Sub-MIC concentrations of cefodizime interfere with various factors affecting bacterial virulence.

The molecular array of the outermost surface of bacteria and their physico-chemical characteristics modulate various functions which, when expressed in terms of the human environment, are generally known as factors of bacterial virulence. The present study investigated the ability of sub-MIC concentrations of cefodizime to interfere with the virulence factors of Escherichia coli. Bacterial adhesiveness to human epithelial cells was inhibited down to 1/32 x MIC of cefodizime, an antibiotic that is also capable of inducing the widespread production of filamentous forms at levels ranging from 1/2 to 1/8 x MIC. Given that this interfered with the correct evaluation of other virulence parameters, the study was extended to consider the effects of 1/16 to 1/128 x MIC. Sub-MIC concentrations of cefodizime inhibit haemagglutination, hydrophobicity and electrophoretic mobility, which are correlated with each other and provide clues relating to the physico-chemical characteristics of the outer surface. Cefodizime also reduces swarming. Phagocytosis was not affected but killing increased significantly. Oxidative bursts investigated by a chemiluminescence procedure were not modified. The interpolation of these pharmacodynamic findings with pharmacokinetic curves indicates that the effect of sub-MIC concentrations of cefodizime can prolong antimicrobial effects on virulence determinants up to 12 h after the antibiotic concentration has fallen below the MIC value.

Effects of erdosteine and its metabolites on bacterial adhesiveness.

Erdosteine (CAS 84611-23-4) is administered as a mucolytic drug in patients with pulmonary disorders who suffer from a thickening of bronchial mucus with altered physico-chemical characteristics. Erdosteine itself does not have a free thiol group but its metabolization produces active metabolites with a -SH group that is capable of breaking disulfide bonds of mucins and improving the mucociliary clearance of the airways, and thus reproducing the effects of the class of muco-active drugs having a thiol group. It has also been reported that muco-active drugs with this group reduce bacterial adhesiveness to human mucosal cells. The aim of this study was to investigate whether erdosteine and its SH-metabolites are capable of interfering with bacterial adhesiveness. Metabolite I significantly reduces both S. aureus and E. coli adhesiveness to human mucosal epithelial cells at concentrations of 2.5, 5 and 10 micrograms/ml. The same concentrations of erdosteine, metabolite II, metabolite III and N-acetylcysteine (as a control drug) were devoid of such activity, whereas the results of hemagglutination and hydrophobicity assays showed that the behaviour of metabolite I overlapped that of bacterial adhesiveness, thus indicating that interference takes place at a fimbrial level. This is confirmed by the fact that the incubation of human buccal cells with drugs does not reduce the adhesiveness of untreated bacteria. The presence of this additional activity in a muco-active drug is useful because bacteria not only adhere to epithelial cells but also to tracheobronchial secretions.

Pharmacodynamic effects of subinhibitory concentrations of rufloxacin on bacterial virulence factors.

It has been reported that subinhibitory concentrations (sub-MICs) of some fluoroquinolones are still capable of affecting the topological characteristics of DNA (inhibition DNA-gyrase) and that this leads to a reduction in some of the factors responsible for bacterial virulence (by means of the disruption of protein synthesis and alterations in phenotype expression), even though the microorganisms themselves are not killed. The present study investigated the ability of sub-MICs of rufloxacin, an orally absorbed monofluorinated quinolone with a long half-life (28 to 30 h), to interfere with the bacterial virulence parameters of adhesiveness, hemagglutination, hydrophobicity, motility, and filamentation, as well as their interactions with host neutrophilic defenses such as phagocytosis, killing, and oxidative bursts. It was observed that Escherichia coli adhesiveness was significantly reduced at rufloxacin concentrations of 1/32 MIC, hemagglutination and hydrophobicity were significantly reduced at concentrations of, respectively, 1/4 MIC and 1/8 MIC, and motility was significantly reduced at concentrations of 1/16 MIC; filamentation was still present at concentrations of 1/4 MIC. Phagocytosis was not affected, but killing significantly increased from 1/2 MIC to 1/8 MIC; oxidative bursts measured by means of chemiluminescence were not affected. The fact that sub-MICs are still effective in interfering with the parameters of bacterial virulence is useful information that needs to be correlated with pharmacokinetic data in order to extend our knowledge of the most effective concentrations that can be used to optimize treatment schedules, for example, single administrations, particularly in noncomplicated lower urinary tract infections.

Influence of sub-minimum inhibitory concentrations of cefodizime on the phagocytosis, intracellular killing and oxidative bursts of human polymorphonuclear leukocytes.

It is generally recognized that sub-minimum inhibitory concentrations (sub-MICs) of antibiotics are still capable of interfering with some bacterial virulence parameters, thus facilitating host neutrophilic defenses such as phagocytosis, killing and oxidative bursts. This study investigated the interaction of Escherichia coli with these neutrophilic functions after pretreatment with various sub-MICs of cefodizime. E. coli exposed to 1/2 to 1/8 MICs of cefodizime showed the extensive production of long and very long filaments that interfere with the precise measurement of phagocytic and killing parameters. Our analysis was consequently extended to the activity of 1/16 to 1/64 MICs. The interesting finding was that, although phagocytosis was unaffected, killing was significantly increased in one strain at 1/16 MIC and in another at 1/32 MIC while in the last strain it was unaffected. Oxidative bursts were not modified by any of the sub-MICs. The knowledge that these sub-MICs are still effective in increasing bacteria killing, correlated with the pharmacokinetic curve of a common single dose of cefodizime 1 g i.m., showed that the killing effects of sub-MICs may last for as long as 12 h after the activity of the MIC value. This integrated information extends our knowledge of the ultimate efficacy of an antibiotic and provides further information for optimizing scheduling.

Age-associated differences in neutrophil oxidative burst (chemiluminescence).

Phagocytic defensive functions consist of a sequence of events, including migration, phagocytosis, secretion, and the release of reactive oxygen species (ROS). The last of these (also called "oxidative burst") has not received due attention in the elderly, even though it can be considered the most important event in the process of killing an invading microorganism. The aim of the present study was to investigate the oxidative burst activity of polymorphonuclear neutrophil leukocytes (PMNs) in relation to age, using a technique that specifically identifies ROS production: luminol-amplified chemiluminescence (LACL). Besides the use of LACL, a particular feature of the study was the use of five rather than just one or two different stimulants: two particulate (Candida albicans and zymosan) and three soluble ones [N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12 myristate 13 acetate (PMA), and polyanetholesulfonate (liquoid)]. This approach allowed us to observe a dichotomy between the effects of Candida and zymosan (particulates), which were not significantly different in the elderly subjects compared to the young controls, and those of fMLP, PMA, and liquoid (solubles), which showed a significant reduction in LACL in the elderly group. Considering the different results obtained with the various stimulants adopted that are all believed to have NADPH oxidase as a common final target of oxidative burst, it may be postulated that aging can influence the different transductional pathways in different ways.

Influence of age on oxidative bursts (chemiluminescence) of polymorphonuclear neutrophil leukocytes.

The release of reactive oxygen species (ROS) during neutrophil oxidative bursts is the last of a sequence of different steps leading to the neutralization of pathogen microorganisms. Using luminol-amplified chemiluminescence (LACL), the oxidative burst activity of neutrophils in elderly people (> or = 75 years) was compared with that in younger controls (39 years on average) after activation with both particulate (Candida albicans) and soluble (formyl-methionyl-leucyl-phenylalanine; fMLP) stimulants. After Candida stimulation, a reduction in LACL was observed in the elderly subjects in comparison with the controls, but the difference did not reach statistical significance. After fMLP stimulation, the reduction in LACL was significant, thus suggesting that the Candida pathway of chemiluminescence production seems to be less affected than the fMLP pathway. This finding raises questions concerning the complex differences in the pathways of cell killing and ROS generation, and their efficacy in the elderly. Various possible explanations are discussed, all of which need further investigation.

Phagocytosis and intracellular killing of rokitamycin-exposed staphylococci by human polymorphonuclear leukocytes.

The exposure of bacteria to antibiotics at even sub-minimum inhibitory concentrations (sub-MICs) induces physicochemical and biochemical modifications that facilitate phagocytosis and intracellular killing by polymorphonuclear leukocytes (PMNs). These PMN functions were investigated by exposing Staphylococcus aureus strains to different sub-MICs (1/2 to 1/32 MIC) of rokitamycin (RKM). Although phagocytosis and the index of phagocytosis of the antibiotic-exposed staphylococci were not significantly modified with respect to controls, the percentage of killing significantly increased after exposure to 1/2 and 1/4 MIC by 31 and 22%, respectively. Taking into consideration the other aspect of a possible direct interaction between RKM and PMNs, it was observed that up to 10 micrograms/ml of RKM did not interfere with phagocytosis but significantly enhanced killing activity by up to 33%. This effect can be correlated with the high uptake of RKM by PMNs (cellular/extracellular ratio congruent to 30.5). The relevance of these in vitro observations to clinical situations remains to be further investigated.