Biomedical subjects
Publications and source records attributed to P Brigidi.
The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.
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A number of N-substituted 3-methyl-4-diazo-5-pyrazolecarboxamides (IVa-p) have been synthesized and tested for their in vitro antibacterial activity. All the compounds have been assayed against several representative Gram-negative and Gram-positive bacteria, as well as against some intestinal bacterial species. Some of the 4-diazopyrazoles IVa-c have shown a quite interesting broad-spectrum activity, while they are ineffective against the "protective" intestinal flora. The structure-activity relationships of the series have been studied quantitatively, via both univariate and multivariate methods: the results are consistent and permit some rationalization of the behaviour of the compounds investigated.
A short series of di-functionalized benzodifurans and phenanthrolines were synthesized for in vitro antimicrobial activity. Dicarboxaldehydes, chiefly those with a phenanthroline supporting moiety, proved to be most effective, showing significant fungal growth inhibition.
A short series of N-monosubstituted (aryl, aminoacyl, dipeptidyl)-2-(2-aminothiazol-4-yl)-(Z) -2-methoxyiminoacetamides was synthesized and tested for antimicrobial activity. A few members showed a somewhat interesting inhibitory action against Cryptococcus neoformans (MIC = 150 micrograms/ml).
Some benzyloxyimio compounds, related to oxiconazole and having a 1H-indole or 1H-benzimidazole moiety, have been synthesized and tested in vitro for their antimycotic activity against Candida tropicalis and C. albicans. The most active was showed to be 0-(2,4-dichlorobenzyl)-1-benzyl-5-nitro-1H-benzimidazole-2-carboxaldehyd e oxime (MIC: 25 micrograms/ml against both microorganisms). A structural feature important for the biological activity of the series appears to be presence of a benzimidazole nucleus substituted by an electron withdrawing group.
Conditions for protoplast formation and cell wall regeneration in the genus Bifidobacterium are described. The ability to form protoplast in high percentage varied according to the species considered. High reversion levels were obtained with the species B. bifidum var. pennsylvanicus, B. thermophilum and B. boum.
The in vitro antimicrobial activity of the title polymers is described. Iodoethylation leads to a sensible improvement of activity only when the amino groups present in the main backbone of the polymers are connected by a long aliphatic chain.
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Some derivatives of N'-(5-nitro-2-furoyl)-N2-(3-chloro-1H-indenyl-2-methylene)hydrazine (I) were synthesized and screened for in vitro antimicrobial activity. None was more active than the parent compound and the greater hydrophilic character was associated with loss of inhibitory activity against gram-negative bacteria.
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The gene encoding beta-galactosidase was isolated by functional complementation of Escherichia coli from Bifidobacterium longum MB219, which exhibited the highest activity among ten Bifidobacterium strains tested of the species B. longum, B. breve, B. adolescentis, B. indicum, B. animalis and B. cuniculi. The nucleotide sequence of the 5.0-kb fragment conferring the positive beta-galactosidase phenotype to E. coli revealed the presence of a lacZ-type gene encoding a 1023-amino-acid protein that was preceded by a ribosome binding site. A sequence showing 72% identity with the proline tRNA of Bacillus subtilis and a gene probably encoding the DNA-3-methyladenine glycosydase I were located downstream from the lacZ gene, after a gap of 30-50 unsequenced base pairs. By primer-extension analysis, the transcription start site of the lacZ gene was mapped 65 nt upstream from the start codon, and it enabled identification of the -10 region of the putative promoter. The nucleotide sequence of lacZ and its deduced amino acid sequence were compared with those of beta-galactosidase genes and enzymes from other microorganisms. High similarity was demonstrated between the B. longum beta-galactosidase and its counterparts in Lactobacillus delbruckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, E. coli, Clostridium acetobutylicum, Leuconostoc lactis, Klebsiella pneumoniae and Kluyveromyces marxianus var. lactis, all belonging to the LacZ family. The B. longum MB219 lacZ gene was cloned in Bifidobacterium and its expression was observed in strains with otherwise low levels of endogenous activity. The expression increased by factors of 1.5-50 and enabled those strains that do not grow on lactose to use this sugar as sole carbon source.
The nucleotide sequence of the 1847-bp Bifidobacterium longum B2577 cryptic plasmid pMB1 was determined. The plasmid had a G+C content of 62.0%, and contained two open reading frames, orf1 and orf2, likely arranged in an operon. The proteins encoded by orf1 and orf2 show the highest degree of similarity with similarly arranged peptide sequences translated from Corynebacterium glutamicum pXZ10142 and Mycobacterium fortuitum pAL5000 plasmids. Recombinant plasmids containing the pMB1 replicon were able to replicate in Bifidobacterium animalis MB209. The successful transformation of this strain with pMB1-based plasmids facilitated characterization of this replicon, results of which showed that both orf1 and orf2 are necessary for plasmid replication. A family of new Escherichia coli-B. animalis shuttle plasmids, based on the pMB1 replicon and expressing a cat and an ery gene, was constructed.
Urease activity in the stomach (fundus and antrum), caecal content and soft faeces of rabbit was studied. Significant differences between fundus and antral content (P less than 0.01) and between caecum and soft faeces (P less than 0.05) were observed. The urease zymograms from caecal content and soft faeces of rabbit presented two different bands. The fundus content and the caecal ureolytic Clostridium innocuum bacterium exhibited only one band. Band B of caecal content was not evident at pH 4, whereas band A, also present in the stomach, was observed both at pH 4 and at pH 6. The optimal pH of urease activity of stomach and caecal content was in the range of 4-5 and 5-6, respectively. A comparison of intestinal urease zymograms with those of the single ureolytic bacterial species was suggested in order to clarify their role in urea metabolism.
The etiology of inflammatory disease is still unknown, but a body of evidence from clinical and experimental observation indicates a role for intestinal microflora in the pathogenesis of this disease. Reduction of microflora using antibiotics, bowel rest and fecal diversion decreases activity in Crohn's disease and in ulcerative colitis. Several trials have been carried out on the use of antibiotic treatment in patients with active ulcerative colitis with contrasting results. A number of trials have been carried out using Rifaximin, a non-absorbable broad-spectrum antibiotic, confirming the absence of systemic bioavalaibility of the drug even when administered at very high doses and for prolonged periods. It may therefore be useful in treatment of ulcerative colitis and pouchitis, since its absorption through inflamed mucosa is negligible, it maintains a topical action without systemic effects and the lack of resistant bacterial strains may allow prolonged and repeated treatments.
The susceptibility pattern of 459 strains of bifidobacteria, representing 15 species, to 16 antimicrobial agents was determined by the broth dilution method. The majority of the strains derived from human faeces. Penicillin G, erythromycin, clindamycin, vancomycin and bacitracin were the most active compounds; they inhibited 90% of the strains at less than 1.6 micrograms/ml. All strains were susceptible to chloramphenicol (MIC90 = 2.0-5.8 micrograms/ml) and also to lincomycin (MIC50 = 0.64-1.5 micrograms/ml). Neomycin, streptomycin and tetracycline presented a great variability in their activity. Most strains were resistant to polymyxin B, nalidixic acid, kanamycin, gentamicin and metronidazole. The only variation in susceptibility which was observed among the different species concerned Bifidobacterium suis, which generally appeared to be more resistant than other species.