[Time-kill curves and evolution of membrane permeability after para-guanidinoethylcalix[4]arene exposure]. / Cinétique d'action du para-guanidinoéthylcalix[4]arène, et évolution de la perméabilité membranaire.

UNLABELLED: Three problems at the moment: multidrug-resistant bacteria, healthcare-associated infections, and decrease of active antibiotics. We have an urgent need of new antibacterials, with an innovative mechanism of action, in order to avoid too quickly bacterial resistance. The first interface between bacteria and antibiotics is the bacterial cell wall. It is a very interesting target, as far as some components or motives are highly conserved between genus or species, and wall destabilization conduct rapidly to bacterial lysis. However, few methods are at our disposal to study rapidly impact of such antibacterials on the structure, composition or functions of the bacterial cell wall. The paraguanidinoethylcalix[4]arene (Cx1) is a new cationic antibacterial drug, with a broad spectrum, not toxic, active on multidrug-resistant bacteria, with a possible parietal target, but with unknown kind of activity (i.e. bactericidal or bacteriostatic). We thus developed, at the same time as the realization of the time-kill curves, a technique to stain bacteria with two dyes: SYTO9 and propidium iodide (PI), to follow the membrane permeability modifications, due to Cx1 exposure. The obtained results demonstrate, for Escherichia coli ATCC 25922, that Cx1 possesses a bactericidal activity, concentration-dependent, with a gradual achievement of membrane permeability, time- and concentration-dependent, with the presence of filamentous bacteria. IN CONCLUSION: the SYTO9-PI double staining, allows a simple and fast detection, easy to implement, of the impact of new antibacterial on the bacterial wall; and Cx1 interacts well with the bacterial wall, pulling in the end a loss of membrane integrity.

In vivo antiviral activity of ribavirin/alpha-cyclodextrin complex: evaluation on experimental measles virus encephalitis in mice.

Intracranial injection of the rodent adapted CAM/RB strain of measles virus (MV) induces encephalitis in CBA/ca mice. It has already been shown that cyclodextrins can be used as carriers to increase the antiviral activity of ribavirin (RBV) against MV in cellular model. In this study, the antiviral activity of a RBV/alpha-cyclodextrin complex has been evaluated in vivo using the above model. CBA/ca mice were treated by intraperitoneal injection of free ribavirin (40 mg/kg) or a RBV/alpha-cyclodextrin complex (molar ratio 1:3). After 21 days, intracerebral injection of CAM/RB resulted in 100% mortality in the mock group. In contrast, mortality rates of 80% and 40%, respectively, were observed in RBV and RBV/alpha-CD-treated mice (p<0.05 and p=0.06 for distilled water and RBV, respectively). The viral load of MV in the mouse brain was monitored daily by real-time PCR until day 6 after infection, to compare virus production in treated and non-treated mice. This data shows that RBV complexation with alpha-cyclodextrin can increase the antiviral activity of ribavirin in a measles virus encephalitis model in mice.

[The Chikungunya virus]. / Le virus Chikungunya.

Chikungunya virus (CHIKV), a member of the Alphavirus genus, represents a real public health problem in tropical regions of the Southeast Asia and Africa. It is transmitted to the man by Aedes mosquitoes and the illness, known as Chikungunya, is characterized by fever, eruptions and invalidating arthralgia. An increased surveillance in tropical and subtropical areas is necessary, as far as we have noticed recently the emergence of this new disease in regions where it had never existed before. The epidemic context is of a high importance for diagnosis. It is very important to know the clinical characteristics of the infection, to detect forms rarely or never described previously. Permanence of a highly technical core in specialized laboratories will allow, fast, specific and differential diagnosis. The knowledge of the epidemiological chain of transmission from reservoir, still unknown, to the host aims to protect populations by limiting the risks of exposure when it is possible. The only prevention measures available are individual protection against mosquitoes and antivectorial fight, in the absence of specific antiviral treatment and vaccine.

Novel bioactive compounds from actinomycetes.

Actinomycetes form an enormous reservoir of secondary metabolites and enzymes. The potential for exploiting rare actinomycetes is highlighted by the discovery of novel compounds from strains of Spirillospora and Nocardioides. Novel compounds of well known classes of antibiotics, such as polyenes, continue to be discovered. For compounds containing a chromophore, the analysis by high-performance liquid chromatography coupled with a diode-array detector enables the elimination of producers of known compounds and facilitates the discovery of novel compounds or derivatives. The complexity of the regulatory mechanisms is illustrated by glutamine synthetase. The characterization of thermostable amylolytic, lignolytic, peroxidase and neuramidase activities, and the isolation of novel cellulolytic actinomycetes clearly demonstrate the potential of Actinomycetes as producers of enzymes.

Influence of the fluoroquinolone ofloxacin on the intrinsic expression of multidrug resistance phenotype in HCT-8 human colon carcinoma cells.

The influence of antibiotics, particularly ofloxacin (OF), a commonly used antimicrobial fluoroquinolone, on the multidrug resistance (MDR) phenotype of the HCT-8 cell line was studied. This cell line was grown in OF containing medium for several months and the expression of the MDR phenotype was followed through the analysis of the expression and functionality of the P-glycoprotein (Pgp), the chemosensitivity to daunorubicin (DNR), and the mRNA expression of mdr-1, multidrug resistance-associated protein (MRP), and topoisomerase IIalpha and IIbeta genes. Replacement of OF by penicillin streptomycin (PS) resulted in a significant decrease in mdr-1 mRNA expression, which was found to correlate with a decrease in the expression and functionality of the Pgp. After antibiotic starvation for 4 weeks, cells grown in antibiotic-free medium were then exposed to PS or OF; these cells showed an increase in mdr-1 mRNA/Pgp and MRP mRNA expression without a decrease in DNR cytotoxicity. OF cultured cells exhibited a significant increase in Pgp expression without evidence of the functionality of the Pgp. An increase in topoisomerase IIalpha mRNA expression was observed with time and with the number of passages of the cell line without any relationship to the presence of antibiotics in the culture medium. These results showed that extensive use of antibiotics, particularly the quinolones, can modify the phenotype of the HCT-8 colon adenocarcinoma cell line.

Improved method for contamination of mussels by poliovirus.

An experimental method for contaminating mussels with poliovirus that approaches natural conditions in a coastal zone is described. A 280 liter aquarium is used to determine optimum conditions for obtaining a maximum titer of virus in mussels. When mussels were kept for 1 h in water contaminated with from 3 to 368 PFU ml-1, between 1 to 628 PFU g-1 tissue were recovered. Mussels exposed to contaminated water for periods of up to 10 days showed decreased titers of virus which seemed to parallel the natural inactivation of viruses in water with time. 1 h of exposure to contaminated water was always sufficient for a maximum adsorption of viral particles by mussels.

Stability of parenteral ceftriaxone disodium solutions in frozen and liquid states: effect of freezing and microwave thawing.

The stability of ceftriaxone disodium solutions stored in glass bottles was tested in two parenteral solvents (0.9% NaCl; 5% dextrose) at two concentration rates (10 and 50 mg/mL) and three temperatures (-22 degrees C, 5 degrees C, room temperature). The solutions, which had been initially frozen, were thawed at room temperature or by exposure to microwave radiations. The stability of each sample was determined by a quantitative bacteriological agar gel diffusion assay and by high-performance liquid chromatography. The results of this study indicate that admixtures of ceftriaxone disodium at the concentration rates tested can be frozen for six months without a significant loss in antibiotic activity. At room temperature, the stability is dependent on the concentration of ceftriaxone disodium. At 5 degrees C, the stability is related to the concentration of ceftriaxone and of the solvent. The results obtained by both analytical methods are very close. In order to perform routine assays, the high-performance liquid chromatography method was chosen on the basis of its rapidity and reproducibility.

Antiseptic properties of two calix[4]arenes derivatives on the human coronavirus 229E.

Facing the lack in specific antiviral treatment, it is necessary to develop new means of prevention. In the case of the Coronaviridae this family is now recognized as including potent human pathogens causing upper and lower respiratory tract infections as well as nosocomial ones. Within the purpose of developing new antiseptics molecules, the antiseptic virucidal activity of two calix[4]arene derivatives, the tetra-para-sulfonato-calix[4]arene (C[4]S) and the 1,3-bis(bithiazolyl)-tetra-para-sulfonato-calix[4]arene (C[4]S-BTZ) were evaluated toward the human coronavirus 229E (HCoV 229E). Comparing these results with some obtained previously with chlorhexidine and hexamidine, (i) these two calixarenes did not show any cytotoxicity contrary to chlorhexidine and hexamidine, (ii) C[4]S showed as did hexamidine, a very weak activity against HCoV 229E, and (iii) the C[4]S-BTZ showed a stronger activity than chlorhexidine, i.e. 2.7 and 1.4log10 reduction in viral titer after 5min of contact with 10⁻³mol L⁻¹ solutions of C[4]S-BTZ and chlorhexidine, respectively. Thus, the C[4]S-BTZ appeared as a promising virucidal (antiseptic) molecule.

Cationic compounds with activity against multidrug-resistant bacteria: interest of a new compound compared with two older antiseptics, hexamidine and chlorhexidine.

Use of antiseptics and disinfectants is essential in infection control practices in hospital and other healthcare settings. In this study, the in vitro activity of a new promising compound, para-guanidinoethylcalix[4]arene (Cx1), has been evaluated in comparison with hexamidine (HX) and chlorhexidine (CHX), two older cationic antiseptics. The MICs for 69 clinical isolates comprising methicillin-resistant Staphylococcus aureus, methicillin-sensitive S. aureus, coagulase-negative staphylococci (CoNS) (with or without mecA), vancomycin-resistant enterococci, Enterobacteriaceae producing various beta-lactamases and non-fermenting bacilli (Pseudomonas aeruginosa, Acinetobacter baumanii, Stenotrophomonas maltophilia) were determined. Cx1 showed similar activity against S. aureus, CoNS and Enterococcus spp., irrespective of the presence of mecA or van genes, or associated resistance genes, with very good activity against CoNS (MIC <1 mg/L). Variable activities were observed against Enterobacteriaceae; the MICs determined seemed to be dependent both on the genus (MICs of 2, 8 and 64 mg/L for Escherichia coli, Klebsiella pneumoniae and Yersinia enterocolitica, respectively) and on the resistance phenotype production of [Extended Spectrum beta-Lactase (ESBLs) or other beta-lactamases; overproduction of AmpC]. Poor activity was found against non-fermenting bacilli, irrespective of the resistance phenotype. CHX appeared to be the most active compound against all strains, with broad-spectrum and conserved activity against multidrug-resistant strains. HX showed a lower activity, essentially against Gram-positive strains. Consequently, the differences observed with respect to Cx1 suggest that they are certainly not the consequence of antibiotic resistance phenotypes, but rather the result of membrane composition modifications (e.g. of lipopolysaccharide), or of the presence of (activated) efflux-pumps. These results raise the possibility that Cx1 may be a potent new antibacterial agent of somewhat lower activity but significantly lower toxicity than CHX.

Effective ribavirin concentration in mice brain using cyclodextrin as a drug carrier: evaluation in a measles encephalitis model.

Ribavirin (RBV) is a water-soluble synthetic nucleoside with broad spectrum antiviral properties, but it is ineffective against major viral encephalitis because of a failure to cross the blood-brain barrier (BBB). The antiviral activity of the complex ribavirin/alpha-cyclodextrin was previously demonstrated to be stronger than free ribavirin, in an in vivo model of measles virus (MV) encephalitis in mice. The role of cyclodextrin (CD) on ribavirin uptake into the brain needs to be defined. Ribavirin specific extraction from brain tissue was developed, based on a solid phase extraction. It was quantified by high performance liquid chromatography at different time points after intraperitoneal injection of single or multiple doses of free ribavirin or of the complex ribavirin/alpha-cyclodextrin. Whatever the tested dose (40 or 100mg/kg), the amount of ribavirin in the brain was significantly higher (p<0.001) when the drug was injected as a complex with alpha-cyclodextrin, in healthy or measles virus-infected mice.

Molecular characterization of African orthobunyaviruses.

The genus Orthobunyavirus is composed of segmented, negative-sense RNA viruses that are responsible for mild to severe human diseases. To date, no molecular studies of bunyaviruses in the genus Orthobunyavirus from central Africa have been reported, and their classification relies on serological testing. Four new primer pairs for RT-PCR amplification and sequencing of the complete genomic small (S) RNA segments of 10 orthobunyaviruses isolated from the Central African Republic and pertaining to five different serogroups have been designed and evaluated. Phylogenetic analysis showed that these 10 viruses belong to the Bunyamwera serogroup. The S segment sequences differ from those of the Bunyamwera virus reference strain by 5-15 % at the nucleotide level, and both overlapping reading frames, encoding the nucleocapsid (N) and non-structural (NS) proteins, were evident in sequenced genomes. This study should improve diagnosis and surveillance of African bunyaviruses.

[Microelectrophoresis and atomic force microscopy: new tools to explore mechanism of action of antibacterial compounds]. / Microélectrophorèse et microscopie à force atomique: deux nouveaux outils d'évaluation de l'effet pariétal d'antibactériens.

Microbial cell surface properties play a central role in controlling phenomena such as bacterial adhesion and biofilm formation (on stent or on prosthesis for example). The quantification of these properties and the understanding of interactions with antibacterial compounds remain difficult, in view of the complex and dynamic nature of the cell wall constituents. Various approaches, macroscopic, microscopic or molecular, have been developed. Two of them interest us today: (i) microelectrophoresis, which permits to evaluate surface modifications by measuring eletrophoretic mobility; and (ii) atomic force microscopy (AFM), a high resolution imaging device, which allows investigations at nanometric scale. After brief presentation of principles and instrumentations, the aim of this article is to present the different applications of these techniques in Microbiology, and to discuss interest of these tools in order to investigate mechanism of action of antibacterial compounds.

[Towards new antibacterial drugs. Interest of para-guanidinoethylcalix[4]arene]. / Vers de nouvelles molécules antibactériennes. Intérêt du para-guanidinoéthylcalix[4]arène.

We present here the results concerning the antibacterial properties evaluation of para-guanidinoethylcalix[4]arene, compared with its constitutive monomer, the para-guanidinoethylphenol, and hexamidine (Hexomédine), an antiseptic from the diamidine family widely used in therapeutic, chosen as a reference in this study for its resemblance in terms of functional groups. Antibacterial activities of those three compounds were evaluated by microdilution methods, in Mueller Hinton broth, onto 5 bacterial strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 & ATCC 29213 and Enterococcus faecalis ATCC 29212, according to CA-SFM and CLSI (formerly NCCLS) approved standards. In parallel, the effects of these three compounds on MRC-5 eukaryotic cell viability were evaluated with MTT assay. The results obtained here confirm a lack of activity for the monomer compound (MIC> or =512 mg/l) and a real antibacterial activity for the calixarene, comparable to hexamidine. This activity is expressed, both on Gram+and Gram- bacteria (MIC=4 mg/l for E. coli, 8 mg/l on both S. aureus strains) and at a lesser degree on E. faecalis and P. aeruginosa (MIC=32 mg/l). Similarly, both compounds, monomer and calixarene, slightly induce any modification on MRC-5 cells viability, and this until 168 h of treatment for concentrations reaching 10(-4) mol/L while hexamidine demonstrates a significant and increasing effect during the time of experiment and this for 100 to 1000 times lower concentrations. Thus, this study tends to confirm the significance of the organization of the para-guanidinoethylphenol monomer into its cyclic calixarenic tetramer for the gain of an antibacterial activity, similar to a widely used antiseptic one.

[Correlation between the intensity of spontaneous bacteriolysis and bacterial pollution in superficial waters]. / Corrélations entre l'intensité de la bactériolyse spontaneé et la pollution bactérienne dans les eaux superficielles.

A study on the determination of the intensity of spontaneous bacteriolysis in different samples of water from the hydrographic basin of Lorraine, has led us to propose a new way of expressing the spontaneous bacteriolytic power: PBSG. Its utilization has permitted us to establish correlations between the intensity of spontaneous bacteriolytic power and the degree of bacteriological pollution found in different places in the two rivers, the Moselle and the Meurthe. A very strong correlation has been shown between the rate of global spontaneous bacteriolysis after 6 days and the number of coliforms in the water.

[Attempt to express the spontaneous global bacteriolytic power of water]. / Essai d'expression du pouvoir bactériolytique spontané global d'une eau.

The utilization of a technique for the determination of spontaneous bacteriolytic power in surface waters has led us to propose a new way of expressing the spontaneous bacteriolytic power of water: PBSG. PBSG allows us to evaluate the intensity of global spontaneous bacteriolysis and to compare the global spontaneous bacteriolytic powers of different water samples.

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization.

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes, were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods.

Improved method for virological analysis of food.

A single, simple method for recovering enteroviruses from several different kinds of food, such as ground beef, fish, oysters, and mussels, has been improved. First, sample contamination technique was studied. It appears that virus adsorption occurs at food pH and varies according to the kind of food and the food-virus exchange surface. Second, virus recovery was evaluated. According to the experimental results obtained, we propose the following method. Samples are submerged in 100 ml of demineralized water adjusted to pH 9 with a conductivity of 8,000 mg of NaCl per liter. Then, viruses are concentrated by ultrafiltration or by ultracentrifugation. This method was efficient for virus recovery from the four kinds of food, even in cases of very low contamination.

Sequence and analysis of BECV F15 matrix protein.

Clones from the bovine enteric coronavirus (F15) cDNA library were cloned in pBR322 and sequenced by the method of Sanger and Coulson. This led to the identification of a sequence of 1,300 bases which contained a single open reading frame of 690 bases yielding a protein having properties of the matrix protein (M). It was comprised of 230 amino acids with a molecular weight of 26,376 Da. It was hydrophobic and had a net charge of +8 at neutral pH. Analysis of its secondary structure could not establish a simple transmembrane arrangement of the amino acids. Comparison of its nucleotide sequence with that of BECV Mebus strain showed only a two-base change resulting in a 100% homology between the two amino acid sequences. Furthermore, a very conserved structure of M appeared on comparison with the Dayoff optimal alignment of MHV-A59, MHV-JHM, TGEV, IBV Beaudette and IBV 6/82M amino acid sequences. As the two strains of BECV, F15 and Mebus present some antigenic differences, this led us to reconsider the role of M in viral antigen specificity. A hypothesis is that, as it seems to possess the necessary information on its transmembrane region, it is an ideal candidate for the viral budding process.

Recognition ability and cytotoxicity of some oligosaccharidyl substituted beta-cyclodextrins.

This paper reports a chemico-enzymatic synthesis of beta-CD derivatives. The recognition properties of these derivatives were tested using flocculating yeast and isolated lectins. It was observed that the substitution of beta-cyclodextrins with galactose end arms induces the better recognition by a cell-linked galactose-specific lectin. The physicochemical effects of the beta-CD derivatives on membranes were estimated using red blood cells and the effects on the viability of yeast and human rectal tumor cells were appreciated by measuring the mitochondrial deshydrogenase activity. The substitutions of the beta-CD ring by sugar antennae decrease the negative physicochemical effects of the beta-CD, ie their hemolytic properties. However, these substitutions induce significant modifications of the biological properties of the molecules, particularly the cytotoxicity and the growth of eukaryotic cells.

Susceptibility of clinical strains of herpes simplex virus to three nucleoside analogues.

The susceptibility of clinical isolates of herpes simplex virus 1 (HSV1) (58 strains) and 2 (HSV2) (17 strains) from the Centre Hospitalier et Universitaire de Nancy to three nucleoside analogues was compared by the dye uptake method. As expected, all strains of HSV2 were resistant to brovavir or sorivudine. Aciclovir and penciclovir activities were comparable; 2 strains of HSV1 were resistant to these two compounds. Four strains isolated from immunocompromised patients gave different results with brovavir as compared to aciclovir; resistance to aciclovir (1 strain of HSV1) did not correlate with resistance to brovavir (3 strains of HSV1). Following up antiviral susceptibility is of interest for the detection of resistant strains in immunocompromised patients receiving prophylactic aciclovir.