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Anthocyanins are extensively studied for their health-related properties, including antibacterial activity against urinary tract infections (UTI). Among common fruits, blueberries, with their remarkable antioxidant capacity, are one of the richest sources. Anthocyanin-rich extracts were obtained from four varieties: Snowchaser, Star, Stella Blue and Cristina Blue, grown in the hot climate of Southern Spain. Their total anthocyanins contents (TAC) were determined spectrophotometrically, and the anthocyanin profile by ultra high performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS). Their antioxidant activity was assessed by oxygen radical absorbance capacity (ORAC) assay, while antibacterial activity against strains isolated from UTI patients was assessed in vitro, helping to select the varieties with the highest bioactive potential. Star showed the highest TAC and antioxidant activity (1663 +/- 159 mg of cyanidin-3-O-glucoside (cy-3-O-glu) equivalents/100 g fresh weight (FW), 6345 +/- 601 mu mol Trolox equivalents (TE)/100 g FW, respectively), followed by Cristina Blue, Stella Blue and Snowchaser. As far as we know, this is the first time that cyanidin-3-rutinoside has been identified in blueberries. The extracts inhibited all the tested strains, MICs ranging from 0.4 mg/mL (for Stella Blue extract against UTIP. aeruginosa) to 9.5 mg/mL (for all extracts against UTIK. pneumoniaessp.pneumoniae). This is the first study that assessed in vitro the antibacterial activity of blueberries againstKlebsiella pneumoniae,Providencia stuartiiandMicrococcusspp. strains isolated from UTI.

Using the Autodisplay system, a recombinant Escherichia coli strain displaying the dimeric nitrilase from Klebsiella pneumoniae subsp. ozaenae (NitKp) on the cell surface was constructed. Localization of the nitrilase in the cell envelope of E. coli was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis and surface exposure was verified by its accessibility to externally added protease. The whole-cell biocatalyst obtained converted the substrates analyzed in the following order: chloroxynil > bromoxynil > ioxynil > 3-bromo-4-hydroxybenzonitrile (1.67, 0.89, 0.13, and 0.09 mM product formation within 72 h, respectively), indicating the same substrate specificity for the displayed enzyme as for the free enzyme. The whole-cell biocatalyst was also able to convert 3-fluoro-4-hydroxybenzonitrile and 3,5-dimethyl-4-hydroxybenzonitrile to the corresponding carboxylic acids. In contrast, it was not possible to detect any enzyme activity when 4-methoxybenzonitrile was used as substrate. The temperature optimum determined was 45 °C for the surface-displayed enzyme instead of 35 °C for the purified enzyme. In addition, the optimum activity of the displayed nitrilase was shifted to more acidic pH in comparison to the free enzyme.

Pyrogallol is a valuable phenolic compound and displays various physiological and pharmaceutical functions. Chemical synthesis of pyrogallol suffered from many issues, including environmental pollution, high cost, and low yield. Here, to address the above drawbacks, an artificial pathway for de novo pyrogallol production was established and this pathway only needed two exogenous enzymes (Y385F/T294A PobA and 3,4-dihydroxybenzoic acid decarboxylase (PDC)). Y385F/T294A PobA is a mutant of PobA which is a hydroxylase from Pseudomonas aeruginosa, while PDC is a decarboxylase from Klebsiella pneumoniae subsp. pneumoniae. First, the conversion efficiency of PDC was tested and 1800 ± 100 mg/L pyrogallol was generated from 4 g/L gallic acid (GA). Subsequently, assembly of the whole pathway enabled 33 ± 6 mg/L pyrogallol production from simple carbon sources. After that, based on the assembling property of CipA (a hydrophobic protein) and to enhance the hydroxylation of 3,4-dihydroxybenzoic acid, CipA was employed to organize its fusion (Y385F/T294A PobA) into protein crystalline inclusions (PCIs). Remarkably, the formation of CipA-Y385F/T294A PobA PCIs increased the pyrogallol production to 60 ± 6 mg/L, a 1.8 ± 0.4-fold higher value as compared to the strain without enzyme self-assembly. Additionally, the titer of pyrogallol was enhanced to 80 ± 1 mg/L through yeast extract concentration optimization. This work not only realizes the biosynthesis of pyrogallol from renewable carbon sources but also demonstrates that using CipA-mediating enzyme self-assembly could reinforce the hydroxylation efficiency of Y385F/T294A PobA, resulting in the enhancement of pyrogallol production.

Background: The opportunistic pathogen Klebsiella pneumoniae is one of the main causes of pediatric bacterial blood stream infections (BSI), which is complicated with sepsis and high mortality.Objectives: To identify atypical Klebsiella species affecting a sample of infected neonates with low antimicrobial response.Methods: Multidrug resistant blood cultures for Klebsiella from a Neonatal Service, were submitted to molecular identification by sequencing analysis of 16S ribosomal RNA.Results: The mean age of the newborns was 14.7 ± 5.6 days. A total of 6 out of 8 cases were sepsis, 1 case of pneumonia, and 1 a catheter-related infection. The molecular identification showed 3 cases of K. pneumoniae subsp. ozaenae, 2 of K. pneumoniae and K. variicola, and 1 case of K. oxytoca. The highest antimicrobial resistance was against cephalosporins and Trimethoprim/sulfamethoxazole.Conclusions: Klebsiella pneumoniae subsp. ozaenae was responsible for multidrug resistant strains of Klebsiella even in 37.5% of cases. In our clinical setting, the use of Amikacin and carbapenems are still useful to treat neonatal infections by Klebsiella even against K. variicola, which is the most resistant.

Febros river water was sampled weekly, during 35 successive weeks, and analyzed for microbiological (total coliforms, faecal coliforms, faecal streptococci and enterococci) and chemical-physical (ammonia and temperature) parameters. All microbiological parameters were highly correlated with each other and with ammonia, suggesting that the simultaneous determination of all variables currently in use in the evaluation of the microbiological quality of waters is probably redundant, and could be simplified, and that ammonia should be tested as a sentinel parameter of the microbiological pollution load of Febros river. From the strains isolated from positive tubes of the faecal coliforms test (multiple tube fermentation technique) and retested in this assay, Escherichia coli, Klebsiella oxytoca and Klebsiella pneumoniae subsp. pneumoniae strains were positive, indicating that the faecal coliforms test is not totally specific for Escherichia coli, and can detect other bacteria. Considering that these Klebsiella spp. are not necessarily of faecal origin, it was concluded that the faecal coliforms test can overestimate true faecal pollution. From the strains isolated from positive tubes of the faecal coliforms procedure, only Escherichia coli strains were clearly positive in the β-D-glucuronidase test. All other species were negative or very weakly positive, suggesting that the assay of the β-D-glucuronidase activity is less prone to false positives than the faecal coliforms test in the quantification of Escherichia coli in environmental waters.

The flower oil of Evodia lunu-ankenda (Gaertn) Merr. (Rutaceae) obtained by hydrodistillation was analyzed by GC/FID and GC/MS. Thirty-one out of 38 constituents comprising 95.1% of the oil were identified. The major constituents identified were evodione (38.9%), (E)-beta-ocimene (12.4%), isolycodolin (11.7%) and alloevodionol (10.6%). The flower oil showed significant antibacterial activity, especially against the Gram-negative bacteria, Salmonella typhi and Klebsiella pneumoniae.

Enterobacter hormaechei, one of the species within the Enterobacter cloacae complex, is a relevant agent of healthcare-associated infections. In addition, it has gained relevance because isolates have shown the capacity to resist several antibiotics, particularly carbapenems. However, knowledge regarding colonization and virulence mechanisms of E. hormaechei has not progressed to the same extent as other Enterobacteriaceae species as Escherichia coli or Klebsiella pneumoniae. Here, we describe the presence and role of the type 3 fimbria, a chaperone-usher assembled fimbria, which was first described in Klebsiella spp., and which has been detected in other representatives of the Enterobacteriaceae family. Eight Chilean E. cloacae isolates were examined, and among them, four E. hormaechei isolates were found to produce the type 3 fimbria. These isolates were identified as E. hormaechei subsp. hoffmannii, one of the five subspecies known. A mutant E. hormaechei subsp. hoffmannii strain lacking the mrkA gene, encoding the major structural subunit, displayed a significantly reduced adherence capacity to a plastic surface and to Caco-2 cells, compared to the wild-type strain. This phenotype of reduced adherence capacity was not observed in the mutant strains complemented with the mrkA gene under the control of an inducible promoter. Therefore, these data suggest a role of the type 3 fimbria in the adherence capacity of E. hormaechei subsp. hoffmannii. A screening in E. hormaechei genomes contained in the NCBI RefSeq Assembly database indicated that the overall presence of the type 3 fimbria is uncommon (5.94–7.37%), although genes encoding the structure were detected in representatives of the five E. hormaechei subspecies. Exploration of complete genomes indicates that, in most of the cases, the mrkABCDF locus, encoding the type 3 fimbria, is located in plasmids. Furthermore, sequence types currently found in healthcare-associated infections were found to harbor genes encoding the type 3 fimbria, mainly ST145, ST78, ST118, ST168, ST66, ST93, and ST171. Thus, although the type 3 fimbria is not widespread among the species, it might be a determinant of fitness for a subset of E. hormaechei representatives.

This study aimed to assess the antibiotic susceptibility and biofilm formation ability of pathogens isolated from braised fish as well as characterize and evaluate the antibacterial, antiadhesive, and antibiofilm activities of the biosurfactant from Lactobacillus paracasei subsp. tolerans N2 against these pathogens. The susceptibility of six pathogens isolated from braised fish (Escherichia coli EM2, Staphylococcus aureus SA1, Salmonella enteritidis PE1, Pseudomonas aeruginosa CT3, Yersinia enterolitica MH5, Proteus mirabilis MR2, and Klebsiella pneumoniae AG5) to 16 antibiotics revealed multiple resistances with an MAR index greater than 0.3. These pathogens were able to form biofilms with S. aureus SA1, which showed the highest ability. Using biochemical and elemental analyses, FTIR, GC-MS, 1H NMR and high-resolution mass spectrometry, the biosurfactant was characterized as a novel glycolipoprotein made of two congeners of mass 482.28 and 507.27 m/z, respectively. They showed bactericidal and antiadhesive activities against all pathogens. The biosurfactant inhibited biofilm formation by these pathogens and eradicated mature biofilms.

The study focused on developing functional yoghurt edible film oral strip encapsulating viable probiotics ( Streptococcus thermophilus ( S. thermophilus ), Lactobacillus bulgaricus ( L. bulgaricus ), and Lactococcus lactis ( L. lactis )) with pectin biopolymer. The oral strip was optimized with Central Composite Design using independent variables such as functional yoghurt powder (2.5% w/v–7.5% w/v), pectin (1.5% w/v–2.5% w/v), and glycerol (55% w/w–65% w/w). The optimum oral strip was prepared at 7.5% (w/v) yoghurt powder, 2.5% (w/v) pectin, and 57.18% (w/w) glycerol, respectively. The oral strip developed with S. thermophilus , L. bulgaricus , and L. lactis had viability of 8.57 ± 0.04, 8.35 ± 0.04 and 8.44 ± 0.04 (log CFU/strip). Antibacterial zone of inhibition against Klebsiella pneumoniae and Pseudomonas aeruginosa were 12.00 ± 1.00 mm and 11.67 ± 0.58 mm accordingly. Optimized oral strip had moisture content of 9.85 ± 0.25 (%), colour difference (∆E) of 48.19 ± 1.30, tensile strength of 2.82 ± 0.07 (MPa), elongation at break of 12.68 ± 0.45 (%) and Youngs modulus as of 0.22 ± 0.01, respectively. Additionally, scanning electron microscopy of oral strip showed well-incorporated yoghurt powder in the film matrix. In the present study, nutritional improvements were developed with acceptable functional properties and probiotic viability.

Spray-drying transforms functional yogurt into yogurt powder quickly and efficiently, maintaining product quality and enhancing storage stability. This process also adds value to the product. The study aimed to develop spray dried functional yoghurt powder encapsulating probiotics ( Streptococcus thermophilus ( S. thermophilus ), Lactobacillus bulgaricus ( L. bulgaricus ), and Lactococcus lactis ( L. lactis )) with longer shelf life. The production of functional yoghurt powder was optimized with Box–Behnken design using independent variables inlet air temperature (110–170 °C), feed temperature (10–20 °C), and pump speed (10–20%). The optimum functional yoghurt powder was obtained at 164.04 (164) °C inlet air temperature, 20 °C feed temperature, and 10.72 (11) % pump speed, respectively. The functional yoghurt powder with amorphous nature had storage viability (log CFU g −1 ) of 8.88 ± 0.03, 6.71 ± 0.12 and 8.16 ± 0.10 for S. thermophilus , L. bulgaricus , and L. lactis correspondingly on 49th day at 4 °C. The functional yoghurt powder demonstrated antibacterial activity, resulting in inhibition zones measuring approximately 18.33 ± 1.15 mm and 11.00 ± 0.00 mm against Klebsiella pneumoniae and Pseudomonas aeruginosa , respectively. Functional yoghurt powder had water activity 0.35–0.52, moisture content 7.27–16.22 (%), process yield 2.29–5.33 (%), hygroscopicity 10.09–16.87 (g/100 g) and degree of caking as 37.46–59.02 (%). The bulk densities, tapped densities, particle density and porosity of yoghurt powder ranged from 242 to 425 (kg m −3 ), 301.9–485.7 (kg m −3 ), 1050–2500 (kg m −3 ), and 58.67–87.85 (%) respectively. Furthermore, the solubility, dispersibility, and wettability of the powder ranged from 33 to 60%, 46.86–86.37%, and 2.13–4.28 min, respectively. The current study successfully achieved optimization at an inlet temperature of 164.04 °C (164 °C), a feed temperature of 20 °C, and a pump speed of 10.72% (11%), with desirability of 72.6%. This study makes a substantial advancement in the production of probiotic yoghurt powder infused with clove, achieving the recommended levels of viability, shelf life, powder characteristics, reconstitution capability, and antibacterial effects. Moreover, the spherical morphology of the functional yoghurt powder was revealed through scanning electron microscopy.