Comparative effect of chlorhexidine and some mouthrinses on bacterial biofilm formation on titanium surface.

The aim of the present study was to evaluate the effectiveness of chlorhexidine digluconate (CHX) and commonly used mouthrinses to single- and poly-species biofilms by S. mutans, S. aureus and P. aeruginosa, on titanium discs of grade IV. The formation of single- and poly-species biofilms at 16.5, 40.5 and 64.5-h incubation on titanium surface was evaluated by plate count (CFU ml⁻¹) before and after exposure to CHX and four mouthrinses (Curasept, Listerine, Meridol and Buccagel) and expressed as percentage of Inhibitory Activity (IA%). The application of the different anti-plaque formulations on biofilm can reduce the adhesion of bacteria to titanium surface with different degrees. The higher efficacy was observed for Listerine that shows IA% = 100 on the biofilm formed by S. mutans at 16.5 h. Log count of CFU was dependent to culture time and four mouthrinses for S. mutans and S. aureus, whilst was not dependent to culture time but to mouthrinses for P. aeruginosa. In general, the efficacy was particularly lesser to poly-species biofilms; no statistical differences were evidenced between all the mouthrinses and CHX as control group. The tested mouthrinses, compared to reference CHX 0.2%, have demonstrated a significant lower antibacterial activity than Listerine towards the experimental biofilms. This "in vitro" biofilm model should prove extremely useful for pre-clinical testing of anti-plaque agents, which inhibit biofilm formation, can prevent subsequent implant failure.

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.

Virion stability is important for the circulative transmission of tomato yellow leaf curl sardinia virus by Bemisia tabaci, but virion access to salivary glands does not guarantee transmissibility.

The capsid protein (CP) of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV), family Geminiviridae, is indispensable for plant infection and vector transmission. A region between amino acids 129 and 152 is critical for virion assembly and insect transmissibility. Two previously described mutants, one with a double Q129P Q134H mutation (PNHD) and another with a further D152E change (PNHE), were found nontransmissible (NT). Another NT mutant with a single N130D change (QDQD) was retrieved from a new mutational analysis. In this study, these three NT mutants and the wild-type (wt) virus were compared in their relationships with the whitefly vector Bemisia tabaci and the nonvector Trialeurodes vaporariorum. Retention kinetics of NT mutants were analyzed by quantitative dot blot hybridization in whiteflies fed on infected plants. The QDQD mutant, whose virions appeared nongeminate following purification, was hardly detectable in either whitefly species at any sampling time. The PNHD mutant was acquired and circulated in both whitefly species for up to 10 days, like the wt virus, while PNHE circulated in B. tabaci only. Using immunogold labeling, both PNHD and PNHE CPs were detected in B. tabaci salivary glands (SGs) like the wt virus, while no labeling was found in any whitefly tissue with the QDQD mutant. Significant inhibition of transmission of the wt virus was observed after prior feeding of the insects on plants infected with the PNHE mutant, but not on plants infected with the other mutants. Virion stability and ability to cross the SG barrier are necessary for TYLCSV transmission, but interactions with molecular components inside the SGs are also critical for transmissibility.

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.

Evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death.

Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 microg ml(-1) of propidium iodide, simultaneous to a 10x concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.

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.

Effects of tumour necrosis factor alpha (TNFalpha) on Mytilus haemocytes: role of stress-activated mitogen-activated protein kinases (MAPKs).

BACKGROUND INFORMATION: Many studies indicate that innate immunity in invertebrates can be modulated by a cytokine network like in vertebrates. In molluscs, the immune response is carried out by circulating haemocytes and soluble haemolymph factors. In the present study, the effects of heterologous TNFalpha (tumour necrosis factor alpha) on cell signalling and function in the haemocytes of the bivalve Mytilus galloprovincialis Lam. were investigated. RESULTS AND CONCLUSIONS: Addition of TNFalpha in the absence of haemolymph serum [in ASW (artificial sea water)] induced cellular stress, as indicated by lysosomal destabilization, and decreased phagocytosis; on the other hand, in the presence of serum, TNFalpha did not affect lysosomal stability and even stimulated phagocytosis. TNFalpha induced rapid phosphorylation of the stress-activated p38 and JNK (c-Jun N-terminal kinase) MAPKs (mitogen-activated protein kinases); both effects were persistent in ASW but transient in serum. Activation of p38 and JNKs in mediating the effects of TNFalpha was confirmed by the use of specific MAPK inhibitors. Moreover, flow cytometric analysis indicated that TNFalpha in the presence of serum induced transient phosphatidylserine exposure on the haemocyte surface, evaluated as annexin V binding; in ASW, the cytokine resulted in a stable increase in the percentage of both annexin- and propidium iodide-positive cells, indicating possible apoptotic/necrotic processes. The results indicate that TNFalpha can affect the function of bivalve haemocytes through conserved transduction pathways involving stress-activated MAPKs and suggest that the haemocyte response to the cytokine is influenced by soluble haemolymph components.

Determination of the viability of Aeromonas hydrophila in different types of water by flow cytometry, and comparison with classical methods.

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.