Innovative scanning electron microscopic investigation in blood samples of patients affected by leukaemia: A physical-chemical-biological approach.

The study proposes an innovative, interdisciplinary observation on blood samples of patients coming from the region Marche (Italy) suffering from acute myeloid leukaemia (AML) by means of a scanning electron microscope coupled with an X-ray microprobe of an Energy Dispersive Spectroscope. A novel protocol of blood preparation was studied and prepared to identify exogenous, potentially toxic foreign bodies coming from an environmental contamination. The results on the four different blood fractions of 38 blood samples (erythrocytes, leukocytes, platelets, and plasma) indicate that the circulating blood of leukaemic patients shows the systematic presence of physical contaminants, with a frequency and concentration which are statistically meaningful as compared to the healthy controls. The chemical compositions of the particles were identified as well as the frequency of the elements they were composed of. The analysis of these chemical compositions demonstrated that these patients had undergone a remarkable environmental, occupational, industrial exposure at some time. A possible pathological mechanism based on a nano-bio-interaction between the internalized environmental particulate matter and the blood components is hypothesized and discussed.

Lactobacillus spp. inhibit the growth of Cronobacter sakazakii ATCC 29544 by altering its membrane integrity.

The effect of the cell-free culture supernatants (CFCSs) from different Lacobacillus spp. on growth ability of Cronobacter sakazakii ATCC 29544 was investigated by time-killing studies. The antimicrobial effect was evaluated using crude and 2.5 × concentrated CFCSs. Most of the CFCSs showed a dose-dependent antimicrobial activity, with the greatest C. sakazakii growth inhibition exerted by the CFCS 2.5 × of Lactobacillus casei rhamnosus ATCC 7469. Indeed, C. sakazakii growth was completely inhibited after 4 h of incubation with the crude CFCSs of L. casei rhamnosus and Lactobacillus acidophilus and after only 2 h using the related 2.5 × CFCSs. The flow cytometric analysis revealed that CFCSs altered the permeability of C. sakazakii cell membrane, showing 55% of live cells after 30 min of treatment with 2.5 × CFCSs of L. casei rhamnosus and L. acidophilus, reaching 1% of live cells after 2 h of exposure. The CFCSs of L. casei rhamnosus and L. acidophilus have showed anti-Cronobacter activity, determining a progressively inhibition of C. sakazakii growth as result of alterations in its membrane permeability.

A dual-species microbial model for studying the dynamics between oral streptococci and periodontal pathogens during biofilm development on titanium surfaces by flow cytometry.

The association of the pioneer organisms Streptococcus mutans ATCC 25175 or Streptococcus oralis ATCC 9811 with secondary colonizers Fusobacterium nucleatum ATCC 25586 or Porphyromonas gingivalis ATCC 33277 during biofilm development on titanium surfaces was evaluated by flow cytometry (FCM) using specific polyclonal antibodies. ELISA and FCM were employed, revealing high antibody sensitivity and specificity. Biofilm formation of four dual-species combinations was analyzed by crystal violet staining, while the association between streptococci and periodontal pathogens was assessed using FCM. Dual-species association between S. oralis and P. gingivalis or F. nucleatum showed a proportional decrease in S. oralis during biofilm development, with a concomitant increase in P. gingivalis or F. nucleatum. This trend was not observed in either of the dual-species associations of S. mutans with the periodontal pathogens. Our dual-species microbial model, which employed FCM, proved to be useful in the study of partnerships between bacteria in oral associations, showing that the presence of primary colonizers is required for the establishment of secondary colonizers in biofilms. The proposed experimental approach is technically simple to prepare and analyze, and also proved to be reproducible; hence, it is well-suited for investigating the development and dynamics of oral communities.

Characterization of biosurfactants produced by Lactobacillus spp. and their activity against oral streptococci biofilm.

Lactic acid bacteria (LAB) can interfere with pathogens through different mechanisms; one is the production of biosurfactants, a group of surface-active molecules, which inhibit the growth of potential pathogens. In the present study, biosurfactants produced by Lactobacillus reuteri DSM 17938, Lactobacillus acidophilus DDS-1, Lactobacillus rhamnosus ATCC 53103, and Lactobacillus paracasei B21060 were dialyzed (1 and 6 kDa) and characterized in term of reduction of surface tension and emulsifying activity. Then, aliquots of the different dialyzed biosurfactants were added to Streptococcus mutans ATCC 25175 and Streptococcus oralis ATCC 9811 in the culture medium during the formation of biofilm on titanium surface and the efficacy was determined by agar plate count, biomass analyses, and flow cytometry. Dialyzed biosurfactants showed abilities to reduce surface tension and to emulsifying paraffin oil. Moreover, they significantly inhibited the adhesion and biofilm formation on titanium surface of S. mutans and S. oralis in a dose-dependent way, as demonstrated by the remarkable decrease of cfu/ml values and biomass production. The antimicrobial properties observed for dialyzed biosurfactants produced by the tested lactobacilli opens future prospects for their use against microorganisms responsible of oral diseases.

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.

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

BACKGROUND: 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). METHODS: The presence of resistance genes was investigated by PCR in 10 strains of E. coli isolated from Foglia River. Bacterial responses to different antibiotics were also tested with FCM techniques by evaluating both the degree of decrease in viability and the light scatter changes in all of the strains. RESULTS: PCR revealed that only one strain exhibits the presence of one resistance gene. Despite this, analyses of strains using FCM evidenced the presence of viable subpopulations after antibiotic treatment. Furthermore, analyses of scatter signals revealed profound changes in the Forward Scatter and Side Scatter of the bacterial populations as a consequence of antibiotic exposure, confirming the viability and membrane potential data. The riverine strains were in general less sensitive to antibiotics than the reference strain (ATCC 25922). CONCLUSIONS: Antibiotic resistance is a widespread phenomena. The multiparametric approach based on FCM used in this study, providing results about different aspects (cell viability, membrane potential, light scatter changes), may overcome the limitation of PCR and could represent an adequate method for the evaluation of bacteria responses to antibiotic exposure.

Role of daptomycin in the induction and persistence of the viable but non-culturable state of Staphylococcus aureus biofilms.

We have recently demonstrated that antibiotic pressure can induce the viable but non-culturable (VBNC) state in Staphylococcus aureus biofilms. Since dormant bacterial cells can undermine anti-infective therapy, a greater understanding of the role of antibiotics of last resort, including daptomycin, is crucial. Methicillin-resistant S. aureus 10850 biofilms were maintained on non-nutrient (NN) agar in the presence or absence of the MIC of daptomycin until loss of culturability. Viable cells were monitored by epifluorescence microscopy and flow cytometry for 150 days. All biofilms reached non-culturability at 40 days and showed a similar amount of viable cells; however, in biofilms exposed to daptomycin, their number remained unchanged throughout the experiment, whereas in those maintained on NN agar alone, no viable cells were detected after 150 days. Gene expression assays showed that after achievement of non-culturability, 16S rDNA and mecA were expressed by all biofilms, whereas glt expression was found only in daptomycin-exposed biofilms. Our findings suggest that low daptomycin concentrations, such as those that are likely to obtain within biofilms, can influence the viability and gene expression of non-culturable S. aureus cells. Resuscitation experiments are needed to establish the VBNC state of daptomycin-exposed biofilms.

A multi-approach study of influence of growth temperature and nutrient deprivation in a strain of Aeromonas hydrophila.

In the present study we investigated the behavior of an Aeromonas hydrophila strain in prolonged nutrient deprivation condition analyzing the possible link among survival, cell morphology and adhesive characteristics and correlating them with the expression of the 43kDa outer membrane protein (OMP). The strain was inoculated in mineral and drinking chlorinated water, and in Nutrient Broth as a control with incubation at 4 and 24°C for 176days. Specimens were analyzed at different times during starvation stress. Viability was assessed by flow cytometry and growth by plate count technique; morphology and adhesivity were detected by optical and electron microscopy. The 43kDa OMP expression at different times was determined after immunoblotting assay using a polyclonal antibody produced in rabbit. The results showed a long-term viability as evidenced by cytofluorimetric analysis; however, the prolonged starvation led to the shift from the normal rod shaped cells to spherical forms in the last phases of incubation especially at 24°C. Concomitantly with the appearance of spherical cells we noted a reduction of the 43kDa OMP content and adhesive ability. Therefore, our results suggest a role of the 43kDa OMP as adhesin in A. hydrophila. In conclusion, we demonstrated that the bacterium can long survive under stress conditions, however adopting strategies which can lead to a loss of some cell surface components involved in the interactions with eukaryotic cells, therefore modifying its virulence properties.

In vitro activity of Carvacrol against titanium-adherent oral biofilms and planktonic cultures.

OBJECTIVE: The aim of this study was to test the effect of Carvacrol against oral pathogens and their preformed biofilms on titanium disc surface. METHODS: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and biofilm inhibitory concentration (BIC) were performed to evaluate Carvacrol antibacterial activity, while flow cytometry (FCM) was used to verify the Carvacrol effect on esterase activity and membrane permeability. Carvacrol was tested in vitro on single- and multi-species biofilms formed on titanium disc by Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277 or Fusobacterium nucleatum ATCC 25586, in different combinations, comparing its effect to that of chlorhexidine. RESULTS: The pathogens were sensitive to Carvacrol with MICs and MBCs values of 0.25 % and 0.50 % and BICs of 0.5 % for S. mutans ATCC 25175 and 1 % for P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586. FCM analysis showed that treatment of planktonic cultures with Carvacrol caused an increase of damaged cells and a decrement of bacteria with active esterase activity. Moreover, Carvacrol demonstrated greater biofilm formation preventive property compared to chlorhexidine against titanium-adherent single- and multi-specie biofilms, with statistically significant values. CONCLUSIONS: Carvacrol showed inhibitory activity against the tested oral pathogens and biofilm formation preventive property on their oral biofilm; then, it could be utilized to control and prevent the colonization of microorganisms with particular significance in human oral diseases. CLINICAL RELEVANCE: This natural compound may be proposed in daily hygiene formulations or as an alternative agent supporting traditional antimicrobial protocols to prevent periodontal diseases in implanted patients.

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

BACKGROUND: In this study, we check for the presence of specific resistance genes by PCR and then we used flow cytometry to evaluate antibiotic-induced effects in different strains of Escherichia coli. METHODS: The presence of resistance genes was investigated by PCR in 10 strains of Escherichia coli isolated from Foglia River. Bacterial responses to different antibiotics were also tested with flow cytometry techniques by evaluating both the degree of decrease in viability and the light scatter changes in all of the strains. RESULTS: PCR revealed that only one strain exhibits the presence of one resistance gene. Despite this, analyses of strains using flow cytometry evidenced the presence of viable subpopulations after antibiotic treatment. Furthermore, analyses of scatter signals revealed profound changes in the Forward Scatter (FSC) and Side Scatter (SSC) of the bacterial populations as a consequence of antibiotic exposure, confirming the viability and membrane potential data. The riverine strains were in general less sensitive to antibiotics than the reference strain (ATCC 25922). CONCLUSIONS: Antibiotic resistance is a widespread phenomena. The multiparametric approach based on flow cytometry used in this study, providing results about different aspects (cell viability, membrane potential, light scatter changes), may overcome the limitation of PCR and could represent an adequate method for the evaluation of bacteria responses to antibiotic exposure. This article is protected by copyright. All rights reserved.

Effects of landfill leachate treatment on hepatopancreas of Armadillidium vulgare (Crustacea, Isopoda).

The major environmental impact of landfills is emission of pollutants via the leachate and gas pathways. The hepatopancreas of the terrestrial isopod Armadillidium vulgare (Isopoda, Crustacea, Latreille 1804) plays an important role in the bioaccumulation of contaminants, such as heavy metals. To evaluate the effects of landfill leachate treatment, 2 different approaches were applied: 1) the detection of accumulation of trace elements (As, Cd, Cr, Cu, Sb, Zn, Pb, Ni, V) in hepatopancreatic cells, and 2) the evaluation of biological effect of contaminants on fresh hepatopancreatic cells by flow-cytometric analyses. The presence of 2 different cell types (herein referred to as "small" [S] cells and "big" [B] cells, in agreement with the literature based on morphological examinations) was detected for the first time by flow cytometry, which also highlighted their different response to stress stimuli. In particular, B cells appeared more sensitive to landfill leachate treatment, being more damaged in the short term, while S cells seemed more adaptive. Furthermore, S cells could represent a pool from which they are able to differentiate into B cells. These findings were also confirmed by principal component analyses, underlining that S SYBR Green I bright cells correlate with specific chemicals (Ca, Cu, Co), confirming their resistance to stress stimuli, and suggesting that the decrease of specific cell types may prime other elements to replace them in a homeostasis-preservation framework.

Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification.

Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.

Detection of environmental Vibrio parahaemolyticus using a polyclonal antibody by flow cytometry.

The aim of this study was to detect and quantify Vibrio parahaemolyticus using flow cytometry (FCM) in combination with a polyclonal antibody developed in our laboratory. Experiments were carried out using V. parahaemolyticus cells in pure and mixed bacteria culture suspensions in either artificial or natural seawater. Using FCM, V. parahaemolyticus cells labelled with the polyclonal antibody and a secondary fluorescein isothiocyanate-conjugated antibody were detected and rapidly quantified at low cell densities (10(3) cells ml(-1) ) in both the pure and mixed cultures. To determine the specificity of our antibody, its cross-reactivity with other ATCC bacterial strains and some environmental Vibrio spp. and Gram-positive isolates was also assessed. Significant immunoreactivity levels above background were obtained for V. harvey 64, V. parahaemolyticus 704 and V. alginolyticus 1407, although the intensities were significantly less than for V. parahaemolyticus Conero. The experiments carried out in natural seawater confirmed the antibody specificity towards V. parahaemolyticus Conero even if a lower proportion of labelled cells was observed. The application of FCM in combination with a primary polyclonal antibody appears to be a promising technique for the detection and quantification of V. parahaemolyticus cells in aquatic environments.

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.

Bacterial cell monitoring in wastewater treatment plants by flow cytometry.

The activated sludge process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment, in which bacteria constitute the majority and represent the main microorganisms responsible for the degradation process in a plant. In this work, we monitored bacterial charge in different wastewater treatment plants by flow cytometry, also evaluating chlorination effects on bacterial viability, both by flow cytometry and traditional plate counts. Maximum values of bacterial charge were registered in the aeration tank of all plants monitored. Cell viability did not show significant differences (p > 0.05) in samples collected in "before chlorination" and "wastewater effluent" treatment steps; this suggests that the chlorination was not able to decrease total viable bacterial charge. In this work, we discuss the need to improve microbiological analyses, both in terms of measuring other potential pathogens and of using new methodological approaches in the traditional evaluation of the microbiological quality of effluents.

State transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow cytometry.

BACKGROUND: Vibrio parahaemolyticus, in response to environmental conditions, may be present in a viable but nonculturable state (VBNC), which can still be responsible for cases of infectious diseases in humans. METHODS: The characterization of the cellular states of V. parahaemolyticus during entry into, persistence in, and resuscitation from the VBNC state, was assessed through plate culture method and epifluorescence microscope evaluation of actively respiring cells. Flow cytometry (FCM) in combination with SYBR Green I (SG) and propidium iodide allowed us to distinguish between viable, dead, and damaged-cells. Immunofluorescence labeling detected by FCM was used to study changes in antibody affinity. RESULTS: Two groups of bacteria, one with High Nucleic Acid (HNA) and one having Low Nucleic Acid (LNA) content, were differentiated using SG and FCM and each was correlated with cell viability. With the aging of the microcosm, the LNA bacteria population increased while the HNA population gradually disappeared. Cytofluorimetric immunofluorescence analyses showed that the bacterial cell levels dropped from 95% at day 0 to 40% at day 26 and by day 29, antibody affinity was virtually lost. FCM analyses of light scatter signals expressed by cell population highlighted morphological changes indicating a reduction in cell size, as also shown by scanning electron microscopy images and variations in cell structure. CONCLUSIONS: The methodology used has provided useful data in relation to the state transitions of V. parahaemolyticus regarding cell viability, antigenic surface components, and the quantification of morphological variations during its entry into the VBNC state.

Comparison of disruption procedures for enumeration of activated sludge floc bacteria by flow cytometry.

BACKGROUND: In a wastewater treatment plant, the degradation process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment. The activated-sludge process is based on the formation of strong microbial flocs where many bacteria are attached to sludge flocs. METHODS: Cytometric analysis requires an homogeneous cell suspension and so detachment of bacteria from flocs is required. In this study, sonication and homogenization were compared to find the most adequate pretreatment method for bacterial cytometric analysis in activated sludge samples. Bacterial viability was tested with a nucleic acid double-staining (NADS) protocol (Barbesti et al., Cytometry 2000;40:214-218) and on flow cytometry. RESULTS: Each method showed a good efficiency in terms of bacterial detachment; thus finally, the choice of which could be the best treatment method was based on both viability results and analysis rapidity. On the basis of the degree of cell detachment and viability, the maximum value was obtained by sonication (2 x 45''). CONCLUSIONS: The use of flow cytometry in conjunction with fluorescent dyes and an adequate pretreatment represents a useful method to rapidly detect and enumerate bacteria in activated sludge samples.