Healthy vaginal microbiota is dominated by Lactobacillus spp., which form a critical line of defence against pathogens, including Candida spp. The present study aims to identify vaginal lactobacilli exerting in vitro activity against Candida spp. and to characterize their antifungal mechanisms of action. Lactobacillus strains were isolated from vaginal swabs of healthy premenopausal women. The isolates were taxonomically identified to species level (L. crispatus B1-BC8, L. gasseri BC9-BC14 and L. vaginalis BC15-BC17) by sequencing the 16S rRNA genes. All strains produced hydrogen peroxide and lactate. Fungistatic and fungicidal activities against C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis and C. lusitaniae were evaluated by broth micro-dilution method. The broadest spectrum of activity was observed for L. crispatus BC1, BC4, BC5 and L. vaginalis BC15, demonstrating fungicidal activity against all isolates of C. albicans and C. lusitaniae. Metabolic profiles of lactobacilli supernatants were studied by 1H-NMR analysis. Metabolome was found to be correlated with both taxonomy and activity score. Exclusion, competition and displacement experiments were carried out to investigate the interference exerted by lactobacilli toward the yeast adhesion to HeLa cells. Most Lactobacillus strains significantly reduced C. albicans adhesion through all mechanisms. In particular, L. crispatus BC2, L. gasseri BC10 and L. gasseri BC11 appeared to be the most active strains in reducing pathogen adhesion, as their effects were mediated by both cells and supernatants. Inhibition of histone deacetylases was hypothesised to support the antifungal activity of vaginal lactobacilli. Our results are prerequisites for the development of new therapeutic agents based on probiotics for prophylaxis and adjuvant therapy of Candida infection.

Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomicproteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.Chlamydia pneumoniae is an obligate intracellular bacterium and a common human pathogen (48). It is a significant cause of pneumonia in both hospital and outpatient settings, accounting for approximately 7 to 10% of cases of community-acquired pneumonia among adults. C. pneumoniae has also been associated with atherosclerotic and cardiovascular disease, as suggested by results of seroepidemiologic studies, detection of the organism in atherosclerotic plaque specimens, experimental in vitro cell culture studies, animal model studies, and two small secondary prevention antibiotic treatment trials (12,13,15,19,20,28,45).Like all obligate intracellular parasites, for its survival and propagation C. pneumoniae must accomplish several essential tasks which include adhering to and entering host cells, creating an intracellular niche for replication, exiting host cells for subsequent invasion of neighboring cells, and also avoiding host defense mechanisms. To carry out all these functions, C. pneumoniae has developed a unique biphasic life cycle involving two developmental forms, a spore-like infectious form (elementary bodies [EBs]) and an intracelluar replicative form (reticulate bodies [RBs]). Adhesion, host cell colonization capabilities, and the ability to cope with host defense mechanisms when outside the cell presumably rely in large part on EB surface organization.Because of the intrinsic difficulty in working with C. pneumoniae and the lack of adequate methods for its genetic ma...

Lactobacillus species dominate the vaginal microbiota of healthy reproductive-age women and protect the genitourinary tract from the attack of several infectious agents. Chlamydia trachomatis, a leading cause of sexually transmitted disease worldwide, can induce severe sequelae, i.e. pelvic inflammatory disease, infertility and ectopic pregnancy. In the present study we investigated the interference of Lactobacillus crispatus, L. gasseri and L. vaginalis, known to be dominant species in the vaginal microbiome, with the infection process of C. trachomatis. Lactobacilli exerted a strong inhibitory effect on Chlamydia infectivity mainly through the action of secreted metabolites in a concentration/pH dependent mode. Short contact times were the most effective in the inhibition, suggesting a protective role of lactobacilli in the early steps of Chlamydia infection. The best anti-Chlamydia profile was shown by L. crispatus species. In order to delineate metabolic profiles related to anti-Chlamydia activity, Lactobacillus supernatants were analysed by 1H-NMR. Production of lactate and acidification of the vaginal environment seemed to be crucial for the activity, in addition to the consumption of the carbonate source represented by glucose. The main conclusion of this study is that high concentrations of L. crispatus inhibit infectivity of C. trachomatis in vitro.

BackgroundOuter membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. MethodsChlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro.ResultsCM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection.ConclusionsWhen expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform.

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis . We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis -infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis -infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4 + /IFN-γ + T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4 + /IFN-γ + –inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4 + T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti- Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.

Treponema denticola and Porphyromonas gingivalis have been identified in atheromatous plaques of two patients suffering from atherosclerosis by PCR and fluorescence in situ hybridization (FISH). The use of the FISH technique suggested that these periodontopathic micro-organisms might be metabolically active within the wall of arteries, under the atherosclerotic lesion. Case reportsCase 1. The patient was a 73-year-old caucasian Italian male with a 12 year history of atherosclerosis and systemic hypertension. The patient also had a previous history of smoking and dislipidemia. He underwent percutaneous transluminal coronary angioplasty (PTCA) of the left coronary artery in 1991, and 2 years later he underwent coronary artery by-pass grafting (CABG). In 2003 an ecotomodoppler study of his carotid arteries showed a bilateral stenosis (70 % in the right vessel and 50 % in the left vessel). Consequently a computerized tomography (CT) scan was performed, which revealed the presence of partially calcified atheromatous plaques located at the carotid bulbs. The patient was then scheduled to undergo an endarterectomy to remove the plaque in the right bulb followed by the application of a dacron patch. Clinical and radiological examinations of the patient immediately before surgery showed a very poor periodontal situation, as indicated by a high value (103) of CPSS (clinical periodontal sum score: the sum of the number of sites with probing pocket depths of 4 mm or greater, the number of gingival sites with bleeding after probing or visible suppuration on probing, and the number of furcation lesions exceeding grade 1), which was the system used to evaluate the periodontal situation (Mattila et al., 2000).The crevicular fluid was sampled by using a paper cone inserted into the periodontal pockets and stored at À80 8C until it was processed for DNA extraction. During surgery, a section of the arterial wall including the atheromatous plaque was removed and longitudinally cut into two sections that were used for PCR and FISH testing.The PCR protocol has previously been reported (Donati et al., 1997) and was performed as described by Mättö et al. (1998) and by Siqueira et al. (2000) for the detection of Porphyromonas gingivalis and Treponema denticola, respectively. DNA extracted from in vitro grown P. gingivalis (ATCC 33277) and T. denticola (ATCC 35405) was used as a positive control; DNA extracted from Treponema pallidum (Nichols strain) (Sambri et al., 2001) was used as a negative control in each PCR reaction set. DNA was extracted from the vessel biopsy as follows: the tissue was treated with a mechanical homogenizer and then 700 mg was incubated with buffer K [10 mM Tris (pH 8 . 3), 50 mM KCl, 1 . 75 mM MgCl 2 , 0 . 01 % (w/v) bovine serum albumin, 0 . 45 % (v/v) Tween 20, 0 . 45 % (v/v) Nonidet P-40 and 100 ìg ml À1Proteinase K) at 56 8C for 7 h. After incubation the sample was extracted with phenol/chloroform, precipitated with 0 . 3 M sodium acetate and 2-propanol, and resuspended in 200 ìl of TE buffer.The PCR analy...

The vaginal microbiota plays a crucial role in maintaining the health and functioning of the female genital tract, preventing the colonization of urogenital pathogens and sexually transmitted infections. In this study, we characterized the vaginal bacterial communities and the metabolome associated to Chlamydia trachomatis infection (CT: 20 women), compared to healthy condition (H: 22 women) and bacterial vaginosis (BV: 19 women). A microarray-based tool (VaginArray), implemented with a real-time PCR for Gardnerella vaginalis, was used to determine the vaginal bacterial composition, whereas the metabolic profiles were assessed by a proton-based nuclear magnetic resonance (1H-NMR) spectroscopy. CT infection was characterized by bacterial and metabolic signatures similar to healthy condition, even though higher amounts of Lactobacillus iners, as well as depletion of some amino acids, biogenic amines, and succinate marked CT infection. Moreover, the frequency of Lactobacillus crispatus was higher in asymptomatic CT-positive patients than in women with CT-correlated symptoms. We also confirmed the marked differences in the microbiome and metabolome between healthy and BV-affected women. In conclusion, we highlighted microbial and metabolic peculiarities of the vaginal ecosystem in the case of CT infection, even though further studies are needed to understand if the observed alterations precede the infection onset or if the pathogen itself perturbs the vaginal environment.

The emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae (GC) underline the need of “antibiotic-free” strategies for the control of gonorrhea. The aim of this study was to assess the anti-gonococcal activity of 14 vaginal Lactobacillus strains, belonging to different species (L. crispatus, L. gasseri, L. vaginalis), isolated from healthy pre-menopausal women. In particular, we performed “inhibition” experiments, evaluating the ability of both lactobacilli cells and culture supernatants in reducing GC viability, at two different contact times (7 and 60 min). First, we found that the acidic environment, associated to lactobacilli metabolism, is extremely effective in counteracting GC growth, in a pH- and time-dependent manner. Indeed, a complete abolishment of GC viability by lactobacilli supernatants was observed only for pH values < 4.0, even at short contact times. On the contrary, for higher pH values, no 100%-reduction of GC growth was reached at any contact time. Experiments with organic/inorganic acid solutions confirmed the strict correlation between the pH levels and the anti-gonococcal effect. In this context, the presence of lactate seemed to be crucial for the anti-gonococcal activity, especially for pH values in the range 4.4–5.3, indicating that the presence of H+ ions is necessary but not sufficient to kill gonococci. Moreover, experiments with buffered supernatants led to exclude a direct role in the GC killing by other bioactive molecules produced by lactobacilli. Second, we noticed that lactobacilli cells are able to reduce GC viability and to co-aggregate with gonococci. In this context, we demonstrated that released-surface components with biosurfactant properties, isolated from “highly-aggregating” lactobacilli, could affect GC viability. The antimicrobial potential of biosurfactants isolated from lactobacilli against pathogens has been largely investigated, but this is the first report about a possible use of these molecules in order to counteract GC infectivity. In conclusion, we identified specific Lactobacillus strains, mainly belonging to L. crispatus species, able to counteract GC viability through multiple mechanisms. These L. crispatus strains could represent a new potential probiotic strategy for the prevention of GC infections in women.