Luigia Sabatini

Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: A comparative study

The distribution of virulence properties in 142 strains of Aeromonas isolated from diarrhoeic patients, food and surface water in Italy and identified by biochemical and molecular methods was investigated. The virulence properties studied were the presence of genes for the aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act); and cytotoxicity for Vero cells and adhesion on Hep-2 cells. A. hydrophila and A. caviae were the species most commonly isolated from clinical and environmental samples (9/30; 30.0% and 5/27; 18.5%, respectively) while mesophilic A. salmonicida was most common in food samples (19/80; 23.7%). Out of 142 strains, 86 (60.6%) were positive for at least one of the virulence properties. All the toxin genes were present in 4/18 (22.3%) of clinical strains. Most of the food isolates (54/55; 98.2%) were cytotoxic and most of the environmental strains (12/13; 92.3%) were adhesive. The aerA gene was present in most toxigenic strains (72/86; 83.7%), irrespective of their origin. The growth temperature affected the expression of cytotoxicity and adhesivity. Aeromonas strains from food and surface water frequently had toxin gene patterns similar to those of clinical strains and expressed virulence properties at human body temperature. These findings indicate that aeromonads have the potential to cause human illness and confirm the role of food and water as vehicles for Aeromonas diseases.

Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

ABSTRACT The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.

Faecal contamination indicators, salmonella, vibrio and aeromonas in water used for the irrigation of agricultural products

The faecal contamination indicators (total coliforms, faecal coliforms, Escherichia coli, enterococci) and the genera Salmonella, Vibrio, Aeromonas were investigated in water samples used for irrigation. During 4 months, 52 samples were taken. The methods used were: multiple tube fermentation method for faecal contamination indicators and membrane filtration techniques for salmonella, aeromonas and vibrio. Two samples were positive for Salmonella spp., fourteen for Aeromonas spp. and no samples for Vibrio spp. No correlation was found between aeromonas and the indicators of faecal contamination. Regarding Aeromonas spp., 21.6% of the strains were adhesive and 12.6% cytotoxic: this confirms the possible role of aeromonas in human pathologies. These results are important to determine the quality of irrigation water in relation to human health. In fact, the spray or sprinkler irrigation produces bioaerosol, which can contaminate the crops that are likely to be eaten uncooked. In addition, the flood or furrow irrigation represents a risk to field workers.

Microbial characteristics of marine sediments in bathing area along Pesaro-Gabicce coast (Italy): a preliminary study

Aims:  This paper evaluates the presence of human pathogen micro‐organisms in marine sediments in a coastal area suitable for bathing. In addition, the grain size analysis was performed in order to correlate the characteristics of the sediments and the microbial content.

Determination of viability of Aeromonas hydrophila in increasing concentrations of sodium chloride at different temperatures by flow cytometry and plate count technique

Aeromonads in waters and foods can represent a risk to human health. Factors such as sodium chloride concentration and temperature can affect growth and viability of several food and water-borne pathogens. The behaviour of an Aeromonas hydrophila strain in the presence of 1.7%, 3.4% and 6% NaCl concentrations at 24 degrees C and 4 degrees C was studied over a 188 day period. Viability and membrane potential were assessed by flow cytometry; growth was evaluated by plate count technique. Flow cytometry evidenced that A. hydrophila retained viability over the period although varying according to temperature and salt concentrations. Colony Forming Units were generally lower in number than viable cells especially in the presence of 6% NaCl, indicating the occurrence of stressed cells which maintain metabolic activity yet are not able to grow on agar plates. In conclusion, A. hydrophila showed a long-term halotolerance even at elevated (6%) NaCl concentrations and a lesser sensitivity to salt at low temperature; therefore, low temperature and salt, which are two important factors limiting bacterial growth, do not assure safety in the case of high initial contamination. Finally, cytometry appears a valid tool for the rapid detection of the viability of pathogenic bacteria in food and environmental matrices to control and prevent health risks.

Changes in adhesion ability of Aeromonas hydrophila during long exposure to salt stress conditions

Stressful environmental conditions influence both bacterial growth and expression of virulence factors. In the present study, we evaluated the influence of NaCl on Aeromonas hydrophila adhesiveness at two temperatures. This agent is often involved in clinical cases; however, its pathogenic potential is still not fully understood.

Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms

Legionnaires’ disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems in order to minimize legionellosis outbreaks.

Role of Biofilm in Protection of the Replicative Form of Legionella pneumophila

The dual nature of Legionella pneumophila enables its survival in free and intracellular environments and underpins its infection and spread mechanisms. Experiments using bacterial cultures and improved RTqPCR protocols were devised to gain fresh insights into the role of biofilm in protecting the replicative form of L. pneumophila. mip gene expression was used as a marker of virulence in sessile (biofilm-bound) and planktonic (free-floating) cells of L. pneumophila serotype 1 ATCC 33152. The ratio of mip gene expression to transcriptionally active Legionella cells increased both in sessile and free-floating cells demonstrating an up-regulation of mip gene under nutrient depletion. However, a different trend was observed between the two forms, in planktonic cells the mip gene expression/transcriptionally active Legionella cells increased until the end of the experiment, while in the biofilm such increase was observed at the end of the experiment. These findings suggest a possible association between the switch to the transmissive phase of Legionella and a mip up-regulation and a role for biofilm in preserving Legionella cells in replicative form. Moreover, it has been shown that improved RTqPCR protocols are valuable tools to explore bacterial virulence.

Evaluation of Escherichia coli viability by flow cytometry: A method for determining bacterial responses to antibiotic exposure.

In this study, we check for the presence of specific resistance genes by polymerase chain reaction (PCR) and then we used flow cytometry (FCM) to evaluate antibiotic‐induced effects in different strains of Escherichia coli (E. coli).

Evaluation of fungal community involved in the bioderioration process of wooden artworks and canvases in Montefeltro area (Marche, Italy)

Microbiological monitoring represents one of the most useful methods to assess potential risks related to the integrity of cultural heritage. The objective of this work was to evaluate the fungal community prevalent in 64 different artworks of Montefeltro area (Marche, central Italy). A total of 293 swabs were collected and, among these, 2.3% resulted negative to cultural method, while 87.7% were positive for the presence of filamentous fungi, yeasts and Actinomycetes. Totally, 11 genera and 12 different species were recovered from Sabouraud Dextrose Agar (SDA), Czapek Dox Agar (CDA), Malt Extract Agar (MEA), including 73 strains of Mycelia sterilia. Penicillium spp. was isolated mostly from canvases (28.8%), while Aspergillus spp. was most present in wooden artworks (25.3%). SDA was the best performing medium with 57.1% of isolations, followed by CDA and MEA with percentages of 24.8 and 18.1% respectively. This study could be useful to better understand the microorganism-related phenomena in cultural heritage of Marche region, identifying the potential risks and defining preventive protecting such as climate control, frequent cleaning and environmental monitoring.