Giulia Amagliani

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.

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.

Microbiological Surveillance of a Bovine Raw Milk Farm Through Multiplex Real-Time PCR

Raw milk is increasingly appreciated by consumers but can be contaminated by a variety of zoonotic pathogens. Therefore, preventive measures, such as on-farm hazard analysis critical control point (HACCP) programs, must be applied to protect consumers. The aim of the present study was the comparison of a multiplex real-time polymerase chain reaction (PCR) assay with a culture-based approach in an on-farm quality assurance program for the detection of Escherichia coli O157, Salmonella spp., and Listeria monocytogenes in bulk tank milk, in-line milk filters, manure, and feces. Results revealed that the real-time PCR was more sensitive in detecting E. coli O157 than the culture method in filters (48% vs. 4% positive), manure (93% vs. 7% positive) and feces (60% vs. 4% positive). The two methods were equally efficient in detecting L. monocytogenes (8% of filters), while Salmonella spp. was not detected in any sample. In conclusion, the real-time PCR, by reducing analysis time to two working days, can be proposed as a useful tool in the raw milk primary production setting as a rapid and user-friendly screening method.

Development of a multiplex PCR assay for Photobacterium damselae subsp. piscicida identification in fish samples.

A multiplex polymerase chain reaction protocol for the detection of Photobacterium damselae and subspecies piscicida and damselae discrimination, with internal amplification control, was developed. Assay specificity was assessed by testing 19 target and 25 non-target pure cultures. The detection limit was 500 fg, corresponding to 100 genome equivalents. The optimized protocol was also prevalidated with spleen, kidney and blood samples from infected and uninfected sea bass, without any culture step, and it can be proposed as a valid alternative to culture standard methods for the rapid and specific diagnosis of photobacteriosis in fish.

High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein.

Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene. After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L. monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli. Thanks to the optimization of the induction protocol, we achieved a high-level LLO synthesis (about 10% of total cell proteins) in hemolytically active form. The expressed hemolysin was then purified to homogeneity, as revealed by SDS-PAGE and Western blot analysis, by a hydroxyapatite adsorption chromatography, followed by an SP Sepharose ion-exchange chromatography. The recombinant protein showed the same properties determined for LLO purified from L. monocytogenes cultures and the characteristics of the sulfhydryl-activated toxins such as inactivation by oxidation and by reaction with cholesterol. By a combination of the pET expression system and the simple purification method, we obtained a significant amount of toxin (4.5 mg/litre cell culture) in a hemolytically active form (1.25 x 10(6)HU/mg protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures, which is a difficult task, mainly owing to the low levels of toxin released in the culture media. The recombinant hemolysin, purified in sufficient quantities, could be very useful for structural studies and for diagnostic and pharmaceutical applications.

Isolation of a novel gene from Photobacterium damselae subsp. piscicida and analysis of the recombinant antigen as promising vaccine candidate

Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 μg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

Antifungal activity of Brassica oleracea var. botrytis fresh aqueous juice

The antifungal activity of fresh, aqueous Brassica oleracea var. botrytis juice against Candida albicans and other pathogenic fungi was investigated. The juice was found to be effective both in inhibiting the growth of blastoconidia and reducing the appearance of C. albicans germ tubes. Furthermore, the juice inhibited the growth of some pathogenic, filamentous fungi.