Carbapenem-hydrolyzing metallo-beta-lactamases in Pseudomonas aeruginosa.
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Bacterial Proteins↗
Biomedical subjects
G Cornaglia
Publications and source records attributed to G Cornaglia.
Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate.
Production of a metallo-beta-lactamase activity was detected in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate (isolate VR-143/97) from an Italian inpatient at the Verona University Hospital (northern Italy). The metallo-beta-lactamase determinant was isolated from a genomic library of VR-143/97, constructed in an Escherichia coli plasmid vector, by screening for clones with reduced susceptibility to imipenem. Sequencing of the cloned gene revealed that it encoded a new class B beta-lactamase that was named VIM-1. At the sequence level VIM-1 was rather divergent from the other class B enzymes (16.4 to 38.7% identity), overall being more similar to members of subclass B1 including the beta-lactamase II of Bacillus cereus (Bc-II), the Bacteroides fragilis CcrA, the Chryseobacterium meningosepticum BlaB, and the cassette-encoded IMP-1 enzymes. Among these, VIM-1 showed the highest degree of similarity to Bc-II. Similarly to blaIMP, blaVIM was also found to be carried on a gene cassette inserted into a class 1 integron. The blaVIM-containing integron was located on the chromosome of P. aeruginosa VR-143/97, and the metallo-beta-lactamase-encoding determinant was not transferable to E. coli by conjugation. Expression of the integron-borne blaVIM gene in E. coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems), revealing a very broad substrate specificity of the VIM-1 enzyme.
Amino Acid Sequence↗
Resistance of enterococci to ampicillin and glycopeptide antibiotics in Italy. The Italian Surveillance Group for Antimicrobial Resistance.
We report the results of the monitoring of resistance to ampicillin, vancomycin, and teicoplanin in enterococci by an Italian network of clinical microbiology laboratories. A total of 16,226 strains were analyzed; 9,169 of these strains were Enterococcus faecalis, 913 were Enterococcus faecium, and 6,144 were Enterococcus species. The average rate of resistance to ampicillin was 1.9% (range, 0-6.5%) for E. faecalis and 70% (range, 33.3%-98.7%) for E. faecium; the average rate of resistance to vancomycin was 1.1% (range, 0.1%-2%) for E. faecalis and 8.5% (range, 0-36.1%) for E. faecium; and the average rate of resistance to teicoplanin was 0.8% (range, 0-2.4%) for E. faecalis and 8.7% (range, 0-36.1%) for E. faecium.
Ampicillin Resistance↗
Resistance of Streptococcus pyogenes to erythromycin and related antibiotics in Italy. The Italian Surveillance Group for Antimicrobial Resistance.
A survey of antimicrobial resistance in Streptococcus pyogenes, performed within the framework of a national surveillance program, has revealed a dramatic increase in resistance of S. pyogenes to erythromycin in most areas of Italy. In virtually all the centers that provided data for 3 consecutive years, the incidence of erythromycin-resistant strains increased twofold to 20-fold from 1993 to 1995 and was greater than 30% in five of the 14 centers participating in the study. The clonality of erythromycin-resistant isolates was studied in 15 strains isolated from different patients at the Institute of Microbiology of Verona University (Verona). The features of the Verona isolates and the substantially different rates of erythromycin and clindamycin resistance observed in most centers suggest that the spread of different resistance genes in multiple clones might be occurring throughout the country.
Anti-Bacterial Agents↗
MEN 10700, a new penem antibiotic: in-vitro activity and its correlation with beta-lactamase stability, PBP affinity and diffusion through the bacterial cell wall.
The in-vitro activity of MEN 10700, a novel penem, was compared with that of imipenem, ritipenem, ampicillin/sulbactam, cefotaxime, ciprofloxacin and amikacin against 1088 strains taken from 21 genera, including Gram-negative, Gram-positive and anaerobic bacteria. MIC data showed that MEN 10700 was very active against staphylococci and streptococci (MIC90 < or = 0.5 mg/L) and against most members of the Enterobacteriaceae (MIC90 < or = 2 mg/L), with reduced activity only against Providencia stuartii (MIC90 = 8 mg/L). MEN 10700 was also active against anaerobic species such as Clostridium perfringens and Bacteroides fragilis as well as Moraxella catarrhalis. It was moderately active against Enterococcus faecalis and inactive against Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Aeromonas spp. and Acinetobacter spp. Its antibacterial spectrum was thus slightly narrower than that of imipenem, but compared favourably with those of a third-generation cephalosporin and ritipenem. MEN 10700 was highly stable to a number of beta-lactamases and was a poor inducer of class I enzymes. It bound penicillin-binding protein 2 with the highest affinity and easily permeated the outer membrane of Escherichia coli.
Anti-Bacterial Agents↗
Interaction of ceftriaxone with penicillin-binding proteins of Escherichia coli in the presence of human serum albumin.
The binding of ceftriaxone, a cephalosporin that exhibits high serum protein binding and prolonged serum half-life, to penicillin-binding proteins (PBPs) of Escherichia coli K12 in the presence of human serum albumin was compared with plasma concentrations of cefotaxime, a cephalosporin with low serum protein binding and a short serum half-life. Ceftriaxone concentrations equivalent to those maintained in plasma for 8 h after an intravenous infusion of 1 g saturated PBPs 2 and 3. Cefotaxime saturated both PBPs at concentrations equivalent to those maintained for 2 h, and PBP 3 only at concentrations maintained for 2-8 h. These results indicate that high serum protein binding does not impair the ability of ceftriaxone to inhibit essential PBPs, and explain the high in-vivo efficacy of the drug.
Bacterial Proteins↗
MIC-based interspecies prediction of the antimicrobial effects of ciprofloxacin on bacteria of different susceptibilities in an in vitro dynamic model.
Multiple predictors of fluoroquinolone antimicrobial effects (AMEs) are not usually examined simultaneously in most studies. To compare the predictive potentials of the area under the concentration-time curve (AUC)-to-MIC ratio (AUC/MIC), the AUC above MIC (AUCeff), and the time above MIC (Teff), the kinetics of killing and regrowth of four bacterial strains exposed to monoexponentially decreasing concentrations of ciprofloxacin were studied in an in vitro dynamic model. The MICs of ciprofloxacin for clinical isolates of Staphylococcus aureus, Escherichia coli 11775 (I) and 204 (II), and Pseudomonas aeruginosa were 0.6, 0.013, 0.08, and 0.15 microg/ml, respectively. The simulated values of AUC were designed to provide similar 1,000-fold (S. aureus, E. coli I, and P. aeruginosa) or 2, 000-fold (E. coli II) ranges of the AUC/MIC. In each case except for the highest AUC/MIC ratio, the observation periods included complete regrowth in the time-kill curve studies. The AME was expressed by its intensity, IE (the area between the control growth and time-kill and regrowth curves up to the point where the viable counts of regrowing bacteria are close to the maximum values observed without drug). For most AUC ranges the IE-AUC curves were fitted by an Emax (maximal effect) model, whereas the effects observed at very high AUCs were greater than those predicted by the model. The AUCs that produced 50% of maximal AME were proportional to the MICs for the strains studied, but maximal AMEs (IEmax) and the extent of sigmoidicity (s) were not related to the MIC. Both Teff and log AUC/MIC correlated well with IE (r2 = 0.98 in both cases) in a species-independent fashion. Unlike Teff or log AUC/MIC, a specific relationship between IE and log AUCeff was inherent in each strain. Although each IE and log AUCeff plot was fitted by linear regression (r2 = 0.97 to 0.99), these plots were not superimposed and therefore are bacterial species dependent. Thus, AUC/MIC and Teff were better predictors of ciprofloxacin's AME than AUCeff. This study suggests that optimal predictors of the AME produced by a given quinolone (intraquinolone predictors) may be established by examining its AMEs against bacteria of different susceptibilities. Teff was shown previously also to be the best interquinolone predictor, but unlike AUC/MIC, it cannot be used to compare different quinolones. AUC/MIC might be the best predictor of the AME in comparisons of different quinolones.
Anti-Infective Agents↗
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↗
Vancomycin-resistant Bacillus circulans carrying the vanA gene responsible for vancomycin resistance in enterococci.
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Anti-Bacterial Agents↗
High incidence of erythromycin-resistant Streptococcus pyogenes in Monza (North Italy) in untreated children with symptoms of acute pharyngo-tonsillitis: an epidemiological and molecular study.
A retrospective analysis of susceptibility data available for Group A streptococcal isolates collected between January 1990 and January 1996 at the Hospital Microbiology Laboratory of Monza (North Italy), showed a sharp rise in the erythromycin resistance rates during the last 3 years. Streptococcus pyogenes resistant to erythromycin accounted for approximately 1% of strains isolated between 1990 and 1992; the percentage then rose from 5% in 1993 to almost 39% in 1995. In January 1996, the resistance rates peaked to 81%. A prospective controlled study performed between March and May of 1996 to determine the percentage of erythromycin-resistant Group A streptococci isolated in Monza from untreated children with acute pharyngo-tonsillitis, gave further confirmation of a high rate of erythromycin resistance (47%) in this area. Molecular characterization by T-serotyping and pulse-field gel electrophoresis analysis of 25 erythromycin-resistant Group A streptococcal isolates, showed a relatively high degree of heterogeneity among these strains, demonstrating that the increased resistance is not caused by the spread of a single clone.
Acute Disease↗
Parameters of bacterial killing and regrowth kinetics and antimicrobial effect examined in terms of area under the concentration-time curve relationships: action of ciprofloxacin against Escherichia coli in an in vitro dynamic model.
Although many parameters have been described to quantitate the killing and regrowth of bacteria, substantial shortcomings are inherent in most of them, such as low sensitivity to pharmacokinetic determinants of the antimicrobial effect, an inability to predict a total effect, insufficient robustness, and uncertain interrelations between the parameters that prevent an ultimate determination of the effect. To examine different parameters, the kinetics of killing and regrowth of Escherichia coli (MIC, 0.013 microg/ml) were studied in vitro by simulating a series of ciprofloxacin monoexponential pharmacokinetic profiles. Initial ciprofloxacin concentrations varied from 0.02 to 19.2 microg/ml, whereas the half-life of 4 h was the same in all experiments. The following parameters were calculated and estimated: the time to reduce the initial inoculum (N0) 10-, 100-, and 1,000-fold (T90%, T99%, and T99.9%, respectively), the rate constant of bacterial elimination (k(elb)), the nadir level (Nmin) in the viable count (N)-versus-time (t) curve, the time to reach Nmin (t(min)), the numbers of bacteria that survived (Ntau) by the end of the observation period (tau), the area under the bacterial killing and regrowth curve (log N(A)-t curve) from the zero point (time zero) to tau (AUBC), the area above this curve (AAC), the area between the control growth curve (log N(C)-t curve) and the bacterial killing and regrowth curve (log N(A)-t curve) from the zero point to tau (ABBC) or to the time point when log N(A) reaches the maximal values observed in the log N(C)-t curve (I(E); intensity of the effect), and the time shift between the control growth and regrowth curves (T(E); duration of the effect). Being highly sensitive to the AUC, I(E), and T(E) showed the most regular AUC relationships: the effect expressed by I(E) or T(E) increased systematically when the AUC or initial concentration of ciprofloxacin rose. Other parameters, especially T90%, T99%, T99.9%, t(min), and log N0 - log Nmin = delta log Nmin, related to the AUC less regularly and were poorly sensitive to the AUC. T(E) proved to be the best predictor and t(min) proved to be the worst predictor of the total antimicrobial effect reflected by I(E). Distinct feedback relationships between the effect determination and the experimental design were demonstrated. It was shown that unjustified shortening of the observation period, i.e., cutting off the log N(A)-t curves, may lead to the degeneration of the AUC-response relationships, as expressed by log N0 - log Ntau = delta log Ntau, AUBC, AAC, or ABBC, to a point where it gives rise to the false idea of an AUC- or concentration-independent effect. Thus, use of I(E) and T(E) provides the most unbiased, robust, and comprehensive means of determining the antimicrobial effect.
Anti-Infective Agents↗
Diffusion of carbapenems through the outer membrane of enterobacteriaceae and correlation of their activities with their periplasmic concentrations.
Scarce information is available on the real mechanism by which carbapenemes penetrate in Enterobacteriaceae, although a considerable amount of evidence suggests that in many species of this family the lack of certain outer membrane proteins is associated with the acquisition of resistance to these antibiotics. The existance of specific pathways for the carbapenems has never been demonstrated, although at times it has been postulated in both wild and mutant strains, on the basis of evident discordances between permeability patterns and suceptibility data. By using the Zimmerman and Rosselet technique, which requires the strain under investigation to harbor a suitable beta-lactamase, the permeability of intact Escherichia coli and Enterobacter cloacae cells to meropenem and imipenem was investigated by transferring a constructed vector carrying the carbapenem hydrolyzing CphA metallo-beta-lactamase gene into the parental strains and their porin-deficient mutants. Reduced amounts of nonspecific porins significantly reduced the penetration of both carbapenems. The virtual absence of porins caused the MICs of meropenem to increase, mostly in Enterobacter cloacae, while it did not affected the MICs of imipenem. No evidence of specific porin pathways of the type described in Pseudomonas aeruginosa was found.
Bacterial Outer Membrane Proteins↗
Characterization of the chromosomal cephalosporinases produced by Acinetobacter lwoffii and Acinetobacter baumannii clinical isolates.
The beta-lactamases produced by Acinetobacter lwoffii ULA-501, Acinetobacter baumannii ULA-187, and A. baumannii AC-14 strains were purified and characterized, and their kinetic interactions with several beta-lactam molecules, including substrates and inhibitors, were studied in detail. The three enzymes appeared to be cephalosporinases with different acylation efficiencies (kcat/Km ratio values), and their hydrolytic activities were inhibited by benzylpenicillin, piperacillin, and cefotaxime, which did not behave as substrates. Carbenicillin was a substrate for the beta-lactamase from A. lwoffii ULA-501, whereas it acted as a transient inactivator of the enzymes produced by the two A. baumannii strains. Clavulanic acid was unable to inactivate the three beta-lactamases, whereas sulbactam behaved as an inactivator only at a high concentration (1 mM) which is difficult to achieve during antibiotic therapy. Analysis of the interaction with 6-beta-iodopenicillanic acid also allowed us to better discriminate the three beta-lactamases analyzed in the present study, which can be included in the group 1 functional class (5).
Acinetobacter↗
Comparative affinities for penicillin-binding proteins of multipolar ionic amphoteric cephalosporins in gram-negative bacteria.
Chemical modification of the highly reactive 7-aminocephalosporanic acid has yielded many compounds with improved activities and expanded clinical spectra. Introduction of an alpha-oxyimino group in C-7 position has given rise to improved activity against Gram-negative bacteria as a consequence of the high affinity of compounds carrying this substituent for the essential penicillin-binding protein (PBP) 3. The spectrum of activity of oxyimino cephalosporins has been further expanded by introduction of substituents with a quaternary nitrogen in C-3 position. These compounds have maintained their high affinity for the essential PBP 3, typical of the third generation cephalosporins and have acquired an improved ability to cross the outer membranes of Gram-negative bacteria. Among these compounds cefepime also exhibits high affinity for PBP 2, a very unusual property among cephalosporins.
Bacterial Proteins↗
A simple method to detect bacteriolytic enzymes produced by enterobacteriaceae.
The production of bacteriolytic enzymes by Enterobacteriaceae in various growth conditions was investigated. Peptone-based media containing killed Gram-negative cells facilitated detection of bacteriolytic enzyme production in the highest number of species. These belonged to the genera Serratia, Proteus, Morganella and Providencia. In contrast, Escherichia coli, Shigella, Salmonella, Klebsiella, Enterobacter and Citrobacter species did not produce bacteriolytic activities in any of the conditions tested.
Bacteriolysis↗
Activities of oral antibiotics on Providencia strains isolated from institutionalized elderly patients with urinary tract infections.
More than 250 Providencia strains isolated from the urine of institutionalized elderly patients were tested against cefaclor, cefuroxime, cefetamet, cefpodoxime, ciprofloxacin, and amoxicillin-clavulanic acid. Our results confirm the strong activities of expanded-spectrum oral cephalosporins against Providencia isolates, f1p4ell as the marked differences in susceptibilities among accurately identified Providencia species.
Administration, Oral↗
Relative importances of outer membrane permeability and group 1 beta-lactamase as determinants of meropenem and imipenem activities against Enterobacter cloacae.
The roles of outer membrane permeability and Bush group 1 beta-lactamase activity in determining Enterobacter cloacae susceptibility to either meropenem or imipenem were investigated. A beta-lactamase-deficient strain was obtained by mutagenesis from a clinical isolate of E. cloacae, and a porin-deficient strain was selected from this mutant with cefoxitin. Both strains were transformed with the plasmid pAA20R, which contained the gene coding for the carbapenem-hydrolyzing CphA beta-lactamase, and the carbapenem permeability coefficients were measured by the Zimmermann and Rosselet technique (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). The permeability coefficient of meropenem was roughly half that of imipenem in the normally permeable strain and almost seven times lower than that of imipenem in the porin-deficient strain. In the porin-deficient strain, the virtual absence of porins caused the MICs of meropenem to increase from 8 to 16 times, while it did not affect the MICs of imipenem. Conversely, the beta-lactamase affected imipenem but not meropenem activity: meropenem showed a similar activity in the parent strain and in the beta-lactamase-deficient mutant with both a low- and high-density inoculum, whereas imipenem was 16 times less active against the parent strain when the high-density inoculum was used. It is concluded that outer membrane permeability and stability to group 1 beta-lactamase have different impacts on the activities of meropenem and imipenem against E. cloacae.
Bacterial Proteins↗
Target for bacteriostatic and bactericidal activities of beta-lactam antibiotics against Escherichia coli resides in different penicillin-binding proteins.
The relationship between cell-killing kinetics and penicillin-binding protein (PBP) saturation has been evaluated in the permeability mutant Escherichia coli DC2 in which the antimicrobial activity of beta-lactams has been described as being directly related to the extent of saturation of the PBP target(s). Saturation of a single PBP by cefsulodin (PBP 1s), mecillinam (PBP 2), and aztreonam (PBP 3) resulted in a slow rate of killing (2.5-, 1.5-, and 0.8-log-unit decreases in the number of CFU per milliliter, respectively, in 6 h). Saturation of two of the three essential PBPs resulted in a marked increase in the rate of killing, which reached the maximum value when PBPs 1s and 2 were simultaneously saturated by a combination of cefsulodin and mecillinam (4.7-log-unit decrease in the number of CFU per milliliter in 6 h). Inactivation of all three essential PBPs by the combination of cefsulodin, mecillinam, and aztreonam further increased the killing kinetics (5.5-log-unit decrease in the number of CFU per milliliter), and this was not significantly changed upon additional saturation of the nonessential PBPs 5 and 6 by cefoxitin. Similar relationships between PBP saturation and killing kinetics were obtained with imipenem and meropenem at concentrations which inhibited only one PBP (PBP 2), only two PBPs (PBP 1s and 2), or all three essential PBPs. Saturation of one or more PBPs also resulted in a different rate of bacteriolysis, the highest rate being obtained by the cefsulodin-mecillinam combination and by 5 micrograms of either imipenem or meropenem per ml. All of these conditions caused saturation of PBP 2 and saturation or extensive binding of PBP 1s. However, none of these conditions caused determined the fastest possible rate of killing, which occurred only when all three essential PBPs were saturated. It was concluded that the actual killing effect of beta-lactams is reflected by killing rates that approach the fastest possible rate for the given microorganism and that the targets for the bactericidal activity are precisely those PBPs whose saturation or binding occurs under conditions.
Anti-Bacterial Agents↗