Giuseppe Satta

Lipoteichoic acid as a new target for activity of antibiotics: mode of action of daptomycin (LY146032).

Daptomycin at the MIC allowed the cell mass increase of enterococcal strains and Bacillus subtilis to continue for 2 to 3 h at rates comparable to those of the controls. During this time the cell shape of the former changed to a rod configuration and that of the latter changed to long rods. In these bacteria, in which cell mass continued to increase, the MIC of daptomycin inhibited peptidoglycan synthesis by no more than 20% after 20 min of incubation and by roughly 50% after 2 h of incubation. Other macromolecules, such as DNA, RNA, and proteins, were only slightly affected. In contrast, incorporation of [14C]acetate into lipids was reduced by about 50% in the various strains after 20 min of treatment with daptomycin at the MIC. When the effect of the major lipid-containing polymers on synthesis was evaluated in detail, it was found that under conditions in which peptidoglycan and the other macromolecules mentioned above were inhibited only slightly (20%) and total lipid synthesis was inhibited by 50%, synthesis of teichoic and lipoteichoic acid was inhibited by 50 and 93%, respectively. Daptomycin was not found to enter the cytoplasm of either bacterial or mammalian cells. It bound, in the presence of calcium ions only, to whole bacterial cells, cell walls (both those that contained and those that did not contain membranes), and isolated membranes of bacterial and mammalian cells. Washing with EDTA removed daptomycin from all cells mentioned above and cell fractions except the bacterial membrane. It is concluded that lipoteichoic acid is most likely the primary target of daptomycin. Images

Distribution of cphA or related carbapenemase-encoding genes and production of carbapenemase activity in members of the genus Aeromonas.

The prevalence of the cphA gene or related carbapenemase-encoding genes was investigated in 114 Aeromonas strains belonging to the six species of major clinical interest. A species-related distribution of cphA-related sequences was observed. Similar sequences were found in A. hydrophila, A. veronii bv. sobria, A. veronii bv. veronii, and A. jandaei, but not in A. caviae, A. trota, or A. schubertii. However, a single A. caviae strain (of 62 tested) was found carrying cphA-related sequences, suggesting the possibility of the horizontal transfer of this gene to species which normally do not carry it. Production of carbapenemase activity was detectable in 83% of the hybridization-positive strains but in none of the hybridization-negative ones. When it was present, carbapenemase activity was always inhibitable by EDTA. Either carbapenemase-producing or not, Aeromonas strains appeared to be susceptible to imipenem when in vitro susceptibility testing was performed with inocula of conventional size (10(5) CFU), for which MICs were always < or = 1 microgram/ml. With a larger inoculum (10(8) CFU), the MICs for carbapenemase-negative strains always remained < or = 1 microgram/ml, while those for carbapenemase-producing strains were always > or = 4 micrograms/ml, being usually higher than the breakpoint for susceptibility. The present results indicate that the production of metallocarbapenemase activity, apparently encoded by cphA homologs, is widespread among some of the Aeromonas species of clinical interest (A. hydrophila, A. veronii bv. sobria, A. veronii bv. veronii, and A. jandaei) and that imipenem MICs for carbapenemase-producing strains are subjected to a relevant inoculum size effect.

Evaluation of ceftriaxone and other antibiotics against Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae under in vitro conditions simulating those of serious infections.

In pursuit of an in vitro system capable of reliably predicting the activities of antibiotics in serious infections and in infections occurring in immunocompromised hosts, we evaluated the abilities of four drugs to achieve virtually complete killing of bacterial cells growing in human body fluids in amounts which are very high and close to those likely to be present in serious infections; drug concentrations varied with time as they vary in human bronchial secretions or blood or urine (dynamic concentrations). The rationale for such a test was (i) to set up in vitro conditions as close as possible to those the antibiotics encounter in serious infections and (ii) to hold the drugs capable of almost completely killing the bacteria used in the assay to be highly active in vitro and likely to be the most efficacious in the treatment of serious infections. Among the antibiotics used, ceftriaxone proved to be highly active under conditions simulating pulmonary infections and septicemias caused by Streptococcus pneumoniae (bacteria grown in bronchoalveolar fluid or blood; antibiotic concentrations varying with time as in human bronchial secretions or blood) and under conditions simulating blood and urinary infections caused by Escherichia coli (bacteria grown in human blood or urine; antibiotic concentrations varying as in the various fluids). Gentamicin (not tested against pneumococci) appeared to be highly active only under conditions simulating urinary infections caused by E. coli; aztreonam (not tested against pneumococci) and ampicillin (tested only against pneumococci) did not appear to be highly active under any of the test conditions. Only the combination of gentamicin plus either ceftriaxone or aztreonam appeared to be highly active under conditions simulating serious septicemias and urinary infections caused by Psudomonas aeruginosa.

In vitro activity of ciprofloxacin against aerobic bacteria isolated in a southern European hospital.

The activity of ciprofloxacin was evaluated against 1,204 isolates freshly isolated in Southern Europe, including 193 isolates of 10 species never studied before. Ciprofloxacin proved more active than other quinolones and very active in absolute terms against the 10 new species and showed against the other species an activity close to that reported for isolates from other geographic areas.

Bacteriostatic and bactericidal activities of beta-lactams against Streptococcus (Enterococcus) faecium are associated with saturation of different penicillin-binding proteins.

The MICs and MBCs of benzylpenicillin, ampicillin, cefotaxime, and methicillin were evaluated against a Streptococcus (Enterococcus) faecium wild-type strain and against three mutants hyperproducing PBP 5 in cells incubated at both optimal and suboptimal temperatures. In the wild-type strain grown at optimal temperature, the MBCs of all beta-lactams were significantly greater than the MICs (bacteriostatic effect). As opposed to this, in the same cells grown at suboptimal temperature and in the mutants hyperproducing PBP 5 at all temperatures, the MICs of all antibiotics coincided with the MBCs (bactericidal effect). Under all conditions in which the MIC and MBC were the same, with all antibiotics, growth inhibition occurred only at the minimal concentration saturating all penicillin-binding proteins (PBPs) (or at higher concentrations). On the contrary, under conditions in which the MIC was lower than the MBC, only some of the PBPs were saturated (or bound) at both the MIC and the MBC, PBP 5 in no case being either saturated or bound. Under all conditions in which saturation of all PBPs was needed for growth inhibition, cells died at all antibiotic MBCs with kinetics which were much faster than those with which they died at the MBCs under conditions in which not all PBPs were saturated (or bound). In addition, under the former conditions, antibiotic concentrations above the MBCs did not significantly accelerate cell death kinetics, while under the latter conditions there was an acceleration in kinetics with increasing antibiotic concentrations up to full saturation of PBPs. It is suggested that the killing that occurs when all PBPs are saturated is a direct consequence of inactivation of PBP functions, while killing occurring when only some of them are saturated or bound is also (or mainly) an indirect consequence of inability of cells to grow and that, in S. faecium, the targets for growth inhibition and cell killing reside in different PBPs: for the latter effect, inactivation of one (or more) of the high-molecular-weight PBPs is sufficient, whereas in the former case inactivation of PBP 5 is necessary (after saturation of all other PBPs). Images

Interference of a Staphylococcus Aureus Bacteriolytic Enzyme with Polymorphonuclear Leucocyte Functions

Staphylococcus aureus is one of the most important human pathogens responsible for serious infections (1). Infections caused by this microorganism are often difficult to treat because of the high frequency with which S. aureus strains are resistant to the most common antibiotics. A way to overcome this problem could be the development of methods for enhancing host defenses against S. aureus. To this purpose, the main virulence factors of this microorganism have been investigated and almost all of the biologically active compounds produced by this microorganism have been analyzed (2). Despite this, an enzyme, the endo-β -N acetylglucosaminidase (SaG) which hydrolyzes peptidoglycan and is produced by all S. aureus strains (3), was never considered as a possible virulence factor.

Evaluation of in vitro antimicrobial activity of lomefloxacin against staphylococci, enterococci, Enterobacteriaceae, and Pseudomonas aeruginosa.

In vitro antimicrobial activity of lomefloxacin and other antibiotics (norfloxacin, beta-lactams, cotrimoxazole, netilmicin, and miokamycin) was evaluated against 317 clinical isolates including Staphylococcus aureus, enterococci, Enterobacteriaceae, and Pseudomonas aeruginosa. Lomefloxacin showed a high in vitro activity against a wide variety of bacterial species. Against Enterobacteriaceae, lomefloxacin displayed the highest activity (MIC90S, less than or equal to 1 microgram/ml), and it was usually more active than ampicillin and netilmicin, and often more active than cotrimoxazole. Lomefloxacin showed a good antimicrobial activity also against both methicillin-susceptible and methicillin-resistant S. aureus, as it was able to inhibit 90% of staphylococcal strains at a concentration less than or equal to 2 micrograms/ml. Against enterococci and P. aeruginosa, lomefloxacin was usually less active (MIC90S, 8 micrograms/ml). As compared to norfloxacin, lomefloxacin displayed an overall similar activity against staphylococci and enterococci, but appeared less active against Gram-negative bacteria. Bactericidal activity of lomefloxacin against E. coli, when assayed by a novel method, proved to be high, and results indicate that an oral dose of 200 mg of lomefloxacin should exert a very high bactericidal activity against E. coli in urinary tract infections, even in those sustained by large bacterial populations.

Evaluation of Bactericidal Activity of Cefotaxime and Other β-Lactams by a Novel Method

SummaryIn previous studies on Streptococcus faecium we proposed that the minimum β-lactam concentration killing 99.9% of a bacterial population within 3 hours be defined as the minimum directly bactericidal concentration (MDBC) of that drug. In the present study we first evaluated the kinetics of cellular killing by various β-lactams as related to penicillin-binding-protein (PBP) binding in Escherichia coli DC2, a hyperpermeable mutant. We concluded that in E. coli the MDBC for β-lactams coincides with the minimum concentration capable of saturating PBPs 1b, 2 and 3. Of the antibacterial drugs we studied, cefsulodin, meciliinam and aztreonam had a much greater affinity for one essential PBP (PBP 1b, 2 and 3, respectively) than for all others, whereas cefotaxime had close affinities for all the above PBPs. MDBC values of >500, 500, >50, Wand 1.5 mg/L were obtained for cefsulodin, meciliinam, aztreonam, ampicillin and cefotaxime, respectively. On the basis of the pharmacokinetic properties of these drugs, our results indicate that meciliinam, ampicillin and cefsulodin may be bactericidal in urine but not at other body sites; aztreonam is probably bactericidal in urine and blood, but not elsewhere; and cefotaxime is bactericidal in all the biological fluids we studied.

Capsular antigenic variations by lysogenic conversion inKlebsiella pneumoniae: Relationship with virulence

Klebsiella pneumoniae lysogenization by phages FR2 and AP3 induces modifications in capsular antigens as evidenced by utilization of K-antisera raised against lysogenic and nonlysogenic strains. After serum absorption, it was found thatK. pneumoniae K59 derivatives lysogenic forKlebsiella phages FR2 and AP3 lack a capsular antigenic determinant present in the nonlysogenic parent and carry a new one. The new antigen, codified by AP3 genes, is different from that codified by FR2. Moreover, strains lysogenic for AP3 phage are less virulent for mice than the FR2 lysogens and the nonlysogenic parent. Phages similar to AP3 for host range and plaque morphology were isolated from 3 of 80 wild-typeKlebsiella strains. In theKlebsiella strain K59, these phages cause the same antigenic modifications observed with AP3.

A Surface Protein of Klebsiella Pneumoniae and Escherichia Coli That Binds Phagocytes and Inhibits Phagocytosis

We have previously described a Klebsiella pneumoniae non-fimbrial adhesin (MIAT: mannose inhibitable adhesin/T7 receptor) which works as a receptor for phage T7 and mediates unencapsulated strain adherence to epithelial cells of the oral cavity, urinary tract and intestine (5–7).