Optimization of lignin precipitation from black liquor using organic acids and its valorization by preparing lignin nanoparticles.

This work focuses on the precipitation of lignin from kraft black liquor (BL) along with its valorization into lignin nanoparticles (LNP). Two organic acids namely, acetic acid, and lactic acid were used for the precipitation of lignin as an alternative to sulfuric acid. An optimization study was carried out to determine the effect of three key variables, namely acid type, temperature, and pH, on the isolation yield and purity of lignin. The study showed that all factors primarily influenced the lignin yield, while the purity of precipitated lignin varied only around 1 % between minimum to maximum purity. Further, the acid precipitation method was selected for the preparation of LNP. The study aimed to observe the effect of pH, lignin concentration, and surfactant concentration over the properties of the prepared nanoparticles. The results showed that a smaller nanoparticle size and maximization of phenolic content was achieved with a lignin concentration of 35 mg/mL, a surfactant concentration of 10 % (w/w lignin), and a pH of 5. Additionally, the antibacterial activity of LNPs against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa bacteria was evaluated. The results showed only minor activity against Staphylococcus aureus. Overall, the study demonstrates the potential method for precipitation and valorization of lignin through the production of LNP with desirable properties.

Exploiting the Crithmum maritimum L. Aqueous Extracts and Essential Oil as Potential Preservatives in Food, Feed, Pharmaceutical and Cosmetic Industries.

Chritmum maritimum, sea fennel, is a facultative halophyte used in salads, soups, and sauces, as well as used to prepare medicinal juices and aqueous extracts (AE) to treat several ailments. Its essential oil (EO) is used as a spice and aromatizing. In this work, the nutritional (crude protein, fiber, lipids, and ashes content) and HPLC-PDA phenolic profiles were determined. Furthermore, the antioxidant potential of the infusion and of the decoction, as well as the antibacterial activity of both, the AE and EO, were assessed against food-contaminating bacteria. The composition of the EO was also established. Sea fennel exhibited considerable fiber (34.3 ± 1.92%) and mineral content (23.6 ± 4.8%). AE contains chlorogenic acid as the major phenolic compound, 49.7 ± 0.8 mg/g in the infusion dry extract and (26.8 ± 0.9 mg/g in the decoction dry extract). EO contains high amounts of monoterpene hydrocarbons, namely γ-terpinene and sabinene. In regards to the antioxidant activity, IC50 values for the infusion and decoction were, respectively: 36.5 ± 1.4 µg/mL and 44.7 ± 4.4 µg/mL in the DPPH assay; 37.3 ± 2.6 µg/mL and 38.4 ± 1.8 µg/mL, in the ABTS assay. EO is particularly active against Bacillus cereus and Lactobacillus plantarum. The results support the use of sea fennel AE and EO as a potential alternative preservative ingredient for feeds, foods, pharmaceutical, and cosmetic industries, due to the antioxidant activity of infusion and decoction, and antibacterial properties of essential oil.

Plasmid-Encoded AmpC and Extended-Spectrum Beta-Lactamases in Multidrug-Resistant Escherichia coli Isolated from Piglets in Portugal.

Considering the concept of "One Health," the aim of this study was to determine susceptibility profiles of Escherichia coli in piglets' intestinal microbiota from different farms in Portugal. Beyond antimicrobial susceptibility, the occurrence of multiple antibiotic resistance and detection of phenotypic/genotypic extended-spectrum beta-lactamases (ESBLs) and plasmid mediated AmpC beta-lactamases (pAmpC) were done. From 10 different pig farms, 340 E. coli isolates were obtained from 75 feces samples. Susceptibility to amoxicillin-clavulanic acid (AMC), piperacillin (PIP), cefoxitin (FOX), ceftazidime (CAZ), cefepime (FEP), aztreonam (AZT), imipenem (IP), amikacin (AK), ciprofloxacin (CIP), and trimethoprim-sulfamethoxazole (SXT) was determined. Five-gene panel for amplification of bla genes was used for ESBL (TEM, SHV, CTX-M) and pAmpC (CMY-2, ACC). Among E. coli isolates, 209 were distributed in three resistance profiles: 57.7% MDR, 3.5% extensively drug-resistance (XDR) (resistant to CIP, SXT, and beta-lactams, except IP, with variability to AK) and 0.3% pandrug-resistance (PDR) (resistant to all antibiotics used). pAmpC and/or ESBLs genes were presented in 65% of the isolates. Presence of different associations of bla genes in the same isolate was the most observed (31%), and the most common were an ESBL (TEM) and a pAmpC (CMY-2). Presence of three or four bla genes in various associations were detected. These isolates were very resistant, especially those with four genes, which were resistant to beta-lactams (except IP), CIP, and SXT. This study showed a surprisingly high rate of MDR E. coli isolated in Portuguese piglets, with enzymes that impair activity of the most used antibiotics in human therapeutic.

Study of rdxA and frxA genes mutations in metronidazole-resistant and -susceptible Helicobacter pylori clinical isolates from the central region of Portugal.

OBJECTIVES: Helicobacter pylori is one of the most prevalent global pathogens colonising an estimated 50% of the world's population. Although metronidazole (MTZ) is an important antibiotic playing a relevant role in various H. pylori eradication therapies, its frequent consumption results in an increased frequency of resistance with a consequent negative impact on treatment efficacy. Mutations on genes encoding NADPH nitroreductases, commonly known as rdxA gene (oxygen-insensitive) and frxA gene (flavinreductase) have been associated to H. pylori resistance to metrodinazole. The aim of this study was to evaluate the mutation profile of rdxA and frxA genes in a population of 38 H. pylori isolates with phenotypic patterns of susceptibility and resistance to this antibiotic. METHODS: Touchdown PCR with the purpose of amplifying rdxA and frxA genes in one PCR was used. Sequence data were made by pair-wise sequence alignment and were examined in terms of codons, and comparison was achieved regarding amino acids. RESULTS: Although repeated mutations occurred in positions 118, 131, 172 and 183 of rdxA and in positions 72, 73, 110, 126 and 193 of frxA, it must be highlighted that the mutations are widespread along these two genes in this population. Furthermore, six MTZ-resistant isolates did not present any mutation in the frxA gene. CONCLUSIONS: This work appears to confirm that mutations in rdxA and frxA alone are unable to explain MTZ resistance in H. pylori isolates and therefore additional mechanisms may exist and should be investigated.

Twelve-day quintuple regime containing four antibiotics as a rescue therapy for Helicobacter pylori eradication in the central region of Portugal.

BACKGROUND: Helicobacter pylori eradication rates with standard triple therapy in many countries are clinically unacceptable. Fluoroquinolone resistance is increasing and jeopardizing second-line regimens. There is a growing need for an effective strategy in patients who failed previous therapies. METHODS: This is a single-center, non-randomized clinical study conducted in the central region of Portugal. Sixty-four patients were included with a positive 13C-urea breath test (UBT) or histology for H. pylori, and at least one failed eradication attempt. The patient cohort included 71.7% of females with a median of age of 52 (range 23-87). They were treated with a twelve-day regimen consisting of a proton-pump inhibitor (PPI) bid, amoxicillin at 1,000 mg 12/12 h and levofloxacin at 500 mg bid during the first seven days, followed by PPI bid, clarithromycin at 500 mg 12/12h and either tinidazole or metronidazole at 500 mg bid/tid for five days. Eradication was assessed by UBT. The local Ethics Committee approved this study. RESULTS: Eradication therapy was prescribed due to dyspepsia (66.7%), peptic ulcer (10%) and thrombocytopenia (8.3%). The median number of failed therapies was one (range 1-4). The eradication rate was 64.6% according to an intention-to-treat analysis (95% CI: 53-77%), and 70% by the per-protocol analysis (95% CI: 58-82%). Age, smoking, indication for eradication, previous therapies and the use of a second-generation or full-dose PPI did not affect success rates. CONCLUSIONS: Even though treatment with four antibiotics was used, this "reinforced" therapy achieved suboptimal results. This fact highlights the lack of effective H. pylori antimicrobials and suggests that second-line treatment in our region should be prescribed according to susceptibility testing.

Levofloxacin or Clarithromycin-based quadruple regimens: what is the best alternative as first-line treatment for Helicobacter pylori eradication in a country with high resistance rates for both antibiotics?

BACKGROUND: Helicobacter pylori eradication rates in Portugal are declining, due to increased resistance of this bacterium to antimicrobial agents, especially Clarithromycin. Quadruple Levofloxacin-containing regimens could be an option for first-line treatment, but its efficacy should be evaluated as fluoroquinolone resistance is rapidly increasing. Our aim was to compare the efficacy of Clarithromycin and Levofloxacin-based sequential quadruple therapies as first-line treatment options and determine factors associated with treatment failure. METHODS: A total of 200 Helicobacter pylori infected patients were retrospectively included (female 57.5%; average age: 53.2 ± 15.7) and received either 10-day sequential therapy (Proton-Pump Inhibitor + Amoxicillin 1 g bid for 5 days and Proton-Pump Inhibitor + Clarithromycin 500 mg + Metronidazole/Tinidazole 500 mg bid/tid in the following 5 days; group A) or a 10-day modified sequential therapy with Levofloxacin 500 mg id instead of Clarithromycin (group B). Eradication was confirmed with urea breath test. Variables that could influence success rate were analyzed. RESULTS: There were no differences between groups in terms of gender, age, smoking habits and indications for treatment. The eradication rate obtained with Clarithromycin-based sequential treatment was significantly higher than with Levofloxacin-based therapy (90%, CI95%: 84-96% vs. 79%, CI95%: 71-87%, p = 0.001). Using full-dose proton-pump inhibitor and high-dose Metronidazole in group A, and full-dose proton-pump inhibitor and prescription from a Gastroenterologist in group B were associated with eradication success. CONCLUSIONS: Ten-day Levofloxacin-based sequential treatment achieved inadequate efficacy rate (<80%) and should not be adopted as first-line therapy. Standard sequential therapy showed significantly better results in this naïve population. Using full-dose proton-pump inhibitor and higher doses of Metronidazole is essential to achieve such results.

Beyond Maastricht IV: are standard empiric triple therapies for Helicobacter pylori still useful in a South-European country?

BACKGROUND: Empiric triple treatments for Helicobacter pylori (H. pylori) are increasingly unsuccessful. We evaluated factors associated with failure of these treatments in the central region of Portugal. METHODS: This single-center, prospective study included 154 patients with positive (13)C-urea breath test (UBT). Patients with no previous H. pylori treatments (Group A, n = 103) received pantoprazole 40 mg 2×/day, amoxicillin 1000 mg 12/12 h and clarithromycin (CLARI) 500 mg 12/12 h, for 14 days. Patients with previous failed treatments (Group B, n = 51) and no history of levofloxacin (LVX) consumption were prescribed pantoprazole 40 mg 2×/day, amoxicillin 1000 mg 12/12 h and LVX 250 mg 12/12 h, for 10 days. H. pylori eradication was assessed by UBT 6-10 weeks after treatment. Compliance and adverse events were assessed by verbal and written questionnaires. Risk factors for eradication failure were determined by multivariate analysis. RESULTS: Intention-to-treat and per-protocol eradication rates were Group A: 68.9% (95% CI: 59.4-77.1%) and 68.8% (95% CI: 58.9-77.2%); Group B: 52.9% (95% CI: 39.5-66%) and 55.1% (95% CI: 41.3-68.2%), with 43.7% of Group A and 31.4% of Group B reporting adverse events. Main risk factors for failure were H. pylori resistance to CLARI and LVX in Groups A and B, respectively. Another independent risk factor in Group A was history of frequent infections (OR = 4.24; 95% CI 1.04-17.24). For patients with no H. pylori resistance to CLARI, a history of frequent infections (OR = 4.76; 95% CI 1.24-18.27) and active tobacco consumption (OR = 5.25; 95% CI 1.22-22.69) were also associated with eradication failure. CONCLUSIONS: Empiric first and second-line triple treatments have unacceptable eradication rates in the central region of Portugal and cannot be used, according to Maastricht recommendations. Even for cases with no H. pylori resistance to the used antibiotics, results were unacceptable and, at least for CLARI, are influenced by history of frequent infections and tobacco consumption.

Correlation of Helicobacter pylori genotypes with gastric histopathology in the central region of a South-European country.

BACKGROUND: Outcome of Helicobacter pylori (H. pylori) infection results from interaction of multiple variables including host, environmental and bacterial-associated virulence factors. AIM: This study aimed to investigate the correlation of cagA, cagE, vacA, iceA and babA2 genotypes with gastric histopathology and disease phenotype in the central region of a South-European country. METHODS: This prospective study involved 148 infected patients (110 female; mean age 43.5 ± 13.4 years) submitted to endoscopy with corpus and antrum biopsies. H. pylori was cultured and DNA extracted from the isolates. Genotypes were determined by PCR. Histopathological features were graded according to the updated Sydney system and OLGA/OLGIM classification. Only patients with single H. pylori genotypes and complete histopathological results were included. RESULTS: Antrum samples presented higher degrees of atrophy, intestinal metaplasia, chronic inflammation and neutrophil activity. Genotype distribution was as follows: cagA-31.8 %; cagE-45.9 %; vacA s1a-24.3 %; vacA s1b-19.6 %; vacA s1c-0.7 %; vacA s2-55.4 %; vacA m1-20.9 %; vacA m2-79.1 %; vacA s1m1-18.9 %; vacA s1m2-25.7 %; vacA s2m1-2 %; vacA s2m2-53.4 %; iceA1-33.8 %; iceA2-66.2 %; babA2-12.2 %. CagA genotype was significantly associated with higher degrees of intestinal metaplasia, neutrophil activity, chronic inflammation and OLGIM stages. BabA2 was linked with higher H. pylori density. Strains with vacA s1m1 or vacA s1m1 + cagA positive genotypes had a significant association with peptic ulcer and vacA s2m2 with iron-deficient anemia. CONCLUSIONS: cagA, vacA s1m1 and babA2 genotypes are relatively rare in the central region of Portugal. cagA-positive strains are correlated with more severe histopathological modifications. This gene is commonly associated with vacA s1m1, and such isolates are frequently found in patients with peptic ulcer.