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Gianfranco Amicosante

Publications and source records attributed to Gianfranco Amicosante.

At least 19 recordsLinked to original sources
PubMed2007

Metallo-beta-lactamases as emerging resistance determinants in Gram-negative pathogens: open issues.

The rapid spread of acquired metallo-beta-lactamases (MBLs) among major Gram-negative pathogens is a matter of particular concern worldwide and primarily in Europe, one of first continents where the emergence of acquired MBLs has been reported and possibly the geographical area where the increasing diversity of these enzymes and the number of bacterial species affected are most impressive. This spread has not been paralleled by accuracy/standardisation of detection methods, completeness of epidemiological knowledge or a clear understanding of what MBL production entails in terms of clinical impact, hospital infection control and antimicrobial chemotherapy. A number of European experts in the field met to review the current knowledge on this phenomenon, to point out open issues and to reinforce and relate to one another the existing activities set forth by research institutes, scientific societies and European Union-driven networks.

Anti-Bacterial Agents↗
PubMed2006

Pseudomonas aeruginosa bloodstream infections: risk factors and treatment outcome related to expression of the PER-1 extended-spectrum beta-lactamase.

BACKGROUND: Bloodstream infection (BSI) due to Pseudomonas aeruginosa (Pa) has relevant clinical impact especially in relation to drug resistance determinants. The PER-1 extended-spectrum beta-lactamase (ESBL) is a common enzyme conferring high-level resistance to anti-pseudomonal cephalosporins. Risk factors and treatment outcome of BSI episodes caused by PER-1-positive Pa (PER-1-Pa) strains were compared to those caused by ESBL-negative Pa isolates (ESBL-N-Pa). METHODS: Twenty-six BSI cases due to ceftazidime-resistant Pa strains have been investigated. MIC values of anti-pseudomonal drugs were determined by the Etest method (AB Biodisk, Solna, Sweden). The double-disk synergy test was used to detect ESBL production. PCR amplification and DNA sequencing were used to characterize ESBL types. Clinical records of BSI-patients were examined retrospectively. Demographic data, underlying diseases (McCabe-Jackson classification and Charlson weighted index), risk factors, antimicrobial therapy, and treatment outcome were evaluated in cases due to ESBL-positive and cases due to ESBL-N-Pa isolates. Unpaired Student's t-test, Mann-Whitney U-test, Fisher's exact test and the chi2 test were used for statistical analysis. RESULTS: Nine Pa isolates expressed the PER-1 ESBL; the remaining 17 isolates did not produce ESBLs. Severe sepsis (P = 0.03), bladder and intravascular catheters (both P = 0.01), immunosuppressive therapy (P = 0.04), and mechanical ventilation (P = 0.03) were significantly associated with BSI due to PER-1-Pa. Empirical treatment (P = 0.02) and treatment after ID/AST (P < 0.01) were rarely adequate in PER-1-Pa cases. With regard to treatment outcome, 77.8% BSI cases due to PER-1-Pa vs. 28.6% cases due to ESBL-N-Pa isolates failed to respond (P < 0.03). All cases due to PER-1-Pa that were treated with carbapenems (alone or in combination with amikacin) failed to respond. In contrast, 7/8 cases due to ESBL-N-Pa given carbapenems were responders. CONCLUSION: Therapeutic failure and increased hospital costs are associated with BSI episodes caused by PER-1-Pa strains. Thus, recognition and prompt reporting of ESBL-production appears a critical factor for the management of patients with serious P. aeruginosa infections.

Adult↗
PubMed2006

Spread of bla(CTX-M-type) and bla(PER-2) beta-lactamase genes in clinical isolates from Bolivian hospitals.

OBJECTIVES: To assess the prevalence and types of genes encoding extended-spectrum beta-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 ESBL-producing 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 ESBL-producing 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.

Acinetobacter↗
PubMed2006

Occurrence of extended spectrum beta-lactamases among isolates of Enterobacteriaceae from urinary tract infections in southern Italy.

The present investigation was undertaken to assess the prevalence of extended-spectrum beta-lactamases (ESbetaLs) among urinary tract infection (UTI) isolates. During 4 months in 2004, a total of 650 Enterobacteriaceae strains from UTIs was collected by five clinical microbiology laboratories located in southern Italy and the beta-lactamase production was investigated. A total of 50 of the 650 isolates were double-disk positive and suspected of producing an ESbetaL; Escherichia coli (36.0%) and Klebsiella pneumoniae (32.0%) were the most common species among all ESbetaL producers. Characterization of ESbetaL determinants was carried out by the colony blot hybridization method, and polymerase chain reaction (PCR) and DNA sequencing in order to identify the presence of bla (TEM), bla (SHV), bla (PER), and bla (CTX-M) determinants. The ESbetaL variants found in this study were the following: TEM-15, TEM-24, TEM-52, TEM-134, SHV-12, CTX-M-1, CTX-M-3, CTX-M-15, and PER-1. As expected, the majority of the isolates were found to be susceptible to imipenem (94%), cefepime (54%) and piperacillin-tazobactam (54%). The results of this survey show the prevalence of ESbetaL enzymes among enterobacterial pathogens causing UTIs in southern Italy.

Anti-Bacterial Agents↗
PubMed2006

CTX-M-type extended-spectrum beta-lactamases in Italy: molecular epidemiology of an emerging countrywide problem.

A nationwide survey of extended-spectrum beta-lactamase (ESBL) production among Enterobacteriaceae, carried out in 2003, showed that CTX-M-type enzymes have achieved a sizeable prevalence among ESBL producers in Italy, mostly in Escherichia coli and, to a lesser extent, in Klebsiella pneumoniae. In this work, we report on the molecular epidemiology of the CTX-M-producing isolates from that survey and on the mechanisms of dissemination of these emerging resistance determinants. The CTX-M-producing isolates were detected in 10 of the 11 participating centers distributed across the Italian national territory, although at remarkably variable rates in different centers (1.2 to 49.5% of the ESBL producers). All CTX-M determinants were of group 1, with CTX-M-15 and CTX-M-1 being the most prevalent variants (60% and 35%, respectively) and CTX-M-32 carried by a minority (5%) of isolates. Each variant was detected both in E. coli and in K. pneumoniae. Genotyping of the CTX-M-producing isolates by random amplification of polymorphic DNA revealed a notable diversity, especially among those producing CTX-M-1, while clonal expansion was evident with some CTX-M-15-producing strains. Mating experiments revealed a higher overall transferability of bla(CTX-M-1) and bla(CTX-M-32) than of bla(CTX-M-15). Coresistance to quinolones and aminoglycosides was overall higher with the CTX-M-15-producing isolates. The present results indicate that CTX-M-producing strains are now widespread across the Italian territory and underscore the emerging role of these ESBL determinants in the European setting. They also reveal notable differences in the dissemination mechanisms of genes encoding different CTX-M variants of the same lineage.

Aminoglycosides↗
PubMed2006

Trends in production of extended-spectrum beta-lactamases among enterobacteria of medical interest: report of the second Italian nationwide survey.

Results of a 2003 survey carried out in Italy to evaluate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing enterobacteria are presented. Eleven Italian Microbiology Laboratories investigated 9,076 consecutive nonreplicate isolates (inpatients, 6,850; outpatients, 2,226). ESBL screening was performed by MIC data analysis. Confirmation was obtained using the double-disk synergy test and the combination disk test based on CLSI methodology. ESBL determinants were investigated by colony blot hybridization and confirmed by sequencing. Results were compared to those of the 1999 Italian survey (8,015 isolates). The prevalence of ESBL producers was 7.4% among isolates from inpatients (in 1999, 6.3%) and 3.5% among outpatients (no data were available for 1999). Among hospitalized patients, the most prevalent ESBL-positive species was Escherichia coli (Klebsiella pneumoniae in 1999). Proteus mirabilis was the most prevalent ESBL-positive species among outpatients. In both groups, most ESBL-positive pathogens were obtained from urinary tract infections. TEM-type ESBLs were the most prevalent enzymes (45.4%). Non-TEM, non-SHV determinants emerged: CTX-M-type in E. coli and K. pneumoniae, and PER-type in P. mirabilis, Providencia spp., and E. coli. With the exception of 3/163 P. mirabilis isolates and 1/44 Providencia stuartii isolate (all of which were intermediate for imipenem), carbapenems were active against all ESBL-positive enterobacteria. Susceptibility to other drugs was as follows: 84.7% for amikacin, 84.4% for piperacillin-tazobactam, 48.0% for gentamicin, and 32.8% for ciprofloxacin. Carbapenems appear to be the drug of choice. Amikacin and beta-lactam/beta-lactamase inhibitor combinations represent an alternative in non-life-threatening infections. The appearance of ESBL-positive enterobacteria in the community makes it mandatory that family physicians learn how to treat these pathogens.

Data Collection↗
PubMed2005

Dynamical aspects of TEM-1 beta-lactamase probed by molecular dynamics.

The dynamical aspects of the fully hydrated TEM-1 beta-lactamase have been determined by a 5 ns Molecular Dynamics simulation. Starting from the crystallographic coordinates, the protein shows a relaxation in water with an overall root mean square deviation from the crystal structure increasing up to 0.17 nm, within the first nanosecond. Then a plateau is reached and the molecule fluctuates around an equilibrium conformation. The results obtained in the first nanosecond are in agreement with those of a previous simulation (Diaz et al., J. Am. Chem. Soc., (2003) 125, 672-684). The successive equilibrium conformation in solution shows an increased mobility characterized by the following aspects. A flap-like translational motion anchors the omega-loop to the body of the enzyme. A relevant part of the backbone dynamics implies a rotational motion of one domain relative to the other. The water molecules in the active site can exchange with different residence times. The H-bonding networks formed by the catalytic residues are frequently interrupted by water molecules that could favour proton transfer reactions. An additional simulation, where the aspartyl dyad D214-D233 was considered fully deprotonated, shows that the active site is destabilized.

Catalytic Domain↗
PubMed2005

Evolution of CTX-M-type beta-lactamases in isolates of Escherichia coli infecting hospital and community patients.

Escherichia coli isolates collected at our Institution from 1999 to 2003 (n=20,258) were studied to evaluate the production of CTX-M-type extended-spectrum beta-lactamases (ESBL). Isolates suspected of producing CTX-M enzymes were analyzed by the double-disk synergy test, hybridization with specific probes, PCR and direct DNA sequencing. Overall, 53 ESBL-positive isolates were found to carry CTX-M-type genes (blaCTX-M-1, n=51; blaCTX-M-15, n=2). The isolation of CTX-M-positive strains increased from 1 per year (1999) to 26 per year (2003). The first isolate carrying the blaCTX-M-15 gene appeared in 2003 and was obtained from a patient previously treated with ceftazidime. CTX-M-positive isolates were characterized by multi-drug resistance and were obtained both from inpatients (n=29) and outpatients (n=24). Most patients were over 60-year-old (n=45), had underlying chronic diseases (n=32), and had been hospitalized more than once (n=33). Strains were frequently isolated from the urinary tract, often after recurrent infections. Our study demonstrates that CTX-M-producing isolates are increasing among E. coli strains. Adequate laboratory detection may help in choosing appropriate treatment and in limiting the spread of this resistance trait.

Aged↗
PubMed2005

Ciprofloxacin, salicylate, and 2,4-dinitrophenol decrease production of AmpC-type beta-lactamase in two Citrobacter freundii clinical isolates.

The effect of ciprofloxacin and two mar RAB inducers on the susceptibility to beta-lactam antibiotics in two AmpC beta-lactamase semi-constitutive producer Citrobacter freundii clinical isolates (the DM 1 and DM 2 strains) was studied. Possible changes in outer membrane protein expression, permeability to cephaloridine, active efflux, and hydrolytic activity of beta-lactamase-crude extracts were evaluated under the influence of ciprofloxacin, sodium salicylate, and 2,4-dinitrophenol. Results were compared with those of the effect of the same three chemicals on a normally beta-lactamase-inducible wild-type C. freundii strain. The three assayed compounds decreased beta-lactamase hydrolysis on cephaloridine in both the two clinical isolates as well as in the wild-type strain. However, only the DM 1 and DM 2 strains showed increased susceptibility to beta-lactams. Sodium salicylate and 2,4-dinitrophenol, but not ciprofloxacin, reduced the expression of a 45-kDa outer membrane protein in the three studied strains, which was accompanied by a 4- to 20-fold diminution in permeability to cephaloridine. In conclusion, two mar RAB inducers and ciprofloxacin induced the Mar phenotype and repressed AmpC beta-lactamase synthesis in the DM 1 and DM 2 clinical isolates.

2,4-Dinitrophenol↗
PubMed2005

Novel TEM-type extended-spectrum beta-lactamase, TEM-134, in a Citrobacter koseri clinical isolate.

A new natural TEM derivative with extended-spectrum beta-lactamase activity, TEM-134, was identified in a ceftazidime-resistant clinical isolate of Citrobacter koseri. Compared to TEM-1, TEM-134 contains the following mutations: Q39K, E104K, R164H, and G238S. The bla(TEM-134) gene was not transferable by conjugation and, apparently, was chromosomally encoded. Expression studies with Escherichia coli revealed efficient cefotaximase and ceftazidimase activity for TEM-134.

Anti-Bacterial Agents↗
PubMed2005

Proteus mirabilis bloodstream infections: risk factors and treatment outcome related to the expression of extended-spectrum beta-lactamases.

Bloodstream infection (BSI) due to Proteus mirabilis strains is a relatively uncommon clinical entity, and its significance has received little attention. This study was initiated to evaluate risk factors and treatment outcome of BSI episodes due to P. mirabilis producing extended-spectrum beta-lactamases (ESBLs). Twenty-five BSI episodes caused by P. mirabilis occurred at our hospital (Ospedale di Circolo e Fondazione Macchi, Varese, Italy) over a 7.5-year period. Phenotypic and molecular methods were used to assess ESBL production. Clinical records of BSI patients were examined retrospectively. Demographic data, underlying diseases (according to McCabe and Jackson classification and Charlson weighted index), risk factors, and treatment outcome were investigated by comparing cases due to ESBL-positive strains to cases due to ESBL-negative strains. Eleven isolates were found to express ESBLs (TEM-52 or TEM-92). The remaining 14 isolates were ESBL negative and were uniformly susceptible to extended-spectrum cephalosporins and monobactams. Comparison of the two groups showed that previous hospitalization in a nursing home (P = 0.04) and use of bladder catheter (P = 0.01) were significant risk factors for infections due to ESBL-positive strains. In addition, cases due to ESBL-positive strains showed a significantly higher mortality attributable to BSI (P = 0.04). BSI cases due to ESBL-negative isolates uniformly responded to therapy, whereas 5/11 cases due to ESBL-positive isolates failed to respond (P < 0.01). Use of carbapenems was associated with complete response independently of ESBL production. Therapeutic failure and mortality may occur in BSI episodes caused by ESBL-positive P. mirabilis isolates. Thus, recognition of ESBL-positive strains appears to be critical for the clinical management of patients with systemic P. mirabilis infections.

Aged↗
PubMed2005

Dissemination of CTX-M-type extended-spectrum beta-lactamase genes to unusual hosts.

A Citrobacter amalonaticus and a Morganella morganii producing the CTX-M-1 extended-spectrum beta-lactamase (ESBL) were isolated from an area where this enzyme is now widespread in Escherichia coli. This is the first report of CTX-M-1 in the former species. In both cases the ESBL determinant was possibly acquired by these unusual hosts in vivo, after coinfection with E. coli strains carrying conjugative plasmids encoding CTX-M-1.

Citrobacter↗
PubMed2004

Overexpression system and biochemical profile of CTX-M-3 extended-spectrum beta-lactamase expressed in Escherichia coli.

An efficient over-expression system was developed for CTX-M-3 extended-spectrum-beta-lactamase. The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme. The N-terminal amino acid sequence was determined to be NH2-QTADVQ... Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates. Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme.

Biotechnology↗
PubMed2004

Salicylate decreases production of AmpC type beta-lactamases and increases susceptibility to beta-lactams in a Morganella morganii clinical isolate.

The effect of salicylate, a marRAB inducer, on the resistance to beta-lactams was characterized in an AmpC beta-lactamase hyperproducer Morganella morganii clinical isolate (the M1 strain). Results were compared with those of the effect of salicylate in a wild-type M. morganii strain. Salicylate induced a decreased susceptibility to nalidixic acid, norfloxacin and tetracycline and simultaneously increased the susceptibility to beta-lactams apparently due to the repression of AmpC beta-lactamase synthesis in the M1 strain. Likewise, salicylate only repressed 46 kDa outer membrane protein expression in the wild-type strain, since the clinical isolate M1 did not express it.

Anti-Bacterial Agents↗
PubMed2004

Prevalence and characterization of metallo-beta-lactamases in clinical isolates of pseudomonas aeruginosa.

The prevalence and the type(s) of metallo-beta-lactamases (MBLs) produced by isolates of Pseudomonas aeruginosa were investigated. During 2001, 506 nonduplicate isolates were obtained from hospitalized patients. Eighty-two strains were selected because of resistance to carbapenems and/or ceftazidime. Screening for MBL production was performed in the latter isolates by the Etest MBL strips (AB Biodisk, Solna, Sweden) and by a broth microdilution method measuring minimum inhibitory concentrations (MICs) of imipenem alone and in the presence of metal-chelating agents (EDTA and o-phenanthroline). Specific DNA probes were used to investigate the presence of genes coding for IMP- or VIM-type enzymes. Overall, four isolates of P. aeruginosa (obtained from independent patients) were found to carry a blaVIM gene. Polymerase chain reaction (PCR) experiments and DNA sequencing revealed that the VIM-2 determinant was present in three cases, whereas VIM-1 was detected in one isolate. Surveillance programs should be adopted to avoid the spread of these worrisome resistance genes.

Anti-Bacterial Agents↗
PubMed2004

Antimicrobial susceptibility of clinical isolates of Enterobacteriaceae producing complex beta-lactamase patterns including extended-spectrum enzymes.

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.

Anti-Bacterial Agents↗
PubMed2004

Enhanced active efflux, repression of porin synthesis and development of Mar phenotype by diazepam in two enterobacteria strains.

The aim of this work was to determine whether diazepam could induce the multiple antibiotic resistance (Mar) phenotype in Klebsiella pneumoniae and Escherichia coli strains. The Mar phenotype is characterized by decreased susceptibility to multiple antibiotics due to the loss of porins and/or increased expression of active efflux systems. The effect of subinhibitory concentrations of diazepam on the susceptibility of different antimicrobial agents, outer-membrane protein expression and norfloxacin intracellular accumulation was studied. The results revealed that diazepam concentrations equal or twice adult dosage induced the same Mar phenotype as two well known E. coli marRAB inducers, sodium salicylate and sodium benzoate. Susceptibility to norfloxacin in a K. pneumoniae clinical isolate and E. coli strain Ag100 decreased due to enhanced active efflux and loss of porin expression. A decreased susceptibility to chloramphenicol, tetracycline, nalidixic acid and beta-lactam antibiotics was also observed. In conclusion, like sodium salicylate or sodium benzoate, diazepam may induce the Mar phenotype.

Anti-Bacterial Agents↗