Giorgio Brandi

A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk

Intoxications and infections caused by food-borne pathogens represent an increasing public health problem, and diagnostic tests in multiplex format are needed for the rapid identification of food contaminations caused by more than one microbial species. We have developed a multiple PCR-based platform for the simultaneous detection of the widespread milk-associated pathogens Salmonella spp., Listeria monocytogenes and Escherichia coli O157. The assay combines an enrichment step in a medium properly formulated for the simultaneous growth of target pathogens, a DNA isolation method, and a multiplex Real-Time PCR detection system based either on dual-labelled probes (mRT-PCR), or on melting curve analysis (mHRM). The second, producing a distinct peak for each amplification product, allows the qualitative assessment of pathogen presence. Moreover, the internal amplification control (IAC) included in the reaction, ensuring the reliability of results, complies with quality management programmes. Inclusivity and exclusivity were 100% each, with a detection limit of 1 CFU for each pathogen in a total of five 25 ml-aliquots of raw milk, and a duration of two working days. The assay represents an alternative approach for the qualitative detection of the cited bacterial species, suitable for a relatively inexpensive screening of several milk samples, reducing the turnaround time and the workload.

Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples.

Aims:  A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out.

Evaluation of the environmental impact of microbial aerosols generated by wastewater treatment plants utilizing different aeration systems

Using three sampler devices (SAS, Andersen Six‐Stages and All Glass Impinger), the environmental impact of bacterial and fungal aerosols generated by municipal wastewater treatment plants operating with different methods of sludge oxygenation were evaluated. The highest microbial concentrations were recovered above the tanks (2247 cfu m−3) and in downwind positions (1425 cfu m−3), where a linear correlation (P < 0·05) was found between the quantity of sewage treated and the entities of microbial aerosol dispersion. Moreover, an exponential increase (P < 0·05) in the bacteria recovered from the air occurred at increasing times of treatment. However, after long‐term plant operation, high bacterial and fungal concentrations were found in almost all of the sites around the plant. Coliforms, enterococci, Escherichia coli and staphylococci were almost always recovered in downwind positions. Considerable fractions (20–40%) of sampled bacteria were able to penetrate the final stages of the Andersen apparatus and thus, are likely to be able to penetrate the lungs. The plant operating with a fine bubble diffused air system instead was found to generate rather low concentrations of bacteria and fungi; moreover, staphylococci and indicator micro‐organisms were almost absent. Finally, salmonellae, Shigella, Pseudomonas aeruginosa and Aeromonas spp. were not detected in either of the plants. The results indicate a remarkable dispersion of airborne bacteria and fungi from tanks in which oxygen is supplied via a mechanical agitation of sludge, and suggest the need to convert them to diffused aeration systems which pose a lesser hazard for human health.

Activity of Brassica oleracea Leaf Juice on Foodborne Pathogenic Bacteria

Many vegetables of the Cruciferae family have been found to possess antimicrobial properties against several microorganisms of clinical importance. In this study, we reported the antibacterial effect of Brassica oleracea juice on several food-borne pathogens. The juice was found to be effective in inhibiting the growth of Salmonella Enteritidis, verotoxigenic Escherichia coli O157:H7, E. coli HB producing thermolabile toxin, nontoxigenic E. coli, and Listeria monocytogenes, but not Enterococcus faecalis. All cauliflower cultivars tested suppressed bacterial growth in a dose-dependent manner after 5 h of treatments, and the reduction in the number of viable cells ranged from 1 log with a 10% juice concentration to more than 3 log with a 20% juice concentration. The foodborne bacteria tested were also markedly reduced by isothiocyanates, natural components abundant in the genus Brassica, indicating that glucosinolate-derived isothiocyanates can play a major role in the antimicrobial activity of cauliflower. The antimicrobial effect of juice was reduced in presence of cysteine, suggesting that one mechanism of action of the juice involves blocking bacterial sulfhydryl groups.

Macrophage protection by addition of glutathione (GSH)-loaded erythrocytes to AZT and DDI in a murine AIDS model

Monocyte-macrophages play a central role in HIV-1 infection because they are among the first cells to be infected and because later they are important reservoirs for the virus. Thus, newly designed therapies should take into account the protection of this cell compartment. Herein, we report the results obtained in a murine AIDS model, by the addition to AZT+DDI of a system (GSH-loaded erythrocytes) able to protect macrophages against HIV-1 infection. Five groups of LP-BM5-infected mice were treated as follows: one group was treated by AZT, one group was treated by DDI, one group was treated by the combination of both, another by GSH-loaded erythrocytes, and finally, one by the combination of all three. After 10 weeks of infection the parameters of the disease were studied and the proviral DNA content in different organs and in macrophages of bone marrow and of the peritoneal cavity was quantified. The results obtained show that mice treated with AZT+DDI+GSH-loaded erythrocytes showed proviral DNA content in the brain and in macrophages of bone marrow that was significantly lower than in mice treated with AZT+DDI. This study may help developing strategies aimed at blocking HIV-1 replication in its reservoirs in the body.

Swimming pools and fungi: An environmental epidemiology survey in Italian indoor swimming facilities

Abstract A growing number of people attend swimming facilities for recreational activities, rehabilitative treatments, or sport. Filamentous fungi and yeast can be isolated from contaminated air, water and surfaces and may represent a biological risk for employees and users. Here we investigated the occurrence of mycotic species, in a sample of Italian swimming pools (n = 10). Detection and identification of isolated species were achieved by cultural and morphological methods. Results revealed moderate mycotic titres and a high biodiversity. Penicillium spp., Aspergillus spp., Cladosporium spp. and Alternaria sp., were constantly detected in air and surfaces sampled by the swimming area, while pathogenic yeast Candida albicans was never detected. Fusarium spp. was the most common taxon isolated from surfaces. For one facility, we typed the genotypic profiles and studied, by genetic typing, the spatial and temporal distribution of isolates. Phylogenetic relationships between species were analysed by alignment of small ribosomal subunit RNA sequences.

A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood

Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1-10(3)cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1 cfu/g, in enriched samples, and higher sensitivity (10(2)-10(3)cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.

Induction of Endoplasmic Reticulum Stress Response by the Indole-3-Carbinol Cyclic Tetrameric Derivative CTet in Human Breast Cancer Cell Lines

Background Indole-3-carbinol and its metabolic products are considered promising chemopreventive and anticancer agents. Previously we have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In the present study, we further characterize the autophagic response and investigate the mechanism through which CTet regulates these events. Methodology/Principal Findings Analysis of gene expression microarray data and subsequent confirmation by quantitative real-time PCR, showed that CTet is able to induce up-regulation of key signaling molecules involved in endoplasmic reticulum (ER) stress response (e.g. DDIT3/CHOP, CHAC1, ATF3, HSPA5/BiP/GRP78, CEBPB, ASNS) and autophagy (e.g. MAP1LC3B), in both MCF-7 and MDA-MB-231 cell lines. Moreover, the monitoring of Xbp-1 splicing confirmed the activation of IRE1/Xbp-1 ER stress response branch after CTet treatment. The role of autophagic processes (known to be induced by ER stress) was investigated further through ATG5 gene silencing and pharmacological inhibition of AVOs formation. CTet was shown to induce an autophagy-related cell death. Moreover, CTet-treated cells stained with Hoechst/PI revealed the presence of necrotic processes without evidence of apoptosis. Conclusions/Significance The ER stress response was identified as the main upstream molecular mechanism through which CTet acts in both hormone-responsive and triple-negative breast cancer cells. Because of its important role in cancer development, ER stress is a potential target in cancer therapy. The abiltiy of CTet to induce ER stress response and subsequently activate a death program in tumor cells confirms this molecule as a promising anticancer agent.

Heterodimer-Loaded Erythrocytes as Bioreactors for Slow Delivery of the Antiviral Drug Azidothymidine and the Antimycobacterial Drug Ethambutol

Disseminated infection with Mycobacterium avium complex (MAC) remains the most common serious bacterial infection in patients with advanced AIDS. The organisms that make up this complex are found ubiquitously in the environment, yet rarely cause disseminated disease in nonimmunocompromised human patients; on the contrary, up to 50% of patients with AIDS may ultimately develop the pathology. Hence, therapeutic strategies able to inhibit HIV and Mycobacterium replication are needed. Because of the rapid plasma elimination and toxicity of the most commonly used drugs, daily multiple-drug therapies must often be continued throughout life, frequently causing major side effects and, as a consequence, poor patient compliance. Therefore, alternative strategies that reduce the toxicity of the drugs and allow prolonged application intervals are sorely needed. Since erythrocytes (RBCs) can behave as bioreactors able to convert impermeant prodrugs to membrane-releasable active drugs, new compounds (AZTpEMB, AZTpEMBpAZT, and AZTp2EMB) consisting of both an antiretroviral and an antimicrobial drug were designed and synthesized. Among these, only AZTp2EMB was hydrolyzed by erythrocyte enzymes and could be encapsulated inside RBCs. AZTp2EMB-loaded RBCs slowly released AZT and EMB in culture medium, reducing its concentration by one-half about every 48 hr of incubation at 37 degrees C. Moreover, when AZTp2EMB-loaded erythrocytes were incubated for 6 days in the presence of human macrophages infected with Mycobacterium avium (M. avium) a marked bactericidal effect (>1 log) was observed. Thus, AZTp2EMB-loaded erythrocytes could be used as endogenous bioreactors for AZT and EMB delivery in the treatment of HIV and M. avium infection.

Bactericidal effect of chlorine on motile Aeromonas spp. in drinking water supplies and influence of temperature on disinfection efficacy

The susceptibility of toxigenic Aeromonas spp. to free chlorine in drinking water supplies, and the influence of environmental temperature on the bactericidal activity of the oxidant, were evaluated. The results showed inactivation curves characterized by an initial phase of rapid reduction of viable cells followed by a slow inactivation of bacteria. The effect of the chlorine compound was markedly influenced by water temperature. At a summer water temperature (20 °C), the efficacy of the chlorine concentrations tested was found to be two to three times lower compared to that found at a winter temperature (5 °C). Resistance was moderately, but significantly, greater in Aer. hydrophilavsAer. caviae and Aer. sobria, but all Aeromonas spp. were more susceptible than Escherichia coli. Selective pressure with free chlorine did not produce Aeromonas cells with higher levels of chlorine resistance.