Anna Maria Schito

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.

Effect of fosfomycin alone and in combination with N-acetylcysteine on E. coli biofilms

Four slime-producing uropathogenic Escherichia coli strains were used to investigate the activity of fosfomycin and N-acetylcysteine (NAC) against biofilms developed on 96-well polystyrene tissue culture plates. Biofilms aged, respectively, 5 (initial) and 48 h (mature) and two fosfomycin concentrations (128 and 2000 mg/l) were used. The effect of various levels (0.007-8 mg/ml) of NAC alone and in combination with fosfomycin on the formation or disruption of biofilms was assessed. Following exposure to the drugs, the percentage of residual slime relative to the control, ranged from 62.5-100 to 26.2-64.1% in the presence of 0.007 and 8 mg/ml of NAC. After treatment of pre-formed biofilms with NAC at the highest concentrations used, the remaining exopolysaccharide matrix was reduced to 25-68% of the amount found with the untreated control. Exposure to fosfomycin at 2000 mg/l reduced biofilms 40-57 and 41-49% for the initial and mature forms, respectively. Fosfomycin was more active at 2000 mg/l combined with NAC 2 mg/ml. Under these conditions initial and mature biofilms were reduced 66-80 and 60-73%, respectively. NAC, when used in combination, enhanced fosfomycin bactericidal activity producing a 99-99.9% reduction in viable cells. Fosfomycin and NAC at concentrations achievable in urine displayed a synergistic effect promoting both the formation of biofilms and reduction of sessile cell viability.

Targeted Expression of SHH Affects Chondrocyte Differentiation, Growth Plate Organization, and Sox9 Expression

The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation.

In vitro activity of fosfomycin against Gram-negative urinary pathogens and the biological cost of fosfomycin resistance

The aim of this study was to reassess the activity of fosfomycin against recently isolated uropathogens circulating in Italy and to evaluate the effect of fosfomycin resistance on the expression of several virulence traits using the rare mutant strains. In vitro activity of fosfomycin was evaluated using 441 Gram-negative organisms isolated from patients with uncomplicated urinary tract infections (UTIs). Fosfomycin was the most active antibiotic against Escherichia coli (99% susceptibility). The activity against Proteus mirabilis was more potent than that of co-trimoxazole and nitrofurantoin (87.5, 67 and 0% susceptibility, respectively). The other microorganisms, accounting for about 7% of all pathogens tested, showed variable susceptibilities to fosfomycin. Compared with susceptible strains, fosfomycin-resistant mutants showed a reduced rate of growth and were impaired in their ability to adhere to uroepithelial cells and to urinary catheters. They were also more resistant to UV irradiation and to phage T7 and showed diminished rates of colicin synthesis and transfer of plasmids.

Antiadhesion and Antibiofilm Activities of High Molecular Weight Coffee Components against Streptococcus mutans

In previous studies we demonstrated that green and roasted coffee contains low molecular weight (LMW) compounds capable of inhibiting the ability of Streptococcus mutans, the major causative agent of human dental caries, to adhere to hydroxyapatite (HA) beads. This study addressed the ability of the whole high molecular weight coffee fraction (cHMW) and of its melanoidin and non-melanoidin components (GFC1-5), applied at concentrations that occur in coffee beverages, to (i) inhibit S. mutans growth; (ii) affect S. mutans sucrose-dependent adhesion to and detachment from saliva-coated HA beads (sHA); and (iii) inhibit biofilm development on microtiter plates. The results indicated that only cHMW is endowed with antimicrobial activity. The cHMW fraction and each of the five GFC components inhibited S. mutans adhesion, the strongest effect being exerted by cHMW (91%) and GFC1 (88%). S. mutans detachment from sHA was four times greater (∼20%) with cHMW and the GFC1 and GFC4 melanoidins than with controls. Finally, biofilm production by S. mutans was completely abolished by cHMW and was reduced by 20% by the melanoidin components GFC2 and GFC4 and by the non-melanoidin component GFC5 compared with controls. Altogether these findings show that coffee beverage contains both LMW compounds and HMW melanoidin and non-melanoidin components with a strong ability to interfere in vitro with the S. mutans traits relevant for cariogenesis.

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.

Antimicrobial Activity of the Ornamental Species Salvia corrugata, a Potential New Crop for Extractive Purposes

As a part of our search for biologically active compounds from cultivated Salvia spp. we investigated Salvia corrugata Vahl. The activity of two isolated icetaxane diterpene quinones, fruticuline A and demethylfruticuline A, was assessed against 46 bacterial pathogens, mostly resistant to several primary antibiotics. The MIC for all the inhibited Gram-positive pathogens tested showed a very narrow distribution and ranged from 32 to 64 mg/L, regardless of their resistance patterns to other antibiotics. Demethylfruticuline A was shown to be highly bactericidal (>3 log(10) CFU decrease within 24 h) against Staphylococcus aureus and S. epidermidis and bacteriostatic against Enterococcus faecalis and E. faecium. Fruticuline A manifested bacteriostatic activity against all tested strains. S. corrugata can be viewed as an interesting source for these diterpenes, which, if well tolerated in vivo, may represent new medical agents useful for the treatment of serious infections caused by resistant Gram-positive pathogens.

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.

The Italian Epidemiological Survey 1997 /1999 Antimicrobial susceptibility data of Haemophilus influenzae, Haemophilus parainfluenzae and Moraxella catarrhalis in Italy

The Italian Epidemiological Survey began a surveillance study with the aim of monitoring the antimicrobial resistance of respiratory pathogens. From 1997 to 1999, 2028 strains of Haemophilus influenzae and 523 strains of Haemophilus parainfluenzae were collected from 59 Clinical Microbiology Laboratories distributed throughout Italy. In 1998, the study was extended to include Moraxella catarrhalis and a total of 360 isolates were collected. There was a significant increase in the beta-lactamase production both for H. influenzae (from 5% in 1997 to 16% in 1999) and for H. parainfluenzae (from 5% in 1997 to 22% in 1999). Beta-lactamase production in M. catarrhalis was 84% in 1998 and 87% in 1999. Beta-lactamase production affected the susceptibility to unprotected penicillins (87% in H. influenzae, 85% in H. parainfluenzae and 34% in M. catarrhalis), and in part the susceptibility to cefaclor (about 98%). Amoxycillin/clavulanate, cefixime, ceftriaxone and ciprofloxacin were active against all strains of H. influenzae, H. parainfluenzae and M. catarrhalis.

Establishment and analysis of in vitro biomass from Salvia corrugata Vahl. and evaluation of antimicrobial activity.

Demethylfruticuline A and fruticuline A, the most abundant compounds from the surface extract of Salvia corrugata Vahl., have shown antibacterial, antitumor and cytotoxic activities. In order to obtain these icetexane diterpenes from in vitro cultures of S. corrugata, protocols were developed for callus production, micropropagation and shoot regeneration. Analysis of the regenerated shoots showed the presence of both icetexanes, micropropagated plants contained only fruticuline A, while the callus contained trace amounts of both diterpenes. The yield of fruticuline A was higher in the methanolic extract of regenerated shoots than in those of fresh leaves and fresh shoot tips. In addition to these diterpenes, the regenerated shoot and micropropagated plant extracts afforded seven other diterpenes, one icetexane and six abietanes, identified by UV, IR, 1D- and 2D-NMR and HR-MS analysis. Five compounds (19-acetoxy-7α-hydroxyroyleanone, 7β,20-epoxy-11,12,19-trihydroxyabieta-8,11,13-triene, 7,20-dihydrofruticuline A, 7β-acetoxy-20-hydroxy-19,20-epoxyroyleanone, 7β-ethoxy-6β,20:19,20-diepoxyroyleanone) were previously undescribed. Although the crude plant surface extract did not possess any antibacterial activity, methanolic extracts of in vitro tissues and two compounds, namely 7β-acetoxy-20-hydroxy-19,20-epoxyroyleanone and 7β-ethoxy-6β,20:19,20-diepoxyroyleanone, isolated in suitable amounts, were active in varying degrees against multidrug resistant clinical strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium, displaying MIC values ranging from 32, 64 to 128μg/mL.