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Escherichia coli K92 is an opportunistic pathogen bacterium able to produce polysialic acid (PA) capsules when grows at 37 degrees C. PA polysaccharides are cell-associated homopolymers tailored from acid sialic monomers that function as virulence factors in different neuroinvasive diseases caused by certain Enterobacteriaceae. Conversely, when grows at 19 degrees C (restrictive conditions), PA synthesis was negligible, whereas in such condition, a slimy substance started to be accumulated in the culture broths. Analysis by uronic acids colorimetric determinations, gas chromatography-mass spectrometry, and Fourier transform infrared spectroscopy allowed the isolation and identification of mucoid substance as colanic acid (CA). CA is a heteropolymer containing glucose, galactose, fucose, and glucuronic acid as monomers which seems to be involved in the protection of this bacterium against environment assaults. The study of physicochemical conditions required for CA synthesis revealed that in E. coli K92, nutrient (carbon and nitrogen sources) modulates CA production, reaching the maximal values when glucose and proline were as carbon and nitrogen sources, respectively. Furthermore, we have found that E. coli K92 is able to produce CA at all temperatures tested (from 42 degrees C to 15 degrees C), whereas PA synthesis only occurred when bacteria were cultured at temperatures higher than 25 degrees C. Additionally, genetic engineering approaches revealed that the CA cluster including several genes required for synthesis was placed into a DNA fragment of 100 kb using polymerase chain reaction methodology.

ObjectiveTo identify, purify, and characterize the proteins responsible for glutenase activity in the feces of healthy subjects and patients with celiac disease (CD).MethodsSixteen subjects were included in this study; 8 were healthy with no known food intolerances, and 8 were treated CD patients on a gluten-free diet. Fecal samples were homogenized, and precipitated proteins were purified by chromatography. Glutenase activity was evaluated by bioassays, zymography, and high-performance liquid chromatography with immunogenic 33-mer, 19-mer, and 13-mer gliadin peptides.ResultsThe gastrointestinal elastase 3B (CEL3B), elastase 2A (CEL2A), and carboxypeptidase A1 (CBPA1) enzymes degraded human gluten. These proteins fully hydrolyzed 13-mer and 19-mer gliadin peptides that trigger immune-mediated enteropathy in individuals genetically predisposed to CD and partially digested a 33-mer. Feces from patients with CD showed more glutenase activity than feces from individuals without CD (171–466% higher). Peptidase activity against the gliadin peptides also increased in patients with CD.ConclusionThe digestive tracts of patients with CD and healthy subjects have enzymatic machinery needed for gluten degradation. Patients with CD showed more gluten hydrolysis than did healthy individuals, although, in both cases, a fraction of 33-mer peptide remained intact. Gliadin peptides derived from gastrointestinal digestion, especially the 33-mer, can potentially be used by commensal microbiota from both CD-positive and CD-negative individuals, and differences in bacterial hydrolysis can modify its immunogenic capacity.

In the present study, the efficacy of generally recognised as safe (GRAS) antimicrobial plant metabolites in regulating the growth of Staphylococcus aureus and S. epidermidis was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against these microorganisms, at a subinhibitory concentration (SIC) of ≤ 50 μg ml(-1). Genistein, hydroquinone and resveratrol showed antimicrobial effects but with a wide concentration range (SIC = 50-1,000 μg ml(-1)), while catechin, gallic acid, protocatechuic acid, p-hydroxybenzoic acid and cranberry extract were the most biologically compatible molecules (SIC ≥ 1000 μg ml(-1)). Genistein, protocatechuic acid, cranberry extract, p-hydroxybenzoic acid and resveratrol also showed anti-biofilm activity against S. aureus, but not against S. epidermidis in which, surprisingly, these metabolites stimulated biofilm formation (between 35% and 1,200%). Binary combinations of cranberry extract and resveratrol with genistein, protocatechuic or p-hydroxibenzoic acid enhanced the stimulatory effect on S. epidermidis biofilm formation and maintained or even increased S. aureus anti-biofilm activity.

Phenylacetic acid (PhAcOH) and 4‐hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonas putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups: those blocked in the gene encoding 4‐hydroxyphenylacetic acid‐3‐hydroxylase; and those blocked in the gene encoding homoprotocatechuate‐2,3‐dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups: those which contain a functional PhAc‐CoA ligase protein; and those lacking this enzyme activity.