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Aims: To characterize and select Lactobacillus strains for properties that would make them a good alternative to the use of antibiotics to treat human vaginal infections. Methods and Results: Ten Lactobacillus strains belonging to four different Lactobacillus species were analysed for properties relating to mucosal colonization or microbial antagonism (adhesion to human epithelial cells, hydrogen peroxide production, antimicrobial activity towards Gardnerella vaginalis and Candida albicans and coaggregation with pathogens). The involvement of electrostatic interactions and the influence of bacterial metabolic state in the binding of lactobacilli to the cell surface were also studied. Adherence to epithelial cells varied greatly among the Lactobacillus species and among different strains belonging to the same Lactobacillus species. The reduction in surface negative electric charge promoted the binding of several Lactobacillus strains to the cell membrane whereas lyophilization reduced the adhesion capacity of many isolates. The antimicrobial activity of lactobacilli culture supernatant fluids was not directly related to the production of H2O2. Conclusions: Three strains (Lactobacillus brevis CD2, Lact. salivarius FV2 and Lact. gasseri MB335) showed optimal properties and were, therefore, selected for the preparation of vaginal tablets. The selected strains adhered to epithelial cells displacing vaginal pathogens; they produced high levels of H2O2, coaggregated with pathogens and inhibited the growth of G. vaginalis. Significance and Impact of the Study: The dosage formulation developed in this study appears to be a good candidate for the probiotic prophylaxis and treatment of human vaginal infections.

Ten strains of lactobacilli were evaluated for the administration of viable microorganisms to restore the normal indigenous flora in the treatment of urogenital tract infections (UTI) in women. As the strains considered are facultative anaerobes, optimization of the production process was particularly critical to preserve bacterial viability. The microorganisms were formulated in single- and double-layer vaginal tablets. The two layers were characterized by different release properties: one is an effervescent composition that ensures a rapid and complete distribution of the active ingredient over the whole vaginal surface; while the second is a sustained release composition capable of releasing the lactobacilli over a longer period of time. Three different retarding polymers were tested, and all the formulations and tablets were evaluated in terms of technological processability, bacterial viability and stability, and cell adhesion properties of the microorganisms. From the results obtained, three out of ten strains appear particularly suitable for their application in the treatment of UTI. A larger batch of tablets made with a mixture of the three strains was then evaluated, confirming the feasibility of their industrial production and a good bacterial viability in the final dosage form.

The mechanism and the kinetics of rubella virus (RV) penetration into Vero cells were studied. By using pronase or acid treatment to inactivate virus which had adsorbed to cell membrane but had not been internalized, it was found that a period of 7 h was required in order for all of the adsorbed virus to enter the host cells. Lysosomotropic agents (monensin, methylamine, ammonium chloride and chloroquine) were used to study the mechanism by which RV penetrates host cells. Virus replication was inhibited if treatment of cells with these compounds was performed for at least 9 h after infection. However, if extracellular adsorbed virions were eliminated by acid treatment following removal of the lysosomotropic compounds, RV replication was completely inhibited by treatment with these drugs for any time period after adsorption. This indicated that the prolonged period of treatment with these compounds necessary to inhibit virus replication is due to the slow rate of RV internalization. None of the compounds had any effect on infection initiated by transfection of RV RNA, confirming that these drugs were exerting their inhibitory activity at penetration. The inhibition of RV replication by lysosomotropic compounds indicates that RV penetrates host cells by the endosomal pathway.

The viral protein 2A of hepatitis A virus (HAV) lacks the conserved 18 aa sequence found in other picornavirus proteases; hence, it is unclear whether the induction of CPE by culture-adapted HAV strains is due to 2A-mediated activity. Moreover, the cleavage sites and actual borders of HAV 2A are not known. Accordingly, a nested series of cDNA sequences encoding the segment of the HAV polyprotein (aa 760–1087) were linked to the 5′-UTR of poliovirus type 2 (Lansing strain) and inserted downstream of the gene encoding human growth hormone (GH). Following transfection of COS-1 cells, levels of GH (translation of which was entirely cap dependent) were determined in culture supernatants. Expression of HAV peptides extending from aa 764, 776 or 791 to 981 strongly inhibited cap-dependent translation of GH, whereas cap-independent expression of a reporter gene (CAT) directed by the poliovirus RNA 5′-UTR was unaffected. The inhibitory effect was absent in constructs expressing either the short peptide encompassing aa 760–836 or proteins initiated downstream of the putative cleavage site 836–837, suggesting that the boundaries of a functional HAV 2A may extend from the Gln/Ser junction 791–792 to residue 981, while peptides initiated at the Gln/Ala pair 836–837 may result from alternative cleavage. Point mutations that substituted members of the triad Ser916, His927 and Asp931 abolished the inhibitory effect on cap-dependent translation, suggesting that the HAV-induced CPE may be mediated by 2A protein.