Alterations of the vaginal microbiota in the third trimester of pregnancy and pPROM.

OBJECTIVE: Preterm premature rupture of membranes (pPROM) is a significant issue in obstetric practice. One of the risk factors for pPROM are vaginal infections in the third trimester of pregnancy. PATIENTS AND METHODS: We performed an observational study on 600 pregnant women, analyzing the lactobacillary grade (LBG) and the presence of any pathogenic bacteria and/or Candida at weeks 28 and 32 of pregnancy and recording any pPROM events at delivery. At week 28, in the case of vaginal infection, the patients were treated for 6 days with a topical association of metronidazole+clotrimazole. RESULTS: At week 28 of pregnancy 54.2% of women had vaginal infection (32.6% bacterial vaginitis, 33.8% candidiasis and 32.4% mixed infection) and/or abnormal vaginal microbiota (67.4% LBG 2a/2b, 32.6% LBG 3). The total number of pPROM was 8 out of 600 (1.3%). The treatment of vaginal infection at week 28 with the topical association of metronidazole+clotrimazole, led to both the eradication of vaginal infections and the restoration of the vaginal microbiota in 72% of the cases, bringing the level of risk of pPROM similar to that of women without vaginal infection at week 28. In addition, the results showed that women with vaginal infections and/or alteration of vaginal microbiota at week 32 of pregnancy had a higher prevalence of pPROM in comparison to the women without vaginal infection at week 32 (p<0.001). CONCLUSIONS: This observational study showed the high prevalence of vaginal infections in the third trimester of pregnancy and its association with pPROM. Furthermore, data suggested the possible benefits of the topical treatment with metronidazole+clotrimazole in pregnancy to eradicate infections, restore the normal microbiota and reduce the risk of pPROM.

Decontamination efficacy of photon-initiated photoacoustic streaming (PIPS) of irrigants using low-energy laser settings: an ex vivo study.

AIM: To assess ex vivo, the antibacterial effectiveness of photon-initiated photoacoustic streaming (PIPS) of irrigants using an Er:YAG laser equipped with a newly designed, stripped and tapered tip in extracted teeth with infected root canals. METHODOLOGY: One hundred and forty-eight single-rooted extracted teeth were prepared to a size 25, 0.06 taper. The specimens were sterilized, and all teeth except ten (negative control group) were inoculated with Enterococcus faecalis and incubated in a CO(2) chamber at 37 °C for 15 days in Eppendorf tubes filled with trypticase soy broth medium changed every 2 days. Infected teeth were then randomly divided into four test groups (n = 32 for each): pulsed erbium/YAG laser at nonablative settings for 30 s with sterile bi-distilled water (Group A) or 5% sodium hypochlorite (NaOCl) (Group B); without laser-activated sterile bi-distilled water irrigation for 30 s (Group C) or 5% NaOCl irrigation for 30 s (Group D); the positive control group received no treatment in infected teeth (n = 10). Colony-forming units (CFUs) were counted from bacteriologic samples taken before (S1) and after treatment (S2). Data were analysed by Kruskal-Wallis and post hoc Dunn's multiple comparison tests. RESULTS: CFU counts were significantly lower in 5% NaOCl groups with or without laser activation than in sterile bi-distilled water without laser activation group (P < 0.001). Moreover, there was a significant difference between bi-distilled water groups with or without laser activation (P < 0.001). Sodium hypochlorite with laser activation group had the greatest CFU reduction, which was significantly greater than that evident in bi-distilled water groups with or without laser activation (P < 0.001). There were no significant differences between 5% NaOCl groups with or without laser activation (P > 0.05). None of the four groups generated negative samples predictably. CONCLUSIONS: Under the conditions of this ex vivo study, there were no significant differences in bacterial reduction between the laser and NaOCl or NaOCl alone groups. [Correction added after online publication, 18th April 2012: The following statement has been deleted: 'Thus, the use of a laser did not improve microbial killing over and above use of NaOCI alone.'].

Antipoliovirus activity and mechanism of action of 3-methylthio-5-phenyl-4-isothiazolecarbonitrile.

Our previous studies described the synthesis and the antiviral activity of 3,4,5-trisubstituted isothiazole derivatives that were found to be particularly effective against enteroviruses. Compound 3-methylthio-5-phenyl-4-isothiazolecarbonitrile (IS-2) exhibited an interesting anti-poliovirus activity with a high selectivity index. In the present study we investigated the mechanism of action of this compound. Studies on the time of IS-2 addition to poliovirus type 1 infected cells suggested that the compound may inhibit some early process of viral replication. In order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [³H]uridine-labeled poliovirus to HEp-2 cells in the presence or absence of IS-2. No effect on poliovirus adsorption and internalization to host cells was detected. We also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral RNA susceptible to ribonuclease. These experiments demonstrated that poliovirus uncoating is influenced by IS-2 action.

In vitro activity of cefditoren versus other antibiotics against S. pneumoniae clinical strains isolated in Italy.

Over the last twenty years there has been an alarming increase in isolation of Streptococcus pneumoniae strains with a reduced susceptibility not only to penicillin, but also to other betalactams and macrolides. This phenomenon justifies the great interest in new antibiotics. Cefditoren, a new aminothiazolyl oral cephalosporin, recently commercialized in Italy, is characterized by an extended activity against penicillin-resistant S. pneumoniae. The aim of this study is to evaluate the incidence of the resistance/susceptibility to various antibiotics in 1000 strains of S. pneumoniae (678 SPSS, 219 SPPI and 103 SPPR), clinically isolated during 2009. The data obtained by our in vitro study show that cefditoren is the most active agent against S. pneumoniae. In fact, the MIC90 values of 0.5 micrograms/ml obtained could be particularly significant in therms of therapeutic predictivity.

Inhibitory activity of cranberry extract on the bacterial adhesiveness in the urine of women: an ex-vivo study.

Strains of uropathogenic E. coli are responsible for approximately 90% of community-acquired, uncomplicated cystitis, and fimbriae represent the adhesive factors enabling E. coli to be anchored to uroepithelial cells in the first step of the infectious process. Recently, a few studies have shown that a correlation between the consumption of cranberry (Vaccinium macrocarpon) and prevention of UTI is related to the ability of proanthocyanidins to reduce the bacterial adhesion to uroepithelial cells. In this study we evaluate the inhibitory activity of urine of healthy women treated with tablets containing cranberry extract on the adhesiveness of E. coli to uroepithelial human cells. Two groups of 12 female volunteers each, aged between 18 and 65 years, were enrolled, one group with negative history and one group with positive history of recurrent cystitis. Subjects were treated with the active product or placebo in a random, cross-over, double-blinded sequence for one week in each of the two treatment sequences. Urine samples were collected at the beginning and the end of each study period. Tests of bacterial adhesiveness were performed with two strains of E. coli (ATCC 25922 and ATCC 35218) on HT1376 human bladder carcinoma cells. Significant reductions of bacterial adhesiveness were observed in women who received cranberry extract (-50.9%; p less than 0.0001), regardless of their medical history and the treatment period in the cross-over sequence. No changes were observed with placebo (-0.29%; n.s.). This ex-vivo study showed that the assumption of cranberry extract in suitable amounts can have an anti-adhesive activity on uropathogenic E. coli.

Antibiotic susceptibility of respiratory pathogens recently isolated in Italy: focus on cefditoren.

The aim of this study was to evaluate the in vitro antibiotic susceptibility of respiratory pathogens recently isolated in Italy to commonly used antibiotics including cefditoren. Six clinical microbiological laboratories collected, between January and September 2009, a total of 2,510 respiratory pathogens from subjects with community-acquired respiratory tract infections (CARTI). Ceftditoren, out of all the beta-lactams studied, had the lowest MIC(90 )against 965 strains of Streptococcus pneumoniae examined, followed by cefotaxime and ceftriaxone (2% resistance in penicillin-resistant S. pneumoniae (PRSP)). Against 470 Haemophilus influenzae , independently of their production of beta-lactamases or ampicillin resistance, cefditoren was the oral cephalosporin with the best in vitro activity, comparable to that of the injectable cephalosporins and levofloxacin. Higher MIC(90)s were found for the macrolides (4 - 16 mg/l) and cefaclor (4 - 32 mg/l). As was foreseeable, Streptococcus pyogenes (225 strains) was uniformly sensitive to all the beta-lactam antibiotics, but the elevated MIC(90 )values reduced (<75%) susceptibility of this pathogen to macrolides. Beta-lactamase-negative Moraxella catarrhalis (100 strains) had reduced susceptibility only to the macrolides, while the 250 beta-lactamase-producing strains also had reduced susceptibility to cefuroxime. Levofloxacin showed the lowest MIC(50)/MIC(90 )values in the producing strains, whereas cefditoren, cefotaxime and ceftriaxone in the non-producers. As regards the enterobacteriaceae, cefditoren and levofloxacin had the lowest MIC(90)s against Klebsiella pneumoniae. Cefditoren and the third-generation injectable cephalosporins had the lowest MIC(90)s against Escherichia coli (100% susceptibility) while levofloxacin was less active (86% susceptibility).In conclusion, cefditoren's wide spectrum and high intrinsic activity, as well as its capacity to overcome most of the resistance that has become consolidated in some classes of antibiotics widely used as empiric therapy for CARTI, allows us to suggest that cefditoren might be included in the european guidelines as one of the first-choice antibiotics in the treatment of CARTI.

Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach.

The polyamines spermine, spermidine and putrescine are ubiquitous cell components. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper-containing amine oxidases. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. In tumors, polyamines and amine oxidases are increased as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H(2)O(2) and aldehydes produced by the reaction. This study demonstrated that multidrug-resistant (MDR) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding wild-type (WT) ones to H(2)O(2) and aldehydes, the products of BSAO-catalyzed oxidation of spermine. Transmission electron microscopy (TEM) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. Increasing the incubation temperature from 37 to 42 degrees Celsius enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumor growth, particularly well if the enzyme has been conjugated to a biocompatible hydrogel polymers. Since both wild-type and MDR cancer cells after pre-treatment with MDL 72527, a lysosomotropic compound, are sensitized to subsequent exposure to BSAO/spermine, it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumor cells. It is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting.

Polyamines: fundamental characters in chemistry and biology.

Polyamines are small cationic molecules required for cellular proliferation and are detected at higher concentrations in most tumour tissues, compared to normal tissues. Agmatine (AGM), a biogenic amine, is able to arrest proliferation in cell lines by depleting intracellular polyamine levels. It enters mammalian cells via the polyamine transport system. Agmatine is able to induce oxidative stress in mitochondria at low concentrations (10 or 100 microM), while at higher concentrations (e.g. 1-2 mM) it does not affect mitochondrial respiration and is ineffective in inducing any oxidative stress. As this effect is strictly correlated with the mitochondrial permeability transition induction and the triggering of the pro-apoptotic pathway, AGM may be considered as a regulator of this type of cell death. Furthermore, polyamine transport is positively correlated with the rate of cellular proliferation. By increasing the expression of antizyme, a protein that inhibits polyamine biosynthesis and transport, AGM also exhibits a regulatory effect on cell proliferation. Methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosyl-L: -methionine decarboxylase, displaying anticancer activity, is a structural analogue of the natural polyamine spermidine. MGBG has been extensively studied, preclinically as well as clinically, and its anticancer activity has been attributed to the inhibition of polyamine biosynthesis and also to its effect on mitochondrial function. Numerous findings have suggested that MGBG might be used as a chemotherapeutic agent against cancer.

Agmatine transport in brain mitochondria: a different mechanism from that in liver mitochondria.

The diamine agmatine (AGM), exhibiting two positive charges at physiological pH, is transported into rat brain mitochondria (RBM) by an electrophoretic mechanism, requiring high membrane potential values and exhibiting a marked non-ohmic force-flux relationship. The mechanism of this transport apparently resembles that observed in rat liver mitochondria (RLM), but there are several characteristics that strongly suggest the presence of a different transporter of agmatine in RBM. In this type of mitochondria, the extent of initial binding and total accumulation is higher and lower, respectively, than that in liver; saturation kinetics and the flux-voltage relationship also exhibit different trends, whereas idazoxan and putrescine, ineffective in RLM, act as inhibitors. The characteristics of agmatine uptake in RBM lead to the conclusion that its transporter is a channel with two asymmetric energy barriers, showing some characteristics similar to those of the imidazoline receptor I(2) and the sharing with the polyamine transporter.

High frequency of Chlamydophila pneumoniae infections: patients with peripheral arterial disease and those with risk factors for cardiovascular diseases compared to normal subjects.

The role of bacterial infections, mainly Chlamydophila pneumoniae, on atherosclerotic processes as well as the therapeutic utility of additional antibiotic treatment is still an open question. In this study we compared the serological profiles of 160 patients (80 with peripheral arterial disease (PAD), diagnosed with an ankle/brachial index (ABI) ≤ 0.9 and 80 with risk factors for cardiovascular disease - CVD) with those of 80 healthy subjects, serum levels of specific C. pneumoniae antibodies using the microimmunofluorescence test. Our results show that PAD patients had a higher frequency of C. pneumoniae infection than those with risk factors for cardiovascular disease. This frequency was lower if compared to the previous two groups in controls. 44 out of the 80 (55%) patients with PAD and 34 out of the 80 (42.58%) subjects with risk factors for cardiovascular disease were seropositive while only 24 of the 80 (30%) healthy subjects showed seropositivity to C. pneumoniae. Furthermore, higher anticorpal titers were also found in patients with peripheral arterial disease and in patients with cardiovascular risk factors if compared to healthy subjects. On the basis of these results, we confirm that C. pneumoniae infection is frequent in peripheral arterial disease patients and we believe that it could be considered as an additional risk factor involved in the pathogenesis of this disease.

In vitro evaluation of the synergistic activity of neomycin-polymyxin B association against pathogens responsible for otitis externa.

The most recent guidelines recommend, for otitis externa antibiotic therapy, the use of topical formulations in that they are very safe, have a quicker effect and do not induce bacterial resistance compared to systemic therapy. The choice of the class of antibiotics in empiric therapy of otitis externa must take into consideration the polymicrobic nature of the infection that includes both bacteria (Grampositive and Gram-negative) and mycetes. For this reason, in this study we evaluated the synergic activity of neomycin in association with polymyxin B against the pathogens commonly responsible for otitis externa, compared to that of a single antibiotic (ciprofloxacin). The polymyxinB/neomycin association shows clear synergic effects with values of both Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) reduced by 3-4 times with respect to the single antibiotic; and in P. aeruginosa the synergistic effect of the neomycin/polymyxin B association with respect to neomycin was more evident (5-6 times), with an intrinsic in vitro activity constantly higher than that of ciprofloxacin alone or in association with hydrocortisone. From the analysis of the data obtained in vitro, we can conclude that the possibility of using a topical formulation containing a synergistic association of antibiotics, such as neomycin-polymyxin B, in such a way as to obtain the maximum effect in the minimum time with an increase in the spectrum of action of non-bacterial pathogens, is an optimal choice for the clinician for the empiric therapy of otitis externa.

The impact of prulifloxacin on vaginal lactobacillus microflora: an in vivo study.

The aim of this study was to evaluate the in vivo effect of a repeated-dose regimen with prulifloxacin in comparison to amoxicillin/clavulanate on vaginal lactobacillus microflora. Thirty healthy female volunteers were treated with prulifloxacin or amoxicillin/clavulanate in this open, randomized, parallel-group, repeated-dose study. Vaginal signs and symptoms were assessed at the first doctor's Visit 0 (3 weeks prior to the start of the study), and subsequent examinations (1, 3, 5, 6, 7 and 8) (followup). Some volunteers treated with amoxicillin-clavulanate showed increased pH values and 73.3% of them had lower lactobacillus flora at Visit 3. this reduction was still present in 66.7% 3 days after the last dose and in 26.7% of subjects at the follow-up, about 7 - 13 days after the last dose. The situation was completely normalized at the second follow-up about one month after treatment stop. On the contrary, the repeated administration of prulifloxacin 600 mg tablets affected neither the pH nor the lactobacillus component of the vaginal flora in healthy fertile women. The oral administration of prulifloxacin may have advantages over some other antimicrobial agents because it preserves the normal vaginal microbiota in healthy women.

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-alpha effects in inducing apoptosis on human cancer cells through the generation of oxidative stress.

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNalpha) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNalpha had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O(2-), nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNalpha could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.

Econazole-polycarbophil, a new delivery system for topical therapy: microbiological and clinical results on vaginal candidiasis.

The aim of this study was to demonstrate that the addition of a bioadhesive polymer to econazole, which increases the duration of the active drug at the site of infection, leads to a greater frequency of negative culture after treatment and probably reduces the recurrence rate of vaginal candidiasis.180 women with vaginal candidiasis were treated with 150 mg vaginal ovules econazole nitrate with (group A) or without (group B) polycarbophil. After 3 days of treatment the negative culture of Candida albicans reached 98.6% in group A and 84.8% in B group, while the overall persistence (C. albicans, C. glabrata, C. krusei, and C. parapsilosis) was 5.6% and 30%, respectively. During a 60-day follow-up, only one case out of 85 (1.2%) in group A reported recurrence while in group B there were 6 out of 63 (9.5%) recurrences. We conclude that, since the women were treated with the same amount of econazole, the better clinical and microbiological results can be attributed to polycarbophil, as confirmed by a significant reduction of recurrences.

Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products.

A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonalpha (IFNalpha) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNalpha and the protein synthesis inhibitor fusion protein TGFalpha/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H2O2 and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.