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.

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.

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.

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.

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.

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.