Biomedical subjects

M Perilli

Publications and source records attributed to M Perilli.

At least 19 recordsLinked to original sources

PubMed2001

Ceftibuten stability to active-site serine and metallo-beta-lactamases.

Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria and shows an improved stability to hydrolysis by several beta-lactamases because of the carboxyethilidine moiety at position 7 of the ss-acyl side chain. The kinetic interactions between ceftibuten and active-site serine and metallo-ss-lactamases were investigated. The activity of several TEM-derived extended spectrum beta-lactamases (ESbetaLs) against ceftibuten, cefotaxime and ceftazidime was compared using K(m), K(cat) and K(cat)/K(m). Ceftibuten behaved as a poor substrate for class A and B beta-lactamases compared with cefotaxime. The chromosomal class C beta-lactamase from Enterobacter cloacae 908R gave a high K(cat) value (21 s(-1)), whereas there was poor activity with enzymes from Acinetobacter baumannii and Morganella morganii and ceftibuten. Ceftibuten resists hydrolysis in the presence of typical respiratory or urogenital-tract pathogens producing beta-lactamases.

Binding Sites↗

PubMed2001

Properties of multidrug-resistant, ESBL-producing Proteus mirabilis isolates and possible role of beta-lactam/beta-lactamase inhibitor combinations.

At our institution, isolation rates of clinical strains of ESBL-producing Proteus mirabilis increased to 8.8% of all P. mirabilis isolates during the period 1997-1999. To evaluate the susceptibility of ESBL-producing P. mirabilis strains against commonly used drugs, we studied 50 non-duplicated isolates selected on the basis of synergy between clavulanate and beta-lactams (ceftazidime, aztreonam, cefotaxime, and ceftriaxone). The presence of ESBL-coding genes was confirmed by colony hybridization with bla(TEM-1) and bla(SHV-1) probes. Minimum inhibitory concentrations of several antimicrobial agents for each isolate were obtained using the Etest method. All strains were encoding for TEM-derived enzymes. Gene sequencing showed that at least three different genes (TEM-15, TEM-20, and TEM-52) were present. These enzymes have not been previously reported in P. mirabilis. Isolates were characterized by: (a) reduced susceptibility or resistance to third- and fourth-generation cephalosporins (MIC > or = 2 mg/l), (b) resistance to piperacillin that was abolished by tazobactam (MIC > or = 256 vs. < or = 2 mg/l, respectively), (c) multiple antibiotic resistance that included gentamicin, fluoroquinolones and co-trimoxazole. Therapeutic failure and lack of eradication of ESBL-positive P. mirabilis by third-generation cephalosporins has been repeatedly observed both at our Institution and elsewhere. Piperacillin-tazobactam, as well as amikacin and meropenem appear to be important therapeutic options for infections due to multidrug-resistant, ESBL-producing P. mirabilis isolates.

Anti-Bacterial Agents↗

PubMed2000

Extended-spectrum TEM- and SHV-type beta-lactamase-producing Klebsiella pneumoniae strains causing outbreaks in intensive care units in Italy.

The aim of the present study was to investigate the production of extended-spectrum beta-lactamases (ESbetaLs) and the epidemiological correlations in a total of 107 Klebsiella pneumoniae strains resistant to third- and fourth-generation cephalosporins. The strains were collected from patients in four intensive care units (3 neonatal and 1 general) in three hospitals in Italy between March 1996 and July 1997. All strains were found to produce ESbetaLs. Phenotypic (antibiotyping and ESbetaL patterns) and genotypic (plasmid profile and pulsed-field gel electrophoresis) analyses showed that a single strain had been responsible for each outbreak in each of the four intensive care units. Isoelectric focusing, activity on substrates and gene sequencing showed that the strains produced SHV-5, SHV-2a, SHV-12 and TEM-52 beta-lactamases. This is not only the first time that ESbetaL-producing Klebsiella pneumoniae strains have been reported as causing epidemics in Italian hospitals, it is also, to the best of our knowledge, the first time that an outbreak caused by a TEM-52 ESbetaL-producing Klebsiella pneumoniae strain has been reported. The data presented here illustrate the complexity of determining the epidemiological pattern of ESbetaL producers in large hospitals that do not have an ESbetaL-monitoring program.

Anti-Bacterial Agents↗

PubMed2000

Bactericidal activity of levofloxacin and ciprofloxacin on clinical isolates of different phenotypes of Pseudomonas aeruginosa.

Levofloxacin has been reported to have in vitro activity against both gram-positive and gram-negative bacteria. A recent survey carried out at our Institution showed clinical isolates of Pseudomonas aeruginosa to be more susceptible to levofloxacin than to ciprofloxacin. The in vitro activity of the two fluoroquinolones was evaluated further by looking at their bactericidal activity against two strains of each of the following antibio-phenotypes of P. aeruginosa: levofloxacin- and ciprofloxacin-susceptible, levofloxacin-susceptible/ciprofloxacin-resistant, levofloxacin-susceptible/ciprofloxacin-susceptible and ceftazidime-resistant, (National Committee for Clinical Laboratory Standards susceptibility breakpoints were used). MIC and MBC values were measured and time-kill experiments were carried out. Drugs were used at susceptibility or resistance breakpoint concentrations in the time-kill experiments and results were recorded over 12 h in an attempt to link in vitro results with the clinical situation The polypeptide profiles of outer membrane preparations of the six strains were examined by gel electrophoresis. Levofloxacin was shown to be more bactericidal than ciprofloxacin in the time-kill experiments. No differences were observed between the outer membrane proteins of the six strains. Levofloxacin showed greater bactericidal activity against P. aeruginosa clinical isolates than ciprofloxacin.

Anti-Infective Agents↗

PubMed2000

PER-1 extended-spectrum beta-lactamase production in an Alcaligenes faecalis clinical isolate resistant to expanded-spectrum cephalosporins and monobactams from a hospital in Northern Italy.

An Alicaligenes faecalis (FL-424/98) resistant to expanded-spectrum cephalosporins and aztreonam was isolated from the urine of an inpatient at the Intensive Care Unit of the Varese Hospital (Northern Italy) after antimicrobial chemotherapy with cefazolin, vancomycin, and amikacin. Clavulanic acid restored the activity of expanded-spectrum cephalosporins, suggesting the production of an extended-spectrum beta-lactamase (ESbetaL). A crude extract of FL-424/98 showed the presence of two beta-lactamase activities focusing at pH 5.3 and 7.6, respectively. The ESbetaL activity, purified by means of three chromatographic steps, was found to correspond to the pI 5.3 enzyme. Determination of kinetic parameters confirmed that the enzyme efficiently hydrolyzed expanded-spectrum cephalosporins and aztreonam. A colony-blot hybridization revealed the presence of blaPER-related sequences in FL-424/98, and sequencing confirmed the identity of this determinant with blaPER-1, previously detected in Pseudomonas aeruginosa, Acinetobacter, and Salmonella clinical isolates from Turkey. Finding of blaPER-1 in a species that can be part of the resident human microbiota raises the possibility that it could be an efficient shuttle for spreading of this resistance gene among other opportunistic pathogens that are normally members of the resident microbiota. Kinetic parameters determined for the PER-1 enzyme with some cephalosporin substrates were somewhat different from those previously reported.

Adult↗

PubMed2000

First identification of an SHV-12 extended-spectrum beta-lactamase in Klebsiella pneumoniae isolated in Italy.

A clinical isolate of Klebsiella pneumoniae highly resistant to third- and fourth-generation cephalosporins, cephamycins and aminoglycosides, was isolated in 1991 from urine. Analysis of a crude extract showed the presence of three beta-lactamases with isoelectric points of 6.6, 7.5 and 8.2. The enzyme with pI 8.2 was transferred by conjugation into Escherichia coli K-12 J53 and was responsible for the resistance to third-generation cephalosporins and monobactams, but not to other antibiotics. Kinetic studies of partially purified beta-lactamase from the transconjugant strain confirmed that the enzyme was able to hydrolyse ceftazidime, cefotaxime and aztreonam but not cephamycins. Analysis of the transconjugant showed the presence of two small non-conjugative plasmids of 14 and 6 kb. A polymerase chain reaction was performed using primers specific for the bla(SHV) gene and a fragment of the expected size (about 961 bp) was obtained with both the K. pneumoniae clinical isolate and the transconjugant. Nucleotide sequence analysis of the fragment showed that it encoded the enzyme SHV-12, derived from SHV-5 (with Gln-35 to Leu). This is the first report of an SHV-12-like enzyme isolated in Italy.

Anti-Bacterial Agents↗

PubMed2000

TEM-72, a new extended-spectrum beta-lactamase detected in Proteus mirabilis and Morganella morganii in Italy.

A new natural TEM-2 derivative, named TEM-72, was identified in a Proteus mirabilis strain and in a Morganella morganii strain isolated in Italy in 1999. Compared to TEM-1, TEM-72 contains the following amino acid substitutions: Q39K, M182T, G238S, and E240K. Kinetic analysis showed that TEM-72 exhibits an extended-spectrum activity, including activity against oxyimino-cephalosporins and aztreonam. Expression of bla(TEM-72) in Escherichia coli was capable of decreasing the host susceptibility to the above drugs.

Anti-Bacterial Agents↗

PubMed2000

A kinetic study on the interaction between tazobactam (a penicillanic acid sulphone derivative) and active-site serine beta-lactamases.

The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.

Acinetobacter↗

PubMed1999

Inhibition of poly(ADP-ribosyl)ation introduces an anomalous methylation pattern in transfected foreign DNA.

The aim of this paper is to verify whether the control played by poly(ADP-ribosyl)ation on genomic DNA methylation, and in particular on CpG islands, can also be seen on foreign DNA transfected in cells where inhibition of the poly(ADP-ribosyl)ation process was obtained by treating them with 2 mM 3-aminobenzamide for 24 h. The CpG island-like pVHCk plasmid containing the bacterial chloramphenicol acyltransferase (CAT) gene under the control of SV40 early promoter was transfected in L929 mouse fibroblast cells. The bisulfite reaction, which is capable of immortalizing the methylation state of cytosine on DNA, was performed before amplification of the plasmid DNA fragment, then used for sequence analysis. Our results have shown that 1) when transfected in control cells, the plasmid maintains its characteristic unmethylated pattern, whereas this pattern is lost when the plasmid is transfected in cells treated with 3-aminobenzamide; and 2) the presence of new methyl groups on plasmid DNA is paralleled by a decrease of CAT reporter gene expression. These data confirm that poly(ADP-ribosyl)ation is a process tightly involved in protecting genomic DNA from full methylation and suggest the use of 3-aminobenzamide as a possible experimental strategy to mime other conditions of DNA hypermethylation in cells.

3T3 Cells↗

PubMed1999

Italian survey on comparative levofloxacin susceptibility in 334 clinical isolates of Pseudomonas aeruginosa.

A national survey on susceptibility patterns of 334 Pseudomonas aeruginosa isolates from intensive care units and hematology and oncology wards from 13 Italian hospitals compared the in vitro activity of levofloxacin, an injectable oral fluoroquinolone, to those of ciprofloxacin, ofloxacin, ceftazidime, imipenem, amikacin, and gentamicin. Amikacin and imipenem had the best susceptibility profiles. The activity of levofloxacin was superior to those of the other quinolones and was comparable to that of ceftazidime. The effect of levofloxacin in vitro on P. aeruginosa clinical isolates suggests that further clinical investigations are warranted.

Anti-Infective Agents↗

PubMed1999

A Kinetic Study on the Interaction between Tazobactam (A Penicillanic Acid Sulphone Derivative) and Active-Site Serine beta-Lactamases.

Journal Article↗

PubMed1998

Characterization and sequence of the Chryseobacterium (Flavobacterium) meningosepticum carbapenemase: a new molecular class B beta-lactamase showing a broad substrate profile.

The metallo-beta-lactamase produced by Chryseobacterium (formerly Flavobacterium) meningosepticum, which is the flavobacterial species of greatest clinical relevance, was purified and characterized. The enzyme, named BlaB, contains a polypeptide with an apparent Mr of 26000, and has a pI of 8.5. It hydrolyses penicillins, cephalosporins (including cefoxitin), carbapenems and 6-beta-iodopenicillanate, a mechanism-based inactivator of active-site serine beta-lactamases. The enzyme was inhibited by EDTA, 1-10 phenanthroline and pyridine-2,6-dicarboxylic acid, with different inactivation parameters for each chelating agent. The C. meningosepticum blaB gene was cloned and sequenced. According to the G+C content and codon usage, the blaB gene appeared to be endogenous to the species. The BlaB enzyme showed significant sequence similarity to other class B beta-lactamases, being overall more similar to members of subclass B1, which includes the metallo-enzymes of Bacillus cereus (Bc-II) and Bacteroides fragilis (CcrA) and the IMP-1 enzyme found in various microbial species, and more distantly related to the metallo-beta-lactamases of Aeromonas spp. (CphA, CphA2 and ImiS) and of Stenotrophomonas maltophilia (L1).

Amino Acid Sequence↗

PubMed1998

Repeated epidemics caused by extended-spectrum beta-lactamase-producing Serratia marcescens strains.

An outbreak of Serratia marcescens involving 42 patients admitted to the general intensive care unit of the Hospital of Varese, Italy, occurred from March 1994 to August 1995. The causative strains were resistant to oxyimino-cephalosporins and monobactams due to their production of an extended-spectrum beta-lactamase. Another outbreak caused by Serratia marcescens strains had occurred in the same unit a few months earlier, from February to October 1993, with the strains involved producing a novel TEM-derived extended-spectrum beta-lactamase. In order to verify whether there were any relationships between isolates from the two epidemics, the strains and their enzymes were characterized. Biochemical data and gene amplification experiments showed that the isolates of the second outbreak harbored a non-conjugative plasmid of approximately 48 kb, codifying for the production of an SHV-derived extended-spectrum beta-lactamase with pI 8.2. Restriction fragment length polymorphism analysis of total genomic DNA by pulsed-field gel electrophoresis of Serratia marcescens isolates unambiguously identified two different bacterial clones responsible for the two epidemics. Epidemiological and microbiological investigations demonstrated the long persistence of Serratia marcescens strains and their circulation in other hospital wards, thus suggesting their possible role as a long-term reservoir for further epidemic spread.

Cross Infection↗

PubMed1998

Ceftazidime and aztreonam resistance in Providencia stuartii: characterization of a natural TEM-derived extended-spectrum beta-lactamase, TEM-60.

A plasmid-encoded beta-lactamase produced from a clinical strain of Providencia stuartii has been purified and characterized. The gene coding for the beta-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 beta-lactamase PER-1. Kinetic parameters relative to penicillins, cephalosporins, and monobactams other than mechanism-based inactivators were related to the in vitro susceptibility phenotype.

Anti-Bacterial Agents↗

PubMed1997

Does poly(ADP-ribosyl)ation regulate the DNA methylation pattern?

The existence of a possible correlation between poly(ADP-ribosyl)ation and DNA methylation processes was investigated. In vivo and in vitro experiments were carried out on L929 mouse fibroblasts preincubated for 24 h with or without 3-aminobenzamide, a well-known inhibitor of poly(ADP-ribose) polymerase. Both experimental approaches evidenced a close relationship between these two important nuclear enzymatic mechanisms, suggesting that the poly(ADP-ribosyl)ated isoform of H1 histone and/or long and branched protein-free ADP-ribose polymers could act as protecting agents against full methylation of the CpG dinucleotides in genomic DNA.

Animals↗

PubMed1997

Characterization of a new TEM-derived beta-lactamase produced in a Serratia marcescens strain.

A natural TEM variant beta-lactamase was isolated from an epidemic strain of Serratia marcescens. Nucleotide gene sequencing revealed multiple point mutations located in the 42-to-44 tripeptide and positions 145 to 146, 178, and 238. In addition, a glutamic acid 212 deletion was also found. The purified enzyme was studied from a kinetic point of view, revealing the highest catalytic efficiency (k[cat]/Km) values for ceftazidime and aztreonam compared with the TEM-1 prototype enzyme. The in vitro resistance correlated with kinetic parameters, and the enzyme also mediated resistance to some penicillins and an ampicillin-clavulanic acid combination. The mutational and kinetic changes are discussed in relation to the three-dimensional crystallographic structure of the wild-type TEM-1 enzyme.

Amino Acid Sequence↗

PubMed1997

Sensitivity of Aeromonas hydrophila carbapenemase to delta3-cephems: comparative study with other metallo-beta-lactamases.

Ceftriaxone and ceftriaxone S-oxide behaved as inactivators against the metallo-beta-lactamase of Aeromonas hydrophila AE036 and as substrates for the zinc beta-lactamase produced by Bacillus cereus (569/H/9) and Stenotrophomonas maltophilia ULA 511. Moreover, RO 09-1428, a catechol-cephalosporin, was not recognized by the A. hydrophila enzyme. Panipenem, cephalosporin C, cephalosporin C-gamma-lactone, and loracarbef were substrates for the three studied beta-lactamases.

Aeromonas hydrophila↗

PubMed1996

Overproduction and purification of the Aeromonas hydrophila CphA metallo-beta-lactamase expressed in Escherichia coli.

The Aeromonas hydrophila CphA metallo-beta-lactamase was overexpressed in a soluble secreted form in Escherichia coli using a T7 RNA polymerase-based expression system, and a simple protocol based on a single cation-exchange chromatographic step was developed, which is suitable for rapid purification of the overexpressed enzyme from E. coli lysates. A yield of up to 30 micrograms of purified enzyme per milliliter of culture was obtained. The purified enzyme preparation showed properties identical to those previously reported in the literature.

Aeromonas hydrophila↗