC (0.05 mg/mL). Bioautography revealed at least two active bands in each plant extract. The 50% lethal concentration (LC50) values ranged between 0.03-0.66 mg/mL. Eugenia zeyheri least cytotoxic (LC50 = 0.66 mg/ml) while E. natalitia had the highest cytotoxicity (LC50 = 0.03 mg/mL). All the bacteria were completely resistant to doxycycline and colistin sulphate and many of the plant extracts significantly reduced adhesion of E. coli to Caco-2 cells. CONCLUSIONS: The extracts of the plants had good antibacterial activity as well as a protective role on intestinal epithelial cells against enterotoxigenic E. coli bacterial adhesion. This supports the potential use of these species in limiting infection causes by E. coli. Some of these plants or extracts may be useful as phytogenic feed additives but it has to be investigated by animal feed trials.

Emergence of Resistance to Quinolones and ß-Lactam Antibiotics in Enteroaggregative and Enterotoxigenic Escherichia coli Causing Traveler's Diarrhea.

The objective of this study was to assess the antimicrobial resistance of enteroaggregative Escherichia coli (EAEC) and enterotoxigenic E. coli (ETEC) strains causing traveler's diarrhea (TD) and to investigate the molecular characterization of antimicrobial resistance genes to third-generation cephalosporins, cephamycins, and quinolones. Overall, 39 EAEC and 43 ETEC clinical isolates were studied. The susceptibilities of EAEC and ETEC against ampicillin, amoxicillin-clavulanic acid, cefotaxime, imipenem, chloramphenicol, tetracycline, co-trimoxazole, nalidixic acid, ciprofloxacin, azithromycin, and rifaximin were determined. All genes encoding resistance determinants were detected by PCR or PCR plus DNA sequencing. The epidemiology of selected EAEC and ETEC strains was studied using multilocus sequence typing (MLST). The resistance to quinolones of EAEC and ETEC strains causing TD has significantly increased over the last decades, and high percentages have been found especially in patients traveling to India and sub-Saharan Africa. Sequence type 38 (ST38) and ST131, carrying the blaCTX-M-15 and blaCTX-M-27 genes, respectively, are highly prevalent among extended-spectrum ß-lactamase (ESBL)-producing EAEC and ETEC strains. The cephamycinase ACT-20 is described in the present study for the first time in EAEC and ETEC strains causing TD in patients who had traveled to Central America. The percentages of resistance to azithromycin in EAEC and ETEC isolates from patients to Southeast Asia/India and Africa are above 25%. Meanwhile, rifaximin is still active against EAEC and ETEC, with the prevalence of resistant strains not being high. In conclusion, fluoroquinolones should no longer be considered the drugs of choice for the prevention or treatment in TD for travelers traveling to India and Africa. Azithromycin and rifaximin are still a good alternative to treat TD caused by EAEC or ETEC.

Rifamycin SV-MMX® for treatment of travellers' diarrhea: equally effective as ciprofloxacin and not associated with the acquisition of multi-drug resistant bacteria.

Background: The novel oral antibiotic formulation Rifamycin SV-MMX®, with a targeted delivery to the distal small bowel and colon, was superior to placebo in treating travellers' diarrhea (TD) in a previous study. Thus, a study was designed to compare this poorly absorbed antibiotic with the systemic agent ciprofloxacin. Methods: In a randomized double-blind phase 3 study (ERASE), the efficacy and safety of Rifamycin SV-MMX® 400 mg twice daily (RIF-MMX) was compared with ciprofloxacin 500 mg twice daily in the oral treatment of TD. Overall, 835 international visitors to India, Guatemala or Ecuador with acute TD were randomized to receive a 3-day treatment with RIF-MMX (n = 420) or ciprofloxacin (n = 415). Primary endpoint was time to last unformed stool (TLUS), after which clinical cure was declared. Stools samples for microbiological evaluation were collected at the baseline visit and the end of treatment visit. Results: Median TLUS in the RIF-MMX group was 42.8 h versus 36.8 h in the ciprofloxacin group indicating non-inferiority of RIF-MMX to ciprofloxacin (P = 0.0035). Secondary efficacy endpoint results including clinical cure rate, treatment failure rate, requirement of rescue therapy as well as microbiological eradication rate confirmed those of the primary analysis indicating equal efficacy for both compounds. While patients receiving ciprofloxacin showed a significant increase of Extended Spectrum Beta Lactamase Producing-Escherichia coli (ESBL-E. Coli) colonization rates after 3-days treatment (6.9%), rates did not increase in patients receiving RIF-MMX (-0.3%). Both drugs were well-tolerated and safe. Conclusion: The novel multi-matrix formulation of the broad-spectrum, poorly absorbed antibiotic Rifamycin SV was found non-inferior to the systemic antibiotic ciprofloxacin in the oral treatment of non-dysenteric TD with the advantage of a lower risk of ESBL-E. Coli acquisition.

Magnolol and Honokiol Attenuate Apoptosis of Enterotoxigenic Escherichia Coli-Induced Intestinal Epithelium by Maintaining Secretion and Absorption Homeostasis and Protecting Mucosal Integrity.

BACKGROUND The cortex of Magnolia officinalis has long been used as an element of traditional Chinese medicine for the treatment of anxiety, chronic bronchitis, and gastrointestinal dysfunction. This study aimed to elucidate the underlying mechanism of its functional ingredients (magnolol and honokiol) in modifying the secretion and absorption homeostasis and protecting mucosal integrity in an Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea mouse model. MATERIAL AND METHODS This study established a diarrhea mouse model infected by ETEC at a dosage of 0.02 ml/g live body weight (BW) in vivo. Magnolol or honokiol was followed by an intraperitoneal administration at dosages of 100, 300, and 500 mg/kg BW according to a 3×3 factorial arrangement. The useful biomarkers for evaluating the integrity of intestinal tract and histologic injury were analyzed and morphological development (including villus height, crypt depth, and ratio of villus height to crypt depth) and the expressions of inflammatory cytokines were determined by real-time PCR. RESULTS The results showed that magnolol and honokiol (500 mg/kg BW) reduced the concentrations of NO, DAO, and DLA, and iNOS activity, and the mRNA expressions of the interferon gamma (IFN-γ) and interleukin 10 (IL-10), and inhibited intestinal epithelial cell apoptosis. Magnolol and honokiol (300 mg/kg BW) elongated the villus height and crypt depth and decreased the number of goblet cells and the ratio of villus height to crypt depth. CONCLUSIONS The current results indicate that magnolol and honokiol enhance the intestinal anti-inflammatory capacities, elongate the villus height and crypt depth, and reduce goblet cell numbers to inhibit the intestinal epithelium apoptosis and effectively protect the intestinal mucosa. These results show that magnolol and honokiol protect the intestinal mucosal integrity and regulate gastrointestinal dysfunction.

L-Glutamine and L-arginine protect against enterotoxigenic Escherichia coli infection via intestinal innate immunity in mice.

Dietary glutamine (Gln) or arginine (Arg) supplementation is beneficial for intestinal health; however, whether Gln or Arg may confer protection against Enterotoxigenic Escherichia coli (ETEC) infection is not known. To address this, we used an ETEC-infected murine model to investigate the protective effects of Gln and Arg. Experimentally, we pre-treated mice with designed diet of Gln or Arg supplementation prior to the oral ETEC infection and then assessed mouse mortality and intestinal bacterial burden. We also determined the markers of intestinal innate immunity in treated mice, including secretory IgA response (SIgA), mucins from goblet cells, as well as antimicrobial peptides from Paneth cells. ETEC colonized in mouse small intestine, including duodenum, jejunum, and ileum, and inhibited the mRNA expression of intestinal immune factors, such as polymeric immunoglobulin receptor (pIgR), cryptdin-related sequence 1C (CRS1C), and Reg3γ. We found that dietary Gln or Arg supplementation decreased bacterial colonization and promoted the activation of innate immunity (e.g., the mRNA expression of pIgR, CRS1C, and Reg3γ) in the intestine of ETEC-infected mice. Our results suggest that dietary arginine or glutamine supplementation may inhibit intestinal ETEC infection through intestinal innate immunity.

Resistance Pattern and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains Isolated in Bangladesh.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children as well as in travellers to ETEC endemic countries. Ciprofloxacin is a broad-spectrum antimicrobial agent nowadays used for the treatment of diarrhea. This study aimed to characterize ciprofloxacin resistant ETEC strains isolated from diarrheal patients in Bangladesh. METHODS: A total of 8580 stool specimens from diarrheal patients attending the icddr,b Dhaka hospital was screened for ETEC between 2005 and 2009. PCR and Ganglioside GM1- Enzyme Linked Immuno sorbent Assay (ELISA) was used for detection of Heat labile (LT) and Heat stable (ST) toxins of ETEC. Antimicrobial susceptibilities for commonly used antibiotics and the minimum inhibitory concentration (MIC) of nalidixic acid, ciprofloxacin and azithromycin were examined. DNA sequencing of representative ciprofloxacin resistant strains was performed to analyze mutations of the quinolone resistance-determining region of gyrA, gyrB, parC and parE. PCR was used for the detection of qnr, a plasmid mediated ciprofloxacin resistance gene. Clonal variations among ciprofloxacin resistant (CipR) and ciprofloxacin susceptible (CipS) strains were determined by Pulsed-field gel electrophoresis (PFGE). RESULTS: Among 1067 (12%) ETEC isolates identified, 42% produced LT/ST, 28% ST and 30% LT alone. Forty nine percent (n = 523) of the ETEC strains expressed one or more of the 13 tested colonization factors (CFs) as determined by dot blot immunoassay. Antibiotic resistance of the ETEC strains was observed as follows: ampicillin 66%, azithromycin 27%, ciprofloxacin 27%, ceftriazone 13%, cotrimaxazole 46%, doxycycline 44%, erythromycin 96%, nalidixic acid 83%, norfloxacin 27%, streptomycin 48% and tetracycline 42%. Resistance to ciprofloxacin increased from 13% in 2005 to 34% in 2009. None of the strains was resistant to mecillinam. The MIC of the nalidixic acid and ciprofloxacin of representative CipR strains were 256 µg/ml and 32µg/ml respectively. A single mutation (Ser83-Leu) in gyrA was observed in the nalidixic acid resistant ETEC strains. In contrast, double mutation in gyrA (Ser83-Leu, Asp87-Asn) and a single mutation in parC (Glu84-Ly) were found in ciprofloxacin resistant strains. Mutation of gyrB was not found in either the nalidixic acid or ciprofloxacin resistant strains. None of the ciprofloxacin resistant strains was found to be positive for the qnr gene. Diverse clones were identified from all ciprofloxacin resistant strains by PFGE analysis in both CF positive and CF negative ETEC strains. CONCLUSION: Emergence of ciprofloxacin resistant ETEC strains results in a major challenge in current treatment strategies of ETEC diarrhea.

Individual-specific changes in the human gut microbiota after challenge with enterotoxigenic Escherichia coli and subsequent ciprofloxacin treatment.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in inhabitants from low-income countries and in visitors to these countries. The impact of the human intestinal microbiota on the initiation and progression of ETEC diarrhea is not yet well understood. RESULTS: We used 16S rRNA (ribosomal RNA) gene sequencing to study changes in the fecal microbiota of 12 volunteers during a human challenge study with ETEC (H10407) and subsequent treatment with ciprofloxacin. Five subjects developed severe diarrhea and seven experienced few or no symptoms. Diarrheal symptoms were associated with high concentrations of fecal E. coli as measured by quantitative culture, quantitative PCR, and normalized number of 16S rRNA gene sequences. Large changes in other members of the microbiota varied greatly from individual to individual, whether or not diarrhea occurred. Nonetheless the variation within an individual was small compared to variation between individuals. Ciprofloxacin treatment reorganized microbiota populations; however, the original structure was largely restored at one and three month follow-up visits. CONCLUSION: Symptomatic ETEC infections, but not asymptomatic infections, were associated with high fecal concentrations of E. coli. Both infection and ciprofloxacin treatment caused variable changes in other bacteria that generally reverted to baseline levels after three months.

[Efficacy of disinfection treatments using essential oils and ultrasound on tomato fruits inoculated with Escherichia coli and impact on antioxidant activity]. / Eficiencia de la desinfección con aceites esenciales y ultrasonido sobre Escherichia coli inoculada en frutos de tomate y el impacto sobre la actividad antioxidante.

Fresh produce often harbors a great number of microorganisms; hence, its growing demand may constitute a risk for consumers. The aim of this study was to evaluate the efficacy of several disinfection procedures against enterotoxigenic Escherichia coli (ETEC) inoculated on tomato fruits and the conservation of the antioxidant properties of these disinfected fruits. Fruits were immersed for 5 or 10min in oregano or thyme essential oil dispersions (5, 10ppm), with or without ultrasound treatment. Antioxidant activity of disinfected fruits was determined as the ability to scavenge 2,2-diphenyl-1-pricrylhydrazyl (DPPH) radicals and was reported as percentage of inhibition (%I). The most efficient disinfectant treatments showing significant differences (p≤.05) between the reductions log10 CFU/g (S) of ETEC were those using 10ppm oregano for 10min, with S=3.05 in individual treatments and S=4.03 in mixed treatments. The highest %I was obtained with individual sonication treatments (69.52 and 72.48), while in combined treatments the %I values increased with thyme oil 5ppm and ultrasound for 5min (51.27%) and 10min (53.31%).

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

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.

Antibacterial activity of silver and zinc nanoparticles against Vibrio cholerae and enterotoxic Escherichia coli.

Vibrio cholerae and enterotoxic Escherichia coli (ETEC) remain two dominant bacterial causes of severe secretory diarrhea and still a significant cause of death, especially in developing countries. In order to investigate new effective and inexpensive therapeutic approaches, we analyzed nanoparticles synthesized by a green approach using corresponding salt (silver or zinc nitrate) with aqueous extract of Caltropis procera fruit or leaves. We characterized the quantity and quality of nanoparticles by UV-visible wavelength scans and nanoparticle tracking analysis. Nanoparticles could be synthesized in reproducible yields of approximately 10(8) particles/ml with mode particles sizes of approx. 90-100 nm. Antibacterial activity against two pathogens was assessed by minimal inhibitory concentration assays and survival curves. Both pathogens exhibited similar resistance profiles with minimal inhibitory concentrations ranging between 5×10(5) and 10(7) particles/ml. Interestingly, zinc nanoparticles showed a slightly higher efficacy, but sublethal concentrations caused adverse effects and resulted in increased biofilm formation of V. cholerae. Using the expression levels of the outer membrane porin OmpT as an indicator for cAMP levels, our results suggest that zinc nanoparticles inhibit adenylyl cyclase activity. This consequently deceases the levels of this second messenger, which is a known inhibitor of biofilm formation. Finally, we demonstrated that a single oral administration of silver nanoparticles to infant mice colonized with V. cholerae or ETEC significantly reduces the colonization rates of the pathogens by 75- or 100-fold, respectively.

Effects of chitosan on intestinal inflammation in weaned pigs challenged by enterotoxigenic Escherichia coli.

The aim of this study was to investigate whether supplementation with chitosan (COS) could reduce diarrhea and to explore how COS alleviates intestinal inflammation in weaned pigs. Thirty pigs (Duroc×Landrace×Yorkshire, initial BW of 5.65±0.27) weaned at age 21 d were challenged with enterotoxigenic Escherichia coli during a preliminary trial period, and then divided into three treatment groups. Pigs in individual pens were fed a corn-soybean meal diet, that contained either 0 (control), 50 mg/kg chlortetracycline, or 300 mg/kg COS for 21 days. The post-weaning diarrhea frequency, calprotectin levels and TLR4 protein expression were decreased (P<0.05) in both the COS and chlortetracycline groups compared with control. Simultaneously, supplemental COS and chlortetracycline had no effect on the mRNA expression of TNF-α in the jejunal mucosa, or on the concentrations of IL-1ß, IL-6 and TNF-α in serum. However, COS supplementation improved (P<0.05) the mRNA expression of IL-1ß and IL-6 in the jejunal mucosa. The results indicate that supplementation with COS at 300 mg/kg was effective for alleviating intestinal inflammation and enhancing the cell-mediated immune response. As feed additives, chitosan and chlortetracycline may influence different mechanisms for alleviating inflammation in piglets.

The suppressive activities of six sources of medicinal ferns known as gusuibu on heat-labile enterotoxin-induced diarrhea.

Diarrheal disease is one of the most important worldwide health problems. Enterotoxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen in diarrheal diseases. In developing countries, a very large number of people, especially children, suffer from diarrhea. To combat this problem, World Health Organization has constituted the Diarrhea Diseases Control Program which guides studies on traditional medicinal practices and preventive measures. Gusuibu, a traditional folk medicine, has been claimed to heal certain types of diarrhea. However, so far no scientific study has been carried out on the anti-diarrheal mechanism of Gusiubu. The present study was performed to examine the suppressive activities of ethanol extracts of six sources of folk medicinal ferns used as Gusuibu on heat-labile enterotoxin (LT)-induced diarrhea. Inhibitory effects of six sources were evaluated on the ETEC LT subunit B (LTB) and monosialotetrahexosylganglioside (GMI) interaction by GM1-enzyme linked immunosorbent assay and patent mouse gut assay. Our results indicated that Drynaria fortunei had no anti-diarrheal effect, while, among the remaining five folk medicinal ferns, four belonging to family Davalliaceae had significant abilities on both the blocking of LTB and GM1 interaction and the inhibition of LT-induced diarrhea. In conclusion, these findings suggested the potential application of Gusuibu as an anti-diarrheal remedy.

Antibacterial and antidiarrheal activities of plant products against enterotoxinogenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) produces two types of enterotoxins: heat-labile (LT) and heat-stable (STa and STb). These molecules are involved in the induction of secretory diarrhea in animals including humans. This condition is currently treated using a fluid replacement therapy and antibiotics. This treatment is often not available to people in developing countries, and several die from the condition provoke by ETEC. Over the years, plants and plant extracts have been use as traditional medicine to treat various gastrointestinal ailments including diarrhea. Many of these plant products have been claimed to be active against diarrhea, however few have been extensively studied. The main objective of this review was to gather the scattered information on the antidiarrheal activities reported for various plant products on ETEC. This includes two major effects: (1) The inhibitory effect on bacterial growth or viability and (2) The interference with ETEC enterotoxins activity upon the intestinal epithelium. We will focus on plant products and extracts for which we have major indications of their biological activity against ETEC and their enterotoxins. Because Vibrio cholerae toxin (CT) is structurally, antigenically and mechanistically related to LT, it will also be discussed in this review.

Selection of Escherichia coli heat-labile toxin (LT) inhibitors using both the GM1-ELISA and the cAMP Vero cell assay.

Weaned piglets are very susceptible to diarrhea caused by enterotoxigenic Escherichia coli. In the past, various natural components were proposed to have beneficial effects by reducing the effects of diarrheal infectious diseases in humans and animals, and thus may represent an alternative for the use of (prophylactic) antibiotics. Alternatives may inactivate enterotoxigenic Escherichia coli heat-labile toxin (LT) by interfering with toxin binding to the cellular receptor GM1. In this study, various plants and other natural substances were tested for inhibitory properties, in the GM1 binding assay, and in the LT-induced cAMP production in Vero cells. The toxic dose of each compound was determined in a cell viability assay, and the highest nontoxic concentrations were used in the GM1 and cAMP assays. Results demonstrated that only d-(+)-galactose, lactose, N-acetyl-d-galactosamine, and two tea extracts were able to inhibit the binding of LT to its GM1 receptor. In the cAMP assay, only the two tea extracts showed inhibitory activity. This shows that d-(+)-galactose, lactose, and N-acetyl-d-galactosamine can indeed inhibit LT binding to GM1 based on structural homology with GM1 in the absence of living cells. However, in the cAMP assay, d-(+)-galactose, and lactose, N-acetyl-d-galactosamine are apparently metabolized to below their effective inhibitory concentration, likely predicting limited practical applicability in vivo. Both tea extracts maintained their activity in the presence of cells. The active compounds in both are probably polyphenols, which are not easily metabolized, and most likely work by aggregating the toxin. In conclusion, the combination of methods used here is a convenient and fast method for preselecting natural substances containing potentially toxin-binding compounds. Furthermore, if antidiarrhea activity is attributed to compounds found inactive here, their activity is unlikely based on interference with toxin binding.

Dietary supplementation with bovine lactoferrampin-lactoferricin produced by Pichia pastoris fed-batch fermentation affects intestinal microflora in weaned piglets.

This work is aimed at investigating the effects of recombinant bovine lactoferrampin-lactoferricin (LFA-LFC) instead of chlortetracycline on intestinal microflora in weaned piglets. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, recombinant LFA-LFC was produced via fed-batch fermentation in recombinant strain Pichia pastoris (KM71) XS10. Uniform design U6(6(4)) was used to optimize the fermentation conditions. The target peptide purified via cation-exchange and size-exclusion chromatography was added into the dietary of weaned piglets. After 21 days, the Lactobacilli, Bifidobacteria, and Enterobacteria in the chyme of the gut were quantified using real-time polymerase chain reaction. The results showed that approximately 82 mg of LFA-LFC was secreted into 1 L of medium under optimized conditions. Moreover, purified peptide showed strong antimicrobial activities against all the tested microorganisms. Compared with the control group, the LFA-LFC group increased the amount of Lactobacilli and Bifidobacteria (P<0.05) in the chyme of the stomach, duodenum, jejunum, ileum, colon, and caecum. These results show that dietary supplementation with LFA-LFC can affect intestinal microflora in weaned piglets.