Gabriella Piatti

Virulence factors in urinary Escherichia coli strains: phylogenetic background and quinolone and fluoroquinolone resistance.

ABSTRACT Quinolone- and fluoroquinolone-resistant Escherichia coli strains harbor fewer virulence factors than susceptible strains. The reasons underlying this correlation are incompletely understood. We investigated the phylogenetic background, the presence of the papC, hlyA, and cnf1 (pathogenicity island IIJ96-associated), fimA, iss, and iutA genes, and the presence of type 1 fimbriae, P fimbriae, and hemolysin in 243 urinary E. coli isolates resistant only to quinolones (8%), resistant to both quinolones and fluoroquinolones (51%), or susceptible to both drugs (41%). Group B2 accounted for 56% of the isolates, showing a significantly higher prevalence among fluoroquinolone-susceptible strains than among resistant strains (65% versus 50% [P = 0.03]). hly and cnf1 were significantly more associated with susceptibility (P < 0.001) and with group B2 (P < 0.001 for group B2 versus groups A and D). However, within group B2, fluoroquinolone-resistant strains showed lower prevalences of papC, hlyA, and cnf1 than their susceptible counterparts (P < 0.001). In contrast, the incidence of iutA appeared higher for refractory isolates, including group B2, than for susceptible isolates (P < 0.001). Only in group B2 did fluoroquinolone-resistant strains reveal a lesser ability to agglutinate Saccharomyces cerevisiae (7%) than quinolone-resistant (87%) and susceptible (80%) isolates, despite uniform possession of fimA genes. No similar contrast emerged for expression of hemolysin and P fimbriae. Mutations conferring quinolone and fluoroquinolone resistance may thus require a particular genetic background, not strictly correlated with phylogenetic groups. More interestingly, the mutational event itself can affect the expression of type 1 fimbriae, at least in the prevalent and complex B2 strains.

Effects of demethylfruticuline A and fruticuline A from Salvia corrugata Vahl. on biofilm production in vitro by multiresistant strains of Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis

In this study, demethylfruticuline A (dfA) and fruticuline A (fA), two quinones representing the major diterpenoid components of the exudate produced by the aerial parts of Salvia corrugata, were assessed for their ability to modify surface characteristics, such as hydrophobicity, and to inhibit synthesis of biofilm in vitro by multiresistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. Five strains of S. aureus (three meticillin-resistant and two meticillin-susceptible), five strains of S. epidermidis (four meticillin-resistant and one meticillin-susceptible) and eight vancomycin-resistant E. faecalis, all recently isolated from clinical specimens and capable of slime production, were studied. fA decrease by at least two-fold the hydrophobic properties of the S. aureus cell membrane but did not affect S. epidermidis or E. faecalis. Biofilm formation on polystyrene plates was quantified spectrophotometrically by established methodologies. Inhibition of biofilm formation was also confirmed by the Congo red agar plate assay. dfA and fA were more effective against S. aureus strains (>70% effect at subinhibitory concentrations) than against S. epidermidis in inhibiting slime synthesis. Against E. faecalis, dfA at subinhibitory concentration induced an inhibition of biofilm production of ca. 60%; fA was less active and more strain-dependent. Moreover, the two compounds were shown to possess chelating activity on divalent and trivalent metal cations. Interactions of fA and dfA with bacteria could be very complex, possibly being species-specific, and could depend not only on inhibition of exopolysaccharide synthesis but also on their chelating activity and on changes in the microorganisms surface, including cell hydrophobicity.

The inhibitory co-receptors: a way to save from anergy the HIV-specific T cells.

The functional impairment of HIV-specific CD4(+) T cells during chronic HIV infection is thought to be closely linked to viral replication and to T cell exhaustion. T cell exhaustion in the presence of ongoing antigen exposure is a common feature of chronic viral infection, in which dysfunctional T cells fail to eliminate the virus. Otherwise, antiviral T cell function impairment is a poorly understood mechanism. Increasing evidences show that HIV-specific T lymphocytes up-regulated inducible co-receptors, such as the Cytoxic T Lymphocyte Antigen-4, (CTLA-4, or CD152) and Programmed Death-1 (PD-1) and that blockade of the CD152 or PD-1 pathway restores HIV-specific CD4(+) T cell function in HIV infection. This review will focus on finding a possible role for inhibitory receptors on virus-specific CD4(+) T cells. The analysis of the role of CD152 and PD-1 in HIV-1 infection could provide important insight into the mechanism of viral induced immune dysfunction and lead to immunotherapeutic strategies to reverse immune suppression in this pathology.

Antibacterial compounds from Salvia adenophora Fernald (Lamiaceae)

From the aerial parts of Salvia adenophora Fernald four derivatives of 12-oxo-phytodienoic acid (1-4) together with five clerodane diterpenoids (5, 6, 8-10), and one known diterpene (7) have been isolated. Compounds 1-6 and 8-10 are described for the first time. The structures were established by extensive 1D, 2D NMR and HRESI-TOFMS spectroscopic methods. Finally, the absolute configuration has been established by comparing of experimental and quantum chemical calculation of ECD spectra. Despite a total lack of antimicrobial activity of the plant extract, hinting to the existence of antagonistic interactions in the crude material, three oxylipins (2-4) displayed a promising inhibition on Gram-positive multidrug-resistant clinical strains including Staphylococcus aureus, Streptococcus agalactiae and, particularly, Staphylococcus epidermidis, while the compounds 9 and 10 revealed a specific and strain-dependent activity against S. epidermidis. Interestingly, the inhibition provided by these compounds was independent of the resistance patterns of these pathogens to classic antibiotics. No action was reported on Gram-negative strains nor on Candida albicans. These results confirm that clerodanes and, particularly, prostaglandin-like compounds can be considered as interesting antimicrobial agents deserving further study.

Human T leukaemia virus type 1 (HTLV-1) specific T-helper cell response: clonal fluctuations and repertoire heterogeneity.

The naive T‐helper (Th) repertoire specific for HTLV‐1 envelope (env) has been examined on antigen specific T‐cell lines and clones from non‐immune individuals. Clonal heterogeneity was determined by analysing the T‐cell receptor (TCR) Vβ gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the Vβ gene usage were determined by comparing the TCR Vβ gene profiles of T‐cell lines at different times. We found that a diverse repertoire for HTLV‐1 env could be triggered in vitro. Diverse Vβ genes were used by the same line tested at different times, suggesting that clonal composition of an antigen‐specific T‐cell line is not constant in vitro. Clones in fact may be up‐ and down‐regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T‐cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen‐specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vβ gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV‐1‐infected patients studied in vivo, the Th cell repertoire specific for HTLV‐1 env may prove very heterogenous, with important implications for vaccine development.

Quinolone/fluoroquinolone susceptibility in Escherichia coli correlates with human polymicrobial bacteriuria and with in vitro interleukine-8 suppression

Urinary tract infections (UTIs) are frequently polymicrobial diseases mainly sustained by Escherichia coli in association with other opportunistic pathogens. Cystitis and pyelonephritis are usually accompanied by an inflammatory response, which includes neutrophil recruitment. Uropathogenic E. coli possess the ability to evade host defenses, modulating the innate immune response. The aim of this study was to determine whether particular E. coli strains correlate with polymicrobial bacteriuria and whether escape from the early host defenses and microbial synergy could lead to mixed UTIs. We evaluated 188 E. coli-positive urine samples and assessed the relationships among polymicrobism, neutrophil presence and several traits of E. coli isolates (virulence factors such as hlyA, fimA, papC and their relative products, i.e. hemolysin, type 1 and P fimbriae, and cnf1, their phylogenetic group) and their ability to suppress cytokine response in 5637 bladder epithelial cells. Escherichia coli susceptibility toward quinolones and fluoroquinolones, known to be linked to the pathogenicity of this species, was also considered. We found significant correlations among polymicrobial bacteriuria, absence of pyuria and quinolone/fluoroquinolone susceptibility of E. coli isolates and their enhanced capability to suppress interleukin-8 urothelial production when compared with the patterns induced by the resistant strains.

Pathogenic potential of Escherichia coli from polymicrobial urinary tract infections.

In a recent issue of this journal, Croxall et al. (2011) described in their work on polymicrobial urinary tract infections (UTIs) data about the pathogenic potential of Escherichia coli isolates. I have several comments on their study regarding the method used by the authors for the research that I think has invalidated the results, which are in discord with the literature. I also believe that the authors omitted consideration of some previous studies that described basic findings on the connection between E. coli pathogenicity and the susceptibility to the antibiotic quinolone. In addition, I think that a more critical view should have been taken in the interpretation of the results.

Handling of retroviral antigens by human antigen-presenting cells

Antigen-specific T helper cells play an important role in retroviral infections. Indeed, they provide help for B-cell activation and antibody production and for clonal expansion of cytolytic lymphocytes. Therefore, we used retrovirus-specific human T helper clones in order to define modes of antigen presentation, antigen-presenting cells and the molecular context of Th epitopes that could be exploited in the design of immunogens aimed at optimizing the Th cell response. In particular, we describe several mechanisms of receptor-mediated antigen uptake that enhance the stimulation of human T-cell clones specific for HIV and HTLV-1 antigens; we report on the differential recognition of Th epitopes depending on the molecular-viral context; we show that dendritic cells are the most efficient presenting cells and are essential for the induction of in vitro primary Th cell responses; and finally, we propose that Th cells specific for internal, conserved antigens of HIV such as reverse transcriptase, may be candidates for intrastructural help resulting in induction of envelope specific antibodies.

Epidemiology of Infection in a Large Hospital in Northern Italy: Questioning the Ward-Based Transmission

Background: Clostridium Difficile infection (CDI) is considered a ward-based nosocomial infection, due to contagion among patients. Molecular studies recently questioned ward-based contact for disease spread. Objective: To investigate whether it is plausible that CDI spread in San Martino Hospital of Genoa was due to a ward-based contact and patient-to-patient diffusion. Methods: We conducted a retrospective cohort study of CDI cases from April 2010 to March 2015. We referred to Hospital data set and Admission Service. Multilevel modelling approach and ecological analysis were used to assess C. difficile infection risk according to wards and time of occurrence. Six representative CD strains were ribotyped to assess a possible equivalence. Results: The assessment of 514 CDI cases showed that the risk of disease and rate of incidence in wards were independent, while frequency of cases and number of wards involved exhibited a positive relationship, excluding the typical epidemic pattern of contagious diffusion, i.e., many cases in few wards. The extra-binomial variability due to ward clustering was not significant, indicating homogeneity in the probability of CDI occurrence across all wards. Three hundred sixty-eight patients changed ward, without showing connection between the frequency of cases in new wards and incidence among new subjects. Trigonometric components described a significant contribution of seasonality, with excess of CDI cases during the winter months. Molecular analysis showed different ribotypes of CD strains from the same ward. Conclusion: From our results it seems unlikely that in our institution CDI occurrence is due to ward-based contact and inter-human contagion of the organism.

Identification of Salmonella enterica Serovar Typhi DNA Fragments with Transcriptional Activity Under Different Growth Conditions

Salmonella enterica serovar Typhi is a human pathogenic microorganism with a very complex infective cycle, involving the transit of bacteria across different microenvironments; to optimize the performance of attenuated Salmonella strains suitable as live carriers of heterologous antigens, fine tuning of wild type bacteria gene expression is essential. Several DNA fragments were obtained from a Salmonella enterica serovar Typhi (vi+, fim+) blood isolate and 18 clones were selected according to the dimension of the insert (range <0.2-1.6 kb). These fragments showed a transcriptional ac- tivity in a promoterless vector cloned in Escherichia coli background, according to homogeneous parameters. The results obtained provide an insight about signals mediating gene activation in vivo, particularly in the microenvironments known to exist during the infectious process, even if the fragments are not promoters sequences. Finally, the functional charac- terization of several fragments showed that they possessed an efficient and homogeneous transcriptional activity, worth to be further investigated.