SYBR green as a fluorescent probe to evaluate the biofilm physiological state of Staphylococcus epidermidis, using flow cytometry

2011 ◽  
Vol 57 (10) ◽  
pp. 850-856 ◽  
Author(s):  
Filipe Cerca ◽  
Gabriela Trigo ◽  
Alexandra Correia ◽  
Nuno Cerca ◽  
Joana Azeredo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Staphylococcus Epidermidis ◽  
Physiological State ◽  
Staining Intensity ◽  
Sybr Green ◽  
Tetrazolium Chloride ◽  
Viable But Nonculturable ◽  
Bacterial Rna ◽  
Nonculturable Cells ◽  
Excess Glucose

Staphylococcus epidermidis biofilms with different proportions of viable but nonculturable bacteria were used to show that SYBR green (SYBR) may be used as a probe to evaluate the bacterial physiological state using flow cytometry. Biofilms grown in excess glucose presented significantly higher proportions of dormant bacteria than biofilms grown in excess glucose with buffered pH conditions or with exponential-phase planktonic cultures. Bacteria obtained from biofilms with high or low proportions of viable but nonculturable cells were further cultured in broth medium and stained with SYBR at different time points. An association between bacterial growth and SYBR staining intensity was observed. In addition, bacteria presenting higher SYBR fluorescence intensity also stained more intensely with cyanoditolyl tetrazolium chloride, used as a probe to evaluate cellular metabolism. Accordingly, planktonic bacteria treated with rifampicin, an inhibitor of bacterial RNA transcription, presented lower SYBR and cyanoditolyl tetrazolium chloride staining intensity than nontreated bacteria. Overall, our results indicate that SYBR, in addition to being used as a component of LIVE/DEAD stain, may also be used as a probe to evaluate the physiological state of S. epidermidis cells.

scholarly journals Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater

2005 ◽  
Vol 3 (3) ◽  
pp. 249-257 ◽  
Author(s):  
James D. Oliver ◽  
Maya Dagher ◽  
Karl Linden
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Stationary Phases ◽  
Health Hazard ◽  
Physiological State ◽  
Treated Wastewater ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Nonculturable Cells ◽  
Nonculturable State

We examined the effects of chlorine disinfection on Escherichia coli and Salmonella typhimurium in secondary-treated wastewater to determine whether such treatment might induce these bacteria into the viable but nonculturable (VBNC) state. In this state, cells lose culturability but retain viability and the potential to revert to the metabolically active and infectious state. To examine the effects of chlorination on cells in different physiological states, cells from the logarithmic and stationary phases, or nutrient starved, or grown in natural wastewater, were studied. Isogenic cells with and without plasmids were also examined. Whereas a mixture of free and combined chlorine, as occurs under typical wastewater disinfection, was found to be rapidly lethal to most cells, regardless of their physiological state or plasmid content, c. 104 of the original 106 cells ml−1 did survive in the VBNC state. While we were not successful in resuscitating these cells to the culturable state, the presence of such nonculturable cells in treated wastewater offers a potential public health hazard.

scholarly journals First Evidence of Division and Accumulation of Viable but Nonculturable Pseudomonas fluorescens Cells on Surfaces Subjected to Conditions Encountered at Meat Processing Premises

2007 ◽  
Vol 73 (9) ◽  
pp. 2839-2846 ◽  
Author(s):  
Sophie Peneau ◽  
Danielle Chassaing ◽  
Brigitte Carpentier
Keyword(s):  
Pseudomonas Fluorescens ◽  
Food Industry ◽  
Striking Difference ◽  
Lag Phase ◽  
Tetrazolium Chloride ◽  
Cleaning Product ◽  
Viable But Nonculturable ◽  
Nonculturable Cells ◽  
Cleaning And Disinfection ◽  
Number Of Cells

ABSTRACT Cleaning and disinfection of open surfaces in food industry premises leave some microorganisms behind; these microorganisms build up a resident flora on the surfaces. Our goal was to explore the phenomena involved in the establishment of this biofilm. Ceramic coupons were contaminated, once only, with Pseudomonas fluorescens suspended in meat exudate incubated at 10°C. The mean adhering population after 1 day was 102 CFU·cm−2 and 103 total cells·cm−2, i.e., the total number of cells stained by DAPI (4′,6′-diamidino-2-phenylindole). The coupons were subjected daily to a cleaning product, a disinfectant, and a further soiling with exudate. The result was a striking difference between the numbers of CFU, which reached 104 CFU·cm−2, and the numbers of total cells, which reached 2 × 106 cells·cm−2 in 10 days. By using hypotheses all leading to an overestimation of the number of dead cells, we showed that the quantity of nonculturable cells (DAPI-positive cells minus CFU) observed cannot be accounted for as an accumulation of dead cells. Some nonculturable cells are therefore dividing on the surface, although cell division is unable to continue to the stage of macrocolony formation on agar. The same phenomenon was observed when only a chlorinated alkaline product was used and the number of cells capable of reducing 5-cyano-2,3-ditolyl tetrazolium chloride was close to the number of total cells, confirming that most nonculturable cells are viable but nonculturable. Furthermore, the daily shock applied to the cells does not prompt them to enter a new lag phase. Since a single application of microorganisms is sufficient to produce this accumulation of cells, it appears that the phenomenon is inevitable on open surfaces in food industry premises.

scholarly journals Dormant bacteria within Staphylococcus epidermidis biofilms have low inflammatory properties and maintain tolerance to vancomycin and penicillin after entering planktonic growth

2014 ◽  
Vol 63 (10) ◽  
pp. 1274-1283 ◽  
Author(s):  
Filipe Cerca ◽  
Ângela França ◽  
Begoña Pérez-Cabezas ◽  
Virgínia Carvalhais ◽  
Adília Ribeiro ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Staining Intensity ◽  
Sybr Green ◽  
Sybr Green I ◽  
Physiological Status ◽  
Dormant Cells ◽  
Planktonic Form ◽  
Pro Inflammatory Cytokines ◽  
Inflammatory Effect

Staphylococcus epidermidis is the most commonly isolated aetiological agent of nosocomial infections, mainly due to its ability to establish biofilms on indwelling medical devices. Detachment of bacteria from S. epidermidis biofilms and subsequent growth in the planktonic form is a hallmark of the pathogenesis of these infections leading to dissemination. Here we showed that S. epidermidis cells collected from biofilms cultured in conditions that promote cell viability present marked changes in their physiological status upon initiating a planktonic mode of growth. When compared to cells growing in biofilms, they displayed an increased SYBR green I staining intensity, increased transcription of the rpiA gene, decreased transcription of the icaA gene, as well as higher susceptibility to vancomycin and penicillin. When bacteria collected from biofilms with high proportions of dormant cells were subsequently cultured in the planktonic mode, a large proportion of cells maintained a low SYBR green I staining intensity and increased resistance to vancomycin and penicillin, a profile typical of dormant cells. This phenotype further associated with a decreased ability of these biofilm-derived cells to induce the production of pro-inflammatory cytokines by bone marrow-derived dendritic cells in vitro. These results demonstrated that cells detached from the biofilm maintain a dormant cell-like phenotype, having a low pro-inflammatory effect and decreased susceptibility to antibiotics, suggesting these cells may contribute to the recalcitrant nature of biofilm infections.

scholarly journals Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

2006 ◽  
Vol 72 (5) ◽  
pp. 3482-3488 ◽  
Author(s):  
M�nica Ordax ◽  
Ester Marco-Noales ◽  
Mar�a M. L�pez ◽  
Elena G. Biosca
Keyword(s):  
Pathogenic Bacteria ◽  
Erwinia Amylovora ◽  
Control Plant ◽  
Viable Cell ◽  
Epifluorescence Microscopy ◽  
Survival Strategy ◽  
Vbnc State ◽  
Tetrazolium Chloride ◽  
Viable But Nonculturable ◽  
Cell Counts

ABSTRACT Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.

SIGNIFICANCE OF VIABLE BUT NONCULTURABLE BACTERIA FOR SAFETY OF FOOD PRODUCTS

2019 ◽  
Vol 1 (2) ◽  
pp. 183-189
Author(s):  
A. M. Abdullaeva ◽  
◽  
L. P. Blinkova ◽  
Yu. D. Pakhomov ◽  
◽  
...  
Keyword(s):  
Food Products ◽  
Active State ◽  
Insufficient Data ◽  
Main Hypothesis ◽  
Viable But Nonculturable ◽  
Nonculturable Cells

In this review data on hazardous influence of nonculturable cells of pathogens on humans and animals, of contamination of foodstuffs is presented and also attention is stressed on properties of such cells and their effect through foodstuffs on humans and animals. Main hypothesis of formation and resuscitation of viable but nonculturable cells are elucidated. Factors that influence shifting bacteria to nonculturability and their conversion into active state are discussed. The conclusion is drawn about biohazard of viable nonculturable cells and insufficient data about their physiology and mechanisms of transition into this state and resuscitation back.

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