Assessment of Zn and Cu in piglets' liver and kidney: impact in fecal Enterococcus spp.?

Zinc and copper have been used as growth promotors in alternative to antibiotics in pig's diet. The aim was the ascertainment of the Zn and Cu concentrations in piglets' liver and kidney and their impact in the reduced susceptibility to Zn, Cu, and antibiotics in enterococci, used as microbiota biomarker. Zn and Cu were determined in the livers and kidneys of 43 piglets slaughtered in Portugal, by flame atomic absorption spectrometry. Enterococci were isolated from feces for determining the identification of species (E. faecalis, E. faecium, and Enterococcus spp.); susceptibility to vancomycin, ciprofloxacin, linezolid, tigecycline, ampicillin, imipenem, and metals; and Cu tolerance genes. In piglets with Zn and Cu high or toxic levels, enterococci had reduced susceptibility to ions, reinforced by the presence of Cu tolerance genes and by resistance to antibiotics. The study relevance is to show the relationship between these metals' levels and decreased susceptibility to Cu, Zn, and antibiotics by enterococci. From the results, it could be supposed that the piglets were being fed with high doses of Zn and Cu which could select more resistant bacteria to both antibiotics and metals that could spread to environment and humans.

Antibiotic Residues and Zinc Concentrations in the Livers and Kidneys of Portuguese Piglets-Relationship to Antibiotic and Zinc Resistance in Intestinal Escherichia coli.

Metal ions such as zinc and copper have been used as alternatives to antibiotics, to improve animal health and growth rates in pig farming. This study aims to determine antibiotic residues and Zn concentration in piglets' livers (n = 56) and kidneys (n = 60); and to examine the correlation between the use of Zn and antibiotics, and resistance to Zn and antibiotics of Escherichia coli isolated from piglets' faeces (n = 60). Samples were collected from randomly selected healthy piglets (n = 60); antibiotic residues were quantified by ultra-high-performance-liquid-chromatography time-of-flight mass spectrometry (UHPLC-ToF-MS); Zn was quantified using flame atomic absorption spectrometry (FAAS); microbiological methods were used for E. coli isolation, antibiotic susceptibility, and Zn minimal inhibitory concentration; and Real-Time PCR was used for gene detection. The presence of antibiotic residues and Zn concentrations in the liver was found to be negatively correlated, whilst no significant difference was observed in the kidney. In E. coli isolated from piglet faeces considered to be susceptible or multi-drug-resistant, no significant difference was found between Zn concentrations in the liver and in the kidney, which appears to indicate that Zn accumulated in the liver and in the kidney does not promote resistance to antibiotics in E. coli. The isolates showed tolerance to Zn which would suggest that antibiotic resistance and phenotypic tolerance to Zn in these isolates are not related. The genes zitB and zntA associated to Zn tolerance, were predominantly found in the more resistant Zn isolates. The findings provide insights on how Zn use in pig production maintains antibiotic resistance and metal tolerance in bacteria, with implications for One Health.

Surveillance in ulcerative colitis: is chromoendoscopy-guided endomicroscopy always better than conventional colonoscopy? A randomized trial.

BACKGROUND: Ulcerative colitis (UC) is associated with an increased risk of colorectal cancer. Chromoendoscopy showed superiority to conventional colonoscopy (CC) in surveillance studies including high-risk patients. We aimed to compare chromoendoscopy-guided endomicroscopy (CGE) with CC for intraepithelial neoplasia (IN) detection in patients with longstanding UC without primary sclerosing cholangitis and/or history of IN. METHODS: One hundred sixty-two patients with longstanding (≥ 8 yr) distal/extensive UC and without primary sclerosing cholangitis and/or history of IN were prospectively randomized to undergo CGE (group A) or CC (group B). Seventeen patients were excluded. In group A (n = 72), circumscribed lesions highlighted by pan-chromoendoscopy were evaluated by endomicroscopy, and targeted biopsy/polypectomy was performed. In group B (n = 73), 4 random biopsies every 10 cm and targeted biopsy/polypectomy of detected lesions were performed. RESULTS: Thirteen IN, all low grade, were detected: 7 IN in group A and 6 in group B (P > 0.05), distributed, respectively, by 6 and 4 patients (P > 0.05). Significantly, more biopsies were performed in group B (4.7 ± 4.9 versus 36.0 ± 6.2, P < 0.001), and the per-biopsy yield of IN was higher in group A (1/48 versus 1/438, P < 0.001). Examination time was 61.5 ± 15.6 minutes in group A and 40.7 ± 8.7 minutes in group B (P < 0.001). The IN detection by endomicroscopy revealed: sensitivity = 85.7%, specificity = 97.9%, positive predictive value = 75.0%, and negative predictive value = 98.9%. CONCLUSIONS: CGE does not improve the detection of IN in the endoscopic screening of patients with longstanding UC without primary sclerosing cholangitis and/or history of IN. CGE takes longer than CC, but it decreases the number of biopsies performed and significantly increases the per-biopsy yield of IN. Endomicroscopy is an accurate tool for IN detection.

NOD2 gene mutations in ulcerative colitis: useless or misunderstood?

PURPOSE: NOD2 mutations have been linked to an increased risk of Crohn's disease and to some of its phenotypes. The association between NOD2 mutations and susceptibility to ulcerative colitis (UC) remains somewhat controversial and potential correlations between these mutations and UC phenotype have not been studied. AIM: To assess whether NOD2 mutations are a risk factor for UC in Portugal and if there are any genotype-phenotype correlations in these patients. METHODS: The three main NOD2 mutations were searched in 200 patients with UC and in 202 healthy controls. RESULTS: NOD2 mutations were present in 28 patients with UC (14.0 %) and in 27 controls (13.4 %) (p = 0.853). Mutation carriers were more likely to receive steroids during the first year of disease than non-carriers (54.2 % vs. 29.6 %, p = 0.018) and among these patients the need for intravenous administration was more frequent in those with the R702W polymorphism (90.0 % vs. 45.5 %, p = 0.014). In patients with severe colitis admitted for intravenous steroids, a greater proportion of mutation carriers was considered intravenous-steroid refractory and required salvage therapy (90.0 % vs. 38.1 %, p = 0.004). Patients with NOD2 mutation were submitted to colectomy more frequently than non-carriers (17.9 % vs. 4.1 %. p = 0.015). No correlation with the need for immunosuppressants/immunomodulators was found. CONCLUSIONS: In the Portuguese population, NOD2 mutations do not increase the risk of UC but are associated with a more aggressive course including greater need of steroids in the first year, increased incidence of intravenous-steroid refractoriness and a higher colectomy rate.

Triple therapy with high-dose proton-pump inhibitor, amoxicillin, and doxycycline is useless for Helicobacter pylori eradication: a proof-of-concept study.

INTRODUCTION: Helicobacter pylori resistance to antibiotics is steadily increasing and multidrug-resistant strains are common and difficult to eliminate, mainly in countries where bismuth, tetracycline, furazolidone, and rifabutin are unavailable. AIM: To evaluate the efficacy and safety of a triple therapy with proton-pump inhibitor (PPI), amoxicillin, and doxycycline in patients with multidrug-resistant H. pylori. PATIENTS AND METHODS: This prospective study involved 16 patients (13 females; mean age - 50 ± 11.3 years) infected by H. pylori with known resistance to clarithromycin, metronidazole, and levofloxacin, but susceptibility to amoxicillin and tetracycline. All patients were previously submitted to upper endoscopy with gastric biopsies for H. pylori culture and susceptibility testing by Etest. Mutations in 23S rRNA and gyrA genes were determined by real-time PCR. A 10-day eradication regimen with PPI (double-standard dose b.i.d.), amoxicillin (1000 mg b.i.d.), and doxycycline (100 mg b.i.d.) was prescribed after pretreatment with PPI during 3 days. Eradication success was assessed by (13) C-urea breath test 6-10 weeks after treatment. Compliance and adverse events were determined through phone contact immediately after treatment and specific written questionnaires. RESULTS: Only one patient did not complete treatment due to adverse events. Another four patients experienced mild side effects not affecting compliance. The control (13) C-urea breath test was positive in all patients. Per-protocol and intention-to-treat eradication rates were 0%. CONCLUSIONS: Although safe, a triple-therapy protocol with high-dose PPI, amoxicillin, and doxycycline is useless for multidrug-resistant H. pylori eradication.

Card15 mutations and gastric cancer in a Portuguese population.

BACKGROUND. CARD15 is involved in the innate immune response and mutations of this gene have been linked with increased risk of Crohn's disease and colorectal cancer. The relation between CARD15 mutations and gastric cancer (GC) remains controversial. AIMS. To assess whether CARD15 mutations are risk factors for GC in Portugal and whether there are genotype-phenotype correlations in these patients. METHODS. The 3 main CARD15 mutations (3020insC, R702W and G908R) were searched in 150 patients with GC and in 202 healthy controls. RESULTS. Overall, CARD15 mutations were found in 28 patients (18.7%) and in 27 controls (13.4%) (p = 0.176). Individually, the incidence of 3020insC was significantly higher in patients than in controls (6.0% vs. 1.0%, p = 0.021). This polymorphism was linked with an increased risk for the intestinal-type of GC (p = 0.002), while no association was found with the diffuse and/or mixed types. Genotype frequencies for R702W (10.0% vs. 7.9%) and G908R (4.0% vs. 4.0%) were not statistically different between the two groups. Similarly, no significant associations were detected between these two polymorphisms and the different histological GC types. No correlations were observed between CARD15 mutations and family history, mean age at diagnosis or GC stage. CONCLUSIONS. The CARD15 3020insC variant is a risk factor for intestinal GC in Portugal. CARD15 variants are not correlated with age of diagnosis or family aggregation of the disease neither with the GC stage.

CARD15 mutations and perianal fistulating Crohn's disease: correlation and predictive value of antibiotic response.

BACKGROUND: CARD15 mutations alter bowel immunity and increase susceptibility to Crohn's disease (CD). However, the relation between these mutations and Crohn's perianal fistulas has not been fully clarified. AIMS: To assess whether CARD15 mutations are associated with risk of developing Crohn's perianal fistulas and whether these mutations are predictors of the response of perianal fistulas to antibiotics. METHODS: CARD15 mutations were investigated in 203 consecutive CD patients. Presence/absence of history of perianal fistula was recorded. Patients with history of perianal fistula were divided into two groups (with/without CARD15 mutations), and response to antibiotics was evaluated in both groups. RESULTS: Of the 203 patients, 60 (29.6%) showed at least one CARD15 mutation and 55 (27.1%) had history of perianal fistula. History of perianal fistula was identified in 13 (21.7%) patients with mutations and in 42 (29.4%) patients without mutations (P = 0.260). Mean age at diagnosis of first perianal fistula was similar in patients with/without CARD15 mutations (28.7 ± 9.8 versus 29.7 ± 10.1 years, P = 0.758). Average time between disease onset and diagnosis of first perianal fistula was also similar in the two groups (4.6 ± 5.1 versus 5.0 ± 5.9 years, P = 0.816). Response of perianal fistulas to antibiotics (metronidazole alone or combined with ciprofloxacin) was significantly higher in patients without CARD15 mutations (7.7% versus 40.5%, P = 0.041). CONCLUSIONS: In CD, CARD15 mutations are not associated with risk of developing perianal fistulas or with time of their outbreak. Nevertheless, patients with perianal fistulas and CARD15 mutations showed worse response to antibiotics.

CARD15 mutations and colorectal cancer in a South European country.

PURPOSE: CARD15 mutations are associated with higher susceptibility to Crohn's disease (CD) and longstanding colonic CD increases the risk of developing colorectal cancer (CRC). The relation between these mutations and sporadic CRC remains controversial. The aim of this study was to assess whether germline and/or somatic CARD15 mutations are risk factors for sporadic CRC in Portugal and whether there are genotype-phenotype correlations in these patients. METHODS: The three main CARD15 mutations (R702W, G908R and 3020insC) were researched in 112 sporadic CRC patients and 152 healthy subjects. RESULTS: Overall, CARD15 mutations were found in 18 patients (16.1%) and in 15 controls (9.9%; p = 0.132). Individually, the incidence of R702W was significantly higher in patients than in controls (12.5% vs. 5.3%, p = 0.035), whereas the genotype frequencies for G908R (2.7% vs. 3.3%) and 3020insC (0.9% vs. 1.3%) were not statistically different between the two groups. Entire genotypic agreement was found in patients genotyped for blood and neoplastic DNA. A significantly higher incidence of CARD15 mutations was detected in patients with CRC diagnosed under 60 years old (28.6% vs. 10.4%, p = 0.015) and in female patients (24.4% vs. 10.4%, p = 0.048). No associations were found between CARD15 mutations and family history, symptoms or CRC pathologic characteristics. CONCLUSIONS: The CARD15 R702W variant might be a predisposing factor to sporadic CRC in Portugal, particularly in patients under 60-years old and in female patients. This susceptibility appears to be linked with germline CARD15 mutations. Nevertheless, we have found no evidence that CARD15 mutations predict the pathologic characteristics of CRC.