Maria Maddalena Scaltrito

Helicobacter pylori: ureA, cagA and vacA expression during conversion to the coccoid form.

As viability of coccoid forms of Helicobacter pylori can only be verified by demonstrating the integrity of the DNA and active protein synthesis, we analysed the expression of ureA, cagA, vacA genes after prolonged incubation in a liquid medium. Exponentially growing and ageing phase cultures were used. Our results showed that, although the coccoid forms had decreased DNA and RNA levels after 31 days, they were not degraded and still expressed the urease, cytotoxic island and vacuolating toxin genes. Coccoid forms are therefore viable and may act as a transmissible agent that plays a crucial role in disease relapses after antibiotic therapy.

Helicobacter pylori: cultivability and antibiotic susceptibility of coccoid forms

Helicobacter pylori is an actively dividing helical bacterium that changes to coccoid morphology as the culture ages. It has been suggested that the coccoid forms may be involved in transmission of infection and in relapses following antimicrobial therapy. The aim of this investigation was to determine the survival and susceptibility of the coccoid forms to amoxycillin, erythromycin, gentamicin and metronidazole. Colony counts and microscopic examination were performed after 1-4 weeks of culture. At 2 and 4 weeks, identical cultures were treated with the antibiotics for 24 h. Our results showed that 4-week cultures of coccoid forms were cultivable after antibiotic treatment.

Bioactive compounds of Crocus sativus L. and their semi-synthetic derivatives as promising anti-Helicobacter pylori, anti-malarial and anti-leishmanial agents

Abstract Crocus sativus L. is known in herbal medicine for the various pharmacological effects of its components, but no data are found in literature about its biological properties toward Helicobacter pylori, Plasmodium spp. and Leishmania spp. In this work, the potential anti-bacterial and anti-parasitic effects of crocin and safranal, two important bioactive components in C. sativus, were explored, and also some semi-synthetic derivatives of safranal were tested in order to establish which modifications in the chemical structure could improve the biological activity. According to our promising results, we virtually screened our compounds by means of molecular modeling studies against the main H. pylori enzymes in order to unravel their putative mechanism of action.

Anti-plasmodial and insecticidal activities of the essential oils of aromatic plants growing in the Mediterranean area

BackgroundSardinia is a Mediterranean area endemic for malaria up to the last century. During a screening study to evaluate the anti-plasmodial activity of some aromatic plants traditionally used in Sardinia, Myrtus communis (myrtle, Myrtaceae), Satureja thymbra (savory, Lamiaceae), and Thymus herba-barona (caraway thyme, Lamiaceae) were collected in three vegetative periods: before, during and after flowering.MethodsThe essential oils were obtained by steam distillation, fractionated by silica gel column chromatography and analysed by GC-FID-MS. Total oil and three main fractions were tested on D10 and W2 strains of Plasmodium falciparum in vitro. Larvicidal and adulticidal activities were tested on Anopheles gambiae susceptible strains.ResultsThe essential oil of savory, rich in thymol, was the most effective against P. falciparum with an inhibitory activity independent from the time of collection (IC50 17–26 μg/ml on D10 and 9–11 μg/ml on W2). Upon fractionation, fraction 1 was enriched in mono-sesquiterpenoid hydrocarbons; fraction 2 in thymol (73-83%); and fraction 3 contained thymol, carvacrol and terpinen-4-ol, with a different composition depending on the time of collection. Thymol-enriched fractions were the most active on both strains (IC50 20–22 μg/ml on D10 and 8–10 μg/ml on W2) and thymol was confirmed as mainly responsible for this activity (IC50 19.7± 3.0 and 10.6 ± 2.0 μg/ml on D10 and W2, respectively). The essential oil of S. thymbra L. showed also larvicidal and adulticidal activities. The larvicidal activity, expressed as LC50, was 0.15 ± 0.002; 0.21 ± 0.13; and 0.15 ± 0.09 μg/ml (mean ± sd) depending on the time of collection: before, during and after flowering, respectively.ConclusionsThis study provides evidence for the use of essential oils for treating malaria and fighting the vector at both the larval and adult stages. These findings open the possibility for further investigation aimed at the isolation of natural products with anti-parasitic properties.

Antimicrobial Susceptibility Testing of Helicobacter pylori Determined by Microdilution Method Using a New Medium

Antibiotic susceptibility testing of Helicobacter pylori isolates was performed by broth microdilution method with MegaCellTM RPMI-1640 Medium (SIGMA). Fifty five clinical isolates of H. pylori were tested against metronidazole, tinidazole, amoxicillin, and clarithromycin. The results were compared to those obtained by standard agar dilution method. The microdilution method performed with new medium, showed excellent correlation with agar dilution results, with 100% agreement for metronidazole, 96.3% for amoxicillin, 90.7% for clarithromycin, and 92.8% for tinidazole. MICs determined by proposed method were highly reproducible: replicate results were variable within one-two-fold dilution by using different inocula and different batches of medium.

Anti-Helicobacter pylori agents endowed with H2-antagonist properties

New anti-Helicobacter pylori (H. pylori) agents endowed with H2-antagonists properties were obtained by combining the lamtidine derived pharmacophoric group with the antibiotic calvatic acid. All the compounds were tested for their irreversible H2-antagonist properties and for their ability to inhibit 20 H. pylori strains, two of them metronidazole resistant. The most active derivative (compound 4) displayed antimicrobial activity similar to metronidazole.

In vitro activity of artemisone and artemisinin derivatives against extracellular and intracellular Helicobacter pylori

The in vitro activity of the new artemisinin derivative artemisone as well as other molecules of the same class against Helicobacter pylori and their effects when combined with standard antibiotics were evaluated. Since H. pylori can be internalised into gastric epithelial cells, the effects of artemisinin, dihydroartemisinin and artemisone against intracellular H. pylori were also investigated. Bacteriostatic [minimum inhibitory concentration (MIC)] and bactericidal [minimum bactericidal concentration (MBC)] activities were assessed against 24 clinical strains of H. pylori with different antibiotics susceptibilities. Artemisone showed MIC50 and MIC90 values of 0.25 mg/L and 0.5 mg/L, respectively, and an MBC50 value of 0.5 mg/L. Artemisone was synergistic with amoxicillin in 60% of strains, with clarithromycin in 40% and with metronidazole in 20%. There was no interaction between artemisone and omeprazole or bismuth citrate. Against intracellular H. pylori, only dihydroartemisinin at 2× MIC caused a 1 log10 CFU decrease after 18 h and 24 h of incubation. This is the first demonstration in vitro of the activity of artemisinin derivatives against intracellular H. pylori and indicates that artemisone has the potential to be efficacious for the treatment of H. pylori infection, especially in combination with antibiotics.

Isolation and Characterization of a New Clostridium difficile Ribotype During a Prospective Study in a Hospital in Italy

Clostridium difficile is one of the most important agent of hospital infection with increasing rate of incidence [4]. During a prospective study conducted between 15 March 2011 and 15 March 2012, 142 C. difficile associated disease (CDAD) cases from four internal medicine wards of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico in Milano, were analyzed. The CDAD was confirmed by laboratory tests. After alcolic shock, the isolates were recovered by inoculating a loopful of diarrheic stool samples onto C. difficile agar (CDA; Oxoid). They were identified with the rapid ID 32A system (bioMerieux) and toxins detected by the enzyme-linked immunosorbent assay (C. diff Quik Chek Complete, Alere); toxigenic culture was also performed using the same enzyme-linked immunosorbent assay. The isolates were typed by ribotyping [2]. The set of primer used were 16S and 23S, corresponding to bases 1482–1501 of the 16S ribosomal RNA gene of C. difficile (EMBL accession number AJI32322) and bases 1-24 of the 23S ribosomal RNA gene of C. difficile (EMBL accession number X65602). DNA was extracted from a single C. difficile colony by the use of a Chelex resin-based commercial kit (InstaGene Matrix; Bio-Rad) as recommended by the manufacturer. Amplification reactions were performed in a 50 ll volume containing 1x reaction buffer, 1.5 mM MgCl2, 200 lM of each dNTP (Sigma), 50 pmol of each primer, 1.25 U of Taq polymerase (Sigma),and 5 ll of DNA extract (or distilled water as negative control). After a denaturation of 5 min at 94 C, the DNA was amplified by 35 cycles of 1 min at 94 C, 1 min at 57 C, and 30 s at 72 C. At the end, samples were held at 72 C for 7 min. Amplification products were electrophoresed in a 3 % agarose (SeaKem LE; Lonza) and visualized under UV light following ethidium bromide staining. After electrophoresis, the clustering of banding patterns was checked visually. Each unique pattern was assigned its own ribotype. A sample of each strain with different ribotypes, was sent to Istituto Superiore di Sanità (ISS), in Rome, in order to code them by matching against reference strains present in the only data bank available on line (https:// webribo.ages.at) after capillary-sequencer-based PCR-ribotyping [6]. It was found that one strain, indicated as Pro7388 was not present. Herein, we report only some data about this new strain. The strain was analyzed by PCR for the detection of the tcdA, tcdB, and cdtB genes with oligonucleotides and conditions reported elsewhere [7, 8] using 5 ll of crude DNA extract. These genes code for toxin A, toxin B, and the binding component of binary toxin, respectively. The toxin A and B genes are part of the pathogenicity locus (PaLoc), a 19.6-kb genetic locus F. Sisto (&) M. M. Scaltrito Department of Biomedical, Surgical and Dental Sciences, University of Milan, Via Pascal 36, 20133 Milan, Italy e-mail: francesca.sisto@unimi.it

‘In vitro’ development of metronidazole, erythromycin, amoxicillin and gentamicin resistance in Helicobacter pylori

Serial passage of 37 Helicobacter pylori clinical isolates on increasing concentrations of metronidazole rapidly produced five strains with MICs up to 512 fold higher than those for the original strains. For these five metronidazole-resistant strains the MICs of erythromycin, gentamicin and amoxicillin were unchanged. When they were submitted to the same technique for these last antimicrobial agents, only one strain developed high level resistance to erythromycin and gentamicin having MIC values respectively up to 32 and 64-fold increased. Finally, no amoxicillin-resistant Helicobacter pylori could be obtained.