Interaction between human polymorphonuclear leucocytes and bacteria released from in-vitro bacterial biofilm models.

The interactions between phagocytic cells (polymorphonuclear leucocytes) and Escherichia coli cells released from a biofilm model formed in vitro on the surface of cotton threads in an artificial medium were compared with those of phagocytes and bacteria released from a newly developed in-vitro biofilm model. This new model of bacterial biofilm on the surface of cotton threads was developed by soaking cotton threads in rat carboxymethylcellulose pouch exudate and culturing E. coli in the exudate. The structure of the biofilm model and the surface structure of the bacteria in the biofilm resembled those observed in vivo in infected pouches, and they were quite different from those observed with the biofilm model in artificial medium. Both bacteria released from biofilm models in an artificial medium and those from biofilms in rat carboxymethylcellulose pouch exudate, in vitro, were almost equally resistant to killing by phagocytes. The sensitivity of these bacteria to phagocytosis was no different from that of normal bacteria grown in artificial medium. Bacteria from both models were also less sensitive to the killing activity of H2O2. Electronmicroscopy showed that bacteria from both models had some products that interacted with ruthenium red on their surfaces, but the respective quantities of these products differed.

Interaction between clarithromycin and biofilms formed by Staphylococcus epidermidis.

Interactions between clarithromycin and biofilms formed by Staphylococcus epidermidis were investigated by using a clarithromycin-resistant strain. Treatment of the colonization with a relatively low concentration of clarithromycin resulted in the eradication of slime-like structure and a decrease in the quantity of hexose. Another result was increased penetration of antibiotics through the biofilm of S. epidermidis.

Interaction between biofilms formed by Pseudomonas aeruginosa and clarithromycin.

Interactions between bacterial biofilms formed by Pseudomonas aeruginosa and clarithromycin, a macrolide having no anti-P. aeruginosa activity, were investigated. P. aeruginosa incubated for 10 days on membrane filters formed biofilms on the surfaces of the filters. The biofilms were characterized by dense colonizations of bacteria and thick membranous structures that covered the colonies. Treatment of the biofilms with a relatively low concentration of clarithromycin for 5 days resulted in an eradication of the membranous structures. Quantitative analysis of alginate and hexose was done to evaluate the quantity of polysaccharides in or on the biofilms. Treatment of the biofilms with clarithromycin decreased the quantity of alginate and hexose and therefore perhaps the quantity of polysaccharides as well. Eradication of the membranous structures of biofilms, or the decrease in the quantity of polysaccharides, resulted in an increase in the rate of penetration of antibiotics through bacterial biofilms. In vivo therapeutic effects of ofloxacin in the rat infection model, in which the biofilm mode of growth of P. aeruginosa is characteristic, were enhanced by oral coadministration of clarithromycin. It is suggested that clarithromycin eradicated glycocalyx produced by P. aeruginosa, or suppressed the production of glycocalyx, by unknown mechanisms and thereby enhanced the therapeutic efficacies of other antimicrobial agents against infections caused by P. aeruginosa.

beta-Lactamase produced by a highly beta-lactam-resistant strain of Bacteroides fragilis: an obstacle to the chemotherapy of experimental mixed infections.

A strain of Bacteroides fragilis, which produces a metallo-beta-lactamase, was inoculated into pouches on the backs of rats together with a beta-lactamase-negative Escherichia coli highly sensitive to beta-lactam antibiotics. The mixed infection rat pouch model was treated with either flomoxef (susceptible to hydrolysis by the beta-lactamase produced by B. fragilis), or cefmetazole (relatively resistant to hydrolysis). In this model of mixed infection flomoxef showed weak in-vivo activity against E. coli, although showing the same strong activity in a model of single infection with E. coli. On the other hand, cefmetazole showed strong activity against E. coli, even in the model of mixed infection. The concentrations of both drugs in the pouches were decreased in infections with the strain of B. fragilis. There was a greater decrease in the concentration of flomoxef than of cefmetazole. Flomoxef was unstable whereas cefmetazole was relatively stable in the pouch exudates that had been infected with B. fragilis. These experimental data suggest that bacteria that produce a metallo-beta-lactamase decrease the in-vivo efficacy of beta-lactam antibiotics against other co-infecting bacteria. Thus, it is suggested that it is important in the chemotherapy of mixed bacterial infections that include these highly resistant beta-lactamase-producing bacteria to use antibiotics that are stable to hydrolysis by these enzymes.

Therapeutic efficacy of granulocyte colony-stimulating factor alone and in combination with antibiotics against Pseudomonas aeruginosa infections in mice.

The therapeutic efficacy of granulocyte colony-stimulating factor (G-CSF) against an experimental intramuscular infection induced by Pseudomonas aeruginosa in mice was confirmed. Bacterial growth in the infected thigh muscle was suppressed by G-CSF treatment. The change in the number of peripheral blood polymorphonuclear leukocytes (PMN) after bacterial challenge was investigated. The results showed that G-CSF could stimulate stronger defense mechanisms after stimulation by bacterial challenge. In the G-CSF-treated group, more clusters of matured PMN were observed in the infected thigh muscle 6 h after bacterial challenge. Next, the correlation between the number of PMN in the blood at the time of infection and the therapeutic efficacy of antibiotics was investigated. The therapeutic efficacy of ceftazidime, a beta-lactam antibiotic, was affected by the number of blood PMN at the time of infection. In particular, a decrease of peripheral blood PMN at the time of infection resulted in a dramatic decrease in the efficacy of ceftazidime. The reduction in leukopenia by G-CSF remarkably strengthened the therapeutic effect of antibiotics in mice.

Effect of human immunoglobulin preparations on experimental Bordetella pertussis infection in mice.

The effects of four kinds of human immunoglobulin preparations for intravenous use [pH-4-treated (IG-100), polyethyleneglycol-treated (PEG-G), sulfonated (S-G) and pepsin-treated (Pep-G)] on intracerebral (i.c.) Bordetella pertussis infection in mice, and on B. pertussis vaccine-induced leukocytosis-promoting factor (LPF) and histamine-sensitizing factor (HSF) were compared with those of human immunoglobulin preparation for intramuscular use (GGN). A prophylactic potential against i.c. B. pertussis challenge was demonstrated in IG-100, PEG-G and GGN. A prophylactic potential was also demonstrated in S-G and Pep-G, although to a lesser extent. Neutralizing activity for LPF was in the following order: GGN = IG-100 = PEG-G greater than S-G = Pep-G; for HSF it was as follows: IG-100 greater than PEG-G greater than GGN = S-G greater than Pep-G. There were no significant differences in antibody titers of the various preparations against B. pertussis antigens. These results suggest that the Fc part of the immunoglobulin molecule is important for protecting against i.c. B. pertussis infection and for neutralizing B. pertussis toxins.

The effect of 2, 4-dinitrophenol on the growth of Bordetella pertussis in chick tracheal organ culture.

Among various metabolic inhibitors tested, only 2, 4-dinitrophenol inhibited the growth of Bordetella pertussis in chick tracheal organ culture at concentrations nontoxic both for bacterial organisms and for ciliary motility of the tracheal fragments. Although this effect of 2, 4-dinitrophenol was reversible in its early stage, longer treatment with this inhibitor resulted in an irreversible inhibition of bacterial growth due to secondary damage of the tracheal fragments. From these observations, it was postulated that the energy required for bacterial growth might be derived from cellular metabolism sensitive to inhibition with 2, 4-dinitrophenol.