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A study on biofilm formation was carried out using five methicillin-sensitive [MSSA] and five methicillin-resistant [MRSA] strains of S. aureus. In each group, there were four strains isolated from patients from Kinshasa (Democratic Republic of Congo, DRC) and one reference strain. All of the strains were hydrophobic. The adherence of the bacteria to an abiotic surface was studied with the Biofilm Ring Test (BFRT®) and the crystal violet staining method (CVSM). Both techniques showed that eight of the strains formed biofilms within 2–3 h. The extent of the biofilm formed by one strain could only be observed with the CVSM. Periodate prevented the formation of biofilms and, in separate experiments, destroyed the biofilm pre-formed by the MSSA reference, but not those pre-formed by the clinical strains. Proteinase K destroyed all pre-formed biofilms. Six of the strains were icaA+; the clinical MSSA strains were not. The results also indicated different mechanisms of biofilm development between MSSA and MRSA clinical strains. The BFRT® and the CVSM are complementary techniques to study the adhesion of bacteria and the development of biofilms. [ABSTRACT FROM PUBLISHER]

Routine typing was performed on a total of 7089 Pseudomonas aeruginosa strains isolated in 16 Belgian hospitals in the period from 1977 to 1986. The annual number of strains received ranged from 318 to 1346. The incidence of serotype O: 12 was less than 2 % until 1981 when it rose to 4 %, steadily increasing to become the predominant serotype in 1984 (22 %), 1985 (18 %) and 1986 (22 %). Since 1980 the O: 12 isolates have exhibited characteristic patterns on pyocin and phage typing, 89 % of O: 12 isolates belonging to pyocin types 1, 39, 43, 45 or 105, whereas only 51 % of isolates of other serotypes belonged to those pyocin types. Ninety-three per cent of serotype O: 12 isolates belonged to phage types 68/119x, 68 or 119x, or were non-typable, whereas only 24.37 % of other serotypes isolates exhibited these phage patterns. These distinctive patterns of pyocin and phage types suggest a high degree of homogeneity within the O: 12 strains isolated in recent years in Belgium. Multi-centre or country-wide survey of Pseudomonas aeruginosa strains isolated in hospitals using epidemiological markers may be of value in identifying a sudden increase in epidemic strains. [ABSTRACT FROM AUTHOR]

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.

Background: European guidelines stress that iron status should be regularly assessed for the optimal management of renal anemia. These guidelines include the hemoglobin content of reticulocytes and the percentage of hypochromic RBC as markers for functional iron deficiency. Recently, equivalents of these indices have become available on the automated hematology analyzer Sysmex XE-2100, these being reticulocyte hemoglobin equivalent (Ret-He) and DF-HYPO XE, respectively., Methods: In a prospective study, we closely monitored these parameters in dialysis-dependent patients with end-stage renal disease during the switch from a first-generation epoetin (EPO) once weekly to a third-generation EPO [continuous erythropoietin receptor activator (CERA)] once monthly. As a control, patients staying on EPO beta were monitored., Results: During follow-up, no changes in erythrocyte indices were noticed in the EPO beta group. By contrast, in the CERA group, a decrease in Ret-He and an increase in DF-HYPO XE were transiently found 7-10 days after administration. The transient state of functional iron deficiency could not be prevented by extra intravenous iron., Conclusion: Fluctuations in Ret-He and DF-HYPO XE have to be taken into account when these parameters are used for the assessment of iron-deficient states. We suggest that a fixed time point in the CERA schedule should be chosen for iron monitoring., (Copyright 2010 S. Karger AG, Basel.)

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.

In several occupational settings, endotoxin, a characteristic external fraction from the outer membrane of Gram negative bacteria (GNB), is an important component of the environmental endotoxin exposure may have a dual influence on atopy and asthma. The aim of this study was to characterise the bacterial and endotoxin composition of settled and airborne dusts from domestic environments. In the nine studied homes, 52 species of GNB were identified on settled dusts and only 13 in the airborne dust. Bacterial levels ranged from 2.76 10(5) to 1.14 10(8) CFU/g in mattresses and from 7.82 10(5) to 6.38 10(7) CFU/g in carpets. In the air 0.39 10(3) to 4.66 10(3) cfu/m3were recorded. Endotoxin varied from 1.85 to 549.0 EU/mg dust and was higher in the carpets compared with the mattresses. The medians of airborne endotoxin levels were 0.239 EU/m3 and 0.062 EU/m3 in inhalable and respirable particles, respectively. Our investigation demonstrates clearly the presence of numerous GNB in domestic environments and shows that domestic environnements contain in some cases relative high amounts of endotoxin.

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.

Twelve HIV-1-infected, nine HIV-2-infected patients and eight HIV-negative subjects were given a 40IU booster dose of tetanus toxoid (TT). Blood was collected on days 0, 7 and 30 after immunization. Changes in HIV-1 or HIV-2 RNA load were evaluated by nested PCR. TT-IgG antibody levels were quantified by ELISA. CD4 cell counts as well as activation, memory and maturation markers of T lymphocyte subsets were determined by flow cytometry. The induction of apoptosis was investigated using 7-aminoactinomycin D (AAD) and propidium iodide (PI) staining. Proliferative responses to TT and pokeweed mitogen (PWM) were determined by the level of [(3)H] thymidine incorporation. Seven and 30 days after immunization, there was no detectable increase in HIV-1 or HIV-2 plasma load. There were also no changes in CD4 cell counts, CD69, HLA-DR and memory CD45RO or naive CD45RA antigens. Immunization did not increase the spontaneous apoptosis of peripheral blood mononuclear cells (PBMCs), CD4+ and CD8+ T cells subsets neither in controls nor in HIV-infected patients. Similarly, apoptosis induced in vitro by PWM or by the specific TT recall antigen did not vary during the study period. The proliferative response to PWM and to the TT recall antigen was decreased both in HIV-1- and HIV-2-infected patients compared to HIV-negative controls. Immunization significantly increased the TT-IgG levels in healthy controls and in HIV-infected patients. However, the anti-TT-IgG response, as measured by the fold-increase index between days 0 and 30, was significantly higher in healthy controls than in HIV-1- (P=0.036) and HIV-2-infected patients (P=0.003). In conclusion, we found no deleterious immunologic or virologic effect was detected in healthy HIV-1- and HIV-2-infected individuals after antigenic challenge with a TT booster. However, the response to TT vaccination was lower in HIV-1- and in HIV-2-infected individuals than in healthy HIV-negative controls.

The line probe assay (LiPA), a rapid molecular method for detecting rifampicin resistance (RMPr) in Mycobacterium tuberculosis, correctly identified all 145 rifampicin-sensitive (RMPs) and 262 (98.5%) of 266 RMPr strains among 411 isolates collected from diverse countries. If used as a marker of multidrug-resistant tuberculosis (MDR-TB), detection of RMPr by LiPA would have detected 236 of the 240 MDR strains in this study but would have wrongly suggested the presence of MDR in 26 RMP-monoresistant isolates (sensitivity 98.3%, specificity 84.8%). Hence, the reliability of using LiPA (or any other rapid RMPr-detection method) as a surrogate marker of MDR-TB largely depends on the prevalence of RMP-monoresistance in the study population. This approach must therefore be validated in each local situation.

Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of [3H]arachidonic acid ([3H]AA) from prelabeled rat submandibular gland (RSMG) ductal cells respectively two- and threefold. Both agonists also increased the release of [3H]AA from acini but at a lower level (+50% and +100% respectively). Carbachol had no significant effect on either cellular population. In ductal cells phorbol myristate acetate, an activator of protein kinase C, slightly increased the basal release of [3H]AA but did not affect the release of [3H]AA in response to ATP. Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines. The removal of calcium from the extracellular medium decreased the response to ATP and Bz-ATP. Only barium could partly substitute for calcium to restore the purinergic response. Zinc inhibited the release of [3H]AA. Permeabilization of the cells with streptolysin O (SLO) activated the calcium-independent phospholipase A2 activity (iPLA2). The iPLA2, not the calcium-dependent PLA2 (cPLA2), released [3H]oleic acid ([3H]OA) from RSMG ductal cells. It is concluded that RSMG ducts have a higher PLA2 activity when compared to acini. This activity is accounted for by iPLA2 and cPLA2. Both enzymes are activated by P2X agonists by a staurosporine-sensitive mechanism. Cells permeabilized with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes. In intact RSMG ductal cells the two activities can be distinguished by rather specific inhibitors, by different ionic conditions and also by the fatty acid used to label the cells.

We have studied the influence of protamine on the sensitivity of three strains of Pseudomonas aeruginosa to various antibiotics: streptomycin, gentamicin, polymyxin B, and beta-lactams. While protamine enhanced the antibacterial action of beta-lactams towards P. aeruginosa, it did not alter the effect of aminoglycosides. The antibacterial power of polymyxin B, on the other hand, was drastically reduced. The observed changes in this strain's sensitivity to antibiotics could be due to changes in the permeability of the outer membrane.