Giulio Bertoloni

Studies on the mechanism of bacteria photosensitization by meso-substituted cationic porphyrins

Cationic porphyrins have been shown to photoinduce the direct inactivation of Gram-positive (G+) and Gram-negative (G-) bacteria, thereby differing from anionic or neutral porphyrins which can photosensitize the G- bacteria only after permeabilization of their outer membrane. The present data show that the differences between these positively and negatively charged porphyrins are not related by a difference in the intrinsic photosensitizing efficiency, as determined by the photo-oxidation of model substrates or the yield of 1O2 generation; moreover, there are only minor differences in the quantum yield of porphyrin photobleaching. Rather, it appears that the positive charge promotes an electrostatic binding of the porphyrin to the outer cell surface inducing an initial limited damage which favours the penetration of the photosensitizer. Actually, the overall photoprocess is inhibited by the preincorporation of the porphyrin into liposomes, while it is enhanced by using amphiphilic dicationic porphyrins which bind to endocellular sites in larger amounts and in a more stable form.

Genital tract infections and infertility.

Infectious agents can impair various important human functions, including reproduction. Bacteria, fungi, viruses and parasites are able to interfere with the reproductive function in both sexes. Infections of male genito-urinary tract account for about 15% of the case of male infertility. Infections can affect different sites of the male reproductive tract, such as the testis, epididymis and male accessory sex glands. Spermatozoa themselves subsequently can be affected by urogenital infections at different levels of their development, maturation and transport. Among the most common microorganisms involved in sexually transmitted infections, interfering with male fertility, there are the Chlamydia trachomatis and Neisseria gonorrhoeae. Less frequently male infertility is due to non-sexually transmitted epididymo-orchitis, mostly caused by Escherichia coli. In female, the first two microorganisms are certainly involved in cervical, tubal, and peritoneal damage, while Herpes simplex cervicitis is less dangerous. The overall importance of cervical involvement is still under discussion. Tubo-peritoneal damage seems to be the foremost manner in which microorganisms interfere with human fertility. C. trachomatis is considered the most important cause of tubal lacerations and obstruction, pelvic inflammatory disease (PID) and adhesions. N. gonorrhoeae, even though its overall incidence seems to decline, is still to be considered in the same sense, while bacterial vaginosis should not be ignored, as causative agents can produce ascending infections of the female genital tract. The role of infections, particularly co-infections, as causes of the impairment of sperm quality, motility and function needs further investigation. Tropical diseases necessitate monitoring as for their diffusion or re-diffusion in the western world.

Photoinactivation of bacterial strains involved in periodontal diseases sensitized by porphycene–polylysine conjugates

Selected bacterial strains that are responsible for periodontal diseases are efficiently inactivated by visible light irradiation in the presence of porphycene-polylysine conjugates. Repeated photosensitization of surviving cells does not induce the selection of resistant bacterial strains and does not modify their sensitivity to antibiotic treatment.

HEMATOPORPHYRIN-SENSITIZED PHOTOINACTIVATION OF Streptococcus faecalis

Abstract— The photosensitizing action of hematoporphyrin (HP) on the cells of some bacterial strains belonging to the species Streptococcus faecalis, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, as well as of strains of yeast Candida albicans, was studied in air‐equilibrated aqueous media at pH 7.2. All Gram‐negative strains appeared to be resistant even to prolonged irradiation both in the presence and in the absence of EDTA. On the other hand. Gram‐positive and yeast strains were photosensitive. In the case of Str. faecalis, whose strains displayed a particularly high photosensitivity, the efficiency of the photoprocess was mainly controlled by the HP concentration in the incubation medium (0.1‐1 μg/ml) and by the irradiation time (0–10 min); temperature (18–37oC) exerted a minor effect, whereas the incubation time (6–30 min) had no detectable influence. The photolability of Str. faecalis cells was only slightly different in the logarithmic and stationary phase of growth. The type of photoinduced ultrastructural modifications and the largely preferential binding of HP to the cytoplasmic membrane, as observed by cell fractionation studies, suggest that one important target of the HP‐photosensitized process is represented by membrane components.

Photosensitizing activity of hematoporphyrin on Staphylococcus aureus cells

The photosensitizing action of hematoporphyrin (Hp) on two Staphylococcus aureus strains was investigated to determine if the photoprocess induces in vivo damage in DNA in addition to that occurring at the level of the cytoplasmic membrane. The results obtained demonstrate that the photokilling is dependent on the Hp dose even though the two strains, having a similar Hp-binding capacity, show different levels of photosensitivity. The electrophoretic analysis of cytoplasmic membrane proteins and DNA (chromosomal and plasmidial) suggests that the membrane represents the primary target of the photoprocess, while the DNA, that is damaged both in vivo and in vitro only at relatively long irradiation time, might be a secondary target. Moreover, the photoprocess results in mutagenesis for Salmonella typhimurium tester strains. This information is particularly important in view of the potential use of photodynamic therapy for the treatment of microbial infections.

Effect of extracellularly generated singlet oxygen on Gram-positive and Gram-negative bacteria

In the separated surface-sensitizer system, a photosensitizer is physically separated from the substrate by a thin air layer under such conditions that only singlet oxygen can reach and oxidize the substrate, preventing the competition by type I photosensitized processes. This method has been used to study the reaction of singlet oxygen with Gram-positive (Streptococcus faecium) and Gram-negative (Escherichia coli) bacterial strains. Studies on cell samples exposed to singlet oxygen for different periods of time show a drastic decrease in survival for S. faecium, while E. coli becomes sensitive only when the integrity of the outer membrane is altered by treatment with CaCl2 or tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid (Tris-EDTA). Biochemical and ultrastructural analyses suggest that the cytoplasmic membrane and the genetic material are the main sites damaged by singlet oxygen.

Laterally transferred elements and high pressure adaptation in Photobacterium profundum strains.

BackgroundOceans cover approximately 70% of the Earths surface with an average depth of 3800 m and a pressure of 38 MPa, thus a large part of the biosphere is occupied by high pressure environments. Piezophilic (pressure-loving) organisms are adapted to deep-sea life and grow optimally at pressures higher than 0.1 MPa. To better understand high pressure adaptation from a genomic point of view three different Photobacterium profundum strains were compared. Using the sequenced piezophile P. profundum strain SS9 as a reference, microarray technology was used to identify the genomic regions missing in two other strains: a pressure adapted strain (named DSJ4) and a pressure-sensitive strain (named 3TCK). Finally, the transcriptome of SS9 grown under different pressure (28 MPa; 45 MPa) and temperature (4°C; 16°C) conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool.ResultsThese studies indicated the presence of a large flexible gene pool in SS9 characterized by various horizontally acquired elements. This was verified by extensive analysis of GC content, codon usage and genomic signature of the SS9 genome. 171 open reading frames (ORFs) were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. Among these genes, six were found to also be up-regulated by high pressure.ConclusionThese data provide information on horizontal gene flow in the deep sea, provide additional details of P. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth.

Inactivation of Bacillus subtilis spores by supercritical CO2 treatment

Bacillus subtilis spores were suspended in saline solution (107 cfu/ml) and treated by both conventional heating and CO2 batch treatment at an operating pressure in the range of 70–150 bar under identical temperature conditions. Temperatures tested were in the range of 36–75 °C. Survival curves indicated significantly higher lethality when spores were treated with supercritical CO2 (SC-CO2) rather than with heating alone. These results appear particularly evident at 60 °C, a temperature at which conventional heating gave no spore-inactivation after a treating time as long as 24 h, whereas a 6 h SC-CO2 treatment led to complete sterilization. At 75 °C spores were partially killed with conventional heating but a treatment of 2 with SC-CO2 hours assured total inactivation. It is concluded that spore-inactivation during SC-CO2 treatment was only in part due to thermal effect (at the higher temperature of 75 °C) and there was a significant additional effect caused by CO2 penetration inside the latent bacteria forms.

Demonstration of Chlamydia pneumoniae in atherosclerotic arteries from various vascular regions

Chlamydia pneumoniae (CP) has been reported to be a pathogenic agent in the mechanism leading to atherosclerosis. The majority of available data is focused mainly on coronary artery disease whereas the distribution of CP in different areas, associated with atherosclerotic disorders, has not been completely clarified. In this study we investigated the presence of CP in atheromasic plaques from five different vascular areas (basilary artery, coronary artery, thoracic aorta, abdominal aorta, renal arteries) using nested polymerase chain reaction (PCR) and immunohistochemical staining (IHC), in order to establish the putative association of CP with atherosclerotic disease. The same atheromasic plaques were also tested for the presence of Helicobacter pylori (HP) and cytomegalovirus (CMV), other putative agents of atherosclerosis, using a nested PCR technique. Our data indicate that the presence of CP can be demonstrated in 100% of patients tested, considering globally the five areas of analysis. On the other hand the presence of HP has been demonstrated in four out of 18 patients (22.2%), and CMV only in three out of 18 (16.6%). Our results strongly suggest an association between CP and atherosclerosis and highlight the need for the detection of CP in multiple vascular areas of the same patient.

Polylysine–porphycene conjugates as efficient photosensitizers for the inactivation of microbial pathogens

Porphycenes are electronic isomers of porphyrins which, when neutral, display no appreciable photosensitizing action towards Gram-negative bacteria. The covalent binding of oligomeric polylysine moieties, which are cationic at physiological pH values, endows porphycenes with a significant phototoxic activity against Gram-negative bacteria while retaining their photoefficiency against a variety of microbial pathogens, including Gram-positive bacteria, fungi and mycoplasmas. The effect of the polylysine moiety is dependent on both the polylysine concentration and the degree of oligomerization. A suitable interplay among the various parameters opens the possibility to obtain either a broad spectrum of antimicrobial activity or a selective action toward a specific pathogen while minimizing the damage to human fibroblasts.