Interaction between tobramycin and CSA-13 on clinical isolates of Pseudomonas aeruginosa in a model of young and mature biofilms.

The bactericidal activity of a cholic acid antimicrobial derivative, CSA-13, was tested against eight strains of Pseudomonas aeruginosa (both reference and clinical strains) and compared with the response to tobramycin. In planktonic cultures, the minimal inhibitory and minimal bactericidal concentrations of CSA-13 and tobramycin were in the 1-25 mg/L range except for one mucoid clinical strain which was much less sensitive to tobramycin (minimal bactericidal concentration, 65-125 mg/L). In young (24 h) biofilms, the sensitivity to CSA-13 was reduced (half-maximal concentration CSA-13 averaged 88 mg/L) and varied among the eight strains. The sensitivity to tobramycin was also very variable among the strains and some were fully resistant to the aminoglycoside. The combination of tobramycin with CSA-13 was synergistic in five strains. Only one strain showed antagonism between the two drugs at low concentrations of CSA-13. One reference and five clinical strains were tested in mature (12 days) biofilms. The effect of CSA-13 was delayed, some strains requiring 9 days exposure to the drug to observe a bactericidal effect. All the strains were tolerant to tobramycin but the addition of CSA-13 with tobramycin was synergistic in three strains. CSA-13 permeabilized the outer membrane of the bacteria (half-maximal concentration, 4.4 mg/L). At concentrations higher than 20 mg/L, it also permeabilized the plasma membrane of human umbilical vein endothelial cells. In conclusion, CSA-13 has bactericidal activity against P. aeruginosa even in mature biofilms and cationic steroid antibiotics can thus be considered as potential candidates for the treatment of chronic pulmonary infections of patients with cystic fibrosis. Considering its interaction with the plasma membrane of eukaryotic cells, less toxic derivatives of CSA-13 should be developed.

Evaluation of long-term co-administration of tobramycin and clarithromycin in a mature biofilm model of cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa colonisation and chronic lung infection associated with biofilm formation is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. There is thus an urgent need to develop alternative ways to treat biofilm-associated clinical infections. A kinetic study of twice-daily co-administration of the antibiotics tobramycin and clarithromycin was performed over 28 days on 12-day-old mature P. aeruginosa biofilms formed on microplate pegs for 23 clinical isolates of various phenotypes and genotypes to simulate the treatment of CF patients with inhaled tobramycin through aerosolisation (TOBI). Drug activity was assessed by enumeration of persistent bacteria before, during and after treatment. A mature (12-day-old) biofilm model was chosen because a previous study suggested that such a biofilm was a more realistic in vitro model than a 24-h-old biofilm. Synergistic activity of the drug combination was confirmed on biofilms of 9/23 P. aeruginosa isolates. Of these nine isolates, total destruction of the biofilm was observed for five of them. Combination treatment was superior or equivalent to treatment with tobramycin alone, as activity was observed on 47.8% of the isolates with the combination versus 26.1% with tobramycin alone. No correlation was observed between drug susceptibility profiles and the phenotype or genotype of the isolates.

Effect of antibiotic co-administration on young and mature biofilms of cystic fibrosis clinical isolates: the importance of the biofilm model.

The prognosis of patients with cystic fibrosis (CF) has improved dramatically over the last three decades although the majority of patients still die in early adulthood. Infection with Pseudomonas aeruginosa has generally been associated with declining lung function and increased mortality in patients. This study aimed to investigate the in vitro activity of tobramycin/clarithromycin combination on biofilms of clinical isolates of P. aeruginosa, meticillin-susceptible and -resistant Staphylococcus aureus, and Burkholderia cepacia. First, the impact of antibiotic co-administration on biofilms at different stages of maturation, i.e. during early formation and on 24-h-old and 12-day-old biofilms, was compared. The 24-h-old biofilms were found to behave differently compared with those aged 12 days, which were more resistant to antibiotics. A kinetic study of antibiotic co-administration twice a day for 9 days on 12-day-old P. aeruginosa biofilms was then performed to simulate the effect of treatment of CF patients by inhaled tobramycin through aerosolisation (TOBI). The results obtained support a synergistic activity of tobramycin/clarithromycin combination on biofilms of P. aeruginosa PY02 and PA01, with a logarithmic bacterial decrease of 3.37 and 3.96, respectively. On the other hand, increased resistance to each of the antibacterial agents used alone was observed. This study highlights the importance of the biofilm stage for in vitro investigations and enabled the development of an in vitro model of mature biofilm that is more appropriate to mimic in vivo conditions in CF patients.

In vitro activity of antibiotic combinations against Pseudomonas aeruginosa biofilm and planktonic cultures.

In order to investigate the hypothesis that the combination of clarithromycin with other antibacterial agents offers a successful treatment in the eradication of Pseudomonas aeruginosa biofilm, we first determined the activity of eight antimicrobial agents against planktonic cultures of P. aeruginosa isolates by the microdilution technique. Second, we determined the in vitro effects of these antimicrobial agents individually and in combination against planktonic cultures and pre-formed biofilms of P. aeruginosa PA01. Drug combinations with marked activity were tested on biofilms of clinical isolates. The percentages of planktonic culture survival and biofilm persistence were determined using spectrophotometry and scanning electron microscopy, respectively. Bacterial enumeration was used as a more quantitative method to assess the viability of bacteria in the biofilm. Among the antibacterial agents tested, tobramycin and polymyxin B had the strongest activity against planktonic cultures when tested alone. Synergistic activity was observed for the combination chitosan/tobramycin against planktonic cultures but not biofilms, and for the combination tobramycin/clarithromycin against biofilms but not planktonic cultures. The results suggest that the combination clarithromycin/tobramycin may be successful for eradicating infections involving bacterial biofilms such as in cystic fibrosis patients chronically infected by P. aeruginosa. Further studies on representative isolates in vivo are warranted to support these results.

Modulation by LL-37 of the responses of salivary glands to purinergic agonists.

The interaction of mice submandibular gland cells with LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a cationic peptide with immunomodulatory properties, was investigated. LL-37 at a concentration that did not affect the integrity of the cells increased the uptake of calcium and activated a calcium-insensitive phospholipase A(2) (PLA(2)). The small release of ATP induced by LL-37 could not account for this stimulation because apyrase did not significantly block the response to LL-37. The divalent cation magnesium inhibited the response to LL-37, but this inhibition was probably nonspecific because it also inhibited the in vitro bacteriostatic effect of the peptide. The increase of calcium uptake by LL-37 was not affected by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a rather specific inhibitor of P2X(7) receptors in mice. LL-37 also increased [Ca(2+)](i) in cells from mice invalidated for these receptors. LL-37 had no effect on the response to carbachol. It inhibited the increase of [Ca(2+)](i) and the activation of phospholipase D by ATP. It potentiated the activation of the PLA(2) by the nucleotide. Finally, LL-37 increased the fluidity of the plasma membrane of submandibular gland cells. In conclusion, our results suggest that LL-37 is an autocrine regulator of submandibular gland cells. It does not stimulate mouse P2X(7) receptors but modulates their responses.

Bacterial contamination of indoor air, surfaces, and settled dust, and related dust endotoxin concentrations in healthy office buildings.

Endotoxin, a characteristic external fraction of the outer membrane from Gram-negative bacteria, continuously shed into the environment, is considered as an important risk factor for human health. Our purpose was to study the bacterial species contaminating healthy working environments. Airborne, working surfaces and carpet dust samples were collected from 25 offices. Bacterial species were identified with biochemical ApiSystem strips. Endotoxin concentrations in settled dust were measured with the kinetic chromogenic Limulus assay. The airborne bacterial level varied from 44-2,511, with a median of 277 cfu/m(3). Bacterial contamination on surfaces ranged from 1-1000, with 33 cfu/25 cm(2) as median value. On carpets, bacterial concentration ranged from 0.73-185 x 10(5) cfu/g, with 7.28 x 10(5) cfu/g as median value. Endotoxin concentration varied from 4.6-116.2 EU/mg, with a median of 20.3 EU/mg. Altogether, 501 bacterial strains were isolated. The species variability was greater in Gram-negative bacteria than in Gram-positive cocci with 41 versus 34 various species. In conclusion, people working in healthy offices can be exposed to large concentrations of airborne and dust bacteria and related endotoxin concentrations, giving a risk of work-related diseases.

Synthesis and enzymatic degradation of epichlorohydrin cross-linked pectins.

The water solubility of pectin was successfully decreased by cross-linking with increasing amounts of epichlorohydrin in the reaction media. The initial molar ratios of epichlorohydrin/ galacturonic acid monomer in the reaction mixtures were 0, 0.37, 0.56, 0.74, 1.00, 1.47, and 2.44. The resulting epichlorohydrin cross-linked pectins were thus referred to as C-LP0, C-LP37, C-LP56, C-LP75, C-LP100, C-LP150, and C-LP250, respectively. Methoxylation degrees ranged from 60.5 +/- 0.9% to 68.0 +/- 0.6%, and the effective cross-linking degrees, determined by quantification of the hydroxyl anions consumed during the reaction, were 0, 17.8, 26.0, 38.3, 46.5, 53.5, and 58.7%. respectively. After incubating the different cross-linked pectins (0.5% w/v) in 25 mL of 0.05 M acetate-phosphate buffer (pH 4.5), containing 50 microL of Pectinex Ultra SP-L (pectinolytic enzymes), between 60 and 80% of the pectin osidic bounds were broken in less than 1 hr. Moreover, increasing the cross-linking degree only resulted in a weak slowing on the enzymatic degradation velocity.