Gianfranco Amicosante - Academia.edu (original) (raw)

Papers by Gianfranco Amicosante

Advances in traditional medicine, Mar 30, 2023

Antimicrobial Agents and Chemotherapy, Jun 1, 1998

A plasmid-encoded ␤-lactamase produced from a clinical strain of Providencia stuartii has been pu... more A plasmid-encoded ␤-lactamase produced from a clinical strain of Providencia stuartii has been purified and characterized. The gene coding for the ␤-lactamase was cloned and sequenced. It appears to be a new natural TEM-derived enzyme, named TEM-60. Point mutations (Q39K, L51P, E104K, and R164S) are present with respect to the TEM-1 enzyme; the mutation L51P has never been previously reported, with the exception of the chromosomally encoded extended-spectrum ␤-lactamase PER-1. Kinetic parameters relative to penicillins, cephalosporins, and monobactams other than mechanism-based inactivators were related to the in vitro susceptibility phenotype.

Abstracts of the Interscience Conference on Antimicrobial Agents & Chemotherapy, May 5, 2003

PubMed, Mar 1, 2002

Beta-lactams represent one of the most important class of antibiotics for the treatment of infect... more Beta-lactams represent one of the most important class of antibiotics for the treatment of infectious diseases due to pathogenic bacteria. The selective pressure exerted from the wide spread use of third generation cephalosporins generated mutant beta-lactamases belonging mainly to the TEM or SHV family that are able to extend the activity spectrum of hydrolysis. Moreover, extended spectrum cephalosporins are often a good choice in clinical practice towards Enterobacteriaceae. Here we report a comparative analysis of stability between cefotaxime, desacetyl-cefotaxime and ceftazidime with some common TEM-derivatives extended spectrum beta-lactamases.

Biochemical and Biophysical Research Communications, Jul 1, 2013

Molecular Dynamics simulations have been carried out in order to provide a molecular rationalizat... more Molecular Dynamics simulations have been carried out in order to provide a molecular rationalization of the biological and thermodynamic differences observed for a class of TEM b-lactamases. In particular we have considered the TEM-1(wt), the single point mutants TEM-40 and TEM-19 representative of IRT and ESBL classes respectively, and TEM-1 mutant M182T, TEM-32 and TEM-20 which differ from the first three for the additional of M182T mutation. Results indicate that most of the thermodynamic, and probably biological behaviour of these systems arise from subtle effects which, starting from the alterations of the local interactions, produce drastic modifications of the conformational space spanned by the enzymes. The present study suggests that systems showing essentially the same secondary and tertiary structure may differentiate their chemical-biological activity essentially (and probably exclusively) on the basis of the thermal fluctuations occurring in their physiological environment.

Advances in traditional medicine, Aug 16, 2020

Gentiana lutea roots have been widely used in the traditional medicine, especially for their stom... more Gentiana lutea roots have been widely used in the traditional medicine, especially for their stomachic properties. In this study, the effect of G. lutea root methanol extract on SH-SY5Y cell survival and on anti-proliferative activity of pro-apoptotic agents was evaluated. In neuroblastoma cell line SH-SY5Y, the extract did not exhibit any cytotoxic activity, but protected cells from vinblastine-induced apoptosis. In the combined treatment with 200 µg/ml extract plus 0.1 µM vinblastine, extract significantly increased cell survival, reduced the percentage of apoptotic cells and decreased caspase-3 activity if compared with the vinblastine alone treatment. The anti-apoptotic activity of the extract was associated to the down-regulation of Sirt-1 protein and to the increase of anti-apoptotic Bcl-2 protein expression and intracellular GSH levels. Since the apoptosis modulation was evidenced on a neuroblastoma cell line, G. lutea extract could have a promising potential as a neuroprotective agent. Moreover, G. lutea root extract exhibited anti-inflammatory activity, since it inhibited LPS-induced expression of TNF-α in macrophage RAW264.7 cells.

International Journal of Antimicrobial Agents, May 1, 2004

The antimicrobial susceptibility of 103 clinical isolates of Enterobacteriaceae to 11 antibiotics... more The antimicrobial susceptibility of 103 clinical isolates of Enterobacteriaceae to 11 antibiotics, was investigated, using a conventional inoculum size (5 x 10(5) CFU) and a higher inoculum size (5 x 10(8) CFU). All the isolates produced complex beta-lactamase patterns, including an extended-spectrum beta-lactamase (ESBL) of the TEM- or SHV-type plus other enzymes (a TEM-type or an SHV-type non-ESBL and/or a class C enzyme). The following repertoire of ESBLs was produced by the isolates: TEM-15, TEM-19, TEM-26, TEM-52, TEM-72, TEM-87, TEM-92, SHV-2a, SHV-5 and SHV-12, as assessed by sequencing. Production of the other enzymes was showed by analytical isoelectric focusing. Overall, meropenem was the most active agent and less influenced by inoculum size, while other beta-lactams showed a lower activity and a significant inoculum size effect. In conclusion, from its in vitro performance, meropenem could be considered as the last resource drug against strains producing complex beta-lactamase patterns including an ESBL.

Clinical and Experimental Immunology, Nov 24, 2003

Tissue transglutaminase (tTG) seems to be the target self-antigen for endomysial antibodies in co... more Tissue transglutaminase (tTG) seems to be the target self-antigen for endomysial antibodies in coeliac disease (CD) and to catalyse the critical deamidation of gliadin which strengthens its recognition by HLA-restricted gut-derived T cells. To date, it has not been demonstrated whether gliadin is cross-linked to tTG within the gut wall, a phenomenon known to occur in vitro. We therefore investigated the putative presence of tTG and gliadin complexes directly in duodenal mucosa. The immunoprecipitation and Western blotting experiments were performed on mucosal biopsies obtained from untreated, treated CD patients and biopsied controls, by using either anti-tTG or anti-gliadin antibodies, in both denaturating/reducing or nondenaturating/nonreducing conditions. A subset of experiments was performed by using anti-tTG antibodies purified by affinity chromatography from sera of untreated coeliac patients. The localization of tTG and gliadin was studied by immunofluorescence at confocal laser microscopy on seriate sections of diseased and normal duodenal mucosa by using the same antibodies of the coimmunoprecipitation section. The amounts of tTG and gliadin coimmunoprecipitated with anti-tTG monoclonal antibody in untreated CD mucosa were significantly increased compared to those of the other two groups. When performing the experiments in nondenaturating/nonreducing conditions, a high molecular weight band formed by both molecules, was evidenciated. Also the anti-tTG antibodies purified from patients' sera turned out to be able to coimmunoprecipitate the two molecules. The analysis by confocal microscopy showed that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD, and only in the lamina propria of the villi in normal mucosa. Our findings firstly demonstrated that gliadin was directly bound to tTG in duodenal mucosa of coeliacs and controls, and the ability of circulating tTG-autoantibodies to recognize and immunoprecipitate the tTG-gliadin complexes.

Journal of Chemotherapy, Jun 1, 1993

Cefodizime (formerly HR221) was tested either for in vitro microbiological activity or for its st... more Cefodizime (formerly HR221) was tested either for in vitro microbiological activity or for its stability to beta-lactamases in the presence of two beta-lactamase inhibitors (clavulanic acid, tazobactam). Cefodizime was a poor substrate of class C enzymes but hyperproducer strains were generally resistant with or without a beta-lactamase inhibitor used in combination. On the contrary, class A enzymes were able to hydrolyze cefodizime. However, strains expressing class A beta-lactamase were susceptible to cefodizime in combination with clavulanic acid.

Antimicrobial Agents and Chemotherapy, Sep 1, 1999

In addition to the BlaB metallo-␤-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CC... more In addition to the BlaB metallo-␤-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine ␤-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA CME gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A ␤-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER ␤-lactamases and the Bacteroides chromosomal cephalosporinases. The blaA CME determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum ␤-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of ␤-lactams, with both K m and k cat values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (k cat /K m ratios) for all types of ␤-lactam substrates.

Journal of Antimicrobial Chemotherapy, Mar 1, 2006

To assess the prevalence and types of genes encoding extended-spectrum b-lactamases (ESBLs) in cl... more To assess the prevalence and types of genes encoding extended-spectrum b-lactamases (ESBLs) in clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. from Bolivia. Methods: A total of 642 clinical isolates were collected consecutively during a 4 month period (September to December 2004). Resistance or reduced susceptibility to cefotaxime and/or ceftazidime and/or aztreonam was assessed using double disc synergy tests using clavulanic acid, cefotaxime, ceftazidime and aztreonam to identify putative ESBL-producing isolates. The ESBL determinants were characterized by colony blot hybridization, PCR and DNA sequencing. Results: Of the 642 isolates, 220 (34.3%) showed resistance or reduced susceptibility to cefotaxime and/or ceftazidime and/or aztreonam, and 150 (23.4%) were putative ESBL producers. A total of 106 ESBLproducing isolates contained the bla CTX-M-2 gene, and 32 isolates had a novel allele, bla CTX-M-43. bla CTX-M alleles were detected in all P. aeruginosa and Acinetobacter spp. studied. In contrast, only 12 ESBLproducing isolates had bla PER-2 , mainly Enterobacteriaceae, although it was also found in a strain of P. aeruginosa. Conclusions: This is the first study on ESBL-producing strains in Bolivia and it reveals a high prevalence of bla CTX-M genes. The PER-2 enzyme was less prevalent, but its gene was detected in several species, including P. aeruginosa, which is consistent with horizontal transfer.

Biochemical Journal, Sep 15, 1988

Both forms of the chromosome-encoded ,I-lactamase of Citrobacter diversus react with ,-iodopenici... more Both forms of the chromosome-encoded ,I-lactamase of Citrobacter diversus react with ,-iodopenicillanate at a rate characteristic of class A /J-lactamases. The active site of form I was labelled with the same reagent. The sequence of the peptide obtained after trypsin hydrolysis is identical with that of a peptide obtained in a similar manner from the chromosome-encoded ,?-lactamase of Klebsiella pneumoniae.

Biochemical Journal, Oct 1, 1988

Various cephalosporins, cefoxitin, moxalactam, imipenem and aztreonam were studied as substrates ... more Various cephalosporins, cefoxitin, moxalactam, imipenem and aztreonam were studied as substrates of six class C /I-lactamases. Nitrocefin, cephaloridine, cefazolin, cephalothin and cephalexin were good substrates, with kcat values ranging from 27 to 5000 s-'. Cefuroxime, cefotaxime and cefoxitin exhibited low kcat values (0.010-1.7s-') and low Km values, which suggested a rate-limiting deacylation. Imipenem and aztreonam were even poorer substrates (kcat 2 x 10-4-3 x 10-2 s-1) and, in the presence of a reporter substrate, behaved as transient inactivators. With moxalactam, biphasic kinetics were observed, indicating a possible rearrangement of the acyl-enzyme.

International Journal of Antimicrobial Agents, 2021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Translational Vision Science & Technology, 2020

This study aims to investigate the antifungal activity and mechanism of action of ozonized oil ey... more This study aims to investigate the antifungal activity and mechanism of action of ozonized oil eye drops in liposomes (Ozodrop), commercialized as eye lubricant for the treatment of dry eye syndrome and eye inflammation. The activity was tested against four clinical Candida species: C albicans, C glabrata, C krusei, and C orthopsilosis. Methods: The antifungal activity of the eye drop solution was ascertained by microdilution method in accordance with EUCAST obtaining the minimum inhibitory concentration for Ozodrop. The mechanism of action was further investigated in C albicans by measuring cell vitality, intracellular reactive oxygen species production, levels of cellular and mitochondrial (m) membrane potential, and the extent of membrane lipid peroxidation. Results: All Candida isolates were susceptible to Ozodrop with minimum inhibitory concentration values ranging from 0.195% (v/v) for C glabrata to 6.25% (v/v) for C orthopsilosis. After 1 hour of exposure at the minimum inhibitory concentration value about 30% of cells were killed, reaching about 70% at the highest Ozodrop value. After Ozodrop exposure, C albicans showed cell membrane depolarization, increased levels of lipid peroxidation, depolarized m , and increased reactive oxygen species generation. Conclusions: The significant increases in reactive oxygen species production cause the accumulation of reactive oxygen species-associated damages leading to progressive Candida cell dysfunction. Translational Relevance: The antifungal activity of Ozodrop was demonstrated at concentrations several times lower than the concentration that can be retrieved in ocular surface after its application. The antifungal activity of the eye drops Ozodrop would represent an interesting off-label indication for a product basically conceived as an eye lubricant.

Diagnostic Microbiology and Infectious Disease, 2019

Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial... more Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial infections. In the present study a molecular characterization of carbapenem resistant K. pneumoniae, isolated from blood samples of hospitalized patients of Verona University Hospital, was performed. The simultaneous presence of SHV-1/CTX-M-15/KPC-3 and SHV-1/CTX-M-15/OXA-48 serin-β-lactamases was ascertained in the 89% and 11% of K. pneumoniae ST512 and K. pneumoniae ST14, respectively. Molecular characterization of bla genes showed that blaKPC-3 was found in Tn4401a transposon with the tnpR, tnpA, ISKpn6, and ISKpn7 mobile elements whereas blaCTX-M-15 was detected downstream ISEcp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aadA2 gene was identified in 33 K. pneumoniae ST512 isolates.

Biochemical Journal, 1988

The interaction between six class C beta-lactamases and various penicillins has been studied. All... more The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.

Biochemical Society Transactions, 1999

Journal of Antimicrobial Chemotherapy, 1998